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Journal of Zhejiang University SCIENCE B
ISSN 1673-1581(Print), 1862-1783(Online), Monthly
2014 Vol.15 No.4 P.322-332
Soil bacterial and fungal community successions under the stress of chlorpyrifos application and molecular characterization of chlorpyrifos-degrading isolates using ERIC-PCR
Abstract: Chlorpyrifos is a widely used insecticide in recent years, and it will produce adverse effects on soil when applied on crops or mixed with soil. In this study, nested polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) were combined to explore the bacterial and fungal community successions in soil treated with 5 and 20 mg/kg of chlorpyrifos. Furthermore, isolates capable of efficiently decomposing chlorpyrifos were molecular-typed using enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR). Under the experimental conditions, degradation of chlorpyrifos in soil was interpreted with the first-order kinetics, and the half-lives of chlorpyrifos at 5 and 20 mg/kg doses were calculated to be 8.25 and 8.29 d, respectively. DGGE fingerprint and principal component analysis (PCA) indicated that the composition of the fungal community was obviously changed with the chlorpyrifos treatment, and that samples of chlorpyrifos treatment were significantly separated from those of the control from the beginning to the end. While for the bacterial community, chlorpyrifos-treated soil samples were apparently different in the first 30 d and recovered to a similar level of the control up until 60 d, and the distance in the PCA between the chlorpyrifos-treated samples and the control was getting shorter through time and was finally clustered into one group. Together, our results demonstrated that the application of chlorpyrifos could affect the fungal community structure in a quick and lasting way, while only affecting the bacterial community in a temporary way. Finally, nine typical ERIC types of chlorpyrifos-degrading isolates were screened.
Key words: Denaturing gradient gel electrophoresis (DGGE), Bacterial community, Fungal community, Chlorpyrifos-degrading isolates, Enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR)
创新要点：通过聚合酶链式反应–变性梯度凝胶电泳（PCR-DGGE）方法，揭示了毒死蜱施用后土壤细菌和真菌群落结构动态变化，并对试验土壤中的降解菌群进行分子鉴定。
研究方法：将农田土壤用终质量分数5 mg/kg和20 mg/kg毒死蜱处理，同时以不施用毒死蜱为对照，定期采集土样进行毒死蜱残留测定并提取土壤DNA。将提取的土壤总DNA作为PCR反应模板，细菌群落结构分析采用对大多数细菌16S rRNA基因V3区具有特异性的引物GC-341F和517R，真菌群落结构分析采用ITS区特异引物对GC-ITS1f和ITS2f。DGGE图谱经Quantity One软件进行数字化分析，各泳道中的条带经标准化处理之后，条带的相对光密度构成一个矩阵，用Matlab软件进行主成分分析（PCA），用Dice法计算相似性指数CS，用MEGA 4.1软件构建系统发育树，进行聚类分析。之后，采用LB、察氏和高氏三种培养基结合的纯培养分离方法，从毒死蜱处理土样中分离具有降解功能的菌株。分别提取纯培养物的染色体DNA，利用ERIC-PCR对分离到的降解菌进行基因组指纹图谱分析，将典型肠杆菌基因间的重复共有序列–聚合酶链式反应（ERIC-PCR）指纹图谱类型的代表菌株进行16S rRNA基因（细菌和放线菌）或ITS区（真菌）扩增并测序，测序结果提交GenBank基因库，并进行BLAST比对，初步确定降解菌株的分子地位。
重要结论：本实验条件下，毒死蜱降解符合一级动力学，5 mg/kg和20 mg/kg毒死蜱浓度下的降解方程分别为y=5.252e−0.09x和y=19.41e−0.08x。DGGE指纹图谱显示，毒死蜱施用后土壤真菌群落结构与对照相比发生很大改变，且毒死蜱处理样品真菌群落结构保持基本稳定，多样性指数明显提高（见图2b）；主成分分析显示，毒死蜱处理样品与对照明显分在2个不同的区域，充分表明毒死蜱对土壤真菌群落结构影响迅速且持久（见图3b）。毒死蜱处理样品细菌群落结构只在试验前30天与对照相比明显不同，60天时已经恢复对照水平（见图2a），同时主成分分析图上，毒死蜱处理样品与对照也越来越靠近，最后聚在一起（见图3a），说明毒死蜱对土壤细菌群落结构影响短暂。最后，获得了9个典型的ERIC型毒死蜱降解菌株。
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DOI:
10.1631/jzus.B1300175
CLC number:
Q93-3
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On-line Access:
2014-04-06
Received:
2013-06-27
Revision Accepted:
2014-01-07
Crosschecked:
2014-03-18