Screening And Identification Of Antagonistic Fungi Against Ralstonia Solanacearum And Research On The Active Products

Tobacco wilt disease caused by Ralatonia solanacearum is a bacterial disease, widely occurred in tropical and subtropical zone. It has happened and led to severe damage in the south of the Yangtze River Basin of China, such as Sichuan, Guangdong, Fujian, Hunan and Guizhou province. Yield loss of tobacco can reach up to80%in most serious tobacco region. Traditional methods of chemical control have severe impacts on ecological environment as well as high costs, therefore, rotation and microbial control were proved to be promising methods. Microbial control has the advantage of pollution-free and long-term, which can fundamentally solve the drug residue in the soil, drug resistance of pathogens and other problems. It not only benefits the environments, but also turns ecological resources into virtuous circle.In order to provide the theoretical basis of biological control of Ralatonia solanacearum, this dissertation focused particularly on studies on the active strain screening, identification, optimization of fermentation conditions, tobacco seedling test in the presence of fermentation broth, and stability of products of active strains, the main results are as follow.1. Fiftyty-nine fungus strains were isolated from soil samples in tobacco fields collected from Qianjiang district, Chongqing. Paper disk method was applied to screen antagonist fungi, and2stains, named3F03and7F06, behaved strong inhibition activities. The diameter of inhibition zone was10.0mm and9.5mm, using the concentration of250μg crude extract per piece paper disk on plate. Repeated plate antagonism test showed the diameter of inhibition zone had no obvious difference.2. According to the morphological, cultural, cellular fatty acid and rDNA ITS sequence analysis, active strains3F03and7F06were identified as Aspergillus fumigatus and Neosartorya aureola, respectively.3. The growth of tobacco seedlings treated with fermentation broth of strains3F03and7F06was in good condition. Compared with controls, the seed germination rate, seedling height, seedling weight, root length and root weight had no obvious changes. Furthermore,10-folds diluted fermentation broth of strain3F03and100-folds diluted fermentation broth of strain7F06had certain growth-promoting effect.4. The fermentation conditions of strains3F03and7F06were studied. The orthogonal experiment indicated that the optimum fermentation condition of strain3F03for producing the most active products was cultured in:fermentation temperature of28℃, fermentation time of96h, inoculation amount of5%, outfit fluid volume of50mL/150mL. The optimum fermentation condition of strain7F06was cultured in: fermentation temperature of28℃, fermentation time of120h, inoculation amount of3%, outfit fluid volume of100mL/150mL.5. Bioassay of the stability of active products of strains3F03and7F06against R.solanacearum. The antibacterial substances from strain3F03showed stability on high temperature, ultraviolet radiation and pH treatment. But protease K and trypsin treatment have certain impact on the activity with the inhibition rate separately decreasing17.0%and14.5%. The activity of strain7F06products had no significant change on ultraviolet radiation and protease treatment. However, the antagonistic activity slightly dropped on acidic conditions, and it entirely lost its antagonistic activity under high temperature (80℃).