Rapid identification of genetic variation of ectomycorrhizal fungi by amplification of ribosomal RNA genes

Abstract

SUMMARYThere is a clear requirement to develop sensitive methods for detecting defined isolates of ectomycorrhizal fungi within the complex microbial communities of natural ecosystems and reforestation sites. We present a method that permits the rapid identification of an ectomycorrhizal isolate using enzymatic amplification (polymerase chain reaction) of DNA extracted either from pure cultures or ectomycorrhizas. A set of oligonucleotide primers capable of amplifying full-length nuclear 17S and 25S ribosomal RNA genes, together with the ribosomal internal transcribed spacer and intergenic spacer, have been designed and could be used for amplifying target sequences from a wide range of ectomycorrhizal genera. Length polymorphism in the amplified rDNA and restriction endonuclease analysis of nearly 6-0 kbp of amplified rDNA provided useful criteria for the rapid typing of isolates from different genera and species. Restriction endonuclease analysis of amplified DNA from 26 isolates representing four species oi Laccaria (L. bicolor, L. laccata, L. proximo, L. tortilis) yielded up to 20 scored RFLPs and revealed interspecific and intraspecific polymorphism. Most of the polymorphisms were located within the regions corresponding to the internal transcribed spacer and intergenic spacer. The degree of variation observed was sufificient to discriminate several isolates from the same species. Genetic variation was correlated to some extent with geographical origin of the isolates. However, RFLPs of the rRNA genes cannot unambiguously discriminate all selected isolates within Laccaria species, requiring the development of additional DNA probes. Alone, or in combination with other DNA probes, the amplified rDNA genes may serve in the determination of pure fungal cultures and in the characterization of genetic variation of field ectomycorrhizal populations.

Genetic diversity of Microdochium nivale isolates from turfgrass

“…This region separates the highly conserved rDNA genes and has been reported to be the most rapidly evolving spacer region (Hillis & Dixon, 1991). Variation within the IGS region has been used to reveal intra-specific variation and to characterize the population structure of several fungi including Fusarium oxysporum (Edel et al, 1995 ;Appel & Gordon, 1995 ;Woudt et al, 1995 ;Woo et al, 1996), Pythium ultimum (Buchko & Klassen, 1990), Colletotrichum lindemuthianum (Fabre et al, 1995), Verticillium chlamydosporium (Arora et al, 1996) and species of Laccaria (Henrion, Letacon & Martin, 1992).…”

Genetic variation and heavy metal tolerance in the ectomycorrhizal basidiomycete Suillus luteus

“…Analyses of the PCR-amplified nuclear ribosomal internal transcribed spacer (ITS) have been successfully applied to ECM community studies. RFLP patterns of the ITS region can be used to distinguish related fungal taxa (Henrion et al, 1992 ;Ka/ re! n et al, 1997 ;Jonsson et al, 1999 ;Sims et al, 1999).…”

Genetic variability in intergenic spacers of ribosomal DNA in Pisolithus isolates associated with pine, eucalyptus and Afzelia in lowland Kenyan forests

“…The basidiocarps were further examined by PCR\ RFLP of the rDNA IGS, a rDNA region known as highly variable in fungi (Henrion et al, 1992(Henrion et al, , 1994Apel & Gordon, 1996 ;Selosse et al, 1996). All basidiocarps of the Pinus-, Eucalyptus-and Afzeliamorphotypes produced a single IGS band of 450 bp (Fig.…”

“…The ITS and IGS1 regions of the rDNA were amplified in duplicate by PCR using the primers ITS1\ITS4 and CNL12\5SA (Henrion et al, 1992) respectively, Taq DNA polymerase, and the buffer provided by the manufacturer (Applige ' ne-Oncor, Illkirch, France) according to Henrion et al (1994). Amplifications were carried out on a GeneAmp2 PCR System 9600 (Perkin Elmer).…”

“…RFLP and sequencing of the intergenic spacers (ITS, IGS1) of the rDNA gene complex of Pisolithus collected beneath A. quanzensis, P. caribaea and E. camaldulensis were undertaken because these regions are known to be variable at the inter-and intraspecific level (Gardes et al, 1991 ;Henrion, Le Tacon & Martin, 1992 ;Appel & Gordon, 1996 ;Kretzer et al, 1996). In addition, ITS region sequences can diagnose phylogenetic relationships at many phylogenetic levels (Hershkovitz & Lewis, 1996).…”