| Diseases in agriculture and forestry were almost caused by insects and fungus.According to studies, we can easily find a very important point in common betweeninsects and fungus, that is the chitin which is a tough, protective, semitransparentsubstance, primarily a nitrogen-containing polysaccharide that occurs in theexoskeletal and gut linings of insects the principal component of the cell walls ofcertain fungi. Therefore, people paid more attention to biocontrol when they found theirreparable disaster caused by the chemical pesticides to environment and humanbeings. Thus unquestionably the chitin-degradation, chitinase, has been studiedextensively. Many kinds of biology can produce chitinase, including insects, plants,microbes, ect. Chitinase from microbes becomes the most popular object researchersdo, because the microbes have many well-known specific properties, for example,small in volume but large in square measure, high absorption and rapid transformation,extreme production, adaptability and variation, widely distributed and vary species,convenient and quick when abstract and transform gene from microbes. Thus, more studies will be researched on the isolation and screening of unknown chitinase formingstrains. Also, the chitinase from microbes must be purified, its characteristics, the wayit works and the effective it produces are the hot spots which researched on biocontroltoday.The subject in our experiment is a typical insect (Bombyx mori), which diednaturally. Outwardly, the dead body had a big bright coeruleus spot on theircephalothorax. A chitinase productive strain was isolated from the naturally deadbody's peritrophic membrane in midgut. The main composition of peritrophicmembrane is chitins, it is the first barrier and plays an important role in protecting thegut epithelium from infection of pathogens. If this place is degradated, there must besomething which can hydrolyze chitins. That's why we chose this place to isolate thechitinase productive microbes. In order to enrich the chitinase productive microbeswhen we culture the mixture sample on the screening medium fiat plate, we usedcolloidal chitin as the only carbon source. Doing this, most microbes which can't usechitin as its carbon source are knocked out, left only the microbes which can use chitinas its carbon source. During these microbes, most can produce chitinase. Under asepsiscondition, we take the sample from the dead body's peritrophic membrane inmidgut,then separated them in space by draw lines on the chitin flat plate. The plates wereincubated at 28℃for 72h. Most microbes on the plate has hydrolytic circle, whichcaused by the chitin degradation. Take note to the ratio of the diameter of thehydrolytic circle to the diameter of the colony. By comparison, we chose a highyielding chitinase-producting strain, named zl1-r. This strain is incubated under thesame condition for five generation, and record the ratio of the diameter of thehydrolytic circle to the diameter of the colony. From this result, we can discover thatthe strain zl1-r has chitinase-producting genetic stability. The method to conserve thestrain is glycerol quick freezing method and petroline slant tube method.In order to conform the function of this strain in biocontrol, we determinated thetoxicity of the strain zl1-r to the Bombyx mori, grasshoppers and cabbage butterflylarva. A pure strain is separated from the LB culture. Then we inoculated the pure strain into the colloidal chitin liquid culture and incubated at 28℃for 72h. Thefermentation broth of zl1-r was fed to the insects. The result showed that lethality rateof the strain to the Bombyx mori, grasshoppers and cabbage butterfly larva wasrespectively 50％, 60％and 73.3％. After the confirmation the function of the strainzl1-r to the insects, we identified the strain by physiological, biochemical test andmolecular systematic analysis. The observation of morphological and culturalcharacteristics, experiments of biochemical tests and the analysis of 16S rDNAsequence are all applied in this identified study. The process of the molecularidentification including the following steps: abstraction and purification of the strain'sgenome; design the primer of 16S rDNA; getting the segment of 16S rDNA by PCRtechnique; recovery of the 16S rDNA segment from the agarose gel; linkage of the 16SrDNA segment and T-vector; translation of the linkage production into the E.coli;selection of the positive colony; extraction of the plasmid; inspection by enzymecutting, sequencing the 16S rDNA segment,et al. The gene sequence was register inGenebank website and compared with other fifteen strains' 16S rDNA sequence inRDPⅡwebsite. The fifteen mode strains have the highest homology with strain zl1-r.On the basis of phenotypic characterization and sequence similarity analysis, strainzl1-r was most closely related to the genus Aeromonas hydrophila. So we initiallyclassified the strain zl1-r into the genus of Aeromonas sp..In order to improve this chitinase-producting bacterium and enhance its chitinaseproduction, this study also optimized the fermentation condition of this strain. Theoptimization factor included the cultural time, the cultural temperature, the initial pH,the volume of the medium, the carbon source, the nitrogen source, et al. The resultshowed that it was the powder chitin and colloid chitin, which can induce theproduction of chitinase, not the foliated chitin. The best one is the powder chitin. Fromthe optimization of the fermentation condition, we can conclude that the optimalcondition of the chitinase-producting cultural medium was as following: powder chitin1.2％(w/w), peptone 0.12％(w/w), K HPO4 0.02％(w/w), inoculated in 30℃,initial pH was 8.0, volume of culture medium was 15％, cultural time was 72 h. We also studied the basic characteristics of the crude chitinase from the zl1-r's fermentationbroth, which laid a solid foundation for the further study of the chitinase from thisstrain. The result showed that this kind of chitinase's optimal activity was at 25℃pH5.5, The chitinase activity decreased when the maintain temperature rised and it wasstable at broad pH range from 5.0-9.0,retaining at least 88％activity.
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