Variation in aggressiveness of Hymenoscyphus fraxineus genotypes amid the ash dieback epidemic

The ascomycete Hymenoscyphus fraxineus causes devastating damage to the European common ash (Fraxinus excelsior). The fungus originates from Asia, where it coexists with native ash species and completes its life cycle by sporulating on degrading ash leaf litter on the forest floor. Given this life cycle of the fungus, genotypes of H. fraxineus with varying degrees of aggressiveness may coexist in infected European ash forests. To test this hypothesis, we cultured 19 single-spore isolates from apothecia collected in a trial of heavily infected ash in Denmark and carried out stem inoculations on young ash seedlings. Microsatellite markers revealed that the 19 isolates were all genetically unique and did not show any genetic structure. High variation was observed among the 19 isolates in pathogenicity on young seedlings. The variation (assessed as necrosis development) was highly significant, but not correlated with the vigour of the fungal isolates when grown on culture media. The results support the hypothesis that aggressiveness of H. fraxineus may not be crucial for its fitness. In this sense, ash dieback disease may differ from other recent emerging infectious diseases on trees. We discuss the variation in aggressiveness of H. fraxineus in relation to durability of resistance and future management of the ash dieback epidemic. The findings of this study call for more research into natural variation in endophytic versus pathogenic behaviour of H. fraxineus on European ash.     

Pathogenicity of Hymenoscyphus fraxineus towards leaves of three European ash species: Fraxinus excelsior,F. angustifolia and F. ornus

This study aimed to demonstrate the association of the ash dieback pathogen Hymenoscyphus fraxineus with leaf symptoms on Fraxinus excelsior and to test its pathogenicity towards leaves of three European ash species, F. excelsior, F. angustifolia and F. ornus, in wound inoculation experiments. On F. excelsior, H. fraxineus was isolated from 94% of leaf rachises with necrotic lesions and from 74% of necrotic leaflet midribs. Following wound inoculation of leaf rachises, in two separate experiments performed in 2010 and 2011, the ash dieback pathogen caused symptoms (necrotic rachis lesions, leaf wilting and premature leaf shedding) on all three ash species, while control leaves remained symptomless. Hymenoscyphus fraxineus was consistently reisolated from fungus‐inoculated rachises. All 10 isolates tested were pathogenic to the three ash species and varied in virulence. Koch's postulates for H. fraxineus as causal agent of leaf symptoms on F. excelsior were fulfilled in this study. Complemented with the isolation of the fungus from naturally infected, symptomatic leaf rachises of F. angustifolia and F. ornus in previous investigations, H. fraxineus was confirmed to be a leaf pathogen of these ash species as well. The leaf inoculation experiments showed that F. excelsior was highly susceptible to H. fraxineus, F. angustifolia was equally or slightly less susceptible, whereas F. ornus was the least affected species; however, F. ornus should also be regarded as a host tree for the ash dieback pathogen. This susceptibility ranking corresponds well with field observations and previous stem inoculation experiments.     

Hymenoscyphus fraxineus is a leaf pathogen of local Fraxinus species in the Russian Far East

Dieback of European ash was first observed in Europe in the early 1990s. The disease is caused by the invasive ascomycete Hymenoscyphus fraxineus, proposed to originate from Far East Asia, where it has been considered a harmless saprotroph. This study investigates the occurrence of H. fraxineus in tissues of local ash species in the Russian Far East, and assesses its population‐specific genetic variation by ITS sequencing. Shoot dieback symptoms, characteristic of H. fraxineus infection on European ash, were common, but not abundant, on Fraxinus mandshurica and Fraxinus rhynchophylla trees in Far East Russia. High levels of pathogen DNA were associated with necrotic leaf tissues of these ash species, indicating that the local H. fraxineus population is pathogenic to their leaves. However, the low levels of H. fraxineus DNA detected in shoots with symptoms, the failure to isolate this fungus from such tissues, and the presence of other fungi with pathogenic potential in shoots with symptoms indicate that local H. fraxineus strains may not be responsible (or their role is negligible) for the observed ash shoot dieback symptoms in the region. Conspicuous differences in ITS rDNA sequences detected between H. fraxineus isolates from Russian Far East and European populations suggest that the current ash dieback epidemic in Europe might not directly originate from the Russian Far East. Revision of the herbarium material shows that the earliest specimen of H. fraxineus was collected in 1962 from the Russian Far East and the oldest H. fraxineus specimen of China was collected in 2004.     

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

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.