辛硫磷降解菌D39的分离鉴定及评价

赵晓燕; 周方园; 吴晓青; 史亚微; 周红姿; 张广志; 范素素; 谢雪迎; 潘美霖; 张新建

doi:10.19303/j.issn.1008-0384.2020.09.011

辛硫磷降解菌D39的分离鉴定及评价

doi: 10.19303/j.issn.1008-0384.2020.09.011

1.
齐鲁工业大学（山东省科学院）生态研究所，山东省应用微生物重点实验室，山东　济南　250103
2.
北京航天威科环保科技有限公司，北京　丰台　100071

基金项目: 山东省重点研发项目（2019GSF109012、2019GSF107086、2019GSF109056、2019GSF107043）；国家自然科学基金（31700426、31901928）；山东省重大科技创新工程项目（2019JZZY020610）；山东省自然科学基金（ZR2019BC064）

详细信息

作者简介:
赵晓燕（1978−），女，硕士，副研究员，研究方向：微生物和面源污染修复

通讯作者:
张新建（1978−），男，博士，副研究员，研究方向：微生物和面源污染修复（E-mail：zhangxj@sdas.org）

中图分类号: X172
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出版历程
- 收稿日期: 2020-08-07
- 修回日期: 2020-09-07
- 刊出日期: 2020-09-28

Isolation, Identification, and Evaluation of Phoxim-degrading Bacterium, D39

1.
Shandong Provincial Key Laboratory of Applied Microbiology,Ecology Institute, Qilu University of Technology (Shandong Academy of Sciences), Jinan　250103, China
2.
Beijing Aerospace wks Environmental Technology Company Limited, Beijing. Fengtai　100071, China

摘要

摘要: 目的为了寻找辛硫磷高效降解菌和新的降解酶系，进行辛硫磷降解菌的分离鉴定，追踪辛硫磷降解菌戴尔福特菌D39（Delftia sp.）的降解活性部位，探讨D39胞内酶在含辛硫磷麸皮上的高效降解作用。为了增加菌株的推广应用价值，进一步研究降解菌D39的防病作用。方法采用富集培养法分离辛硫磷降解菌，通过16S rDNA序列分析，结合菌落形态、生理生化特征对菌株进行鉴定。通过平板活性试验确定降解活性部位。经HPLC法检测D39胞内酶对含辛硫磷麸皮的降解作用。D39对5种植物病原菌的抑菌试验采用对峙培养法。结果从农田土壤分离到1株能以辛硫磷为唯一碳源生长的降解菌D39，初步将其鉴定为戴尔福特菌属（Delftia sp.）。D39的降解活性部位主要是胞内酶。进一步提取胞内粗酶液，并将提取的胞内酶喷雾加入含300 mg·kg⁻¹辛硫磷的麸皮，麸皮中酶液质量浓度为0.10 mg·kg⁻¹，25℃反应11 h后经HPLC检测，胞内酶对麸皮上辛硫磷的降解率为100%。D39对5种植物病原菌的抑菌试验结果表明D39对5种病原菌均有不同程度的抑制作用，其中D39对小麦纹枯病菌的平均抑菌率最高（50.00%），对玉米弯孢叶斑病菌的平均抑菌率最低（21.05%）。结论 D39的胞内酶能高效降解麸皮中的辛硫磷残留，未来采用酶降解粮食和农副产品中的辛硫磷残留也更加安全，本研究为粮食及农副产品中辛硫磷的降解奠定理论基础。
- 辛硫磷降解菌 /
- 分离 /
- 鉴定 /
- 抑菌率 /
- 胞内酶
Abstract: Object Bacteria capable of degrading phoxim were isolated from field soils and identified by 16S rDNA sequencing prior to characterization and evaluation for pollution abatement of the organophosphate insecticide on farmland. Possibility of utilizing the bacterium for disease prevention was also explored. Method Phoxim-degrading bacteria were isolated on the enrichment culture and identified by 16S rDNA sequences analysis combined with morphological, physiological, and biochemical characteristics. Specific agent in the bacterium responsible for the degradation was located by a plate activity test, and its degrading effect on phoxim-containing wheat bran determined by HPLC. For possible application in disease preventions, the efficacy of the isolated bacteria on fungal phytopathogens was tested with a dual-culture agar plating technique. Result A phoxim-degrading bacterium designated as D39 was isolated from the soil polluted by the pesticide and primarily identified as Delftia sp. The endoenzyme in D39 was determined to be the key agent responsible of the insecticide degradation. The enzyme was, thus, extracted to spray at a concentration of 0.10 g·kg⁻¹ onto wheat bran spiked with 300 mg·kg⁻¹ of phoxim. As measured by HPLC, the treatment produced a 100% phoxim reduction on the bran after 11 h at 25 ℃. Separately, the antagonism study by the dual-culture plating showed that D39 affected 5 tested phytopathogens in varying degrees. The highest average inhibition rate exerted by D39 was 50.00% on Rhizotonia cerealis, and the lowest 21.05% on Curvularia lunata. Conclusion D39 could not only efficiently degrade phoxim residues on wheat bran but also inhibit the growth of a certain phytopathogens. It appeared that the extracted endoenzyme from D39 could be applied on grains to decompose the residual insecticide for food safety improvement and served as a disease prevention bioagent as well.
- Phoxim-degrading bacterium /
- isolation /
- identification /
- inhibition rate /
- endoenzyme

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图 1 各菌株在基础盐培养基上产生的水解圈

Figure 1. Hydrolytic halos generated by bacteria on mineral salt medium with phoxim as sole carbon source

下载: 全尺寸图片幻灯片

图 2 3株菌（39、25、26）对液体SMM中辛硫磷的降解

Figure 2. Degradation of phoxim in liquid SMM by bacterial strains 39, 25 and 26

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图 3 菌株D39在基础盐培养基上产生的水解圈

Figure 3. Hydrolytic halos generated by D39 on mineral salt medium with phoxim as sole carbon source

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图 4 D39的显微形态

Figure 4. Microscopic morphology of D39

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图 5 16S rDNA PCR 产物电泳图

注：1、2为D39的16S rDNA PCR产物；M为D₂₀₀₀ marker；3、阴性对照H₂O

Figure 5. 16S rDNA PCR product of D39

Note: 1 and 2: 16S rDNA PCR products of D39; M: D2000 marker; 3: H₂O as negative control.

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图 6 菌体和胞内酶活性测定

注：A为D39降解活性定位，B为酶活部位检测

Figure 6. Bacterial and endoenzyme activities

Note: A: activities of bacteria and supernatant; B: activities of endoenzymes and extracellular enzyme

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图 7 辛硫磷标准曲线

Figure 7. Standard curve of phoxim

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图 8 D39的胞内酶对麸皮上辛硫磷的降解作用

Figure 8. Phoxim degradation on wheat bran by endoenzyme from D39

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图 9 胞内酶及对照11 h对麸皮上辛硫磷降解的HPLC图谱

Figure 9. HPLC chromatograms of phoxim degradations on wheat bran by endoenzyme from D39 and by CK

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表 1 菌株D39对植物病原菌的抑菌作用

Table 1. Inhibition activities of D39 on phytopathogens

植物病原菌 plant pathogens	5 d平均抑菌率 average inhibition activity after 5 d/%
白菜黑腐病菌 Xanthomonas campestris pv. campestris	27.59±0.67 c
玉米弯孢叶斑病菌 Curvularia Lunata	21.05±0.91 e
黄瓜枯萎病菌 Fusarium oxysporum f.sp. cucumerinum	30.33±0.33 b
苹果轮纹病菌 Botryosphaeria dothidea	24.53±1.28 d
小麦纹枯病菌 Rhizotonia cerealis	50.00±0.84 a
注：抑菌率后面不同的小写字母表示各处理之间差异显著（P＜0.05） Note: Inhibition rates with different lowercase letters denote significant differences between treatments （P＜0.05）.

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参考文献(15)

[1]	周云全, 刘春宇, 曲冠男, 等. 农药辛硫磷的密度泛函理论计算及拉曼光谱分析 [J]. 原子与分子物理学报, 2020, 37(3):331−336. ZHOU Y Q, LIU C Y, QU G N, et al. Density functional theory calculation and Raman spectrum analysis of the pesticidephoxim [J]. Journal of Atomic and Molecular Physics, 2020, 37(3): 331−336.(in Chinese
[2]	刘飞翔, 王小梅, 吴文睿, 等. 高效液相色谱法测定小麦和大米中辛硫磷残留的快速前处理方法 [J]. 食品安全质量检测学报, 2019, 10(10):2989−2994. doi: 10.3969/j.issn.2095-0381.2019.10.025 LIU F X, WANG X M, WU W R, et al. Rapid pretreatment for the determination of phoxim residues in wheat and rice by high performance liquid chromatography [J]. Journal of Food Safety & Quality, 2019, 10(10): 2989−2994.(in Chinese doi: 10.3969/j.issn.2095-0381.2019.10.025
[3]	崔新刚, 李百祥. 辛硫磷与高效氯氰菊酯对雄性大鼠精子的联合作用 [J]. 牡丹江医学院学报, 2016, 37(6):1−4. CUI X G, LI B X. Joint toxic effect of phoximand and beta-cypermethrin on the sperm system of male rats [J]. Journal of Mudanjiang Medical University, 2016, 37(6): 1−4.(in Chinese
[4]	李宏辉, 刘秀芳, 马潇, 等. 辛硫磷和灭多威混配对雄性小鼠生殖细胞的毒性作用 [J]. 宁夏医科大学学报, 2009, 31(5):582−584. doi: 10.3969/j.issn.1674-6309.2009.05.008 LI H H, LIU X F, MA X, et al. Toxic effects of phoxim and methomyl on male reproductive cells [J]. Journal of Ningxia Medical University, 2009, 31(5): 582−584.(in Chinese doi: 10.3969/j.issn.1674-6309.2009.05.008
[5]	李松花, 金文红, 李铁骥. 辛硫磷对小鼠心脏CHE和SOD活性的影响 [J]. 中国卫生工程学, 2011, 10(1):12−13, 17. LI S H, JIN W H, LI T J. Activity of cordis cholinesterase and superoxide dismutase in mice exposed to phoxim [J]. Chinese Journal of Public Health Engineering, 2011, 10(1): 12−13, 17.(in Chinese
[6]	王运儒, 秦玉燕, 农耀京, 等. 高效液相色谱法测定马铃薯和土壤中的辛硫磷残留 [J]. 食品安全质量检测学报, 2017, 8(2):421−427. WANG Y R, QIN Y Y, NONG Y J, et al. Determination of phoxim residues in potato and soil by high performance liquid chromatography [J]. Journal of Food Safety & Quality, 2017, 8(2): 421−427.(in Chinese
[7]	张其才, 饶钦雄, 汤倩倩, 等.三种有机磷农药在双孢蘑菇及其栽培基质中的残留动态[J]. 农药学学报, 2020, 22: 1-11. ZHANG Q C, RAO Q X, TANG Q Q, et al. The residual dynamics of three organophosphorus pesticides in Agaricus bisporus fruits and cultivation substrates[J]. Chinese Journal of Pesticide Science, 2017, (8)2: 421-427.
[8]	沈雨佳, 洪源范, 洪青, 等. 辛硫磷降解菌XSP-1的分离、鉴定及其降解特性研究 [J]. 环境科学, 2007, 28(12):2833−2837. doi: 10.3321/j.issn:0250-3301.2007.12.029 SHEN Y J, HONG Y F, HONG Q, et al. Isolation, identification and characteristics of a phoxim-degrading bacterium XSP-1 [J]. Chinese Journal of Environmental Science, 2007, 28(12): 2833−2837.(in Chinese doi: 10.3321/j.issn:0250-3301.2007.12.029
[9]	李芳, 黄素芳, 刘波. 淡紫拟青霉对辛硫磷的降解效应 [J]. 应用与环境生物学报, 2006, 12(1):104−107. doi: 10.3321/j.issn:1006-687X.2006.01.025 LI F, HUANG S F, LIU B. Degradation of phoxim by paecilomyces lilaciuns [J]. Chinese Journal of Applied & Environmental Biology, 2006, 12(1): 104−107.(in Chinese doi: 10.3321/j.issn:1006-687X.2006.01.025
[10]	张瑞福, 朱卫, 崔中利, 等. 辛硫磷降解菌X-1的分离鉴定及降解性状的初步研究 [J]. 环境科学学报, 2003, 23(3):411−413. doi: 10.3321/j.issn:0253-2468.2003.03.025 ZHANG R F, ZHU W, CUI Z L, et al. Isolation, identification and characters of phoxim degrading bacterium [J]. Acta Scientiae Circumstantiae, 2003, 23(3): 411−413.(in Chinese doi: 10.3321/j.issn:0253-2468.2003.03.025
[11]	于婷, 侯长明, 王海荣, 等. 嗜铁细菌枯草芽胞杆菌Bs-15对辛硫磷的降解特性 [J]. 应用与环境生物学报, 2014, 20(2):179−184. YU T, HOU C M, WANG H R, et al. Characteristics in degradation of phoxim by siderophore producing bacterium Bacillus subtilis Bs-15 [J]. Chinese Journal of Applied & Environmental Biology, 2014, 20(2): 179−184.(in Chinese
[12]	东秀珠, 蔡妙英. 常见细菌系统鉴定手册[M]. 北京:科学出版社, 2001.
[13]	周永丹, 张勇, 李存雄, 等. HPLC法同步测定谷物中的吡虫啉和辛硫磷 [J]. 江西师范大学学报(自然科学版), 2015, 39(6):566−569. ZHOU Y D, ZHANG Y, LI C X, et al. The determination of imidacloprid and phoxim residues in cereals by HPLC [J]. Journal of Jiangxi Normal University (Natural Sciences Edition), 2015, 39(6): 566−569.(in Chinese
[14]	侯晓慧, 薛科宇, 魏珂, 等. 超高效液相色谱-飞行时间质谱法测定蔬菜中辛硫磷残留量 [J]. 食品安全质量检测学报, 2018, 9(17):4645−4649. doi: 10.3969/j.issn.2095-0381.2018.17.029 HOU X H, XUE K Y, WEI K, et al. Determination of phoxim residue in vegetables by ultra performance liquid chromatography-quadrupole time of flight mass spectrometry [J]. Journal of Food Safety & Quality, 2018, 9(17): 4645−4649.(in Chinese doi: 10.3969/j.issn.2095-0381.2018.17.029
[15]	赵晓燕, 洪永聪, 郭凯, 等. TL2的鉴定及其对植物病原菌的抑制作用 [J]. 福建农业学报, 2013, 28(4):353−356. doi: 10.3969/j.issn.1008-0384.2013.04.010 ZHAO X Y, HONG Y C, GUO K, et al. Identification of TL2 and the inhibitive effect of TL2 to fungal pathogens [J]. Fujian Journal of Agricultural Sciences, 2013, 28(4): 353−356.(in Chinese doi: 10.3969/j.issn.1008-0384.2013.04.010