Preliminary Study On Interaction Between Bacillus Subtilis NCD-2 And Verticillium Dahliae WX-1

Cotton verticillium wilt,caused by Verticillium dahliae,is a typical soilborne disease responsible for significant losses in cotton.Bacillus subtilis strain NCD-2 is a promising biocontrol agent against cotton verticillium wilt,and has been used as the main active ingredient of the bio-fungicide registered in China.Previous studies showed strain NCD-2 could produce lipopeptides fengycin and surfactin.However,the mechanism of lipopeptides against V.dahliae and the molecular mechanism of interaction between V.dahliae and strain NCD-2 need to be further clarified.In this study,the inhibitory abilities of strain NCD-2 wild type,the fengycin deficient mutant(MF)and surfactin deficient mutant(MS)against V.dahliae were compared.By transcriptome analysis,the differentially expressed genes were identified from V.dahliae during interaction with strain NCD-2.The results preliminary analyzed mechanism of lipopeptide extracts of strain NCD-2 inhibit V.dahliae and the response mechanism of V.dahliae during the interaction with strain NCD-2,which laid the foundation for clarifying the inhibitory mechanism of strain NCD-2 on cotton verticillium wilt.1.Inhibitory abilities of strain NCD-2,mutant MF and MS against V.dahliae were compared by double layer medium method.Results showed that clear inhibitory zone could be formed around the wild type strain and the mutant MS,but not around the mutant MF.Further research found that the lipopeptides from strain NCD-2 wild type and MS showed strong inhibitory abilities against V.dahliae,however,the lipopeptide from mutant MF almost lost the inhibitory ability.The results indicated that fengycin contributed to the antifungal activity against V.dahliae.2.Germination inhibition of V.dahliae conidia by lipopeptides extracted from the wild type strain and mutants of NCD-2 were evaluated.At 0.1 mg/mL concentration,the lipopeptide extracted from the wild type strain could inhibit the conidial germination by 46.30%,however,only 18.48%inhibitory rate was achieved by the lipopeptide from mutant MF.The inhibitory effect of lipopeptide extract from mutant MS was similar to that of the wild type strain.The results showed that fengycin from strain NCD-2 played an important role in inhibiting the spore germination of V.dahliae.3.Effects of lipopeptides from the different strains on microsclerotia formation were evaluated.Results showed that the lipopeptides from the wild type strain and mutant MS significantly suppressed microsclerotia formation.However,the lipopeptide from mutant MF almost lost the inhibitory ability on microsclerotia formation.Further RT-qPCR experiment convinced that the lipopeptides from the wild type strain and mutant MS could inhibit expression of genes related to microsclerotia formation.However,the lipopeptide from mutant MF lost the inhibition ability.It was indicated that fengycin was the main active compound of strain NCD-2 inhibited the microsclerotia formation.4.Transcriptome identified the differentially expressed genes from V.dahliae,after interacted with strain NCD-2.The differentially expressed genes were mainly enriched in the middle and later stages of the interaction.In the middle stage,there were 336 genes were up-regulated and 145 genes were down-regulated.In the later stage,there were 342 genes were up-regulated,236 genes were down-regulated.The function and pathways of differentially expressed genes were predicted using GO,KEGG databases.In the middle stage of the interaction,the differentially expressed genes were mainly enriched in cellulose catabolic,(3-glucan metabolism,and calcium transmembrane transport.In the later stage,differentially expressed genes were mainly enriched in the melanin synthesis process,pyridoxal binding,pathogenicity related genes,cellulose binding,and gluconeogenesis pathways.The domain analysis of differentially expressed genes in the middle and later stages was analyzed using Pfam database.It was found that the differentially expressed genes were mainly responsible for encoding MFS transporters,ABC transporters,short-chain dehydrogenase or reductase families,glycoside hydrolase families,and cytochromes P450 family(CYP)and hydrophobins.Results suggested that related domains played an important role in the interaction process.5.Genes associated with colonization in hosts and cell wall degradation enzyme including cellulase and pectinase were significantly inhibited during the interaction between V.dahliae and strain NCD-2.6.Genes associated with stress resistance were induced in V.dahliae during interaction with strain NCD-2.Genes associated with melanin synthesis during secondary metabolism,including laccase,polyketide synthase,VdT3HR,VDSCD and VdT4HR were induced in the later stage of the interaction.In addition,genes associated with ABC transporter,laccase β-lactamase,and the gluconeogenesis pathway-related enzymes were also induced in the presence of strain NCD-2.It was shown that during the interaction process,the candidate defense genes of V.dahliae were up-regulated under the stress of strain NCD-2.7.Genes encoding transcription factors,including C2H2 type transcription factors,Zn2Cys6 type transcription factors,and HLH type transcription factors were up-regulated in V.dahliae in the middle and later stages of interaction.Zn2Cys6 type transcription factors were up-regulated during the middle and later stages of the interaction.C2H2 type transcription factors were involved in pathogenicity-related genes and were also differentially expressed during the interaction.These results indicated that transcription factors play an important role in the interaction between V.dahliae and B.subtilis.