Structural, physical characteristics and biological activities assessment of scleroglucan from a local strain Athelia rolfsii TEMG.

Athelia rolfsii TEMG (MH 236106) an exopolysaccharide (EPS) producing fungal strain was isolated and identified. Extraction, purification, characterization, antimicrobial, antioxidant, antiviral and antitumor activities of the polysaccharide were investigated. It characterized as a homopolysaccharide of glucose with a molecular weight of 345.622 kDa. The identification of the polysaccharide was conducted using scanning electron microscopy, energy dispersive X-ray analysis, 1H and 13C NMR spectra. The existence of ß-1,3 and ß-1,6 linkages suggests that EPS could be scleroglucan. The purified scleroglucan showed considerable antibacterial and antioxidant activities. The results indicated that, there was no cytotoxicity on normal cell (W138) and no effect on tumor cell lines including HepG2 and PC3 showing IC50 of 5096.83, 5885.80 and 4803.90 µg/mL, respectively. The results showed also that Sclg could reduce the cytopathic effect by 50% (EC50) at 15 and 50 µg/mL of herpes simplex virus type-1 (HSV-1) and influenza virus (H5N1), respectively.

Phylogeny of the genus Fuscoporia and taxonomic assessment of the F. contigua group.

The genus Fuscoporia of the Hymenochaetaceae is characterized by resupinate to pileate basidiocarps, a dimitic hyphal system with fine crystal aggregates and encrusted generative hyphae in dissepiment edge and tube trama, the presence of hymenial setae, and hyaline, thin-walled, smooth basidiospores. Members of the F. contigua group are easy to distinguish from other species of Fuscoporia because of the moderately large pores, presence of mycelial setae, and large hymenial setae. Here, we explore phylogenetic relationships among 20 species of Fuscoporia based on examination of some 90 collections sampled worldwide. Seven new species are recognized in the F. contigua group-F. americana, F. centroamericana, F. costaricana, F. latispora, F. monticola, F. septiseta, and F. sinica-described from China, Costa Rica, Mexico, and the United States. Phylogenetic analyses inferred from DNA sequences of the nuc rDNA internal transcribed spacer (ITS1-5.8S-ITS2 = ITS), D1-D2 domains of nuc 28S rDNA (28S), and translation elongation factor EF-1 alpha (tef1) support the F. contigua group as one of two major clades within Fuscoporia comprising nine species worldwide.

Assessment of white rot fungus mediated hardwood degradation by FTIR spectroscopy and multivariate analysis.

Evaluating the biomass degradation using fast, validate and sensitive techniques for exploratory purposes of biofuel production has been developed since last decade. Thus, we assessed the degradation of two Indian hardwoods using FTIR and chemometric approaches. Two white rot fungi, namely Pseudolagarobasidium acaciicola AGST3 and Tricholoma giganteum AGDR1, were selected among twenty-one fungal isolates for higher hardwood degradation. In the screening, P. acaciicola AGST3 and T. giganteum AGDR1 depicted the dry woody mass loss of 20.51% and 22.38%, respectively. Cellulose crystallinity of P. acaciicola AGST3 treated hardwoods was 4-fold lower than untreated hardwoods, showing the higher cellulose degradation efficiency. P. acaciicola AGST3 treated samples exhibited maximum deviation of guaiacyl units of lignin, cellulose and hemicelluloses. T. giganteum AGDR1 treated hardwoods showed maximum deviation of guaiacyl- and syringyl- units of lignin and hemicelluloses. Multivariate approach revealed the degradation patterns and preferences are varied based on the fungi and hardwood. The approach used in the present study can certainly distinguish the variations among the different biomass samples that having similar composition. Additionally, higher lignin degradability of these fungi can be used in biomass pretreatment, which significantly utilized to produce second-generation biofuels.

Phylogeny, biogeography and taxonomic re-assessment of Multifurca (Russulaceae, Russulales) using three-locus data.

Multifurca is a small genus newly established to accommodate lactarioid and russuloid species with some characters reminiscent of corticoid members of Russulaceae. It shows an amphi-pacific distribution with strong preference for the tropical zone of the Northern Hemisphere and thus has particular significance for biogeographical study. Using worldwide samples and three loci (ITS, 28S rDNA and rpb2), we demonstrated that Multifurca is split into two highly supported major clades that are here recognized at the subgeneric level: subg. Furcata subg. nov. exclusively includes lactarioid species, while subg. Multifurca includes species with a russuloid habit. Using phylogenetic species recognition and comparison of genetic distances we recognize five new and six previously described species, almost double the known number of species before this study. Molecular dating using a Bayesian method suggested that Multifurca originated in early Paleocene and diversified in the Eocene. The most recent interspecific divergences occurred both in Asia and America, roughly at the same time around the Pliocene. Ancestral area reconstruction and comparisons of genetic distances and morphology suggested an early divergence within Australasia or tropical Asia. From the early Miocene to Pliocene, multiple dispersals/migrations to Australasia and North America by island hopping or land bridge likely happened. Vicariance at the late Tertiary might be the most likely mechanism accounting for the eastern Asia-southeastern North America and Australasia-tropical Asia disjunct distributions. The shared polymorphisms in the ITS alignment, numerous degenerated base pairs in the rpb2 sequences and weak conflict between the ITS and LSU genealogies of M. subg. Furcata suggest recent speciation. Host specificity of Multifurca species or species pairs is relatively low. Host shifts are believed to have aided establishment in new territories during the dispersals and migrations.

Study on Lipid Accumulation in Novel Oleaginous Yeast Naganishia liquefaciens NITTS2 Utilizing Pre-digested Municipal Waste Activated Sludge: a Low-cost Feedstock for Biodiesel Production.

The economical production of lipids is considered as an appropriate renewable alternative feedstock for biodiesel production because of the contemporary concerns on fuel crisis, climate change and food security. In this study, lipid accumulation potential of a novel oleaginous yeast isolate Naganishia liquefaciens NITTS2 by utilizing pre-digested municipal waste activated sludge (PWAS) was explored. Optimization of culture conditions was performed using response surface methodology coupled with genetic algorithm and maximum lipid content of 55.7% was obtained. The presence of lipid was visually confirmed by fluorescence microscopy and its characteristic profile was determined by GC-MS. The yeast lipid was recovered and converted into biodiesel by garbage lipase with the efficiency of 88.34 ± 1.2%, which was further analyzed by proton nuclear magnetic resonance spectroscopy. Hence, the results of this study strongly suggest the possibility of using PWAS as an efficient and low-cost resource for the production of biodiesel from the oleaginous yeast.

The discovery of novel LPMO families with a new Hidden Markov model.

BACKGROUND: Renewable biopolymers, such as cellulose, starch and chitin are highly resistance to enzymatic degradation. Therefore, there is a need to upgrade current degradation processes by including novel enzymes. Lytic polysaccharide mono-oxygenases (LPMOs) can disrupt recalcitrant biopolymers, thereby enhancing hydrolysis by conventional enzymes. However, novel LPMO families are difficult to identify using existing methods. Therefore, we developed a novel profile Hidden Markov model (HMM) and used it to mine genomes of ascomycetous fungi for novel LPMOs. RESULTS: We constructed a structural alignment and verified that the alignment was correct. In the alignment we identified several known conserved features, such as the histidine brace and the N/Q/E-X-F/Y motif and previously unidentified conserved proline and glycine residues. These residues are distal from the active site, suggesting a role in structure rather than activity. The multiple protein alignment was subsequently used to build a profile Hidden Markov model. This model was initially tested on manually curated datasets and proved to be both sensitive (no false negatives) and specific (no false positives). In some of the genomes analyzed we identified a yet unknown LPMO family. This new family is mostly confined to the phyla of Ascomycota and Basidiomycota and the class of Oomycota. Genomic clustering indicated that at least some members might be involved in the degradation of ß-glucans, while transcriptomic data suggested that others are possibly involved in the degradation of pectin. CONCLUSIONS: The newly developed profile hidden Markov Model was successfully used to mine fungal genomes for a novel family of LPMOs. However, the model is not limited to bacterial and fungal genomes. This is illustrated by the fact that the model was also able to identify another new LPMO family in Drosophila melanogaster. Furthermore, the Hidden Markov model was used to verify the more distant blast hits from the new fungal family of LPMOs, which belong to the Bivalves, Stony corals and Sea anemones. So this Hidden Markov model (Additional file 3) will help the broader scientific community in identifying other yet unknown LPMOs.

An assessment of ectomycorrhizal fungal communities in Tasmanian temperate high-altitude Eucalyptus delegatensis forest reveals a dominance of the Cortinariaceae.

Fungal diversity of Australian eucalypt forests remains underexplored. We investigated the ectomycorrhizal (EcM) fungal community characteristics of declining temperate eucalypt forests in Tasmania. Within this context, we explored the diversity of EcM fungi of two forest types in the northern highlands in the east and west of the island. We hypothesised that EcM fungal community richness and composition would differ between forest type but that the Cortinariaceae would be the dominant family irrespective of forest type. We proposed that EcM richness would be greater in the wet sclerophyll forest than the dry sclerophyll forest type. Using both sporocarps and EcM fungi from root tips amplified by PCR and sequenced in the rDNA ITS region, 175 EcM operational taxonomic units were identified of which 97 belonged to the Cortinariaceae. The Cortinariaceae were the most diverse family, in both the above and below ground communities. Three distinct fungal assemblages occurred within the wet and dry sclerophyll forest types and two geographic regions that were studied, although this pattern did not remain when only the root tip data were analysed. EcM sporocarp richness was unusually higher than root tip richness and EcM richness did not significantly differ among forest types. The results are discussed in relation to the importance of the Cortinariaceae and the drivers of EcM fungal community composition within these forests.

Phylogenetic assessment of global Suillus ITS sequences supports morphologically defined species and reveals synonymous and undescribed taxa.

The genus Suillus represents one of the most recognizable groups of mushrooms in conifer forests throughout the Northern Hemisphere. Although for decades the genus has been relatively well defined morphologically, previous molecular phylogenetic assessments have provided important yet preliminary insights into its evolutionary history. We present the first large-scale phylogenetic study of the boundaries of each species in the genus Suillus based on the most current internal transcribed spacer (ITS) barcode sequences available inPUBLIC databases, as well as sequencing of 224 vouchered specimens and cultures, 15 of which were type specimens from North America. We found that species boundaries delimited by morphological data are broadly congruent with those based on ITS sequences. However, some species appear to have been described several times under different names, several species groups cannot be resolved by ITS sequences alone, and undescribed taxa are apparent, especially in Asia. Therefore, we elevated S. tomentosus var. discolor to S. discolor; proposed synonymies of S. neoalbidipes with S. glandulosipes, S. borealis with S. brunnescens, Boletus serotinus and B. solidipes with Suillus elbensis, S. lactifluus with S. granulatus, S. himalayensis with S. americanus; and proposed usage of the names S. clintonianus in the place of the North American S. grevillei, S. weaverae for North American S. granulatus, S. ampliporus in the place of the North American S. cavipes, and S. elbensis in place of the North American S. viscidus. We showed that the majority of Suillus species have strong affinities for particular host genera. Although deep node support was low, geographic differentiation was apparent, with species from North America, Eurasia, and Asia often forming their own clades. Collectively, this comprehensive genus-level phylogenetic integration of currently available Suillus ITS molecular data and metadata will aid future taxonomic and ecological work on an important group of ectomycorrhizal fungi.

Molecular inference, multivariate morphometrics and ecological assessment are applied in concert to delimit species in the Russula clavipes complex.

Species of Russula subsect. Xerampelinae are notoriously difficult to identify and name and have not been subject to molecular study. A group of species, referred to here as the R. clavipes complex, growing in association with Salix, Betula and Populus as well as coniferous tree species from temperate to arctic and alpine habitats, were examined. Analyses of the nuc rDNA internal transcribed spacer (ITS) region and a numerical analysis of morphological characters were used. The R. clavipes complex is a monophyletic group within Russula subsect. Xerampelinae, according to molecular results. The complex includes three species: R. nuoljae is a phylogenetically and morphologically well-supported species while the other two, R. clavipes and R. pascua, are similar based on ITS data and morphology but separate based on their ecology. Russula pseudoolivascens is conspecific with R. clavipes Several combinations of characters traditionally used in the taxonomy of R. subsect. Xerampelinae are inappropriate for species delimitation in this group and the adequacy of the ITS for species identification in this group is discussed. Detailed microscopic observations on the type collection of R. nuoljae are presented and illustrated, along with a key to the European members of R. subsect. Xerampelinae.

Assessment of ectomycorrhizal biodiversity in Tuber macrosporum productive sites.

Tuber macrosporum Vittad. is a truffle with superb organoleptic properties, whose cultivation is still in its infancy. For the first time we have aimed to provide information on ectomycorrhizal communities in natural and cultivated T. macrosporum sites. Ectomycorrhizal morphotypes were identified using ITS nrDNA sequencing and sorted into molecular operational taxonomic unit (MOTU). We detected 16 MOTUs in the T. macrosporum cultivated plantation. Ascomycota were the most abundant (86.4%) with Helvellaceae, Pyronemataceae and Pezizaceae the most common. Twenty-two MOTUs were collected in the natural T. macrosporum site. Basidiomycota morphotypes were plentiful (70.6%) and Thelephoraceae dominated. Each site had different taxa belowground with only T. macrosporum in common, being more abundant in the natural (18.2%) than in the cultivated (14.4%) site. Species richness, Simpson and Shannon diversity indices, taxonomic diversity, distinctness and variation of taxonomic distinctness were lower in the cultivated than in the natural site.

Critical assessment of the Lactarius gerardii species complex (Russulales).

This paper investigates species delimitation within the Lactarius gerardii species complex and explores its taxonomic and geographical extent. A combined molecular phylogeny based on ITS, LSU and rpb2 gene sequences is constructed and morphological characters are evaluated. While L. gerardii was originally described from North America, it has later been reported from all over Asia. Therefore a worldwide sampling range was aimed at, including species exhibiting morphological affinities with L. gerardii. The phylogenetic analyses indicate that intercontinental conspecificity in L. gerardii is absent. Thirty strongly supported clades are retrieved of which 18 are morphologically identifiable species. The group is elevated to Lactarius subg. Gerardii stat. nov. It includes, apart from L. gerardii s.l., L. atrovelutinus, L. bicolor, L. ochrogalactus, L. petersenii, L. reticulatovenosus, L. sepiaceus, L. subgerardii and L. wirrabara, as well as the pleurotoid L. uyedae. The paraphyletic nature of the genus Lactarius is confirmed. Lactarius subg. Gerardii appears not affiliated with L. subg. Plinthogalus and this can be substantiated morphologically. No representatives are known from Europe, Africa or South America. The high frequency of intercontinental sister relationships observed between America, Asia and the Australian region, suggests multiple migration and speciation events have occurred across continents.

Assessment of soil fungal communities using pyrosequencing.

Pyrosequencing, a non-electrophoretic method of DNA sequencing, was used to investigate the extensive fungal community in soils of three islands in the Yellow Sea of Korea, between Korea and China. Pyrosequencing was carried out on amplicons derived from the 5' region of 18S rDNA. A total of 10,166 reads were obtained, with an average length of 103 bp. The maximum number of fungal phylotypes in soil predicted at 99% similarity was 3,334. The maximum numbers of phylotypes predicted at 97% and 95% similarities were 736 and 286, respectively. Through phylogenetic assignment using BLASTN, a total of 372 tentative taxa were identified. The majority of true fungal sequences recovered in this study belonged to the Ascomycota (182 tentative taxa in 2,708 reads) and Basidiomycota (172 tentative taxa in 6,837 reads). The predominant species of Ascomycota detected have been described as lichen-forming fungi, litter/wood decomposers, plant parasites, endophytes, and saprotrophs: Peltigera neopolydactyla (Lecanoromycetes), Paecilomyces sp. (Sordariomycetes), Phacopsis huuskonenii (Lecanoromycetes), and Raffaelea hennebertii (mitosporicAscomycota). The majority of sequences in the Basidiomycota matched ectomycorrhizal and wood rotting fungi, including species of the Agaricales and Aphyllophorales, respectively. A high number of sequences in the Thelephorales, Boletales, Stereales, Hymenochaetales, and Ceratobasidiomycetes were also detected. By applying high-throughput pyrosequencing, we observed a high diversity of soil fungi and found evidence that pyrosequencing is a reliable technique for investigating fungal communities in soils.

Molecular phylogenetic biodiversity assessment of arctic and boreal ectomycorrhizal Lactarius Pers. (Russulales; Basidiomycota) in Alaska, based on soil and sporocarp DNA.

Despite the critical roles fungi play in the functioning of ecosystems, especially as symbionts of plants and recyclers of organic matter, their biodiversity is poorly known in high-latitude regions. In this paper, we discuss the molecular diversity of one of the most diverse and abundant groups of ectomycorrhizal fungi: the genus Lactarius Pers. We analysed internal transcribed spacer rDNA sequences from both curated sporocarp collections and soil polymerase chain reaction clone libraries sampled in the arctic tundra and boreal forests of Alaska. Our genetic diversity assessment, based on various phylogenetic methods and operational taxonomic unit (OTU) delimitations, suggests that the genus Lactarius is diverse in Alaska, with at least 43 putative phylogroups, and 24 and 38 distinct OTUs based on 95% and 97% internal transcribed spacer sequence similarity, respectively. Some OTUs were identified to known species, while others were novel, previously unsequenced groups. Non-asymptotic species accumulation curves, the disparity between observed and estimated richness, and the high number of singleton OTUs indicated that many Lactarius species remain to be found and identified in Alaska. Many Lactarius taxa show strong habitat preference to one of the three major vegetation types in the sampled regions (arctic tundra, black spruce forests, and mixed birch-aspen-white spruce forests), as supported by statistical tests of UniFrac distances and principal coordinates analyses (PCoA). Together, our data robustly demonstrate great diversity and nonrandom ecological partitioning in an important boreal ectomycorrhizal genus within a relatively small geographical region. The observed diversity of Lactarius was much higher in either type of boreal forest than in the arctic tundra, supporting the widely recognized pattern of decreasing species richness with increasing latitude.

Assessment of inter- and intra-specific variability in the main species of Boletus edulis complex by ITS analysis.

The Boletus edulis species complex includes ectomycorrhizal fungi producing edible mushrooms appreciated worldwide. However, species delineation is very difficult in these fungi, because it is based exclusively on a few, highly variable morphological features. As a consequence, a high number of taxa--including several varieties, subspecies and/or species sensu stricto--have been described in this species complex. In this paper we report on an extensive analysis of internal transcribed spacer of the nuclear rDNA region on a large sample of species of the B. edulis complex, mainly harvested in Italy, and representative of the European variability of this group. The molecular analysis allowed us to discriminate among and within B. edulis, B. aestivalis, B. pinophilus and B. aereus spp. and resolve their phylogenetic relationship.