Isolation and Identification of Tomato Bacterial Wilt Pathogen (Ralstonia solanacearum) and Screening of Disease-Resistant Germplasm

【Objective】To isolate and identify strong pathogenic strains in high-incidence area for tomato bacterial wilt in Zhanjiang City, western Guangdong Province, and screen tomato disease-resistant lines, in order to lay the foundation for development of germplasm resources resistant to Ralstonia solanacearum and breeding new tomato cultivars.【Method】Infected tomato plants were collected from Guangdong Ocean University in Zhanjiang City. The pathogenic bacteria were isolated through the tissue isolation method, and identified by combining morphological, physiological, biochemical and molecular biological techniques. The stem puncture method and root-wounding inoculation method were used to identify the physiological races and pathogenicity of the strains. The highly pathogenic strain was inoculated onto 12 elite inbred lines resistant to bacterial wilt to verify and re-screen disease-resistant materials with stable traits.【Result】The isolated strains (h2 and h4) were both Gram-negative bacteria, with colonies exhibiting an irregular, nearly circular shape featuring pale red centers and milky white edges, demonstrating viscous and fluid properties. The strains h2 and h4 were identified as biochemical type Ⅳ, physiological race 1, and both amplified specific fragments of R. solanacearum (280 bp) and evolutionary type Ⅰ (144 bp), indicating that both strains belong to phylotype Ⅰ. 16S rDNA analysis showed that the two strains clustered in the same branch as R. solanacearum, with sequence homologies of 99.51% and 99.65%, respectively. Both strains exhibited strong pathogenicity, with h4 demonstrating higher virulence. At 4 days and 6 days post-inoculation, disease incidence rates in 'ZS-5' and 'GZ-1' inoculated with h4 reached 67.7% and 4.4% (4 d), 92.2% and 13.3% (6 d), respectively, consistently higher than those of h2. Ten disease-resistant lines were screened from 12 elite inbred lines using the strain h4.【Conclusion】Both strains h2 and h4 are Gram-negative bacteria belonging to evolutionary type Ⅰ, biochemical type Ⅳ, and physiological race 1. Strain h4 has stronger pathogenicity than h2, and ten highly resistant lines were selected using strain h4.