Mechanisms And Identification Of Growth-promoting And Antibacterial Substances Produced By Bacillus Subtilis

Plant growth-promoting rhizobacteria(PGPR)are naturally occurring soil microorganisms that aggressively colonize plant roots and benefit plants by increasing plant growth or reducing disease.The proposed mechanisms for plant growth promotion by PGPR include increasing mineral,nitrogen,and phosphorus availability in the soil;synthesizing plant hormones such as indole-3-acetic acid,cytokinins and gibberellins;decreasing plant ethylene levels by producing ACC deaminase and releasing volatiles such as 2,3-butanediol.Several Bacillus spp.representing typical PGPR have been widely applied due to their ability to form heat-and desiccation-resistant spores and safe for environment and human,but the mechanisms for plant-growth-promotion and biocontrol remain unclear.This research identified genes involved in promoting plant growth through transcription microarray and mutant library,and then explored the plant-growth-promotion mechanisms by mutant construction,HPLC,real-time PCR and Elisa technology.Moreover,antibacterial activity of Bacillus volatile compounds against Xanthomonas oryzae pv.oryzae was also estimated in this research.The main research results are as follows:Bacillus subtilis OKB105 significantly increased rice growth.Microarray analysis of B.subtilis OKB 105 after an interaction period of 2 h was used to reveal the plant-growth-promotion mechanisms.A total of 176 genes representing 3.8%of the B.subtilis strain OKB 105 transcriptome showed significantly altered expression levels in response to rice seedlings.Among these differentially expressed genes,122 had known functions such as involvement in aspects of metabolism,transport,mobility,and chemotaxis.Of these,four groups of genes were particularly strongly affected by rice seedlings,such as:genes involved in metabolism or transport of carbohydrates or amino acids;genes associated with stress response transcriptional regulators;genes associated with chemotaxis,motility and sporulation;genes associated with teichuronic acid biosynthesis.The results had indicated that during the interaction process(1)Bacillus could utilize carbohydrates and amino acids released by rice as energy sources;(2)Bacillus may enhance self-adaptability in rhizosphere by inducing the expression of some transcriptional regulators;(3)plant may indirectly affect sporulation and cell wall components by providing energy.Basic properties of the promoting active substance produced by B.subtilis OKB105 were also detected.These results indicated that the promoting active substance was highly stable and the molecular mass was less than 3 kDa.After screening 700 mutants,4 mutants defective in promoting tobacco and rice growth were isolated.To identify the gene loci present in the mutants,the inserted transposon and its flanking regions were cloned by inverse PCR and sequenced.The insertions of the transposon in the 4 mutants were located in the yecA,yusV,sigH and comX gene.The functions of these genes are respectively relative to encoding putative amino acid/polyamine permease,iron(?)-siderophore transporter,RNA polymerase factor sigma and competence pheromone precursor.We found that the differentially expressed gene speA in microarray data and gene yecA selected from mutant library were both associated with polyamine.The speA gene,which encodes arginine decarboxylase,is a key gene involved in the biosynthesis of polyamine,while yecA gene is associated with polyamine transport.These results suggest that the growth-promoting effect of B.subtilis OKB 105 is related to polyamines.The site-directed mutants OKB105?yecA and OKB105?speB were constructed to confirm this speculation.The genes yecA(encoding putative amino acid/polyamine permease)and speB(encoding agmatinase)were involved in the secretion or synthesis of polyamine in B.subtilis OKB105.Disruption of either gene abolished the growth-promoting activity of the bacterium,which was restored when polyamine synthesis was complemented.HPLC analysis of culture filtrates of OKB 105 and its derivatives demonstrated that spermidine,a common polyamine,is the pivotal plant-growth-promoting compound.Moreover,spermidine in concentrations ranging from 5 to 100?M enhanced root elongation In addition,real-time PCR analysis revealed that treatment with OKB 105 and the wild-type-complemented transformant as well as synthetic spermidine induced expansin gene(Nt-EXPA1 and Nt-EXPA2)expression and inhibited the expression of the ethylene biosynthesis gene ACO1.Furthermore,Elisa analysis showed that the ethylene content in plant cells also decreased.Therefore,during plant interactions,OKB 105 may produce and secrete spermidine,which induces expansin production and lowers ethylene levels.The antibacterial activity of Bacillus volatile compounds against Xanthomonas oryzae pv.oryzae was also estimated in this research.Bacillus subtilis OKB105 and Bacillus cereus D13,which is antagonistic to Xanhomonas oryzae pv.oryzae,were isolated and screened from 50 Bacillus spp.strains.Volatile compounds emitted from OKB 105 and D13 reduced the colony diameter and cell motility of Xanthomonas oryzae pv.oryzae cultured in divided Petri plates.Scanning electron micrograph and transmission electron micrograph analysis showed concentration of the protoplasts and altered surface morphology in the majority of Xanthomonas cells exposed to volatiles.The function and transcriptional expression of virulence-associated genes in Xanthomonas oryzae pv.oryzae were both repressed by D13 volatiles.Based on the gas chromatography-mass spectrometry(GC-MS)analysis,7 volatile compounds produced by OKB 105 and 12 volatile compounds produced by D13 were identified.Among them,decyl alcohol and 3,5,5-trimethylhexanol inhibited the growth of Xanthomonas oryzae pv.oryzae.Xanthomonas oryzae pv.oryzicola(Xoc),Psudomonas syringae pv.tomato DC3000(Pst)and Ralstonia solanacearum pv.tomato(Rst)were all inhibited by 3,5,5-trimethylhexanol,indicating that 3,5,5-trimethylhexanol exert general toxicity against pathogenic bacteria.