Indian Phytopath. 53 (4) : 384-394 (2000)
Biological management of bacterial leaf spot of mungbean caused by Xanthomonas axonopodis pv. vignaeradiatae
P. K. BORAH*, lK. JINDAL and J.P. VERMA Division 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 axonopodis pv. 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, exhibiting inhibition zones ranging from 6 mm to 25 mm. The most promising antagonistic phylloplane bacteria (Plb-2) was identified to be a strain of Bacillus sp. Effectiveness of Plb-2 and XMP-l as biocontrol agents in the suppression of Xav from seed, seed coat and cotyledon of mugbean was assessed. The pathogen was effectively eliminated from mugbean seeds when these were dipped in a mixture of Xav and Plb at 1:1 ratio. The phage multiplied in mugbean seeds in presence 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 to challenge inoculation of Xav at 1:2. No protection was obtained when Xav was challenged by Plb at all concentrations tested. 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 in stimulating germination and restricting seedling infection to 10.0 and 15.9 per cent, respectively, as compared to 68 per cent in control. The results indicated that Plb and phage strains antagonistic against Xav, during the present study could be 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, phylloplane bacteria (Plb) and bacteriophage (phages) has been re- ported to be effective when used as seed treatment to control seedling diseases in many crop plants (Labedeva, 1937; Rao, 1970; Verma et al., 1978; Santhi et al., 1987; Liao, 1989; Parashar et al., 1992). Xanthomonas axonopodis pv. vignaeradiatae, the incitant of bacte- rial leaf spot of mungbean and phages of Xav are widely distributed 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 of the pathogenic bacteria, their precise role in disease development process(es) is not clearly understood. Some
* Present address: Department of Plant Pathology, Assam Agricultural University, Jorhat 785 013
information are available with regard to the manage- ment of bacterial leaf spot disease of mungbean using Plb 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 bactriophages in management of bacterial leaf spot of mungbean. This communication deals with the interaction of Plb and bacteriophage with Xav with a view to resolve their potential as biocontrol agents.
MATERIALS AND METHODS
Isolation of Xav Diseased leaves of mungbean plants (Vigna ra-
diata), were collected from the experimental fields of Indian 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 of individual 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 colony culture was maintained on Yeast Glucose Chalk Agar (YGCA) slants (Yeast extract - 10.0 g, Glucose - 10.0 g, 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 vigorously to obtain bacterial suspension. This was allowed to stand for 5 min for the agar bits to settle down and only the supernatant was used. This suspension is reffered to as concentrated bacterial suspension in present study.
Isolation of Plb
Apparently healthy leaves were collected from different positions of susceptible mungbean cultivar Pusa Baisakhi (upper, middle and lower) grown in the field of Indian Agricultural Research Institute. The Plb were isolated by using leaf washing method (Hoa, 1991). Five gram leaves were taken in a conical flask with 100 ml sterile water and stirred vigorously with sterile glass rod for 10 min. Serial dilutions were pre- pared by transferring I ml of leaf washing to 9 ml sterile distilled water blanks. An aliquot of 0.1 ml of each 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 and colour were picked up on YGCA slants. These colo- nies were further purified by streaking on NSA plates. Single colonies were picked up and maintained on YGCA slants for further studies.
In vitro antagonism of Plb against Xav
For testing antagonists against Xav, 1 ml of Xav suspension (0.1 O.D. at 620 nm wave length) was mixed with 25 ml melted and cooled (40° - 45°C) SPA and poured into petriplates. The solidified plates were spot inoculated with the 24 h old growth of Plb isolates. After 3 days of incubation at 26° ± 1°C, the plates were examined for antagonistic action indicated by the ap- pearance of inhibition zones and the diameter of clear zones were measured. Plates without Plb served as control.
Indian Phytopathology 385
Morphological, cultural and physiological charac- ters of Plb
Staining procedures for the study of morphology was followed as recommended in the "Manual of Mi- crobiological Methods" published by the Society of American Bacteriologists (Anon., 1957). The shape and arrangement of bacterial cells were determined by simple staining with crystal violet. Hucker's identifica- tion of gram stain was used to determine the gram reaction. For spore and capsule staining, Domer's methods modified by Sayder and Liefson's method, respectively, were used. Unless otherwise stated, the media, test reagents, indicators were the same as given by Dye (1962).
To determine the shape, size and flagellation of the phylloplane bacteria, electron microscopy was done following standard methods.
Isolation of phage The isolation and characterisation of phage was
done following the methods of Duttta Majumdar and Verma (1988). One gram diseased chopped leaves of mungbean collected from tile experimental farm of In- dian Agricultural Research Institute, New Delhi, was suspended in 50 ml sterile water in 100 ml conical flasks and incubated at 26° ± 1°C for 48 h to release the phage particles. The supernatant was then decanted and 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 for 12 h in the refrigerator. The individual supernatants were tested for the presence of phage by spot test method. One ml of concentrated Xav suspension was mixed 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) and poured in a sterile petriplate. On the solidified agar surface, 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 were observed for lytic action of the phage, indicated by the clearing of bacterial growth at the site of seeding.
When lytic areas developed, 0.5 em agar piece from the lytic spot was suspended in 5 ml sterile water and lO-fold dilutions were made. One ml of the phage suspension at the desired dilution was mixed with I ml concentrated bacterial suspension of Xav in a sterile tube. The phage bacterial mixture was mixed with 25 ml melted cooled (40° - 45°C) 1.5 per cent SPA in 100 ml 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 plaque material, I representative plaque was used for prepar- ing the phage stock solution.
Preparation of high titer phage stock
Purified single plaque phage suspension was plated in a dilution series with Xav. Five plates that had the proper phage concentration for achieving almost confluent lysis were processed for making the high titer phage stock suspension. Five ml of the sterile distilled water was added to each plate and allowed to stand for 30 min. After that sterile glass spreader was gently moved over the confluent lytic area. The phage suspension thus obtained was filtered through Whatman filter 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 tubes and stored in refrigerator.
Interaction between Plb and Xav in seeds Surface 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: 1 and 4: 1. Ten seeds were kept for each treatment. Population of Xav and Plb were estimated after drying the seeds for 1 h after the treatment. Five whole seeds, seed coats and cotyledons of other five seeds were thoroughly macerated separately (in sterilised water).
From the above suspensions, 0.1 ml was plated on SPA plates and incubated at 26° ± 1°C, for 4 days. Bacterial population on seeds, seed coat and cotyledon was estimated on the basis of number of colonies (cfu) recovered.
Interaction between phage and Xav in seeds Surface 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 drying the seeds 1 h after the treatment. Five seeds, seed coats and the cotyledons from each treatment were thoroughly macerated separately in 2 ml sterillsed water. From each suspension 0.1 ml was poured on SPA plates and spread with a sterillsed glass spreader. Inoculated plates were incubated at 26° ± 1°C for 4 days and bacterial population on seed coat and cotyledons was estimated on the basis of number of colonies developed. For es- timation of phage population, the seeds and its parts were macerated in sterile pestle and mortar with 10 ml sterile 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, which was allowed to settle down at the bottom of the tube. Supernatant thus obtained was serially diluted and plated on SPA seeded with Xav and incubated at 26° ± 1°C for 24 h. The plates were observed for the formation of plaques. The clear zones produced in plates were due to the lytic activity of phage particles.
Interaction between Plb and Xav in plants
Thirty days old mungbean plants (cv. Pusa Baisakhi) were used in the experiment. Plb (Bacillus sp.) [0.5 x 107 cfulml] and Xav (1.0 x 107cfu/ml) were used for pre-inoculation and challenge inoculation of the 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. The inoculations were done by the syringe infiltration method (Klement, 1963) during 8.00 A.M. - 11.00 A.M. when most of the stomata remained open. Water soaked areas (about 2 ern) were developed on the adaxial side of the leaflets of 2 youngest trifoliates in each plant by injecting desired amount of bacterial suspension with the help of a Glassvan 2 ml syringe fitted with 22 gauge hypodermic needle. The challenge inoculations were 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 September when the temperature ranged between 20°C and 37°C and humidity was around 80 per cent. Disease severity was recorded 2, 4, 6 and 10 days after inoculation following the scale described by Hoa (1991). Per cent reduction in susceptible reaction was calculated with the following formula of Verma et al. (1994):
Grade obtained Reduction = x 100 - 100
Maximum grade
Interaction between Xav and phage in plants The bacteriophage XMP-I (6.0 x 109 pfu/ml) and
Xav (1.0 x 107 cfu/ml) were inoculated on 30 day old mungbean (cv. Pusa Baisakhi) by syringe infiltration method (Klement, 1963) in pots. The inoculation of one 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 was recorded 2, 4, 6 and 10 days after inoculation follow- ing the scale described earlier and per cent reduction in susceptible 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 preinoculation of one followed by the challenge inoculation by the other and vice-versa. The challenge time was 24h and the ratio of Plb and Xav was 1:2. Population of both Plb and Xav were estimated 0, 1, 2, 3, 4 and 5 days after challenge inoculation by maceration method. The tests were run in triplicates and leaves of similar posi- tion and age were selected for inoculation on plants raised in pots. Tissue discs (5 mm) were cut (3 discs/ leaf and 3 leaves/estimation) with the help of cork borer from inoculated area of the leaf of each treatment and macerated separately in sterile water. From the above suspension, 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 on the basis of number of colonies (cfu) recovered. Si- multaneously, ooze test was also performed to corrobo- rate the visual symptoms with the multiplication of the pathogen in plant tissues.
Population of Xav and its phage in mungbean plants
Inoculations using syringe infiltration technique (Klement, 1963) were carried out with Xav, phage and in combinations as challenge inoculation. After 24 h of inoculation, it was challenged with either Xav or ph- age. The ratio of phages and Xav was 60: 1. Population of 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 inoculated area of the leaf at different time intervals and macer- ated separately. For estimation of Xav population, 0.1 ml suspension from each treatment was poured on a SPA plate (containing 0.05 g cycJohexamide/l) and spreadwith a sterilised glass spreader. Inoculated plates were incubated at 260 ± I°C for 4 days and bacterial population in leaves was estimated on the basis of number 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 sterile phosphate buffer (pH 7.0). After shaking well, it was centrifuged at 15,000 rpm for 15 min. The supernatant thus obtained was mixed with chloroform, which was allowed to settle down at the bottom of the tube. Su- pernatant thus obtained was serially diluted and plated on SPA seeded with Xav and inoculated at 260 ± I°C for 24 h. The plates were observed for the formation of plaques (pfu). The clear zones produced in plates were due to the lytic activity of phage particles. Sirnulta-
Indian Phytopathology 387
neously, ooze test was also performed to corroborate the visual symptoms with the multiplication of the pathogen inplant tissue. The tests were run in triplicate and leaves of similar position and age were selected for inoculation on plants raised in pots.
Effect of seed treatment with Plb and phage on germination and seedling infection in mungbean
Surface sterillsed mungbean seeds (cv. Pusa Baisakhi) were soaked for 6 h in bacterial suspension of 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 for I h under the shade, the seeds were sown in 8 inch earthen pots. The observations were recorded on per- centage germination and seedling infection after 20 days.
RESULTS AND DISCUSSION
In vitro antagonism of phylloplane bacteria against Xav
During isolations, 4 different types of colony, i.e., white, yellow, pink and other types were observed and 110 bacterial cultures were established from mungbean phylloplane 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 were found antagonistic causing inhibition zones ranging from 6 mm to 22 mm (Table I). Plb-I and Plb-2 produced larger inhibition to 22 mm zones of 22 mm followed by Plb-3 with 20 mm (Fig. I). Hoa (1991) also isolated Plb from mungbean, which produced maximum inhibition zone of 17 mm. The antagonistic behaviour of different mungbean phylloplane bacteria was attributed to the production of chemical substances in the culture medium (Jindal and Thind, 1993). In contrast, none of the Plb of cotton possessing pre-in- oculative protective ability was antagonistic against X a. pv. malvacearum in vitro, indicating non-production of inhibitory substances by the Plb in culture media (Chowdhury and Verma, 1980b; Verma, 1986).
Table 1. In vitro antagonism of phylloplane bacteria against Xanthomonas axonopodis pv. vignaeradiatae.
SI. Original New Diameter of No. isolate isolate inhibition
number number zone (mm)
1 Plb-S Plb- I 22 2. Plb-13 Plb-2 22 3. Pib-26 Plb-3 20 4. Plb-32 Plb-4 8 S. Plb-30 Plb-S 6
388 Indian Phytopathology
Fig.1. Antagonistic effect of Phylloplane bacteria against Xav. 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 in the first edition of the Bergey's Manual of systematic Bacteriology (Anon, 1984), the Plb-2 isolate was ten- tatively identified upto generic level as Bacillus sp. It was 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 smooth but the surface was rough and wrinkled, irregular, flat, opaque, margin irregular, creamy white, filiform growth on slants, facultive anaerobe, catalase positive and pro- duced ammonia. The results were summarized in the Table 2. Jindal and Thind (1989), Hoa (1991) and Bora et al. (1993) also reported different genera of Plb in- cluding Bacillus sp. from mungbean.
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Table 2. Morphological, cultural and biochemical characters of phylloplane bacteria of mungbean (Plb-2) an- tagonistic to Xanthomonas axonopodis pv. vignaeradiatae
SI. Characters No.
Plb-2
A. Morphological I Shape 2. Size 3. Arrangement of cell 4. Gram reaction 5. Spore 6. Flagella 7. Capsule
B. Cultural
I. Colony characters after 48 h at 28°C (on NNSPA) and growth on solid medium
2. Oxygen requirement
I. Catalase activity +ve 2. Production of NHJ +ve
Short rod 2.0 x 0.5-1 ILm Scattered or in pairs +ve +ve +ve (peritrichous) +ve
Colonies irregular, flat, margin irregular, opaque, smooth, filiform, creamy white (18-20) mm in dia.
Facultative anaerobe
Isolation of phages Xav bacteriophage isolate (XMP-I) was established
from infected mungbean leaves (cv. Pusa Baisakhi). The lytic activity of the phage isolate was tested against Xanthomonas axonopodis pv. vignaeradiatae on SPA plates by spot test method which showed clear lytic zone on the spotted area.
Interaction between Xav and Plb in seeds Not a single colony forming unit (cfu) of Xav could
be recovered from seeds and seed parts in treatments of 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 in mungbean seeds
Treatments Ratio of Xav: Plb Seed
Number of cfu recovered/seed/seed parts after 6 h of treatment
Cotyledon Xav ' Plb
1:1 2:1 4:1 1:0 (Xav alone) 0: I (Plb alone)
o 1.5 x I(}'
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 seeds by 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 in eradicating seed infection of mungbean by Xav. Ear- lier, Vasudeva et at. (1958) isolated an antibiotic pro- duced by B. subtilis and named as bulbiformin. Thind and lindal (1988); lindal and Thind (1989) and lindal and Thind (1990) also found significant control of seedbome infection of mungbean (Xav) and cowpea (X.a. pv. vignicola) using Plb viz., Erwinia herbicola, Bacillus spp., Pseudomonas fluorescens and Acenetobacter calcoaceticus.
Interaction between Xav and phage in seeds
The results (Table 4) of interaction between Xav and phage (XMP-l) in mungbe.an seeds revealed that the phage could not eliminate Xav completely from seed, seed coat, and cotyledons, even at an application ratio 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 to control (phage alone), indicating that in presence of host bacterium (Xav) phage multiplied in seed. Bacte- riophages have also been tried earlier for the control of seed-borne bacterial pathogens. Thomas (1935) found that seed com from infected crop yielded bacterioph- ages and the seed developed only 5 per cent infected plants. 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 18 per cent in the phage untreated samples. In addition to their roles in biocontrol, bacteriophages have been widely used in detection of seed-borne pathogens of various crop plants (Keil and Wilson, 1963; Soni and Thind, 1991, Kahveci and Maden, 1994).
Indian Phytopathology 389
Interaction between Xav and Plb in plants
Results of interaction between Xav and Plb (Bacil- lus sp.) in plants are presented in Table 5. Maximum reduction (60%) of susceptible reaction (SR) was ob- served at 1:2 ratio of Plb and Xav after 24 h challenge by Xav. The reduction in SR was also recorded in challenge inoculations of Xav at 0, I and 24 h at all ratios tested except in case of on challenge in the ratio 1:8. Density dependent mechanisms such as coloniza- tion of sites and/or nutrient acquisition may be likely explanation for the reduction of pathogen population following preinoculation of antagonists on the phylloplane. The time gap further helped the antago- nists to produce antibacterial substances in sufficient quantity to inhibit the pathogen. Similar observations were made by Sinha and Verma (1984), Jindal and Thind (1993) and Bora et al. (1993). This also indi- cated ineffectiveness of pre-inoculation treatment of Xav 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 by Xav.
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 of mungbean caused by Xav (Bora et al., 1993) and cowpea 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 as compared 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 (Plb Xam).
Table 4. Interaction between Xav and Phage (XMP-I) in mungbean seeds
No. of cfu recovered No. of pfu recovered Ratio 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 100 Control (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/mJ Phages = 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 reduction plants 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 cent Pre- Challenge challenge Days after challenge reduction inoculation inoculation (h) inocualtion in SR*
2 4 6 10
Control (Xav alone) I 5 5 5 Plb Xav 1:2 0 I 2 3 4 20 Plb Xav 1:4 0 1 3 4 4 20 Plb Xav 1:8 0 I 3 5 5 0 Plb Xav 1:2 I I 2 2 3 40 Plb Xav 1:4 1 I 2 2 3 40 Plb Xav 1:8 I I 2 2 4 20 Xav Plb 2:1 I I 3 5 5 0 Xav Plb 4:1 I I 3 5 5 0 Xav Plb 8:1 I I 3 5 5 0 Plb Xav 1:2 24 0 I I 2 60 Plb Xav 1:4 24 0 I 2 3 40 Plb Xav 1:8 24 0 I 2 3 40 Xav Plb 2:1 24 I 3 5 5 0 Xav Plb 4:1 24 I 3 5 5 0 Xav 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 cent Pre- Challenge challenge Days after challenge reduction inoculation inoculation (h) inocualtion in SR*
2 4 6 10
Control 5 5 5 (Xav alone)
.Xav Phages 1 : I 0 I 3 5 5 0 Xav Phages I : 30 0 I 3 5 5 0 Xav Phages 1 : 60 0 I 3 5 5 0 Phages Xav 1 : 1 I 0 2 4 5 0 Phages Xav 30 : I I 0 2 4 5 0 Phages Xav 60: I 1 0 2 4 4 20 Yay Phages I: 1 24 1 3 5 5 0 Xav Phages 1 : 30 24 1 3 5 5 0 Xav Phages 1 : 60 24 1 3 5 5 0 Phages Xav I: I 24 0 2 4 5 0 Ii ..ges Xav 30 : 1 24 0 2 4 4 20 l 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, phage could delay the susceptible reaction atleast upto fourth day in all these treatments. Similar results were also obtained by Verma et al. (1994) in interaction between X 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 of phage and Xam, whereas in the present studies maxi- mum protection (40 per cent) of Xav in mungbean leaves was obtained on 24 h challenge inoculation of Xav at 60: 1 ratio of phage and Xav. It was observed that when the bacterial pathogen was applied first, the bacterium occupied the infective sites in the host within the intercellular spaces, which made them relatively non-accessible to the phages. Earlier workers also sug- gested that phage could reduce plant infections of sev- eral phytopathogenic bacteria when bacteriophages were applied first (Tanaka et al., 1990; Zaccardelli et al., 1992).
Population of Xav and Plb in plants The results (Table 7) showed that in mono-inocu-
lation of Xav, a population of 1.5 x 109 cfu/cm? leaf was reached after 5 days. At this stage, the susceptible symptoms were fully developed in the mungbean leaves. The appearance of symptoms was also corroborated with ooze test. Profuse bacterial ooze was recorded when the susceptible symptoms were fully expressed as compared to little ooze after I day of inoculation.
The Plb in mono-inoculation did not multiply and the population remained almost at the same level upto fifth 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) when Plb multiplied to a population of 4.5 x 105 on fifth day,
Indian Phytopathology 391
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 from the previous interaction studies (between Xav and Plb), where maximum reduction (60 per cent) of susceptible reaction was observed when Plb was challenged by Xav in plants. This clearly indicated the reduced rate of 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 to 3.1 x 10M cfu/cm? leaf on fifth day with expression of susceptible 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? leaf and 4.5 x 105 cfu/cm? leaf in control (Plb alone) and challenge inoculation, respectively. The increased popu- lation of Plb might be due to release of nutrients from hosts 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 that Plb-6 (Flavobacterium sp.) multiplied in cotton in pres- ence of X. axonopodis, pv. malvacearum only. Dake (1991) also observed that Plb-51 (Pseudomonas sp.) in its mono-inoculation did not multiply in susceptible as well as in immune cultivars of cotton. However, popu- lation of Pseudomonas sp. increased in the presence of X. a. pv. malvacearum both in susceptible and immune cultivars and reduced X. a. pv. malvacearum popula- tion. It was concluded that Plb and saprophytes present on 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 area Days after Xav alone Plb alone Plb challenged with Xav Xav challenged with Plb inoculation Popula- Ooze Popula- Ooze Plb Xav Ooze Xav Plb Ooze
tion test tion test popula- Popula- test popula- popula- test tion 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
392 Indian Phytopathology
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 cful cmvleaf area after 5 days whereas phage population in mono-inoculation decreased from 5.0 x 102 to 1.0 X
101 pfu/cmvleaf area thereby indicating inability of phages to multiply in the absence of host bacteria (Table 8). When the receptors (Xav) were present phages could multiply and established higher population to survive for a longer period on the leaf tissues. As expected, Verma et at. (1994) also reported that phages multiply only 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 population reached only 3.0 x 107 cfu/cmvleaf area when chal- lenged by Xav at 24 h, which was not sufficient to produce disease symptoms. On the other hand, when Xav was challenged by phages, both phages and Xav multiplied. Thus, it was concluded that less number of phages in the host in mono-inoculation was probably due to absence of receptors (Xav). However, in the present study phages survived upto 5 days with de- creasing population in mono-inoculation, whereas Verma et at. (1994) observed survival of phages up to 48 h only in mono-inoculation and 72 h in challenge inoculations. It was also reported by the various work- ers (Keil and Wilson, 1963; Civerolo, 1970) that the protection from disease was better when bacterioph- ages were applied first.
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Effect of seed treatment with Plb and phage lysate on germination and seedling infection in mungbean
Results (Table 9) showed that seed treatment with Plb (Bacillus sp.).at 2 : 1 ratio (Xav : Plb) and with phage lysate (XMP-l) at 1 : 60 (Xav : phage) ratio were found effective not only in stimulating germina- tion but also in restricting the seedling infection arising out of seed inoculation to 10 per cent as compared to 68 per cent in control (seed treated with Xav alone). The seeds soaked in water alone showed no infection indicating that the seeds used for treatment were free from internal as well as external seed-borne infection. This clearly indicated that the Plb (Bacillus sp.) and bacteriophage could be exploited as potential biocontrol agents against bacterial leaf spot of mungbean. Singh et at. (1965) observed that seedlings of pigeon pea gained resistance to Fusarium udum infection when the 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.) and phage (XMP-I) on germination and seedling infec- tion in mungbean variety Pusa Baisakhi
Treatments No. of Percentage seedlings of infected infected
Percentage of germi-
nation
I. Seeds + water 2. Seeds + Xav (Control) 3. Seeds + Xav + Plb 4. Seeds + Xav + phage
90 75 80 82
o 51 8 13
0.0 68.0 10.0 !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 = 6.0 x 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 area Days after Xav alone Phage Phages challenged with Xav Xav challenged with phages inoculation Popula- Ooze alone Phage Xav Ooze Xav Phage Ooze
tion test popula- popula- test popula- popula- test tion tion tion tion
0 1.0x I06 5.0xlO' 5.0xlO' 1.0x 106 I 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 and provided a protective zone around the roots of pigeon pea 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).
ACKNOWLEDGEMENT The authors are grateful to Head, Division of Plant
Pathology, I.A.R.I., New Delhi for providing facilities and for critical discussion.
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Received for publication September 4, 1997