Volume 3, Issue 3 (2016) Tropical Plant Research

TROPICAL PLANT RESEARCH
An International Journal
Society for tropical Plant Research

ISSN (E): 2349 1183
ISSN (P): 2349 9265
Volume 3, Issue 3
S.No.
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https://doi.org/10.22271/tpr.2016.v3.i3
31 December 2016
Contents
An inoculum of endophytic fungi for improved growth of a traditional ricevariety
in Sri Lanka
W.A.D.K. Wijesooriya and N. Deshappriya*
Morphological and molecular characterization of Colletotrichum capsici causing
leaf-spot of soybean
Rajyasri Ghosh*, Sreetama Bhadra and Maumita Bandyopadhyay
Structural study of Gilbertiodendron dewevrei mono-dominant forest based on
mature individuals in the Masako forest reserve (Tshopo province, Democratic
Republic of the Congo)
Francine K. Botelanyele, Patience K. Kahola, Jean-Leon K. Kambale, Nicole S.
Assani, Esther I. Yokana, Prosper S.Yangayobo, Honorine N. Habimana, Monizi
Mawunu and Koto-te-Nyiwa Ngbolua*
First report on three new diatom species from the Hooghly District, West Bengal
Nilu Halder
Effect of various dormancy breaking treatments on seed germination, seedling
growth and seed vigour of medicinal plants
Ashwani Kumar Bhardwaj, Sahil Kapoor, Avilekh Naryal, Pushpender Bhardwaj,
Ashish Rambhau Warghat, Bhuvnesh Kumar and Om Prakash Chaurasia*
Some ethnomedicinal plants used against high blood pressure in Bargarh district
in Western Odisha (India)
S. K. Sen* and L. M. Behera
Uncultivated fodder grass for cattle
R. Prameela* and M. Venkaiah
Ecological studies of mangroves species in Gulf of Khambhat, Gujarat
Vandna Devi and Bhawana Pathak*
Floristic assessment of different habitats of Parvati Aranga wildlife sanctuary
and adjacent Tikri forest area, Gonda, Uttar Pradesh, India
Vineet Singh*, S. K. Srivastava and L. M. Tewari
Intra-specific variation in response of Neem (Azadirachta indica A. Juss) to
elevated CO2 levels and biochemical characterization of differently responding
plants
C. Buvaneswaran*, K. Arivoli, T. Sivaranjani, E. Menason, K. Vinothkumar, S.
Padmini and S. Senthilkumar*
Morphological characters of Chaetoceros lorenzianus (Bacillariophyceae)
isolated from North Arabian Sea after Tasman Spirit oil spill
Asma Tabassum*, Hina Baig and Aliya Rehman
Diversity and distribution of Litsea in Chikkamagaluru, Karnataka
S. G. Srinivas and Y. L. Krishnamurthy*
Alternaria polypodiicola, a new foliicolous fungus discovered on Microsorum
punctatum from Uttar Pradesh, India
Shambhu Kumar* and Raghvendra Singh
Floristic composition and biological spectrum of weeds in agro-climatic zone of
Nalbari district, Assam, India
D. K. Bhattacharjya* and S. K. Sarma
Pages
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31.
Contents
Differential responses of pea seedlings to salicylic acid under UV-B stress
Chanda Bano, N. B. Singh* and Sunaina
Exotic species invasion threats to forests: A case study from the Betla national
park, Palamu, Jharkhand, India
Preeti Kumari and A. K. Choudhary*
Disease progression in potato germplasm from different reaction groups against
potato virus Y in relation to environmental factors
Ata-ul-Haq, Yasir Iftikhar, Muhammad Irfan Ullah, Mustansar Mubeen*, Qaiser
Shakeel, Faheema Bakhtawar and Iram Bilqees
A note on precociouspollen germination in Woodfordia fruticosa (L.) Kurz.
Kanak Sahai*, Krishna Kumar Rawat and Dayanidhi Gupta
New species and new records of Graphis (Ostropales: Graphidaceae) from
Eastern Ghats, India
Satish Mohabe, Anjali Devi B., Sanjeeva Nayaka and A. Madhusudhana Reddy*
Seed priming with spermine ameliorates salinity stress in the germinated
seedlings of two rice cultivars differing in their level of salt tolerance
Saikat Paul and Aryadeep Roychoudhury*
Isolation and characterization of lectin from the leaves of Euphorbia
tithymaloides (L.)
Aruna A. Jawade, Shubhangi K. Pingle*, Rajani G. Tumane, Anvita S. Sharma,
Archana S. Ramteke and Ruchika K. Jain
Nutritional composition and fungi deterioration of canned tomato products
collected from Ibadan, South-western Nigeria
S. G. Jonathan, B. J. Babalola, O. J. Olawuyi, J. A. Odebode* and A. O. Ajayi
Taxonomic account of an Indian endemic, monotypic genus Adenoon Dalzell with
a note on lectotypification of Adenoon indicum Dalzell.
Bandana Bhattacharjee*, Sobhan Kumar Mukherjee and P. Lakshminarasimhan
Floristic composition and vegetation analysis of Hulikal Ghat region, central
Western Ghats, Karnataka
Vinayaka K. S.* and Krishnamurthy Y. L.
Role of dicot angiosperms in the livelihood of Mishing community in Sonitpur
district, Assam, India
Jintu Sarma and Ashalata Devi*
Finger millet (Eleusine coracana L.) grain yield and yield components as
influenced by phosphorus application and variety in Western Kenya
Wekha N. Wafula*, Korir K. Nicholas, Ojulong F. Henry, Moses Siambi and Joseph
P. Gweyi-Onyango
Bidens bachulkarii (Asteraceae-Heliantheae): A new species from Western Ghats,
India
D. G. Jagtap* and M. Y. Cholekar
A checklist of succulent plants of Ahmedabad, Gujarat, India
Ruchi M. Patel, Umerfaruq M. Qureshimatva*, Rupesh R. Maurya and Hitesh A.
Solanki
Lichens in 50 ha permanent plot of Mudumalai Wildlife Sanctuary, Tamil Nadu,
India
Komal K. Ingle, Sanjeeva Nayaka* and H. S. Suresh
Development and characterization of microsatellite markers for Osyris lanceolata
Hochst. & Steud., an endangered African sandalwood tree species
John O. Otieno*, Stephen F. Omondi, Annika Perry, David W. Odee, Emmanuel
T. Makatiani, Oliver Kiplagat and Stephen Cavers
Rice false smut [Ustilaginoidea virens (Cooke) Takah.] in Paraguay
Lidia Quintana*, Susana Gutirrez, Marco Maidana and Karina Morinigo
Pages
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ISSN (E): 2349 1183

ISSN (P): 2349 9265
3(3): 470480, 2016
DOI: 10.22271/tpr.2016.v3.i3.063
Research article
An inoculum of endophytic fungi for improved growth of a

traditional ricevariety in Sri Lanka
W.A.D.K. Wijesooriya and N. Deshappriya*
Department of Botany, Faculty of Science, University of Kelaniya, Kelaniya, Sri Lanka
*Corresponding Author: deshapri@kln.ac.lk
[Accepted: 12 September 2016]
Abstract: Use of chemical fertilizers and pesticides in rice cultivation has incurred many
environmental and health problems in Sri Lanka. Therefore, there is renewed interest in cultivating
traditional rice varieties as they are more amenable to organic farming practices. However, as the
yield of these varieties is comparatively low, strategies to enhance their performance should be
investigated. As endophytes of plants are reported to promote growth and yield of a number of
crop varieties, this study was aimed at studying the endophytic fungal assemblage present in the
traditional rice variety Kuruluthuda with a view to evaluate their capacity to enhance plant growth
and yield. Twenty seven endophytic fungal species were isolated from different parts of
Kuruluthuda rice plants collected from a paddy field cultivated using organic fertilizers in the
Gampaha district, Sri Lanka. Two frequently isolated endophytic fungal spp. i.e. Acremonium and
Arthrobotrys (frequencies of isolation 60% and 38.6% respectively) were introduced separately
and in combination torice seedlings using spore suspension and plate methods to determine their
effect on growth and yield under green house and field conditions. All endophyte inoculated plants
showed a significant difference (P 0.05) in plant growth (height, fresh weight and dry weight),
number of tillers and yield when compared withnon-inoculated plant sunder both green house and
field conditions. The effect of Acremonium and Arthrobotrys when introduced in combination
showed a significant difference (P 0.05) in the fresh weight, dry weight, tiller number and yield
(weight of seeds harvested) when compared to their individual effects under field conditions which
indicates that the two endophytes in combination can be used as a better inoculum to improve
biomass and yield of the plants of rice variety tested. This is the first report of the endophytic
mycoflora of the rice variety Kuruluthuda and their potential use for growth promotion and yield
enhancement.
Keywords: Fungal endophytes - Acremonium - Arthrobotrys - Kuruluthuda - Growth enhancement.
[Cite as: Wijesooriya WADK & Deshappriya N (2016) An inoculum of endophytic fungi for improved growth
of a traditional ricevariety in Sri Lanka. Tropical Plant Research 3(3): 470480]
INTRODUCTION
Rice (Oryza sativa L.) is the staple food of a large part of the world population (FAO 1994). Improvement of
rice grain yield has been dependent on the introduction of new high yielding varieties that are heavily dependent
on the use of chemical fertilizers and pesticides. The need for long term, indiscriminate application of chemical
fertilizers has led to hazardous influences on the environment as well as on human and animal health.
Traditional rice varieties are highly amenable to organic farming practices that cause minimum damage to the
environment and sustain soil health (Rathnabharathi 2009). Therefore, there is renewed interest in cultivating
traditional rice varieties combined with organic farming practices. However, these varieties are known to be low
producers of yield and thus their performance has to be improved to meet the demand of the highly expanding
population. Recent studies aimed at improving the productivity of plants using alternative cultivation practices
have focused on the possibility of using microorganisms to increase root growth and nutrient uptake of plants,
fix nitrogen as well as decrease plant stress and disease incidence (Montesinos 2003).
Endophytes are microorganisms that can colonize internal plant tissues without causing apparent harm to the
host (Petrini 1991). They are symbionts in plants and benefit the plant by enhancing growth directly by releasing
www.tropicalplantresearch.com
470
Received: 11 May 2016
Published online: 31 October 2016

https://doi.org/10.22271/tpr.2016.v3.i3.063
Wijesooriya & Deshappriya (2016) 3(3): 470480

.
phyto-hormones (i.e. auxins, giberallin and cytokinin) which are biosynthesized through their own pathways
(Contreras-Cornejo 2009, You et al. 2012). These additional concentrations of hormones improve root and shoot
biomass and reinforce the root system of plants which facilitates the acquisition of mineral nutrients from the
soil (Feng et al. 2000) and enable plant roots to increase nitrogen and phosphorus uptake (Ryan et al. 2008).
Indirectly, they increase the availability of the nutrients in the rhizosphere by mobilization of phosphate from
organic phosphate sources (Tarafdar & Claassen 1998) and producelow molecular weight metabolites (i.e.
mycotoxins, altechromones and flavonoids) which are required for the normal functioning of plants under
adverse conditions (Strobel 2003).
Diazotrophic bacterial and actinomycete populations of rice are reported to promote plant growth promotion
and to possess the potential for nitrogen fixation and disease resistance (Barraquio et al. 1997, Tian et al. 2004).
Fungal endophytes also have been detected in cultivated rice and they have shown to possess antagonistic or
plant growth stimulating properties (Yuan et al. 2009, Naik et al. 2009). Previous studies in Sri Lanka have
shown that some endophytes present in the traditional rice varieties Suwandel, Kaluheenati and Herath Banda
have the ability to increase the growth of plants significantly (Atugala & Deshappriya 2015, Ponnawila &
Deshappriya 2014). However, the endophytic fungal assemblage of the rice variety Kuruluthuda and their effect
on plant growth and yield has not been reported in Sri Lankapreviously.
Therefore, the present study was carried out with the objectives:
To isolate the endophytic fungal assemblage present in different plant parts of the rice variety
Kuruluthuda.
To develop method(s) of introducing endophytic fungi into seedlings.
To evaluate the effect of the most frequently isolated fungal species on the performance of plants under
green house and field conditions with a view to develop an inoculum for improved plant growth and
productivity.
MATERIALS AND METHODS
Sample collection and microscopic observation of roots
Nine week old healthy, intact plants of the traditional rice variety Kuruluthuda were randomly collected from
a field treated with organic fertilizers at Eldeniya, Sri Lankaduring the Maha season (September to February)
and transported to the laboratory in clean polythene bags. Roots were cut into 7 cm long pieces and washed
under running tap water for 20 minutes. Squashed preparations of root segments were observed under the light
microscope and the presence of root inhabiting endophytic fungi was confirmed by trypan blue staining method
(Yuan et al. 2009).
Isolation and identification of endophytic fungi of therice variety Kuruluthuda
Leaf pieces stem pieces, root segments and seeds of Kuruluthuda were used to isolate endophytic fungi.
Each plant part was cleaned separately by rinsing under running tap water for 10-15min and the cleaned plant
parts were surface sterilized using the sterilization protocols reported previously (Atugala & Deshappriya 2015).
After treatments with surface sterilizing agents, each plant part and seeds were washed with three
consecutive changes of sterilized distilled water (SDW). The edges of the surface sterilized leaf, stem and root
segments were trimmed and twenty five pieces of each of the plant parts and seeds were transferred aseptically
into petri plates containing Malt Extract Agar (MEA) supplemented with tetracycline (50 mg.l-1). After 10 days
incubation at room temperature (RT) (30C), endophytic fungal speciesthat grew out were subcultured onto
fresh Potato Dextrose Agar (PDA) plates supplemented with tetracycline (50 mg.l-1). Pure cultures of each
isolated colony was obtained by hyphal tip method (Tutte 1969) and stored at 4C. The fungal cultures were
identified based on morphological characters (i.e. features of hyphae and sporulating structures, colony
morphology, texture and color) to genus level using standard keys of identification (Domschet al. 1993).
Isolation frequencies and percentages of dominant fungi were calculated as following (Fisher & Petrini
1991, Goveas et al. 2011):
Isolation frequency = (Total number of isolates yielded in a given trial/ Total number of sample segments
in the same trial) 100
Percentage dominance = (Number of isolates collected from the samples/ Total number of leaf/stem/root/seed
samples) 100
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Inoculation of endophytic fungal species into seedlings
The effect of most frequently isolated fungal endophytes i.e. Acremonium and Arthrobotrys on rice plant
growth and yield was determined by inoculating each fungus separately and together to three day old seedlings
of the rice variety Kuruluthuda.
Seed germination
Seeds were surface sterilized as described in 2.2 above. The surface sterilized seeds were soaked overnight
in SDW and allowed to germinate by wrapping with a wet cloth for three days at RT (30C).
Inoculation of seedlings
Inoculation of seedlings was carried out using two methods, (a) spore suspension method and (b) plate
method.
(a) Spore suspension method:
10 day old cultures of Acremonium and Arthrobotrys grown on PDA plates were used to prepare the spore
suspensions. 5 ml of SDW was added to each culture and the spores were dislodged using a sterilized glass
spreader. The resulting spore suspensions were transferred to sterilized petri dishes and spore concentrations
adjusted to 1106 spores/ml.
Inoculation of the seedlings was done in the following manner:
(i) Immersion of seedlings in 40 ml of each quantified fungal spore suspension separately in a petri plate.
(ii) Immersion of seedlings in a mixture of 20 ml each of spore suspensions (40 ml in total) in a petri plate.
(iii) Immersion of seedlings in 40 ml of SDW in a petri plate (control).
Ten seedlings were used for each treatment and they were immersed in the spore suspensions/SDW
overnight at RT (30C).There were ten replicates for each treatment.
(b) Plate method:
10 day old Acremonium and Arthrobotrys cultures grown on PDA were used for the plate method and the
inoculation was carried out as follows;
(i) Seedlings were placed on each fungal culture plate separately and incubated for 23 days at RT (30C).
(ii) Seedlings were placed on Acremonium culture plates for 2 consecutive days and subsequently on
Arthrobotrys culture platesfor 2 consecutive daysat RT (30C).
(iii) Seedlings transferred onto PDA+tetracycline plates without the fungus served as the controls.
Ten seedlings were used for each treatment and there were ten replicates for each treatment.
Confirmation of entry and establishment of inocula
To test whether the inoculations were successful, randomly selected 15 seedlings subjected to each treatment
of both methods including controls were surface sterilized (75% ethanol for 30 s, 1% NaOCl for 10 min and
70% ethanol for 30 s) and transferred onto PDA plates supplemented with tetracycline (50 mg.l-1).
Determination of the effect on plant growth and yield
The effect of the endophytes was evaluated under greenhouse conditionas pot experiment and in the field
using seedlings inoculated with the fungal species separately and together by both methods of inoculation.
Pot experiments in greenhouse
Fifty seedlings selected randomly from the five replicates of each treatment were washed with SDW and
planted in polythene bags (31 cm of diameter and 24.4 cm of height) filled with soil treated with organic
fertilizers transported from a paddy field, which had been autoclaved for 20 min (121C and 15 lb.in-2). Overall
eighty bags were prepared at a rate of five seedlings per bag. Plants were watered regularly and maintained
under a temperature range 30C (day) and 20C (night) in the greenhouse for 3 months.
Field trials
Autoclaved (at 121C and 15 lb.in-2 for 20 min) coconut coir was layered on plastic trays (31 cm 20 cm)
and seedlings subjected to each treatment were planted in the trays (150 seedlings per tray). Plants were allowed
to grow under greenhouse conditions for 2 weeks with regular watering of the coconut coir. 14 day old plants
were introduced into a pre-leveled field prepared for planting and they were hand-planted separating the groups
of plants subjected to different treatments according to randomized block design (Fig. 1).
Plants were allowed to grow in the field for 4 months until maturation under controlled conditions:the water
depth was maintained evenly at 5 inches with 2 weekly additions of a liquid organic fertilizer mixture
supplemented with dolomite (green manure, cow dung and dolomite, 7:2:1).
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Figure 1. Lay out of the field trials.
Parameters Evaluated
Height, fresh and dry weights of the plants under greenhouse and field conditions were measured for each
treatment separately at 2 weeks, 4 weeks and 10 weeks after planting. The length of the crown to the end point
of the flag leaf was measured as plant height. Dry weight of the plant was measured by oven drying at 60C until
a constant weight was reached. Number of tillers per plant of the plants grown under field conditions was
measured at the panicle initiation stage (10 weeks after planting). Weight of the seeds per plant was measured at
harvesting stage. The results were analyzed statistically using ANOVA and pair wise comparisons of treatments
were done using T-test.
RESULTS
Microscopic observations of endophytic fungi in Kuruluthuda
Figure 2. Endophytic fungal structures in Kuruluthuda roots: AB, Trypan blue stained hyphae of endophytic fungal species
(10402); CD, Sclerotium-like structures of colonized endophytic fungi (10403).
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Root segments stained withtrypan blue were examined for the presence of endophytic fungal structures. Intra
and intercellular hyphae were seen in the root cortex parallel to the longitudinal axis of the roots (Fig. 2A & B),
and intra-cellular, sclerotium-like structures were observed in roots (Fig. 2C & D).
Table 1. Occurrence of endophytic fungi in different parts of Kuruluthuda plants.
Total number of
Number of fungal
Percentage frequency of
samples
species isolated
isolation
Root
25
13
52
Stem
25
6
24
Leaf
25
5
24
seed
25
3
12
Total
125
27
21.6
Note: Fungi isolated from surface sterilized plant parts placed on MEA plates. There were 25
replicates for each plant part.
Plant part
Isolation and identification of endophytic fungi from Kuruluthuda rice variety

A total of 27 endophytic fungi were isolated from 125 samples of root, stem, leaf and seeds of plants of
Kuruluthuda assessed. Endophytic fungi were isolated at a higher frequency from root segments (Table 1).
Acremonium, Arthrobotrys, Colletotrichum and Humicola showed ubiquitous colonization as they were isolated
at least from 3 different plant parts and Acremonium was the most dominant endophytic fungus present in
Kuruluthuda (Table 2).
Table 2. Dominant endophytic fungal percentage and their colonized plant part of Kuruluthuda.
Endophytic fungi
Acremonium
Arthrobotrys
Aspergillus sp1
Aspergillus sp2
Aureobasidium
Chaetomium
Colletotrichum
Curvularia
Fusarium
Humicola
Penicillium sp1
Penicillium sp2
Phoma sp1
Phoma sp2
Rhizoctonia
Rhizopus sp1
Trichoderma sp1
Trichoderma sp2
Unidentified genus 1
Unidentified genus 2
Sterile Mycelia (SM) 1
SM 2
SM 3
SM 4
SM 5
SM 6
Colonized plant part

seed , stem, leaf
root, stem, leaf
root
root
root
root
stem, leaf, seed
root
seed
root, stem, leaf
root
stem
root
stem
leaf
stem
root
root
root
root
stem
root
root
leaf
root, leaf
seed
Percentage dominance
60.0
38.6
10.4
20.8
0.8
10.4
26.4
13.6
3.2
33.6
6.4
8.0
8.0
4.8
0.8
4.0
5.6
1.6
13.6
8.8
4.0
0.8
2.4
4.8
9.6
0.8
Effectiveness of the methods used for endophyte inoculation to rice seedlings

Two of the most frequently isolated fungal endophytes i.e. Acremonium and Arthrobotrys were inoculated to
seedlings separately and in combination to test their effect on the growth and productivity of the rice variety
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Kuruluthuda using spore suspension and plate methods. To test for successful entry and colonization of
seedlings, re-isolations were carried out. Fungal species identical to those that were inoculated were re-isolated
from the seedlings inoculated using both methods. The inoculated fungal species were not isolated from the noninoculated seedlings treated with SDW (controls). These results confirmed that both methods of inoculation of
the endophytes into seedlings were successful.
Figure 3. Variation in shoot length after 10 weeks: AD, Kuruluthuda plants grown under field conditions; EH,
Kuruluthuda plants grown under greenhouse conditions; A,E, Acremonium inoculated plants; B,F, Arthrobotrys inoculated
plants; C,G, Acremonium&Arthrobotrys inoculated plats; D,H, non-inoculated plants. Plants inoculated with both
endophytic fungi show a significant increase of plant height (arrowed).
Effect of Acremonium and Arthrobotrys on growth and yield of Kuruluthuda

A significant difference (P 0.05) in plant height as well as fresh and dry weights was observed for the
plants subjected to all 3 treatments (i. Acremonium inoculation, ii. Arthrobotrys inoculation, iii. Acremonium
and Arthrobotrys combined inoculation) when compared with non-treated plants under both field and
greenhouse conditions after two, six and ten weeks (Table 3,4 & 5) (Fig 3). Plant height, fresh weight and dry
weight of all treated and non-treated plants increased with time up to 10 weeks and finally became constant at
panicle initiation stage.
Table 3. Effect of endophyte inoculation on plant height 2, 6 and 10 weeks after planting.
Treatment
After 2 weeks
Field
Greenhouse
18.7 0.28a 17.9 0.37a
18.5 0.38a 19.1 0.50a b
19.2 0.37a 20.0 0.45b
Mean plant height (cm) SE

After 6 weeks
After 10 weeks
Field
Greenhouse
Field
Greenhouse
71.8 0.98a 64.9 1.44a 106.0 1.21a
93.6 2.03a
79.5 0.91b 78.5 1.64a b 113.7 0.74b
108.5 1.93b
b
b
b
81.6 1.01 84.0 0.90
117.1 0.84
110.2 1.88b
Acremonium inoculation
Arthrobotrys inoculation
Acremonium &
Arthrobotrys inoculation
Non inoculation
14.5 0.32b 14.3 0.41c 58.3 0.93c 51.0 1.16c
98.3 0.84c
73.6 1.24c
Note: n=20; mean SE; Mean values sharing common letters in each row are not significantly different p 0.05.
Plants inoculated with Arthrobotrys showed a significant difference(P 0.05) in plant height, fresh weight,
dry weight, tiller number and yield compared to the plants inoculated with Acremonium (Table 3,4,5,6 & 7) (Fig
3 A,B & E,F). There was a significant difference (P 0.05) in fresh and dry weights in plants inoculated with
both fungal species separately and in combination in both greenhouse and field trials as compared with noninoculated plants. This was shown as early as 6 weeks (Table 4 & 5). There was no significant difference (P
0.05) in plant height and in tiller number between Arthrobotrys single inoculation and combined inoculation of
both fungal species. However it was significantly different (P 0.05) when compared to Acremonium
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inoculated plants after 6 and 10 weeks of planting (Table 3 & 6). Combined effect of two endophytic fungi
showed an increase of the fresh weight and dry weight than their individual effects after 6 and 10 weeks of
planting (Table 4 & 5).
Table 4. Effect of endophyte inoculation on fresh weight 2, 6 and 10 weeks after planting.
Mean fresh weight (g) SE

After 2 weeks
After 6 weeks
After 10 weeks
Field
Greenhouse
Field
Greenhouse
Field
Greenhouse
0.28 0.01a
0.18 0.02a 3.41 0.98a 1.71 0.38a 7.25 0.22a 5.20 019a
0.39 0.02b
0.20 0.02 a 3.85 0.78b 1.82 0.51b 7.86 0.20b 5.25 022a
b
0.40 0.03
0.21 0.02a 4.21 0.53c 2.15 0.48c 9.31 0.19c 5.74 013b
Treatment
Acremonium inoculated
Arthrobotrys inoculated
Acremonium & Arthrobotrys
inoculated
Non inoculated
0.13 0.01c
0.08 0.01b 2.24 0.37d 1.14 0.36d 6.24 0.12d 3.87 0.17c
Table 5. Effect of endophyte inoculation on dry weight 2, 6 and 10 weeks after planting.
Treatment
Mean dry weight (g) SE

After 2 weeks
After 6 weeks
Field
Greenhouse
Field
Greenhouse
0.049 0.002a
0.034 0.002a 1.095 0.23a
0.478 0.02a
a
b
b
0.050 0.003
0.050 0.02 1.214 0.16 0.584 0.035a b
a
0.052 0.003
0.055 0.02b 1.586 0.12c
0.748 0.11c
After 10 weeks
Field
Greenhouse
2.478 0.05a
1.115 0.05a
b
2.707 0.021 1.244 0.036b
3.421 0.002c
1.586 0.01c
Acremonium &
Arthrobotris inoculated
Non inoculated
0.030 0.001b 0.014 0.001c 0.894 0.08d 0.361 0.013d
1.987 0.04d 0.758 0.02d
A significant difference (P 0.05) in tiller number and yield per plant was observed in the plants subjected
to all 3 treatments compared with non-treated plants under field conditions (Table 6 & 7). Combined inoculation
of Acremonium and Arthrobotrys showed an increase of yield than the plants subjected to other two treatments
(Table 7). However data indicated that the overall effect of Arthrobotrys was higher than that of Acremonium.
There was no significant difference in plant height between plants grown under greenhouse and field condition
but the fresh weight and dry weight increased in all treated and non-treated plants under field condition when
compared to greenhouse condition (Table 3).
Table 6. Mean number of tillers at panicle initiation stage. (After10 weeks of planting)
Treatment
Mean number of tillers SE
5.5 0.14a
6.0 0.14b
Acremonium & Arthrobotrys inoculated
7.3 0.16b
Non inoculated
4.6 0.11c
Note: n=20; mean of tillers SE; mean values that do not share a letter are significantly different p 0.05.
Table 7. Mean yield/plant (g) of Kuruluthuda variety. (After 12 weeks)
Treatment
Mean yield per plant (g) SE
5.99 0.12a
7.57 0.12b
Acremonium & Arthrobotrys inoculated
9.65 0.23c
Non inoculated
4.54 0.17d
Note: n=20; mean of yield/plant SE; mean values that do not share a letter are
significantly different p 0.05. The mean weight of seeds per plant is considered as yield.
DISCUSSION
There is renewed interest in the cultivation of traditional rice varieties using organic farming practicesin Sri
Lanka due to numerous health problems such as the Chronic Kidney Disease of Unknown Etiology (CKDU)
which is spreading fast amongst farmers in the country as well as various environmental problems. The
traditional rice varieties, whilst responding well to organic cultivation practices, produce low yields and thus,
finding means of yield improvementis important. Research based on the development of effective organic
fertilizers consisting of applicable microorganism preparations for increased yield is a viable solution to the
problem. Endophytic fungi that grow as symbiontsin plants have been reported to enhance growth in a number
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of crops as well as in organic rice cultivation (Angelard et al. 2010).The ability of endophytic fungi to improve
plant growth and reduce rice blast incidence in traditional rice varieties of Sri Lanka has also been reported.
(Atugala & Deshappriya 2015, Ponnawila & Deshappriya 2014).
Therefore, the present study was carried out to test whether fungal endophytes of the Sri Lankan traditional
rice variety Kuruluthuda would show a similar capacity for growth and yield enhancement of rice plants under
green house and field conditions. Determination of the endophytic association was initialized by isolation of the
endophyticfungal assemblage present in different parts of plants of the variety Kuruluthuda. As the use of fresh
plant material is a critical requirement for successful isolation, fresh plant samples collected from the field were
used for fungal isolations within 48 hours. Direct observation of trypan blue stained squashed root preparations
indicated the co-existence of endophytic fungi inside roots without causing apparent damage to plant tissue.
Since the endophytes are a group of microorganisms thatcan spend a part of their life cycle in different parts
of the same plant (Petrini et al. 1992), most parts of the plant i.e. roots, stem, leaves and seeds were used for the
isolations. A suitable surface sterilization regime is essential to eliminate epiphytic microbes present on the
plant surface whilst maintaining the viability of the endophytes. Therefore, the most effective surface sterilizing
regimes developed in a previous study (Atugala & Deshappriya 2015) were used for the effective surface
sterilization of the plant parts used for isolations. Once surface sterilized,the plant parts were plated on MEA
medium supplemented with tetracycline (50 mg/L) as soil and endophytic fungi are reported to grow more
effectively on MEA resultingin a higher fungal yield and species richness (Bills & Polishook 1993).
Tetracycline added to the medium restrained bacterial growth until emergence of fungal colonies from the plant
segment.
Isolation of endophytic fungi from healthy Oryza sativa plants has been reported previously to yield a few
fungal genera such as Fusarium, Aspergillus, Curvularia, Penicillium, Gilmaniella and Arthrobotrys foliicola at
a high frequency (Zakaria et al. 2010). Most fungal genera isolated from Sri Lankan traditional rice varieties
Suwandel and Kaluheenati belonged to the class ascomycetes and they have been identified as being members
of commonly observed genera of soil fungi, e.g. Fusarium, Penicillium, Rhizopus, Rhizoctonia, Absidia,
Aspergillus and Paecilomyces (Atugala & Deshappriya 2015). In a comparison of endophytic assemblages
between traditional and newly improved rice varieties in Sri Lanka, higher fungal colonization and species
richness have been shownby the traditional rice varieties Suwandel and Herath Banda compared to the newly
improved variety Bg 352. Fusarium and Arthrobotrys have been identified as the most frequently colonized
endophytic fungi in Herath Banda traditional variety (Ponnawila & Deshappriya 2014). In the present study, 27
different fungal genera wereisolated from the variety Kuruluthuda. However, unlike in the previous studies, the
most frequently isolated genera were Acremonium, Arthrobotrys, Colletotrichum and Humicola. Acremonium
was mostly isolated from leaf and stem, Arthrobotrys from roots, Colletotrichum from stem and Humicola from
leaves. Colonization and isolation frequencies of isolates in each plant part revealed that Kuruluthuda has a rich
endophytic fungal diversity that is ubiquitous within the whole plant. Nevertheless, most isolates were restricted
to specified areas of the plants. A surprisingly rich endophytic community was associated with roots but
Fusarium and sterile mycelia species were restricted only to seeds.
The combined and individual effects of the two most frequently isolated fungal endophytes Acremonium and
Arthrobotryson growth and production of variety Kuruluthuda was tested under field and greenhouse conditions.
Previous studies have shown that fungal inoculation into immature roots of seedlings was successful when they
were in direct contact with fungal cultures (Fischer 1962, Herre et al. 2007) or immersed in a spore suspension
(Champion et al. 1973, Abdel-Motaal et al. 2010). In this study, both methods were used and the ability to
reisolate the same fungal species from the seedlingsas that of the inoculated species confirmed the success of
both spore suspension and plate methods in introducing the endophytes to plant seedlings. This is significant as
successful and easy introduction of inoculum into plants is an essential aspect of development of a biofertilizer.
In the present study,endophyte inoculated plants showed a significant increase of plant height, fresh weight
and dry weight (P 0.05) when compared with non-inoculated plants during a 10 week period under both field
and greenhouse conditions. These results are in agreement with previous studies carried out in Sri Lanka
(Ponnawila & Deshappriya 2014, Atugala & Deshappriya 2015) and indicate that rice plant growth can be
enhanced by introducing their endophyticmyco flora.
Mechanisms of growth enhancement were not studied in the present study. However evidence from previous
studies have indicated that fungal endophytes promote plant growth and performance in a number of ways
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including the production of siderophores, supplying biologically fixed nitrogen and by secretion of plant growth
regulators (Herre et al. 2007, Kedar et al. 2014). The endophytic fungi of Potentilla fulgensis is reported to
have the metabolic machinery to secrete plant growth hormones such as cytokinins, auxins and gibberellins
which promote seed germination and growth (Bhagobaty & Joshi 2009).
Khan et al. (2015) reported that two fungal endophytes, Fusarium and Alternaria isolated from the leaves of
Solanumnigrum, increased the chlorophyll content, root-shoot length, and biomass production of Dongjin rice
plantswhen inoculated at the seedling stage. Gas chromatography/ mass spectrometry analysis for the culture
filtrates of each fungal endophytic isolate revealed that both Fusarium and Alternaria could produce 54 and 30
.gm.L-1 of indole acetic acid, respectively.
In addition, Kedar et al. (2014) revealed a significant effect on shoot height, root length, fresh and dry
weights of shoots and roots of experimental maize plants inoculated with the endophytic fungus Phoma
compared to non-inoculated plants. It was also found that the roots of germinated seeds treated with the Phoma
extract showed development of additional root hairs allowing the absorption of more nutrients which
subsequently resulted in increased growth and biomass of plants.
Based on these reports, it can be concluded that endophytes have a number of mechanisms to enhance plant
growth and the similar trend of growth enhancement observed in the present study may also be attributed to one
or more of such mechanisms.
In most previous studies, individual effects of two endophytic fungi on host plant performances have been
tested. Waqas et al. (2012) reported that the endophytic fungi Phomaglomerata and Penicillium sp. significantly
promoted the shoot growth of rice as well as increased plant biomass and related growth parameters of
cucumber plants compared to non-inoculated plants. However, the combined effect of two endophytes has not
been evaluated in these studies. In the present study, the combined effect of two endophytic fungal species
showed an increase of the fresh and dry weights when compared with their individual effects on Kuruluthuda
plants under both field and greenhouse conditions. It can be concluded that Acremonium and Arthrobotrys may
have a synergistic effect that results inimproved plant weight. A significant difference of plant height could not
be observed between Arthrobotrys single inoculation and combined inoculation with both fungal species.
However, there was a significant increase in the height of plants inoculated with Arthrobotrys when compared
with those inoculated with Acremonium indicating that Arthrobotrys might have the ability to improve plant
height than Acremonium.
Although tillers of the Kuruluthuda variety were formed 4 weeks after planting, the number of tillers was
counted at the panicle initiation stage to obtain an accurate count of tillers per one bush. An increased number of
tillers could be observed for the plants inoculated with both fungi. It is reported that endophytic fungi associated
with many agricultural crops improve crop production significantly (Yuan et al. 2009). In this study also
endophytes inoculated Kuruluthuda plants yielded a significantly higher amount of seeds when compared with
non-inoculated plants. Plants inoculated with both Acremonium and Arthrobotrys gave a higher yield as well as
improved fresh and dry weights. Therefore it can be concluded that the combination of two endophytic fungi
can result in increased biomass and rice yield. These results were evident as early as six weeks after planting
and remained to be so uptothe harvesting stage (12 weeks). There was no difference in plant height between
plants grown under greenhouse and field conditions but increased fresh weight and dry weight could be
observed under field conditions when compared to greenhouse condition. The enhanced effect of endophyte
inoculation shown by plants grown under field conditions when compared with those grown under greenhouse
conditions was both an interesting and encouraging result.
Based on these very promising results, an inoculum consisting of the two fungal endophytes i.e.
Acremonium and Arthrobotrys could be utilized to enhance the biomass and yield of the rice variety
Kuruluthuda. Thisis the first report on the endophytic fungal assemblage present in the varietyKuruluthuda and
their potential to be used for improved productivity of the rice variety under field conditions. The possibility of
developing such an inoculum as a biofertiliser for other rice varieties after more stringent testscould be the
solution to the many problems associated with the use of chemical fertilizers in rice cultivation in Sri Lanka and
worldwide.
CONCLUSIONS
Kuruluthuda traditional rice variety consists of a high diversity of fungal endophytesin most parts of the
plant i.e. root, stem, leaf, and seeds.
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Spore suspension method and plate method can be used for successful inoculation of fungal endophytes
into seedling roots.
Acremonium and Arthrobotrys can improve the growth of rice plants of the variety Kuruluthuda and yield
under field conditions.
Combined effect of the two endophytes enhances the fresh weight, dry weight, number of tillers and yield
of rice plants significantly as compared to their effect when inoculated separately.
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ISSN (E): 2349 1183

ISSN (P): 2349 9265
3(3): 481490, 2016
DOI: 10.22271/tpr.2016.v3.i3.064
Research article
Morphological and molecular characterization of Colletotrichum

capsici causing leaf-spot of soybean
Rajyasri Ghosh1*, Sreetama Bhadra2 and Maumita Bandyopadhyay2
1
Department of Botany, Scottish Church College, 1 & 3 Urquhart Square, Kolkata, West Bengal, India
Department of Botany, University of Calcutta, 35 Ballygunge Circular Road, Kolkata, West Bengal, India
*Corresponding Author: rajyasri_5@rediffmail.com
2
Abstract: In the present investigation an attempt was made to carry out morphological and
molecular characterization of Colletotrichum isolate (CI) associated with leaf spot disease of
soybean. Pathogenicity test was carried out with the isolate and symptoms of leaf spot were found
to appear after 7 days of inoculation. The pathogen was compared with 2 isolates of
Colletotrichum capsici (CII and CIII) and one isolate of Colletotrichum gloeosporoides (CIV)
from chilli. On the basis of morphological and cultural characteristics the pathogen was identified
as C. capsici. For molecular identification random amplified polymorphic DNA (RAPD) primers
and internal transcribed spacer (ITS2) primers were tested on the genome of Colletotrichum
isolates from soybean and chilli plants and the data provided characteristic genetic evidence of
100% similarity within the CI isolate from soybean and C. capsici isolates from chilli (CII and
CIII). To confirm the molecular identification of the pathogen species specific primers
(CcapF/CcapR) were used and it was established that the pathogen is indeed C. capsici. Results of
present investigation revealed the first report of C. capsici in soybean plant.
Keywords: Soybean - leaf-spot - Colletotrichum - RAPD - ITS.
[Cite as: Ghosh R, Bhadra S & Bandyopadhyay M (2016) Morphological and molecular characterization of
Colletotrichum capsici causing leafspot of soybean. Tropical Plant Research 3(3): 481490]
INTRODUCTION
The genus Colletotrichum contains many morphologically similar taxa comprising endophytic, saprobic and
plant pathogenic fungi (Photita et al. 2004, Damm et al. 2009). Anthracnose disease caused by the
Colletotrichum species is a major problem worldwide. Among these species, C. gloeosporioides (Penz.) Penz. &
Sacc. and C. capsici (Syd.) E.J. Butler & Bisby are most frequently cited as causal agents of anthracnose (Than
et al. 2008, Gautam 2014, Ramdial & Rampersad 2015). C. capsici also has been reported to have a wide
putative host range associated with symptoms of foliar blight and leaf spot diseases (Shenoy et al. 2007). Leaf
spot disease caused by Colletotrichum capsici is the most important economic constraint which hamper,
turmeric (Curcuma longa L.) production in major turmeric growing regions of the India, and often results in
high yield losses (Adhipathi 2013, Uma Devi 2008).
The occurrence of different virulent strains of Colletotrichum species has been well documented in India
(Sharma et al. 2005, 2013). Numerous cases have been reported in which several Colletotrichum species or
biotypes are associated with a single host (Peres et al. 2002) making their identification by morphological and
physiological methods more difficult. The use of molecular marker techniques has improved the accuracy and
speed of identification of Colletotrichum spp. (Cai et al. 2009). Among these molecular techniques, RAPD
technique has been extensively used to investigate relationships among isolates of Colletotrichum spp.
(Madhavan et al. 2010, Sangdee et al. 2011). Similarly nucleotide sequence information for the 5.8S rDNA gene
and the internal transcribed spacer (ITS) region of ribosomal DNA (rDNA) have been used to design species
specific primers of members of Colletotrichum for diagnostic purposes and for phylogenetic analysis
(Thalhinhas et al. 2002).
Soybean is an important global crop that is grown in tropical, subtropical and temperate climate.
Anthracnose is a major fungal disease of soybean which is caused by Colletotrichum capsici and several related
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species. Anthracnose of soybean can cause severe symptoms and yield loss in southern Taiwan. Strains of C.
truncatum and C. gloeosporioides derived from diseased pods were noted as causal agents (Chen et al.1986).
Purkayastha & Banerjee (1990) reported anthracnose of soybean in India caused by C. dematium. Soybean
foliage is also susceptible to a number of fungal and bacterial pathogens causing leaf spots and blights. Among
leaf spot fungal diseases, brown spot disease caused by Septoria glycines and frogeye disease caused by
Cercospora sojina are most frequently encountered (Hershman 2013).
In the present investigation an attempt was made to carry out morphological and molecular characterization
of Colletotrichum isolate associated with leaf spot disease of soybean plants.
Isolation and identification of pathogens from infected plants
Fungal isolate (CI) was collected from leaves of infected soybean plants grown in the field of experimental
garden. The diseased leaf parts were cut at the advanced margin of lesions into small pieces (55 mm) and then
surface disinfected with 0.1% HgCl2 for 1 min, followed by rinsing with sterile distilled water two times. The
pieces were then transferred to potato dextrose agar (PDA) medium and incubated at 302C for 23 days. Pure
cultures of the fungi were obtained by single spore isolation method. The isolate was first identified to species
by morphological observation under a compound microscope. Two other isolates of C. capsici (CII and CIII)
and one isolate of C. gloeosporoides (CIV) from chilli were used for identification of C. capsici.
Pathogenicity test
In order to prove Kochs postulate pathogenicity test was carried out. One month old soybean plants were
sprayed with spore suspension of CI (106 conidia/ml) and covered with moist polythene bags for 48 hours under
natural condition of light and temperature (2030C). Disease intensity was measured after 5, 7, 10, 14, 21 days
after inoculation following the method of Purkayastha & Banerjee (1990). Disease symptom is necrotic spots
(lesion) on the leaves of plant. Six leaves from each plant were selected randomly and the number and size of
the lesions on each leaf were noted. On the basis of visual observation, the necrotic lesions were graded into
small, medium and large groups and a value of 0.5, 1 and 2 were given to lesions denoting their diameter of 1, 2
and more than 2 mm respectively. The number of spots in each group was multiplied by value assigned to it.
The sum total values taken together for all leaves divided by the number of test plants.
Cultural characters of Colletotrichum isolates
Growth characteristics of fungal isolates were measured in potato dextrose agar (PDA) solid medium. The
mycelial diameter and morphological nature of mycelia was recorded in solid medium. Shape and size of
conidia of the fungal isolates were also observed.
Fungal DNA extraction and RAPD analysis
For DNA extraction, each isolate was grown in 100 ml conical flasks, containing 30 ml of potato dextrose
broth, for 5 days at room temperature (282C). The mycelia were harvested by filtration and stored at -20C
for 23 days. Freeze-dried mycelium (0.5 g) was ground to a fine powder and DNA was extracted using CTAB
(hexa-decyltrimethylammonium bromide) method described by Bhadra & Bandyopadhyay (2015). RAPD
analysis was performed using 25 10-mer primers initially, among which 10 were chosen for final analysis due to
high reproducibility of the banding pattern generated (Table 1). Amplification was performed in 25 l reaction
volume consisting of 100 ng template DNA, 100 pmole of primer, 10 milimole dNTPs (Applied Biosystems,
Carlsbad, California, USA), 1.5 U Taq DNA polymerase and 10X reaction buffer containing 15 mM MgCl2
(Merck Genei, Bangalore, India). PCR was performed using Veriti 96-Well Thermal Cycler (Applied
Biosystems, Carlsbad, California, USA) with an initial denaturation step for 5 minutes at 94 oC followed by 40
cycles of 1 minute at 94oC, 1 minute at 37oC and 2 minutes at 72oC with a final extension for 10 minutes at
72oC. 1.5 % (W/V) agarose gel was used to analyse the PCR product. Size of the amplified fragments was
determined using 100bp DNA ladder (Applied Biosystems, Carlsbad, California, USA). The gel was
photographed using E-Gel Imager (Applied Biosystems, Carlsbad, California, USA).
The amplified bands thus obtained were scored as present (1) or absent (0) for data analysis. Regardless of
the intensity of the amplified band, each was treated as an independent entity. The Rp (resolving power) of each
primer was calculated using the formula Rp= 1-[2*(0.5-p)] (where p is the proportion of taxa containing the
band) (Prevost & Wilkinson 1999). The scored binary matrix was then subjected to analysis using NTSYSpc
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software, version 2.02i (Rohlf 1999). Jaccard's similarity co-efficient was calculated using SIMQUAL program
using the formula NAB / (NAB+NA+NB) where NAB is the number of amplified fragments shared by samples, NA
is the number of fragments in sample A, and, NB denotes those in sample B (Souframanien & Gopalakrishna
2004).
Amplification and Sequencing of ITS1-5.8S-ITS2 of rDNA
The four Colletotrichum isolates were further characterized by nucleotide sequence analysis after
amplification of the ITS1-5.8S-ITS2 regions using the universal primers ITS4 (5-TCC TCC GCT TAT TGA
TAT GC-3) and ITS5 (5-GGA AGT AAA AGT CGT AAC AAG G-3) (White et al. 1990), and soybean
isolate CI was used as positive control. PCR reactions were performed in reaction volumes of 50 l containing
25 ng of genomic DNA, 1PCR buffer (Merck Genei, Bangalore, India), 0.5 mM of each dNTP (Applied
Biosystems, Carlsbad, California, USA), 1 M primers, and 1U Taq polymerase (Merck Genei, Bangalore,
India). DNA amplification was performed in a Veriti 96-Well Thermal Cycler (Applied Biosystems, Carlsbad,
California, USA), and the program consisted of an initial denaturing step at 95C for 3 min, followed by 25
cycles of 30 s at 95C, 30 s at 54C, and 60 s at 72C; and a final extension step of 10 min at 72C. PCR
products, approximately 600 bp, were separated by electrophoresis in 1.5% (W/V) agarose gels and visualized
by ethidium bromide staining and were photographed using the gel documentation system. Specific bands were
then cut off from gel and the amplified DNA was extracted using Silica Bead DNA Gel Extraction Kit (Thermo
Fisher Scientific Inc., Waltham, MA USA).
DNA sequencing was performed with two primers (ITS4 and ITS5) in both directions to ensure that there
was no misreading. PCR products were sequenced by ABI 3500 Genetic Analyzer (Applied Biosystems,
Carlsbad, California, USA). Alignment and edition were carried out with the CAP3 Sequence Assembly
Program (Huang & Madan 1999) and visually corrected. Sequences were then compared using Mega 6.0
software program (Tamura et al. 2013).
PCR using Species-Specific Primers
Colletotrichum capsici specific primer pairs CcapF (5-GTA GGC GTC CCC TAA AAA GG-3) and
CcapR (5-CCC AAT GCG AGA CGA AAT G-3) previously reported by Torres-Calzada et al. (2011) were
used for rDNA amplification of all the samples. Amplification reactions were performed using protocol reported
earlier. PCR products were visualized by electrophoresis in 1.5% (W/V) agarose gels, stained with ethidium
bromide and documented using gel documentation system.
RESULTS AND DISCUSSION
Identification of pathogen
Pathogen associated with leaf spot disease of soybean was identified as C. capsici on the basis of
morphological characteristics.
Pathogenicity test
Healthy soybean plants were inoculated after one month of sowing and disease index was noted after 5, 7,
10, 14 and 21 days of inoculation. Symptoms were found to appear 7 days of inoculation and disease intensity
was found to increase with the incubation period. Disease symptom was noted as necrotic spots (lesion) on the
leaves of the plant. The results are presented in the table 1 and figures 1A & B.
Table 1. Pathogenicity test of Colletotrichum capsici (CI) on healthy soybean cultivar.
Incubation period (days)a

Disease index / plantb
5
0
7
3.5
10
5.75
14
7.25
21
8
Note: a - Days after inoculation; b - Average of 4 plants. Age of plant at the
time of inoculation: 1 month; Inoculation potential: 106 conidia/ml
Cultural characteristics
The mycelial growth of Colletotrichum isolates in solid PDA medium was measured after 12 days of
incubation. The CI, CII and CIII isolates showed dense white to greyish brown mycelia with ring like zonations
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in solid oat meal medium (Fig. 1C). C. gloeosporoides (CIV) produced pinkish white colony with cylindrical
conidia (Table 2).
Table 2. Morphological and cultural characters of strains of Colletotrichum capsici.
Cultural characters
Isolate
Colony colour Margin Topography
Zonation
Grayish brown smooth Mycelium flat growth Concentric zonation

CI
CII
CIII
Pinkish white smooth Mycelium flat growth Concentric zonation
CIV
Note: Incubation period: 12 days. a - Average of three replicates.
Colony
diametera
7.130.19
8.40.21
7.660.04
7.85 0.16
Morphological
characters
Conidia shape
Falcate, fusiform
Falcate, fusiform
Falcate, fusiform
cylindrical
Figure 1. Symptoms of leaf spot disease on infected leaves of soybean and morphological and cultural characters of the
pathogen.
Morphological characters
The conidia of Colletotrichum isolates from soybean and chilli (CI, CII and CIII) is falcate, fusiform with
acute apices. (Fig. 1D) The size ranges from 19.6521.00 m in length and 3.03.5 m in breadth. In C.
gloeosporiodes (CIV) conidia are hyaline, one celled, cylindrical. The size ranges from 812 m in length and
46 m in width.
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Molecular identification of the pathogen
i. RAPD-PCR analysis
25 RAPD primers were tested on the genome of CI, CII, CIII and CIV. The generated DNA fingerprints
were evaluated for assessing genetic similarity among the four isolates. Data generated by 10 primers were
analyzed as they reliably and reproducibly detected polymorphism among the selected isolates (Table 3). Figure
2 shows typical profile and polymorphic bands generated after amplification with the primers.
Table 3. List of primers used, their sequences, and amplified products generated.
Total number
of bands
OPA 04 5'-AATCGGGCTG-3' 35
OPA 11 5'-CAATCGCCGT-3' 29
OPB 01 5'-GTTTCGCTCC-3' 16
OPB 07 5'-GGTGACGCAG-3' 27
OPC 09 5'-CTCACCGTCC-3' 22
OPD 04 5'-TCTGGTGAGG-3' 15
OPE 03 5'-CCAGATGCAC-3' 37
OPG 08 5'-TCACGTCCAC-3' 26
OPK 17 5'-CCCAGCTGTG-3' 32
OPL 03 5'-CCAGCAGCTT-3' 37
Primer
Sequence of primers
Number of
polymorphic bands
31
24
14
23
19
14
36
23
30
36
Number of
monomorphic bands
4
5
2
4
3
1
1
3
2
1
Percentage
polymorphism
88.6
82.8
87.5
85.2
86.4
93.3
97.3
88.5
93.8
97.3
Resolving
power (Rp)
35.5
33
16.5
31.5
23.5
15
35.5
29.5
35.5
40
Figure 2. RAPD amplification patterns of four Colletotrichum isolates using the primers OPC09 and OPA11. L: Ladder; CI,
CII, CIII & CIV: Colletotrichum isolates; C: negative control.
The 10 RAPD primers produced a total of 276 amplified fragments with 250 polymorphic and 26
monomorphic bands. The average polymorphism of these primers varied from 82.8% to 97.30% with an average
of 90.05%. The resolving power (Rp) of the primers varied from 15 to 40. Table 3 summarizes the above data.
The dendrogram generated from the RAPD data using Jaccard's similarity coefficient showed a variation
from 0.10 to 1.00 among the four isolates. While the three Colletotrichum isolates (CI, CII and CIII) separated
out in a single cluster showing 0.85 similarity coefficient among them, C. gloeosporoides (CIV) showed
similarity coefficient of 0.10 with them. Based on the high similarity value of 1.0 between CI and CII it may be
assumed that both of them belong to the same species (Fig. 3).
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Figure 3. Dendogram generated using Jaccards similarity distance obtained from RAPD primers. CI, CII, CIII & CIV:
Colletotrichum isolates.
ii. Amplification and Sequencing of ITS1-5.8S-ITS2 of rDNA

To further confirm the result obtained using RAPD marker-based study, PCR amplification of the ITS region
of all the studied samples were also done using universal primers ITS4 and ITS5. A single band of 620 bp was
obtained in all cases (Fig. 4). No nonspecific banding was found. The amplified fragments were, thus, purified
and sequenced.
Figure 4. Products of PCR amplification of ITS region using primer set ITS 4and ITS 5. Lane L: Marker DNA ; CI,CII,CIII
& CIV: Colletotrichum isolates; C: negative control.
The sequence data, thus obtained, were compared using Mega 6.06 software. The sequence analysis of the
ITS region revealed 100% sequence similarity within the three isolates namely CI, CII and CIII. The ITS2
sequence of C. gloeosporoides (CIV), on the other hand, revealed significant difference with other three isolates
(Fig. 5). The sequences were also used to generate a dendrogram revealing phylogenetic relationship among the
studied taxa using UPGMA method (Fig. 6).
486
C C C C T A A A A A G G A C G T C T C C C G G C C C T C T C C C G T C C G C G G G T G G G G C G C C C G C C G G A G G A T A A C C A A A C T C T G A T T T A A C G A C G
G
A
A
A
A
T A A C T T C T G A G T A A A A C C A T A A A T A A A T C A A A A C T T T C A A C A A C G G A T C T C T T G G T T C T G G C A T C G A T G A A G A A C G C A G C A
T G C G A T A A G T A A T G T G A A T T G C A G A A T T C A G T G A A T C A T C G A A T C T T T G A A C G C A C A T T G C G C C C G C C A G T A T T C T G G C G G G C A
-
T G C C T G T T C G A G C G T C A T T T C A A C C C T C A A G C T C T -
T G C C T G T T C G A G C G T C A T T T C A A C C C T C A A G C C C C C G G G T T T G G T G T T G G G G A T C G G C G A G C C C T T G C G G C A A G C C G G C C C C G A
T T T C T T C T G A G T G A C A C A A G C A A A T A A T C A A A A C T T T T A A C A A C G G A T C T C T T G G T T C T G G C A T C G A T G A A G A A C G C A G C G A A
T G C G A T A A G T A A T G T G A A T T G C A G A A T T C A G T G A A T C A T C G A A T C T T T G A A C G C A C A T T G C G C C C G C C A G C A T T C T G G C G G G C A
A T
A A G G T A G T G G C G G A C C C T C T C G G A G C C T C C T T T G C G T A G T A A C A T T T C G T C T C G C A T T G G G A T T C G G A G G G A C T C T A G C C G T A
A A T C T A G T G G C G G T C T C G C T -
A A C C C C C A A T T T T A C T A A G G T T G A C C T C G G A T C A G G T A G G A A T A C C C G C T G A A C T T A A G C A T A T C A A T A A G C G G A G G A A
A A C C C C C A A C T T C -
Contig S4
Contig S1
Contig S2
Contig S3
Contig S4
Contig S1
Contig S2
Contig S3
Contig S4
Contig S1
Contig S2
Contig S3
Contig S4
Contig S1
Contig S2
Contig S3
Contig S4
Contig S1
Contig S2
Contig S3
Contig S4
G C T T G G T G T T G G G G C T C T A C G G -
T T G A C G T A -
T T G A C G T A -
T T G A C G T A -
G G C C C T T
G G C C C T T
G G C C C T T
A A
C A A G C C G T T
G G A C G G C C C G C C A G A G G A C C C C T A A A C T C T G T T T C T A T -
G C A G C T T C C A T T G C G T A G T A G T A A A A C C C T C G C A A C T G G T A C G C G G C G C G G C -
C A G C C C G C T C C C G G T A A A A C G -
T G A A T G T T G A C C T C G G A T C A G G T A G G A A T A C C C G C T G A A C T T A A G C A T A T C A A A A A G C G G A G G A A
Figure 5. Alignment of ITS1-5.8S-ITS2 sequences of the rRNA gene region of all accessions used in the present investigation using ClustalW. Contig S1= CI; Contig S2=CII; Contig S3:
CIII; Contig S4: C. gloeosporoides.
Contig S3
Contig S2
Contig S1
A A T T G T T G C C T C G G C G G A T -
Contig S4
T T K T A G T C G C G G R G G W C A T T A C T G A G T T A C C G C T C A T C A A C C C T T T G T G A A C A T A C C T C A A C T G T T G C T T C G G C G G G T A G G C G T
Contig S3
T K G G T C G C G G A G G W C A T T A C C G A G T T T A C A A C T C C C A A A C C C C T G T G A A C A T A C C -
T K K G G T C G C G G A G G W C A T T A C T G A G T T A C C G C T C A T C A A C C C T T T G T G A A C A T A C C T C A A C T G T T G C T T C G G C G G G T A G G C G T
Contig S2
T K K G G T C G C G G A G G W C A T T A C T G A G T T A C C G C T C A T C A A C C C T T T G T G A A C A T A C C T C A A C T G T T G C T T C G G C G G G T A G G C G T
Contig S1
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Figure 6. Dendogram generated using the UPGMA method obtained from ITS sequences. CI, CII, CIII & CIV:
Colletotrichum isolates.
iii. PCR using Species-Specific Primers

When the primer pair CcapF/CcapR were used to amplify the genomic DNA in the present study, the two
chilli isolates of C. capsici (CII and CIII) and the CI isolate from soybean tested positive with a single amplified
fragment in each taxa. The size of this fragment was 394 bp in all of the C. capsici tested. The primers,
however, used failed to amplify genomic DNA of C. gloeosporoides (Fig. 7).
Figure 7. Products of PCR amplification using C.capsici specific primer pairs Ccap F / Ccap R. Lane L: Marker DNA; CI,
CII, CIII & CIV: Colletotrichum isolates; C: negative control.
DISCUSSION
In the present investigation results of pathogenicity test indicated that Colletotrichum sp. (CI isolate) can
establish leaf spot disease in susceptible soybean cultivar. Traditional identification and characterization of
Colletotrichum species relies primarily on difference in morphological features such as colony, colour, size and
shape of conidia and appressoria. In this study morphological characterization of Colletotrichum isolates from
chilli and soybean plants were done. Results revealed that CI, CII and CIII isolates showed similar
characteristics, whereas CIV is morphologically different from the other 3 isolates. This result is in agreement
with a previous study by Sandgee et al. (2011) who found a morphometric overlap of conidial size among
Colletotrichum species. Thangamani et al. (2011) performed morphological and physiological characterization
of Colletotrichum musae, the causal organism of banana anthracnose. However morphological characterizations
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are not always adequate for reliable differentiation among species of Colletotrichum. Thus molecular methods
have been employed successfully to differentiate between populations of Colletotrichum from many hosts. In
the present investigation, RAPD primers were tested on genome of four Colletotrichum isolates. Analysis of
data generated using 10 RAPD primers exhibited polymorphism varying from 82.76% to 97.30% with an
average of 90.05%. Cluster analysis using Jaccards coefficient clearly separated the isolates of chilli and
soybean from C. gloeosporioides. RAPD analysis was also performed by Ratancherdchai et al. (2007) on 18
isolates belonging 2 species, C. gloeosporiodes and C. capsici isolated from three varieties of chilli, i.e. chilli
pepper (Capsicum annum), long cayenne pepper (C. annuum var. acuminatum) and Birds eye chilli (C.
frutescens). Their study showed that a clear difference could be established between C. gloeosporiodes and C.
capsici. The three C. capsici isolates analysed in the present study show similarity of 0.851.00 among
themselves. The existence of molecular variability among isolates of C. capsici that differed in virulence was
earlier established by Srinivasan et al. (2010) using RAPD markers. The result of RAPD in the present
investigation was further confirmed by sequence analysis of the ITS region which revealed 100% sequence
similarity between the two C. capsici isolates of chilli and CI isolate of soybean. C. gloeosporoides, on the other
hand, revealed significant sequence difference with C. capsici isolates CI. Freeman (2008) used sequence
analysis of ITS region to establish similarity between Colletotrichum acutatum isolates from almond and
strawberry. Shi et al. (2008) had used ITS universal primer pair ITS1-F/ITS4 to characterize Colletotrichum
acutatum and C. gloeosporioides isolates from owering dogwood (Cornus orida). Katoch et al. (2016)
carried out Metageographic population analysis of Colletotrichum truncatum associated with chili fruit rot and
other hosts using ITS region nucleotide sequences. Molecular characterization of the soybean isolate was finally
established by using species specific primer which revealed that this isolate and two other C. capsici isolates
tested positive with a single amplified fragment in each taxa. C. gloeosporoides, however, failed to produce any
amplified DNA fragment using these primers. The results of molecular characterization and morphological
identification thus established that the CI isolate from soybean is C. capsici. C. capsici was not reported in
soybean till date. Results of present investigation revealed the first report of C. capsici in soybean plant.
ACKNOWLEDGEMENT
Scientific assistance received from UGC, Govt. of India is gratefully acknowledged.
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ISSN (E): 2349 1183

ISSN (P): 2349 9265
3(3): 491500, 2016
DOI: 10.22271/tpr.2016.v3.i3.065
Research article
Structural study of Gilbertiodendron dewevrei mono-dominant

forest based on mature individuals in the Masako forest reserve
(Tshopo province, Democratic Republic of the Congo)
Francine K. Botelanyele1, Patience K. Kahola1, Jean-Leon K. Kambale1, Nicole S.
Assani1, Esther I. Yokana1, Prosper S.Yangayobo2, Honorine N. Habimana2,
Monizi Mawunu3 and Koto-te-Nyiwa Ngbolua4*
1
University of Kisangani, Biodiversity monitoring centre, Department of Ecology and Botanical

Resources Management, P.O. Box 2012 Kisangani, Democratic Republic of the Congo
2
University of Kisangani, Faculty of Science, Department of Ecology and Botanical Resources
Management, P.O. Box 2012 Kisangani, Democratic Republic of the Congo
3
Department of Agronomy, College of Uige Polytechnic, University Kimpo Vita, Republic of Angola
4
University of Kinshasa, Faculty of Science, Department of Biology, Democratic Republic of the Congo
*Corresponding Author: jpngbolua@unikin.ac.cd
Abstract: The goal of the study is to determine the vegetal composition and structure based on
mature trees in the Masako Forest Reserve. A transect was installed in a mono-dominant forest
with Gilbertiodendron dewevrei at 5 km from the guest house. For transect of 2,100 m, we placed
a 500 m perpendicular line to the survey to a plot of 2,500 square meters in a G. dewevrei forest.
An inventory was carried out in an area of 3 ha, consisting of 12 plots of 50 50 m. All trees with
a diameter equal or greater than 30 cm were inventoried, and divided into eight size classes
according to their dbh (diameter at breast height) dimension: 40 cm; 40 to 49.9 cm; 50 to 59.9
cm; 60 to 69.9 cm, 70 to 79.9 cm; 80 to 89.9 cm; 90 to 99.9 cm and 100 cm. To facilitate the
inventory, the survey plots were divided in two subplots of 1,250 m2. For the entire forest, we
found an overall density of 86.3 stems per hectare of which 63.3 stems/ha belonged to G.
dewevrei. We also found that the basal area for an individual tree was on average 29.5 m2.ha-1 for
the entire forest, and 24.2 m2.ha-1 for G. dewevrei.
Keywords: Mono-dominant forest - Gilbertiodendron dewevrei - Masako Forest Reserve.
[Cite as: Botelanyele FK, Kahola PK, Kambale J-LK, Assani NS, Yokana EI, Yangayobo PS, Habimana HN,
Mawunu M & Ngbolua K-t-N (2016) Structural study of Gilbertiodendron dewevrei mono-dominant forest
based on mature individuals in the Masako forest reserve (Tshopo province, Democratic Republic of the
Congo). Tropical Plant Research 3(3): 491500]
INTRODUCTION
Tropical rainforests are among to the most diverse terrestrial ecosystems of the world (Borah 2016), but they
also contain zones which are dominated by a single species (mono-dominant forests). These zones pose a great
enigma in tropical ecology. The mono-dominance leads to a change in the vegetal composition of the forest in
which sun-loving species dominate (Fonty 2011). The high diversity of tree species, which is characteristic for
tropical forests, is as well a permanent source of scientific questions as a strong constraint to improve our
knowledge of the functioning of the forest ecosystem. The answers explaining the preservation of this diversity
oppose the deterministic or stochastic mechanisms maintaining this high diversity (Blanc et al. 2003). However,
in tropical rainforests, there are areas of low diversity too (Richards et al. 1952), where the canopy trees are
dominated by one species (Richards et al. 1996). When this species reaches 50% of the relative diversity, it is
considered to be a mono-dominant species (Hart et al. 1989), single-dominant Forest sensu (Connel et al. 1989).
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This is already known and documented in the tropical forests of Asia, where Drybalonopsa romatica
(Dipterocarpaceae) is considered a mono-dominant species (Reitsma 1988).
In the Congo basin, Gilbertiodendron dewevrei (De Wild.) J. Leonard forms extensive stands, which, in
some cases, are virtually monophyletic (Whitmore 1984, Gerard 1960, Kouob 2009). Unlike heterogeneous
forests, interest on the origin and preservation of mono-dominant tropical forests is recent (Toft et al. 2003). It is
assumed that the massive and synchronous fruiting, low diaspores predation, tolerance to low light levels, and
ecto-mycorrhizal symbiosis would be the basis for the survival of this forest type. Although they only represent
a small fraction of the area of tropical forests, mono-dominant stands were described from all around the tropics.
These settlements do not conform to the general pattern and it can be assumed that one or more processes
controlling diversity have been altered there. Also the understanding of the mechanisms leading to these species
imposing their mono-dominance can shed a (negative) light on the processes allowing the coexistence of many
species (Gross et al. 2000).
According to McGuire (2007), the structure of a G. dewevrei forest is the result of the impact of the port and
the social temperament of this species on the light factor (Louis 1947). Therefore, we decided to approach the
problems by focusing on the G. dewevrei forest, where changes are easily noticeable, not only because of its
wide distribution in the tropical forest, but also because of its great vegetation diversity.
The structural profiles pose a number of problems, which are sometimes difficult to solve, requiring a lot of
time and resources (Mabay 1994). This is also the reason why we based our description of the morphological
appearance of the forests on the vegetation composition, vertical and horizontal structure of mature individuals
(dbh 30 cm) in the G. dewevrei mono-dominant forest. As such, given the extent to which inventories were
made, we expected to be closer to the physionomical reality, offering one or two structural profiles. This
research was motivated by the need to identify the basic data on the structural composition and plant species
diversity of the G. dewevrei forest, based on mature individuals (dbh 30 cm).
METERIALS AND METHODS
Study site
Our study was performed in the Masako Forest Reserve, which is located about 14 km northeast from the
city of Kisangani (DRC) on the old road towards Buta (0 36' 30.4" N 25 15' 38.9" E) at an altitude of about
500 m (Sonk 1998). It covers an area of about 2,104 ha of which is occupied by primary forest (Northeast) and
at least 2/3 by secondary forests (Northwest). The area was visited during three field trips of ten days (March,
April and May 2009).
The structure of mature individuals (dbh 30 cm) and dominant species (depending on the distribution of
tree frequencies classes and the diameter structure) are aspects that have been selected to meet our goal:
understanding the particular physiological nature of the primary G. dewevrei forest in Masako, as these aspects
allow the easy identification of the balanced state of a plant formation.
Location of the transect
The transect was traced in the Masako monodominant G. dewevrei forest with the main axis directed in a NS direction and reaching 5 km from the camp site. The forest area was divided into plots and transects arranged
to pass through a series of tributaries to characterize and visualize the key areas in the various topographical
conditions (Mboengongo 1999).
Description of the transect
Along the 2,100 m main axis of the transect, we drew 500 m long perpendicular lines at 100 m intervals,
giving a total of 12 lines (Fig. 1). Along these lines, random plots were selected to survey for G. dewevrei.
Each of these plots measured 50 50 m (2,500 m2).
Data collection
All trees with dbh 30 cm were surveyed and their coordinates (x,y) within the plot, their diameters at a
height of 1.30 m above the ground (= breast height [dbh]) were registered.
Data analysis
The structure diversity is defined by a set of parameters (plant diversity, density, distribution, vertical
distribution, etc.) and dimensions in the plots as well as by the relationships that may exist between these
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parameters. The latter were calculated using the model proposed by (Makana 1989, Gillet et al. 1991). Besides
of these quantitative analyses, biodiversity indices were also calculated.
Diversity indices
1. Margalef diversity index (DMg)
This index is calculated by the following formula:
DMg = (S1) / ln (N).
Where, S number of species and N = total number of individuals.
2. Menhinick diversity index (DMn)
This index is calculated as follows:
DMn = S /N
Where, S = number of species, N = total number of individuals.
3. Simpson diversity index (DS)
This index measures the probability that two individuals, that were selected by chance, belong to the same
species:
DS = pi2
with pi ni/N
Where, ni = number of individuals belonging to a given species i, N = total number of individuals.
4. Shannon-Wiener diversity index
According to Lejoly (1993) and Danais (1982), the Shannon-Wiener index measures the average amount of
information presented by the indication of the species of an individual to the collection.
H' = -(piln pi) with pi ni/N
Where, N = total number of individuals (i.e. trunks), ni = number of individuals belonging to a given
species i (between 0 and N), pi ranges from 0 to 1. S
Figure 1. Schematic representation of the sampling plots in the Masako Gilbertiodendron dewevrei mono-dominant forest.
Variation Coefficient (VC)

This coefficient is used to compare two standard deviations, especially when the means are different. It also
allows us to evaluate the magnitude of dispersion of data. The high value of VC indicates the larger of the
dispersion around the mean (Frontier 1993, Legendre et al. 1998).
VC = 100/M
Where, - variance, M - mean
If VC 15%, the dispersion is low or less pronounced and the series is homogeneous. If VC is between
15% and 30%, the dispersion is somewhat weak and the series is relatively homogeneous, However, if VC >
30%, the dispersion is most pronounced or high and the distribution is heterogeneous.
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Diametric and structural analysis
The total diametric structure or distribution of stems per diameter class is determined by taking into account
the individuals of every species (Magurran 2004). This value gives information on the stability of the stands. It
can also be calculated for specific species and in that case, it represents the specific structure. The diametric
structure indicates the number of stems inventoried by diameter classes. The Cartesian coordinates (x, y) of all
individuals Gilbertiodendron dewevrei (with dbh 30 cm) were registered to accurately characterize the
organization of the trees in each plot. The basal area is expressed in m2 (Rollet 1974) is calculated for each
individual by means of the formula ST = D / 4 where D is the dbh.
Quantitative study
Quantitative determination of the data results in their structural nature consisting of a set of parameters,
including the spatial distribution, species density and the relationships that interfere with these (Gounot 1969).
The abundance or relative density of a species and a family is calculated as the total number of individuals of a
species or family in the sample multiplied by 100:
RDs = (ns / N) x 100
Where, ns is the number of individuals for a given species and N is the total number of individuals in the
sample.
RDf = (nf / N) x 100
Where, nf is the number of individuals for a given family and N is the total number of individuals in the
sample.
The relative dominance of a species or family is determined by the basal area occupied by a species or
family in total basal area and is multiplied by 100 according to following formula:
ts st100
and for the family tf st100
Where, ts is the basal area of a species, tf the basal area of a family and St the total basal area in the
sample
Density
Density is defined as the number of stems per surface unit. There are several expressions of density.
However, the most commonly used is the number of stems per hectare (N/ha). Unfortunately, this expression of
the density does not take into account the size of trees.
Vegetation composition and density
The floristic study in the Masako G. dewevrei mono-dominant forest resulted in an overall number of 259
individuals with dbh 30 cm, divided over 36 species, 34 genera, 19 families (Fig. 2).
Figure 2. Floristic composition of Gilbertiodendron dewevrei mono-dominant forest at the Masako Forest Reserve.
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The Gilbertiodendron dewevrei dominate the sample with 75%, followed by the Anonidium mannii and
Trilepisium madagascariensis with 3% each, followed by the Funtumia africana with 2%, Klainedoxa
gabonensis (1%) and other species are each represented with 16%.
D
Figure 3. Gilbertiodendron dewevrei (De Wild.) J.Leonard: A, Seeds; B, Seedling; C, Stem with bark; D, Habitat.
Stocking densities of individuals in the Masako G. dewevrei mono-dominant forest

The 3 ha sample area in the Masako G. dewevrei mono-dominant forest contained 259 trees with dbh 30
cm. On average, there were 86.3 trees per ha. Of these trees, 190 belonged to G. dewevrei, giving an average
per hectare of 63.3.
Quantitative analysis of the floral data
The global basal areas for the Masako G. dewevrei mono-dominant forest is 29.5 m/ha and 24.2 m/ha for
G. dewevrei individuals.
The species abundance in the G. dewevrei mono-dominant forest at Masako
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The current study indicates that the dominant species at Masako are: Gilbertiodendron dewevrei, Musanga
cecropioides, Anonidium mannii, Trilepisium madagascariensis, Funtumia africana, Klainedoxa gabonensis,
Petersianthus macrocarpus and Panda oleosa (Fig. 3).
Vegetation structure by diametric class
The diameter distribution of all species combined (total structure) is one of the stand's characteristics
reflecting an equilibrium constant, which is the existence wherever moist evergreen forests are in their original
state (Fournier et al. 1983). To highlight the disturbed state of forest plant community proposed a structural
analysis for the species that account for the unbalanced or balanced state of the forest.
Diversity indices
The alpha () diversity was calculated using the following indices: Shannon-Wiener, Menhinick, Margalef
and Simpson. This allows assessing the diversity of each group, according to the species distribution, more
accurately. Margalev index gives the highest value (6.299). This shows that the Masako G. dewevrei monodominant forest is more diverse in species.
It has a good evenness between the species studied. This demonstrates the dominance of the G. dewevrei
forest on others in terms of species richness. The Simpson index, meanwhile, shows that the diversity of sites is
not so variable because the value obtained (0.458) represents a very low diversity. On the other hand, the
Menhinick index, which is based on species richness, presents a distinctly lower value (2.237) relative to
Margalef index, where the number of individuals is relatively low. Comparing the Menhinick index with the one
from Margalef reveals that both indices almost evolve in the same way.
There is a minor difference at the species level, where the Margalef index results in higher values if there is a
higher number of individuals, while that of Menhinick is low. In the current study, the Shannon index is
relatively low as it represents the sum of the information given by the frequency of the various species over the
3 ha sampled surface.
Statistical parameters
The data for the G. dewevrei trees occurring in the twelve 50 50 m sample plots of the Masako
Gilbertiodendron dewevrei dominated forest are presented in tables 1 and 2.
Table 1. Number of Gilbertiodendron dewevrei trees with dbh 30 cm and distribution of diameter registered in the twelve
50 50 m sample plots.
Sample plot
P1
P2
P3
P4
P5
P6
P7
P8
P9
P10
P11
P12
Total
Average
Variance
Standard deviation
CV(%)
Number of trees
14
16
16
12
21
23
17
17
17
12
17
8
190
15,8
16,15
4,01
25,37
Diametric Class
30-39,9
40-49,9
50-59,9
60-69,9
70-79,9
80-89,9
90-99,9
100
Total
Moyenne
Variance
Ecart-type
CV(%)
Number of trees
31
25
28
30
35
13
10
18
190
23,75
82,21
9,06
38,14
The average number of G. dewevrei trees over the sampled plots is 15.8 (Table 1). The values for the
individual sample plots are more or less around this average value, with one lower value for P12 and two higher
ones for P5 and P6. The coefficient variation is between 15 and 30% indicating that the dispersion is somewhat
weak. The distribution of individuals in the different plots is considered relatively homogeneous. This indicates
that on average, there are 23.7 G. dewevrei trees in each diametric class. However, in most of the diametric
classes the number of trees differs considerably from that average. The coefficient variation exceeds 30%,
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indicating that the dispersion is strong and that the diametric distribution of G. dewevrei should be considered
heterogeneous.
Over the 12 sampled plots, there were on average 21.6 trees with dbh 30 cm. The low value registered for
P12 confirms the low value for G. dewevrei trees. This is also the case for the high values in P5 and P6,
although these are less extreme than found for G. dewevrei. The coefficient variation is low (less than 15 %),
indicating that the plots form a homogenous sample.
Table 2 shows that in the G. dewevrei forest, there were on average 32.4 trees in each of the diametric
classes. With the exception of classes 50 to 79.9 cm, most classes have a value which is quite different from the
average value. As was already shown in figure 4 the smaller classes have a much higher number of trees, and
the larger classes have a much lower number. The tree class of 100 cm contains an elevated number of trees,
which might be explained as being the result of including a much wider range of diameters, with trees up to 136,
6 cm the coefficient variation exceeds 30%, confirming the heterogeneity of the sampled plots in relation to the
diameter of the trees.
Table 2. Number of trees with dbh 30 cm and diameter distribution registered in the twelve 50 50 m sample plots.
Sample plot
P1
P2
P3
P4
P5
P6
P7
P8
P9
P10
P11
P12
Total
Average
Variance
Standard deviation
CV(%)
Number of trees
22
23
20
23
24
26
19
21
19
21
23
18
259
21,58
5,53
2,35
10,88
Diametric Class
30-39,9
40-49,9
50-59,9
60-69,9
70-79,9
80-89,9
90-99,9
100
Total
Moyenne
Variance
Ecart-type
CV(%)
Number of trees
65
40
35
33
35
18
10
23
259
32,375
124,07
11,13
34,37
Number of trees
70
60
50
40
30
20
10
0
30-39,9
40-49,9
50-59,9
60-69,9
70-79,9
Diametric classes
80-89,9
90-99,9
100
Figure 4. Vegetation structure by diametric class at Masako forest reserve.
The present study revealed that the sample area (3 ha) in the Masako G. dewevrei mono-dominant forest
contained 259 trees with dbh 30 cm. On average, there were 86.3 trees per ha. Of these trees, 190 belonged
to G. dewevrei, giving an average per hectare of 63.3. Lomba (2007), working in the G. dewevrei monodominant forest at Yoko, found 97 trees with dbh 30 cm in a 3 ha plot, resulting in an average of 32.3 threes
per ha. 74 of the trees belonged to G. dewevrei (or 24.7 ha-1). These numbers indicate that at Yoko fewer mature
trees were occurring, both for all species combined and for G. dewevrei separately. This is mainly due to human
action as farmers harvest the average diameter ( 40 dbh) of species for commercial interests.
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Quantitative analysis of the floral data revealed that the global basal areas for the Masako G. dewevrei
monodominant forest is 29.5 m.ha-1 and 24.2 m.ha-1 for G. dewevrei individuals. For the Yoko forest, Masiala
(2009) calculated these as 24.4 m.ha-1 and 22.7 m.ha-1, respectively. This allows us to hypothesise that the
forest at Yoko is still evolving and that this is no longer the case for the forest at Masako, which also has a much
more disturbed character.
By comparing the species abundance in the G. dewevrei mono-dominant forest at Masako with the work of
other researchers, the current study indicates that the dominant species at Masako are: Gilbertiodendron
dewevrei, Musanga cecropioides, Anonidium mannii, Trilepisium madagascariensis, Funtumia africana,
Klainedoxa gabonensis, Petersianthus macrocarpus and Panda oleosa. At Yoko, Masiala (2009) found the
dominant species to be: Gilbertiodendron dewevrei followed by Scorodophloeus zenkeri, Julbernardia seretii,
Gilbertiodendron kisantuensis and Grossera sp. In the forest of Uma, Katembo (2013) found that the dominant
species are: Gilbertiodendron dewevrei (29%), followed by Cola griseiflora (8%), Diospiros sp (6%) and other
species. The species composition varies from site to site.
In the three sites (Masako, Yoko and Uma) the same trend in abundance of Gilbertiodendron dewevrei was
encountered. However, in the two following locations, this was not the case. Indeed, in Mbiye Island, Kambale
(2009) found that Coelocaryon botryoides was the most abundant species, followed by Gilbertiodendron
dewevrei, Diospyros boala, Pycnanthus angolensis and Cleistanthus mildbraedii.
The diameter distribution of all species combined (total structure) is one of the stand's characteristics
reflecting an equilibrium constant, which is the existence wherever moist evergreen forests are in their original
state (Fournier et al 1983). To highlight the disturbed state of forest plant community proposed a structural
analysis for the species that account for the unbalanced or balanced state of the forest.
Nshimba (2008) indicated that Gilbertiodendron dewevrei forest at Uma and the mixed forest on the Mbiye
Island have the form of a "mirrored J" for the stems with dbh 30 cm, whereby the class size 3039.9 cm
accounts for the highest number of trees and subsequent diametric classes all have lower values. These forests
have the typical diametric structure of natural forests, which is contrary to the Masako Gilbertiodendron
dewevrei.
Forest showing that the histogram has the form of a "mirrored J with a bulbous tail". The diametric classes
between 40 and 70 cm dbh contain much higher numbers of trees, which indicate that the Masako forest is
unbalanced and this is due to human action. Farmers harvest the average diameter of species for commercial
interests.
CONCLUSION
The current study has focused on the forest structure of a Gilbertiodendron dewevrei monodominant forest
based on tree species in the Masako Forest Reserve. An inventory of the mature trees (dbh 30 cm) was made
on a 3 ha area, which was composed of 12 plots of 50 50 m each, where all trees meeting this condition were
surveyed.
o In the 3 ha sample area, 259 meeting the dbh condition were registered. The number of trees is 259 for the
forest G. dewevrei in 3 ha. Trees belonging to the Fabaceae family were found to be the most commonly
occurring group (85% of species).
o The results showed that the overall tree density is 83.3 stems/ha and 63.3 stems/ha for G. dewevrei.
o In the forest, the overall basal area is 29.5 m.ha-1 and for G. dewevrei this is 24.2 m.ha-1.
o The vegetation was classified in eight diameter classes: 3039.9 cm; 4049.9 cm; 5059.9 cm; 6069.9 cm;
7079.9 cm; 8089.9 cm; 9099.9 cm and 100 cm.
o As illustrated by the high values for the variance coefficient (exceeding 30%), the diametric distribution is
heterogeneous for both the overall sample and G. dewevrei alone.
Regarding the distribution of the number of trees over the sampled plots, the variation coefficient has values
between 15 and 30 %, indicating that the forest is relatively homogeneous. Additional research is needed to find
ways to stop the decrease and to determine measures enabling to prevent the forest overexploitation.
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ISSN (E): 2349 1183

ISSN (P): 2349 9265
3(3): 501507, 2016
DOI: 10.22271/tpr.2016.v3.i3.066
Research article
First report on three new diatom species from the Hooghly

district, West Bengal
Nilu Halder*
Department of Botany, Raja Peary Mohan College, Uttarpara-712258, Hooghly, West Bengal, India
*Corresponding Author: niluhalder1@gmail.com
Abstract: The present paper was communicated with the morpho-taxonomic description of three
diatom taxa belonging to the order Pennales of the class Bacillariophyceae namely Fragilariopsis
cylindrus, Achnanthidium lineare and Rhopalodia gibba var. ventricosa. Among them, the former
species is rare in occurrence within freshwater ecosystem while the latter two species are found
sparsely to abundant in pond, water reservoirs and other types of aquatic bodies. The limnological
characteristics that supported their occurrences in water bodies were recorded and found to be
congenial for their growth. The pH of water in studied aquatic bodies was observed alkaline and
acceptable quantity of phosphate, nitrate-nitrogen and silica along with other physico-chemical
parameters of waters were also noted. The above mentioned all three diatom taxa are new
taxonomic reports from this district of West Bengal, India.
Keywords: New report - Diatoms - Hooghly - West Bengal - India.
[Cite as: Halder N (2016) First report on three new diatom species from the Hooghly district, West Bengal.
Tropical Plant Research 3(3): 501507]
INTRODUCTION
Diatoms are a major group of microscopic algae and the most common types of phytoplankton which are
found in every habitat where water is present (Stoermer & Smol 1999) and their (both fossils and living forms)
well preserved siliceous frustules /cell walls make them ideal tools for various applied applications. Recently,
they have been using in the other fields of studies as indicators of oil and gas exploration processes. They are
now successfully used in comprehensive forensic examinations (Dwivedi & Misra 2015) to detect the suspicious
persons or murderers for crime investigations. It has been reported that their growth rate is faster in comparison
to other phytoplanktonic taxa (Wetz & Wheeler 2007). Thus, diatoms are important algal flora among the
eukaryotic algae. They are beautiful microscopic organisms with various kinds of fabulous characteristics
ornamentations on their frustules or cell walls. Especially, the presence of accessory pigments like fucoxanthin
and universal -carotene give them characteristic golden coloration. Generally, they grow in single cells as
unicellular forms and sometimes form chains or simple colonies of various shapes like filaments or ribbons,
fans, zigzags or stars in the aquatic bodies. They are capable of growing in different trophic levels of the water
bodies. As the siliceous cell walls contain hydrated silicon dioxide (SiO2) they do not decompose after death and
henceforth, diatom beds or diatomite are being used as an important tool to study paleoecology, correlation
analysis and to interpret or predict the phylogenetic evolutionary lineages as well as to calculate relative age
dating of rocks being an important constituent of rock-forming microfossils.
The diatom flora is diverse in fresh water bodies, brackish and marine ecosystems and one of the richest
algal groups in India due to having wide range of climate, topology and natural habitats. Their abundant growth
is controlled by the physical as well as chemical conditions of water. According to You et al. (2009) their
diversity depends on gradients of water, nutrients availability, pH, temperature and altitudes of the habitats.
Therefore, analysis of water has a great importance in the ecological study of diatoms.
Fragilariopsis Hust. is a planktonic diatom comprising of living as well as fossil species (Lundholm & Hasle
2008). It is characteristically ribbon-shaped and valves outlines are linear with rounded apical ends. In the recent
years, considerable taxonomic revisions have been made on the nomenclatural concept and generic placement of
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Halder (2016) 3(3): 501507

.
monoraphid achnanthoid diatoms since the publication of Lange-Bertalot & Krammer (1989). They first
removed the genus Achnanthes Bory from the family Achnanthaceae Ktz. and placed into two genera
Achnanthidium Ktz. and Eucocconeis Cl. ex Meist. under the family Achnanthidiaceae D.G. Mann based on
morphological and ecological features (Round et al. 1990, Potapova 2012). After that, a number of new genera
have been created by splitting of Achnanthes Bory sensu lato. However, there are still a number of species, that
have not yet been studied by LM (light microscopy) and SEM (scanning electron microscopy) or not transferred
within this genus. The genus is abundant in rivers, streams and springs. They inhabit in clean and polluted
waters. Their cells are small linear-lanceolate to lanceolate-elliptic with less than 30 m long and less than 5 m
broad; consists of a concave raphe valve (RV) and a convex rapheless valve (RLV); uniseriate striae present on
the RV which are comparatively denser towards the apices and the fine raphe is either straight or turned to one
side (Ponader & Potapova 2007).
The genus Rhopalodia O. Mll. includes 37 species of which at least 26 species are known only from the
type samples from all over the world (VanLandingham 1967, Krammer 1988). It exhibited broader range of
distributions, including fresh water, brackish and even marine environments. The valves are bracket-shaped in
valve view with swollen in middle, indented at the central nodule, the apices are bent acutely. In girdle view,
valves are lanceolate-elliptical, strongly swollen in the middle of the valve with broadly rounded apices. Raphe
is excentric and chromatophore is single, laminate with irregularly margins.
The present work is focused on the morpho-taxonomic investigation of the fresh water diatom flora of the
class Bacillariophyceae from Hooghly district, West Bengal, India. Few taxonomic works had been reported
earlier from India (Venkataraman 1939, Biswas 1949, Gonzalves & Gandhi 1952, Gandhi 1958, Sreenivasa &
Duthie 1973, Anand & Kant 1976, Sarode & Kamat 1979, 1980, Barhate & Tarar 1981, Das & Santra 1982,
Patel & Patel 1982, Venkateswarlu 1983, Prasad et al. 1984, Somshekar 1983, 1984, Chaturvedi 1985, Roy &
Sen 1985, Pal et al. 1986, Maity et al. 1987, Shukla & Shukla 1987, Santra et al. 1989, Pal & Santra 1990,
Banerjee & Santra 2001, Misra 2005, Bhakta et al. 2011, Das & Adhikary 2012, Tripathi et al. 2012, Dwivedi &
Misra 2014). Except a single report (Halder & Sinha 2015) there is no work in relation to exploration of diatom
flora from this locality of West Bengal. Therefore, the present work was undertaken from this area. The main
objectives of the present work were to identify, explore the diversity of diatom algal flora and documentation of
them in respect of ecology from this state.
The diatom specimens had been collected from different places viz. Chinsurah (22.90' N; 88.39' E), Ganga
river at Tribeni (22.99' N; 88.39' E) and Kuntighat (23.01' N; 88.41' E) of Hooghly district in West Bengal,
India. The light microscopic (LM) taxonomic study of the cleaned diatom specimens was made under Olympus
compound microscope with camera attachment (Model No. CH20i) and photographs were taken using Canon
A480 camera. Samples were preserved in 4% formalin. The organic contents particularly calcium and irons
were removed from the diatom samples by the acid digestion method in which 4 ml. of concentrated HCl (30%)
and 2 ml. of saturated potassium permanganate (KMnO4) solution were added with 2 ml. of diatom sample as
mentioned in materials & methods (MitiKopanja et al. 2014). Cleaned diatoms were mounted with DPX
mounting medium. The ecological study was carried out following the standard method described earlier by the
author/s (Halder 2015a,b,c,d, Halder & Sinha 2014,2015, Halder 2016a,b). Identification of those algal species
were done by following standard monographs and scientific literature viz. Hustedt (1930), Hirano (1955), Foged
(1977), Lundholm & Hasle (2008), Cefarelli et al. (2010), Van De Vijver et al. (2011), Al-Hassany & Hassan
(2014) etc.
A total number of three diatom taxa belonging to the order Pennales of the class Bacillariophyceae had been
morpho-taxonomically described with author citation, habitat, collection number, date of collection,
significance, species abundance and ecology for the first time. Each currently accepted names had been
provided with its author(s) name. All the limnological parameters except temperature and pH were expressed as
mg.l-1.
1. Fragilariopsis cylindrus (Grunow) Helm. & Krieg. in Diatomeenschalen 2: 17, pl. 187189, 1954; Hasle in
Skr. Norske Vidensk Akad. I. Mat. Nat. Kl. NS. 21: 34-37, pl.12, figs. 612; pl. 14, figs. 110; pl. 17, figs. 24,
502
Halder (2016) 3(3): 501507

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1965; Lundholm & Hasle in Nov. Hedw., Beiheft 133, 237241, figs. 16, 1214, 17, 19, 20, 2223, 2008;
Cefarelli et al., in Pol. Biol. 33: 1467, 70, 81: figs. 2ei, 7ce, 2010. (Fig. 1A)
Synonym(s): Fragilaria cylindrus Grunow; Nitzschia cylindrus (Grunow) Hasle
Description: Planktonic, cells solitary or chain-forming, ribbon-shaped, attached by the valve surfaces and each
cell contains two rectangular chloroplasts in the girdle view; valve shape linear and isopolar with broadly
rounded ends; apical axis 14.023.0 m; transapical axis 2.03.0 m; transapical striae 1216 in 10 m; at the
ends, the striae become nearly parallel to the apical axis; striae perforated and consist of 2 or rarely 3 rows of
minute poroid areolae, each with 4060 poroids in 10 m; fibulae (continuations of costae) occur at
approximately the same density as the striae; raphe canal is eccentric.
Habitat: Ganga river water at Tribeni site, Hooghly district, West Bengal.
Collection No: NH 804; Dated: 03.01.2011
Significance: Primary producer and a component of food chain in aquatic ecosystem.
Species abundance: Rare in Hooghly district, West Bengal.
2. Achnanthidium lineare W. Sm. in Ann. & Mag. Nat. Hist. 2(15): 8, pl.1, fig. 9, 1855; Van De Vijver et al. in
Algol. Stud. 136/137: 170180, figs. 135, 2011. (Fig. 1B)
Synonym(s): Achnanthes linearis (W. Sm.) Grunow; Achnanthes minutissima Ktz. partim sensu Krammer &
Lange-Bertalot, Rossithidium lineare (W. Sm.) Round et Bukht.
Description: Frustules in girdle view rectangular; valves linear or linear-lanceolate with almost parallel
margins; valve apices broadly rounded, non-protracted; rapheless and raphe valves are linear; valve length 12.0
13.5 m, width 2.52.8 m; axial area narrower, linear, weakly widening towards the central area; central area
rectangular fascia; raphe filiform straight with raphe endings; striae radiate to weakly radiate throughout the
entire valve; 2832 in 10 m; striation pattern slightly to densely spaced near apices; numbers of areolae per
stria 23 but in this specimen striae and areolae not visible.
Habitat: Pond water at Kuntighat, Hooghly district, West Bengal.
Significance: Primary producer and a component of aquatic food chain in this pond.
Species abundance: Sparsely present in Hooghly district, West Bengal.
Figure 1. A. Fragilariopsis cylindrus; B. Achnanthidium lineare; C-D. Rhopalodia gibba var. ventricosa (valve & girdle views).
3. Rhopalodia gibba var. ventricosa (Ktz.) H. Perag. & M. Perag. in Diat. Mar. France 302, pl. 77, figs. 35,
1900; Patrick & Reimer, The diatom of the United States, 190, pl. 28, figs. 34, 1975; Foged, Fresh Water
Diatoms in Ireland, 106, pl. 43, fig. 7, 1977; Czarnecki & Blinn in Biblioth. Phycol. 102 , pl. 22 , fig. 12, 1978;
Hadi et al. in Nov. Hedw., 534, pl. 12, fig. 217, pl. 37, fig. 3, 1984; Al-Hassany & Hassan, Asian J. Natl. &
Appl. Sci. 3(1): 2, pl. 1, fig. 1, 2014. (Fig. 1CD)
Description: Planktonic, frustules bracket shaped in valve view with swollen middle, apices acutely bent and
margin convex; in girdle view, valves linear-elliptical, inflated in median portion with broadly rounded poles;
valves 50.554.5 m long and 9.510.0 m broad, having sometimes median constrictions; ventral margin
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Halder (2016) 3(3): 501507

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straight, arcuate or curve at the ends; dorsal margin convex; chromatophore is single in each cell, laminate with
irregular margins; striae slightly radiate to parallel; costae 8 and striae 15 in 10 m.
Habitat: Water reservoir in rice field at Chinsurah, Hooghly, West Bengal.
Significance: Primary producer in water bodies.
Species abundance: Abundant in Hooghly district, West Bengal.
In the recent work, three freshwater diatom taxa had been morpho-taxonomically described from this IndoGangetic plain of West Bengal, India. Fragilariopsis cylindrus sensu lato is a cold-water diatom species and
was documented from polar and subpolar regions in the Arctic, Antarctic and open water as well as in ice
although, a small number of species have been recorded exclusively from the higher latitudes of the Northern
Hemisphere (Witkowski et al. 2000, Lundholm & Hasle 2008). It could dominate the water column, sea ice and
ice edge communities (Hegseth & von Quillfeldt 2002, Cefarelli et al. 2010). Therefore, the species of the genus
Fragilariopsis Hust. is abundant particularly in the sea, Arctic and Antarctic ice waters. But here, author
collected the species of F. cylindrus (Grunow) Helm. & Krieg. from the lower stretch (downstream) of river
Hooghly (Ganga) at Tribeni site during winter when water temperature was below the level of 20C from West
Bengal, India. It is morphologically similar to F. curta (Van Heurck) Hust. and F. kerguelensis (OMeara) Hust.
but differs it from those two species by having isopolar apical axes. The shape, measurements of valves and
other identifying characteristics of this species also agreed with the type specimen and other published reports
(von Quillfeldt 2001, Lundholm & Hasle 2008, Cefarelli et al. 2010). It was collected as planktonic form like
Kang & Fryxell (1992). The description of species Achnanthidium lineare W. Sm. is exactly coincided with the
type specimens and the valve length and width of the present specimen is matched with the European type
materials especially of Scotland and France (Van De Vijver et al. 2011). The taxon Rhopalodia gibba var.
ventricosa (Ktz.) H. Perag. & M. Perag. is differentiated from R. gibba (Ehr.) O. Mll. by i) its marked
swelling in the middle of the valve ii) more elliptical nature of frustule in girdle view and iii) length: breadth
ratio is much less than in R. gibba (Ehr.) O. Mll.
Table 1. Physico-chemical characteristics of different lentic aquatic bodies during the algae sampling times (MeanSE).
Limnological
parameters
Reservoir at Chinsurah
Temp. (C)
31C0.18
pH
8.10.05
7.40.11
DO (mg.l-1)
4.00.05
BOD (mg.l-1)
90.05.77
COD (mg.l-1)
0.250.05
NO3-N (mg.l-1)
-1
30.340.11
PO4 (mg.l )
3.60.13
Silicate (mg.l-1)
6.00.22
SO42- (mg.l-1)
-1
220.00.20
Total alkalinity (mg.l )
Different Sampling Sites

River Ganga at Tribeni
19C0.13
7.30.05
7.00.12
4.50.11
120.02.88
0.120.05
0.180.12
6.60.13
6.80.17
164.00.22
Pond at Kuntighat
30C0.17
7.80.05
7.10.11
4.30.13
1102.88
0.300.08
0.280.15
5.40.14
7.00.20
184.00.22
The physico-chemical characteristics of different types of water bodies during the diatom sampling times
were measured and depicted in table 1. The pH of the studied aquatic ecosystems was found to be alkaline.
Kamat (1965) reported that the diatoms are usually abundantly found in the alkaline water bodies. Thus, the
present investigation confirmed the earlier finding. The ranges of nitrate-nitrogen and phosphate values were
measured from 0.120.30 mg.l-1 and 0.180.34 mg.l-1 respectively while; silicate was recorded as 3.66.6 mg.l-1.
This study revealed that presence of adequate amount of nitrate-nitrogen, phosphate and silicate along with
other selected physico-chemical parameters favored the growth of those diatom species in the above said water
bodies. The investigation also revealed that they can tolerate varying degrees of temperatures (1931C), pH
(pH = 7.38.1) and total alkalinity (164.0220.0 mg.l-1) values. Other parameters like BOD and SO42- except
COD values were found in lower amounts in the water bodies. DO was observed between 7.0 mg.l-1 and 7.4
mg.l-1 which is higher that might be due to maximum abundance of diatom and other plankton flora. Henceforth,
it can be summarized that these species appear in those aquatic ecosystems which are enriched with sufficient
essential nutrients. This documentation of diatom species of fresh water habitats from poorly studied region has
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a great significance for future investigations on algal taxonomy and freshwater ecology.
ACKNOWLEDGEMENTS
The author is grateful to Dr. Sankar Narayan Sinha, Dept. of Botany, University of Kalyani, Nadia, West
Bengal, India for providing opportunity to work under his guidance, co-operations and valuable suggestions.
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ISSN (E): 2349 1183

ISSN (P): 2349 9265
3(3): 508516, 2016
DOI: 10.22271/tpr.2016.v3.i3.067
Research article
Effect of various dormancy breaking treatments on seed

germination, seedling growth and seed vigour of medicinal plants
Ashwani Kumar Bhardwaj, Sahil Kapoor, Avilekh Naryal, Pushpender Bhardwaj,
Ashish Rambhau Warghat, Bhuvnesh Kumar and Om Prakash Chaurasia*
Defence Institute of High Altitude Research, Defence Research & Development Organization, Leh-Ladakh,
Jammu & Kashmir, India
*Corresponding Author: opchaurasia1998@gmail.com
Abstract: Study was conducted during 201314 to examine the role of various dormancy breaking
treatments, viz. hot water treatment, scarification, stratification, concentrated acids (H2SO4, HNO3
and HCl), gibberellic acid, potassium nitrate, alcohol, and acetone and gamma-rays irradiation on
the percentage germination, seedling growth and seed vigour. Dried seeds were incubated in the
plant growth chambers for 2028 days at constant temperature of 252 C under continuous light
(16 hrs) photoperiod after its treatments. Maximum percent germination 97.2% was obtained in
Innula racemosa followed by Rheum webbianum (95.1%), Carum carvi (93.4%), Saussurea lappa
(90.01%) and Bunium persicum (81.4%) when seeds were pretreated with acid (H2SO4 for 5
minutes). According to results obtained in present study, all studied species found best
germination with H2SO4 for 5 minutes in duration of 30 days. The seedlings derived from seeds
exposed to the various treatments performed well when grown in a green house. Maximum length
of seedlings were found in 24.3 cm in S. lappa followed by R. webbianum (23.8 cm) and C. carvi
(22.2 cm) when seeds were pretreated with H2SO4 for 5 minutes, on the other side B. persicum
(19.4cm) in hot water treatment at 80C for 20 minutes and I. racemosa (17.4 cm) in 0.2 KNO3 for
10 minutes. Highest value of seed vigour index (2263) and lowest seed vigour index (390) was
found in R. webbianum and B. persicum. The well developed seedlings were observed in 90 days
and transplanted it for further developments. The data have implications for conservation and
cultivation of the species studied.
Keywords: Acid treatment - Dormancy - Gibberellic acid - Nitric acid - Seed germination.
[Cite as: Bhardwaj AK, Kapoor S, Naryal A, Bhardwaj P, Warghat AR, Kumar B & Chaurasia OP (2016) Effect
of various dormancy breaking treatments on seed germination, seedling growth and seed vigour of medicinal
plants. Tropical Plant Research 3(3): 508516]
INTRODUCTION
Seed germination is a complex physiological processes that response to environmental signals such as water
potential, light and other factors. Poor seed germination is the major limiting factor of threatened medicinal
plants for large scale production and cultivation under cold desert conditions. Seed germination in general can
be controlled by many factors like natural germination (growth) inhibitors (Angevine & Chabot 1979). These
are the derivatives of benzoic acid, cinnamic acid, coumarin, naringenin, jasmonic and abscisic acid (ABA). It
has been postulated that seed coat (testa) of many plant species contains considerable amount of germination
inhibitor, which prevent their seed germination (Arora & Bhojwani 1989). The first stage of germination
consists of ingesting water and an awakening or activation of the germplasm. Protein components of the cells
that were formed as the seed developed became inactive as it matured. Seed germination is important to know
the germination pattern of a plant, more particularly the medicinal ones that might need to bring under
cultivation for the primary healthcare system. The significance of the seedling in plant population ecology has
long been recognized (Baskin & Baskin 1998). The germination response pattern of seeds is also regarded as a
key characteristic in plant life history strategy (Baskin et al. 1993, Baskin & Baskin 1972). The variation in seed
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.
dormancy and the subsequent patterns of seedling emergence are controlled by environmental conditions.
Important factors controlling the variation seed dormancy within species include the environment of the mother
plant during the time of seed maturation and environmental conditions after the seeds have been released
(Bewley 1997). Certain environmental conditions may be required to break dormancy, and other conditions are
often required to permit germination after dormancy is broken (Bewley & Black 1994). Seeds of many species
require days, weeks, or months at low temperatures to break dormancy (Bradbeer 1992), whereas others require
warm temperatures for after-ripening to germinate when permissive conditions arrive (Chauhan & Johnson
2008).
In some temperate species, dormancy is broken by a period of warm temperatures followed by cold
stratification. This response is most often associated with morpho-physiological dormancy; however, seeds with
morphophysiological dormancy have under developed embryos (Durrani et al. 1997). In order to accelerate this
method, it can be combined with some treatments such as chemical applications or mechanical seed coat
removal (El-Barghathi & El-Bakkosh 2005, Fenner & Thompson 2005). Many investigators have studied the
effects of exogenous growth regulators on seed germination. Gibberellins eliminated the chilling requirements
of peach and apple seeds and increased their germination (El-Barghathi & El-Bakkosh 2005). Recent studies
have revealed that cold stratification has a direct effect on production of gibberellins (GAs) in seeds of
Arabidopsis thaliana (Fernandez et al. 2002, Hartmann et al. 1997). Exogenously applied GA overcomes seed
dormancy in several species (Hassan & Fardous 2003, Hradilik & Cisarova 1975) and promotes germination in
some species that normally require cold stratification, light, or after-ripening (Kandari et al. 2012). Pre-chilling,
scarification, and treatments with gibberellic acid (GA3) or nitric acid (KNO3) are the standard procedures used
to enhance seed germination of dormant seeds.
However, many attempts have been made to investigate seed germination and seedling emergence of
different annual and perennial species including medicinal plants (Bewley 1997, Liebst &scheneller2008, Liza
et al. 2010; Martinez-Gomez &dicenta 2001, Mayer & Poljakoff-mayber 1989, Mehanna et al. 1985). However,
no study has surveyed germination patterns in medicinal plants from Ladakh region of India.
Seed source
Fruits of S. lappa, R. webbianum, I. racemosa, C. carvi and B. persicum were collected from their localities
at an altitude of 15004000 m of Ladakh region of India in August 2012 (Table 1). The seeds were later air
dried and stored at room temperature (25C) before experimentation.
Table 1. Brief description of plant species studied.
Name/Family
Saussurea lappa C.B
Clarke (Asteraceae)
Local name
Habit
Kuth
Perennial
shrub
Habitat
Altitude (m)
Uses
Cultivated land & waste 26003600 Roots used as anti-arthritic,
land (Lahaul valley)
antiseptic, aphrodisiac,
carminative and digestive
agent
Rheum webbianum
Lachoo
Perennial
Moist slopes & open
33005200 Roots, stem and petioles used
Royle (Polygonace)
herb
slopes (Zanskar valley)
as appetizer, astringent and
in the treatment of asthma,
bronchitis, eye diseases,
piles, etc.
Inula racemosa Hook. Pushkarmool Perennial
Cultivated land (Indus & 15952800 Roots used as anthelmintic,
(Asteraceae)
shrub
Lahaul valley)
antiseptic, anti-inflammatory
and diuretic agents
Carum carvi Linn.
Gonyor,
Biennial or Cultivated land or water 36503900 Fruits/seeds used as spice,
(Apiaceae)
jirah
annual herb streams (Indus & spiti
carminative, back pain, liver
valley)
problem and stimulant
Bunium persicum
Kala jirah
Perennial
Rocky slopes
18003500 Fruits/seeds used as spice,
(Boiss.) B. Fedtsch.
herb
(Suru valley)
carminative, back pain, liver
(Apiaceae)
problem and stimulant
Seed viability assessment
To ensure that the seeds used for the experiment were viable and of high quality, the sample lots were
subjected to viability test using the tetrazolium technique. Three replicates (20 seeds each replicate) were
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subjected to 2, 3, 5, triphenyl tetrazolium chloride (TTC) test after 15, 30, 45, 60 days of storage at 4C. In this
method, seeds were longitudinally sectioned and the sections were immersed in a 0.5% aqueous solution of TTC
(pH 6.5) for 24 hrs at room temperature (25C) under controlled dark conditions. The TTC solution was drained
and sections were rinsed 3 times with tap water. The topographical staining pattern of the embryos and
cotyledons were studied under a dissection microscope.
Seed surface sterilization and germination assessments
Seeds of S. lappa, R. webbianum, I. racemosa, C. carvi and B. persicum were sterilized using 0.04%
aqueous solution of mercuric chloride (HgCl2) for 15 sec. to remove any fungal infection and then rinsed with
distilled water. Three replications of 30 seeds each were prepared for each treatment and control. T1, Seeds were
dipped in concentrated acids, i.e. H2SO4 for 5 min; T2, gamma rays irradiation of seeds at different doses (i.e.
1050 KR) using the 60Co gamma cell irradiator facility at the Physics Department, RTM, Nagpur University,
Nagpur followed by dipping in concentrated H2SO4 for 5 min. ; T3, seeds were first pretreated in concentrated
H2SO4 for 10 min. further dipped in GA3 solutions (i.e. 200 ppm) for a period of 1 hr; T4, seeds were soaked at 3
different doses of KNO3 (i.e. 0.1, 0.2 and 0.3 %) for 10 min. after presoaking in concentrated H2SO4 for 10 min;
T5, scarification of seeds by P320A sandpaper (sand grain.cm-2) then dipped in GA3 solutions (i.e. 200 ppm) for
a period of 1 hr; T6, seeds were stratified at -20C for 130 days; T7, seeds were dipped in the hot water at 80C
for 20 minutes. and T8, seeds were first soaked in absolute alcohol and acetone for 10 minutes. All the treated
seeds were placed in closed 9 cm Petridishes ( 9 cm) which were lined with 2 sheets of filter papers Whatman
No.1 and moistened with sterilized MilliQ water. Treated seeds were placed on the moist paper for germination
for 2028 days and light was provided by philips daylight lamps (324 mol.m-2.s-1). A clear labeled lid was
placed on top of each Petri-dish denoting the treatment, temperature and replication. All these Petri-dish were
then kept in plant growth chamber at 252 C with relative humidity of 65% and 16 hr of light. Petri dishes
were checked daily for germinated seeds and filter paper was moistened with sterilized MilliQ water as needed.
Germination was determined by observing a visible radical or shoot. The number of seeds used for the
germination tests were 3 replications 90seeds/replication for each treatments.
Seedling growth & vigour
Seedling were incubated in plant growth chamber and monitored weekly. The growth of seedling was
measured by vernier caliper in cm after 30 days of incubation. The well developed seedlings were potted in
potting mixture containing Coconut + vermiculite + perlite (1:1:1) under controlled green house conditions.
Initially, for 510 days the developed seedlings were covered with glass jars to provide sufficient moisture for
growth of new shoots. During transplanting process, jars were taken off every day for 12 hr to acclimatize the
plantlets to the external conditions. Seed vigour Index (SVI): Germination percentage Seedling length (cm).
Experiments were performed in triplicate.
Data analysis
The data were statistically analyzed as a factorial experiment based on completely randomized design with
three replicates. Means were compared by one-way ANOVA using SPSS for windows (Version 21.0) and
differences between the means were compared by Duncans multiple range test (DMRT). A probability of
0.05 was considered significant.
Seed viability
Tetrazolium chloride test showed the percentage viability of S. lappa, R. webbianum, I. racemosa, C. carvi
and B. persicum as 88, 72, 78, 81 and 75 at the day of harvesting, which remains 55, 51, 55, 58 and 57 percent
by 60 days of storage, showing a continuous decline with storage period (Fig. 1). A continuous decrease in seed
viability was observed of different rhizomatous herbs of Himalayan region with storage period (Pupalla &
Fowler 2002, Sharma et al. 2006) which supplemented the present observation.
Seed germination assessments
Mean and standard error comparisons of each treatment are based on Duncans test as presented in table 2.
The effect of various durations of concentrated H2SO4 showed a positive effect of H2SO4 on seed germination,
while no significant germination was observed with concentrated HCl. In case of concentrated HNO3 treatment
there was no seed germination at all. Maximum germination of 97.2% was observed in I. racemosa with 5
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minutes of soaking in concentrated H2SO4 followed by 95.1%, 93.4%, 90% and 81.4% in R. webbianum, C.
carvi, S. lappa and B. persicum (Table 2 & Fig. 2). However, any increase or decrease in acid soaking time
significantly reduced the seed germination which can be attributed to embryo damage. Poor germination or no
germination in case of concentrated HCl and HNO3 respectively might be due to the inability of these treatments
to break the physical dormancy. Also, the positive effect of gamma rays irradiation on seed germination is
already known in many crops. Therefore, seeds were also treated with different doses of gamma rays along with
concentrated H2SO4 treatment for 5 minutes. Maximum germination of 81.96% was observed when seeds of C.
carvi were treated with 30 Kr gamma rays, followed by 5 min. concentrated H2SO4 treatment, followed by 77%,
76.4%, 73.04% and 45.6% in B. persicum, S. lappa, I. racemosa and R. webbianum. Obviously, any further
increase or decrease dose of gamma rays or H2SO4 duration showed negative effect on the overall percent
germination (Table 2 & Fig. 2).
Figure 1. Decrease in seed viability of some medicinal plants during storage at 4C with increase in storage period.
Figure 2. Seed germination of some selected medicinal plants.
Gibberellic acid is also known to play an essential role in seed germination, stem elongation and flower
development (Sharma et al. 2006). However, we have tried 100500 ppm solution of GA3 out of which 200 ppm
showed significant results. 200 ppm of GA3 treatment showed the highest germination of 69.20% in C. carvi
when seed treated with 1 hr soaking in GA3 alongwith 10 min. of H2SO4 pre-soaking followed by 62%, 59.6%,
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53.6% and 52.3% in R. webbianum, S. lappa, B. persicum and I. racemosa (Table 2 & Fig. 2). This means that
the regulation of endogenous gibberellic levels after seed imbibitions along with specific H2SO4 presoaking time
of 10 minutes is crucial factor in determining the seed germination. Lesser or longer treatment time was
inhibitory in each case. A chemical treatment such as pre-treatment with H2SO4 for 1530 minutes was found to
be an effective method to increase germination. While seed treatments with KNO3 or GA3 is known to enhance
the germination percentage (Silvertown & Lovett Doust 1993). When H2SO4 pre-treated seeds of I. racemosa
were treated with KNO3 in different combinations the highest germination of 78.4% (Table 2 & Fig. 2) was
obtained in case of pre-soaking in concentrated H2SO4 (for 10 minutes), followed by dipping in KNO3 (0.2% for
10 minutes). While, decreasingly 69%, 68.4%, 65% and 62.3% found in C. carvi, S. lappa, B. persicum and R.
webbianum. Any increase or decrease in the concentration of KNO3 or soaking duration along with further
increase in the presoaking time showed negative effect on the overall percent germination. Unsatisfactory
germination percentage i.e. less than 20 was found in 0.3% KNO3 for 10 min. along with 10 min. H2SO4
presoaking.
Scarification, stratification and hot water treatments were also found satisfactory results. Application of
alcohol, acetone and HNO3 although broke the seed coat but not found better germination as compared to other
treatments. Sulfuric acid treatment to remove mucilage and soaking in either of KNO3, GA3 or gamma ray
treatment was found effective to allow penetration of oxygen from the surroundings to the embryos and
increased germination of seeds. Germination in each case was superior over the control (1020%). Maximum
germination percentage i.e. 69.03% was found in C. carvi when seeds were stored at -20C for 30 days followed
by 67.02%, 61.23%, 59.5% and 57.23% in R. webbianum, I. racemosa, B. persicum and S. lappa. On the other
hand, maximum percent germination (79.2%) found in R. webbianum when seeds treated with hot water at 80C
for 20 min. followed by 72.02%, 65.6%, 65.6% and 61.5% in S. lappa, C. carvi, B. persicum and I. racemosa
while, 82.12% germination found in S. lappa when seeds treated with alcohol and acetone for 10 min. followed
by 80.67%, 78%, 66.02% and 58.02% in C. carvi, I. racemosa, B. persicum and R. webbianum (Table 2 & Fig.
2). The responses of seeds to different treatments were strongly species-specific. It is obvious from the present
data and similar work reported by other authors that the responses of dormant seeds of the same species to
different factors are variable, depending upon the habitat of collection and duration of storage. However, some
reported 70% germination in S. lappa when seeds were treated with gibberellic acid (GA) (3 M) (Tairu et al.
2007). 89% germination was observed in R. webbianum when seeds were treated with GA3 and KNO3 (Taiz &
Zeiger 2010). Similar to our observations, effectiveness of low temperature in causing dormancy removal has
also been reported in other populations of C. carvi (Vleeshouwers et al. 1995) and B. persicum (Warghat et al.
2014). The low temperature requirement appeared to be replaced by GA3 in I. racemosa and C. carvi, but not in
B. persicum, further signifying the species specificity in responses even of closely related species. Lowtemperature treatment of seeds could be easily adopted for the cultivation of species wherever it proved
effective. According to results obtained in present study, all studied species found best germination with H 2SO4
for 5 minutes in duration of 30 days and significantly different as compared to other treatments at 5% level.
Seedling growth and vigour index
The effect of various durations of concentrated H2SO4 showed a positive effect of H2SO4 on seedling
growth. The maximum length of seedling of S. lappa was observed 24.3 cm, when seeds were treated with
H2SO4 for 5 minutes while, minimum length found 8.9 cm when seeds treated with alcohol and acetone for 10
minutes. Whereas, in R. webbianum maximum (23.8 cm) & minimum (9.7 cm), I. racemosa maximum (17.4
cm) in KNO3 & H2SO4 treatment and minimum (11.7 cm) in GA3 treatment, C. carvi maximum (22.2 cm) and
minimum (10.2 cm) in H2SO4 + GA3 treatment and in B. persicum maximum (19.4 cm) in hot water treatment
and minimum (5.9 cm) in alcohol + acetone for 10 minutes. Similar type of effects of treatments on seedlings
growth of Q. coccifera was also found (Arora & Bhojwani 1989). There was a significant difference among the
different treatments and medicinal plant seeds on seedling vigour (Table 3). R. webbianum significantly
produced the best seedling vigour index 2263 which is statistically different from the other medicinal plants and
treatments. However, B. persicum produced seedlings with low vigour index of 390. The bigger sized seed of R.
webbianum with H2SO4 treatment for 5 min. statistically produced seedling of very high vigour index of 2263
compared to small sized seed of B. persicum with alcohol + acetone treatment for 10 min. which produced
seedling that have low vigour index of 390. However, bigger sized seeds of R. webbianum and S. lappa
produced the more stem girth compared to the small sized seed of B. persicum that gave less stem girth. The
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better seedling growth with H2SO4 treatment for 5 min. could be as a result of the early germination recorded
and the maintenance of continuous growth and vigour with these treatments, during the period of observation.
Table 3. Seed vigour index of medicinal plants.
Treatments
T1
T2
T3
T4
T5
T6
T7
T8
Control
S. lappa
2187
1406
1168
807
575
950
900
731
143
R. webbianum
2263
757
1265
1215
809
865
1386
563
622
I. racemosa
1264
1176
654
1364
683
765
720
975
46
C. carvi
2073
844
706
1145
665
849
1207
1146
94
B. persicum
1490
1078
992
780
657
779
1273
390
483
As the seed size increases there was more food reserved in cotyledon of the seed to sustain the seedling
growth than the smaller seed sizes whose food reserved could be exhausted thus affecting the seedling growth
and vigour. This is agreed with the work previously done (Yamaguchi & Kamiya 2000). The significantly
maximum seedling length obtained by S. lappa and R. webbianum could be that there were enough spaces
within the treatments that allow growth. The maximum seedling length produced by the big sized seeds could be
as results of its radicles where roots can easily attach themselves (Yamaguchi & Kamiya 2002). After the 90 th
days, the well developed seedlings were transplanted to green house condition at DIHAR, Leh and placed under
shade for further growth and development. However, result accomplished that 80% survival rate in green house
condition and 70% in open condition of herbal garden with well developed healthy plantlets (Fig. 3). Similarly,
It was found that survival rate of seedlings in the green house was high in S. lappa, R. webbianum and low in I.
racemosa, C. carvi and B persicum (Yamaguchi & Kamiya 2002). Ten month old plants of these species are
shown in figure 3. The transplanted seedlings of B. persicum and C. carvi did not survive beyond three month.
Figure 3. A & E, Seed-derived seedlings in petridish. BD, Ten-month old plants growing under green house conditions.
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CONCLUSIONS
Plant propagation multiplies plants in bulk and preserves their essential genetic characteristics. Acid
scarification, followed by gamma rays, addition of GA3 or KNO3 solution and hot water is a simple, efficient
and cost-effective method for ensuring better seed germination and well developed seedlings. The outcomes of
the present study can be gainfully utilized for multiplication of the species. The information will prove
beneficial not only for conservation of species but also in boosting rural economy and the information will prove
beneficial for other related species.
ACKNOWLEDGEMENTS
Authors are thankful to Defence Research and Development Organization (DRDO), Ministry of Defence,
Government of India for providing financial assistance for the research. We are also thankful to Mr. Sandeep
Singh Rawat for laboratory help.
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ISSN (E): 2349 1183

ISSN (P): 2349 9265
3(3): 517521, 2016
DOI: 10.22271/tpr.2016.v3.i3.068
Research article
Some ethnomedicinal plants used against high blood pressure in

Bargarh district in Western Odisha (India)
S. K. Sen1* and L. M. Behera2
1
Department of Botany, Panchayat College, Bargarh - 768028, India

Ex-Reader in Botany, Modipara (Near Water Tank), Sambalpur - 768002, India
*Corresponding Author: sunilsen06@rediffmail.com
2
Abstract: The knowledge and usage of herbal medicine for the treatment of various ailments
among the rural people still a major part of their life and culture. An ethnomedicinal survey was
conducted during 201013 in different forest pockets and rural areas to collect ethnobotanical
information on plant species from the local inhabitants of Bargarh district. Out of a number of
collected plant species some are reported to be very useful against high blood pressure. The
outcome of this survey is that the local people of the study area still have a strong faith in the
efficacy and success of the herbal medicine.
Keywords: Ethnomedicine - High blood pressure - Tribals - Bargarh district.
[Cite as: Sen SK & Behera LM (2016) Some ethnomedicinal plants used against high blood pressure in Bargarh
district in Western Odisha (India). Tropical Plant Research 3(3): 517521]
INTRODUCTION
Medicinal plants have been used since time immemorial for the treatment of human as well as animal
diseases and ailments. Traditional medicine practice is an important part of health care delivery in moist of the
developing countries (Akerel 1998, Truyen et al. 2105, Ngbolua et al. 2016). According to World Health
Organization, approximately 80% population in developing countries depends on traditional medicine for the
primary healthcare (Mehra et al. 2014). A major portion of these involves the use of medicinal plants. Use of
medicinal plants are known by ethnic tribes who resides in the forest area pay a vital role in using forest
vegetation of food, cloth, shelter, for the treatment of common ailments, utilize the plants and manage to
conserve it to some extent for future use.
Odisha State is situated in the eastern part of India having 30 districts. Bargarh is one among them located in
western part of Odisha. Prior to 5th November 2011, Bargarh was a subdivision under the district Sambalpur.
The district lies between 20402149 N and 82458348 E. The District is surrounded by Chhattisgarh state
on the north, Sambalpur District on the east, Bolangir and Subarnapur on the south and Nuapada District on the
west. The Bargarh district experiences extreme type of climate with hot and dry summer followed by humid
monsoon and chilling winter. The temperature varies between 10C to 46C. The average annual rainfall in the
district is 1527 mm.
The tribals such as Sahanra (Soara), Binjhal, Gond, Kondh, Munda, Kuli, Kalanga, Oran, Mirdha, Dharua,
Kisan, Kharia and Parja are inhabited in the Bargarh district. The tribals mostly dependent on their traditional
healing system for healthcare and for treating various diseases (Bajpai et al. 2016). Tradition and beliefs are the
only basis of use of the herbal medicine. During the ethnobotanical survey in the district, it has been observed
that many plants species are being used by the tribals and other rural people for various purposes including
herbal medicine to cure and or preventive diseases. Generally the plant parts such as root, leaf, bark, stem,
flower, fruit, gum and resin are used as paste, powder, extract, decoction etc. by the people to cure diseases and
ailments (Deepa et al. 2016).
Some valuable research works have been contributed in the last four to five decades from this locality
(Panigrahi 1963, Brahmam & Saxena 1990, Misra et al. 1994, Misra 2004, Pradhan et al. 1999, Sen & Pradhan
1999, Behera & Sen 2007, 2008, Sen & Behera 2003, 2008). But no work on the present subject so far has
been contributed from the study area. Therefore, an attempt has been made in this paper to highlights on some
ethnomedicinal plants used for the treatment of high blood pressure in Bargarh district.
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Field trips were conducted to different forest and rural area of Bargarh district during 2010 2013. The local
traditional healers, herbal medicine practitioners, village headman, experienced old men and women were
contacted and interviewed to record the ethnomedicinal uses of the plants and their local names (Bajpai et al.
2016). Mostly collections were made from local forests, scrub jungle, village suburbs and cultivated fields.
Repeated quarries were made to confirm the authenticity of the information gathered from the local inhabitants.
He collected plant specimens were identified with the help of flora books (Haines 1921 25, Saxena & Brahmam
1994 96). The latest botanical nomenclature has been checked with the world renowned and widely accepted
website http://www.the plantlist.org. (The Plant List 2010). The herbarium specimens have been deposited in
the herbarium of Botany Department, Panchayat College, Bargarh, Odisha.
RESULTS
Enumeration of species
In the enumeration, the names of 10 plant species by the ethnic people of Bargarh district for the treatment of
high blood pressure have been arranged alphabetically along with their correct botanical name, family in
parenthesis, local names in inverted comma, locality and collection number, parts used, mode of preparation,
details of ethnomedicinal uses.
1. Allium sativum L. (Amaryllidaceae), Lesun, Udepali-529 (Fig. 1A)
2 3 cloves of bulb are soaked in water overnight and either it is swallowed or crushed to paste is taken with a
little warm water once daily in empty stomach in the morning. Fresh leaf paste (5 10 g) is taken once daily in
empty stomach in the morning.
2. Catharanthus roseus (L.) G. Don (Apocynaceae), Baramasi, Beherapali-234 (Fig. 1B)
Petals (5 numbers) are chewed once in the morning in empty stomach.
3. Cuscuta reflexa Roxb. (Convolvulaceae), Nirmuli, Khandijharan-276 (Fig. 1C)
Whole plant juice (2 teaspoonful) is taken once daily in stomach in the morning.
4. Hygrophila auriculata (Schumach.) Heine, (Acanthaceae), Kuilekha, Ramkhol-534 (Fig. 1D)
Leaf juice (1 teaspoon) and Piper nigrum fruit (5 7 in number) powder are mixed together and taken once
daily in empty stomach for 15 days to get relief from high blood pressure at least for a period of one year.
5. Moringa oleifera Lam. (Moringaceae), Munga, Ramkhol-256 (Fig. 1E)
Leaf juice (one teaspoonful) is taken once daily in empty stomach for one month to get relief from
hypertension at least for period of one year. Tender leaves are chewed early in the morning for one month to
cure blood pressure for one year.
6. Psydrax dicoccos Gaertn. (Rubiaceae), Benimanj, Ramkhol-376 (Fig. 1F)
Equal amount of leaf and bark are crushed together and the juice (5 ml) is taken 12 times daily in empty
stomach.
7. Rauvolfia serpentina (L.) Benth. ex Kurz (Apocynaceae), Patalgarud, Nrusinghnath- 519 (Fig. 1G)
Root powder/ paste (1.0 1.5 g) is given once daily regularly 15 days and after three days break once again
the medicine is continue for 15 days.
8. Senna occidentalis (L.) Link. (Caesalpiniaceae), Chakunda, Kharmunda-217 (Fig. 1H)
Fresh leaf paste (510 g) is taken once daily in empty stomach in the morning.
9. Terminalia arjuna (Roxb. ex DC.) Wt. & Arn. (Combretaceae), Ka, Khandijharan-316 (Fig. 1I)
Bark (10 g) is boiled in a glass (250 ml) of water to obtain decoction is taken once daily in empty stomach
early in the morning for one month. Bark powder (50 gm) and old jaggery (50 gm) are mixed together and made
into pills of 1 cm size. One pill is taken once daily in warm water.
10. Trigonella foenum-graecum L. (Fabaceae), Methi, Beherapali-236 (Fig. 1J)
Seed powder (5g) is taken with warm water in empty stomach once daily.
DISCUSSION
The present study focuses on traditional medicines used by the tribals to cure or get relief from high blood
pressure. Ten plant species from 9 families have been identified with 13 ethnomedicinal prescriptions used by
the tribals of the present study. The observation revealed that the herbal medicines used by the tribals are mostly
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Figure 1. Plants used by the ethnic people for the treatment of high blood pressure: A, Allium sativum; B, Catharanthus
roseus; C, Cuscuta reflexa; D, Hygrophila auriculata; E, Moringa oleifera; F, Psydrax dicoccos; G, Rauvolfia serpentina;
H, Senna occidentalis; I, Terminalia arjuna; J, Trigonella foenum-graecum.
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administered in the form of paste, powder, juice, decoction, prepared in a crude method from different plant
parts such as of root, bulb, bark, leaves, flower, seed and whole plant. Of the various plant parts used, the leaves
were most commonly used. Leaf is used maximum (4 prescriptions), followed by bark (3 prescriptions), root (2
prescriptions)and other plant parts are used once in each case and in one case both leaf and bark are used in one
prescription (Fig. 2).
Figure 2. Plant parts used in the treatment high blood pressure.
It has also been highlighted that after observing their mode of treatment by different herbal medicine
practitioners, which are compared with some of the scientific literatures (Jain 1991, Kirtikar & Basu 1991,
Chopra et al. 1992, Ambasta et al. 1992, Warrier 1996, Pal & Jain 1998, Joshi 2004, Paria 2005, Singh 2013)
and it has been recorded that out of 13 prescriptions 7 from 6 plant species (with asterisk mark) are new report.
CONCLUSION
The tribals depend on the plants around them which made them acquire knowledge of economic medicinal
properties of many plants by trial and error. Consequently, they act as knowledge saviour for the utilization of
many useful plants accumulated and enriched through generations and passed to one another without any written
documents. But careful approaches should be followed before administration these drugs. The lack of proper
documentation and carelessness towards the knowledge of ethnomedicine are forcing depletion of the traditional
knowledge, which has to be preserved for the future benefit of the human civilization. Proper documentation
and digitalization of tribal information is utmost importance. However, the present study may create some
awareness among the people which might help to conserve their rich and effective ethnomedicinal knowledge in
this region.
ACKNOWLEDGEMENTS
Authors are thankful to Prof N. B. Pradhan, Retired Reader in Botany and Mr. Pareswar Sahu for their kind
help during field collection and identification of plant materials. Authors are also thankful to the local
informants for sharing their valuable ethnomedicinal knowledge about the plants.
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ISSN (E): 2349 1183

ISSN (P): 2349 9265
3(3): 522535, 2016
DOI: 10.22271/tpr.2016.v3.i3.069
Research article
Uncultivated fodder grass for cattle

R. Prameela1* and M. Venkaiah2
1
M.R.College for women, Vizianagaram, Andhra Pradeah, India

Department of Botany, Andhra University, Visakhapatnam, India
*Corresponding Author: prameelachris@yahoo.com
2
Abstract: Vizianagaram district, one of the northern districts of Andhra Pradesh lies on the East
Coast of India. The district is located between 17o15' to 19o15' N and 83o00' to 83o45' E.
Agriculture is the key occupation of the people of this district. Geographically vizianagaram
district divided in to 3 regions, i.e 1) The hilly region 2) The plains and 3) The coasts. The present
study has concentrated to generate the information on uncultivated fodder grass. The grass family
Poaceae is of a major economic and ecological importance. It is the single most important family
of flowering plants for survival of mankind. The grasses form a natural homogenous group of
plants with remarkable diversity playing a significant role in the lives of human beings and
animals. Studies on grasslands and wild grasses, especially of fodder value have become very
important for development of dairy industry, productions of meat and restoration of degraded
ecosystems. The grasses have good potentials in sustainable development of the country as well as
conservation of both plant and animal diversity. Study areas in vizianagaram district are
undisturbed places, open grounds, strip lands, unused rice fields, orchards and sandy areas. During
rainy season grass seeds germinate and grow very fast. All these grasses are annuals. Where as in
summer, we can see very little pasture. Many grasses are perennials; they can survive in drought
conditions, because they possess thick rhizomatous or stoloniferous root system and tufted growth.
Perennial grasses form a valuable pasture.
Keywords: Annuals - Herbivores - Pasture - Perennials - Strip lands - Vizianagaram.
[Cite as: Prameela R & Venkaiah M (2016) Uncultivated fodder grass for cattle. Tropical Plant Research 3(3):
522535]
INTRODUCTION
Hookers Flora of British India accounted for the known grasses of the country (Hooker 1896). Cooke
(19011908) provided an account of grasses along with other families in his Flora of the Presidency of
Bombay. Gamble (1896) published The Bambuseae of British India. Blatter & McCann (1935) published an
excellent illustrated account of Bombay grasses while Achariyar & Mudaliyar (1921) published an account of
South Indian grasses. Fischer (1934) contributed account for the grasses of Madras Presidency. Bor (1960) made
extensive studies on grasses of Assam, Uttar Pradesh and published some 125 papers on Indian grasses and
finally published a consolidated concise account of grasses of the whole Indian subcontinent The grasses of
Burma, Ceylon, India and Pakistan. Several studies were conducted on the flora of North Coastal Andhra
Pradesh. Subba rao (1977) studied on the flora of Visakhapatnam; Sriramulu (1986) studied flora of Srikakulam
and Venkaiah (2004) flora of Vizianagaram district. In the present studies an attempt made to study the
uncultivated fodder grasses of Vizianagaram district.
Everybody knows that all herbivorous animals depend upon the grass for their food. We can differentiate
herbivorous animals into two categories namely, wild and domesticated or livestock animals. We need not take
care about wild herbivores, because they feed on wild grass or green pastures of the forest areas. But we have to
take a special care for livestock. Cultivated grass is compulsory for feeding the livestock at home. Generally
cultivated grass is growing at the farms for livestock. It is very expensive and limited. In Vizianagaram there are
no grasslands, but farmers are growing some types of grasses like Pennisetum spp., Sorghum spp., and also used
straw of Rice, Maize, Sugarcane, Finger millet, Proso millet etc. It is limited and not sufficient for livestock,
thats why they are being taken to open areas, where there are some pastures available. The present study
concentrated on these uncultivated grasses growing in open lands and used as fodder.
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Study areas in vizianagaram are undisturbed places, open grounds, Striplands, unused rice fields, orchards
and Sandy areas. During rainy season grass seeds germinate and grow very fast. All these grasses are annuals.
Examples of annual grasses are Brachiaria spp, Digitaria, Echinochloa, Eriochloa, Ischaemum, Paspalidium,
Rottboellia, Setaria, Sorghum, Sporobolus and Urochloa etc. These grasses collectively form in a good pasture
for the cattle. Where as in summer we can see very little pasture. Many grasses are perennials, they can survive
in drought conditions, because they possess thick rhizomatous or stoloniferous root system and tufted growth.
Perennial grasses form a valuable pasture. Examples of perennial grasses are Aristida, Chloris, Chrysopogon,
Cenchrus, Cynodon, Dactyloctenium, Dichanthium, Eragrostis, Heteropogon, Paspalum, Paspalidium, Perotis
and Themeda etc.
Regular field trips have been undertaken to the study areas of the district and collected the uncultivated
grasses. The collected grasses are identified with the help of regional floras and Herbarium, BSI, Coimbatore.
RESULTS
Enumeration of Species
1. Alloteropsis cimicina (L.) Stapf in Prain, Fl. Trop. Afr. 9: 487.1919; GS. 357; Bor 276; chowdhary in Ind.
For. 86: 90.1960; Gam vol. 3.1766; MV. 197; V. Fl. 213; HS. 52.1972.
Milium cimicinum L., Mant. Pl. 2: 184.1771.
Herbs, annuals, tufted, decumbent, hairy with bulbous based hairs; nodes hairy, internodes smooth leafy
sheaths, leaf blades hairy, 7 cm long, 11.3 cm wide, ovate lanceolate, base cordate; base of the internodes
purple colour; spikes 45, terminal 6 cm long, 2024 spikelets in each spike. Spikelets 0.3 cm long, elliptic,
green, upper glume ciliate, acuminate, upper lemma aristate, keeled, upper palea membranous contains bisexual
floret, lower glume mucronate, smaller than the upper one, lower lemma 3 nerved, shortly mucronate and its
palea smaller membranous, contains malefloret, stamens 3.
2. Aristida adscensionis L., Sp. Pl. 82.1753; FBI 7: 221; var. adscensionis Bor 407.
A. depressa Retz., Obs. Bot 4. 22.1786; GS. 360; AP. Fl. 1130; Gam vol. 3: 1809.
Annual, tufted, branched, grows on rocky places; culms erect or decumbent, slender, rootstock creeping,
nodes glabrous, leaf blades very narrow or linear; inflorescence contracted panicle, spikelets single, pedicel
scaberulous, callus present, glumes 2 unequal, aristate, keel spinuscent, lemma base fringe of hairy, awns 3,
unequal, stamens 3, stigmas plumose.
3. Aristida funiculate Trin. & Rupr., Sp. Gram. Stip. 159.1842; var. funiculate; Bor 410.1960; Gam vol. 3:
1809; FBI 7: 226.1896.
An annual, tufted grass, culms upto 50 cm tall, geniculately ascending; nodes glabrous, ligule a small ciliate
membrane, blade 616 0.10.2 cm linear convolute, base cordate, apex acuminate; inflorescence panicle, lax,
narrow, spikelets single, pedicelled, 1-flowered, glumes linear, hyaline, lemma truncate, awn trifid, subequal,
stamens 3, caryopsis spiny.
4. Aristida hystrix L.f., Suppl. Pl. 113.1781; FBI 7: 225.1896; Gam vol. 3.1809; Bor 410; AP. Fl. 1131. (Fig.
1A)
Annual, small herbs upto 30 cm high; stems shows pseudo dichotomous branching, branches spreading;
leaves small, linear, ligule gringe of hairs, axis angled, axis and nodes hairy; inflorescence panicle, effuse,
dichotomously branches, spikelets single, pedicelled; glumes unequal, aristate, awns 3, double the length of the
spikelets, stamens 3, base of the spikelet black coloured, dry spikelets straw coloured.
5. Bothriochloa pertusa (L.) A. Camus., Soc. Lyon. 1930, Gam vol. 3.1731; Bor 109.1960.
A perennial, stoloniferous grass, culms upto 50 cm tall, ascending, nodes sparsely hairy, leaf sheaths 26 cm
long, glabrous, ligule membranous, blades 3.010 0.20.4 cm, linear, base truncate, apex acuminate, glabrous;
racemes 26, 5 cm long, slender, digitate, silky hairy, spikelets 6 mm, 2-nate, pitted, sessile spikelet: 4 mm,
oblong-lanceolate, pitted, callus bearded, lower glume elliptic-oblong, bearded below, dorsally pitted,
subchartaceous, upper glume ovate-lanceolate, finely pointed at the tip, lower lemma lanceolate, nerveless,
hyaline, upper lemma reduced to a slender awn, stamens 3, stigmas plumose, caryopsis oblong, pedicelled
spikelet: 4mm, male or barren, similar to sessile, spikelet, but awnless.
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6. Brachiaria reptans (L.) C.A. Gardner & C.E. Hubb., Hookers Icon. Pl. 34: t. 3363.1938; Fl. Trop. Afr. 9:
601.1920.
Annuals, culms prostrate or creeping, slender, branched; leaves ovate-lanceolate, cordate at base, margin
scaberulous, mouth ciliate, ligule a fringe of hairs, leaf sheath clasping the stem, shorter than the internode;
inflorescence panicle, branches up to 9; spikelets 2-nate, both are pedicelled, bears 45 setae.
7. Capillipedium assimile (Steud.) A.Camus., Fl. India 15: 6.1984. (Fig. 1B)
Andropogon assimillis Steud. in Zoll. Syst. Verz. 48.1854. et in Syn. Pl. Glum. 1: 397.1854; FBI 7: 179.1896.
Capillipedium assimile (Steud.) Camus in Lecomte, Fl. Gen. del Indo-chine 7: 314.1922; Bor 110.1960.
C.glaucopsis (Steud.) Stapf in Hook., lc. Pl. Sub. tab. 3085.1922; Gam vol. 3. 1730.
A perennial, tufted grass, culms upto 50 cm long, decumbent, often branched, nodes glabrous or bearded,
leaf sheaths usually glabrous, ciliate at mouth, ligule a short membrane, apex ciliate, blades 820 36 cm,
linear-lanceolate, apex acuminate, slightly hairy; panicle lax, branches slender, capillary, with long hairs on the
axils, spikelets few, distant, joints sparsely ciliate, sessile spikelet: 3 mm, lower glume oblong, ciliate, not pitted,
upper glume lanceolate, lower lemma linear, shorter, obtuse, upper lemma reduced to a scale, flattened based
awn, pedicelled spikelet: 4mm, not awned, pedicels sparsely ciliate, lower glume lanceolate, lower lemma
obovate-oblong, apex ciliate, lower glume lanceolate, lower lemma obovate-oblong, apex ciliate, hyaline, upper
lemma absent, stamens 3.
8. Cenchrus ciliaris L., Mant. Alt. 302.1771; Bor 287.
Pennisetum cenchroides Rich. in Pers., Syn. Pl. 1: 72.1805, nom. Superfl. FBI 788.1896; AP Fl. 1146; Gam vol.
3.1793; Maha Fl. 421.
Tufted herbs, erect or decumbent; leaves linear with tubercle based hairs; racemes cylindric, dense, pale
purple, bristles connate at base only, scabrid; involucral spikelets 2, unequal, 2-flowered, large spikelet contains
bisexual and male florets, small spikelet contains 2 male florets; stamens 3, green.
9. Chloris barbata Sw., Fl. Ind. Occ. 1: 200.1797; Bor 465; FBI 7: 292; Gam vol. 3.1836; AP Fl. 1148; HS.
508; MV. 199. (Fig. 1C)
Annual tufted herbs, base creeping; leaves linear-lanceolate, tip acuminate, ligule membranous; racemes
umbellate, spikes 812, rachis scaberulous, spikelets 3-flowered, perfect floret 1, glumes linear, densely ciliate
on the margin; fertile lemma obovate, densely stiff ciliate on margins, awned; both sterile lemmas boat-shaped,
awned, stamens 3, caryopsis oblong, compressed.
10. Chrysopogon aciculatus (Retz.) Trin., Fund. Agrose. 188.1820; Bot. Bihar & Orissa 1035.1924; Gam vol.
3. 1738.1934; Bor 115.1960; V Fl. 209. (Fig. 1D)
Andropogon aciculatus Retz., Obs. Bot. 5: 22.1789; FBI 7: 188.1896; AP. Fl. 1155.
Perennials with creeping rhizomes, culms decumbent, glabrous, leaf blades sub basal, upto 7 cm long, 0.5
cm wide, glabrous; inflorescence panicle, branches in whorled, in each whorl 56 spikes, each spike has long
peduncle, tip of the peduncle has 3 spikelets, middle one sessile, remaining two pedicelled, sessile spikelet base
hairy awned, bisexual, lower glume bidentate, 3-nerved, 2-keeled, upper glume awned, ciliate, lower lemma
epaleate, empty, upper lemma aristate, paleate, contains bisexual floret.
11. Chrysopogon orientalis (Desv.) A. Camus., Bor 118.1960. (Fig. 1E)
Rhaphis orientalis Desv., Opuse. 69.1831.
Andropogon wightianus Nees ex Steud., Syn. Pl. Glum. 1: 395.1854. FBI 7: 191.1896; AP. Fl. 1155; Gam vol.
3. 1736.
A perennial, tufted grass, culms upto 80 cm tall, slender ascending at base, nodes glabrous, leaf sheaths 59
cm, shortly ciliate-glabrous at mouth, ligule short, villous; inflorescence panicle, 15 cm long, whorled branches,
branches with reddish-brown hair tip, spikelets 3, middle sessile lateral 2 pedicelled, 3 spikelets awned;
peduncle very long, slender, spiral, tip hairy, sessile spikelet, lemma awn, very long, as long as peduncle, glume
awn as long as spikelet, sessile spikelet contains bisexual floret, pedicelled spikelet lanceolate, pubescent
pedicel truncate, margins shortly ciliate, glumes bright red/purple, lower glume ciliate awned, upper glume
lanceolate, acuminate, lower lemma, upper lemma also ciliate, staments 3.
12. Cynodon dactylon (L.) Pers., Syn. Pl. 1: 85.1805; FBI 7: 288.1960; Gam vol. 3.1835; Bor 469.
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Panicum dactylon L., Sp. Pl. 58.1753.
Perennial, stoloniferous, culms creeping or decumbent; leaves small, linear, base rounded, tip acuminate,
distichous; spikes 45, digitate; spikelets sub sessile, laterally compressed; glumes sub equal, lanceolate, 1nerved, lemma oblong, aristate, keels ciliate, palea linear, stamens 3, caryopsis oblong.
13. Dactyloctenium aegyptium (L.) Willd., Ess. Agrost. Expl. t. 15.1812; Gam vol. 3: 1840 (1273). 1934; Bor
489.1960. (Fig. 1F)
Cynosurus aegypticum L., Sp. Pl. 72.1753.
Eleusine aegyptia (L) Roxb., Fl. Ind. 1: 345.1820; FBI 7: 295.1896.
Dactyloctenium aegyptiacum Willd., Enum. Pl. Hort. Berol. 1029.1809.
An annual grass, culms erect or creeping, roots at the nodes, branches geniculately ascending, nodes bearded
or glabrous, leaf sheaths 26 cm long, hairy near mouth, ligule a ciliate rim, blades 210 0.30.8 cm, linear,
flat, base rounded, apex acuminate, glabrous or hairy with bulbous hairs; spikes 26, digitately radiating, upto 7
cm long, spikelets 35 flowered, laterally compressed, sessile, glumes subequal, folded, in lower keels scabrid,
in upper keel smooth or hispid, awned, lemmas aristate, palea hyaline, winged on keels, stamens 3, stigmas
plumose; caryopsis obovate.
14. Dichanthium annulatum (Forssk.) Stapf., Fl. Trop. Afr. 9: 178.1917; Gam vol. 3.1740; Bor 133.1960;
Deshp in Fasc. Fl. India 15: 5.1984.
Andropogon annulatus Forssk., Fl. Aegy-Arab. 173.1775; FBI 7: 196.1896.
A perennial, tufted grass, culms upto 70 cm tal, erect or ascending, nodes bearded, leaf sheaths 510 cm
long, glabrous, ciliate near the mouth, ligule membranous, blades 620 0.30.5 cm, linear, base rounded, apex
acuminate; racemes 6 cm long, sub digiatately fascicled, peduncles glabrous, spikelets 4 mm, 2-nate, sessile
spikelet: 4mm, bisexual, elliptic-oblong, lower glume elliptic-oblong, truncate, keeled, ciliate, upper glume,
nerveless, upper lemma narrow, with a scabrid, slender awn, stamens 3, stigmas plumose, caryopsis oblongslightly compressed, pedicelled spikelet: 4mm, male or barren, lower glume elliptic-oblong, 711 nerved keeled,
upper glume narrow, 3-nerved, lower lemma ciliate, upper lemma small or obsolete.
15. Dichanthium caricosum (L.) A. Camus., Gam vol. 3. 1741; Bor 134.1960; Deshp. in Fasc. Fl. India 15:
7.1986.
Andropogon caricosus L., Sp. Pl. 2: 1480. 1763; FBI 7: 196.1896.
A perennial, tufted grass, ascending, nodes bearded or with short hairs, leaf sheaths 3-10 cm long, glabrous,
ligule shortly ciliate membrane, blades 420 0.30.5cm linear, base rounded, apex acuminate, glabroussparsely pubscent, racemes 16, upto 8 cm long, both terminal and axillary; joints and pedicels of racemes with
short hairs, spikelets 4mm, 2-nate, sessile spikelets: 2mm, oblong, bisexual, lower glume oblong-obvate, 4nerved, truncate, pilose, upper glume elliptic, 3-nerved, hairy, lower lemma lanceolate, nerveless, hyaline, upper
narrow, awned, stamens 3, pedicelled spikelet, lower glume obovate, many nerved, pilose, upper glume and
lemmas similar to sessile spikelet.
16. Digitaria ciliaris (Retz.) Koeler., Descr. Gramin. 27.1802. (Fig. 1G)
Digitaria marginata Link var. fimbriata (Link) Stapf in Prain, Fl. Trop. Afr. 9: 440.1919; Gam vol. 3: 1764
(1222). 1934.
D. adscendens (H.B.K.) Henr. in Blumea 1: 92.1934; Bor 298.1960.
Panicum adscendens H.B.K. Nov., Gen. Pl. 1: 102.1821.
An annual, tufted grass, culms upto 90 cm tall, usually ascending from geniculate or prostrate base,
branching from lower nodes, nodes glabrous, leaf sheaths 412 cm long, glabrous, ligule membranous, truncate,
blades 410 0.40.6 cm, linear-lanceolate, base cordate, apex acute, glabrous, racemes 49, 10 cm long,
digitate, rachis serrate, trigonous, spikelets 3mm, elliptic-lanceolate, 2-nate, apperssed to the rachis, glabroussparsely ciliate, homomorphous, lower glume reduced to a triangular scale, upper glume linear-lanceolate, 35
nerved, pubescent, lower lemma oblong-lanceolate, 5-nerved, marginal nerves pubescent, upper lemma oblonglanceolate, obscurely 3-nerved, stamens 3, stigmas plumose, caryopsis oblong.
17. Digitaria longiflora (Retz.) Pers., Gam vol. 3: 1765; Bor 302.1960.
Paspalum longiflorum Retz., Obs. Bot. 4: 5.1786; FBI 7: 17.1896.
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Figure 1. A, Aristida hystrix L.f.; B, Capillipedium assimile (Steud.) A. Camus.; C, Chloris barbata Sw.; D, Chrysopogon
aciculatus (Retz.) Trin.; E, Chrysopogon orientalis (Desv.) A. Camus.; F, Dactyloctenium aegyptium (L.) Willd.; G,
Digitaria ciliaris (Retz.) Koeler.; H, Eragrostiella bifaria (Vahl) Bor.; I, Eriochloa procera (Retz) C.E. Hubb.
An annual, tufted grass, culms upto 60 cm tall, ascending, rooting at the nodes, nodes glabrous, leaf sheaths
24 cm long, glabrous, ligule membranous, blades 27 0.20.5 cm, linear-lanceolate, base rounded, apex
acuminate, glabrous, racemes 25, 7 cm long, digitate, rachis serrate, spikelets 2 mm long, elliptic,
homomorphous, sparsely pubscent, lower glume absent, upper glume oblong-ovate, 35 nerved, hairy, lower
526

.
lemma similar to upper glume, upper lemma ovate-oblong, subchartaceous, nerveless, palea similar to upper
lemma, stamens 3, stigmas plumose, caryopsis ellipsoid.
18. Digitaria sanguinalis (L.) Scop., Fl. Carn. ed. 2.1. 552.1772; Bor 304.1960; T.A. Cope in Nasir & Ali Fl.
Pak. 143. 231.1982.
Panicum sanguinalis L., Sp. Pl. 57.1753.
Paspalum sanguinale (L.) Lam., III. 1: 176.1791; FBI 7: 13.1896.
Digitaria sanguinalis (L.) Scop. ssp. aegyptica var. frumentacea Henr. Monogr.
Digitaria 985.1950; Bor 304.1960.
Digitarua sabguinalis ssp. vulgaris var. rottleriana Henr., 1.c. 986; Bor 304.1960.
An annual, tufted grass, culms up to 40 cm tall, erect or ascending from a creeping, branching base, leaf
sheaths glabrous, hairy near the mouth, blades 2.5180.40.8 cm, linear; spikes, slender, 310, 8 cm long,
digitate, spikelets 2-nate on abbreviated peduncles, imbricate, lower glume 35 nerved, lateral nerves marginal,
lower lemma lanceolate or oblong-lanceolate, smooth; caryopsis oblong, whitish.
19. Dinebra retroflexa (Vahl) Panz., Gam vol. 3: 1841 (1274). 1934; Bor 491.1960.
Cynosurus retroflexus Vahl, Symb. Bot. 2. 20.1791.
Dinebra arabica Jacq., Fragm. Bot. 77. t. 121. f. 1.1807; FBI 7: 297.1896; AP. Fl. 1182.
An annual, tufted grass, culms up to 70 cm tall, geniculately ascending below, nodes glabrous, leaf sheaths
59 cm long, loose, glabrous, ligule narrow membrane; blades 510 0.20.4 cm linear, base cordate, apex
acuminate, glabrous, spikes racemosely arranged along the axis of an inflorescence, up to 20 cm long, rachis
stiff, serrate, winged, spikelets 5mm, alternate, 2-flowered, glumes persistent, lanceolate, 1-nerved, strongly
keeled, slightly recurved, scaberulous awn, lemma ovate-oblong, hyaline; palea ovate, hyaline, 2-keeled,
minutely sparsely ciliate on the keel, stamens 3, stigmas plumose, caryopsis ellipsoid.
20. Echinochloa colonum (L.) Link., Gam vol. 3: 1776; Bor 308.1960.
Panicum colonum L., Syst. Nat. (ed.10) 810.1759; FBI 7: 32.1896.
An annual, tufted grass, culms up to 70 cm tall, branching from lower nodes, nodes glabrous, leaf sheaths 5
15 cm long, glabrous, ligule obsolete, blade 525 0.30.6 cm, linear-lanceolate, base cordate, apex acuminate,
glabrous; spike like panicle, racemes 820, appressed to the rachis, rachis angular, scaberulous, spikelets 2.5
cm, broadly ovate, acute or sub-cuspidate, crowded in rows, second, glumes unequal, lower glume half the
length of the upper glume, broadly ovate-orbicular, upper glume broadly ovate, mucronate, puberulous, lower
lemma broadly ovate, cuspidate, upper lemma broadly ovate, obtuse, palea oblong, stamens 3, stigmas plumose;
caryopsis broadly ellipsoid.
21. Echinochloa crusgalli (L.) Gam, vol. 3: 1777 (1231). 1934; Bor 310.1960.
Panicum crusgalli L., Sp. Pl. 56.1753; FBI 7: 30.1896.
An annual, tufted grass, culms 70 cm tall, branching from lower nodes, nodes glabrous, leaf sheaths 525
cm, glabrous, ligule obsolete, blades 520 0.40.8 cm, linear-lanceolate, base cordate, apex, acuminate,
glabrous; spikelike panicle 10 cm long, racemes spreading, rachis rtiquetrous, scabrid, spikelets broadly ovoid, 5
mm, crowded, acute-cuspidate, awned, lower glume broadly ovoid or orbicular, margin ciliate, half as long as
the upper glume, upper glume broadly ovate-oblong, concave, cuspidate, hispid on back, lower lemma ovateoblong, stamens 3, stigmas plumose, caryopsis broadly ellipsoid.
22. Echinochloa frumentacea Link., Bor 311.1960.
Panicum crusgalli var. frumentaceum Hook.f. FBI 7: 31.1896.
Echinochloa colona Link var. frumentacea Ridl., Fl. Malay Penin 5: 223.1925; Gam vol. 3: 1777.
An annual, robust grass, culms up to 1 m, leaf sheaths 12 cm, ligule absent, blades to 20 2 cm, racemes 18
cm, several seriate, spikelets 4 mm, ovoid-broadly ellipsoid, hispid, glumes subequal, upper with a short
mucronate apex, lower lemma mucronate, stiff-hispid outside, upper lemma crustaceous, short mucronate, palea
similar to its lemma, caryopsis broadly ellipsoid.
23. Echinochloa stagnina (Retz.) Beauv., Gam vol. 3: 1777 (1231). 1934; Bor 311.1960.
Panicum stagninum Retz., Obs. Bot. 5: 17.1789.
P.crusgalli sensu Hook.f., FBI 7: 30.1896 p.p. non L. 1753.
527

.
An annual stoloniferous grass, culms up to 50 cm tall, rooting at the nodes, nodes glabrous, leaf sheaths 5
10 cm long, loose, glabrous, ligule a fringe of stiff hairs, blades 415 0.40.8 cm, linear-lanceolate, base
cordate, apex acuminate, midrib prominent; spikelike panicle 510 cm long, racemes interrupted, rachis angular,
scabrid, spikelets ovate, crowded, acutely acuminate, awned, secung, pubescent, lower glume broadly ovate,
bidentate, pubescent, upper glume broadly ovate, acuminate, hairy at apex and margins, lower lemma ovate,
hairy and scabrid at apex, awned, palea oblong-ovate, scabrid margin, male; upper lemma ovate, scabrid along
the margins, acute, chartaceous, palea similar to upper lemma, bisexual, stamens 3, stigmas plumose; caryopsis
broadly ellipsoid.
24. Enteropogon dolichostachya (Lag.) Keng., Gen. Sp. Pl. 5.1816; Gam vol. 3: 1838 ; Bor. 466.
Chloris incompleta Roth, Nov. Pl. Sp. 60.1821; FBI 7: 290.1896; Bot. Bihar & Orissa 968.1924; Gam vol.
3.1838; Suppl. Bot. Bihar & Orissa 164.1950; GS. 374; AP. Fl. 1149; MV. 199; Maha Fl. 426.
Slender perennials, upto 93 cm long, culms erect, sometimes decumbent, nodes glabrous, leaf blades linear
acuminate, ligule comprise of long hairs, spikes 24 digitate, rhachis angled, scabrid on angles, spikelets
unilateral, 2-flowered, 2-awned, 2 seriats, lower glume membranous 1-nerved, upper glume short awned, lower
lemma membranous, 2-toothed at apex, awned, callus beareded.
25. Eragrostiella bifaria (Vahl) Bor., FBI 7: 325.1896; Gam vol. 3.1828. (Fig. 1H)
A perennial, tufted grass, culms up to 50 cm tall, slender, leaf sheaths glabrous, ligule a pubescent line,
blades 520 1.01.5 cm, linear, filiform, base narrow, apex acute, glabrous, spike 525 cm long, spikelets
5mm long, oblong, second, alternately arranged on the rachis, laterally compressed, 540 flowered, glumes
subequal, deciduous, lanceolate, 1-nerved, keeled, lemma broadly ovate, 1-nerved, keeled, palea lanceolate,
winged on keels, stamens 3, styles plumose; caryopsis globose.
26. Eragrostis cilianensis (All.) Janch., Gam vol. 3.1827; Bor 503.1960.
Poa cilianensis All., Fl. Pedem. 2: 246. t. 91. f. 2, 1785.
Eragrostis major Host., Icon. Descr. Gram Austr. 4: 14. t. 24.1809; FBI 7: 320.1896; HS. 516.
An annual, tufted grass, culms up to 90 cm high, leafy, branched, erect or geniculately ascending, nodes
glabrous, leaf sheaths 24 cm long, loose ciliate at mouth, ligule a ciliate ridge, blades 512 0.40.6 cm,
lanceolate, flat, prominently nerved, base cordate, glandular along the margins, apex acuminate; panicle up to 20
cm long, spreading or contracted, rhachis stiff, spikelets 1 cm long, oblong, breaking up from down wards, 10
30 flowered, glumes subequal, linear, ovate-lanceolate, scabrid on the nerves at back sides, lemma broadly
ovate, acute, 3-nerved, palea linear, ciliate on the margins near the apex, persistent, stamens 3, stigmas plumose,
caryopsis subglobose.
27. Eragrostis ciliaris (L.) R.Br., FBI 7:314.1896; Gam vol. 3.1825; Bor 506.1960.
Poa cilliaris L., Syst. Nat. ed. 10. 2. 875.1759; HS. 516.
An annual, tufted grass, culms up to 40 cm tall, slender, geniculately ascending below, nodes glabrous, leaf
sheaths 2.5 cm long, glabrous, ciliate at the mouth, ligule ciliate, blades 25 0.20.4 cm, linear, convolute,
base rounded, apex acuminate, glabrous, panicle up to 8 cm long, interrupted, cylindrical, branches short,
spikelets 2.5 mm, oblong, subsessile, glumes subequal, ovate-lanceolate, scabrid on the nerve on the backside,
lemma ovate, 3-nerved, lateral nerves close to the margin, ciliate, palea obovate, truncate, with soft ciliate,
stamens 3, stigmas plumose, caryopsis ovoid.
28. Eragrostis coarctata Stapf., FBI 7: 313.1896; Gam vol. 3.1825; Bor 507.1960; GS. 396.
Annual, tufted, 34 culms in a tuft, erect, slender, nodes, internodes glabrous, leaf sheath, leaves hairy;
panicle dense 1520 cm long, grayish-purple, spikes up to 15, short 56 cm long, villous at nodes, spikelets up
to 50 arranged in distichous manner, pedicel short scaberlous, spikelets single contains a bisexual floret, glumes
unequal, scaberulous on margins, tip acute or acuminate, 1-nerved, lemma & palea ovate, tip obtuse, margins
ciliate, pink, stamens 3, lodicules 2, grain oblong, reddish brown.
29. Eragrostis japonica (Thunb.) Trin., Gam vol. 3.1826; Bor 509.1960.
Poa japonica Thunb., Fl. Jap. 51.1784.
Eragrostis interrupta Beauv. var. tenuissima Stapf in. FBI 7: 316.1896; GS. 397.
528

.
Perennial, tufted grass, culms 1.01.5 m high, geneculately ascending at base, plants glabrous, ligule
membranous, leaf blade linear-lanceolate, tip acuminate, base cordate or rounded; panicle up to 50 cm long,
branches whorld, lax, inturrepted, internodes long, spikelets 29 flowered, glumes subequal, narrow, 1-nerved,
lemma 3-nerved, palea boat shaped, scaberulous, stamens 3 or 2, caryopsis obovoid.
30. Eragrostis tenella (L.) Beauv. ex Roem. & Schult., FBI 7: 315.1896; Bor 513.1960. var. tenella; HS. 518;
GS. 398.
Annual, densely tufted grass, culms 50 cm high, leaf sheaths glabrous long ciliate at mouth, panicle 20 cm
long, decompound, spreading or contracted, spikelets 39 flowered, small, palea keeled ciliate, caryopsis ovate.
31. Eragrostis viscosa (Retz.) Trin. Acad. Sci. Petersb. 6, 1: 397.1830; Gam vol. 3.1826; Bor 515.1960.
Poa viscose Retz., Obs. Bot. 4: 20.1786/87.
Eragrostis tenella var. viscosa (Retz.) Stapf in FBI 7: 315.1896.
Perennial, tufted, viscid, scented grass, culms 50 cm high, glandular patches scattered throughout, nodes
glabrous ligule fimbriate membrane, panicle 20 cm long, cylindric, dense glandular, branches numerous,
spreading spikelets 520 flowered, glumes unequal, linear, 1-nerved, lemmas ellipsoid, palea linear, ciliate on
the keels, caryopsis ovoid.
32. Eriochloa procera (Retz) C.E. Hubb., Gam vol. 3.1767; Bor 312; AP. Fl. 1202. (Fig. 1I)
Eriochloa polystachya Hook.f., FBI 7: 20.1896; Maha Fl. 494.
Perennials, densely tufted, up to 1 m high, root stock short; leaves linear, base cordate, ligule rim of hairs.
33. Eriochloa fatmensis (Hochst. & Steud.) Clayton., Bor 312; Maha Fl. 493.
Annual, terrestrial, culms 3060 cm high, geneculately ascending and rooting at lower nodes, ligule hairy;
panicle of simple or branched racemes, 310 cm long; spikelets paired or rarely solitary, ellipsoid, hispid.
34. Heteropogon contortus (L.) Beauv. ex Roem. & Schult., Gam vol. 3: 1743; Bor 163.1960. (Fig. 2A)
Andropogon contortus L., Sp. Pl. 1045.1753; FBI 7: 199.1896; AP. Fl. 1206.
An annual, tufted grass, culms up to 60 cm high, erect or decumbent below; nodes glabrous, leaf sheaths 5
10 cm, glabrous; ligule membranous, fimbriate; blades 410 0.20.4 cm, lanceolate, flat, base rounded, apex
acute, scaberulous; racemes solitary, 6 cm, awns forming a twisted spire; lower spikelets homogamous,
unawned, either neuter or male; upper spikelets heterogamous, awned, 2-nate, sessile hermaphrodite, pedicelled
male or barren; pedicel glabrous; callus pungent, bearded with brown hairs; sessile spikelets: 7 mm, linear,
hispidulous, female; callus bearded with brown hairs, lower glume linear-oblong, hispidulous; upper glume
lenear, obtuse, margin hyaline, dark brown, hispidulous; lower lemma oblong, truncate, epaleate; upper lemma
linear, stipitoform, awned, epaleate; stamens 3, stigma plumose, caryopsis linear; pedicelled spikelets: oblong,
hispidulous, lower glume lanceolate, dorsally hispidulous with long bulbous based hairs, winged, serrulate;
upper glume oblong-lanceolate, margin hyaline; lemmas hyaline, nerveless, hairy.
35. Oplismenus burmanni (Retz.) P. Beauv., FBI 7.66; Gam vol. 3: 1777; V. Fl. 216.
Panicum burmanii Retz., Obs. 3. 10.1783; MV.205; HS. 521; GS. 412.
A small procumbent herb, lower nodes bears roots, leaf sheaths, leaf blades hairy, leaf blade ovate
lanceolate, base cordate, ligule fringe of hairs; inflorescence panicle, racemes 4, distant, rhachis trigonous,
villous, spikelet 2 nate, unequal, sessile spikelet small, pedicelled spikelet large, pedicel villous, 2 flowered,
lower glume membranous, ovate, obtuse, pubescent margins ciliate, 3 nerved, awned, awn scaberulous, upper
glume similar to lower glume 7 nerved, awn short, lower lemma awned, empty, membranous, 9 nerved, 2
keeled, epaleate; upper lemma chartaceous, its palea similar, contains bisexual floret, stamens 3, stigmas
plumose.
36. Oplismenus compositus (L.) P. Beauv., FBI 7: 66; Bor. 317; S. 317; Gam vol. 3.1777; Bot. Bihar & Orissa
3.1045; MV. 205; HS. 521.
Pannicum compositus L., Sp. Pl. 1: 57.1753.
Oplismenus lanceolatus Kunth, Rev. Gram. 45.1829.
529

.
Perennial, stoloniferous up to 60 cm tall, creeping, rooting at nodes, nodes glabrous, leaf sheath 29 cm
long, margin density liliate, ligule fimbriate membrane; inflorescence terminal, racemes 20 cm long, spikelets 2
flowered, awned, lower glume awned, upper glume aristate.
37. Panicum maximum Jacq., Gam vol. 3.1783; Bor 327.
Perennial tufted grass, culms up to 1 m high, stout, nodes hirsute; ligule membranous, blades linear, flat,
base cordate, margin scaberulous, apex acuminate, glabrous, mid rib prominent; panicle lax, spikelets linearoblong, dense, lower glume orbicular, half the length of spikelet, 1-nerved, upper glume broadly oblong,
margins incurved, 5-nerved; lower lemma oblong, 5-nerved, palea oblong, 2-nerved, male, upper lemma ovate
oblong, 3-nerved, transversely rugose, its palea similar, nerve less, bisexual.
38. Panicum notatum(Retz.) Bor, 329; Gam vol. 3.1783.
Perennial slender grass, culms up to 1 m high, erect from woody stock, branched; nodes glabrous; leaf
sheaths ciliate on margin, ligule of soft long hairs, blades linear-oblong, base amplexicaul, margin with
tubercled based hairs, apex acuminate, pubescent on both surfaces; panicle up to 25 cm long, lax, open,
branches spreading, spikelets few, pedicels very long, filiform; lower glume ovate, 35 nerved, pilose, upper
glume broadly ovate, 5-nerved; lower lemma ovate, 5-nerved, hairy on outsite, epaleate, upper lemma ovate,
crustaceous, indurated, palea coriaceous with involute margins.
39. Paspalidium flavidum (Retz.) A. Camus., Fl. Indo-Chine 7.419.1922; Bot. Bihar & Orissa 1000.1924; Gam
vol. 3.1774; Bor 333.1960. (Fig. 2B)
Panicum flavidum Retz., obs. Bot. 4: 15.1786; FBI 7. 28.1896; GS. 419; MV. 206; HS. 523; V. Fl. 214.
Perennials, tufted, up to 80 cm high, culms erect base decumbent, striate, glabrous, nodes glabrous, leaf
blades linear, lanceolate, acute or obtuse, cordate at base, margins scabrid, ligule membranous, base narrow
ciliate, sheaths glabrous, striate; inflorescence racemose, racemes distant, up to 2 cm long, rhachis flattened,
minutely ciliate, spikelets biseriate, sub sessile, spikelets 2 flowered, lower floret empty, upper floret bisexual,
lower glume hyaline, suborbicular, 3-nerved, lower lemma ovate-oblong, subcoriaceous, 5- nerved, palea sililar
to its lemma, bidentate, upper glume membranous, sub orbicular, rounded at apex, 7-nerved, upper lemma
ovate, acute, coriaceous, granulose, palea similar to its lemma 2 keeled, lodicules 2, broadly cuneate, stamens 3,
yellow, ovary triquetrous, styles 2, laterally exserted.
40. Paspalidium geminatum (Forssk.) Stapf., Fl. Trop. Africa 9.583.1920; Bot. Bihar & Orissa 1001.1924; Gam
vol. 3.1774.1934; Susppl. Bot. Bihar & Orissa 176.1950; Bor. 333.1960.
Panicum geminatum Forssk., Fl. Heg-Arab. 18.1775.
P. paspaloides Pers., Syn. 1. 81.1805; FBI 7: 30.1896; GS. 420.
Perennial, culms up to 50 cm high, creeping below, branches from the lowernodes, nodes glabrous, ligule a
ciliate rim; panicle, spike like, racemes 1015, alternate on the rhachis; spikelets solitary, 2- flowered, lower
male, upper bisexual.
41. Paspalidium punctatum (Burm.) A. Camus., Gam vol. 3.1774; Bor 333.
Panicum punctatum Burm., f. Fl. Ind. 26.1768; FBI 7: 29.1896; AP. Fl. 1233.
A perennial, tufted grass, culms up to 1m tall, prostrate, rooting at lower nodes, spongy, leaf sheaths 10 cm
long, ligule a ridge of hairs, blades 1015 0.51.0cm, flat or convolute, apex acuminate, glabrous; spike like
panicle 30 cm long, racemes 20 cm long, distant, sessile, compressed, appressed to the rachis, spikelets 3mm
long, 1840 per raceme, ovoid, lower glume truncate; upper glume broadly oblong; lower lemma acuminate,
palea empty, upper lemma punctate, apiculate, palea coriaceous, margins incurved, stamens 3, stigmas plumose,
caryopsis ovoid.
42. Paspalum canarae (Steud.) Veldkamp., Blumea 21: 72 1973.
Panicum canarae Steud., Syn. Pl. Glumac. 1: 58.1853.
Paspalum compactum sensu FBI 7: 12.1896, non Roth 1821; Gam. vol. 3.1772; Bor 336.1960; GS. 421.
Annuals, slender, tufted, culms decumbent glabrous nodes bearded, leaf blades ovate-lanceolate, obtuse,
base rounded, hirsute with bulbous based hairs, margins ciliate ligule a rim of hirsute hairs, inflorescence
racemose, rhachis angled, winged, hairy at the base of branches, spikelets 2-nate, plano convex, lower glume
530

.
absent, lower lemma empty as long as the spikelet, 3-nerved epaleate, upper glume as long as the spikelet,
broadly ovate, convex, 5-nerved, margins hyalime, upper lemma bisexual, chartaceous, apiculate, palea similar
its lemma, 2-keeled, stamens 3, grain reddish-brown, plano convex, granulate.
43. Paspalum scrobiculatum L., FBI 7: 10.1896; Bot. Bihar & Orissa 1000. 1924; Gam vol. 3. 1772; Suppl.
Bot. Bihar & Orissa 176.1950; Bor 340.1960; GS. 422; HS. 524.
Perennial tufted, culms erect or decumbent, nodes glabrous, leaf blades linear-lanceolate, acuminate,
margins white and scabrid, ligules membranous, short, sheaths compressed, keeled, racemes 23, spikelets 2
nate, single obovate or suborbicular, rhachis ribbon like, ciliate, pedicels flattened, ciliate, lower glume wanting,
lower lemma nerved, epaleate membranous, upper glume membranous, glabrous concave, orbicular or broadly
ovate, 7 nerved, upper lemma broadly ovate, turning brown with age, palea similar to its lemma, margins
inflexed with flaps on either side at base, lodicules 2, quadrangular, stamens 3.
44. Paspalum vaginatum Sw., Prodr. Veg. Ind. Occ. 21.1788; Bor 341. (Fig. 2C)
Aquatic, perennial, rhizomatous, tufted; leaves linear-lanceolate, glabrous, margin puberulous, tip
acuminate, sheath hairy, ligule membranous, young leaves 7-nerved, old leaves 1-nerved; spikes 2, sub digitate;
spikelets 2-ranked, pedicelled, single, 2-flowered, lower floret empty and upper floret bisexual; pedicel
glabrous; Spikelets planoconvex, glabrous; lower glume absent, lower lemma membranous, 1-nerved, its palea
7-nerved, thick, stamens 2, orange, lodicules 2.
45. Pennisetum americanum (L.) Leeke., FBI 7: 82.1986; Gam vol. 3.1792; Bor 350.1960; AP. Fl. 1236.
An annual, cultivated, tufted grass, culms up to 2 m tall, nodes bearded, leaf sheaths overlapping, sparsely
tubercled based hairs, ligule a ciliate rim, blades 3090 0.55.0 cm, linear-lanceolate, base rounded, apex
acuminate, pilose below, midrib prominent, panicle up to 25 cm long, spiciform, cylindric, dense; rhachis stout,
villous below the inflorescence; involucral bristles 4 mm, often purplish-coloured, spikelet 4 mm, oblong, lower
glume absent, upper glume minute, truncate, 3-nerved, lower lemma oblong, obtuse, 5-nerved, epaleate, upper
lemma ovate-oblong, membranous, 2-nerved; palea very broad, truncate, ciliate at the tip, stamens 3, apex
penicillate, stigmas plumose, caryopsis oblong-obovoid or fusiform; glabrous, top exposed.
46. Pennisetum pedicellatum Trin., Mm. Acad. Imp. Sci. Saint-Ptersbourg, Sr. 6, Sci. Math., Seconde Pt.
Sci. Nat. 3(2): 184.1834; FBI 1: 86.1896; Gam vol. 3.1792; Bor 346.1960; AP. Fl. 1237.
An annual, tufted grass, culms up to 90 cm tall, branches from the base, leafy, leaf sheaths glabrous; ligule a
shortly ciliate membrane; blades 1020 39 cm, linear, flat, glabrous-sparsely hairy, racemes 13 cm long,
cylindric, densely flowered, involucral bristles outer few, slender, short, 3 mm, inner long, 1 cm, numerous,
densely villous below the middle, unequal, free from the base, spikelets 4 mm, solitary in the involucral, sub
sessile, lower glume very small, woolly; upper glume oblong-lanceolate, hyaline, apiculate; lower lemma
oblong, truncate, 3-lobed, hyaline; upper lemma ovate-oblong, obtuse, apex fimbriate ciliate, coriaceous,
shining; palea lanceolate, toothed.
47. Pennisetum polystachyon (L.) Schult., Gam vol. 3: 1792 (1241). 1934; Bor 346.1960.
Panicum polystachyon L., Syst. Nat. ed. 10. 870.1759; AP. Fl. 1238.
An annual, tufted grass, culms up to 40 cm tall, much branched, leaf blades 7.035 0.51.5 cm, panicle 20
cm long, linear, slender; rachis slender, puberulous; bristles densely plumose, especially the inner, spikelets
solitary, rarely 2 in an involucre, pedicelled, lower glume minute or suppressed; upper glume puberulous; lower
lemma similar to upper glume, 3-toothed; upper lemma chartaceous, fimbriate; palea similar to its lemma.
48. Pennisetum purpureum Schumach., Bor 348.1960; AP. Fl. 1238.
A perennial, stoloniferous grass, culms up to 2 m tall, erect, branching from lower nodes; nodes bearded, leaf
sheaths 1015 cm long, glabrous, ligule a dense fringe of hairs; blades 3060 0.71.5 cm, linear-lanceolate,
base rounded, margins scaberulous, apex acuminate, scabrid, midrib prominent, spikelike raceme up to 18 cm
long, solitary, cylindric, yellowish or purplish; involucral bristles numerous of unequal length, one usually very
much longer, 8 mm, scabrid, ciliate, inner most scabrid, 6 mm, sparingly plumose towards the base, spikelets
solitary, sessile, if in fascicles 24, lateral pedicelled, all lanceolate, 5mm, lower lemma absent or if present
ovate-lanceolate, truncate, scabrid; upper glume triangular, margin incurved, 3-nerved, scabrid; lower lemma
531

.
oblong-lanceolate, scabrid on outside surface, 5-nerved, male or barren; upper lemma lanceolate, rolled, scabrid
at apex, 2-nerved, stamens 3, penicillate at apex, style united throughout, stigmas plumose, caryopsis ovoid.
49. Pennisetum setosum(Sw) Rich. FBI 7: 87.1896; Bor 340.1960.
Cenchrus strosum Sw., Prodr. Veg. Ind. Occ. 26.1788; AP. Fl.1239.
A perennial, tufted grass, culms up to 80 cm high, often fastigiately branched at the nodes, leaf sheaths
glabrous; ligule with fringed long, soft hairs, blades 1020 510 cm, linear, apex acuminate, slightly hairy;
racemes 10 cm long, purplish brown, involucral bristles unequal, the outer not ciliate, short, 3mm, the inner
longer, long silky hairy below the middle, 1.3 cm, spikelet 4 mm, solitary within the involucral, lower glume
minute or completely absent; upper glume ovate-oblong, cuspidate, hyaline; lower lemma oblong, obtuse,
bidentate, palea narrowly oblong, hyaline, male: upper lemma ovate-oblong, truncate, coriaceous, shining; palea
oblong, truncate, toothed or ciliate at the apex, stamens 3.
50. Perotis indica (L.) Kuntze., Gam vol. 3.1814; Bor 611. (Fig. 2D)
Anthoxanthum indicum L., Sp. Pl. 28.
Perotis latifolia Ait., Hort. Kew Bull. 1: 96; AP. Fl. 1239.
Perennial, tufted, creeping or geniculately ascending from base; ligule absent, blades ovate-lanceolate, base
amplexical, margin spinulose, tip acute; inflorescence spike like raceme, terminal, spikelets small, subsessile;
glumes subequal, linear, 1-nerved, margin scabrid, awned, lemma and palea contains a bisexual floret, stamens
3.
51. Rottboellia exaltata (L.) L.f., FBI 7: 156.1896; Bot. Bihar & Orissa 1059.1924; Gam vol. 3.1759; Suppl.
Bot. Bihar & Orissa 194.1950; Bor. 206; V. Fl. 211; MV. 207; HS. 525. (Fig. 2E)
Annuals, erect, tufted, clinging roots from lower nodes, nodes and inter nodes glabrous purple colour, leaf
blades up to 60 cm, much longer than inter nodes, acuminate tip, margins & upper surface scaberlous, lower
surface smooth, mid nerve prominent white, ligule small membranous, sheaths scaberlous; racemes cylindric,
tapering towards the apex into an appendage, greenish yellow, spikelets 2 nate, sessile and pedicelled, sessile
spikelet sunk in the receptacle, awnless, lower glume coriaceous, 2 keeled, up to 11-nerved, lower lemma male,
ovate-lanceolate, 3-nerved, palea similar to its lemma, lodicules 2, stamens 3, upper glume boat shaped,
chartaceous, upper lemma bisexual, hyaline, 1-nerved, palea hyaline, lanceolate, pedicelled spikelet green, 2
flowered, both are male, compressed.
52.Setaria parviflora (Poir.) M. Kerguelen, Lejeunia 120: 161.1987.
S. geniculata P. Beauv., Bor 360.
Perennial herb, tufted, rhizome short; culms short, flat, procumbent, up to 25cm high; leaf sheath purple,
blade 10cm long, 1cm wide, margin purple, ligule membranous; inflorescence panicle cylindric, dense, 4.0
4.3cm long; spikelets single, if 2-nate, lower spikelets reduced to bristles (about 10), upper half part purple;
spikelets ovate, planoconvex, middle part of the pedicel hirsute, lower glume small 3- nerved, half of the
spikelet, lower lemma membranous margins inflexed, epaleate, empty; upper glume membranous, upper lemma
boat shaped, crustaceous rugose, its palea similar, containing a bisexual floret, stamens 3.
53. Setaria pumila (Poir.) Roem & Schult., Gam vol. 3.1789; Bor 363; AP. Fl. 1254. (Fig. 2F)
Annual tufted grass, culms up to 50 cm high, nodes glabrous; leaf sheaths glabrous, ligule a fringe of hairs,
blades linear-lanceolate, flat, base cordate, apex acuminate; panicle spiciform, cylindric, densely flowered,
spikelets ovoid; lower glume, upper glume and higher lemma ovate, hyaline; upper lemma crustaceous,
transversely rugose, stamens 3.
54. Setaria verticellata (L.) P. Beauv., FBI 7: 80; Gam vol. 3.1789; Bor 365.
Panicum verticillatum L., Sp. Pl. ed. 2.82; AP. Fl. 1255.
Annual, tufted, 24 culms in a tuft, ererct, culms up to 2 m high; stem flat, nodes swollen, straite, solid;
leaves up to 40 cm long, 3 cm wide, linear, base narrow, upper surface scabrid, 10 side nerves, lower surface
white, no nerves, margin scaberulous, ligule small membranous; inflorescence 2023 cm long, rhachis ends with
a setae, lower 3 spikelets 3-nate, remaining all 2-nate, spikelet having 1-long setae, setae downward barbelleate.
532

.
Figure 2. A, Heteropogon contortus (L.) Beauv. ex Roem. & Schult.; B, Paspalidium flavidum (Retz.) A. Camus.; C,
Paspalum vaginatum Sw.; D, Perotis indica (L.) Kuntze.; E, Rottboellia exaltata (L.) L.f.; F, Setaria pumila (Poir.) Roem
& Schult.; G, Sporobolus indicus (L.) R.Br.; H, Urochloa panicoides P.Beauv.; I, Urochloa setigera (Retz.) Stapf.
55. Sporobolus coromandelianus (Retz.) Kunth., FBI 7: 252.1896; Gam vol. 3: 1817 (1258). 1934; Bor
627.1960.
Agrostis coromandeliana Retz., Obs. Bot. 4: 1786.
Sporobolus commutatus (Trin.) Kunth, Enun. Pl. 1: 214.1833.
533

.
Vilfa commutate Trin., Diss. Bot. 156.1824; AP. Fl. 1258.
Annual, tufted grass, decumbent, nodes glabrous, ligule membranous, blades lanceolate, flat, base cordate,
margins cartilaginous spinulosely tooth, apex acute, sparsely tuberculate based hairs, inflorescence panicle,
decompound, branches capillary, horizontal, whorled at base, spikelets minute, pedicelled, lower glume minute,
suborbicular, upper glume, lemma similar, palea hyaline contains bisexual floret, stamens 3, caryopsis ellipsoid.
56. Sporobolus indicus (L.) R.Br., FBI 7: 247.1896; Gam vol. 3.1817; Bor 629.1960; AP. Fl. 1259. (Fig. 2G)
Perennial, tufted grass, culms 60 cm high, nodes, nodes glabrous, leaf sheaths glabrous, ligule fringe of
hairs, leaf blades linear flat, base truncate, apex acuminate, inflorescence contracted panicle, branches filiform,
spreading, spikelets small, single flowers, lower glume minute, ovate, hyaline, upper glume broadly elliptic,
hyaline, acuminate, lemma lanceolate, its palea small hyaline, stamens 2, stigmas plumose, caryopsis obovoid.
57. Themeda laxa (Anderson) A. Camus., Gam vol. 3.1746; Bor 251. AP. Fl. 1264.
Perennial, culms up to 40 cm high, tufted; leaf blades filiform, apex acuminate; panicle up to 8 cm long,
leafy, few racemes, shortly peduncled outer spathes 2.5 cm, proper spathes glabrous, longer than the spikes;
involucral spikelet: 4, all on the same level, male; lower glume glabrous except few bristles near the apex;
pedicelled spikelets: similar to involucral spikelet; sessile spikelet: solitary, bisexual, awned.
58. Themeda quadrivalvis (L.) Kuntze., Bot Bihar & Orissa 1050.1924; Gam. vol. 3: 1746.1934; Bor 252; FBI
7: 213.1896; GS. 442; HS. 528; MV. 2008; V. Fl. 210.
Annual, erect, culms and nodes glabrous, leaf blades flat, linear lanceolate, tip acuminate, rounded at base,
mouth ciliate, ligule membranous; inflorescence leafy panicle, fascicled, erect, spatheoles boat shaped
acuminate, tip 35 nerved, involucral spikelets pairs situated at the same leaf, middle sessile spikelet awned,
contains bisexual floret remaining four spikelets sessile, contains male florets, stamens 3, glumes of male florets
2 keeled with scarious and ciliate margin on one side, pedicelled spikelets 2, linear lanceolate, contains male
floret or empty.
59. Themeda triandra Forssk., Gam. vol. 3: 1746.1934; Bor 254: FBI 7: 211.1896.
Themeda imberbis (Retz.) Bomb., Fl. 2: 993; Bot. Bihar & Orissa 1049.1924.
Perennial, erect, up to 170 cm high, culms & nodes glabrous, leaf-blades flat, up to 41 cm long, linearlanceolate, acute, ligule membranous, rounded, truncate ciliate; inflorescence up to 40 cm long, panicles leafy
fascicled, erect or drooping, spathes long, leaf like, acuminate, margins with long hairs, involucral spikelets 2
pairs situated at the same level, shortly acuminate, each pair contains 5 spikelets, middle one sessile awned and
contains bisexual floret, callus bearded, sessile spikelets, surrounded by 4 male spikelets, involucral spikelets;
lower glume reddish brown, membranous, 2-toothed at apex, teeth unequal, 2-keeled with scarious margins on
one side, 11-nerved, covered with stiff bristles, upper glume margins ciliate in the upper half, 3-nerved, 2keeled, lemma male, hyaline, obtuse, margins ciliate, 1-nerved, epaleate, stamens 3, sessile spikelets, lower
glume coriaceous truncate at apex, 9-nerced, upper glume coriaceous, obtuse, 3-nerved, lower lemma hyaline,
epaleate, upper lemma reduced to an awn, epaleate, pedicelled spikelets male, pedicels glabrous.
60. Urochloa panicoides P. Beauv., Gam vol. 3: 1775; Bor 372. FBI 7: 35 p.p; HS. 530; V. Fl. 214. (Fig. 2H)
Annual, tufted, herbs sub erect, nodes swollen; leaves hairy, ligule fringe of hairs or glabrous, cordate at the
base, leaf sheath clasping the stem; inflorescence panicle, racemes 56, spikelets 2-nate, spikelets ovate to
lanceolate, glumes membranous, unequal, upper lemma cuspidate.
61. Urochloa setigera (Retz.) Stapf., Fl. Trop. Africa 9: 598.1920; Bot. Bihar & Orissa 1003.1924; Gam. vol. 3:
1775. (Fig. 2I)
Panicum setigerum Retz., Obs. Bot. 4: 15.1786; FBI 7: 36.
Brachiaria setigera (Retz.) Hubbard in Hookers Icon. Pl. 34. sub. t. 3363.1938; Suppl. Bot. Bihar & Orissa
177.1950; Bor 286.
Annuals up to 75 cm high, culms decumbent, rooting at lower nodes, geniculately ascending, nodes
pubescent, leaf sheaths hairy, leaf blade lanceolate, 1214 cm long, wide 2.6 cm, leaf base cordate, acute tip,
ligule a ring of white hairs; inflorescence panicle, 1015 cm long, racemes distant 1020, crowded 23, 26 cm
long, rhachis triquentrous, hairy on the angles interposed with 35 long hairs, each spikelet bears 35 long hairs,
534

.
spikelets 2 nate, sessile and pedicelled, spikelets 0.3 cm pedicels pubescent, glumes dissimilar, lower glume one
fourth of the spikelet, pink.
CONCLUSION
Uncultivated grasses are drought tolerant, fast growing, disease resistant, no need to sow, no need to
watering, no need to use fertilizers because these soils enriched with humus (cattle dung), nutrients and
moisture. They are helpful in controlling soil erosion. Over grazing leads the pastures to disappear, so retire the
land from the use by livestock at least till the green cover is sufficiently restored.
REFERENCES
Achariyar KR & Mudaliyar CT (1921) A Handbook of South Indian Grasses. Madras.
Blatter E & McCann C (1935) The Bombay Grasses. Sci. Monogr. No. 5. Imp. Counc. Agric. Rec. India.
Bor NL (1960) The Grasses of Burma, Ceylon, India and Pakistan. Pergamon Press, London.
Cooke T (19011908) The Flora of the Presidency of Bombay. London.
Fischer CEC (1934) Gramineae in J.S. Gambles Flora of Madras. London.
Gamble JS (1896) The Bambuseae of British India. Annals of Royal Botanical Garden Calcutta 7(1): 1133.
Hooker JD & Stapf O (1896) Flora of British India, Vol. 7. Gramineae International, U.K.
Sriramulu HS (1986)
Flora of Srikakulam district, Andhra Pradesh, India (Flora of India series). Indian
Botanical Society, Meerut.
Subba rao GV (1977) Flora of Visakhapatnam district, Andhra Pradesh, India (Flora of India series). Indian
Botanical Society, Meerut.
Venkaiah M (2004) Studies on the Vegetation and Flora of Vizianagaram District, Andhra Pradesh. Andhra
University, Visakhapatnam.
535
ISSN (E): 2349 1183

ISSN (P): 2349 9265
3(3): 536542, 2016
DOI: 10.22271/tpr.2016.v3.i3.070
Research article
Ecological studies of mangroves species in Gulf of Khambhat,

Gujarat
Vandna Devi and Bhawana Pathak*
School of Environment and Sustainable Development, Central University of Gujarat, Gandhinagar, India
*Corresponding Author: bhawana.pathak@cug.ac.in
Abstract: Mangrove forests are of utmost importance due to their role in preventing extreme
weather events like tsunamis and cyclones etc. The present study aimed to observe the mangrove
plant diversity and edaphic characteristics from Gulf of Khambhat, Gujarat. Ecological parameters
and edaphic characteristics were studied for different sites i.e. Navsari, Surat and Bhavnagar.
Avicennia marina was found as dominant species at all study sites. Plant species diversity shows
increasing tendency with the decrease in plant density. Important Value Index, Shannon-Weaver
diversity index and Simpson index of dominance of the mangrove species across the study area
were also determined. The present study provides the baseline data of mangrove species and
concludes the need of detail study for mangrove species in Gulf of Khambhat, Gujarat for
conservation and management strategies.
Keywords: Mangrove plants - Avicennia marina - IVI - Plant density - Conservation.
[Cite as: Devi V & Pathak B (2016) Ecological studies of mangroves species in Gulf of Khambhat, Gujarat.
INTRODUCTION
Mangrove forest ecosystems are significant for the biodiversity, protection of coastal area from erosion and
provision of protected nursery breading areas for marine fauna. Ecological study of any area or habitat helps to
understand the inter-relationship of all biotic (plants, microbes, other organisms) and abiotic (temperature,
moisture and soil etc.) components of environment. Globally mangrove forest cover around 1,46,500.00 km2 of
coastline (Alongi 2008) while total mangrove cover India is about 4,662.56 km2. This represents 0.14% of the
total geographical area of country and 3% of the global mangrove area. Mangroves are worlds most productive
ecosystems, found at the interface between land and sea in tropical and subtropical latitudes. Mangrove forests
are only forest on earth where land, freshwater and sea mix together. These forests are specially adapted to high
salinity, extreme tides, strong winds, high temperatures, low oxygen and muddy soil (Kathiresan 2010).
Gujarat is situated in the west coast of India which is surrounded by Arabian Sea. In maritime states of India;
Gujarat has largest coastal area around 28,000 km2 or longest coast line around 1650 km supports variety of
marine flora and fauna. The area under mangrove cover (1058 km) along the Gujarat coast is the second largest
block of tidal forest in India, next only to the Sunderbans (2155 km) (MoEF 201314). This state has two gulfs
out of three gulfs in India and the coastal area is spread from south Gujarat (high rainfall area about 2500
mm) to northwest of Kachchh (low rain area about 250 mm only). Different range of tides, waves, cyclones
and currents in the sea affect the physical as well as the biological conditions of the marine ecosystem whereas
clear cutting, hydrological changes, oil spills and climate change are creating more pressure on mangrove forests
sites (Blasco et al. 2001). In Gujarat 1103 km2 area is under mangrove which includes 175 km2 moderately
dense mangroves (15.86% of mangrove area of state), 928 km2 open mangrove (84.13%). The present research
study deals with the ecological status of mangrove species in Gulf of Khambhat, Gujarat.
Study area
Gujarat state is situated on the west coast of India between 2006 N to 2442 N latitude and 6810 E to
7428 E longitude. It is bounded by the Arabian Sea on the west. Ghogha from Bhavnagar (2140 N, 7217
E), Dumas from Surat (214N, 7242 E), Dandi from Navsari (2055' N, 7247' E), Dahej from Bharuch were
selected from Gulf of Khambat (Cambey) are selected for the present research work (Fig. 1 & 2).
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Figure 1. Aerial view of four selected sites (Source: Google Earth)
Figure 2. Habitat of mangrove vegetation: A & B, Bhavnagar; C, Navsari, D, Bharuch; E, Surat.
Field methods
The mangrove vegetation study was carried out from selected sites during low tide. Quantification of
mangrove vegetation at each site was done by quadrate method. 10 quadrates (33m) were laid randomly at
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each site and in each quadrate, total numbers of trees were counted; tree height and Girth at Breast Height
(GBH) was measured for all trees. Quantity parameters like frequency, relative frequency, density, relative
density, abundance, dominance, relative dominance, Important Value Index (IVI) was determined Curtis (1959).
The distribution pattern of species was determined by ratio of abundance to frequency if the ratio is below
0.025 then it indicates regular distribution, between 0.0250.050 indicates random distribution and when
exceeds 0.050 indicates contagious distribution (Whitford 1949). Species diversity was determines by using
Shannon index (H), Simpsons index of diversity (1-D) were calculated using standard methods (Shannon &
Weaver 1963, Simpson 1949, Kerkhoff 2010).
Collection and authentication of plant samples
Plant samples (leaves, flowers, stem, seeds and roots) were collected from selected sites for authentication
and preparation of herbarium. Authentication and identification of collected plant samples was done with the
help of Scientist, GEER foundation, Gujarat.
Soil sampling and chemical analysis
Soil samples were collected from 010 cm depths from each site during October and November of the year
2014. Five sets of samples were collected from each study site and mixed together to form a composite soil
sample and from which three replicate samples were brought to the laboratory. Collected soil samples were air
dried and sieved through a 2 mm mesh and was subjected to routine chemical analysis. Physicochemical
characteristics of soil samples were determined using standard methods (APHA 1998). pH of soil was
determined using method described by Black (1973). Total organic carbon was determined using method
described by Walkey & Black (1934).
RESULTS
Mangrove plant status
Mangrove
associates
True
mangrove
Table 1. Mangrove diversity of selected site of Gujarat.

Vernacular
Species Name
name
Avicennia marina (Forsk.) Vierh Tivar, Tavarian
var. acutissima Mold.
Bruguiera gymnorhiza (L) Lam
Tavar
Family
Avicenniaceae
Status in various sites

Bhavnagar Surat Navsari Bharuch
+
+
+
+
Rhizophoraceae
Sonneratia apetala Buch.-Ham
Motitavar
Lythraceae
Acanthus illicifolius
Ipomoea pes-carpae (L.) Sw.
Kantaliyo
Maryada-vel
Acanthaceae
Convolvulaceae
+
+
Sesuvium portulacastrum (L.) L.
Shore purslane
Aizoaceae
Salvadora persica L.
Toothbrush tree
Salvadoraceae
Suaeda sp.
Seepweeds
Amaranthaceae
Note: +, indicates presence; - indicates absence.

The patterns of mangroves and associated species distribution in selected study area depict little variation in
the species composition (Table 1). At Bhavnagar a total of 886 plants representing two species were identified
within 90 m2 area survey. Avicennia marina (Forsk.) Vierh (Avicenniaceae) showed the highest density (87.5
plants/90m2) with 83.33% relative frequency 99.99% relative dominance and 282.09 important value index.
Other species recorded with their ecological parameters is presented in table 2. Only two species (Avicennia
marina and Sonneratia apetala Buch.-Ham.) were found at Surat site. The maximum number of species was
recorded at Navsari site such as Avicennia marina, Sonneratia apetala, Bruguiera gymnorhiza (L.) Lam., and
Acanthus ilicifolius L. (Fig. 3). Only one species (Avicennia marina) was recorded at Bharuch site.
Avicennia marina has been found common in all selected sites exhibiting maximum density at Bhavnagar
(87.5 plants/90m2). Among all species the mean height was maximum of Sonneratia apetala (230.86 cm)
followed by Avicennia marina (127.28 cm) and Bruguiera gymnorhiza (109.82 cm). Avicennia marina is
dominating species at all selected sites with higher important value index at all sites. In general the distribution
pattern of all species was contagiously distributed at all study sites except Sonneratia apetala, which showed
random pattern of distribution at Surat and Navsari site. The Shannon-Weaver diversity index and Simpson
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Figure 3. Some mangrove plants: A, Bruguiera gymnorhiza (L.) Lam.; B, Sonneratia apetala Buch.-Ham.; C, Avicennia
marina (Forsk.) Vierh; D, Ipomoea pes-caprae (L.) R.Br.; E, Acanthus illicifolius L.
index of dominance was analyzed for dominance and species diversity (Shannon & Weaver 1963, Simpson
1949). The Shannon-Weaver index showed highest diversity at Navsari site (1.20) followed by Surat (0.20),
Bhavnagar (0.07) whereas Simpson index of diversity (1-D) was higher in Navsari (0.66) followed by Surat
(0.09), Bhavnagar (0.03) and the value of this index ranges between 0 and 1, the greater the value, the greater
the plant diversity. Result shows that Navsari site have been found with more diversity than other sites (Table 3
& 4).
13.7
1.75
Avicennia marina
Bruguiera gymnorhiza
Sonneratia apetala
14.9
3.9
3.1
12.4
43.44
11.37
9.04
36.15
14.9
4.88
3.44
13.77
Avicennia marina
18.5
100
18.5
83.33
16.66
71.68
3.30
471.59
0.006
99.99
0.001
0.88
0.28
282.09
17.91
71.43
28.57
86.87
77.01
61.63
2.02
96.83
3.17
0.13
0.04
263.39
36.60
27.03
21.62
24.32
27.03
127.28
109.82
230.86
63.75
122.39
27.56
119.63
-
44.74
10.07
43.73
-
0.15
0.06
0.04
0.14
115.21
43.07
77.09
-
100
114.6
43.09
100
0.185
300
Important
value index
(IVI)
95.14
4.86
A/F ratio
13.7
0.7
Relative
Dominance (%)
Avicennia marina
Sonneratia apetala
94.09 100
5.914 20
Site 2 Surat
88.67 100
11.32 40
Site 3 Navsari
40.27 100
13.18 80
9.31
90
37.24 100
Site 4 Bharuch
100
100
Total Basal
area (cm2/90m2)
87.5
5.5
Mean Height
(cm)
98.76
1.24
Relative
Frequency (%)
Abundance
(plants/90m2
87.5
1.1
Frequency (%)
Relative density
(%)
Avicennia marina
Sesuvium portulacastrum
Relative
Abundance (%)
Density
(plants/90m2)
Table 2. Vegetation characteristics of different selected sites.

Site 1 Bhavnagar
Table 3. Diversity indexes of selected mangrove sites.
Diversity indexes
Bhavnagar
Surat
Navsari
Shannon-Weaver Index (H)
0.07
0.20
1.20
Simpson index of Diversity (1-D)
0.03
0.09
0.66
Note: At Bharuch site only single species Avicennia marina was recorded in selected sampling area.
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Table 4. Species diversity of different mangrove sites.
Species Name
Sites
Total
species
Bhavnagar
Surat
Avicennia marina
Avicennia marina
Sesuvium portulacastrum Sonneratia apetala
Salvadora persica
Suaeda sp.
2
Navsari
Bharuch
Avicennia marina
Avicennia marina
Sonneratia apetala
Bruguiera gymnorhiza
Ipomoea pes-carpae
Edaphic characteristics
Soil characteristics of different selected sites have been presented in table 5. Soil mean temperature was
recorded from 29.933.5 C. pH ranges from 8.37 to 8.68. Similarly, the values of electrical conductivity were
from 4.25 mS.cm-1 to 12.28 mS.cm-1. In this study it was found that more water contents were present in soil at
Navsari than Bhavnagar and Surat.
Table 5. Physicochemical characteristics of different mangrove sites.
Navsari
Surat
Mean SD
SE
Mean SD
SE
29.9
0.581 0.259
33.5
0.757 0.338
Temperature (C)
8.376 0.034 0.015 8.680 0.147 0.066
pH
4.248 0.190 0.085 6.626 1.755 0.785
EC (mS/cm)
60.1
3.021 1.351
23.7
6.485 2.90
M.C. (%)
0.17
0.02
0.01
1.09
0.07
0.04
O.Carbon (%)
0.29
0.04
0.02
1.88
0.12
0.07
O.M. (%)
2.438
--1.208
--Total Nitrogen (%)
4.73
2.73
0.011
0.58
0.33
Av. Phosphorous (%) 0.019
0.081 0.012 0.007 0.111 0.003 0.002
Sulphate (%)
12.23
0.02 0.013 18.24
0.44 0.253
Cl (ppm)
265.33 2.31
1.33 538.67 4.62
2.67
Total Hardness
(mg CaCO3/kg)
Note: SD, Standard deviation; SE, standard error; n, 3; Av., Average.
Soil parameters
Bhavnagar
Mean SD
32.5
1.155
8.396 0.096
12.282 4.228
44.2
0.932
1.52
0.04
2.62
0.07
4.734
-0.022
3.61
0.119 0.001
38.16
1.05
1588 41.57
SE
0.516
0.043
1.891
0.417
0.02
0.04
-2.08
0.001
0.604
24.00
DISCUSSION
Gujarat has second largest area of mangroves in India (1058 km2). About 99.4% mangrove forest area is
represented by three mangrove areas; Gulf of Kachchh (15.2%), Gulf of Khambhat (10.1%) and Kachchh
district including Kori creek (74.1%) and remaining 0.6% in Valsad and Navsari district. Even with fewer
mangroves area Gulf of Khambhat was reported to have rare mangrove species. The Gulf of Khambhat includes
Bharuch, Surat, Navsari and Bhavnagar districts mainly. Therefore coastal area Ghogha from Bhavnagar,
Dumas from Surat, Dandi from Navsari, and Dahej from Bharuch has been selected for ecological study. In
present study total eight species (Avicennia marina (Forsk.) Vierh, Bruguiera gymnorhiza (L.) Lam., Sonneratia
apetala Buch.-Ham., Acanthus ilicifolius, Ipomoea pes-caprae (L.) R.Br., Sesuvium portulacastrum, Salvadora
persica and Suaeda sp.) were recorded from Gulf of Khambhat which includes both true mangrove and
associate species. Fourteen species of mangrove have already been reported i.e. Avicennia marina (Forsk.)
Vierh, Avicennia officinalis L., Avicennia alba Bl., Aegiceras corniculatum (L.) Blanco, Ceriops tagal (Perr.)
Robinson, Ceriops decandra (Griff.) Ding Hou, Excoecaria agallocha L., Sonneratia apetala Buch.-Ham.,
Acanthus ilicifolius L., Bruguiera cylindrica (L.) Bl., Bruguiera gymnorhiza (L.) Savigny, Lumnitzera racemosa
Wild, Rhizophora mucronata Lamk., Kandelia candel (L.) Druce (Bhatt et al. 2011). The abundance to
frequency ratio indicated that most of the species were contagiously distributed except Sonneratia apetala at
two sites (Surat and Navsari), which showed random distribution pattern. Smith (1957), Kershaw (1973), Kumar
& Bhatt (2006) have also reported contagious distribution in natural vegetation. In most of places landward
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mangrove are under pressure due to clearing of mangrove plants for fodder and conversion of mangrove area to
other forms of land use (Farnsworth & Ellison 1997, Ashton & Macintosh 2002).
Present study revealed that Avicenniaceae is most dominant mangrove family in all selected sites Avicennia
marina was the most frequent, most abundant and most dominant species in all selected sites. Average GBH of
selected mangrove species ranged from 0.28 cm to 22 cm. A similar kind of work has also been reported where
Avicenniaceae was the dominant mangrove family with 100% frequency and 72.55% relative frequency.
Average GBH of mangroves was 21.69 cm. Studies also showed that Avicennia marina can withstand more
harsh environmental conditions such as high salinity, high temperature etc (Lunar & Laguardia 2013). It was
found that mean height of Avicennia marina was maximum at Navsari (127.28 cm) followed by Bharuch (114.6
cm), Surat (86.87 cm) and Bhavnagar (71.68 cm). Among all species the mean height was maximum of
Sonneratia apetala (230.86 cm) followed by Avicennia marina (127.28 cm) at Navsari site and then by
Avicennia marina (114.6 cm) of Bharuch site and Bruguiera gymnorhiza (109.82 cm) of Navsari site. In this
study it was observed that where plant density is more there plant mean height is less. This may be due to the
reason that higher density of Avicennia marina at Bhavnagar may cause reduction in plant growth due to
competition for limited resources (Volin et al. 2005, Li et al. 2014).
Soil mean temperature was recorded in the range between 29.9C to 33.5C. A similar study has been done
where mean temperature of soil was recorded 31.87C at degrading mangrove habitat and 28.26C at luxuriant
mangrove habitat (Kathiresan 2002). The pH was recorded 8.37 (Navsari), 8.39 (Bhavnagar) and 8.68 (Surat)
which is supported by study of Rao & Rao (2014). Study showed that alkaline pH (8.358.79) in similar type of
habitat. pH have been reported from 7.118.52 in Pondicherry mangroves (Satheeshkumar & Khan 2009). In
present study it was found that more water contents were present in Navsari mangrove soil than Bhavnagar and
Surat mangrove soil. The moisture contents (mean values) were 23.7% (Surat), 44.4% (Bhavnagar) and 60.1%
(Navsari). High moisture content at Navsari can either be due to freshwater input of Purna river or frequent tidal
inundation (Ashton & Macintosh DJ 2002). It has been observed that 44.4% moisture content is more suitable
for plant density at Bhavnagar site where as on other sites with less and more moisture content plant density was
less. Kathiresan (2002) also found moisture content 31.49% at degrading sites and 42.2% at luxuriant mangrove
habitat. Total nitrogen was recorded from 1.21% to 4.7%. Similar results were observed by Hossain et al.
(2012), where total nitrogen has been reported from 0.057% to 0.158% in Sunderban mangrove soil whereas
available nitrogen at same site was reported from 0.504 to 2.016 g.g-1. Available nitrogen has been reported
from 29.4 to 81.2 ppm (Rao & Rao 2014). Available phosphorous was found maximum at Bhavnagar (0.022%)
followed by Navsari (0.019%) and Surat (0.011%). Available phosphorous of mangrove soil has been reported
3.32 ppm to 5.89 ppm (Rao & Rao (2014). Phosphates have been reported 0.06 mg L-1 by Dogiparti et al.
(2014). The values of organic carbon were observed 0.17% at Navsari, 1.09% at Surat and 1.52% at Bhavnagar
site and it has been found that with increase in organic carbon plant density is also increasing. Organic carbon
has been reported 4.28% and 3.12% at two mangrove areas of Andhra Pradesh (Dogiparti et al. 2014). The
organic matter was recorded from 0.29% to 2.62%, which shows similar results studied by Satheeshkumar &
Khan (2009) where organic matter has been reported from 0.94% to 3.94% in mangrove soil.
CONCLUSION
Present study revealed that Avicenniaceae is only dominant family in the Gulf of Khambhat coastal area,
Gujarat and due to anthropogenic disturbance i.e. clear cutting, hydrological changes, oil spills and climate
change are creating more pressure on mangrove forests in these sites and the other mangrove species either
present in very low number or disappeared from that site. The distribution pattern of mangrove species at Gulf
of Khambhat is mostly contagious. Availability of eight species but in less number indicated that in near future
the coastal region will be dominated by monotonous species (Avicennia marina). Other species regeneration
should be focused while making conservation plans. This study will provide baseline for further study in this
area and for development of conservation as well as management strategies for mangrove species.
ACKNOWLEDGMENTS
We are thankful to UGC for providing non-net fellowship. We extend our thanks to Dean, School of
Environment and Sustainable Development, CUG, Gandhinagar, India, for providing all necessary facility. We
also thank Dr. Harshad Salvi, Scientist, GEER foundation, Gujarat, India for extending help in identifying the
plant species. We would like to thanks my colleagues of the development, Central University of Gujarat.
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Satheeshkumar P & Khan BA (2009) Seasonal Variations in Physico-Chemical Parameters of Water and
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Volin HS, Novoplansky A, Goldberg DE & Turkington R (2005) Density regulation in annual plant
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ISSN (P): 2349 9265
3(3): 543550, 2016
DOI: 10.22271/tpr.2016.v3.i3.071
Research article
Floristic assessment of different habitats of Parvati Aranga

wildlife sanctuary and adjacent Tikri forest area,
Gonda, Uttar Pradesh, India
Vineet Singh1*, S. K. Srivastava2 and L. M. Tewari3
1*
Botanical Survey of India, Central Regional Centre, Allahabad, Uttar Pradesh, India
Botanical Survey of India, Northern Regional Centre, Dehradun, Uttarakhand, India
3
Department of Botany, DSB Campus, Kumaon University, Nainital, Uttarakhand, India
*Corresponding Author: vineet.singh332@gmail.com
[Accepted: 20 October 2016]
2
Abstract: The Parvati Aranga wildlife sanctuary and adjoining Tikri reserve forest in northeastern Terai region of Uttar Pradesh with its varied ecological habitats and occurrence of patchy
wetlands in form of River and Tals sustains a variety of plant communities. The area also
harbours a rich diversity of economical and medicinal plant species, mainly confined to the
peripheral region of the forest. A large component of the forest is occupied by diverse forest stands
and a number of special habitats portray remarkable vegetational diversity. The present
communication reveals that the plant community with special habitat especially in protected and
reserve forest area may plays a vital role in the future sustenance of the forest vegetation. Rarity
and regeneration pattern of the flora is also discussed.
Keywords: Plant community - Special habitats - Terai region.
[Cite as: Singh V, Srivastava SK & Tewari LM (2016) Floristic assessment of different habitats of Parvati
Aranga wildlife sanctuary and adjacent Tikri forest area, Gonda, Uttar Pradesh, India. Tropical Plant Research
3(3): 543550]
INTRODUCTION
The comprehension of relationship between plants and environmental factors can be used as an indicator of
environment, in this context a number of plants species used as ecological indicators. In a plant community
some plants are dominant and found in abundance, these are important markers because they bear full impact of
surroundings. In general, plant communities are better indicators than individual plants and are used to
determine the types of soil and other conditions of the environment in a given area. Sometimes these also
indicate past or future conditions of the environment. Community structure and composition with special
habitats immensely affects the plant diversity pattern in any forest area in terms of the sustenance of a particular
community.
Forest composition, community structure and diversity pattern are important ecological attributes
significantly correlated with prevailing environmental as well as anthropogenic variables (Gairola et al. 2008).
The region free from anthropogenic disturbances continues to provide a platform for the microhabitats for an
array of local floral elements Wildlife protected areas in India have had a relatively long history of forest
management and exploitation as majority of these areas were originally reserved or other categories of
government owned forests where focus on management was timber production, meeting the biomass demands
of local communities or soil or water conservation (Rodgers & Sawarkar 1988). The special habitats of any
forest plays a key role for the state of natural or reserve forest in the area and to suggest conservation measures
for the concerned elements.
The Terai expanse of eastern Uttar Pradesh is an assortment of human settlement, cultivation fields, natural
and semi-natural vegetations comprising of grasslands and forests. In this area most of the primary forests have
been substituted by economically and commercially important plants particularly tree species and agricultural
fields (Bajpai et al. 2012a). This landscape is listed among the important ecoregions of the world, well known
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for its unique biodiversity and productivity (Tripathi & Singh 2009). The region is an ecotone between bhabar
tract of foot hills of Himalayas and the Gangetic plains (Bajpai et al. 2015a). Several authors have dealt with the
vegetation of Terai region of the state (Panigrahi et al. 1969, Maliya 2007, Bajpai et al. 2015a, Kumar et al.
2015) and recently some authors also explored the ecological parameters of some forests of the region (Pandey
& Shukla 2003, Chauhan et al. 2008, Behera et al. 2012, Bajpai et al. 2012b, Bajpai et al. 2015b). However the
baseline data related to typical vegetation community of the Parvati Aranga wildlife sanctuary (PAWS) has not
been documented yet. Thus, this communication deals with the species composition and indicator taxa with
special habitats of PAWS.
The Study Area, biota and Climate
The study was conducted in Parvati Aranga wildlife Sanctuary (PAWS) and adjacent Tikri forest area at a
26 48'27 N longitude and 81 37'82 37' E latitude located in Gonda district of north-east Uttar Pradesh
(Fig. 1). It is established in 1990, spread over an area of 10 km2 of total 80 km2 area and remaining 70 km2 is of
the reserve forest area characterized by typical terai landscape. The sanctuary harbours a rich floral and faunal
diversity and is the home for many rare and migratory avifaunas (Singh 2015). The reserve forest is dominated
by Shorea robusta as climax species along with other tree species viz. Haldina cordifolia (Roxb.) Ridsdale,
Syzygium cumini (L.) Skeels, S. salicifolium (Wight) J. Graham, Tectona grandis L.f., Acacia catechu (L.f.)
Willd., Streblus asper Lour., Aegle marmelos (L.) Correa, Madhuca longifolia (J. Koenig ex L.) J. F. Macbr.,
Barringtonia acutangula (L.) Gaertn., Ficus racemosa L. etc. Understorey species were represented by
Clerodendrum serratum (L.) Moon, C. infortunatum L., Mallotus philippensis (Lam.) Muell. Arg., Glycosmis
pentaphylla (Retz.) DC. and Carrisa spinarum L. accompanied with climbers and lianas viz. Ichnocarpus
frutescens (L.) W.T.Aiton, Tiliacora racemosa Colebr., Bauhinia vahlii Wight & Arn., Cissampelos pareira L.
var. hirsuta (Buch. Ham. ex DC.) Forman, Cocculus hirsutus (L.) W. Theob., Abrus precatorius L., Tinospora
cordifolia (Willd.) Hook. f. & Thompson.
Figure 1. Location of the study area i.e. Parvati Aranga wildlife sanctuary and Tikri forest area, Gonda.
Along with affluent flora the reserve forest is also endowed with many mammalian fauna viz. Wild boar (Sus
scrofa), spotted deer (Axis axis), blue bull (Boselaphus tragocamelus), Indian porcupine (Hystrix indica),
Rhesus macaque (Macaca mulata) and grey langur (Semnopithecus ajax) with many reptilian species viz.
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Bengal monitor (Varanus benghalensis), Indian Cobra (Naja naja), Krait (Bungarus caeruleus), Rat snake
(Ptyas mucosa) and Indianpython (Python molurus). The wildlife sanctuary consists of a large wetland in form
of lake, rich in avifaunal diversity and different species of fishes. The area also harbours manyrare, threatened
and common native and migratory bird viz. Black drongo (Dicrurus macrocercus), Green bee eater (Merops
orientalis), Red- wattled Lapwing (Vanellus indicus), Purple swamphen (Porphyrio porphyrio), Sarus crane
(Grus antigone), Pied kingfisher (Ceryle rudis), Grey headed fish eagle (Ichthyophaga ichthyaetus) and Red
vented Bulbul (Pycnonotus cafer) along with many other species. The people residing near by the sanctuary and
the reserve forest are mainly depend upon the natural resources such as fuel wood, fodder, thatched grass and
Non- timber forest products for their livelihood and for sacred rituals (Singh & Srivastava 2014). The area is
also rich in many medicinal and economically valuable angiosperms and pteridophytes (Singh & Srivastava
2015).
The climate is typical monsoon type with three different seasons viz. summer (MarchJune), Rainy (July
September) and winter (OctoberFebruary). Mean annual rainfall is about 1240 mm. The driest month is
November with 2 mm of rain. The greatest amount of precipitation occurs in July with an average of 356 mm.
May is the warmest month of the year; the average temperature is about 34C during this month. The lowest
temperature in the year occurs during January it measures around 15.5C. The forest of the area have been
classified as Eastern Heavy Alluvium plains Sal forest with some part located along the river in swampy areas
fall under 4D/SS2- Barringtonia swamp forests and 4D/SS2- Syzygium cumini swamp low forests (Champion &
Seth 1968).
During the course of exploration (20142016), the various ecological habitats were visited in different
seasons of the year and the dominant species growing in different communities, which act as a keystone species
with special importance as indicator, have been collected randomly along with their field data, dried, preserved
and mounted by following the standard herbarium techniques (Jain & Rao 197778). These plant specimens
were finally identified with the help of floras (Hooker 18721897, Duthie 19031929) assisted by matching
with herbarium specimens for authentication and deposited in BSA and the correct nomenclature of the plants
has been provided after consulting recent floras and website like IPNI and The PLANTLIST. The vegetation of
the area was observed under different categories viz. top canopy tree species, under-storey, ground flora, lianas
and climbers. The relevant information regarding habit, habitat, relative abundance, association, flowering,
period, GPS data etc. were collected in the field.
There are certain plant species which portrays the nature and disposition of habitats commonly referred to as
plant indicator. It is found that certain species have one or more specific requirements which may limit their
distribution and the occurrence, character and behavior of a plant are thus indicator of the combined effect of all
factors prevailing in a habitat. These plant species establish themselves according to their environmental
requirement where conditions are favourable. The knowledge of relationship between plants and ecological
factors can be used as an indicator of environment. The characteristic species are collectively the best indicators
of ecological conditions of the community (Braun-Blanquet 1932). The plants are admittedly a measure of the
environment and although the community indicates the nature of the surroundings, only a few key species which
are restricted to their habitats are of special importance (Santapau 1958a).
The indicator implication of one group of plants must be inferred and applied to an entirely different group
of plants. Generally, forest indicators are herbs or shrubs as compared to trees. In the broad sense, forest
indicators are site indicators, but rarely do they suggest more than a portion of the several factors that contribute
to site. Some plants indicate the characteristic types of forest and they grow in an area which is not disturbed.
Narenga porphyrocoma is a grass which binds the soil in which sal (Shorea robusta) can be cultivated. Viola
species in eastern Himalayas is a suitable indicator for plantation of Cedrus deodara and Pinus wallichiana. If
we know that a particular forest grows better in certain area of specific soil the productivity can be increased.
Physical or chemical characteristics of soil moisture relationships, aeration or erosion may be indicated by some
species.
The nature and composition of flora is manifestation of their cumulative effects of all aspects functioning in
a particular habitat. It is usually accepted that a set of species or a whole community is steadier as an indicator
than a solitary species and that dominants, particularly of the climax species are more useful indicators than
lesser species. Species which are less tolerant to many varying conditions are usually indicators since their
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growth requirements are exacting hence, the dominant species from different communities of selected habitats,
are of special importance as indicators of the nature of habitat. The vegetation of the area can be broadly
classified in to two grops consisting of various types of plant associations, A. Common vegetation and B.
Vegetation of special habitats.
A. Common Vegetation
The common vegetation of the study area is of moist deciduous type with some evergeen and semi-evergreen
tree species. The flora under this category dominates the physiognomy of the forest area by forming different
phytoassociations which ultimately leads to a healthy forest in this terai region. Some of the important plant
associations (Fig. 2) are discussed below.
Figure 2. Number of important plant associates in general vegetation community.
1. Shorea robusta - Mallotus philippensis community: Under this category the other associates are Bridelia
retusa (L.) A.Juss., Carrisa spinarum L., Rotheca serrata (L.) Steane & Mabb., Clerodendrum infortunatum L.,
Curculigo orchioides Gaertn., Ceriscoides turgida (Roxb.) Tirveng., Glycosmis pentaphylla (Retz.) DC. and
Haldina cordifolia (Roxb.) Ridsdale.
2. Shorea robusta - Terminalia chebula Community: The other important associates are Aegle marmelos (L.)
Correa, Desmodium gangeticum (L.) DC., Phyllodium pulchellum (L.) Desv., Diospyros montana Roxb.,
Bauhinia vahlii Wight & Arn., Tiliacora racemosa Colebr. and Oplismenus compositus (L.) P. Beauv.
3. Tectona grandis - Steblus asper community: The other important co-existing species are Abrus precatorius
L., Alangium salvifolium (L.f.) Wangerin, Cissampelos pareira L. var. hirsuta (Buch.-Ham. ex DC.) Forman,
Dioscorea bulbifera L., Cocculus hirsutus (L.) W. Theob., Clerodendrum infortunatum L. and Elephantopus
scaber L.
4. Shorea robusta - Terminalia alata community: The other phytoassociates are Abutilon indicum (L.) Sweet,
Oroxylum indicum (L.) Kurz., Ailanthus excelsa Roxb., Sida cordata (Burm.f.) Borss. Waalk., Madhuca
longifolia (J. Koenig. ex. L.) J.F.Macbr. and Chrysopogon zizanoides (L.) Roberty.
5. Dalbergia sissoo - Acacia catechu community: The other phytoassociates are Ailanthus excelsa Roxb.,
Albizia procera (Roxb.) Benth., Ampelocissus latifolia (Roxb.) Planch., Kydia calycina Roxb., Abrus
precatorius L. and Cardiospermum halicacabum L.
6. Barringtonia acutangula - Syzygium spp. association: In this type of association there may be individual
stands of these species or mixed stands at some places. Syzygium cumini (L.) Skeels and S. salicifolium (Wight)
J. Graham. are two important species of syzygium in the study area. The other associates are Calamus tenuis
Roxb., Saccharum spontaneum L., Oxystelma secamone K. Schum., Tiliacora racemosa Colebr., Smilax
zeylanica L., Helminthostachys zeylanica (L.) Hook., and Lygodium flexuosum (L.) Sw.
7. Syzygium spp. - Ficus spp. association: Mainly consists of Syzygium cumini (L.) Skeels and S. salicifolium
(Wight) J. Graham. of Syzygium and Ficus racemosa L., F. heterophylla L.f. and F. virens Aiton. Other
phytoassociates are Pongamia pinnata (L.) Pierre, Terminalia arjuna (Roxb. ex DC.) Wight & Arn. and Vitex
negundo L.
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Aquatic flora
The common habitations of the hydrophyte are tals, nalas and other water reservoir with low-lying areas.
Most of the tals and the Parvatiaranga lakes in the area hold water throughout the year, only a few smaller and
less deeper ones may dry up during summer season. During rainy season these get filled with water as a part of
Saryu river flood area. As the flood water recedes, these water bodies get roofed with a number of hydrophytes
viz. Eichhornia crassipes (Mart.) Solms, Hygroryza aristata (Retz.) Nees ex Wight & Arn., Pistia stratiotes L.
& Spirodela polyrrhiza (L.) Schleid., Ceratophyllum demersum L., Hydrilla verticillata (L.f.) Royle, Najas
graminea Delile, Nechamandra alternifolia (Roxb. ex Wight) Thwaites, Ottelia alismoides (L.) Pers.,
Potamogeton crispus L., Nelumbo mucifera Gaertn., Nymphaea nouchali Burm.f., Nymphoides indica (L.)
Kuntze., Ipomoea aquatica Forssk., Ludwigia adscendens (L.) Hara, Bacopa monnieri (L.) Wettst., Hygrophila
auriculata (Schumach.) Heine, Phragmitis karka (Retz.) Trin. ex Steud., Ranunculus sceleratus L., Rumex
dentatus L., Typha angustifolia L. and Veronica anagallis-aquatica L.
B. Vegetation of Special Habitats
The study area is also harbours a rich population of flora with special habitats. There are almost 5 categories
(Fig. 3) of special habitats have been observed along with aquatic flora under which the species from the unique
and characteristic phytoassociation forms the habitat conditions.
Figure 3. Frequency and percentage of vegetation of special habitats.
1. On marshy shady conditions: Under this situation the following phytoassociates are growing together viz.
Bacopa monnieri (L.) Wettst., Centella asiatica (L.) Urb., Oldenlandia corymbosa L., Laphangium luteoalbum
(L.) Tzvelev,. Ceratopteris thalictroides (L.) Brongn.,Pycreus pumilus (L.) Nees, Peperomia pellucida (L.)
Kunth, Ranunculus muricatus L. and R. scleratoides Perfil. ex Ovczinn.
2. On dry shady situations: In these conditions scattered poulation of Abutilon indicum (L.) Sweet, Desmodium
gangeticum (L.) DC., Phyllodium pulchellum (L.) Desv., Leea indica (Burm.f.) Merr., Scoparia dulcis L., Aerva
sanguinolenta (L.) Blume and Ageratum conyzoides (L.) L. associations.
3. On drying up beds: In such areas plant species forming large clumps and patches under these associates viz.
Coldenia procumbens L., Glinus lotoides L., Grangea maderaspatana (L.) Poir., Heliotropium supinum L.,
Polycarpon prostratum (Forssk.) Aschers & Schweinf., Polygonum plebeium R. Br., Rumex dentatus L. and
Sphaeranthus indicus L.
4. On the bank of water bodies: The following important plants and their associates are observed under this
situation viz. Ammannia baccifera L., Lippia javanica (Burm.f.) Spreng., Chrysopogon zizaniodes (L.) Roberty,
Arundo donax L., Typha domingensis Pers., Persicaria lapathifolia (L.) Delarbre species. There are also some
woody species found along the water bodies in the forest area viz. Barringtonia acutangula (L.) Gaertn.,
Syzygium cumini (L.) Skeels, S. salicifolium (Wight) J. Graham and Pongamia pinnata(L.) Pierre along with
other associates like Oxystelma secamone K. Schum., Tiliacora racemosa Colebr. and Vitex negundo L.
5. On open situations: These conditions support a rich wealth of grasslend flora with some woody species viz.
Alangium salvifolium (L.f.) Wang subsp. decapetalum (Lam.) Wang, Alysicarpus monilifer (L.)DC., Apluda
mutica L., Biophytum sensitivum (L.) DC., Boerhavia diffusa L., Bothriochloa pertusa (L.) A. Camus, Carissa
spinarum L., Chrozophora rotleri A. Juss.Dalz., Clerodendrum infortunatum L., Cynodon dactylon (L.) Pers.,
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Imperata cylindrica (L.) Raeusch., Indigofera linifolia (L.f.) Retz., Solanum virginianum L., Urena lobata L.,
Chrysopogon zizanioides (L.) Nash, Woodfordia fruticosa (L.) Kurz and Ziziphus nummularia (Burm.f.) Wight
& Arn.
Parasitic and Epiphytic associations
Along with general floral association some unique association in form of parasitic and epiphytic are also
found in the area:
1. Cuscuta reflexa Roxb. found on a variety of host plants viz. Streblus asper Lour., Glycosmis pentaphylla
Retz. DC., Ipomoea fistulosa Mart. ex Choisy, Vitex negundo L. and on Ziziphus spp.
2. Dendrophthoe falcata (L.f.) Ettingsh. on Bombax ceiba L., Butea monosperma (Lam.) Taub., Mangifera
indica L., Dalbergia sissoo DC., Syzygium cumini (L.) Skeels at some places on Tectona grandis L.f. .
3. Vanda tessellata (Roxb.) Hook. ex G. Don mainly growing as an epiphytes on Madhuca longifolia (J.
Koenig ex L.) J.F. Macbr., Tectona grandis L.f. and on Shorea robusta Gaertn. (Fig. 4).
Figure 4. Epiphytic association of Vanda tessellata (Roxb.) Hook. ex G. Don on Madhuca longifolia (J. Koenig ex L.) J.F.
Macbr.
Pattern of rareness and regeneration potential in the study area

Rarity and regeneration of plant species in any forest area plays a significant role in maintenance of a healthy
forest. Plumbago zeylanica L., Oroxylum indicum (L.) Kurz., Hymenodictyon orixense (Roxb.) Mabb.,
Clerodendrum indicum (L.) Kuntze, Gloriosa superba L., Terminalia chebula Retz., Helicteres isoraL.,
Bauhinia vahlii Wight & Arn., Bacopa monnieri (L.) Wettst., Leea alata Edgew., Habenaria plantaginea
Lindl., Tylophora indica (Burm.f.) Merr., Cheilocostus speciosus (J. Koenig) C. D. Specht, Holarrhena
pubescens Wall. ex G.Don, Heminthostachys zeylanica were considered most rare plant species found during
collection and survey of the area (Table. 1). There are also some common specious occurs frequently in the
entire area due to their capacity to produce seedling rapidly viz. Bridelia retusa (L.) A.Juss., Mallotus
philippensis (Lam.) Mull. Arg., Mitragyna parvifolia (Roxb.) Korth., Terminalia alata Wall., Rotheca serrata
(L.) Steane & Mabb., Clerodendrum infortunatum L., Cayratia trifolia (L.) Domin, Cissampelos pareira L.,
Ichnocarpus frutescens (L.) W. T. Aiton, Elephantopus scaber L.and Lygodium flexuosum (L.) Sw (Table. 2).
Certain rhizomatous species like Curculigo orchioides Gaertn., Typhonium trilobatum (L.) Schott,
Helminthostachys zeylanica (L.) Hook. and Gloriosa superba L. growing even in highly stochastic environment.
This type of rarity and regeneration among various species are indicative of their ability to reproduce and
establish effiently in frequently distributed environment (Shukla 2009).
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.
Table 1. Rare species occurring in the study area.
Name of the species

Bacopa monnieri (L.) Wettst
Bauhinia vahlii Wight & Arn.
Clerodendrum indicum (L.) Kuntze
Cheilocostus speciosus (J. Koenig) C.D. Specht
Gloriosa superba L.
Habenaria plantaginea Lindl.
Helminthostachys zeylanica (L.) Hook.
Holarrhena pubescens Wall. ex G.Don
Hymenodictyon orixense (Roxb.) Mabb.
Leea alata Edgew.
Oroxylum indicum (L.) Kurz.
Passiflora foetida L.
Plumbago zeylanica L.
Schleichera oleosa (Lour.) Merr.
Strychnos nux-vomica L.
Terminalia chebula Retz.
Tylophora indica (Burm.f.) Merr.
Family
Plantaginaceae
Leguminosae
Verbenaceae
Costaceae
Liliaceae
Orchidaceae
Ophioglossaceae
Apocynaceae
Rubiaceae
Vitaceae
Bignoniaceae
Passifloraceae
Plumbaginaceae
Sapindaceae
Loganiaceae
Combretaceae
Apocynaceae
Habit
Herbs
Lianas
Shrubs
Shrubs
Climbers
Herbs
Herbs
Large Shrubs
Trees
Shrubs
Trees
Climbers
Shrubs
Trees
Trees
Trees
Climbers
Phenology
JulyMarch
Sept.Jan.
AprilDec.
Aug.Jan.
JulyNov.
Aug.Nov.
Oct.Jan.
MayFeb.
JulyFeb.
JuneSept.
JuneMarch
Nov.Jan.
Aug.Oct.
AprilAug.
MarchFeb.
MarchOct.
MaySept.
Table 2. Most common species occurring in the study area.
Name of the species

Aerva sanguinolenta (L.) Blume
Bridelia retusa (L.) A.Juss.
Cayratia trifolia (L.) Domin
Cissampelos pareira L.
Rotheca serrata (L.) Steane
Clerodendrum infortunatum L.
Phyllodium pulchellum (L.) Desv.
Dioscorea bulbifera L.
Elephantopus scaber L.
Glycosmis pentaphylla (Retz.) DC.
Hemidesmus indicus (L.) R.Br. ex Schult.
Holoptelea integrifolia Planch.
Ichnocarpus frutescens (L.) W.T. Aiton
Mallotus philippensis (Lam.) Mull. Arg.
Mitragyna parviflora (Roxb.) Korth.
Streblus asper Lour.
Terminalia alata Wall.
Family
Amaranthaceae
Phyllanthaceae
Vitaceae
Menispermaceae
Lamiaceae
Lamiaceae
Leguminosae
Dioscoreaceae
Asteraceae
Rutaceae
Apocynaceae
Ulmaceae
Apocynaceae
Euphorbiaceae
Rubiaceae
Moraceae
Combretaceae
Habit
Herbs
Trees
Climbers
Climbers
Shrubs
Shrubs
Shrubs
Climbers
Herbs
Shrubs
Climbers
Trees
Climbers
Small trees
Trees
Trees
Trees
Phenology
JulyApril
JulyMarch
Aug.Nov.
JuneDec.
Aug.Oct.
MarchJune
Aug. April
JuneNov.
Jan.March
Dec.March
Aug.Jan.
Dec.April
JulyFeb.
Oct.May
Sept.Jan.
MaySept.
AprilNov.
An extensive ecological and floristic study has been conducted in the north-eastern terai region of the Uttar
Pradesh with respect to the floral diversity and documentation of vegetational phytosociology. The present
communication reveals that the plant community with special habitat specially in protected and reserve forest
area may plays a vital role in the future sustenance of the forest vegetation. The area also harbours a rich
diversity of economical and medicinal plant species, mainly confined to the peripheral region of the forests.
There is need of continued monitoring of various ecological parameters with the help of more accurate and
sophasticated ecological tools for the betterment of the plant community of the study area.
ACKNOWLEDGEMENTS
The authors are thankful to Director, Botanical Survey of India, Kolkata and Scientist- E and Head of
Office, BSI, CRC, Allahabad for facilities and encouragement. The authors are also grateful to Range Officers
and field staffs of Parvati Aranga Wildlife Sanctuary and Tikri reserve forest, Gonda for providing necessary
help during field exploration. The authors are thankful to the Head, Department of Botany, D.S.B. campus,
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Kumaon University, Nainital for valuable suggestions.
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ISSN (E): 2349 1183

ISSN (P): 2349 9265
3(3): 551557, 2016
DOI: 10.22271/tpr.2016.v3.i3.072
Research article
Intra-specific variation in response of Neem (Azadirachta indica

A. Juss) to elevated CO2 levels and biochemical characterization
of differently responding plants
C. Buvaneswaran*, K. Arivoli, T. Sivaranjani, E. Menason,
K. Vinothkumar, S. Padmini and S. Senthilkumar
Institute of Forest Genetics and Tree Breeding, (ICFRE), R.S.Puram, Coimbatore, Tamil Nadu, India
*Corresponding Author: buvanesc@icfre.org
Abstract: Global climate change the looming environmental threat is mainly due to the increase in
atmospheric CO2 levels, which was increasing earlier by about 1.55 ppm per year and currently by
2.76 ppm per year. Thus, CO2 concentration has reached 400.16 ppm in 2015. To understand the
response of various tropical tree species to such an elevated CO2, experiments were conducted in
Automated Open Top Chambers (AOTC) facility at Institute of Forest Genetics and Tree Breeding
(IFGTB), Coimbatore (India). The results of initial studies indicated the scope for exploring intraspecific variation in response of tropical trees to elevated CO2. Subsequently, experiments were
carried out to assess intra-specific variation in Neem (Azadirachta indica). The selected
phenotypes of Neem (varieties or clones) were exposed to four treatments viz., i) CO2 of 600 ppm,
ii) CO2 of 900 ppm, iii) chamber control- without any CO2 enrichment and iv) ambient conditions.
The parameters studied were shoot length, root length, dry matter accumulation in shoot and root.
The results of the study showed that there existed significant variation among different treatments
of CO2 as well as among various phenotypes or clones in terms of growth characteristics. This
intra-specific variation in biomass accumulation under elevated CO2 levels could be exploited for
future breeding programmes in developing climate ready genotypes having greater potential to
sequester more carbon and produce greater biomass under forecasted elevated levels of
atmospheric CO2. Another objective in this study was to analyze intra-specific variation in selected
biometrical and biochemical
characteristics of leaf samples of neem trees in relation to their
differential response to elevated CO2. Among parameters of leaf, Fumaric acid, Malic acid and
Oxalic acid, leaf Nitrogen and Specific Leaf Weight may be considered as a biochemical and
biometrical marker to categorize the plants adapted to elevated CO2 environments.
Keywords: Response to elevated CO2 - Neem (Azadirachta indica) - Open Top Chambers Within species variation in growth.
[Cite as: Buvaneswaran C, Arivoli K, Sivaranjani T, Menason E, Vinothkumar K, Padmini S & Senthilkumar S
(2016) Intra-specific variation in response of Neem (Azadirachta indica A. Juss) to elevated CO2 levels and
biochemical characterization of differently responding plants. Tropical Plant Research 3(3): 551557]
INTRODUCTION
Even the most environment friendly emission scenarios lead to an increase in atmospheric CO2 concentration
over the next 100 years, to about double the pre-industrial levels up to 550 ppm (IPCC 2007). Currently, CO2
concentration has reached 400.16 ppm in 2015 Mike McGee (2015). This increasing concentration of carbon
dioxide (CO2) in the atmosphere may have a direct effect on the physiology of plants: higher CO2 tends to
suppress plant transpiration through reduced stomatal conductance (Field et al. 1995). In tree species, short term
experiments with Pinus ponderosa, Quercus coccinea, Pinus radiata and Populus deltoides have shown a
definite increase in photosynthesis rate up to 4080% under 600 ppm levels of CO2 (Couteaux et al. 1992).
Studies conducted at Institute of Forest Genetics and Tree Breeding (IFGTB) showed the existence of greater
inter- and intra-specific variation in tropical tree species in response to elevated CO2 and temperature
(Buvaneswaran et al. 2010). With reference to intra-specific clonal variation in tree species, Johanna et al.
(2003) reported that two clones of silver birch (Betula pendula) responded differently to elevated CO2 levels. In
one clone, total biomass increased by 40% under elevated CO2, but in another clone no response was found.
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Buvaneswaran et al. (2016) 3(3): 551557

Similar such intra-specific clonal variations have been reported in Sitka spruce (Murray et al. 1994), Poplar
(Ceulemans et al. 1995), Hevea brasiliensis (Devakumar et al. 1998), Populus tremuloides (Isebrands et al.
2003). However, not much study has been conducted on intra-specific variation in response of tree species to
elevated CO2 levels, particularly the plantation forestry species of India. Hence, the present study was conducted
to generate knowledge on intra-specific variation in important tropical plantation species besides helping to
screen assembled productive clones of Neem (Azadirachta indica A. Juss) for higher carbon sequestration
potential under elevated CO2. This selection of clones adapted to elevated CO2 levels may also be used for
developing climate ready genotypes in future through biotechnological interventions. The information
generated could also help in identification of biometrical and biochemical markers, if any, for higher carbon
sequestration potential of the selected species under elevated CO2.
The present research experiment was conducted using Automated Open Top Chambers (AOTC) facility at
Institute of Forest Genetics and Tree Breeding (IFGTB), Coimbatore, India. This study site is located on
1159'0.69" N, 7657'2.32" E and 437 m MSL. The facility of AOTCs has chambers which are cubical type
structures of 3 3 3 m dimension, fabricated with galvanized iron (GI) pipe frames and covered with
polyvinyl chloride (PVC) sheet of 120 micron gauge to have more than 90% transmittance of light. The upper
portion of the chamber is kept open to maintain near-natural conditions. A software facility called Supervisory
Control and Data Acquisition (SCADA) was used to continuously control record and display the actual and
desired CO2 level, relative humidity and temperature in each OTC by feedback control loop passing through
Programmable Logical Controllers (PLC).
The species selected for the present study was Neem (Azadirachta indica A. Juss) in which 12 clones
(varieties) were selected and subjected to various treatments. The initial shoot length was recorded for all the
plants. Then, these plants were exposed to four treatments with the different levels of CO2 viz., i) chamber with
600 ppm of CO2, ii) chamber with 900 ppm concentration of CO2, iii) chamber without any elevation of CO2 and
iv) ambient control- in open area. CO2 enrichment was done by using CO2 cylinders. The design of the
experiment adopted is Completely Randomized Block Design (CRBD).
At end of exposure period under various treatments, plants were sampled and growth parameters were
measured like shoot length, root length, and fresh weight of leaf, shoot and root. Then, the plant samples were
kept in hot air oven at 60C till the constant dry weight is obtained. Then dry weight was recorded for in root,
shoot and leaves. The data on growth and dry matter production were subjected to analysis of variance for
completely randomized design with replications.
To analyze intra-specific variation in few biometrical and biochemical
characteristics of Neem trees in
relation to their differential response to elevated CO2, Four different clones (varieties) of Neem viz. IFGTB-AI9, IFGTB-AI-11, IFGTB-AI-3 and IFGTB-AI-5 were used to assess intra-specific variation in biochemical
characteristics in neem trees in relation to their differential response to elevated CO2. Among these four clones
selected for the present study, two clones IFGTB-AI-9 and IFGTB-AI-11 were responding positively under
elevated CO2 environments in the earlier experiments conducted under Open Top Chambers at Institute of
Forest Genetics and Tree Breeding, Coimbatore. On the other hand, clones IFGTB-AI-3 and IFGTB-AI-5 were
responding poorly to the elevated CO2 conditions by recording lesser dry matter accumulation in biomass of
various plant parts.
The mother plants of these four selected clones of neem, belonging to two categories with respect to their
response to elevated CO2 environments - were studied for variation in chlorophyll content (chlorophyll - a,
chlorophyll - b and total chlorophyll), Organic acids (Fumaric acid, Malic acid, Citric acid and Oxalic acid),
Nutrient elements (Nitrogen, Phosphorus, Potassium, Calcium and Magnesium) and also for leaf weight, leaf
area and Specific Leaf Weight. All these parameters were assessed using leaf samples collected from the
respective clones available in the germplasm assemblage of neem in IFGTB campus, Coimbatore.
RESULTS AND DISCUSSIONS
A) Response of Azadirachta indica to elevated CO2 in Open Top Chambers
A set of 12 phenotypes (clones) of Neem (Azadirachta indica A. Juss) were used in the experiments
conducted in Automated Open Top Chambers (AOTC) facility at IFGTB, Coimbatore. The results of the study
showed that there existed significant variation among different treatments of CO2 as well as among various
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clones in terms of growth characteristics. Overall mean shoot length was 59.73 cm for plants exposed to 600
ppm of CO2 and it was only 28.25 cm under ambient conditions, irrespective of clones (Table 1). Root length
was greater (39.33 cm) under 900 ppm CO2 level than that recorded in ambient conditions (30.07 cm). Dry
matter accumulation in shoots was 78% more under 600 ppm CO2 level than that of in ambient conditions.
Similarly, 58% more dry matter accumulation was recorded in roots of plants grown under 600 ppm of CO 2
levels when compared to the plants grown in ambient conditions (Table 1).
Among 12 clones studied, clone IFGTB-AI-12 recorded 86% more total dry matter accumulation in biomass
of plants grown under 600 ppm of CO2 as compared to that observed for the same clone under ambient
conditions. Clone IFGTB-AI-9 accumulated greater amount of total dry matter in biomass under 900 ppm CO 2
levels that is 89% over and above the total dry matter accumulated in the same clone under ambient
environments (Fig 1). In Neem, only one clone that is IFGTB-AI-5 showed negative response in term of total
dry matter accumulation under 900 ppm CO2 level by registering 3% less dry matter accumulation in biomass
when compared to that of ambient grown plants of the same clone. Followed by clone IFGTB-AI-3 which
recorded lesser accumulation of total dry weight when compared to all other positively responding clones.
Table 1. Shoot and root characteristics of Azadirachta indica A. Juss plants exposed to various levels of CO2 in
open top chamber experiments conducted in IFGTB, Coimbatore, India .
600 ppm
Mean shoot length (cm)
59.73 3.48
Mean root length (cm)
36.10 3.11
Leaf dry weight (g per plant)
6.22 0.26
Shoot dry weight (g per plant) 12.24 0.80
Root dry weight (g per plant)
9.12 0.90
Note: Values shown are Mean Standard Error
900 ppm
Chamber control
54.69 2.12
62.05 2.56
39.33 2.15
33.20 2.79
5.52 0.48
6.85 0.40
10.72 0.61
11.50 0.55
8.29 0.61
7.54 0.57
Ambient
28.25 1.59
30.07 1.01
4.53 0.29
6.86 0.54
5.78 0.49
Figure 1. Increase or decrease in total dry weight (%) under different levels of CO2 as percentage over that of ambient
grown respective clones of Azadirachta indica A. Juss.
In the present study, shoot length was increased under elevated CO2 conditions when compared to ambient
conditions. As observed in the present study, Pokorny et al. (2012) observed that the shoot length was increased
frequently under elevated CO2 conditions. With regard to increased root length under elevated CO2
environment, it is reported that the responses of roots to CO2 are dependent on experimental conditions
(Ceulemans & Mousseau 1994). When the plants exposed to increased CO2, root have observed to become more
numerous, longer, thicker and faster growing in crops (Chandhuri et al. 1990) with increased root length in
many plant species (Norby 1994, Pritchard & Rogers 2000, Bernecchi et al. 2000). Lengths and volumes of tap
root and fine roots were higher for CO2 enhanced cotton plants (Rogers et al. 1993). 110% increase in root
length of soybean was observed as CO2 concentration increased (Rogers et al. 1992). Increased root length and
number under elevated CO2 environments has also been reported in sweet potato (Bhattacharya et al. 1990) and
Phaseolus acutifolius and P. vulgaris (Salsman et al. 1999).
In the present study, dry matter accumulation both in shoot and root biomass was greater in plants grown
under elevated CO2 environments as compared to that in ambient grown plants. Similar findings were reported
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by Cao et al. (2008) in Gossypium hirsutum and the above ground biomass was increased under 600 ppm of
elevated CO2 level. Lukac et al. (2003) revealed that the CO2 enrichment increases the belowground biomass
allocation in three populus species were investigated and the standing root biomass enhanced by 4776%.
Moreover the growth of Minjiang fir showed significantly positive responses of elevated CO2 with greater
increases of total biomass than the control conditions (Hou et al. 2011). Ghasemzadeh & Jaafar (2011) reported
in two varieties of Zingiher officinale were exposed to different CO2 concentration (400 and 800 ppm) resulted
in increasing total plant biomass over the ambient conditions. Mohamed (2013) reported that there is no
significant intra specific difference in dry matter production in the tropical dry land forest species of Neem. But
when the plants were exposed on increased atmospheric CO2 the final plant biomass, above ground biomass and
below ground biomass was significantly increased in tree species (Madhu & Hatfield 2013). Similarly, the shoot
biomass was approximately 35% greater for creeping bentgrass plants grown under elevated CO2 compared to
plants maintained under ambient CO2, while the root biomass increased by 37% due to elevated CO2 (Burgess &
Huang 2014). Wang et al. (2015) studied the responses of rice production under elevated CO2, there was
significant stimulation in above ground biomass 28 per cent and below ground biomass of rice was 42 per cent
increases. Similar findings of root biomass was higher under elevated CO2 levels has been reported by several
authors and it was increased of 55 per cent in Pinus sylvestris (Jach et al., 2000), 32 per cent in Pinus taeda by
Jackson et al. (2009).
With reference to intra-specific clonal variation in tree species, it is observed in the present study that huge
variation existed among the clones in terms of total dry matter accumulation in Neem. Similarly, Johanna et al.
(2003) also reported that two clones of silver birch (Betula pendula) responded differently to elevated CO2
levels. In one clone, total biomass increased by 40% under elevated CO2, but in another clone no response was
found. Similar such intra-specific clonal variations have been reported in Sitka spruce (Murray et al.1994),
Poplar (Ceulemans et al. 1995), Hevea brasiliensis (Devakumar et al. 1998), Populus tremuloides (Isebrands et
al. 2003).
B) Foliar analysis of differently responding clones for biometrical and biochemical traits
In the present study, the existing variation in chlorophyll contents among the clones studied were not
corresponding to the growth response observed for the respective clones under the elevated CO2 environments
(Table 2). Similarly, Nicolas et al. (2007) also reported that there is no link of chlorophyll with tree growth
irrespectively of site and family in hybrids of poplar. However, Kumar & Paramathama (2005) reported that all
the traits studied, via., plant height, collar diameter. Number of branches, survival percent, chlorophyll content
and suitability index were strongly associated with volume index in 44 clones of Casuarina equisetifolia
assembled from Tamilnadu, Andhra Pradesh, Orissa and Pondicherry. Reddy et al. (2003) also reported that
chlorophyll a, b and total chlorophyll showed significant positive correlation with leaf area and yield in different
genotype of Mulberry.
Table 2. Intra-specific Variation in Chlorophyll contents (mg chlorophyll per g leaf tissue) in selected Neem clones.
Clones
Chlorophyll-a
IFGTB-AI-9
0.0135 0.0016
IFGTB-AI-11
0.0074 0.0025
IFGTB-AI-3
0.0049 0.0023
IFGTB-AI-5
0.0140 0.0014
Note: Values shown are Mean Standard Error.
Chlorophyll-b
0.0115 0.0037
0.0155 0.0046
0.0048 0.0025
0.0196 0.0028
Total Chlorophyll
0.0359 0.0072
0.0298 0.0049
0.0245 0.0079
0.0490 0.002
Among various organic acids, Fumaric and Malic acid was in greater quantity in clones positive to elevated
CO2 when compared to other two clones which are responding poorly to elevated CO2. On the contrary, Oxalic
acid content was observed to be lesser in quantity in clones adaptive to elevated CO2 than in clones less
adaptive to elevated CO2. However, there is not much difference among clones in respect of Citric acid content
(Table 3). Similar variation in organic acid contents under exposure to stress environments has been reported by
Silva et al. (2004) who reported that Gas chromatography/mass spectrometry and ion chromatography analyses
indicated that root exposure to AI led to a greater than 200% increase in Malic acid concentration in the root tips
of all eucalypt species. The increase in Malate concentration in response to AI treatment is correlated with the
tree species. A small increase in citric acid `concentration was also observed in all species, but there were no
consistent changes in the concentration of other organic acids in response to AI treatment. In all eucalypt
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species, AI treatment induced the secretion of citric acid and Malic acid in root exudates. But no trend with
respect to AI tolerance was observed. Thus, although malate and citrate exudation by roots may partially
account for the overall high AI tolerance of these eucalypt species, it appears that tolerance is mainly derived
from the internal detoxtification of AI by complexation with malic acid.
Table 3. Intra-specific Variation in organic acids (mg per g leaf tissue) in selected Neem clones.
Clones
Fumaric Acid
Malic acid
IFGTB-AI-9
11.56 0.022
11.70 0.322
IFGTB-AI-11
10.36 0.093
11.34 0.305
IFGTB-AI-3
9.40 0.066
10.40 0.907
IFGTB-AI-5
8.32 0.057
10.54 0.636
Citric acid
1.80 0.027
2.04 0.002
1.79 0.032
2.42 0.099
Oxalic acid
0.049 0.001
0.2697 0.012
0.4767 0.065
0.3967 0.085
Significant observation in the present study was that among various nutrient elements studied, Nitrogen
content was greater in amount in clones which are categorized as adaptive to elevated CO2 (0.112 to 0.14%)
when compared to that in clones which are not adaptive to elevated CO2 environments (0.028 to 0.056%) (Table
4). It is reported that the largest single pool of nitrogen in leaves is RuBisCO and hence it can be inferred that
the greater amount of Nitrogen in the leaves of adaptive clones may indicate greater amount of RuBisCO
availability. This higher RuBisCO availability is related to higher biomass production, as photosynthesis is
catalysed by this RuBisCO, which fixes carbon to form carbohydrates. Moore et al. (1998) also reported that
short term exposure of elevated CO2 for plants generally leads to increased rates of leaf-level photosynthesis due
to enhanced activity of RuBisCO. On the other hand, photosynthesis down regulation is characterized at the
biochemical and leaf levels by reduced chlorophyll content, reduced RuBisCO content and activity and
decreased leaf nitrogen concentration on a leaf mass basis (Sage 1994, Tissue et al. 1995). It warrants further
research to understand the reason for higher leaf N in some species and lower N levels in leaf in other species
when plants are exposed to elevated CO2.
Table 4. Intra-specific Variation in nutrient elements (% in leaf tissue) in selected Neem clones.
Clones
IFGTB-AI-9
N%
P%
IFGTB-AI-11
K%
Ca
IFGTB-AI-3
%
Mg %
IFGTB-AI-5
N%
0.140
0.112
0.056
0.028
P%
0.196
0.136
0.143
0.135
K%
2.00
2.87
1.97
2.22
Ca
0.56
%
0.28
1.24
1.16
Mg %
0.08
0.21
1.87
1.74
Another important observation made in the present study was that the actual dry weight and leaf area of
clones were not differentiated in terms of clonal variation in respect of their response to elevated CO2. However,
when Specific Leaf Weight (SLW) was worked out, this SLW parameter clearly got differentiated in clones.
Greater SLW was recorded in clones which were responding positively to the elevated CO2 enforcing. Hence,
clones IFGTB-AI-9 and IFGTB-AI-11 recorded greater SLW values of 13.06 and 10.75 mg per cm2 respectively
(Table 5). On the other hand, clones IFGTB-AI-3 and IFGTB-AI-5 recorded lesser SLW, which were the clones
responded poorly to the elevated CO2 environments in the earlier experiments. Sicher et al. (1994) who studied
the photosynthetic acclimation to elevated CO2 occurs in transformed Tobacco and reported that the dry weight
gain was due to increased specific leaf weight. Specific leaf weight was shown to be a valuable index for
comparing photosynthesis various parts of a tree canopy over a season or throughout an entire year. Mean
annual photosynthetic rate in five separate portions of a spruce canopy was directly proportional to observed
differences in specific leaf weight (Ram 1984).
In brief, among various parameters of leaf, organic acids particularly Fumaric acid, Malic acid and Oxalic
acid, leaf Nitrogen content and Specific Leaf Weight may be considered to act as a biochemical and biometrical
marker to categorize the plants adapted to elevated CO2 environments, specifically to neem trees.
Table 5. Intra-specific variation in leaf dry weight, leaf area and Specific Leaf Weight in clones of Neem.
Dry weight
Clones
(mg
leaf)
IFGTB-AI-9
389.6per
0.129
IFGTB-AI-11
451.7 0.075
IFGTB-AI-3
519.7 0.087
IFGTB-AI-5
221.2 0.037
Leaf area (cm2) Specific Leaf Weight (mg per cm2)

13.06 2.16
31.67 5.89
43.00 7.00
10.75 2.31
56.67 6.64
9.36 0.84
28.00 3.05
7.99 0.68
555

CONCLUSION
The present study evidently proves that there exists significant intra-specific variation in Azadirachta indica
in response to elevated CO2. Greater variation existed in dry matter accumulation in biomass among different
clones under elevated CO2 conditions. This variation could be explored in all other commercially important
tropical tree species and superior tree varities can be identified for higher productivity and carbon sequestration
potential under forecasted elvated CO2 levels of the future envirnment.
When four different clones (varieties) of Neem (Azadirachta indica) viz. IFGTB-AI-9, IFGTB-AI-11,
IFGTB-AI-3 and IFGTB-AI-5 were used to assess intra-specific variation in biochemical characteristics in
neem trees in relation to their differential response to elevated CO2, it is observed that among various
parameters of leaf, organic acids particularly Fumaric acid, Malic acid and Oxalic acid, leaf Nitrogen content
and Specific Leaf Weight may be considered to act as a biochemical and biometrical marker to categorize the
plants adapted to elevated CO2 environments, specifically to neem trees. Further research is suggested to
confirm these observations in other tropical tree species which will aid in large scale screening of various tree
species for their adaptation potential to ever increasing atmospheric CO2.
ACKNOWLEDGMENTS
The authors very much thankful to the Director, Institute of Forest Genetics and Tree Breeding, Coimbatore
and Director General, Indian Council of Forestry Research and Education, Dehra Dun for providing the
opportunity to conduct the experiments in Automated Open Top Chambers.
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557
ISSN (E): 2349 1183

ISSN (P): 2349 9265
3(3): 558563, 2016
DOI: 10.22271/tpr.2016.v3.i3.073
Research article
Morphological characters of Chaetoceros lorenzianus

(Bacillariophyceae) isolated from North Arabian Sea after
Tasman Spirit oil spill
Asma Tabassum1*, Hina Baig2 and Aliya Rehman1
1
Department of Botany, University of Karachi, Karachi, Pakistan

2
National Institute of Oceanography, Karachi, Pakistan
*Corresponding Author: centricdiatomist@gmail.com
Abstract: The present study investigated the morphology and taxonomy of marine centric diatom
Chaetoceros lorenzianus from Karachi Harbor, Pakistan for the first time for the first time during
the incident of Tasman Spirit Oil Spill (2003) in the area. Phytoplankton samples were collected
from 5 different locations from the study area. Chaetoceros lorenzianus was found only at one
station collected after Tasman Spirit Oil Spill. Moreover morphometric measurements of present
record showed narrow range as compared to the records investigated by other workers.
Keywords: Chaetoceros lorenzianus - Diatom - Phytoplankton - Tasman Spirit oil spill.
[Cite as: Tabassum A, Baig H & Rehman A (2016) Morphological characters of Chaetoceros lorenzianus
(Bacillariophyceae) isolated from North Arabian Sea after Tasman Spirit oil spill. Tropical Plant Research 3(3):
558563]
INTRODUCTION
Phytoplankton are the major primary producers of marine and fresh water environment (Baliarsingh et al.
2012). Among phytoplankton, the Bacillariophyta (diatoms) contributes at least 40% of the global annual
primary productivity (Field et al. 1998). These diatoms are ubiquitous occurrence in marine environment
(Sunesen et al. 2008). Genus Chaetoceros Ehrenberg is considered as most diverse and wide spread centric
diatom (Cupp 1943, Rines 1999, Hasle & Syvertsen 1997). It comprises of about 400 marine species with few
fresh water records (Round et al. 1990). Morphological and taxonomical studies of this genus contributed new
records time to time from various parts of the world oceans (Hernandez-Becerril 1993, Hernandez-Becerril
1999, Rines 1999, Trigueros et al. 2002, Murthy et al. 2012, Ozgur et al. 2013). A number of studies have been
conducted on distribution and composition of Chaetoceros (Hargraves 1972, Fanuko & Valic, 2009, Tabassum
& Saifullah 2010). It was observed to be one of the most frequently occurring genus among centric diatoms
(Nwankwo & Onyema 2003, Tabassum & Saifullah 2010). Records of Chaetoceros have also been well
observed in sediments with special reference to their resting spores (Stockwell 1991, Witak et al. 2011, Ferrario
et al. 1998, Moazzam & Baig 1994). Variation in physiological behavior and their responses to hydrological
parameters have also been studied (Johansen et al. 1990).
Chaetoceros lorenzianus, is considered to be a harmful bloom forming species (Sunesen et al. 2008). This
species was studied by a number of scientists (Cupp 1943, Subrahmanyan 1946, Hendey 1964, Moazzam 1973,
Hasle & Syvertsen 1997, Shevchenko et al. 2006, Sunesen et al. 2008, Tabassum & Saifullah 2010). Moreover
the lysis of this species by a single stranded DNA virus has also been investigated (Tomaru et al. 2011). The
occurrence of Chaetoceros lorenzianus has been discussed from various parts of the world ocean (Cupp 1943,
Sunesen et al. 2008, Shevchenko et al. 2006, Wood 1963, Subrahmanyan 1946, Rajasekar et al. 2010, Hendey
1964) It is known from North Arabian Sea bordering Pakistan (Moazzam 1973, Saifullah & Chaghtai 2005,
Tabassum & Saifullah 2010). This is the first attempt to study the impact of oil spill in North Arabian Sea on
morphological characters of this species.
558
Received: 01 July 2016

Tabassum et al. (2016) 3(3): 558563

.
Phytoplankton samples were collected by net (50 m) hauls of 5 minute duration at speed of 2 km at 5
sampling stations which were selected in the area affected by oil spill (Fig. 1 and Table 1). Samples were fixed
with 10 % buffered formalin immediately after collection. Observations on oceanographic parameters like
temperature, salinity and pH were also measured at each station.
Figure 1. Map showing sampling stations of Tasman Spirit Oil Spill effected area.
Table 1. Sampling stations in off Karachi Harbour.
Station No.
1
2
3
4
5
Sampling date
19-11-03
19-11-03
19-11-03
19-11-03
20-11-03
Latitude N
2480248N
2480816N
2480771N
2479753N
2477204N
Longitude E
6689938E
6699215E
6701087E
6702718E
6705435E
Samples were observed in light microscope LABX N-400M. Prior to scanning electron microscopy (SEM)
the samples were cleaned by cold H2O2 method (Karthick et al. 2010). Cleaned material were coated up to 300
A with auto coater using JEOL # JFC 1500 having gold targets. The coated samples were then scanned with
JEOL # JSM 6380A microscope. Present paper manifests the light and electron microscopic structures probably
for the very first time in this study area.
RESULTS
Enumeration of species
Chaetoceros lorenzianus Grunow, 1863, 157, pl. 5: fig. 13; Cupp 1943, p. 118, Fig. 71; Subrahmanyan 1946, p.
131, Figs. 198199, 202204, 206209 (p. 132); Hendey 1964, p. 124, Plate 16, Fig. 1; Hasle & Syvertsen
1997, p. 204, Plate 42; Shevchenko et al. 2006, p. 249, Figs. 8489; Sunesen et al. 2008, p. 317 & 318, Fig.
11AF; Tabassum & Saifullah 2010, p. 1144 & 1146, Fig. 13 (1145). (Fig. 2)
Chains straight, cells rectangular, apical axis 1115 m. Apertures wide, elliptical to lanceolate, foramina
hexagonal, ranges from 1012 m. Setae thick, long, spiny, polygonal in cross section, fuse just near the
margin, divergent with slight curve forming an angle of 3545 to the chain axis.
Distribution: During the present study this species was collected only from Station 2.
This species is reported by various parts of the world ocean. West Coast of North America (Cupp 1943);
Madras, India (Subrahmanyan 1946); Chaleurs Bay Canada (Brunel 1962); Indian Ocean (Wood 1963); British
Coastal Waters (Hendey 1964); Manora Channel Karachi (Moazzam 1973); Indian Ocean (Simonsen 1974);
Peter the Great Bay, Sea of Japan (Shevchenko et al. 2006); Buenos Aires Argentina (Sunesen et al. 2008).
559
Tabassum et al. (2016) 3(3): 558563

.
Observation from the study area: The valve profile of Chaetoceros lorenzianus is nearly close to the findings of
numerous workers (Cupp 1943, Subrahmanyan 1946, Hendey 1964, Moazzam 1973, Hasle & Syvertsen 1997,
Shevchenko et al. 2006, Sunesen et al. 2008, Tabassum & Saifullah, 2010) except the size of apical axis which
is within a narrow range (Table 2). This may be attributed to their presence in the environment which was
polluted with the crude oil because of Tasman Spirit oil spill which might have affected the cell metabolism.
Parab et al. (2008) observed morphological changes in another centric diatom Thalassiosira because of oil
exposure.
Figure 2. Chaetoceros lorenzianus (SEM, pair of sibling cells in girdle view): A, Scale bar: 10 m; B, Sibling cells with
long setae. Scale bar: 20 m; C, Sibling valves with wide aperture. Scale bar: 5 m.
Table 2. Comparison of morphometric data among Chaetoceros lorenzianus of present study with the previous records.
Apical axis
Pervalvar
axis
Cupp,
1943
(Pacific
Ocean)
Subrahman
yan, 1946
(Indian
Ocean)
Hendey,
1964
(Atlantic
Ocean)
7 m
48 m
-
16 m
58 m
-
26 m
60 m
-
Moazzam
, 1973
(North
Arabian
Sea)
5 m
40 m
-
Hasle and
Syvertsen
, 1997
7 m
80 m
-
Sunesen
et al.,
2008
(Atlantic
Ocean)
16 m
36 m
-
Tabassum and
Saifullah,
2010
(North
Arabian Sea)
15 m
35 m
10 m
20 m
Present
study
(North
Arabian
Sea)
10 m
15 m
7 m
11 m
DISCUSSION
Any change in the ecosystem of an aquatic environment can be determined by analysis of phytoplankton
composition of that area (Guilloux et al. 2013). An extensive amount of studies have been conducted on effects
of oil pollution on ecosystem of water bodies which showed deleterious effects on growth of phytoplankton
community structure (Parab et al. 2008, Jiang et al. 2010).
560
Tabassum et al. (2016) 3(3): 558563

.
Genus Chaetoceros is termed as fast growing diatom and its domination among other members of
phytoplankton was also observed during the studies conducted in the other parts of the world during stress
condition of oil spill (Hallare et al. 2011). Chaetoceros lorenzianus collected from North Arabian Sea bordering
Pakistan belongs to sub-genus Hyalochaete (Hasle & Syvertsen 1997). In the present study morphometric data
including apical axis and pervalver axis of Ch. lorenzianus was found in narrow range but at the same time
within the range of the results recorded earlier by other workers (Table 2). It is recorded that the traces of oil
(raw or refine) are lethal to autotrophic life forms as they can cease metabolic activities by limiting their
enzymatic activities (Lewis & Pryor 2013) and decrease chlorophyll a concentration (Lee et al. 2009). As it is
evident that the toxicity of crude oil is concentration dependent (Sheekh et al. 2000) and the species were
recorded during the initial days of spill so recorded narrow range of morphometric measurements of the cells
may accounted due to the effect of crude oil.
Moreover previous findings showed that Kuzmenko (1975), Tabassum & Saifullah (2010) observed 16
species of Chaetoceros in the month of February from Arabian Sea whereas seven species of this genus
including C. lorenzianus were reported in the month of October from Kuwait Bay, Arabian Sea (Heil et al.
2001). Present study manifests sporadic occurrence of the species in the month of November immediate after
Tasman Spirit Oil Spill which may have attributed to the effect of oil spill in this area.
ACKNOWLEDGEMENTS
Authors are deeply indebted to National Institute of Oceanography, Pakistan for providing the samples of
project entitled Tasman Spirit Oil Spill for this study. And we are also thankful to Dr. Ines Sunesen from
Departamento Cientifico Ficologia, Universidad Nacional de La Plata, Argentina for constant support in this
study.
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DOI: 10.22271/tpr.2016.v3.i3.074
Research article
Diversity and distribution of Litsea in

Chikkamagaluru, Karnataka
S. G. Srinivas and Y. L. Krishnamurthy*
Dept. of P.G Studies and Research in Applied Botany, Kuvempu University, Jnanasahyadri,
Shankaraghatta-577451, Shivamogga, Karnataka, India
*Corresponding Author: murthy_ylk@yahoo.co.in
Abstract: The study gives a report on the diversity of Litsea (Lauraceae) occur in Chikkamagaluru
district of Karnataka, India. Study was conducted in two habitats of Kemmannugundi and
Mullayyanagiri regions. Extensive field surveys were conducted for survey of the species by
laying six belt transects of 2504 m size. The data indicated that four species of Litsea occurred in
the study sites; namely, Litsea floribunda, Litsea stocksii, Litsea glabrata and Litsea mysorensis.
L. floribunda showed higher density when compared with other species, all the four species
distributed frequently in Kemmannugundi whereas; in Mullayyanagiri only L. floribunda species
is present. These trees commonly associated with other tree species are Cinnamomum verum,
Neolitsea cassia, Maesa indica, Memecylon malabaricum and Syzizium cumini.
Keywords: Associated species - Kemmannugundi - Lauraceae - Mullayyanagiri - Western Ghats.
[Cite as: Srinivas SG & Krishnamurthy YL (2016) Diversity and distribution of Litsea in Chikkamagaluru,
Karnataka. Tropical Plant Research 3(3): 564568]
INTRODUCTION
The genus Litsea consists of about 400 species which is largest genus in the family Lauraceae distributed in
tropical and subtropical Asia, Australia, New Zealand, North America and subtropical South America (Chaing
et al. 2012). In India about 45 species are distributed in evergreen and semi evergreen forests of the Western
Ghats (Bhuniya et al. 2010), 12 species are also found in Meghalaya, Manipur, Assam and Sikkim. Among 45
species 40 of which are endemic to peninsular India, 11 species are found in Karnataka (Saldanha 1996).
The Litsea trees are evergreen dioecious with alternate or whorled leaves, inflorescence is pedunculate
axillary umbellate or corymbose racemes. Bracts are present, 46 in numbers perianth tube companulate, anthers
four celled. Ovary free or coverved by perianth, style curved, stigma dilated, fruit ovoid or globose (Gamble &
Fischer 1998).
Leaves and barks of Litsea stocksii and L. glutinosa are used as medicines. Essential oils like citral, lauric
acid and oleic acid extracted are used commercially for the preparation of insecticides, perfumes, flavours and
colognes. Oil extracted from Litsea cubeba is a good competitor of Chinese lemon oil due to its low cost of
production and easy method of cultivation of the species. Decoction of different parts of the plant used to cure
burns, sprains, cough, bronchitis and paralysis (Bhuniya et al. 2009).
The taxonomy of the family Lauraceae is still not settled compare to other families. It is poorly understood
due to its great diversity, inadequate morphological characters and lack of investment in taxonomic work. Litsea
is a very interesting tree species in Western Ghats of India occur in evergreen and semi evergreen forests,
information on its diversity, distribution and genetic relatedness within populations are not fully explored.
Hence in this present study we focussed to study the diversity and distribution of Litsea species in
Chikkamagaluru district, Karnataka.
Study area
The study area covers Kemmannugundi, Mullayyanagiri in Chikkamagaluru district situated between 1254'
to 1353' N and 7504' to 7621' E in the Western Ghats regions of Karnataka (Fig. 1). The sampling sites have
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Srinivas & Krishnamurthy (2016) 3(3): 564568

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rich forest vegetation such as evergreen and semi evergreen forests, the wide range of ecological conditions and
altitudinal variation resulted in diverse vegetation in study area. Mullayyanagiri is the highest elevated region in
Karnataka. In Kemmannugundi, Mullayyanagiri region the temperature varies between 10 to 32 C across the
different months of the year.
Figure 1. Map showing sampling sites in Chikkamagaluru District, Karnataka.
Tree sampling and Data analysis

Extensive field surveys carried out throughout the year to know the diversity, distribution and phenology of
the Litsea species. Stratified random sampling method is used to collect the tree data, three belt transects of
2504 m was laid in each study sites and girth was measured at breast height using a girth tape. Species density,
frequency, abundance, importance value index and basal area of plant were calculated by following Mishra
(1968), Mueller-Dambois & Ellenberg (1974). The importance value index was calculated by summing of
relative density, frequency and relative dominance. Species diversity index was calculated by Shannon Wiener
index (1963); the species dominance index was calculated by using Simpson (1949).
The four species of Litsea occurred in the two study sites; namely, Litsea floribunda, Litsea stocksii, Litsea
glabrata and Litsea mysorensis (Fig. 2). These four species collected from the study sites, identified through
some morphological characters using standard floras and herbarium samples were prepared. L. floribunda is
present in Kemmannugundi and Mullayyanagiri, but the L. stocksii, L. glabrata, L. mysorensis only present in
Kemmannugundi region absent in Mullayyanagiri (Table 1).
The results showed that L. floribunda frequently present in all transects, the frequency of L. glabrata is 0.67,
L. mysorensis 0.33, L. stocksii 1.0. The L. floribunda showed highest density 46.67 and 33.67 it covers a basal
area of 1904.79 m2.ha-1 and 885.27 m2.ha-1 (Table 1) in Mullayyanagiri and Kemmannugundi respectively, L.
mysorensis showed lesser density and basal area compare to all the species. Abundance and frequency (A/F)
ratio of all the Litsea species in the study sites is >0.05, it showed a clumped or contagious pattern of
distribution this is because it is a dioecious tree, clumping of individuals of the same species is often clearly
related to gap formation and dispersal, pollination mechanism of the species. Upadhaya et al. (2003) investigated
on the same family members Cinnamomum and Neolitsea it also showed clumped pattern of distribution.
565

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Figure 2. Litsea species in Chikkamagaluru district, Karnataka: AB, Litsea floribunda; CD, Litsea stocksii; EF, Litsea
mysorensis; GH, Litsea glabrata.
A total of 15 associated species belongs to 10 families were recorded in both Kemmannugundi and
Mullayyanagiri study sites (Table 2). Five species belongs to family Lauraceae, this is because of preference of
same environmental factors from the genera. Callicarpa, Cinnamomum, Cryptocarya, Neolitsea and Syzygium
species are frequently distributed in all the transects of Kemmannugundi. Cinnamomum showed high density
(21.33) per transect in Mullayyanagiri whereas Memecylon, Ochlandra, Psychotria showed low density (0.33)
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in both the study sites (Table 2). Actinodaphne, Cryptocarya, Macaranga and Neolitsea cassia only present in
Kemmannugundi absent in Mullayyanagiri region.
Table 1. Frequency (Fre), density per transect (Den), abundance (Abun), IVI, A/F ratio of Litsea spp.
Species
Kemmannugundi
Litsea floribunda
Litsea glabrata
Litsea mysorensis
Litsea stocksii
Mullayyanagiri
Litsea floribunda
Fre
Den
Abun
RF
RD
RA
IVI
A/F Basal area m2/ha
1
0.67
0.33
1
33.67
2
1
7
33.67
3
3
7
7.32
4.88
2.44
7.32
37.14
2.21
1.1
7.72
29.93
2.67
2.67
6.22
74.39
9.76
6.21
21.26
4.6
0.61
1.23
0.96
885.27
1.77
0.48
39.48
47.67
47.67
10
50
44.9
104.9
4.77
1904.79
Table 2. Frequency (Fre), density per transect (Den), abundance (Abun), IVI, A/F ratio of the major associated species.
Species
Fre
Actinodaphne sp.
0.33
Callicarpa tomentosa
1
Cinnamomum verum
1
Cryptocarya sp.
1
Glochidion sp.
0.67
Macaranga peltata
0.67
Maesa indica
0.67
Memecylon malabaricum 0.33
Neolitsea cassia
1
Neolitsea zeylanica
0.67
Nothapodytes foetida
0.33
Ochlandra travancorica 0.33
Psychotria nigra
0.33
Syzygium cumini
1
Vernonia arborea
0.33
Kemmannugundi
Den Abun RF
RD
1.67
5 2.44 1.84
2.67 2.67 7.32 2.94
11
11 7.32 12.13
4.67 4.67 7.32 5.15
3.67
5.5 4.88 4.04
1.67
2.5 4.88 1.84
4.33
6.5 4.88 4.78
0.33
1 2.44 0.37
5
5 7.32 5.52
2.33
3.5 4.88 2.57
1.33
4 2.44 1.47
0.33
1 2.44 0.37
0.33
1 2.44 0.37
4
4 7.32 4.41
1
3 2.44
1.1
RA
4.44
2.37
9.78
4.15
4.89
2.22
5.78
0.89
4.44
3.11
3.56
0.89
0.89
3.56
2.67
IVI
8.72
12.63
29.23
16.62
13.81
8.94
15.44
3.7
17.28
10.56
7.47
3.7
3.7
15.29
6.21
A/F
2.05
0.36
1.5
0.64
1.13
0.51
1.33
0.41
0.68
0.72
1.64
0.41
0.41
0.55
1.23
Fre
1
1
0.67
1
0.33
1
0.67
0.33
0.33
0.67
0.33
Mullayyanagiri
Den Abun RF
RD
1.67 1.67
10 1.75
21.33 21.33
10 22.3
3
4.5 6.67 3.15
3.67 3.67
10 3.85
0.33
1 3.33 0.35
4.33 4.33
10 4.55
2.33
3.5 6.67 2.45
0.33
1 3.33 0.35
0.33
1 3.33 0.35
4.67
7 6.67
4.9
0.33
1 3.33 0.35
RA
IVI
1.57
20
4.24
3.45
0.94
4.08
3.3
0.94
0.94
6.59
0.94
13.32
52.3
14.06
17.3
4.62
18.63
12.42
4.62
4.62
18.16
4.62
Shanon index is a diversity index taking into account of number of individuals as well as number of taxa.
The Shanon and Simpson index of Kemmannugudi is 2.32, 0.82 respectively and 1.65, 0.69 in Mullayyanagiri
respectively (Fig. 3). According to Shanon and Simpson indices Kemmannugundi has highest species richness
area compare to the Mullayyanagiri region. The Shanon index of the Kemmannugundi region is lower (2.32)
compare to Sulimudi forests of Western Ghats, Kerala (2.64) (Magesh & Menon 2011) and Simpson value
higher (0.82) compare to Vagamon region (0.36) (Brilliant et al. 2012).
Figure 3. Shanon and Simpson diversity index in Kemmannugundi and Mullayyanagiri.
CONCLUSION
567
A/F
0.17
2.13
0.68
0.37
0.3
0.43
0.53
0.3
0.3
1.05
0.3

.
This study revealed that the two study sites harboured four Litsea species and Litsea floribunda showed
good species richness. Litsea stocksii, Litsea glabrata and Litsea mysorensis showed low species richness in
Kemmannugundi whereas these species absent in Mullayyanagiri. A total of 15 associated species belongs to 10
families were recorded; Laural members are the dominant associated species in both the study sites.
ACKNOWLEDGEMENTS
The authors are thankful to the Department of Science and Technology (DST) New Delhi, for providing
financial assistance as an Inspire Fellowship (IF140097) to Srinivas SG and authors also acknowledge thanks to
Kuvempu University to providing research facilities. The author also acknowledges special thanks to Shravan
Kumar S., Avinash K.S., Ashwini H.S., for their help in field collections in the studies.
REFERENCES
Bhuniya T, Singh P & Mukherjee SK (2009) Distribution of the genus Litsea Lam. (Lauraceae) in India with
special reference to rare and endemic species. Phytotaxonomy 9: 116121.
Bhuniya T, Singh P & Mukherjee SK (2010) An account of the species of Litsea Lam. (Lauraceae) endemic to
India. Bangladesh Journal of Plant Taxon 17: 183191.
Brilliant R, Varghese VM, Paul J & Pradeepkumar AP (2012) Vegetation analysis of montane forest of Western
Ghats with special emphasis on RET species. International journal of Biodiversity and Conservation 4: 652
664.
Chaing YC, Huei CS, Min CH, Li PJ & Hsiang HK (2012) Characterization of microsatellite loci from Litsea
hypophaea, a tree endemic to Taiwan. American Journal of Botany 99(6): e251e254.
Gamble JS & Fischer CEC (1998) Flora of Presidency of Madras, Vol. 13. Adlard and Son Limited, 21, Hart
street, WC.
Magesh G & Menon ARR (2011) Vegetation status, species diversity and endemism of Sulimudi forests in
southern Western Ghats of Kerala, India. The Indian Forester 2: 304311.
Mishra R (1968) Ecology work book. Oxford and IBH publishing company Calcutta India.
Mueller DD & Ellenberg H (1974) Aims and methods of vegetation ecology. John Wiley and Sons New York
USA.
Saldanha CJ (1996) Flora of Karnataka, Vol. 14. Oxford and IBH publishing Ltd, New Delhi.
Shanon CE & Weiner W (1963) The Mathematical theory of communication. University of Illinois press
Urbana.
Simpson EH (1949) Measurement of diversity. Nature 163: 688.
Upadhaya K, Pandey HN, Law PS & Tripathi RS (2003) Tree diversity in sacred grooves of the Jaintia hills in
Meghalaya, Northeast India. Biodiversity and Conservations 12: 583597.
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3(3): 569572, 2016
DOI: 10.22271/tpr.2016.v3.i3.075
Research article
Alternaria polypodiicola, a new foliicolous fungus discovered on

Microsorum punctatum from Uttar Pradesh, India
Shambhu Kumar1* and Raghvendra Singh2
1
Department of Forest Pathology, Kerala Forest Research Institute, Thrissur, Kerala, India
Centre of Advanced Study in Botany, Institute of Sciences, Banaras Hindu University, Varanasi.
Uttar Pradesh, India
*Corresponding Author: skumartaxon@gmail.com
Abstract: Alternaria polypodiicola sp. nov., is described, illustrated and discussed, causing foliar
disease on a pteridophytic plant Microsorum punctatum (Polypodiaceae) from Uttar Pradesh,
India. The present species was compared with closely similar species based on morphological
characters. This species is characterized by having well developed stromata, unbranched and
shorter conidiophores with shorter smooth conidia. A key is provided to all species of Alternaria
reported on Polypodiaceae. The description and nomenclatural novelty details were deposited in
Mycobank.
Keywords: Taxonomy - Foliicolous - Hyphomycetes - Microsorum - Alternaria - New species.
[Cite as: Kumar S & Singh R (2016) Alternaria polypodiicola, a new foliicolous fungus discovered on
Microsorum punctatum from Uttar Pradesh, India. Tropical Plant Research 3(3): 569572]
INTRODUCTION
Microsorum punctatum (L.) Copel. is a small evergreen ornamental pteridophytic plant belongs to family
Polypodiaceae of Plant kingdom. It is a common fern species in Africa and Asia and occurs naturally in various
forest types of tropics and subtropics from sea level up to 2800 m elevation (Nooteboom 1997, Bosman 1991).
The plant shows good medicinal properties. The leaf and juice are used as purgative, diuretic and for healing
wounds (May 1978, Sharma & Pegu 2011).
During the regular observation of plants of the BSIP garden, Lucknow, the living leaves of Microsorum
punctatum exhibiting foliar blights was encountered. However, it differs morphologically from previously
described Alternaria species and therefore is proposed here as new based on critical microscopic examination
and comparison of morphological features with those of the closely similar forms. The details description and
illustration of Alternaria polypodiicola is presented here.
The diseased plant leaves samples were collected from BSIP Campus, Lucknow during September 2012.
The photographs of the infection spots were taken by using a Sony DSC-5730 camera during the time of
collection. The collected samples were carried to the laboratory and processed by following the standard
techniques (Castaeda-Ruiz 2005, Hawskworth 1974, Savile 1962). The sun dried and pressed leaf specimens
were placed in air tight polyethylene bags and then kept in ziplock polythene bag along with collection details.
The surface scrapping and free hand cut sections of infected leaf samples were taken through infection spots and
mounted in cotton-blue lactophenol mount mixture for microscopic examination. Detailed observations of
morphological characters were carried out by means of an Olympus CX31 light microscope (400) and
measurement was done by micrometry. Morphotaxonomic determination was made with the help of current
literature pertaining to Alternaria. The holotype specimen has been deposited in Ajrekar Mycological
Herbarium (AMH), Agharkar Research Institute, Pune, India future reference. Description and nomenclatural
detail were deposited in MycoBank (www.MycoBank.org). The systematics position of the taxa is given in
accordance with Cannon & Kirk (2007), Kirk et al. (2008), Seifert et al. (2011), Farr & Rossman (2015) and the
Index Fungorum (www.indexfungorum.org; accessed 30 April 2015).
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RESULTS
Taxonomic descriptions
Alternaria polypodiicola Sham. Kumar & Raghv. Singh sp. nov. (Fig. 1, 2)
MycoBank MB 817343
Anamorphic fungus, hyphomycetes, Foliicolous, Infection spots amphigenous, initially circular to irregular
(5-25 mm diam.), brown, but later on severe infection it spreading on entire surface of the leaves. Colonies
amphiphyllous, effuse, brown. Mycelium internal. Stromata present (15m in diam), pseudoparenchymatous.
Conidiophores macronematous, fasciculatous (57 in a fascicle), straight to curved, simple, cylindrical,
unbranched, thick walled, smooth, 13 septate, brown, 1055 35 m. Conidiogenous cells integrated,
terminal, monotretic, scars thickened. Conidia simple, acropleurogenous, solitary to catenate, dry, smooth
obclavate to ellipsoidal to ovoid (muriform), rostrum present, 24 transversely septate and 23 obliquely
septate, brown, base obtuse, 2050 1018 m, hilum thickened (1.52.0 m), germinating conidium present.
Material examined: India, Uttar Pradesh, Lucknow, BSIP Campus, on living leaves of Microsorum punctatum
(L.) Copel. (Polypodiaceae), 2nd September, 2012, coll. Shambhu Kumar, AMH-9515 (holotype).
Etymology: The specific epithet polypodiicola in reference to host family.
Teleomorph: Undetermined.
Figure 1. Microsorum punctatum: A, Host Plant; B, Infection spots on upper surface of leaf (Scale bars: B = 20 mm); C,
Infection spots on lower surface of leaf.
Identification key to Alternaria spp. reported on Polypodiaceae

1 Stromata absent.....2
1* Stromata present......3
2 Conidiophores up to 50 36 m, branched........4
2*Conidiophores up to 115 46 m, simple or unbranched.........5
3 Conidiophores 1055 m 35 m, unbranched.........6
4 Conidia 2063 918 m, 8 transversely septate and several obliquely septate, verruculose......... A. alternata
5 Conidia 2295 819 m, 47 transversely septate and several obliquely septate,verruculose A. tenuissima
6 Conidia 2050 1018 m, 24 transversely septate and 23 obliquely septate, smooth. A. polypodiicola
DISCUSSION
Perusal of literatures indicated that there was no record of Alternaria on this host (Farr & Rossman, 2015,
Bilgrami et al. 1991, Jamaluddin et al. 2004). Alternaria polypodii (invalidly published) on Polypodium sp. and
Alternaria sp. on Platycerium bifurcatum and Platycerium sp. from Florida (Farr & Rossman 2015) have been
reported on Polypodiaceae which were very similar to A. alternata. From India, A. alternata was previously
reported on Polypodium vulgare L. (Narang et al. 1978) from Allahabad and A. tenuissima (Kunze ex Pers)
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Wilt. on Polypodium multilineatum L. (Kanaujia et al. 1978) from Faizabad respectively on the family
Polypodiaceae. Therefore, the present fungus was compared with these two earlier reported species.
The stromata is present in A. polypodiicola while absent in earlier described species. The conidiophores are
unbranched and very much shorter (1055 35 m) in A. polypodiicola while branched and longer (up to 50
36 m) in A. alternata and A. tenuissima (simple or unbranched and up to 115 46 m). The Conidia of A.
polypodiicola are shorter (2050 1018 m) than both previously described A. alternata (2063 918 m)
and A. tenuissima (2295 819 m). The conidia of novel species are smooth while verruculose in both the
earlier described species. Thus, A. polypodiicola is treated as a new species.
Figure 2. Alternaria polypodiicola (AMH-9515, holotype): AD, Conidiophores; EH, Conidia. (Scale bars AH = 5 m)
ACKNOWLEDGEMENTS
Authors are thankful to the Director Kerala Forest Research Institute, Peechi for encouragement and
necessary facilities. Thankfulness is also due to the Curator, Ajrekar Mycological Herbarium (AMH), Agharkar
Research Institute, Pune (MS), India for depositing specimen and providing accession number thereof. We are
also grateful to Dr. Ajit Pratap Singh, Senior Scientist, CSIR-NBRI, Lucknow, for the host identification.
Shambhu Kumar is grateful to Science and Engineering Research Board (SERB), Department of Science and
Technology (DST), Government of India, New Delhi for financial support (SB/YS/LS-288/2013).
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572
ISSN (E): 2349 1183

ISSN (P): 2349 9265
3(3): 573585, 2016
DOI: 10.22271/tpr.2016.v3.i3.076
Research article
Floristic composition and biological spectrum of weeds in agroclimatic zone of Nalbari district, Assam, India
D. K. Bhattacharjya1* and S. K. Sarma2
1
Corresponding author, Dept. of Botany, M.C. College, Barpeta-781301, Assam, India

2
Department of Botany, Gauhati University, Guwahati-781014, Assam, India
*Corresponding Author: dipkrbhatta@gmail.com
[Accepted: 02 November 2016]
Abstract: Present paper deals with the study of floristic composition and biological spectrum of
the agro-climatic zone of Nalbari district of Assam. Field observation encompasses a total of 217
weed species from both angiosperms and pteridophytes belonging to 150 genera under 60 families.
Asteraceae, being the largest family possesses 19 genera and 22 species. Out of the total species,
183 species (84.33 %) showed the annual life span as against 34 species (15.67 %) showing the
perennial life span. One hundred thirty eight species (63.59 %) were recorded to be propagated by
seeds/spores; 66 species (30.41 %) were found to opt for both seeds and vegetative propagules and
only 13 species (5.99 %) were listed to be propagated exclusively by vegetative propagules. The
phytoclimate of the agro-climatic zone can be regarded as thero-cryptophytic since major
percentage of the species falls under the life forms Therophytes and Cryptophytes which has been
assessed after comparison with the normal world spectrum as proposed by Raunkiaer.
Keywords: Agro-climatic zone - Weed flora - Life span - Phytodiversity - Propagation.
[Cite as: Bhattacharjya DK & Sarma SK (2016) Floristic composition and biological spectrum of weeds in agroclimatic zone of Nalbari district, Assam, India. Tropical Plant Research 3(3): 573585]
INTRODUCTION
Agro-climatic condition includes such basic factors of plant growth like soil types, temperature, rainfall and
water availability which directly influence the vegetation of an area. Agro-climatic zone is a unit of land use in
terms of major climates and the zone is favourable for certain range of crops and cultivars. Such climatic
division is meant for effective and efficient management of local phytoresources, i.e. crops to meet the growing
demand of food, fodder, fibre, fuel wood, timber etc. without adversely affecting the environment and causing
any sort of loss to the nature (http://vikaspedia.in). India comprises a total of 15 agro-climatic zones and Assam
belongs to the Eastern Himalayan zone of 15 such zones (Singh 2012).
Nalbari district of Assam is basically an agriculture dependent district. The richness of the flora of this
district with a healthy combination of both primitive and advanced families is supported by the geographical
position of the district and also the favourable climatic conditions. Varieties of weed species are found to infest
the crop-lands, waste lands, aquatic bodies etc. in the district throughout the year. Particularly in the crop-lands,
the obnoxious weeds create severe problems interfering the yield of the crops in the area. Varieties of crop
cultivations are practiced throughout the year in the agro-climatic zones in Nalbari district of Assam. Along with
the crops, the crop fields in the agro-climatic zones are found to be infested by a variety of weed species
throughout the year.
Weeds are the plants grown in the places where they are not desired. Weeds may include all types of
undesirable plants like grasses, sedges, forbs, aquatic plants, parasitic angiosperms, pteridophytic plants etc.
Such plants may be the constant associates of the cultivated plants also, comprising the crop-field weeds, i.e. the
vegetation infesting the human maintained crop-fields. Still the weeds have been occupying an integral part of
the phytodiversity of a region.
The vegetation infesting the crop-fields of the agro-climatic zones is very often ignored and not included in
the study in terms of ecology and taxonomy in comparison to forest, grassland, wetland and others. Weeds,
573

Bhattacharjya & Sarma (2016) 3(3): 573585

.
although are very frequently termed as obnoxious, harmful to both human and animals and reducer of crop
yield, yet a lot of weed species are useful in many ways (Bhattacharjya & Borah 2006, Bhattacharjya et al.
2006). Apart from these, weeds have an important contribution towards the phytodiversity of a region.
Survival of mankind is directly related with the survival of phytodiversity. Being rich in phytodiversity the
entire country in general and the state of Assam and entire North-East in particular possess a large number of
plant species growing in a variety of climatic and edaphic conditions resulting in the formation of a wide range
of habitats (Reddy et al. 2008). Floristic richness of a region enables one to have the idea about the design and
functioning of the communities along with the pattern and process of the community structure (Thakur 2015).
Study of floristic composition is considered as fundamental and regarded as prerequisite for all kinds of
ecological research. All other works revolve around the floristic study (Naveed et al. 2012).
Climatic, edaphic and biotic factors prevailing in an area influence the formation of vegetation of that area
(Shahid & Joshi 2015). Existing vegetation is also an indicator of the climate, soil and anthropogenic influences
occurring in a region (Sharma et al. 2014). The major community description and its appearance depend upon
the occurrence of life forms which are based on the position and degree of protection of regenerating parts with
respect to the ground surface (Cain 1950). The physical appearance of vegetation chiefly depends on the life
form of dominant plant species (Hanson & Churchill 1961). Life form pattern of the species and the proportion
of life forms in an area reflect a complete ecological picture of the community as well as provide a good
indication of the climatic zone of the community (Cain 1950, Kershaw 1973).
The plant species of any community can be classified in one or the other life forms. The ratio of the life
forms of different species in terms of numbers or percentages in any floristic community is the biological or
phytoclimatic spectrum. The biological spectrum is also regarded as the indicative of the prevailing environment
as the life forms are related to the environment around the plants (Sudhakar Reddy et al. 2011).
Plants life form is one among its most striking characteristics. In classifying vegetation, Raunkiaers life
form system is very widely used as such or with a few modifications (Braun-Blanquet 1951, Dansereau 1957a,
Mueller-Dombois & Ellenberg 1974). Analyzing the life forms of various regions of the world and comparing
them with a normal spectrum based on 1,000 species selected at random, Raunkiaer reported a predominance of
Phanerophytes in tropical moist regions. Following the same principle, a high preponderance of Therophytes
and not insignificant proportions of Chamaephytes and Hemicryptophytes may be found from a desert area
(Raunkiaer 1934).
There has been an increasing effort on the study of vegetation in connection with floristic composition, life
forms and biological spectrum in different times (Gillespie 2004, Batalha & Martins 2004, Reddy et al. 2011,
Theilade et al. 2011, Saikia et al. 2012, Naveed et al. 2012, Burja et al. 2013, Aye et al. 2014, Thakur 2015,
Alemu et al. 2015). However, no such study has been conducted hitherto in the Nalbari district of Assam.
Accordingly, no work is reported from the agro-climatic zone of the district in this regard. The present work,
therefore, aims at to study the floristic composition of the agro-climatic zone of the district along with the
preparation of biological spectrum of the agro-zone.
The study area
Nalbari districtof Assam lies between 26 10' N to 26 47' N latitude and 90 15' E to 91 10' E longitude
which occupies an area of about 1009.57 km2. The area is mainly plain. The northern side of the district is
bounded by the Baksa district. The southern side by the mighty Brahmaputra. The Kamrup District falls in the
east and the Barpeta District in the west. The entire area of the District is situated at the plains of the
Brahmaputra Valley. The tributaries of the Brahmaputra, Nona, Buradia, Pagaldia, Borolia and Tihu which are
originated from the foothills of the Himalayan Range are wild in nature and have enormous contribution
towards the agrarian economy of the district. The Soil condition of the district is heterogeneous one. The Soil of
the northern part is clayey and loamy, whereas middle part is loamy and sandy. The southern part is
characterized by sandy soil (Industrial Profile of Nalbari district, ministry of MSME, Govt. of India). The soil
pH varies from 5.05 to 7.22. The District has a sub-tropical climate with semi dry hot summer and cold winter.
During summer, generally during the months from May to August, heavy rainfall occurs for which the district
experiences flood. The District experiences annual (average) rainfall of 1500 mm and its humidity hovers
around 80%. The average temperature during summer and winter are 27.00 C and 16.21 C. respectively
(Office of the Deputy Commissioner, Nalbari, Assam, India). Several crop cultivations belonging to both
574

.
summer and winter seasons are practiced throughout the year in the district. The major crops include rice,
wheat, lentil, pea, mustard, jute, sugarcane, chillis, onion, turmeric, vegetable yielding species etc.
Data collection
Frequent field visits were made for a period of three years from 2013 to 2015 to collect the weed species
infesting the crop-fields under the agro-climatic zone of Nalbari district of Assam. Cultivated crop species were
not taken into consideration as the species are fully human maintained and not part of natural vegetation.
Method(s) of propagation and the position of the perennating buds of each weed species were recorded on the
spot by thorough observation. Life spans of the species were recorded after close observation of the life cycle
right from seedling stage till death. For determination of the life-forms and analysis of the biological spectrum,
Raunkiaers system as modified by Braun-Blanquet (1951) has been followed. The percentage of each life form
was calculated by using the following formula:
Number of species in any life form
% Life form =
100
Total number of species of all life forms
The data recorded have been presented in tabular form by arranging the families according to Bentham &
Hookers system of classification.
RESULTS
A total of 217 weed species belonging to 150 genera and 60 families were encountered during the field study
in the agro-climatic zone of Nalbari district of Assam. Normally highest number of angiospermic species (210)
has been recorded from the study area in comparison to the pteridophytic species (7) (Table 1, 2; Fig. 1, 2).
Table 1. Enumeration of Angiospermic weed species and their basic ecological features. (Abbreviations: A: annual, Bl: bulbil,
Ch: chamephyte, Cr: cryptophyte, Hm: hemicryptophyte, P: perennial, Of: offset, Ph: phanerophyte, R: runner, Rs: root stock,
Rz: rhizome, S: seed, Sc: sucker, Sm: stem, St: stolon, Tb: tuber, Th: therophyte)
Family
Nymphaeaceae
Species
Euryale ferox Salisb.

Nelumbo nucifera Geartn.
Nymphaea alba L.
N. nouchali Burm.f.
N. rubra Roxb. ex Salisb.
Nymphoides cristata (Roxb.) Kuntze
Papaveraceae
Argemone mexicana L.
Brassicaceae
Capsella bursa-pastoris (L.) Medikus.
Rorippa benghalensis (DC.) H. Hara.
Capparaceae
Cleome viscosa L.
Caryophyllaceae Drymaria diandra Blume.
Polycarpon prostratum (Forsk.) Asch. & Schweinf.
Stellaria media (L.) Vill.
S. wallichiana Haines.
Portulacaceae
Portulaca oleracea L.
Hypericaceae
Hypericum japonicum Thunb. ex Murray
Malvaceae
Abutilon indicum (L.) Sweet.
Malvastrum coromandalianum (L.) Garcke
Sida cordifolia L.
Sida rhombifolia L.
Tiliaceae
Corchorus aestuans L.
Grewia sapida Roxb.
Triumfetta rhomboidea Jacq.
Linaceae
Linum ustitatissimum L.
Balsaminaceae
Impatiens glandulifera Royle
Oxalidaceae
Oxalis corniculata L.
O. debilis H.B.K. var. corymbosa
Sapindaceae
Cardiospermum halicacabum L.
Fabaceae:
Mimosoideae
Mimosa pudica L.
Caesalpinioideae Cassia sophera L.
C. tora L.
Life span
P
P
P
P
P
P
A
A
A
A
A
A
A
A
A
A
A
P
P
P
A
P
P
A
A
A
A
A
A
A
A
Method of
Life form
Propagation
S, Rz
Cr
S, Rz
Cr
Rz, St
Cr
Rz, St
Cr
Rz, St
Cr
Rz
Cr
S
Th
S
Ph
S
Th
S
Th
S
Ch
S
Th
S
Th
S
Th
S
Th
S
Th
S
Ph
S
Ph
S
Th
S
Th
S
Ph
S
Ph
S
Ph
S
Th
S
Th
S, R
Hm
S, Bl
Hm
S
Ph
S
S
S
Th
Th
Ph
575

.
Papilionatae
Aeschynomene aspera L.
A
S
Cr
Aeschynomene indica L.
A
S
Ph
Crotalaria juncea L.
P
S
Ph
Desmodium gangeticum (L.) DC.
A
S
Th
D. laxiflorum DC.
A
S
Th
D. triflorum (L.) DC.
A
S
Th
D. triquetrum (L.) DC. ssp. pseudotriquetrum
A
S
Ph
Lathyrus aphaca L.
A
S
Th
Tephrosia purpurea (L) Pers.
P
S
Ph
Rosaceae
Duchesnea indica (Andrews) Focks.
A
S, R
Hm
Haloragaceae
Callitriche stagnalis Scop.
A
S
Th
Lythraceae
Ammannia baccifera L.
A
S
Th
A. multiflora Roxb.
A
S
Th
Cuphea carthagenensis (Jacq.) J.F.Macbr.
A
S
Th
Rotala indica (Willd.) Cochne.
A
S
Th
Onagraceae
Ludwigia adscendens (L.) Hara.
A
S, Of
Cr
L. octavalvis (Jacq.) Raven.
A
S
Th
L. perennis L.
P
S
Th
Trapaceae
Trapa bispinosa (Roxb.) Makino
P
S, Rz
Cr
T. natans L.
P
S, Rz
Cr
Molluginaceae
Glinus lotoides L.
A
S
Th
Mollugo pentaphylla L.
A
S
Th
Apiaceae
Centella asiatica (L.) Urban.
A
S, R
Hm
Hydrocotyle javanica thunb.
A
S, R
Hm
H. sibthorpioides Lam.
A
S, R
Hm
Oenanthe javanica (Blume) Dc.
A
S
Th
Rubiaceae
Dentella repens (L.) J.R. & G. Forst.
A
S
Th
Oldenlandia diffusa (Willd.) Roxb.
A
S, R
Ch
Richardia scabra L.
A
S, R
Th
Asteraceae
Blumea densiflora DC.
A
S
Th
B. lacera (Burm. f.) DC.
A
S
Ph
Cosmos sulfureus Cav.
A
S
Th
Cotula hemisphaerica Wall.
A
S
Th
Dichrocephala integrifolia (L.f.) O.Ktze.
A
S
Th
Eclipta prostrata L.
A
S
Th
Elephantopus scaber L.
A
S
Th
Enhydra fluctuans Lour.
A
S
Cr
Chromolaena odorata (L.) R.M.King & H.Rob.
A
S
Ph
Gnaphalium luteo-album L.
A
S
Th
G. pensylvanicum Willd.
A
S
Th
G. polycaulon Pers.
A
S
Th
Grangea maderaspatana (L.) Poiret.
A
S
Ch
Mikania micrantha Kunth.
A
S, R
Ph
Parthenium hysterophorus L.
P
S
Th
Sonchus wightianus DC.
A
S
Th
Sphaeranthus indicus L.
A
S
Th
Spilanthes paniculata Wallich ex DC.
A
S, Sc
Ch
Taraxacum officinale Wigg.
A
S
Ph
Vernonia cinerea (L.) Less.
A
S
Th
Xanthium indicum Koenig. in Roxb.
A
S
Th
Youngia japonica (L.) DC.
A
S
Th
Campanulaceae Lobelia zeylanica L.
A
S
Th
Wehlandbergia marginata (Thunb.) DC.
A
S
Th
Hydrophyllaceae Hydrolea zeylanica (L.) Vahl.
A
S
Cr
Boraginaceae
Cynoglossum zeylanicum (Vahl.) Brand
A
S
Ph
Heliotropium indicum L.
A
S
Th
Convolvulaceae Evolvulus mummularis L.
A
S, R
Hm
Ipomea aquatica Forst.
A
S
Cr
I. carnea (Mart. ex Choisy) Austin.
P
S, Sm.
Ch
576
Solanaceae
Scrophulariaceae
Lentibulariaceae
Acanthaceae
Verbenaceae
Lamiaceae
Amaranthaceae
Chenopodiaceae
Polygonaceae
Euphorbiaceae
Urticaceae
Cannabinaceae
Ceratophyllaceae
Hydrocharitaceae
Nicotiana plumbaginifolia Viv.

Solanum nigrum L.
S. torvum Swartz.
Limnophila hirsuta Benth.
Limnophila indica (L.) Druce
L. heterophylla (Roxb.) Ben
Lindernia anagallis (Burm. f.) Pennell.
L. antipoda (L.) Alston.
L. ciliata (Colsm.) Pennell.
L. cordifolia (Colsm.) Merr.
L. crustacea (L.) Muell.
L. parviflora (Roxb.) Haines.
L. ruelloides (Colsm.) Pennell.
L. tenuifolia (Colsm.) Alston.
L. viscosa (Hornem) Boldingh.
Mazas pumilus (Burm.f.) Steen.
Mecardonia procumbens (Mill.) Small
Scoparia dulcis L.
Torenia diffusa D. Don.
Utricularia aurea Lour.
Hygrophila polysperma (Roxb.) T. Anders.
Lepidagthis incurva D. Don.
Rostellularia japonica (Thunb.) Ellis.
Rungia pectinata (L.) Nees.
Clerodendrum viscosum Vent.
Phyla nodiflora (L.) Greene.
Leonurus japonicus Houtt.
Leucas Plukenetii (Roth.) Spreng.
Ocimum Basilicum L.
Pogostemon Fraternus Mig.
P. Strigosus Benth.
Achyranthes aspera L.
Alternanthera philoxeroides (Mart.) Griseb.
A. sessilis (L.) R.Br. ex DC.
Amaranthus hybridus L.
A. spinosus L.
A. viridis L.
Celosia argentea (L.) Schinz.
Chenopodium album L.
Polygonum barbatum L.
P. glabrum Willd.
P. chinense L.
P. hydropiper L.
P. orientale L.
P. plebeium R.Br.
P. strigosum Br. Prodr.
Rumex dentatus L.
Rumex maritimus L.
Rumex nepalensis Spreng.
Acalypha indica L.
Croton bonplandianum Baill.
Euphorbia hirta L.
E. thymifolia L.
Phyllanthus fraternus Webster.
Pouzolzia zeylanica (L.) Bennett.
Cannabis sativa L.
Ceratophyllum demersum L.
Hydrilla verticillata (L.f.) Royle.
Ottelia alismoides (L.) Pers.
Valisnaria spiralis L.

.
A
S
Ph
A
S
Th
A
S
Ph
A
S
Th
A
S
Cr
A
S
Cr
A
S
Th
A
S
Th
A
S
Th
A
S
Th
A
S
Th
A
S
Th
A
S
Th
A
S
Cr
A
S
Th
A
S
Th
A
S
Th
A
S
Th
A
S
Th
A
S, Of
Cr
A
S
Cr
A
S
Ph
A
S
Th
A
S
Th
P
S
Ph
A
S
Th
A
S
Ph
A
S
Th
P
S
Ph
A
S
Th
A
S
Th
A
S
Th
A
S
Ch
A
S, R
Ch
A
S
Th
A
S
Th
A
S
Th
A
S
Th
A
S
Th
A
S
Th
A
s
Th
A
S
Th
A
S
Th
A
S
Th
A
S
Th
A
S
Th
A
S
Th
A
S
Th
A
S
Th
A
S
Th
A
S
Th
A
S, R
Ch
A
S
Th
A
S
Th
A
S
Th
A
S
Th
A
S, Rs
Cr
A
Rz
Cr
A
S
Cr
P
S, Rz
Cr
577
Zingiberaceae
Pontederiaceae
Alpinia allughas (Retz.) Rosc.

Eichhornia crassipes (Mart.) Solms.
Monochoria hastata (L.) Solms.
M. vaginalis C.Presl.
Commelinaceae Commelina benghalensis L.
C. diffusa Burm.
Cyanotis axillaris (L.) Don.
Floscopa scandens Lour.
Murdania nudiflora (L.) Brenan.
Typhaceae
Typha elephantina Roxb.
Araceae
Amorphophallus campanulatus (Roxb.) Bl.
Colacasia esculenta (L.) Schott.
Lasia spinosa Thw.
Pistia stratiotes L.
Lemnaceae
Lemna purpusilla Torrey
Spirodela polyrrhiza (L.) Schl.
Najadaceae
Najas indica (Willd.) Cham.
Najas minor All.
Aponogetonaceae Aponogeton appendiculatus H. Brug
Potamogetonaceae Potomogeton crispus L.
Alismataceae
Alisma plantago L.
Sagittaria guayanensis H.B. & K.
Sagittaria sagittifolia L.
Eriocaulaceae
Eriocaulon viride Koern.
Cyperaceae
Cyperus bulbosus Vahl.
C. corymbosus Rottb.
C difformis L.
C. halpan L.
C. iria L.
C. pilosus Vahl.
C. pumilus L.
C. rotundus L.
C. sanguinolentus Vahl.
C. tenuispica Steud.
Elaeocharis dulcis (Burm.f.) Henschel.
Fimbristylis aestivalis (Retz.) Vahl.
F. dichotoma (L.) Vahl.
F. littoralis Gaud.
F. miliacea (L.) Vahl.
F. tomentosa Vahl.
Kyllinga monocephela Roxb.
Schoenoplectus articulatus (L.)
S. grossuss (L.f.)
Scirpus articulatus L.
S. juncoides Roxb
Poaceae
Andropogon ascinoidis C.B.Clarke.
Axonopus compressus (Sw.) Beauv.
Brachiaria distachya (L.) Stapf.
Cynodon dactylon (L.) Pers.
Dactyloctenium aegyptium (L.) P.Beauv.
Digitaria ciliaris (Retg.) Koel.
D. sanguinalis Scop.
Echinochloa colonum Link.
Eleusine indica (L.) Gaertn.
Eragrostis coarctata Stapf.
E. tenella (L.) P. Beauv.
E. unioloides (Retz.) Nees ex Steud.
E. viscosa Trin.
Eriochloa procera (Retz.) C.E.Hubb
Phragmites karka Trin. ex Steud.

.
P
Rz
Cr
A
Of
Cr
A
Rz
Cr
A
Rz
Cr
A
S,R
Ch
A
S,R
Ch
A
S,R
Ch
A
S,R
Ch
A
S
Ch
P
S, Rz
Cr
P
Tb
Ch
P
Sc, Rz
Hm
P
S, St
Ch
A
S, St
Cr
A
S
Cr
A
S, B
Cr
A
S
Cr
A
S
Cr
A
S, Rs
Cr
A
S,B
Cr
P
S
Cr
A
Tb
Cr
P
Tb
Cr
A
S. Tb
Cr
A
S, Tb
Cr
P
S, Tb
Cr
A
S, Tb
Ch
A
S, Tb
Ch
A
S, Tb
Ch
A
S, Tb
Ch
A
S, Tb
Ch
A
S, Tb
Ch
A
S, Tb
Ch
A
S, Tb
Ch
A
S
Cr
A
S, Tb
Hm
A
S, Tb
Ch
A
S, Tb
Hm
A
S, Tb
Hm
P
S, Tb
Ch
A
S, Rs
Cr
P
S, Tb
Cr
P
S, Tb
Cr
A
S, Tb
Hm
A
S, Tb
Hm
A
S
Ch
A
S,R
Hm
A
S
Hm
A
S,R
Hm
A
S, Tb
Hm
A
S
Ch
A
S
Ch
A
S
Ch
A
S, Rs
Hm
A
S
Hm
A
S, Tb
Ch
A
S, Tb
Hm
A
S, Tb
Ch
A
S, Rz
Ch
A
S
Th
578

.
Pteridophytes,
7
Angiosperms:
Monocot, 63
Angiosperms:
Dicot, 147
Figure 1. Plant groups and constituent number of species.

Table 2. Enumeration of Pteridophytic weed species and their basic ecological features. (Abbreviations: A: annual, Ch:
chamephyte, Cr: cryptophyte, Hm: hemicryptophyte, P: perennial, R: runner, Rs: root stock, Sp: spore)
Family
Species
Selaginellaceae
Equisetaceae
Dryopteridaceae
Thelypteridaceae
Marsiliaceae
Selaginella descipiens Warb.

Equisetum ramosissimum Desf. ssp. debile Hauke.
Diplazium esculentum (Retz.) Swartz.
Christella parasitica (L.) Holttum
Marsilea minuta L.
M. quadrifolia L.
Azolla pinnata R.Br.
Azollaceae
Life span
A
A
P
P
A
A
A
Method of
Propagation
Sp
Sp, Rs
Sp, Rs
Sp, Rs
Sp, R
Sp, R
Sp
Life form
Ch
Ch
Hm
Hm
Cr
Cr
Cr
Among the angiosperms, 147 species belonging to 104 genera and 40 families fall under dicotyledons, whereas
63 species belonging to 40 genera and 14 families undergo monocotyledons comprising a dicot-monocot ratio of
2:1, 3:1 and 3:1 for species, genera and families respectively. Among the pteridophytes, 7 species could be
collected from the study area belonging to 6 genera under 6 families (Table 2). Among all the families
(Angiospermic and Pteridophytic), Asteraceae (Dicot) was found to be the largest one comprising 19 genera
(12.67 %) followed by Poaceae (Monocot) comprising 11 genera (7.33 %), Fabaceae (Dicot) comprising 7
genera (4.67 %), Scrophulariaceae (Dicot) comprising 6 genera (4.00 %) etc. (Table 3). Regarding species
content, the family Asteraceae (Dicot) was found to be the largest comprising 22 species (10.14 %) followed by
Cyperaceae (Monocot) comprising 21 species (9.68%), Scrophulariaceae (Dicot) comprising 16 species (7.37
%), Poaceae (Monocot) comprising 15 species (6.91), Fabaceae (Dicot) comprising 12 species (5.53 %) etc.
(Table 3). Thus the family Asteraceae has been found to possess highest number of both genera and species and
can be regarded richest of all observed families in the agro-climatic zone of the district.
Table 3. Different taxa and their constituent numbers in the study area.
Sl. No.
Family
Angiosperms (Dicotyledons):
1
Nymphaeaceae
2
Papaveraceae
3
Brassicaceae
4
Capparaceae
5
Caryophyllaceae
6
Portulacaceae
7
Hypericaceae
8
Malvaceae
9
Tiliaceae
10
Linaceae
11
Balsaminaceae
12
Oxalidaceae
13
Sapindaceae
No. of genera
No. of species
4
1
2
1
3
1
1
3
3
1
1
1
1
2.67
0.67
1.33
0.67
2.00
0.67
0.67
2.00
2.00
0.67
0.67
0.67
0.67
6
1
2
1
4
1
1
4
3
1
1
2
1
2.76
0.46
0.92
0.46
1.84
0.46
0.46
1.84
1.38
0.46
0.46
0.92
0.46
579

.
14
Fabaceae:
Mimosoideae
Caesalpinioideae
Papilionatae
15
Rosaceae
16
Haloragaceae
17
Lythraceae
18
Onagraceae
19
Trapaceae
20
Molluginaceae
21
Apiaceae
22
Rubiaceae
23
Asteraceae
24
Campanulaceae
25
Hydrophyllaceae
26
Boraginaceae
27
Convolvulaceae
28
Solanaceae
29
Scrophulariaceae
30
Lentibulariaceae
31
Acanthaceae
32
Verbenaceae
33
Lamiaceae
34
Amaranthaceae
35
Chenopodiaceae
36
Polygonaceae
37
Euphorbiaceae
38
Urticaceae
39
Cannabinaceae
40
Ceratophyllaceae
Angiosperms (Monocotyledons):
41
Hydrocharitaceae
42
Zingiberaceae
43
Pontederiaceae
44
Commelinaceae
45
Typhaceae
46
Araceae
47
Lemnaceae
48
Najadaceae
49
Aponogetonaceae
50
Potamogetonaceae
51
Alismataceae
52
Eriocaulaceae
53
Cyperaceae
54
Poaceae
Pteridophytes:
55
Selaginellaceae
56
Equisetaceae
57
Dryopteridaceae
58
Thelypteridaceae
59
Marsiliaceae
60
Azollaceae
Total
1
1
5
1
1
3
1
1
2
3
3
19
2
1
2
2
2
6
1
4
2
4
4
1
2
4
1
1
1
4.67
0.67
0.67
2.00
0.67
0.67
1.33
2.00
2.00
12.67
1.33
0.67
1.33
1.33
1.33
4.00
0.67
2.62
2.62
2.67
2.67
0.67
1.33
2.67
0.67
0.67
0.67
1
2
9
1
1
4
3
2
2
4
3
22
2
1
2
3
3
16
1
4
2
5
7
1
10
5
1
1
1
5.53
0.46
0.46
1.84
1.38
0.92
0.92
1.84
1.38
10.14
0.92
0.46
0.92
1.38
1.38
7.37
0.46
1.84
0.92
2.30
3.23
0.46
4.61
2.30
0.46
0.46
0.46
3
1
2
4
1
4
2
1
1
1
2
1
6
11
2.00
0.67
1.33
2.67
0.67
2.67
1.33
0.67
0.67
0.67
1.33
0.67
4.00
7.33
3
1
3
5
1
4
2
2
1
1
3
1
21
15
1.38
0.46
1.38
2.30
0.46
1.84
0.92
0.92
0.46
0.46
1.38
0.46
9.68
6.91
1
1
1
1
1
1
150
0.67
0.67
0.67
0.67
0.67
0.67
100.00
1
1
1
1
2
1
217
0.46
0.46
0.46
0.46
0.92
0.46
100.00
Regarding life span of the species, 183 species (84.33 %) were found to be annual as against 34 (15.67 %)
perennial species (Table 1; Fig. 3). Mode of propagation reveals the predominance of seed producing species.
One hundred thirty eight (63.59 %) species were found to propagate only by seeds and spores (in case of
pteridophytes), 66 species (30.41 %) by both seed/spore and vegetative propagules. Only 13 species (5.99 %)
were found to propagate exclusively by vegetative means (Fig. 4). The vegetative propagules were recorded to
be runner, sucker, corm, tuber, offset, bulbil, rhizome, stolon etc. (Table 1).
580

.
Figure 2. A, Acalypha indica; B, Ageratum conyzoides; C, Alternanthera sessilis; D, Amaranthus spinosus; E, Amaranthus viridis;
F, Andropogon ascinoidis; G, Cassia tora; H, Commelina benghalensis; I, Cyanotis axillaris; J, Cyperus bulbosus; K, Cyperus iria;
L, Echinochloa colonum; M, Eclipta alba; N, Eichhornia crassipes; O, Eleusine indica; P, Eragrostis viscosa; Q, Euphorbia hirta;
R, Evolvulus nummularis; S, Ipomea aquatica; T, Leucas plukenetii; U, Ludwigia octavalvis; V, Mikania micrantha; W, Mimosa
pudica; X, Oldenlandia diffusa; Y, Persicaria hydropiper; Z, Ricinus communis; Aa, Scoperia dulcis; Ab, Urena lobata.
581

.
Perennial,
15.67%
Annual,
84.33%
Figure 3. Life span of the species.
Only by
vegetative
propagules,
5.99%
Both by
seeds/spores
and vegetative
propagules,
30.41%
Only by
seeds/spores,
63.59%
Figure 4. Propagation method of the species.
The current work was based on extensive explorations of the agro-climatic zone of the district. Out of the
217 species collected from different locations of the district, 23 species (10.60 %) belong to the life-form class
Phanerophyte, 34 species (15.67 %) to Chamaephyte, 22 species (10.14 %) to Hemicryptophyte, 47 species
(21.65 %) to Cryptophyte and 91 species (41.94 %) to Therophyte (Table 1, 4). The analysis clearly indicates
the total deviation of the biological spectrum from the normal spectrum as proposed by Raunkiaer (Table 4).
Study reveals the Therophytes to have the highest percentage followed by Cryptophytes and Chamephytes. On
the other hand, Hemicryptophytes showed the lowest percentage. Thus, Therophytes are more abundant in the
study area of the district and in contrary; the hemicryptophytes are the rare life form in the study area (Fig. 5).
Table 4. Comparative Biological spectrum.
No. of species in Percentage distribution of the species among Raunkiaers normal

each life form
different life forms (Observed spectrum)
spectrum
Phanerophytes (Ph)
23
10.60
46
Chamaephytes (Ch)
34
15.67
9
Hemicryptophytes (Hm)
22
10.14
26
Cryptophytes (Cr)
47
21.65
6
Therophytes (Th)
91
41.94
13
Total
217
100.00
100.00
Life forms
DISCUSSION AND CONCLUSION

Agro-climatic zones are rich in weed diversity. Apart from the cultivated crops, the zones are good habitat
for a variety of weed species grown throughout the year (Sarma & Bhattacharjya 2006, Bhattacharjya & Sarma
2007, Bhattacharjya & Sarma 2008, Padal et al. 2013, Rana & Masoodi 2013, Dhole et al. 2013, Talukdar
2013). Present study reveals the predominance of annual weed species over the perennial ones. This is due to the
anthropogenic activities including various cropping practices, weeding, collecting food and fodder species and
overall control on the engineered ecosystem to gain maximum output which may reduce the growth of perennial
species. Annual weeds produce very high amount of seeds to ensure propagation and survival. Sufficient amount
of small seeds also ensures high probability of dispersal and re-infestation (Shivakumar et al. 2014). Thus in the
582
% of Species

.
study area, predominance of seed producing annuals is well marked. The dual method of propagation including
seeds and vegetative propagules offer few weed species extra advantages to survive even in the extremes of
environmental conditions.
50
45
40
35
30
25
20
15
10
5
0
Observed spectrum
Raunkiaers normal spectrum
Life forms
Figure 5. Comparison of observed & normal spectrum.
The dominant life forms in biological spectrum of a region indicate the phytoclimate of that region (Yadava
& Singh 1977, Dagar & Balakrishnan 1984, Al-Yemeni & Sher 2010, Reddy et al. 2011, Sharma et al. 2014,
Thakur 2015, Shahid & Joshi 2015). Since the present observation indicates a higher percentage of the
Therophytes, hence, the weed flora of the study area is a therophytic one which, in turn, indicates a therophytic
phytoclimate prevailing in the agro-climatic zone of the district.
The rich therophytic flora is due to the grazing, weeding or other human interference in the area during the
process of cultivation which reduces the number of other life forms. However the human interference has not
affected the dominance of the therophytes as they produce a large number of viable seeds which, in turn, are
able to establish themselves to continue their generations. Although the Hemicryptophytes are able to withstand
various adverse climatic conditions along with the biotic pressure, their lower percentage value (10.14 %) kept
them less significant in the area. This is due to the constant human interference in the area through ploughing,
hoeing, slashing, burrowing etc. associated with the agricultural practices.
It can be concluded that the vegetation of the agro-climatic zone of Nalbari district is mostly seasonal and
annual weeds predominate, majority of which continue to survive in subsequent periods by their seeds or
vegetative propagules and therefore their presence remains almost unchanged. Agricultural practices, grazing,
scrapping by animals, collection of plants for different purposes etc. are the disturbing factors operating in the
area which contribute to higher the number of therophytes and overall deviation of the biological spectrum from
the normal one.
ACKNOWLEDGEMENT
Authors are thankful to Mrs. Kaberi Saikia Das, Head, Dept. of Botany, M.C. College, Barpeta (Assam) for
her encouragement to conduct the work.
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ISSN (E): 2349 1183

ISSN (P): 2349 9265
3(3): 586591, 2016
DOI: 10.22271/tpr.2016.v3.i3.077
Research article
Differential responses of pea seedlings to salicylic acid

under UV-B stress
Chanda Bano, N. B. Singh* and Sunaina
Plant Physiology Laboratory, Department of Botany, University of Allahabad, Uttar Pradesh, India
*Corresponding Author: singhnb.au2016@gmail.com
Abstract: In nature, plants are continuously exposed to solar light. They cannot avoid exposure to
UV-B radiation. The purpose of this investigation was to examine how UV-B radiation affects
seed germination, seedling growth, protein and sugar contents and activities of antioxidant
enzymes in pea (Pisum sativum) seedlings. Salicylic acid mediated physiological responses in UVB stressed pea seedlings. UV-B exposure adversely affected germination and physiology of Pisum
sativum L. Salicylic acid mitigated the impacts of UV-B stress. Seed germination decreased with
increased duration of UV-B exposure. Enhanced activities of antioxidant enzymes in response to
UV-B radiation played a protective role against UV-B radiation.
Keywords: Antioxidants - Oxidative stress - Pisum sativum L. - UV-B radiation - Salicylic acid.
[Cite as: Bano C, Singh NB & Sunaina (2016) Differential responses of pea seedlings to salicylic acid under
UV-B stress. Tropical Plant Research 3(3): 586591]
INTRODUCTION
Plants are exposed to various abiotic and biotic stresses including solar ultraviolet-B (UV-B) radiation in the
natural environment. UVB radiation is the biggest challenge to life forms on the earth. Over the last few
decades degradation of ozone layer leads to enhanced solar UV-B radiation on the earth (McKenzie et al. 2003,
Liu et al. 2013). The damage of the ozone layer caused by human activities may result in increased level of
ultraviolet radiation reaching the earth surface which is harmful for all living beings including crop plants
(Shaukat et al. 2011). UV-B exposure alters biochemical processes in crop plants and affects the morphological
parameters (Casati & Walbot 2003, Zu et al. 2010). The exposure of plant to UV-B damages macromolecules
viz., nucleic acid, lipid and proteins (Ries et al. 2000, Frohnmeyer & Staiger 2003) decreases photosynthetic
rate (Feng et al. 2003) and alters activities of several antioxidant enzymes (Agrawal & Mishra 2009). The UV-B
radiation has adverse impact on growth and decreases productivity of crop plants (Shaukat et al. 2011).
Plant growth regulators mitigate adverse effects of various environmental stresses. Salicylic acid (SA), a
secondary phenolic metabolite, is considered as plant hormone. It is naturally found in plants and acts as a
signaling molecule (Davies 2004, Amin et al. 2013). SA plays an important role in regulating the metabolic
activities of plants (Davies 2004, Amin et al. 2013). SA application enhances the biomass production and yield
in a variety of plants like maize (Amin et al. 2013), wheat (Arfan et al. 2007) under adverse conditions. SA also
activates the defense system of plants to protect them from deleterious impact of abiotic stress.
Pisum sativum L. (pea) is one of the most economically important pulse crops in India because of its high
protein content. Cultivation of pea is common in India due to its ability to fix atmospheric nitrogen. It is used as
soil primer for other crops.
The aim of the present study was to examine the impact of UV-B exposure on seed germination and growth
of pea and the stress responses of SA in UV-B stressed pea seedlings. This study may help regulate the
tolerance of abiotic stress like UV-B by plant growth hormone.
Seeds and chemical
Seeds of Pisum sativum L. cv. Rachana were procured from the Seed Agency at Allahabad, Uttar Pradesh,
586
Received: 04 August 2016

Bano et al. (2016) 3(3): 586591

.
India. Salicylic acid (molecular weight 138.121 g.mol-1) was purchased from Loba Chemie Pvt. Ltd., Mumbai,
India.
Salicylic acid and ultraviolet-B treatments
Salicylic acid was prepared by dissolving a requisite amount of SA in 1.0 mL of ethanol and the volume was
made 100 mL with double distilled water and used for treatment. Seeds were exposed to UV-B radiation for 15,
30, 60 and 90 min. Fluorescent UV-B tube (TL 40 W/12 Philips, Holland) was used for UV-B irradiation. The
UV-B tube was wrapped with cellulose acetate filter (Johnston Industrial Plastics, Toronto, Canada) to avoid all
incidents of UV-C (< 280 nm). The UV-B irradiation intensity was measured with the help of power meter
(Spectra Physics, USA model 407, A-2).
Petri-plate assay
Seeds were soaked in two groups each in 100 mL of SA (0.5 mM). The seeds soaked in double distilled
water (DDW) for 4 hours, separately. After completion of course of time the seeds of the two groups were
further divided into five sets. Out of five, four sets of each group was exposed to UV-B radiation for 15, 30, 60
and 90 min. The complete experimental setup has ten different combinations such as: control (without
treatment), SA (0.5 mM), UV-B1 (15 min), UV-B2 (30 min), UV-B3 (60 min), UV-B4 (90 min), UV-B1+SA,
UV-B2+SA, UV-B3+SA, UV-B4+SA. Ten seeds of each treatment were placed at equal distance in sterilized
petriplates (dia 9 cm, depth 1.5 cm ) lined with double layer of Whatman No. 1 filter papers. Filter papers were
moistened with 5 mL of SA for respective treatments and DDW in control and incubated at 282 C for
germination in growth chamber. The experiment was conducted in replicate of three. Germination was
initiated at 48 hrs after sowing. Germination and seedling growth were recorded till 7 days after sowing at the
interval of 24 hours. Radicle and plumule length was recorded with the help of metric scale.
Protein content
Protein content was determined according to the method of Lowry et al. (1951). The amount of protein was
calculated with reference to the standard curve obtained from bovine serum albumin.
Sugar content
The determination of total soluble sugars (TSS) was done following Hedge & Hofreiter (1962). About 0.1 g
radicle was homogenized in 5 mL 95% (v/v) ethanol. After centrifugation, 1 mL supernatant was mixed with 4
mL anthrone reagent and heated on boiling water bath for 10 min. Absorbance was recorded at 620 nm after
cooling. The amount of sugar was determined by the standard curve prepared from glucose.
Extraction and assay of antioxidant enzymes
Fresh sample of radicle (0.25 g) was homogenized with 0.1 M sodium phosphate buffer containing 1% (w/v)
polyvinyl pyrrolidone (pH 7.0) in a pre-cooled mortar and pestle. The extract was centrifuged at 4 C at 14,000g
for 30 min in cooling centrifuge (Remi instruments C 24). The supernatant was used for the assay of antioxidant
enzymes viz., superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and guaiacol
peroxidase (GPX) activities.
The SOD activity (EC 1.15.1.1) was estimated by the nitroblue tetrazolium (NBT) photochemical assay
following Beyer and Fridovich (1987). The reaction mixture (4 mL) consisted of 20 mM methionine, 0.15 mM
ethylene diamine-tetra acetic acid (EDTA), 0.12 mM NBT and 0.5 mL supernatant. The test tubes were exposed
to fluorescent lamps for 30 min and identical unilluminated assay mixture served as blank. One unit of enzyme
was measured as the amount of enzyme which caused 50% inhibition of NBT reduction.
Catalase (CAT, EC1.11.1.6) activity was assayed following the method by Cakmak & Marschner (1992).
Assay mixture (2 mL) contained 25 mM sodium phosphate buffer (pH 7.0), 10 mM H 2O2, and 0.5 mL enzyme
extract. The rate of H2O2 decomposition for 1 min was monitored at 240 nm and calculated using extinction
coefficient of 39.4 mM 1cm 1 and expressed as enzyme unit mg 1 protein. One unit of CAT was determined as
the amount of enzyme required to oxidize 1 mM H2O2 min 1.
Ascorbate peroxidase (APX, EC1.11.1.11) activities were assayed following Nakano & Asada (1981). Assay
mixture (2 mL) contained 25 mM sodium phosphate buffer (pH 7.0), 0.1 mM EDTA, 0.25 mM ascorbate, 1.0
mM H2O2, and 0.2 mL enzyme extract. H2O2 was the last component to be added. The absorbance was recorded
for 1 min at 290 nm (extinction coefficient of 2.8 mM 1cm 1). Enzyme specific activity was measured as
enzyme unit per one milligram protein as the amount of enzyme required to oxidize 1 mM H2O2 min 1.
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Guaiacol peroxidase (GPX, EC 1.11.1.7) activities were assayed following Hemeda and Klein (1990). The
reaction mixture (2 mL) contained 25 mM sodium phosphate buffer (pH 7.0), 0.1 mM EDTA, 0.05% (v/v)
guaiacol, 1.0 mM H2O2, and 0.2 mL of enzyme extract. The increase in absorbance due to oxidation of guaiacol
was monitored at 470 nm. The enzyme activities were measured using extinction coefficient of 26.6 mM 1cm 1
and expressed as enzyme unit per mg protein.
RESULTS
UV-B decreased seed germination in dose dependent manner. However, in combination with SA gradual
increase in germination was recorded (Table 1). Maximum 53.84% reduction in seed germination was observed
in the pea seedlings exposed in UV-B for 90 min. Growth was measured in terms of radicle and plumule length.
UV-B radiation induced impact on growth of pea seedlings was measured and results are presented in tables 1.
UV-B significantly declined the radicle and plumule length which was concentration dependent. Maximum
59.11% and 90.66% decrease in radicle and plumule length in the pea seedlings over control was recorded
respectively in 90 min single UV-B treatment. Application of SA with UV-B exposure exhibited positive effects
on radicle and plumule length of the seedlings.
Table 1. Mitigating effects of salicylic acid on UV-B stressed Pisum sativum L.
Plumule length
Protein
Sugar
(cm)
(mg.g-1 FW)
(mg.g-1 FW)
C
97.50.21a
9.050.31b
3.750.02b
21.470.05b
29.660.26b
S
100.00.23a
10.30.57a
4.41.0a
25.801.48a
32.800.29a
UV1
82.50.33b
7.850.20c
2.050.60bc
16.340.36c 28.200.53bcd
UV2
72.51.43d
7.10.17c
1.650.83c
9.331.48d
26.330.26de
UV3
57.51.33f
5.20.05de
1.250.54de
6.030.014e 24.100.27fg
UV4
45.00.76g
3.70.34f
0.350.14e
4.230.014f
20.670.26h
UV1+S
85.00.32b
8.10.41c
3.40.28abc
19.341.45b 29.160.02bc
UV2+S
77.51.43c
7.850.02c
2.60.80cd
10.661.47d 27.640.08cde
UV3+S
62.501.3e
6.10.23d
2.70.11cd
7.950.34e
25.720.20ef
UV4+S
55.00.67f
4.350.54ef
0.550.25e
5.750.08f
22.590.32g
Note: MeanSE values followed by same letters within each column are not significantly different at 0.05
(ANOVA and Duncans multiple range test), n =3. C= control, S= 0.5mM concentration of salicylic acid, UV 1=
15, UV2 = 30, UV3 = 60 and UV4 = 90 min treatment of ultraviolet-B radiation, UV1+S, UV2+S, UV3+S and
UV4+S are combined treatments of UV-B and salicylic acid.
Treatments
Seed germination %
Radicle length (cm)
The results pertaining to protein and sugar in seedlings are depicted in table 1. The increase in duration of
UV-B radiation from 15 min to 90 min caused progressive decrease in protein and sugar contents. A steep
decline of 80.29% protein and 30.33% sugar in pea was recorded following 90 min of UV-B exposure.
The seedlings showed significant increase in activities of antioxidant enzymes viz., SOD, CAT, APX and
GPX following UV-B exposure from 15 to 90 min (Fig. 1). The seedlings exposed to UV-B alone exhibited an
increase in the activities of SOD, CAT APX and GPX by 59.78%, 88.76%, 81.56% and 62.80% respectively
over the control. Simultaneous treatment with SA and UV-B caused significant improvement in the activities of
SOD, CAT, APX and GPX. SA manifested positive impact on UV-B stressed seedlings to avoid oxidative
damage caused by UV-B exposure.
DISCUSSION
UV-B radiation is one of the important abiotic stress factors manifesting significant impact on growth and
physiological processes in plants (Mishra et al. 2009, Dwivedi et al. 2015). This problem is aggravated due to
further increase in UV-B radiation in troposphere. Previously it has been demonstrated that SA priming reduced
susceptibility towards different kinds of abiotic stresses by modulating defense system (Horvath et al. 2007)
Decline in seed germination under UV-B exposure was reported (Shaukat et al. 2011).Our results are in
agreement with the previous findings of Shaukat et al. (2011) on Helianthus annuus. SA in low doses
significantly increased seed germination in Arabidopsis under different abiotic stresses (Rajjou et al. 2006). The
positive effect of SA on seed germination under abiotic stress is due to decreased level of oxidative damage
(Peykarestan et al. 2012) and it also initiates translation and elongation factors, proteases and two subunits of
the 20S proteasome which consequently revamp seed germination by encouraging protein synthesis that are
indispensable for seed germination and the movement or protein breakdown gather during the course of seed
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.
4
10
a
a
b
b
c
e
f
CAT(EU g-1 FW )
SOD(EU g-1 FW )
bc
de
cd
ef
f
2
0
0
1
UV
2
UV
3
UV
4
S
S
S
S
UV V1+ V2+ V3+ V4+
U
U
U
U
14
1
UV
2
UV
3
UV
4
S
S
S
S
UV V1+ V2+ V3+ V4+
U
U
U
U
14
ab
abc
10
abcd
abcde
abcde
bcde
ab
12
GPX(EU g-1 FW )
APX(EU g-1 FW )
12
ab
abc
10
bc
bc
cde
de
bc
c
e
4
1
UV
2
UV
3
UV
4
S
S
S
S
UV V1+ V2+ V3+ V4+
U
U
U
U
Treatments
Treatment
1
UV
2
UV
3
UV
4
S
S
S
S
UV V1+ V2+ V3+ V4+
U
U
U
U
Treatments
Treatment
Figure 1. Mitigating effects of salicylic acid on antioxidant enzyme activities of UV-B stressed Pisum sativum L. MeanSE
values followed by same letters within each column are not significantly different at 0.05 (ANOVA and Duncans multiple
range test), n =3. C= control, S= 0.5mM concentration of salicylic acid, UV1= 15, UV2 = 30, UV3 = 60 and UV4 = 90 min
treatment of ultraviolet-B radiation, UV1+S, UV2+S, UV3+S and UV4+S are combined treatments of UV-B and salicylic
acid.
maturation and also in the synthesis of various enzymes used in several metabolic pathways like gycolysis,
glyoxylate cycle, pentose phosphate pathway, and gluconeogenesis (Rajjou et al. 2006). The results of the
present study showed that UV-B alone decreased growth of pea seedlings in dose-dependent manner. The
declined growth in Helianthus annuus exposed to UV-B is reported (Shaukat et al. 2011). In contrast, SA
priming of seedlings /plants ameliorated the toxic effect of UV-B and promoted growth as compared to the pea
seedlings exposed to UV-B treatments alone. SA pretreatment may mask impact of the UV-B response of the
seedlings by modulating the antioxidant system. UV-B induced reduction in protein content of pea at higher
duration of UV-B exposure may be linked with UV-B induced disturbance in protein synthesis. Like protein
content we recorded reduced sugar content under UV-B stress. Seed germination and early development of
seedlings exploit their storage food material (Rosa et al. 2009) and when plants are exposed to stress, some
protective mechanisms can also form in plants and it requires energy and resources and the resource of energy in
plants is mostly sugar (Ho 1988). Similar to our results decreased sugar content observed in maize (Correia et
al. 2005) Different metabolic pathways in plants continuously produced reactive oxygen species as byproduct.
Plants exposed to increased UV-B radiation induced ROS accumulation which includes not only free radicals
such as superoxides but also hydrogen peroxide and singlet oxygen which caused oxidative damage to nucleic
acid and proteins (Foyer et al. 1994) and metabolic system. The plants have efficient antioxidative defense
system to mitigate the harmful impact of oxidative stress induced by UV-B stress. The plant defense system
provides protection against free radicals and reactive oxygen species.SA pre-treatment alleviates the adverse
effect of UV-B by modulating activity of antioxidant (Horvath et al. 2007). The activities of antioxidant
enzymes induced by UV-B exposure varied with plant species. Generally, activities of antioxidants like SOD,
CAT, APX and GPX were found to be increased by UV-B radiation (Mishra et al. 2009, Dwivedi et al. 2015).
SA treatment inhibits oxidative damage by modulating antioxidant enzymes activity and detoxifies superoxide
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Bano et al. (2016) 3(3): 586591

.
radicals (Farooq et al. 2008). SOD present in different part of cell acts as first line of defense in plant. It acts
first on free radicals and converts to hydrogen peroxide, CAT in peroxisomes and APX in the cell as whole
have potential to convert hydrogen peroxide into water and oxygen (Noctor & Foyer 1998). Kondo and
Kawashima have also demonstrated the enhanced antioxidant activity to UV-B exposure (Kondo & Kawashima
2000). The growth of plant is the result of equilibrium between ROS produced and antioxidant enzymes
induced. Enhanced activities of antioxidant evinced the plant tolerance against UV-B radiation. Several studies
reported the effect of UV-B and salicylic acid on plants but here we studied the effect of UV-B and salicylic
acid on seed germination and metabolic activity of pea seedlings.
CONCLUSION
The present study showed that UV-B exposure exhibited adverse effects on seed germination, seedlings
growth of Pisum sativum and their defence system. The seed priming with salicylic acid, as plant growth
regulator, in appropriate concentration alleviate the adverse effects of UV-B radiation in pea seedlings.
ACKNOWLEDGMENTS
The authors are thankful to the UGC, New Delhi and University of Allahabad, Allahabad for providing
financial assistance to Chanda Bano.
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ISSN (E): 2349 1183

ISSN (P): 2349 9265
3(3): 592599, 2016
DOI: 10.22271/tpr.2016.v3.i3.078
Research article
Exotic species invasion threats to forests: A case study from the

Betla national park, Palamu, Jharkhand, India
Preeti Kumari and A. K. Choudhary*
Department of Botany, Ranchi University, Ranchi-834002, Jharkhand, India
*Corresponding Author: ashokchoudhary.ru@gmail.com
Abstract: Exotic Species Invasion (ESI) cause a little recognized, but very substantial impact to
forest ecosystems worldwide. Climatic variability, physiographic range, increasing trade, travel
and tourism have accelerated the spread of unwanted non-native species to conservation areas,
making vulnerable to the establishment of ESI. Exotic Invasive Plants (EIPs) are known to
displace native plants, alter ecosystems processes, hydrology, primary productivity, nutrient
cycling and soil structure and most importantly reduce native biodiversity. There is evidence to
suggest that the threats due to ESI may increase with climate change and associated changes in
habitats. In this paper, we assess the threat of EIPs to natural forests in Betla National Park (BNP),
Palamu in Jharkhand State, India. Based on intensive field surveys and using quadret method we
identified 142 EIPs in the BNP forest. 21 plots of 20 20 m for trees, 5 5 m for shrubs and 1 1
m for herbs were laid randomly adjoining the forest at 10 to 100 m distance from the road and
settlement area. Total 14 EIPs were recorded among which Lantana camara and Parthenium
hysterophorus were found to be the most dominant species. . The survey revealed that apart from
the ecological harm, invasive plants adversely affect the livelihood of all those who are dependent
on forests. The paper identifies impact, early detection and rapid control, prevention of spread and
habitat restoration as urgent measures for combating the threats.
Keywords: Exotic invasive plants - Risk assessment - BNP forest - Climatic change impact.
[Cite as: Kumari P & Choudhary AK (2016) Exotic species invasion threats to forests: A case study from the
Betla national park, Palamu, Jharkhand, India. Tropical Plant Research 3(3): 592599]
INTRODUCTION
Exotic Species that become invasive are considered to be main direct drivers of biodiversity loss across the
globe. Management of Exotic Species Invasion (ESI) is seen as major challenge in the field of biodiversity
conservation. ESI, the non-native species threaten ecosystems, destroy habitats and create problems to other
native species through invasion. It is considered as the second greatest agent of species endangerment and
extinction. The ecological cost is often the irretrievable loss of native species and ecosystems. It also causes
heavy economic loss, in terms of reduced crop and livestock production, reduced native biodiversity, increased
production costs and so forth. Biodiversity has become one of the most popular topics for discussion at local,
national and global level. Biodiversity entails all forms of biological entities inhabiting the earth including
prokaryoteswild plants and animals, microorganisms, domesticated animals and cultivated plants and even
genetic material like seeds and germ-plasma (Kothari 1993). Exotic Invasive Species (EIS) are species, native to
one area or region, that have been introduced into an area outside their normal distribution, either by accident or
on purpose and which have colonized or invaded their new home, threatening biological diversity, ecosystems
and habitats, and human wellbeing (CBD 2000). Biological invasion worldwide threatens biodiversity,
ecosystem dynamics, resource availability, national economy and human health studied by (Ricciardi et al.
2000). The spread of EIS is now recognized as one of the greatest threat to the ecosystem. Exotic invasive
species (animal pests, viruses, pathogens and plants) have become one of the most serious threats to the
ecological and economic well-being of every habitat and region on the Earth (Boy et al. 2013). The introduction
of alien species to a new location can either be accidental or intentional studied by (Enserink 1999). Accidental
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Kumari & Choudhary (2016) 3(3): 592599

.
introductions are helped by travel across countries and continents and import of various items such as timber,
food grains, fodder etc. (Shimono et al. 2008). Intentional introductions are for a variety of purposes such as
agriculture, horticulture, forestry and ornamental (Cremer 2003). All invasive species possess certain biological
attributes which contribute to their success as invaders in a new habitat. For invasive alien plants (IAPs), these
attributes include production of a large number of easily dispersible, light weight seeds, fast growth rate and
better competitive resource capture and utilization abilities compared to native plants (Burns 2006). The
economic damage due to ESI is estimated to be to the tune of 1.4 trillion dollars globally. In many countries, the
overall loss due to invasions is over 1% of the GDP. In the United States alone, for example, the annual costs of
containing the spread of IAS are reported to be more than US$ 135 billion (Boy et al. 2013). The impacts of
IAPs include displacement of native plant species, change of soil chemical profile, rewarding pollinators better
than the native species thereby reducing the reproductive success of local species, changing hydrological
regimes, making the new habitat fire prone and limiting the photosynthetic efficiency of the local species by
reducing light availability (Nilsson & Grelsson1995). Subsequent impacts would happen by reduced availability
of forest resources like medicinal plants from natural forests and timber from forest plantations. As in the
classical case of Kaziranga National Park (Assam, India) where in the movement of the endangered one horned
rhinoceros was limited by thickets of Mimosa diploticha var. diplotricha, the impact on fauna would also be
critical studied by (Vattakkavan et al. 2002). Indirect impacts occur by way of complete elimination of food
plants of the fauna and by making the habitat fire prone. It was believed that the threat of IES would be much
low in natural habitats as compared to disturbed habitats. Forests were considered to be immune to large scale
plant invasions. Climate change and the emergence of invasive alien plant species (IAPS), which are commonly
referred as weeds, are two of the greatest threats to biodiversity and ecosystem services (Burgiel & Muir 2010,
IUCN 2000) defines IAPS as plants that have become established in natural or semi-natural ecosystems or
habitats, an agent of change, and threatens native biological diversity. A study of (IPCC 2007) identified that
climate change is one of the factors for emergence of invasive species. Increase in atmospheric temperatures and
CO2 concentrations are likely to increase opportunities for the introduction of invasive species because of their
adaptability and ability to disturb a broader range of bio geographic conditions and environments (Mooney
2000). A study by (Lodge et al. 2006) showed that IAPS endanger the environment, the economy and human
welfare. It also reduces biodiversity, replaces important native species and increases investment in agriculture
and silviculture operations (Ricciardi et al. 2000) and disrupts prevailing vegetation dynamics and nutrient
cycling (Richardson & Higgins 1998). The estimated damage from IAPS worldwide totals more than US $1.4
trillionna year (5 percent of the global economy). Impacts affect a wide range of sectors including agriculture,
forestry, aquaculture, transportation, trade, energy and recreation (Stern 2006).
The prime objective of the present paper is to report the Exotic Species Invasion (ESI) Threats to Forests in
Betla National Park (BNP) Palamu, Jharkhand, India. We have also reported effect of ESI on different plant
species, environment and different ecosystem etc. In addition to the above the preventive measures of ESI of
National Park have been also studied.
MATERIALS AND METHODOLOGY
Study Area
Betla National Park (BNP) situated between latitude 2325' N to 2355' N and longitude 8350' to 8436' E,
was notified in 1973 as one of Indias first nine tiger reserves established under Project Tiger . It is located in
the western part of the Chhotanagpur Plateau and spans an area of 1129.93 km2 comprising the Palamau
Wildlife Sanctuary and Betla National Park is spread over Latehar, Palamau and Garhwa District in Jharkhand
(Fig. 1). It is also part of the Central India Landscape and extends into the Sanjay-Dubri Tiger reserve and
Achanakmar-Kanha tiger landscape through the Jashpur and Mahan forest of Chhattisgarh. The vegetation types
mainly categorized as dry moist forest, dry Sal forest, moist Sal forest, high level plateau Sal forest and moist
forest. BNP is also becoming home to many unwanted non-native plants. Very limited research has been carried
out about ESI in BNP.
Data collection
Questionnaire survey was carried out from October 2015 to June 2016. Total 140 individuals who have long
been inhabited in the study area and utilizing the local resources for their livelihood were interviewed to explore
their perception regarding ESI. Total 21 sampling plots of various sized quadrates 20 20 m for trees, 5 5 m
593

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for shrubs and 1 1 m for herbs were laid at 10 to 100 m distance in adjoining forest from road and human
settlement area. Nested plots 5 5 m and 1 1 m quadrates were allocated randomly in two corners of 20 20
m plot (Tiwari et al. 2005, Bajpai et al. 2015). Community consultations, individual interviews, field
observations, literature review, group discussions were conducted to collect data.
Figure 1. Study Area of Betla National Park, Palamu, Jharkhand, India.
Data analysis
Both quantitative and qualitative techniques were used for data analysis. By using following method density
and frequency were calculated. The analysis was interpreted in a simple and understandable chart form. Density
is an expression of numerical strength of a species in a community studied by using formula (Mishra 1968).
( )
Frequency denotes the number of sampling unite in which, a particular species occurs and so it expresses the
distribution of dispersion of various species in community.
( )
ESI in study area
All together 14 EIPs were encountered in the sampled areas (Table 1). Among the ESI observed, Lantana
camara & Parthenium hysterophorus was most common in the study area. It has the highest cover in the
adjoining forest, near settlement where human disturbances were high. Based on household survey Lantana
camara & Parthenium hysterophorus was found to be the most problematic ESI. An area where tree canopy is
dense and the undergrowth do not find sufficient sunlight, invasion of species is low compared to open and
degraded land. Therefore, with increasing tree canopy there is decreasing invasion of unwanted species. After
Lantana camara & Parthenium hysterophorus, Agertum houstonianum and Ipomoea purpurea, Ipomoea
hederfolia were other to ESI found with high density in the study area. The spread of EIPs especially
Perthanium hysterophorus complex and is threatening both the natural biological richness and livelihood of
inhabitants. Many locals have stopped grazing their livestocks in forest as the palatable grasses in the forest like
Imperata cylindrical, Cynodon dactylon are rapidly being replaced by the ESI especially by Perthanium
hysterophorus.
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Table 1. Exotic Species Invasion (ESI) Observed in Betla National Park, Palamu, Jharkhand, India.
Specie
Alternanthera brasiliana (L.) Kuntze
Alternanthera philoxeroides (Mart.) Griseb.
Amaranthus spinosus L.
Ipomoea purpurea (L.) Roth
Ipomoea hederifolia L.
Jatropha gossypifolia L.
Lantana camara L.
Leucaena leucocephala (Lam.) de Wit
Merremia vitifolia (Burm.f.) Hallier f.
Mimosa pudica L.
Parthenium hysterophorus L.
Prosopis juliflora (Sw.) DC.
Sphagneticola trilobata (L.) Pruski
Synedrella nodiflora (L.) Gaertn.
Introduction
Intentional
Unknown
Accidental
Intentiona
International
Intentional
Intentional
Intentional
Accidental
Intentional
Accidental
Intentional
Intentional
Accidental
Purpose
Ornamental
Unknown
NA
Ornamental
Ornamental
Hedge plant
Ornamental
Social forestry
NA
Ornamental
NA
Fire wood
Ornamental
NA
Habitat
Subshrub
Herb
Herb
Climber
Climber
Shrub
Shrub
Tree
Climber
Herb
Herb
Tree
Herb
Herb
Origin
Central & South America
South America
South & Central America
Central America
Cenral America
Tropical America
Mexico & Central America
South Asia
Tropical America
North & South America
South America & West Indies
Tropical America
Effect of Exotic Species Invasion

Effects on ecosystem
After studies we found that invasive exotic plant species (IEPS) threaten the environment, reduce
biodiversity, replace economically important plant species and increase the investment in agriculture and
silviculture practices, prevail vegetation dynamics and alter nutrient cycling (Richardson & Higgins 1998). They
can promote hazards like forest fire. Plant invasions dramatically affect the distribution, abundance and
reproduction of many native species (Sala et al. 1999). In the study area too, impacts of Invasive Exotic Plant
species especially Parthenium hysterophorus was well observed and none of the ecosystems were free from
their impact. Edges of forests, agricultural lands and wetlands have been severe IEPS intrusion. Although all
ecosystems are susceptible to invasion, ecosystems entwined with higher level of human interventions (e.g.
forestry, agriculture, wetland and rangelands) are likely to pose greater susceptibility (Yelenik et al. 2007). In
the study site, rangeland, agriculture land as well as fallow lands and roadsides were highly susceptible to
invasion of Ipomoea purpurea (L.) & Ipomoea hederifolia L. IEPS (Fig. 2).
Both IEPs
Lantana
Perthanium
20
18
Number of site observed
S.N.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
16
14
12
10
8
6
4
Forest
Agriculture Grazing land

Wetland
Road side
Different types of ecosystem
Figure 2. showing impact of invasive exotic plant species on important ecosystem.
Effects on Forest
As per surveys revealed that the forest, roadsides and fall low lands previously dominated by Alternanthera,
Ipomoea, Jatropha, Lantana etc. were invaded by IEPS. Some plants failed to maintain their biomass in the
changing climate and their dominance was fairly slacked off by IEPS. As a result, dense and diverse forests are
595

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more resistant to ecological invasion (Pimm 1984). However, forests in studied area has invaded about 20% area
particularly forest edges and plantation forests of the studied ecosystem. These species suppress the growth of
native trees, shrubs and grasses growing beneath or close to them. Subsequently foliage for wildlife animals is
reduced leading to starvation and death of the animals.
Effects on human ecosystem
The full costs of invasions also include the social and health impacts of exotic invasive species on humans,
in particular to the rural communities depending on forests. As a result of the impacts of exotic invasive species
on native forest biodiversity, a loss of food sources and traditional medicines may be experienced thereby
compromising not only the health of local people but also the livelihoods of those dependent on the collection
and sale of such items for income. For small-scale landowners, exotic invasive species can also decrease the
value of their land. Forest workers, as part of their jobs, and people living in and around forests are more
exposed to exotic invasive species such as the reservoirs and hosts of many emerging infectious diseases.
Examples of such diseases include Lyme disease, hay fever also known rhinitis, some immune disorders,
eczema, Ebola and Marburg hemorrhagic fevers, malaria, yellow fever, leishmaniasis, trypanosomiasis and
Kyasanur forest disease. People living in and around invaded forest areas may also suffer allergic or other
negative reactions to the exotic invasive species themselves or to the measures used to control them such as
chemical and biological pesticides. A commonly planted tree for land restoration and as a source of forest
products, mesquite (Prosopis juliflora) is a major cause of allergies in India. Sensitivity to mesquite pollen has
been shown to result in asthma, rhinitis and conjunctivitis. In some places, children living close to areas infested
with Dendrolimus sibiricus have experienced significant allergic reactions to the hairy caterpillars that have
entered their homes. The hairs on larvae and egg masses of gypsy moth (Lymantria dispar) also cause allergies
in some people. All these invasive plants and trees have had serious socio-economic impacts and ultimately
increased poverty in the local communities. Some area of the study site is densely populated by subsistence
farmers and livestock rearing is an integral part of their livelihood.
30
PNS
PIS
25
20
15
10
5
Achyranthus aspera
Anagallis arvensis
Ageratum conyzoides
Allium cepa
Alternanthera paronichyoides
Argemone mexicana
Blumea sp.
Borreria stricta
Cajanus cajan
Chrysophogon aciculatus
Commelina benghalensis
Cynodon dactylon
Desmodium triflorum
Dichanthium sp.
Dichanthium sp2
Digitaria ciliaris
Dolichos lablab
Heteropogon contortus
Leucas aspera
Lindernia crustata
Parthenium hysterophorus
Raphanus sativus
Solanum surratense
Tridax procumbans
Triticum astevum
Vernonia cinneria
Xanthium strumarium
Zea mays
Figure 3. Reperesenting the comparative account of parthenium (exotic species invasion) non invaded and invaded study
sites.
Impacts on Wetland and Rangeland Ecosystems

P. hysterophorus and I. purpurea is distributed throughout the study site; it can be just in any type of soil
and environment. Nearby wetlands and throughout the rangeland is the most favourable environment for the
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invasion of invasive species. Apart from those species, water bodies of the study area were invaded by
Jalkumbhi (Pistia stratiotes L.) and unwanted water favoring plants. I. purpurea mostly prefer damps areas such
as wetland margins, drainage lines and gullies.
Impact on Agricultural Ecosystem
IEPS are also considered biological polluters and are capable of hybridizing with native plant relatives that
result in genetically modified to a plants genetic make-up results into great peril of biodiversity, which has also
same impact in the study site. Parthenium hysterophorus has adverse impacts on most of the agricultural crops
as nutrients and fertilizers supplied to the main crop are being exploited by this species (Fig. 3).
Agricultural crops, particularly ginger, millet, rice and grasses, were outcompeted by others and their
productivity declined. Reduction in production of cereals and grasses in studied area as a result of invasion by
Lantana, Perthanium. According to (Oerke et al. 1995) there was a loss of 13 percent of agricultural outputs due
to weeds. Many grasses species, such as Artemisia spp, Solanum xanthocarpum and Urtica sp. of fallow lands
and Scrophularia species, Hypoxis aurea etc. of agricultural lands were threatened by invasion of Perthaniums,
Mimosa, and Sphagneticola invasive exotic plant species in the study site. Impact of P. hysterophorus on
livestock is more severe in the study site. There are a number of livestock mortalities, particularly of buffaloes.
The cases generally happened in the spring when the plant flowers are in full bloom. Similarly, IEPS affect the
dynamics and composition of soil and have impacts on ecosystem functions, such as soil nutrient cycling and
soil chemistry. In the study site, IEPS were growing in a wide range of soils but not flourishing in shade. Soil
texture in agriculture land of the study area is silt clay.
Methods to control of Exotic Species Invasion
The following steps are proposed to manage existing EIPs and prevent any new incursions.
Preventive measures
This study revealed the presence of invasion exotic plants in all the forest areas surveyed. It also showed
direct and indirect impacts due to these invasions. It is recommended that a more comprehensive forest
surveillance covering all the forest divisions in the State needs to be carried out before evolving proper control
strategies. To prevent new incursions of ESI into forests, the following steps are to be adopted:
i. All plants, plant prop gules and soil intended for transportation into forest areas (soil for civil works,
seedlings of forest tree species) should be thoroughly monitored for the presence of seeds and other prop gules
of ESI.
ii. Import of seeds, seedlings and other prop gules of all plant species should be done only after risk
assessment and observing proper quarantine procedures.
iii. Forest areas, especially those which are tourist destinations, need to have water filled dips at the entry
point so as to wash agricultural implements and tires of vehicles free of ESI propagules before entering into
forest areas.
Early detection and rapid control
The most economical way to contain ESI is to establish an efficient surveillance system so as to detect ESI
soon after their arrival and eradicate them when their population is small and the spread is limited. To achieve
this, sea ports, airports and tourist and pilgrimage routes into forest areas are to be monitored regularly for new
invasive species using proper tools and methods. The staff of quarantine/customs and forest department need be
trained to identify ESI which are potential threats so as to adopt measures to stop incursions and contain the
population.
Prevention of spread
For ESI which have already established in some areas and immediate eradication is difficult, efforts should
be focused on preventing their spread by:
1) Restricting the movement of soil and plant parts from infested areas to un-infested areas and
2) Removing the weeds manually or mechanically (cutting or pulling) before flowering and fruiting and
burning them at the site.
Habitat restoration
Manual/mechanical control may be difficult, costly and unsustainable for exotic weeds which have
established in large areas. In such cases, systematic restoration strategies should be taken up. To achieve this,
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remove the weeds manually or mechanically (pulling along with roots/tubers) in small areas at a time and
subsequently plant the area with fast growing native species. Assisted regeneration may also be attempted in
such areas.
CONCLUSION
Exotic Species Invasion (ESI) is a serious threat to forests of Jharkhand since they impact heavily on native
biodiversity, productivity and result in landscape level changes. In this observation we have found that total 14
IEPs in covered area and some species like Lantana, Perthanium and Ipomoea purpurea are highly dominated
over native resources. The invasion is more serious in agriculture land followed by disturbed sites, such as
newly constructed earthen roads. The invasion is seriously affecting agriculture productivity. A few households
are using these species for making compost manure and producing bio-briquettes for energy. Promoting biobriquette production and market linkage would help to improve the health condition of different ecosystems and
provide additional income to local community. Furthermore, it supports controlling forest degradation and loss
of biodiversity. The problem demands urgent attention. Prevention of new incursions can be achieved by
adopting risk assessments before import of plants and planting material. Forest surveillance, early detection and
rapid control, manual/mechanical removal of weeds followed by planting of native species and assisted
regeneration are suggested as immediate steps to control invasion and reduce impacts. Herbicidal application in
forest areas need to be avoided as far as possible.
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Lodge DM, Williams S, MacIsaac HJ, Hayes KR, Leung B, Reichard S, Mack RN, Moyle PB, Smith M, Andow
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Mishra R (1968) Ecology Work Book. Oxford and IBH Company, New Delhi, India pp. 244
Mooney HA (2000) Invasive Species in a Changing World. Island Press, Washington DC.
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677692.
Oerke EC, Dehne H-W, Schnbeck F & Weber A (1995) Crop Production and Crop Protection: Estimated
Losses in major Food and Cash Crops. Elsevier, Amsterdam and New York.
Pimm SL (1984) the complexity and stability of ecosystems. Nature 307(5949): 321326.
Ricciardi A, Steiner WWM, Mack RN & Simerloff D (2000) Towards a global information system for invasive
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Richardson DM & Higgins SI (1998) Pines as invasion in the Southern hemisphere. In: Richardson DM (ed)
Ecology and biogeography of Pinus. Cambridge: Cambridge University Press.
Sala OE et. al. (1999) Global change, biodiversity and ecological complexity. In: Walker B, Steffen WL,
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Shimono Y & Konuma A (2008) Effects of human-mediated processes on weed species composition in
internationally traded grain commodities. Weed Research 48(1): 1018
Stern N (2006) The Economics of Climate Change. HM Treasury, London, UK.
Tiwari S, Adhikari B, Siwakoti M & Subedi K (2005) An inventory and assessment of invasive alien plant
species of Nepal. IUCN, The World Conservation Union, Nepal.
Vattakkavan J, Vasu NK, Varma S, Gureja N & Aiyadurai A (2002) Silent stranglers: Eradication of Mimosa in
Kaziranga National Park, Assam. Wildlife Trust of India, New Delhi.
Yelenik SG, Stock WD & Richardson DM (2007), Functional group identity does not predict invader impacts,
Differential effects of nitrogen-fixing exotic plants on ecosystem function. Biological Invasions 9(2): 117
125.
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ISSN (E): 2349 1183

ISSN (P): 2349 9265
3(3): 600605, 2016
DOI: 10.22271/tpr.2016.v3.i3.079
Research article
Disease progression in potato germplasm from

different reaction groups against potato virus Y in relation
to environmental factors
Ata-ul-Haq1, Yasir Iftikhar1, Muhammad Irfan Ullah2, Mustansar Mubeen3*,
Qaiser Shakeel3, Faheema Bakhtawar1 and Iram Bilqees1
1
Department of Plant Pathology, University College of Agriculture, University of Sargodha, Pakistan

Department of Entomology, University College of Agriculture, University of Sargodha, Sargodha, Pakistan
3
State Key Laboratory of Agricultural Microbiology and Key Laboratory of Plant Pathology of Hubei Province,
Huazhong Agricultural University, Wuhan 430070, China
2
*Corresponding Author: mustansar01@yahoo.com
Abstract: Germplasm of 10 potato varieties/lines were screened out against PVY. Out of 10, five
(Tota-704, FD 71-1, FSD WHITE, FD 8-1, FD 76 24) were found susceptible, three (FD 74-41,
FD 74-50 and N-96-25) moderately susceptible and two (Kuruda and SH 216 A) moderately
resistant varieties. The mean incidence of PVY was 61%. ELISA confirmed virus in the samples
from all varieties showing moderately yellow to yellow color in the ELISA plate. There was
significant correlation between PVY and maximum, minimum temperature and relative humidity.
Disease severity was maximum in the range of 2029C for maximum temperature, 56C for
minimum temperature in all three groups of Varieties. 8283% relative humidity favors the disease
severity.
Keywords: Correlation - Disease severity - Potyvirus - Relative humidity - Temperature.
[Cite as: Ata-ul-Haq, Iftikhar Y, Ullah MI, Mubeen M, Shakeel Q, Bakhtawar F & Bilqees I (2016) Disease
progression in potato germplasm from different reaction groups against potato virus Y in relation to
environmental factors. Tropical Plant Research 3(3): 600605]
INTRODUCTION
Potato (Solanum tuberosum L.) is an important vegetable crop all over the world. It is a staple food in
different part of the world because of its high nutrition value. The production of potato is threatened by different
biotic and abiotic factors not only in the world but also in Pakistan Qamar et al. (2015). Potato has very low
production in Pakistan as compared to world production due to biotic and abiotic factors. Among those biotic
factors viruses are the most devastating pathogens. Potato crop is affected by 40 viruses Valkonen (2007). In
Pakistan, Eight potato viruses viz., Potato virus Y (PVY), Potato virus X (PVX), Potato leaf roll virus (PLRV),
Potato mop top virus (PMTV), Potato virus S (PVS), Potato virus A (PVA), Alfalfa mosaic virus (AMV) and
Potato virus M (PVM) have been reported Mughal et al. (1988). Among these PVY, PVX and PLRV are widely
distributed in Pakistan. PVY alone or along with PVX cause a huge loss to potato crop. Potato virus Y belongs
to family Potyviridae, which contains economically most important and largest group of plant viruses Buchen &
Osmend (1987). Ahmad et al. (2003) during a survey declared the PVY, PVX and PVS as major diseases in
fields of potato. Losses due to PVY are up to 83% in Pakistan Mughal & Khalid (1985) and Khalid et al. (2000).
Potato virus Y is ssRNA Potyvirus having different strains; PVYN, PVYO and PVYC de Box & Huttinga (1981).
It is transmitted mechanically and through insect vector in non-persistent manners. Green peach Aphid (Myzus
persicae Sulzer) plays vital role in disease transmission. Environmental factors play an important role in disease
development and their information will not only be helpful in understanding importance of epidemiological
factors but also in formulating management strategies. Ahmad et al. (2011) studied the relation of environmental
factors (rainfall, temperature and humidity) with incidence of PVY and PVX. They concluded that rainfall and
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high humidity help to spreading of incidence. Environmental factors have positive relation with PVY and PVX
incidence. PVY have significant and non-significant result at high and low temperature but it is negatively
correlated Qamar et al. (2003). The current research work was carried out to monitor the disease incidence of
PVY and influence of environmental factors on disease severity.
MATERIALS & METHODS
Screening of Potato germplasm
The research trial comprising of 10 potato varieties was planned to screen the potato germplasm against
potato virus Y (PVY). Symptoms were observed carefully for screening using the disease rating scale Qamar et
al. (2003) with slight modification (Table 1). Disease incidence was calculated as follows;
Percent disease incidence (%) =
Table 1. Rating scale for the screening of potato germplasm.
Rating Scale
0
1
2
3
4
Response
No visible symptom
Mosaic pattern starts on leaves( 25% leaves showing symptoms)
Mosaic and Mottling (50% leaves have symptoms)
Dwarfing, rugosity and mottling of leaves (75% leaves affected)
Leaf drooping severe mosaic and mottling (100)
Reaction
Immune
Resistance
Moderately resistance
Moderately susceptible
Susceptible
Enzyme Linked Immunosorbent Assay (ELISA)

Samples from 10 varieties on the basis of symptoms were collected and confirmed through DAS-ELISA by
Iftikhar et al. (2009). Polyclonal antibodies diluted in coating buffer were coated in ELISA plate and incubation
at 4C for 24 hours was followed by washing for three times at five minutes interval. After washing wells of
ELISA plate were charged with antigens. Incubation and washing were repeated prior to add conjugate
antibody. After incubation and washing pNP (para nitro phenyl) was added as substrate. Reaction was observed
visually after the incubation of 3060 minutes at room temperature.
Correlation of Environmental factors
Environmental factors (Maximum and minimum temperature, rainfall and relative humidity) were recorded
for crop duration (OctoberJanuary).
Statistical Analysis
Correlation between environmental factors and disease severity in different varieties was analyzed by using
R for Windows.
RESULTS
Screening and Incidence
Incidence (%)
80
70
70
63
63
60
FSD
WHITE
FD 8-1
63
60
60
63
60
57
57
50
40
30
20
10
0
Tota-704 FD 71-1
FD 76 24 FD 74-41 FD 74-50 N-96-25
Kuruda SH 216 A
Varieties
Figure 1. Incidence of PVY in different potato varieties.
Ten Varieties/lines were screened out against PVY. The disease incidence was in range from 70% (Tota704) to 57% in two varieties Kuruda and SH 216A (Fig. 1). Varieties were grouped into three categories;
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Susceptible (Tota-704, FD 71-1, FSD White, FD 8-1 and FD 76-24) Moderate susceptible (FD 74-41, FD 74-50
and N-96-25) and Moderate resistant (Kuruda and SH 216 A) on the basis of disease rating scale (Fig. 2).
4.5
Rating Scale
4
3.5
3
2.5
2
1.5
1
0.5
0
Tota-704 FD 71-1
FSD
WHITE
FD 8-1 FD 76 24 FD 74-41 FD 74-50 N-96-25 Kuruda SH 216 A

Varieties
Figure 2. Disease severity in different potato varieties.
Enzyme Linked Immunosorbant Assay (ELISA)

ELISA confirmed the PVY in the samples collected from ten varieties on the basis of symptoms resembling
to the virus. Light yellow to moderate yellow colour was observed in virus infected samples. The samples were
tested against PVY and PVX antibodies separately on a same microplate and found the positive results (Fig. 3).
Figure 3. ELISA plate (wells) showing the reaction against positive samples of PVY.
Correlation of environmental factors with disease severity

All the environmental factors such as maximum, minimum temperature and relative humidity had highly
significantly correlation with disease progression in susceptible varieties while maximum and minimum in
moderately susceptible and only minimum temperature in moderately resistant (Table 2). Disease was
progressed in susceptible varieties maximum severity was observed at temperature of 1922C and started
decrease gradually as the temperature increases. Similarly high disease severity was observed minimum
temperature of 56C and started decrease with the increase in temperature. 8283% relative humidity was
found favorable for disease severity (Fig. 4).
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Table 2. Correlation between varieties and environmental factors.
Varieties
Tota-704
FD 71-1
FSD WHITE
FD 8-1
FD 76-24
FD 74-41
FD 74-50
N-96-25
Kuruda
SH 216 A
Group
Max. temperature
Min. temperature
Humidity
Susceptible
0.00063 ***
2.31e-09 ***
0.01192 *
Moderate Susceptible
0.00361 **
3.41e-06 ***
0.28519
Moderate Resistance
0.10435
0.00182 **
0.13293
Figure 4. Disease progression in susceptible varieties against PVY in Sargodha.
Moderately susceptible varieties showed that highest disease severity at 22C in case of maximum
temperature and increase in temperature reduced the disease severity. In minimum temperature disease severity
was maximum at 6C and minimum disease severity was recorded at 10C. Intensity of disease decreased with
the increase in relative humidity (Fig. 5). Moderately resistant varieties showed minimum temperature was
highly significant correlation with disease intensity. Disease severity was at peak at 19C in case of maximum
temperature while at 5C in minimum temperature. Maximum disease intensity was recorded at the 83% relative
humidity (Fig. 6).
DISCUSSION
Potato virus potyvirus (PVY) is one of the most economically declared threatening viruses in potato not only
in Pakistan also all over the world. Variety of symptoms exhibited by PVY depends upon the viral strains.
PVYO is the most prevailing strain. Potato varieties/lines were screened out to record the disease incidence and
disease severity according to disease rating scale Qamar et al. (2003) with slight modification. Ten varieties
were divided to three groups i.e. moderately resistant, moderately susceptible and Susceptible. Out of ten
varieties no variety showed immune or resistance response with the mean disease incidence of 62% was
recorded. Out of ten varieties five were susceptible, three moderately susceptible and two moderately resistant.
Our results were in accordance with the results of Abbas et al. (2012). They reported disease incidence of 55%
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during a survey in the different localities of Punjab. Similarly Ahmed et al. (2013) recorded the incidence of
PVY up to 67% in Egypt. They reported that PVYO and PVYN were most prevalent strains.
Figure 5. Disease progression in moderately susceptible varieties against PVY in Sargodha.
Figure 6. Disease progression in moderately resistant varieties against PVY in Sargodha.
Mughal et al. (1988) detected the presence of PVY through ELISA. Similarly Ahmed et al. (1995)
confirmed the PVY, PLRV and PVX in potato samples through ELISA. Susceptible Verities significantly
correlated with maximum, minimum temperature and relative humidity. While maximum and minimum
temperature showed significant correlation with moderately susceptible varieties. Whereas moderately resistant
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varieties had significantly correlated only with minimum temperature. Our results confirmed the work of Qamar
et al. (2003) who studied the correlation of environmental variables (maximum and minimum air temperature,
pan evaporation, wind velocity, wind speed, clouds and relative humidity) with PVY disease on six Potato
varieties viz; 394017-45, TPS-8901, 394029-129, TPS- 9620, 3912202-103 and TPS 9804. Maximum disease
severity was recorded at 2428C as maximum temperature and 912C as minimum temperature. He also
indicated the increasing trend of PVY disease development at minimum temperature of 513C and 1531C
maximum temperature. He also indicated the increasing trend of PVY at minimum temperature of 513C.
Therefore it is concluded that environmental factors have significant impact on disease severity and will be
helpful not only in formulating management strategies but also finding a resistant source against PVY.
ACKNOWLEDGEMENTS
Authors are highly thankful to Dr. S.M. Mughal for providing polyclonal antibodies against PVY and
Director, Potato Research Institute, Sahiwal, Pakistan, for providing us potato germplasm.
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de Box JA & Huttinga H (1981) CMI/AAB description of plant viruses. Potato virus Y 242. Available from:
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Khalid S, Iftikhar S, Munir A & Ahmad I (2000) Potato diseases in Pakistan. PARC Islamabad, Pakistan pp.
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Mughal SM, Khalid S, Gillani TS & Devaux A (1988) Detection of potato viruses in Pakistan. In: Proceeding of
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3(3): 606610, 2016
DOI: 10.22271/tpr.2016.v3.i3.080
Research article
A note on precocious pollen germination in

Woodfordia fruticosa (L.) Kurz.
Kanak Sahai*, Krishna Kumar Rawat and Dayanidhi Gupta
CSIR-National Botanical Research Institute, Rana Pratap Marg, Lucknow-226001, India
*Corresponding Author: sahaikanak@rediffmail.com
Abstract: The unusual phenomenon of pollen germination prior to their release from anthers was
studied in detail in different samples of flowers collected from different geographical regions of
India viz. Eastern Himalayan region (Teesta valley near Darjeeling, West Bengal), Western
Himalayan region (Yamuna bridge near Mussoorie and Pauri Garhwal district), Gangetic plains
(Katerniaghat Wildlife Sanctuary, Uttar Pradesh) and arid region of Rajasthan (Mount Abu).
Though pollen germination prior to their release was regularly observed during the study, it varied
at different level of anther and stigma and in different geographical regions. Highest i.e. 7.31% of
such type of pollen germination was recorded in the samples of Pauri Garhwal of Western
Himalayan region followed by 3.45% from Mussoorie of the same region when flower was at its
initial stage of opening and pistil was short and below the level of anther. This phenomenon of
pollen germination was regularly absent in the samples of Eastern Himalayan region.
Keywords: Precocious pollen germination - Himalayan - Gangetic plain - Arid region.
[Cite as: Sahai K, Rawat KK & Gupta D (2016) A note on precocious pollen germination in Woodfordia
fruticosa (L.) Kurz. Tropical Plant Research 3(3): 606610]
INTRODUCTION
Precocious pollen germination inside the anther loculi are commonly reported to occur in cleistogamous
flowers (Maheshwari 1962, Frankel & Galun 1977, Lord 1979). However, It has also been observed in some
chasmogamous flowers by many workers like Trigonella foenum-graecum (Joshi & Raghuvanshi 1967),
Lathyrus sativus (Verma & Grewal 1971), Bergenia crassifolia (L.) Fritsch, Citrus limon (L.) Burm. Fil,
Cucumis melo L. cv. Giant stide (Tezier), Prunus avium L. cv. Mora di Cazzano, (Pacini & Franchi 1982);
Malus pumila, Prunus amygdalus (Koul et al. 1985), Catharanthus roseus (Mishra & Kumar 2001),
Arabidopsis thaliana (Johnson & McCormick 2001, Xie et al. 2010), Biophytum sensitivum (Sreedevi & Rekha
2003), Glycine max (Kaur et al. 2005) etc. While working on various samples of Woodfordia fruticosa (L.)
Kurz. collected from different parts of the country, the interesting phenomenon of pollen germination inside
indehisced anther lobe was observed and investigated further in detail for the present study. Since Woodfordia
fruticosa is an important dye yielding (Dymock et al. 1890, Uphof 1959, Das et al. 2007) and medicinally
important species (Kuramochi-Motegi et al. 1992, Liu et al. 2004, Chandan et al. 2008), its overexploitation
reported it threatened species (Kokkirala et al. 2012) and even now listed as Low risk/Least concerned species
(IUCN 1998). A perusal of literature reveals though, multifarious properties of Woodfordia fruticosa were
highlighted from time to time, studies on reproductive behavior are almost lacking so far. Woodfordia fruticosa
with chasmogamous flowers regularly showed pollen germination inside anther lobes. Hence, this phenomenon
not reported so far in this plant species was considered interesting for the present study.
MATERIAL AND METHODS
Study species
Woodfordia fruticosa (Lythraceae) commonly known as Fire flam bush due to brilliant red flowers is a
shrub/small tree. It is endemic to India and also distributed in Bhutan, China, Indonesia, Laos, Madagascar,
Myanmar, Nepal, Pakistan, Sri Lanka, Thailand and Tropical Africa (Abbasi et al. 2012, Meena & Kumar
2015). The plant is known for various medicinal properties including anti-tumor, antioxidant,
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immunomodulatory, antiproliferative, antihyperglycemic, antimicrobial, anti-inflammatory and analgesic,
hepatoprotective, cytotoxic, preventive & curative and anti-ulcer activities (Rani et al. 2015). The flowers have
wider use in Ayurvedic formulations like arishta and asava (Kores et al. 1993). Of the 18 aristas, 17 have
been found to contain Woodfordia fruticosa (Meena & Kumar 2015).
Study sites
Flowers and buds of various stages of Woodfordia fruticosa were collected from four different geographical
regions of the country viz. Eastern Himalayan (Teesta valley near Darjeeling, West Bengal), Western Himalayan
(Yamuna bridge near Mussorie and Pauri Garhwal district), Gangetic plain (Uttar Pradesh: Katerniaghat wildlife
sanctuary) and Arid region of Rajasthan (Mount Abu).
Sample preparation and observation
The randomly collected flowering material from different geographical regions of India was fixed in FAA
and later preserved in 70% alcohol. Flowers and buds of different stages were observed under dissecting
microscope for morphological studies. Indehiscent anthers and stigmas of different developmental stages were
selected, dissected and examined under microscope. Since stigma was present at the different level of anthers,
the pistil was categorized as short (stigma below the anther level), medium (stigma equal to anther level) and
long (stigma above the anther level) and the stigma receptivity was tested for each category with Benzedine test
(Galen et al. 1985). Pollen grains were extracted from anther lobes corresponding to the entire three pistil
category and stained with Alexanders stain (Alexander 1980). Observations were made under stereoscopic
zoom microscope to check the pollen viability, sterility as well as their germination inside the anther lobe and
photographed with the help of Nikon Eclipse 80i microscope. Percentage of fertile pollen grains and their
germination inside anther was assessed by counting the pollen grains in randomly selected microscopic fields.
Average score was calculated from 25 such ramdom counts for each sample. For histological observation,
samples were dehydrated in tertiary-butyle-alcohol series and infiltrated and embedded in paraffin wax. Thin
sections of 10m were cut through rotary microtome, double stained with Safranine and Fast green and finally
mounted onto Canada balsam. Microphotographs were taken with the help of microscopic camera attachment.
Study species
The plant has simple, opposite, sessile or shortly petiolate, ovate-lanceolate or lanceolate leaves. Flowers are
irregular, born in axillary cymes, bright red and bisexual; calyx tubular; petal small; stamen 12; ovary superior
with numerous ovule. Fruit membranous capsule; seeds numerous, wedge-shaped, brown and smooth
(Jayaweera 1981, Napagoda & Yakandawala 2008).
Table 1. Pollen status and its perecocious germination along with different developmental stages of stigma in
Woodfordia fruticosa of different geographical region. (S = Short pistil, M = Medium pistil, L = Long pistil)
Geographical regions
Pollen/ anther
Mount Abu, Rajasthan

(Arid region)
59375-146875,
avg. 9307530824
Katerniaghat Wildlife Sanctuary

(Gangetic plains)
33125-111250,
avg. 6687521308
Pauri Garhwal
(W. Himalaya)
38000-91667,
avg. 6420014051
Teesta valley, near Darjeeling

(E. Himalaya)
46667-112667
avg. 7366718522
Near Yamuna bridge, Moosoorie

(W. Himalaya)
37666-85666,
avg. 5876014180
Fertile pollens
(%)
91.28 (S)
87.33 (M)
50.00 (L)
98.87 (S)
97.00 (M)
40.90 (L)
83.94 (S)
88.82 (M)
94.93 (L)
99.27 (S)
98.09 (M)
91.67 (L)
99.37 (S)
89.35 (M)
93.53 (L)
Precocious pollen
germination (%)
0.89 (S)
0.66 (M)
0.68 (L)
0.00 (S)
1.19 (M)
0.00 (L)
7.31 (S)
0.43 (M)
0.53 (L)
0.00 (S)
0.00 (M)
0.00 (L)
3.45 (S)
0.00 (M)
0.00 (L)
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.
Figure 1. Precocious pollen germination in Woodfordia fruticosa. A, section of anther lobe showing germinated pollen
grains; BD, germinating pollen grains with pollen tubes of varying length; E, germinated pollen grains forming cluster.
Stigma became receptive along the flower opening and the receptivity reached at peak at the level of anthers
(medium sized pistil), which gradually decreased when stigma passes the anthers and reached above the anther
level (long pistil). However, pollen viability has no concern with the in-situ pollen germination (Table 1). Pollen
germination inside anther (Fig. 1AE) was observed in all the samples of three different eco-geographic regions,
viz. western Himalayan and Arid region and Gangetic plain except in the samples of eastern Himalayan region
where this phenomenon was absent in spite of repeated visits and random collections (Table 1). Pollen
germination prior to their release from anther (precocious pollen germination) greatly varied provenance to
provenance. It was highest i.e. 7.31% in the samples collected from Pauri Garhwal of western Himalayan
region followed by 3.45% from Mussoorie of the same region (Table 1). Interestingly in both the samples, the
maximum in-situ germination was recorded at the initial stage of flower opening when pistil was short and
below the level of anthers. However, in open flower stage, when pistil reached either at the level of the stamen
(medium sized pistil) or above them (long pistil), the pollen germination was poor or even absent in some
samples (Table 1). Such phenomenon of pollen germination did not occur at the same rate in all the samples; it
varied at different levels of anthers in respect to stigma and in different geographical regions (Table 1). In some
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cases pollen germination inside the anther was observed in clusters and therefore, eventually released together
(Fig. 1E). These pollen clusters may also create obstruction for other pollen to discharge freely at once from
anther and thus act as a hindrance to selfing (Verma & Grewal 1971). On the other hand, as a result of in-situ
germination and subsequent obstruction, the pollen will come out slowly and therefore, pollen discharge may
lost longer, which may attribute to long lasting chances of selfing as suggested by Kaur et al. (2005) who
described in-situ pollen germination in Glycine max as a strategy to facilitate a high degree of selfing. However,
Koul et al. (1985) suggested that in-situ pollen germination is probably controlled by genetic factors as they
observed the phenomenon in specific varieties of apples growing along with many other varieties in the same
environment. According to them such type of pollen germination does not occur year after year which indicates
that certain environmental condition is necessary for the expression of particular gene or genes responsible for
precocious pollen germination in particular plant species. They speculated the reason of limited number of
precocious pollen germination either they do not carry the same potential of germination or not exposed strictly
to similar conditions inside the anthers. Similar observations were made by Mishra & Kumar (2001) in
Catharanthus roseus and found this mechanism as genetically controlled. Dhar et al. (2002) co-related
precocious pollen germination with high relative humidity and high soil moisture. This fact is evident from our
observation as the maximum pollen germination inside the anther lobe was observed in Western Himalayan
samples where high humid conditions were usual. Interestingly, contrary to them, the samples collected from
dry arid area (Rajasthan) regularly revealed precocious pollen germination at all the stages of pistil, though the
percentage was not very high (Table 1).
During the normal case of pollen germination the moisture content of pollen grains as well as of the
microsporangium is reduced as the anther ages (Koul et al. 1985). The pollen grains get partially dehydrated and
the locular fluid disappears before anthesis to promote anther opening and pollen grain dispersal. Reaching onto
the stigma, the pollen grains rehydrate before germination by absorbing the water provided by the receptive
stigma (Heslop-Harrison 1979a, 1979b). The extent of dehydration varies plants to plants according to
pollination strategies and meterological conditions at anthesis (Baranas & Rajki 1976). Partially dehydrated
pollen grains could easily germinate in-situ, where probably the locules remain wet. Precocious pollen
germination with partly dehydrated anther has been observed in seagrass Thalassodendron ciliatum (Ducker et
al. 1978). The wet loculus and absence or delay of pollen dehydration, was speculated to trigger pollen
germination inside anthers by Pacini & Franci (1982). However, in our opinion pre dispersal pollen maturity is
more responsible for precocious pollen germination. Since all the pollen grains inside the anther lobe did not
take part in germination (Fig. 1A), only mature pollen grains that got moisture might be able to germinate.
Though, it is very difficult to speculate whether the pre- germinated pollen grains takes part in self or cross
pollination/fertilization, once the pre-germinated pollen or pollen cluster reaches to the receptive stigma of
same flower, it can enhance the possibility of self-pollination to some extent due to already grown pollen tubes.
Cross pollination due to precocious pollen germination cannot be successful as the dispersal of germinated
pollen grains and their landing on to the stigma surface in a healthy condition cannot be expected because they
can be damaged mechanically by exposure to dry air. However, this mechanism of precocious pollen
germination needs further analysis to find out its real causes and significance.
ACKNOWLEDGEMENTS
The authors are grateful to the Director, CSIR-National Botanical Research Institute, Lucknow for kindly
providing the necessary facilities. The study was conducted with the financial support from Council of Scientific
and Industrial Research (CSIR), New Delhi under project head OLP-0083.
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ISSN (E): 2349 1183

ISSN (P): 2349 9265
3(3): 611615, 2016
DOI: 10.22271/tpr.2016.v3.i3.081
Research article
New species and new records of Graphis (Ostropales:

Graphidaceae) from Eastern Ghats, India
Satish Mohabe1, Anjali Devi B.1, Sanjeeva Nayaka2 and A. Madhusudhana Reddy1*
1
Department of Botany, Yogi Vemana University, Vemanapuram, Kadapa-526003, Andhra Pradesh, India
2
Lichenology Laboratory, CSIR-National Botanical Research Institute, Rana Pratap Marg,
Lucknow-226001, Uttar Pradesh, India
*Corresponding Author: grassced@yahoo.com
Abstract: A new species Graphis neeladriensis, and two new records, G. plumierae and G.
subalbostriata are described from the Eastern Ghats of India. The newly described species is
characterized by crustose, UV+ yellow thallus, sub-immersed to erumpent, short to elongate and
simple to sparingly branched lirellae, 24 striate labia, laterally carbonized exciple, clear
hymenium and terminally muriform ascospores.
Keywords: Rayalaseema - Seshachalam Biosphere Reserve - Lichens - Taxonomy.
[Cite as: Mohabe S, Anjali DB, Nayaka S & Reddy AM (2016) New species and new records of Graphis
(Ostropales: Graphidaceae) from Eastern Ghats, India. Tropical Plant Research 3(3): 611615]
INTRODUCTION
Recent studies on the global diversity within the lichen family Graphidaceae indicates that there are large
numbers of undiscovered species in the family and at least 175 species have been discovered since 2002. Further
analysis predicts that geographically Graphidaceae have a concentrated diversity in a few regions of the world
including Southern India (Lcking et al. 2014). Graphis Staiger (2002) is a major genus under the lichen family
Graphidaceae, comprising of around 370 species worldwide (Kirk et. al. 2008, Lucking 2009, Joshi et al. 2010,
Brcenas-Peta et al. 2014, Singh et al. 2014, Joshi et al. 2014). 111 species of Graphis are known from
tropical, subtropical and temperate regions of India (Singh & Sinha 2010) and recent studies added 17 more
species to the genus (Chitale et al. 2011, Singh & Swanlatha 2011a,b, Gupta & Sinha 2012, Singh et al. 2014).
Within the genus Graphis species having lichexanthone are rare and so far only five species such as G. stipitata
A. W. Archer, G. sauroidea Leight., G. haleana R. C. Harris, G. lucifica R. C. Harris and G. flavopalmicola Y.
Joshi, Lcking & Hur are reported (Lcking 2009, Joshi et al. 2010).
During the exploration on lichen in Rayalaseema forest of Andhra Pradesh a total of 126 species have been
reported (Reddy et al. 2011, Nayaka et al. 2013, Anjali et al. 2013, Mohabe et al. 2014a,b & 2016) out of which
only a single species Diorygma junghuhnii (Mont. & Bosch) Kalb, Staiger & Elix belonged to Graphidaceae.
The explorations resulted in collection of several interesting specimens belonging to Graphidaceous taxa of
which recently Mohabe et al. (2015a) described a new species Diorygma kurnoolensis Mohabe, Nayaka &
Reddy. In the present communication a new species Graphis neeladriensis and new records for India, G.
plumierae Vain. and G. subalbostriata Lcking are reported. The new species is unique in having
lichexanthone in chemistry and terminally muriform ascospores.
MATERIALS & METHODS
The present study is based on recently collected specimens from Neeladri range of Tirumala hills (Fig. 1A)
and Mallaiah Konda hills of Thambalapalli from Chittoor district which comes under Seshachalam Biosphere
Reserve in Andhra Pradesh of India. The external morphology of the specimens were observed under a Magns
MS 24/13 stereo-zoom microscope while anatomical characters of the thallus and apothecia were observed
under a ZEISS Axiostar plus compound microscope. Thin hand cut sections of the thallus and apothecia were
initially mounted in water to record the colour and measurements of various structures. The apothecial sections
were then observed after applying aqueous 10% KOH solution while Lugols solution (I) was used for iodine
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reactions. The colour tests were performed by using routine reagents; aqueous solution of KOH (K), calcium
hypochlorite (C), and paraphenylenediamine (P). The lichen substances were identified by thin-layer
chromatography following literature (White & James 1985, Orange et. al. 2001) and specimens were identified
through world key to the genus Graphis (Lcking et al. 2009) and by comparison with the protologues. The
nomenclature of Lcking et al. (2009) was followed for lirellae morphology. The specimens of the new species
have been deposited in the Herbarium of CSIR-National Botanical Research Institute Lucknow (LWG), Uttar
Pradesh, India and specimens of new records have been deposited in the Lichen Herbarium, Department of
Botany, Yogi Vemana University (YVUH) Kadapa, Andhra Pradesh, India.
RESULTS & DISCUSSION
NEW SPECIES
Graphis neeladriensis Mohabe S, Anjali DB & Nayaka S sp. nov.
(Fig. 1AF)
MycoBank No.: MB 819499
This species is characterized by sub-immersed to erumpent, short to elongate and simple to branched lirellae,
24 striate labia, laterally carbonized exciple, clear hymenium, 8spored ascus, transversely 412 and terminally
12(3) vertically septate ascospores and presence of lichexanthone in thallus.
Type: INDIA, Andhra Pradesh, Chittoor district, Tirumala hills, Neeladri range, on bark of Artocarpus
heterophyllus, alt. ca. 650 m, 06.07.2014, Satish Mohabe & Anjali Devi B. 4097 (holotype-LWG).
Thallus greenish grey to grey, smooth to cracked, shiny, 80160 m thick, corticated; cortex hyaline, 2035
m thick; algal layer continuous 75120 m thick, medulla with oxalate crystals, 1025 m thick; prothallus
indistinct or white.
Ascomata lirellate, variable, numerous, sub-immersed to erumpent, simple, short to elongate (towards centre)
and sparingly branched (towards periphery), 0.23.5 mm long, 0.10.4 mm wide, end acute to obtuse, laterally
covered by thalline margin in younger parts; labia epruinose, 24 striate; disc slit like closed, rarely open,
epruinose; exciple dark brown to black, 5090 m thick, convergent, laterally carbonized; hymenium hyaline,
clear, without oil globules, 175225 m wide, 85200 m high; hypothecium hyaline; ascus cylindrical, 40150
1420 m, 8spored, I ; ascospores hyaline, transversely 412 and vertical cells 13 in end locules
(terminally muriform), 2477 712 m, I+ blue.
Table 1. Comparison of Graphis species containing lichexanthone. (New species is in bold)
Species name
1. Graphis
flavopalmicola
Lirellae morph
handelii-morph
Labia Exciple
Hymenium Septation
entire completely clear
transversely
carbonized
septate
No. of septa
59-septate
Spore length Substance

Unknown
1927 m
2. Graphis haleana striatula-morph
striate completely clear

carbonized
transversely
septate
919-septate
5085 m
Nil
3. Graphis lucifica striatula-morph
striate completely clear

carbonized
transversely
septate
59-septate
2040 m
Nil
4. Graphis
neelladriensis
sp. nov.
tenella-morph
striate laterally
clear
carbonized
terminally
muriform
412 transverse 2477 m

to 13 vertical septate
Nil
5. Graphis
sauroidea
hossei-morph
entire completely clear

carbonized
transversely
septate
45-septate
4560 m
Nil
6. Graphis
stipitata
hossei-morph
entire laterally
clear
carbonized
transversely
septate
715-septate
1520 m
Norstictic,
connorstictic
acid
Chemistry: Thallus K , P , C , KC , UV+ yellow; TLC: lichexanthone present.

Distribution and Ecology: Graphis neeladriensis is found growing on bark of Arctocarpous heterophylla trees
growing in the Insitu conservation garden of medicinal plants in Neeladri range of Tirumala hills, Chittoor
district of Andhra Pradesh at an altitude of around 650 m. It was found growing in association with other
crustose lichens such as Bacidia sp., Graphis sp., Lecanora achroa Nyl. and a foliose species Hyperphyscia
adglutinata (Flrke) H. Mayrhofer & Poelt.
Etymology: The specific epithet is named after type locality Neeladri range which is the highest peak of
Tirumala hills.
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Figure 1. Graphis neeladriensis (holotype): AC, Habit showing variations in lirellae; D, transverse section of lirellae; E,
ascus with ascospores; F, terminally muriform ascospores in iodine.
Remark: Graphis neeladriensis resembles Graphis neoelongata Lcking and Graphis dichotoma (Mll. Arg.)
Lcking in having striate labia, laterally carbonized exciple, clear hymenium, terminally muriform or
submuriform ascospores but both the species differs chemically by lacking lichexanthone in thallus. The
lichexanthone containing species, G. haleana, and G. lucifica differs from new species with completely
carbonized exciple and transversely septate ascospores, while G. stipitata differs in having entire labia, smaller
ascospores and presence of norstictic and connorstictic acid. Further G. flavopalmicola and G. sauroidea have
entire labia, completely carbonized exciple, transversely septate and smaller to medium ascospores. The
comparative status of the new species among other lichexanthone containing species are given in table 1.
Additional specimen examined: INDIA, Andhra Pradesh, Chittoor district, Tirumala hills, Neeladri range, on
bark of Artocarpus heterophyllus, alt. ca. 650 m, 06.07.2014, Satish Mohabe & Anjali Devi B. 4098 (isotypeLWG).
613
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.
NEW RECORDS
1. Graphis plumierae Vain. Ann. Acad. Sci. fenn., Ser. A 6(7): 161 (1915).
(Fig. 2A)
Thallus whitish to greenish grey, smooth to cracked, corticated, 190297 m thick; cortex hyaline, 2339
m thick; medulla white with many crystals, 1028 m wide; algal layer continuous 7595 m thick;
prothallus indistinct to brownish.
Apothecia lirellate, numerous, immersed to sub-immersed, simple to branched, 1.02.5 mm long, 0.30.7
mm wide, with rounded to acute ends; labia pruinose, laterally covered by thalline margin; disc concealed,
epruinose; epihymenium 1419 m, exciple dark brown to black, laterally carbonized, 2562 m thick;
hymenium inspersed, with oil globules, 122149 m wide; hypothecium hyaline to yellowish brown 2036 m
high; ascus cylindrical, 64100 916 m, 8spored; ascospores colourless, transversely 511 septate, 1858
610 m.
Chemistry: K+ yellow turning red, P+ yellow, KC, C; TLC: Norstictic acid, stictic and salazinic acid present.
Distribution: It is a Neotropical species earlier known from Guadeloupe, Mexico and in the present study it is
found on tree trunks in tropical forests of Eastern Ghats in India.
Remark: G. plumierae Vain. has resemblance to G. brevicarpa M. Nakan., Kashiw. & K.H. Moon and G.
crebra but differs by its concealed disc, white pruinose labia, immersed lirellae with lateral thalline margin and
presence of norstictic acid with stictic and salazinic acid. Further G. brevicarpa differs by its apically thick
complete thalline margin, epruinose labia and smaller ascospores while G. crebra has erumpent lirellae with
lateral thalline margin, exposed disc with white pruina (scripta-morph).
Specimens examined: INDIA, Andhra Pradesh, Chittoor district, Thambalapalli, Mallaiah Konda Hills, alt. ca.
956 m, on bark, 05.01.2013, A. Madhusudhana Reddy & Satish Mohabe 2812 (LWG); Tirumala hills,
Dharmagiri, alt. ca. 937 m, on bark, 07.02.2013, Anjali Devi B. & Satish Mohabe 3414 (YVUH).
Figure 2. Habit of Graphis species A, Graphis plumierae Vain.; B, Graphis subalbostriata Lcking.
2. Graphis subalbostriata Lcking, Lichenologist 41(4): 363452 (2009).

(Fig. 2B)
Thallus crustose, corticolous, whitish grey, thin, shiny, smooth to rough, continuous to discontinuous
ecorticated; prothallus indistinct or absent.
Apothecia lirellate, lirellae simple, small, rarely branched, 0.51.5 mm long, 0.10.5 mm wide, erumpent to
prominent, laterally covered by thallus; disc concealed, epruinose; labia partially black, with 78 striation,
distinct white lines in-between striation, formed by clusters of calcium-oxalate crystals; exciple apically to
peripherally carbonized, 2127 m thick; hymenium clear, without oil globules; ascus cylindrical, 125235
2030 m, 8spored; ascospores colourless, fusiform, transversely 511septate, 4595 812 m.
Chemistry: Thallus K , KC , C , P ; TLC: No chemicals.
Distribution: It is a Neotropical species earlier recorded from Guadeloupe and in the present study it was found
on tree trunks in tropical forest of Eastern Ghats in India.
Remark: G. subalbostriata is close to G. patwardhanii Kulk. but latter species has isidiate thallus. G.
subalbostriata is also close to G. olivacea but it has dark olive-grey thallus, erumpent lirellae with apically thin
and complete thalline margin, elongate and irregularly branched lirellae.
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Mohabe et al. (2016) 3(3): 611615

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Specimen examined: INDIA, Andhra Pradesh, Chittoor district, Tirumala hills, Dharmagiri, on bark, alt. ca.
937 m, 07.02.2013, Anjali Devi B. & Satish Mohabe 3396, 3449, 3402, 3420 (YVUH); Shilathoranam, on bark,
alt. ca. 958 m, 05.07.2013, Satish Mohabe & Anjali Devi B. 4034 (YVUH).
ACKNOWLEDGMENTS
The authors are very grateful to Council of Scientific and Industrial Research, New Delhi and Department of
Science and Technology (INSPIRE), New Delhi for financial support; to the Director and Dr. D.K. Upreti, Chief
Scientist, CSIR-National Botanical Research Institute, Lucknow for providing laboratory facilities; to Forest
Officials of Andhra Pradesh for their cooperation during the exploration.
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(Ascomycota: Ostropales: Graphidaceae) from Mexico, with updates to taxonomic key entries for 41 species
described between 2009 and 2013. Lichenologist 46: 6982.
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Mohabe S, Reddy MA, Anjali DB, Nayaka S & Chandramati PS (2014a) Further new addition to the lichen mycota of
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Rayalaseema Forest of Andhra Pradesh, India. In: National Conference on Plant Biology-2014. held at
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Mohabe S, Anjali DB, Reddy AM, Nayaka S & Chandramati PS (2016) An appraisal of lichen biota in Chittoor
district of Andhra Pradesh, India. In: Pulaiah T, Sandhya R (eds) Biodiversity in India. pp. 247296.
Nayaka S, Reddy MA, Ponmurugan P, Devi BA, Ayyappadasan G & Upreti DK (2013) Eastern Ghats, biodiversity
reserves with unexplored lichen wealth. Current Science 104(7): 821825.
Orange A, James PW & White FJ (2001) Microchemical methods for the identification of lichens. British Lichen
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Reddy MA, Nayaka S, Shankar PC, Reddy SR & Rao BRP (2011) New distributional records and checklist of lichens
for Andhra Pradesh, India. The Indian Forester 137: 13711376.
Singh KP & Swarnlatha G (2011a) New records of Graphis (Lichenized fungi) from India. Indian Journal of
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ISSN (E): 2349 1183

ISSN (P): 2349 9265
3(3): 616633, 2016
DOI: 10.22271/tpr.2016.v3.i3.082
Research article
Seed priming with spermine ameliorates salinity stress in the

germinated seedlings of two rice cultivars differing in
their level of salt tolerance
Saikat Paul and Aryadeep Roychoudhury*
Post Graduate Department of Biotechnology, St. Xaviers College (Autonomous), 30,
Mother Teresa Sarani, Kolkata-700016, West Bengal, India
*Corresponding Author: aryadeep.rc@gmail.com
[Accepted: 02 December 2016]
Abstract: The present study was aimed to assess the efficacy of the tetramine, spermine (Spm) as
a seed priming agent in attenuating oxidative damages and improving salt tolerance in salt-stressed
seedlings of IR-64 (salt-sensitive) and Nonabokra (salt-tolerant) rice cultivars. The extent of
damages was lesser in Nonabokra due to higher cysteine and ascorbic acid (AA), reducing power
ability, concomitant with unaltered ascorbic acid oxidase (AAO) activity, and elevated ascorbate
peroxidase (APX) and -amylase activity. Spm priming alleviated salt stress injury by lowering
the malondialdehyde and H2O2 content and avoiding chlorophyll degeneration in both the
cultivars, the effect being more pronounced in IR-64 in terms of H2O2 reduction. The intrinsic
property of Spm in stress amelioration was highly evident with respect to the reduction in the
levels of anthocyanin, total phenolics and cysteine, and activity of AAO and superoxide dismutase
(SOD) in IR-64, whereas lowered guaiacol peroxidase (GPX), catalase (CAT) and SOD activity in
Nonabokra, as compared to Spm non-primed stressed-seedlings. However, Spm priming enhanced
the reducing power ability, GPX, -amylase and polyphenol oxidase (PPO) activities in IR-64, and
anthocyanin, AA and CAT activity in Nonabokra, as means of mitigating cellular NaCl toxicity. A
clear-cut variation in GPX, CAT, SOD and esterase isozyme profile was discernible between the
two cultivars during salinity stress, with specific isoform(s) being up regulated or down regulated
with Spm pre-treatment. In terms of osmolyte regulation, Spm priming appeared to be more
promising in Nonabokra, because of the enhanced levels of reducing sugar, amino acids and
proline. All these results indicated that seed priming with Spm at the pre-sowing stage can
promote salinity tolerance with varying degrees in the two rice cultivars by attenuating oxidative
damages, triggering the antioxidants and osmolytes, and activating the antioxidative enzymes at
the protein level.
Keywords: Antioxidants - Osmolytes - Salt stress - Seed priming - Spermine.
[Cite as: Paul S & Roychoudhury A (2016) Seed priming with spermine ameliorates salinity stress in the
germinated seedlings of two rice cultivars differing in their level of salt tolerance. Tropical Plant Research 3(3):
616633]
INTRODUCTION
Soil salinity is one of the brutal abiotic stress factors affecting crop productivity worldwide. Rice sensitivity
to salt varies according to growth stage and also among the cultivars. The high yielding rice cultivars like IR-29,
IR-64, IR-72, M-1-48 are salt-sensitive, whereas cultivars like Pokkali, Nonabokra, Oormundakon, etc. are low
yielding, but salt-tolerant (Soda et al. 2013). Salt stress involves a combination of dehydration or osmotic stress
damages due to excess accumulation of Na+ ions and loss of K+ ions, which adversely affects plant growth and
development. Oxidative damages, invoked under salinity stress, as an early rapid response is due to the
formation of reactive oxygen species (ROS), like superoxide anion (O2-), singlet oxygen (1O2), hydroxyl radicals
(OH-) and hydrogen peroxide (H2O2). The ROS also trigger peroxidative reactions and cause serious damages to
phospholipids, nucleic acids and proteins. To ward off such damages, plants have evolved a complex
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Received: 07 September 2016
Published online: 31 December 2016

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antioxidative system, participated by the non-enzymatic and enzymatic antioxidants (Gill & Tuteja 2010). The
enzymatic antioxidants typically include guaiacol peroxidase (GPX, EC 1.11.1.7), superoxide dismutase (SOD,
EC 1.15.1.1) and catalase (CAT, EC 1.11.1.6) and enzymes belonging to the ascorbate-glutathione cycle (AsAGSH cycle) such as ascorbate peroxidase (APX, EC 1.11.1.11). In perennial rye grass (Lolium perenne L.),
NaCl treatment enhanced the expression of SOD, CAT and peroxidase (POD) (Hu et al. 2011). Four forms of
CAT isozymes, three APX isozymes and seven GPX isozymes were identified in plants under salinity stress
(Lee & An 2005). Differential changes in the level of isozyme forms might be important signals in salt stress
response (Parida et al. 2004).
Under osmotic stress, the compatible solutes or osmolytes accumulate intracellularly in order to lower the
osmotic potential, so as to drag more water within the cell. The common osmolytes include sucrose, proline
(Pro) and glycine betaine, in addition to other molecules accumulating to high concentrations in certain species
(Munns & Tester 2008). Accumulation of Pro and mannitol has been reported under drought stress (Sickler et
al. 2007). Polyamines (PAs) constitute another class of low molecular weight, nitrogeneous, aliphatic
osmoprotectants. The common PAs in plants are spermidine (Spd3+), spermine (Spm4+) and their precursor,
putrescine (Put2+). Being polycationic in nature at physiological pH, PAs can readily bind to the negatively
charged phospholipid head group or other anionic sites on the membrane, regulating the integrity of the
membrane. They also play vital role during multiple abiotic stresses including salinity, drought, low or high
temperature and heavy metal toxicity (Roychoudhury et al. 2008). Being a tetramine, Spm is presumed to be a
protective agent against various oxidative damages. Yamaguchi et al. (2006) found that a Spm-deficient
Arabidopsis mutant exhibited hypersensitivity to NaCl stress. The NaCl-hypersensitivity of the mutant could be
cured by Spm but not by Put and Spd, suggesting a close link between NaCl hypersensitivity and Spm
deficiency. Spm was found to have a protective role in detached rice leaves during water stress induced by the
application of polyethylene glycol 6000 (Cheng & Kao 2010).
Numerous attempts have been made to improve the salinity tolerance of various crops by traditional
breeding program, but the progress has been quite slow with limited commercial success. Exogenous PA
application increased endogenous PA levels and alleviated salt stress damages in vegetative tissues of several
plants including rice. Such protective roles have been attributed to the reduction of salinity stress-induced
damages (Liu et al. 2006) by inducing the activity of antioxidative enzymes and increasing the synthesis of nonenzymatic antioxidants and compatible osmolytes (Roychoudhury et al. 2011, Saleethong et al. 2011).
Transgenic plants overproducing PAs, via modulating PA-metabolic pathways, rendered protection against
abiotic stress conditions, while reduced in vivo PA levels resulted in decreased stress tolerance (Alet et al.
2012). Exogenously applied PAs have also been shown to effectively alleviate stress injury caused by acid rain,
ozone, heavy metals, chilling and water stress (Alczar et al. 2011). Shi et al. (2010) found that Spm pretreatment confers dehydration tolerance of Citrus plants through modulation of antioxidative potential and
stomatal regulation. Since the distinct functional regulation of PAs largely depend on plant species as well
varieties of the same species, deciphering the precise significance of PAs in stress response is quite complicated.
The use of some simple, cost-effective methods, such as seed priming is regarded as a pragmatic approach to
overcome plant growth retardation under saline condition. Seed quality, seed germination rate and seedling
vigor are altogether vital factors for sustainable crop production, particularly under adverse environmental
conditions (Sun et al. 2007). Priming is a process by which seeds are exposed to restricted water availability
under controlled conditions, allowing some of the pre-germination metabolic activities (physiological and
chemical) to proceed, before completion of germination. This is followed by short-term storage through redrying before ultimate sowing (Farooq et al. 2010a, b). There are adequate reports showing that under diverse
environmental stresses such as salinity, water deficit and extreme temperatures, osmopriming leads to cellular,
subcellular and molecular changes in seeds, subsequently promoting seed vigor during germination and seedling
emergence in different plant species (Beckers & Conrath 2007). However, the efficacy of different priming
agents varies under different stresses as well as in different crop species.
Although the effects of seed osmopriming with different compatible solutes and growth regulators on
different plant species have been reported from time to time, little evidence exists on the effects of seed priming
with Spm on germinated rice seedlings, subsequently exposed to salt stress. Using two rice cultivars with
varying degrees of salt tolerance, viz., IR-64 (salt-sensitive) and Nonabokra (salt-tolerant) as experimental
models, the focus of this study was to investigate if pre-soaking the seeds with Spm has the potentiality to
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ameliorate the oxidative damages in the germinated seedlings exposed to salinity stress. The efficacy of Spm as
a seed priming agent in improving salt tolerance was investigated on the basis of measuring certain biochemical
stress-markers (lipid peroxidation, hydrogen peroxide formation and chlorophyll degradation), analyzing the
level or activity of antioxidants (non-enzymatic and enzymatic), osmolyte regulation, isozyme expression
patterns and activities of the enzymes like -amylase and polyphenol oxidase.
Plant materials, growth conditions and stress treatment
The seeds of Oryza sativa L. cv. IR-64 were obtained from Chinsurah Rice Research Station (Hooghly, West
Bengal, India) and Nonabokra seeds from Central Soil Salinity Research Institute (Canning, West Bengal,
India). The initial seed moisture content ranged between 8.5-9.5% (on a dry weight basis). The seeds were
surface sterilized with 0.1% (w/v) HgCl2 for 20 min, and washed extensively with sterile water. The seeds were
pre-treated with 2.5 mM of Spm, the concentration of Spm was chosen on the basis of earlier work by Iqbal et
al. (2006). Healthy rice seeds (500 seeds for each treatment) were primed separately in 50 ml solution of Spm or
in distilled water for 8 h at room temperature (25oC) in plastic cups. After pre-soaking, the seeds were surface
dried on filter paper and allowed to dry for 12 h at room temperature (25oC). The air-dried seeds of both the
cultivars were placed on two layers of filter paper and supplemented with 75 mM NaCl for stress treatment,
while distilled water was used as control (untreated). Solutions were renewed every two days. Four sets of
samples were maintained:
Set 1. Water-primed seed without salt stress
Set 2. Water-primed seed with salt (75 mM NaCl) stress
Set 3. Spm-primed seed without salt stress
Set 4. Spm-primed seed with salt (75 mM NaCl) stress
The 10 day-old seedlings from each of the above sets were germinated at 32oC, under 16 h light and 8 h dark
photoperiodic cycles with 50% relative humidity and 700 mol photons m-2 s-1. The seedlings were harvested,
frozen in liquid nitrogen, and 0.5 g of each sample was used for the following estimation.
Estimation of oxidative damages
The malondialdehyde (MDA) content was estimated from 0.5 g of each of the samples, using MDA
extinction coefficient 155 mM-1cm-1 (Roychoudhury et al. 2012). Hydrogen peroxide levels from 0.5 g of
samples were determined spectrophotometrically at 390 nm according to Velikova et al. (2000). Total
chlorophyll content from 0.5 g of leaf samples was estimated according to Roychoudhury et al. (2007).
Estimation of non-enzymatic antioxidant parameters [anthocyanin, cysteine (Cys), total phenolic content (TPC)
ascorbic acid (AA), reducing power] and ascorbic acid oxidase (AAO, EC 1.10.3.3) activity
For anthocyanin determination, 0.5 g of samples was extracted with acidified [1% (v/v) HCl] methanol (25
mg ml-1) for 24 h at 4oC with occasional shaking. The absorbance of the extract was recorded at 525 nm and the
amount of anthocyanin was calculated using a millimolar extinction coefficient of 31.6 (Roychoudhury et al.
2007). For Cys estimation, 0.5 g of samples was homogenized in 5% (v/v) chilled perchloric acid (PCA) and
centrifuged at 10,000 g for 10 min at 4oC. The absorbance of the supernatant was measured using acidninhydrin reagent at 560 nm (Roychoudhury et al. 2007). The TPC in the extract was determined according to
Jayaprakasha et al. (2001) with some modifications. About 0.5 ml of the aqueous extract of each sample was
mixed with 2.5 ml of 10-fold-diluted FolinCiocalteu reagent and 2 ml of 7.5% (w/v) sodium carbonate. The
mixture was allowed to stand for 30 min at room temperature (25oC) and the absorbance was measured at 760
nm. The final results were expressed as tannic acid equivalents. The assay of AA was performed by macerating
the leaf tissues with metaphosphoric acid and titrating with 0.1% 2, 6-dichlorophenolindophenol (DCPIP)
(Roychoudhury et al. 2007). The AAO activity was assayed following the method of Oberbacher & Vines
(1963). The reducing power of the rice extract was determined following Kumaran & Karunakaran (2006), with
some modifications and the absorbance was measured at 700 nm.
Estimation of activity of antioxidant enzymes: guaiacol peroxidase (GPX, EC 1.11.1.7), ascorbate peroxidase
(APX, EC 1.11.1.11), catalase (CAT, EC 1.11.1.6) and superoxide dismutase (SOD, EC 1.15.1.1)
Total protein was extracted from the samples by the method of Anderson et al. (1995). The GPX activity
was determined using the method of Srinivas et al. (1999) following the formation of tetraguaiacol by
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measuring the absorbance at 470 nm and using an extinction coefficient 26.6 mM-1cm-1. The activity of APX
was measured according to the method of Nakano & Asada (1981) by estimating the rate of ascorbate oxidation
(extinction coefficient 2.8 mM-1 cm-1). The CAT activity was assayed by measuring the initial rate of H2O2
disappearance at 240 nm using the extinction coefficient 40 mM-1cm-1 for H2O2 (Velikova et al. 2000). The
SOD activity, the basis of which is its ability to inhibit the photochemical reduction of nitroblue tetrazolium
(NBT) (Beauchamp & Fridovich 1971), was assayed following Alonso et al. (2001) with certain modifications.
The unit of SOD activity was defined as that amount of enzyme which caused 50% inhibition of the initial rate
of reaction in the absence of enzyme.
In gel analysis of isozymes of GPX, CAT, SOD and esterase (EST, EC 3.1.1.43)
For in gel analysis of the isozymes of GPX, 50 g of protein was run in a non-denaturing 10%
polyacrylamide gel in dark under cold condition. The specific bands were detected by submerging the gel in a
staining solution containing 50 mM potassium phosphate buffer pH 7.0, 0.46% (v/v) guaiacol and 13 mM H2O2
until red bands appeared. For in gel studies of CAT, 80 g of protein was loaded in non-denaturing 10%
polyacrylamide gel under cold condition. The electrophoresed samples in the gel was incubated in 0.05% H2O2
(v/v) for 10 min and subsequently developed in 2% (w/v) FeCl3 and 2% (w/v) K3FeCN6 solution for 10 min. For
in gel staining of isozymes for SOD, 80 g of protein was run through 10% native polyacrylamide gel
electrophoresis in dark under cold conditions, followed by completely submerging the gel in freshly prepared
staining buffer, containing 50 mM phosphate buffer pH 7.0, 0.1 ml EDTA, 28 mM TEMED, 0.003 mM
riboflavin and 0.25 mM nitroblue tetrazolium for 30 min in dark condition. Thereafter, the gel was placed on an
illuminated glass plate until the bands become visible. For detection of isozymes for EST, about 50 g of
protein was run in a non-denaturing 10% polyacrylamide gel in dark under cold condition. For staining of the
bands, -naphthyl acetate was used as a substrate and Fast Blue RR salt as a dye coupler. The staining solution
consisted of 100 ml of 0.1 M sodium phosphate buffer pH 6.0, 100 mg Fast Blue RR salt and 0.1 g of naphthyl acetate (acetone : water, 1: 1). The gel was incubated in the filtered solution for 30 min at 37oC in dark,
and then fixed in 50% (v/v) ethanol.
Estimation of reducing sugars, total amino acids and proline
The reducing sugar content from 0.5 g of samples was determined spectrophotometrically at 630 nm with
freshly prepared anthrone reagent (Irigoyen et al. 1992). The total amino acids were quantified by the ninhydrin
method according to Moore (1968). Free proline (Pro) content from the leaf samples was determined at 520 nm
according to the procedure of Bates et al. (1973).
Estimation of -amylase (EC 3.2.1.1) and polyphenol oxidase (PPO, EC 1.14.18.1) activity
The assay of -amylase activity was performed from 1 g of tissues (Tarrago & Nicolas 1976) after
inactivating -amylase by heating at 70C for 5 min with 9 mM CaCl2 and performing the assay following the
standard method (Chrispeels & Varner 1967). The PPO activity was assayed spectrophotometrically at 480 nm
using 1 g of samples (Mayer & Harel 1979).
Protein estimation
For all the enzyme assays, protein contents were estimated using bovine serum albumin (BSA) as standard
(Lowry et al. 1951). Equal amount of total protein from all the test samples were used in our assay.
Statistical analysis
The experiments were carried out in a completely randomized design (CRD) with three replicates; each
replication comprised an average of 50 seeds, and the results presented as means standard error (SE). The data
and significant differences among mean values were compared by descriptive statistics ( SE) followed by
Studentst-test. The statistical significance was calculated at P 0.05.
RESULTS
MDA, H2O2 and chlorophyll content
During salt stress, the MDA content increased in both the cultivars raised from water pre-treated seeds, viz.,
2.2 times and 1.2 times respectively in IR-64 and Nonabokra, when compared to the control. When Spm pretreated seeds were subjected to stress imposition, the MDA content in the seedlings was reduced 1.7 folds and
1.4 folds respectively in IR-64 and Nonabokra, as compared with seedlings raised from Spm non pre-treated
seeds under salinity stress conditions (Fig. 1A). Salinity stress increased H2O2 content 1.7 times and 1.1 times
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respectively in IR-64 and Nonabokra seedlings germinated from water pre-treated seeds, as compared with
control. The induction of H2O2 by salt was reduced in the seedlings raised from Spm pre-treated seeds, viz., 1.9
times and 1.2 times in IR-64 and Nonabokra respectively, as compared to the stressed seedlings without Spm
pre-treatment (Fig. 1B). The chlorophyll content in saltstressed seedlings decreased 58% and 49% respectively
in IR-64 and Nonabokra, as compared with unstressed seedlings. However, seedlings raised from Spm pretreated seeds could overcome the salinity-induced chlorophyll loss by 32% and 33% respectively in IR-64 and
Nonabokra, as compared to the seedlings raised from Spm non-pretreated seeds (Fig. 1C).
Figure 1. Effect of Spm (2.5 mM) pre-treatment of seeds (8 h) on oxidative damage indices, viz., MDA content (A), H2O2
content (B) and chlorophyll degeneration (C), in IR-64 and Nonabokra seedlings under 75 mM NaCl; the stress was imposed
for 10 days. The untreated seedlings (with or without Spm pre-treatment of seeds) served as experimental control. Data are
the mean value (n = 3) SE. The SE in each case is represented by the vertical bar in each graph. Statistical differences at P
0.05 have been calculated for each t-test.
Non-enzymatic antioxidant levels and AAO activity

Salinity stress triggered the anthocyanin level almost 1.6 times in IR-64 and 1.2 times in Nonabokra, as
compared with control seedlings. The stressed seedlings from Spm-treated seeds showed 2.1 times reduction in
anthocyanin in IR-64, whereas 1.3 times increment in Nonabokra, relative to the Spm non-treated stressed
seedlings. The anthocyanin level was considerably higher in Nonabokra (1.5 times) than IR-64 in Spm pretreated stressed seedlings (Fig. 2A). For Cys, a decrease in the level was recorded for both the cultivars during
salinity stress, viz., 1.4 times and 1.1 times in IR-64 and Nonabokra respectively. Following Spm priming, the
stressed seedlings showed further reduction in Cys level in both the cultivars, viz., 1.6 times and 1.8 times in IRwww.tropicalplantresearch.com
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64 and Nonabokra respectively, as compared with stressed seedlings without Spm pre-treatment (Fig. 2B). Salt
stress increased the AAO activity by 1.2 times in IR-64, whereas the activity remained unchanged in
Nonabokra, relative to the control seedlings. Spm pre-treatment lowered the AAO activity 1.3 times in IR-64,
while remaining unchanged in Nonabokra seedlings, when subjected to salt stress (Fig. 2C). The TPC in saltstressed seedlings showed 1.3 times and 1.1 times increase in IR-64 and Nonabokra respectively, as compared
with control seedlings. However, exposure of seedlings from Spm pre-treated seeds to salt stress lowered the
TPC, viz., 1.8 times and 1.4 times respectively, relative to the stressed seedlings in absence of Spm (Fig. 2D).
With salinity stress, the AA content remained unaltered in IR-64, whereas it was induced 1.2 times in
Nonabokra, as compared with non-stressed seedlings. When Spm-primed seeds were grown under salt stress, the
AA content again remained almost unchanged in IR-64, whereas Nonabokra showed 1.2 times enhancement,
with respect to Spm-non primed salt-treated seedlings (Fig. 2E). The reducing power in salt-stressed seedlings
of IR-64 decreased drastically by 11.4 times, while 1.8 times decrease was recorded in Nonakora seedlings, with
respect to the unstressed seedlings. The reducing power was enhanced particularly in IR-64 seedlings by 1.5
times, when Spm-pre-treated seeds were used, as compared with Spm non-primed samples (Fig. 2F).
Figure 2. Effect of Spm (2.5 mM) pre-treatment of seeds (8 h) on antioxidant parameters, viz., anthocyanin content (A), Cys
content (B), TPC (C), AA content (D), AAO activity (E) and reducing power (F), in IR-64 and Nonabokra seedlings under
75 mM NaCl; the stress was imposed for 10 days. The untreated seedlings (with or without Spm pre-treatment of seeds)
served as experimental control. The data represented are means of three observations (n = 3) SE. Statistical differences at
P 0.05 have been calculated for each t-test.
Antioxidant enzyme activity

Salinity stress increased the GPX activity 1.8 times in both IR-64 and Nonabokra, relative to the control.
Spm pre-treatment drastically induced further the GPX activity, viz., 5.8 times in IR-64, while in the tolerant
cultivar Nonabokra, the GPX activity was lowered 1.5 times, as compared with stressed seedlings in absence of
Spm pre-treatment (Fig. 3A). The APX activity, on the contrary, showed differential response in the two
cultivars. While the activity decreased 1.8 times in IR-64, it increased 1.3 times in Nonabokra after salinity
stress. Seed priming with Spm induced the activity slightly in IR-64, while the activity was lowered by 1.7 times
in Nonabokra during salinity stress, as compared to stressed seedlings in absence of Spm (Fig. 3B). Salinity
stress increased the CAT activity 2.1 times and 1.6 times in IR-64 and Nonabokra respectively, when compared
to the control. Spm pre-treatment of seeds increased the CAT activity particularly in Nonabokra (1.3 times),
while in IR-64, it was almost unaltered, as compared with Spm non-treated plants under salinity stress condition
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(Fig. 3C). Salinity stress raised the SOD activity 3.7 times in IR-64 and 4.4 times in Nonabokra seedlings
relative to the control (unstressed seedlings). Spm pre-treatment of seeds, however, lowered the SOD activity in
the salt-stressed seedlings of both the cultivars, viz., 3.9 times and 1.6 times respectively in IR-64 and
Nonabokra, when compared to the stressed seedlings raised from Spm non-primed seeds (Fig. 3D).
Figure 3. Effect of Spm (2.5 mM) pre-treatment of seeds (8 h) on the activity of antioxidative enzymes, viz., GPX (A), APX
(B), CAT (C) and SOD (D) in IR-64 and Nonabokra seedlings under 75 mM NaCl; the stress was imposed for 10 days. The
untreated seedlings (with or without Spm pre-treatment of seeds) served as experimental control. The data represented are
means of three observations (n = 3) SE. Statistical differences at P 0.05 have been calculated for each t-test.
Isozyme profile for the antioxidative enzymes

While five GPX isozymes (GPX1, GPX2, GPX3, GPX4 and GPX5) were noted in IR-64, GPX5 was
uninduced in Nonabokra, so that four isozymes (GPX1, GPX2, GPX3 and GPX4) were prominent. The GPX1
expression was more prominent in IR-64, while lesser induced only in the stressed seedlings of Nonabokra,
whether in absence or presence of Spm pre-treatment. However, GPX2 expression was higher in Nonabokra,
especially in the stressed seedlings, while weakly induced in IR-64 (Fig. 4A, 4B). Three CAT isozymes (CAT1,
CAT2 and CAT3) were found in Nonabokra, while CAT3 was undetected in IR-64. The most abundant isozyme
was CAT1, whose expression was almost constitutive in Nonabokra, while in IR-64, it was better induced after
salinity stress and with Spm pre-treatment. Both salinity stress and Spm pre-treatment increased the intensity of
CAT2 in IR-64, but undetected under control condition. The CAT2 and CAT3 inductions were higher in
Nonabokra during salinity stress, while the expression was lower after Spm pre-treatment (Fig. 4C, 4D). Four
SOD isozymes (SOD1, SOD2, SOD3 and SOD4) were observed in Nonabokra, of which SOD4 was undetected
in IR-64. The abundance of SOD1, SOD2 and SOD3 was enhanced in IR-64 stressed seedlings raised from Spm
pre-treated seeds (Fig. 4E, 4F). In case of EST, three isozymes, namely EST1, EST2 and EST3 were noted in
both the cultivars. The most abundant isozyme was EST2, which was constitutively expressed in both IR-64 and
Nonabokra under all the experimental conditions. The EST3 isozyme induction was considerably higher in
Nonabokra than IR-64, where EST3 induction was noted upon salinity stress, with or without Spm pretreatment. Of all the isozymes, the EST1 was the most feebly induced in both the cultivars; the expression was
raised after salinity stress in Nonabokra, whereas Spm pre-treatment induced the isozyme appreciably in IR-64
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(Fig. 4G, 4H).
Figure 4. Effect of Spm (2.5 mM) pre-treatment of seeds (8 h) on isozyme profile of GPX, CAT, SOD and EST in IR-64 (A,
C, E and G respectively) and Nonabokra (B, D, F and H respectively) seedlings under 75 mM NaCl; the stress was imposed
for 10 days. The untreated seedlings (with or without Spm pre-treatment of seeds) served as experimental control. The bands
were resolved in non-denaturing polyacrylamide gel.
Reducing sugar, total amino acid and proline levels

Salinity stress lowered the reducing sugar content in both the cultivars, viz., 1.1 times and 1.3 times
respectively in IR-64 and Nonabokra, as compared with unstressed seedlings. When Spm pre-treated seeds were
germinated in presence of salt, the reducing sugar level in IR-64 seedlings remained unaffected, while that in
Nonabokra seedlings increased 1.2 times, compared with the stressed seedlings without Spm pre-treatment (Fig.
5A). Upon salt stress exposure, the total amino acid level in Nonabokra decreased 1.3 times, while it remained
unaltered in IR-64, with respect to the control conditions. Seedlings raised from Spm pre-treated seeds showed
differential response during salinity stress. While in IR-64, the amino acid level was lowered 1.1 times, the level
enhanced almost 1.8 times in Nonabokra, as compared with stressed seedlings in absence of Spm (Fig. 5B). The
Pro level in both the cultivars increased with salinity stress, 1.6 times in IR-64 and 1.9 times in Nonabokra, as
compared with non-stressed seedlings. Nonabokra seedlings raised from Spm primed seeds showed further
enhancement in Pro level, viz., 1.2 times, while IR-64 seedlings registered 1.2 times lowered Pro level, with
respect to stressed seedlings without Spm pre-treatment (Fig. 5C).
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Figure 5. Effect of Spm (2.5 mM) pre-treatment of seeds (8 h) on osmolyte regulation, viz., reducing sugar content (A),
amino acid content (B) and Pro content (C), in IR-64 and Nonabokra seedlings under 75 mM NaCl; the stress was imposed
for 10 days. The untreated seedlings (with or without Spm pre-treatment of seeds) served as experimental control. The data
represented are means of three observations (n = 3) SE. Statistical differences at P 0.05 have been calculated for each ttest.
-amylase and PPO activity

The -amylase activity showed a differential pattern of activity in the two cultivars during salt stress. While
a decreased activity was noted in IR-64, Nonabokra seedlings registered 1.4 times enhanced activity, as
compared with unstressed seedlings. When Spm-primed seeds were exposed to salinity stress, the enzyme
activity increased 1.1 times in IR-64, whereas it remained unaltered in Nonabokra, with respect to the salttreated seedlings without Spm pre-treatment (Fig. 6A). Salinity stress lowered the PPO activity 1.8 times in
Nonabokra, while the activity was slightly triggered in IR-64, with respect to control seedlings. When Spmprimed seeds were grown under salt treatment, the PPO activity was lowered to a small extent (1.1 times) in
Nonabokra, while showing a small increment (1.2 times) in IR-64, relative to the stressed seedlings without
Spm application (Fig. 6B).
DISCUSSION
Priming, a prior encounter with a particular type of chemical or stress condition, is known to endow plants
with greater tolerance to subsequent stress exposure of the same or different kind. Seed priming is a pre-sowing
strategy that influences the seedling development at a later stage, via modulation of pre-germination metabolic
activity, preceding the protrusion of the radicle (Patane et al. 2009). During priming, the seeds may be partially
treated with water or various chemical solutions, so that the pre-germination metabolic activity starts, but the
radicle emergence is prevented, followed by drying the seed. Improved growth and stress tolerance of the
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primed seedlings have been reported for wheat, maize, cucumber, sugarcane, lentil and capsicum (Yadav et al.
2011). Although the phenomenon is known for decades, the underlying mechanism responsible for the better
performance of the primed plants under stress regimes is not well explained or understood.
Figure 6. Effect of Spm (2.5 mM) pre-treatment of seeds (8 h) on -amylase (A) and PPO (B) activity in IR-64 and
Nonabokra seedlings under 75 mM NaCl; the stress was imposed for 10 days. The untreated seedlings (with or without Spm
pre-treatment of seeds) served as experimental control. The data represented are means of three observations (n = 3) SE.
Statistical differences at P 0.05 have been calculated for each t-test.
The PA accumulation in many plants is the immediate response observed in different crop species after
exposure to saline conditions (Roychoudhury & Das 2014). Most significant changes in PA levels upon
salinization appear to be those of Spm, according to the data reported in rice (Maiale et al. 2004), maize
(Jimenez-Bremont et al. 2007) and wheat (El-Shintinawy 2000). In most cases, Spm is more potent than Spd
and considerably more efficient than Put. The more pronounced protective effect of Spm in comparison with
other PAs could be accounted for by its longer chain and greater number of positive charges which allows
greater neutralizing and membrane stabilizing ability. The Spm-deficient mutant Arabidopsis was found to be
hypersensitive to high salt stress and this phenotype was abrogated by exogenously applied Spm (Yamaguchi et
al. 2006). The ameliorative role of exogenous Spm in reproductive phase of soybean during polyethylene glycol
(PEG)-induced osmotic stress has been shown, thereby improving the plant reproductive health (Radhakrishnan
& Lee 2013). Since chemical priming is one of the prominent pre-germination strategies to overcome the
detrimental effects associated with osmotic stresses, the effect of seed priming with Spm was assessed in the
present study on the performance of seedlings of two rice cultivars, IR-64 and Nonabokra, during subsequent
exposure to salinity stress.
Salinity stress led to a significant decline in the seedling quality of both the varieties by causing chlorophyll
degeneration, increased MDA content and higher production of H2O2, though the salt-tolerant cultivar suffered
lesser damages. Both chlorophyll degradation and increment of MDA content, which is a product of lipid
peroxidation, are associated with the accumulation of ROS, pointing towards the damages incurred by the cell
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membrane and chloroplast membrane in plants (Halliwell 2006). Gill & Tuteja (2010) reported that higher
accumulation of MDA declines the membrane fluidity, inactivates the receptors and degrade the membrane
proteins, enzymes and ion channels. However, seed pre-treatment with Spm considerably lowered the MDA and
H2O2 content, along with partial recovery of chlorophyll during salinity stress. The effect of Spm was more
prominent in IR-64 in terms of considerable lowering of the H2O2 production under saline conditions. All these
results are consistent with the previous studies in Arabidopsis, sunflower and soybean (Kusano et al. 2007,
Radhakrishnan & Lee 2013) where exogenous Spm could effectively overcome the deleterious effects of
salinity stress.
Among the non-enzymatic antioxidants, the levels of anthocyanin and TPC showed increment with salinity
stress in both the cultivars, with somewhat greater enhancement in the susceptible cultivar IR-64. Such increase
is well supported by our earlier observations in rice during salinity (Roychoudhury et al. 2008) and drought
(Basu et al. 2010) stress. The concerted action of low molecular weight antioxidants like anthocyanins (ChalkerScott 1999) and polyphenols (Sgherri et al. 2004) can effectively scavenge harmful radicals and stabilize the
membranes against lipid oxidation under stressed conditions. Genisel et al. (2015) have reported the mitigating
effect of Cys on growth inhibition in salt-stressed barley seedlings related to its own antioxidant properties. The
lowered Cys content in both the cultivars, especially in IR-64, with salinity stress could be explained by the fact
that more and more endogenous Cys pool is being utilized probably for the synthesis of thiol-related
compounds, like glutathione or -glutamylcysteine-containing homologues, which maintain cellular redox
homeostasis by quenching of ROS (Das & Roychoudhury 2014). Shalata & Neumann (2001) have shown the
role of AA in increasing resistance to salt stress in tomato. Exogenous AA also increased the endogenous AA
content, thereby reducing oxidative damages, with more pronounced effect in the tolerant cultivar Pokkali than
in Peta, the sensitive cultivar (Wang et al. 2014). These data are in agreement with our observation where
salinity stress enhanced the AA content only in the tolerant cultivar Nonabokra, concomitant with unaltered
AAO activity. On the other hand, the AA content remained unaltered in IR-64, with a slightly enhanced AAO
activity, signifying the better protective role of AA in Nonabokra. The suppressed expression of AAO in
Nonabokra also justifies greater tolerance to salt stress than IR-64 (Yamamoto et al. 2005). The higher reducing
power capability of Nonabokra during salinity stress depicts more efficient antioxidative mechanism in this
cultivar in terms of free radical scavenging. The reduction in anthocyanins, TPC and Cys levels in stressed
seedlings raised from Spm-primed seeds (as compared to stressed seedlings in absence of Spm) in IR-64 clearly
highlights the role of Spm itself as antioxidant in mitigating oxidative stress in the salt-sensitive cultivar.
However, Spm priming further elevated the anthocyanin and AA content in the stressed Nonabokra seedlings as
a means to ward off the damaging symptoms. This increase is well supported by the earlier observation where
Spm application induced an increment in certain antioxidants like reduced glutathione and polyphenols
(Radhakrishnan & Lee 2013). The lowered AAO activity and increased reducing power in stressed IR-64
seedlings raised from Spm-primed seeds also reflect the importance of Spm in maintaining higher AA pool with
better defense mechanism in IR-64, the sensitive cultivar.
To prevent ROS from damaging various cellular components, plants have developed multiple detoxification
mechanism, including the activation of various antioxidative enzymes like SOD, POD, CAT, APX and
glutathione reductase. In our case, salinity stress increased the activities of GPX and CAT in both the cultivars.
The elevated activities of CAT and POD were also observed in salinity-stressed cucumber (Duan et al. 2008).
The activity of APX, that uses AA as the specific electron donor, increased in the salt-tolerant cultivar during
stressed conditions, concomitant with the increase in AA. A similar increase in APX activity was observed in
Hordeum vulgare, Plantago maritima and Brassica campestris in response to salt stress (Hernndez et al. 2010).
However, in our study, a decreased APX activity was recorded in the salt-sensitive cultivar IR-64. Such
decrease is in accordance with earlier studies in wheat (Heidari & Mesri 2008) and Eugenia (Acosta-Motos et
al. 2015). In response to salt stress, the SOD activity increased in both IR-64 and Nonabokra seedlings, the
activity being more pronounced in the sensitive cultivar. A similar increase in SOD activity was observed in
salt-stressed pea, beet, maize and tomato (Azevedo-Neto et al. 2005, Koca et al. 2006). Seed priming with Spm
followed by seedling exposure to salinity stress showed quite a contrasting response of all the antioxidative
enzymes examined in the two cultivars. With respect to the GPX, Spm priming during salinity stress was
particularly significant in the sensitive cultivar, showing a direct elevation or stimulatory effect of GPX activity,
thereby rendering the tolerance mechanism in IR-64. The GPX activity, on the contrary, was down regulated by
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Spm in Nonabokra. A similar trend in Nonabokra was noteworthy with respect to APX and SOD as well, where
the stressed seedlings from Spm-primed seeds showed lowered enzyme activity, compared to the stressed
seedlings without Spm. This showed that Spm at the applied concentration has an intrinsic protective role during
salinity stress, so that the much heightened activity of such enzymes observed during salt stress is not that prerequisite in presence of Spm. The same explanation could also hold for SOD in Spm-primed, salt-treated IR-64
seedlings, where the down regulation is strikingly noteworthy. Our observation differs from many of the earlier
observations, where exogenous PA application in the form of foliar spray or hydroponic culture during salinity
or drought stress, mostly increased the activity of CAT, SOD, APX and POD (Roychoudhury et al. 2011, Shi et
al. 2013, Zhu et al. 2014). However, Velikova et al. (2000) showed that pre-treatment with PAs led to a
reduction of POD activity in acid rain-treated bean plants. Spm priming possibly pre-disposes the seedlings to
and mimics a stress-like condition and enables them for better acclimatization when they encounter the actual
stress situation, so that the higher activity of the antioxidant enzymes is not obligatory even in presence of NaCl.
Yet the seedlings can manage to survive with a lowered activity of the majority of antioxidant enzymes. Thus,
the anti-stress effect of PA pre-treatment during salinity stress is actually supported by the reduced defensive
response rendered by the antioxidative enzymes in presence of Spm, as reported in the present work. The
enhanced CAT activity with Spm in salt-treated Nonabokra is, however, in accordance with the earlier
observation by Farooq et al. (2009).
Several investigations have reported varietal differences in isozyme profile with salinity stress. While four
SOD activity bands were identified in the leaves of salt-tolerant Plantago maritima, only two bands were
observed in salt-sensitive P. media. Likewise, five POD activity bands were identified in the leaves of P.
maritima, whereas only two bands in P. media (Sekmen et al. 2007). A differential POD and SOD isozyme
activities among the four potato cultivars, Agria, Kennebec (relatively salt tolerant), Diamant and Ajax
(relatively salt sensitive) were noted under saline conditions (Rahnama & Ebrahimzadeh 2006). Short-term
salinity induced the expression of POD and SOD isozymes in cucumber seedlings (Du et al. 2010). In our
experiment, five isozymes of GPX (GPX1-5) were noted in the sensitive cultivar, while four (GPX1-4) in the
tolerant cultivar. The effect of Spm pre-treatment during salinity stress was evident from the higher induction of
GPX1 in IR-64. GPX1 was however feebly induced in the stressed Nonabokra seedlings, even after Spm pretreatment. GPX5 was almost undetected in IR-64 upon Spm priming, as compared to control. Unlike IR-64, the
GPX2 expression was sharper in the stressed Nonabokra seedlings, whether Spm pre-treated or not. Thus, the
two varieties responded differentially with respect to GPX isozyme induction during Spm pre-treatment, with
GPX1 playing more vital role in IR-64 and GPX2 in Nonabokra. In case of SOD, SOD4 was undetected in IR64, and the expression of SOD1, SOD2 and SOD3 was enhanced in the stressed seedlings of IR-64, raised from
Spm pre-treated seeds. SOD3 expression was higher in Nonabokra under salinity stress. El-baky et al. (2003)
observed that POD and CAT isozymes for different onion cultivars differed in number and relative
concentration due to salt stress. In our study, the induction of CAT2 and CAT3 was higher in Nonabokra during
salinity stress, while the expression was lower after Spm pre-treatment. The CAT1 expression in IR-64 was saltinducible and dependent on Spm treatment, whereas CAT3 expression was sharper in Nonabokra. Puyang et al.
(2015) showed that pre-treatment with Spd increased the intensity of APX and CAT isozymes in both the
cultivars, Kenblue and Midnight, and POD isozymes only in Kenblue cultivar of Poa pratensis. Exogenous Spd
elevated the intensities of isozymes of APX, POD and SOD in alfalfa (Zhu et al. 2014). Application of
exogenous Spd could also overcome oxidative damages in cucumber during salt and heat stress by causing
certain changes in the zymogram expression of some antioxidant enzymes (Du et al. 2010, Tian et al. 2012).
Our observation on EST showed that EST3 induction was considerably higher in Nonabokra than IR-64, where
the induction was noted only after salinity stress, with or without Spm pre-treatment. EST1 was also expressed
at a higher level in Nonabokra during stress, either in absence or presence of Spm. Such EST isozyme variation
was reported earlier by Swapna (2002) in four rice cultivars, viz., Pokkali (moderately salt-tolerant) and M-1-48,
Annapoorna and Jyothi (salt-sensitive), which indicated that this isozyme is also linked with salt tolerance.
Mung bean and Sueada maritima plants, cultured under in vitro conditions, exhibited the highest EST activity
between 150 and 400 mM NaCl. Overall, a clear-cut variation in GPX, CAT, SOD and EST isozyme profile was
discernible between the two cultivars in our work, with specific isoform(s) being up regulated or down regulated
with Spm pre-treatment.
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One of the strategies to regain turgor and resume growth during ionic stresses is to accumulate osmolytes
like reducing sugar, including major carbohydrates, total free amino acids and especially Pro, which
synergistically reduce the osmotic potential of the cytosol to facilitate water uptake (Roychoudhury et al. 2015).
Zahra et al. (2010) reported that sugar levels in rice leaves increased significantly under salt stress. However,
Alamgir & Ali (1999) observed that salinity reduced sugar content in four varieties, but increased sugar content
in five other varieties. Pro and free amino acid content in the salt-stressed tissues of Pennisetum glaucum
increased with increase in salt concentration as well as with duration of salt stress, thereby protecting the
cellular macromolecules, maintaining the osmotic balance and also scavenging the free radicals (Sneha et al.
2013). Chutipaijit et al. (2009) reported that free Pro content of rice varieties was significantly increased with
increasing salinity levels. Our observation showed a reduction in the level of reducing sugars in both the
cultivars, as well as decrease in total amino acids in Nonabokra during salinity stress. Salinity stress might have
led to a significant decrease in the efficiency of photosynthesis, thereby reducing the supply of soluble sugars.
The soluble sugars, under such challenging situations, are probably utilized extensively for growth and
maintenance of the osmotic homeostasis of cells. The reduced amino acid level could be accounted for by the
fact that the amino acids are probably channelized towards the production of novel proteins during stress,
rendering better tolerance in Nonabokra. Increase in free Pro level in the salt-stressed cultivars could either be
due to enhanced synthesis, decreased degradation or both. The implication of Spm in increasing the level of
reducing sugar in Nonabokra during salt stress is clear from our observation. Amino acids, especially Pro
detoxifies plants by scavenging ROS or preventing them from damaging cellular structures (Roychoudhury et
al. 2015). The constructive role of Spm priming in stress alleviation in terms of enhanced amino acid and Pro
accumulation seems to be more promising in Nonabokra. The reverse trend, viz., decrease in amino acid or Pro
content during salinity stress with Spm pre-treatment in IR-64 suggests an alternate mechanism by which Spm
priming reduce the impact of salinity stress in the sensitive cultivar.
The influence of NaCl on -amylase activity was different in the two rice cultivars. While the activity was
lowered in the sensitive cultivar, it increased to an appreciable extent in Nonabokra, signifying that sugar
mobilization and hence germination was not compromised in the latter. Ashraf et al. (2002) have suggested that
salt stress led to a decrease in -amylase activity and break down of starch into reducing and non-reducing
sugars in cotton. The amylase activity decreased with increasing salinity in Phaseolus vulgaris and increased in
maize. In case of rice, salt stress significantly inhibited the activity of -amylase during germination stage (Hualong et al. 2014), supporting our observation with IR-64. The reduction in -amylase activity in IR-64 could
account for the reduction in concentration and translocation of free sugars into the embryo axes during
germination and early growth. Exogenous Put treatment during salinity stress increased the -amylase activity
in the growing seeds of P. vulgaris, thereby increasing the germination percentage of salt-stressed seeds (Zeid
2004). Tipirdamaz et al. (1995) also reported that Put, Spd and Spm significantly increased -amylase activity
in barley seeds. Our data, showing slightly improved -amylase activity in stressed seedlings of IR-64 raised
from Spm-primed seeds, correlates with earlier observations, indicating that adverse effect of salt stress on
germination could be partially rectified in the salt-sensitive cultivar. The PPO activities were higher in bean and
maize seedlings treated with NaCl (Tuna et al. 2013). A higher PPO activity in the leaf tissues of groundnut var.
TAG-24 than in the variety TG-26 during water stress ensured better drought tolerance mechanism in the former
(Shinde & Laware 2015). Quite a contrasting result was derived in maize and Phaseolus mungo where PPO
activity was decreased with increasing salinity (Dash & Panda 2001). In our case, the PPO activity followed a
different trend in the two cultivars during salinity stress, with a significant decrease in the tolerant cultivar,
thereby enabling conservation of phenolics as antioxidants, while increasing to a small extent in the sensitive
cultivar. Earlier reports have shown that application of Spd increased the PPO activity in the leaves of sugarbeet
during salinity stress (Hajiboland & Ebrahimi 2013). Treatment with Spd also caused PPO activation and
mitigated the salinity effect in cucumber plants (Radhakrishnan & Lee 2014). In our case, the small increment in
PPO activity in salt-stressed IR-64 seedlings raised from Spm-primed seeds is in agreement with the available
reports.
Although enhanced stress tolerance via exogenous PA application as foliar spray or in hydroponic culture
are well documented in the available literatures, the mechanism of the protective effect of Spm pre-soaking of
seeds on the performance of NaCl-stressed, germinated seedlings is poorly studied and understood. The present
communication showed that oxidative damages encountered in the two examined rice cultivars could be
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effectively attenuated with varying degrees, if the Spm pre-treated seeds are germinated and grown under salt
stress. Spm itself exerts a direct antioxidative role, particularly in IR-64, thereby reducing the detrimental effect
of salt stress by lowering the MDA and H2O2 content, and retrieving the endogenous chlorophyll content. The
protective effect of Spm in Nonabokra was evidenced from the increase in osmolyte levels, rather than
regulating the antioxidant machinery. Spm also effectively rendered protection against salt stress in IR-64 by
enhancing the -amylase and PPO activity. In the nutshell, seed priming with Spm at the pre-sowing stage holds
a great promise as a traditional method of agriculture in growing major staple food crop like rice under salinity
regimes, so as to prevent cumulative damages and widespread crop losses.
ACKNOWLEDGEMENTS
Financial assistance from Science and Engineering Research Board (SERB), Department of Science and
Technology (DST), Government of India through the research grant (SR/FT/LS-65/2010) and from Council of
Scientific and Industrial Research (CSIR), Government of India, through the research grant [38(1387)/14/EMRII] to Dr. Aryadeep Roychoudhury is gratefully acknowledged.
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ISSN (E): 2349 1183

ISSN (P): 2349 9265
3(3): 634641, 2016
DOI: 10.22271/tpr.2016.v3.i3.083
Research article
Isolation and characterization of lectin from the leaves of

Euphorbia tithymaloides (L.)
Aruna A. Jawade, Shubhangi K. Pingle*, Rajani G. Tumane, Anvita S. Sharma,
Archana S. Ramteke and Ruchika K. Jain
National Institute of Miners Health, JNARDDC Campus, Wadi Nagpur-440023, Maharashtra, India
*Corresponding Author: pingle.shubhangi@gmail.com
Abstract: Lectins or glycoproteins from the leaves of Euphorbia tithymalo were isolated after
screening of different flora from Central India. The crude extract of leaves was dialyzed
andammonium sulfate precipitation done and followed by dialysis for the purification of lectins.
Protein concentration in the purified extract was 10 mg.ml-1 measured by Biuret Method. The
purified lectins were able to agglutinate human erythrocytes of ABO blood group system.
Agglutination was also visible with animal erythrocytes. Lectin of ET was Galactose/Lactose
specific and shows maximum activity at pH-7 and temperature between 4060 C. Molecular
weight of purified extract of ET was determined by 1D- SDS Polyacrylamide gel electrophoresis
which was found to be 70.24, 28.53 and 14.68 kDa.
Keywords: Agglutination - Blood group - Electrophoresis - Euphorbia - Glycoprotein.
[Cite as: Jawade AA, Pingle SK, Tumane RG, Sharma AS, Ramteke AS & Jain RK (2016) Isolation and
characterization of lectin from the leaves of Euphorbia tithymaloides (L.). Tropical Plant Research 3(3): 634
641]
INTRODUCTION
Lectins are proteins or glycoproteins of non-immune origin which possess the ability to agglutinate
erythrocytes or precipitate glycoconjugates by binding to the recognized and specific carbohydrate residue
present on cell surface (Ramteke 2009). Lectins are widely distributed in nature and can be found in many
plants, animals and microorganisms. Plant lectin contains at least one catalytic domain and has the ability to
recognize complex glycoconjugates (Peumans & Damme 1998). Lectins present in leaves, roots, stems and
seeds of plants perform different biological activities and help in secondary metabolism such as defense
mechanism. Due to specific binding capabilities, lectins involve in endocytosis, intracellular translocation of
glycoproteins, cellular regulation, migration and adhesion, phagocytosis, binding of microorganisms to target
tissues, control of morphogenesis, metastasis and many other activities (Sharon & Lis 2004, Abreu & Matthew
2006). ABO blood group system with Rhesus factor comprises of distinct determinant and lectins agglutinate
with specific type of antigen present on RBCs (Ajit & Kanjaksha 2016).
Euphorbia tithymaloid (ET) is a perennial succulent spurge. It is native to tropical and sub-tropical North
America and Central America. These shrubs are 6 to 8 feet long and 18 to 24 inch wide. ET grows in fertilized
sandy soil rich in metal concentration like boron, copper, iron, manganese, molybdenum and zinc. Their leaves
are alternate, sessile, glabrous and acuminate in shape. It is a carcinogenic plant thus has the ability to grow in
toxic soil very easily and rapidly. Sometimes, ET is also used to remediate soil and can be used as border of
garden. Lectins present in this plant have many medicinal use and help in curing many diseases also. It shows
anti-inflammatory, anti-bacterial, anti-septic, anti-hemorrhagic, anti-viral, anti-tumor and abortive properties. In
this study lectins from the leaves of ET were characterized in terms of their physical, chemical and biological
properties.
Leaves of ET were collected from Nagpur, Maharashtra in Central India and used as source of lectins.
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Human blood of groups A Rh+, B Rh+ and O Rh+ were collected from healthy persons. Animal bloods were
collected from veterinary hospital of Nagpur, Maharashtra. Ammonium sulfate, sodium chloride, dialysis
membrane-50, total protein kit (BioSystem-Reagent and chemicals), different sugars, pH buffers (pH 212),
acrylamide, bis-acrylamide, -Mercaptoethanol, coomassie brilliant blue, urea, thiourea, DDT, CHAPS, IPG
strips, bromophenol blue, glycerol, SDS, TEMED and other chemicals were purchased from Himedia and Serva.
Preparation of crude extract
Leaves of ET were collected from the road side area of Nagpur, Maharashtra. Leaves were washed 23 times
with tap water and then with distilled water and soaked in tissue paper. Leaves were homogenized with
minimum amount of saline by using mortar and pestle at 4C. Homogenized extract was allowed to filter by
using muslin cloth. The filtrate was centrifuged at 5000 rpm for 20 minutes. The obtained supernatant was
stored at 4C and used for further analysis (Patil & Despande 2015).
Purification of crude extract
The crude extract was dialyzed for the separation of proteins and removal of impurity and small moleculesby
dialysis membraneinnormal saline at 4C. This membrane contains micro pores through which the small
molecules easily escaped. Therefore, protein molecules having dimensions significantly greater than the pore
diameter are retained inside the dialysis bag. Ammonium sulfate (AS) was used to precipitate lectins from the
dialyzed extract by gradualaddition of ASat 4C. The precipitation obtained after 0100% saturation of AS was
centrifuged at 6,000 rpm for 30 minutes. Then, precipitate was dissolved in normal saline and again dialyzed till
the solution was free from ammonium sulfate fraction (ASF).
Protein Estimation
Protein was estimated by using commercially available protein kit.
Preparation of 2% erythrocytes
The human blood samples werecollected in heparinized tubes and stored at 4C. Erythrocytes were washed
34 times with normal saline. Washed RBCs were used for preparation of 2% erythrocytes solution (Olsen
1944). Similarly, animal bloodswere also preceded as mentioned above. This 2% erythrocytes solution was used
for determination of hemagglutination activity.
Agglutination assay
Agglutination test of purified extract was done by using 2% suspension of erythrocytes (Deshpande & Patil
2003). Hemagglutination activity was determined in 96 wells plate by using serial dilution. Agglutination was
observed visually with carpet and button pattern after 5 hrs. Hemagglutination unit (HAU) which represents the
titer strength was calculated with the reciprocal of last dilution of agglutination. Specific activity (SA) which is
HAU per mg protein was also calculated.
Agglutination inhibition assay
Agglutination inhibition assay was done by testing the ability of different carbohydrates like disaccharides,
pentoses, hexoses, oligosaccharides etc. to inhibit the agglutination (Kurokawa et al. 1976). 100 l of 500 mM
sugar solutions were incubated with 100 l lectin for 30 minutes at room temperature. The agglutination in the
presence of sugar was examined with 2% erythrocytes by the above described method. Minimum inhibitory
concentration was taken which did not agglutinate the erythrocytes.
pH stability studies
The pH dependence of ET leaves lectin was determined by using buffer ranging from pH 113. For pH 1:
0.1N HCl, for pH 2 & 3: 0. 2M glycine - HCl buffer, for pH 4 & 5: 0.2M sodium acetate buffer, for pH 6 & 7:
0.2M sodium phosphate buffer, for pH 8: 0.2M Tris HCl buffer, for pH 9: 0.2M glycine-NaOH buffer and for
pH 10 & 13: 0.2M carbonate-bicarbonate buffers were used. 100 l of lectin was incubated with 100 l of
different buffer solutions for 30 minutes at room temperature and then assayed for agglutination with 2%
erythrocytes (Suseelan et al. 1997).
Temperature stability study
Effect of temperature on the lectins of ET was observed by incubating 100 l of leaves extract at different
temperature ranging from 10100C for 30 minutes. Agglutination test was carried out with 2% erythrocytes
solution.
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SDS polyacrylamide gel electrophoresis (SDS-PAGE)
SDS-PAGE was done to determine the molecular weight of lectins of ET by the method of Weber & Osborn
(1969). A 10% stacking gel and 5% running gel was used in SDS-PAGE with a standard marker protein
(Aprotinin-6.5kDa, Lyzozyme-14.3 kDa, Soyabean Trypsin Inhibitor-20.1 kDa, Carbonic Anhydrase-29 kDa,
Ovalbumin-43 kDa, Bovine Serum Albumin-66 kDa and Phosphorylase b-97.4 kDa). After electrophoresis the
gel was stained with 0.2% coomassie brilliant blue (R250) and then destained in 10% acetic acid.
Isoelectric focusing
In 2-D electrophoresis, IPG (Immobilized pH gradient) strip of pH ranging 310 was used to perform
isoelectric focusing of the sample. The strip containing sample were rehydrated using rehydrating buffer (6M
Urea, 4% CHAPS, ampholyte, 0.1% Bromophenol blue) at room temperaturefor 18 hrs. 1D was performed
according to the standard method after which the strip was treated with equilibration buffer (1.5 M TrisHCl, 6M
Urea, 30% glycerol, 2% SDS, 0.01% Bromophenol blue and 200 mg of dithiothreitol) and with blocking buffer
(1.5 M TrisHCl, 6M Urea, 30% glycerol, 2% SDS, 0.01% Bromophenol blue and 250 mg of iodoacetamide).
Then 2D SDS-PAGE was allowed to run with 10% running gel at 15C. The spots appeared after 0.2%
Coomassie blue stain was destained by using 10% acetic acid.
RESULTS
A total 120 herbs and shrubs from Nagpur District were randomly selected and screened for identification of
agglutination activity. The crude extract of 25 plants showed agglutination activity with erythrocytes of different
blood groups of human and animals. On the basis of literature survey and information obtained from civilian
about medicinal valuesthree plantswere selected for further study. ET was selected as it showed good
agglutination activity against blood group system (ABO).
Protein estimation
Protein content in the dialyzed extract of ET was found to be 10 mg.ml-1 by using Biuret kit.
Agglutination assay
Table 1. Agglutination study of lectins of Euphorbia tithymaloides leaves with human and animal erythrocytes.
Erythrocytes
Human O
Human B
Human A
Cow
Dog
Fish
Hen
Agglutination
+++
++
+
+
-
Hemagglutination results in table 1 revealed that human blood group O showed strong agglutination as
compared to A and B with lectin from the leaves of ET. In figure 1, blood group A, B and O gives
carpet pattern till 6, 7 and 8 times dilutions respectively and on further dilution button pattern starts appearing
which represents no more further precipitation of lectins. Plant lectin has also showed agglutination against cow
erythrocyte but no agglutination was found in case of dogs, fish and hen (table 1). On the basis of result it was
observed that minimum concentration of lectin was required for agglutination of group O erythrocyte and was
followed by group B and group A. Hemagglutination unit (HAU) and specific activity (SA) were also
calculated and depicted in the table 2.
Figure 1. Hemagglutinationtiterwith ABO blood group.
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Table 2. Protein concentration in leaves of Euphorbia tithymaloides.
HAU/ml
A
B
O
10
320
640
1280
Dialyzed extract
Note: HAU-Hemagglutination Unit, SA-Specific activity.
Protein (mg.ml-1)
A
32
SA
B
64
O
128
Agglutination inhibition assay

Agglutination activity of lectin from leaves of ET was inhibited by D-Galactose and Lactose as showed in
table 3. Result indicated inhibition of lectin was due to galactose/lactose specific sugars.
Table 3. Inhibition of agglutination with different sugars by lectins of Euphorbia tithymaloides leaves.
Sugars
D-Glucose
Sucrose
Lactose
Sorbitol
D-Fructose
D-Maltose
D-Arabinose
D-Galactose
D-Xylose
D-Mannose
D-Ribose
Minimum concentration required to inhibit the

hemagglutination (mM)
No inhibition
No inhibition
500
No inhibition
No inhibition
No inhibition
No inhibition
500
No inhibition
No inhibition
No inhibition
pH stability
According to the result the optimum pH for maximum agglutination by leaves of ET was found to be neutral
(pH 7). The activity varies in pH ranging from 3 to 11. Agglutination activity was lost below pH 4 and above pH
11 as mentioned in figure 2.
Figure 2. Effect of pH on Agglutination activity of lectin of Euphorbia tithymaloides leaves.
Effect of temperature and thermal inactivation

Lectin from leaves of ET shows 100% agglutination between temperatures ranging from 40- 60C when
tested after heated for 1 h at temperature above 20C. Below 40C and above 60C till 70C agglutination
activity was half of initial and as the temperature increases the activity decreases and finally lost at 100C.
According to the observed result lectin was stable for long period of time but it thermally inactive after heating
at 100 C as showed in figure 3.
Figure 3. Effect of temperature on Agglutination activity of lectin of Euphorbia tithymaloides leaves.
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1D-SDS polyacrylamide gel electrophoresis
SDS-PAGE resulted into the appearance of bands with molecular weight of 70.24, 28.53 and 14.68 kDa in
the purified extract from leaves of ET showed in figure 4. Thus, these may be the molecular mass of lectins
present in ET.
Figure 4. Band pattern of lectins of ET on 1-D SDS-PAGE.
2D-SDS polyacrylamide gel electrophoresis

On the basis of analyses of 12 observed spots, which were ranging in between pIs of 6.5 to 29 as showed in
the figure 5.
Figure 5. Isoelectric focusing of lectins of ET on 2-D PAGE.
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DISCUSSION
This study focused on identification and characterization of the lectin activities from ET plant species.
Lectins were extracted and purified by using conventionalammonium sulfate precipitation method. The
purification procedure consists of 0100% ammonium sulfate saturation, which was followed by dialysis. The
conventional ammonium sulfate precipitation method was very useful technique for initial purification of lectins
from crude extract of Tridax procumben leaves (Ramteke & Patil 2005) and Volvariella volvacea (Mothong
2009).
The extract agglutinates the erythrocytes of ABO blood group which resembles the characteristics of most of
the glycoproteins. Nagata & Burger (1974) had reported that wheat germ lectins (WGL) were non blood group
specific lectins. The present study also convinced with the above report as lectins of ET agglutinated RBC of all
blood groups with similar competence indicating that there is no specificity towards blood groups. This may be
due to absence of lectins specific receptors on the surface of RBSs. Lectins of many plant species also
agglutinate with erythrocytes of different animals. Ramteke & Patil (2005) noted that lectin of Tridax
procumbans agglutinates dog erythrocyte. Similar results were also observed in case of lectins of ET which can
be also supported by the study of Ahmed & Chatterjee (1987).
Lectins have the specific ability to bind carbohydrates which was examined by hemagglutination inhibition
assay. Goldstein & Hayes (1978) has reported that the lectins of Euphorbiaceae family are galactose specific. In
the same line Irazoqui et al. (2005) and Lubaki et al. (1983) noted that E. miliiand, E. heterophylla species are
galactose specific lectins. Our findings are also on the same line as lectins of ET were inhibited by D-galactose
and lactose which shows galactose specificity. Similar inhibition was observed in Tridax procumbens (Ramteke
& Patil 2005), tubers of Dioscorea opposite (Chan & Ng 2013) and Zizyphus oenoplia (Butle & Patil 2015).
Optimum pH for maximum activity of lectins varies in different plant species. In present study lectins of ET
shows optimum activity at neutral pH-7 and at the same time it is inactive at extreme acidic and basic pH.
Similar activity had been observed in many species like Spatholobus parviflorus (Geethanandan 2010),
Volvariella volvacea lectins (Mothong 2009), bioactive lectin from Zizyphus oenoplia (Butle & Patil 2015),
calyx lectin of Tridax procumbens (Ramteke et al. 2005), Apios tuber lectin (Kenmochi et al. 2015) and
Jackfruit (Artocarpus integrifolia) lectin (Ahmed & Chatterjee 1987).
Thermal stability of lectins was studied in ET shows maximum activity at temperature range from 4060C
respectively and loses its activity at 100C. Similar results were observed in case of Jackfruit (Artocarpus
integrifolia) lectins (Ahmed & Chatterjee 1987) and Apios tuber lectin (Kenmochi et al. 2015) in which the
activity was lost after 85C. Geethanandan (2010) and Pereira et al. (2015) mentioned that lectins of
Spatholobus parviflorus and Colocasia esculenta respectively, lose their activity after 100C.
The purified lectins from ET were processed on One Dimensional SDS-PAGE to get molecular mass as
shown in figure 4. Lectins with molecular mass 70.24, 28.53 and 14.68 kDa were obtained. MW of lectin
present in E. heterophylla (Lubaki et al. 1983) resembles with the result as it also possesses dimeric protein with
two identical subunits of 32 kDa. Thus, it may be possible that ET have subunits of 33.91 and 31.33 kDa.
Two dimensional SDS-PAGE isoelectric points (pI) of ET were obtained as shown in figure 5. According to
Pereira et al. (2015) in Colocasia esculenta16 spots with pIs ranging from 6.5 to 9.5 were reported. Similarly, in
our study 12 spots with pIs ranging from 6.5 to 29 were obtained. Similar results were also obtained in
Volvariella volvacea (Mothong 2009) and Cyphomandra betacea (Xu 1991).
CONCLUSION
Partial purified ET plants lectins exhibited strong agglutination with erythrocytes of different species
however, the titer against O Rh + was higher than B Rh + and A Rh + and cow. In contrast, no haem
agglutination of goat, dog and fish erythrocyte was observed. Result would be explained by differences in
glycosylation of the surface protein in different species of erythrocytes. Agglutination activity of ET was
completely inhibited by Galactose and lactose sugars it exhibited that lectins of ET are galactose specific.
Plant lectins showed optimum activity at different pH and temperature range. Extreme acidic and basic pH
inhibits the lectin activity of ET whereas at neutral pH gives optimum activity. ET lectins works between
temperatures range 4060 C and gradually loses its activity at higher temperature and completely loss after
100C. It indicated that this lectin is not thermostable. Based on one dimensional study, bands with molecular
mass 70.24, 28.53 and 14.68 kDa were expressed in purified lectins samples. Galactose specific lectins isolated
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from other plants have been reported to be dimeric or tetremeric proteins. Thus, it may be possible that ET have
subunits of 33.91 and 31.33 kDa.
From the literature surveys and reports it can be said that the importance of lectin is widely spread in cellular
and molecular biology. There are several applications of lectins including treatment of various diseases, as a
potential therapeutic agent and lectins also act as markers. The role of lectins in research has been steadily
increasing these days. There are many unidentified flora lectins. So, those plants should be explored for
experimental investigation to make lectins next generations medicine and food.
ACKNOWLEDGEMENTS
Authors are thankful to Director of National Institute of Miners Health for their valuable support and
encouragement. We are also grateful to local people for their help during the field study.
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Irazoqui FJ, Hamp MMV, Lardone RD, Villarreal MA, Sendra VG, Montich GG, Trindade VM, Clausen H &
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Mothong N (2009) Lectins from straw mushroom cultivated in north- eastern Thailand, Ph.D. Thesis. Suranaree
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York City, USA.
Patil MB & Deshpande KV (2015) Isolation and characterization of lectin from leaves of Dregea volubilis.
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Pereira PR, Winter HC, Vericimo MA, Meagher JL, Stuckey JA, Goldstein IJ, Paschoalin VMF & Silva JT
(2015) Structural analysis and binding properties of isoform of tarin, the GNA-related lectin from Colocasia
esculenta. Biochimica et Biophysica Acta 1854(1): 2030.
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Ramteke AP & Patil MB (2005) Purification and characterization of Tridax procumbens calyx lectin.
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Ramteke AP (2009) Studies on the lectins of some medicinal plants, Ph.D. Thesis. Nagpur University, Nagpur,
Maharashtra, India.
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Vignamungo. Journal of Biosciences 22 (4): 439455.
Weber K & Osborne M (1969) The reliability of molecular weight determination by sodium dodecyl
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ISSN (E): 2349 1183

ISSN (P): 2349 9265
3(3): 642648, 2016
DOI: 10.22271/tpr.2016.v3.i3.084
Research article
Nutritional composition and fungi deterioration of canned tomato

products collected from Ibadan, South-western Nigeria
S. G. Jonathan1, B. J. Babalola1, O. J. Olawuyi2, J. A. Odebode3* and A. O. Ajayi4
1
Mycology & Biotechnology Unit, Department of Botany, University of Ibadan, Ibadan, Nigeria
Genetics and Molecular Biology, Department of Botany, University of Ibadan, Ibadan, Nigeria
3
Mycology Unit, Department of Botany, University of Lagos, Akoka Nigeria
4
Department of Microbiology, Federal University of Oye-Ekiti, Ekiti State, Nigeria
*Corresponding Author: odebode04@yahoo.co.uk
Abstract: The present study was conducted in order to evaluate fungi and proximate analysis in
three popularly consumed canned tomato products in Ibadan, Nigeria, Neurospora crassa was
isolated from Pomo and Terra products while Aspergillus flavus and Macrophomina phaseolina
was only isolated from Terra tin tomato products. The presence of Saccharomyces cerevisiae and
Cercospora sp. was also observed in Pomo tin tomato products. Penicillium chrysogenum and
Fusarium oxysporum was observed in Gino tin tomato products and Terra also shows the presence
of F. oxysporum. The proximate analysis shows that the crude protein, ash content, ether extract
and dry matter compositions of canned tomato products were significantly influenced by the
brands of tomato product analyzed and it was indicted that the tomato products were very rich in
nutrient. The Gino tin tomato presented the highest mean ash concentration with significant
differences with respect to the Pomo and Terra tin products. There were no significant differences
between the ether extract content when compared and significant differences were found within
the replicates of the three tomato tin tomato products. The Gino tin products had the least mean
value of crude protein which might be as a result of only two fungi isolates present while the high
crude protein in Terra tin products is as a result more fungi contaminants that were present during
isolation. The variations in aflatoxins levels in all the three mouldy tomato products indicates that
they pose a threat to human health since there was invasion by toxigenic fungi after three weeks of
storage. However, the opening of tin tomato products allows easy colonization of fungi and this
has health implications on human being, Therefore, tin tomato products should be used
immediately after opening.
Keywords: Proximate analysis - Tomato - Nutrient - Aflatoxins - Toxigenic fungi.
[Cite as: Jonathan SG, Babalola BJ, Olawuyi OJ, Odebode JA & Ajayi AO (2016) Nutritional composition and
fungi deterioration of canned tomato products collected from Ibadan, South-western Nigeria. Tropical Plant
Research 3(3): 642648]
INTRODUCTION
Tomato is a herbaceous plant (Solanum lycopersicum L.) and a member of the Solanaceae. It products are
widely consumed by humans all over the world as processed products such as canned tomato, sauce, juice
ketchup, stews and soup (Lenucci et al. 2006). Tomato products are essential source of vitamin A, vitamin C,
potassium, fiber (Herson & Hulland 1980, USDA 2012) and are considered as one of the most important
ingredient in many dishes. It is desirable as dietary choices for vulnerable population groups such as the elderly
(Banwart 1981, 2001, Buchann 2008) and it is associated with a reduced risk of chronic degenerable diseases
(Agarwa & Aai 2000, Rao & Agarwal 1998).Tomato seeds contain high quality plant proteins that can be
supplemented into various food products (Sogi et al. 2005). In recent years, tomato has received a considerable
increment in its horizontal and vertical total annual production (FAO 1999).
In Nigeria, the demand for canned tomato products has increased considerably, because of its prevention of
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heart diseases and prostate cancer (Jones 2008) and in the lowering of high blood pressure and because of its
fresh taste for salad. Tomatoes are now consumed world over. Tomatoes also contain calystegine alkaloids
(polyhydroxylated nortropane alkaloids) (Asano et al. 1997, 2001). Tomato products make a significant
contribution to human nutrition due to the concentration and availability of several nutrients in these products
and to their widespread consumption (Sahlin et al. 2004). The processing of canned tomato paste seems to
increase nutrient bioavailability, which could be due to the fact that the nutrients are detached or extracted from
their structures. This is particularly true for lycopene (Rao et al. 1998, Shi & Le 2000). Tomatoes and its
byproducts serve as raw materials for several secondary products. A very valuable constituent of tomato is the
red pigment carotenoid lycopene, an exceptionally efficient quencher of singlet oxygen and therefore an
important anti-oxidant. Lycopene, as well as other valuable substances such as beta-carotene, alphacarotene,
alpha-tocopherol, gamma-tocopherol and delta-tocopherol can be effectively extracted from tomato skins, seeds,
and other by-products using supercritical fluid extraction technology (Baysal et al. 2000, Rozzi et al. 2002).
Canned tomato pastes are packed in tin or steel cans, an air-tight container for distribution, storage or
preservation. Fungi may be found in canned tomato paste due to corrosion and leakage of the metals or from tin
foils used in packaging. These canned containers have a high potential of harboring toxigenic fungi. In this
study, the aim was to determine the nutritional analysis and the common fungi associated with the deterioration
of canned tomato pastes
Study area and sample collection
This study was conducted at the Mycology/pathology unit of the Department of Botany, University of
Ibadan, Ibadan, Nigeria. Gino, Pomo and Terra, three popular brands of canned tomato products which are
widely consumed among the University of Ibadan students were used in this research study and were purchased
at Bodija markets in Ibadan, Nigeria. These samples were collected in sterile nylon and transported to the
laboratory immediately.
Sterilization of Materials and Media Preparation
The canned tomato products were aseptically opened using a sterile tin cutter in a microbial free
environment. The media used were sterilized at 121C for 15minutes in an autoclave and were prepared
according to the manufacturers instruction. Culture media generally used for the study is potato dextrose agar
(PDA). All glass ware were sterilized in the hot air oven at 160C for two hours. The inoculating needle were
sterilized by flaming in the spirit lamp until red hot, working surface were sterilized by the application of
sodium hypochlorite and absolute ethanol
Isolation of pure cultures
5 ml of the canned tomato products was measured into each of the sterilized McCartney bottles labeled
accordingly. This was vigorously shaken and 1 ml of sample was pipette into a sterile McCartney bottles
containing 9 ml of distilled water. The sample was serially diluted and 1 ml each of aliquots of 106 and 107 were
added to molten PDA plates. The plates were allowed to solidify and incubated at 30C for 35 days. The fungal
colonies were counted every 24 hours. Successive hyphae tip were transferred until pure cultures of each of
fungus was obtained. Pure culture were obtained by picking distinct colonies of fungi from the pour plate using
inoculating needle and subculture into freshly prepared plates of PDA. The plates were incubated at room
temperature. After which the pure culture was transferred into slant.
Morphological and Microscopic Identification
With the aid of the sterile inoculating needle, pure fungi isolates were inoculated into the centre of sterile
potato dextrose agar plate to allow uniform growth distribution, hyphae formation with the colour and shape. A
sterile inoculating needle was used to pick a thin films 4872 hours old mycelium from a pure culture and was
transferred to a drop of Lactophenol cotton blue in a clean, grease free glass slide and was gently teased in the
stain to ensure mixing by using an inoculating needle. The slide was covered with a cove slip. Identification was
done with the aid of microscope X10 and X40 objective lenses. The shape and arrangement of the fruity body
was noted. This was done for the different isolates and the observations were recorded.
Analysis of Nutrient Composition of Kilishi
The crude protein, ether extract, ash content, and dry matter of the canned tomato products were determined
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according to AOAC (2005). The experimental plates were arranged in triplicates. Screening for aflatoxin B1
was also carried out using the procedure of AOAC Offical methods of analysis.
Data analyses
The data obtained were subjected to Analysis of Variance (ANOVA) using SPSS version 16.0. Duncan
Multiple Range Test (DMRT) was further used to separate treatment means where there was significant
difference. Tables, plates and graphs were also used to illustrate results as appropriate.
RESULTS
Different fungi were isolated from the various canned tomato products obtained from Bodija market, Ibadan.
Neurospora crassa Shear & B.O. Dodge was found in Pomo and Terra canned tomato products but was not
present in Gino products. Aspergillus flavus Link and Macrophomina phaseolina (Tassi) Goid. was only
isolated from terra canned tomato products. Saccharomyces cerevisiae Meyen ex E.C. Hansen and Cercospora
sp. was also isolated from Pomo canned tomato products but was not found in Terra and Gino products.
Penicillium chrysogenum Thom and Fusarium oxysporum Schlecht. emend. Snyder & Hansen was observed in
Gino tomato products (Table 1). Terra also shows the presence of Fusarium oxysporum but did not occur in
Table 1. Fungi isolated from canned tomato products purchased from Bodija market in Ibadan.
Fungi
Neurospora crassa Shear & B.O. Dodge
Aspergillus flavus Link
Macrophomina phaseolina (Tassi) Goid.
Aspergillus terreus Thom
Saccharomyces cerevisiae Meyen ex E.C. Hansen
Penicillium chrysogenum Thom
Cercospora sp.
Fusarium oxysporum Schlecht. emend. Snyder & Hansen
Note: +, - indicates present and not present respectively.
Pomo
+
+
+
-
Terra
+
+
+
+
+
Gino
+
+
Pomo products. The mean square effect of replicate, day after inoculation on the growth area of fungi found in
POMO, TERRA and GINO Tomato Canned products in presented in table 2. The effect of replicate is nonsignificant for the growth area of the fungi isolated from Pomo and Terra tomato canned products but highly
significant for the growth area of fungi found in Gino tomato canned products. The effect of day after
inoculation is also significant for the growth area of the fungi found in Pomo tomato canned products but nonsignificant for the growth area of the fungi isolated from Gino tomato canned products but highly significant for
the growth area of the fungi found in Terra Tomato canned products. The effect of replicates on the growth area
of fungi found in POMO, TERRA and GINO Tomato Canned products is shown in table 3. Replicate 1 is
significantly different from second replicate and third replicate. The least growth is of fungi isolated from Pomo
products were found in third replicate.
Table 2. Effect of Mean Square of Replicate, Day after inoculation on the growth area of fungi found in POMO, TERRA
and GINO Tomato Canned products.
Source of variation
Df
GAP
GAT
2
19.27ns
4.50ns
Rep
*
4
19.74
8.48**
DAI
38
6.72
3.00
Error
45
Total
44
Corrected total
Note: GAT= Growth area of fungi found in Terra, GAG= Growth area of fungi found in GINO.
*= P< 0.01 highly significant, **= P< 0.05 significant, ns= Non-significant.
GAG
10.20**
6.48ns
5.77
Table 3. Effect of Replicates on the growth area of fungi found in POMO, TERRA and GINO Tomato Canned products.
Replicate
1
2
3
GAP
6.79a
6.08ab
4.57b
GAT
7.35a
8.06a
6.98a
GAG
4.69b
3.28b
7.19a
Note: GAP= Growth area of fungi found in Pomo, GAT= Growth area of fungi found in Terra, GAG= Growth
area of fungi found in GINO. Means with the same letter in the same column are not significantly different at P<
0.05 using Duncans Multiple Range Test (DMRT).
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Table 4. Effect of Day After Inoculation on the growth area of fungi found in POMO, TERRA and GINO Tomato
Canned products.
DAI
GAP
GAT
GAG
3.80b
4.89c
3.89a
3
4.75ab
6.74b
4.72a
6
6.30ab
7.94ab
4.97a
9
7.03a
8.87a
5.53a
12
7.18a
8.87a
6.14a
15
Note: DAI= Day ater inoculation, GAP=Growth area of fungi found in Pomo, GAT= Growth area of
fungi found in Terra, GAG= Growth area of fungi found in GINO Means with the same letter in the
same column are not significantly different at P< 0.05 using Duncans Multiple Range Test (DMRT).
There are non-significance differences exhibited by the replicate on the growth area of all the fungi isolated
from Gino tomato canned products. However, third replicate is significantly different from first replicate and
second replicate which are non-significantly different from each other for the growth area of fungi isolated from
Gino Tomato Canned products. The Effect of Day after inoculation on the growth area of fungi isolated in
POMO, TERRA and GINO Tomato Canned products is shown in table 4. There is non-significance differences
between the growth area of the fungi isolated from Pomo tomato canned products at 12 and 15 DAI, but
significantly different from 6 and 9 DAI which are non-significantly different from each other. The growth are
of fungi found in Pomo products at 3DAI is significantly different with the least mean value of 3.80. Also, the
growth area of the fungi isolated from terra at 12 and 15 DAI are non-significantly different from each other but
significantly different from 3, 6 and 9 DAI which are significantly different from each other. There are nonTable 5. Mean square table of Tomato product showing the proximate analysis.
Source
df
CP (%)
AS (%)
EE (%)
DM (%)
2
57.26**
22.71**
0.00ns
0.00ns
Tomato
ns
2
2.95
6.02**
0.05**
1676.30**
Rep
4
4.77
0.18
6.11
1.88
Error
9
Total
8
Corrected total
Note: CP= Crude protein, AS= Ash, EE= Ether extract, DM= Dry matter.
significant differences between all the growth areas of the fungi isolated from Gino for all the days after
inoculation. The mean square effect shows that the Crude protein, Ash content were highly significant for the
tomato products but Ether extract, and dry moisture were non-significant for the tomato paste products. The
mean effect on the replicate also shows that it is highly significant for ash content, ether extract and dry
moisture but non-significant for crude protein (Table 5).
Table 6. Effect of Tomato Canned products on the proximate analysis.
Tomato products
CP (%)
AS (%)
EE (%)
DM (%)
3.11c
9.50a
0.86a
49.99a
Gino
5.26b
7.08b
0.87a
50.00a
Pomo
11.52a
4.01c
0.88a
50.00a
Terra
Table 6 shows the effect of tomato products on the proximate analysis. The Gino tomato product shows the
least mean value for CP and it is significantly different from the CP of POMO and TERRA. The highest mean
value for crude protein was shown in Terra. There is a significant difference among the ash content of Gino,
Pomo and Terra Tomato products. The least ash content was obtained from terra. For the ether extract and
moisture content, there were non-significant differences between Gino, Pomo and Terra. Table 7 shows the
Table 7. Effect of replicate on the proximate analysis of tomato products.
Replicate
CP (%)
AS (%)
EE (%)
DM (%)
7.67a
8.33a
1.00a
26.36bc
1
6.54ab
5.51c
0.74c
73.63a
2
5.69b
6.75b
0.87b
50.00b
3
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effect of replicate on the proximate analysis of tomato products. First replicate has the highest crude protein and
its significantly different from second replicate, third replicate has the least protein value of 5.69. For Ash
content, Replicate 1 is significantly different from second replicate and third replicate while second replicate
shows the least value for the ash content 5.51. The ether extract of first replicate is also significantly different
from second replicate and third replicate, second replicate shows the least value of 0.74. The effect of second
replicate on dry weight is significantly different from first and third replicates. First replicate has the least mean
value of 26.36. Figure 1 shows the presence of aflatoxin B1 in a mouldy kilishi.
Figure 1. Aflatoxin B1(ug/kg) Assay in Mouldy Tomato Canned products.
DISCUSSION AND CONCLUSION

The analysis carried out showed that various fungi can be isolated from canned tomato products. This study
agrees with the work of Kalyoncu et al. (2005) who reported the presence of Aspergillus flavus, A.
terreus Thom, Fusarium oxysporum, Penicillium ochraceus in home-made tomato paste samples from
the fields and markets in Manisa Province of Turkey. High rate of fungi found in Pomo and Terra canned
tomato products could be as a result of leakage in the packaging tin can. There might be two factors such as
packaging and processing method that influence growth of fungi and the proximate composition of tomatoes.
The result of this study is in accordance with the report made by Alabi & Esan (2013) who identified
Aspergillus flavus, A. fumigatus Fresenius, A. niger van Tieghem and Fusarium sp. associated with the spoilage
of the industrial tomato paste. The processing method is a more influential factor than production method. Some
differences in the ripening stage could decisively influence the studied proximate parameters. The aflatoxins
detected in all the three mouldy tomato products indicates that they pose a threat to human health since there
was invasion by toxigenic fungi after three weeks of storage. Microorganisms isolated from the tin tomato
products are in accordance with previous report where enzymes of A. flavus and A. fumigatus were found to be
responsible for the deterioration of tomato fruit (Adisa 1985). Aspergillus sp. is very common and is involved in
spoilage of food items. This work is also in line with the work of Kolawole et al. (2010) who reported the
presence of Aspergillus sp., Aspergillus niger, Rhizopus stolonifer (Ehrenb.: Fr.) Vuill. and Penicillium
chrysogenum in dried tomato products. The health status of man can be compromised with aspergillosis if after
large amounts of spores are inhaled. ANOVA reveals that the tomato pastes were very rich in nutrient and can
easily out rank all other vegetables in total contribution to human nutrition (Grubben & Denton 2004). The
proximate analysis showed that the crude protein, ash content, ether extract and dry matter compositions of
tomato fruits were significantly influenced by the brands of tomato product analysed. The Gino tin tomato
presented the highest mean ash concentration with significant differences with respect to the Pomo and Terra tin
products. The high ash content obtained in Gino tomato products might be due to the phosphorus fertilizer
supplementation that acted on the tomato fruit on the field (Oke et al. 2005). However, there were no significant
differences between the ether extract content when compared and significant differences were found within the
replicates of the three tomato tin tomato products. The mean content of total crude protein in the analyzed Terra
tin tomatoes was 11.52 which were significantly higher than other Gino tomato products. The GINO tin
products had the least mean value of crude protein (3.11) possibly as a result of the low level of fungi present
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(fungi isolates from Gino were Penicillium chrysogenum and Fusarium oxysporum) while the high crude protein
in Terra tin products is as a result more fungi contaminants (Cotran et al. 1999, Diane 2004). Atteh (2002)
reported a similar increase in the level of crude protein and ash of dried tomato. The result of the proximate
analysis showed that, there was significant difference (p<0.05) in the dry matter at replicate level of the tomato
tin product. The dry matter content of the tomato tin product ranged from 49.9950.00 for Gino, Pomo and
Terra respectively.
ACKNOWLEDGEMENTS
Special thanks to Department of Botany, University of Ibadan for access to the laboratory. There is no
conflict of interest whatsoever.
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3(3): 649653, 2016
DOI: 10.22271/tpr.2016.v3.i3.085
Research article
Taxonomic account of an Indian endemic, monotypic genus

Adenoon Dalzell with a note on lectotypification of
Adenoon indicum Dalzell
Bandana Bhattacharjee1*, Sobhan Kumar Mukherjee2 and P. Lakshminarasimhan3
1
Central National Herbarium, Botanical Survey of India, Howrah-711103, West Bengal, India
2
Department of Botany, University of Kalyani, Kalyani-741235, West Bengal, India
3
Botanical Survey of India, Western Regional Centre, 7-Koregaon Road, Pune-411001, Maharashtra, India
*Corresponding Author: bandanabsi@rediffmail.com
Abstract: The genus Adenoon is endemic to India with its only species A. indicum distributed in
Western Ghats of Goa, Maharashtra, Karnataka, Kerala and Tamil Nadu. A taxonomic account of
the genus is provided here along with detailed descriptions and photo-plates and a lectotype is
designated for A. indicum to fix the application of the name.
Keywords: Asteraceae - Endemic - Monotypic - Vernonieae - Lectotype.
[Cite as: Bhattacharjee B, Mukherjee SK & Lakshminarasimhan P (2016) Taxonomic account of an Indian
endemic, monotypic genus Adenoon Dalzell with a note on lectotypification of Adenoon indicum Dalzell.
INTRODUCTION
The genus Adenoon Dalzell (Asteraceae: Cichorioideae: Vernonieae) was described by Dalzell (1850) with a
single species A. indicum Dalzell. The genus is characterized by herbaceous (perennial) habit, alternate, sessile
leaves with ovate to obovate-elliptic lamina and irregularly serrated margins, homogamous heads with long
peduncles in terminal corymbose panicles, purple florets devoid of pappus, terete to narrowly oblong-ovoid
strongly 10-ribbed achenes. The genus is monotypic as any other species under the same is yet to be described.
The present communication is a part of the revisionary study on the tribe Vernonieae Cass. for Flora of
India scheme of Botanical Survey of India which is mainly based on thorough scrutiny of authentic literature
(including protologue), the study of herbarium specimens (including types) deposited at various Indian as well
as foreign herbaria and study of live-specimens collected during the field surveys. Morphological details of the
specimens are critically observed under Olympus SZ-51 dissecting microscope. Flowers from fresh collections
and herbarium/dry specimens are dissected for drawing descriptions and preparation of photo-plate. After
completion of the study, the collected specimens are poisoned, pressed, mounted on standard herbarium sheets
and deposited at CAL with field-data written on the herbarium-labels for future reference.
RESULTS
Taxonomic account of the genus Adenoon Dalzell:
Adenoon Dalzell in Hookers J. Bot. Kew Gard. Misc. 2: 344. 1850; Benth. in Benth. & Hook. f., Gen. Pl. 2(1):
170. 1873; C.B. Clarke, Compos. Ind. 5. 1876; Hook. f., Fl. Brit. India 3: 229. 1882; Uniyal in Hajra & al.
(eds.), Fl. India 13: 331. 1995; H. Rob. in Kubitzki (ed.), Fam. Gen. Vas. Pl.: 171. 2007.
Type: Adenoon indicum Dalzell
Erect, terrestrial, rigid, perennial herbs. Stem simple below, branched above, angled, grooved, scabrous,
hairy. Leaves alternate, sessile; lamina ovate to obovate-elliptic, margins irregularly serrated. Heads in
corymbose panicles, homogamous, long-pedunculate, bractetate at the forks and below the head. Involucre
campanulate; phyllaries numerous. Florets actinomorphic, purple. Pappus absent. Corolla slender, tubular, limbs
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narrowly 5-cleft. Anther-base sagittate, shortly tailed. Style branched at apex, arms subulate, hairy. Achenes
terete to narrowly oblong-ovoid, subcompressed, with numerous glands on wall, 10-ribbed.
Distribution: The genus is monotypic and endemic to India (Goa, Maharashtra, Karnataka, Kerala, Tamil Nadu).
Etymology: The name of the genus is derived from the Greek words aden (glands), because of the presence of
numerous glands in between the ridges of the achene wall.
Adenoon indicum Dalzell in Hookers J. Bot. Kew Gard. Misc. 2: 344. 1850; Dalzell & A. Gibson, Bombay Fl.:
121. 1861; Hook. f., Fl. Brit. India 3: 229. 1881; T. Cooke, Fl. Bombay 2: 9. 1904; Gamble, Fl. Madras 2: 668.
1921; B.D. Sharma & al., Fl. Karnataka Anal.: 134. 1984; V. Chandrasekaran in A.N. Henry & al. (eds.), Fl.
Tamil Nadu India Anal. 2: 28. 1987; R.R. Rao & al., Fl. Ind. Enum. Asterac.: 1. 1988; S.D. Deshp. & al., Fl.
Mahabaleshwar 1: 299. 1993; Sivar. & P. Mathew, Fl. Nilambur: 357. 1997; M.R. Almeida, Fl. Maharashtra
3A: 66. 2001; Singh & al. (eds.), Fl. Maharashtra St. 2: 180. 2001; T.S. Nayar & al., Flow. Pl. Kerala: 98. 2006;
Karthik. & al., Fl. Pl. India Dicot. 1: 241. 2009.
Types: Bombay, Dalzell s.n. [lectotype, designated here: K (K000814618), photo! (Fig. 1)]; Mihi?, Ex Herb
Dr Stocks, Dalzell s.n. [syntype: K (K000814616), photo!].
Vernacular/Common names: Blue Sonki, Motha Sonki, Dhuni, Kusamb
Erect, terrestrial, rigid, perennial herbs, 2550 cm tall. Stem simple below, branched above, angled, grooved,
scabrous, pubescent, hairs simple, multiseptate. Leaves alternate, sessile; lamina ovate to obovate-elliptic, 110
0.56.5 cm, light green above, pale beneath, margins irregularly serrate, serrations ending in mucro, acute to
short-mucronate at apex, trinerved, prominently nerved beneath, scabrous, with thick hairs on both sides, hairs
articulated, glandular. Heads 11.5 cm across with purple florets; peduncle up to 10 cm long; bracts oblonglinear-lanceolate, cartilaginous, mucronate, rough, trinerved, hirsute, hairs jointed, glandular. Phyllaries
numerous, 45-seriate, 26 1.02.2 mm, imbricate, glandular hairy on the back, ciliate on the margins in the
upper half, outer shorter, oblong-lanceolate to ovate-lanceolate, progressively increasing in length, inner oblongelliptic, thinner. Florets up to 50, purple. Pappus absent. Corolla regular, slender, tubular, limbs narrowly 5cleft, each ca. 2.0 0.5 mm, tube 56 mm long. Anthers ca. 3 mm long, base sagittate, shortly tailed; filaments
1.52.0 mm long. Style 911 mm long, base with ca. 0.5 mm long knob like structure, bifid at apex, arms 1.5
2.0 mm long bearing stigmatic surfaces, subulate, hairy. Achenes terete to narrowly oblong-ovoid with obtuse
ends, 23 0.71.2 mm, subcompressed, glabrous, 10-ribbed, ribs very stout, glandular between ribs. (Fig. 2)
Flowering & Fruiting: AugustApril.
Habitat: Common in open forest areas.
Distribution: INDIA (Goa, Maharashtra, Karnataka, Kerala, Tamil Nadu); Endemic.
Etymology: The species was named after India, the country from where the types were collected.
Specimens examined: INDIA, Goa, Castle Rock, ca. 610 m, October 1908, A. Meebold 9863 (CAL). Karnataka,
Coorg district, Brahmagiri hill slope, ca. 1372 m, 30.11.1976, T.A. Rao & B.C. Banerjee 11669 (CAL);
Chikkamagaluru district, Kulhutty, Bababoodan, ca. 12201524 m, October 1908, A. Meebold 9403 (CAL);
Chikkamagaluru, Bidertallu?, Mudigere, 18.10.1965, R.K. Arora 5069 (CAL); Dakshina Kannada, South
Cauasa ghats, 1873, R.H. Beddome (MH); Mysore district, Megarvalli, Agumbe, 14.02.1961, R.S. Raghavan
68161 (CAL); Uttara Kannada district, Thiai Ghant?, 15.09.1891, W.A. Talbot 2518 (CAL). Kerala, Wayanad
district, Brahmagiri, ca. 12201585 m, 05.12.1907, C.E.C. Fischer 185 (CAL); Brahmagiri, 1000 m,
08.04.1988, C. Ramesan 41348 (CALI); 900 m, September 1988, C. Ramesan 41348 (CALI); Kottayam,
Ithithassum, Changanacheday, 25 m, 04.02.1984, V.T. Antony 262 (MH). Maharashtra, Kolhapur district,
Kondosh, 27.09.1998, S.R. Yadav 5817 (SUK); Kondosh, M.M. Sardesai 613 (SUK); Satara District,
Mahabaleshwar, T. Cooke 88 (CAL); March 1883, T. Cooke s.n. (CAL); Mahabaleshwar, T. Cooke 105 (CAL);
Mahabaleshwar, T. Cooke s.n. (MH); Mahabaleshwar, Niwar, Fitzerald Ghat?, c. 1067 m, 29.12.1950, P.V. Bole
220 (CAL); Kas, October 1992, M.P. Bachulkar & Cholekar 5459 (SUK); Mahabaleshwar, V.D. Vartak s.n.
(AHMA); Mahabaleshwar, 29.10.13, B. Bhattacharjee 62150, 62151, 62152 (CAL); Sindhudurg district,
Amboli area, 30.11.1982, K. Kulkarni 27370 (AHMA); Amboli, 650 m, October 2012, S. Raskar s.n. (CAL);
Thane district: Thane, Sadsya Ghat top, Tok Range, 16.11.1968, K.V. Billore 115455 (CAL); Thane, Kedarnath
hill slope, Takavada Range, 16.11.1968, K.V. Billore 115487 (CAL); Without precise locality, Concan, Dr.
Stocks 140 [P (P02837341, P02837342, P02837343), photo!)].
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Figure 1. Adenoon indicum Dalzell: Lectotype (K000814618) [ copyright of the Board of Trustees of the Royal Botanic Gardens,
Kew]
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Figure 2. Adenoon indicum Dalzell: A, Habit; B, Heads; C, Involucre; D, Phyllaries; E, Floret; F, Gynoecium; G, Close-up of
ovary; H, Close-up of style apex; I, Stamens; J, Achenes [Prepared from B. Bhattacharjee 62150 (CAL) before mounting].
652

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Note on lectotypification of Adenoon indicum Dalzell:
While describing A. indicum, Dalzell (l.c.) mentioned that the plants were collected from Phonda Ghaut,
Syhadree which flowered in September. He further mentioned Every part of this plant, including corolla and
anthers, is covered with white, smooth, oval glands, like the eggs of some insects. Two specimens of A.
indicum collected by Dalzell were traced at K and both of these were indicated as nov. gen. by Dalzell. The
precise locality of collections, date of collection etc. are not provided on the label-data of these specimens.
However, Dalzell provided a crude description and an illustration of a single floret in a separate label which was
mounted on a K-specimen (K000814618). Further, Dalzell wrote sp. scabrum and Bombay on the same
sheet. All these informations provided in K-specimen (K000814618) clearly indicate that this particular
specimen was emphasized by Dalzell while describing A. indicum. Therefore, the K-specimen (K000814618) is
selected here as lectotype of A. indicum according to Art. 9.2 of ICN (McNeill et al. 2012) which also in full
agreement with the description provided in the protologue. The specimens Dr. Stocks 140 presently deposited
at P (studied by J.D. Hooker) were not examined by Dalzell while describing A. indicum and thus not considered
as original material. Two more specimens were traced at CAL and as per the label-data these specimens were
purchased from Dalzells herbarium in 1878 (Herb. of N. Dalzell Bombay: purchased 1878). However,
collection locality, date of collection and name of the collector were not written on the labels/sheets. Further,
there is no indication that Dalzell studied these specimens before publishing A. indicum. Therefore, these two
CAL-specimens, which most probably postdate the protologue of A. indicum, are also not considered as
original material of A. indicum.
ACKNOWLEDGEMENT
The authors are thankful to Dr. Paramjit Singh, Director, Botanical Survey of India (BSI), Kolkata for
providing research facilities.
REFERENCES
Dalzell NA (1850) Contribution to the Botany of Western India. Hooker's Journal of Botany and Kew Garden
Miscellany 2: 336344.
McNeill J, Barrie FR, Buck WR, Demoulin V, Greuter W, Hawksworth DL, Herendeen P, Knapp S, Marhold K,
Prado J, Prudhomme Van Reine WF, Smith GF, Wiersema JH & Turland NJ (eds) (2012) International
Code of Nomenclature for algae, fungi, and plants (Melbourne Code): Adopted by the Eighteenth
International Botanical Congress Melbourne, Australia, July 2011. Regnum Vegetabile 154. Knigstein:
Koeltz Scientific Books, 140 pp.
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3(3): 654661, 2016
DOI: 10.22271/tpr.2016.v3.i3.086
Research article
Floristic composition and vegetation analysis of Hulikal Ghat

region, central Western Ghats, Karnataka
Vinayaka K. S.1* and Krishnamurthy Y. L.2
1
Department of Botany, Kumadvathi First Grade College, Shikaripura-577427, Shimoga, Karnataka

2
Department of Studies and Research in Applied Botany, Kuvempu University,
Shankaraghatta-577451, Shimoga, Karnataka
*Corresponding Author: ks.vinayaka@gmail.com
Abstract: Forest ecosystem is one of the most important terrestrial ecosystems of the world.
Biodiversity is a dynamic process among living organisms exhibiting different degrees of activities
according to their placement in nature. The present study was carried out in the Hulikal forest
(1346'15" N to 1342'30" N and 751'30" E to 755'15" E) Hosanagar taluk of Shimoga district,
Karnataka. Study areas are located at an elevation range of 560 MSL to 800 MSL above the sea
level and it is having highest rainfall receiving area in Karnataka. The Hulikal forest consists of
almost evergreen forest with swamp/marshy vegetation. Major tree species found in the study are
Litsea floribunda, Garcinia gummi-gutta, Cinnamomum verum, Myristica malabarica etc. with
shrubs like Carissa carandas, Croton malabaricus, Memecylon malabaricum, Maesa indica, Leea
indica etc. In Hulikal forest a total of 2172 samples were recorded from 30 quadrats. They are
belongs to 231 species and 60 families, among them 53 herb, 51 shrubs, 31 climbers and 96 were
trees species. Lower diversity of herbaceous plants is present here because of closed canopy. The
Hulikal forest showed more canopy trees with evergreen forests. The trees are tall and long in
height. Fahrenheitia zeylanica is an important species with Impotence Value Index (IVI) of 10.4
and basal area of 0.7. The Shannon diversity index value of the herbaceous species, climbers and
tree species were 3.6, 3.8 & 4.3 respectively. The study revealed that this region is very rich in
species composition.
Keywords: Forest ecosystem - Tree diversity - Diversity indices - Hulikal - Evergreen.
[Cite as: Vinayaka KS & Krishnamurthy YL (2016) Floristic composition and vegetation analysis of Hulikal
Ghat region, central Western Ghats, Karnataka. Tropical Plant Research 3(3): 654661]
INTRODUCTION
Forests are renewable resources encompassing millions of living organisms both plants and animals living in
perfect harmony with nature. Forest ecosystem is one of the most important terrestrial ecosystems of the world.
Biodiversity is a dynamic process among living organisms exhibiting different degrees of activities according to
their placement in nature. Tropical forests often referred to as one of the most special diverse terrestrial
ecosystem (Ashish et al. 2006). The Western Ghats are considered as the 18th mega biodiversity centre of the
world and it is recognized as one of the biological Hot spots of the world. These forests are unique ecosystem
due to their rich plant and animal diversity (Mayer et al. 2000). Correct inventorization and assessment of
biodiversity in different habitats is also necessary for evolving a long term strategy for rehabilitation of
endangered species in similar alternate habitats when original habitat gets destroyed (Ali et al. 2006). The forest
types in India ranges from thorny scrubby jungle to moist ever green forest along with moist grasslands and
characteristic shoal vegetation. In each of these different types of forests, a very diverse plant species are found
growing naturally. Identification of species and their diversity is a difficult task and it is virtually impossible to
have a complete inventory of Indian biodiversity. However, there has been a limited investigation to
characterize species at regional levels. Western Ghats is threatened by the catastrophe and anthropogenic
activities to larger extent are frequently responsible for endangering species through various ways such as
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degradation and fragmentation of habitats over exploitation and effect of pest and diseases. The diversity of
Western Ghats regions of Karnataka depleted in fast rate. For example, in the Uttar Kannada regions the forest
area has condensed from 8000 km-2 to 600 km-2 in about 40 years (Myens et al. 2000, Ananth & Potter 1997).
This constitutes an enormous loss of biodiversity in a small area over a short period of time. So for the
conservation and sustainable development of the Western Ghats, elaborate and long term research study is
necessary. Biodiversity losses have been alarmed in the developing countries in the tropics (FAO 1990)
estimated that about 60% of the tropical forests of the world have already been destroyed. As for the
biodiversity of Western Ghats is concern limited attempts has been made by certain group of researcher to
document floristic diversity (Ramkumar & Parthasarthy 2001). There are certain areas within the Western Ghats
which remains to be explored for their biodiversity. The endemic species are in threat due to damming and
clearing diversity of KMTR Western Ghats region (Ganesan 2002). Forest degradation not only cause reduction
in biodiversity but also leads to the change in the community composition (Prem Kumar & Uma Nandri 2003).
Climate is probably the most important determinant of vegetation patterns globally and has significant
influence on the distribution, structure and ecology of forests. The global assessment has shown that future
climate change is likely to significantly impact forest ecosystem (Ravindranath et al. 2006). Species population
shows two kinds of mature phase, where the topography is raised and gently sloping the vertical structure of the
stand is discontinuous and where the stand is lower vertical structure of the stand is continuous (Pascal &
Pellissier 1996, Utkarsh et al. 1998, Pelissier 1998, Seetharam et al. 2000). Increasing human population in the
last few decades, demanding development in various areas has resulted directly or indirectly in sudden and often
for reaching disturbances in natural ecosystem (Raizada & Vaid 1957). Hulikal forest has been facing many
problems, various mining activities; power projects, luxuriant growth of exotic species and others are rendering
the existence of rich floristic diversity. In this attempt we carry out the vegetational analysis of the area.
MATERIALS AND METHODOLOGY
Study area
The present study was carried out in the Hulikal state forest 1346'15" N to 1342'30" N latitude and
751'30" E to 755'15" E longitude of Hosanagar taluk of Shimoga district, Karnataka. Study areas are located at
an elevation range of 560 MSL to 800 MSL above the sea level. The rainfall varies from 850 to 900 mm per
annum with a temperature range of 15 to 30 C. Hulikal is recently recognized as highest rainfall area in south
India. Most of the rainfall occurs in the month of June to September carried by south west monsoon different
weather parameters which are recorded in study areas during the last 10 years (20032013) are given (Fig.1).
Figure 1. Average Rainfall and Temperature at Hulikal Forest region (20032013).
Vegetation type
The Hulikal forest consists of almost all evergreen forest with swamp/marshy vegetation (Fig. 2). Major tree
species found in the study are Litsea floribunda, Garcinia gummi-gutta, Cinnamomum verum, Myristica
malabarica etc., with shrubs like Carissa carandas, Croton malabaricus, Memecylon malabaricum, Maesa
indica, Leea indica etc., and herbs like Habenaria longicorniculata, Impatiens scapiflora, Costus speciosus,
Cassia tora, Mimosa pudica etc., as major ground species.
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Figure 2. Forest types in Hulikal Ghat region: A, GIS map showing forest types; B, View of evergreen forest; C, View of
Swamps.
Vegetation Analysis
The study site comprises of different types of vegetation. A total of 30 belt transects each measuring 255 m
were laid in Hulikal forest in which plants were documented. All the plants above 10 cm GBH (girth at breast
height) were considered as trees whose girth has been measured for the basal area. Shrubs and herbs were
recorded in 5m 5m and 1mx1m were identified and confirmed by using various region floras (Gamble 1935,
Pascal & Ramesh 1987, Yoganarasimhan & Razi 1981, Ramaswamy 2001, Neginhal 2004, Ganeshaiah et al
2002). The vegetational data was qualitatively and quantitatively analysed for abundance, density, frequency,
dominance and basal area following (Cottam & Curtis 1956). The Important value index (IVI) for the species
was determined as the sum of relative frequency, relative density and relative dominance (Pielou 1975). Based
on the data of the occurrence of species within the transects by Shannons diversity index (H) and Simpsons
diversity index were calculated as per (Magurran 1988).
Density and relative density is calculated as
Frequency and relative frequency is calculated as
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Abundance is calculated as
Relative dominance is calculated by
Basal area is calculated as

Basal area = BA= r2
Important value index (IVI) is calculated as
IVI = Relative frequency + Relative Density + Relative BA
Based on the data of the occurrence of species in the transects by Shannons diversity index was calculated
which is represented below
H' = -pi lnpi
where pi = (ni/N)
Other diversity indices such as Simpsons values (D), Simpsons diversity index (E), was calculated as
follows.
Where ni = Number of individuals of the ith species; N = Total number of individuals

RESULTS
In Hulikal forest a total of 2172 individuals recorded from 30 quadrats. They belong to 231 species and 60
families. Lower diversity of herbaceous plants is present because of closed canopy. The Impatiens scapiflora is
high diversity represented by 20 individuals with IVI of 9.0 and Girardinia diversifolia, Mimosa pudica shows
IVI of 8.7 and 7.0 respectively. Rauvolfia serpentina shows less frequency with IVI of 1.2 (Table 1). Shrubs
diversity is represented by Lantana camara with 30 individuals and IV of 10.3 which is followed by Maesa
indica, Leea indica with IVI of 9.8 and 8.3 respectively. Maytenus emarginata shows less IVI of 0.8 (Table 2).
This forest region shows less number of climbers. Naravelia zeylanica is an important species with IVI of 14.8
followed by Thunbergia mysorensis, Piper hookeri with IVI of 14.0 and 13.8 respectively. Clematis gouriana
shows less IVI of 1.5 (Table 3).
Table 1. Herb species composition with their Re-density, Re-frequency and Importance Value Index in Hulikal Forest. (RDRelative Density, RF-Relative Frequency, IVI- Importance Value Index)
S.N.
Herb Species
1
2
3
4
5
6
Impatiens scapiflora Heyne ex Roxb.

Girardinia diversifolia (Link) Friis
Mimosa pudica L.
Justicia montana (Nees.) Wall.
Curculigo orchioides Gaertner
Chassalia curviflora var. ophioxyloides (Wall.) Deb &
B.Krishna
7 Ophiorrhiza rugosa var. prostrata (D.Don) Deb &
Mondal
8 Zingiber cernuum Dalzell
9 Triumfetta rhomboidea N. Jacq.
10 Laportea interrupta (L.) Chew
Balsaminaceae
Urticaceae
Fabaceae
Acenthaceae
Zingiberaceae
Rubiaceae
Number of
individuals
20
20
17
15
15
16
Rubiaceae
12
2.4
3.5
6.0
Zingiberaceae
Tiliaceae
Urticacae
12
13
12
2.4
2.7
2.4
3.5
3.2
2.8
6.0
5.8
5.3
Family
RD
RF
IVI
4.1
4.1
3.5
3.1
3.1
3.3
5.0
4.6
3.5
3.9
3.9
3.2
9.0
8.7
7.0
7.0
7.0
6.5
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Table 2. Shrub species composition with their Re-density, Re-frequency and Importance Value Index in Hulikal Forest.
(RD- Relative Density, RF-Relative Frequency, IVI- Importance Value Index)
Sl. No.
1
2
3
4
5
6
7
8
9
10
Shrub Species
Family
Lantana camara L.
Maesa indica (Roxb.) DC.
Leea indica (Burm. f.) Merrill
Memecylon malabaricum Cogn.
Rotheca serrata (L.) Steane & Mabb
Pandanus canaranus Warb.
Chromolaena odorata (L.) King & Robinson
Scleropyrum pentandrum (Dennst.) Mabb.
Memecylon umbellatum Burm. f.
Psydrax dicoccos Gaertn.
Verbinaceae
Myrsinaceae
Vitaceae
Melastomaceae
Verbinaceae
Pandanacae
Asteraceae
Santalaceae
Melastomaceae
Rubiaceae
Number of
individuals
30
27
24
30
20
20
30
21
18
15
RD
RF
IVI
5.0
4.5
4.0
5.0
3.3
3.3
5.0
3.5
3.0
2.5
5.3
5.3
4.3
3.3
4.7
4.3
1.3
2.7
3.0
3.0
10.3
9.8
8.3
8.3
8.0
7.6
6.3
6.1
6.0
5.5
Table 3. Climber species composition with their Re-density, Re-frequency and Importance Value Index in Hulikal Forest.
(RD- Relative Density, RF-Relative Frequency, IVI- Importance Value Index)
Sl. No.
1
2
3
4
5
6
7
8
9
10
Climber Species
Family
Naravelia zeylanica (L.) DC.

Thunbergia mysorensis (Wight) T. Anderson
Piper hookeri Miq.
Rubia cordifolia L.
Piper hymenophyllum (Miq.) Wight
Dioscorea bulbifera L.
Dioscorea pentaphylla L.
Gymnema sylvestre (Retz.) R. Br. ex Schultes
Celastrus paniculatus Willd.
Schefflera venulosa (Wight & Arn.) Harms
Rhamnaceae
Acanthaceae
Piparaceae
Rubiaceae
Piperiaceae
Dioscoraceae
Dioscoraceae
Asclepadeaceae
Celastraceae
Araliaceae
Number of
individuals
25
22
23
19
17
21
19
14
15
13
RD
RF
IVI
7.1
6.3
6.6
5.4
4.8
6.0
5.4
4.0
4.3
3.7
7.7
7.7
7.2
4.3
4.8
3.4
3.8
4.8
4.3
4.3
14.8
14.0
13.8
9.7
9.7
9.3
9.3
8.8
8.6
8.0
Table 4. Tree species composition with their Re-density, Re-frequency and Importance Value Index in Hulikal Forest. (BA- basal
Area, RD- Relative Density, RF-Relative Frequency, Re-BA- Relative basal area, IVI- Importance Value Index)
S.N.
Tree Species
1
2
3
4
5
6
7
8
9
10
Fahrenheitia zeylanica (Thw.) Airy Shaw

Hopea ponga (Dennst.) Mabberly
Murraya paniculata (L.) Jack
Garcinia xanthochymus Hook. f. ex T. Anderson
Leea indica (Burm. f.) Merrill
Holigarna arnottiana Hook. f.
Madhuca neriifolia (Moon) H. J. Lam
Litsea floribunda (Blume) Gamble
Aporosa lindleyana (Wt.) Baill.
Heynea trijuga Roxb. ex Sims
Family
Number of
BA
individuals
RD
RF
Re-BA IVI
Euphorbiaceae
Dipterocarpaceae
Meliaceae
Clusiaceae
Leeaceae
Anacardaceae
Sapotaceae
Lauraceae
Euphorbaceae
Meliaceae
30
30
22
25
19
16
19
14
14
9
4.1
4.1
3.0
3.4
2.6
2.2
2.6
1.9
1.9
1.2
4.5
3.6
3.4
3.6
2.7
1.8
3.0
1.8
2.7
1.8
1.7
0.2
1.1
0.4
1.8
2.3
0.7
1.8
0.9
2.3
0.7
0.1
0.5
0.2
0.8
1.0
0.3
0.8
0.4
1.0
10.4
8.0
7.5
7.5
7.1
6.3
6.3
5.6
5.5
5.3
The Hulikal forest showed more canopy trees with evergreen forests. The trees are tall and long in height.
Fahrenheitia zeylanica is an important species with IVI of 10.4 and basal area of 0.7. Lagerstroemia lanceolata,
Mangifera indica, Symplocos cochinchinensis had the basal area of 1.0. Hopea ponga, Murraya paniculata
showed IVI of 8.0 and 7.5 respectively. Cassia fistula showed less basal area and less IVI of 0.1 and 0.8
respectively (Table 4).
658
Species composition of Hulikal

.
Number of species
120
100
80
60
40
20
0
Herbs
Shrubs
Climbers
Trees
Life forms
Figure 3. Graph showing diversity of different life forms in the study area.
The Hulikal forest had 53 herb species, 51 shrubs, 31 climbers and 96 trees species they are representing a
total of 2172 individuals of 60 families this indicates Hulikal forest is rich in floristic diversity (Fig. 3). The
Shannon diversity index of herbaceous species of Hulikal forest is 3.8. The diversity index of the climbers and
tree species like Shannon index value of Hulikal is 4.3. The Simpson diversity index similarly shows following
results (Table 5). Hulikal Herbaceous, Shrubs and climbers diversity is 25%, 73% and 69% respectively. The
tree diversity of Hulikal is 81% and this indicates Hulikal forest have rich in tree species diversity.
Table 5. Showing the diversity index vales.
Life forms
Herbs
Shrubs
Climbers
Trees
Shannon (H I)
3.9
3.8
3.3
4.3
Diversity indices
Simpson (D)
0.019
0.025
0.040
0.015
Simpson (1/d)
51.03
39.84
25.00
67.11
DISCUSSION
The present study revealed the species richness in the Hulikal forests of central Western Ghats region. The
study revealed that this region is very rich in species composition. The number of individuals species in the area
is formed to be very high when compared to Mudumalai deciduous forest which accounts for 71 species in 50
hectare permanent plot (Sukumar et al.1992) it was moderate against the transect study at 3.82 hectare done in
Kalakad-Mundanthurai Tiger reserve (Ganesh et al. 1996 ). It is also higher diversity when compared to the total
of 74 species recorded in Kudremukh National Park (Nagaraja et al. 2005). They also mentioned that
Achranthes aspera is the dominant herb, Lantana camara is the dominant shrub, Ichnocarpus frutenscens is the
dominant climber and also represented by rich in epiphytic, orchids and mosses diversity same pattern of
vegetation occurs in Hulikal forest region.
Conservation of the whole spectrum of biological diversity is a new challenge. This calls for an in-depth
understanding of the patterns of distribution of many different parameters that can be used to characterise
abundance and diversity at different taxonomic hierarchies and at differential spatial scales. Then such an
understanding has to be applied towards assigning conservation values to particular taxa, localities and habitats.
CONCLUSION
The indiscriminate use and over exploitation of natural plant wealth for minor causes results in deterioration
of ecosystem and a great loss to mankind. The developing countries concentrate on the development of
industries and technique. This will lead to a great decrease in global biodiversity. The Hulikal forest of Western
Ghats, Hosanagar region shows high level of plant diversity compared with the other forests in the Western
Ghats. The total of one hectare vegetative plot from these forests. The present study is highlighting the rich
species composition of the forest and fragmentation of the forest for the different commercial plantation is
threatening the forest balance. While in case of Hulikal state forest, Government prone developmental activities
659

.
like dam construction across the river Varahi near Hulikal and clearing of forest for the power lined are
damaging the plant diversity. Several forest management activities are needed for the conservation of diversity
existing in these forests and also there is a need of educating the people regarding the loss of biodiversity from
these forests. In the context, present study would serve as a baseline data for the effective forest management for
the policy makers.
ACKNOWLEDGMENTS
Authors are thankful to the Chairman, Department of Applied Botany, Kuvempu University, Shankarghatta,
Shimoga and Secretary, Swamy Vivekananda Vidya Samsthe, Shikaripura, Shimoga and Karnataka Forest
Department for permitting and encouraging the carryout the present work.
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ISSN (E): 2349 1183

ISSN (P): 2349 9265
3(3): 662672, 2016
DOI: 10.22271/tpr.2016.v3.i3.087
Research article
Role of dicot angiosperms in the livelihood of Mishing community

in Sonitpur district, Assam, India
Jintu Sarma and Ashalata Devi*
Ecology and Biodiversity Lab., Department of Environmental Science, Tezpur University,
Napaam, Tezpur-784028, Assam, India
*Corresponding Author: ashalatadevi12@gmail.com
Abstract: Assam, a state in Northeast India holds and supports a high percentage of tribal
population, highly distinguished in terms of ethno-lingual physiognomies as well as livelihood
status in their habitats. The Mishing people or Misng, also called Miri, is a major ethnic tribal
community inhabiting in the Sonitpur district of Assam and the second largest tribal group in
Northeast India. The livelihoods of Mishing community are closely associated with several plant
species. The present study was carried out in the Sonitpur district of Assam, India to trace out the
role of dicot angiosperm in the livelihood pattern of Mishing community living in the area.
Through a series of extensive survey a total number of ethnobotanically important dicot plant
species under 64 genera and 45 families were recorded. 48 dicot plant species were exclusively
used for medicinal purposes and 25 dicot plant species were found to be marketed for different
purposes as NTFPs. The uses and marketing of different Non Timber Forest Products (NTFPs)
were also recorded to determine their economic reliance. While, 11 species of dicot angiosperm
were recorded that are used for the preparation of Rogjin Apong, an ethnic alcoholic rice bear.
Keywords: Dicot Angiosperms - Mishing Community - Livelihood - Sonitpur - Assam.
[Cite as: Sarma J & Devi A (2016) Role of dicot angiosperms in the livelihood of Mishing community in
Sonitpur district, Assam, India. Tropical Plant Research 3(3): 662672]
INTRODUCTION
India has about 10.05 crore tribal population, consisting 8.60% of countrys total population (Census 2011).
The tribal population in India contains approximately 250 groups, speaking about 105 languages and 225
subsidiary languages. In the context of socio-economic development, the tribals in India vary from primitive life
style to modern way of living. The primitive tribal economy is intimately connected with forests and its
resources. Tribals and their symbiotic relationship with forest and their surrounding environment has been seen
through nature worship since ancient times. Nature gives many things such as food, medicine, raw materials,
shelter, fertilizer, fuel, timber etc. Thus tribal people are dependent on these natural resources for their
livelihood and sustenance. Many workers reported that tribal peoples are highly dependent on NTFPs for the
source of income and livelihood (Rao 1987, Gauraha 1992, Chopra 1993, Mallik 2000). About 60 per cent of
NTFPs is consumed by about 7 crore tribals or ethnic communities in the country and reported to contribute
about 10 to 40 % of their household earnings (Shiva 1993). About 70% of Indian population dwells in rural
areas and many of them rely on various non-timber forests products for their sustenance (Datta et al. 2014).
Medicinal plants have a long-standing history among indigenous communities and are an integral part for
treating various diseases, particularly to curb/cure daily ailments and this practice of traditional medicine is
based on experience of hundreds of years of belief and observations. Indigenous healing practices have been
culturally accepted during all phases of human culture and environmental evolution. Traditional medicine is
widely used since prehistoric period (Singh & Lahiri 2010) and accounts for about 40% of all health care
delivered (WHO 2005). It has also been estimated that about 85% of worldwide traditional medicines are
derived from plants (Fransworth 1988). Majority of different tribal community of Indian population depend
directly or indirectly on about 7500 different medicinal plants for the treatments of their various health ailments.
662

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India is one of the great treasures of ethnobotanical wealth having ultimate multiplicity of ethnic community and
highly engrossed biological (Kala 2005) resources. In developing countries, the use of herbal treatment is
enormously increasing day by day and many modern researchers are trying to intricate and explore the huge
potential of ethnobotanical knowledge of medicinal plants for treatment of various diseases (Kala 2005, Dutta &
Dutta 2005, Jain et al. 2010, Jeyaprakash et al. 2011, Mehra et al. 2014, Bajpai et al. 2016, Ngbolua et al.
2016). However, the ethnomedicinal plants in their natural habitat are under threat due to deforestation,
overgrazing and their reckless harvesting and utilization. Several workers carried out, ethnobotanical studies
with reference to the uses of medicinal plants by different tribal communities from various places of Assam
(Jain & Borthakur 1980, Jain 1987, Jain 1989, Das 2008, Saikia et al. 2010, Rout 2012, Teron & Borthakur
2014). Among other tribes Mishing is a major tribal community of Sonitpur district whose livelihood is highly
associated with the plants for medicine, food and for daily basic needs. The main source of livelihood for the
Mishing community is agriculture. An attempt has been made to examine the dependency of Mishing
community on several plants for medicinal uses values and NTFPs for marketing as one of their livelihood
options in Sonitpur district of Assam, India.
Study area
Figure 1. Map of study area, Sonitpur district of Assam, India.
Sonitpur is the second largest district of state Assam after Karbi Anglong district, spread over an area of
5324 km2 on the north bank of Brahmaputra river with a population about 1.926 million (Census 2011). The
Sonitpur district lies between 26 40' 25.9860'' N and 92 51' 27.7560'' E and is bounded by Arunachal Pradesh
in north, River Brahmaputra in south; Lakhimpur in east and Darrang in the west (Fig. 1). There are five major
Rivers of Sonitpur namely, Brahmaputra, Jiabharali, Gabharu, Borgang and Buroi. The total forest area of
663
Sarma & Devi (2016) 3(3): 662672

Sonitpur is 1420 km2. The major forest area of district Sonitpur comprises Nameri National Park (NP);
Burhachapori and Sonai Rupai Wildlife Sanctuaries (WLS) and 11 Reserve Forests (RF) distributed in two
broad divisions Sonitpur East and West. As per census 2011 data the tribal population of Sonitpur is about
2,32,000 which comprises of different tribal groups, among them Mishing, Bodo, Rabhas, Mechs, Nyishis,
Garos, Adis, Munda, Apatanis, Lamas etc. are some important tribal communities.
Methodology
An extensive field survey was carried out in order to document ethnobotanical plant species associated with
Mishing community in seventeen (17) different villages during 2013 2015. A series of informal meeting was
conducted with the village heads of each Mishing village and villagers with the age group of 1585. A standard
semi-structured questioner was used for the proposed survey which was prepared following the guideline of
Convention on Biological Diversity (CBD). Vernacular names (Mishing) of plants were recorded while for
medicinal plants part used, mode of administration, dose recommended for human care, nature and name of
diseases were properly recorded through personal interaction with medicine men. For the record of NTFPs
available in markets several small local markets (Bhalukpong, Khonamukh, Chariduar, near Gohpur, etc.) were
also visited at frequent interval. Whole plants and different plant parts sold in the local markets were recorded
and prices of each item were quantified. Herbarium were prepared following Jain & Rao (1977) for the
recorded plant species and were identified checking the specimens at the herbarium of Gauhati University,
Assam, Botanical Survey of India, Shillong and consulting relevant literature such as Flora of British India
(Hooker 18751894), Flora of Assam (Kanjilal et al. 19341940), etc. and also consulted with some highly
experienced taxonomists like Dr. Gajen Chandra Sarma, Gauhati University, Assam, India. The analysed
herbariums were submitted to the Tezpur University Herbarium (TUH) at Ecology and Biodiversity laboratory
for preservation.
RESULTS
The results of the present study highlight the ethno-medicine and NTFPs used by Mishing community of
Sonitpur districts. The survey was carried out in 17 villages viz. Dharikati, Khonamukh, Kathani, Rangajan,
Rongajan miri, Baligaon, Sotaimiri, Toupamiri, Bamunipam, Bordikorai, Sikomgaon, Silenighat, Morikhuti,
Bokagaon, Kekokoli, Tinighoria and Gudamghat. A total of 194 villagers (149 male and 45 female) were
interviewed. 55 respondents in the interview process belonged to age group of 32 - 45 years; 70 individuals in
the range of 4655 years; 33 individuals of 5665 years; 24 individuals in 6675 years and only 12 individuals
in between 7685 years. During this study a total number of 74 ethnobotanically important dicot plant species
under 64 genera and 45 families has been recorded. Among this, 48 species were medicinal plant belonging to
46 genera under 34 families that were exclusively used for the ethno-medicinal purposes by the Mishing
community (Table 1). Trees contributed highest (42%) number having 20 species belonging to 18 genera and 15
families followed by shrubs (31%; 15 species under 15 genera and 11 families), herbs (17%; 8 species, 8 genera
and 8 families) and climber (10%) with 5 species, 5 genera and 5 families. Among the families Acanthaceae
shows highest species diversity (4 genera and 4 species) followed by Rutaceae (3 genera and 3 species),
Verbenaceae and Moraceae (2 genera and 3 species each) and Combretaceae has 3 species under 1 genera.
While Euphorbiaceae, Lythraceae, Meliaceae, Solanaceae, Lamiaceae, Rubiaceae holds 2 genera and 2 species
each. The other families comprise 1 genera and single species (Fig. 2). The different plant parts used for the
different treatment of human diseases are also recorded during the study. Among the plant part, leaves (50%) is
extensively used as medicines followed by fruit (17%), whole plant, root and stem (6%), bark (7%) , gum/resin
(4%), flower and seed consists (2%), as given in figure 3. During the study a detailed note on the species used
for different diseases were also estimated. As per record, 19% of species were utilised for stomach problems;
13% utilised for dysentery; 10% were used for skin problems; 8% for hair therapy, worm and cough; 4% were
used in liver disorder, gynaecological, urinary problems, infections and as health tonic each; and 2% of recorded
species were used for diabetes, dog-bite, pains, teeth problems, abortion and fractures (Fig. 4). During the
interview it has also been observed that the majority of the medicine prepared by herbal medicine-men was
administrated internally contributing 81% and some 19% medicine were administrated externally. Another part
of this study was to trace out different NTFPs specially dicots from Sonitpur district used by Mishing tribe.
After a multi phased field observation a total of 23 species belonging to 17 genera under 15 families were
664
Sarma & Devi (2016) 3(3): 662672
Figure 2. Analysis of dominant families (Dicots) for the recorded plant species.
Figure 3. Percentage contribution of different plant parts used in preparation of medicine.
Figure 4. Contribution of medicinal plants used for treatments of certain human health ailments.
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Table 1. Details of the recorded ethno-medicinal plant species used by Mishing tribe. (Note: T- Tree, CL- Climber, SH- Shrub, H- Herb)
Sl.
No.
1.
Botanical name
of the plants
Acacia nilotica (L.) Delile
Mimosaceae
Vern name
(Mishing)
Babul
2.
3.
4.
Adhatoda vasica Nees

Aegle marmelos (L.) Corra
Ananas comosus (L.) Merr.
Acanthaceae
Rutaceae
Bromeliaceae
Bahek phul
Bel
Matikothal
5.
Acanthaceae
Kalmegh
6.
Andrographis paniculata
(Burm. f.) Nees
Asparagus racemosus Willd.
Asperagaceae
Satmul
7.
Averrhoa carambola L.
Oxalidaceae
Kordoi
8.
Azadirachta indica A.Juss.
Meliaceae
Mohaneem
9.
Scrophulariaceae
Brahmi
Crassulaceae
Duportenga
Fabaceae
13.
Bacopa monnieri (L.)

Wettst.
Bryophyllum pinnatum
(Lam.) Oken
Butea monosperma (Lamk.)
Taub
Calotropis gigantea (L.)
Dryand.
Capsicum annuum L.
14.
Family
Uses
Life
form
T
Part used
Dose/
Leaf, bark
I: decoction of leaf and bark used after meal.
H
T
SH
Leaf
Leaf
Leaf
SH
Leaf
Upset stomach
(dyspepsia),
constipation
Cough
CL
Root
Fruit
Skin infection
/measles
Brain tonic
Leaf
I: decoction of leaf used after meal.

E: pest of young leaf used in the small pox.
I: 2/3 young leaf taken and grind then the juice is
directly taken with a small salt.
I: young leaf smashed and the distilled juice is
taken daily for liver problem.
I: root boiled with water or dried first and then
powder boiled in water and taken for stomach
problems.
I: ripen fruit roasted in wood fire and eaten: juice
eaten directly
E: mature leaves boiled with water and the water
used to bath.
I: whole plant grind and the juice drink.
Dry cough,
kidney trouble
Cough etc.
Small pox
Vomiting
/indigestion
Liver tonic
administration
(E/I)*
Palas
Urinal
infection
Diarrhoea
Whole
plant
Leaf
Gum/resin
I: used directly
Apocynaceae
Aah: Kam
Pain
SH
Leaf
Solanaceae
Surging mirsi
SH
Fruit
Centella asiatica (L.) Urban.
Apiaceae
Manimuni
15.
Cissus quadrangularis L.
vitaceae
Harjora
Stomach
problem/gastric
Vomiting
/indigestion
Bone fracture
E: mature leaves kept over fire and then with

mastered oil and wrapped the pained area.
I: eaten directly
16.
Citrus maxima (Burm.)

Rutaceae
Merr.
Clerodendron colebrokianum Verbenaceae
L.
Corchorus capsularis L.
Tiliaceae
Singliang
Skin
Whole
plant
Whole
plant
Fruit
I: leaf juice with water or chewed

the whole plant.
E: the plant used as bandage or plastering for bone
fracture.
I: fruit eaten directly
Pakkom
Weight
loss/fever
SH
Leaf
I: young leaf consumed as vegetable
Mura
Stomach problem
/vomiting
SH
Leaf
I: tender Leaf dried with smoke and then boiled

and eaten
10.
11.
12.
17.
18.
CL
T
I: young leaf consumed directly.
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22
Sl.
No.
19.
Botanical name
of the plants
Datura stramonium L.
Solanaceae
Vern name
(Mishing)
Dhatura
Dillenia indica L.
Dilleniaceae
Champa
Bite by mad
dog
Hair therapy
20.
Caryophyllaceae
Laijabori
Dermatitis
leaf
22.
Drymaria cordata (L.)

Willd. ex Schult.
Eupatorium odoratum L.
E: the seeds are grind and the then seeds are used
over hair for smooth and to reduce hair fall.
I: leaf consumed directly.
21.
Asteraceae
Ayapan
SH
Leaf/root
I: Decoction
23.
Ficus hirta Vahl
Moraceae
Taksek
High blood
pressure
Urine problem
Fruit
24.
Hedyotis diffusa Willd.
Rubiaceae
Sarpajiva
Leaf
25.
26.
27.
Jatropha curcas L.
Justicia adhatoda L.
Lawsonia inermis L.
Euphorbiaceae
Acanthaceae
Lythraceae
Votera
Bahaka
Jetuka
T
SH
SH
Resin
Leaf
Leaf
I: resin with milk consumed for 2/3 days

I: leaf juice used for dry cough
E: leaf paste used in hair and skin
28.
29.
Leucas aspera (Willd.) Link

Mangifera indica L.
Lamiaceae
Anacardiaceae
Dorun
Ke: di milong
Stomach pain/
nerve tonic
Abortion
Cough
Skin and hair
diseases
Sinus
Dysentery
I: ripen fruit used directly or cooked for urine

problem.
I: juice or cooked vegetable
H
T
Leaf
Bark /seed
30.
31.
Melia azadirachta L.
Moringa oleifera Lam.
Meliaceae
Moringaceae
Ghoraneem
Munga
T
T
32.
Murraya koenigii (L.)

Spreng.
Nyctenthis arbor-tristis L.
Rutaceae
Norhing
Skin infection
Stomach
problem
Dysentery
Leaf
Fruit/flowe
r/leaf
Leaf
I: 2/3 drop of leaf juice used per nose.

I: decoction of bark taken in empty stomach; twice
daily before meal till complete relief.
The seeds are eaten raw or roasted.
E: Leaf boiled and water used to bath.
I: cooked a vegetable
Oleaceae
Sewali
Worm
Flower
I: flower fry eaten
Ocimum basilicum L.
Paederia foetida L.
Phlogacanthus thyrsiflorus
Nees
Phyllanthus acidus Skeel
Lamiaceae
Rubiaceae
Acanthaceae
Tulsi
Vedeli
Titaphul
Cough
Indigestion
Bronchitis
SH
CL
SH
Euphorbiaceae
Pora amlokhi
White
discharge of
women
Leaf
Leaf
Bark &
Leaf
Leaf
I: leaf juice with honey taken for cough.

I: leaf juice used directly or cooked with fish.
I: decoction of bark & leaf taken till complete
relief.
I: leaf juice with sugar. Daily in empty stomach for
15 days to 3 months.
33.
34.
35.
36.
37.
Family
Uses
Life
form
SH
Part used
Dose/
administration
Root
I: root decoction
Fruit
(E/I)*
I: cooked a vegetable
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Sl.
No.
38.
Botanical name
of the plants
Piper betel Blanco.
Piperaceae
Vern name
(Mishing)
Paan
39.
40.
Psidium guajava L.
Punica granatum L.
Myrtaceae
Lythraceae
Madhuri
Anar
41.
42.
43.
44.
Scoparia dulcis L.
Sesamum orientalae L.
Streblus asper Lour.
Swertia chirata Buds-Ham.
Plantaginaceae
Padaliaceae
Moraceae
Gentinaceae
Tisilkosa
Tanam
Namhoi
Sirata
45.
Terminalia chebula Retz.
Combretaceae
Silika
46.
Terminalia arjuna (Roxb. ex

DC.) Wight & Arn.
Terminalia bellirica
(Gaertn.) Roxb.
Tylophora indica (Burm f.)
Merr.
Combretaceae
Arjun
Combretaceae
Bhomora
Asclepiadaceae
Anantamul
47.
48.
Family
Uses
Boil/cut injury
Stomach pain
Diarrhoea/
anaemia
Diabetes
Hair fall
Teeth problem
Worm /allergy
/vegetable /
Hair growth
/constipation
/heart problem
Heart/liver
tonic
Hair growth
/constipation
Liver Tonic/
Jaundice
Life
form
CL
T
SH
SH
SH
T
H
Part used
Dose/
stem
E: stem is taken and dipped in the hot mustard oil

and then touched the boil or cut injury for relief of
pain and quick recovery
I: 2/3 young leaf grind and spoon juice taken.
I: fruit eaten directly; flower and leaf cooked; leaf
juice used
I: eaten directly.
I: seed cooked or grind and used in hair.
E: stem used as tooth brass.
2/3 stem soaked for some hours or overnight and
the soaked water is taken (2 spoon) for two days
I: Fruit dried and grind and the powder used
directly; or wholly eaten
Leaf
Leaf/flowe
r/fruit
Leaf
Seed
Stem
Stem/ Leaf
Fruit
Bark /root
Fruit
CL
Root
administration
(E/I)*
I: decoction of root and bark used; powder of bark

mixed with hot water for heart diseases.
I: fruit dried and the inner part grind and the
powder used directly
I: root eaten directly
Table 2. List of NTFPs (Dicots) marketed by Mishing tribe of Sonitpur, Assam. (Note: T- Tree, CL- Climber, SH- Shrub, H- Herb)
Sl.
Botanical name
No. of the plants
Fruits
1. Baccaurea sapida (Roxb.)
Muell.-Arg.
2. Citrus grandis (L.) Osbeck
3. Garcinia paniculata Roxb.
4. Phyllanthus acidus (L.)
Skeels
Family
Vernacular name
(Mishing)
Part
collected
Uses
Life
forms
Euphorbiaceae
Buri aaye
Fruit
Fruit
SH
Rutaceae
Clusiaceae
Phyllanthaceae
Sinkin
Tepor tenga
Pomlokhi /Pora aamlokhi
Fruit
Fruit
Fruit
Fruit
Fruit
Fruit
T
T
T
Local market Source of

Availability
price (INR) collection
15-20
/dozen
5/fruit
10/fruit
15-20/kg
Forest
MarchJuly
Forest
Forest
Forest
All seasons
JulyOctober
MarchMay
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Sl.
Botanical name
No. of the plants
Medicine
5. Adhatoda vasica Nees
Family
Vernacular name
(Mishing)
Part
collected
Acanthaceae
Bahek phul
Leaf
Medicine
cough etc.
10/ bundle
Forest
All seasons
Burseraceae
Dhuna
Resin
Resin
70-100/kg
Forest
All seasons
Piperaceae
Jaluk
Seed
Spice
CL
Forest
All seasons
Piperaceae
Pipali
Seed
Spice
CL
120-150/
100 gm
120-150/
100 gm
Forest
All seasons
Amaranthaceae
Geyag
Vegetable
SH
20/bundle
Forest
All seasons
10. Amaranthus virdis L
Amaranthaceae
Datha
Vegetable
SH
10/bundle
Forest
All seasons
11. Dillenia indica L.
Dillenaceae
Champa
Leaf with
stem
Leaf with
stem
Fruit
vegetable
5-7/pair
Forest
JuneOctober
12. Dioscorea alata L.
Dioscoreaceae
Nimti
vegetable
CL
20-25 /kg
Forest
All seasons
13. Solanum nigrum L.
Solanaceae
Bangko
Tuberous
root
Fruit
vegetable
SH
Forest
All seasons
14. Murraya koenigii (L) Spreng
Rutaceae
Narasingha
vegetable
SH
Forest
All seasons
15. Alternanthera sessilis

(L.) R.Br. ex DC.
16. Solanum indicum L.
17. Solanum torvum Sw.
18. Garcinia cowa L.
19. Ficus glomerata Roxb.
Amaranthaceae
Morisha
Tender
leaf
Leaf
10/ poa (250

gm)
5/bunch
vegetable
Forest
All seasons
Solanaceae
Solanaceae
Clusiaceae
Moraceae
Banko
Sitabanko
Kuji Thekera
Tejing /taksek
Leaf
Fruit
Fruit
Leaf
SH
SH
T
T
Forest
Forest
Forest
Forest
All season
All season
Almost all season
All season
20. Dioscorea alata L.
Dioscoreaceae
Al
Tuber
Vegetable
Vegetable
Vegetable
Vegetable
specially with
Pork
Vegetable
1015/bundle
20/ bunch
30/ 250 gm
20/fruit
20/packet
CL
50/kg
Forest
All season
Resins
6. Boswellia serrata Roxb.
Spices
7. Piper nigrum L.
8.
Piper longum L.
Vegetables
9. Amaranthus spinosus L.
Uses
Life
forms

Availability
669
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Sl.
Botanical name
No. of the plants
Vegetable And Medicine
21. Paederia foetida L.
Family
Vernacular name
(Mishing)
Part
collected
Rubiaceae
Bunka fore
Young
Leaf
22. Centella asiatica (L.) Urban
Apiceae
Manimuni
23. Leucas aspera (Willd) Link
Lamiaceae
Durum
Whole
plant
Young
leaf
Life
forms
Uses
Vegetable and
medicine for
stomach
problem.
medicine and
vegetable
vegetable and
medicine for
nose problem

Availability
CL
10/bundle
Forest
All seasons
5/bundle
Forest
All seasons
5/bunch
Forest
All seasons
Table 3. List of plant species (dicots) used for the preparation of Epop, used for preparation of Nogjin Apong. (Note: T- Tree, CL- Climber, SH- Shrub, H- Herb)
1.
Botanical name
of the plants
Clerodendrum infortunatum L.
Verbenaceae
Vern name
(Mishing)
Pakkom
2.
Coriandrum sativum L.
Apiaceae
3.
4.
5.
6.
Costus speciosus (J.Koenig) Sm.

Cuscuta reflexa Roxb.
Flemingia strobilifera (L.) W.T.Aiton
Hibiscus rosa-sinensis L.
7.
Sl. No.
Family
Life form
Part used
Use /mode
SH
Leaf
I: Leaf dried and powder used in the

preparation of Epop
Dhania
Stem/leaf
I: stem/ Leaf dried and powder used

in the preparation of Epop
Costaceae
Convolvulaceae
Fabaceae
Malvaceae
Jomlakhuti
Rabonlota
Makhioti
Leunaapum
SH
CL
SH
T
Leaf
Root
Leaf
Leaf
Lippia javanica (Burm.f.) Spreng.
Verbenaceae
-----
Leaf/flower
8.
Polygonum hydropiper L.
Polygonaceae
Leubo
Leaf/stem
9.
10.
11.
Polygonum microcephalum D. Don

Tinospora cordifolia (Willd.) Miers
Zanthoxylum nitidum (Roxb.) DC.
Polygonaceae
Menispermaceae
Rutaceae
Nekungkune
Amrita
Jabrang
H
CL
SH
Leaf
Leaf
Leaf
I: root dried and powder used in the

preparation of Epop
I: Leaf dried and powder used in the
preparation of Epop
I: Leaf/flower dried and powder
used in the preparation of Epop
I: Leaf/stem dried and powder used
in the preparation of Epop
----I: Leaf dried and powder used in the
preparation of Epop
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Sarma & Devi (2016) 3(3): 662672

recorded as NTFPs that has been marketed extensively by this community in different regions of the district
(Table 2). It has been observed that species used for vegetables contributes maximum (52%) followed by fruits
(18%); species used for both vegetable and medicine (13%); spices (9%); and resins and medicine 4% each (Fig.
5). Different plant parts were collected by the villagers for marketing and to be used for vegetables, fruits,
resins, medicine, etc. The majority of the plant parts are available throughout the year and some are found to be
seasonal. One more interesting fact about the marketing of the NTFPs is that they generally do not use the
weighing scale for measuring quantity of items instead they sold the items in some bundles/bunch or in pairs.
Another most important traditional practice of Mishing community is the preparation of Nogin or Nogjin
Apong, a traditional rice alcoholic beverage of Mishing Community. For the preparations of the Nogin Apong
they use many plants species (both dicots and monocots). During the preparations of the Apong first they
prepare Epop. Epop is the tablet or ball shaped cake prepared with the dry powder of plant materials mixing
with the rice powder and is used for the preparation of Nogin or Nogjin Apong. During the study a total of 11
dicot plant species (Clerodendrum infortunatum, Coriandrum sativum, Costus speciosus, Cuscuta reflexa,
Flemingia strobilifera, Hibiscus rosa-sinensis, Lippia javanica, Polygonum hydropiper, P. microcephalum,
Tinospora cordifolia and Zanthoxylum nitidum) under 10 genera and 9 families (Table 3) were recorded that
were exclusively used for the preparation of Epop.
DISCUSSIONS
The main objective of the present study was to investigate the different plant species (especially dicots) that
are exclusively used by the Mishing community in their day-to-day life for different purposes like medicine,
vegetable and importantly other NTFPs i.e. vegetables, fruits, medicine, spices, gum/resin etc. as their one of the
important income sources. From the result it has been observed that the majority of the plant species were used
mainly for medicinal purpose. Leaves exhibited major part of the plant used for treatment of majority of
diseases contributing 50% while flower shows lowest used (2%). Saikia et al. (2010) studied ethnobotany of
Bodo tribes in Sonitpur district and enumerated 20 species and analysed briefly the degree of dependency on
medicinal plants and common health concern. Another etnobotanical study reported a total number of 20 plant
species out of which 12 dicot species from Gahpur area of Sonitpur district (Saikia 2006). In the present study a
total of 48 plant species were recorded for medicinal uses, 23 species were recorded as NTFPs and 11 species
recorded that are used for the preparation of Apong. Majority of the plants like Ananas comosus, Centella
asiatica, Psidium guajava, Hedyotis diffusa, Moringa oleifera, Corchorus capsularis, Capsicum annuum,
Streblus asper and Asparagus racemosus are used for stomach problems while Butea monosperma, Punica
granatum, Acacia nilotica, Mangifera indica, Murraya koenigii, etc are used for dysentery. Averrhoa
carambola, Justicia adhatoda, Ocimum basilicum and Adhatoda vasica are commonly used for cold and cough
by the community. Fruits of Baccaurea sapida, Citrus grandis, Garcinia paniculata, Phyllanthus acidus, etc are
marketed as NTFPs while resins of Boswellia serrate and fruit/seeds of Piper nigrum and P. longum are marked
as spices.
CONCLUSION
From the present study it can be concluded that the Mishing tribe is highly dependent upon the dicot
angiosperms as their source of income in addition to fulfilling their various day to day requirements. Thus
NTFPs plays a major role in the livelihood status of Mishing tribe by supplying domestic requirement and
marketing goods. Understanding the present days environmental health issues related to chemicals in the form
of pesticides, weedicide, etc. peoples generally prefer to buy the NTFPs sold in the local markets by tribal
peoples which are collected from the forest. This marginal marketing also serves as a source of income to this
community.
ACKNOWLEDGEMENTS
Authors are thankful to the villagers of Dharikati, Khonamukh, Kathani, Rangajan, Rongajan miri,
Baligaon, Sotaimiri, Toupamiri, Bamunipam, Bordikorai, Sikomgaon, Silenighat, Morikhuti, Bokagaon,
Kekokoli, Tinighoria and Gudamghat villages of Sonitpur district of Assam, India. Author offers their sincere
thanks to Ms. N Pamegam (ATIO, Tezpur), Mr. M Das (ATIO, Bhalukpong) for their tremendous help
throughout the field work. Authors deeply acknowledged the help of Comission Mili, Podeswar Mili, Noipani
Doley, Kameswar Mili, Dimbeswar Mili, Bismita Doley and Joinath Gum for their assistance during survey.
671
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Special thanks to Dr. GC Sarma, Dept. of Botany, G.U. for identification of plant species and Dr. Rajesh Kumar
Sah of GIS Lab., Dept. of Environmental Science, Tezpur University for their kind help.
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ISSN (E): 2349 1183

ISSN (P): 2349 9265
3(3): 673680, 2016
DOI: 10.22271/tpr.2016.v3.i3.088
Research article
Finger millet (Eleusine coracana L.) grain yield and yield

components as influenced by phosphorus application
and variety in Western Kenya
Wekha N. Wafula1*, Korir K. Nicholas1, Ojulong F. Henry2,
Moses Siambi2 and Joseph P. Gweyi-Onyango1
1
Department of Agricultural Science and Technology, Kenyatta University, PO Box 43844-00100 Nairobi, Kenya
2
ICRISAT, ICRAF house, UN Avenue, Gigiri, PO BOX 39063-00623, Nairobi, Kenya
*Corresponding Author: nelwaf@gmail.com
Abstract: Finger millet is one of the potential cereal crops that can contribute to the efforts of
realization of food security in the Sub-Saharan Africa. However, scientific information available
with regards to improving soil phosphorus supply and identification of P efficient varieties for the
crops potential yield is limited. In order to investigate the effects of P levels on yield components
and grain yield On-station field experiments were conducted in two sites of western Kenya during
the long and short rain seasons of 2015. The experiment was laid out in a Randomized Complete
Block Design in factorial arrangement with four levels of P (0, 12.5, 25 and 37.5 kg P2O5 ha-1 and
three finger millet varieties (U-15, P-224 and a local check-Ikhulule) and the treatments replicated
three times. The increase of phosphorus levels significantly (P0.05) increased the grain yield
over the control up to 25 kg P2O5 ha-1 during the long rain seasons and 25 kg P2O5 ha-1 during the
short rain seasons in both sites. Interactions at P0.05 were revealed on the grain yield where
improved variety P-224 at 25 kg P2O5 ha-1 produced the highest grain yield of 4.74 t.ha-1 in Alupe
and 4.77 t.ha-1 in Kakamega and the consistent results suggest that the combination is highly
recommended. Therefore the use of judicious and proper rates of P fertilizers can markedly
increase the grain yield of finger millet in western Kenya.
Keywords: Finger millet - Potential cereal - Food security - Phosphorus supply - Judicious.
[Cite as: Wafula WN, Nicholas KK, Henry OF, Siambi M & Gweyi-Onyango JP (2016) Finger millet (Eleusine
coracana L.) grain yield and yield components as influenced by phosphorus application and variety in Western
Kenya. Tropical Plant Research 3(3): 673680]
INTRODUCTION
Finger millet (Eleusine coracana L. Geartn) belongs to family Poaceae and is one of the most important
food cereals in the Sub-Saharan Africa. Indigenous to the highlands of Uganda and Ethiopia, finger millet is
widely produced by small scale landholders and consumed locally (Adugna et al. 2011). The crop possesses
greater impact on the poor in Africa through the provision of food security and economic growth. Its grains can
be stored for several years in local storage conditions without damage by insect pests and this cushion against
vulnerability to unpredictable famine and drought. It is well adapted to heat, drought and poor soil stress that
prevail in marginal and degraded soils (Okalebo et al. 1990) and has a relatively better nutritional value with
high calcium content as well as crucial amino acids that are deficient in most cereals. These attributes plus its
high market value compared to other cereals makes finger millet one of the salient crops among resource poor
communities living in food insecure areas (NRC 1996). The cultivation of the crop covers around 65,000 ha in
Kenya with an average yield of 1 t.ha-1 which is below the potential as observed in other parts of the world like
in India where between 56 kg.ha-1 were realized under ideal irrigated conditions (Oduori 2005, NRC 1996).
One of the main problems faced by the farmers is inherent low soil P and degradation due to continued
cultivation. Notwithstanding the importance of finger millet, the research attention given to the crop has been
673

Wafula et al. (2016) 3(3): 673680

limited and the information is scarce on the P management of the crop. Phosphorus is an essential nutrient for
the life of plants whereby without adequate supply plants cannot reach their maximum yield. Adoption of proper
agronomic management practices to existing varieties can lead to achieving potential yields. Therefore, it is
necessary to know the optimum dose of the P fertilizer to be applied for maximum yields without compromising
the environment. Thus, the present study was conducted to determine the effect of phosphorus fertilizer rates on
the yield components and grain yield of finger millet in the western region of Kenya where most of the crop is
grown.
Study area
Two On-station experiments were conducted in the crops research stations in Kakamega and Busia Counties.
The Kenya Agricultural and Livestock Research Organization (KALRO)-Kakamega lies on Longitude 4450
E and Latitude 01660 N with an elevation of 1523 metres above sea level. The soils were predominantly
sandy loam (53.55% sand, 32.18% silt and 14.27% clay). The Kenya Agricultural and Livestock Research
Organization (KALRO)-Alupe lies within latitude 030 N and Longitude 340750 E with an elevation of
1157 metres above sea level. The soils were sandy loam with 47.57% sand, 35.76% silt/loam and 16.67% clay.
The two sites have two growing seasons in a calendar year between March and August termed the long rain
season and between September and December which is termed the short rain season. The experiment was
carried out in the two seasons of 2015.
Experimental design and treatments
The experiments were laid out in Randomized Complete Block Design in factorial arrangement with two
factor sets of four levels of P (P0=Control, P1=12.5 kg.ha-1 P2O5, P2=25 kg.ha-1 P2O5 and P3=37.5 kg.ha-1 P2O5)
and three varieties (U-15, P-224 and Ikhulule). Ikhulule was the local check variety and no phosphorus applied
for the control. Each treatment was replicated three times while the other cultural practices were kept constant
with a blanket application of N (50 kg.ha-1 N) at four weeks after emergence.
Land management and cultural operations
Analysis of composite surface soil samples collected from the experimental fields indicated the soils were
moderately acidic (pH in 1:2.5 soil: water ratio of 5.45 in Alupe and 5.60 in Kakamega). The top soils were
moderately low in Total N (0.12% in Alupe and 0.18% in Kakamega), high in organic carbon (2.26% in Alupe
and 3.40% in Kakamega), low in Olsen extractable P (6 ppm in Alupe and 5 ppm in Kakamega), and moderate
in available K contents (0.30% in Alupe and 0.75% in Kakamega).
Land preparation was done prior to the onset of rains during both seasons. Planting was done by hand
drilling seeds obtained from ICRISAT in rows spaced 40 cm between rows and later thinned after four weeks
between plants to an intra-row spacing of 10 cm. Phosphorus fertilizer was applied wholly by hand drilling at
planting according to the treatments as Triple Superphosphate (TSP). All other agronomic operations were done
as recommended for the crop. Harvesting on the net plot was done when the fingers had achieved 90% brown
color.
Data collection and analysis
The finger length of the longest spike from at least five main tillers from each plot was measured and the
average recorded. The finger width was measured across the center of the longest five spikes from each plot and
the average recorded. The number of grains per spikelets was counted at dough stage after removing five
spikelets from the middle portion of the rachis of five main tillers in each plot and the average recorded. The
number of panicles per finger was counted at dough stage on the main ears of five plants and the average
recorded. The grain yield from the net plot of every experimental unit (3 m2) was weighed on a Tronix Avery
digital scale at 13% moisture content and extrapolated to yield per hectare.
Analysis of variance (ANOVA) was carried out for the grain yield data collected following statistical
procedures appropriate for the design using SAS computer software. Whenever significant differences between
treatments were observed, the means were separated using Fishers Protected LSD at 5% probability.
Regression analysis was carried out to estimate the relationship between the grain yields as influenced by the
applied phosphorus fertilizer.
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Influence of phosphorus and variety on yield and yield components
Phosphorus did not have a significant role on the finger length and width in both sites for both seasons
(Table 1). However, numerically the control had the shortest fingers in Kakamega for both seasons and in Alupe
during the long rain season. The varieties had significant differences (P0.05) for both seasons in Kakamega
and Alupe where P-224 had the longest fingers (spikes). Similarly, the widest fingers were observed on variety
P-224 in both sites for both seasons. The interaction between phosphorus application levels and variety was only
revealed on the finger width in Kakamega during the long rain season where P-224 at 12.5 kg.ha-1 P2O5 had the
widest fingers with 1.189 cm.
Table 1. The influence of phosphorus and variety on the finger length and width (cm) in Alupe and Kakamega.
Finger Length (cm)

Finger Width (cm)
Fertilizer
Kakamega
Alupe
Kakamega
Alupe
Level
2015 SR 2015 LR 2015 SR 2015 LR 2015 SR 2015 LR 2015 SR 2015 LR
0
6.7a
5.3a
7.9a
7.7a
1.00a
1.05a
1.20a
1.10a
12.5
6.8a
5.7a
7.7a
8.0a
0.98a
1.06a
1.11a
1.09a
25.0
6.8a
5.9a
8.3a
8.1a
0.99a
1.02a
1.1a
1.10a
37.5
6.7a
5.8a
7.8a
8.2a
0.98a
1.03a
1.09a
1.08a
F pr.
0.928
0.211
0.671
0.175
0.836
0.71
0.427
0.856
LSD (0.05)
0.650
0.750
0.993
0.504
0.061
0.058
0.155
0.046
Variety
U-15
6.6b
5.5b
8.4a
8.2b
0.94b
0.98b
1.12b
1.01b
P-224
7.3a
6.4a
8.5a
9.0a
1.04a
1.10a
1.18a
1.15a
Ikhulule
6.3b
5.1b
6.89b
6.9c
0.99b
1.04b
1.08c
1.11a
F pr.
0.003
0.001
0.003
0.002
0.036
.001
.001
.001
LSD (0.05)
0.560
0.685
0.860
0.437
0.053
7.300
0.035
0.040
CV%
9.8
10
12.8
6.4
6.3
7.3
14.1
4.3
Interaction
PV
NS
NS
NS
NS
NS
*
NS
NS
Note: Means within a column followed by different letter (s) are significantly different at P0.05. LR=Long
rain season; SR=Short rain season; **=Significant at P0.05; NS=Not significant at P0.05
Table 2. Effect of phosphorus levels on the number of fingers per panicle and grains per spikelet in Alupe and Kakamega.
Fingers per panicle

Grains per spikelet
Fertilizer
Kakamega
Alupe
Kakamega
Alupe
Level
2015 SR 2015 LR 2015 SR 2015 LR 2015 SR 2015 LR 2015 SR 2015 LR
0
6a
7b
7b
7c
8a
7b
7a
7a
12.5
6a
7b
8a
9a
8a
8a
7a
7a
25
6a
8a
8a
8b
8a
8a
7a
7a
37.5
6a
8a
8a
9a
8a
7b
7a
7a
F pr.
0.834
0.043
0.022
0.003
0.51
0.007
0.799
0.128
LSD (0.05)
0.500
0.842
0.558
0.531
1.000
0.543
0.544
0.576
Variety
U-15
7a
8a
8a
9a
8a
7b
6b
6b
P-224
7a
7b
8a
8b
8a
8a
7a
7a
Ikhulule
6b
7b
7b
8b
8a
7b
6b
6b
F pr.
0.036
0.002
0.005
0.497
0.037
0.004
.001
.001
LSD (0.05)
0.500
0.448
0.483
0.460
0.800
0.541
0.471
0.499
CV%
9
14.4
7.3
6.6
12.2
7.4
8.4
9
Interaction
PV
NS
NS
NS
NS
NS
NS
NS
NS
Note: Means within a column followed by different letter (s) are significantly different at P0.05. LR=Long
rain season; SR=Short rain season; **=Significant at P0.05; NS=Not significant at P0.05
Application of phosphorus fertilizer led to increased number of fingers per panicle for both seasons in Alupe
and Kakamega during the long rain season compared to the control (Table 2). The highest number of fingers per
panicle was observed during the long rain season in Kakamega with a mean of nine fingers per panicle (head).
The number of grains in a spikelet only showed significant differences (P0.05) during the long rain season in
Kakamega where the 12.5 and 25.0 kg.ha-1 P2O5 treatments had the highest while the control and the highest rate
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had the lowest as shown in table 2. The number of fingers per panicle and grains per spikelet varied significantly
(P0.05) among the varieties in both sites for both seasons. U-15 showed the highest number of fingers per
panicle in both sites for both seasons while P-224 had the highest number of grains per spikelet for both seasons
across the sites.
Interactive effect of phosphorus and variety on grain yield
The interaction between variety and phosphorus fertilizer exerted significant influence (P0.05) on the grain
yield for both seasons in both sites (Fig. 1). In Alupe during the long rain season, improved variety P-224 at a
phosphate rate of 25 kg.ha-1 P2O5 had significantly (P0.05) the highest yield of 4.738 t.ha-1 while the local
variety Ikhulule with the highest rate had the lowest grain yield of 2.993 t.ha-1. At 12.5 kg.ha-1 P2O5, the local
variety Ikhulule showed the highest grain yield of 4.549 t.ha-1. Variety U-15 had the highest grain yield on the
25 kg.ha-1 P2O5 rate of 4.172 t.ha-1. The lowest grain yield in the improved varieties was observed on the control.
The same pattern shown in the long rain season of the highest yielding combination was observed in the short
rain season with 3.695 t.ha-1. The grain yield was however generally lower compared to the long rain season
(Fig. 1). Variety U-15 showed the highest grain yield on the 25 kg.ha-1 P2O5 rate while the local variety Ikhulule
had the highest grain yield on the two highest rates. The lowest grain yield among the varieties was observed on
the control.
Figure 1. The influence of variety and phosphorus fertilizer on the grain yield of finger millet in Alupe during the long (A)
and short (B) rain seasons.
Figure 2. The influence of variety and phosphorus fertilizer on the grain yield of finger millet in Kakamega during the long
(A) and short (B) rain seasons.
In Kakamega during the long rain season significant interactions (P0.05) between variety and phosphorus
fertilizer were observed where variety P-224 showed the highest yield at 25 kg.ha-1 P2O5 (4.770 t.ha-1) as well as
variety U-15 (4.650 t.ha-1) which was however insignificantly different from that at 12.5 kg.ha-1 P2O5 rate and
the highest rate (Fig. 2). The local variety Ikhulule had the highest grain yield at the highest rates of phosphorus
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fertilizer as observed in the short rain season in Alupe. Irrespective of the varieties, the lowest grain yield was
observed in the control. In the short rain season, the highest grain yield (4.0 t.ha-1) was observed on variety P224 at the 25 kg.ha-1 P2O5 rate while the same variety had the least grain yield on the 12.5 kg.ha-1 P2O5 rate and
the control. The local variety Ikhulule showed the highest grain yield at the 25 kg.ha-1 P2O5 while improved
variety U-15 showed the highest grain yield at the highest rate of phosphorus fertilizer (Fig. 2).
Figure 3. Mean grain yield as a polynomial function of phosphate rates for finger millet during the short (A) and long (B)
rain seasons in Alupe.
Figure 4. Polynomial regression characterizing the relationship between the grain yield response of finger millet varieties to
applied phosphorus fertilizer in Kakamega during the short (A) and long (B) rain seasons.
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The influence of phosphorus was significant (P0.05) on the finger millet varieties grain yield for both
seasons in the two sites. In Alupe, grain yield significantly increased from 2.6 t.ha-1 on the control to 3.2 t.ha-1
with an increase in the level of phosphorus fertilizer up to 25 kg.ha-1 and started decreasing with further increase
as shown in figure 3 during the short rains season. During the long rain season, the increase of phosphorus
fertilizer up to 20 kg.ha-1 showed significantly (P0.05) the highest grain yield with a 20.56% increase over the
control. During the same period, the highest rate and the control elicited the lowest grain yield (Fig. 3). In
Kakamega, a linear trend was observed with the increment of the phosphorus fertilizer dose up to 25 kg.ha -1 and
thereafter decreased in the short rain season while in the long rain season an increase of up to 20 kg.ha-1 showed
the peak on the grain yield and a drop followed thereafter. The highest yield of 4.0 t.ha-1 was observed during
the short rain season and 4.98 t.ha-1 in the long rain season while the control had the lowest grain yield for both
seasons (Fig. 4).
DISCUSSION
The numerical increase in the finger length in Kakamega is probably to the enhanced metabolic processes
due to optimum phosphorus and lack of reduced metabolic activity. However, the insignificant response to
applied P might be due to the optimal availability of N and supplement of Urea during study period which led to
the luxurious vegetative growth and induced the hidden P hunger. Similar findings were reported by Dixon
(2003) who found that in low soil P, foliar application of P only corrected the deficiencies and maintained
higher yields but did not increase the spike length or width of winter wheat due to excessive application and
availability of nitrogen. The increase in the number of fingers per panicle was due to the synthesis of amino acid
and chlorophyll and better carbohydrates transformation which resulted in better growth and a higher number of
panicles which ultimately produced more number of grains per spikelet. These findings were in conformity with
that by Parmer (2007) who observed an increase in the number of spikes per head due to an increase of
phosphorus up to 100 kg P2O5 per hectare of super phosphate in Tuberose Cv. Double. The improved varieties
produced higher number of grains per spikelet during the same period due to their ability to respond positively
to applied phosphorus through efficient utilization and also due to their genetic potential. Similar spikelet count
differences among rice varieties were reported by Obaidullah (2007).
The positive response of finger millet varieties on the applied P was due to the low available tested P in the
study soils. It has also been reported that in many parts of western Kenya P stress is a major limiting nutrient
and also the response of other nutrients is usually inhibited under this limitation (Opiyo 2004). Phosphorus
efficiency of the identified variety P-224 was probably due to its genetic ability to produce high yield per unit P
acquired. Ozturk (2005) reported that the identification of P efficient varieties with great ability to grow and
yield in soil with limited phosphorus supply and efficiently use applied P improves the sustainability of crop
production. This reduces the production cost for the small holder farmers which is associated with high P
applications and minimizes environmental pollution resulting from run-off of excess P fertilizer. The capacity
for P uptake has been documented to be affected by several environmentally or genetically controlled factors
that differ among varieties (Pearse et al. 2006). Pearse et al. (2006) also introduced a model that roots in some
varieties have a positive effect on P uptake, which enhances P status and efficiency. Different varieties have
inherent differences in the maximum growth rate and P uptake which leads them to differ in their productivity
(Shen et al. 2005). Ren et al. (2007) also found that different species of ryegrass with inherent low growth rates
accumulated higher levels of phosphorus in their shoots at low P-levels which led to lower grain yields probably
due to reduced efficiency of P for grain filling. The difference in P uptake among the genotypes showed the
diversity in efficiency with which plants are able to absorb P from soils with varying P availability (Hinsinger
2009). During the short rain season in Kakamega the control had the same grain yield as the 12.5 kg.ha-1 P2O5
rate and it is probably due to the availability of phosphorus that was earlier in its organic form. Organic forms of
P are found in humus and other organic material (Masinde et al. 2009). Phosphorus in organic material is
released by a mineralization process involving soil organisms (Shen et al. 2005) and thus the increased growth
on the control as that of mildly P applied treatment.
The increase in grain yield was probably due to the positive influence of phosphorus in initiation and growth
of roots that in turn sped up and increased the uptake of essential elements and moisture from the soil. Supply of
optimal phosphorus also ensured that the plants inhibited the formation of excess roots that would have led to
the excessive loss of carbon from the plants rooting system and reducing the efficiency in energy production
(Shane & Lambers 2005). Several investigators have reported the same findings that either initiation or
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inhibition of root formation is linked to internal P concentration, uptake and availability in the soil and
significantly influences the productivity of crops (Li & Liang 2005, Shen et al. 2005). Alam et al. (2003) also
reported similar findings that the application of phosphorus fertilizer (SSP) to wheat crop significantly increased
the grain yield as well as P-uptake over control. A limited supply of P reduces leaf area and therefore reducing
the supply of photosynthates to the grain and subsequently reducing the grain filling. Optimal phosphorus also
increased the synthesis of cytokinins which enhanced the flowering process that supported the higher formation
of grains. Various scientists (Rehman et al. 2006, Yousaf 2004) have also reported that appropriate and balanced
phosphorus fertilization on wheat and rice not only caused grain yield enhancement but also had a positive
impact on phosphorus and another nutrient uptake by these crops. Finger millet varieties efficiency on the use of
available phosphorus might also be due to the plants mechanism to secrete phosphatases which are key drivers
of rhizosphere activities that influenced P uptake. These chemical and biological processes in the rhizosphere
determine the mobilization and acquisition of soil nutrients, microbial dynamics, and controls nutrient use
efficiency of crops, thus profoundly influencing crops productivity (Hinsinger 2009, Richardson et al. 2009,
Wissuwa et al. 2009). Optimal availability of P resulted in an in increase of nutrient use efficiency by the
provision of adequate energy and an early proliferation of growth attributes which increased the grain yield
potential.
CONCLUSION
Application of phosphorus affected positively the productivity of the finger millet varieties in Kakamega and
Alupe for both seasons. An increase of more than 20% was observed on the 20 kg.ha-1 P2O5 rate during the long
rain seasons in both sites and at 25 kg.ha-1 P2O5 in the short rain seasons. The maximum yield was achieved
under the improved variety in both sites due to its superiority in efficiency in utilizing applied phosphorus
fertilizer and superior yield attributes. The application of phosphorus fertilizer beyond 25 kg.ha-1 P2O5 was
found to be detrimental to finger millet grain yield in the study regions of Kenya.
ACKNOWLEDGEMENT
The authors are grateful to the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT)Nairobi for the financial support. The Kenya Agricultural and Livestock Research Organization and ICRISAT
staff in Kakamega and Alupe are also highly appreciated for their support.
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ISSN (E): 2349 1183

ISSN (P): 2349 9265
3(3): 681685, 2016
DOI: 10.22271/tpr.2016.v3.i3.089
Research article
Bidens bachulkarii (Asteraceae-Heliantheae): A new species

from Western Ghats, India
D. G. Jagtap* and M. Y. Cholekar
Shri Vijaysinha Yadav Arts and Science College, Peth-Vadgaon, Kolhapur-416112, Maharashtra, India
*Corresponding Author: jagtapdg6139@gmail.com
Abstract: Bidens bachulkarii a new species in Asteraceae (Tribe: Heliantheae) from Western
Ghats (Maharashtra and Karnataka), India is described and illustrated. The species is closely
related with the Bidens pilosa and Bidens pilosa var. minor. However, it differs in several
characters viz., glabrous and ribbed stem, head terminal and axillary in position, tri-lobed, bisexual
and fertile ray florets, achenes dimorphic, pappus awns of ray and disc achenes half covered with
retrose bristles.
Keywords: Bidens bachulkarii - Heliantheae - New species - Maharashtra - Karnataka.
[Cite as: Jagtap DG & Cholekar MY (2016) Bidens bachulkarii (Asteraceae-Heliantheae): A new species from
Western Ghats, India. Tropical Plant Research 3(3): 681685]
INTRODUCTION
Bidens L. is one of the largest genus of family Asteraceae- Heliantheae, having ca. 150235 species
distributed in different habitats of world (Serff 1937, Chowdhery 1995, Strother & Weedon 2006). Amongst
these 10 species and one verity were reported from India, of which only two species and two varieties were
reported from Maharashtra & four species from Karnataka, two major states of Western Ghats (Hajra et al.
1995, Shirodkar & Lakshminarsimhan 2001, Jagtap et al. 2014, Sharma et al. 1984). The authors had collected
an interesting species of Bidens from Western Ghats of Maharashtra and Belgaum district of Karnataka. The
critical taxonomic investigations and relevant literature revealed that it is new species belonging to genus Bidens
L. which is described and illustrated here.
RESULTS
Taxonomic description:
Bidens bachulkarii Jagtap & Cholekar sp. nov.
(Fig. 1&2)
Annual herbs, erect, up to 1.01.1 m tall; stems glabrous, branched. Leaves variable, imparipinnate, ternatepinnate or ternately-trifoliate, 49 3.04.5 cm, petiolate; leaflets ovate to elliptic-lanceolate, serrate, sharply
acuminate, cuneate, glabrous, 0.75.0 0.32.5 cm; base obtuse or oblique. Heads radiate, terminal or axillary,
lax corymbose, in the axial of leaves, 56 1012 mm across. Peduncles 1.510.0 cm long, glabrous, ribbed,
with scaly or leafy bractlets. Involucral bracts biseriate; outer linear-spathulate, distinctly 8, connate at the base,
56 1.01.5 mm across; inner linear, lanceolate, ciliate at apex, 56 1.52.0 mm across. Receptacle convex,
paleaceous; pale 57 mm long, linear lanceolate. Ray florets distinctly 5, fertile, bisexual; corolla creamy white,
bilobed; lower lip completely dissected, 3.54.5 mm long. Anthers black, appendiculate, ca 1 mm long, linear or
obtuse at base. Style 3.2 mm long; arms ciliate or papillose, 2 mm long. Achenes flat, somewhat curved, ribbed,
5.56.0 mm long, fully covered with barbellate tubercles. Pappus awns 23, unequal, 2.02.5 mm long, smooth
at lower part; retrosely barbed bristles at upper half. Disc florets 2630, fertile, bisexual; corolla tubular, yellow,
5-dentate, glabrous, 4.55.0 mm long. Anther black, appendiculate, linear, 1.61.7 mm long, sagittate. Style
5.56.0 mm long; arms ciliate or pappilose, 2 mm long. Achenes blackish brown, tetragonous, ribbed, truncate
at apex, 79 mm long, covered with barbellate tubercles. Pappus awns 23, 2.53.0 mm long, upper half is
covered with retrosely barbed bristles.
Type: INDIA, Maharashtra, Satara district, Yewteshwar, 500600 m, 168374.19 N, 743233.59 E,
10.08.2015, D.G. Jagtap 1001, (Holotype: CAL, Isotype: SUK, BSI Pune, VYMP).
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Jagtap & Cholekar (2016) 3(3): 681685
Figure 1. Bidens bachulkarii Jagtap & Cholekar: A, Habit; B, Portion of twig; C, Heads in lax corymb; D, Single head; E,
Single head (top view); F, Mature head; G, Ray floret; H, Style of ray floret; I, Single stamen of ray floret; J, Stamen of disc
floret; K, Achene on mature head; L, Achene of ray & disc floret; M, Style of disc floret; N, Mature disc floret; OR,
Variable leaves; S, Single achene of ray floret.
Flowering and Fruiting: AugustDecember.

Habitat: it grows in moist places, ditches, fallow lands or along the roadside of Kolhapur, Satara Sangli districts
of Maharashtra and Belgaum district of Karnataka.
Etymology: The species is named in the honour of Dr. M.Y. Bachulkar, well known Plant Taxonomist, Social
worker, Environmentalist and Principal, of Shri Vijaysinha Yadav Arts and Science, College, Peth-Vadgaon,
Kolhapur (Maharashtra) India, in recognition of his contribution in the Angiosperm Taxonomy.
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Jagtap & Cholekar (2016) 3(3): 681685
Figure 2. Bidens bachulkarii Jagtap & Cholekar: A, Habit; B, Single head; C, Outer involucral bract; D, Inner involucral
bract; E, Single ray floret; F, Gynoecium of ray florets; G, Single stamen of ray floret; H, Achene of ray floret; I, Disc
floret; J, Gynoecium of disc floret; K, Stamen of disc floret; L, Achene of disc floret.
Conservation Status: Bidens bachulkarii Jagtap & Bachulkar was collected from Peth-Vadgaon, Gaganbawada
road and Malkapur of Kolhapur district, Yewteshwar, Kas and Koyananagar of Satara district, Chandoli and
Bhivghat of Sangli district; Rakaskop and Bijgarni of Belgaum district also. A population of about 500
individuals was found in each locality. The area of occupancy is 0.51.0 km2/per locality. Hence, it is assessed
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as Endemic (E), Vulnerable (VN) species following the IUCN categories and criteria (IUCN 2001).
DISCUSSION
In India 10 species and 02 varieties of Bidens L. were reported. The species viz., Bidens comosa, B. cernua,
B. minima, B. tetraspinosa are having simple leaves and leafy or foliaceous involucral bracts whereas others
viz., B. tripartita, B. tripartite var. repens, B. pilosa, B. pilosa var. minor, B. biternata, B. bipinnata, B.
sulphurea and B. humilis are having pinnately compound leaves and linear-lanceolate or narrowly spathulate
involucral bracts. The species Bidens bachulkarii is having spathulate involucral bract which is allied with the
species Bidens pilosa L. and Bidens pilosa var. minor (Bl.) Sherff. However, it differs in several characters viz.,
glabrous and ribbed stem; leaves glabrous; head terminal and axillary in position; receptacle convex, alveolate;
outer involucral bracts spathulate, densely hairy, distinctly 8, connate at base, acute at apex, 45 mm long; inner
involucral bracts larger than outer, 56 mm long, linear-lanceolate, obtuse, hairy at apex; ray florets distinctly 5,
tri-lipped, upper lip 2-nerved, creamy-white, bisexual, fertile; achenes dimorphic; achenes of ray florets curved
with paired, barbellate tubercles, tetragonous, fertile, hairy; achenes of disc florets linear, truncate at both end,
covered with paired barbellate tubercles, tetragonous, hairy; pappus awns of ray and disc achenes half covered
with retrose bristles (Table 1). These differences helped us to describe the collected species from Kolhapur,
Satara, Sangli and Belgaum districts as a new species.
Table 1. Comparative account of Bidens bachulkarii with its allied species.
Characters
Habit
Stem
Leaves
Receptacle
Head
Involucral
bracts
Ray florets
Disc florets
Achenes of
rays
Achenes of
disc
Pappus of
awns
Bidens bachulkarii sp. nov.

Annual herb
Glabrous
Very variable, 25 partite,
glabrous
Convex, alveolate
Radiate, 510 mm in diam.,
terminal and axillary in
position
Bi-seriate; outer spathulate,
densely hairy, distinctly 8,
connate at base, acute at apex,
45 mm long; inner larger
than outer, 56 mm long,
linear-lanceolate, obtuse, hairy
at apex
Distinctly 5, tri-lipped, upper
lip 2-nerved, creamy-white,
bisexual, fertile
Corolla yellow, 5-lobed,
papillose, 45 mm long
Curved with paired, barbellate
tubercles, tetragonous, fertile,
hairy
Linear, truncate at both end,
covered with paired barbellate
tubercles, tetragonous, hairy
23, unequal, smooth at base,
retrose bristles at apex
Bidens pilosa L.
Annual herb
Pilose
35 partite, sparsely hairy
Bidens pilosa var. minor (Bl.) Sherff.

Annual herb
Pilose
35 partite, sparsely hairy
Convex, necked
Radiate, 515 mm in diam.,
terminal in position
Convex, necked
Discoid, 815 mm in diam.,
terminal in position
Bi-seriate; outer narrowly

spathulate, 510, sparsely
hairy at base, obtuse, 57
mm long; inner smaller or
equal to outer, 45 mm
long, ovate, glabrous, acute
at apex
38, ligulate, white, 5nerved, female, sterile
unisexual
Corolla yellow, 5-lobed,
hairy on throat
Flat and sterile, smooth
Bi-seriate; outer narrowly

spathulate, 46 mm long, hairy at
apex, obtuse; inner smaller than
outer, glabrous, linear-lanceolate,
apex acute,
Obconic, fusiform,
tetragonous, glabrous or
sparsely hairy
25, unequal, fully covered
with retrose bristle
Linear, compressed, tetragonous,

sparsely hairy
Absent, if present ligulate or

tubercular, white, female, unisexual
Corolla dark yellow, 5-lobed, hairy
on throat
Flat and sterile and flat
25, unequal, fully covered with

retrose bristle
ACKNOWLEDGMENTS
Authors are very much thankful to Shri. Gulabrao Pol, President, of Shahu Shikshan Prasarak Seva Mandal for
encouraging and providing the laboratory facilities. Authors are also thankful to Dr. M. Tadesse, Addis Ababa
University, Ethiopia, for helping in the identification of species.
REFERENCES
Chowdhery HJ (1995) Heliantheae in Hajra PK, Rao RR, Singh DK & Uniyal BP (eds) Flora of India.Vol.12
(Asteraceae: Anthemideae- Heliantheae). B.S.I., Calcutta. pp. 358431.
684
Jagtap & Cholekar (2016) 3(3): 681685

Hajra PK, Rao RR, Singh DK & Uniyal BP (1995) Flora of India (Heliantheae), Vol.12. B.S.I. Calcutta, pp.
365377.
Jagtap DG, Bachulkar MY & Bhamburdekar SB (2014) Bidens pilosa var. pilosa (Asteraceae): A new record
for Maharashtra. In: Proceeding of National Conference on Environment and Biotechnology pp.198200.
Sharma BD, Singh NP, Raghavan RS & Despande UR (1984) Flora of Karnataka. Analysis. Seris-2. P. 135.
Sherff EE (1937) The genus Bidens. Publication of field Museum of natural History, Botanical Seris 16: pp. 1
709.
Shirodkar DL & Lakshminarsimhan P (2001) In: Singh NP, Karthikeyan S et al. (eds) Flora of Maharashtra
State. Dicotyledones. Vol. 2 (Asteraceae), B.S.I.Calcutta, pp.184.
Strother JL & Weedon RR (2006) Bidens. In Flora of North America Editorial Committee (eds) Flora of North
America 21. New York and Oxford, pp. 205218.
685
ISSN (E): 2349 1183

ISSN (P): 2349 9265
3(3): 686693, 2016
DOI: 10.22271/tpr.2016.v3.i3.090
Research article
A checklist of succulent plants of Ahmedabad, Gujarat, India

Ruchi M. Patel1, Umerfaruq M. Qureshimatva2*, Rupesh R. Maurya2
and Hitesh A. Solanki2
1
Government Science College, Vankal, Mangrol Taliuka, Surat, Gujarat, India

Department of Botany, University School of Sciences, Gujarat University, Ahmedabad, Gujarat, India
*Corresponding Author: ufmqureshi@yahoo.in
Abstract: The study deals with the diversity of succulent plants in Ahmedabad. Succulent plants
are increasingly popular among plant collectors, home gardeners and professional landscapes for
colorful leaves, sculptural shapes, simple care, etc. Succulents are widely used for the indoor
gardening as well as outdoor gardening for their outstanding appearance. The study reported 73
species of succulent plants from Ahmedabad.
Keywords: Diversity - Succulent - Cactus - Ahmedabad.
[Cite as: Patel RM, Qureshimatva UM, Maurya RR & Solanki HA (2016) A checklist of succulent plants of
Ahmedabad, Gujarat, India. Tropical Plant Research 3(3): 686693]
INTRODUCTION
Succulent plants have a global distribution and represented in nearly all habitat type. Over 30 botanical
families have succulent plant species, ranging from tiny annuals plants to huge tree (IUCN 1997). Succulence is
an adaptive response to drought, rapid drainage in rocky and sandy soil, high evaporation in windy, hot
environments and in salty or alkaline habitats. There are probably more than five thousand species worldwide
(Newton & Chan 1998).
The "Succulent Karoo" of South Africa and Namibia boasts the richest succulent flora of Earth. Mexico is
the country with the highest diversity of cactus in the American continent (Ortega & Hctor 2006). More than
60 species are listed in the Red Data Book of the International Union for the Conservation of Nature (IUCN)
(IUCN 2003). Many of these species has an outstanding biological, cultural, and economical importance.
Several species of cactus are among the most dominant plants in different vegetation types, where they interact
with a large variety of animal and plant species (Hctor et al. 2003).
All the succulents evolved from other related plants growing in a normal environment by adaptation to the
changing climatic conditions of their habitat, especially the regularity and amount of rainfall. This process of
adaptation varied in every family and doubtless many plants succumbed in the struggle for survival. Water is
essential for the growth and life of all vegetation, including the succulents, which have mastered the art of
economizing water (Rudolf 1980). In geological times, the earths climate changed becoming drier as the
mountains were pushed up to create rain shadows and deserts. Other plant families adapted similarly to these
conditions and there are thousands of succulent species (Edwards & Donoghue 2006).
Succulent plants are increasingly popular among plant collectors, home gardeners and professional
landscapes for a number of reasons. With their colorful leaves, sculptural shapes and simple care, succulents are
beautiful yet forgiving plant for pots (Debra 2010). Succuelents are highly diverse. The present study shows
diversity in the succulents. They are highly ignored by taxonomist in gujarat just because many of them are
ornamental. Now days succulents are used in outdoor and indoor gardening at various place like malls,
industries, colleges, hospitals and gardens in the city of Ahmadabad. So there is urgent need to have
documentation of such ornamental groups also.
Study area
Ahmedabad is located 23.01 N Latitude and 72.61 East Longitude covering 8,086.81 km2 area at an
altitude of 55 m above the sea level (Qureshimatva et al. 2016). Ahmedabad has a hot semi-arid climate, with
686

Patel et al. (2016) 3(3): 686693

marginally less rain than required for a tropical savanna climate. There are three main seasons: summer,
monsoon and winter. Aside from the monsoon season, the climate is extremely dry. The weather is hot through
the months of March to June; the average summer maximum is 41C (106F), and the average minimum is 27C
(81F). From November to February, the average maximum temperature is 30C (86F), the average minimum
is 15C (59F), and the climate is extremely dry. Cold northerly winds are responsible for a mild chill in
January. The southwest monsoon brings a humid climate from mid-June to mid-September. The average annual
rainfall is about 800 millimeters (31 in), but infrequent heavy torrential rains cause local rivers to flood and it is
not uncommon for droughts to occur when the monsoon does not extend as far west as usual. The highest
temperature recorded is 48.5C (119.3F) (Anonymous 2012).
Field work
Field wok was carried out during 2013 to 2014. The visits were conducted across various gardens; nursery
besides theses also visited some people who has a personal collection of cactus and succulent. During field work
the photos of each succulent were taken along with habit, stem, leaves and other floral parts. Field notes were
taken to have information on habit, habitat and characteristics of succulents. All the specimens were identified
with the help of available literature (Anderson 2012, Mary 2000, Qureshimatva 2016, Rudolf 1980, Scott 2012,
Shah 1978).
In the present study 45 genera and 73 species with 1 sub sp., 5 varieties and 2 cultivated varieties belonging
to 15 families have been reported from the study area (Fig. 1; Table 1). In the present study 5 succulents were
reported as indigenous and endemic to India. Other succulents were introduced from the Madagascar, Southern
Africa, Brazil, Europe, Mexico, Tropical America, etc. (Fig. 2).
Table 1. List of succulent plants occurring in Ahmedabad.
S.N. Botanical Name
Family
Portulaca oleracea L.
Portulacaceae
Portulaca pilosa L.
Portulacaceae
Common
Name
Purslane,
Lunia
Native
Propag
ation
Western Asia Stem
cutting
Kiss me quick North

America
Central Asia Seeds or
Common in
Ahmedabad
Gurjar vaani, Gujarat
University Campus.
Commiphora wightii (Arn.)

Bhandari
Burseraceae
Cissus quadrangularis L.
Vitaceae
Veldt Grape,
Devil's
Backbone
Southern
and eastern
Africa,
Arabia to
India
Stem
cutting
Gujarat University
Campus
Cissus rotundifolia Vahl
Vitaceae
Venezuelan
Treebine,
Arabian wax
cissus
Africa
Stem
cutting
K. H. Bhatts house.
Bryophyllum delagoense
(Eckl. & Zeyh.) Druce
Crassulaceae
Mother of
Millions
Madagascar
Leaf
cutting
Gujarat University
Campus
Bryophyllum fedtschenkoi
(Raym.-Hamet & H.Perrier)
Lauz.-March.
Crassulaceae
Air plant,
Cathedral
bells
Madagascar Leaf
and Southern cutting
Africa
Kalanchoe blossfeldiana
Poelln
Crassulaceae
Flaming Katy, Madagascar

Christmas
Kalanchoe,
Florist
Kalanchoe
Gugal
stem
cutting
Distribution
Serenity
stem
cutting
Stem
cutting
Common in
Ahmedabad
Common in
Ahmedabad
687
Patel et al. (2016) 3(3): 686693

Deserts
Cabbage
Paddle plant
South Africa Stem
Crassulaceae
Panda plant,
Plush plant,
Pussy ears.
chocolate
soldier
Madagascar
Sedum album L.
Crassulaceae
Coral Carpet, Europe

White
stonecrop
12
Sedum morganianum
E.Walther
Crassulaceae
Burro's tail or South Africa Stem

donkey tail
cutting
Some private and

Home gardens
13
Acanthocereus tetragonus
(L.) Hummelinck
Cactaceae
Triangle
cactus
Gujarat University
Campus
14
Astrophytum myriostigma
Lem
Cactaceae
Bishop's Cap, Highlands of Seeds or

Bishop's Hat central and Grafting
Kalanchoe tetraphylla H.
Perrier
Crassulaceae
10
Kalanchoe tomentosa Baker
11
cutting
Gujarat University
Campus
Leaf
cutting
Common in
Ahmedabad
Stem
cutting
Gujarat University
Campus
South Texas Seeds or

to Venezuela, grafting
on the
Atlantic
coast of
Central
America
K. H. Bhatts house.
northern
Mexico
Brazil
15
Brasiliopuntia brasiliensis
(Willd.) A.Berger
Cactaceae
Brazilian
Prickly Pear
16
Cereus hexagonus (L.) Mill
Cactaceae
Hedge cactus, South

America
spiny tree
cactus
Stem
cutting
K. H. Bhatts house.
17
Echinocactus grusonii Hildm. Cactaceae
Golden Barrel Mexico

Cactus
Seeds
K. H. Bhatts house.
18
Echinocactus texensis Hopffe Cactaceae
Horse
Crippler,
Devils head
Mexico
Seeds
Gujarat University
Campus
19
Epiphyllum anguliger (Lem.) Cactaceae

G.Don
Rickrack
Cactus
Mexico
Leaf
cutting
The Sarabhai
Foundation Botanical
Garden
20
Epiphyllum oxypetalum (DC.) Cactaceae

Haw.
Brahmakamal Mexico
21
Ferocactus peninsulae
Cactaceae
(A.A.Weber) Britton & Rose
Barral cactus
Mexico
22
Hamatocactus sp.
Cactaceae
Turk's head
Native of
Seed or
Argentina
cutting
and Paraguay
Dept. of Botany
Gujarat University
23
Harrisia martinii (Labour.)

Britton
Cactaceae
Harrisia
cactus,
Moonlight
cactus
Argentina
Stem
and Paraguay cutting
Gujarat University
Campus
Seeds and Gujarat University

stem
Campus
cuttings
Rhizome, Private gardens

herbaceous
stem and
leaf cutting
or layering
Seeds
Gurjar vaani
688
Patel et al. (2016) 3(3): 686693

24
Mammillaria beneckei
Ehrenb
Cactaceae
Mammillaria
Mexico
Seeds
Gujarat University
Campus
25
Myrtillocactus geometrizans
(Mart. ex Pfeiff.) Console
Cactaceae
Blue Candle
Northern
central
Mexico
down to
Oaxaca
Seeds,
Gujarat University
Seeds or
stem
cutting
cuttings in Campus
summer
26
Opuntia cochenillifera (L.)

Miller
Cactaceae
Cochineal
nopal cactus,
warm hand
Mexico
27
Opuntia cylindrica (Lam.)

DC
Cactaceae
Cane cactus
Ecuador and Stem

Per
cutting
28
Opuntia elatior Mill
Cactaceae
Prickly Pear, Central

Slipper Thorn, America
Phafdo Thor
29
Opuntia microdasys (Lehm.) Cactaceae

Pfeiff
Branching
Beavertail
Mexico
Stem
Serenity, Gujarat
cutting or University Campus
Seeds
30
Pereskia grandifolia Haw
Cactaceae
Rose cactus
Brazil
(Uncertain)
Cutting or Serenity
seed
31
Schlumbergera kautskyi
(Horobin & McMillan)
N.P.Taylor
Cactaceae
Christmas
Cactus
Brazil
Stem
(Esprito
cutting
Santo, Minas
Gerais)
Private gardens
32
Aptenia cordifolia (L.f. )

Schwantes
Cactaceae
Baby Sun
Rose
North
America
Stem
cutting
Private gardens
33
Adenium obesum Roem. &

Schult
Apocynaceae
Desert rose
Africa
Seeds,
K. H. Bhatts house.
grafting
and cutting
34
Pachypodium lamerei Drake Apocynaceae
Madagascar
Palm, Club
Foot
Madagascar
Seeds
Science city garden

and K. H. Bhatts
house.
35
Plumeria alba L.
White
South
America
Seed
Gujarat University
Campus
Gujarat University
Campus
Apocynaceae
champa
Seeds or
stem
cutting
Serenity Library, Bhat
Private garden near

Sardar Stadium, Gujarat
University Campus
Gujarat University
Campus
36
Plumeria rubra L.
Apocynaceae
Champa
America
Seeds
37
Ceropegia bulbosa Roxb.

var. lushii (Grah.) Hook.f.
Asclepiadaceae
Bulbous
Ceropegia
Africa
Nodal
Gujarat University
segments Campus
38
Sarcostemma acidum Voigt
Asclepiadaceae
Soma
India and
Africa
Seed
Serenity Library,
Bhat.
39
Bacopa monnieri (L.) Wettst Scrophulariaceae
Brahmi,
India
Stem
cutting
Gujarat University
Campus
Baam
40
Basella alba L.
Basellaceae
Red vine
spinach
Asia
Seed
Gujarat University
Campus
41
Alluaudia procera (Drake)

Drake
Didiereaceae
Madagascan
Ocotillo
Southern &
Stem
Gujarat University
Campus
south-western cutting
Madagascar
(Toliara)
689
Patel et al. (2016) 3(3): 686693

42
Aemaralmeidia fusiformis
Euphorbiaceae
(Buch.-Ham) S.M. Almeida
& S. Dutta ex Santosh Yadav
& Rashmi Sharma
Thor
Stem
cutting
Gujarat University
Campus
43
Euphorbia antiquorum L.
Euphorbiaceae
Thor
Peninsular
India
Seed
Common in
Ahmedabad
44
Euphorbia cristata B.Heyne

ex Roth
Euphorbiaceae
Thor,
Stem
cutting
Science city
45
Euphorbia milii Des Moul.
Euphorbiaceae
Thor
Stem
cutting
Gujarat University
Campus, St. Xavier
College Campus
46
Euphorbia milii var.

splendens (Bojer ex Hook.)
Ursch & Leandri
Euphorbiaceae
Crown of
thorns
Stem
cutting
Gujarat University
Campus, St. Xavier
College Campus.
47
Euphorbia neriifolia L.
Euphorbiaceae
Thor,
India
Stem
cutting
Common in
Ahmedabad
Neurang
Neurang
48
Jatropha podagrica Hooker
Euphorbiaceae
Buddha belly
plant, Bottle
plant shrub
Tropical
America
Seeds
Common in
Ahmedabad
49
Pedilanthus tithymaloides
(Linn.) Poit
Euphorbiaceae
Nivali,
Vilayati-sher
Tropical
America
Stem
cutting
Common in
Ahmedabad
50
subsp. smallii (Millsp.)
Dressler
Euphorbiaceae
Nivali,
Vilayati-she
Stem
cutting
Common in
Ahmedabad
51
var. varigatus (L.) Poit
Euphorbiaceae
Nivali,
Vilayati-sher
Stem
cutting
Law garden
52
Synadenium grantii Hook.f.
Euphorbiaceae
African Milk
Bush
East Central
Africa.
Stem
cutting
Private garden
53
Tirucalia indica Raf.
Euphorbiaceae
Sher, Indian
Tree Spurge,
saptala (in
Sanskrit)
South Africa Stem
cutting
Private garden near

SG highway
54
Aechmea distichantha Lem
Bromeliaceae
Brazilian
vaseplant
Brazil
Offsets
Private garden
55
Aechmea fasciata (Lindl.)

Baker
Bromeliaceae
Silver vase
plant
Brazil
Offsets
Gujarat University
Campus
56
Deuterocohnia scapigera
(Rauh & L.Hrom.)
M.A.Spencer & L.B.Sm.
Bromeliaceae
Seed
Private garden
57
Agave americana L.
Agavaceae
Century plant, Mexico
Suckers
Common in
Ahmedabad
American aloe
58
Agave sisalana Perrine ex

Engelm
Agavaceae
Baby Sun
Rose,
Mexico
Suckers
Common in
Ahmedabad
59
Agave victoriae-reginae
T.Moore
Agavaceae
Baby Sun
Rose
Mexico
Seeds or
Suckers
Common in
Ahmedabad
60
Agave vivipara L.
Agavaceae
Caribbean
Century Plant
Suckers
Serenity
690
Patel et al. (2016) 3(3): 686693

61
Nolina recurvata (Lem.)

Hemsl
Agavaceae
Pony Tail
Palm
Tamaulipas
(Mexico)
Seeds
K. H. Bhatts house.
62
Yuccaa loifolia L.
Agavaceae
Spanish
bayonet
Seeds
Gujarat University
Campus
63
Sansevieria cylindrica Bojer

ex Hook
Sansevieriaceae
Sasevieria
Native to
Leaf
Gujarat University
Campus
Tropical Asia cutting
and natal
Blue
sansevieria,
sword
sansevieria
Africa
Leaf
cutting
Serenity
Sanseneria hyacinthoides (L.) Sansevieriaceae

Hort ex Staud
Piles root
Africa
Leaf
cutting
Gujarat University
Campus
66
Sansevieria kirkii Baker
Sansevieriaceae
Snake plant
Kenya
(Africa)
Leaf
cutting
Serenity
67
Sansevieria trifasciata Prain
Sansevieriaceae
Mother-inlaw's tongue
Africa
Leaf
cutting
Gujarat University
Campus
68
Sansevieria trifasciata cv
golden hahnii
Sansevieriaceae
Africa
Leaf
cutting
Private gardens
69
Sansevieria trifasciata cv
hahnii Graff
Sansevieriaceae
Africa
Leaf
cutting
Private gardens
70
Sansevieria trifasciata var.

laurentii (De Wild.) N.E.Br
Sansevieriaceae
Belgian
Congo
Leaf
cutting
Gujarat University
Campus
71
Aloe deltoideodonta Baker
Aloeaceae
Baby Sun
Rose
Madagascar
Offshoots Private gardens
72
Aloe maculata All
Aloeaceae
Baby Sun
Rose
South Africa Rhizome, Private gardens
Baby Sun
Rose
South Africa Offshoots Common in
64
Sansevieria ehrenbergii
Schweinf. ex Baker
65
73
Aloe vera (L.) Burm. f.
Sansevieriaceae
Aloeaceae
tubers,
corns or
bulbs
Ahmedabad
74
Gasteria batesiana
G.D.Rowley
Aloeaceae
South Africa Offshoots Serenity
75
Gasteria obliqua (Aiton)

Duval.
Aloeaceae
South Africa, Offshoots Serenity

Lesotho and
Swaziland.
76
Haworthia coarctata var.

adelaidensis (Poelln.)
M.B.Bayer
Aloeaceae
South Africa Offshoots K. H. Bhatts house.
77
Haworthia fasciata (Willd.)

Haw
Aloeaceae
Variegated
Zebra plant
South Africa Offsets,
78
Haworthia glauca Baker
Aloeaceae
Eastern Cape Seeds and Private gardens

of South
offsets
Africa
79
Haworthia limifolia Marloth
Aloeaceae
File Leafed
Haworthia
South Africa Seeds,
K. H. Bhatts house.
leaves
Shraddha house and

Offsets or Serenity
leaf
cutting
691
Patel et al. (2016) 3(3): 686693
Figure 1. Photographs of some succulents: A, Portulaca pilosa Linn. Spp. Grandiflora(Hook.) Gesink; B, Portulaca pilosa
L.; C, Cissus rotundifolia Vahl; D, Bryophyllum fedtschenkoi (Raym.-Hamet & H.Perrier) Lauz.-March; E, Kalanchoe
blossfeldiana Poelln; F, Echinocactus grusonii Hildm; G, Opuntia microdasys (Lehm.) Pfeiff.; H, Ferocactus peninsulae
(A.A.Weber) Britton & Rose; I, Plectranthus amboinicus (Lour.) Spreng; J, Aechmea fasciata (Lindl.) Baker.; K, Agave
victoriae-reginae T.Moore Gard.; L, Aloe maculata All.
ACKNOWLEDGEMENTS
Authors are thankful to Head of the Department of Botany, Dr. A. U. Mankad and Dr. Santosh L. Yadav,
Chief Botanist, The Serenity Library for their constant help and support.
692
Patel et al. (2016) 3(3): 686693
Figure 2. Number of succulent species in dominant families.
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Anonymous (2012) Hong Kong Observatory.
Debra LB (2010) Succulent Container Gardens: Design Eye-Catching Displays with 350 Easy-care plants.
Timber Press, Portland.
Edward FA (2001) The Cactus Family. Portland, Ore. Timber Press, United States of America.
Edwards EJ & Donoghue MJ (2006) Pereskia and the Origin of the Cactus Life- Form. The American Naturalist
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Hctor G-, Teresa V & Pablo O-B (2003) Demographic trends in the Cactaceae. The Botanical Review 69:
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IUCN (1997) Cactus and Succulent Plants. International Union for the Conservation of Nature and Natural
Resources. Available from: http://www.iucnredlist.org/ (accessed: 16 Mar. 2014)
IUCN (2003) Red List of Threatened Species. International Union for the Conservation of Nature and Natural
Resources. Available from: http://www.iucnredlist.org/ (accessed: 16 Mar. 2014)
Mary I (2000) Agave, Yuccas and Related Plants: A Gardeners Guide. Timber Press, United States of America.
Newton DJ & Chan J (1998) South Africas Trade in Southern African Succulent Plants. TRAFFIC
East/Southern Africa, Johannesburg, Republic of South Africa.
Ortega BP & Hctor G- (2006) Global diversity and conservation priorities in the Cactaceae. Biodiversity and
Conservation 15: 817827.
Qureshimatva UM, Gamit SB, Maurya RR, Solanki HA & Yadav SL (2016) Cheklist of palms in Ahmedabad.
Gujarat, India. International Journal of Recent Scientific Research 7(3): 94139417.
Rudolf S (1980) London Cacti and Succulents. The Hamlyn Publishing Group Ltd., U.K.
Scott DC (2012) The Gardener's Guide to Cactus: The 100 Best Paddles, Barrels, Columns, and Globes.
Timber Press, United States of America.
Shah GL (1978) Flora of Gujarat State. Sardar Patel University, Vallabh, Vidyanagar, Anand, Gujarat.
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3(3): 694700, 2016
DOI: 10.22271/tpr.2016.v3.i3.091
Research article
Lichens in 50 ha permanent plot of Mudumalai Wildlife

Sanctuary, Tamil Nadu, India
Komal K. Ingle1, Sanjeeva Nayaka1* and H. S. Suresh2
1
Lichenology Laboratory, CSIR-National Botanical Research Institute, Rana Pratap Marg,

Lucknow-226001, India
2
Centre for Ecological Sciences, Indian Institute of Science, Bangalore-560012, India
*Corresponding Author: nayaka.sanjeeva@gmail.com
Abstract: Mudumalai Wildlife Sanctuary and National Park is a protected area located in the
Tamil Nadu state within the track of Western Ghats. The very popular, permanent 50 ha
'Mudumalai Forest Dynamics Plot' is situated in Compartment 17 of the Kargudi Range in the
sanctuary. The sanctuary has rich diversity of vascular plants while information regarding
cryptogams is scarce. Recently, 10th plot of 50 ha plot was visited for lichen study and it was
noticed that the trunks of the trees did not support any lichens. However, luxuriant growths of
lichens were observed on the fallen twigs indicating their presence in the canopy. The
identification of these lichens revealed the occurrence of 66 species belonging to 27 genera and 16
families. The crustose lichens are dominant in the area with 32 species which is followed by
foliose with 27 species. The lichen family Physciaceae is most diverse in the plot with 6 genus and
16 species, while Pertusaria with 15 species is the most dominant genus. Among the trees
growing within the plot, Terminalia crenulata harboured maximum number of 39 species of
lichens. Parmotrema tinctorum, P. crinitum, Pertusaria concinna and Pyxine coralligera are the
most common lichens in the plot. There are as many as 28 rare lichens in the plot with one time
encounter.
Keywords: Lichenized fungi - Nilgiri Biosphere Reserve - Western Ghats - Biodiversity.
[Cite as: Ingle KK, Nayaka S & Suresh HS (2016) Lichens in 50 ha permanent plot of Mudumalai Wildlife
Sanctuary, Tamil Nadu, India. Tropical Plant Research 3(3): 694700]
INTRODUCTION
Lichen is symbiotic association between two organisms, an alga (or cyanobacteria) and a fungus. In the
world there are about 20,000 species of lichens are known to occur and India is represented by 2350 species
(Singh & Sinha 2010). Among various states Tamil Nadu is represented by maximum number of lichens with
760 species. Location of lichen rich sites such as Nilgiri and Palni Hills, and major portion of Western Ghats
falling in Tamil Nadu are the reasons for lichen richness in the state. In addition the Eastern Ghats part of Tamil
Nadu also has rich diversity of lichens (Nayaka et al. 2013). However, there are many interesting localities and
protected areas such as Mudumalai Wildlife Sanctuary and National Park in Tamil Nadu that are unexplored
for the lichens.
Mudumalai Wildlife Sanctuary and National Park (11 36' N, 76 32' E) abuts the northern flank of the
Nilgiri mountain range in the Western Ghats and is contiguous with the protected areas Bandipur and Wynaad.
It is also declared 'Tiger Reserve' in the recent times. The popular, permanent 50 ha 'Mudumalai Forest
Dynamics Plot' is located in Compartment 17 of the Kargudi Range of the sanctuary (Fig. 1) at the transition
zone between dry and moist deciduous forest. The 50 ha plot receives average rain fall of 1200 mm/yr. The
flowering plants and fauna of the sanctuary including the 50 ha plot are well documented. The sanctuary has a
total of 187 tree species (Suresh et al. 1996), over 200 birds, at least 17 species of amphibians, 42 species of
reptiles and 35 species of mammals. The 50 ha permanent plot provides an opportunity to conduct regular
vegetation dynamics, climate and several studies related to temporal and seasonal changes. The literature survey
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Ingle et al. (2016) 3(3): 694700

clearly indicates the studies on cryptogams are rare or lacking for the Mudumali Wildlife Sanctuary (Satish et
al. 2007). The lichens being a sensitive to air pollution and microclimate conditions their documentation could
very well supplement the climate change studies being carried out in the 50 ha plot. Recently, one of the authors
(SN) paid a short visit to the sanctuary and the preliminary observation on lichen biota is presented here.
Figure 1. Map of Mudumalai Wildlife Sanctuary showing the location of 50 ha permanent Forest Dynamic Plot, from where
lichens were collected.

The 10th plot within the 50 ha plot was surveyed for lichens and it was noticed that the trunks of the trees did
not support any lichens. However, luxuriant growths of lichens were observed on the fallen twigs indicating
their presence in the canopy. Such fallen twigs are gathered, cut in to smaller pieces and dried thoroughly under
sun and preserved in lichen herbarium packets with detailed label. The twigs belongs to following tree species,
Cassia fistula, Ficus tsjakela, Grewia tiliifolia, Lagerstroemia microcarpa, Schleichera oleosa, Syzygium
cumini, Tectona grandis, and Terminalia crenulata.
The lichen specimens investigated morphologically, anatomically and chemically following recent literature
(Awasthi 1991, 2007). The colour tests were performed with the routine reagent i.e. K (5% potassium
hydroxide), C (aqueous solution of calcium hypochloride) and P (paraphenylene diamene). Some lichen
specimens were investigated with Thin Layer Chromatography (TLC) in solvent system A (toluene: dioxine:
acetic acid) by Walker & James (1980) method. After the identification of specimens, the samples are preserved
at lichen herbarium of CSIR-National Botanical Research Institute, Lucknow (LWG). The classification of
Lumbusch and Huhndorf (2007) followed for arranging species under various families.
RESULTS AND DISCUSSIONS
The identification of these lichens revealed the occurrence of 66 species belonging to 27 genera and 16
families (Table 1). The crustose lichens are dominant in the area with 32 species followed by foliose and
squamulose with 27 and 5 species respectively (Fig. 2). Chrysothrix chlorina is the only leprose and Ramalina
pacifica is the only fruticose lichen recorded from the 17th ha plot. The lichen family Physciaceae is most
diverse in the plot with 6 genera and 16 species while Ramalinaceae with 3 genera and 8 species and
Parmeliaceae with 2 genera 8 species are other dominant families (Fig. 3). Pertusaria with 15 species is the
most dominant genus and it is followed by Parmotrema (7 spp.), Heterodermia (5 spp.) and Phyllopsora (5 spp.).
695
Ingle et al. (2016) 3(3): 694700
Figure 2. Pie chart showing number of species under different growth forms in the study area.
Figure 3. Bar diagram showing the diversity of species within genera and families represented in the study area.
Among the trees growing within the plot, Terminalia crenulata harboured maximum number of 39 species
of lichens, followed Tectona grandis with 30 species, Syzygium cumini 25 spp., Ficus tsjakela 19 spp., and
Lagerstroemia microcarpa 16 spp. The tree Grewia tiliifolia harboured only one lichen species Leptogium
austroamericanum (Fig. 4). Further, L. austroamericanum is rare lichen found only on this tree.
Table 1. List of lichens recorded from 10th plot of 50 ha plot of Mudumalai Wildlife Sanctuary and their distribution on various tree
species.
Lichen taxa
Chrysotrichaceae
1. Chrysothrix chlorina (Ach.) J.R. Laundon
Collemataceae
2. Leptogium austroamericanum (Malme) C.W. Dodge
3. L. cyanescens (Rabenh.) Krb.
Graphidaceae
4. Diorygma junghuhnii (Mont. & Bosch) Kalb & al.
5. Graphis nigroglauca Leight.
Lecanoraceae
6. Lecanora helva Stizenb.
7. L. perplexa Brodo
GF
Tree species
4 5 6
F
F
C
C
C
C
Remarks
+ Common
Rare
+ Common
+
+
Rare
Common
+
+
+ Common
+ Most common
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Lobariaceae
8. Pseudocyphellaria argyracea (Bory ex Delise) Vain.
Ochrolechiaceae
9. Ochrolechia subpallescens Verseghy
Parmeliaceae
10. Bulbothrix isidiza (Nyl.) Hale
11. Parmotrema crinitum (Ach.) M. Choisy
12. P. hababianum (Gyeln.) Hale
13. P. indicum Hale
14. P. melanothrix (Mont.) Hale
15. P. nilgherrense (Nyl.) Hale
16. P. reticulatum (Taylor) M. Choisy
17. P. tinctorum (Despr. ex Nyl.) Hale
Pertusariaceae
18. Pertusaria albidella Nyl.
19. P. cf. cryptocarpa Nyl.
20. P. concinna Erichsen
21. P. coronata (Ach.) Th. Fr.
22. P. idukkiensis D.D. Awasthi & Preeti Srivast.
23. P. leioplacella Nyl.
24. P. leucosorodes Nyl.
25. P. melastomella Nyl.
26. P. multipuncta (Turn.) Nyl.
27. P. neilgherrensis (Mll. Arg.) D.D. Awasthi & Preeti
Srivast. in D.D. Awasthi
28. P. punctata Nyl.
29. P. rigida Mll. Arg.
30. P. subdepressa Mll. Arg.
31. P. trisperma Mll. Arg.
32. P. tuberculifera Nyl.
Physciaceae
33. Dirinaria aegialita (Afzel.) Moore
34. D. consimilis D.D. Awasthi in D.D. Awasthi & M.R.
Agarwal
35. Heterodermia diademata (Taylor) D.D. Awasthi
36. H. dissecta (Kurok.) D.D. Awasthi
37. H. isidiophora (Nyl.) D.D. Awasthi
38. H. obscurata (Nyl.) Trevis.
39. H. speciosa (Wulfen) Trevis.
40. Hyperphyscia adglutinata var. adglutinata H. Mayrhofer
& Poelt in Hafellner & al.
41. H. adglutinata var. pyrithrocardia (Mll. Arg.) D.D.
Awasthi
42. Phaeophyscia hispidula (Ach.) Moberg
43. Physcia tribacoides Nyl.
44. Pyxine austroindica D.D. Awasthi
45. P. cocoes (Sw.) Nyl.
46. P. coralligera Malme
47. P. cylindrica Kashiw.
48. P. retirugella Nyl.
Pilocarpaceae
49. Lopadium pulchrum (Mll. Arg.) Zahlbr.
Pyrenulaceae
50. Anthracothecium maculatum Nagarkar & Patw.
51. Arthopyrenia grisea (Schleich. ex Schaer.) Krb.
52. A. keralensis Upreti & G. Pant
53. Pyrenula interducta (Nyl.) Zahlbr.
Ramalinaceae
54. Bacidia laurocerasi (Delise ex Duby) Vain. in Zahlbr.
55. B. medialis (Tuck. ex Nyl.) Zahlbr.
+ Rare
Rare
F
F
F
F
F
F
F
F
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Most common
Most common
Common
Rare
Common
Rare
Common
Most common
C
C
C
C
C
C
C
C
C
C
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Common
Common
Most common
Rare
Rare
Rare
Common
Common
Common
Common
C
C
C
C
C
+
+
Rare
Rare
Common
Rare
Rare
F
F
+ Rare
+ Most common
F
F
F
F
F
F
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Common
F
F
F
F
F
F
F
+
+
+
+
+
+
+
+
+
+
+
+
+ Common
C
C
C
C
C
C
+ Rare
+ Rare
Common
Most common
Common
Common
Most common
Rare
Rare
Common
Common
Common
Most common
Common
Rare
Rare
Rare
Rare
Common
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56. Phyllopsora corallina var. subglaucella G.K. Mishra,
S
+ + Common
Upreti & Nayaka
57. Phyllopsora isidiotyla (Vain.) Riddle
S
+ Rare
58. P. kalbii Brako
S
+ Rare
59. P. mauritiana (Taylor) Gotth. Schneid
S
+ Rare
60. P. nemoralis Timdal & Krog
S
+ + Rare
61. Ramalina pacifica Asahina
Fr
+ Rare
Roccellaceae
62. Opegrapha bengalensis Upreti & Ajay Singh
C
+ Rare
Teloschistaceae
63. Caloplaca bassiae (Willd. ex Ach.) Zahlbr.
C
+ + + + Most common
Thelotremataceae
64. Myriotrema rugiferum (Harm.) Hale
C
+ Common
Trypetheliaceae
65. Trypethelium flavocinereum Makhija & Patw.
C
+ + Common
66. T. tropicum (Ach.) Mll. Arg.
C
+ + Common
Note: GF = Growth Form, C = Crustose, F = Foliose, Fr = Fruticose, S = Squamulose, 1 = Cassia fistula, 2 = Ficus tsjakela,
3 = Grewia tiliifolia, 4 = Lagerstroemia microcarpa, 5 = Schleichera oleosa, 6 = Syzygium cumini, 7 = Tectona grandis, 8 =
Terminalia crenulata: = Absent, + = Present.
Figure 4. Pie chart showing number of lichen species on different host plant in the study area.
Parmotrema tinctorum, P. crinitum, Pertusaria concinna, and Pyxine coralligera, are the most common
lichens in the plot (Fig. 5). Ten very common lichens as many as 28 rare lichens with one time in the plot are
marked in the table 1. Some prominent examples of rare lichens are Arthopyrenia keralensis, Diorygma
junghuhnii, Ochrolechia subpallescens, Parmotrema indicum, Phaeophyscia hispidula, Pseudocyphellaria
argyracea, and Ramalina pacifica.
Mudumalai has high density of furgivorous mammals causing damage to woody plants through their feeding
activity. Asian elephants are the most notorious animals that destroy the trees by peeling off their bark and also
by felling. Mudumalai has a long history of human settlement (Hockings 1989, Prabhakar 1994). Huntersgatherer societies such as the Kurubas, Irulas, Paniys, and Kotas have inhabited this region for several centuries,
but their number was always fluctuating due to outbreaks of disease and strife. In the recent times due better
medical facilities the population size has increased and they are invading the forest area for settlement, hence
anthropogenic disturbance to the forest is inevitable. The anthropogenic disturbance involves mainly logging,
systematic extraction of timber (Ranganathan 1941) and collection of non-timber forest products. However, the
major threat to the forest in general and to the lichen in particular is frequent fires during month dry season as
the grasses are desiccated and highly flammable. Intense and widespread fires usually burn the understory
vegetation and tree trunks spreading up to a considerable height. The fire completely destroys the epiphytic
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Ingle et al. (2016) 3(3): 694700
Figure 5. Some common lichen species reported from the study area: A, Dirinaria consimilis; B, Heterodermia dissecta; C,
H. speciosa; D, Lecanora perplexa; E, Parmotrema crinitum; F, P. tinctorum; G, Pertusaria concinna; H, Pyxine
coralligera. [Scale bars: AF = 2 mm; G & H = 1 mm]
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plants including lichens. Unlike other epiphytes the lichens are slow growing organisms and take several years
to colonize a barren habitat. The frequent firing in the Mudumalai is not giving enough time for lichens to
establish on the tree trunk. However, high diversity and luxuriant growth of lichens are observed in the canopy
of the forest and are collected from the fallen twigs as an example. The lichen diversity in turn indicate the
healthy micro-climatic condition of the forests suitable for the growth climate sensitive plants and as well as
inhabitation of animals.
CONCLUSION
Undoubtedly, the occurrence of 66 lichen species in just one ha plot clearly indicates the huge and diverse
lichen reserve within the Mudumalai Sanctuary. The thorough lichen study in the area would yield many more
species. The data provided in the present study will be helpful to plan and execute future lichenological studies
in the area. It would be a great idea to systematically survey the 50 ha plot and quantitatively document the
lichen diversity within the plot. The lichens being sensitive to micro-climatic changes such quantitative data
would be helpful to monitor the effect of climate change on lichens and forest in general. The data can also be
correlated with tree species and forest dynamics due herbivory by large mammals, and forest fires.
ACKNOWLEDGMENTS
We are thankful to Director, CSIR-NBRI, Lucknow for providing the laboratory facilities, to Dr. D.K. Upreti
for his cooperation, to Prof. R. Sukumar, CES, IISc, Bengaluru for his kind permission to use the facilities at
Mudumalai Field Station, to all members of Field Station for their cooperation during the collection.
REFERENCES
Awasthi DD (2007) A Compendium of the Macrolichens from India, Nepal and Sri Lanka. Bishen Singh
Mahendra Pal Singh, Dehra Dun, India, 580 p.
Awasthi DD (1991) A key to the microlichens of India, Nepal and Sri Lanka. Bibliotheca Lichenologica 40: 337 p.
Hockings P (1989) Blue Mountains: The Ethnography and Biography of a South Indian Region. Oxford
University Press, New Delhi, 406 p.
Lumbsh HT & Huhndord SM (2007) Notes on ascomycetes, Nos. 4408-4750. Myconet 13: 5999.
Nayaka S, Reddy AM, Ponmurugan P, Devi A, Ayyappadasan G & Upreti DK (2013) Eastern Ghats
biodiversity reserves with unexplored lichen wealth. Current Science 104(7): 821825.
Prabhakar R (1994) Resource Use, Culture and Ecological Change: A Case Study of the Nilgiri Hills of
Southern India, Ph.D. thesis. Indian Institute of Science, Bangalore, India.
Ranganathan CR (1941) Working Plan for the Nilgiris Division. Government Press, Madras.
Satish N, Sultana S & Nanjundiah V (2007) Diversity of soil fungi in a tropical deciduous forest in Mudumalai,
Southern India. Current Science 93(5): 669677.
Singh KP & Sinha GP (2010) Indian Lichens, An Annonated Checklist. Botanical Survey of India, Kolkata, 571 p.
Suresh HS, Dattaraja HS & Sukumar R (1996) Tree ora of Mudumalai Sanctuary, southern India. Indian
Forester 122: 507519.
Walker FJ & James PW (1980) A revised guide to microchemical techniques for the identification of lichen
substances. British Lichen Society Bulletin 46:1329.
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3(3): 701703, 2016
DOI: 10.22271/tpr.2016.v3.i3.092
Short communication
Development and characterization of microsatellite markers for

Osyris lanceolata Hochst. & Steud., an endangered
African sandalwood tree species
John O. Otieno1,3*, Stephen F. Omondi1, Annika Perry2, David W. Odee1,2,
Emmanuel T. Makatiani1, Oliver Kiplagat3 and Stephen Cavers2
1
Kenya Forestry Research Institute, P.O. Box 20412-00200, Nairobi Kenya

Centre for Ecology and Hydrology, CEH Edinburgh, Bush Estate Penicuik, Midlothian, United Kingdom
3
University of Eldoret, P.O. Box 1125-30100, Eldoret, Kenya
*Corresponding Author: jochiengo@gmail.com
[Cite as: Otieno JO, Omondi SF, Perry A, Odee DW, Makatiani ET, Kiplagat O & Cavers S (2016)
Development and characterization of microsatellite markers for Osyris lanceolata Hochst. & Steud., an
endangered African sandalwood tree species. Tropical Plant Research 3(3): 701703]
Osyris lanceolata Hochst. & Steud. is a multipurpose tree species widely spread in many of the sub-Saharan
countries ranging from Algeria to Ethiopia all the way to South Africa. In Kenya, the species is endemic to the
Arid and Semi-Arid Lands (ASALs). It is highly valued for its essential oils used in the cosmetic and
pharmaceutical industries. Despite its endangered status and economic importance, little is known about its
genetic diversity status and only few conservation strategies exist for the species. Overexploitation of the
species has resulted in the decline of its population and reduced availability of its products. The mode of
harvesting of sandalwood is destructive and unsustainable. This is because the whole tree is usually uprooted to
get the heartwood from the stem, stump and roots. The exploitation of African sandalwood could soon drive the
species to extinction unless proper control measures are put in place through regulation of its trade and
development of conservation strategies. Despite its endangered status and economic importance, no genetic
study has been carried out on the species to provide information vital for conservation strategies. This paper
reports the development and characterization of a set of 12 polymorphic and five (5) monomorphic
microsatellite markers isolated and characterized of O. lanceolata.
One plant leaf sample was used as the source of DNA for genomic library construction. Total genomic DNA
was extracted from silica gel dried leaf using DNeasy Plant Mini Kit (QIAGEN, Hilden, Germany). The DNA
sample was then sent to The Gene Pool Institute of Evolutionary Biology, University of Edinburgh for
sequencing. Simple sequence repeats (SSRs) were extracted through PAL Finder software version 0.02.04
(Castoe et al. 2012) and primer pairs developed. Identified microsatellites and designed primers were assembled
using QDD (Meglcz et al. 2010) with parameters given in set_qdd_default.ini.file. The gaps emerging during
the scaffolding process were closed using GapCloser (vs. 1.12). The contigs >1000 bp of the draft assembly
were analyzed and functionally annotated using Blast2GO (Conesa et al. 2005). Based on this information, 48
primer pairs consisting of either di- or trinucleotide repeats were selected. After testing, 17 primer pairs were
identified and used to characterize 84 samples of O. lanceolata from three natural populations, namely Mt.
Elgon (28), Gachuthi (27) and Kitui (29). The PCR analysis was performed using Multiplex PCR Mater Mix
(QIAGEN) and 10 ng of DNA as described by (Omondi et al. 2015). The PCR mix contained a fluorescently
labelled M13 primer, M13-tailed forward primer and a reverse primer in the concentration ratio of
0.15:0.01:0.15 M. For all loci, a touchdown thermal cycling program was used with annealing temperature
ranging between 5755C. The cycling profile consisted of initial denaturation of 95C for 15 min followed by
10 cycles at 94C for 30 s, 57C for 90 s and 72C for 60 s (annealing temperature decreasing by 1C per cycle);
and 22 cycles at 94C for 30 s, 55C for 90 s and 72C for 60 s and a single final cycle at 60C for 30 min using
Verity 96 well thermocycler (Applied Biosystems).
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Otieno et al. (2016) 3(3): 701703

Table 1. Descriptive statistics over all loci for the three natural populations of Osyris lanceolata Hochst. & Steud.
DDBJ
Locus
GenBank
accession no.
LC126834
KFOL2
LC154965
KFOL7
LC154966
KFOL8
LC126835
KFOL13
LC154967
KFOL15
LC154968
KFOL16
LC126836
KFOL17
LC154969
KFOL19
LC126838
KFOL24
LC154970
KFOL27
LC126839
KFOL28
LC154961
KFOL29
LC126840
KFOL30
LC126841
KFOL37
LC126843
KFOL42
LC126844
KFOL47
LC126845
KFOL48
Primer sequences (5 3 )
F:AGAATGTCATTTGAAGGCTCGA
R:CCTTTCCTCCGTTCTCCTCG
F: CTGTGCAATGGAGAAGGCCA
R:CGCGGGATTGGGATGTCATA
F:GCTGCTTCTACGGTCACTGT
R:GTGGTGGATATGGAGGTGGC
F:TCCGAGGAACAGGGACTCTT
R:AGCGAAGAACTCATGAGCGAA
F:CATTGACGAATTGCATCCCGT
R:CGTGAAGTTCAGTGCAAACC
F:TGGAGCCCATTCTCTTTCCTT
R:TGCACGTATTCCACATTTCCA
F:CATTGACGAATTGCATCCCGT
R:CGTGAAGTTCAGTGCAAACC
F:GGTAGCGAGCGGTGATATGT
R:ACCTAACAACTTGAAGCTCTCCC
F:CAACTCGATCGTGCATTGGC
R:TCCGCATATCCATTTGGCCG
F:CTAAACTGTCAGGGCTTGCT
R:ATACCTTAGCTCCCGTTGCG
F:ATAAAGGCCCACGAGCTCAG
R:AACATCGCCATGCAGAACAG
F:GCTGAATCAGGGACAGGCAT
R:GGCCTCGAACAAAGTGCATG
F:CTAAACTGTCAGGGCTTGCT
R:ATACCTTAGCTCCCGTTGCG
F:TTTCTAGAGCTAACATACCTCTGAA
R:ATGACCTGGGTGCTTTGCTG
F:AGGTCCTCCTGCCTGAGAAT
R: CATAGGGCTGTGATGCGTCA
F:TTTGATCGTAAATTATAGATGTCCACA
R:CCCTTGCTTGATCTCCAGGTA
F:GAGTGCATGGAATTATGTGCGT
R:TCGCCATGAGAAGGGTTACT
Allele
Repeat size
Na
motif range
(bp)
CGTC 178-194 5
ATT
HO
HO
HO
HE
HE
HE
Mt.
Mt.
Gachuthi Kitui
Gachuthi Kitui
Elgon
Elgon
0.393
0.556
0.483
0.572
0.626
0.569
115-120
0.043
0.000
0.000
0.043
0.000
0.000
CCG 120-130
0.000
0.200
0.462
0.073
0.184
0.434
139-165
0.556
0.148
0.069
0.552
0.139
0.067
CGC 145-150
0.000
0.000
0.000
0.000
0.000
0.000
AC
GT
130-160
0.107
0.333
0.107
0.103
0.352
0.166
AG
178-220 21
0.893
0.741
0.793
0.879
0.824
0.863
TC
200-230
0.259
0.000
0.000
0.338
0.000
0.000
CT
219-263 15
0.821
0.192
0.276
0.902
0.286
0.452
0.000
0.000
0.000
0.000
0.000
0.000
ATG 225-230
CT
245-255
0.714
0.000
0.069
0.605
0.000
0.067
GA
230-250
0.000
0.074
0.034
0.000
0.073
0.034
TC
270-306 12
0.643
0.333
0.483
0.614
0.471
0.663
TG
300-340 17
0.889
0.185
0.517
0.853
0.278
0.609
TG
315-337
0.308
0.037
0.000
0.277
0.036
0.000
CA
353-387 15
0.393*
0.731
0.759
0.791
0.771
0.826
TC
369-393 12
0.357
0.519
0.621
0.343
0.666
0.519
Note: 5 M13 tail: TGTAAAACGACGGCCAGT; F, forward sequence; R, reverse sequence; Na, number of observed alleles per
locus, HO heterozygosity observed with P-values for the Hardy Weinberg equilibrium test and significance threshold adjusted
using the Bonferroni correction: *P < 0.05, HE heterozygosity expected.
Amplified fragments were analyzed against an internal standard (Liz 600 size standard) on an ABI 3500
(Applied Biosystems). Alleles were visualized and scored using GeneMapper version 5 (Applied Biosystems).
The genetic parameters were determined using GenAlex software v 6.4 (Peakall & Smouse 2012). Deviations
from HardyWeinberg equilibrium (HWE) and linkage disequilibrium (LD) was determined using Genepop
online software version (http://wbiomed.curtin.edu.au/genepop/).
The number of alleles per locus across the three populations ranged from one (KFOL27) to 21 (KFOL17).
Expected heterozygosity ranged from 0.00 (KFOL15, KFOL27 and KFOL29) to 0.902 (KFOL24) in Mt. Elgon
population, from 0.00 (KFOL28) to 0.824 (KFOL7, KFOL15, KFOL17, KFOL19 and KFOL27) in Gachuthi
population, 0.00 (KFOL7, KFOL15, KFOL19, KFOL27 and KFOL42) to 0.863 (KFOL17) in Kitui population
(Table 1). Total paternity exclusion probability (Pe) over all loci was 0.989. Only one pair of loci (KFOL16 KFOL37) showed significant LD at the 5 % level after Bonferroni correction. Deviation from HWE was
detected for one locus (KFOL47) in Mt. Elgon population (Table 1). Out of the 17 markers developed, 12 were
polymorphic while five (KFOL7, KFOL8, KFOL15, KFOL27 and KFOL29) were monomorphic.
The 17 microsatellite markers developed are the first reported for O. lanceolata and are suitable for
population genetic studies due to their high polymorphic characteristics. The markers will be used for studying
genetic diversity and population structure across the distribution range, and to assess levels of gene flow
702
Otieno et al. (2016) 3(3): 701703

between populations. These studies will be important in designing sustainable management and conservation
strategies for the species.
ACKNOWLEDGMENTS
This study was funded and conducted by collaboration between Kenya Forestry Research Institute (KEFRI)
and Centre of Ecology and Hydrology (CEH) of UK, under International Timber Tracking Organization
(ITTO) project contract No.: 2005-34135-16007. We wish to thank Mr. John Gicheru and Mr.
Charles Oduor of KEFRI for helping in sample collection and laboratory work.
REFERENCES
Castoe TA, Poole AW, de Koning APJ, Jones KL, Tomback DF, Oyler-McCance SJ, Fike JA, Lance SL,
Streicher JW, Smith EN & Pollock DD (2012) Rapid Microsatellite Identification from Illumina paired-end
genomic sequencing in two birds and a snake. PLoS ONE 7: e30953.
Conesa A, Gtz AS, Garcia-Gomez JM, Terol J, Talon JM & Robles M. (2005) Blast2GO: a universal tool for
annotation, visualization and analysis in functional genomics research. Bioinformatics 21: 36743676.
Meglcz E, Costedoat C, Dubut V, Gilles A, Malausa T, Pech N & Martin JF (2010) QDD: a user-friendly
program to select microsatellite markers and design primers from large sequencing projects. Bioinformatics
26: 403404.
Omondi SF, Machua J, Gicheru JM &, So Hanaoka (2015). Isolation and characterization of microsatellite
markers for Acacia tortilis (Forsk.) Hayne. Conservation Genetics Resources 7 (2): 529531.
Peakall R & Smouse PE (2012) GenAlEx 6.5: genetic analysis in Excel. Population genetic software for
teaching and research-an update. Bioinformatics 28: 25372539.
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ISSN (E): 2349 1183

ISSN (P): 2349 9265
3(3): 704705, 2016
DOI: 10.22271/tpr.2016.v3.i3.093
Short communication
Rice false smut [Ustilaginoidea virens (Cooke) Takah.] in Paraguay

Lidia Quintana1*, Susana Gutirrez2, Marco Maidana1 and Karina Morinigo1
1
Facultad de Ciencias Agropecuarias y Forestales, Universidad Nacional de Itapa, Encarnacin, Paraguay

2
Facultad de Ciencias Agrarias, Universidad Nacional del Nordeste, Corrientes, Argentina
*Corresponding Author: lviedmaq@gmail.com
[Cite as: Quintana L, Gutirrez S, Maidana M & Morinigo K (2016) Rice false smut [Ustilaginoidea virens
(Cooke) Takah.] in Paraguay. Tropical Plant Research 3(3): 704705]
False smut of rice, caused by Ustilaginoidea virens (Cooke) Takah., is a common disease in rice panicles.
Disease was first reported in India (1878) and was considered as a secondary disease due to their sporadic
occurrence (Ladhalakshmi et al. 2012). In the 20142015 growing season disease survey was conducted in
different rice producting areas of the country. Rice plants of IRGA 424 cultivar were observed, whose panicles
had grains replaced by globose yellowish green masses of spores. These symptoms were visible after crop
flowering, when the fungus transforms individual grains of the panicle into globose green-yellow mass that
subsequently acquire greyish-black color.
In the national bibliography no history published about this disease was found, thus the objective of this
study was to determine the etiology of this new disease in Paraguay. One hundred and twenty panicles taken
from fields with symptoms and signs of false smut were collected in the districts of General Delgado, General
Artigas and Coronel Bogado (Itapa Department), districts of Santa Maria, San Juan Bautista and San Juan de
eembuc (Misiones Department). The symptoms and signs were observed with naked eye and under an
stereomicroscope (40x). Reproductive structures (spores) from the affected panicles were examined and
measured using a compound microscope (400x). Subsequently, the fungus spores were seeded on potato
dextrose agar for observation of the colonies.
The false smut was detected in green rice plants at ripening stage in IRGA 424 cultivar, grown in the
departments of Itapa and Misiones. The disease incidence in panicles was 40%, with at least 23 galls per
symptomatic panicles.
This disease has been described in all rice producing countries in the world such as India, Australia,
Pakistan, the United States, Mexico, the Philippines and Peru (Ou 1985, Webster & Gunnel 1992). In Argentina,
the disease was reported by Gutirrez et al. (2000). Its occurrence is related to soils with high fertilization, rainy
periods with humidity higher than 90%, especially during the crop flowering stage (Ahonshi et al. 2000). These
weather conditions during 2015 growing season, coincided with the El Nio phenomenon.
The symptoms observed were similar with those described by (Padwick 1950, Ou 1985, Webster and Gunnel
1992). The symptoms are visible only after flowering when the fungus transforms the individual grains of the
panicle into globose structures or yellowed carbonaceous masses. These masses are dusty representing more
than twice the diameter of normal grain and at early development are yellow and then acquire dark green or
almost black color, and explode releasing the spores of the fungal causal agent (Fig. 1).
Isolation and identification of the causal agent: Grain samples infected with false smut were collected from
different monitores localities. Infected kernels were washed with tap water to remove dust particles and surface
disinfestated with sodium hypochlorite solution (2.5%) for 12 minutes, dried and then transferred to potatodextrose-agar culture medium and incubated at 2528C for 710 days.
Morphometric characteristics of the pathogen found in the panicle were similar to those described by several
authors (Mew & Misra 1994, Mulder & Holliday 1985, Ou 1985, Webster & Gunnel 1992). Chlamydospores
formed in the masses of spores are spherical to elliptical, warty, of olive color, and 3 to 5 4 to 6 m. Colonies
on PDA developed in approximately 1415 days. The causal agent of rice false smut was identified as
Ustilaginoidea virens (Cooke) Tak. This is the first report of rice false smut in Paraguay.
704

Quintana et al. (2016) 3(3): 704705
Figure 1. Symptoms and signs of false smut caused by Ustilaginoidea virens (Cooke) Takah. in rice panicle: A, Spore galls
on panicle; B, Chlamydospores.
REFERENCES
Ahonshi MO, Adeoti ID, Erinkle TA, Alegrejo BN & Singh AA (2000) Effect of variety and sowing date of
false smut incidence in upland rice in Edo State. Nigeria IRRI Notes 25: pp. 14
Gutirrez de Arriola SA, Cndom MA & Mazzanti de Castaon MA (2000) Caracterizacin del Falso Carbn
(Ustilaginoidea virens). Enfermedad de reciente aparicin, en cultivos de arroz de Argentina. En: Reunin
de Comunicaciones Cientficas y Tecnolgicas, SGCYT, UNNE.
Ladhalakshmi D, Laha GS, Singh R, Karthikeyan A, Mangrauthia SK, Sundaram RM, Thukkaiyannan P &
Viraktamath BC (2012) Isolation and characterization of Ustilaginoidea virens and survey of false smut
disease of rice in India. Phytopara 40: 171176.
Mew TW & Misra JK (eds) (1994) A Manual of rice seed testing. Philippines IRRI, Los Baos, 113p.
Mulder JL & Holliday P (1985) Ustilaginoidea virens. Descriptions of Pathogenic Fungi and Bacteria. CAB
International, Wallingford, 30: 296 p.
Ou SH (1985) Rice Diseases. 2nd Edition. Commonwealth Mycological Institute, U.K., 385 p.
Padwick GW (1950) Manual of rice diseases. Commonwealth Mycological Institute, Kew, 198 pp.
Webster RK & Gunnell PS (1992) Compendium of rice diseases. St. Paul. American Phytopalogical Society.
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Society for tropical Plant Research

ISSN (E): 2349 1183

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