| Cotton is an important economic crop.Verticillium wilt is the most devastating disease of cotton in China and a challenge for producers to find effective means of control.At present,we mainly rely on chemical fungicides to control the disease,which not only has little control effect,but also has a huge potential threat to the ecological environment and social stability due to its massive abuse.For soil-borne fungal diseases of crops,the most effective strategy is to use resistant germplasm resources,while most dicotyledonous crops lack effective resistance genes to soil-borne diseases and the few disease-tolerant varieties available in production face the bottleneck of rapid loss of resistance.Therefore,we urgently need to carry out application research around environment-friendly biological agents.This research takes the hypovirulent Gibellulopsis nigrescens CEF08111 as the research object,predicts the main metabolites through whole-genome sequencing,screens the active substances through the method of active substance tracking,and then combines transcriptome analysis to reveal the induced resistance mechanism of CEF08111 on cotton Verticillium wilt,laying a foundation for the control of cotton Verticillium wilt.The research results mainly include the following aspects:The genome size of G.nigrescens is 33.96 Mb,the GC content is 57.13%,the predicted transcriptional genes are 9785,the encoded proteins are 9785,of which 2145 proteins have transmembrane helical structure,and the predicted secretory proteins are 995;The prediction analysis of secondary metabolite synthesis gene clusters showed that 47 secondary metabolite synthesis gene clusters were obtained from the genome of G.nigrescens CEF08111.Among them,24 gene clusters encoding non-ribosomal peptide synthase and saccharide accounted for 51% of the total gene clusters,and 3 gene clusters were 100% similar to the known gene clusters,choline,biotin and microperfuranone gene clusters,respectively.The active substances of CEF08111 were screened according to the active substance tracking method.Through sterilization or protease K treatment of CEF08111 culture solution,it is speculated that the active substances of CEF08111 belong to non-protein substances;Through the induced disease resistance test of proteins,lipids,polysaccharides and glycopeptides in CEF08111 culture medium,it is speculated that the active substances of CEF08111 belong to saccharide substances;Through the GC-MS analysis of saccharide substances with different molecular sizes,10 different saccharide metabolites were identified.Combined with the induced disease resistance test,it was speculated that melezitose was the main active substance of CEF08111.The effects of the filtrate of CEF08111 strain on the growth and development of Verticillium dahliae were analyzed.After co-culture of Vd076 and CEF08111,the growth and development of 30 single spore strains were detected.Compared with Vd076,the growth rate,sporulation,conidia,microsclyptosis and pathogenicity were significantly reduced.The transcriptome of the treated strain Vd076 was analyzed and 728 differentially expressed genes were found,of which 443 were up-regulated and 285 were down-regulated.According to gene function annotation,28 genes were found to be related to growth,sporulation,melanin synthesis and metabolism,and disease,and 15 of them were downregulated.In order to clarify the molecular mechanism of the induced resistance of G.nigrescens to cotton,the RNA-seq data at three time points,3 hours,12 hours and 48 hours after inoculation of cotton with G.nigrescens culture solution dipped in the root were analyzed.RNA-seq analysis showed that there were 249,3559 and 33 DEGs at 3 hours,12 hours and48 hours after inoculation with CEF08111.KEGG enrichment analysis showed that 3 hours after CEF08111 inoculation,the DEGs were mainly concentrated in glucose metabolism and phenylalanine biosynthesis pathways,such as glycolysis/gluconeogenesis,phenylalanine biosynthesis,keratin,cork and wax biosynthesis.At 12 hours after CEF08111 inoculation,the DEGs were mainly concentrated in the plant-pathogen interaction,MAPK signal pathway and plant hormone signal transduction pathway.GO enrichment analysis showed that these DEGs were mainly concentrated in response to external stimuli,systemic acquired resistance,kinase activity,phosphotransferase activity,xylan and xylan transferase activity,xylan metabolism process,cell wall polysaccharide metabolism process and hemicellulose metabolism process.In addition,we also found that the DEGs of CDPK,FLS2,RPM1,MYC2,et al,which regulate the key nodes of defenserelated pathways in cotton,may be related to the resistance of cotton to V.dahliae.Through the weighted gene co-expression network(WGCNA)analysis,7 genes related to lignin synthesis were found to be differentially expressed.The physiological mechanism of defense response of cotton induced by G.nigrescens was elucidated.After inoculation with CEF08111,cotton dipped root could increase the content of reactive oxygen,alicylic acid and jasmonic acid in leaves,activate the salicylic acid and jasmonic acid pathways,and the expression of genes related to the metabolism of chitin,glucan and phenylpropanes.For example,the expression of genes PR10,NPR1,MYC2,AOS,COI1,JAR1,PR1,4CL,PAL and β-1,3-glucanase was significantly upregulated.To explore the effect of different application methods of G.nigrescens CEF08111 on the control of cotton Verticillium wilt.CEF08111 was used to control Verticillium wilt in V.dahliae nursery by seed immersion,root irrigation,spray and coating.The results showed that the four methods had certain control effects on Verticillium wilt,and the effect of soaking was the best,which was 40.0%-60.0%.In conclusion,this study analyzed the "cross protection" mechanism of G.nigrescens CEF08111 on cotton Verticillium wilt,and explored the application technology of CEF08111 to control cotton Verticillium wilt.The results laid a foundation for further studying the biological characteristics and mechanism of G.nigrescens,and provided a new approach and theoretical basis for the control of cotton Verticillium wilt. |
PDF Full Text Request