Polyphasic characterisation of three novel species of <Emphasis Type="Italic">Paraboeremia</Emphasis>

Paraboeremia was recently introduced for a distinct lineage in the family Didymellaceae. Currently, three species are included, i.e. P. adianticola, P. putaminum and P. selaginellae, all of which are plant pathogens. Paraboeremia is morphologically similar to Phoma but phylogenetically distinct. In this paper, three new species, i.e. Paraboeremia camelliae isolated from Camellia sp., P. litseae from Litsea sp., and P. oligotrophica from cave limestone, are described, illustrated and compared with closely related taxa. Phylogenetic analysis based on the multi-locus sequences of the internal transcribed spacer regions 1 and 2 and 5.8S nuclear ribosomal RNA gene (ITS), partial large subunit 28S nrDNA region (LSU), partial β-tubulin (TUB2) gene and RNA polymerase II (RPB2) gene regions confirmed the distinction of these species in Paraboeremia. These three new species were discovered from habitats and hosts that are previously unknown from this genus.     

<Emphasis Type="Italic">Cortinarius majestaticus</Emphasis> comb. nov.: phylogenetic evidence for the transfer of <Emphasis Type="Italic">Descolea majestatica</Emphasis> to <Emphasis Type="Italic">Cortinarius</Emphasis>

Descolea majestatica is an agaric with features described as intermediate between the genera Descolea Singer and Rozites P. Karst. (≡ Cortinarius). Molecular phylogenetic analysis of nuclear ribosomal internal transcribed spacer (ITS), large ribosomal subunit (LSU) and RNA polymerase second largest subunit (RPB2) sequences indicates that D. majestatica is nested within the genus Cortinarius, with its closest relative a sequestrate Cortinarius species from Argentina. Taxonomic recombination is made to restore the monophyly of Cortinarius and Descolea.     

Taxonomy and multi-gene phylogeny of <Emphasis Type="Italic">Haploporus</Emphasis> (Polyporales,Basidiomycota)

Taxonomic and phylogenetic studies of Haploporus were carried out. Three species in Haploporus, H. cylindrosporus, H. septatus and H. subpapyraceus, are described as new based on morphological differences and molecular phylogenetic analyses inferred from the internal transcribed spacer (ITS), the large subunit nuclear ribosomal RNA gene (nLSU), the small subunit mitochondrial rRNA gene (mtSSU), the second subunit of RNA polymerase II (rpb2) and the translation elongation factor 1-α gene (TEF1) sequences. Haploporus cylindrosporus is characterized by big irregular crystals occasionally present in the subiculum, an abundant oily substance among hyphae and typically cylindrical basidiospores 10–11.5?×?4.5–5 μm; H. septatus differs from other species in the genus by its leathery to corky basidiomata when dry, small round pores (5–6 per mm), simple septate skeletal hyphae at the edge of the dissepiments, and oblong to ellipsoid basidiospores 8.5–11?×?5–6 μm; H. subpapyraceus is separated by white to cream basidiomata, numerous apically simple septate cystidioles and ellipsoid basidiospores 9–12?×?5.5–8 μm. An identification key to accepted species of Haploporus is provided.     

<Emphasis Type="Italic">Helicocollum</Emphasis>, a new clavicipitalean genus pathogenic to scale insects (Hemiptera) in Thailand

A new genus pathogenic to scale insects (Coccidae, Hemiptera), Helicocollum, is described from Thailand. It includes the three species, H. chanthaburiensis, H. krabiensis and H. surathaniensis. Phylogenetic inferences on the basis of partial sequences of the large subunit of the nuclear ribosomal DNA, translation elongation factor 1-α and RNA polymerase II subunit 1 place the new genus in the Clavicipitaceae, although morphological features of the phialides resemble Hirsutella in the Ophiocordycipitaceae. Within the Clavicipitaceae, Helicocollum is closely related to Aschersonia, Moelleriella, Regiocrella, and Samuelsia, all known as pathogens of scale insects. Helicocollum differs from other scale insect pathogens in having phialides with helical necks and synnematous or sporodochial conidiomata, but pycnidia. Morphological characteristics are evaluated and compared with other asexual morphs producing twisted, undulated phialidic necks or occurring on scale insects.     

Multilocus phylogeny of <Emphasis Type="Italic">Clonostachys</Emphasis> subgenus <Emphasis Type="Italic">Bionectria</Emphasis> from Brazil and description of <Emphasis Type="Italic">Clonostachys chloroleuca</Emphasis> sp. nov.

Phylogenetic analyses based on protein-encoding gene exons and introns of ATP citrate lyase (ACL1), beta tubulin (TUB), the largest subunit of RNA polymerase II (RPB1), and translation elongation factor 1-α (TEF1) are used for inferring the existence of a new Clonostachys species from the Cerrado biome in Brazil, described here as C. chloroleuca. The species produces dimorphic, primary, and secondary conidiophores that form consistently greenish conidial masses on artificial media. It resembles therefore C. rosea f. catenulata although it differs from this species by less adpressed branches in the secondary conidiophores. The new species is also phylogenetically related to C. byssicola and C. rhizophaga. Our inventory suggests that C. byssicola, C. chloroleuca, C. pseudochroleuca, C. rhizophaga, C. rogersoniana, and C. rosea commonly occur in native and agriculturally used soils of the Cerrado and Amazon Forest. Using sequences available from two genome-sequenced strains employed as biological control agents, we confirm the identity of the European strain IK726 as C. rosea and identify strain 67-1 from China as C. chloroleuca.     

Multi-locus phylogeny and morphology reveal five new species of <Emphasis Type="Italic">Fomitiporia</Emphasis> (Hymenochaetaceae) from China

Five newly identified species of Fomitiporia (F. alpina, F. gaoligongensis, F. hainaniana, F. subrobusta and F. subtropica) and their morphological and molecular characterisation are described in this paper. Fomitiporia alpina sp. nov. is distinguished by its pileate basidiomata, parallel tramal hyphae and large basidiospores (6.5–8 × 6–8 μm), and by its gymnosperm wood-living habitat. Fomitiporia gaoligongensis sp. nov. is distinct from other species due to its semicircular pileus and subglobose to globose basidiospores (6.5–7.6 × 6–7.4 μm). Fomitiporia hainaniana sp. nov. is marked by its resupinate basidiomata, the presence of setae and small globose basidiospores (4–5 × 3.8–4.4 μm). Fomitiporia subrobusta sp. nov. is characterised by its triquetrous basidiomata, small pores (6–9 per mm) with entire and thick dissepiments, and subglobose to obovoid basidiospores (6.2–6.8 × 5.2–6 μm). Fomitiporia subtropica sp. nov. can be differentiated by its resupinate basidiomata, smaller pores (6–10 per mm) and smaller basidiospores (5.2–6 × 4.4–5 μm). Phylogenetic analysis, based on multi-gene comparison of the internal transcribed spacer regions (ITS), nuclear large subunit ribosomal RNA gene regions (nLSU), the translation elongation factor 1-α gene (tef1α) and the second subunit of RNA polymerase II (rpb2), confirmed affinity with the Fomitiporia species and showed association with similar fungi in the genus.     

Trojan Horse Strategy for Non-invasive Interference of <Emphasis Type="Italic">Clock</Emphasis> Gene in the Oyster <Emphasis Type="Italic">Crassostrea gigas</Emphasis>

RNA interference is a powerful method to inhibit specific gene expression. Recently, silencing target genes by feeding has been successfully carried out in nematodes, insects, and small aquatic organisms. A non-invasive feeding-based RNA interference is reported here for the first time in a mollusk bivalve, the pacific oyster Crassostrea gigas. In this Trojan horse strategy, the unicellular alga Heterocapsa triquetra is the food supply used as a vector to feed oysters with Escherichia coli strain HT115 engineered to express the double-stranded RNA targeting gene. To test the efficacy of the method, the Clock gene, a central gene of the circadian clock, was targeted for knockout. Results demonstrated specific and systemic efficiency of the Trojan horse strategy in reducing Clock mRNA abundance. Consequences of Clock disruption were observed in Clock-related genes (Bmal, Tim1, Per, Cry1, Cry2, Rev.-erb, and Ror) and triploid oysters were more sensitive than diploid to the interference. This non-invasive approach shows an involvement of the circadian clock in oyster bioaccumulation of toxins produced by the harmful alga Alexandrium minutum.     

Communities of arbuscular mycorrhizal fungi under <Emphasis Type="Italic">Picconia azorica</Emphasis> in native forests of Azores

Arbuscular mycorrhizal fungi (AMF) from the rhizosphere of the endemic Laurisilva tree, Picconia azorica, were characterised at two sites in each of two Azorean islands (Terceira and São Miguel). Forty-six spore morphotypes were found, and DNA extraction was attempted from individual spores of each of these. DNA was obtained from 18 of the morphotypes, from which a 1.5 kb long fragment of the nuclear ribosomal RNA gene (SSU-ITS-LSU) was sequenced. A total of 125 AMF sequences were obtained and assigned to 18 phylotypes. Phylogenetic analysis revealed sequences belonging to the families, Acaulosporaceae, Archaeosporaceae, Claroideoglomeraceae, Gigasporaceae and Glomeraceae. Phylotype richness changed between islands and between sampling sites at both islands suggesting that geographical and historical factors are determinant in shaping AMF communities in native forest of Azores. Ecological analysis of the molecular data revealed differences in AMF community composition between islands. In Terceira, the rhizosphere of P. azorica was dominated by species belonging to Acaulosporaceae and Glomeraceae, while São Miguel was dominated by members of Glomeraceae and Gigasporaceae. This is the first molecular study of AMF associated with P. azorica in native forest of the Azores. These symbiont fungi are key components of the ecosystem. Further research is needed to develop their use as promoters of plant establishment in conservation and restoration of such sites.     

The complete chloroplast genome sequence of <Emphasis Type="Italic">Pseudoroegneria libanotica</Emphasis>, genomic features,and phylogenetic relationship with <Emphasis Type="Italic">Triticeae</Emphasis> species

Pseudoroegneria libanotica is an important herbage diploid species possessing the St genome. The St genome participates in the formation of nine perennial genera in Triticeae (Poaceae). The whole chloroplast (cp) genome of P. libanotica is 135 026 bp in length. The typical quadripartite structure consists of one large single copy of 80 634 bp, one small single copy of 12 766 bp and a pair of inverted regions (20 813 bp each). The cp genome contains 76 coding genes, four ribosomal RNA and 30 transfer RNA genes. Comparative sequence analysis suggested that: 1) the 737 bp deletion in the cp of P. libanotica was specific in Triticeae species and might transfer into its nuclear genome; 2) hot-spot regions, indels in intergenic regions and protein coding sequences mainly led to the length variation in Triticeae; 3) highly divergence regions combined with negative selection in rpl2, rps12, ccsA, rps8, ndhH, petD, ndhK, psbM, rps3, rps18, and ndhA were identified as effective molecular markers and could be considered in future phylogenetic studies of Triticeae species; and 4) ycf3 gene with rich cpSSRs was suitable for phylogeny analysis or could be used for DNA barcoding at low taxonomic levels. The cpSSRs distribution in the coding regions of diploid Triticeae species was shown for the first time and provided a valuable source for developing primers to study specific simple sequence repeat loci.     

Novel fungi from an ancient niche: lachnoid and chalara-like fungi on ferns

A survey was conducted in Brazil to collect fungi on ferns. Based on morphology and inferred phylogeny from DNA sequences of two loci, namely the internal transcribed spacer (ITS) regions and the large subunit nuclear ribosomal RNA gene (LSU), several species belonging to chalara-like genera and lachnoid fungi were recognized. Eighteen fungal isolates, collected from five host species, representing 10 different localities were studied. Three novel genera (Lachnopsis, Scolecolachnum and Zymochalara), and six novel species (Bloxamia cyatheicola, Lachnopsis catarinensis, Lachnopsis dicksoniae, Scolecolachnum pteridii, Zymochalara lygodii and Zymochalara cyatheae) are introduced. Furthermore, two new combinations (Erioscyphella euterpes and Erioscyphella lushanensis) are proposed. Two novel taxa (Lachnopsis catarinensis and Lachnopsis dicksoniae) may be included in the list of potentially endangered fungal species in Brazil, if proven to be restricted to their tree-fern host, Dicksonia sellowiana, which is included in the official list of endangered plant species in Brazil.     

Morphological and molecular taxonomy of novel species <Emphasis Type="Italic">Pleurotheciaceae</Emphasis> from freshwater habitats in Yunnan,China

The diversity of lignicolous freshwater fungi of the Greater Mekong Subregion are currently being studied. In this paper, 18 fresh collections of Pleurotheciaceae from submerged wood in freshwater are characterized based on morphological characters and analyses of ITS, LSU, SSU and RPB2 sequence data. Six new species of Pleurotheciella (P. aquatica, P. fusiformis, P. guttulata, P. lunata, P. saprophytica, P. submersa), one new Phaeoisaria species (Ph. aquatica) and one new Pleurothecium species (Pl. aquaticum) are introduced based on their distinct morphology and evidence from molecular phylogeny. Pleurotheciella uniseptata, Phaeoisaria clematidis and Pleurothecium pulneyense are also redescribed and phylogenetic relationships assessed herein, these species are the first records for China.     

Two new species of <Emphasis Type="Italic">Conidiobolus</Emphasis> occurring in Anhui,China

Two new species of Conidiobolus were isolated from Anhui Province, China. A polyphasic taxonomic approach comprising morphological characteristics and molecular data (the nuclear large subunit of ribosomal DNA and the translation elongation factor 1 alpha gene sequences) was applied to determine their novel taxonomic status in the genus Conidiobolus. The new species C. mirabilis is a sister group to C. thromoides, but morphologically differs by smaller primary conidia and a unique formation of zygospores among two to four adjacent hyphal segments. The other new species C. pachyzygosporus characterized with thick-walled zygospores is phylogenetically closely related to C. antarcticus, C. couchii, and C. osmodes.     

Pine wilt disease and possible causes of its incidence in Russia

Surveys of forests and stockpiled timber of pine, spruce, larch, and silver fir in 14 administrative subjects of the Russian Federation revealed widespread occurrence of the coniferous wood parasitic nematode Bursaphelenchus mucronatus. Twenty species of bacteria belonging to 13 genera have been detected in 25 B. mucronatus isolates, and their identity has been determined by direct sequencing of the 16S RNA gene. The most frequently occurring were bacteria from the genera Pseudomonas, Stenotrophomonas, Pantoea, Bacillus, Burkholderia, and Serratia. Prevalence of Pseudomonas brenneri and P. fluorescence, which were also found in the nematode dauer larva (L_IV) isolated from the fir sawyer beetle Monochamus urussovi, have also been assessed. Two nematode B. xylophilus isolates from Portugal and one isolate from the United States have been examined, and 10 symbiotic bacteria species have been isolated, including Agrobacterium tumefacience, P. fluorescens, P. brenneri, Rahnella aquatilis, Stenotrophomonas maltophilia, S. rhizophila, and Yersinia mollaretii.     

Enhancers in the <Emphasis Type="Italic">Peril</Emphasis> lincRNA locus regulate distant but not local genes

Background

Recently, it has become clear that some promoters function as long-range regulators of gene expression. However, direct and quantitative assessment of enhancer activity at long intergenic noncoding RNA (lincRNA) or mRNA gene bodies has not been performed. To unbiasedly assess the enhancer capacity across lincRNA and mRNA loci, we performed a massively parallel reporter assay (MPRA) on six lincRNA loci and their closest protein-coding neighbors.

Results

For both gene classes, we find significantly more MPRA activity in promoter regions than in gene bodies. However, three lincRNA loci, Lincp21, LincEnc1, and Peril, and one mRNA locus, Morc2a, display significant enhancer activity within their gene bodies. We hypothesize that such peaks may mark long-range enhancers, and test this in vivo using RNA sequencing from a knockout mouse model and high-throughput chromosome conformation capture (Hi-C). We find that ablation of a high-activity MPRA peak in the Peril gene body leads to consistent dysregulation of Mccc1 and Exosc9 in the neighboring topologically associated domain (TAD). This occurs irrespective of Peril lincRNA expression, demonstrating this regulation is DNA-dependent. Hi-C confirms long-range contacts with the neighboring TAD, and these interactions are altered upon Peril knockout. Surprisingly, we do not observe consistent regulation of genes within the local TAD. Together, these data suggest a long-range enhancer-like function for the Peril gene body.

Conclusions

A multi-faceted approach combining high-throughput enhancer discovery with genetic models can connect enhancers to their gene targets and provides evidence of inter-TAD gene regulation.

     

Taxonomic revisions in the Microstromatales: two new yeast species,two new genera,and validation of <Emphasis Type="Italic">Jaminaea</Emphasis> and two <Emphasis Type="Italic">Sympodiomycopsis</Emphasis> species

Environmental sampling yielded two yeast species belonging to Microstromatales (Exobasidiomycetes, Ustilaginomycotina). The first species was collected from a leaf phylloplane infected by the rust fungus Coleosporium plumeriae, and represents a new species in the genus Jaminaea, for which the name Jaminaea rosea sp. nov. is proposed. The second species was isolated from air on 50% glucose media and is most similar to Microstroma phylloplanum. However, our phylogenetic analyses reveal that species currently placed in Microstroma are not monophyletic, and M. phylloplanum, M. juglandis and M. albiziae are not related to the type species of this genus, M. album. Thus, Pseudomicrostroma gen. nov. is proposed to accommodate the following species: P. glucosiphilum sp. nov., P. phylloplanum comb. nov. and P. juglandis comb. nov. We also propose Parajaminaea gen. nov. to accommodate P. albizii comb. nov. and P. phylloscopi sp. nov. based on phylogenetic analyses that show these are not congeneric with Jaminaea or Microstroma. In addition, we validate the genus Jaminaea, its respective species and two species of Sympodiomycopsis and provide a new combination, Microstroma bacarum comb. nov., for the anamorphic yeast Rhodotorula bacarum. Our results illustrate non-monophyly of Quambalariaceae and Microstromataceae as currently circumscribed. Taxonomy of Microstroma and the Microstromataceae is reviewed and discussed. Finally, analyses of all available small subunit rDNA sequences for Jaminaea species show that J. angkorensis is the only known species that possess a group I intron in this locus, once considered a potential feature indicating the basal placement of this genus in Microstromatales.     

Constitutive expression of <Emphasis Type="Italic">SlTrxF</Emphasis> increases starch content in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

The plastidic thioredoxin F-type (TrxF) protein plays an important role in plant saccharide metabolism. In this study, a gene encoding the TrxF protein, named SlTrxF, was isolated from tomato. The coding region of SlTrxF was cloned into a binary vector under the control of 35S promoter and then transformed into Arabidopsis thaliana. The transgenic Arabidopsis plants exhibited increased starch accumulation compared to the wild-type (WT). Real-time quantitative PCR analysis showed that constitutive expression of SlTrxF up-regulated the expression of ADP-glucose pyrophosphorylase (AGPase) small subunit (AtAGPase-S1 and AtAGPase-S2), AGPase large subunit (AtAGPase-L1 and AtAGPase-L2) and soluble starch synthase (AtSSS I, AtSSS II, AtSSS III and AtSSS IV) genes involved in starch biosynthesis in the transgenic Arabidopsis plants. Meanwhile, enzymatic analyses showed that the major enzymes (AGPase and SSS) involved in the starch biosynthesis exhibited higher activities in the transgenic plants compared to WT. These results suggest that SlTrxF may improve starch content of Arabidopsis by regulating the expression of the related genes and increasing the activities of the major enzymes involved in starch biosynthesis.     

<Emphasis Type="Italic">Talaromyces heiheensis</Emphasis> and <Emphasis Type="Italic">T. mangshanicus</Emphasis>, two new species from China

Two new species, Talaromyces heiheensis from rotten wood and T. mangshanicus isolated from soil, are illustrated and described as new to science in sections Trachyspermi and Talaromyces. The phylogenetic positions of the two new species inferred from the internal transcribed spacer, beta-tubulin, calmodulin and RNA polymerase II second largest subunit regions were carried out. Talaromyces heiheensis is phylogenetically closely related to T. albobiverticillius, T. rubrifaciens, T. solicola and T. erythromellis, and characterised by slow growth on Czapek yeast autolysate agar at 25 °C, orange conidia en masse on malt extract agar at 25 °C, biverticillate and terverticillate conidiophores, acerose phialides and subglobose to ellipsoidal, smooth-walled conidia. Talaromyces mangshanicus is related to T. kendrickii, T. qii and T. thailandensis, and characterised by slow-growing colonies with absent or sparse sporulation on CYA agar at 25 °C, conidia en masse greyish purple, purplish red soluble pigment on yeast extract agar (YES) at 25 °C, biverticillate conidiophores, ampulliform phialides and subglobose to ellipsoidal conidia with echinulate walls. They are distinguished from the known species in culture characteristics on four standard media, microscopic features and sequence data.     

Comparative pan genome analysis of oral <Emphasis Type="Italic">Prevotella</Emphasis> species implicated in periodontitis

Prevotella is part of the oral bacterial community implicated in periodontitis. Pan genome analyses of eight oral Prevotella species, P. dentalis, P. enoeca, P. fusca, P. melaninogenica, P. denticola, P. intermedia 17, P. intermedia 17-2 and P. sp. oral taxon 299 are presented in this study. Analysis of the Prevotella pan genome revealed features such as secretion systems, resistance to oxidative stress and clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems that enable the bacteria to adapt to the oral environment. We identified the presence of type VI secretion system (T6SS) in P. fusca and P. intermedia strains. For some VgrG and Hcp proteins which were not part of the core T6SS loci, we used gene neighborhood analysis and identified putative effector proteins and putative polyimmunity loci in P. fusca and polymorphic toxin systems in P. intermedia strains. Earlier studies have identified the presence of Por secretion system (PorSS) in P. gingivalis, P. melaninogenica and P. intermedia. We noted the presence of their homologs in six other oral Prevotella studied here. We suggest that in Prevotella, PorSS is used to secrete cysteine proteases such as interpain and C-terminal domain containing proteins with a “Por_secre_tail” domain. We identified subtype I-B CRISPR-Cas system in P. enoeca. Putative CRISPR-Cas system subtypes for 37 oral Prevotella and 30 non-oral Prevotella species were also predicted. Further, we performed a BLASTp search of the Prevotella proteins which are also conserved in the red-complex pathogens, against the human proteome to identify potential broad-spectrum drug targets. In summary, the use of a pan genome approach enabled identification of secretion systems and defense mechanisms in Prevotella that confer adaptation to the oral cavity.