Semi-Quantitative Analysis of Arbuscular Mycorrhizal Colonization of Onion Roots Inoculated with Single or Mixed Species Based upon PCR-RFLP

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Arbuscular mycorrhizal (AM) fungi colonizing roots cannot be identified based on their hyphal morphology. We applied the PCR-RFLP technique to distinguish the AM fungal species inoculated to plants and to quantify the biomass in roots. Seedlings of onion were inoculated with single or mixed AM fungal species, Gigaspora margarita and / or Scutellospora cerradensis, and were raised in a growth chamber. About 1.2 kbp of the fungal 18S rRNA gene in the roots were amplified with the primer VANS1 which is specific to AM fungi and the non-specific primer NS4. The restriction enzyme digestion with HincII of the PCR products enabled to distinguish these species. The amounts of PCR products were quantified based upon the relative brightness of the bands on agarose gel stained with ethidium bromide. In single species inoculation, the band brightness was correlated with the colonization percentage. In mixed species inoculation, the relative proportion of each species could be estimated. These results suggest that the current semi-quantitative PCR-RFLP technique is effective to analyze the interaction between these two fungal species in roots.
社団法人日本土壌肥料学会の論文