Development of an assay to detect Xanthomonas oryzae pathovars from rice seeds – the application of a molecular tool in facilitating the global movement of “clean” rice seeds

The inclusion of Xanthomonas oryzae pathovars oryzae (Xoo) and oryzicola (Xoc), causal organisms of bacterial blight and bacterial leaf streak, respectively, as select agents calls for a universally accepted protocol for their detection. This is very important in facilitating global movement of rice seeds. Thus, this study aims to develop a method to detect these pathogens from seeds, which incorporates the use of diagnostic multiplex PCR primers developed by Lang and co-workers. Development and refinement of the protocol focuses on preparation of extracts from seed samples in a simple and cost-effective manner, but still obtaining reliable results. Offering flexibility to the users, the developed protocol provides an option for pathogen detection without bacterial isolation, termed direct assay, which involves sonication, addition of sodium sulfite (Na2SO3) and polyvinylpolypyrrolidone (PVPP), filtration, and freezing, as steps to obtain PCR-ready samples. When applied to artificially inoculated seeds and seeds from naturally infected plants, Xoo DNA is detected from the latter after 10 months of storage in room temperature and 4oC. The other option combines multiplex PCR and microbiological methods to isolate, identify and confirm virulence of the pathogens. Validation of the qualitative assay was done by 24 participants from 13 countries who are involved in germplasm-related work during a training-workshop conducted to harmonize detection strategies for the two pathogens. Providing a validated, standard protocol for detecting these pathogens from rice seeds ensures understanding of the end-users, whether the technology is employed in quarantine, research, or institutions involved in the global movement of rice seeds.