Capture of Ralstonia solanacearum species complex strains directly from plant tissue sampled on FTA cards for molecular characterization

Abstract

Proper diagnosis of the causal agents and their characterization are critical steps in the management of bacterial wilt of tomato and other solanaceous vegetable crops. As the species and phylotypes of Ralstonia are different in different geographical regions, it is important to be able to identify the phylotypes present locally quickly and reliably when identifying sources of resistance to bacterial wilt and when deploying host resistance in different regions. Traditional methods for diagnosis and characterization of the causal agents of bacterial wilt require several steps including pathogen isolation from the infected tissues, culturing in a growth media, genomic DNA extraction from the pathogen culture, and few additional steps for molecular identification. In this study, we used a multiplex PCR method to identify the causal species/phylotypes of the pathogen trapped directly from infected plant tissue on FTA (Flinder Technology Associates) cards. Previously characterized R. pseudosolanacearum strain Pss4 (phylotype I) and R. solanacearum strain Pss1632 (phylotype II) separately were inoculated in tomato and eggplant seedlings. Bacterial ooze either directly squeezed from infected plants or streamed in water or 70% ethanol was directly applied on FTA cards and the samples were amplified using a multiplex polymerase chain reaction (PCR) assay. We evaluated four standard methods for processing bacterial wilt samples on FTA cards, which were rapid, sensitive, and reliable for the identification of the species and phylotypes of R. solanacearum complex. These methods will be useful for the characterization/identification of R. solanacearum complex species and phylotypes from diverse locations worldwide since as yet there is no restriction and no import permit is required for the international movement of bacterial wilt sample DNA immobilized and preserved on FTA cards.