A combined morphological and molecular approach to characterize isolates of arbuscular mycorrhizal fungi in Gigaspora (Glomales)

Morphological features of resting spores and information from nucleotide sequences of ribosomal RNA were used to characterize seven mycorrhizal fungal isolates in Gigaspora from different geographical areas. Detailed observations were made under the light microscope on single spores mounted in Melzer's reagent and polyvinyl alcohol-lactic acid-glycerol medium to resolve size, colour and cell wall structures. Neighbour-joining analyses were carried out on a portion of the 18S gene and on the internal transcribed spacer (ITS) region amplified by PCR from multisporal DNA preparations. Combined data allowed us to design oligonucleotides that unambiguously distinguished Gi. rosea from Gi. margarita and Gi. gigantea and also identified two isolates as Gi. rosea that had been previously diagnosed as Gi. margarita. ITS sequences revealed substantial genetic variability within clones of a single isolate of Gi. rosea as well as among geographically disjunct Gi. rosea isolates. The results show how complementary morphological and molecular data can clarify relationships among species of low morphological divergence. Sequence information allowed the extent of genetic divergence within these species to be investigated and provided useful PCR primers for detection and identification.