Detection of Ralstonia solanacearum in natural substrates using phage amplification integrated with real-time PCR assay

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.