Despite the importance of arbuscular mycorrhizal fungi (AMF) to ecosystem processes, few experimental tools are available to quantify AMF contributions to process rates. In this study we examine the efficacy of an experimental system consisting of wildtype (WT) and different non-mycorrhizal (Myc−) genotype pairs of tomato (Solanum lycopersicum L.), specifically focusing on cv Micro-Tom. Two conditions necessary to make such a system useful were examined; (1) that the Myc− genotype(s) do not get colonized in a full soil AMF community background, while the WT does, and B) that there are no non-target effects of the Myc− phenotype on soil microbes. We assessed the second condition by growing Myc− genotypes and WT in non-mycorrhizal soil, monitoring plant growth (root, shoot biomass; root length; root diameter size distribution) and soil microbial community structure (PLFA analysis) as indicators of any changes in root tissue quality or rhizodeposition. All tested Myc− genotypes showed a drastically reduced colonization in mycorrhizal soil. However, in non-mycorrhizal soil, M161 had greater root biomass and M20 greater microbial biomass compared to WT. Only one of the Myc− mutants examined fully met the criteria. We conclude that the BC1/WT pair is a powerful experimental system and recommend caution when using Myc− mutants in mycorrhizal ecology.