Characterisation of dark septate endophytic fungi (DSE) using inter-simple-sequence-repeat-anchored polymerase chain reaction (ISSR-PCR) amplification

Suitability and reproducibility of ISSR-PCR to find strain-specific and tax on specific markers for strains of the free-root endophytes Phialocephala f ortinii and 'Type 1', a non-sporulating dematiaceous mycelium, were examine d. The results were compared with data previously generated by isozyme anal yses. P. fortinii and 'Type 1' are two DSE taxa and are abundant colonisers of coniferous forest-tree roots in the North Temperate zone. 'Type 1' was never observed to sporulate in pure culture but is well defined by its cult ural characteristics. DNA of 14 strains per taxon was amplified with three short, 17-18-nucleotide-long, tandemly-repeated primers (CCA, CGA, ACA) wit h two (CCA) or three degenerated bases at their 5'-ends. The resulting DNA products were separated by agarose gel electrophoresis. The bands were scor ed for absence/presence, respectively, and the binary matrix subjected to m ultiple correspondence analysis (MCA). ISSR-PCR was found to be highly repr oducible since amplification of DNA from several single-hyphal-tip cultures of the same strain resulted in identical banding patterns. Eighty-five (92 .4 %) of the 92 DNA fragments were polymorphic. The fragments ranged from 3 20 to 4100 bp. ISSR-PCR was found to be a powerful tool to find strain-spec ific and taxon-specific markers. Each strain showed a unique banding patter n and diagnostic bands for the two taxa could be identified. ISSR-PCR data correlated neither with the geographical nor the host origin of the strains . The strains grouped into similar clusters independently of whether MCA wa s performed with ISSR-PCR or isozyme data. However, ISSR-PCR allowed the di fferentiation of strains with the same allozyme phenotype.