Development of a qPCR Assay for the Detection and Quantification of the Fungal Pathogen Calonectria canadiana on Conifers

IF 1.3 4区 农林科学 Q3 FORESTRY Forest Pathology Pub Date : 2024-09-09 DOI:10.1111/efp.12885
Philippe Tanguay, Nathan Benoit, Amélie Potvin, Louis Bernier
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Abstract

A real-time PCR TaqMan assay was developed for the detection of Calonectria canadiana, a fungal pathogen responsible for damping off, root rot and seedling blight in conifer forest nurseries in central and eastern North America. While highly significant in Quebec Forest nurseries, coniferous seedling mortality decreased significantly when nurseries transitioned from bare root to container seedling production. However, over the past few years, this pathogen has re-emerged as a threat and millions of container white spruce seedlings were culled in two nurseries in eastern Quebec. A sensitive detection and quantification assay for C. canadiana was essential to investigate the biological and environmental factors driving this new epidemic. We designed primers and a TaqMan probe targeting the internal transcribed spacer (ITS) of C. canadiana. The resulting Ccan TaqMan assay successfully differentiated C. canadiana from other soil-borne pathogens of the Nectriaceae encountered in Quebec Forest nurseries. The limit of detection of the assay was established at eight copies of C. canadiana ITS. The Ccan TaqMan assay quickly identified the presence of the pathogen in both symptomatic and asymptomatic white spruce (Picea glauca) seedlings. Furthermore, we demonstrated that the pathogen was more easily detected when DNA was extracted from necrotic needles at the base of the stem rather than from necrotic roots. This molecular tool will greatly aid in understanding the biology and epidemiology of C. canadiana.

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开发用于检测和定量针叶树上真菌病原体 Calonectria canadiana 的 qPCR 分析方法
我们开发了一种实时 PCR TaqMan 分析方法,用于检测北美中部和东部针叶林苗圃中的一种真菌病原体 Calonectria canadiana,这种病原体可导致苗木受潮、根腐病和枯萎病。虽然针叶树苗死亡率在魁北克森林苗圃中非常高,但当苗圃从裸根苗生产过渡到容器苗生产时,针叶树苗死亡率明显下降。然而,在过去几年中,这种病原体再次成为威胁,在魁北克东部的两个苗圃中,数百万株容器白云杉幼苗被剔除。要研究导致这种新流行病的生物和环境因素,就必须对 C. canadiana 进行灵敏的检测和定量分析。我们设计了针对 C. canadiana 内部转录间隔区 (ITS) 的引物和 TaqMan 探针。由此产生的 Ccan TaqMan 检测方法成功地将 C. canadiana 与魁北克森林苗圃中遇到的其他花椰菜科土传病原体区分开来。该检测方法的检测限被确定为 C. canadiana ITS 的 8 个拷贝。Ccan TaqMan 检测法可快速确定有症状和无症状的白云杉(Picea glauca)幼苗中是否存在病原体。此外,我们还证明,从茎基部坏死针叶中提取 DNA 比从坏死根部提取 DNA 更容易检测到病原体。这种分子工具将大大有助于了解 C. canadiana 的生物学和流行病学。
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来源期刊
Forest Pathology
Forest Pathology 农林科学-林学
CiteScore
2.70
自引率
0.00%
发文量
62
审稿时长
12-24 weeks
期刊介绍: This peer reviewed, highly specialized journal covers forest pathological problems occurring in any part of the world. Research and review articles, short communications and book reviews are addressed to the professional, working with forest tree diseases caused by fungi, bacteria, nematodes, viruses, and phytoplasms; their biology, morphology, and pathology; disorders arising from genetic anomalies and physical or chemical factors in the environment. Articles are published in English. Fields of interest: Forest pathology, effects of air pollution and adverse environmental conditions on trees and forest ecosystems.
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