| For preventing chive maggot flooding, peasent have to use lots of chemical pesticides during production. Chlorpyrifos is one of organophosphate pesticides, which has many advantages such as high efficiency, low toxicity, broad-spectrum, low-residue, low resistance over other insecticides. But exceeded using of chlorpyrifos caused pesticide residues over normal upper limit of leek, which greatly threatened human health and resulted in deterioration of eco-environment. It is well known that microorganisms play an important role in reducing pesticide residues of crop and soil, and a large number of pesticide-degrading microorganisms have developed and applied. Thus, we isolate the chlorpyrifos-degrading bacteria from leek and its rhizosphere soil. It is certain to contribute to pollution-free production of leek. The specific results are as follows:1.One dominant endophytic bacterial strain W7 was isolated from the root of fresh leek,which had a broad inhibition spectrum against several vegetable pathogenic fungi. Its antagonistic mechanism was different from those of other biocontrol bacteria reported previously. The extracellular metabolites of strain W7 had no effect on pathogenic fungi, but the somatic polysaccharose which obtained from cells by ultrasonic disruption and organic solvent precipitation could inhibit the growth of mycelium. And also, strain W7 was found to be capable of utilizing beta-cypermethrin as the sole source of carbon for growth, and its degradation rate at initial concentration of 100mg/L was 51.3% within 7 days. Strain W7 was identified as Paenibacillus spp. based on morphological, physiological-biochemical properties and 16S rDNA sequence analysis.2. A chlorpyrifos-degrading bacteria strain D10 was isolated from rhizosphere soil of leek. It was capable of utilizing chlorpyrifos as the sole source of carbon for growth, and the degradation rate was 57.9% (100mg/L) within 6 days. By morphological, physiological, biochemical and 16S rDNA sequence homology analysis, it was identified as Acinetobacter spp.3. In order to research the degradation characteristics of D10, the effects of carbon source concentration, pH, temperature, inoculum size and chlorpyrifos concentration on degradation and growth was determined. The results indicated that the optimal conditions for degradation were pH 8.0, temperature 30~35℃, inoculum size 5%, chlorpyrifos concentration below of 200mg/L; The optimal conditions for growth were pH 8.0 and above, more than 1% carbon, temperature 35℃, inoculum 10%, chlorpyrifos concentration below of 200mg/L.4. The location and property of pesticide-degrading enzyme which obtained from strain D10 by ultrasonic disruption and organic solvent precipitation was studied. The result showed that endoenzyme had higher velocity than ectoenzyme. The pH optimum was 8.0 for enzymatic degradation of chlorpyrifos, and enzyme activity was relatively stable under alkaline conditions. The optimum temperature was 30℃, and had the stability of endure for temperature.5. To measure practical degrading rate of D10 in soil, the contaminated soil was simulated under laboratory conditions. The results showed that the degradation rate was 82.5% in non-sterilized soil added strain D10 within 20 days but 26.7% in control, so strain D10 could effectively decrease chlorpyrifos residues of soil.6. As the dominant endophytic bacteria W7 couldn't degrade chlorpyrifos, the degrading enzyme gene of D10 was studied in order to make strain W7 to obtain chlorpyrifos-degrading ability by transgene. It was reported that organophosphate hydrolase gene located in plasmid or genomic DNA. But after several plasmid extraction of strain D10, plasmid bands were not detected, so the gene encoding chlorpyrifos degrading enzyme was regarded on the genomic DNA. Acoording to coding region of previous reported mpd gene, designed primers and obtained a 900bp fragments by PCR. |
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