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A sensitive, selective, and rapid protocol for detecting Ralstonia solanacearum from soil and plant tissues was developed based on the integration of the rapid self-replicating ability of bacteriophages with quantitative PCR (q-PCR). Six bacteriophages were isolated and selected for their ability to specifically infect and lyse R. solanacearum. Sixty-three strains of R. solanacearum and 72 isolates of other bacterial species were tested for their susceptibility to the bacteriophages. Based on the large host range and observed replication speed and reproductive burst sizes in ginger infecting R. solanacearum strain GW-1, phage M_DS1 was selected for the development of the phage-based indirect assay. With primers based on the phage genome, the protocol was used to detect R. solanacearum from a number of substrates. In pure R. solanacearum cultures, the protocol consistently detected approximately 3.3 CFU/ml after an hour's incubation with 5.3 × 102 PFU/ml M_DS1. We used the protocol to confirm the presence of the pathogen in infected potted ginger plants, detecting levels near 102 CFU/g in 0.1 g of leaf tissue and levels near 103 CFU/ml in drainage water from the pots. In soils emended with the bacteria, we observed detection limits down to approximately 102 CFU/g.

An adaptation of the most probable number (MPN) method and use of microfluor plates for monitoring a bioluminescent strain (171LIIH-7) of Xanthomonas campestris pv. campestris (Pammel) Dowson in debris-inoculated soil, is described. Microfluor plates with liquid culture medium were inoculated with a dilution series of soil extract previously infested with cabbage debris containing 171LIIH-7. Light produced by the bacteria was detected on X-ray film, and absorbance readings of the images were converted to a binary set of values for estimation of MPN by the computer program MPNES (Woomer et al., 1990). The MPN-microfluor plate system was compared with direct dilution plating on FS medium and MPN-miniplate enrichment ELISA with a pathovar-specific monoclonal antibody. The detection limit of X. c. pv. campestris with FS medium was about 104 cfu g−1 soil. The detection threshold of MPN-microfluor plates (102 bacteria g−1 non-sterile soil) was equivalent to the MPN-miniplate enrichment ELISA and 100 times more sensitive than direct dilution plating. The MPN-microfluor plate system is an easy, accurate, sensitive and inexpensive method to detect and enumerate bioliminescent X. c. pv. campestris in soil, and permits ecological studies in the soil environment that will provide the rationale for changes in crop rotations or other cultural practices.

Summary A total of 70 presumptive non-pathogenic yellow-pigmented strains from different origins were identified to the genus Xanthomonas with the genus-specific monoclonal antibodies (XI and XII), and fatty acid methyl ester (FAME) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) protein databases. On the basis of FAME profiles and SDS-PAGE protein patterns, these non-pathogenic xanth- omonads formed a heterogeneous population. Fourty-two strains were identified as the same species by both methods, whereas five strains were identified as different species. Only eight strains remained unidentified by both methods, whereas in 15 cases the identification was ambiguous. The identification at pathovar level was usually ambiguous and not concordant, which indicated that the non-pathogenic xanthomonads cannot be classified in the described pathovars. None of the non-pathogenic xanth- omonads was identified as belonging to the pathovars of the plant from which they were isolated.