Enhancement of polysaccharides production using microparticle enhanced technology by Paraisaria dubia.

BACKGROUND: Polysaccharides are important active ingredients in Ophiocordyceps gracilis with many physiological functions. It can be obtained from the submerged fermentation by the anamorph (Paraisaria dubia) of Ophiocordyceps gracilis. However, it was found that the mycelial pellets of Paraisaria dubia were dense and increased in volume in the process of fermentation, and the center of the pellets was autolysis due to the lack of nutrient delivery, which extremely reduced the yield of polysaccharides. Therefore, it is necessary to excavate a fermentation strategy based on morphological regulation for Paraisaria dubia to promote polysaccharides accumulation. RESULTS: In this study, we developed a method for enhancing polysaccharides production by Paraisaria dubia using microparticle enhanced technology, talc microparticle as morphological inducer, and investigated the enhancement mechanisms by transcriptomics. The optimal size and dose of talc were found to be 2000 mesh and 15 g/L, which resulted in a high polysaccharides yield. It was found that the efficient synthesis of polysaccharides requires an appropriate mycelial morphology through morphological analysis of mycelial pellets. And, the polysaccharides synthesis was found to mainly rely on the ABC transporter-dependent pathway revealed by transcriptomics. This method was also showed excellent robustness in 5-L bioreactor, the maximum yields of intracellular polysaccharide and exopolysaccharides were 83.23 ± 1.4 and 518.50 ± 4.1 mg/L, respectively. And, the fermented polysaccharides were stable and showed excellent biological activity. CONCLUSIONS: This study provides a feasible strategy for the efficient preparation of cordyceps polysaccharides via submerged fermentation with talc microparticles, which may also be applicable to similar macrofungi.

Comparative Analysis of Mitochondrial Genome Features among Four Clonostachys Species and Insight into Their Systematic Positions in the Order Hypocreales.

The mycoparasite fungi of Clonostachys have contributed to the biological control of plant fungal disease and nematodes. The Clonostachys fungi strains were isolated from Ophiocordyceps highlandensis, Ophiocordycepsnigrolla and soil, which identified as Clonostachyscompactiuscula, Clonostachysrogersoniana, Clonostachyssolani and Clonostachys sp. To explore the evolutionary relationship between the mentioned species, the mitochondrial genomes of four Clonostachys species were sequenced and assembled. The four mitogenomes consisted of complete circular DNA molecules, with the total sizes ranging from 27,410 bp to 42,075 bp. The GC contents, GC skews and AT skews of the mitogenomes varied considerably. Mitogenomic synteny analysis indicated that these mitogenomes underwent gene rearrangements. Among the 15 protein-coding genes within the mitogenomes, the nad4L gene exhibited the least genetic distance, demonstrating a high degree of conservation. The selection pressure analysis of these 15 PCGs were all below 1, indicating that PCGs were subject to purifying selection. Based on protein-coding gene calculation of the significantly supported topologies, the four Clonostachys species were divided into a group in the phylogenetic tree. The results supplemented the database of mitogenomes in Hypocreales order, which might be a useful research tool to conduct a phylogenetic analysis of Clonostachys. Additionally, the suitable molecular marker was significant to study phylogenetic relationships in the Bionectriaceae family.

The 'pears and lemons' protein UvPal1 regulates development and virulence of Ustilaginoidea virens.

Ustilaginoidea virens is an economically important fungus causing a devastating grain disease, rice false smut. An insertional mutagenesis screen was used to explore biological mechanisms underlying infection process of U. virens. T184, a new mutant was identified, with abnormal conidial morphology and deficient virulence. Analysis of the T-DNA inserted gene UvPal1 in the mutant confirmed it as a putative homologue of a cellular morphogenetic protein in yeast, Pal1, whose function has not been well characterized. Deletion of UvPal1 affected hyphal growth, cell morphology, stress adaptation and virulence. UvPal1 could interact with the endocytic proteins, UvEde1 and UvSla2, but was not required for receptor-mediated endocytosis. A yeast two-hybrid (Y2H) analysis was further carried out to screen the UvPal1-interacting proteins, resulting in the identification of 16 putative interacting proteins. Interestingly, UvPal1 interacted with a septin protein, UvCdc11 in vivo and in vitro, and also affected subcellular localization of UvCdc11 protein. Deletion of the four core septins impaired the growth, morphogenesis, stress response and virulence. Collectively, effects on cell morphology, oxidative stress response and virulence are similar to those of UvPal1, suggesting that UvPal1 physically interacts with UvCdc11 to mediate the septin complex to maintain the cellular morphology and virulence of U. virens.

Three new species of Gliocephalotrichum causing fruit rot on different hosts from Brazil.

The genus Gliocephalotrichum (Nectriaceae), originally described as a soil-borne fungus, has been associated with postharvest diseases, especially of tropical fruits. Taxonomic studies using both morphological and molecular phylogenetic analyses have contributed to recognition of novel species in several countries. However, in Brazil, only three isolates of Gliocephalotrichum have been collected from soil samples and roots since the late 1970s. Our study expands the sample range using many Gliocephalotrichum isolates obtained from rotting fruits of tropical plant species in different states of Brazil. Polyphasic taxonomy was assessed with phylogenetic analyses of DNA sequences from four nuclear loci, morphological comparisons, and pathogenicity tests. As a result, three known species (G. bulbilium, G. longibrachium, and G. simplex) were identified from new hosts and locations in Brazil. In addition, three new species are described-G. abrachium, G. brasiliense, and G. caryocaris. A key to all Gliocephalotrichum species worldwide is provided. Although species of Gliocephalotrichum have not been considered to be important plant pathogens, this study shows they may cause postharvest fruit rot in tropical fruits and therefore have an impact in communities that depend economically on the harvest and sale of these fruits.

Ophiocordyceps desmidiospora, a basal lineage within the "Zombie-Ant Fungi" clade.

The genus Ophiocordyceps contains the most diverse assemblage of fungi that attack ants worldwide and are remarkably well adapted to the specific ecologies of their hosts. Desmidiospora myrmecophila Thaxt. is closely related to other ant-pathogenic species within Ophiocordyceps, possibly specific to queens, but the sheer infrequency of encounters and previously unsuccessful attempts to culture this fungus has precluded any meaningful assessment until now. A new record of Desmidiospora myrmecophila from Louisiana was found infecting a foundress Camponotus pennsylvanicus queen, the same host species favored by the more common and ubiquitous ant-pathogenic Ophiocordyceps unilateralis clade found in the same geographic locality. To evaluate a long-held assumption that these fungi represent synanamorphs of a single species, we sampled our Desmidiospora specimen along with the local O. unilateralis population for molecular comparison. We are able to present for the first time the in vitro characteristics and morphology of Desmidiospora myrmecophila, as well as a phylogenetic context for this fungus based on combined molecular analysis of representative members of the Ophiocordycipitaceae. Our results place the Desmidiospora myrmecophila lineage within the genus Ophiocordyceps, with a basal affiliation to the Ophiocordyceps unilateralis core clade; thus, in accordance to the "One Fungus-One Name" (1F1N) rule, we propose a new synonym to suppress Desmidiospora in protection of Ophiocordyceps, i.e., O. desmidiospora. These results further implicate this species as an important and quintessential example of cryptic diversity among an already taxonomically diverse and ecologically important group of fungi.

Commelinaceomyces, gen. nov., for four clavicipitaceous species misplaced in Ustilago that infect Commelinaceae.

A fungus causing false smut in the flowers of Murdannia keisak (Commelinaceae, Commelinales, Monocots) in Japan was morphologically identical to Ustilago aneilematis. The fungus infected ovaries of most flowers of host plants. Infected flowers were filled with yellow to orange thick-walled conidia that became olivaceous green at maturity. However, multilocus phylogenetic analysis of DNA sequences (18S, 28S, translation elongation factor 1α [TEF], the largest [RPB1] and the second largest [RPB2] subunit of RNA polymerase II) showed that the fungus belonged to the tribe Ustilaginoideae (Clavicipitaceae, Hypocreales, Ascomycota). Microscopic examination showed that the fungus developed conidia at the apex of conidiogenous cells, in contrast to other species in the Ustilaginoideae that develop conidia pleurogenously. A new genus, Commelinaceomyces, is formally proposed in the Ustilaginoideae to accommodate this fungus. Four species previously misplaced in Ustilago (Ustilaginales, Basidiomycota) are transferred to Commelinaceomyces, including the type of the genus, C. aneilematis, on Murdannia keisak. This is the first report of a clavicipitaceous species infecting host plants in the Commelinaceae.

Optimised red- and green-fluorescent proteins for live cell imaging in the industrial enzyme-producing fungus Trichoderma reesei.

The filamentous fungus Trichoderma reesei is a major source of cellulolytic enzymes in biofuel production. Despite its economic relevance, our understanding of its secretory pathways is fragmentary. A major challenge is to visualise the dynamic behaviour of secretory vesicles in living cells. To this end, we establish a location juxtaposing the succinate dehydrogenase locus as a "soft-landing" site for controlled expression of 4 green-fluorescent and 5 red-fluorescent protein-encoding genes (GFPs, RFPs). Quantitative and comparative analysis of their fluorescent signals in living cells demonstrates that codon-optimised monomeric superfolder GFP (TrmsGFP) and codon-optimised mCherry (TrmCherry) combine highest signal intensity with significantly improved signal-to-noise ratios. Finally, we show that integration of plasmid near the sdi1 locus does not affect secretion of cellulase activity in RUT-C30. The molecular and live cell imaging tools generated in this study will help our understanding the secretory pathway in the industrial fungus T. reesei.

Entomopathogenic effect of Trichothecium roseum (Pers.) Link (Hypocreales: Ascomycota) against Pauropsylla buxtoni (Psylloidea: Hemiptera) infesting Ficus carica leaves and its potential use as biocontrol agent of the insect.

AIMS: To isolate and characterize a native strain of Trichothecium roseum infecting the immatures of Pauropsylla buxtoni on fig leaves, to study the morphological features of the isolated strain, then to test the entomopathogenic effect of the isolated strain against the immatures of P. buxtoni on fig leaves. METHODS AND RESULTS: The isolated strain of T. roseum produced pink mycelial growth on culture medium with septate mycelium and conidiophores. It also produced two-celled conidia with elliptical to pyriform shape born at the tip of conidiophores. Molecular characterization of the isolated strain confirmed the identity of the strain as T. roseum. In bioassays, application of conidial suspension of the isolated strain against the 4th instar of P. buxtoni immatures infesting fig leaves showed an obvious entomopathogenic effect of the applied fungus strain against the targeted insect. This effect was exhibited by the death of treated P. buxtoni immatures with the fungus. The dead insects were characterized by the presence of pinkish mycelial growth on the outer surface which is characteristic to the fungus, in addition to the positive isolation of the fungus from internal tissues of treated insects after a proper external disinfection. Moreover, significant differences (at P < 0·018) were obtained between the means of mortality % of P. buxtoni immatures treated with different concentrations of conidial suspension of the fungus. CONCLUSIONS: The overall results confirm the entomopathogenic effect of T. roseum against P. buxtoni immatures infesting fig leaves. Significant mortalities of P. buxtoni immatures were obtained when the different concentrations of the fungus conidial suspension were bio-assessed against the insect. SIGNIFICANCE AND IMPACT OF THE STUDY: The tested strain of T. roseum can be applied as biocontrol agent of P. buxtoni on fig leaves within an integrated control programme to reduce the impact of pest on fig trees.

Genomic characterization of Parengyodontium americanum sp. nov.

Modern genome analysis and phylogenomic methods have increased the number of fungal species, as well as enhanced appreciation of the degree of diversity within the fungal kingdom. In this context, we describe a new Parengyodontium species, P. americanum, which is phylogenetically related to the opportunistic human fungal pathogen P. album. Five unusual fungal isolates were recovered from five unique and confirmed coccidioidomycosis patients, and these isolates were subsequently submitted to detailed molecular and morphological identification procedures to determine identity. Molecular and morphological diagnostic analyses showed that the isolates belong to the Cordycipitaceae. Subsequently, three representative genomes were sequenced and annotated, and a new species, P. americanum, was identified. Using various genomic analyses, gene family expansions related to novel compounds and potential for ability to grow in diverse habitats are predicted. A general description of the genomic composition of this newly described species and comparison of genome content with Beauveria bassiana, Isaria fumosorosea and Cordyceps militaris shows a shared core genome of 6371 genes, and 148 genes that appear to be specific for P. americanum. This work provides the framework for future investigations of this interesting fungal species.

Novel halotolerant species of Emericellopsis and Parasarocladium associated with macroalgae in an estuarine environment.

Macroalgae of the genera Fucus, Ulva, and Enteromorpha are typically abundant in estuaries. Endophytic fungi may have beneficial effects on the hosts affecting their ability to cope with stress. They are also a source of biologically active compounds. However, little is known about the endophytic fungi that colonize these macroalgae. Endophytic isolates were obtained from macroalgae from various sites in the estuary Ria de Aveiro (Portugal), as well as from saline water and sponges. Six Acremonium-like species could not be affiliated to any known species. Phylogenetic analyses based on internal transcribed spacer (ITS) region of the ribosomal DNA and ß-tubulin (tub2) and actin (act1) genes placed these species in the genera Emericellopsis and Parasarocladium, but distinct from all currently known species. Although sharing morphological characteristics with the most closely related species, these genera differ in micromorphological and molecular characters. Thus, three novel species of Emericellopsis (E. cladophorae, sp. nov., E. enteromorphae, sp. nov., and E. phycophila, sp. nov.) and three novel species of Parasarocladium (P. aestuarinum, sp. nov., P. alavariense, sp. nov., and P. fusiforme, sp. nov.) are proposed.

Occurrence and Diversity of Black-Foot Disease Fungi in Symptomless Grapevine Nursery Stock in Spain.

In this study, 3,426 grafted grapevines ready to be planted from 15 grapevine nursery fields in Northern Spain were inspected from 2016 to 2018 for black-foot causing pathogens. In all, 1,427 isolates of black-foot pathogens were collected from the asymptomatic inner tissues of surface sterilized secondary roots and characterized based on morphological features and DNA sequence data of the nuclear ribosomal DNA-internal transcribed spacer region, histone H3, translation elongation factor 1-alpha and ß-tubulin genes. Eleven species belonging to the genera Dactylonectria, Ilyonectria, Neonectria, and Thelonectria were identified, including Dactylonectria alcacerensis, D. macrodidyma, D. novozelandica, D. pauciseptata, D. torresensis, Ilyonectria liriodendri, I. pseudodestructans, I. robusta, Neonectria quercicola, Neonectria sp. 1, and Thelonectria olida. In addition, two species are newly described, namely D. riojana and I. vivaria. Twenty-four isolates representing 13 black-foot species were inoculated onto grapevine seedlings cultivar 'Tempranillo'. The pathogenicity tests detected diversity in virulence among fungal species and between isolates within each species. The most virulent species was D. novozelandica isolate BV-0760, followed by D. alcacerensis isolate BV-1240 and I. vivaria sp. nov. isolate BV-2305. This study improves our knowledge on the etiology and virulence of black-foot disease pathogens, and opens up new perspectives in the study of the endophytic phase of these pathogens in grapevines.

Coccinonectria pachysandricola, Causal Agent of a New Foliar Blight Disease of Sarcococca hookeriana.

Woody plants of the Buxaceae, including species of Buxus, Pachysandra, and Sarcococca, are widely grown evergreen shrubs and groundcovers. Severe leaf spot symptoms were observed on S. hookeriana at the U.S. National Arboretum in Washington, DC, in 2016. Affected plants were growing adjacent to P. terminalis exhibiting Volutella blight symptoms. Fungi isolated from both hosts were identical based on morphology and multilocus phylogenetic analysis and were identified as Coccinonectria pachysandricola (Nectriaceae, Hypocreales), causal agent of Volutella blight of Pachysandra species. Pathogenicity tests established that Co. pachysandricola isolated from both hosts caused disease symptoms on P. terminalis and S. hookeriana, but not on B. sempervirens. Artificial inoculations with Pseudonectria foliicola, causal agent of Volutella blight of B. sempervirens, did not result in disease on P. terminalis or S. hookeriana. Wounding enhanced infection by Co. pachysandricola and Ps. foliicola on all hosts tested but was not required for disease development. Genome assemblies were generated for the Buxaceae pathogens that cause Volutella diseases: Co. pachysandricola, Ps. buxi, and Ps. foliicola; these ranged in size from 25.7 to 28.5 Mb. To our knowledge, this foliar blight of S. hookeriana represents a new disease for this host and is capable of causing considerable damage to infected plants.

Oxidative functionalization of a halimane diterpenoid achieved by fungal transformation.

Regio and stereoselective activation of sp3 CH bonds remain one of the major advantages of biocatalysis over traditional chemocatalytic methods. Herein, we describe the oxy-functionalization of halimane diterpenoid 1 by whole cells of three filamentous fungi, aiming to obtain derivatives with desirable biological properties. After incubating 1 with Fusarium oxysporum, Myrothecium verrucaria, and Rhinocladiella similis at different concentrations and incubation times, four known (3, 5, 6, and 7) and three new (2, 4, and 8) halimane derivatives were obtained and characterized. F. oxysporum catalyzed the hydroxylation of positions C-2 (2) and C-7 (4), while R. similis simultaneously mediated the 2-oxo-functionalization and the hydration of 13,14-(CC)double bond belonging to an α,ß-unsaturated carbonyl system (8). Compounds 1-7 were non-cytotoxic against HCT-116 and MCF-7 cancer cell lines at tested concentrations. However, substrate 1 displayed moderate reduction ability against biofilm produced by Staphylococcus epidermidis ATCC35984 (84% at 1.6 mM), and this effect was retained to some extent by derivatives 4 and 7. These results emphasize the prominent potential of filamentous fungi associated with the microbiota of medicinal plants as versatile catalysts for singularly useful reactions through their complex enzymatic machinery, as well as the high susceptibility of halimane-diterpenoid substrates.

Discovery of a second species of Hyalocylindrophora and the phylogenetic position of the genus in Bionectriaceae.

A new holomorphic species, Hyalocylindrophora bispora, is described and illustrated based on a collection on rotten branches from Guangdong Province, China. The fungus is characterized by fleshy perithecia that become deeply cupulate when dry, covered with long and stiff hairs on the surface, and not change color in KOH or lactic acid. Asci are two-spored and evanescent at maturity. Ascospores are ellipsoidal to elongate-ellipsoidal, unicellular, and warted. Conidiogenous cells are phialidic and cylindrical. Conidia are thick-walled, unicellular, ellipsoidal to somewhat lemon-shaped. This is the first report of sexual state for Hyalocylindrophora. The phylogenetic position of the genus in Bionectriaceae is confirmed by sequence analyses of the combined nuc rDNA 28S, α-actin, and DNA-directed RNA polymerase II subunit 1 regions. Distinctions between the new taxon and the only known species of the genus are compared.

Survey, Identification, and Characterization of Cylindrocarpon-Like Asexual Morphs in Spanish Forest Nurseries.

Cylindrocarpon-like asexual morphs infect herbaceous and woody plants, mainly in agricultural scenarios, but also in forestry systems. The aim of the present study was to characterize a collection of Cylindrocarpon-like isolates recovered from the roots of a broad range of forest hosts from nurseries showing decline by morphological and molecular studies. Between 2009 and 2012, 17 forest nurseries in Spain were surveyed and a total of 103 Cylindrocarpon-like isolates were obtained. Isolates were identified based on DNA sequences of the partial gene regions histone H3 (his3). For the new species, the internal transcribed spacer and intervening 5.8S nrRNA gene (ITS) region, ß-tubulin (tub2), and translation elongation factor 1-α (tef1) were also used to determine their phylogenetic position. Twelve species belonging to the genera Cylindrodendrum, Dactylonectria, and Ilyonectria were identified from damaged roots of 15 different host genera. The species C. alicantinum, D. macrodidyma, D. novozelandica, D. pauciseptata, D. pinicola, D. torresensis, I. capensis, I. cyclaminicola, I. liriodendri, I. pseudodestructans, I. robusta, and I. rufa were identified. In addition, two Dactylonectria species (D. hispanica sp. nov. and D. valentina sp. nov.), one Ilyonectria species (I. ilicicola sp. nov.), and one Neonectria species (N. quercicola sp. nov.) are newly described. The present study demonstrates the prevalence of this fungal group associated with seedlings of diverse hosts showing decline symptoms in forest nurseries in Spain.

Disentangling cryptic species with isaria-like morphs in Cordycipitaceae.

A new genus and eight new species, all with isaria-like phialides, are described in Cordycipitaceae from Thailand. The new genus, Samsoniella, is segregated from Akanthomyces based on morphological and molecular evidence. Samsoniella differs from Akanthomyces in producing orange cylindrical to clavate stromata with superficial perithecia and orange conidiophores with isaria-like phialides and white to cream conidia. A new combination for CBS 240.32, originally identified as Paecilomyces farinosus (Isaria farinosa), and CBS 262.58, originally identified as Penicillium alboaurantium, respectively, is made in Samsoniella. Two new species, Samsoniella aurantia and S. inthanonensis, are described from lepidopteran larvae. Two new species of Cordyceps, C. blackwelliae and C. lepidopterorum, were also found on coleopteran and lepidopteran larvae. Both produce isaria-like morphs with globose phialides and attenuated long necks and white mycelium in culture. The authors established a sexual-asexual link for Cordyceps javanica (= Isaria javanica) on lepidopteran larvae. Four new species, Akanthomyces kanyawimiae, A. sulphureus, A. thailandicus, and A. waltergamsii, were pathogenic on spiders, with some strains of A. kanyawimiae also found on unidentified insect larvae. These four species of Akanthomyces occur on the underside of leaves and produce white to cream white powdery conidia, whereas S. aurantia and S. inthanonensis were found in leaf litter and produce bright orange stromata and synnemata with white conidia. Another new combination, Akanthomyces ryukyuensis, is proposed. Phylogenetic analyses based on a combined data set comprising the nuc rDNA region encompassing the internal transcribed spacers 1 and 2 along with the 5.8S rDNA (ITS), nuc 28S rDNA (28S), partial sequences of translation elongation factor 1-α gene (TEF1), and the genes for RNA polymerase II largest (RPB1) and second-largest (RPB2) subunits strongly support the delimitation of these new species of Cordyceps, Akanthomyces, and in a new genus Samsoniella in Cordycipitaceae.

Psychronectria hyperantarctica, gen. nov., comb. nov., epitypification and phylogenetic position of an Antarctic bryophilous ascomycete.

The holotype of Thyronectria hyperantarctica was re-examined, redescribed, and compared with new collections of an Antarctic bryophilous ascomycete from a similar area. Because the condition of type material was insufficient for molecular studies, the authors designated an epitype from newly collected material with a high degree of morphological similarity to the holotype and paratype material. Phylogenetic analysis of the epitype revealed that its closest phylogenetic affinity was with the family Tilachlidiaceae and it formed a monophyletic group in this lineage within other collections of the species. Therefore, the new monotypic genus Psychronectria is described to accommodate Thyronectria hyperantarctica. The fungus is superficially similar to Thyronectria species in Nectriaceae, but the ascospores differ in color, size, and type of septation.

Effect of plant resistance and BioAct WG (Purpureocillium lilacinum strain 251) on Meloidogyne incognita in a tomato-cucumber rotation in a greenhouse.

BACKGROUND: The effectiveness of combining resistant tomato with BioAct WG (Purpureocillium lilacinum strain 251, Pl251) against Meloidogyne incognita was assessed in a tomato-cucumber rotation in a greenhouse over 2 years. Additionally, the enzymatic activity of the fungus, the percentage of fungal egg and juvenile parasitism, cardinal temperatures and the effect of water potential on mycelial growth and the soil receptivity to Pl251 were determined in vitro. RESULTS: Plant resistance was the only factor that suppressed nematode and crop yield losses. Percentage of egg parasitism in plots treated with BioAct WG was less than 2.6%. However, under in vitro conditions, Pl251 showed protease, lipase and chitinase activities and parasitised 94.5% of eggs, but no juveniles. Cardinal temperatures were 14.2, 24-26 and 35.4 °C. The maximum Pl251 mycelial growth was at -0.25 MPa and 25 °C. Soil temperatures and water potential in the greenhouse were in the range of the fungus. However, soil receptivity was lower in greenhouse soil, irrespective of sterilisation, than in sterilised sand. CONCLUSION: Plant resistance was the only factor able to suppress nematode densities, disease severity and yield losses, and to protect the following cucumber crop. Environmental factors involved in soil receptivity could have negatively affected fungus effectiveness. © 2016 Society of Chemical Industry.

Revision of the genus Thyronectria (Hypocreales) from China.

Recent collections and herbarium specimens of Thyronectria from different regions in China were examined. Using combined analyses of morphology and molecular data, we recognized eight species. Among them, Thyronectria atrobrunnea, T. orientalis, and T. sinensis are described and illustrated as new species. Thyronectria atrobrunnea is characterized by blackish brown perithecia that become cupulate when dry, and 8-spored asci containing ellipsoidal to broadly fusiform or subcylindrical ascospores that bud to form bacillar to subellipsoidal ascoconidia within the asci. Thyronectria orientalis can be easily recognized by stromata that are erumpent through the epidermis of the host, immersed or semi-immersed perithecia covered with yellowish green scurf, and ellipsoidal to subfusiform, muriform ascospores. Thyronectria sinensis on Pinus features solitary ascomata that are rarely aggregated, and 8-spored asci giving rise to subcylindrical to vermiform, multiseptate ascospores that form bacillar to allantoid ascoconidia that fill the asci. The new species and their close relatives are compared and differences between them are discussed. Thyronectria strobi is reported for the first time in China. Name changes for the previously recorded species are noted. Phylogenetic analyses inferred from 28S, ITS, RPB1, TEF1, and TUB2 hint that phenotypic characters, viz. stromata, ascospores, appendage of perithecial wall, and host specificity may carry phylogenetic information as previous papers discussed.