| Bacterial wilt of tomato, Lycopersicon esculentum Miller, caused by Ralstonia solanacearum (Yabuuchi et al.), is a destructive soil-borne disease and a serious threat for agricultural production in many provinces of Southern China. So far, Ralstonia wilt control approaches, including field sanitation, crop rotation and application of resistant varieties, have proven limited success. Applying chemical pesticides is generally considered as the most effective and fastest strategy for plant diseases management, however, no effective chemical product has yet been available for Ralstonia wilt. Therefore, more and more efforts need to be devoted to biological control with living microbes. Biological control is one of the most promising approaches to reduce the disease severity and yield loss caused by the diseases. Although several products have been commercialized, living microbial BCAs have not been widely accepted by farmers, since their biocontrol efficiency is not quite stable. This study was focused on development of a novel screen strategy of antagonist against R.solanacearum, evaluation of antagonists identified by the strategy and investigation of mechanism of the antagonistic strain XY21.1. Evaluation of the strains of Acinetobacter and Enterobacter as potential biocontrol agents against ralstonia wilt of tomatoBased on antagonistic activity against R. solanacearum and three other soil-borne fungal pathogens as well as biocontrol efficiency in greenhouse, we select from a total of14candidates two bacterial strains Xa6(Acinetobacter sp.) and Xy3(Enterobacter sp.) as potential biocontrol agents. To seek a suitable antagonist inoculation method, root-dipping and drenching treatments were compared for bacterial rhizocompetence, biocontrol efficiencies and plant growth promotion potential under greenhouse conditions. Drenching treatment resulted in higher rhizocompetence, biocontrol efficiency and plant growth promotion, and this method was easier to operate in field in large scale. Field trails were conducted for further evaluation of these two antagonists. In both greenhouse and field experiments, strain Xy3had a better control effect against bacterial wilt than Xa6did, while Xa6caused more yield increase. Biocontrol efficiencies of Xy3were both about65%, and the yield increases caused by Xa6were32.4%and40.7%at the75th day after treatment in two field experiments. This is the first report of an Acinetobacter sp. strain used as a BCA agaisnt R. solanacearum of tomato.2. Rhizocompetence and antagonistic activity towards a range of genetically diverse Ralstonia solanacearum strains-a novel approach for selecting biocontrol agentsEight antagonistic bacterial strains were tested for their inhibition towards a range of genetically diverse R. solanacearum strains from China and for their efficiency to control Ralstonia wilt of tomato under greenhouse condition. The results indicated that the same antagonists showed markedly different in-vitro antagonistic activity towards different Ralstonia strains, and biocontrol efficiency. From the antagonists tested, Serratia sp. strain XY21was selected because of its good in-vitro inhibition of all ten R. solanacearum strains and its good rhizocompetence on tomato plants. Biocontrol efficacy of XY21towards seven R. solanacearum strains ranged from19%to70%. A good correlation of in-vitro antagonistic activity and the actual biocontrol efficiency towards different R. solanacearum strains was found. Efficient biocontrol and yield increases were observed in field experiments with pepper and tomato. This is the first report to select potential biocontrol strains of R. solanacearum based on their rhizocompetence and their ability to antagonize a range of genetically diverse R. solanacearum isolates.3. Mechinisms of antagonist XY21against Ralstonia solanacearum strainsIn this study, antagonistic strain XY21was labeled with RFP firstly. The antagonistic activity towards genetically diverse Ralstonia solanacearum strains and enzyme activity of strain XY21-RFP were tested and compared to that of wild type strain XY21. No difference was found between wild type strain XY21and mutants XY21-RFP and XY21-rif. The results of first greenhouse experiment showed that strain XY21-RFP colonized surface of primary root after seed inoculation, and after root inoculation, the strain XY21-RFP colonized root surface and soil particles. The expression of florescence of antagonistic strain was unstably, but the expression of florescence of pathogen strain Rs-GFP was stable and the vessel of the wilt plant was full of the pathogen bacteria. The results of second greenhouse experiment displayed that the CFU counts of Serratia sp. XY21-rif and XY21-RFP in the rhizosphere of tomato plants determined by selective plating at all sampling time were around107g-1RFW (root fresh weight). DGGE analysis of16S rRNA gene fragments amplified from total community DNA was used to detect shifts in the relative abundance of ribotypes in the tomato rhizosphere as a consequence of inoculation with antagonists. The comparison of the bacterial DGGE fingerprints revealed that at10and17days after seed inoculation, the patterns of all four replicates of both treatments were highly similar except that strong bands corresponding to the electrophoretic mobility16S rRNA gene products of XY21were detected in the profiles of XY21-mutants-inoculated plants. At24and41days after seed inoculation, several differentiating bands were detected. Some of these bands were only observed in the control or the inoculated plants. Remarkably,27days after the last drenching a dominant band resembling XY21mutants was still detected in three of four replicates suggesting that the inoculant established as dominant rhizosphere population. Highly similar Bacillus profiles were found for both the inoculated and control plants indicating that the inoculation had no effect on the Bacillus community composition, while the inoculation with XY21mutants clearly affected the abundance of some Pseudomonas populations. |
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