| Rubber tree diseases is an important factor to hinder the smooth development of natural rubber industry, along with the enhancement of environmental protection consciousness of people, the biological control of rubber tree plant disease will be more and more important. Bacillus subtilis Czkl isolated from the root xylem of rubber tree, shows a good inhibitory effect on rubber tree root disease, anthracnose and watermelon fusarium wilt disease. The identification, characteristic study and produce of active substance is the important foundation for developing bio-control product.In order toidentify the active substance from Bacillus subtilis Czkl,a system including from the biocontrol bacteria screening, research of the metabolites substance to the separation and purification of antibacterial substance was build up to provide theoretical basis for the development of new microbial pesticides for prevention of rubber tree disease. Specific research contents are as follows:1.The cups and saucers and growth rate method was adopted to study the active substance from rubber tree Bacillus subtilis strains Czkl fermentation, the results showed that the active substance belongs to the exocytosis metabolites, existing in the fermentation supernatant. Further research on the physical and chemical properties of fermented liquid showed that the antimicrobial substance has resistance to high temperature, strong acid but not alkali.and is not sensitive to uv, proteinase K and 5 kinds of common metal ions, organic solvent; Through acid precipitation after methanol extraction to extract the active substance; Fermentation supernatant by ninhydrin and biuret reagent of color reaction, antibacterial substance is a peptide bond and annular structure, according to a study in the nature of the fermentation supernatant, speculated that its active substance is peptide antibiotics.2.The single factor experiments to study the strain Czk1 culture conditions shows that YPD medium used as the strains of the fermented liquid culture medium, the initial pH value of 7.0, with 7% quantity of inoculation, shaking table culture (28℃, rpm 180) in 3 d, Czkl showed best activity of antagonism to pathogens.3.With four specific primers of lipopetide antibiotics synthesis related genes, the Bacillus sub til is Czkl genome genetic was tested, and the results show that the strain Czkl genome of surface active element (Surfactin) synthesis related genes sfp, fengycin element synthesis related genes fenB (Fengycin), develop the hay cephalosporins (Iturin) synthesis related genes ituD and lpa-14, proved that B. subtilis Czkl has the ability to produce fat of peptide antibiotics on a molecular level.4.According to extraction-precipitation method, B. subtilis Czkl active substance was extracted, to obtain the crude extraction. The crude extraction by silica gel column and gel column chromatography for separation and purification, get five active component. Bacteriostatic experiment and spore germination experiment showed strongest component F3 activity. Five active component for MALDI-TOP-MS detection analysis, comprehensive analysis of the results speculated that Bacillus subtilis Czkl peptide may have produced by lipopeptide C)3Surfactin or C14SurfactinB、C16Surfactin or C16SurfactinA, C16IturinB、C17ItuririB、C18turinB, C16IturinA、C17IurinA, and C14FengycinB、C18FengycinB、C15FengycinB or C17FengycinA, may be produced C14BacillomycinD、C16BacillomycinD.5.The bacteriostasis study In vitro shows that the minimum inhibitory concentration of crude extracts of lipopeptide is 0.04g/L, and the value of EC 50 of the lipopeptide to inhibite rubber tree anthrax conidia germination is 3.8mg/L.And the occurence and development of anthracnose on rubber tree leaves both in vitro and in vivo can be partly ibhibited by 1.0g/L of the lipopeptide.The pot experiments also indicate that anticipating apply of the lipopeptide can have a control effect of 45.6% on rubber tree powdery milde.The disease-resistant mechanism showed that spraying crude extracts of lipopeptide antibiotic substances, on the one hand, will change the permeability of cell membrane or cell wall, causing plasma leaked, causing dry cell death, finally can’t finish the whole process of differentiation; on the other hand, can improve the rubber tree leaf peroxidase (POD, PPO, and PPO) plant enzyme phenylalanine of PAL activity, enhance the resistance of the outside world, or spraying crude extracts of lipopeptide antibacterial substance occupied a certain ecological sites, so as to achieve the purpose of disease prevention. Can say, Bacillus subtilis Czkl prevention mechanism is a combination of a variety of ways. |
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