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AIMS: To study the phenotypic and genetic diversity of culturable bacteria associated with rice seed and to asses the antagonistic and pathogenic potential of the isolated bacteria. METHODS AND RESULTS: Seed of rice cultivar PSBRc14 was collected from farmers' fields of irrigated lowland in southern Luzon, Philippines. Isolations of distinct colonies yielded 498 isolates. Classification of the isolates according to similarities in cellular characteristics, whole-cell fatty acid composition, and colony appearance differentiated 101 morphotype groups. Predominant bacteria were Coryneform spp., Pantoea spp. and Pseudomonas spp. Other bacteria regularly present were Actinomycetes spp., Bacillus pumilus, B. subtilis, Burkholderia glumae, Enterobacter cloacae, Paenibacillus polymyxa, Staphylococcus spp. and Xanthomonas spp. The genetic diversity among isolates was assessed by BOX-PCR fingerprinting of genomic DNA and represented 284 fingerprint types (FPTs). Most FPTs (78%) were not shared among samples, while eight FPTs occurred frequently in the samples. Seven of these FPTs also occurred frequently in a previous collection made from rainfed lowlands of Iloilo island, Philippines. Sixteen per cent of the isolates inhibited in vitro the mycelial growth of the rice pathogens Rhizoctonia solani and Pyricularia grisea, whereas 4% were pathogens identified as Burkholderia glumae, Burkholderia gladioli and Acidovorax avenae ssp. avenae. CONCLUSIONS: This study reveals a broad morphological and genetic diversity of bacteria present on seed of a single rice cultivar. SIGNIFICANCE AND IMPACT OF THE STUDY: This line of work contributes to a better understanding of the microbial diversity present on rice seed and stresses its importance as a carrier of antagonists and pathogens.

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A variety of methods that included natural and synthetic media for isolating bacteria and fungi were used to isolate the causal agent of the red stripe disease of rice. A fungus was isolated consistently from leaves with red stripe symptoms using the blotter method. In this method, surface-sterilized leaves were placed on sterile petri plates, lined with moistened filter paper, and the plates were incubated for 14 days at 28 to 30°C. The fungal isolate was reisolated from the lesions of inoculated plants and reinoculated on test plants that became symptomatic, thus fulfilling Koch's postulates. Based on the morphological characteristics of the fungus, the causal agent of red stripe was identified as a species of Gonatophragmium. Colonies of the latter are slow-growing, reaching an average of 29.0 ± 0.9 mm in diameter after incubation on potato dextrose agar (PDA) for 18 days at 28 to 30°C in the dark. It can be overgrown easily by saprophytes when infected leaves are incubated using the blotter method without sterilization. Yellow-orange pigmentation was observed on PDA with mycelial growth and on filter paper with infected leaves, a unique characteristic which facilitates the isolation of the fungus. Studies are in progress using traditional and molecular techniques to identify the pathogen to the species level.

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With the use of a seed washing technique, more than 4000 Gram negative bacteria were isolated by two improved isolation methods from 446 batches of 1 kg rice seed samples obtained from 22 provinces in the Philippines. They were initially characterized on the basis of colony morphology and results of biochemical and pathogenicity tests. Six hundred and fifty-two strains were further identified by Biolog, from which 133 were selected for fatty acid methylester (FAME) analysis together with 80 standard reference strains. Sixteen species or types of Pseudomonas and 17 genera of non-pseudomonads were identified, more than one third of which have not been recorded in rice. The most predominant species observed were P.putida and P. fulva. About 17% of the strains of Pseudomonas and 2% of the non pseudomonads were antagonistic to one or more fungal or bacterial pathogens of rice. Rice seed is an important source of biological control agents.

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Genetic transformation has been attempted for management of rice sheath blight disease, caused by Rhizoctonia solani. We introduced a PR-3 rice chitinase gene (RC7), isolated from R. solani-infected rice plants, into indica rice cultivars IR72, IR64, IR68899B, MH63, and Chinsurah Boro II by the biolistic and PEG-mediated transformation system. Inheritance was studied up to the T(2) generation by Southern blot analysis. Western blot analysis of transgenic plants with polyclonal antibody revealed the presence of chitinase protein with a molecular weight of 35 kDa that reacts with chitinase antibody. The transformants synthesized different levels of chitinase proteins constitutively and progeny from the plants containing the chitinase gene showed different levels of enhanced resistance when challenged with the sheath blight pathogen R. solani.

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ABSTRACT During the 1995 wet season, harvested rice seed was collected from farmers' fields at different locations in Iloilo, Philippines. Bacterial isolations from crushed seed yielded 428 isolates. The isolates were characterized by BOX-polymerase chain reaction fingerprinting of total genomic DNA and represented 151 fingerprint types (FPT). Most FPTs were found on a single occasion, although matching fingerprints for isolates from different samples also were found. Identifications were made by cellular fatty acid methyl ester analysis and additional use of Biolog GN/GP MicroPlates and API 20E/50CHE systems. The predominant bacteria were Enterobacteriaceae (25%), Bacillus spp. (22%), and Pseu-domonas spp. (14%). Other bacteria regularly present were identified as Xanthomonas spp., Cellulomonas flavigena, and Clavibacter michiganense. Of the total number of isolated bacteria, 4% exhibited in vitro antifungal activity against Rhizoctonia solani or Pyricularia grisea. Two percent of isolates were pathogens identified as Burkholderia glumae and Burkholderia gladioli. Five percent of isolates induced sheath necrosis on only 50 to 90% of inoculated plants and were related to Bacillus pumilus, Paenibacillus spp., Pseudomonas spp., and Pantoea spp.

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Durability of plant disease resistance (R) genes may be predicted if the cost of pathogen adaptation to overcome resistance is understood. Adaptation of the bacterial blight pathogen, Xanthomonas oryzae pv. oryzae (Xoo), to virulence in rice is the result of the loss of pathogen avirulence gene function, but little is known about its effect on aggressiveness under field conditions. We evaluated the cost in pathogenic fitness (aggressiveness and persistence) associated with adaptation of Xoo to virulence on near-isogenic rice lines with single R genes (Xa7, Xa10, and Xa4) at two field sites endemic for bacterial blight. Disease severity was high in all 3 years on all lines except the line with Xa7. Of two Xoo lineages (groups of strains inferred to be clonally related based on DNA fingerprinting) detected, one, lineage C, dominated the pathogen population at both sites. All Xoo strains were virulent to Xa4, whereas only lineage C strains were virulent to Xa10. Only a few strains of lineage C were virulent to Xa7. Adaptation to virulence on Xa7 occurred through at least four different pathways and was associated with a reduction in aggressiveness. Loss of avirulence and reduced aggressiveness were associated with mutations at the 3' terminus of the avrXa7 allele. Strains most aggressive to Xa7 were not detected after the second year, suggesting they were less persistent than less aggressive strains. These experiments support the prediction that Xa7 would be a durable R gene because of a fitness penalty in Xoo associated with adaptation to Xa7.

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Crop heterogeneity is a possible solution to the vulnerability of monocultured crops to disease. Both theory and observation indicate that genetic heterogeneity provides greater disease suppression when used over large areas, though experimental data are lacking. Here we report a unique cooperation among farmers, researchers and extension personnel in Yunnan Province, China--genetically diversified rice crops were planted in all the rice fields in five townships in 1998 and ten townships in 1999. Control plots of monocultured crops allowed us to calculate the effect of diversity on the severity of rice blast, the major disease of rice. Disease-susceptible rice varieties planted in mixtures with resistant varieties had 89% greater yield and blast was 94% less severe than when they were grown in monoculture. The experiment was so successful that fungicidal sprays were no longer applied by the end of the two-year programme. Our results support the view that intraspecific crop diversification provides an ecological approach to disease control that can be highly effective over a large area and contribute to the sustainability of crop production.

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ABSTRACT Among the 171 strains of Xanthomonas oryzae pv. oryzae (the bacterial blight pathogen of rice) collected from eight rice-producing zones in Nepal, 31 molecular haplotypes were distinguished using two polymerase chain reaction-based assays. Six common haplotypes represented nearly 63% of the strains, and some haplotypes were geographically dispersed. Multiple correspondence analysis divided the collection into five putative genetic lineages. Lineages 1, 2, and 4 were the most frequently detected and occurred in diverse geographic populations. Twenty-six pathotypes (virulence phenotypes) of X. oryzae pv. oryzae were identified using 11 near-iso-genic rice lines, each containing a single gene for resistance. The 26 pathotypes grouped into five clusters, and cluster 1 contained wide virulence spectrum strains from all geographic populations. Although molecular variation was greatest between strains of different virulence phenotypes, some variation was observed among strains with identical virulence. There was a weak correlation (r = 0.52) between molecular haplotypes and virulence phenotypes. There are two major groups of X. oryzae pv. oryzae in Nepal. One group consists of strains with high molecular polymorphism and many pathotypes that are either virulent to the 11 major resistance genes or avirulent only to Xa21. Strains in the second group have low molecular polymorphism and are avirulent to Xa4, xa5, Xa7, and Xa21.

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The progress of bacterial blight epidemics, caused by Xanthomonas oryzae pv. oryzae, varies with environment. The irrigated lowland rice production environment in central Terai (plain) is less conducive to the disease than the irrigated lowland rice production environment in eastern Terai in Nepal. The effect of partial resistance on bacterial blight was studied in central Terai during the wet seasons of 1994, 1995, and 1996. Three partially resistant rice cultivars, Sabitri, Laxmi, and IR54 (possessing the Xa4 gene), and susceptible check IR24 were included in this study. Analysis of pooled data from the 3 years of experiments indicated that rice cultivars differed in resistance based on three epidemiological parameters: disease severity (DS), area under the disease progress curve (AUDPC), and rate of disease increase (r). Estimates of DS, AUDPC, and r were reduced and yield loss was negligible in the partially resistant cultivar Laxmi compared with the susceptible check IR24. IR54, which has partial resistance to X. oryzae pv. oryzae, also showed low DS, AUDPC, and r, which prevented yield loss due to bacterial blight. The locally adapted cultivar Sabitri showed intermediate estimates of DS, AUDPC, r, and yield loss. Earlier and more severe disease developed in the susceptible check IR24, resulting in a yield loss of 22%. Yield losses were mainly due to reductions in number of tillers, number of grains per panicle, and 1,000-grain weight. These data indicate that the use of rice cultivars with high levels of partial resistance will be an effective tool for the management of bacterial blight in central Terai and similar environments in Nepal.

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Fifty isolates of Xanthomonas oryzae pv. oryzae were collected from different rice-producing districts of Nepal and evaluated for their virulence on these 11 rice lines having from one to four resistance genes: IRBB4 (Xa4), IRBB5 (xa5), IRBB7 (Xa7), IRBB8 (xa8), IRBB10 (Xa10), IRBB14 (Xa14), and IRBB21 (Xa21), two-gene combination AY4+5 (Xa4 and xa5), three-gene combinations NH21-37-1-1 (Xa4, xa5, and xa13) and NH24-10-1-3 (Xa4, xa5, and Xa21), and four-gene combination NH56-1-44-4 (Xa4, xa5, xa13, and Xa21). The ability of an isolate to cause lesions with different lengths across the lines was interpreted as virulence. Isolates that were consistently associated with high or low virulence were statistically differentiated. Most isolates produced large lesions on near-isogenic lines with single genes and small lesions on lines with different gene combinations. Based on infection responses on the two-, three-, and four-gene combinations, five virulence groups were identified. Isolates in virulence group I were widely distributed. The line × isolate interactions were generally not significant with gene combinations, indicating a low possibility of specificity. A line with a four-gene combination, NH56, showed wider spectrum and higher level of resistance to X. oryzae pv. oryzae than the other lines. The results of this study will facilitate the deployment of effective resistance to X. oryzae pv. oryzae in Nepal.

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Molecular tools were used to analyze the genetic diversity and population structure of Xanthomonas oryzae pv. oryzicola, the bacterial leaf streak pathogen of rice in the Philippines. Representative pathogen strains were selected and used to assess resistance in rice germplasm. A partial genomic library of X. oryzae pv. oryzicola was constructed, and a 459-bp clone containing the repetitive DNA element R41 was selected as a probe for restriction fragment length polymorphism (RFLP) analysis and sequenced. R41 shared 44% sequence homology with the putative transposase gene of IS1112, an insertion element cloned from X. oryzae pv. oryzae. Using R41 as a probe for RFLP analysis, 26 band profiles were discerned in a collection of 123 strains of X. oryzae pv. oryzicola. Analysis of PstI digestion patterns of DNA from the same collection resolved 36 haplotypes. Several clusters of strains were detected after grouping of data based on either pR41 as a probe or Pst1 digestion patterns. However, based on bootstrap analysis, the clusters were not robust. Genetic diversity was high for the entire collection as well as within spatially and temporally defined subsets of strains. Even a set of strains collected from a single site at a single time was highly diverse. Strains representing the different DNA types were inoculated to a set of diverserice cultivars. Consistent rice varietal groupings were obtained from disease reaction data, but there was no correlation between pathogen isolate cluster and host reaction across inoculation trials. Isozyme group I of rice, representing tropical japonica and javanica germplasm, is a promising source of resistance to bacterial leaf streak.

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ABSTRACT The effect of a soil amendment (SA) composed of urea (200 kg of N per ha) and CaO (5,000 kg/ha) on the survival of Ralstonia solanacearum in four Philippine soils was investigated in a series of laboratory experiments. Within 3 weeks after application, the SA either caused an initial decrease, a final decline, or no change in the pathogen population, depending on the particular soil type. An initial decrease occurred in a soil with a basic pH and resulted in a significantly (P < 0.001) lower pathogen population immediately and at 1 week after amending the soil. This decrease was probably due to the high pH in the soil during urea hydrolysis. A final decline in the R. solanacearum population after 3 weeks occurred in two soils in which nitrite accumulated after 1 week. In these soils, no decline in bacterial levels occurred when nitrite formation was inhibited by 2-chloro-6-trichloromethylpyridine. In the soil with low pH, no nitrite accumulated and the R. solanacearum population did not decline. The suppressive effects of pH and nitrite on R. solanacearum growth were confirmed by in vitro experiments. Ammonium reduced the growth of R. solanacearum, but was not suppressive. Interactions of pH with ammonium and nitrite also occurred, whereby ammonium reduced growth of R. solanacearum only at pH 9 and nitrite was suppressive only at pH 5. Nitrate had no effect on R. solanacearum growth in vitro.

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A leaf-inoculation method for detecting Xanthomonas oryzae pv. oryzicola in rice seed was developed and tested. The method is based on inoculating leaf segments on agar with seed washings in a moist chamber. Freshness of inoculated leaf segments is maintained by addition of 75 to 100 ppm benzimidazole to the 1% water agar after sterilization. Using cells from pure cultures, the minimum inoculum concentration to initiate leaf streak lesions on the segments was found to be 103 CFU/ml. Inoculum prepared from seeds harvested from severely infected mother plants induced lesions typical of leaf streak, followed by bacterial ooze. With 4 g of seed, the incubation period was 3.5 days and bacterial ooze appeared within 5 days. The minimum amount of seed needed to detect the bacterium based on seed washings varied according to disease severity of the mother plants. The bacteria isolated from lesions on the leaf segments were confirmed as X. oryzae pv. oryzicola after bacteriological, serological, and Biolog tests were conducted. The results confirm that the detached-leaf method is as simple and reliable as the immuno-radiometric assay and inoculation on intact pot-grown rice plants in the greenhouse.

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The potential of antagonistic bacteria from paddy water, rhizosphere soils, sclerotia, and rice plants to control "bakanae" caused by Fusarium moniliforme was assessed. Experiments were conducted to determine the in vitro antibiosis and effect of bacteria on seed germination. Out of 441 isolates, 113 were inhibitory to mycelial growth of the pathogen. Bacterial strains were classified into three groups based on effect on seed germination: (i) those that promoted germination and enhanced seedling vigor; (ii) those that had no effect on germination; and (iii) those that were deleterious and inhibited germination. Bacterization of naturally infected seeds reduced bakanae incidence in seedbox and seedbed tests. In a seedbed experiment with IR 58 seeds soaked in suspensions of bacterial strains, bakanae incidence and disease control ranged from 0.9 to 6.8% and 71.7 to 96.3%, respectively. From the 3 years of field trials, 10 strains reduced bakanae. Five strains consistently reduced bakanae, but the other five exhibited variable effects among trials. Specificity of suppression by antagonistic bacteria against different pathogenic isolates of F. moniliforme from various locations in the Philippines was observed.

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Fifty seed samples of different rice varieties stored in warehouses for varying periods (1-28 months) were collected and screened for their fungal flora, using standard blotter and agar plate methods, resulting in the isolation of 36 fungal forms. The samples were also studied for moisture content, germinability, and seedling abnormalities. Significant correlations were found among the numbers of fungi, storage period, and germinability. Fourteen samples, seven each of IR64 and IR66 were studied with regard to moisture content, germination test, abnormal seedlings, speed of germination, conductance of leachates, total dehydrogenase activity, total free amino acids, total soluble sugar, fat acidity, gelatinization temperature, gel consistency, amylose content, translucency, and per cent whiteness. Significant relationships were found between the fungi and the parameters studied.

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Restriction fragment length polymorphism and virulence analyses were used to evaluate the population structure of Xanthomonas oryzae pv. oryzae, the rice bacterial blight pathogen, from several rice-growing countries in Asia. Two DNA sequences from X. oryzae pv. oryzae, IS1112, an insertion sequence, and avrXa10, a member of a family of avirulence genes, were used as probes to analyze the genomes of 308 strains of X. oryzae pv. oryzae collected from China, India, Indonesia, Korea, Malaysia, Nepal, and the Philippines. On the basis of the consensus of three clustering statistics, the collection formed five clusters. Genetic distances within the five clusters ranged from 0.16 to 0.51, and distances between clusters ranged from 0.48 to 0.64. Three of the five clusters consisted of strains from a single country. Strains within two clusters, however, were found in more than one country, suggesting patterns of movement of the pathogen. The pathotype of X. oryzae pv. oryzae was determined for 226 strains by inoculating five rice differential cultivars. More than one pathotype was associated with each cluster; however, some pathotypes were associated with only one cluster. Most strains from South Asia (Nepal and India) were virulent to cultivars containing the bacterial blight resistance gene xa-5, while most strains from other countries were avirulent to xa-5. The regional differentiation of clusters of X. oryzae pv. oryzae in Asia and the association of some pathotypes of X. oryzae pv. oryzae with single clusters suggested that strategies that target regional resistance breeding and gene deployment are feasible.

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Several transposable elements were isolated from the genome of Xanthomonas oryzae pv. oryzae. These elements and an avirulence gene isolated from X. oryzae pv. oryzae were used as hybridization probes for a collection of X. oryzae pv. oryzae strains from the Philippines. Each of the sequences was present in multiple copies in all strains examined and showed distinct patterns of hybridizing bands. Phenograms were derived from the restriction fragment length polymorphism data obtained for each of the individual probes and for pooled data from multiple probes. The phenograms derived from the different probes differed in topology and, on the basis of bootstrap analysis, were not equally robust. For all of the probes, including the avirulence gene, some groups (even some haplotypes) consisted of multiple races. The strains were grouped into four major clusters on the basis of the two probes giving the highest bootstrap values. These groups were inferred to represent phylogenetic lineages. Three of the six races of X. oryzae pv. oryzae appeared in more than one of the lineages, and another was present in two sublineages. For three of the races, strains representing different phenetic groups were inoculated on rice cultivars carrying 10 resistance genes. Two new races were differentiated, corresponding to pathogen lineages identified by DNA typing. On the basis of DNA and pathotypic analyses, together with information on the spatial and temporal distribution of the pathogen types from this and other studies, a general picture of X. oryzae pv. oryzae evolution in the Philippines is presented.

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A repetitive DNA element cloned from Xanthomonas oryzae pv. oryzae was used to assess the population structure and genetic diversity of 98 strains of X. oryzae pv. oryzae collected between 1972 and 1988 from the Philippine Islands. Genomic DNA from X. oryzae pv. oryzae was digested with EcoRI and analyzed for restriction fragment length polymorphisms (RFLPs) with repetitive DNA element as a probe. Twenty-seven RFLP types were identified; there was no overlap of RFLP types among the six races from the Philippines. Most variability (20 RFLP types) was found in strains of races 1, 2, and 3, which were isolated from tropical lowland areas. Four RFLP types (all race 5) were found among strains isolated from cultivars grown in the temperate highlands. The genetic diversity of the total population of X. oryzae pv. oryzae was 0.93, of which 42% was due to genetic differentiation between races. The genetic diversities of strains collected in 1972 to 1976, 1977 to 1981, and 1982 to 1986, were 0.89, 0.90, and 0.92, respectively, suggesting a consistently high level of variability in the pathogen population over the past 15 years. Cluster analysis based on RFLP banding patterns showed five groupings at 85% similarity. The majority of strains from a given race were contained within one cluster, except for race 3 strains, which were distributed in three of the five clusters.

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Two R1 IR8 plants derived from somatic cells cultured in 25% Helminthosporium oryzae toxin-medium, and one IR54 plant derived from a control (toxin free medium) were found to have mutated resistance to brown spot disease of rice. In the second generation (R2, the offspring of the R1 generation) of the three resistant populations, the segregation of resistance and susceptibility to the disease was observed. This is the first report about a disease resistant mutation obtained successfully in rice by tissue culture and in vitro screening with phytotoxin.