Isolation?Identification And Biocontrol Potential Analysis Of Endophytic Bacillus BEB17 Antagonistic Banana Fusarium Wilt

In this study,56 strains of endophytic bacteria were isolated and identified from the roots tissues of healthy banana plants collected from the banana wilt disease area in Hainan and Guangxi province,of which 21 strains of endophytic bacteria had antimicrobial activity.A plate antagonistic method and a double dish confrontation method were used to screen and acquire an endophytic bacteria with antagonistic and fumigation inhibition that was antagonistic to a variety of plant pathogenic fungi such as Fusarium oxysporum f.sp.cubense race 4(Foc4),Pyricularia oryzae,Corynespora cassiicola,Colletotrichum capsici.And the antagonistic inhibition rate was 32.24-54.69%,the fumigation inhibition rate was 33.92-66.86%,in which the antimicrobial activity of Foc4 was the highest,reaching up 54.69%and 66.86%respectively.Through colony morphological observation,physiological and biochemical characteristics analysis,this strain was identified as Bacillus amyloliquefaciens,and numbered BEB17 combined with 16S rDNA(Genbank:MF444158)and gyrB gene sequence(Genbank:MF471351).And the PCR test showed that this strain had the genes encoding the synthesis of multiple lipopeptide and polyketide compounds.It had the potential to synthesize many kinds of lipopeptide and polyketone.By using the two extraction methods(acid precipitation and macroporous resin extraction)and two fermentation medium(M9 and Landy medium)to extract the fermentation broth of this strain,the results showed that the acid precipitation method was more efficient,and Landy medium was more prone to the fermentation of lipopeptide and polyketide by the strain BEB17.The activity tests showed that the crude extracts extracted by the two extraction methods had a good inhibitory effect on banana fusarium wilt,which the crude extracts extracted by macroporous resin extraction method had the best inhibitory effect.When the concentration was 300 ?g/mL,the inhibition rate could reach 80.75%.The further research showed that it could cause the mycelium of Foc4 swelling and deformity,and inhibit its spore germination.It was found that this strain could produce three classes of polyketides Bacillibactin,Difficidin,Bacillaene,three known lipopeptides including Surfactin,Fengycin A,Fengycin B,Iturin,and an unknown lipopeptide compound,which was obtained to analyze the extracts by MALDI-TOF-MS and LC-MS.Through the double dish confrontation experiment,it was found that the volatile organic compounds by this strain BEB17 could inhibit the growth of a variety of pathogenic fungi.After treatment with the VOCs produced by strain BEB17,the mycelium of Foc4 expanded and constricted,the cell membranes were destroyed and the spore germination could be inhibited significantly.And 10 kinds of 33 compounds from the VOCs produced by BEB17 were identified through headspace solid phase microextraction and GC-MS.According to the different nature of the compounds can be divided into alkanes(6 species),alkenes(9 species),phenols(2 species),ketones(2 species),esters(5 species),alcohols halogenated hydrocarbons(2 species),heterocyclic compounds(1 species),amine compounds(2 species)and benzothiazoles(1 species).In this study,the colonization of BEB17 in the different cartivars of banana plants was observed by dilution plate coating method.And we found the strain BEB17 could be stably colonized in four banana cultivars.Among them,the highest colonization amounts reached 6.43 x 104 CFU/g after 10 days of colonization in the roots of the Nan Tian Huang,the peak values of the colonization amounts reached respectively 1.58×103 CFU/g and 1.43×103 CFU/g after 10 days and 20 days of colonization in the stem and leaf part of the Gui Jiao No.1,and the number of colonization increased first and then decreased.The comparison of cartivars showed that strain BEB17 had stronger affinity with the Nan Tian Huang and the Gui Jiao No.1.The direct root irrigation was used to evaluate the growth promoting effect of the strain on four different banana cartivars and on the control of banana fusarium wilt.The results showed that the average plant height,number of leaves and fresh weight of the banana cup seedings returned by BEB17 were significantly better than those of the control.Among them,the growth promoting effect of the Nan Tian Huang was the best,the plant height increased 48.24%,and the fresh weight increased 111.19%.After the banana cup seedlings transferred back to the strain BEB17 were inoculated with of the banana fusarium wilt,the incidence and disease index of the banana cup seedlings were significantly lower than those of the control.Among them,the strain BEB17 had the most obvious effect on the control of the Nan Tian Huang and the Gui Jiao No.1,and the relative control effect was up to 81.51%and 77.46%.