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The use of ascospores of the dieback fungus Hymenoscyphus fraxineus for infection reveals a period of biotrophic interaction in penetrated ash cells

John Mansfield: Imperial College London

<div>Ascospores, discharged naturally from apothecia growing on rachis debris, were used as inoculum to examine the invasion of ash tissues by the dieback fungus in order to understand the critical, but poorly understood, early interactions between host and pathogen. Methods were developed to collect ascospores for controlled infection assays on detached leaves, petiole and stem internode tissues. Light microscopy, including plasmolytic techniques, allowed the invasion of living plant cells to be observed. Ascospores were readily available from late May to September. On the plant surface, most spores differentiated directly to form appressoria without germ-tube growth. Direct penetration was followed by a significant period of biotrophic fungal growth before lesions developed. Following the formation of a vesicle-like structure after penetration, bulbous and elongated intracellular hyphae were produced in living plant cells. The use of ascospore inoculum, rather than mycelia, will allow natural and rapid screening of ash genotypes for resistance to the devastating dieback disease. The identification of the biotrophic phase of infection suggests that virulence is controlled by effector triggered immunity. Consequently, the emergence of the ongoing epidemic may be through loss of an avirulence determinant that has allowed a “host jump”, comparable to the wheat blast outbreak across Asia.</div>