Fernández-Ortuño D, Schnabel G
School of Agricultural, Forest, & Environmental Sciences, Clemson University, Clemson SC 29634.
Plant Dis. 2012 Nov;96(11):1700. doi: 10.1094/PDIS-06-12-0557-PDN.
Botrytis cinerea Pers.:Fr. is the causal agent of gray mold disease and one of the most important plant-pathogenic fungi affecting strawberry (Fragaria× ananassa). Control of gray mold mainly depends on fungicides, including the methyl benzimidazole carbamate (MBC) thiophanate-methyl. In 2011, strawberries with gray mold symptoms were collected from commercial fields near Chesnee, Florence, Lexington, McBee, Monetta, and North Augusta, all in South Carolina. MBC fungicides were used in most of these fields for gray mold control during the last 3 years. A total of 124 single spore B. cinerea isolates were obtained, each from a different fruit. Resistance to thiophanate-methyl (Topsin M 70WP, Cerexagri-Nisso LLC, King of Prussia, PA) was determined using a conidial germination assay as described previously (1). The majority of isolates (81.4%) were resistant; the rest were sensitive. Resistant isolates were found in all locations with some populations (Chesnee, McBee, and Lexington) revealing no sensitive isolates. Genomic DNA from 35 resistant isolates (representing all locations) and 10 sensitive isolates (from Chesnee, Monetta, and North Augusta, SC) was extracted, and the molecular basis of MBC fungicide resistance was determined as described previously (2). All MBC-resistant isolates possessed the E198A mutation known to confer high levels of MBC fungicide resistance in many fungi, including B. cinerea (2,3). Disease was assessed using a detached strawberry fruit assay. Commercially grown strawberry fruit (24 in total for each isolate and fungicide treatment) were rinsed with water, dried, and sprayed 4 h prior to inoculation with either water or 2.4 g/liter of Topsin M to runoff using a hand mister. Fruit was stab-wounded with a sterile syringe and inoculated with a 30-μl droplet of a conidial suspension (10 spores/ml) of either a sensitive or resistant isolate. After inoculation, the fruit were kept at 22°C for 4 days. The sensitive isolate developed gray mold disease in untreated but not Topsin M-treated fruit. The resistant isolate developed gray mold disease of equal severity in both, the control and fungicide-treated fruit. This experiment was repeated once. The results of the study show that resistance to MBC fungicides is common and widespread in B. cinerea from strawberry in South Carolina. Prior to this study, resistance to MBCs has only been reported in B. cinerea from ornamental crops grown in greenhouses in South Carolina (4). References: (1) J. E. Luck and M. R. Gillings. Mycol. Res. 99:1483, 1995. (2) R. W. S. Weber and M. Hahn. J. Plant Dis. Prot. 118:17, 2011. (3) O. Yarden and T. Katan. Phytopathology 83:1478, 1993. (4) L. F. Yourman and S. Jeffers. Plant Dis. 83:569, 1999.
灰葡萄孢菌（Botrytis cinerea Pers.:Fr.）是灰霉病的病原菌，也是影响草莓（Fragaria×ananassa）的最重要的植物病原真菌之一。灰霉病的防治主要依赖于杀菌剂，包括甲基苯并咪唑氨基甲酸酯（MBC）类的甲基托布津。2011年，从南卡罗来纳州切斯尼、佛罗伦萨、列克星敦、麦克比、莫内塔和北奥古斯塔附近的商业种植园采集了有灰霉病症状的草莓。在过去3年中，这些种植园中的大多数都使用MBC类杀菌剂来防治灰霉病。共获得了124个单孢灰葡萄孢菌分离株，每个分离株来自不同的果实。按照之前描述的方法（1），使用分生孢子萌发试验测定了对甲基托布津（Topsin M 70WP，Cerexagri-Nisso LLC，宾夕法尼亚州普鲁士王镇）的抗性。大多数分离株（81.4%）具有抗性，其余为敏感株。在所有地点都发现了抗性分离株，有些种群（切斯尼、麦克比和列克星敦）没有敏感分离株。从35个抗性分离株（代表所有地点）和10个敏感分离株（来自南卡罗来纳州的切斯尼、莫内塔和北奥古斯塔）中提取了基因组DNA，并按照之前描述的方法（2）确定了MBC类杀菌剂抗性的分子基础。所有对MBC有抗性的分离株都具有E198A突变，已知该突变会使包括灰葡萄孢菌在内的许多真菌对MBC类杀菌剂产生高水平抗性（2,3）。使用离体草莓果实试验评估病害情况。将商业种植的草莓果实（每个分离株和杀菌剂处理各24个）用水冲洗、晾干，在接种前4小时用手动喷雾器喷清水或2.4克/升的甲基托布津直至径流。用无菌注射器刺伤果实，接种30微升敏感或抗性分离株的分生孢子悬浮液（10个孢子/毫升）。接种后，将果实置于22°C下4天。敏感分离株在未处理但经甲基托布津处理的果实中均未引发灰霉病。抗性分离株在对照和经杀菌剂处理的果实中引发的灰霉病严重程度相同。该实验重复了一次。研究结果表明，在南卡罗来纳州草莓的灰葡萄孢菌中，对MBC类杀菌剂的抗性普遍且广泛存在。在本研究之前，仅报道过南卡罗来纳州温室种植的观赏作物中的灰葡萄孢菌对MBCs有抗性（4）。参考文献：（1）J. E. Luck和M. R. Gillings。《真菌学研究》99:1483，1995年。（2）R. W. S. Weber和M. Hahn。《植物病害防治杂志》118:17，2011年。（3）O. Yarden和T. Katan。《植物病理学》83:1478，1993年。（4）L. F. Yourman和S. Jeffers。《植物病害》83:569，1999年。
