First report of Ralstonia pseudosolanacearum phylotype I sequevar 14 causing bacterial wilt on tomato (Solanum lycopersicum) and eggplant (Solanum melongena) in North Carolina, USA.

Abstract

Ralstonia solanacearum species complex (RSSC) consists of three species, including R. solanacearum, R. pseudosolanacearum, and R. syzygii. The K60-type strain of R. solanacearum was isolated from a wilted 'Marglobe' tomato in Raleigh, North Carolina (NC) in 1953 (Kelman 1954). It is classified as phylotype IIA, sequevar 7 (Prior and Fegan 2005). In July 2023, during a field visit in Eastern NC, patches of >50 eggplant (Solanum melongena cv. Pingtung Oriental) and tomato (Solanum lycopersicum cv. Saybrook) plants showing wilt symptoms were observed in a 1 ha field. Two plants from each host were collected from this site and tested positive for bacterial streaming in sterile deionized water for 2 min. One plant from each host was used for bacterial isolation by plating a 10 μl aliquot of the resulting bacterial streaming suspension on triphenyl tetrazolium chloride (TZC) medium (Kelman 1954) and incubated at 28°C for 48 hr. Multiple fluidal white colonies with a pink center and irregularly round morphology reminiscent of strains in the RSSC were observed on all plates. Only one colony from each plant host, NG-RL and EP-RL from tomato and eggplant, respectively, was selected for molecular characterization. Neither strain amplified the 357 bp band and was not R. solanacearum Select Agent (Opina et al. 1997). Genomic DNA from both NG-RL and EP-RL generated the 280 bp and 144 bp bands and confirmed as R. pseudosolanacearum phylotype I using the RSSC multiplex PCR (Fegan and Prior 2005). To determine sequevar, the primers Endo-F/Endo-R (Poussier et al. 2000; Fegan and Prior 2005) were used to sequence the partial endoglucanase (egl) gene from EP-RL and NG-RL (GenBank accessions: PQ554799 and PQ554800). These sequences were compared to publicly available egl sequences from GenBank and Cellier et al. (2023). A maximum likelihood phylogenetic tree showed that both NG-RL and EP-RL clustered with reference strains PSS81, MLI71-15, and Zo4 with 100% identity, confirming NG-RL and EP-RL are R. pseudosolanacearum phylotype I sequevar 14. To fulfill Koch's postulates, NG-RL and EP-RL inoculant was prepared from 48 h cultures grown on TZC plates at 28°C. Plates were flooded with sterile deionized water and then transferred to a falcon tube, adjusting O.D. 600 to 0.2 (~1×108 CFU/ml). The roots of six-week-old eggplants (cv. Black Beauty) and tomatoes (cv. Bonny Best) were wounded by running a scalpel through the soil 2 cm from the stem. This was repeated on six plants for both NG-RL and EP-RL. Mock-inoculated plants treated with sterile deionized water (SDW) served as controls. Plants incubated at 28°C in the greenhouse showed bacterial wilt symptoms nine days post-inoculation. R. pseudosolanacearum phylotype I sequevar 14 was confirmed from all bacteria-inoculated plants using the multiplex PCR and egl sequencing methods described above. No symptoms or bacteria were isolated from SDW mock-inoculated plants. There have been no prior reports of R. pseudosolanacearum phylotype I in North Carolina. To our knowledge, this is the first report on R. pseudosolanacearum phylotype I and the sequevar 14 in both tomato and eggplant in NC. This phylotype I is endemic to Asia and was first reported in the US in 2003 on pepper in Florida (Ji et al. 2006) and in 2015 on tomatoes in Louisiana (Jimenez Madrid et al. 2019). This finding highlights the need for a nationwide RSSC survey program, which can undoubtedly inform pathogen spread and management for crops in NC and beyond.