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 taxon specific markers for strains of the free-root endophytes Phialocephala fortinii and 'Type 1', a non-sporulating dematiaceous mycelium, were examined. The results were compared with data previously generated by isozyme analyses. 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 cultural characteristics. DNA of 14 strains per taxon was amplified with three short, 17-18-nucleotide-long, tandemly-repeated primers (CCA, CGA, ACA) with two (CCA) or three degenerated bases at their 5'-ends. The resulting DNA products were separated by agarose gel electrophoresis. The bands were scored for absence/presence, respectively, and the binary matrix subjected to multiple correspondence analysis (MCA). ISSR-PCR was found to be highly reproducible 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 320 to 4100 bp. ISSR-PCR was found to be a powerful tool to find strain-specific and taxon-specific markers. Each strain showed a unique banding pattern 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 was performed with ISSR-PCR or isozyme data. However, ISSR-PCR allowed the differentiation of strains with the same allozyme phenotype.