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

Abstract

Ascospores, discharged naturally from apothecia growing on rachis debris, were used as inoculum to examine the invasion of ash tissues by Hymenoscyphus fraxineus 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, petioles and stem internode tissues. Light microscopy, using 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 host range is controlled by effector‐triggered immunity.

A versatile method for assessing pathogenicity of Hymenoscyphus fraxineus to ash foliage

“…This study adds to previous observations on development of symptoms following ash inoculations reported by Schwanda and Kirisits (2016) and Mansfield et al (2018) by noting the "pre-top dead" symptom, which is clearly the precursor to wilting of the distal end of the leaf. In two inoculations, this phenotype occurred when no rachis lesion was visible.…”

“…The fungus is grown on malt agar for 3 weeks, then small ash wood sticks are placed on the fungal colony and incubated for another 3 weeks. More recently, Mansfield, Galambos, and Saville (2018) were successful in inducing infection of leaves following inoculation with quantified ascospores numbers in a water suspension. Commonly, lesion development is assessed by destructive sampling several months after inoculation.…”

Life at the edge – the cytology and physiology of the biotroph to necrotroph transition in Hymenoscyphus fraxineus during lesion formation in ash

“…Electron microscopy used tissue 6 and 8 days after inoculation, prior to and after the appearance of macroscopic lesions (Mansfield et al ., ). Slices of epidermis were examined to confirm that inoculations were progressing as expected from earlier work on living tissues.…”

“…The progress of colonization by H. fraxineus was examined here by light and electron microscopy, extending the time period of observations described by Mansfield et al (2018). The main aim of the study was to characterize the cytology of the biotroph to necrotroph transition during infection of ash stems.…”

“…The spread of ash dieback disease across the United Kingdom follows a pattern largely consistent with the distribution of ascospores of Hymenoscyphus fraxineus from the east (Timmermann et al ., ). However, there have been few attempts to follow infection from ascospore inocula (Cleary et al ., ; Fones et al ., ; Mansfield et al ., ; Orton et al ., ). The symptoms of the disease, involving death and browning of leaves and stems, suggest that the pathogen has a distinctive necrotrophic mode of infection (Gross et al ., ; Stenlid et al ., ).…”