Survival of Ralstonia solanacearum race 4 in drainage water and soil and detection with immunodiagnostic and DNA-based assays

Abstract

Ralstonia solanacearum (Rs) race 4 strains cause bacterial wilt of edible ginger (Zingiber officinale). The survival of the pathogen was studied in plant-free soil and potting medium in the presence of plants inoculated by different methods (non-wounded, rhizome-wounded, and stem-wounded) and irrigated on different schedules (alternate and daily). Detection thresholds for Rs were determined for an enzyme-linked immunoabsorbent assay (ELISA), immunostrip assay, and polymerase chain reaction (PCR) using drainage water from soil and potting medium containing known concentrations of Rs. In the absence of a plant or in the presence of non-wounded plants, Rs populations declined rapidly in drainage water from potting medium during the first 9 days and were undetectable after 81 days. When plants were stem- or rhizome-wounded, Rs populations increased by two to three orders of magnitude from the initial population levels for the first 9 to 19 days and then gradually declined and became undetectable after 89 days. Results were similar in experiments with soil except for non-wounded ginger plants, where the initial decline in Rs populations was followed by an abrupt increase after day 11, reaching 7 log cfu/mL on day 21, then declining gradually to non-detectable levels after 137 days. The increase was attributed to natural infection of the plants followed by release of high populations of Rs into the irrigation water when plants wilted. When rhizome-inoculated plants were watered on alternate days, Rs was recovered from 97 to 129 days in soil and potting medium, but when the plants were watered daily, Rs was recovered in soil and potting medium up to 153 days after plant inoculation. ELISA using Ps1a monoclonal antibody detected the pathogen from >95% of the samples from soil and potting medium when viable populations were >5 log cfu/mL. The immunostrip assay (using the same antibody) detected the pathogen from 100% of the samples when viable populations were >3 log cfu/mL. PCR based on the flagellin gene fliC detected the pathogen from >95% of the samples from soil and from >74% of the samples from potting medium when viable populations were >4 log cfu/mL.