Analysis Of Tomato Rhizosphere Microbiome And Screening Of Functional Bacteria For Disease Control And Growth Promotion

Tomato is a very important economic crop with various nutrients such as vitamins.In the process of tomato production,chemical fertilizers and pesticides are often applied to increase production and control pests and diseases.However,long-term excessive application of chemical fertilizers and pesticides has brought pollution problems such as excessive pesticide residues and eutrophication of water bodies,harm to human health and ecological environment.Microorganisms play an important role in promoting crop nutrient absorption and maintaining crop health.Functional microorganisms can not only promote plant growth and increase yield,but also improve plant resistance and prevent diseases,so that they can partially replace chemical fertilizers and pesticides to reduce pollution and protect ecology.However,traditional studies only focus on the function of microorganisms and ignore the colonization and competence of strains,resulting in unstable practical application effect.In recent years,with the rapid development of related technologies in microbiomics and culturomics,powerful techniques have been provided for obtaining highly active functional strains with strong colonization ability.In this study,the 16 S rRNA amplicon sequencing technology was used to analyze the composition and structure of tomato rhizosphere bacterial communities in different variety and growth stage.High-throughput isolation and cultivation of healthy tomato rhizosphere bacteria was by using culturomics technology,and the rhizosphere bacteria members were identified through bioinformatics pipelines.Comparing with 16 S rRNA amplicon sequences,the target strains were selected based on relative abundance and literature analysis.Then the functional screening of the strains was carried out through the detection of phosphate-solubilizing activity,siderophore activity and antagonism test against plant pathogens.Finally,the colonization ability in the tomato rhizosphere,the disease control and growth promotion effects and the influence on the rhizosphere microbiota of target strains were studied by pot tests.The main findings are as follows:(1)Through 16S rRNA amplicon analysis of tomato rhizosphere bacteria of different variety and growth stage,it was found that the variety and growth stage had impacts on the diversity,richness and community structure of rhizosphere microbiota,among which the community structure was affected variously.The bacterial community composition under different conditions is similar,Proteobacteria,Actinobacteria,Firmicutes,Bacteroidota are the main dominant bacterial phyla,the relative abundance of Cyanobacteria is higher in F85 and F689,and the dominant bacterial genus are Streptomyces,Lechevalieria,Microbacterium,Devosia,Pseudomonas,Shinella,Bacillus,Flavobacterium,Sphingobium,Ensifer,Mitsuaria,etc.(2)High-throughput isolation and culture technique was used to isolate bacterial members from healthy tomato roots.Through bioinformatic pipeline identification,a total of 702 ASVs were isolated,belonging to 8 phyla,14 class,33 order,75 family,161 genus and 175 species.The dominant phyla were Proteobacteria,Actinobacteria,Bacteroidetes,Firmicutes,and the frequent genera were mainly Pseudoxanthomonas,Microbacterium,Sphingobium,Xanthomonas,etc.Comparing with the sequences of16 S rRNA amplicons,it was found that there were 145 ASVs with relative abundance bigger than 0.1%,of which 113 ASVs were isolated and cultured.Combined with the purity,abundance and previous literature analysis,a total of nine bacteria were selected for follow-up research,namely P1E3(Pseudomonas piscium),P2B4(Bacillus velezensis),P2H4(Shinella kummerowiae),P3E5(Mitsuaria noduli),P4C8(Brevundimonas vesicularis),P5A12(Chitinophaga niabensis),P5C5(Bacillus velezensi),P5H4(Sphingobium mellinum)and P6F7(Ensifer sesbaniae).(3)Through plate screening,the results showed that all 9 target bacteria had phosphate-solubilizing activity,and six of them could produce siderophore.Through the confrontation test,6 strains were found to have significant inhibitory effect on Fusarium oxysporum,and 8 strains on Botrytis cinerea.Two strains had inhibitory effect on Ralstonia solanacearum.(4)Rifampicin resistance was used to label the target bacteria,and then the colonization ability of the strains was tested.The results showed that 6 strains could still colonize stably in 28 days.The target strains were found to promote plant growth by measuring stem diameter,plant height,root-shoot ratio,underground fresh weight and seedling index of tomato seedlings.Through pot experiment,the result showed that the control efficiency of P2B4 and P5C5(Bacillus velezensi)on tomato bacterial wilt was50%,and the disease index decreased by about 30.Thirty days after inoculation of target strains in tomato rhizosphere,16 S rRNA amplicon sequencing was performed.The results showed that all beneficial bacteria except P1E3(Pseudomonas piscium)improved the richness and diversity of tomato rhizosphere bacterial community.The composition of the dominant bacterial phyla and genera did not vary,but the relative abundance changed,with an overall increase in the relative abundance of beneficial microorganisms was found.