Analyses of Multilocus Sequences and Morphological Features Reveal Ilyonectria Species Associated with Black Rot Disease of Gastrodia elata.

Black rot is a common disease of Gastrodia elata, causing serious threats to G. elata production. In this study, a total of 17 Cylindrocarpon-like strains were isolated from G. elata black rot tissues. Multilocus sequence analyses based on ITS, HIS, TEF, and TUB combined with morphological characterizations were performed to identify six Ilyonectria species, including four new species, Ilyonectria longispora, I. sinensis, I. xiaocaobaensis, and I. yunnanensis, and two known species, I. changbaiensis and I. robusta. The pathogenicity of 11 isolates comprising type strains of the four new species and representative isolates from each of the six species was tested on healthy tissues of G. elata. All isolates were pathogenic to G. elata tissues, and symptoms were identical to black rot disease, confirming that our isolates were the causal agents of black rot disease of G. elata.

Identification of and Mechanistic Insights into SARS-CoV-2 Main Protease Non-Covalent Inhibitors: An In-Silico Study.

The indispensable role of the SARS-CoV-2 main protease (Mpro) in the viral replication cycle and its dissimilarity to human proteases make Mpro a promising drug target. In order to identify the non-covalent Mpro inhibitors, we performed a comprehensive study using a combined computational strategy. We first screened the ZINC purchasable compound database using the pharmacophore model generated from the reference crystal structure of Mpro complexed with the inhibitor ML188. The hit compounds were then filtered by molecular docking and predicted parameters of drug-likeness and pharmacokinetics. The final molecular dynamics (MD) simulations identified three effective candidate inhibitors (ECIs) capable of maintaining binding within the substrate-binding cavity of Mpro. We further performed comparative analyses of the reference and effective complexes in terms of dynamics, thermodynamics, binding free energy (BFE), and interaction energies and modes. The results reveal that, when compared to the inter-molecular electrostatic forces/interactions, the inter-molecular van der Waals (vdW) forces/interactions are far more important in maintaining the association and determining the high affinity. Given the un-favorable effects of the inter-molecular electrostatic interactions-association destabilization by the competitive hydrogen bond (HB) interactions and the reduced binding affinity arising from the un-compensable increase in the electrostatic desolvation penalty-we suggest that enhancing the inter-molecular vdW interactions while avoiding introducing the deeply buried HBs may be a promising strategy in future inhibitor optimization.

Keqinzhangia aquatica gen. et sp. nov. and Pseudocoronospora hainanense gen. et sp. nov., isolated from freshwater in southern China.

During an investigation of the diversity of aquatic hyphomycetes from southern China, two interesting isolates were collected. These two isolates were cultured and sequenced, and a BLAST search of their LSU sequences against data in GenBank revealed that the closest related taxa were in the genus Microthyrium. Phylogenetic analyses, based on the combined sequence data from the internal transcribed spacer (ITS) and large nuclear subunit ribosomal DNA (LSU), revealed that our isolates belong to the Microthyriaceae. Combined morphological characters allowed us to describe our isolates as two new genera and species in Microthyriaceae, named as: Keqinzhangia aquatica and Pseudocoronospora hainanense. The full descriptions, illustrations, and a phylogenetic tree showing the position of the two new genera were provided in this paper.

Cordana yunnanensis sp. nov., Isolated from Desertified Rocky Soil in Southwest China.

During a survey of soil fungal diversity in a rocky desertification area in Yunnan Province, China, a new dematiaceous hyphomycete, Cordana yunnanensis was identified. Morphologically, this species is characterized by macronematous, mononematous conidiophores with discrete, polyblastic conidiogenous cells arranged in a whorl at the apices. Phylogenetic analysis of the combined sequences of the internal transcribed spacer and the large nuclear ribosomal RNA subunit confirmed the phylogenetic position of C. yunnanensis within the genus Cordana, in Cordanaceae.

Colletotrichum litangense sp. nov., Isolated as an Endophyte of Hippuris vulgaris, an Aquatic Plant in Sichuan, China.

An unknown endophytic fungus was isolated from the aquatic plant Hippuris vulgaris in Litang county, Sichuan province, China. Phylogenetic analyses inferred from combined ITS, Sod2, Apn2, and TUB2 sequences revealed that the endophyte is a new species belonging to the Colletotrichum graminicola species complex. Morphological characteristics showed that Colletotrichum litangense is characterized by its falcate, lunate to sublunate conidia, and ellipsoidal, ovoid, or lobed appressoria. Pathogenicity tests on several fruits showed that C. litangense could induce anthracnose lesions. As a result of the phylogenetic, morphological, and pathogenicity analyses, we proposed the name Colletotrichum litangense for the new species.

Anacraspedodidymum submersum sp. nov. (Chaetosphaeriaceae, Chaetosphaeriales), a new species of freshwater hyphomycetes from southwest China.

During a mycological survey of freshwater hyphomycetes on submerged leaves in southwest China, a new species, Anacraspedodidymum submersum, was isolated. The new species A. submersum is characterized by having monophialidic conidiogenous cells with funnel shaped collarettes and globose or subglobose, aseptate and hyaline conidia. Morphologically, A. submersum is somewhat similar to A. aquaticum and A. hyalosporum in conidiophores and conidiogenous cells, but can be easily distinguished by its subglobose to globose and smaller conidia. Phylogenetic analysis of combined ITS and LSU sequences show that the new species of Anacraspedodidymum clustered together with the genus Thozetella in Chaetosphaeriaceae. A full description, illustrations and a phylogenetic tree showing the position of A. submersum are provided herein.

Exophiala pseudooligosperma sp. nov., a novel black yeast from soil in southern China.

Exophiala is an important genus, with several species associated with infections in humans and animals. In a survey of soil fungal diversity in Yunnan province, PR China, a novel taxon, Exophiala pseudooligosperma sp. nov., was identified based on combined morphological and molecular phylogenetic features. Morphologically, this species is characterized by having torulose, septate hyphae and swollen, terminal or intercalary conidiogenous cells arising at acute angles from aerial hyphae. Phylogenetic analysis of the combined sequences of the internal transcribed spacer, the small and large nuclear subunit of the rRNA gene and part of the ß-tubulin gene confirmed the phylogenetic position of the new species within the genus Exophiala.

Remotididymella ageratinae sp. nov. and Remotididymella anemophila sp. nov., two novel species isolated from the invasive weed Ageratina adenophora in PR China.

To determine if Ageratina adenophora can accumulate diverse pathogens from surrounding native plants, we intensively sampled fungal communities, including endophytes, leaf spot pathogens and canopy air fungi, associated with Ag. adenophora as well as native plants in its invasive range. In total, we collected 4542 foliar fungal strains from 10 geographic sites, including 1340 from healthy leaves of Ag. adenophora, 2051 from leaf spots of Ag. adenophora and 1151 from leaf spots of 56 species of native plants and crops. Taxonomically, the common fungal genera included Colletotrichum, Diaporthe, Alternaria, Nemania, Xylaria, Neofusicoccum, Nigrospora, Epicoccum, Gibberella, Pestalotiopsis, Irpex, Schizophyllum and Clonostachys. We also isolated the cultivable fungi from 12 air samples collected from six areas in Yunnan Province, PR China. Among the total of 1255 air fungal isolates, the most common genera were Cladosporium, Trichoderma and Epicoccum. Among them, two new Remotididymella species, Remotididymella ageratinae from leaf spot of Ag. adenophora and Remotididymella anemophila from canopy air of Ag. adenophora were found. The two species showed both asexual and sexual reproductive structures. The conidia of R. ageratinae and R. anemophila are larger than those of R. anthropophila and R. destructiva. The size of ascospores of R. ageratinae and R. anemophila also differ from R. bauhiniae. Phylogenetic analysis of the combined ITS, LSU rRNA, rpb2 and tub2 sequences showed that R. ageratinae and R. anemophila each formed a distinct clade, separated from all species previously described in Remotididymella and confirmed them as new species belonging to Remotididymella. Full descriptions of R. ageratinae and R. anemophila are provided in this study.

Ramichloridium endophyticum sp. nov., a novel species of endophytic fungus from Potamogeton pectinatus.

During a survey of endophytic fungi in aquatic plants collected from Tibet, PR China, a novel species, Ramichloridium endophyticum, was isolated from Potamogeton pectinatus. This novel species differs from other species of the genus Ramichloridium by its finely verrucose, obovoid, ellipsoidal-obovoid and occasionally subglobose conidia. Phylogenetic analysis of the combined sequences of the internal transcribed spacers (ITS) and the translation elongation factor 1-alpha gene (tef1-α) confirmed that the isolated strain represents a member of the genus Ramichloridium. A full description, illustrations and a phylogenetic tree showing the position of R. endophyticum are provided.

Two new species of Leptographium associated with Tomicus spp. infesting Pinus spp. in Southwestern China.

Leptographium panxianense and L. puerense are proposed as new taxa based on sequence data and morphological characters. The phylogenetic analyses based on ITS2-partial LSU rDNA region, ß-tubulin and elongation factor 1-α genes showed that L. panxianense and L. puerense formed well-supported clades and were closely related to L. yunnanense, L. wushanense and L. conjunctum, and then nested within the L. lundbergii complex. The two species differ in their conidial size and shape. The conidia of L. panxianense are larger than those of L. puerense while the conidial shape of L. puerense is more ovovoid. The optimal growth temperature of both L. panxianense and L. puerense is at 20 °C, which is different from those of L. yunnanense, L. wushanense and L. conjunctum. Comparison of sequence data and morphological characters confirmed the placement of the two undescribed taxa in the genus of Leptographium.

Morphological and molecular characterization of Orbilia pseudopolybrocha and O. tonghaiensis, two new species of Orbiliaceae from China.

This study examined over 20 fungal specimens of the Orbiliaceae (Orbiliomycetes) from different regions in China. Our analyses based on morphological traits and the ITS rDNA sequences revealed two new Orbilia species with drechslerella-like asexual morphs. These new species are able to trap nematodes with constricting rings. In addition, Orbilia cf. orientalis is reported as a new cryptic Chinese variant of European collections of O. orientalis. All three species are described and illustrated in detail in this paper. Their phylogenetic relationships with other orbiliaceous species were identified based on their ITS sequences.

Beltrania sinensis sp. nov., an endophytic fungus from China and a key to species of the genus.

During a survey of endophytic fungi in plant roots in secondary forests in Yunnan, China, a novel ascomyceteous taxon, Beltrania sinensis, was isolated from Quercus cocciferoides Hand.-Mazz. and Fraxinus malacophylla Hemsl. This novel species is characterized by having oval or obovoid conidiogenous cells with several apical, flat-tipped denticles, and biconic, aseptate, smooth, pale brown conidia with a hyaline to subhyaline equatorial transverse band and apical tubular appendage. Phylogenetic analysis of the combined sequences of the internal transcribed spacer and the LSU rRNA gene confirmed its novel species status within the genus Beltrania. Here, the novel species is described and illustrated, and a taxonomic key to species in the genus Beltrania is provided.

Staphylotrichum sinense sp. nov., a new hyphomycete (Chaetomiaceae) from China.

Staphylotrichum sinense, a new hyphomycete classified in the Chaetomiaceae (Ascomycota), was isolated from soil in Jianshui county, Yunnan Province. It is characterized by globose and ochreous conidia born laterally on aerial hyphae, and micronematous, unbranched and 0-1-septate conidiophores, sometimes reduced to conidiogenous cells. Morphologically, Staphylotrichum sinense is similar to Staphylotrichum boninense, but it can be distinguished by lacking of macronematous conidiophores and having larger conidia. Phylogenetically Staphylotrichum sinense formed a single clade within Staphylotrichum species, and is closely related to Staphylotrichum boninense and Staphylotrichum brevistipitatum.

Memnoniella sinensis sp. nov., a new species from China and a key to species of the genus.

During a survey of fungal diversity in a deserted rocky area in Yunnan, PR China, a new species, Memnoniella sinensis, was identified. This new species is characterized by having phialidic conidiogenous cells with conspicuous collarettes, and aseptate, verrucose, ellipsoidal to sometimes ovoid, olivaceous brown to dark brown conidia. Morphologically, M. sinensis is similar to M. dichroa, but can be easily distinguished due to its hyaline conidiophores and smaller conidia. Phylogenetic analysis based on DNA sequences at five loci showed that our strain grouped together with M. dichroa and M. oenanthes. Here, the new species is described and illustrated, and a key to the species of the genus Memnoniella is provided.

The contribution of Pir protein family to yeast cell surface display.

Proteins with internal repeats (Pir) in the Baker's yeast are located on the cell wall and include four highly homologous members. Recently, Pir proteins have become increasingly used as anchor proteins in yeast cell surface display systems. These display systems are classified into three types: N-terminal fusion, C-terminal fusion, and inserted fusion. In addition to the GPI (glycosylphosphatidyl inositol) and the FL/FS anchor proteins, these three Pir-based systems significantly increase the choices for target proteins to be displayed. Furthermore, Pir proteins can also be used as a fusion partner for target proteins to be effectively secreted into culture medium. Here, we summarize the development and application of Pir proteins as anchor proteins.

Endosymbiotic bacteria within the nematode-trapping fungus Arthrobotrys musiformis and their potential roles in nitrogen cycling.

Endosymbiotic bacteria (ESB) have important effects on their hosts, contributing to its growth, reproduction and biological functions. Although the effects of exogenous bacteria on the trap formation of nematode-trapping fungi (NTF) have been revealed, the effects of ESB on NTF remain unknown. In this study, we investigated the species diversity of ESB in the NTF Arthrobotrys musiformis using high-throughput sequencing and culture-dependent approaches, and compared bacterial profiles to assess the effects of strain source and culture media on A. musiformis. PICRUSt2 and FAPROTAX were used to predict bacterial function. Our study revealed that bacterial communities in A. musiformis displayed high diversity and heterogeneity, with Proteobacteria, Firmicutes, Bacteroidetes and Actinobacteria as the dominant phyla. The ESB between A. musiformis groups isolated from different habitats and cultured in the same medium were more similar to each other than the other groups isolated from the same habitat but cultured in different media. Function analysis predicted a broad and diverse functional repertoire of ESB in A. musiformis, and unveiled that ESB have the potential to function in five modules of the nitrogen metabolism. We isolated nitrogen-fixing and denitrifying bacteria from the ESB and demonstrated their effects on trap formation of A. musiformis. Among seven bacteria that we tested, three bacterial species Bacillus licheniformis, Achromobacter xylosoxidans and Stenotrophomonas maltophilia were found to be efficient in inducing trap formation. In conclusion, this study revealed extensive ESB diversity within NTF and demonstrated that these bacteria likely play important roles in nitrogen cycling, including nematode trap formation.

DNA methylation plays important roles in lifestyle transition of Arthrobotrys oligospora.

Trap formation is the key indicator of carnivorous lifestyle transition of nematode-trapping fungi (NTF). Here, the DNA methylation profile was explored during trap induction of Arthrobotrys oligospora, a typical NTF that captures nematodes by developing adhesive networks. Whole-genome bisulfite sequencing identified 871 methylation sites and 1979 differentially methylated regions (DMRs). This first-of-its-kind investigation unveiled the widespread presence of methylation systems in NTF, and suggested potential regulation of ribosomal RNAs through DNA methylation. Functional analysis indicated DNA methylation's involvement in complex gene regulations during trap induction, impacting multiple biological processes like response to stimulus, transporter activity, cell reproduction and molecular function regulator. These findings provide a glimpse into the important roles of DNA methylation in trap induction and offer new insights for understanding the molecular mechanisms driving carnivorous lifestyle transition of NTF.

Genomic and proteomic analyses of the fungus Arthrobotrys oligospora provide insights into nematode-trap formation.

Nematode-trapping fungi are "carnivorous" and attack their hosts using specialized trapping devices. The morphological development of these traps is the key indicator of their switch from saprophytic to predacious lifestyles. Here, the genome of the nematode-trapping fungus Arthrobotrys oligospora Fres. (ATCC24927) was reported. The genome contains 40.07 Mb assembled sequence with 11,479 predicted genes. Comparative analysis showed that A. oligospora shared many more genes with pathogenic fungi than with non-pathogenic fungi. Specifically, compared to several sequenced ascomycete fungi, the A. oligospora genome has a larger number of pathogenicity-related genes in the subtilisin, cellulase, cellobiohydrolase, and pectinesterase gene families. Searching against the pathogen-host interaction gene database identified 398 homologous genes involved in pathogenicity in other fungi. The analysis of repetitive sequences provided evidence for repeat-induced point mutations in A. oligospora. Proteomic and quantitative PCR (qPCR) analyses revealed that 90 genes were significantly up-regulated at the early stage of trap-formation by nematode extracts and most of these genes were involved in translation, amino acid metabolism, carbohydrate metabolism, cell wall and membrane biogenesis. Based on the combined genomic, proteomic and qPCR data, a model for the formation of nematode trapping device in this fungus was proposed. In this model, multiple fungal signal transduction pathways are activated by its nematode prey to further regulate downstream genes associated with diverse cellular processes such as energy metabolism, biosynthesis of the cell wall and adhesive proteins, cell division, glycerol accumulation and peroxisome biogenesis. This study will facilitate the identification of pathogenicity-related genes and provide a broad foundation for understanding the molecular and evolutionary mechanisms underlying fungi-nematodes interactions.

New Species of Didymellaceae within Aquatic Plants from Southwestern China.

Members of Didymellaceae have a wide geographical distribution throughout different ecosystems, and most species are associated with fruit, leaf, stem and root diseases of land plants. However, species that occur in aquatic plants are not clearly known. During a survey of the diversity of endophytes in aquatic plants in Yunnan, Sichuan, and Guizhou provinces, we obtained 51 isolates belonging to Didymellaceae based on internal transcribed spacer region (ITS) sequences. Further, the phylogenetic positions of these isolates were determined by combined sequences composed of ITS, partial large subunit nrRNA gene (28S nrDNA; LSU), RNA polymerase II second largest subunit (rpb2) and partial beta-tubulin gene (tub2). Combining morphological characteristics and multi-locus phylogenetic analyses, two new varieties belong to Boeremia and 12 new species distributed into seven genera were recognized from 51 isolates, i.e., Cumuliphoma, Didymella, Dimorphoma, Ectophoma, Leptosphaerulina, Remotididymella, and Stagonosporopsis. Among these species, only one species of Stagonosporopsis and two species of Leptosphaerulina show teleomorphic stages on OA, but have no anamorphic state. Each new species is described in detail, and the differences between new species and their phylogenetically related species are discussed here. The high frequency of new species indicates that aquatic plants may be a special ecological niche which highly promotes species differentiation. At the same time, the frequent occurrence of new species may indicate the need for extensive investigation of fungal resources in those aquatic environments where fungal diversity may be underestimated.

Aspergillus niger produces lethal compounds against nematodes.

BACKGROUND: Aspergillus niger is generally considered safe and is widely used across a broad range of industries from food to pharmaceuticals. The metabolites of some Aspergillus species are effective in killing nematodes. The active ingredients that play a role are citric acid and oxalic acid. RESULTS: The effective nematocidal metabolite in the fermentation broth of the A. niger Ym16 is a calcium oxalate coordination compound (C2 H2 O4 •2(C2 HO4 )•C2 O4 •4(H2 O)•2(Ca)). The mortality of fermentation broth and compound solution to second-stage juveniles (J2s) of Meloidogyne incognita were 94% (8 h) and 95% (2 h), respectively. The hatching inhibition rates of fermentation broth and compound solution to eggs of M. incognita were 86% (24 h) and 87% (12 h), respectively. Furthermore, the Ym16 strain promoted plant growth. CONCLUSION: Strain Ym16 and its metabolites should be the first choice for nematode control because it is an effective agent against soil nematodes. © 2023 Society of Chemical Industry.