Anderson Ryan G, McDowell John M
Department of Plant Pathology, Physiology and Weed Science, Virginia Tech, Blacksburg, VA, 24061-0329, USA.
Mol Plant Pathol. 2015 Oct;16(8):893-8. doi: 10.1111/mpp.12247. Epub 2015 May 4.
The accurate quantification of disease severity is important for the assessment of host-pathogen interactions in laboratory or field settings. The interaction between Arabidopsis thaliana and its naturally occurring downy mildew pathogen, Hyaloperonospora arabidopsidis (Hpa), is a widely used reference pathosystem for plant-oomycete interactions. Current methods for the assessment of disease severity in the Arabidopsis-Hpa interaction rely on measurements at the terminal stage of pathogen development; namely, visual counts of spore-producing structures or the quantification of spore production with a haemocytometer. These assays are useful, but do not offer sensitivity for the robust quantification of small changes in virulence or the accurate quantification of pathogen growth prior to the reproductive stage. Here, we describe a quantitative real-time polymerase chain reaction (qPCR) assay for the monitoring of Hpa growth in planta. The protocol is rapid, inexpensive and can robustly distinguish small changes in virulence. We used this assay to investigate the dynamics of early Hpa mycelial growth and to demonstrate the proof of concept that this assay could be used in screens for novel oomycete growth inhibitors.
疾病严重程度的准确量化对于在实验室或田间环境中评估宿主与病原体的相互作用至关重要。拟南芥与其天然存在的霜霉病病原体——寄生霜霉（Hyaloperonospora arabidopsidis，Hpa）之间的相互作用，是用于植物与卵菌相互作用研究的一种广泛使用的参考病理系统。目前评估拟南芥 - Hpa相互作用中疾病严重程度的方法依赖于病原体发育末期的测量；即，对产孢结构进行目视计数或用血球计数板对孢子产量进行量化。这些测定方法很有用，但对于毒力微小变化的可靠量化或生殖阶段之前病原体生长的准确量化缺乏敏感性。在此，我们描述了一种用于监测植物中Hpa生长的定量实时聚合酶链反应（qPCR）测定方法。该方法快速、廉价，并且能够可靠地区分毒力的微小变化。我们使用该测定方法研究了Hpa早期菌丝体生长的动态，并证明了该测定方法可用于新型卵菌生长抑制剂筛选的概念验证。
