This article was downloaded by:[University of Torino]On: 10 August 2007Access Details: [subscription number 778576145]Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK

Archives Of Phytopathology AndPlant ProtectionPublication details, including instructions for authors and subscription information:http://www.informaworld.com/smpp/title~content=t713454295

Rhizobacterial bioformulation for the effectivemanagement of Macrophomina root rot in mungbean

Online Publication Date: 01 October 2007To cite this Article: Saravanakumar, D., Harish, S., Loganathan, M.,Vivekananthan, R., Rajendran, L., Raguchander, T. and Samiyappan, R. (2007)'Rhizobacterial bioformulation for the effective management of Macrophomina rootrot in mungbean', Archives Of Phytopathology And Plant Protection, 40:5, 323 - 337To link to this article: DOI: 10.1080/03235400600587326URL: http://dx.doi.org/10.1080/03235400600587326

PLEASE SCROLL DOWN FOR ARTICLE

Full terms and conditions of use: http://www.informaworld.com/terms-and-conditions-of-access.pdf

This article maybe used for research, teaching and private study purposes. Any substantial or systematic reproduction,re-distribution, re-selling, loan or sub-licensing, systematic supply or distribution in any form to anyone is expresslyforbidden.

The publisher does not give any warranty express or implied or make any representation that the contents will becomplete or accurate or up to date. The accuracy of any instructions, formulae and drug doses should beindependently verified with primary sources. The publisher shall not be liable for any loss, actions, claims, proceedings,demand or costs or damages whatsoever or howsoever caused arising directly or indirectly in connection with orarising out of the use of this material.

© Taylor and Francis 2007

Dow

nloa

ded

By:

[Uni

vers

ity o

f Tor

ino]

At:

08:2

5 10

Aug

ust 2

007

Rhizobacterial bioformulation for the effective managementof Macrophomina root rot in mungbean

D. SARAVANAKUMAR, S. HARISH, M. LOGANATHAN,

R. VIVEKANANTHAN, L. RAJENDRAN, T. RAGUCHANDER, &

R. SAMIYAPPAN

Department of Plant Pathology, Centre for Plant Protection Studies, Tamil Nadu Agricultural University,

Coimbatore, India

(Received 19 October 2005)

AbstractFluorescent pseudomonads based bioformulation was evaluated for their ability to controlMacrophomina root rot disease in mungbean (Vigna mungo). P. fluorescens isolate Pf1 showed themaximum inhibition in mycelial growth of Macrophomina phaseolina under in vitro conditions.Bioformulation of Pf1 with chitin was effective in reducing the root rot incidence in green gram bothunder glasshouse and field conditions. The rhizosphere colonization of P. fluorescens was observedappreciable with the green gram plants. However, Pf1 amended with chitin colonized effectively.Furthermore, the induction of defence-related enzymes and chemicals in plants by Pf1 amended with orwithout chitin and neem were tested. Increased accumulation of defence enzymes viz., phenylalanineammonia lyase (PAL), peroxidase (PO), polyphenol oxidase (PPO), chitinase, b-1,3-glucanse andphenolics were observed in Pf1 bioformulation amended with chitin, pre-treated plants challengeinoculated with M. phaseolina under glasshouse conditions. The present study reveals that in addition todirect antagonism and plant-growth promotion, PGPR strains amended with chitin bioformulationinduced defence-related enzymes and pathogenesis related (PR) proteins which collectively enhance theresistance in green gram against the infection of M. phaseolina.

Keywords: Defence enzymes, mungbean, Macrophomina phaseolina, pathogenesis related(PR) proteins, Pseudomonas fluorescens

Introduction

In a country like India where a large population is vegetarian, the cheap and best sources of

protein are pulses, among which mungbean or green gram (Vigna mungo) is an important

pulse, contributing 20% to overall world pulse production. It is severely affected by root rot

disease caused by the soil-borne plant pathogen Macrophomina phaseolina (Tassi) Goid.

and distributed worldwide causing disease on more than 500 hosts (Sinclair 1984). Its

destructive nature on many crop plants has been well documented (Dhingra & Sinclair

1978). It is one of the destructive plant pathogens known in the tropics and sub-tropics

Correspondence: D. Saravanakumar, Department of Plant Pathology, Centre for Plant Protection Studies, Tamil Nadu Agricultural

University, Coimbatore, India. Fax: 091 422 2431672. E-mail: patsara@rediffmail.com

Archives of Phytopathology and Plant Protection

October 2007; 40(5): 323 – 337

ISSN 0323-5408 print/ISSN 1477-2906 online ª 2007 Taylor & Francis

DOI: 10.1080/03235400600587326

Dow

nloa

ded

By:

[Uni

vers

ity o

f Tor

ino]

At:

08:2

5 10

Aug

ust 2

007

(Dhingra & Sinclair 1978). Hence, management of this disease with suitable measures has

become indispensable while considering the severe incidence of the disease. Though,

fungicides have shown promising results in controlling the disease, their use has recently been

restricted due to phytotoxicity and residue problems besides causing environmental pollution

and human health hazards. In this context, biocontrol approaches using PGPR strains help to

develop an eco-friendly control strategy for managing pathogens in crop plants.

The utilization of a plant’s own defence mechanism is a fascinating arena of research which

can be systemically activated upon exposure of plants to PGPR strains or infection by

pathogen (Baker et al. 1997). This phenomenon is called induced systemic resistance

(ISR) (Tuzun & Kuc 1991). This mechanism is facilitated by PGPR organism and

operates through the activation of multiple defence compounds at sites distant from the point

of pathogen attack (Dean & Kuc 1985). Among the PGPR, fluorescent pseudomonads are

the most exploited bacteria for biological control of soil-borne and foliar plant pathogens.

In addition to disease control, it promotes the growth and development of crop plants.

In the past three decades numerous strains of fluorescent pseudomonads have been

isolated from the rhizosphere soil and plant roots by several workers and their biocontrol

activity against soil-borne and foliar pathogens was reported (Vidhyasekaran & Muthamilan

1999; Nandakumar et al. 2001a,b; Vivekananthan et al. 2004). Fluorescent pseudomonads

are non-pathogenic rhizobacteria which suppress the soil-borne pathogens

through rhizosphere colonization, antibiosis (Pierson & Weller 1994), iron chelation by

siderophore production (Jagadeesh et al. 2001) and ISR (Van Loon et al. 1998).

Earlier studies indicated that seed treatment and soil application of P. fluorescens prevented

the pathogen infection and reduced the disease incidence (Ramamoorthy & Samiyappan

2001). However, the induction of defence-related enzymes by the regulation of PGPR strains

amended with chitin and their role in phenyl propanoid metabolism is limited. The present

study has been carried out to evaluate the efficacy of PGPR strains with or without the

addition of chitin against dry root rot pathogen incited by M. phaseolina both under glass-

house and field conditions. In addition, the induction of defence-related genes in conferring

resistance against the root rot pathogen under glasshouse conditions was studied.

Materials and methods

Isolation of pathogen and biocontrol agent

The root rot pathogen M. phaseolina was isolated from infected green gram roots of the

susceptible cultivar. Pseudomonas strains were isolated from the rhizosphere of green gram. In

addition, P. fluorescens (Pf-1) culture was obtained from the Department of Plant Pathology,

Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu, India. Pure cultures of

fluorescent pseudomonads were maintained on KB slants at 48C.

In vitro testing of PGPR strains on inhibition of mycelial growth of M. phaseolina

PGPR strains were tested for their inhibition on mycelial growth of M. phaseolina by following

the dual culture technique (Dennis & Webster 1971). The bacterial culture was streaked at

one side of a Petri dish (1 cm from the edge of the plate) plated with PDA medium.

A mycelial disc (8 mm diameter) of seven days old culture of M. phaseolina was placed on

the opposite side in the Petri dish perpendicular to the bacterial streaks. The plates were

incubated at room temperature (28+ 28C) for four days. The mycelial inhibition of pathogen

was measured in millimeters when the fungus covered the full plate in control.

324 D. Saravanakumar et al.

Dow

nloa

ded

By:

[Uni

vers

ity o

f Tor

ino]

At:

08:2

5 10

Aug

ust 2

007

Efficacy of PGPR strains on plant growth under laboratory conditions

Seed bacterization. P. fluorescens strains were grown in conical flasks (250 ml) containing

100 ml of KB broth for 48 h on a rotary shaker (150 rev min71) at 28+ 28C. Cells

were collected by centrifugation at 8000 rpm for 10 min at 48C and the pellet was

resuspended in a small quantity of sterile distilled water to get bacterial of population of

36 108 cfu/ml. Seeds of susceptible green gram (cv. Co6) were surface-sterilized with 2%

sodium hypochlorite for 30 sec, rinsed in sterile distilled water and dried overnight under a

sterile air stream. One gram of seeds with 100 mg of carboxy methyl cellulose (as adhesive

material) were soaked in 10 ml of bacterial suspension for 2 h and dried overnight in a sterile

Petri dish.

Plant growth promotion. Plant growth-promoting activity of PGPR strains was assessed based

on the seedling vigour index by the standard roll towel method (ISTA 1993). Three

replications were maintained for each treatment. The root length and shoot length of

individual seedlings were measured and the germination percentage of seeds was also

calculated. The vigour index was calculated by using the formula as described by Abdul Baki

and Anderson (1973):

Vigour Index ¼ ðMean root lengthþMean shoot lengthÞ �Germination ð%Þ:

Talc-based formulation. A loopful of P. fluorescens was inoculated into the KB and incubated in

a rotary shaker at 150 rpm for 72 h at room temperature (28+ 28C). After 72 h of

incubation, the broth containing 96 108 cfu/ml was used for the preparation of talc-based

formulation. To the 400 ml of bacterial suspension, 1 kg of sterilized talc-powder, 15 g

calcium carbonate (to adjust the pH to neutral) and 10 g carboxymethyl cellulose (adhesive)

were mixed and dried under shade for 12 h as described by Vidhyasekaran and Muthamilan

(1995).

Chitin amendment with talc-based formulation of P. fluorescens. A total of 5 g of crab shell chitin

was slowly added into 100 ml of cold 0.25 N HCL with vigorous stirring and kept overnight at

48C. The mixture was filtered through glass wool into 200 ml of ice cold ethanol at 48C with

rapid stirring. The resultant chitin suspension was centrifuged at 1000 rpm for 20 min and

the chitin pellets were washed repeatedly with distilled water until pH becomes neutral. The

concentration of colloidal chitin was adjusted to 10 mg per ml by gravimetric dry method

analysis. Colloidal chitin was incorporated into KB broth at 1% (v/v). After sterilization,

P. fluorescens was inoculated in KB broth and incubated at room temperature (28+ 28C) for

72 h. The bioformulation was prepared with the 400 ml culture grown in chitin amended KB

medium, 1 kg of talc-powder, calcium carbonate 15 g and carboxymethyl cellulose 10 g as

described by Radjacommare et al. (2004).

Neem amendments with talc-based formulation of P. fluorescens. Seeds from mature dehisced

fruits of Azadirachta indica were oven dried for 2 days at 608C. The seeds were ground using

liquid nitrogen in pestle and mortar. The powdered neem was extracted with cold distilled

water (Amadioha 2000). One ml of neem seed kernel extract was added into 100 ml of King’s

B broth. After sterilization, P. fluorescens (Pf1) was inoculated into neem-based media,

incubated in a shaker and prepared formulations. P. fluorescens amended with chitin and neem

bioformulation were prepared by adding the equal quantity of Pf1 þ chitin and Pf1 þ neem-

amended broth with the talc powder.

Rhizobacterial bioformulation against root rot in mungbean 325

Dow

nloa

ded

By:

[Uni

vers

ity o

f Tor

ino]

At:

08:2

5 10

Aug

ust 2

007

Glasshouse study

Treatment of bioformulation. Seeds were soaked in double the volume of sterile distilled water

containing talc-based formulation (10 g/kg of seed). After 24 h, the suspension was drained

off and the seeds were dried under shade for 30 min and used for sowing (Vidhyasekaran et al.

1997a). Carbendazim at 2 g/kg of seed was applied as chemical treatment. Five g of talc-based

formulation per kg of soil was added 30 days after sowing. Carbendazim at 0.1% was applied

as soil-drench to the pots.

Pathogen inoculation. Pure culture of the pathogen obtained by single hyphal tip method was

used for inoculating plants (Rangaswami 1972). Sand-maize medium containing sand and

maize at 19:1 respectively was sterilized at 1218C, 15 psi for 2 h. M. phaseolina was inoculated

in sand-maize medium and incubated for 15 days at room temperature 28+ 58C for

multiplication (Riker & Riker 1936). Potting soil (redsoil:sand:cowdung manure at 1:1:1 w/w/

w) was autoclave sterilized at 1218C, 15 psi for 2 h in two consecutive days. The potting soil

incorporated with the pathogen was filled in 30-cm diameter pots. Seeds of green gram

treated with different bioformulations were sown at 20 seeds per pot separately. The pots were

watered at weekly intervals and observations were made 30 days after sowing.

Disease and yield assessment. The percent disease incidence (PI) was assessed using the

following formula:

PI ¼ Number of infected plants

Total number of seeds sown� 100

The yield attributing parameters, viz., number of pods/plant, number of seeds/pod, root

nodulations, root length and shoot length were recorded. Pulse grain yield recorded after

harvesting the crop.

Protein analysis. Protein content in the green gram root extracts were determined by the

method of Bradford (1976). Ten milligram of Coomassie brilliant blue G-250 were dissolved

in 4.7 ml of absolute alcohol and 10 ml of concentrated phosphoric acid and the volume was

made up to 100 ml with distilled water. A sample of 50 ml added to 950 ml of dye solution

and the mixture was incubated for 5 min at room temperature. The absorbance recorded

at 595 nm in GS5703 AT spectrophotometer. Bovine serum was albumin used as the standard.

Enzyme extraction

Green gram root samples were taken at 7 days interval starting from 0 day to 49 days after

inoculation of pathogen in the plants pretreated with and without Pseudomonas bioformula-

tions. Four plants were sampled from each replication of the treatment for analysis. One g of

root sample was homogenized with 2 ml of 0.1 M sodium citrate buffer (pH 5.0) at 48C.

The homogenate was centrifuged for 20 min. at 10000 rpm. The supernatant was used as

crude enzyme extract for assaying chitinase activity. Enzyme extracted with 0.1 M sodium

phosphate buffer (pH 7.0) was used for the estimation of peroxidase (PO), polyphenol

Oxidase (PPO) and phenylalanine ammonia lyase (PAL).

Phenylalanine ammonia lyase (PAL). The PAL assay was carried out as per the method

described by Ross and Sederoff (1992). The assay mixture containing 100 ml of

326 D. Saravanakumar et al.

Dow

nloa

ded

By:

[Uni

vers

ity o

f Tor

ino]

At:

08:2

5 10

Aug

ust 2

007

enzyme, 500 ml of 50 mM Tris HCl (pH 8.8) and 600 ml of 1 mM L-phenylalanine was

incubated for 60 min and the reaction was arrested by adding 2 N HCl. To the mixture,

1.5 ml of toluene was added, vortexed for 30 sec and centrifuged (1000 rpm, 5 min, 48C).

The toluene fraction containing trans-cinnamic acid was separated and measured at 290 nm

against toluene as toluene the blank. Standard curve for comparison was drawn with cinnamic

acid in toluene. The enzyme activity was expressed as nmoles of cinnamic acid min71 mg71

of protein.

Peroxidase (PO). Assay of PO activity was carried out as per the procedure described by

Hammerschmidt et al. (1982). The reaction mixture consisted of 2.5 ml of mixture

containing 0.25% (v/v) guaiacol in 0.01 M sodium phosphate buffer, pH 6.0 and 0.1 M

hydrogen peroxide. Enzyme extract (0.1 ml) was added to initiate the reaction and read at

470 nm. The boiled enzyme preparation served as blank. Activity was expressed as the

increase in absorbance at 470 nm min71 mg71 of protein.

Polyphenol oxidase (PPO). PPO activity was determined as per the procedure given by Mayer

et al. (1965). The reaction mixture consisted of 1.5 ml of 0.1 M sodium phosphate buffer (pH

6.5) and 200 ml of the enzyme extract. The reaction was started with 0.5ml of 0.01 M catechol

and the activity was expressed as changes in absorbance (in units) min71 mg71 of protein.

Phenolics content. Phenol content was estimated as per the procedure given by Zieslin and Ben-

Zaken (1993). One gram of tissue was homogenized in 10 ml of 80% methanol and agitated

for 15 min at 708C. One ml of the methanol extract was added to 5 ml of distilled water and

250 ml of Folin Ciocalteau reagent (1 N) and the solution was kept at 258C. After 3 min, 1 ml

of saturated Na2CO3 and 1 ml of distilled water was added and the reaction mixture was

incubated for 1 h at 258C. The absorption of the developed blue colour was measured using

GS 5703 AT spectrophotometer at 725 nm. The content of the total soluble phenols was

calculated according to a standard curve obtained from with catechol and expressed as

catechol equivalents mg71 of protein.

Chitinase. The colorimetric assay of chitinase was carried out as per Boller and Mauch (1988).

The reaction mixture consisted of 10 ml of 0.1 M sodium acetate buffer (pH 4.0), 0.4 ml

enzyme solution and 0.1 ml colloidal chitin (10 mg/ml). After incubation for 2 h at 378C,

the reaction was stopped by centrifugation at 1000 rpm for 3 min. An aliquot of the

supernatant (0.3 ml) was pipetted into a glass reagent tube containing 30 ml of 1 M potassium

phosphate buffer (pH 7.0) and incubated with 20 ml of 3% (w/v) snail gut enzyme. After 1 h,

the reaction mixture was brought to pH 8.9 by the addition of 70 ml of 0.1 M sodium borate

buffer (pH 9.8). The mixture was incubated in a boiling water bath for 3 min and then rapidly

cooled in an ice-water bath. Two ml of DMAB was added to the mixture, incubated

for 20 min at 378C and immediately thereafter the absorbance was measured at 585 nm.

N-acetylglucosamine (GlcNac) was used as a standard. The enzyme activity was expressed as

nmoles GlcNac equivalents min71 mg71 of protein.

b-1,3-glucanase. b-1,3-glucanase activity was assayed according to Pan et al. (1991).

The reaction mixture consisted of 62.5 ml of 4% laminarin and 62.5 ml of enzyme extract.

The reaction was carried out at 408C for 10 min. The reaction was then stopped by adding

375 ml of dinitrosalicylic acid and heated for 5 min in boiling water, vortexed and the

absorbance was measured at 500 nM. The enzyme activity was expressed as mg glucose

released min71 mg71 protein.

Rhizobacterial bioformulation against root rot in mungbean 327

Dow

nloa

ded

By:

[Uni

vers

ity o

f Tor

ino]

At:

08:2

5 10

Aug

ust 2

007

Population density

Soil samples were randomly collected from the green gram rhizosphere treated with various

bioformulations and population dynamics were analysed on 30, 45 and 60 days after sowing.

Population of Pseudomonas per g of soil was assessed using serial dilution technique.

Field study

Treatments of bioformulations. Seeds were soaked in double the volume of sterile distilled water

containing talc-based formulation (10 g/kg of seed). After 24 h, the suspension was drained off

and the seeds were dried under shade for 30 min and used for sowing (Vidhyasekaran et al.

1997a). Carbendazim at 2 g/kg of seed was applied as chemical control. In field, biocontrol

agents were applied at 2.5 kg/ha on 30 days after sowing (DAS) and carbendazim at 0.1% was

applied as soil-drench.

Disease and yield assessment. From each plot, 1 m2 area was selected randomly from three

locations. Total number of plants and infected plants were taken into account and the per

cent disease incidence (PI) was calculated using the following formula:

PI ¼ Number of infected plants

Total number of plants� 100

The yield attributing parameters, viz., number of pods/plant, number of seeds/pod were

recorded. The grain yield per ha was calculated.

Statistical analysis. The glasshouse and field trial data were analysed using the IRRISTAT

version 92-1 programme developed by the biometrics unit, International Rice Research

Institute, The Philippines. Percentage infection, growth and yield data were analysed

independently by trial. Data were subjected to analysis variances (ANOVA). Disease

incidence data were arcsine transformed before analysis. The treatment means were

compared by Duncan’s Multiple Range test (DMRT) (Gomez & Gomez 1984).

Results

In vitro screening of the PGPR strains against the pathogen

Different strains of PGPR were tested against the mycelial growth of M. phaseolina. Among

the different PGPR strains, P. fluorescens Pf1 showed the highest inhibition of M. phaseolina

with per cent inhibition of 44.55 whereas in case of PfG2 per cent inhibition was 8.44

(Table I) (Figure 1).

Efficacy of PGPR strains on plant-growth promotion

Green gram seeds treated with the different bacterial suspensions showed improvement

in plant growth parameters over untreated seeds. Pseudomonas fluorescens isolates (Pf1,

PfG1 and PfG2) were found to increase the vigour index of green gram seedlings

significantly. The increase in mean root length (12.3 cm) and shoot length (11.5 cm) due

to P. fluorescens Pf1 was significantly higher compared to the seedlings from untreated

control. The maximum vigour index of 2372 was observed in green gram seedlings treated

328 D. Saravanakumar et al.

Dow

nloa

ded

By:

[Uni

vers

ity o

f Tor

ino]

At:

08:2

5 10

Aug

ust 2

007

with Pf1 suspension and less vigour index of 1191 was recorded from untreated control

(Table II).

Glasshouse experiments

The PGPR strain which showed significantly higher vigour index and inhibition over the

mycelial growth of M. phaseolina were selected and screened against dry root rot disease

under glasshouse conditions. Among the different combinations of bioformulations used,

application of Pf1 with chitin as seed treatment and soil application resulted in less root rot

Table I. Efficacy of PGPR strains in inhibiting mycelial growth of M. phaseolina.

Treatments

Mycelial growth

of pathogen (mm)

Percent

inhibition

P. fluorescens (Pf1) 49.9 44.55a

Pf G1 62.5 31.11b

Pf G2 82.4 8.44c

Control 90.0 0.00d

Values are mean of four replications; In a column, means followed by a common letter are not significantly different

(p¼0.05) by DMRT.

Figure 1. In vitro antibiosis of PGPR strain Pf1 against M. phaseolina.

Table II. Effect of seed treatment with PGPR strains on growth attributes of green gram.

Treatments

MSL

(cm)

MRL

(cm)

Germination

(%)

Vigour

index

Percent

increase

over control

P. fluorescens (Pf1) 12.3a 11.5a 99.7a (86.99) 2372.86a 99.08

Pf G1 8.1b 7.6b 93.7b (75.46) 1471.09b 18.96

Pf G2 8.2b 7.8b 93.0b (74.65) 1488.00b 24.93

Control 7.2c 6.5c 87.0c (68.58) 1191.9c –

MSL, Mean shoot length; MRL, Mean root length; Values in parentheses are arcsine transformed; In a column,

means followed by a common letter are not significantly different (p¼ 0.05) by DMRT.

Rhizobacterial bioformulation against root rot in mungbean 329

Dow

nloa

ded

By:

[Uni

vers

ity o

f Tor

ino]

At:

08:2

5 10

Aug

ust 2

007

incidence of 0.0, 2.5 and 7.5% at 30, 45 and 60 days after sowing (DAS) respectively which

showed significant different from the untreated control plants (Table III).

The biometrical observations were taken at 30, 45 and 60 days after sowing. Again Pf1 with

chitin showed significantly higher root and shoot length throughout the study period

(Table IV). Higher yield was recorded in P. fluorescens Pf1 amended with chitin (9.25 g/plant)

bioformulation followed by Pf1 combination with chitin and neem (7.10 g/plant). Untreated

control recorded the lowest yield of 3.06 g/plant. The yield parameters, viz., number of seeds,

number of pods per plant were found to be higher in the bioformulation of Pf1 amended with

chitin treatments and recorded 201.63% yield increase over the untreated control (Table V).

Table III. Chitin amended rhizobacterial efficacy on root rot incidence of green gram under glasshouse conditions.

Treatments 30 DAS 45 DAS 60 DAS Mean

Percent

reduction

over control

Pf1 2.5b (9.05) 7.5b (15.88) 12.5c (20.70) 7.5b 74.20

Pf1þChitin (C) 0.0a (0.00) 5.0a (12.90) 7.5a (15.88) 4.1a 85.62

Pf1þNeem (N) 2.5b (9.05) 12.5d (20.70) 17.5e (24.72) 10.8d 62.78

Pf1þCþN 2.5b (9.05) 10.0e (18.42) 10.0b (18.42) 7.5c 74.20

Carbendazim 0.0a (0.00) 5.0a (12.90) 7.5a (15.88) 4.1a 85.62

Control 15.0c (22.78) 25.0f (29.99) 47.5g (43.56) 29.1f 0.00

DAS, Days After Sowing; Values in parentheses are arcsine transformed; In a column, means followed by a common

letter are not significantly different (p¼ 0.05) by DMRT.

Table V. Chitin amended rhizobacterial effect on yield of green gram under greenhouse conditions.

Treatments

Number of

seeds/pod

Number of

pods/plant

Grain yield

(g/plant)

Percent increase

over control

Pf1 (P) 12.5bc 14.5b 6.25c 104.24

Pf1þChitin (C) 15.5a 16.5a 9.23a 201.63

Pf1þNeem (N) 10.0c 13.0cd 4.20d 37.25

Pf1þCþN 13.5b 15.0ab 7.10ab 132.02

Carbendazim 12.5bc 12.0d 5.00d 63.39

Control 9.0d 8.5e 3.06e 0.00

Values in parentheses are arcsine transformed; In a column, means followed by a common letter are not significantly

different (p¼0.05) by DMRT.

Table IV. Chitin amended rhizobacterial effect on plant biometrics of green gram under glasshouse conditions.

30 DAS 45 DAS 60 DAS

Treatments

Root

length (cm)

Shoot

length (cm)

Root

length (cm)

Shoot

length (cm)

Root

length (cm)

Shoot

length (cm)

Pf1 (P) 13.4bc 38.7cd 15.1c 48.1de 18.2c 55.2ef

Pf1þChitin (C) 16.2a 42.3a 18.4a 53.9a 22.8a 67.9a

Pf1þNeem (N) 12.5d 36.8de 15.2d 47.1cd 18.2c 54.6de

Pf1þCþN 15.7a 40.2ab 17.5b 49.9b 20.3b 60.2bc

Carbendazim 11.24e 31.4f 13.2e 40.7f 16.8d 52.6f

Control 8.5f 26.8g 11.0f 35.1g 14.0e 46.9g

Values are mean of four replications; In a column, means followed by the same letter are not differ significantly (p¼0.05)

by DMRT.

330 D. Saravanakumar et al.

Dow

nloa

ded

By:

[Uni

vers

ity o

f Tor

ino]

At:

08:2

5 10

Aug

ust 2

007

P. fluorescens colonization in rhizosphere of green gram

The survival of P. fluorescens strain Pf1 was estimated from the rhizosphere of green gram at

30, 45 and 60 DAS. The maximum population of P. fluorescens (cfu) was recorded in

the treatment Pf1 with chitin. Pf1 with chitin and neem was observed to the next best

treatment for the survival and colonization of Fluorescent pseudomonads and less bacterial

population was recorded in untreated control (Table VI).

Field experiments

Efficacy of bioformulations on dry root rot of green gram. Field experiments revealed the minimum

root rot incidence of 1.3, 2.6 and 9.3% in the trial plots applied with the bioformulation of

P. fluorescens Pf1 amended with chitin at 30, 45 and 60 DAS respectively. Though Pf1 alone

significantly reduced the disease incidence when compared to untreated control, its

amendment with chitin showed highest percent reduction (82.13) in disease control followed

by Pf1 amended with chitin and neem (69.55) (Table VII).

Efficacy of PGPR strains on yield of green gram. A significantly higher number of seeds per pod

and number of pods per plant were recorded from the trial plots treated with Pf1 along with

chitin followed by Pf1 in combination with chitin and neem bioformulation. The parameters

were positively correlated with the grain yield. Pf1 mixed with chitin treated plots recorded

Table VII. Chitin amended rhizobacterial efficacy on dry root rot of green gram under field conditions.

Treatments 30 DAS 45 DAS 60 DAS Mean

Percent reduction

over control

Pf1 (P) 2.6b (9.27) 5.3c (13.30) 16.0d (23.57) 7.96c 67.65

Pf1þChitin (C) 1.3a (6.53) 2.6a (9.27) 9.3a (17.75) 4.40a 82.13

Pf1þNeem (N) 4.0c (11.53) 6.6d (14.88) 18.6e (25.54) 9.73e 60.48

Pf1þCþN 2.6b (9.27) 5.3c (13.30) 14.6c (22.45) 7.50d 69.55

Carbendazim 1.3a (6.53) 4.0b (11.53) 13.3b (21.38) 6.20b 74.83

Control 11.3a (19.64) 20.0e (26.56) 42.6f (40.74) 24.63f 0.00

Values are mean of four replications; Values in parentheses are arcsine transformed; In a column, means followed by

the same letter are not differ significantly (p¼ 0.05) by DMRT.

Table VI. Rhizosphere colonization of P. fluorescens on green gram roots under green house conditions.

Pseudomonas population/g of soil (6106)

Treatments 30 DAS 45 DAS 60 DAS

Pf1 7.14c 8.91c 8.01c

Pf1þChitin (C) 8.54a 9.85a 9.20a

Pf1þNeem (N) 5.91d 6.24d 5.92d

Pf1þCþN 7.24b 9.01b 8.62b

Carbendazim 4.02e 5.91e 5.51e

Control 1.96f 3.83f 3.61f

Values are mean of four replications; In a column, means followed by the same letter are not differ significantly (p¼0.05)

by DMRT.

Rhizobacterial bioformulation against root rot in mungbean 331

Dow

nloa

ded

By:

[Uni

vers

ity o

f Tor

ino]

At:

08:2

5 10

Aug

ust 2

007

the highest yield of 1218 kg/ha which is significantly different from all other treatments

including chemical treatment (800 kg/ha) and lower yield was recorded in the untreated

control plots (583 kg/ha). In general, all the treatments were effective in increasing the

grain yield of green gram and Pf1 combination with chitin maintained its superiority both

in increasing the yield and in reducing the disease incidence over the untreated control

(Table VIII).

PGPR-induced defence-related protein and chemicals against the root rot pathogen. The induction

of defence enzymes, viz., PO and PPO activity in green gram was studied at 7 days interval

starting from 0 – 49 days after challenge inoculation with M. phaseolina pretreated with Pf1

amended chitin bioformulation. The enzyme activity was increased up to 21 days after

challenge inoculation and the maximum induction was observed during this period. The

enzyme activity declined at 35 days after challenge inoculation in PGPR-treated plants. In

control plants, the enzyme activity started declining drastically from 21 days after challenge

inoculation (Figures 2a, 2b). The PAL enzyme activity induced by Pf1 along with chitin

bioformulation reached the maximum at 35 days after challenge inoculation and thereafter it

decreased. Moreover, PAL activity was significantly higher in PGPR treatment when

compared to untreated control (Figure 2c).

Higher levels of accumulation of phenolics was observed in green gram roots up to 28 days

in plants treated with the bioformulation of Pf1 with chitin after challenge inoculated with

M. phaseolina. In PGPR untreated plants increased phenolic content was noticed up to

21 days after challenge inoculation and thereafter it decreased drastically and remained at

lower levels throughout the experimental period (Figure 2d).

The activity of lytic enzymes such as chitinase and b-1,3-glucanse was observed at higher

levels in roots of green gram plants pretreated with Pf1 bioformulation containing chitin after

challenge inoculation with root rot pathogen. The maximum activity was recorded at 21 days

after challenge inoculation and it started decline 35 days after challenge inoculation towards

the end of experiment. Indeed, the activity was significantly higher in Pf1 with chitin treat-

ment than the untreated control (Figures 2e, 2f).

Discussion

The development of biological techniques using PGPR amended with suitable mixture

bioformulations is an emerging arena in crop protection to reduce the damage caused by plant

pathogens in economically important crops. Production of antibiotics, viz., HCN,

Table VIII. Chitin amended rhizobacterial effect on yield of green gram under field conditions.

Treatment

Number of

seeds/pod

Number of

pods/plant

Grain

yield (kg/ha)

Percent

increase

over control

Pf1 10.33c 15.33c 889c 52.48

Pf1þChitin (C) 14.66a 19.66a 1218a 108.91

Pf1þNeem (N) 9.33d 14.33d 857d 46.90

Pf1þCþN 12.33b 17.33b 984b 68.78

Carbendazim 9.33d 13.66e 800e 37.22

Control 8.66e 9.33f 583f –

Values are mean of four replications; In a column, means followed by the same letter are not differ significantly (p¼0.05)

by DMRT.

332 D. Saravanakumar et al.

Dow

nloa

ded

By:

[Uni

vers

ity o

f Tor

ino]

At:

08:2

5 10

Aug

ust 2

007

pyrrolnitrin, phenazine and 2,4-diacetyl phloroglucinol and lytic enzymes by P. fluorescens

against fungal pathogens were reported by many workers (Ramamoorthy & Samiyappan

2001; Viswanathan & Samiyappan 2001b). The above facts suggest that the inhibition of root

rot pathogen, M. phaseolina by P. fluorescens Pf1 may be due to the production of antibiotics,

siderophore mediated competition and lytic enzymes, viz., chitinase, b-1,3-glucanase which

degraded the fungal cell wall and restricted the growth of fungus under in vitro conditions.

The vigour index of green gram plants was conspicuously increased on Pf1 treatment.

Many workers have evidenced that Pseudomonas spp. has the ability to produce IAA, which is

effective for plant growth promotion (Kumar Dileep & Dube 1992; O’Sullivan & O’Gara

1992). The ipdc gene encodes for indolepyruvate decarboxylase, a key enzyme in the

indolepyruvic acid pathway, was isolated from Pseudomonas putida GR12-2 and revealed

Figure 2. Induction of defence enzymes in PGPR (strain Pf1) treated green gram plants challenge inoculated with

M. phaseolina. (a) Peroxidase; (b) Polyphenol oxidase; (c) PAL; (d) Phenolics; (e) Chitinase; (f) b-1,3-glucanase.

Rhizobacterial bioformulation against root rot in mungbean 333

Dow

nloa

ded

By:

[Uni

vers

ity o

f Tor

ino]

At:

08:2

5 10

Aug

ust 2

007

the impact of bacterial IAA in the development of the host plant root system (Patten &

Glick 2002).

In addition, PGPR increased plant growth directly by mediating the production of

secondary metabolites and phytohormones such as auxins, cytokinins or gibberellic acid

(Arshad & Frankenberger 1991; Beyeler et al. 1999) or indirectly either by suppression of

well-known diseases caused by major pathogens or by reducing the deleterious effects of

minor pathogens (Schippers et al. 1987). Glick and Bashan (1997) reported the role of ACC

deaminase from Pseudomonas in reducing the production of ethylene during stress conditions.

Plant-growth promotion by PGPR strain Pf1 in green gram seedlings might have attributed to

the induction of plant-growth hormones and suppression of deleterious pathogens or a

combination of both from the rhizosphere and spermosphere.

Seed treatment and soil application of bioformulation Pf1 amended with chitin reduced the

disease incidence of Macrophomina root rot and increased the yield both under glasshouse and

field conditions. Chitin has wider applications against various fungal pathogens (Chirkov

2002) and this justifies its use as an amendment in bioformulation mixture in the present

study. Chitin amendment increased the growth and multiplication of chitinolytic microflora

(Bell et al. 1998). Viswanathan and Samiyappan (2001a) reported that when chitin was

substituted with glycerol as a carbon source, it resulted in enhanced growth and

multiplication of pseudomonads. Yuen et al. (2001) also found that incorporation of chitin

in the medium increased bacterial population when compared to the non-chitin amended

medium and improved the efficiency of PGPR strains in reducing the severity of rust disease

in bean plants. Chitin amendment of soil may have effects in the rhizosphere, such as the

stimulation of growth of chitinolytic microorganisms (De Boer et al. 1999; Ahmed et al.

2003), their increased biocontrol activity and elicitation of plant defense proteins (Bharathi

et al. 2004). All these effects may culminate in enhancing plant protection.

Higher level expression of defence-related proteins and timely accumulation of chemicals at

the infection site certainly prevent the colonization of pathogen in green gram seedlings

treated with the bioformulation amended with chitin. PGPR strains induce the defence-

related enzymes, viz., phenylalanine ammonia lyase (PAL) that is involved in catalyzing the

deamination of L-phenylalanine to trans-cinnamic acid, which is the first step in the

biosynthesis of large class of plant secondary metabolites including phenolic compounds and

phytolexins. The maximum accumulation of PAL at 21 days of challenge inoculation

coincides with the time which is normally favourable for the pathogen to infect green gram

plants. The increased activity of PAL constituted for enhancing the resistance in green gram

plants against fungal pathogen, M. phaseolina. Induction of PAL by fluorescent pseudomo-

nads was reported in cucumber against P. aphanidermatum (Chen et al. 2000), tomato against

F.oxysporum f. sp. lycopersici (Ramamoorthy et al. 2002) and bean against Botrytis cinerea

(Zdor & Anderson 1992).

Peroxidase (PO) and polyphenol oxidase (PPO) is involved in the formation of lignin to

restrict the entry and movement of fungal pathogens in the plant system. Increased PO and

PPO activity has been shown in a number of resistant interactions involving plant pathogenic

fungi, bacteria and viruses (Chen et al. 2000; Nandakumar et al. 2001a,b; Kandan et al.

2002). Peroxidases have been implicated in a number of physiological functions that may

contribute to resistance including exudation of hydroxy-cinnamyl alcohol into free radical

intermediates (Gross 1980), phenol oxidation (Schmidt & Feucht 1980), polysaccharide cross

linking (Fry 1986), cross linking of extensin monomers (Everdeen et al. 1988) and

lignification (Walter 1992) and also associated with deposition of phenolic compounds into

plant cell walls during resistant interactions (Graham & Graham 1991). Phenolic compounds

enhance the mechanical strength of host cell wall and also inhibit the invading pathogenic

334 D. Saravanakumar et al.

Dow

nloa

ded

By:

[Uni

vers

ity o

f Tor

ino]

At:

08:2

5 10

Aug

ust 2

007

organisms. Seed treatment with P. fluorescens 63 induced the accumulation of phenolics in

tomato root tissue (M’Piga et al. 1997).

Other defence enzymes include pathogenesis related proteins such as b-1,3-glucanase and

chitinases which degrade the fungal cell wall and cause lysis of fungal cell. Chitin and glucan

oligomers released during degradation of fungal cell wall act as elicitors that elicit various

defence mechanisms in the plants. In general, fungal cells contain chitin and glucan as their

cell wall constituents. The main mode of antagonistic activity of microbes is production of

lytic enzymes (chitinases and b-1,3-glucanases) which act on cell walls of organisms with

chitin and glucan as their cell wall component (Singh et al. 1999) and also through induced

systemic resistance (Van Loon et al. 1998) in plant systems.

In field experiments, the talc-based bioformulation mixture containing Pf1 along with chitin

showed significantly less disease incidence and correspondingly resulted in enhanced yield

(Nandakumar et al. 2001a; Ahmed et al. 2003; Bharathi et al. 2004). P. fluorescens Pf1 along

with chitin amendment was effective for survival and colonization of bacteria under field

conditions. The colonization ability of Pf1 was correlated with its competitive ability against the

deleterious organisms thereby inhibiting the colonization of pathogenic organisms around the

plant system. Root colonization by rhizobacteria appears to be an important factor in plant

growth promotion and biological control (Knudsen et al. 1997; Roberts et al. 1999). Jayashree

et al. (2000) reported that disease suppression by fluorescent pseudomonads depends on its

ability to colonize rhizosphere. The colonization of roots by inoculated bacteria is an important

step in the interaction between beneficial bacteria and the host plant to reduce the attack of

deleterious pathogen (Benizri et al. 2001). The present study implies that direct and indirect

mechanisms of chitin-based rhizobacterial bioformulation are associated with the reduction in

disease incidence of root rot in green gram plants both under glasshouse and field conditions.

Hence, enhancement of disease resistance by developing newer bioformulation, viz., chitin

amended rhizobacteria requires intensive research in the field of plant disease management.

References

Abdul Baki AA, Anderson JD. 1973. Vigour determination in soybean seed by multiple criteria. Crop Sci

13:630 – 633.

Ahmed AS, Ezziyyani M, Sanchez CP, Candela ME. 2003. Effect of chitin on biological control activity of Bacillus

spp. and Trichoderma harzianum against root rot disease in pepper (Capsicum annuum) plants. Eur J Plant Pathol

109:633 – 637.

Amadioha AC. 2000. Controlling rice blast in vitro and in vivo with extracts of Azadirachta indica. Crop Prot

19:287 – 290.

Arshad M, Frankenberger WT Jr. 1991. Microbial production of plant hormones. In: Keister CDL, Cregan PB,

editors. The rhizosphere and plant growth. Dordrecht, The Netherlands: Kluwer Academic Publishers.

p 327 – 334.

Baker B, Zambryski P, Staskaawicz B, Dineshkumar SP. 1997. Signaling in plant-microbe interaction. Science

276:726 – 733.

Bell AA, Hubbard JC, Liu L, Davis RM, Subbarao KV. 1998. Effects of chitin and chitosan on the incidence and

severity of Fusarium yellows in celery. Plant Dis 82:322 – 328.

Benizri E, Baudoin E, Guckert A. 2001. Root colonization by inoculated plant growth promoting rhizobacteria.

Biocontrol Sci Technol 11:557 – 574.

Beyeler M, Keel C, Michaux P, Hass D. 1999. Enhanced production of indole-3-acetic acid by a genetically modified

strain of Pseudomonas fluorescens CHAO affects root growth of cucumber, but does not improve protection of the

plant against Pythium root rot. FEMS Microbiol Ecol 28:225 – 233.

Bharathi R, Vivekananthan R, Harish S, Ramanathan A, Samiyappan R. 2004. Rhizobacteria-based bio-formulations

for the management of fruit rot infection in chillies. Crop Prot 23:835 – 843.

Boller T, Mauch F. 1988. Colorimetric assay for chitinase. Meth Enzymol 161:430 – 435.

Bradford MM. 1976. A rapid and sensitive method for quantification of microgram quantities of protein utilizing the

principle of protein dye binding. Anal Biochem 72:248 – 254.

Rhizobacterial bioformulation against root rot in mungbean 335

Dow

nloa

ded

By:

[Uni

vers

ity o

f Tor

ino]

At:

08:2

5 10

Aug

ust 2

007

Chen C, Belanger RR, Benhamou N, Paulitz TC. 2000. Defense enzymes induced in cucumber roots by treatment

with plant-growth promoting rhizobacteria (PGPR). Physiol Mol Plant Pathol 56:13 – 23.

Chirkov SN. 2002. The antiviral activity of chitosan. Appl Biochem Microbiol 38:1 – 8.

De Boer W, Gerards S, Gunnewiek PJAK, Modderman R. 1999. Response of the chitinolytic microbial community

to chitin amendments of dune soils. Biol Fertility Soils 29(2):170 – 177.

Dean RA, Kuc J. 1985. Induced systemic protection in plants. Trends BioTechnol 3:125 – 129.

Dennis C, Webster J. 1971. Antagonistic properties of species groups of Trichoderma 1. Production of non-volatile

antibiotics. Trans Br Mycol Soc 57:25 – 39.

Dhingra OD, Sinclair JB. 1978. Biology and pathology of Macrophomina phaseolina. Vicosa, MG, Brazil: Universidade

Federal de Vicosa Press. p 125.

Everdeen KE, Kiefer S, Willard JJ, Muldoon EP, Dey PM, Li XB, Lamport DTA. 1988. Enzymic cross linkage of

monomeric extensin precursors in vitro. Plant Physiol 87:616 – 621.

Fry SC. 1986. Cross-linking of matrix polymers in the growing cell walls of angiosperms. Ann Rev Plant Physiol

37:165 – 186.

Glick BR, Bashan Y. 1997. Genetic manipulation of plant growthpromoting bacteria to enhance biocontrol of fungal

phytopathogens. Biotechnol Adv 15:353 – 378.

Gomez KA, Gomez AA. 1984. Statistical procedure for agricultural research. New York: John Wiley and Sons.

Graham MY, Graham TL. 1991. Rapid accumulation of anionic peroxidases and phenolic polymers in soybean

cotyledon tissues following treatment with Phytopthora megasperma f. sp. glycinea wall glucan. Plant Physiol

97:1445 – 1455.

Gross GG. 1980. The biochemistry of lignification. Adv Bot Res 8:25 – 63.

Hammerschmidt R, Nuckles EM, Kuc J. 1982. Association of enhanced peroxidase activity with induced systemic

resistance of cucumber to Colletotrichum lagenarium. Physiol Plant Pathol 20:73 – 82.

ISTA. 1993. Proceedings of International Seed Test Association, International rules for seed testing. Seed Sci

Technol 21:1 – 152.

Jagadeesh KS, Kulkarni JH, Krishnaraj PU. 2001. Evaluation of the role of fluorescent siderophore in the biological

control of bacterial wilt in tomato using Tn5 mutants of fluorescent Pseudomonas sp. Curr Sci 81:882 – 889.

Jayashree K, Shanmugam V, Raguchander T, Ramanathan A, Samiyappan R. 2000. Evaluation of Pseudomonas

fluorescens (Pf1) against black gram and sesame root-rot disease. J Biol Control 14:55 – 61.

Kandan A, Ramiah R, Radja Commare R, Nandakumar R, Raguchander T, Samiyappan R. 2002. Induction of

phenyl propanoid metabolism by Pseudomonas fluorescens against tomato spotted wilt virus in tomato. Folia

Microbiol 47:121 – 129.

Knudsen IMB, Hockenhull J, Jensen DF, Gerhardson B, Hokeberg M, Tahvonen R, Teperi E, Sundheim L,

Henriksen B. 1997. Selection of biological control agents for controlling soil and seed-borne diseases in the field.

Eur J Plant Pathol 103:775 – 784.

Kumar Dileep BS, Dube HC. 1992. Seed bacterization with fluorescent pseudomonads for enhanced plant growth,

yield and disease control. Soil Biol Biochem 24:539 – 542.

Mayer AM, Harel E, Shaul RB. 1965. Assay of catechol oxidase a critical comparison of methods. Phytochemistry

5:783 – 789.

M’Piga P, Belanger RR, Paulitz TC, Benhamou N. 1997. Increased resistance to Fusarium oxysporum f. sp. lycopersici

in tomato plants treated with the endophytic bacterium Pseudomonas fluorescens strain 63-28. Physiol Mol Plant

Pathol 50:301 – 320.

Nandakumar R, Babu S, Viswanathan R, Raguchander T, Samiyappan R. 2001a. Induction of systemic resistance

in rice against sheath blight disease by plant growth promoting rhizobacteria. Soil Biol Biochem 33:

603 – 612.

Nandakumar R, Babu S, Viswanathan R, Sheela J, Raguchander T, Samiyappan R. 2001b. A new bioformulation

containing plant growth promoting rhizobacterial mixture for the management of sheath blight and enhanced

grain yield in rice. Biocontrol 46:493 – 510.

O’Sullivan DJ, O’Gara F. 1992. Traits of fluorescent Pseudomonas spp. involved in suppression of plant root

pathogens. Microbiol Rev 56:662 – 676.

Pan Q, Te YS, Kuc J. 1991. A technique for detection of chitinases, b-1,3-glucanases and protein patterns after single

separation using PAGE or isoelectric focusing. Phytopathology 81:970 – 974.

Patten CL, Glick BR. 2002. Role of Pseudomonas putida indoleacetic acid in development of the host plant root

system. Appl Environ Microbiol 68:3795 – 3801.

Pierson EA, Weller DM. 1994. Use of mixtures of fluorescent pseudomonads to suppress take-all and improve the

growth of wheat. Phytopathology 84:940 – 947.

Radjacommare R, Ramanathan A, Kandan A, Sible GV, Harish S, Samiyappan R. 2004. Purification and anti-fungal

activity of chitinase against Pyricularia grisea in finger millet. World J Micro Biotech 20:251 – 256.

336 D. Saravanakumar et al.

Dow

nloa

ded

By:

[Uni

vers

ity o

f Tor

ino]

At:

08:2

5 10

Aug

ust 2

007

Ramamoorthy V, Samiyappan R. 2001. Induction of defense-related genes in Pseudomonas fluorescens treated chilli

plants in response to infection by Colletotrichum capsici. J Mycol Plant Pathol 31:146 – 155.

Ramamoorthy V, Raguchander T, Samiyappan R. 2002. Induction of defense protein in tomato roots treated with

Pseudomonas fluorescens Pf1 and Fusarium oxysporum f. sp. lycopersici. Plant Soil 239:55 – 68.

Rangaswami G. 1972. Diseases of crop plants in India. New Delhi: Prentice Hall of India Pvt. Ltd. p 520.

Riker AJ, Riker RS. 1936. Introduction to research on plant diseases. New York: John Swift. p 117.

Roberts DP, Dery PD, Yeel I, Buyer JS, Holtman MA, Kobayashi DY. 1999. Role of pfkA and general carbohydrate

catabolism in seed colonization by Enterobacter cloacae. Appl Environ Microbiol 65:2513 – 2519.

Ross WW, Sederoff RR. 1992. Phenylalanine ammonia lyase from loblolly pine: Purification of the enzyme and

isolation of complementary DNA clones. Plant Physiol 98:380 – 386.

Schippers B, Bakker AW, Bakker PAHM. 1987. Interactions of deleterious and benefical rhizosphere microorganisms

and the effect of cropping practices. Ann Rev Phytopathol 25:339 – 358.

Schmidt PS, Feucht W. 1980. Tissue specific oxidation browning of polyphenols by peroxidase in cherry shoots.

Gartenbauwissenschaft 45:68 – 73.

Sinclair JB. 1984. Compendium of soybean disease. 2nd ed. St Paul, MN: American Phytopathology Society.

Singh PP, Shin YC, Park CS, Chung YR. 1999. Biological control of Fusarium wilt of cucumber by chitinolytic

bacteria. Phytopathology 89:92 – 99.

Tuzun S, Kuc J. 1991. Plant immunization: An alternative to pesticides for control of plant diseases in greenhouse

and field. In: Bay-Peterson J, editor. The biological control of plant diseases. Taiwan: Food and Fertilizer

technology centre. p 30 – 40.

Van Loon LC, Baker PAHM, Pieterse CMJ. 1998. Systemic resistance induced by rhizosphere bacteria. Ann Rev

Phytopathol 36:453 – 483.

Vidhyasekaran P, Muthamilan M. 1995. Development of formulations of Pseudomonas fluorescens for control of

chickpea wilt. Plant Dis 79:782 – 786.

Vidhyasekaran P, Muthamilan M. 1999. Evaluation of powder formulation of Pseudomonas fluorescens Pf1 for control

of rice sheath blight. Biocontrol Sci Technol 9:67 – 74.

Vidhyasekaran P, Rabindran R, Muthamilan M, Nayar K, Rajappan K, Subramanian N, Vasumathi K. 1997a.

Development of powder formulation of Pseudomonas fluorescens for control of rice blast. Plant Pathol

46:291 – 297.

Viswanathan R, Samiyappan R. 2001a. Role of chitinases in Pseudomonas spp. induced systemic resistance against

Colletotrichum falcatum Went in sugarcane. Ind Phytopathol 54:418 – 423.

Viswanathan R, Samiyappan R. 2001b. Antifungal activity of chitinase produced by some fluorescent pseudomonads

against Colletotrichum falcatum Went causing red rot disease in sugarcane. Microbiol Res 155:309 – 314.

Vivekananthan R, Ravi M, Ramanathan A, Samiyappan R. 2004. Lytic enzymes induced by Pseudomonas fluorescens

and other biocontrol organisms mediate defence against the anthracnose pathogen in mango. World J Micro

Biotech 20:235 – 244.

Walter MH. 1992. Regulation of lignification in defense. In: Boller T, Meins F, editors. Genes involved in plant

defences. New York: Springer-Verlag. p 327 – 352.

Yuen GY, Steadman JR, Lindgren DT, Schaff D, Jochum C. 2001. Bean rust biological control using bacterial

agents. Crop Prot 20:395 – 402.

Zdor RE, Anderson AJ. 1992. Influence of root colonizing bacteria on the defence responses in bean. Plant Soil

140:99 – 107.

Zieslin N, Ben-Zaken R. 1993. Peroxidase activity and presence of phenolic substances in peduncles of rose flowers.

Plant Physiol Biochem 31:333 – 339.

Rhizobacterial bioformulation against root rot in mungbean 337