土壤微生物对手性氯代乙酰胺类除草剂的对映体选择性降解机制

Chloroacetanilide herbicides are one of the widest used chiral herbicides in agriculture. To effectively control weeds, a major-use of chloroacetanilide herbicides exists in China, which have achieved high detection rates and may led to a potentially carcinogenic risk. As the soil managed agent, the effects of chloroacetanilide herbicides on soil microorganisms and how to remove the herbicide residues have increasingly become a research focus in recent years. Based on our previous work, the purpose of the present study is to (1) ascertain the enantioselective degradation and accumulation of chiral chloroacetanilide herbicides after repeated applications, (2) define the enantioselective ecological effects of chiral chloroacetanilide herbicides, and (3) to further clarify the enantioselective degradation mechanism of chiral chloroacetanilide herbicides by soil microorganisms. Lab experiments will be carried out to assess: (1) the enantiomer residues of chloroacetanilide herbicides by high-performance liquid chromatography−tandem mass spectrometry (HPLC− MS/MS), (2) soil bacterial and fungal community structure, (3) the diversity and abundance of chloroacetanilide herbicide-degrading genes in soil by Illumina high-throughput sequencing, and (4) isolation, whole-genome sequencing by PacBio sequencing and functional annotation of chloroacetanilide herbicide-degrading microorganisms. The results will be important to reveal the inherent mechanism for degradation of chiral chloroacetanilide herbicides in soil, but also provide fundamental information about the ecological risk assessment of chiral chloroacetanilide herbicides in long-term contaminated soil .

氯代乙酰胺类除草剂是应用最广泛的手性除草剂种类。农业生产中使用量大，检出率高，具有潜在的致癌风险。作为土壤处理剂，氯代乙酰胺类除草剂对土壤微生物的生态效应及其残留去除日益受到人们的关注。本项目在前期研究基础上，研究重复处理土壤中手性氯代乙酰胺类除草剂不同对映体的残留变化，探明对映体在土壤中的选择性降解与累积性；分析不同对映体重复处理土壤中的细菌、真菌群落结构变化，明确手性氯代乙酰胺类除草剂的对映体选择性生态效应；以Illumina高通量测序法分析不同对映体重复处理土壤中的降解基因多样性和相对丰度变化，结合降解菌的分离鉴定、全基因组测序和功能注释，阐明土壤微生物对手性氯代乙酰胺类除草剂的对映体选择性降解机制。研究结果对于揭示土壤中手性氯代乙酰胺类除草剂降解的内在机制具有重要意义，也为手性氯代乙酰胺类除草剂长期污染土壤的生态风险评价提供基础数据。

在现代农业种植中，手性除草剂的长期大量使用对土壤造成严重污染。因此，手性除草剂在长期污染土壤中的选择性降解特征以及参与选择性降解的功能微生物菌群和降解基因成为当前的研究热点。本研究以典型氯代乙酰胺类除草剂乙草胺和精异丙甲草胺为目标化合物，首先对其对映体进行了拆分与鉴定，并建立了残留分析方法。采用对映体重复处理土壤的策略，研究了乙草胺和精异丙甲草胺对映体在重复处理土壤中的选择性降解特征，并挖掘了相关的土著降解微生物菌群和降解基因。结果显示，乙草胺和精异丙甲草胺对映体在土壤中的降解均较为缓慢，S-对映体的降解明显快于R-对映体。通过宏基因组测序方法，发现了与乙草胺和精异丙甲草胺对映体降解明显相关的8个细菌属Amycolatopsis、Saccharomonospora、 Mycoplasma、Myroides、Mycobacterium、Burkholderia、Afipia和Kribbella。其中Amycolatopsis、Saccharomonospora和Kribbella与乙草胺S-对映体的优先降解相关，而精异丙甲草胺S-对映体的优先降解主要依赖于Amycolatopsis和Saccharomonospora。通过宏基因组测序和荧光定量PCR的方法，进一步发现了ppah、alkb、benA和 p450是参与乙草胺和精异丙甲草胺S-对映体优先降解的主要降解基因。网络分析结果显示，细菌属Amycolatopsis、Saccharomonospora、Mycoplasma、Myroides和Mycobacterium是上述4种降解基因潜在的寄主。综上所述，细菌属Amycolatopsis和Saccharomonospora在乙草胺和精异丙甲草胺S-对映体的优先降解中发挥着重要作用。本项目在对映体水平上，揭示了乙草胺和精异丙甲草胺在长期污染土壤中的选择性降解特征以及潜在的微生物选择性降解机制，对长期受乙草胺和精异丙甲草胺污染农田土壤的微生物修复提供了基础研究数据。