Inoculum of arbuscular mycorrhizal fungi, with growing use in horticulture, is produced mainly in two technically different cultivation systems: in vivo culture in symbiosis with living host plants or in vitro culture in which the fungus life cycle develops in association with transformed roots. To evaluate the effectiveness and the infectivity of a defined isolate obtained by both production methods, a replicated comparative evaluation experiment was designed using different propagules of Rhizophagus irregularis produced in vivo on leek plants or in vitro in monoxenic culture on transformed carrot roots. The size of the spores obtained under both cultivation methods was first assessed and bulk inoculum, spores, sievings, and mycorrhizal root fragments were used to inoculate leek plantlets. Spores produced in vitro were significantly smaller than those produced in vivo. Although all mycorrhizal propagules used as a source of inoculum were able to colonize plants, in all cases, leek plants inoculated with propagules obtained in vivo achieved significantly higher mycorrhizal colonization rates than plants inoculated with in vitro inocula. Inoculation with in vivo bulk inoculum and in vivo mycorrhizal root fragments were the only treatments increasing plant growth. These results indicate that the production system of arbuscular mycorrhizal fungi itself can have implications in the stimulation of plant growth and in experimental results.