Bacteriophage-mediated detection of Ralstonia solanacearum. K. KUTIN (1), D. Borthakur (1), A. M. Alvarez (1), D. M. Jenkins (1)
(1) University of Hawaii
Phytopathology 98:S85

Abstract: This project aimed to exploit bacteria-bacteriophage interactions for the development of sensitive techniques for the detection of Ralstonia solanacearum. We coupled the rapid self-replicating ability of bacteriophages with quantitative PCR (q-PCR) in an indirect assay for R. solanacearum. We are also exploring the possibility of using phage lytic enzymes for the selective disruption of bacterial cell membrane for further analysis of cell contents. Six R. solanacearum selective bacteriophages (ISO_2, M_DS1, M_DL, S3_S, S6_S1 and S5_5) were isolated from farms on the Hawaiian island of Oahu. These were tested for their generation times and their fecundities. Based these criteria, phage M_DS1 was selected as the optimal candidate for the indirect detection of R. solanacearum. A combination of phage amplification and q-PCR resulted in a detection limit of 2.3 CFU/ml R. solanacearum after incubation for an hour with 1.9 × 106 PFU/ml M_DS1 particles. The sensitivity of the approach is being tested further with drainage water collected from pots growing ginger infected with R. solanacearum. The lysis gene of phage M_DS1 has been isolated and expressed as a 18 KD protein and efforts are under way to evaluate its lytic activity against R. solanacearum and its effect on standard methods of gene replication and detection.