Phylogenomic Insights on the Complex, and Development of a TaqMan Real-Time Assay for Specific Detection of pv. on Barley.

The reemergence and spread of , the causal agent of bacterial leaf streak in cereal crops and wilt in turfgrass and forage species, is a concern to growers in the United States and Canada. The pathogen is seedborne and listed as an A2 quarantine organism by EPPO, making it a major constraint to international trade and exchange of germplasm. The pathovar concept of the group is confusing due to overlapping of plant host ranges and specificity. Here, comparative genomics, phylogenomics, and 81 up-to-date bacterial core gene set (ubcg2) were used to assign the pathovars of into three genetically and taxonomically distinct clusters. The study also showed that whole genome-based digital DNA-DNA hybridization unambiguously can differentiate the pvs. and . Orthologous gene and proteome matrix analyses suggest that the cluster consisting of , , , , and is very divergent. Whole-genome data were exploited to develop the first pathovar-specific TaqMan real-time PCR tool for detection of pv. s on barley. Specificity of the TaqMan assay was validated using 62 and non- strains as well as growth chamber-inoculated and naturally infected barley leaves. Sensitivity levels of 0.1 pg (purified DNA) and 23 CFUs per reaction (direct culture) compared favorably with other previously reported real-time PCR assays. The phylogenomics data reported here suggest that the clusters could constitute novel taxonomic units or new species. Finally, the pathovar-specific diagnostic tool will have significant benefits to growers and facilitate international exchange of barley germplasm and trade.