Nuclear Ribosomal RNA Research Articles

Marked genetic diversity within Blastocystis in Australian wildlife revealed using a next generation sequencing–phylogenetic approach

Blastocystis is a genus of intestinal stramenopiles that infect vertebrates, and may cause disease of the alimentary tract. Currently, at least 40 genotypes (“subtypes”) of Blastocystis are recognised worldwide based on sequence data for the small subunit of the nuclear ribosomal RNA (SSU-rRNA) gene. Despite the numerous studies of Blastocystis worldwide, very few studies have explored Blastocystis in wild animals, particularly in Australia. Here, we used a PCR-based next generation sequencing (NGS)–phylogenetic approach to genetically characterise and classify Blastocystis variants from selected wildlife in the Australian state of Victoria. In total, 1658 faecal samples were collected from nine host species, including eastern grey kangaroo, swamp wallaby, common wombat, deer, European rabbit, canines and emu. Genomic DNA was extracted from these samples, a 500 bp region of the SSU-rRNA gene amplified by polymerase chain reaction (PCR) and, then, a subset of samples sequenced using Illumina technology. Primary PCR detected Blastocystis in 482 of the 1658 samples (29%), with the highest percentage in fallow deer (63%). Subsequent, Illumina-based sequencing of a subset of 356 samples revealed 55 distinct amplicon sequence variants (ASVs) representing seven currently-recognised subtypes (STs) [ST13 (prominent in marsupials), ST10, ST14, ST21, ST23, ST24 and ST25 (prominent in deer)] and two novel STs (ST45 and ST46) in marsupials. Mixed infections of different STs were observed in macropods, deer, emu and canids (fox, feral dog or dingo), but no infection was detected in rabbits or wombats. This study reveals marked genetic diversity within Blastocystis in a small number of species of wild animals in Australia, suggesting complexity in the genetic composition and transmission patterns of members of the genus Blastocystis in this country.

Molecular and morphological evidence of hybridization between two dimorphic sympatric species of Fuchsia (Onagraceae).

Hybridization is commonly reported in angiosperms, generally based on morphology, and in few cases confirmed by molecular markers. Fuchsia has a long tradition of ornamental cultivars with different hybrids produced by artificial crosses, so natural hybridization between sympatric Fuchsia species could be common. Natural hybridization between F. microphylla and F. thymifolia was tested using six newly developed microsatellites for F. microphylla in addition to other molecular markers with codominant and maternal inheritance. Geometric morphometrics of leaves and floral structures were also used to identify putative hybrids. Hybrids showed a different degree of genetic admixture between both parental species. Chloroplast DNA (cpDNA) sequences indicated that hybridization occurs in both directions, in fact, some of the hybrids showed new haplotypes for cpDNA and ITS (internal transcriber spacer of nuclear ribosomal RNA genes) sequences. The morphology of hybrid individuals varied between the two parental species, but they could be better identified by their leaves and floral tubes. Our study is the first to confirm the hybridization in natural populations of Fuchsia species and suggests that hybridization has probably occurred repeatedly throughout the entire distribution of the species. Phylogeographic analysis of both species will be essential to understanding the impact of hybridization throughout their complete distribution.

Plasmopara echinaceae, a new species of downy mildew affecting cone flowers (Echinacea purpurea) in the United States.

Downy mildew is one of the most important diseases of commercial sunflower and other Asteraceae hosts, including ornamental Rudbeckia. Plasmopara halstedii has historically been identified as the causal agent of this disease, considered a complex of species affecting nearly 35 genera in various tribes. However, with the use of molecular DNA characters for phylogenetic studies, distinct lineages of P. halstedii in the Asteraceae have been identified, confirmed as distinct or segregated as new species. During August of 2022, a downy mildew was observed on potted Echinacea purpurea grown in a retail greenhouse in Jefferson County, Wisconsin, USA. Phylogenetic analyses of the cytochrome c oxidase subunit 2 (cox2) and nuclear large subunit ribosomal RNA (nc LSU rDNA) gene regions indicated these Plasmopara sp. isolates are not conspecific with P. halstedii. Based on phylogenetic evidence and new host association, the Plasmopara isolates from E. purpurea are here described as Plasmopara echinaceae. Diagnostic morphological characters for this new species were not observed when compared with other isolates of P. halstedii or other Plasmopara species found on Asteraceae hosts, and therefore a list of species-specific substitutions in the cox2 region are provided as diagnostic characters. As this study corresponds to the first observation of downy mildew in cone flowers, it is recommended to follow the required disease prevention guidelines to prevent outbreaks and the establishment of this plant pathogen in production sites. Citation: Salgado-Salazar C, Romberg MK, Hudelson B (2023). Plasmopara echinaceae, a new species of downy mildew affecting cone flowers (Echinacea purpurea) in the United States. Fungal Systematics and Evolution 12: 203-217. doi: 10.3114/fuse.2023.12.10.

Eucalyptus leaf spot disease caused by Coniella eucalyptorum in Sri Lanka

Coniella has a wide distribution as a plant pathogen in Eucalyptus species. Here we report a leaf spot disease caused by Coniella eucalyptorum on Eucalyptus camaldulensis in Sri Lanka. The pathogen was isolated from symptomatic leaf samples of E. camaldulensis plants from Polonnaruwa District in Sri Lanka. The isolated fungus was identified as C. eucalyptorum based on morphological characteristics and multi-gene phylogenetic analysis of combined sequences of the internal transcribed spacer region (ITS), the partial translation elongation factor 1-α gene (tef1-α), and large subunit of nuclear ribosomal RNA (LSU). The pathogenicity test was performed to confirm its pathogenicity to the host, which produced symptoms similar to those observed in the field. This is the first report of C. eucalyptorum associated with Eucalyptus plantations in Sri Lanka. This finding is important to expand the knowledge of plant pathogen diversity in Eucalyptus species and other economically important crops in Sri Lanka.

Redescription and Molecular Characterisation of Derogenes ruber Lühe, 1900 (Hemiuroidea: Derogenidae) from Chelidonichthys lastoviza (Scorpaeniformes: Triglidae) in the Western Mediterranean

PurposeDerogenes ruber Lühe, 1900, the type-species of the genus Derogenes Lühe, 1900, is a poorly known derogenid digenean. The original description of this species was not illustrated and aspects of the morphology of the parasite from the type-host remain scarce. Available records of this species were brief and/or lacked illustrations and were based on morphology alone. Additionally, molecular data for Derogenes spp. are warranted to untangle species complexes as they provide a better assessment of interspecific genetic divergence.MethodsDerogenes ruber is redescribed based on newly collected specimens from the gall bladder of its type-host Chelidonichthys lastoviza (Bonnaterre, 1788) collected in the Western Mediterranean off the Algerian coast during 2017–2019 and molecular data are provided using a partial fragment of the nuclear 28S ribosomal RNA gene (28S rRNA), the internal transcribed spacer 2 (ITS2) and a fragment of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene.ResultsWe herein provide a detailed illustrated redescription and morphometric data of D. ruber from its type-host C. lastoviza. We report a new geographical record (off Algeria) for it. Derogenes ruber is also genetically characterised for the first time. Species/lineages of Derogenes were recovered in five strongly supported reciprocally monophyletic clades: (i) D. ruber from C. lastoviza off Algeria; (ii) D. lacustris from Galaxias maculatus (Jenyns) off Argentina; (iii) Lineage “D. varicus DV1” (D. varicus sensu stricto) from fish hosts in the White and Barents seas and the North Sea; (iv) Lineage “D. varicus DV2” from mollusc hosts in the White Sea; and (v) Lineage “D. varicus DV3” from Eumicrotremus fedorovi Mandrytsa. in the Pacific Ocean. Hence, comparison of the newly generated sequences with other available data for Derogenes species supports the distinction of D. ruber confirming its taxonomic status and helping assess interspecific variation. Comparison of D. ruber with the closely related species Derogenes latus revealed overlaps in morphometric data and the validity of the latter species is questioned.ConclusionThe combination of morphological and molecular data provided for D. ruber provides a firm foundation for further investigations of Derogenes spp. Although we do describe herein material of D. ruber from the type-host, given that the occurrence of a single Derogenes species in various hosts has been challenged by molecular data, and both D. lacustris and D. varicus sensu stricto had been genetically proven to occur in various hosts, D. ruber and D. latus may be indeed synonymous. Additional sequencing effort on Derogenes spp. will strengthen systematic comparative studies and evolutionary relationships within the Derogenidae in general.

Mitochondrial genome sequence of the protist Ancyromonas sigmoides Kent, 1881 (Ancyromonadida) from the Sugluk Inlet, Hudson Strait, Nunavik, Québec.

There is little information on evolutionarily ancient eukaryotes, which are often referred to as basal eukaryotes, in Arctic waters. Despite earlier studies being conducted in the Russian White Sea, only few have been reported. Following a shotgun sequence survey of diatom cultures from Sugluk Inlet off the Hudson Strait in Northern Québec, we obtained the complete mitochondrial genome and the operon of nuclear ribosomal RNA genes from a strain that matches that of Ancyromonas sigmoides (Kent, 1881). The sequence of the mitogenome retrieved was 41,889 bp in length and encoded 38 protein-coding genes, 5 non-conserved open-reading frames, and 2 rRNA and 24 tRNA genes. The mitogenome has retained sdh2 and sdh3, two genes of the succinate dehydrogenase complex, which are sometimes found among basal eukaryotes but seemingly missing among the Malawimonadidae, a lineage sister to Ancyromonadida in some phylogenies. The phylogeny inferred from the 18S rRNA gene associated A. sigmoides from Sugluk Inlet with several other strains originating from the Arctic. The study also unveiled the presence of a metagenomic sequence ascribed to bacteria in GenBank, but it was clearly a mitochondrial genome with a gene content highly similar to that of A. sigmoides, including the non-conserved open-reading frames. After re-annotation, a phylogeny was inferred from mitochondrial protein sequences, and it strongly associated A. sigmoides with the misidentified organism, with the two being possibly conspecific or sibling species as they are more similar to one another than to species of the genus Malawimonas. Overall our phylogeny showed that the ice associated ancryomonads were clearly distinct from more southerly strains.

Revealing zooplankton diversity in the midnight zone

Zooplankton diversity in the deep “midnight zone” (>1000 m), where sunlight does not reach, remains largely unknown. Uncovering such diversity has been challenging because of the major difficulties in sampling deep pelagic fauna and identifying many (unknown) species that belong to these complex swimmer assemblages. In this study, we evaluated zooplankton diversity using two taxonomic marker genes: mitochondrial cytochrome oxidase subunit 1 (COI) and nuclear 18S ribosomal RNA (18S). We collected samples from plankton net tows, ranging from the surface to a depth of 5000 m above the Atacama Trench in the Southeast Pacific. Our study aimed to assess the zooplankton diversity among layers from the upper 1000 m to the ultra-deep abyssopelagic zone to test the hypothesis of decreasing diversity with depth resulting from limited carbon sources. The results showed unique, highly vertically structured communities within the five depth strata sampled, with maximal species richness observed in the upper bathypelagic layer (1000–2000 m). The high species richness of zooplankton (>750 OTUS) at these depths was higher than that found in the upper 1000 m. The vertical diversity trend exhibited a pattern similar to the well-known vertical pattern described for the benthic system. However, a large part of this diversity was either unknown (>50%) or could not be assigned to any known species in current genetic diversity databases. DNA analysis showed that the Calanoid copepods, mostly represented by Subeucalanus monachus, the Euphausiacea, Euphausia mucronata, and the halocypridade, Paraconchoecia dasyophthalma, dominated the community. Water column temperature, dissolved oxygen, particulate carbon, and nitrogen appeared to be related to the observed vertical diversity pattern. Our findings revealed rich and little-known zooplankton diversity in the deep sea, emphasizing the importance of further exploration of this ecosystem to conserve and protect its unique biota.

Mycobiome Analysis of Tall Fescue Grass Under Drought Stress Using the Illumina MiSeq and Oxford Nanopore Technology MinION

The effects that mycobiomes have on physiological traits in turfgrasses are poorly understood. Drought tolerance, an economically and ecologically important trait, can be influenced by symbiotic fungi. In this two-year study, we evaluated the mycobiome associated with tall fescue exposed to prolonged periods of drought stress in a rainout shelter. Twelve plants, comprising six sets of half-sibs (progenies having one parent in common), one exhibiting a drought-tolerant phenotype and the other a drought-susceptible phenotype, were selected for analysis each year. The mycobiomes associated with the shoot, root, and rhizosphere soil were evaluated for each tall fescue half-sib pair using both short-read Illumina MiSeq and long-read Oxford Nanopore Technology (ONT) MinION sequencing pipelines. Both platforms sequenced portions of the fungal nuclear ribosomal RNA genes. The Illumina MiSeq sequenced the internal transcribed spacer region (ITS, 600 bp), while the ONT MinION covered the small subunit, ITS, and partial large subunit (4,600 bp). Both sequencing pipelines revealed that the mycobiomes associated with the roots, shoots, and soil were significantly different. The ONT MinION pipeline identified more diverse fungal lineages and had higher taxonomic resolution than the Illumina pipeline. Our results also indicated that root pathogens may play a more important role than endophytic or mycorrhizal symbionts in tall fescue drought stress tolerance.

Morphology and molecular phylogeny of Neolentinus in northern China.

Neolentinus is a significant genus, belonging to Gloeophyllaceae, with important economic and ecological values, which are parasites on decaying wood of broad-leaf or coniferous trees, and will cause brown rot. However, the taxonomic study is lagging behind to other groups of macrofungi, especially in China. In view of this, we conducted morphological and molecular phylogenetic studies on this genus. We have discovered new types of cheilocystidia and with extremely long lamellae in Neolentinus, and, thus proposed it as a new species-Neolentinus longifolius. At the same time, we clarified the distribution of Neolentinus cyathiformis in China and provided a detailed description. Moreover, we also described two common species, viz. Neolentinus lepideus and Neolentinus adhaerens. All the species are described based on the Chinese collections. The key to the reported species of Neolentinus from China is provided. And the phylogeny of Neolentinus from China is reconstructed based on DNA sequences of multiple loci including the internal transcribed spacer (ITS) regions, the large subunit nuclear ribosomal RNA gene (nLSU), and the translation elongation factor 1-α gene (tef-1α). In addition, full morphological descriptions, illustrations, color photographs, taxonomic notes, and all the available sequences of Neolentinus species are provided.

Altered RNA Export in SF3B1 Mutants Increases Sensitivity to Nuclear Export Inhibition

SF3B1 mutations are the most frequent spliceosomal alterations across cancers, yet no successful therapy exists to target this pathway. Previous findings from a phase 2 clinical trial of the XPO1 inhibitor selinexor in patients with high-risk myelodysplastic syndrome (MDS) relapsed or refractory to hypomethylating agents (HMA) revealed increased activity in patients with SF3B1 mutations. XPO1 (Exportin-1) is responsible for the nuclear export of over 200 proteins, but also plays a role in the transport of multiple RNA species, including small nuclear RNAs (snRNAs), ribosomal RNAs (rRNAs), and select messenger RNAs (mRNAs) out of the nucleus. Given the role of XPO1 in exporting snRNAs, which form the catalytic portion of the spliceosome, we hypothesized that XPO1 inhibition may preferentially affect SF3B1-mutant cells via altered splicing, and that SF3B1-mutant high-risk MDS patients would have a better response to rational targeted drug combinations with next generation XPO1 inhibitors. To evaluate the mechanism underlying the preferential sensitivity of SF3B1-mutant cells to XPO1 inhibition, we performed nuclear and cytoplasmic fractionation followed by RNA sequencing (subcellular RNA-seq) before and after XPO1 inhibition in SF3B1 wildtype and SF3B1 K666N mutant cells. Whole transcriptomic analysis of subcellular RNA-seq data revealed increased global retention of RNA transcripts in the nucleus after XPO1 inhibition in the SF3B1 mutant cell line ( Figure 1). Validation assays using qPCR confirmed nuclear retention of several key transcripts such as Salt Inducible Kinase 1 (SIK1) and Cancer/Testis Antigen Family 45 Member A2 (CT45A2) in SF3B1-mutants (both SF3B1 K666N and K700E). Similarly, we performed subcellular RNA-seq for small RNAs and found snRNAs to be increased in the nucleus after XPO1 inhibition in the SF3B1 mutant cells. We then performed total cellular RNA sequencing to understand the effect of XPO1 inhibition on global RNA expression and RNA splicing. Differential gene expression analysis revealed that XPO1 inhibition had a more pronounced effect on cell cycle in SF3B1 wildtype cells, but differentiation pathways were more significantly affected in SF3B1-mutant cells. Alternative splicing analysis showed increased 3' alternative splicing events in SF3B1 mutant after XPO1 inhibition. These results suggest that the preferential sensitivity of SF3B1 mutant cells to nuclear export inhibition arises through increased nuclear retention of spliceosomal snRNAs and select mRNAs that results in perturbation of differentiation pathways. Despite the promising efficacy of XPO1 inhibition in SF3B1 mutated MDS, toxicity limits its dose escalation. Combination therapies may allow for greater synergism with lower doses of XPO1 inhibitor. In order to identify novel drug combination targets, we employed whole genome CRISPR screens in two myeloid leukemia cell lines treated with eltanexor, a next generation XPO1 inhibitor with lower toxicity than selinexor ( Figure 2). These analyses identified several novel targets, which we validated by testing for synergy in combination with eltanexor in wildtype and SF3B1-mutant cell lines. Using the Bliss independence model to calculate synergy, we identifed two drugs that greatly synergized with eltanexor specifically in the SF3B1 mutant cell lines: venetoclax (a BCL2 inhibitor), and navitoclax (a BCL2/BCL-XL inhibitor). We further validated these combinations in vivo using competitive transplant assays in Sf3b1 K700E conditional knock-in mice. The combination of eltanexor with venetoclax showed a significant decrease in the Sf3b1-mutant burden in the peripheral blood and bone marrow progenitor compartments, suggesting a preferential sensitivity of the combination for SF3B1 mutation. In conclusion, by analyzing the effects of XPO1 inhibition on RNA export, our study provides insight on the mechanisms behind the increased efficacy of XPO1 inhibition in SF3B1-mutant MDS/AML. Our findings may also contribute to the development of potentially synergistic therapeutic combinations. In this regard, recent human data have shown that venetoclax can overcome the poor prognosis of spliceosomal mutant AML patients (Senapati et al, Blood 2023); therefore, combining eltanexor with venetoclax could represent a promising SF3B1-specific therapy.

Discovery of Encyclometra bungara (Digenea: Encyclometridae) in a new host (Enhydris enhydris) from Thailand and Cambodia through morphological and molecular identification.

The genus Encyclometra is one of the two genera in family Encyclometridae, known for parasitising the oesophagus, stomach and intestine of snakes. Among Encyclometra, the species present are: Encyclometra colubrimurorum, Encyclometra japonica, Encyclometra asymmetrica and Encyclometra bungara. Species discrimination within Encyclometra has predominantly relied on morphological differences, such as the length of the caeca and the position of the testes. Morphological overlaps exist among these species making species discrimination challenging. Additionally, the use of molecular information has been limited for Encyclometra. To determine the Encyclometra species infecting Enhydris enhydris from Thailand and Cambodia, morphological and molecular identification was conducted. Morphological characters and measurements were obtained from 30 Encyclometra adults, and they were compared with previous studies of other Encyclometra species. Novel sequences of E. bungara were generated using the nuclear 18S and 28S ribosomal RNA genes, and the mitochondrial cytochrome c oxidase subunit 1 gene. Our results revealed that the specimens could be morphologically identified as E. bungara, with support from molecular information obtained from the phylogenies of the 3 genetic markers employed. Molecular analysis showed that the Encyclometra specimens were distinct from E. colubrimurorum and E. japonica. Through morphological and molecular identification of the Encyclometra specimens found in E. enhydris from Thailand and Cambodia, we describe and provide a record of E. bungara in a new host and new locality. Additionally, novel molecular sequences were generated, revealing the phylogenetic position of E. bungara within the superfamily Gorgoderoidea.

Small but strong: the emerging role of small nucleolar RNA in cardiovascular diseases.

Cardiovascular diseases (CVDs) are the leading cause of mortality and disability worldwide. Numerous studies have demonstrated that non-coding RNAs (ncRNAs) play a primary role in CVD development. Therefore, studies on the mechanisms of ncRNAs are essential for further efforts to prevent and treat CVDs. Small nucleolar RNAs (snoRNAs) are a novel species of non-conventional ncRNAs that guide post-transcriptional modifications and the subsequent maturation of small nuclear RNA and ribosomal RNA. Evidently, snoRNAs are extensively expressed in human tissues and may regulate different illnesses. Particularly, as the next-generation sequencing techniques have progressed, snoRNAs have been shown to be differentially expressed in CVDs, suggesting that they may play a role in the occurrence and progression of cardiac illnesses. However, the molecular processes and signaling pathways underlying the function of snoRNAs remain unidentified. Therefore, it is of great value to comprehensively investigate the association between snoRNAs and CVDs. The aim of this review was to collate existing literature on the biogenesis, characteristics, and potential regulatory mechanisms of snoRNAs. In particular, we present a scientific update on these snoRNAs and their relevance to CVDs in an effort to cast new light on the functions of snoRNAs in the clinical diagnosis of CVDs.

Morphological and molecular characterization of Stomachicola muraenesocis Yamaguti, 1934 (Digenea: Hemiuridae) from the daggertooth pike conger Muraenesox cinereus (Forsskål).

Hemiurid digeneans conspecific with Stomachicola muraenesocis Yamaguti, 1934 (the type species of the genus Stomachicola Yamaguti, 1934) were collected from the stomach of the daggertooth pike conger Muraenesox cinereus (Forsskål) off the Persian Gulf of Iran. This study aimed to provide a detailed characterization of Stom. muraenesocis, including measurements, illustrations and scanning electron microscopy (s.e.m.) representations. Comparisons with the original and previous descriptions revealed morphological and metrical variations in several features (i.e. body size and shape, arrangement of reproductive organs, soma to ecsoma length ratio, position of genital opening, number of vitelline tubules and extension of uterine coils) between Stom. muraenesocis from different hosts and localities. This study presents the first molecular sequence data associated with the small (18S) and large (28S) subunit nuclear ribosomal RNA genes (rDNA) for Stom. muraenesocis. Phylogenetic analyses of the 18S dataset placed Stom. muraenesocis as sister lineage to a clade formed of a group of species of Lecithaster Lühe, 1901 (Lecithasteridae Odhner, 1905). In contrast, phylogenetic analyses based on the 28S consistently recovered a sister relationship between Stom. muraenesocis and representatives of the Hemiuridae Looss, 1899. Further comprehensive phylogenetically based classification in light of morphology and taxonomic history of the Hemiuridae and Lecithasteridae is required to infer phylogenetic affinities and historical biogeography of Stomachicola. A comprehensive list of previously reported species of Stomachicola together with their associated hosts, localities and morphometric data is provided.

An unexpected discovery of a new species in Alloclavaria (Rickenellaceae, Hymenochaetales) from China

A newly discovered fungal species associated with conifers in China, Alloclavaria orientalis, is formally described and illustrated. A molecular phylogenetic analysis of concatenated internal transcribed spacer and nuclear ribosomal RNA large subunit sequences supported the monophyly of A. orientalis as a distinct lineage within the Alloclavaria clade.

A Rare Fish Amphistome Revisited: The Phylogenetic Position of Kalitrema kalitrema (Trematoda: Cladorchiidae) Found in Hypostomus spp. (Siluriformes: Loricariidae) from Brazil

Despite recent advances in the molecular knowledge of amphistome trematodes, most genera known from fish remain to be genetically characterized. This is the case for Kalitrema, a genus of the speciose family Cladorchiidae and the type of Kalitrematinae. The type and only species of this genus, Kalitrema kalitrema Travassos, 1933, was originally proposed based on two specimens found in an armored suckermouth catfish from Brazil, and its phylogenetic position has not been evaluated. In this study, paramphistomes found in Hypostomus alatus (2/9; 22.2%) and Hypostomus francisci (4/143; 2.8%) from the Paraobepa River (São Francisco River basin), Minas Gerais, Brazil, between December 2019 and November 2021, were subjected to morphological study. The parasites were identified in low intensity of infection [1.2 (1–2)] and redescribed as K. kalitrema. This species exhibits unique features such as a linguiform body with a circular ridge near the anterior end and a deep, median notch present at the posterior extremity of the body, apparently dividing the body into two lobes. A subset of specimens was further subject to phylogenetic analyses based on the most densely sampled markers, the nuclear ribosomal RNA (28S and ITS2) and mitochondrial cox1, which revealed the inclusion of K. kalitrema in a Neotropical clade of fish paramphistomes. The most comprehensive phylogenetic tree, based on the 28S dataset, confirmed K. kalitrema as an independent, early diverging lineage among Neotropical fish cladorchiids. However, the monophyly of Kalitrematinae was not sustained, given that species of the other kalitrematine genera Pseudocladorchis and Iquitostrema included in the phylogenetic analysis fell in a distinct clade with other fish cladorchiids. As a result, we propose here a narrower concept for Kalitrematinae sensu stricto, accommodating only Kalitrema (type genus) until a more natural subfamilial or familial classification is provided.

Unveiling the Diversity of Hydnum in the Republic of Korea with One New Species, Hydnum paucispinum

Hydnum is a genus of ectomycorrhizal fungi belonging to the Hydnaceae family. It is widely distributed across different regions of the world, including North America, Europe, and Asia; however, some of them showed disjunct distributions. In recent years, with the integration of molecular techniques, the taxonomy and classification of Hydnum have undergone several revisions and advancements. However, these changes have not yet been applied in the Republic of Korea. In this study, we conducted an integrated analysis combining the morphological and molecular analyses of 30 specimens collected over a period of approximately 10 years in the Republic of Korea. For molecular analysis, the sequence data of the internal transcribed spacer (ITS) region, the large subunit of nuclear ribosomal RNA gene (nrLSU), and a portion of translation elongation factor 1-α (TEF1) were employed as molecular markers. Through this study, we identified eight species that had previously not been reported to occur in the Republic of Korea, including one new species, Hydnum paucispinum. A taxonomic key and detailed descriptions of the eight Hydnum species are provided in this study.

First Report of Pseudopithomyces palmicola Causing Leaf Spot on Italian Ryegrass in China.

Italian ryegrass (Lolium multiflorum) is widely cultivated for the production of forage, hay and silage worldwide, due to its high nutritional value and palatability (Feng et al. 2021). It has been infected by a number of foliar fungal diseases caused by different fungal pathogens (Xue et al. 2017, 2020; Victoria Arellano et al. 2021; Liu et al. 2023). In August 2021, three Pseudopithomyces isolates with similar colony characteristics were isolated from fresh samples of leaf spots on Italian ryegrass that was collected from the Forage Germplasm Nursery, Maming town of Qujing city, Yunnan province, China (25.53833°N, 103.60278°E). For specific isolation, tissue pieces (about 0.5 cm × 1 cm) from symptomatic leaves were surface-disinfested in 75% ethanol solution for 40s, rinsed three times in sterilized distilled water and air dried, plated on potato dextrose agar (PDA), and incubated at 25°C for 3 to 7 days in the dark. Following initial isolation, a representative isolate, KM42, was selected for further study. Colonies growing on PDA were cottony, white to grey, 53.8 to 56.9 mm diameter after 6 days in the dark at 25°C, with white and regular edge; in reverse, colonies were brown to dark brown in the center, and pale yellow toward the periphery with concentric rings. Under near-UV light at room temperature (20 ± 3°C), colonies were incubated on PDA for ten days to produce conidia. Conidia were globose or ellipsoid to amygdaloid, 1- to 3-transversely septate, 0- to 2-vertical septate, light brown to brown, measuring 11.6 to 24.4 × 7.7 to 16.8 µm (avg. 17.3 × 10.9 µm). The internal transcribed spacer regions 1 and 2 and 5.8S nuclear ribosomal RNA(ITS), large subunit nrRNA (LSU), and partial DNA-directed RNA polymerase II second largest subunit (RPB2) genes were amplified with primers described by Chen et al. (2017). Sequences were deposited in GenBank (OQ875842 for ITS, OQ875844 for LSU, and OQ883943 for RPB2). BLAST analysis of all these three segments showed 100% (ITS: MF804527), 100% (LSU: KU554630), and 99.4% (RPB2: MH249030) identity with sequences of reported isolate CBS 143931 (= UC22) of Pseudopithomyces palmicola (Lorenzini et al. 2016; Liu et al. 2018). To fulfill Koch's postulates, four 12-week-old healthy Italian ryegrass plants were spray-inoculated separately with a mycelial suspension of about 5.4 × 102 CFU/ml of an isolate of P. palmicola. In addition, four control plants were sprayed with sterilized distilled water. All plants were individually covered with transparent polyethylene bags for 5 days to maintain high relative humidity and placed in a greenhouse at 18 to 22°C. Small brown to dark brown spots developed on leaves 10 days after inoculation; symptoms were not observed on control plants. Pathogenicity tests were conducted three times by the same method. The same fungus was re-isolated from the lesions, and confirmed by morphological and molecular methods as described above. To the best of our knowledge, this is the first report of P. palmicola causing leaf spot on Italian ryegrass in China or worldwide. This information will be useful to forage grass managers and plant pathologists in recognizing the disease and developing effective control measures.

A new omega-3 rich euglenoid Eutreptiella sp. from the Korean coastal waters

Euglenoids, the primary producers of fatty acids (FAs), are used in functional foods, feeds, and biofuels. Long-chain polyunsaturated FAs (LC-PUFAs) such as eicosapentaenoic acid and docosahexaenoic acid (DHA) are crucial for human health. The primary commercial source of LC-PUFA is fish; however, its heavy metal bioconcentration is concerning. Globally declining wild-harvested fish stocks is another recently recognized serious problem. Herein, the morphology, molecular phylogeny, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectroscopy, and FA compositions of an unspecified euglenoid strain KCTC 19016P isolated from the Korean coastal waters were examined. The vegetative cells were longitudinally spindle-shaped with a truncated apex and a tail-like extension at the anterior and posterior ends, respectively and had two unequal flagella. They had distinct red eyespots, numerous chloroplasts, and paramylon granules. Pellicular cell striations were easily observed using a scanning electron microscope. Phylogenetic analyses based on nuclear and chloroplast small-subunit ribosomal RNA sequences revealed that Eutreptiella sp. KCTC 19016P was nested within the Eutreptiella clade and was closely related to Eutreptiella marina (AY703209). Using the MALDI-TOF spectrum analysis, we differentiated between Eutreptiella sp. KCTC 19016P and the related NIES strains, thereby confirming their taxonomic similarities. Eutreptiella sp. KCTC 19016P growth was observed for 30 days; the maximum growth rate and doubling time was 0.50 ± 0.05 day−1 and 1.40 ± 0.15 day, respectively. Eutreptiella sp. KCTC 19016P had a relatively high monounsaturated FA percentage compared to the other species. The omega-3 FA content in Eutreptiella sp. KCTC 19016P was significantly higher than that of the other strains, as determined using ordinary one-way analysis of variance (P < 0.0001). Among the four euglenoids, Eutreptiella sp. KCTC 19016P had the highest α-linolenic acid and DHA contents. Thus, this new isolate could potentially be used in aquaculture and nutraceuticals without the risks associated with consuming fish.

Morphological and phylogenetic analyzes reveal two new species of Melanconiella from Fujian Province, China.

Species of Melanconiella include a diverse array of plant pathogens as well as endophytic fungi. Members of this genus have been frequently collected from the family Betulaceae (birches) in Europe and North America. Little, however, if known concerning the distribution of Melanconiella and/or their potential as pathogens of other plant hosts. Fungi were noted and isolated from diseased leaves of Loropetalum chinense (Chinese fringe flower) and Camellia sinensis (tea) in Fujian Province, China. Genomic DNA was extracted from fungal isolates and the nucleotide sequences of four loci were determined and sued to construct phylogenetic trees. Morphological characteristics of fungal structures were determined via microscopic analyses. Four strains and two new species of Melanconiella were isolated from infected leaves of L. chinense and C. sinensis in Fujian Province, China. Based on morphology and a multi-gene phylogeny of the internal transcribed spacer regions with the intervening 5.8S nrRNA gene (ITS), the 28S large subunit of nuclear ribosomal RNA (LSU), the second largest subunit of RNA polymerase II (RPB2), and the translation elongation factor 1-α gene (TEF1-α), Melanconiellaloropetali sp. nov. and Melanconiellacamelliae sp. nov. were identified and described herein. Detailed descriptions, illustrations, and a key to the known species of Melanconiella are provided. These data identify new species of Melanconiella, expanding the potential range and distribution of these dark septate fungi. The developed keys provide a reference source for further characterization of these fungi.

The 18S rRNA genes of Haemoproteus (Haemosporida, Apicomplexa) parasites from European songbirds with remarks on improved parasite diagnostics

BackgroundThe nuclear ribosomal RNA genes of Plasmodium parasites are assumed to evolve according to a birth-and-death model with new variants originating by duplication and others becoming deleted. For some Plasmodium species, it has been shown that distinct variants of the 18S rRNA genes are expressed differentially in vertebrate hosts and mosquito vectors. The central aim was to evaluate whether avian haemosporidian parasites of the genus Haemoproteus also have substantially distinct 18S variants, focusing on lineages belonging to the Haemoproteus majoris and Haemoproteus belopolskyi species groups.MethodsThe almost complete 18S rRNA genes of 19 Haemoproteus lineages of the subgenus Parahaemoproteus, which are common in passeriform birds from the Palaearctic, were sequenced. The PCR products of 20 blood and tissue samples containing 19 parasite lineages were subjected to molecular cloning, and ten clones in mean were sequenced each. The sequence features were analysed and phylogenetic trees were calculated, including sequence data published previously from eight additional Parahaemoproteus lineages. The geographic and host distribution of all 27 lineages was visualised as CytB haplotype networks and pie charts. Based on the 18S sequence data, species-specific oligonucleotide probes were designed to target the parasites in host tissue by in situ hybridization assays.ResultsMost Haemoproteus lineages had two or more variants of the 18S gene like many Plasmodium species, but the maximum distances between variants were generally lower. Moreover, unlike in most mammalian and avian Plasmodium species, the 18S sequences of all but one parasite lineage clustered into reciprocally monophyletic clades. Considerably distinct 18S clusters were only found in Haemoproteus tartakovskyi hSISKIN1 and Haemoproteus sp. hROFI1. The presence of chimeric 18S variants in some Haemoproteus lineages indicates that their ribosomal units rather evolve in a semi-concerted fashion than according to a strict model of birth-and-death evolution.ConclusionsParasites of the subgenus Parahaemoproteus contain distinct 18S variants, but the intraspecific variability is lower than in most mammalian and avian Plasmodium species. The new 18S data provides a basis for more thorough investigations on the development of Haemoproteus parasites in host tissue using in situ hybridization techniques targeting specific parasite lineages.