Study On The Prevention And Control Of Tomato Bacterial Wilt Using Microbiome

The bacterial wilt caused by Ralstonia solanacearum is one of the most serious plant diseases wordwide.R.solanacearum has very various hosts,including tomato,an important vegetable crop.Existing control methods for bacterial wilt have disadvantages such as poor effect,poor specificity,and susceptibility to R.solanacearum variants.Soiland plant-microbiome are crucial for plant disease resistance and are eco-friendly biocontrol resources.Although the relationship between tomato bacterial wilt and soil microorganisms is gradually being revealed,most of them are limited to laboratory and greenhouse studies,and large-scale comprehensive investigations are still lacking.Furthermore,the microbes that are heritable and are resistant to bacterial wilt in the tomato rhizosphere microbiome are yet to be unveiled.Therefore,this study focused on tomato and adopted the following two approaches for research: 1)Performing comparative analysis of the microbial composition and function between diseased and healthy soil in major tomato planting areas nationwide by large-scale outdoor natural environment sampling.Validating the function of potentially important bacteria.2)Exploration and functional validation of the rhizosphere microbiome composition and function of the tomatoes with clear kinship and different resistance to bacterial wilt.The aim is to explore microbes that are heritable and able to resist the bacterial wilt.The main results are following:1)We found that a minority of high-abundance taxa were the core microbiome of the main tomato planting areas in China.Results of the amplicon data showd that Proteobacteria,Actinobacteria,Chloroflexi,Acidobacteria,and Gemmatimonadetes were the core microbes.2)We revealed the main influencing factor and the characteristics of healthy soil.Further analysis of the amplicon data showed that the climate type was the main influencing factor,which had a greater impact on the microbiome than plan health status.Compared with the diseased samples,healthy samples had more diversified microbiota and enriched potential probiotics such as Actinomycetales,Myxococcales,and Acidimicrobiales.3)We revealed the different functional composition of microbiome between healthy and diseased soil.We found that healthy soil enriched potential beneficial functions,such as “antibiotic biosynthesis”,“Starch and sucrose metabolism”,and “Replication and repair”.These functions were mainly contributed by Chitinophaga,Bacteroidetes,Sphingobacterium.Diseased samples enriched “Cell motility” function that was largely contributed by Ralstonia and Cupriavidus,and functions including “Lipid metabolism”and “Xenobiotics biodegradation and metabolism” that were mainly contributed by Cupriavidus,Burkholderiaceae,and Hydrogenophaga.4)We validated the disease resistance potential of Chitinophaga eiseniae and Leifsonia aquatica,which owned potential disease-resistant function and were enriched in the healthy samples.C.eiseniae could antagonize R.solanacearum directly with high specificity.L.aquatica could not antagonize R.solanacearum directly,however,it was able to reduce the expression of virulence-related genes of R.solanacearum.These two bacteria could compete resources with R.solanacearum and they had biocontrol effect.5)We found that bacteria enriched in the diseased soil could work with R.solanacearum synergistically to lead to disease.The diseased-enriched bacteria Hydrogenophaga pseudoflava and Pseudogulbenkiania subflava could enhance the expression of motorial and chemotactic genes of R.solanacearum,and could accelerate the occurrence of bacterial wilt.6)We built a pipeline for amplicon data process based on QIIME2.This pipeline enables basic analysis of raw sequencing data with a single command.The R package micro Visu used for subsequent visulization included conventional visualization and statistical analysis functions,which was easy to use.7)We validated the different resistance of different tomato cultivars to bacterial wilt and the important role of soil microorganisms in the resistance process of resistant tomato to bacterial wilt.The susceptible cultivars were highly susceptible to bacterial wilt.The resistant parent and offspring were highly resistant to bacterial wilt,however,the resistance needed the participation of natural soil or natural soil extract which included soil microbes.8)The resistant cultivars had more robust rhizosphere microbiome than susceptible cultivars.What is more,at almost all sampling time point,the rhizosphere microbiome of the resistant offspring was more similar to that of the resistant parent.The function of the rhizosphere microbiome in all cultivars changed largely after the challenge of R.solanacearum,however,the function of rhizosphere microbiome of resistant and susceptible cultivars was commonly similar.Only a few functions differed in abundance,for example,compared with the resistant tomato cultivars,“Tetracycline biosynthesis”was enriched in the susceptible tomato rhizosphere.9)Sphingomonas sp.Cra20 and Pseudomonas putida KT2440 were heritable rhizosphere bacteria that confer resistance against bacterial wilt.They were enriched in both the resistant parent and offspring compared to the susceptible cultivars.Although they did not directly antagonize the pathogen,they could help the susceptible parent HG70 resist bacterial wilt.Additionally,they also assisted the susceptible tomato Moneymaker in resisting bacterial wilt in sterilized soil.However,when combined,their protective effect is almost lost.Cra20,isolated in this study,was a short rod-shaped bacterium,and genome annotation suggested it was a potential novel species with the capability to adapt to and colonize plant roots effectively.These two bacteria could help tomato plants resist bacterial wilt by reducing the expression of virulence genes in the pathogen and influencing the tomato root transcriptome.This study found microbes enriched in the healthy soil could resist bacterial wilt effectively by multiple strategies.Microbes enriched in the diseased soil could lead to disease with R.solanacearum synergistically,which needed to be taken into account as well.This study also unearthed two heritable tomato rhizosphere bacteria that could resist bacterial wilt and revealed their diversified resistance strategies.These provided strain resources for biological control and also laied a solid theoretical foundation for restoring healthy soil microbial ecology.