杀菌剂多菌灵高频投入对土壤微生物群落的影响及其生物修复
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摘要
多菌灵作为一种高效低毒的广谱内吸性杀菌剂，在我国大棚蔬菜以及中药材生产中频繁施用，对环境造成一定程度污染。为评价多菌灵施用的安全性及其对土壤质量的影响，本文研究了多菌灵重复施药对土壤微生物群落功能和结构的影响以及在多菌灵胁迫下土壤微生物功能的抗性和结构稳定性。在此基础上，进行了多菌灵高效降解菌的分离、土壤中多菌灵降解的生物强化、生物强化对土壤微生物群落功能以及结构的影响等方面的研究。主要研究结果如下：
1．以浙江大学华家池校区蔬菜田土为材料，采用BIOLOG方法系统研究了多菌灵重复施用对土壤微生物群落功能多样性的影响以及多菌灵胁迫下土壤微生物功能的抗性。研究结果表明，多菌灵使用初期，对土壤微生物群落多样性会产生抑制作用，Simpson指数和McIntosh指数显著降低，说明优势种群和物种均一性受到一定程度的影响。随着施药频率的增加，多菌灵对土壤微生物群落多样性的抑制作用逐步减弱。在第四次喷药后15 d，0.94和1.88 kg a．i．ha~(-1)多菌灵处理土壤微生物多样性已经超过对照水平，而4.70kg a．i．ha~(-1)多菌灵处理土壤微生物多样性与对照没有显著性差异。
AWCD值、Simpson指数、McIntosh指数和Shannon指数的变化能够很好的表征多菌灵胁迫下土壤微生物的功能抗性。AWCD值和Simpson指数的表征效果最好。在田间第二次施药后土壤微生物对多菌灵的胁迫已经具有一定的抗性。在第三次施药后，土壤微生物功能对多菌灵的抗性完全形成，后续投入的多菌灵对土壤微生物功能基本不产生负面影响。
2．在BIOLOG研究基础上，采用温度梯度凝胶电泳(TGGE)方法，对多菌灵重复施用后土壤细菌群落结构变化及其结构稳定性进行了系统的研究。研究发现，单次施药对微生物群落结构不会产生明显影响，而重复施药对土壤微生物群落结构会产生一定程度的影响，并且随着施药频率、施药浓度的增加，影响越来越明显。在第二次施药后，处理土壤样品中出现了一条对照土壤中没有的新增条带，说明土壤中存在能够在多菌灵浓度下生长良好的微生物种群，经序列测定及与GenBank中核酸数据进行网上比对分析，与一株uncultured gamma proteobacterium clone，CRE-PA17具有98％的相似率，初步确定为一不可培养细菌种。
在第二次喷药后，土壤微生物群落结构稳定性较差，容易受外界胁迫的影响而发生改变。随着施药频率的增加，结构稳定性又有所增强。在第三次和第四次喷药后，土壤微生物群落结构均比较稳定，但该结构与对照不同。经过1年后，处理土壤微生物群落结构能够恢复到对照水平。
土壤微生物群落结构稳定性与多菌灵施药频率关系的结果证实多菌灵施药次数的增加会改变土壤微生物结构，而这一变化并未准确、及时反映在土壤整体功能上(BIOLOG)。因此，分子生物学方法的应用有助于更加深入解析农药对生态系统的影响。
3．从浙江大学华家池校区土壤筛选到能够以多菌灵作为唯一碳源和能源进行生长的混合菌。该混合菌主要由Bacillus subtilis、Paracoccus sp．、Flavobacterium omnivorum和Pseudomonas sp．四个优势菌组成。培养1 d后，1、10、100 mg l~(-1)多菌灵的降解率分别为64.25％、20.59％和17.47％。该混合菌在不同pH值和温度下对多菌灵的降解效果为pH 9.0＞pH 7.0＞pH 5.0，35℃＞25℃＞15℃。
在本地的气候条件下，可以通过生物强化的手段治理生态系统中的多菌灵残留污染。多菌灵施用后11 d，在喷菌一次、两次和三次土壤中的降解速率分别为0.16、0.20和0.23 mgd~(-1) kg~(-1)，比对照土壤快1.07、1.33和1.53倍。随着时间的推移，接种降解菌土壤中多菌灵的降解速率逐渐降低，在多菌灵施用后18 d，其降解速率接近于对照土壤中多菌灵的降解速率。
4．采用BIOLOG方法监测土壤中多菌灵降解生物强化过程中微生物群落功能多样性的变化。研究发现，在未施用多菌灵的情况下，接种降解菌对土壤微生物多样性不会产生明显影响；而多菌灵施用后，降解菌的添加能缓解多菌灵对土壤微生物抑制影响的负面效应。多菌灵施用后11 d，接种降解菌，尤其是接种3次降解菌的土壤微生物整体活性、Simpson指数显著高于对照土壤，而Shannon指数与对照相差不大。多菌灵施用后18 d，降解菌处理土壤微生物多样性逐渐恢复到对照水平。
5．采用PCR-TGGE方法对生物强化过程中降解菌以及土著微生物的变化动态进行了研究。研究发现，接种降解菌对未施用多菌灵的土壤微生物群落结构不会产生明显影响。施用多菌灵后11d，接种降解菌的土壤微生物群落结构受到明显影响。随着时间的推移，接种降解菌土壤的微生物群落结构得到一定程度的恢复，尤其是接种一次的土壤微生物群落结构在多菌灵施用后18 d恢复到对照水平。
Carbendazim is a broad-spectrum fungicide widely used for the control of plant fungal diseases on arable crops, fruits, vegetables and ornamentals which results in potential environmental contamination. To evaluate the safety of carbendazim applications, studies were conducted concerning its influences on soil microbial functional diversity and genetic structure. The soil functional resilience and structural stability exposed to carbendazim were also investigated. One bacterial consortium capable of degrading carbendazim was isolated. The bioaugmentation of carbendazim in soil by this bacterial consortium, its effect on microbial functional diversity and genetic structure were further studied. The results were summarized as follows:
1. Microbial functional diversity and functional resilience in soil after repeated carbendazim applications were estimated with BIOLOG Ecoplates. An obvious inhibitory effect was observed in microbial utilization of carbon substrates and diversity after the first introduction of carbendazim. With subsequent carbendazim applications, this effect disappeared gradually. Soil bacterial diversity in all treatments recovered to the control level 15 d after the fourth applications. AWCD and diversity index, such as Simpson, McIntosh and Shannon indexes can be used to indicate the soil functional resilience to carbendazim. The best results can be obtained by using AWCD and Simpson index to indicate the functional resilience. Soil functional resilience to carbendazim appeared after the second application and formed completely after the third application.
2. The effects of repeated applications of carbendazim on bacterial community structure as well as the structural stability exposed to carbendazim was studied under field conditions using TGGE and partial sequence analysis of PCR-amplified 16S rRNA genes. The results showed that bacterial community structure was not changed after the first carbendazim application, but gradually altered after the second application. With the application frequency and carbendazim concentration increased, the effect on bacterial community structure became much more obvious. At the same time, an application-responsive band from the carbendazim treatments was sequenced and aligned. The sequence of this band was related to members of theγsubdivision, showing 98% similarity with an uncultured gamma proteobacterium clone, CRE-PA17.
Soil microbial structural stability was weak after the second carbendazim application and became stronger after the third and fourth applications. However, the microbial community structure in the carbendazim-treated soil was different from that in the control soil after the third and fourth applications. After one year, the bacterial community in carbendazim-treated soil recovered to that in the control.
3. A bacterial consortium capable of utilizing carbendazim as sole carbon and energy sources was isolated from Huajiachi campus, Zhejiang University, Hangzhou, China. This bacterial consortium was mainly composed of Bacillus subtilis、Paracoccus sp.、Flavobacterium omnivorum and Pseudomonas sp. The ability of the bacterial consortium to degrade carbendazim in pure cultures depends on pesticide concentration, pH and temperature. The degradation of carbendazim at concentrations of 1, 10 and 100 mg l~(-1) within 1 d by this consortium were measured to be 64.25%、20.59% and 17.47%, respectively. The degradation rates of carbendazim were affected by pH and temperature following an order of pH 9.0 > pH 7.0 > pH 5.0 and 35℃> 25℃> 15℃, respectively.
Degradation of carbendazim in soil was significantly accelerated by the inoculation with the carbendazim-degrading bacterial consortium. Compared with the uninoculated soil, the degradation rates of carbendazim in the soil inoculated one, two and three times 11d after the carbendazim application were accelerated by 1.07, 1.33 and 1.53 times, respectively.
4. The effect of repeatedly inoculating a soil with the carbendazim-degrading bacterial consortium on the soil microbial functional diversity was studied by BIOLOG method. The results showed that soil microbial functional diversity was not affected by the inoculation with the carbendazim-degrading bacterial consortium before carbendazim application, but influenced after carbendazim application. Microbial utilization of carbon substrates and Simpson index were stimulated in inoculated soil, especially in the soil inoculated three times with the carbendazim-degrading bacterial consortium 11d after carbendazim application. After initial variations, soil microbial diversity recovered to similar levels of the controls 18 d after carbendazim application.
5. TGGE method was applied to determine the relative genetic complexity of microbial communities in uninoculated and inoculated soils. The result showed that soil microbial community structure was not changed by the inoculation with the carbendazim-degrading bacterial consortium before carbendazim application, but was altered 11 d after carbendazim application. Microbial community structure of inoculated soil, especially the soil inoculated only one time recoverd to the control level 18 d after carbendazim application.
1．以浙江大学华家池校区蔬菜田土为材料，采用BIOLOG方法系统研究了多菌灵重复施用对土壤微生物群落功能多样性的影响以及多菌灵胁迫下土壤微生物功能的抗性。研究结果表明，多菌灵使用初期，对土壤微生物群落多样性会产生抑制作用，Simpson指数和McIntosh指数显著降低，说明优势种群和物种均一性受到一定程度的影响。随着施药频率的增加，多菌灵对土壤微生物群落多样性的抑制作用逐步减弱。在第四次喷药后15 d，0.94和1.88 kg a．i．ha~(-1)多菌灵处理土壤微生物多样性已经超过对照水平，而4.70kg a．i．ha~(-1)多菌灵处理土壤微生物多样性与对照没有显著性差异。
AWCD值、Simpson指数、McIntosh指数和Shannon指数的变化能够很好的表征多菌灵胁迫下土壤微生物的功能抗性。AWCD值和Simpson指数的表征效果最好。在田间第二次施药后土壤微生物对多菌灵的胁迫已经具有一定的抗性。在第三次施药后，土壤微生物功能对多菌灵的抗性完全形成，后续投入的多菌灵对土壤微生物功能基本不产生负面影响。
2．在BIOLOG研究基础上，采用温度梯度凝胶电泳(TGGE)方法，对多菌灵重复施用后土壤细菌群落结构变化及其结构稳定性进行了系统的研究。研究发现，单次施药对微生物群落结构不会产生明显影响，而重复施药对土壤微生物群落结构会产生一定程度的影响，并且随着施药频率、施药浓度的增加，影响越来越明显。在第二次施药后，处理土壤样品中出现了一条对照土壤中没有的新增条带，说明土壤中存在能够在多菌灵浓度下生长良好的微生物种群，经序列测定及与GenBank中核酸数据进行网上比对分析，与一株uncultured gamma proteobacterium clone，CRE-PA17具有98％的相似率，初步确定为一不可培养细菌种。
在第二次喷药后，土壤微生物群落结构稳定性较差，容易受外界胁迫的影响而发生改变。随着施药频率的增加，结构稳定性又有所增强。在第三次和第四次喷药后，土壤微生物群落结构均比较稳定，但该结构与对照不同。经过1年后，处理土壤微生物群落结构能够恢复到对照水平。
土壤微生物群落结构稳定性与多菌灵施药频率关系的结果证实多菌灵施药次数的增加会改变土壤微生物结构，而这一变化并未准确、及时反映在土壤整体功能上(BIOLOG)。因此，分子生物学方法的应用有助于更加深入解析农药对生态系统的影响。
3．从浙江大学华家池校区土壤筛选到能够以多菌灵作为唯一碳源和能源进行生长的混合菌。该混合菌主要由Bacillus subtilis、Paracoccus sp．、Flavobacterium omnivorum和Pseudomonas sp．四个优势菌组成。培养1 d后，1、10、100 mg l~(-1)多菌灵的降解率分别为64.25％、20.59％和17.47％。该混合菌在不同pH值和温度下对多菌灵的降解效果为pH 9.0＞pH 7.0＞pH 5.0，35℃＞25℃＞15℃。
在本地的气候条件下，可以通过生物强化的手段治理生态系统中的多菌灵残留污染。多菌灵施用后11 d，在喷菌一次、两次和三次土壤中的降解速率分别为0.16、0.20和0.23 mgd~(-1) kg~(-1)，比对照土壤快1.07、1.33和1.53倍。随着时间的推移，接种降解菌土壤中多菌灵的降解速率逐渐降低，在多菌灵施用后18 d，其降解速率接近于对照土壤中多菌灵的降解速率。
4．采用BIOLOG方法监测土壤中多菌灵降解生物强化过程中微生物群落功能多样性的变化。研究发现，在未施用多菌灵的情况下，接种降解菌对土壤微生物多样性不会产生明显影响；而多菌灵施用后，降解菌的添加能缓解多菌灵对土壤微生物抑制影响的负面效应。多菌灵施用后11 d，接种降解菌，尤其是接种3次降解菌的土壤微生物整体活性、Simpson指数显著高于对照土壤，而Shannon指数与对照相差不大。多菌灵施用后18 d，降解菌处理土壤微生物多样性逐渐恢复到对照水平。
5．采用PCR-TGGE方法对生物强化过程中降解菌以及土著微生物的变化动态进行了研究。研究发现，接种降解菌对未施用多菌灵的土壤微生物群落结构不会产生明显影响。施用多菌灵后11d，接种降解菌的土壤微生物群落结构受到明显影响。随着时间的推移，接种降解菌土壤的微生物群落结构得到一定程度的恢复，尤其是接种一次的土壤微生物群落结构在多菌灵施用后18 d恢复到对照水平。
Carbendazim is a broad-spectrum fungicide widely used for the control of plant fungal diseases on arable crops, fruits, vegetables and ornamentals which results in potential environmental contamination. To evaluate the safety of carbendazim applications, studies were conducted concerning its influences on soil microbial functional diversity and genetic structure. The soil functional resilience and structural stability exposed to carbendazim were also investigated. One bacterial consortium capable of degrading carbendazim was isolated. The bioaugmentation of carbendazim in soil by this bacterial consortium, its effect on microbial functional diversity and genetic structure were further studied. The results were summarized as follows:
1. Microbial functional diversity and functional resilience in soil after repeated carbendazim applications were estimated with BIOLOG Ecoplates. An obvious inhibitory effect was observed in microbial utilization of carbon substrates and diversity after the first introduction of carbendazim. With subsequent carbendazim applications, this effect disappeared gradually. Soil bacterial diversity in all treatments recovered to the control level 15 d after the fourth applications. AWCD and diversity index, such as Simpson, McIntosh and Shannon indexes can be used to indicate the soil functional resilience to carbendazim. The best results can be obtained by using AWCD and Simpson index to indicate the functional resilience. Soil functional resilience to carbendazim appeared after the second application and formed completely after the third application.
2. The effects of repeated applications of carbendazim on bacterial community structure as well as the structural stability exposed to carbendazim was studied under field conditions using TGGE and partial sequence analysis of PCR-amplified 16S rRNA genes. The results showed that bacterial community structure was not changed after the first carbendazim application, but gradually altered after the second application. With the application frequency and carbendazim concentration increased, the effect on bacterial community structure became much more obvious. At the same time, an application-responsive band from the carbendazim treatments was sequenced and aligned. The sequence of this band was related to members of theγsubdivision, showing 98% similarity with an uncultured gamma proteobacterium clone, CRE-PA17.
Soil microbial structural stability was weak after the second carbendazim application and became stronger after the third and fourth applications. However, the microbial community structure in the carbendazim-treated soil was different from that in the control soil after the third and fourth applications. After one year, the bacterial community in carbendazim-treated soil recovered to that in the control.
3. A bacterial consortium capable of utilizing carbendazim as sole carbon and energy sources was isolated from Huajiachi campus, Zhejiang University, Hangzhou, China. This bacterial consortium was mainly composed of Bacillus subtilis、Paracoccus sp.、Flavobacterium omnivorum and Pseudomonas sp. The ability of the bacterial consortium to degrade carbendazim in pure cultures depends on pesticide concentration, pH and temperature. The degradation of carbendazim at concentrations of 1, 10 and 100 mg l~(-1) within 1 d by this consortium were measured to be 64.25%、20.59% and 17.47%, respectively. The degradation rates of carbendazim were affected by pH and temperature following an order of pH 9.0 > pH 7.0 > pH 5.0 and 35℃> 25℃> 15℃, respectively.
Degradation of carbendazim in soil was significantly accelerated by the inoculation with the carbendazim-degrading bacterial consortium. Compared with the uninoculated soil, the degradation rates of carbendazim in the soil inoculated one, two and three times 11d after the carbendazim application were accelerated by 1.07, 1.33 and 1.53 times, respectively.
4. The effect of repeatedly inoculating a soil with the carbendazim-degrading bacterial consortium on the soil microbial functional diversity was studied by BIOLOG method. The results showed that soil microbial functional diversity was not affected by the inoculation with the carbendazim-degrading bacterial consortium before carbendazim application, but influenced after carbendazim application. Microbial utilization of carbon substrates and Simpson index were stimulated in inoculated soil, especially in the soil inoculated three times with the carbendazim-degrading bacterial consortium 11d after carbendazim application. After initial variations, soil microbial diversity recovered to similar levels of the controls 18 d after carbendazim application.
5. TGGE method was applied to determine the relative genetic complexity of microbial communities in uninoculated and inoculated soils. The result showed that soil microbial community structure was not changed by the inoculation with the carbendazim-degrading bacterial consortium before carbendazim application, but was altered 11 d after carbendazim application. Microbial community structure of inoculated soil, especially the soil inoculated only one time recoverd to the control level 18 d after carbendazim application.
引文
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