Biological management of bacterial leaf spot of mungbean

Indian Phytopath. 53 (4) : 384-394 (2000)

Biological management of bacterial leaf spot of mungbean causedby Xanthomonas axonopodis pv. vignaeradiatae

P. K. BORAH*, lK. JINDAL and J.P. VERMADivision of Plant Pathology, Indian Agricultural Research Institute, New Delhi 110 012

ABSTRACT: As many as 110 bacterial cultures were established from mungbean (Vigna radiata L.) phylloplane show-ing distinct colour and colony characters. Besides, a bacteriophage was also isolated from the Xanthomonas axonopodispv. vignaeradiatae (Xav) infected mungbean plants. Antagonistic nature of these phylloplane bacteria (Plb) and phage(XMP"') against the bacterial leaf spot pathogen were tested in vitro. Five isolates were found antagonistic, exhibitinginhibition zones ranging from 6 mm to 25 mm. The most promising antagonistic phylloplane bacteria (Plb-2) wasidentified to be a strain of Bacillus sp. Effectiveness of Plb-2 and XMP-l as biocontrol agents in the suppression of Xavfrom seed, seed coat and cotyledon of mugbean was assessed. The pathogen was effectively eliminated from mugbeanseeds when these were dipped in a mixture of Xav and Plb at 1:1 ratio. The phage multiplied in mugbean seeds inpresence of Xav, but it failed to eliminate Xav completely from seed, seed coat and cotyledons even at 1:60 (Xav : phage)concentration. Maximum reduction in susceptible reaction was observed when Plb was pre-inoculated 24 h prior tochallenge inoculation of Xav at 1:2. No protection was obtained when Xav was challenged by Plb at all concentrationstested. The phage was also effective in providing protection when challenge inoculation (24 h) was done with Xav.Application of the phage reduced the Xav population in plants to levels insufficient to induce susceptible reaction.Increased population of Plb (4.5 x 10' from 5.5 x 103 cfulcm') in challenge inoculation by Xav or vise-versa (4.5 x 10'from 6.0 x 10" cfulcm') were recorded, which suggested that Plb could multiply in presence of Xav in mungbean leaves.Seed treatment with Plb at 2:1 ratio (Xav : Plb) and with phage at 1:60 (Xav : phage) ratio were found effective instimulating germination and restricting seedling infection to 10.0 and 15.9 per cent, respectively, as compared to 68 percent in control. The results indicated that Plb and phage strains antagonistic against Xav, during the present study couldbe exploited as potential biocontrol agents in mangement of bacterial leaf spot of mungbean.

Key words: Mungbean, Vigna radiata, phylloplane bacteria, phage, biocontrol, Xanthomonas axonopodis pv. vignaeradiatae,bacterial leaf spot.

Among various boiocontrol agents, phylloplanebacteria (Plb) and bacteriophage (phages) has been re-ported to be effective when used as seed treatment tocontrol seedling diseases in many crop plants (Labedeva,1937; Rao, 1970; Verma et al., 1978; Santhi et al.,1987; Liao, 1989; Parashar et al., 1992). Xanthomonasaxonopodis pv. vignaeradiatae, the incitant of bacte-rial leaf spot of mungbean and phages of Xav are widelydistributed in India and are easily isolated from soil,infected seeds and diseased plants (Jindal and Patel,1981). Although phylloplane bacteria and bacterioph-ages influence the important factors in the ecology ofthe pathogenic bacteria, their precise role in diseasedevelopment process(es) is not clearly understood. Some

* Present address: Department of Plant Pathology, AssamAgricultural University, Jorhat 785 013

information are available with regard to the manage-ment of bacterial leaf spot disease of mungbean usingPlb in seeds and plants (Thind and Jindal, 1988; Hoa,1991 et al., 1993; Jindal and Thind, 1994). However,no information is available on the use of bactriophagesin management of bacterial leaf spot of mungbean. Thiscommunication deals with the interaction of Plb andbacteriophage with Xav with a view to resolve theirpotential as biocontrol agents.

MATERIALS AND METHODS

Bacterial strains, phage and culture media

Isolation of XavDiseased leaves of mungbean plants (Vigna ra-

diata), were collected from the experimental fields ofIndian Agricultural Research Institute, New Delhi. Iso-lation was done from young growing spots by streak

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plate method (Jindal, 1976) on modified Sucrose Pep-tone Agar (SPA) media (Na2HP0412HP - 2.0 g, Ca(N0))2 4Hp - 0.5 g, FeS04 - 0.5 g, Peptone - 5.0 g,Sucrose - 20.0 g, Agar - 20.0 g, pH 6.8). A number ofindividual colonies developed after 5-6 days at 26° +1°C. These were picked up and purified twice by dilu-tion plate technique and finally purified single colonyculture was maintained on Yeast Glucose Chalk Agar(YGCA) slants (Yeast extract - 10.0 g, Glucose - 10.0g, calcium carbonate - 20.0 g, Agar - 20.0 g) at 5° -10°C in the refrigerator by periodic transfer.

To a 24 h actively growing YGCA slant culture,10 ml sterile water was added and shaken vigorouslyto obtain bacterial suspension. This was allowed to standfor 5 min for the agar bits to settle down and only thesupernatant was used. This suspension is reffered to asconcentrated bacterial suspension in present study.

Isolation of Plb

Apparently healthy leaves were collected fromdifferent positions of susceptible mungbean cultivarPusa Baisakhi (upper, middle and lower) grown in thefield of Indian Agricultural Research Institute. The Plbwere isolated by using leaf washing method (Hoa,1991). Five gram leaves were taken in a conical flaskwith 100 ml sterile water and stirred vigorously withsterile glass rod for 10 min. Serial dilutions were pre-pared by transferring I ml of leaf washing to 9 mlsterile distilled water blanks. An aliquot of 0.1 ml ofeach dilution was spread with glass spatula on the Nu-trient Sucrose Agar (NSA) media (Beef extract - 3.0 g,Peptone 5.0 g, Sucrose - 5.0 g, Agar - 20.0 g, pH -6.8)containing 0.05 g cycloheximide per litre to avoid fun-gal contamination. The plates were incubated at 26°±1°C for 48 h. Colonies of different shapes, size andcolour were picked up on YGCA slants. These colo-nies were further purified by streaking on NSAplates. Single colonies were picked up and maintainedon YGCA slants for further studies.

In vitro antagonism of Plb against Xav

For testing antagonists against Xav, 1 ml of Xavsuspension (0.1 O.D. at 620 nm wave length) was mixedwith 25 ml melted and cooled (40° - 45°C) SPA andpoured into petriplates. The solidified plates were spotinoculated with the 24 h old growth of Plb isolates.After 3 days of incubation at 26° ± 1°C, the plates wereexamined for antagonistic action indicated by the ap-pearance of inhibition zones and the diameter of clearzones were measured. Plates without Plb served ascontrol.

Indian Phytopathology 385

Morphological, cultural and physiological charac-ters of Plb

Staining procedures for the study of morphologywas followed as recommended in the "Manual of Mi-crobiological Methods" published by the Society ofAmerican Bacteriologists (Anon., 1957). The shape andarrangement of bacterial cells were determined bysimple staining with crystal violet. Hucker's identifica-tion of gram stain was used to determine the gramreaction. For spore and capsule staining, Domer'smethods modified by Sayder and Liefson's method,respectively, were used. Unless otherwise stated, themedia, test reagents, indicators were the same as givenby Dye (1962).

To determine the shape, size and flagellation ofthe phylloplane bacteria, electron microscopy was donefollowing standard methods.

Isolation of phageThe isolation and characterisation of phage was

done following the methods of Duttta Majumdar andVerma (1988). One gram diseased chopped leaves ofmungbean collected from tile experimental farm of In-dian Agricultural Research Institute, New Delhi, wassuspended in 50 ml sterile water in 100 ml conicalflasks and incubated at 26° ± 1°C for 48 h to releasethe phage particles. The supernatant was then decantedand passed through Whatman filter paper No. 42. Chlo-roform was added to the filtrate so as to give 1:9 ratio(CHCl) : filtrate). It was then shaken well and kept for12 h in the refrigerator. The individual supernatantswere tested for the presence of phage by spot testmethod. One ml of concentrated Xav suspension wasmixed with 25 ml of the melted cooled (40°-45°C)Sucrose Peptone Agar (SPA) media (Na2HP04 12Hp- 2.0 g, Ca (NO))2 4Hp - 0.5 g, Fe S04 • 0.5 g, pep-tone 5.0 g, Sucrose-20.0 g, Agar - 20>Qg, pH-6.8) andpoured in a sterile petriplate. On the solidified agarsurface, the supernatant was seeded by placing a loopful(3 mm diameter) of suspension. The plates were incu-bated at 26° ± 1°C for 24 h, after which they wereobserved for lytic action of the phage, indicated by theclearing of bacterial growth at the site of seeding.

When lytic areas developed, 0.5 em agar piecefrom the lytic spot was suspended in 5 ml sterile waterand lO-fold dilutions were made. One ml of the phagesuspension at the desired dilution was mixed with I mlconcentrated bacterial suspension of Xav in a steriletube. The phage bacterial mixture was mixed with 25ml melted cooled (40° - 45°C) 1.5 per cent SPA in 100ml flasks. The contents were mixed thoroughly and

386 Indian Phytopathology

poured in a sterile plate. After solidification of agar,the plates were incubated at 26° ± 1°C for plaque de-velopment. After 3 successive platings of single plaquematerial, I representative plaque was used for prepar-ing the phage stock solution.

Preparation of high titer phage stock

Purified single plaque phage suspension was platedin a dilution series with Xav. Five plates that had theproper phage concentration for achieving almostconfluent lysis were processed for making the hightiter phage stock suspension. Five ml of the steriledistilled water was added to each plate and allowed tostand for 30 min. After that sterile glass spreader wasgently moved over the confluent lytic area. The phagesuspension thus obtained was filtered through Whatmanfilter Paper No. 42. Chloroform was added to 1:9 ratio(CHCI, : filtrate). After keeping for 12 h in the refrig-erator the supernatant was transferred in screw cap tubesand stored in refrigerator.

Interaction between Plb and Xav in seedsSurface sterilised seeds of mungbean (cv. Pusa

Baisakhi) were soaked for 6 h in suspension of Xav(1.0 x 107 cfulml) and Plb (0.5 x 107cfu/ml), tenta-tively identified as Bacillus sp, in the ratio of 1:1, 2: 1and 4: 1. Ten seeds were kept for each treatment.Population of Xav and Plb were estimated after dryingthe seeds for 1 h after the treatment. Five whole seeds,seed coats and cotyledons of other five seeds werethoroughly macerated separately (in sterilised water).

From the above suspensions, 0.1 ml was plated onSPA plates and incubated at 26° ± 1°C, for 4 days.Bacterial population on seeds, seed coat and cotyledonwas estimated on the basis of number of colonies (cfu)recovered.

Interaction between phage and Xav in seedsSurface sterilised seeds of mungbean (cv. Pusa

Baisakhi) were soaked for 6 h in Xav : phage suspen-sion at 3 different ratios, i.e., 1:1, 1:30 and 1:60. Popu-lation of Xav in each case was ascertained by dryingthe seeds 1 h after the treatment. Five seeds, seed coatsand the cotyledons from each treatment were thoroughlymacerated separately in 2 ml sterillsed water. Fromeach suspension 0.1 ml was poured on SPA plates andspread with a sterillsed glass spreader. Inoculated plateswere incubated at 26° ± 1°C for 4 days and bacterialpopulation on seed coat and cotyledons was estimatedon the basis of number of colonies developed. For es-timation of phage population, the seeds and its partswere macerated in sterile pestle and mortar with 10 mlsterile phosphate bufer (pH-7.0). After shaking well, it

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was centrifuged at 15000 rpm for 15 min. The super-natant thus obtained was mixed with chloroform, whichwas allowed to settle down at the bottom of the tube.Supernatant thus obtained was serially diluted and platedon SPA seeded with Xav and incubated at 26° ± 1°Cfor 24 h. The plates were observed for the formation ofplaques. The clear zones produced in plates were dueto the lytic activity of phage particles.

Interaction between Plb and Xav in plants

Thirty days old mungbean plants (cv. PusaBaisakhi) were used in the experiment. Plb (Bacillussp.) [0.5 x 107 cfulml] and Xav (1.0 x 107cfu/ml) wereused for pre-inoculation and challenge inoculation ofthe plants in the ratio of 1:2, 1:4 and 1:8 (Plb : Xav)and 3 challenge time intervals of 0, 1 and 24 h. Theinoculations were done by the syringe infiltrationmethod (Klement, 1963) during 8.00 A.M. - 11.00 A.M.when most of the stomata remained open. Water soakedareas (about 2 ern) were developed on the adaxial sideof the leaflets of 2 youngest trifoliates in each plant byinjecting desired amount of bacterial suspension withthe help of a Glassvan 2 ml syringe fitted with 22gauge hypodermic needle. The challenge inoculationswere done at the same spot on the preinoculated leaf.Leaves inoculated with Xav alone served as control.The study was carried out during July to Septemberwhen the temperature ranged between 20°C and 37°Cand humidity was around 80 per cent. Disease severitywas recorded 2, 4, 6 and 10 days after inoculationfollowing the scale described by Hoa (1991). Per centreduction in susceptible reaction was calculated withthe following formula of Verma et al. (1994):

Grade obtainedReduction = x 100 - 100

Maximum grade

Interaction between Xav and phage in plantsThe bacteriophage XMP-I (6.0 x 109 pfu/ml) and

Xav (1.0 x 107 cfu/ml) were inoculated on 30 day oldmungbean (cv. Pusa Baisakhi) by syringe infiltrationmethod (Klement, 1963) in pots. The inoculation ofone organism was followed by the challenge inocula-tion of the second and vice- versa in the ratio of I: 1,1:30 and 1:60 (Xav : phage). The challenge inocula-tion was done 0, 1 and 24 h after the first inoculation.Co-inoculation with phage and Xav served as 0 h chal-lenge inoculation. The inoculated plants were main-tained at 20° - 37°C in pots and observed for the ap-pearance of disease symptoms. Disease severity wasrecorded 2, 4, 6 and 10 days after inoculation follow-ing the scale described earlier and per cent reduction insusceptible reaction was calculated similarly.

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Population of Xav and its Plb in plants

Inoculations were also done with Xav and Plb(Bacillus sp.) individually, as well as the preinoculationof one followed by the challenge inoculation by theother and vice-versa. The challenge time was 24h andthe ratio of Plb and Xav was 1:2. Population of bothPlb and Xav were estimated 0, 1, 2, 3, 4 and 5 daysafter challenge inoculation by maceration method. Thetests were run in triplicates and leaves of similar posi-tion and age were selected for inoculation on plantsraised in pots. Tissue discs (5 mm) were cut (3 discs/leaf and 3 leaves/estimation) with the help of cork borerfrom inoculated area of the leaf of each treatment andmacerated separately in sterile water. From the abovesuspension, 0.1 ml was plated on SPA plate (contain-ing 0.05 g cyclohexamide/l) and incubated at 260 ±10Cfor 4 days. Bacterial population was estimated onthe basis of number of colonies (cfu) recovered. Si-multaneously, ooze test was also performed to corrobo-rate the visual symptoms with the multiplication of thepathogen in plant tissues.

Population of Xav and its phage in mungbeanplants

Inoculations using syringe infiltration technique(Klement, 1963) were carried out with Xav, phage andin combinations as challenge inoculation. After 24 h ofinoculation, it was challenged with either Xav or ph-age. The ratio of phages and Xav was 60: 1. Populationof both phages and bacteria was estimated at 0, 1, 2, 3,4 and 5 days after challenge inoculation. Tissue discs(5 mm size) were cut (3 discs/ leaf and 3 leaves/esti-mation) with the help of cork borer from inoculatedarea of the leaf at different time intervals and macer-ated separately. For estimation of Xav population, 0.1ml suspension from each treatment was poured on aSPA plate (containing 0.05 g cycJohexamide/l) andspreadwith a sterilised glass spreader. Inoculated plateswere incubated at 260 ± I°C for 4 days and bacterialpopulation in leaves was estimated on the basis ofnumber of colonies (cfu) recovered. The phage popu-lation was estimated by macerating the inoculated tis-sue discs in sterile pestle and mortar with 10 ml sterilephosphate buffer (pH 7.0). After shaking well, it wascentrifuged at 15,000 rpm for 15 min. The supernatantthus obtained was mixed with chloroform, which wasallowed to settle down at the bottom of the tube. Su-pernatant thus obtained was serially diluted and platedon SPA seeded with Xav and inoculated at 260 ± I°Cfor 24 h. The plates were observed for the formation ofplaques (pfu). The clear zones produced in plates weredue to the lytic activity of phage particles. Sirnulta-


neously, ooze test was also performed to corroboratethe visual symptoms with the multiplication of thepathogen inplant tissue. The tests were run in triplicateand leaves of similar position and age were selectedfor inoculation on plants raised in pots.

Effect of seed treatment with Plb and phage ongermination and seedling infection in mungbean

Surface sterillsed mungbean seeds (cv. PusaBaisakhi) were soaked for 6 h in bacterial suspensionof Xav (1.0 x 107 cfu/ml) and Plb (0.5 x 107 cfu/ml),i.e., Bacillus sp. in the ratio of 2: I and phage lysate(6.0 x 109 pfu/ml) in the ratio of I:60, After drying forI h under the shade, the seeds were sown in 8 inchearthen pots. The observations were recorded on per-centage germination and seedling infection after 20days.

RESULTS AND DISCUSSION

In vitro antagonism of phylloplane bacteriaagainst Xav

During isolations, 4 different types of colony, i.e.,white, yellow, pink and other types were observed and110 bacterial cultures were established from mungbeanphylloplane showing distinct colour and colony char-acters. The isolated Plb were tested for their antagonis-tic effect against Xav in vitro. Of these, 5 isolates werefound antagonistic causing inhibition zones rangingfrom 6 mm to 22 mm (Table I). Plb-I and Plb-2produced larger inhibition to 22 mm zones of 22 mmfollowed by Plb-3 with 20 mm (Fig. I). Hoa (1991)also isolated Plb from mungbean, which producedmaximum inhibition zone of 17 mm. The antagonisticbehaviour of different mungbean phylloplane bacteriawas attributed to the production of chemical substancesin the culture medium (Jindal and Thind, 1993). Incontrast, none of the Plb of cotton possessing pre-in-oculative protective ability was antagonistic against Xa. pv. malvacearum in vitro, indicating non-productionof inhibitory substances by the Plb in culture media(Chowdhury and Verma, 1980b; Verma, 1986).

Table 1. In vitro antagonism of phylloplane bacteria againstXanthomonas axonopodis pv. vignaeradiatae.

SI. Original New Diameter ofNo. isolate isolate inhibition

number number zone (mm)

1 Plb-S Plb- I 222. Plb-13 Plb-2 223. Pib-26 Plb-3 204. Plb-32 Plb-4 8S. Plb-30 Plb-S 6


Fig.1. Antagonistic effect of Phylloplane bacteria againstXav. A = Plb - I (22 mm), B = Plb - 2 (22 mm),C = Plb - 3 (20 mm)

Morphological, cultural and physiological charac-ters of Plb

On the basis of the morphological, cultural, bio-chemical and electron microscopic studies described inthe first edition of the Bergey's Manual of systematicBacteriology (Anon, 1984), the Plb-2 isolate was ten-tatively identified upto generic level as Bacillus sp. Itwas short rod with rounded end (2.0 x 0.5 J.(m) in size,scattered or in pairs, gram positive, peritrichous flagel-lation, spore forming and capsulated, Colony smoothbut the surface was rough and wrinkled, irregular, flat,opaque, margin irregular, creamy white, filiform growthon slants, facultive anaerobe, catalase positive and pro-duced ammonia. The results were summarized in theTable 2. Jindal and Thind (1989), Hoa (1991) and Boraet al. (1993) also reported different genera of Plb in-cluding Bacillus sp. from mungbean.

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Table 2. Morphological, cultural and biochemical charactersof phylloplane bacteria of mungbean (Plb-2) an-tagonistic to Xanthomonas axonopodis pv.vignaeradiatae

SI. CharactersNo.

Plb-2

A. MorphologicalI Shape2. Size3. Arrangement of cell4. Gram reaction5. Spore6. Flagella7. Capsule

B. Cultural

I. Colony charactersafter 48 h at 28°C(on NNSPA) andgrowth on solidmedium

2. Oxygen requirement

C. Biochemical

I. Catalase activity +ve2. Production of NHJ +ve

Short rod2.0 x 0.5-1 ILmScattered or in pairs+ve+ve+ve (peritrichous)+ve

Colonies irregular, flat, marginirregular, opaque, smooth,filiform, creamy white(18-20) mm in dia.

Facultative anaerobe

Isolation of phagesXav bacteriophage isolate (XMP-I) was established

from infected mungbean leaves (cv. Pusa Baisakhi).The lytic activity of the phage isolate was tested againstXanthomonas axonopodis pv. vignaeradiatae on SPAplates by spot test method which showed clear lyticzone on the spotted area.

Interaction between Xav and Plb in seedsNot a single colony forming unit (cfu) of Xav could

be recovered from seeds and seed parts in treatmentsof Xav : Plb (Bacillus sp.) at I: I ratio (Table 3), indi-

Table 3. Recovery of Xanthomonas axonopodis pv. vignaeradiatae and phylloplane bacteria (Bacillus sp.) after 6 h soaking inmungbean seeds

TreatmentsRatio ofXav: Plb Seed

Number of cfu recovered/seed/seed parts after 6 h of treatment

CotyledonXav ' Plb

Seed coat Cotyledon Seed Seed coat

1:12:14:11:0 (Xav alone)0: I (Plb alone)

o1.5 x I(}'

3.4 x 105

9.6 X 106

0 0 1.0 x 10" 1.0 x 105

9.0 x 10" 1.5 x 10' 7.0 x 10" 3.0 x 10'1.7 X 105 5.5 X 10' 3.5 x 10" 1.5 x 10'5.6 x 10" 1.5 x 106

8.0 X 106 3.7 X 106

5.0 x 10"1.5 x 10"1.0 x 10"

1.0 x 106

Initial concentration = 0.1 0.0. of bacteria (1.0 x 107 cfulml) at 620 nm, 0 = No cfu could be recovered

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eating complete inactivation of Xav on mungbean seedsby antagonistic action of Plb. At 2 : 1 and 4 : 1 ratios(Xav : Plb) the population of Xav could not be elimi-nated completely but was reduced considerably. There-fore, Xav : Plb at 1 : 1 ratio proved highly effective ineradicating seed infection of mungbean by Xav. Ear-lier, Vasudeva et at. (1958) isolated an antibiotic pro-duced by B. subtilis and named as bulbiformin. Thindand lindal (1988); lindal and Thind (1989) and lindaland Thind (1990) also found significant control ofseedbome infection of mungbean (Xav) and cowpea(X.a. pv. vignicola) using Plb viz., Erwinia herbicola,Bacillus spp., Pseudomonas fluorescens andAcenetobacter calcoaceticus.

Interaction between Xav and phage in seeds

The results (Table 4) of interaction between Xavand phage (XMP-l) in mungbe.an seeds revealed thatthe phage could not eliminate Xav completely fromseed, seed coat, and cotyledons, even at an applicationratio of 1:60 (Xav : phage), however, the bacterial popu-lation was reduced to a considerable level. In each treat-ment, the phage population increased as compared tocontrol (phage alone), indicating that in presence ofhost bacterium (Xav) phage multiplied in seed. Bacte-riophages have also been tried earlier for the control ofseed-borne bacterial pathogens. Thomas (1935) foundthat seed com from infected crop yielded bacterioph-ages and the seed developed only 5 per cent infectedplants. On treating 18 commercial seed lots with bac-teriophages and then inoculation with Xc. pv. stewartii,he obtained 1.47 per cent infection as compared to 18per cent in the phage untreated samples. In addition totheir roles in biocontrol, bacteriophages have beenwidely used in detection of seed-borne pathogens ofvarious crop plants (Keil and Wilson, 1963; Soni andThind, 1991, Kahveci and Maden, 1994).


Interaction between Xav and Plb in plants

Results of interaction between Xav and Plb (Bacil-lus sp.) in plants are presented in Table 5. Maximumreduction (60%) of susceptible reaction (SR) was ob-served at 1:2 ratio of Plb and Xav after 24 h challengeby Xav. The reduction in SR was also recorded inchallenge inoculations of Xav at 0, I and 24 h at allratios tested except in case of on challenge in the ratio1:8. Density dependent mechanisms such as coloniza-tion of sites and/or nutrient acquisition may be likelyexplanation for the reduction of pathogen populationfollowing preinoculation of antagonists on thephylloplane. The time gap further helped the antago-nists to produce antibacterial substances in sufficientquantity to inhibit the pathogen. Similar observationswere made by Sinha and Verma (1984), Jindal andThind (1993) and Bora et al. (1993). This also indi-cated ineffectiveness of pre-inoculation treatment ofXav irrespective of its concentration. It may be con-cluded that, among the three concentrations evaluated,1:2 ratio of Plb and Xav was most effective in check-ing susceptible reaction when challenged at 24 h byXav.

Pre-inoculation of Plb followed by challenge in-oculation of Xav after 24 h at a ratio of 49: 1 (Plb :Xav) could effectively control the bacterial leaf spot ofmungbean caused by Xav (Bora et al., 1993) andcowpea caused by X.a. pv. vignicola (Jindal and Thind,1993). This clearly showed that Plb (Bacillus sp.) iso-lated in the present studies was more aggressive ascompared to Erwinia herbicola reported by Bora et·al.(1993). Maximum protection of bacterial blight of soy-bean caused by P. glycinea was observed at 9: I ratio(plb : P. glycinea) but not at I : 1 ratio (Scherff, 1972).Sinha (1981) also reported almost JOO per cent protec-tion against bacterial blight of cotton at 2: 1 ratio (PlbXam).

Table 4. Interaction between Xav and Phage (XMP-I) in mungbean seeds

No. of cfu recovered No. of pfu recoveredRatio of Xav Phage (XMP-l)Xav : Phage Seed Seed coat Cotyledon Seed Seed coat Cotyledon

Control (Xav alone) 8.8 x 106 4.0 x 100 1.1 x 100Control (Phage alone) 1.5 x 103 1.0 x 10' 2.5 x 102

Xav : Phage (I : I) 2.9 x 105 9.0 x 10" 1.2 X 105 6.2 X 103 2.3 x 10' 5.0 x 102

Xav : Phage (I : 30) 2.0 x 105 8.0 x (()" 1.0 x 10' 9.5 x 103 6.0 x 10' 7.0 x 103

Xav : Phage (I : 60) 1.4 x 105 5.0 x (()" 4.5 x 10" 1.0 x 10" 6.3 x 103 1.2 X 10'

Xav = 1.0 x 107 cfu/mJPhages = 6.0 x 109 pfu/ml

390 Indian Phytopathology [Vol. 53(4) 2000]

Interaction between Xav and bacteriophages in served at 60 : 1 ratio followed by 20 per cent reductionplants at 30 : 1 ratio when the phage was challenged by Xav

The results (Table 6) indicated that degree of pro- at 24 h. Similarly, 20 per cent reduction in susceptible

tection increased with increase in concentration of phage reaction was observed when phage was challenged at

from 1 : 1 to 60 : I ratio (Phage : Xav). Maximum 1 h with 60: 1 ratio of phage and Xav. No protection

reduction (40 per cent) in susceptible reaction was ob- from susceptible reaction was observed when the Xav

Table 5. In Planta interaction between mungbean Phylloplane bacteria (Plb) and Xav using injection-infiltration method

Treatments Ratio Time of Disease grades Per centPre- Challenge challenge Days after challenge reductioninoculation inoculation (h) inocualtion in SR*

2 4 6 10

Control (Xav alone) I 5 5 5Plb Xav 1:2 0 I 2 3 4 20Plb Xav 1:4 0 1 3 4 4 20Plb Xav 1:8 0 I 3 5 5 0Plb Xav 1:2 I I 2 2 3 40Plb Xav 1:4 1 I 2 2 3 40Plb Xav 1:8 I I 2 2 4 20Xav Plb 2:1 I I 3 5 5 0Xav Plb 4:1 I I 3 5 5 0Xav Plb 8:1 I I 3 5 5 0Plb Xav 1:2 24 0 I I 2 60Plb Xav 1:4 24 0 I 2 3 40Plb Xav 1:8 24 0 I 2 3 40Xav Plb 2:1 24 I 3 5 5 0Xav Plb 4:1 24 I 3 5 5 0Xav Plb 8:1 24 I 3 5 5 0

Xav = 1.0 x 10' cfulml; Plb = 0,5 x 10' cfulml, SR* - Susceptible Reaction

Table 6. Interaction between Phages and Xav in planta using injection-infiltration method

Treatments Ratio Time of Disease grades Per centPre- Challenge challenge Days after challenge reductioninoculation inoculation (h) inocualtion in SR*

2 4 6 10

Control 5 5 5(Xav alone)

.Xav Phages 1 : I 0 I 3 5 5 0Xav Phages I : 30 0 I 3 5 5 0Xav Phages 1 : 60 0 I 3 5 5 0Phages Xav 1 : 1 I 0 2 4 5 0Phages Xav 30 : I I 0 2 4 5 0Phages Xav 60: I 1 0 2 4 4 20Yay Phages I: 1 24 1 3 5 5 0Xav Phages 1 : 30 24 1 3 5 5 0Xav Phages 1 : 60 24 1 3 5 5 0Phages Xav I: I 24 0 2 4 5 0Ii ..ges Xav 30 : 1 24 0 2 4 4 20l nages Xav 60: 1 24 0 I 2 3 40

Phage Iystate = 6,0 x 109 pfulml; Xav = 1.0 x 1.0' cfulml; SR* = Susceptible Reaction

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was challenged by phage at 0 to 24 h. However, phagecould delay the susceptible reaction atleast upto fourthday in all these treatments. Similar results were alsoobtained by Verma et al. (1994) in interaction betweenX c. P'f. malvacearum (Xav) and its phages. Pre-inoculation of phage at 1 h provided maximum protec-tion (40 per cent) of Xam on Acala 44 at 30: 1 ratio ofphage and Xam, whereas in the present studies maxi-mum protection (40 per cent) of Xav in mungbeanleaves was obtained on 24 h challenge inoculation ofXav at 60: 1 ratio of phage and Xav. It was observedthat when the bacterial pathogen was applied first, thebacterium occupied the infective sites in the host withinthe intercellular spaces, which made them relativelynon-accessible to the phages. Earlier workers also sug-gested that phage could reduce plant infections of sev-eral phytopathogenic bacteria when bacteriophages wereapplied first (Tanaka et al., 1990; Zaccardelli et al.,1992).

Population of Xav and Plb in plantsThe results (Table 7) showed that in mono-inocu-

lation of Xav, a population of 1.5 x 109 cfu/cm? leafwas reached after 5 days. At this stage, the susceptiblesymptoms were fully developed in the mungbean leaves.The appearance of symptoms was also corroboratedwith ooze test. Profuse bacterial ooze was recordedwhen the susceptible symptoms were fully expressedas compared to little ooze after I day of inoculation.

The Plb in mono-inoculation did not multiply andthe population remained almost at the same level uptofifth days as evident from the initial (3.1 x 103 cfu/cm?leaf) and fifth day (1.5 x 103 cfu/cm? leaf population.In cross inoculation, (i.e., Plb challenged by Xav) whenPlb multiplied to a population of 4.5 x 105 on fifth day,


the Xav also multiplied to 1.8 x 106 cfu/cm? leaf. How-ever, the rate of multiplication was slow and the popu-lation could not reach the minimum required to ex-press susceptible reaction. This was also evident fromthe previous interaction studies (between Xav and Plb),where maximum reduction (60 per cent) of susceptiblereaction was observed when Plb was challenged byXav in plants. This clearly indicated the reduced rateof multiplication of Xav due to pre-inoculation of Plb.When Xav was challenged by Plb at 24 h, Xav popu-lation increased rapidily from 1.2 x 106 cfu/cm? leaf to3.1 x 10M cfu/cm? leaf on fifth day with expression ofsusceptible symptoms and profuse ooze. The Plb popu-lation also increased and reached to 4.5 x 106 cfu/cm?leaf on fifth day as compared to 1.5 x 103 cfu/cm? leafand 4.5 x 105 cfu/cm? leaf in control (Plb alone) andchallenge inoculation, respectively. The increased popu-lation of Plb might be due to release of nutrients fromhosts by the pathogen and their utilization by Plb.Sinclair et al. (1970) found a lO-fold increase in car-bohydrates, amino acids and proteins in their intercel-lular fluid of susceptible pepper leaves after inocula-tion with compatible X. vesicatoria.

Chowdhury and Verma (1980b) demonstrated thatPlb-6 (Flavobacterium sp.) multiplied in cotton in pres-ence of X. axonopodis, pv. malvacearum only. Dake(1991) also observed that Plb-51 (Pseudomonas sp.) inits mono-inoculation did not multiply in susceptible aswell as in immune cultivars of cotton. However, popu-lation of Pseudomonas sp. increased in the presence ofX. a. pv. malvacearum both in susceptible and immunecultivars and reduced X. a. pv. malvacearum popula-tion. It was concluded that Plb and saprophytes presenton the host surface could not multiply profusely due to

Table 7. Population of Xanthomonas axonopodis pv. vignaeradiatae and Plb (Bacillus sp.) in mono and challenge (at 24 h)inoculation in mungbean

Number of cfu/ em? leaf areaDays after Xav alone Plb alone Plb challenged with Xav Xav challenged with Plbinoculation Popula- Ooze Popula- Ooze Plb Xav Ooze Xav Plb Ooze

tion test tion test popula- Popula- test popula- popula- testtion tion tion tion

0 1.2x I Q6 3.lxlOl 3.1 X IOl 1.2xlO6

I 9.7xlQ6 + 4.0x1Ol 5.5xlOl 5.6xW' 1.7xlO6 6.0x104 +2 3.7x107 ++ 1.5xlOl 2.0xlQ4 5.lxlOS + 2.0xJ07 4.5xlO' ++3 1.4xl0' +++ 3.0xlOl 3.0xJQ4 9.8xJOS + 2.8x107 6.5xlO' ++4 1.6xlOB ++++ 1.5xlOl 2.0xJOS 1.0xJQ6 + 1.4xlOB 5.0x106 ++++5 1.5x lOB ++++ 1.5xlOl 4.5xJOS 1.8x JQ6 + 3.lxlOB 4.5x106 ++++

o = Immediately after inoculation, Xav = 1.0 x 107 cfu/rnl, Plb = 0.5 x 107 cfu/ml, - = no ooze, + = little ooze, ++ =moderate ooze, ++-+ r-; good ooze, ++++ = profuse ooze


non-availability of certain essential nutrients, however,due to diseased condition, substantial increase in nutri-ent release takes place and helps in quick multiplica-tion of host surface bacteria (Young and Paton, 1972;Chowdhury and Verma, 1980a).

Population of Xav and its phages in plants

The recoverable population of Xav in mono-in-oculation increased from 1.0 x 106 to 1.0 X 109 cfulcmvleaf area after 5 days whereas phage population inmono-inoculation decreased from 5.0 x 102 to 1.0 X

101 pfu/cmvleaf area thereby indicating inability ofphages to multiply in the absence of host bacteria (Table8). When the receptors (Xav) were present phages couldmultiply and established higher population to survivefor a longer period on the leaf tissues. As expected,Verma et at. (1994) also reported that phages multiplyonly in the presence of host bacterium. The phage popu-lation however, increased from 5.0 x 102 to 1.9 X 107

pfu/cm? leaf area after 5 days, but the Xav populationreached only 3.0 x 107 cfu/cmvleaf area when chal-lenged by Xav at 24 h, which was not sufficient toproduce disease symptoms. On the other hand, whenXav was challenged by phages, both phages and Xavmultiplied. Thus, it was concluded that less number ofphages in the host in mono-inoculation was probablydue to absence of receptors (Xav). However, in thepresent study phages survived upto 5 days with de-creasing population in mono-inoculation, whereasVerma et at. (1994) observed survival of phages up to48 h only in mono-inoculation and 72 h in challengeinoculations. It was also reported by the various work-ers (Keil and Wilson, 1963; Civerolo, 1970) that theprotection from disease was better when bacterioph-ages were applied first.

[Vol. 53(4) 2000)

Effect of seed treatment with Plb and phagelysate on germination and seedling infection inmungbean

Results (Table 9) showed that seed treatment withPlb (Bacillus sp.).at 2 : 1 ratio (Xav : Plb) and withphage lysate (XMP-l) at 1 : 60 (Xav : phage) ratiowere found effective not only in stimulating germina-tion but also in restricting the seedling infection arisingout of seed inoculation to 10 per cent as compared to68 per cent in control (seed treated with Xav alone).The seeds soaked in water alone showed no infectionindicating that the seeds used for treatment were freefrom internal as well as external seed-borne infection.This clearly indicated that the Plb (Bacillus sp.) andbacteriophage could be exploited as potential biocontrolagents against bacterial leaf spot of mungbean. Singhet at. (1965) observed that seedlings of pigeon peagained resistance to Fusarium udum infection whenthe seeds were bacterized with B. subtilis before sow-ing. They concluded that bulbiformin (antibiotic pro-

Table 9. Effect of seed treatment with Plb (Bacillus sp.) andphage (XMP-I) on germination and seedling infec-tion in mungbean variety Pusa Baisakhi

Treatments No. of Percentageseedlings ofinfected infected

Percentageof germi-

nation

I. Seeds + water2. Seeds + Xav (Control)3. Seeds + Xav + Plb4. Seeds + Xav + phage

90758082

o51813

0.068.010.0!5.9

Ratio of Xav and Plb = 2: I, Ratio of Xav and phage = 1:60,Xav = 1.0 x 10' cfu/ml, Plb = 0.5 x 10' cfu/ml., Phage = 6.0x 10· pfu/ml, 0 = No infection

Table 8. Recoverable population of Xav and phages in mono and challenge (at 24 h) inoculation in mungbean

Number of cfU/cfu ern' leaf areaDays after Xav alone Phage Phages challenged with Xav Xav challenged with phagesinoculation Popula- Ooze alone Phage Xav Ooze Xav Phage Ooze

tion test popula- popula- test popula- popula- testtion tion tion tion

0 1.0x I06 5.0xlO' 5.0xlO' 1.0x 106I 4.6x106 + 2.0x101 1.4xlO' 2.5x105 2.3x106 1.7xlO' +2 3.lxlO' ++ 4.0x101 1.5x 10' 3.0x106 + 2.6x1O' 2.0x1O' ++3 1.5xlO' +++ 3.0xI0' 1.7xl0' 4.0x106 + 1.0x 10' 2.6x105 +++4 1.9xlO' ++++ 2.0xI0' 2.0x 106 2.4x1O' ++ 1.3x 10' 1.7x I06 ++++5 LOxl09 ++++ LOx10' L9x 10' 3.0x1O' ++ 4.0xI0' 3.7x1O' ++++o = Immediately after inoculation, Xav = 1.0 x 10' cfu/ml, Phage = 6.0 x 10' pfu/ml, - = no ooze, + = little ooze, ++ =moderate ooze, +++ = good ooze, ++++ = profuse ooze

[Vol. 53(4) 2000]

duced by B. subtilis) became systemic in the plant andprovided a protective zone around the roots of pigeonpea seedlings. Earlier, control of various bacterial dis-eases of plants using phylloplane bacteria and bacte-riophage as seed treatment has been reported by vari-ous workers including mungbean (Labedeva 1937; Rao,1970; Thind and Jindal, 1988; Jindal and Thind, 1990;Dake, 1991; Verma et al., 1994).

ACKNOWLEDGEMENTThe authors are grateful to Head, Division of Plant

Pathology, I.A.R.I., New Delhi for providing facilitiesand for critical discussion.

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Received for publication September 4, 1997

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Biological management of bacterial leaf spot of mungbean