A new multilocus approach for a reliable DNA-based identification of Armillaria species

Abstract

In this paper we highlight and critically discuss limitations to molecular methods for identification of fungi via the example of the basidiomycete genus Armillaria. We analyzed a total of 144 sequences of three DNA regions commonly used for identifying fungi (ribosomal IGS-1 and ITS regions, translation elongation factor-1 alpha gene) from 48 specimens of six Armillaria species occurring in Europe (A. cepistipes, A. ostoyae, A. gallica, A. borealis, A. mellea, A. tabescens). Species were identified by comparing newly obtained sequences with those from the NCBI database, phylogenetic analyses and PCR-RFLP analyses of the three regions considered. When analyzed separately, no single gene region could unambiguously identify all six Armillaria species because of low interspecific and high intrasequence variability. We therefore developed a multilocus approach, which involves the stepwise use of the three regions. Following this scheme, all six species could be clearly discriminated. Our study suggests that, to improve the reliability of DNA-based techniques for species identification, multiple genes or intergenic regions should be analyzed.

• fungi
• molecular methods
• morphology
• public databases
• sequencing
• PCR-RFLP

Acknowledgments

We thank the WSL institute and the Swiss National Science Foundation for financial support and Brigitte Commarmot for successfully leading the Ukrainian-Swiss scientific cooperation project. We also thank Esther Jung and Hélène Blauenstein for technical support in the laboratory, Silvia Dingwall for English revision of the manuscript and the anonymous reviewers for helpful comments.