Molecular Detection Of Ralstonia Solanacearum And Analysis Of Tobacco Resistance To R. Solanacearum

Tobacco bacterial wilt disease, caused by Ralstonia solanacearum, which is a kind of soil-borne pathogen, has become a destructive disease to tobacco throughout the world. R. solanacearum entries into plants mostly by injured roots, and moves rapidly in the vascular bundles, then results in wilt. In the field, the disease becomes more serious when occurs with tobacco black shank and root nematode disease. Since now agricultural control, chemical control and biological control are adopted to control this disease, but the effects are not remarkable. In the long run, the basic way to control this disease is selecting and breeding resistant varieties. Quickly and exactly detecting the number of R. solanacearum in its latent form in soil is an urgent measure to prevent the development of the tobacco bacterial wilt disease. In this research, we did molecular detection of R. solanacearum and resistance identification of varieties.In this study, total DNA of R. solanacearum strains from Shandong province was extracted. The specific primer pair RS-1/RS-2 was designed based on the unique nucleotide sequence of R. solanacearum. The PCR was developed with 290bp of amplification product from R. solanacearum. The result showed that only R. solanacearum separated from tobacco was detected by PCR and other strains were not. R. solanacearum was also specifically detected in tobacco tissues and soil with RS-1/RS-2, and the sensitivity of detection was up to 100 fg/μL.12 soil samples from tobacco-cultivated fields in Mengyin, Yishui and Juxian areas of Shandong Province were detected with the system. The results illustrated that the system we have established was stable and reliable.Different inoculation contents of bacterium and seedling ages were performed to determine the resistance to R. solanacearum during seedling stage. A quick and accurate method for identifying resistance applied to tobacco seedling stage was filtered. 6-week-old tobaccos were inoculated with 1×10~8 cfu/mL R. solanacearum suspension, and the disease index was detected after 30-day-inoculation which could reflect the different resistance to R. solanacearum among different tobacco varieties.Identification of 24 tobacco varieties resistance indicated that all of the tested varieties did not show immune responses and the resistance was different among them. 24 tobacco varieties did not exhibited highly resistance and immunity, RG17 showed the best resistance, the disease index was 26.11, moderate resistance was 20.8% of total materials, moderate susceptibility was 54.2% and high susceptibility was 25.0%.4 tobacco materials with different resistance levels from the 24 tobacco varieties were inoculated with R. solanacearum, sterile water as control. The activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), polyphenol oxidase (PPO) and phenylalanine ammonia lyase (PAL) were determined in the inoculated leaves. After infection by the pathogen, SOD activity remained stable; POD activity increased significantly; PPO activity was a positive correlation with the performance of resistant varieties; PAL activity in different species was different and increased after inoculation; there were small differences of CAT activity in different species and the activity in strong resistant varieties dropped after reaching to the peak.