Arbuscular mycorrhizal (AM) fungi are key soil symbiotic microorganisms, intensively studied for their roles in improving plant fitness and their ubiquity in terrestrial ecosystems. Research on AM fungi is difficult because their obligate biotrophic nature makes it impossible to culture them in the absence of a host. Over the last three decades, Ri T-DNA transformed roots have been the gold standard to study AM fungi under in vitro conditions. However, only two host plant species (Daucus carota and Cichorium intybus) have been routinely used to in vitro propagate less than 5% of the known AM fungal species. There is much evidence that host identity can significantly affect AM symbioses, therefore, we investigated any potential host-specific effects of two novel Ri T-DNA transformed root species, Medicago truncatula and Nicotiana benthamiana, by associating them with seven AM fungal species selected based on their contrasting behaviors when grown with Ri T-DNA transformed D. carota roots. To evaluate the performance of new Ri T-DNA transformed roots to host and propagate AM fungal species, a factorial set-up was used to generate nine unique pairs of hosts (M. truncatula, N. benthamiana, D. carota) and AM fungi (Rhizophagus irregularis, R. clarus, Glomus sp.). Using statistical modeling, all pairs of hosts and AM fungi were compared by their symbiosis development (SD) and sporulation patterns in the hyphal compartments (HCs) of two-compartment Petri dishes. Our results show that 1) most of the variation between host and AM fungus pairs relating to SD or HC sporulation was explained by an interaction between host and AM fungal identity, i.e., host identity alone was not sufficient to explain AM fungal behaviour, 2) AM symbioses involving different combinations of symbiont identities trigger heterogenous fungal behaviours. This work provides a robust framework to develop and evaluate new Ri T-DNA roots for the in vitro propagation of AM fungi, an important asset for germplasm collections and biodiversity preservation.