生防细菌的农药降解特性及其降解机制的研究
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摘要
长期以来,植物病虫害防治中大量化学农药的使用造成的病原菌和害虫的抗药性以及土壤和作物中的农药残留,给人类健康和生态环境造成了严重的危害。近年来,生物防治以其无毒无害,不污染环境,不易引起抗药性等特点备受社会各界的广泛关注,许多生防菌剂已得到成功地应用,与此同时,利用微生物进行土壤修复的研究也取得了很大的进展。据报道,有些生防细菌也具有农药降解作用,若能分离到兼具生防和农药降解作用的微生物,将对农业的可持续发展具有重要意义。而目前国内外在此领域的研究却少有开展。
为此,我们以多种土传植物病原真菌为靶标,以3种常用的杀虫剂为筛选底物,从花生根际土壤中筛选到对多种植物病原真菌具有拮抗作用且对多种农药具有降解作用的细菌,并对其降解特性和降解机制进行了初步研究,具体结果如下:
1.从山东泰安的花生根际土壤中分离到54株细菌,从中筛选到对棉花枯萎病菌(Fusarium oxysporum f.sp. vasinfectum)等多种植物病原真菌具有明显拮抗作用的生防细菌B3、H10,通过形态特征、生理生化及16S rDNA同源性序列分析,将其分别鉴定为多粘类芽孢杆菌(Paenibacillus polymyxa)、苍白杆菌(Ochrobactrum spp.)。
2.抑菌实验发现,H10发酵液及其80%硫酸铵饱和度的粗蛋白质沉淀对西瓜枯萎病菌(Fusarium oxysporum f. sp. niveum)和黄瓜枯萎病菌(Fusarium oxysporum f. sp. cucumerinum)的生长均具有明显的抑制作用。镜检发现,病原菌菌丝严重扭曲、变形膨大成珠状、原生质浓缩,并在粗蛋白质沉淀中检测到了蛋白酶、几丁质酶和β-1,3-葡聚糖酶活力,表明H10产生的胞外水解酶破坏了病原真菌的细胞壁。
3.为了研究生防菌的农药降解特性,本研究采用紫外分光光度法分别测定生防细菌B3、H10对3种常用杀虫剂(合成拟除虫菊酯类的高效氯氰菊酯、有机磷类的毒死蜱、烟碱类的吡虫啉)的降解率。在原药含量为100 mg/L时,菌株B3对3种农药7 d的降解率分别为54.42%、57.44%和49.24%,菌株H10对3种农药60 h的降解率分别为83.51%、79.82%、59.12%,当高效氯氰菊酯浓度在400mg/L时降解率达到最大,为75.55%,但底物浓度高至700mg/L,大多数微生物不能忍受,而该菌仍可生长,降解率达60.05%;接种量为5%时降解率最高,达89.61%;在农药培养基中添加少量碳源或氮源,均有利于菌株H10对高效氯氰菊酯的降解。
4.为了深入研究生防菌的农药降解机制,本实验对H10拟除虫菊酯降解酶进行了分离纯化。采用超声波细胞破碎、DEAE-Sepharose Fast Flow离子交换层析、Sephadex G-75凝胶过滤层析,纯化出一种拟除虫菊酯降解酶。经SDS-PAGE电泳染色后呈单带,分子量约为65.0 kD。降解实验发现,150μL该酶液在30 min内可将10 mL 50 mg/L的高效氯氰菊酯完全降解。
5.为进一步研究该酶的性质,本实验以对硝基苯乙酸酯为底物测定了温度、pH、金属离子对酶活性的影响。结果表明:酶作用的最适温度为40℃,在40℃以下稳定性良好;最适pH为8.0,在pH 6.0～9.0之间具有较高的稳定性,酶活力维持在70%以上;Na~+、EDTA对酶活无影响,Ca~(2+)、Mg~(2+)能够降低酶的活性,而过渡态金属离子Zn~(2+)、Fe~(3+)、Cu~(2+)有强烈的抑制作用。
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.
为此,我们以多种土传植物病原真菌为靶标,以3种常用的杀虫剂为筛选底物,从花生根际土壤中筛选到对多种植物病原真菌具有拮抗作用且对多种农药具有降解作用的细菌,并对其降解特性和降解机制进行了初步研究,具体结果如下:
1.从山东泰安的花生根际土壤中分离到54株细菌,从中筛选到对棉花枯萎病菌(Fusarium oxysporum f.sp. vasinfectum)等多种植物病原真菌具有明显拮抗作用的生防细菌B3、H10,通过形态特征、生理生化及16S rDNA同源性序列分析,将其分别鉴定为多粘类芽孢杆菌(Paenibacillus polymyxa)、苍白杆菌(Ochrobactrum spp.)。
2.抑菌实验发现,H10发酵液及其80%硫酸铵饱和度的粗蛋白质沉淀对西瓜枯萎病菌(Fusarium oxysporum f. sp. niveum)和黄瓜枯萎病菌(Fusarium oxysporum f. sp. cucumerinum)的生长均具有明显的抑制作用。镜检发现,病原菌菌丝严重扭曲、变形膨大成珠状、原生质浓缩,并在粗蛋白质沉淀中检测到了蛋白酶、几丁质酶和β-1,3-葡聚糖酶活力,表明H10产生的胞外水解酶破坏了病原真菌的细胞壁。
3.为了研究生防菌的农药降解特性,本研究采用紫外分光光度法分别测定生防细菌B3、H10对3种常用杀虫剂(合成拟除虫菊酯类的高效氯氰菊酯、有机磷类的毒死蜱、烟碱类的吡虫啉)的降解率。在原药含量为100 mg/L时,菌株B3对3种农药7 d的降解率分别为54.42%、57.44%和49.24%,菌株H10对3种农药60 h的降解率分别为83.51%、79.82%、59.12%,当高效氯氰菊酯浓度在400mg/L时降解率达到最大,为75.55%,但底物浓度高至700mg/L,大多数微生物不能忍受,而该菌仍可生长,降解率达60.05%;接种量为5%时降解率最高,达89.61%;在农药培养基中添加少量碳源或氮源,均有利于菌株H10对高效氯氰菊酯的降解。
4.为了深入研究生防菌的农药降解机制,本实验对H10拟除虫菊酯降解酶进行了分离纯化。采用超声波细胞破碎、DEAE-Sepharose Fast Flow离子交换层析、Sephadex G-75凝胶过滤层析,纯化出一种拟除虫菊酯降解酶。经SDS-PAGE电泳染色后呈单带,分子量约为65.0 kD。降解实验发现,150μL该酶液在30 min内可将10 mL 50 mg/L的高效氯氰菊酯完全降解。
5.为进一步研究该酶的性质,本实验以对硝基苯乙酸酯为底物测定了温度、pH、金属离子对酶活性的影响。结果表明:酶作用的最适温度为40℃,在40℃以下稳定性良好;最适pH为8.0,在pH 6.0～9.0之间具有较高的稳定性,酶活力维持在70%以上;Na~+、EDTA对酶活无影响,Ca~(2+)、Mg~(2+)能够降低酶的活性,而过渡态金属离子Zn~(2+)、Fe~(3+)、Cu~(2+)有强烈的抑制作用。
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.
引文
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