溴氰菊酯降解菌的筛选和鉴定研究

doi:10.11853/j.issn.1003.8280.2024.05.002

摘要/Abstract

摘要： 目的筛选和鉴定稻田中可降解溴氰菊酯的细菌菌群,并测定其降解效能。方法采用富集驯化、分离纯化获取溴氰菊酯降解菌群,基于形态学和分子生物学特征鉴定其种类。对单个菌落进行16S rRNA全长序列测定和分析,同时利用MiSeq高通量测序技术测定菌群的16S rRNA V4区片段,对序列进行注释和分析,统计细菌物种和属水平的组成和丰度。在溴氰菊酯降解菌中培养3种菊酯类杀虫剂降解菌,根据杀虫剂的残留浓度,计算菌群降解溴氰菊酯、联苯菊酯和三氟氯氰菊酯杀虫剂的效率。结果本研究从稻田水中分离筛选了可降解溴氰菊酯的菌群,单菌落分子鉴定为：广温拟无枝酸菌、鲍氏不动杆菌、屈曲杆菌、红球菌、拟无枝酸菌、苍白杆菌和黄色杆菌。MiSeq高通量分子鉴定结果显示,在属水平丰度>4%的菌群分别为假单胞菌属（26.78%）,不动杆菌属（7.00%）,苍白杆菌属（6.82%）,无色杆菌属（5.00%）,克雷伯菌属（4.39%）和微杆菌属（4.12%）。降解菌群对3种菊酯类杀虫剂的降解率从第2天开始持续增加,第8天（最长观察时间）时达到高峰,分别为76.75%（联苯菊酯）、63.65%（溴氰菊酯）和74.20%（三氟氯氰菊酯）,其中三氟氯氰菊酯在第6天达到平台期。结论成功分离并获得了可降解溴氰菊酯的细菌菌群,其中假单胞菌属种类为优势菌群。降解菌群对菊酯类杀虫剂有广泛的降解效能,该研究为其在现场应用以及促进杀虫剂的快速降解方面提供了基础资料。

关键词: 溴氰菊酯, 降解菌, 降解效能

Abstract: Objective To select and identify deltamethrin-degrading bacteria in paddy fields, and to determine their degradation efficiency. Methods A bacterial flora capable of degrading deltamethrin was obtained through domestication, enrichment, isolation, and purification. The species was identified based on morphological and molecular characteristics. For single colonies, the full length of the 16S rRNA gene was sequenced and analyzed, and for the flora, the 16S rRNA V4 region was sequenced using the MiSeq high-throughput method. Sequence annotation and analysis were conducted. Bacterial composition and abundance were described at species and genus levels. The flora was cultured with deltamethrin, bifenthrin, and cyhalothrin separately, and the degrading efficiency was calculated based on the residual concentrations of the insecticides. Results A bacterial flora capable of degrading deltamethrin was obtained from the water of paddy fields. The identified single colonies included Amycolatopsis eurytherma, Acinetobacter baumannii, Ancylobacter sp., Rhodococcus spp., Amycolatopsis spp., Ochrobactrum sp., and Xanthobacter sp. The MiSeq high-throughput sequencing results showed that bacteria >4% in abundance at the genus level included Pseudomonas （26.78%）, Acinetobacter （7.00%）, Ochrobactrum （6.82%）, Achromobacter （5.00%）, Klebsiella（4.39%）, Microbacterium（4.12%）. The degradation rates of the three pyrethroid insecticides by the bacterial flora continued to increase since day 2, peaking on day 8 the longest observation period at 76.75% （bifenthrin）, 63.65% （deltamethrin）, and 74.20% （cyhalothrin； reaching the plateau on day 6）. Conclusions A deltamethrin-degrading bacterial flora was successfully obtained, in which Pseudomonas was the dominant population, showing wide degradation efficiency for pyrethroid insecticides. This study provides basic data for the field application of degrading bacteria and the studies about rapid degradation of insecticides.

Key words: Deltamethrin, Degrading bacteria, Degradation efficiency

中图分类号:

R378

周秋明, 南春燕, 袁浩, 单文琪, 陶峰, 董昊炜, 彭恒, 马雅军. 溴氰菊酯降解菌的筛选和鉴定研究[J]. 中国媒介生物学及控制杂志, 2024, 35(5): 517-522.

ZHOU Qiu-ming, NAN Chun-yan, YUAN Hao, SHAN Wen-qi, TAO Feng, DONG Hao-wei, PENG Heng, MA Ya-jun. Selection and identification of deltamethrin-degrading bacteria[J]. Chinese Journal of Vector Biology and Control, 2024, 35(5): 517-522.

参考文献

[1] Field LM,Emyr Davies TG,O'Reilly AO,et al. Voltage-gated sodium channels as targets for pyrethroid insecticides[J]. Eur Biophys J,2017,46（7）：675-679. DOI：10.1007/s00249-016-1195-1.
[2] Li HZ,Cheng F,Wei YL,et al. Global occurrence of pyrethroid insecticides in sediment and the associated toxicological effects on benthic invertebrates：An overview[J]. J Hazard Mater,2017,324：258-271. DOI：10.1016/j.jhazmat.2016.10.056.
[3] Zhang WL,Peng H,Shan WQ,et al. Research progress on safety of repellents[J]. Chin J Vector Biol Control,2022,33（4）：601-607. DOI：10.11853/j.issn.1003.8280.2022.04.029.（in Chinese）张婉莉,彭恒,单文琪,等. 驱避剂安全性研究进展[J]. 中国媒介生物学及控制杂志,2022,33（4）：601-607. DOI：10.11853/j.issn.1003.8280.2022.04.029.
[4] Cycoń M,Piotrowska-Seget Z. Pyrethroid-degrading microorganisms and their potential for the bioremediation of contaminated soils：A review[J]. Front Microbiol,2016,7：1463. DOI：10.3389/fmicb.2016.01463.
[5] Williams IH,Brown MJ. Persistence of permethrin and WL 43775 in soil[J]. J Agric Food Chem,1979,27（1）：130-132. DOI：10.1021/jf60221a017.
[6] Huang YC,Xiao LJ,Li FY,et al. Microbial degradation of pesticide residues and an emphasis on the degradation of cypermethrin and 3-phenoxy benzoic acid：A review[J]. Molecules,2018,23（9）：2313. DOI：10.3390/molecules23092313.
[7] Wang XH,Shang WX,Xu HY,et al. Research progress in microbial degradation of lambda-Cyhalothrin[J]. Chem Bioeng,2020,37（3）：7-14. DOI：10.3969/j.issn.1672-5425.2020.03.002.（in Chinese）王晓慧,商文贤,徐宏英,等. 高效氯氟氰菊酯的微生物降解研究进展[J]. 化学与生物工程,2020,37（3）：7-14. DOI：10.3969/j.issn.1672-5425.2020.03.002.
[8] Yang JJ,Feng YM,Zhan H,et al. Characterization of a pyrethroid-degrading Pseudomonas fulva strain P31 and biochemical degradation pathway of D-phenothrin[J]. Front Microbiol,2018,9：1003. DOI：10.3389/fmicb.2018.01003.
[9] Deng WQ,Lin DR,Yao K,et al. Characterization of a novel β-cypermethrin-degrading Aspergillus niger YAT strain and the biochemical degradation pathway of β-cypermethrin[J]. Appl Microbiol Biotechnol,2015,99（19）：8187-8198. DOI：10.1007/s00253-015-6690-2.
[10] Zhou DQ,Xu DQ,Hu BL. Experimental microbiology[M]. 3rd ed. Beijing：Higher Education Press,2013：87-90.（in Chinese）周德庆,徐德强,胡宝龙. 微生物学实验教程[M]. 3版. 北京：高等教育出版社,2013：87-90.
[11] Hu Q,Tang J,Lei D,et al. Isolation,identification and optimization of degradation conditions of a deltamethrin degrading strain[J]. Microbiol China,2020,47（3）：699-709. DOI：10.13344/j.microbiol.china.190633.（in Chinese）胡琼,唐洁,雷丹,等. 一株溴氰菊酯降解菌的分离鉴定及其降解条件优化[J]. 微生物学通报,2020,47（3）：699-709. DOI：10.13344/j.microbiol.china.190633.
[12] Wang Y,Qian PY. Conservative fragments in bacterial 16S rRNA genes and primer design for 16S ribosomal DNA amplicons in metagenomic studies[J]. PLoS One,2009,4（10）：e7401. DOI：10.1371/journal.pone.0007401.
[13] Chen HP,Liu X,Wang QH,et al. Determination of 88 pesticide residues in tea using gas chromatography-tandem mass spectrometry[J]. Chin J Chromatogr,2011,29（5）：409-416. DOI：10.3724/SP.J.1123.2011.00409.（in Chinese）陈红平,刘新,汪庆华,等. 气相色谱-串联质谱法测定茶叶中88种农药残留量[J]. 色谱,2011,29（5）：409-416. DOI：10.3724/SP.J.1123.2011.00409.
[14] Wang Y,Yu WQ,Shi H,et al. Historical survey of the kdr mutations in the populations of Anophelessinensis in China in 1996-2014[J]. Malar J,2015,14：120. DOI：10.1186/s12936-015-0644-0.
[15] Maloney SE,Maule A,Smith AR. Microbial transformation of the pyrethroid insecticides：Permethrin,deltamethrin,fastac,fenvalerate,and fluvalinate[J]. Appl Environ Microbiol,1988,54（11）：2874-2876. DOI：10.1128/aem.54.11.2874-2876.1988.
[16] Pankaj,Sharma A,Gangola S,et al. Novel pathway of cypermethrin biodegradation in a Bacillus sp. strain SG2 isolated from cypermethrin-contaminated agriculture field[J]. 3 Biotech,2016,6（1）：45. DOI：10.1007/s13205-016-0372-3.
[17] Ma Y,Chen LS,Qiu JG. Biodegradation of beta-cypermethrin by a novel Azoarcus indigens strain HZ5[J]. J Environ Sci Health,Part B,2013,48（10）：851-859. DOI：10.1080/03601234.2013.795843.
[18] Zhang SB,Yin LB,Liu Y,et al. Cometabolic biotransformation of fenpropathrin by Clostridium species strain ZP3[J]. Biodegradation,2011,22（5）：869-875. DOI：10.1007/s10532-010-9444-y.
[19] Guo P,Wang BZ,Hang BJ,et al. Pyrethroid-degrading Sphingobium sp. JZ-2 and the purification and characterization of a novel pyrethroid hydrolase[J]. Int Biodeter Biodegr,2009,63（8）：1107-1112. DOI：10.1016/j.ibiod.2009.09.008.
[20] Liang WQ,Liu YH,Li H. Isolation,identification and characterization of a cypermethrin-degrading strain J-2[J]. J Guangdong Coll Pharm,2007,23（2）：199-202,204. DOI：10.3969/j.issn.1006-8783.2007.02.033.（in Chinese）梁卫驱,刘玉焕,李荷. 氯氰菊酯降解菌的分离鉴定及其降解特性研究[J]. 广东药学院学报,2007,23（2）：199-202,204. DOI：10.3969/j.issn.1006-8783.2007.02.033.
[21] Cycoń M,Żmijowska A,Piotrowska-Seget Z. Enhancement of deltamethrin degradation by soil bioaugmentation with two different strains of Serratia marcescens[J]. Int J Environ Sci Technol,2014,11（5）：1305-1316. DOI：10.1007/s13762-013-0322-0.
[22] Paingankar M,Jain M,Deobagkar D. Biodegradation of allethrin,a pyrethroid insecticide,by an Acidomonas sp.[J]. Biotechnol Lett,2005,27（23/24）：1909-1913. DOI：10.1007/s10529-005-3902-3.
[23] Saikia N,Das SK,Patel BKC,et al. Biodegradation of beta-cyfluthrin by Pseudomonas stutzeri strain S1[J]. Biodegradation,2005,16（6）：581-589. DOI：10.1007/s10532-005-0211-4.
[24] Li QY,Gu BQ,Liu YY,et al. Isolation,identification and characteristics of a cypermethrin-degrading bacterium GF31[J]. Microbiology,2009,36（9）：1334-1339.（in Chinese）李青云,顾宝群,刘幽燕,等. 氯氰菊酯降解菌GF31的分离鉴定及其降解特性[J]. 微生物学通报,2009,36（9）：1334-1339.
[25] Lee S,Gan JY,Kim JS,et al. Microbial transformation of pyrethroid insecticides in aqueous and sediment phases[J]. Environ Toxicol Chem,2004,23（1）：1-6. DOI：10.1897/03-114.
[26] Xu YX,Sun JQ,Li XH,et al. Study on cooperating degradation of cypermethrin and 3-phenoxybenzoic acid by two bacteria strains[J]. Acta Microbiol Sin,2007,47（5）：834-837. DOI：10.3321/j.issn：0001-6209.2007.05.017. 许育新,孙纪全,李晓慧,等. 两株菌对氯氰菊酯及其降解产物3-PBA的协同代谢研究[J]. 微生物学报,2007,47（5）：834-837. DOI：10.3321/j.issn：0001-6209.2007.05.017.
[27] Tang WX,Wang D,Wang JQ,et al. Pyrethroid pesticide residues in the global environment：An overview[J]. Chemosphere,2018,191：990-1007. DOI：10.1016/j.chemosphere.2017.10.115.
[28] Tang MQ,Tian P,You MS. Isolation,identification and growth characteristics of a fenvalerate degradation bacterium[J]. Microbiol China,2010,37（5）：682-688. DOI：10.13344/j.microbiol.china.2010.05.014.（in Chinese）汤鸣强,田盼,尤民生. 氰戊菊酯降解菌FDB的分离鉴定及其生长特性[J]. 微生物学通报,2010,37（5）：682-688. DOI：10.13344/j.microbiol.china.2010.05.014.（in Chinese）
[29] Wang ZS,Lin G,You MS,et al. Isolation and character of synthetic pyrethroid insecticides-degrading bacteria from tea leaves[J]. Acta Ecol Sin,2005,25（7）：1824-1827. DOI：10.3321/j.issn：1000-0933.2005.07.041.（in Chinese）王兆守,林淦,尤民生,等. 茶叶上拟除虫菊酯类农药降解菌的分离及其特性[J]. 生态学报,2005,25（7）：1824-1827. DOI：10.3321/j.issn：1000-0933.2005.07.041.
[30] Chen SH,Geng P,Hu MY,et al. Effects of Stenotrophomonas sp. strain ZS-S-01 on the degradation of β-cypermethrin and fenvalerate residue on flowering Chinese cabbage[J]. J South China Agric Univ,2011,32（4）：47-52. DOI：10.7671/j.issn.1001-411X.2011.04.011.（in Chinese）陈少华,耿鹏,胡美英,等. 菌株Stenotrophomonas sp. ZS-S-01去除菜心中残留高效氯氰菊酯和氰戊菊酯农药的作用[J]. 华南农业大学学报,2011,32（4）：47-52. DOI：10.7671/j.issn.1001-411X.2011.04.011.
[31] Wei YJ,Liu YY,Zhang HW,et al. Biodegradation of cypermethrin by Pseudomonas aeruginosa GF31 in the soil environment[J]. Environ Pollut Control,2011,33（10）：55-58,100. DOI：10.3969/j.issn.1001-3865.2011.10.012.（in Chinese）韦军,刘幽燕,张洪威,等. 铜绿假单胞菌GF31降解土壤中氯氰菊酯的性能研究[J]. 环境污染与防治,2011,33（10）：55-58,100. DOI：10.3969/j.issn.1001-3865.2011.10.012.
[32] Ding HT,Li SP,Shen B,et al. Isolation of pyrethroids degrading strain and its physiological characteristics[J]. Acta Pedol Sin,2003,40（1）：123-129. DOI：10.11766/trxb200012250117.（in Chinese）丁海涛,李顺鹏,沈标,等. 拟除虫菊酯类农药残留降解菌的筛选及其生理特性研究[J]. 土壤学报,2003,40（1）：123-129. DOI：10.11766/trxb200012250117.
[33] Dong HW,Nan CY,Zhou QM,et al. Composition of microbiota in Anopheles sinensis and water in larvae breeding site：A study based on 16S rRNA high-throughput sequencing[J]. Chin J Vector Biol Control,2021,32（5）：533-540. DOI：10.11853/j.issn.1003.8280.2021.05.005.（in Chinese）董昊炜,南春燕,周秋明,等. 基于16S rRNA高通量测序的现场中华按蚊及其孳生地水体菌群结构研究[J]. 中国媒介生物学及控制杂志,2021,32（5）：533-540. DOI：10.11853/j.issn.1003.8280.2021.05.005.
[34] Wang HY,Liu HM,Peng H,et al. A symbiotic gut bacterium enhances Aedes albopictus resistance to insecticide[J]. PLoS Negl Trop Dis,2022,16（3）：e0010208.