Quantitative in planta PCR assay for specific detection of Xanthomonas oryzae pv. oryzicola using putative membrane protein based primer set

Molecular-based techniques are becoming desirable as tools for identification and detection of plant diseases. Amongst the Xanthomonas oryzae pathovars, there is a need to differentiate X. oryzae pv. oryzicola from X. oryzae pv. oryzae, as misidentification could lead to improper treatment of rice crops. Therefore, we developed a novel SYBR green real-time PCR assay to target a putative membrane protein gene of X. oryzae pv. oryzicola BLS256. The specificity of the primer set was tested using purified DNA from eleven X. oryzae pv. oryzicola strains, three X. oryzae pv. oryzae strains, and twenty-six other reference pathogenic bacteria. The primer set amplified a single band of expected size from genomic DNA obtained from X. oryzae pv. oryzicola strains, but not from X. oryzae pv. oryzae or from other bacterial strains. The assay was also able to detect at least two genome equivalents of cloned amplified target DNA using purified DNA, or 0.1 × 10−5 OD600 unit of cells per reaction, respectively. This assay represents a novel approach to bacterial leaf streak diagnosis that will advance the field of rice disease research.

► The specificity of the primer set for Xanthomonas oryzae pv. oryzicola was confirmed.
► The assay can detect at least two genome equivalents of cloned amplified target DNA.
► PCR products derived from only infected leaves showed fluorescence signals.