Fungal-specific Primers Research Articles

Sequence Analyses of PCR Amplified Partial SSU of Ribosomal DNA for Identifying Arbuscular Mycorrhizal Fungi in Plant Roots

The genomic DNAs were extracted from roots of Glycine max and Sorghum bicolor, and compared with those from spores of two arbuscular mycorrhizal (AM) fungi, Glomus mosseae and Scutellospora heterogama. The partial small subunit (SSU) of ribosomal RNA genes were synthesized and amplified by polymerase chain reaction with the fungal specific primers, AM1 and NS31. By the recent molecular techniques, the presence of another AM fungal DNA were confirmed in the roots of two plants, and three sequences of rDNA fragments amplified were identified to be close to those of G. caledonium, G. fasic- ulatum and G. proliferum The two AM fungi, both, were found to colonize at the cortical layers of plant roots collected in the fields, together.

Identification of fungi from dairy products by means of 18S rRNA analysis

The role of fungi as cause of spoilage of dairy products, such as cheese and yoghurt, has been clearly demonstrated. Despite of this, there is still a lack in rapid methods for the identification of food-associated fungi. In the course of the present work, molecular taxonomical techniques were developed and used to identify yeasts involved in the spoilage of yoghurt and moulds responsible for spoilage of vacuum-packaged hard cheese. Three methods for DNA extraction and purification were evaluated and the fungus-specific primers TR1 and TR2 were used to amplify a 581-bp fragment within the gene, coding for the small ribosomal subunit (18S rRNA) of fungi. The 18S rRNA sequence analysis of fungi isolated from yoghurt and packaged cheese allowed to identify yeast belonging to Zygosaccharomyces microellipsoides and moulds belonging to Penicillium chrysogenum and Cladosporium cladosporoides.

Detection and Taxonomic Placement of Endophytic Fungi within Frond Tissues of Livistona chinensis Based on rDNA Sequences

The 5.8S gene and flanking internal transcribed spacers (ITS1 and ITS2) of the rDNA were amplified from total DNA extracted from frond tissues of Livistona chinensis with universal and fungal-specific primers. These amplified fragments were cloned and sequenced. Phylogenetic analysis based on the 5.8S gene sequences indicated that the six clone sequences obtained were of different origins. Five sequences, P1-9, P2-6, P4-4, P4-5, and P4-7, belonged to the fungi and one sequence, P3-2, belonged to the plants. P1-9 was inferred to belong to the Basidiomycota based on the phylogenetic analysis of the 5.8S gene sequences but could not be identified to lower taxonomic levels. Further identification of the other four fungal clones to lower taxonomic levels was attempted based on phylogenetic analysis and sequence comparison of both the conserved 5.8S gene and the variable ITS regions. The origin of P2-6 was identified to be Glomerella and its anamorph Colletotrichum, the origins of P4-5 and P4-7 were Mycosphaerella and its anamorph Cladosporium, and the origin of P4-4 was the Herpotrichiellaceae. The direct approach to detection and taxonomic placement of endophytic fungi within host tissue without the need for conventional in vitro culturing is discussed.

Development of Molecular Methods for Identification of Schizophyllum commune from Clinical Samples

In the last 50 years, to our knowledge, only 16 cases of diseases caused by Schizophyllum commune in humans have been reported. Within only 6 months, we found four isolates of this basidiomycetous fungus, obtained from patients suffering from chronic sinusitis. The cultures of the isolated fungi showed neither clamp connections nor fruiting bodies (basidiocarps), which are distinctive features for S. commune, but fast-growing cottony white mycelium only. This was harvested, and DNA was extracted. The internal transcribed spacer region of the ribosomal DNA (rDNA) was amplified with fungus-specific primers, and the PCR products were sequenced. Two strains of S. commune, collected from branches of a European hornbeam (Carpinus betulus) and a tree of heaven (Ailanthus altissima), respectively; four specimens from the herbarium of the Institute of Botany, Karl-Franzens-University Graz; and two strains from internationally known culture collections (CBS 340.81 [ATCC 44201] and CBS 405.96) were investigated in the same way. The sequence data of all strains were compared and showed homology of over 99% in this 660-bp-long fragment of rDNA. With these results, a map of restriction enzyme cutting sites and a primer set specific for S. commune were created for reliable identification of this human pathogenic fungus.

Variation in the ribosomal ITS-sequence of the lichens Buellia frigida and Xanthoria elegans from the Vestfold Hills, eastern Antarctica

Abstract:Thalli of the lichens Buellia frigida and Xanthoria elegans were collected from five different locations each 5–15km apart in the Vestfold Hills, Princess Elizabeth Land, eastern Antarctica. A further collection was made from Mawson Station, Mac Robertson Land, eastern Antarctica, 660km away. DNA was extracted from whole thalli and the ribosomal ITS region amplified by PCR using fungal specific primers. Resulting products were sequenced to gain an indication of whether or not variation was present within populations of lichen-forming fungi from continental Antarctica, and therefore of the availability of genetic resources to react to pressures such as climate change. Three genotypes ofB. frigida and two of X. elegans were detected in the Vestfold Hill collections. However, these differed by only one nucleotide position suggesting the presence of relatively little genetic variation, if the ITS region is indicative of the overall genome. Buellia frigida collected from Mawson Station had an identical ITS region sequence to the most common Vestfold Hills genotype, indicating that this species may have a low level of genetic variation across much of eastern Antarctica. In contrast, X. elegans collected from Mawson showed considerable genetic variation from the Vestfolds thalli, differing at 14·2% of nucleotide positions and had an identical ITS region sequence to an isolate from maritime Antarctica 4960km away. Samples from the Vestfold Hills formed a distinct cluster in a phylogenetic analysis of ITS sequences from a worldwide collection of X. elegans isolates.

Molecular diversity of ericoid mycorrhizal fungi

Using restriction fragment length polymorphism (RFLP) patterns from two ribosomal internal transcribed spacers (ITS) and DNA sequences from ITS2, we characterized ericoid mycorrhizal fungal isolates from culture collections.With a synoptic key to RFLP patterns, we divided 34 mycorrhizal or root-associated isolates into 16 groups. RFLP patterns were identical when fungal specific primers were used to amplify DNA from pure fungal cultures and in vitro mycorrhizae. Sequence analysis clustered 23 of 24 mycorrhizal isolates into two larger groups: the Oidiodendron group and the Hymenoscyphus group. The Oidiodendron group included genetically uniform, conidiating fungi. The Hymenoscyphus group encompassed more diversity and included other discomycetes (Leotiales) as well as sterile, unidentifiable mycorrhizal isolates from four RFLP groups. Results from our field site on Vancouver Island, British Columbia, Canada, suggest that several ericoid mycorrhizal fungi can coexist in a single root of Gaultheria shallon Pursh and that our molecular data base is not yet complete. From sixty 3-mm root sections, we cultured four genetically different fungi that formed mycorrhizae in resynthesis experiments and sequence analysis showed that one of these differed from all previously known ericoid mycorrhizal fungi.

Direct detection of Histoplasma capsulatum in soil suspensions by two-stage PCR

Histoplasmosis is the most common pulmonary mycosis in the United States. The responsible fungal pathogen, Histoplasma capsulatum , grows in soils contaminated with bird or bat droppings. Inhalation of dust from contaminated areas containing H. capsulatum spores is a primary route of infection. The ability to detect H. capsulatum in soil samples has been limited by the lack of fast, reliable and inexpensive methods. A polymerase chain reaction (PCR) method was developed that allows the direct detection of H. capsulatum in soil. A two-stage PCR protocol was followed employing both fungal-specific primers and nested primers specific for the internal transcribed spacer (ITS) region of the 5·8S rRNA gene of H. capsulatum . The estimated limit of detection of this method is 10 spores. In contrast to the more expensive and indirect mouse inoculum assay, which requires 6–8 weeks for sample analysis, PCR analysis of soil contaminated with H. capsulatum can be completed in less than 2 days.

Rapid identification of fungi by using the ITS2 genetic region and an automated fluorescent capillary electrophoresis system.

Invasive fungal disease often plays an important role in the morbidity and mortality of immunocompromised patients. The poor sensitivity of current fungal blood culture and histological practices has led to the development of highly sensitive and specific molecular techniques, such as the PCR. Sequence variability of the internal transcribed spacer 2 (ITS2) region of fungi is potentially useful in rapid and accurate diagnosis of clinical fungal isolates. PCR with fungus-specific primers targeted toward conserved sequences of the 5.8S and 28S ribosomal DNA (rDNA) results in amplification of the species-specific ITS2 regions, which are variable in amplicon length. We have made use of the ABI PRISM 310 genetic analyzer and the ABI PRISM 310 GeneScan analysis software for the determination of variable size differences of the ITS2 region of clinically important fungi, including Candida and non-Candida yeasts, Aspergillus species, and a variety of dermatophytes. No cross-reaction occurred when samples were tested against human and bacterial genomic DNA. We have found that most clinically significant fungal isolates can be differentiated by this method, and it therefore serves to be a promising tool for the rapid (<7 h) diagnosis of fungemia and other invasive fungal infections.

Laboratory diagnosis of Pneumocystis carinii infections by PCR directed to genes encoding for mitochondrial 5S and 28S ribosomal RNA

PCR with 5S mitochondrial ribosomal RNA (5S) target is a sensitive and specific assay for the detection of Pneumocystis cariniiin clinical specimens from the respiratory tract. We developed an oligonucleotide probe directed to a 200 bp amplicon generated by fungal-specific universal primers that anneals with sequrences specific for P. carinii in the 28S ribosomal RNA gene (28S). Of 50 archived bronchoalveolar lavage (BAL) specimens, 46 of 50 samples (92% agreement) gave the same result (23 positive, 23 negative) by PCR directed to the 5S and 28S assays. Results of calcofluor white staining of BAL smears on slides indicated agreement with the molecular results in 43 of 46 (93.5%) assays. PCR detection of P. carinii by amplification of 28S ribosomal gene target by fungal-specific primers and an organism-specific probe provides an alternate genomic target for the laboratory diagnosis of this organism.

Isolation of fungal rDNA from bottlenose dolphin skin infected with Loboa loboi

Fungal-specific primers targeted for highly conserved genomic nucleic acid sequences were used in a polymerase chain reaction (PCR) to amplify DNA from lobomycosis lesions in a bottlenose dolphin. Sequence alignments of this DNA possessed high homology to fungal ribosomal DNA sequences found in the genus Cladosporium. When used for in situ hybridization, the riboprobe transcribed from a cloned PCR-generated fragment bound to Loboa loboi cells. These results support the hypothesis that L. loboi in dolphin tissue is a fungus.

A protocol for PCR in situ hybridization of hyphomycetes.

A protocol for application of Polymerase Chain Reaction (PCR) in situ hybridization for the detection of hyphomycetes is presented. The experiments are exemplary carried out with strains of the genera Penicillium and Cladosporium. The small ribosomal subunit is amplified in situ by PCR using fungal specific primers. The amplicon is used as target region for a fluorescein-marked probe. The permeability of the fungal cell wall for the primers and the probe can be successfully achieved by enzymatic treatment with beta-glucanase. The protocol can be used as a basis for further development of in situ hybridization with taxon specific probes.

Isolation of fungal rDNA from bottlenose dolphin skin infected withLoboa loboi

Fungal-specific primers targeted for highly conserved genomic nucleic acid sequences were used in a polymerase chain reaction (PCR) to amplify DNA from lobomycosis lesions in a bottlenose dolphin. Sequence alignments of this DNA possessed high homology to fungal ribosomal DNA sequences found in the genus Cladosporium. When used for in situ hybridization, the riboprobe transcribed from a cloned PCR-generated fragment bound to Loboa loboi cells. These results support the hypothesis that L. loboi in dolphin tissue is a fungus.

Molecular analysis of ectomycorrhizal fungal communities

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. Key words: molecular ecology, fungal community structure, PCR.

ITS primers with enhanced specificity for basidiomycetes ‐ application to the identification of mycorrhizae and rusts

We have designed two taxon-selective primers for the internal transcribed spacer (ITS) region in the nuclear ribosomal repeat unit. These primers, ITS1-F and ITS4-B, were intended to be specific to fungi and basidiomycetes, respectively. We have tested the specificity of these primers against 13 species of ascomycetes, 14 of basidiomycetes, and 15 of plants. Our results showed that ITS4-B, when paired with either a 'universal' primer ITS1 or the fungal-specific primer ITS1-F, efficiently amplified DNA from all basidiomycetes and discriminated against ascomycete DNAs. The results with plants were not as clearcut. The ITS1-F/ITS4-B primer pair produced a small amount of PCR product for certain plant species, but the quantity was in most cases less than that produced by the 'universal' ITS primers. However, under conditions where both plant and fungal DNAs were present, the fungal DNA was amplified to the apparent exclusion of plant DNA. ITS1-F/ITS4-B preferential amplification was shown to be particularly useful for detection and analysis of the basidiomycete component in ectomycorrhizae and in rust-infected tissues. These primers can be used to study the structure of ectomycorrhizal communities or the distribution of rusts on alternate hosts.