Plant and Soil 251: 73–82, 2003. © 2003 Kluwer Academic Publishers. Printed in the Netherlands.

73

Biological suppression of rice diseases by Pseudomonas spp. under saline soil conditions Sunita Rangarajan1 , Lilly M. Saleena1 , Preeti Vasudevan2 & Sudha Nair1,3 1 M.

S. Swaminathan Research Foundation, III Cross Street, Taramani Institutional Area, Taramani, Chennai-600 113, India. 2 Center for Advanced Studies in Botany, University of Madras, Guindy Campus, Guindy, Chennai-600 025, India. 3 Corresponding author∗ Received 20 November 2001. Accepted in revised form 26 August 2002

Key words: bacterial leaf blight, biological control, Oryza sativa, Pseudomonas, salinity, sheath blight

Abstract The aim of this study was to develop antagonistic strains specific for the coastal agricultural niche in Southern India. Indigenous Pseudomonas strains isolated from rhizosphere of rice cultivated in the coastal agri-ecosystem were screened for in vitro antibiosis against Xanthomonas oryzae pv. oryzae and Rhizoctonia solani – the bacterial leaf blight (BB) and sheath blight (ShB) pathogens of rice (Oryza sativa) respectively. The strains exhibiting antibiosis were tested in the greenhouse under normal and saline soil conditions. The antagonists suppressed BB by 15 to 74% in an unamended soil. The efficient strains were tested under saline soil conditions and found to suppress disease by 46 to 82%. Similarly, incidence of ShB was also suppressed by 30 to 57% in the unamended soil by the efficient strains which, under saline soil conditions, were found to suppress ShB by 19 to 51%. Four strains of Pseudomonas tested suppressed both BB and ShB diseases in rice, of which three were efficient under both natural and saline soil conditions. Introduction The search for alternatives to chemical control of plant pathogens, such as biological control, has gained momentum in the recent years due to the emergence of fungicide-resistant pathogens and health concerns for the producer and the consumer. Plant growth promoting rhizobacteria (PGPR) and plant associated bacteria (PAB) are known to rapidly colonize the rhizosphere, compete with and suppress the deleterious microorganisms as well as soil-borne pathogens at the root surface. Further, plant growth promotion afforded by biocontrol agents resembles the beneficial effect of a biofertilizer. Pseudomonas spp. have been studied mainly because of their widespread distribution in the soil, their ability to colonize the rhizosphere of host plants and ability to produce a wide range of compounds inhibitory to a number of serious plant patho∗ FAX No: 91-44-2541319.

E-mail: [email protected]

gens (Anjaiah et al., 1998; Rodriguez and Pfender, 1997; Thomashow et al., 1997). Plant-associated pseudomonads are accomplished growth promoters capable of simultaneously increasing crop productivity (Cook et al., 1995). Gnanamanickam et al. (1998) showed that a strain of P. fluorescens afforded 79–82% control of rice blast and sheath blight and simultaneously enhanced the grain yield in rice (Oryza sativa L.). Rice is highly vulnerable at all stages of growth to pathogens that affect the quality and quantity of its yield. Of the 80 biotic and abiotic diseases described in rice, bacterial leaf blight (BB) caused by Xanthomonas oryzae pv. oryzae and sheath blight (ShB) caused by Rhizoctonia solani are among the most destructive. Yield losses due to BB range from 74 to 81% in areas of India where it is epidemic. This loss is relatively higher than those reported in other parts of the world which is generally around 20–30% and occasionally going up to 50% (Mew, 1992). ShB was considered to be a minor disease till recently but has now become

74 a major production constraint. This is because of the increased use of nitrogen-responsive, semi-dwarf rice cultivars which provide a good canopy for the development of R. solani. ShB is distributed in 1.2 to 1.4 million ha which is about 32–50% of the world’s total rice cropping area (Thara, 1994). Although there are several studies conducted on the biological suppression of rice diseases, all have been designed for non-saline agricultural soils and do not address the problems associated with coastal ecosystems. Although numerous efficient biocontrol Pseudomonas strains have already been identified, their efficiency under conditions of abiotic stress has not been adequately elucidated. The abiotic environment has however been recognized as the main criterion determining the efficiency of antagonistic bacteria in soil (Lewis and Papavizas, 1987). Hence, the present study addresses this issue and aims at identifying bacterial strains that can perform equally efficiently under saline soil conditions. This study deals with the potential of Pseudomonas strains to biologically control rice diseases under saline soil conditions.

Materials and methods Rice seeds The seeds of rice cultivars IR50 and CO43, used in this study were obtained from Tamil Nadu Agricultural University, Coimbatore, India. Source of pathogens The Rhizoctonia solani (PTB-99-023) and Xanthomonas oryzae pv. oryzae (PXO61- International Race 1 strain) pathogens used in the biocontrol studies were from the laboratory collection of S. S. Gnanamanickam, Center for Advanced Studies (CAS) in Botany, University of Madras, India. These pathogens were isolated from naturally infected rice tissues collected from rice growing regions of Southern India. Isolation of bacterial strains Five sites cropped to rice with varying salinity levels were chosen along the coastline of Tamil Nadu in Southern India. Healthy, mature rice plants were collected by sampling at regular intervals and pooled together. From these, the intact root systems were collected and shaken gently to remove all but the

soil closely adhering to the roots. These root portions with just a layer of closely adhering rhizosphere soil were then transferred to conical flasks with 100 mL sterile water and shaken at 120 rpm for 30 min. Suspensions from all five samples were serially diluted up to 10−4 with three replications for each sample. From 10−3 and 10−4 dilutions, 1 mL was pour-plated on Pseudomonas Isolation Agar (Hi-Media, India) for fluorescein (casein enzyme hydrolysate-10.0; proteose peptone-10.0; dipotassium phosphate-1.5; magnesium sulphate-1.5; agar-15.0 (g l−1 ); glycerol-10.0 mL; final pH 7.0±0.2) and pyocyanin (peptic digest of animal tissue-20.0; potassium sulphate-10.0; magnesium chloride-1.4; agar-15.0 (g l−1 ); glycerol-10.0 mL; final pH 7.0±0.2) with three replications for each dilution. Identification of Pseudomonas strains Total genomic DNA was isolated using standard procedure (Graves and Swaminathan, 1993) and the 16S rDNA region amplified using the Pseudomonas genusspecific 16S rRNA gene PCR primers. The forward primer Ps-for (20-mer [5 GGTCTGAGAGGATGAT CAGT]) and reverse primer Ps-rev (18-mer [5 TTAGC TCCACCTCGCGGC]) (Alm et al., 1996) were used to amplify the total genomic DNA (Widmer et al., 1998). To estimate the size, the products were electrophoresed on 1.2% agarose gels along with 1 Kb ladder as marker (Gibco-BRL) and stained with ethidium bromide. For the species identification, the NEFERM test kit (Medispan Ltd., Chennai) based on the differential utilization of 25 substrates was used (Boeufgras et al., 1987). Salt tolerance assays for bacterial strains Pure cultures of all the Pseudomonas strains were plated on a Minimal Media Davis (Hi-Media, India) plate composed of dextrose-1.0 g l−1 , ammonium sulphate-1.0 g l−1 , dipotassium phosphate-7.0 g l−1 , monopotassium phosphate-2.0 g l−1 , sodium citrate0.5 g l−1 and magnesium sulphate-0.1 g l−1 and amended with 0.5 to 2.0 M NaCl with an increment of 0.5 M. Control plates, without NaCl amendment were also included for all strains. All plates were incubated at 30 ◦ C overnight. Laboratory assay for pathogen inhibition All the Pseudomonas strains isolated were screened in vitro for the inhibition of X. oryzae pv. oryzae

75 and R. solani. A fresh culture of X. oryzae pv. oryzae was inoculated into Peptone Sucrose Broth (Hi-Media, India) (peptone-10.0; sucrose-10.0; monosodium glutamate-1.0 (g l−1 )) and grown for 72 h at 30 ◦ C. From this culture, 100 µL was pipetted onto Peptone Sucrose Agar (Hi-Media, India) medium and spread plated. The Pseudomonas strains to be tested for antagonistic activity were grown in nutrient broth medium (Peptic digest of animal tissue-10.0; Sodium chloride-5.0; Beef extract-5.0; Agar-15.0 (g l−1 )) overnight at 30 ◦ C on a rotary shaker at 120 rpm. From this, three aliquots of 20 µL each were spotted on each PSA plate already spread with X. oryzae pv. oryzae. These plates were incubated at 30 ◦ C for 48 h and antibiosis recorded by measuring the diameter of the inhibition zone of pathogen’s growth. Those strains that tested positive were checked again individually with three replications for each strain. For in vitro screening against R. solani, a fresh culture was grown in potato dextrose agar (PDA) medium (potato infusion-200.0; dextrose-20.0; agar-18.0 (g l−1 )). A plug from the growing edges of the R. solani colony was placed in the center of a fresh PDA plate. Five Pseudomonas strains grown overnight were spotted per plate along the periphery, equidistant from the mycelial plug. These plates were incubated at 30 ◦ C for 48 h. Antibiosis was recorded by measuring the diameter of the inhibition zones. Those strains that inhibited the mycelial growth of R. solani alone were tested again separately on single plates. In this assay, three plugs of mycelia were placed equidistant on a PDA plate and the Pseudomonas strain was spotted in the center. Three replicate plates were maintained for each strain tested.

nas strains were selected on the basis of the in vitro inhibition of R. solani and X. oryzae pv. oryzae. Preparation of bacterial strains The bacterial strains showing antagonistic activity in vitro were inoculated into 50 mL nutrient broth and grown with continuous shaking on a rotary shaker at 120 rpm for 48 h at 30 ◦ C. Their OD600 was adjusted between 0.3 and 0.5 after the incubation period with an aqueous solution of 1% carboxy methyl cellulose (CMC) to obtain a cell concentration of 109 cfu/mL and used for bacterization of seeds, seedlings and foliage of rice plants. Cultivation of rice and treatment with bacteria The rice seeds were surface sterilized with 4% sodium hypochlorite for 15 min and rinsed repeatedly with sterile water. The sterilized seeds were soaked overnight in the respective bacterial cultures (Gnanamanickam and Mew, 1992; Mew and Rosales, 1986). The excess bacterial suspension was drained off and the seeds allowed to pre-germinate on moistened filter paper in petri plates. On the 5th day, the germinating seedlings were transferred to cups containing 1:1 ratio of vermiculite and sand weighing a total of 100 g. When the seedlings were 21 d old, they were transplanted into pots containing 5 kg of soil prior to which a root dip treatment was given with their respective formulated cultures for 6 h. On the 43rd and 44th days, the plants were given foliar spray treatment with their respective bacterial cultures (109cfu/mL) made in 1% aqueous CMC. Plants sprayed with sterile 1% CMC solution served as the control. Preparation of pathogen inoculum

Greenhouse evaluation of antagonistic bacteria Two greenhouse experiments were conducted for testing the biocontrol potential of the Pseudomonas strains. In the first experiment, a salt-sensitive and disease susceptible rice variety (IR50) was used and the experiment carried out with natural soil conditions. In the second experiment, a salt-tolerant and disease susceptible variety (CO43) was used and saline soil conditions were imposed by amending the soil with 25% artificial sea water throughout the experiment (Flowers et al., 1990). In both the experiments, the soil used was from the coastal agri-ecosystem. The greenhouse experiments were repeated to check for consistency of the results obtained. Pseudomo-

X. oryzae pv. oryzae The pathogen was grown on PSA plates for 72 h at 30 ◦ C. A suspension of this culture was prepared in sterile water and the OD600 adjusted to 0.1–0.2 to obtain 106 colony-forming units (cfu)/mL. This inoculum was used to clip inoculate the leaves of rice plants on the 45th day as per the method of Kaufmann et al., (1973). R. solani A rice grain:rice hull (1:3) substrate was prepared (Thara, 1994) and sterilized. Mycelial plugs of R. solani were introduced into this substrate and allowed to grow for 10 days. Before inoculation onto the rice

76 Table 1. Scale for greenhouse testing of bacterial leaf blight Disease Score

Infected Leaf Area (%)

1 2 3 4 5 6 7 8 9

0–3 4–6 7–12 13–25 26–50 51–75 76–87 88–94 95–100

plants, the culm region of the rice tillers of each plant was covered with aluminium foil to form a cone. For the artificial induction of ShB, 40 g of the above inoculum was dropped into the cone at the base of each rice plant. Scoring of disease severity The results on severity of disease (bacterial leaf blight and sheath blight) development were scored 15 days after pathogen inoculation. BB suppression by Pseudomonas treatments Suppression of bacterial leaf blight was measured in terms of a reduction in the mean bacterial blight lesion length measured on treated leaves against that of the untreated control by using the following formula: % Diseased leaf area (%DLA) = Average BB lesion length in bacterial treatment / Average leaf length in control × 100 BB disease severity was also scored using the Standard Evaluation System (Standard Evaluation System for Rice, IRRI) (Table 1). The percentage disease suppression was calculated using the formula: % Disease suppression = Difference in mean BB lesion length between control and treated leaves − − − − − − − − − − − − ×100 Mean lesion length in control leaves

ShB suppression by Pseudomonas treatments Percentage severity of sheath blight disease was calculated using the formula: % Disease incidence = (Lesion height/Plant height) × 100 The percent disease suppression was calculated using the following formula: % Disease suppression = Difference in disease incidence between control and treated plants − − − − − − − − − − − × 100 Disease incidence in control Data analysis The data for both diseases were analyzed for statistical significance using the Least Significant Difference (LSD) test.

Results Identification of Pseudomonas spp. A total of 256 strains (MSP330 to 585) showed a single distinct amplified product of approximately 950 bp to 1Kb, thereby confirming that they belong to the genus Pseudomonas (data not shown). The presence of this fragment correlates with a specific PCR primer pair especially for the detection of 16S (small-subunit) rRNA genes exclusive to the genus Pseudomonas. In vitro screening of Pseudomonas spp. for inhibition of X. oryzae pv. oryzae Of the 256 rice rhizosphere Pseudomonas strains isolated, 122 strains (47.7%) showed antagonism to X. oryzae pv. oryzae. The zone of inhibition ranged from 4 mm to 32 mm (Table 2). With regard to the salt tolerance of the antagonistic strains, 44 (36.0%) could grow in 0.5 M NaCl concentration, 20 (16.4%) in 1.0 M NaCl and 4 (3.3%) even in 1.5 M NaCl concentration. None of the strains tolerated NaCl concentrations higher than 1.5 M. A total of 20 strains that were antagonistic to X. oryzae pv. oryzae in the laboratory assays and could tolerate a minimum of 1.0 M NaCl were chosen for the greenhouse tests (Table 2).

77 Table 2. Suppression of bacterial leaf blight (BB) by bacterial antagonists MSP

In vitro

In vivo Non-saline soil conditions#

Acc. No

Inhibition zone (mm)

Severity grade

% Area infected

% Disease suppression

Control 352a 374b 377a 412a 477c 478a 480a 483a 496a 497c 498b 500a 504a 522a 531e 538c 566e 572d 573a 581a

27 15 7 13 21 24 18 32 12 20 6 31 12 9 16 8 13 16 11 15

7 4 4 3 4 5 4 5 5 5 5 5 4 4 5 6 4 4 6 4 4

85 14 23 11 19 30 22 27 31 36 27 33 25 16 35 53 15 25 69∗ 23 22

71 62 74 66 55 63 58 54 49 58 51 60 69 50 32 70 60 15 62 63

Saline soil conditions@ Severity % Area % Disease grade infected suppression 8 4 4 4 5 4 5 5 4 5 3 4 4 3 5 5 3

89 18 16 21 27 15 29 43 21 32 11 13 14 7 28 30 11

71 73 68 62 74 60 46 68 57 78 76 75 82 61 59 78

# LSD @ LSD 0.05 = 11; LSD0.01 = 14 0.05 = 13; LSD0.01 = 18. ∗ Significant at 5% (LSD ) 0.05 ; The other values are significant at 1% (LSD0.01 ). (Each value is a mean of four replications). a -P. fluorescens;b -P. aeruginosa;c -P. putida;d -P. alcaligenes;e -P. pseudoalcaligenes.

In vitro screening of Pseudomonas spp. for inhibition of R. solani

Greenhouse experiments for bacterial leaf blight suppression

When 256 strains were screened for antagonism towards R. solani in the laboratory assays, 84 strains (32.8%) inhibited fungal growth. The zone of inhibition ranged from 1 mm to 19 mm (Table 3). With regard to the salt tolerance of this set of antagonistic strains, 38 (45.2%) could grow in 0.5 M NaCl, 16 (19%) could grow in 1.0 M NaCl and 7 (8.3%) even in 1.5 M NaCl concentration. Sixteen strains which showed in vitro antibiosis towards R. solani and could tolerate a minimum of 1.0 M NaCl were chosen for evaluation of sheath blight suppression in greenhouse tests (Table 3). There were four strains which inhibited both pathogens in the in vitro assays. They were MSP377, MSP497 and MSP504 and MSP573. Of these, strain MSP497 alone could tolerate 1.5 M NaCl while the others could only tolerate 1.0 M NaCl.

Greenhouse experiment under non-saline soil conditions When the short-listed 20 bacterial strains were evaluated for the suppression of bacterial leaf blight on susceptible rice plants and compared with untreated control, it was observed that they suppressed the incidence of BB disease by 15 to 74% (Table 2). The percent leaf area infected in the untreated control plants was 85, which corresponded to a disease score of 7. In the treated plants, this was reduced to 11– 69% that corresponded to a disease score of 3 to 6. Sixteen strains suppressed disease incidence by more than 50%. The strain MSP377 afforded the highest level of disease suppression. It reduced BB by 74% and reduced the leaf area infected to 11%. In thein vitro plate assay this strain exhibited an inhibition zone of only 7 mm diameter. The largest zone of inhibi-

78 Table 3. Suppression of sheath blight (ShB) by bacterial antagonists MSP Acc. No.

Control 343b 349f 351a 368c 377a 390c 393a 494c 495d 497c 503a 504a 534e 555d 573a 584e

In vitro

In vivo Non-saline soil#

Saline soil@

Inhibition zone (mm)

% Disease severity

% Disease suppression

% Disease severity

% Disease suppression

16 17 8 6 4 11 18 19 14 7 11 10 5 13 10 14

64 28 34 16 9 13 32 23 11 28 22 7 14 30 31 30 20

36 30 48 5 51 32 41 53 36 42 57 50 33 33 33 44

56 18 24 17 15 5 8 37∗ 8 7 6 11

38 32 40 41 51 48 19 49 49 50 45

# LSD @ 0.05 = 14; LSD0.01 = 19. LSD0.05 = 17; LSD0.01 =22. ∗ Significant at 5% (LSD 0.05 ); The other values were significant at1% (LSD0.01 ). (Each value is a mean of four replications). a -P. fluorescens;b -P. aeruginosa;c -P. putida;d -P. alcaligenes;e -P. pseudoalcaligenes; f -P. cepacia.

tion was 32 mm diameter induced by strain MSP483 that suppressed BB disease by 54%. The performance of all except one strain, MSP 572, was found to be statistically significant (at LSD0.01 ). A majority of the bacterial antagonists were identified as strains of P. fluorescens (60%), including MSP377 which afforded maximum BB suppression. The other antagonists were strains of P. aeruginosa (10%), P. putida (15%), P. alcaligenes (5%) and P. pseudoalcaligenes (10%).

line conditions than under non-saline conditions. Eight strains showed disease suppression of more than 70%. The strain that performed best under saline conditions was MSP538 from site V which suppressed the BB disease by 82%. This strain was identified as P. putida. The percent leaf area infected in the plants treated with this strain was 7% which had a disease score of 3. The performance of all sixteen strains was found to be statistically significant (at LSD0.01 ).

Greenhouse experiment under saline soil conditions

Greenhouse experiment for sheath blight (ShB) suppression

The 16 strains that afforded disease suppression of more than 50% under non-saline conditions were chosen for this experiment conducted under saline soil conditions. The percent BB suppression varied from 46 to 82%. The disease symptoms in untreated and treated leaves of CO43 plants are shown in Figure 1. The percent leaf area infected in the untreated control was 89% which had a disease score of 8. In the treated plants this was 7 to 43% which corresponded to a disease score of 3 to 5 (Table 2). All 16 strains chosen for this experiment performed well with ten strains showing higher levels of disease suppression under sa-

Greenhouse experiment under non-saline soil conditions The 16 bacterial strains tested for their suppression of sheath blight showed 30 to 57% reduction under nonsaline conditions in the greenhouse. The percent ShB disease severity in the untreated control was 64%. In the plants that received bacterial treatments, the reduction in disease severity ranged from 7 to 34% (Table 3). The most effective bacterial strain was MSP 503, and it afforded 57% ShB suppression. This strain was identified as P. fluorescens. The species that were able

79

Figure 1. Symptoms of bacterial leaf blight in CO43 leaves. (a) Untreated, uninoculated negative control; (b) untreated, pathogen inoculated positive control; (c) bacteria treated leaves showing disease suppression.

to suppress ShB disease were P. fluorescens (38%), P. putida (25%), P. cepacia (6%), P. aeruginosa (6%), P. alcaligenes (12.5%) and P. pseudoalcaligenes (12.5%). The reduction in disease severity by all strains tested was statistically significant (at LSD0.01 ). Greenhouse experiment under saline soil conditions Eleven strains that afforded a disease suppression of 35% or more under non-saline soil conditions were chosen for greenhouse studies under saline soil conditions. The percent ShB severity in the untreated control was 56%. This was reduced by 5 to 36% in the bacteria treated plants (Table 3). The disease symptoms in untreated and treated plants of CO43 plants are shown in Figures 2 and 3. Of the 11 strains chosen for study under saline conditions, the performance of six strains was comparable to their efficacy under normal soil conditions. Their suppression of ShB disease ranged from 19 to 51%. The strain that afforded the

Figure 2. Symptoms of rice sheath blight in IR50 rice plants. Severe symptoms seen in untreated control (left), and restriction of sheath blight symptoms in treated plants (right).

maximum disease suppression was MSP393. The reduction in percent disease severity exhibited by 17 of 18 strains tested (except MSP495) was significant at 1% (LSD0.01). The levels of disease suppression of five of the strains increased marginally under saline conditions.

Discussion Most biocontrol studies have been directed towards normal environmental conditions and fail to take into account the stress factors that operate in problematic ecosystems (Ross et al., 2000). Saline environments such as those in the coastal regions of Tamil Nadu in Southern India have not received any attention before. However, such studies are important in these areas as they are known hot-spots for natural incidence of blight and blast diseases of rice. A systematic search for biocontrol agents efficient under saline conditions

80

Figure 3. Sheath blight suppression in CO43 rice plants by strain MSP538. Untreated plants showing severe sheath blight symptoms on 60th day (above) and treated plants showing disease suppression (below).

would bring about significant suppression of rice diseases and prove as a suitable strategy for the small income group, resource-poor rice farmers. Salinity was used in our study as the selection factor while screening for efficient Pseudomonas strains. This stress assumes greater proportions in rice fields as they are subjected to excessive acidity and herbicide usage (Gopalaswamy, 2001). Native strains are found to perform better and are less prone to inhibition by stress factors than introduced ones. The efficiency of these strains can also be improved. (Gopalaswamy, 2001). All 256 strains isolated from the coastal rice rhizospheres in this study were screened for antagonism towards both R. solani and X. oryzae pv. oryzae. Of the strains tested under in vitro conditions, only those exhibiting an inhibition zone of >1 mm against R. solani and >2 mm against X. oryzae pv. oryzae were chosen. Though some researchers have found no correlation between in vitro antagonism exhibited by the

bacteria and their ability to suppress disease under greenhouse or field conditions (Papavizas and Lewis, 1983), there are other reports suggesting it is possible to identify efficient biocontrol agents based on their inhibition in dual plate assays in the laboratory. In the past, researchers working in this area (Gnanamanickan and Mew, 1992; Mew and Rosales, 1986) have used in vitro antibiosis as a criterion for their selection of biological control strains. In this study, it was found that strains which showed in vitro antibiosis could also suppress disease in the greenhouse experiments. However, there was no definite correlation between the in vitro antibiosis and greenhouse suppression. Strain MSP352 that showed a zone of inhibition of 27 mm was able to suppress disease by 71% under both nonsaline and saline greenhouse conditions, while strain MSP483 that showed an inhibition zone of 32 mm, but could suppress disease by 54 and 68%. Similar examples can be cited also with sheath blight suppression. Overall, 47.7% of the strains inhibited X.

81 oryzae pv. oryzae, and 32.8% inhibitedR. solani. For the greenhouse experiments, only those strains tolerant to a minimum level of 1.0 M NaCl were chosen. This is in keeping with the main aim of this study which was to identify efficient strains that can compete and survive in the South Indian coastal environment. It is well known that the adaptability of a bacterial inoculant to soil condition is crucial to its ultimate fate in the soil (Van Overbeek and Van Elsas, 1995). Most of the strains that could successfully inhibit bacterial leaf blight and sheath blight disease development in the greenhouse were found to belong to the fluorescent pseudomonads group. The most common species encountered was P. fluorescens followed by P. putida, P. cepacia and P. aeruginosa. Nonfluorescent species like P. alcaligenes and P. pseudoalcaligenes also showed disease suppression although their performance was relatively poor. Earlier studies have linked the presence of fluorescent pseudomonads to the inherent ability of soils for disease suppressiveness. The performance of strains varied with soil conditions. The strain which afforded maximum disease suppression against bacterial leaf blight and sheath blight under non-saline conditions were MSP377 (74%) and MSP503 (57%), respectively. Under saline soil conditions, MSP538 (82%) and MSP393 (51%) performed best against bacterial leaf blight and sheath blight, respectively. The ability of P. fluorescens strains to suppress rice diseases has already been well established (Gnanamanickam et al., 1999, 2001). Previously, P. putida strains have also been shown as good biocontrol agents for other plants diseases (Dupler and Baker, 1984). P. cepacia, present in the rhizoplane of rice has been found to suppress various pathogens such as Penicillium expansum and Botrytis cinerea (Janisiewicz and Roitman, 1988). With regard to application of bacterial antagonistic strains, foliar spray applications have been found to give the best results for BB reduction (Kavitha, 1999). However, it is also known that deployment of biocontrol agents at the seedling stage may prevent early infection, resulting in increased levels of disease suppression (Gnanamanickam et al., 1999). In consideration of these facts, antagonistic strains were applied as seed treatments, root dip treatments and as foliar spray applications to confer the maximum protection possible to the plants. With respect to the disease symptoms, the bacteria-treated plants showed smaller ShB lesions restricted to the lower portions of the stem (Figure 3). In plants treated with bacterial strains for bacterial leaf blight, the BB lesions on the

leaves were restricted to the tips (Figure 1). They were also restricted to the lower leaves alone indicating their failure to spread to new leaves. One of the most useful observations to emerge from this study is the multiple protection exhibited by strains MSP377 (P. fluorescens), 497 (P. putida), 504 (P. fluorescens) and 573 (P. fluorescens) to both rice diseases tested. Three of them could suppress both bacterial leaf blight and sheath blight diseases under non-saline as well as saline soil conditions. These strains hold great potential for development as biological control agents most suited for the coastal environment. There have been many reports of a single biocontrol bacterium being active against more than one pathogen (Nautiyal, 1999). The consistent performance of Pseudomonas spp. as antagonists under both natural and saline soil conditions is an important finding for the development of commercially viable biocontrol strains. Several new strains of Pseudomonas, which are effective suppressors of two rice pathogens, have been described in this study. Further field tests need to be undertaken to ascertain their full potential under saline conditions. More insight into the mechanisms that govern interactions between rhizobacteria, host and pathogen is also needed. Though this study has been aimed at the coastal agri-ecosystem of Tamil Nadu, it may be possible to use these strains in other target regions of similar nature elsewhere in the world as well.

Acknowledgements We are very grateful to the Department of Biotechnology, Government of India for providing the financial assistance to carry out this work, and also to Prof. S. S. Gnanamanickam for his valuable input.

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