Molecular investigation of genetic diversity in ericoid mycorrhizal endophytes associated with Woollsia pungens (Cav.) F. Muell (Epacridaceae)

Two hundred and forty three fungal isolates were obtained from roots of four Woollsia pungens (Cav.) F. Muell plants collected from a field site in New South Wales, Australia. 175 sterile isolates were slow growing and dark-coloured on 2 percentage malt agar and were selected for further analysis. Microsatellite-primed PCR using the primers (GACA)4 and (GTG)5 separated these isolates into 50 genets. Isolates representative of 43 genets (including 168 isolates in total) formed typical ericoid mycorrhizal structures when inoculated onto roots of Vaccinium macrocarpon Ait (Ericaceae), confirming their status as mycorrhizal endophytes. It was estimated that a minimum of 43 genetically-distinct mycorrhizal mycelial genets were present in the root systems of the sampled W. pungens population with 7 to 15 distinct endophytic genets identified in each host plant, indicating that considerable genetic diversity exists within the endophyte population. While most genets were represented by less than 8 isolates, 3 genets contained up to 41 isolates, suggesting that root system colonisation by some mycelia may be extensive. While most fungal genets were shown to be confined to individual plants, 2 genets (genets 32 and 33), however, were present within the root systems of 2 adjacent plants (plants C and D), suggesting that the two root systems were interconnected by the endophyte mycelia. The ITS region of 13 mycorrhizal endophytes and a non-mycorrhizal isolate selected from the endophyte population were sequenced and compared to the sequence of Hymenoscyphus ericae as well as sequences from the GenBank database. A phylogenetic tree was generated from the nucleotide sequence data. This analysis revealed that 6 putative taxa were present in the root systems of 4 host plants. No isolates were positively identified to genus or species level. Closest matches with fungal sequences in the database indicated that most isolates probably belonged to the order Leotiales. Cluster analysis on the basis of the ITS sequences indicated that H. ericae was not clustered together with any endophytes from W. pungens, suggesting that endophytes of W. pungens are not identical to the known ericoid mycorrhizal fungus H. ericae. H. ericae had a low degree of sequence similarity with isolates from W. pungens, with similarities ranging from 68.3-80.6%. Cluster analysis based on DNA sequences of the ITS region did not fully support the groupings inferred from microsatellite-based fingerprinting