Scanning electron microscopic examination of a pellet formulation of Bacillus megaterium and B. pumilus,antagonists of Rhizoctonia solani,and survival during storage

Bacterial formulations, produced using both Bacillus megaterium and B. pumilus individually with pharmaceutical technology, were formulated using a wet granular method. Viability testing in the laboratory revealed that bacterial populations rapidly declined during storage at room temperature (26–30 °C) for 6 months. The scanning electron microscope (SEM) was used to observe bacterial formulations. Both endospores and vegetative cells of B. megaterium and B. pumilus were detected on the formulation surfaces. This revised version was published online in July 2006 with corrections to the Cover Date.     

Preparation and evaluation of Bacillus megaterium-alginate microcapsules for control of rice sheath blight disease

Bacillus megaterium encapsulated in calcium alginate microcapsules was prepared and tested for its efficacy against sheath blight disease of rice. In laboratory conditions, the aqueous suspension (1:100, v/v in potato dextrose agar) of the bacterial microcapsules (10¹⁰ spores/ml) inhibited mycelial growth of Rhizoctonia solani (>99 %) after the microcapsules were produced and stored for 12 months at room temperature (28 ± 2 °C). The survival of the bacterium in the microcapsules in response to ultraviolet (u.v.) irradiation and high temperature was investigated. The survivability of the bacterium in the encapsulated form was greater than that of the fresh cells when it was subjected to u.v. (20-W General electric u.v. lamp from a 25 cm distance for 48 h) and a high temperature treatment (80 °C for 48 h). Cells of the bacterium were detected by scanning electron microscope on both the leaf sheath and the leaf blade (in pot tests in a greenhouse) after spraying encapsulated product. The number of bacteria on the surface of both rice tissues (5 Log. number/g of plant) after spraying with encapsulated product was not significantly different from that after spraying with fresh cells onto the rice seedlings. Spraying the encapsulated B. megaterium on rice plants in the greenhouse was as effective as spraying a chemical fungicide for suppressing rice sheath blight disease.     

Water-soluble granules containing <Emphasis Type="Italic">Bacillus megaterium</Emphasis> for biological control of rice sheath blight: formulation,bacterial viability and efficacy testing

Endospores of B. megaterium were formulated in granule formulations with sodium alginate, lactose and polyvinylpyrrolidone (PVP K-30) by the wet granulation technique. The granule formulation exhibited good physical characteristics, such as high-water solubility and optimal viscosity, that would be suitable for spray application. The bacteria remained viable in the dry granule formulation at 10⁹ c.f.u./g after 24 months storage at room temperature. Under laboratory conditions, aqueous solutions of the formulation showed high activity against mycelial growth of R. solani (99.64 ± 0.14% mycelial inhibition). High viability of the bacterial antagonist on leaf sheath and leaf blade at day 7 after spraying with the formulation was observed (approximately 10⁶ c.f.u./g of plant). Application of an equivalent number of un-formulated endospores resulted in much loss of the bacterial endospores even 1 day after application. In a small pilot field study, an aqueous solution of the formulation (3%w/v) applied by spraying at days 1, 5 and 10 after pathogen inoculation of the rice plants was more effective in suppressing rice sheath blight disease than one application of a fungicide (Iprodione) at day 1. Additionally, rice plants sprayed with the aqueous solution of the granule formulation had higher panicle and whole kernel weights than those of fungicide-treated and control (untreated) plants.     

Development of a spore-based formulation of microbial pesticides for control of rice sheath blight

An effective formulated biopesticide for controlling sheath blight in rice was developed using three microbial antagonists (Bacillus megaterium, Bacillus subtilis and Aspergillus niger) isolated from the rice sheath. The efficiency of spore-based formulations of the above microbial antagonists was investigated and their effectiveness in controlling sheath blight was demonstrated. Application of talc-based formulations of individual antagonists and mixtures of the three antagonists as spray treatments or soil applications were effective in reducing the incidence by up to 45% at 27 days after inoculation of the pathogen of sheath blight and increased rice yield. The use of spores of a fungal antagonist (A. niger), in comparison to commonly used bacterial antagonists, is a novel feature of the present study. Optimum sporulation conditions of the antagonists for preparation of spore-based formulations and their commercially desirable features such as the ability to maintain spore viability in storage were also determined. Culturing in the synthetic replacement sporulation medium (SRSM-2) for 72 hours was the most effective for sporulation of the two bacterial antagonists while culturing in potato dextrose broth (PDB) for 7 days was the most effective for sporulation of the fungal antagonist. It was demonstrated that talc-based formulations of all antagonists, either in refrigerated storage (4°C) or at room temperature (28±2°C), were able to maintain greater spore viability over a longer period (>6 months) than spore suspensions. In view of the relatively shorter life spans of formulations based on vegetative cells, spore-based formulations have a distinct advantage in achieving longer-lasting control, especially under harsh field conditions.     

Characterisation,screening and selection of Bacillus subtilis isolates for its biocontrol efficiency against major rice diseases

A Soil bacterium, Bacillus subtilis, isolated from the rhizosphere of rice, showed high biocontrol activity against blast, sheath blight and bacterial blight. In the present study, four B. subtilis strains isolated from paddy soil were studied under laboratory, greenhouse and field conditions. Among the four strains assayed, UASP17 gave maximum inhibition of paddy pathogens and was validated under field trials. B. subtilis (UASP17) under different doses and methods of applications was evaluated for two seasons at Agriculture Research Station (UAS, Raichur). UASP17 was effective in reducing the severity of blast (9.00% and 15.57%), bacterial leaf blight (BLB) (5.00% and 6.11%) and sheath blight (11.93% and 4.17%) diseases for two seasons. The application of bioagent also increased the paddy grain yield (61.00 and 64.30?Q/ha) in the two seasons, respectively. Taken together, these results indicate that B. subtilis UASP17 as seedling dip for 30?min (10?mL/L of water) prior to transplanting and 2.50?L/ha foliar application was effective in managing the diseases of paddy.     

Biological suppression of rice diseases by Pseudomonas spp. under saline soil conditions

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. oryzaeand 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.     

Bacterial Antagonist as Seed Treatment to Control Leaf Blight Disease of Bambara Groundnut (Vigna subterranea)

Summay Soil samples were taken from 48 fields in the southern part of Thailand in which either bambara groundnut (Vigna subterranea) or groundnut (Arachis hypogeae) had been planted. Bacillus spp. were isolated using soil dilution plates and heat treatment to screen for endospore-producing bacteria. Among 342 Bacillus spp. isolates tested, 168 isolates were not antagonistic to Bradyrhizobium sp. strain NC-92 using dual culture technique. Further testing found 16 isolates of Bacillus spp. had the ability to inhibit mycelial growth of Rhizoctonia solani, a causal agent of leaf blight of bambara groundnut. Among these isolates, Bacillus spp. isolate TRV 9-5-2 had the greatest activity in anti-microbial tests against R. solani. This isolate was later identified as B. firmus. A powder formulation of B. firmus was developed by mixing bacterial endospores, talcum, sodium carboxymethylcellulose (SCMC) and polyvinylpyrolidone (PVP). The formulations contained bacterial levels ranging from 10⁸ to 10¹⁰ c.f.u./g and the viability of bacteria in all formulations remained high after 1 year storage at room temperature (26–32 °C). All formulations showed satisfactory effectiveness in vitro in suppressing mycelial growth of R. solani using dual culture technique. The application of formulations as seed treatment showed that these formulations did not cause abnormality of seedling shape and had no effect on the germination of bambara groundnut seeds.     

Plant growth promotion abilities and formulation of <Emphasis Type="Italic">Bacillus megaterium</Emphasis> strain B 388 (MTCC6521) isolated from a temperate Himalayan location

Bacillus megaterium strain B388, isolated from rhizosphere soil of pine belonging to a temperate Himalayan location has been characterized. The plant growth promotion and biocontrol properties of the bacterium have been evaluated through petridish and broth based assays. The isolate solubilized tricalcium phosphate under in vitro conditions; maximum activity (166 μg/ml) was recorded at 28°C after 15 days of incubation. Production of indole acetic acid demonstrated in broth assays was another important plant growth promoting character. The bacterium produced diffusible and volatile compounds that inhibited the growth of two phytopathogens viz. Alternaria alternata and Fusarium oxysporum. The carrier based formulations of the bacterium resulted in increased plant growth in bioassays. The rhizosphere colonization and the viability of the cells entrapped in alginate beads were greater in comparison to coal or broth based formulations. The bacterium showed maximum similarity with Bacillus megaterium by 16S rRNA analysis.     

利用纤维二糖差向异构酶制备乳果糖的研究进展

乳果糖是由D-半乳糖和D-果糖两个基团通过β-1,4糖苷键连接而成的还原型二糖;乳果糖口服液具有治疗慢性便秘和肝性脑病的功效,在100多个国家作为常见非处方药(OTC)使用,需求量十分巨大;乳果糖还可以作为益生元改善人体肠道菌群关系.乳果糖的生产依赖化学法,其催化剂对人体有害,下游分离难度大.近年来,纤维二糖差向异构酶...     

Phenotypic and molecular characterization of rhizobacterium <Emphasis Type="Italic">Burkholderia</Emphasis> sp. strain R456 antagonistic to <Emphasis Type="Italic">Rhizoctonia solani</Emphasis>, sheath blight of rice

The effect of rhizobacterium Burkholderia sp. strain R456 on the inhibition of Rhizoctonia solani, sheath blight of rice was examined. Results from this study indicated that strain R456 not only suppressed the in vitro mycelial growth of R. solani, but also reduced the incidence and severity of rice sheath blight under greenhouse conditions. However, similar to plant pathogenic strain LMG 1222^T of Burkholderia cepacia, the type species of the genus, infiltration of tobacco leaves with cell suspension of strain R456 resulted in typical hypersensitivity reactions while the two bacterial strains were unable to cause disease symptoms on rice seedlings. The fatty acid methyl ester profile, sole carbon source utilization, and biochemical tests confirmed that the antagonistic rhizobacterium R456 is a member of the genus Burkholderia. Furthermore, strain R456 was differentiated from B. cepacia LMG 1222^T and was identified as Burkholderia seminalis based on recA gene sequence analysis and multilocus sequence typing. In addition, this rhizobacterium had a lower proteolytic activity compared with that of the pathogenic B. cepacia LMG 1222^T while no cblA and esmR marker genes were detected for the two bacterial strains. Overall, this is the first characterization of rhizobacterium B. seminalis that protected rice seedlings from infection by R. solani.     

Selection and evaluation of the potential of choline-metabolizing microbial strains to reduce Fusarium head blight

Choline and betaine are found in wheat flower tissues and have been implicated in stimulating hyphal growth of the primary causal agent of Fusarium head blight (FHB), Gibberella zeae. Choline metabolizing strains (CMS) from wheat anthers may therefore be a useful source of antagonists of G. zeae. One-hundred twenty-three of 738 microbial strains that were recovered from wheat anthers collected from plants grown in Illinois and Ohio were CMS as determined by growth in a liquid medium containing choline as a sole carbon and nitrogen source and a colorimetric, choline oxidase-based assay of culture filtrate. Thirty-one out of 123 CMS reduced FHB disease severity by at least 25% in greenhouse tests on wheat and 17 reduced FHB severity by at least 50%. All five CMS selected for field testing in 2003 reduced disease severity compared to the untreated check at both field locations on moderately resistant cultivar Freedom. Freedom wheat treated with Pseudomonas sp. AS 64.4 had 63% and 46% less FHB severity than untreated wheat at the two sites. Three of five CMS reduced severity at both locations on susceptible cultivar Pioneer Brand 2545. Disease control was comparable to that obtained using the fungicide Folicur 3.6F. Selection of wheat anther colonists for ability to utilize choline as a sole carbon and nitrogen source has utility as a screening tool in the search for efficacious antagonists of G. zeae although choline utilization does not insure that an isolate will be an effective biocontrol agent against Fusarium head blight.     

Biological control of Phytophthora blight in red pepper (Capsicum annuum L.) using Bacillus subtilis

Phytophthora blight is one of the most important devastating diseases of red pepper plants. Forty-one bacterial isolates were obtained from rhizosphere soil and subsequently tested for antagonistic activity under in vitro and in vivo conditions. Among the 41 isolates tested, 12 exhibited a maximum antagonistic activity in dual culture assay. These 12 isolates were further screened for disease suppression on red pepper plants in both natural and greenhouse conditions. All the antagonists showed varying levels of antagonism, whereas the isolates R33 and R13 exhibited the maximum (86.8 and 71%) ability to reduce the disease severity in in vivo conditions. Based on the 16S rDNA sequencing, the most effective isolates were identified as Bacillus subtilis. In addition, the isolates were also screened for siderophores, hydrogen cyanide and hydrolytic enzymes. Further, the isolates increased the root and shoot length of the red pepper, which is an added advantage of the isolates while performing the desired function.     

Identification of rice sheath blight and blast quantitative trait loci in two different O. sativa/O. nivara advanced backcross populations

Two accessions of Oryza nivara, a wild ancestral species of rice (O. sativa) identified as being moderately resistant to sheath blight and leaf blast disease, were used as donor parents to develop two advanced backcross populations with the US rice cultivar, Bengal, as the recurrent parent. The O. nivara donor parent for Wild-1 (252 BC2F1 lines) was acc. IRGC100898 and for Wild-2 (253 BC2F1 lines) was acc. IRGC104705. Both populations were genotyped with 131 simple sequence repeat markers and the linkage maps covered 1,567.5 cM (Wild-1) and 1,312.2 cM (Wild-2). Sheath blight (ShB) disease was evaluated in both inoculated greenhouse and field conditions. Days to heading (DH), plant height (PH), and plant type (PT), confounding factors for sheath blight disease under field conditions, were recorded. Leaf blast disease was rated under inoculated greenhouse conditions. Multiple interval mapping identified qShB6 with resistance to sheath blight disease attributed to the O. nivara parent in the greenhouse. In the field, qShB6 also was the most significant ShB quantitative trait locus (QTL) in all trials, with resistance attributed to O. nivara. In addition, qShB1 and qShB3 were identified in all trials but were not always attributed to the same parent. The qShB6 QTL is in the same region as the DH-QTL, qDH6, and qShB1 is in the same region as the major PH-QTL, qPH1, suggesting that these ShB-QTL may be confounded by other traits. Although qShB3 did not have as large an effect as other loci, it was not confounded by either DH or PH. For leaf blast, qBLAST8-1 was found in both populations providing resistance to races IB1 and IB49, whereas qBLAST12 providing resistance to both races, was only found in Wild-2. Resistance was attributed to O. nivara for both QTL, and blast resistance genes have been previously reported in these regions.     

Biological control of bacterial spot of tomato under field conditions at several locations in North America

Following the relatively successful biological control of bacterial speck of tomato under field conditions at several locations (Phytopathology 92 (2002) 1284), similar selection and testing strategies were employed in an effort to isolate an effective biological control agent for bacterial spot of tomato. Fifty potential biological control agents were isolated from tomato foliage in Alabama (AL) and Florida (FL) and tested under greenhouse conditions in AL for the ability to reduce the foliar severity of bacterial spot of tomato (Lycopersicon esculentum), which is caused by either Xanthomonas campestris pv. vesicatoria or Xanthomonas vesicatoria. Three pseudomonads that provided protection against bacterial speck also were included in the tests. The strains which were most efficacious (i.e., high mean percentage reduction) and consistent (i.e., low standard deviation) in reducing bacterial spot severity in repeated greenhouse experiments were selected for field experiments conducted over the period 1996–1998. Among these strains were Cellulomonas turbata BT1, which provided the highest mean reduction in disease severity [45.2% (SD = 21.0)], and Pseudomonas syringae Cit7 [36.4% (SD = 12.2)], which was the most consistent. Field experiments were conducted in Shorter, AL; Bradenton and Sanford, FL; Clinton, North Carolina; Wooster, Ohio; and London, Ontario, Canada. The highest mean reductions in severity of bacterial spot on foliage, averaged across all locations, were provided by P. syringae Cit7 [28.9% (SD = 11.6)] and Pseudomonas putida B56 [23.1% (SD = 7.4)]. The efficacy and consistency of P. syringae Cit7 against bacterial spot were very similar to those achieved against bacterial speck [28.3% (SD = 12.7)] (Phytopathology 92 (2002) 1284). Unfortunately, neither the bacterial strains nor the standard copper bactericides consistently reduced disease incidence on fruit.     

Biological control of common blight of bean (Phaseolus vulgaris) causedby Xanthomonas axonopodis pv. phaseoli by using the bacteriumRahnella aquatilis

The aim of this study was to evaluate the bacterium Rahnella aquatilis (Ra) for protection of bean plants against common blight disease caused by Xanthomonas axonopodis pv. phaseoli (Xap). Xap isolates were isolated from a naturally blighted leaves of bean plants grown in Assiut governorate. The blight symptoms were produced by all three isolates, but the isolates differed in their degree of the pathogenicity. Xap1 was the most virulence one against bean plants. The effect of Ra against common blight of bean plant was tested. In vitro studies, Ra exhibited inhibitor effect against the pathogen. Under greenhouse and field conditions, beanvariety “Giza 6” treated by Ra resulted in marked disease suppression. Ahigh decrease of the disease was correlated with a reduction of the bacterial multiplication. In physiological studies, bean plants treated by Ra exhibited higher phenolic compounds contents and higher activity of peroxidase (PO) enzyme than untreated plants. In conclusion, application of Ra was effective and could be recommended for controlling the bean common blight disease.     

Pyramiding transgenic resistance in elite indica rice cultivars against the sheath blight and bacterial blight

Elite indica rice cultivars were cotransformed with genes expressing a rice chitinase (chi11) and a thaumatin-like protein (tlp) conferring resistance to fungal pathogens and a serine-threonine kinase (Xa21) conferring bacterial blight resistance, through particle bombardment, with a view to pyramiding sheath blight and bacterial blight resistance. Molecular analyses of putative transgenic lines by polymerase chain reaction, Southern Blot hybridization, and Western Blotting revealed stable integration and expression of the transgenes in a few independent transgenic lines. Progeny analyses showed the stable inheritance of transgenes to their progeny. Coexpression of chitinase and thaumatin-like protein in the progenies of a transgenic Pusa Basmati1 line revealed an enhanced resistance to the sheath blight pathogen, Rhizoctonia solani, as compared to that in the lines expressing the individual genes. A transgenic Pusa Basmati1 line pyramided with chi11, tlp, and Xa21 showed an enhanced resistance to both sheath blight and bacterial blight. S. Maruthasalam and K. Kalpana have contributed to this article equally.     

Suppression of Botrytis cinerea sporulation by Clonostachys rosea on rose debris: a valuable component in Botrytis blight management in commercial greenhouses

Botrytis blight, caused by Botrytis cinerea (Bc), is an important disease on roses grown in plastic greenhouses in Brazil. Biocontrol with Clonostachys rosea (Cr) applied to leaves and crop debris to reduce pathogen sporulation can complement other control measures for disease management. Two experiments, each with a rose cultivar, were conducted in a plastic greenhouse. For ‘Red Success,’ four treatments were compared: (1) control; (2) fortnightly sprays of Cr; (3) weekly sprays of mancozeb; and (4) weekly sprays of either Cr or mancozeb to the lower third of the plants and the debris. For ‘Sonia,’ treatment 4 was not included. Samples were taken from debris (leaves and petals) at ten 15-day intervals and plated on PCA medium. Sporulation of fungi and incidence of Botrytis blight on buds were assessed. For both cultivars, C treatments significantly (P=0.05) reduced Bc sporulation. However, disease incidence was not consistently reduced, probably because the applications of C. rosea started when Botrytis blight epidemic was advanced and no sanitation practices were performed on nontreated plots. From the present and previous studies, continuous application of Cr on debris, associated with sanitation practices, has the potential to reduce Bc sporulation and disease incidence in the buds.     

Identification and characterization of rhizosphere fungal strain MF‐91 antagonistic to rice blast and sheath blight pathogens

Aim

To examine the inhibition effects of rhizosphere fungal strain MF‐91 on the rice blast pathogen Magnaporthe grisea and sheath blight pathogen Rhizoctonia solani.

Methods and Results

Rhizosphere fungal strain MF‐91 and its metabolites suppressed the in vitro mycelial growth of R. solani. The inhibitory effect of the metabolites was affected by incubation temperature, lighting time, initial pH and incubation time of rhizosphere fungal strain MF‐91. The in vitro mycelial growth of M. grisea was insignificantly inhibited by rhizosphere fungal strain MF‐91 and its metabolites. The metabolites of rhizosphere fungal strain MF‐91 significantly inhibited the conidial germination and appressorium formation of M. grisea. Moreover, the metabolites reduced the disease index of rice sheath blight by 35·02% in a greenhouse and 57·81% in a field as well as reduced the disease index of rice blast by 66·07% in a field. Rhizosphere fungal strain MF‐91 was identified as Chaetomium aureum based on the morphological observation, the analysis of 18S ribosomal DNA internal transcribed spacer sequence and its physiological characteristics, such as the optimal medium, temperature and initial pH for mycelial growth and sporulation production.

Conclusions

Rhizosphere fungus C. aureum is effective in the biocontrolling of rice blast pathogen M. grisea and sheath blight pathogen R. solani both in in vitro and in vivo conditions.

Significance and Impact of the Study

This study is the first to show that rhizosphere fungus C. aureum is a potential fungicide against rice blast and sheath blight pathogens.     

Evaluation of microbial strains for linoleic acid hydroxylation and reclassification of strain ALA2

In previous studies, a new microbial strain ALA2 was isolated which produced many new products from linoleic acid [Gardner H.W., Hou C.T., Weisleder D. and Brown W. 2000. Lipids 35: 1055–1060; Hou C.T. 1998. 12,13,17-Trihydroxy-9(Z)-Octodecenoic acid and derivatives and microbial isolate for production of the acid. US Patent No. 5, 852, 196]. Strain ALA2 was preliminary identified as Clavibacter sp. based on its physiological and fatty acid profiles. To determine if strain ALA2 is the optimal strain for industrial applications, other related strains were screened for their abilities to convert linoleic acids. Two strains from Clavibacter and 20 type strains from the phylogenetically related genus Microbacterium were studied. Surprisingly, all of these strains tested showed very little or no activity in converting linoleic acid. On reexamination of the identification of strain ALA2, the sequence of the 16S ribosomal RNA gene of ALA2 was found to be 99% identical to that of Bacillus megaterium and the strain was also found to have 76.3% DNA homology to the B. megaterium type strain. Therefore, strain ALA2 is now reclassified as B. megaterium. Screening of 56 strains of B megaterium strains showed that many of them were able to produce reasonable amounts of hydroxyl fatty acids from linoleic acid, although strain ALA2 possessed the greatest activity.     

Biological Control Activities of Rice-Associated Bacillus sp. Strains against Sheath Blight and Bacterial Panicle Blight of Rice

Potential biological control agents for two major rice diseases, sheath blight and bacterial panicle blight, were isolated from rice plants in this study. Rice-associated bacteria (RABs) isolated from rice plants grown in the field were tested for their antagonistic activities against the rice pathogens, Rhizoctonia solani and Burkholderia glumae, which cause sheath blight and bacterial panicle blight, respectively. Twenty-nine RABs were initially screened based on their antagonistic activities against both R. solani and B. glumae. In follow-up retests, 26 RABs of the 29 RABs were confirmed to have antimicrobial activities, but the rest three RABs did not reproduce any observable antagonistic activity against R. solani or B. glumae. According to16S rDNA sequence identity, 12 of the 26 antagonistic RABs were closest to Bacillus amyloliquefaciens, while seven RABs were to B. methylotrophicus and B, subtilis, respectively. The 16S rDNA sequences of the three non-antagonistic RABs were closest to Lysinibacillus sphaericus (RAB1 and RAB12) and Lysinibacillus macroides (RAB5). The five selected RABs showing highest antimicrobial activities (RAB6, RAB9, RAB16, RAB17S, and RAB18) were closest to B. amyloliquefaciens in DNA sequence of 16S rDNA and gyrB, but to B. subtilis in that of recA. These RABs were observed to inhibit the sclerotial germination of R. solani on potato dextrose agar and the lesion development on detached rice leaves by artificial inoculation of R. solani. These antagonistic RABs also significantly suppressed the disease development of sheath blight and bacterial panicle blight in a field condition, suggesting that they can be potential biological control agents for these rice diseases. However, these antagonistic RABs showed diminished disease suppression activities in the repeated field trial conducted in the following year probably due to their reduced antagonistic activities to the pathogens during the long-term storage in -70C, suggesting that development of proper storage methods to maintain antagonistic activity is as crucial as identification of new biological control agents.