Amplification Of Ribosomal RNA Genes Research Articles

THIS ARTICLE HAS BEEN WITHDRAWN: Glomerella acutata

THIS ARTICLE HAS BEEN WITHDRAWN: <i>Glomerella acutata</i>

The first isolation in Japan of Trichophyton mentagrophytes var. erinacei causing tinea manuum

The first isolation in Japan of <i>Trichophyton mentagrophytes</i> var. <i>erinacei</i> causing tinea manuum

P9 Proband-mediated information dissemination does not meet the expressed wishes in families with a BRCA1/2 gene mutation

Ribosomal RNA gene amplification has been demonstrated in the rat XC sarcoma cell line. Cells of this rat line have a fairly stable karyotype with several unusual features, which have been clarified by in situ hybridization, silver staining, and binding of antibodies to 5-methylcytosine. There are one or two tiny acrocentric chromosomes containing transcriptionally active 18S and 28S ribosomal RNA (rRNA) genes. The short arm of one chromosome No. 12 has been replaced by a GC-rich, C-band negative, differentially staining region (DSR) containing an increased number of rRNA genes; most of these are transcriptionally inactive and located in regions containing highly methylated DNA. The short arm of a small chromosome, probably a No. 20, has been replaced by a GC-rich, C-band negative, homogeneously staining region (HSR) that presumably represents amplification of a DNA sequence other than the 18S and 28S rRNA coding sequences. The DNA in this HSR is not enriched in 5-methylcytosine and neither is that in the HSRs of methotrexate-resistant Syrian hamster cells.

Identification of Propionibacterium acnes by polymerase chain reaction for amplification of 16S ribosomal RNA and lipase genes

Propionibacterium acnes belongs to the cutaneous flora and is present in sebaceous follicles. The fatty acids that are released from sebum triglycerides by the action of this bacterial lipase play an important role in the pathogenesis of acne vulgaris. P. acnes is also involved in postoperative disorders and opportunistic infections in immunosuppressed hosts. Recently, it has been proposed that P. acnes causes sarcoidosis. Therefore, rapid isolation and identification of P. acnes is important. This study evaluated the polymerase chain reaction (PCR) for the detection of the 16S rRNA and lipase genes of P. acnes. The PCR used to detect the 16S rRNA gene could amplify the gene of P. acnes, but not the genes of the other tested strains of P. avidum, P. granulosum, P. lymphophilum, P. jensenii, P. acidipropionici and P. thoenii. The PCR to detect the lipase gene of P. acnes, however, could amplify not only the gene of P. acnes but also that of P. avidum. The PCR product of this lipase gene was not found in the strains of the other species tested. Therefore, the organism that has both the 16S rRNA gene and lipase gene was identified as P. acnes, while the strain with the lipase gene but not the 16S rRNA gene of P. acnes was characterized as P. avidum. These findings were confirmed by the conventional biochemical tests including lipase activity. Furthermore, out of the seven clinical isolates from acne vulgaris, four were identified as P. acnes and three as P. avidum by the PCR method and biochemical tests. The combination of two PCR, one for the detection of the 16S rRNA and the other of lipase genes was shown to be an easier, faster and more accurate method to identify P. acnes and P. avidum than conventional methods.

Bleeding peptic ulcers and presence of Helicobacter pylori by various tests: a case-control study.

Virulence factors of Helicobacter pylori are associated with peptic ulcer disease and may be also associated with bleeding peptic ulcers (BPU). To determine whether H. pylori and/or the cytotoxin-associated gene (cagA) can increase the risk of bleeding in peptic ulcers. Sixty-seven patients were studied. Thirty had BPU, 20 had non-bleeding peptic ulcers (NBPU), and 17 were control subjects (NPU). The prevalence of H. pylori was assessed by the urease fast test, histological examination, serology, and 16S ribosomal RNA and cagA gene amplification by polymerase chain reaction (PCR). Histology and PCR showed greater sensitivity for diagnosis of H. pylori under bleeding circumstances when compared with other tests. Association of H. pylori was greater in the NBPU group (odds ratio [OR] 4.91, P = 0.06) than in the BPU group (OR 1.27, P = NS) when compared with the control group. When the BPU and NBPU groups were compared, H. pylori was found more often in the NBPU group (OR 0.26, P < 0.10 ). The cagA-positive gene showed a similar distribution in the three groups. The titres for anti-CagA immunoglobulin A (IgA) antibodies were higher in NBPU patients (83%) than in BPU or control patients. Furthermore, anti-urease immunoglobulin G (IgG) was detected more frequently among BPU and NBPU patients. NBPU patients had the highest prevalence of H. pylori by PCR. It seems unlikely that either H. pylori or the cagA-positive gene act as significant risk factors for bleeding in peptic ulcers. The lower prevalence of the microorganism among patients who bleed cannot be explained as an artificial finding.

Identification of novel Crenarchaeota and Euryarchaeota clusters associated with different depth layers of a forest soil

Abstract Archaea have been shown to be ubiquitous among soil microbial communities. However, our knowledge on their diversity and spatial distribution in soil ecosystems is still limited. This study was conducted to investigate archaeal community changes along a forest soil depth profile in Unterehrendingen, Switzerland. From four consecutive soil depth layers, bulk soil DNA was extracted. Archaea-specific PCR amplification of small subunit ribosomal RNA genes (rDNA) was performed and combined with restriction fragment length polymorphism (RFLP) analysis with restriction endonuclease HaeIII [Bundt et al., Soil Biol. Biochem. 33 (2001) 729-738]. Significant changes of the RFLP fingerprints were reproducibly observed from the soil surface to 1 m depth. From the surface soil layer (0-9 cm) and the bottom soil layer (50-100 cm), libraries of PCR-amplified archaeal rDNA fragments were constructed. Screening of the libraries yielded various clones of different HaeIII RFLP types from the surface and the bottom soil layers, revealing shifts in major archaeal components along the soil depth profile. Clones of all RFLP types were sequenced and phylogenetically affiliated. These analyses revealed even more pronounced Archaea community shifts along the depth gradient. Several novel soil archaeal clusters were identified and some appeared predominantly associated to either the surface or the bottom soil layer. Euryarchaeal rDNA sequences, not yet reported from aerated soils, were found in the surface soil layer and were affiliated to the order Thermoplasmales and relatives. Novel crenarchaeal soil clusters were identified that included sequences only retrieved from the bottom soil layer. In this study, a this far unreported variety of archaeal groups was found in a forest soil ecosystem. The distinct depth-related community shift suggested the occurrence of different archaeal types that depend on environmental parameters that change along the soil depth profile.

Identification of novel Crenarchaeota and Euryarchaeota clusters associated with different depth layers of a forest soil

Archaea have been shown to be ubiquitous among soil microbial communities. However, our knowledge on their diversity and spatial distribution in soil ecosystems is still limited. This study was conducted to investigate archaeal community changes along a forest soil depth profile in Unterehrendingen, Switzerland. From four consecutive soil depth layers, bulk soil DNA was extracted. Archaea-specific PCR amplification of small subunit ribosomal RNA genes (rDNA) was performed and combined with restriction fragment length polymorphism (RFLP) analysis with restriction endonuclease HaeIII [Bundt et al., Soil Biol. Biochem. 33 (2001) 729–738]. Significant changes of the RFLP fingerprints were reproducibly observed from the soil surface to 1 m depth. From the surface soil layer (0–9 cm) and the bottom soil layer (50–100 cm), libraries of PCR-amplified archaeal rDNA fragments were constructed. Screening of the libraries yielded various clones of different HaeIII RFLP types from the surface and the bottom soil layers, revealing shifts in major archaeal components along the soil depth profile. Clones of all RFLP types were sequenced and phylogenetically affiliated. These analyses revealed even more pronounced Archaea community shifts along the depth gradient. Several novel soil archaeal clusters were identified and some appeared predominantly associated to either the surface or the bottom soil layer. Euryarchaeal rDNA sequences, not yet reported from aerated soils, were found in the surface soil layer and were affiliated to the order Thermoplasmales and relatives. Novel crenarchaeal soil clusters were identified that included sequences only retrieved from the bottom soil layer. In this study, a this far unreported variety of archaeal groups was found in a forest soil ecosystem. The distinct depth-related community shift suggested the occurrence of different archaeal types that depend on environmental parameters that change along the soil depth profile.

First Report of Grapevine "Bois Noir" Disease and a New Phytoplasma Infecting Solanaceous Plants in Lebanon.

During a 2001 survey to evaluate the incidence of phytoplasma diseases in Lebanon, samples were collected from plants showing symptoms suggestive of phytoplasmal infections. Samples were also collected from symptomless plants. Sampled hosts from the Bekaa Valley included: 3 samples of tomato (Lycopersicum esculentum), 4 samples of pepper (Capsicum annuum), 10 samples of grapevine (Vitis vinifera) cvs. Chardonnay and Alicante Bouschet; 7 samples of ornamental periwinkle (Catharantus roseus) from the Tyr area; and 4 samples of weeds (Lactucca serratia). DNA was extracted from leaf midveins of diseased and symptomless plants, and from healthy periwinkle, grapevine, tomato, and pepper plants grown in a greenhouse in France. Polymerase chain reaction (PCR) with universal primers for the amplification of phytoplasma ribosomal RNA genes (3) only produced a 1.8-kbp rDNA fragment from symptomatic samples. The amplified DNAs were analyzed by restriction fragment length polymorphism (RFLP) with several restriction enzymes and sequenced. The analysis showed extracts of diseased grapevines, and two periwinkle plants had identical rDNA sequences and restriction profiles of the stolbur cluster (4). The sequences had 98% identity with two European stolbur isolates from grapevine and periwinkle (GenBank Accession Nos. X76428 and AF248959, respectively). In grapevine, the disease induced by the stolbur phytoplasma is "bois noir." Bois noir is present in Europe where its incidence is predominant in northern vineyards and has been reported in Israel (2). To our knowledge, this is the first report of the stolbur/bois noir disease in Lebanon. In tomato and pepper, the restriction profiles and sequences of the phytoplasma rDNAs were identical. Sequencing and phylogenetic analysis indicated that the phytoplasma belonged to the clover proliferation (CP) cluster, as does the eggplant little leaf phytoplasma of solanaceous plants in Asia. They differed from the stolbur phytoplasma, known to infect solanaceaous plants in Europe. Lastly, a phytoplasma belonging to the pigeon pea witches' broom (PPWB) cluster was found in L. serratia and in some periwinkle plants. A phytoplasma of the PPWB cluster was recently shown to be responsible for an emerging lethal disease of almond trees in Lebanon (1).

Molecular and functional diversity in soil micro-organisms

Traditional approaches to the study of microbial diversity have relied on laboratory cultivation of isolates from natural environments and identification by classical techniques, including analysis of morphology, physiological characteristics and biochemical properties. These approaches provide information on fine-scale diversity but suffer from bias, resulting from the media and cultivation conditions employed, and from the inability to grow and isolate significant proportions of natural communities in the laboratory. An alternative approach is the amplification of ribosomal RNA and functional genes from nucleic acids extracted directly from environmental samples, with subsequent analysis by ‘fingerprinting’ methods or by sequencing and phylogenetic analysis. This approach avoids the need for laboratory cultivation and has provided major insights into species and functional diversity of bacterial and archaeal populations. This article reviews molecular approaches to the characterisation of prokaryote diversity in natural environments, their more recent application to fungal diversity and the advantages and limitations of molecular analyses.

Variable selection in classification of environmental soil samples for partial least square and neural network models

Two variable selection methods were evaluated by comparing their predictions with respect to differentiating among environmental soil samples. The focus of this work is to determine which input variables are most relevant for prediction of soil sources using discriminant partial least square (D-PLS) and back-propagation artificial neural network (BP-ANN) models. The methods investigated were stepwise variable selection method and genetic algorithms (GAs). Microbial community DNA was extracted from 48 environmental soil samples derived from different field crops and soil sources. After amplification of bacterial ribosomal RNA genes by polymerase chain reaction (PCR), the products were separated by gel electrophoresis. Characteristic complex band patterns were obtained, indicating high bacterial diversity. Two hundred and twenty-three DNA band patterns produced in the gels of the soil samples were used in the analysis, after removal of included DNA standard markers. Based on the brightness of the bands, densitometric curves of the selected DNA band pattern were extracted from the gel images. The curves were smoothed using Savitsky–Golay method and scaled to the DNA standard markers. The prediction results based on the two variable selection methods for PLS and ANN models are presented and compared. Both methods gave good results before any variable selection methods, with the ANN being better than D-PLS. The prediction performance of both methods specially the D-PLS were improved by applying the stepwise variable selection and the GA variable selection method. The study also shows that GA variable selection had a significant improvement of the predictive ability than the stepwise variable selection method.

Amplification of ribosomal RNA genes in acute myeloid leukemia.

Gene amplification is a relatively rare event in acute myeloid leukemia (AML). Double minutes (dmin) and homogeneously staining regions are well established phenomena as cytogenetic correlates of gene amplification. Recently, however, two additional mechanisms leading to gene amplification, i.e., segmental jumping translocations and formation of ring chromosomes, have been described. We report four patients with AML, in whom bone marrow cells exhibited amplifications of ribosomal RNA (rRNA) genes in the form of ring chromosomes or a hsr. In two patients, the MLL gene, and in one patient the CBFA2 gene were shown to be co-amplified with rRNA genes. In two of the four patients, multiple copies of alpha-satellite sequences of the centromeres 13 and 21, respectively, were also demonstrated. In three of the four patients, the clinical course was very aggressive, leading to death within 2-8 months. In these three patients, complex karyotype abnormalities were found, whereas the karyotype of Patient 4 was characterized only by supernumerary ring 21 chromosomes of different sizes and a trisomy 8 in half of the metaphases. Modes of origin and clinical significance of the amplification of rRNA genes are discussed.

First Report of a Phytoplasma Disease of Almond (Prunus amygdalus) in Lebanon.

During a survey conducted in October 1999 to establish the sanitary status of stone fruits in Lebanon, almond trees with symptoms of leaf yellowing, shoot proliferation, and dieback were observed in the Bekaa region. Because such symptoms are often associated with phytoplasma infections, samples were collected for analysis by PCR using universal primers for amplification of phytoplasma ribosomal RNA genes (2). DNA was extracted from the leaf midveins and/or bark phloem tissue from nine symptomatic trees and one symptomless tree in four different orchards as well as from healthy almond trees collected in France. PCR resulted in amplification of an expected 1.8 kbp rDNA fragment from all symptomatic samples but not from the healthy or symptomless samples. For characterization, the amplified DNA was analyzed by RFLP. Even though the restriction profiles were different from those published for other phytoplasmas and in particular from those infecting almond trees in Western Europe (1), sequence analysis of the amplified DNA revealed that it belongs to the pigeon pea witches' broom cluster (PPWB) (2). This is the first report of a phytoplasma infection in Lebanon and the first report for a PPWB group phytoplasma in almond trees.

Symbiotic spirochetes in the termite hindgut: phylogenetic identification of ectosymbiotic spirochetes of oxymonad protists.

Some species of protists inhabiting the hindgut of lower-termites have a large number of ectosymbiotic spirochetes on the cell surface. The phylogenetic positions of the ectosymbiotic spirochetes of three oxymonad protists, Dinenympha porteri in the gut of Reticulitermes speratus, and Pyrsonympha sp. and Dinenympha sp. in Hodotermopsis sjoestedti, were investigated without cultivation of these organisms. Protist fractions carefully collected with a micromanipulator were used as templates for the amplification of small subunit ribosomal RNA genes (SSU rDNA). The phylogenetic tree inferred from the nucleotide sequences of the SSU rDNA showed that they were affiliated with the Treponema cluster of spirochetes and they were divided into two clusters. One was grouped together with the spirochetal sequences reported previously from the gut of termites and the other was related to the Treponema bryantii subgroup of treponemes (denoted as termite Treponema clusters I and II, respectively). Whole-cell in situ hybridization using a fluorescent-labeled oligonucleotide probe specific for the group of sequences in cluster II identified most of the ectosymbiotic spirochetes of the oxymonad protists in the gut of R. speratus and H. sjoestedti. However, not all of the ectosymbiotic spirochetes could be detected by means of this cluster II group-specific probe and the population of ectosymbiotic spirochetes of cluster II was different among the oxymonad species. In the case of D. porteri, an oligonucleotide probe specific for one member of cluster II recognized a portion of the ectosymbiotic spirochetes of cluster II, and their population was also different depending on the cell-type of D. porteri in terms of the attachment of ectosymbiotic spirochetes. The results indicate that the spirochetes of cluster II and probably those of a part of cluster I can be assigned to ectosymbiotic species of oxymonad protists and that the population of ectosymbiotic spirochetes associated with a single protist consists of at least three species of phylogenetically distinct spirochetes.

Symbiotic spirochetes in the termite hindgut: phylogenetic identification of ectosymbiotic spirochetes of oxymonad protists

Some species of protists inhabiting the hindgut of lower-termites have a large number of ectosymbiotic spirochetes on the cell surface. The phylogenetic positions of the ectosymbiotic spirochetes of three oxymonad protists, Dinenympha porteri in the gut of Reticulitermes speratus, and Pyrsonympha sp. and Dinenympha sp. in Hodotermopsis sjoestedti, were investigated without cultivation of these organisms. Protist fractions carefully collected with a micromanipulator were used as templates for the amplification of small subunit ribosomal RNA genes (SSU rDNA). The phylogenetic tree inferred from the nucleotide sequences of the SSU rDNA showed that they were affiliated with the Treponema cluster of spirochetes and they were divided into two clusters. One was grouped together with the spirochetal sequences reported previously from the gut of termites and the other was related to the Treponema bryantii subgroup of treponemes (denoted as termite Treponema clusters I and II, respectively). Whole-cell in situ hybridization using a fluorescent-labeled oligonucleotide probe specific for the group of sequences in cluster II identified most of the ectosymbiotic spirochetes of the oxymonad protists in the gut of R. speratus and H. sjoestedti. However, not all of the ectosymbiotic spirochetes could be detected by means of this cluster II group-specific probe and the population of ectosymbiotic spirochetes of cluster II was different among the oxymonad species. In the case of D. porteri, an oligonucleotide probe specific for one member of cluster II recognized a portion of the ectosymbiotic spirochetes of cluster II, and their population was also different depending on the cell-type of D. porteri in terms of the attachment of ectosymbiotic spirochetes. The results indicate that the spirochetes of cluster II and probably those of a part of cluster I can be assigned to ectosymbiotic species of oxymonad protists and that the population of ectosymbiotic spirochetes associated with a single protist consists of at least three species of phylogenetically distinct spirochetes.

DNA probe for Lactobacillus delbrueckii subsp. lactis.

Lactobacilli are of great commercial value because they are widely used as starters in food fermentation. The species Lactobacillus delbrueckii, which comprises the three subspecies bulgaricus, lactis and delbrueckii, is important in dairy products and vegetables. The subspecies bulgaricus is present in yogurt, and subspecies lactis is recovered from whey, starter cultures and cheeses (Stiles &amp; Holzapfel, 1997). It is unusual to recover Lb. delbrueckii subsp. delbrueckii (Lb. delbrueckii) from dairy products, from which Lb. delbrueckii subsp. lactis (Lb. lactis) and subsp. bulgaricus (Lb. bulgaricus) are typically isolated. However, given the similarity of these two latter subspecies, a clear identification of isolates on the basis of phenotype criteria alone is often problematic (Dellaglio, 1989; Millière et al. 1996).The use of DNA probes and methods based on polymerase chain reaction (PCR) have greatly facilitated diagnostic identification of bacteria. For the lactobacilli, DNA probes have been described for Lb. helveticus, Lb. acidophilus, Lb. fermentum, Lb. plantarum and most of the other Lactobacillus species (Pot et al. 1994; Tailliez et al. 1994; Quere et al. 1997). For Lb. delbrueckii, an EcoRI DNA fragment of the plasmid pY85 was used as a probe for this species, although it was not able to discriminate its three subspecies (Delley et al. 1990).In our laboratory, a specific amplification of a DNA fragment of ∼1·7 kbp using universal primers for the amplification of ribosomal RNA (rRNA) genes was found only for dairy isolates of Lb. lactis and not for Lb. bulgaricus, Lb. delbrueckii, Lb. helveticus and Lb. acidophilus (G. Giraffa, P. de Vecchi and L. Rossetti, unpublished results). This prompted us to test the possible use of this fragment as a specific DNA probe for Lb. lactis. To this end, Southern and dot blot hybridization experiments were carried out with total DNA of several strains belonging to different lactic acid bacteria (LAB) species.

Whipple’s syndrome (uveitis, B27-negative spondylarthropathy, meningitis, and lymphadenopathy) associated with Arthrobacter sp. infection

Objective To report an unusual case of Whipple’s disease, including uveitis, seronegative spondylarthropathy, meningitis, and lymphadenopathy, associated with an Arthrobacter sp. infection. Design Interventional case report. Patient and intervention A 60-year-old white man presenting with severe chronic uveitis and systemic inflammatory manifestations was treated efficiently for Whipple’s disease after histopathologic analysis of vitreous and inguinal adenopathy biopsy specimens. The authors performed a retrospective, laboratory-based evaluation of stored tissue specimens. Measurements Molecular analysis based on 16S ribosomal RNA gene amplification was applied to pretreatment biopsy specimens of inguinal lymph node to identify a causative bacterial agent. Results Tropheryma whippelii genome was not detected in these specimens. However, an amplification product was obtained after the first polymerase chain reaction run and subsequently was sequenced. It corresponded to an Arthrobacter sp., a gram-positive agent presenting diagnostic patterns and therapeutic management similar to those of Whipple’s disease caused by T. whippelii. Conclusion The absence of T. whippelii identification by molecular amplification during a clinically and histologically oriented Whipple’s syndrome should not rule out the diagnosis. Arthrobacter infection may represent a new bacterial etiology of systemic inflammatory disorders involving the eye and associated with periodic acid-Schiff-positive inclusions.

Monitoring of fluorescence during DNA melting as a method for discrimination and detection of PCR products in variety identification

DNA melting curves of genotype-specific PCR fragments were used to differentiate between species and amongst varieties of cereals. Melting curves were generated by ramping the temperature of PCR fragments through their dissociation temperature in the presence of a double-stranded DNA binding dye. Genotypes were discriminated by differences in the position and shape of the melting curve which is a function of the fragment's sequence, length and GC content. Amplification of 5S ribosomal RNA genes generated species-specific fragments for six of the major cereal crops. Of the 15 possible pairwise comparisons, 13 distinctions could be reliably made using melting curve position data. Wheat varieties were identified by the melting profiles of PCR products generated using microsatellite primers. DNA melting curve analysis was conveniently coupled with capillary-PCR using a LightCycler instrument to provide a rapid method of genotyping in cereals.

DNA probes for the identification of microorganisms

The detection and identification of microorganisms is being carried out increasingly using DNA. Each organism has a unique DNA sequence which can be used to distinguish closely related organisms. Using PCR amplification and sequencing of ribosomal RNA genes we have developed DNA probes for a number of pathogenic bacteria and fungi. The development of DNA assays based on PCR has resulted in new questions which must be addressed including process carry-over contamination and inhibition of the PCR amplification reaction once the problems associated with the implementation of DNA assays are ironed out.

Structure of the large ribosomal subunit RNA of Phytophthora megasperma, and phylogeny of the oomycetes

The 5.8S and 28S rRNA sequences of the oomycete Phytophthora megasperma were determined in order to study the secondary structure of these molecules and to assess the phylogenetic position of the oomycetes among the eukaryotes. Preliminary results point to an affiliation between the oomycetes, dinoflagellates and ciliates, a cluster which seems related to the fungi. In the course of this work, we developed a set of primers which allow sequencing and PCR amplification of eukaryotic large ribosomal subunit RNA genes of a wide range of phylogenetically distant organisms.

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

There is a clear requirement to develop sensitive methods for detecting denned 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 of Laccaria (L. bicolor, L. laccata, L. proxima, 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 sufficient 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.