Isolation, Identification, and Characterization of Omethoate-degrading Pseudomonas abietaniphila ZZY-C13-1-9
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摘要:
目的 从设施棚室土壤中筛选获得可降解氧化乐果的微生物菌株，研究降解菌对氧化乐果在土壤中残留量的降解效果。 方法 通过唯一碳源筛选法筛选土壤样本中的本源菌株，生理生化及16S rDNA鉴定种属类型，通过钼胺蓝显色法及HPLC法验证菌株对氧化乐果的降解效果。在土壤中添加一定量的氧化乐果，再添加微生物菌剂并保持湿度温度以验证微生物菌剂在土壤中的应用效果。 结果 筛选获得一株可分解利用氧化乐果的微生物菌株ZZY-C13-1-9，该菌株可在含1 000 mg·L−1的氧化乐果无机盐培养基中正常生长。经鉴定确定该菌为嗜松香假单胞菌（Pseudomonas abietaniphila）。经HPLC证实，在含氧化乐果400 mg·L−1发酵培养基中，30 ℃、180 r·min−1摇瓶培养240 h，氧化乐果的降解率达92.3%。ZZY-C13-1-9亦可在敌百虫与敌敌畏中正常生长。土壤室内试验证明：土壤水分含量保持25%～30%，室内温度25～34 ℃，45 d后嗜松香假单胞菌ZZY-C13-1-9可将土壤中200 mg·L−1的氧化乐果降解59.2%。 结论 筛选获得一株对土壤中残留的氧化乐果具有降解作用的菌株。 Abstract:Objective Omethoate-degrading bacteria were isolated from shed soil to determine their capacities in decomposing the pesticide for possible application in the field. Method Single carbon source culture was used to screen microbes for the potential purpose followed by morphological observation and 16S RNA sequencing for identification. The pesticide-degrading ability of the isolates was determined by molybdate blue spectrophotometry and HPLC, then verified by a shaking flask method in the laboratory. The culture broth for the challenge test containing the selected isolates, one at a time, was artificially added with omethoate at various concentrations. Result A strain, ZZY-C13-1-9, could grow normally when the pesticide was at 1 000 mg·L−1. It was identified as Pseudomonas abietaniphila and capable of degrading 92.3% of omethoate at 400 mg·L−1 level in the nutrient broth within 240 h at the condition of 30 ℃ and 180 r·min−1. In addition, the strain was found to tolerant DDVP and Dipterex in high concentration as well. In a laboratory test at 25–34 ℃ on soil with a moisture content of 25%–30% and omethoate at 200 mg·L−1, ZZY-C13-1-9 decomposed 59.2% of the pesticide in 45 d. Conclusion A bacterium capable of effectively degrading omethoate in soil was successfully isolated, identified, and characterized. -
Key words:
- Prokaryotic /
- Pseudomonas abietaniphila /
- omethoate /
- biodegradation
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图 2 ZZY-C13-1-9菌株形态鉴定
注：a.菌落形态；b.革兰氏染色。
Figure 2. Morphology of ZZY-C13-1-9
Note：a. colony morphology; b. gram stain.
表 1 土壤样本信息
Table 1. Information on soil sample
样本编号
Sample ID样本来源
Source经度
Longitude（E）纬度
Latitude（N）海拔
Altitude/m土壤类型
Soil type植被
PlantTY21 陕西省西安市周至县田峪 108.20.38° 34.3.20° 660 壤土 Loam 核桃树 Walnut TY 44 陕西省西安市周至县田峪 108.20.38° 34.3.20° 660 壤土 Loam 柿子树 Persimmon QL107 陕西省宝鸡市太白县太白山 107.47.52° 33.59.18° 3510 壤土 Loam 猕猴桃 Kiwi QL106 陕西省宝鸡市太白县太白山 107.47.53° 33.59.25° 3495 壤土 Loam 猕猴桃 Kiwi BF19 陕西省西安市周至县厚畛子镇花耳坪村 107.49.79° 33.49.75° 1317 壤土 Loam 细辛 Asarum BF12 陕西省西安市周至县厚畛子镇黑河口 107.49.72° 33.49.75° 1312 壤土 Loam 七叶一枝花 Paris polyphylla ZZ34 陕西省西安市周至县尚村镇涧里村 107.31° 34.05° 1543 壤土 Loam 枣树 Jujube ZZ37 陕西省西安市周至县终南镇毓兴村 107.31° 34.05° 1543 壤土 Loam 枣树 Jujube WN1-2 陕西省渭南市大荔县埝桥乡东埝村 109.87° 34.82° 354 壤土 Loam 黄瓜 Cucumber WN1-1 陕西省渭南市大荔县埝桥乡东埝村 109.87° 34.82° 354 壤土 Loam 黄瓜 Cucumber WN1-3 陕西省渭南市大荔县埝桥乡东埝村 109.87° 34.82° 354 壤土 Loam 黄瓜 Cucumber WN1-4 陕西省渭南市大荔县埝桥乡东埝村 109.87° 34.82° 354 壤土 Loam 黄瓜 Cucumber WN2-1 陕西省渭南市大荔县埝桥乡东埝村 109.87° 34.82° 354 壤土 Loam 黄瓜 Cucumber WN2-2 陕西省渭南市大荔县埝桥乡东埝村 109.87° 34.82° 354 壤土 Loam 黄瓜 Cucumber WN2-3 陕西省渭南市大荔县埝桥乡东埝村 109.87° 34.82° 354 壤土 Loam 黄瓜 Cucumber WN2-4 陕西省渭南市大荔县埝桥乡东埝村 109.87° 34.82° 354 壤土 Loam 黄瓜 Cucumber T-13 陕西省西安市周至县楼观镇羊坡村 107.31° 34.05° 1546 壤土 Loam 枣树 Jujube T-20 陕西省西安市周至县马召镇郭家寨 107.31° 34.05° 1527 壤土 Loam 枣树 Jujube T-24 陕西省西安市周至县竹峪镇兰塬村 107.31° 34.05° 1532 壤土 Loam 枣树 Jujube T-45 陕西省西安市周至县楼观镇下三清寸 107.31° 34.05° 1555 壤土 Loam 枣树 Jujube T-46 陕西省西安市周至县 107.31° 34.05° 1528 壤土 Loam 枣树 Jujube T-51 陕西省西安市周至县集贤镇大曲村 107.31° 34.05° 1574 壤土 Loam 枣树 Jujube T-64 陕西省西安市周至县 107.31° 34.05° 1544 壤土 Loam 枣树 Jujube T-67 陕西省西安市周至县 107.31° 34.05° 1543 壤土 Loam 枣树 Jujube 
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表 2 ZZY-C13-1-9对其他有机磷农药的耐受
Table 2. Tolerance of ZZY-C13-1-9 to organophosphorus pesticides
测定值 Values 农药 Pesticide 质量浓度 Concentration/（mg·L−1） 1000 500 100 0 OD600nm 敌百虫 Dipterex 0.45±0.05 0.40±0.01 0.50±0.05 0.45±0.01 敌敌畏 DDVP 0.73±0.40 0.59±0.09 0.75±0.03 -
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表 3 土壤室内试验氧化乐果残留量及降解率（t-test，P=0.016）
Table 3. Residues and degradation rate of omethoate in laboratory soil (t-test, P=0.016)
处理
Treatment残留量
Residual quantity/（mg·L−1）降解率
Degradation rate/%0 d 45 d 对照 CK 224.10 169.58 24.33 B ZZY-C13-1-9 213.42 87.07 59.20 A 注：不同大写字母表示差异极显著（P＜0.01）。
Note: Data with different uppercase letters indicate extremely significant difference (P＜0.01).
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[1] SCOY A, PENNELL A, ZHANG X Y. Environmental fate and toxicology of dimethoate [J]. Reviews of Environmental Contamination and Toxicology, 2016, 237: 53−70. doi: 10.1007/978-3-319-23573-8_3 [2] 和文祥, 郑粉莉, 田海霞. 氧化乐果对土壤酶活性的影响 [J]. 中国农业科学, 2009, 42(12):4282−4287. doi: 10.3864/j.issn.0578-1752.2009.12.020HE W X, ZHENG F L, TIAN H X. Effect of omethoate on soil enzyme activities [J]. Scientia Agricultura Sinica, 2009, 42(12): 4282−4287.(in Chinese) doi: 10.3864/j.issn.0578-1752.2009.12.020 [3] CALATAYUD-VERNICH P, CALATAYUD F, SIMÓ E, et al. A two-year monitoring of pesticide hazard in-hive: High honey bee mortality rates during insecticide poisoning episodes in apiaries located near agricultural settings [J]. Chemosphere, 2019, 232: 471−480. doi: 10.1016/j.chemosphere.2019.05.170 [4] 宋志慧, 刘冰. 氧化乐果对小球藻的毒性研究 [J]. 生态毒理学报, 2014, 9(3):483−489.SONG Z H, LIU B. Toxic effects of omethoate on Chlorella vulgaris [J]. Asian Journal of Ecotoxicology, 2014, 9(3): 483−489.(in Chinese) [5] 张元园. 氧化乐果对斑马鱼的毒性作用[D]. 青岛: 青岛科技大学, 2016.ZHANG Y Y. Toxicity effect of omethoate to Danio rerio[D]. Qingdao: Qingdao University of Science & Technology, 2016.(in Chinese) [6] LOTTI M, MORETTO A. Organophosphate-induced delayed polyneuropathy [J]. Toxicological Reviews, 2005, 24(1): 37−49. doi: 10.2165/00139709-200524010-00003 [7] HUO D, JIANG S L, QIN Z, et al. Omethoate induces pharyngeal cancer cell proliferation and G1/S cell cycle progression by activation of Akt/GSK-3β/cyclin D1 signaling pathway [J]. Toxicology, 2019, 427: 152298. doi: 10.1016/j.tox.2019.152298 [8] COSTA L G. Organophosphorus compounds at 80: Some old and new issues [J]. Toxicological Sciences, 2018, 162(1): 24−35. doi: 10.1093/toxsci/kfx266 [9] KATSIKANTAMI I, COLOSIO C, ALEGAKIS A, et al. Estimation of daily intake and risk assessment of organophosphorus pesticides based on biomonitoring data-The internal exposure approach [J]. Food and Chemical Toxicology, 2019, 123: 57−71. doi: 10.1016/j.fct.2018.10.047 [10] SINGH B K, WALKER A. Microbial degradation of organophosphorus compounds [J]. FEMS Microbiology Reviews, 2006, 30(3): 428−471. doi: 10.1111/j.1574-6976.2006.00018.x [11] 张娜娜, 姜博, 邢奕, 等. 有机磷农药污染土壤的微生物降解研究进展 [J]. 土壤, 2018, 50(4):645−655.ZHANG N N, JIANG B, XING Y, et al. Microbial degradation of organophosphorus pesticide contaminated soils [J]. Soils, 2018, 50(4): 645−655.(in Chinese) [12] MATSUMURA F, BOUSH G M. Malathion degradation by Trichoderma viride and a Pseudomonas species [J]. Science, 1966, 153(3741): 1278−1280. doi: 10.1126/science.153.3741.1278 [13] SHI Y H, REN L, JIA Y, et al. Genome sequence of organophosphorus pesticide-degrading bacterium Pseudomonas stutzeri strain YC-YH1 [J]. Genome Announcements, 2015, 3(2): 192. doi: 10.1128/genomea.00192-15 [14] ERMAKOVA I T, SHUSHKOVA T V, SVIRIDOV A V, et al. Organophosphonates utilization by soil strains of Ochrobactrum anthropi and Achromobacter sp [J]. Archives of Microbiology, 2017, 199(5): 665−675. doi: 10.1007/s00203-017-1343-8 [15] SUN L N, LIU H M, GAO X H, et al. Isolation of monocrotophos-degrading strain Sphingobiumsp. YW16 and cloning of its TnopdA [J]. Environmental Science and Pollution Research, 2018, 25(5): 4942−4950. doi: 10.1007/s11356-017-0718-3 [16] 顾欣, 刘文辉, 杨环羽, 等. 有机磷农药广谱降解菌A1A18菌株(Brevundimonas sp.)的筛选、鉴定与降解特性分析 [J]. 西北农业学报, 2019, 28(11):1896−1905.GU X, LIU W H, YANG H Y, et al. Screening, identification and degradation characteristics of a broad- spectrum organophosphorus pesticide-degrading bacteria strain A1A18 (Brevundimonas sp.) [J]. Acta Agriculturae Boreali-Occidentalis Sinica, 2019, 28(11): 1896−1905.(in Chinese) [17] ABO-AMER A. Biodegradation of diazinon by Serratia marcescens DI101 and its use in bioremediation of contaminated environment [J]. Journal of Microbiology and Biotechnology, 2011, 21(1): 71−80. doi: 10.4014/jmb.1007.07024 [18] 黄高凌, 蔡慧农, 曾琪, 等. 碱水解-分光光度法快速检测有机磷农药的研究 [J]. 集美大学学报(自然科学版), 2009, 14(4):366−371.HUANG G L, CAI H N, ZENG Q, et al. Study on an alkalinehydrolysis-spectrophotometry for detecting organophosphate pesticide [J]. Journal of Jimei University (Natural Science Edition), 2009, 14(4): 366−371.(in Chinese) [19] 石成春. 有机磷农药曲霉生物降解特性及其动力学的研究[D]. 福州: 福州大学, 2005.SHI C C. Study on the characteristics and kinetics of biodegradation of organophosphate pesticides by the aspergillus[D]. Fuzhou: Fuzhou University, 2005.(in Chinese) [20] 汪耀明, 陶玉贵, 叶连斌, 等. 毛细管电泳仪测定土壤中氧化乐果的含量 [J]. 安徽工程科技学院学报(自然科学版), 2007, 22(4):38−41.WANG Y M, TAO Y G, YE L B, et al. Determination of omethoate in soil by capillary electrophoresis [J]. Journal of Anhui University of Technology and Science (Natural Science Edition), 2007, 22(4): 38−41.(in Chinese) [21] 杨慧. 有机磷农药降解菌的分离、鉴定及固定化研究[D]. 哈尔滨: 黑龙江大学, 2008.YANG H. Isolation, identification and immobilization of organophosphorus pesticide degrading bacteria[D]. Harbin: Helongjiang University, 2008.(in Chinese) [22] LU Y L, SONG S, WANG R S, et al. Impacts of soil and water pollution on food safety and health risks in China [J]. Environment International, 2015, 77: 5−15. doi: 10.1016/j.envint.2014.12.010 [23] CHOUDRI B S, CHARABI Y. Pesticides and herbicides [J]. Water Environment Research, 2019, 91(10): 1342−1349. doi: 10.1002/wer.1227 [24] VAN BRUGGEN A H C, HE M M, SHIN K, et al. Environmental and health effects of the herbicide glyphosate [J]. The Science of the Total Environment, 2018, 616/617: 255−268. doi: 10.1016/j.scitotenv.2017.10.309 [25] UWIZEYIMANA H, WANG M, CHEN W P, et al. The eco-toxic effects of pesticide and heavy metal mixtures towards earthworms in soil [J]. Environmental Toxicology and Pharmacology, 2017, 55: 20−29. doi: 10.1016/j.etap.2017.08.001 [26] RICHARDSON J R, FITSANAKIS V, WESTERINK R H S, et al. Neurotoxicity of pesticides [J]. Acta Neuropathologica, 2019, 138(3): 343−362. doi: 10.1007/s00401-019-02033-9 [27] ISLAM F, WANG J, FAROOQ M A, et al. Potential impact of the herbicide 2, 4-dichlorophenoxyacetic acid on human and ecosystems [J]. Environment International, 2018, 111: 332−351. doi: 10.1016/j.envint.2017.10.020 [28] JIN Y X, WU S S, ZENG Z Y, et al. Effects of environmental pollutants on gut microbiota [J]. Environmental Pollution, 2017, 222: 1−9. doi: 10.1016/j.envpol.2016.11.045 [29] CYCON M, MROZIK A, PIOTROWSKA-SEGET Z. Bioaugmentation as a strategy for the remediation of pesticide-polluted soil: A review [J]. Chemosphere, 2017, 172: 52−71. doi: 10.1016/j.chemosphere.2016.12.129 [30] HUANG Y C, XIAO L J, LI F Y, 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 [31] CYCOŃ M, PIOTROWSKA-SEGET Z. Pyrethroid-degrading microorganisms and their potential for the bioremediation of contaminated soils: A review [J]. Frontiers in Microbiology, 2016, 7: 1463. [32] CHUN M, CHNGCHUN S, YANGHAO G, et al. Study on characteristics of biocometabolic removal of omethoate by the Aspergillus spp [J]. Water Research, 2004, 38(5): 1139−1146. doi: 10.1016/j.watres.2003.11.026 [33] LI C K, MA Y Z, MI Z H, et al. Screening for Lactobacillus plantarum strains that possess organophosphorus pesticide-degrading activity and metabolomic analysis of phorate degradation [J]. Frontiers in Microbiology, 2018, 9: 2048. doi: 10.3389/fmicb.2018.02048 -
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