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
{"title":"针对线粒体基因的实时荧光定量PCR超灵敏检测雨疫霉。","authors":"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","doi":"10.1094/PHYTO-01-25-0020-R","DOIUrl":null,"url":null,"abstract":"<p><p><i>Phytophthora pluvialis</i> 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 (<i>ypt1</i>). A qPCR assay targeting a multiple-copy mitochondrial gene (Cytochrome c oxidase subunit 2 (<i>cox2</i>)) was designed to increase sensitivity of <i>P. pluvialis</i> 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 <i>ypt1</i> assay. In New Zealand samples, the assay was found to consistently detect <i>P. pluvialis</i> in all stages of needle disease symptoms from early asymptomatic infection through to fully cast needles. The new assay allowed for asymptomatic detection of <i>P. pluvialis</i> 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 <i>P. pluvialis</i>. 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 <i>P. pluvialis</i> in affected forests.</p>","PeriodicalId":20410,"journal":{"name":"Phytopathology","volume":" ","pages":""},"PeriodicalIF":3.1000,"publicationDate":"2025-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Ultra-Sensitive Detection of <i>Phytophthora pluvialis</i> by Real-Time PCR Targeting a Mitochondrial Gene.\",\"authors\":\"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\",\"doi\":\"10.1094/PHYTO-01-25-0020-R\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p><i>Phytophthora pluvialis</i> 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 (<i>ypt1</i>). A qPCR assay targeting a multiple-copy mitochondrial gene (Cytochrome c oxidase subunit 2 (<i>cox2</i>)) was designed to increase sensitivity of <i>P. pluvialis</i> 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 <i>ypt1</i> assay. In New Zealand samples, the assay was found to consistently detect <i>P. pluvialis</i> in all stages of needle disease symptoms from early asymptomatic infection through to fully cast needles. The new assay allowed for asymptomatic detection of <i>P. pluvialis</i> 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 <i>P. pluvialis</i>. 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 <i>P. pluvialis</i> in affected forests.</p>\",\"PeriodicalId\":20410,\"journal\":{\"name\":\"Phytopathology\",\"volume\":\" \",\"pages\":\"\"},\"PeriodicalIF\":3.1000,\"publicationDate\":\"2025-08-20\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Phytopathology\",\"FirstCategoryId\":\"97\",\"ListUrlMain\":\"https://doi.org/10.1094/PHYTO-01-25-0020-R\",\"RegionNum\":2,\"RegionCategory\":\"农林科学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q2\",\"JCRName\":\"PLANT SCIENCES\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Phytopathology","FirstCategoryId":"97","ListUrlMain":"https://doi.org/10.1094/PHYTO-01-25-0020-R","RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"PLANT SCIENCES","Score":null,"Total":0}
Ultra-Sensitive Detection of Phytophthora pluvialis by Real-Time PCR Targeting a Mitochondrial Gene.
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.
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.