The fungal predominance in stem collar necroses of Fraxinus excelsior: a study on Hymenoscyphus fraxineus multilocus genotypes

Over the past decades, European ash trees in Germany have been affected by ash dieback, reducing their vigour and mechanical resistance. Those trees that also have stem collar necroses and the resulting stem rot are particularly affected. In this study, multilocus genotypes (MLGs) of Hymenoscyphus fraxineus and their interactions with other fungi from stem collar necroses were analysed. Ten ash trees from three different adjacent forest stands in central Germany were sampled. A total number of 716 isolates were obtained from stem collar necroses from these ten trees. Microsatellite analysis was successfully performed on 274 isolates identified as H. fraxineus and 26 MLGs were revealed. The number of MLGs varied from one to seven per tree and did not correspond to the number or severity of necroses. A striking result was that five of the MLGs occurred in two trees. All other MLGs occurred independently in only one tree, as expected. Our data show that when multiple MLGs were observed in a tree, one of the MLGs outnumbered the others, indicating that H. fraxineus is a primary coloniser of stem collar necroses. A total of 61 morphotypes, including H. fraxineus, were identified and discussed, comprising endophytic, saprotrophic and pathogenic fungi. Between five and 19 different fungi were found per stem collar necrosis. The majority of all isolated morphotypes were Ascomycota (82%), with the most common orders being Xylariales and Hypocreales. The most frequently isolated morphotypes, apart from H. fraxineus, were Armillaria sp. and Diplodia fraxini. Together they account for more than three quarters of all assigned isolations. Apart from H. fraxineus, only Diplodia fraxini was isolated from all ten trees.