385

ISSN: 0974 - 0376

KEYWORDS

Proceedings of National Conference onHarmony with Nature in Context of

Bioresources and Environmental Health(HORMONY - 2015)

November 23 - 25, 2015, Aurangabad,

organized byDepartment of Zoology,

Dr. Babasaheb Ambedkar Marathwada UniversityAurangabad (Maharashtra) 431 004

in association withNational Environmentalists Association, India

www.neaindia.org

NSave Nature to Survive

: Special issue, Vol. VII: 385-389: 2015

www.theecoscan.in

AN INTERNATIONAL QUARTERLY JOURNAL OF ENVIRONMENTAL SCIENCES

Girija Shankar et al.,

Soybean

Xanthomonas

Biological control

Epiphyt bacteria

Anatagonist

IN VITRO INVESTIGATION ON BIOCONTROL OF XANTHOMONAS

AXONOPODIS PV. GLYCINES CAUSE OF SOYBEAN BACTERIAL

PUSTULE BY SOYBEAN ANTAGONISTIC BACTERIA

386

*Corresponding author

GIRIJA SHANKAR*, PUSHPANJALI WAGH, MANOJ SAHU AND R. K. DANTREDepartment of Plant Pathology*,

College of Agriculture, Indira Gandhi KrishiVishwavidyalaya, Raipur - 492012 (Chhattisgarh)

e-mail: girijasinha23@gmail.com

INTRODUCTION

Soybean [Glycine max. (L.) Merril] belonging to family Leguminaceae is designatedas ‘miracle bean’, established its potential as an industrially vital and viable oilseedcrop in many areas of India. Soybeans are the world’s largest source of animalprotein feed and the second largest source of vegetable oil (Patil, 2014). Globalarea and production of soybean is 111.27 m ha and 276.4 million tonnesrespectively (Anonymous, 2013).

Bacterial pustule caused by Xanthomonas axonopodis pv. glycines is one of themajor bacterial diseases of soybean. The disease occurs typically on soybeanfoliageand symptoms include small, pale green spots with elevated pustules, whichmay develop into large necrotic lesions leading to premature defoliation (Narvel etal., 2001).Yield losses of up to 40% have been reported in certain parts of theworld (Prathuangwong and Amnuaykit, 1987). The control of bacterial pustule hasbeen difficult due to the high variability of the pathogen, high ability to survive indiverse environments and its extremely wide host range.

Antagonism is the balance wheel of nature. Where there is life there is antagonism.Bio-control or bio-management of pest and pathogen relies on this very fact ofnature. In the bio management, different groups of organisms from viruses, bacteria,

fungi and insects, depending on the target organisms are put into service for

management of diseases. Pseudomonas have shown effectiveness in the bio-

management of different crops. Among the bio-control bacteria, fluorescent

Pseudomonas have become the bacterium of the choice for its versatility and

ability to contain a large number of plant pathogens in diverse target

environments.Bacterial antagonists have the twin advantage of faster multiplication

and higher rhizosphere competence hence, P. fluorescens have been successfully

used for biological control of several plant pathogens (Ramamoorthy et al., 2002)

and biological control using PGPR strains especially from the genus Pseudomonas

is an effective substitute for chemical pesticides to suppress plant diseases (Compant

et al., 2005). Their applicability as bio control agents has drawn wide attention

because of the production of secondary metabolites such as siderophore, antibiotics,

volatile compounds, HCN, enzymes and phytohormones (Dewangan et al., 2014)

However, chemical control of plant diseases is usually expensive and may have a

negative impact on the environment and on public health. Biological control makesmanagement of plant diseases less dependent on the use of high-risk chemicalsand it is environmentally friendly

The objective of this investigation is to assess the antibacterial activity of theantagonists Fluorescent Pseudomonas against Xanthomonas axonopodis pv.glycines causing bacterial pustule in soyabean under in vitro and in vivo conditionboth.

MATERIALS AND METHODS

Evaluation of Fluorescent Pseudomonas against Xanthomonas axonopodis pv.glycines under in vitro condition

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Soybean bacterial pustule disease caused byXanthomonas axonopodis pv.glycines is oneof the most important diseases of soybean. Invitro evaluation, among the sixteen isolates,Pf 18 was found to be most significantly

effective in inhibiting the growth of the

pathogen and with inhibition zone of 37.33

mm, 41.00 mm and 46.00 mm after 24, 48

and 72 hrs of incubation period, respectively.

This was followed by Pf 34 (34.33 mm), Pf 33

(37.00 mm) and Pf 35 (43.00 mm) after 24,

48 and 72 hrs of incubation period,

respectively. Bio efficacy of Fluorescent

Pseudomonas was evaluated under field

condition. Highest per cent disease control of

pustule was recorded for variety Monatta

(17.80%) inhibition followed by NRC-7

(15.10%) inhibition after foliar application of

Fluorescent Pseudomonas. Different tested

strains of Fluorescent Pseudomonas bio agent,

Pf 18 was found to be most significantly

effective in inhibiting the growth of the

pathogen after 24, 48 and 72 hrs of incubation

period, respectively.

ABSTRACT

387

IN VITRO INVESTIGATION ON BIOCONTROL

Well diffusion method

The antagonistic activity of sixteen isolates of FluorescentPseudomonas towards Xanthomonas axonopodis pv. glycineswas tested by the well diffusion technique suggested by Zellerand Brulez (1987) and Vignolo et al. (1993). A suspension ofXanthomonas axonopodis pv. glycines prepared from anovernight shaken culture, was spread over the surface ofplates containing King’s B agar medium. After drying, 0.05 mlof an overnight culture of Fluorescent Pseudomonas waspipetted into 8 mm well. Plates were then incubated at 25±20Cfor 1day and examined for inhibition zones. There were fourreplicates for each treatment. The diameter of zone of inhibitionwas measured at different intervals viz., 24, 48 and 72 hrs.Finally data were statistically analyzed by using completelyrandomized design (CRD).

Bio efficacy of Fluorescent Pseudomonas against bacterialpustule under natural field condition

Three varieties viz., Panjab-1, Monatta and NRC-7 wereselected for studies on bio efficacy of FluorescentPseudomonas against bacterial pustule under natural fieldcondition. Single spray of isolates Pf 18 (FluorescentPseudomonas) was given at the time of first appearance of thesymptoms, Selected on the basis of in vitro performance.Fluorescent Pseudomonas suspension (@ 108cfu/ml) (Ran etal 2005) was thoroughly sprayed on the foliage with the helpof automizer. The treatment was continued until the finedroplets of isolates Pf 18 appeared on the foliage (Mew andRosales, 1986). The disease intensity/index was recorded after10 days of spray. Per cent disease control (PDC) was calculatedby the following formula:

Data of five randomly infected plants were recorded in eachplot after initiation of the disease, than plant is tagged. Per

plant total number of leaves and infected number of leavescounted before and after foliar spray.

RESULTS AND DISCUSSION

In vitro evaluation of Fluorescent Pseudomonas againstXanthomonas axonopodis pv. glycines

Antagonistic activity of sixteen isolates of Fluorescent

Pseudomonas were evaluated against Xathomonas

axonopodis pv. glycines under in vitro condition. All the

bacterial isolates significantly inhibited the growth of the test

pathogen. Among the sixteen isolates, Pf 18 was found to be

most significantly effective in inhibiting the growth of the

pathogen and with inhibition zone of 37.33 mm, 41.00 mm

and 46.00 mm after 24, 48 and 72 hrs of incubation period

respectively. This was followed by Pf 34 (34.33 mm) Pf 33

(37.00 mm) and Pf 35 (43.00 mm) after 24, 48 and 72 hrs of

incubation period respectively. Isolates such as Pf 25 (30.00

and 35.33 mm), Pf 33 (31.00 and 37.00 mm), Pf 34 (34.33

and 36.67 mm) and Pf 35 (30.00 and 36.33 mm) were at par

with Pf 18 (37.33 and 41.00 mm) after 24 and 48 hrs of

incubation respectively. Similarly isolated such as Pf 22 (36.33

mm), Pf 25 (39.33 mm), Pf 27 (39.00 mm) and Pf 33 (41.33

mm), Pf 34 (41.00 mm) and Pf 35 (43.00 mm) were at par to Pf

18 (46.00 mm) after 72 hrs of incubation. Least inhibition

zone was achieved by Pf 4 (8.00, 9.33 and 12.33 mm) followed

by Pf 36 (17.33, 20.33 and 23.67 mm) after 24 ,48 and 72 hrs

of incubation period respectively. The results are coincided

with the findings of Gangwar and Sinha (2012). They evaluated

seven isolates of Pseudomonas fluorescens and found that all

the isolates significantly reducing disease severity over the

control. Manmeet and Thind (2002) reported that

Pseudomonas fluorescens was inhibitory to the testXanthomonas oryzae pv. oryzae. Kumar et al. (2009) evaluatedfour bioagents against Xanthomonas oryzae pv. oryzae (BLBof rice) and results revealed that among bioagent Pseudomonas

Table 1: In vitro evaluation of Fluorescent Pseudomonas against Xanthomonas axonopodis pv. glycines

Treatment Fluorescent Pseudomonas isolates no. Zone of inhibition (mm), at different incubation period

24 hrs 48 hrs 72 hrs

T1 Pf 3 22.67 27.00 28.67T2 Pf 4 8.00 9.33 12.33

T3 Pf 8 22.00 26.00 33.00

T4 Pf 18 37.33 41.00 46.00T5 Pf 22 24.33 30.67 36.33T6 Pf 24 20.33 22.67 24.67

T7 Pf 25 30.00 35.33 39.33

T8 Pf 27 26.00 30.33 39.00T9 Pf 29 22.67 27.00 31.67T10 Pf 30 24.67 30.00 35.33

T11 Pf 33 31.00 37.00 41.33

T12 Pf 34 34.33 36.67 41.00T13 Pf 35 30.00 36.33 43.00T14 Pf 36 17.33 20.33 23.67

T15 Pf 38 20.33 23.33 27.33T16 Pf 39 28.00 31.67 34.67

T17 Control 0.00 0.00 0.00Mean 23.470 27.33 31.61Sem (+/-) 2.543 3.07 3.566

CD (5%) 7.346 8.86 10.299

x 100

Infection index in control plants – infectionindex in treatment plants

PDC =Infection index in control plants

388

Table 2: Bio efficacy of Fluorescent Pseudomonas against bacterial pustule under field condition

S. No. Varieties PDI Per cent disease controlBefore application After application

1. Punjab-1 11.60 32.81 12.072. Monatta 10.19 30.86 17.803. NRC-7 10.93 31.66 15.10

Control 12.03 37.30 0Mean 11.18 33.15 14.99

fluorescens and Trichoderma harzianum restricted maximumgrowth of pathogen. Raut et al. (2010) observed thatPseudomonas fluorescens (Pf-1) significantly inhibited thegrowth of Xanthomonas axonopodis pv. malvacearum(Bacterial blight of cotton). Workers like Giri et al. (2008) andYenjerappa et al. (2013) observed almost similar type of findings.

Bio efficacy of Fluorescent Pseudomonas against bacterialpustule under field condition

Bio efficacy of Fluorescent Pseudomonas was evaluatedagainst bacterial pustule of soybean under field condition. Itwas revealed from the experimental data presented in the Table2 that highest inhibition of pustule was recorded for varietyMonatta with (17.80%) inhibition followed by NRC-7, (15.10%)inhibition after foliar application of Fluorescent Pseudomonas.Least inhibition was found in case of Punjab-1 (12.07%). Itwas also revealed from the data that only a single variety,Monatta was least infected with the disease. Hence we maysay that variety, Monatta is the best over two other varieties.The results are coincided with the findings of Sain (2010) whoreported that Pseudomonas fluorescens significantly inhibitedthe pustules in soybean plant under field condition. Lodha(2001) recorded inhibition of bacterial blight intensity andincrement of seed yield in cluster bean after foliar applicationof bacterial antagonists. Ganeshan and Kumar (2005)suggested that application of Pseudomonas fluorescensreduces severity of various diseases. The results are inagreement with the findings of workers like Manonmani et al.(2007), Singh and Singh (2007), Raut et al. (2010) andYenjerappa et al. (2013).

ACKNOWLEDGEMENT

This research is dedicated to Dr. R. K. Dantre and Dr. A.S.Kotasthane College of Agriculture, Indira Gandhi KrishiVishwavidyalay, Raipur (Chhattisgarh). This paper is part ofdegree of Master of Science in Dept. of Plant Pathology,College of Agriculture Indira Gandhi Krishi Vishwavidyalaya,Raipur (Chhattisgarh) India.

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