LAMP detection of the genetic element ‘Mona’ associated with DMI resistance in Monilinia fructicola

IF 4.2 1区农林科学 Q1 AGRONOMY

Pest Management Science Pub Date : 2018-08-19 DOI:10.1002/ps.5178

Shuning Chen, Guido Schnabel, Huizhu Yuan, Chaoxi Luo

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引用次数: 12

Abstract

BACKGROUND

The increasing use of demethylation inhibitor (DMI) fungicides for the control of peach brown rot has resulted in resistance in Monilinia fructicola. Resistance in the southeastern USA is caused by overexpression of the MfCYP51 gene due to the presence of a 65-bp inserted element ‘Mona’ located in the upstream regulatory region of MfCYP51. A rapid diagnostic assay would be useful to detect the presence and monitor further spread of this resistance mechanism.

RESULTS

A loop-mediated isothermal amplification (LAMP) method was developed for rapid detection of ‘Mona’-based DMI resistance. The assay was optimized for specificity and sensitivity, and was shown to detect the presence of 10 fg of purified target DNA per reaction within 85 min. Only DNA isolated from DMI-resistant isolates containing ‘Mona’ resulted in a fluorescent signal after LAMP assay amplification. DNA from sensitive isolates from China and the USA and six other common fungal species of peach did not yield a signal. The method also positively identified ‘Mona’ from crude DNA extracts (using Lyse and Go reagents heated to 100 °C for 10 min) obtained from the mycelium and conidia of symptomatic fruit.

CONCLUSION

Considering its specificity, stability and repeatability, the LAMP assay could be a valuable tool for rapid on-site diagnosis of M. fructicola isolates resistant to DMI fungicides in the southeastern USA. © 2018 Society of Chemical Industry

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山核桃念珠菌抗DMI相关遗传因子“Mona”的LAMP检测

背景随着去甲基化抑制剂(DMI)杀菌剂在桃褐腐病防治中的应用越来越广泛，桃褐腐病对桃褐腐病产生了抗性。在美国东南部，抗性是由于MfCYP51基因的过表达引起的，这是由于MfCYP51上游调控区存在一个65 bp的插入元件“Mona”。快速诊断试验将有助于发现这种耐药机制的存在并监测其进一步扩散。结果建立了一种环介导等温扩增(LAMP)方法，用于快速检测“Mona”基DMI耐药性。该方法对特异性和敏感性进行了优化，每次反应可在85分钟内检测到10 fg纯化的目标DNA。只有从含有' Mona '的dmi耐药分离株中分离的DNA在LAMP扩增后产生荧光信号。从中国和美国的敏感分离株和其他六种常见的桃真菌中提取的DNA没有产生信号。该方法还从有症状果实的菌丝体和分生孢子的粗DNA提取物(使用Lyse和Go试剂加热至100℃10 min)中鉴定出“Mona”。结论LAMP法具有特异性、稳定性和可重复性，可作为快速诊断美国东南部地区对DMI杀菌剂耐药的果分枝杆菌的有效方法。©2018化学工业协会

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来源期刊

Pest Management Science 农林科学-昆虫学

CiteScore

7.90

自引率

9.80%

发文量

553

审稿时长

4.8 months

期刊介绍： Pest Management Science is the international journal of research and development in crop protection and pest control. Since its launch in 1970, the journal has become the premier forum for papers on the discovery, application, and impact on the environment of products and strategies designed for pest management. Published for SCI by John Wiley & Sons Ltd.