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Nectriaceae is a highly diverse family, and members have a worldwide distribution, particularly in warm temperate to tropical regions. During the survey of fungal diversity in different habitats in Yunnan province, China, two new species isolated from soil and air respectively, namely Atractium yunnanense sp. nov. and Nalanthamala xishuangbannaensis sp. nov., were proposed based on morphological comparisons and the multi-gene phylogenetic analyses of combined ITS, LSU, rpb2, and tub2 sequence data. Phylogenetically, both species clustered in a monophyletic clade within Nectriaceae with strong support. A. yunnanense is characterized by synnematous conidiophores, pale olivaceous-green, clavate to oblong-ellipsoidal, multi-septate conidia, and pale olivaceous-green chlamydospores. N. xishuangbannaensis has acremonium-like or penicillium-like conidiophores and either obovate or ellipsoidal, cylindrical or fusiform conidia. Full descriptions, illustrations, and a phylogenetic tree showing the phylogenetic position of the two new species were provided.

Abstract 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.

IntroductionTrichoderma species establish symbiotic relationships with plants through both parasitic and mutualistic mechanisms. While some Trichoderma species act as plant pathogenic fungi, others utilize various strategies to protect and enhance plant growth.MethodsPhylogenetic positions of new species of Trichoderma were determined through multi-gene analysis relying on the internal transcribed spacer (ITS) regions of the ribosomal DNA, the translation elongation factor 1-α (tef1-α) gene, and the RNA polymerase II (rpb2) gene. Additionally, pathogenicity experiments were conducted, and the aggressiveness of each isolate was evaluated based on the area of the cross-section of the infected site.ResultsIn this study, 13 Trichoderma species, including 9 known species and 4 new species, namely, T. delicatum, T. robustum, T. perfasciculatum, and T. subulatum were isolated from the diseased tubers of Gastrodia elata in Yunnan, China. Among the known species, T. hamatum had the highest frequency. T. delicatum belonged to the Koningii clade. T. robustum and T. perfasciculatum were assigned to the Virens clade. T. subulatum emerged as a new member of the Spirale clade. Pathogenicity experiments were conducted on the new species T. robustum, T. delicatum, and T. perfasciculatum, as well as the known species T. hamatum, T. atroviride, and T. harzianum. The infective abilities of different Trichoderma species on G. elata varied, indicating that Trichoderma was a pathogenic fungus causing black rot disease in G. elata.DiscussionThis study provided the morphological characteristics of new species and discussed the morphological differences with phylogenetically proximate species, laying the foundation for research aimed at preventing and managing diseases that affect G. elata.

Species of Trichoderma are widely distributed around the world. In this study, two new species in Trichoderma, named as T. albidum and T. variegatum, were introduced and illustrated. These species were isolated from diseased tubers of Gastrodia elata in China and identified based on morphological characteristics and multi-gene sequence analyses of three loci that is the internal transcribed spacer regions of the ribosomal DNA (ITS), the translation elongation factor 1-α encoding gene (tef1-α) and the gene encoding the second largest nuclear RNA polymerase subunit (rpb2). Distinctions between the new species and their close relatives were discussed. According to results of the phylogenetic analyses, T. albidum belonged to the Harzianum clade and T. variegatum are grouped with species of the Spirale clade. The expansion of two clades provided research foundations for the prevention and control of tuber diseases in G. elata.

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.

During a survey of soil fungi collected from Yunnan Province, China, two new species of Exophiala, E. yunnanensis and E. yuxiensis, were isolated from the soil of karst rocky desertification (KRD). The DNA sequences of these respective strains, including internal transcribed spacers (ITS), large subunit nuclear ribosomal RNA (LSU rRNA), partial small subunit (SSU) and β-tubulin (tub2) were sequenced and compared with those from species closely-related to Exophiala. Exophiala yunnanensis differs from the phylogenetically closely related E. nagquensis and E. brunnea by its smaller aseptate conidia. Exophiala yuxiensis is phylogenetically related to E. lecanii-corni, E. lavatrina and E. mali, but can be distinguished from them by its larger conidia. Full descriptions, illustrations and phylogenetic positions of E. yunnanensis and E. yuxiensis were provided.

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.

Members of Plectosphaerella inhabit different substrates, including plants, soil and insects, and most species are pathogens causing large losses in agriculture. During a survey of endophytic fungi in aquatic plants in southwest China, 112 strains of Plectosphaerella were isolated, representing two new species, P. endophytica sp. nov. and P. sichuanensis sp. nov., as well as two known species, P. cucumerina and P. pauciseptata. The novel taxa are described and illustrated here using combined morphological and multi-locus phylogenetic (LSU-ITS-TEF-1α-TUB2) analyses. Our result revealed Plectosphaerella species inhabiting within aquatic plants in southwest China, and the separation frequency of each species was presented.

The asexual taxon Phialolunulospora vermispora gen. et sp. nov., collected from submerged dicotyledonous leaves in Hainan, China, is described and illustrated herein. Phialolunulospora gen. nov. is characterized by macronematous, semimacronematous, septate and pigmented conidiophores and acrogenous, long lunate, vermiform to sigmoid, hyaline conidia with an eccentric basal appendage. Complete sequences of internal transcribed spacer (ITS) and partial sequences of nuclear large subunits ribosomal DNA (LSU) genes are provided. Phylogenetic analyses of combined ITS and LSU sequences revealed its placement in the Chaetosphaeriaceae. The new fungus is compared with morphologically similar genera.

During a mycological survey of aquatic hyphomycetes on submerged decaying leaves in southwest China, a distinct new fungus was collected. The collection was cultured and sequenced and a BLAST search of its ITS and LSU sequence against data in GenBank revealed a dothideomycetous affiliation, with the closest related taxa in the genus Neodactylaria. Phylogenetic analyses of a multigene matrix containing sequences from four genes (LSU, SSU, rpb2, and tef1), representing broad groups of Dothideomycetes, revealed its placement within Dothideomycetes, but without a supported familial or ordinal affiliation. Based on further phylogenetic analyses and morphological investigations, the new fungus is described here as a new species of Neodactylaria, N. simaoensis sp. nov., and placed in a new family Neodactylariaceae fam. nov. and a new order Neodactylariales ord. nov.

Aquatic ecosystems contain tremendous plant and microbial diversity. However, little is known about endophyte diversity in aquatic plants. In this study, we investigated the diversity of endophytic fungi in aquatic plants in southwest China using both culture-based and culture-independent high-throughput sequencing methods. A total of 1,689 fungal isolates belonging to three phyla and 154 genera were obtained from 15,373 plant tissue segments of 30 aquatic plant species. The most abundant endophytic fungi were those in ascomycete genera Aspergillus, Ceratophoma, Fusarium, Penicillium, Phoma and Plectosporium. No difference in fungal isolation rates was observed among tissues from roots, stems, and leaves. Twenty tissue samples from three most common plant species were further subjected to culture-independent meta-barcode sequencing. The sequence-based analyses revealed a total of 1,074 OTUs belonging to six fungal phyla and 194 genera. Among the three plants, Batrachium bungei harbored the highest number of OTUs. Besides, a total of 66 genera were detected by two methods. Both the culture-dependent and independent methods revealed that aquatic plants in southwest China have abundant endophytic fungal diversity. This study significantly expands our knowledge of the fungal community of aquatic plants.

A new genus and species, Blastosporium persicolor, is described and illustrated from leaves of mildewed tobacco. It is characterised by branched, septate hyphae from which arise macronematous, unbranched or spaced branched conidiophores and mono- or polyblastic conidiogenous cells that produced solitary and blastocatenate, obovoid, oblong, ellipsoidal, allantoid, broad fusiform to irregular, unicellular, hyaline conidia. The phylogenetic analyses, based on the combined sequence data from the small and large nuclear subunit ribosomal DNA (SSU and LSU), placed B. persicolor in the Leotiomycetes class, Helotiales order.

Two new species of the genus Verruconis, V. hainanensis and V. pseudotricladiata, were described using combined morphological and DNA sequence data. The DNA sequences of respective strains including nuclear ribosomal DNA genes (nuSSU, ITS, nuLSU) and fragments of three protein-coding genes (ACT1, BT2, TEF1) were sequenced and compared with those from closely-related species to genera Ochroconis and Verruconis (Family Sympoventuriaceae, Order Venturiales). Morphologically, both species showed typical ampulliform conidiophores and conidiogenous cells, features not seen in other species of Verruconis. The conidia of V. hainanensis are fusiform and those of V. pseudotricladiata are Y or T shaped, similar to old members of a closely-related genus Scolecobasidium. The addition of these two new species provides a new perspective on the heterogeneity of Scolecobasidium.

Ageratina adenophora is one of the most invasive weeds in China. Following an outbreak in Yunnan in the 1960s, A. adenophora has been spreading in Southwest China at tremendous speed. Previous research indicated A. adenophora contained many Colletotrichum species as endophytes. In this study, we investigated the diversity of Colletotrichum in healthy and diseased leaves of the invasive plant A. adenophora and several surrounding crops in Yunnan, Guangxi, and Guizhou provinces in China, and obtained over 1000 Colletotrichum strains. After preliminary delimitation using the internal transcribed spacer region (ITS) sequences, 44 representative strains were selected for further study. Their phylogenetic positions were determined by phylogenetic analyses using combined sequences of ITS, actin (ACT), chitin synthase (CHS-1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and beta-tubulin (TUB2). Combined with morphological characteristics, 14 new Colletotrichum species were named as C. adenophorae, C. analogum, C. cangyuanense, C. dimorphum, C. gracile, C. nanhuaense, C. nullisetosum, C. oblongisporum, C. parvisporum, C. robustum, C. simulanticitri, C. speciosum, C. subhenanense, and C. yunajiangense.

Summary: Understanding how fungi interact with other organisms has significant medical, environmental, and agricultural implications. Nematode-trapping fungi (NTF) can switch to pathogens by producing various trapping devices to capture nematodes. Here we perform comparative genomic analysis of the NTF with four representative trapping devices. Phylogenomic reconstruction of these NTF suggested an evolutionary trend of trapping device simplification in morphology. Interestingly, trapping device simplification was accompanied by expansion of gene families encoding adhesion proteins and their increasing adhesiveness on trap surfaces. Gene expression analysis revealed a consistent up-regulation of the adhesion genes during their lifestyle transition from saprophytic to nematophagous stages. Our results suggest that the expansion of adhesion genes in NTF genomes and consequential increase in trap surface adhesiveness are likely the key drivers of fungal adaptation in trapping nematodes, providing new insights into understanding mechanisms underlying infection and adaptation of pathogenic fungi.

Predatory fungi in Orbiliaceae (Ascomycota) have evolved a diversity of trapping devices that enable them to trap and kill nematodes, other small animals, and protozoans. These trapping devices include adhesive hyphae, adhesive knobs, adhesive networks, constricting rings, and non-constricting rings. Their diversity and practical importance have attracted significant attention from biologists, making them excellent model organisms for studying adaptative evolution and as biological control agents against parasitic nematodes. The putative origins and evolutionary relationships among these carnivorous fungi have been investigated using nuclear protein-encoding genes, but their patterns of mitogenome relationships and divergences remain unknown. Here we analyze and compare the mitogenomes of 12 fungal strains belonging to eight species, including six species representing all four types of nematode trapping devices and two from related but non-predatory fungi. All 12 analyzed mitogenomes were of circular DNA molecules, with lengths ranging from 146,101 bp to 280,699 bp. Gene synteny analysis revealed several gene rearrangements and intron transfers among the mitogenomes. In addition, the number of protein coding genes (PCGs), GC content, AT skew, and GC skew varied among these mitogenomes. The increased number and total size of introns were the main contributors to the length differences among the mitogenomes. Phylogenetic analyses of the protein-coding genes indicated that mitochondrial and nuclear genomes evolved at different rates, and signals of positive selection were found in several genes involved in energy metabolism. Our study provides novel insights into the evolution of nematode-trapping fungi and shall facilitate further investigations of this ecologically and agriculturally important group of fungi.

Colletotrichum species are plant pathogens, saprobes, and endophytes in many economically important hosts. Many studies have investigated the diversity and pathogenicity of Colletotrichum species in common ornamentals, fruits, and vegetables. However, Colletotrichum species occurring in aquatic plants are not well known. During the investigation of the diversity of endophytic fungi in aquatic plants in southwest China, 66 Colletotrichum isolates were obtained from aquatic plants there, and 26 of them were selected for sequencing and analyses of actin (ACT), chitin synthase (CHS-1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), the internal transcribed spacer (ITS) region, and β-tubulin (TUB2) genomic regions. Based on morphological characterization and multi-locus phylogenetic analyses, 13 Colletotrichum species were recognized, namely, C. baiyuense sp. nov., C. casaense sp. nov., C. demersi sp. nov., C. dianense sp. nov., C. fructicola, C. garzense sp. nov., C. jiangxiense, C. karstii, C. philoxeroidis sp. nov., C. spicati sp. nov., C. tengchongense sp. nov., C. vulgaris sp. nov., C. wuxuhaiense sp. nov. Two species complexes, the C. boninense species complex and C. gloeosporioides species complex, were found to be associated with aquatic plants. Pathogenicity tests revealed a broad diversity in pathogenicity and aggressiveness among the eight new Colletotrichum species.

Classicula sinensis, isolated from decaying leaves from Mozigou, Chongqing Municipality, China, is described as a new species. The new species is a member of basidiomycetous aquatic hyphomycetes which represent a small proportion of all aquatic hyphomycetes. This species falls within the genus Classicula (Classiculaceae, Pucciniomycotina) and is closely related to C. fluitans, based on multiple gene sequence analyses. Morphologically, it is characterised by the apical, hyaline, obclavate or navicular conidia with several hair-like lateral appendages and by its holoblastic and monoblastic conidiogenesis, with a flat un-thickened conidiogenous locus. Clamp connections and haustorial branches were often observed in culture.

During the investigation of endophytic fungi diversity in aquatic plants and the fungal diversity in soil in southwest China, we obtained 208 isolates belonging to Trichoderma, including 28 isolates as endophytes from aquatic plants and 180 isolates as saprobes from soil, respectively. Finally, 23 new species of Trichoderma are recognized by further studies. Their phylogenetic positions are determined by sequence analyses of the combined partial sequences of translation elongation factor 1-alpha (tef1) and gene encoding of the second largest nuclear RNA polymerase subunit (rpb2). The results revealed that the 23 new species are distributed in nine known clades. The morphology and culture characteristics are observed, described and illustrated in detail. Distinctions between the new species and their close relatives were compared and discussed. These include: Trichoderma achlamydosporum, T. amoenum, T. anaharzianum, T. anisohamatum, T. aquatica, T. asiaticum, T. asymmetricum, T. inaequilaterale, T. inconspicuum, T. insigne, T. obovatum, T. paraviride, T. pluripenicillatum, T. propepolypori, T. pseudoasiaticum, T. pseudoasperelloides, T. scorpioideum, T. simile, T. subazureum, T. subuliforme, T. supraverticillatum, T. tibetica, and T. uncinatum.

In this study, the complete mitochondrial DNA sequence was obtained from Dactylellina leptospora using next-generation sequencing technology. The mitogenome is a circular molecule of 201,677 bp in length. The overall nucleotide composition was 38.54% A, 36.34% T, 11.21% C, and 13.9% G, with a higher A + T content (74.88%) than other species. In the D. leptospora mitogenome, we identified 36 genes including 15 protein-coding genes, 2 ribosomal RNA (rRNA), 19 transfer RNA (tRNA). Phylogenetic analysis showed D. leptospora was genetically divergent from species Pyronema omphalodes within Pezizomycetes.

Starch is promising candidate material for enhancing the catalytic activity of α-amylase during the crosslinking process. To help meet industrial needs, here we tested the influence of bovine serum albumin (BSA) and starch on the performance of crosslinked α-amylase aggregates (CLEA), α-amylase-prepared as CLEA with starch (CLEA-S), and BSA (CLEA-BSA). Our results showed that the activities of CLEA, CLEA-S, and CLEA-BSA were 1.1-, 1.0-, and 0.74-fold higher than the free α-amylase, respectively. The stability of the immobilized enzyme slightly changed. After immobilization, the enzyme increased its pH and temperature ranges with the optimal pH values of 5.5, 7.5, 5.5, respectively for CLEA, CLEA-S, and CLEA-BSA, and an upper temperature limit of 50 °C for all three immobilized forms. Among the three immobilized forms, the CLEA-S was the most thermostable, losing only 3% of its initial activity during 390 min incubation at 50 °C. Our microscopic observations of CLEA-S showed that porous structures were formed and such structures could help substance diffusion. In addition, there was excellent affinity between CLEA-S and the substrate. The results suggest that CLEA-S have great potential for industrial application, including for use in starch-based alcohol fermentation.

Mushrooms that are morphologically indistinguishable from Russula virescens (Schaeff.) Fr. are among the most popular wild edible mushrooms in Yunnan province, southwestern China. However, almost nothing is known about their biology. This study investigated the diversity and population genetics of a R. virescens ally from Yunnan. A total of 210 samples were collected from 13 geographical locations throughout the main distribution range in Yunnan. The patterns of genetic variation within and among these geographic populations were analyzed using sequences from three nuclear and two mitochondrial DNA fragments. Analysis of the ITS sequences revealed that samples from Yunnan showed 3-6% sequence difference from R. virescens samples from North America and Europe and formed a distinct clade. Our multilocus population genetic analyses suggested frequent gene flow among geographic populations of the R. virescens ally in Yunnan. Interestingly, the nuclear and mitochondrial genes exhibited different levels of gene flow and recombination. We discuss the implications of our results for understanding speciation, reproduction and conservation of this important biological resource.

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.