Characterisation of arbuscular mycorrhizal fungi in roots by means of epifluorescence microscopy and molecular methods

Abstract

In this work we characterised different species of the arbuscular mycorrhizal (AM) fungal genus Glomus by using Restriction Fragment Length Polymorphisms (RFLPs), combining epifluorescence microscopy and nested PCR, for the easy retrieval of AM fungal DNA in planta. Epifluorescence microscopy allowed us to select highly colonised root segments for DNA extraction, enhancing the chance of fungal DNA amplification by PCR. The primer pair used in nested PCR after a first reaction performed by using NS31/AM1 primers, showed a high specificity for Glomus DNA amplification and yielded enough fungal DNA for RFLP analyses. RFLP patterns of PCR products from colonised roots matched with reference profiles obtained from spores and discriminated the four Glomus species tested. The method described may be useful for checking the establishment of mycorrhizal colonisation by the inoculated fungal species in controlled experimental conditions.