Screening And Mechanism Of Antagonistic Bacteria Against Root Rot Of Panax Notoginseng

Panax notoginseng(Burk.)F.H.Chen is a kind of valuable medicinal herb used commonly in China,which have many pharmacological activities such as promote blood circulation and detoxification.P.notoginseng need higher requirements for planting conditions.In addition,the increasing severe soil obstacle to continuous cropping on P.notoginseng,which leads to many diseases mainly root rot occur frequently.At present,the main prevention and control methods of P.notoginseng root rot involves spraying various chemical pesticides.Although chemical pesticides can reduce the occurrence of diseases to a certain extent,the use of a large number of pesticides leads to the development of drug resistance of pathogens.In addition,the use of a large number of pesticides causes environmental pollution,and various chemical pesticides remain in P.notoginseng.Therefore,the prevention and control of P.notoginseng root rot urgently needs more safe and effective control measures.In recent years,biological control methods have been developed rapidly,environment-friendly,safe and effective,and a lot of achievements have been made in the prevention and control of various diseases.The aim of this study was to screen the antagonistic bacteria of P.notoginseng root rot and explore its antifungal mechanism.The experimental work and results of this paper are as follows:1.Fusarium solani is the primary pathogen of P.notoginseng root rot.In order to find an ideal biological control method for this fungus,antagonistic bacteria Pn1 were screened,which had inhibitory effect on mycelial growth of 8 fungal pathogens,such as Fusarium oxysporum and Alternaria alternata.Pn1 was identified as Bacillus subtilis by morphological observation and molecular biological analysis.Enzyme activity analysis showed that Pn1 could produce protease,β-1,3-glucanase,siderophores and cellulase.The cell free filtration of Pn1 showed antifungal activity.We selected several crucial genes involved in the development and pathogenesis of F.solani,F.oxysporum and A.alternata.The expression level of Pn1 after treatment was analyzed by q RT-PCR,which indicated that Pn1 could down-regulate the expression of these genes.The experiment of disease prevention and growth promotion showed that the growth condition of P.notoginseng was good and the symptoms of root rot caused by F.solani of P.notoginseng could be reduced after Pn1 treatment.In addition,the POD activity of P.notoginseng root increased after Pn1 treatment.High performance liquid chromatography analysis showed that Pn1 treatment promoted the synthesis of saponins in P.notoginseng.2.The extracellular protein of Pn1 was extracted by precipitation method of ammonium sulfate precipitation,which showed that it had antifungal effect on the hyphal growth of F.solani and A.alternata,and also had inhibitory effect against spore germination of fungal pathogens.The lipopeptides produced by Pn1 were extracted by acid precipitation and methanol extraction,and the antifungal experiment showed that the crude lipopeptide extracts had inhibitory effect against mycelial growth of F.solani,F.oxysporum and A.alternata.The extracellular proteins produced by Pn1 strain were identified by nano ultra high performance liquid chromatography-quadrupole orbitrap mass spectrometry.The analysis showed that Pn1 could produce a variety of proteins related to antibiotic production,such as surfactin synthase,plipastatin synthase and polyketide synthase,etc.In addition,possible protein-based antitoxins were detected;The hydrolase and alkaline serine protease with antifungal activity were obtained.3.Non-targeted metabolomics was used to analyze the extracellular metabolome of Pn1.Global metabolome analysis showed that different accumulations of compounds from several metabolites,including organic acids and derivatives,lipids and lipid-like molecules,organoheterocyclic compounds,organic oxygen compounds,phenylpropanoids and polyketides,benzenoids.The plant hormone gibberellin,3-indoleacrylic acid and ethephon were detected.Betaine,hypoxanthine,nohaman and other antifungal substances were detected.KEGG enrichment analysis showed that small molecule metabolites mainly clustered in alkaloid synthesis and phenylpropanoid biosynthesis pathways.4.Transcriptomics was used to analyze the molecular mechanism of Pn1 against the fungal pathogens of P.notoginseng root rot.RNA-seq analysis showed that 1258up-regulated and 1831 down-regulated differentially expressed genes(DEGs)were detected in the Pn1 treatment group compared with the control group of P.notoginseng root rot.Enrichment analysis of KEGG pathway indicated that a large number of DEGs expressions were up-regulated in the metabolism of starch and sucrose,plant signal transduction,plant pathogen interaction and phenylpropanoid biosynthesis pathways,including beta-amylase and beta-glucosidase genes,plant hormone-related auxinresponsive protein IAA,gibberellin receptor GID1,WRKY transcription factor,pathogenesis related protein gene,and phenylalanin ammonia-lyase gene.It indicated that Pn1 had positive regulatory effects on the growth and development,carbohydrate metabolism and disease resistance of P.notoginseng.Compared with P.notoginseng inoculated with F.solani,the expression of DEGs in MAPK signaling pathway,plantpathogen interaction,JA/SA signaling pathway and flavonoid biosynthesis pathway in P.notoginseng inoculated with F.solani after Pn1 treatment were relatively upregulated,indicating that Pn1 treatment inhibited the infection of F.solani and alleviated the symptoms of P.notoginseng.Eight DEGs associated with disease resistance were verified by q RT-PCR,which was consistent with transcriptome sequencing results.In conclusion,Bacillus subtilis strain Pn1 is an antagonistic bacteria with obvious antifungal effect on fungal pathogens,which can produce a variety of antibacterial active substances,promote the growth of P.notoginseng,and improve the disease resistance of P.notoginseng.