微孔板法检测番茄灰霉病菌对杀菌剂的敏感性

doi:10.3864/j.issn.0578-1752.2012.15.008

摘要/Abstract

摘要： 【目的】利用酶标仪建立番茄灰霉病菌（Botrytis cinerea）对杀菌剂敏感性的快速、高效测定方法。【方法】以OD增加值作为评价指标，确立微孔板法检测灰霉病菌敏感性的测试条件，并对啶酰菌胺、嘧菌酯、乙霉威、甲基硫菌灵、异菌脲、苯醚甲环唑6种常用杀菌剂对灰霉病菌孢子萌发和菌丝生长的抑制效果进行评价，将结果与孢子萌发法和菌丝生长速率法所得结论进行比对。【结果】微孔板法测定条件为：酶标仪测定波长为630 nm，测试培养基为灰霉分生孢子萌发刺激液（PDE），孢子悬浮液浓度为8×105—1.6×106个/mL，培养条件为21℃黑暗条件下振荡（150 r/min），孔中加入孢子悬浮液与药剂培养24 h作为药剂对孢子萌发抑制作用的测试时间，孔中孢子悬浮液培养12 h后再加入药剂培养12—24 h为药剂对菌丝生长抑制作用的测试时间。测试结果与孢子萌发法和菌丝生长速率法所得结论基本一致。用该方法测得啶酰菌胺对山东省3个地区57株灰霉病菌分生孢子萌发及菌丝生长的EC50均值分别为1.9311和5.6488 μg•mL-1。【结论】微孔板法综合了孢子萌发法和菌丝生长速率法的优势，测试速度快、结果准确、重现性好，具有良好的实用性。

关键词: 微孔板法, 灰霉病菌, 敏感性, 测试条件

Abstract: 【Objective】The objective of this study is to establish a rapid and efficient method for testing the sensitivity of B. cinerea to fungicides by using the enzyme-labelling measuring instrument. 【Method】With △OD value as an evaluation index, the conditions of microtiter method to test the sensitivity of B. cinerea to fungicides were optimized. The effect of six widely-used fungicides (boscalid, azoxystrobin, diethofencarb, thiophanate-methyl, iprodione and difenoconazole) against B. cinerea spore germination and mycelial growth was tested, and the results were compared with the conclusions of spore germination method and mycelium growth rate method.【Result】The conditions of microtiter method were optimized. The determination wavelength was 630 nm, the medium was PDE, the concentration of conidial suspension was 8×105-1.6×106 spores/mL, the optimum culture conditions were under shaking culture (150 r/min) at 21℃ in the dark, the text time for the inhibition of spore germination was 24 h after adding fungicide and conidial suspension in the wells, and the testing time for the inhibition of mycelial growth was 12-24 h after adding fungicide (conidial suspension in wells was incubated for 12 h before adding fungicide). The conclusion was almost consistent with that from conventional spore germination method or mycelium growth rate method. Fifty-seven isolates of B. cinerea from three regions of Shandong Province were tested by this method, and the mean EC50 values were 1.9311 and 5.6488 μg•mL-1 for inhibition of conidial germination and mycelial growth, respectively.【Conclusion】Microtiter method combines the advantages of both conventional methods together, it has characteristics of fast testing speed, high measuring accuracy and good reproducibility, so it possesses better practicability.

Key words: microtiter method, Botrytis cinerea, sensitivity, testing conditions

杨晓楠, 王猛, 申瑞平, 刘峰. 微孔板法检测番茄灰霉病菌对杀菌剂的敏感性[J]. 中国农业科学, 2012, 45(15): 3075-3082.

YANG Xiao-Nan, WANG Meng, SHEN Rui-Ping, LIU Feng. Microtiter Method to Test the Sensitivity of Botrytis cinerea to Fungicides[J]. Scientia Agricultura Sinica, 2012, 45(15): 3075-3082.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2012.15.008

https://www.chinaagrisci.com/CN/Y2012/V45/I15/3075

参考文献

[1]朱丽华. 田间灰葡萄孢菌株对杀菌剂抗性的机理. 世界农药, 2003, 25(2): 32-35.

Zhu L H. The mechanism of fungicide resistance of Botrytis cinerea. World Pesticides, 2003, 25(2): 32-35. (in Chinese)

[2]Rosslenbroich H J, Stuebler D. Botrytis cinerea-history of chemical control and novel fungicides for its management. Crop Protection, 2000, 19(8/10): 557-561.

[3]周明国, 叶钟音, 刘经芬. 杀菌剂抗性研究进展. 南京农业大学学报, 1994, 17(3): 33-41.

Zhou M G, Ye Z Y, Liu J F. Progress of fungicide resistance research. Journal of Nanjing Agricultural University, 1994, 17(3): 33-41. (in Chinese)

[4]张传清, 张雅, 魏方林, 刘少颖, 朱国念. 设施蔬菜灰霉病菌对不同类型杀菌剂的抗性检测. 农药学学报, 2006, 8(3): 245-249.

Zhang C Q, Zhang Y, Wei F L, Liu S Y, Zhu G N. Detection of resistance of Botryotinia fuckeliana from protected vegetables to different classes of fungicides. Chinese Journal of Pesticide Science, 2006, 8(3): 245-249. (in Chinese)

[5]Latorre B A, Spadaro I, Rioja M E. Occurrence of resistant strains of Botrytis cinerea to anilinopyrimidine fungicides in table grapes in Chile. Crop Protection, 2002, 21(10): 957-961.

[6]田丽娟, 叶非. 杀菌剂生物测定技术的发展. 农药科学与管理, 2007, 28(6): 19-23.

Tian L J, Ye F. Development of fungicidal bioassay method. Pesticide Science and Administration, 2007, 28(6): 19-23. (in Chinese)

[7]慕立义, 吴文君, 王开运. 植物化学保护研究方法. 第二版. 北京: 中国农业出版社, 1994: 72-82.

Mu L Y, Wu W J, Wang K Y. Chemical Methods of Plant Protection Research. 2nd ed. Beijing: China Agriculture Press, 1994: 72-82. (in Chinese)

[8]邱立红, 张文吉, 王成菊, 郑明奇. 高通量筛选在新农药创制研究中的应用. 农药科学与管理, 2002, 23(5): 20-24.

Qiu L H, Zhang W J, Wang C J, Zheng M Q. High-throughput screening and it’s application in pesticide innovation. Pesticide Science and Administration, 2002, 23(5): 20-24. (in Chinese)

[9]Gisi U, Walder F, Rwsheat-Eini Z, Edel D, Siherotzki H. Changes of genotype, sensitivity and aggressiveness in Phytophthora infestans isolates collected in European countries in 1997, 2006 and 2007. Journal of Phytopathology, 2011, 159: 223-232.

[10]张晓, 王满意, 于淑晶, 王力钟, 李宝聚, 赵卫光. 抑制番茄早疫病菌活性化合物的高通量筛选方法研究. 农药学学报, 2010, 12(4): 468-471.

Zhang X, Wang M Y, Yu S J, Wang L Z, Li B J, Zhao W G. High throughput screening as a tool for discovering agrochemicals effective on Alternaria solania. Chinese Journal of Pesticide Science, 2010, 12(4): 468-471. (in Chinese)

[11]Stammler G, Benzinger G, Speakman J. A rapid and reliable method for monitoring the sensitivity of Sclerotinia sclerotiorum to boscalid. Journal of Phytopathology, 2007, 155: 746-748.

[12]Sargent J M, Taylor C G. Appraisal of the MTT assay as a rapid test of chemosensitivity in acute myeloid leukaemia. British Journal of Cancer, 1989, 60: 206-210.

[13]唐静, 谈满良, 赵江林, 汪冶, 周立刚. 多孔板-MTT比色法测定植物抗菌成分对细菌的抑制活性. 天然产物研究与开发, 2008, 20: 949-952.

Tang J, Tan M L, Zhao J L, Wang Y, Zhou L G. Detection of plant antibacterial components by using microplate-MTT colorimetric assay. Nature Product Research and Development, 2008, 20: 949-952. (in Chinese)

[14]Stammler G, Speakman J. Microtiter method to test the sensitivity of Botrytis cinerea to boscalid. Journal of Phytopathology, 2006, 154: 508-510.

[15]马筱玲, 周红艳, 黄尊波. 酶标仪性能检测和质量控制. 江西医学检验, 2000, 18(3): 147-149.

Ma X L, Zhou H Y, Huang Z B. Function evaluate and quality control testing on the microplate reader. Jiangxi Medical Laboratory Science, 2000, 18(3): 147-149. (in Chinese)

[16]方中达. 植病研究方法. 第三版. 北京: 中国农业出版社, 1998: 46.

Fang Z D. Research Methods of Plant Pathology. 3rd ed. Beijing: China Agriculture Press, 1998: 46. (in Chinese)

[17]于昕, 邓婷婷, 雷高鹏, 赵慧军, 邓洁, 乔代蓉, 白林含, 曹毅. 草莓灰霉孢子萌发条件研究. 应用与环境生物学报, 2006, 12(1): 9-12.

Yu X, Deng T T, Lei G P, Zhao H J, Deng J, Qiao D R, Bai L H, Cao Y. Factors influencing the germination of strawberry Botrytis cinerea conidia. China Journal of Applied and Environmental Biology, 2006, 12(1): 9-12. (in Chinese)

[18]Edwards S G, Seddon B. Selective media for the specic isolation and enumeration of Botrytis cinerea conidia. Letters in Applied Microbiology, 2001, 32: 63-66.

[19]Zhang C Q, Yuan S K, Sun H Y, Qi Z Q, Zhou M G, Zhu G N. Sensitivity of Botrytis cinerea from vegetable greenhouses to boscalid. Plant Pathology, 2007, 56: 646-653.

[20]冯学忠, 吴广辉, 方炳虎, 温子明, 盘苑芷. 紫外分光光度法测定泰乐菌素注射液含量方法的建立. 动物医学进展, 2009, 30(6): 63-66.

Feng X Z, Wu G H, Fang B H, Wen Z M, Pan Y Z. Using UV spectrophotometer to determine the content of tylosin. Progress in Veterinary Medicine, 2009, 30(6): 63-66. (in Chinese)

[21]张从宇, 高智谋, 岳永德. 番茄灰霉病菌生物学性状研究. 安徽技术师范学院学报, 2002,16(3): 10-14.

Zhang C Y, Gao Z M, Yue Y D. Research of the biological characteristics of Botrytis cinerea. Journal of Anhui Technical Teachers College, 2002, 16(3): 10-14. (in Chinese)

[22]潘金菊, 刘峰, 慕卫, 杨连来, 魏光. 黄瓜炭疽病菌对杀菌剂敏感性的优化测定方法研究. 农药科学与管理, 2005, 26(6): 20-22.

Pan J J, Liu F, Mu W, Yang L L, Wei G. Study on optimized measurement of the sensitivity of Gloeosporium orbiculare Arx to fungicide. Pesticide Science and Administration, 2005, 26(6): 20-22. (in Chinese)

[23]李良孔, 翟利利, 王洪预, 王岩. 番茄灰霉病菌对几种杀菌剂的敏感性测定. 中国农学通报, 2010, 26(2): 230-233.

Li L K, Zhai L L, Wang H Y, Wang Y. Sensitivity of Botrytis cinerea to six fungicides. Chinese Agricultural Science Bulletin, 2010, 26(2): 230-233. (in Chinese)

[24]Ziogas B N, Baldwin C, Young J E. Alternative respiration: a biochemical mechanism of resistance to azoxystrobin (ICIA5504) in Septoria tritici. Pesticide Science, 1997, 50(1): 28-34．

[25]Kerssies A. A selective medium for Botrytis cinerea to be used in a spore-trap. Netherlands Journal of Plant Pathology, 1990, 96: 247-250.

[26]Kritzman G, Netzer D. A selective medium for isolation and identification of Botrytis spp. from soil and onion seed. Phytoparasitica, 1978, 6(1): 3-7.