| Fusarium crown and root rot of tomato is one of the most serious soilborne diseases caused by Fusarium oxysporum f.sp.radices-lycopersici,which threatens the development of Chinese tomato industry and has a significant negative effect on the yield and quality of tomato.Chinese tomato industry has developed rapidly,the output ranks the first in the world,and the position in the world tomato trade is more and more important.The rhizosphere microbial community plays an important role in coordinating plant resistance to pathogen invasion.However,the relationship between the rhizosphere microbial community and the occurrence of Fusarium crown and root rot of tomato has not been analyzed,and the microbial resources for controlling Fusarium crown and root rot of tomato are short.Therefore,the rhizosphere soil of healthy and diseased tomato was collected from the area of perennial Fusarium crown and root rot of tomato as the research object,and the characteristics of rhizosphere microbial community of healthy and diseased tomato were analyzed by 16 S r RNA and ITS amplification technology.PICRUSt and FUNGuild were used to predict the functions of rhizosphere fungal and bacterial communities of tomato,and the potential relationships between the occurrence of Fusarium crown and root rot of tomato and the community structure and composition of tomato rhizosphere fungal and bacterial communities were identified by comparing the characteristics and diversity of the rhizosphere microbial communities of healthy and diseased tomatoes.Through untargeted metabolomics,the characteristics of rhizosphere soil metabolic profiles of healthy and diseased tomato were further clarified,functional strains of Fusarium crown and root rot of tomato prevention and control were screened,the functional flora was established and evaluated,and soil metabolites with potential regulation of functional flora and regulatory network on functional flora were analyzed.The effects of functional bacteria and regulatory factors on the incidence of Fusarium crown and root rot of tomato and tomato growth were analyzed.On this basis,the mechanism of promoting tomato growth by functional flora was explored.The main results are as follows:(1)By analyzing the characteristics of rhizosphere microflora of healthy and diseased tomato soil,it was found that the contents of total potassium and available phosphorus in healthy tomato rhizosphere soil were higher than those in diseased tomato rhizosphere soil,while the contents of total nitrogen,total phosphorus,organic matter and available potassium in healthy tomato rhizosphere soil were lower.The results of high-throughput sequencing showed that there were significant differences in rhizosphere bacterial and fungal community structure between healthy and diseased tomato.Meanwhile,compared with the healthy rhizosphere bacterial and fungal communities,the Simpson microbial diversity index of the diseased tomato rhizosphere bacterial community was significantly increased,while the Shannon microbial diversity index of the diseased tomato rhizosphere fungal community was significantly decreased.By the difference analysis,we discovered in tomato rhizosphere community enrichment of the Fusarium,Plectosphaerella,unclassified＿k＿Fungi and Aspergillus.According to the functional classification analysis of fungal in tomato rhizosphere fungal community,the relative abundance of plant pathogens in diseased tomato rhizosphere fungal community was significantly higher than that in healthy tomato rhizosphere fungal community.There were significant differences in Fusarium community structure between health and disease tomato rhizosphere.And the relative abundance of unclassified＿g＿Fusarium OTU68 was the highest in tomato rhizosphere Fusarium communities.Through phylogenetic tree analysis,it was found that it was closely related to the pathogen of Fusarium crown and root rot of tomato.Therefore,it was speculated that the imbalance of Fusarium community led to the occurrence of Fusarium crown and root rot of tomato.In tomato bacterial community,the dominant flora of healthy tomato rhizosphere bacterial community was norank＿f＿norank＿o＿norank＿c＿Subgroup＿6,Gaiella,norank＿f＿norank＿o＿norank＿c＿KD4-96,but their variation range was smaller than that of Fusarium.It was found that the relative abundance of defense mechanism in the rhizosphere bacterial community of healthy tomato increased significantly.Therefore,there may be microbial resources in the rhizosphere bacterial community of healthy tomato which can prevent and control the Fusarium crown and root rot of tomato.(2)The bacterial strains in rhizosphere soil of healthy tomato were isolated and screened,and the ability of candidate strains to control Fusarium crown and root rot of tomato was verified by pot experiment.The results showed that two functional strains B-1 and B425-1,which were antagonistic to Fusarium oxysporum f.sp.radices-lycopersici.Pot experiment verified that functional strains B-1 and B425-1 could significantly reduce the incidence of Fusarium crown and root rot of tomato.The functional strains B-1 and B425-1 were identified as Bacillus velezensis and Pseudomonas aeruginosa by morphological,biochemical and 16 S r DNA sequence analysis.Phylogenetic tree analysis showed that functional strains B-1 and B425-1 were closely related to Pseudomonas OTU245 and Bacillus OTU224.(3)The untargeted metabolomics was used to study the metabolite composition and difference characteristics of rhizosphere soil of healthy and diseased tomato.The results showed that the metabolite composition of rhizosphere soil of healthy and diseased tomato was significantly different,with the relative content of 285 metabolites significantly different.KEGG enrichment analysis showed that 75 metabolites were enriched in the metabolic pathways of terpenoid and polyketone alkaloids biosynthesis and anthocyanin biosynthesis.By predicting the sources of these different metabolites,it was found that 16 metabolites came from plants,and 12 metabolites came from plant-microbial interactions.These metabolites were mainly composed of alkaloids,sugars and other substances.It also indicated that the different metabolites of tomato rhizosphere soil may directly or indirectly affect the activity and function of beneficial microorganisms or pathogens.(4)Through phylogenetic tree analysis,functional strains B-1,B425-1 and Fusarium oxysporum f.sp.radices-lycopersici had the closest relationship with Pseudomonas OTU245,Bacillus OTU224 and Fusarium OTU68.Sucrose was positively correlated with Pseudomonas OTU245 and Bacillus OTU224,and negatively correlated with Fusarium OTU68,suggesting that sucrose may directly or indirectly regulate the interaction between functional flora and pathogen of Fusarium crown and root rot of tomato.It was found that,compared with the single application of functional flora S＿P(composed of functional strains B-1 and B425-1),the mixed application of functional flora S＿P and sucrose could more effectively control Fusarium crown and root rot of tomato,and sucrose made the functional flora S＿P interaction change from the competitive relationship to the cooperative relationship.Sucrose could significantly increase the amount of biofilm formation and rhizosphere colonization of functional flora S＿P in tomato.Through the construction of genomic metabolic network model of Bacillus velezensis and Pseudomonas aeruginosa,sucrose may be involved in biofilm formation by Bacillus velezensis and Pseudomonas aeruginosa through fructin biosynthesis pathway.In addition,it was found that tomato defensive substance tomatidine was negatively correlated with Fusarium OTU68,and further found that tomatidine could inhibit Fusarium oxysporum f.sp.radices-lycopersici,and the application of functional flora S＿P and functional flora S＿P mixed with sucrose could induce the up-regulation of tomato defensive substance tomatidine synthesis gene expression.(5)In addition,pot experiment showed that application of functional flora S＿P,mixed application of functional flora S＿P and sucrose could significantly promote tomato growth,and mixed application of functional flora S＿P and sucrose could better promote tomato growth.The application of functional flora S＿P and the combination of functional flora S＿P and sucrose could induce the up-regulation of putrescine synthesis gene.It was found that after spraying the putrescine sythetic inhibitor,the application of functional flora S＿P could not significantly promote the growth of tomato,but the combination of functional flora S＿P and sucrose could still significantly promote the growth of tomato.In addition,plant height and biomass of tomato increased significantly after exogenous injection of putrescine.Therefore,the promotion of tomato growth by functional flora S＿P depends on the synthetic pathway of putrescine.In summary,this study analyzed the characteristics of rhizosphere microbial community of healthy and diseased tomato,and found that the imbalance of Fusarium community in the rhizosphere of tomato may lead to the occurrence of Fusarium crown and root rot of tomato,and that there may be microbial resources in the healthy rhizosphere bacterial community that can prevent and control Fusarium crown and root rot of tomato.The functional strains were screened for the prevention and control of Fusarium crown and root rot of tomato and the functional flora was established,and the soil metabolites for the potential regulation of functional flora were analyzed.The combination of functional flora and sucrose could be applied to the prevention and control of Fusarium crown and root rot of tomato and promote the growth of tomato.Therefore,this study provides beneficial thinking for the prevention and control of Fusarium crown and root rot of tomato and the research and development of complex microbial agents,which has certain directive significance and scientific value for vegetable safety. |
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