| Bacterial wilt of tobacco caused by Ralstonia solanacearum (RS) is a soil-borne plant disease, which seriously threatened the production of tobacco, and resulted in huge economic losses. Traditional controlling managements are inefficient and threaten the environment in agriculture production. Therefore, using the rhizobacteria as biological control agents could be a new and effective method. Functional microorganisms were added to the organic fertilizer to produce bio-organic fertilizer by solid fermentation technology which was used in agricultural production. This was not only to achieve the purpose of prevention disease, but also reduce chemical fertilizer use and pesticide contamination. This technology is found to be environmental friendly and can promote efficiency and green agriculture development.In the present study, plate dilution and plate confrontation methods was used for screening efficiently antagonistic strains against R. solanacearum. Identification of the antagonistic bacteria strains was carried out by physiological and biochemical experiments and16S rRNA sequence analysis. The antibiotic biosynthetic genes was cloned and conducted through a preliminary study, and the conditions for the production of antagonistic substances, physical and chemical properties of the antagonistic substances were also studied. The biocontrol effect of the selected strains were detected in continuous pot experiments under greenhouse conditions, In the third pot experiment, the rhizoshpere soil, bulk soil and various plant organisms were random sampled in the healthy and wilted plants in each treatment, and the population of the pathogens and antagonistic bacteria were detected and analyzed to expound a preliminary biocontrol mechanism. In order to improve the biocontrol effects, the effect of different second solid fermentation substrates and conditions on the growth of selected biocontrol agents were investigated. The concrete results are listed as follows:(1) Total of30strains with different colony morphologies were isolated in this research, five obvious and stable antagonist bacillus strains named SQR11, SQR33, SQR55SQR99, SQR110were selected for fouther study The selected five strains were used to produce bio-organic fertilizers for pot experiments to test the biocontrol efficiency of each strain. The results showed that strain SQR11possessed the best biocontrol efficiency. The biocontrol index of strain SQR11was more than47%in the first pot experiment, SQR33showed45%, the others are less than30%.(2) Strains SQR11was identified as Bacillus amyloliquefaciens based on the16S rDNA sequence together with morphological, biochemical and physiological characteristics.(3) The best condition of Bacillus amyloliquefaciens SQRll for production of antagonistic substances was as following:Mannitol10g, peptone30g, MgSO4,2.0g, K2HPO4,1.0g, pH7.0, deionized water1L, with bottling amount of1/5,190rpm, cultured at30℃for48h.(4) Significant inhibition effect was observed from the crude extracts which were extracted by ethyl acetate from the liquid fermentation of Bacillus amyloliquefaciens SQR11against R. solanacearum. The inhibition zones of the crude extracts secreted by Bacillus amyloliquefaciens SQRll were not changes obviously after treated for2h at80℃, which were also stability in UV treatment. Antagonistic activity of crude extracts was stable ranging from pH2.0～9.0, and more sensitive to the strong alkaline conditions. Proteinase K and pepsin did not significantly affect the activity of antagonistic substance, however, the trypsin could degrade the antagonistic substance under certain conditions.(5) According to the results of the first pot experiments, Bacillus amyloliquefaciens SQR11was selected in the second and third pot experiment to verify the stability of the biocontrol effect. The biocontrol efficiency of serially pot experiments were listed as following:47%in the first experiment,69%in the second experiment, and89%in the third experiment. The pot experiment results indicated that Bacillus amyloliquefaciens SQR11could significantly suppress the bacterial wilt of tobacco.(6) The third pot experiment results showed that disease symptoms occurred when the population of pathogen in the plant rhizosphere soil arrived2x105CFU-g-1dry soil, with the number of pathogens increasing, the disease symptoms could be more seriously. The growth of pathogen could be effectively suppressed when the density of antagonistic bacteria more than2x107CFU-g-1dry soil in the rhizosphere soil, then the plant disease can be effectively prevented.In contrast, when the population of antagonistic bacteria was below107CFU-g-1dry soil, the pathogen could growth well, and the plants showed disease symptoms. The entophytic bacteria in the various organisms of the plant were also detected, the antagonistic bacteria was4×104CFU-g-1plant tissue in the stems of healthy plants, while which was only6×103CFU-g-1plant tissue in the stems of infected plants. The populations of pathogen were1.5×102CFU-g-1plant tissue and3×103CFU-g-1plant tissue in corresponding stems of plants, respectively. Meanwhile, the plant biomass increased by50%in the bio-organic fertilizer treatment compared to chemical fertilizer treatment. Thus, all of these proved that the strain SQR11could suppress the growth of R. solanacearum, and the bio-organic fertilizer produced by strain SQR11could promote the growth of tobacco plant.(7) The second solid fermentation substrates contained feather powder, rapeseed meal amino acids, raw rapeseed meal, pig manure, and algae mud were used in the solid fermentation optimization experiments. The results showed that, the highest population of strain SQRll was found in the the production ingredient of bio-organic fertilizer containing feather powder, rapeseed meal amino acids, and pig manure, and strain SQRll number in the new product could keep stability for long time. Furthermore, the pot experiment also showed that new products significantly promoted plant growth than the chemical fertilizer.(8) Antagonist genes was cloned and analyzed from Bacillus amyloliquefaciens SQR11, the results showed that surfactin, iturin synthesis genes were involved in the genome. And both of the two antagonistic substances were also detected in the fermentation broth by high performance liquid chromatography (HPLC) analysis. |
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