| Of all the agricultural pests and diseases that threaten citrus crops, citrus bacterial canker disease (CBCD) is one of the most devastating, which caused by the bacterium Xanthomonas axonopodis pv. citri. More than 30 countries and areas worldwide which produce citrus have suffered from CBCD including China. Severe infection of the disease produces a variety of effects including defoliation, dieback, severely blemished fruit, reduced fruit quality and premature fruit drop.Control of canker in countries or regions where the disease is not present include quarantine or regulatory programme to prohibit introduction of infected citrus plant material and fruit, as well as continuous and strict surveying in the field and the immediate destruction of infected trees. In countries where canker is present, integrated systems of compatible cultural practices and phytosanitary measures consisting of resistant hosts, removal of inoculum sources, properly designed windbreak systems, timely application of protective copper-containing and/or antibiotic sprays are generally the most effective means of disease management. With the further knowledge to chemical pesticides, however, many concerns, such as the pollution of soil, water and atmosphere, the increase of the leftover of germicides in produce which imperils human health and life directly, ecological environment resulting from the pesticides, phytopathogenic fungi and bacteria to germicides, the agricultural unbalance of and resistance of have caused the withdrawal of many of them. So, new and safe alternative control measures are much sought after. Biological control by natural antagonistic organisms is a potential nonchemical tool for crop protection against phytopathogenic fungi and bacteria and offers a promising alternative to synthetic pesticides; in part because it is perceived as safe to the environment and human health and can avoid the appearance of drug-resistance.Because of good antibiosis against phytopathogenic fungi and bacteria, such as Xanthomonas axonopodis pv. citri, Colletotrichum gloeosporioides, Fusarium oxysporum f.sp.vasinfectum, Botrytis cinerea Pers. and Penicillium digitatum, bacterium CQBS03 had the potential as biocontrol agent, and so further study on it was carried out.In order to determine the taxonomic placement, the identification of CQBS03 was performed with traditional bacterial systemic identification method. The result showed that the physiological and biochemical character of CQBS03 is similar to that of Bacillus subtilis. Then, the molecular identification of CQBS03 was performed. Through the comparative 16s rDNA sequences analysis, the 16s rDNA sequence of CQBS03 had high homology with typical Bacillus subtilis (99%).Through single factor and orthogonal test, the optimal fermentation conditions of producing antibiotic products of Bacillus subtilis CQBS03 were given out. The optimal culture conditions of the bacteria for the antibacterial protein producing are pH 8.0, 28℃in yeast peptone glucose liquid medium with shaking for 72h.Most antimicrobial products can be precipipitated by 70% saturated ammonium sulphate. The antibacterial crude substance from CQBS03 could designate as one kind of protein based on the large molecular weight (more than 10kd), unstable to the high temperature (60℃to 70℃), partly sensitive to chloroform, insensitive to proteinase K and trypsin. PI value is 5.0 by IEF-PAGE.Through SDS-PAGE, antagonistic activity tracing, t-Butyl HIC chromatography, the antibacterial protein was prepared and purified. It was demonstrated to be one single protein band by SDS-PAGE with a molecular weight of 55KD.
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