Synergistic Regulation Of Plant Systemic Resistance And Growth And Development By Bacillus Subtilis J-15

Microbial-plant interactions are of interest to many researchers,especially for the development of bio-organic fertilizers and crop disease control using the regulatory effects of inter-root beneficial microorganisms on plant induced systemic resistance(ISR)and growth and development as important directions.And revealing the mechanism of beneficial microbial-plant interactions is the necessary foundation for it.In the previous stage,the laboratory screened Bacillus subtilis J-15 from cotton inter-rhizosphere soil,which is able to antagonize various plant species such as Botrytis cinerea,Verticillium dahliae and Fusa Hum graminearum and other plant pathogens,and can colonize plant roots,with good inter-rooted probiotic properties,and is a good strain resource for crop fungal disease control,but the mechanism of interaction between Bacillus subtilis J-15 and plants is not clear,and there is a lack of theoretical guidance for its use.In this study,we firstly clarified that J-15 and its fermentation broth acid precipitation extract could enhance the systemic resistance effect and regulate the growth of Arabidopsis thaliana in the model plant,Arabidopsis thaliana.The material basis and mechanism of action for growth regulation and resistance induction were further investigated,and the main results are as follows:(1)J-15 and fermentation broth,which can rapidly activate the expression of PRs,PDF1.2,NPR1 genes in the Arabidopsis SA,JA / ET signaling pathway and FRK1 / WRKY33 genes in the MAPK signaling pathway,and can induce their reactive oxygen species(ROS)and callus(Call)deposition,suggesting that it may be able to participate in microbial-associated molecular patterns through these two pathways(MAMP)-triggered immunity(PTI),induction of ISR and regulation of plant growth.(2)Metabolomic analysis showed that J-15 secondary metabolites included a variety of substances capable of inhibiting pathogenic bacteria,regulating plant growth and development,inducing plant systemic resistance and immunity related to plants and animals,such as amino acids,alkaloids,abscisic acid,5-methoxysalicylic acid,3-indoleacetic acid,7-epijasmonic acid,acetylcholine,palmitic acid and other phytohormones and growth regulating substances.This is consistent with the experimental results that J-15 and its metabolites can induce plant systemic resistance and regulate plant growth.(3)The resistant primary active substance was isolated from the secondary metabolites of the fermentation by HPLC and other techniques,and identified as Mycosubtilin by chromatography-mass spectrometry and other techniques,using resistance to Verticillium dahliae as an indication.(4)The primary antifungal active fraction was isolated from the secondary metabolites of fermentation by HPLC and identified as Mycosubtilin by liquid mass spectrometry,nuclear magnetic resonance and infrared spectroscopy.The biochemical assay showed that it could induce ROS burst and Call deposition;the Western-blotting assay showed that it could induce MAPK(MPK3/MPK6)cascade phosphorylation up-regulation expression;the real-time fluorescence quantitative analysis showed that Mycosubtilin treatment could enhance Arabidopsis PR1 and PDF1.2 gene expression;the resistance assay Mycosubtilin can be involved in multiple PTI(ROS,Call,MAPK and resistance genes)components,MAPK and other signaling pathways to induce plant resistance.MAPK and other signaling pathways to induce ISR in plants.(5)Based on transcriptome analysis,we investigated the effects of Mycosubtilin on the regulation of plant gene expression,and the results showed that Mycosubtilin acts on Arabidopsis similarly to the plant-pathogen interaction: it induces the biosynthesis of phenylpropanoids and triggers Arabidopsis ISR by regulating metabolic pathways such as plant MAPK signaling pathway and phytohormone signaling.where SA,JA and ET signaling pathways were activated and transcription factors related to IAA and BR signaling pathways were down-regulated,suggesting that Mycosubtilin is regulating plant systemic resistance and growth and development through multiple signaling pathways.(6)Comparative metabolomic analysis was used to show that Mycosubtilin regulates plant systemic resistance and growth and development by affecting some regulatory substance synthesis and energy metabolism pathways in Arabidopsis.(7)Combined RNA-Seq and GC-MS analysis revealed that Mycosubtilin mediates the regulation of plant systemic resistance and growth and development by affecting relevant transcription factors and metabolites in the pathways of Arabidopsis thaliana phenylpropanoid biosynthesis,glycolysis and sugar metabolism,pentose phosphate pathway,tricarboxylic acid cycle and amino acid metabolism.In summary,J-15 can ferment and produce a variety of metabolites that regulate plant resistance and growth,among which the main effective resistance substance against plant pathogenic fungi is Mycosubtilin,which can mediate the improvement of plant systemic resistance and growth and development regulation by affecting the synthesis of regulators in the regulatory pathways of plant material metabolism and energy metabolism,providing an experimental basis for elucidating the mechanism of microbial-plant interactions.It also provides an important theoretical guidance for the development and use of new biological control agents.