Intra- and intersporal diversity of ITS rDNA sequences in Glomus intraradices assessed by cloning and sequencing, and by SSCP analysis

Abstract

The level of molecular variability in the arbuscular mycorrhizal fungus (AMF) Glomus intraradices was assessed. We established several monoxenically growing single-spore cultures of the G. intraradices collected from a long-term soil tillage experiment in Tänikon (Switzerland). They were grown in symbiosis with Ri T-DNA transformed carrot roots. The ITS region was amplified from genomic DNA of fungal spores using ITS1 and ITS4 primers. The amplified ITS region of three of the fungal isolates was cloned and sequenced. All sequences obtained from our samples closely resembled those previously reported for G. intraradices, and no evidence for genetic exchange was found on the interspecific or intergeneric level. Phylogenetic analysis shøwed the presence of at least two different sequence families within a single-spore isolate. The single strand conformation polymorphism (SSCP) method detected different sequence types in a single PCR sample. This technique enabled us to detect several distinct ITS sequences within each of the single-spore isolates of the AMF. Estimates of intra- and intersporal sequence diversity were obtained by comparing diversity within and among the single-spore isolates. A higher level of diversity was found among spores than within a single spore.