Volume 5, Issue 3 (2018) Tropical Plant Research

ISSN (Online): 2349-1183; ISSN (Print): 2349-9265
TROPICAL PLANT RESEARCH 5(3): 267–274, 2018

The Journal of the Society for Tropical Plant Research DOI: 10.22271/tpr.2018.v5.i3.034
Research article
Population structure and regeneration potential of Sal dominated

tropical dry deciduous forest in Chhattisgarh, India
Abhishek Raj
Department of Forestry, College of Agriculture, Indira Gandhi Krishi Vishwavidyalaya,
Raipur-492012, Chhattisgarh, India
*Corresponding Author: ranger0392@gmail.com [Accepted: 05 October 2018]
Abstract: Several biotic and abiotic factors including poor regeneration, changing environment
and edaphic factor along with poor regeneration affects health and establishment of Shorea
robusta nowadays. No systematic attempts were made to understand the dynamism of its natural
regeneration and to suggest management inputs to encourage its regeneration. The present study
deals with the regeneration status and population structure of four sites of Sal dominating tropical
dry deciduous forest during 2016–18. Regeneration status of the forest was determined based on
population size of seedlings and saplings. A total of 24 species of 19 families were encountered.
Regeneration status in all the study sites is dissimilar. In the entire four sites, site quality I was
good regenerating because of the high density of seedlings and saplings in forest site. The results
indicated that the average number of regeneration of Sal seedlings per hectare worked out to be
2562 ha-1, which are quite adequate. It was also observed that Shorea robusta showed
uninterrupted type of distribution pattern along with abundant regeneration in the forest stand
which showed healthy sign of establishment and growth of this species in the past. Other
associates showed different growth patterns. Efforts are needed to conserve the forest for their
diversity and existence.
Keywords: Population structure - Regeneration - Dry deciduous forest - Shorea robusta.
[Cite as: Raj A (2018) Population structure and regeneration potential of Sal dominated tropical dry deciduous
forest in Chhattisgarh, India. Tropical Plant Research 5(3): 267–274]
INTRODUCTION
Tropical forests are one of the richest and complex terrestrial ecosystems supporting a variety of life forms
and have a tremendous intrinsic ability for self-maintenance. The growth of forest trees is often observed to vary
with changes in the soil profile. This diversity of soil and climate where forests grow determines different site
qualities, where the most productive sites combine the best weather conditions, topography and soil
characteristics (Skovsgaard & Vanklay 2008). This implies differences in structure and composition of
vegetation, its regeneration potential and population structure of vegetation. Sal is deciduous, light demanding,
gregarious and dominant tree species in its stand (Champion & Osmaston 1962, Troup 1986) comes under
Dipterocarpaceae family, mainly distributed in the Northern, central and eastern region of Indian subcontinents.
Sal forests extend into the tropical and subtropical regions, and to the zones where precipitation ranges from
1000 to 2000 mm and above, and the dry period does not exceed 4 months (Tewari 1995, Chaudhary et al.
2016). Sal forest is relatively rich in ground flora diversity. Besides tree and shrub, ground flora of Sal forest
included fern, herb, grass and liana. Environmental conditions such as light, temperature, soil quality, moisture
availability and drainage system affect the regeneration of Sal forest and tend into various form of even-aged,
relatively pure and mixed type of vegetation (Troup 1986, Rautiainen & Suoheimo 1997, Mishra et al. 2013).
However, there are very few attempts have been made in the context of studying and understanding the dynamic
nature of natural regeneration of Sal forest and required suggestions and input for encouraging its regeneration
in India. The objective of the present study was to assess the regeneration status, population structure and rarity
or commonness of the species of Sal dominating forest at different sites in the tropical dry deciduous forest in
Chhattisgarh.
www.tropicalplantresearch.com 267
Received: 05 July 2018 Published online: 31 October 2018
https://doi.org/10.22271/tpr.2018.v5.i3.034
Raj 2018
MATERIAL AND METHODS

In order to assess the status of Sal regeneration and population structure of standing crop, total four
contiguous sites were selected along the gradient of site quality i.e. edaphic quality in large tract of the Sal based
mixed, dry deciduous forest located in the Dugli forest range of Dhamtari forest division (23° 21′ N and 82.85°
E with an average elevation of 527 m) situated in Dhamtari district (Chhattisgarh). In each of these sites, one
100 × 100 m (1 ha.) plot, visually representative of the overall vegetation, was delineated for detailed study. The
forest stand on each site was analyzed using ten randomly placed quadrats (each 10 × 10 m in size) within the
representative plot of 1 ha. Girth at breast height (GBH) of each individual tree in each quadrat was measured
and recorded. To show the regeneration pattern of tree species, the population structures were developed based
on different tree girth classes in addition to seedlings and saplings. The total number of individuals belonging to
these girth classes was calculated for each species on each site following Saxena & Singh (1984). In addition to
seedling (A) and sapling (B) classes, three more size classes based on girth at breast height (GBH) i.e., C) 30.1–
60.0 cm; 60.1–90.0 cm; D) and E) >90 were arbitrarily established for each tree species. The total number of
individuals belonging to above girth classes was computed for each species. This database is further used to
determine the trend of establishment and growth of each species. The number of individuals in each girth class,
for each species, was divided by the total number of individuals in all girth classes of that species. The resultant
value was further multiplied by 100 to yield percent density for each girth class for different species.
The regeneration status of species was determined according to Shankar (2001) which is based on population
size of seedling and sapling. The status of sampled species was assessed based on one time phytosociological
data in the following categories. (a) good, if seedlings > saplings > adult; (b) fair, if seedlings > saplings ≤
adults; (c) poor, if a species survives in only sapling stage, but not as seedlings (though saplings may be less,
more or equal to adults); (d) none, if a species is absent both in sapling and seedling stages, but present in adults;
and (e) new, if a species has no adults, but only saplings and/or seedlings.
Raunkiaer’s (1934) frequency class analysis was used to assess the rarity or commonness of the tree species
(Hewit & Kellman 2002). In this classification, the percentage frequency of the species was classified as A, B,
C, D, and E, where A represents rare (0–20%), B represents low frequency (20–40%), C represents intermediate
frequency (40–60%), D represents moderately high frequency (60–80%), and E represents high frequency or
common (80–100%).
RESULTS
Population Structure
Sal represented uninterrupted type of regeneration from saplings to mature stage of the entire growth phase.
This is the good indication of formation and development of Sal. In the site I, Shorea robusta Gaertn. showed all
the size classes of population structure (Fig. 1). The proportion of seedling size class (A) was dominant except
the species like Buchanania cochinchinensis (Lour.) M.R.Almeida, Desmodium oojeinense (Roxb.) H.Ohashi,
Mitragyna parvifolia (Roxb.) Korth. and Terminalia chebula Retz. The size class (A) was dominated by Shorea
robusta, Cleistanthus collinus (Roxb.) Benth. ex Hook.f., Phyllanthus emblica L., Terminalia tomentosa Wight
& Arn. and Anogeissus latifolia (Roxb. ex DC.) Wall. ex Guillem. & Perr., respectively with the contribution of
more than half (60–90%) proportion in this class. The sapling size class (B) proportion of Cleistanthus collinus
was higher. In size class C and D the highest proportion were represented by Shorea robusta, Terminalia
tomentosa and Anogeissus latifolia. The older tree size class (E) is represented Shorea robusta as the highest
contributors butnegligible or nil for Terminalia tomentosa and Schleichera oleosa (Lour.) Merr. The proportion
of seedling size class (A) to a higher class (D) decreased gradually (e.g., Shorea robusta, Cleistanthus collinus,
Schleichera oleosa, Anogeissus latifolia etc). The population structure of site II (Fig. 2) indicated that seedling
size class (A) was higher in which was mostly contributed by Shorea robusta, Anogeissus latifolia and
Phyllanthus emblica of the total population. The sapling size class (B) was represented by Phyllanthus emblica
and Schleichera oleosa. The young tree size class (C) was found almost for all the species on this site except for
Cleistanthus collinus. The older tree size class (D) and (E) were negligible or nil in case of most of the species.
The size class (E) was represented only by Shorea robusta, Terminalia tomentosa and Schleichera oleosa. In
this zone Shorea robusta represented by all the size classes, whereas Cleistanthus collinus representing all size
class except class (E). Phyllanthus emblica and Shorea robusta were reported maximum contribution in the
seedling size class (A). The size class (C) and (D) were reported for all major species which is dominating class
over the (E) whereas Cleistanthus collinus, Phyllanthus emblica and Schleichera oleosa were absent in older
size class (E) (Fig. 3). In the site IV Shorea robusta showed all the size classes of population structure.
Tropical Plant Research (2018) 5(3): 267–274
Figure 1. Population structures of major tree species of site quality I.
Figure 2. Population structures of major tree species of site quality II.
Raj 2018
Figure 3. Population structures of major tree species of site quality III.
Figure 4. Population structures of major tree species of site quality IV.

Cleistanthus collinus and Phyllanthus emblica were represented by all classes except older class (E). Almost all
the species were represented by seedling size class (A). Size class (B) was dominated by Shorea robusta,
Cleistanthus collinus, Phyllanthus emblica but absent for Terminalia bellirica (Gaertn.) Roxb., Schleichera
oleosa and Anogeissus latifolia. Moreover, size class (B) and (C) were dominated by Cleistanthus collinus.
Most of the recorded species were not represented by the older size class i.e., (E). The older tree size class (E)
was represented by Terminalia bellirica but absent for Cleistanthus collinus, Phyllanthus emblica and
Schleichera oleosa (Fig. 4). The interpretation of population structure of tree species was based on the
assumption that size class corresponds with the age of individuals. Though the size class distribution often
differs from the age class distribution, the former in case analyzed properly can also be useful for interpretation
of patterns of population changes.
Regeneration status
The renewal of the tree crop by natural means in the form of the crop is the indication of the health of site,
recruitment and establishment of regenerating plants of tree species. In the study site I out of 17 species, three
species, viz. Shorea robusta, Diospyros melanoxylon Roxb. and Schleichera oleosa showed good regeneration
as all these species having a good number of seedlings and saplings and seven species showed not abundant and
poor regeneration. In study site II, out of 17 tree species, only one species i.e. Phyllanthus emblica showed good
regeneration and one species (Diospyros melanoxylon) showed fair and 7 species recorded poor regeneration
viz., Anogeissus latifolia, Cleistanthus collinus, Madhuca longifolia var. latifolia (Roxb.) A.Chev., Desmodium
oojeinense, Terminalia chebula and Terminalia tomentosa while seven species were not abundant (Table 1).
One species, viz. Mitragyna parvifolia was not regenerating. In study site III, out of 16 species, only two species
viz., Shorea robusta and Cleistanthus collinus showed good regeneration and 8 species showed poor
regeneration viz. Buchanania cochinchinensis, Diospyros melanoxylon, Phyllanthus emblica, Madhuca
longifolia var. latifolia, Desmodium oojeinense, Schleichera oleosa, Semecarpus anacardium L.f. and
Terminalia chebula, respectively. Six species (Butea monosperma (Lam.) Taub., Catunaregam spinosa
(Thunb.) Tirveng., Lannea coromendelica, Syzigiumcumini, Tectona grandis L.f. and Terminalia tomentosa) on
this site exhibited not abundant (Table 1). In study site IV out of 15 species, only one species viz. Shorea
robusta showed good and 6 species showed poor regeneration viz. Anogeissus latifolia, Butea monosperma,
Schleichera oleosa, Tectona grandis, Terminalia chebula and Terminalia tomentosarespectively.Six species
viz., Catunaregam spinosa, Diospyros melanoxylon, Lannea coromendelica, Madhuca longifolia var. latifolia,
Semecarpus anacardium and Firmiana simplex (L.) W.Wight on this site exhibited not abundant (Table 1).
Raunkiaer’s frequency classes
In most of the plant life forms, there was a high number of species that occurred singly. The distribution of
sapling species into Raunkiaer’s frequency classes reflected that most of the species were of rare category and of
low-frequency class; intermediate with moderately high and high or common frequency classes were totally
absent in the study area while high-frequency class was represented in all sites of tree layers only (Table 2).
DISCUSSIONS
In all the different site qualities studied showed individuals with small girth class A (<10 cm) were high. In
the seedling layer, Shorea robusta was the dominant species among the qualities of the entire site except site III.
This species was successfully reached as a dominant species in the sapling and the tree layer due to less
mortality and high density that showed Sal based mixed forest. Therefore, Species with a nearly equal
distribution of individuals in the three life stages are expected to remain dominant in the near future. The Sal
showed adequate regeneration with uninterrupted growth pattern in most of the stands studied, indicating a
healthy sign of establishment and growth of Sal crop in the past. Thus, the growth phase of Sal showed an
uninterrupted trend of regeneration from saplings to mature stage. This indicates the establishment and
development of Sal in the study site. The population structure of certain species is characterized by gap phase
type regeneration (interrupted). Interrupted regeneration of species may indicate that one or more climatic
and/or bio-edaphic factors inhibited the regeneration completely for certain periods of time, and with the return
of favorable conditions, the species was able to regenerate again. Other species comprises Ixora parviflora Lam.
and Gardenia latifolia Ait. were unable to reach in the sapling and tree stages. This is because the grazing by
domestic animals and cattle’s due to good fodder quality of Gardenia species. Another cause is seedlings are not
comings up frequently the species might have produced the seeds but, the environment is not supporting their
proper establishment. Due to this heavy stress, the species could not reach in the tree or sapling layer. Therefore,
the rising anthropological pressure of human and cattle in these forests has become a serious problem for their
Raj 2018
sustainability as they are the main source of timber, fuelwood and other non-timber forest products (NTFPs).
The fewer number of saplings recorded in relation to seedlings in this study implies that most of the saplings are
transiting into young trees. Intense competition leads to mortality of seedling at very early stage (Weidelt 1988).
Among the various girth classes, saplings and seedlings reflect high regeneration potential of the stand (Mishra
et al. 2005, Khumbongmayum et al. 2006).
Table 2. Distribution of vegetation layers according to Raunkiaer’s classification scheme.
Layers Sites A B C D E
Tree I 5 2 2 0 1
II 5 2 0 2 1
III 4 4 1 0 1
IV 4 4 0 0 1
Sapling I 6 0 0 0 0
II 5 1 0 0 0
III 4 1 0 0 0
IV 6 0 0 0 0
Seedling I 8 5 1 1 0
II 4 7 1 0 0
III 6 5 2 0 0
IV 9 5 0 0 0
Another type of uninterrupted growth pattern of Sal associated species was Schleichera oleosa and
Phyllanthus emblica in all sites quality I, II, III and IV. These tree species are successfully crosses the sapling
layer and attained the first tree size class. Similarly, one species, viz. Mitragyna parvifolia was not regenerating
in site quality II. It might be due to thick litter accumulation which reduced seed germination of most canopy
species (Janzen 1970, Singh & Singh 1992). The forest stands characterized by the abundance of only adults of
the species or absence or very low population of seedlings and saplings are expected to face local extinction
(Dalling et al. 1998). This type of condition was observed for tree species Anogeissus latifolia in site quality III.
There is another pattern which consists of individuals in lower and middle girth classes but absence of seedlings.
This type of population distribution was reported in Schleichera oleosasite II. Therefore, the densities of
seedlings are influenced by the densities of large trees (Rao et al. 1990). Similarly, the low IVI may be
attributed towards the rarest occurrence of species (Oyun et al. 2009), as confirmed by Raunkiaer’s frequency
distribution of the tree species. The rarity of species may be attributed to the occurrence of abundant sporadic
species with low frequency in the stands (Oyun et al. 2009). The density values of seedlings and saplings are
considered as regeneration potential of the species. The presence of good regeneration potential shows the
suitability of a species to a given environment (Dhaulkhandi et al. 2008). Edhapo-climatic factors and biotic
interference influence the regeneration of different species in the vegetation. Higher seedling density values get
reduced to sapling due to soil characteristics, competition for space and nutrients and biotic disturbance.
CONCLUSIONS
It is concluded that tree canopy cover, site characteristics, availability of sunlight on the ground surface and
important associated species impacted the regeneration of Sal and other associated species in the region. The
absence of tree species may lead to the availability of higher solar insolation to the younger plants especially
light loving trees as Sal. The Sal showed adequate regeneration with uninterrupted growth pattern in most of the
stands studied, indicating a healthy sign of establishment and growth of Sal crop in the past.
ACKNOWLEDGEMENTS
Authors are thankful to Department of Forestry, Dean College of agriculture, Director Research Services,
Director Instructions and Hon’ble Vice-Chancellor, Indira Gandhi Krishi Vishwavidyalaya, Raipur for
encouragement, inspiration and providing necessary assistance during the study.
REFERENCES
Champion H & Osmaston FC (eds) (1962) E. P. Stebbing’s The Forests of India, Volume IV. Oxford University
Press, London.
Chaudhary LB, Kushwaha AK & Bajpai O (2016) Trees of Uttar Pradesh (Part 1). Bennett, Coleman & Co.
Ltd., Lucknow, India, pp 308. [ISBN: 978-93-5267-070-3]
Dalling JW, Hubbel SP & Silvera K (1998) Seed dispersal, seedling establishment and gap partitioning among
tropical pioneer trees. Journal of Ecology 86: 674–689.
Raj 2018
Dhaulkhandi M, Dobhal A, Bhatt S & Kumar M (2008) Community structure and regeneration potential of
natural forest site in Gangotri, India. Journal of Basic and Applied Sciences 4(1): 49–52.
Hewit N & Kellman M (2002) True seed dispersal among forest fragments: dispersal ability and
biogeographical controls. Journal of Biogeography 29(3): 351–363.
Janzen DH (1970) Herbivores and the number of tree species in tropical forest. American Naturalist 104: 501–
528.
Khumbongmayum AD, Khan ML & Tripathi RS (2006) Biodiversity conservation in sacred groves of Manipur,
Northeast India: population structure and regeneration status of woody species. Biodiversity and
Conservation 15: 2439–2456.
Mishra AK, Bajpai O, Sahu N, Kumar A, Behera SK, Mishra RM & Chaudhary LB (2013) Study of Plant
Regeneration Potential in Tropical Moist Deciduous Forest in Northern India. International Journal of
Environment 2(1): 153–163.
Mishra BP, Tripathi OP & Laloo RC (2005) Community characteristics of a climax subtropical humid forest of
Meghalaya and population structure of ten important tree species. Tropical Ecology 46: 241–251.
Oyun MB, Bada, SO & Anjah GM (2009) Comparative analysis of the floral composition at the edge and
interior of Agulii Forest reserve, Cameroon. Journal of Biological Sciences 9(5): 431–437.
Rao P, Barik SK, Pandey HN & Tripathi RS (1990) Community composition and tree population structure in a
sub-tropical broadleaved forest along a disturbance gradient. Vegetatio 88: 151–162.
Raunkiaer C (1934) The Life Form of Plants and Statistical Plant Geography. Claredon Press, Oxford. [ISBN
9978–40–943-2]
Rautiainen O & Suoheimo J (1997) Natural regeneration potential and early development of Shorea robusta
Gaertn.f. forest after regeneration felling in the Bhabar-Terai zone in Nepal. Forest Ecology and
Management 92: 243–251.
Saxena AK & Singh JS (1984) Tree population structure of central Himalayan forest associations and
implications concerning their future composition. Vegetatio 54: 61–69.
Shankar U (2001) A case of high tree diversity in a sal (Shorea robusta) dominated lowland forest of eastern
Himalaya: Floristic composition, regeneration and conservation. Current Science 81(7): 776–786.
Singh JS & Singh SP (1992) Forest of Himalaya: Structure and Functioning and Impact of Man. Gyanodya
Prakashan, Nainital.
Skovsgaard JP & Vanklay JK (2008) Forest site productivity: a re-view of the evolution of dendrometric
concepts for even-aged stands. Forestry 81: 13–31.
Tewari DN (1995) A Monograph on Sal (Shorea robusta Gaertn. f.). International Book Distributors, Dehradun,
India.
Troup RS (1986) The Silviculture of Indian Trees. International Book Distributors, Dehradun, India.
Weidelt HJ (1988) On the diversity of tree species in tropical Rain Forest Eosystems. Plant Research and
Development 28: 110–125.
Research article
Fruits seasonality in selected markets at Mont-Ngafula district in

Democratic Republic of the Congo: Biodiversity and food values
Mike Mukendi Tshibangu, Gédéon Bongo Ngiala, Anthony Kikufi Batoba, Eric Bukaka Wakini Yeto,
Koto-te-Nyiwa Ngbolua, Henry Mbale Kunzi and Félicien Lukoki Luyeye*
Department of Biology, Faculty of Science, University of Kinshasa, B.P. 190, Kinshasa XI, Kinshasa,
Democratic Republic of the Congo
*Corresponding Author: gedeon.bongo@unikin.ac.cd [Accepted: 15 October 2018]
Abstract: Fruit seasonality remains so far one of the priorities of agricultural research and
development in the Democratic Republic of the Congo. However, due to the lack of reliable
statistics and a schedule, it seems unappreciated and underestimated. This situation limits the
prospect of fruit resource promotion and justifies the initiation of the current study of which the
objective was to assess the biodiversity and fruit food value available according to the season in
Kinshasa. The survey was carried out between January and June 2016 in the Lukunga district,
particularly in Mont-Ngafula township where 100 vendors of fruits in selected markets of this
township were interviewed. In the current survey, about twenty fruits sold on these markets were
recorded and these fruits belong to 22 families dominated by Anacardiaceae and Rutaceae
families. It was observed that the greater varieties of fruits were available in February than in June.
It was noted that papaya, banana, coconut and apples were present from January to June while
mangosteens, fewer passion fruits, rambutan and pink apples were only present in January and
February. These findings showed that the sale of fruit is a very profitable activity. All these
elements are important for they serve as the basis for the development of a research strategy for the
promotion of fruit resources in DRC.
Keywords: Biodiversity - Fruits - Seasonality - Socio economic - DRC.
[Cite as: Tshibangu MM, Ngiala GB, Batoba AK, Yeto EBW, Ngbolua K-te-N, Kunzi HM & Luyeye FL (2018)
Fruits seasonality in selected markets at Mont-Ngafula district in Democratic Republic of the Congo:
Biodiversity and food values. Tropical Plant Research 5(3): 275–285]
INTRODUCTION
Given the socio-economic realities of various developing countries (insufficient or non-existent income,
scarcity of employment, chronic food insecurity, etc.) among which is included the Democratic Republic of the
Congo (DRC), the population in order to maintain its survival has developed diverse lucrative activities among
which the exploitation of non-timber forest products (NWFPs) (Ndjebet 1997, Sene 2001, Biloso 2006).
According to Tollens (2003), the economic growth for the poor appears as a prescriptive side of neoliberal
epistemology for the improvement of the well-being of populations, accompanied by a reduction of poverty. In
this perspective, the promotion of economic sectors in which the poor are active has become a miracle recipe for
poverty reduction. In Sub-Saharan Africa, a significant proportion of poor lives on the exploitation of natural
resources. Besides lumber, energy wood, forest, grassland and aquatic ecosystems deliver products that are
commonly needed and sold in most major consumption centers. These products are resources of various and
indispensable utilities (Tchatat et al. 1999).
The current food crisis and the continuing deterioration of the health status of the Congolese population in
general and of Kinshasa city, in particular, are among the causes of strong pressure on natural environments
including forests. In the long run, these may create scarcity or disappearance of certain resources and cause
drought, erosion and desertification. On the other hand, according to FAO, the uncontrolled exploitation of
natural resources could severely limit future prospects for the economic growth for the poor and development
(FAO 2001). This suggests that the sustainable and rational exploitation of natural resources could contribute,
Received: 14 May 2018 Published online: 31 October 2018
Tshibangu et al. 2018
not only to the preservation of a significant part of the biodiversity of ecosystems in peri-urban and rural areas
of Kinshasa city but also to the improvement of the fate of many local communities. Through the income
generation and the equitable distribution of richness from the exploitation of natural resources. The country is
going through a crisis that has reached a critical nutritional threshold today. The Congolese population is
malnourished and, lacking enough food in quantity and quality, draws what they need from their natural
environment: these are the products of the biodiversity provided by wild flora and fauna as well as agricultural
commodities. This production has been tested for more than 20 years by the climatic changes that the country
undergoes, without forgetting the agricultural production threatened in the same way with consequently the fall
in yield resulting from the disturbance of the agricultural calendar. Fruits are essential to the body and contribute
to its development with its vitamin intake including vitamin C which is in large quantities, minerals precisely
electrolytes, dietary fibers and antioxidants (phytochemicals) which prevent against cancer and ageing (Muraki
et al. 2013, Dhandevi & Jeewon 2015). The only problem with fruits is that most people do not know how to
consume them in order to effectively assimilate nutrients (Combe 1982). Several studies reported that there is an
association between low intake of fruits with chronic diseases like cardiovascular diseases, blood pressure,
hypercholesterolemia, osteoporosis, many cancers, chronic obstructive pulmonary diseases, respiratory
problems as well as mental health (Adebawo et al. 2006, Park et al. 2011, Payne et al. 2012, Dhandevi &
Jeewon 2015).
Fruits are the best foods that are consumed in order to protect the body against heart disease for instance and
many others diseases; they contain bioflavonoids that can prevent the thickening of blood as well as the
obstruction of arteries. In addition, they strengthen capillaries and fragile capillaries are almost the cause of
internal bleeding and heart attacks (Merlin 2012). Fruits are mainly composed of fructose as the main sugar
(which can be easily converted into glucose), the rest is mainly water (90 to 95%) (Merlin 2012). Fruits are a
group of foods that should not be missed in your diet because of their high fiber, vitamin and mineral contents
and are excellent for maintaining regular bowel movements and skin health, hair and all our organs (Merlin
2012). Fruits are consumed by many groups of animals, with many examples of coevolution and most of the
terrestrial mammals like fruits, even among which are supposed to be carnivorous. In the great apes, their share
in the diet may suggest that which concerned the Australopithecus and the first Homo (Muraki et al. 2013). This
is why in the present work, it was a question of knowing the seasonality of fruits available and marketed in
different markets of Kinshasa, which was oriented in the identification of the fruits on the markets as well as
their source of origin. The main objective was to know the seasonality of fruits present in various markets of
Kinshasa. The specific objectives were to make an inventory of the fruits sold in four markets of Mont-Ngafula
township (Kinshasa city) each month pending 2016, establish the list of the fluctuation of fruits as well as
identify their sources of origin. The seasonality of fruits in the markets of Kinshasa is not scientifically
determined; this shows the significance of this research. Practically, this study allows to know the correct period
(the availability) of having a fruit in the market.

Study area
The Mont-Ngafula township is located in the southwest of Kinshasa city. It is bounded in the North by
Makala, Selembao, Lemba and Kisenso townships; in the South by Kasangulu Territory (Kongo-Central
Province); in the East by Ndjili, Kimbanseke and N'sele townships; and in the West by Ngaliema township and
the Republic of Congo (Brazzaville). Mont-Ngafula township has an area of 358.90 km2 and has a mild tropical
warm and humid tropical climate that originates from hill breezes at a certain time of the day, blow from low
valleys to peaks in there bringing certain freshness. Two types of soils characterize the aforementioned
township, namely the soils of hills and those of valleys. In fact, the hilly soils are covered by the soft, whitish
sandstone where there is a predominance of fine ocher-yellow sand while the second seems to be the most fertile
and therefore consist of silt. This explains the high concentration of agricultural activities in valleys such as
Funa, Lukaya and Lukunga. Mont-Ngafula township has a wooded and grassy savannah and it is a derived
savannah of anthropic origin. It has to be noted that Mont-Ngafula township is drained by a multitude of small
rivers South-North direction that flow into the Congo River in the west and the N'djili River in the east.
Material
The biological material used for the current survey was samples of food fruits collected in four markets in
the western part of Kinshasa city notably UPN, Matadi-Kibala, Cité Pumbu and Masanga-Mbila.
Methodology
For the current study, the direct interview survey and direct observation were used as well as the literature
search. After the site exploration, the next step was the interview, using a pre-elaborated questionnaire. The use
of more than one market was an appropriate strategy in order to verify the veracity of the information received
from various informants. In our surveys, the following information was needed: the fruits found on the market,
their use, the techniques for preserving products, the ecological environment in which the products (fruits) were
collected, the provenance, and finally any other relevant additional information. The survey was carried out
between January and June 2016 in the Lukunga district, particularly in Mont-Ngafula township and 100 vendors
of fruits in selected markets were interviewed in total.
Collection and plant identification for herbarium formation
The constitution of a reference herbarium is a necessary basis in any floristic inventory study. We bought
leafy plants sold by different informants to construct a herbarium, this allowed us to identify some plants on the
field. To ensure the correct determination of these plants, the main floristic works of the neighboring countries
and Central Africa namely the Flora of Gabon, the Central African Flora, the Flora of Cameroon and the Flora
of the Belgium-Congo was used. The ascertaining of our determinations were compared with specimens kept at
the Herbarium, in the Department of Biology, Faculty of Science, University of Kinshasa.
RESULTS
Socio-demographic characteristics of informants
The socio-demographic characteristics of informants considered in the current survey are gender and marital
status (Fig. 1). The above figure shows that the majority of vendors are female because they are predominant in
selected markets. The percentage of female vendors of the UPN, Matadi Kibala, Cité Pumbu and Masanga-
Mbila markets are 100%, 80%, 100% and 75% respectively. This explains that the exploitation and sale of fruits
are the responsibility of females than males.
Figure 1. Distribution of vendors according to gender in selected markets.

The figure 2 shows that married people represent the large group of vendors of NWFPs in these markets and
their proportion is as follows: UPN market (46.9%), Matadi-Kibala market (40%), Cité Pumbu market (53%)
and Masanga-Mbila market (50%).
The other groups namely bachelors and divorced are represented as follows in different markets. According
to the markets, the proportion of bachelors ranges between 26.9% and 40.6% while the proportion of divorced
ranges between 9.4% and 30%. Unfortunately, during the interview, no widows were recorded. It is obvious that
the fruit trade is a function of the marital status of the vendor. Married people have significant social security
contributions; henceforth the need to resort to an income-generating activity compared to bachelors and
divorced people.
Diversity and nomenclature of fruits
In the current survey, about twenty fruits on sale in different markets were recorded (Table 1; Fig. 3) and
these fruits belong to 22 families dominated by Anacardiaceae and Rutaceae families. The general situation of
fruits recorded over six months is presented in tables 1 and 2 below.
Figure 2. Distribution of vendors according to marital status in selected markets.

Table 1. Names of different fruits found in selected markets.
Scientific Name Vernacular Name Botanical Family Frequency of fruits
in selected markets
Aframomum alboviolaceum K.Schum. Ntundulu Zingiberaceae ++
Ananas comosus (L.) Merill. Ananas (F) Bromeliaceae +++
Anisophyllea quangensis Engl. ex Henriques Mbila esobé (L) Anisophylleaceae +++
Annona muricata L. Mbundu ngombe (K) Annonaceae ++
Averrhoa carambola L. Carambole (F) Oxalidaceae +
Carica papaya L. Papaye (F) Caricaceae +++
Citrullus lanatus (Thunb.) Matsum.& Nakai Pastèque (F) Cucurbitaceae +++
Citrus limon L. Burm. f Citron (L) Rutaceae ++
Citrus reticulata Blanco Mandarine (L) Rutaceae ++
Citrus sinensis L. Osbeck. Orange (F) Rutaceae ++
Cocos nucifera L. Noix de coco (F) Arecaceae +++
Cola acuminata (P.B.) Schott & Endl Noix de cola (F) Malvaceae ++
Dacryodes edulis (G.Don) H.J.Lam Safou (L) Burseraceae ++
Garcinia mangostana L. Mangoustan (F) Clusiaceae ++
Landolphia owariensis P. Beauv. Litongé (L) Apocynaceae ++
Malus domestica Borkh. Pomme(F) Rosaceae +
Mangifera indica L. Mangue (F) Anacardiaceae ++
Musa sp. Banane (F) Musaceae +++
Musa x paradisiaca L. Banane plantain (F) Musaceae +++
Nephelium lappaceum L. Poilus (L) Sapindaceae +
Passiflora edulis Sims. Maracuja (L) Passifloraceae +
Persea americana Mill. Avocat (F) Lauraceae ++
Solanum lycopersicum L. Tomate (F) Solanaceae ++
Solanum melongena L. Aubergine (F) Solanaceae +
Spondias cytherea Som Prune cythère (F) Anacardiaceae +
Spondias mombin L. Mombin (F) Anacardiaceae +
Strychnos cocculoides Baker Makalakonki (K) Loganiaceae ++
Syzygium malaccense (L.) Merr. & L.M.Perry Pomme rose (F) Myrtaceae +
Vitis vinifera L. Raisin (F) Vitaceae +
Note: (L), Lingala; (K), Kikongo; (F), French; (+), Less frequent; (++), Frequent; (+++), More frequent.
Figure 3. Some fruits found in four markets of Mont-Ngafula township, Kinshasa City: A, Aframomum alboviolaceum; B,
Ananas comosus; C, Annona muricata; D, Carica papaya; E, Citrullus lanatus; F, Citrus limon; G, Citrus reticulata; H,
Citrus sinensis; I, Cola acuminata; J, Dacryodes edulis; K, Elaeis guineensis (Inset showing pulp of fruits); L, Garcinia
mangostana; M, Malus domestica; N, Mangifera indica; O, Musa paradisiaca; P, Nephelium lappaceum; Q, Persea
americana; R, Syzygium malaccense.
It was observed that in February presents a greater variety of fruits than the month of June. It should also be
noted that papaya, banana, coconut and apples were present from January to June while mangosteens, fewer
passion fruits, rambutan and pink apples were only present in January and February.
Characteristics of vendors activities
Commercial activity: To understand the commercial activity taking place in different markets surveyed is
necessary because it allows knowing if these vendors sell only fruits or fruits along with other products
(Table 3).
Table 2. General situation of fruits seasonality for six months.

January February March April May June
Pineapple Pineapple Pineapple Pineapple Pineapple Banana
Avocado Avocado Avocado Avocado Avocado Plantain
Banana Banana Banana Banana Banana Custard apple
Plantain Plantain Plantain Plantain Plantain Lemon
Custard apple Custard apple Custard apple Lemon Lemon Mango
Mangosteen Lemon Lemon Coconut Orange Apple
Mango Mango Grape-seeded amomum Papaya Apple Coconut
Passion fruit Passion fruit Orange Orange Coconut Tangerine
Coconut Mangosteen Coconut Coconut Watermelon Orange
Kola nut Kola nut Papaya Tangerine Papaya Papaya
Papaya Coconut Tangerine Grapefruit Tangerine Watermelon
Watermelon Palm nut Grapefruit Apple Bush butter tree Grapefruit
Rambutan Rambutan Orange Mango Grape-seeded amomum Carambola
Apple Apple Apple Grape-seeded amomum Grapefruit
Bush butter tree Red apple Watermelon Carambola Mango
Watermelon
Grapefruit
Golden apple
Papaya
Bush butter tree
Table 3. Commercial activity.

Commercial Activity Frequency Percentage
Sale of fruits exclusively 32 100
Sale of fruits and other products 0 0
Total 32 100
It can be observed from the above table that 100% of fruit vendors sell exclusively fruits and they don’t
sell any other products than fruits.
Duration in the activity: The duration in the activity explains the time that the respondents spent in this activity.
The distribution following the duration of vendors’ activity is presented in figure 4.
Figure 4. Duration of activity in selected markets.

As shown in the above figure, a large majority of vendors were in this activity for more than 10 years as
observed at Matadi-Kibala market (70%), Cité-Pumbu market (53.8%) and Masanga-Mbila market (37.5%)
except for the UPN market where a proportion of 21% was found for this category. The UPN market was
dominated by vendors (31.3%) having an experience of less than 5 years in exerting this activity. This
explains the deterioration of socio-economic conditions that are driving many people into the informal
commercial sector for their survival.
Storage method: This allows to determine how various fruits identified are preserved from the purchase to sale.
Some fruits are still immature at the time of purchase and are preserved so that they can ripe at the time of
sale (Fig. 5).
Figure 5. Storage method of fruits sold in selected markets.

The figure above gives the distribution in the set of fruits according to their preservation method. It
shows that that most of fruits sold in these markets are preserved in the bag (UPN market, 28.1%; Matadi
Kibala market, 70%; Cite Pumbu market, 46%; Masanga Mbila market, 37.5%). Meanwhile, a smaller
portion of these fruits are kept in the plastic bag as follows: 3.1%, (UPN); 10%, (Matadi Kibala), 2%, (Cite
Pumbu) and 6.3% (Masanga Mbila). Fruits cannot be stored for a long time for fear that they will rot.
It should be noted that some fruits such as safou and cola nuts have special preservation methods that are
not mentioned in the figure above. Safou are sensitive to heat that is why they are kept outdoors while cola
nuts prefer humidity, thus they are buried to be well preserved. Bananas, Plantains and papaya can also be
stored in the open air but the only problem with this method is that it takes longer.
Duration of fruit conservation: The duration of fruit conservation sold in selected markets is given in figure 6
below.
Figure 6. Time of conservation of fruits sold in selected markets.

From the above figure, it is clearly shown that a large proportion of fruit is stored in less than 5 days
distributed as follows in different markets respectively: UPN (40.6%), Matadi-Kibala (90%), Cité Pumbu
(80%) and 56.6%, Masanga-Mbila market (56.6.%). Fruits kept for less than 2 days have a high proportion
in the UPN market (40.6%) as well.
Source of fruit: During the 6-month survey period, a question was asked to the vendors about the origin of fruit
sold. In general, these fruits originate from the province of Kongo Central which is the nearest province to
Kinshasa city especially to Mont-Ngafula township.

Availability of fruits in selected markets
The availability of fruits in selected markets is presented in tables (4–7) below.
Table 4. Distribution of available fruits per month at UPN market.
SN Fruits January February March April May June
1 African Eggplant + + + + + +
2 Avocado + + + + + +
3 Banana + + + + + +
4 Bush butter tree + + + - - -
5 Carambola - - - + + +
6 Coconut + + + + + +
7 Congo rubber + + - - - -
8 Custard apple + + + - - +
9 Golden apple + + - - - -
10 Grape - - - - - -
11 Grapefruit - - + + + -
12 Green/red apple + + - - - -
13 Guava - - - - - -
14 Kola nut + + - - - -
15 Lemon - - + + + -
16 Mangosteen + + + - - -
17 Meleguette - - + + + -
18 Orange - - + + + -
19 Papaya + + + + + +
20 Passion fruit - - + + - -
21 Pear - - - - - -
22 Pineapple + + + + + +
23 Plantain + + + + + +
24 Rambutan + + - - - -
25 Tangerine - - + + + -
Note: (-), Absent in the market; (+), Present in the market.
Table 5. Distribution of available fruits per month at Matadi-Kibala market.

1 African Eggplant - - - - - -
2 Avocado - - - - - -
3 Banana + + + + + +
4 Bush butter tree - - - - - -
5 Carambola - - - - - -
6 Coconut + + + + + +
7 Congo rubber - - - - - -
8 Custard apple - - - - - -
9 Golden apple - - - - - -
10 Grape - - - - - -
12 Guava - - - - - -
13 Kola nut - - - - - -
14 Lemon - - + + + -
15 Mangosteen - - - - - -
16 Meleguette - - - - - -
17 Orange - - + + + -
18 Papaya - - - - - -
19 Passion fruit - - - - - -
20 Pear - - - - - -
21 Pineapple + + + + - -
22 Plantain + + + + + +
23 Rambutan - - - - - -
24 Red/Green apple - - - - - -
25 Tangerine - - + + + -
Note: (-), Absent on the market; (+), Present on the market.
Table 6. Distribution of available fruits per month at Cité-Pumbu market.

2 Avocado - + - - - -
3 Banana + + + + + +
4 Bush butter tree + + + - - -
5 Carambola - - - - - -
6 Coconut - - - - - -
10 Grape - - - - - -
12 Green or red apple - - - - - -
13 Guava - - - - - -
14 Kola nut - - - - - -
15 Lemon - - + + + -
16 Mangosteen + + + - - -
17 Meleguette - - + + + -
18 Orange - - + + + -
19 Papaya - - - - - -
21 Pear - - - - - -
22 Pineapple + + + + - -
23 Plantain + + + + + +
24 Rambutan + + - - - -
25 Tangerine - - + + + -
Table 7. Distribution of available fruits per month at Masanga-Mbila market.

2 African pear + + + - - -
3 Avocado + + - - - -
4 Banana + + + + + +
5 Carambola - - - - - -
6 Coconut - - - - - -
10 Grape - - - - - -
11 Grape fruit - - + + + -
12 Guava - - - - - -
13 Kola nut - - - - - -
14 Lemon - - - - - -
15 Mangosteen + + - - - -
16 Meleguette - - - - - -
17 Orange - - + + + -
18 Papaya - - - - - -
20 Pear - - - - - -
21 Pineappple + + + - - -
22 Plantain + + + + + +
23 Rambutan + + - - - -
24 Red or green apple + + - - - -
25 Tangerine - - + + + -
From the table 4, it is clearly shown that only six fruits were present on the UPN market. These include
African Eggplant, Avocado, Banana, Coconut, Pineapple and Plantain. While the others were present for only a
few months, hence their seasonality. Table 5 reveals that some fruits were present at the Matadi-Kibala market
during all the six months of the survey namely Banana, Coconut and Plantain. Compared to the UPN market,
there are many fruits missing at this market. This may be due to the quality of the customer base found in this
market. In the market of Cité-Pumbu only two fruits are present throughout the six months i.e. Banana and
Plantain (Table 6). This also proves that the clientele is not interested in fruits, hence the lack of interest of
vendors to devote themselves to this activity. The market of Masanga-Mbila also has only two fruits (Banana
and Plantain) for the six months (Table 7).
The almost same situation in the markets of Matadi-Kibala, Cité-Pumbu and Masanga-Mbila shows that
customers of these markets do not have much interest in fruit consumption. These results do not prove that the
fruits are absent, since they are found in the UPN market, but they only prove the quality of the present
customer base, which is that UPN vendors have a different customer base than three other markets.
DISCUSSION
NWFPs remains one of the main sources of food, medicine and income for rural and urban populations in
Central Africa. However, different approaches namely the study of marketing and sustainable management have
been used in sub-tropical Africa, Asia and America. There are various approaches used for the exploitation of
NWFPs, and our focus was on the ethnoecology approach. This survey showed us that the sale of fruits is more
exercised by women than men. However, it emphasizes that in production areas, men are responsible for the
production of annual and perennial crops; while women are content to practice annual crops. It should be noted
that women are increasingly interested in the collection of NWFPs and especially fruits that bring back
substantial income for the household. Men are found in the middle links: (Matadi Kibala, 20% and Masanga
Mbila, 25%). It was noted a total absence of men at the UPN and Cite Pumbu markets. This leads to the
conclusion that fruit harvesting and retailing are the responsibility of women rather than men. This is because
collecting just like retail requires a lot of time that men are not ready to spend. These findings are similar to
Biloso (2006) who reported that 49.3% of the heads of households surveyed are men while 50.7% are women.
It is clear that the fruit trade is a function of the vendor marital status. Married people have significant social
security contributions, hence the need to resort to an income-generating activity. These results resemble those
of Biloso (2006) in valuing NWFPs which show that farmers living together with their partner represent the
largest class of farmers (74% to 82.1%), it is evident that the exploitation of NWFPs is a function of the marital
status of the farmer. This finding is consistent with Biloso & Lejoly (2006) reported that there were more
women than men in the performance of this activity.
Concerning the diversity of fruits, this survey identified twenty-nine different fruits belonging to 23 families
dominated by Anacardiaceae and Rutaceae families. These results are consistent with Kouebou et al. (2013)
who identified about twenty fruits on sale belonging to 14 families dominated by Rutaceae (citrus fruits).
Regarding the frequency of fruit on the market, the findings of this survey showed that banana, plantain,
papaya, coconut are the most common because they appear for six months while citrus fruits become common
from March. There is a diversity of fruits in the UPN market while in other markets surveyed (Cité Pumbu,
Matadi Kibala and Masanga Mbila), it was observed that the sale of fruits is selective such as: bananas, plantain,
coconut (all year), pineapple (January–May) and citrus (March–June).
CONCLUSION
Preliminary observations were initiated at the Department of Biology to assess the biodiversity of different
fruits sold in Kinshasa markets and their seasonality. Twenty-nine fruits were inventoried in UPN, Matadi-
Kibala, Cité Pumbu and Masanga Mbila markets between January and June 2016. The major groups of these
fruits come from Kongo Central province which is the nearest province of Kinshasa city. They belong to
twenty-three botanical families dominated by Anacardiaceae and Rutaceae families.
The findings of this study showed that more women are interested in selling fruits than men; while the
permanent presence of plantain, banana, papaya and watermelon on the market during the six months of the
survey, followed by citrus fruits (lemon, orange, grapefruit and mandarin) which appear from the third month of
the year, followed by the disappearing of safou and rambutan, found more in the first two months i.e. January
and February, while in June appears the carombola. These fruits are kept in various ways from purchase to sale.
The sale of fruits is an old activity for the majority of the respondents and it was showed in the time that vendors
have spent in this activity. Knowing that the sale of fruits is a very profitable activity, it allows the vendors to
provide to their own needs and the one of their families. Considering the Congolese vegetal biodiversity, all
these elements are important for they serve as the basis for the development of a research strategy for the
promotion of fruit resources in DRC.
ACKNOWLEDGEMENTS
We express our gratefulness to the GBIF (Congo Democratic Biodiversity Information Facility) for its
support to the realization of this research as well as to the Laboratory of Systematic Botany and Plant Ecology,
Department of Biology, Faculty of Sciences for the identification of species.
REFERENCES
Adebawo O, Salau B, Ezima E, Oyefuga O, Ajani E, Idowu G, Famodu A & Osilesi O (2006) Fruits and
vegetables moderate lipid cardiovascular risk factor in hypertensive patients. Lipids in Health and Disease 5:
14.
Biloso A (2006) Exploitation et marché des Produits Forestiers Non Ligneux : Cas de la fougère (Pteridium
centrali-africanum Hieron.) à Kinshasa, Gepac Newsletter n°8, Université Libre de Bruxelles (Belgique), 4
p.
Biloso A & Lejoly J (2006) Etude l’exploitation et du marché des produits forestiers non ligneux à Kinshasa.
Tropicultura 24(3) :183–188.
Dhandevi PEM & Jeewon R (2015) Fruit and Vegetable Intake: Benefits and Progress of Nutrition Educattion
Interventions: Narrative Review Article. Iran Journal of Public Health 44(10): 1309–1321.
FAO (2001) Le rôle de la diversité biologique dans l’alimentation de l’humanité.
Kouebou C, Fadi G, Bourou S, Djakissam PK, Layla H, Zenabou G, Barbi M, Vunyingah M & Woin N (2013)
Biodiversité et valeur alimentaire des fruits au Cameroun: Observations préliminaires dans le Département
de la Bénoué (Région du Nord). Journal of Applied Biosciences 69: 5510–5522.
Merlin (2012) Manger le fruit avec l’estomac vide l’estomac vide. 37 p.
Muraki I, Imamura F, Manson JE, Frank BH, Willett WC, Dam RMvan & Sun Q (2013) Fruit consumption and
risk of type 2 diabetes: results from three prospective longitudinal cohort studies. British Medical Journal
347: f5001.
Ndjebet NC (1997) Spatial distribution of non timber forest product collection, (a case study of South
Cameroon). Wageningen Agricultural University (department of forestry), 65 p.
Park HM, Heo J & Park Y (2011) Calcium from plant sources is beneficial to lowering the risk of osteoporosis
in postmenopausal Korean women. Nutrition Research 31: 27–32.
Payne ME, Steck SE, George RR & Steffens DC (2012) Fruit, Vegetable, and Antioxidant Intakes Are Lower in
Older Adults with Depression. Journal of the Academy of Nutrition and Dietetics 112: 2022–2027.
Sene A (2001) Exploitation et valorisation des produits forestiers non ligneux dans la région de Kolda :
Caractérisation des acteurs de base. Convention ISRA, BAME et UICN, Dakar.
Tchatat M, Ndoye O & Nasi R (1999) Produits forestiers autres que le bois d’oeuvre (PFAB) : Place dans
l’aménagement durable des forêts denses humides d’Afrique Centrale. FORAFRI, pp. 94.
Combe J (1982) Agroforestry techniques in tropical countries: potential and limitations. Agroforestry Systems 1:
13–27.
Tollens E (2003) L’Etat Actuel de la Sécurité Alimentaire en R.D. Congo : Diagnostic et Perspectives, Working
Paper 277. Département d’Economie Agricole et de l’Environnement, Katholieke Universiteit Leuven, pp.
48.
Research article
Determination of nutrients and fiber contents of seven invasive

plants and their decomposition rates
N. Hewavitharana1*, S. D. P. Kannangara1, L. R. Jayasekera1 and P. Weerasinghe2
1
Department of Botany, Faculty of Science, University of Kelaniya, Sri Lanka
2
Horticultural Crop Research and Development Institute (HORDI), Gannoruwa, Sri Lanka
*Corresponding Author: nalakahewavitharana@gmail.com [Accepted: 22 October 2018]
Abstract: Nutrients (C, N, P, K, Mg, Ca, Cu, Fe Mn and Zn), fiber contents and decomposition
rates of seven invasive plants (Mikania scandens, Tithonia diversifolia, Lantana camara,
Sphagneticola trilobata, Chromolaena odorata, Mimosa pigra and Panicum maximum) were
analyzed aiming at their potentiality to prepare cost effective, organic compost for crop cultivation.
Litter bag technique was used to measure the decomposition rates. Significantly the highest
nutrient contents; N (3.44%), Mg (1.3%), Cu (34 mg kg-1), Fe (393 mg kg-1), Mn (150 mg kg-1)
and Zn (671 mg kg-1) were found in M. scandens. T. diversifolia had significantly higher P
(0.37%) and Ca (4.92%) contents than that of others. S. trilobata showed significantly higher K
content (4.32%). Whereas, M. scandens and T. diversifolia showed significantly lower organic
carbon contents (16.8% and 19.8%), crude fiber contents (3.5% and 4.8%) and C:N ratio (4.8 and
6.1) respectively. Significantly higher decomposition rates were observed in M. scandens (k=
12.91 per year) and T. diversifolia (k= 10.77 per year). Although the nutrient contents and
decomposition rate (k= 3.41 per year) in P. maximum were significantly lower, but its carbon
(33.7%), crude fiber content (20.42%) and C:N ratio (26.5) were significantly higher than that of
others. T. diversifolia and M. scandens have the potential to use in low cost organic compost
preparation, due to their comparatively higher nutrients and decomposition rates. P.maximum can
also be incorporated in preparing compost for its higher crude fiber content to improve the soil
physical properties.
Keywords: Nutrients - Crude fiber - Invasive plants - Decomposition.
[Cite as: Hewavitharana N, Kannangara SDP, Jayasekera LR & Weerasinghe P (2018) Determination of
nutrients and fiber contents of seven invasive plants and their decomposition rates. Tropical Plant Research
5(3): 286–291]
INTRODUCTION
Sri Lanka is a tropical country with invasive plants distributed all over the country. At present limited
information is available on the distribution and abundance of invasive plant species in many areas of Sri Lanka
(Weerakoon 2008). Invasive plants alter ecosystem functions, reduce profitability, productivity long term
sustainability of agriculture and national resources. (Veeragurunthan 2009). Even though, these invasive plants
are problematic, some of them have the potential to be utilized as green manure based on their nutrient
compositions. Preparation of the composts by using these plants; and it would be a possible way to manage
invasive plants by sustainable manner. It is important to know the nutrient status, rate of decomposition,
abundance and growth rates of these plants for better selection.
Organic farming is important now a day because of the residual effect of chemicals (fertilizers and
substances) used in agriculture leading the environmental pollution and human health as well (Hsieh et al.
1996). Therefore researchers showed considerable interest in promoting the use of green manures in crop
cultivation. Many plant species have the ability to serve as good organic manure and the addition of such plants
to the field can enhance soil fertility (Akanbi et al. 2007).
In the central region of Sri Lanka, use of green leaves and twigs of certain common trees and plants, such as
Kekuna (Canarium zeylanicum (Retz.) Blume), Dadap (Erythrina variegata L.), Keppetiya (Croton laccifer L.),
Received: 20 May 2018 Published online: 31 October 2018
Hewavitharana et al. 2018
Karanda (Pongamia pinnata (L.) Pierre) and wild sunflower (Tithonia diversifolia (Hemsl.) A.Gray) as green
manure is a common practice in rice cultivation (Sangakkara et al. 2004). In addition, Sesbania spp. and
Crotalaria spp. have the highest nitrogen contents and the lowest C:N ratios among the species that exhibit wide
spread distribution in Sri Lanka (Weerakoon 2008).
The amount of nutrients contributed by green manure will depend on the chemical composition of plant parts
and the amount added (Sariyildiz & Anderson 2003). However, many researchers have indicated that a
significant increase of total soil nitrogen, available phosphorus exchangeable potassium, magnesium content and
soil organic carbon with the application of green manure (Jama et al. 2000, Akanbi et al. 2005, Nziguheba et al.
2005, Olabode et al. 2007, Shokalu et al. 2010). Although many studies (Alexander 1977, Swift et al. 1979,
Johansson 1995) have been carried out to investigate the effects of leaf quality variables on decomposition using
different plant species, but best indicator of leaf variable for the decomposition is still unknown. The objectives
of the present study were to determine the leaf chemical composition of selected widely available invasive plant
species and to investigate their decomposition rates.
MATERIALS AND METHOD

Collection of invasive plants
Leaf samples of Mikania scandens (L.) Willd., Tithonia diversifolia (Hemsl.) A.Gray, Lantana camara L.,
Sphagneticola trilobata (L.) Pruski, Chromolaena odorata (L.) R.M.King & H.Rob., Mimosa pigra L. and
Panicum maximum Jacq. (Table 1) were collected from Madawachchiya, Nikaweratiya, Ulapane, Horana and
Godagama areas of Sri Lanka. Three replicate of leaf samples from each site was collected separately in
polythene bags and transported to the laboratory in University of Kelaniya. Sri Lanka.
Table 1. Selected invasive plants (with their family and common names) in Sri Lanka.
S.N. Plant species Family Common name
1 Mikania scandens (L.) Willd. Asteraceae Watupalu
2 Tithonia diversifolia (Hemsl.) A.Gray Asteraceae Naththasuriya
3 Lantana camara L. Verbenaceae Gandapaana
4 Sphagneticola trilobata (L.) Pruski Asteraceae Arunadevi/Kaha karabu
5 Chromolaena odorata (L.) R.M.King & H.Rob. Asteraceae Podisinghomarang
6 Mimosa pigra L. Fabaceae Yodha nidikumba
7 Panicum maximum Jacq. Poaceae Gini thana
Samples preparation
Sample were washed properly in running tap water followed by rinsing in distilled water, air dried at room
temperature (30°C) for two days and oven dried at 80°C until a constant weight was obtained. The oven dried
samples were ground using a mortar and pestle and were sieved using a 2 mm mesh. The homogenized leaf
samples were transferred to clean glass bottles and stored at room temperature.
Determination of nutrients contents
Nutrients and fiber contents of invasive plants were analyzed at the Horticultural Research and Development
Institute (HORDI) in Gannoruwa and at CIC Soil, Plant and Water Analytical Laboratory in Dambulla. Organic
carbon contents were determined by Walkley- Black method (Walkley & Black 1934). The total nitrogen (N) and
phosphorus (P) contents were determined by Kjeldahl (Kjeldahl 1883) and Vanado-molybdate method (Bernhart
& Wreath 1955). Contents of potassium (K), calcium (Ca), magnesium (Mg), copper (Cu), iron (Fe), manganese
(Mn) and zinc (Zn) were determined on a dry weight basis by atomic absorption spectrophotometry (Model:
Spectraa 110) following a wet digestion with a tri acid mixture (HNO3:H2SO4:HClO4; 9:4:1) at 200°C (Jackson
1973). A blank solution was prepared with tri acid mixture without adding samples. Crude fiber contents were
determined gravimetrically after chemical digestion and solubilization of the materials following Weende
method (AOAC 1990).
Rate of decomposition
Litter bag technique was used to determine the decomposition rates of the selected invasive plants in order to
identify fast decomposition species. Fresh leaves of the seven invasive plants were washed properly in running
tap water and rinsed in distilled water then air dried. Five grams of the air-dried leaves of each plant species was
placed in 1 mm mesh litter bags (20 × 20 cm). They were randomly placed on the ground in the Botanical
Garden (6° 58.428' N, 79° 54.851' E) University of Kelaniya. The precipitation is it affects more or less
similarity to the decomposition of all the litter materials. The average soil depth is 15 cm and the stagnant water
has been always drained out naturally from the study site. Physiochemical properties of study site was studied.
Soil was sandy clay loam in texture, having a moderate drainage and pH range of 5.5–6.0. Microbial activities
was relatively low. The mean annual precipitation is 2400 mm, with 50–75 mm per month during the dry season
of experiment period (January to April). Average monthly temperature ranges 28–32°C and relative humidity is
65%. Forty replicates were used per plant species. Five litter bags of each leaf species were randomly collected
in every 2 weeks and roots, adhering soil particles were removed gently without damaging the litter materials
inside. Leaf residues were oven dried at 70°C for a constant weight was obtained. Finally the percentage of mass
remaining was calculated using following equation and then % of mass remaining was plotted at intervals of two
weeks (Guendehou et al. 2014).
Data analysis
Data obtained were analyzed statistically with the MINITAB version 16 using one-way analysis of variance
(ANOVA, p < 0.05) followed by Tukey’s pair-wise multiple comparison test to determine whether the nutrient
content of the 7 plant species were significantly different.
Single negative exponential decay model (Hartemink & Sullivan 2001) applied in decomposition studies was
fitted to the observations on remaining mass in the seven invasive plant species studied using following equation
(Loranger et al. 2002).
Y = e-k × t
Y = X t / X0
Where Y is the proportion initial leaf mass remaining at time t and k is the decomposition factor. Xt is the
remaining mass of the litter at time t, X0 the initial mass of the leaf placed in the litter bags. The linear
regression of the lnY vs. time (t) was further done for the calculation of k value.
RESULTS AND DISCUSSION

Nutrients and fiber contents of the invasive plants
According to the results of nutrient analysis, Mikania scandens and Tithonia diversifolia exhibited
comparatively higher macro nutrient contents compared to that in the others (Table 2). However, M. scandens
and T. diversifolia had a significantly lower amount of organic carbon (16.8%; 19.8%) and fiber content (3.5%;
4.8%) compared to Panicum maximum (20.4 %). P. maximum had the highest carbon (33.7%) and fiber content
(20.4%), but lower in other nutrient contents (N; 1.27%, P; 0.24%, Ca; 1.55% and Mg; 0.54%). The C/N ratio
M. scandens (4.8) and T. diversifolia (6.1) were significantly lower than that of the other plant species studied (p
< 0.05).
Table 2. Macro nutrient and fiber contents (%) of selected invasive plants.
Plant species C% N% P% K% Ca% Mg% Fiber % C:N
Mikania scandens 16.8c 3.44a 0.36a 3.30a 3.39a 1.33a 3.5c 4.8c
Tithonia diversifolia 19.8c 3.28a 0.37a 2.53b 4.92a 0.83ab 4.8c 6.1bc
Lantana camara 21.3cb 2.51ab 0.28a 1.90b 3.85a 0.64b 10.6ab 8.4b
Sphagneticola trilobata 23.5cb 2.15ab 0.32a 4.32a 2.81b 0.57b 10.2b 10.9b
Chromolaena odorata 20.2cb 2.94ab 0.28a 1.62b 3.49a 0.92a 8.7b 6.9b
Mimosa pigra 30.6ab 2.98ab 0.23b 0.29bc 3.08a 0.47b 15.2ab 10.7b
Panicum maximum 33.7ab 1.27cb 0.24b 1.44b 1.55c 0.54b 20.4a 26.5a
Note: Each data point represents the mean of ten replicates. Means sharing a common letter (s) in each column are not
significantly different p > 0.05 by Tukey’s multiple comparison test.
The highest N and Mg contents were observed in M. scandens (3.44% and 1.33% respectively); and the
lowest N (1.27%) and Mg (0.47%) contents were observed in P. maximum and Mimosa pigra plant leaves
respectively. The highest K content (4.32%) was found in Sphagneticola trilobata while M. pigra had the lowest
(0.29%). The highest P (0.37%) and Ca (4.92%) contents were found in T. diversifolia whereas the lowest P
(0.23%) and Ca (1.55%) contents were found in M. pigra and P. maximum respectively (Table 2). M. scandens
had the highest Cu (34 mg kg-1), Fe (393 mg kg-1), Mn (150 mg kg-1) and Zn (671 mg kg-1) contents. The lowest
Cu content was found in P. maximum (9 mg kg-1) while the lowest Fe (76 mg kg-1), Mn (70 mg kg-1) and Zn
(267 mg kg-1) contents were detected in S. trilobata, T. diversifolia and P. maximum respectively (Table 3).
Olabode et al. (2007) found 1.76%, 0.82%, 3.907%, 0.005% and 5.53% of N, P, K, Ca, Mg and crude fiber
respectively in T. diversifolia and 1.12%, 1.62%, 1.49%, 1.42%, 0.205% and 17.30% respectively in P.
maximum. Agbede et al. (2013) reported the chemical composition of T .diversifolia as C (4.8 %), N (1.88%), P
(0.79%), K (3.89%), Ca (3.41%) and Mg (0.004%). The nitrogen content for T. diversifolia in the present study
was significantly higher than the values reported by Agbede et al. (2013). Sariyildiz & Anderson (2003) and
Reddy & Venkataiah (1989) were also reported that the nutrient content in green manure varies with the age of
the plants, soil fertility, climatic conditions, season and proportion of the leaf to stem. However, Jama et al.
(2000) reported more or less similar values for N (3.50%), P (0.37%) and K (4.10%) contents of T. diversifolia
in comparison to the present study.
Table 3. Some micro nutrient contents (mg kg-1) of selected invasive plants.
Plant species Cu (mg kg-1) Fe (mg kg-1) Mn (mg kg-1) Zn (mg kg-1)
Mikania scandens 34a 393a 150a 671a
Tithonia diversifolia 10b 296ab 70b 517a
Lantana camara 10b 285ab 118a 365b
Sphagneticola trilobata 24a 76c 130a 510a
Chromolaena odorata 19b 375a 77b 363b
Mimosa pigra 10b 172c 136a 394b
Panicum maximum 9b 181c 72b 267b
Note: Each data point represents the mean of ten replicates. Means sharing a common letter (s) in each column are
not significantly different p > 0.05 by Tukey’s multiple comparison test.
In a study to increase the yield of cauliflower, Hafifah et al. (2016) had observed an enhancement of soil
physical and chemical properties, by the application of T. diversifolia as a green manure along with cow
manure. Green manure of T. diversifolia contributed an increase in soil organic carbon, total N, total porosity, P
availability and K exchange and a decrease in soil density either singly or in combination with other treatments.
These findings are also applicable for introduction of T. diversifolia as a green manure in organic farming.
Dense growth of T. diversifolia and M. scandens probably makes them ideal sources of green manures along
with a wide availability in Sri Lanka.
Decomposition rates
The mass loss patterns of the selected invasive plants are presented in figure 1, where the mass remaining is
expressed as a percentage of the initial mass of the leaves. After 112 days, the remaining mass was constant for
all the invasive plants (Fig. 1). During the study period, M. scandens and T. diversifolia showed a rapid decrease
in mass in the initial phase than that in the others. As decomposition proceeds, the decomposers use the soluble
components and relatively easily degradable components like sugars, starches, and proteins. Therefore, during
the initial phases, the decomposition rate is higher than in the later phases, the more recalcitrant materials like
lignin, tannins, celluloses, and hemicelluloses being decomposed at much slower rates (Loranger et al. 2002).
Figure 1. Mean remaining mass (%) of invasive plants during decomposition period. [Error bars represent the mean of five
replicates ± standard error]
A higher decomposition factor (k value) indicates a higher decomposition rate (Hartemink & Sullivan 2001).
M. scandens (k= 12.91 per year) and T. diversifolia (k= 10.77 per year) exhibited significantly higher (p<0.05)
decomposition rates compared to that of other specie P. maximum showed the lowest decomposition rate (k=
3.41 per year) than that of the other plant species (Table 4). Several studies have adopted the litterbag technique
to determine the effect of litter quality on decomposition rates (Swarnalatha & Reddy 2011, Guendehou et al.
2014).
Table 4. Leaf litter decomposition rates (k values) of the selected invasive plants calculated
using first odder exponential decay modal (Y =e-k ×t) and correlation coefficient (R2).
Plant species k- value R2
Mikania scandens 12.91a 0.965
Tithonia diversifolia 10.77a 0.947
Lantana camara 4.83bc 0.936
Sphagneticola trilobata 5.11bc 0.974
Chromolaena odorata 8.00ac 0.881
Mimosa pigra 4.14bc 0.968
Panicum maximum 3.41b 0.940
Note: Means sharing a common letter (s) in each column are not significantly different p >
0.05 by Tukey’s multiple comparison test.
Leaf litter of different plant species has diverse nutrient release patterns, which are related to leaf quality of
initial nitrogen contents and C: N ratio (Swarnalatha & Reddy 2011). Higher decomposition rates were observed
with low C:N ratio and high nitrogen content, in comparison to high C:N ratio with low nitrogen content. This
suggests that the C:N ratio and initial N content could be the main factors for efficient litter decomposition.
Previous studies have revealed that the total nitrogen content accelerates litter decomposition (Swarnalatha &
Reddy 2011). M. scandens and T. diversifolia also showed higher nitrogen contents (3.44% and 3.28%
respectively) and low C:N ratio (4.8 and 6.1 respectively) which accelerate the decomposition rates in this study.
Even though, the decomposition rate of P. maximum was significantly lower compared to that of other species,
due to the higher fiber content (20.4%) low nitrogen content (1.27%) with high C:N (26.5) ratio values. M.
scandens and T. diversifolia contained higher nutrient contents and displayed higher decomposition rates. Rapid
nutrient release from added organic materials is beneficial, especially for short duration crops.
CONCLUSION
Based on the nutrient content and decomposition rates of the selected invasive plants, Mikania scandens and
Tithonia diversifolia could be suggested as plant base substrates for the formulation of compost. Panicum
maximum has low nutrients, but significantly a higher content of crude fiber, can also be suggested to
incorporate with other selected species in preparation of compost, for improving the soil physical properties.
Combination of M. scandens, T. diversifolia and P. maximum can be recommended for a cost effective nutrient
rich compost preparation and also as one of the solution for removal of invasive plants from ecosystems in
another aspect.
ACKNOWLEDGMENTS
The Horticultural Research and Development Institute (HORDI) in Gannoruwa and CIC Soil, Plant and
Water Analytical Laboratory in Dambulla are acknowledged for providing necessary research facilities.
Financial support from the Ministry of Higher Education is also highly appreciated.
REFERENCES
Agbede TM, Adekiya AO & Ogeh JS (2013) Effects of Chromolaena and Tithonia mulches on soil properties,
leaf nutrient composition, growth and yam yield. West African Journal of Applied Ecology 21(1): 15–29.
Akanbi WB, Adebayo TA, Togun OA, Adeyeye AS & Olaniran OA (2007) The use of compost extract as foliar
spray nutrient source and botanical insecticide in Telfairia occidentalis. World Journal of Agricultural
Sciences 3(5): 642–652.
Akanbi WB, Akande MO & Adediran JA (2005) Suitability of composted maize straw and mineral nitrogen
fertilizer for tomato production. Journal of Vegetable Science 11(1): 57–65.
Alexander M (1977) Introduction to soil microbiology, 2nd edition. John Wiley and Sons, New York, pp. 68–70.
AOAC (1990) Appendix A, Analytical Techniques, Determination of Crude Fiber content. Available from:
http://studentsrepo.um.edu.my/2346/12/LAMPIRAN.pdf (accessed: 02 Mar. 2018).
Bernhart DN & Wreath AR (1955) Colorimetric determination of phosphorus by modified phosphomolybdate
method. Analytical Chemistry 27(3): 440–441.

Guendehou GHS, Liski J, Tuomi M, Moudachirou M, Sinsin B & Makipaa R (2014) Decomposition and
changes in chemical composition of leaf litter of five dominant tree species in a West African tropical forest.
Tropical Ecology 55(2): 207–220.
Hafifah H, Sudiarso S, Maghfoer MD & Prasetya B (2016) The potential of Tithonia diversifolia green manure
for improving soil quality for cauliflower (Brassica oleracea var brotrytis L.). Journal of degraded and
mining lands management 3(2): 499–506.
Hartemink AE & Sullivan JNO (2001) Leaf litter decomposition of Piper aduncum, Gliricidia sepium and
Imperata cylindrica in the humid lowlands of Papua New Guinea. Plant and Soil 230: 115–124.
Hsieh CF, Fang HC, Nan K & Hsu KN (1996) Effect of continuous use of organic manures on the growth and
yield of vegetable soybean and cabbage. Organic Agriculture 46: 1–10.
Jackson ML(1973) Soil Chemical Analysis. Prentice Hall of India Pvt. Ltd., New Delhi, pp. 42–48.
Jama B, Palm CA, Buresh RJ, Niang A, Gachengo C, Nziguheba G & Amadalo B (2000) Tithonia
diversifolia as a green manure for soil fertility improvement in western Kenya. Agroforestry Systems 49(2):
201–221.
Johansson MB (1995) The chemical composition of needle and leaf litter from Scots pine, Norway spruce and
white birch in Scandinavian forests. Forestry 68: 49–62.
Kjeldahl J (1883) A New Method for the Determination of Nitrogen in Organic Substances. Zeitschrift für
Analytische Chemie 22: 366–382.
Loranger G, Ponge JF, Imbert D & Lavelle P (2002) Leaf decomposition in two semi-evergreen tropical forests:
influence of litter quality. Biology Fertile Soils 35: 247–252.
Nziguheba G, Merckx R, Palm CA & Mutuo P (2005) Combined use of Tithonia diversifolia and inorganic
fertilizers for improving maize production in a phosphorus deficient soil in Western Kenya. Agroforestry
Systems 55(3): 165–174.
Olabode OS, Ogunyemi SWB, Akanbi GO & Babajide PA (2007) Evaluation of Tithonia diversifolia (Hemsl.)
A gray for soil improvement. World Journal of Agricultural Sciences 3(4): 503–507.
Reddy MV & Venkataiah B (1989) Influence of microarthropod abundance and climatic factors on weight loss
and mineral nutrient content of Eucalyptus leaf litter during decomposition. Biology and Fertility of Soils 8:
319–324.
Sangakkara UR, Liedgens M & Soldati A (2004) Root and shoot growth of maize (Zea mays) as affected by
incorporation of Crotalaria juncea and Tithonia diversifolia as green manures. Journal of Agronomy and
Crop Science 190: 339–346.
Sariyildiz T & Anderson JM (2003) Interactions between litter quality, decomposition and soil fertility: a
laboratory study. Soil Biology and Biochemistry 35: 391–399.
Shokalu AO, Ojo AO, Adewoin DT E & Azeez JA (2010) Comparing the use of Tithonia diversifolia and
compost as soil amendments for growth and yield of Celosia argentea. New York Science Journal 3(6): 13–
3.
Swarnalatha B & Reddy M (2011) Leaf litter breakdown and nutrient release in three tree plantations compared
with a natural degraded forest on the coromandel coast (puducherry, india). Eco-tropical 17: 39–51.
Swift MJ, Heal OW & Anderson JM (1979) Decomposition in terrestrial ecosystems. 3rd edition. Blackwell
Scientific publications, Oxford, pp.75–78.
Veeragurunthan V (2009) Effect of commercial fertilizer (Urea) and S.L.F. of Entermorpha intestinalis on the
growth of Capsicum annum L. Seaweed Research and Utilization 31(1): 158–163.
Walkley A & Black IA (1934) An examination of Degtjareff method for determining soil organic matter and a
proposed modification of the chromic acid titration method. Soil Science 37: 29–37.
Weerakoon D (2008) Management of invasive plants in protected areas by the Department of Wildlife
Conservation: Lessons learned and the way forward. National symposium on invasive Alien species, Sri
Lanka, pp. 57–62.
Research article
Effect of fungal endophytes of rice variety Ld 368 on growth

and brown spot disease incidence of rice
C. D. N. Priyadarshani1, N. Deshappriya2* and T. G. I. Sandamali3
1
Department of Botany, University of Kelaniya, Sri Lanka
2
Department of Botany, University of Sri Jayewardenepura, Sri Lanka
3
Rice Research Station, Bentota, Sri Lanka
*Corresponding Author: nelum@sci.sjp.ac.lk [Accepted: 28 October 2018]
Abstract: Use of chemicals for growth enhancement and disease control in plants has resulted in
hazardous influences to the environment and human health. Therefore, less harmful methods
should be implemented and the possibility of using microbes for this purpose has been
investigated. Endophytic fungal assemblages have been known to enhance plant growth and
decrease disease incidence in some crops including rice and thus can be used as an alternative to
chemicals. Therefore, this study was aimed to isolate the endophytic fungal communities
associated with the rice variety Ld 368 with a view to examine the possibility of using them for
plant growth enhancement and management of brown spot disease incidence. Brown spot disease
caused by Bipolaris oryzae is one of the major rice diseases prevalent in Sri Lanka. Healthy plant
parts of variety Ld 368 were used for the isolation of endophytes. 31 endophytic fungal species
were isolated, and eight of the most frequently isolated fungal species were tested for their ability
to inhibit the growth of B. oryzae using dual culture assays. From the fungal species tested,
Trichoderma sp.1, Trichoderma sp.2 and Chaetomium sp. inhibited the colony growth of Bipolaris
Oryzae significantly (P ≤ 0.05) under in-vitro conditions. Based on the results of in-vitro tests,
spore suspensions of the more effective endophytes were inoculated separately to healthy Ld 368
seedlings to evaluate their efficacy in controlling brown spot disease and to determine their effect
on rice plant growth under greenhouse conditions. Two inoculation methods (i.e. seedling and soil
inoculation) were used to identify the best approach to introduce the endophytic fungi into the
plants. Plants inoculated with Trichoderma sp.1 and Chaetomium sp. using seedling inoculation
method showed the lowest disease incidence as well as a significant difference (P ≤ 0.05) in shoot
length and fresh and dry weight of plants. These results indicated that the tested endophytic fungal
sp. have the ability to control brown spot disease incidence and enhance plant growth of rice
variety Ld 368.
Keywords: Endophytes - Bipolaris oryzae - Bio-control - Growth enhancement.
[Cite as: Priyadarshani CDN, Deshappriya N & Sandamali TGI (2018) Effect of fungal endophytes of rice
variety Ld 368 on growth and brown spot disease incidence of rice. Tropical Plant Research 5(3): 292–302]
INTRODUCTION
Rice (Oryza sativa L.) is one of the most important cereal crops in the world and is the staple diet of Sri
Lanka occupying 34% of the total cultivated area. Brown spot disease caused by Bipolaris oryzae (Breda de
Haan) Shoemaker is an important seed-borne disease of rice, causing 6–90% losses in grain yield in both wet
and dry seasons (Archana et al. 2014). Brown spot disease has become prevalent in some districts in Sri Lanka
due to unusual rainfall patterns and climatic changes (RRDI-Bathalagoda 2016).
Bipolariz oryzae attacks the crop from seedling to milk stage (Arshad et al. 2013, Sunder et al. 2014) and
infects leaves, leaf sheath, coleoptile, panicle branches, glumes, and spikelets (Arshad et al. 2013) and results in
seedling blight, lesions on coleoptile, leaves and glumes and eventually leads to seedling death (Schwanck et al.
2015). In severe infections, germination failure or poor germination of seeds, rotting of seeds, roots and
coleoptiles (Kamal & Mia 2009), as well as loss in weight, may occur (Kumar et al. 2016). When the disease is
Received: 24 March 2018 Published online: 31 October 2018
Priyadarshani et al. 2018
severe or when the cultivar is more susceptible to the disease, spots become larger and may cover the entire leaf,
reducing photosynthetic area, nutrient absorption and result in the decrease of tillering nodes (Archana et al.
2014, Sunder et al. 2014).
The available management strategies for brown spot disease include the use of partially resistant rice
cultivars, appropriate plant nutrition management and fungicide application (Dallagnol et al. 2014). However,
long-term application of fertilizers and fungicides may result in hazardous influences to the environment and
human health (Tian et al. 2004) and use of partially resistant rice cultivars and plant nutrition management is not
feasible due to higher costs (Mithrasena et al. 2012). Therefore, use of a bio-control method as an alternative to
these methods is a promising approach. Plants host diversity of plant-associated microorganisms that play
beneficial roles such as, enhance plant growth, tolerance to abiotic stresses, decrease plant stress and disease
incidence (Parsa et al. 2016). An example group of such microorganisms is endophytic fungi, which are
reported to possess many beneficial effects on plants.
Endophytic fungi are effective in controlling diseases in a number of crops (Kusari et al. 2012) and has been
reported to control rice blast disease in the traditional rice variety Suwandel in Sri Lanka (Atugala &
Deshappriya 2015). Endophytic fungi are also reported to enhance the growth of plants by several mechanisms
including the release of auxins, gibberellin and cytokinin (You et al. 2012). Although endophytic fungi of rice
have been reported to enhance growth and yield of a number of rice varieties (Atugala & Deshappriya 2015,
Wijesooriya & Deshappriya 2016), the endophytic fungal assemblage of the rice variety Ld368 and their effect
on brown spot disease development and plant growth has not been reported in Sri Lanka previously.
Therefore, the present study carried out to isolate the endophytic fungi present in different healthy plant parts
of the Ld 368 rice variety, and to evaluate the effect of the most frequently isolated fungal endophytes on brown
spot disease development and rice plant growth under greenhouse conditions.
MATERIALS AND METHODS

Sample collection
10-week old healthy rice plants and seeds of rice variety Ld 368 and 10-week old plants showing brown spot
symptoms were randomly collected from the paddy fields at the Rice Research Station, Bentota, Sri Lanka.
Isolation of endophytic fungi from healthy Ld 368 rice variety
Healthy leaf, stem and root pieces, seeds and seedlings of Ld 368 rice variety were used to isolate
endophytic fungi. Each plant part was cleaned by rinsing under running tap water for 10 min and the cleaned
plant parts were surface sterilized separately according to the protocols described by Atugala & Deshappriya
(2015). After surface sterilization, three consecutive rinses in sterilized distilled water were carried out for each
of the plant parts, seeds and seedlings and dried using sterilized filter papers. The edges of the surface sterilized
leaf, stem and root segments were trimmed and placed (5 per plate) separately on Potato Dextrose Agar (PDA)
plates containing tetracycline (50 mg L-1). 5 replicate plates were prepared for each plant part and incubated at
room temperature.
Fungi that grew out from each plant part, seeds and seedlings were sub-cultured onto a fresh PDA medium
containing tetracycline (50 mg L-1) and incubated at room temperature. Pure cultures of all isolates were
prepared using the Hyphal tip method (Dhingra & Sinclair 1995). The isolated endophytic fungi were identified
to the genus level using their macroscopic and microscopic features and identification keys (Domsch et al. 1993,
Barnett & Barry 1998).
Colonization frequencies (CF), isolation frequencies and dominant fungi percentages were calculated
for each isolated endophytic fungal species using the following (Atugala & Deshappriya 2015);
Isolation of brown spot pathogen from diseased leaves of Ld 368 rice variety
Leaf pieces of 7 cm length, which contained the brown spot necrotic lesions were cut and cleaned under
running tap water for 10 minutes. Sections of approximately 1 cm2 size were cut from the diseased leaf from the
edge of the area showing symptoms. Leaf pieces were surface sterilized 70% (v/v) ethanol for 1 min, 0.25%
(w/v) NaOCl for 20 min and 70% (v/v) ethanol for 30 s and washed three times with sterilized distilled water
and dried on sterilized filter paper. The edges of the sections were trimmed using a sterile scalpel and under
aseptic conditions, and the pieces were placed on PDA containing tetracycline (50 mg L-1) distributing 4 pieces
on one plate. After 7 days incubation, resultant fungal colonies were separately sub-cultured into PDA plates
and incubate at room temperature. The colony morphology and sporulating structures of the brown spot
pathogen were observed and identified using identification keys (Domsch et al. 1993, Barnett & Barry 1998).
Identification of Morphological features
Colony characteristics such as front and reverse side colony color, colony shape, elevation, texture, type of
mycelium, margin, zonation and pigment formation were observed visually after incubation at room
temperature.
Identification using microscopic features
Pure cultures of isolated endophytic fungi were observed under light microscope for their microscopic
characters, such as sporulating structures, spores, features of hyphae, presence or absence of asci and paraphyses
using sticky tape method, in which piece of sticky tape was gently pressed on the colony and placed on a glass
slide containing drop of cotton blue.
Identification using molecular methods
i. DNA extraction: For the further identification of endophytic fungal species that showed to have an
antagonistic activity aginst the pathogen i.e. Trichoderma sp.1, Trichoderma sp.2, Chaetomium sp. and
pathogen Bipolaris oryzae, genomic DNA were extracted according to the protocol described by Cenis
(1992) and PCR amplified.
ii. PCR amplification: Amplification of the ribosomal DNA, ITS 1 and 2 was done to the extracted fungal DNA
with the primers of ITS-5 and ITS-4 primers. PCR was carried out in 25 reaction volumes using 2 mM
MgCl2 (Promega Inc, USA), 0.12 mM dNTP (Promega Inc, USA), 1 µM each primer, 0.05 unit µM -1 Taq
polymerases (Go Taq flexi, Promega Inc, USA) on a thermal cycler (Techne, flexigene, England). PCR was
done under the conditions of initial denaturation at 95°C for 5 min, followed by 30 cycles, of denaturing at
95°C for 1 min, annealing at 55°C for 1 min and extension at 72°C for 2 min and the final extension at 72°C
for 10 min.
Pathogenicity tests of the fungal sp isolated from diseased plants
Pathogenicity of the isolated fungal sp was confirmed on healthy rice plants grown under greenhouse
conditions using a spore suspension. A suspension of, 1× 105 spores ml-1 of the isolated organism was prepared
using a 14 days old culture maintained in PDA. 0.05% of tween 80 was added to the suspension.
Leaves of 45 days old rice plants maintained under greenhouse conditions, were wiped with 70% ethanol
and were slightly wounded using a sterilized needle and sprayed with 2 ml of the prepared spore suspension.
Plants sprayed with sterilized distilled water containing 0.05% Tween 80 served as the controls. All treated
plants were covered with transparent polythene bags with wetted cotton wool to provide adequate humidity and
observed after 7 days for disease symptom development.
Antagonistic activity of isolated endophytic fungi
i. Dual culture assay: A 5 mm diameter disc from growing edge of a 5-day old Bipolariz oryzae and each
endophytic fungal culture maintained on PDA were placed at the two opposite ends of a PDA plate. A sterile
PDA disc replaced the endophytic fungal disc in the control plates. There were 5 replicates for each
endophyte tested. All plates were incubated at room temperature and the effect of endophytic fungi on
mycelial growth of B. oryzae was evaluated 2 days after incubation by measuring the diameter of the B.
oryzae colony (R2) on the test and the diameter of the B. oryzae colony in the control plate (R1).
ii. Percentage growth inhibition (I%) was calculated using the equation, (Rahman
et al. 2009, Khalili et al. 2012).
Effect of volatile metabolites of endophytes on the growth of Bipolaris oryzae
To determine the effects of volatile metabolites of endophytic fungi on mycelial growth of Bipolaris oryzae,
discs (5 mm diameters) cut from the margin of the 7 days old endophytic fungi and Bipolaris oryzae cultures
were placed in the center of two separate bottom portions of petri dishes containing PDA, plates with the
pathogen were placed in an inverted position over the plate containing each endophytic fungal sp. The two
bottoms held together with the pathogen at top, was sealed with parafilm and incubated at room temperature for
7 days. A control plate was maintained without endophytic fungi in the bottom plate. There were 5 replicates for
each fungus and the percentage of growth inhibition was calculated.
Tests for the effect of endophytes on brown spot disease incidence under greenhouse conditions
Three endophytic fungal isolates i.e. Trichoderma sp.1, Trichoderma sp.2 and Chaetomium sp. that showed
a significant antagonistic activity against the brown spot pathogen under in vitro conditions were used for the
greenhouse experiments. Healthy Ld 368 rice seeds were surface sterilized (75% ethanol for 30 s, 1% NaOCl for
10 min and 70% ethanol for 30 s) and soaked overnight in sterile distilled water. The seeds were germinated by
wrapping them with a wet sterilized cloth and incubating under room temperature for five days. Inoculation of
endophytes to plants was carried out using two methods:
i. Method I- Seedling Inoculation- plate method: Seedlings were inoculated with endophytes Trichoderma
sp.1, Trichoderma sp.2 and Chaetomium sp. by using the plate method (Wijesooriya & Deshappriya 2016)
and incubated at room temperature. Seedlings were placed on fresh PDA plates as a control.
The success of inoculation was confirmed by placing 15 randomly selected seedlings subjected to each
treatment including controls on PDA plates supplemented with tetracycline (50 mg L-1) after surface
sterilization (75% ethanol for 30 s, 1% NaOCl for 10 min and 70% ethanol for 30 s). Trichoderma sp.1,
Trichoderma sp.2 and Chaetomium sp. inoculated seedlings and control seedlings were planted in pots (8 cm
in height and 9.5 cm in width) filled with sterilized soil which had been autoclaved for 20 min (121°C and
15 lb in-2) separately.
ii. Method II- Soil inoculation method: Soil samples collected from a field were autocalved for 20 min at 121°C
and 15 lb in-2. 15 ml (Tarafdar & Gharu 2006) of spore suspensions (1× 105 spores ml-1) of Trichoderma
sp.1, Trichoderma sp.2 and Chaetomium sp. were added to pots (8 cm in height and 9.5 cm in width) filled
with sterilized soil (250 g) for planting and control was prepared adding sterilized distilled water. 6 days old
healthy non-inoculated Ld 368 seedlings germinated as mentioned above were planted in pots (5 seedlings
per pot). There were 5 replicates for each inoculation and plants were maintained under the average
temperature of 30 ± 5°C during day and 20 ± 5°C during night in the greenhouse.
To prepare the pathogen inoculum a spore suspension of 1× 107 spores/ ml of Bipolaris oryzae was
prepared using a 14-day old culture maintained in PDA. 0.05% tween 80 was added to the spore suspension.
Leaves of the 21 days old endophyte treated and control plants were wiped with 70% ethanol and leaves
were slightly wounded using a sterilized needle and sprayed with B. oryzae spore suspensions. Control
plants were sprayed with sterilized distilled water added to fresh uncontaminated PDA plate. Each plant was
covered separately with clean, transparent polythene bags and wet cotton wool was incorporated to provide
moisture. Disease development on the rice leaves was observed daily, and the disease incidence was
calculated using the following formula,
Effect of fungal endophytes on rice plant growth

The effect of the most frequently isolated fungal endophytes i.e. Trichoderma sp.1, Trichoderma sp.2 and
Chaetomium sp., on the growth of the rice variety Ld 368 was tested. Ld 368 healthy seeds were surface
sterilized and germinated as mentioned earlier and the fungal endophyte isolates were inoculated using Seedling
method (plate method) and soil method.
Treated and control seedlings were planted in pots (5 seedlings per pot) filled with autoclaved soil. There
were 5 replicates for each treatment and plants were maintained under the average temperature of 30±5°C
during the day and 20±5°C during the night in the greenhouse. Fresh weight, dry weight and shoot lengths of
randomly selected 5 plants from each treatment were measured at 1 week interval. First initial fresh weights of
the plants were measured and dry weights were measured by drying plants in an oven at 60°C for 24 hours until
a constant weight was obtained (Vibhuti et al. 2015). The length between the starting point of the shoot and end
point of the flag leaf was measured as shoot length. Results were statistically analyzed using two-way ANOVA
and Tukey’s pairwise comparisons.
RESULTS
A total of 31 fungal species was isolated from 150 samples of the root, stem, leaf, seeds and seedlings of
healthy Ld 368. Higher colonization (92%) and isolation frequency (40 %) were shown by seeds while lowest
colonization (20%) and isolation (4%) frequency shown by leaves (Table 1). Chaetomium sp. was the dominant
fungus present in Ld 368 rice variety (Table 2).
Table 1. Frequency of isolation and colonization of endophytic fungi from different parts of healthy Ld
368 rice plants.
Total number of Isolation frequency Colonization frequency
Plant part
samples (%) (%)
Seed 25 40 92
shoot 25 48 20
Seedling {
root 25 36 8
Root 25 20 96
Stem 25 32 60
Leaves 25 4 20
Total 150 20.7 58.7
Table 2. List of isolated endophytic fungi, % dominance and colonized plant part of healthy Ld 368.
Endophytic fungi % Dominance
Chaetomium sp. 28.8
Sterile mycelia (SM 1) 23.3
Trichoderma sp. 1 16.7
SM 2 16.7
Penicillium 13.7
Trichoderma sp. 2 13.3
SM 6 13.3
Aspergillus 12.5
Fusarium sp.1 9.6
SM 3 6.7
Fusarium sp.2 4.0
SM 4 3.3
SM 5 3.3
SM 7 3.2
Colletotrichum 2.5
SM 8 1.6
Curvularia 1.3
In vitro tests for the effect of endophytes on the brown spot pathogen
Figure 1. Endophytes and Bipolaris oryzae in dual culture showing antagonism (10 days old cultures) colonies on the right
hand side represent endophytes: A, Trichoderma sp. 1; B, Trichoderma sp. 2; C, Chaetomium and colony on the left hand
side represent Bipolaris oryzae.
Figure 2. Structures formed by endophytes in mycoparasitism of Bipolaris oryzae: A, Fungal endophytes hyphae coiling
around the thicker hyphae of Bipolaris oryzae (10×40×2); B, Loops formed by endophytes to trap Bipolaris oryzae
(10×40×3); C, Endophytes forming clamps on pathogen mycelia (10×40×4).
i. Dual culture assays: All fungal isolates tested showed a significant inhibition (P≤ 0.05) of the growth of
Bipolaris oryzae on PDA. However, Trichoderma sp.1, Trichoderma sp.2and Chaetomium sp. showed the
highest inhibitory effects (Fig. 1A, B & C) with inhibitory percentages of 64.4%, 47.0% and 29.5% on the
colony growth of Bipolaris oryzae respectively (Table 3). When the dual cultures were observed under the
microscope, hyphae of Trichoderma sp.1 showed coiling around the hyphae of Bipolaris oryzae (Fig. 2A)
and Trichoderma sp.2, Chaetomium sp. showed formation of loops (Fig. 2B) and clamps (Fig. 2C), which
would trap the pathogen hyphae.
Table 3. Results of dual plate method.
Endophytic fungal isolate Diameter of Bipolaris oryzae colony (cm) % inhibition
Control 5.53 ± 0.77 0
Trichoderma sp. 1 1.97± 0.21 64.4a
Trichoderma sp. 2 2.93 ± 0.15 47.0b
Chaetomium 3.90 ± 0.26 29.5c
Fusarium sp. 2 4.00 ± 0.20 27.7cd
SM 7 4.13 ± 0.30 25.3cd
Fusarium sp. 1 4.20 ± 0.20 24.0cd
SM 8 4.20 ± 0.72 24.0cd
SM 2 4.33 ± 0.30 21.7cd
Note: n=5; mean ± SD; Mean values sharing common letters in each row are not significantly different p ≤ 0.005.
ii. Effect of volatile compounds produced by endophytic fungi on pathogen growth: All fungal endophytes
tested inhibited the growth of B. oryzae colony. Trichoderma sp.1 showed the maximum % inhibition, while
Chaetomium sp. showed the minimum inhibition of pathogen growth (Table 4). One way ANOVA showed a
significant difference (P ≤ 0.05) in colony growth inhibition between control and all fungal endophytes.
Table 4. Effect of metabolites on the growth of Bipolaris oryzae.
Fungal endophyte Diameter of pathogen growth (cm) % Inhibition of the pathogen
Control 7.97 ± 0.51 0
Trichoderma sp. 1 5.70 ± 0.80 28.5a
Trichoderma sp. 2 6.30 ± 0.47 20.9a
Chaetomium 7.90 ± 0.24 0.8b
Note: n=5; mean ± SD; Mean values sharing common letters in each row are not significantly different P ≤ 0.005.
DNA extraction and PCR amplification of fungal DNA
PCR amplification was performed to DNA extracted from Trichoderma sp.1, Trichoderma sp.2,
Chaetomium sp. and pathogen Bipolaris oryzae using the rDNA primers ITS 5 and ITS 4. PCR amplified
products and a 100 bp ladder were visualized as shown in figure 3. All the endophytes and pathogen were given
approximately 600–700 bp size bands in the gel except Trichoderma sp.1.
1 2 3 4 5 6 7 8
Figure 3. Gel image of PCR products of fungal endophytes and pathogen amplified using ITS 5 and ITS 4. [Well 1: 100 bp
ladder, Well 2: Trichoderma sp. 1, Well 3: Trichoderma sp. 2, Well 4: Unidentified sp. 4, Well 5: Chaetomiumsp., Well 6:
PCR (-) control, Well 7: PCR (+) control, Well 8: Bipolarizoryzae (pathogen)]
In planta tests for the effect of endophytes on the development of brownspot disease
Symptoms developed on the plants onto which only Bipolaris oryzae was inoculated. Disease symptoms
were also observed in all plants treated with each endophyte Trichoderma sp.1, Trichoderma sp.2 and
Chaetomium sp. using soil and seedling inoculation methods when inoculated with Bipolaris oryzae. However,
the disease incidence was lower in plants inoculated with Trichoderma sp. 1 and Chaetomium using both soil
and seedling inoculation methods (Table 5). There was no disease development in control plants sprayed with
sterilized distilled water. Plants grown from seedlings treated with Trichoderma sp. 1 and inoculated with
Bipolaris oryzae showed the lowest disease incidence compared to other treatments.
Table 5. Disease incidence in the Ld 368 plants of each endophyte treated plants.
Disease incidence %
Method of
Plants sprayed with pathogen Plants sprayed with
inoculation of Treatment
spore suspension sterilized distilled water
endophytes
( 1× 107 spores ml-1) (Controls)
Soil Trichoderma sp. 1 10 0
inoculation Trichoderma sp. 2 75 0
Chaetomium 50 0
Sterilized distilled water 83 0
Seedling Trichoderma sp. 1 5 0
inoculation Trichoderma sp. 2 25 0
Chaetomium 15 0
Sterilized distilled water 80 0
Effect of fungal endophytes on the growth of Ld 368 rice variety
A significant increase (P ≤ 0.05) in plant height, fresh weight and dry weight were observed in plants
inoculated with the endophytes compared with non-treated plants. The highest increase in plant height, fresh
weight and dry weight was shown in plants inoculated with Trichoderma sp.1.
Table 6. Mean shoot length (cm) of plants after 2 and 4 weeks.
Inoculation method
Week Treatment
Soil inoculation Seedling inoculation
After 2 weeks Trichoderma sp.1 13.42 ± 1.24a 14.40 ± 0.62a
a
Trichoderma sp.2 13.26 ± 1.39 13.72 ± 0.81a b
a
Chaetomium 13.68 ± 0.64 12.58 ± 0.43a b
a
non-inoculated 12.60 ± 0.66 12.02 ± 0.66b
a
After 4 weeks Trichoderma sp.1 19.86 ± 0.61 21.90 ± 0.58a
c
Trichoderma sp.2 14.90 ± 0.55 16.72 ± 0.45b
ab
Chaetomium 18.68 ± 0.83 16.94 ± 0.55b
c
non-inoculated 13.60 ± 0.29 13.10 ± 0.29c
Note: n=5; mean ± SE; Mean values sharing common letters in each row are not significantly different P ≤ 0.05.
Plants inoculated with endophytes did not show a significant increase in their shoot length after the second
week depending on the inoculation method but after four weeks endophytes inoculated via seedlings plants
showed a significant increase in their shoot length compared to plants inoculated using soil inoculation method
(Table 6).
DISCUSSION
In this study, the fungal endophytes present in different parts of the plant and in seeds of the rice variety Ld
368 was isolated and identified and subsequently their effect on rice plant growth and their ability to control
brown spot disease was studied.
In order to determine the entire endophytic fungal assemblage of Ld 368 rice variety, isolations were carried
out using roots, stems, leaves, seeds and seedlings of Ld 368. Use of fresh samples is required for successful
isolation, therefore, samples collected from the field were used for isolations within 48 hours. Leaf pieces of
wheat (Larran et al. 2002), stems and leaf pieces of maize (Fisher et al. 1992) have used to isolate endophytic
fungi with different surface sterilizing protocols. In order to isolate a maximum number of colonized endophytes
while eliminating microbes present on the plant surface, the surface sterilization regimes developed in a
previous study (Atugala & Deshappriya 2015) were used for effective surface sterilization of the plant parts
used for the isolations. Endophytic fungi of Ld 368 were isolated placing the surface sterilized tissues on PDA
supplemented with tetracycline. Tetracycline was added as an antibiotic agent to prevent the bacterial growth
until the emergence of fungal colonies from the plant segments.
Thirtyone different fungal genera were able to isolate from roots, stems, leaves, seeds and seedlings of
healthy disease free Ld 368 rice variety. Trichoderma sp.1, Trichoderma sp.2, Chaetomium sp., Fusarium sp.1,
Fusarium sp.2, sterile mycelia (SM 2), SM 7 and SM 8 were the most commonly isolated fungal genera while
sterile mycelia were the most common inhabitors of seeds of Ld 368. Isolation of endophytic fungi from healthy
rice plants has been reported to yield 35 and 31 different genera of endophytic fungi associated with 80 samples
of leaves, stems, roots and seeds of traditional rice varieties Suwandel and Kaluheenati respectively (Atugala &
Deshapriya 2015).
Fungal endophytes thus isolated from various crops have been reported to enhance plant growth as well as to
reduce disease incidence. Endophytic Fusarium moniliforme isolated from roots, nodes and stems of Zea mays
enhanced root growth and histological modifications in leaves and shoots of corn seedlings (Yates et al. 1997)
and Gliocladium catenulatum the endophytic fungus isolated from the Theobroma cacao L. reduced the
incidence of Witches' Broom Disease in cacao seedlings (Rubini et al. 2005).
Fungal endophytes could be used to enhance the growth of rice plants (Wijesooriya & Deshappriya 2016)
due to their plant growth promoting ability (Yates et al. 1997, Maciá‐Vicente et al. 2009). Therefore, in this
study, the effect of more frequently isolated fungal endophytes on Ld 368 rice plant growth was tested under
greenhouse conditions. The endophytes were inoculated into soil and seedlings to determine their effect on plant
growth. Previous studies have shown that successful inoculation of endophytes into seedlings can be done
through placing them on the respective cultures grown on artificial media (Atugala & Deshapriya 2015) or by
inoculating endophytes into the sterile soil (Tarafdar & Gharu 2006). Endophytes inoculated plants using both
soil and seedling inoculation methods showed a significant increase (P ≤ 0.05) in plant height, fresh weight and
dry weight when compared with non-inoculated plants after 4 weeks.
Even though the during second week plant did not show a significant increase in plant height, fresh weight
and dry weight when compared with non-inoculated plants depending on the inoculation method, plants showed
a significant increase after 4 weeks, that could be due to the colonization rate of the endophytic fungi (Maciá‐
Vicente et al. 2009). These results are in accordance with the previous studies carried out in Sri Lanka
(Ponnawila & Deshappriya 2015, Wijesooriya & Deshappriya 2016) and it indicates that the plant growth can be
enhanced significantly by inoculating endophytic fungi.
The sterilized soil was used to grow the rice plants in the greenhouse experiments and thus, the direct or
indirect impact of endophytes on rice plant growth may be correlated with this significant increase in plant
height, fresh weight and dry weight. Trichoderma sp.1 and Chaetomium sp. inoculated plants showed a
significant increase in plant height, fresh weight and dry weight when compared to other treatments.
Therefore, these endophytes may have some mechanisms to enhance the plant growth by inducing the plant
nutrient uptake and producing chemicals which can enhance the plant growth. It has found that Trichoderma sp.
have the ability to enhance plant growth by solubilizing many plant nutrients from their solid phase compounds
and increasing the plant's nutrients uptake efficiency (Altomare et al. 1999). Wheat seedlings grown in sterilized
soil mixed with 15 ml of Chaetomium globosum culture result a significant improvement in plant biomass, root
length, plant phosphorous (P) concentration, seed and straw yield and seed P content, therefore Tarafdar &
Gharu (2006) suggested that C. globosum produces phosphatases and phytases, which mobilize phosphorous (P)
and enhance the plant growth.
In the present study, inoculating the seedlings with the endophytes using seedling method was more
successful than introducing the inoculum through soil, the concentrations of inoculants in the inoculated soils
might have an impact on the results or the biotic factors of a sterile soil may have an influence on the fungal
growth in the soil compared to non-sterile soil (Tefera & Vidal 2009).
Endophytic fungi can be potentially used as effective bio-control agents and they can play an important role
in ecological agriculture (Mejía et al. 2008). Therefore, In this study, fungal endophytes isolated from healthy
plant organs of Ld 368 were tested for their ability to control the growth of the brown spot pathogen, Bipolaris
oryzae both in vitro and in planta.
Dual culture method was used to assess the effect of endophytic fungi on the growth of pathogen mycelium
by measuring the radial growth of the pathogen colonies. Percentage of inhibition was the important parameter
to evaluate the antagonistic activity of the endophyte against the pathogen. Among the screened isolated strains,
some endophytes showed inhibitory activity against the test fungal pathogen through mechanisms such as
competition, compared to the control. Trichoderma sp. 1 and Trichoderma sp. 2 showed the most effective
inhibition 64% and 47% respectively. Competition between Trichoderma spp and Bipolaris oryzae plays an
important role. These fungi showed competition for nutrients and space with Bipolaris oryzae pathogen in the
dual culture test as the evident antagonism. Trichoderma isolates have the ability to grow faster while efficiently
competing for space and nutrients than the pathogenic fungi, inhibiting the growth of the target organisms
(Khalili et al. 2012). Similarly, Abdel-Fattah et al. (2007) used a dual culture method to study the antagonistic
behavior of Trichoderma harzianum against Bipolaris oryzae and Trichoderma harzianum showed 48%
inhibition. Chaetomium sp. showed 29.5% inhibition of the pathogen. Shanthiyaa et al. (2013) found that
Chaetomium sp. produces several fungistatic metabolites such as chaetomin, chaetocin, chaetoglobosin A and B,
chaetoviridin A and B that are active against many soilborne plant pathogens and exhibit higher exo- and endo-
glucanase activity in vitro. Other antifungal mechanisms may also be involved during the inhibition. The
mycoparasitic activity of bio controlling fungal endophytes was observed under the high power of the light
microscope and various structures that used by the antagonist to parasitize the pathogen were observed in this
study.
Loops formation to trap pathogen mycelia, coiling around thick pathogen mycelia to obtain nutrients were
observed and coiling may result in the formation of haustoria which develop into pathogen hyphae and facilitate
the nutrient uptake.
Volatile compounds produced by Trichoderma sp.1 and Trichoderma sp.2 showed 28.5% and 20.9%
inhibition percentages respectively against the pathogen B. oryzae. Khalili et al. (2012) tested the effect of
volatile compounds produced by 45 Tichoderma isolates obtained from paddy fields against the B. oryzae. T.
harzianum strains, T. virens and T. atroviride showed 72%, 66% and 75% inhibitory action against B. oryzae
respectively.
Gel electrophoresis of the PCR-amplified rDNA gene of the fungal genus exhibited a single band for each
genus which varied in size corresponding to the primer pairs used. Different sizes of PCR products were
obtained for Trichoderma sp. 2, Chaetomium sp. and Bipolaris oryzae ITS region amplified using ITS 5 and 4
primers but primers may have not worked for the Trichoderma sp.1, since there was no amplified band in the
gel. Amplification of ITS region was successful for the fungal isolates and brown spot pathogen. The size of the
PCR product of ITS region of Bipolaris oryzae with the use of ITS 5/ITS 4 primers was visualized as 600–680
bp in size by Goh et al. (1998). Green-house experiments were carried out to test the ability of fungal
endophytes to reduce disease incidence.
According to Abdel-Fattah et al. (2007), diseased rice seedlings treated with Trichoderma harzianum
showed 53.5% and 46.9% brown spot disease incidence after 7 and 21 days of T. harzianum application
respectively. In the present study lowest disease incidences 5% and 15% were observed with rice seedlings
treated with Trichoderma sp. 1 Chaetomium sp. using the seedling inoculation method after 10 days, therefore,
these endophytes may be having a potential to prevent pathogen growth in rice plants. So it is possible to
consider that these isolates are endophytic strains as nonpathogenic mutualistic endophytes on rice, but further
experiments are needed to determine their genetic relationships and mutualistic associations with the host plant
and effect against the brown spot.
The ability of isolated endophytes to inhibit brown spot pathogen growth, promote host plant growth and
reduce disease severity was attempted in this study. Further in-vitro and field trials have to be carried out to
obtain effective results and gain knowledge about their bio-control strategies and crop performance. Based on
the results, optimized an inoculum consisting of fungal endophytes i.e. Trichoderma sp. 1, Trichoderma sp. 2
and Chaetomium sp. could be developed to control the brown spot disease as well as to enhance the growth of
Ld 368 rice variety.
CONCLUSION
 Higher diversity of fungal endophytes is present in the most parts (i.e. root, stem, leaf and seed) of the Ld
368 rice variety.
 Seedling application of endophytic fungi is more effective than soil application.
 Endophytic fungi, Trichoderma sp.1, Trichoderma sp.2 and Chaetomium sp.can increase rice plant
growth and reduce the disease incidence of the brown spot disease of rice caused by Bipolaris oryzae.
ACKNOWLEDGEMENTS
The first author wishes to thank Mrs. T. G. I. Sandamali, Assistant Director of Rice Research Station,
Bentota for providing the rice samples and the Department of Botany, University of Kelaniya, Sri Lanka for
facilities provided during the research.
REFERENCES
Abdel-Fattah GM, Shabana YM, Ismail AE & Rashad YM (2007) Trichoderma harzianum: a biocontrol agent
against Bipolaris oryzae. Mycopathologia 164(2): 81–89.
Altomare C, Norvell WA, Björkman T & Harman GE (1999) Solubilization of phosphates and micronutrients
by the plant-growth-promoting and biocontrol fungus Trichoderma harzianum Rifai 1295-22. Applied and
environmental microbiology 65(7): 2926–2933.
Archana B, Kini KR & Prakash HS (2014) Genetic diversity and population structure among isolates of the
brown spot fungus, Bipolaris oryzae, as revealed by inter-simple sequence repeats (ISSR). African Journal
of Biotechnology 13(2): 238–244.
Arshad HM, Hussain N, Ali S, Khan JA, Saleem K & Babar MM (2013) Behavior of Bipolaris oryzae at
different temperatures, culture media, fungicides and rice germplasm for resistance. Pakistan Journal of
Phytopathology 25(1): 84–90.
Atugala DM & Deshappriya N (2015) Effect of endophytic fungi on plant growth and blast disease incidence of
two traditional rice varieties. Journal of the National Science Foundation of Sri Lanka 43(2): 173–187.
Barnett HL & Barry B (1998) Illustrated genera of imperfect fungi. The American Phytopathological Society.
Cenis JL (1992) Rapid extraction of fungal DNA for PCR amplification. Nucleic acids research 20(9): 2380.
Dallagnol LJ, Rodrigues FA, Mielli MV & Ma JF (2014) Rice grain resistance to brown spot and yield are
increased by silicon. Tropical Plant Pathology 39(1): 56–63.
Dhingra OD & Sinclair JB (1995) Basic plant pathology methods. CRC press, pp. 31– 49.
Domsch KH, Gams W & Anderson TH (1993) Key to the genera, Compendium of Soil Fungi. Eching.
Germany: IHW.
Fisher PJ & Petrini O (1992) Fungal saprobes and pathogens as endophytes of rice (Oryza sativa L.). New
Phytologist 120(1): 137–143.
Goh TK, Hyde KD & Lee DK (1998) Generic distinction in the Helminthosporium-complex based on restriction
analysis of the nuclear ribosomal RNA gene. Fungal Diversity 1: 85–107.
Kamal MM & Mia MAT (2009) Diversity and pathogenicity of the rice brown spot pathogen, Bipolaris oryzae
(Breda de Haan) Shoem. in Bangladesh assessed by genetic fingerprint analysis. Bangladesh Journal of
Botany 38(2): 119–125.
Khalili E, Sadravi M, Naeimi S & Khosravi V (2012) Biological control of rice brown spot with native isolates
of three Trichoderma species. Brazilian Journal of Microbiology 43(1): 297–305.
Kumar A, Solanki IS, Akhtar J & Gupta V (2016) Morpho-molecular diversity of Bipolaris oryzae causing
brown spot of paddy. The Indian Journal of Agricultural Sciences 86(5): 615–620.
Kusari S, Hertweck C & Spiteller M (2012) Chemical ecology of endophytic fungi: origins of secondary
metabolites. Chemistry & Biology 19(7): 792–798.
Larran S, Perello A, Simon MR & Moreno V (2002) Isolation and analysis of endophytic microorganisms in
wheat (Triticum aestivum L.) leaves. World Journal of Microbiology and Biotechnology 18(7): 683–686.
Maciá‐Vicente JG, Rosso LC, Ciancio A, Jansson HB & Lopez‐Llorca LV (2009) Colonisation of barley roots
by endophytic Fusarium equiseti and Pochonia chlamydosporia: effects on plant growth and disease. Annals
of Applied Biology 155(3): 391–401.
Mejía LC, Rojas EI, Maynard Z, Van Bael S, Arnold AE, Hebbar P, Samuels GJ, Robbins N & Herre EA (2008)
Endophytic fungi as biocontrol agents of Theobroma cacao pathogens. Biological Control 46(1): 4–14.
Mithrasena YJPK, Silva JN, Adikari AAWP, Weerasingha WMSK & Sumanasingha HPD (2012) Identification
and management of brown leaf spot and grain discolouration diseases of rice (Oryza sativa L.). in Sri Lanka.
Annals of the Sri Lanka department of agriculture 14: 77–86.
Parsa S, García-Lemos AM, Castillo K, Ortiz V, López-Lavalle LAB, Braun J & Vega FE (2016) Fungal
endophytes in germinated seeds of the common bean, Phaseolus vulgaris. Fungal biology 120(5): 783–790.
Ponnawila PVAR & Deshappriya N (2015) Effect of fungal endophyte Arthrographis on growth of rice varieties
Herath Banda and Bg 352. Proceedings of 35th Annual session of The Institute of Biology, pp. 30.
Rahman MA, Begum MF & Alam MF (2009) Screening of Trichoderma isolates as a biological control agent
against Ceratocystis paradoxa causing pineapple disease of sugarcane. Mycobiology 37(4): 277–285.
RRDI-Bathalagoda (2016) Rice Research and Development Institute, Department of Agriculture, Sri Lanka.
Available from: https://www.doa.gov.lk/rrdi/index.php?option=com_sppagebuilder&view=page&id=42&lang=en
(accessed: 05 Jul. 2016).
Rubini MR, Silva-Ribeiro RT, Pomella AW, Maki CS, Araújo WL, Dos Santos DR & Azevedo JL, (2005)
Diversity of endophytic fungal community of cacao (Theobroma cacao L.) and biological control of
Crinipellis perniciosa, causal agent of Witches' Broom Disease. International Journal of Biological
Sciences 1(1): 24.
Schwanck AA, Meneses PR, Farias CR, Funck GR, Maia AH & Del Ponte EM (2015) Bipolaris oryzae seed
borne inoculum and brown spot epidemics in the subtropical lowland rice-growing region of Brazil.
European Journal of Plant Pathology 142(4): 875–885.
Shanthiyaa V, Saravanakumar D, Rajendran L, Karthikeyan G, Prabakar K & Raguchander T (2013) Use of
Chaetomium globosum for biocontrol of potato late blight disease. Crop Protection 52: 33–38.
Sunder S, Singh R & Agarwa R (2014) Brown spot of rice: an overview. Indian Phytopathology 67(3): 201–
215.
Tarafdar JC & Gharu A (2006) Mobilization of organic and poorly soluble phosphates by Chaetomium
globosum. Applied Soil Ecology 32(3): 273–283.
Tefera T & Vidal S (2009) Effect of inoculation method and plant growth medium on endophytic colonization
of sorghum by the entomopathogenic fungus Beauveria bassiana. BioControl 54(5): 663–669.
Tian XL, Cao LX, Tan HM, Zeng QG, Jia YY, Han WQ & Zhou SN (2004) Study on the communities of
endophytic fungi and endophytic actinomycetes from rice and their antipathogenic activities in vitro. World
Journal of Microbiology and Biotechnology 20(3): 303–309.
Vibhuti V, Shahi C, Bargali K & Bargali S (2015) Seed germination and seedling growth parameters of rice
(Oryza sativa) varieties as affected by salt and water stress. The Indian Journal of Agricultural Sciences
85(1): 102–108.
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): 470–480.
Yates IE, Bacon CW & Hinton DM (1997) Effects of endophytic infection by Fusarium moniliforme on corn
growth and cellular morphology. Plant Disease 81(7): 723–728.
You YH, Yoon H, Kang SM, Shin JH, Choo YS, Lee IJ, Lee JM & Kim JG (2012) Fungal diversity and plant
growth promotion of endophytic fungi from six halophytes in Sun cheon Bay. Journal of Microbiology and
Biotechnology 22: 1549–1556.
Short communication
Eleutherine bulbosa (Mill.) Urb. (Iridaceae): A new distributional

record to the flora of Eastern Ghats, India
R. Prameela1*, J. Swamy2 and M. Venkaiah3
1
Department of Botany, M.R. Degree College, Vizianagaram, Andhra Pradesh-535002, India
2
Botanical Survey of India, Deccan Regional Centre, Hyderabad, Telangana-500048, India
3
Department of Botany, Andhra University, Visakhapatnam, Andhra Pradesh-530003, India
*Corresponding Author: prameelachris@yahoo.com [Accepted: 20 November 2018]
[Cite as: Prameela R, Swamy J & Venkaiah M (2018) Eleutherine bulbosa (Mill.) Urb. (Iridaceae): A new
distributional record to the flora of Eastern Ghats, India. Tropical Plant Research 5(3): 303–305]
The family Iridaceae Juss. contains 70 genera and 2000 species having a cosmopolitan distribution, with the
highest diversity in Southern Africa, East Mediterranean, Central and South America (Mabberley 2008). The
genus Eleutherine Herb. is a member of the new world tribe Tigridieae of Iridaceae and comprises low-growing
bulbous plants with pleated lanceolate leaves and small white, evening-blooming flowers (Goldblatta & Snow
1991), and comprises four species. Eleutherine angusta Ravenna native range is Mato Grosso do Sul (Brazil) to
Paraguay of South America. E. bulbosa (Mill.) Urb. is distributed in Mexico, Caribbean, and Central and South
America; it is introduced and cultivated in several parts of Africa and Asia, and now naturalized in Indochina,
Philippines, and in some parts of India. E. citriodora (Ravenna) Ravenna from northern Argentina, and
E. latifolia (Standl. & L.O.Williams) Ravenna is distributed in northern Central America and subtropical South
America. (Goldblatta & Snow 1991, The Plant List 2013).
During a short visit to the Vizianagaram district in Andhra Pradesh, the authors collected an Eleutherine
Herb. species in flower from Eastern Ghats (Mantrajola forest of Andhra Pradesh), which was later identified as
Eleutherine bulbosa (Mill.) Urb., is distributed in Mexico, Caribbean, and Central and South America. It is
introduced and cultivated in several parts of Africa and Asia, and now naturalized in Indochina, Philippines, and
in some parts of India (Pradeep 1995). Scrutiny of Indian literature reveals that the species was first collected
from the Bengal by Prain (1903), and reported as a Cipura paludosa Aubl. Since then Santapau & Henry
(Santapau & Henry 1973), Karthikeyan et al. (1989) have followed Prain (1903) in the nomenclature of this
species. Pradeep (1995) observed this species under cultivation in many gardens of India and reported from
Kerala, and not from the Eastern Ghats of Andhra Pradesh and hence the present collection is reported as a new
distributional record for the Eastern Ghats of Andhra Pradesh. Detailed description, updated citation, habitat,
distribution and colour photographs are provided to facilitate easy identification.
TAXONOMIC TREATMENT
Eleutherine bulbosa (Mill.) Urb., Report. Spec. Nov. Regni Veg, 15: 305.1918; Godblatt & Snow, Ann.
Missouri Bot. Gard. 78(4): 946. 1991; Pradeep, Rheedea 5 (2):181–183. 1995. (Fig. 1)
Bermudiana bulbosa Molina Hist. Chil., ed. angl. i. 113, 293 (1809).
Cipura paludosa Aubl., Hist. Pl. Guiane. 1: 38, t. 13. 1775; Bengal Pl. 2: 1055. 1903; Karthik. et al., Fl. Ind.
Enum. Monocot. 83. 1989, non. Aubl., 1775.
Perennial bulbous herbs, 50–60 cm high. Bulbs 5–7 cm long and 3–4 cm width, ellipsoid, fleshy, red in
colour and acrid. Basal leaves 20–37 × 0.8–1.5 cm, narrowly lanceolate, plicate, entire along the margin,
glabrous, many nerved. Stem terete, comprising one long internode, with a large cauline leaf at the apex,
subtending the inflorescence. Inflorescences of several stalked rhipidia, the peduncles 2.0–5.5 cm long, borne
both in the axil of the cauline leaf and in umbellate fashion on a secondary axis. Spathes 11–15 mm long, green,
glabrous, margins slightly scarious. Flowers white, stellate, 2.0–2.9 cm long, 2.0 cm in diam.; pedicels ca. 10
mm long; bracts membranous, hyaline, as long as pedicel; tepals 6, white, free, in two whorls of three each;
Received: 22 May 2018 Published online: 31 December 2018
Prameela et al. 2018
outer tepals ca. 12 × 5.0 mm, obovate; inner ones narrowly obovate, ca. 10 × 4.0 mm. Stamens 3, adnate to the
outer tepal segments; filaments 2–3 mm long, free, filiform; anthers 3–4 mm long, linear to oblong, orange
yellow. Ovary 4 mm long, obovoid, glabrous, tri-carpellary, tri-locular, 2–4 ovules in each locule; ovules
stalked, arranged in axile placentation; style filiform, yellow, 2 mm long, 3-branched; branches as long as or
slightly longer than the anthers, subulate. Fruit not seen.
Figure 1. Eleutherine bulbosa (Mill.) Urb.: A, Habit; B, Flowering branches; C, Bulb with transverse section; D, Stamen; E,
Pistil.
Flowering & fruiting: April to September.
Habitat: Rarely found in thickets of Eastern Ghats.

Distribution: Eleutherine bulbosa is distributed in Mexico, Caribbean, and Central and South America. It is
introduced and cultivated in several parts of Africa and Asia, and now naturalized in Indochina, Philippines, and
in some parts of India (Pradeep 1995).
Specimen examined: Eastern Ghats, Andhra Pradesh, Vizianagaram Gummalaxmipuram mandal, Mantrajola,
06.04.2018, R. Prameela & J. Swamy 009258 (BSID!).
ACKNOWLEDGEMENTS
Authors are thankful to Director, Botanical Survey of India, Kolkata and Dr. L. Rasingam, Scientist In-
charge, Botanical Survey of India, Deccan Regional Centre, Hyderabad for facilities.
REFERENCES
Mabberley DJ (2008) Mabberley’s Plant-book A portable dictionary of plants - their classification and uses (3rd
edition). Cambridge University Press, Cambridge, U.K., p. 433.
Goldblatta P & Snow N (1991) Systematics and Chromosome Cytology of Eleutherine Herbert (Iridaceae).
Annals of the Missouri Botanical Garden 78(4): 942–949.
The Plant List (2013) Eleutherine Herb. Available from: http://www.theplantlist.org/ version 1.1 (accessed: 16
Apr. 2018).
Pradeep AK (1995) Eleutherine bulbosa (Mill.) Urb. (Iridaceae-Tigridieae): a little understood exotic in India.
Rheedea 5(2): 180–183.
Prain D (1903) Bengal Plants (Apocynaceae- Selaginellaceae). Vol. II, Culcutta, p. 1055.
Santapau H & Henry AN (1973) A Dictionary of flowering plants in India. New Delhi.
Karthikeyan S, Jain SK, Nayar MP & Sanjappa M (1989) Florae Indicae Enumeratio-Monocotyledonae.
Botanical Survey of India, Calcutta, India.
Research article
C3 and C4 plants as potential phytoremediation and bioenergy

crops for stabilization of crude oil and heavy metal co-contaminated
soils-response of antioxidative enzymes
Songita Sonowal1, Majeti Narasimha Vara Prasad2 and Hemen Sarma1*
1
Department of Botany, N. N. Saikia College, Titabar-785630, Assam, India
2
Department of Plant Science, University of Hyderabad, Hyderabad-500046, Telengana, India
*Corresponding Author: hemens02@yahoo.co.in [Accepted: 28 November 2018]
Abstract: Metal accumulation in 15 (C3 and C4) plants growing on crude oil spill laden soil and
the responses of antioxidative enzymes were examined. In this study, the synergistic effect of four
different metals was examined to find out the antioxidative stress responses. Plants were collected
from their natural habitat (crude oil spill laden soil) during the rainy season at the vegetative stage
(before flowering) and analyzed for shoot metal concentrations and activities of catalase (CAT),
superoxide dismutase (SOD), and malondialdehyde (MDA). The shoot metal concentrations (mg
kg-1) of all the individual metals (Mn, Co, Cd, and Zn) were found in different concentration. All
the metal accumulating plants, CAT and SOD activities were found to be high in comparison to
the control plants. The highest SOD activity was found in Cynodon dactylon (47 µg-1 FW) whereas
the lowest was found in Fimbristylis dichotoma (13 µg-1 FW). The SOD activity increased
considerably in all the metal accumulating plants, and the increase ranges 13–47 µg-1
FW. Catalase activity was also found to be high (2–18 µg-1 FW) in all the grass and sedges, of
which the highest was recorded in Echinochloa colonna (18 µg-1 FW) and lowest in Arundo donax
(2 µg-1 FW). The significant decrease in MDA activity (between 1–0.04 nmol g-1 FW) in the leaves
of all metal accumulating plants, suggested metals in soil induced oxidative damage. The
antioxidant responses among the species grown in a contaminated site displayed higher levels of
activity in all the enzymes compared to no-polluted plants. Therefore, it can be assumed that
the heavy metal uptake and bio-productivity (the coordinated manifestation of the efficiency that
operates at various molecular and cellular level of these species) is sustained through antioxidative
defense system in the examined plants.
Keywords: C3 and C4 plants - Metals - Crude oil - Antioxidative stress - Bioenergy.
[Cite as: Sonowal S, Prasad MNV & Sarma H (2018) C3 and C4 plants as potential phytoremediation and
bioenergy crops for stabilization of crude oil and heavy metal co-contaminated soils-response of antioxidative
enzymes. Tropical Plant Research 5(3): 306–314]
INTRODUCTION
The plants have a certain potential cellular mechanism through which they defend too many abiotic stresses.
These include exposures to heavy metals (inorganics) and hydrocarbons (organics). The heavy metal stress
induces free radicals which damage different plant macromolecules inside the plant body (Fu & Huang 2001).
Plant accumulation of metals from contaminated media (soil, air, water) induce the overproduction of reactive
oxygen species (ROS); these ROS (superoxide anion (O-2), hydroxyl radical (•OH), as well as nonradical
molecules like hydrogen peroxide (H2O2), singlet oxygen (1O2),) which is harmful for plant metabolism, growth
and development. The overproduction of ROS is accountable for oxidative damage on plant macromolecules
that includes lipids, proteins and nucleic acids as substantiated by research evidence (Yadav 2010, Pospíšil et al.
2017). Furthermore, the presence of organic compounds in soil is also detrimental for plants growth causing a
damaging effect on their metabolism (Sarma et al. 2017). Thus, the investigated plants (grass and sedges)
exhibit the combined stress tolerance of organics and inorganics since they are adopted in crude oil and heavy
metal co-contaminated soil.
Received: 12 August 2018 Published online: 31 December 2018
Sonowal et al. 2018
These grass and sedges can accumulate metals and hydrocarbons from polluted fields through a cluster of the
process that helps the restoration of such degraded soil (Fig. 1). One of the important criteria for these plants is
that they generate the high amount of biomass that could have a potential of application viz., a) direct
combustion for bioenergy b) enzymatic digestion of the biomass for making a second-generation biofuel (Sarma
et al. 2017). Therefore, the growth and adaptation of such plants in polluted fields not only help the
phytoextraction of metals and degradation of hydrocarbon but their harvested biomass could be a profitable
business for making the lignocellulosic product (Sarma & Lee 2018).
Figure 1. Grasses and sedges accumulate metals and degrades hydrocarbon from soil through a variety of biogeochemical
process.
Table 1. Phytoremediation and energy crops for possible application in filed.
Plant species Family Pollutant remediation aspects
Arundo donax L. Poaceae Phytoremediation of soil contaminated with Cd,
As and Ni (Papazoglou et al. 2005, Mirza et al.
2005)
Axonopus compressus (Sw.) P. Beauv. Poaceae Phytoaccumulator of Cd (Sao et al. 2007)
Cannabis sativa L. Cannabaceae Phytoremediation of soil contaminated with Cd,
Cu, Pb and Zn, Cd, Cr and Ni and other heavy
metals (Kos et al. 2002, Arru et al. 2014)
Cynara cardunculus L. Asteraceae Phytoremediation of soil contaminated with Cd
and as (Ge et al. 2012)
Cynodon dactylon (L.) Pers. Poaceae Phytoextractors of Cr (Sampanpanish et al. 2010)
Cyperus esculentus L. Cyperaceae Phytoaccumulator of Fe (Chandra & Yadav 2011)
Cyperus rotundus L. Cyperaceae Phytoaccumulator of Cd (Kos et al. 2002)
Dactyloctenium aegyptium (L.) Wild. Poaceae Phytoaccumulator of heavy metals (Irshad et al.
2015)
Echinochloa colonum (L.) Link Poaceae Phytoextraction of Cr, Cd, P (Subhashini &
Swamy 2015)
Eucalyptus sp. Myrtaceae Phytoremediation of soil contaminated with As
and other heavy metals and bioaccumulation of
Pb, Zn and Cr (Zhuang et al. 2007, Li et al. 2011)
Fimbristylis dichotoma (L.) Vahl Cyperaceae Phytoextraction of Cu, Ni (Das & Maiti 2007)
Hibiscus cannabinus L. Malvaceae Phytoremediation of soil contaminated with As,
Fe, Pb and Cd (Ho et al. 2008, Meera & Agamuth
2012)
Jatropha curcas L. Euphorbiaceae Phytoremediation of Al, Fe, Cr, Mn, Ar, Zn, Cd
and Pb (Pandey et al. 2012)
Leersia hexandra Swartz. Poaceae Phytoextraction of Cr (Liu et al. 2011)

Linum usitatissimum L. Linaceae Phytoremediation of soil contaminated with Cd,
Ni and other heavy metals (Vrbova et al. 2013)
Miscanthus sp. Poaceae Phytoextraction of As, Sn, Cd, Cr, Cu, Ni, Pb, Zn
and Al (Pidlisnyuk et al. 2014)
Paspalum conjugatum Berg. Poaceae Phytoaccumulator of Pb (Paz-Alberto 2007).
Phalaris arundinacea L. Poaceae Phytoremediation of soil contaminated with
different heavy metals and trace metal (Kacprzak
et al. 2014, Polechonska et al. 2014)
Poa annua L. Poaceae Phytoaccumulator of Zn, Pb, Cd (Barbafieri et al.
2011)
Populus sp. Salicaceae Phytoremediation of Cd, Cr, Cu, Se, Pb and Zn
(Pilon-Smits et al. 1998, Robinson et al. 2000,
Sebastiani et al. 2004, Chen et al. 2015)
Ricinus communis L. Euphorbiaceae Phytoremediation of Cd, Pb, Zn, As (Meloa et al.
2009, Costa et al. 2012, Olivares et al. 2013,
Kiran et al. 2017)
Salix sp. Salicaceae Phytoremediation of Cd and Zn and
Bioaccumulation of Cd, Cr, Cu, Ni, Pb and Zn
(Meers et al. 2007, Miller et al. 2011)
The C3 and C4 (biomass crops) having phytoremediation potentials were listed in the table (Table 1) showing
amazing phytoextraction of wide-ranging metals in their biomass. The C4 plants are more tolerant and better
adapted in contaminated sites, unlike C3 plants; the C4 plants possess a bundle sheath cells (a special type of
cell around the vascular bundle), while C3 plants do not possess these cells. In addition to such anatomical
difference, the cells of C4 plants, produces a special type of enzyme phosphoenolpyruvate carboxykinase
(PEPCK) that plays a key role in the C4 cycle. Therefore, the C4 plants are much more efficient at capturing
carbon dioxide for photosynthesis which helps more biomass production. In addition to that C4 plants are better
for application in phytoremediation and biomass production reported in the current literature. However, it is also
emphasized that some C3 plants have been explored as an ideal candidate for metal remediation and biomass
production (Zhuo et al. 2017).
Plant cells are able to respond to elevated levels of reactive oxygen species (ROS) by activating their
antioxidative defense systems (Noctor et al. 2017). To protect the lipids, proteins and nucleic acids (cells
macromolecules) and counteract oxidative damage, plant activated endogenous antioxidants defense system.
Therefore, the expression of antioxidant enzymes (in C3 and C4 plants) suggested as more reliable and sensitive
indicators of exposure to the abiotic stress including metals (Yadav 2010). The antioxidative system includes
metabolites GSH, ascorbate and the enzymes ascorbate peroxidase (APX), glutathione reductase
(GR), monode hydro ascorbate reductase (MDHAR), superoxide dismutases (SOD), catalases (CAT) and
peroxidases (PODs) are involved in detoxification of O2−, and H2O2 respectively; thereby, preventing the
formation of OH- radicals (Noctor et al. 1998).
The oxidative stress induced by chromium was tolerated by many plants through the hyperactivity of their
antioxidant defense system (Yadav 2010). Related experiments confirmed that chromium was inhibited by
superoxide generation and mitochondrial electron transport and can seriously interrupt normal metabolism
through oxidative damage to cellular components in higher plants (Liu et al. 2011), but data remain scanty in
case of herbs and shrubs. The Lead is recalcitrant in the environment and can cause damage to biota (Zeng et al.
2017). Some plants accumulate Pb, although it is not necessary for growth and development. It was established
that Pb accumulation reduced the total chlorophyll and carotenoids content and cause oxidative damage in
plants. However, unlike Pb and Cr, the synergistic effect of Mn, Co, Cd, and Zn in C3 and C4 plants that cause
oxidative damage by increasing lipid peroxidation still remain scanty. Therefore, in this study, the synergistic
effect of four different metals was examined to find out the antioxidative stress responses in 15 biomass crop
(C3 and C4) that can be used for stabilization of crude oil spill laden soils.

In this study, 15 species that were grown in the metals and hydrocarbon contaminated habitat were collected
from crude oil drilling sites of Borhola, Assam (Table 2). The sample collection site is a hydrocarbon-rich agro-
ecosystem, located in the north-eastern region of India with an area of 5 km2, which is surrounded by tea garden
(Sharma et al. 2018). The species were identified using flora of India. The total of 45 individuals (n=3) were
taken to the laboratory, washed with distilled water and stored in the refrigerator before analysis. Leaves (0.2 g)
Sonowal et al. 2018
were taken from the -20°C refrigerator, ground in a mortar and pestle. The crude extracts of each part were
homogenized in 3 mL 50 mM potassium phosphate buffer (pH 7.8). The homogenate was centrifuged at 8000
rpm for 20 min at 4°C. The supernatant (i.e., the enzyme extract) was used for determinations of enzyme
activities viz. superoxide dismutase (SOD), catalase (CAT) and malondialdehyde (MDA).
Table 2. The grass and sedges collected from crude oil and heavy metal co-contaminated soils for analysis.
Species Family
Arundo donax L. Poaceae
Axonopus compressus (Sw.) P. Beauv. Poaceae
Cynodon dactylon (L.) Pers. Poaceae
Cyperus esculentus L. Cyperaceae
Cyperus rotundus L. Cyperaceae
Dactyloctenium aegyptium (L.) Willd. Poaceae
Digitaria sanguinalis (L.) Scop. Poaceae
Echinochloa colona (L.) Link Poaceae
Fimbristylis dichotoma (L.) Vahl Cyperaceae
Imperata cylindrica (L.) Raeusch. Poaceae
Kyllinga brevifolia Rottb. Cyperaceae
Leersia hexandra Sw. Poaceae
Paspalum conjugatum P.J.Bergius Poaceae
Poa annua L. Poaceae
Saccharum spontaneum L. Poaceae
A SOD assay was performed according to the method of Giannopolitis & Ries (1977) with slight
modifications. Briefly, 3 mL reaction mixtures contain 0.3 mL of each: 750 μmol L−1 nitro blue tetrazolium, 20
μmol L−1 riboflavin, 130 mmol L−1 methionine, and 100 μmol L−1 EDTA Na, 1.5 mL of 50 mmol L−1 phosphate
buffer (pH 7.8), 0.25 mL of deionized water, and 0.05 mL of enzyme extract. The test tubes were placed under
light for 20 min, and the absorbance of the reaction mixture was measured at 560 nm. A reaction solution placed
in the dark was used as the control. The enzyme activity (one unit) was well defined such as the amount of the
enzyme that resulted in inhibition (50%) of the rate of nitro blue tetrazolium (NBT) reduction.
A CAT assay was modified from the method of Brennan & Frenkel (1977). An enzyme extract (0.1 mL) was
added to mixture solution of 1 mL 0.3% H2O2 and 1.9 mL 50 mmol L−1 phosphate buffer (pH 7.0) to initiate the
reaction. The activity of CAT was measured by determining the rate of change of H2O2 absorbance at 240
nm. One unit of enzyme activity was defined as the amount of the enzyme that resulted in 1% absorbance
reduction. Lipid peroxides were measured by the thiobarbituric acid test for MDA according to Heath & Packer
(1968). Supernatant (1.5 mL) was added to 2.5 mL of 20% trifluoroacetic acid containing 0.5% thiobarbituric
acid. The mixture was kept at 100°C in a water bath for 20 min, then cooled quickly in an ice bath followed by
centrifugation at 10,000 rpm for 10 min. The absorbance of the resulting solutions was determined using UV-
Vis at 450, 532 and 600 nm.
In this study SOD, CAT and MDA were also analyzed in the same species organically grown in the
experimental garden of the institute. There are numerous studies on the impact of individual environmental
factors on plants but few have tried to explore their combined effect on antioxidant defense system in plants.
The present study seeks to address this particular aspect that has hitherto remained unexplored, by looking at a
combination of hydrocarbons and heavy metals impact on plant antioxidant defense system.
The oven dried samples (0.03 g) were then put inside a heating flask and heated in a mixture of 4mL
HNO3 (70%, Merck India, Mumbai) and 1 mL HClO4 (30%) and digested at 140°C for 2 hours. The digested
plant samples were treated with 30 mL water and 2.5 g H3BO3 and filtered through Whatman no.1 filter paper
and analyzed for metal concentration using atomic absorption spectrophotometer.
To assess analytical precision and quality control, the Trace CERT certified standards (Sigma Aldrich)
reference material, reagent blanks and duplicates of every third sample were applied. The limit of detection
(LOD) and limit of quantification (LOQ) of HMs were carried out by recovery experiments. The results showed
a recovery of metals in the range of 90.6–98.5 %. The data obtained were then analyzed using statistical
software.

In this study, four metals viz. Mn, Co, Cd, and Zn were analyzed in plants to correlate it with their
antioxidative responses. The results illustrated that individual metal and species exhibited dissimilar trends of
accumulation (Table 3). The test species continue to survive in extreme polymetallic environment. The
considerable accumulation of Co was recorded in C. esculentus (16.46), D. sanguinalis (15.34), E. colona

(13.36), and I. cylindrical (12.36). The limited uptake of Cd was seen only two species within the range of 2–8
mg kg-1 which are C. esculentus (8.67) and K. brevifolia (2.60). Again, in this study, the exceptional ability to
uptake moderately high concurrent ternary metals (Mn, Co and Cd) in C. esculentus were recorded and for (Mn,
Co, and Zn) uptake potential was shown by in E. colona. The Mn (mg kg-1) accumulations were recorded within
the range of (0.09–26.33) in all the species. The species which exhibited maximum accumulation of Mn were
presented in chronological orders from highest to lowest: L. hexandra (26.33), E. colona (21.25), C. dactylon
(14.5), C. esculentus (11.40), and S. Spontaneum (7.52). Similarly, highest accumulation of zinc found in D.
sanguinalis (893.45) while lest uptake was recorded in E. colona (26.15). Metal contents in aerial parts of
different plant samples showed significant differences (P<0.01).
Table 3. Accumulation of heavy metals (mg kg-1) in 15 bioenergy crops collected from crude oil exploration site, Assam.
Plants Family Common name Mn Co Cd Zn
Arundo donax L. [C3] Poaceae Giant reed 0.46 0.43 2.6 162.15
Axonopus compressus (Sw.) P. Beauv. [ C4] Poaceae American carpet grass 0.51 0.57 0.6 340.25
Cynodon dactylon (L) Pers. [ C4] Poaceae Bermuda grass 14.56 0.63 0.6 306.95
Cyperus esculentus Linn [ C4] Cyperaceae Nut sedge 16.41 16.5 8.7 784.15
Cyperus rotundus L. [ C4] Cyperaceae Nutgrass 0.21 0.53 0.4 127.75
Dactyloctenium aegyptium (L.) Wild. [ C4] Poaceae Egyptian crowfoot grass 0.46 0.52 0.3 148.65
Digitaria sanguinalis (L.) Scop [ C4] Poaceae Hairy crabgrass 0.1 15.3 0.6 893.45
Echinochloa colona (L.) Link [ C4] Poaceae Jungle rice 21.25 13.4 0.3 26.15
Fimbristylis dichotoma (L.) Vahl [ C4] Cyperaceae Tall fringe rush 0.07 0.64 1.6 167.75
Imperata cylindrica (L.) Raeusch. [ C4] Poaceae Japanese bloodgrass 0.4 12.4 0.4 235.75
Kyllinga brevifolia Rottb. [ C4] Cyperaceae Green kyllinga 0.46 35 2.6 82.95
Leersia hexandra Swartz. [C3] Poaceae Clubhead cutgrass 26.33 0.63 0.6 152.15
Paspalum conjugatum P.J. Bergius. [ C4] Poaceae Buffalo grass 0.41 3.37 1.6 115.55
Poa annua L. [ C3] Poaceae Annual bluegrass 0.34 12.4 0.6 212.15
Saccharum spontaneum L. [ C4] Poaceae Wild sugarcane 7.52 0.63 1.3 93.55
Note: Value are mean of ± standard deviation of (n=3).
The antioxidant stress responses among the species grown in a contaminated site displayed higher levels of
activity in all the enzymes compared to plants grown in an experimental garden (normal) (Figs. 2–4). This is
because of abiotic stress produced by hydrocarbon and metals. Plants under stress from individual
environmental factors may produce antioxidative enzymes SOD and CAT that directly or indirectly detoxify
reactive oxygen species (ROS). Plants respond to changes in the environment by changing their antioxidative
systems. Environmental factors such as salinity, drought and elevated levels of metals/metalloids may regulate
antioxidant defense systems in plants; this is the reason that we recorded higher leaf SOD and CAT activities
among the examined species (Panda et al. 2017). The SOD was highest in the leaves of C. dactylon (47µg-1 FW)
and lowest in F. dichotoma (13 µg-1 FW). All the energy crops populating the study site showed increments of
SOD within the range of 13–47 µg-1 FW. Elevated SOD activities are beneficial for the protection of plants
against oxidative stresses (Schwanz et al. 1996). Similarly, CAT activities were (µg-1 FW) within the range of
2–18 of which is even higher in comparison to control species. This is routinely used as an index of lipid
peroxidation caused by heavy metals (HMs) toxicity. Previous studies have shown the respective consequences
of salinity and HMs stress on the MDA level (Neto et al. 2006, Zhang et al. 2007) but, as stated earlier, little
research has been conducted on the combined effects of hydrocarbons (HCs) and multiple metals. Therefore, in
this study, there was a significant decrease in MDA activities (between 1–0.04 nmol g-1 FW) in the leaves of all
the energy crops in the contaminated site; suggesting significant alleviation of heavy metals induced oxidative
damage in them.
The application of high biomass yielding plants for the remediation of metals and hydrocarbon contaminated
soil is a groundbreaking low-cost strategy to derive added profits (i.e. biomass for bioenergy) from such
remediation approaches. There are a wide variety of grasses and sedges that have been explored for concomitant
phyto-management of polymetallic sites, besides obtaining beneficial phytoproduct such as second-generation
energy, biodiesel, bioethanol, wood, fiber, biochar, charcoal, and bioplastics during the remediation process.
Recently, the production of bioenergy from biomass is getting international attention as it assumed as a clean
and a multipurpose alternate source of energy. Nevertheless, there are several challenges to be achieved before
the large volume commercial production of bioenergy from a contaminated site (Tripathi et al. 2016).
Sonowal et al. 2018
Figure 2. Malondialdehyde (MDA) activity of selected grasses and sedges in polluted and control sites.
Figure 3. Catalase (CAT) activity of selected grasses and sedges in polluted and control sites.
Figure 4. Super oxide dismutase (SOD) activity of selected grasses and sedges in polluted and control sites.
CONCLUSIONS
The antioxidative stress responses of 15 C3 and C4 plants grown in the heavy environmental metal-rich crude
oil spill laden soils has been examined. All the plants examined were found to accumulate metals (Mn, Co, Cd,
and Zn) in their biomass. The antioxidant responses among the species grown in a contaminated site displayed
higher levels of activity in all the enzymes compared to nopolluted plants. Therefore, it can be assumed that
the heavy metal uptake and bioproductivity is sustained through antioxidative defense system in the examined
plants. The C4 plants were better adapted in contaminated sites, whereas C3 plants were less adapted as we
recorded only 3 species of C3 plants in the examined site. This is because C4 plants are much more efficient at
capturing carbon dioxide and biomass production and more resilient by chance, not the choice. However, the
study established that the synergistic effect of four different metals (Mn, Co, Cd, and Zn) and how they created
a negative effect of antioxidative stress responses for some biomass crops, although they are tolerant to heavy
metals.
ACKNOWLEDGMENTS
This research was supported by the Department of Biotechnology, Ministry of Science and
Technology, Govt. of India, Grants No BT/489/NE/TBP/2013. The authors would like to acknowledge the
Rebecca Palacio for technical support
REFERENCES
Arru L, Rognon S, Baroncini M, Bonatti P M & Perata P (2004) Copper localization in Cannabis sativa L.
grown in a copper-rich solution. Euphytica, 140: 33–38.
Barbafieri M, Dadea C, Tassi E, Bretzel F & FanfaniI (2011) Uptake of heavy metals by native species growing
in a mining area in Sardinia, Italy: discovering native flora for phytoremediation. International Journal of
Phytoremediation 13: 985–999.
Brennan T & Frenkel C (1977) Involvement of hydrogen peroxide in the regulation of senescence in pear. Plant
Physiology 59: 411–416.
Chandra R & Yadav S (2011) Phytoremediation of Cd, Cr, Cu, Mn, Fe, Ni, Pb and Zn from aqueous solution
using Phragmitescummunis, Typhaangustifolia and Cyperusesculentus. International Journal of
phytoremediation 13(6): 580–591.
Chen L, Hu X, Yang W, Xu Z, Zhang D & Gao S (2015) The effects of arbuscular mycorrhizal fungi on sex-
specific responses to Pb pollution in Populus cathayana. Ecotoxicology and Environmental Safety 113: 460–
468.
Costa ETS, Guilherme LRG, Melo EEC, Ribeiro BT, Inácio ESB, Severiano EC, Faquin V & Hale BA (2012)
Assessing thetolerance of castor bean to Cd and Pb for phytoremediation purposes. Biological Trace
Element Resesarch 145: 93–100.
Das M & Maiti S K (2007) Metal accumulation in A. baccifera growing naturally on abandoned copper tailings
pond. Environmental Monitoring and Assessment 127: 119–125.
Fu J & Huang B (2001) Involvement of antioxidants and lipid peroxidation in the adaptation of two cool-season
grasses to localized drought stress. Environmental Experimental Botany 45: 105–114.
Ge Z-M, Kellomaki S, Zhou X, Peltola H, Wang K-Y & Martikainen PJ (2012) Seasonal physiological
responses and biomass growth in a bioenergy crop (Phalaris arundinacea L.) under elevated temperature
and CO, subjected to different water regimes in boreal conditions. Biological Energy Research 5(3): 637–
642.
Giannopolitis CN & Ries SK (1977) Superoxide dismutase I. Occurrence in higher plants. Plant Physiology 59:
309–314.
Heath RL & Packer L (1968). Kinetics and stoichiometry of fatty acid peroxidation. Archives of Biochemistry
and Biophysics 125: 189–198
Ho WM, Ang LH & Lee DK (2008) Assessment of Pb uptake, translocation and immobilization in kenaf
(Hibiscus cannabinus L.) for phytoremediation of sand tailings. Journal of Environmental Sciences 20(11):
1341–1347.
Irshad M, Ahmad S, Pervez A & Inoue M. (2015) Phytoaccumulation of heavy metals in natural plants thriving
on wastewater effluent at hattar industrial estate, Pakistan. International Journal of Phytoremediation 17 (1–
6): 154–158.
Kacprzak MJ, Rosikon K, Fijalkowski K & Grobelak A (2014) The effect of Trichoderma on heavy
metalmobility and uptake by Miscanthus giganteus, Salix sp., Phalaris arundinacea, and Panicum virgatum.
Applied and Environmental Soil Science 10: 240–249.
Kiran BR & Prasad MNV (2017) Ricinus communis L. (Castor bean), a potential multi-purpose environmental
crop for improved and integrated phytoremediation. The EuroBiotech Journal 1(2): 101–116.
Kos B, Grcman H & Lestan D (2003) Phytoextraction of lead, zinc and cadmium from soil by selected plants.
Sonowal et al. 2018
Plant, Soil and Environment 49(12): 548–553.

Li C, Wang, Q-H, Xiao B & Li Y-F (2011) Phytoremediation potential of switchgrass (Panicum virgate.) for
Cr-polluted soil. In: Proceedings of international symposium on water resource and environmental
protection (ISWREP). Xi'an, Shaanxi Province, China, pp. 1731–1734.
Liu J, Duan C & Zhang X (2011) Potential of Leersia hexandraswartz for phytoextraction of Cr from soil.
Journal of Hazardous Materials 188(1–3): 85–91.
Meera M & Agamuth P (2012) Phytoextraction of As and Fe using Hibiscus cannabinus L. from soil polluted
with landfill leachate. International Journal of Phytoremediation 14(2):186–199.
Meers E, Vandecasteele B, Ruttens A, Vangronsveld J & TackF (2007) Potential of five willow species (Salix
spp.) for phytoextraction of heavy metals. Environmental and Experimental Botany 60: 57–68.
Meloa EEC, Costa ETS, Guilhermea LRC, Faquina V & Nascimento CWA (2009) Accumulation of arsenic and
nutrients by castorbean plants grown on an As- enriched nutrient solution. Journal of Hazardous Materials
168: 479–483.
Miller RS, Khan Z & Doty SL (2011) Comparison of trichloroethylene toxicity, removal, and degradation by
varieties of Populus and Salix for improved phytoremediation applications. Journal of Bioremediation &
Biodegradation 27: 231–239.
Mirza N, Mahmood Q, Pervez A, Ahmad R, Farooq R, Shah MM & Azim MR (2010) Phytoremediation
potential of Arundodonax in arsenic contaminated synthetic wastewater. Bioresource Technology 101:
5815–5819.
Neto ADA, Prisco JT, Ene as-Filho J, Abreu CEB & Gomes-Filho E (2006) Effect of salt stress on antioxidative
enzymes and lipid peroxidation in leaves and roots of salt-tolerant and salt-sensitive maize genotypes.
Brazilian Journal of Plant Physiology 56: 87–94.
Noctor G, Arisi AC, Jouanin L & Foyer CH (1998) Manipulation of glutathione and amino acid biosynthesis in
the chloroplast. Plant Physiology 118: 471–482.
Noctor G, Reichheld Jean-Philippe & Foyer Christine H (2017) ROS-related redox regulation and signaling in
plants. Seminars in Cell & Developmental Biology 80: 3–12.
Olivares AR, Carrillo-González R, González-Chávez Ma CA & Hernández RMS (2013) Potential of castor bean
(Ricinus communis L.) for phytoremediation of mine tailings and oil production. Journal of Environmental
Management 114: 316–323.
Panda A, Jaykumar R, Kumari A & Parida AK (2017) Efficient regulation of arsenic translocation to shoot
tissue and modulation of phytochelatin levels and antioxidative defense system confers salinity and arsenic
tolerance in the Halophyte Suaeda maritime. Environmental and Experimental Botany 143: 149–171.
Pandey VC, Singh K, Singh JS, Kumar A, Singh B & Singh RP (2012) Jatropha curcas: a potential biofuel
plant for sustainable environmental development. Renewable & Sustainable Energy Reviews 16: 2870–2883.
Papazoglou EG, Karantounias GA, Vemmos SN & Bouranis DL (2005) Photosynthesis and growth responses of
giantreed (Arundodonax L.) to the heavy metals Cd and Ni. Environment International 31(2): 243–249.
Paz-Alberto AM (2007) Phytoextraction of lead-contaminated soil using vetiver grass (Vetiveria zizanioides L.),
cogon grass (Imperata cylindrica L.) and carabao grass (Paspalum conjugatum L.). Environmental Science
and Pollution Research 14(7): 498–504.
Pidlisnyuk V, Stefanovska T, Lewi EE, Erickson LE & Davis LC (2014) Miscanthus as a productive biofuel
crop for phytoremediation. Critical Reviews in Plant Sciences 33(1): 1–19.
Pilon-Smits EAH, Dsouza Lytle CM, Shang C, Lugo T & Terry N (1998) Selenium volatilization and
assimilation by hybrid poplar (Populus tremula-alba). Journal of Experimental Botany 49: 1889–1892.
Polechonska L & Klink A (2014) Trace metal bioindication and phytoremediation potentialities of Phalaris
arundinacea L. (reed canary grass). Journal of Geochemical Exploration 146: 27–33.
Pospíšil P & Yamamoto Y (2017) Damage to photosystem II by lipid peroxidation products Biochimica et
Biophysica Acta - General Subjects 186: 457–466.
Robinson BH, Mills TM, Petit D, Fung LE, Green SR & Clothier BE (2000) Natural and induced cadmium-
accumulation in poplar and willow: implications for phytoremediation. Plant and Soil 227: 301–306.
Sampanpanish P, Tippayasak K & Chairat-Utai P (2010) Chromium accumulation by phytoremediation with
monocot weed plant species and a hydroponic sand culture system. Journal of Environmental Research and
Development 4(3): 654–666.
Sao V, Nakbanpote W & Thiravetyan P (2007) Cadmium accumulation by Axonopus compressus (Sw.) P.
Beauv and Cyperus rotundas Linn growing in cadmium solution and cadmium-zinc contaminated soil.
Songklanakarin Journal of Science and Technology 29(3): 881–892.

Sarma H & Lee WY (2018) Bacteria enhanced lignocellulosic activated carbon for biofiltration of bisphenols in
water. Environmental Science and Pollution Research 25(18): 17227–17239.
Sarma H, Islam NF & Prasad MNV (2017) Plant-microbial-association in petroleum and gas exploration sites in
Assam, Northeast-Indian State significance for remediation. Environmental Science and Pollution Research
24: 8744–8758.
Schwanz P, Haberle KH & Polle A (1996) Interactive effects of elevated CO2, ozone and drought stress on the
activities of antioxidative enzymes in needles of Norway spruce trees [Picea abies, (L.) Karsten] growth
with luxurious N-supply. Journal of Plant Physiology 148: 351–355.
Sebastiani L, Scebba F & Tognetti R (2004) Heavy metal accumulation and growth responses in poplar clones
Eridano (Populusdeltoides maximowiczii) and I- 214 (P. euramericana) exposed to industrial waste.
Environmental and Experimental Botany 52: 79–88.
Sharma D, Sarma H, Hazarika S, Islam NF & Prasad MNV (2018) Agro-Ecosystem Diversity in Petroleum and
Natural Gas Explored Sites in Assam State, North-Eastern India: Socio-Economic Perspectives. In:
Lichtfouse E (eds) Sustainable Agriculture Reviews 27. Springer.
Shen Z, Dong XM, Gao ZF, Chao Q & Wang BC (2017) Phylogenic and phosphorylation regulation difference
of phosphoenolpyruvate carboxykinase of C3 and C4 plants. Journal of Plant Physiology 213: 16–22.
Subhashini V & Swamy AVVS (2015) Phytoremediation of lead, cadmium and chromium contaminated soils
using selected weed plants. Acta Biologica Indica 4(2): 205–212.
Tripathi V, Edrisi SA & Abhilash PC (2016) Towards the coupling of phytoremediation with bioenergy
production. Renewable and Sustainable Energy Reviews 57: 1386–1389.
Vrbova M, Kotrba P, Horacek J, Smykal P, Svabova L, Vetrovcova M, Smyka-lova I & Griga M (2013)
Enhanced accumulation of cadmiumin Linum usitatissimum L. plants due to overproduction of metallothione
in α-domain as a fusion to β- glucuronidase protein. Plant Cell, Tissue and Organ Culture 112(3): 321–330.
Yadav SK (2010) Heavy metals toxicity in plants: an overview on the role of glutathione and phytochelatins in
heavy metal stress tolerance of plants. South African Journal of Botany 76: 167–179.
Zeng G, Wan J, Huang D, Hu L, Huang C, Cheng M, Xue W, Gong X, Wang R & Jiang D (2017) Precipitation,
adsorption and rhizosphere effect: The mechanisms for Phosphate-induced Pb immobilization in soils-A
review. Journal of Hazardous Materials 339: 354–367.
Zhang FQ, Wang YS, Lou ZP & Dong JD (2007) Effect of heavy metal stress on antioxidative enzymes and
lipid peroxidation in leaves and roots of two mangrove plant seedlings (Kandelia candel and Bruguiera
gymnorrhiza). Chemosphere 67: 44–50.
Zhuang X, Chen J, Shim H & Bai Z (2007) New advances in plant growth-promoting rhizobacteria for
bioremediation. Environment International 33: 406–413.
Research article
Habitat characterization and plant community classification of

Surajpur Reserve Forest: a potential bird sanctuary in
National Capital Region, India
Nasim Ahmad Ansari
Wildlife Institute of India, Post Box # 18, Chandrabani, Dehradun-248001, Uttarakhand, India
*Corresponding Author: dr.ansari.nasim@gmail.com [Accepted: 10 December 2018]
Abstract: Surajpur Reserve Forest is a prominent forested wetland site in the National Capital
Region, India, known for its rich floral and faunal biodiversity. The present study was conducted
to assess the habitat characteristics, vegetation composition and plant community classification
from March 2010 to February 2013. Stratified random sampling techniques applied for sampling
of vegetation in circular and quadrat plots and TWINSPAN analysis was used in PC-ORD
Software for classification of plant communities. A total of 257 vascular plants belonging to 214
genera and 65 families were recorded, including a comprehensive herbarium of 267 plant
specimens have been recorded from 3 major habitats (woodland, grassland and wetland) and 9
microhabitats. A maximum of 157 plants in woodland, 73 plants in grassland and 65 plants in
wetland habitat were recorded. Flowering and fruiting plants recorded maximum in monsoon
followed by summers and winters. Various life-forms include 144 herbs, 39 trees, 31 grasses, 20
climbers, 12 shrubs and 11 sedges. A total of 51% plant species as abundant and 14% plant species
as rare have been recorded. Woodland habitat recorded maximum density and diversity of herbs
and shrubs. Five dominant plant communities have been identified in terrestrial and 3 in wetland
habitat. The results indicate that Surajpur wetland supports a mosaic of habitat which enables the
conservation and protection of threatened flora and fauna in an urban environment. The
conservation implications are discussed in light of the results hitherto unreported.
Keywords: Flora - Greater Noida - Habitat - Surajpur wetland - TWINSPAN - Vegetation
structure.
[Cite as: Ansari NA (2018) Habitat characterization and plant community classification of Surajpur Reserve
Forest: a potential bird sanctuary in National Capital Region, India. Tropical Plant Research 5(3): 315–330]
INTRODUCTION
Forests are renewable resources and have contributed substantially to the economic development of the
country by providing goods and services to the mankind. Forests also have a major role in enhancing the quality
of the environment and balancing the ecosystem. However, this forest wealth is dwindling due to overgrazing,
overexploitation, encroachments, unsustainable practices, forest fire and indiscriminate sitting of development
projects in the forest areas. Tropical forests constitute diverse ecosystems supporting rich biodiversity. These
forests are disappearing at alarming rates owing to deforestation for extraction of timber and other forests
products (Devi & Yadava 2006). Vegetation is an integral part of the landscape and is required for management
planning (Singh & Rawat 1999). The classification and mapping of vegetation are fundamental tools for
obtaining knowledge about habitat characteristics (Mueller-Dombois 1984) and its relationship with various
flora and fauna existing in that forest habitat. Forest structure and composition have been used to assess the
habitat characteristics (Gillespie & Walter 2001), which provides the suitable place for breeding and resting of
various flora and fauna and in conserving the biodiversity. Forest structure, such as diversity, the density of
plants and species composition, has been significantly correlated with faunal distribution patterns. These
patterns of distribution have been shaped by historical and ecological factors that play different roles at both
temporal and spatial scales (Vuilleumier & Simberloff 1980, Hutto et al. 1986, Cherril & McClean 1997,
Gaston & Fuller 2009).
Received: 22 February 2018 Published online: 31 December 2018
Ansari 2018
Surajpur is one such example of Reserve Forest in outskirts of National Capital Region, representing the
mosaic of habitats, which supports a diverse range of flora and fauna. It represents an excellent example of
urban biodiversity conservation and reported 186 species of birds in different habitats (Ansari & Nawab 2015)
hence recognized as a potential bird sanctuary in the National Capital Region, India. The inventorisation studies
on flora of National Capital Region has done by several authors (Maheshwari 1963, Srivastava 2004, Vardhana
2007, Dash & Ahmedullah 2012, Chaudhary et al. 2012, Manral et al. 2013, Mishra et al. 2014, Ansari 2015,
Ansari et al. 2016), but there is no systematic study has been made on habitat characterization in the National
Capital Region or in Surajpur Reserve Forest, although it supports luxuriant growth of angiospermic flora and
plays an important role in the plant species conservation. Therefore, the present study has been made to assess
the habitat characterization, vegetation composition and plant community classification of Surajpur Reserve
Forest, National Capital Region, India.

Study area
Surajpur wetland (28°31.425′ N, 77°29.714′ E) is located in Dadri Tehsil of the district Gautam Budh Nagar,
Uttar Pradesh and it comes under the purview of Delhi- National Capital Region (NCR) India (Fig. 1). The NCR
comprises an urban conglomerate including Delhi, Faridabad, Gurugram, Ghaziabad and Gautam Budh Nagar
(Noida and Greater Noida). The Greater Noida City is just 3 kilometers from Surajpur wetland is one of the
best-planned cities and is the largest industrial townships of Asia (Joshi 2009). The study area falls in the Upper
Gangetic Plain Biogeographic Zone (Rodgers et al. 2002) at an elevation of 184.7 m above mean sea level. The
area is a reserve forest and spreads over 308 hectare including a water body of 108 hectares. The wetland is
mainly rain-fed and other sources for water recharge are Hawaliya drain which is attached to Hindon River and
Tilapta irrigation canal. The climate is tropical monsoon type and maximum rainfall occurs from July to October
ranging from 400–500 mm and normally the rain depends on north-west monsoon. The minimum and maximum
temperatures ranged between 6.86°C and 41.69°C, and highest temperature was observed during June and the
lowest during January.
Figure 1. Map of the Surajpur Reserve Forest.
Habitat characterization
The present study was carried for the period of three years from March 2010 to February 2013 to explore the
floral diversity of Surajpur forest. Intensive floristic surveys were made on monthly basis to collect the plant
specimens and to prepare a comprehensive herbarium following Jain & Rao (1977) and Singh & Subramaniam
(2008). The modern system of classification followed (APG III 2009, Haston et al. 2009, IPNI 2013, The Plant
List 2013). The frequency of occurrence of plant species was assigned into 4 abundance categories, A=
Abundant (>50), F= Frequent (30–50), O= Occasional (10–30) and R= Rare (<10).
On the basis of dominant floristic composition and soil type, the study area was categorized into 3 major
habitats: woodland, grassland and wetland. These major habitats further divided into several microhabitats (Fig.
2). The woodland habitat characterized by trees, grassland habitat by grasses and wetland habitat by various
aquatic species. Wetland area has been further characterized by marshland and deep water area. The habitat
condition varies according to different seasons. The floristic composition also changes accordingly, from
summer to monsoon, from monsoon to winter and from winter to summer and monsoon respectively. The
various habitats have been quantified based on surveys of flora, water quality, soil quality, air quality and
availability of water from season to season in the wetland area. The measurements were done on monthly basis
by standard techniques to calculate the changes in habitat conditions and subsequent maps have been prepared.
Figure 2. Micro-habitat map of Surajpur Reserve Forest.

Vegetation sampling was carried out in broadly classified habitats i.e. woodland, grassland and wetland by
stratified random sampling. Terrestrial vegetation (woodland and grassland habitat) sampling was conducted in
January - February 2012, while aquatic vegetation (wetland and marshland habitat) sampling was conducted in
January - February 2013. In the woodland habitat, trees were sampled by placing circular plots of 10 m radius
with an interval of 50 m. Shrubs were sampled within 3m radius while four 0.5 m × 0.5 m quadrats were laid for
herbs and grasses in each plot. In all 52 plots were laid (4 plots in Terminalia arjuna (Roxb. ex DC.) Wight &
Arn, 8 plots in Syzygium cumini (L.) Skeels and 20 plots each in Phoenix sylvestris (L.) Roxb. and Prosopis
juliflora (Sw.) DC. habitat, respectively). In the grassland habitat, grasses/sedges and herbs were sampled by
placing 20 quadrats of 0.5 m × 0.5 m size and four quadrats of 0.5 m × 0.5 m were laid in each 10 m circular
plot with an interval of 50 m distance.
Vegetation sampling for aquatic macrophytes was carried out by stratifying wetland area into 10 sampling
blocks on the basis of water availability. Hydrophytic plants were further divided into 5 subtypes on the basis of
their habitat and adaptation, Emergent (rooted erect herbs stand above the level of water), Rooted- floating
(aquatic herbs floating or creeping), Submerged (rooted aquatic plants totally submerged in water), Free-floating
(plants which are not rooted, their roots are suspended in water) and Amphibious (plants living partially in water
Ansari 2018
and partially above the surface of water) (Vijayan 1983). The abundance of these plant species was recorded by
laying random sampling plots in each stratum. Quadrats of 0.5 m × 0.5 m were laid in each block with an
interval of 5 m between two quadrats. In each sampling plot name of the plant species, number of individuals,
water depth and percentage vegetation cover were recorded.
The data collected were compiled in MS-Excel software. Replicate data were pooled separately for
individual sampling sites to compute estimates of population parameters. Overall mean density was estimated
for respective habitats following Mueller-Dombois & Ellenberg (1974). Shannon Diversity Index and
Margalef’s Richness Index were used by DOS based Programme SPECDIVER.EXE programme (Ludwig &
Reynolds 1988) for calculations of diversity and richness.
Plant Community Classification
PC-ORD (version 4.34) was used for classification of plant communities. Data were analysed by
multivariate technique as Two Way Indicator Species Analysis (TWINSPAN), a computer based programme,
commonly used for ecological studies (Hill 1979, Waite 2000). A thorough description about this method can be
found in Dillon & Goldstein (1984). Plant community classification was done habitat-wise separately for all the
three major habitats- woodland, grassland and wetland, respectively. The vegetation classification analysis was
carried out using the polythetic divisive clustering technique in TWINSPAN (Siddiqui et al. 2010). The plant
communities were classified and named into individual groups. This involved formation of groups of definite
floristic composition and physiognomy. In the present analysis, uniform habitat condition was not taken into
consideration. Five pseudo species cut levels (0, 2, 5, 10, and 20) were given according to the frequency of
cover scale for all the habitats. The maximum level of divisions was 6, minimum group size for the division was
5 with the maximum number of indicators per division equal to 5. The total number of species and pseudo
species calculated from TWINSPAN analysis. Relative Euclidean distance was measured and Nearest-
neighbour linkage method (also called single linkage method) was adopted for cluster analysis (Hamid 2009).
The homogenous groups were identified from the cluster made by TWINSPAN analysis and it has been done for
plant community classification separately for terrestrial and aquatic habitats.
RESULTS
Habitat quantification
Figure 3. Mapping of various habitats of Surajpur Reserve Forest.

Different vegetation structures of the study area classified into three major habitats on the basis of dominant
floristic composition and soil type as, woodland, grassland and wetland habitats (Fig. 3). These major habitats
further divided into microhabitats, woodland includes Phoenix sylvestris, Terminalia arjuna, Syzygium cumini
and Prosopis juliflora, grassland are dominant with Sachharum sp., Vetiveria zizanioides (L.) Nash and
Desmostachya bipinnata (L.) Stapf species, whereas wetland includes clear water with submerged aquatic
vegetation of Ceratophyllum demersum L., Hydrilla verticillata (L.f.) Royle, Vallisneria spiralis L., emergent
aquatic vegetation of Eichhornia crassipes (Mart.) Solms, Alternanthera philoxeroides (Mart.) Griseb., Ipomoea
sp., Typha angustata Bory & Chaub., and marshland with Phoenix sylvestris, Terminalia arjuna, Syzygium
cumini vegetation. The mosaics of habitat formed the 9 microhabitat types representing the woodland habitat as
dominant habitat type and Nature trail (tracks) represents the minimum area of microhabitat (Table 1). The
details of each microhabitat can be described as:
Table 1. Different habitat types in Surajpur reserve forest.
S.N. Habitat types Area (ha) Percentage %)
1. Woodland (Prosopis juliflora) 87.56 26.38
2. Woodland (Phoenix sylvestris) 56.89 17.14
3. Shrub-land (Prosospis juliflora) 33.40 10.06
4. Grassland 27.91 08.41
5. Water (Wetland area) 32.42 09.77
6. Vegetation within water body (Marshy area) 31.94 09.62
7. Open (Bare ground) 33.69 10.15
8. Track (Nature trail) 04.19 01.26
Total 308.00 92.79
9. Agricultural land (outside Forest) 23.93 07.21
(i) Woodland (Prosopis juliflora): It is the most dominant microhabitat type (87.56 ha) in the study area, with
26.38% of the total area and reflects dark green colour with smooth texture. This microhabitat is found only in
the Gulistanpur Reserve Forest area. It is distributed widely on the southern side around the water body. In the
extreme western side of the reserve area, this habitat class can be seen as a dense patch.
(ii) Woodland (Phoenix sylvestris): It is the second most dominant microhabitat type (56.89 ha), with 17.14%
of the total area in Khodna Khurd Reserve Forest area. This habitat class is represented by cyan colour in the
map with smooth texture. It is distributed densely towards northern side and sparsely towards southern side
intermixed with Prosopis juliflora woodland. The northern side of this microhabitat touches the wetland area.
(iii) Shrub-land (Prosopis juliflora): Shrub-land (other than trees) are the third most abundant microhabitat
type (33.40 ha) in the study area representing 10.06% of the total area. It is represented with lightest green
colour with course texture in map intermixed with Prosopis juliflora woodland. It is distributed mainly in the
Gulistanpur Reserve Forest area followed by Khodna Khurd Reserve Forest area.
(iv) Grassland: This area (27.91 ha) is intermixed with woodland habitat and represents by 8.41% of the total
area. This microhabitat type is shown by light green colour with course texture in the map, mostly towards the
south western side of the area. This microhabitat type can be seen in Gulistanpur Reserve Forest and Khodna
Khurd Reserve Forest blocks respectively.
(v) Water (wetland area): This is one of the major habitat types in the study area and account for about
19.39% (64.36 ha) of the total forest cover, including marshy area i.e. vegetation within water body. The
wetland area is demarcated by a white coloured nature track with blue and green colour, as smooth texture in
the map. This forest cover type falls in both Gulistanpur Reserve Forest and Khodna Khurd Reserve Forest
blocks.
(vi) Marshy Area (Vegetation within water body): Small woodland patches of Terminalia arjuna along the
south-eastern side and Syzygium cumini along the north-eastern side are located with respect to the wetland
serves as marshland. The blue colour also represents the Yamaha canal which is passing through the
Gulistanpur Reserve Forest along the Prosopis juliflora woodland microhabitat type.
(vii) Open (bare ground): The areas devoid of vegetation are depicted as open or bare ground. This
microhabitat class is distributed towards the periphery and the area is represented by 10.15% (33.69 ha) of the
total area. It is shown by light pink colour with course texture in the map.
(viii) Nature Trail (Track): There are 3 nature trails made passing through all the major habitats. The nature
trail also serves as embankment to the wetland area. The complete nature trail includes the 1.26% of the total
area (4.19 ha) and is depicted by white colour in map.
(ix) Agriculture land: This area lies outside, adjacent to the forest and is 7.21% of the total area (23.93 ha).
The agriculture land belongs to Khodna Village Panchayat towards northern side of the forest and is depicted
by yellow colour in map.
Ansari 2018
Vegetation structure and composition

During the study period, a total of 257 vascular plant taxa pertaining to 214 genera belonging to 29 orders
and 65 families were recorded. During the study period, a comprehensive herbarium of 267 plant specimens
including 229 plant species was prepared and arranged family-wise alphabetically from Acanthaceae to
Zygophyllaceae, indexed in 09 herbarium files and was deposited at Department of Forestry and Environmental
Sciences, Kumaun University, Nainital, Uttarakhand, India for future use. The analysis of flora shows a
comparatively higher representation of herbaceous species (144) followed by 39 trees, 31 grasses, 20 climbers,
12 shrubs and 11 species of sedges during the study period (Table 2). The occurrence status of plant species
recorded as Abundant by 51% (n= 130 plant species), followed by Frequent 19% (n= 50 plant species),
Occasional as 16% (n= 42 plant species) and Rare as 14% (n= 35 plant species) of the total recorded plant
species.
Table 2. Plant species composition in different taxonomic groups and in various life forms.
Angiosperms
Habit Pteridophytes Total
Dicotyledons Monocotyledons
Trees 38 1 0 39
Shrubs 12 0 0 12
Climbers 20 0 0 20
Herbs 129 12 3 144
Grasses and Sedges 0 42 0 42
Total 199 55 3 257
Habitat association of each plant species was recorded. Woodland habitat recorded maximum of 157 plant
species followed by 73 plant species in grassland habitat and 65 plant species in wetland habitat. The aquatic
plant species found in wetland habitat was further classified into 5 subtypes on the basis of their nature of
occurrence as amphibious (26 species), emergent (25 species), rooted-floating (6 species), submerged (4
species) and free-floating (4 species). The flowering and fruiting period of the plant species recorded for all
seasons. Monsoon recorded maximum plant species (177 plant species), followed by summer (87 plant species)
and winters (74 plant species) in the flowering and fruiting period.
The overall mean tree density in woodland habitat was determined as 76.52 individual ha-1, however
maximum tree density was recorded for Phoenix sylvestris (129.95 individual ha-1) while Terminalia arjuna
recorded least tree density (36.73 individual ha-1) (Table 3). Overall mean shrub density in woodland habitat
was determined as 219.69 individual ha-1, wherein maximum density (769.23 individual ha-1) was determined
for Ziziphus jujuba Mill. and least density (20.40 individual ha-1) was determined for Abutilon indicum (L.)
Sweet (Table 3).
Table 3. Trees and Shrub density (individual ha-1) in woodland habitat.
S.N. Tree Species Individual (ha-1)
1. Phoenix Sylvestris (L.) Roxb. 129.77
2. Prosopis juliflora (Sw.) DC. 72.84
3. Syzygium cumini (L.) Skeels 66.72
4. Terminalia arjuna (Roxb. ex DC.) Wight & Arn. 36.73
Mean Density ha-1 76.52
S.N. Shrub/Seedling/Sapling species Individual (ha-1)
1. Abutilon indicum (L.) Sweet 20.40
2. Acacia nilotica (L.) Delile 34.01
3. Phyllanthus reticulatus Poir. 54.41
4. Phoenix sylvestris (L.) Roxb. 163.24
5. Prosopis juliflora (Sw.) DC. 476.10
6. Syzygium cumini (L.) Skeels 20.40
7. Ziziphus jujuba Mill. 769.23
Mean Density ha-1 219.69
Overall mean herb density in woodland habitat was determined as 0.50 individual m-2, wherein maximum
herb density (2.00 individual m-2) was determined for Alternanthera sp. and least density (0.02 individual m-2)
was determined for Achyranthes aspera L. (Table 4). Overall mean grass/sedge density in woodland habitat was
determined as 5.31 individual m-2, wherein maximum grass/sedge density (30.71 individual m-2) was determined
for Desmostachya bipinnata (L.) Stapf and least density (0.04 individual m-2) was determined for Echinochloa
crusgalli (L.) P. Beauv. and Eleocharis species (Table 5).
Table 4. Herb density (individual m-2) in woodland habitat.

S.N. Herbs species Individual (m-2)
1. Achyranthes aspera L. 0.02
2. Ageratum conyzoides (L.) L. 0.27
3. Alternanthera sp. 2.00
4. Cannabis sativa L. 0.15
5. Chenopodium sp. 0.69
6. Ipomoea sp. 0.04
7. Launaea nudicaulis (L.) Hook.f. 0.17
8. Marsilea quadrifolia L. 0.35
9. Dicliptera paniculata (Forssk.) I.Darbysh. 0.04
10. Polygonum sp. 0.54
11. Ranunculus sceleratus L. 1.96
12. Rumex dentatus L. 0.17
13. Sida sp. 0.50
14. Solanum americanum Mill. 0.04
15. Tridax procumbens (L.) L. 0.08
16. Urena lobata L. 0.12
17. Veronica anagallis-aquatica L. 1.29
Mean Density m-2 0.50
Table 5. Grass/Sedge density (individual m-2) in woodland and grassland habitat.

S.N. Grass/Sedge species Individual (m-2)
Woodland Habitat
1. Cynodon dactylon (L.) Pers. 12.92
2. Cyperus sp. 2.88
3. Desmostachya bipinnata (L.) Stapf 30.71
4. Echinochloa crus-galli (L.) P.Beauv. 0.04
5. Eleocharis sp. 0.04
6. Imperata cylindrica (L.) Raeusch. 1.00
7. Paspalum distichum L. 2.12
8. Saccharum ravennae (L.) L. 0.48
9. Sporobolus diandrus (Retz.) P.Beauv. 1.10
10. Chrysopogon zizanioides (L.) Roberty 1.81
Grassland Habitat
1. Chrysopogon zizanioides (L.) Roberty 3.00
3. Desmostachya bipinnata (L.) Stapf 19.25
4. Saccharum ravennae (L.) L. 0.38
5. Sporobolus diandrus (Retz.) P.Beauv. 0.50
The overall mean grass/sedge density in grassland habitat was determined as 5.90 individual m-2, wherein
maximum density (19.27 individual m-2) was determined for Desmostachya bipinnata and least density (0.38
individual m-2) was determined for Saccharum ravennae (L.) L. Only one species of herb i.e. Chenopodium
album L. was recorded in the grassland habitat, the density of which was determined as 1.6 individual m-2
(Table 5).
The overall mean herbs, grasses and sedge density in wetland habitat was determined as 9.92 individual m-2,
wherein maximum density (38.20 individual m-2) was determined for Alternanthera philoxeroides and least
density (0.08 individual m-2) was determined for Ceratophyllum demersum and Ranunculus sceleratus L. (Table
6).
Among three habitats, woodland habitat represents the maximum Margalef’s species richness (4.72) and
Shannon-Weiner’s diversity index (0.98) and grassland habitat represents minimum Margalef’s species richness
(0.80) and Shannon-Weiner’s diversity index (0.40). Whereas wetland habitat represents median values for both
the indices (Table 7).
Plant Community Classification
TWINSPAN analysis was done for plant community classification. The analysis was made separately for
terrestrial and aquatic habitats, woodland-grassland and wetland habitat, respectively.
Ansari 2018
Table 6. Aquatic herbs, grasses and Sedge density (individual m-2) in wetland habitat.
S.N. Aquatic herbs and grasses Individual (m-2)
1. Alternanthera philoxeroides (Mart.) Griseb. 38.20
2. Azolla caroliniana Willd. 12.75
3. Ceratophyllum demersum L. 0.08
5. Eichhornia crassipes (Mart.) Solms 14.88
6. Hydrilla verticillata (L.f.) Royle 0.28
7. Ipomoea aquatica Forssk. 0.12
8. Marsilea quadrifolia L. 3.16
9. Paspalum distichum L. 35.32
10. Ranunculus sceleratus L. 0.08
11. Spirodela polyrrhiza (L.) Schleid. 21.28
12. Typha domingensis Pers. 0.88
13. Utricularia stellaris L.f. 1.76
Table 7. Diversity Indices for different habitats in the Surajpur wetland.

Diversity Indices
Habitat No. of Species
Margalef’s Richness (S) Shannon Diversity (H´)
Woodland 40 4.72 0.98
Grassland 6 0.80 0.40
Wetland 13 1.50 0.75
Groups
Figure 4. Cluster dendrogram by TWINSPAN analysis in woodland habitat.
In the terrestrial habitats, a total of 52 sampling plots were laid- 4 plots in Terminalia arjuna, 8 plots in
Syzygium cumini and 20 plots each in Phoenix sylvestris and Prosopis juliflora habitats respectively. A total of
34 plant species comprising trees, shrubs, climbers, herbs, grasses and sedges recorded in all the 52 sampling
plots were pooled together for TWINSPAN analysis. The total number of species and pseudo species is 115.
The application of TWINSPAN technique made it possible to divide the set of 52 sampling plots inside the
woodland habitat into 14 clusters/groups at level 6 of the hierarchical classification (Fig. 4). The first
TWINSPAN dichotomy of Hierarchical classification separated all the 52 plots into two clusters, that in the left
direction exists 41 plots with Desmostachya bipinnata as indicator species where as in right direction exists 11
plots with Cyperus sp. as indicator species. The second division, 41 plots have divided into two clusters that in
left direction exists 12 plots with Prosopis juliflora as indicator species and right direction exists 29 plots with
Desmostachya bipinnata as indicator species. The third division, 11 plots have divided into two clusters that in
left direction exists 3 plots with Terminalia arjuna as indicator species and right direction exists 8 plots with
Cyperus sp. and Paspalum distichum L. as indicator species. The fourth division, 12 plots have divided into two
clusters of 6 left and 6 right directions with Prosopis juliflora as indicator species. The fifth division, 29 plots
have divided into two clusters that in left direction 8 plots with Cynodon dactylon (L.) Pers. and Prosopis
juliflora as indicator species and right direction exists 21 plots with Desmostachya bipinnata, Cynodon dactylon
and Phoenix sylvestris as indicator species. The sixth division, 8 plots have divided into two clusters that in left
direction two plots with Paspalum distichum as indicator species and right direction exists 6 plots with
Ranunculus scleratus and Sizigium cumini as indicator species (Table 8). Thus by applying TWINSPAN
analysis technique, five homogenous groups have been identified representing five plant communities in
woodland habitat (Table 9).
Table 8. TWINSPAN classification of plant species in woodland habitat.
Main groups I II III IV V
Plot numbers
Species code
Groups code
In the aquatic area, wetland habitat, a total of 100 quadrats were laid in 10 blocks (10 quadrats in each
block). Out of the 100 quadrats, only 67 quadrats recorded plant species. Therefore, only 67 quadrats are
considered for TWINSPAN plant community classification. A total of 11 plant species (aquatic herb species)
recorded in all the 67 sampling quadrats were pooled together for TWINSPAN analysis. The total number of
species and pseudo species is 41. The application of TWINSPAN technique made it possible to divide the set of
67 sampling quadrats inside the wetland habitat into 8 clusters/groups at level 6 of the hierarchical classification
(Fig. 5). The first TWINSPAN dichotomy of Hierarchical classification separated all the 67 plots into two
clusters, that in the left direction exists 65 quadrats with Alternanthera philoxeroides as indicator species
Ansari 2018
whereas in right direction exists only 2 quadrats with Hydrila verticillata as indicator species. The second
division, 65 quadrats have divided into two clusters that in left direction exists 17 quadrats with Mersilea
quadrifolia L. as indicator species and right direction exists 48 quadrats with Eichornea crassipes and
Alternanthera philoxeroides as indicator species. The third division, 17 quadrats have divided into two clusters,
which in left direction two quadrats with Typha domingensis Pers. as indicator species and in right directions
exists 15 quadrats with Marsilea quadrifolia as indicator species. The fourth division, 48 quadrats have divided
into two clusters of that is in left direction 28 quadrats with Alternanthera philoxeroides as indicator species
whereas in the right direction exists 20 quadrats with Spirodela polyrrhiza (L.) Schleid. and Eichornea crassipes
as indicator species (Table 10). Thus by applying TWINSPAN analysis technique, three homogenous groups
have been identified representing three plant communities in wetland habitat (Table 11).
Table 9. Plant communities identified by TWINSPAN analysis in woodland habitat.
S.N. Dominant plant Associated plant species Plots number in which
community species reported
1. Prosopis Desmostachya bipinnata, Cynodon dactylon, 48, 46, 49, 50, 51 and
community Chenopodium sp., Dicliptera paniculata, Sida sp. 47
2. Prosopis-Sida Sporobolus diander, Desmostachya bipinnata, Urena 33, 39, 41, 34, 35 and
community lobata, Dicliptera paniculata, Achyranthes aspera, 36
Ageratum conizoides and Cynodon dactylon
3. Desmostachya– Prosopis juliflora, Phoenix sylvestris, Terminalia 01, 52, 40, 42, 45, 37,
Cynodon arjuna, Chrysopogon zizanioides, Abutilon indicum, 38 and 44
community Ziziphus sp., Ageratum conizoides, Sida sp., Urena
lobata, Sporobolus dianadrus, Chenopodium sp., and
Cyperus sp.
4. Desmostachya– Chrysopogon zizanioides, Sachharum sp., Alternanthera 27, 28, 29, 43, 3, 16, 17,
Cynodon-Phoenix philoxeroides, Prosopis juliflora, Acacia sp., Eleocharis 18, 19, 20, 21, 23, 24,
community sp., Paspalum distichum and Cyperus sp. 25, 26, 31, 14, 15, 22,
30 and 32
5. Cyperus-Paspalum- Terminalia arjuna, Alternanthera philoxeroides, 02, 03, 04, 10, 12, 05,
Syzygium Paspalum distichum, Ranunculus scleratus, Prosopis 08, 09, 06, 07 and 11
community juliflora, Phoenix sylvestris, Cynodon dactylon,
Cannabis sativa, Phyllanthus reticulatus, Chenopodium
sp., Marsilea quadrifolia, Veronica anagallis-aquatica,
Polygonum sp., Launea procumbens, Sida sp., Solanum
nigrum, Echinochloa crus-galii, Tridex procumbens,
Rumex dentatus and Ipomoea sp.
Groups
Figure 5. Cluster dendrogram by TWINSPAN analysis in wetland habitat.
DISCUSSION
Vegetation play an important role for the existence of various biodiversity and wildlife especially avian
fauna in an area as it provides the suitable habitat for survival. Surajpur wetland is also one such habitat, of
which vegetation structure forms the integral part of wildlife habitat in the area and is pre-requisite for the better
ecological understanding and management of the area. The different varieties of vegetation provide favorable
habitat conditions to survive the avian fauna in the study area.
Table 10. TWINSPAN classification of plant species in wetland habitat.
Surajpur wetland revealed a total of 257 vascular plants that represents about 47% of the total NCR flora
(Maheshwari 1963) and 10% of the total Uttar Pradesh flora (Srivastav 2004) respectively. High diversity of
vascular plants in the region is mainly attributed to climatic, topographic and edaphic factors. Similar floristic
studies were also conducted in Indian region, Manral et al. (2013) reported 192 plant species in Okhla Bird
Sanctuary in Noida, NCR, Chaudhary et al. (2012) reported 95 species only from Poaceae and Cyperaceae
family in Noida, NCR, Mishra & Narain (2010) reported 129 plant species in Bakhira wetland Uttar Pradesh,
Adhikari & Babu (2008) reported 178 plant species in Baanganaga wetland, Uttarakhand.
The outcome of habitat mapping includes eight microhabitat types with the maximum area of Prosopis
juliflora woodland (26.38% of the total area) and an additional land cover type, i.e. agricultural land (7.21%)
outside the protected area serves as buffer of the reserve forest area. Prosopis juliflora is a fast-growing,
nitrogen-fixing and tolerant to arid conditions, saline soils (Mwangi & Swallow 2005) and has a deep to very
deep, well-meshed root system. It can grow in adverse climatic conditions also. It is also not palatable to
livestock and does not require any proper maintenance to sustain. Large-scale plantation work can be
undertaken on such habitats without any fencing.
Out of three major habitats (woodland, grassland and wetland), woodland recorded the maximum number of
plant species (157 plant species). Ecologically it is very good for wildlife, as it serves as a refuge for threatened
faunal diversity. It is particularly important for conserving declining woodland birds that are found in the region,
including the Greater Spotted Eagle (Clanga clanga Pallas, 1811), Steppe Eagle (Aquila nipalensis Hodgson,
1833), Orange-headed Thrush (Geokichla citrine Latham, 1790), Black-rumped Flamback (Dinopium
benghalense L., 1758), Brown-headed Barbet (Megalaima zeylanica Gmelin, 1788), Eurasian Golden Oriole
(Oriolus kundoo Sykes, 1832), Rufous Treepie (Dendrocitta vagabunda Latham, 1790) and Red Collared-dove
(Streptopelia tranquebarica Hermann, 1804). These are resident birds and prefer woodland habitat for nesting
and roosting in Surajpur wetland.
The species richness of different life forms, such as trees, shrubs and herbs, is one of the major
considerations in recognizing the importance of an area for conservation (Negi et al. 2008). The various life
forms are the constituents of the various habitats, that attracts many wildlife species specially birds and insects.
Margalef’s species richness index and Shannon-Weiner’s diversity index recorded maximum for woodland
habitat and least for grassland habitat due to larger area occupied by woodland habitat (43.52% of the total area)
and less area occupied by grassland habitat (8.41% of the total area). Different life forms such as trees, shrubs
and herbs have different diversity patterns (Grubb 1977). Out of total 257 plant species, maximum number herbs
species (144 plants) recorded, which has clearly indicates that the marshy land habitat supporting rich herb
diversity. The difference in species diversity between communities generally results from variation in site
Ansari 2018
quality (Denslow 1980). Pant & Samant (2007) reported that high richness may be due to diverse habitats and
suitable edaphic and climatic factors supporting growth and survival of the species. As Surajpur wetland also
showing high species richness due to the mosaic of habitats and suitable climatic factors that enhances the
growth and survival of species. The monsoon season, recorded maximum plants in flowering stage (177 plants),
because of higher soil moisture content (Bajpai et al. 2017), which results high humic acid and organic content
as well as low temperature in the soil. On the other hand, the minimum value in summer season indicates higher
heterogeneity in climatic conditions which results poor plant growth (Patel & Pandya 2014).
Table 11. Plant communities identified by TWINSPAN analysis in wetland habitat.
Plots no. in which
S.N. Plant communities Associated plant species
species reported
1. Marsilea community Typha aungustata, Eichornea crassipes, 43, 57, 46, 47, 48, 49,
Utricularia stellaris, Ipomoea sp. and 50, 54, 55, 56, 60, 61,
Spirodela polyrriza 62, 63, 64, 65 and 32
2. Alternanthera Eichornea crassipes, Utricularia stellaris, 32, 24, 27, 28, 38, 41,
community Typha domingensis, Marsilea quadrifolia, 51, 66, 01, 03, 04, 05,
Spiroldela polyrriza, Azolla pinnata and 06, 07, 09, 10, 11, 12,
Ceratophyllum demersum 13, 31, 34, 37, 39, 40,
42, 44, 45 and 59
3. Eichornea-Spirodela Azolla pinnata, Alternanthera philoxeroides, 02, 08, 14, 15, 16, 17,
community Hydrila verticillata, Utricularia stellaris and 18, 19, 20, 21, 22, 25,
Ranunculus scleratus 26, 33, 35, 63, 52, 53,
58, 67, 29 and 30
In woodland habitat, Terminalia arjuna recorded maximum tree density while Prosopis juliflora recorded
least density because Terminalia arjuna was densely planted only at one place that is on the edge of water body,
whereas Prosopis juliflora was grown naturally and sparsely in the entire southern range of the study area.
Among shrub species in woodland habitat, Ziziphus jujuba recorded maximum density and Abutilon indicum
was determined least density, because Ziziphus jujuba is a fast growing shrub species and can flourish even in
very extreme temperatures and thrives under rather dry conditions (Orwa et al. 2009). Among herb species in
the woodland habitat, Alternanthera sp. recorded maximum density and Achyranthes aspera recorded least
density because Alternanthera sp. is a moisture loving species and ground cover was wet during sampling that
enhanced the growth of this species in comparison to Achyranthes aspera, as it is a herb species which grows
mostly in dry areas.
Among grass species, Desmostachya bipinnata is dominating in both the habitats i.e. woodland and
grassland, as this grass species is a drought and salt-tolerant C4 grass of desert or semi-desert conditions with a
deep, strong rhizome, making it an excellent sand-binder (Pandey et al. 2013). Though the study area falls in the
upper Gangetic plain, it shows similarities with semi-arid region, because it is very close to Delhi, which is a
semi-arid region. Grassland habitat is very important in terms of providing shelter for threatened faunal
biodiversity especially grassland birds, including Bristled Grassbird (Chaetornis striata Jerdon, 1841), Black
Francolin (Francolinus francolinus L., 1766), Crested Lark (Galerida cristata L., 1758), Booted Warbler (Iduna
caligata M.H.C. Lichtenstein, 1823), Ashy Prinia (Prinia socialis Sykes 1832) and Zitting Cisticola (Cisticola
juncidis Rafinesque, 1810).
Invasive plants are widely recognised as one of the most important threats to native plant biodiversity (Kolar
& Lodge 2001). Surajpur wetland is a marshland type habitat supported mostly amphibious and emergent
category of aquatic herbs. Excessive growth of herbaceous weed such as Alternanthera philoxeroides and
Eichhornea crassipes are issues of concern in wetland habitat. This weed becomes a growing menace in water
bodies in India (Varshney et al. 2008). These both the plants are very harmful invasive, exotic and obnoxious
weeds reported across the world by many workers (Bassett 2008, Ruiz et. al. 2008, Mandal & Mondal 2011,
Bassett et al. 2012, Chen et al. 2013, Chatterjee & Dewanji 2014).
Plant communities are defined as the collection of plant species growing together in a particular location that
show a definite association with each other (Mueller-Dombois & Ellenberg 1974). The species in a community
grow together in a particular environment because they have a similar requirement for existence in terms of
environmental factors such as light, temperature, water drainage and soil nutrients (Billings 1974, Mueller-
Dombois & Ellenberg 1974, MacAlister 1997).
To know the details of various plant species enumerating in the various habitats, TWINSPAN analysis was
performed and dominant plant communities were identified habitat wise separately. In TWINSPAN analysis,
five plant communities were identified in terrestrial habitat, Prosopis community, Prosopis-Sida community,
Desmostachya–Cynodon community, Desmostachya–Cynodon-Phoenix community and Cyperus-Paspalum-
Syzygium community, and three plant communities were identified in aquatic habitat, Marsilea community,
Alternanthera community and Eichornea-Spirodela community. Thus, with the help of TWINSPAN analysis,
we can come to know what are the various plant communities existing in the various habitats. Subsequently, the
analysis helps in planning for protections and conservation measures of prevailing wildlife faunal diversity.
The diverse floral composition and the better management practices have made the site a safe haven for
particularly waterbirds in the region. The excessive growth of Eichhornia crassipes and Alternanthera
philoxeroides are issues of concern and appropriate measures have been implemented to check their growth. As
the hydrological system is the major factor to control the plant composition of Surajpur wetland, the
management of natural flooding is required to ensure the sustenance of mosaic of habitats.
CONCLUSION
The present study concludes that the Surajpur Reserve Forest represents mosaic of habitats supports rich
avifauna and other biodiversity. The record of 13 species of mammals (Ansari 2017a), 186 species of avifauna
(Ansari & Nawab 2015, Ansari 2017b), 19 species of herpetofauna (Ansari 2018a), 15 species of fishes (Ansari
2018b), 53 species of butterflies (Ansari et al. 2015) and 36 species of odonates (Ansari 2017c) from Surajpur
wetland ecosystem corroborates the fact. Surajpur area is very important to biodiversity conservation as it
provides an opportunity to conserve and preserve the native flora, fauna and biodiversity amidst a densely
populated urban area without hindering the development of social and economical structures. There is a need to
monitor these habitats for long-term protection and conservation of various groups of flora and fauna in the area.
The present findings can be used as a baseline for future studies and a comparison with previous works suggests
that steps should be taken to curtail the growth of invasive species and plantation of native species should be
encouraged. Appropriate measures need to be taken to check the growth of invasive species. The diverse floral
composition and the better management practices have made the site a safe haven for particularly waterbirds in
the region. As the hydrological system is the major factor to control the plant composition, the maintenance of
natural flooding is required for sustenance of mosaic of habitats at Surajpur. The urban and industrial
development across the Greater Noida city which is resulting in habitat destruction is a matter of great concern.
This small piece of marshy land with stagnant water has very rich biodiversity creating a small biodiversity
hotspot. This area should therefore be conserved and kept pollution free across the city limits as they support a
good congregation of aquatic/semi aquatic insects.
ACKNOWLEDGEMENTS
I express my gratitude to the Greater Noida Industrial Development Authority and WWF-India for financial
support to this study. I would like to thank Uttar Pradesh Forest Department for granting the permission to carry
out this study. I am obliged to Mr. R. Singh, Dr. S. Worah, Dr. P. Gautam, Dr. A. Nawab and Dr. A. Pant
(WWF-India) for their constant encouragement and support. I would also like to thank Dr. Athar A. Khan
(Aligarh Muslim University) for plant taxonomic help, Dr. F. Khudsar (Yamuna Biodiversity Park) for
designing the study and Dr. Jeet Ram (Kumaun University) for their overall guidance. The help rendered by our
colleagues at Wildlife Institute of India (Dehradun) is also appreciated.
REFERENCES
Adhikari BS & Babu MM (2008) Floral diversity of Baanganga Wetland, Uttarakhand, India. Check List 4:
279–290.
Ansari NA (2015) A study on bird communities and its relationship with habitat structure in Surajpur Wetland,
Uttar Pradesh, India, (Ph.D. Thesis). Kumaun University, Nainital, Uttarakhand, India.
Ansari NA (2017a) Status of mammals with special reference to population estimation of Nilgai Boselaphus
tragocamelus and suggest mitigation measures to prevent crop damage in and around Surajpur reserve
forest, Uttar Pradesh, India. Journal of Entomology and Zoology Studies 5: 1085–1091.
Ansari NA (2017b) Monitoring of Water bird population with an account of Heronry at Surajpur Lake, an Urban
Wetland in National Capital Region, India. Indian Journal of Animal Sciences 87(12): 1504–1512.
Ansari NA (2017c) Diversity of Odonate Fauna in Surajpur Lake: An Urban Wetland of Upper Gangetic Plain,
Northern India. International Journal of Ecology and Environmental Sciences 43: 73–79.
Ansari NA (2018a) Enumeration of herpetofaunal assemblage of Surajpur Wetland, National Capital Region
(India). Amphibian & Reptile Conservation 12(2): 90–97.
Ansari 2018
Ansari NA (2018b) Assessment of scope for fish biodiversity conservation in relation to environmental
variables at Surajpur Lake, an urban wetland of the Upper Gangetic Plain, Northern India. Indian Journal of
Fisheries 65(3): 16–24.
Ansari NA, Khan A & Ram J (2016) Vascular Plants of Surajpur Wetland, National Capital Region India.
Indian Journal of Plant Sciences 5: 54–69.
Ansari NA, Ram J & Nawab A (2015) Structure and Composition of Butterfly (Lepidoptera: Rhopalocera)
fauna in Surajpur wetland, National Capital Region, India. Asian Journal of Conservation Biology 4: 43–53.
Ansari NA & Nawab A (2015) Avifauna of Surajpur Wetland. Journal of Threatened Taxa 7: 7776–7785.
APG III (2009) An update of the Angiosperm Phylogeny Group classification for the orders and families of
flowering plants: APG III. Botanical Journal of Linnean Society 161: 105–121.
Bajpai O, Pandey J & Chaudhary LB (2017) Periodicity of different phenophases in selected trees from
Himalayan Terai of India. Agroforestry Systems 91: 363–374.
Bassett I, Paynter Q, Hankin R & Beggs JR (2012) Characterizing Alligator Weed Alternanthera philoxeroides,
Amaranthaceae invasion at a northern New Zealand lake. New Zealand Journal of Ecology 36: 216–222.
Bassett IE (2008) Ecology and Management of Alligator Weed Alternanthera philoxeroides, (Ph.D. Thesis).
University of Auckland, New Zealand.
Billings WD (1974) Arctic and alpine vegetation plant, adaptations to cold summer climates. In: Ives JD &
Barrey RG (eds) Arctic and Alpine Environments. Methuen and Company Limited, London, pp. 403–444.
Chatterjee A & Dewanji A (2014) Effect of varying Alligator Weed Alternanthera philoxeroides cover on the
macrophyte species diversity of pond ecosystems: a quadrat–based study. Aquatic Invasions 9: 343–355.
Chaudhary S, Gupta SK & Kumar L (2012) The Sedges and Grasses of Gautambudhnagar (Noida) U.P. India.
International Multidisciplinary Research Journal 2: 45–48.
Chen Y, Zhou Y, Yin TF, Liu CX & Luo FL (2013) The Invasive Wetland Plant Alternanthera philoxeroides
shows a higher tolerance to water logging than its native congener Alternanthera sessilis. PLoS ONE 8:
e81456.
Cherril A & McClean C (1997) The impact of landscape and adjacent land cover upon linear boundary features.
Landscape Ecology 12: 255–260.
Dash SS & Ahmedullah M (2012) Distribution of some plants in the National Capital Territory of Delhi. Indian
Forester 138: 403–406.
Denslow JS (1980) Gap partitioning among tropical rain forest trees. Biotropica (Supplement) 12: 47–55.
Devi LS & Yadava PS (2006) Floristic diversity assessment and vegetation analysis of tropical semi-evergreen
forest of Manipur, northeast India. Journal of Tropical Ecology 47: 89–98.
Dillon WR & Goldstein M (1984) Multivariate Analysis: Methods and Applications. John Wiley and Sons, New
York.
Gaston KJ & Fuller RA (2009) The sizes of species geographic ranges. Journal of Applied Ecology 46: 1–9.
Gillespie TW & Walter H (2001) Distribution of bird species richness at a regional scale in tropical dry forest of
Central America. Journal of Biogeography 28: 651–662.
Grubb PJ (1977) The maintenance of species richness in plant communities. The importance of regenerational
niche. Biological Review 52: 107–145.
Hamid F (2009) A Study on Waterfowl Population and Human Use of Hokersar and Hygam Wetlands of
Kashmir Valley for Conservation Planning, (Ph.D. Thesis). Wildlife Institute of India, Saurashtra
University, Gujarat.
Haston E, Richardson JE, Stevens PF, Chase MW & Harris DJ (2009) The Linear Angiosperm Phylogeny
Group (LAPG) III: A linear sequence of the families in APG III. Botanical Journal of Linnean Society 161:
128–131.
Hill MO (1979) Twinspan. A Fortran Program for arranging multivariate data in an ordered two-way table by
classification of the individuals and attributes. Ecology and Systematic, Cornell University Ithaca, New
York, United States of America.
Hutto RL, Pletschet SM & Hendricks P (1986) A fixed radius point count method for nonbreeding and breeding
season use. The Auk 103: 593–602.
IPNI (2013) The International Plant Names Index. Available from: http://.ipni.org.html. (accessed: 10 Mar.
2013).
Jain SK & Rao RR (1977) A Handbook of Field and Herbarium Methods. Today and Tomorrows Printers and
Publishers, New Delhi, India.
Joshi BC (2009) District brochure of Gautam Budh Nagar, Uttar Pradesh. Central Ground Water Board,
Ministry of Water Resources, Government of India.
Kolar CS & Lodge DM (2001) Progress in invasion biology: predicting invaders. Trends in Ecology and
Evolution 16: 199–204.
Ludwig JA & Reynolds JF (1988) Statistical Ecology: A Primer on Methods and Computing. John Wiley and
Sons, New York.
MacAlister S (1997) Cryptogams communities on fallen logs in the Duke Forest, North Carolina. Journal of
Vegetation Science 8: 115–125.
Maheshwari JK (1963) The flora of Delhi. Council of Scientific and Industrial Research, New Delhi, India.
Mandal A & Mondal AK (2011) Taxonomy and Ecology of Obnoxious Weed Alternanthera Philoxeroides
Grisebach (Family Amaranthaceae) on Spore Germination in Ampelopteris Prolifera (Ketz.) Cop. Advances
in Bioresearch 2: 103–110.
Manral U, Raha A, Solanki R, Hussain SA, Babu MM, Mohan D, Veeraswami GG, Sivakumar K & Talukdar G
(2013) Plant species of Okhla Bird Sanctuary: A wetland of Upper Gangetic Plains, India. Check List 9:
263–274.
Mishra AK, Mir SA, Sharma MP & Singh HB (2014) Addition to the Flora of Delhi. Indian Journal of Plant
Sciences 3: 64–67.
Mishra S & Narain S (2010) Floristic and Ecological Studies of Bakhira Wetland, Uttar Pradesh, India. Indian
Forester 136: 375–381.
Mueller-Dombois D & Ellenberg H (1974) Aims and Methods of Vegetation Ecology. John Wiley and Sons,
New York.
Mueller-Dombois D (1984) Classification and Mapping of Plant Communities: a Review with Emphasis on
Tropical Vegetation. In: Woodwell GM (ed) The Role of Terrestrial Vegetation in the Global Carbon Cycle:
Measurement by Remote Sensing. John Wiley & Sons Ltd., pp. 21–88.
Mwangi W & Swallow B (2005) Invasion of Prosopis juliflora and local livelihoods: Case study from the lake
Baringo area of Kenya. ICRAF Working Paper no. 3. Nairobi: World Agroforestry Centre.
Negi BS, Chauhan DS & Todaria NP (2008) Comperative diversity between Panchayat and adjoining reserve
forests in Garhwal Himalaya. Indian Journal of Forestry 31: 585–593.
Orwa C, Muta A, Kindt R, Jamnads R & Anthony S (2009) Agro-forestry Database: a tree reference and
selection guide version 4.0. Available from: http:/www.worldagrofrestry.org/sites/tredbs/ tredatbase.asp
(accessed: Feb. 2014).
Pandey A, Sharma SK, Singh L & Singh T (2013) An overview on Desmostachya bippinata. Journal of Drug
Discovery and Therapeutics 1: 67–68.
Pant S & Samant SS (2007) Assessment of Plant diversity and prioritization communities for conservation in
Mornaula. Applied Ecology and Environmental Research 5: 151–166.
Patel AP & Pandya NR (2014) Assessment of temporal and spatial variation in species richness and Diversity of
butterfly host plants. International Journal of Plant, Animal and Environmental Sciences 4: 235–245.
Rodgers WA, Panwar HS & Mathur VB (2002) Wildlife Protected Area Network in India: A Review, Executive
Summary. Wildlife Institute of India, Dehradun, India.
Ruiz TT, López EM, Granado GL, Pérez EL, López RM & Guzmán JMS (2008) The Water Hyacinth
Eichhornia crassipes: an invasive plant in the Guadiana River Basin (Spain). Aquatic Invasions 3: 42–53.
Siddiqui MF, Ahmed M, Shaukat SS & Khan N (2010) Advanced Multivariate Techniques to Investigate
Vegetation-Environmental Complex of Pine Forests of Moist Temperate Areas of Pakistan. Pakistan Journal
of Botany 42: 267–293.
Singh HB & Subramaniam B (2008) Field Manual on Herbarium Techniques. National Institute of Science and
Information Resources, CSIR. Oxford University Press, New Delhi, India.
Singh SK & Rawat GS (1999) Floral Diversity and Vegetation Structure in Great Himalayan National Park,
Western Himalaya. Wildlife Institute of India, Dehradun, India.
Srivastava SK (2004) Floristic diversity in Uttar Pradesh- an overview. Journal of Economic and Taxonomic
Botany 28: 292–334.
The Plant List (2013) The Plant List. Available from: http://www.theplantlist.org.html. (accessed: 31 Mar.
2013).
Vardhana R (2007) Flora of Ghaziabad District. Shree Publishers and Distributors, New Delhi, India.
Varshney JG, Kumar S & Mishra JS (2008) Current Status of Aquatic Weeds and their Management in India.
Ansari 2018
In: Sengupta S & Dalwani R (eds) Proceedings of Taal 2007: The 12th World Lake Conference Jaipur,
Rajasthan, pp. 1039–1045.
Vijayan VS (1983) Hydrobiological (Ecological) Research report of Keoladeo National Park, Bharatpur. First
Interim Report, Bombay Natural History Society, Bombay.
Vuilleumier F & Simberloff D (1980) Ecology versus history as determinants of patchy and insular distributions
in high Andean birds. In: Hecht MK & Steere WC (eds) Evolutionary Biology. Plenum Publishing
Corporation, New York, pp. 235–379.
Waite S (2000) Statistical Ecology in Practice: A guide to analyzing environmental and ecological field data.
Pearson Education Limited.
Research article
Salinity would be an option to control Eichhornia crassipes

(Mart.) Solms [Water Hyacinth]: Sri Lanka perspective
T. Mathiventhan*, T. Jayasingam and M. Umaramani
Department of Botany, Faculty of Science, Eastern University, Sri Lanka
*Corresponding Author: tmathiventhan@gmail.com [Accepted: 12 December 2018]
Abstract: Eichhornia crassipes, commonly known as Water hyacinth, is an aquatic plant. It has
been listed as one of the worst weeds mostly in the tropics and subtropics and listed as Invasive
Alien Species (IAS) in Sri Lanka. Many efforts had been made to eradicate this species using both
manual and biological control methods over the last 100 years, but Eichhornia still shows wide
distribution, posing a tremendous threat to aquatic biota in many inland water systems. The study
would investigate to control of the species by using salinity as a tool. Experiments were set up to
study the growth of the species and to study the role of salinity on the growth and survival of the
species. The growth of the species was measured in terms of the production of leaves in fresh
water tanks for a period of three months. Salinity values of 1, 2, 3, 4, 5, 6, 7, 10, 15 and 20 ppt was
made up and plants were placed. The control treatment was the water with zero salinity. Two
plants were placed in each tray and observed over the next 16 days. The number of leaves
increases with time up to 40 days. It showed a sudden declined in the production of leaves, 55 days
onwards. Leaves that were produced were getting rotten after 40 days onwards. The experiment
showed that the E. crassipes survive at 0 ppt saline water throughout the experiment, the
appearance of the percentage of green shoots existed as 100%. The shoots were becoming brown
and subsequently dead after 4 days of the experiment at the salinity level 7 ppt. While the plants
showed a gradual decline in their appearance of green shoot between 0 and 6 ppt with the
increasing salinity over time.
Keywords: Survival - Growth - Impact - Invasive plants - Salinity.
[Cite as: Mathiventhan T, Jayasingam T & Umaramani M (2018) Salinity would be an option to control
Eichhornia crassipes (Mart.) Solms [Water Hyacinth]: Sri Lanka perspective. Tropical Plant Research 5(3):
331–335]
INTRODUCTION
Eichhornia crassipes (Mart.) Solms, commonly known as water hyacinth, is an aquatic plant, which is on the
IUCN's list of the 100 most dangerous invasive species (Fig. 1). It can spread rapidly through the waterways of
catchments, populations to double in size in as little as 6 to 18 days (Gettys 2014), likely to be 400–700 tons per
hectare per day (Parson & Cuthbertson 2001) and four times the loss of water from normal water surface
evaporation due to its high rates of transpiration during summer (Téllez et al. 2008). It reduces temperature, pH,
biological oxygen demand (organic load), and nutrient levels (Rai & Datta 1978). It is a major global challenge
that requires urgent action (Xu et al. 2012).
Eichhornia crassipes (Mart.) Solms [Water hyacinth] - Sri Lankan context
Sri Lanka is reported to be a biodiversity hotspot in which over 30 invasive alien species (IAS) has been
recorded. Water hyacinth is listed as one of the worst invaders in aquatic ecosystems in Sri Lanka (Pradeepa et
al. unknown). The dry zone water tanks have been invaded or under threat of invasion of water hyacinth
(Villamgna & Murphy 2010) and also can affect the functions of these freshwater tanks. Water Hyacinth has
been introduced in 1905 and has spread in all parts of the country mainly in the dry zone and has become a
significant weed in the aquatic ecosystems. The water hyacinth reaches the lagoons/rivers from freshwater tanks
in the catchments or from irrigation channels. During the rainy periods flood water to move the plants across the
area and spread it to the entire region. An ordinance was declared for the eradication of same in 1909 called the
Mathiventhan et al. 2018
„water hyacinth ordinance, an ordinance 4 of 1909. This has been manually removed in many water bodies,
which have been expensive. Biological control also has been tried by using Neochetina eichhorniae Warner,
which was introduced in 1988 and this beetle is breeding well but its effectiveness against water hyacinth is
unlikely to be evident before 1994 (Room & Fernando 1992).
Figure 1. Habit of Eichhornia crassipes (Mart.) Solms.

Many efforts had been made to eradicate this species using both manual and biological control methods over
the last 100 years, but Eichhornia still shows wide distribution, posing a tremendous threat to aquatic biota in
many inland water systems. The study would investigate to control of the species by using salinity as a tool.

Data collection
A reconnaissance visit was made to the entire district of Batticaloa (Eastern region of Sri Lanka). Sites with
high abundance of Water Hyacinth, more than 50% of cover, were chosen (nine sites) for collecting water and
the plants. Readings were recorded in two ways: (i) amidst the plants; (ii) open waters where Eichhornia
present. Salinity, pH and temperature were measured on site.
Growth experiment
Experiments were set up to study the growth of the species. Units of single plants (SP) and units of two
plants linked by stolen/off-set (DP) were used as treatments for the study. Plants were placed in freshwater tanks
and observed daily for a period of three months. Plants were floated in triplicates in the tank. The setup was in
the open and was subjected to temperature changes and rainfall as in the field. The tank was large enough, and
not to be affected considerably by the heat from the sunlight. The growth of water hyacinth was measured in
terms of production of leaves.
Effect on salinity on growth
An experiment was set up with three replicates each, to study the role of salinity on the growth and survival
of the species. The design was completely randomized. Salinity values of 1, 2, 3, 4, 5, 6, 7, 10, 15 and 20 ppt
were made up using sodium chloride in 4 litres of water taken from the water bodies in the field, with zero
salinity. The control treatment was the water taken as zero salinity. Two plants were placed in each tray and
observed over the next 16 days. The recording was done at 4-day intervals. Percentage of green in the shoots
was recorded in these trays. The experiment was conducted in a shaded place to avoid direct overheating of
water by the sunlight.

Eichhornia crassipes (Mart.) Solms distribution is mainly identified in the lagoon, water bodies and
irrigation channels. The temperature of the study sites ranges between 27–32°C, pH between 6–8. Both the
temperature and pH gives optimum and tolerance ranges for the Eichhornia respectively (James 1983). Salinity
showed variation between 0 and 32 ppt. The growth of E. crassipes was recorded in sites where the salinity was
0 ppt (sites 6, 7 and 8) whereas dead plants were noted in sites where the salinity was above 2 ppt (Fig. 2)
(James 1983, Muramoto et al. 1991).
Water samples: amidst the plants Water samples: open waters where Eichhornia
present
Figure 2. Variation of Temperature, pH and Salinity of the water where Eichhornia crassipes (Mart.) Solms. noticed. [The
plant was noticed in places where the salinity less than 1ppt]
Growth experiment
The number of leaves increases with time up to 40 days. It showed a sudden declined in the production of
leaves, 55 days onwards. Leaves that were produced were getting rotten after 40 days onwards (Fig. 3).
Single Plant
Average number of leaves
Double Plant
Number of days
Figure 3. A comparison of the growth of Eichhornia crassipes (Mart.) Solms: Plants were grown as single plant and double
plants (where two plants are connected by a stolon).
The plant grew in terms of a number of leaves to a larger level and then they rescued themselves to around 5
leaves per plant. As there were no new offsets produced and there was no addition of nutrients in the tank the
only possible explanation would be intraspecific competition, very similar to self-thinning where the number of
leaves reduces for survival. This is similar to the reduction of tillers certain plants under a competitive
environment. It could also be a check based on its morphology where more stem leaves could not be
accommodated without expansion. This is also similar to branches in a tree where when one grows the other is
seen to reduce or arrested growth, forming the architecture of the plants that are seem in trees. The culm seems
to be happy with around 5 leaves per plant in individuals and pairs.
Effect of salinity on growth
The experiment showed that the E. crassipes survives at 0 ppt saline water throughout the experiment, the
appearance of the percentage of green shoots existed as 100%. The Results of the performance of the species
under different salinity levels are given at 4, 8, 12 and 16 day periods are given in graphs 1,2,3,4 respectively
(Fig. 4). The shoots were becoming brown and subsequently dead after 4 days of the experiment at the salinity
level 7 ppt. While the plants showed a gradual decline in their appearance of green shoot between 0 and 6 ppt
with the increasing salinity over time.
Existing green shoot after 4 days of treatment (A) Existing green shoot after 8 days of treatment (B)
100 100
80 80
Mathiventhan et al. 2018
60 60
www.tropicalplantresearch.com
40 40
Presence of green shoot (% )

Percentage of green shoot (% )
20 20
0 0
0 1 2 3 4 5 6 7 10 15 20 0 1 2 3 4 5 6 7 10 15 20
Salinity (ppt) Salinity (ppt)
Existing green shoot after 12 days of treatment (C) Existing green shoot after 16 days of treatment (D)
100 100
80 80
60 60
40 40

20 20
0 0
0 1 2 3 4 5 6 7 10 15 20 0 1 2 3 4 5 6 7 10 15 20
Salinity (ppt) Salinity (ppt)
Figure 4. The performance of Eichhornia crassipes (Mart.) Solms in different salinity strengths at different time intervals. [A mean connect line is showing the general trend of declining
334
green shoot with salinity changes over time]
There is a sharp decrease of survival (% green) to the salinity of 3 ppt and then a gradual decrease is seen up
to 6 ppt where almost all are dead (yellow), in 4-day period itself. One ppt concentration seems to have no
different from control. But with time, even 1ppt and 2 ppt decrease in survival. It had been shown in our study
that they cannot survive beyond 3 ppt and even at 1 ppt they seem the whiter off faster.
CONCLUSION
The salinity experiment confirmed that the Eichhornia seems to be affected by salinity levels as low as 3 ppt
itself and even in levels below. Therefore, the salinity would be an option to control the water hyacinth but its
practical applicability in the larger water bodies would make a great challenge when considering the ecosystem
and its biodiversity.
ACKNOWLEDGEMENT
This study is part of a project of “Distribution and socio economic impacts of Eichhornia crassipes (Water
Hyacinth) in Batticaloa District” by the Biodiversity Secretariat, Ministry of Mahaweli Development and
Environment, Sri Lanka.
REFERENCES
Gettys LA (2014) Waterhyacinth: Florida’s Worst Floating Weed. SS-AGR-380, UF/IFAS extension,
University of Florida. Available from: https://link.springer.com/article/10.1007/BF00012564 (accessed: 25
Dec. 2017)
James AD (1983) Eichhornia crassipes (Mart.) Solms. Handbook of Energy Crops. Unpublished.
Muramoto S, Aoyama I & Oki Y (1991) Effect of salinity on the concentration of some elements in water
hyacinth (Eichhornia crassipes) at critical levels. Journal of Environmental Science and Health. Part A:
Environmental Science and Engineering and Toxicology 26(2): 205–215.
Parson WT & Cuthbertson EG (2001) Noxious Weeds of Australia, 2nd Ed, CSIRO Publishing. Available from:
www.publish.csiro.au (accessed: 10 Nov. 2017).
Pradeepa P, Sudeera R & Devaka W (unknown) A study of some ecological and biological aspects of the water
hyacinth (Eichhornia crassipes). Ministry of Mahaweli Development and Environment, Sri Lanka.
Rai DN & Datta MJ (1978) The influence of thick floating vegetation (Water hyacinth: Eichhornia crassipes)
on the physicochemical environment of a freshwater wetland. Hydrobiologia 62: 65–69.
Room PM & Fernando IVS (1992) Weed invasion countered by biological control: Salvinia molesta and
Eichhornia crassipes in Sri Lanka. Aquatic Botany 42: 99–107.
Téllez TR, López EMR, Granado GL, Pérez EA, López RM and Guzmán JMS (2008) The Water Hyacinth,
Eichhornia crassipes: an invasive plant in the Guadiana River Basin (Spain). Aquatic Invasion (3)1: 42–53.
Villamgna AM & Murphy BR (2010) Ecological and socio-economic impacts of invasive water hyacinth
(Eichhornia crassipes): a review. Fresh Water Biology 55: 282–298.
Xu H, Qiang S, Genovesi P, Ding H, Wu J, Meng L, Han Z, Miao J, Hu B, Guo J, Sun H, Huang C, Lei J, Le Z,
Zhang X, He S, Wu Y, Zheng Z, Chen L, Jarošík V & Pyšek P (2012) An inventory of invasive alien species
in China. NeoBiota 15: 1–26.
Research article
Effect of biochar on seed germination, early growth of

Oryza sativa L. and soil nutrients
Montasir Shamim*, Narayan Saha and Farhana Bintay Hye
Department of Forestry and Environmental Science, School of Agriculture and Mineral Sciences,
Shahjalal University of Science and Technology, Sylhet-3114, Bangladesh
*Corresponding Author: shanto.fes.sust@gmail.com [Accepted: 14 December 2018]
Abstract: Biochar application to soil has been recognized worldwide for enhancing plant
productivity, soil properties as well as long term carbon storage. But very few studies related to
biochar have been undertaken in the tropical region. This study has been undertaken in the nursery
of Department of Forestry and Environmental Science, Shahjalal University of Science and
Technology in Sylhet, Bangladesh to assess the impact of various treatments of three different
biochar on germination and early growth of paddy (Oryza sativa). The selected species used as
feedstock for biochar production are Albizia saman (Raintree), Neolamarckia cadamba (Kadam),
and Albizia richardiana (Chambul). Biochar was produced by using Kon-Tiki kiln. Two
treatments viz. 10 t ha-1 and 15 t ha-1 for each biochar were applied along with Control. Complete
Randomized Block Design (CRBD) was followed as experimental design. Data were analyzed by
using Tukey HSD post hoc test and ANOVA. In the case of germination percentage biochar
treatments did not show significant (P < 0.05) increase compared to control. The 15 t ha-1
application rate of Raintree biochar showed significant (P < 0.05) increase in root length compared
to control. For shoot dry weight 15 t ha-1 application rate of Raintree and 10 t ha-1 application rate
of Chambul showed significant (P < 0.05) increase than control. While 10 t ha-1 of Kadam biochar
showed significant (P < 0.05) increase in RWC than control. Soil chemical test showed that
Chambul biochar’s 15 t ha-1 application rate shows strongly significant (P <0.001) increase of
NPK than control.
Keywords: Biochar - Germination - Early growth - Oryza sativa - Soil NPK.
[Cite as: Shamim M, Saha N & Hye FB (2018) Effect of biochar on seed germination, early growth of Oryza
sativa L. and soil nutrients. Tropical Plant Research 5(3): 336–342]
INTRODUCTION
Biochar is a product of pyrolysis which is rich in carbon content and produced by heating biomass such as
wood, manure or leaves in a closed container with little or no available air. In other words, it is produced by the
thermal decomposition of organic material under a limited supply of oxygen (O2), and at relatively low
temperatures (< 700oC) (Lehmann & Joseph 2009). Biochar has a high stability potential, can remain in the soil
for a very long period of time and can contribute in sequestration of atmospheric carbon. Thus the longer period
of the stability of biochar can play an important role in reducing the emission of CO 2 to the atmosphere (Ahmed
et al. 2014). Biochar contains a high concentration of stable organic carbon (C) as well as eluated carbon and
ash. Several macro and microelements can be stored in the mineral fraction of biochar which may act as a
source of mineral substances for microorganisms in soil (Saletnik et al. 2016).
Biochar has been accepted for having the potentiality to sequestrated carbon (C) as well as having a
beneficial impact on soil quality parameters (Free et al. 2010). Biochar application to soil has been reported to
increase soil pH, cation exchange capacity (CEC) and soil nutrient availability (Kamara et al. 2014). Biochar
has also been reported to increase water holding capacity (WHC) of sandy soils, improve soil structure and
enhance soil chemical fertility (Free et al. 2010).
Generally, biochar can act as a renewable bio resource and has the potentiality to exhibit positive impact on
plant growth (Kamara et al. 2014). However, some biochar may have unexpected materials such as crystalline
Received: 09 August 2018 Published online: 31 December 2018
Shamim et al. 2018
silica, dioxin, polyaromatic hydrocarbons (PAHs), phenolic compounds and heavy metals that can be harmful to
plants, microorganisms and human (Solaiman et al. 2012).
Early growth is an important factor for the survival and production of any plant species. Biochar application
significantly increases the early growth of seedlings (Thomas & Gale 2015). So it is crucial to study the impact
of biochar on early growth of seedlings. Generally, biochar has the ability to enhance crop productivity. But
some biochar may have substances that can negatively affect seed germination and early growth of seedlings
(Solaiman et al. 2012). So it is necessary to test the impact of any biochar on seed germination and early growth,
before large scale use. The objective of the study is to assess the effect the various dosages of biochar from
different feedstocks on the germination and early growth of paddy and to evaluate the impact of biochar on soil
nutrients availability.

The study was conducted at the nursery of the Department of Forestry and Environmental Science in
Shahjalal University of Science and Technology, Sylhet, Bangladesh. For the preparation of biochar, the
selected tropical tree species were Albizia saman (Jacq.) Merr. (Raintree), Neolamarckia cadamba (Roxb.)
Bosser (Kadam) and Albizia richardiana (Voigt) King & Prain (Chambul). The wood of these three species
were collected from sawmill and dried in sunlight to reduce excess moisture. The biochar kiln which was used
to produce biochar was an open fire kiln. Its name is Kon-Tiki biochar kiln which was invented by Ithaka
Institute.
The soil was collected from an agricultural field near the University campus. Germination of Paddy seeds
were done in clay pots. In each pot 10 paddy seeds were sown. Each biochar application has been applied in
three treatments for the study and these are control (c), 10 t ha-1 and 15 t ha-1. Each treatment had three
replications. The treatments were named according to table 1. The Complete Randomized Block Design
(CRBD) was used in this study.
Table 1. Biochar treatments name with application rate.
Treatment Name Feedstock Name of the Biochar Application rate (t ha-1)
Cont. Control 0
R 10 Albizia saman (Jacq.) Merr. 10
R 15 Albizia saman (Jacq.) Merr. 15
K 10 Neolamarckia cadamba (Roxb.) Bosser 10
K15 Neolamarckia cadamba (Roxb.) Bosser 15
C 10 Albizia richardiana (Voigt) King & Prain 10
C 15 Albizia richardiana (Voigt) King & Prain 15
Germination data was collected at three days interval and in this study germination data of 7th day was used.
Shoot length, root length was measured by measuring the scale and fresh and oven dry weight of seedling were
measured by digital balance.
Germination percentage was determined by the formula of Close & Wilson (2002) formula:
Seed vigor of the seedling was calculated by following Abdul & Anderson (1973) formula:
The water content respective to the fresh weight was determined by following Weatherley (1950) formula:
At the end of the initial data collection, soil samples were collected from each plot and analysed in the
laboratory of Soil Research Development Institute (SRDI) in Sylhet, Bangladesh to measure the amount of NPK
in each soil sample.
Data was analysed statistically at 5% probability level by using one way ANOVA test in SPSS (IBM SPSS
Version 23, Armonk, NY; IBM Corp) and Microsoft Excel 2013. In case of obtaining a significant result,
multiple comparisons of the mean was obtained by Tukey Honest Significant Difference (HSD) post hoc test.
RESULTS
Germination percentage
Results from Tukey HSD post hoc test shows that there is no significant difference in seed germination of
paddy between the treatments and control (Fig. 1).
Shoot and root length
The result shows that there is no significant increase in shoot length compared to control (Fig. 2). On the
other hand a significant (P < 0.05) increase in root length of paddy was observed in case of 15 t ha -1 application
rate of Raintree biochar compared to control (Fig. 3).
Figure 1. Biochar impact on seed germination percentage. [Cont.= Control, R 10= 10 t ha-1 of Raintree biochar, R 15= 15 t
ha-1 of Raintree biochar, K 10= 10 t ha-1 of Kadam biochar, K 15= 15 t ha-1 of Kadam biochar, C 10= 10 t ha-1 of Chambul
biochar, C 15= 15 t ha-1 of Chambul biochar]
Figure 2. Biochar impact on shoot length of Paddy seedlings. [Cont.= Control, R 10= 10 tha-1 of Raintree biochar, R 15= 15
t ha-1 of Raintree biochar, K 10= 10 t ha-1 of Kadam biochar, K 15= 15 t ha-1 of Kadam biochar, C 10= 10 t ha-1 of Chambul
Shoot and root dry weight
The result shows that 15 t ha-1 application rate of Raintree (P < 0.05) and 10 t ha-1 application rate of
Chambul (P < 0.01) biochar have a significant increase in shoot dry weight compared to control (Fig. 4). But
biochar application did not show any significant impact (α > 0.05) on root dry weight of paddy.
Seed vigor
The application rate of Raintree biochar (10 t ha-1 and 15 t ha-1) have significant (P < 0.05) increase in seed
vigor compared to control treatment (Fig. 5).
Shamim et al. 2018
Figure 3. Biochar impact on root length of Paddy seedlings. [Cont.= Control, R 10= 10 t ha-1 of Raintree biochar, R 15= 15 t
ha-1 of Raintree biochar, K 10= 10 t ha-1 of Kadam biochar, K 15= 15 t ha-1 of Kadam biochar, C 10= 10 t ha-1 of Chambul
Figure 4. Biochar impact on shoot dry weight of Paddy seedlings. [Cont.= Control, R 10= 10 t ha-1 of Raintree biochar, R
15= 15 t ha-1 of Raintree biochar, K 10= 10 t ha-1 of Kadam biochar, K 15= 15 t ha-1 of Kadam biochar, C 10= 10 t ha-1 of
Chambul biochar, C 15= 15 t ha-1 of Chambul biochar]
Figure 5. Biochar impact on seed vigor of paddy. . [Cont.= Control, R 10= 10 t ha-1 of Raintree biochar, R 15= 15 t ha-1 of
Raintree biochar, K 10= 10 t ha-1 of Kadam biochar, K 15= 15 t ha-1 of Kadam biochar, C 10= 10 t ha-1 of Chambul biochar,
C 15= 15 t ha-1 of Chambul biochar]
Figure 6. Biochar impact on RWC of paddy seedlings. [Cont.= Control, R 10= 10 t ha-1 of Raintree biochar, R 15= 15 t ha-1
of Raintree biochar, K 10= 10 t ha-1 of Kadam biochar, K 15= 15 t ha-1 of Kadam biochar, C 10= 10 t ha-1 of Chambul
Figure 7. Impact of time and treatment on soil N. [T= 0 means beginning of the experiment and T= 1 means end of the
experiment. Cont.= Control, R 10= 10 t ha-1 of Raintree biochar, R 15= 15 t ha-1 of Raintree biochar, K 10= 10 t ha-1 of
Kadam biochar, K 15= 15 t ha-1 of Kadam biochar, C 10= 10 t ha-1 of Chambul biochar, C 15= 15 t ha-1 of Chambul biochar]
Figure 8. Impact of time and treatment on soil K. [T= 0 means beginning of the experiment and T= 1 means end of the
Shamim et al. 2018
Figure 9. Impact of time and treatment on soil P. [T= 0 means beginning of the experiment and T= 1 means end of the
Relative water content (RWC)
The result from Tukey HSD post hoc test shows that 10 t ha-1 application rate of Kadam biochar shows
significant (P < 0.05) increase in comparison with control (Fig. 6).
Soil NPK changes over time
Results shows that all the treatment has shown an increase soil N where 15 t ha-1 application rate of Chambul
biochar shows highest changes in soil N and 10 t ha-1 application rate of Kadam biochar shows lowest changes
in soil N over time.
Except 10 t ha-1 of Chambul and Kadam biochar, every biochar treatment shows an increase in soil N in
comparison to control treatment (Fig. 7).
In case of soil K, all biochar treatment has shown a better increase over time compared to control, among
them 15 t ha-1 of Chambul biochar shows the highest increase in soil K over time (Fig. 8).
Besides all the treatments of biochar has shown the better increase in soil P over time compared to control.
Among this treatment, 15 t ha-1 of Chambul biochar shows the highest increase over time compared to the other
biochar treatments (Fig. 9).
DISCUSSION
Seed germination is very crucial for the production of the crop. Some biochar may contain phytotoxic
substances like dioxins, furans, polyaromatic hydrocarbons, phenolic compounds and heavy metals which can
be harmful to crop, soil and even human (Solaiman et al. 2012).
In this study, the germination percentage of paddy increased in case of Raintree and Chambul biochar was
above the control level but the difference was not significant. On the other hand, Kadam biochar shows decrease
in germination percentage than control. The study revealed that Raintree and Chambul biochar did not show any
negative impact on seed germination while Kadam biochar caused a decrease in germination of paddy seeds.
Other reports on the related study found that there is no negative impact of biochar on the germination of
paddy seeds (Kamara et al. 2014). Other report based on forest seeds found that biochar application increases
germination of seeds (Robertson et al. 2012).
The greatest increase in early growth was observed in the case of 15 t ha-1 application of Raintree biochar.
The root length, shoot dry weight, and seed vigor increased with the application of 15 t ha -1 of Raintree biochar.
On the other hand, 10 t ha-1 application rate of Chambul biochar showed a significant increase in shoot dry
weight of paddy.
The Kadam biochar had shown a decrease in germination percentage, shoot length and root length than
control but it had shown significantly higher RWC compared to control.
The study found that both the application rate of Raintree and Chambul biochar showed an increase in root
length and Kadam biochar show slightly decrease over control. Report of the relevant study showed that root
length of paddy seedlings decreased with the application of biochar (Kamara et al. 2014). Although the
difference between the treatments were not significant.
A significant effect of treatments was found on soil Potassium (K) and Phosphorus (P) and Nitrogen (N).
The 15 t ha-1 application rate of Chambul biochar showed an increase in soil N, K and P.
On the other hand, Kadam biochar showed relatively less changes in soil NPK compared to other two
biochar. The study also revealed that there is no significant difference among the application rates of each
biochar.
Other related study found that biochar application increase soil NPK compared to control (Abdul & Abdul
2017). On the other hand (Ghosh et al. 2015) found that biochar had no significant effect on soil N.
CONCLUSION
Germination percentage of paddy was not affected by biochar application. However, results showed that
application of Raintree and Chambul biochar significantly affect the early growth of paddy seedlings. On the
other hand, Kadam biochar showed a decrease in the early growth of paddy seedlings compared to control. But,
the soil analysis showed that biochar had a significant impact on soil nutrient properties.
It seems that biochar has the potentiality to enhance seed germination and plants growth and can increase
soil nutrient, while some biochar could have a negative influence. More research is needed to identify the
biochars and also their application rates which have beneficial impacts on plant and soil.
ACKNOWLEDGEMENTS
The authors wish to express their gratitude to Sonchita Biswas, Rupon Kumar Nath and Tasnima Mukit of
Department of Forestry and Environmental Science, Shahjalal University of Science and Technology,
Bangladesh for their help in biochar production, soil collection and early growth and soil data collection.
REFERENCES
Abdul B & Anderson J (1973) Vigor determination in soybean seed by multiple criteria. Crop Science 13: 630–
633.
Abdul N & Abdul N (2017) The Effect of Biochar Application on Nutrient Availability of Soil Planted with
MR219. Journal of Microbial and Biochemical Technology 9(2): 583–586.
Ahmed M, Rajapaksha A, Lim J, Zhang M, Bolan N, Mohan D & Ok Y (2014) Biochar as a sorbent for
contaminant management in soil and water: a review. Chemosphere 99: 19–33.
Close D & Wilson S (2002) Provenance effects on pregermination treatments for Eucalyptus regnans and
Eucalyptus delegatensis seed. Forest Ecology and Management 170: 299–305.
Free H, McGill C, Rowarth J & Hedley M (2010) The effect of biochars on maize (Zea mays) germination. New
Zealand Journal of Agricultural Research 53(1): 1–4.
Ghosh S, Ow LF & Wilson B (2014) Influence of biochar and compost on soil properties and tree growth in a
tropical urban environment. International Journal of Environmental Science and Technology 12(4): 1303–
1310.
Kamara A, Kamara A, Mansaray M, & Sawyerr P (2014) Effects of biochar derived from maize stover and rice
straw on the germination of their seeds. American Journal of Agriculture and Forestry 2(6): 246–249.
Lehmann J & Joseph S (2009) Biochar for environmental management. Earthscan publishing, London.
Robertson S, Rutherford P, López-Gutiérrez J & Massicotte H (2012) Biochar enhances seedling growth and
alters root symbioses and properties of sub-boreal forest soils. Canadian Journal of Soil Science 92(2): 329–
340.
Saletnik B, Bajcar M, Zagula G, Czernicka M, & Puchalski C (2016) Influence of biochar and biomass ash
applied as soil amendment on germination rate of Virginia mallow seeds (Sida hermaphrodita R.).
Econtechmod 5(3): 71–76.
Solaiman Z, Murphy D & Abbott L (2012) Biochars influence seed germination and early growth of seedlings.
Plant Soil 353: 273–287.
Thomas S & Gale N (2015) Biochar and forest restoration: a review and meta-analysis of tree growth responses.
New Forests 46: 931–946.
Weatherley P (1950) Studies in water relations of cotton plants. I.The field measurement of water deficit in
leaves. New Phytologist 49: 81–87.
Research article
Sorption behaviour of thermally and chemically

modified selected wood species
F. A. Faruwa1 and E. A. Iyiola2*
1
Department of Forestry and Wildlife Management, Federal University of Technology, Oweri, Nigeria
2
Department of Forestry and Wood Technology, Federal University of Technology, Akure, Ondo State Nigeria
*Corresponding Author: eaiyiola@futa.edu.ng [Accepted: 15 December 2018]
Abstract: This study investigates the sorption behaviour of thermally and chemically modified
selected wood species. Wood samples of dimensions 20 mm × 20 mm × 20 mm were used in this
study. The samples were oven dried and thermally treated at temperatures of 160°C, 180°C and
200°C for 30 minutes. Another set of wood samples were prepared for chemical treatment
(Acetylation) inside an oven at 80oC for 180 min. The colour varied from light cream to slightly
brown at 160°C and very brown at 200°C for thermally modified wood. The color of the chemical
modified wood changes from being yellowish of the untreated wood to pale yellow in colour. The
percentage weight loss increases with temperature from 22.62% at 160°C to 26.46% at 180°C and
20.8% for Percentage Weight Gain (PWG). The average value of water absorption ranged from
8.60 to 16 %; 26 to 40.78 %; 35 to 50.35 % and 42.88 to 57.53 % for 1 hour to 78 hrs respectively.
The value for the chemically modified wood ranged between 5.22 and 5.59 %, with RH of 97%
and 7% having the lowest and highest value respectively. The study revealed that there was a
reduction in the weight and density of thermally treated wood as a result of thermolysis and weight
was gained after chemical modification.
Keywords: Sorption - Water absorption - Temperature - Colour - Density.
[Cite as: Faruwa FA & Iyiola EA (2018) Sorption behaviour of thermally and chemically modified selected
wood species. Tropical Plant Research 5(3): 343–348]
INTRODUCTION
Wood is a hard organic fibrous material found in the stems, branches and roots of trees and other woody
plants. Wood is composed of three intimately-associated structural polymers; cellulose, hemicelluloses and
lignin material which are vulnerable to degradation due to microbial and termite activities causing significant
losses of wood in service (Shupe et al. 2007).
Wood due to its exceptional properties like good aesthetic values, easy workability, high strength to volume
ratio, and good finishing (painting and polishing) has been used for various purposes like; construction,
engineered wood products, furniture and utensils, arts and in sports and recreational equipment. In Nigeria, more
than 80% of timber products are used for constructional purposes such as building, furniture, fuel, railways,
sleepers, transmission poles, pulp and paper, plywood, veneer, composite boards, matches and fuelwood
(Akanbi & Ashiru 2002).
Although wood continues to be used for many excellent material properties, it also suffers from a number of
disadvantages. These disadvantages among others are; dimensional changes in response to altering atmospheric
conditions, susceptibility to biological attack and changes in appearance when exposed to weathering. All these,
place restrictions on the potential end-usage of wood (Hill 2006). As reported by Stamm (1964), biological
degradation experienced in wood is on the account of moisture gained by wood through the hydrogen bonding
on exposure to moisture, which makes the wood dimensionally unstable and also spawning biological agents.
Wood exposed in exterior conditions is subject to degradation by the ultraviolet component of the solar
spectrum. This degradation is essentially confined to the lignin component of wood, resulting in the gradual
release of polysaccharide-rich wood cells, which are subsequently removed from the wood surface by wind and
rain erosion (Hill 2006). Therefore, because of these negativities confronting wood, there is a need to enhance
Received: 13 March 2018 Published online: 31 December 2018
Faruwa & Iyiola 2018
its usability through modification via chemical modification and/or plasma modification.
Chemical modification is a reaction between some reactive part of the wood and a simple single chemical
reagent, with or without the catalyst, to form a covalent bond between the two (Rowell 2005).This process
exclude impregnation, coating among others. The most abundant single site for reactivity in these polymers is
the hydroxyl group which makes the wood dimensionally unstable, as regards to its sorption behaviour and
durability.
Thermal modification of wood is controlled pyrolysis process of wood to include some chemical changes to
the chemical structures of the cell wall component and to increase its durability (Tiemann 1915). Kollmann &
Fengel (1965) reported that, thermal modification of wood affects predominantly hemicelluloses; this process of
thermal degradation of compounds begins at 120°C, and its intensity is proportional to the temperature gradient.
Also, Boonstra et al. (2007) and Windeisen et al. (2009) observed that acetic acid is released during thermal
modification via the hydrolysis of hemicelluloses. This Acetic acid plays a major role in the depolymerisation of
cellulose and thus, increases its crystallinity. As observed and reported by Mazela et al. (2004), Peters et
al. (2009) and Mohareb et al. (2012), the major impact of thermal treatments of wood is to decrease the number
of hydroxyl groups responsible for bonding water molecules. When the hemicelluloses content are significantly
decreased, the capacity for moisture adsorption of the material decreases accordingly and this will improves the
dimensional stability and resistance to microbiological degradation.
Therefore, with these two major types of wood modification, these negativities befalling wood and
especially dimension stability (sorption behaviour) would be combated. Hence, this study focused on the
sorption behaviour of thermally and chemically modified selected Entandrophragma utile (Dawe & Sprague)
Sprague wood species. Entandrophragma utile has light brown sapwood and is clearly differentiated from the
heartwood. When newly cut, the heartwood is pinkish-brown, but it darkens to a deep red-brown on exposure.
The grain is typically interlocked with a medium texture. Quarter sawn surfaces can display irregular wide stripe
or ribbon figure. E. Utile is more durable and often used for external joinery such as windows and doors.
METHODOLOGY
Preparation of Wood Samples
Commercial lumber of Entandrophragma utile (Dawe & Sprague) Sprague wood used for this study was
obtained from a commercial sawmill (Njoku and Sons Ltd. Naze, Owerri Imo State, Nigeria).Wood samples of
dimensions 20 mm × 20 mm × 20 mm were used in this study. The substrate (wood samples) that were used
were determined based on density determination of different species of wood in the market. The chemical used
for different relative humidity test were: sodium hydride, potassium nitrate, sodium nitrate, ammonium sulphate,
copper sulphide while the chemicals used for modification were acetic acid and acetic anhydride.
The wood samples were air-dried to approximately 20% moisture content and machined into the required
dimensions in the direct parallel to grain with a circular saw. Twenty specimens of dimensions 20 mm × 20 mm
× 20 mm (length × breadth × thickness) were prepared. The specimens were oven dried at 105 oC until the
constant weight of 12 % was achieved; thereafter, the dimensions and weight of specimens were measured.
Heat Treated Process
Heat treatment was carried out in a closed process vessel a muffle furnace with a temperature controlled
heating unit. Wood conditioning went through thermal modification at different temperature. The furnace was
ramped to desire temperature (160°C, 180°C and 200°C) before introducing wood samples. The samples were
heated for 30 mins. At the end of the furnace conditioning, the conditioned wood samples were cooled in a
desiccator over silica gel before they were subjected to further analysis.
Chemical Modification of Wood Specimens
In the chemical modification, set of selected wood samples were submerged in a preheated 70% of acetic
anhydride and 30% of acetic acidic solution contained in a closed stainless container placed inside an oven at
80oC for 180 min, under the atmospheric pressure and in the air. The wood samples were air-dried for 2 mins to
remove excessive moisture content in the wood sample and cooled.
The volume and the weight of specimen were determined after the samples had been cooled over silica gel in
desiccators for their dry weight. The percentage weight gain was determined using equation 1.
( ) Equation 1
Where, wo (g) is the oven-dry weight of specimens before treated and wt is the oven-dry weight of specimens
after treatment. The reaction for acetylation of wood is shown below:
Wood-OH + CH3-C (=O)-O-C(C=O)-CH3 Wood-O-C (=O)-CH3 +CH3-C (=O)-OH

Wood Acetic Anhydride Acetylated Wood Acetic Acid
Percentage weight gain (PWG) is the mass of chemical reagent retained in a sample as a percent of the
original theoretical oven-dry mass of the sample.
Physical Properties Test
i. Dimensional Stability Tests: Modified and untreated wood were submerged in distilled water in a plastic
container. A metal sheet was placed over the samples to hold them at approximately 2.5 cm below the
surface but this did not add or have any impact on the samples. Mass and volume measurement were
measured. Weight and volume gained were measured after 24, 48 and 72 hours respectively. At the end of
72 hours, the sample were removed drained of its excess water, oven dry and then cooled over silica gel in a
desiccator.
ii. Sorption Behaviour Test: For evaluation of sorption behaviour tests, fifteen substrates were used and
subjected to different humidity (Sodium hypochloride 7%, Potassium nitrate 48%, Sodium nitrate 65%,
ammonium sulphate 80% and copper sulphide 97%). The samples used in this study (control and modified
wood) were subjected to the same humidity so as to maintain one source of variation Wood samples were
kept in the desiccator that contains saturated solution and left in there until it has attained equilibrium
moisture content then moved to the next solution. Data in form of weight and volume were taken as samples
from one solution to the other. The dimension of wood samples for the test was 20 mm × 20 mm × 25 mm.
Data collected were then processed. Randomized Complete Block Design (CRD) was used to test for the
significance of the different treatment variables. Treatment means were separated using the Analysis of
Variance (ANOVA). For data obtained from this study using Excel and Statistical Package for Social
Sciences (SPSS) Range test at a= 0.05. The follow-up test was conducted to determine the difference
between means and choose the best treatment.

Results of the sorption behaviour of thermally and chemically modified Entandrophragma utile (Dawe &
Sprague) Sprague wood for the experiment were presented using statistical models, tables and percentages as
relevant to the objectives of the study.
Physical Properties
The results of the visual observation of colour changes for the thermally modified wood at two different
temperature and duration levels are presented. The results of the water absorption (WA), Percentage Weight
Gain (PWG) and Percentage Weight Loss (PWL) of the modified Entandrophragma utile wood were presented
in tables.
Appearance
The visual observation revealed that thermally treated Entandrophragma utile varied in relation to
temperature. Also, the colour after chemical modification was altered. The colour varied from light cream to
slightly brown at 160°C and very brown at 200°C for thermally modified wood. The color of the chemical
modified wood changes from being yellowish of the untreated wood to pale yellow in colour. The colour of the
control remained creamy. This degree of colour change can be attributed to some factors such as the chemical
composition of the extractives, wood pH, drying temperature, time and heating medium which can cause
hydrolysis and/or oxidation of wood components which corroborate the report of Sehlstedt-Persson (2003),
Owoyemi & Iyiola (2016). This colour change can be attributed to some chemical reactions that took place
during the heat process. The color change was noted throughout the cross-section, with the intensity being
greater on the surfaces than in the core. For a given treatment time, the amount of color change was greater for
specimens treated at 200ºC than for specimens treated at 160ºC at the same time duration. However, as observed
in this study, temperature regimes were found to have profound impact that time used on all the samples. The
changes in the color of thermally modified wood are attributed to oxidative changes, which predominate over
hydrolysis reactions (Owoyemi & Iyiola 2016). Furthermore, as reported by Owoyemi & Iyiola (2016), the un-
extracted and extracted acetone presents in the heat-treated wood could be the reason for varying colour after the
treatment. They further conclude that both the extractives and the structural component (hemicelluloses and/or
lignin) possibly took part in colour change experienced. Their observation also held for this study; the treated
wood was affected by thermal degradation which made the wood darker due to thermal degradation of lignin.
Percentage Weight Loss and Percentage Weight Gain
The result of Percentage Weight Loss (PWL) and Percentage Weight Gain (PWG) is presented in table 1.
The result gave a clear indication that PWL increases with temperature i.e. from 22.62% at 160°C to 26.46% at
180°C and 20.8% for PWG. This result agreed with the finding of Iyiola et al. (2017) on the impact of heat
treatment on physico-mechanical properties of thermally modified Anthocleistha djalonensis A.Chev. wood that
weight loss increases with increase in temperature and time. Statistically, the result of ANOVA revealed that
there were no significant differences recorded within the temperature range at P values ≤ 0.05 (Table 2). The
result of this change can be attributed to the various chemical changes that occur during thermolysis of wood
such as degradation of hemicelluloses, loss of water and volatile extractives and cellular breakdown of cell wall
polymers as reported by Hill (2006). The weight gain was as a result of replacing of hydroxyl group with acetyl
group at the sorption site and reduction in monolayer water sorption for acetylated wood. Several studies have
shown that if the wood is chemically modified well enough it will be highly durable compared to durability
class 1 i.e. it will be able to stand rottenness for up to 25 years.
Table 1. Percentage Weight Loss for thermally modified Entandrophragma utile (Dawe & Sprague) Sprague wood.
Treatment Mean Std. Error
160°C 22.62 1.12
180°C 26.47 0.97
200°C 24.53 1.73
Chemical Treatment (PWG) 20.84 3.05
Table 2. ANOVA of percentage weight loss for thermally modified Entandrophragma utile
(Dawe & Sprague) Sprague wood.
Sum of Squares Df Mean Square F Significance
PWL 110.961 2 55.480 2.137 0.131
Error 1090.515 42 25.965
Total 1201.476 44
Dimension Stability
The result of water absorption is presented in table 3. The result revealed that the value ranged from 8.60 to
16 %; 26 to 40.78 %; 35 to 50.35 % and 42.88 to 57.53 % for 1 hour to 78 hrs respectively. The result showed
that all through the period of water immersion, the woods that were chemically modified had the lowest rate of
water absorption. This is closely followed by thermally modified wood at 200°C. The result of ANOVAs as
shown in table 4 showed that there was a significant difference among various treatment and duration of
immersion. While the relationship between time and treatment as no significant difference in water absorption at
P value ≤ 0.05. The decrease in the hygroscopicity of thermal and chemical modified wood is attributed to the
decrease in the hemicellulose content as evident in the infrared spectra in the as shown by studies. Although in
the hygroscopic ranges, the moisture content of specimens is dominated by the number of hydrophilic sites in
wood, especially hydroxyl groups of carbohydrate, WA after immersion is mainly determined by the
permeability of wood (Ilker & Arif 2000). The main factor influencing the permeability of wood is the size and
volume of the gross capillary system comprising the vessels and pits. When wood is subjected to thermal
modification, lignin softens and blocks the cell pores probably decreasing the radius and number of effective
openings on pit membranes (Kocaefe et al. 2008). This could be one of the contributing factors to the reduction
in water absorption of thermally modified wood.
Table 3. Water absorption of thermally modified Entandrophragma utile (Dawe & Sprague) Sprague wood.
Hours/WA (%)
Treatment
1 24 48 72
Control 16.00±6.54 40.78±4.70 48.57±3.62 51.49±3.57
11.96±3.04 37.85±9.10 50.35±11.46 57.53±11.92
9.75±2.75 33.12±10.23 42.69±10.96 47.90±9.89
11.52±0.99 33.09±3.76 43.38±4.41 49.19±4.43
Chemical 8.60±0.95 26.11±1.54 35.79±0.46 42.88v0.46
Note: Values are mean ± Stdv.
Adsorption
The result of the rate of adsorption for modified and unmodified wood is presented in table 5. The result
showed an increase increases relative humidity. For the chemically modified wood the value ranged between
5.22% and 5.59%, with RH of 97% and 7% having the lowest and highest value respectively. The ANOVAs as
presented in table 6 showed that there was a significant difference on the effect of treatment on rate of
adsorption. While the different RH used as no significant effect on adsorption rate.
The weight of the wood increases as it moves from low relative humidity to a higher one, which contains
different salt constant like sodium hypochloride, potassium nitrate, sodium nitrate, ammonium sulphate, copper
sulphide. The effect of the chemical modification of wood on different relative humidity after the chemical
modified wood has been modified with a mixture of 30% of acetic acid and 70% acetic anhydride and exposed
to different relative. Ranging from 7% of sodium hypochloride to 48% of potassium nitrate, 65% of sodium
nitrate, 80% of ammonium sulphate and 97% of copper-sulphate. The weight of the wood increases as it move
from a low relative humidity to a higher one for thermally modified wood and decreases for chemically
modified wood untreated wood samples.
Table 4. ANOVA of water absorption for chemically and thermally modified Entandrophragma utile (Dawe &
Sprague) Sprague wood.
Source Sum of Squares Df Mean Square F Sig.
Treatment 1015.876 4 253.969 4.726 0.003
Time 12788.203 3 4262.734 79.322 0.000
Treatment *time 187.887 12 15.657 0.291 0.987
Error 2149.597 40 53.740
Total 16141.563 59
This is an indication that thermal modification Entandrophragma utile at different relative humidity has an
effect on the lignin concentration. The results clearly indicate that thermal modification significantly reduces
moisture adsorption in wood. In general, the WA of thermally modified wood increases with an increase in the
relative humidity. However, at different relative humidity, there was no significant difference (p>0.05). This
indicates that an increase in the different relative humidity has more impact on water absorption of chemically
modified wood compared to the thermally modified wood.
Table 5. Adsorption Rate for chemically and thermally modified Entandrophragma utile (Dawe & Sprague)
Sprague wood Under Different Relative Humidity.
Relative Humidity (%)
Treatment
7 48 65 80 97
Control 5.58±0.09 5.38±0.07 5.19±0.16 5.24±0.37 5.21±0.35
4.34±0.33 4.57±0.40 4.53±0.31 4.65±0.39 4.71±0.48
4.41±0.39 4.45±0.29 4.56±0.08 4.59±0.08 5.62±0.08
4.36±0.20 4.37±0.04 4.48±0.04 4.54±0.08 4.65±0.09
Chemical 5.59±0.45 5.38±0.36 5.22±0.30 5.22±0.30 5.22±0.30
Table 6. ANOVA of Percentage Weight Gain for chemically and thermally modified
Entandrophragma utile (Dawe & Sprague) Sprague wood.
Source Sum of Squares Df Mean Square F Sig.
Treatment 10.852 4 2.713 34.541 0.000
RH 0.028 4 0.007 0.089 0.986
Error 5.184 66 0.079
Total 16.064 74
CONCLUSION
Sorption behaviour of modified wood was investigated in this study using heat and chemical. The study
revealed that there was a reduction in the weight and density of thermally treated wood as a result of thermolysis
and the weight gain from chemical modification was above 20%. This is an indication that wood properties were
found to be improved in terms of water absorption and bulkiness. It was found also that, water permeability is
based on the moisture content which is influenced by modification and not the relative humidity. From the result
obtained in this study, it was clear that chemical and thermal modification control can be used to increase
morphological properties of wood which utilization.
ACKNOWLEDGEMENTS
The authors sincerely acknowledge the Technologist of the Department of Forestry and Wildlife
Technology, Owerri, Nigeria for their time and advice in the course of this study.
REFERENCES
Akanbi MO & Ashiru MO (2002) A handbook of Forest and Wood Insects of Nigeria. Agbo Area Publishers,
Ibadan, pp. 66.
Boonstra MJ, Van Acker J, Tjeerdsma BF & Kegel EF (2007) Strength properties of thermally modified
softwoods and its relation to polymeric structural wood constituents. Ann. Forest Sci. 64(7): 679–690.
Hill CAS (2006) Wood modification, thermal and other processes. John Wiley and Sons, Ltd.
Ilker USTA & Arif GURAY (2000) Comparison of the Swelling and Shrinkage Characteristics of Corcisan Pine
(Pinus nigra var. Mantima) Turkish Journal of Agriculture and Forestry 24: 461–464.
Iyiola EA, Olufemi B, Owoyemi JM & Fuwape JA (2017) Impact of Heat Treatment on Physico-Mechanical
Properties of Thermally modified Anthocleistha djalonensis Wood. Journal of Materials Sciences and
Applications 3(2): 28–34.
Kocaefe D, Shi JL, Yang DQ & Bouazara M (2008) Mechanical properties, dimensional stability, and mold
resistance of heat-treated jack pine and aspen. Forest Products Journal 58: (6): 88–93.
Kollmann F & Fengel D (1965) Changes in the chemical composition of wood by thermal treatment. Holz als
Roh- und Werkstoff 23(12): 461–468.
Mazela B, Zakrzewski R, Grzeskowiak W, Cofta G & Bartkowiak M (2004) Preliminary research on the
biological resistance of thermally modified wood. In: Abstracts of the First European Conference on Wood
Modification. Ghent, Belgium.
Mohareb A, Sirmah P, Pétrissans M & Gérardin P (2012) Effect of heat treatment intensity on wood chemical
composition and decay durability of Pinus patula. European Journal of Wood and Wood Products 70(4):
519–524.
Owoyemi JM & Iyiola EA (2016) Effects of Thermal Treatment on Selected Physical and Mechanical
Properties of Rubber (Hevea brasiliensis) Wood. Journal of Applied Tropical Agriculture 21(1): 190–195.
Peters J, Pfriem A, Horbens M, Fischer S & Wagenführ A (2009) Emissions from thermally modified beech
wood, their reduction by solvent extraction and fungicidal effect of the organic solvent extracts. Wood
Materials Science and Engineering 4(1-2): 61–66.
Rowell RM (2005) Chemical modification of wood. In: Handbook of wood chemistry and wood composite.
CRC Press, USA. [ISBN: 9780849315886]
Sehlstedt-Persson M (2003) Colour responses to heat treatment of extractives and sap from pine and spruce. In:
8th International IUFRO Wood Drying Conference. Brasov, Romania, pp. 459–464.
Shupe T, Lebow S & Ring D (2008) Causes and control of wood decay, degradation and stain. Louisiana State
University Agriculture Center, Baton Rouge, LA. Publication, 27 p.
Stamm AJ (1964) Wood and cellulose science. Ronald Press, New York, USA.
Tiemann HD (1915) The effect of different methods of drying on the strength of wood. Lumber World Review
28(7): 19–20.
Windeisen E, Bächle H, Zimmer B & Wegener G (2009) Relations between chemical changes and mechanical
properties of thermally treated wood. Holzforschung 63(6): 773–778.
Research article
Antibacterial properties of Ipomoea staphylina Roem & Schult.

plant extracts with comparing its preliminary qualitative
phytochemical and quantitative GC-MS analysis
Padmashree M. S. 1, Roopa B.1, Ashwathanarayana R.2* and Raja Naika2
1
Department PG Studies and Research in Botany, Tumkur University, Tumkur-572103, Karnataka, India
2
Department PG Studies and Research in Applied Botany, Jnanasahyadri, Kuvempu University,
Shankaraghatta, Shimoga-577451, Karnataka, India
*Corresponding Author: ashwinjamadagni497@gmail.com [Accepted: 18 December 2018]
Abstract: Ipomoea staphylina plant ethanolic extract was subjected to preliminary qualitative
phytochemical and quantitative GC-MS analysis with analyzing its antibacterial activity by the
standard method. The preliminary qualitative phytochemical analysis is done by standard
procedure and quantitative GCMS analysis is done by subjecting it to GC Model: Thermo Trace
GC Ultra model instrument. Antibacterial activity is done by the standard agar well diffusion
method. Preliminary qualitative phytochemical analysis for pet ether and chloroform extract
confirms for fewer phytochemicals but ethanolic extract confirms for the presence of alkaloids,
saponins, flavonoids, steroids, glycosides, phenols and sterols. GC-MS analysis of I. staphylina
ethanolic leaf extract confirms the presence of 79 compounds, out of these 24 compounds were
unknown and 55 compounds were known for its medicinal properties. The antibacterial
experiment revealed that I. staphylina plant ethanolic extract has appreciable antibacterial activity
in all tested concentrations against selected bacterial pathogens but comparably less with the
standard ciprofloxacin used but pet ether and chloroform extract showed negligible antibacterial
activity. I. staphylina plant could be exploited as a valuable source of antibacterial agent enriching
with known antibacterial compounds.
Keywords: Ipomoea staphylina - Phytochemical analysis - Antibacterial activity - ciprofloxacin.
[Cite as: Padmashree MS, Roopa B, Ashwathanarayana R & Naika R (2018) Antibacterial properties of
Ipomoea staphylina Roem & Schult. plant extracts with comparing its preliminary qualitative phytochemical
and quantitative GC-MS analysis. Tropical Plant Research 5(3): 349–369]
INTRODUCTION
Ipomoea staphylina Roem & Schult. is a climber plant grows near water resources and distributed
throughout India, China, and Sri Lanka Deciduous forests. Leaves were stout stragglers. 15 × 10 cm dimension,
broadly ovate, base cordate, apex acute, membranous, nerves oblique; petiole 6.5 cm. Flower is Panicle of
cymes axillary, to 15 cm; pedicels 0.5–1.0 cm; bracts minute; outer sepals 5 × 4 mm, oblong, obtuse, inner
obovate with hyaline margins, 6 × 5 mm; corolla 2 cm long, shallowly 5-lobed, funnel-shaped, pink; stamens 5,
included, base dilated, hairy, filaments 7–8 mm; anthers 3 mm; ovary 2 mm; style 1.5 cm, stigma 2, globose. In
axillary or subterminal panicles; white with a purple throat. Flowering from December–March. Fruit is
subglobose capsule; seeds oblong, subtrigonous, hairy at top. Fruiting is from January-April. (Gamble: Ipomoea
staphylina Vol- II, 1883; Fig. 1)
PLANT DESCRIPTION
Ipomoea staphylina Roem. & Schult, Syst. Veg. 4: 249 1819.
Ipomoea dichroa Choisy, Prodr. 9: 364 1845.
Common names: Clustered Morning Glory, Kannada: Ugina kodi, Unang kodi, Sunang kodi, Tamil:
Onaankodi, Onan Kodi.
Padmashree et al. 2018
Taxonomic Hierarchy
Kingdom Plantae
Subkingdom Viridiplantae
Infrakingdom Streptophyta
Superdivision Embryophyta
Division Tracheophyta
Subdivision Spermatophytina
Class Magnoliopsida
Superorder Asteranae
Order Solanales
Family Convolvulaceae
Genus Ipomoea
Species Ipomoea staphylina Roem. & Schult
Figure 1. Ipomoea staphylina Roem & Schult.: A, Plant habit; B, Plant flower; C, Collection of plant material; D, Shade
drying of the plant sample; E, Plant sample grinded; F, Soxhlet extraction with different solvents; G, Extracts collected in
glass container; H, Priliminary phytochemical analysis of plant extracts.
Traditional Uses
The plant I. staphylina has been used in different systems of traditional medication for the treatment of
diseases and ailments of human beings. It has been reported for its analgesic (Nagariya et al. 2010), anti-
inflammatory (Kirtikar & Basu 1999, Sarvalingam et al. 2011), anti-diarrheal, gastro protective properties
(Suresh et al. 2011).
In Tamil Nadu Kanikkars tribal people of Tirunelveli District, used I. staphylina leaf latex to cure foot
crack (Muralidharan et al. 2012), in Coimbatore, Tamil Nadu Irulas and Palliyars tribes were eaten the plant
leaves in raw and roots were used as an antidote for snake-bite (Balasubramanian et al. 1997, Arinathan et al.
2007, Sarvalingam et al. 2014), root tubers were rich with starch were eaten in raw (Mohan & Kalidass 2010),
Valaiyans tribes of Karandamalai, Dindigul District, Tamil Nadu Ipomea plant leaves were boiled and made
decoction used in the treatment of stomach disorders (Kottaimuthu 2008), In Andhra Pradesh, the tribes called
Chenchus used the plant leaves in the treatment of piles (Kumar & Pullaiah 1999).
I. staphylina is used as purgative, dyspepsia, anthelmintic, bronchitis (Savitramma et al. 2015) and the I.
staphylina is used for respiratory disorders (Reddy et al. 2013). A literature review reveals anti-inflammatory
activity (Firdous & Koneri 2012a,b), 5-lipoxygenase, α-glucosidase and α-amylase inhibitory activity of I.
staphylina. Analgesic activity of water and the methanolic extract of I. staphylina was also reported against
acetic acid-induced writhing test (Kumar et al. 2013). Some report proved that I. staphylina has anti-ulcer
properties (Banerjee & Firdous 2015), Antidiabetic properties (Firdous & Singh 2016), Hepatoprotective and
nephroprotective properties (Bag & Mumtaz 2013).
Scientific work done
It has been reported as a analgesic (Nagariya et al. 2010, Kumar et al. 2013), anti-inflammatory (Kirtikar &
Basu 1999, Sarvalingam et al. 2011); Firdous & Koneri 2012a,b), anti-diarrheal, gastroprotective effect (Suresh
et al. 2011), anti-ulcer properties (Banerjee & Firdous 2015), Antidiabetic properties (Firdous & Singh 2016), α-
amylase inhibitory activity (Kumar et al. 2013), antioxidant (Firdous & Singh 2016), Hepatoprotective and
nephroprotective activity (Bag & Mumtaz 2013). Bioactive chemical constituents like Sitosteryl-3-O-β-D-
glucoside and chiro deoxy inositol were reported from the leaves of I. staphylina (Reddy et al. 2013).
The aim of the research topic is to evaluate the antibacterial properties along with the confirmation of
phytoconstituents present in it by preliminary qualitative phytochemical and quantitatve GC-MS analysis.

Plant collection and authentication
The plant materials were collected from 13.367190° N, 77.101176° E, near Tomlinson church, Tumkur
District, Karnataka in January 2018. The plant was authenticated by Dr. Y. N. Seetharam, Tumkur University
and the voucher specimen was conserved under the reference number TU/BD/PMS/001 (Fig. 2).
Figure 2. location where experiment plant was collected.

Plant preparation and extraction
The plant samples were dried in shade for 20 to 25 days, mechanically powdered and subjected to Soxhlet
extraction using petroleum ether, chloroform and ethanol (De-Castro & Ayuso 1998) The crude extracts were
stored at 4 C in air-tight plastic containers.
Preliminary phytochemical screening
Soxhlet extracted solvent crude extracts were screened for the presence of tannins, alkaloids, saponin,
glycosides, flavonoids, steroids/sterols and phenols using standard methods (Harborne 1998, Ajaiyeoba 2000).
Each extract was subjected to phytochemical investigation, to study the presence of the following constituents
viz., Alkaloid, Flavonoids, Glycosides, Saponin, Steroids, Tannins and Phenols.
GC-MS analysis
Plant extracts were subjected to Gas Chromatography and mass spectroscopy (GC-MS) obtained spectra was
analyzed. GC Model: Thermo Trace GC Ultra, MS Model: Thermo DSQ II, Ionization: Electron Impact
Ionisation (EI), Chemical Ionisation (CI), Mass Range: 1 - 1074 m z-1. The column used is HP-5MS UI (cross-
linked 5% methyl phenyl Silox) capillary column (30 m × 0.25 mm) and the film thickness is 0 25 μm The oven
temperature was increased from 50–200 °C at a rate of 10ºC min-1. Then, continued 200–300 °C at rate 30 °C
min-1. Then post run for 10 minutes in 300°C. Pure helium gas was used as the carrier gas with flow rate of 1
mL min-1. Injector and detector temperatures were 250°C. GC-MS was done by injecting 1 μL of sample (0 1 %
in absolute methanol).
Antibacterial activity
i. Microorganisms used: Pathogenic bacterial strains like Xanthomonas campestris (MTCC-2286),
Pseudomonas syringae (MTCC-1604), Klebsiella pneumonia (MTCC-7028), Escherichia coli (MTCC 1559),
Salmonella typhi (MTCC-734), Pseudomonas aeruginosa (MTCC-1934), Staphylococcus aureus (MTCC-902)
obtained from the Institution of Microbial Technology (IMTECH), Chandigarh, India were used.
ii. Medium used and composition: Nutrient agar (NA) media used for the culturing of experimental bacterial
pathogens. All the ingredients used in the preparation of the nutrient agar medium is of analytical grade.
Composition of nutrient agar media-
Beef extract 3.00 gm
Peptone 5.00 gm
Sodium Chloride (NaCl) 5.00 gm
Agar 15.00 gm
Distilled water 1000 ml
pH 7.4
iii. Preparation of media: Nutrient agar was prepared by adding 3 gm. of beef extract, 5gm of sodium chloride,
15 gm agar was dissolved in 1000 ml of distilled water, pH of the solution was adjusted to 7.4 and then
sterilized for 15 min at 15 lbs. pressure in an autoclave.
iv. Preparation of subcultures: One day prior to the experiment, the microorganisms were inoculated into the
sterilized tubes containing nutrient broth and incubated at 35ºC for 24 hrs.
v. Sterilization of media and glassware: The media used in the present study is nutrient agar and nutrient broth,
were sterilized in the conical flask of the suitable capacity of autoclaving at 15 lbs.
vi. Standard drug: Antibacterial drug Ciprofloxacin (1 mg ml-1 of sterile distilled water) were used as a standard
antibiotic to compare with the plant crude extracts.
vii. Preparation of bacterial cultures: The test pathogenic bacteria were aseptically inoculated in sterile test tubes
using nutrient broth and incubated at 37ºC for 24 hours. The plant crude extracts were diluted with 10% DMSO
make it to desired concentrations of 12.5, 25, 50 and 100 mg ml-1 respectively. The drug Ciprofloxacin was used
as a standard antibiotic (1 mg ml-1 of sterile distilled water) to compare with the plant crude extracts. Nutrient
agar plates were uniformly swabbed with respected bacterial strains cultured in nutrient broth. 0.6 cm diameter
wells were punched in the inoculated plates using a sterile cork borer. 100 µl of different concentrations of
crude extracts and standard (Ciprofloxacin, 1 mg ml-1 of sterile distilled water) and DMSO (10%) were filled
into the respectively labeled wells and incubate for 24 hours at 37ºC.
RESULTS
Extracts yield of Ipomoea staphylina leaf
Table 1. The percentage yield of crude extracts.
Organic Solvent used Yield of extracts in grams % of Yield
Petroleum ether 1.305 3.95
Chloroform 2.88 8.76
Ethanol 28.693 87.28
Soxhlet extraction of Ipomoea staphylina leaves (700 grams) with solvents like petroleum ether gives 1.30
grams (3.95%), with chloroform 2.88 grams (8.76%) , and with ethanol 28.69 grams (87.28%). The result shows
that the highest yield is obtained in ethanol followed by chloroform and least is petroleum ether (Table 1; Fig.
3).
Figure 3. Yield of crude extracts in percentage.

Preliminary qualitative phytochemical analysis of Ipomoea staphylina leaf extracts
The preliminary phytochemical analysis of Ipomoea staphylina leaf extracts were analyzed results showed
that, the analysis of petroleum ether extract reveals the presence of saponins, steroids, glycosides and sterols.
The chloroform extracts shows the presence of saponins, steroids, sterols. The ethanolic extract give positive
result for alkaloids, saponins, flavonoids, steroids, glycosides, phenols and sterols (Table 2).
Table 2. Preliminary qualitative phytochemical analysis of Ipomoea staphylina Roem & Schult. leaf extracts.
S.N. Secondary Name of the Test Petroleum Chloroform Ethanolic
Metabolites ether Extract Extract Extract
1 Alkaloids Mayer’s test - - +
Wagner’s test - - +
2 Saponins Foam test + + +
3 Tannins Ferric chloride test - - -
Ferric chloride test - - +
Shenoda test - - +
4 Flavonoids Zinc HCl reduction test - - -
Alkaline reagent test - - +
Lead acetate test - - +
5 Steroids Salkowski test + + +
Keller-Kiliani test + + +
6 Glycosides Legal's test + - -
Ferric chloride test - - +
7 Phenols Ellagic acid test - - -
8 Sterols Liebermann Burchard test + + +
Note: -, Negative results; +, Positive results.
Quantitative GC-MS analysis of Ipomoea staphylina leaf extracts
Due to the less extract yield and less secondary metabolites present in petroleum ether and chloroform
extracts, we took only leaf ethanolic extract of Ipomoea staphylina for Gas chromatography mass spectroscopy
(GC-MS) analysis.
GC-MS analysis of I. staphylina ethanolic leaf extract confirms the presence of 79 compounds, out of these
24 compounds were unknown and 55 compounds were known for its medicinal properties, most of them were
antimicrobial agents 18 in numbers, followed by 16 food additive and flavoring agents, 15 compounds were
antioxidant, 14 compounds have anticancer properties, 14 compounds were Anti-hypercholesterolemic, 12
compounds were anti-inflammatory agents, 6 compounds were cytotoxic, 6 compounds were used in cosmetics
and perfumeries, 6 compounds have hepatoprotective properties, 5 compounds have antiviral properties, 5
compounds were analgesic, 4 compounds were insect pheromones, rest of them were Allergenic, Anesthetic,
Antimutagenic, Antispasmodic, Choleretic, Dermatitigenic, Fungicide, Herbicide, Laxative, Pesticide,
Lipoxygenase-Inhibitor, Pesticide, Tyrosinase Inhibitor, Vermifuge etc. major compound is Dodecanoic acid, 3-
hydroxy- (10.41%), followed by 9-Hexadecen-1-ol (9.52%), 9-Octadecen-1-ol (8.56%), Hydroperoxide, 1-
ethylbutyl (5.88%) etc. and the least percentage is 3,3,7,11-Tetramethyltricyclo[5.4.0.0(4,11)] undecan-1-ol
(0.06%) (Table 3; Figs. 4–5).
Table 3. GC-MS analysis of Ipomoea staphylina Roem & Schult. ethanolic extract. [%- Percentage in the crude extract]
S.N. % Chemical name Properties References
1 0.11 2-Furanmethanol Moderately toxic, Flavoring Agents, Fuster et al. (2000),
important constituent of urine, present Yanagimoto et al.
in the aroma of coffee, tea, wheat (2002), PubChem-
bread, crispbread, soybean, cocoa, rice, Furfuryl
potato chips, Adhesives and Sealants, alcohol/C5H6O2
anti-oxidative activity.
2 0.4 Propane, 1-(1-methylethoxy)- Inhibitors of Hepatitis C Virus, used in PubChem- Propane, 1-
the treatment of mental disorders. (1-
methylethoxy)/C6H14O
3 0.98 2-Propanone, 1,3-dihydroxy- Used in the treatment of vitiligo, in PubChem-
cosmetics, antifungal agent in creams, Dihydroxyacetone/
Flavoring Agents, intermediate of C3H6O3
bacterial metabolism, less toxic
commonly derived from sugar beets
and sugar cane.
4 0.06 Butyrolactone cdc2 and cdk2 kinases inhibitor, anti- Giarman & Schmedit
cancer activity, antimicrobial, (1963), Nishio et al.
antidepressant, Flavoring Agents. (1996), Kitagawa et
al. (1994), PubChem-
Butyrolactone/C4H6O2
5 0.23 2,4-Dihydroxy-2,5-dimethyl-3(2H)-furan-3-one Food-grade flavor ingredients. PubChem- 2,4-
Dihydroxy-2,5-
dimethyl-3(2H)-
furanone/C6H8O4
6 0.54 Diglycerol Skin moisturizers, Hand cleaners, Nelson et al. (1989),
Insect repellent lotions and sprays, PubChem-
Deodorants, Chewing gums, Diglycerin/C6H14O5
combinational drugs used in the
treatment of respiratory and urinary
track disorders.
7 0.1 2,3-Dioxabicyclo[2.2.1]heptane, 1-methyl- Unknown
8 0.06 Pentanoic acid, 4-oxo- Hepatoprotective, Flavoring Agents. PubChem- Levulinic
acid/C5H8O3
9 0.86 Cyclopentane, 1-acetyl-1,2-epoxy- Anti-inflammatory, antiviral and Awakan et al. (2018)
bronchodilatory properties.
10 0.23 Tetrahydro-4H-pyran-4-ol Hepatitis C virus inhibitors, treatment PubChem- Oxan-4-ol/
of respiratory and urinary track C5H10O2
disorders.
11 0.08 Uracil, 1-methyl- Antiviral Compounds. PubChem- 1-
Methyluracil/
C5H6N2O2
12 0.1 Alpha-amino-gamma-butyrolactone Unknown
13 1.92 4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6- Mutagen, Antimicrobial, anti- Hiramoto et al.
methyl- inflammatory and antioxidant capacity. (1997), Kumar et al.
(2010), Yu et al.
(2013)
14 0.21 1,2-Benzenediol Antibacterial, Flavoring Agents, Xu et al. (2003),
Antioxidants, moderately toxic, PubChem-
treatment of respiratory and urinary Pyrocatechol/ C6H6O2
track disorders.
15 0.21 2-[2-(5-Norbornenyl)oxy]-tetrahydropyran Unknown
16 2.02 2-Furancarboxaldehyde, 5-(hydroxymethyl)- Antimicrobial, Preservative. Gopalakrishnan et al.

(2011)
17 0.97 1,2,3-Propanetriol, monoacetate Food Additives PubChem- 1,2,3-
Propanetriol,
monoacetate/ C5H10O4
18 5.88 Hydroperoxide, 1-ethylbutyl Unknown
19 2.68 1-Deoxy-d-arabitol Unknown
20 0.18 2-Methoxy-4-vinylphenol Can induce cell cycle arrest, Jeong & Jeong (2010),
antibacterial, Anti-inflammatory, Silici et al. (2005),
antioxidant, flavoring agent, also acts Jeong et al. (2011),
as insect pheromones. Fukai et al. (2009),
Pherobase.com- 2-
methoxy-4-
vinylphenol;
PubChem- 2-
Methoxy-4-
vinylphenol/ C9H10O2
21 0.08 3,4-Altrosan Bacteriostat, Fungicide. Jadhav et al. (2014),
22 0.4 Caryophyllene Local anaesthetic, Non-Steroidal Anti- Ghelardini et al.
inflammatory, Anticancer, Analgesic, (2001), Tambe et al.
Gastric cytoprotective, Antimicrobial, (1996), Sabulala et al.
induces apoptosis, moderate cytotoxic, (2006), Yang et al.
antioxidant, anticancer antipyretic, (2000), Huang et al.
platelet-inhibitory and Inhibition of (2012), Park et al.
prostaglandin synthesis, sedative, (2011), Calleja et al.
fungicide. (2013), Dahham et al.
(2015), Kumar et al.
(2010), PubChem –
Caryophyllene/C15H24
23 0.58 Benzaldehyde, 2-hydroxy-6-methyl- Pheromone of the Acarid Mite Leal et al. (1988),
Tyrophagus perniciosus, Collohmannia PubChem- 6-
gigantea, Dermatophagoides farinae, Methylsalicylaldehyde
Acarus siro, Tyrophagus neiswanderi. / C8H8O2
Used in the treatment Cancer, sexual
or genital disorder, antipyretic, anti-
inflammatory, analgesic, treatment in
immunological or allergic disorder.
24 1.54 Sucrose As a sweetener in foods and soft PubChem – Sucrose /
drinks, in the manufacture of syrups, in C12H22O11
invert sugar, confectionery, preserves
and jams, demulcent, beverages,
medications, pharmaceutical products,
and caramel.
25 0.36 alpha.-Caryophyllene Essential oil present in Humulus Fernandes et al. (2007),
lupulus, anti-inflammatory, Antitumor Legault et al. (2003),
activity, analgesic, anti-inflammatory, Legault & Pichette
antiseptic, immunostimulant, (2007), PubChem-
perfumes. Humulene / C15H24
26 0.47 DL-Arabinitol Indicator of liver cirrhosis, Wong et al. (1990)
gastrointestinal candidiasis etc in
serum and urine.
27 10.41 Dodecanoic acid, 3-hydroxy- In the treatment of Fatty Acid Jones et al. (2000)
Oxidation disorder, intermediate of
liver fatty acid metabolism.
28 0.37 4-((1E)-3-Hydroxy-1-propenyl)-2- Antimicrobial, Antioxidant, Gopalakrishnan et al.

methoxyphenol Antiinflammatory, Analgesic. (2011)
29 0.17 Cyclohexanol, 1R-4-trans-acetamido-2,3-trans- Camphorlike odor and are used in Cyclohexanols / CTD
epoxy- making soaps, insecticides, germicides,
dry cleaning, and plasticizers.
30 1.2 3,7,11,15-Tetramethyl-2-hexadecen-1-ol Treatment in asthma, antimicrobial, Ogunlesi et al. (2009),
cancer preventive, Anti-inflammatory. Yu et al. (2013),
Ponnamma &
Manjunath (2012),
Srinivasan et al. (2014)
31 0.37 Oxirane, hexadecyl- Unknown
32 0.15 Hexadecanoic acid, 15-methyl-, methyl ester Unknown
33 0.17 Oleic Acid Antimicrobial, edible oils, Fish Oil Dilika et al. (2000),
Supplementation, Colorectal Cancer PubChem- Oleic acid/
Prevention, Flavoring Agents, C18H34O2
Insecticide, Acaricide, Herbicide, Plant
growth regulator, Surfactants
Lubricants, Paint additives.
34 5.53 Pentadecanoic acid Adhesives and sealant chemicals, PubChem-
Agricultural chemicals (non- Pentadecanoic acid/
pesticidal), Finishing agents, C15H30O2
Lubricants and lubricant additives,
Surface active agents.
35 0.15 Hexadecanoic acid, ethyl ester Antioxidant, lubricant, Kumar et al. (2010),
hypocholesterolemic nematicide, Maruthupandian &
pesticide, anti-androgenic, flavoring Mohan (2011), Dr.
agent, hemolytic, 5-Alpha reductase Duke's Phytochemical
inhibitor. and Ethnobotanical
Databases
36 0.12 d-Mannose Protein quality control in human body. PubChem- D-
Mannose/ C6H12O6
37 2 Oleyl Alcohol Savory, emulsion stabilizers, surfactant PubChem- Oleyl
- emulsifying agents, antifoaming alcohol/ C18H36O
agents, and skin conditioning agents,
cosmetics, protect the outer surface of
plants and animals from water loss,
chemical intermediate, automotive
lubricant, defoamer, cosolvent and
plasticizer for printing ink, Oleyl
alcohol is a natural product in fish oils.
38 0.12 1-Heptadecanol Flavoring Agents, Insect sex Butler & McDonough
pheromone, antiacne agents, antibiotic. (1981), Kubo et al.
(1994), PubChem- 1-
Heptadecanol/ C17H36O
39 0.92 Phytol Antimicrobial, Anti-inflammatory. Srinivasan et al. (2014)
40 2.3 9,12-Octadecadienoic acid (Z,Z)- Anticoronary, Antialopecic, Ponnamma &
Antiarteriosclerotic, Antiarthritic, Manjunath (2012),
antianaphylactic, Antieczemic, Cancer Maruthupandian &
preventive, antiprostatic, Mohan (2011),
hepatoprotective, Hypocholesterolemic, Kalaivani et al. (2012)
Metastatic, Nematicide, Insectifuge,
Antihistaminic, Antieczemic, Antiacne,
5-Alpha reductase inhibitor
Antiandrogenic, Antiarthritic,
Anticoronary,
41 3.54 9,12,15-Octadecatrienoic acid, methyl ester, Anti-inflammatory, Srinivasan et al.

(Z,Z,Z)- Hypocholesterolemic, Antihistaminic. (2014)
42 1.21 Octadecanoic acid Cosmetic, Flavor, Ponnamma &
Hypocholesterolemic, Lubricant, Manjunath (2012)
Perfumery, Propecic, Suppository.
43 8.56 9-Octadecen-1-ol, (E)- pheromonal component from the sting Pickett et al. (1982)
of the honey bee.
44 0.72 1-Nonadecanol Unknown
45 0.68 12-Chlorobicyclo[8.2.0]dodecan-11-one Unknown
46 0.15 Z,E-2,13-Octadecadien-1-ol Insect sex pheromone component. Schwarz et al. (1983)
47 9.52 9-Hexadecen-1-ol, (Z)- Cosmetics, anti-hair fall agent. PubChem-9-Hexadecen-
1-ol/ C16H32O
48 0.44 1-Tetracosanol Unknown
49 2.85 Hexadecanoic acid, 2-hydroxy-1- Unknown
(hydroxymethyl)ethyl ester
50 0.14 Ethanol, 2-(9-octadecenyloxy)-, (Z)- Unknown
51 0.25 cis-9-Hexadecenal Insect pheromone. Berg et al. (2005)
52 1.63 9,12-Octadecadienoic acid (Z,Z)-, 2,3- hypocholesterolemic, antieczemic, Gnanavel & Saral
dihydroxypropyl ester Nematicide, hepatoprotective. (2013)
53 1.28 Methyl (Z)-5,11,14,17-eicosatetraenoate Unknown
54 0.5 Octadecanoic acid, 2,3-dihydroxypropyl ester Food additive in Dairy, Surfactants, PubChem- Glyceryl
Antiviral. monostearate/ C21H42O4
55 0.15 Octacosanoic acid, 2,4,6,8-tetramethyl-, methyl Unknown
ester, [2R-(2R*,4R*,6R*,8R*)]-
56 1.4 2,6,10,14,18,22-Tetracosahexaene, Antibacterial, Antioxidant, Cancer- Ponnamma &
2,6,10,15,19,23-hexamethyl-, (all-E)- preventive,Antitumor, Manjunath (2012)
Immunostimulant, perfumery,
Pesticide, Sunscreen.
57 0.06 1,5,9,9-Tetramethyl-2- Unknown
oxatricyclo[6.4.0.0(4,8)]dodecane
58 0.5 1-Hexacosanol naturally in the epicuticular wax and Wikipedia - 1-
plant cuticle of many plant species. Hexacosanol
59 0.16 Oxirane, 2,2-dimethyl-3-(3,7,12,16,20- Unknown
pentamethyl-3,7,11,15,19-heneicosapentaenyl)-,
60 0.06 3,3,7,11- Fungistatic, Ginsenol is found in tea. Aleu et al. (2001),
Tetramethyltricyclo[5.4.0.0(4,11)]undecan-1-ol Ginsenol is isolated from ginseng plant PubChem- Ginsenol/
rootlets. C15H26O
61 0.34 gamma.-Tocopherol Anticancer, Antioxidant, Antitumor, Ponnamma &
Antiinflammatory, Manjunath (2012)
Hypocholesterolemic,
Cardioprotective.
62 0.28 1-Triacontanol Plant growth stimulator. Jones et al. (1979),
Khandaker et al. (2013)
63 0.56 Vitamin E Antiaging, Antialzheimeran, Ponnamma &
Antidermatitic, Antidiabetic, Manjunath (2012),
Antioxidant, Antitumor, Cancer- Srinivasan et al.
preventive, Hypocholesterolemic, (2014), Kumar et al.
Immunostimulant, analgesic, anti- (2010)
inflammatory, antioxidant,
antidermatitic, antileukemic,
hepatoprotective, ypocholesterolemic,
antiulcerogenic, vasodilator,
antispasmodic, antibronchitic,
anticoronary.
64 0.15 beta.-Sitosterol Anti-hypercholesterolemia, Reduces Kalaivani et al. (2012)

blood levels of cholesterol, antioxidant
activity, anticancer.
65 1.03 Campesterol Antioxidant, Hypocholesterolemic. Ponnamma &
Manjunath (2012)
66 1.77 Stigmasterol Antihepatotoxic, Antiviral, Ponnamma &
Antioxidant, Cancer preventive, Manjunath (2012)
Hypocholesterolemic.
67 0.69 4-Acetoxycinnamic acid Plant growth inhibitor. Hiradate et al. (1999)
68 0.1 Squalene Antibacterial, Antioxidant, Srinivasan et al.
Immunostimulant. (2014)
69 3.77 gamma.-Sitosterol used to treat Hyperlipidemias, Venkata et al. (2012),
Antioxidant, antibacterial and Akpuaka et al. (2013),
prophylactic activities. Pubchem- gamma-
Sitosterol/ C29H50O
70 0.16 Cholest-5-en-3-ol, 24-propylidene-, (3.beta.)- Unknown
71 0.11 Cyclohexanecarboxylic acid, 4-butyl-, 4- Unknown
pentylphenyl ester
72 0.95 4,4,6a,6b,8a,11,11,14b-Octamethyl- Unknown
1,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,14,14a,14b-
oc
73 0.28 9,19-Cyclolanost-24-en-3-ol, acetate Unknown
74 2.19 Lupeol Antibacterial, Antioxidant, Antitumor, Maruthupandian &
Cancer preventive, Immunostimulant, Mohan (2011)
Chemo preventive, Lipoxygenase
inhibitor, Pesticide.
75 0.1 D:B-Friedo-B':A'-neogammacer-5-en-3-ol, Unknown
(3.beta.)-
76 4.9 Cyclohexane, 1,2-dimethyl-3,5-bis(1- Unknown
methylethenyl)-, (1.alpha.,2.beta.,3.beta.,5.alpha.)-
77 0.33 2-Propenoic acid, 3-(4-hydroxyphenyl)- Antibacterial, flavor, Aldose- Ponnamma &
Reductase-Inhibitor, Allergenic, Manjunath (2012),
Anesthetic, Antiinflammatory, Kumar et al. (2010)
Antimutagenic, Antispasmodic, Cancer
Preventive; Choleretic;
Dermatitigenic,Fungicide, Herbicide,
Laxative, Pesticide, Lipoxygenase-
Inhibitor, Pesticide, Tyrosinase
Inhibitor, Vermifuge.
78 2.46 D:A-Friedooleanan-3-ol, (3.alpha.)- Unknown
79 0.53 Friedelan-3-one Unknown
Antibacterial activity of Ipomoea staphylina leaf extracts
Antibacterial activity of Ipomoea staphylina leaf extracts were measured in millimeters and expressed in
Mean ± standard error of mean (SEM) and the experiment is triplicated to know the exact concentration value.
Petroleum ether extract of I. staphylina leaf showed nil antibacterial activity in all tested concentrations along
with the control the standard Ciprofloxacin showed excellent antibacterial activity against all the tested bacterial
pathogens. For accurate values the experiment is triplicated and the values were expressed in terms of Mean ±
standard error of mean (SEM). The values obtained from the petroleum ether extract were shown in the table 4.
Chloroform extract of I. staphylina leaf showed nil antibacterial activity in all the tested concentrations. The
control showed nil activity, the standard Ciprofloxacin showed excellent antibacterial activity against all the
tested bacterial pathogens. For accurate values the experiment is triplicated and the values were expressed in
terms of Mean ± standard error of mean (SEM). The values obtained from the antibacterial activity of of I.
staphylina leaf chloroform extract which is showed in the table 5.
Figure 4. GC-MS analysis of Ipomoea staphylina Roem & Schult., leaf ethanolic extract (i).
Figure 5. GC-MS analysis of Ipomoea staphylina Roem & Schult., leaf ethanolic extract (ii).
Table 4. Antibacterial activity of Ipomoea staphylina Roem & Schult. leaf petroleum ether extract.
Petroleum ether
Zone of Inhibition in mm (Mean±SEM)
extract
12.50 25 50 100 Control Standard
Test Organisms
mg ml-1 mg ml-1 mg ml-1 mg ml-1
Xc 0±0 0±0 0±0 0±0 0±0 30.66±0.66
Ps 0±0 0±0 0±0 0±0 0±0 28.66±0.66
Kp 0±0 0±0 0±0 0±0 0±0 30.33±0.33
Ec 0±0 0±0 0±0 0±0 0±0 32±0.57
St 0±0 0±0 0±0 0±0 0±0 32.33±0.33
Pa 0±0 0±0 0±0 0±0 0±0 31±0.57
Sa 0±0 0±0 0±0 0±0 0±0 33.66±0.33
Note: Xc- Xanthomonas campestris, Ps- Pseudomonas syringae, Kp- Klebsiella pneumoniae, Ec- Escherichia
coli, St- Salmonella typhi, Ps- Pseudomonas aeruginosa, Sa- Staphylococcus aureus.
Table 5. Antibacterial activity of Ipomoea staphylina Roem & Schult. leaf chloroform extract.
Chloroform
extract
Test 12.50 25 50 100 Control Standard
Organisms mg ml-1 mg ml-1 mg ml-1 mg ml-1
Xc 0±0 0±0 0±0 0±0 0±0 30.66±0.66
Ps 0±0 0±0 0±0 0±0 0±0 28.66±0.66
Kp 0±0 0±0 0±0 0±0 0±0 30.33±0.33
Ec 0±0 0±0 0±0 0±0 0±0 32±0.57
St 0±0 0±0 0±0 0±0 0±0 32.33±0.33
Pa 0±0 0±0 0±0 0±0 0±0 31±0.57
Sa 0±0 0±0 0±0 0±0 0±0 33.66±0.33
Note: Xc- Xanthomonas campestris, Ps- Pseudomonas syringae, Kp- Klebsiella pneumoniae, Ec-
Escherichia coli, St- Salmonella typhi, Ps- Pseudomonas aeruginosa, Sa- Staphylococcus aureus.
Table 6. Antibacterial activity of Ipomoea staphylina Roem & Schult. ethanolic extract.
Ethanolic
extract
Test 12.50 25 50 100 Control Standard
Organisms mg ml-1 mg ml-1 mg ml-1 mg ml-1
Xc 9.66±0.88 14±1.15 14.66±0.88 21.33±0.88 0±0 30.66±0.66
Ps 9±1.15 12.33±1.45 15.33±1.45 17±0.57 0±0 28.66±0.66
Kp 9.33±1.45 10.33±0.88 13.66±0.88 20.33±1.2 0±0 30.33±0.33
Ec 0±0 11.33±1.2 12±1.15 16±0.57 0±0 32±0.57
St 10.33±1.45 17.33±0.88 19.33±1.45 21±1.54 0±0 32.33±0.33
Pa 9.33±88 19.33±0.88 21.33±0.88 23.33±0.66 0±0 31±0.57
Sa 10.33±0.88 16.66±0.88 21±0.57 22±1.15 0±0 33.66±0.33
Note: Xc- Xanthomonas campestris, Ps- Pseudomonas syringae, Kp- Klebsiella pneumoniae, Ec- Escherichia coli,
St- Salmonella typhi, Ps- Pseudomonas aeruginosa, Sa- Staphylococcus aureus.
Ethanolic extract of I. staphylina leaf showed appreciable antibacterial activity in all tested concentrations
(12.50, 25, 50, 100 mg ml-1) which is showed in the table 4.4.3. In 100 mg ml-1 concentration maximum zone of
inhibition showed by Pseudomonas aeruginosa (23.33±0.66) followed by Staphylococcus aureus (22±1.15),
Xanthomonas campestris (21.33±0.88), Salmonella typhi (21±1.54), Klebsiella pneumoniae (20.33±1.2),
Pseudomonas syringae (17±0.57) and least was Escherichia coli (16±0.57) (Table 6; Figs. 6 & 8). The control
DMSO shows nil zone and standard ciprofloxacin showed maximum zone of inhibition for all the pathogenic
bacteria (Table 6; Fig. 7).
DISCUSSION
Soxhlet extraction
Soxhlet extraction is a common procedure to extract phytoconstituents which is essential to mankind. The
leaf sample (700 grams) of Ipomoea staphylina yields more percentage of extract in ethanol (87.28%) when
compared to other solvents like petroleum ether (3.95%) and with chloroform (8.76%) so it is revealed that, the
plant leaf sample is having more alcohol soluble extractive than other solvents which is more essential in
extraction of good phytoconstituent (Table 1; Fig. 3).
Preliminary phytochemical analysis
The preliminary phytochemical analysis of I. staphylina leaf extracts also revealed the presence of more
phytoconstituent in the ethanolic extracts like alkaloids, saponins, flavonoids, steroids, glycosides, phenols and
sterols when compared to petroleum ether extract which only confirms the presence of soponins, steroids,
glycosides and sterols, similarly chloroform extracts confirms only the presence of saponins, steroids, sterols.
So, we took only ethanolic extract for Gas Chromatography and Mass Spectroscopic (GC-MS) analysis for
confirmation of different constituents (Table 2).
GC-MS analysis
GC-MS analysis of I. staphylina leaf ethanolic extract was analysed in the instrument GC Model: Thermo
Trace GC Ultra, MS Model: Thermo DSQ II, Ionization: Electron Impact Ionisation (EI), Chemical Ionisation
(CI), Mass Range: 1 - 1074 m/z and obtained spectra was analysed, revealed the presence of 79 compounds in
that 24 compounds were unknown and 55 compounds were known for its medicinal properties. Major
percentage of compound is Dodecanoic acid, 3-hydroxy- (10.41%) used in the treatment of Fatty Acid
Oxidation disorder and it also intermediate of liver fatty acid metabolism (Jones et al. 2000), followed by 9-
Hexadecen-1-ol (9.52%) used as Cosmetics, anti-hair fall agent (PubChem- 9-Hexadecen-1-ol/ C16H32O), 9-
Octadecen-1-ol (8.56%), Hydroperoxide, 1-ethylbutyl (5.88%) etc. and the least percentage is 3,3,7,11-
Tetramethyltricyclo [5.4.0.0(4,11)] undecan-1-ol (0.06%).
Figure 6. Antibacterial activity of Ipomoea staphylina Roem & Schult. ethanolic crude extract with: A, Escherichia coli; B,
Klebsiella pneumonia; C, Pseudomonas aeruginosa; D, Pseudomonas syringae; E, Salmonella typhi; F, Staphylococcus
aureus; G, Xathomonas campestris.
Eighteen compounds were antimicrobial agents such as Butyrolactone; 4H-Pyran-4-one, 2,3 - dihydro - 3, 5
- dihydroxy - 6- methyl-; 1,2-Benzenediol; 2 Furancarboxaldehyde, 5-(hydroxymethyl)-; 2-Methoxy-4-
vinylphenol; 3,4-Altrosan; Caryophyllene; 4-((1E)-3-Hydroxy-1-propenyl)-2-methoxyphenol; 3,7,11,15-
Tetramethyl-2-hexadecen-1-ol; Oleic Acid; 1-Heptadecanol; Phytol; 2,6,10,14,18,22-Tetracosahexaene,
2,6,10,15,19,23-hexamethyl-, (all-E)-; 3,3,7,11-Tetramethyltricyclo [5.4.0.0(4,11)] undecan-1-ol; Squalene;
gamma.-Sitosterol; Lupeol; 2-Propenoic acid, 3-(4-hydroxyphenyl)-.
Fourteen compounds was found to have anticancer properties, in that compounds such as Butyrolactone;
Caryophyllene; alpha.-Caryophyllene; 3,7,11,15-Tetramethyl-2-hexadecen-1-ol; Oleic acid; 9,12-
Octadecadienoic acid (Z,Z)- ; 2,6,10,14,18,22-Tetracosahexaene, 2,6,10,15,19,23-hexamethyl-, (all-E)-;
gamma.-Tocopherol; Vitamin E; beta.-Sitosterol; Stigmasterol; Lupeol; 2-Propenoic acid, 3-(4-hydroxyphenyl)-
; 2-Methoxy-4-vinylphenol were in major percentage
Fourteen compounds have Anti-hypercholesterolemic properties such as 2-Propenoic acid, 3-(4-
hydroxyphenyl)-; Lupeol; gamma.-Sitosterol; Stigmasterol; beta.-Sitosterol; Campesterol; Vitamin E; gamma.-
Tocopherol; 9,12-Octadecadienoic acid (Z,Z)-, 2,3-dihydroxypropyl ester; 9,12-Octadecadienoic acid (Z,Z)-;
9,12,15-Octadecatrienoic acid, methyl ester, (Z,Z,Z)-; Octadecanoic acid; Hexadecanoic acid, ethyl ester;
Dodecanoic acid, 3-hydroxy-.
Figure 7. Antibacterial activity of standard ciprofloxacin and control with: A, Escherichia coli; B, Klebsiella pneumonia; C,
Pseudomonas aeruginosa; D, Pseudomonas syringae; E, Salmonella typhi; F, Staphylococcus aureus; G, Xathomonas
campestris.
Sixteen compounds were used as food additive and flavoring agent such as 2-Furanmethanol; 2-Propanone,
1,3-dihydroxy-; Butyrolactone; 2,4-Dihydroxy-2,5-dimethyl-3(2H)-furan-3-one; Diglycerol; Pentanoic acid, 4-
oxo-; 1,2-Benzenediol ; 2-Furancarboxaldehyde, 5-(hydroxymethyl)- ; 1,2,3-Propanetriol,
monoacetate; 2-Methoxy-4-vinylphenol; Sucrose; Oleic Acid; Hexadecanoic acid, ethyl ester; Octadecanoic
acid; Octadecanoic acid, 2,3-dihydroxypropyl ester; 2-Propenoic acid, 3-(4-hydroxyphenyl)-.
Fifteen compounds have antioxidant properties such as 2-Furanmethanol; 4H-Pyran-4 - one, 2,3-dihydro-
3,5-dihydroxy-6-methyl-; 1,2-Benzenediol; 2-Methoxy-4-vinylphenol; Caryophyllene; 4-((1E)-3-Hydroxy-1-
propenyl)-2-methoxyphenol; Hexadecanoic acid, ethyl ester; 2,6,10,14,18,22-Tetracosahexaene,
2,6,10,15,19,23-hexamethyl-, (all-E)-; gamma.-Tocopherol; Vitamin E; beta.-Sitosterol; Campesterol; Squalene;
gamma.-Sitosterol; Lupeol.
Twelve compounds have anti-inflammatory properties such as 2-Propenoic acid, 3-(4-hydroxyphenyl)-;
Vitamin E; gamma.-Tocopherol; 9,12,15-Octadecatrienoic acid, methyl ester, (Z,Z,Z)-; Phytol; 3,7,11,15-
Tetramethyl-2-hexadecen-1-ol; 4-((1E)-3-Hydroxy-1-propenyl) -2- methoxyphenol; alpha – Caryophyllene);
Caryophyllene; 2-Methoxy-4-vinylphenol; 4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl-;
Cyclopentane, 1-acetyl-1,2-epoxy-.
Six compounds have hepatoprotective properties such as Pentanoic acid, 4-oxo-; Caryophyllene; 9,12-
Octadecadienoic acid (Z,Z)-; 9,12-Octadecadienoic acid (Z,Z)-, 2,3-dihydroxypropyl ester; gamma.-Tocopherol;
Vitamin E.
Five compounds have antiviral properties such as Cyclopentane, 1-acetyl-1,2-epoxy-; Uracil, 1-methyl-;
Stigmasterol; Tetrahydro-4H-pyran-4-ol; Octadecanoic acid, 2,3-dihydroxypropyl ester.
Five compounds have analgesic properties such as Caryophyllene; Benzaldehyde, 2-hydroxy-6-methyl-;
alpha.-Caryophyllene; 4-((1E)-3-Hydroxy-1-propenyl)-2-methoxyphenol; Vitamin E. (Table 3; Figs. 4–5).
Antimicrobial properties
Petroleum ether and chloroform extracts of I. staphylina leaf showed negligible antibacterial activity against
tested bacterial pathogen but ethanolic extract of I. staphylina leaf showed appreciable antibacterial activity in
all tested concentrations (12.50, 25, 50, 100 mg ml-1) which is due to the phytochemical dissolved in it and
antibacterial compounds present in it. In 100 mg ml-1 concentration maximum zone of inhibition showed by
Pseudomonas aeruginosa followed by Staphylococcus aureus, Xanthomonas campestris, Salmonella typhi,
Klebsiella pneumoniae, Pseudomonas syringae and least was Escherichia coli. All the tested pathogens were
belongs to plant and animal pathogens, some of them were opportunistic pathogens. In recent years drug
resistance bacterial strains were dangerous threat to human beings and new antibacterial drug synthesis is
necessary to get rid these drug resistance bacterial strains so, drug which has multiple pathogenic target and
safer to consume is appreciable, in that concern plant origin compounds were safety as well as cost effective.
Control DMSO shows nil zone inturn confirm the positive effect of ethanolic extract (Table 4–6; Figs. 6–8).
Figure 8. Antibacterial activity of ethanolic crude extract of Ipomoea staphylina Roem & Schult. leaf. [ISE - Ipomoea
staphylina ethanolic extract]
CONCLUSION
After the present investigation, it can be concluded that leaf ethanolic extract of Ipomoea staphylina can act
as good antibacterial agents. Because the leaf and bark of the plant tested on different organisms and some of
the organisms have controlled in high concentration of plants extract, but few showed negative results, this
bioactivity is due to the presence of secondary metabolites in the plants. It is proved that as the concentration of
secondary metabolites increases, the bioactivity will also increase. GC-MS analysis of ethanolic extract revealed
the presence of 79 compounds in that 55 compounds were known for its medicinal properties, most of them
were Antimicrobial agents followed by food additive and flavoring agents, Antioxidant, Anticancer agents,
Anti-hypercholesterolemic compounds, Anti-inflammatory agents, Hepatoprotective agents, Antiviral agents,
analgesic, Allergenic, Anesthetic, Antimutagenic, Antispasmodic, Choleretic, Dermatitigenic, Fungicide,
Herbicide, Laxative, Pesticide, Lipoxygenase-Inhibitor, Pesticide, Tyrosinase Inhibitor, Vermifuge etc.
Ethanolic extracts is very powerful due to the high efficiency attributed to its intermediate polarity leading to
the extraction of polar and non-polar compounds. Elemental composition of the plant is tested gives positive
results for macro as well as micro nutrients in that plant is rich with iron which is an essential nutrient for the
human being.
The overall study on antimicrobial reports that the plant species contains many active compounds which by
their synergistic effect may reduce or check the growth of microbial colonies. So, it is finally concluded that leaf
ethanolic extract of Ipomoea staphylina can be explored for potential antimicrobial compounds with rich full of
phytoconstituents.
ACKNOWLEDGEMENT
The authors thankful to Department of Applied Botany, Kuvempu University and Department of Botany,
Tumkur University Karnataka for providing facilities to conduct our experimental work.
REFERENCES
Ajaiyeoba EO (2000) Phytochemical and antimicrobial studies of Gynandropsis gynandra and Buchholzia
coriaceae extracts. African Journal of Biomedical Research 3: 161–165.
Akpuaka A, Ekwenchi MM & Dashak DA (2013) Biological Activities of Characterized Isolates of n-Hexane
Extract of Azadirachta Indica A. Juss (Neem) Leaves. Nature and Science 11(5): 141–147.
Aleu J, Hanson JR, Galán RH & Collado IG (2001) Biotransformation of the fungistatic sesquiterpenoids
patchoulol, ginsenol, cedrol and globulol by Botrytis cinerea. Journal of Molecular Catalysis B: Enzymatic
11(4–6): 329–334.
Arinathan V, Mohan VR, Britto AJD & Murugan C (2007) Wild edibles used by Palliyars of western ghats,
Tamil Nadu. Indian Journal of Traditional Knowledge 6(1): 163–168.
Awakan OJ, Malomo SO, Adejare AA, Igunnu A, Atolani O, Adebayo AH & Owoyele BV (2018) Anti-
inflammatory and bronchodilatory constituents of leaf extracts of Anacardium occidentale L. in animal
models. Journal of Integrative Medicine 16(1): 62–70.
Bag AK & Mumtaz SMF (2013) Hepatoprotective and nephroprotective activity of hydroalcoholic extract of
Ipomoea staphylina leaves. Bangladesh Journal of Pharmacology 8: 263–268.
Balasubramanian P, Rajasekaran P & Prasad SN (1997) Folk medicine of the Irulas of Coimbatore forests.
Ancient Science of Life 16(3): 222–226.
Banerjee A & Firdous SM (2015) Antiulcer activity of hydroalcoholic extract of Ipomoea staphylina plant in
rats. Bangladesh Journal of Pharmacology 10: 652–653.
Berg BG, Almaas TJ, Bjaalie JG & Mustaparta H (2005) Projections of male-specific receptor neurons in the
antennal lobe of the oriental tobacco budworm moth, Helicoverpa assulta: A unique glomerular organization
among related species. Journal of Comparative Neurology 486: 209–220.
Butler LI & McDonough LM (1981) Insect sex pheromones: Evaporation rates of alcohols and acetates from
natural rubber septa. Journal of Chemical Ecology 7(3): 627–633.
Calleja M, Vieites J, Montero-Meterdez T, Torres M, Faus M, Gil A & Suárez A (2013) The antioxidant effect
of β-caryophyllene protects rat liver from carbon tetrachloride-induced fibrosis by inhibiting hepatic stellate
cell activation. British Journal of Nutrition 109(3): 394–401.
Cyclohexanols / CTD. Available from: ctdbase.org/detail.go?type=chem&acc=D003511 (accessed: 10 Feb.
2018).
Dahham SS, Tabana YM, Iqbal MA, Ahamed MBK, Ezzat MO, Majid ASA & Majid AMSA (2015) The
Anticancer, Antioxidant and Antimicrobial Properties of the Sesquiterpene β-Caryophyllene from the
Essential Oil of Aquilaria crassna. Molecules 20(7): 11808–11829.
De-Castro MDL & Ayuso LEG (1998) Soxhlet extraction of solid materials: an outdated technique with a
promising innovative future. Analytica Chimica Acta 369: 1–10.
Dilika F, Bremner PD & Meyer JJM (2000) Antibacterial activity of linoleic and oleic acids isolated from
Helichrysum pedunculatum: a plant used during circumcision rites. Fitoterapia 71(4): 450–452.
Dr. Duke's Phytochemical and Ethnobotanical Databases, USDA. Available from:
https://phytochem.nal.usda.gov/ (accessed: 10 Feb. 2018).
Fernandes ES, Passos GF, Medeiros R, da Cunha FM, Ferreira J, Campos MM, Pianowski LF & Calixto JB
(2007) Anti-inflammatory effects of compounds alpha-humulene and (−)-trans-caryophyllene isolated from
the essential oil of Cordia verbenacea. European Journal of Pharmacology 569(3): 228–236.
Firdous SM & Singh A (2016) Effect of Ipomoea staphylina leaves on Streptozotocin- Nicotinamide Induced
Type-II Diabetes in Wistar Rats. Asian Pacific Journal of Health Science 3 (3): 30–44.
Firdous SM & Koneri R (2012a) In vivo and in vitro anti-inflammatory activity of leaves of Ipomoea
staphylina. International Journal of Pharmacy and Pharmaceutical Sciences 4(5): 339–343.
Firdous SM & Koneri R (2012b) Antiinflannatory and membrane-stabilizing activity of ethanolic extract and its
ethylacetate and n-butanol fractions of Ipomoea staphylina. Asian Journal of Pharmaceutical and Clinical
Research 5(4): 50–53.
Fukai S, Tanimoto S, Maeda A, Fukuda H, Okada Y & Nomura M (2009) Pharmacological Activity of
Compounds Extracted from Persimmon Peel (Diospyros kaki Thunb.). Jounal of Olea Science 58(4): 213–219.
Fuster MD, Mitchell AE, Ochi H & Shibamoto T (2000) Antioxidative activities of heterocyclic compounds
formed in brewed coffee. Journal of Agricultural and Food Chemistry 48: 5600–5603.
Gamble: Ipomoea staphylina Roem. & Schult., Syst. Veg. 4: 249. 1819; Hook. f., Fl. Brit. India 4: 210. 1883;
Gamble, Fl. Pres. Madras 917(643). 1923; Manilal & Sivar., Fl. Calicut 181. 1982; Babu, Fl. Malappuram
Dist. 489. 1990; Vajr., Fl. Palghat Dist. 312. 1990; Sivar. & Mathew, Fl. Nilambur 455. 1997; Sasidh., Fl.
Chinnar WLS 215. 1999.
Ghelardini C, Galeotti C, Mannelli DC, Mazzanti G & Bartolini A (2001) Local anaesthetic activity of β-
caryophyllene. Il Farmaco 56(5–7): 387–389.
Giarman NJ & Schmidt KF (1963) Some Neurochemical Aspects of the Depressant Action of γ-Butyrolactone
on The Central Nervous System. British Journal of Pharmacology and Chemotherapy 20: 563–568.
Gnanavel V & Saral AM (2013) GC-MS analysis of petroleum ether and ethanol leaf extracts from Abrus
precatorius LINN. International Journal of Pharma and Bio Sciences 4(3): 37–44.
Gopalakrishnan S & Vadivel E (2011) GC-MS analysis of some bioactive constituents of Mussaenda frondosa
LINN. International Journal of Pharma and Bio Sciences 2(1): 313–320.
Harborne JB (1998) Phytochemical Methods: A Guide to Modern Techniques of Plant Analysis, 3rd edition.
Chapman and Hall Co. New York, pp. 1–302.
Hiradate S, Yada H, Ishii T, Nakajima N, Kameyama MO, Sugie H, Zungsontiporn S & Fujii Y (1999) Three
plant growth inhibiting saponins from Duranta repens. Phytochemistry 52(7): 1223–1228.
Hiramoto K, Nasuhara A, Michikoshi K, Kato T & Kikugawa K (1997) DNA strand-breaking activity and
mutagenicity of 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one (DDMP), a Maillard reaction product
of glucose and glycine. Mutation Research/Genetic Toxicology and Environmental Mutagenesis 395(1–5):
47–56.
Huang M, Sanchez-Moreiras AM, Abel C, Sohrabi R, Lee S, Gershenzon J & Tholl D (2012) The major volatile
organic compound emitted from Arabidopsis thaliana flowers, the sesquiterpene (E)-β-caryophyllene, is a
defense against a bacterial pathogen. New Phytologist Journal 193(4): 997–1008.
Jadhav V, Kalase V & Patil P (2014) GC-MS analysis of bioactive compounds in methanolic extract of
Holigarna grahamii (wight) Kurz. International Journal of Health and Medicine 2(4): 35–39.
Jeong JB, Hong SC, Jeong HJ & Koo JS (2011) Anti-inflammatory effect of 2-methoxy-4-vinylphenol via the
suppression of NF-κB and MAPK activation, and acetylation of histone H3 Archives of Pharmacal
Research 34(12): 2109–2116.
Jeong JB & Jeong HJ (2010) 2-Methoxy-4-vinylphenol can induce cell cycle arrest by blocking the hyper-
phosphorylation of retinoblastoma protein in benzo[a]pyrene-treated NIH3T3 cells. Biochemical and
Biophysical Research Communications 400(4): 752–757.
Jones J, Wert V & Ries S (1979) Specificity of 1-triacontanol as a plant growth stimulator and inhibition of its
effect by other long-chain compounds. Planta 144(3): 277–282.
Jones PM, Quinn R, Fennessey PV, Tjoa S, Goodman SI, Fiore S, Burlina AB, Rinaldo P, Boriack RL &
Bennett MJ (2000) Improved stable isotope dilution-gas chromatography-mass spectrometry method for
serum or plasma free 3-hydroxy-fatty acids and its utility for the study of disorders of mitochondrial fatty
acid beta-oxidation. Clinical Chemistry 46(2): 149–155.
Kalaivani CS, Sathish SS, Janakiraman N & Johnson M (2012) GC-MS studies on Andrographis paniculata
(Burm.f.) Wall. ex Nees- A medicinally important plant. International Journal of Medicinal and Aromatic
Plants 2(1): 69–74.
Khandaker MM, Faruq G, Rahman MM, Azirun MS & Boyce AN (2013) The Influence of 1-Triacontanol on
the Growth, Flowering, and Quality of Potted Bougainvillea Plants (Bougainvillea glabra var “Elizabeth
Angus”) under Natural Conditions The Scientific World Journal 2013: Article ID 308651.
Kirtikar KR & Basu BD (1999) Ipomoea: Indian Medicinal Plants. International book distributors, Dehradun,
1720 p.
Kitagawa M, Higashi H, Takahashi IS, Okabe T, Ogino H, Taya Y, Hishimura S & Okuyama A (1994) A
cyclin-dependent kinase inhibitor, butyrolactone I, inhibits phosphorylation of RB protein and cell cycle
progression. Oncogene 9(9): 2549–2557.
Kottaimuthu R (2008) Ethnobotany of the Valaiyans of Karandamalai, Dindigul District, Tamil Nadu, India.
Ethnobotanical Leaflets 12: 195–203.
Kubo I, Muroi H & Kubo A (1994) Naturally Occurring Antiacne Agents. Journal of Natural Products 57(1):
9–17.
Kumar CAS, Kumar GS, Bakyaraj M, Kumar RV & Anbuchelvan PE (2013) Evaluation of Analgesic Activity
of Ipomoea staphylina in Acetic Acid Induced Writhing Reflux in Mice. International Journal of
Pharmaceutical Sciences Review and Research 22(1): 67–69.
Kumar PP, Kumaravel S & Lalitha C (2010) Screening of antioxidant activity, total phenolics and GC-MS study
of Vitex negundo. African Journal of Biochemistry Research 4(7): 191–195.
Kumar TD & Pullaiah T (1999) Ethno-Medico-Botany of Chenchus of Mahaboobnagar District, Andhra
Pradesh. Ancient Science of life 19(2): 31–35.
Leal WS, Nakano Y, Kuwahara Y, Nakao H & Suzuki T (1988) Pheromone Study of Acarid Mites XVII. :
Identification of 2-Hydroxy-6-Methyl Benzaldehyde as the Alarm Pheromone of the Acarid Mite
Tyrophagus perniciosus (Acarina: Acaridae), and Its Distribution among Related Mites. Applied Entomology
and Zoology 23(4): 422–427.
Legault J, Dahl W, Debiton E, Pichette A & Madelmont J (2003) Antitumor Activity of Balsam Fir Oil:
Production of Reactive Oxygen Species Induced by α-Humulene as Possible Mechanism of Action. Planta
Medica 69(5): 402–407.
Legault J & Pichette A (2007) Potentiating effect of β-caryophyllene on anticancer activity of α-humulene,
isocaryophyllene and paclitaxel. Journal of Pharmacy and Pharmacology 59: 1643–1647.
Maruthupandian A & Mohan VR (2011) GC-MS analysis of some bioactive constituents of Pterocarpus
marsupium Roxb. International Journal of ChemTech Research 3(3): 1652–1657.
Mohan VR & Kalidass C (2010) Nutritional and Antinutritional evaluation of some unconventional wild edible
plants. Tropical and Subtropical Agroecosystems 12: 495–506.
Muralidharan R & Narasimhan D (2012) Plants used for topical application from Gingee hills, Tamil Nadu,
India. Current Botany 3(4): 49–52.
Nagariya AK, Meena AK, Jain D, Gupta BP, Yadav AK, Gupta MR, Pathak AK & Neelam (2010) Medicinal
plants used in the healing of skin diseases in different regions of India: A Review. International Journal of
Chemical and Analytical Science 1(5): 110–113.
Nelson BJ, Melnik JD & Schmitt WH (1989) Glycerol and diglycerol mixtures for skin moisturizing. US Patent
4,829,092, 1989 - Google Patents.
Nishio K, Ishida T, Arioka H, Kurokawa H, Fukuoka K, Nomoto T, Fukumoto H, Yokote H & Saijo N (1996)
Antitumor effects of butyrolactone I, a selective cdc2 kinase inhibitor, on human lung cancer cell lines.
Anticancer Research 16(6B): 3387–3395.
Ogunlesi M, Okiei W, Ofor E & Osibote AE (2009) Analysis of the essential oil from the dried leaves of
Euphorbia hirta Linn (Euphorbiaceae), a potential medication for asthma. African Journal of Biotechnology
8(24): 7042–7050.
Park K, Nam D, Yun H, Lee S, Jang H, Sethi G & Cho SK (2011) β-Caryophyllene oxide inhibits growth and
induces apoptosis through the suppression of PI3K/AKT/mTOR/S6K1 pathways and ROS-mediated
MAPKs activation. Cancer letters 312(2): 178–188.
Pherobase.com-2-methoxy-4-vinylphenol. Pheromone Study of Acarid Mites XVII. : Identification of 2-
Hydroxy-6-Methyl Benzaldehyde as the Alarm Pheromone of the Acarid Mite Tyrophagus perniciosus
(Acarina: Acaridae) and Its Distribution among Related Mites. Available from:
www.pherobase.com/database/compound/compounds-detail-2-methoxy-4-vinylphenol.php (accessed: 10
Feb. 2018).
Pickett JA, Williams IH & Martin AP (1982) (Z)-11-eicosen-1-ol, an important new pheromonal component
from the sting of the honey bee, Apis mellifera L. (Hymenoptera, Apidae ) Journal of Chemical Ecology
8(1): 163–175.
Ponnamma SU & Manjunath K (2012) GC-MS analysis of phytocomponents in the methanolic extract of
Justicia wynaadensis (nees) t. anders. International Journal of Pharma and Bio Sciences 3(3): 570–576.
PubChem- Caryophyllene/C15H24 Available from: https://pubchem.ncbi.nlm.nih.gov/compound/beta-
caryophyllene (accessed: 10 Feb. 2018).
PubChem- Sucrose / C12H22O11. Available from: https://pubchem.ncbi.nlm.nih.gov/compound/sucrose
(accessed: 10 Feb. 2018).
PubChem- 1,2,3-Propanetriol, monoacetate/ C5H10O4. Available from: https://pubchem.ncbi.nlm.nih.gov/
compound/Acetin (accessed: 10 Feb. 2018).
PubChem- 1-Heptadecanol/ C17H36O. Available from: https://pubchem.ncbi.nlm.nih.gov/compound/1-
Heptadecanol (accessed: 10 Feb. 2018).
PubChem- 1-Methyluracil/ C5H6N2O2. Available from: https://pubchem.ncbi.nlm.nih.gov/compound/1-

methyluracil (accessed: 10 Feb. 2018).
PubChem- 2,4-Dihydroxy-2,5-dimethyl-3(2H)-furanone/C6H8O4. Available from: https://pubchem.ncbi.nlm.
nih.gov/compound/538757 (accessed: 10 Feb. 2018).
PubChem- 2-Methoxy-4-vinylphenol/ C9H10O2. Available from: https://pubchem.ncbi.nlm.nih.gov/compound
/2-Methoxy-4-vinylphenol (accessed: 10 Feb. 2018).
PubChem- 6-Methylsalicylaldehyde / C8H8O2. Available from: https://pubchem.ncbi.nlm.nih.gov/compound/2-
Hydroxy-6-methylbenzaldehyde (accessed: 10 Feb. 2018).
PubChem- 9-Hexadecen-1-ol/ C16H32O. Available from: https://pubchem.ncbi.nlm.nih.gov/compound
/5283282 (accessed: 10 Feb. 2018).
PubChem- Butyrolactone/C4H6O2. Available from: https://pubchem.ncbi.nlm.nih.gov/compound/gamma-
Butyrolactone (accessed: 10 Feb. 2018).
PubChem- Diglycerin/C6H14O5. Available from: https://pubchem.ncbi.nlm.nih.gov/compound/Diglycerol
PubChem- Dihydroxyacetone/ C3H6O3. Available from: https://pubchem.ncbi.nlm.nih.gov/compound/
Dihydroxyacetone (accessed: 10 Feb. 2018).
PubChem- D-Mannose/ C6H12O6. Available from: https://pubchem.ncbi.nlm.nih.gov/compound/mannose
PubChem- Furfuryl alcohol/C5H6O2. Available from: https://pubchem.ncbi.nlm.nih.gov/compound/furfuryl
_alcohol (accessed: 10 Feb. 2018).
Pubchem- gamma-Sitosterol/ C29H50O. Available from: https://pubchem.ncbi.nlm.nih.gov/compound/
Clionasterol (accessed: 10 Feb. 2018).
PubChem- Ginsenol/ C15H26O. Available from: https://pubchem.ncbi.nlm.nih.gov/compound/610072
PubChem- Glyceryl monostearate/ C21H42O4. Available from: https://pubchem.ncbi.nlm.nih.gov/compound/
Glyceryl_monostearate (accessed: 10 Feb. 2018).
PubChem- Humulene / C15H24. Available from: https://pubchem.ncbi.nlm.nih.gov/compound/Humulene
PubChem- Levulinic acid/C5H8O3. Available from: https://pubchem.ncbi.nlm.nih.gov/compound/4-
Oxopentanoic_acid (accessed: 10 Feb. 2018).
PubChem- Oleic acid/ C18H34O2. Available from: https://pubchem.ncbi.nlm.nih.gov/compound/oleic_acid
PubChem- Oleyl alcohol/ C18H36O. Available from: https://pubchem.ncbi.nlm.nih.gov/compound/oleyl
_alcohol (accessed: 10 Feb. 2018).
PubChem- Oxan-4-ol/ C5H10O2. Available from: https://pubchem.ncbi.nlm.nih.gov/compound/Tetrahydro-4-
pyranol (accessed: 10 Feb. 2018).
PubChem- Pentadecanoic acid/ C15H30O2. Available from: https://pubchem.ncbi.nlm.nih.gov/compound/
pentadecanoic_acid (accessed: 10 Feb. 2018).
PubChem- Propane, 1-(1-methylethoxy)/C6H14O. Available from: https://pubchem.ncbi.nlm.nih.gov/
compound/Isopropyl_propyl_ether (accessed: 10 Feb. 2018).
PubChem- Pyrocatechol/ C6H6O2. Available from: https://pubchem.ncbi.nlm.nih.gov/compound/catechol
Reddy DP, Kota R, Renuka S, Anarthe SJ & Raghavendra NM (2013) Isolation, characterization of
phytoconstituents and pharmacological screening of Ipomoea staphylina. Asian Journal of Pharmaceutical
and Clinical Research 6(1): 30–33.
Sabulala B, Dan M, John JA, Kurupa R, Pradeep NS, Valsamma RK & George V (2006) Caryophyllene-rich
rhizome oil of Zingiber nimmonii from South India: Chemical characterization and antimicrobial activity
Phytochemistry. 67(22): 2469–2473.
Sarvalingam A, Rajendran A & Aravindhan V (2011) Curative climbers of Maruthamalai Hills in the Southern
Western Ghats of tamil Nadu, International Journal of Medicinal and Aromatic Plants 1(3): 326–332.
Sarvalingam A, Rajendran A & Sivalingam R (2014) Wind edible plant resouces used by the Irulas of the
maruthamalai hills, Southern Western Ghats, Coimbatore, Tamil Nadu. Indian Journal of Natural Products
And Resources 5(2): 198–201.
Savitramma N, Yugandhar P & Suhrulatha D (2015) Traditional Medicinal Plants Used By Local People of
Kailasakona- A Sacred Grove of Chittoor District, Andhra Pradesh, India. International Journal of
Pharmacy and Pharmaceutical Sciences 7(3): 407–411.
Schwarz M, Klun JA., Leonhardt BA & Johnson DT (2000) (E,Z)-2,13-Octadecadien-1-ol acetate. A new
pheromone structure for sesiid moths. Tetrahedron Letters 24: 1007–1010.
Silici S & Kutluca S (2005) Chemical composition and antibacterial activity of propolis collected by three
different races of honeybees in the same region. Journal of Ethnopharmacology 99(1): 69–73.
Srinivasan K, Sivasubramanian S & Kumaravel S (2014) Phytochemical profiling and GCMS study of
Adhatoda vasica leaves. International Journal of Pharma and Bio Sciences 5(1): 714–720.
Suresh K, Kottaimuthu R, Normen TSJ, Kumuthakalavalli R & Simon SM (2011) Ethnobotonical study of
medicinal plants used by malayali Tribals In kolli hills of Tamilnadu, India. International Journal of
Research in Ayurveda and Pharmacy 2(2): 502–508.
Tambe Y, Tsujiuchi H, Honda G, Ikeshiro Y & Tanaka S (1996) Gastric Cytoprotection of the Non-Steroidal
Anti-Inflammatory Sesquiterpene, β-Caryophyllene. Planta Medica 62(5): 469–470.
Venkata R, Samuel L, Pardha SM, Narasimhan R, Naga VKA, Sudhakar M & Radhakrishnan TM (2012)
Antibacterial, antioxidant activity and GC-MS Analysis of Eupatorium odoratum. Asian Journal of
Pharmaceutical and Clinical Research 5(2): 0974–2441.
Wikipedia- 1-Hexacosanol. Available from: https://en.wikipedia.org/wiki/1-Hexacosanol (accessed: 10 Feb.
2018).
Wong B, Brauer KL, Clemens JR & Beggs S (1990) Effects of gastrointestinal candidiasis, antibiotics, dietary
arabinitol, and cortisone acetate on levels of the Candida metabolite D-arabinitol in rat serum and urine.
Infect. Immun 58(2): 283–288.
Xu N, Fan X, Yan X, Li X, Niu R & Tseng CK (2003) Antibacterial bromophenols from the marine red alga
Rhodomela confervoides. Phytochemistry 62(8): 1221–1224.
Yanagimoto K, Lee KG, Ochi H & Shibamoto T (2002) Antioxidative activity of heterocyclic compounds found
in coffee volatiles produced by Maillard reaction. Journal of Agricultural and Food Chemistry 50: 5480–
5484.
Yang D, Michel L, Chaumont JP & Millet-Clerc J (2000) Use of caryophyllene oxide as an antifungal agent in
an in vitro experimental model of onychomycosis. Mycopathologia 148(2): 79–82.
Yu X, Zhao M, Liu F, Zeng S & Hu J (2013) Identification of 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-
one as a strong antioxidant in glucose–histidine Maillard reaction products. Food Research International
51(1): 397–403.
Research article
Effects of leaf mulch application of Acacia crassicarpa Benth.,

Albizia adianthifolia (Schum.) W. Wight
and Albizia zygia (DC.) J. F. Macbr. on soil properties
F. E. Adesuyi*, C. I. Arinzechi, F. O. Iyagin, B. E. Omomoh,
A. S. Akinbowale and H. B. Dafiewhare
Department of Forestry and Wood Technology, Federal University of Technology Akure,
P.M.B 704, Ondo State, Akure, Nigeria
*Corresponding Author: adesuyifisola5@gmail.com [Accepted: 20 December 2018]
Abstract: This study was conducted to assess soil nutrient changes following application of mulch
materials from Acacia crassicarpa, Albizia zygia, and Albizia adianthifolia. Complete
Randomized Design was used for experimental layout. The mulch materials constituted the
treatments. The experimental plot (3 m × 4 m) was partitioned into 12 micro-plots of 1 m × 1 m in
dimension. Adjacent micro-plots were separated from each other by a buffer 0.5 m wide. Each
treatment including a control (without mulch) was replicated three times and treatments were
randomly allocated to the micro-plots. Ambient and soil temperatures were measured daily for six
weeks following treatment application. Soil samples were collected from soil depth of 0 to 15 cm
after six weeks of mulch applications in both mulched and control plots and analyzed for pH, %
of Organic matter, % of Nitrogen (N), available Phosphorus and exchangeable cations. The study
revealed that the mean levels of the various nutrients were not significantly different. The analysis
showed that the application of the various mulch materials tends to reduce the temperature in hot
weather condition and also temperature had a very important role to play in the decomposition
process. In low-temperature condition decomposition tends to be slow while in high-temperature
decomposition becomes fast.
Keywords: Soil nutrient - Soil temperature - Mulch - Decomposition - Exchangeable cations.
[Cite as: Adesuyi FE, Arinzechi CI, Iyagin FO, Omomoh BE, Akinbowale AS & Dafiewhare HB (2018) Effects
of leaf mulch application of Acacia crassicarpa Benth., Albizia adianthifolia (Schum.) W. Wight and Albizia
zygia (DC.) J. F. Macbr. on soil properties. Tropical Plant Research 5(3): 370–374]
INTRODUCTION
Intensive soil cultivation has worldwide resulted in the degradation of agricultural soils, with decreases in
soil organic matter and loss of soil structure, adversely affecting soil functioning and causing a long-term threat
to future yields Pagliai et al. (2004). Moreover, intensive tillage operations over a long period cause a
detrimental effect on surface soil as well as hastening the decomposition of soil organic matter Slentel et al.
(2007). Tillage degrades the fertility of soils, limits the availability of air and water, intensifies drought stress,
consumes fuel and contributes to global warming.
Agroforestry is a land use system which involves deliberate integration of trees and crop components on the
same piece of land. One of the key acceptance of agroforestry is that trees enrich soil fertility and also the major
recognized avenue of soil fertility improvement in tropical agroforestry is the recycling of nutrients through
decomposition of tree biomass mainly litterfall or pruning, but also root that is added to the soil. Obviously, the
extent of benefits derived will depend on the quantity and nutrient content of the biomass added, and the rate at
which it decomposes. Soil mulching with organic material is one method of soil water protection and also helps
maintain a constant soil temperature within the root system of crops (Awal & Khan 2000, Sinkevičienė et al.
2009, Samaila et al. 2011).
Acacia crassicarpa Benth. is a fast-growing tropical tree that grows on a wide variety of soil types. It fixes
nitrogen and tolerates fire, weed competition, and low soil fertility. It has recently been introduced in several
Adesuyi et al. 2018
countries of Southeast Asia and Africa, and it has proved to be one of the most promising new exotic plantation
species for degraded lands (Turnbull 1987, Boland 1989, Turnbull 1991, Awang & Taylor 1992, Awang &
Taylor 1993). Many materials have been published which states that Albizia species can be used to improve soil
fertility. Albizia zygia (DC.) J. F. Macbr. and Albizia adianthifolia (Schum.) W. Wight have been stated to
improve the soil organic matter, exchange calcium, magnesium, cation exchange capacity and available
phosphorous of soil under its canopy in Cameroon (Prinz 1986).
Organic farming is the alternate method to reduce the cost of fertilizer and also improve the soil. Leaf
biomass is a very important organic source of soil fertility improvement. The amount of N availability for plant
uptake is influenced by decomposition of leaf litters. Leaf litter supplies the nutrient necessary for plants such as
organic carbon, nitrogen, phosphorus, potassium and other nutrients in soil which are further considered as
important indicators of soil productivity and the ecosystem health. Acacia crassicarpa Benth., Albizia zygia,
Albizia adianthifolia can help to replace soil organic matter and nutrient status by protecting the soil against soil
erosion, enhancing humus and nutrient accumulation in the soil through litter fall, mineralization and by
recycling nutrients leached into the sub-soil back to topsoil. Thus, there is a need to determine soil nutrient
changes following application of mulch materials from the tree species.
The soil is continuously depleting as a result of numerous human activities mostly continuous cultivation of
land. This has been a serious problem that needs to be addressed. Hence a change to natural soil nutrient
replenishing through litterfall. There is also an increase in the use of inorganic fertilizers which on the long run
affects the yield of crops and quality of soil nutrient. To recover the lost soil nutrient, there must be a
mechanism that can effectively return nutrients (soil carbon, nitrogen) to the soil in proportion to the quantity
they have taken up. To contribute to this research gap, the effect of the application of leaf biomass of Acacia
crassicarpa, Albizia zygia, and Albizia adianthifolia on soil temperature and the effects of the various mulch
materials on soil organic matter were examined.
MATERIALS AND METHOD

Study area
The experiment was carried out at the Agroforestry Site of the Teaching and Research Farm, Federal
University Of Technology Akure, Nigeria (latitude 7° 17′ N, longitude 5° 10′ E, 350 m above sea level). The
bimodal rainfall pattern of the area has an annual mean of 1500 mm and a mean annual temperature of 26°C.
The area has a bimodal rainfall pattern with an annual mean of 1500mm and a mean annual temperature of
26°C. The main growing season is from August to October, followed by a long dry season from November to
April.
Experimental Design
Complete Randomized Design was used for experimental layout. The mulch materials constituted the
treatments. The whole experiment plot (3 m × 4 m) was partitioned into 12 micro-plots of 1mx1m in dimension.
Adjacent micro-plots were separated from each other by a buffer 0.5 m wide. Each treatment including a control
(without mulch) was replicated three times and treatments were randomly allocated to the micro-plots.
The following plant materials which have different physical and chemical characteristics were chosen for the
field study; Acacia crassicarpa, Albizia adianthifolia, Albizia zygia. Matured leaves of the species were
collected as mulch materials for the study. Fresh mulch materials were applied at the rate of 1 kg per micro-plot.
Measurement of Soil Temperature and Ambient Temperature.
Measurement of soil and ambient temperature were done daily at 6:00 pm. Soil temperature was measured
using a liquid crystal display (LCD) thermoprobe set at 10cm depth and ambient temperature was measured
using a digital infrared thermometer.
Soil Nutrient Analysis
Soil samples were collected from each micro plot six weeks after application of mulch. The soil samples
were dried sieved and labeled accordingly. The pH of the soil was determined using an electronic pH meter. pH
was determined by immersing the glass electrode of the pH meter into a partly settled suspension of soil.
Walkley Black wet oxidation method was used to determine soil organic carbon. Samples for Nitrogen were
digested using the micro-Kjeldahl method with selenium catalyst. The digested samples were distilled after the
addition of sodium hydroxide and the ammonia thus released was determined acid-base titration. Phosphorus
was determined by the molybdenum blue method. Calcium and magnesium content was determined by ethylene
diamine tetra acetic acid (EDTA) titration method and expressed in kg mol-1. Potassium and sodium were
determined by flame photometer and expressed in kg mol-1. The phosphorus content of the soil was also
determined using a 410 nm wavelength spectrometer.
Statistical Analysis of Data
Graphs were used to summarize the data for the soil temperature and ambient temperature. The data obtained
from the various nutrient analyses were subjected to analysis of variance (ANOVA) to determine the degree of
variation among the treatments used. Follow up test was carried out using Duncan’s multiple range test (DMRT)
to identify means that were significantly different.
RESULTS
Effect of Mulch Type on Soil Temperature
The graphical illustrations of changes in soil temperature are presented in figure 1. During the 6 weeks
period of this study, soil temperature showed fluctuation over time. For all the mulched plots the soil
temperature was low compared to the plots without mulch which was high. In comparison with the ambient
temperature, the mulch had an effect in moderating the soil under various treatments but the range was a little
bit different with the highest soil temperature being recorded as 28°C and the ambient temperature was 31°C.
32 Acacia crassicarpa Albizia adiantifolia Albizia zygia

Control Ambient Temperature
31
30
Temperature (°C)
29
28
27
26
25
24
23
1 2 3 4 5 6
Time (Weeks)
Figure 1. Effects of leaf mulch of the various species on soil temperature.
Table 1. ANOVA test of significant for soil properties of the species and control. [Sig. Value- Value of
Significance]
Source SS df MS F Sig. Value
pH Treatment 0.024 3 0.008 0.393 0.761
Error 0.163 8 0.020
Total 0.187 11
OM Treatment 0.014 3 0.005 0.541 0.667
Error 0.070 8 0.009
Total 0.085 11
Ca Treatment 6.129 3 2.043 0.222 0.879
Error 73.633 8 9.204
Total 79.763 11
Mg Treatment 9.003 3 3.001 0.624 0.619
Error 38.473 8 4.809
Total 47.477 11
Phos Treatment 0.752 3 0.251 0.173 0.911
Error 11.558 8 1.445
Total 12.310 11
K Treatment 9.180 3 3.060 2.491 0.134
Error 9.827 8 1.228
Total 19.007 11
N Treatment 0.001 3 0 1.326 0.332
Error 0.002 8 0
Total 0.002 11
Adesuyi et al. 2018
The results of the One-way Analysis of Variance for assessing the presence of the significant difference in
the soil chemical properties among species and control are represented in table 1. The results shows that there is
no significant difference (p<0.05) among the species except for potassium where a significant difference was
discovered to exist between Albizia adianthifolia and the control.
Table 2. Comparison of soil chemical properties with Duncan multiple range test (DMRT).
Soil properties Acacia crassicarpa Albizia adianthifolia Albizia zygia Control
pH 6.52a ± 0.1 6.58a ± 0.3 6.60a ± 0.1 6.50a± 0.1
Organic Matter 1.58a ± 0.1 1.52a ± 0.1 1.48a ± 0.6 1.53a ± 0.8
Calcium 7.47a ± 2.8 9.00a ± 3.6 7.10a ± 3.1 7.73a ± 2.5
Magnesium 5.70a ± 2.9 3.53a ± 2.2 4.40a ± 1.4 3.63a ± 1.9
Phosphorus 3.06a ± 1.6 2.54a ± 1.3 2.39a ± 1.1 2.61a ± 0.7
Potassium 4.50ab ± 0.4 5.80b ± 2.0 4.00ab±0.5 3.45a ± 0.6
Nitrogen 1.04a ± 0.0 1.03a ± 0.0 1.01a ± 0.1 1.03a ± 0.0
The follow-up procedure for the mean separation indicates that values in the same row followed by the same
letter(s) are not significantly different at 0.05 level of significance (Table 2). From this table, Acacia crassicarpa
has the highest mean value of organic matter (1.58a ± 0.1), magnesium (5.70a ± 2.9), phosphorus (3.06a ± 1.6)
and nitrogen (1.04a ± 0.0) while Albizia zygia has the lowest mean value of organic matter (1.48a ± 0.6), calcium
(7.10a ± 3.1), phosphorus (2.39a ± 1.1) and nitrogen (1.01a ± 0.1).
DISCUSSION
During the six weeks period of this study, the entire measured variable (soil temperature, ambient
temperature and soil nutrients) showed fluctuation with the climatic condition. This is due to certain climatic
conditions such as rainfall, temperature and humidity. The highest temperature was observed in soil under
Albizia adianthifolia mulch. This may be due to their fast decomposition rate and also the soil under control had
a high temperature.
Soil temperature may increase with depth below the surface, which makes the surface temperature undergo
fluctuation at the different time of the year due to change in climatic condition. The amount of heat energy
reaching the surface of the soil and its thermal properties are factors responsible for the change in temperature
(Taylor & Parkinson 1988). The temperature of the surface layer during the sunny day may be very much
different for a bare soil as compared to the one with a vegetative cover. The temperature a soil attains depends
on how much heat that reaches the soil surface (Nye & Greenland 1960). The result of the nutrient analysis
showed that the nutrient level of the soil were not that affected by the various treatments. The nutrients were not
significantly increased after weeks of mulch application. The reason to the nutrient not being significant maybe
due to the low temperature of the season and also since decomposition is an enzyme-mediated biological
process carried out by bacteria and fungi; it is very sensitive to temperature. In most soils, the decomposition
rate peaks at about 25°C and declines as temperature varies from this maximum. A study by Swift & Prichet
(1984) showed that the rate of decomposition is usually controlled by several factors such as the physiochemical
environment including climate and chemical composition of the organic matter and nature of the decomposer.
Therefore change in soil nutrient content after mulch application can be attributed to the decomposition and
mineralization of the mulch materials. Soil moisture also affects the activity of microorganisms. Very dry or
very wet (flooded) conditions tend to reduce decomposition rates (Hanson et al. 1993).
CONCLUSION
This study has examined the effect of the application of leaf mulch of Acacia crassicarpa, Albizia
adianthifolia, Albizia zygia on soil properties. These results did not support the hypothesis that the application of
the various mulch materials has the significant impact on soil properties. Since the use of organic mulch affects
majorly the soil temperature, which influences soil chemical, physical and biological processes. It also reduced
soil temperature at the different climatic condition. Hence there is need for proper selection of mulch materials
which can bring about a direct impact on the soil.
From this study, it is recommended that selection of mulch material should be based on species richness in
nutrients, decomposition and mineralization rates which is necessary for basic plant nutrition.
ACKNOWLEDGEMENTS
We wish to appreciate the Department of Forestry and Wood Technology, Akure for giving us the
authorization to access the Agroforestry Site of the Teaching and Research Farm, Federal University Of
Technology Akure, Nigeria within their Jurisdictions where data was collected for this work.
REFERENCES
Awang K & Taylor DA (1992) Tropical Acacias in East Asia and the Pacific. Proceedings of the First Meeting,
Consultative Group for Research and Development of Acacias (COGREDA), Phuket, Thailand, Winrock
International Institute for Agricultural Research,105 p.
Awal MA & Khan MAH (2000) Mulch induced ecophysiological growth and yield of maize. Pakistan Journal
of Biological Sciences 3: 61–64.
Awang K & Taylor DA (1993) Acacias for rural, industrial and environmental development. Proceedings,
Second Meeting, Consultative Group for Research and Development of Acacias (COGREDA), Udorn Thani,
Thailand, Winrock International Institute for Agricultural Research and FAO, 258 p.
Boland DJ (1989) Trees for the Tropics. Growing Australian multipurpose trees and shrubs in developing
countries. ACIAR Monograph No. 10, Canberra, Australia.
Hanson PJ, Wullschleger SD, Bohlman SA & Todd. DE (1993) Seasonal and topographic patterns of forest
floor CO2 efflux from an upland oak forest. Tree Physiology 13: 1–15.
Nye PA & Greenland DJ (1960) Soil under shifting cultivation. Technical communication 51, Farmharm,
Common Wealth Bureau of Soils, pp. 147.
Pagliai M, Vignozzi N & Pellegrini S (2004) Soil structure and the effect of management practices. Soil and
Tillage Research 79: 131–143.
Prinz D (1986) Ecosystem fertility and fallow function Agroforestry system. Journal of diary science 86: 4070–
4078.
Samaila AA, Amans EB, Abubakar IU, Babaji B (2011) Yield and fruit quality of tomato (Lycopersicon
esculentum Mill) as influenced by mulching, nitrogen and irrigation interval. International Research Journal
of Agricultural Science and Soil Science 1(3): 90–95.
Sinkevičienė A, Jodaugienė D, Pupalienė R & Urbonienė M (2009) The influence of organic mulches on soil
properties and crop yield. Agronomy Research 7(1): 485–491.
Slentel I, Rogasik J, Funder U & Panten K( 2007) Effect of tillage system and P fertilization on soil physical
and chemical properties, crop yield and nutrient uptake. Soil and Tillage Research 32(2): 50–57.
Swift M J & Prichet T S (1984) Tropical Soil Biology and Fertility (TSBF): planning for research biology
international special size 9: 24.
Taylor BR & Parkinson D (1988) Aspen and Pine leaf litter decomposition in laboratory microorganism
interaction of temperature and moisture level. Journal of Botany, 66(10): 1966–1973.
Turnbull JV (1987) Australian acacias in developing countries. Proceedings of an international workshop held
at the Forestry Training Centre, Gympie, Qld., Australia, ACIAR proceedings No. 16, 196 p.
Turnbull JV (1991) Advances in Tropical Acacia research. International workshop, Bangkok, Thailand, ACIAR
proceedings No. 35, 234 p.
Research article
Arbuscular mycorrhizal fungi inoculation with organic matter

and phosphorus supplementation enhance nutrient contents
of Amaranthus tricolor L. and Basella alba L.
by improving nutrients uptake
Md. Tahjib-Ul-Arif1*, Aporna Ghosh2, S. G. Chamely2, M. R. Haque3
and Md. Mokhlesur Rahman2
1
Department of Biochemistry and Molecular Biology, Bangladesh Agricultural University,
Mymensingh, Bangladesh
2
Department of Agricultural Chemistry, Bangladesh Agricultural University, Mymensingh, Bangladesh
3
Graduate School of Science, Kyushu University, Japan
*Corresponding Author: arif1002215@gmail.com [Accepted: 22 December 2018]
Abstract: Most of the vegetable crops retain an association with arbuscular mycorrhizal fungi
(AMF) which can improve the growth, nutrition, water supply and tolerance to biotic and abiotic
stresses of host plants. Therefore, the main objective of this study was to investigate whether the
association of red amaranth (Amaranthus tricolor) and Indian spinach (Basella alba) with AMF,
increase the shoot mineral nutrient contents and whether supplementation of organic matter and
phosphorus enhance AMF association. The experiment was laid out in a factorial design
considering three-factor viz. arbuscular mycorrhiza (AM), cowdung (CD) and phosphorus (P).
There were five replications and total 40 treatment combinations. Results showed that in both
vegetable maximum N, P, K, S, Mg, Fe, Mn, Cu and Zn and protein contents were observed due to
the application of AMF and cowdung with phosphorus. The combined application of AMF with
cowdung and phosphorus enhanced the association with plants and was more effective than only
AMF application to enhance nutrient contents. Also, the uptake of almost all macro- and
micronutrients increased due to the application of cowdung and phosphorus with arbuscular
mycorrhiza.
Keywords: Arbuscular mycorrhizal fungi - Mineral nutrients - Red amaranth - Indian spinach -
Nutrient uptake - Cowdung - Phosphorus.
[Cite as: Tahjib-Ul-Arif M, Ghosh A, Chamely SG, Haque MR, Rahman MM (2018) Arbuscular mycorrhizal
fungi inoculation with organic matter and phosphorus supplementation enhance nutrient contents of Amaranthus
tricolor L. and Basella alba L. by improving nutrients uptake. Tropical Plant Research 5(3): 375–384]
INTRODUCTION
The utilization of natural resources for sustainability and exponential increase in agricultural production has
now become a demand of the globalized era to fulfil the nutritional needs of the thriving population. The
phenomenon of arbuscular mycorrhizal symbiosis has paved the way towards a new window of enhanced
agricultural production as well as nutritional security. All arbuscular mycorrhizal fungi (AMF or AM fungi) are
obligate symbionts and are grouped in the phylum Glomeromycota, with almost 220 species actually described
with the majority of representatives belong to the order Glomerales (Schüβler et al. 2001).
The diversity of AM fungal community may have an influence on plant communities and various fungal
species preferentially associate with different plant species (Pringle & Bever 2002). AMF form a symbiotic and
mutualistic interconnection with thin roots of most terrestrial plants (Yoshimura et al. 2013). The AMF
mycelium interacts with the plant roots and helps plants to uptake more nutrients from soil-water system that are
distant from roots (Smith et al. 2000). Furthermore, fungal hyphae are much thinner than roots and are which
facilitated to penetrate smaller pores (Allen 2011).
Tahjib-Ul-Arif et al. 2018
The symbiotic relation provides plant many benefits, including enhanced uptake of poorly mobile soil
nutrients and reduced susceptibility of roots to soil-borne pathogens (Quilambo 2003). The fungus also receives
carbohydrate and growth factors from the plant (Berruti et al. 2016). AMF hyphae exclusively colonize the root
cortex and form highly branched structures inside the cells, i.e., arbuscules, which are considered the functional
site of nutrient exchange (Balestrini et al. 2015). Consequently, AMF association helps to update adequate
nutrients and enhance plant growth (Nouri et al. 2014).
Mycorrhizal symbioses not only profit to plant growth but also to plant protection, especially against
environmental stresses. Scarcity of essential nutrients especially phosphorus is primarily limits crop growth and
productivity (Nagarathna et al. 2007). The AMF have been suggested as having a role in mediating the uptake
of water at times of drought stress and of metals on the contaminated ground (Farahani et al. 2008). The scarcity
of inorganic fertilizers in some poor developing countries considerably hamper crop cultivation. The
incorporation of factors that enable plants to withstand nutrient deficiency and toxicity as well as drought stress
would, therefore, be helpful to improve crop production.
Leafy vegetables that lack natural AMF association as such red amaranth and Indian spinach, can be
incorporated with the inoculation of AMF along with cowdung and phosphorus to increase their nutrient
content. AMF accept the hexoses sugars from the host plant. Carbon transfer occurs from plant to fungi may
occur through the arbuscular or intraradical hyphae. The host plants invest considerable carbon in the
mycorrhizal network and contribute to the below-ground organic carbon pool. The main benefit of mycorrhizas
to plants has been attributed to increased uptake of nutrients, especially phosphorous.
The level of minerals in vegetables depends on a number of factors including genetic properties of the crop
species, climatic conditions, soil characteristics and the degree of maturity of the plant at the moment of
harvesting (Carvajal et al. 2014). As vegetables constitute the main source of minerals in the human diet, one of
the most important challenges for agriculture, besides enhancing food production, is to provide almost all the
essential minerals and organic nutrients to humans for maintenance of health and proper organ function.
Humans need more than 22 mineral elements. Some are required in large amounts, such as Fe, Zn, Cu, I and Se,
some are required in trace amounts (Welch & Graham 2004) but their absence renders human life impossible.
Concentrations of several essential elements in modern crops are insufficient for optimal human nutrition, thus
contributing to the huge “hidden hunger” problem (Graham et al. 2001). This is the case of iron (Fe), whose
deficiency affects more than 3.5 billion people, mostly in the developing world and impairs the cognitive
development of children, causes productivity and educational losses and increases morbidity and maternal
mortality (ACC/SCN 2000). Other important examples are zinc (Zn), a micronutrient whose levels of intake in
the diet may be inadequate for nearly half of the world’s population and copper (Cu), essential for human health
but consumed in inadequate quantities in developing countries (Brown & Wuehler 1990).
Considering the above-mentioned facts, this study was carried out to evaluate the potential of arbuscular
mycorrhizal fungi inoculation with organic matter and phosphorus supplementation to produce mineral nutrients
rich leafy vegetables i.e. red amaranth and Indian spinach that lack natural arbuscular mycorrhizal fungi
association.

Experimental site
The experiment was conducted in the net house of the Department of Agricultural Chemistry, Bangladesh
Agricultural University, Mymensingh.
Collection and preparation of soil sample
The soil used in this experiment was collected from Central Farm of Bangladesh Agricultural University,
Mymensingh. The soil was dried and the clods were broken and sieved to remove weeds, stubbles and hard
clods. Before starting the experiment, the soil was analyzed primarily for its physical and chemical properties.
Organic carbon, soil pH, soil texture, cation exchange capacity, total nitrogen, available phosphorus,
exchangeable potassium, available sulphur were analyzed (Table 1).
Table 1. Different properties of experimental soil.
pH CEC Total N Organic Organic Available P Exchangeable K Available S
(%) (%) carbon matter (µg g-1) (cmol kg-1) (µg g-1)
(%) (%)
6.18 8.4 0.10 0.71 1.16 12.6 0.14 10.9
Analysis of cowdung
Cowdung manure was analyzed for assessing the constituents of N, P, K, S, Ca, Mg and Fe following the
standard methods used for analysis. Total N (%)=1.28, P (%)=0.60, K (%)=1.65, Ca (%)=0.43, Mg (%)=0.28, S
(%)=0.16, Fe(%)=14.47.
Test crops
Red amaranth cv. BARI Lalshak-1 and Indian spinach cv. BARI Puishak-1 were used as plant materials for
the experiment. The seeds were collected from Horticulture Research Centre, Bangladesh Agricultural Research
Institute, Joydebpur, Gazipur.
Treatment combination and experimental design
There were eight treatment combinations for each test crop. The experiment was laid out in a factorial design
considering three factor arbuscular mycorrhiza (AM), cowdung (CD) and phosphorus (P). There were five
replications and total treatment combinations were 40. T1=Control, T2 = AM, T3=CD, T4=P, T5=AM+CD,
T6=CD+P, T7=AM+P, T8=AM+CD+P. (Here, AM= Arbuscular mycorrhiza, CD= Cowdung, P= Phosphorus).
Fertilizer dose- For red amaranth: AM=80 g polybag-1, CD= 20 g polybag-1, P=0.30 g TSP polybag-1
Basal dose- Urea @ 110 kg ha-1 (0.95 g polybag-1), MOP @ 25 kg ha-1 (0.20 g polybag-1) and Gypsum @ 4
kg ha-1 (89 mg polybag-1)
For Indian spinach: AM=80 g polybag-1, CD= 32 g polybag-1, P=0.30 g TSP polybag-1
Basal dose- Urea @ 120 kg ha-1 (1.04 g polybag-1), MOP @ 50 kg ha-1 (0.40 g polybag-1) and Gypsum @ 13
kg ha-1 (290 mg polybag-1)
Per polybag 8 kg soil was used. Basal doses were added from Fertilizer Recommendation Guide-2005.
Application of arbuscular mycorrhizal (AM) fungi in soil and sowing of seed
Soil-based AM inoculum was used in the seed furrows of about 3 cm depth at the rate of 80 g polybag-1. The
seeds of each test crop were sown in each furrow on AM inoculums and covered them with side soil. After
sowing the seed, the soil was saturated with water.
Preparation of the plant extract for different chemical analyses
Plant extract was prepared by wet oxidation method using a di-acid mixture (Estefan et al. 2013). This
solution was used for the determination of P, K, S, Ca, Mg, Fe, Mn, Cu and Zn.
Analytical methods
The phosphorus content was determined by spectrophotometer (Model- LT-31) at 660 nm wavelength (Page
et al. 1987). The content of K was determined separately with the help of flame emission spectrophotometer
(Model- Jenway PET 7) (Ghosh et al. 1983). Calcium and magnesium content was determined by the
complexometric method of titration using Na2EDTA (Page et al. 1987). Sulphur content was analyzed
turbidimetrically with the help of spectrophotometer (Model- LT-31) as described by (Wolf 1982) and (Tandon
1995). The contents of Fe, Zn, Cu and Mn were determined by atomic absorption spectrophotometer setting
wavelengths at 248.3, 213.9, 324.8, 279.5 nm, respectively (Allen et al. 1974). Total N was estimated by semi-
micro Kjeldahl method (Jackson 1973, Page et al. 1987).
i. Calculation of protein content
A protein present in plant samples was calculated by the following formula.
( )
ii. Nutrient uptake
After chemical analyses of plant samples, the nutrient contents were calculated and from the value of
nutrient contents, nutrient uptakes were also calculated by the following formula:
( ) ( )
( )
Statistical analyses
Analysis of variance was done following the factorial design with the help of computer package Minitab
17.0. The data analysis was performed by F-test (Gomez & Gomez 1984). The mean of different treatment
groups were compared using Fisher’s least significant difference (LSD) test.
RESULTS
Effect of arbuscular mycorrhiza, cowdung and phosphorus
i. Macronutrient contents in red amaranth: In red amaranth, application of AM+CD+P showed maximum N, P,
K, S and Mg content which was significantly higher than control and only application of AM, CD and P.
The nitrogen content was found in AM + CD application was statistically similar to AM+CD+P. The
combined application of CD+P and AM+P also increased nitrogen content significantly than the control
condition. Application of AM+CD and AM+P increased P content statistically similar to AM+CD+P and
AM+CD also increased P content significantly than control. The combined application of AM+CD, CD+P
and AM+P increased potassium content significantly than control condition which was similar to T8. The
effect of arbuscular mycorrhiza, cowdung and phosphorus on calcium content was statistically not
significant. When AM was treated with phosphorus statistically similar Mg content was found compared to
AM+CD+P.
ii. Micronutrient contents in red amaranth: The combined application of AM, CD and P showed maximum Fe,
Mn, Cu and Zn content in red amaranth which was significantly higher than control. AM + P application
showed statistically similar Fe and Zn content to AM+CD+P. The combined application of AM+CD, CD+P
and AM+P also increased Cu contents significantly than control condition which was statistically similar to
AM+CD+P.
iii. Macronutrient content in Indian spinach: The combined application of AM bio-fertilizer, cowdung and
phosphorus showed maximum P, K, S, Ca and Mg content in Indian spinach which was significantly higher
than control. Application of AM with phosphorus and AM with cowdung achieved the highest nitrogen
content. Application of AM with phosphorus showed similar P and K content compared to AM+CD+P.
Application of AM with CD or P also increased S and Mg content which was statistically similar to
AM+CD+P.
iv. Micronutrient content in Indian spinach: The combined application of AM bio-fertilizer, cowdung and
phosphorus showed maximum Fe, Mn, Cu and Zn content in Indian spinach which was significantly higher
than control. The treatment CD + P achieved Fe content which was statistically similar to only CD and P
application. Application of AM gave the statistically higher result to control. Application of phosphorus
with cowdung produced Mn content which was statistically similar to AM + CD + P. The lowest Mn content
was found in AM applied condition. Application of AM with cowdung and AM with phosphorus achieved
Cu content which was statistically similar to AM + CD + P. The application of arbuscular mycorrhiza and
phosphorus which was statistically similar with AM+CD+P.
v. Protein content: The results showed that protein content in Indian spinach was higher with phosphorus
application than control treatment. Application of AM with phosphorus achieved 4.31% protein which was
statistically similar to T5 (CD + AM) and T4 (P). In red amaranth, the maximum protein content was
observed in AM, CD and P applied the treatment, which was statistically similar with AM+CD, CD+P and
AM+P treatments.
Nutrient uptake by Red amaranth and Indian spinach and correlation among the nutrients
AM inoculation, organic matter and phosphorus supplementation increased N, P, K, S, Ca, Mg, Fe, Mn, Cu
and Zn uptake. Always the maximum nutrient uptake was noticed in combined application of AM, CD & P. AM
inoculation with CD or P also increased nutrient uptake than only CD or P application. Most of the nutrient
contents were strongly correlated among themselves.
DISCUSSION
Our previous study showed that arbuscular mycorrhiza, cowdung and phosphorus enhanced growth and
yield contributing characters of red amaranth and Indian spinach (Ghosh et al. 2017).
Application of AMF remarkably elevated the plant mineral nutrition, mainly with nitrogen and phosphorus
(Salvioli et al. 2012, Colella et al. 2014). This dependence seems to be confirmed by results of our studies, in
which application of AM supplemented with CD and P showed higher N and P content as compared to red
amaranth and Indian spinach plants grown without AMF inoculation (Table 2 & 3). Guo et al. (2006) reported
that in generally mycorrhizal colonization resulted in increased shoot N and P contents in onion. Arbuscular
mycorrhizal fungus Glomus intraradices actively mobilize P from phosphates and increase the take-up of more
in roots inoculated with G. intraradices than those un-inoculated (Antunes et al. 2007). In this study, the
application of cowdung and phosphorus showed statistically identical phosphorus uptake. The results further
indicated that arbuscular mycorrhiza biofertilizer, cowdung and phosphatic fertilizer treated pots performed
better with respect to phosphorus uptake (Table 2 & 3). Similarly, Harrison et al. (2010) reported that
phosphorus was often the key element for increased growth or fitness of mycorrhizal plants because it was
transported in hyphae in large amounts compared to the plant phosphorus demand. Moreover, (Kehri & Chandra
2001) suggested that it was possible to reduce the phosphate recommendation for black gram with inoculation of
effective AM fungi.
Table 2. Effect of arbuscular mycorrhiza (AM), cowdung (CD) and phosphorus (P) on nutrients content of red amaranth.
N P K S Ca Mg Fe Mn Cu Zn Protein
Treatments
(%) (%) (%) (%) (%) (%) (µg g-1) (µg g-1) (µg g-1) (µg g-1) (%)
T1: Control 0.63g 0.22d 0.23c 0.07d 0.31 0.35d 4.08e 3.32e 1.74b 2.60c 3.96d
T2: AM 0.82f 0.22d 0.28b 0.07d 0.33 0.43c 5.60c 4.00d 2.02b 3.00bc 5.13c
T3: CD 0.87e 0.39c 0.28b 0.11c 0.32 0.45c 6.36b 3.20e 2.03b 2.60c 5.41c
T4: P 0.94d 0.47b 0.27b 0.12c 0.33 0.37d 6.12b 3.80e 2.53a 2.80bc 5.89bc
T5: AM+CD 1.09a 0.39c 0.33a 0.12c 0.33 0.44c 6.42b 5.40e 2.59a 3.20b 6.83a
T6: CD+P 1.01c 0.52a 0.33a 0.13c 0.33 0.52b 5.18d 6.40b 2.80a 3.00bc 6.33ab
T7: AM+P 1.05b 0.49ab 0.35a 0.17b 0.34 0.55ab 6.50ab 5.52c 2.96a 3.80a 6.56ab
T8: AM+CD+P 1.09a 0.53a 0.34a 0.23a 0.34 0.59a 6.88a 7.60a 2.95a 4.00a 6.80a
LSD (0.05) 0.012 0.04 0.027 0.022 0.022 0.040 0.39 0.67 0.49 0.57 0.87
CV (%) 16.47 14.27 17.65 10.27 16.48 15.15 7.21 10.91 17.45 15.59 6.59
Level of sig. ** ** * * NS ** ** ** ** ** **
Note: * Significant at 5% and ** significant at 1% level of probability.
In a column, the figure(s) having the same letter are not significantly different at 5% or 1% level of probability by DMRT.
Table 3. Effect of arbuscular mycorrhiza (AM), cowdung (CD) and phosphorus (P) on nutrients content of Indian spinach.
N P K S Ca Mg Fe Mn Cu Zn Protein
Treatments
(%) (%) (%) (%) (%) (%) (µg g-1) (µg g-1) (µg g-1) (µg g-1) (%)
T1: Control 0.53e 0.31d 0.57c 0.10b 0.40e 0.40c 3.44f 1.12d 2.52f 6.13f 3.30f
T2: AM 0.59c 0.43bc 0.61bc 0.10b 0.43de 0.41bc 4.20e 1.12d 2.63f 6.24ef 3.66de
T3: CD 0.61cd 0.37cd 0.60bc 0.12ab 0.45d 0.40c 4.62c 1.32b 3.18bcd 6.44d 3.84cd
T4: P 0.66ab 0.40c 0.59bc 0.12ab 0.50bc 0.41bc 4.32d 1.22c 2.97e 6.42ed 4.13ab
T5: AM+CD 0.69a 0.40c 0.62abc 0.13ab 0.47cd 0.44ab 4.90b 1.32b 3.29bc 7.03c 4.33a
T6: CD+P 0.57d 0.48b 0.60bc 0.14a 0.52ab 0.41bc 4.66c 1.34b 3.17cd 7.25b 3.54e
T7: AM+P 0.69a 0.63a 0.64ab 0.14a 0.55a 0.45a 4.48c 1.30b 3.30b 7.61a 4.31a
T8: AM+CD+P 0.63bc 0.66a 0.66a 0.14a 0.55a 0.46a 5.36a 1.38a 3.48a 7.60a 3.96bc
LSD (0.05) 0.04 0.07 0.05 0.03 0.04 0.03 0.184 0.041 0.12 0.18 0.23
CV (%) 15.73 10.75 13.76 9.25 19.91 15.78 6.77 10.23 7.85 4.48 6.35
Level of sig. ** ** ** * ** ** ** ** ** ** **
Note: * Significant at 5% and ** significant at 1% level of probability.
In a column, the figure(s) having the same letter are not significantly different at 5% or 1% level of probability by DMRT.
In the opinion of Candido et al. (2015), cooperation with mycorrhizal mycelium also improves the nutrition
of a host plant with potassium. In the present study, the significant influence of mycorrhization on improved
potassium supply was confirmed for red amaranth and Indian spinach was grown on cowdung and phosphorus
supplemented condition (Table 2 & 3). A similar result was found by Giri et al. (2005) and Zuccarini (2007),
who observed that mycorrhizal symbiosis stimulated K content of Cassia siamea and lettuce, respectively.
When only AM fungi were inoculated no change in S content was noticed in both vegetables. But when AM
fungi applied with cowdung or phosphorus or with both, the S content increased in both vegetables. It is clear
from the present study that, solo application of AM fungi was less effective than a combined application with
cowdung or phosphorus (Table 2 & 3). This result was totally different to those of (Allen & Shachar-Hill 2009)
and (Azcón-Aguilar et al. 2003), who described that AMF enhanced S content in carrot and lettuce,
respectively. Further study on red amaranth and Indian spinach are required to clarify this issue. AM inoculation
with organic matter and phosphorus also increased Mg content in both vegetables and a similar result was found
by Azcón-Aguilar et al. (2003).
Our study revealed a non-significant effect of AM fungi on Ca contents compared to non-colonized red
amaranth plants (Table 2). Similarly, Kothari et al. (1990) found a decreased concentration of Ca in the shoot of
maize under AMF inoculated condition. Several previous studies showed inconsistent results regarding Ca
contents in different species due to mycorrhizal inoculation (Clark 1997, Alloush et al. 2000, Bagayoko et al.
2000). AMF associated plants usually maintain relatively low Ca content because the Ca-loaded polyphosphates
might hamper the functioning of AM fungi (Marschner & Dell 1994). Li et al. (2007) observed that inoculation
with AM fungi significantly increased the contents of calcium in taro. On the contrary, calcium content
increased in Indian spinach due to the application of AM supplemented with cowdung and phosphorus (Table
3). A similar result was found by (Rosen et al. 1994), who observed that the calcium content in spinach beets
significantly increased by municipal solid waste over chemical fertilizer.
In the current study, we found a positive impact of AMF inoculation on the Fe, Mn, Cu and Zn contents in
red amaranth (Table 4). Our result is in line with some previous studies (Hirata et al. 1988, Gavito et al. 2000).
In other experiments, similar results were found where the application of AM fungi increase Fe (Farzaneh et al.
2011), Mn (Farzaneh et al. 2011), Cu (Giri et al. 2005) and Zn (Ortas & Akpinar 2006) content in different plant
shoots. In Indian spinach, AMF inoculation with CD or P or both increased Fe, Cu and Zn content (Table 5).
They are in line with (Bi et al. 2003) and (Nogueira et al. 2007). (Al-Karaki 2006) also observed that shoot
contents of Fe were higher in AM compared with non-AM tomato plants grown under non-saline and saline
water conditions. On the other hand, application of AM solely or combined with CD did not increase Mn
content, but when AM applied with P than Mn content enhanced (Table 4 & 5). A totally different result was
found by (Farzaneh et al. 2011), who stated that AMF colonization resulted in a significantly higher Mn
concentration in chickpea. Detailed studies regarding this issue are necessary for a broad understanding of this
process.
Table 4. Correlation matrix among nutrient contents of red amaranth.
Correlation coefficient (r value)
Characters Fresh N P K S Ca Mg Fe Mn Cu
weight
N 0.609**
P 0.544** 0.799**
K 0.572** 0.811** 0.639**
S 0.427** 0.737** 0.791** 0.687**
Ca 0.580** 0.510** 0.384* 0.549** 0.465**
Mg 0.632** 0.651** 0.625** 0.739** 0.795** 0.477**
Fe 0.345* 0.755** 0.583** 0.567** 0.641** 0.396** 0.537**
Mn 0.568** 0.627** 0.590** 0.629** 0.702** 0.269NS 0.566** 0.280NS
Cu 0.214NS 0.832** 0.797** 0.694** 0.759** 0.587** 0.696** 0.573** 0.614**
Zn 0.300NS 0.364* 0.302 NS 0.442** 0.514** 0.238NS 0.348* 0.358* 0.551** 0.438**
Note: ** Significant at 1% and * Significant at 5% level of probability, NS = Not significant.
Tabulated value of r with 38 df are 0.404 at 1% level and 0.313 at 5 % level.
Table 5. Correlation matrix among nutrient contents of Indian spinach.

Correlation coefficient (r value)
Characters Fresh N P K S Ca Mg Fe Mn Cu
weight
N 0.630**
P 0.662** 0.378*
K 0.501** 0.368* 0.529**
S 0.507** 0.398* 0.399** 0.221NS
Ca 0.706** 0.434** 0.712** 0.297NS 0.632**
Mg 0.501** 0.269NS 0.606** 0.342* 0.300* 0.500**
Fe 0.814** 0.506** 0.562** 0.546** 0.385** 0.504** 0.415**
Mn 0.755** 0.482** 0.502** 0.277NS 0.614** 0.637** 0.379* 0.738**
Cu 0.772** 0.522** 0.503** 0.435** 0.402** 0.636** 0.507** 0.711** 0.767**
Zn 0.741** 0.458** 0.785** 0.541** 0.537** 0.768** 0.672** 0.645** 0.705** 0.745**
Note: ** Significant at 1% and * Significant at 5% level of probability, NS = Not significant.
Tabulated value of r with 38 df are 0.404 at 1% level and 0.313 at 5 % level.
The present investigation showed that AM-inoculated red amaranth and Indian spinach plants had higher
contents of protein than nonmycorrhizal plants (Table 4 & 5). The observed increase in protein contents in
mycorrhizal plants is in a good conformity with the results of other researchers (Abdel-Fattah et al. 2014).
Again, in our research, always the protein content in both the vegetables was higher when phosphorus was
applied with AM fungi (Table 1 & 2). This may demonstrate the role of phosphorus in the regulation of
symbiosis through synthesis, simulation, or activation of proteins and enzymes (Lambais & Mehdy 1995).
However, Epstein & Bloom (1999) emphasizes that phosphorus has a direct effect on diverse enzymatic plant
processes, very often acting as a biochemical co-factor. This protein increment leads to membrane stabilization
and helps plants to grow and develop under adverse conditions (Goudarzi & Pakniyat 2009).
AMF are capable of up taking and transporting almost all the 15 essential macro- and micronutrients. AMF
mobilize firm or rock-bound nutrients such as phosphorous, iron, and other tightly arrested mineral nutrients in
the soil (Teotia et al. 2017). In the present study, strong mycorrhizal effects on red amaranth and Indian spinach
were also observed when looking at the nutrient uptake. We found an increase of N, P, K, S, Mg, Fe, Mn, Zn,
and Cu uptake after AMF inoculation. Only changes in Ca uptakes with AM-red amaranth were relatively small
(Table 6 & 7). Similar positive effects of AMF on nutrient uptake in legumes were reported earlier (Ilbas &
Sahin 2005, Lin et al. 2007). Our results are also in accordance with those of previous work on chickpea by
Akhtar & Siddiqui (2006), showing increased P and K uptake in mycorrhizal plants. AMF produces an array of
siderophores and act as chelating agents that ultimately increase the nutrient uptake and transport in plants
(Caris et al. 1998). In higher plants the AMF association also helps plant by increasing the surface area of the
root by penetrating their hyphae deep into the soil (Schnepf et al. 2011). AMF interactions also may influence
plant growth through the improvement in nutritional attainment by mobilizing nutrient from the organic
substrates, by improving the fertilizer use efficacy, or by an advantageous alliance with other soil microbes
(Finlay 2008).
Table 6. Effect of AM, cowdung and phosphorus on macro- and micronutrients uptake (per plant) of red amaranth.
N P K S Ca Mg Fe Mn Cu Zn
Treatments
(mg) (mg) (mg) (mg) (mg) (mg) (µg) (µg) (µg) (µg)
T1: Control 1.10e 0.36c 0.40e 0.10e 0.54c 0.58d 0.007f 0.006e 0.003d 0.005g
T2: AM 6.44d 1.76c 2.24d 0.56d 2.56b 3.40c 0.045e 0.030d 0.016c 0.026de
T3: CD 8.26bcd 3.80b 2.74cd 1.00cd 3.04b 4.30b 0.061bc 0.031d 0.019c 0.025ef
T4: P 7.46c 3.80b 2.10d 0.90cd 2.64b 3.04c 0.049de 0.026d 0.020c 0.019f
T5: AM+CD 11.7b 4.14b 3.58bc 1.24c 3.58b 4.68bc 0.068b 0.056c 0.027b 0.034c
T6: CD+P 10.7bc 5.42b 3.48bc 1.42bc 3.54b 5.50b 0.055cd 0.067b 0.030b 0.032cd
T7: AM+P 11.8b 5.50b 3.96b 1.88b 3.86b 6.20b 0.073b 0.061bc 0.033b 0.042b
T8: AM+CD+P 19.7a 9.58a 6.14a 4.16a 6.18a 10.6a 0.124a 0.135a 0.053a 0.069a
LSD (0.05) 4.14 1.81 1.21 0.59 1.35 2.02 0.006 0.006 0.006 0.006
CV (%) 9.97 15.77 17.29 11.48 16.30 14.63 13.29 18.22 13.05 15.14
Level of sig. ** ** ** ** ** ** ** ** ** **
Note: ** = Significant at 1% level of probability.
In a column, the figure(s) having the same letter are not significantly different at 1% level of probability by DMRT.
Table 7. Effect of AM, cowdung and phosphorus on macro- and micronutrients uptake (per plant) of Indian spinach.
N P K S Ca Mg Fe Mn Cu Zn
Treatments
(mg) (mg) (mg) (mg) (mg) (mg) (µg) (µg) (µg) (µg)
T1: Control 4.86d 2.90e 5.22e 0.94d 3.78e 3.70e 0.03e 0.01c 0.02e 0.06e
T2: AM 12.3c 9.00d 12.8d 2.16c 8.98d 8.62d 0.09d 0.02bc 0.06d 0.13d
T3: CD 12.6c 7.62d 12.4d 2.54b 9.24d 8.26d 0.09d 0.03b 0.07cd 0.13d
T4: P 13.8c 8.34d 12.2d 2.60b 10.4cd 8.46d 0.09d 0.03b 0.06d 0.13d
T5: AM+CD 16.3b 9.48cd 14.7c 2.98b 11.0bc 10.3c 0.12c 0.03b 0.08c 0.17c
T6: CD+P 13.6c 11.5c 14.3c 3.28b 12.4b 9.88c 0.11c 0.03b 0.08c 0.17c
T7: AM+P 24.9a 22.8b 23.0b 5.22a 19.9a 16.1b 0.16b 0.05a 0.12b 0.27b
T8: AM+CD+P 24.9a 25.8a 26.1a 5.46a 21.6a 18.0a 0.21a 0.05a 0.14a 0.30a
LSD (0.05) 1.77 2.17 1.67 0.75 1.67 1.32 0.006 0.006 0.006 0.006
CV (%) 6.57 9.02 6.64 15.65 7.80 8.04 19.72 16.07 16.85 14.60
Level of sig. ** ** ** ** ** ** ** ** ** **
Note: ** = Significant at 1% level of probability.
In a column, the figure(s) having the same letter are not significantly different at 1% level of probability by DMRT.
CONCLUSIONS
In summary, the capability of AMF to increase mineral nutrition in both the plant species is clearly
positively influenced. AMF could be used for the production mineral rich leafy vegetables to provide almost all
the essential minerals and organic nutrients at a higher quantity to humans for maintenance of health. On the
other side, mycorrhizal fungi not merely assist in the uptake of the major plant nutrients such as P, K and N but
also help in captivating other micronutrients like Fe, Cu, Zn, Mn, etc. Consequently, mycorrhiza is established
as a significant association for nutrient management in the ecosystem.
ACKNOWLEDGEMENTS
The authors are thankful to Department of Biochemistry & Molecular Biology and Department of
Agricultural Chemistry, Bangladesh Agricultural University, Mymensingh, Bangladesh and Graduate School of
Science, Kyushu University, Japan for providing required facilities to conduct the research work.
REFERENCES
Abdel-Fattah GM, Asrar AA, Al-Amri SM & Abdel-Salam EM (2014) Influence of arbuscular mycorrhiza and
phosphorus fertilization on the gas exchange, growth and phosphatase activity of soybean (Glycine max L.)
plants. Photosynthetica 52: 581–588.
ACC/SCN (2000) ACC/SCN (Administrative Committee on Coordination, Subcommittee on Nutrition);
International Food Policy Research Institute. Fourth Report on the World Nutrition Situation: United
Nations.
Akhtar MS & Siddiqui ZA (2006) Effects of phosphate solubilizing microorganisms on the growth and root-rot
disease complex of chickpea. Mikologiya I Fitopatologiya 40: 246–254.
Al-Karaki GN (2006) Nursery inoculation of tomato with arbuscular mycorrhizal fungi and subsequent
performance under irrigation with saline water. Scientia Horticulturae 109: 1–7.
Allen ES, Grimshaw HW, Parkinson JA & Quarmbly C (1974) Chemical Analyses of Ecological Materials.
Blackwell Publication, Oxford.
Allen JW & Shachar-Hill Y (2009) Sulfur transfer through an arbuscular mycorrhiza. Plant Physiology 149:
549–560.
Allen MF (2011) Linking water and nutrients through the vadose zone: a fungal interface between the soil and
plant systems. Journal of Arid Land 3: 155–163.
Alloush GA, Zeto SK & Clark RB (2000) Phosphorus source, organic matter, and arbuscular mycorrhiza effects
on growth and mineral acquisition of chickpea grown in acidic soil. Journal of Plant Nutrition 23: 1351–
1369.
Antunes PM, Schneider K, Hillis D & Klironomos JN (2007) Can the arbuscular mycorrhizal fungus Glomus
intraradices actively mobilize P from rock phosphates? Pedobiologia 51: 281–286.
Azcón-Aguilar C, Palenzuela J, Roldán A, Bautista S, Vallejo R & Barea JM (2003) Analysis of the mycorrhizal
potential in the rhizosphere of representative plant species from desertification-threatened Mediterranean
shrublands. Applied Soil Ecology 22: 29–37.
Bagayoko M, George E, Romheld V, Buerkert A & Stuttgart D (2000) Effects of mycorrhizae and phosphorus
on growth and nutrient uptake of millet, cowpea and sorghum on a West African soil. Journal of
Agricultural Science 135: 399–407.
Balestrini R, Lumini E, Borriello R & Bianciotto V (2015) Plant-Soil Biota Interactions. In: Soil Microbiology,
Ecology and Biochemistry, 4th edition. Academic Press.
Berruti A, Lumini E, Balestrini R & Bianciotto V (2016) Arbuscular mycorrhizal fungi as natural biofertilizers:
Let’s benefit from past successes. Frontiers in Microbiology 6: 1–13.
Bi YL, Li XL, Christie P Hu ZQ & Wong MH (2003) Growth and nutrient uptake of arbuscular mycorrhizal
maize in different depths of soil overlying coal fly ash. Chemosphere 50: 863–869.
Brown KH & Wuehler SE (1990) Zinc and Human Health: The Results of Recent Trials and Implications for
Programmed Interventions and Research. The Micronutrient Initiative/International Development Research
Centre, Ottawa, Canada.
Candido V, Campanelli G, D’Addabbo T, Castronuovo D, Perniola M & Camele I (2015) Growth and yield
promoting effect of artificial mycorrhization on field tomato at different irrigation regimes. Scientia
Horticulturae 187: 35–43.
Caris C, Hördt W, Hawkins HJ, Römheld V & George E (1998) Studies of iron transport by arbuscular
mycorrhizal hyphae from soil to peanut and sorghum plants. Mycorrhiza 8: 35–39.
Carvajal M, Martinez-Ballesta MC, Moreno DA, Bernabeu J & García-Viguera C (2014) Seed Coating Increase
Broccoli Nutrient Content and Availability after Cooking. Journal of Agricultural Science 7: 182–191.
Clark RB (1997) Arbuscular mycorrhizal adaptation, spore germination, root colonization, and host plant
growth and mineral acquisition at low pH. Plant Soil 192: 15–22.
Colella T, Candido V, Campanelli G, Camele I & Battaglia D (2014) Effect of irrigation regimes and artificial
mycorrhization on insect pest infestations and yield in tomato crop. Phytoparasitica 42: 235–246.
Epstein E & Bloom AJ (1999) Mineral nutrition of plants: principles and perspectives. Sinauer Associates, Inc.
Sinauer Associates, Inc.
Estefan G, Sommer R & Ryan J (2013) Methods of Soil, Plant, and Water Analysis. A manual for the West Asia
and North Methods of Soil, Plant, and Water Analysis: A manual for the West Asia and North Africa region,
ICARDA (International Center for Agricultural Research in the Dry Areas) Box 114/5055, Beirut, Lebanon.
Farahani A, Lebaschi H, Hussein M, Hussein SA, Reza VA & Jahanfar D (2008) Effects of arbuscular
mycorrhizal fungi, different levels of phosphorus and drought stress on water use efficiency, relative water
content and proline accumulation rate of Coriander (Coriandrum sativum L.). Journal of Medicinal Plants
Research 2: 125–131.
Farzaneh M, Vierheilig H, Lössl A & Kaul HP (2011) Arbuscular mycorrhiza enhances nutrient uptake in
chickpea. Plant, Soil and Environment 57: 465–470.
Finlay RD (2008) Ecological aspects of mycorrhizal symbiosis: With special emphasis on the functional
diversity of interactions involving the extraradical mycelium. Journal of Experimental Botany 59: 1115–
1126.
Gavito ME, Curtis PS, Mikkelsen TN & Jakobsen I (2000) Atmospheric CO2 and mycorrhiza effects on
biomass allocation and nutrient uptake of nodulated pea (Pisum sativum L.) plants. Journal of Experimental
Botany 51: 1931–1938.
Ghosh A, Tahjib-Ul-Arif M, Chamely SG, Haque MR, Rahman MM & Bhuiyan MAH (2017) Comparative
effect of arbuscular mycorrhiza, cowdung and phosphorus on growth and yield contributing characters of red
amaranth (Amaranthus tricolor L.) and Indian spinach (Basella alba L.). Tropical Plant Research 4: 254–
263.
Ghosh AB, Bajoy JC, Hasan R & Singh D (1983) Soil and Water Testing Method. A Laboratory Manual,
Division of Soil Science and Agricultural Chemistry, IARI, New Delhi, India.
Giri B, Kapoor R & Mukerji KG (2005) Effect of the arbuscular mycorrhizae Glomus fasciculatum and G.
macrocarpum on the growth and nutrient content of Cassia siamea in a semi-arid Indian wasteland soil. New
Forests 29: 63–73.
Gomez AA & Gomez KA (1984) Statistical procedures for agricultural research. John Wiley & Sons. John
Wiley & Sons.
Goudarzi M & Pakniyat H (2009) Salinity causes increase in proline and protein contents and peroxidase
activity in wheat cultivars. Journal of Applied Sciences 9: 348–353.
Graham RD, Welch RM & Bouis HE (2001) Addressing micronutrient malnutrition through enhancing the
nutritional quality of staple foods: Principles, perspectives and knowledge gaps. Advances in Agronomy 70:
77–142.
Guo ZH, Zhang XD, Huang LL, Ju GS & Chen J (2006) Solar energy and water utilization of Quercus
mongolica, a deciduous broadleaf tree, in different light regimes across the edge of a deciduous broad-leaved
forest. Acta Ecologica Sinica 26: 1047–1056.
Harrison MJ, Pumplin N, Breuillin FJ, Noar RD & Park H-J (2010) Phosphate Transporters in Arbuscular
Mycorrhizal Symbiosis. In: Koltai H & Kapulnik Y (eds) Arbuscular Mycorrhizas: Physiology and Function.
Springer Netherlands, Dordrecht, pp. 117–135.
Hirata H, Masunaga T & Koiwa H (1988) Response of chickpea grown on ando-soil to vesicular-arbuscular
mycorrhizal infection in relation to the level of phosphorus application. Soil Science and Plant Nutrition 34:
441–449.
Ilbas AI & Sahin S (2005) Glomus fasciculatum inoculation improves soybean production. Acta Agriculturae
Scandinavica, Section B - Soil & Plant Science 55: 287–292.
Jackson ML (1973) Soil Chemical Analysis. Hall of India Pvt. Ltd., New Delhi.
Kehri H & Chandra S (2001) Performance of black gram with VAM inoculation and phosphate fertilization.
Philippine Journal of Science 130: 33–38.
Kothari SK, Marschner H & Romheld V (1990) Direct and indirect effects of VA mycorrhizal fungi and
rhizosphere microorganisms on acquisition of mineral nutrients by maize (Zea mays L.) in a calcareous soil.
New Phytologist 116: 637–645.
Lambais MR & Mehdy MC (1995) Differential Expression of Defense-Related Genes in Arbuscular
Mycorrhiza. Canadian Journal of Botany 73: 533–540.
Li M, Liu R, Christie P & Li X (2007) Influence of Three Arbuscular Mycorrhizal Fungi and Phosphorus on
Growth and Nutrient Status of Taro. Communications in Soil Science and Plant Analysis 36: 2383–2396.
Lin AJ, Zhang XH, Wong MH, Ye ZH, Lou LQ, Wang YS & Zhu YG (2007) Increase of multi-metal tolerance
of three leguminous plants by arbuscular mycorrhizal fungi colonization. Environmental Geochemistry and
Health 29: 473–481.
Marschner H & Dell B (1994) Nutrient uptake in mycorrhizal symbiosis. Plant Soil 159: 89–102.
Nagarathna TK, Prasad TG, Bagyaraj DJ & Shadakshari YG (2007) Effect of arbuscular mycorrhiza and
phosphorus levels on growth and water use efficiency in Sunflower at different soil moisture status.
International Journal of Agricultural Technology 3: 221–229.
Nogueira MA, Nehls U, Hampp R, Poralla K & Cardoso EJBN (2007) Mycorrhiza and soil bacteria influence
extractable iron and manganese in soil and uptake by soybean. Plant Soil 298: 273–284.
Nouri E, Breuillin-Sessoms F, Feller U & Reinhardt D (2014) Phosphorus and Nitrogen Regulate Arbuscular
Mycorrhizal Symbiosis in Petunia hybrida. PLoS One 9: e90841.
Ortas I & Akpinar C (2006) Response of kidney bean to arbuscular mycorrhizal inoculation and mycorrhizal
dependency in P and Zn deficient soils. Acta Agriculturae Scandinavica, Section B - Soil & Plant Science
56: 101–109.
Page AL, Miller RH & Keeney DR (1987) Methods of Soil Analysis, Part-2, 2nd edition. Amer. Soc. Agron. Inc.
Medison, Washington, USA.
Pringle A & Bever JD (2002) Divergent phenologies may facilitate the coexistence of arbuscular mycorrhizal
fungi in a North Carolina grassland. American Journal of Botany 89: 1439–1446.
Quilambo OA (2003) The vesicular-arbuscular mycorrhizal symbiosis. African Journal of Biotechnology 2:
539–546.
Rosen CJ, Olson D & Bierman PM (1994) Swiss Chard and Alfalfa Responses to Soils Amended with
Municipal Solid Waste Incinerator Ash: Growth and Elemental Composition. Journal of Agricultural and
Food Chemistry 42: 1361–1368.
Salvioli A, Zouari I, Chalot M & Bonfante P (2012) The arbuscular mycorrhizal status has an impact on the
transcriptome profile and amino acid composition of tomato fruit. BMC Plant Biology 12: 44.
Schnepf A, Jones D & Roose T (2011) Modelling Nutrient Uptake by Individual Hyphae of Arbuscular
Mycorrhizal Fungi: Temporal and Spatial Scales for an Experimental Design. Bulletin of Mathematical
Biology 73: 2175–2200.
Schüβler A, Schwarzott D & Walker C (2001) A new fungal phylum, the Glomeromycota: phylogeny and
evolution. Mycological Research 105: 1413–1421.
Smith F, Jakobsen I & Smith S (2000) Spatial differences in acquisition of soil phosphate between two
arbuscular mycorrhizal fungi in symbiosis with Medicago truncatula. New Phytologist 147: 357–366.
Tandon H (1995) Methods of Analysis of Soils, Plants, Water and Fertyilizers. Fertilizer Development and
Consultation Organization, New Delhi. pp. 44–45.
Teotia P, Kumar M, Prasad R, Kumar V, Tuteja N & Varma A (2017) Mobilization of Micronutrients by
Mycorrhizal Fungi. In: Varma A, Prasad R & Tuteja N (eds) Mycorrhiza - Function, Diversity, State of the
Art. Springer International Publishing, Cham, pp. 9–26.
Welch RM & Graham RD (2004) Breeding for micronutrients in staple food crops from a human nutrition
perspective. Journal of Experimental Botany 55: 353–364.
Wolf B (1982) A comprehensive system of leaf analyses and its use for diagnosing crop nutrient status.
Communications in Soil Science and Plant Analysis 13: 1035–1059.
Yoshimura Y, Ido A, Iwase K, Matsumoto T & Yamato M (2013) Communities of Arbuscular Mycorrhizal
Fungi in the Roots of Pyrus pyrifolia var. culta (Japanese Pear) in Orchards with Variable Amounts of Soil-
Available Phosphorus. Microbes and Environments 28: 105–111.
Zuccarini P (2007) Mycorrhizal infection ameliorates chlorophyll content and nutrient uptake of lettuce exposed
to saline irrigation. Plant, Soil and Environment 53: 283–289.
Research article
Molecular identification based on the sequence of internal

transcribed spacer (ITS) of the ribosomal nuclear DNA (rDNA) of
pathogenic fungus Pythium aphanidermatum (Edson) Fitzp.
isolated from soil and its morphology
Maneesha George and Pramod W. Ramteke*
Sam Higginbottom University of Agriculture, Technology and Sciences, Post Agricultural Institute,
Rewa Road, Naini, Prayagraj-211007, Uttar Pradesh, India
*Corresponding Author: pramod.ramteke@shiats.edu.in [Accepted: 25 December 2018]
Abstract: Soil-borne fungi Pythium aphanidermatum causes damping-off of cucumber, coriander

and other economically important plants in India and presumably in many other countries. The
objective of the present investigation was to characterize the pathogen morphologically in vitro
and to confirm its molecular identity by the sequence of ITS region of rDNA. P. aphanidermatum
was isolated from soil and cultured in PDB broth and PDA solid media. The characteristic of
growth was monitored and the reproductive structures were analyzed. Hyphae were grown with a
daily increment of 2.5-3.0 cm on PDB. Sporangia were swollen, multinucleate and usually
measure 10-50 μm in diameter. Oogonia were found mostly terminal, spherical, 22-27 µm in
diameter. Fungus was also characterized using molecular methods based on ITS-PCR. The
ampliﬁed sequence was compared with the available sequences in the NCBI GenBank. The
sequence showed 99% similarity with other species of Pythium. Based on the morphological and
molecular characters isolate was confirmed as Pythium aphanidermatum. The isolation,
morphological characterization and sequencing of ITS region of rDNA will add knowledge to the
scientific community for proper identification and in-depth research on the management of this
plant pathogen.
Keywords: ITS sequence - Morphological characteristics - Oomycete - Plant pathogen - Pythium
aphanidermatum.
[Cite as: George M & Ramteke PW (2018) Molecular identification based on the sequence of internal
transcribed spacer (ITS) of the ribosomal nuclear DNA (rDNA) of pathogenic fungus Pythium aphanidermatum
(Edson) Fitzp. isolated from soil and its morphology. Tropical Plant Research 5(3): 385–390]
INTRODUCTION
The fungus Pythium is a facultative parasite and lives saprophytically on the moist humus in soil and attacks
seedlings at the soil level. It causes damping-off, soft-rot, crown-rot, wheat-rot or foot-rot diseases and
sometimes causing death (van der Plaats‐Niterink 1981, El‐Tarabily et al. 2009). One of the species of Pythium,
P. aphanidermatum (Edson) Fitzp. causes extensive damage to economically important crops worldwide
(Martin & Loper 1999). It causes considerable damage to cucumber crops worldwide by damping‐off disease of
seedlings and root and Crown-rots of mature plants (Zitter et al. 1996, Chen et al. 2000, Al‐Sa’di et al. 2007). It
is also a causal pathogen of damping-off disease of coriander in India (Ashwathi et al. 2017). Several studies
have also been carried out in India. Muthukumar (2016) isolated twelve different species of Pythium from
different soil types, water types, vegetables and ornamental plants of Delhi.
The genus Pythium was first created by Pringsheim in 1858. Taxonomic details of the genus had been
described by the end of the 19th century and many new species had been added. Furthermore, the genus was
included in a new family, Pythiaceae, by Schroter in 1897 under the kingdom of Stramenopila, subdivision of
Mastigomycotina, class of Oomycetes and order of Peronosporales (Domsch et al. 1980). Afterwards, there are
almost 307 species of Pythium were morphologically and taxonomically described by different scientist and
Received: 05 September 2018 Published online: 31 December 2018
George & Ramteke 2018
submitted in www.mycobank.org (Middleton 1943, Waterhouse 1968, Plaats-Niterink 1981). An account of this
genus in India was given by Butler (1907).
Rapid and safe identification is crucial to implement proper diagnosis and effective treatment to the diseased
plants (Schurko et al. 2003). Though the identification and classification of Pythium species are based on
morphological and physiological characteristics; but taxonomists faced problems due to a lack of sexual
structures and failure to induce in vitro zoosporogenesis (Schurko et al. 2003). Thus the use of molecular
technologies has become an indispensable tool for accurate identification of this pathogen (Grooters & Gee
2002, Schurko et al. 2003, Pannanusorn et al. 2007, Ashwathi et al. 2017). The polymerase chain reaction
(PCR) for the amplification of the ribosomal genes are used for the genetic identification of many organisms
because they comprise both highly conserved sequences during evolution.
In the present study, we have isolated the fungal pathogen Pythium aphanidermatum. We have identified the
pathogen morphologically based on its reproductive structures. We have also obtained the sequence of the
internal transcribed spacer (ITS) of the ribosomal nuclear DNA (rDNA) with the PCR method using universal
primers (Chen et al. 1992) for the molecular identification and deposited the sequence in Genbank (NCBI).

Collection and isolation of the fungus
The Fungus was isolated from Lucknow (26º 50′ N latitude, 80º 56′ E longitude, 128 m above the sea level),
the capital of Uttar Pradesh, is spread over an area of 310 Km2 in the central plain of the Indian subcontinent.
The sample was collected from soil samples together with plant root debris in sterile capped bottles. The fungus
was isolated from these samples by the usual baiting techniques (Middleton 1943, Paul et al. 1998). The
collected sample was purified in sterile distilled water and maintained on solid media like PDA (potato dextrose
agar) at 25ºC (Booth 1971). The isolate was identified with the help of keys provided by Middleton (1943),
Waterhouse (1967), Plaats-Niterink (1981) and also by the sequence obtained from ITS-PCR.
DNA isolation and PCR
The culture conditions, DNA isolation and the PCR amplification of the ITS of the rDNA were the same as
described earlier (Paul et al. 1999, Paul 2000). The fungus was grown in potato dextrose broth medium,
incubated at 25ºC on a rotary shaker for 5 days. The mycelium was then washed in TE buffer (10 mM Tris-HCl
pH 8, 1 mM EDTA) and was kept at 320ºC for 24 h and DNA was extracted. Polymerase chain reactions were
performed in 50 μl volumes containing 100 pmol of each of the universal primers ITS1 (TCC GTA GGT GAA
CCT GCG G) and ITS4 (TCC TCC GCT TAT TGA TAT GC); 200 μM of each of the four dNTPs; 1.5 U of
Taq polymerase (Invitrogen) and 300 ng DNA template in a PCR buffer (50 mM KCl, 1.5 mM MgCl 2, 10 mM
Tris‐HCl). The temperature cycling parameters were 95°C for 3 min for denaturation the first cycle and 1 min
for subsequent cycles, primer annealing for 1 min at 55°C and primer extension at 72°C for 1 min with a total of
35 cycles and a final extension at 72°C for 3 min 25.
Sequencing and phylogenetic analysis
The PCR product was purified and sequenced (Chromous Biotech Pvt. Ltd., Bangalore). The sequence was
compiled by ApE software (A plasmid Editor). The rDNA sequence was submitted to NCBI gene bank. The
sequence was also blasted (NCBI) to determine the percentage of similarity with related sequences. The BioEdit
sequence alignment editor was used to obtain multiple alignments of nucleotides with related sequences for ITS.
The blast output was processed for generating the phylogenetic tree in software MEGA7 by using ‘Maximum
Likelihood’ method based on the Tamura-Nei model (Tamura & Nei 1993, Kumar et al. 2016). ITS sequence
was aligned with other reported sequence of P. aphanidermatum (Genbank accession no. MF040822.1,
KY646468.1, MF347709.1) to obtained the percentage of similarity using the online software ‘Multalin’, which
creates a multiple sequence alignment by progressive pairwise alignments (Corpet 1988).

Morphological descriptions
Pythium is a representative of class Oomycetes, which bear large spherical Oogonia or female gametangia
(Fig. 1) (van der Plaats-Niterink 1981). The class comprises mostly water molds. In the present study, P.
aphanidermatum was collected from soil and their hyphae were grown with a daily increment of 2.5–3.0 cm at
25ºC on PDA. Asexual reproduction occurred under favourable environmental conditions through sporangia
which developed into vesicles containing zoospores; which were liberated to give rise to hyphae (Stanghellini &
Burr 1973). Sporangia were swollen, multinucleate and measured 10–50 μm in diameter (Fig. 1). The structure
of sporangia differs from species to species it can be spherical, filamentous slightly inflated and filamentous
inflated. In this study, sporangia were found inflated filamentous, readily produced zoospores on transfer to
water at 20–30ºC, which is a typical feature of P. aphanidermatum. After maturation, the sporangia get
separated from the rest of hyphae by means of a septum as observed here (Fig. 1). When they were matured, a
tubular structure called Papilla was developed at the apex or laterally which soon developed into a sac-like thin
walled vesicle (Fig. 1). Papilla helps in the dispersal of uninucleate, naked, pyriform, biflagellate zoospores
(Stanghellini & Burr 1973). As Pythium is homothallic, the male and female sex organs were developed in close
proximity of each other either on the same or different hypha embedded in the host tissue (Fig. 1). Oogonia were
found mostly terminal, spherical, 22–27 µm diameter; oospores aplerotic, 17–19 µm diameter, moderately thick-
walled; antheridia were barrel or dome-shaped or cylindrical with a dimension of 11–19 µm long and 10–14 µm
wide monoclinous, intercalary or terminal and found 1–2 per oogonium. Ashwathi et al. (2017) observed the
similar structure of oogonia (terminal, globose and smooth 20–25 µ diameter) and antheridia (broadly sac-
shaped 10–14 µ long and 10–14 µ wide, 2 per oogonium) in P. aphanidermatum isolated from coriander
growing regions of Tamil Nadu, India. Pythium species have been traditionally identified and classified based
on the morphology of asexual and sexual structures (Van der Plaats-Niterink 1981) as structures of sporangia
and oospore vary between the species (Schroeder et al. 2013). Considering the reproductive structures
morphologically the isolated fungus was identified as P. aphanidermatum as the similar morphology found in
earlier reposts (Ashwathi et al. 2017) and with the help of keys provided by Middleton (1943), Waterhouse
(1967), Plaats-Niterink (1981).
Figure 1. Vegetative and reproductive structures of Pythium aphanidermatum (Edson) Fitzp.: A–B, Oogonia; C–D, Inflated
filamentous Sporangia; E, Sporangia with laterally placed Papilla; F, Papilla was developed at the apex of Sporangia.
Molecular identification based on the sequence of ITS region of rDNA
In the present study, the partial ITS sequence of the flanking regions of the rDNA of P. aphanidermatum
was PCR amplified using the specific forward and reverse primers with an amplicon size 820 base pairs. The
sequence was submitted to NCBI GenBank (Accession number MK158217). The sequence of amplified ITS
region is given below.
1 tttcgtatcc gattcgcgcc gggtttcgag cgtgtttgta ttcgttactg tgtaatgcag
61 tgatagtgca agcaatgcga ggagctttgg ctgatcgaag gtcgttgcgc aagtatttat
121 atgcgcgctt cggctgactt atactttcaa accccttact ttaaaaactg atcaatactg
181 tgaggacgaa agtctttgct ttaaaactag ataacaactt tcagcagtgg atgtctaggc
241 tcgcacatcg atgaagaacg ctgcgaactg cgatacgtaa tgcgaattgc agaattcagt
301 gagtcatcga aattttgaac gcatattgca ctttcgggtt atacctggaa gtatgtctgt
361 atcagtgtcc gtacatcaac cttgcctctc tttgtcggtg tagtccggtt tgtagcatgt
421 gcagatgtga ggtgtctcgc ggcgtgtgtg tgtgctgtaa aatgcatacg cttgctgcga
481 gtccctttaa aacgacacga tctttctatt tgctttctat ggagcgcgta tctcgaacgc
541 ggcggtcctc ggatcgctcg cagtcgacag cgacttcagc ggagacatat ggaagaaacc
601 actattcgcg gtacgttagg cttcggctcg acaatgttgc gttttagtgt gtggattccg
661 ttttcgcttt gaggtgtact gttcggttgt gagcttgaac cttgtgtctc gctttgttag
721 tagaggtgtg tcgatttctg tggtttgatt ccgcacttta tgtgtgggta gagagactcc
781 atttgggaaa cattgtactg cgcgtacgct ttcgggtgt
Figure 2. Molecular Phylogenetic analysis of Pythium aphanidermatum (Edson) Fitzp. based on the Tamura-Nei model by
Maximum Likelihood method. The analysis is based on 14 nucleotide sequences from NCBI BLAST result.
The ampliﬁed sequence was blasted in the NCBI. The sequence showed 99% similarity to partial sequence
of ITS-1 of 5.8S ribosomal RNA gene and ITS-2 of large subunit ribosomal RNA gene (MK026903.1,
KF831231.1, KF802196.1, KU715058.1 etc.) of different isolates of Pythium sp. (Fig. 2). Phylogenetic analysis
based on NCBI Genbank database confirmed the genus as Pythium and morphological observation predicted the
species as P. aphanidermatum.
The ITS sequence was aligned with other reported sequence of P. aphanidermatum (Genbank accession no.
MF040822.1, KY646468.1, MF347709.1) and found maximum 90% of similarity (Fig. 3).
Figure 3. Multiple sequence alignment of the ITS sequence of Pythium aphanidermatum (Edson) Fitzp. with previously
reported different isolates of Pythium aphanidermatum (Genbank accession no. MF040822.1, KY646468.1, MF347709.1).
The small difference in the ITS region is significant for the genus Pythium. The results of the sequence
alignment support the morphological observations as found in earlier reports (Grooters & Gee 2002, Schurko et
al. 2003, Pannanusorn et al. 2007, Ashwathi et al. 2017).
CONCLUSION
Pathogenic fungus Pythium aphanidermatum was isolated from Lucknow, India. The taxonomic
identification was carried out by morphological diagnosis and molecular analysis. After the analysis of the
characteristics and dimensions of hyphae, sporangia and sexual reproductive structures, this fungal pathogen
was identified as Pythium aphanidermatum. The partial ITS sequence of the rDNA of P. aphanidermatum was
obtained and submitted to NCBI GenBank (Accession number MK158217). Phylogenetic analysis revealed its
similarity with other Pythium species. The isolation and identification of this fungus will help in further research
on accurate biological control for this plant pathogen.
ACKNOWLEDGEMENTS
Authors are grateful to Sam Higginbottom University of Agriculture, Technology and Sciences, Post
Agricultural Institute of Prayagraj, Uttar Pradesh for throughout support to carry out the work.
REFERENCES
Al‐Sa'di AM, Drenth A, Deadman ML, De Cock AW & Aitken EA (2007) Molecular characterization and
pathogenicity of Pythium species associated with damping‐off in greenhouse cucumber (Cucumis sativus) in
Oman. Plant Pathology 56: 140–149.
Ashwathi S, Ushamalini C, Parthasarathy S & Nakkeeran S (2017) Morphological, pathogenic and molecular
characterisation of Pythium aphanidermatum: A causal pathogen of coriander damping-off in India. The
Pharma Innovation 6: 44.
Booth C (1971) Fungal Culture Media. In: Methods in microbiology. Academic Press, Elsevier, United States,
Vol. 4, Chapter II, pp. 49–94.
Butler EJ (1907) An account of the genus Pythium and some Chytridiaceae. Memoirs of the Department of
Agriculture in India (Botanical series) 1: 1–160.
Chen C, Belanger RR, Benhamou N & Paulitz TC (2000) Defense enzymes induced in cucumber roots by
treatment with plant growth-promoting rhizobacteria (PGPR) and Pythium aphanidermatum. Physiological
and Molecular Plant Pathology 56: 13–23.
Chen W, Schneider RW & Hoy JW (1992) Taxonomic and phylogenetic analyses of ten Pythium species using
isozyme polymorphisms. Phytopathology 82: 1234–1244.
Corpet F (1988) Multiple sequence alignment with hierarchical clustering. Nucleic Acids Research 16: 10881–
10890.
Domsch KH, Gams W & Anderson TH (1980) Pythium Pringsheim 1858. Compendium of soil fungi. Academic
Press, Elsevier, New York, United States, pp. 678–697.
El‐Tarabily KA, Nassar AH, Hardy GS & Sivasithamparam K (2009) Plant growth promotion and biological
control of Pythium aphanidermatum, a pathogen of cucumber, by endophytic actinomycetes. Journal of
Applied Microbiology 106: 13–26.
Grooters AM & Gee MK (2002) Development of a nested polymerase chain reaction assay for the detection and
identification of Pythium insidiosum. Journal of Veterinary Internal Medicine 16: 147–152.
Kumar S, Stecher G & Tamura K (2016) MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for
bigger datasets. Molecular Biology and Evolution 33: 1870–1874.
Martin F & Loper JE (1999) Soilborne diseases caused by Pythium species ecology, epidemiology and prospects
for biological control. Critical Reviews in Plant Sciences 18: 111–181.
Middleton JT (1943) The taxonomy, host range and geographic distribution of the genus Pythium. Memoirs of
the Torrey Botanical Club 20: 1–171.
Muthukumar A, Udhayakumar R & Naveenkumar R (2016) Ecofriendly management of damping-off of
solanaceous crops caused by Pythium species. In: Current Trends in Plant Disease Diagnostics and
Management Practices. Springer, United States, pp. 49–90.
Pannanusorn S, Chaiprasert A, Prariyachatigul C, Krajaejun T, Vanittanakom N, Chindamporn A,
Wanachiwanawin W & Satapatayavong B (2007) Random amplified polymorphic DNA typing and
phylogeny of Pythium insidiosum clinical isolates in Thailand. Southeast Asian Journal of Tropical
Medicine and Public 38: 383.
Paul B (2000) ITS1 region of the rRNA of Pythium megacarpum sp. nov., its taxonomy, and its comparison
with related species. FEMS Microbiology Letters 183: 105–110.
Paul B, Galland D & Masih I (1999) Pythium prolatum, isolated from soil in the Burgundy region: a new record
for Europe. FEMS Microbiology Letters 173: 69–75.
Paul B, Galland D, Bhatnagar T & Dulieu H (1998) A new species of Pythium isolated from the Burgundy
region of France. FEMS Microbiology Letters 158: 207–213.
Plaats-Niterink AJ (1981) Monograph of the genus Pythium. Studies in Mycology 21: 1–242.
Pringsheim N (1858) Contribution to morphology and systematics of algae. 2, The Saprolegniales. Jb Wiss
Botany 1: 284–306.
Schroeder KL, Martin FN, de Cock AWAM, Lévesque CA, Spies CFJ, Okubara PA & Paulitz TC (2013)
Molecular detection and quantification of Pythium species: evolving taxonomy, new tools, and challenges.
Plant Disease 97: 4–20.
Schroter J (1897) Pythiaceae. Engler & Prantl nat PflFam 11: 104–105.
Schurko AM, Mendoza L, Lévesque CA, Désaulniers NL, De Cock AW & Klassen GR (2003) A molecular
phylogeny of Pythium insidiosum. Mycological Research 107(5): 537–544.
Stanghellini ME & Burr TJ (1973) Germination in vivo of Pythium aphanidermatum oospores and sporangia.
Phytopathol 63: 1493–1496.
Tamura K & Nei M (1993) Estimation of the number of nucleotide substitutions in the control region of
mitochondrial DNA in humans and chimpanzees. Molecular Biology and Evolution 10: 512–526.
van der Plaats-Niterink AJ (1981) Monograph of the genus Pythium. Baarn: Centraalbureau voor
Schimmelcultures, Studies in mycology, Vol. 21, pp. 1–242.
Waterhouse GM (1967) Key to Pythium Pringsheim. Mycology Papers 109: 1–15.
Zitter TA, Hopkins DL & Thomas CE (1996) Compendium of Cucurbit Diseases. American Phytopathological
Society Press, St Paul MN.
Research article
Effect of disturbance on mangrove species diversity in Delta

Tumpat, Kelantan, Malaysia
Z. Nor Syahirah1, J. N. J. Noor2* and A. M. Syafinie2
1
Natural Resource Science Program, Faculty of Earth Science, Universiti Malaysia Kelantan, 17600 Jeli,
Kelantan, Malaysia
2
Department of Natural Resource and Sustainability, Faculty of Earth Science, Universiti Malaysia Kelantan,
17600 Jeli, Kelantan, Malaysia
*Corresponding Author: janatunnaim@umk.edu.my [Accepted: 26 December 2018]
Abstract: The aim of this study is to determine the diversity of mangrove trees species and forest
content such as number of species, trees height, diameter breast height (DBH) and trees density of
mangrove species in disturbed and undisturbed area at delta Tumpat, Kelantan, Malaysia. The site
selection of undisturbed mangrove areas were the area that far from local settlements and free from
any development which is located at Layang-layang Island, Bedal Island, Nelayan Island and
Tanjung Duff Island. Whereas, Tujuh Island and Kambing Island were selected as disturbed
mangrove area as their location was near to the settlements. A total of ten rectangular plots were
established randomly at both area and each plot size is set at 20 x 10m. The sampling area was 0.2
hectare (ha). From the findings, there were five mangrove species identified at both disturbed and
undisturbed mangrove area which are Avicennia marina, Bruguiera gymnorrhiza, Rhizophora
apiculata, Rhizophora mucronata and Sonneratia caseolaris. The average DBH at undisturbed
mangrove area ranges from 3 to12cm, compared to 4 to 8cm in disturbed mangrove area. The
range of average height of mangrove species in undisturbed area is 3 to 10m and 1 to 6m for
disturbed area. In delta Tumpat, the diversity of mangrove in undisturbed area is higher than in
disturbed area calculated using Shannon-Weiner index (H’) with 1.54 and 0.38. Species richness
community increase as the Shannon-Weiner index increase.
Keywords: Kelantan delta - Distribution - Mangrove - Shannon-Weiner - Disturbance.
[Cite as: Nor Syahirah Z, Noor JNJ & Syafinie AM (2018) Effect of disturbance on mangrove species diversity
in Delta Tumpat, Kelantan, Malaysia. Tropical Plant Research 5(3): 391–395]
INTRODUCTION
Along the intertidal zones and coastal area in subtropical and tropical countries, there were a colony of
mangrove tress with particular root system and adaptation to high salinity nad brackish water condition (Beh et
al. 2012). Mangroves are adapted to saltwater and flooded soils. It can adapt in much extreme condition but
adapt less in cold weather condition. Generally, mangroves do not grow in climate with annual average
temperature of less than 19°C. The special ability of mangrove is to regulate salt that allows them to compete
with others trees species in the tropical and subtropical tidal environment. Mangrove absorbs the energy of tidal
current, storm wind and wave action in order to protect coastal land. Mangrove provides ecosystem purposes on
tropical coasts (Gilman et al. 2008, Walters et al. 2008). Many studies had recorded the biodiversity richness of
mangrove area in term of plants and animals (Cannicci et al. 2008, Nagelkerken et al. 2008). Unfortunately, this
forest ecosystem was found degraded by times due to natural and anthropogenic disturbances (Dahdouh-Guebas
et. al. 2005, Ellison 2008).
DISTRIBUTION OF MANGROVE
The length of Malaysia coastline is estimated at 4,810 km. It is distribute along the West Coast Peninsular
Malaysia (1,110 km), East coast Peninsular Malaysia (860 km), Sabah (1,800 km) and Sarawak (1,040 km).
There is 641,886 ha of mangrove forest of Malaysia. Sabah consist of 57% of the mangrove area, while Sarawak
Received: 09 June 2018 Published online: 31 December 2018
Nor Syahirah et al. 2018
occupied 26% of it. The remaining 17% is located at Peninsular Malaysia (Shukor 2004). Figure 1 shows the
wetlands (mangrove and peat swamp forest) areas in Malaysia. Out of the total mangrove area, there are
441,092 ha or 69% of the mangrove land has been gazette as forest reserved (about 74 mangrove forests
reserved) in Peninsular Malaysia. Among 74 mangrove forests reserved, 54 areas are on the West Coast, 13 on
the East Coast and remaining seven are on the Straits of Johor (Shukor 2004).
Figure 1. Mangrove Area in Malaysia.

Based on satellite QuickBird data obtained from the Malaysian Remote Sensing Agency, recently mangrove
forest area in Tumpat is estimated at 339.6 ha (Satyanarayana et al. 2011). Due to seasonal rainfall at delta
Tumpat, Kelantan, the bay, mangrove and estuary waterways experienced run-off and offshore currents at north
and south that regularly modify the coastal morphology and hydrographical condition in this area (Mohd-
Suffian et al. 2004).
TYPES OF MANGROVE TREES SPECIES

Mangroves can survive in areas where the water has low content of oxygen, salt water, freshwater and the
mixture of fresh and salt water. Mangrove only takes several years to grow and it can reach up to 25 m when
they are fully grown. The characteristic of mangrove trees are strong root system, special bark and leaf
structures and able to survive in harsh conditions (Jusoff 2013). There are several types of mangrove species
found in delta Tumpat which are Avicennia marina (Forssk.) Vierh., Bruguiera gymnorrhiza (L.) Lam.,
Rhizophora mucronata Lam. and Sonneratia caseolaris (L.) Engl. (Satyanarayana et al. 2010). Based on
previous study by Satyanarayana et al. (2011), the overall structure of mangrove forest at Tumpat generally is
determined largely by the distribution of Sonneratia caseolaris, but the specific area was not stated.

Study area
This study was undertaken in delta Tumpat, Kelantan. The delta Tumpat located in Tumpat district. The
delta is located at the east cost of Peninsular Malaysia, between latitude and longitude of 06º 11.00′ N to 06º
13.00′ N and 102º 10.00′ E to 102º 14.00′ E respectively (Fig. 2). The climate was influenced mainly by the
northeast and southeast monsoons, with a mean temperature of 26.8ºC and 83.7% of relative humidity. The delta
Tumpat consist of 17 smalls islands with an estimated total area of 1,200 ha where 339.6 ha of the area is
covered by mangrove forest. The delta area consist of important forests ecosystem that contributed to social,
economic and environmental of local community.
Filed work and sampling plots
There were two types of sampling sites chosen in this study; disturbed and undisturbed areas. The disturbed
area is represented by developed, disturbed or manipulated by human which is located at Tujuh Island and
Kambing Island, while the undisturbed mangrove areas is located distant from local settlement, consist of intact
forest area without any human destruction. The undisturbed area was located at Layang-layang Island, Bedal
Island, Nelayan Island and Tanjung Duff Island.
Figure 2. Location of Delta Tumpat, Kelantan.

Sampling plot with size of 20 m × 10 m following method by Wah et al. (2011) were set up at the selected
sampling sites. The random sampling method was applied in this study. Ten plots were set up at each disturbed
and undisturbed areas based on random points created from geographical information system (GIS) map using
ArcGIS version 10.2 software by ESRI™.
In the plot, number of individual mangrove trees, species, diameter-at-breast-height (DBH) and tree height
were recorded. In this study, mangrove tree species identification is referred to the nomenclatures by Tomlinson
(1986) and Duke et al. (2010). The tree height measurement was taken using laser distance meter, while DBH
were measured using DBH tape. A hand held Global Positioning System (GPS) by GARMIN was utilized to
find and record the position of each plot.
Data analysis
The diversity of mangrove trees was calculated using Shannon-Weiner index (H’). The H’ is calculated
using the formula as in Equation 1:
∑( ) ( )
Where:
H = Shannon diversity index
Pi = fraction of the entire population made up of species i
s = numbers of species encountered

The total area covered of both study sites were 0.4 ha. In the disturbed areas, where the area is occupied by
local and near to development, the number of mangrove species is lower than in undisturbed area. Only two
mangrove species was found in disturbed area which is Avicennia marina (Forssk.) Vierh. and Rhizophora
apiculata Blume. Meanwhile, five dominant species were found in undisturbed area namely Avicennia marina,
Bruguiera gymnorrhiza (L.) Lam., Rhizophora apiculata, Rhizophora mucronata Lam. and Sonneratia
caseolaris (L.) Engl. Table 1 summarized the mangrove vegetation composition structure for different category
of sites. The diversity index (H’) of disturbed area was found lower compared to undisturbed area with 0.38 and
1.54 respectively. Tree density of disturbed and undisturbed area was found at 1,035 trees/ha and 1,155 trees/ha
respectively.
Meanwhile, the details of mangrove tree composition at each category of sites were presented in table 2.
Number of individual in undisturbed area is higher than disturbed area. This is because these areas did not
encroached by any activity such as land settlement and cutting of trees. Result showed that in undisturbed area,
Nor Syahirah et al. 2018
Avicennia marina has higher number of individuals whereas Sonneratia caseolaris has a lower number of
individual. Avicennia marina has high tolerance to hyper saline conditions (Duke et al. 2010). It also a fast-
growing species and occasionally planted along with Rhizophora and Sonneratia species. In disturbed area,
Rhizophora apiculata has higher number of species than Avicennia marina. Rhizophora sp. is dominant in
mangrove forest in Malaysia due to strong propagule that can grow in sediment with accumulated deposit of
mud. Meanwhile, Avicennia sp. was found dominant at the seaward sediments, where there is soft and
bottomless mud (FAO 1981).
Table 1. Summary of data at disturbed and undisturbed area at Delta Tumpat.
Disturbed Undisturbed
Parameter
area area
Number of tree 207 231
Number of species 2 5
Average tree height (m) 5.54 6.40
Average DBH (cm) 7.37 7.72
H’ Index 0.38 1.54
The average DBH at undisturbed mangrove area ranges from 3 to 12 cm, as compared to 4 to 8 cm in
disturbed mangrove area. It showed that the DBH in undisturbed mangrove area is higher than disturbed
mangrove area. The average height of mangrove species in undisturbed area is higher than disturbed area. The
range of trees height for undisturbed area is 3 to 10 m and 1 to 6 m for disturbed area. In both areas, Rhizophora
apiculata is recorded as the dominant species.
Table 2. Diversity of mangrove in undisturbed and disturbed area at Delta Tumpat, Kelantan.
No. of Average Average
Category
individuals DBH (cm) Height (m)
Undisturbed area
Avicennia marina (Forssk.) Vierh. 73 7.06 3.99
Bruguiera gymnorrhiza (L.) Lam. 44 3.76 3.87
Rhizophora apiculata Blume. 58 9.40 9.70
Rhizophora mucronata Lam. 32 11.19 8.52
Sonneratia caseolaris (L.) Engl. 24 8.36 7.58
Disturbed area
Avicennia marina (Forssk.) Vierh. 26 4.33 1.96
Rhizophora apiculata Blume. 181 7.81 6.05
Based on H’ value calculated, undisturbed mangrove areas in delta Tumpat are more diverse compared to
disturbed areas comparable to the study by Wah et al. (2011). Decreased of diversity in disturbed area at delta
Tumpat was predicted due to sand dredging activity. Based on observation made during field survey, several
anthropogenic activities were actively threatening the biodiversity of mangrove ecosystem in delta Tumpat. The
main threats were the sand dredging activity to deeper the nearest river, development of port, conversion of land
for agriculture activity and introduction of invasive species to the area. These activities essentially disturbed the
growth of mangrove trees in the area as they dig the sand from the bottom of the river and pool the soil to the
land that have mangrove trees. Therefore, the mangrove trees cannot grow well in this condition and being a
main reason of the decreasing of species diversity and abundance of mangrove trees in the area.
CONCLUSION
The diversity of mangrove tree species and the forest content in undisturbed and disturbed areas at delta
Tumpat, Kelantan are variance. Physically, trees height, DBH and trees density in undisturbed area is higher
than disturbed area. Meanwhile, diversity of mangrove in delta Tumpat was analysed using Shannon-wiener
index. It showed that the diversity of mangrove in undisturbed area is higher than disturbed area with a value of
1.54 and 0.38 respectively. As the Shannon-wiener index increased, it showed that the place has higher species
richness community. It was also found that the main threats of anthropogenic disturbance in this delta was from
sand dredging activity to deeper the nearest river, development of port, conversion of land for agriculture
activity and introduction of invasive species to the area. Based on this study, delta Tumpat consists of five
mangrove species which are Avicennia marina, Bruguiera gymnorrhiza, Rhizophora apiculata, Rhizophora
mucronata and Sonneratia caseolaris. Conservation of these species is very crucial since mangroves plays an
important role as habitats for numerous species of coastal and aquatic flora and fauna as well as preventing
coastal erosion and as insulator for tidal and waves.
ACKNOWLEDGEMENTS
We would like to thanks the Kelantan State Forestry Department of Malaysia for their assistance and help in
the completion of this study.
REFERENCES
Beh BC, MatJafri MZ & Lim HS (2012) Temporal Changes Monitoring of Mangroves Distribution in Penang
Island from 2002-2010 by Remote Sensing. Journal of Applied Sciences 12(9): 2044–2051.
Cannicci S, Burrows D, Fratini S, Smith TJ, Offenberg J & Dahdouh-Guebas F (2008) Faunal impact on
vegetation structure and ecosystem function in mangrove forests: a review. Aquatic Botany 89(2): 186–200.
Dahdouh-Guebas F, Hettiarachchi S & Lo Seen D (2005) Transitions in ancient inland freshwater resource
management in Sri Lanka affect biota and human populations in and around coastal lagoons. Current
Biology 15: 579–586.
Duke N, Kathiresan K, Salmo III SG, Fernando ES, Peras JR, Sukardjo S, Miyagi T, Ellison J, Koedam NE,
Wang Y, Primavera J, Jin Eong O, Wan-Hong Yong J & Ngoc Nam V (2010) Avicennia marina. The IUCN
Red List of Threatened Species 2010. Available from: e.T178828A7619457.
http://dx.doi.org/10.2305/IUCN.UK.2010-2.RLTS.T178828A7619457.en. (accessed: 22 Jan. 2019).
Ellison AM (2008) Managing mangroves with benthic biodiversity in mind: Moving beyond roving banditry.
Journal of Sea Research 59: 2–15.
FAO U (1981) Tropical Forest Resourcess Assesment Project, Forest Resources of Tropical Asia. FAO, UNEP,
Rome.
Gilman EL, Ellison J, Duke NC & Field C (2008) Threats to mangroves from climate change and adaptation
options: a review. Aquatic Botany 89(2): 237–250.
Jusoff K (2013) Malaysian Mangrove Forests and Their Significance to the Coastal Marine Environment. Polish
Journal of Environmental Studies 22(4): 979–1005.
Mohd-Suffian I, Antonina AN, Sulong I, Mohd-Lokman H & Suhaila SI (2004) Monitoring the short-term
changes of the Kelantan Delta using remote sensing and GIS applications. In: Proceedings of the KUSTEM
3rd annual seminar on sustainability science and management. pp. 4–5.
Nagelkerken I, Blaber SJM, Bouillon S, Green P, Haywood M, Kirton LG & Somerfield PJ (2008) The habitat
function of mangroves for terrestrial and marine fauna: a review. Aquatic Botany 89(2): 155–185.
Satyanarayana B, Idris IF, Mohamad KA, Husain ML, Shazili NA & Dahdouh-Guebas F (2010) Mangrove
species distribution and abundance in relation to local environmental settings: a case-study at Tumpat,
Kelantan Delta, East coast of peninsular Malaysia. Botanica Marina 53(1): 79–88.
Satyanarayana B, Mohamad KA, Idris IF, Husain ML & Dahdouh-Guebas F (2011) Assessment of mangrove
vegetation based on remote sensing and ground-truth measurements at Tumpat, Kelantan Delta, East Coast
of Peninsular Malaysia. International Journal of Remote Sensing 32(6): 1635–1650.
Shukor AH (2004) The use of mangroves in Malaysia. In: Promotion of mangrove-friendly shrimp aquaculture
in Southeast Asia. Tigbauan, Iloilo, Philippines: Aquaculture Department, Southeast Asian Fisheries
Development Center. pp. 136–144.
Tomlinson PB (1986) The Botany of Mangroves. Cambridge University Press, Cambridge, 419 p.
Wah LM, Mojiol AR & Saleh E (2011) Diversity of mangroves ecosystem in Semporna mangrove
forest. Borneo Science 28(8): 17.
Walters BB, Rönnbäck P, Kovacs JM, Crona B, Hussain SA, Badola R & Dahdouh-Guebas F (2008)
Ethnobiology, socio-economics and management of mangrove forests: a review. Aquatic Botany 89(2): 220–
236.
Research article
Qualitative estimation of amylase enzyme activity of fungal

species isolated from iron ore mined overburden soil
Poonam Verma1* and R. K. Verma2
1
Bio-Design Innovation Center, Vigyan Bhawan, Rani Durgawati University Jabalpur-482001,
Madhya Pradesh, India
2
Forest Pathology Division, Tropical Forest Research Institute, Jabalpur-482021, Madhya Pradesh, India
*Corresponding Author: poonamverma8624@gmail.com [Accepted: 27 December 2018]
Abstract: The enzyme is a biocatalyst and processes many biological activities. In the present
investigation soil samples were collected from iron ore mined overburden soil and fungal flora
were isolated. For qualitative estimation of fungi for amylase activity, plate assay method was
used. Out of 99 fungi, only 21 test fungi were found to produce amylase. Most of the amylase
producers identified belonged to Aspergillus sp., Penicillium sp., Periconia sp., Scytalidium sp.,
Memmoniella sp., Trichoderma sp., Phoma sp. and Fusarium sp. followed by Alternaria sp. Other
78 test fungi were found to grow on medium, but were unable to produce amylase. Maximum 44
fungi were isolated from Trichocomaceae family and one fungus from Chaetomiaceae and
Myxotrichaceae family. Isolated fungi of Chaetomiaceae, Mucoraceae, Mycosphaerelleaceae,
Myxotrichaceae and Nectriaceae family were unable to produce enzyme. Maximum numbers of
enzyme producing fungi belongede to Trichocomaceae family, followed by Incertae sedis. In the
present investigation observed that Penicillium sp. 1 give the highest relative enzyme activity
index.
Keywords: Chhattisgarh - Degraded land - Dalli-Rajhara - Fungal family - Relative enzyme
activity index - Soil fertility.
[Cite as: Verma P & Verma RK (2018) Qualitative estimation of amylase enzyme activity of fungal species
isolated from iron ore mined overburden soil. Tropical Plant Research 5(3): 396–404]
INTRODUCTION
The fungi present in mine overburden soil have the capacity to tolerate the unfavorable condition, by
improving some special character like biosorption of heavy metal, secretion of enzyme etc., and these fungi
degrade the plant material and help in improving biogeochemical cycle (Verma et al. 2015a, Verma et al. 2017).
The group of fungi has for a long time been known to excrete extracellular enzyme capable of breaking down
complex substrate such as proteins, starch, cellulose and lipids (Barbesgaard 1992). Fungi are sources of
amylolytic enzymes suitable for the industrial conversion of starch into maltose or glucose. But the proof is
required that these fungi are physiologically capable of breaking down and utilizing the complex
polysaccharides of plant cell walls.
Amylase (α-amylase or α-1,4 glucan glucanohydrolase; EC 3.2.1.1) are enzymes that break down starch or
glycogen (Mishra & Maheshwari 1996). Amylase enzyme can be derived from several sources, such as plants,
animals and microorganisms. Microorganism secretes amylase to the outside of their cells to carry out extra-
cellular digestion. These enzyme breakdowns the plant debris in soil and converted into soluble starch.
Amylase constitutes a class of industrial enzymes having approximately 25% of the enzyme market (Sidhu
et al. 1997, Rao et al. 1998). Amylase have potential application in a number of industrial processes such as in
the textiles (12%), starch (11%), baking (8%), animal feed (6%), ethanol, food, paper industries, bread making,
glucose and fructose syrups, detergents (37%), fuel ethanol from starch, fruit juices, alcoholic beverages,
sweeteners, digestive aid and spot remover in dry cleaning which use about 75% of industrially produced
enzymes (Kang & Cottrell 1979, Tiwari et al. 2015). The estimated value of the world market is presently about
US$ 2.7 billion and is estimated to increase by 4% annually through 2012 (Das et al. 2011). The aim of this
Received: 10 July 2018 Published online: 31 December 2018
Verma & Verma 2018
study is to isolate and identify the fungal flora in iron ore mine overburden soil and determine the qualitative
measurement of amylolytic activity by plate assay method.

Study site
Dalli-Rajhara is located on a hill range bounded by 20º 33'0" and 20º 34'30" N latitude and 81º 1'0" and 81º
4'30" E longitude under Balod district in Chhattisgarh.
Soil sampling, isolation and identification of fungi
Soil samples were collected from rhizosphere of planted trees in iron ore mined overburden dump. Sample
used for fungal quantification were taken from rhizosphere zone by removing one cm soil from the surface. A
soil auger was used, which was washed thoroughly before starting of the sampling procedure. Samples were
collected from 10–20 cm depth and were carefully placed in polyethylene bags and their mouth were tied with
rubber bands. In the laboratry, soil samples were homogenized and spread on paper to remove plant material,
they were air dried, and stored at 4ºC for further experiment (Parkinson 1979). Soil dilution was prepared and 1
ml of the sample was placed in a sterile Petri-dish and 10 ml of sterile cooled (40ºC) PDA was added, mixed
and the plates were incubated at 27ºC for 3 to 7 days in BOD incubator (Warcup 1950).
After incubation distinct colonies were identified. The cultures were identified on the basis of macroscopic
and microscopic characteristics. Pure cultures of fungi isolates were identified with the help of literature
(Gilman 1957, Booth 1971, 1977, Ellis 1971, 1976, Barnett & Hunter 1972, Nagmani et al. 2006, Verma et al.
2008). After the identification pure culture was stored in the refrigerator for further use and preservation.
Screening by enzymatic assay of isolated soil fungi
Prepared the starch agar medium (Starch 20 g; Peptone 5 g; Beef extract 3 g; Agar 15 g; D/W 1000 ml),
poured into the sterile Petri dishes. Using needle a bit of the fungal culture (4 mm) cut by cock borer was taken
and placed onto the center of media without further disturbing its position. The labeled plate then incubated
plate for 72–96 hours at 25ºC in an inverted position. After incubation flood the surface of the starch agar
medium plates with iodine solution with a dropper for 30 seconds and pour off the excess iodine solution. The
starch hydrolysis around the line of growth of each organism, i.e. the colour changes of the medium because
amylase is a starch hydrolyzing enzyme (Ross 1976).
Index of relative enzyme activity
The enzymatic activities were estimated according to Hankin & Anagnostakis (1975) method and results
were concluded by Relative Enzyme Activity Index (REA) (Goldbeck et al. 2012, Choudhary & Jain 2012).
Growth simulation/inhibition index

Different isolates were cultured on growth media (Potato Dextrose Agar) and enzymatic activity test media
and observed growth simulation/inhibition index (Bradner et al. 1999).
RESULT AND DISCUSSION

138 soil samples were collected in replication from the different plantation and natural soil. A total of 99
fungal forms were obtained from the samples.
Screening of amylase producing fungi
Out of 99 fungi, only 21 test fungi were found to produce amylase enzyme as indicated by the production of
the zone on starch agar medium around the fungal colonies grown at pH 7 after 5 days of incubation at 28±3°C.
Most of the amylase producers identified belonged to Aspergillus sp., Penicillium sp., Periconia sp., Scytalidium
sp., Memmoniella sp., Trichoderma sp., Phoma sp. and Fusarium sp. followed by Alternaria sp. However, some
isolates of Alternaria sp., Aspergillus sp., Fusarium sp., Penicillium sp., Phoma sp., Scytalidium sp. and
Trichoderma sp. did not show any amylase activity. Other 78 test fungi Absidia sp., Acremonium sp.,
Biopolarus sp., Botryotrichum sp., Cephalosporium sp., Cladosporium sp., Clamydomyces sp., Curularia sp.,
Emericella sp., Eupenicillium sp., Gliocladium sp., Mucor sp., Nigrospora sp., Non sporolating hypomyctes,
Oidiodendron sp., Paecilomyces sp., Rhizopus sp., Verticillum sp. and Sterile fungi followed by Tritriachium sp.
were found to grow on medium (pH 7) but were unable to produce zone of hydrolysis. Results presented in table
1 indicate that highest amylase activity was detected in only seven isolates viz. Penicillium sp. 1 (0.93 cm),
Aspergillus fumigatus (0.6 cm), Periconia hispidula (0.59 cm), Aspergillus sp. 3 (0.57 cm), Penicillium
citreonigrum (0.52 cm), Penicillium roseopurpureum (0.47 cm), Memmoniella echinata (0.42 cm), Phoma sp.
(0.42 cm), Aspergillus parasiticus, Aspergillus terreus, Penicillium funiculosum, Scytalidium lignicola,
Trichoderma polysporum (0.4 cm). The enzymatic activity was considerably low in other fungi such as
Aspergillus nidulars var. echinulatus, Fusarium chlamydosporum (0.3), Penicillium rugulosum (0.29),
Aspergillus sp. 4 (0.27), Aspergillus janus (0.23), Aspergillus versicolor (0.2), Alternaria alternata (0.17) and
Aspergillus awamori (0.1).
Table 1. Growth and amylase enzyme activity of Fungal isolates.
S.N. Fungal isolates Family HZ CD REA CDP GS/ II
(cm) (cm) (cm)
1 Absidia fuca Mucoraceae 0 6.36 0 3.22 1.976
2 Acremonium strictum Nectriaceae 0 2.63 0 3.83 0.687
3 Alternaria alternata Pleosporaceae 6.23 6.06 1.028 6.32 0.959
4 Alternaria humicola Trichocomaceae 0 3.86 0 4.94 0.781
5 Alternaria tenuissima Trichocomaceae 0 7.03 0 4.75 1.48
6 Alternaria awamori Trichocomaceae 3.56 3.46 1.029 4.95 0.699
7 Alternaria candidus Trichocomaceae 0 2.63 0 4.36 0.603
8 Alternaria clavatus Trichocomaceae 0 4.44 0 3.35 1.325
9 Alternaria flavus Trichocomaceae 0 7.06 0 4.32 1.634
10 Alternaria flavus var. columnaris Trichocomaceae 0 4.46 0 3.17 1.407
11 Alternaria flavus var. oryzae Trichocomaceae 0 2.85 0 4.67 0.61
12 Alternaria fumigatus Trichocomaceae 3.33 2.73 1.219 7.27 0.376
13 Alternaria humicola Trichocomaceae 0 4.29 0 4.98 0.861
14 Alternaria janus Trichocomaceae 2.86 2.63 1.087 4.74 0.555
15 Alternaria nidulars var. echinulatus Trichocomaceae 6.16 5.86 1.051 4.46 1.313
16 Alternaria niger Trichocomaceae 0 5.36 0 4.74 1.13
17 Alternaria parasiticus Trichocomaceae 3.26 2.86 1.139 5.74 0.498
18 Alternaria repens Trichocomaceae 0 5.06 0 4.83 1.048
19 Alternaria restrictus Trichocomaceae 0 4.36 0 3.86 1.129
20 Aspergillus sp. 1 Trichocomaceae 0 6.54 0 5.46 1.198
21 Aspergillus sp. 2 Trichocomaceae 0 2.76 0 4.67 0.591
22 Aspergillus sp. 3 Trichocomaceae 5.73 5.16 1.11 4.26 1.211
23 Aspergillus sp. 4 Trichocomaceae 5.33 5.06 1.053 5.22 0.969
24 Aspergillus sydowii Trichocomaceae 0 7.66 0 5.15 1.487
25 Aspergillus terreus Trichocomaceae 3.93 3.53 1.113 4.24 0.832
26 Aspergillus unguis Trichocomaceae 0 6.83 0 4.92 1.389
27 Aspergillus versicolor Trichocomaceae 4.36 4.16 1.048 5.67 0.733
28 Biopolaris halodus Pleosporaceae 0 4.83 0 3.56 1.357
29 Botryotrichum piluliferum Chaetomiaceae 0 2.96 0 4.74 0.624
30 Cephalosporium indicum Incertae sedis 0 6.57 0 3.16 2.079
31 Cladosporium oxysporum Mycosphaerelleaceae 0 3.36 0 4.77 0.704
32 Cladosporium variabile Mycosphaerelleaceae 0 4.43 0 3.25 1.363
33 Clamydomyces palmarum Incertae sedis 0 3.86 0 2.96 1.304
34 Curularia indica Pleosporaceae 0 3.83 0 6.15 0.623
35 Emericella nidulans Trichocomaceae 0 2.63 0 4.34 0.605
36 Eupenicillium sp. Trichocomaceae 0 6.56 0 7.12 0.921
37 Fusarium oxysporum Nectriaceae 0 6.13 0 5.36 1.144
38 Fusarium chlamydosporum Nectriaceae 5.66 5.36 1.056 7.63 0.702
39 Fusarium javanicum Nectriaceae 0 4.65 0 4.92 0.945
40 Fusarium moniliforme Nectriaceae 0 5.73 0 5.75 0.997
41 Fusarium poae Nectriaceae 0 2.16 0 3.16 0.683
42 Fusarium roseum Nectriaceae 0 5.14 0 6.38 0.752
43 Fusarium sp. Nectriaceae 0 5.43 0 4.81 1.129
44 Fusarium solani Nectriaceae 0 3.87 0 3.26 1.187
45 Fusarium udum Nectriaceae 0 5.56 0 4.65 1.195
46 Gliocladium deliquescens Hypocreaceae 0 2.83 0 3.43 0.825
47 Memmoniella echinata Incertae sedis 3.26 2.84 1.148 5.11 0.556
48 Mucor hiemalis Mucoraceae 0 5.76 0 6.56 0.878
49 Nigrospora oryzae Hypocreaceae 0 7.36 0 4.27 1.724
50 Nigrospora padwicki Hypocreaceae 0 6.33 0 6.17 1.025
51 Nigrospora panici Hypocreaceae 0 5.47 0 6.39 0.856
Verma & Verma 2018
52 Non sporolating hypomyctes - 0 6.45 0 2.83 2.279

53 Oidiodendron mais Myxotrichaceae 0 5.43 0 2.56 2.121
54 Paecilomyces lilacinus Trichocomaceae 0 2.56 0 3.75 0.682
55 Penicillium adametzi Trichocomaceae 0 2.63 0 2.54 1.035
56 Penicillium asperum Trichocomaceae 0 3.56 0 3.75 0.949
57 Penicillium aurantiogriseum Trichocomaceae 0 3.21 0 2.71 1.185
58 Penicillium citreonigrum Trichocomaceae 3.14 2.62 1.198 5.14 0.509
59 Penicillium citrinum Trichocomaceae 0 3.65 0 4.32 0.845
60 Penicillium commune Trichocomaceae 0 2.87 0 3.92 0.732
61 Penicillium funiculosum Trichocomaceae 5.26 4.86 1.082 4.98 0.975
62 Penicillium nigricans Trichocomaceae 0 3.16 0 3.16 0
63 Penicillium novae Trichocomaceae 0 3.98 0 3.84 1.036
64 Penicillium oxalicum Trichocomaceae 0 6.13 0 4.25 1.442
65 Penicillium restrictum Trichocomaceae 0 5.53 0 5.27 1.049
66 Penicillium roseopurpureum Trichocomaceae 3.93 3.46 1.135 5.26 0.658
67 Penicillium rugulosum Trichocomaceae 4.84 4.55 1.063 5.64 0.807
68 Penicillium sp. 1 Trichocomaceae 4.93 4 1.224 3.76 1.0638
69 Penicillium sp. 2 Trichocomaceae 0 3.55 0 5.12 0.693
70 Penicillium sp. 3 Trichocomaceae 0 7.56 0 4.81 1.571
71 Periconia hispidula Incertae sedis 4.13 3.54 1.167 4.85 0.729
72 Phoma glomerata Incertae sedis 0 3.39 0 4.66 0.727
73 Phoma sp. Incertae sedis 4.96 4.54 1.092 5.75 0.789
74 Phoma sp. 1 Incertae sedis 0 4.54 0 4.95 0.917
75 Rhizopus sp. Mucoraceae 0 3.13 0 5.14 0.609
76 Rhizopus stolonifer Mucoraceae 0 5.33 0 6.15 0.867
77 Scytalidium lignicola Incertae sedis 2.96 2.56 1.157 3.22 0.795
78 Scytalidium thermophilum Incertae sedis 0 7.86 0 5.96 1.319
79 Sterile fungi 1 - 0 4.74 0 4.75 0.998
80 Sterile fungi 2 - 0 5.76 0 3.68 1.565
81 Sterile fungi 3 - 0 3.76 0 3.15 1.193
82 Sterile fungi 4 - 0 2.87 0 4.68 0.613
83 Sterile fungi 5 - 0 2.34 0 2.68 0.873
84 Sterile fungi 6 - 0 5.46 0 4.93 1.108
85 Sterile fungi 7 - 0 3.03 0 2.23 1.359
86 Sterile fungi 8 - 0 5.13 0 3.21 1.644
87 Sterile fungi 9 - 0 3.34 0 4.74 0.705
88 Sterile fungi 10 - 0 5.16 0 4.65 1.109
89 Trichoderma aureoviride Hypocreaceae 0 6.83 0 5.37 1.272
90 Trichoderma polysporum Hypocreaceae 3.33 2.93 1.137 4.25 0.689
91 Trichoderma pseudokoningii Hypocreaceae 0 6.56 0 6.87 0.955
92 Trichoderma reesi Hypocreaceae 0 3.21 0 5.42 0.592
93 Trichoderma sp. Hypocreaceae 0 4.65 0 5.18 0.898
94 Trichoderma sp. 1 Hypocreaceae 0 3.36 0 3.43 0.979
95 Trichoderma strctipilis Hypocreaceae 0 6.26 0 6.17 0.932
96 Trichoderma viride Hypocreaceae 0 7.64 0 4.21 1.814
97 Tritriachium dependens Trichocomaceae 0 2.43 0 3.64 0.668
98 Verticillum sp. Plectorsphaerellaceae 0 6.03 0 6.17 0.977
99 Verticillum terrestre Plectorsphaerellaceae 0 5.06 0 6.39 0.791
Note: REA= Relative enzyme activity index (values more than 0 showed positive amylase enzymatic activity), HZ=
Hydrolysis zone, CD= Colony diameter, CDP= Colony diameter on potato dextrose agar, GS/II= Growth
simulation/inhibition index.
Growth simulation (G.S.) /inhibition index (I.I.) was computed as the colony diameter on starch hydrolysis
agar/colony diameter on control agar ratio (Verma & Verma 2016a). The index value <1, represented substrate
inhibited fungal growth, while the index value >1, exhibited substrate rendered growth stimulation. Range of
growth stimulation (GS) 2.279–1.025. Test fungi give highest growth stimulation on an enzymatic medium such
as Non sporolating hypomyctes (2.279) followed by Oidiodendron mais (2.121), Cephalosporium indicum
(2.079), Absidia fuca (1.976), Trichoderma viride (1.814), Nigrospora oryzae (1.724) Range of inhibition index
(II) 2.279–1.025. Fungi give highest I.I. Aspergillus fumigates (0.376), Aspergillus parasiticus (0.498),
Penicillium citreonigrum (0.509), Aspegillus janus (0.555), Memmoniella echinata (0.556), Aspergillus sp. 2
(0.591) and Trichoderma reesi (0.592). However, hydrolysis zone diameters were equal to colony diameter in
case Penicillium nigricans (0).
Furthermore, the colony growth of 41 tested fungi on starch hydrolysis agar was found to be greater as
compared to control, slightly greater in 13 test fungi, growth was slightly inhibited in 37 test fungi and colony
diameter was reduced in 7 fungi (Fig. 1). None of the isolates were found to be resistant for starch hydrolysis
agar (Table 1).
Figure 1. Amylase enzymatic activity: A, Control plate; B, Zone of hydrolysis activity; C, Negative result of amylase
activity.
Isolated 29 genera were mainly belonging to 10 families. Maximum 44 fungi were isolated from
Trichocomaceae family and one fungus from Chaetomiaceae and Myxotrichaceae family. Isolated fungi of
Chaetomiaceae, Mucoraceae, Mycosphaerelleaceae, Myxotrichaceae and Nectriaceae family were unable to
produced enzyme. Maximum numbers of fungi were produced enzyme belonging to Trichocomaceae family,
followed by Incertae sedis (Fig. 2).
Figure 2. Family of amylase enzyme producing fungi.
DISCUSSION
Screening of fungi on the basis of amylase activity
The saprophytic fungi represent the largest proportion of soil fungi and they perform a crucial role in the
decomposition of plant structural polymers, such as cellulose, hemicelluloses, lignin and starch, thus
contributing to the maintenance of global carbon cycle. The distribution of these organisms is influenced by the
abundance and nature of the organic content of the soil, as well as by other soil texture (Waksman 1944). Fungi
are known agents of decomposition of organic matter, by producing extracellular enzymes (Reese & Levinson
1952, Lynd et al. 2002, Chi et al. 2007, Gupta et al. 2008). Due to wide diversity, fungi have been recognized as
a source of the new enzyme with useful and /or novel characteristics.
In the present findings maximum amylase enzyme was produced by Penicillium sp. 1 and Yamasaki et al.
(1977), Ertan et al. (2006), Ogbonna et al. (2014), Verma et al. (2015b) were also observed similar results but
the source of isolation of fungi was different. Aspergillus sp. was produced enzyme in medium this finding was
Verma & Verma 2018
supported by Razzaque & Ueda (1978), Abe et al. (1988), Kunamneni et al. (2005), Ragunathan &
Swaminathan (2005), Bakri et al. (2009), Toye (2009), Ominyi et al. (2013), Ogbonna et al. (2014), Verma et
al. (2015b) but source of isolation of fungi was different. Trichoderma polysporum was produced the enzyme in
medium this finding was supported by Mishra & Maheshwari (1996), but source of isolation of fungi was
different. Fusarium chlamydosporum was produced the enzyme in medium these finding was supported by
Pathak et al. (2014) but the source of isolation of fungi was different. Scytalidium lignicola, Memmoniella
echinata and Periconia hispidula were isolated from leaf litter of Eucalyptus (Dorai 1988) Phoma sp. and
Alternaria alternata was produced enzyme in medium this finding was supported by Masumi et al. (2014) but
source of isolation of fungi was different.
Amylase activity of Bacillus cereus isolated from a coal mine was studied by Bhatt et al. (2015). A similar
study was also done by Parida et al. (2014) in iron ore mine bacterial isolates. Microbial enzymes like amylase
and cellulase are extracellular enzymes that play a vital role in the nutrient cycling of the soil (Tabatabai 1994).
However, soil microbial enzymes are considered to be indicative measures of soil fertility and bioremediation
activities (Margesin et al. 2000). In this investigation, it was found that mining soils harbor fewer
microorganisms with less diversity. Therefore, microbial function in the mining soil could be attributable to low
microbial diversity and confined to a specific group of microorganisms (Khan & Joergensen 2009, Parida et al.
2014) (Table 1).
In the present finding Acremonium strictum, Penicillium oxalicum, Aspergillus niger, Rhizopus sp. and
Aspergillus flavus give negative result, But Yamasaki et al. (1977), Takahashi et al. (1978), Purushotham et al.
(1996), Jain et al. (2012) and Sundar et al. (2012) observed Acremonium sp., Penicillium oxalicum, Aspergillus
niger and Aspergillus flavus produced amylase enzyme. Hence, there is an increasing worldwide interest in the
screening of new microorganisms producing amylases suitable for industrial applications (Burhan et al. 2003).
Studies on fungal amylases especially in developing countries have concentrated mainly on ubiquitous fungi
like, Aspergillus, Penicillium, Alternaria etc. probably because of their ubiquitous nature and nonfastidious
nutritional requirements of these organisms (Azevedo 2000, Suganthi et al. 2011, Sidkey et al. 2011, Khan &
Yadav 2011).
Fungi give negative results in amylase medium
Out of 99 fungi, 78 fungi were unable to produced amylase enzyme (Table 1). Thus the lack of a positive
result could mean that either the enzyme is not produced, or that it is produced intracellular and not released
from the mycelium, or that it is produced and released, but the medium inhibits its detection. The relationship
between the ability to grow on a particular test medium and to produce the corresponding enzyme to digest the
substrate incorporated into the medium is not well correlated (Egger 1986, Pointing 1999, Abdel-Raheem &
Shearer 2002). This may be because fungus uses other material from the medium and another carbon source
rather than substrates added (Yuen et al. 1998, Abdel-Raheem & Shearer 2002, Verma & Verma 2016a, Verma
& Verma 2016b).
CONCLUSION
Penicillium sp. 1 showed the highest amylase activity, which may be applied in mine overburden soil along
with amendment of organic matter to improve fertility of overburden.
ACKNOWLEDGEMENT
Authors are thankful to Director, Tropical Forest Research Institute, Jabalpur for providing laboratory
facilities.
REFERENCES
Abdel-Raheem A & Shearer CA (2002) Extracellular enzyme production by freshwater ascomycetes. Fungal
Diversity 11: 1–19.
Abe J, Nakajima K, Nagao H, Hizukuri S & Obata K (1988) Properties of the raw-starch digesting amylase of
Aspergillus sp. K-27: A synergistic action of glucoamylase and α-amylase. Carbohydrates Research 175:
85–92.
Azevedo JL, Maccheroni Jr W, Pereira JO & Araujo WL (2000) Endophytic microorganisms: a review on insect
control and recent advances on tropical plants. Electronic Journal of Biotechnology 3(1): 3–4.
Bakri Y, Magali M & Thonart P (2009) Isolation and identification of a new fungal strain for amylase
biosynthesis. Polish Journal of Microbiology 58(3): 269–273.
Barbesgaard P, Heldt Hansen HP & Diderichsen B (1992) On the safety of Aspergillus oryzae: a review.
Applied Journal of Microbiology and Biotechnology 36: 569–572.
Barnett HL & Hunter BB (1972) Illustrated genera of imperfect fungi, 3rd edition. Burgess Publishing Co., 273
p.
Bhatt RA, Dar MA, Kumar U, Varghese S & Kumari S (2015) Characterization and production of amylase by
using Bacillus cereus isolated from coal mines. International Journal of Research in Biological Sciences
5(3): 17–20.
Booth C (1971) Genus Fusarium. Commonwealth Mycological Institute (CMI), Kew, Surry, England, 273 p.
Booth C (1977) Fusarium. Commonwealth Mycological Institute, Kew, Surrey, England, 58 p.
Bradner JR, Gillings M & Nevalainen KMH (1999) Qualitative assessment of hydrolytic activities in Antarctic
microfungi grown at different temperatures on solid media. World Journal of Microbiology and
Biotechnology 15: 131–132.
Burhan AL, Nisa U, Gokhan C, Omer C, Ashabil A & Osman G (2003) Enzymatic properties of a novel
thermostable, thermophilic, alkaline and chelator resistant amylase from an alkaliphilic Bacillus sp. isolate
ANT-6. Process Biochemistry 38: 1397–1403.
Chi HLZ, Wang X, Duan X, Ma L & Gao L (2007) Purification and characterization of extracellular amylase
from the marine yeast Aureobasidium pullulans N13d and its raw potato starch digestion. Enzyme and
Microbial Technology 40: 1006–1012.
Choudhary V & Jain PC (2012) Screening of alkaline protease production by fungal isolates from different
habitats of Sagar and Jabalpur district (M.P.). Journal of Academia and Industrial Research 1(4): 215–220.
Das S, Singh S, Sharma V & Soni ML (2011) Biotechnological applications of industrially important amylase
enzyme. International Journal of Pharma and Bio Sciences 2(1): 486–496.
Dorai M (1988) Taxonomic and ecological studies of fungi colonizing leaves and litter of Eucalyptus species in
South India, (Ph.D. Thesis). University of Madras.
Egger KN (1986) Substrate hydrolysis patterns of post-fire ascomycetes (Pezizales). Mycologia 78: 771–780.
Ellis MB (1971) Dematiaceous hyphomycetes. Commonwealth Mycological Institute, Kew, Surry, England,
608 p.
Ellis MB (1976) More dematiaceous hyphomycetes. Commonwealth Mycological Institute, Kew, Surry,
England, 507 p.
Ertan F, Yagar H & Balkan B (2006) Some properties of free and immobilized α-amylase from Penicillium
griseofulvum by solid state fermentation. Preparative, Biochemistry and Biotechnology 36: 81–91.
Gilman JC (1957) A manual of soil fungi, Revised 2nd edition. Oxford and IBH publishing Co., 220 p.
Goldbeck R, Andrade CCP, Pereira GAG & Filho FM (2012) Screening and identification of cellulase
producing yeast-like microorganisms from Brazilian biomes. African Journal of Biotechnology 11(53):
11595–11603.
Gupta A, Gupta VK, Modi DR & Yadava LP (2008) Production and characterization of α amylase from
Aspergillus niger. Biotechnology 1: 1–6.
Hankin L & Anagnostakis SL (1975) The use of solid media for detection of enzyme production by fungi.
Mycologia 47: 597–607.
Jain P, Aggarwal V, Sharma A & Pundir RK (2012) Screening of endophytic fungus Acremonium sp. for
amylase production. Journal of Agricultural Technology 8(4): 1353–1364.
Khan JA & Yadav SK (2011) Production of alpha amylases by Aspergillus niger using cheaper substrates
employing solid state fermentation. International Journal of Plant, Animal and Environmental Sciences
1(3): 100–108.
Khan KS & Joergensen RG (2009) Changes in microbial biomass and P fractions in biogenic household waste
compost amended with inorganic P fertilizers. Bioresource Technology 100: 303–309.
Kunamneni A, Permaul K & Singh S (2005) Amylase production in solid state fermentation by the thermophilic
fungus Thermomyces lanuginosus. Journal of Bioscience and Bioengineering 100: 168–171.
Lynd LR, Weimer PJ, Van-Zyl WH & Pretorius IS (2002) Microbial cellulose utilization: fundamentals and
biotechnology. Microbiology and Molecular Biology Reviews 66(3): 506–577.
Margesin R, Zimmerbauer A & Schinner F (2000) Monitoring of bioremediation by soil biological activities.
Chemosphere 40: 339–346.
Masumi, Mirzaei S, Kalvandi R & Zafari D (2014) Asparaginase and amylase activity of thyme endophytic
fungi. Journal of Crop Protection 3(Supplementary): 655–662.
Verma & Verma 2018
Mishra RS & Maheshwari R (1996) Amylases of the thermophilic fungus Thermomyces lanuginosus: Their
purification, properties, action on starch and response to heat. Journal of Bioscience 21(5): 653–672.
Nagmani A, Kunwar IK & Manoharachary C (2006) Hand book of soil fungi. International Pvt. Ltd. New Delhi,
477 p.
Ogbonna CN, Okpokwu NM, Okafor CU & Onyia CE (2014) Isolation and screening of amylase producing
fungi obtained from garri processing site. International Journal of Biotechnology and Food Science 2(5):
88–93.
Ominyi MC, Ogbonna JC, Nwoba EG, Nwagu KE & Ukachi R (2013) Isolation and screening of α-amylase and
glucoamylase producing fungi and their application in bioethanol production. International Journal of
Science and Nature 4(1): 44–50.
Parida D, Jena SK & Rath CC (2014) Enzyme activities of bacterial isolates from iron mine areas of Barbil,
Keonjhar district, Odisha, India. International Journal of Pure and Applied Bioscience 2(3): 265–271.
Parkinson D (1979) Soil microorganisms and plant roots. In: Burges A & Raw F (eds) Soil Biology. Academic
Press, New York, pp. 449–478.
Pathak SS, Kumar S, Rajak RC & Sandhu SS (2014) Study of effect of temperature on amylase production by
soil mycotic flora of Jabalpur region. World Journal of Pharmacy and Pharmaceutical Sciences 3(9): 1448–
1458.
Kang KS & Cottrell IW (1979) Polysaccharides. In: Peppler HJ & Perlman D (eds) Microbial Technology, 2nd
edition. Academic Press, New York, 417–481.
Pointing SB (1999) Qualitative methods for the determination of lingo-cellulolytic enzyme production by
tropical fungi. Fungal Diversity 2: 17–33.
Purushotham SP, Kesha V L, Patkar HS, Prakash & Shetiy HS (1996) Storage fungi and their influence on rice
seed quality. Indian Phytopathology 49(2): 152–156.
Ragunathan R & Swaminathan K (2005) Growth and amylase production by Aspergillus oryzae during solid
state fermentation using banana waste as substrate. Journal of Environment Biology 26: 653–656.
Rao MB, Tanksale AM, Gathe MS & Deshpande VV (1998) Molecular and Biotechnological aspects of
microbial proteases. Microbial Review 62: 597–635.
Razzaque A & Ueda S (1978) Glucoamylase of Aspergillus oryzae. Journal of Fermentation Technology 56:
296–302.
Reese ET & Levinson HS (1952) A comparative study of the breakdown of cellulose by microorganism.
Physiologia Plantarum 5: 345–366.
Ross DJ (1976) Invertase and amylase activities in ryegrass and white clover plants and their relationships with
activities in soils under pasture. Soil Biology and Biochemistry 8: 351–356.
Sidhu GS, Sharma P, Chakrabarti T & Gupta JK (1997) Strain improvement for the production of a
thermostable alpha amylase by Bacillus species. Enzyme Microbial Technology 21: 525–530.
Sidkey NM, Abo-Shadi MA, Balahmar R, Sabry R & Badrany G (2011) Purification and characterization of α-
amylase from a newly isolated Aspergillus flavus F2Mbb. International Research Journal of Microbiology
2(3): 96–103.
Suganthi R, Benazir JF, Santhi R, Ramesh Kumar V, Anjana H, Nitya M, Nidhiya K A, Kavitha, G & Lakshmi
R (2011) Amylase production by Aspergillus niger under solid state fermentation using agroindustrial
wastes. International Journal of Engineering Science and Technology 3(2): 1756–1763.
Sundar R, Liji T, Rajila C & Suganyadevi P (2012) Amylase production by Aspergillus niger under submerged
fermentation using Ipomoea Batatas. International Journal of Applied Biology and Pharmaceutical
Technology 3(2): 175–182.
Takahashi T, Tsuchida Y & Irie M (1978) Purification and some properties of three forms of glucoamylase from
Rhizopus species; Journal of Biochemistry 84: 1183–1194.
Tabatabai MA (1994) Soil enzymes. Microbiological and biochemical properties. In: Weaver RW, Angle S,
Bottomley P, Bezdicek D, Smith S, Tabatabai A & Wollum A (eds) Methods of soil analysis, Part 2. Soil
Science Society of America, Madison, pp. 775–833.
Tiwari SP, Srivastava R, Singh CS, Shukla K, Singh RK, Singh P, Singh R, Singh NL & Sharma R (2015)
Amylase: An overview with special reference to Alpha amylase. Journal of Global Bioscience 4(1): 1886–
1901.
Toye E (2009) Laboratory production and assay of amylase by fungi and bacteria. Buscar Manuals 2: 1–30.
Verma P & Verma RK (2016a) Cellulase activity of soil fungi (Aspergillus, Fusarium, Penicillium,
Trichoderma) isolated from rhizosphere region of iron ore mine overburden soil. International Journal of
Basic and Applied Biology 3(2): 115–120.
Verma P & Verma RK (2016b) Screening of cellulase production by fungal isolates from rhizosphere region of
mine degraded land in Dalli-Rajhara (Chhattishgarh). International Journal of Basic and Applied Biology
3(2): 162–165.
Verma P, Dahayat A, Chandrakar V & Jamaluddin (2015b) Studies on enzymatic potential fungi isolated from
municipal solid waste in Jabalpur. International Journal of Recent Scientific Research 6(8): 5927–5932.
Verma P, Singh S & Verma RK (2015a) Heavy metal biosorption by Fusarium strains isolated from iron ore
mines overburden soil. International Journal of Environmental Science and Toxicology Research 4(4): 61–
69.
Verma P, Singh S & Verma RK (2017) Impact of plantation on iron ore mined overburden at Durg in
Chhattisgarh (India). International Research Journal of Environment Science 6(1): 1–12.
Verma RK, Sharma N, Soni KK & Jamaluddin (2008) Forest fungi of central India. International Book
Distributing Co., Lucknow, 418 p.
Waksman SA (1944) Three decades with soil fungi. Soil Science 58: 89–114.
Warcup JH (1950) The soil plate method for isolation of fungi from soil. Nature 166: 117–118.
Yamasaki Y, Suzuki Y & Ozawa J (1977) Purification and properties of two forms of glucoamylase from
Penicillium oxalicum. Agricultural and Biological Chemistry 41: 755–762.
Yuen IK, Hyde KD & Hodgkiss IJ (1998) Physiological growth parameters and enzyme production in tropical
freshwater fungi. Material and Organismen 32: 2–16.
Research article
Diversity and uses of medicinal plants in Chandra Prabha

Wildlife Sanctuary, Chandauli district, Uttar Pradesh
Nitisha Srivastava and Achuta Nand Shukla*
Botanical Survey of India, Central Regional Centre, 10, Chatham Lines, Allahabad-211002, Uttar Pradesh, India
*Corresponding Author: achutbsi@gmail.com [Accepted: 29 December 2018]
Abstract: Protected areas play a very significant role in the conservation of medicinal plants and
traditional knowledge. Chandra Prabha Wildlife Sanctuary (CPWLS) is situated in the district
Chandauli. The presented study was carried out in the area of CPWLS for survey and collection of
medicinal plants. Information on medicinal properties of plants encountered in the present study
was generated through surveys and relevant literature. A total of 121 medicinally important plant
species were reported. The present study aimed to document the traditional uses of different plant
parts of medicinal plants. Present study of diversity of medicinal plant in CPWLS is helpful for
information on medicinal values of plant species will also be helpful in conservation of these plant
resources.
Keywords: Chandra Prabha Wildlife Sanctuary - Medicinal plants - Traditional knowledge.
[Cite as: Srivastava N & Shukla AN (2018) Diversity and uses of medicinal plants in Chandra Prabha Wildlife
Sanctuary, Chandauli district, Uttar Pradesh. Tropical Plant Research 5(3): 405–418]
INTRODUCTION
Sustainable use of natural resources refers to the process of making a balance between the unlimited desire
of human being and limited natural resources. In order to obtain the greatest benefit for the present generation
and maintaining the potential for future, conservation is the only way. Conservation of plant resources is of
global concern because we don't know what we are losing and what we will need in the future. However,
conservation methods may vary globally.
Medicinal plants are an important natural resource. To meet the requirements of expanding regional and
international markets healthcare products and the needs of growing populations, large quantities of medicinal
plants are harvested from forests (de Silva 1997). There are many factors which pose threat to many medicinal
plants. The threats are degradation of habitat due to expanding human activity, decline of forest areas,
destructive collections of plant species, invasion of exotic species, diseases, overexploitation, grazing by
animals, industrialization, shifting cultivation, excessive use of fertilizers and agrochemicals, natural and
manmade calamities, etc.
In India history of uses of medicinal plants goes back to 300 BC, in this year Charak Samhita, a document
on herbal therapy by Charak reports on the production of 340 herbal drugs and their uses (Vedprakash 1991,
Mehra et al. 2014). These herbal medicines always attracted peoples due to their low cost and minimal side
effects (Bajpai et al. 2016). Medicinal plants which are in use traditionally by rural people for curing various
diseases are now moving into mainstream. Other peoples are also aware of their therapeutic uses for treating
various diseases as well as maintaining proper health conditions. Thus today not only the rural and ethnic
peoples but peoples in urban areas also rely upon these medicinal plants. In the country, large numbers of
medicinal plants are extracted from the wild to meet the increasing demand for increasing population and export
business. This has created rapid loss of medicinal plant genetic resources (Gadgil 1989). Therefore it is very
important to ensure conservation of knowledge of these traditional medicinal plants as well as it also very
essential to conserve them for the betterment of our future (Deepa et al. 2016). For that, more information is
required on medicinal plant production, utilization, trade, monitoring the stock of medicinal plants, development
of sustainable harvesting practices, preservation of traditional knowledge and intellectual property rights.
Protected Areas are one of the most widely accepted and practical approaches to biodiversity conservation
Received: 02 September 2018 Published online: 31 December 2018
Srivastava & Shukla 2018
worldwide. However ex-situ conservation methods such as plant tissue culture, seed storage and tissue banking
etc. also play an important role in the conservation of plant genetic resources. Today almost every country in the
world has designated protected areas for a range of conservation objectives, such as maintenance of the integrity
and diversity of ecosystems, protection of flora and fauna, protection of cultural heritage and unique landscape,
etc.
At present in India there are 104 designated national parks, 544 wildlife sanctuaries, which play a very
significant role in the conservation of medicinally important plant species. As per Uttar Pradesh forest report,
Uttar Pradesh shares one national park and 25 wildlife sanctuaries. Out of these 25 wildlife sanctuaries, Chandra
Prabha Wildlife Sanctuary (CPWL) is the oldest and first declared wildlife sanctuary of Uttar Pradesh and was
designated in 1957. Since wildlife sanctuaries play a very important role in the conservation of medicinal plants
of the region, therefore it is important to study the medicinal wealth of these sanctuaries. The present study was
undertaken to study the medicinal floristic diversity of Chandra Prabha Wildlife Sanctuary. The botanical
explorations were done for study and collection of existing medicinal plant species. The study was focused on
medicinal plant diversity, distribution and uses of medicinal plants and also on conservation status and
preferences for the medicinal plants of the area.

Study area
Chandra Prabha Wildlife Sanctuary (CPWLS) is situated in District Chandauli of Uttar Pradesh and covers
an area of about 78 km2 (Fig. 1). The area lies between the latitudes 24° 52´0´´ N to 25° 3´55´´ and 83° 03´24´´
E to 83° 22´55´´ longitudes. It was famous for the Asiatic Lion from 1957 to 1970. The place has also been
gifted with attractive natural sceneries, picnic spots, intense forest, river and beautiful waterfalls. Two waterfalls
namely Rajdari and Devdari are famous for the picnic spot. It lies on the Naugarh and Vijaygarh hillocks on the
North Slope of the Kaimur range. The Karamnasha River, a tributary of the Ganges, flows through the
sanctuary, as does the Chandraprabha River, a tributary of Karamnasha. Raj Dari waterfall is surrounded by the
forest area, this stepped waterfall is the main attraction point for the tourists. Deodari water fall is about 500 m
down the stream below Raj Dari waterfall. Chandra Prabha dam has been constructed by the Irrigation
Department. Chandra Prabha dam is located upstream on Chandra Prabha River near the sanctuary and is the
source of water for both the waterfalls. The sanctuary forest is the typically dry deciduous type with dominant
shrubby vegetation.
Figure 1. Location of study area (Chandra Prabha Wildlife Sanctuary, Chandauli, Uttar Pradesh).
Data collection
In present work the study materials consists of medicinal plant diversity of Chandra Prabha Wildlife
Sanctuary. Besides the specimens collected from the sanctuary some information was also taken from
previously collected specimens and related documentation about the sanctuary (Maurya et al. 2015). Extensive
survey has been carried out to record the information and the specimens were identified with the help of
herbarium and published literature. The voucher specimens were deposited in the herbarium of Botanical Survey
of India, Central Regional Centre, Prayagraj (BSA).
RESULTS
During the botanical excursion, the sanctuary area was explored for medicinal floristic diversity. After
critical examination 121 medicinal plants (Table 1) belonging to 51 families were reported with scientific
names, common names, habit, life form and their medicinal uses. Among these 5 species belong to Pteridophyta
group. Out of total 116 Angiopserms 10 genera and 10 species belongs to monocot and 106 species and 86
genera belongs to dicot families. Fabaceae (13 species), Caesalpiniaceae (11 species), Rubiaceae (07 species),
Malvaceae (6 species), Poaceae (06 species) were the most dominant medicinal plant families of the area. The
medicinal plants were dominant in low altitude areas. Other families are Cucurbitaceae, Acanthaceae,
Asclepiadaceae, Rutaceae, Rhamnaceae, etc. The pteridophyte belongs to 5 families viz., Sellaginellaceae,
Isoetaceae, Schizaceae, Pteridaceae and Marsiliaceae. Monocot families were represented by only Poaceae,
Araceae, Asparagaceae, Hypoxidaceae and Hydrocharitaceae. Some of the important medicinal plants in
Chandra Prabha Wildlife Sanctuary shown in figure 2.
Table 1. List of medicinal plants of Chandra Prabha Wildlife Sanctuary with their medicinal uses, habit, life form, local names and parts used.
S.N. Name Field Family Habit Life Local Name Medicinal Uses Plant
number form parts used
1. Selaginella bryopteris (L.) 76376 Selaginellaceae Herb Th Sanjeevani Anti-inflammatory and Leaf
Baker cures venereal disease
2. Calamaria coromandelina 75082 Isoetaceae Herb Cry Quilworts Spleen and liver Corm
(L.f.) Kuntze disease
3. Lygodium flexuosum (L.) Sw. 76278 Schizaeaceae Climber Ph - Roots are used in Root
rheumatism, sprains,
scabies, eczema and
cut wounds
4. Ceratopteris thalictroides 76210 Pteridaceae Herb Cry Indian fern Leaf and roots are used Leaf,
(L.) Brongn. in skin diseases Root
5. Marsilea minuta L. 76295 Marsiliaceae Herb Cry Susnisak For the treatment of Frond
psychopathy, diarrhea,
respiratory diseases,
and skin diseases.
6. Tinospora cordifolia (Willd.) Maurya Menispermaceae Vine Ph Giloe Used in debility, Twig,
Miers et al. dyspepsia, fever and Bark
(2015) urinary diseases.
7. Nymphaea nouchali Burm.f. 75050 Nympheaceae Herb Cry Berra Seeds used in Seeds,
cutaneous diseases. Rhizome
Rhizomes used in
dysentery, diarrhea,
dyspepsia.
8. Cleome viscosa L. 75039 Capparaceae Herb Th Hulhul, Whole herb is used in Whole
Hurhur the treatment of plant
ringworm, flatulence,
colic, dyspepsia,
cough, bronchitis,
cardiac disorders.
9. Hybanthus enneaspermus (L.) 75131, Violaceae Herb Ch Ratan purus Used in urinary Roots
F.Muell. 75013, affections and bowel
75106 complaints of children.
10. Polygala chinensis L. 75090, Polygalaceae Herb Th Meradu Leaves used in Leaf
75055, asthama and chronic
75162 bronchitis.
11. Abutilon indicum (L.) Sweet 76334 Malvaceae Small Ph Kanghi Used in urinary Root
Shrub troubles. Roots used as
nervine tonic and also
used in piles.
12. Sida acuta Burm.f. Maurya Malvaceae Herb Th Bariara Leaves demulcent and Leaf,
et al. (l.c.) diuretic, used in Root
Elephantiasis. Roots in
urinary disorder and
bowel disorder.
13. Sida cordata (Burm.f.) Borss. 75063, Malvaceae Herb Th Bhumi- Plant extract used in Whole
Waalk. 75145, bariara Dysentery. Leaves in plant
75172, leucorrhoea. Roots
75004, used in joint pains and
76292, weakness.
76241
14. Sida cordifolia L. Maurya Malvaceae Sub Th Kungyi Diuretic, used in Whole
et al. (l.c.) Shrub jaundice, urinary plant
problems.
15. Sida rhombifolia L. 76327 Malvaceae Herb Th Lalbarela Used in rheumatism Whole
and tuberculosis plant
16. Urena lobata L. 76275, Malvaceae Shrub Ph Bachita Used as expectorant in Flower
76318 sore throat
17. Helicteres isora L. 75044 Sterculeaceae Shrub Ph Marorphali Fruits used in colic Fruit
diarrhoea, dysentery
and improve appetite.
18. Waltheria indica L. 75095, Sterculeaceae Shrub Ph - Analgesic, anti- Whole
76264 inflammatory, plant
antibacterial,
antifungal,
antimalarial, anti-
anemic, anti-oxidant,
sedative and
anticonvulsant
activities.
19. Corchorus capsularis L. 75128 Tiliaceae Shrub Ph Jute Leaves used in Leaf,
dysentery. Roots and Root, Fruit
unripe fruits in
diarrhoea.
20. Grewia hirsuta Vahl 75153, Tiliaceae Shrub Ph Sita-Chabeni Twig used in Whole
76365 stomachache. Fruits plant
used in diarrhoea and
dysentery. Powdered
roots used in
leucorrhoea.
21. Grewia tiliifolia Vahl 75002 Tiliaceae Tree Ph Dhamni Bark used in Bark
dysentery.
22. Tribulus terrestris L. Maurya Zygophyllaceae Herb Ch Gokhru Used in diabetic Whole
et al. (l.c.) mellitus, cardiac plant
disorders, anticancer &
anti-infertility agent.
23. Biophytum sensitivum (L.) 75103 Oxalidaceae Herb Th Lakhshan Plant decoction used in Whole
DC. asthama, diabetes, plant
Roots used in Fever
and urinary disorders
24. Aegle marmelos (L.) Corrêa 75003 Rutaceae Tree Ph Bel Unripe or half-ripe Fruit
fruits astringent,
digestive and used for
diarrhea and dysentery.
25. Murraya paniculata (L.) Jack 76357 Rutaceae Tree Ph Kamini Leaves used in Leaf
diarrhea and dysentery,
applied on cuts, and
also in cough and
rheumatism
26. Naringi crenulata (Roxb.) 76341 Rutaceae Tree Ph Belsaundha Fruit pulp used in Fruit
Nicolson dysentery
27. Ailanthus excelsa Roxb. Maurya Simaroubaceae Tree Ph Ajan, Arua Leaves used in cuts, Leaf,
et al. (l.c.) sores and used as Bark
antiseptic. Bark is
anthelmintic.
28. Azadirachta indica A.Juss. Maurya Meliaceae Tree Ph Nim Bark used in skin Whole
et al. (l.c.) trouble. Leaves plant
considered antiseptic,
decoction given for
ulcers and eczema.
Flowers tonic and
stomachic.
29. Celastrus paniculatus Willd. 76361 Celastraceae Shrub Ph Malkangni Fruit paste used as Bark,
stimulant nerve tonic, Fruit, Seed
rejuvenant, sedative,
tranquilizer and
diuretic. Seeds used in
cough and cold.
30. Ventilago denticulata Willd. 76221 Rhamnaceae Climber Ph Karia bouri Bark paste used in Whole
dyspepsis and fever. plant
Stem pulp in eye
inflammation. Roots
used in Snake bites.
31. Ziziphus jujuba Mill. 76206, Rhamnaceae Shrub Ph Pitni-ber, Used in treatment of Fruit
75054 ban-ber chronic fatigue, loss
of appetite, diarrhea,
anemia, irritability
and hysteria.
32. Ziziphus oenopolia (L.) Mill. 75045, Rhamnaceae Shrub Ph Makai Roots anthelmintic. Whole
76226 Fruits are used in plant
coryza, aphrodisiac,
tonic and fevers.
33. Ampelocissus latifolia (Roxb.) 75066 Vitaceae Vine Ph Panibel Juice of leaves are used Leaf, Root
Planch. in dental problems and
roots used in
dysentery.
34. Cayratia trifolia (L.) Domin 75032 Vitaceae Vine Ph Amal-bel, Roots astringent, Root
Ramchana ground with pepper
applied to boils.
35. Abrus precatorius L. 75096, Fabaceae Climber Ph Gunchi, Rati Roots diuretic, tonic Root, Seed
75137, and emetic. Seeds used
76375 in affections of
nervous system and
stiffness of shoulder,
joints
36. Aeschynomene indica L. 75138, Fabaceae Shrub Ph Phulan Plant extract has Whole
75130 spermicidal activity plant
37. Alysicarpus vaginalis (L.) DC. 75099 Fabaceae Herb Th Chukalai Anti-inflammatory in Leaf
stomachache, used in
skin diseases and as a
diuretic. Leaves used
in fever, jaundice.
38. Butea monosperma (Lam.) 76291, Fabaceae Tree Ph Dhak Leaves used in Whole
Taub. 76374 dysentery, heat stroke, plant
Gums used in
diarrhoea and
dysentery. Fruit extract
used in urinary
troubles.
39. Cajanus scarabaeoides (L.) 75104, Fabaceae Climber Ph Bankulthi Plant decoction used Whole
A.Thouars 75141, for dysentery. plant
76326,
76358
40. Desmodium triflorum (L.) DC. 75065, Fabaceae Herb Ch Kudaliya Plants used in Whole
76300, toothache. Leaves used plant
76328, in dysentery and
76300 diarrhoea.
41. Indigofera cassioides Rottler 75181 Fabaceae Shrub Ph Kathi Roots used in the Roots
ex DC. treatment of coughs.
42. Indigofera tinctoria L. Maurya Fabaceae Herb Th Nil Extract used in Whole
et al. (l.c.) epilepsy and other plant
nervous disorders, in
form of ointment used
for sores, old ulcers,
and piles. Roots used
in urinary complains
43. Pongamia pinnata (L.) Pierre Maurya Fabaceae Tree Ph Karanja Used in cutaneous Seed,
et al. (l.c.) diseases. Juices of Root
roots used for cleaning
ulcers, sores and also
for cleaning teeth.
44. Pterocarpus marsupium Maurya Fabaceae Tree Ph Beejasal Leaf paste used in skin Leaf, Heart
Roxb. et al. (l.c.) disease. Heartwood wood,
used in asthama and Bark
diabetes. Bark used in
dysentery
45. Rhynchosia minima (L.) DC. 75135 Fabaceae Herb Ph Tinpatia Leaves used on Leaf, Root
wounds. Roots used in
urinary diseases
46. Tephrosia purpurea (L.) Pers. Maurya Fabaceae Shrub Th Dhamasia, Used in Bronchitis, Whole
et al. (l.c.) Sarphonka pimples, boils. Roots plant
and seeds are
insecticidal. Decoction
of roots are given in
dyspepsis, diarrhea,
rheumatism, asthama
and urinary disorders.
47. Uraria picta (Jacq.) Desv. Maurya Fabaceae Shrub Ph - Leaves used on Leaf, Root
et al. (l.c.) wounds. Powdered
root used in bodyache.
48. Bauhinia purpurea L. 76204 Caesalpiniaceae Tree Ph Khairwal Bark used in diarrhea. Whole
Flower buds eaten as plant
pot-herb, laxative and
antihelmintic
49. Bauhinia racemosa Lam. 75048, Caesalpiniaceae Tree Ph Kachnal Bark astringent, used Bark, Leaf
76237 in dysentery. Leaves
used for diarrhoea.
50. Bauhinia vahlii Wight & Arn. 76262 Caesalpiniaceae Tree Ph Mahurayan Bark used in chronic Bark, Root
stomach pain,
dysentery and cholera.
Roots used in syphilis
51. Bauhinia variegata L. 76204 Caesalpiniaceae Tree Ph Kachnar Twigs in diarrhoea, Flower,
toothache and mouth Twig,
ulcer. Barks used in Bark, Root
dysentery. Roots in
dyspepsia.
52. Cassia fistula L. Maurya Caesalpiniaceae Tree Ph Amaltas Leaves used in burn Leaf, Fruit,
et al. (l.c.) and skin diseases, Fruit Seed, Root
in dysentery; Ash used
in cough, fever,
gastritis, liver disorder.
Root used in fever.
53. Chamaecrista absus (L.) H.S. 75151 Caesalpiniaceae Herb Th Bhatwas Leaves used in cough. Leaf, Seed
Irwin & Barneby Seed in skin diseases.
54. Senna alata (L.) Roxb. Maurya Caesalpiniaceae Shrub/Sm Ph Ergaj Used in ringworms, Leaf, Root
et al. (l.c.) all Tree goiter, hook worm Bark,
infestation, sexually Flower
transmitted diseases,
constipation and other
skin diseases.
55. Senna obtusifolia (L.) Irwin 75185 Caesalpiniaceae Herb Th Panevar Leaves used in ulcer. Leaf, Root
& Barneby Roots in ringworm.
56. Senna occidentalis (L.) Link Maurya Caesalpiniaceae Herb Th Kasondi Leaves and seeds Leaf, Seed
et al. (l.c.) purgative, used in skin
troubles.
57. Senna tora (L.) Roxb. Maurya Caesalpiniaceae Herb Th Chakunda Leaves are purgative Leaf
et al. (l.c.) and used in skin
troubles.
58. Tamarindus indica L. 75168 Caesalpiniaceae Tree Ph Imli Leaves used in injury, Leaf, Seed
ringworm, leucoderma
and eye inflammation.
Seeds used in snake
bites.
59. Acacia catechu (L.f.) Willd. 75016, Mimosaceae Tree Ph Kattha Plants used in Bark,
75140, diarrhoea, sore throat. Heart
76339 Bark used in skin wood
diseases. Kattha from
heartwood is used in
wounds.
60. Albizia lebbeck (L.) Benth. 76202 Mimosaceae Tree Ph Shirisha Antitoxic and Bark
antiallergic.
61. Anogeissus latifolia (Roxb. ex 76254 Combretaceae Tree Ph Dhaw Bark used in diarrhoea, Bark, Root
DC.) Wall. ex Bedd. headache, scorpion
bite. Roots used in
toothache
62. Combretum album Pers. 76273 Combretaceae Large Ph Paibal Leaves are used in Leaf
shrub diarrhoea, acne,
eczema and other skin
diseases.
63. Terminalia arjuna (Roxb. ex 75015, Combretaceae Tree Ph Arjuna Styptic, tonic, Leaf, Bark
DC.) Wight & Arn. 76346 febrifuge and
antidysenteric,
pulverized bark
relieves hypertention
and act as diuretic in
liver cirrhosis. Leaves
used in ear-ache.
64. Terminalia bellirica (Gaertn.) Maurya Combretaceae Tree Ph Bahera Bark used in fever, Bark, Fruit
Roxb. et al. (l.c.) cold, cough, cholera.
Fruits used in
stomachache, liver
diseases,
pilesdiarrhoea, fever
and rheumatic pain.
65. Terminalia chebula Retz. Maurya Combretaceae Tree Ph Harra Fruits form a Fruit, Brak
et al. (l.c.) constituent of triphala,
fruit used in
dentifrices, fruit
powder smoked in
asthama, Bark diuretic
and cardiotonic.
66. Syzygium cumini (L.) Skeels 76218 Myrtaceae Tree Ph Jaman Decoctions of bark and Fruit, Bark
that of powdered seeds
are used in diabetes.
67. Barringtonia acutangula (L.) Maurya Lecythidaceae Tree Ph Injar Decoction in blood Bark, Stem
Gaertn. et al. (l.c.) deficiency and vitality.
68. Woodfordia fruticosa (L.) Maurya Lythraceae Shrub Ph Dawi Used in bowel Flower,
Kurz et al. (l.c.) complaints, Flower
hemorrhages, bud, Fruit
menorrhagia.
69. Ludwigia octovalvis (Jacq.) 76234, Onagraceae Herb Th Tilijuria Plants astringent, Whole
P.H.Raven 76301 laxative, diuretic, used plant
in leucorrhoea and
diarrhoea.
70. Diplocyclos palmatus (L.) 76332 Cucurbitaceae Climber Th Ban-Kakra Plant paste used in Whole
C.Jaffrey rheumatism. Leaves plant
used in fever, seeds
used in paralysis.
71. Mukia maderaspatana (L.) M. 75038 Cucurbitaceae Climber Th Madras pea Leaves used in Leaf
Roem. pumpkin diabetes.
72. Trichosanthes cordata Roxb. Maurya Cucurbitaceae Climber Ph - Roots used as tonic, Root
et al. (l.c.) given in enlargement
of liver, spleen and
disorder of other
viscera.
73. Trichosanthes cucumerina L. 75035 Cucurbitaceae Climber Ph Jangali- Roots used in Root
Chichinda bronchitis, headache
and boils.
74. Mollugo pentaphylla L. 75108 Molluginaceae Herb Th - Leaves are stomachic, Leaf
antiseptic, also used for
poultices for sore legs.
75. Ceriscoides turgida (Roxb.) 75075, Rubiaceae Tree Ph Kharhar Roots used in Root
Tirveng. 76364 toothache.
76. Gardenia gummifera L.f. 75067 Rubiaceae Small Ph Dikamali Used in nervous Young
Tree disorder of children shoots
during dentition, used
for dyspepsis.
77. Gardenia latifolia Sol. 75037, Rubiaceae Tree Ph - Used in treatment of a Whole
75047, wide range of ailments plant
76303 such as snake bite, skin
diseases, stomach
pains, inflammatory
pain, caries,
haemorrhages.
78. Haldina cordifolia (Roxb.) 76285 Rubiaceae Tree Ph Haldu Bark is used as Bark
Ridsdale antiseptic for killing
worms in sores and
also used in malarial
fever and dysentery.
79. Hymenodictyon orixense 75049, Rubiaceae Tree Ph Bhaulan Bark paste used is Bark, Fruit
(Roxb.) Mabb. 76230 swelling and pain.
Fruits used in asthama.
80. Mitragyna parvifolia (Roxb.) 75160, Rubiaceae Tree Ph Kadamb Bark and roots used to Bark, Root
Korth. 76236, treat fever, colic, and Leaf
76349 muscular pain, burning
sensation, poisoning,
gynecological
disorders, cough,
edema.
81. Oldenlandia corymbosa L. 76216, Rubiaceae Herb Th Pitpapra Used in Jaundice, and Leaf
76369 other diseases of the
liver, heat eruptions.
82. Caesulia axillaris Roxb. 76249 Asteraceae Herb Th Phulave Flowers used in boils. Flower,
Roots used in mouth Root
sore.
83. Eclipta prostrata (L.) L. 76315 Asteraceae Herb Th Bhangra, Plant paste used in Whole
Bhringraj Elephantiasis, plant
leucoderma, liver and
spleen diseases, Leaves
used in treatment of
dandruff, wounds,
snake bites, migraine,
high blood pressure.
84. Sphaeranthus indicus L. Maurya Asteraceae Herb Th Gorakhmundi Plant paste used in Whole
leucorrhoea. Leaves plant
et al. (l.c.)
used in eye diseases.
Roots used in madness.
85. Tridax procumbens L. 75113 Asteraceae Herb Th Mewadi Plant paste used in Whole
cuts, wounds, scorpion plant
bites, Leaves used in
cuts and leucorrhoea.
86. Calotropis gigantea (L.) R.Br. 75121 Asclepiadaceae Shrub Ph Akauva Warmed leaves used in Leaf,
skin inflammation, Flower,
flowers used in mental Root,
disorders, latex used in Latex
snake bites and roots in
jaundice.
87. Calotropis procera (Aiton) Maurya Asclepiadaceae Shrub Ph Akauva, Leaves used in sores, Leaf,
W.T.Aiton Madar skin diseases. Juice in Flower,
et al. (l.c.)
mouth ulcer and Root,
asthama, flowers used Latex
in epilepsy, cholera,
cough, cold.
88. Gymnema sylvestre (Retz.) 75037, Asclepiadaceae Woody Ph Gurmar Leaves used in Leaf
R.Br. ex Schult. 75184 vine diabetes, asthama,
cough and cold.
89. Hemidesmus indicus (L.) 76325, Asclepiadaceae Semierect Cry Anantmool, Plant paste in Whole
R.Br. ex Schult. 76335 Shrub Kali papri leucoderma, psorosis plant
and rheumatism.
Leaves used in cough
and gonorrhea. Roots
in eczema, snake bites,
diabetes, ringworm
infection and fever.
90. Heliotropium strigosum 75110 Boraginaceae Herb Th - Powdered plants used Whole
Willd. in mothers for plant
increasing lactation.
91. Evolvulus alsinoides (L.) L. 75026, Convolvulaceae Herb Cha Sankh- Plants ash used in Whole
75034, dermitis, used as nerve plant
pushpi
75070, tonic, vermifuge,
75148, antispasmodic,
75157 Powdered leaves used
in bronchitis, asthama,
bleeding and for hair
growth.
92. Ipomoea carnea Jacq. 75101 Convolvulaceae Shrub Ph Behaya Powdered plants used Whole
in conception. Leaves plant
paste used as an
antidote to scorpion
sting. Warm leaves
used in rheumatism
and gout.
93. Merremia hederacea 76280 Convolvulaceae Herb Th - Leaves used for Leaf
(Burm.f.) Hallier f. preparing poultice.
94. Datura innoxia Mill. 76286 Solanaceae Shrub Ph Datura Leaves paste used in Leaf, Seed
boils, pimples ad
asthama. Seeds used in
arthritis.
95. Solanum americanum Mill. 76246 Solanaceae Herb Th Kali Makoi Decoction of root is Root
used in fever.
96. Solanum virginianum L. 75094 Solanaceae Herb Th Berkateli Fresh root are applied Root
with lemon for
preventive measure of
cataract.
97. Scoparia dulcis L. 75006, Scrophulariaceae Herb Th Meethi patti, Plant extract used in Whole
76331 Ban mircha toothache, diabetes. plant
Leaves in diarrhea and
irregular menstruation.
Fruits in headache and
roots in menorrhagia
and in excessive
menstruation.
98. Oroxylum indicum (L.) Kurz Maurya Bignoniaceae Tree Ph Arlu, Bark decoction used in Bark, Leaf,
et al. (l.c.) Sheonak jaundice and bone Root
fracture. Leaves in
diarrhea and
rheumatism. Roots in
stomachache,
dysentery, and powder
for snake bites.
99. Sesamum indicum L. 75068, Pedaliaceae Herb Th Til Powdered seeds in Seed
75177 increasing lactation
and spermatorrhoea
and oil in urinary
troubles.
100. Andrographis paniculata Maurya Acanthaceae Herb Th Kalmegh Plant paste used for Whole
(Burm.f.) Nees et al. (l.c.) blood purification, plant
dysentery, diarrhea,
piles, jaundice,
bronchitis, lever
diseases.
101. Barleria cristata L. 76281 Acanthaceae Herb Cha Subhaga Plants used in cough Whole
and bronchitis. Leaves plant
in toothache. Roots and
leaves in bronchial
problems. Stem used as
toothbrush in
gummosis.
102. Barleria prionitis L. Maurya Acanthaceae Herb Cha Katsaraya Plant used in boils, Whole
et al. (l.c.) glandular swelling and plant
cough, cancer. Leaves
in tootache and
rheumatism.
103. Justicia adhatoda L. Maurya Acanthaceae Shrub Ph Adusa, Arusa Leaves used as Leaf
expectorant,
et al. (l.c.)
antispasmodic, in
chronic bronchitis,
asthama and
tuberculosis
104. Gmelina arborea Roxb. Maurya Verbenaceae Tree Ph Gumare Leaves carminative. Bark, Leaf,
Bark antiviral used in Root
et al. (l.c.)
swelling, choak throat,
rheumatism. Roots
used in weakness and
constipation
105. Ocimum americanum L. 75119, Lamiaceae Herb Th Bantulsa, Leaves juice used in Leaf, Seed
76248 Mamari headache. Seed paste
in animal ulcer to kill
worm
106. Amaranthus spinosus L. Maurya Amaranthaceae Herb Th Kateli-chauli Fresh leaves used in Whole
spermatorrhoea. Stem plant
et al. (l.c.)
in rheumatic pain.
Roots in gonorrhea,
diarrhoea.
107. Euphorbia hirta L. 75024 Euphorbiaceae Herb Th Dudhi Plant paste used for Whole
killing stomach worm, plant
in dysentery, viral
fever and cough. Latex
in snake bite and in
wounds.
108. Jatropha curcas L. Maurya Euphorbiaceae Tree Ph Ratanjot Leaves in Leaf, Seed,
inflammation of body Twig, Bark
et al. (l.c.)
and chest congestion.
Twig in toothache and
gummosis. Bark is
dysentery and
tuberculosis
109. Mallotus philippensis (Lam.) 76266 Euphorbiaceae Tree Ph Rohini, Bark used in Bark, Leaf,
Müll.Arg. Sinduri abdominal pain and Seed
jaundice. Seeds in
rheumatism. Roots
used as antiseptic.
Powdered seeds in
dysentery and
constipation.
110. Ricinus communis L. Maurya Euphorbiaceae Shrub Ph Arandi Leaves used in Leaf, Seed,
pneumonia fever, Root
et al. (l.c.)
jaundice and menstrual
disorder. Seeds in
rheumatism and
constipation. Roots in
boils.
111. Ficus racemosa L. Maurya Moraceae Tree Ph Gular Leaves used in boils, Leaf,
paralysis. Leaves and Latex,
et al. (l.c.)
latex in fever and Fruit, Root
dysentery. Fruits in
sunstroke, diarrhoea,
leucorrhoea and
diabetes. Roots sap in
diabetes.
112. Hydrilla verticillata (L.f.) 76245 Hydrocharitaceae Herb Cry - Improves Whole
Royle gastrointestinal, blood plant
circulatory problems.
Helps in detoxification
and neurological
disorders
113. Curculigo orchioides Gaertn. 75074, Hypoxidaceae Herb Cry Kali-musli Anticancer activity. Whole
75167 Plant paste used in plant
gonorrhea, asthama,
jaundice, diarrhoea,.
Seeds in body pain.
Rhizome paste in
itching and other
diseases.
114. Asparagus racemosus Willd. 76368, Asparagaceae Herb Cry Satawar Roots in nervous Root
75109 disorder, jaundice,
diabetes, leucorrhoea,
night blindness and
rheumatic pains.
115. Amorphophallus paeoniifolius Maurya Araceae Herb Cry Oal, Sooran Used in piles, Bulb
(Dennst.) Nicolson et al. (l.c.) dyspepsia, dysentery,
boils.
116. Apluda mutica L. 76344 Poaceae Herb Hemi Sutara Plant juice used in Whole
dysentery plant
117. Root 75087, Poaceae Herb Cha Khas, Barhni Roots in menstrual Root
76283 disorders, conceptions,
headache.
118. Cynodon dactylon (L.) Pers. 75165 Poaceae Herb Hemi Doob ghas Roots used in Root
leucorrhea
119. Desmostachya bipinnata (L.) 75014 Poaceae Herb Th Dhab Roots used in jaundice Root
Stapf and urinary troubles
120. Oplismenus burmannii (Retz.) 76220, Poaceae Herb Th - Used in epilepsy Root
P.Beauv. 76348
121. Saccharum spontaneum L. Maurya Poaceae Herb Cry Kans Extract expels
et al. (l.c.) abdominal worms
DISCUSSION
These plants are reported to cure various diseases such as asthama, bronchitis, tuberculosis, headache,
rheumatic pains, jaundice, constipation, diarrhoea, diabetes, leucorrhoea, urinary troubles, epilepsy, fever,
elephantiasis, skin diseases, snake bites, cough, gummosis, gastritis, liver disorder, scorpion bites, eczema,
cholera, cold, menorrhagia, boils, dysentery, sore, toothache, cardiac disorders, cancer, inflammation. And there
are many plant species with antibacterial, antifungal, anti-inflammatory, antioxidant, laxative, antiseptic and
sedative properties as also observed by other workers (Yadav et al. 2012, Chakraborty et al. 2013, Srilatha &
Ananda 2014, Arya et al. 2016, Choudhary & Jain 2016, Dutta et al. 2016, Singh et al. 2016, Sarkar & Devi
2017, Umadevi & Srinathrao 2017, Sundar & Habibur 2018, Venkanna et al. 2018). It was also a common
observation that many plants are used to treat the same diseases.
Out of total 121 medicinal plant species collected from the sanctuary the most commonly used parts of
medicinal plants are leaves (in 78 species), roots (in 75 species) and bark with 22 species only. The 78 species
of leaves includes the plants whose whole plant and twigs are used and 75 species of roots also includes the
plants whose whole plant is used for therapeutic treatment. Most of the medicinal plants are herb followed by
tree, shrub, climber and vine. The study indicated that Chandra Prabha Wildlife Sanctuary is rich in medicinal
plant diversity and it is an urgent need to conserve them. Uncontrolled human interference in sanctuary may
lead to great loss of medicinal biodiversity of the area. The traditional knowledge of medicinal plant species and
their therapeutic uses is also vanishing rapidly; therefore it is important to study their medicinal properties
which will definitely increase their conservation processes.
Figure 2. Some medicinal plants of Chandra Prabha Wildlife Sanctuary: A, Abrus precatorious L.; B, Helicteres isora L.; C,
Mallotus philippensis (Lam.) Mull. Arg.; D, Woodfordia fruticose (L.) Kurz; E, Gardenis latifolia Sol.; F, Aegle marmelos
(L.) Correa; G, Tinospora cordifolia (Willd.) Miers; H, Bauhinia vahlii Wight & Arn; I, Sida cordifolia L.; J, Andrographis
paniculata (Burm. F.) Nees; K, Butea monosperma (Lam.) Taub.; L, Trichosanthes cucumerina L.; M, Barleria cristata L.;
N, Indigofera cassoides Rottler ex DC.
ACKNOWLEDGEMENTS
Authors are thankful to the Director, Botanical Survey of India, Kolkata for facilities. Thanks are also due to
Head of office, Botanical Survey of India, Central Regional Centre, Allahabad for resources.
REFERENCES
Arya P, Mehta JP & Kumar S (2016) Antibacterial action of medicinal plant Alysicarpus vaginalis against
respiratory tract pathogens. International Journal for Environmental Rehabilitation and Conservation 7(2):
25–32.
Bajpai O, Pandey J & Chaudhary LB (2016) Ethnomedicinal Uses of Tree Species by Tharu Tribes in the
Himalayan Terai Region of India. Research Journal of Medicinal Plant 10(1): 19–41.
Chakraborty R, De B, Devanna N & Sen S (2013) Antitussive, expectorant activity of Marsilea minuta L., an
Indian vegetable. Journal of Advanced Pharmaceutical Technology & Research 4(1): 61–64.
Choudhary GP & Jain AP (2016) A Review on Mitragyna parvifolia (Roxb.) Korth. - An Indian Medicinal
Plant. International Journal of Pharmacy and Pharmaceutical Research 7 (1): 175–184.
de Silva T (1997) Industrial utilization of medicinal plants in developing countries. In: FAO medicinal plants for
forest conservation and health care. FAO, Rome, Italy, pp. 34–44.
Deepa MR, Sheema Dharmapal P & Udayan PS (2016) Floristic diversities and medicinal importance of
selected sacred groves in Thrissur district, Kerala. Tropical Plant Research 3(1): 230–242.
Dutta G, Baruah G & Devi A (2016) Wild food plants of Mishing tribe- An ethnobotanical survey. Tropical
Plant Research 3(1): 221–223.
Gadgil M (1989) Deforestation: Problems and Prospects. Foundation Day Lecture.Published in supplement to
Wasteland News. Vol V No. 4. Society for Promotion of Wastelands Development, New Delhi, India.
Maurya SK, Seth A, Nath D, Singh G & Singh AK (2015) Biodiversity and Indigenous Uses of Medicinal Plant
in the Chandra Prabha Wildlife Sanctuary, Chandauli District, Uttar Pradesh. International Journal of
Biodiversity 2015: Article ID 394307.
Mehra A, Bajpai O & Joshi H (2014) Diversity, utilization and sacred values of Ethno-medicinal plants of
Kumaun Himalaya. Tropical Plant Research 1(3): 80–86.
Sarkar M & Devi A (2017) Analysis of medicinal and economic important plant species of Hollongapar Gibbon
wildlife sanctuary, Assam, northeast India. Tropical Plant Research 4(3): 486–495.
Singh V, Khanna KK & Sinha GP (2016) Flora of Uttar Pradesh (Ranunculaceae to Apiaceae). Vol 1,
Botanical Survey of India, pp. 60–86.
Srilatha BR & Ananda S (2014) Antidiabetic effects of Mukia maderaspatanaand its phenolics: An in
vitro study on gluconeogenesis and glucose uptake in rat tissues. Pharmaceutical Biology 52 (5): 597–602.
Sundar RA & Habibur RC (2018) Pharmacognostic, phytochemical and antioxidant studies of Gardenia latifolia
Aiton: An ethnomedicinal tree plant. International Journal of Pharmacognosy and Phytochemical Research
10(5): 216–228.
Umadevi V & Srinathrao (2017) Tribulus terrestris (Linn): botany, medicinal uses, chemical composition and in
vitro regeneration a review. European journal of pharmaceutical and medical research 4(9): 324–330.
Vedprakash (1991) In: Samant SS, Dhar U & Palni LMS (eds) Indian Medicinal Plant: Current Status in
Himalayan Medicinal Plants: Potential and Prospects. Gramodaya Prakashan, Nainital, pp. 45–63.
Venkanna P, Sudheer Kumar K & Seetaram Swamy S (2018) Evaluation of antibacterial, anthelmintic activities
of leaves of Ziziphus oenoplia (L.) Mill. International Journal of Advanced Pharmaceutical Sciences 1(07):
25–34.
Yadav E, Mani M, Chandra P, Sachan N & Ghosh AK (2012) A review on therapeutic potential of Lygodium
flexuosum Linn. Pharmacognosy Review 6(12): 107–114.
Research article
Phytosociological and floristic evaluation of Kuldiha Wildlife

Sanctuary, Odisha, India
S. D. Rout1, S. K. Panda1 and T. Panda2*
1
Department of Wildlife and Biodiversity Conservation, North Orissa University, Takatpur, Baripada-757003,
Odisha, India
2
Department of Botany, Chandbali College, Chandbali, Bhadrak-756133, Odisha, India
*Corresponding Author: taranisenpanda@yahoo.co.in [Accepted: 30 December 2018]
Abstract: The present paper documents the findings of phytosociological attributes which have
been carried out in tropical moist deciduous forest of Kuldiha Wildlife Sanctuary, Odisha, India.
The main objectives of this study are to identify, characterize and classify the vegetation
community which is naturally distributed in the forest reserve. The vegetation sampling and data
analysis were done following standard procedures. One hundred and eight plant species belonging
to 38 families in the form of 38 species of trees, 38 species of shrubs and 32 species of herbs are
documented. The most common plant species based on importance value in tree, shrub and herb
layers are Terminalia tomentosa (IVI-292.27), Shorea robusta (RVI-50.89) and Croton roxburghii
(RVI- 17.11) respectively. Euphorbiaceae is found to be most dominant family. The incidence of
fire, livestock grazing and other anthropogenic disturbance are responsible for depletion of the old
and uneven age structure of forest. This study provides baseline information on the dry forests of
Kuldiha Wildlife Sanctuary. Appropriate conservation and management can considerably improve
the botanical value of Kuldiha Wildlife Sanctuary, and consequently their value for other life
forms.
Keywords: Dry deciduous forest - IVI - Long-term management - Sustainable utilization -
Vegetation community.
[Cite as: Rout SD, Panda SK & Panda T (2018) Phytosociological and floristic evaluation of Kuldiha Wildlife
Sanctuary, Odisha, India. Tropical Plant Research 5(3): 419–430]
INTRODUCTION
Plant species diversity is complex in nature and its structure and composition differs from place to place
because of varying climatic condition and topography (Raturi 2012). Compared to the other ecosystems, tropical
forest ones, the most complex of all the terrestrial ecosystems are harshly exploited ecosystems of the biosphere
(Bahuguna 1999). It covers 7 % of the earth’s land surface, but harbors more than half of the world’s plant and
animal biodiversity (Wilson 1988). Despite its direct services for sustainable human life, they are disappearing
at an overall rate of 0.8 to 2 % per year (May & Stumpf 2000, Sagar et al. 2003) and particularly dry deciduous
forests are the most disturbed and least protected ecosystems on the earth (Murphy & Lugo 1986). Even with a
national policy aimed at conserving and improving nature, biodiversity is still decreasing. In addition to
eutrophication, acidification and desiccation; habitat destruction, deforestation, human settlements,
globalization, agricultural expansion, and other infrastructure related to development over the last century have
accelerated the rapid decline of tropical forests throughout the world, which in turn bring about negative impacts
on biodiversity, climate change, ecological services, soil productivity and the livelihoods of forest dwelling as
well as rural people (Myers 1992, Raghubanshi & Tripathi 2009). Biodiversity has become the issue of global
attention because of growing awareness of its importance on the one hand as ecosystem energy and on the other
hand it allows building complex tropical networks and functions as insurance for ecosystem stability and
resilience (Gaston & Spicer 2004). The health of ecosystems, especially in mountainous regions, is closely
allied to its plant biodiversity (Schafer 2011) and vegetation classification is therefore the first step towards
ecosystem conservation. Such studies may become a vital tool in the estimation of the level of adaptation to the
Received: 18 March 2018 Published online: 31 December 2018
Rout et al. 2018
environment and their ecological significance (Pascal & Pelissier 1996). Phytosociological analysis indicates the
organization and structure of plant diversity which determines the distribution pattern of individuals among the
species in a particular habitat. In connection to this, Warger & Morrel (1976) noted that phytosociological
analysis is important for understanding the functioning of any community. It provides useful basic data for
ecology, geography, landscape science, conservation and environmental science because the data represent
integrated units in vegetation systems (Fujiwara 1987). Intensive studies concerning the phytosociology of the
tropical forests of India and also other parts of the world (Timilsina et al. 2007, Wahab et al. 2008, Tripathi &
Singh 2009, Slimani et al. 2010, Tel et al. 2010, Badshah et al. 2010, Bhat et al. 2011, Hegde et al. 2011, Bajpai
et al. 2012, Dangwal et al. 2012, Sahu et al. 2012, Verma et al. 2013, Ahmed & Sharma 2014, Jehad et al.
2014, Srinivasa et al. 2014, Pradhan & Rahman 2015, Knight 2015, Sundarapandian & Subbiah 2015, Shahid
& Joshi 2016, Bajpai et al. 2017, Iyagin & Adekunle 2017, Masens et al. 2017, Shiferaw et al. 2018) have been
highlighted. A perusal of literature reveals that phytosociological studies in different parts of Odisha are well
studied (Sahu et al. 2007, Ekka & Behera 2011, Behura et al. 2015, Nayak et al. 2016, Paul 2017). In this
context, there have, however, been no reports in Kuldiha Wildlife Sanctuary, Odisha, India. In the present
investigation an attempt has been made to document the structure of plant communities, composition and
diversity of tropical moist deciduous forest of Kuldiha Wildlife Sanctuary, Odisha, India which will help in
management and conservation of forest vegetation in future.
METERIALS AND METHODS

Study site
Figure 1. Location of study area.

Kuldiha Wildlife Sanctuary (21º 20′ N to 21º 30′ N latitude and 86º 30′ E to 86º 45′ E longitude) is situated
in Baleswar district of Odisha, India (Fig. 1). It spreads over 2505 km2 and is linked with the Similipal
Biosphere Reserve of Mayurbhanj district through the Sakhuapada and Nato Hill ranges. The landscape is hilly
with moderate to steep slopes having ranges of altitude between 169 to 682 m. The maximum temperature in the
warmest month and the minimum temperature in the coldest month are 42ºC and 6ºC, respectively. The mean
annual rainfall is 1,568mm. Ecologically the sanctuary falls within the Biogeographic zone of Deccan Plateau
and within the Biogeographic Province of Chhotanagpur Plaleau (Rodger & Panwar 1988). Three small rivers,
Tangna, Kamala and Usatal nala are the main water sources of the sanctuary. Adjoining to Nilagiri forest in the
north and Mayurbhanj forest in the west, the vegetation of the sanctuary is mostly tropical deciduous forest type
(Champion & Seth 1968). The rock of the sanctuary comprises with khondolite (grayish or reddish-brown in
colour), Pyrogene granites (dark in colour), charnockite (greenish grey in colour) and garnetiferrous Granites. A
good exposure of laterite has been found in the south-west portion of the sanctuary. This is an alternation
product of khondolytes. Alluvial soil is restricted to bank of nalas, rivers, and other water sources.
Data collection
Phytosociological studies were carried out during June 2016 to May 2017 to cover all spectrum of
vegetation. The surveys of area have been done by sampling method .Vegetation surveys was carried out by
quadrate methods (Misra 1968). For this purpose the entire area of 100 ha was divided into 10 segments. In each
segment a sampling area of 400 m2 with length and breadth of 20 m and 20 m respectively were measured. All
plants above 3 m tall were recorded by measuring girth at breast height (GBH) species wise. For bushes, shrubs
(less than 3 m tall) a sampling area of 25 m2 (5 m × 5 m) was nested inside the aforementioned 400 m2 plot. The
sampling plot of 1 m2 (1 m × 1 m) area was also nested inside the 25 m2 plots to make inventory of all
herbaceous vegetation (Fig. 2).
Figure 2. Sample plot structure, used in the study.

The vegetation data were quantitatively analysed following standard procedures (Curtis & McIntosh 1950,
Philips 1959, Misra 1968). Girth was measured using 2 m tape. Height of small trees and shrubs was measured
using a 5 m graded pole. When the height exceeded 5 m it was estimated visually. For calculating the basal area
of multi-stemmed trees, the girth of each stem was measured individually and added up. The economic uses of
plant species if any were discussed with the local people. Plant samples were identified or confirmed with
available regional floras (Haines 1925, Saxena & Brahmam 1996). Phytosociological parameters indicated
below were analyzed by the following methods and formulas (Curtis 1959, Misra 1968).
( )
For non-timber species the importance value is called Relative Importance Value (RVI) and calculated as
followed:
Rout et al. 2018
Figure 3. Plot wise distribution of plant diversity in Kuldiha Wildlife Sanctuary, Baleswar, Odisha.
Table 1. Phytosociological analysis of tree species in Kuldiha Wildlife Sanctuary, Baleswar, Odisha.
S. Botanical Local NOI NPSO D RD F RF AB CV RCV IVI AB/F
N. Name Name
1 Emblica officinalis Aonla 4 3 0.4 1.55 30 29.707 1.33 584229.1 8.510 39.764 0.044
2 Terminalia tomentosa Asana 60 9 6.0 23.26 90 89.108 6.67 12349795 179.905 292.270 0.074
3 Terminalia bellerica Bahada 4 3 0.4 1.55 30 29.702 1.33 2059774 30.005 61.259 0.040
4 Ziziphus oenoplia Borakoli 3 2 0.3 1.16 20 19.801 1.50 492542.4 7.175 28.139 0.075
5 Buchanania lanzan Char 1 1 0.1 0.39 10 9.900 1.00 218541.8 3.183 13.472 0.100
6 Oroxylum indicum Phanphena 6 3 0.6 2.33 30 29.702 2.00 735122.2 10.708 42.737 0.067
7 Anogeissus latifolia Dhaura 22 7 2.2 8.53 70 69.306 3.14 8381885 122.103 199.937 0.045
8 Polyalthia cerasoides Gandhasal 5 4 0.5 1.94 40 39.603 1.25 484262.4 7.054 48.596 0.031
9 Mitragyna parvifolia Godikoima 2 1 0.2 0.78 10 9.900 2.00 279752.7 4.075 14.751 0.200
10 Randia dumetorum Patua 5 2 0.5 1.94 20 19.801 2.50 1088583 15.857 37.597 0.125
11 Terminalia chebula Harida 5 4 0.5 1.94 40 39.603 1.25 1491308 21.724 63.266 0.031
12 Syzygium cumini Jamu 1 1 0.1 0.39 10 9.900 1.00 306795.9 4.469 14.757 0.100
13 Bridellia retusa Kashi 12 6 1.2 4.65 60 59.405 2.00 8521081 124.130 188.188 0.033
14 Diospyros melanoxylon Kendu 2 2 0.2 0.78 20 19.801 1.00 479686.5 6.987 27.565 0.050
15 Casearia elliptica Khakada 3 2 0.3 1.16 20 19.801 1.50 407993.5 5.943 26.908 0.075
16 Diospyros malabarica Kala kendu 1 1 0.1 0.39 10 9.900 1.00 384844.8 5.606 15.894 0.100
17 Xylia xylocarpa Kongada 3 1 0.3 1.16 10 9.900 3.00 928339.9 13.524 24.587 0.300
18 Hollarhena Kuluchi 3 2 0.3 1.16 20 19.801 1.50 487344 7.099 28.064 0.075
antydysenterica
19 Careya arborea Kumbhi 2 2 0.2 0.78 20 19.801 1.00 515903.2 7.515 28.092 0.050
20 Protium seeatum Rimuli 1 1 0.1 0.39 10 9.900 1.00 218541.8 3.184 13.472 0.100
21 Haldinia cordifolia Kurma 3 2 0.3 1.16 20 19.801 1.50 728775.3 10.616 31.581 0.075
22 Schleichera oleosa Kusuma 7 5 0.7 2.71 50 49.504 1.40 5961148 86.839 139.057 0.028
23 Madhuca indica Mahula 2 2 0.2 0.78 20 19.801 1.00 1116914 16.270 36.847 0.050
24 Antidesma acidum Nunannunia 1 1 0.1 0.39 10 9.900 1.00 253388.1 3.691 13.979 0.100
25 Dalbergia latifolia Pahadia sisso 2 1 0.2 0.78 10 9.900 2.00 221521.3 3.227 13.903 0.200
26 Butea parviflora Palasha 1 1 0.1 0.39 10 9.900 1.00 0 0 10.288 0.100
27 Millusa velutina Parashi 12 4 1.2 4.65 40 39.603 3.00 3227790 47.020 91.275 0.075
28 Pterocarpus marsupium Piasal 1 1 0.1 0.39 10 9.900 1.00 95936.5 1.397 11.686 0.100
30 Antidesma ghaesembilla Matha saga 12 4 1.2 4.65 40 39.603 3.00 2152429 31.355 75.610 0.075
31 Croton roxburghii Putuli 1 1 0.1 0.39 10 9.900 1.00 47143.48 0.687 10.975 0.100
32 Shorea robusta Sal 54 9 5.4 20.93 90 89.108 6.00 11010969 160.402 270.441 0.067
33 Largerstroemia Sidha 3 2 0.3 1.16 20 19.801 1.50 556303.7 8.103 29.068 0.075
parviflora
34 Albizia lebbeck Sirisa 3 3 0.3 1.163 30 29.702 1.00 593628 8.647 39.513 0.033
35 Dalbergia sisso Sisso 4 2 0.4 1.55 20 19.801 2.00 413417.6 6.022 27.374 0.100
36 Cassia fistula Sunari 1 1 0.1 0.39 10 9.900 1.00 26446.1 0.385 10.673 0.100
37 Soymida febrifuga Rohini 1 1 0.1 0.39 10 9.900 1.00 55571.59 0.809 11.098 0.100
38 Zizyphus mauritiana Barkoli 2 1 0.2 0.78 10 9.900 2.00 120072.4 1.749 12.425 0.200
Note: NOI- Number of individuals, NPSO- Number of plots in which species occurred, D- Density, RD- Relative density, F- Frequency, RF-
Relative frequency, AB- Abundance, CV- Coverage, RCV- Relative coverage, IVI- Importance value index.
Table 2. Phytosociological analysis of shrub species in Kuldiha Wildlife Sanctuary.

S. Botanical Local NOI NPSO D RD F RF AB AB/F RVI
N. Name Name
1 Emblica officinalis Anla 6 1 0.6 1.333 10 1.14 6.00 0.600 2.469
2 Combretum roxburghii Atundi 10 4 1.0 2.222 40 4.55 2.50 0.062 6.767
3 Aegle marmelos Bela 1 1 0.1 0.222 10 1.14 1.00 0.100 1.358
4 Flacourtia jangomas Boinchakoli 1 1 0.1 0.222 10 1.14 1.00 0.100 1.358
5 Buchanania lanzan Char 7 3 0.7 1.555 30 3.41 2.33 0.077 4.964
6 Dillenia pentagyna Rai 1 1 0.1 0.222 10 1.14 1.00 0.100 1.358
7 Ageratum conyzoides Dahanimari 14 2 1.4 3.111 20 2.27 7.00 0.350 5.383
8 Acacia pennata Dantari 1 1 0.1 0.222 10 1.14 1.00 0.100 1.358
9 Flemingia chapper Rani dant kathi 2 1 0.2 0.444 10 1.14 2.00 0.200 1.580
10 Nyctanthes arbor-tristis Gangasiuli 6 2 0.6 1.333 20 2.27 3.00 0.150 3.606
11 Pterospermum acerifolium Giringa 4 1 0.4 0.888 10 1.14 4.00 0.400 2.025
12 Clerodendrum viscosum Gobra 4 3 0.4 0.888 30 3.41 1.33 0.044 4.297
13 Syzygium cumini Jamu 4 3 0.4 0.888 30 3.41 1.33 0.044 4.297
14 Mallotus philippensis Kamalagundi 1 1 0.1 0.222 10 1.14 1.00 0.100 1.358
15 Ziziphus oenopila Kankoli 6 3 0.6 1.333 30 3.41 2.00 0.066 4.742
16 Diospyros melanoxylon Kendu 8 3 0.8 1.777 30 3.41 2.67 0.088 5.186
17 Casearia elliptica Khakada 4 3 0.4 0.888 30 3.41 1.33 0.044 4.297
18 Mimosa himalayana Khirkichikanta 6 1 0.6 1.333 10 1.14 6.00 0.600 2.469
19 Zyzyphus xylopyrus Barokoli 2 1 0.2 0.444 10 1.14 2.00 0.200 1.580
20 Xylia xylocarpa Kongada 2 1 0.2 0.444 10 1.14 2.00 0.200 1.580
21 Hollarhena antidyesenterica Kulchi 24 6 2.4 5.333 60 6.82 4.00 0.066 12.15
22 Haldinia cordifolia Kurma 1 1 0.1 0.222 10 1.14 1.00 0.100 1.358
23 Schleichera oleosa Kusuma 1 1 0.1 0.222 10 1.14 1.00 0.100 1.358
24 Madhuca indica Mahula 10 3 1.0 2.222 30 3.41 3.33 0.111 5.631
25 Helicteres isora Modimudica 5 3 0.5 1.111 30 3.41 1.67 0.055 4.520
26 Smilax zeylanica Mutri 3 2 0.3 0.666 20 2.27 1.50 0.075 2.939
27 Butea supeba Noi palaso 1 1 0.1 0.222 10 1.14 1.00 0.100 1.358
28 Cleistanthus collinus Koarada 1 1 0.1 0.222 10 1.14 1.00 0.100 1.358
29 Gardenia gummifera Bhurudu 2 1 0.2 0.444 10 1.14 2.00 0.200 1.580
30 Butea parviflora Polash 4 2 0.4 0.888 20 2.27 2.00 0.100 3.161
31 Bauhinia vahlii Siali 20 5 2.0 4.444 50 5.68 4.00 0.080 10.12
32 Albizzia lebbek Sirisha 2 1 0.2 0.444 10 1.14 2.00 0.200 1.580
33 Cassia fistula Sunari 8 2 0.8 1.777 20 2.27 4.00 0.200 4.050
34 Shorea robusta Sal 183 9 18.3 40.666 90 10.23 20.33 0.225 50.89
35 Cycas circinalis Veru 2 2 0.2 0.444 20 2.27 1.00 0.050 2.717
36 Randia dumetorum Potua 2 2 0.2 0.444 20 2.27 1.00 0.050 2.717
37 Croton roxburghii Putuli 87 7 8.7 19.333 70 7.95 12.43 0.177 27.28
38 Lantana camara Putush 1 1 0.1 0.222 10 1.14 1.00 0.100 1.358
Note: NOI- Number of individuals, NPSO- Number of plots in which species occurred, D- Density, RD- Relative density, F-
Frequency, RF- Relative frequency, AB- Abundance, RVI- Relative value index.
All over the world, plant biodiversity in terrestrial ecosystems have diverse biological communities due to
their rapidly changing landscape and geo-climatic history (Herben et al. 2003, Fosaa 2004). The distribution
pattern of plant species in an ecosystem is always representative of the function of that system because it
signifies the nature of its biomass (Enquist 2002, Myklestad & Sætersdal 2004). In an ecosystem plant species
assemble in a definite fashion and hence can assist in vegetation quantification and evaluation. Thus knowledge
of floristic composition of natural ecosystems and habitat types is a key factor for improving the long-term
management of natural resources (Mucina 1997, Ewald 2003, Kumar et al. 2015). During the study period a
total of 108 plant species belonging to 38 families of vascular plants were recorded. Plot wise distribution of
species is depicted in figure 3. The floristic composition of the reserve forest reveals 38 species in the upper
storey, 38 species in the middle storey and 32 species in the ground flora (Table 1, 2 & 3). The findings of the
present study are comparable with that of different ecosystems under tropical climates (Bhadra et al. 2010,
Behera et al. 2012, Jaykumar & Nair 2012, Mishra et al. 2013, Rabha 2014, Bajpai et al. 2015, Borah et al.
2016, Barua et al. 2018). Krishnamurthy et al. (2010) reported 46 species from a tropical dry deciduous forest in
Bhadra Wildlife Sanctuary, Karnataka. Sahu et al. (2012) recorded 57 species in dry deciduous forests of
Eastern Ghats. Studies of Thakur (2015) in tropical dry deciduous forest in Sagar district reported total 36 trees,
8 shrubs and 34 herbs. Pradhan & Rahaman (2015) recorded a total of 65 species belong to 36 families from
three tropical dry deciduous forests of Birbhum District, West Bengal. Working on phytosociology of Hulikal
Rout et al. 2018
state forest Vinayaka & Krishnamurthy (2016) reported a total of 231plant species out of which 96 are trees
followed by 53 herbs, 51 shrubs and remaining 31 are climbers. Sukumaran et al. (2018) has been recorded 36
trees, 18 shrubs, 26 herbs and 22 climbers in Muppuram sacred grove of Kollencode, Tamilnadu.
Table 3. Phytosociological analysis of herb species in Kuldiha Wildlife Sanctuary.
S. Botanical Local NOI NPSO D RD F RF AB AB/F RVI
N. Name Name
1 Hemidesmus indicus Anantamula 5 2 0.5 2.688 20 0.25 2.50 2.935 0.125
2 Combretum roxburghii Atundi 10 4 1.0 5.376 40 0.49 2.50 5.870 0.062
3 Urginea indica Banapiaja 1 1 0.1 0.537 10 0.12 1.00 0.661 0.100
4 Flacourtia jangomas Boinchakoli 6 3 0.6 3.225 30 0.37 2.00 3.596 0.067
5 Buchanania lanzan Char 6 3 0.6 3.225 30 0.37 2.00 3.596 0.067
6 Ageratum conyzoides Dahanimari 7 3 0.7 3.763 30 0.37 2.33 4.133 0.078
7 Elephantopus scaber Mayurachulia 10 1 1.0 5.376 10 0.12 10.00 5.499 1.000
8 Dioscoea bulbifera Pita alu 2 1 0.2 1.075 10 0.12 2.00 1.198 0.200
9 Icnocarpus frutescens Dudhilata 6 3 0.6 3.225 30 0.37 2.00 3.596 0.067
10 Andrographis paniculata Bhuinimba 2 1 0.2 1.075 10 0.12 2.00 1.198 0.200
11 Indigofera cassioides Gileri 3 3 0.3 1.612 30 0.37 1.00 1.983 0.033
12 Lygodium flexuosum Indrajal 4 4 0.4 2.150 40 0.49 1.00 2.644 0.025
13 Imperata cylindrica Juna 8 4 0.8 4.301 40 0.49 2.00 4.794 0.050
14 Ziziphus oenopila Kankoli 6 2 0.6 3.225 20 0.24 3.00 3.472 0.150
15 Diospyros melanoxylon Kendu 4 3 0.4 2.150 30 0.37 1.33 2.520 0.044
16 Mimosa himalayana Khirkhichi 1 1 0.1 0.537 10 0.12 1.00 0.661 0.100
17 Hollarhena antydesenterica Kuluchi 1 1 0.1 0.537 10 0.12 1.00 0.661 0.100
18 Haldinia cordifolia Kurma 1 1 0.1 0.537 10 0.12 1.00 0.661 0.100
19 Scleichera oleosa Kusuma 8 2 0.8 4.301 20 0.25 4.00 4.547 0.200
20 Ixora parviflora Luhajangi 2 2 0.2 1.075 20 0.25 1.00 1.322 0.050
21 Dioscorea pentaphylla Bana alu 3 2 0.3 1.612 20 0.25 1.50 1.859 0.075
22 Helicteres isora Modimudica 4 2 0.4 2.150 20 0.25 2.00 2.397 0.100
23 Cleistanthus collinus Parasi 1 1 0.1 0.537 10 0.12 1.00 0.661 0.100
24 Pterocarpus marsupium Piasal 2 1 0.2 1.075 10 0.12 2.00 1.198 0.200
25 Catunaregam spinosa Potua 3 2 0.3 1.612 20 0.25 1.50 1.859 0.075
26 Croton roxburghii Putuli 30 8 3.0 16.129 80 0.99 3.75 17.110 0.046
27 Dillenia pentagyna Rai 7 5 0.7 3.763 50 0.62 1.40 4.380 0.028
28 Smilax zeylanica Ramdantuni 3 2 0.3 1.612 20 0.25 1.50 1.859 0.075
29 Shorea robusta Sal 28 7 2.8 15.053 70 0.86 4.00 15.910 0.057
30 Asparagus racemosus Satabari 2 2 0.2 1.075 20 0.24 1.00 1.322 0.050
31 Albizzia lebbek Sirisha 5 3 0.5 2.688 30 0.37 1.67 3.058 0.050
32 Curculigo orchioides Talmuli 5 1 0.5 2.688 10 0.12 5.00 2.811 0.500
Note: NOI- Number of individuals, NPSO- Number of plots in which species occurred, D- Density, RD- Relative density, F-
Frequency, RF- Relative frequency, AB- Abundance, RVI- Relative value index.
Figure 4. Girth classes of tree species in Kuldiha Wildlife Sanctuary.

The upper storey vegetation was covered by tall trees with epiphytic growth of lichens, bryophytes, ferns and
orchids. In the present study, Terminalia tomentosa (Roxb. ex DC.) Wight & Arn. was the dominant tree species
having maximum relative density (23.26) and relative frequency (89.11) followed by Shorea robusta Gaertn.f.,
Anogeissus latifolia (Roxb. ex DC.) Wall. ex Guill. & Perr and Bridelia retusa (L.) Spreng. in terms of
density/100 hector (Table 1). The importance value of Terminalia tomentosa (Roxb. ex DC.) Wight & Arn. was
the highest (292.30) followed by Shorea robusta Gaertn.f. (270.40), Anogeissus latifolia (Roxb. ex DC.) Wall.
ex Guill. & Perr (199.90), Bridelia retusa (L.) Spreng. (188.20) and Schleichera oleosa (Lour.) Oken (139.10).
In the middle storey, some of the shrubs i.e. Croton roxburghii Balak, Cleistanthus collinus (Roxb.) Benth. ex
Hook.f., Polyalthia cerasoides (Roxb.) Bedd., Gardenia gummifera L.f. and Hollarhena pubescens (Buch.-
Ham) Wall. ex G.Don. were found to grow in dense in interior forests. In shrub layer the highest value of RVI
(50.89) was recorded for Shorea robusta Gaertn.f. whereas lowest (RVI-1.36) was recorded for Aegle marmelos
(L.) Corr., Adina cordifolia (Roxb.) Hook.f. ex Brandis and Flacourtia species (Table 2). A good number of
lianas and woody climber were present in plots such as Bauhinia vahlii Wight & Arn., Combretum albidum
G.Don., Hemidesmus indicus (L.) R. Br., Smilax zeylanica L. and Veltilago species. In the herb layer the most
dominant species was Croton roxburghii Balak (RVI, 17.11) followed by Shorea robusta Gaertn.f. (15.91) and
Combretum decandrum Roxb. (5.87) (Table 3). The family with the most species present in the study area is
Euphorbiaceae, followed by Rubiaceae and Combretaceae. Twelve families are monospecific. For the analysis
of individuals per GBH class, all tree species were taken into consideration. For individuals below 2 cm GBH
were surveyed only in the 5 m × 5 m subplot. Ninety tree species were recorded having GBH between 51–100
whereas only two species like Madhuca indica Gmel. and Dillenia pentagyna Roxb. showed >200 GBH (Fig.
4). Medicinal plant species such as Aegle marmelos (L.) Corr., Andrographis paniculata (Burm.f.) Wall. ex
Nees, Asparagus recemosus Willd., Curculigo orchioides Gaertn., Emblica officinalis Gaertn., Madhuca indica
Gmel., Nyctanthes arbor-tristis L., Syzygium cumini (L.) Skeels, Terminalia bellerica (Gaertn.) Roxb. and
Terminalia chebula Retz. were harvested in bulk for preparation of medicines by the local people (Fig. 5).
Similar use has been reported in other studies of India (Kassam et al. 2011, Khan et al. 2011, Mehra et al. 2014,
Bajpai et al. 2016) and also other parts of the world (Jones 2000, Maurer et al. 2006, Chowdhury & Koike
2010). The medicinal plant resources are depleting rapidly due to unsustainable harvesting, lack of awareness
and unrestricted grazing by domestic animals from nearby villages. Unsustainable collection of above medicinal
plants has placed them in threatened and vulnerable categories in Conservation Assessment and Management
Plan (CAMP) of Odisha (Pattanaik et al. 2009). Sustainable utilization and conservation of biodiversity are
essential for the continuation of ecosystem functioning (Srivastava & Vellend 2005). The indigenous people in
the study area gave less attention to the long term ecosystem goods and services since they were focused on
their marginal and short time benefits. They illicitly utilized plants for a number of uses including timber, fuel,
medicines, food, grazing and fodder. Extensive use of natural vegetation in the sanctuary in the past has
decreased the provisioning services (Stewart & Pullin 2008, Giam et al. 2010). This diminution is fairly
remarkable in the categories of food, fodder, timber fuel and medicines. The consequence of the imbalance in
supply of these services and the increasing human demands has been deterioration in the condition of the natural
habitats and increasing rarity of plant biodiversity (Giam et al. 2010). These effects are becoming worse as the
indigenous people neither possess enough services locally nor can they compete in the urban societies. Due to
the depletion of important species, the traditional indigenous knowledge of the people is decreasing day by day.
The impact of these problems on single species and ecosystems are likely to be complex. According to Bhandari
et al. (1999), any species in a community plays a specific role and there is a definite quantitative relationship
between abundant and rare species. Plant biodiversity can be restored and the risks of degradation may be
combated, if measures like reforestation, greater awareness by the people and ex-situ conservation of rare
species are initiated (Pereira et al. 2005, Muzaffar et al. 2011).
In the shrub layer Sal samplings were more in the plots. Consequently, these will be large trees after some
years, if can be preserved in healthy condition. This is a natural state as trees are competing for space and light
and not all of the seedlings and saplings reach the state of adult trees. Still one has to be aware of the fact that
matured and adult trees are attractive for wood smugglers as well as villagers, searching for construction
material. The relatively low percentage of those trees is pointing to this direction. Out of total 38 tree species
recoded in the 10 plots, 36 number of matured good class tree shows 100% adult (>100 GBH). So, the
protection is needed to preserve these highest GBH trees. Regeneration pattern of some species with good and
healthy offspring are found in the forest floor. Examples include Michelia champaca L., Shorea robusta
Gaertn.f., Scleichera oleosa (Lour.) Oken, Smilax macrophylla Roxb., Aegle marmelos (L.) Corr., Bauhinia
vahlii Wight & Arn., Combretum decandrum Roxb. and Hemidesmus indicus (L.) R. Br. Few numbers of
bamboos with dispersed distribution along with few grass species was observed in some sites of the forest.
However, while the forest shows a good regeneration status, their further development is prevented by the
various anthropogenic activities like lumbering, overgrazing, forest encroachments and fire. In the present study
Rout et al. 2018
under storey fires and grazing are major regulatory factors controlling species distribution. In all the plots dung
were found, so livestock are destroying the new germinated seedlings as they prefer soft leaves of seedlings and
saplings. Livestock grazing have gradually caused a reduction in vegetation cover. Continuations of overgrazing
will not only endanger the sustainability of forest ecosystems, but also will increase the challenge of sustainable
forest management (Pour 2012, Hailu 2017). Fire is one of the most important disturbance factors in natural
ecosystems throughout the world (Moretti & Barbalat 2004, Pashaki et al. 2013). Frequent firing may remove
vegetation species that rely on seed production for their persistence (Fox & Fox 1986). Changes in habitat
structure as a consequence of frequent burning are likely to disadvantage many native species (Whelan 1995).
Studies of Peterson et al. (2007) and Peterson & Reich (2008) indicated that repeated fires not only reduced
biological diversity but also play a crucial role in eradication of species.
Figure 5. Some medicinal plant species of Kuldiha Wildlife Sanctuary: A, Aegle marmelos (L.) Corr.; B, Asparagus
racemosus Willd.; C, Madhuca indica Gmel.; D, Nyctanthes arbor-tristis L.; E, Syzygium cumini (L.) Skeels; F, Terminalia
bellirica (Gaertn.) Roxb.
CONCLUSIONS
Considering over all phytosociological status of Kuldiha Wildlife Sanctuary Odisha, India, it reveals that
there is a big gap between the values of various parameters like IVI, density, frequency and abundance. There
are many tree species having very low values of IVI and other parameters and these species deserve more
attention. A special care should be taken for growth of immature tree species growing in these forest areas.
Further, this forest exhibits good regeneration status, and offer opportunities to investigate forest dynamics and
changes in species relative abundances in the future. Although the study site is protected, this forest is
experiencing destruction because of the frequent visits of people from nearby villages for their daily
requirements (fuel, medicine, fodder, bamboo and other non-timber forest produce) and over grazing by
livestock. This has resulted in the fragmentation of the forest, thereby causing damage to biodiversity. Further,
educating the local people and effective implementation of the rules would be helpful in decreasing the
depletion of natural forest produce. The present study in the Kuldiha forest is preliminary, and subsequent re
census will be helpful for the restoration and management of mountainous regions in the larger-scale
conservation planning of the Kuldiha and adjacent ranges.
ACKNOWLEDGEMENTS
The authors wish to convey their sincere gratitude to the people concerned for their help in field work.
REFERENCES
Ahmed J & Sharma S (2014) Spatial pattern, diversity and phytosociological analysis of woody plant species in
ponda watershed, Rajouri, J&K, India. International Journal of Current Research 6(6): 7022–7027.
Badshah L, Hussain F & Akhtar N (2010) Vegetation of subtropical forest of Tabai, South Waziristan,
Pakistan. Frontier of Agriculture in China 4(2): 232–236.
Bahuguna VK (1999) Forest fire prevention and control strategies in India. International Forest Fire News 20:
5–9.
Bajpai O, Kumar A, Mishra AK, Sahu N, Pandey J, Behera SK & Chaudhary LB (2012) Recongregation of tree
species of Katerniaghat Wildlife Sanctuary, Uttar Pradesh, India. Journal of Biodiversity and Environmental
Sciences 2(12): 24–40.
Bajpai O, Kushwaha AK, Srivastava AK, Pandey J & Chaudhary LB (2015) Phytosociological status of a
monotypic genus Indopiptadenia: A Near Threatened Tree from the Terai-Bhabar Region of Central
Himalaya. Research Journal of Forestry 9(2): 35–47.
Bajpai O, Pandey J & Chaudhary LB (2016) Ethnomedicinal uses of tree species by Tharu tribes in the
Himalayan Terai region of India. Research Journal of Medicinal Plant 10(1): 19–41.
Bajpai O, Suman S & Upadhyay N (2017) Ecological exploration of Kuwana forest A tropical moist deciduous
forest of eastern Terai, India. Annals of Plant Sciences 6(12): 1811–1816.
Barua KN, Gogoi G & Hazarika P (2018) Comparative study on structural composition and community
association of Nambor Wildlife Sanctuary and its South-Westward extended Bornewria forest, Assam, India.
Tropical Plant Research 5(2): 233–242.
Behera SK, Mishra AK, Sahu N, Kumar A, Singh N, Kumar A, Bajpai O, Chaudhary LB, Khare PB & Tuli R
(2012) The study of microclimate in response to different plant community association in tropical moist
deciduous forest from northern India. Biodiversity and Conservation 21: 1159–1176.
Behura S, Kar M & Upadhyay VP (2015) Phytosociological Study of herbs species at two reclaimed sites of
Sukinda Chromite mining region of Odisha, India. Geo-Eco-Trop 39(2): 329–342.
Bhadra AK, Dhal NK, Rout NC & Raja Reddy V (2010) Phytosociology of the three community of
ghandhmardan hill ranges. Indian Forester 137 (5): 610–620.
Bhandari BS, Nautiyal DC & Gaur RD (1999) Structural attributes and productivity potential of an alpine
pasture of Garhwal Himalaya. Journal of Indian Botanical Society 78: 321–329.
Bhat DM, Hegde GT, Shetti DM, Patgar SG, Hegde GN, Furtado RM, Shastri CM, Bhat PR & Ravindranath
NH (2011) Impact of disturbance on composition, structure and floristics of tropical moist forests in Uttara
Kannada District, Western Ghats, India. Ecotropica 17(2): 1–14.
Borah N, Rabha D & Athokpam FD (2016) Tree species diversity in tropical forests of Barak valley in Assam,
India. Tropical Plant Research 3(1): 1–9.
Champion HG & Seth SK (1968) A revised survey of the forest types of India. Manager Publications, New
Delhi, pp. 16–17.
Chowdhury MSH & Koike M (2010) Towards exploration of plant-based ethno-medicinal knowledge of rural
community: Basis for biodiversity conservation in Bangladesh. New Forests 40: 243–260.
Curtis JT & McIntosh RP (1950) The interrelations of certain analytic and synthetic phytosociological
characters. Ecology 31: 434–455.
Curtis JT (1959) The vegetation of Wisconsin. An ordination of plant Communities. University of Wisconsin
Press, Madison Wisconsin.
Dangwal LR, Singh T, Singh A & Sharma A (2012) Species composition of woody plants in forest of block
Nowshera, district Rajouri (J&K), India. Journal of Current Research 4(5): 5–10.
Ekka NJ & Behera N (2011) Species composition and diversity of vegetation developing on an age series of
coal mine spoil in an opencast coal field in Odisha, India. Tropical Ecology 52: 337–343.
Enquist BJ (2002) Universal scaling in tree and vascular plant allometry: Toward a general quantitative theory
linking plant form and function from cells to ecosystems. Tree Physiology 22: 1045–1064.
Ewald J (2003) A critique for phytosociology. Journal of Vegetation Science14: 291–296.
Fosaa AM (2004) Biodiversity patterns of vascular plant species in mountain vegetation in the Faroe Islands.
Diversity and Distributions10: 217–223.
Fox MD & Fox BJ (1986) The effect of fire frequency on the structure and floristic composition of a woodland
understory. Australian Journal of Ecology 11: 77–85.
Fujiwara K (1987) Aims and methods of phytosociology of ‘vegetation science. In: Plant Ecology and
Rout et al. 2018
Taxonomy. Kobe: The Kobe Geobotanical Society, pp. 607–628.

Gaston KJ & Spicer JI (2004) Biodiversity: an introduction, 2nd edition. Blackwell Publishing.
Giam X, Bradshaw CJA, Tan HTW & Sodhi NS (2010) Future habitat loss and the conservation of plant
biodiversity. Biological Conservation 143: 1594–1602.
Hailu H (2017) Analysis of Vegetation Phytosociological characteristics and soil physico-chemical conditions in
Harishin rangelands of Eastern Ethiopia. Land 6(4): 1–17.
Haines HH (1925) The Botany of Bihar and Orissa. Adland and Son, West Newman Ltd., London.
Hegde GT, Murthy IK, Bhat PR, Swarnim S, Alipuria AK & Ravindranath NH (2011) Vegetation status in
degraded forest, community and private lands of Himanchal Pradesh. Indian Forester 137(5): 544–553.
Herben T, Krahulec F, Hadincová V, Pecháčková S &Wildová R (2003) Year-to-year variation in plant
competition in a mountain grassland. Journal of Ecology 91: 103–113.
Iyagin FO & Adekunle VAJ (2017) Phytoecological studies of some protected and degraded forest areas of
Lowland Humid forest, Ondo state Nigeria: a Comparative approach. Tropical Plant Research 4(3): 496–
513.
Jaykumar R & Nair KKN (2012) Beta diversity of angiosperms in the tropical forests of Nilgiri Biosphere
Reserve, India. Tropical Ecology 53(2): 125–136.
Jehad MHI, Cano-Ortiz1 A, Asma AAS, Mohammed MHI & Cano E (2014) Phytosociology with other
characteristic biologically and ecologically of plant in Palestine. American Journal of Plant Sciences 5:
3104–3118.
Jones A (2000) Effects of cattle grazing on North American arid ecosystems: A quantitative review. Western
North American Naturalist 60: 155–164.
Kassam K, Karamkhudoeva M, Ruelle M & Baumflek M (2011) Medicinal Plant Use and Health Sovereignty:
Findings from the Tajik and Afghan Pamirs. Human Ecology 38: 817–829.
Khan SM, Zeb A & Ahmad H (2011) Medicinal Plants and Mountains: Long-Established Knowledge in the
Indigenous People of Hindu Kush Germany. VDM Verlag Dr. Müller.
Knight DH (2015) From Phytosociology and Plant Evolution to Ecosystem Analysis. Ecological Society of
America Bulletin 96(2): 209–210.
Krishnamurthy YL, Prakasha HM, Nanda A, Krishnappa M, Dattaraja HS & Suresh HS (2010) Vegetation
structure and floristic composition of a tropical dry deciduous forest in Bhadra Wildlife Sanctuary,
Karnataka, India. Tropical Ecology 51(2): 235–246.
Kumar A, Bajpai O, Mishra AK, Sahu N, Behera SK, Bargali SS & Chaudhary LB (2015) A checklist of the
flowering plants of Katerniaghat Wildlife Sanctuary, Uttar Pradesh, India. Journal of Threatened Taxa 7(7):
7309–3408.
Masens da-Musa YB, Ngbolua K-t-N, Masens M, Tambu TM & Gédéon NB (2017) Phytoecologicalstudy of
Nzundu massif forest of Imbongo city, Kwilu Province, Democratic Republic of the Congo. Tropical Plant
Research 4(3): 363–375.
Maurer K, Weyand A, Fischer M & Stocklin J (2006) Old cultural traditions, in addition to land use and
topography, are shaping plant diversity of grasslands in the Alps. Biological Conservation 130: 438–446.
May RM & Stumpf MPH (2000) Species area relations in tropical forests. Science 290: 2084–2086.
Mehra A, Bajpai O & Joshi H (2014) Diversity, utilization and sacred values of Ethno-medicinal plantsof
Kumaun Himalaya. Tropical Plant Research 1(3): 80–86.
Misra R (1968) Ecology Work-Book. Oxford and IBH Publishing Co., New Delhi.
Mishra AK, Bajpai O, Sahu N, Kumar A, Behera SK, Mishra RM & Chaudhary LB (2013) Study of Plant
Regeneration Potential in Tropical Moist Deciduous Forest in Northern India. International Journal of
Environment 2(1): 153–163.
Moretti M & Barbalat M (2004) The effects of wildfire on wood eating beetles in deciduous forests on the
southern slope of the Swiss Alps. Forest Ecology and Management 187: 85–103.
Mucina L (1997) Classification of vegetation: Past, present and future. Journal of Vegetation Science 8(6): 751–
760.
Murphy PG & Lugo AE (1986) Ecology of tropical dry forests. Annual Review of Ecology and Systematics 17:
67–88.
Muzaffar SB, Islam MA, Kabir DS, Khan MH, Ahmed FU, Chowdhury W, Aziz MA, Chakma S & Jahan I
(2011) The endangered forests of Bangladesh: Why the process of implementation of the Convention on
Biological Diversity is not working. Biodiversity and Conservation 20: 1587–1601.
Myers N (1992) Population/environment linkages: discontinuities ahead. Ambio 21: 116–118.

Myklestad Å & Sætersdal M (2004) The importance of traditional meadow management techniques for
conservation of vascular plant species richness in Norway. Biological Conservation 118: 133–139.
Nayak S, Bhuyan BK, Satapathy GK, Das S & Kumar V (2016) Phytosociological studies on biodiversity of
Bonaigarh forest division, Sundergarh district, Odisha. Journal of Plant Development Sciences 8(6): 261–
273.
Pascal JP & Pelissier R (1996) Structure and floristic composition of tropical evergreen forest in south-west
India. Journal of tropical Ecology 12(2): 191–214.
Pashaki MS, Daryayi MG, Adel MN & Kuhestani JS (2013) Effect of repeated fire on understory plant species
diversity in Saravan forests, northern Iran. Folia Forestalia Polonica Series A 55(3): 137–145.
Pattanaik C, Reddy CS & Reddy KN (2009) Ethno-medicinal survey of threatened plants in Eastern Ghats,
India. Our Nature 7: 122–128.
Paul S (2017) Why foresters should care about social sector schemes-case of Ghumsur forests of Ganjam district
of Odisha. The Indian Forester 143(12): 1213–1220.
Pereira E, Queiroz C, Pereira HM & Vicente L (2005) Ecosystem services and human well-being: A
participatory study in a mountain community in Portugal. Ecology and Society 10(2): 14.
Peterson DW & Reich PB (2008) Fire frequency and tree canopy structure influence plant species diversity in a
forest grassland ecotone. Plant Ecology 194: 5–16.
Peterson DW, Reich PB & Wrage KJ (2007) Plant functional group responses to fire frequency and tree canopy
cover gradients in oak savannas and woodlands. Journal of Vegetation Science 18: 3–12.
Philips EA (1959) Methods of Vegetation Study. Henry Holt Co. Inc New York, USA, pp. 107.
Pour MJ (2012) Effects of grazing on natural regeneration of tree and herb species of Kheyroud forest in
northern Iran. Journal of Forestry Research 23(2): 299–304.
Pradhan B & Rahman CH (2015) Phytosociological study of plant species in three tropical dry deciduous forests
of Birbhum District, West Bengal, India. Journal of Biodiversity and Environmental Sciences 7(2): 22–31.
Rabha D (2014) Species composition and structure of Sal (Shorea robusta Gaertn. f.) forests alongdistribution
gradients of Western Assam, Northeast India. Tropical Plant Research 1(3): 16–21.
Raghubanshi AS & Tripathi A (2009) Effect of disturbance, habitat fragmentation and alien invasive plants on
floral diversity in dry tropical forest of Vindhyan Highlands: a review. Tropical Ecology 50(1): 57–69.
Raturi GP (2012) Forest community structure along an altitudinal gradient of district Rudraprayag of Garhwal
Himalaya, India. Ecologia 2(3): 76–84.
Rodgers WA & Panwar SH (1988) Biogeographical classification of India. In: New Forest. Dehradun, India.
Sagar R, Raghubansi AS & Singh JS (2003) Trees species composition, dispersion and diversity along a
disturbance gradient in a dry tropical forest region of India. Forest Ecology and Management 186: 61–71.
Sahu SC, Dhal NK & Mohanty RC (2012) Tree species diversity, distribution and population structure in a
tropical dry deciduous forest of Malygiri hill ranges, Eastern India. Tropical Ecology 53(2): 163–168.
Sahu SC, Dhal NK, Reddy CS, Pattanaik C & Brahman M (2007) Phytosociological study of tropical dry
deciduous forest of Boudh district, Orissa, India. Research Journal of Forestry 1(2): 66–72.
Saxena HO & Brahmam M (1996) The Flora of Orissa. Vol. I-IV. Orissa Forest Development Corporation,
Bhubaneswar.
Schafer RB (2011) Biodiversity, ecosystem functions and services in environmental risk assessment:
Introduction to the special issue. Science of the Total Environment 15: 1–2.
Shahid M & Joshi SP (2016) Phytosociological assessment and distribution patterns of tree species in the forests
of Doon Valley, Shivalik hills of lower Himalaya. Tropical Plant Research 3(2): 263–271.
Shiferaw W, Lemenih M & Gole TWM (2018) Analysis of plant species diversity and forest structurein Arero
dry Afromontane forest of Borena zone, South Ethiopia. Tropical Plant Research 5(2): 129–140.
Slimani H, Aidoud A & Rozé F (2010) 30 years of protection and monitoring of a steppic rangland undergoing
desertification. Journal of Arid Environments 74: 685–691.
Srinivasa RD, Murthy P & Aniel KO (2014) Distribution of Soil Types, Vegetation and tree species diversity in
Eastern Ghats of Srikakulam district, Andhra Pradesh, India. International Journal of Biodiversity and
Conservation 6(6): 488–494.
Srivastava DS & Vellend M (2005) Biodiversity-ecosystem function research: Is it relevant to conservation?
Annual Review of Ecology, Evolution, and Systematics 36: 267–294.
Stewart GB & Pullin AS (2008) The relative importance of grazing stock type and grazing intensity for
Rout et al. 2018
conservation of mesotrophic 'old meadow' pasture. Journal for Nature Conservation 16: 175–185.
Sukumaran S, Pepsi A, SivaPradesh DS & Jeeva S (2018) Phytosociological studies of the sacredgrove of
Kanyakumari district, Tamilnadu, India. Tropical Plant Research 5(1): 29–40.
Sundarapandian SM & Subbiah S (2015) Diversity and tree population structure of tropical dryevergreen forests
in Sivagangai district of Tamil Nadu, India. Tropical Plant Research 2(1): 36–46.
Tel AZ, Tatli A & Varol O (2010) Phytosociological structure of Nemrut Mountain (Adiyaman/Turkey).
Turkish Journal of Botany 34: 417–434.
Thakur AS (2015) Floristic composition, life-forms and biological spectrum of tropical dry deciduousforest in
Sagar District, Madhya Pradesh, India. Tropical Plant Research 2(2): 112–119.
Timilsina N, Ross MS & Heinen JT (2007) A community analysis of Sal (Shorea robusta) forests in the western
Terai Nepal. Forest Ecology and Management 241(1-3): 223–234.
Tripathi KP & Singh B (2009) Species diversity and vegetation structure across various strata in
naturalandplantation forests in KaterniaGhat Wildlife Sanctuary, North India. Tropical Ecology 50(1): 191–
200.
Verma MK, Niranjan RK & Pal A (2013) Phytosociological attributes of a tropical dry deciduous forest of
Bundelkhand region of Uttar Pradesh, India. Journal of Biodiversity and Environmental Sciences 3 (10): 86–
99.
Vinayaka KS & Krishnamurthy YL (2016) Floristic composition and vegetation analysis of HulikalGhat region,
central Western Ghats, Karnataka. Tropical Plant Research 3(3): 654–661.
Wahab M, Ahmed M & Khan N (2008) Phytosociology and dynamics of some pine forests of
Afghanistan. Pakistan Journal of Botany 40(3): 1071–1079.
Warger MJA & Morrel VE (1976) Plant species and plant communities: Some conclusion. In: Proceedings of
the International Symposium, Nijmegen. The Netherlands, pp. 167–175.
Whelan RJ (1995) The Ecology of fire. Cambridge University Press.
Wilson EO (1988) The current state of biological diversity. In: Wilson EO & Peter FM (eds) Biodiversity.
National Academy Press, Washington DC, USA, pp. 3–18.

Volume 5, Issue 3 (2018) Tropical Plant Research

Încărcat de

Informații document

Drepturi de autor

Partajați acest document

Partajați sau inserați document

Opțiuni de partajare

Vi se pare util acest document?

Este necorespunzător acest conținut?

Drepturi de autor:

Volume 5, Issue 3 (2018) Tropical Plant Research

Încărcat de

Drepturi de autor:

ISSN (Online): 2349-1183; ISSN (Print): 2349-9265

TROPICAL PLANT RESEARCH 5(3): 267–274, 2018

Population structure and regeneration potential of Sal dominated

MATERIAL AND METHODS

Figure 1. Population structures of major tree species of site quality I.

Figure 2. Population structures of major tree species of site quality II.

Figure 3. Population structures of major tree species of site quality III.

Figure 4. Population structures of major tree species of site quality IV.

Fruits seasonality in selected markets at Mont-Ngafula district in

MATERIAL AND METHODS

Figure 1. Distribution of vendors according to gender in selected markets.

Figure 2. Distribution of vendors according to marital status in selected markets.

Table 2. General situation of fruits seasonality for six months.

Table 3. Commercial activity.

Figure 4. Duration of activity in selected markets.

sale (Fig. 5).

Figure 5. Storage method of fruits sold in selected markets.

Figure 6. Time of conservation of fruits sold in selected markets.

Kinshasa city especially to Mont-Ngafula township.

Table 5. Distribution of available fruits per month at Matadi-Kibala market.

Table 6. Distribution of available fruits per month at Cité-Pumbu market.

Table 7. Distribution of available fruits per month at Masanga-Mbila market.

Determination of nutrients and fiber contents of seven invasive

MATERIALS AND METHOD

RESULTS AND DISCUSSION

method. Analytical Chemistry 27(3): 440–441.

Effect of fungal endophytes of rice variety Ld 368 on growth

MATERIALS AND METHODS

Effect of fungal endophytes on rice plant growth

Eleutherine bulbosa (Mill.) Urb. (Iridaceae): A new distributional

Habitat: Rarely found in thickets of Eastern Ghats.

C3 and C4 plants as potential phytoremediation and bioenergy

Leersia hexandra Swartz. Poaceae Phytoextraction of Cr (Liu et al. 2011)

MATERIALS AND METHODS

RESULTS AND DISCUSSION

considerable accumulation of Co was recorded in C. esculentus (16.46), D. sanguinalis (15.34), E. colona

Plant, Soil and Environment 49(12): 548–553.

Songklanakarin Journal of Science and Technology 29(3): 881–892.

Habitat characterization and plant community classification of

MATERIALS AND METHODS

Figure 1. Map of the Surajpur Reserve Forest.

Figure 2. Micro-habitat map of Surajpur Reserve Forest.

Figure 3. Mapping of various habitats of Surajpur Reserve Forest.

Vegetation structure and composition

Table 4. Herb density (individual m-2) in woodland habitat.

Table 5. Grass/Sedge density (individual m-2) in woodland and grassland habitat.

Table 7. Diversity Indices for different habitats in the Surajpur wetland.

Table 10. TWINSPAN classification of plant species in wetland habitat.

Salinity would be an option to control Eichhornia crassipes

Figure 1. Habit of Eichhornia crassipes (Mart.) Solms.

MATERIALS AND METHODS

RESULTS AND DISCUSSION

Presence of green shoot (% )

Percentage of green shoot (% )

Effect of biochar on seed germination, early growth of

MATERIALS AND METHODS

Sorption behaviour of thermally and chemically

after treatment. The reaction for acetylation of wood is shown below:

Wood-OH + CH3-C (=O)-O-C(C=O)-CH3 Wood-O-C (=O)-CH3 +CH3-C (=O)-OH

RESULTS AND DISCUSSION

Antibacterial properties of Ipomoea staphylina Roem & Schult.

MATERIALS AND METHODS

Figure 2. location where experiment plant was collected.

Figure 3. Yield of crude extracts in percentage.

16 2.02 2-Furancarboxaldehyde, 5-(hydroxymethyl)- Antimicrobial, Preservative. Gopalakrishnan et al.

28 0.37 4-((1E)-3-Hydroxy-1-propenyl)-2- Antimicrobial, Antioxidant, Gopalakrishnan et al.

41 3.54 9,12,15-Octadecatrienoic acid, methyl ester, Anti-inflammatory, Srinivasan et al.