ABSTRACT

Induction of Systemic Resistance against bacterial blight of cotton

caused by Xanthomonas axonopodis pv. malvacearum
(E.F. Smith) Vauterin et al.
Gayathri Subbiah* and R. Bhaskaran**

*Assistant Professor, Department of Biotechnology, St. Peters Engineering

College, Avadi, Chennai.

** Professor, Department of Plant Pathology, Tamil Nadu Agricultural University,

Coimbatore.

Bacterial blight caused by Xanthomonas axonopodis pv. malvacearum is the

serious problem in cotton cultivation in India and elsewhere. Resistance can be
systemically induced in plants lacking the gene for resistance by inoculation with nonpathogens, by restricted inoculation with pathogens, or by treatment with chemicals.
A study was undertaken to evolve a management practice for bacterial blight of
cotton caused by Xanthomonas axonopodis pv. malvacearum by induced systemic
resistance using biotic (Phylloplane micro organisms and bacterial endophytes) and
abiotic elicitors. Among the phylloplane micro organisms, Trichoderma viride (plf 7) and
Pseudomonas sp. plb 7 were effective in inhibiting the growth of the pathogen in vitro.
Endophytes were isolated from root, stem and leaf of two to six week old cotton
seedlings. Endo R3, Endo S4 and Endo L4 was effective under in vitro conditions.

Among the abiotic elicitors (salicylic acid, benzoic acid, dipotassium phosphate,
napthalene acetic acid, glycine, hydrogen peroxide, oxalic acid, copper sulphate and
indole acetic acid), tested against the bacterial blight disease, salicylic acid 250 ppm
controlled the disease by producing the least number of lesions per leaf compared to other
chemicals.
Pot culture and field experiments revealed that the per cent disease intensity was
reduced in salicylic acid sprayed and Pseudomonas sp. plb 7 sprayed plants. This
treatment also produced increase in the biochemical constituents (total sugars, reducing
sugars, non-reducing sugars, protein, phenol, activities of phenylalanine ammonia lyase,
polyphenol oxidase, peroxidase and catalase) in treated plants.

INTRODUCTION
Cotton is one of the most important commercial crop of the world. It constitutes nearly 70 per cent
of the raw material for the textile industry. In India, it is cultivated in an area of 77.85 lakh ha with a
production of 177.00 lakh bales in the year 2003-2004. Tamil Nadu has an area of 1.03 lakh ha with a
production of 3.50 lakh bales in the year 2003-2004. Diseases are one of the chief constraints in cotton
production. Among the several fungal, bacterial and viral diseases occurring in cotton, bacterial blight
caused by Xanthomonas axonopodis pv. malvacearum (Smith) Vauterin et al. is a major disease causing
considerable yield losses both in rainfed and summer irrigated cotton. In India, the annual loss ranges from
5 to 25 per cent.
Management of the disease using chemicals like copper oxychloride and antibiotics like
streptomycin sulphate and agrimycin is possible but apart from hazards of pollution, the chances of
selection of resistant mutants of the pathogen is high. Biological control of the plant diseases employing
phylloplane and rhizosphere microflora and more recently by indigenous endophytic bacterial flora is
gaining momentum especially with increasing awareness on the need for use of ecofriendly management
practices. The plants have evolved several lines of defense besides pre-existing physical and chemical
barriers (Osbourn, 1996). Inducible resistance mechanisms can be activated upon infection by pathogen by
means of chemical sprays, avirulent pathogen and antagonistic organisms.
Endophytes colonize systemically in plants, which is well documented by Patriquin et al. (1983),
Sharrock et al. (1991) and McInroy and Kloepper (1995a and 1995b). Several studies have shown that the
interaction between plants and some endophytic bacteria was associated with beneficial effects such as
plant growth promotion and biocontrol potential against plant pathogens. Moreover, their unique ability to
survive in plants with no or little microbial competition makes them potential candidates for biological
control (Misaghi and Donndelinger, 1990).
A new technology for plant disease control is based on the activation of the plants own defense
system with the aid of low molecular weight synthetic molecules that induce systemic acquired resistance
in plants against a wide range of microbial pathogens. The systemic acquired resistance (SAR) is an
important component of plants defense against diseases where initial infection provides systemic
resistance to subsequent infection by a variety of bacterial, fungal and viral pathogens (Gaffney et al.,
1993). Induced resistance depends on recognition of a pathogen or stress by the plant. This generates a
cascade of events, eventually leading to the expression of defense mechanisms, which include physical
barriers, metabolites and proteins that interfere with spread of the invading microorganism. In recent years,

a large number of chemicals, (viz., salicylic acid, actigard, phosphates, calcium, hydrogen peroxide,
jasmonic

acid,

methyl

jasmonate,

ethylene,

acetyl

salicylic

acid,

benzothiadiazole,

2,

6-

dichloroisonicotinic acid, etc.) which mimic the function of the pathogen in induction of systemic acquired
resistance have been reported (Ryals et al., 1996). Induced resistance is expressed locally at the site of
infection or systemically at sites remotely located from the initial infection. The present study for
induction of systemic resistance against bacterial blight of cotton caused by Xanthomonas axonopodis pv.
malvacearum was carried out with phylloplane microorganisms, endophytic bacteria and chemicals.

MATERIALS AND METHODS

Collection of isolates of Xanthomonas axonopodis pv. malvacearum
Cotton leaves of the cultivars LRA 5166, SVPR 2 and four local varieties showing the typical
symptoms of bacterial blight (angular leaf spot and vein blight) were collected from different conventional
cotton growing areas of Tamil Nadu viz., Checkanuarani (Madurai district), Coimbatore, Bodi, Madurai,
Rajapalayam, Srivilliputhur and Theni during 2002-2003.
From the infected portions of the leaf showing the typical symptoms the bacterial pathogen (viz., I1,
I2, I3, I4, I5, I6, and I7) were obtained and subcultured.
In all the experiments, cotton seeds of the variety SVPR 2 (obtained from the Cotton Research
Station, Srivilliputhur) susceptible to bacterial blight were used.
Pathogenicity test was performed according to the methods described by Mahmood and Hussain,
1993.
Nutrient agar (Allen, 1953), Kings B (King et al., 1954), Potato Dextrose Agar (Ainsworth, 1961),
and Trichoderma selective medium (Elad and Chet, 1983) were used for subculturing pathogen and fungal
and bacterial antagonists.
The per cent disease index (PDI) was calculated using the formula proposed by Mc Kinney (1923).
Sum of all numerical ratings

PDI = Total number of leaves graded x maximum grade 100

Based on the symptoms produced, the most virulent isolate was selected and used throughout the
study.Management of the disease.
Use of phylloplane organisms
Isolation of phylloplane organisms
From the healthy cotton leaves (1.0 g) phylloplane microbes were isolated and identified based on
biochemical reactions (Saha et al., 2001).
Efficacy of phylloplane microflora against Xanthomonas axonopodis pv. malvacearum
The phylloplane isolates were tested against the pathogen by paper disc assay method. The isolates
were grown in their respective culture broth (bacteria for 48 h and fungi for 5 days). The sterilized paper
discs of 5 mm diameter were placed on the nutrient agar medium seeded with X. axonopodis pv.
malvacearum by means of sterilized forceps. The culture broths were passed through the bacterial filter
before wetting the paper discs to avoid bacterial contaminations. A quantity of 20 l of culture filtrate was
added to the paper disc and the plates were kept for incubation at room temperature. The inhibition zone

was measured after 48 h. The filter paper disc treated with sterile distilled water served as control and
streptomycin sulphate 100 ppm and culture filtrate of Pseudomonas fluorescens (Pf 1) served as standard
checks.
Use of endophytes
Isolation of bacterial endophytes
Isolation of endophytic bacteria was made from root, stem and leaves of healthy cotton seedlings of
SVPR 2 variety following those given by Misaghi and Donndelinger, 1990.
Efficacy of bacterial endophytes against Xanthomonas axonopodis pv. malvacearum
The endophytic bacteria were tested against the pathogen by paper disc assay method as described
earlier.
Screening of abiotic elicitors under glass house conditions against bacterial blight of cotton
The abiotic elicitors viz., dipotassium phosphate, salicylic acid, indole acetic acid, glycine,
hydrogen peroxide, napthalene acetic acid, oxalic acid, copper sulphate and benzoic acid were selected for
this study. In order to find out the phytotoxic effects, if any, the plants were sprayed with the abiotic
elicitors at different concentrations ranging from 50 ppm to 250 ppm and the symptoms of toxicity if any
were recorded after a week. To assess their potential for the management of bacterial blight in cotton, the
selected chemicals were sprayed at 3-4-leaf stage and after 24 h, the plants were inoculated with the
bacterial blight pathogen. The symptom expression was observed from 15 days after inoculation and
number of lesions formed were assessed. The chemical that produced less number of lesions/leaf was
taken for further study.
Preparation of formulation of antagonistic organisms for pot culture and field studies
Talc based formulation of Pseudomonas, (Vidhyasekaran and Muthamilan, 1995), peat based
formulation of Bacillus (Raguchander et al., 1998) and talc based formulation of Trichoderma were
prepared.
Effect of biocontrol agents and abiotic elicitors on bacterial blight of cotton in pot culture
The effective phylloplane microorganisms, endophytic bacteria and abiotic elicitor were taken for
pot culture experiments. The pots of 30-cm diameter were filled with three kg of black soil and fifteen
cotton seeds were sown. Seed treatment were done with rifampicin mutant endophytic bacteria 24 h before
sowing. When the plants were 30 days old, the following spray treatments were given and 24 h later
pathogen inoculation was done.

T1
- Spraying of phylloplane Bacillus sp.
T2
- Spraying of phylloplane Pseudomonas sp. plb 7
T3
- Spraying of phylloplane Trichoderma viride
T4
- Seed treatment with root endophytic bacterium (Endo R3)
T5
- Seed treatment with stem endophytic bacterium (Endo S4)
T6
- Seed treatment with leaf endophytic bacterium (Endo L4)
T7
- Salicylic acid spray
T8
- Spraying of Pseudomonas fluorescens (Pf 1)
T9
- Streptomycin sulphate (0.1%) spray
T10
- Control (Pathogen alone)
Three replications were maintained. The disease intensity was assessed 15 days after treatment and
the per cent disease index (PDI) was calculated.
Management of bacterial blight of cotton under field conditions
Two field trials were laid out for the management of bacterial blight of cotton in randomised block
design at Checkanuarani (Madurai district) and Venkatachalapuram (Theni district). The trial plot size was
4.5 x 3.0 m and spacing adopted was 75 cm x 30 cm. The same set of treatments tested under pot culture
conditions were tested in the field trial in both the locations with three replications.
Seed treatment with rifampicin resistant mutant of respective endophytes was done by soaking the
seeds in streptomycin sulphate solution (0.1%) for 6 h and washing the seeds with water thrice followed by
seed treatment with the formulation product of the antagonists. The treated seeds were shade dried and
then sown. Spraying was given 45 days after sowing and repeated thrice at 15 days interval. Disease
intensity was recorded on 60th day after sowing. Five pickings were done and the total kapas yield per plot
was recorded and the cost benefit ratio was calculated.
Biochemical changes induced in plants by biotic and abiotic elicitors after inoculation with the
bacterial blight pathogen
Samples were collected from the plants treated with biotic and abiotic elicitors in pot culture
experiment and used for biochemical analysis. After inoculation of the pathogen, post inoculation changes
were recorded at 0, 1, 3, 6, 9, 12 and 15 days after inoculation. Ethanol extraction of leaf samples were
used for the estimation of sugars and phenols (Mahadevan et al., 1965), reducing sugars (Nelson, 1944),
total sugars (Inman,1965) and non-reducing sugars (Mahadevan and Sridhar, 1986), protein (Lowry et al.,
1951).
Phenylalanine ammonia lyase (PAL) (Zucker, 1965), Polyphenol oxidase (PPO) (Meyer et
al.,1965), Peroxidase (PO) (Srivastava, 1987) and Catalase (Chance and Machly, 1955) were estimated.

For the analysis of the results, the procedures described by Gomez and Gomez (1984) were
followed.
RESULTS
The pathogenicity conducted stated that the Coimbatore isolate (I2) produced the maximum disease
grade of 3.6 followed by isolate from Checkanuarani (I1).
Seven fungal and seven bacterial isolates were obtained from the phylloplane of healthy cotton
leaves.
Efficacy of phylloplane microflora against Xanthomonas axonopodis pv. malvacearum.
Among the fungal isolates, plf 7 (Trichoderma viride) recorded the maximum inhibition zone of
18.30 mm followed by commercial formulation isolate of Trichoderma viride (17.00 mm) this was on par
with streptomycin sulphate. Among the bacterial isolates, plb 7 (Pseudomonas sp. 5) recorded the
maximum inhibition zone of 18.5 mm (Table 1).
Efficacy of bacterial endophytes against Xanthomonas axonopodis pv. malvacearum
Eight bacterial endophytes each from root and stem and seven leaf endophytes were isolated.
The endophytic bacteria were tested against the pathogen by paper disc assay method.
Pseudomonas fluorescens (Pf 1) was used as standard check. Data presented in Table 2 revealed that Endo
R3 (18.5 mm), Endo S4 (18.35 mm) and Endo L4 (18.40 mm) recorded the maximum inhibition zone.
Screening of abiotic elicitors under glass house conditions against bacterial blight of cotton
In order to find out an effective abiotic elicitor, various chemicals were screened under glasshouse
at different concentrations. None of the chemicals tested as elicitors showed any phytotoxicity. The data
presented in Table 3, show that salicylic acid at 250 ppm produced least number of lesions per leaf (5.00)
followed by SA 200 ppm (12.00). Hydrogen peroxide at 250 ppm produced 13.67 lesion per leaf followed
by 200 ppm (14.33). Salicylic acid at 250 ppm concentration was used in pot culture and field studies.
Pot culture experiments
Pot culture studies were conducted in completely randomized block design to assess the efficacy of
biotic and abiotic elicitors in the management of bacterial blight of cotton and to assess the mechanism of
induced resistance. The effective phylloplane microorganisms (Trichoderma viride, Bacillus sp.,
Pseudomonas sp. plb 7), endophytic bacteria (Endo R3, Endo S4 and Endo L4) and abiotic elicitor
salicylic acid (250 ppm) were taken for pot culture experiments. P. fluorescens (Pf 1) and streptomycin

sulphate spray 0.1 per cent were taken as standard checks. In order to record the biochemical changes
induced in plants, the leaf samples were collected at different intervals of time and disease intensity was
recorded 15 days after inoculation.
Effect of biotic and abiotic elicitors on bacterial leaf blight
Data presented in Table 4 show that 250 ppm salicylic acid spray significantly reduced the lesion
number per leaf (9.0) and percent disease index (25.00), followed by spray application of phylloplane
Pseudomonas (plb 7) (10.5 lesion number/leaf with 30 per cent disease index). P. fluorescens (Pf 1) and
spray application of Trichoderma viride were on par. The disease intensity in all the three endophyte
treatments were on par (PDI = 45.00).
Biochemical changes in elicitor treated plants in response to inoculation with the bacterial blight
pathogen
Twenty four h after treatment of biotic and abiotic elicitors, the leaves were inoculated with
X. axonopodis pv. malvacearum. Biochemical changes were recorded from zero day to 15 days after
pathogen inoculation.
Sugars
The data presented in the Fig 1 show that, all the treatments recorded decrease in total sugar
content upto 9 days after inoculation. Among the treatments, salicylic acid spray showed very high
decrease in total sugars on 9th and 12th day after pathogen inoculation when compared to the initial level.
The initial level of total sugars in this treatment was 38.60 mg/g, on 9 th day 25.80 mg/g and on 12 th day
24.80 mg/g. In phylloplane Pseudomonas sp. plb 7 spray the initial level was 32.50 mg/g which decreased
to 24.40 mg glucose/g leaf tissue on 9 th day and subsequently increased to 38.90 mg glucose/g leaf tissue
on 15th day after inoculation. Spray application of Pf 1 recorded a decrease from 34.50 at zero day to 23.60
mg glucose/g leaf tissue at 9th day and with increase in days after inoculation the total sugar increased to
29.50 mg/g on 15th day after inoculation. In contrast to elicitors treatment and pathogen inoculated plants,
control plants recorded increase in total sugars from 31.30 at zero day to 44.60 mg glucose/g leaf tissue at
9th day after pathogen inoculation and subsequently a reduction in total sugars was noted on 15th day (40.70
mg glucose/g leaf tissue). Seed treatment with leaf endophytic bacteria (Endo L4) recorded high per cent
decrease in total sugars over control.
Analysis of reducing sugar content of elicitors treated and pathogen inoculated leaves (Fig 2) show
that there was gradual reduction in reducing sugar content in all the treatments from zero to 9 th day after
inoculation. Salicylic acid spray showed the highest decrease in reducing sugar content from the initial

level of 33.50 to 17.00 mg/g on 12th day after inoculation of X. axonopodis pv. malvacearum and an
increase on 15th day (18.70 mg glucose/g leaf tissue). Phylloplane Pseudomonas sp. plb 7 spray recorded
decrease in reducing sugar content from the initial level of 27.00 to 17.20 mg glucose/g leaf tissue on 9th
day and later increased to 21.10 mg /g on 15th day after inoculation. Spray application of Pf 1 recorded
decrease in sugar content on 9th day (17.90 mg /g) and increase on 15th day (21.60 mg/g). Control
(pathogen inoculation alone) recorded increase in total sugars from the initial level of 26.80 (at zero day)
to 38.30 mg glucose/g leaf tissue on 9th day after X. axonopodis pv. malvacearum inoculation and
decreased to 32.50 mg /g on 15 th day. Seed treatment with stem endophytic bacteria (endo S4) recorded
high per cent decease in reducing sugar content over control.
From the data presented in the Fig 3, it can be seen that in most of the treatments (except salicylic
acid spray and control) there was decrease in non-reducing sugar content on 3 or 6 days after inoculation
and increase from 9th day onwards. Streptomycin sulphate (0.1 %) spray showed maximum decrease in
non-reducing sugar content (mean value of 4.90 mg/g leaf tissue) followed by seed treatment with stem
endophyte (Endo S4) (4.95 mg/g leaf tissue). There was no significant difference in non-reducing sugar
content in salicylic acid spray upto 9th day after inoculation and the content increased only on 12 th and 15th
day (5.90 mg glucose/g leaf tissue at the end of 15 th day after inoculation) Phylloplane Pseudomonas sp.
plb 7 spray recorded decrease in non-reducing sugar content (4.90 mg glucose/g leaf tissue) on 6th day
after inoculation and later increased to 5.80 mg/g at the end of 15 th day after inoculation. There was no
significant difference in non-reducing sugar content between the different sampling periods in Pf 1
treatment. Control recorded increase in non-reducing sugars from the initial level of 4.50 to 8.20 mg/g on
15th day. Streptomycin sulphate (0.1 %) spray showed high per cent decrease in non-reducing sugar
content over control.
Protein
There was increase in protein content in all the treatments upto 9 days after inoculation. Salicylic
acid spray showed the maximum increase (mean value of 47.34 g /g leaf tissue) followed by phylloplane
Pseudomonas sp. plb 7 spray (42.24 g /g leaf tissue). Among the sampling period, 9 th day after pathogen
inoculation recorded significantly higher amount of protein. In spray application of salicylic acid, protein
content increased from the initial level of 36.30 to 56.20 g/g on 9 th day after pathogen inoculation and
decrease in content was noted at the end of 15th day (49.50 g/g) after X. axonopodis pv. malvacearum
inoculation. This treatment also recorded higher per cent increase in protein content over control.
Phylloplane Pseudomonas sp. plb 7 spray recorded increase in content from the initial level of 31.75 (zero

day) to 51.25 g/g fresh leaf tissue on 9th day after inoculation and subsequently decreased to 43.50 g/g at
the end of 15th day. Spray application of Pf 1 recorded increased protein content of 50.50 g/g on 9th day
from the initial level of 31.20 g/g Control recorded increase in protein content on 9 th day (31.25 g/g
fresh leaf tissue) and later decreased to 28.60 g/g fresh leaf tissue on 15 th day after X. axonopodis pv.
malvacearum inoculation (Fig. 4).
Total phenols
Changes in total phenol content recorded at different intervals of time after inoculation showed that
the phenol content increased in all the treatments upto 9 th day after inoculation. Among the treatments,
spray application of salicylic acid recorded the maximum increase (mean value of 1.270 mg/g fresh leaf
tissue) followed by phylloplane Pseudomonas sp. plb 7 spray (1.240 mg/g). Salicylic acid spray recorded
significantly higher content of phenol from the beginning showing a level of 1.005 mg/g on zero day and
1.425 mg/g on 9th day. The level subsequently decreased to 1.215mg/g at the end of 15 th day. Phylloplane
Pseudomonas sp. plb 7 spray recorded increase in total phenols from 0.980 to 1.405 mg/g from zero to 9 th
day and a decrease in content was noticed (1.205 mg/g) on 15 th day after pathogen inoculation. Spray
application of Pf 1 recorded increase in total phenol from the initial level of 0.965 to 1.400 mg/g at the end
of 9th day and later decreased to 1.201 mg/g on 15th day. Control recorded 1.233 mg/g on 9th day as
compared to the initial level of 0.815 mg/g and decreased to 1.008 mg/g on 15th day after inoculation (Fig.
5).
Phenylalanine ammonia lyase (PAL)
There was steady increase in PAL activity in all the treatments from zero to 9 th day after inoculation
(Fig. 6). Salicylic acid spray recorded significantly high amount of PAL activity (mean value of 92.29 m
transcinnamic acid/g leaf tissue) followed by phylloplane Pseudomonas sp. plb 7 spray (90.84 m
transcinnamic acid/g leaf tissue). In salicylic acid spray treatment, PAL activity at the time of inoculation
was 67.12 m transcinnamic acid/g leaf tissue and the activity reached to 110.14 m/g on 9 th day followed
by decrease on 15th day (101.21 m/g) after inoculation. This treatment also recorded high per cent
increase over control. In the treatment phylloplane Pseudomonas sp. plb 7 the initial PAL activity was
66.81 m/g which increased to108.24 m/g on 9th day after inoculation and later decreased to 99.80 m/g
at the end of 15th day. Spray application of Pf 1 recorded PAL activity of 104.12 m/g on 9 th day which
later decreased to 97.15 m/g leaf tissue (15 th day after inoculation). In control (pathogen inoculation
alone) there was a drastic decrease in PAL activity one day after inoculation, but from 3 rd day onwards the
enzyme activity gradually increased upto 9th day and again decreased on 15th day after inoculation.

Polyphenol oxidase (PPO)

Data presented in the Fig 7 show that PPO activity increased in all the treatments from zero to 9 th
day after inoculation. Among the treatments, spray application of salicylic acid, phylloplane Pseudomonas
plb 7 spray and Pf 1 spray were on par with each other. PPO activity was significantly high on 9 th day after
pathogen inoculation when compared to other sampling periods. Salicylic acid spray treatment recorded
the highest PPO activity (mean value 0.069) and this was on par with phylloplane Pseudomonas plb 7
spray (0.068) and Pf 1(0.064). In X. axonopodis pv. malvacearum alone inoculated plants (control), PPO
activity was lower than the elicitor treated plants at all the sampling periods. But in control plants also, the
trend of increase in PPO activity upto 9th day and a subsequent decrease was observed.
Peroxidase (PO)
Data presented in the Fig 8 show that in all the treatments PO activity increased. Among the
treatments, spray application of salicylic acid and phylloplane Pseudomonas sp. plb 7 spray and Pf 1 spray
were on par. PO activity was significantly high on 9 th day after pathogen inoculation when compared to
other sampling days. Salicylic acid spray recorded 0.791 PO activity on 9 th day as compared to the initial
level of 0.071. The activity decreased to 0.718 on 15th day after inoculation. The increase in PO activity in
salicylic acid treatment was 92.88 per cent over control.
Catalase
Changes in catalase activity at different intervals of time after X. axonopodis pv. malvacearum
inoculation showed that, there was increase in enzyme activity in all the treatments when compared to
control. Among the treatments salicylic acid spray recorded very high catalase activity (mean value 0.032)
and this was statistically on par with plb 7 spray (mean value of 0.031). In all the treatments, catalase
activity increased upto 9th day after inoculation and decreased thereafter. In the pathogen alone inoculated
plants (control) the same trend was observed. Though the catalase activity decreased in all the treatments
after 9th day of inoculation, the enzyme activity on 15th day in all the treatments was much higher than the
initial level (Fig. 9).
Management of bacterial leaf blight of cotton in the field
A field experiment was conducted in randomized block design with three replications. The disease
intensity was recorded 60 days after sowing and the total kapas yield from the five pickings was recorded.
The results showed that the least disease intensity was recorded in spray application of salicylic acid (10.3
and 9.25 PDI respectively in field I and field II (Table 5) followed by spray application of phylloplane
Pseudomonas sp. plb 7 (11.1 and 10.3 PDI in field I and II respectively)) P. fluorescens (Pf 1) recorded

12.4 and 13.6 PDI in field I and II followed by streptomycin sulphate (0.1 %) spray (15.90 in field I and
16.70 PDI in field II) as compared to the PDI of 31.20 and 33.20 in control in the two fields. Kapas yield
was the highest in salicylic acid spray treatment (20.9 q/ha) followed by phylloplane Pseudomonas sp. plb
7 spray (20.8 q/ha) and both were on par. Pf 1 treatment recorded 18.5 q/ha and streptomycin sulphate
spray 16.7 q/ha as compared to 12.2 q/ha in control.
DISCUSSION
Management of the disease
Efficacy of phylloplane microflora against Xanthomonas axonopodis pv. malvacearum
In the present study, plf 7 (18.30 mm) and plb 7 (18.33 mm) recorded maximum inhibition zone
against X. axonopodis pv. malvacearum
Similarly Saha et al. (2001) reported that cotton phylloplane bacteria, Pseudomonas is highly
antagonistic to X. axonopodis pv. malvacearum in vitro with inhibition zone of 1.2 cm. Sanjay and
Parashar (2002a) reported the in vitro antagonistic activity of Bacillus subtilis, P. fluorescens and Bacillus
sp. towards X. axonopodis pv. malvacearum.
Efficacy of bacterial endophytes against Xanthomonas axonopodis pv. malvacearum
Presence of endophytic bacteria in cotton plants has been reported earlier. In the present study,
Endo R3 (18.50 mm), Endo L4 (18.40 mm) and Endo S4 (18.35 mm) showed maximum inhibition zone in
the paper disc assay experiment. Bhowmik et al. (2002) studied in vitro antagonism of Endo PR 8 (P.
fluorescens) against X. axonopodis pv. malvacearum which gave an inhibition zone of 15.0 mm. The
findings of the present study and the earlier published reports clearly indicate that the endophytic bacteria
have a great potential as biocontrol agents for the management of bacterial blight of cotton.
Induction of systemic resistance by biotic and abiotic elicitors
In the present study, abiotic elicitors were sprayed on cotton plants at different
concentrations ranging from 50 ppm to 250 ppm and after 24 h X. axonopodis pv. malvacearum was
inoculated. Symptom production (lesion number per leaf) was observed after 15 days of artifical
inoculation. It was noted that, salicylic acid spray at 250 ppm recorded least number of lesions per leaf (5)
followed by salicylic acid spray at 200 ppm (12). Salicylic acid spray which produced least number of
lesions per leaf compared to other chemicals under glass house conditions was selected since salicylic acid
is the natural signal molecule in the induction of resistance in the host against disease causing pathogens.

Enyedi et al. (1992) reported that salicylic acid induced genes comparable with biologically
induced resistance acting as a signal molecule and co-ordinating plant defense gene expression. Muller and
Weltzien (1990) succeeded in inducing resistance in oats against Drechslera avenae using salicylic acid.
Shulaev et al. (1995), Molders et al. (1996) and Mur et al. (2000) demonstrated the systemic nature of
salicylic acid and showed that salicylic acid moved from its production site in the infected leaf of tobacco
and cucumber to the upper leaves by phloem.
Hammerschmidt et al. (2001) reported that salicylic acid is important for the establishment of both
localized acquired resistance (LAR) and systemic acquired resistance (SAR). Salicylic acid also induced
PR protein in plants which give resistance against blackgram diseases (Ramanathan and Vidhyasekaran,
2002). Similar to the effect of salicylic acid in reducing disease symptom, another phenolic compound
benzoic acid also acted as abiotic elicitor in reducing the disease incidence and severity in stem rot of rice
(Kumar et al., 2003).
Foliar application of synthetic activators 2,6 dichloroisonicotinic acid or benzo (1,2,3) thiadiazole
7- carbothioic acid S- methyl ester in cotton plants significantly reduced the infection of X. axonopodis pv.
malvacearum (Colson et al., 2000).
In the present study, phylloplane bacteria Bacillus sp., Pseudomonas sp. plb 7 and bacterial
endophytes (Endo R3, Endo S4 and Endo L4) were selected as the antagonists based on in vitro
antagonism. Pre-inoculation of non-pathogen has been utilized for induction of resistance in plants
(Gregersen and Petersen, 1989). MPiga et al. (1997) and Van Wees et al. (1997) stated that fluorescent
pseudomonads induce resistance against many plant pathogens. Maurhofer et al. (1998) demonstrated that
salicylic acid produced by P. fluorescens in rhizosphere is involved in induced systemic resistance. They
stated that expression of salicylic acid biosynthetic genes (pch A and pch B) in P. fluorescens strain P 3
significantly improved its ability to induce systemic resistance in tobacco against tobacco necrosis virus.
Wei et al. (1996) treated cotton seeds with plant growth promoting rhizobacteria, which exhibited
biological control activity, including induced systemic resistance against various pathogens in green house
and field trials in cucumber. Wilson et al. (1996) used plant growth promoting rhizobacteria to elicit
systemic resistance against tomato bacterial speck (Pseudomonas syringae pv. tomato) and bacterial leaf
spot (X. campestris pv. vesicatoria).

Biochemical changes induced in cotton plants by biotic and abiotic elicitors

The accumulation of phenol and activities of enzymes are known to be associated with
biochemical disease resistance. The biochemical mechanisms involved in plant disease resistance is a
complex phenonmenon.
Sugars
Sugars are the building blocks of the defense chemicals phenols and their derivatives
(Vidhyasekaran, 1988). Induction of high level of sugar by biotic and abiotic elicitors treatment, provides
the substrate needed for the synthesis of defense chemicals. The results obtained in the present study,
supports this hypothesis, since the phenol level increased throughout the sampling period (upto 24 h) in the
elicitor treated cotton plants. Kehlenbuk et al. (1994) reported that the culture filtrates of Bacillus subtilis
induced resistance against biotrophic leaf pathogens, by altering the carbohydrate metabolism in barley
after the attack by powdery mildew. The application of culture filtrates led to increased movement of
assimilates from flag leaf into the ears, where sugar content also increased, which was used for the
synthesis of defense related compounds.
Protein
Salicylic acid spray and plb 7 showed increase in protein content upto 9 days after pathogen
inoculation. Induced systemic resistance in tobacco after inoculation with Pseudomonas tabaci was
followed by an increase in the concentration of PR proteins (Tuzun et al., 1989). Boller (1985) expressed
that proteins are associated in the defense of plant against fungi and bacteria by their action on the cell
walls of invading pathogens. Ward et al. (1991) stated that salicyclic acid application induced gene
families of PR proteins (PR-1, PR-2, PR-3, PR-4, PR-5, PR-Q 1) in tobacco against fungal and bacterial
pathogens.
Phenols
Phenylalanine ammonia lyase (PAL) is the key enzyme of phenylpropanoid metabolism in higher
plants leading to the synthesis of phenols (Massala et al., 1980). Salicylic acid spray and phylloplane
Pseudomonas plb 7 spray recorded the maximum increase in phenol content after pathogen inoculation.
The production of phenolic compounds depends upon phenylalanine ammonia lyase enzyme activity
(Graham and Graham, 1991). Thangavelu et al. (2003) reported increase in phenolic content in leaf tissues
3 to 6 days after P. fluorescens strain Pf 10 treatment when challenge inoculated with Fusarium
oxysporum in banana.

Enzyme assay
Increase in enzyme activities due to elicitor treatment is one of the resistance response against the
invading pathogens. The enzymes phenylalanine ammonia lyase (PAL), peroxidase (PO), polyphenol
oxidase (PPO) and catalase play an important role in lignin biosynthesis (Vance et al., 1980). These
enzymes also bring about the production of molecules like phytoalexins and phenolic compounds that
elicit the steps of induction of resistance (Keen and Yoshikawa, 1983; Mauch et al., 1988; Van Loon et al.,
1994). A large number of enzymes have been associated with ISR including PO, PAL, lipoxygenase, 1, 3
glucanase and chitinase (Ye et al., 1990; Koch et al., 1992; Schneider and Ullrich, 1994 and Van Loon,
1997).
Phenylalanine ammonia lyase
Salicylic acid or plb 7 treated cotton plants were inoculated with the bacterial blight pathogen,
there was increase in PAL activity. Podile and Laxmi (1998) stated that the product of PAL, cinnamic acid
is directly linked to cell lignification processes and the highest level of PAL activity usually occur about 24
h after initial infection. Harllen et al. (2004) studied the effect of rhizobacteria B101R, B212R and AO68R
in induction of systemic resistance against Pseudomonas syringe pv. tomato.
Polyphenol oxidase
Among the elicitor treatments, salicylic acid spray and phylloplane Pseudomonas sp. plb 7 spray
showed increased PPO activity from the time of treatment to ninth day after inoculation. Earlier reports
given by Chen et al. (2000) showed that treatment of cucumber roots with Pseudomonas corrugata 13 or
P. aureofaciens 63-28 increased PPO activity after bacterization and inoculation with Pythium
aphanidermatum.
Peroxidase
Peroxidase is an important enzyme in the synthesis of lignin (Hammerschmidt et al., 1982 and
Kuc, 1990). This enzyme is also known to catalyze the oxidation of many mono and diphenols and
aromatic amines to the highly toxic quinones in the presence of hydrogen peroxide (Lobenstein and
Linsey, 1961). increase in PO activity was detected.
In the elicitor treated and pathogen inoculated cotton leaves, salicylic acid spray and phylloplane
Pseudomonas sp. plb 7 spray were on par with regard to increasing the activity of PO upto 9 days after
inoculation. Podile and Laxmi (1998) observed peak peroxidase activity when pea plants were treated with
Bacillus subtilis, seven days after inoculation with Fusarium udum. Delannoy et al. (2003) suggested that
class III peroxidase activity is involved in the defense response to Xanthomonas infections. Analysis of

gene expression showed variation in transcript accumulation during both compatible and incompatible
interactions for pod 2, pod 3, pod 4 and pod 6 genes. Pod 4 and pod 6 were more intensely up regulated
during resistance than during disease and in control, while in pod 3, it is specifically down regulated
during hypersensitive reaction (HR) after oxidative burst. Pod 2 was induced by the pathogen infection
and weakly stimulated in the control. The increased PO activity may oxidise phenols to toxic quinones and
such oxidation products may inhibit the pathogen development. Peroxidase itself was found to inhibit the
pathogen growth (Joseph et al., 1998). PO along with PAL may also accelerate lignin synthesis.
Lignification has been suggested to inhibit the pathogens in a number of ways:
i.

by providing resistance to enzymes of the pathogen.

ii.

by restricting diffusion of nutrients and water from the plant cell to the bacterium or

iii.

by inhibiting the bacterial growth due to accumulation of phenolic precursors and the

subsequent free radicals produced during polymerization (Ride, 1978).

Catalase
In the elicitor treated and pathogen inoculated cotton leaves, salicylic acid spray and phylloplane
Pseudomonas sp. plb 7 spray were on par with regard to increasing the catalase activity. Earlier reports
also suggest that, catalase activity was enhanced after 3 days of Uromyces phaseoli inoculation in bean
plants (Montalbini, 1991). Amaresh et al.(2001) found increase in catalase activity four days after salicylic
acid treatment in Rhizoctonia solani inoculated cowpea plants. However, Scandalios (1993) has reported
that catalase may act as a free radical scavenger and as a suppressor in a number of host-pathogen and
elicitor interactions.
Management of bacterial leaf blight of cotton in field
Field experiments conducted showed that the spray application of salicylic acid and
phylloplane Pseudomonas sp. plb 7 recorded least disease incidence compared to control followed by
spray application of Pseudomonas fluorescens (Pf 1) and streptomycin sulphate. Preinoculation of
phylloplane bacteria protect the plants from pathogen infection (reduction in disease severity both in terms
of time and intensity). Sanjay and Parashar (2002a) reported that foliar spray of Pseudomonas fluorescens
provided the maximum disease control of cotton bacterial blight (4.6% disease intensity) followed by seed
treatment and foliar application of the antagonist 12D (12.6% disease intensity) under greenhouse and
field conditions. Samuel and Patro (2003) reported that bioformulations of Bacillus sp. were effective
against X. axonopodis pv. malvacearum, X. campestris pv. mangifer and X. campestris pv. campestris
under field conditions.

LITERATURE CITED

Ainsworth, G.C. 1961. Dictionary of fungi. Commonwealth mycological institute, Kew, Surrey. England. p 547.
Allen, O.N. 1953. Experiments in soil bacteriology. Burgess Publishing Co., Minneapolis, Minn., USA. p 127. King, E.O.,
Ward, M.K. and D.E. Raney. 1954. Two simple media for demonstration of pyocyanin and fluorescin. Journal of
Laboratory and Clinical Medicine. 44: 301-307.
Amaresh, C., Anjali. A., Pranab, K.M. and S. Pradeep. 2001. Influence of salicylic acid on protein content and catalase
activity in relation to systemic acquired resistance in cowpea against root rot. Indian Phytopath., 54: 284-287.
Bhowmik, B., Singh, R.P., Jayaraman, J. and J.P. Verma. 2002. Population dynamics of cotton endophytic Pseudomonas,
their antagonism and protective action against the major pathogens of cotton. Indian Phytopath., 55: 124-132.
Boller, T. 1985. Induction of hydrolases as defense reaction against pathogens. In: Cellular and molecular biology of plant
stress. Edited by Key, J.L. and T. Kosuge. pp 247-262. ULCA symposia on Molecular and cellular biology, New
Series, volume 22 Alan R. Liss, Inc., New York.
Chance, B. and A.C. Machly. 1955. Assay of catalases and peroxidases. Methods in Enzymology, 2: 764-775.
Delannoy, E., Jalloul, A., Assigbetse, K., Marmey, P., Geiger, J.P., Lherminier, J., Daniel, J.F., Martinez, C and N. Nicole.
2003. Activity of class III peroxidases in the defense of cotton to bacterial blight. Phytopathology, 93: S31 (Abstr).
Elad, Y.I. and I. Chet. 1983. Improved selective media for isolation of Trichoderma sp. and Fusarium spp. Phytoparasitica,
11: 55-58.
Enyedi, A.J., Yalpani, N., Paul, S. and I. Raskin. 1992. Signal molecules in systemic plant resistance to pathogens and pests.
Cell, 70: 879-886.
Gaffney, T., Friedrich, L., Vernooji, B., Negrotto, D., Nye, G., Vkes, S., Ward, E., Kessmann, H. and J. Ryals. 1993.
Requirement of salicylic acid for the induction of systemic resistance. Science, 261: 754-756.
Gomez, A.K. and A.A. Gomez. 1984. Statistical Procedures for Agricultural Research. John Wiley and Sons, New York.
p 680.
Graham, T.L. and M.Y. Graham. 1991. Cellular coordination of molecular responses in plant defense. Mol. Plant-Microbe
Interact., 4: 415-421.
Gregersen, P.L. and S.V. Petersen. 1989. Induction of resistance in barley against Erysiphe graminis f. sp. hordei after
preinoculation with the saprophytic fungus, Cladosporium macrocarpum. J. Phytopathol., 137: 128-136.
Hammerschmidt, R., Metraux, J.P. and L.C. Van Loon. 2001. Inducing resistance: a

summary of papers presented at the

First International Symposium on Induced Resistance to Plant Diseases, Corfu, May 2000. Eur. J. Plant
Pathol.,106: 1-6.
Harllen, S.A. Silva, Reginaldo de Silva, R., Dirceu, M., Bernardo de, A.H.V., Maria, C.B.P. and A. Mounteer. 2004.
Rhizobacterial induction of systemic resistance in tomato plants: non-specific protection and increase in enzyme
activities. Biol. Control, 29: 12 (Abstr).

Inman, R.E. 1965. Quantitative sugar changes in barley infected with a facultative parasite. Phytopathology, 55: 341-345.
Joseph, L.M., Tan, T.K. and S.M. Wong. 1998. Antifungal effects of hydrogen peroxide and peroxidase on spore germination
and mycelial growth of Pseudocercospora species. Can. J. Bot., 76: 2119-2124.
Keen, N.T. and M. Yoshikawa. 1983. 1, 3 endo glucanases from soybean releases elicitor active carbohydrates from fungus
cell

walls.

Plant

Physiol.,

71:

460-465. Mauch, F., Mauch-Mani, B. and T. Boller. 1988. Antifungal hydrolases in pea tissue: inhibition of fungal
growth by combinations of chitinases and 1, 3 glucanase. Plant Physiol., 88: 936-942.
Kehlenbuk, H., Krone, C., Oerke, E.C. and F. Shonbeck. 1994. The effectiveness of induced resistance on yield of mildewed
barley. Z. Pflkrankh. Pflschutz., 101: 11-21.
Kumar, A. Singh, R. and B.L. Jalali. 2003. Management of stem rot of rice with resistance inducing chemicals and
fungicides.

Indian

Phytopath.,

56:

Colson, H.E.S., Allen, S.J. and B.J. Deverall. 2000. Effect of 2, 6-dichloroisonicotinic acid or benzothiadiazole on
Alternaria leaf spot, bacterial blight and Verticillium wilt in cotton under field conditions. Australasian Plant
Pathol.,
Lowry, O.H., Rosebrough, N.J., Fam, A.L. and R.J. Raandall. 1951. Protein measurement with Folin-phenol reagent. J. Biol.
Chem., 193: 265-275.
MPiga, P., Belanger, R. R., Paulitz, T. C. and N. Benhanou. 1997. Increased resistance to Fusarium oxysporum f.sp. radicislycopersici in tomato plants treated with endophytic bacterium Pseudomonas fluorescens strain 63-28. Physiol. Mol.
Plant Pathol., 50: 301-320.
Mahadevan, A. and R. Sridhar. 1986. Methods in physiological plant pathology. Sivakagami publications, Madras, India. p
237.
Mahadevan, A., Kuc, J. and E.B. Williams. 1965. Biochemistry of resistance in cucumber against Cladosporium
cucumerinum: presence of pectinase inhibitor in resistant plants. Phytopathology, 55: 100-103.
Massala, R., Legrand, M. and B. Fritig. 1980. Effect of amino oxyacetate, a competitive inhibitor of phenylalanine ammonia
lyase on hyper sensitive reaction of tobacco mosaic virus. Physiol. Plant Pathol., 16: 213-226.
Maurhofer, M., Reimmann, C., Schmidli-sacherer, P., Heeb, S., Haas, D. and G. Defago. 1998. Salicylic acid biosynthetic
genes expressed in Pseudomonas fluorescens P3 improve the induction of systemic resistance in tobacco against
tobacco necrosis virus. Phytopathology, 88: 678-684.
Mc Kinney, H. H. 1923. Influence of soil temperature and moisture on infection of wheat seedlings by Helminthosporium
sativum. J. Agri. Res., 26: 195-217.
McInroy, J.A. and J.W. Kloepper. 1995a. Population dynamics of endophytic bacteria in field grown sweet corn and cotton.
Can. J. Microbiol., 41: 895-901.

McInroy, J.A. and J.W. Kloepper. 1995b. Survey of indigenous bacterial endophytes from cotton and sweet corn. Plant Soil,
173: 337-342. Ryals, J.A., Neuenschwander, U.H., Willits, M.G., Molina, A., Steiner, Henry York and M.D. Hunt.
1996. Systemic acquired resistance. Plant cell, 8: 1809-1819.
Meyer, A.H. Harel, F. and R.B. Shaul. 1965. Assay of catechol oxidase a critical comparison of methods. Phytochemistry,
5: 783-789.
Meyer, G. de. and M. Hofte. 1997. Salicylic acid produced by rhizobacterium Pseudomonas aeruginosa FNSK 2 induces
resistance to leaf infection by Botrytis cinerea on bean. Phytopathology, 87: 588-593.
Meyer, J.M. and M.A. Abdallah. 1978. The fluorescent pigment of Pseudomonas fluorescens, biochemical purification and
physiochemical properties. J. Gen. Microbiol., 107: 319-328.
Millar, R.L. and V.J. Higgins. 1970. Association of cyanide with infection of birdsfoot trefoil by Stemphylium loti.
Phytopathology, 60: 104-110.
Misaghi, I.J. and C.R. Donndelinger. 1990. Endophytic bacteria in symptom free cotton plants. Phytopathology, 80: 808811.
Molders, W., Buchala, A. and J.P. Mitraux. 1996. Transport of salicylic acid in tobacco necrosis virus infected cucumber
plants. Plant Physiol., 112: 787-792.
Montalbini, P. 1991. Effect of rust infection on levels of uricase, allantoinase and ureides in susceptible and hypersensitive
bean leaves. Physiol. Mol. Plant Pathol., 39: 173-188.
Muller, V. and A.C. Weltzien. 1990. Resistenzinduktion in gerste und hafer dun vorinokulati;on mit apathogenein
befalsminadernder mechanismus in getreibestenden. Z. Pflkrankh. Pflschutz., 97: 532.
Mur, L., Maddison, A.L., Darby, R.M. and J. Draper. 2000. Systemically translocated salicylic acid is vital in establishing
systemic acquired resistance in tobacco. First International congress on systemic induced resistance. Corfu.
Nelson, N. 1944. A photometric adaptation of the Somogyi method for the determination of glucose. J. Bot. Chem., 153:
375-380.
Osbourn, A.E. 1996. Preformed antimicrobial compounds and plant defense against fungal attack. Plant Cell, 8: 1821-1823.
Patriquin, D.G., Taber, R.A. and D.K. Jain. 1983. Sites and processes of association between diazotrophs and grasses. Can.
J. Microbiol., 29: 900-915.
Podile, A.R. and V.D.V. Laxmi. 1998. Seed bacterization with Bacillus subtilis AF1 increases phenylalanine ammonia lyase
and reduces the incidence of Fusarial wilt in pigeonpea. J. Phytopathol., 146: 255-259.
Raghuchander, T., Samiyappan, R. and G. Arjunan. 1998. Biocontrol of Macrophomina root rot of mungbean. Indian
Phytopath., 51: 379-382.
Reeves, M.L., Pine, J.B. and A.B. Neilands. 1983. Absence of siderophore activity in Legionella sp. grown in iron deficient
media. J. Bacteriol., 154: 324-329.
Ride, J.P. 1978. The role of cell wall alterations in resistance to fungi. A. Appl. Biol., 89: 302-306.

Saha, S., Singh, R.P., Verma, J. P. and J. Jayaraman. 2001. Population dynamics of cotton phylloplane bacteria antagonistic
towards Xanthomonas campestris pv. malvacearum. Indian Phytopath., 54: 409-413.
Samuel, S.K. and T. Patro. 2003. Evaluation of biocontrol potential of phylloplane bacteria against bacterial leaf spot of
greengram. Abst. of research papers presented during 55 th annual meeting at Osmania University, Hyderabad 500
007, Jan 16-18, 2003.
Sanjay, A. and R.D. Parashar. 2002a. Biological control of cotton bacterial blight with phylloplane bacterial antagonists.
Tropical Agriculture, 79: 51-55.
Scandalios,

J.G.

1993.

Oxygen

stress

and

superoxide

dismutases.

Plant

Physiol.,

101: 7-12. Sanjay, A. and R.D. Parashar. 2002a. Biological control of cotton bacterial blight with phylloplane
bacterial antagonists. Tropical Agriculture, 79: 51-55.
Schneider, S. and W.R. Ullrich. 1994. Differential induction of resistance and enhanced enzyme activities in cucumber and
tobacco caused by treatment with various abiotic and biotic inducers. Physiol. Mol. Plant Pathol., 43: 57-67.
Sharrock, K.R., Parkes, S.L., Javk, H.K., Rees-George, J. and B.T. Hawthorne. 1991. Involvement of bacterial endophytes in
storage rots of buttercup squash (Cucurbita maxima D. hybrid Delica). N. Z. J. Crop. Hortic. Sci. 19: 157-165.
Shulaev, V., Leon, J. and I. Raskin. 1995. Is salicylic acid a translocated signal of systemic acquired resistance in tobacco.
Plant Cell, 7: 1691-1701.
Sivakumar, G. and R.C. Sharma. 2003. Induced biochemical changes due to seed bacterization by Pseudomonas fluorescens
in maize plants. Indian Phytopath., 56: 134-137.
Srivastava, S.K. 1987. Peroxidase and polyphenol oxidase in Brassica juncea plants infected with Macrophomina
phaseolina (Tassi) Goid and their implications in disease resistance. Phytopathology, 77: 249-254.
Thangavelu, R., Palaniswami, A., Doraiswamy, S. and R. Velazhahan. 2003. The effect of Pseudomonas fluorescens and
Fusarium oxysporum f.sp. cubense on induction of defense enzymes and phenolics in banana. Biologia Plantarum,
46: 107-112.
Tuzun, S. Rao, M.N., Vogeli, U. Schardi, C.C. and J. Kuc. 1989. Induced systemic resistance to blue mold: early induction
and accumulation of 1, 3 glucanases, chitinases and other PR proteins ( proteins) in immunized tobacco.
Phytopathology, 79: 979-983.
Van Loon, L.C. 1997. Induced resistance in plants and the role of pathogenesis related proteins. Eur. J. Plant Pathol., 103:
753-765.
Van Loon, L.C., Pierpoint, W.S., Boller, T. and V. Conejero. 1994. Recommendations four naming plant pathogenesis-related
proteins.

Plant

Mol.

Biol.

12:

245-264.
Van Wees, S.C.M., Mirjam, L., Inge, S., Van Loon, L.C. and C.M.J. Pieterse. 1999. Rhizobacteria-mediated induced
systemic resistance (ISR) in Arabidiopsis is not associated with a direct effect on expression of known defenserelated genes Atvsp upon challenge. Plant. Mol. Biol., 41: 537-549.

Van Wees, S.C.M., Pieterse, C.M.J., Tdrijssenaar, Y.A., Van t Westerde, Hartog, F. and L.C. Van Loon. 1997. Differential
induction of systemic resistance in Arabidiopsis by biocontrol bacteria. Mol. Plant-Microbe Interac., 10:
716-724.
Vidhyasekaran, P. 1988. Physiology of disease resistance in plants. Volume I. CRC press, Florida, p 128.
Vidhyasekaran, P. and M. Muthamilan. 1995. Development of formulation of Pseudomonas fluorescens for control of
chickpea

wilt.

Plant

Dis.,

79:

782-790.
Ward, E.R., Payne, G.P., Moyer, M.B. Williams, S.C., Dincher, S.S., Sharkey, K.C., Beck, J.J., Taylor, H.T., Ahl-Goy, P.,
Meins, F.Jr. and J.A. Ryals. 1991. Differential regulation of 1, 3-glucanase messenger RNAs in response to
pathogen infection. Plant Physiol., 96: 390-397.
Wei, G., Kloepper, J.W. and S. Tuzun. 1991. Induction of systemic resistance of cucumber to Colletotrichum orbiculare by
select strains of plant growth promoting rhizobacteria. Phytopathology, 81: 1508-1512.
Wilson, M., Ji, P., Camphell, H.L. and J.W. Kloepper. 1996. Development of an integrated biocontrol strategy for bacterial
speck of tomato caused by Pseudomonas syringae pv. tomato. In: Advances in biological control of plant
diseases. Edited by Tang, W., Cook, J. and A. Rovra. Beijing Agricultural University press, Beijing. pp. 387-389.
Ye, X.S., Pan, S.Q. and J. Kuc. 1990. Association of pathogenesis related proteins and activities of peroxidase, 1, 3,
glucanase and quitinase with systemic induced resistance to blue mould tobacco but not to systemic tobacco mosaic
virus. Physiol. Mol. Plant Pathol., 41: 523-531. Koch, E., Meier, B.M., Eihen, H.G. and A. Slusarenko. 1992. A
lipoxygenase from leaves of tomato (Lycopersicon esculentum Mill) is induced in response to plant pathogenic
pseudomonads. Plant Physiol., 99: 571-576.
Zucker, M. 1965. Induction of phenylalanine ammonia lyase by light and its relation to chlorogenic acid synthesis in potato
tuber

tissue.

Plant

Physiol.,

40:

779-781.

Table 1. Efficacy of phylloplane fungi and bacteria against Xanthomonas axonopodis pv. malvacearum in vitro (paper disc method)
Fungal isolates
plf 1

Inhibition zone (mm)*

Bacterial isolates
13.60
(3.75)c

plb 1

Inhibition zone (mm)*

16.30
(4.09)c

plf 2

16.66
(4.14)b

plb 2

7.10
(2.76)e

plf 3

12.70
(3.63)d

plb 3

15.70
(4.02)d

plf 4

8.30
(2.97)f

plb 4

17.40
(4.23)b

plf 5

12.70
(3.63)d

plb 5

5.00
(2.35)f

plf 6

9.50
(3.16)e

plb 6

2.30
(1.67)g

plf 7

18.30
(4.34)a

plb 7

18.50
(4.36)a

Trichoderma viride

17.00
(4.18)b

Pseudomonas fluorescens (Pf

17.10
(4.20)b

Streptomycin sulphate (100 ppm)

18.70
(4.38)a

Streptomycin sulphate (100 ppm)

18.70
(4.38)a

Control

0.00
(0.71)g

Control

0.00
(0.71)h

0.04

CD (P=0.05)

0.05

CD (P=0.05)

* Mean of three replications. Means carrying same letters are not significantly different according to DMRT

Table 2. Efficacy of bacterial endophytes against Xanthomonas

vitro (paper disc method)

axonopodis pv. malvacearum in

Bacterial strains Inhibition zone (mm)*

Bacterial

(Root endophytes)

endophytes)

Inhibition
strains zone(Stem
(mm)*

Bacterial Inhibition
strainszone (Leaf
(mm)*
endophytes)

Endo R1

Endo
S1
6.00

Endo
2.40L1
e

6.60

Endo R2

(2.55)
Endo
15.30
S2

(1.70)
Endo
15.20L2

(2.66)
2.50

Endo R3

(3.97)c
Endo
18.50
S3

(3.96)b
Endo
6.80L3

(1.73)
6.30

Endo R4

(4.36)a
Endo
5.20
S4

(2.70)d
Endo
18.35L4

(2.61)
18.40

Endo R5

(2.39)g
Endo
1.20
S5

(4.34)a
Endo
1.20L5

(4.35)
12.00

Endo R6

(1.30)h
Endo
13.30
S6

(1.30)f
Endo
12.70L6

(3.53)
8.40

Endo R7

(3.71)d
Endo
8.00
S7

(3.63)c
Endo
8.00L7

(2.98)
13.30

(2.92)d
3.00

(3.71)

Endo R8

(2.92)e
Endo
16.70
S8

Pseudomonas fluorescens (Pf

(4.15)b
Pseudomonas
17.10
fluorescens
(4.20)
0.00

CD (P=0.05)

(0.71)i
CD 0.05
(P=0.05)

* Mean of three replications

Means carrying same letters are not significantly different according to DMRT
Values in parentheses are

x 0.5 transformed values

(1.87)e
Pseudomonas
17.10
fluorescens
(4.20)
0.00

(0.71)g
CD
0.23
(P=0.05)

17.10
(4.20)
0.00
(0.71)
0.13

Table 3. Effect of chemicals on bacterial leaf blight of cotton

Lesion number per leaf*

Chemicals

Benzoic acid
Copper sulphate
Di-Potassium phosphate
Glycine
Hydrogen peroxide
Indole acetic acid
Napthalene acetic acid
Oxalic acid
Salicylic acid

* Mean of three
replications
C.D(P=0.05)
Conc
- 0.99
Chemicals
- 1.41
Conc
- 3.15 X
chemicals

Concentration (ppm)
50
27.33
26.67
24.00
21.33
22.00
23.00
23.33
25.33
18.33
30.00
23.83

100
27.67
25.67
24.00
16.00
20.67
19.00
21.67
23.67
16.33
32.00
22.67

150
26.33
23.00
23.33
23.00
24.00
19.33
21.00
26.33
13.33
33.50
23.32

200
25.00
21.33
21.33
24.00
14.33
20.00
20.00
21.33
12.00
34.00
21.33

250
21.67
19.00
20.00
20.33
13.67
19.33
19.00
21.33
5.00
33.60
19.29

Mean
25.60
23.13
22.53
20.93
18.13
20.13
21.00
23.60
13.00
32.62
22.07

Table 4. Effect of biotic and abiotic elicitors on lesion number per leaf and per cent disease index (PDI)
Treatments

Lesion number/leaf*

Spraying of phylloplane Bacillus sp.

Spraying of phylloplane Pseudomonas sp. plb 7
Spraying of phylloplane Trichoderma viride
Seed treatment of root endophytic bacteria (Endo R3)
Seed treatment of stem endophytic bacteria (Endo S4)
Seed treatment of leaf endophytic bacteria (Endo L4)
Salicylic acid spray
Spraying of Pseudomonas fluorescens (Pf 1)
Streptomycin sulphate (0.1%) spray
Control (pathogen alone)
CD (P=0.05)
* Mean of three replications * *Mean of 10 leaf ratings

Per

cent

disease

(PDI) **
15.08b
10.5a
13.42b
21.67c
28.0d
26.33d
9.00a
13.08b
20.92c
30.33e
2.29

40.00 (39.23)
30.00 (33.21)
35.00(36.27)bc
45.00(42.13)d
45.00(42.13)d
45.00(42.13)d
25.00(30.00)a
32.50(34.76)bc
37.50(37.76)c
75.00(60.00)e
3.41

Values in parenthesis are arc sine transformed values

Means carrying same letters are not significantly different according to DMRT

Table 5. Effect of biotic and abiotic elicitors on disease intensity in field

Sl.No.

Treatments

1Spraying of phylloplane Bacillus sp.

index

Per cent disease index*

Field I (Checkanuarani)
FieldII (Venkatachalapuram)
22.90
21.75

2Spraying of phylloplane Pseudomonas sp. plb 7

(28.59)d
11.10

(27.80)e
10.30

3Spraying of phylloplane Trichoderma viride

(19.46)ab
26.90

(18.72)b
25.30

4Seed treatment of root endophytic bacteria (Endo R3)

(31.24)e
27.80

(30.20)g
26.50

5Seed treatment of stem endophytic bacteria (Endo S4)

(31.82)ef
29.00

(30.98)g
28.50

6Seed treatment of leaf endophytic bacteria (Endo L4)

(32.58)f
25.80

(32.27)h
23.75

7Salicylic acid spray

(30.53)e
10.30

(29.17)f
9.25

8Spraying of Pseudomonas fluorescens (Pf 1)

(18.72)a
12.40

(17.71)a
13.60

9Streptomycin sulphate (0.1%) spray

(20.62)b
15.90

(21.64)c
16.70

10

(23.50)c
31.20

(24.12)d
33.20

(35.79)g
1.68

(35.18)i
0.83

CD (P=0.05)
* Mean of three replications
Values in parenthesis are arc sine transformed values
Means carrying same letters are not significantly different

according to DMRT

1. Spraying of phylloplane Bacillus sp.

2. Spraying of phylloplane Pseudomonas sp. plb 7
3. Spraying of phylloplane Trichoderma viride
4. Seed treatment of root endophytic bacteria (Endo R3)
5. Seed treatment of stem endophytic bacteria (Endo S4)
6. Seed treatment of leaf endophytic bacteria (Endo L4)
7. Salicylic acid spray
8. Spraying of Pseudomonas fluorescens (Pf 1)
9. Streptomycin sulphate spray (0.1%)
10. Control (pathogen alone)

1. Spraying of phylloplane Bacillus sp.

2. Spraying of phylloplane Pseudomonas sp. plb 7
3. Spraying of phylloplane Trichoderma viride
4. Seed treatment of root endophytic bacteria (Endo R3)
5. Seed treatment of stem endophytic bacteria (Endo S4)
6. Seed treatment of leaf endophytic bacteria (Endo L4)
7. Salicylic acid spray
8. Spraying of Pseudomonas fluorescens (Pf 1)
9. Streptomycin sulphate spray (0.1%)
10. Control (pathogen alone)

1. Spraying of phylloplane Bacillus sp.

2. Spraying of phylloplane Pseudomonas sp. plb 7
3. Spraying of phylloplane Trichoderma viride
4. Seed treatment of root endophytic bacteria (Endo R3)
5. Seed treatment of stem endophytic bacteria (Endo S4)
6. Seed treatment of leaf endophytic bacteria (Endo L4)
7. Salicylic acid spray
8. Spraying of Pseudomonas fluorescens (Pf 1)
9. Streptomycin sulphate spray (0.1%)
10. Control (pathogen alone)

1. Spraying of phylloplane Bacillus sp.

2. Spraying of phylloplane Pseudomonas sp. plb 7
3. Spraying of phylloplane Trichoderma viride
4. Seed treatment of root endophytic bacteria (Endo R3)
5. Seed treatment of stem endophytic bacteria (Endo S4)
6. Seed treatment of leaf endophytic bacteria (Endo L4)
7. Salicylic acid spray
8. Spraying of Pseudomonas fluorescens (Pf 1)
9. Streptomycin sulphate spray (0.1%)
10. Control (pathogen alone)

1. Spraying of phylloplane Bacillus sp.

2. Spraying of phylloplane Pseudomonas sp. plb 7
3. Spraying of phylloplane Trichoderma viride
4. Seed treatment of root endophytic bacteria (Endo R3)
5. Seed treatment of stem endophytic bacteria (Endo S4)
6. Seed treatment of leaf endophytic bacteria (Endo L4)
7. Salicylic acid spray
8. Spraying of Pseudomonas fluorescens (Pf 1)
9. Streptomycin sulphate spray (0.1%)
10. Control (pathogen alone)

1. Spraying of phylloplane Bacillus sp.

2. Spraying of phylloplane Pseudomonas sp. plb 7
3. Spraying of phylloplane Trichoderma viride
4. Seed treatment of root endophytic bacteria (Endo R3)
5. Seed treatment of stem endophytic bacteria (Endo S4)
6. Seed treatment of leaf endophytic bacteria (Endo L4)
7. Salicylic acid spray
8. Spraying of Pseudomonas fluorescens (Pf 1)
9. Streptomycin sulphate spray (0.1%)
10. Control (pathogen alone)

1. Spraying of phylloplane Bacillus sp.

2. Spraying of phylloplane Pseudomonas sp. plb 7
3. Spraying of phylloplane Trichoderma viride
4. Seed treatment of root endophytic bacteria (Endo R3)
5. Seed treatment of stem endophytic bacteria (Endo S4)
6. Seed treatment of leaf endophytic bacteria (Endo L4)
7. Salicylic acid spray
8. Spraying of Pseudomonas fluorescens (Pf 1)
9. Streptomycin sulphate spray (0.1%)
10. Control (pathogen alone)

1. Spraying of phylloplane Bacillus sp.

2. Spraying of phylloplane Pseudomonas sp. plb 7
3. Spraying of phylloplane Trichoderma viride
4. Seed treatment of root endophytic bacteria (Endo R3)
5. Seed treatment of stem endophytic bacteria (Endo S4)
6. Seed treatment of leaf endophytic bacteria (Endo L4)
7. Salicylic acid spray
8. Spraying of Pseudomonas fluorescens (Pf 1)
9. Streptomycin sulphate spray (0.1%)
10. Control (pathogen alone)