Spatio-Temporal Monitoring and Ecological Significance of Retrievable Pelagic Heterotrophic Bacteria in Kongsfjorden,an Arctic Fjord

Kongsfjorden is a glacial fjord in the Arctic that is influenced by both Atlantic and Arctic water masses. In the present report retrievable heterotrophic bacteria isolated from two distinct zones (outer and inner fjord) of Kongsfjorden was studied during summer to fall of 2012. 16S rRNA gene sequences of the retrievable heterotrophic bacteria corresponded to γ-proteobacteria (13 phylotypes), α-proteobacteria (3 phylotypes), Bacteroidetes (4 phylotypes) and Actinobacteria (2 phylotypes). The heterotrophic bacterial community structure was fundamentally different in different months which could be linked to changes in the water masses and/or phytoplankton bloom dynamics. It is hypothesized that monitoring the retrievable heterotrophic bacterial assemblage in the fjord would give valuable insights into the complex ecological role they play under extreme and dynamic conditions.     

Detection of <Emphasis Type="Italic">Helicobacter</Emphasis> DNA in different water sources and penguin feces from Greenwich,Dee and Barrientos Islands,Antarctica

Helicobacter spp. colonize the gastrointestinal tract of humans and animals and have been associated with gastrointestinal diseases. Antarctic habitats are considered pristine ecosystems, but the increase in human activity could be introducing human bacteria hosted into waters and wildlife. However, Helicobacter spp. occurrence has not been studied in Antarctica. The aim of our study was to detect the Helicobacter DNA in different water sources and penguin feces from Greenwich, Dee and Barrientos Islands during summer of 2012 and 2013. High Helicobacter proportion was observed in water sources amplifying the 16S rRNA (33/40) and 23S rRNA genes (37/40) by semi-nested PCR. Similar results were observed in feces from Gentoo penguins (16S rRNA: 32/39, and 23S rRNA: 28/39) and Chinstrap penguins (16S rRNA: 16/17, and 23S rRNA: 15/17) by PCR. The phylogenetic relationship of 16S rRNA and 23S rRNA sequences from penguin feces was closely related to Helicobacter brantae. Analyses of 16S rRNA sequences showed that the majority of water samples are related to penguin (3/6) and Helicobacter pylori (2/6) sequences, but the 23S rRNA sequences matched with Campylobacter and Arcobacter. These results show for the first time the presence of the genus Helicobacter in different Antarctica water sources and in Gentoo and Chinstrap penguin feces. A few 16S rRNA sequences are very closely related to H. pylori, but specific glmM and ureA H. pylori genes were not detected. More studies are needed to determine the Helicobacter species present in this ecosystem and to establish the human impact in these Antarctic Islands.     

Polymorphic Sites in ITS1-5.8S rDNA-ITS2 Region in Hybridogenic Genus × <Emphasis Type="Italic">Elyhordeum</Emphasis> and Putative Interspecific Hybrids <Emphasis Type="Italic">Elymus</Emphasis> (Poaceae: Triticeae)

The genus Elymus L. is a complicated aggregate of ecological and geographical races, species, subspecies, varieties, and hybrids. We suggest that comparative analysis of intragenomic polymorphism of internal transcribed spacers ITS1 and ITS2 of 35S rRNA genes in the supposed hybrids and their possible “parents” can be one of the approaches to verification of hybrid origin of the samples collected in nature to confirm or reject the hypotheses about their possible “parents.” Polymorphic sites (PS) in ITS of 23 Elymus species, as well as in two supposed interspecific Elymus hybrids and in a supposed intergeneric hybrid between Elymus × Hordeum determined as × Elyhordeum sp., were analyzed in the work. We collected all hybrids in the Altai. There were 2 and 5 PS in two samples of E. dahuricus and 1 and 4 PS in two studied samples of E. schrenkianus in the ITS1-5.8S rDNA-ITS2 region. From 0 to 4 (modes 0 and 3) PS were detected in 32 samples relating to 21 tetraploid Elymus species. More PS (14) were found in the × Elyhordeum sp. sample. A large number of single nucleotide substitutions were found in 5.8S rRNA in × Elyhordeum. It was shown that about half of them do not change the secondary structure of the 5.8S rRNA molecule, so these molecules probably retain the ability to work as a component of large subunit of a ribosome. On the other hand, the absence or weakening of 5.8S rDNA homogenization in × Elyhordeum indirectly suggests that a significant part of 5.8S rDNA is not transcribed. Paradoxically, ITS sequences of × Elyhordeum sp. are less polymorphic than 5.8S rDNA. There are no ITS sequences derived from Hordeum among × Elyhordeum ITS sequenced by Sanger method. No traces of the H subgenome and a subgenome originating from Agropyron (P-subgenome) are seen in the Alt 10–278 plant genome (a chimera, combining the morphological traits of Elymus, Elytrigia, and Agropyron). In this plant, as well as in the supposed intersectional hybrid Alt 11–60 distinguished by a mosaic of the traits typical for the E. caninus × E. mutabilis species, only 4 and 5 PS, respectively, are detected when sequencing by Sanger method. The comparison of ITS sequences of the supposed Elymus Alt 10–278 hybrid and its probable “parents” demonstrates that one of the species of the Elymus macrourus kinship circle, as well as the Elytrigia geniculata, could be one of its ancestors. The comparison of the ITS sequence of the supposed parental species with ITS of Alt 11–60 samples and five PS of the supposed Alt 11–60 hybrid does not contradict the hypothesis that this is an intersectional hybrid of the first generation that emerged with the involvement of E. caninus and E. mutabilis common in the Altai.     

Analysis of the ITS1/ITS2 nuclear spacers and the secondary structure of <Emphasis Type="Italic">5.8S</Emphasis> rRNA gene in endemic species <Emphasis Type="Italic">Bellevalia sarmatica</Emphasis> (Pall. ex Georgi) Woronow and related species of the subfamily scilloideae

Sequence variability of the ITS spacers and 5.8S rRNA gene was examined in 11 accessions of the subfamily Scilloideae, including seven accessions of rare and endangered species Bellevalia sarmatica from Volgograd region. The intraspecific polymorphism level of the examined ITS1–5.8S–ITS2 sequence of B. sarmatica accessions constituted 1.3%. The phylogenetic position of B. sarmatica within the genus Bellevalia was determined. It was demonstrated that B. sarmatica belonged to the section Nutantes, and the most closely related species were B. webbiana and B. dubia. Nucleotide substitutions in the 5.8S rRNA gene sequence of the analyzed Scilloideae accessions were identified and studied. The predicted secondary structure of 5.8S rRNA gene was constructed. It was demonstrated that in the examined accessions, mutations in the 5.8S rRNA gene were mainly localized in the third hairpin region and had no effect on the secondary structure of the 5.8S rRNA molecule.     

Bird feather fungi from Svalbard Arctic

Despite feather fungi being an important component of the Arctic fungal flora, their ecological role and diversity are not fully known. In the current study, fungal cultures were isolated from feathers (barnacle goose, common eider, and glaucous gull) collected in the Ny-Ålesund region, Svalbard. Isolates were identified by ITS region sequences, which include the ITS1, ITS2, and 5.8S rRNA. The result showed culturable yeast and filamentous fungi belonging to three classes: Ascomycota (Pyrenochaetopsis pratorum, Cladosporium herbarum, Thelebolus microsporus, Aspergillus versicolor, Penicillium commune, and Venturia sp.), Basidiomycota (Mrakia blollopis and Rhodotorula mucilaginosa), and Zygomycota (Mucor flavus). Most of the fungal isolates appeared to be cold-tolerant, and about 60 % of the isolates showed keratinase activity. The reasonably low fungal diversity colonizing feathers indicates that the birds of Svalbard are casual carriers of fungi which may result in a negligible impact on their health. To the best of our knowledge, this is the first record of fungal communities present on the feathers of birds in the high Arctic.     

Secondary structure prediction of ITS rRNA region and molecular phylogeny: an integrated approach for the precise speciation of <Emphasis Type="Italic">Muscodor</Emphasis> species

Muscodor is a non-sporulating, volatile organic compounds producing endophytic fungi that has been extensively explored as a bio-fumigant and bio-preservative. Novel species of this genus have been mainly identified using ITS sequences. However, the ITS hyper-variability hinders the creation of reproducible alignments and stable phylogenetic trees. Conserved structural data of the ITS region represents as a vital auxiliary information for accurate speciation of fungi. In the present study, secondary structural data of ITS1, 5.8S, and ITS2 region of all Muscodor species were generated using LocaRNA web server. The predicted secondary structural data displayed greater variability in ITS1 region in comparison to ITS2. The structural data of all sequences exhibited characteristic conserved features of eukaryotic rRNA. Evolutionary conserved motifs were found among all 5.8S and ITS2 sequences. Profile neighbor joining (PNJ) tree based on combined sequence-structural information of ITS region was generated in ProfDists. The PNJ tree resolved into four major groups whereby M. fengyangenesis and M. albus species formed monophyletic clades. However, three M. albus species along with other Muscodor species emerged as sister branches to the existing clades, thereby, improving the precision of phylogenetic analysis for identification of novel species of Muscodor genus. Hence, the results indicated that structural analysis along with primary sequence information can provide new insights for precise identification of Muscodor species.     

<Emphasis Type="Italic">Salinispora</Emphasis><Emphasis Type="Italic">arenicola</Emphasis> from temperate marine sediments: new intra-species variations and atypical distribution of secondary metabolic genes

The obligate marine actinobacterium Salinispora arenicola was successfully cultured from temperate sediments of the Pacific Ocean (Tosa Bay, offshore Kochi Prefecture, Japan) with the highest latitude of 33°N ever reported for this genus. Based on 16S rRNA gene sequence analysis, the Tosa Bay strains are of the same phylotype as the type strain S. arenicola NBRC105043. However, sequence analysis of their 16S-23S rRNA intergenic spacer (ITS) revealed novel sequence variations. In total, five new ITS sequences were discovered and further phylogenetic analyses using gyrase B and rifamycin ketosynthase (KS) domain sequences supported the phylogenetic diversity of the novel Salinispora isolates. Screening of secondary metabolite genes in these strains revealed the presence of KS1 domain sequences previously reported in S. arenicola strains isolated from the Sea of Cortez, the Bahamas and the Red Sea. Moreover, salinosporamide biosynthetic genes, which are highly homologous to those of Bahamas-endemic S. tropica, were detected in several Tosa Bay isolates, making this report the first detection of salinosporamide genes in S. arenicola. The results of this study provide evidence of a much wider geographical distribution and secondary metabolism diversity in this genus than previously projected.     

Diversity of <Emphasis Type="Italic">Colletotrichum</Emphasis> spp. isolated from chili pepper fruit exhibiting symptoms of anthracnose in Thailand

Colletotrichum spp. are causal agents of anthracnose disease in chili fruits and other tropical crops. The disease is increasing in chili fruits in Thailand and significantly reduces fruit quality and fruit production. Forty-eight isolates of Colletotrichum spp. associated with chili anthracnose were collected from different areas of Thailand during 2010–2015. Based on morphological characteristic identification, 10 isolates were shown to belong to the C. gloeosporioides species complex, 24 isolates belong to the C. acutatum species complex and 14 isolates to C. capsici. For molecular identification, two primer sets, ITS1/ITS4 and ACT528/ACT738, were used for amplification of the internal transcribed spacer of rRNA gene (ITS1–5.8S–ITS2) and partial region actin gene (ACT), respectively. The phylogenetic analysis of individual and combined ITS region and actin nucleotide sequences identified the collected isolates into 4 species: C. gloeosporioides, C. siamense, C. acutatum and C. capsici. The pathogenicity test demonstrated that all four species were pathogenic on intact unwounded and healthy fruits. These results indicated that C. capsici, C. acutatum, C. gloeosporioides and C. siamense were the causal agents of chili anthracnose disease.     

Relationships within <Emphasis Type="Italic">Capitotricha bicolor</Emphasis> (Lachnaceae,Ascomycota) as inferred from ITS rDNA sequences,including some notes on the <Emphasis Type="Italic">Brunnipila</Emphasis> and <Emphasis Type="Italic">Erioscyphella</Emphasis> clades

DNA sequences of Capitotricha bicolor from Quercus, Fagus sylvatica, Alnus alnobetula, and Nothofagus, and C. rubi from Rubus idaeus were obtained from apothecia to establish whether specimens from different hosts belong to separate species. The obtained ITS1–5.8S–ITS2 rDNA sequences were examined with Bayesian and parsimony phylogenetic analyses. Intra- and interspecific variation was also investigated based on molecular distances in the ITS region. The phylogenetic analyses supported the specific distinctness of Capitotricha rubi and the Capitotricha from Nothofagus, but also suggest specific distinctness between samples from Quercus, Fagus, and Alnus. The interspecific distances were larger than intraspecific distances for all examined units. The smallest distance was found between the “Alnus alnobetula” and “Fagus sylvatica” units. Two new sequences of Brunnipila are published. Capitotricha, Lachnum, and Erioscyphella are compared to each other based on hair and excipulum characteristics.     

<Emphasis Type="Italic">Kondoa gutianensis</Emphasis> f.a. sp. nov., a novel ballistoconidium-forming yeast species isolated from plant leaves

Two strains, GT-165^T and GT-261, isolated from plant leaves collected from Gutian Mountain in Zhejiang province in China were identified as a novel species of the genus Kondoa by the sequence analysis of the internal transcribed spacer (ITS) region, the D1/D2 domains of the large subunit of rRNA (LSU rRNA) and the RNA polymerase II second largest subunit (RPB2), complemented by physiological tests. Phylogenetic analysis based on the concatenated sequences of ITS, D1/D2 and RPB2 showed that the closest known relatives of the new species are three undescribed Kondoa species and Kondoa thailandica. The ITS and D1/D2 sequences of the new species differ from the closely related species by 11–22% and 2–9%, respectively. The name Kondoa gutianensis f.a. sp. nov. (MB 820648, holotype = CGMCC 2.5703^T; isotype: CBS 14811^T = CGMCC 2.5703^T) is proposed to accommodate the new taxon.     

Evolution of blue-flowered species of genus <Emphasis Type="Italic">Linum</Emphasis> based on high-throughput sequencing of ribosomal RNA genes

Background

The species relationships within the genus Linum have already been studied several times by means of different molecular and phylogenetic approaches. Nevertheless, a number of ambiguities in phylogeny of Linum still remain unresolved. In particular, the species relationships within the sections Stellerolinum and Dasylinum need further clarification. Also, the question of independence of the species of the section Adenolinum still remains unanswered. Moreover, the relationships of L. narbonense and other species of the section Linum require further clarification. Additionally, the origin of tetraploid species of the section Linum (2n?=?30) including the cultivated species L. usitatissimum has not been explored. The present study examines the phylogeny of blue-flowered species of Linum by comparisons of 5S rRNA gene sequences as well as ITS1 and ITS2 sequences of 35S rRNA genes.

Results

High-throughput sequencing has been used for analysis of multicopy rRNA gene families. In addition to the molecular phylogenetic analysis, the number and chromosomal localization of 5S and 35S rDNA sites has been determined by FISH.Our findings confirm that L. stelleroides forms a basal branch from the clade of blue-flowered flaxes which is independent of the branch formed by species of the sect. Dasylinum. The current molecular phylogenetic approaches, the cytogenetic analysis as well as different genomic DNA fingerprinting methods applied previously did not discriminate certain species within the sect. Adenolinum. The allotetraploid cultivated species L. usitatissimum and its wild ancestor L. angustifolium (2n?=?30) could originate either as the result of hybridization of two diploid species (2n?=?16) related to the modern L. gandiflorum and L. decumbens, or hybridization of a diploid species (2n?=?16) and a diploid ancestor of modern L. narbonense (2n?=?14).

Conclusions

High-throughput sequencing of multicopy rRNA gene families allowed us to make several adjustments to the phylogeny of blue-flowered flax species and also reveal intra- and interspecific divergence of the rRNA gene sequences.

     

<Emphasis Type="Italic">Papiliotrema phichitensis</Emphasis> f.a., sp. nov., a novel yeast species isolated from sugarcane leaf in Thailand

Strain DMKU-SP105^T representing a novel yeast species was isolated from the external surface of a sugarcane leaf (Saccharum officinarum L.) collected from a sugarcane plantation field in Phichit province, Thailand. On the basis of sequence analysis of the D1/D2 region of the large subunit (LSU) rRNA gene and the internal transcribed spacer (ITS) region, the strain DMKU-SP105^T differed by 7–16 substitutions in the D1/D2 region of LSU rRNA gene and 6–22 substitutions in the ITS region from a group of related species, Papiliotrema aspenensis, Papiliotrema odontotermitis, Papiliotrema rajasthanensis and Papiliotrema laurentii. A phylogenetic analysis based on the concatenated sequences of ITS region and the D1/D2 region of the LSU rRNA gene indicated that strain DMKU-SP105^T belongs to the laurentii clade of Papiliotrema in the Tremellales and is distinct from other related species in the clade. It therefore represents a novel species of the genus Papiliotrema although the formation of basidiospores was not observed. The name Papiliotrema phichitensis f.a., sp. nov. is proposed. The type is DMKU-SP105^T (=?CBS 13390^T?=?BCC 61187^T?=?NBRC 109699^T).     

<Emphasis Type="Italic">Melampsora salicis-bakko</Emphasis>, a new species on willows in Japan evidenced by morphological and molecular phylogenetic analyses

Through the morphological and molecular examinations of Melampsora species on willows, we clarified the taxonomic identity of the rust specimens on Salix bakko, S. hultenii and S. leucopithecia from Japan and described the following rust fungus as a new species, Melampsora salicis-bakko. This rust fungus resembled M. caprearum in morphology of teliospores, but it differed from M. caprearum mainly in the density of spines on the urediniospores. Molecular phylogenetic analyses using the rDNA ITS region (complete ITS1, 5.8S rRNA gene and ITS2) revealed that M. salicis-bakko was monophyletic, and that this rust fungus was distinct from other Melampsora species, including M. caprearum.     

Molecular phylogenetics and anti-<Emphasis Type="Italic">Pythium</Emphasis> activity of endophytes from rhizomes of wild ginger congener, <Emphasis Type="Italic">Zingiber zerumbet</Emphasis> Smith

Zingiber zerumbet, a perennial rhizomatous herb exhibits remarkable disease resistance as well as a wide range of pharmacological activities. Towards characterizing the endophytic population of Z. zerumbet rhizomes, experiments were carried out during two different growing seasons viz., early-June of 2013 and late-July of 2014. A total of 34 endophytes were isolated and categorized into 11 morphologically distinct groups. Fungi were observed to predominate bacterial species with colonization frequency values ranging from 12.5 to 50 %. Among the 11 endophyte groups isolated, molecular analyses based on ITS/16S rRNA gene sequences identified seven isolate groups as Fusarium solani, two as F. oxysporum and one as the bacterium Rhizobium spp. Phylogenetic tree clustered the ITS sequences from Z. zerumbet endophytes into distinct clades consistent with morphological and sequence analysis. Dual culture assays were carried out to determine antagonistic activity of the isolated endophytes against Pythium myriotylum, an economically significant soil-borne phytopathogen of cultivated ginger. Experiments revealed significant P. myriotylum growth inhibition by F. solani and F. oxysporum isolates with percentage of inhibition (PoI) ranging from 45.17 ± 0.29 to 62.2 ± 2.58 with F. oxysporum exhibiting higher PoI values against P. myriotylum. Using ZzEF8 metabolite extract, concentration-dependent P. myriotylum hyphal growth inhibition was observed following radial diffusion assays. These observations were confirmed by scanning electron microscopy analysis wherein exposure to ZzEF8 metabolite extract induced hyphal deformities. Results indicate Z. zerumbet endophytes as promising resources for biologically active compounds and as biocontrol agents for soft rot disease management caused by Pythium spp.     

Phylogenetic relationships in Iranian <Emphasis Type="Italic">Scutellaria</Emphasis> (Lamiaceae) based on nuclear ribosomal ITS and chloroplast <Emphasis Type="Italic">trn</Emphasis>L-F DNA data

The genus Scutellaria (Lamiaceae, Scutellarioidae) includes taxa with a wide range of pharmacological traits, which make this genus economically important. Previous taxonomical studies on Scutellaria were mainly based on morphological and chromosomal characters. The infrageneric classification of the genus has been the subject of a long controversy among authors, and delimitation of taxa remains problematic. In this study, the phylogeny of the genus was evaluated using nrDNA ITS and trnL-F sequences, with a focus on the 42 Iranian taxa belonging to subgenera Apeltanthus and Scutellaria. In both ITS and trnL-F trees, there were two main clades within the genus corresponding to the two subgenera Scutellaria and Apeltanthus. In the subg. Scutellaria, the sect. Anaspis was monophyletic, but the monophyly of sect. Scutellaria was not supported. In this subgenus, S. galericulata was located away from other species in both trees, supported the isolation of this species from the sect. Scutellaria and inclusion in the sect. Galericularia as was previously considered by some authors. In subg. Apeltanthus, sect. Apeltanthus and sect. Lupulinaria were not monophyletic; furthermore, the monophyly of subsect. Lupulinaria was not supported. Based on the results of this study, it can be suggested that the sectional classification of both subgenera Apeltanthus and Scutellaria should be revised.     

<Emphasis Type="Italic">Sordaria fimicola</Emphasis>-like ascomycete isolated from <Emphasis Type="Italic">Pinus coulteri</Emphasis> needles in Slovakia

This is the first report of Sordaria fimicola-like ascomycete which was encountered during a diversity study of injured tissues of coulter pine in Slovakia. The fungus was identified as Sordaria fimicola by morphological analyses. Sequence analysis of internal transcribed spacer region (ITS) showed that the fungus is highly related to the ITS sequences of several S. fimicola isolates documenting wide ecological valence and geographical distribution of S. fimicola-like ascomycetes.