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The genus Prototheca (alga) comprises a unique group of achlorophyllic saprotrophic and mammalian pathogen species. Despite its rare occurrence in humans and animals, protothecosis is considered an emerging clinical entity with relevance in immunocompromised patients. In this study, the characterization of spherical structures with endospores recovered from a blood culture in an HIV patient was investigated using phenotypic and molecular methodologies. On 2% Sabouraud dextrose agar, the isolate displayed morphological and biochemical characteristics found on isolates identified as Prototheca wickerhamii. To validate these analyses, molecular phylogeny of the internal transcript space (ITS) partial gene confirmed the identity of the isolate as P. wickerhamii. This is the first case of systemic human protothecosis in Brazil. The present case of human Prototheca and those reported in the medical literature highlight the need for novel methodologies to identify pathogenic algae in the clinical laboratory, improving in this way the diagnosis and treatment of this group of neglected pathogens., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Duarte-Silva et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Efficient and accurate classification of DNA barcode data is crucial for large-scale fungal biodiversity studies. However, existing methods are either computationally expensive or lack accuracy. Previous research has demonstrated the potential of deep learning in this domain, successfully training neural networks for biological sequence classification. We introduce the MycoAI Python package, featuring various deep learning models such as BERT and CNN tailored for fungal Internal Transcribed Spacer (ITS) sequences. We explore different neural architecture designs and encoding methods to identify optimal models. By employing a multi-head output architecture and multi-level hierarchical label smoothing, MycoAI effectively generalizes across the taxonomic hierarchy. Using over 5 million labelled sequences from the UNITE database, we develop two models: MycoAI-BERT and MycoAI-CNN. While we emphasize the necessity of verifying classification results by AI models due to insufficient reference data, MycoAI still exhibits substantial potential. When benchmarked against existing classifiers such as DNABarcoder and RDP on two independent test sets with labels present in the training dataset, MycoAI models demonstrate high accuracy at the genus and higher taxonomic levels, with MycoAI-CNN being the fastest and most accurate. In terms of efficiency, MycoAI models can classify over 300,000 sequences within 5 min. We publicly release the MycoAI models, enabling mycologists to classify their ITS barcode data efficiently. Additionally, MycoAI serves as a platform for developing further deep learning-based classification methods. The source code for MycoAI is available under the MIT Licence at https://github.com/MycoAI/MycoAI., (© 2024 The Author(s). Molecular Ecology Resources published by John Wiley & Sons Ltd.)

Here, we report on a Cordyceps species entering into a multi-trophic, multi-kingdom association. Cordyceps cateniannulata, isolated from the stem of wild Coffea arabica in Ethiopia, is shown to function as an endophyte, a mycoparasite and an entomopathogen. A detailed polyphasic taxonomic study, including a multilocus phylogenetic analysis, confirmed its identity. An emended description of C. cateniannulata is provided herein. Previously, this species was known as a pathogen of various insect hosts in both the Old and New World. The endophytic status of C. cateniannulata was confirmed by re-isolating it from inoculated coffee plants. Inoculation studies have further shown that C. cateniannulata is a mycoparasite of Hemileia vastatrix, as well as an entomopathogen of major coffee pests; infecting and killing Hypothenemus hampei and Leucoptera coffeella. This is the first record of C. cateniannulata from Africa, as well as an endophyte and a mycoparasite. The implications for its use as a biocontrol agent are discussed., (Copyright © 2024 British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

Candida auris represents one of the most urgent threats to public health, although its ecology remains largely unknown. Because amphibians and reptiles may present favorable conditions for C. auris colonization, cloacal and blood samples (n = 68), from several snake species, were cultured and molecularly screened for C. auris using molecular amplification of glycosylphosphatidylinositol protein-encoding genes and ribosomal internal transcribed spacer sequencing. Candida auris was isolated from the cloacal swab of one Egyptian cobra (Naja haje legionis) and molecularly identified in its cloaca and blood. The isolation of C. auris from wild animals is herein reported for the first time, thus suggesting the role that these animals could play as reservoirs of this emerging pathogen. The occurrence of C. auris in blood requires further investigation, although the presence of cationic antimicrobial peptides in the plasma of reptiles could play a role in reducing the vitality of the fungus., (© The Author(s) 2024. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

During an epidemiological survey, a potential novel species within the basidiomycetous yeast genus Trichosporon was observed. The clinical strain was obtained from a urine sample taken from a Brazilian kidney transplant recipient. The strain was molecularly identified using the intergenic spacer (IGS1) ribosomal DNA locus and a subsequent phylogenetic analysis showed that multiple strains that were previously reported by other studies shared an identical IGS1-genotype most closely related to that of Trichosporon inkin. However, none of these studies provided an in-depth characterization of the involved strains to describe it as a new taxon. Here, we present the novel clinically relevant yeast for which we propose the name Trichosporon austroamericanum sp. nov. (holotype CBS H-24937). T. austroamericanum can be distinguished from other siblings in the genus Trichosporon using morphological, physiological, and phylogenetic characters., (© 2024. The Author(s).)

Species in the Melastomataceae (Myrtales) include trees and woody shrubs that are amongst the most common hosts of Chrysoporthe and related fungi. These fungi cause stem cankers, branch death and in extreme cases, kill their hosts. Chrysoporthe-like fungi were observed on Miconia spp. and Rhynchanthera grandiflora (Melastomataceae) plants during tree disease surveys in south-eastern Brazil including the states of Minas Gerais and Rio de Janeiro. The aims of this study were to isolate and identify the fungi utilising morphological characteristics and phylogenetic analyses. This led to the identification of a new species of Chrysoporthe described here as Chrysoporthe brasilensis sp.nov. Inoculations were conducted on R. grandiflora and M. theaezans, showing that C. brasiliensis is an aggressive pathogen. This study adds to a growing number of reports of new and pathogenic species of Chrysoporthe that potentially threaten native Myrtales globally, including important trees such as Eucalyptus, both in natural ecosystems and in planted forests., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2024 British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

Starting in the fall of 2019, mortality, blight symptoms, and signs of white fungal mycelia were observed on external host tissues of non-native landscape trees as well as numerous native trees, understory shrubs, and vines throughout northern and central Florida, USA. We determined that the fungus is an undescribed species of Basidiomycota based on morphological characteristics and DNA sequence analysis. Phylogenetic analyses of the internal transcribed spacer (ITS), large subunit (LSU), and translation elongation factor 1-alpha (tef1) regions revealed that this novel plant pathogen is an undescribed taxon of the genus Parvodontia (Cystostereaceae, Agaricales). We propose the name Parvodontia relampaga sp. nov. which describes its unique morphological features and phylogenetic placement. We confirmed the pathogenicity of P. relampaga in greenhouse inoculations on host plants from which strains of this novel pathogen were isolated, including the non-native gymnosperm Afrocarpus falcatus, the non-native and commercially important Ligustrum japonicum, and the native tree Quercus hemisphaerica. P. relampaga was also detected on a total of 27 different species of woody host plants, including such economically and ecologically important hosts as Fraxinus, Ilex, Magnolia, Persea, Prunus, Salix, Vitis, and Vaccinium. For this new plant disease, we propose the name "relampago blight," which refers to the lightning-like rhizomorph growth (relámpago means 'lightning' in Spanish). This study presents a newly discovered fungal taxon with a wide host range on both angiosperms and gymnosperms that may be an emerging pathogen of concern in Florida and the Gulf Coast region., Competing Interests: Declaration of competing interest The authors have no conflict of interest to declare., (Copyright © 2024 British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

Gremmenia abietis (Dearn.) Crous (syn: Phacidium abietis ) was originally described in North America to accommodate the species associated with snow blight of Abies and Pseudotsuga spp. In Japan, this species was first observed on the dead needles on Abies sachalinensis and Picea jezoensis var. jezoensis in 1969. However, the identity of Japanese species was unclear due to the lack of molecular data and the absence of anamorph description. In this study, we collected fresh specimens from various conifer species ( A. sachalinensis, A. veitchii, Pic. jezoensis var. jezoensis, Pic. jezoensis var. hondoensis, Pinus koraiensis , and Pin. pumila ) in Japan and revised the taxonomy based on morphological and phylogenetic analyses. Phylogenetic analyses based on nuc rDNA internal transcribed spacer ITS1-5.8S-ITS2 (ITS), nuc 28S rDNA (28S), and RNA polymerase II second largest subunit ( RPB2 ) regions indicated that the species belongs to Phacidiaceae. Conidiomata formed in vitro produced pyriform, hyaline conidia without mucoid appendage, which distinguished the species from phylogenetically related genera. Consequently, we established Chionobium takahashii to accommodate the snow blight fungus in Japan. Further phylogenetic analyses also indicated that C. takahashii includes several distinct clades corresponding to the host genera ( Abies, Picea, Pinus ). Morphological differences among those clades were unclear, suggesting that C. takahashii may contain host-specific cryptic species.

Purpose: The objective of this study is to study the secondary structure analysis of Fasciola flukes from a rare mithun host from Manipur. Fascioliasis, a neglected tropical trematodiasis, is poorly studied in India and is widely believed to be predominantly caused by F. gigantica. Through this study, we want to assess the flukes from the rare semi-wild ruminants of Northeast India. This study is important as the mithun population is semi-wild and its population is declining in Manipur., Methods: Sample collected from the difficult and challenging terrain of Northeast India. The sample was collected from mithun and observed under the microscope. DNA was isolated, sequenced, and analyzed using various bioinformatics tools. The secondary structure analysis of the Internal Transcribed Spacer 2 (ITS2) region was also performed., Results: The secondary structure species tree corroborated the Bayesian inference and, hence, strengthened the phylogeny reconstructed. The annotated ITS2 sequence and RNA secondary of the Manipur isolate displayed the typical four-helix or four-domain model. Helix III reveals the presence of the UGGU motif with other deviations like UGG and GGU., Conclusion: This is an in-depth analysis of the secondary structure of Fasciola species. The present study has demonstrated the usefulness of ITS2 and its secondary structures for characterizing parasites. The information on fascioliasis in the mithun's population presents itself useful with regards to their conservation strategy as their populations in both Manipur and Nagaland are dwindling., (© 2024. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

The majority of Candida species are known as non-pathogenic yeasts and rarely involved in human diseases. However, recently case reports of human infections caused by non-albicans Candida species have increased, mostly in immunocompromised hosts. Our study aimed to describe and characterize as thoroughly as possible, a new species of the Metschnikowia clade, named here Candida massiliensis (PMML0037), isolated from a clinical sample of human sputum. We targeted four discriminant genetic regions: "Internal Transcribed Spacers" of rRNA, D1/D2 domains (28S large subunit rRNA) and part of the genes encoding Translation Elongation Factor 1-α and β-tubulin2. The genetic data were compared to morphological characters, from scanning electron microscopy (TM 4000 Plus, SU5000), physiological, including the results of oxidation and assimilation tests of different carbon sources by the Biolog system, and chemical mapping by Energy-Dispersive X-ray Spectroscopy. Lastly, the in vitro antifungal susceptibility profile was performed using the E-test™ exponential gradient method. The multilocus analysis supported the genetic position of Candida massiliensis (PMML0037) as a new species of the Metschnikowia clade, and the phenotypic analysis highlighted its unique morphological and chemical profile when compared to the other Candida/Metschnikowia species included in the study., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

Haemonchus contortus is the most pathogenic and economically restrictive gastrointestinal nematode in the small ruminant industry globally. Morbidity, poor cross-bodily state, and mortality of sheep in Lesotho suggest the presence of H. contortus. The present study investigated the morphological, molecular, and population genetics of H. contortus third-stage larvae infecting sheep in four ecological zones (EZ) of Lesotho. Coprocultures were prepared for larval morphological identification and PCR determination. Larvae were identified morphologically as 100% H. contortus. The Second Internal Transcribed Spacer (ITS-2) gene of the ribosomal DNA of H. contortus isolates in the present study revealed nucleotide homology ranging from 97 to 100% when compared with selected GenBank reference sequences. Pairwise evolutionary divergence among H. contortus isolates was low, with 0.01318 recorded as the highest in the present study. Five haplotypes resulted from 14 Lesotho sequences. Haplotype diversity and nucleotide diversity were 0.76923 and 0.00590, respectively. Genetic differentiation among isolates was low but not statistically significant. An analysis of molecular variance revealed that most molecular variation was distributed within topographic populations at 94.79% (F ST  = 0.05206, p > 0.05) and 5.21% among populations. There was high gene flow and no definite population genetic structure among Lesotho isolates., Competing Interests: Declaration of Competing Interest The authors do not declare any competing interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Four yeast strains were isolated from the gut of stingless bee, collected in Churdhar, Himachal Pradesh, India. Physiological characterization, morphological examination, and sequence analysis of small subunit ribosomal RNA (18S rRNA) genes, internal transcribed spacer (ITS) region, and D1/D2 domain of the large subunit rRNA gene revealed that the four strains isolated from the gut of stingless bee belonged to the Debaryomyces clade. Strain CIG-23H T showed sequence divergence of 7.5% from Debaryomyces nepalensis JCM 2095 T , 7.8% from Debaryomyces udenii JCM 7855 T , and Debaryomyces coudertii JCM 2387 T in the D1/D2 domain. In the ITS region sequences, strain CIG-23H T showed a 15% sequence divergence from Debaryomyces nepalensis JCM 2095 T and Debaryomyces coudertii JCM 2387 T . In 18S rRNA gene sequence, the strain CIG-23H T showed 1.14% sequence divergence from Debaryomyces nepalensis JCM 2095 and and Debaryomyces coudertii JCM 2387, and 0.83% sequence divergence from Debaryomyces hansenii NRRL Y-7426. Strain CIG-23H T can utilize more carbon sources than closely related species. The findings suggest that strain CIG-23H T is a novel species of the genus Debaryomyces, and we propose to name it as Debaryomyces apis f.a., sp. nov. The holotype is CBS 16297 T , and the isotypes are MTCC 12914 T and KCTC 37024 T . The MycoBank number of Debaryomyces apis f.a., sp. nov. is MB836065. Additionally, a method using cresol red and Bromothymol blue pH indicator dyes was developed to screen for lipase producers, which is more sensitive and efficient than the currently used phenol red and rhodamine B dye-based screening methods, and avoids the problem of less differentiable zone of hydrolysis., (© 2023. The Author(s).)

Phylogenetic and morphological analyses on Rigidoporus were carried out. The genus Rigidoporus ( Hymenochaetales , Basidiomycota ), typified by R. microporus (Fr.) Overeem. (synonym Polyporus micromegas Mont.), was established by Murrill in 1905. The genus is mainly characterized by annual to perennial, resupinate, effused-reflexed to pileate or stipitate basidiomata with azonate or concentrically zonate and sulcate upper surface, a monomitic to pseudo-dimitic hyphal structure, simple-septate generative hyphae, and ellipsoid to globose basidiospores. Phylogeny on species of the genus is reconstructed with two loci DNA sequences including the internal transcribed spacer regions and the large subunit. Three new species in Rigidoporus are described and illustrated from Asia, and one new combination in the genus is proposed. The main morphological characteristics of the currently accepted species of Rigidoporus are provided., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Wang, Vlasák, Jin and Si.)

Two new species of Scytinostroma viz. S. acystidiatum and S. macrospermum , are described from southwest China. Phylogeny based on ITS + nLSU dataset demonstrates that samples of the two species form two independent lineages and are different in morphology from the existing species of Scytinostroma . Scytinostroma acystidiatum is characterized by resupinate, coriaceous basidiomata with cream to pale yellow hymenophore, a dimitic hyphal structure with generative hyphae bearing simple septa, the absence of cystidia, and amyloid, broadly ellipsoid basidiospores measuring 4.7-7 × 3.5-4.7 μm. Scytinostroma macrospermum is characterized by resupinate, coriaceous basidiomata with cream to straw yellow hymenophore, a dimitic hyphal structure with generative hyphae bearing simple septa, numerous cystidia embedded or projecting from hymenium, and inamyloid, ellipsoid basidiospores measuring 9-11 × 4.5-5.5 μm. The differences between the new species and morphologically similar and phylogenetically related species are discussed., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Zhang, Liu, Bian and Chen.)

This study looked for correlations between molecular identification, clinical manifestation, and morphology for Trichophyton interdigitale and Trichophyton mentagrophytes. For this purpose, a total of 110 isolates were obtained from Czech patients with various clinical manifestations of dermatophytosis. Phenotypic characters were analyzed, and the strains were characterized using multilocus sequence typing. Among the 12 measured/scored phenotypic features, statistically significant differences were found only in growth rates at 37 °C and in the production of spiral hyphae, but none of these features is diagnostic. Correlations were found between T. interdigitale and higher age of patients and between clinical manifestations such as tinea pedis or onychomychosis. The MLST approach showed that internal transcribed spacer (ITS) genotyping of T. mentagrophytes isolates has limited practical benefits because of extensive gene flow between sublineages. Based on our results and previous studies, there are few taxonomic arguments for preserving both species names. The species show a lack of monophyly and unique morphology. On the other hand, some genotypes are associated with predominant clinical manifestations and sources of infections, which keep those names alive. This practice is questionable because the use of both names confuses identification, leading to difficulty in comparing epidemiological studies. The current identification method using ITS genotyping is ambiguous for some isolates and is not user-friendly. Additionally, identification tools such as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry fail to distinguish these species. To avoid further confusion and to simplify identification in practice, we recommend using the name T. mentagrophytes for the entire complex. When clear differentiation of populations corresponding to T. interdigitale and Trichophyton indotineae is possible based on molecular data, we recommend optionally using a variety rank: T. mentagrophytes var. interdigitale and T. mentagrophytes var. indotineae., (© The Author(s) 2023. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

The genes coding for the rRNAs seem evolutionary conserved on the first glance, but astonish one with their variability in the structure and a variety of functions on closer examination. The non-coding parts of rDNA contain regulatory elements, protein binding sites, pseudogenes, repetitive sequences, and microRNA genes. Ribosomal intergenic spacers are not only in charge with the nucleolus morphology and functioning, namely, the rRNA expression and ribosome biogenesis, but also control nuclear chromatin formation thus mediating cell differentiation. The alterations in the expression of these non-coding regions of rDNA in response to environmental stimuli underlie the keen sense of a cell to various types of stressors. Malfunctioning of this process may result in a wide range of pathologies from oncology to neurodegenerative disease and mental illness. Here, we observe to-date materials on the structure and transcription of the ribosomal intergenic spacer in humans and its role in rRNA expression, in-born disease development, and cancer.

At present, 25 species are accepted in Haploporus and are distributed in Asia, Europe, North America, South America, Australia, and Africa. In this study, two new species, Haploporus ecuadorensis from Ecuador and H. monomitica from China, are described and illustrated based on morphological examination and phylogenetic analyses. H. ecuadorensis is characterized by annual, resupinate basidiomata with pinkish buff to honey yellow hymenophore when dry, round to angular pores of 2-4 per mm, a dimitic hyphal structure with generative hyphae bearing clamp connections, hyphae at dissepiment edge usually with one or two simple septa, the presence of dendrohyphidia and cystidioles, and oblong to ellipsoid basidiospores measuring 14.9-17.9 × 6.9-8.8 µm. Haploporus monomitica differs from other Haploporus species in that it has a monomitic hyphal system and strongly dextrinoid basidiospores. The differences between the new species and morphologically similar and phylogenetically related species are discussed. In addition, an updated key to 27 species of Haploporus is provided., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Man, Dai, Bian, Zhou, Zhao and Vlasák.)

Anisakis nematodes infecting Indian mackerel ( Rastrelliger kanagurta ) were initially discovered in Thailand in our preliminary investigation. Nevertheless, the species of Anisakis collected has not been determined nor has its genetic variation been researched. Thus, this study aimed to molecularly identify the species of Anisakis specimens using the internal transcribed spacer (ITS) region of ribosomal DNA sequences. In addition, the intraspecific genetic variation was also determined using mitochondrial cytochrome oxidase subunit II (COII) gene sequences. The phylogenetic relationships of the ITS region classified all samples into Anisakis typica ; however, the genetic variation between them could not be distinguished. By contrast, the phylogenetic tree analysis of the COII region identified all samples as A . typica , with 17 different haplotypes by 66 polymorphic sites and five of the substitutions resulted in amino acid change. Additionally, the distribution pattern of the COII region can be separated into two groups between South America and Asian countries. All our haplotypes belong to Asian countries. Compared with the two genetic markers used in this investigation, COII appears to be a better candidate for studying genetic variation sensitive to environmental changes and intermediate or definitive host behavioral changes., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2022 Tanawat Chaiphongpachara et al.)

Background: There is limited clinical or epidemiological knowledge regarding Bartonella infection in cats, and no serological studies have compared the presence of antibodies against different Bartonella species. Moreover, there are limited feline Bartonella studies investigating co-infections with other vector-borne pathogens and the associated risk factors. Therefore, the objective of this study was to investigate Bartonella spp. infections and co-infections with other pathogens in cats from Barcelona (Spain) based on serological and/or molecular techniques and to determine associated risk factors., Methods: We studied colony and owned cats (n = 135). Sera were tested for Bartonella henselae-, Bartonella quintana-, and Bartonella koehlerae-specific antibodies using endpoint in-house immunofluorescence antibody assays. Bartonella real-time PCR (qPCR) and conventional PCR (cPCR) were performed. In addition, cPCR followed by DNA sequencing was performed for other pathogenic organisms (Anaplasma, Babesia, Cytauxzoon, Ehrlichia, Hepatozoon, hemotropic Mycoplasma, and Theileria spp.)., Results: From 135 cats studied, 80.7% were seroreactive against at least one Bartonella species. Bartonella quintana, B. koehlerae, and B. henselae seroreactivity was 67.4, 77.0, and 80.7%, respectively. Substantial to almost perfect serological agreement was found between the three Bartonella species. Colony cats were more likely to be Bartonella spp.-seroreactive than owned cats. Moreover, cats aged ≤ 2 years were more likely to be Bartonella spp.-seroreactive. Bartonella spp. DNA was detected in the blood of 11.9% (n = 16) of cats. Cats were infected with B. henselae (n = 12), B. clarridgeiae (n = 3), and B. koehlerae (n = 1). Mycoplasma spp. DNA was amplified from 14% (n = 19) of cat blood specimens. Cats were infected with Mycoplasma haemofelis (n = 8), Candidatus M. haemominutum (n = 6), Candidatus Mycoplasma turicensis (n = 4), and Mycoplasma wenyonii (n = 1). Anaplasma, Babesia, Cytauxzoon, Ehrlichia spp., Hepatozoon, and Theileria spp. DNA was not amplified from any blood sample. Of the 16 Bartonella spp.-infected cats based on PCR results, six (37%) were co-infected with Mycoplasma spp., Conclusions: Bartonella spp. and hemoplasma infections are prevalent in cats from the Barcelona area, whereas infection with Anaplasma spp., Babesia, Cytauxzoon, Ehrlichia spp., Hepatozoon, and Theileria infections were not detected. Co-infection with hemotropic Mycoplasma appears to be common in Bartonella-infected cats. To our knowledge, this study is the first to document M. wenyonii is infection in cats., (© 2021. The Author(s).)

Neopsilotrema is a small genus of psilostomid digeneans parasitic in the intestine of birds in the Palearctic and Nearctic. At present, the genus includes 4 species: Neopsilotrema lisitsynae from the Palearctic and Neopsilotrema affine, Neopsilotrema lakotae, and Neopsilotrema marilae from the Nearctic. Herein, we describe a new species, Neopsilotrema itascae n. sp., from lesser scaup Aythya affinis collected in Minnesota. The species can be distinguished from congeners on the basis of the ventral sucker:oral sucker width ratio, body width:length ratio, and cirrus sac size, along with other characters. We generated new 28S ribosomal deoxyribonucleic acid (DNA) and NADH dehydrogenase (ND1) mitochondrial DNA sequence data of a variety of psilostomids from the Palearctic and Nearctic along with sequences of the ribosomal internal transcribed spacer (ITS) region (ITS1 + 5.8S + ITS2) from 3 Neopsilotrema species. The molecular phylogenetic affinities of a variety of psilostomid taxa were studied using 28S sequence data. The 28S sequences of psilostomids demonstrated 1-7.9% intergeneric divergence, whereas the sequences of ND1 had 17.7-34.1% intergeneric divergence. The interspecific divergence among members of Neopsilotrema was somewhat lower (0.2-0.5% in 28S; 0.3-0.4% in ITS; 12-15.7% in ND1). Our comparison of DNA sequences along with morphologic study suggests Holarctic distribution of N. lisitsynae., (© American Society of Parasitologists 2021.)

A freshwater dwelling cyanobacterium (strain MKW3) was isolated from a sample collected from a water logged sugarcane field located in Malkapur, Karad, Maharashtra, India, and was characterized using a polyphasic approach. In the 16S rRNA gene phylogenetic analysis, strain MKW3 clustered with two misidentified strains-Nostoc sp. CENA239 and Calothrix sp. NIES2100. The phylogenetically related members included strains identified as Nostoc, Aulosira, Calothrix, Tolypothrix, Camptylonemopsis and Microchaete. The phylogenetic and the morphological analysis of the strain MKW3 indicated that it does not belong to any of the above mentioned genera. Furthermore, the 16S-23S ITS secondary structure analysis provided clear evidence indicating that strain MKW3 is different from Nostoc sp. CENA239 and Calothrix sp. NIES2100. Based on the morphological, phylogenetic and 16S-23S ITS secondary structure analysis we describe our strain as Constrictifilum karadense gen. et sp. nov. in accordance with the International Code of Nomenclature for algae, fungi and plants., (© The Author(s) 2021. Published by Oxford University Press on behalf of FEMS. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Species of the genus Coleps are one of the most common planktonic ciliates in lake ecosystems. The study aimed to identify the phenotypic plasticity and genetic variability of different Coleps isolates from various water bodies and from culture collections. We used an integrative approach to study the strains by (i) cultivation in a suitable culture medium, (ii) screening of the morphological variability including the presence/absence of algal endosymbionts of living cells by light microscopy, (iii) sequencing of the SSU and ITS rDNA including secondary structures, (iv) assessment of their seasonal and spatial occurrence in two lakes over a one-year cycle both from morphospecies counts and high-throughput sequencing (HTS), and, (v) proof of the co-occurrence of Coleps and their endosymbiotic algae from HTS-based network analyses in the two lakes. The Coleps strains showed a high phenotypic plasticity and low genetic variability. The algal endosymbiont in all studied strains was Micractinium conductrix and the mutualistic relationship turned out as facultative. Coleps is common in both lakes over the whole year in different depths and HTS has revealed that only one genotype respectively one species, C. viridis, was present in both lakes despite the different lifestyles (mixotrophic with green algal endosymbionts or heterotrophic without algae). Our results suggest a future revision of the species concept of the genus Coleps.

Despite possible drawbacks (intraspecific polymorphisms and possible fungal contamination), sequencing of the ITS region of the ribosomal RNA genes remains one of the most popular nuclear sequences used for plant taxonomy and phylogeny. A protocol for PCR amplification and sequencing of this region using universal plant primers is provided.

Many early studies of ribosomal RNA gene (rDNA) suggested that rDNA tandem repeats within species are homogeneous. However, increasing number of reports have found intra-individual rDNA polymorphism across a range of taxa. Here, we reported a high level of intra-individual polymorphism of 18S-ITS1-5.8S rDNA in the genome of Cynoglossus melampetalus (Pleuronectiformes: Cynoglossidae), indicating a non-concerted evolution manner. Sequence alignments found two distinct types of 18S and 5.8S (Type A and B) and five types of ITS1 sequence (Type A - E) coexisted in the genome differing in length, GC content, secondary structure stability and minimum free energy. Based on the unique features of pseudogene and comparison of the conserved 18S rDNA sequence and 5.8S secondary structure of 22 flatfishes revealed that Type B sequences of 18S, 5.8S and their linked ITS1 were putative pseudogenes. So far, detection of rRNA pseudogenes from the multiple rDNA copies has been an intricate puzzle. Our results, as a result, provide a new ideal for rRNA pseudogene identification., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The crucial function of the internal transcribed spacer 2 (ITS2) region in ribosome biogenesis depends on its secondary and tertiary structures. Despite rapidly evolving, ITS2 is under evolutionary constraints to maintain the specific secondary structures that provide functionality. A link between function, structure and evolution could contribute an understanding to each other and recently has created a growing point of sequence-structure phylogeny of ITS2. Here we briefly review the current knowledge of ITS2 processing in ribosome biogenesis, focusing on the conservative characteristics of ITS2 secondary structure, including structure form, structural motifs, cleavage sites, and base-pair interactions. We then review the phylogenetic implications and applications of this structure information, including structure-guiding sequence alignment, base-pair mutation model, and species distinguishing. We give the rationale for why incorporating structure information into tree construction could improve reliability and accuracy, and some perspectives of bioinformatics coding that allow for a meaningful evolutionary character to be extracted. In sum, this review of the integration of function, structure and evolution of ITS2 will expand the traditional sequence-based ITS2 phylogeny and thus contributes to the tree of life. The generality of ITS2 characteristics may also inspire phylogenetic use of other similar structural regions.

A new fast-growing mycobacterium, designated strain QGD101T, was isolated from the sputum of an 84-year-old man suspected of tuberculosis in Wuhan Medical Treatment Center, Hubei, China. This strain was a gram-staining-negative, aerobic, non-spore-forming and catalase-positive bacterium, which was further identified as the NTM by PNB and TCH tests. The moxifloxacin and levofloxacin exhibited strong suppressing function against QGD101T with MIC values of 0.06 and 0.125 µg/ml after drug susceptibility testing of six main antimicrobial agents on mycobacteria. Based on the sequence analysis of 16S rRNA, rpoB, hsp65 and 16S-23S rRNA internal transcribed spacer, the strain QGD101T could not be identified to a species level. Mycobacterium moriokaense ATCC43059T that shared the highest 16S rRNA gene sequence similarity (98%) with strain QGD101T was actually different in genomes average nucleotide identity (78.74%). In addition, the major cellular fatty acids of QGD101T were determined as C18:1ω9c, C16:0 and C18:2ω6c. The DNA G + C content was 64.9% measured by high performance liquid chromatography. Therefore, the phenotypic and genotypic characterisation of this strain led us to the conclusion that it represents a novel species of mycobacteria, for which the name Mycobacterium hubeiense sp. nov. (type strain QGD101T = CCTCCAA 2017003T = KCTC39927T) was proposed. Thus, the results of this study are very significant for the clinical diagnosis of tuberculosis and future personalised medicine.

Giardia duodenalis is one of the most important intestinal parasites globally, especially in children, and in Cuba is the leading cause of chronic paediatric diarrhoea in this population. G. duodenalis is composed of eight genetic groups (or assemblages), two of which (A and B) are apparently zoonotic, occurring in both humans and other animals. However, consensus on the most appropriate genotyping scheme for optimal characterization of G. duodenalis isolates is lacking. In this article we present the results of three descriptive observational studies conducted in Havana, Cuba between 2010 and 2013, with the aim of comparing the results from molecular (PCR) approaches targeting different genes in order to assign with confidence 224 isolates of G. duodenalis to the correct assemblages. In each sub-study, following DNA isolation by the phenol/chloroform/isoamyl alcohol extraction method, PCR targeting the triose phosphate isomerase (tpi) gene was used for molecular characterization, as well as one additional PCR-method targeting another gene or pair of genes. DNA amplification was obtained in 87%, 83%, and 80% in the three sub-studies. Although excellent agreement (kappa index = 1) was recorded between results from some pairs of genes, for other combinations only moderate or substantial agreement was achieved. These results highlight the importance of interpretation of genotyping data, especially when single genetic markers are used. From the results of our studies, PCR targeting a combination of the tpi gene and the intergenic spacer region of rDNA may be a useful approach for the molecular characterization of G. duodenalis isolates., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Outbreaks of avian trichomonosis are being reported worldwide; meanwhile, the genetic and virulence variations are under investigation. In this study, the occurrence and genetic variability of oral or faecal trichomonads among various avian species were investigated. Samples obtained from either the oropharyngeal cavity, crop/oesophagus, droppings/cloaca, or conjunctival swabs of avian species were inspected for flagellates. Phylogenetic analysis of partial ITS1-5.8s rRNA-ITS2 sequences from selected samples was performed to investigate the genetic diversity of the isolates. Investigation of 737 birds revealed an infection rate of 15.7% in the upper gastrointestinal tract, 7.3% in the faecal samples, and 0.7% involvement of the conjunctiva. Phylogenetic analysis of partial ITS1-5.8s rRNA-ITS2 sequences from selected samples, identified genotypes A and B of Trichomonas gallinae and genogroups A-C and E of Tetratrichomonas gallinarum . A novel ITS genotype of intestinal trichomonads was also detected in hooded crow ( Corvus cornix ) and common mynah ( Acridotheres tristis ). In the present study, in addition to Columbiformes and Falconiformes, trichomonads were detected in Passeriformes and Galliformes with the involvement of organs other than the gastrointestinal tract. Genotype A T. gallinae was detected in domestic pigeons ( Columba livia domestica ), a laughing dove ( Spilopelia senegalensis ), a common kestrel ( Falco tinnunculus ), a budgerigar ( Melopsittacus undulates ), and a canary ( Serinus canaria ). Distinct genotype B was detected in a common mynah and a budgerigar. Genogroups A-C of T. gallinarum were also demonstrated in Galliformes and Anseriformes. Furthermore, two novel trichomonad ITS genotypes were detected in hooded crows and a common mynah warranting detailed multi-locus molecular analysis. RESEARCH HIGHLIGHTS ITS diversity of trichomonads was shown in various avian species.Diversity of the parasites' target organ and clinical manifestations was demonstrated.Two novel ITS genotype trichomonads from common mynah and hooded crow were identified.

The genus Eustrongylides includes nematodes known as the etiological agent of the "big red worm disease." The aim of this work was to identify Eustrongylides spp. larvae from fish and adults from great cormorant ( Phalacrocorax carbo ) sampled at Lake Trasimeno, Italy, by morphological and molecular analysis. Histopathological description of the lesions in birds was also provided. We described adults of Eustrongylides excisus for the first time in Italy, and we also linked larval stages 3 and 4 to adults. The use of molecular tools combined with the traditional taxonomy will help the identification of the species, including species inquirendae. Moreover, molecular analysis can also help to investigate the role of intermediate and paratenic hosts, to deepen the knowledge about geographical distribution of the different Eustrongylides spp. and to define the zoonotic potential of E. excisus , which has not yet been identified as causal agent of human cases.

The diversity of the fungal community associated with adults of Dryocosmus kuriphilus following emergence was examined using HTS analysis. Ascomycota dominated the fungal core-biome community. The functional guilds of the 90 taxa forming the core-biome were assessed, demonstrating three main groups: saprotrophs, plant pathogens and entomopathogens. Twenty-nine OTUs out of 90 were resolved to species level identifying 26 different fungal species. Among these species, many were cosmopolitan or previously recorded in Europe. Ten taxa were previously recorded on chestnut, including some recognized plant pathogens associated with foliage and green tissues such as Epicoccum nigrum, Gnomoniopsis castanea, Colletotrichum acutatum, Stromatoseptoria castaneicola, Ramularia endophylla. Beauveria bassiana; within the core microbiome, Fusarium larvarum represented the most abundant entomopathogenic species. Some of these species are known to impact directly or indirectly the vitality of the insects in the galls. The chestnut blight pathogen, Cryphonectria parasitica, was never found associated with D. kuriphilus. Based on the present study, an active role for D. kuriphilus as a vector of chestnut fungal endophyte/pathogens cannot be demonstrated but neither ruled out., (Copyright © 2019 British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

For decades entomopathogenic fungi have garnered interest as possible alternatives to chemical pesticides. However, their ecology outside of agroecosystems demands further study. We assessed the diversity and abundance of entomopathogenic and insect-associated fungi at a loblolly pine forest in North Carolina, USA using culture-dependent and next-generation sequencing libraries. Fungi were isolated using Galleriamellonella larvae, as well as from soil dilutions plated on a selective medium. Isolates were identified using Sanger sequencing of the ITS and LSU rRNA gene regions, and represented 36 OTUs including Metarhizium, Lecanicillium, and Paecilomyces. Additionally, we assessed the chitinolytic potential of isolates and found widespread, variable ability to degrade chitin within and between genera. Phylogenetic analyses resolved several isolates to genus, with some forming clades with other insect-associated taxa, as well as with fungi associated with plant tissues. Saprophytes were widely distributed in soil, while entomopathogens were less abundant and present primarily in the top two cm of the soil. The similarity between culture-dependent and next-generation sequencing results demonstrates that both methods can be used concurrently in this system to study the ecology of entomopathogenic fungi., (Copyright © 2019 British Mycological Society. All rights reserved.)

Acacia koa and A. heterophylla are commonly occurring native trees on the Hawaiian Islands and La Réunion, respectively. A recent phylogenetic study suggested that A. heterophylla renders A. koa paraphyletic, and that the former likely arose from the Hawaiian Islands around 1.4 million years ago. An intriguing question is whether their microbiota is similar, although they occur naturally in two very distant geographical locations. In this study, we compared the fungi in the Botryosphaeriaceae isolated from natural populations of A. koa and A. heterophylla. These fungi were chosen because they commonly occur on woody plants and some are important pathogens. They are also known to have been moved globally on asymptomatic plant materials. Isolates were identified based on comparisons of DNA sequence data for the rDNA-ITS, TEF1-α and β-tubulin loci. Ten Botryosphaeriaceae species were identified, of which four species were specific to A. koa from the Hawaiian Islands and five to A. heterophylla in La Réunion. Only one species, Neofusicoccumparvum, which is known to have a wide global distribution, was common to both hosts. The overall results of this study suggest that although A. koa and A.heterophylla share a recent evolutionary history, they have established independent microbiota, at least in terms of the Botryosphaeriaceae., (Copyright © 2019 British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

Paragnomonia fragariae is a plant pathogenic ascomycete causing root rot and petiole blight of perennial strawberry in northern Europe. This paper provides a revised description of Paragnomonia and P. fragariae with lecto- and epitypification based on the species original description, recent collections from four European countries, examination of specimens used in the previous taxonomic studies and phylogenetic analyses of DNA sequences of LSU, ITS/5.8S and tef1-α. This study presents the first report of P. fragariae on cultivated strawberry in Finland and Lithuania. Our study on growth rate showed that P. fragariae is a cold-adapted fungus growing almost equally at 5 °C as at 20 °C and attaining maximal growth at 15 °C. New primers were designed for amplification of ca. 0.8 kb fragment of tef1-α of Sydowiella fenestrans. Additionally, newly generated sequences of tef1-α were obtained for the first time from 21 isolates of seven species belonging to five genera of Sydowiellaceae, including the type species S. fenestrans, therefore considerably contributing to the current knowledge on phylogenetic relationships of this insufficiently studied group of fungi. The phylogenetic analysis has also revealed that the recently described species "S." centaureii is genetically distant from the generic type S. fenestrans and other Sydowiella., (Copyright © 2019 British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

A PCR targeting mitochondrial cytochrome oxidase subunit III (cox3) for molecular detection of Babesia gibsoni infection in dogs has been developed in this study. Fifty blood samples from suspected clinical cases from dogs, brought to the veterinary college clinics, were examined for presence of B. gibsoni using conventional diagnosis by microscopic examination of Giemsa stained thin blood smears. In addition, species specific PCRs targeting ITS-1 region (BgITS-1 PCR) and nested PCR targeting 18S ribosomal RNA gene (Bg18SnPCR) were carried out. A 634 bp PCR fragment of B. gibsoni cox3 gene was amplified in positive samples from three geographical locations of Satara, Wai and Pune in Maharashtra state of India. From analysis of the sequence of the B. gibsoni cox3 gene, we found that the Indian isolate had 96-98% similarity to the isolate from Japan and China. Post sequencing, de-novo diagnostic primer pair for species specific amplification of 164 bp fragment of B. gibsonicox3 was designed and the PCR was standardized. The diagnostic results of de-novo Bgcox3 PCR were compared with BgITS-1 PCR and Bg18S nPCR. Thin blood smears detected 22% (11/50) samples positive for small form of Babesia species. The BgITS-1 PCR detected 25% samples (15/50) as positive and Bg18S nPCR detected 80% (40/50) B. gibsoni positive samples. The de-novo Bgcox3 PCR detected 66% (33/50) samples positive for B. gibsoni (at 95% CI). The analytical sensitivity of cox3 PCR was evaluated as 0.000003% parasitaemia or 09 parasites in 100  μl of blood. The de-novo diagnostic cox3 PCR did not cross react with control positive DNA from other haemoprotozoa and rickettsia like B. vogeli, Hepatozoon canis, Trypanosoma evansi, Ehrlichia canis and Anaplasma platys. Statistically, cox3 PCR had better diagnostic efficiency than ITS-1 PCR in terms of sensitivity (p = 0.0006). No statistically significant difference between results of cox3 PCR and 18S nPCR was observed (p = 0.1760). Kappa values estimated for each test pair showed fair to moderate agreement between the observations. Specificity of Bgcox3 PCR was 100% when compared with microscopy or BgITS-1 PCR. Sensitivity of Bgcox3 PCR was 100% when compared with that of Bg18S nPCR., (Copyright © 2019 Elsevier Inc. All rights reserved.)

We have validated ITS-2 rDNA nemabiome next-generation amplicon sequencing to determine relative species abundance of gastrointestinal nematode species in ovine fecal samples. In order to determine species representation biases, ITS-2 rDNA amplicon sequencing was applied to mock communities or field populations with known proportions of L3 for eight of the major ovine gastrointestinal nematode species: Teladorsagia circumcincta, Trichostrongylus vitrinus, Haemonchus contortus, Cooperia curticei, Trichostrongylus axei, Trichostrongylus colubriformis, Chabertia ovina and Oesophagostumum venulosum. Correction factors, calculated from this data, were shown to reduce species representation biases when applied to an independent set of field samples of known composition. We compared ITS-2 rDNA amplicon sequencing data that was generated from harvested eggs, freshly hatched L1 or L3 larvae following fecal culture and no statistically significant differences were found for the more abundant parasite species. We then applied the validated ITS-2 rDNA nemabiome amplicon sequencing assay to a set of archived L1 gastrointestinal nematode populations, collected in 2008 from fecal samples from 93 groups of 20 ewes and 61 groups of 20 lambs derived from 99 UK sheep farms. The presence of the major gastrointestinal nematode species had previously been determined on this large sample set by species-specific PCR. We show how the ITS-2rDNA amplicon sequencing data provided much more detailed information on species abundance than the previous species-specific PCR. This new data represents the most comprehensive overview of the relative abundance of the major gastrointestinal nematode species across UK sheep farms to date. Substantial variance in the relative abundance of both T. circumcincta and T. vitrinus between farms was revealed with the former species being of statistically significantly higher abundance in all three regions sampled (England, Scotland and Wales). The data also revealed that the relative abundance of T. circumcinta was statistically significantly higher in ewes than in lambs with the opposite pattern being the case for T. vitrinus. The nemabiome sequencing data also clearly illustrated the sporadic nature and skewed distribution of H. controtus across UK sheep farms as well as a higher relative abundance on farms from England compared to Wales and Scotland. The nemabiome survey also provides the first widescale data on the relative abundance of the two major large intestinal nematodes C. ovina and O. venulosum. This work validates ITS-2 rDNA nemabiome sequencing for use in sheep and illustrates the power of the approach for large scale surveillance of ovine gastrointestinal nematodes., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Seaweeds have been used as a source of natural fertilizer and biostimulant in agriculture for centuries. However, their effects on soil and crop root microbiota remain unclear. Here, we used a commercially available Ascophyllum nodosum extract (ANE) to test its effect on bacterial and fungal communities of rhizospheric soils and roots of pepper and tomato plants in greenhouse trials. Two independent trials were conducted in a split-block design. We used amplicon sequencing targeting fungal ITS and bacterial 16S rRNA gene to determine microbial community structure changes. We find that productivity parameters of root, shoot and fruit biomass were positively and significantly influenced by the ANE amendment. In addition, a-diversity differed significantly between amended and control plants, but only in some of the experimental conditions. Species composition among sites (b-diversity) differed according to the amendment treatment in all four communities (fungal-root, fungal-soil, bacterial-root and bacterial-soil). Finally, we identified a number of candidate taxa most strongly correlated with crop yield increases. Further studies on isolation and characterization of these microbial taxa linked to the application of liquid seaweed extract may help to enhance crop yield in sustainable agro-ecosystems., (© 2019 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.)

We explore the growing issue of cryptic speciation in the Digenea through study of museum material and newly collected specimens consistent with the enenterid genus Koseiria from five species of the Kyphosidae and Chaetodontoplus meredithi Kuiter (Pomacanthidae) collected in the Indo-Pacific. We use an integrated approach, employing traditional morphometrics, principal components analysis (PCA), and molecular data (ITS2 and 28S rDNA). Our results support recombination of Koseiria allanwilliamsi Bray & Cribb, 2002 as Proenenterum allanwilliamsi (Bray & Cribb, 2002) n. comb. and transfer of Koseiria huxleyi Bray & Cribb, 2001 to a new genus as Enenterageitus huxleyi (Bray & Cribb, 2002) n. comb. Molecular data indicate the presence of four further species consistent with Koseiria, one from Western Australia (sequence data only) and three from eastern Australia. All three eastern Australian species are morphologically consistent with Koseiria xishaensis Gu & Shen, 1983, but distinct from all other previously described species. Although K. xishaensis has been reported from Australia, we conclude that the similarity of the present forms to the original description of K. xishaensis means records of this species from Japan, Palau and Australia are unreliable. Because the eastern Australian forms cannot be reliably ascribed to K. xishaensis, we describe Koseiria argalea n. sp., Koseiria laiphopharophora n. sp., and Koseiria pyknophora n. sp., following application of PCAs and iterative refinement of species concepts and type series. These analyses did not allow convincing identification hypotheses for all specimens examined. In this genus, both morphological and molecular data, together with reliable host identifications, are essential for species recognition, and thus we refrain from attempting to name samples lacking molecular data. The issues presented by these taxa encapsulate those of trematodes in the region as a whole. Many records require dramatically improved supporting data, leading to substantial uncertainly in the identification of this fauna., (Copyright © 2019 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)

Background: Ribosomal DNA (rDNA) repeats are situated in the nucleolus organizer regions (NOR) of chromosomes and transcribed into rRNA for ribosome biogenesis. Thus, they are an essential component of eukaryotic genomes. rDNA repeat units consist of rRNA gene clusters that are transcribed into single pre-rRNA molecules, each separated by intergenic spacers (IGS) that contain regulatory elements for rRNA gene cluster transcription. Because of their high repeat content, rDNA sequences are usually absent from genome assemblies. In this work, we used the long-read sequencing technology to describe the chicken IGS and fill the knowledge gap on rDNA sequences of one of the key domesticated animals., Methods: We used the long-read PacBio RSII technique to sequence the BAC clone WAG137G04 (Wageningen BAC library) known to contain chicken NOR elements and the HGAP workflow software suit to assemble the PacBio RSII reads. Whole-genome sequence contigs homologous to the chicken rDNA repetitive unit were identified based on the Gallus_gallus-5.0 assembly with BLAST. We used the Geneious 9.0.5 and Mega software, maximum likelihood method and Chickspress project for sequence evolution analysis, phylogenetic tree construction and analysis of the raw transcriptome data., Results: Three complete IGS sequences in the White Leghorn chicken genome and one IGS sequence in the red junglefowl contig AADN04001305.1 (Gallus_gallus-5.0) were detected. They had various lengths and contained three groups of tandem repeats (some of them being very GC rich) that form highly organized arrays. Initiation and termination sites of rDNA transcription were located within small and large unique regions (SUR and LUR), respectively. No functionally significant sites were detected within the tandem repeat sequences., Conclusions: Due to the highly organized GC-rich repeats, the structure of the chicken IGS differs from that of IGS in human, apes, Xenopus or fish rDNA. However, the chicken IGS shares some molecular organization features with that of the turtles, which are other representatives of the Sauropsida clade that includes birds and reptiles. Our current results on the structure of chicken IGS together with the previously reported ribosomal gene cluster sequence provide sufficient data to consider that the complete chicken rDNA sequence is assembled with confidence in terms of molecular DNA organization.

The wild legume Lotus japonicus engages in mutualistic symbiotic relationships with arbuscular mycorrhiza (AM) fungi and nitrogen-fixing rhizobia. Using plants grown in natural soil and community profiling of bacterial 16S rRNA genes and fungal internal transcribed spacers (ITSs), we examined the role of the Lotus symbiosis genes RAM1 , NFR5 , SYMRK , and CCaMK in structuring bacterial and fungal root-associated communities. We found host genotype-dependent community shifts in the root and rhizosphere compartments that were mainly confined to bacteria in nfr5 or fungi in ram1 mutants, while symrk and ccamk plants displayed major changes across both microbial kingdoms. We observed in all AM mutant roots an almost complete depletion of a large number of Glomeromycota taxa that was accompanied by a concomitant enrichment of Helotiales and Nectriaceae fungi, suggesting compensatory niche replacement within the fungal community. A subset of Glomeromycota whose colonization is strictly dependent on the common symbiosis pathway was retained in ram1 mutants, indicating that RAM1 is dispensable for intraradical colonization by some Glomeromycota fungi. However, intraradical colonization by bacteria belonging to the Burkholderiaceae and Anaeroplasmataceae is dependent on AM root infection, revealing a microbial interkingdom interaction. Despite the overall robustness of the bacterial root microbiota against major changes in the composition of root-associated fungal assemblages, bacterial and fungal cooccurrence network analysis demonstrates that simultaneous disruption of AM and rhizobium symbiosis increases the connectivity among taxa of the bacterial root microbiota. Our findings imply a broad role for Lotus symbiosis genes in structuring the root microbiota and identify unexpected microbial interkingdom interactions between root symbionts and commensal communities. IMPORTANCE Studies on symbiosis genes in plants typically focus on binary interactions between roots and soilborne nitrogen-fixing rhizobia or mycorrhizal fungi in laboratory environments. We utilized wild type and symbiosis mutants of a model legume, grown in natural soil, in which bacterial, fungal, or both symbioses are impaired to examine potential interactions between the symbionts and commensal microorganisms of the root microbiota when grown in natural soil. This revealed microbial interkingdom interactions between the root symbionts and fungal as well as bacterial commensal communities. Nevertheless, the bacterial root microbiota remains largely robust when fungal symbiosis is impaired. Our work implies a broad role for host symbiosis genes in structuring the root microbiota of legumes., (Copyright © 2019 Thiergart et al.)

Operational taxonomic units 94%-95% similar to the known Pedinophyceae were found as a result of high-through sequencing of 18S rDNA V4 amplicons of environmental DNA from the summer picophytoplankton samples from the White Sea. Partial sequence of a ribosomal operon (the 5,298 bp includes partial 18S and 28S rDNA, complete 5.8S rDNA, ITS1, and ITS2 sequences) and a partial 2,112 bp chloroplast 23S rDNA sequence White Sea Pedinophyceae was amplified from metagenomic DNA by specific primers and sequenced. A new phylotype was designated as uncultured Pedinophyceae WS. On Chlorophyta phylogenetic trees the discovered phylotype occupies a basal position in the Marsupiomonadales clade. The synapomorphic base substitutions in rRNA hairpins confirm the relationship of Pedinophyceae WS to Marsupiomonadales and its difference from known genera of the order. The obtained results extend knowledge of picophytoplankton diversity in subarctic waters., (© 2019 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

Himalayan range of Pakistan is one of the diversity rich hotspots of the world. Many areas are yet to be explored here to discover new species of organisms including fungi. During present research, Swat District of Pakistan was explored for fungal diversity. One new species of mushroom Russula swatica (Russulales) is described from Himalayan range of Pakistan and analyzed by light microscopy and scanning electron microscopy as well as by molecular markers. A comprehensive description, photographs, and comparisons with morphologically similar and phylogenetically related species are provided. Conclusions of its phylogenetic relationships within the genus are provided based on the sequence of the nuclear internal transcribed spacer region., (© 2019 Wiley Periodicals, Inc.)

Mycelial basidiomycetes rarely produce mycoses in animals including humans. We report a case of a 9-year-old female mongrel dog with lesions in the prescapular lymph nodes. The histopathology of a lymph node sample showed flexuous septate hyphae, and a sterile mold grew in culture from that specimen. DNA sequencing of the ITS region allowed us to identify the fungus as Tropicoporus tropicalis. The dog was treated with itraconazole, but it was euthanized six months later due to an unfavorable clinical outcome. Tropicoporus tropicalis is an infrequent pathogen of pets, and the use of molecular tools is needed for its identification. Animal infections due to T. tropicalis were not previously been reported in Argentina.

Internal Transcribed Spacer structures are important in preserving accessibility to specific enzymes for the maturation of rRNAs. ITS1 sequences reported in the literature in Crustaceans range between 182 and 820 bp and are characterized by the absence of repeats or the presence of only a limited number of microsatellites. Here, we sequenced ITS1 for a range of shrimp families (infraorder Caridea) and show that most taxa have much larger ITS1 sequences. We find a high number of microsatellites in Alpheus hebes and Crangon crangon and we report repeat units in Pandalidae, Palaemonidae and mainly in Alpheidae species. Up to four repeats were found in A. vanderbilti (1915 bp), A. rostratus (1635 bp) and A. lottini (1625 bp). In general, four helices were found in ITS1. Repeat units led to extra hairpins and loops. No conserved positions occurred except in helix 4. Three clades were defined in A. lottini for the first time. We estimated the ITS1 divergence rate for the three clades of A. lottini collected in French Polynesia using existing calibrations of substitution rates. Rates of sequence evolution are largely influenced by repeat units, which likely evolve separately. By comparison with COI marker, we estimated the divergence rate of the whole ITS1 sequence to range from 0.5 to 1.4% Pmy and between 0.12 and 0.5% for the 3' end of ITS1 located outside the repeat units. Given the degree of identity between repeats, we suggest that a duplication event recently occurred in A. floridanus (98% identity) whereas an ancient duplication happened in A. sulcatus (50% identity) early at the origination of the group Alpheidae, approximately 50 mya ago. In conclusion, our results highlight an over representation of shorter ITS1 sequences in public repositories, and underlines the importance to further understand patterns of molecular evolution of this functionally important gene.

Truttaedacnitis truttae is a cucullanid nematode of primarily salmonine fishes. Brown trout ( Salmo trutta ) in Europe reportedly become parasitized by ingesting lampreys ( Lampetra planeri ) carrying infective larvae. However, our field and laboratory observations suggested that North American specimens of T. truttae have an alternative life cycle. High abundances and potential impact of T. truttae in rainbow trout, Oncorhynchus mykiss , in the Colorado River drainage in Grand Canyon, where there are no lampreys, prompted a study on the transmission dynamics of this nematode. Eggs of T. truttae , collected from live gravid females, were incubated in the laboratory. Snails, Physa gyrina and Lymnaea sp., were exposed to T. truttae larvae 3-4 wk later. Active larvae of T. truttae were observed penetrating the intestinal wall of exposed snails, and worm larvae were found in the visceral tissues when examined 1 wk after exposure. Larvae in snails showed little growth and development 2 wk later and corresponded to L3 larvae. Infected snails were fed to hatchery-reared juvenile rainbow trout. Developing stages were subsequently found in the mucosal lining and lumen of trout intestines. Adult male and female (gravid) worms were found in the ceca of trout examined 5-6 mo after consuming infected snails. Larvae found in pepsin/trypsin digests and mucosal scrapings from wild, naturally infected, trout corroborate laboratory findings. Screening of Physa sp. and gammarids collected from Colorado River, Grand Canyon, for natural infections with T. truttae using the ITS1 rDNA marker gave positive results. Truttaedacnitis truttae is the second species, after Truttaedacnitis clitellarius of lake sturgeon, capable of using a snail first intermediate/paratenic host and is similar to several other cucullanids in having a histotropic phase of development in the definitive fish host.

Semi-aquatic freshwater earthworms in the genus Glyphidrilus from Southeast Asia are characterized by both an extreme morphological crypsis among divergent phylogenetic lineages and a high morphological variability within the same phylogenetic lineages. The present study provides a new taxonomic framework for this problematic genus in SE Asia by integrating DNA sequence and morphological data. When single-locus and multilocus multispecies coalescent-based (MSC) species delimitation methods were applied to DNA sequence data, they usually yielded highly incongruent results compared to morphology-based species identifications. This suggested the presence of several cryptic species and high levels of intraspecific morphological variation. Applying reciprocal monophyly to the cytochrome c oxidase subunit 1 (COI) gene tree allowed us to propose the existence of 33 monophyletic species. Yet, often substantially more molecular operational taxonomic units (MOTUs) were obtained when species delimitation was based on COI and 16S rRNA sequences. In contrast, the ITS1 and ITS2 sequences suggested fewer MOTUs and did not recover most of the monophyletic species from the Mekong basin. However, several of these latter taxa were better supported when MSC species delimitation methods were applied to the combined mtDNA and ITS datasets. The ITS2 secondary structure retrieved one unnamed Mekong basin species that was not uncovered by the other methods when applied to ITS2 sequences. In conclusion, based on an integrative taxonomic workflow, 26 Glyphidrilus candidate species were retained and two remained to be confirmed. As such, this study provides evidence to suggest nine species new to science and to synonymize 12 nominal morphospecies. It also illustrates that the uncritical use of COI as a universal DNA barcode may overestimate species diversity because COI may be unable to distinguish between divergent conspecific lineages and different candidate species., (Copyright © 2019 Elsevier Inc. All rights reserved.)

In human cells, the intergenic spacers (IGS), which separate ribosomal genes, are complex approximately 30 kb-long loci. Recent studies indicate that all, or almost all, parts of IGS may be transcribed, and that at least some of them are involved in the regulation of the ribosomal DNA (rDNA) transcription, maintenance of the nucleolar architecture, and response of the cell nucleus to stress. However, since each cell contains hundreds not quite identical copies of IGS, the structure and functions of this locus remain poorly understood, and the dynamics of its products has not been specially studied. In this work, we used quantitative PCR to measure the expression levels of various rDNA regions at different times after inhibition of the transcription by Actinomycin D applied in high doses. This approach allowed us to measure real or extrapolated half-life times of some IGS loci. Our study reveals characteristic dynamic patterns suggestive of various pathways of RNA utilization and decay.

To meet a global demand for timber, tree plantations were established in South America during the first half of the 20th century. Extensive plantings of non-native species now are found in Brazil, Chile, Argentina, and Uruguay. In Colombia, miscellaneous plantations were established in the 1950s, during a period of intensive local logging, when policies to limit deforestation in native Quercus humboldtii forests were established. One unforeseen consequence of planting non-native trees was the simultaneous introduction and subsequent persistence of ectomycorrhizal fungi. We sought to document the origins and spread of the introduced Amanita muscaria found in Colombian plantations of the Mexican species Pinus patula , North American species P. taeda , and Australian species Acacia melanoxylon and Eucalyptus globulus . In Colombia, Amanita muscaria is establishing a novel association with native Q. humboldtii and has spread to local Q. humboldtii forests. According to a Bayesian phylogeny and haplotype analysis based on the nuclear rDNA internal transcribed spacer region ITS1-5.8-ITS2 (ITS barcode), A. muscaria individuals found in four exotic plant species, and those colonizing Q. humboldtii roots, have a Eurasian origin and belong to two Eurasian haplotypes. This is the first time the spread of an introduced mutualist fungus into native Colombian Q. humboldtii forests is reported. To arrest its spread, we suggest the use of local inocula made up of native fungi, instead of inocula of introduced fungi.

Ganoderma is a cosmopolitan genus that encompasses species with cultural, economic, and pathogenic importance. Despite the importance of this genus, knowledge pertaining to the species diversity of Ganoderma in South Africa is limited. This study aimed at elucidating the identity and phylogenetic placements of Ganoderma samples obtained during a survey of wood-rotting fungi in the Garden Route National Park (GRNP) of South Africa, supplemented with isolates obtained from other localities across the country. Identification was achieved by means of multilocus phylogenetic inference combined with morphological evaluation. In total, eight distinct species of Ganoderma were recovered from different hosts and localities across the country. Of these, Ganoderma cf. cupreum and Ganoderma cf. resinaceum represent possible new records for South Africa. Two novel species are described, namely, G. eickeri . and G. knysnamense. Ganoderma eickeri , sp. nov., is characterized by a triquetrous and broadly attached basidiome, a sulcate or zonate yellowish brown to brown pilear surface, and ovoid to ellipsoid basidiospores. Ganoderma knysnamense is distinguished by an applanate to ungulate, sometimes convex, dimidiate to broadly attached basidiome, a chocolate-brown pilear surface covered with a hard woody-like crust and ellipsoid, broadly ellipsoid to ovoid basidiospores. The discovery of two new Ganoderma species in this study raises the known Ganoderma species in South Africa to 13.

We present an account of Rhizopogon introduced from plantings of exotic pine plantations in Argentine Patagonia. Nuc rDNA internal transcribed spacer ITS1-5.8S-ITS2 (ITS) and nuc 28S rDNA (28S) sequences were used to identify specimens from Argentina and examine their relationships with geographically different Rhizopogon species. Based on phylogenetic analyses, we confirm that four species of Rhizopogon occur in pine plantations across Patagonia. Several Rhizopogon collections from Pinus ponderosa plantations across different provinces cluster with R. arctostaphyli , a species within R . subg. Amylopogon . The majority of Patagonian Rhizopogon , however, form three different lineages in R . subg. Roseoli . The first of these, R. roseolus sensu Trappe, includes numerous collections from Pinus ponderosa, P. contorta , and P. radiata stands of North American affiliation. The second, R. roseolus sensu Martin and Garcia from P. ponderosa plantations, clusters in clade IIIa of the R. roseolus complex, which also includes the holotype collection of R. mohelnensis from the Czech Republic. The third species in R . subg. Roseoli , and fourth species overall from Patagonia, is R. granuloflavus from Pinus ponderosa plantations. Multiplex polymerase chain reaction (PCR) of numerous Roseoli samples failed to produce an amplicon indicative of either Japanese or New Zealand shoro.

Two new species and a new combination of Hypoxylon from Texas were identified and described based on morphological, multigene phylogenetic (ITS [nuc rDNA internal transcribed spacer region ITS1-5.8S-ITS2], 28S [5' 1200 bp of nuc 28S rDNA], RPB2 [partial second largest subunit of the DNA-directed RNA polymerase II], TUB2 [partial β-tubulin ] ), and chemotaxonomic data. Hypoxylon olivaceopigmentum is characterized by its pulvinate to glomerate stromata, olivaceous KOH-extractable pigments, equilateral ascospores, and indehiscent perispore. Hypoxylon texense can be distinguished from morphologically similar species by its rust to dark brick KOH-extractable pigments and the high-performance liquid chromatography (HPLC) profile of its stromatal secondary metabolites. Hypoxylon hinnuleum is proposed as the sexual morph of Nodulisporium hinnuleum , featuring dark vinaceous glomerate stromata with dark brick KOH-extractable pigments composed of cohaerin-type azaphilones and smooth equilateral ascospores with indehiscent perispore. Based on these diagnostic characters, H. hinnuleum forms a complex with H. croceum and H. minicroceum . More than 50 ITS sequences with high identity originating from North American and East Asian environmental isolates formed a well-supported clade with the type of N. hinnuleum , demonstrating the widespread distribution of the species complex. In addition, updated descriptions and comprehensive illustrations with detailed information on the diagnostic features of H. fendleri and H. perforatum are provided. The multilocus phylogenetic reconstruction of Hypoxylon supported the status of the new species and broadened the knowledge about intergeneric relationships.