Focus on Bacterial Blight of Rice Bacterial blight of rice, caused by Xanthomonas or-pw. aryzap (37), was first reported in Japan over a century

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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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Four genes of rice, Oryza sativa L., conditioning resistance to the bacterial blight pathogen Xanthomonas oryzae pv. oryzae (X. o. pv. oryzae), were tagged by restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD) markers. No recombinants were observed between xa-5 and RFLP marker loci RZ390, RG556 or RG207 on chromosome 5. Xa-3 and Xa-4 were linked to RFLP locus XNpbl81 at the top of chromosome 11, at distances of 2.3 cM and 1.7 cM, respectively. The nearest marker to Xa-lO, also located on chromosome 11, was the RAPD locus 0072o00 at a distance of 5.3 cM. From this study, the conventional map [19,28] and two RFLP linkage maps of chromosome 11 [14,26] were partially integrated. Using the RFLP and RAPD markers linked to the resistance genes, we selected rice lines homozygous for pairs of resistance genes, Xa-4 + xa-5 and Xa-4 + Xa-lO. Lines carrying Xa-4 + xa-5 and Xa-4 + Xa-lO were evaluated for reaction to eight strains of the bacterial blight pathogen, representing eight pathotypes and three genetic lineages. As expected, the lines carrying pairs &genes were resistant to more of the isolates than their single-gene parental lines. Lines carrying Xa-4 + xa-5 were more resistant to isolates of race 4 than were either of the parental lines ('quantitative complementation'). No such effects were seen for Xa-4 + Xa-lO. Thus, combinations of resistance genes provide broader spectra of resistance through both ordinary gene action expected and quantitative complementation.

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.

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 ofX. 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

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.