Transcriptome Analysis Of Fusarium Oxysporum In Response To Soybean Root Rot Biocontrol Bacteria HSY21 And Functional Verification Of FoxERG3 Gene

Soybean root rot caused by Fusarium oxysporum（F.oxysporum）is one of the important diseases in soybean production.In order to avoid the harm caused by the use of azole fungicides,bacillus has gradually become an alternative strategy for chemical control of plant diseases as biological agents.In this study,Bacillus subtilis HSY21 with good biocontrol effect against F.oxysporum was isolated.Transcriptome sequencing was used to analyze the molecular changes of F.oxysporum under the antagonistic action of strain HSY21,and the differentially expressed genes were explored to verify the function of candidate gene FoxERG3 affecting F.oxysporum infection.At the same time,the similarity between ergosterol biofunction damage and FoxERG3 gene deletion caused by liquid treatment of strain HSY21 was compared and analyzed,to explore whether the regulation of ergosterol biosynthesis of F.oxysporum was one of the antibacterial targets of strain HSY21.The main results are as follows:1.Prevention of plant harvest and prevention of fruit priceA strain named HSY21 with obvious antagonistic effect against F.oxysporum was isolated from soybean rhizosphere soil,and the inhibition rate was 81.30%.It was identified as Bacillus subtilis.The fermentation broth of strain HSY21 could inhibit the growth of mycelium and spores of F.oxysporum.Under greenhouse conditions,the control effect on soybean root rot reached 63.83%,and significantly increased soybean plant height and chlorophyll content.In the field,the control effect on soybean root rot reached 57.07%.Soybean plant height,root length,dry weight,fresh weight and other biomass indicators,pods,100-seed weight and other yield indicators were improved in varying degrees.2.Molecular changes of F.oxysporum under the antagonism of strain HSY21RNA-seq analysis of F.oxysporum after antagonistic treatment with strain HSY21（48h and 72h）showed that there were 2991 differential genes at two time points,including1561 and 1445 differentially expressed genes up-regulated and down-regulated,respectively.In GO clustering and KEGG enrichment analysis,they were mainly clustered under GO Terms such as Integral component of membrane and Transmembrane transport,and pathway such as Starch and sucros emetabolism,Nitrogen metabolism and Alanine metabolism.Most of the down-regulated genes are involved in the maintenance of mycelial structure,regulation of carbon and nitrogen metabolism,virulence transport factors and synthesis of plant cell wall degrading enzymes.Gene FOXG＿10530（FoxERG3）which may participate in ergosterol biosynthesis and decrease the infectivity of F.oxysporum was selected from these genes as the object of follow-up study.3.Functional verification of gene FoxERG3 in F.oxysporumGene FoxERG3 was 2793 bp in length,encoding 347 amino acids and containing fatty acid hydroxylase domain.The growth rate of the mutantΔFoxERG3 slowed down on PDA plate and the penetration growth ability of mycelia decreased,while the growth of the mutantΔFoxERG3 produced almost no fungal pigment and significantly reduced the number of spores in PDB.The deletion of gene FoxERG3 blocked ergosterol biosynthesis in F.oxysporum,resulting in increased membrane permeability,increased extracellular K⁺and intracellular Cyt C release,and increased sensitivity to four cell stress agents（Na Cl,D-sorbitol,Congo Red and H₂O₂）.4.Analysis of similarity between HSY21 treatment and gene FoxERG3 deletion on ergosterol-involved biological function damageThe treatment of strain HSY21 and the deletion of gene FoxERG3 also led to the decrease of ergosterol content in F.oxysporum.Compared with the wild type,both treatments showed changes in mycelia swelling,distortion and membrane permeability,and decreased infectivity to soybean.The activities of four plant cell wall degrading enzymes（cellulase,pectingalacturonase,α-amylase andβ-glucosidase）were significantly decreased.Inoculation with HSY21 could significantly improve the activities of soybean leaf defense enzymes（SOD,CAT,POD）,and mutantΔFoxERG3could also induce the improvement of soybean leaf defense enzyme activities.However,the time of defense response was delayed obviously.To sum up,Bacillus subtilis HSY21 has good biological control of soybean root rot effect,its antibacterial mechanism is a complex process of multiple targets,may be affected by destroying hyphae cell structure,carbon and nitrogen metabolism and active oxygen removal mechanism,slow down the virulence of transshipment and CWDEs secretion of infection must be reduced fusarium oxysporum infection of soybean.Gene FoxERG3 is involved in the regulation of ergosterol biosynthesis of F.oxysporum,and plays an important role in the normal functioning of F.oxysporum infection.Both the deletion of gene FoxERG3 and the treatment of strain HSY21 can damage the biological function of ergosterol involved in F.oxysporum.These results indicated that strain HSY21 was expected to replace the use of azole fungicides which also inhibit ergosterol biosynthesis.This study provided strong theoretical support for the development of biocontrol agents.