二甲四氯植物内生降解菌的筛选、鉴定及降解特性研究

【目的】筛选可降解二甲四氯除草剂的内生真菌，研究内生真菌对二甲四氯的降解特性和途径，为除草剂污染的微生物治理提供理论依据。【方法】采用平板培养法从被二甲四氯严重污染的飞机草中筛选可降解二甲四氯除草剂的内生真菌；采用形态学方法观察内生真菌在培养基上的形态，结合分子生物学方法对内生真菌的ITS、TUB和LUS序列进行克隆和测序，对内生真菌进行鉴定；通过单因素法优化内生真菌在无机盐培养基中对二甲四氯的降解条件（温度、pH和营养源）；并采用液相色谱标准品比对、气相色谱—质谱联用仪（GC-MS）和液相色谱—质谱联用仪（LC-MS）鉴定内生真菌在无机盐培养基中降解二甲四氯的产物。在30℃恒温培养箱中，分别添加内生真菌到不灭菌土壤和灭菌土壤中，同时设不添加内生真菌的土壤为对照组，测定二甲四氯在土壤中的降解速率。【结果】从飞机草中初筛发现1株内生真菌可很好地降解二甲四氯，编号为E68，结合形态学和基因序列分析可将E68鉴定为树状炭角菌（Xylaria arbuscula）。在二甲四氯初始浓度为50.0 mg/L条件下，E68降解二甲四氯的最优条件是pH 5.0、温度28℃和添加0.5%的葡萄糖，7 d后其降解率为97.03%。E68在无机盐培养基中降解二甲四氯的主要产物为4-氯-2-甲基苯。在含有2.5 mg/kg二甲四氯的土壤中添加E68，可明显提高二甲四氯在土壤中的降解率；与不添加E68的土壤相比，在不灭菌和灭菌土壤中分别接入E68后，二甲四氯的降解半衰期分别提高2.8和2.5倍。【结论】从飞机草中分离出的内生真菌E68在无机盐培养基和土壤中可有效降解二甲四氯，具有修复环境中二甲四氯污染的应用潜力。

Screening,identification and characteristics of 2-methyl-4-chlorophenoxyacetic acid-degrading endophytic fungus from plants

【Objective】This experiment aimed to screen and identify 2-methyl-4-chlorophenoxyacetic acid(MCPA) -degrading endophytic fungi from plants and studied the degradation characteristics and pathway of the isolated endophytic fungi to MCPA. The study provided atheoretical basis for the bioremediation of herbicide-contaminated environment.【Method】Used plating method, endophytic fungus for degradation of MCPA were isolated from MCPA-contaminated Eupatorium odoratum. The isolated endophytic fungus was identified through morphological feature observed on the medium, combined with molecular biology method, cloning and sequencing of ITS, TUB and LUS sequences were conducted. The degradation conditions including temperature, pH, nutrition source were also optimized in the mineral salt medium by single-factor test. The degradation product of MPCA by endophytic fungi was identified in the mineral salt medium using high performance liquid chromatography(HPLC), gas chromatography-mass spectrometry(GC-MS) and liquid chromatography-mass spectrometry(LC-MS). In constant temperature incubator(30℃), endophytic fungi were added into nonsterile soil and sterile soil, and soil adding no endophytic fungi was as control group, and degradation rate of MCPA in soil were detected.【Result】An endophytic fungus strain(numbered E68), screened from E. odoratum, could effectively degrade MCPA.The E68 was identified as Xylaria arbuscula according to the analysis of its phenotypic feature and gene sequence. When the initial concentration of MCPA was 50.0 mg/L, the optimum degradation condition of MCPA was pH 5.0, temperature 28℃ and adding 0.5% glucose, the degradation rate was 97.03% after 7 d. The main production of strain E68 degrading MCPA was 4-chloro-2-methylphenol. Adding E68 into soil with 2.5 mg/kg MCPA could increase the degradation rate of MCPA in soil. Compared with soil without E68, the half-lives of MCPA increased 2.8 times in non-sterilized soil adding strain E68 and increased 2.5 times in sterilized soil adding strain E68.【Conclusion】The endophytic fungus E68 isolated from E. odoratum can effectively degrade MCPA in inorganic salt media and soils, and has potential value for the bioremediation of MCPA-contaminated environment.