Characterization of juvenile maritime pine (Pinus pinaster Ait.) ectomycorrhizal fungal community using morphotyping, direct sequencing and fruitbodies sampling

Using ectomycorrhizal root tip morphotyping (anatomical and morphological identification), molecular analysis (internal transcribed spacer region amplification and sequencing), and fruitbody sampling, we assessed diversity and composition of the ectomycorrhizal fungal community colonizing juvenile Pinus pinaster Ait. under natural conditions in NW Spain. Overall, we found 15 Basidiomycetes and two Ascomycetes. Members of the family Thelephoraceae represented up to 59.4% of the samples. The most frequent species was Tomentella sublilacina followed by Thelephora terrestris, Russula drimeia, Suillus bovinus, and Paxillus involutus, while the less frequent were Pseudotomentella tristis, Lactarius subdulcis, Russula ochroleuca, and Entoloma conferendum. From October 2007 to June 2008, we sampled 208 sporocarps belonging to seven genera and nine species: Thelephora terrestris, Paxillus involutus, Suillus bovinus, Xerocomus badius, Scleroderma verrucosum, Amanita gemmata, A. rubescens, Amanita sp., and Russula sp. The species belonging to the genus Amanita, X. badius and S. verrucosum were not found on root samples. By comparing our results with a bibliographic review of papers published from 1922 to 2006, we found five genera and six species which have not been previously reported in symbiosis with P. pinaster. This is the first time that the diversity of the ectomycorrhizal fungal community associated with P. pinaster was investigated using molecular techniques. Considering that only 38% of the genera found by sequencing were found as fruitbodies, we conclude that integrating morphotyping and sporocarps surveys with molecular analysis of ectomycorrhizas is important to documenting the ectomycorrhizal fungus community.