氟磺胺草醚对土壤微生物多样性的影响
摘要
氟磺胺草醚主要用于防除大豆田阔叶杂草。然而，氟磺胺草醚在土壤中在土壤中半衰期较长，易对后茬作物产生影响。除草剂大量残留易导致土壤质量的下降。土壤微生物的变化可以直接反映土壤生态系统的健康状况。本研究中，通过室内培养和田间调查相结合的方式，采用土壤生态环境评价的微生物指标体系，从微生物呼吸作用、微生物量碳、群落结构和功能多样性等阐述氟磺胺草醚影响土壤生态环境的微生物学机制。主要研究结果如下：
1、利用QuEChERS提取结合超高液相色谱-串联四级杆质谱法（UPLC-MS/MS），检测氟磺胺草醚在土壤中残留量。样品经乙腈（含0.5%甲酸）提取，N-丙基乙二胺（PSA）净化，UPLC-MS/MS检测。在5min内实现氟磺胺草醚的检测。在0.05mg kg-1～5mg kg-1范围内，此方法显示了良好的线性（r=0.9945)。在0.25mg kg-1、10mg kg-1、100mg kg-1三个水平上的平均回收率为91.5～100.3%，变异系数为5.9～11.7%。
2、室内培养实验，选择2种土壤（粉砂质壤土和黑土），添加3种浓度的氟磺胺草醚（推荐剂量、10倍推荐剂量、100推荐剂量），培养箱中恒温培养,样品采集时间7天、15天、30天、60天和90天。实验结果表明，培养过程中，氟磺胺草醚降解呈现出先快后慢的趋势。在2种土壤中，高浓度氟磺胺草醚的施用（375mg kg-1；T100），显著性抑制了土壤微生物总体活性，土壤基础呼吸、诱导呼吸、微生物量碳及土壤总菌PLFA，而且抑制了土壤总细菌，总真菌，革兰氏阴性菌（GN）和革兰氏阳性菌（GP）。同时，氟磺胺草醚显著改变了2种土壤的GN/GP和bacteria/fungi（B/F），显著提高了2种土壤的压力指数。然而，高浓度氟磺胺草醚的处理却增加了土壤表征细菌的氧化代谢的能力。主成分分析（PCA）结果分析表明，氟磺胺草醚尤其是高浓度（375mg kg-1；T100）下，严重改变了土壤微生物的群落结构和功能多样性(Shannon, McIntoshand Simpson)，而且整个培养时间内，土壤微生物的群落结构和功能多样性未恢复。而且，高浓度氟磺胺草醚处理（37.5mg kg-1；375mg kg-1），显著抑制2种土壤固氮菌nifH的基因丰度。且375mg kg-1处理下，固氮菌nifH的基因丰度一直被被抑制。
3、田间调查实验，样品采自连续施用氟磺胺草醚的东北大豆连作区。氟磺胺草醚施药年限为3年、5年和10年，推荐使用量是375g a.i. hm-2。随着氟磺胺草醚施药年限的增加，微生物量碳和总PLFA显著下降。施药历史10年的土壤中，GN、GN/GP和真菌显著增加；GP和bacteria/fungi（B/F）显著低于对照。PCA分析结果表明即使氟磺胺草醚推荐剂量下，随着氟磺胺草醚施用年限的增加，同样改变了土壤微生物的群落结构。然而，氟磺胺草醚不同施药历史的土壤中，微生物功能多样性没有改变。
Fomesafen, as selective post-emergence herbicide, are used agriculturally for broad-leaved weedscontrol in soybean field in China. However, fomesafen degrades slowly in soils and has been linked tocrop damage. The long-term pesticide residues lead to decline of the quality of soil. The changes ofmicrobial diversity directly reflect the health of the soil ecosystem. In this work, I focus on cultureexperiments, field investigation of contaminated soil and using microbial indicators of system of soilhealth evaluation, and the project clarify the microbiology mechanism of long residual herbicidefomesafen on soil health from the intensity of soil respiration, soil microbes microbial biomass carbon,community structure and functional diversity. The results are as follows:
1: An effective and rapid analytical method for the determination of fomesafen residues in soilwas developed by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS)using the modified quick, easy, cheap, effective, rugged, and safe （QuEChERS） extraction concept.The fomesafen was extracted from soil matrices with0.5%(V/V) formic acid aqueous solution andacetonitrile then cleaned by dispersive solid phase extraction (dSPE) with PSA. The determination ofthe fomesafen was achieved within a5minute run time by using an UPLC C18column connected to anelectrospray ionization source (ESI, negative ion mode). The method showed excellent linearity (r=0.9945). The mean recoveries from soil matrices ranged from91.5to100.3%, with intra-day relativestandard deviations (RSDr) in the range of5.9～11.7%. The proposed method is a useful tool in routinefomesafen residue monitoring in soil.
2: Culture experiments was conducted to evaluate the impact of different concentrations offomesafen on [1-fold,10-fold, and100-fold of the recommended field rate (T1, T10, T100)] on soilmicrobial activity, the structure of the soil microbial community, and functional diversity of the soilmicrobial community using the soil microbial respiration, microbial biomass, phospholipid fatty acids(PLFAs), community level physiological profiles (CLPPs), and real-time PCR in two different soils.Soil sampling was performed after7,15,30,60, and90days of application to determine the fomesafenconcentration. Fomesafen degradation appeared to consist of a rapid initial phase and a slower or morestagnant second phase. Our results indicate that applying100times the recommended dose offomesafen (T100) adversely affects soil microbial activity and stresses soil microbial communities asreflected by the reduced respiratory quotient (qCO2, QR). The PLFA analysis showed that high levels offomesafen treatment (T100) decreased the total amount of PLFAs and both bacterial (bothGram-positive (GP) bacteria and Gram-negative (GN) bacteria) and fungal biomass but increased themicrobial stress level. However, the BIOLOG results are not consistent with our other results. Theaddition of fomesafen significantly increased the average well color development, substrate utilization,and the functional diversity index (Shannon, McIntosh and Simpson). Additionally, the abundance ofthe nifH (N2-fixing bacteria) gene was reduced in the presence of high concentrations of fomesafen (T100). Taken together, these results suggest that the addition of fomesafen can alter the microbialcommunity structure and functional diversity of the soil, and these parameters do not recover even aftera90-day incubation period.
3: Fomesafen has been widely used for the soybean production of China, but less information isavailable on the possible risk of long-term application of fomesafen. Soil samples were collected fromthe plots having been received375g active component of fomesafen/ha per year for3,5and10yearsin a continuously cropped soybean field of Northeast China with their microbial biomass, the structureof the soil microbial community, and functional diversity of the soil microbial community analyzed byphospholipid fatty acids (PLFAs) and community level physiological profiles (CLPPs). The microbialbiomass and total PLFA were suppressed with the increasing year of fomesafen application. Analysis ofthe PLFA profiles revealed that Gram-negative (GN), the GN/GP ratio and as well as total fungi in soilwith3,5and10year history of fomesafen application were much higher than blank soil. However, thebacterial biomass, Gram-positive (GP) bacteria and bacteria/fungi (B/F) had a significant decrease in theplot having been received fomesafen for10years. A principal component analysis (PCA) of the PLFAsrevealed that fomesafen treatment significantly shifted the microbial community structure. However,regarding AWCD, Shannon, McIntosh and Simpson, no clear differences were observed betweenfomesafen treatments and controls.
1、利用QuEChERS提取结合超高液相色谱-串联四级杆质谱法（UPLC-MS/MS），检测氟磺胺草醚在土壤中残留量。样品经乙腈（含0.5%甲酸）提取，N-丙基乙二胺（PSA）净化，UPLC-MS/MS检测。在5min内实现氟磺胺草醚的检测。在0.05mg kg-1～5mg kg-1范围内，此方法显示了良好的线性（r=0.9945)。在0.25mg kg-1、10mg kg-1、100mg kg-1三个水平上的平均回收率为91.5～100.3%，变异系数为5.9～11.7%。
2、室内培养实验，选择2种土壤（粉砂质壤土和黑土），添加3种浓度的氟磺胺草醚（推荐剂量、10倍推荐剂量、100推荐剂量），培养箱中恒温培养,样品采集时间7天、15天、30天、60天和90天。实验结果表明，培养过程中，氟磺胺草醚降解呈现出先快后慢的趋势。在2种土壤中，高浓度氟磺胺草醚的施用（375mg kg-1；T100），显著性抑制了土壤微生物总体活性，土壤基础呼吸、诱导呼吸、微生物量碳及土壤总菌PLFA，而且抑制了土壤总细菌，总真菌，革兰氏阴性菌（GN）和革兰氏阳性菌（GP）。同时，氟磺胺草醚显著改变了2种土壤的GN/GP和bacteria/fungi（B/F），显著提高了2种土壤的压力指数。然而，高浓度氟磺胺草醚的处理却增加了土壤表征细菌的氧化代谢的能力。主成分分析（PCA）结果分析表明，氟磺胺草醚尤其是高浓度（375mg kg-1；T100）下，严重改变了土壤微生物的群落结构和功能多样性(Shannon, McIntoshand Simpson)，而且整个培养时间内，土壤微生物的群落结构和功能多样性未恢复。而且，高浓度氟磺胺草醚处理（37.5mg kg-1；375mg kg-1），显著抑制2种土壤固氮菌nifH的基因丰度。且375mg kg-1处理下，固氮菌nifH的基因丰度一直被被抑制。
3、田间调查实验，样品采自连续施用氟磺胺草醚的东北大豆连作区。氟磺胺草醚施药年限为3年、5年和10年，推荐使用量是375g a.i. hm-2。随着氟磺胺草醚施药年限的增加，微生物量碳和总PLFA显著下降。施药历史10年的土壤中，GN、GN/GP和真菌显著增加；GP和bacteria/fungi（B/F）显著低于对照。PCA分析结果表明即使氟磺胺草醚推荐剂量下，随着氟磺胺草醚施用年限的增加，同样改变了土壤微生物的群落结构。然而，氟磺胺草醚不同施药历史的土壤中，微生物功能多样性没有改变。
Fomesafen, as selective post-emergence herbicide, are used agriculturally for broad-leaved weedscontrol in soybean field in China. However, fomesafen degrades slowly in soils and has been linked tocrop damage. The long-term pesticide residues lead to decline of the quality of soil. The changes ofmicrobial diversity directly reflect the health of the soil ecosystem. In this work, I focus on cultureexperiments, field investigation of contaminated soil and using microbial indicators of system of soilhealth evaluation, and the project clarify the microbiology mechanism of long residual herbicidefomesafen on soil health from the intensity of soil respiration, soil microbes microbial biomass carbon,community structure and functional diversity. The results are as follows:
1: An effective and rapid analytical method for the determination of fomesafen residues in soilwas developed by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS)using the modified quick, easy, cheap, effective, rugged, and safe （QuEChERS） extraction concept.The fomesafen was extracted from soil matrices with0.5%(V/V) formic acid aqueous solution andacetonitrile then cleaned by dispersive solid phase extraction (dSPE) with PSA. The determination ofthe fomesafen was achieved within a5minute run time by using an UPLC C18column connected to anelectrospray ionization source (ESI, negative ion mode). The method showed excellent linearity (r=0.9945). The mean recoveries from soil matrices ranged from91.5to100.3%, with intra-day relativestandard deviations (RSDr) in the range of5.9～11.7%. The proposed method is a useful tool in routinefomesafen residue monitoring in soil.
2: Culture experiments was conducted to evaluate the impact of different concentrations offomesafen on [1-fold,10-fold, and100-fold of the recommended field rate (T1, T10, T100)] on soilmicrobial activity, the structure of the soil microbial community, and functional diversity of the soilmicrobial community using the soil microbial respiration, microbial biomass, phospholipid fatty acids(PLFAs), community level physiological profiles (CLPPs), and real-time PCR in two different soils.Soil sampling was performed after7,15,30,60, and90days of application to determine the fomesafenconcentration. Fomesafen degradation appeared to consist of a rapid initial phase and a slower or morestagnant second phase. Our results indicate that applying100times the recommended dose offomesafen (T100) adversely affects soil microbial activity and stresses soil microbial communities asreflected by the reduced respiratory quotient (qCO2, QR). The PLFA analysis showed that high levels offomesafen treatment (T100) decreased the total amount of PLFAs and both bacterial (bothGram-positive (GP) bacteria and Gram-negative (GN) bacteria) and fungal biomass but increased themicrobial stress level. However, the BIOLOG results are not consistent with our other results. Theaddition of fomesafen significantly increased the average well color development, substrate utilization,and the functional diversity index (Shannon, McIntosh and Simpson). Additionally, the abundance ofthe nifH (N2-fixing bacteria) gene was reduced in the presence of high concentrations of fomesafen (T100). Taken together, these results suggest that the addition of fomesafen can alter the microbialcommunity structure and functional diversity of the soil, and these parameters do not recover even aftera90-day incubation period.
3: Fomesafen has been widely used for the soybean production of China, but less information isavailable on the possible risk of long-term application of fomesafen. Soil samples were collected fromthe plots having been received375g active component of fomesafen/ha per year for3,5and10yearsin a continuously cropped soybean field of Northeast China with their microbial biomass, the structureof the soil microbial community, and functional diversity of the soil microbial community analyzed byphospholipid fatty acids (PLFAs) and community level physiological profiles (CLPPs). The microbialbiomass and total PLFA were suppressed with the increasing year of fomesafen application. Analysis ofthe PLFA profiles revealed that Gram-negative (GN), the GN/GP ratio and as well as total fungi in soilwith3,5and10year history of fomesafen application were much higher than blank soil. However, thebacterial biomass, Gram-positive (GP) bacteria and bacteria/fungi (B/F) had a significant decrease in theplot having been received fomesafen for10years. A principal component analysis (PCA) of the PLFAsrevealed that fomesafen treatment significantly shifted the microbial community structure. However,regarding AWCD, Shannon, McIntosh and Simpson, no clear differences were observed betweenfomesafen treatments and controls.
引文
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