Basidiomycete Species Research Articles

Mitogenomes of Two Phallus Mushroom Species Reveal Gene Rearrangement, Intron Dynamics, and Basidiomycete Phylogeny.

Phallus indusiatus and Phallus echinovolvatus are edible bamboo mushrooms with pharmacological properties. We sequenced, assembled, annotated, and compared the mitogenomes of these species. Both mitogenomes were composed of circular DNA molecules, with sizes of 89,139 and 50,098 bp, respectively. Introns were the most important factor in mitogenome size variation within the genus Phallus. Phallus indusiatus, P. echinovolvatus, and Turbinellus floccosus in the subclass Phallomycetidae have conservative gene arrangements. Large-scale gene rearrangements were observed in species representing 42 different genera of Basidiomycetes. A variety of intron position classes were found in the 44 Basidiomycete species analyzed. A novel group II intron from the P. indusiatus mitogenome was compared with other fungus species containing the same intron, and we demonstrated that the insertion sites of the intron had a base preference. Phylogenetic analyses based on combined gene datasets yielded well-supported Bayesian posterior probability (BPP = 1) topologies. This indicated that mitochondrial genes are reliable molecular markers for analyzing the phylogenetic relationships of the Basidiomycetes. This is the first study of the mitogenome of the genus Phallus, and it increases our understanding of the population genetics and evolution of bamboo mushrooms and related species.

Assessment of Polysaccharide and Biomass Production from Three White-Rot Fungi by Solid-State Fermentation Using Wood and Agro-Industrial Residues: A Kinetic Approach

Research Highlights: For the first time, a model was developed and applied for polysaccharide production from Trametes versicolor grown in agro-industrial and woody residues under solid-state fermentation (SSF) conditions. Background and Objectives: Fungal biomass is an important biological resource for biotechnological applications. Basidiomycetes fungi can be grown and developed on lignocellulosic materials such as forestry, wood, and agro-industrial residues in order to produce value-added products like bioactive polysaccharides. The objectives of this study were to evaluate the effects of the C/N ratio and copper concentration on biomass and polysaccharide production during solid state fermentation (SSF), as well as on the consumption of cellulose and hemicellulose, and lignin degradation, and to propose and validate a mathematical model to describe the overall SSF process. Materials and Methods: This research was carried out by growing three Basidiomycetes species (T. versicolor, Lentinula edodes, and Pleurotus ostreatus) on twelve formulations of solid substrates using mixtures of different inexpensive lignocellulosic residues such as oak sawdust, coconut fiber (hairs), coffee husks, and corn bran plus soybean oil, calcium carbonate, and two levels of copper(II) sulfate. Results: The three fungal species grew well on all substrate formulations. The statistical analysis of experimental data showed no significant effects on polysaccharide production, in the range of C/N and copper concentrations evaluated. Taking into account that the best polysaccharide production was obtained with T. versicolor (96.09 mg/g solid substrate), a mathematical model was proposed for this fungus to describe the behavior of the fermentation system from the obtained data of all the resulting combinations to reach the highest polysaccharide production by the fungus. Conclusions: The mathematical model disclosed in this work enabled to describe the growth and development of a higher basidiomycete under solid-state fermentation conditions on lignocellulosic substrates as well as the production of value-added products like polysaccharides with medicinal properties.

Biocatalytic Potential of Native Basidiomycetes from Colombia for Flavour/Aroma Production

Aromas and flavours can be produced from fungi by either de novo synthesis or biotransformation processes. Herein, the biocatalytic potential of seven basidiomycete species from Colombia fungal strains isolated as endophytes or basidioma was evaluated. Ganoderma webenarium, Ganoderma chocoense, and Ganoderma stipitatum were the most potent strains capable of decolourizing β,β-carotene as evidence of their potential as biocatalysts for de novo aroma synthesis. Since a species’ biocatalytic potential cannot solely be determined via qualitative screening using β,β-carotene biotransformation processes, we focused on using α-pinene biotransformation with mycelium as a measure of catalytic potential. Here, two strains of Trametes elegans—namely, the endophytic (ET-06) and basidioma (EBB-046) strains—were screened. Herein, T. elegans is reported for the first time as a novel biocatalyst for the oxidation of α-pinene, with a product yield of 2.9 mg of cis-Verbenol per gram of dry weight mycelia used. The EBB-046 strain generated flavour compounds via the biotransformation of a Cape gooseberry medium and de novo synthesis in submerged cultures. Three aroma-producing compounds were identified via GC–MS—namely, methyl-3-methoxy-4H-pyran-4-one, hexahydro-3-(methylpropyl)-pyrrolo[1,2-a]pyrazine-1,4-dione, and hexahydro-3-(methylphenyl)-pyrrolo[1,2-a]pyrazine-1,4-dione.

First Report of Wheat Common Bunt Caused by Tilletia laevis in Henan Province, China.

Wheat common bunt is a serious disease that may lead to yield losses of 75-80% in many wheat regions of the world (Mathre 1996). The disease may reduce yield and flour quality by producing trimethylamine, a compound that smells like rotting fish (Castlebury et al. 2005; Hoffmann 1981; Mathre 1996). Two closely related basidiomycete species, Tilletia caries (DC.) Tul. & C. Tul. [syn. T. tritici (Bjerk.) Wint.] and T. laevis J. G. Kühn [syn. T. foetida (Wallr.) Liro], cause wheat common bunt. Teliospore morphology is used to differentiate the two species. Teliospores of T. caries have reticulates on the surface while teliospores of T. laevis have a smooth surface (Pieczul et al. 2018). T. laevis was reported in Liaoning, Shaanxi, Shandong, Beijing, Hebei, Shanxi, Jilin, Heilongjiang, Jiangsu, Gansu, Xinjiang, Sichuan, Yunnan, Inner Mongolia, and Tibet (Guo 2011; Wang 1963), but not in Henan, the biggest wheat production province in China, before the present study. In July 2019, we found wheat common bunt in three fields grown with cultivar Zhengmai 618 in Yugong Mountain, Henan province. The diseased wheat heads had bunt balls filled with black powder with fishy smell. The disease incidences in these fields were 20-50%, but no common bunt was found in other nearby fields. About 200 diseased heads were sampled from the three fields. Teliospores from each head were observed under a microscope, and they all had smooth surface. Observations using a scanning electron microscope also showed smooth-surfaced teliospores. Teliospores were measured 13.5 to 18.5 μm in diameter. After surface sterilization of diseased heads using 0.25% NaClO for 5 min, teliospore suspension (1×106/ml) was made using sterilized distilled water and spread on water agar (200 μl per plate), and the plates were kept at 15°C with 24 h light (Goates and Hoffman 1987). On the 6th days, teliospores were germinated. Based on the disease symptoms, teliospore morphology, and germination, the bunt fungus was identified as T. laevis. To fulfill Koch's postulates, 1 ml of germinating teliospore suspension at the concentration of 106 spores/ml was injected into the heads of susceptible wheat cultivar (Dongxuan 3) at the boot stage with a syringe, and the plants injected with sterile ddH2O were used as control. The inoculated plants were grown in a growth chamber at 17°C with 50% humidity and 24 h light (300 μmol/m2/s). After one month at the ripening stage, the kernels of the inoculated plants were filled with black teliospores releasing fishy smell, and the control plants did not have bunt heads. Under a scanning electron microscope, teliospores from the inoculated heads had smooth surface and were measured 13.5 to 18.5 μm in diameter, similar to the teliospores of bunt heads from the fields. The fungus was also confirmed through molecular characterization using sequence characterized amplification region (SCAR) markers specific for T. laevis, and the expected 660 bp (Yao et al., 2019) and 286 bp (Zhang et al. 2012) bands were obtained separately from the teliospore samples from both the fields and growth chamber. The collection named as CGMCC 3.20112 was deposited in China General Microbiological Culture Collection Center. To the best of our knowledge, this is the first report of T. laevis causing wheat common bunt in Henan Province of China. Because the pathogen is seedborne and soilborne, the disease may become a high risk to wheat production in Henan and other provinces of China.

Soil Yeast Communities in Revegetated Post-Mining and Adjacent Native Areas in Central Brazil.

Yeasts represent an important component of the soil microbiome. In central Brazil, mining activities are among the main anthropogenic factors that influence the dynamics of the soil microbiota. Few studies have been dedicated to analysis of tropical soil yeast communities, and even fewer have focused on Brazilian hotspots influenced by mining activity. The aim of the current study was to describe soil yeast communities in a post-mining site with revegetated and native areas, along Neotropical Savanna and Atlantic Forest biomes. Yeast communities were described using a culture-based method and estimator-based species accumulation curves, and their associations with environmental characteristics were assessed using multivariate analysis. The results indicate a greater species richness for yeast communities in the revegetated area. We identified 37 species describing 86% of the estimated richness according to Chao2. Ascomycetous yeasts dominated over basidiomycetous species. Candida maltosa was the most frequent species in two phytocenoses. Red-pigmented yeasts were frequent only in the summer. The main soil attributes affecting yeast communities were texture and micronutrients. In conclusion, each phytocenosis showed a particular assemblage of species as a result of local environmental phenomena. The species richness in a Revegetated area points to a possible ecological role of yeast species in environmental recovery. This study provided the first comprehensive inventory of soil yeasts in major phytocenoses in Minas Gerais, Brazil.

Mrakia stelviica sp. nov. and Mrakia montana sp. nov., two novel basidiomycetous yeast species isolated from cold environments.

Five yeast strains were isolated from soil and sediments collected from Alps and Apennines glaciers during sampling campaigns carried out in summer 2007 and 2017, respectively. Based on morphological and physiological tests and on phylogenetic analyses reconstructed with ITS and D1/D2 sequences, the five strains were considered to belong to two related but hitherto unknown species within the genus Mrakia, in an intermediate position between Mrakia cryoconiti and Mrakia arctica. The names Mrakia stelviica (holotype DBVPG 10734T) and Mrakia montana (holotype DBVPG 10736T) are proposed for the two novel species and a detailed description of their morphological, physiological and phylogenetic features are presented. Both species fermented glucose, sucrose and trehalose, which is an uncommon feature in basidiomycetous yeasts, and showed septate hyphae with teliospore formation.

Naganishia floricola sp. nov., a novel basidiomycetous yeast species isolated from flowers of Sorbaria sorbifolia.

Two yeast strains representing a novel species in the basidiomycetous yeast genus Naganishia were isolated from flowers of Sorbaria sorbifolia collected in Beijing Olympic Forest Park, PR China. Results of multi-gene phylogenetic analysis indicated that the two strains were closely related to the type strains of Naganishia bhutanensis (CBS 6294T) and Naganishia antarctica (CBS 7687T). However, the new isolates differed from N. bhutanensis CBS 6294T by 1.79 % sequence divergence in the D1/D2 domain (11 nt substitutions and three indels), and 2.42 % (15 nt differences and one indel) to N. antarctica CBS 7687T. In the ITS region, the new isolates showed 1.15 % divergence (7 nt substitutions and one indel) to N. bhutanensis CBS 6294T and 0.92 % divergence (5 nt substitutions and no indels) to N. antarctica CBS 7687T. A phylogenetic analysis employing the sequences of six genes (D1/D2 domain of large subunit rDNA, ITS, small subunit rDNA, two subunits of the RNA polymerase II and elongation factor-1α) indicated that the novel species belonged to the genus Naganishia and formed a well-supported clade with N. bhutanensis, N. antarctica and N. indica. Moreover, the two strains differed from their closest relatives by the ability to grow on distinct carbon and nitrogen sources and ability to grow at 30 °C. On the basis of these findings, we propose a novel species in the genus Naganishia (Filobasidiales), Naganishia floricola sp. nov. (holotype CGMCC 2.5856).

Comparative mitogenome analysis of two ectomycorrhizal fungi (Paxillus) reveals gene rearrangement, intron dynamics, and phylogeny of basidiomycetes

In this study, the mitogenomes of two Paxillus species were assembled, annotated and compared. The two mitogenomes of Paxillus involutus and P. rubicundulus comprised circular DNA molecules, with the size of 39,109 bp and 41,061 bp, respectively. Evolutionary analysis revealed that the nad4L gene had undergone strong positive selection in the two Paxillus species. In addition, 10.64 and 36.50% of the repetitive sequences were detected in the mitogenomes of P. involutus and P. rubicundulus, respectively, which might transfer between mitochondrial and nuclear genomes. Large-scale gene rearrangements and frequent intron gain/loss events were detected in 61 basidiomycete species, which revealed large variations in mitochondrial organization and size in Basidiomycota. In addition, the insertion sites of the basidiomycete introns were found to have a base preference. Phylogenetic analysis of the combined mitochondrial gene set gave identical and well-supported tree topologies, indicating that mitochondrial genes were reliable molecular markers for analyzing the phylogenetic relationships of Basidiomycota. This study is the first report on the mitogenomes of Paxillus, which will promote a better understanding of their contrasted ecological strategies, molecular evolution and phylogeny of these important ectomycorrhizal fungi and related basidiomycete species.

Methods and Difficulties of Identifying Species in Studies on the Ecology and Distribution Patterns of Spore Organisms

The use of molecular biology methods for identifying species of fungi and myxomycetes (DNA barcoding) has shown the necessity of reconsidering our views on the ecology and habitats of many species of spore organisms. Spores of basidiomycetes and myxomycetes can spread over considerable distances by water, wind, and insects, resulting in their resettlement to various distant habitats, where the only limiting factors are microenvironment and suitable substrates. The possibility of inhabiting various “island” habitats, for example, such as large tree debris in steppe regions or special microclimatic conditions on the bottoms of ravines and gullies in the forest steppe, where the humidity is much higher than on the plain areas, allows spore organisms to expand their habitats and settle in natural areas that are not typical for them. Our original research, an analysis of literature sources, and GenBank data have shown that, when studying the ecology and distribution patterns of spore-like organisms, issues of correctly identifying species are quiet questionable. This article discusses examples using the influence of the hypothesis Everything is everywhere, but, the environment selects (EiE hypothesis) on understanding the ecology and ranges of some species of myxomycetes and basidiomycetes. Data on the distribution and ecology of nivicolous myxomycetes Lamproderma pseudomaculatum on the plain territory of Western Siberia are presented for the first time. Using the myxomycetes Arcyria imperialis and A. stipata, as well as fungi Disciseda hyalothrix and Pleurotus pulmonarius, we envisage the possibility of a DNA barcoding application for studies of the species diversity of spore organisms and the morphological differentiation of similar species and for correctly interpreting their ranges and ecology. rDNA sequences have been studied for all these species; for myxomycetes, photos have been taken on a scanning electron microscope.

Critical Factors Involved in Primordia Building in Agaricus bisporus: A Review.

The button mushroom Agaricus bisporus is an economically important crop worldwide. Many aspects of its cultivation are well known, except for the precise biological triggers for its fructification. By and large, for most basidiomycete species, nutrient availability, light and a drop in temperature are critical factors for fructification. A. bisporus deviates from this pattern in the sense that it does not require light for fructification. Furthermore its fructification seems to be inhibited by a self-generated factor which needs to be removed by microorganisms in order to initiate fruiting. This review explores what is known about the morphogenesis of fruiting initiation in A. bisporus, the microflora, the self-inhibitors for fruiting initiation and transcription factors involved. This information is subsequently contrasted with an overall model of the regulatory system involved in the initiation of the formation of primordia in basidiomycetes. The comparison reveals a number of the blank spots in our understanding of the fruiting process in A. bisporus.

List of mushrooms found in Dhikura village and its adjoining Rotepakho community forest in Arghakhanchi district, Central Nepal

The study was carried out for higher fungi, especially mushrooms, found in Dhikura village and its adjoining Rotepakho community forest, Arghakhanchi district, Central Nepal from October 25 to 26, 2014. A total of 33 species, including both Ascomycetes (3 species) and Basidiomycetes (30 species) fungi were collected from the study area. The documented Ascomycetes species were from three orders belonging to three families and three genera, whereas Basidiomycetes species were from eight orders belonging to 17 families and 26 genera. Polyporales were found to be the dominant order in the study area, with 11 species followed by Agaricales (6 species) and Boletales, Hymenochaetales, and Russullales (3 species). Similarly, Polyporaceae was found to be the dominant family represented by nine species, Hymenochaetaceae (3 species), and followed equally by Exobasidiaceae, Sclerodermataceae, and Steriaceae (2 species). Litsea monopetalous was found to be the primary host plant for three different mushroom species (including one Ascomycetes and two Basidiomycetes species) followed by Shorea robusta, Grewia asiatica, Mangifera indica, Machillus odoratissima, Terminalia bellirica, Wedlandia coriacea and Terminalia chebula(2 Basidiomycetes species).

Ethanol extract of Pycnoporus sanguineus relieves the dextran sulfate sodium-induced experimental colitis by suppressing helper T cell-mediated inflammation via apoptosis induction

Inflammatory bowel disease (IBD) is a chronic relapsing inflammation involving the gut system, and disequilibrium of T helper (Th) cell paradigm has been recognized as critical pathogenesis. Pycnoporus sanguineus (L.) Murrill is a species of the white-rot basidiomycetes listed as food- and cosmetic-grade microorganisms. In this study, anti-inflammatory activity of the ethanol extract from P. sanguineus (PSE) was investigated in dextran sulfate sodium (DSS)-induced experimental colitis model. PSE recovered the DSS-caused weight loss, reversed the colon shortening, and ameliorated the histopathological lesion in colon, resulting in lower disease activity index (DAI). Levels of serumal lipopolysaccharide (LPS), colonic myeloperoxidase (MPO) in the colitis-suffering mice were declined by PSE treatment. PSE also improved the mucosal integrity by enhancing the expression of tight junction and adherens junction proteins in the colon, including ZO-1, occludin, claudin-1, and E-cadherin. Besides, PSE reduced helper T cells (Th) in the colon, together with an evident decrease of several Th cell-related cytokines. Moreover, it was found that in vitro, PSE suppressed T cells and the Th subset upon Concanavalin A (ConA)-stimulation by inducing apoptosis. In summary, PSE displayed a remission on the colitis-related inflammation, which would possibly rely on the epithelial barrier restoration by suppressing Th cells via apoptosis induction, highlighting a promising potential in the treatment for IBD.

Corn stover induces extracellular laccase activity in Didymosphaeria sp. (syn. = Paraconiothyrium sp.) and exhibits increased in vitro ruminal digestibility when treated with this fungal species.

Fungi can improve stover digestibility due to their ability to secrete oxidative enzymes that depolymerize lignin, allowing the rumen microorganisms to access the polysaccharides of the plant cell wall. Some ascomycetes have shown good delignification capability; however, they have been scarcely evaluated for their ability to improve corn stover (CS) ruminal digestibility. We evaluated the laccase induction by CS of the CMU-196 strain of the ascomycete fungus Didymosphaeria sp. (syn. = Paraconiothyrium sp.). Also, we analyzed the capacity of such strain to modify the cell wall of CS and to improve its digestion by the ruminal microbiota. The CMU-196 strain showed a maximum extracellular laccase activity of 39.74 ± 0.24U/L when an aqueous stover extract (SE, 10% v/v) was added to the growth medium. The addition of ground stover (GS, 2% w/v) increased the activity to a maximum of 262.27 ± 0.58U/L. In solid-state fermentation (SSF) assays of GS, the strain degrades cell walls, destabilizing the vessels and tracheids of plant biomass; the protein content reaches a maximum of 33.2g/kg dry matter (DM) at 70days, while the crude fiber content shows the highest level of 314g/kg DM at 14days. SSF treatment of the CS increased the in vitro ruminal production of gas in a fraction that was considered nondigestible at 18h, and gas production increased by 14% with respect to the untreated GS at 14days. The CMU-196 strain can digest the plant cell wall and improve ruminal CS digestibility at a level equivalent to several basidiomycete species.

Vib-PT, an Aromatic Prenyltransferase Involved in the Biosynthesis of Vibralactone from Stereum vibrans.

Vibralactone, a hybrid compound derived from phenols and a prenyl group, is a strong pancreatic lipase inhibitor with a rare fused bicyclic β-lactone skeleton. Recently, a researcher reported a vibralactone derivative (compound C1) that caused inhibition of pancreatic lipase with a half-maximal inhibitory concentration of 14 nM determined by structure-based optimization, suggesting a potential candidate as a new antiobesity treatment. In the present study, we sought to identify the main gene encoding prenyltransferase in Stereum vibrans, which is responsible for the prenylation of phenol leading to vibralactone synthesis. Two RNA silencing transformants of the identified gene (vib-PT) were obtained through Agrobacterium tumefaciens-mediated transformation. Compared to wild-type strains, the transformants showed a decrease in vib-PT expression ranging from 11.0 to 56.0% at 5, 10, and 15 days in reverse transcription-quantitative PCR analysis, along with a reduction in primary vibralactone production of 37 to 64% at 15 and 21 days, respectively, as determined using ultra-high-performance liquid chromatography-mass spectrometry analysis. A soluble and enzymatically active fusion Vib-PT protein was obtained by expressing vib-PT in Escherichia coli, and the enzyme's optimal reaction conditions and catalytic efficiency (Km /kcat) were determined. In vitro experiments established that Vib-PT catalyzed the C-prenylation at C-3 of 4-hydroxy-benzaldehyde and the O-prenylation at the 4-hydroxy of 4-hydroxy-benzenemethanol in the presence of dimethylallyl diphosphate. Moreover, Vib-PT shows promiscuity toward aromatic compounds and prenyl donors.IMPORTANCE Vibralactone is a lead compound with a novel skeleton structure that shows strong inhibitory activity against pancreatic lipase. Vibralactone is not encoded by the genome directly but rather is synthesized from phenol, followed by prenylation and other enzyme reactions. Here, we used an RNA silencing approach to identify and characterize a prenyltransferase in a basidiomycete species that is responsible for the synthesis of vibralactone. The identified gene, vib-PT, was expressed in Escherichia coli to obtain a soluble and enzymatically active fusion Vib-PT protein. In vitro characterization of the enzyme demonstrated the catalytic mechanism of prenylation and broad substrate range for different aromatic acceptors and prenyl donors. These characteristics highlight the possibility of Vib-PT to generate prenylated derivatives of aromatics and other compounds as improved bioactive agents or potential prodrugs.

Obesity preventive function of novel edible mushroom, Basidiomycetes-X (Echigoshirayukidake): Manipulations of insulin resistance and lipid metabolism

sEchigoshirayukidake is an edible mushroom found in Uonuma, Japan in 1994. It was assigned to a new species of Basidiomycetes (BDM-X) but is uniquely defect of forming bashidium. The high antioxidant activity and β-glucan content of BDM-X suggest possible functions preventing type 2 diabetes. In the present study, anti-obesity and insulin resistance preventive functions of BDM-X were examined using genetically defined obese model rat, OLETF (Otsuka Long Evans Tokushima Fatty) by feeding regular diet with and without supplementation of 5% dried BDM-X powder (BDMP) for 15 weeks. BDMP supplementation to the diet significantly (p < 0.01) suppressed the body weight gain and also visceral fat accumulation during the feeding period compared to control diet. Simultaneously, the insulin resistance and the plasma levels of adiponectin and triglycerides were significantly (p = 0.003) ameliorated in the BDMP supplemented diet group. A statistical multivariate analysis showed the weight of three types of adipose tissue (epididymal, retroperirenal, and mesenteric fat) positively correlated with HOMA-IR (Homeostasis Model Assessment of Insulin Resistance), and negatively correlated with plasma adiponectin. These results indicate BDM-X is a new resource applicable to the functional foods or the complementary biomedicines to prevent metabolic syndromes leading to type 2 diabetes.

XYLOTROPHIC BASIDIOMYCETES OF SMALL-LEAVED FORESTS OF THE KOSTROMA REGION

This paper provides an overview of the biology and species diversity of xylotrophic basidiomycetes, as well as the biotopic relationships of xylotroph mycobiota with phytocenoses. Ecological groups of these organisms are considered in connection with substrate specialization and their species similarity in birch and aspen small-leaved forests of different pharmacy. A brief assessment of the influence of the anthropogenic factor on the biological diversity of the represented group of organisms is given. For the first time, the Kostroma region is divided into southern, central, and northern parts to assess species diversity depending on weather, climate, and phytocenotic factors. The data on the study of changes in the degree of integration of mycocenoses of birch and aspen forests within the studied areas of the region are presented. In total, over 60 species of xylotrophic basidiomycetes were found in the small-leaved forests in the region, among which 38 species primarily affected birch forests and 40 species of pathogenic xylotrophic basidiomycetes have tropism for P. tremula. It was established that species belonging to the Coriolaseae family predominate in the species composition of wood-destroying mycobiota; they comprise about 27% of the total species composition in birch forests and about 24% of the species composition in aspen forests.

Fluorescent protein expression in the ectomycorrhizal fungus Laccaria bicolor: a plasmid toolkit for easy use of fluorescent markers in basidiomycetes.

For long time, studies on ectomycorrhiza (ECM) have been limited by inefficient expression of fluorescent proteins (FPs) in the fungal partner. To convert this situation, we have evaluated the basic requirements of FP expression in the model ECM homobasidiomycete Laccaria bicolor and established eGFP and mCherry as functional FP markers. Comparison of intron-containing and intronless FP-expression cassettes confirmed that intron-processing is indispensable for efficient FP expression in Laccaria. Nuclear FP localization was obtained via in-frame fusion of FPs between the intron-containing genomic gene sequences of Laccaria histone H2B, while cytosolic FP expression was produced by incorporating the intron-containing 5' fragment of the glyceraldehyde-3-phosphate dehydrogenase encoding gene. In addition, we have characterized the consensus Kozak sequence of strongly expressed genes in Laccaria and demonstrated its boosting effect on transgene mRNA accumulation. Based on these results, an Agrobacterium-mediated transformation compatible plasmid set was designed for easy use of FPs in Laccaria. The four cloning plasmids presented here allow fast and highly flexible construction of C-terminal in-frame fusions between the sequences of interest and the two FPs, expressed either from the endogenous gene promoter, allowing thus evaluation of the native regulation modes of the gene under study, or alternatively, from the constitutive Agaricus bisporus gpdII promoter for enhanced cellular protein localization assays. The molecular tools described here for cell-biological studies in Laccaria can also be exploited in studies of other biotrophic or saprotrophic basidiomycete species susceptible to genetic transformation.

Diversity and phylogeny of basidiomycetous yeasts from plant leaves and soil: Proposal of two new orders, three new families, eight new genera and one hundred and seven new species.

Nearly 500 basidiomycetous yeast species were accepted in the latest edition of The Yeasts: A Taxonomic Study published in 2011. However, this number presents only the tip of the iceberg of yeast species diversity in nature. Possibly more than 99 % of yeast species, as is true for many groups of fungi, are yet unknown and await discovery. Over the past two decades nearly 200 unidentified isolates were obtained during a series of environmental surveys of yeasts in phyllosphere and soils, mainly from China. Among these isolates, 107 new species were identified based on the phylogenetic analyses of nuclear ribosomal DNA (rDNA) [D1/D2 domains of the large subunit (LSU), the small subunit (SSU), and the internal transcribed spacer region including the 5.8S rDNA (ITS)] and protein-coding genes [both subunits of DNA polymerase II (RPB1 and RPB2), the translation elongation factor 1-α (TEF1) and the mitochondrial gene cytochrome b (CYTB)], and physiological comparisons. Forty-six of these belong to 16 genera in the Tremellomycetes (Agaricomycotina). The other 61 are distributed in 26 genera in the Pucciniomycotina. Here we circumscribe eight new genera, three new families and two new orders based on the multi-locus phylogenetic analyses combined with the clustering optimisation analysis and the predicted similarity thresholds for yeasts and filamentous fungal delimitation at genus and higher ranks. Additionally, as a result of these analyses, three new combinations are proposed and 66 taxa are validated.

Aerosol measurement methods to quantify spore emissions from fungi and cryptogamic covers in the Amazon

Abstract. Bioaerosols are considered to play a relevant role in atmospheric processes, but their sources, properties, and spatiotemporal distribution in the atmosphere are not yet well characterized. In the Amazon Basin, primary biological aerosol particles (PBAPs) account for a large fraction of coarse particulate matter, and fungal spores are among the most abundant PBAPs in this area as well as in other vegetated continental regions. Furthermore, PBAPs could also be important ice nuclei in Amazonia. Measurement data on the release of fungal spores under natural conditions, however, are sparse. Here we present an experimental approach to analyze and quantify the spore release from fungi and other spore-producing organisms under natural and laboratory conditions. For measurements under natural conditions, the samples were kept in their natural environment and a setup was developed to estimate the spore release numbers and sizes as well as the microclimatic factors temperature and air humidity in parallel to the mesoclimatic parameters net radiation, rain, and fog occurrence. For experiments in the laboratory, we developed a cuvette to assess the particle size and number of newly released fungal spores under controlled conditions, simultaneously measuring temperature and relative humidity inside the cuvette. Both approaches were combined with bioaerosol sampling techniques to characterize the released particles using microscopic methods. For fruiting bodies of the basidiomycetous species, Rigidoporus microporus, the model species for which these techniques were tested, the highest frequency of spore release occurred in the range from 62 % to 96 % relative humidity. The results obtained for this model species reveal characteristic spore release patterns linked to environmental or experimental conditions, indicating that the moisture status of the sample may be a regulating factor, whereas temperature and light seem to play a minor role for this species. The presented approach enables systematic studies aimed at the quantification and validation of spore emission rates and inventories, which can be applied to a regional mapping of cryptogamic organisms under given environmental conditions.

UvCom1 Is an Important Regulator Required for Development and Infection in the Rice False Smut Fungus Ustilaginoidea virens.

Ustilaginoidea virens is an economically important biotrophic fungal pathogen that causes rice false smut worldwide. However, the regulatory mechanisms of smut ball formation under U. virens infection remain unclear. Here, we identified an important transcription factor, UvCom1, from this pathogen, which regulates the formation of smut balls on rice spikelets. UvCom1 contains two conserved internal repeat 1 (RPT) domains and is found only in fungi, with specific conservation in species of Basidiomycetes and Ascomycetes. UvCom1 protein N- or C-terminal fusion vectors both showed transactivation activity in yeast. Deletion of UvCom1 significantly affected the vegetative growth and conidiation of U. virens. UvCom1 negatively regulated the responses to oxidative, osmotic, and cell wall stresses. Remarkably, UvCom1 was found to be essential for the formation of rice smut balls, and UvCom1 deletion mutants lost the ability to stably utilize nutrients from the rice host. UvCom1 was also highly expressed at the mycelial expansion stage. Transcriptomic analysis and quantitative real-time PCR revealed that UvCom1 could affect the expression of genes significantly enriched in transmembrane transport. This study demonstrates that UvCom1 is a key transcription factor governing smut ball formation of this biotrophic fungus.