Research About The Antifungal Mechanism Of Bacillomycind From Bacillus Amyloliquefaciens FZB42 Fusarium Graminiarum

Plant-growth promoting bacteria（PGPR）,which live within or in the vicinity of plants roots,can suppress soil-borne plant pathogens and promote plant growth.These beneficial biological activities have led to development of many PGPR strains to commercial biocontrol agents able to control plant diseases.Among them,Bacillus species are considered as the best candidates for developing efficient biocontrol agents because of their production of a wide range of bioactive compounds,and their ability to form highly adversity-resistant endosporesThe filamentous fungus Fusarium graminearum can cause Fusarium head blight（FHB）on wheat and barley,stalk and ear rot diseases on maize,and seedling blight on maize and wheat.In addition to severe yield losses and grain quality reduction,F.graminearum can produce several mycotoxins,such as deoxynivalenol（DON）and zearalenone,in infested grains.Despite the high economic and health impacts of FHB,no highly resistant varieties of wheat and barley are available.In fact,FHB management relies heavily upon synthetic antifungal agents.However,the continuous use of these synthetic antifungal agents has resulted in the emergence of resistant F.graminearum,and pose a potential risk to the environment and human health.Therefore,it is essential to develop alternative methods and agents that show low toxicity and are environmentally friendly for the efficient control of FHB.Among them,biocontrol agents that are friendly to the environment and ecosystems have attracted increasing attention worldwide.In the present study,the commercially available strain Bacillus amyloliquefaciens FZB42 shows strong antifungal activity against F.graminearum.In this study,we find out the specific secondary metabolites with antimicrobial and nematocidal activities,then we use a series of biological methods to clarify the mechanism underlying its antifungal activity.Antagonism assays showed that both FZB42 and its crude extract of secondary metabolites could suppress the growth of F.graminearum.To identify the fungicidal agents acting against F.graminearum,the corresponding mutants of FZB42 were used.In FZB42,The Sfp protein,4’-phosphopantetheine transferase,acts as the peptidyl carrier protein,and is essential for the production of three lipopeptides and three polyketides.When the sfp deficient mutant CH03 was used in the assays,the strain and its secondary metabolites showed no antifungal activity.When the bacillomycin D and fengycin double deficient mutant AK3 was tested,the antifungal activity was abolished.However,the mutants AK1（surfactin and fengycin producer）and AK2（surfactin and bacillomycin D producer）,which can produce two lipopeptides,had similar antifungal activities to FZB42.We then tested two double mutants,CH02 and AK1S.CH02 only produces one lipopeptide,bacillomycin D,and AK1S only synthesizes one lipopeptide,fengycin.Both mutants could suppress the growth of F.graminearum.Thus,the results indicated that both bacillomycin D and fengycin acted as the fungicidal factors,and were responsible for in vitro suppression of F.graminearum growth.Fengycin,which is a lipodecapeptide with a β-hydroxy fatty acid in its side chain and different from the structure of bacillomycin D,has attracted more attention.So,in this study,we focus on the mechanism of bacillomycin D,which was earlier shown as being the main antifungal agent produced by FZB42.Bacillomycin D belongs to the Iturin lipopeptides family,which have strong antifungal and hemolytic activities.This compound is composed with heptapeptides and β-amino fatty acids,and its ring structure is formed through the condensation between the carboxyl of the 7th-L-Thr and the P-amino fatty acid.In FZB42,the biosynthesis of bacillomycin D is attributed to the bmy gene cluster.Until now,the mechanism of bacillomycin D’s activity was unknown.Clarification of the mechanisms of bacillomycin D’s antifungal activity in biocontrol Bacillus strains is vitally important for the efficient application of such compounds in agriculture.The preparation method of bacillomycin D was constructed for the following research on the antifungal merchanism of bacillomycin D against F.graminearum.Bacillomycin D was separated a from the fermentation broth of B.amyloliquefaciens CH02 by acid precipitation,then we used silica gel column to conduct a preliminary purification of bacillomycin D,and the bacillomycin D was separated when the ratios of methanol and methylene chloride was 3:1.Finally,we used preparative high performance liquid chromatography（HPLC）to purify bacillomycin D,HPLC analysis showed five peaks between 21 min to 30 min.The extracted substances from these five peaks all sho-wed inhibitory activity against F.graminearum.All of these molecules containing the same ion had a 14 Da difference in molecular weights,suggesting the presence of varied lengths of fatty acid chains within bacillomycin D The purity of bacillomycin D collected from peak I was determined as being 96.6%,was used for further study.Purified bacillomycin D showed strong fungicidal activity against F.graminearum,and its 50%effective concentration（EC50）was determined as approximately 30 μg/mL.Analyses using scanning and transmission electron microscopy revealed that bacillomycin D caused morphological changes in the plasma membrane and cell wall of F.graminearum hyphae and conidia.Fluorescence microscopy combined with different dyes showed that bacillomycin D induced the accumulation of reactive oxygen species（ROS）and caused cell death in F.graminearum hyphae and conidia.F.graminearum secondary metabolism also responded to bacillomycin D challenge by inducing greater production of deoxynivalenol.Biological control experiments demonstrated that bacillomycin D could suppress disease symptoms in different plant tissues caused by F.graminearum.Bacillomycin D downregulated the expressions of genes FGSG₀2881,FGSG₀6554,FGSG₀6733,FGSG₀2974 and FGSG₁2369)involved in scavenging ROS,but upregulated the expressions of genes（TRI5,TRI6,TRI10,and TRI12）involved in synthesis of deoxynivalenol.In this study,both the formation and germination of conidial spores were inhibited strongly by bacillomycin D.When the conidial spores of F.graminearum were used as the resource to infect wheat germ and wheat heads,the damage to the conidia and the inhibition of conidial germination caused by bacillomycin D resulted in markedly reduced disease symptoms in these two tissues.In addition,because of the negative effect of this compound on the hyphae,bacillomycin D could also inhibit the infection by F.graminearum hyphae on corn silks.Bacillomycin D increased the phosphorylation of two mitogen-activated protein kinases,Mpkl and Hog1.Taken together,these findings highlight the potential of B.amyloliquefaciens FZB42 as a biocontrol agent against F.graminearum,and the utility of bacillomycin D as antifungal compound.