JZUS - Journal of Zhejiang University SCIENCE

Journal of Zhejiang University SCIENCE B

Accepted manuscript available online (unedited version)

Soil bacterial and fungal community successions under the stress of chlorpyrifos application and molecular characterization of chlorpyrifos-degrading isolates using ERIC-PCR

Author(s): Lie-zhong Chen^1;2, Yan-li Li², Yun-long Yu¹
Affiliation(s): 1. Department of Plant Protection, College of Agriculture & Biotechnology, Zhejiang University, Hangzhou 310029, China;2. State Key Laboratory Breeding Base for Zhejiang Sustainable Pest and Disease Control, Institute of Plant Protection and Microbiology, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China
Corresponding email(s): zwsclz@163.com
Key Words: Denaturing gradient gel electrophoresis (DGGE), Bacterial community, Fungal community, Chlorpyrifos-degrading isolates, Enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR)

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Lie-zhong Chen, Yan-li Li, Yun-long Yu. Soil bacterial and fungal community successions under the stress of chlorpyrifos application and molecular characterization of chlorpyrifos-degrading isolates using ERIC-PCR[J]. Journal of Zhejiang University Science B,in press.Frontiers of Information Technology & Electronic Engineering,in press.https://doi.org/10.1631/jzus.B1300175

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author="Lie-zhong Chen, Yan-li Li, Yun-long Yu",
journal="Journal of Zhejiang University Science B",
year="in press",
publisher="Zhejiang University Press & Springer",
doi="https://doi.org/10.1631/jzus.B1300175"
}

%0 Journal Article
%T Soil bacterial and fungal community successions under the stress of chlorpyrifos application and molecular characterization of chlorpyrifos-degrading isolates using ERIC-PCR
%A Lie-zhong Chen
%A Yan-li Li
%A Yun-long Yu
%J Journal of Zhejiang University SCIENCE B
%P 322-332
%@ 1673-1581
%D in press
%I Zhejiang University Press & Springer
doi="https://doi.org/10.1631/jzus.B1300175"

TY - JOUR
T1 - Soil bacterial and fungal community successions under the stress of chlorpyrifos application and molecular characterization of chlorpyrifos-degrading isolates using ERIC-PCR
A1 - Lie-zhong Chen
A1 - Yan-li Li
A1 - Yun-long Yu
J0 - Journal of Zhejiang University Science B
SP - 322
EP - 332
%@ 1673-1581
Y1 - in press
PB - Zhejiang University Press & Springer
ER -
doi="https://doi.org/10.1631/jzus.B1300175"

Abstract
Chinese Summary
Academic Network
Reviewer Comment

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.

毒死蜱胁迫下土壤细菌和真菌群落演替及其降解菌群的分子鉴定

研究目的：评价毒死蜱施用后对土壤细菌和真菌群落结构的影响，并对降解功能菌株进行分析。
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。

关键词组：变性梯度凝胶电泳（DGGE）；细菌群落结构；真菌群落结构；毒死蜱降解菌群；ERIC-PCR

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References

[1] Anwar,S, Liaquat,F.M, Khan,Q.M, 2009, Biodegradation of chlorpyrifos and its hydrolysis product 3,5,6-trichloro-2-pyridinol by strain C2A1 J Hazard Mater, 168(1):400-405.

[2] Borges,L.G.D, Dalla,V.V, Corao,G, 2003, Characterization and genetic diversity via REP-PCR of isolates from polluted waters in southern Brazil FEMS Microb Ecol, 45(2):173-180.

[3] Chu,X.Q, Fang,H, Pan,X.D, 2008, Degradation of chlorpyrifos alone and in combination with chlorothalonil and their effects on soil microbial populations J Environ Sci, 20(4):464-469.

[4] da Mota,F.F, Gomes,E.A, Paiva,E, 2005, Assessment of the diversity of species in environmental samples by a novel rpoB-based PCR-DGGE method FEMS Microbial Ecol, 53(2):317-328.

[5] Devashis,S, Ramen,K.K, 2005, Metabolism of chlorpyrifos in relation to its effect on the availability of some plant nutrients in soil Chemosphere, 61(9):1273-1280.

[6] Fang,H, Yu,Y.L, Wang,X.G, 2006, Dissipation of chlorpyrifos in pakchoi-vegetated soil in a greenhouse J Environ Sci, 18(4):760-764.

[7] Fang,H, Yu,Y.L, Wang,X.G, 2008, Effects of repeated applications of chlorpyrifos on its persistence and soil microbial functional diversity and development of its degradation capability Bull Environ Contam Toxicol, 81(4):397-400.

[8] Fang,H, Yu,Y.L, Chu,X.Q, 2009, Degradation of chlorpyrifos in laboratory soil and its impact on soil microbial functional diversity J Environ Sci, 21(3):380-386.

[9] Gillings,M, Holley,M, 1997, Repetitive element PCR fingerprinting (rep-PCR) using enterobacterial repetitive intergenic consensus (ERIC) primers is not necessarily directed at ERIC elements Lett Appl Microbiol, 25(1):17-21.

[10] Kulkarni,A.R, Soppimath,K.S, Dave,A.M, 2000, Solubility study of hazardous pesticide (chlorpyrifos) by gas chromatography J Hazard Mater, 80(1-3):9-13.

[11] Li,X.Y, Zhang,H.W, Wu,M.N, 2008, Effect of methamidophos on soil fungi community in microcosms by plate count, DGGE and clone library analysis J Environ Sci, 20(5):619-625.

[12] Maya,K, Singh,R.S, Upadhyay,S.N, 2011, Kinetic analysis reveals bacterial efficacy for biodegradation of chlorpyrifos and its hydrolyzing metabolite TCP Process Biochem, 46(11):2130-2136.

[13] Muyzer,G, de Waal,E.C, Uitterlinden,A.G, 1993, Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S ribosomal RNA Appl Environ Microbiol, 59(3):695-700.

[14] Pandey,S, Singh,D.K, 2006, Soil dehydrogenase, phosphomonoesterase and arginine deaminase activities in an insecticide treated groundnut ( L.) field Chemosphere, 63(5):869-880.

[15] Robertson,L.N, Chandler,K.J, Stickley,B.D.A, 1998, Enhanced microbial degradation implicated in rapid loss of chlorpyrifos from the controlled-release formulation suSCon Blue in soil Crop Prot, 17(1):29-33.

[16] Sampaio,J.L.M, Viana-Niero,C, de Freitas,D, 2006, Enterobacterial repetitive intergenic consensus PCR is a useful tool for typing and isolates Diagn Microbiol Infect Dis, 55(2):107-118.

[17] Sardar,D, Kole,R.K, 2005, Metabolism of chlorpyrifos in relation to its effect on the availability of some plant nutrients in soil Chemosphere, 61(9):1273-1280.

[18] Sebiomo,A, Ogundero,V.W, Bankole,S.A, 2011, Effect of four herbicides on microbial population, soil organic matter and dehydrogenase activity Afr J Biotechnol, 10(5):770-778.

[19] Valkov,V, Baldrian,P, 2009, Denaturing gradient gel electrophoresis as a fingerprinting method for the analysis of soil microbial communities Plant Soil Environ, 55(10):413-423.

[20] Vidya,L.C, Kumar,M, Khanna,S, 2009, Biodegradation of chlorpyrifos in soil by enriched cultures Curr Microbiol, 58(1):35-38.

[21] Wang,F, Yao,J, Chen,H.L, 2010, Comparative toxicity of chlorpyrifos and its oxon derivatives to soil microbial activity by combined methods Chemosphere, 78(3):319-326.

[22] Xie,H, Zhu,L.S, Ma,T.T, 2010, Immobilization of an enzyme from a fungus WZ-I for chlorpyrifos degradation J Environ Sci, 22(12):1930-1935.

[23] Yu,Y.L, Fang,H, Wang,X, 2006, Characterization of a fungal strain capable of degrading chlorpyrifos and its use in detoxification of the insecticide on vegetables Biodegradation, 17(5):487-494.

[24] Yuan,W, Chai,T.J, Miao,Z.M, 2010, ERIC-PCR identification of the spread of airborne in pig houses Sci Total Environ, 408(6):1446-1450.

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毒死蜱胁迫下土壤细菌和真菌群落演替及其降解菌群的分子鉴定

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References