| Cotton is an important economic crop.About 60%of the world’s cotton is produced in Asia.Cotton Verticillium wilt is one of the most serious diseases occurring in cotton.To further expand the biocontrol resources of Cotton Verticillium Wilt and provide scientific basis for its basic research and development and application,in this study,endophytic bacteria were isolated and screened from Fraxinus hupehensis for control of Cotton Verticillium wilt and the biocontrol mechanisms were investigated.The following results were obtained:1.A total of 67 endophytic bacteria were isolated from seven tissues of F.hupehensis,and one strain,YZU-SG146（G146）,which showed 89.22%growth inhibition on Verticillium dahliae,was selected for further study.The strain was identified as Bacillus amyloliquefaciens by morphological features and 16S r DNA and gyr B sequence analysis.2.Strain G146 had broad-spectrum antifungal activity,and its inhibition rates against 14 pathogenic fungi including Bipolaris maydis,Alternaria solani and Phytophthora nicotiana were 63.50%-94.34%.The inhibition rates of G146 culture filtrates on V.dahliae spores germination were 100%,52.50%and 38.33%when its concentration was 75%,50%and 25%,respectively.When the concentration of G146culture filtrates was 40%,30%,20%and 10%,the inhibition rates of V.dahliae mycelium growth were 72.20%,56.11%,54.93%and 42.67%,respectively.The inhibition rate of G146 culture filtrates on germination of V.dahliae micro sclerotia was 68.20%.3.Treatment of cotton seeds with fermentation broth or bacterial suspension of G146 resulted in significant increases in root length,shoot length,and total root fresh weight of young shoots.Fermentation broth of G146 also resulted in significant increases in root length,seedling length,and seedling fresh weight,whereas root fresh weight and SPAD values did not change significantly.In pot experiment,strain G146exhibited 84.21%control efficacy on cotton Verticillium wilt,which was significantly higher than that of the positive control（ethylicin）.4.Strain G146 could secrete siderophores,protease,amylase,cellulase and IAA,but it did not produce chitinase,had no nitrogen fixation ability and could not dissolve phosphate.Strain G146 could form biofilm,and when the OD600value of its bacterial culture was between 0.2 and 1.0,the number of biofilms showed an increasing trend with the increase of the OD value.5.After cotton leaves treatment with strain G146 suspension,the toxin produced by V.dahliae was reduced significantly.After cotton seeds treatment with G146suspension,the damage degree of V.dahliae to radicle was reduced;after cotton seedlings treatment with G146 suspension,the damage degree of V.dahliae to cotton leaves was also reduced.Meanwhile,it deepened the degree of lignification of hypocotyl cells and induced the explosion of reactive oxygen species in leaves,which made the infection degree of cotton leaves significantly reduced.6.The whole genome size of strain G146 was 3914255 bp containing 79 tandem repeats（TR）,and there were 4105 coding genes.The total length of all coding genes was 3453777 bp.A total of 2970,2383,2154,4105,3496 and 3297 genes were annotated in COG,GO,KEGG,NR,Swiss-Prot and Pfam databases respectively.It is predicted that there are 15 secondary metabolite synthesis gene clusters in the genome,which encode surfactant,bacilysin,fengycin,bacillaene,macrolactin and difficidin,among others.In addition,strain G146 may encode four unknown secondary metabolites.7.Transcriptome sequencing and data analysis of V.dahliae induced by strain G146 revealed 3633 DEGs in V.dahliae after treatment with G146 culture filtrates.Among them,genes related to cellular material metabolism,ribosome synthesis,and cellular component synthesis were highly variable.8.LC-MS analysis showed that there were 13 known active substances in the n-butanol extract of G146 culture filtrates,such as Macrolactin A,Macrolactin B,Macrolactin E,Difficidin,bacillysin and so on. |
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