Álvaro Fonseca

Biodiversity and systematics of basidiomycetous yeasts as determined by large-subunit rDNA D1/D2 domain sequence analysis

The molecular systematics of 337 strains of basidiomycetous yeasts and yeast-like fungi, representing 230 species in 18 anamorphic and 24 teleomorphic genera, was determined by sequence analysis of the D1/D2 region of the large-subunit rDNA. The data were compared with published sequences of other basidiomycetous fungi. The results demonstrated that the yeast species and genera are phylogenetically distributed among the Microbotryum, Sporidiobolus, Agaricostilbum and Erythrobasidium clades of the Urediniomycetes; the Tremellales, Trichosporonales ord. nov., Filobasidiales and Cystofilobasidiales clades of the Hymenomycetes; and the Ustilaginales, Microstromatales and Malasseziales clades of the Ustilaginomycetes. Genera such as Bensingtonia, Cryptococcus, Rhodotorula and Sporobolomyces are polyphyletic, i.e. they occur in two or more clades. In contrast, other genera, e.g. Bullera, Cystofilobasidium, Fellomyces, Filobasidiella, Filobasidium, Kondoa, Kurtzmanomyces, Leucosporidium, Rhodosporidium, Sporidiobolus and Udeniomyces, are monophyletic. The majority of the species can be identified using D1/D2 analyses, although the internal transcribed spacer region is required to distinguish closely related species. The intergenic spacer region is recommended for additional differentiation of species and strains.

In Situ Accessibility of Small-Subunit rRNA of Members of the Domains Bacteria, Archaea, and Eucarya to Cy3-Labeled Oligonucleotide Probes

ABSTRACT Low accessibility of the rRNA is together with cell wall impermeability and low cellular ribosome content a frequent reason for failure of whole-cell fluorescence hybridization with fluorescently labeled oligonucleotide probes. In this study we compare accessibility data for the 16S rRNA of Escherichia coli (gamma Proteobacteria, Bacteria) with the phylogenetically distantly related organisms Pirellula sp. strain 1 (Planctomycetes, Bacteria) and Metallosphaera sedula (Crenarchaeota, Archaea) and the 18S rRNA accessibility of Saccharomyces cerevisiae (Eucarya). For a total of 537 Cy3-labeled probes, the signal intensities of hybridized cells were quantified under standardized conditions by flow cytometry. The relative probe-conferred fluorescence intensities are shown on color-coded small-subunit rRNA secondary-structure models. For Pirellula sp., most of the probes belong to class II and III (72% of the whole data set), whereas most of the probes targeting sites on M. sedula were grouped into class V and VI (46% of the whole data set). For E. coli, 45% of all probes of the data set belong to class III and IV. A consensus model for the accessibility of the small-subunit rRNA to oligonucleotide probes is proposed which uses 60 homolog target sites of the three prokaryotic 16S rRNA molecules. In general, open regions were localized around helices 13 and 14 including target positions 285 to 338, whereas helix 22 (positions 585 to 656) and the 3′ half of helix 47 (positions 1320 to 1345) were generally inaccessible. Finally, the 16S rRNA consensus model was compared to data on the in situ accessibility of the 18S rRNA of S. cerevisiae.

Polyphasic taxonomy of the basidiomycetous yeast genus Rhodosporidium: Rhodosporidium kratochvilovae and related anamorphic species

The phenotypic and genetic heterogeneity of the basidiomycetous yeast species Rhodosporidium kratochvilovae was investigated in a group of recent isolates and collection strains. A polyphasic taxonomic approach was followed which included micromorphological studies, nuclear staining, determination of sexual compatibility, physiological characterization, comparison of electrophoretic isoenzyme patterns, PCR fingerprinting, determination of mol% G+C, DNA-DNA reassociation experiments and 26S and ITS rDNA sequence analysis. The results allowed a more natural circumscription of the species, both from the genetic and phenotypic perspectives. The relationships with anamorphic species of the genus Rhodotorula were studied and isolates previously identified as Rhodotorula glutinis were found to belong to Rhodosporidium kratochvilovae. Other isolates included in the study were found to represent members of Rhodotorula glutinis var. dairenensis. Rhodosporidium kratochvilovae was found to include heterothallic strains, besides those already known to be self-sporulating. A total of 17 isolates, which were found to belong to this species, were heterothallic, self-sporulating and anamorphic strains. It is anticipated that integrated polyphasic studies of basidiomycetous yeasts will provide a more coherent classification system and the basis for accurate identification schemes, which in turn are essential for detailed ecological studies.

Estimation and Diversity of Phylloplane Mycobiota on Selected Plants in a Mediterranean–Type Ecosystem in Portugal

Mediterranean ecosystems have not been consistently investigated as natural habitats for microbes in general, and fungi in particular. Here we present the results of a survey of epiphytic mycobiota (filamentous fungi and yeasts) on the phylloplane of selected plants in the Arrábida Natural Park, an ecosystem of Mediterranean characteristics in Portugal, using conventional culture-dependent isolation methods. Leaves from the species Acer monspessulanum and Quercus faginea (deciduous trees) and Cistus albidus, Pistacia lentiscus, and Osyris quadripartita (evergreen shrubs) were collected twice a year for two consecutive years, at two distinct locations of Serra da Arrábida: the more humid northern slope and the drier southern slope. A total of 1029 strains of filamentous fungi and 540 strains of yeasts were isolated, which represented at least 36 and 46 distinct species, respectively. Total counts were higher on the plants from the northern slope and there was a general increase from spring to autumn, notably on the deciduous trees for the yeasts. Plant species that had higher numbers of leaf colonists (A. monspessulanum, C. albidus, and Q. faginea) also yielded a wider range of species. Among the filamentous fungi there was a predominance of species of ascomycetous affinity, whereas basidiomycetous species dominated among yeast isolates. Some of the taxa recovered were common to other phylloplane studies (e.g., ubiquitous molds and yeasts such as Cladosporium spp. and Cryptococcus spp., respectively), but less common species were also found, some of which appeared to represent undescribed taxa. Interestingly, a few species seemed to be associated with a particular plant, notably in the case of the evergreen shrub C. albidus. However, for a considerable number of fungi and yeasts the same taxon was recovered throughout the year from more than one plant and at both sites, suggesting that such species might be genuine phylloplane inhabitants (or at least of aerial plant surfaces) even though they appeared not to display host specificity.

One fungus, which genes? Development and assessment of universal primers for potential secondary fungal DNA barcodes

The aim of this study was to assess potential candidate gene regions and corresponding universal primer pairs as secondary DNA barcodes for the fungal kingdom, additional to ITS rDNA as primary barcode. Amplification efficiencies of 14 (partially) universal primer pairs targeting eight genetic markers were tested across > 1 500 species (1 931 strains or specimens) and the outcomes of almost twenty thousand (19 577) polymerase chain reactions were evaluated. We tested several well-known primer pairs that amplify: i) sections of the nuclear ribosomal RNA gene large subunit (D1–D2 domains of 26/28S); ii) the complete internal transcribed spacer region (ITS1/2); iii) partial β -tubulin II (TUB2); iv) γ-actin (ACT); v) translation elongation factor 1-α (TEF1α); and vi) the second largest subunit of RNA-polymerase II (partial RPB2, section 5–6). Their PCR efficiencies were compared with novel candidate primers corresponding to: i) the fungal-specific translation elongation factor 3 (TEF3); ii) a small ribosomal protein necessary for t-RNA docking; iii) the 60S L10 (L1) RP; iv) DNA topoisomerase I (TOPI); v) phosphoglycerate kinase (PGK); vi) hypothetical protein LNS2; and vii) alternative sections of TEF1α. Results showed that several gene sections are accessible to universal primers (or primers universal for phyla) yielding a single PCR-product. Barcode gap and multi-dimensional scaling analyses revealed that some of the tested candidate markers have universal properties providing adequate infra- and inter-specific variation that make them attractive barcodes for species identification. Among these gene sections, a novel high fidelity primer pair for TEF1α, already widely used as a phylogenetic marker in mycology, has potential as a supplementary DNA barcode with superior resolution to ITS. Both TOPI and PGK show promise for the Ascomycota, while TOPI and LNS2 are attractive for the Pucciniomycotina, for which universal primers for ribosomal subunits often fail.

Cryptococcus Vuillemin (1901)

Publisher Summary This chapter discusses the genus Cryptococcus. In the determination of the asexual reproduction it is seen that cells are spheroidal, ovoid, ellipsoidal, or elongate. A polysaccharide capsule is present in most species. Reproduction is by multilateral or polar, enteroblastic budding; pseudohyphae or true hyphae may develop. In species with true hyphae, septa have dolipores with or without parenthesomes. In the sexual reproduction it is found that some species are anamorphic states of teleomorphic genera in the Cystofilobasidiales, Filobasidiales, and Tremellales. The chapter also discusses physiology/biochemistry and phylogenetic placement of the genus. The type species taken is Cryptococcus neoformans. In the systematic discussion of the species phylogenetic placement, growth on malt extract, soytone, yeast extract (MYP) agar, growth in YM broth, Dalmau plate culture on corn meal agar, gene sequence accession numbers, type strain, origin of the strains studied, and systematics are determined.

Phylogeny and phenotypic characterization of pathogenic Cryptococcus species and closely related saprobic taxa in the Tremellales.

ABSTRACT The basidiomycetous yeasts Cryptococcus neoformans and Cryptococcus gattii are closely related sibling species that cause respiratory and neurological disease in humans and animals. Within these two recognized species, phylogenetic analysis reveals at least six cryptic species defined as molecular types (VNI/II/B, VNIV, VGI, VGII, VGIII, and VGIV) that comprise the pathogenic Cryptococcus species complex. These pathogenic species are clustered in the Filobasidiella clade within the order Tremellales. Previous studies have shown that the Filobasidiella clade also includes several saprobic fungi isolated from insect frass, but information evaluating the relatedness of the saprobes and pathogens within this cluster is limited. Here, the phylogeny encompassing a subset of species in the Tremellales lineage that clusters closely with the pathogenic Cryptococcus species complex was resolved by employing a multilocus sequencing approach for phylogenetic analysis. Six highly conserved genomic loci from 15 related basidiomycete species were sequenced, and the alignments from the concatenated gene sequences were evaluated with different tree-building criteria. Furthermore, these 15 species were subjected to virulence and phenotype assays to evaluate their pathogenic potential. These studies revealed that Cryptococcus amylolentus and Tsuchiyaea wingfieldii, two nonpathogenic sibling species, are the taxa most closely related to the pathogens C. neoformans and C. gattii and together with Filobasidiella depauperata form a Cryptococcus sensu stricto group. Five other saprobic yeast species form the Kwoniella clade, which appears to be a part of a more distantly related sensu lato group. This study establishes a foundation for future comparative genomic approaches that will provide insight into the structure, function, and evolution of the mating type locus, the transitions in modes of sexual reproduction, and the emergence of human pathogenic species from related or ancestral saprobic species.

Discussion of Teleomorphic and Anamorphic Basidiomycetous Yeasts

Publisher Summary The presence of ballistoconidia in Sporobolomyces led Kluyver and van Niel to suggest that this yeast is basidiomycetous, based on the morphological resemblance of ballistoconidia with actively discharged basidiospores. The life cycles of basidiomycetous yeasts include heterothallic and homothallic systems, which may occur within a single species. In the heterothallic system, compatible mating types conjugate and give rise to dikaryotic hyphae, usually with clamp connections. In many dimorphic basidiomycetes the dikaryotic hyphal state may be parasitic on either green plants or fungi. However, putative host associations have not been identified from any yeast taxa and their sexual states are only known from observations on culture media. Basidiomycetous yeasts predominantly form soft, often slimy, or mucoid colonies. The yeast state usually commences with basidiospore germination. In some Tremella species and in species of Mycogloea, basidial cells can occasionally bud directly The unicellular haploid state ends at the initiation of the dikaryotic state in those yeasts with known teleomorphs (i.e., following mating of compatible cells). However, development of hyphae and basidia from single haploid cells (i.e., self-fertile or monokaryotic fruiting) or fusion of same sex haploid cells occurs in some species, such as Filobasidiella neoformans.

In Situ Accessibility of Saccharomyces cerevisiae 26S rRNA to Cy3-Labeled Oligonucleotide Probes Comprising the D1 and D2 Domains

ABSTRACT Fluorescence in situ hybridization (FISH) has proven to be most useful for the identification of microorganisms. However, species-specific oligonucleotide probes often fail to give satisfactory results. Among the causes leading to low hybridization signals is the reduced accessibility of the targeted rRNA site to the oligonucleotide, mainly for structural reasons. In this study we used flow cytometry to determine whole-cell fluorescence intensities with a set of 32 Cy3-labeled oligonucleotide probes covering the full length of the D1 and D2 domains in the 26S rRNA of Saccharomyces cerevisiae PYCC 4455T. The brightest signal was obtained with a probe complementary to positions 223 to 240. Almost half of the probes conferred a fluorescence intensity above 60% of the maximum, whereas only one probe could hardly detect the cells. The accessibility map based on the results obtained can be extrapolated to other yeasts, as shown experimentally with 27 additional species (14 ascomycetes and 13 basidiomycetes). This work contributes to a more rational design of species-specific probes for yeast identification and monitoring.

Genome Sequencing of the Plant Pathogen Taphrina deformans, the Causal Agent of Peach Leaf Curl

ABSTRACT Taphrina deformans is a fungus responsible for peach leaf curl, an important plant disease. It is phylogenetically assigned to the Taphrinomycotina subphylum, which includes the fission yeast and the mammalian pathogens of the genus Pneumocystis. We describe here the genome of T. deformans in the light of its dual plant-saprophytic/plant-parasitic lifestyle. The 13.3-Mb genome contains few identifiable repeated elements (ca. 1.5%) and a relatively high GC content (49.5%). A total of 5,735 protein-coding genes were identified, among which 83% share similarities with other fungi. Adaptation to the plant host seems reflected in the genome, since the genome carries genes involved in plant cell wall degradation (e.g., cellulases and cutinases), secondary metabolism, the hallmark glyoxylate cycle, detoxification, and sterol biosynthesis, as well as genes involved in the biosynthesis of plant hormones. Genes involved in lipid metabolism may play a role in its virulence. Several locus candidates for putative MAT cassettes and sex-related genes akin to those of Schizosaccharomyces pombe were identified. A mating-type-switching mechanism similar to that found in ascomycetous yeasts could be in effect. Taken together, the findings are consistent with the alternate saprophytic and parasitic-pathogenic lifestyles of T. deformans. IMPORTANCE Peach leaf curl is an important plant disease which causes significant losses of fruit production. We report here the genome sequence of the causative agent of the disease, the fungus Taphrina deformans. The genome carries characteristic genes that are important for the plant infection process. These include (i) proteases that allow degradation of the plant tissues; (ii) secondary metabolites which are products favoring interaction of the fungus with the environment, including the host; (iii) hormones that are responsible for the symptom of severely distorted leaves on the host; and (iv) drug detoxification enzymes that confer resistance to fungicides. The availability of the genome allows the design of new drug targets as well as the elaboration of specific management strategies to fight the disease. Peach leaf curl is an important plant disease which causes significant losses of fruit production. We report here the genome sequence of the causative agent of the disease, the fungus Taphrina deformans. The genome carries characteristic genes that are important for the plant infection process. These include (i) proteases that allow degradation of the plant tissues; (ii) secondary metabolites which are products favoring interaction of the fungus with the environment, including the host; (iii) hormones that are responsible for the symptom of severely distorted leaves on the host; and (iv) drug detoxification enzymes that confer resistance to fungicides. The availability of the genome allows the design of new drug targets as well as the elaboration of specific management strategies to fight the disease.