Amiri A, Scherm H, Brannen P M, Schnabel G
Department of Entomology, Soils, and Plant Sciences, Clemson University, Clemson, SC 29634.
Department of Plant Pathology, University of Georgia, Athens 30602.
Plant Dis. 2008 Mar;92(3):415-420. doi: 10.1094/PDIS-92-3-0415.
Three rapid, agar-based assays were compared with a traditional petri dish method for assessing the sensitivity of Monilinia fructicola to propiconazole (0.3 and 2.0 μg/ml), thiophanate-methyl (1.0 and 50 μg/ml), and azoxystrobin (1.0 and 35 μg/ml) in the laboratory. The three assays were based on mycelial growth inhibition on agar disks sliced from lipbalm tubes filled with fungicide-amended potato dextrose agar (PDA), on PDA-coated cotton swabs, or in PDA-filled microcentrifuge tubes. Mycelial growth inhibition of eight previously characterized isolates (two resistant to propiconazole, two highly resistant to thiophanate-methyl, two with low levels of resistance to thiophanate-methyl, and two sensitive to all three fungicides) was determined visually 24, 48, and 72 h after inoculation. The 48-h time point was the earliest suitable time to collect data for all methods because insufficient growth was recorded in the petri dish and tube assays after 24 h. With the exception of the swab assay, all methods classified the isolates previously determined to be fungicide sensitive correctly (i.e., no fungal growth was observed for these isolates). For propiconazole-resistant isolates, the lipbalm assay resulted in levels of growth inhibition very similar to the petri dish method, whereas the swab assay and the tube assay overestimated and underestimated, respectively, the level of resistance. Both the lipbalm and the swab assays classified isolates correctly as being thiophanate-methyl resistant, and both were able to discriminate the isolates previously classified as having low versus high levels of resistance when treated with this fungicide at 50 μg/ml, as was the petri dish method. None of the eight isolates which previously were determined to be azoxystrobin sensitive grew on azoxystrobin-amended media, regardless of the assay type. Overall, the average percentage of correct isolate classifications (relative to their previously determined resistance status) on propiconazole- and thiophanate-methyl-amended media after 48 h ranged from 87.5 to 100, 85.3 to 100, 63.2 to 94.5, and 50.5 to 81.0% for the petri dish, lipbalm, swab, and tube assays, respectively. The lipbalm assay provided the most accurate assessments (85.3 to 100%) after only 24 h of incubation, supporting its use as a rapid and simple tool to monitor resistance levels in M. fructicola field populations.
在实验室中，将三种基于琼脂的快速检测方法与传统培养皿法进行比较，以评估褐腐病菌对丙环唑（0.3和2.0μg/ml）、甲基托布津（1.0和50μg/ml）以及嘧菌酯（1.0和35μg/ml）的敏感性。这三种检测方法分别基于对从装有杀菌剂改良马铃薯葡萄糖琼脂（PDA）的唇膏管切下的琼脂圆盘上的菌丝生长抑制、PDA包被的棉拭子上的菌丝生长抑制或PDA填充的微量离心管中的菌丝生长抑制。接种后24、48和72小时，通过肉眼观察测定了8个先前已鉴定特征的分离株（2个对丙环唑耐药、2个对甲基托布津高度耐药、2个对甲基托布津低水平耐药以及2个对所有三种杀菌剂敏感）的菌丝生长抑制情况。48小时时间点是所有方法收集数据的最早合适时间，因为24小时后在培养皿和试管检测中记录的生长不足。除棉拭子检测外，所有方法都能正确分类先前确定为对杀菌剂敏感的分离株（即这些分离株未观察到真菌生长）。对于丙环唑耐药分离株，唇膏检测产生的生长抑制水平与培养皿法非常相似，而棉拭子检测和试管检测分别高估和低估了耐药水平。唇膏检测和棉拭子检测都能正确将分离株分类为对甲基托布津耐药，并且当用50μg/ml这种杀菌剂处理时，二者都能区分先前分类为低水平与高水平耐药的分离株，培养皿法也是如此。无论检测类型如何，先前确定对嘧菌酯敏感的8个分离株在嘧菌酯改良培养基上均未生长。总体而言，48小时后在丙环唑和甲基托布津改良培养基上，相对于先前确定的耐药状态，培养皿、唇膏、棉拭子和试管检测正确分离株分类的平均百分比分别为87.5%至100%、85.3%至100%、63.2%至94.5%和50.5%至81.0%。唇膏检测在仅培养24小时后就能提供最准确的评估（85.3%至100%），这支持了其作为监测褐腐病菌田间种群耐药水平的快速简便工具的应用。
