Screening,Identification,Characterization And Exploration Of Antibiotic Potential Of Volatile Compounds Produced By Endophytic Bacillus Species Against Necrotrophic Phytopathogen Sclerotinia Sclerotiorum

Bacterial endophytes are evidently now that they can produce metabolites and emit volatile gaseous substances which are useful in the agricultural and non-agricultural sectors.In line with agriculture,these bacterial endophytes play a vital role in protecting plants against phytopathogens through reducing inoculum level or total inhibiting their virulence mechanisms.Most research studies have focused on investigating the mechanism of virulence effectors during the pathogen-host interaction,and have thus largely ignored the investigation of biogenic compounds that could weaken and suppress the severity of virulence repertoires of necrotrophic pathogens including Sclerotinia sclerotiorum.Therefore,this study aimed to explore the beneficial effects of volatile and non-volatile compounds of endophytic Bacillus species on the physiological functions of Sclerotinia scleroliorum,the causal agent of sclerotinia rot disease.The first part of the study was to isolate,characterize,identify and evaluate the effectiveness of metabolites produced by the bacterial isolates against growth of fungal mycelia.Among the five randomly picked bacterial colonies,three of them showed significant inhibitory activity against S.sclerotiorum in in vitro dual culture assay,and thus they were selected for identification and characterization.The analysis based on 16S rRNA and gyrA gene sequencing revealed the isolates had high similarity with B.amyloliquefaciens and B.velezensis.For the antibiotic test,both bacterial isolates were sensitive to various degrees in ampicillin,chloramphenicol,gentamicin,kanamycin,and streptomycin.The metabolites produced by bacterial endophytes were identified by Matrix-assisted laser desorption/ionization-time of flight-mass spectrometry(MALDI-TOF-MS)analysis,whereby they commonly found to produce bacillomycin L while VM10 and VM 42 were also producing bacillomycin D and L.Further analysis on agar plates revealed that the isolates could hydrolyze starch,lipids,celluloses,proteins and the bacterial strain VM11(B.velezensis)mediated a very strong hemolytic activity,suggesting that,extracellular products and bacillomycin contributed to the antagonistic activity observed in confrontation plate assay.The antagonistic activity against mycelial growth of S.sclerotiorum prompted us to investigate futher whether the isolates could produce volatile organic compounds(VOCs)with potential synergy against the pathogen.The assay performed in bicompartment Petri dishes showed that the VOCs produced by B.velezensis(VM11)had a substantial inhibitory effect on fungal mycelial growth by limiting fungal growth by 86.6%compared to the control.Solid phase micro extraction(SPME)-gas chromatography-mass spectrometry(GC-MS)analysis,revealed that the bacterial endophytes could produce a cocktail of 16 volatile compounds,where 8 of those were commercially available,thus purchased and individually tested for their antifungal ability against S.sclerorotiorum.Eight of the tested volatiles exerted adverse effects on fungal mycelia while the other four triggered the accumulation of reactive oxygen species(ROS)in the fungal cells.The damage of fungal mycelia and their organelle membranes observed through transmission electron microscopy(TEM)and scanning electron microscopy(SEM)analysis indicated that the fungal membranes could be the main target of the VOCs emitted by Bacillus endophyte VM11.Through bromophenol blue assay,the VOCs of Bacillus spp showed to reduce the levels of oxalic acid(OA)produced by S.sclerotiorum which is the key pathogenicity factor of this pathogen.The further experiment proved this phenomenon.However,the pathogenicity test on the detached leaves of host plants vividly showed that the funga1 mycelial plugs previously exposed to the VOCs of bacterial endophytes produced limited disease lesions compared to the control.The synergistic activity of the VOCs emmited by bacterial endophytes on S.sclerotiorum was active in a range of temperatures(10?-40?),suggesting that the VOCs of bacterial-endophytes can be effective against the pathogen in various environmental conditions.The findings in this report open a new possibility of controlling S.sclerotiorum using the volatile substances produced by bacterial endophytes.