Screening, Identification And Characterization Of The Antifungal Substance Of Antagonistic Endophytic Bacteria Against Verticillium Dahilae

Cotton Verticillium Wilt is a world-epidemic disease caused by Verticillium dahliae. It has a serious influence on the quality of cotton and cotton farmers'income. Nowadays the disease is mainly controlled by chemical germicide which will bring about such problems as environment pollution, drug-fast and so on. So it is considered to use biological means instead of chemical germicide to solve the problem. Endophytic bacteria have good prospects in biocontrol application. Existing in plants, endophytic bacteria take a favorable biocontrol position, so they can endure the plant self-defense reaction and directly act on pathogens, which can provide comprehensive and effective protection for the host plants.The aim of the study was to screen strains which had the higher antagonistic activity with better controlling effect on Verticillium dahliae. The best fermentation condition of the strain was chosen and its antifungal substance was isolated and extracted, then its inhibitory mechanism was studied.The main results were as following:120 bacterial strains were isolated in cotton from Xinji, Boye, Chengan region in Hebei. The antagonistic assay in plates showed that there were 36 strains which had antagonistic activity to Verticillium dahliae among them. After second screening, two strains with higher antagonistic activity.were selected for identification. They were identified as Bacillus subtilis and Bacillus pumilus based on the results of morphologic characteristics, physiological and biochemical properties and phylogenetic analysis of 16S rDNA sequence and were named for BSD-2 and BPT-18. The antifungal spectrum of strain BSD-2 was superior to that of strain BPT-18, so strain BSD-2 was further studied.The condition of producing antifungal substance in liquid fermentation was studied. The results showed that optimal liquid ferment medium was composed of beef extract 0.5%, peptone 1%, NaCl 0.5%, glucose 2%. The optimal cultural condition was pH 7.0-7.5, inoculation quantity 5% with 50 mL medium in 250 mL flask, 30℃, 200 r/min, and culture time 38 h.The antifungal substance A could be isolated from other groups by salting out with 40% (NH4)2SO4. The saturation was purified by Sephadex G-75 and Sephadex G-50. The properties of the antifungal substance of strain BSD-2 were studied. The results suggested that it had high stability against heat, acid and alkali, and it was unsensible to Trypsin, Proteinase K, Pepsin and chloroform and light as well. The antifungal substance had the ultraviolet absorption peak in 220 nm and 280 nm. It is preliminarily determined as antimicrobial peptides.The study of antimicrobial mechanism demonstrated that BSD-2 could secrete proteinase and cellulase which could digest the content of cellulose and protein in the fungal cell wall so that the fungal hyphal were destroyed and its growth was inhibited.The antifungal substance could inhibit the hyphal's growth and spore's sprouting of pathogen Verticillium Wilt. Volatile metabolites from BSD-2 could inhibit the hyphal's growth.