The Mechanism Of Inhibition Of Bacillus Subtilis ZA1S Metabolites Against Colletotrichum Coccodes

China is the leading producer of potatoes in the world,with more than 4.81 million hectares of production space and about 180.64 million tons of potatoes produced yearly.These numbers correspond to 27.36%and 24.53%of global potato growing area and production,respectively.Colletotrichum coccodes,a disease that not only reduces potato yields but also negatively affects the commercial viability of potatoes,is one of the problems that come along with a growing potato industry.To minimize the harm that C.coccodes does to the potato industry,we examined the metabolites generated by Bacillus subtilis with bacteriostatic effects and the mechanism of metabolite inhibition.In this study,B.subtilis ZA1S was discovered on the fodder plant Polygonum pearlum in the alpine grassland of the East Qilian Mountains.According to the results of earlier studies,B.subtilis ZA1S demonstrated outstanding antagonistic power against C.coccodes,and its ethyl acetate extract likewise exhibited anti-fungal activity.Column chromatography and multiple spectrum data were used in this study to define the structure of metabolites,and a combination of transcriptomics and metabolomics was used to mine strain ZA1S functional genes implicated in disease resistance.The results were as follows:1.Study on the antimicrobial activity of strain ZA1SThe endophytic strain ZA1S exhibited antagonistic effects on all 11 pathogenic fungi,inhibiting C.coccodes and C.tabaci spore germination by 81.93%and 78.82%,respectively.Its ethyl acetate extract inhibited C.coccodes by 31.48%,resulting in a decrease in spore production from 1.1×10⁶ spores/m L to 5.8×10⁵ spores/m L,and the inhibition of spore germination of C.tabaci was as high as 83.67%.The strain ZA1S was identified as B.subtilis based on its morphological traits and the results of a molecular clustering analysis.The results showed that ZA1S can play an anti-fungal function in three areas,including mycelial growth,spore generation,and spore germination and has the potential to become an excellent biological control agent.2.Isolation and identification of metabolites of strain ZA1SA total of six compounds were isolated from the metabolites of endophytic strain ZA1S and identified as cyclo（Leu-Tyr）（Bs1）,cyclo（Tyr-Phe）（Bs2）,thymine（Bs3）,genistin（Bs4）,daidzein（Bs5）and genistein（Bs6）by multiple spectroscopic methods.The Bs1-Bs6 were found to have a significant effect on C.coccodes with minimum inhibitory concentration（MIC）of 0.8 mg·m L^-1,0.8 mg·m L^-1,2 mg·m L^-1,1 mg·m L^-1,1 mg·m L^-1 and 0.5 mg·m L^-1,respectively,and compared with the MIC value of tetramethylthiuram disulfide for 1.25 mg·m L^-1,except for thymidine,the The compounds were all more potent,further confirming the reliability of strain ZA1S developed as a biocontrol agent.This further confirms the development of strain ZA1S as the reliable of the anti-pathogenic agent.3.In vitro antioxidant activity study of the metabolites Bs1-6All six metabolites had some ability to scavenge 1,1-diphenyl-2-trinitrophenylhydrazine（DPPH）radicals,and the compounds with the most obvious scavenging advantage was genistein,with a scavenging rate of 67.19%.Except for thymidine,the other metabolites were more capable of scavenging 2,2-biazo-bis（3-ethyl-benzothiazole-6-sulfonic acid）diammonium salt（ABTS+）radicals,with scavenging rates above 99%,which were positively correlated with metabolite concentrations.the scavenging ability of the six compounds for·OH was relatively consistent,with scavenging rates ranging from 63%to 72%,showing a stoichiometry-dependent characteristic.The highest O₂^-·scavenging rate of 67.84%was achieved when the concentration of genistein was 1 mg·m L^-1,and the other five metabolites showed insignificant scavenging effect on O₂^-·.This result indicated that the ethyl acetate extract of strain ZA1S has good antioxidant activity in vitro,which provides a test basis for the development and application of additional products of strain ZA1S.4.Mechanistic studies on the inhibition of C.coccodes by metabolites of strain ZA1SThe mycelium displayed deformation,increased inclusions,halted growth,mycelial swelling,and increased branching after the six metabolites affected C.coccodes.When genistin,daidzein,and genistein were used at doses of 0.5 mg·m L^-1,0.25 mg·m L^-1,and 0.5 mg·m L^-1,respectively,and malondialdehyde（MDA）concentrations of 0.98 mmol·L^-1,0.73 mmol·L^-1,and 0.86 mmol·L^-1 were detected,respectively,the most damage to mycelial cell membranes was caused,while the after treatment with the other three metabolites,the highest value of MDA was only 0.33mmol·L^-1,showing that there was less damage to the pathogenic mycelial cell membrane.When the concentrations of thymidine,genistin,daidzein,and genistein were 0.25 mg·m L^-1,0.125 mg·m L^-1,0.125 mg·m L^-1 and 0.0625 mg·m L^-1,respectively,the synthesis of ergosterol was blocked,and as the concentration increased,ergosterol could not participate in the synthesis of cell membrane.The mycelium synthesized the lowest protein content of 298μg·m L^-1,301μg·m L^-1,334μg·m L^-1,328μg·m L^-1 and337μg·m L^-1,respectively,when cyclo（Leu-Tyr）,cyclo（Tyr-Phe）,genistin,daidzein and genistein were used at concentrations of 0.1 mg·m L^-1,0.1 mg·m L^-1,0.125 mg·m L^-1,0.125 mg·m L^-1 and 0.0625 mg·m L^-1,respectively.The extracellular cellulase（Cx）activity of mycelium reduced 3.0-fold,1.2-fold,and 1.4-fold,at genistin,daidzein,and genistein doses of 1 mg·m L^-1,1 mg·m L^-1,and 0.5 mg·m L^-1 respectively.This discovery suggests that the antifungal activity of metabolites generated by endophytic strain ZA1S may be due to disruption of mycelial structure,which causes mycelium cell leaking.5.Combined transcriptomics and metabolomics reveal key genes of strain ZA1S for suppression of C.coccodesTo identify the genes that play a regulatory function in strain ZA1S’s ability to control C.coccodes,transcriptome and metabolome studies of strain ZA1S were carried out in the current work.Transcriptome sequencing was used to screen 4168differentially expressed genes,of which 1026 showed up-regulation and 2130 showed down-regulation.1911 differentially expressed genes were enriched in biological responses,cellular components,and molecular functions,with 768（up-regulated 141,down-regulated 111）,235（up-regulated 119,down-regulated 116）and 874（up-regulated 210,down-regulated 202）,respectively,playing major roles in oxidation-reduction processes,developmental processes,membrane action,and redox enzyme activity.Seven KEGG signaling pathways,including cofactor biosynthesis and purine metabolism,were strongly enriched in differential genes,with 188 differential genes with regulatory functions,of which 95 were up-regulated and 93 were down-regulated.Metabolomics was used to screen 529 differential metabolites,of which 35showed up-regulated and 28 showed down-regulated.Up-regulated metabolites included D-mannitol,galactitol,9,12,13-trihydroxy-10,15-octadecadienoic acid,cis-aconitate and adenosine-5’-monophosphate,while down-regulated metabolites included 3-indoleacrylic acid,L-pyroglutamic acid,L-piperidinic acid,N6-（2-hydroxyethyl）adenosine,etc.,enriched in 88 KEGG pathways The representative pathways included lysine degradation,nucleotide metabolism,citric acid cycle（TCA cycle）,metabolism of glyoxylate and dicarboxylic acid and phosphotransferase system（PTS）and other related pathways.The analysis showed that 5 metabolites and 18 genes were annotated in the ethoxylate and dicarboxylate pathways,8 metabolites and 86 genes were annotated in the biosynthetic pathway of cofactors,and 6 metabolites and 11 genes were annotated in the degradation pathway of lysine in the top 25 KEGG pathways that were jointly enriched by differential genes and differential metabolites.Chlorite dismutase,aldehydedehydrogenasefamily,Pyridinenucleotide-disulphide oxidoreductase|Pyridine nucleotide-disulphide oxidoreductase,dimerisation domain and thiolase,N-terminal domain|Thiolase,C-terminal domain,respectively,were used to screen three typical differential metabolites,cis-aconitate,adenosine-5’-monophosphate,and L-piperidinic acid.The suppression of C.coccodes during growth is caused by high concentrations of cis-aconitic acid and adenosine-5’-monophosphate and low amounts of L-piperidinic acid.The expression of the genes hem Q,ald Y,ald X,lpd A1,lpd A2,aco L,and yhf S,which control three separate metabolites,was greater than that of the control,suggesting that these genes are crucial for inhibiting C.coccodes.