Distinction of species in Aureobasidium and related genera by PCR-ribotyping

Taxonomic markers for differentiation of the anamorph genera Aureobasidium, Hormonema and Kabatiella were developed using PCR-ribotyping with the primers 5.8S-R and LR7 for amplification and the restriction enzymes Alul, DdeI, Hhal, MspI and RsaI for digestion. Aureobasidium and Hormonema are optimally differentiated with MspI; DdeI is particularly useful to distinguish aureobasidium, Kabatiella and Selenophoma. Relationships of the anamorph genera Aureobasidium, Hormoneng and Kabatiella with the teleomorph genera Pringsheimia and Dothiora are discussed.     

Conidiogenesis,nutritional physiology and taxonomy ofAureobasidium andHormonema

Diagnostic criteria for the distinction of the slightly osmophilic species,Aureobasidium pullulans, and the opportunistic pathogen on conifers,Hormonema dematioides, are provided. Reliable identification requires a combination of characters of conidiogenesis, expansion growth and assimilative abilities. Relationships with species ofKabatiella, and with the teleomorph generaPringsheimia, Dothidea andDothiora, all havingHormonema-like cultural states, are discussed. An identification key is provided.     

Intraspecific variability and exopolysaccharide production inAureobasidium pullulans

Forty seven strains of the black yeasts,Aureobasidium pullulans andHormonema dematioides, and the type strain ofHormonema macrosporum were examined using PCR-ribotyping and universally primed PCR with subsequent hybridization. Four groups (populations) were distinguished withinA. pullulans with PCR-ribotyping, which largely coincided with UP-PCR/hybridization groups. The UP-PCR technique revealed a greater degree of heterogeneity between the groups studied. Five strains identified asHormonema dematioides on the basis of physiological and morphological data formed a group recognizable with PCR-ribotyping and UP-PCR/hybridization, which also includedH. macrosporum. Aureobasidium pullulans is characterized by the absence of RsaI restriction sites in rDNA amplified with primers 5.8S-R and LR7, whileHormonema species possessed several bands after RsaI digestion. For analysis of distance between populations, PCR-ribotyping with AluI and MspI is sufficient. Strains ofA. pullulans produce exopolysaccharides in liquid media with different nitrogen sources, while the strains ofHormonema synthesize minor amounts of polysaccharides in media with peptone. Populations ofA. pullulans differ slightly from each other in their optimal, medium-dependent production of polysaccharides.     

A new fungal genus, Teracosphaeria, with a phialophora-like anamorph (Sordariomycetes, Ascomycota)

The new genus and species Teracosphaeria petroica is described for a perithecial ascomycete and its anamorph occurring on decayed wood collected in New Zealand. The fungus produces immersed, non-stromatic ceratosphaeria-like perithecia in nature, with hyaline, septate ascospores produced in unitunicate, non-amyloid asci. The anamorph produced in vitro is phialophora-like with lightly pigmented phialides terminating in flaring, deep collarettes that are often noticeably brown with conspicuous periclinal thickening. Phylogenetic analysis of LSU rDNA sequence data indicates that this fungus is distinct from morphologically similar fungi classified in the Chaetosphaeriales, the Trichosphaeriales or the Magnaporthaceae. It forms a monophyletic group with recently described, chaetosphaeria-like ascomycetes, such as the pyrenomycete genus Mirannulata, and shows affinity with the anamorphic species Dictyochaeta cylindrospora. The usefulness of describing anamorph genera for morphologically reduced anamorphs, when anamorph characteristics are actually part of the holomorph diagnosis, is discussed. An apparently contradictory example of the so-called Cordana and Pseudobotrytis anamorphs of Porosphaerella spp. is also discussed.     

Phenotypic and genotypic diversity among strains of <Emphasis Type="Italic">Aureobasidium pullulans</Emphasis> in comparison with related species

Intra-specific diversity of 200 Aureobasidium pullulans strains isolated from different sources and their relatives Kabatiella lini CBS 125.21 T and Hormonema prunorum CBS 933.72 T were studied by assessment of macromorphological, and physiological tests, sodium dodecyl sulfate-polyacrylamide gel electrophoresis technique (SDS–PAGE) of whole-cell proteins as well as enterobacterial repetitive intergenic consensus (ERIC)-, repetitive extragenic palindromic (REP)- and BOX-PCR techniques (collectively known as rep-PCR). Rep-PCR is an efficient procedure for discrimination of A. pullulans in terms of simplicity and rapidity. RFLP-PCR technique was applied for the identification of A. pullulans isolates and distinction from related species. This technique was insufficient for investigation of intra-specific diversity. The tested strains of A. pullulans could be divided into two groups based on their macromorphological, protein patterns obtained after SDS-PAGE as well as rep-PCR patterns. The first group of strains shared similar characteristics and was very different from the second one, designated as “complex group”, consisting of strains with very little similarities within the group. Phenetic analysis of ERIC banding patterns failed to group the isolates on the basis of their substrate or geographical origin. Using 18S rDNA gene sequence analysis of selected isolates, three strains: HoHe3 km, A. pullulans DSM 62074 and H. prunorum CBS 933.72 T were distinguished from all other analysed members of genera Aureobasidium and Kabatiella. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Extensive host range of an endophytic fungus, <Emphasis Type="Italic">Guignardia endophyllicola</Emphasis> (anamorph: <Emphasis Type="Italic">Phyllosticta capitalensis</Emphasis>)

Isolation of endophytic species of Guignardia (anamorph: Phyllosticta) from healthy leaves of 94 plants (91 species and 3 varieties) in 69 genera, 42 families, was carried out in a test site (Kyoto Herbal Garden) to investigate the host range of Guignardia endophyllicola (anamorph: Phyllosticta capitalensis). Species of Guignardia and Phyllosticta were isolated from the leaves of 67 plants (66 species and 1 variety) belonging to 54 genera, 38 families. Among them, 53 isolates from different plants belonging to 43 genera in 36 families were similar in morphology, and sequence analysis of internal transcribed spacer (ITS) regions of ribosome DNA revealed these isolates to be conspecific with G. endophyllicola. In addition, this fungus was isolated from leaves of various plants collected in different places in Japan and Thailand. Thus, this endophytic fungus has been revealed to live within various vascular plants, angiosperms, gymnosperms, and pteridophytes.     

Ceratorhiza oryzae-sativae,a new combination for the anamorph of Cerabasidium setariae

Rhizoctonia oryzae-sativae, the anamorph of Ceratobasidium setariae, is transferred to Ceratorhiza. A discussion of teleomorph and anamorph taxonomy and nomenclature is also given and it is concluded that, for the present, there is only one holomorph species.     

The type species of Apiognomonia, A. veneta, with its Discula anamorph is distinct from A. errabunda

Species of Apiognomonia with their Discula anamorphic states in the Gnomoniaceae, Diaporthales, are known throughout the temperate Northern Hemisphere and cause diseases such as sycamore or plane tree anthracnose. The genus Apiognomonia was described based on A. veneta as the type species; however, there has been disagreement about whether or not A. veneta is a synonym of A. errabunda. Using morphological, ecological, and DNA sequence data we conclude that A. errabunda and A. veneta are different species, although very closely related; thus, A. veneta is the correct name for the type species of Apiognomonia. This conclusion is based on a combined analysis of sequences from the ITS regions of nuclear rDNA for 51 isolates from host plants of eight genera and intron regions from actin, calmodulin and translation elongation factor 1-alpha for over 25 isolates. The type species of the genus Discula is D. nervisequa, the earliest available epithet for D. platani, the lectotype of Discula. D. nervisequa is the anamorph of A. veneta. Based on an examination of the type specimen, we determined that the commonly used name for the anamorph of A. errabunda, D. umbrinella, refers to another species. A. veneta and A. errabunda including their anamorphs are described and illustrated. An account of all synonyms and excluded synonyms is presented.     

Revision of some Thecaphora species (Ustilaginomycotina) on Caryophyllaceae

Specimens of Thecaphora saponariae s. lat. from several caryophyllacean host plants belonging to the genera Cerastium, Dianthus, Petrorhagia, Saponaria, Silene, and Stellaria were studied by means of both LM, SEM, and molecular phylogenetic analyses using ITS and LSU rDNA sequences. The data show that T. saponariae s. lat. is not uniform but represents several taxa. Molecular phylogenetic analyses, correlated with morphology of the sori, spore balls, and spores, permitted the recognition of five species. Two new species, Thecaphora italica and T. cerastii are described, and two new combinations, T. alsinearum and T. melandrii are proposed. The anamorph of Thecaphora saponariae is reported for the first time. A lectotype is designated for Sorosporium silenes-inflatae. Evolutionary aspects are discussed.     

Three new <Emphasis Type="Italic">Ophiostoma</Emphasis> species isolated from Japanese red pine

Three new species of Ophiostoma found on Japanese red pine are described as Ophiostoma pusillum sp. nov., O. botuliforme sp. nov., and O. nigrogranum sp. nov. Ophiostoma pusillum is characterized by oblong ascospores and a Hyalorhinocladiella anamorph. Ophiostoma botuliforme has ostioles covered with a hyaline gelatinous cap, allantoid ascospores, and a Pesotum anamorph with hyaline to pale brown stipes. Ophiostoma nigrogranum has hyaline ostiolar hyphae with rounded tips, allantoid ascospores, and sclerotium-like structures.Contribution no. 172, Laboratory of Plant Pathology and Mycology, Institute of Agriculture and Forestry, University of Tsukuba     

Phylogenetic relationships of Nemania plumbea sp. nov. and related taxa based on ribosomal ITS and RPB2 sequences

During a survey fungal diversity of xylariaceous fungi in Thailand, a new Nemania species, N. plumbea, was identified. Nemania plumbea is characterized by soft-textured grey stromata on a persistent mat of white hyphae, pale brown ascospores with a short germ-slit on the more convex side. It also produces a Geniculosporium-like anamorph in culture. In order to evaluate its phylogenetic relationships among related species and genera, ITS-5.8S rDNA and RPB2 were analysed separately and simultaneously. Results from the phylogenetic analyses indicate that there is close phylogenetic association between N. plumbea and N. aenea. A preliminary account into the natural grouping of Xylariaceae based on ITS-5.8S rDNA and RPB2 sequences is also discussed.     

Mycosphaerella buna sp. nov. with aPseudocercospora anamorph isolated from the leaves of Japanese beech

A species ofMycosphaerella with aPseudocercospora anamorph was collected on overwintered fallen leaves of Japanese beech,Fagus crenata. Based on comparison of morphology withMycosphaerella species on Fagaceae, the fungus was newly described asMycosphaerella buna. ThePseudocercospora anamorph derived from a single ascospore of the fungus was morphologically identical to an endophytic anamorph isolated from asymptomatic living leaves of Japanese beech. Contribution No. 150, Laboratory of Plant Parasitic Mycology, Institute of Agriculture and Forestry, University of Tsukuba     

Survey of bovine mycotic mastitis in dairy herds in the State of São Paulo,Brazil

The purpose of this study was to isolate fungi from the quarter milk of cow udders from several dairy herds and to identify the different genera and species involved in mastitis. A total of 2078 milk samples from normal, clinical and subclinical mastitis quarters from 22 dairy herds of 16 districts in the State of São Paulo, Brazil was utilized in this survey. Two hundred and fifty one (12.07%) fungi were isolated from the samples. Two hundred and eight of these (82.86%) were yeasts and 30 (11.95%) were moulds. The fungi were isolated in pure culture (24.77%) or in cultures mixed with bacteria (72.22%). The yeasts isolated were:Cryptococcus spp. (71 strains),Rhodotorula spp. (40),Candida spp. (68),Trichosporon cutaneum (21),Aureobasidium pullulans (7), andPichia ohmeri (1). Moulds classified in following genera were also isolated:Aspergillus (3),Penicillium (3),Alternaria (3),Phoma (3),Epicoccum (2), andGeotrichum (16).     

Two new species ofTalaromyces from Taiwan and Japan

Two new species ofTalaromyces, isolated from soils in Taiwan and Japan, are described and illustrated.Talaromyces eburneus, associated with aGeosmithia anamorph, is characterized by off-white colony on oatmeal agar, small pale yellow ascomata, and subglobose to ovoid ascospores with a smooth wall.Talaromyces muroii is characterized by restricted growth on Czapek agar, luteous ascomata, which are initiated by paired gametangia like those seen in members of the seriesFlavi, ellipsoidal and nearly smooth ascospores, and the absence of an anamorph.     

Comparison of the influence of different elicitors on hyoscyamine and scopolamine content in hairy root cultures of <Emphasis Type="Italic">Brugmansia candida</Emphasis>

Summary Hairy roots of Brugmansia candida were exposed to different elicitors, such as pectinase, B. candida root homogenate, Hormonema ssp. homogenate, and the acetate control buffer. Pectinase increased intracellular hyoscyamine (200–300%) and the release of both alkaloids up to 1500% (scopolamine) and 1100% (hyoscyamine). However, the increment observed in both alkaloids in roots with the acetate control buffer was superior than with pectinase, obtaining increases of 700% in hyoscyamine and 200% in scopolamine. The B. candida root homogenate enhanced the accumulation (50–600%) and specific production of both alkaloids (ca. 150%). Hormonema ssp. homogenates induced different responses according to the original medium in which the fungus was cultured. The effect of each elicitor is discussed.     

Molecular Systematics of Zopfiella and allied genera: evidence from multi-gene sequence analyses

This study aims to reveal the phylogenetic relationships of Zopfiella and allied genera in the Sordariales. Multiple gene sequences (partial 28 S rDNA, ITS/5.8 S rDNA and partial β-tubulin) were analysed using MP and Bayesian analyses. Analyses of different gene datasets were performed individually and then combined to infer phylogenies. Phylogenetic analyses show that currently recognised Zopfiella species are polyphyletic. Based on sequence analyses and morphology, it appears that Zopfiella should be restricted to species having ascospores with a septum in the dark cell. Our molecular analysis also shows that Zopfiella should be placed in Lasiosphaeriaceae rather than Chaetomiaceae. Cercophora and Podospora are also polyphyletic, which is in agreement with previous studies. Our analyses show that species possessing a Cladorrhinum anamorph are phylogenetically closely related. In addition, there are several strongly supported clades, characterised by species possessing divergent morphological characters. It is difficult to predict which characters are phylogenetically informative for delimiting these clades.     

Isolation and characterization of endophytic taxol-producing fungi from <Emphasis Type="Italic">Taxus chinensis</Emphasis>

This study investigated the endophytic fungi diversity of Taxus chinensis and screened the taxol-producing fungi in the host. A total of 115 endophytic fungi isolates obtained from bark segments of T. chinensis were grouped into 23 genera based on the morphological traits and sequence analysis of the internal transcribed spacers (ITS1-5.8S-ITS2), indicating endophytic fungi in T. chinensis are diverse and abundant. Diaporthe, Phomopsis (anamorph of Diaporthe), Acremonium, and Pezicula were the dominant genera, whereas the remaining genera were infrequent groups. The 13 representative species of the distinct genera were capable of producing taxol verified by reverse-phase high performance liquid chromatography (HPLC). Among the taxol-producing fungi, the yield of taxol produced by the Metarhizium anisopliae, H-27 was 846.1 μg l⁻¹ in reformative potato dextrose liquid medium, and the fungal taxol was further validated by mass spectrometry (MS). The taxol-producing fungi (92.3%) were infrequent communities, suggesting that infrequent fungi associated with T. chinensis might be a fascinating reservoir of taxol-generating fungi.