毒死蜱农药降解菌及其降解特性的研究
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摘要
当前,韭菜地下害虫韭蛆的肆虐,迫使菜农在韭菜生产中大量使用化学杀虫剂。毒死蜱是常用的有机磷杀虫剂之一,虽然具有高效、低毒、广谱、低残留和低抗药性等优点,但大量使用造成了韭菜农药残留超标,严重危害了人类健康并造成了土壤生态环境的日益恶化。众所周知,微生物在降低作物及土壤农药残留中发挥了重要作用,许多农药降解微生物已得到开发应用。针对上述现状,本文从韭菜植株和韭菜根际土壤中分离并筛选具有毒死蜱降解能力的生防菌株,对于韭菜的无公害生产具有重要的现实意义和实践意义。具体结果如下:
1.从新鲜韭菜的根中分离到一株对多种蔬菜病原真菌有抑制作用的优势内生细菌W7,其拮抗机制与已报道的其它生防细菌不同,其胞外代谢物对病原真菌无抑制作用,而菌体经超声波破碎及有机溶剂沉淀得到的菌体多糖粗提液可明显抑制病原菌菌丝生长。而且,该菌能以100mg/L高效氯氰菊酯为唯一碳源生长,7d的降解率为51.3%,但不能降解毒死蜱。通过对其形态特征、生理生化及16S rDNA同源性序列分析,将其鉴定为类芽孢杆菌(Paenibacillus spp.)。
2.从受农药污染的韭菜根际土壤中获得了一株高效毒死蜱降解细菌D10,该菌能以毒死蜱为唯一碳源生长,6d内对100mg/L的毒死蜱降解率达59.7%。通过形态特征、生理生化及16S rDNA同源性序列分析,鉴定该菌为不动杆菌(Acinetobacter.spp)。
3.为进一步研究毒死蜱降解菌D10的降解特性,测定了外加碳源浓度、pH、温度、接种量及毒死蜱浓度对菌株降解率和生长量的影响。结果表明,当pH为8.0,温度为30～35℃,接种量在5%,毒死蜱浓度为200mg/L以下时,菌株D10的降解效果较好;在pH为8.0以上,外加碳源1%以上,温度为35℃,接种量10%,毒死蜱浓度为200mg/L以下时,菌株D10的生长量最大。
4.采用超声波破碎、有机溶剂沉淀等方法从菌株D10中提取粗酶液,用于研究毒死蜱降解酶在细胞中的定位和性质。结果表明:毒死蜱降解酶主要位于胞内。该酶对毒死蜱的酶促降解最适pH为8.0,在碱性条件下相对稳定;最适温度为30℃,且具有较好的热稳定性。
5.实验室模拟受污染土壤,研究菌株D10在土壤中的实际降解效果。结果表明,在添加了菌株D10的未灭菌土壤中,20d降解率达82.5%,而未灭菌土壤的自然降解率仅有26.7%,表明在土壤中添加菌株D10可明显降低土壤中的毒死蜱残留。
6.由于韭菜优势内生细菌W7不能降解毒死蜱,为使其今后能通过转基因的方式获得毒死蜱降解能力,对菌株D10的毒死蜱降解酶基因进行初步研究。目前,已报道的有机磷降解酶基因位于质粒或者基因组DNA上。本研究对菌株D10多次进行质粒提取,电泳检测无质粒条带,从而可以判断菌株降解毒死蜱的相关基因位于基因组DNA上。根据已报道的甲基对硫磷降解酶(mpd)基因的编码区设计引物,PCR得到一条约900bp的片段。
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.
1.从新鲜韭菜的根中分离到一株对多种蔬菜病原真菌有抑制作用的优势内生细菌W7,其拮抗机制与已报道的其它生防细菌不同,其胞外代谢物对病原真菌无抑制作用,而菌体经超声波破碎及有机溶剂沉淀得到的菌体多糖粗提液可明显抑制病原菌菌丝生长。而且,该菌能以100mg/L高效氯氰菊酯为唯一碳源生长,7d的降解率为51.3%,但不能降解毒死蜱。通过对其形态特征、生理生化及16S rDNA同源性序列分析,将其鉴定为类芽孢杆菌(Paenibacillus spp.)。
2.从受农药污染的韭菜根际土壤中获得了一株高效毒死蜱降解细菌D10,该菌能以毒死蜱为唯一碳源生长,6d内对100mg/L的毒死蜱降解率达59.7%。通过形态特征、生理生化及16S rDNA同源性序列分析,鉴定该菌为不动杆菌(Acinetobacter.spp)。
3.为进一步研究毒死蜱降解菌D10的降解特性,测定了外加碳源浓度、pH、温度、接种量及毒死蜱浓度对菌株降解率和生长量的影响。结果表明,当pH为8.0,温度为30～35℃,接种量在5%,毒死蜱浓度为200mg/L以下时,菌株D10的降解效果较好;在pH为8.0以上,外加碳源1%以上,温度为35℃,接种量10%,毒死蜱浓度为200mg/L以下时,菌株D10的生长量最大。
4.采用超声波破碎、有机溶剂沉淀等方法从菌株D10中提取粗酶液,用于研究毒死蜱降解酶在细胞中的定位和性质。结果表明:毒死蜱降解酶主要位于胞内。该酶对毒死蜱的酶促降解最适pH为8.0,在碱性条件下相对稳定;最适温度为30℃,且具有较好的热稳定性。
5.实验室模拟受污染土壤,研究菌株D10在土壤中的实际降解效果。结果表明,在添加了菌株D10的未灭菌土壤中,20d降解率达82.5%,而未灭菌土壤的自然降解率仅有26.7%,表明在土壤中添加菌株D10可明显降低土壤中的毒死蜱残留。
6.由于韭菜优势内生细菌W7不能降解毒死蜱,为使其今后能通过转基因的方式获得毒死蜱降解能力,对菌株D10的毒死蜱降解酶基因进行初步研究。目前,已报道的有机磷降解酶基因位于质粒或者基因组DNA上。本研究对菌株D10多次进行质粒提取,电泳检测无质粒条带,从而可以判断菌株降解毒死蜱的相关基因位于基因组DNA上。根据已报道的甲基对硫磷降解酶(mpd)基因的编码区设计引物,PCR得到一条约900bp的片段。
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.
引文
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