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Early detection of Neophysopella tropicalis in grapevine leaves and on spore traps by qPCR
Plant Pathology ( IF 2.3 ) Pub Date : 2020-10-15 , DOI: 10.1111/ppa.13295 Isabela V. Primiano 1, 2 , Mariana C. Cia 1 , Luis E. A. Camargo 1 , Lilian Amorim 1
Plant Pathology ( IF 2.3 ) Pub Date : 2020-10-15 , DOI: 10.1111/ppa.13295 Isabela V. Primiano 1, 2 , Mariana C. Cia 1 , Luis E. A. Camargo 1 , Lilian Amorim 1
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Neophysopella tropicalis, one of the causal agents of Asian grapevine leaf rust (AGLR), can cause severe epidemics in Brazil that lead to yield losses in commercial vineyards. An early detection of the pathogen by air sampling of urediniospores on spore traps or in symptomless leaves would be valuable to multiple studies, such as epidemics modelling, risk forecasting, monitoring of pathogen introductions in rust‐free areas, and predicting the beginning of epidemics. This study developed a quantitative PCR (qPCR) protocol to quantify N. tropicalis urediniospores attached to adhesive tapes and in grapevine leaves before symptom appearance. A specific primer pair was designed based on the internal transcribed spacer (ITS) sequence region of the AGLR pathogen. Standard amplification curves using genomic DNA from urediniospores of N. tropicalis and from urediniospores attached to adhesive tapes were established. Grapevine leaves inoculated with N. tropicalis were collected at 2, 5, and 7 days postinoculation (dpi). One primer pair (580F/720R) amplified a 140 bp product in all AGLR isolates but did not amplify products of other rust genera, such as Phakopsora, Puccinia, Hemileia, Tranzschelia, Cerotelium, and Coleosporium. As little as 0.1 pg DNA and 10 urediniospores of N. tropicalis attached to adhesive tapes could be detected. qPCR enabled the detection of the pathogen as early as 2 dpi, before symptom appearance. This method can be used to monitor N. tropicalis inoculum in grapevine‐growing areas and to quantify symptomless infections of the AGLR pathogen.
中文翻译：
qPCR早期检测葡萄叶和孢子陷阱中的热带新藻
热带新藻，亚洲葡萄叶锈病（AGLR）的病因之一，可在巴西引起严重的流行病，导致商业性葡萄园的产量下降。通过对孢子阱或无症状叶片上的雷公孢子进行空气采样来早期发现病原体，对于多种研究具有重要的价值，例如流行病模型，风险预测，监测无锈病地区的病原体传入以及预测流行病的开始。本研究中开发的定量PCR（qPCR）协议来量化Ñ。热带在症状出现之前，用胶带和葡萄叶中附着的urediosiospores。基于AGLR病原体的内部转录间隔区（ITS）序列区域设计了特异性引物对。使用来自夏孢子基因组DNA标准扩增曲线Ñ。建立了热带鱼，并从附着在胶带上的海胆孢子建立。接种葡萄叶ñ。在接种后第2、5和7天（dpi）收集热带病。一个引物对（580F / 720R）在所有AGLR分离物中扩增了一个140 bp的产物，但未扩增其他锈菌属的产物，例如Phakopsora，Puccinia，Hemileia，Tranzschelia，Cerotelium和Coleosporium。少至0.1 pg DNA和10个N的indusiosiospores 。可以检测到附着在胶带上的tropicalis。qPCR能够在症状出现之前最早检测到2 dpi的病原体。此方法可用于监测Ñ。热带接种在葡萄种植区和量化AGLR病原体感染无症状。
更新日期：2020-10-15
中文翻译：
qPCR早期检测葡萄叶和孢子陷阱中的热带新藻
热带新藻，亚洲葡萄叶锈病（AGLR）的病因之一，可在巴西引起严重的流行病，导致商业性葡萄园的产量下降。通过对孢子阱或无症状叶片上的雷公孢子进行空气采样来早期发现病原体，对于多种研究具有重要的价值，例如流行病模型，风险预测，监测无锈病地区的病原体传入以及预测流行病的开始。本研究中开发的定量PCR（qPCR）协议来量化Ñ。热带在症状出现之前，用胶带和葡萄叶中附着的urediosiospores。基于AGLR病原体的内部转录间隔区（ITS）序列区域设计了特异性引物对。使用来自夏孢子基因组DNA标准扩增曲线Ñ。建立了热带鱼，并从附着在胶带上的海胆孢子建立。接种葡萄叶ñ。在接种后第2、5和7天（dpi）收集热带病。一个引物对（580F / 720R）在所有AGLR分离物中扩增了一个140 bp的产物，但未扩增其他锈菌属的产物，例如Phakopsora，Puccinia，Hemileia，Tranzschelia，Cerotelium和Coleosporium。少至0.1 pg DNA和10个N的indusiosiospores 。可以检测到附着在胶带上的tropicalis。qPCR能够在症状出现之前最早检测到2 dpi的病原体。此方法可用于监测Ñ。热带接种在葡萄种植区和量化AGLR病原体感染无症状。
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