Effects And Mechanisms Of Suppression Of Tomato Bacterial Wilt By Stem Injection Of Biocontrol Agents

Biological control of plant diseases has been investigated as a hot topic because of its environmental-friendly and safe characteristics.However,soil inoculation of bio-control agents showed limited capability due to the uncertain effects of indigenous microorganisms and complex soil abiotic environment,which reduces their root colonization ability and competitiveness with pathogen.Bacterial wilt,caused by a bacterial pathogen Ralstonia solanacearum,is one of the most devastating plant diseases in the tropical and subtropical regions of the world.In this study,bio-control agents Bacillus amyloliquefaciens T-5 and Pseudomonas fluorescens CHAO were injected into tomato plant stem to evaluate their biocontrol efficacy of tomato bacterial wilt.Stem injection can avoid the effects of the complex soil biotic and abiotic environment due to the relatively simple environment in plant stem,increasing the possibility and intensity of bio-control efficacy.Further,we conducted an experiment to study the ability of bio-control agents and pathogen to use stem sap and chemical carbon resources which were used to mimic the nutrients in stem sap,to explore the nutrient competition mechanisms between bio-control agents and pathogen.These results were attributed to the nutrient competition research between bio-control agents and pathogen in plant stem.The main results obtained were shown below:1.Compared with soil-drenching method,field experiment showed that stem injection of T-5 and CHAO had higher bio-control efficacy(BCE)of tomato bacterial wilt.Greenhouse experiment was conducted to explore the effects of injection on control efficacy of bacterial wilt during different tomato growth stages(seeding stage,bloom stage and fruit stage).Our results showed that the control efficacy of T-5 was 70.32%both in bloom stage and fruit stage,and the BCE of T-5 in seeding stage was only 22.54%.There was no significant difference in the BCE of the bio-control bacterium CHAO among different tomato plant growth stages,which was always 39.8%.Based on these results,we found that the best injecting time of T-5 was at bloom stage and fruit stage of tomato plant,which is consistent with the outbreak of bacterial wilt from other literatures.2.Nutrient competition between bio-control bacteria and the pathogen in tomato plant stem played an important role in disease suppression.Nutritional contents of stem sap were different at different tomato plant growth stages.Nutrient competition was mimicked in vitro by co-culturing bio-control agent and the pathogen using tomato plant stem sap from different tomato plant growth stages.Our results showed that the growth of bio-control bacteria and pathogens in stem sap from different tomato growth stages was significantly different.The bio-control bacterium CHAO showed higher growth density than the pathogen in the stem sap from flowering and fruit stages.The pathogen inhibition ability of T-5 in stem sap from bloom stage of tomato plant was significantly higher than in seedling stage.These results suggested that CHAO and T-5 could be stem-injected to inhibit the proliferation of pathogens at reproductive stages of tomato plant,which was in line with the bio-control results from field experiment.3.The composition of tomato plant stem sap is complex and still unknown.We used 48 kinds of chemical resources in this study to mimic nutrients of tomato plant stem.We measured the resource utilization ability of bio-control bacteria and QL-RFP(RS marked with red fluorescence),and calculated the niche overlap index(NOI)between them.The ability of bio-control bacteria to inhibit the growth of QL-RFP while using different kinds of resources was also measured.The results showed that T-5 and CHAO both had nutrient competition with QL-RFP.The resource utilization intensity of T-5 and CHAO was also higher than that of the QL-RFP.NOI of CHAO with QL-RFP was higher than T-5 with QL-RFP.The growth of QL-RFP was significantly inhibited by T-5 when under the condition of resources which could be both used by T-5 and QL-RFP.Inhibitory efficiency of bio-control bacteria T-5 in different resources conditions had big variations.T-5 had the highest inhibitory efficiency on QL-RFP,which was 74.5%when using glutamine as the single resource.In addition,we found that inhibitory efficiency on pathogen of bacteria is generally better while using organic acid than the other resources.4.The inhibitory efficiency of bio-control bacteria T-5 and CHAO on QL-RFP were both high while using organic acid,but the composition and content of organic acids in tomato plant stem sap is unknown.The composition and contents of organic acids in tomato plant stem sap were determined by high performance liquid chromatography(HPLC).The concentration of organic acids in tomato plant stem was mimicked by chemicals.We studied on the effects of the growth and the inhibitory efficiency of bio-control bacteria T-5 and CHAO on QL-RFP while using different single organic acids.The results showed that the concentration of organic acids would influence the growth of T-5,CHAO and QL-RFP differently.As a result,the inhibitory efficiency would be also influenced.Malic acid and succinic acid could increase the ability of CHAO to inhibit QL-RFP.In conclusion,injecting tomato plant stem with bio-control bacteria could effectively inhibit the wilted disease of tomato plants.More studies about the competitive mechanisms between bio-control bacteria and pathogen in the plant stem needed to be done in the future.