A soil-free method for assessing pathogenicity of fungal isolates from apple roots

Apple replant disease is a problem in tree nurseries and apple orchards worldwide. Its cause is still unknown, but fungi are discussed to contribute to a complex of causal biotic factors. Several fungi are claimed to be replant disease pathogens but have not been significantly confirmed in experiments. Therefore, it seems indispensable to study fungal root endophytes in pathogenicity tests. Bioassays conducted in green house pot cultures using peat substrate or disinfected natural soil have been time and labor intensive and often resulted in only low infection rates. A quick biotest using the inert material perlite under controlled conditions is presented as an improved method to assess the effects of fungal isolates from replant-diseased root tissue. In vitro cultivated M26 rootstock plantlets were grown for 3 weeks in a Petri dish growth box with perlite substrate inoculated with selected fungal isolates. Symptom ratings for shoot wilting started after only 2 days; root symptoms appeared later and were assessed microscopically. Necroses in root tissue as well as hyphae, chlamydospores, and macroconidia could be detected. The tested endophytic isolates led to the following plant reactions: (1) negative (Cadophora, Calonectria, Dactylonectria, Ilyonectria, and Leptosphaeria) or (2) neutral (Plectosphaerella, Pleotrichocladium, and Zalerion). After re-isolation, most of the Nectriaceae isolates were confirmed as pathogens for M26 plants by fulfilling Koch’s postulates in a subsequent test. We recommend this perlite biotest to facilitate studies on root endophyte interactions with their hosts.