Genetic analysis of Ralstonia solanacearum strains from different hosts in Thailand using PCR-Restriction fragment length polymorphism

Genetic relatedness of 108 strains of Ralstonia solanacearum (RS) from different hosts in Thailand and other countries were assessed by restriction fragment length polymorphism (RFLP) technique using a pair of primers 759f (5- GTCGCCGTCAACTCACTTTCC -3) and 760r (5- GTCGCCGTCAGCAATGCGGAATCG -3). An amplified DNA fragment of 281 base pairs was obtained from all RS strains and showed three and five patterns after digested with HaeIII and MspI restriction enzymes, respectively. Six RFLP groups were designated A, B, C, D, E and F based on the eight patterns of HaeIII and MspI products. Cluster analysis by UPGMA with Dices coefficient divided into two clusters at 13 percent similarity. Cluster I contained group A (biovar 1/race 1), group B (biovar 2/race 3) and group C (biovar 1/race 1 and race 2) while cluster II consisted of group D (biovars 3, 4/race1, biovar N2 and the blood disease bacterium, BDB), group E (biovar 1/race 1) and group F (biovars 1, 3, 4/race 1 and biovar 5/race 5). RS strains of Thailand were mainly typed into race 1 biovars 3 and 4 and few strains were race 3 biovar 2 from potato in the highland. The strains of race 1 biovar 3 from Thailand could be separated into two groups (D and F) in which group D consisting of strains from pepper, tomato and marigold and group F containing only strains from sesame. The strains of race 1 biovar 4 from ginger and patumma, and race 3 biovar 2 from potato were designated into group D and B, respectively. Comparing with foreign strains, most of biovar 3 and all of biovar 4 from Thailand shared with the strains of biovars 3 and 4 from other countries, biovar N2 strains from Japan including the BDB from Indonesia except biovar 3 of sesame strains which comprised with strains of biovars 1, 4 and 5 from other countries, while biovar 2 strain from highland potato grouped together with the potato strain from other countries. In addition, this technique demonstrated a rapid and efficient identification strains of RS biovars 3, 4 and N2 from biovars 1, 2 and 5 within approximately 6 hr and provided a similar result of RFLP analysis with hrp probe encoded for hypersensitive response and pathogenicity which consumed times and expenses.