# Early-Stage Growth Restriction of Hymenoscyphus fraxineus on Tolerant Fraxinus excelsior Is Associated with Constitutive Chemical Defenses

## TL;DR

European ash trees in Japan resist ash dieback disease due to natural chemical defenses that limit the pathogen's early growth.

## Contribution

The study identifies constitutive chemical defenses, including fraxetin, as key factors in early-stage resistance to Hymenoscyphus fraxineus in tolerant ash trees.

## Key findings

- Fungal growth was restricted on Fraxinus excelsior at 7 days post inoculation.
- Fraxetin was detected in F. excelsior but not in F. angustifolia.
- Fraxetin treatment reduced ascospore germination in vitro.

## Abstract

Hymenoscyphus fraxineus is the causal agent of ash dieback, a devastating disease of European ash (Fraxinus excelsior). Although the pathogen is believed to have originated in East Asia and has been confirmed in Japan, European ash trees cultivated in the Sapporo Experimental Forest of Hokkaido University remain asymptomatic despite the presence of H. fraxineus. In this study, we investigated the early infection behavior of H. fraxineus and associated host defense responses by comparing asymptomatic F. excelsior with the susceptible control species F. angustifolia. Leaflets were inoculated with ascospores, and fungal development as well as host responses were examined microscopically during early infection stages. In addition, we analyzed the accumulation of selected coumarins, which have been proposed as candidate compounds associated with ash dieback tolerance, and assessed their effects on ascospore germination. We found that fungal growth was consistently restricted on F. excelsior at 7 days post inoculation, particularly at the stage of invasion into adjacent epidermal cells. Fraxetin was detected in F. excelsior leaflets but not in F. angustifolia, and fraxetin treatment significantly reduced ascospore germination in vitro. While typical markers of induced resistance were not clearly detected at the examined time points, these results indicate that constitutive chemical traits, including fraxetin accumulation, may contribute to early-stage suppression of H. fraxineus growth in F. excelsior. Together, our findings provide insight into early host–pathogen interactions associated with ash dieback tolerance and highlight the potential role of constitutive defenses during initial infection.