Mitochondrial Small Subunit Research Articles

Two new species and three new records of Diachea (Physarales) from China based on morphological and molecular evidence

Diachea is an important genus of myxomycetes, recognized for its ecological role and wide distribution. This study aimed to expand knowledge of species diversity within this genus in China. We collected Diachea specimens from various locations in Shaanxi and Sichuan provinces and characterized them through morphological analysis and phylogenetic analysis using four genetic markers: small subunit ribosomal RNA (nSSU), translation elongation factor 1-alpha (EF-1α), mitochondrial small subunit (mtSSU), and alpha-tubulin gene (α-Tub). Based on these analyses, we describe two new species, namely, Diachea plectophylla and D. sichuanensis, discovered in the Shaanxi and Sichuan provinces, respectively. Diachea plectophylla is distinguished by its dense, rigid capillitium, spore warts, and distinct separation of capillitium ends from the peridium. Diachea sichuanensis, closely related to D. leucopodia, is identified by its blunt-headed columella, clustered spore warts, and robust stalks. In addition to these new species, we recorded five previously documented species, including D. bulbillosa in Gansu province, D. leucopodia in Yunnan and Sichuan provinces, and D. subsessilis in Sichuan province. Detailed descriptions, micrographs, taxonomic comparisons, and an identification key are provided to aid in accurate identification. The discovery of these new species not only enhances the known diversity of slime molds in the region but also provides valuable information for future studies on their geographical distribution and ecological relationships.

Catalogue of fungi in China 2. Ramaria from northern China

ABSTRACT The species of Ramaria are coralloid-fungi with intricately branched and vividly coloured basidiomata. There are many studies on the genus Ramaria in China, but molecular phylogenetic analysis is rarely employed. In this study, we performed a multigene phylogenetic analysis to identify Ramaria species in Shanxi Province of northern China. Phylogenetic analyses based on four loci, the internal transcribed spacer region of nuclear ribosomal DNA (ITS), the nuclear large subunit ribosomal DNA (nrLSU), ATPase subunit 6 (atp6), and mitochondrial small subunit ribosomal DNA (mtSSU), revealed 13 Ramaria species from our collections. Combined with morphological examinations, 12 of them were identified as new species, plus a new report to China. The thirteen species were described and illustrated in this paper.

Morphological and Phylogenetic Analyses Reveal Three Novel Species of Sanguinoderma (Ganodermataceae, Basidiomycota) from Yunnan Province, China.

Sanguinoderma (Ganodermataceae) is recognized as a valuable medicinal resource in Taiwan, China. Additionally, it serves as a traditional folk medicine for treating neurotic epilepsy in Malaysia. This study involved the collection of six specimens of Sanguinoderma from Yunnan Province, China. Employing multigene phylogenetic analysis of DNA sequences, including internal transcribed spacer (ITS), nuclear large subunit (LSU), RNA polymerase II second largest subunit (rpb2), translation elongation factor 1-alpha (tef1-α), mitochondrial small subunit (mtSSU), nuclear small subunit (nSSU) and morphological examinations, three new species, viz. Sanguinoderma concentricum, S. dehongense and S. ovisporum, are introduced. Sanguinoderma concentricum is characterized by a central stipe basidiomata, an orbicular to suborbicular pileus, a grayish-yellow surface with alternating concentric zones and wavy margin-like petals and regular pileipellis cells (4-8 × 17-28 μm). Sanguinoderma dehongense is characterized by a long stipe and flabelliform basidiomata, a dark-grayish yellow-to-dark-yellow pileus surface, irregular pileipellis cells and wavy margin and ellipsoid basidia (8-11 × 9-13 μm). Sanguinoderma ovisporum is characterized by a reniform basidiomata, a heterogeneous context and ovoid basidiospores (7.5-8.6 × 5.5-7.2 µm). A detailed description and illustrations of these new species are provided, as well as a morphological comparison with similar taxa.

Phylogenetic and morphological re-evaluation of Camptophora.

The genetic variety and habitats of Camptophora species, generally known as black yeast, have not been clarified. In this study, we re-evaluated Camptophora based on morphological observations and phylogenetic analyses. Because prior investigations on Camptophora only included a few strains/specimens, 24 Camptophora-related strains were newly obtained from 13 leaf samples of various plant species to redefine the genetic and species concepts of Camptophora. Their molecular phylogenetic relationships were examined using small subunit nuclear ribosomal DNA (nSSU, 18S rDNA), the internal transcribed spacer (ITS) rDNA operon, the large subunit nuclear ribosomal DNA (LSU, 28S rDNA), β-tubulin, the second largest subunit of RNA polymerase II (rpb2), and mitochondrial small subunit DNA (mtSSU). Single- and multi-locus analyses using nSSU-ITS-LSU-rpb2-mtSSU revealed a robust phylogenetic relationship among Camptophora species within Chaetothyriaceae. Camptophora species could be distinguished from other chaetothyriaceous genera by their snake-shaped conidia with microcyclic conidiation and loosely interwoven mycelial masses. Based on the results of phylogenetic analyses, two undescribed lineages were recognized, and Ca. schimae was excluded from the genus. ITS sequence comparison with environmental DNA sequences revealed that the distribution of the genus is restricted to the Asia-Pacific region. Camptophora has been isolated or detected from abrupt sources, and this was attributed to its microcycle. The mechanisms driving genetic diversity within species are discussed with respect to their phyllosphere habitats.

First report of Fusarium commune Causing Bulb Rot on Lily (Lilium brownii var. Viridulum Baker) in Hunan Province of China.

Lily (Lilium brownii var. Viridulum Baker) is a well-known edible plant with large, white and sweet bulb scales that has important medicinal value (Zhou et al. 2021) and is grown mainly in the Hebei, Shanxi and Henan provinces of China. In May 2021, a case of bulb rot was discovered in a 3.33 hm2 plantation in Huaihua, Hunan Province, affecting 20% of the area (27°59'30″N, 110°32'20″E). The disease is most severe during the rainy season in May and June. In the early stage, irregular brown spots appeared on the lily scales, the necrosis was depressed and gradually enlarges, and in the later stage, the scales were scattered from the base of the disc and slough off. Ten samples were taken randomly from different plants in the plantation area to isolate the pathogens. After washing with sterile water, they were cut into small pieces and sterilised with 3% hydrogen peroxide for 30 s, 75% ethanol for 90 s, rinsed three times with sterile water and dried on sterile filter paper, then placed on a water agar plate and incubated in the dark in a constant temperature incubator at 28℃ for 3 to 5 days. After 2 days, the mycelium at the edge of the colony was transferred to a PDA plate and incubated for 3-5 days at 28°C in the dark to obtain pure fungal isolates. Eighteen purified fungal isolates were obtained, of which sixteen looked like Fusarium (88.9% isolation rate) and three representative isolates (BHBR2, BHBR3 and BHBR5) were selected for further study. The surface of this fungus was white with dense aerial mycelium. Some had an orange centre in the medium. Microconidia were oval in shape and appeared either straight or slightly curved. These microconidia were colourless, had 0-1 septa and measured 3.334 to 14.724 × 2.216 to 5.385 μm (n=100). Macroconidia were predominantly three-septate, crescent-shaped structures that were thin-walled and slightly curved. Cells at the apex and base were similarly curved. Macroconidia measured 17.956 to 32.150 × 2.788 to 4.492 μm (n=100). The mitochondrial small subunit (mtSSU) and translation elongation factor 1-α (TEF1) genes were amplified and sequenced using the NMS1/NMS2 and TEF-R/TEF-F primers to verify the identity of the pathogens (Stewart et al. 2006). The sequences were submitted to GenBank (BHBR2: mtSSU, PP273435; TEF, OR900976; BHBR3: mtSSU, PP277729; TEF, OR900977; BHBR5: mtSSU, PP277728; TEF, OR900978). A concatenated phylogenetic tree of the two genes was constructed and analysis showed that BHBR2, BHBR3 and BHBR5 were significantly clustered with Fusarium commune. Based on the results of morphological identification and phylogenetic tree analysis, the three isolates were identified as Fusarium commune. We carried out pathogenicity tests using two methods, one in which 6 × 6 mm fungal blocks were inoculated on lily (L. brownie var. viridulum Baker) scales and controls inoculated with sterile blocks, and the other in which strain BHBR2 was selected to carry out pathogenicity tests on bulbs of live plants soaked with 50 ml of a 1 × 106 conidial suspension and bulbs of control plants soaked with sterile water, all in three replicates. They were placed in a growth chamber at 28°C and 80% relative humidity, and the scales were moistened with moistened sterile filter paper. After 3 days of rearing treated scales, lesions appeared on lily scales inoculated with mycelial blocks and expanded with time, whereas no lesions appeared on lily scales inoculated with sterile blocks. One month later, whole plants soaked in the spore suspension wilted, while the control plants grew well. The pathogens re-isolated from the diseased tissues had the same morphological characteristics as representative isolates. This confirms Koch's hypothesis. Fusarium commune has been shown to be the most important pathogenic fungus causing root rot in Alfalfa (Medicago sativa) (Yang et al. 2022) and blueberry (Vaccinium uliginosum L.) (Li et al. 2023) in China. To our knowledge, this is the first report of Fusarium commune causing lily bulb rot in the world, which will lay the foundation for future control of lily bulb rot.

New insights into the DNA extraction and PCR amplification of minute ascomycetes in the genus Laboulbenia (Pezizomycotina, Laboulbeniales)

Molecular studies of fungi within the order Laboulbeniales (Ascomycota, Pezizomycotina) have been hampered for years because of their minute size, inability to grow in axenic culture, and lack of reliable and cost-efficient DNA extraction protocols. In particular, the genus Laboulbenia is notorious for low success with DNA extraction and polymerase chain reaction (PCR) amplification. This is attributed to the presence of melanin, a molecule known to inhibit PCR, in the cells. We evaluated the efficacy of a standard single cell-based DNA extraction protocol by halving the recommended amount of reagents to reduce the cost per extraction and adding bovine serum albumin (BSA) during the multiple displacement amplification step to reverse the effect of melanin. A total of 196 extractions were made, 111 of which were successful. We found that halving the reagents used in the single cell-based extraction kit did not significantly affect the probability of successful DNA extraction. Using the halved protocol reduces cost and resource consumption. Moreover, there was no significant difference in the probability of successfully extracting DNA based on whether BSA was added or not, suggesting that the amount of melanin present in cells of the thallus has no major inhibitory effect on PCR. We generated 277 sequences from five loci, but amplification of the internal transcribed spacer region, the mitochondrial small subunit rDNA, and protein-coding genes remains challenging. The probability of successfully extracting DNA from Laboulbeniales was also impacted by specimen storage methods, with material preserved in > 95% ethanol yielding higher success rates compared to material stored in 70% ethanol and dried material. We emphasize the importance of proper preservation of material and propose the design of Laboulbeniales-specific primers to overcome the problems of primer mismatches and contaminants. Our new insights apply not only to the genus Laboulbenia; Laboulbeniales generally are understudied, and the vast majority of species remain unsequenced. New and approachable molecular developments will benefit the study of Laboulbeniales, helping to elucidate the true diversity and evolutionary relationships of these peculiar microfungi.

Aptrootia khaoyaiensis (Trypetheliaceae), a new corticolous lichen from the dry dipterocarp forest in central Thailand

Abstract Aptrootia khaoyaiensis Polyiam & Lücking is described as new to science from dry dipterocarp forest in central Thailand, based on morphological assessment and sequence data of the mitochondrial small subunit (mtSSU). The new species is characterized by a corticolous habit, a corticate thallus, ascomata typically immersed in the thallus, with a brown to blackish ostiolar region, an inspersed hamathecium, and dark brown, muriform ascospores occurring 1–2 per ascus. Phylogenetic analyses support placement of the new species in Aptrootia; it is morphologically close to A. elatior but differs in the smaller ascospores and the inspersed hamathecium.

The phenotypic spectrum of PTCD3 deficiency.

The PTCD3 gene product (protein PTCD3 or MRPS39) forms the entry channel of the mitochondrial small ribosomal subunit and binds to single-stranded mRNA. Here, we expand on the clinical manifestations of PTCD3 pathogenic variants by describing an early-onset patient with Leigh-like syndrome and two patients with milder form of disease, with combined oxidative phosphorylation deficiency. A 34-year-old male and his 33-year-old sister both have horizontal nystagmus, pronounced rough tremor, truncal ataxia, dysmetria, spasticity and hyperreflexia. The basal respiration rate decreased significantly for the male patient and his mother (p < 0.0001) compared to the controls. The whole genome sequencing analysis revealed two heterozygous variants in the PTCD3: c.1182T>A, p.(Tyr394Ter) and c.805C>T, p.(His269Tyr). Tyr394Ter variant ablates the C-terminal half of the protein, including a significant portion of the central fold. In silico modelling for the variant His269Tyr shows that the inclusion of the slightly larger tyrosine sidechain is well tolerated, with no significant change in either the position or the movement of the surrounding area. The third case is a 9-year-old boy, who has a global developmental delay, central hypotonia, hyperreflexia and abnormal MRI. PTCD3 pathogenic variant c.538+4A>G was identified by whole exome sequencing. To test the variant's effect on splicing, an RT-PCR experiment was performed, which revealed skipping of an out-of-frame exon 7.

Initial study and phylogenetic analysis of hard ticks (Acari: Ixodidae) in Nantong, China along the route of avian migration.

The growing concern about migratory birds potentially spreading ticks due to global warming has become a significant issue. The city of Nantong in this study is situated along the East Asia-Australasian Flyway (EAAF), with numerous wetlands serving as roosting sites for migratory birds. We conducted an investigation of hard ticks and determined the phylogenetic characteristics of tick species in this city. We utilized three different genes for our study: the mitochondrial cytochrome oxidase subunit 1 (COX1) gene, the second internal transcribed spacer (ITS2), and the mitochondrial small subunit rRNA (12S rRNA) gene. The predominant tick species were Haemaphysalis flava (H. flava) and Haemaphysalis longicornis (H. longicornis). Additionally, specimens of Haemaphysalis campanulata (H. campanulata) and Rhipicephalus sanguineus (R. sanguineus) were collected. The H. flava specimens in this study showed a close genetic relationship with those from inland provinces of China, as well as South Korea and Japan. Furthermore, samples of H. longicornis exhibited a close genetic relationship with those from South Korea, Japan, Australia, and the USA, as well as specific provinces in China. Furthermore, R. sanguineus specimens captured in Nantong showed genetic similarities with specimens from Egypt, Nigeria, and Argentina.

Characterization of Mitoribosomal Small Subunit unit genes related immune and pharmacogenomic landscapes in renal cell carcinoma.

Mitoribosomes are essential for the production of biological energy. The Human Mitoribosomal Small Subunit unit (MRPS) family, responsible for encoding mitochondrial ribosomal small subunits, is actively engaged in protein synthesis within the mitochondria. Intriguingly, MRPS family genes appear to play a role in cancer. A multistep process was employed to establish a risk model associated with MRPS genes, aiming to delineate the immune and pharmacogenomic landscapes in clear cell renal cell carcinoma (ccRCC). MRPScores were computed for individual patients to assess their responsiveness to various treatment modalities and their susceptibility to different therapeutic targets and drugs. While MRPS family genes have been implicated in various cancers as oncogenes, our findings reveal a contrasting tumor suppressor role for MRPS genes in ccRCC. Utilizing an MRPS-related risk model, we observed its excellent prognostic capability in predicting survival outcomes for ccRCC patients. Remarkably, the subgroup with high MRPS-related scores (MRPScore) displayed poorer prognosis but exhibited a more robust response to immunotherapy. Through in silico screening of 2183 drug targets and 1646 compounds, we identified two targets (RRM2 and OPRD1) and eight agents (AZ960, carmustine, lasalocid, SGI-1776, AZD8055_1059, BPD.00008900_1998, MK.8776_2046, and XAV939_1268) with potential therapeutic implications for high-MRPScore patients. Our study represents the pioneering effort in proposing that molecular classification, diagnosis, and treatment strategies can be formulated based on MRPScores. Indeed, a high MRPScore profile appears to elevate the risk of tumor progression and mortality, potentially through its influence on immune regulation. This suggests that the MRPS-related risk model holds promise as a prognostic predictor and may offer novel insights into personalized therapeutic strategies.

Multi-locus phylogenies revealed a new record of Entoloma species (Basidiomycota, Agaricales) responsible for gastrointestinal poisoning

The frequency of mushroom poisoning has increased worldwide. In Thailand, reports on mycetism between 2013 and 2021 included 18 cases of Entoloma poisoning. Incorrect identification of toxic Entoloma species occurs during wild mushroom foraging, and their consumption results in gastrointestinal irritation. Morphological distinction of mushroom leftovers is difficult; however, we aimed to characterize 40 clinical samples to the genus level using macroscopic and microscopic analyses. Furthermore, species identification of the poisonous Entoloma was performed based on multi-locus phylogenetic analyses of the nuclear internal transcribed spacer region, nuclear large subunit ribosomal DNA, and mitochondrial small subunit ribosomal DNA. Phylogenetic trees reconstructed using maximum likelihood and Bayesian methods demonstrated that forty samples of mushroom remnants formed eight robust clades with six identified species. The findings confirmed the taxonomic identity of E. griseolazulinum as a new record of species causing gastrointestinal poisoning. The use of molecular annotation provided a foundation for the development of accurate methods for identification of toxic mushrooms.

Structural insights into the specific recognition of mitochondrial ribosome-binding factor hsRBFA and 12 S rRNA by methyltransferase METTL15

Mitochondrial rRNA modifications are essential for mitoribosome assembly and its proper function. The m4C methyltransferase METTL15 maintains mitochondrial homeostasis by catalyzing m4C839 located in 12 S rRNA helix 44 (h44). This modification is essential to fine-tuning the ribosomal decoding center and increasing decoding fidelity according to studies of a conserved site in Escherichia coli. Here, we reported a series of crystal structures of human METTL15–hsRBFA–h44–SAM analog, METTL15–hsRBFA–SAM, METTL15–SAM and apo METTL15. The structures presented specific interactions of METTL15 with different substrates and revealed that hsRBFA recruits METTL15 to mitochondrial small subunit for further modification instead of 12 S rRNA. Finally, we found that METTL15 deficiency caused increased reactive oxygen species, decreased membrane potential and altered cellular metabolic state. Knocking down METTL15 caused an elevated lactate secretion and increased levels of histone H4K12-lactylation and H3K9-lactylation. METTL15 might be a suitable model to study the regulation between mitochondrial metabolism and histone lactylation.

Three New Species of Russulaceae (Russulales, Basidiomycota) from Southern China.

The characterization of natural fungal diversity impacts our understanding of ecological and evolutionary processes and can lead to novel bioproduct discovery. Russula and Lactarius, both in the order Russulales, represent two large genera of ectomycorrhizal fungi that include edible as well as toxic varieties. Based on morphological and phylogenetic analyses, including nucleotide sequences of the internal transcribed spacer (ITS), the 28S large subunit of ribosomal RNA (LSU), the second largest subunit of RNA polymerase II (RPB2), the ribosomal mitochondrial small subunit (mtSSU), and the translation elongation factor 1-α (TEF1-α) gene sequences, we here describe and illustrate two new species of Russula and one new species of Lactarius from southern China. These three new species are: R. junzifengensis (R. subsect. Virescentinae), R. zonatus (R. subsect. Crassotunicatae), and L. jianyangensis (L. subsect. Zonarii).

Unveiling species diversity within the family Conidiobolaceae (Entomophthorales) in China: Descriptions of two new species and reassessment of the taxonomic position of Conidioboluspolyspermus.

In the present study, two new Conidiobolus s.s. species were described relying on the morphological studies and phylogenetic analysis utilizing nuclear large subunit of rDNA (nucLSU), mitochondrial small subunit of rDNA (mtSSU), and elongation-factor-like gene (EFL) sequences. Conidiobolusjiangxiensissp. nov. is distinguished by its short primary conidiophores, a feature not commonly observed in other Conidiobolus s.s. species. Conversely, Conidiobolusmarcoconidiussp. nov. is characterized by larger primary conidia and the emergence of 2-5 secondary conidia from each branched secondary conidiophores. Additionally, the taxonomic reassessment of C.polyspermus confirms its distinct status within the genus Conidiobolus s.s. Moreover, molecular analyses, incorporating the nucLSU, mtSSU, and EFL sequences, provide robust support for the phylogenetic placement of the two newly described species and the taxonomic identity of C.polyspermus. This investigation contributes valuable insights into the species diversity of Conidiobolaceae in China, enhancing our understanding of the taxonomy within this fungal family.

A phylogenetic assessment of a fungicolous lineage in Coniocybomycetes: Chaenotricha, a new genus of Trichaptum-inhabiting species.

The globally distributed genus Trichaptum is one of the most species-rich among polypores in terms of hosting other fungi. Among Trichaptum-associates, there is a group of mazaediate lichenized fungi (Coniocybomycetes, Ascomycota) that previously had an uncertain phylogenetic position. DNA sequences - mitochondrial small subunit (mtSSU), nuclear large subunit rDNA (nuLSU), and internal transcribed spacer (ITS) - were obtained from 29 specimens collected from Europe and North America. Maximum likelihood and Bayesian inference analyses of these three gene loci were used to infer phylogenetic position and relationships among lineages. Statistical tests were used to find which phenotypical characteristics distinguish species. The molecular sequence data provide evidence that the fungicolous specimens form a distinct lineage within Coniocybomycetes sister to the combined clade of Chaenotheca s. lat. and Sclerophora. Considering its phylogenetic placement and strict specialization, we describe a new genus - Chaenotricha. This fungicolous lineage contains three species based on molecular characteristics. Morphological characters mostly overlap except for spore size and stalk length of apothecia. We provide a new combination, Chaenotricha obscura, for the only previously described species for which we designate an epitype, and introduce a new species - Chaenotricha cilians. The third lineage remains undescribed because of a small sample size, which did not allow us to clearly delineate species boundaries. Citation: Suija A, McMullin RT, Lõhmus P (2023). A phylogenetic assessment of a fungicolous lineage in Coniocybomycetes: Chaenotricha, a new genus of Trichaptum-inhabiting species. Fungal Systematics and Evolution 12: 255-269. doi: 10.3114/fuse.2023.12.13.

Multi-locus phylogeny of Bryoria reveals recent diversification and unexpected diversity in section Divaricatae

AbstractIn recent years, the genus Bryoria (Parmeliaceae, Lecanoromycetes) has been the subject of considerable phylogenetic scrutiny. Here we used information on six gene regions, three nuclear protein-coding markers (Mcm7, GAPDH and Tsr1), two nuclear ribosomal markers (ITS and IGS) and a partial mitochondrial small subunit (mtSSU), to examine infrageneric relationships in the genus and to assess species delimitation in the Bryoria bicolor/B. tenuis group in section Divaricatae. For this purpose, phylogenetic analyses and several of the available algorithms for species delimitation (ASAP, GMYC single, GMYC multiple and bPTP) were employed. We also estimated divergence times for the genus using *BEAST. Our phylogenetic analyses based on the combined data set of six gene loci support the monophyly of sections Americanae, Divaricatae and Implexae, while section Bryoria is polyphyletic and groups in two clades. Species from Bryoria clade 1 are placed in an emended section Americanae. Our study reveals that section Divaricatae is young (c. 5 My) and is undergoing diversification, especially in South-East Asia and western North America. Separate phylogenetic analyses of section Divaricatae using ITS produced a topology congruent with the current species concepts. However, the remaining gene regions produced poorly resolved phylogenetic trees and the different species delimitation methods also generated highly inconsistent results, congruent with other studies that highlight the difficulty of species delimitation in groups with recent and rapid radiation. Based on our results, we describe the new species B. ahtiana sp. nov., characterized by its bicolorous, caespitose, widely divergent thallus, conspicuously thickening main stems, well-developed secondary branches, and rather sparse third-order branchlets. Another new lineage, referred to here as B. tenuis s. lat., is restricted to western North America and may represent a new species recently diverged from B. tenuis s. str., though further work is needed.

First Report of Fusarium Root Rot of Tobacco Caused by Fusarium fujikuroi in Guangdong Province, China.

From March to June 2022, Fusarium tobacco root rot broke out in Shaoguan Guangdong Province, China, affecting approximately 15% of tobacco production fields, with an incidence of 24% to 66%. In the early stage, the lower leaves showed chlorosis, and the roots became black. In the later stage, the leaves became browned and withered, the root cortices were broken and shed, and only a small number of roots were left. Eventually, the entire plant died. Six diseased plant samples (cv. yueyan 97) from Shaoguan (113.8°E, 24.8°N) were collected as test materials. The diseased root tissues (4×4 mm) were surface-sterilized using 75% ethanol for 30 s and 2% NaOCl for 10 min, rinsed 3 times with sterile water and incubated for 4 days on potato dextrose agar (PDA) medium at 25 °C. Fungal colonies were subcultured on fresh PDA, grown for the next 5 d and purified by single-spore separation. Eleven isolates with similar morphological characteristics were obtained. Their colonies were white and fluffy, and the bottoms of the culture plates were pale pink after 5 days of incubation. The macroconidia were slender, slightly curved and measured 18.54~45.85 µm×2.35~3.84 µm (n=50), with 3 to 5 septa. The microconidia were oval or spindle shaped, with one to two cells, and measured 5.56~16.76 µm×2.32~3.86 µm (n=50). Chlamydospores were absent. Such characteristics are typical of the genus Fusarium (Booth C, 1971). The SGF36 isolate was chosen for further molecular analysis. The TEF-1α and β-tubulin genes (Pedrozo et al.2015) were amplified. Based on a phylogenetic tree (neighbor-joining method and 1,000 bootstrap values) obtained using multiplex alignments of concatenations of these two genes from 18 Fusarium species, SGF36 was grouped into a clade with Fusarium fujikuroi strain 12-1 (MK443268.1/MK443267.1) and F. fujikuroi isolate BJ-1 (MH263736.1/MH263737.1). To further identity the isolate, five additional gene sequences (rDNA-ITS (OP862807.1), RPB2, histone 3, calmodulin, and mitochondrial small subunit) (Pedrozo et al.2015), were subjected to BLAST searches in GenBank, and the results indicated that they were most similar to F. fujikuroi sequences, with sequence identities greater than 99%. The phylogenetic tree obtained using six genes except mitochondrial small subunit gene showed that SGF36 was grouped together with four F. fujikuroi strains to form a single clade. Pathogenicity was determined by the inoculation of wheat grains with fungi in potted tobacco plants. The SGF36 isolate was inoculated onto sterilized wheat grains, which were then incubated at 25 °C for 7 d. Thirty wheat grains with fungi were added to 200 g of sterilized soil, which was then mixed well and placed into pots. One six-leaf-stage tobacco seedling (cv. yueyan 97) was planted in each pot. A total of 20 tobacco seedlings were treated. Another 20 control seedlings were treated with wheat grains without fungi. All seedlings were placed in a greenhouse at 25 °C with 90% relative humidity. After 5 d, the leaves of all inoculated seedlings showed chlorosis, and the roots became discolored. No symptoms were observed in the controls. The fungus was reisolated from symptomatic roots and confirmed to be F. fujikuroi based on the TEF-1α gene sequence. No F. fujikuroi isolates were recovered from control plants. F. fujikuroi was previously reported to be associated with rice bakanae disease (Ram et al., 2018), soybean root rot (Zhao et al., 2020) and cotton seedling wilt (Zhu et al., 2020). To our knowledge, this is the first report of F. fujikuroi causing root wilt on tobacco in China. The identification of the pathogen may help to establish appropriate measures for controlling this disease.

Isolation, characterization and virulence of Metanophrys sp. (Ciliophora: Scuticociliatida) from large yellow croaker (Larimichthys crocea) in China

In recent years, an outbreak of large yellow croaker (Larimichthys crocea) disease caused by unknown ciliate infections has occurred along the southeast coast of China, resulting in substantial economic losses. To investigate the taxonomic status and pathogenic characteristics of the ciliate, we isolated the ciliate from the brain tissue of naturally diseased L. crocea larvae, established in vitro monoclonal culture, carried out molecular and morphological studies, and observed the host histopathology. In the present study, we characterized three linked genes (ITS1–5.8S-ITS2, LSU rDNA, SSU rDNA) of the ciliate. The mitochondrial cytochrome oxidase subunit I (mt-COI rDNA) and mitochondrial small subunit ribosome DNA (mt-SSU rDNA) were also applied. The phylogenetic analysis based on ITS1–5.8S-ITS2, LSU, SSU, mt-COI, and mt-SSU rDNA molecular markers showed that the ciliate had the closest genetic relationship with Metanophrys sinensis, and the sequence similarity was 89.95%, 98.22%, 98.74%, 94.63%, and 94.08%, respectively. It was also clustered in a branch with M. sinensis on the phylogenetic tree. Morphological observation showed that the ciliate was melon seed shaped, with a slightly sharp front end and a blunt rear end; a tail cilium, approximately 10 um long; and an oval macronucleus. There were 10–11 somatic kineties, and mitochondria were distributed beneath the cell surface membrane, forming a mitochondrial system that conforms to the morphological characteristics of Metanophrys. After artificially infecting the healthy L. crocea with the ciliate, a large number of ciliates with identical molecular markers and morphology were isolated from the infected L. crocea. The fins and skin of infected fish were partially ulcerated, and the tail or fin base was white, which is the same as the symptoms of the above-mentioned naturally diseased L. crocea. In addition, the results of histopathological observation showed that the ciliate could cause local necrosis and dissolution of L. crocea muscle fibers, infiltration of inflammatory cells in muscle spaces, increased swelling cells in gills and skin, and vacuolation of liver and spleen cells. Therefore, the ciliate that causes the above disease can be classified into the genus Metanophrys, which has been isolated and identified for the first time.

Identification and pathogenicity analysis of Fusarium spp. on peach in China

BackgroundVascular system is affected by diseases that can seriously damage plant health by inducing browning and death of branches. This study aimed to identify and analyze the pathogenicity of Fusarium spp. isolates obtained from diseased peach branches in several peach-producing areas of China.ResultsWe obtained and confirmed nine Fusarium isolates based on morphological and molecular characteristics. Phylogenetic relationships using a combination of rDNA-internal transcribed spacer (ITS), elongation factor (EF)-1α, and mitochondrial small subunit (mtSSU) gene sequences were analyzed. GJH-Z1, GJH-6, and GJH-1 were identified as F. avenaceum; HYR-Z3, and ZLZT-6 as F. concentricum, HH-2020-G2, and HYTZ-4 as F. solani, GG-2020-1 as F. asiaticum, SYGZ-1 as F. equiseti. Through acupuncture comparison, the pathogenicity of F. equiseti (SYGZ-1) was highest amongst nine strains. Meanwhile, F. concentricum (HYR-Z3 and ZLZT-6), and F. solaini (HYTZ-4) had a higher level of pathogenicity as revealed by impregnation.ConclusionsOur study shed light on the findings that Fusarium spp. can inflict vascular bundle browning of peach plants. Our results will extend the understanding of pathogenic diseases in China’s peach industry.

A New Lichenized Fungus, Lendemeriella luteoaurantia, with a Key to the Species of Lendemeriella

Lendemeriella luteoaurantia B.G. Lee is described as a new lichen species from South Korea. The new species is identified by smaller, yellow-orange apothecia, larger ascospores with wider septum width, and the absence of Cinereorufa-green pigment and teloschistin, different from the closest species, L. aureopruinosa I.V. Frolov, Vondrák, Arup, Konoreva, S. Chesnokov, Yakovczenko and Davydov in morphology and chemistry. Molecular phylogeny employing internal transcribed spacer (nuITS), nuclear large subunit ribosomal RNA (nuLSU), and mitochondrial small subunit (mtSSU) sequences strongly supports the new species as nonidentical in the genus Lendemeriella. A preliminary key is provided to assist in the identification of all 10 species of Lendemeriella.