微孔板法检测番茄灰霉病菌对杀菌剂的敏感性

Microtiter Method to Test the Sensitivity of Botrytis cinerea to Fungicides

【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630nm,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5.6488μg.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。

[ Objective ]【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 EC50values were 1.9311 and 5.6488 μg.mL-1for 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.