Yabing Duan1, Ying Yang1, Tao Li1, Donglei Zhao1, Junhong Cao1, Yiyuan Shi1, Jianxin Wang1, Mingguo Zhou2. 1. College of Plant Protection, State and Local Joint Engineering Research Centre of Green Pesticide Invention and Application, Nanjing Agricultural University, Nanjing, China. 2. College of Plant Protection, State and Local Joint Engineering Research Centre of Green Pesticide Invention and Application, Nanjing Agricultural University, Nanjing, China. mgzhou@njau.edu.cn.
Abstract
BACKGROUND: The point mutation at codon 200 (TTC→TAC, F200Y) of the β2 -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. RESULTS: 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. CONCLUSION: 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.
BACKGROUND: The point mutation at codon 200 (TTC→TAC, F200Y) of the β2 -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. RESULTS: 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. CONCLUSION: 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.
Authors: Hao Zhang; Balázs Brankovics; Theo A J van der Lee; Cees Waalwijk; Anne A D van Diepeningen; Jin Xu; Jingsheng Xu; Wanquan Chen; Jie Feng Journal: PeerJ Date: 2016-10-25 Impact factor: 2.984
Authors: Katherine G Zulak; Belinda A Cox; Madeline A Tucker; Richard P Oliver; Francisco J Lopez-Ruiz Journal: Front Microbiol Date: 2018-04-13 Impact factor: 5.640