New genus of trichomatous coelomycete on Myrcia fenzliana from the Brazilian Cerrado.

Megacoelomyces (type species: Megacoelomyces sanchezii), an ascomycete asexual morph infecting Myrcia fenzliana (Myrtaceae) from the Brazilian Cerrado, is described as a new genus in the Phaeosphaeriaceae (Pleosporales, Dothideomycetes, Ascomycota), based on multilocus phylogeny (three nuclear ribosomal DNA and two protein-coding genes) in addition to morphological (light and scanning electron microscopy) and ecological data.

Especies oportunistas de importancia clínica de los géneros bipolaris shoemaker y curvularia boedijn: su caracterización bajo los nuevos criterios taxonómicos / Opportunistic species of clinical relevance in the bipolaris shoemaker & curvularia boedijn genera: its characterization under newest taxonomic criteria

Los géneros fúngicos Bipolaris y Curvularia,incluyen numerosas especies fitopatógenas,saprotrofas y algunas oportunistas emergentes enel ser humano como en otros animales. La distribuciónecológica de ambos taxas es cosmopolita enla gran mayoría de sus integrantes, encontrándosefrecuentemente en hojas, tallos y raíces de diferentespastos, en especial Poaceae, pero tambiénen una gran variedad de dicotiledoneas, aire y sueloen todos los continentes. Cochliobolus, Bipolarisy Curvularia integran un complejo de especiestaxonómicamente confuso, debido a los constantescambios en la nomenclatura de algunos de sus integrantesasexuales (Bipolaris y Curvularia), loscuales se han diferenciado principalmente en basea la morfología de sus conidios, situación a vecesmuy dificultosa debido a que en ambos génerosalgunas especies presentan similares característicasconidiales. Una cuidadosa identificación y unanominación precisa de las especies es crucial parael acceso a la información referente en la literaturamoderna, que gracias a la biología molecularha resuelto muchas de las dudas de la taxonomíabasada solo en el fenotipo.Las especies de ambos géneros (principalmenteCurvularia) pueden afectar a pacientesinmunocomprometidos e inmunocompetentes,en especial B. cynodontis, C. australiensis C.hawaiensis y C. spicifera, causando cuadros clínicosdiversos ya sea superficiales o profundos enmuchas áreas geográficas...

The fungal genera Bipolaris and Curvularia,include numerous plant pathogenic species,saprophitic and some emerging opportunistic inman and other animals. The ecological distributionof both taxa is cosmopolitan in the vast majority ofits members, often being in leaves, stems and rootsof different grasses, especially Poaceae, but also ina variety of dicotyledonous, air and soil on all continents. Cochliobolus, Bipolaris and Curvulariaintegrate a complex of species taxonomically confusingdue to the constant changes in the nomenclatureof some of its asexual members (Bipolarisand Curvularia), which are differentiated mainlybased on the morphology of its conidia, a situationsometimes very difficult because in both generaconidial of some species have similar characteristics. Careful identification and a precise nominationof species is crucial for access to informationconcerning in modern literature, actually thanks tomolecular biology has solved many of the doubtsof taxonomy based only on the phenotype.The species of both genera (mainly Curvularia)can affect immunocompetent or debilitatepatients, especially B. cynodontis, C. australiensis,C. hawaiensis and C.spicifera, causing varioussuperficial or systemic clinical cases in manygeographic areas...

A century later: rediscovery, culturing and phylogenetic analysis of Diploöspora rosea, a rare onygenalean hyphomycete.

Nearly 100 years after its first discovery, Diploöspora rosea was detected on biologically damaged parchment paper in Rome, Italy and isolated from house dust collected in Micronesia. The isolation of this culture permitted morphological study of colony characters, conidium and conidiophore development, and phylogenetic investigations using sequences of nuc 18S rDNA, internal transcribed spacers, and 28S rDNA. The results indicate that D. rosea is an onygenalean fungus, of uncertain taxonomic position, basal or sister to the Gymnoascaceae. Based on observations of the parchments using SEM-Energy Dispersive Spectroscopy, we speculate that the fungus occurs in archival and domestic environments subject to periodic wetting. Its ability to grow on all low water activity media used in the study, including malt extract agar amended with 60% sucrose, confirms its xerophilic nature.

Paleomycology of the Princeton Chert II. Dark-septate fungi in the aquatic angiosperm Eorhiza arnoldii indicate a diverse assemblage of root-colonizing fungi during the Eocene.

Tissues of the extinct aquatic or emergent angiosperm, Eorhiza arnoldii incertae sedis, were extensively colonized by microfungi, and in this study we report the presence of several types of sterile mycelia. In addition to inter- and intracellular proliferation of regular septate hyphae, the tissues contain monilioid hyphae with intercalary branching. These filamentous mycelia are spatially associated with two distinct morphotypes of intracellular microsclerotia. These quiescent structures are morphologically similar to loose and cerebriform microsclerotia found within the living tissues of some plants, which have been attributed to an informal assemblage of dematiaceous ascomycetes, the dark-septate endophytes. While there are significant challenges to interpreting the ecology of fossilized fungi, these specimens provide evidence for asymptomatic endophytic colonization of the rooting structures of a 48.7 million year old aquatic angiosperm.

Paleomycology of the Princeton Chert I. Fossil hyphomycetes associated with the early Eocene aquatic angiosperm, Eorhiza arnoldii.

The Eocene (~ 48.7 Ma, Ypresian-Lutetian) Princeton Chert of British Columbia, Canada, has long been recognized as a significant paleobotanical locality, and a diverse assemblage of anatomically preserved fossil plants has been extensively documented. Co-occurring fossil fungi also have been observed, but the full scope of their diversity has yet to be comprehensively assessed. Here, we present the first of a series of investigations of fossilized fungi associated with the silicified plants of the Princeton Chert. This report focuses on saprotrophic, facultative-aquatic hyphomycetes observed in cortical aerenchyma tissue of an enigmatic angiosperm, Eorhiza arnoldii. Our use of paleontological thin sections provides the opportunity to observe and infer developmental features, making it possible to more accurately attribute two hyphomycetes that were observed in previous studies. These comprise multiseptate, holothallic, chlamydospore-like phragmoconidia most similar to extant Xylomyces giganteus and basipetal phragmospore-like chains of amerospores like those of extant Thielaviopsis basicola. We also describe a third hyphomycete that previously has not been recognized from this locality; biseptate, chlamydosporic phragmoconidia are distinguished by darkly melanized, inflated apical cells and are morphologically similar to Brachysporiella rhizoidea or Culcitalna achraspora.

Beauveria bassiana: endophytic colonization and plant disease control.

Seed application of Beauveria bassiana 11-98 resulted in endophytic colonization of tomato and cotton seedlings and protection against plant pathogenic Rhizoctonia solani and Pythium myriotylum. Both pathogens cause damping off of seedlings and root rot of older plants. The degree of disease control achieved depended upon the population density of B. bassiana conidia on seed. Using standard plating techniques onto selective medium, endophytic 11-98 was recovered from surface-sterilized roots, stems, and leaves of tomato, cotton, and snap bean seedlings grown from seed treated with B. bassiana 11-98. As the rate of conidia applied to seed increased, the proportion of plant tissues from which B. bassiana 11-98 was recovered increased. For rapid detection of B. bassiana 11-98 in cotton tissues, we developed new ITS primers that produce a PCR product for B. bassiana 11-98, but not for cotton. In cotton samples containing DNA from B. bassiana11-98, the fungus was detected at DNA ratios of 1:1000; B. bassiana 11-98 was detected also in seedlings grown from seed treated with B. bassiana 11-98. Using SEM, hyphae of B. bassiana11-98 were observed penetrating epithelial cells of cotton and ramifying through palisade parenchyma and mesophyll leaf tissues. B. bassiana11-98 induced systemic resistance in cotton against Xanthomonas axonopodis pv. malvacearum (bacterial blight). In parasitism assays, hyphae of B. bassiana 11-98 were observed coiling around hyphae of Pythium myriotylum.

Quantification and characterisation of IgG binding to mould spores by flow cytometry and scanning electron microscopy.

The concentration of mould-specific IgG antibodies in serum may objectively indicate mould exposure and can help identifying exposed individuals. Although inhaled spores probably are the most important source of mould exposure, the commonly used methods for detecting mould-specific IgG antibodies are based on extracts from all mould components, with only low contribution from spores. We have developed a flow cytometric method using surface antigens on mould spores for quantifying mould-specific IgG antibodies in serum. Flow cytometric results were evaluated by comparison with ImmunoCap and ELISA measurements. The flow cytometric assay showed a broad linear dose-dependency and correlated moderately to strongly (r=0.41-0.97) with ImmunoCap and ELISA measurements. The IgG antibody binding was studied in detail by immunolabelling in scanning electron microscopy (SEM), revealing that morphology and IgG antibody binding differed among spores, both within and between mould strains. Germination studies by flow cytometry and SEM showed that IgG antibody binding to mould spores was altered during germination due to loss of coat. The present spore based antibody assay are simple and suitable for quantification of mould-specific IgG antibodies in serum, and includes specificity to other and possibly more relevant antigens than existing methods.

A synnematous species of Dictyoarthrinium from Thailand.

Genus Dictyoarthrinium is reviewed. Dictyoarthrinium synnematicum, collected on decaying banana leaves in Thailand, is illustrated, described as a new species and compared with related taxa. The fungus differs from other Dictyoarthrinium species in having a synnematous structure. This species is the first synnematous record of an anamorphic fungus with basauxic conidiophores. A key to species of Dictyoarthrinium is provided.

Endophytic colonisation of opium poppy, Papaver somniferum, by an entomopathogenic Beauveria bassiana strain.

Beauveria bassiana strain EABb 04/01-Tip isolated from stem-borer larvae of Timaspis papaveris (Hymenoptera: Cynipidae), a serious pest of opium poppy in Spain, was shown to be able to become established endophytically in this pharmaceutical crop. Microbiological, molecular and light and electron microscopic methods were used to study fungal colonisation and to describe its mode of penetration. After inoculation with a foliar spray of conidia, microbiological methods showed 100% of plants examined 24, 48, 72 and 144 h after treatment to be colonised endophytically by the fungus, although the percentage of previously surface sterilised leaf pieces showing fungal growth was 100% at 24 and 48 h, and 80 and 75% at 72 and 144 h after treatment, respectively. The fungus was also observed in leaf pieces obtained from newly formed leaves, indicating that it could spread from treated leaves to leaves formed after fungal application. For molecular studies, a polymerase chain reaction (PCR) protocol was used to amplify the ITS1-5.8S-ITS2 regions of the rDNA of the plant and the fungus. This procedure allowed the detection of the fungus on the surface of the leaves and also endophytically, but only at 72 h after treatment. A nucleotide BLAST search revealed that the ITS1-5.8S-ITS2 sequence of strain EABb 04/01-Tip showed 100% homology with a similar sequence from Cordyceps bassiana. SEM images revealed that although numerous conidia were observed on the leaf surface, few germinated and penetrated. Intracellular colonisation by B. bassiana was not observed, but hyphae were detected growing into the xylem vessels. The fungus was found to colonise 40.5 +/- 4.3% of seedlings (with two cotyledons and the two first real leaves) from seeds dressed with a fungal spore suspension. These results may have implications in the biological control of T. papaveris, including the possible systemic protection of the plant against this cynipid.

A case of tinea nigra palmaris in Okinawa, Japan.

We report a case of tinea nigra on the left palm of a 13-year-old girl. She had noticed a pigmented, asymptomatic macule on the left palm approximately 4-5 years prior to her first visit to our hospital. The color of the lesion tended to change before and after a bath; it became lighter after a bath and darkened some time later. Physical examination revealed that the macule was 4 cm x 5 cm in size, dark brown in color and irregularly shaped. Direct potassium hydroxide (KOH) microscopic examination from skin scrapings revealed branched brown hyphae with light brown septa. A fungal culture on Sabouraud's agar media produced wet, medium brown, yeast-like colonies, the surface of which later became black and shiny. A slide culture disclosed light brown, elliptic or peanut-shaped conidia comprised of one to two ampullaceous cells. Scanning electron microscopic examination of the conidia showed both annellation conidia with lunate bud scars and sympodial conidiogenesis. Using extracted DNA from separately cultured fungi, we performed polymerase chain reaction with the primers specific to Hortaea werneckii. The results showed positive bands. We performed direct sequencing with the DNA segments from the positive bands. The causative fungus in our case was determined to be type C of H. werneckii on the grounds of the base sequences obtained. The final diagnosis of the present case was made as tinea nigra by H. werneckii. We also report a brief survey of all the cases of tinea nigra reported in Japan to date.

Morphology and productivity of filamentous fungi.

Cultivation processes involving filamentous fungi have been optimised for decades to obtain high product yields. Several bulk chemicals like citric acid and penicillin are produced this way. A simple adaptation of cultivation parameters for new production processes is not possible though. Models explaining the correlation between process-dependent growth behaviour and productivity are therefore necessary to prevent long-lasting empiric test series. Yet, filamentous growth consists of a complex microscopic differentiation process from conidia to hyphae resulting in various macroscopically visible appearances. Early approaches to model this morphologic development are recapitulated in this review to explain current trends in this area of research. Tailoring morphology by adjusting process parameters is one side of the coin, but an ideal morphology has not even been found. This article reviews several reasons for this fact starting with nutrient supply in a fungal culture and presents recent advances in the investigation of fungal metabolism. It illustrates the challenge to unfold the relationship between morphology and productivity.

The mycological and molecular study of Hortaea werneckii 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 degrees 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.

Paecilomyces fumosoroseus (Deuteromycotina: Hyphomycetes) as a potential mycoparasite on Sphaerotheca fuliginea (Ascomycotina: Erysiphales).

Hyphomycete Paecilomyces fumosoroseus that is well known as saprophytic and entomopatogenic fungus was investigated for its mycoparasitism on the cucumber powdery mildew pathogen. Mycoparasitism was documented by using standard bioassay and SEM. Effects of mycoparasitism were evaluated in three types of experiments. Paecilomyces fumosoroseus was applied in the form of graded suspensions into a colony of powdery mildew on a leaf segment. Interaction between both fungi was observed as the percentage of colonized area vs. experimental time. In the second experiment, young cucumber plants were sprayed with a suspension of Paecilomyces fumosoroseus 24 h before inoculation of Sphaerotheca fuliginea. Pre-treatment with P. fumosoroseus reduced development and spreading of powdery mildew infection significantly 15 days post-inoculation in contrast to pre-treatments with sulfur fungicide and distilled water. The development of pure culture powdery mildew under determined experimental conditions was observed and compared with treated variants. In the third experiment, mildewed plants were treated with a suspension of P. fumosoroseus. The control treatments with sulfur fungicide and distilled water were tested. Effects of P. fumosoroseus on the dispersion of powdery mildew during a 21-day period were observed. P. fumosoroseus suppressed the development and spread of cucumber powdery mildew significantly during the time of the experiment. The mechanical and physical damages and disruptions of vegetative and fruiting structures of powdery mildew were recorded under light microscopy and S.E.M. Results were concluded in pursuance to differences between the natural behaviour and development of S. fuliginea on cucumber plants treated with P. fumosoroseus and non-treated plants.

Endosporoideus gen. nov., a mitosporic fungus on Phoenix hanceana.

Endosporoideus pedicellata gen. et sp, nov. is described and illustrated from decaying petioles of Phoenix hanceana collected from grassland in Tai Mo Shan, Hong Kong. The genus is unique in producing solitary, phragmosporous conidia. The conidia comprise a brown to dark brown inner-wall layer and thick, hyaline outer-wall layer and are produced holoblastically from determinate conidiogenous cells on micronematous, mononematous conidiophores. Cells of conidia may disarticulate at the septa. Representative steps in conidiogenesis of E. pedicellata are illustrated with light micrographs, and details of the conidiogenous events are interpreted schematically.

Formation of structures resembling ericoid mycorrhizas by the root endophytic fungus Heteroconium chaetospira within roots of Rhododendron obtusum var. kaempferi.

A resynthesis study was conducted to clarify the relationship between the root endophyte, Heteroconium chaetospira and the ericaceous plant, Rhododendron obtusum var. kaempferi. The host plant roots were recovered 2 months after inoculation, and the infection process and colonization pattern of the fungus were observed under a microscope. The hyphae of H. chaetospira developed structures resembling ericoid mycorrhizas, such as hyphal coils within the host epidermal cells. These structures were morphologically the same as previously reported ericoid mycorrhizal structures. The frequencies of hyphal coils within the epidermal cells of host roots ranged from 13 to 20%. H. chaetospira did not promote or reduce host plant growth. This is the first reported study that H. chaetospira is able to form structures resembling mycorrhizas within the roots of ericaceous plants.

Purification and characterization of a lipopeptide produced by Bacillus thuringiensis CMB26.

AIMS: To isolate an antagonist for use in the biological control of phytopathogenic fungi including Colletotrichum gloeosporioides, then to purify and characterize the biocontrol agent produced by the antagonist. METHODS AND RESULTS: Bacteria that exhibited antifungal activity against the causative agent pepper anthracnose were isolated from soil, with Bacillus thuringiensis CMB26 showing the strongest activity. A lipopeptide produced by B. thuringiensis CMB26 was precipitated by adjusting the pH 2 with 3 n HCl and extracted using chloroform/methanol (2:1, v/v) and reversed-phase HPLC. The molecular weight was estimated as 1447 Da by MALDI-TOF mass spectrometry. Scanning electron and optical microscopies showed that the lipopeptide has activity against Escherichia coli O157:ac88, larvae of the cabbage white butterfly (Pieris rapae crucivora) and phytopathogenic fungi. The lipopeptide had cyclic structure and the amino acid composition was L-Glu, D-Orn, L-Tyr, D-allo-Thr, D-Ala, D-Val, L-Pro, and L-Ile in a molar ratio of 3:1:2:1:1:2:1:1. The purified lipopeptide showed the same amino acid composition as fengycin, but differed slightly in fatty acid composition, in which the double bond was at carbons 13-14 (m/z 303, 316) and there was no methyl group. CONCLUSION: A lipopeptide was purified and characterized from B. thuringiensis CMB26 and found to be similar to the lipopeptide fengycin. This lipopeptide can function as a biocontrol agent, and exhibits fungicidal, bactericidal, and insecticidal activity. SIGNIFICANCE AND IMPACT OF THE STUDY: Compared with surfactin and iturin, the lipopeptide from B. thuringiensis CMB26 showed stronger antifungal activity against phytopathogenic fungi. This lipopeptide is a candidate for the biocontrol of pathogens in agriculture.

Molecular studies on terricolous microfungi reveal novel anamorphs of two Tuber species.

This study reports novel terricolous mitosporic fungal morphs nested in the genus Tuber according to molecular phylogenetic analysis. Fungal DNA was amplified directly from field-collected anamorph samples. Nuclear ribosomal DNA (nrDNA) sequences including the ITS regions and the D1 and D2 domains of the LSU identify the anamorphs as mitosporic Tuber borchii and Tuber oligospermum. The link of the novel anamorphs to the genus Tuber is confirmed by the comparative analysis of five collections from four sampling sites. Ectomycorrhizas with characteristic features of Tuber borchii ectomycorrhizas were found in the soil volume collected with one of the mitosporic T. borchii collections. A nrDNA sequence amplified from these ectomycorrhizae is identical with the corresponding anamorph sequence. The possible role of the newly discovered anamorphs in the Tuber life-cycle and the potential significance of anamorphs for the propagation of ectomycorrhizal fungi are discussed.

Nuclei, micronuclei and appendages in tri- and tetraradiate conidia of Cornutispora and four other coelomycete genera.

The distribution and behaviour of nuclei in conidia of 11 coelomycete species with tri- and tetraradiate conidia and belonging to five genera has been investigated: Cornutispora (C. ciliata, C. intermedia, C. lichenicola, C. limaciformis, and C. pittii), Eriosporella (E. calami), Furcaspora (F. abieticola, F. pinicola), Suttoniella (S. eriobotryae, S. gaubae), and Tetranacrium (T. gramineum). They have been studied by the HCl-Giemsa technique using dried, preserved material including holotypes and isotypes with ages ranging from 3 to 116 yr. Conidia of Cornutispora species showed different ploidy levels, and C. limaciformis showed a very high (> 90%) frequency of stable and viable micronuclei with an unusual type of ploidy level, occurring naturally. Frequency of ploidy levels in nuclei within conidia of Cornutispora species appeared to be associated with changes in gross conidial morphology. This is the first report of micronuclei in coelomycetes. The types of appendages on arms or parts of conidia have been studied using various stains including erythrosin in ammonia and a modified Leifson's flagella staining technique. In Furcaspora species the apical and basal conidial appendages are cellular maintaining protoplasmic continuity with the arms on which they are sited. The results have been compared with those of Crucellisporium species which have tetraradiate conidia. The new species, Cornutispora intermedia, C. pittii and Furcaspora abieticola spp. nov. are described, and illustrated, and a key to all known Cornutispora species is provided.

Monacrosporium janus sp. nov., a new nematode-trapping hyphomycete parasitizing sclerotia and hyphae of Sclerotinia sclerotiorum.

During an investigation of mycoparasitic fungi on sclerotia of Sclerotinia sclerotiorum in China, a new fungal species was consistently encountered and isolated from natural soils taken from soybean fields of Shandong and Jiangsu Provinces. The fungus is featured by its sphaeroid conidia with 1-2 transverse septa, but mostly (>65%) with only one septum at the base. It resembles Monacrosporium indicum, M. sphaeroides and M. sinense, but can be distinguished from the first two species by lack of basal hila and large vacuoles on its conidia, respectively, and from M. sinense by its typically two-celled and broadly turbinate to napiform conidia. Colonization frequencies on S. sclerotiorum sclerotia by the new species were 10% and 33.3% in the two field soils, respectively, when the sclerotia were introduced into soils and coincubated at 22-24 degrees C for 4 wk. Reinoculation tests by placing surface-sterilized sclerotia onto the tested isolate colony for 2 wk and then surface-sterilized again resulted in 23.3% sclerotia colonized. Microscopic observations indicated that the fungus coiled around hyphae of Rhizoctonia solani and grew along and appressed to hyphae of Fusarium solani f.sp. pisi, S. sclerotiorum and Phytophthora cactorum when dual-cultured in slides. Tests on agar plates demonstrated that the fungus formed adhesive networks and was an active predator of Panagrellus redivivus. This study indicated the diverse mechanisms for the fungus to survive in soil. For expression of its mycoparasitic and nematode-trapping capacities, the fungus is named as Monacrosporium janus.