Isolation And Identification Of Actinomycetes Against Fusarium Oxysporum F.sp. Cubense And Optimization Of It's Fermentation Condition

Fusarium wilt of banana is a destructive soil-borne disease of bananas,especially of Fusarium oxysporum f.sp.cubense race 4(FOC4),which can infect virtually all species of bananas.Fusarium wilt of banana has occurred in Guangdong,Guangxi,Hainan,Fujian,Yunnan and other places of China.Now,we haven't found the effective chemical measures to control fusarium wilt of banana.Therefore,it becomes a hot topic that Fusarium wilt of banana is controlled by biocontrol microorganisms.Rhizosphere actinomycete is a kind of highly diverse microorganism,which can produce a variety of beneficial metabolites to inhibit pathogenic microorganism,and has a good developed and applied prospect in biological control of plant diseases.In this study,two actinomycetes strains have been isolated and purified by antagonistic experiments against FOC4 from the rhizosphere soil of cassava.In addition,the two strains have been classified and identified,whose fermentation conditions have been optimized.These studies can lay a foundation for the development and utilization of rhizosphere actinomycetes and new pesticide discovery on biological control banana fusarium wilt.The main results are as follows:(1)In this study,63 strains of actinomycetes were isolated from the rhizosphere soil of cassava,and the strains MS13 and 1-14 has obvious antagonistic activities against FOC4.The results of plate antagonistic experiments in petri dishes showed that strain 1-14 had broad-spectrum antimicrobial activity,which had a powerfully antagonistic ability against 11 pathogens,and antibacterial rate of more than 77%.Specifically,inhibition rate against Colletotrichum acutatum was as high as 97.59%,and inhibition against FOC4 was about 87.65%.(2)Strains MS 13 and 1-14 were respectively identified as Catenulispora and Streptomyces,according to the morphological,biochemical and physiological characteristics,identification results of 16S rDNA and analysis results of phylogenetic tree of strains MS13 and 1-14,.(3)It was analyzed that inhibition rates of crude extracts of the strain 1-14 fermented liquid in MB fermentation medium,soybean meal fermentation culture medium and based fermentation medium against 10 target pathogens,and we found that it of based fermentation medium was obviously superior to it of the other two medium,the based fermentation medium's antibacterial rate of more than 36.15%,and inhibitiona rate against FOC4 was 43.80%.Therefore,the based fermentation medium was the best optimization for 1-14 strain.(4)We optimized fermentation conditions of actinomycete strain 1-14 through response surface methodology,including the single factor experiment,Plackett-Burman design,Steepest ascent design and Box-Behnken design.The optimum fermentation medium of the 1-14 strain was composed of glucose 38.877 g/L,Soy flour 20 g/L,CaCl2·2H2O 0.161 g/L,MgSO4 0.2 g/L,NaH2PO4 0.5 g/L,K2HPO4 0.5 g/L,pH of medium 8,fermentation temperature 29.97 ?,inoculums size 8.93%.Under the best fermentation conditions for biological activity determination,the actinomycete strain 1-14 inhibition rate reached 56.13%,which was 12.33%higher than before(43.80%).(5)According to the stability determination of actinomycete strain 1-14 fermentation crude extracts,it was found that the fermentation crude extracts were relatively stable to heat(50-100 ?),but it was poorer stability to pH and ultraviolet irradiation,it being more stable under 4 ? than room temperature.(6)1-14 strain's quantity was determine in the sterilized and unsterilized soil to inoculate 1-14 strain alone or mixture of 1-14 strain with FOC4 by using laboratory simulation.The results showed that:firstly,the populations of 1-14 strain in sterilized soil were significantly higher than the unsterilized treatment,so high temperature sterilization could significantly improve the survival rate of 1-14 strain in the soil.The increase of 1-14 strain population was inhibited by indigenous microorganisms in soil.Secondly,in sterilized soil where 1-14 strain was inoculated alone,its population changes trend was increased before decreased,which mould be associated with changes of soil moisture,nutrients,temperature or other environmental conditions.The end,FOC4 didn't almost grow in the sterilized and unsterilized soil,which indicated that 1-14 strain have a strong inhibitory effect on FOC4.