Determination of Ralstonia (Pseudomonas) solanacearum rDNA subgroups by PCR tests

Rapid and sensitive polymerase chain reaction (PCR) methods ape described f or determination of the two 16 S rDNA subgroups of Ralstonia solanacearum, the causal agent of bacterial wilt. A third subgroup consisting of Indonesi an R. solanacearum isolates belonging to Division II, the blood disease bac terium and Pseudomonas syzygii can also be identified. Primers were designe d to sequences within R, solanacearum 16 S rDNA (equivalent to Escherichia coli 16 S rDNA positions 74-97, 455-475, 1454-1474), and the internal trans cribed spacer region between the 16 S and 23 S rDNA genes. Different combin ations of forward and reverse primers allowed selective PCR amplification o f (a) R. solanacearum Division I (biovars 3, 4 and 5), (b) Division TI (bio vars 1, N2, and 2) including the blood disease bacterium and P. syzygii, or (c) amplification of Division II only except for five biovar 1, 2 or N2 is olates of R. solanacearum from Indonesia, P. syzygii and the BDB. A total o f 104 R. solanacearum, 14 blood disease bacterium and 10 P. syzygii isolate s were tested. Simultaneous detection of species and subdivision was achiev ed by designing a multiplex PCR test in which a 288-base pair (bp) band is produced by all R. solanacearum isolates, and an additional 409-bp band in Division I strains.