Physiological and Molecular Plant Pathology 65 (2004) 91–100

www.elsevier.com/locate/pmpp

Antimicrobial activity and induction of systemic resistance

in rice by leaf extract of Datura metel against Rhizoctonia solani
and Xanthomonas oryzae pv. oryzae
Sateesh Kagalea,*, T. Marimuthua, B. Thayumanavanb, R. Nandakumara, R. Samiyappana
a
Department of Plant Pathology, Tamil Nadu Agricultural University, Coimbatore-641 003, India
b
Department of Biochemistry, Tamil Nadu Agricultural University, Coimbatore-641 003, India
Accepted 8 November 2004

Abstract
The leaf extracts of Datura metel significantly reduced the in vitro growth of Rhizoctonia solani (RS7, Anastomosis group AG1) and
Xanthomonas oryzae pv. oryzae (Xoo). Methanol extract exhibited the best control of the pathogens recording 10–35% more toxicity than
aqueous extract. Foliar application of leaf extracts effectively reduced the incidence of sheath blight and bacterial blight diseases of rice
under greenhouse condition. Pre-inoculation applications of leaf extract were found better than post-inoculation applications. Induction of
systemic resistance as evident from the increased accumulation of pathogenesis related (PR) proteins and other defense related compounds
was observed in rice plants following application of leaf extract of D. metel and challenge inoculation with either R. solani or Xoo. The
methanol extract of D. metel when subjected to thin layer chromatography (TLC) showed a spot with relative front (Rf) value of 0.705. Well
pronounced antibacterial activity against Xoo was demonstrated by the compound eluted from the region corresponding to this spot. The
molecular ion peak and the fragmentation peaks of the eluted compound analyzed by mass spectrometry correspond to the already published
mass spectrum of daturilin (Siddiqui et al., Phytochemistry 26: 2641-2643, 1987), a withanolide compound from D. metel.
q 2005 Elsevier Ltd. All rights reserved.

Keywords: Datura metel; Rhizoctonia solani; Xanthomonas oryzae pv. oryzae; Induced systemic resistance; Daturilin

1. Introduction Rhizoctonia solani which causes rice sheath blight is both

Rice (Oryza sativa) is the staple food crop of over half of
the world’s population. Losses due pests and diseases are
one of the major constraints in rice production. Sheath blight
and bacterial blight diseases of rice cause considerable loss,
especially, in areas where high yielding varieties are grown.
Abbreviations: Xoo, Xanthomonas oryzae pv. oryzae; PR, pathogenesis
related; PDA, potato dextrose agar; ISR, induced systemic resistance; SES,
standard evaluation system; TLC, thin layer chromatography; HPLC, high
performance liquid chromatography; MS, mass spectrometry; Rf, relative
front; PAL, phenylalanine ammonia-lyase; TMV, tobacco mosaic virus;
SRV, sunhemp rosette virus.
* Corresponding author. Present address: Department of Biology, The
University of Western Ontario, Biological and Geological Sciences
building, 1151, Richmond St. N, London, Ont., Canada N6A 5B7.
Tel.: C1 519 661 2111x86473; fax: C1 519 661 3935.
E-mail address: skagale@uwo.ca (S. Kagale).
0885-5765/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.pmpp.2004.11.008
soil and water-borne and management of the disease is
difficult [57]. Moreover, R. solani has a wide host range
infecting more than 27 families in both monocots and dicots
[50]. The seed-borne Xanthomonas oryzae pv. oryzae (Xoo)
severely affects the production of rice cultivated in
Asia, Australia, Latin America, Africa, and the United
States [25,30,37,38], incurring crop loss of upto 50% [14].
The existence of different races/pathotypes of the bacterium
with high variability [37], poses serious problems in
formulating foolproof management strategies.
Several broad spectrum fungicides and bactericides have
been recommended for the control of sheath blight and
bacterial blight of rice, respectively. However, the chemical
methods of disease management are expensive and can
affect the beneficial microbial population present in the
ecosystem. The obvious pollution problems due to indis-
criminate use of synthetic pesticides and their toxic effect on
92 S. Kagale et al. / Physiological and Molecular Plant Pathology 65 (2004) 91–100

non-target organisms have prompted investigations on Table 1

exploiting pesticides of plant origin. Natural plant products Plant species used in the study
are important sources of new agrochemicals for the control Scientific name Family
of plant diseases [5,18]. Furthermore, biocides of plant Abutilon indicum Malvaceae
origin are non-phytotoxic, systemic and easily biodegrad- Annona squamosa Annonaceae
able [34,47,54]. Investigations on mechanisms of disease Argemone mexicana Papaveraceae
suppression by plant products have suggested that the active Azadirachta indica Meliaceae
Bougainvillaea spectabilis Nyctaginaceae
principles present in them may either act on the pathogen
Calotropis gigantea Asclepiadaceae
directly [1,2], or induce systemic resistance in host plants Cyperus rotundus Cyperaceae
resulting in reduction of disease development [41,43,46,51]. Datura metel Solanaceae
Induced systemic resistance (ISR) activates multiple Glyricidia maculata Papilionaceae
defense mechanisms that include increased activity of Ipomoea carnea Convolvulaceae
Ocimum sanctum Labiatae
pathogenesis related (PR) proteins like chitinase, b-1,3-
Prosopis juliflora Mimosaceae
glucanase and peroxidase [35,59], and also the accumu- Tridax procumbens Asteraceae
lation of low molecular weight substances called Vinca rosea Apocynaceae
phytoalexins [56]. Chitinases and b-1,3-glucanases are a Vitex negundo Verbanaceae
structurally and functionally diverse group of hydrolytic Zizyphus jujuba Rhamnaceae
enzymes involved in defense reactions of plants against
pathogens [23]. Peroxidase and phenylalanine ammonia- through double-layered cheesecloth, followed by centrifu-
lyase (PAL) are the key enzymes involved in phenyl- gation at 5000 rpm for 10 min. To obtain hot water extract,
propanoid metabolism [57]. fresh leaves of the selected plant species were shade dried
Evaluation of botanicals against sheath blight and for 4 days followed by oven drying at 45 8C overnight. One
bacterial blight in rice has been attempted earlier under hundred grams of dried powder was added with 100 ml of
both greenhouse and field conditions [16,28]. However, no sterile water (1:1 w/v) and then heated over water bath at
attempts have been made to understand the mechanisms of 60 8C for 10 min. The remnant was then ground using sterile
disease resistance induced by plant products, and to identify pestle and mortar and strained through cheesecloth,
the compounds responsible for their biological activity. The followed by centrifugation at 5000 rpm for 10 min. To
objectives of the present study are (1) to evaluate the obtain the solvent extracts, 100 g of finely powdered dry
antimicrobial activity of leaf extracts of various plant samples were homogenized in 100 ml of solvent (methanol,
species against R. solani and Xoo under in vitro and petroleum ether, or chloroform) (1:1 w/v). The mixture was
greenhouse conditions, (2) to study the potential for then left at room temperature (28G2 8C) for at least 3 h.
induction of systemic resistance in rice by leaf extract of Supernatants obtained by centrifugation at 5000 rpm for
Datura metel, and (3) to identify the biologically effective 10 min were evaporated completely, under reduced pressure
compound(s) against R. solani and Xoo from the D. metel using a rotavapour R-114 (Bucchi), and the residue was
leaf extract. D. metel, a member of solanaceae family is a dissolved in 100 ml of sterile distilled water to obtain
sub-glabrous shrubby herb found in all parts of the world. aqueous extract (1 g/ml). Leaf extracts were filter sterilised
using 0.22 mm filters (BD Biosciences, Bawal, India). The
supernatants thus obtained were designated as concentrated
2. Materials and methods leaf extract.
2.1. Plant materials and pathogens
2.3. In vitro assay of antimicrobial activity of leaf extracts
Rice cultivars, IR50 and T(N)-1, and the virulent isolates against R. solani and Xoo
of R. solani (RS7, Anastomosis Group AG1) [55], and Xoo
(kindly provided by Dr. P. Balasubramanian) were used in The technique of Nene and Thapliyal [42] was followed
all the experiments. All the sixteen plant species (Table 1), to study antifungal activity of leaf extracts. Ten millilitres of
tested for their antimicrobial activity were collected from cold water, hot water, methanol, petroleum ether, and
the southern parts of Tamil Nadu, India. chloroform leaf extracts (1 g/ml), of various plant species
listed in Table 1 were mixed separately with 90 ml of potato
2.2. Preparation of leaf extracts dextrose agar (PDA) medium to obtain a 1:10 dilution of
each leaf extract. Twenty ml of this mixture were poured
Aqueous extracts were obtained as described by into the sterile Petri plates and a 5 mm mycelial disc of the
Kurucheve et al. [28]. Briefly, 100 g fresh leaves of selected test fungus was placed at the centre of the plate, followed by
plant species were collected and washed with distilled water incubation at room temperature (28G2 8C). PDA without
and then sterile water. They were ground with 100 ml of leaf extract and with either sterile distilled water or
sterile water (1:1 w/v), with pestle and mortar and filtered carbendazim (0.1%) served as the control treatments.
 S. Kagale et al. / Physiological and Molecular Plant Pathology 65 (2004) 91–100
The observations on the linear growth of the fungus were
recorded after 96 h of incubation and the number of sclerotia
were counted at 164 h after incubation. Each treatment was
replicated 3 times with 5 plates per replication. Three
independent experiments were performed with similar
results.
To study antibacterial activity, a loopful of bacterium
(Xoo) was added to 90 ml Wakimoto broth (1 L of boiled
extract of 300 g potato tubers, 5 g sucrose, 0.5 g calcium
nitrate, 2.0 g peptone, pH 6.8), and incubated in a gyrotary
shaker at 150 rpm for 12 h at room temperature (28G2 8C).
Then, 10 ml of each of the sterile leaf extracts (1 g/ml), were
poured separately into 90 ml of the broth to get 1:10
dilution, followed by incubation in a gyrotary shaker at
150 rpm for an additional 12 h at room temperature (28G
2 8C). Using 1 ml of the above mixture, serial dilutions were
made to obtain 10K5, 10K6 and 10K7 suspension dilutions.
One ml of each dilution and 20 ml of Wakimoto’s semi
synthetic potato sucrose agar medium (1 L of boiled extract
of 300 g potato tubers, 5 g sucrose, 0.5 g calcium nitrate,
2.0 g peptone, 20 g agar, pH 6.8), were mixed and poured
into the sterile Petri plates. The plates were incubated at
room temperature (28G2 8C) and the number of colonies
formed were recorded after 24 h. Media amended with
either sterile distilled water or streptomycin (100 ppm)
served as control treatments. Each treatment was replicated
3 times with 5 plates per replication and the experiment was
repeated 3 times.
2.4. Greenhouse experiments
Among the various aqueous and solvent extracts tested,
methanol extracts of all the plant species were found highly
effective in suppressing the in vitro growth of R. solani and
Xoo, and hence only methanol extracts were used to assess
their effect on severity of sheath blight and bacterial blight
diseases under greenhouse conditions. Seeds of the rice
cultivar IR-50 (susceptible to sheath blight) and T(N)-1
(susceptible to bacterial blight) were sown in the earthen
pots. Fourty-five days after planting, seedlings were
inoculated with sclerotia of R. solani as previously
described [39]. For inoculation with bacterial blight
pathogen, the bacteria were suspended in water at 108
cells/ml. The leaves were then inoculated by scissors-dip
method [24]. After the inoculation, the seedlings were kept
in a moist chamber at 25 8C for 2 days before being
transferred to a greenhouse at 25–30 8C. Pre and post-
inoculation applications (two days prior to- and after-
inoculation, respectively) with methanol extracts (1:10
dilution of concentrated leaf extract) of leaves of selected
plant species, carbendazim at 0.1% or streptomycin at
100 ppm, or sterile water (control) were made at 45 days
after sowing. Fourteen days after inoculation, observations
on development of blight symptoms were made on at least
20 seedlings per treatment. The experiment was repeated
once and similar results were observed. The severity of
93
sheath blight disease was evaluated on 0–5 scale [55]. The
assessment of severity of bacterial blight disease was done
as per the standard evaluation system (SES) for rice [22].
The percent disease index (PDI) was estimated using the
formula suggested by McKinney [36]. For further studies
leaf extract of D. metel alone was chosen as it exhibited
better control of both the pathogens under in vitro and
greenhouse conditions.
2.5. Assay of induced enzymes
2.5.1. Tissue collection
The sheath (inoculated with R. solani) and leaf
(inoculated with Xoo) portions of rice plants sprayed with
leaf extracts of D. metel or water were collected at various
time intervals (0, 24, 48, 72, 96 and 164 h) after pathogen
inoculation and quickly frozen in liquid nitrogen and stored
at K70 8C.
2.5.2. Assay of chitinase, b-1,3-glucanase, peroxidase, and
phenylalanine ammonia-lyase
One gram of sample was ground using a chilled pestle
and mortar with 0.1 M sodium citrate buffer (pH 5.0) at
4 8C. The homogenate was centrifuged at 10000 rpm for
20 min. The supernatant was used as a crude enzyme extract
for assaying chitinase activity. Instead of sodium citrate
buffer, 0.05 M sodium acetate buffer (pH 5.0), 0.1 M
phosphate buffer (pH 7.0) and 0.1 M sodium borate buffer
(pH 7.0) at 4 8C were used for extraction of b-1,3-glucanase,
peroxidase, and PAL, respectively.
The changes in the chitinase and peroxidase activities
were determined by colorimetric assays described by Boller
and Mauch [4], and Hammerschmidt et al. [20], respect-
ively. b-1,3-glucanase activity was assayed by the lami-
narin-dinitrosalicylic acid method [45]. PAL activity was
determined as the rate of conversion of L-phenylalanine to
trans-cinnamic acid at 290 nm as described by Dickerson et
al. [8]. The amount of trans-cinnamic acid synthesized was
calculated using its extinction coefficient of 9630 MK1.
2.5.3. Estimation of phenolic substances
One gram of fresh sample was homogenized with 10 ml
of 80% methanol and agitated for 15 min at 70 8C [61]. One
millilitre of the methanolic extract was added to 5 ml of
distilled water and 250 ml of Folin-Ciocalteau reagent (1 N)
and the solution was kept at 25 8C. The absorbance of the
blue color was measured using a spectrophotometer at
725 nm. Catechol was used as the standard.
2.6. Purification of the bioactive compound(s) from D. metel
2.6.1. Thin layer chromatography (TLC)
TLC was carried out on 20!20 cm glass plate coated
with 0.5 mm thickness silica gel. Twenty ml of D. metel leaf
extracts (1 g/ml) were applied on each plate. The chromato-
gram was developed in a mixture of chloroform
94 S. Kagale et al. / Physiological and Molecular Plant Pathology 65 (2004) 91–100
and methanol (1:1). The developed chromatogram was
observed under visible, UV light, and after being exposed to
iodine vapours. Preparative TLC was carried out using
2 mm thickness silica gel. The spot corresponding to Rf
value 0.705 was scraped off and eluted using methanol. The
eluted compound was tested for its antimicrobial activity
using the inhibition zone technique [3]. About 15–20 ml of
the medium seeded with broth culture of R. solani and Xoo
were poured into sterilized Petri plates and allowed to
solidify. Filter paper discs of 6 mm diameter were
thoroughly moistened with the eluted compound and kept
at the centre of the plate and incubated at room temperature
(28G2 8C). The zone of inhibition (mm) was measured after
96 h (R. solani) and 48 h (Xoo).
2.6.2. High performance liquid chromatography (HPLC)
The partially purified compound from TLC was sub-
jected to HPLC analysis as described by Grayer et al. [17].
A RP-HPLC system consisting of multisolvent delivery
pump and programmable photodiode array detector was
used. The size of the m Bondapack C-18 column was 3.9
mm!30 mm I.D. The solvent gradient employed started
with 70% A (methanol) and 30% B (water) which was
changed to 0% A and 100% B at 15 min. The flow rate was
1.0 ml per min. The sample was screened at 262 nm.
2.6.3. Mass spectrometry (MS)
The electro spray mass spectra of the purified compound
were recorded on a MICROMASS QUATTRO II tripple
quadrupole mass spectrometer available at the Regional
Sophisticated Instrumentation Centre, CDRI, Lucknow,
India. The sample was dissolved in methanol and introduced
into the ESI source through a syringe pump at the rate of 5 ml
per min. The ESI capillary was set at 3.5 kV and the cone
voltage was 40 V. The spectra were collected in 6S scans and
the print outs obtained were averaged spectra of 6–8 scans.
2.7. Statistical analysis
The data on effect of the treatments on the growth of
pathogens, severity of diseases, and activity of enzymes in
rice seedlings were analyzed by analysis of variance
Table 2
Effect of water and solvent extracts of different plant species on mycelial growth and sclerotia production of R. solani
Leaf extractsa Colony diameter (mm)
Hot water Cold water
Azadirachta indica 32.0b 23.3e
Datura metel 14.0d 12.6f
Ipomoea carnea 26.3bc 35.0bcd
Vitex negundo 32.6b 29.3d
Zizyphus jujuba 9.2d 19.6e
Carbendazim (0.1%) 0.0e 0.0 g
Control (DW) 45.0a 45.0a
DW, distilled water. Means followed by a common letter within a column are not significantly different (PZ0.05) by Duncan’s Multiple Range Test.
a
All the leaf extracts were used at 1:10 dilution.
(ANOVA), and treatment means were compared by
Duncan’s Multiple Range Test (DMRT). The data on
disease severity was arcsine transformed before undergoing
statistical analysis [15]. The package used for analysis was
IRRISTAT version 92–1 developed by the International
Rice Research Institute, Biometrics unit, the Philippines.
3. Results
3.1. Effect of leaf extracts on the growth of R. solani and Xoo
Sixteen plant species (Table 1), belonging to 16 different
families were selected and evaluated for the antimicrobial
activity. The leaf extracts of all the plant species tested,
except Abutilon indicum, Argemone mexicana, Cyperus
rotundus, and Ocimum sanctum, significantly reduced the
growth of R. solani and Xoo. Data has been presented for the
most effective plant species (Tables 2 and 3). The leaf
extracts of D. metel and Zizyphus jujuba were most effective
in inhibiting the mycelial growth of R. solani. Azadirachta
indica, Ipomoea carnea, and Vitex negundo also had
strong inhibitory activity against R. solani. Methanol extract
of D. metel exhibited 10 to 35% more toxicity than aqueous
and other solvent leaf extracts. The effects of aqueous and
various solvent extracts on sclerotia production were also
tested. Methanol extract was found most effective in
restricting the sclerotia production of R. solani and hence
data has been presented for methanol extract only (Table 2).
The leaf extract of D. metel strongly inhibited the sclerotia
production of R. solani. Similarly, the aqueous and
methanol extracts of D. metel followed by Z. jujuba, and
I. carnea significantly suppressed the growth of Xoo, in
comparison to the control. Greater than 90% inhibition of
the bacterial colonies compared to the control was observed
in medium amended with leaf extract of D. metel.
Streptomycin at 100 ppm concentration completely inhib-
ited the growth of Xoo, whereas, leaf extract of Vitex
negundo showed least antibacterial activity among the
various plant species tested (Table 3).
Sclerotia production
(methanol)
Methanol Chloroform Petroleum ether
25.3b 23.0b 19.3c 64.3b
7.0c 21.0bc 15.0c 20.6c
26.3b 17.6c 23.0c 67.3b
36.3a 43.0a 37.6b 108.6a
9.2b 26.3b 18.0c 52.6b
0.0d 0.0d 0.0d 0.0c
45.0a 45.0a 45.0a 110.6a
 S. Kagale et al. / Physiological and Molecular Plant Pathology 65 (2004) 91–100 95

Table 3
Effect of water and solvent extracts of different plant species on growth of Xanthomonas oryzae pv. oryzae

Leaf extractsa Number of cfu/ml

Hot water Cold water Methanol Chloroform Petroleum ether
Azadirachta indica 3.2!106d 6.4!107c 6.6!106c 5.4!10 b 8
6.5!107c
Datura metel 2.9!105f 5.4!106d 6.2!106c 5.3!106d 8.0!105e
Ipomoea carnea 5.4!105e 2.6!106d 3.7!105d 4.6!106d 6.8!106d
Vitex negundo 6.9!108b 4.9!108b 7.5!107b 4.8!107c 4.7!108b
Zizyphus jujuba 3.7!107c 5.6!106cd 5.9!107b 5.9!107c 5.7!105e
Streptomycin (100 ppm) 1.0!100 g 1.0!100e 1.0!100e 1.0!100e 1.0!100f
Control (DW) 9.4!109a 9.4!109a 6.5!109a 6.5!109a 6.5!109a

DW, distilled water; cfu, colony forming units. Means followed by a common letter within a column are not significantly different (PZ0.05) by Duncan’s
Multiple Range Test.
a
All the leaf extracts were used at 1:10 dilution.

3.2. Effect of methanol leaf extracts on severity of sheath
blight and bacterial blight
Foliar application of methanol leaf extracts of D. metel
significantly reduced the severity of sheath blight and
bacterial blight diseases under greenhouse condition
(Table 4). There were 53 and 41% reduction in severity of
sheath blight disease over the control in rice plants sprayed
with leaf extract of D. metel in pre- and post-inoculation
spraying, respectively. More than 50% reduction in the
severity of bacterial blight was observed in rice seedlings
treated with leaf extract of D. metel (Table 4). The leaf
extracts of Z. jujuba and I. carnea were equally effective as
that of D. metel in restricting the spread of sheath blight but
not the bacterial blight. Application of leaf extracts prior to
inoculation was found better in restricting the symptom
development than post-inoculation application. For further
studies D. metel alone was chosen as it showed better
antimicrobial activity than other plant species tested in both
in vitro and in vivo conditions.
3.3. Activity of pathogenesis related (PR) proteins and other
defense related compounds
Application of leaf extract of D. metel induced resistance
in rice against R. solani and Xoo. Two- to five-fold increase
Table 4
Effect of pre and post-inoculation spraying of methanol leaf extracts on the incidence of sheath blight and bacterial blight of rice
in accumulation of PR-proteins such as chitinase, b-1,3-
glucanase, and peroxidase was recorded in rice plants
treated with leaf extracts of D. metel, and challenged with
either of the pathogens (Fig. 1). Although their activity
began to increase 24 h after challenge inoculation, the
activity increased further and remained significantly higher
from 48 to 96 h in the plants treated with leaf extract than
the control. The activities of b-1,3-glucanase and peroxi-
dase declined drastically 96 h after challenge inoculation
(Fig. 1C–F); however, such a decline was not observed in
chitinase activity as the maximum activity was noted at
164 h (Fig. 1A and B). There was no change in the activity
of PR-proteins in control plants. Similarly, plants
that were either challenged with the pathogen or sprayed
with D. metel leaf extract alone did not show an appreciable
change in PR-protein accumulation.
A significant increase in the activity of PAL and
phenolic substances was observed in rice plants following
treatment of D. metel leaf extract and inoculation with
either R. solani or Xoo (Fig. 2). PAL activity increased
24 h after challenge inoculation and reached a maximum
at 72 h in R. solani inoculated plants and declined
thereafter, but in case of Xoo inoculated plants maximum
activity was observed at 164 h after inoculation (Fig. 2A
and B). In plants treated with Xoo alone maximum
activity of PAL was observed at 48 h after challenge

Leaf extractsa Per cent disease indexb

Pre-inoculation spraying Post-inoculation spraying
Sheath blight Bacterial blight Sheath blight Bacterial blight
Azadirachta indica 50.0ab 25.0b 43.3bc 34.2a
Datura metel 33.3b 13.8c 35.0c 18.5c
Ipomoea carnea 46.6b 16.6c 41.6bc 21.2bc
Vitex negundo 55.0ab 31.4ab 60.0ab 28.7ab
Zizyphus jujuba 40.0b 24.8b 40.0c 31.4a
Carbendazim (0.1%)/Streptomycin (100 ppm) 11.6c 7.4d 18.3d 9.0d
Control (DW) 71.6a 33.0a 68.3a 38.8a

DW, distilled water. Means followed by a common letter within a column are not significantly different (PZ0.05) by Duncan’s Multiple Range Test.
a
All the leaf extracts were used at 1:10 dilution.
b
The percentage data were arcsine transformed prior to the analysis.
96 S. Kagale et al. / Physiological and Molecular Plant Pathology 65 (2004) 91–100

Fig. 1. Time course activity of chitinase, b-1,3-glucanase and peroxidase in Datura metel leaf extract treated rice plants. (A) and (B) Total chitinase activity in
rice plants with challenge inoculation of Rhizoctonia solani and Xanthomonas oryzae pv. oryzae, respectively. (C) and (D) Total b-1,3-glucanase activity in
rice plants with challenge inoculation of R. solani and Xoo, respectively. (E) and (F) Total peroxidase activity in rice plants with challenge inoculation of R.
solani and Xoo, respectively. The inoculation was done 45 days after planting and the samples were collected from both un-inoculated as well as inoculated
plants after specified times. The spraying of leaf extract was taken 2 days prior to inoculation.
 S. Kagale et al. / Physiological and Molecular Plant Pathology 65 (2004) 91–100 97

Fig. 2. Time course activity of phenylalanine ammonia-lyase (PAL) and phenolic substances in Datura metel leaf extract treated rice plants. (A) and (B) PAL
activity in rice plants with challenge inoculation of Rhizoctonia solani and Xanthomonas oryzae pv. oryzae, respectively. (C) and (D) Total phenol activity in
rice plants with challenge inoculation of R. solani and Xoo, respectively. The inoculation was done 45 days after planting and the samples were collected from
both un-inoculated as well as inoculated plants after specified times. The spraying of leaf extract was taken 2 days prior to inoculation.

inoculation and thereafter declined drastically, whereas,
in plants inoculated with R. solani alone the activity of
PAL remained at same level after 48 h of inoculation.
Phenolic substances started to accumulate 24 h after
inoculation and reached maximum at 164 h in the plants
treated with leaf extract of D. metel (Fig. 2C and D).
Untreated control, or plants treated with leaf extract
alone, or those inoculated with pathogen alone did not
show any remarkable change in the activity of PAL and
phenolic substances.
3.4. Purification of the bioactive compound
Methanol extract of D. metel leaves, when subjected
to TLC using chloroform: methanol (1:1) as solvent
yielded a spot with Rf value 0.705 under visible light
and when exposed to iodine vapours. The compound
eluted from the region corresponding to this spot showed
remarkable antibacterial activity against Xoo (Fig. 3).
However, it was not effective against R. solani. Inhibition
zone technique was followed to study the antimicrobial
activity. An inhibition zone of 10.2 mm was observed in
case of Xoo. Reverse phase HPLC analysis of the eluted
compound showed a major peak with retention time of
1.54 min at 262 nm wavelength. The compound isolated
through TLC on silica gel and showing inhibition against
the pathogens was identified with the help of mass
spectrometry. The molecular ion peak at m/e 487
(MCH)C, and fragmentation peaks at m/e 269 (M-side
chain), 227, and 171 correspond to the already published
98 S. Kagale et al. / Physiological and Molecular Plant Pathology 65 (2004) 91–100
Fig. 3. Antibacterial activity of daturilin, a withanolide compound from
Datura metel. Inhibition zone technique was followed to assay the
antibacterial activity. 1. streptomycin (100 ppm), 2. daturilin, 3. distilled
water.
mass spectrum of daturilin [53], a withanolide compound
from D. metel.
4. Discussion
In the current study, we evaluated the antimicrobial
activity of leaf extracts of some of the plant species
commonly available in South East Asia against R. solani
and Xoo. Leaf extracts of D. metel, Z. jujuba, and I. carnea
were found highly effective in inhibiting the growth of both
the organisms. Other leaf extracts which performed well in
terms of inhibiting the growth of these pathogens included
A. indica and V. negundo. Our results are in accordance with
the previous findings by Shivpuri et al. [52], who have
demonstrated the antifungal activities of D. stramonium
against R. solani. Yossry et al. [60] also reported that the
leaf extract of D. stramonium gave maximum reduction in
the growth of R. solani and four other soil borne fungi.
Similarly, aqueous extracts of leaves and fruits of A. indica
have been found to be effective against a range of
pathogenic fungi, bacteria, and viruses [11,29]. The leaf
extract of Z. jujuba was found to restrict the growth of
Pyricularia oryzae, that causes blast disease in rice [33].
Many plant species have been reported to be antibacterial
and this property can be utilized for the management of
bacterial diseases [40]. In the present study, we have shown
that the leaf extracts of D. metel followed by I. carnea, and
Z. jujuba have profound inhibitory effect on the growth of
Xoo. The efficiency of plant products in inhibiting the
growth of phytopathogenic bacteria has been examined
earlier. For instance, leaf extracts of Artabotrys hexapetalus
highly inhibited the growth of Xoo [16]. There is
considerable evidence that solvents improve the solubility
of biologically active compounds compared to water [31,44,
49]. In our study, methanol proved to be a good solvent in
extracting the inhibitory substances from the tested plant
species as it showed maximum inhibitory activity against
both the organisms.
Management of plant diseases by application of plant
products has previously been suggested [1,2]. In the present
study, leaf extract of D. metel was found to be more
effective in reducing the spread of sheath blight and
bacterial blight diseases in rice. Kumudini et al. [27] have
reported that the leaf extract of D. metel exhibited 80%
protection against the downy mildew pathogen, Sclerospora
graminicola, and induced resistance in the highly suscep-
tible HB3 cultivar of pearl millet. Moreover, in induced
resistant seedlings, appearance of hypersensitive response
was quicker than in non-induced susceptible seedlings.
Various other plant species have been tested for the control
of sheath and bacterial blight diseases. Ansari [2] suggested
the use of Trachispermum ammi and Ocimum sp. to control
sheath blight of rice without any harmful effects to the plant.
Eswaramurthy et al. [12] observed reduction in the spread of
bacterial blight disease in rice seedlings treated with leaf
extract of A. indica. Similarly, Gangopadhyay [13] reported
the suppression of symptom development in turmeric
(Curcuma longa) extract sprayed rice plants when inocu-
lated with Xoo.
In this study, induction of systemic resistance in rice
seedlings treated with leaf extract of D. metel was evident
from the increased accumulation of PR-proteins and other
defense related compounds. Changes in the PR-protein
levels have been correlated with altered disease resistance in
rice [39], barley [21], and tomato [48]. In a previous study,
Kumudini and Shetty [26] used the aqueous leaf extract of
D. metel to induce systemic resistance against S. gramini-
cola in pearl millet and showed similarity in the structural
resistance responses in ISR and host cultivar resistance of
pearl millet against the downy mildew pathogen. Plant
products have been considered as one of the major groups of
compounds that induce ISR. Application of spinach and
rhubarb leaf extracts induced systemic resistance in
cucumber to anthracnose disease caused by Colletotrichum
lagenarium [10]. Schneider and Ullrich [51] reported that
treatment with extracts of Reynoutria sachalinensis led to an
increase in the activities of chitinase, b-1,3-glucanase,
peroxidase, and polyphenol oxidase in cucumber, and
tobacco and additionally in tobacco an increase in lysozyme
activity was noticed. Narwal et al. [41] observed induction
of systemic resistance by an antiviral protein from
Bougainvillea xbuttiana in Nicotiana glutinosa and Cya-
mopsis tatragonoloba against tobacco mosaic virus (TMV)
and sunhemp rosette virus (SRV). Biologically active
compounds present in plant products act as elicitors and
induce resistance in host plants resulting in reduction of
disease development [58].
 S. Kagale et al. / Physiological and Molecular Plant Pathology 65 (2004) 91–100
Our attempts to identify biologically active compounds
from D. metel revealed the presence of a withanolide
compound, daturilin. The presence of daturilin in D. metel
has been reported, previously, by Siddiqui et al. [53], but
its antimicrobial activity was not noted. Other with-
anolides have been identified in D. metel [19,53].
Withanolides are a strong category of active compounds
of therapeutical importance with antimicrobial [6], anti-
feedant [32], and antagonistic [9] activities. They have
been found to be active against a number of potentially
pathogenic fungi [7]. In this work, we have demonstrated
the antibacterial activity of daturilin. We are currently
investigating the involvement of daturilin in induction of
systemic resistance in rice against R. solani and Xoo. In
conclusion, the results from our study suggest that the leaf
extract of D. metel has strong inhibitory effect against R.
solani and Xoo under both in vitro and in vivo conditions,
and has the potential to induce systemic resistance in rice
against these organisms. We have also demonstrated the
direct inhibitory effects of daturilin, a withanolide
compound isolated from D. metel against Xoo. The
isolation and testing of other withanolides produced by
D. metel could identify their antimicrobial activity and the
potential use in controlling fungal and bacterial diseases
in crop plants.
Acknowledgements
We thank Dr. K.P. Madhusudanan, Head, Regional
Sophisticated Instrumentation Centre, CDRI, Lucknow,
India for recording the mass spectrum.
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