First Report of Bacterial Wilt of Tomato Caused by Ralstonia pseudosolanacearum (Ralstonia solanacearum phylotype I) in Cambodia

Abstract

In January 2019, wilted tomato plants were discovered in fields in the Chi Kraeng district of Cambodia, 60 km south of Siem Reap. We observed vascular browning from cut stems from symptomatic plants, which tested positive for Ralstonia solanacearum with the Rs Immunostrip (Agdia, Elkhart, IN). Although bacterial wilt was noted in Cambodia (UK-CAB-International 1977), there has been no official report, and tomato farmers have reported historical losses up to 100% from a disease called sropoun sros (“fresh wilt” or in Khmer). Fluidal, irregular bacterial colonies typical of the R. solanacearum species complex (RSSC) were isolated from cut tomato stems on casamino peptone glucose (CPG) plates amended with 1% 2,3,5-triphenyl tetrazolium chloride (Schaad et al. 2001). To confirm the bacterial identification and to subclassify isolates by phylotype, genomic DNA from eight distinct isolates was amplified with the diagnostic RSSC multiplex PCR primers 759/760, Nmult21:1F, Nmult21:2F, βNmult23:AF, Nmult22:InF, and Nmult22:RR (Fegan and Prior 2005; Opina et al. 1997). Positive controls for each RSSC phylotype were genomic DNA from RSSC strains GMI1000 (phylotype I), K60 (phylotype II), CMR15 (phylotype III), and PSI07 (phylotype IV). Each Cambodian isolate yielded the 280-bp RSSC-specific DNA fragment and the 144-bp phylotype I-specific DNA fragment from this diagnostic multiplex PCR. Therefore, all Cambodian isolates were identified as phylotype I, which was recently renamed R. pseudosolanacearum (Prior et al. 2016; Safni et al. 2014). To perform Koch’s postulates, a single fresh colony from each isolate was cultured overnight in CPG broth at 28°C with 220 rpm shaking, pelleted, and resuspended in sterile water to O.D.₆₀₀ ₙₘ = 0.1 (∼1 × 10⁸ CFU/ml). Unwounded 33-day-old tomato plants (wilt-susceptible cultivar Bonny Best) were inoculated via soil soak with 50 ml of inoculum to a final density of ∼3.1 × 10⁷ CFU/g of soil. Two plants were inoculated per isolate, and four plants were treated with sterile water as negative controls. Fourteen out of 16 tomato plants showed wilting symptoms after 14 days, and all plants were wilted after 21 days. These results are representative of two replicates. No water-treated control plants wilted. R. pseudosolanacearum colonies were successfully reisolated from the wilted tomato plants. Sropoun sros or bacterial wilt has likely been a longstanding problem for Cambodian farmers. Characterizing and reporting bacterial wilt of tomato in Cambodia will be the foundation for future management strategies and the development of tomato varieties specifically resistant to Cambodian R. pseudosolanacearum.