Pesticide-degradation Characteristics and Degradation Mechanism of Biocontrol Bacteria

Abstract: Chemical insecticides have been used widely all over the world to control a wide range of plant diseases and insect pests. Extensive application of these pesticides results in insects and pathogens resistance to these insecticides, pollution of soil and crops, potential hazards to environment and human health. While biological control has attracted wide attention, as it’s non-toxic and harmless, nonexistent pollution to the environment, unlikely to cause drug resistance, etc. Some biocontrol preparations have been successfully applied in production. On the other hand, the research of soil remediation by microorganisms has made great progress. Many biocontrol bacteria have been reported to also degrade pesticides. It will be of great significance to isolate microorganism that can be applied in biological control and soil bioremediation for continuable development of agriculture. However, study in this field is rarely carried out both in China and abroad.Therefor, bacteria were isolated from peanut rhizosphere soil which antagonize against various phytopathogenic fungi with a variety of soil-borne plant pathogens as targets, and among them pesticide-degradation bacteria were screened with three kinds of pesticides used extensively as substrates. The pesticide-degradation characteristics and mechanisms were studied further. The main results and conclusions are as follows:1. 54 isolates were obtained from peanut rhizosphere at Taian, Shandong Province of China. Strains B3 and H10 were two potential biocontrol bacteria against broad-spectrum plant pathogenic fungi. According to the characteristics of morphology, physiology, biochemistry tests and the comparison of 16S rDNA sequence, strains B3 and H10 were identified as Paenibacillus polymyxa, Ochrobactrum spp. respectively.2. It was found that fermentation filtrate of H10 and its 80% saturated (NH4)2SO4 precipitate had obvious antagonism against Fusarium oxysporum f. sp. niveum and Fusarium oxysporum f. sp. cucumerinum by antagonistic experiment. Its cell-free culture filtrate showed great inhibition to mycelial growth, which were swelling, seriously distortion hyphae and concentrated protoplasm under light microscope. Activities of protease, chitinase andβ-1.3-glucanase were tested in protein precipitate. The results indicated that extracellular hydrolase produced by strain H10 destroyed the cell-wall of pathogens.3. In order to study the degradation characteristics of bacteria, the degradation rates of B3 and H10 against insecticides (beta-cypermethrin in synthetic pyrethroids, chlorpyrifos in organophosphate pesticides and imidacloprid in neonicotinoid insecticides) were determined by UV-spectrophotometry. The extent of degradation of them at initial concentration of 100 mg/L was 54.42%, 57.44% and 49.24% respectively by strain B3 within 7 days, and 83.51%, 79.82%, 59.12% by strain H10 within 60 hours. When substrate beta-cypermethrin was at 400 mg/L, the extent of degradation by H10 was the maximum, 75.55%, when substrate was at 700 mg/L, most microorganisms could not live, but the degradation rate by H10 still reached to 60.05%. When the amount of inoculation was at 5%, the degradation rate was 89.61%, as the hignest. Adding a little carbon source or nitrogen source could improve the degradation ability of H10 against beta-cypermethrin.4. To research the pesticide-degradation mechanisms of bacteria, a pyrethroid hydrolase was purified by ultrusonic cell disrupter, following by DEAE Sepharose Fast Flow chromatography and Sephadex G-75. It appeared as a single band corresponding to molecular weight (MW) of approximately 65.0 kDa on SDS-PAGE with commassie blue staining. 150μL purified enzyme degraded 10 mL 50 mg/L beta-cypermethrin in 30 minutes completely.5. In order to ascertain the nature of the pyrethroid hydrolase concerned, effects of temperature, pH and different metal ions on enzyme activities were assayed, with 4-nitrophenyl acetate as a substrate. The optimum temperature for enzyme activitis was 40℃and pH was 8.0. The enzyme was more stable when temperature was lower than 40℃and pH ranged from 6.0 to 9.0. Na+ and EDTA had no effect on enzyme activities, while Ca~(2+) and Mn~(2+) inhibited in some degree, and Zn~(2+), Fe~(3+), Cu~(2+) can strongly retard enzyme activities.
Key words: Paenibacillus polymyxa; Ochrobactrum spp.; antagonism; pyrethroid hydrolase; purification;

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