Succession Of Microbial Community In Tomato Rhizosphere During Continuous Cropping And Control Mechanism Of Tomato Bacterial Wilt By Combined Application Of Chemical And Biocontrol Agents

Tomato continuous cropping is a common farming practice in facility agriculture.The incidences of tomato bacterial wilt are significantly increased,even no harvest under continuous cropping conditions.There were lots of studies about microbial community of tomato rhizosphere soil in continuous cropping conditions.In general,tomato continuous cropping decreased soil microbial diversity and enriched pathogens,which resulted in breakout of tomato bacterial wilt disease.However,it was not clear that the succession pattern of tomato rhizosphere microbial community and the effects of enriched microbes on the infection of Ralstonia solanacearum.Here,we combined culture-dependent and high throughput sequencing methods to reveal the succession pattern of rhizosphere bacterial community in different soils during tomato continuous cropping,the composition of gradually enriched bacteria and their effects on the infection of R.solanacearum and determine the effects of tomato root residues and exudates on the bacterial community in soil.At present,there are no effective methods to control tomato bacterial wilt disease caused by R.solanacearum.Because of their environment friendly characteristic,biocontrol has been paid more and more attentions in control of plant diseases.However,biocontrol is easy to be affected by environmental factors that may result in a poor colonization in rhizosphere.The environmental friendly bactericide potassium phosphite can inhibit the growth of plant pathogens and induce the defensed responses of plants.Here,we combined potassium phosphite and biocontrol agent(Bacillus amyloliquefaciens F8)to manage tomato bacterial wilt and deciphered their control mechanism.The results are listed as follows:(1)Three different soils were used to plant tomatoes in greenhouse experiment for three cycles.Although the structures of bacterial community in tomato rhizosphere were different in the three tested soils during tomato continuous cropping,the pattern of succession in bacterial community of tomato rhizosphere was similar.Moreover,the differences in rhizosphere bacterial community before planting were greater than that after planting suggesting that tomato plant could recruit similar bacterial community from different soils.However,the pattern of succession in bacterial community of tomato rhizosphere in fields was not obvious,but the disease index of bacterial wilt was as high as 42.7%.In addition to pathogen R.solanacearum,certain beneficial bacteria,such as,Rhizobium,Massilia,Pseudoxanthomonas,Flavobacterium,Leifsonia,Sphingobium,Arthrobacter and Burkholderia were significantly enriched in the tomato rhizosphere.We isolated 82 strains from rhizosphere soil and determined their classification based on the sequences of 16S rRNA.The 16S rRNA sequences of isolates Bacillus niacini,Flavobacterium daejeonense and Beijerinckia fluminensis were clustered to the sequences of OTU3,OTU44 and OTU182 at the 97%similarity level.These OTUs were significantly and gradually enriched in the rhizosphere soil in continuous cropping.In addition,these isolates can control bacterial wilt disease or stimulate the growth of tomato plant.(2)Tomato root residues and root exudates could significantly affect soil bacterial community and diversity.At phylum level,Proteobacteria and Bacteroidetes were significantly enriched approximately 1.92-fold and 2.62-fold,but Actinobacteria was significantly depleted in the soil amended with tomato root residues.At genus level,the gradually enriched Massilia,Favobacterium and Sphingobium in rhizosphere soils in continuous cropping were positively affected by tomato root residues.Moreover,Proteobacteria was significantly enriched approximately 1.13-fold,but Actinobacteria and Bacteroidetes were significantly depleted in the soil amended with tomato root exudates.R.solanacearum,Hydrogenophaga,Rhizobium and Pseudoxanthomonas that were gradually enriched in rhizosphere soil in continuous cropping,were positively affected by both root residues and root exudates.We determined the preference of the enriched bacteria in rhizosphere.The results showed that the number of preferred carbon sources was most in B.fluminensis GR2.Sucrose and galactose strongly promoted the growth of B.fluminensis GR2.The number of preferred carbon sources was 11 in B.niacin S16.Galactose and glucose strongly promoted the growth of B.niacin S16.The number of preferred carbon sources was 9 in F.daejeonense HA5.Sucrose and galactose strongly promoted the growth of F.daejeonense HA5.The number of preferred carbon sources was 11 in R.solanacearum.Aspartic acid and glutamic acid strongly promoted the growth of R.solanacearum.(3)The infection of R.solanacearum significantly changed the bacterial and fungal community composition and decreased the bacterial and fungal diversity in the rhizosphere soil of tomato plant.In bacterial community,at the phylum level,Proteobacteria and Bacteroidetes were enriched 1.43-fold and 2.25-fold in the rhizosphere soil of diseased plants than that of healthy plants.However,the relative abundances of Firmicutes and Actinobacteria were increased 8.29-fold and 3.19-fold in the rhizosphere soil of healthy plants than that of diseased plants.At the genus level,Cellvibrio,Chryseobacterium,Duganella,Dyadobacter,Flavobacterium,Pseudomonas,Ralstonia and Sphingobacterium were the indicator taxa in the diseased rhizosphere soils.Specifically,Ralstonia was greatly enriched 23.71-fold in the diseased rhizosphere soils.In fungal community,at the order level,the relative abundances of Hypocreales and Sordariomycetidae were increased approximately 2.91-fold and 8.79-fold in the diseased rhizosphere soils than in healthy rhizosphere soils.The potential pathogens Fusarium,Gibellulopsis,Nectria and Plectosphaerella were the indicator taxa in the diseased rhizosphere soils.Specifically,Fusarium solani was greatly enriched in the diseased rhizosphere soils.R.solanacearum infection significantly decreased the network complex in bacterial and fungal community.There were 7585 and 772 links in the bacterial and fungal networks of the healthy rhizosphere soils,respectively.However,there were 3079 and 554 links in the bacterial and fungal networks of the diseased rhizosphere soil,respectively.In addition,R.solanacearum infection significantly decreased the relative abundance of Bacillus and copy numbers of antagonism genes(srf,itu and fen)related to the production of lipopeptide.The contents of phenolic acids in the rhizosphere soil of diseased plants were significantly increased,and the increased phenolic acids could significantly enrich the abundance of Fusarium solani in soil.(4)After application of potassium phosphite in soil,the composition of soil bacterial community was significantly changed,and the copy numbers of filC(R.solanacearum)and 16S rRNA gene(total bacteria)were significantly decreased.Actinobacteria was significantly enriched approximately 1.68-fold,but Proteobacteria was significantly depleted approximately 3.83-fold in the soil amended with potassium phosphite at 7 days.Δt genus level,the relative abundances of and were significantly increased,but the relative abundances of Sphingobium,Chitinophaga and Ensifer was significantly decreased at 7 days.In addition,the abundances of antagonistic bacteria Streptomyces coelicoflavus and Paenibacillusfavisporus and antagonism genes(srf,itu and fen)related to the production of lipopeptide were significantly increased in the soil amended with potassium phosphite.Moreover,the abundances of antagonistic bacteria S.coelicoflavus and P.favisporus as well as antagonism gene(srf)were significantly increased,but the copy number of fliC(R.solanacearum)was significantly decreased in the soil amended with the potassium phosphite-modulated microbiome.(5)Potassium phosphite did not inhibit the growth of Bacillus amyloliquefaciens F8,but greatly inhibited the growth of R.solanacearum in vitro at the concentrations of 0.05%-0.15%.0.3%potassium phosphite slightly inhibited the growth of B.amyloliquefaciens F8.Moreover,the growth of other three gram positive bacteria(Bacillus amyloliquefaciens,Bacillus velezensis and Staphylococcus aureus)was not affected by 0.05%potassium phosphite,but the growth of two gram negative bacteria(Pseudomonas fluorescens and Sphingomonas melonis)was significantly inhibited by 0.05%potassium phosphite.Thus,gram negative bacteria were mainly inhibited by potassium phosphite.Moreover,the result of RT-PCR showed that the relative expression of genes(dfn,srf,fen and itu)related to the production of lipopeptide and polyketide antagonistic substances were significantly upregulated by 0.05%potassium phosphite in vitro.The result of transcriptome showed that the relative expression of gene degU of two-component regulatory system of strain F8 was significantly upregulated by potassium phosphite.It is known that degU was related to regulate the genes for production of antagonistic substances(difficidin,surfactin,fengcin and iturin).Moreover,the expression levels of yceC,D,E,F,G(resistant to oxidation or reduction environment)from cell membrane resistance genes of strain F8 were significantly upregulated by potassium phosphite.Greenhouse experiment and outdoor experiment were performed to determine whether combined potassium phosphite and strain F8 could control tomato bacterial wilt disease.Application of strain F8 could not control tomato bacterial wilt disease in greenhouse experiment and outdoor experiment.Combined potassium phosphite and strain F8 could control tomato bacterial wilt disease in both greenhouse(disease index of combined treatment 34.2%,control 70.3%)and outdoor experiment(disease index of combined treatment 53.2%,control 90.8).Potassium phosphite could significantly change the composition of bacterial community,but could not change the diversity of bacterial community in the rhizosphere soil.The copy numbers of srf,fen and dfn in rhizosphere soils were increased 4.1-fold,10.43-fold and 5.24-fold in the combined treatment than strain F8 treatment.In addition,the relative abundance of Ralstonia was significantly decreased in the combined treatment than control.Moreover,Flavobacterium was significantly enriched in the rhizosphere soil amended with potassium phosphite.The isolate Flavobacterium daejeonense could significantly inhibit the growth of R.solanacearum in vitro.When applied salicylic acid and potassium phosphite to the tomato leaves,Flavobacterium was enriched in the rhizosphere soils of both treatments suggesting that the enrichment of Flavobacterium was related to the defense response of salicylic acid signal induced by potassium phosphite.Overall,the pattern of succession in bacterial community of tomato rhizosphere was similar in different initial soils.In addition to R.solanacearum,beneficial bacteria B.niacin,F.daejeonense and B.fluminensis were enriched in the rhizosphere and could protected host from R.solanacearum infection.Aspartic acid and glutamic acid strongly promoted the growth of R.solanacearum,that may be the main reason for the enrichment of R.solanacearum in continuous cropping system.R.solanacearum infection decreased the microbial diversity and complex of microbial networks,but increased the relative abundances of fungal pathogens,such as Fusarium,Gibellulopsis,Nectria and Plectosphaerella.Potassium phosphite could not inhibit gram positive bacteria,but significantly inhibit gram negative bacteria.Moreover,potassium phosphite could enhance the antagonistic ability of Bacillus amyloliquefaciens F8 and induce host to recruit beneficial F.daejeonense to protect host from R.solanacearum infection.