Development of a Loop-Mediated Isothermal Amplification Method for the Rapid Detection of Phytopythium vexans.

Jianping Xu,Yongting Yu,Xiaojie Wang,Tuhong Wang,Jia Chen,Zhimin Li,Chunsheng Gao,Litao Guo,Haojun Ji,Yi Cheng

doi:10.3389/fmicb.2021.720485

Abstract

Brown root rot caused by Phytopythium vexans is a new destructive root disease on many plants such as Gingko, Citrus, kiwifruit, and ramie. The establishment of loop-mediated isothermal amplification (LAMP) technology for detecting P. vexans can help monitor and control brown root rot quickly, efficiently, and accurately. LAMP technology is known for its simplicity, sensitivity, and speed; and it does not require any specialized equipment – a water bath or a thermoblock is sufficient for isothermal amplifications. LAMP products can be visualized by using hydroxy naphthol blue (HNB) dye or agarose gel electrophoresis. In this study, by searching and comparing the internal transcribed spacer (ITS) sequences of P. vexans and the related species in oomycete genera Pythium, Phytopythium, and Phytophthora, we designed specific primers targeting the ITS gene region of P. vexans. Using HNB dye, we established a LAMP technique for rapid detection of P. vexans by visible color change. In addition, we optimized the protocol to enhance both sensitivity and specificity for P. vexans detection. Under the optimized condition, our protocol based on LAMP technology could detect as low as 24 copies of the P. vexans genomic DNA, which is ∼100 times more sensitive than conventional PCR. This method can successfully detect P. vexans using cell suspensions from P. vexans – infected ramie root tissues.

Highlights

Oomycetes belong to the Kingdom Stramenopila (Baldauf et al, 2000; Yoon et al, 2002), which includes diverse microorganisms living in marine, freshwater, and terrestrial environments (Sparrow, 1960; Karling, 1981)
Two P. vexans isolates, two isolates of two other Phytopythium species, 11 isolates of 11 Pythium species, one P. capsici isolate, and four isolates of four Fusarium species obtained from different provinces in China (Table 1) were used to test the specificity of the loopmediated isothermal amplification (LAMP) primers and reaction conditions
Brown root rot of ramie caused by P. vexans has become an increasingly important disease endangering ramie production in China, resulting in >40% yield loss in some ramie plantations (Zhu et al, 2014; Yu et al, 2016)

Summary

Introduction

Oomycetes belong to the Kingdom Stramenopila (Baldauf et al, 2000; Yoon et al, 2002), which includes diverse microorganisms living in marine, freshwater, and terrestrial environments (Sparrow, 1960; Karling, 1981). A few Phytopythium species have been observed to cause diseases in plants, such as Phytopythium litorale infecting the Old-World sycamore Platanus orientalis (Dervis et al, 2020); Phytopythium helicoides causing citrus fruit and root rot R. et al, 2016); LAMP Technique for Phytopythium vexans and Phytopythium vexans (de Bary) (Lévesque et al, 2008; Bala et al, 2010) causing root rot in many economically important plants (Adhikari et al, 2013; de Cock et al, 2015). P. vexans can cause patch canker, damping-off, and crown rot, stem rot, and root rot in many economically important fruit trees, such as durian, kiwifruit, apple, and avocado in Vietnam, Turkey, Morocco, and Mexico, respectively (Polat et al, 2017; Hernández et al, 2019; Jabiri et al, 2020; Thao et al, 2020). The increasing reports of diseases caused by P. vexans suggest that this pathogen has the potential to cause large-scale disease outbreaks across multiple plants in the future

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