Background & Aims: Better understanding of belowground interactions in agroforestry systems is crucial for the success of plant co-existence. Beyond root competition, associated arbuscular mycorrhizal (AM) fungi can also be involved in plant to plant interactions. Thus far, the contribution of each agroforestry component (trees, herbaceous vegetation beneath trees -HVbT- and crops) in the establishment and maintenance of AM communities is poorly documented, particularly in temperate areas. This study investigates the spatio-temporal dynamics of both roots and AM fungi in two alley-cropping sites located in southwestern France. Methods: Over a one-year period, (i) root length density, production and distribution, (ii) AM activity (root mycorrhization rate and extra-radical hyphal production) and (iii) AM diversity (metabarcoding) were assessed at different distances from tree rows in two agroforestry systems. Results: The mycorrhization rate and hyphal production increased at the interface between tree rows and cultivated alleys, showing a positive effect of the presence of a perennial system (tree and HVbT) and of plant diversity. Compared to HVbT, tree roots colonized farther into superficial layers of the cultivated alleys. However, due to higher root densities and well-established AM fungi observed throughout all the year, HVbT appeared to be more relevant in maintaining an active source of AM inoculum for newly developing crop roots in winter. Conclusion: The spatial proximity of roots and common AM fungi provides new perspectives in deciphering the significance of arbuscular mycorrhizal communities in crop nutrition and yield in agroforestry systems.