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.

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.

Two new asexual genera and six new asexual species in the family Microthyriaceae (Dothideomycetes, Ascomycota) from China.

The family Microthyriaceae is represented by relatively few mycelial cultures and DNA sequences; as a result, the taxonomy and classification of this group of organisms remain poorly understood. During the investigation of the diversity of aquatic hyphomycetes from southern China, several isolates were collected. These isolates were cultured and sequenced and a BLAST search of its LSU sequences against data in GenBank revealed that the closest related taxa are in the genus Microthyrium. Phylogenetic analyses, based on the combined sequence data from the internal transcribed spacers (ITS) and the large subunit (LSU), revealed that these isolates represent eight new taxa in Microthyriaceae, including two new genera, Antidactylaria gen. nov. and Isthmomyces gen. nov. and six new species, Antidactylariaminifimbriata sp. nov., Isthmomycesoxysporus sp. nov., I.dissimilis sp. nov., I.macrosporus sp. nov., Triscelophorusanisopterioideus sp. nov. and T.sinensis sp. nov. These new taxa are described, illustrated for their morphologies and compared with similar taxa. In addition, two new combinations are proposed in this family.

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.

Morphology, Phylogeny and Pathogenicity of Colletotrichum menglaense sp. nov., Isolated from Air in China.

A new species, Colletotrichum menglaense, isolated from air in Mengla, Xishuangbanna, Yunnan Province, China, was characterized and described combining morphological characteristics and multigene phylogenetic analysis. Morphologically, it is characterized by oblong, sometimes slightly constricted, micro-guttulate conidia and simple obovoid to ellipsoidal appressoria. Phylogenetic analysis of the ITS, ACT, CHS, and GAPDH sequences showed that C. menglaense belongs to the C. gloeosporioides complex. The pathogenicity of C. menglaense on fruits of several crop plants, including strawberry, orange, grape, tomato, and blueberry, was tested and confirmed by the re-isolation of C. menglaense.

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.

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.

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.

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.

Phalangispora sinensis sp. nov. from Yunnan, China and two new members of Wiesneriomycetaceae.

Phalangispora sinensis, an aquatic hyphomycete collected from south-western PR China, is described as a new species. This new species is characterized by having multicellular branched conidia composed of a curved main axis and one or two laterals, with the laterals arising from the third or fourth cell of the base of the main axis. Combined analyses of the LSU, SSU, RPB2 and TEF1 gene sequence data revealed that Phalangispora and another aquatic hyphomycete genus, Setosynnema, belonged to Wiesneriomycetaceae, Tubeufiales, Dothideomycetes, Ascomycota.

The complete mitochondrial genomes of Dactylella tenuis, a fungus phylogenetically close to nematode-trapping fungus.

In our study, we sequenced the complete mitochondrial genome of Dactylella tenuis and obtained the complete mitochondrial DNA sequence. This mitogenome is a typical circular molecule of 186,056 bp in length, which is rich in AT (73.79%), including 14 protein-coding genes, 24 transfer RNA genes, and 2 ribosomal RNA genes. Phylogenetic analysis showed the evolutionary relationship between D. tenuis and other species of nematode-trapping fungus.

Bark Beetle-Associated Blue-Stain Fungi Increase Antioxidant Enzyme Activities and Monoterpene Concentrations in Pinus yunnanensis.

Yunnan pine is the most important tree species in SW China in both economical and ecological terms, but it is often killed by pine shoot beetles (Tomicus spp.). Tomicus beetles are secondary pests in temperate regions and the aggressiveness of the beetles in SW China is considered to be due to the warm subtropical climates as well as the beetles' virulent fungal associates. Here, we assessed the virulence of three blue-stain fungi (Leptographium wushanense, L. sinense and Ophiostoma canum) associated with pine shoot beetles to Yunnan pine (Pinus yunnanensis) in SW China. Following fungal inoculation, we measured necrotic lesion lengths, antioxidant enzyme activities and monoterpene concentrations in the stem phloem of Yunnan pine. Leptographium wushanense induced twice as long lesions as L. sinense and O. canum, and all three fungi induced significantly longer lesions than sterile agar control inoculations. The activity of three tested antioxidant enzymes (peroxidase, polyphenol oxidase, and superoxide dismutase) increased after both fungal inoculation and control inoculation. However, L. wushanense and L. sinense generally caused a greater increase in enzyme activities than O. canum and the control treatment. Fungal inoculation induced stronger increases in six major monoterpenes than the control treatment, but the difference was significant only for some fungus-monoterpene combinations. Overall, our results show that L. wushanense and L. sinense elicit stronger defense responses and thus are more virulent to Yunnan pine than O. canum. The two Leptographium species may thus contribute to the aggressiveness of their beetle vectors and could damage Yunnan pine across SW China if they spread from the restricted geographical area they have been found in so far.

New bioactive compounds from aquatic endophyte Chaetomium globosum.

Two new oxidation products-related aureonitol and cytochalasan were isolated from Chaetomium globosum fermented in Chinese yam (Dioscorea opposita) and determined as 10,11-dihydroxyl- aureonitol (1) and yamchaetoglobosin A (2). Compound 2 indicated significant inhibitory effect on nitric oxide production in LPS-activated macrophages, anti-acetylcholinesterase activity with the inhibition ratios of 92.5, 38.2% at 50 µM, and cytotoxicity to HL-60, A-549, SMMC-7721, MCF-7 and SW480 with the range of inhibition ratio at 51-96% for a concentration of 40 µM. Compounds 1, 2 showed weak anticoagulant activity with PT at 16.8 s. Few work was reported on the anti-acetylcholinesterase, and anticoagulant activities of aureonitol, and cytochalasan derivatives. The preliminary structure-activity relationship stated that the oxidation ring-opening in yamchaetoglobosin A can retain the inhibitory effect against NO production and tumor cell.

Bacteria can mobilize nematode-trapping fungi to kill nematodes.

In their natural habitat, bacteria are consumed by bacterivorous nematodes; however, they are not simply passive preys. Here we report a defensive mechanism used by certain bacteria to mobilize nematode-trapping fungi to kill nematodes. These bacteria release urea, which triggers a lifestyle switch in the fungus Arthrobotrys oligospora from saprophytic to nematode-predatory form; this predacious form is characterized by formation of specialized cellular structures or 'traps'. The bacteria significantly promote the elimination of nematodes by A. oligospora. Disruption of genes involved in urea transport and metabolism in A. oligospora abolishes the urea-induced trap formation. Furthermore, the urea metabolite ammonia functions as a signal molecule in the fungus to initiate the lifestyle switch to form trap structures. Our findings highlight the importance of multiple predator-prey interactions in prey defense mechanisms.

Genetic diversity and recombination in natural populations of the nematode-trapping fungus Arthrobotrys oligospora from China.

Nematophagous fungi can trap and capture nematodes and other small invertebrates. This unique ability has made them ideal organisms from which to develop biological control agents against plant- and animal-parasitic nematodes. However, effective application of biocontrol agents in the field requires a comprehensive understanding about the ecology and population genetics of the nematophagous fungi in natural environments. Here, we genotyped 228 strains of the nematode-trapping fungus Arthrobotrys oligospora using 12 single nucleotide polymorphic markers located on eight random DNA fragments. The strains were from different ecological niches and geographical regions from China. Our analyses identified that ecological niche separations contributed significantly, whereas geographic separation contributed relatively little to the overall genetic variation in our samples of A. oligospora. Interestingly, populations from stressful environments seemed to be more variable and showed more evidence for recombination than those from benign environments at the same geographic areas. We discussed the implications of our results to the conservation and biocontrol application of A. oligospora in agriculture and forestry.

Pseudotripoconidium, a new anamorph genus connected to Orbilia.

A new anamorphic fungus is described based on four isolates from ascospores of Orbilia aff. luteorubella. This fungus differs from previously known Orbilia anamorphs in producing inversely pyramidal, unicellular conidia with several protuberances at their distal end. Conidia produce 1-7 prominent denticles that emerge from a node at the conidiophore apex. Conidiogenesis is holoblastic. Because phylogenetic analysis indicated greater than 90% ITS sequence similarities among the four isolates they are treated here as a single species. In the sequence analysis of the internal transcribed spacer region (ITS) these isolates and other sequences identified as O. aff. luteorubella were nested within Orbilia and formed a clade with 99% bootstrap support. This clade is separated from nematode-trapping species of Orbilia. Based on both morphological and molecular analyses, we propose a new genus, Pseudotripoconidium.

Divergence and dispersal of the nematode-trapping fungus Arthrobotrys oligospora from China.

Nematode-trapping fungi are of significant agricultural, forestry and ecological importance. However, relatively little is known about the patterns of genetic variation for any nematode-trapping fungus through its broad geographic and ecological contexts. Here, we analysed DNA sequence variation among strains of the nematode-trapping fungus Arthrobotrys oligospora from China. Our analyses revealed that the Chinese A. oligospora is a species complex with at least three divergent lineages (cryptic species). In addition, there was significant geographic structuring with unambiguous evidence for localized recombination within two of the three lineages in nature. However, evidence for clonal reproduction was also found. We discuss the implications of our results to the conservation and biocontrol application of A. oligospora in agriculture and forestry.

Stereumin A-E, sesquiterpenoids from the fungus Stereum sp. CCTCC AF 207024.

Five cadinane sesquiterpenoids, named stereumin A (1), B (2), C (3), D (4) and E (5) were isolated from the CHCl(3) extract of the culture broth of the fungal strain CCTCC AF 207024. Based on the sequences at the internal transcribed spacer (ITS) region and partial 28S rDNA, this fungus was identified as a Stereum sp. The structures of the five compounds were elucidated using spectroscopic data from 1D, 2D NMR and HRESIMS experiments, and the structures of 1 and 2 were further confirmed by single-crystal X-ray diffraction analysis. Compounds 1-5 showed nematicidal activities against the nematode Panagrellus redivivus at 400 mg l(-1). Among these five compounds, compounds 3 and 4 killed 84.4% and 94.9% of P. redivivus, respectively in 48 h.