ITS-RFLP and sequence analysis of endophytes from Acianthus, Caladenia and Pterostylis (Orchidaceae) in southeastern Queensland

Abstract

We used ITS-RFLP and sequence analysis to determine the identities of the fungal endophytes of six terrestrial orchid species from southeastern Queensland, a region previously unexplored in this context. Pure cultures of orchid--colonising fungi were obtained and fungal identities were assessed by means of ITS-PCR, RFLP analysis, sequence comparison, and protocorm colonisation tests. ITS-PCR and RFLP analysis resulted in five main groupings. Sequencing and GenBank comparison of these five groups showed that the fungal endophytes isolated from the three Pterostylis species were probably Thanatephorus species. There was close sequence identity (90%) of the fungus isolated from Acianthus spp. to Epulorhiza repens, suggesting these may be the same fungal species. However, that only E. repens succeeded in colonising protocorms of Thelymitra pauciflora suggests these may be different species of Epulorhiza. Analysis of the ITS and LSU sequences of the fungus isolated from Caladenia carnea showed high identities with a sequence from a Sebacina vermifera originally isolated from Caladenia dilatata. These results show that there is specificity for fungal partners within the orchid genera Acianthus, Caladenia and Pterostylis.