Jurnal

South African Journal of Botany 100 (2015) 132–140
Phytotoxic interference of volatile organic compounds and water extracts of Tinospora

tuberculata Beumee on growth of weeds in rice fields
F. Aslani a,⁎, A.S. Juraimi a, M.S. Ahmad-Hamdani a, M.A. Alam a, F.S.Golestan Hashemi c, D. Omar
b, M.A. Hakim c
a Department of Crop Science, Faculty of Agriculture, Universiti Putra Malaysia (UPM), 43400 Serdang, Selangor, Malaysia b
Department of Plant Protection, Faculty of Agriculture, Universiti Putra Malaysia (UPM), 43400 Serdang, Selangor, Malaysia c
Institute of Tropical Agriculture, Universiti Putra Malaysia (UPM), 43400 Serdang, Selangor, Malaysia
a r t i c l e i n f o a b s t r a c t Article history: Received 4 September 2014 Received in revised form 26 February 2015 Accepted
23 April 2015 Available online 14 June 2015
Edited by L Sebastiani
Keywords: Allelopathy Aqueous water extract GC–MS HS-SPME-GC–MS Natural herbicide Volatile organic compounds
⁎ Corresponding author at: Department of Crop Science, Faculty of Agriculture, Universiti Putra Malaysia (UPM), 43400
Serdang, Selangor, Malaysia. Tel.: +60 129134508.
E-mail address: aslani.farzad.1362@gmail.com (F. Aslani).
Tinospora tuberculata Beumee is an aromatic shrub with pharmacological properties and belongs to the Menispermaceae family.
We assessed T. tuberculata for its ability to suppress growth of weeds in rice fields. In seedling bioassays, the rate of seed
germination and the radicle and hypocotyl lengths of barnyardgrass, rice and weedy rice seedlings were reduced as the
concentration of aqueous water extracts and volatile organic compounds increased compared to the control. In a glasshouse
experiment, T. tuberculata leaf powder also exhibited effective reduction in the emergence and growth of weeds in rice fields.
Applications at rates of 4 t ha−1 completely suppressed emergence of barnyardgrass, sprangletop and smallflower umbrella. The
chemical compositions of extracts and volatiles were analyzed by GC–MS and HS-SPME-GC–MS. The results showed that 7
of 43 components in the leaf aqueous extract, as compared to 5 of 15 compounds in the stem aqueous extract, are known to be
toxic compounds. Moreover, eight main volatile organic compounds were identified, which accounted for 97 and 95% of the
whole fresh and dry leaf volatiles, respectively. We observed qualitative and quantitative differences in the compounds present in
the dry and fresh leaf. Confocal laser scanning microscope images showed that the volatile emissions and aqueous extracts also
inhibited the main root diameter, and the number and elongation of root hairs of tested weeds in rice fields. These results suggest
that T. tuberculata con- tains a significant source of plant growth inhibitors. Hence, it can be used as a mulch to suppress weeds
in rice fields and for developing new herbicide based on phytotoxic compounds release by this plant.
© 2015 SAAB. Published by Elsevier B.V. All rights reserved.
1. Introduction
Rice (Oryza sativa L.) is the most widely consumed food for more than 557 million people in Southeast Asia (IRRI, 2009).
Weed infesta- tions are recognized as a serious biological constraint to rice production in both lowland and upland ecosystems
and in all seasons (Ni et al., 2009). The reduction in manual weeding because of the high labor costs has been accompanied
globally by intensive use of synthetic herbicides. However, the continuous use of herbicides has led to the evolution of
herbicide resistance in weed species (Beltran et al., 2012), increasing costs of production, environmental contamination (Mahajan
and Chauhan, 2013), unsafe agricultural products and adverse effects on human health (Khanh et al., 2005). These problems have
caused an interest among researchers to explore alternative methods for weed management with a lower environmental
impact.
Allelopathy is defined as an interaction among plants by chemical pathways. Living or dead plants with allelopathic properties
release toxic substances, collectively known as allelochemicals, through root exudation, volatilisation, leaching and
decomposition of plant materials (Rice, 1984). Volatilization from the aboveground parts of the plant is one of the major
releasing modes (Li et al., 2011). The incorporation of plant parts that release allelopathic substances as a part of a weed man-
agement program can be exploited for sustainable cropping systems (Chon and Kim, 2004).
Natural compounds offer alternative weed control options that may contribute to the reduction in the use of commercial
herbicides (Haig et al., 2009). Hence, there has been increasing interest in natural plant products in herbicide discovery programs
(Duke et al., 2002).
Malaysia has been classified as one of the top rich countries in terms of biodiversity (Eswani et al., 2010). There are 15,000
plant species, of which about 10% are known to be medicinal (Hanum et al., 2001). Nevertheless, only a few studies have been
reported regarding the identification of allelopathic plants and associated allelochemicals in this country (Ismail and Sugau,
1993; Ismail and Chong, 2009; Toosi and Bakar, 2012). The phytotoxic effects of volatile compounds released by
http://dx.doi.org/10.1016/j.sajb.2015.04.011 0254-6299/© 2015 SAAB. Published by Elsevier B.V. All rights reserved.
Contents lists available at ScienceDirect
South African Journal of Botany

journal homepage: www.elsevier.com/locate/sajb

plant issues have been scarcely examined (Barney et al., 2005; Eom et al., 2006; Zhang et al., 2012).
Tinospora tuberculata (Tinospora rumphii Boerl or Tinospora crispa), Malay name: Batawali, belongs to the family
Menispermaceae, and is a traditional medicinal plant (Forman, 1981). It is a wild plant that grows in primary rainforests in all
parts of Malaysia, Thailand and Indonesia. We speculated that T. tuberculata has enough allelopathic potential to contribute
towards weed management in rice cultivation systems.
Water soluble and volatile allelochemicals released by the aerial organs of this plant have not been known. Although, there
are a few studies that dealt with allelopathic activity of methanol extracts of T. tuberculata plant parts and identification of
phytotoxic compounds (Pukclai and Kato-Noguchi, 2012; Kato-Noguchi et al., 2014; Aslani et al., 2013; Aslani et al., 2014). To
date, no experiments have been conducted to assess the allelopathic potential of T. tuberculata for weed suppression in paddy
soils. Therefore, a combination of the laboratory and glasshouse bioassays was performed to evaluate the potential of allelo-
pathic activity of T. tuberculata mulch on the emergence and growth of natural weeds in rice fields and identify the phytotoxic
volatile components and water soluble compounds derived from T. tuberculata tissues for developing a new herbicide.
2. Materials and methods
2.1. Test materials
T. tuberculata Beumee plants used in this study were obtained from the Herbal Garden of Universiti Putra Malaysia (02°59′
N, 101°43′ E and 64 m above sea level), Selangor, Malaysia. Mature plants (excluding roots) were harvested, cleaned several
times with tap water and air- dried for 3 weeks. The plants were separated and bulked into the two major parts, leaf and stem.
Both bulked plant parts were then ground into a fine powder in a laboratory blender and sieved through a 40 mesh sieve.
Two common weeds in rice fields were tested for inhibitory activity of T. tuberculata in the seedling bioassays. Commercial
seeds of barnyardgrass (Echinochloa crusgalli (L.) Beauv.) and weedy rice (O. sativa L.) were purchased from Herbiseed
Company, London, United Kingdom. The germination rates of the seeds were between 80 and 100%.
The soil used in the glasshouse experiment was collected from Tanjung Karang rice growing area, Selangor, Malaysia, at 10
cm depth. The soil was mixed well to uniformly spread the weed seeds density in the whole paddy soil.
2.2. Extraction procedure
The phytotoxic activity of T. tuberculata Beumee on the selected weeds was investigated using aqueous (water) extracts.
Leaves and stems of T. tuberculata (each measuring 200 g dry weight) were soaked in 1000 mL of distilled water in conical
flasks. The conical flasks were then wrapped with parafilm, and shaken in an orbital shaker at room temperature (24–26 °C) for
48 h. The solution was filtered through four layers of cheese cloth to remove the debris and then centrifuged at 3000 rpm for 1 h.
The supernatant was filtered through Whatman no. 42 filter paper. To prevent microorganism growth, the solution was filtered
again through 0.2 u 15 mm syringe filters (Phenex).
Each stock extract of the stem and leaf of T. tuberculata was diluted with sterile distilled water to obtain extract
concentrations of 6.25, 12.5, 25, 50, and 100 g L−1 for seedling bioassay testing.
2.3. Laboratory bioassays
Seedling development bioassay was carried out to determine differential phytotoxicity of aerial parts of T. tuberculata
Beumee (leaf and
133 F. Aslani et al. / South African Journal of Botany 100 (2015) 132–140
stem) on germination and early growth of the tested weeds. Seeds were surface sterilized in a 1:10 (v/v) dilution of commercial
hypochlo- rite bleach for 5 min and rinsed several times with distilled water. Thirty seeds of weedy rice and barnyardgrass were
placed in two layers of filter paper in sterilized 90 × 15 mm petri dishes. 5 mL of the extracts at the different concentrations
(6.25, 12.5, 25, 50, and 100 g L−1) or distilled water as control was applied to the petri dishes.
The phytotoxic activity of volatile compounds released by dry and fresh leaves of T. tuberculata was evaluated. The volatile
bioassay was carried out based on the method of Zhang et al. (2012). Briefly, thirty seeds of barnyardgrass and weedy rice were
placed on Whatman no. 1 filter paper wetted with 5 mL distilled water in a 500 mL glassflask. Col- lected mature leaves were
hand-crushed and directly wrapped in cheesecloth at amounts of 0, 20, 40, and 60 g. Leaf water content was calculated based on
the method of Sims and Gamon (2002). Ground dry leaves at the amounts of 0, 7, 14 and 21 g which were equivalent to 0, 20, 40,
60 g fresh leaves respectively, were wrapped in cheesecloth as mentioned above. Each wrapped sample was suspended in a glass
flask, which was then sealed with parafilm.
All petri dishes and sealed flasks were placed in a growth chamber at a 12 h day/12 h night photoperiod with temperatures of
25 and 20 °C, respectively. Germination percentage, radicle and hypocotyl lengths were measured on all seedlings in each petri
dish and sealed flask at 7 days after seeding. The inhibitory effect on germination, and radicle and hypocotyl lengths was
calculated using the following equation:
I = 100 C−A ( )/C
(Kordali et al., 2009)where I is the amount of inhibition (%), C is the mean radicle or hypocotyl length of the control, and A is
the mean radicle or hypocotyl length exposed to the aqueous extract and volatile treatments. Both bioassay tests were arranged
in a completely randomized design with four replications and the experiment was repeated twice.
2.4. Root anatomy
On day 7 postplanting, confocal laser scanning microscopy (CLSM) was used to observe and monitor anatomical differences
of radicles of weedy rice and barnyardgrass treated with leaf volatiles and aqueous extracts of T. tuberculata in comparison with
the control. For analysis by confocal microscopy, one living radicle per treatment was randomly collected and washed with
distilled water and then mounted on glass microscope slides and observed using a Leica TCS SP5 II confocal microscope
(Wetzlar, Germany) equipped with a 5× (NA 0.3) objective lens at medium scan speed (400 MHz) at excitation wavelength of
405 nm and emission range of 415–485 nm.
2.5. Effects of T. tuberculata Beumee leaf powder on natural weed growth in rice field soil
The paddy soil (six kilogram per experimental unit) was added into the plastic containers (25 cm up diameter × 20 down
diameter × 23 cm length) and saturated with tap water. Leaf powder of T. tuberculata was applied at 1, 2 and 4 t ha−1 by
scattering the powder onto the soil surface in the containers. Pots receiving only tap water were used as con- trols. The plastic
containers were maintained for 60 days at ambient temperature (25–30 °C) in a glasshouse at field 16, Faculty of Agricul- ture,
Universiti Putra Malaysia, Selangor, Malaysia. The containers were watered with tap water at two day intervals. The treatments
were arranged in a randomized complete block design with four replications. All plants were harvested and plant numbers,
above ground dry weight (oven-dried at 75 °C for 48 h) and weed species were determined.

2.6. Collection of volatile compounds by headspace solid phase microextraction technique
Headspace solid phase microextraction (HS-SPME) technique was used to collect the volatile compounds and GC–MS
analysis was utilized to identify the volatile compounds in the atmosphere surrounding the T. tuberculata dry and fresh leaves.
The mature leaves were harvested, cut with scissors (1–2 cm long) and then crushed by hand. After weighing, foliar tissues (1 g)
of T. tuberculata were placed into a 40 mL clear vial and sealed with a PTFE-faced silicone septum (Supelco, Bellefonte, PA,
USA). For equilibration of headspace volatile constituents, the vial was placed in a thermo block (Supelco, Bellefonte PA,
USA) at 35 °C for 45 min. The SPME fiber (100 μm) coated with PDMS was inserted into the vial for 20 min at room
temperature to absorb the leaf headspace compounds. After extraction, the fiber was injected into the gas chromatography–mass
spectroscopy for 25 min.
2.7. Analysis of volatile and extract components by gas chromatography–mass spectrometry
The volatiles and aqueous extract compositions were determined by gas chromatography–mass spectroscopy (GC–MS).
Before injecting, the water extracts were evaporated to dryness under vacuum at 40 °C using a rotary evaporator and diluted with
80% HPLC-grade methanol. GC–MS was carried out using an Agilent 6890 + Agilent 5973 equipped with a 30 m × 250 μm ×
0.25 μm HP-5MS (5% phenyl methyl siloxane) column and a Hewlett Packard 5973 Mass Selective Detector. The operating con-
ditions were as follows:
The column was temperature-programmed from 50 °C (held for 3 min), raised at 5 °C/min to 200 °C (held for 3 min) and then
increased to 300 °C (held for 5 min) at a rate of 10 °C/min. Helium (He) at a split ratio of 1:20 and a constant flow rate of 1 mL
min−1 was used as carrier gas. The mass spectral range was recorded from m/z 40 to 600 amu. For GC–MS detection, an electron
ionization system with ionization voltage of 70 eV and an ion source temperature of 200 °C were used. The injec- tor and MS
transfer line temperatures were fixed at 230 °C and 250 °C, respectively.
The relative amounts of the different compounds were calculated automatically from the peak areas on the ion chromatograms
without any correction factor. Peaks obtained were compared to data obtained from the GC–MS analysis. The constituents were
identified on the basis of computer matching of mass spectra with those of the internal reference using the library search system
HP-5872 (Hewlett-Packard).
2.8. Statistical analysis
In the seedling bioassay and glasshouse experiments, treatments were arranged in a completely randomized design (CRD) and
randomized complete block design (RCBD) with four replicates, respectively. In order to determine any significant differences
among treatments, a one- way ANOVA was carried out using the PROC GLM procedure in SAS soft- ware (SAS 9.3, SAS
Institute Inc. Cary, NC). Separation of treatment means from the control at 0.05 probability level was conducted using the LSD
test, as outlined in the SAS procedure (version 9.3). Effective doses capable of inhibiting 50% of germination, radicle growth and
hypocotyl growth were calculated as EC
g
50, EC
r
50 and EC
h
50, respectively. EC
g
50, EC
r
50 and EC
h
50 values were calculated by Probit analysis based on percentage of inhibition. Ranking for each of the most
active extracts and volatiles was determined as an index (R
e
) using the following equation for each plant tested:
R
e
= EC
g
50n ( germination ) + EC
r
50n ( radicle ) + EC
h
50n ( hypocotyl )
where R
e
is the rank of the weeds (barnyardgrass and weedy rice) or tested indices (germination, radicle and hypocotyl lengths)
or treatments (dry leaf and fresh leaf volatiles, leaf and stem aqueous extracts).
50n The EC
g
are the concentrations of extract or volatile of T. tuberculata that inhibits 50% of germination,
radicle and hypocotyl growth, respectively. The tissue extract, volatile, tested indices and tested weed species with the lowest R
e
values are the most phytotoxic treatments and the most sensitive plant. The graphs were
drawn with
Fig. 1. Inhibition percentage of initial growth of weedy rice (a), barnyardgrass (b) and rice (c) at 7 days after treatment in a dose
response to leaf and stem water extracts.
50n, EC
r
50n and EC
h

SigmaPlot (version 12.5). The GC–MS analysis, laboratory and glasshouse experiments were repeated twice and mean values
were pooled.
3. Results and discussion
3.1. Inhibitory effects of T. tuberculata Beumee volatiles and aqueous extracts on test weed species
The potential allelopathic contribution of aqueous water extracts of the aerial parts of T. tuberculata at different concentrations
was assessed based on the germination and initial growth of barnyardgrass (E. crusgalli (L.) Beauv.), rice (O. sativa L.) and
weedy rice (O. sativa L.) to investigate its extensibility and possibility for use as natural weed inhibitors. The results revealed
that the application of both T. tuberculata leaf and stem aqueous extracts significantly reduced percentage of germination, as
well as radicle and hypocotyl lengths of the tested plants. The reductions increased with the increase in extract concentration
when compared to the control (Fig. 1).
The sprouting of barnyardgrass was significantly reduced (P b 0.05) at all tested concentrations of leaf aqueous extracts.
Conversely, only the highest concentration resulted in the 90% reduction of germination in weedy rice. Meanwhile, no significant
reduction was observed in germination when the T. tuberculata stem extract was applied at low concentrations of 6.25, 12.5
and 25 g L−1. On the contrary, when the concentration increased to 100 g L−1, the germination was reduced to a range of 8–20%
in the indicator weed species.
Considerable allelopathic effects on radicle and hypocotyl growth of tested weed species were exerted by water extracts at
various concentrations. The radicle elongation of species was significantly reduced (P b 0.05) by both aqueous extracts at
concentrations equal or higher than 6.25 g L−1. The highest concentration (100 g L−1) of the leaf extract resulted in no radicle
development for barnyardgrass and weedy rice. On the other hand the 100 g L−1 of stem extract resulted in radicle reductions of
64, 43 and 72% in weedy rice, rice and barnyardgrass, respectively (Fig. 1).
The present study was designed to investigate whether the volatile compounds obtained from T. tuberculata dry and fresh
leaves are capable of inhibiting the growth of the neighboring plant. Inhibition of barnyardgrass, rice and weedy rice initial
growth was evaluated with 7, 15 and 21 g dry foliage and 20, 40 and 60 g fresh leaves of T. tuberculata. Dry and fresh foliar
tissue exhibited adverse effects on the initial development of seedlings. Increasing inhibition was observed with increasing rates
of dry and fresh foliage placed in the enclosed environment (Fig. 2).
All the applied concentrations of the dry leaf had no significant effects on the germination of the target weeds. In contrast,
fresh leaves at 40 and 60 g caused significant reductions in germination of barnyardgrass by 30% and 32%, respectively.
However, the fresh leaves did not significantly reduce the germination of rice and weedy rice.
Significant inhibitory effects on radicle and hypocotyl length (P b 0.05) in both weed species were observed in the presence of
dry and fresh leaves of T. tuberculata as compared to the control (Fig. 2).
The radicle elongation varied considerably corresponding to the different dry and fresh leaf concentrations (Fig. 2). All
doses diminished radicle growth of barnyardgrass, rice and weedy rice. Fresh leaf caused reductions of 23–67%, 53–83% and
54–97% in the radicle growth of rice, weedy rice and barnyardgrass, respectively. However, dry leaf volatiles inhibited radicle
growth of target species by an average of 61% at the highest concentration.
Hypocotyl growth inhibition increased by 9, 21 and 28% in rice, 2, 8 and 43% in weedy rice, and by 11, 45 and 44% in
barnyardgrass with 20, 40 and 60 g fresh leaf, whereas it was 7, 10 and 20% in rice, 28, 29 and 3% in weedy rice and 13, 23 and
36% in barnyardgrass when the seedlings were exposed to the dry leaf at concentrations of 7, 14 and 21 g dry leaf, respectively.
Fig. 2.Inhibition percentage of initial growth of weedy rice (a), barnyardgrass (b) and rice (c) at 7 days after treatment in a dose
response to dry and fresh leaf volatiles.

The results indicate the phytotoxic importance of secondary metab- olites produced by T. tuberculata aerial parts on seed
germination, radicle growth and hypocotyl growth of barnyardgrass, rice and weedy rice. The emitter density showed a positive
correlation with the growth inhibition percentage. It could be a reflection of plant growth inhibitor concentrations being released
by plant tissues. Higher concentrations of the aqueous extracts would contain greater amounts of inhibitory substances, and thus
had a higher degree of inhibition. Similarly, Han et al. (2008) and Maharjan et al. (2007) reported that the degree of the
phytotoxicity of ginger (Zingiber officinale) and Parthenium hysterophorus extracts was dependent on the concentration and that
phytotoxicity increased with incremental extract concentration. It was also reported that with increasing concentrations of the
Croftonweed (Ageratina adenophora) foliage, a higher herbicide impact of volatiles was observed in rice seedling roots (Zhang et
al., 2012). The role of volatiles in growth reduction of neighboring plants has been evaluated by several researchers (Barney et
al., 2005; Eom et al., 2006).
The 50% inhibitory concentrations (EC50) of each tested indices for both species are shown in Table 1. The comparative
analysis of extracts derived from the aerial parts of T. tuberculata showed the plant parts- dependent effects on seed germination
and initial growth of tested weeds (Table 1). The effectiveness of the stem extract was lower than the leaf extract, as the rank
value of stem extract (10440) was much higher than the leaf rank value (841) in both weed species. In addition, EC50 and rank
values of target weeds treated by dry and fresh leaves showed that the fresh leave had a high allelopathic effect in comparison to
that of the dry leave for all tested indices and plants (Table 1).
Therefore, the magnitude of the phytotoxicity effects at the same concentration levels in the test plants was dependent on the
source of the extract (either leaf or stem) and the volatile fraction contained in the dry and fresh leaves. Khanh et al. (2005)
similarly reported that the leaves of five medicinal and leguminous plants possessed the greatest herbicidal impact on the control
of weeds in rice fields, follow- ed by roots and stems. Zhang et al. (2012) revealed that volatile allelopathic interference of A.
adenophora had significant adverse effects on the seedling growth of upland rice. However, there is no study on the comparison
of the allelopathic behavior between dry and fresh leaf.
Analysis of the EC
50
results showed that rice was only affected at higher concentrations of both extracts (630 and 1563 in leaf
and stem extracts, respectively) and volatiles (1143 and 1207 in fresh and dry leaf, respectively) as compared to barnyardgrass
and weedy rice. There- fore, it was apparent that different concentrations were needed to suppress different species of plants.
There are several reasons for the different sensitivity of various plant species to inhibitory compounds. It could be due to the
physiological and biochemical characteristics of
each species (Kobayashi, 2004), the seed structure (Hodgson and Mackey, 1986), seed coat penetrability (Hanley and Whiting,
2005) and seed size (Machado, 2007).
3.2. Effects of T. tuberculata Beumee leaf powder on natural weed growth in rice field soil
Bioassays showed the allelopathic activity of T. tuberculata against germination and growth of two common weeds in rice
fields under laboratory condition, but the presence of allelochemicals in the plant tissues may not imply a significant toxic
effect in the soil (de Bertoldi et al., 2012). Therefore, a soil study performed to determine the weed suppressive ability of T.
tuberculata leaves powder on emergence and growth of natural weeds in rice field soil.
Incorporation of T. tuberculata leaf powder to paddy soil has resulted in effective suppression of natural weed growth in rice
field soil (Fig. 3). In the untreated pots, the emergence of five major weeds in rice fields was observed, including barnyardgrass
(E. crusgalli (L.) Beauv.), smallflower umbrella (Cyperus difformis L.), weedy rice (O. sativa L.), sprangletop (Leptochloa
chinensis (L.) Nees) and flatsedge (Cyperus iria L.). At the lowest application rate (1 t ha−1), the reduction in the total number of
weed was significant. However, the number of O. sativa increased. The growth of L. chinensis was completely controlled (Fig.
3). Furthermore, the total weed biomass was greatly decreased. The number of O. sativa grown on 2 t ha−1 leaf powder displayed
a significant reduction compared to the control. In addition, complete inhibition of E. crusgalli occurred at 2 t ha−1. At the
highest dose (4 t ha−1), a significant reduction in number of plant and total biomass of all weed species was observed. O. sativa
and C. iria plants were reduced to only 3 and 2 plants per pot, respectively. While, a complete control of the other three weed
species was recorded at this rate.
The results confirm that the natural allelopathic substances present in the incorporated leaf powders had significant effects on
plants. The extent of growth inhibition was proportional to the applied dose and was species dependent. O. sativa and C. iria
appeared to be the most re- sistant species to control. Volatile and water soluble compounds released by leaf powder applied as
mulch affected emergence and growth of weeds in rice fields. Volatile organic compounds can perme- ate into the soil easily.
Therefore, these may play a noticeable role in the suppression of natural weed growth. The mulch of T. tuberculata can reduce
weed growth by release of allelochemical compounds. This study showed a new application of T. tuberculata in addition to its
use as a medicinal plant. Other researchers have also introduced medicinal plants as natural rice field herbicides (Fujii et al.,
2003; Khanh et al., 2005; Nazir et al., 2007).
Table 1 Allelopathic activity of leaf water extract and volatiles of fresh leaf compared to the stem water extract and dry leaf,
respectively.
Target plants
Leaf water extract Rank Stem water extract Rank
EC
g50
EC
r50
EC
h50
EC
g50
EC
r50
EC
h50
Values in g L−1 Barnyard 40.5 9.4 36.0 85.9 8049.3 52.1 144 8245.4 Rice 486.0 51.9 92.2 630.1 1286 105.4 171.9 1563.3
Weedy 70.5 21.3 33.5 125.3 347.6 73.84 210.8 632.24 Rank 597 82.6 161.7 841.3 9682.9 231.3 526.7 10440.9
Target plants
Fresh leaf Rank Dry leaf Rank
EC
g50
EC
r50
EC
h50
EC
g50
EC
r50
EC
h50
Values in g Barnyard 86.9 18.9 58.7 164.5 124.5 22.6 108.2 255.3 Rice 993.7 46.1 103.9 1143.7 993.7 61.7 152.1 1207.5 Weedy
193.3 19.40 70.3 283 123.8 27.4 130.1 281.3 Rank 1273.9 84.4 232.9 1591.2 1242 111.7 390.4 1744.1
Fig. 3. Effects of T. Tuberculata leaf powder on natural weed growth in paddy soil.

3.3. Anatomical effects of treatments on the radicle development of weeds
The epidermis and ephemeral roots have received great attention because they perform essential functions in the plant. They
make close
Fig. 5.Confocal laser scanning microscope images of barnyardgrass radicles treated by a) control, b) fresh leaf volatile (the dose
of 40 g), c) dry leaf volatile (the dose of 14 g), d) leaf extract (the dose of 25 g L−1), and e) stem extract (the dose of 25 g L−1).
Fig. 4. Confocal laser scanning microscope images of weedy rice radicles treated by a) control, b) fresh leaf volatile (the dose of
40 g), c) dry leaf volatile (the dose of 14 g), d) leaf extract (the dose of 25 g L−1), and e) stem extract (the dose of 25 g L−1).
contact with the soil and provide minerals and water to plants (Kramer and Boyer, 1995).
The radicles of weedy rice (Fig. 4) and barnyardgrass (Fig. 5) grown with the fresh and dry leaves placed in the enclosed
environment and

that aqueous extracts exhibited remarkable morphological anomalies when
water soluble leaf exudates of Dittrichia viscose
suppressed root compared to the control. The effects became more and more intense
length, lateral root development, cell division and the
formation of when they were treated with fresh leaf volatiles and leaf water extract.
root hairs in lettuce. Allelochemical stress reduced the
main root diameter and the number and lengths of the lateral roots in both the target species. Reduced
3.4. Identified substances in aqueous extracts and
volatiles root hair density, diameter and length of the roots may have adverse effects on the seedling's ability to compete and
their attempts of establish-
The observed allelopathic effects of the T.
tuberculata Beumee sug- ment especially under limited water and nutrition resources (Levizou
gested that the stem and leaf contain phytotoxic
compounds. Identifica- et al., 2002).
tion of the inhibitor components is important to explore
new herbicide These results are in agreement with the findings reported by Zhang
based on chemical compounds that can be extracted
from T. tuberculata et al. (2012), who observed that the volatiles of A. adenophora fresh fo-
tissues. Moreover, it can be useful to determine the
phytotoxic interfer- liage showed an inhibitory effect on the number and length of root hairs
ence mechanism on receiver plants. GC–MS analyses
of the different of upland rice seedlings. Moreover, Levizou et al. (2002) also observed
plant parts (stem and leaf) of T. tuberculata revealed the presence of
Table 2 Relative percentage of components identified in the water extract of leaf and stem of T. tuberculata.
Retention time Relative percentage Compound Literature Plant part
4.69 43.69 2,3-Butanediol Ji et al. (2011), Reynolds (1977), Streiblová et al. (2012) Leave 4.69 29.81 2,3-Butanediol Ji et al.
(2011), Reynolds (1977), Streiblová et al. (2012) Stem 5.73 0.89 Silane, ethoxytriethyl- Leave 8.05 0.99 Butyrolactone Leave
8.10 0.38 2(5H)-furanone Leave 8.17 0.52 Butanoic acid, 4-hydroxy- Stem 8.42 0.56 2(3H)-furanone, 5-methyl- Leave 8.53 0.81
2(3H)-furanone, 5-methyl- Stem 10.39 1.30 Phenol Blum et al. (1999) Leave 12.09 0.45 Oxazole, 2,4-dimethyl- Leave 13.03
2.30 2-Pyrrolidinone Seal et al. (2009), Sun and Tang (2013) Leave 13.43 1.29 Furancarboxylic acid, methyl ester Gu et al.
(2007) Leave 13.48 0.51 Furancarboxylic acid, methyl ester Gu et al. (2007) Stem 13.78 0.40 Hexane, 1,6-dibromo- Leave 14.96
0.42 2,5-Pyrrolidinedione Leave 15.63 2.11 Thiazole Yang et al. (2014) Leave 15.62 1.01 Thiazole Yang et al. (2014) Stem
16.15 0.55 1,2-Benzenediol, 3-methoxy- Leave 16.37 0.55 Oxime-, methoxy-phenyl- Leave 16.37 1.86 Oxime-,
methoxy-phenyl- Stem 16.82 0.57 Oxirane, butyl- Leave 17.04 4.80 Indole Seal et al. (2004) Stem 17.05 5.07 Indole Seal et al.
(2004) Leave 17.29 1.39 1,3-Benzenedicarboxylic acid Leave 17.29 1.57 1,3-Benzenedicarboxylic acid Stem 18.66 0.40
Flamenol Leave 22.83 0.67 2-Methoxy-4-vinylphenol Leave 24.41 0.37 Phenol, 2,6-dimethoxy- Leave 24.57 0.49 1-Heptene,
4-methyl- Stem 25.12 0.41 1,3-Diazine Leave 25.34 1.81 4-Methyleneproline Leave 25.94 9.31 3-Pentenoic acid, 4-methyl- Stem
27.77 0.63 Silane, dimethylphenyl- Leave 27.88 1.24 Silane, dimethylphenyl- Stem 30.70 2.27 4-Hexenoic acid Leave 33.50 0.54
Guaifenesin Stem 33.52 1.69 Guaifenesin Leave 35.69 0.68 1H-indole-3-ethanol Leave 35.93 0.51 Pyrazine,
2-butyl-3,5-dimethyl- Stem 36.33 0.57 2,4-Heptadiene, 2,6-dimethyl- Leave 36.44 4.84 Guaifenesin Leave 39.18 0.81
2-Pyrrolidinone, 1-(4-methylphe...) Leave 42.29 0.79 5-Amino-1-methyl-3-phenylpyrazole Leave 44.90 4.16
3,4-Pyridinediamine Leave 46.51 0.58 Benzenemethanol, 3-amino- Leave 47.95 0.61 4-(Bromomethyl)cyclohexene Leave 50.02
0.53 1-Dodecyne Yang and Lee (2012) Leave 50.46 0.45 9-Oxabicyclo[4.2.1]non-7-en-3-ol Leave 51.19 1.12 1-Dodecyne Leave
52.36 0.79 5-Butyl-2-methylpyrroline Leave 52.69 1.12 Benzene, 1-fluoro-4-methyl- Leave 52.99 0.42 Norbornane, 2-isobutyl-
Leave 53.23 2.00 3-Bromocamphor Leave 55.54 3.98 Ledol Kamatou et al. (2008), Sahaf et al. (2008) Leave 56.48 0.80
Di-t-butylacetylene Leave 56.68 3.70 1-Nonyne Leave 56.92 0.85 Eicosane Liang et al. (2012), Jia et al. (2011), Zhang et al.
(2010) Stem 59.75 0.83 Benzonitrile, 2-methoxy-6-methyl- Stem

58 compounds that appeared between 4 and 59 min (Table 2). The components of each plant-part extract demonstrated that the
composition of components differed in the stem and leaf water extracts of T. tuberculata. The leaf water extract contained 43
compounds, while the stem water extract had 15 compounds. At the concentration of 50 g L−1, the number of known toxic
constituents in the leaf extract was 7 compounds (60.27% of the total), while stem extract contained only 5 known toxicity
compounds (36.98% of the total).
To date, solid-phase microextraction (SPME) has been used exten- sively for sample volatile emissions emitted by plants
(Musteata and Vuckovic, 2012). Volatile emissions of dry and fresh leaves of T. tuberculata were analyzed to better understand
plant chemotaxo- nomic studies and for the identification of allelopathic volatile components emitted by the leaf.
The analyses of dry and fresh leaves enabled identification of 8 volatile substances that appeared between 4 and 40 min
(Table 3). Six main volatile components of the fresh leaf amounting to 97.6% of the total leaf volatiles, were identified as
ethanolamine (92.84%), Ala-Gly (2.38%) and thymol (1.51%), diphenyl ether (0.54), (−)-carvone (0.28) and 3-carene (0.05). The
five principal constituents of the dry leaf were hydrazinecarboxamide (92.74%),
D
-limonene (0.23%), 3-carene (0.18%), thymol (1.27%), and diphenyl ether (0.66%).
There were quantitative and qualitative variations in the volatile substances identified from the fresh and dry leaf. It may be
suggested that volatiles of fresh leaf (ethanolamine, Ala-Gly and (−)-carvone) were vapourised or converted to other substances
during the drying process. de Gouw et al. (1999) noted that enzymatic processes induced by the wounding of plant tissue are also
involved in the volatile compounds release from the drying vegetation. Other workers have also reported that some chemical
transformations occur during the drying process (Asekun et al., 2007). Díaz-Maroto et al. (2004) believed that the cell damage
during drying could be a reason for the change in the volatile organic compound emissions.
Other researchers have observed alterations in the volatile composition of Ocimum basilicum (Díaz-Maroto et al., 2003),
Mentha longifolia (Asekun et al., 2007), Laurus nobilis (Díaz-Maroto et al., 2002) and Mentha spicata (Díaz-Maroto et al., 2003)
after drying under different methods.
Variations in the identified substances could explain the stronger inhibitory effect of the leaf extract and the fresh leaf
compared to the stem and dry leaf. These differences lead to differential inhibitory and biological effects on the growth of
weeds in rice fields. Verdeguer et al. (2009) also suggested that the suppressive effect of the extracts is dependent on the
chemical composition and the plants on which they are applied. The allelopathic activity of each sample was found to be
proportional to the number and type of compounds present.
The GC–MS and HS-SPME-GC–MS analyses showed that water extracts of T. tuberculata leaves produced different results
in comparison with the volatiles. This study is the first to demonstrate the chemical composition of the aqueous water extract and
leaf volatile profile of T. tuberculata.
Table 3 Relative percentage of main volatile components identified in the dry and fresh leaf of T. tuberculata.
Retention time Relative percentage Compound Literature Volatile emissions
1.25 92.84 Ethanolamine Elmore (1980), Walsh et al. (2014) Fresh leaf 1.25 92.74 Hydrazinecarboxamide Feng (2003) Dry leaf
3.47 2.38 Ala-Gly Dadon et al. (2004), Hao et al. (2010) Fresh leaf 8.6 0.05 3-Carene Dudai et al. (2004), Zahed et al. (2010),
Singh et al. (1999) Fresh leaf 8.6 0.18 3-Carene Dudai et al. (2004), Zahed et al. (2010), Singh et al. (1999) Dry leaf 9.15 0.23
D
-Limonene Barney et al. (2005) Dry leaf 15.64 0.28 (−)-Carvone Azirak and
Karaman (2008) Fresh leaf 17.17 1.51 Thymol Szczepanik et al. (2012), Azirak and Karaman (2008) Fresh leaf 17.17 1.27
Thymol Szczepanik et al. (2012), Azirak and Karaman (2008) Dry leaf 28.51 0.54 Diphenyl ether Towers and Arnason (1988)
Fresh leaf 28.51 0.66 Diphenyl ether Towers and Arnason (1988) Dry leaf
T. tuberculatais capable of producing and releasing a large number of water soluble and volatile toxic compounds into the soil
and thereby may also affect the growth of other plants in the vicinity. The incorporation of dry leaf could increase weed
biomass reduction and may de- crease the use of synthetic herbicides. Hence, this offers a promising source of eco-friendly
natural herbicide for sustainable agriculture. However, additional studies are required to: a) validate the present results on T.
tuberculata under actual paddy field conditions, in order to provide applicable recommendations to farmers and using the
allelochemicals produced by T. tuberculata as structural leads for developing future new herbicides. Moreover, experiments are
also needed to establish the appropriate growth stage and agronomic and environmental conditions under which this plant will
produce maximum quan- tities of allelochemicals.
Acknowledgement
Many thanks go to Dr. Samira Bagheri for her assistance in conducting the study. The authors would also like to thank
Long-term Research Grant Scheme (LRGS), Food Security Project, Ministry of Higher Education, Malaysia (5525001) and
Fundamental Research Grant Scheme (07-01-13-1241FR) for providing financial support.
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South African Journal of Botany 100 (2015) 132–140

Phytotoxic interference of volatile organic compounds and water extracts of Tinospora

South African Journal of Botany

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