Identifying carbohydrate-active enzymes of Cutaneotrichosporon oleaginosus using systems biology.

BACKGROUND: The oleaginous yeast Cutaneotrichosporon oleaginosus represents one of the most promising microbial platforms for resource-efficient and scalable lipid production, with the capacity to accept a wide range of carbohydrates encapsulated in complex biomass waste or lignocellulosic hydrolysates. Currently, data related to molecular aspects of the metabolic utilisation of oligomeric carbohydrates are sparse. In addition, comprehensive proteomic information for C. oleaginosus focusing on carbohydrate metabolism is not available. RESULTS: In this study, we conducted a systematic analysis of carbohydrate intake and utilisation by C. oleaginosus and investigated the influence of different di- and trisaccharide as carbon sources. Changes in the cellular growth and morphology could be observed, depending on the selected carbon source. The greatest changes in morphology were observed in media containing trehalose. A comprehensive proteomic analysis of secreted, cell wall-associated, and cytoplasmatic proteins was performed, which highlighted differences in the composition and quantity of secreted proteins, when grown on different disaccharides. Based on the proteomic data, we performed a relative quantitative analysis of the identified proteins (using glucose as the reference carbon source) and observed carbohydrate-specific protein distributions. When using cellobiose or lactose as the carbon source, we detected three- and five-fold higher diversity in terms of the respective hydrolases released. Furthermore, the analysis of the secreted enzymes enabled identification of the motif with the consensus sequence LALL[LA]L[LA][LA]AAAAAAA as a potential signal peptide. CONCLUSIONS: Relative quantification of spectral intensities from crude proteomic datasets enabled the identification of new enzymes and provided new insights into protein secretion, as well as the molecular mechanisms of carbo-hydrolases involved in the cleavage of the selected carbon oligomers. These insights can help unlock new substrate sources for C. oleaginosus, such as low-cost by-products containing difficult to utilize carbohydrates. In addition, information regarding the carbo-hydrolytic potential of C. oleaginosus facilitates a more precise engineering approach when using targeted genetic approaches. This information could be used to find new and more cost-effective carbon sources for microbial lipid production by the oleaginous yeast C. oleaginosus.

Geometric morphometric analysis of spore shapes improves identification of fungi.

Morphology of organisms is an essential source of evidence for taxonomic decisions and understanding of ecology and evolutionary history. The geometric structure (i.e., numeric description of shape) provides richer and mathematically different information about an organism's morphology than linear measurements. A little is known on how these two sources of morphological information (shape vs. size) contribute to the identification of organisms when implied simultaneously. This study hypothesized that combining geometric information on the outline with linear measurements results in better species identification than either evidence alone can provide. As a test system for our research, we used the microscopic spores of fungi from the genus Subulicystidium (Agaricomycetes, Basidiomycota). We analyzed 2D spore shape data via elliptic Fourier and principal component analyses. Using flexible discriminant analysis, we achieved the highest species identification success rate for a combination of shape and size descriptors (64.7%). The shape descriptors alone predicted species slightly better than size descriptors (61.5% vs. 59.1%). We conclude that adding geometric information on the outline to linear measurements improves the identification of the organisms. Despite the high relevance of spore traits for the taxonomy of fungi, they were previously rarely analyzed with the tools of geometric morphometrics. Therefore, we supplement our study with an open access protocol for digitizing and summarizing fungal spores' shape and size information. We propagate a broader use of geometric morphometric analysis for microscopic propagules of fungi and other organisms.

Reusable System for Phenol Detection in an Aqueous Medium Based on Nanodiamonds and Extracellular Oxidase from Basidiomycete Neonothopanus nambi.

A reusable system for phenol determination in an aqueous medium was obtained by adsorption of extracellular oxidase from fungus Neonothopanus nambi onto modified nanodiamonds (MND) synthesized by detonation. It was found that the enzyme strongly binds to MND and exhibits catalytic activity in the reaction of co-oxidation of phenol with 4-aminoantipyrine without the addition of hydrogen peroxide. In the presence of the MND-oxidase complex, a significantly (by an order of magnitude) higher yield of the reaction product is recorded as compared to the yield in the presence of a free enzyme; the mechanism of the revealed effect is discussed. Model experiments have demonstrated the multiple use of the MND-oxidase complex for testing phenol in aqueous samples. The immobilized enzyme exhibits functional activity during long-term (2 months) storage of the MND-oxidase complex at 4°C. The data obtained create the prerequisites for using the created system in environmental monitoring of water pollution with phenol.

Phosphoinositide signaling plays a key role in the regulation of cell wall reconstruction during the postharvest morphological development of Dictyophora indusiata.

The potential signaling mechanism of Dictyophora indusiata during postharvest morphological development was investigated through quantitative phosphoproteomic analyses. A total of 1566 phosphorylation sites changed significantly (872 upregulated and 694 downregulated) in the mature stage compared with those in the peach-shaped stage of D. indusiata. Bioinformatics analysis showed that the upregulated differentially phosphorylated proteins were mainly involved in the "phosphatidylinositol signaling system" and "mitogen-activated protein kinase signaling pathway-yeast", while the downregulated differentially phosphorylated proteins were related mainly to "starch and sucrose metabolism". Further mining of the phosphoproteome data revealed that upregulated phosphoinositide signaling activated the cell wall integrity pathway and then regulated the synthesis of the main components of the cell wall. The results suggested that phosphoinositide signaling could be a potential target pathway for the regulation of the postharvest morphological development of D. indusiata.

New species of Tulasnella associated with Australian terrestrial orchids in the Cryptostylidinae and Drakaeinae.

Many orchids have an obligate relationship with Tulasnella mycorrhizal fungi for seed germination and support into adulthood. Despite the importance of Tulasnella as mycorrhizal partners, many species remain undescribed. Here, we use multiple sequence locus phylogenetic analyses to delimit and describe six new Tulasnella species associated with Australian terrestrial orchids from the subtribes Cryptostylidinae and Drakaeinae. Five of the new species, Tulasnella australiensis, T. occidentalis, T. punctata, T. densa, and T. concentrica, all associate with Cryptostylis (Cryptostylidinae), whereas T. rosea associates with Spiculaea ciliata (Drakaeinae). Isolates representing T. australiensis were previously also reported in association with Arthrochilus (Drakaeinae). All newly described Tulasnella species were delimited by phylogenetic analyses of four loci (nuc rDNA internal transcribed spacer region ITS1-5.8S-ITS2 [ITS], C14436 [ATP synthase], C4102 [glutamate synthase], and mt 16S rDNA [mtLSU]). The pairwise sequence divergence between species for the ITS region ranged from 5.6% to 25.2%, and the maximum sequence divergence within the newly described species ranged from 1.64% to 4.97%. There was a gap in the distribution of within- and between-species pairwise divergences in the region of 4-6%, with only one within-species value of 4.97% (for two T. australiensis isolates) and one between-species value of 5.6% (involving an isolate of T. occidentalis) falling within this region. Based on fluorescence staining, all six new Tulasnella species are binucleate and have septate, cylindrical hyphae. There was some subtle variation in culture morphology, but colony diameter as measured on 3MN+vitamin medium after 6 wk of growth did not differ among species. However, T. australiensis grew significantly (P < 0.02) slower than others on ½ FIM and » potato dextrose agar (PDA) media. Formal description of these Tulasnella species contributes significantly to documentation of Tulasnella diversity and provides names and delimitations to underpin further research on the fungi and their relationships with orchids.

Species diversity, taxonomy, and phylogeny of Gymnosporangium in China.

Gymnosporangium is a group of plant fungal pathogens that cause rust diseases on many economically important fruit trees. Most Gymnosporangium are heteroecious and demicyclic, producing four morphologically diverse spore stages on two taxonomically unrelated host plants, the Cupressaceae and Rosaceae. The complex life cycle and heteroecism make it difficult to investigate the species within Gymnosporangium. To determine the taxonomy, phylogeny, and species diversity of Gymnosporangium in China, a large collection of 672 specimens were analyzed using a combination of morphological observations and phylogenetic analyses. In total, 27 Gymnosporangium species from China are documented here, including 22 known species, one new combination, one new record, and three new species. The study also documents a novel aeciospore surface structure with an irregular surface that is described here as "surfy."

Cell death in Ustilago maydis: comparison with other fungi and the effect of metformin and curcumin on its chronological lifespan.

Ustilago maydis is a Basidiomycota fungus, in which very little is known about its mechanisms of cell survival and death. To date, only the role of metacaspase1, acetate and hydrogen peroxide as inducers of cell death has been investigated. In the present work, we analyzed the lifespan of U. maydis compared with other species like Sporisorium reilianum, Saccharomyces cerevisiae and Yarrowia lipolytica, and we observed that U. maydis has a minor lifespan. We probe the addition of low concentrations metformin and curcumin to the culture media, and we observed that both prolonged the lifespan of U. maydis, a result observed for the first time in a phytopathogen fungus. However, higher concentrations of curcumin were toxic for the cells, and interestingly induced the yeast-to-mycelium dimorphic transition. The positive effect of metformin and curcumin appears to be related to an inhibition of the mechanistic Target of Rapamycin (mTOR) pathway, increase expression of autophagy genes and reducing of reactive oxygen species. These data indicate that U. maydis may be a eukaryotic model organism to elucidate the molecular mechanism underlying apoptotic and necrosis pathways, and the lifespan increase caused by metformin and curcumin.

Poroid Hymenochaetaceae associated with trees showing wood-rot symptoms in the Garden Route National Park of South Africa.

Poroid Hymenochaetaceae associated with wood rots of trees in three timber-harvesting compartments of the Garden Route National Park (GRNP), South Africa, were investigated using multilocus phylogenetic analyses and morphology of the basidiomes. Results revealed the presence of 10 species belonging to five genera. Six of the species are known, but four are described as new. The known species include Fomitiporia capensis, Fuscoporia gilva, Sanghuangporus microcystideus, Tropicoporus tropicalis, Inonotus rickii, and Inonotus setuloso-croceus. The new species are described as Fomitiporia tsitsikamensis, Fulvifomes elaeodendri, Fuscoporia pulviniformis, and Phellinus guttiformis.

Diversity trapped in cages: Revision of Blumenavia Möller (Clathraceae, Basidiomycota) reveals three hidden species.

Basidiomata of Phallales have a diversified morphology with adhesive gleba that exudes an odor, usually unpleasant that attracts mainly insects, which disperse the basidiospores. The genus Blumenavia belongs to the family Clathraceae and, based on morphological features, only two species are currently recognized: B. rhacodes and B. angolensis. However, the morphological characters adopted in species delimitations within this genus are inconsistent, and molecular data are scarce. The present study aimed to review and identify informative characters that contribute to the delimitation of Blumenavia species. Exsiccates from America and Africa were analyzed morphologically, and molecularly, using ITS, LSU, ATP6, RPB2 and TEF-1α markers for Maximum Parsimony, Bayesian and Maximum likelihood analyses, and also for coalescent based species delimitations (BP&P), as well as for bPTP, PhyloMap, Topo-phylogenetic and Geophylogenetic reconstructions. According to our studies, seven species can be considered in the genus: B. rhacodes and B. angolensis are maintained, B. usambarensis and B. toribiotalpaensis are reassessed, and three new species are proposed, B. baturitensis Melanda, M.P. Martín & Baseia, sp. nov., B. crucis-hellenicae G. Coelho, Sulzbacher, Grebenc & Cortez, sp. nov., and B. heroica Melanda, Baseia & M.P. Martín, sp. nov. Blumenavia rhacodes is typified by selecting a lectotype and an epitype. Macromorphological characters considered informative to segregate and delimit the species through integrative taxonomy include length of the basidiomata, color, width and presence of grooves on each arm as well as the glebifer position and shape. These must be clearly observed while the basidiomata are still fresh. Since most materials are usually analyzed after dehydration and deposit in collections, field techniques and protocols to describe fugacious characters from fresh specimen are demanded, as well as the use of molecular analysis, in order to better assess recognition and delimitation of species in Blumenavia.

Uromyces rebeccae, sp. nov., a newly described rust on the federally endangered plant, California sea-blite (Suaeda californica).

Rust disease was observed on populations of Suaeda californica near Morro Bay, California. The pathogen was identified as a species of Uromyces based on teliospore and urediniospore morphology and nuc 28S rDNA sequence analysis. The isolate was compared with previously described species of Uromyces that infect members of Chenopodiaceae, prompting a taxonomic reevaluation of Uromyces species on Suaeda. Herein, Uromyces rebeccae is described. It can be differentiated from the closely related U. chenopodii (syn.: Aecidium chenopodii-fruticosi; U. giganteus) based on host range, teliospore morphology, and 28S sequence data. The new combination, Uromyces chenopodii-fruticosi, is made for Aecidium chenopodii-fruticosi, the oldest name for Eurasian Suaeda rust. Finally, it was determined that U. giganteus likely does not occur in the United States and that the rust of S. taxifolia in the United States likely comprises a third, yet unnamed taxon, different from both U. rebeccae and U. chenopodii-fruticosi. This is the first record of a rust fungus on S. californica. An identification key for Uromyces species reported on Chenopodiaceae is provided.

Bambusicolous Fomitiporia revisited: multilocus phylogeny reveals a clade of host-exclusive species.

FOMITIPORIA: is a genus of wood-inhabiting Hymenochaetaceae (Agaricomycetes) that includes pathogens and decomposers occurring on a wide array of plant substrates. Some species decay culms of woody bamboos, a poorly known biotic interaction. Four bambusicolous species of Fomitiporia are currently known. However, no studies concerning their phylogenetic relationships have been performed. In order to assess species boundaries and their relationships to each other, we conducted molecular phylogenetic analyses of nuc rDNA internal transcribed spacer region ITS1-5.8S-ITS2 (ITS) and portions of nuc 28S rDNA (28S), RNA polymerase II second largest subunit (RPB2), and translation elongation factor 1-α (TEF1), as well as morphological analyses. Four species that occur on culms of woody bamboos, F. bambusarum, F. spinescens, F. uncinata, and the new species F. bambusipileata, grouped together in an exclusive clade within a primarily Neotropical lineage. The new species differs from all other species in the group by the pileate basidiomata. Hypotheses regarding host-exclusivity are discussed.

Taxonomic revision of species of Kuehneola and Phragmidium on Rosa, including two new species from China.

Kuehneola japonica and K. warburgiana are the only species of genus Kuehneola that parasitize plants belonging to genus Rosa (Rosaceae). Systematic revision of the genus Phragmidium and related genera by molecular phylogenetic analyses using nuc rDNA internal transcribed spacer (5.8S-ITS2 = ITS2) and nuc rDNA 28S (28S) sequences indicated that K. japonica and K. warburgiana belong to a group of Phragmidium species that also occur on Rosa. Morphological and molecular phylogenetic analyses revealed that these Kuehneola species were transferred to Phragmidium and renamed as P. japonicum and P. warburgianum. Two new Phragmidium species were also discovered on Rosa from China that could be distinguished from other species in the genus based on aeciospore or urediniospore morphology and phylogenetic placement. The first species, P. jiangxiense, is characterized by a urediniospore surface structure with stout spines that are basally embedded in the wrinkled spore wall. The second species, P. leucoaecium, is characterized by an aeciospore surface structure with irregularly elongated verrucae. Taxonomic descriptions and illustrations are provided.

Expansion of the genus Imleria in North America to include Imleria floridana, sp. nov., and Imleria pallida, comb. nov.

Imleria is a small genus of Boletaceae found primarily in the Northern Hemisphere characterized by the light to dark brown hymenophore that stains blue. In Florida, specimens of Imleria were collected that resembled I. badia, a species known primarily from northeastern North America and Europe. Five nuclear loci of these Florida specimens were sequenced and phylogenetically analyzed. A nuc rDNA internal transcribed spacer ITS1-5.8S-ITS2 (ITS) phylogeny of Imleria was generated using primarily uncultured environmental sequences to confirm ectomycorrhizal associations. Based on morphological and molecular data, we describe a new species, Imleria floridana. Results from these analyses also support the new combination Imleria pallida. A lectotype of I. pallida is also established from Charles H. Peck's original material. We discuss the distinguishing characters and species of Imleria found in North America and provide a key to the known worldwide species of Imleria.

Revision of leccinoid fungi, with emphasis on North American taxa, based on molecular and morphological data.

The leccinoid fungi are boletes and related sequestrate mushrooms (Boletaceae, Basidiomycota) that have traditionally been placed in Leccinum, Boletus, Leccinellum, and a handful of other less familiar genera. These mushrooms generally feature scabers or scaber-like dots on the surface of the stipe, and they are often fairly tall and slender when compared with other boletes. They are ectomycorrhizal fungi and appear to be fairly strictly associated with specific trees or groups of related trees. In the present study, we investigate the phylogenetic relationships among the leccinoid fungi and other members of the family Boletaceae using portions of three loci from nuc 28S rDNA (28S), translation elongation factor 1-α (TEF1), and the RNA polymerase II second-largest subunit (RPB2). Two DNA data sets (combined 28S-TEF1 and 28S-TEF1-RPB2), comprising sequences from nearly 270 voucher specimens, were evaluated using two different phylogenetic analyses (maximum likelihood and Bayesian inference). Five major clades were obtained, and leccinoid fungi appeared in four of them. Taxonomic proposals supported by our results, representing a broadly circumscribed Leccinum that includes several sequestrate genera, along with Leccinellum, are made.

Psiloboletinus is an independent genus sister to Suillus.

The genus Psiloboletinus was proposed by Rolf Singer in 1945 based on Phylloporus lariceti, a species that associates with Larix in the Altai Mountains of central and eastern Asia. However, this classification has been controversial due to the morphological similarity to known genera Boletinus and Fuscoboletinus. Because of the lack of fresh material to study, the phylogenetic position of Psiloboletinus has remained unknown since its publication. However, the recently described species Suillus foetidus reported from northeast China allows this issue to be reexamined and resolved. Through morphological observations and comparison, we find that S. foetidus is a heterotypic synonym of Ps. lariceti. Furthermore, Psiloboletinus should be retained as an independent genus sister to Suillus based on molecular phylogenetic evidence and morphological features.

A comparative study of liquid holding restitution of viability after oxidative stress in Ustilago maydis and Saccharomyces cerevisiae cell populations.

The ability of Saccharomyces cerevisiae to reconstitute viability after strong peroxide-induced oxidative stress during liquid holding (LH) in non-nutrient medium has been compared with that of Ustilago maydis. It was found that like U. maydis, S. cerevisiae was capable of reconstituting viability through multiplication of the survivors. However, differences were observed in the pattern of their response: (i) the reconstitution of viability was slower in S. cerevisiae; (ii) before the viability was progressively increasing the treated samples of this fungus reproducibly passed through a phase of additional decrease of the surviving fraction and (iii) the final yields of viable cells attained in S. cerevisiae were below those achieved by U. maydis. The reason for the relative superiority of U. maydis is twofold: (1) early initiated and faster degradation and leakage of the intracellular biomolecules and (2) greater ability of U. maydis cells to recycle damaged and released intracellular compounds. Conceptually similar studies extended to another oxidative-stress-inducing condition, namely desiccation, indicated that the marked differences between these fungi in their patterns of the post-stress regrowth, cellular leakage and macromolecule decomposition are reproduced during LH of desiccated cells. The concordance of the findings obtained upon these two approaches was also corroborated by an analysis of the post-desiccation LH response of U. maydis mutants (adr1, did4, kel1 and tbp1) that were previously identified as defective in post-peroxide LH restitution of viability. We discuss the findings in terms of their possible relevance to the mechanisms of the ecological and evolutionary adaptation of free-living microorganisms to fluctuating and severely inhospitable environments.

Tradeoffs in hyphal traits determine mycelium architecture in saprobic fungi.

The fungal mycelium represents the essence of the fungal lifestyle, and understanding how a mycelium is constructed is of fundamental importance in fungal biology and ecology. Previous studies have examined initial developmental patterns or focused on a few strains, often mutants of model species, and frequently grown under non-harmonized growth conditions; these factors currently collectively hamper systematic insights into rules of mycelium architecture. To address this, we here use a broader suite of fungi (31 species including members of the Ascomycota, Basidiomycota and Mucoromycota), all isolated from the same soil, and tested for ten architectural traits under standardized laboratory conditions. We find great variability in traits among the saprobic fungal species, and detect several clear tradeoffs in mycelial architecture, for example between internodal length and hyphal diameter. Within the constraints so identified, we document otherwise great versatility in mycelium architecture in this set of fungi, and there was no evidence of trait 'syndromes' as might be expected. Our results point to an important dimension of fungal properties with likely consequences for coexistence within local communities, as well as for functional complementarity (e.g. decomposition, soil aggregation).

Russula swatica: A new species of Russula based on molecular, light microscopy, and scanning electron microscopy analyses from Swat Valley of Khyber Pakhtunkhwa province of Pakistan.

Himalayan range of Pakistan is one of the diversity rich hotspots of the world. Many areas are yet to be explored here to discover new species of organisms including fungi. During present research, Swat District of Pakistan was explored for fungal diversity. One new species of mushroom Russula swatica (Russulales) is described from Himalayan range of Pakistan and analyzed by light microscopy and scanning electron microscopy as well as by molecular markers. A comprehensive description, photographs, and comparisons with morphologically similar and phylogenetically related species are provided. Conclusions of its phylogenetic relationships within the genus are provided based on the sequence of the nuclear internal transcribed spacer region.

Investigation of urediospore morphology, histopathology and epidemiological components on wheat plants infected with UV-B-induced mutant strains of Puccinia striiformis f. sp. tritici.

Planting resistant cultivars is the most economical and effective measure to control wheat stripe rust caused by Puccinia striiformis f. sp. tritici (Pst), but the cultivars often lose their resistance due to the emergence of new physiological races. The UV-B-irradiated urediospores of the Pst physiological race CYR32 in China were inoculated on wheat cultivar Guinong 22 for screening virulence-mutant strains. CYR32 and mutant strains (CYR32-5 and CYR32-61) before and after UV-B radiation were used to conduct urediospore morphological and histopathological observations and an investigation of epidemiological components. The results showed that UV-B radiation affected the urediospore morphology of each strain. UV-B radiation inhibited urediospore invasion and hyphal elongation, which mainly manifested as decreases in germination rate, quantities of hyphal branches, haustorial mother cells and haustoria and hyphal length. After wheat cultivar Mingxian 169 was inoculated with the UV-B-irradiated urediospores, the incubation period was prolonged, and the infection efficiency, lesion expansion rate, total sporulation quantity and area under the disease progress curve were reduced. The results demonstrated that CYR32-5 and CYR32-61 may have more tolerance to UV-B radiation than CYR32. The results are significant for understanding mechanisms of Pst virulence variations and implementing sustainable management of wheat stripe rust.