Screening, Identification And Antibacterial Mechanism Of Antagonistic Microorganisms Against The Leaf Black Blight Pathogen Of Cyclocarya Paliurus

Cyclocarya paliurus is a rare and endangered tree species in China,which has high medicinal and health care value.This laboratory found,for the first time,the disease of Cyclocarya paliurus caused by Diaporthe eucommiae in a field study,named as leaf black blight.The disease is mainly harmful to the leaves of Cyclocarya paliurus,and seriously affecting the development of related industries and species conservation.Biological control has shown a broad application prospect with many advantages such as environmental friendliness,specific target and low cost.However,there is still no research on biological control of the leaf black blight of Cyclocarya paliurus.In this paper,the rhizosphere microbes of Cyclocarya paliurus were isolated and purified by serial dilution method and spread plate method.Then the antagonistic microorganisms which could significantly inhibit the pathogenic bacteria were screened by plate confrontation method.Also,the bacteriostatic spectrum of antagonism was analyzed.The antagonistic strains were identified based on morphological,physiological and biochemical characteristics and 16 s r RNA gene sequence analysis.Single factor design was used to optimize the fermentation conditions of the antagonistic strains,and the best fermentation conditions were determined by venomous plate method.The mechanisms of antagonistic action of antagonistic strains were also discussed.The results and conclusions were obtained as follows:1.A total of 32 microorganisms,including 8 strains of bacteria,18 strains of fungi and 6 strains of actinomycetes,were isolated from Cyclocarya paliurus rhizosphere soil.The bacterial strain QJK-3 and the actinomycete strain QJK-26 showed good antibacterial activity,and the inhibitory rates were 72.22% and 84.67%,respectively.Strain QJK-26 could also inhibit pathogenic fungi(Rhizoctonia solani and Magnaporthe oryzae)and Bacillus bacteria,indicating a broad-spectrum bacteriostatic effect.2.The antagonistic strain QJK-3 was Gram-positive,catalase positive,short rod-shaped and can produce spores.On the LB medium,the colony grew irregularly and protruding around with wavy edge and wrinkled surface,opaque yellowish.Comprehensive phenotypic and genotypic identification revealed that the QJK-3 strain belonged to Bacillus sp.The antagonistic strain QJK-26 was Gram-positive.The hyphae were free of cross-septum and had branches.The spore filaments exhibited flexible or hook-like growth patterns.By comparing with the model strains with the highest similarity,it was identified as a potential new species of Streptomyces,named as Streptomyces tanzhouensis sp.nov..3.Millet medium was the optimal fermentation one for the production of antibacterial active substances from the antagonistic strain QJK-26.The optimum fermentation conditions were as follows: p H 8,rotation speed 180 rpm,inoculum concentration 5%,temperature 32?,fermentation time 2 days,and the volume of liquid 2/5.4.Both antagonistic strains QJK-3 and QJK-26 had Antibacterial effects.They could inhibit the hypha growth of the Diaporthe eucommiae sp.CB3 and influenced the germination of the spores,and could secrete certain volatile organic compounds(VOCs)to inhibit the normal growth and metabolism of the Diaporthe eucommiae sp.CB3.Also,they had the very strong ability to produce extracellular protease.They could not produce chitinase,glucanase and cellulase.