Ash dieback (ADB), caused by the invasive fungus Hymenoscyphus fraxineus, has spread extensively across Europe, resulting in considerable economic and ecological damage to European forests. Resistance screening is crucial for selecting and breeding common ash genotypes that are resistant to the ADB pathogen. This requires a standardised and effective method for infecting ash trees with the pathogen and subsequently measuring the infection success. However, to date, there is a lack of scientifically sound comparisons of available methods. In this study, three different methods for testing the resistance of ash genotypes against H. fraxineus (i.e., stem infections, rachis infections, and spore germination assays) were compared and benchmarked against crown defoliation and stem infection assessments. All three resistance assays were performed on 12 ash genotypes covering a wide intraspecific variation in ADB resistance. Stem infections correlated best with crown defoliation observed in the field. However, this method requires a long preparation time and experimental duration. The easiest and fastest method in terms of preparation and execution was the rachis infection assay. Although not completely equivalent to stem infections, the results of the rachis infection assay significantly correlated with the stem lesion lengths measured. Spore germination assays were also quick to carry out, but the timing of sporulation and the sampling of the leaves, followed by agar-medium preparation, proved to be very difficult to synchronise. Following the very small variations in spore germination rates, there was no significant correlation with the stem lesion lengths measured. To obtain meaningful results on the resistance of ash genotypes to ADB, the stem infection method still proved to be the best and most accurate assay. Although new indirect methods for testing resistance in ash trees are arising (e.g., chemistry, spectroscopy, or genetics), direct resistance testing of ash genotypes remains the basis for resistance and breeding research for ash conservation.
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