Biocontrol And Its Mechanism Of Bacillus Amyloliquefaciens And B.Subtilis In Soybean Phytophthora Blight

Phytophthora sojae can cause the decay of soybean roots and stems,posing a huge risk to soybean cultivation.The control of soybean Phytophthora Blight is mainly achieved by rationally planting soybean resistant varieties,spraying effective fungicide and improving field management measures.However,with the improper application of the fungicide,P.sojae gradually developed resistance to the fungicide,and the control effect of some fungicides decreased significantly.Therefore,in the context of vigorously promoting green agriculture,improving agricultural production methods,and achieving negative growth in the use of chemical fertilizers and pesticides,it is of great significance to achieve the prevention and control of soybean Phytophthora Blight with biological control technologies.This paper screened the biological control bacteria,and studied their control mechanism against soybean Phytophthora Blight.Transcriptome and metabolome were combined with to reveal the mechanism of antibacterial mechanism from two aspects:gene expression and metabolites changes.Candidate genes were mined by bioinformatics analysis,and the functions of them were verified by gene knockout techniques.This study would provide a theoretical basis for the effective development of targeted biologic agents for the biological control of soybean Phytophthora Blight.The main research results are as follows:1.A strain JDF3 with good control effect against R sojae was isolated from healthy soybean soil.This strain could inhibit the growth of P.sojae,the swimming of spores and germination of the cyst.The colonies of isolates JDF3 were light yellow,round,non-embossed,and the edges were not smooth on LB medium.The strain JDF3 is rod-shaped,Gram-positive,and can form spores.It was initially identified as Bacillus by physiological and biochemical tests.After that,we amplified and sequenced with universal primers '?27F and 1492R?and gyrB gene-specific primers.Blasted in the NCBI database and isolates JDF3 was identified as Bacillus amyloliquefaciens.In addition,the Bacillus subtilis RSS-1 preserved in our laboratory also has a good inhibitory effect on P.sojae.Similar to strain JDF3,it could also inhibit the growth of P.sojae,the swimming of spores and germination of the cyst.Both B.amyloliquefaciens JDF3 and B.Subtilis RSS-1 fermentation broth can inhibit soybean Phytophthora Blight.2.B.subtilis RSS-1 and B.amyloliquefaciens JDF3 had good control effects on soybean blight,and the control effect was in the range of 65.5-70.7%.Their control mechanisms mainly include inhibition on the growth of P.sojae and improvement of plant disease resistance,including active oxygen burst,callose accumulation,increased lignin,stomatal closure and up-regulation of soybean resistance genes,were closely related to the prevention and treatment mechanism of soybean disease.For example,B.amyloliquefaciens JDF3 could cause HR responses in N.benthamiana leaves,which could induce active oxygen bursts,stomata closure,and NO production.Moreover,both strains of Bacillus,RSS-1 and JDF3 could induce different degrees of accumulation of callose in soybean as well as the ligninification of soybean.In addition,more and more evidences indicated that the synergy between the leaves and roots of plants and microorganisms in the planting process has a positive effect on improving plant disease resistance,stress resistance,and promoting plant growth and yield.And watering the fermentation broth of B.subtilis RSS-1 and B.amyloliquefaciens JDF3 in the root of soybean respectively,could promote the development of soybean roots and the fresh weight of soybean.3.We used a combination of comparative transcriptomics and metabolomics to analyze the molecular mechanism of two strains of Bacillus.The results showed that a total of 1616 differentially expressed genes?DEGs?were detected under the treatments of Bacillus,and these DEGs were divided into two groups by Venn diagram and were mainly involved in the regulation of two types,namely,"specificity regulation " and "common regulation".DEGs involved in the common regulation mainly involve the function of ribosomes,and almost all of them are down-regulated.They might inhibit the growth of P.sojae mainly by inhibiting the activity of ribosomes.The P.sojae samples treated by B.subtilis RSS-1 yielded specifically regulated DEGs,enriched in fat metabolism and energy metabolism pathways,while B.amyloliquefaciens JDF3 treated samples yielded specific regulation of DEGs,enriched in peroxisomes,riboflavin metabolism,folate synthesis and autophagy processes.Metabolome results showed that most of the metabolites from the inhibited P.sojae were significantly different from the control,and their amino acid,fatty acid and carbon metabolic pathways were inhibited or interfered,which was consistent with the transcriptome results,that is to say,the functions of synthesis protein in ribosome and tRNA transport process were likely to be inhibited,resulting in abnormal amino acid metabolism.Down-regulation of the DEGs in fat metabolism pathway might result in a decrease in the content of palmitic acid and stearic acid,which was confirmed by gas chromatography-mass spectrometry.Fatty acid is the main component of the cell membrane in fungi.When its content is decreased,it would cause partial damage to the cell membrane and cause the growth of P,sojae to be inhibited.Utilization of transcriptome and metabolome combined technology,the TOS1-like gene family,the small RNA editing gene Dicer-like and ricin-B lectin protein-like genes were explored,which was probable involved in the inhibition of the growth of P.sojae by Bacillus.At the same time,the gene family might also be related to the pathogenicity of P.sojae,which deserved attention.4.A gene family with a conserved domain DUF2401,glycosyl hydrolase?GH?and glycine-rich protein were identified from the P.sojae genome.The sequence and conserved domain of this gene family are similar to those of the TOS1 family,tentatively designated as the TOS1-like gene family.A putative protein gene PHYSODRAFT₅55423 with a glycine-rich protein and a glycosyl hydrolase conserved domain in this family was named PsGRGH.Bioinformatics predicts that this gene may be involved in the growth and pathogenic regulation of P.sojae.We knocked out the gene using the CRISPR-Cas9 method,resulting in a decrease in the growth rate and virulence of P.sojae,which confirmed the speculation,and transformants were more sensitive to the stress of Bacillus.In addition,the gene was localized on the cell membrane and had elicitor activity,which could cause hypersensitive response?HR?on Arabidopsis and tomato leaves,and improve their resistance to Sclerotinia sclerotiorum.In addition,the results were similar to the functions of protein PeaT1 and effector molecule CRN 115 previously reported,and the protein PsGRGH could also improve the drought resistance of Nicotiana benthamiana.5.We used transcriptome and metabolome techniques to screen out two candidate genes,PHYSODRAFT₄78479?ricin-B lectin protein-like gene,temporarily named PsRBLGH1?and PHYSODRAFT₄89360?Dicer-like gene,tentatively named For PsDCL₄89360?,and by gene silencing and knockout techniques,the two genes were silenced and knocked out respectively,and their transformants were obtained.Then,these transformants were used as experimental materials to study the functions of these two genes.The results showed that the gene PsRBLGH1 was involved in the growth of P.sojae and the process of infecting soybean.After silencing the gene,the growth rate of P.sojae mycelium slowed down,and its pathogenicity was greatly reduced.However,this gene had no significant effect on the mycelial morphology and sporangia production of P.sojae.The gene PsDCL₄89360 also had no significant effect on the production of sporangia,but its knockout could lead to the division and distortion of the hyphae of P.sojae.The KOTs obtained by knocking out the gene were more sensitive to B.subtilis RSS-1 and B.amyloliquefaciens JDF3 fermentation broth than WT and CK,suggesting that the gene PsDCL₄89360 may be involved in the regulation of P.sojae against biocontrol stress of Bacillus.We hypothesize that this regulatory process may be related to the editing of small RNAs,and further research is needed.6.The genetic structure of the plant pathogen population reflects its evolutionary process and potential of pathogens.Deeper understanding of the genetic knowledge of pathogens has a positive effect on the prevention and control of diseases,especially the P.sojae with rich diversity.Using ISSR-PCR technology,we analyzed the genetic structure of P.sojae in the main soybean growing areas of Anhui Province.The genetic structure of P.sojae in the main planting areas of soybean in Anhui Province was analyzed by ISSR-PCR.Thertheen pairs of ISSR primers were screened,and genome DNA of 62 strains of P.sojae were amplified by ISSR-PCR.The polymorphic bands accounted for 80.6%,which is rich in genetic diversity of P.sojae in Anhui Province.The genetic similarity coefficient between the strains ranged from 0.72 to 0.96,with an average of 0.85,and the genetic variation was higher.We used NJ cluster analysis to classify them into five clusters.Mantel's test showed?r=0.3938?that the genetie distance and genetic differentiation coefficient were not related to spatial distance.The gene flow between different geographical sources ranged from 0.623 to 2.773,with a mean value of 1.325,and gene communication was very frequent.In Anhui province,Improper farming operations such as the return of pathogen,soybean straw,and uncleaned agricultural machinery may be the main reason for frequent gene exchanges.Therefor,strengthening the quarantine of soybean crops and seeds and cutting off long-distance transmission channels are necessary measures in the prevention and control of soybean phytophthora blight in our province and preventing their spread.The genetic diversity researchs of P.sojae in Anhui Province had been guiding significance for the effective prevention and control of soybean phytophthora blight in our province.