IDENTIFICATION OF RIBOSOMAL DNA POLYMORPHISMS AMONG AND WITHIN SPORESOF THE GLOMALES - APPLICATION TO STUDIES ON THE GENETIC DIVERSITY OF ARBUSCULAR MYCORRHIZAL FUNGAL COMMUNITIES

Little information currently exists on species diversity in communitie s of arbuscular mycorrhizal fungi (AMF), mainly owing to difficulties in identification of field extracted spores on the basis of morphology . The possibility was explored to identify individual AMF spores from the field on the basis of a molecular marker, namely the nuclear ribos omal DNA encoding the highly conserved 5.8S rRNA with the two flanking internal transcribed spacers (ITS region), known to vary between spec ies. A technique involving polymerase chain reaction followed by restr iction fragment length polymorphism analysis (PCR-RFLP) was developed to amplify and characterize the ITS region from single AMF spores. PCR reactions with extracts from single spores of three AMF species, rais ed under glasshouse conditions, yielded reproducibly a single amplific ation product of the ITS region in sufficient amounts to allow cleavag e with several restriction enzymes. The size of the ITS region, c. 600 base pairs, varied only slightly between species. Digestion of the PC R products with the restriction enzymes HinfI and TaqI resulted in ban ding patterns that were reproducible for different individual spores o f a given species, but showed clear differences between the three spec ies tested. The sum of the fragment sizes was sometimes greater than t he size of the original PCR product, e.g. in Glomus mosseae. Clones of the amplification product from a single spore of this fungus were obt ained and sequenced. This yielded two closely related but different se quences, indicating that two different ITS regions co-existed in the s pore. The RLFP pattern of the amplification product of the spore was a result of an amalgamation of these two sequences. The technique was a pplied to AMF spores collected from a species-rich grassland. Spores w ere sorted into morphological groups on the basis of their colour, siz e, and shape, and then subjected to PCR-RFLP analysis. In some morphol ogical groups, a large percentage of spores failed to yield an amplifi cation product, probably because they had lost their contents. A group of Glomus spores yielding amplification products in the majority of c ases was further investigated: PCR-RFLP analysis on 10 individual spor es from the field produced 10 different patterns. Similar results were obtained with other groups of spores. The results suggest that the di versity in natural AMF communities and the genetic diversity within in dividual spores might be much greater than previously thought.