• Root morphological response to experimentally induced soil heterogeneity is reported here on three grassland species (Luzula campestris, Poa angustifolia and Plantago lanceolata) under field conditions.
• Nutrient application was combined with suppression of mycorrhizal infection and with substrate structure modification in experimental patches. For each isolated root, we determined five dimensional characteristics and two topological parameters, including a newly introduced topological index (dichotomous branching index).
• Nonmycorrhizal L. campestris responded little to nutrient application, but strongly to benomyl application, in all characteristics measured. Mycorrhizal P. angustifolia produced the longest, most branched roots but exhibited limited sensitivity to nutrients and benomyl application. Strongly mycorrhizal P. lanceolata was the most sensitive to nutrient application, but showed little response to benomyl application. It was the only one among the species studied with root characteristics influenced (negatively) by increased production of total root biomass in the patches. Substrate structure influenced dimensional characteristics of Poa and Luzula roots, but not the topological indices.
• Results indicate different exploitation of soil microsites by L. campestris, P. angustifolia and P. lanceolata. Root topology seems to play a limited role in this process.