Four strains of Phellinus spp. was identified based on internal transcribed spacer (ITS) region of rDNA sequence analysis and morphological characteristics. Basidiocarps of all strains were effused-reflexed and hymenial surface was poroid. Hyphal system was dimitic and basidiospore was globose to ellipsoid. The amplification of ITS regions produced a DNA fragment of 500 to 780 bp in all strains examined. The determined sequences were analyzed for the reconstruction of phylogenetic tree. From these results, Phellinus sp. KM-1, KM-2, and KM-4 was identified as P. hartigii, P. baumii, and P. linteus, respectively.
DNA ; DNA, Ribosomal ; Fruiting Bodies, Fungal ; Sequence Analysis

Flammulina velutipes was transformed efficiently by Agrobacterium-mediated transformation system. The transformation frequency was about 16% with the gill tissues of the fungal fruiting body. Southern hybridization and genetic analysis suggest that the introduced DNA was inserted onto different locations of the fungal genome, and inherited stably to the next generation via basidiospores. Transformation or gene tagging with Agrobacterium T-DNA based vector should be useful for wide ranges of genetic or molecular biological studies of the mushroom.
Agaricales* ; Agrobacterium ; Animals ; DNA ; Flammulina* ; Fruiting Bodies, Fungal ; Genome, Fungal ; Gills

A very reliable and specific method for the identification of fungi in ectotrophic mycorrhizal symbiosis was developed using a specific PCR assay based on the amplification of the ITS1 region. To obtain specific data, an ITS-diagnostic assay was carried out that reveals genera and species specific sequences. Here, an application of one method is presented, which covers the identification of pure mycelia, basidiocarps as well as mixed samples such as ectomycorrhizal roots that were mingled with remains of the host plant. For this purpose a protocol was established that allowed the extraction of DNA from single mycorrhizal roots. In order to perform a specific ITS analysis we generated a new ITS-primer (ITS8) by a multiple alignment of five different genera and species of mycorrhizal fungi. The utilization of ITS1 and ITS8 resulted in specific PCR amplicons, which were characterized by sequencing without purification steps, even when the template DNA was associated with roots.
DNA ; Fruiting Bodies, Fungal ; Fungi* ; Plants ; Polymerase Chain Reaction* ; Symbiosis

Isolates of Trichoderma spp. collected from Pleurotus ostreatus and P. eryngii beds, which included loosened substrate compactness and development of green colour, were grouped into three species. The occurrence of different species of Trichoderma was as T. cf. virens (70.8%), T. longibrachiatum (16.7%) and T. harzianum (12.5%). The conidia of Trichoderma spp. were ellipsoidal, obovoid and phialides were bowling pins, lageniform and the length of phialides was 3.5~10.0 x 1.3~3.3 micromm. Phialides of T. cf. virens and T. harzianum were tending clustered, but it was solitary disposition in T. longibrachiatum. T. cf. virens was characterized by predominantly effuse conidiation, sparingly branched, and fertile to the apex and it was penicillate type. RAPD analysis could detect variability amongst three different species of Trichoderma using two newly designed URP-primers. However, intra-specific variation could not be detected in all the isolates except for rDNA sequence data classified Trichoderma isolates into three distinct groups representing three species. The profiles of rDNA sequences of isolates representing a species showed high similarity in T. cf. virens and T. harzianum. However, there was a variation in rDNA sequences of isolates representing T. longibrachiatum. The results of present study reveals that molecular techniques of RAPD and rDNA sequencing can greatly aid in classification based on morphology and precise identification of fast evolving species of Trichoderma.
Classification ; DNA, Ribosomal ; Fungi* ; Ostreidae* ; Pleurotus* ; Spores, Fungal ; Trichoderma*

Amylosporus sulcatus sp. nov. is described from Nonggang Nature Reserve, southern China, on the basis of morphological and molecular data. The morphological description and illustrations for the new species are provided. The species is characterized by pileate and stipitate basidiocarps. The pileus surface is obviously concentrically and radiately sulcate and tomentum, and the pore surface is snow white. Phylogenetic analyses based on sequences of the internal transcribed spacer and nuclear large subunit ribosomal DNA confirmed it to be a new species.
China* ; Classification ; DNA, Ribosomal ; Fruiting Bodies, Fungal ; Snow

To accelerate the breeding process of cultivated Ophiocordyceps sinensis and increase its yield, it is important to identify molecular fingerprint of dominant O. sinensis. In the present study, we collected 3 batches of industrially cultivated O. sinensis product with higher yield than the others and compared their internal transcribed spacer (ITS) sequences with the wild and the reported. The ITS sequence was obtained by bidirectional sequencing and analyzed with molecular systematics as a DNA barcode for rapid and accurate identification of wild and cultivated O. sinensis collected. The ITS sequences of O. sinensis with detailed collection loci on NCBI were downloaded to construct a phylogenetic tree together with the sequences obtained from the present study by using neighbor-joining method based on their evolution relationship. The information on collection loci was analyzed with ArcGIS 10.2 to demonstrate the geographic distribution of these samples and thus to determine the origin of the dominant samples. The results showed that all wild and cultivated samples were identified as O. sinensis and all sequences were divided into seven phylogenetic groups in the tree. Those groups were precisely distributed on the map and the process of their system evolution was clearly presented. The three cultivated samples were clustered into two dominant groups, showing the correlation between the industrially cultivated samples and the dominant wild samples, which can provide references for its optimized breeding in the future.
Breeding ; DNA, Fungal ; genetics ; DNA, Intergenic ; genetics ; Genes, Mating Type, Fungal ; Hypocreales ; chemistry ; classification ; genetics ; growth & development ; Phylogeny

BACKGROUND: Culture based technique, a traditional method for extraction of DNA from a cultured colony, was complex in culture conditions and was associated with a lower chance of successful culture. Recently, non-culture based technique, which skipped the culture process and directly extracted fungal DNA and differentiated Malassezia species, has been introduced. OBJECTIVE: Using 26S rDNA PCR-RFLP, the authors identified Malassezia yeasts and compared the yield of Malassezia DNA by the traditional culture based technique and the non-culture based technique via Op-site adhesive tape. METHODS: DNA of Malassezia yeasts were extracted using the culture based technique and the non-culture based technique from normal adults. Comparison was performed in order to clarify the differences between these two techniques. RESULTS: Use of the culture based technique resulted in a culture rate of 57.8% (78 out of 135 samples). On the other hand, using the non-culture based technique, fungal species were identified from all 135 samples. Using both techniques, M. globosa was the most identified species. The identification rate of the non-culture based technique was 100%; however, 7 repeats of PCR were required to reach 100% identification. Among samples from five body sites, those from the thigh required 5.5 repeats of PCR. CONCLUSION: The non-culture based technique was better than the culture based technique. However, due to the low amount of DNA extracts from the body sites with low habitation of Malassezia yeasts, repeated PCR was required for differentiation of Malassezia species.
Adhesives ; Adult ; DNA ; DNA, Fungal ; DNA, Ribosomal ; Hand ; Humans ; Malassezia ; Polymerase Chain Reaction ; Skin ; Thigh ; Yeasts

To establish a simple, sensitive and effective technique for the identification of six common dermatophytes, polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (RFLP) targeting Topoisomerase II gene were used. The DNA of 6 common dermatophytes was amplified by primer dPsD1 and then primers dPsD2. The products generated by dPsD2 were digested with restriction enzyme Hinc II and Hinf I separately. A DNA fragment of about 3390 bp was amplified by using primer dPsD1 from the genomic DNA of each dermatophyte species. The product of dPsD2 was 2380 bp and the restriction profiles of Hinc II and Hinf I were between 58-1670 bp. By using PCR-RFLP, all of the 6 dermatophytoses were diagnosed to species level and no obvious difference identification between Hinc II and Hinf I. It is concluded that the PCR-RFLP identification of dermatophytes by Hinc II or Hinf I is efficient and rapid in clinical practice.
Arthrodermataceae/*classification ; Arthrodermataceae/genetics ; Arthrodermataceae/*isolation & purification ; DNA Topoisomerases, Type II/genetics ; DNA, Fungal/analysis ; DNA, Fungal/genetics ; Dermatomycoses/*microbiology ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length

OBJECTIVESTo investigate the DNA polymorphism of Sporothrix schenckii (S. schenckii) and to find the relationship between DNA patterns and geographic areas and clinical manifestations.

METHODThe total DNA was extracted with hexadecyltrimethyl-ammonium bromide. Random Amplification of Polymorphic DNA (RAPD) assay was used to study DNA typing of 24 strains of S. schenckii collected from different areas and isolated from different clinical types.

RESULTSOf seven random primers used, three primers (OPAA11, OPD18 and OPB07) gave good reactions, the sequences of which were 5'-ACCCGACCTG-3', 5'-GAGAGCCAAC-3', 5'-GGTGAC~GCAG-3' respectively. The RAPD patterns of the 24 isolates were not completely identical, showing certain degrees of hereditary variability. Different isolates showed a common conserved DNA band with the same primer. Different clinical types showed different genotypes.

CONCLUSIONRAPD analysis is useful in DNA typing of S. schenckii, the DNA band type of which is related to geographic origin and Clinical manifestation.

Arbuscular mycorrhizas (AM) have mutualistic symbiosis with plants and thus efforts have been placed on application of these symbiotic relationships to agricultural and environmental fields. In this study, AM fungi were collected from 25 sites growing with 16 host plant species in Korea and cultured with Sorghum bicolor in greenhouse condition. AM fungal spores were extracted and identified using both morphological and molecular methods. Using morphological characters, total 15 morpho-speices were identified. DNA was extracted from single spore of AM fungi and a partial region on 18S rDNA was amplified using nested PCR with AM fungal specific primers AML1/AML2. A total of 36 18S rDNA sequences were analyzed for phylogenetic analysis and 15 groups of AM fungi were identified using both morphological and molecular data of spores. Among the species, 4 species, Archaeospora leptoticha, Scutellospora castanea, S. cerradensis, S. weresubiae were described for the first time in Korea and two species in Glomus and a species in Gigaspora were not identified. Morphological and molecular identification of AM fungal spores in this study would help identify AM fungal community colonizing roots.
Colon ; DNA ; DNA, Ribosomal ; Fungi ; Korea* ; Mycorrhizae ; Plants ; Polymerase Chain Reaction ; Sorghum ; Spores ; Spores, Fungal* ; Symbiosis