Identification of Hortaea werneckii Isolated from Mangrove Plant Aegiceras comiculatum Based on Morphology and rDNA Sequences

Hortaea werneckii is a black yeast-like ascomycetous fungi associated with the human superficial infection tinea nigra, which commonly occurs in tropical and subtropical countries. Now, this fungus has been found in the halophilic environment all over the world and recognized as a new model organism in exploring the mechanisms of salt tolerance in eukaryotes. During a survey of endophytic fungi of mangrove forest at South China Sea, two isolates of H. werneckii were recovered from medicinal plant of Aegiceras comiculatum. The isolates were identified by morphological characters and phylogenetic analyses (e.g., ITS rDNA, LSU rDNA and translation elongation factor EF1α). Some physiological tests such as thermotolerance, acid tolerance (pH) and NaCl tolerance as well as pathogenicity test in vitro for the strains of Hortaea were performed. It is the first report that H. werneckii was isolated from medicinal plant of A. comiculatum in south sea of China as the endophytic fungi.     

Erysiphe corylopsidis sp. nov., a new powdery mildew fungus found on Corylopsis spicata and C. pauciflora

A powdery mildew fungus occurring on leaves of Corylopsis pauciflora and C. spicata in Japan is described as a new species, Erysiphe corylopsidis. This species is characterized by fewer than 15 appendages on a chasmothecium, primary branches of the appendages occasionally elongated, and a relatively small number (2–5) of ascospores per ascus. Molecular phylogenetic analyses based on rDNA ITS and 28S rDNA sequences indicate that this fungus forms an independent lineage in the genus Erysiphe.     

A new species of Leptobacillim,L. symbioticum,isolated from mites and sori of soybean rust

A verticillium-like fungus forming a whitish colony and mainly solitary phialides that produced fusiform to cylindrical conidia in chains was isolated from uredinia of Phakopsora pachyrhizi, a causal agent of soybean rust. In addition, two similar looking isolates were obtained from Prostigmata mites. Our taxonomic study based on morphology and phylogenetic analysis using ITS rDNA and LSU rDNA D1/D2 region sequences revealed that the three isolates were the same species and assigned to the genus Leptobacillium. These isolates represent a new species, morphologically and phylogenetically distinguished from the type species, L. leptobactrum, and we propose L. symbioticum sp. nov. In addition, Simplicillium chinense and S. coffeanum are assigned to the genus Leptobacillium.     

Phylogeny and taxonomic revision of the genus Candelabrum,aero-aquatic fungi

The genus Candelabrum, an aero-aquatic fungus, produces chandelier-shaped conidia that can float on the water. Seven species have been described in the genus. Our phylogenetic analysis based on the D1/D2 regions of the large subunit rDNA (LSU) revealed that the genus Candelabrum contains two separate lineages, C. spinulosum group (C. clathrosphaeroides, C. desmidiaceum, C. japonense, and C. spinulosum, the type species) nested in Leotiomycetes, and C. brocchiatum group (C. brocchiatum and C. microsporum) in Sordariomycetes. The two groups were distinctive in conidial morphology, especially for the presence/absence of the conidial basal plate. To accommodate the latter group, a new genus Brocchiosphaera is proposed along with a new species, B. bulbiformis. Furthermore, the phylogenetic tree inferred from LSU indicated that C. spinulosum group was closely related to Hyaloscypha in Helotiales, whereas Brocchiosphaera (the C. brocchiatum group) belonged to Pisorisporiales. Strains of H. occulta (NBRC 108595 and SANK 13397) clustered with C. japonense and C. clathrosphaeroides in phylogenetic tree inferred from combined sequence data of the RNA polymerase II gene, the internal transcribed spacer regions and 5.8S rDNA (ITS) and LSU, and NBRC 108595 produced conidia of C. japonense in submerging culture. Thus, H. occulta was proved to be a teleomorph of C. japonense. This finding warrants transferring all species of the C. spinulosum group (Candelabrum sensu stricto) to Hyaloscypha and proposing four new combinations H. desmidiacea, H. japonensis, H. macrospora, and H. spinulosa.     

Melanin is crucial for growth of the black yeast Hortaea werneckii in its natural hypersaline environment

Melanin has an important role in the ability of fungi to survive extreme conditions, like the high NaCl concentrations that are typical of hypersaline environments. The black fungus Hortaea werneckii that has been isolated from such environments has 1,8-dihydroxynaphthalene-melanin incorporated into the cell wall, which minimises the loss of glycerol at low NaCl concentrations. To further explore the role of melanin in the extremely halotolerant character of H. werneckii, we studied the effects of several melanin biosynthesis inhibitors on its growth, pigmentation and cell morphology. The most potent inhibitors were a 2,3-dihydrobenzofuran derivative and tricyclazole, which restricted the growth of H. werneckii on high-salinity media, as shown by growth curves and plate-drop assays. These inhibitors promoted release of the pigments from the H. werneckii cell surface and changed the medium colour. Inhibitor-treated H. werneckii cells exposed to high salinity showed both decreased and increased cell lengths. We speculate that this absence of melanin perturbs the integrity of the cell wall in H. werneckii, which affects its cell division and exposes it to the harmful effects of high NaCl concentrations. Surprisingly, melanin had no effect on H. werneckii survival under H₂O₂ oxidative stress.     

Phylogenetic Relationships Among Selected Heteroderoidea Based on 18S and ITS Ribosomal DNA

In a study of relationships among selected cyst-forming and noncyst-forming species of Heteroderoidea, combined sequences comprised of DNA from part of the conserved 18S ribosomal RNA gene (rDNA) plus the complete ITS rDNA segment were more similar to analyses based on the ITS data alone than to analyses based on the 18S data alone. One of the two noncyst-forming species, Ekphymatodera thomasoni, grouped with cyst-forming species of Heteroderoidea. Bilobodera flexa, also a noncyst-forming species, was separated from all the other taxa by a long branch. Afenestrata koreana, with a weakly sclerotized cyst, grouped closely with H. bifenestra. These observations suggest that phylogenetic analyses using molecular data may aid in our understanding of the evolution of cyst formation in nematodes, including the possibility of secondary loss. The usefulness of molecular phylogenetic analyses in nematodes may depend more on the particular selection of taxa than on mere addition of data from additional genes.     

Setoidium castanopsidis,a new species of anamorphic Cystotheca (Ascomycota,Erysiphales) from Indonesia

A powdery mildew fungus belonging to the genus Setoidium (anamorph of Cystotheca) was found on Castanopsis javanica in Cibodas Botanical Garden and mount Tangkuban Perahu, West Java, Indonesia. The fungus is considered as a new species, namely, Setoidium castanopsidis. Phylogenetic analyses of the 28S and ITS rDNA regions showed that S. castanopsidis formed a distinct lineage separated from Cy. tjibodensis, Cy. lanestris, and Cy. wrightii. Setoidium castanopsidis also differs morphologically from Cy. tjibodensis in having distinct appressoria (nipple-shaped), longer conidiophores with longer foot-cells, larger conidia, and being found on Ca. javanica. The teleomorphic state has not been found during the collection.     

<Emphasis Type="Italic">Melampsora salicis-bakko</Emphasis>, a new species on willows in Japan evidenced by morphological and molecular phylogenetic analyses

Through the morphological and molecular examinations of Melampsora species on willows, we clarified the taxonomic identity of the rust specimens on Salix bakko, S. hultenii and S. leucopithecia from Japan and described the following rust fungus as a new species, Melampsora salicis-bakko. This rust fungus resembled M. caprearum in morphology of teliospores, but it differed from M. caprearum mainly in the density of spines on the urediniospores. Molecular phylogenetic analyses using the rDNA ITS region (complete ITS1, 5.8S rRNA gene and ITS2) revealed that M. salicis-bakko was monophyletic, and that this rust fungus was distinct from other Melampsora species, including M. caprearum.     

Expanding evolutionary diversity in the Ustilaginomycotina: Fereydouniaceae fam. nov. and Fereydounia gen. nov., the first urocystidalean yeast lineage

The systematic position of two strains of a yeast-like fungus isolated from plant remnants on the Kharg Island in the Persian Gulf of Iran is evaluated using morphological, physiological and phylogenetic analyses. In culture, this fungus produced cylindrical cells that reproduced by polar budding on short stalks. Production of ballistoconidia and blastospores was observed. The carbon source assimilation spectrum was broad, but fermentation ability was absent. Phylogenetic analyses of the nuclear SSU, LSU (D1/D2 domain), and ITS rDNA revealed that this fungus represents a new lineage in the Urocystidales of the subphylum Ustilaginomycotina. Based on the comparison of phenotypic characters, physiology, and DNA sequences, a new genus and species Fereydounia khargensis (IBRC-M 30116^T?=?CBS 13305^T) is described for this fungus and accommodated in the novel family Fereydouniaceae. This is the first report of anamorphic saprobic fungus residing in the Urocystidales, stressing the remarkable evolutionary diversity in the subphylum Ustilaginomycotina.     

Phylogeny and morphology of Hirsutella tunicata sp. nov. (Ophiocordycipitaceae), a novel mite parasite from Peru

A new species of Hirsutella was isolated from unidentified mites on Petri plates inoculated with soil and root fragments collected from asparagus rhizosphere at Virú, Northern Peru. The fungus differs from other Hirsutella species by an envelope surrounding the conidium, conidia dimension and DNA sequences. In PDA cultures, the mycelium produced aerial hyphae with conidiogenous cells mainly at right angles, occasionally showing a secondary conidiophore. The solitary conidia are cymbiform, slightly apiculate, 5.0–6.0 × 3.0–4.0 μm. Phylogenetic analyses with partial rRNA and β-tubulin gene sequences confirmed the fungus as an Hirsutella (Ophiocordycipitaceae). Closest species shown by maximum likelihood and neighbor-joining trees were H. nodulosa and H. aphidis, from which the new species differs for conidium or conidiogenous cells dimensions, lack of synnemata and host type. A recombination event was also detected in the rRNA of the holotype strain, involving Ophiocordyceps sinensis as major parent and O. cochlidiicola as minor parent. A complement, inverted insertion was also found in its rRNA, involving part of the ITS2 and 5.8S regions, flanked by two short nucleotide arrays. Due to conidia dimension and phylogenetic position, the fungus is described as Hirsutella tunicata sp. nov. A review of mononematous Hirsutella species is provided.     

Gluconacetobacter maltaceti sp. nov., a novel vinegar producing acetic acid bacterium

Comparison of HaeIII- and HpaII-restriction profiles of PCR-amplified 16S-23S rDNA ITS regions of Gluconacetobacter sp. LMG 1529^T and SKU 1109 with restriction profiles of reference strains of acetic acid bacteria described by Tr?ek and Teuber [34] revealed the same but unique restriction profiles for LMG 1529^T and SKU 1109. Further analyses of nearly complete 16S rRNA gene sequences, nearly complete 16S-23S rDNA ITS sequences, as well as concatenated partial sequences of the housekeeping genes dnaK, groEL and rpoB, allocated both strains to a single phylogenetic cluster well separated from the other species of the genus Gluconacetobacter. DNA–DNA hybridizations confirmed their novel species identity by 73% DNA–DNA relatedness between both strains, and values below the species level (<70%) between SKU 1109 and the type strains of the closest phylogenetic neighbors. The classification of strains LMG 1529^T and SKU 1109 into a single novel species was confirmed also by AFLP and (GTG)₅-PCR DNA fingerprinting data, as well as by phenotypic data. Strains LMG 1529^T and SKU 1109 can be differentiated from their closely related Gluconacetobacter species, Gluconacetobacter entanii and Gluconacetobacter hansenii, by their ability to form 2-keto-d-gluconic acid from d-glucose, their ability to use d-mannitol, d-gluconate and glycerol as carbon source and form acid from d-fructose, and their ability to grow without acetic acid. The major fatty acid of LMG 1529^T and SKU 1109 is C_18:1ω7c (60.2–64.8%). The DNA G + C content of LMG 1529^T and SKU 1109 is 62.5 and 63.3 mol% respectively. The name Gluconacetobacter maltaceti sp. nov. is proposed. The type strain is LMG 1529^T (= NBRC 14815^T = NCIMB 8752^T).     

Bensingtonia pseudonaganoensis sp. nov., a novel ballistoconidium-forming yeast species isolated from plant leaves

Among the basidiomycetous yeasts isolated from plant leaves collected in different regions of China, two ballistoconidium-forming strains were revealed to represent an undescribed species of the genus Bensingtonia by conventional, chemotaxonomic and molecular phylogenetic characterization. Sequence analysis of the 26S rDNA D1/D2 domains and the internal transcribed spacer (ITS) region indicated that the novel species was located in the Agaricostilbum lineage and closely related to Bensingtonia naganoensis and Bensingtonia ciliata, with the former as its closest relative. The name Bensingtonia pseudonaganoensis sp. nov. is proposed (type strain: AS 2.2601^T = CBS 10121^T)     

Natural hybridization between Paragonimus bangkokensis and Paragonimus harinasutai

Paragonimus bangkokensis and Paragonimus harinasutai, which are morphologically distinguishable species, often co-infect in the same crab intermediate hosts. Recent molecular phylogenetic analyses revealed that these two species are genetically close to each other and are considered as the sister species. While we have been studying Paragonimus adult worms obtained from the lungs of a cat experimentally infected with Paragonimus metacercariae which were morphologically identified as P. harinasutai collected from central Viet Nam, one out of 6 adult worms has grouped cuticular spines, which is a feature of P. bangkokensis. By molecular analyses, the CO1 sequence of this specimen was identical with that of P. bangkokensis, but the ITS2 and the D2 region of 28S rDNA sequences showed a two peak pattern. Then, PCR products of the ITS2 and the D2 region of 28S rDNA sequences were ligated to TOPO vector and subcloned to determine the heterozygosity. Two types of sequences were obtained from each ITS2 and D2 region of 28S; one was identical with P. harinasutai and the other with P. bangkokensis. Taking all these morphological and molecular data together, we identified this adult worm as a hybrid specimen of P. bangkokensis and P. harinasutai.     

Rhodotorula lamellibrachii sp. nov., a new yeast species from a tubeworm collected at the deep-sea floor in Sagami Bay and its phylogenetic analysis

A new species of the genus Rhodotorula was isolated from a tubeworm (Lamellibrachia sp.) collected at a depth of 1156 m in Sagami Bay, Japan. Strain SY-89 had physiological properties quite similar to R. aurantiaca. Two phylogenetic trees, one based on internal transcribed spacer (ITS) regions and 5.8S rDNA sequences and the other based on the D1/D2 region of the large subunit (26S) rDNA sequences, united strain SY-89 to the type strain of Sakaguchia dacryoides through a considerable evolutionary distance. Strain SY-89 was differentiated from S. dacryoides by the G+C content of the nuclear DNA and differences in the ability to utilize specific carbon and nitrogen compounds. The low complementarity of strain SY-89 DNA to that of the type strain of S. dacryoides confirmed that this strain was genetically unrelated to previously known species. The tubeworm isolates are described as R. lamellibrachii sp. nov. The type strain of R. lamellibrachii is strain SY-89 (= JCM 10907). R. lamellibrachii formed a cluster with Erythrobasidium hasegawianum, R. lactosa, S. dacryoides and Sporobolomyces elongatus on the ITS and 5.8S rDNA phylogenetic tree. These five species shared a signature sequence in 26S rDNA, although this relationship was not supported by phylogeny based on the D1/D2 region of 26S rDNA.     

Characterization and Sequence Variation in the rDNA Region of Six Nematode Species of the Genus Longidorus (Nematoda)

Total DNA was isolated from individual nematodes of the species Longidorus helveticus, L. macrosoma, L. arthensis, L. profundorum, L. elongatus, and L. raskii collected in Switzerland. The ITS region and D1-D2 expansion segments of the 26S rDNA were amplified and cloned. The sequences obtained were aligned in order to investigate sequence diversity and to infer the phylogenetic relationships among the six Longidorus species. D1-D2 sequences were more conserved than the ITS sequences that varied widely in primary structure and length, and no consensus was observed. Phylogenetic analyses using the neighbor-joining, maximum parsimony and maximum likelihood methods were performed with three different sequence data sets: ITS1-ITS2, 5.8S-D1-D2, and combining ITS1-ITS2+5.8S-D1-D2 sequences. All multiple alignments yielded similar basic trees supporting the existence of the six species established using morphological characters. These sequence data also provided evidence that the different regions of the rDNA are characterized by different evolution rates and by different factors associated with the generation of extreme size variation.     

The mycological and molecular study of <Emphasis Type="Italic">Hortaea werneckii</Emphasis> isolated from blood and splenic abscess

Hortaea werneckii is an environmental dematiaceous fungus found in the halophilic environment. It causes tinea nigra. We report the isolation of H. werneckii from blood and splenic abscess of two patients with acute myelomonocytic leukaemia. H. werneckii grew at room temperature but not at 37 °C, it was identified by biochemical tests, growth characteristics and the presence of conspicuous collarette intercalary on dividing yeast cells. The use of specific oligonucleotide primer Hor-F (5′-TGGACACCTTCA TAACTCTTG-3′) and Hor-R (5′-TCACAACGCTTAGAGACGG-3′) confirmed the two isolates were H. werneckii. The sequence for 281 nucleotide of HW299 and HW403 were 99% identical but differed only in one nucleotide. In vitro anti-fungal susceptibility testing showed that the isolates were resistant to amphotericin B and flucytosine.     

GENETIC DIVERSITY AND INTROGRESSION IN TWO CULTIVATED SPECIES (PORPHYRA YEZOENSIS AND PORPHYRA TENERA) AND CLOSELY RELATED WILD SPECIES OF PORPHYRA (BANGIALES,RHODOPHYTA)1

We investigated the genetic variations of the samples that were tentatively identified as two cultivated Porphyra species (Porphyra yezoensis Ueda and Porphyra tenera Kjellm.) from various natural populations in Japan using molecular analyses of plastid and nuclear DNA. From PCR‐RFLP analyses using nuclear internal transcribed spacer (ITS) rDNA and plastid RUBISCO spacer regions and phylogenetic analyses using plastid rbcL and nuclear ITS‐1 rDNA sequences, our samples from natural populations of P. yezoensis and P. tenera showed remarkably higher genetic variations than found in strains that are currently used for cultivation. In addition, it is inferred that our samples contain four wild Porphyra species, and that three of the four species, containing Porphyra kinositae, are closely related to cultivated Porphyra species. Furthermore, our PCR‐RFLP and molecular phylogenetic analyses using both the nuclear and plastid DNA demonstrated the occurrence of plastid introgression from P. yezoensis to P. tenera and suggested the possibility of plastid introgression from cultivated P. yezoensis to wild P. yezoensis. These results imply the importance of collecting and establishing more strains of cultivated Porphyra species and related wild species from natural populations as genetic resources for further improvement of cultivated Porphyra strains.     

A new species of Pithoascus and first report of this genus as endophyte associated with Ferula ovina

A newly described species of Pithoascus from roots of Ferula ovina differs from other Pithoascus species by producing larger ascomata than all described species except P. exsertus. The shape of its ascospores is similar to that of P. lunatus, but differs from it by having an asexual state. This new species differs from P. ater by having a sexual state. Phylogenetic analyses based on concatenated ITS rDNA, LSU rDNA and partial translation elongation factor 1 alpha gene datasets also confirmed the generic placement in Pithoascus and showed its close phylogenetic relationships to P. ater and P. lunatus.