| Zanthoxylum Bungeanum ear blight is a new plant disease. It caused leaf spot and ear necrosis. The disease occurs faster, serious impact on its yield and quality, and cause economic losses. In order to knowedge the pathogen, tissue culture method and the Koch’s Postulation were employed.Dendranthema morifolium is the traditional medicinal materials, has medicinal, edible, and other multiple values. In the production practice, the chrysanthemum is infectted by a variety of diseases, such as brown spot, root rot, and stunting disease of chrysanthemum. These two kinds of diseases occured widespread and caused serious damage resulting in economic losses. Biological control is one of the safely methods for reducing the demage caused by this disease. Endophytic antagonistic bacteria with antibacterial effect on brown spot of chrysanthemum were screened. And the physiological and biochemical properties, 16S rDNA sequence, were used to identify the biological strain. Marking and colonization, optimization of fermentation, and antibacterial mechanism were carried out for laying a theoretical foundation for the biological control of brown spot of chrysanthemum. The main results are as follows:1 The fungal E-1 was determined to be responsible for this disease by the Koch’s Postulation. Morphological and the phylogenetic tree constructed based on ITS sequences showed that the morphological characters were the same as Alternaria alternata and the homologous between E-1 and A. alternata up to 100 percent. It indicated that pathogen was A. alternate.2 One hundreds and fifty-eight plant endophytic bacteria strains were isolated from the chrysanthemum, Coix lacryma-jabi, and other plants. Ten strains of them showed antagonistic activity to the fungi of brown spot of chrysanthemum and Z. bungeanum ear blight. A strain Y-1 with a rather higher antagonistic activity was obtained after primary and secondary screening. The morphology characteristics, physiological and biochemical properties and 16S rDNA sequence of this strain were studied. The similarity of the 16S rDNA sequence between bacteria strain Y-1 and Bacillus sp. were up to 99%. The strain Y-1 was finally identified as Bacillus.3 The colonization of Y-1 was studied with dual-resistant label. The Y-1 strains was cultured in shaking flask containing NB mediumin with 160μg·mL-1 of rifampicin, at 28℃, 180 r·min-1, culture liquid with a population of 3×105 cfu·mL-1 was used to detect the colonization of the bicontrol agent by irrigation soil inoculation method in sterile soil. After inoculation, antagonistic bacteria were recovered from the chrysanthemum plant, colony morphology, physiological and biochemical identification results of recovered strains are consistent with the original strain, antagonistic effect is also similarity with the original strain. Thus, the strain Y-1 is an endophytic bacterium, and can colonize in the Chrysanthemum plant.4 In order to improve the yield of antibacterial substance produced by Bacillus, single factor tests were applied to determine the optimum fermentation conditions. The results are as follows: the optimum medium is LB medium, fermentation medium 50 mL in a 250 mL erlenmeyer flask, inoculums size is up to 2%, culture temperature is at 34℃, culture for 32 h, initial pH of fermentation medium is 7.0 and vibration speed is 200 r·min-1. Under the above optimal conditions antimicrobial substances produced the strongest effect.5 Based on the determination of volatile antimicrobial substances of antagonistic bacteria, including chitinase, cellulase, protease production ability of antagonistic bacteria, were detected, and found that the antagonistic bacterium Y-1 can produce cellulase and protease. Microscopic observation showed that the fermentation of strain Y-1 can effectively inhibit the mycelial growth, and cause the mycelium dry, broken, uneven thickness, damaged cell wall, protoplasm overflow, a cavity and other phenomena. The results show that the obtained Y-1 is endophytic biocontrol bacterium. Its metabolites with cellulase activity and protease activity, and has good antagonism in the pathogen interaction. |
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