Visualizing Early Infection Sites of Rice Blast Disease (Magnaporthe oryzae) on Barley (Hordeum vulgare) Using a Basic Microscope and a Smartphone.

Abstract

Understanding how plants and pathogens interact, and whether that interaction culminates in defense or disease, is required to develop stronger and more sustainable strategies for plant health. Advances in methods that more effectively image plant-pathogen samples during infection and colonization have yielded tools such as the rice leaf sheath assay, which has been useful in monitoring infection and early colonization events between rice and the fungal pathogen, Magnaporthe oryzae. This hemi-biotrophic pathogen causes severe disease loss in rice and related monocots, including millet, rye, barley, and more recently, wheat. The leaf sheath assay, when performed correctly, yields an optically clear plant section, several layers thick, which allows researchers to perform live-cell imaging during pathogen attack or generate fixed samples stained for specific features. Detailed cellular investigations into the barley-M. oryzae interaction have lagged behind those of the rice host, in spite of the growing importance of this grain as a food source for animals and humans and as fermented beverages. Reported here is the development of a barley leaf sheath assay for intricate studies of M. oryzae interactions during the first 48 h post-inoculation. The leaf sheath assay, regardless of which species is being studied, is delicate; provided is a protocol that covers everything, from barley growth conditions and obtaining a leaf sheath, to inoculation, incubation, and imaging of the pathogen on plant leaves. This protocol can be optimized for high-throughput screening using something as simple as a smartphone for imaging purposes.