Abstract

Sweet cherries (Prunus avium) are an economically important crop in British Columbia, Canada. Cherries are harvested and distributed locally and overseas, where seemingly healthy fruit can succumb to postharvest diseases if disease conditions are met. Disease mitigation includes pre-harvest controls such as disease prediction models, disease monitoring, and fungicide applications. Development of disease-prediction models requires an understanding of how host and environmental conditions can affect the quantity of pathogens; therefore, quick, sensitive and accurate methods for pathogen quantification are required. This study has identified Alternaria spp. and Botrytis cinerea as major contributors to sweet cherry rot in Kelowna, British Columbia, in 2016 and developed a novel duplex droplet digital PCR assay for the rapid, concurrent quantification of the two pathogens. The assay involves the amplification of two abundant target regions, the internal transcribed spacer, and the intergenic spacer, in Alternaria spp. and B. cinerea, respectively. The detection limit was 0.1 pg of DNA for each target. The assay was validated during the 2016 and 2017 growing seasons at the bud break (2017 only), full bloom, petal fall, onset of straw colour and harvest stages of sweet cherry. In general, pathogen quantities were lowest at petal fall and highest during late season. The method can be used in future studies to evaluate pathogen quantities during the growing season and to facilitate the development of disease-prediction models and mitigation practices for growers.

摘要

甜樱桃（Prunus avium）是加拿大不列颠哥伦比亚省重要的经济作物。樱桃在本地和海外收获和分销，如果满足疾病条件，看似健康的水果可能会死于收获后的疾病。疾病缓解包括收获前控制，例如疾病预测模型、疾病监测和杀菌剂应用。疾病预测模型的开发需要了解宿主和环境条件如何影响病原体的数量；因此，需要快速、灵敏和准确的病原体定量方法。该研究已鉴定出链格孢属（Alternaria spp）。和灰葡萄孢作为 2016 年不列颠哥伦比亚省基洛纳甜樱桃腐病的主要贡献者，他们开发了一种新型双链液滴数字 PCR 检测方法，用于快速、同时定量两种病原体。该测定涉及在Alternaria spp 中扩增两个丰富的目标区域，即内部转录间隔区和基因间间隔区。和B. cinerea， 分别。每个目标的检测限为 0.1 pg DNA。该测定在 2016 年和 2017 年的萌芽期（仅限 2017 年）、盛开、花瓣落下、稻草色开始和甜樱桃收获阶段得到验证。一般来说，病原体数量在花瓣落下最低，在晚季最高。该方法可用于未来的研究，以评估生长季节的病原体数量，并促进疾病预测模型的开发和种植者的缓解措施。