Functions Of Volatile Organic Compounds In Interaction Of Biocontrol Bacteria Bacillus And Pathogenic Fungus Fusarium Graminearum

Plant diseases are a major threat to food security all around the world.Around 70%of these diseases are caused by a diverse group of fungi.Fusarium spp.are notorious to cause many plant diseases,like Fusarium head blight(FHB)also known as the scab is a destructive disease of wheat and barley.The causal organism of FHB is Fusarium graminearum which also infects other small grain crops like maize,oats,and rice.F.graminearum causes yield loss by reducing the quality and quantity of grains in favorable conditions.Wheat spikes are most susceptible to F.graminearum infection at anthesis.Besides causing yield losses,Fusarium spp.also produces some toxic secondary metabolites including deoxynivalenol(DON)and nivalenol which is a very important factor in pathogenicity and is a potent inhibitor of protein synthesis in eukaryotic organism.Mycotoxins accumulation is harmful to human and animals.TRI genes encode the enzymes that are responsible for DON biosynthesis.Fusarium spp.can produce a number of volatile organic compounds(VOCs)which are important for long term communication and it may help Fusarium to make the host or rival organisms weak for infection and competition.Many strategies are adopted to manage Fusarium diseases,like agronomic practices,chemical control and biological control.Bacillus spp.are considered as the most efficient biocontrol agents(BCA).Many phytopathogens are being controlled by Bacillus spp.Important biological potentials of Bacillus are the production of lipopeptides,production of exoenzymes and release of volatile organic compounds(VOCs).First of all,we analyzed the adverse effects of fungus on plants by conducting In vitro and growth chamber experiments to investigate the effect of volatile organic compounds(VOCs)produced by the plant pathogenic fungi F.graminearum strain PH-1 on seed germination and growth of wheat plants.We observed a significant decrease in seed germination and plant growth under VOCs produced by F.graminearum.Volatile organic compounds were identified by SPME coupled with GC-MS.Mass spectra data of the volatile compounds were analyzed using the data in the NIST/EPA/NIH Mass Spectrum Library.Twenty-six volatile compounds were identified from F.graminearum.The identified VOCs included alcohols and acids,an ester(Benzoic acid,3,5-difluophenyl ester),and ethers and phenols.Four pure synthetic chemicals(DL-2-Aminoadipic acid,1,3-Dimethyl-5-n-decyclohexane,Phenol,2,4-bis(1,1-dimethylethyl)-,Diisooctyl phthalate)were purchased and tested for seed germination.These compounds were tested in I-plate containing sterilized seeds in one part and pure chemical in the other.One compound out of four i.e.Phenol,2,4-bis(1,1-dimethylethyl)(commercial name;2,4 di-tert-butyl phenol)was found to suppress the seed germination as well as adversely affecting the root morphological parameters.Similar results were seen when wheat plants were grown under the influence of this chemical.In order to assess the biocontrol potential of bacterial compounds,two mutants of Bacillus amyloliquefaciens FZB42 i.e.AK1S,which can only produce Fengycin and AK2,which has the ability to produce only bacillomycin D and surfactin but can't produce fengycin,were used for suppression of pathogen causing FHB in wheat by the production of vital lipopeptides.The crude extract of these mutants exhibited antifungal activity against F.graminearum.Fengycin was purified from the crude extract by performing HPLC analysis and further analyzed by MALDI-TOF-MS.Its application resulted in structural deformations in fungal hyphae as observed by Scanning Electron Microscopy.In-planta experiment revealed the ability of fengycin to suppress pathogen and highlighted-its capacity to combat the plant disease incidence,fengycin also reduced the mycotoxin biosynthesis by F.graminearumTo explore the role of fungal VOCs against Bacillus spp.three Bacillus strains i.e.(Bacillus amyloliquefaciens FZB42,Bacillus.subtilis OKB105 and SYST-2)were selected.Our results revealed that F.graminearum VOCs significantly suppressed the Bacillus colony growth,cell viability,biofilm formation,root colonization ability and motility of biocontrol bacteria.Out of 26 VOCs detected by GC-MS,2,4 di-tert-butyl phenol,Di-heptyl ether,and DL-2-aminoadipic acid significantly reduced the colony growth of Bacillus.Furthermore,the expression profiling of bacterial genes responsible for biofilm formation i.e.yuaB,epsO,and those responsible for motility i.e.fli and swrA was down-regulated.The present study revealed that the fungal VOCs have adverse effects on growth-related traits and antagonistic properties of Bacillus strains i.e.B.amyloliquefaciens strain FZB42,B.subtilis strains OKB105 and SYST-2.The 4th part of the study was done to explore the effect of Fusarium graminearum VOCs on the most important and significant biocontrol factors of Bacillus i.e.production of extracellular enzymes and lipopeptides.Our results revealed that the production of extracellular enzymes was stimulated by Fusarium graminearum VOCs.The expression of extracellular enzymes such as cellulase,protease,and amylase(Egls,ClpP and Amy E)was enhanced as compared to the reference gene.lipopeptides production in Bacillus strains was positively influenced by F.graminearum VOCs.To the best of our knowledge,the current study is the first report of positive effects of VOCs produced by pathogenic fungus on biocontrol bacterial exoenzymes.