Duan Yabing, Yang Ying, Li Tao, Zhao Donglei, Cao Junhong, Shi Yiyuan, Wang Jianxin, Zhou Mingguo
College of Plant Protection, State and Local Joint Engineering Research Centre of Green Pesticide Invention and Application, Nanjing Agricultural University, Nanjing, China.
Pest Manag Sci. 2016 Nov;72(11):2128-2135. doi: 10.1002/ps.4243. Epub 2016 Mar 3.
The point mutation at codon 200 (TTC→TAC, F200Y) of the β -tubulin gene confers resistance to benzimidazole fungicide in Fusarium asiaticum. These isolates with this mutation have been detected mainly by determining the minimum inhibitory concentration (MIC) of fungicides, which is always time consuming, tedious and inefficient.
A visual, rapid and efficient method with high specificity was developed, based on loop-mediated isothermal amplification (LAMP). Six sets of LAMP primers were designed, and one set was optimised specifically to distinguish the F200Y mutant genotype. With the optimal LAMP primers, concentrations of LAMP components were optimised. The optimal reaction conditions were 57-64 °C for 75 min. The feasibility of the LAMP assay for detection of the F200Y mutant genotype of F. asiaticum was demonstrated by assaying diseased wheat spikelets that were artificially inoculated in the field.
The new LAMP assay had good specificity, sensitivity, stability and repeatability. It will be useful for assessing the risk of F. asiaticum populations with carbendazim resistance developing in the field, and will also provide important reference data for integrated control of Fusarium head blight caused by F. asiaticum. © 2016 Society of Chemical Industry.
亚洲镰孢菌β-微管蛋白基因第200位密码子的点突变（TTC→TAC，F200Y）使其对苯并咪唑类杀菌剂产生抗性。这些具有该突变的分离株主要通过测定杀菌剂的最小抑菌浓度（MIC）来检测，这一过程总是耗时、繁琐且效率低下。
基于环介导等温扩增技术（LAMP）开发了一种可视化、快速且高效的高特异性方法。设计了6组LAMP引物，并优化了一组引物以特异性区分F200Y突变基因型。使用优化后的LAMP引物，对LAMP反应体系的各组分浓度进行了优化。最佳反应条件为57 - 64℃反应75分钟。通过对田间人工接种病害小麦小穗的检测，证明了LAMP检测方法用于检测亚洲镰孢菌F200Y突变基因型的可行性。
新的LAMP检测方法具有良好的特异性、敏感性、稳定性和重复性。它将有助于评估田间亚洲镰孢菌对多菌灵产生抗药性群体的风险，也将为亚洲镰孢菌引起的赤霉病综合防治提供重要参考数据。© 2016化学工业协会。
