| Microbial volatiles play an expedient role in the agricultural ecological system by enhancing plant growth and inducing systemic resistance against plant pathogens,without hazardous effects on the environment.Volatile organic compounds(VOCs)produced by various bio-control species can control phytopathogens and stimulate plant growth by suppressing the inoculum levels and inducing systemic resistance in plants against plant pathogens.Similarly,these VOCs play a significant role in promoting plant growth by regulating the synthesis or metabolism of phytohormones.However,the underlying mechanisms and pathways involved during the interaction between pathogens and microbial VOCs are not yet clear.Therefore,the aim of this study was to explore the mechanism of interaction between plant and microbial VOCs,and wilt pathogen Ralstonia solanacearum(Rs)and VOCs,and to evaluate their beneficial effects on plant health and growth by using bacillus species.In the first part of this study,six of the most effective antagonistic Bacillus spp.were used against Ralstonia solanacearum(Rs)TBBS1,the causal agent of bacterial wilt disease in tobacco.Bacillus amyloliquefaciens FZB42 and Bacillus artrophaeus LSSC22 had the strongest inhibitory effect against Rs.Thirteen VOCs produced by FZB42 and 10 by LSSC22 were identified using gas chromatography-mass spectrometry analysis.Benzaldehyde,1,2-benzisothiazol-3(2H)-one and 1,3-butadiene significantly inhibited the colony size,cell viability,and motility of pathogens and negatively influenced chemotaxis.Transmission and scanning electron microscopy revealed severe morphological and ultra-structural changes in cells of Rs.Furthermore,VOCs altered the transcriptional expression level of PhcA(a global virulence regulator),type ? secretion system(T3SS),type IV secretion system(T4SS),extracellular polysaccharides and chemotaxis-related genes,which are major contributors to pathogenicity,resulting in decreased wilt disease.The VOCs significantly up-regulated the expression of genes related to wilt resistance and pathogen defense.Over-expression of EDS1 and NPR1 suggest the involvement of SA pathway in induction of systemic resistance.In order to explore the underlying mechanism,transcriptomic changes in Rs were observed in response to VOCs by a famous biocontrol bacteria Bacillus amyloliquefaciens FZB42.Transcriptional response of Rs to FZB42-VOCs was observed using the Illumina RNA-seq system after a three days exposure to VOCs,resulting in the generation of approximately 22 million high-quality reads.These reads were then aligned to the Rs reference genome and analyzed to quantify gene expression levels.A total of 2094 genes of the Rs transcriptome showed significantly altered expression levels in response to FZB42-VOCs including 593 up-regulated and 1501 down-regulated genes.The Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)databases were used to annotate DEG functions.According to the results of sequence alignments,BP category covered most of the GO annotations(3817;49.42%)followed by MF(2056;26.62%)and CC(1850;23.95%).Furthermore,these classes were categorized into 38 functional groups.A total of 1564 DEGs were assigned to 139 KEGG pathways involving the highest number of DEGs(502,32.1%)in metabolic pathways.A significant alteration was observed in directly or indirectly virulence related characters including bacterial invasions of epithelial cells,flagellar assembly,plant-pathogen interactions,bacterial chemotaxis and bacterial secretion systems.To confirm RNA-Seq results,transcriptional expression of 10 genes was observed using RT-qPCR.In vitro and growth chamber experiments were conducted to investigate the effect of volatile organic compounds(VOCs)produced by the plant growth promoting rhizobacterium(PGPR)Bacillus subtilis strain SYST2 on hormone regulation and growth promotion in tomato plants.We observed a significant increase in plant biomass under both experimental conditions;we observed an increase in photosynthesis and in the endogenous contents of gibberellin,auxin,and cytokinin,while a decrease in ethylene levels was noted.VOCs emitted by SYST2 were identified through gas chromatography-mass spectrometry(GC-MS)analysis.Of 11 VOCs tested in glass jars containing plants in test tubes,only two,albuterol and 1,3-propanediole,were found to promote plant growth.Furthermore,tomato plants showed differential expression of genes involved in auxin(SIIAA1,SIIAA3),gibberellin(GA20ox-1),cytokinin(SlCKX1),expansin(Exp2,Exp9,Exp 18),and ethylene(ACO1)biosynthesis or metabolism in roots and leaves in response to B.subtilis SYST2 VOCs.Our findings suggest that SYST2-derived VOCs promote plant growth by triggering growth hormone activity,and provide new insights into the mechanism of plant growth promotion by bacterial VOCs.To explore the effect of VOCs by Ralstonia solanacearum TBBS1(Rs)on tobacco plant growth and on plant growth promoting efficiency of VOCs produced by Bacillus subtilis SYST2,experiments were conducted both in vitro and in planta.VOCs produced by SYST2 significantly enhanced plant growth and induced systemic resistance(ISR)against wilt pathogen Rs in all experiments.The SYST2-VOCs significantly increased PPO and PAL activity and over-expressed the genes relating to expansin,wilt resistance,and defense-related proteins while repressed the genes relating to ethylene production.More interestingly,VOCs produced by pathogen,Rs had no significant effect on plant growth;however,Rs-VOCs decreased the growth promoting potential of SYST2-VOCs when plants were exposed to VOCs produced by SYST2 and Rs together.Co-culture of SYST2 and Rs revealed that they inhibited the growth of each other;however,inhibition of Rs by SYST2-VOCs appeared to be greater than SYST2 by Rs-VOCs.Our findings provide new insights regarding the interaction among SYST2-VOCs,Rs-VOCs and plant,resulting in growth promotion and induced systemic resistance against the bacterial wilt pathogen Rs.This is the first report on effect of VOCs produced by pathogenic microorganism on plant growth and on plant growth-promoting and systemic resistance-inducing potential of PGPR strain SYST2.Overall,our findings provide new insights regarding the potential of antibacterial VOCs as a biocontrol tool against bacterial wilt diseases. |
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