Molecular analysis of ectomycorrhizal fungal communities

Abstract

Despite advances in mycorrhizal identification, the goal of elucidating the structure and development of mycorrhizal communities remains elusive. Fruit body production can be sporadic, morphological typing of mycorrhizae is subject to variation with environmental conditions or host, and cultural studies are labor intensive and miss fungi that cannot be isolated. Molecular techniques for identification of fungal symbionts can supplement these techniques and offer an approach that is rapid, is independent of environmental variation, and can be applied directly to large numbers of samples. Molecular approaches to mycorrhizal community analysis attempt to distinguish taxonomic groups so they can be monitored and their interactions studied. Initial characterization of community structure involves enzymatic amplification of DNA directly from mycorrhizal roots using fungus-specific primers, followed by restriction endonuclease digestion to produce taxon-specific restriction fragment patterns. Comparison of these patterns with those obtained from fungal fruit bodies or reference cultures facilitates identification of fungal symbionts. Phylogenetic relationships of fungi that cannot be matched to reference isolates can be inferred by sequencing enzymatically amplified DNA. Future directions that will result from molecular approaches include development of sampling strategies, resolution of species complexes, field observations of host specificity, elucidation of the dynamics of replacement processes (succession), and determination of the role of dispersal in community development. As additional techniques are developed for population analysis, resolution of questions related to genetic structure, variation, and gene flow will become feasible.RCsumC : En depit des progrks sur l'identification des mycorhizes, l'objectif de comprendre la structure et le dCveloppement des communautCs mycorhiziennes demeure difficile. La production des fructifications peut &tre sporadique, la caracttrisation des mycorhizes est sujette aux variations des conditions du milieu et de I'hBte, et les Ctudes culturales exigent beaucoup de travail et ntgligent les champignons qu'on ne sait pas cultiver. Les techniques molCculaires pour l'identification des symbiotes fongiques peuvent supplCer a ces dCficiences, et ouvrent une avenue rapide, indCpendante des variations du milieu, qui peut &tre appliquCe directement a un grand nombres dlCchantillons. Les mtthodes molCculaires appliquCes a l'analyse des communautCs visent distinguer les groupes taxonomiques, de facon a ce qu'on puisse les suivre et Ctudier leurs interactions. La caractCrisation initiale de la structure communautaire implique l'amplification enzymatique de l'ADN, directement 21 partir des racines mycorhizCes, en utilisant une amorGe spCcifique au champignon, suivi d'une digestion par des endonuclCases de restriction pour produire des patrons de restriction spCcifiques aux taxons. Une comparaison de ces patrons avec ceux obtenus a partir de fructifications de champignons ou de cultures de ...

Characterization of Mycobionts of Photomorph Pairs in the Peltigerineae (Lichenized Ascomycetes) Based on Internal Transcribed Spacer Sequences of the Nuclear Ribosomal DNA

“…The amplification reaction was performed in 50 µl vol including 50 mM KCl, 10 mM Tris-HCl, 0.1% Triton, 5% glycerol, 2.5 mM MgCl 2 , 20 µM of each dNTP, 0.2 µM of the universal primer ITS5 (White et al, 1990) and the ascomycete-specific primer NL6A (Egger, 1995; Fig. 1), and 0.2 to 30.0 ng of template DNA.…”

“…In order to address the ''one-fungus-two photomorphs'' hypothesis using actual DNA sequence data, we used fungus-specific primers for the amplification of a portion of the nuclear DNA from a total lichen DNA extract. Fungus-specific primers were initially designed by Gargas and Taylor (1992) and Gardes and Bruns (1993) for studying symbiotic or parasitic fungi, and recently, Egger (1995) designed primers specific to ascomycetes for studying ectomycorrhizal communities.…”

“…The main objectives of this study are to: (1) test the applicability of fungus-specific primers designed for mycorrhizal ascomycetes (Egger, 1995) for the amplification of the ITS region of lichenized fungi using a total lichen DNA template; and (2) test the hypothesis ''one fungus-two photomorphs'' based on nucleotide sequences of the ITSs and 5.8S gene of members of photomorph pairs in the Peltigerineae.…”

Spatial distribution of discrete RAPD phenotypes of a root endophytic fungus, Phialocephala fortinii, at a primary successional site on a glacier forefront

“…The ITS-region was amplified with primers ITS1 (White et al, 1990) and NL6Amun (Egger, 1995) with the cycle parameters described in and digested according to the manufacturer's instructions with 5 units of restriction enzymes, MspI, RsaI and TaqI (Promega Corporation, Madison, WI, USA). Only two of the macroscopically prescreened isolates possessed an RFLP pattern different from that of the type culture (data not shown) ; these were omitted from further analyses.…”

Polymorphism in the internal transcribed spacer (ITS) of the ribosomal DNA of 26 isolates of ectomycorrhizal fungi

“…Identification of ectomycorrhizal fungi is mainly based on the analysis of sporocarps whose presence is triggered by specific environmental conditions (Egger, 1995;Gardes and Bruns, 1996). Usually the identification of the mycosymbiont by morphological analyses of the mycorrhiza is more feasible than sporocarp analyses, although special skills are required to directly analyze a root-attached symbiont (Karén et al, 1997).…”