| Tobacco bacterial wilt, which was caused by Ralstonia solanacearum, spread widely and caused very severe damages in Changjiang River and Southwest tobacco field in China. The wilt disease can decrease tobacco product and quality or even cause total destruction. In a word, tobacco bacterial wilt has been one of the mort crucial limiting factors in Chinese tobacco production. From the viewpoint of host-pathogen-environment ecological interaction triangle, this study investigated the biological control of tobacco bacterial wilt, bioremediation of soils, the bio-film protection from antagonists to R. solanacearum and the effet on the growth of pathogen and antagonists of tobacco root exudates on the basis of pathogenesis of tobacco bacterial wilt.Rhizosphere soil or infested tobacco plants were collected from a severely wilt-diseased field. One hundred and nineteen colonies similar to R. solanacearum was screened on a semi-selective medium (SMSA) from roots and stem spots of infested plants and rhizosphere soils. A strain of R. solanacearum with strong virulence was identified by its colony and microscopic morphology,16S rRNA sequence homologies and its physo-biochemical characteristics. The isolated strain was identified as Ralstonia solanacearum E.F.Smith f. sp. Nicotianae (Rs) by re-inoculating pathogen to healthy tobacco plant and other four kinds of solanaceous plants. The results of pathogenic factors for R. solanacearum in soil showed that tobacco plants were more susceptible to wilt disease when the population of R. solanacearum reached to10cfu g-1soil, the environmental temperature was30℃and the humidity kept75%above.In lab condition, two antagonists, L-9and L-25, were isolated and screened from uninfected tobacco plants and rhizosphere soils. Through16S rRNA sequence homologies and physo-biochemical analysis, one strain was Brevibacillus brevis, the other was Streptomyces rochei. These two strains not only had antagonictic effect on R. solanacearum with antagonism zone11mm and15mm, but also suppressed other kinds of soil borne disease on plates in out lab, such as tomato bacterial wilt, eggplant bacterial wilt, pepper bacterial wilt and tobacco black shank.The carbon and nitrogen sources used by antagonists were analysed by BIOLOG phenotype microarray microplates. In microplates, the average well color development (A WCD) of amino acids, which were mainly contained in rapeseed meal, wheat barn and oat bran, was the highest in rapeseed meal among these three solid wastes. These results indicated that the rapeseed meal was the best substance for reproducetion of antagonists. Considering the comprehensive factors for tobacco plants, an organic fertilizer consisted of rapeseed meal and dairy manure at the proportion of1:1(w:w) was the best compost for the secondary solid fermentation of antagonists.Biological experiment was conducted using antagonistis cell suspention or bioorganic fertilizer (BOF) to proof bio-control efficacy of antagonists. The results showed that the mixed BOF treatment had the best biocontrol effect in all antagonists applied treatments in pot experiment. The control efficacy of mixed BOF treatment was up to100%in pot experiment and that was95.4%in Anhui field experiemt and30.1%in Guizhou field experiment. The effect of BOF applied treatments was better than antagonists-cell-suspension applied treatments. Besides, the result of treatment where the two BOF second solid fermentated were applied to the soil together at the proportion of1:1, was better than those of sigle BOF applied treatments. The disease incidence and index were the lowest in mixed BOF treatment among all treatments. Moreover, in mixed BOF treatment, tobacco biomass was the largest while R. solancearum population was the least and kept106cfu g-1soil.The counts of bacteria and actinomycetes were significantly higher while populations of fungi and R. solanacearum were much lower in BOF applied treatment than that of control treatment (infected soil). Furthermore, defence enzymes in leaves and roots, such as POD, CAT, PPO, SOD and so on, and the enzymes in soil were significantly different between the BOF applied treatments and the control treatment (infected soil). Observations of vascular bundle of healthy tobacco plant, wilt plant in control treatment (infected soil) and uninfected plant in antagonists applied treatment were made by scanning electron microscope (SEM). The SEM results showed that there was viscous material deforming and blocking vessel of vascular bundle in wilt tobacco while the vascular bundle of healthy tobacco in control and BOF treatments grew well with a normal shape. Denaturing Gradient Gel Electrophoresis (DGGE) and BIOLOG-Ecoplate were used to analyze the microbial changees in rhizosphere soil in pot experiment. Soil was transformed from fungal type to bacterial type, which indicated that continuous cropping soil was gradually recovered to a new microbial system, sustainable soil ecology and good productive functions. The Shannon index, Simpson index and McIntosh index were higher in BOF treatment than that of control treatment (infected soil). The application of BOF could not only suppress pathogen, but also have a promotion to tobacco yield. Biomass was increased3.43times and3.27times in pot and field experiment, respectivily.Rhizosphere colonization of antagonists experiment showed that, antagonistic strain adhered on root and root hairs along with the root growth due to the exudates of roots after cell suspensions of antagonists were inoculated to tobacco seeds before its germination. After R. solanacearum was inoculated, antagonists could colonize in rhizosphere firstly, forming a protecting biofilm to suppress R. solanacearum and prevent pathogens from infecting roots. Plasmid pJZ383with green fluorescent protein (gfp) was successfully transformed to R. solanacearum. Antagonists could colonize on the root surface prior to pathogen, thus suppressing R. solanacearum in the rhizosphere. Phenolic acids in tobacco root exudates (TRE) were isolated and identified by high performance liquid chromatography (HPLC). Benzoic acid with the concentration of0.25μg g-1dry roots and3-phenylpropanoic acid with the concentration of1.15μg g-1dry roots were identified in TRE. Exogenous phenolic acids were used to investigate the effect of some phenolic acids, which existed in TRE, on the growth of pathogen and antagonists. It revealed that lower concentration of benzoic acid and3-phenylpropanoic acid promoted the growth of pathogen and antagonists, while higher concentration of those phenolic acids inhibited pathogen and antagonists. Besides, the promotion concentration of these phenolic acids for R. solanacearum (benzoic acid:300mg L-1and3-phenylpropanoic acid:600mg L-1) were higher than that for antagonists. This suggested that R. solanacearum made a better use of tobacco root exudates.Above all, two antagonistic microbes, Brevibacillus brevis and Streptomyces rochei were screened. They were applied to aan orgnic fertilizer consisted of rapeseed meal and dairy manure at the proportion of1:1(w:w) for the secondary solid fermentation to make a BOF. The mixed BOF had the best control efficacy and the antagonists could colonize on root surface prior to pathogen. BOF could biocontrol tobacco bacterial wilt and increase yield through improve soil microbial diversities and strengthened tobacco self-resistance. Tobacco root exudates had a larger role in growth promoting of R. solanacearum than antagonists. This was one of the most important reseaons for tobacco obstacle of continuous mono-cropping. |
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