关键词: 青枯雷尔氏菌, 雷帕链霉菌, 放线菌, 生物防治

Abstract:

【Objective】Tobacco bacterial wilt caused by Ralstonia solanacearum is the main disease of tobacco in cash crops. In this paper, the actinomycetes with strong antagonism against R. solanacearum were screened from the soil of different habitats, and their antibacterial mechanism and biocontrol effect were determined, which provided a theoretical basis for the further development of anti-disease microbial agents.【Method】After screening the target actinomycetes by co-culture method and Oxford cup method, the target strain was identified by morphological studies, physiological and biochemical experiments and polygenic phylogenetic analysis. The minimum inhibitory concentration (MIC) of actinomycetes crude extract against R. solanacearum was determined by the 96-well plate method. After co-culture treatment of R. solanacearum with crude extract, the growth dynamics of R. solanacearum were detected and the morphological changes of R. solanacearum were observed. The effects on membrane permeability and membrane composition were observed by measuring β-galactosidase activity and propidium iodide (PI) fluorescence experiments and Fourier transform infrared spectroscopy. The antibacterial activity and mechanism of target actinomyces antagonistic R. solanacearum were preliminarily explored by measuring protein synthesis, extracellular polysaccharides (EPS), intracellular reactive oxygen species (ROS), and so on. The effect of the target actinomycetes on tobacco bacterial wilt control was determined by pot experiments.【Result】According to the morphological characteristics, physiological and biochemical experimental results and sequencing results, the target actinomyces strain Sa-21 was identified as Streptomyces rapamycinicus, and the diameter of the inhibition zone against R. solanacearum was 47.9 mm. The results of mechanism experiments showed that the minimum inhibitory concentration of Sa-21 strain crude extract inhibiting R. solanacearum was 0.5 μg·mL^-1, which also had a significant inhibitory effect on bacterial proliferation, and the inhibitory effect increased with the increase of crude extract concentration in the test range. Scanning electron microscopy showed that the structure of R. solanacearum changed after crude extract treatment, resulting in perforation and shrinkage of the bacteria, and with the increase of the concentration of crude extract in the test range, the number of ruptured bacteria increased, and the degree of shrinkage increased. After crude extract treatment, propidium iodide could pass through the cell membrane and bind to intracellular substances to fluoresce, and the activity of β-galactosidase was significantly increased, indicating that the permeability of the cell membrane of R. solanacearum was improved. Further studies showed that crude extract treatment could lead to the accumulation of intracellular reactive oxygen species and the decrease of extracellular polysaccharide production in R. solanacearium, indicating that the cell membrane was damaged to a certain extent. The results of potted plant disease control test showed that the relative control effect of 10-fold dilution of fermentation filtrate of treatment group was 67.61%, while the relative control effect of 10-fold dilution of fermentation filtrate of prevention group was 85.89%.【Conclusion】Streptomyces Sa-21 can inhibit the proliferation of R. solanacearum and destroy the cell membrane structure, which has a good control effect on tobacco bacterial wilt, and has certain development potential and application value.

Key words: Ralstonia solanacearum, Streptomyces rapamycinicus, actinomycete, biological control