First Report of Ralstonia pesudosolanacearum Causing Bacterial Wilt of Canna edulis in China

Canna edulis is a perennial herb, belonging to the Canna, Cannaceae. Native to South America and the West Indies, it was introduced into China in the 1950s, and widely planted in China (Liu et al, 2013). The planting area of C. edulis was about 20 hm2 in Datian town, Taining county, Sanming city, Fujian Province, China, and a new bacterial disease of Canna was found in July 2022, the disease incidence was about 20% in the field. The apical or middle leaves of the affected plants initially showed withering symptoms during the daytime and recovered at night. As the disease progressed, the whole plant withered, but the leaves' color remained green. Blackening and rotting of the roots and basal stem tissues were observed, leading to plant death (Figure S1). The transverse section of the basal stem or roots showed that the vascular tissues became brown, and when compressed, a milky whitish ooze could be observed. Eight bacterial strains (B1-B8) were isolated and purified from six diseased plants on nutrient agar medium (NA) by streak plate method. The pathogenicity of the isolated strains was tested by wounding inoculation on roots, that is, first, we cut off the roots of healthy Canna seedlings with a sterile knife (avoiding lumpy roots), and then watered 50 mL bacterial solution at a concentration of 3×108 CFU·mL-1 per seedling, took sterile water as a negative control, inoculated three plants with each bacterial strain, and repeated three times. The results indicated that the Canna seedlings wilted on the 6th day and began to die on the 14th day after inoculation, the symptoms were consistent with those in the field. The water treatment produced no symptoms. The bacterial strains whose colony morphology was similar to those of test strains were re-isolated on NA medium from inoculated plants, in which the colonies were irregular, milky-white with the production of sticky substances and strong motility. So, Koch's rule proved all the test strains as the causal pathogens of bacterial disease. The physiological and biochemical assays of eight bacterial strains were consistent with those of the control strains, R. pseudosolanacearum RS-5 and FQRS1. The 16S rDNA gene was amplified with the universal primer 27F/1492R (Lane, 1991), and the phylogenetic tree showed that the tested strains clustered with R. pseudoslanacearum (Figure S2A), indicating the test strains to be R. pseudoslanacearum. All the test bacteria could obtain two specific bands (280-bp and144-bp) by PCR amplification with composite primers [Nmult21:1F, Nmult21:2F, Nmult23:AF, Nmult22:InF, Nmult22:RR] (Fegan and Prior, 2005), and the bacterial strains could be identified as phylotype I (Asia group). In addition, the bacteria could use lactose, maltose, cellobiose, mannitol, sorbitol and dulcitol, according to the classification standard of Ralstonia biovar by Hayward (1964), the test bacterial strains could be identified as biovar III. PCR amplification of the endoglucanase gene (egl) by the primer of Endo-F / Endo-R (Fegan and Prior, 2006), and the sequences were compared on NCBI, the phylogenetic tree showed that the tested strains B1, B4, B7, and B8 belonged to sequevar 17, and strain B2, B3, B5 and B6 belonged to sequevar 15, phylotype I (Figure S2B). To our knowledge, this is the first report of R. pseudosolanacearum causing bacterial wilt of C. edulis in Fujian, China. Identifying the pathogen of this disease is crucial for developing effective management strategies.