Manici, L. M., Kelderer, M., Caputo, F., Nicoletti, F., De Luca Picione, F. and Topp A. R. 2015. Impact of cover crop in pre-plant of apple orchards: relationship between crop health, root inhabiting fungi and rhizospheric bacteria. Can. J. Plant Sci. 95: 947-958. Replant disease of fruit tree orchards has a multifactorial etiology, mainly due to the decline in soil biodiversity along with an increase in root rot pathogens, which can be principally countered with appropriate cropping practices. Therefore, a study on the impact of cover crops on plant health of young fruit trees in long-term orchards was performed. Bioassays were performed over two consecutive growing cycles using soil from a multigeneration apple orchard affected by replant disease. First, a cycle was performed with three cover crops (alfalfa, barley, marigold) and apple rootstock plantlets; at the end, the above-ground part of the plant was removed and root residues left in the soil. In the second cycle, an apple orchard planting was simulated upon the first experimental design. Changes of diversity and composition of root inhabiting fungi and rhizospheric bacteria were evaluated as well as apple plant growth response to the pre-plant treatments. Results suggest that one cycle with alternate plants was sufficient to induce changes at the rhizosphere level, despite soil microbial resilience caused by the same long-term soil management. Rhizospheric bacteria were generally affected by plant genotype. Findings suggest that all three different cover crops can harbor almost all fungal species that colonize apple in replanted orchards (Fusarium spp., Pythum spp., binucleate Rhizoctonia sp., Cylindrocarpon-like-fungi and a several nonpathogenic saprophytic fungi named “other”), but their infection frequency varied according to the host plant. A single pre-plant break treatment did not overall differ significantly in plant growth of subsequent apple tree; however, break with marigold, which increased abundance of nonpathogenic root inhabiting fungi more than other cover crops, gave significantly higher plant growth than obtained after barley. This study provides evidence about cover-crop potential to increase soil diversity in long-term permanent cropping systems and to manipulate root colonizing fungi involved in crop health.