Spray inoculation and image analysis-based quantification of powdery mildew disease severity on pea leaves

Pea (Pisum sativum) is an important agricultural legume crop, but powdery mildew disease caused by the biotrophic fungus Erysiphe pisi regularly limits its annual yield. Assays to evaluate the efficacy of potential antifungal compounds or resistance genes for disease control require a simple fungal inoculation method that provides control over the initial inoculum concentration and enables uniform inoculum distribution within a leaf and across replicates as well as a method for the quantitative assessment of disease severity. Here, we present an easy spray inoculation method for the uniform distribution of a defined concentration of E. pisi conidia on the leaves of pea plants and a semi-automated image analysis-based quantification of disease symptoms. The uniformity in conidial distribution was validated using a novel grading system termed the uniformity index. In addition, RT-qPCR was used to validate the reproducibility of the spray inoculation method and image analysis-based disease quantification. These procedures permit the accurate quantification of powdery mildew disease severity at macroscopic and molecular levels.

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  Uniform and reproducible inoculum distribution on leaves using a simple and inexpensive spray device
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  Rapid and reproducible quantification of powdery mildew disease symptoms using open-source software without the requirement of computational expertise