Opportunities for maintaining and rejuvenating strain collections of Hymenoscyphus fraxineus.

The ash dieback pathogen (Hymenoscyphus fraxineus) is a major threat to ash populations across Europe. This study examined how strain handling can affect in vitro growth and in planta virulence of H. fraxineus, with implications for maintaining culture collections for laboratory and greenhouse studies. Ten H. fraxineus strains were selected: seven from a strain collection and three newly isolated individuals. Growth characteristics of the strains were evaluated in vitro on malt extract agar (MEA) and ash-amended malt extract agar (AMEA). Virulence was also investigated in planta following artificial inoculations into Fraxinus excelsior saplings. In vitro growth rate did not correlate with virulence in planta. The strains were subsequently reisolated to investigate changes in in vitro growth and in planta virulence due to reintroduction to the host. Reintroduction to the host led to increased virulence of strains in planta, including a significant increase in necrosis growth rate following two reintroductions. There was a significant increase in necrosis growth rate following two reintroductions to the host. These observations were more distinct in newly isolated strains previously cultured on AMEA. Moreover, more strains were virulent when previously cultured on AMEA, compared with MEA. These results indicate not only that strains of H. fraxineus from collections can be rejuvenated, but also that the virulence of newly isolated strains can be increased. Overall, these results provide insights into the impacts of laboratory protocols on in vitro growth characteristics and in planta virulence of H. fraxineus strains. Furthermore, these results provide methodological opportunities for maintaining growth characteristics in strains intended for long-term use and storage in collections, as well as a possible method for rejuvenating strain virulence.