Antifungal Activity And The Mechanism Effect Of Bacillus Amyloliquefaciens XJ5 On Alternaria Solani

In order to obtain the dominant biocontrol strains which can effectively inhibit Alternaria solani,the antifungal spectrum,the types of antifungal substances and the antifungal mechanism of Bacillus amyloliquefaciens XJ5 were studied in this paper.The main results are as follows:1.The antifugnal protein of B.amyloliquefaciens XJ5 was crudely extracted by ammonium sulfate salting out,and its antifugnal substance was separated and purified by high performance liquid chromatography.Its inhibitory zone against A.solani can reach 28 mm.SDS-PAGE electrophoresis showed that the molecular weight of the antifugnal protein was about 70 KDa.2.The results of antifugnal activity test of volatile compounds of XJ5 strain showed that the inhibition rate of volatile compounds of XJ5 strain against 9 kinds of plant pathogenic fungi was47.9%-84.8%.SPME-GC-MS test showed that the main volatile substances secreted by strain XJ5 were dodecaldehyde,and the main volatile substances with antifugnal activity were 2-nonyl alcohol,2-ethylhexanol and 2-undecanol.Among them,the EC50 of 2-nonyl alcohol against apple brown rot was 3.43 μg/ml.Fumigation in vitro could effectively inhibit the mycelium growth and plaque expansion of apple brown rot.3.In order to further analyze the molecular basis of the antifugnal effect of B.amyloliquefaciens XJ5,the genome of the XJ5 strain was determined by the third-generation sequencing technology.The results showed that the genome of the XJ5 strain consisted of 1 circular chromosome and 2circular plasmids,with sizes of 4,160,003 bp,215,881 bp and 63,621 bp,respectively.It encoding a total of 4586 genes.The KEGG annotation results show that the XJ5 strain can secrete various protein substances such as glucanase,chitinase,xylanase,lipase,phosphatase,and Tas A;analysis of the secondary metabolic gene cluster shows that the XJ5 strain can produce Surfactin,Macrolactin,Bacillaene,Fengycin,Difficidin,Bacillibactin,Bacilysin and other lipopeptide antibiotics,polyketides and polypeptide compounds and other antifugnal substances.At the same time,XJ5 strain also has the ability to promote plant growth and induce plants genes related to resistance and biofilm and chemotactic behavior.4.In order to analyze the molecular basis of XJ5 strain inhibiting A.solani,RNA-seq technology was used to analyze the changes in transcriptional levels of A.solani treated with XJ5 antifugnal protein for 6h,12 h,24h,and 48 h.The measurement results showed that 15 samples produced an average of 6.5 Gb sequence,and the comparison rate of Clean Reads of each sample to the reference genome was between 82.27% and 86.79%.A total of 250 genes were differentially expressed in 4 periods.GO enrichment analysis showed that: 117 DEGs can be enriched to 67 GO entries,mainly including oxidoreductase activity,carbohydrate binding,lipase activity,cell wall structure,Chitin metabolism process,siderophore biosynthesis process,etc.KEGG enrichment analysis shows that 37 differential genes can be enriched into 51 KEGG pathways,mainly amino sugar and nucleotide sugar metabolism,starch and sucrose metabolism,glycosphingolipid biosynthesis,steroid biosynthesis,glutathione metabolism The related metabolic pathways that maintain energy production and participate in the growth and development of organisms,among which the chitin synthase gene CC77DRAFT₉82264 is significantly down-regulated,the β-hexosaminidase encoding gene CC77DRAFT₁032488 is down-regulated,the oxidative phosphorylation pathway gene CC77DRAFT₁91163 is up-regulated,and glutathione The peptide sulfur transferase encoding gene CC77DRAFT₆87848 responds to stress and so on.The q RT-PCR expression changes of 10 differential genes were consistent with the results of transcriptome sequencing analysis.Studies have shown that B.amyloliquefaciens XJ5 antifugnal protein can inhibit the growth of the hyphae and the production of spores of A.solani by inhibiting the synthesis of chitin in the cell wall and destroying the phospholipid bilayer structure of the cell membrane.The expression of many types of genes to reduce or alleviate the stress of antifugnal substances.