Sensitive and Specific Detection of Xanthomonas oryzae pv. oryzae by Real-Time Bio-PCR Using Pathovar-Specific Primers Based on an rhs Family Gene

Abstract

Cho, M. . 2011. Sensitive and specific detection of Xanthomonas oryzae pv. oryzae by real-time bio-PCR using pathovar-specific primers based on an rhs family gene. Plant Dis. 95:589-594.The present study describes bio-polymerase chain reaction (PCR) assays to detect bacterial leaf blight caused by Xanthomonas oryzae pv. oryzae in rice. Successful control of X. oryzae. pv. oryzae requires a specific and reliable diagnostic tool. However, other X. oryzae pathovars are detected by currently available molecular and serological methods. In this study, SYBR Green real-time and conventional PCR primer sets were designed based on an rhs family gene of X. oryzae pv. oryzae KACC10331 because these genes are structurally diverse. The specificity of the primers was evaluated using purified DNA from 11 isolates of two X. oryzae pathovars. 21 other Xanthomonas species, and 4 other reference phytopathogenic bacteria and fungi. The assay was also able to detect at least two genome equivalents of cloned amplified target DNA using purified DNA. Thus, the SYBR Green real-time PCR-based method can be used for the rapid and specific detection of X. oryzae pv. oryzae and will potentially simplify and facilitate diagnosis and monitoring of this pathogen and guide plant disease management.Bacterial blight caused by Xanthomonas oryzae pv. oryzae is the major rice disease in all tropical and subtropical Asian countries. The disease commonly produces leaf blight symptoms, and the pathogen invades the host tissue through leaf hydathodes or through mechanical injuries of the leaf blades and multiplies in the vascular system. Yield losses of 10 to 20% are common, and losses of 50 to 70% have been recorded in severely infected fields (9,10,14). The control of bacterial blight is difficult, and the only practical methods for disease management rely on appropriate culture practices and the use of pathogen-free seed and resistant cultivars. Therefore, the specific detection of this pathogen in seed and plants is essential for effective disease control (16,21). Diagnosis of X. oryzae pv. oryzae infection is based on isolation of the pathogen, followed by biochemical identification, pathogenicity tests, or serological tests consisting of fatty acid and other metabolic profiling (2,3,5,19).Currently, molecular assays based on repeated element, siderophore receptor gene, and 16S-23S rDNA spacer region are widely used for the detection of X. oryzae pv. oryzae strains but there have been critical defects in the diagnosis and identification of X. oryzae pv. oryzae isolates, in that these assays also detect X. oryzae pv. oryzlcola ( 1,7,16,21 ). Therefore, in this study, a pathovar-specific primer set based on the rhs family gene of X. oryzae pv. oryzae KACC 10331 was designed. It was reported that Rhs repertoires (YD repeat proteins) are highly dynamic among enterobacterial genomes, due to repeated gene gains and losses. In contrast, the primary structures of rhs genes are evolutionarily conserved, indicating that rhs sequence diversity is driven not by...

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