Isolation And Identification Of Endogeny Actinomycetes Against Banana Wilt Disease And Study On Antifungial Mechanism Of Streptomyces Sp.5-4

Banana wilt disease is caused by Fusarium oxysporum f.sp.cubeme(Foc)infestation,and has become a major problem from the global banana planting industry.Biocontrol is an important way to manage the banana wilt disease.In this study,we regarded the Fusarium oxysporum f.sp.cubeme race 4(Foc4)as the target fungi to assess the biocontrol effect of endophytic actinomycetes from medicinal plants of Wuzhishan,optimized the fermentation of Streptomyces sp.5-4 by response surface methodology,study on the antifungal mechanism and control effect on banana wilt disease of Streptomyces sp.5-4.This research will lay the foundation for the development and application of Streptomyces sp.5-4,and provide a reference for the green control of banana wilt.The research results are as follows:1.A total of 144 endophytic actinomycetes were isolated from 35 medical plants from Wuzhishan.Of these endophytic actinomycetes,43 strains had antifungal activity against Foc4,especially the strain 5-4 and the strain 5-10 showed strong antifungal activity,and most of them belong to the genus Streptomyces.Combining the morphological and biochemical characteristics,the strain 5-4 belongs to Streptomyces hygroscopicus subsp.hygroscopicus,and strain 5-10 belongs to Streptomyces sp.2.Using the response surface methodology to establish the fermentation model:Y=87.29-1.52X₁+7.99X₂+3.16X₃+2.52X₁X₂+3.55X₁X₃+1.97X₂X₃-14.36X₁²-12.74X₂²-5.32X₃².Finally,the optimal fermentation of Streptomyces sp.5-4 were determined as follows:soluble starch 21.512 g/L,soy flour 15.0 g/L,yeast extract 5.0 g/L,peptone 2.0 g/L,Ca CO₃4.0 g/L,Na Cl 4.734 g/L,p H 7.0,and inoculation amount(3.217%).Predicted the maximum antifungal activity was 89.24%.Through verification experiments were showed that the antifungal activity under the optimized condition(88.36%)was higher than that before optimization(54.96%).It indicating that the optimized fermentation medium was more conducive to the production of active substances in crude extracts.3.Streptomyces sp.5-4 exhibited good antifungal activity on 13 target fungus of 6genus,and had broad-spectrum antifungal activity.The crude extract of Streptomyces sp.5-4 can dissolve the mycelium of Foc4 and the mycelium were significant showed that swelling,shrinkage,dryness and breakage.In addition,the cell wall of Foc4 was irregularly thickened,and showing a multi-layered cell walls,most of the organelles were damaged and hydrolyzed,the cell matrix was reduced and concentrated that causing the obvious vacuoles.Foc4 can demonstrate a series of changes in biochemistry after treating by crude extract of Streptomyces sp.5-4.The results showed that the content of extracellular reducing sugar and malondialdehyde is all increase,the content of soluble protein and the activity of NADH oxidase is all decrease.And all above them will become more serious when the drug concentration and time has increased.The cell membrane of Foc4 showed oxidative damage,leading to a series of cell lesion such as reduced extracellular regulatory capacity and cell membrane lipid peroxidation.The crude extract of Streptomyces sp.5-4 could also induced the chitinase and?-1,3-glucanase to damage the cell walls of Foc4,thereby inhibiting the normal growth of Foc4.It indicated that the crude extract of Streptomyces sp.5-4 had a toxic and inhibitory effect on Foc4,which affected the normal growth of mycelium and sporangium.4.Control effect on banana wilt disease was showed that the disease index was74.07%of inoculated the GFP-Foc4 alone,the corms had obvious blackening phenomenon,and a large number of GFP-Foc4 mycelia and conidia were observed in the banana root by laser confocal scanning microscope.It indicating that the GFP-Foc4 could quickly entered in banana plant and cause disease.The control effect of Streptomyces sp.5-4 treatment was71.36%,and only a few of GFP-Foc4 was attached to the banana root.The results showed that Streptomyces sp.5-4 had a certainly inhibitory effect for pathogen to infect banana root.Pot experiments showed that Streptomyces sp.5-4 had a growth-promoting effect on banana plants.5.The total genome sequence of Streptomyces sp.5-4 is 9,929,690 bp and the content of(G+C)mol%was 72.094%.Among them,the COG functional annotation showed that280 genes were involved in the synthesis,transport and metabolism of secondary metabolites,and accounting for 4.38%of total annotated genes.KEGG databases analysis showed that the pathways most involved in genes including metabolism of terpenoids and polyketides(119 genes),biosynthesis of other secondary metabolites(122 genes),xenobiotics biodegradation and metabolism(146 Genes),carbohydrate metabolism(636genes),and amino acid metabolism(485 genes).The anti SMASH software predicted that there are 51 secondary metabolite biosynthetic gene clusters in Streptomyces sp.5-4.It mainly embrace genes synthesized by NRPS,T1 PKS,T2 PKS,terpene,bacteriocin,indole,ectoine,siderophore,etc.6.Through metabonomics analysis of Streptomyces sp.5-4 during the fermentation process,it showed that the fermentation has significant differences in their metabolites,and most of the differential metabolites appeared in the early and late culture period.Differential metabolites were further screened under the condition that VIP value>2 and p<0.05,a total of 990 metabolites were detected,among of them only 46 known substances.It indicated that the components of the fermentation were complex and have a large number of unknown metabolites.The metabolites such as hygromycin B,sarmentosin,betavulgaroside VIII,and pubescenol might be the key metabolites for anti-Foc4.The most important differential metabolic pathways including arginine and proline metabolism,glycine,serine and threonine metabolism,histidine metabolism,beta-Alanine metabolism,glycerophospholipid metabolism,porphyrin and chlorophyll metabolism,aminoacyl-t RNA biosynthesis,antibiotic biosynthesis,and biosynthesis of plant secondary metabolites.In conclusion,the above results indicated that Streptomyces sp.5-4 is one of great potential to be used as a biocontrol agent to suppress Fusarium wilt disease with both of its antagonistic activity and growth promoting ability.