Ultra-Sensitive Detection of Phytophthora pluvialis by Real-Time PCR Targeting a Mitochondrial Gene.

IF 3.1 2区 农林科学 Q2 PLANT SCIENCES
R O'Neill, E McLay, L Nunes Leite, P Panda, A Perez-Sierra, A Eacock, J M LeBoldus, E A Stamm, S Fraser, R L McDougal
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Abstract

Phytophthora pluvialis is a pathogen present in USA, New Zealand, UK and Belgium forests. Reported hosts include Douglas-fir in the USA, New Zealand, UK and Belgium, as well as tanoak in the USA, radiata pine in New Zealand, Japanese larch and western hemlock in the UK. Disease symptoms range from needle lesions and casting on radiata pine through to twig and stem cankers, and crown dieback on western hemlock, Douglas-fir and Japanese larch. Current detection methods rely on isolation and culture, or PCR targeting a single-copy gene (ypt1). A qPCR assay targeting a multiple-copy mitochondrial gene (Cytochrome c oxidase subunit 2 (cox2)) was designed to increase sensitivity of P. pluvialis detection, critical for early diagnostics. The resulting assay has a detection limit of 12.8 fg mycelial DNA, detecting the pathogen on average 6.12 qPCR cycles before the ypt1 assay. In New Zealand samples, the assay was found to consistently detect P. pluvialis in all stages of needle disease symptoms from early asymptomatic infection through to fully cast needles. The new assay allowed for asymptomatic detection of P. pluvialis in pine needles four weeks before visual symptoms of disease were observed. The availability of a highly sensitive assay has enabled diagnostic support of the biosecurity response in the UK during recent detection of P. pluvialis. The assay has been used in applications requiring detection at low pathogen titre levels including asymptomatic infection, stream baiting, cast needles and biosecurity response, demonstrating its efficacy for early detection of P. pluvialis in affected forests.

针对线粒体基因的实时荧光定量PCR超灵敏检测雨疫霉。
雨疫霉是一种存在于美国、新西兰、英国和比利时森林中的病原体。据报道,寄主包括美国、新西兰、英国和比利时的道格拉斯冷杉,以及美国的tanoak,新西兰的辐射松,日本落叶松和英国的西部铁杉。疾病症状包括从针叶损伤和辐射松到树枝和茎溃烂,以及西部铁杉、道格拉斯冷杉和日本落叶松的树冠枯死。目前的检测方法依赖于分离和培养,或针对单拷贝基因(ypt1)的PCR。设计了一种针对多拷贝线粒体基因(细胞色素c氧化酶亚基2 (cox2))的qPCR检测方法,以提高雨孢假单胞菌检测的灵敏度,这对早期诊断至关重要。该方法的检出限为12.8 fg,在ypt1检测之前平均检测6.12个qPCR循环。在新西兰的样本中,发现该检测方法在从早期无症状感染到完全铸造针头的针病症状的所有阶段都能始终检测到雨淋假单孢菌。新的检测方法允许在观察到疾病的视觉症状之前4周在松针中无症状地检测到雨孢假单孢菌。在英国最近发现雨形假单胞虫期间,一种高度敏感的检测方法的可用性为生物安全反应提供了诊断支持。该方法已用于需要在低病原体滴度水平下检测的应用,包括无症状感染、溪流诱饵、投针和生物安全响应,证明了其在受影响森林中早期检测雨淋假蝇的有效性。
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来源期刊
Phytopathology
Phytopathology 生物-植物科学
CiteScore
5.90
自引率
9.40%
发文量
505
审稿时长
4-8 weeks
期刊介绍: Phytopathology publishes articles on fundamental research that advances understanding of the nature of plant diseases, the agents that cause them, their spread, the losses they cause, and measures that can be used to control them. Phytopathology considers manuscripts covering all aspects of plant diseases including bacteriology, host-parasite biochemistry and cell biology, biological control, disease control and pest management, description of new pathogen species description of new pathogen species, ecology and population biology, epidemiology, disease etiology, host genetics and resistance, mycology, nematology, plant stress and abiotic disorders, postharvest pathology and mycotoxins, and virology. Papers dealing mainly with taxonomy, such as descriptions of new plant pathogen taxa are acceptable if they include plant disease research results such as pathogenicity, host range, etc. Taxonomic papers that focus on classification, identification, and nomenclature below the subspecies level may also be submitted to Phytopathology.
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