Assessment of soil fungal communities using pyrosequencing.

Pyrosequencing, a non-electrophoretic method of DNA sequencing, was used to investigate the extensive fungal community in soils of three islands in the Yellow Sea of Korea, between Korea and China. Pyrosequencing was carried out on amplicons derived from the 5' region of 18S rDNA. A total of 10,166 reads were obtained, with an average length of 103 bp. The maximum number of fungal phylotypes in soil predicted at 99% similarity was 3,334. The maximum numbers of phylotypes predicted at 97% and 95% similarities were 736 and 286, respectively. Through phylogenetic assignment using BLASTN, a total of 372 tentative taxa were identified. The majority of true fungal sequences recovered in this study belonged to the Ascomycota (182 tentative taxa in 2,708 reads) and Basidiomycota (172 tentative taxa in 6,837 reads). The predominant species of Ascomycota detected have been described as lichen-forming fungi, litter/wood decomposers, plant parasites, endophytes, and saprotrophs: Peltigera neopolydactyla (Lecanoromycetes), Paecilomyces sp. (Sordariomycetes), Phacopsis huuskonenii (Lecanoromycetes), and Raffaelea hennebertii (mitosporicAscomycota). The majority of sequences in the Basidiomycota matched ectomycorrhizal and wood rotting fungi, including species of the Agaricales and Aphyllophorales, respectively. A high number of sequences in the Thelephorales, Boletales, Stereales, Hymenochaetales, and Ceratobasidiomycetes were also detected. By applying high-throughput pyrosequencing, we observed a high diversity of soil fungi and found evidence that pyrosequencing is a reliable technique for investigating fungal communities in soils.

The Wood-rotting Fungal Flora of Three Islands in the Yellow Sea, Korea.

The wood-rotting fungi of three Korean islands in the Yellow Sea, Soyeonpyung-do (SY), Oeyeon-do (OY), and Gageo-do (GG), were investigated from October 2004 to November 2005. During four sampling trips on each island, a total of 46, 44, and 44 specimens were collected from SY, OY, and GG, respectively. The identified wood-rotting fungi from SY included 29 species of 22 genera and nine families; OY had 31 species of 26 genera and 10 families; and GG had 34 species of 27 genera and 11 families. The majority of the fungi were in the Polyporaceae, which was represented by 23 genera and 30 species. Auricularia polytricha, Daedaleopsis tricolor, Daldinia concentrica, Hymenochaete cinnamomea, Hymenochaete yasudai, Hyphoderma setigerum, Lopharia mirabilis, Schizopora paradoxa, and Trametes versicolor were collected from all three islands.

Morphological discretion of basidiospores of the puffball mushroom Calostoma by electron and atomic force microscopy.

Comparative morphology among species of the genus Calostoma, including C. cinnabarina, C. ravenelii, and C. japonicum, was investigated by scanning electron microscopy and atomic force microscopy. Spore morphology of cinnabarina and C. ravenelii showed no dramatic differences by light microcopy and scanning electron microscopy. To differentiate these species, atomic force microscopy was employed. Quantitative analysis of the surface roughness basidiospores revealed subtle differences in height fluctuation at the nanometer scale between the species of Calostoma. Basidiospores of C. cinnabarina had a relatively rougher surface than those of C. ravenelii at 2.0 x 2.0 micro m2 scan areas.

Fomitopsis incarnatus sp. nov. based on generic evaluation of Fomitopsis and Rhodofomes.

A new polypore in the genus Fomitopsis was discovered in Kangwon Province, Korea. The species was morphologically similar to Fomitopsis rosea and F. cajanderi, but the pinkish white pore surface, the size and shape of the pores and the number of sterigmata were different enough for it to be distinguished from the recorded species of Fomitopsis. Based on the results of morphological and phylogenetic analyses, this new polypore is proposed as Fomitopsis incarnatus sp. nov.

Phaeomoniella zymoides and Phaeomoniella pinifoliorum spp. nov., new acid-tolerant epiphytic fungi isolated from pine needles in Korea.

Two new epiphytes of anamorphic ascomycetes, Phaeomoniella zymoides sp. nov. and Phaeomoniella pinifoliorum sp. nov., were isolated from the needle surface of Pinus densiflora in Korea. The new taxa were characterized by acid-tolerant, slow, partially yeast-like growth and extensive production of emerging cells on convex wrinkled mycelial colonies. Phaeomoniella zymoides produced mycelium with large numbers of intercalary and lateral or terminal vesicles or swollen cells. Large conidiogenous cells had a swollen base and appeared to be phialidic, and many phialoconidia also were produced from lateral hyphal apertures. Maturing colonies of Ph. zymoides were made up of dark green to blackish areas and produced a Phoma-like synanamorph. Primary conidia became elongate mother cells giving rise to polar or lateral secondary conidia. Phaeomoniella pinifoliorum was characterized by reduced, swollen, phialide-like cells, lateral production of conidia from hyphae and terminal or subterminal, or less commonly lateral, secondary production of conidia from yeast-like primary conidia. When ITS and 28S rDNA sequences were compared and analyzed with those of best matching GenBank taxa, the Phaeomoniella group consisted of three lineages, "zymoides", "pinifoliorum" and "chlamydospora" clades, which again showed a complete sister relationship to Moristroma quercinum ined.

Taxonomic study on Korean Aphyllophorales (5) - on some unrecorded genera and species -.

A total of 149 species and 209 strains of Korean Aphyllophorales in Seoul National University Fungus Collection (SFC) were analyzed by taxonomic and phylogenetic methods. Among those examined fungal specimens, 9 genera Abundisporus, Antrodiella, Cyphellopsis, Dendrothele, Dichomitus, Laxitextum, Piloderma, Skeletocutis and Tubulicrinis, and 23 species, Abundisporus fuscopurpureus, Antrodiella semisupina, Auriporia pileata, Cantharellus subalbidus, Clavulina cinerea, Cyphellopsis confusa, Dendrothele acerina, Dichomitus campestris, Haplotrichum aureum, Heterobasidion annosum, Hyphoderma argillaceum, Hyphodontia tropica, Inonotus dryophilus, Ischnoderma benzoinum, Laxitextum bicolor, Phanerochaete radicata, Phellinus lonicericola, Piloderma byssinum, Skeletocutis nivea, Tomentella terrestris, Trametes elegans, Trametes tenuis, and Tubulicrinis accedens were confirmed as new to Korea and registered here with descriptions.

Pseudomonas panacis sp. nov., isolated from the surface of rusty roots of Korean ginseng.

A Gram-negative, aerobic bacterium, designated CG20106(T), was isolated from the surface tissues of rusty root lesions of Korean ginseng. Phylogenetic analysis of the 16S rRNA gene sequence revealed that this isolate represents a hitherto unknown subline within the genus Pseudomonas. Strain CG20106(T) was catalase- and oxidase-positive, motile and rod-shaped. The overall phenotypic features of the ginseng isolate were similar to those of Pseudomonas cedrina, Pseudomonas migulae and Pseudomonas azotoformans. However, several physiological and chemotaxonomic properties can be weighted to distinguish the isolate from these organisms. The major fatty acids were C(16:1)omega7c and/or iso-C(15:0) 2-OH (summed feature 3, 36.4+/-0.4%), C(16:0) (27.5+/-0.7%) and C(18:1)omega7c (19.4+/-0.2%). The DNA G+C content was 61.4 mol%. On the basis of the polyphasic results revealed in this study, the name Pseudomonas panacis sp. nov. is proposed for strain CG20106(T). The type strain is CG20106(T) (=IMSNU 14100(T)=CIP 108524(T)=KCTC 12330(T)).

Wood-rotting Fungal Flora of Kanghwa Island.

Through ten field surveys in Kanghwa Island from August of 1997 to March of 2002, total 107 specimens of wood-rotting fungi belonging to the Aphyllophorales were collected and identified to the species. They taxonomically amounted to 10 families, 31 genera, and 48 species. Among them, one family, Steccherinaceae, and four genera, Australohydnum, Castanoporus, Dacryobolus and Haplotrichum were confirmed as new to Korea. Five unrecorded species, Australohydnum dregeanum, Castanoporus castaneus, Dacryobolus karstenii, Haplotrichum conspersum and Hyphoderma odontiiforme were registered here with descriptions.

Evaluation of the monophyly of Fomitopsis using parsimony and MCMC methods.

To evaluate the monophyly of Fomitopsis and elucidate phylogenetic relationships of its members, partial nuclear large subunit (partial 28S) ribosomal RNA genes were sequenced from 10 species of Fomitopsis and 15 related species. Phylogenetic analyses indicated that Fomitopsis was phylogenetically heterogeneous and its members were divided into three subgroups. The constrained tree excluding F. palustris (the type species of Pilatoporus) from Fomitopsis core group was rejected, thus rejecting the taxonomic concept to segregate Pilatoporus from Fomitopsis. The monophyly of taxa belonging to F. rosea complex was rejected, thus rejecting the complex definition based on morphological similarities. The exclusion of Piptoporus betulinus (the type species of Piptoporus) from Fomitopsis core group was rejected and Piptoporus proved to be heterogeneous in both best MP and MAP trees. The monophyly of F. officinalis with Fomitopsis core group also was rejected. Fomitopsis officinalis was closely related to Antrodia xantha and formed an independent lineage from Fomitopsis core group at the basal position of brown rotting fungi comprising Antrodia, Daedalea, Fomitopsis, Piptoporus and Postia. The MAP tree topologyobtained from MCMC computation of Bayesian inference was similar to the one of the best MP tree based on the parsimony analysis but showed a higher likelihood score in the Kishino-Hasegawa test and reflected better evolutionary patterns for the phylogeny of Fomitopsis.

Phylogenetic analysis of Ganoderma based on nearly complete mitochondrial small-subunit ribosomal DNA sequences.

Characteristics and structures of mt SSU rDNA were investigated for the phylogenetic study of Ganoderma. Phylogenetic information was concentrated mostly in the V1, V4, V5, V6 and V9 variable domains, but informative sites in conserved domains also significantly contributed in resolving phylogenetic relationships between Ganoderma groups. Secondary structure information of variable domains was found to be a useful marker in delineation of phylogenetic groups. Strains of Ganoderma species used in this study were divided into six monophyletic groups. Ganoderma colossus made a distinct basal lineage from other Ganoderma species and Tomophagus, created for G. colosuss, appeared to be a valid genus. Ganoderma applanatum and G. lobatum classified in subgenus Elfvingia made a monophyletic group. Ganoderma tsugae from North America and G. valesiacum from Europe, both living on conifers, were closely related. Ganoderma oregonense and strains labeled G. lucidum from Europe and Canada were grouped with G. tsugae and G. valesiacum. Strains labeled G. lucidum living on hardwoods from the United States and Taiwan were grouped with G. resinaceum, G. pfeifferi and G. subamboinense var. laevisporum, and they all produced chlamydospores. Two strains labeled G. lucidum and three strains labeled G. resinaceum from America were concluded to be conspecific. Strains labeled G. lucidum from Korea and Japan were monophyletic and were distinguished from strains labeled G. lucidum from Europe and North America. Host relationships and the presence of chlamydospores in culture proved to be important characteristics in the systematics as well as the phylogenetic relationships of Ganoderma.

Chaga mushroom extract inhibits oxidative DNA damage in human lymphocytes as assessed by comet assay.

The Chaga mushroom (Inonotus obliquus) is claimed to have beneficial properties for human health, such as anti-bacterial, anti-allergic, anti-inflammatory and antioxidant activities. The antioxidant effects of the mushroom may be partly explained by protection of cell components against free radicals. We evaluated the effect of aqueous Chaga mushroom extracts for their potential for protecting against oxidative damage to DNA in human lymphocytes. Cells were pretreated with various concentrations (10, 50, 100 and 500 microg/mL) of the extract for 1 h at 37 degrees C. Cells were then treated with 100 microM of H2O2 for 5 min as an oxidative stress. Evaluation of oxidative damage was performed using single-cell gel electrophoresis for DNA fragmentation (Comet assay). Using image analysis, the degree of DNA damage was evaluated as the DNA tail moment. Cells pretreated with Chaga extract showed over 40% reduction in DNA fragmentation compared with the positive control (100 micromol H2O2 treatment). Thus, Chaga mushroom treatment affords cellular protection against endogenous DNA damage produced by H2O2.

Irpex hydnoides, sp. nov. is new to science, based on morphological, cultural and molecular characters.

Irpex, one of the most common polypore genera, is easily identified by macro- and microscopic characters. During field trips to Korea's Kangwon Province, some Irpex specimens with conspicuous morphological differences from I. lacteus were collected. Cultural characters and molecular evidence differentiated this new strain from I. lacteus, and this taxon is proposed as I. hydnoides sp. nov.

Three nonorthologous ITS1 types are present in a polypore fungus Trichaptum abietinum.

To explore phylogenetic relationships of Trichaptum species, internal transcribed spacer (ITS) regions of nuclear ribosomal DNAs were sequenced and analyzed. Gene trees from ITS1 and ITS2 sequences showed striking discrepancy in relationships of eight T. abietinum strains. All strains of T. abietinum had a single orthologous ITS2 type, but there were three paralogous types in the ITS1 region, which were designated Types I, II, and III. PCR amplification tests using type-specific primers showed that Types I and II are present in all strains of T. abietinum. The results suggest that gene duplication of the ancestral ITS1 region might have occurred prior to evolutionary radiation of Trichaptum and both types have been maintained in Trichaptum. However, Type III was amplified only in three T. abietinum strains collected from Korea, indicating that a new local geographic subtype has arisen in Korean strains.

Amplification of mitochondrial small subunit ribosomal DNA of polypores and its potential for phylogenetic analysis.

There has been a systematic need to seek adequate phylogenetic markers that can be applied in phylogenetic analyses of fungal taxa at various levels. The mitochondrial small subunit ribosomal DNA (mt SSU rDNA) is generally considered to be one of the molecules that are appropriate for phylogenetic analyses at a family level. In order to obtain universal primers for polypores of Hymenomycetes, mt SSU rRNA genes were cloned from Bjerkandera adusta, Ganoderma lucidum, Phlebiopsis gigantea, and Phellinus laevigatus and their sequences were determined. Based on the conserved sequences of cloned genes from polypores and Agrocybe aegerita, PCR primers were designed for amplification and sequencing of mt SSU rDNAs. New primers allowed effective amplification and sequencing of almost full-sized genes from representative species of polypores and related species. Phylogenetic relationships were resolved quite efficiently by mt SSU rDNA sequences, and they proved to be more useful in phylogenetic reconstruction of Ganoderma than nuclear internal transcribed spacer (ITS) rDNA sequences.