Microbial Ecological Mechanism Of Disease Suppressive Bacterial Community In Tomato Rhizosphere Driven By Bio-Organic Fertilizer Application

Bacterial wilt disease caused by the pathogen Ralstonia solanacearum has been regarded as a global soil-borne disease,due to its high genetic diversity,board hosts,and strong adaptability.Soil microbiome especially the rhizosphere microbiome plays an important role in enhancing crop resistance and maintaining plant health.In this study,we firstly evaluated the biocontrol effect of long-term continuous application of bio-organic fertilizer on tomato bacterial wilt in the field,and the inhibition effect of the soil from the field experiment on the Ralstonia solanacearum invasion was confirmed by pot experiments.Secondly,transplantation experiment was applied to investigate the characteristics of tomato rhizosphere microbiome assembly under the influence of disease-suppressive soils.Thirdly,the dynamics of the rhizo-bacterial community and metabolite composition along with the developmental stages of tomatoes were monitored in different initial soil treatments using high-throughput sequencing and non-targeted metabolomics detection technology.Finally,we investigated the ecological mechanisms of the rhizosphere microbial community assembly by analyzing the correlations among representative compounds,keystone bacteria and the relative abundance of Ralstonia in rhizosphere.The main results obtained are as follows:1.The biocontrol effect of continuous application of bio-organic fertilizer on tomato bacterial wilt and soil microbiomes were evaluated in a 7-season field experiment.Results showed that compared with chemical fertilizer（CF）and organic fertilizer（OF）treatments,application of bio-organic fertilizer（BF）could significantly decrease the disease incidence of tomato bacterial wilt（50%,45%and 27%,respectively）in the 7th season.The bacterial community composition of BF treatment was distinctly changed and the relative abundances of Bacillus,Steroidobacter,Nitriliruptor,Lysinibacillus,Pirellula and Pseudolabrys were significantly increased.Pot experiments showed that the population of Ralstonia in BF treatment rhizosphere was the lowest.The inhibition effect on the pathogen invasion vanished when the BF soil was sterilized,which was similar to the CF and OF treated soils.Results indicate that the soil microbiome cultured by continuous application of bio-organic fertilizer obtained the ability to resist the invasion of tomato bacterial wilt pathogen.2.The three distinct soils varied in different level of disease supprssion in the field experiment were selected to conduct a transplanting experiment.The 30-day plants were transplanted from the three soils（BF,CF and OF）to the disease-suppressive soil（BF）.After another 15 days,the plants obtained the disease suppressive rhizosphere microbiome.Results suggested the main rhizo-microbial community of the plants after transplanting into BF soil was recruited from the disease suppressive soil.In detail,compared to rhizosphere that transplanting to the original soils,the rhizosphere of transplanting into BF treatments recruited more Pseudomonas,Anoxybacillus,Flavobacterium,Permianibacter and Erythrobacter,and these genera were significantly negatively correlated with the abundance of R.solanacearum.3.The original bulk soil and rhizosphere samples of BF,CF and OF treatments were continuously collected along tomato developmental stages（Seedling,Flowering and Fruiting stages）.The bacterial community were analyzed by high-throughput sequencing to explore the succession features of rhizosphere microbiome started in different original bulk soil.The bacterial communities of tomato rhizosphere were significantly distinct among seedling,flowering and fruiting stages,indicating that tomato selected the rhizosphere microbiomes independently according to its growing stages.The linear models predicted that the disease incidences of fruiting tomato was detemined by the bacterial community from the original bulk soil.Compared to CF and OF treatments,the relative abundance of Actinobacteria（Streptomyces）,γ-proteobacteria（Luteimonas,Lysobacter）was significantly enriched in suppressive rhizosphere soil at fruiting stage.The rhizo-microbial community in BF treatment had a more complex and closely related bacterial community network at all different growing stages.Both random forest and "netshift" prediction indicated that ASV 3530（Lysobacter）played a key role in the assembly of disease-suppressive rhizosphere.The relative abundance of AS V₃530 showed significantly negative correlation with R.solanacearum at the fruiting stage.Moreover,the Lysobacter gummosus L17 was furhter isolated from the suppressive rhizosphere and its inhibition ability on the growth of Ralstonia solanacearum within 24 h was confirmed.4.Rhizosphere soil metabolome of tomato in the three treatments at different growth stages was detected by non-targeted metabolomics（GC-MS）.There are significant differences in the metabolic components of tomato rhizosphere at seedling,flowering and fruiting stages.The root exudate at seedling stage was dominated by organic acids and amiano acids while it was predominated by esters at flowering stage and sugars and sugar alcohols at fruiting stage.Compared with conducive rhizosphere soil,suppressive rhizosphere soil accumulated mannose,threonic acid,lyxose,2-hydroxybutanoic acid,αsantonin,sophorose at flowering stage;and caprylic acid,catechol and D-alanyl-D-alanine at fruiting stage.In addition,the rhizosphere soil metabolome showed significantly positive correlation with rhizosphere microbial communities.The relative content of catechol showed significant negative correlation with the relative abundance of Ralstonia solanacearum.In addition,the catechol has a significant inhibitory effect on the growth of Ralstonia solanacearum under the concentration of 1 mM,.The relative abundance of threonic acid,mannose,and α-santalin were significantly positive correlated with the relative abundance of potential keystone ASV₃530（Lysobacter）at flowering stage.While D-mannitol has a obvious growth-promoting effect on Lysobacter gummosus L17 within 16 hours under the concentration of 1 mM.The disease-suppressive soil induced by continuous application of bio-organic fertilizer can effectively control the occurrence of tomato bacterial wilt.The bacterial community and metabolites in rhizosphere were dynamic with the development of tomato,however,significant differences with bacterial community and metabolites of tomato rhizosphere were found among different original soils.Furthermore,the results showed that the keymetabolites interacted with the key microorganisms to resist pathogen invasion.Collectively,we tried to reveal the microbial ecological mechanism of disease suppressive bacterial community in tomato rhizosphere driven by bio-organic fertilizer application.