Abstract
A better understanding of ectomycorrhizal symbiosis leads to numerous advancements in forest management and environmental protection. The morphological identification of the ectomycorrhizae often proves to be misleading. For this reason, in order to study the ectomycorrhizal fungi communities, a number of molecular methods that require the isolation of nucleic acids are being used. However, ectomycorrhizal root tips, low mass heterogenic material rich in inhibitors, are a recalcitrant substrate in DNA isolation. It is common for published studies to include some number of unidentified root tips in their results, in spite of diverse isolation protocols being available to researchers. This study aims to analyze the relationship between the collected fungal material and later isolation results, and to propose a DNA isolation protocol specifically optimized for ectomycorrhizal root tips. It was found that the taxonomic position can be used to predict the potential isolation efficiency, with Ascomycota being generally more difficult from which to isolate DNA. After a number of cell lysis and lysate purification methods were evaluated, the joined approach of mechanical and chemical lysis, followed by silica column purification, was found to provide the best results, even with recalcitrant material.
Highlights
Ectomycorrhizal (ECM) fungi, involved in obligatory symbiosis with a number of major tree families, are invaluable to most forest ecosystems [1,2,3]
The lowest fresh mass tips belonged to Elaphomyces muricatus Fr. and the highest fresh mass to Suillus variegatus (Sw.)
The only approach used to produce a visible band for each fungal species was mechanical and a chemical lysis accompanied by silica columns purification (Figure S1)
Summary
Ectomycorrhizal (ECM) fungi, involved in obligatory symbiosis with a number of major tree families, are invaluable to most forest ecosystems [1,2,3]. ECM symbiosis improves tree tolerance to biotic and abiotic stress [5,6]. Continuous research of this interaction provides insight into forest ecology, enhancing nature protection and forestry [6]. The complexity of ECM symbiosis is partially due to the great variety of ECM fungi At this time 18 fungal orders, five belonging to Ascomycota and 12 belonging to Basidiomycota, as well as one Zygomycota, are known to contain ECM species [3,7,8]. The size and shape of the ECM mantle covering a root tip may vary from, barely distinguishable from a naked root like Elaphomyces muricatus Fr., to a full and rounded example like Tuber puberulum Berk. & Broome; its surface can be smooth like Lactarius aurantiacus (Pers.) Grey, hirsute like Cortinarius torvus (Fr.) Fr
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