Gusberti Michele, Patocchi Andrea, Gessler Cesare, Broggini Giovanni A L
Institute of Integrative Biology Zurich, Plant Pathology Group, Swiss Federal Institute of Technology, CH-8092 Zürich, Switzerland.
Agroscope Changins Wädenswil ACW Research Station, Schloss, CH-8820 Wädenswil, Switzerland.
Plant Dis. 2012 Dec;96(12):1791-1797. doi: 10.1094/PDIS-12-11-1058-RE.
A quantitative real-time polymerase chain reaction (qPCR) was developed and validated for quantification of Venturia inaequalis in infected leaf tissue of Malus × domestica. The method is based on dual-labeled hybridization probes, allowing simultaneous detection of host and pathogen DNA within one single reaction. Limit of quantification for the pathogen was 0.5 pg per reaction and, for the host, reached 5 pg per reaction. The fungal growth measured in four apple cultivars 2 weeks after inoculation significantly correlated with their different level of scab resistance and allowed the observation of ontogenic resistance. After sporulation on the youngest leaf, fungal biomass in susceptible 'Gala' was 118 times higher than in resistant 'Florina' and 'Discovery' while intermediate values were found with the intermediate susceptible 'Milwa'. Correlation was also observed between severity classes obtained by visual scoring of symptoms and qPCR results. Moreover, qPCR demonstrated validity of the developed method as a disease severity forecast tool 10 days after the pathogen's inoculation and prior to the appearance of the symptoms. Applications of the methodology can include the quantification of scab resistance during breeding programs, evaluation of fungicide and biocontrol efficacy, and quantification of the fitness of different pathogenic strains.
开发并验证了一种定量实时聚合酶链反应（qPCR）方法，用于定量分析苹果属植物感染叶片组织中的苹果黑星病菌。该方法基于双标记杂交探针，可在单个反应中同时检测宿主和病原体DNA。病原体的定量限为每个反应0.5 pg，宿主的定量限为每个反应5 pg。接种两周后，在四个苹果品种中测量的真菌生长与它们不同的抗黑星病水平显著相关，并能够观察到个体发育抗性。在最幼嫩叶片上形成孢子后，感病品种“嘎啦”中的真菌生物量比抗病品种“弗洛里纳”和“发现”高118倍，而中度感病品种“米尔瓦”的真菌生物量处于中间值。通过症状目视评分获得的严重程度等级与qPCR结果之间也存在相关性。此外，qPCR证明了所开发方法作为一种在病原体接种后10天且症状出现之前的病害严重程度预测工具的有效性。该方法的应用可包括在育种计划中定量抗黑星病性、评估杀菌剂和生物防治效果以及定量不同致病菌株的适合度。
