Alternaria solani effectors AsCEP19 and AsCEP20 reveal novel functions in pathogenicity and conidiogenesis.

Previous work identified a pair of specific effectors AsCEP19 and AsCEP20 in Alternaria solani as contributors to the virulence of A. solani. Here, we constructed AsCEP19 and AsCEP20 deletion mutants in A. solani strain HWC168 to further reveal the effects of these genes on the biology and pathogenicity of A. solani. Deletion of AsCEP19 and AsCEP20 did not affect vegetative growth but did affect conidial maturation, with an increase in the percentage of abnormal conidia produced. Furthermore, we determined the expression patterns of genes involved in the conidiogenesis pathway and found that the regulatory gene abaA was significantly upregulated and chsA, a positive regulator for conidiation, was significantly downregulated in the mutant strains compared to the wild-type strain. These results suggest that AsCEP19 and AsCEP20 indirectly affect the conidial development and maturation of A. solani. Pathogenicity assays revealed significantly impaired virulence of ΔAsCEP19, ΔAsCEP20, and ΔAsCEP19 + AsCEP20 mutants on potato and tomato plants. Moreover, we performed localization assays with green fluorescent protein-tagged proteins in chili pepper leaves. We found that AsCEP19 can specifically localize to the chloroplasts of chili pepper epidermal cells, while AsCEP20 can localize to both chloroplasts and the plasma membrane. Weighted gene co-expression network analysis revealed enrichment of genes of this module in the photosynthesis pathway, with many hub genes associated with chloroplast structure and photosynthesis. These results suggest that chloroplasts are the targets for AsCEP19 and AsCEP20. IMPORTANCE: Alternaria solani is an important necrotrophic pathogen causing potato early blight. Previous studies have provide preliminary evidence that specific effectors AsCEP19 and AsCEP20 contribute to virulence, but their respective functions, localization, and pathogenic mechanisms during the infection process of A. solani remain unclear. Here, we have systematically studied the specific effectors AsCEP19 and AsCEP20 for the first time, which are essential for conidial maturation. The deletion of AsCEP19 and AsCEP20 can significantly impair fungal pathogenicity. Additionally, we preliminarily revealed that AsCEP19 and AsCEP20 target the chloroplasts of host cells. Our findings further enhance our understanding of the molecular mechanisms underlying the virulence of necrotrophic pathogens.

CgNis1's Impact on Virulence and Stress Response in Colletotrichum gloeosporioides.

Pepper anthracnose caused by Colletotrichum gloeosporioides infection is an important fungal disease and represents a serious threat to pepper yield and quality. At present, the pathogenic molecular mechanism of C. gloeosporioides is not very clear. In our study, we characterized the function of C. gloeosporioides CgNis1, a homolog of Magnaporthe oryzae MoNis1. We found that the ∆Cgnis1 mutant reduced the growth rate and was defective in conidiation. Although the rate of appressorium formation was unaffected, the germ tube was found to be abnormal. CgNis1 was shown to be involved in the H2O2 stress response and maintaining cell membrane permeability. The pathogenicity assays performed in this study indicated that the deletion of CgNIS1 is associated with virulence. Our results indicate that CgNis1 is necessary for the growth, development, and pathogenicity of the fungus. This work provides an in-depth analysis of the Nis1 protein, helps to enhance studies on pathogen-related molecular mechanisms, and provides a theoretical basis for the prevention and control of C. gloeosporioides in peppers.

A feedback regulation of FgHtf1-FgCon7 loop in conidiogenesis and development of Fusarium graminearum.

The transcription factor FgHtf1 is important for conidiogenesis in Fusarium graminearum and it positively regulates the expression of the sporulation-related gene FgCON7. However, the regulatory mechanism underlying its functions is still unclear. The present study intends to uncover the functional mechanism of FgHtf1 in relation to FgCon7 in F. graminearum. We demonstrated that FgCON7 serves as a target gene for FgHtf1. Interestingly, FgCon7 also binds the promoter region of FgHTF1 to negatively regulate its expression, thus forming a negative-feedback loop. We demonstrated that FgHtf1 and FgCon7 have functional redundancy in fungal development. FgCon7 localizes in the nucleus and has transcriptional activation activity. Deletion of FgCON7 significantly reduces conidia production. 4444 genes were regulated by FgCon7 in ChIP-Seq, and RNA-Seq revealed 4430 differentially expressed genes in FgCON7 deletion mutant, with CCAAT serving as a consensus binding motif of FgCon7 to the target genes. FgCon7 directly binds the promoter regions of FgMSN2, FgABAA, FgVEA and FgSMT3 genes and regulates their expression. These genes were found to be important for conidiogenesis. To our knowledge, this is the first study that unveiled the mutual regulatory functions of FgCON7 and FgHTF1 to form a negative-feedback loop, and how the loop mediates sporulation in F. graminearum.

MoMih1 is indispensable for asexual development, cell wall integrity, and pathogenicity of Magnaporthe oryzae.

Asexual spore serves as essential inoculum of rice blast during the disease cycle, and differentiation of young conidium from conidiophore is intimately regulated by cell cycle. Mih1 encodes a dual-specificity phosphatase that involved in the G2/M transition of the mitotic cell cycle by regulating the Cdk1 activity in eukaryotes. Till now, the roles of Mih1 homologue, however, remain unclear in Magnaporthe oryzae. We here functionally characterized the Mih1 homologue MoMih1 in M. oryzae. MoMih1 is localized to both the cytoplasm and nucleus and can physically interact with the CDK protein MoCdc28 in vivo. Loss of MoMih1 led to delayed nucleus division and a high level of Tyr15 phosphorylation of MoCdc28. The MoMih1 mutants showed retarded mycelial growth with a defective polar growth, less fungal biomass, and shorter distance between diaphragms, compared with the KU80. Asexual reproduction altered in MoMih1 mutants, with both abnormal conidial morphogenesis and decreased conidiation. The MoMih1 mutants severely attenuated the virulence to host plants due to the impaired ability of penetration and biotrophic growth. The incapability of scavenging of host-derived reactive oxygen species, which was possibly ascribed to the severely decreased extracellular enzymes activities, were partially associated with deficiency of pathogenicity. Besides, the MoMih1 mutants displayed also improper localization of retromer protein MoVps26 and polarisome component MoSpa2, and defects of cell wall integrity (CWI), melanin pigmentation, chitin synthesis, and hydrophobicity. In conclusion, our results demonstrate that MoMih1 plays pleiotropic roles during fungal development and plant infection of M. oryzae.

New genera and species with chloridium-like morphotype in the Chaetosphaeriales and Vermiculariopsiellales.

In this study, we investigated the morphological and genetic variability of selected species belonging to the genus Chloridium sensu lato, some also referred to as chloridium-like asexual morphs and other undescribed morphologically similar fungi. These species do not conform to the revised generic concept and thus necessitate a re-evaluation in terms of taxonomy and phylogeny. The family Chaetosphaeriaceae (Chaetosphaeriales) encompasses a wide range of asexual morphotypes, and among them, the simplest form is represented by Chloridium sect. Chloridium. The morphological simplicity of the Chloridium morphotype has historically led to the amalgamation of numerous unrelated species, thereby creating a heterogeneous genus. By conducting phylogenetic reconstruction of four DNA loci and examining a set of 71 strains, including all available ex-type and other non-type strains as well as holotypes and other herbarium material, we were able to gain new insights into the relationships between these taxa. Phylogenetic analyses revealed that the studied species are distantly related to Chloridium sensu stricto and can be grouped into two orders in the Sordariomycetes. Within the Chaetosphaeriales, they formed nine well-separated genera in four clades, such as Cacumisporium, Caliciastrum gen. nov., Caligospora gen. nov., Capillisphaeria gen. nov., Curvichaeta, Fusichloridium, Geniculoseta gen. nov., Papillospora gen. nov., and Spicatispora gen. nov. We also established Chloridiopsiella gen. nov. and Chloridiopsis gen. nov. in Vermiculariopsiellales. Four new species and eight new combinations are proposed in these genera. Our study provides a clearer understanding of the genus Chloridium, its relationship to other morphologically similar fungi, and a new taxonomic treatment and molecular phylogeny to facilitate their accurate identification and classification in future research. Taxonomic novelties: New genera: Caliciastrum Réblová, Caligospora Réblová, Capillisphaeria Réblová, Chloridiopsiella Réblová, Chloridiopsis Réblová, Geniculoseta Réblová, Papillospora Réblová, Spicatispora Réblová; New species: Caliciastrum bicolor Réblová, Caligospora pannosa Réblová, Chloridiopsis syzygii Réblová, Gongromerizella silvana Réblová; New combinations: Caligospora dilabens (Réblová & W. Gams) Réblová, Capillisphaeria crustacea (Sacc.) Réblová, Chloridiopsiella preussii (W. Gams & Hol.-Jech.) Réblová, Chloridiopsis constrictospora (Crous et al.) Réblová, Geniculoseta preussii (W. Gams & Hol.-Jech.) Réblová, Papillospora hebetiseta (Réblová & W. Gams) Réblová, Spicatispora carpatica (Hol.-Jech. & Révay) Réblová, Spicatispora fennica (P. Karst.) Réblová; Epitypifications (basionyms): Chaetosphaeria dilabens Réblová & W. Gams, Chloridium cylindrosporum W. Gams & Hol.-Jech. Citation: Réblová M, Nekvindová J (2023). New genera and species with chloridium-like morphotype in the Chaetosphaeriales and Vermiculariopsiellales. Studies in Mycology 106: 199-258. doi: 10.3114/sim.2023.106.04.

BsTup1 is required for growth, conidiogenesis, stress response and pathogenicity of Bipolaris sorokiniana.

Tup1, a conserved transcriptional repressor, plays a critical role in the growth and development of fungi. Here, we identified a BsTup1 gene from the plant pathogenic fungus Bipolaris sorokiniana. The expression of BsTup1 showed a more than three-fold increase during the conidial stage compared with mycelium stage. Deletion of BsTup1 led to decrease hyphal growth and defect in conidia formation. A significant difference was detected in osmotic, oxidative, or cell wall stress responses between the WT and ΔBsTup1 strains. Pathogenicity assays showed that virulence of the ΔBsTup1 mutant was dramatically decreased on wheat and barely leaves. Moreover, it was observed that hyphal tips of the mutants could not form appressorium-like structures on the inner epidermis of onion and barley coleoptile. Yeast two-hybrid assays indicated that BsTup1 could interact with the BsSsn6. RNAseq revealed significant transcriptional changes in the ΔBsTup1 mutant with 2369 genes down-regulated and 2962 genes up-regulated. In these genes, we found that a subset of genes involved in fungal growth, sporulation, cell wall integrity, osmotic stress, oxidation stress, and pathogenicity, which were misregulated in the ΔBsTup1 mutant. These data revealed that BsTup1 has multiple functions in fungal growth, development, stress response and pathogenesis in B. sorokiniana.

Aspartate Transaminase AST2 Involved in Sporulation and Necrotrophic Pathogenesis in the Hemibiotrophs Magnaporthe oryzae and Colletotrichum graminicola.

Aspartate family includes five additional amino acids other than aspartate, among which most except aspartate have been reported for their action in pathogenesis by amino acid biosynthesis. However, how aspartate, the initial substrate of this family metabolic pathway, is involved in pathogenesis remains unknown. Here, we focused on aspartate transaminase (AST) that catalyzes transamination reaction between glutamate-aspartate in Magnaporthe oryzae. Three MoAST genes were bioinformatically analyzed, of which MoAST2 was uniquely upregulated when invasive hyphae switched to necrotrophic pathogenesis. MoAST2 deletion (ΔMoast2) caused a drastic reduction in conidiogenesis and appressorium formation. Particularly, ΔMoast2 was observed to be proliferated at the biotrophic phase but inhibited at the necrotrophic stage, and with invisible symptoms detected, suggesting a critical role in necrotrophic phase. Glutamate family restored the ΔMoast2 defects but aspartate family did not, inferring that transamination occurs from aspartate to glutamine. MoAST2 is cytosolic and possessed H2O2 stress tolerance. In parallel, Colletotrichum graminicola AST2, CgAST2 was proven to be a player in necrotrophic anthracnose development. Therefore, conserved AST2 is qualified to be a drug target for disease control.

Proteomic and Phosphoryproteomic Investigations Reveal that Autophagy-Related Protein 1, a Protein Kinase for Autophagy Initiation, Synchronously Deploys Phosphoregulation on the Ubiquitin-Like Conjugation System in the Mycopathogen Beauveria bassiana.

Autophagy is a conserved intracellular degradation mechanism in eukaryotes and is initiated by the protein kinase autophagy-related protein 1 (Atg1). However, except for the autophosphorylation activity of Atg1, the target proteins phosphorylated by Atg1 are largely unknown in filamentous fungi. In Beauveria bassiana (a filamentous insect-pathogenic fungus), Atg1 is indispensable for autophagy and is associated with fungal development. Comparative omics-based analyses revealed that B. bassiana Atg1 (BbAtg1) has key influence on the proteome and phosphoproteome during conidiogenesis. In terms of its physiological functions, the BbAtg1-mediated phosphoproteome is primarily associated with metabolism, signal transduction, cell cycle, and autophagy. At the proteomic level, BbAtg1 mainly regulates genes involved in protein synthesis, protein fate, and protein with binding function. Furthermore, integrative analyses of phosphoproteomic and proteomic data led to the identification of several potential targets regulated by BbAtg1 phosphorylation activity. Notably, we demonstrated that BbAtg1 phosphorylated BbAtg3, an essential component of the ubiquitin-like conjugation system in autophagic progress. Our findings indicate that in addition to being a critical component of the autophagy initiation, Atg1 orchestrates autophagosome elongation via its phosphorylation activity. The data from our study will facilitate future studies on the noncanonical targets of Atg1 and help decipher the Atg1-mediated phosphorylation networks. IMPORTANCE Autophagy-related protein 1 (Atg1) is a serine/threonine protein kinase for autophagy initiation. In contrast to the unicellular yeast, the target proteins phosphorylated by Atg1 are largely unknown in filamentous fungi. In this study, the entomopathogenic fungus Beauveria bassiana was used as a representative of filamentous fungi due to its importance in the applied and fundamental research. We revealed that Atg1 mediates the comprehensive proteome and phosphoproteome, which differ from those revealed in yeast. Further investigation revealed that Atg1 directly phosphorylates the E2-like enzyme Atg3 of the ubiquitin-like conjugation system (ULCS), and the phosphorylation of Atg3 is indispensable for ULCS functionality. Interestingly, the phosphorylation site of Atg3 is conserved among a set of insect- and plant-pathogenic fungi but not in human-pathogenic fungi. This study reveals new regulatory mechanisms of autophagy and provides new insights into the evolutionary diversity of the Atg1 kinase signaling pathways among different pathogenic fungi.

Consolidation of Chloridium: new classification into eight sections with 37 species and reinstatement of the genera Gongromeriza and Psilobotrys.

Chloridium is a little-studied group of soil- and wood-inhabiting dematiaceous hyphomycetes that share a rare mode of phialidic conidiogenesis on multiple loci. The genus has historically been divided into three morphological sections, i.e. Chloridium, Gongromeriza, and Psilobotrys. Sexual morphs have been placed in the widely perceived genus Chaetosphaeria, but unlike their asexual counterparts, they show little or no morphological variation. Recent molecular studies have expanded the generic concept to include species defined by a new set of morphological characters, such as the collar-like hyphae, setae, discrete phialides, and penicillately branched conidiophores. The study is based on the consilience of molecular species delimitation methods, phylogenetic analyses, ancestral state reconstruction, morphological hypotheses, and global biogeographic analyses. The multilocus phylogeny demonstrated that the classic concept of Chloridium is polyphyletic, and the original sections are not congeneric. Therefore, we abolish the existing classification and propose to restore the generic status of Gongromeriza and Psilobotrys. We present a new generic concept and define Chloridium as a monophyletic, polythetic genus comprising 37 species distributed in eight sections. In addition, of the taxa earlier referred to Gongromeriza, two have been redisposed to the new genus Gongromerizella. Analysis of published metabarcoding data showed that Chloridium is a common soil fungus representing a significant (0.3 %) proportion of sequence reads in environmental samples deposited in the GlobalFungi database. The analysis also showed that they are typically associated with forest habitats, and their distribution is strongly influenced by climate, which is confirmed by our data on their ability to grow at different temperatures. We demonstrated that Chloridium forms species-specific ranges of distribution, which is rarely documented for microscopic soil fungi. Our study shows the feasibility of using the GlobalFungi database to study the biogeography and ecology of fungi. Taxonomic novelties: New genus: Gongromerizella Réblová; New sections: Chloridium section Cryptogonytrichum Réblová, Hern.-Restr., M. Kolarík & F. Sklenar, Chloridium section Gonytrichopsis Réblová, Hern.-Restr., M. Kolarík & F. Sklenar, Chloridium section Metachloridium Réblová, Hern.-Restr., M. Kolarík & F. Sklenar, Chloridium section Volubilia Réblová, Hern.-Restr., M. Kolarík & F. Sklenar; New species: Chloridium bellum Réblová & Hern.-Restr., Chloridium biforme Réblová & Hern.-Restr., Chloridium detriticola Réblová & Hern.-Restr., Chloridium gamsii Réblová & Hern.-Restr., Chloridium guttiferum Réblová & Hern.-Restr., Chloridium moratum Réblová & Hern.-Restr., Chloridium peruense Réblová & Hern.-Restr., Chloridium novae-zelandiae Réblová & Hern.-Restr., Chloridium elongatum Réblová & Hern.-Restr., Chloridium volubile Réblová & Hern.-Restr.; New varieties: Chloridium bellum var. luteum Réblová & Hern.-Restr., Chloridium detriticola var. effusum Réblová & Hern.-Restr., Chloridium chloridioides var. convolutum Réblová & Hern.-Restr.; New combinations: Chloridium section Gonytrichum (Nees & T. Nees) Réblová, Hern.-Restr., M. Kolarík & F. Sklenar, Chloridium section Mesobotrys (Sacc.) Réblová, Hern.-Restr., M. Kolarík & F. Sklenar, Chloridium section Pseudophialocephala (M.S. Calabon et al.) Réblová, Hern.-Restr., M. Kolarík & F. Sklenar, Chloridium simile (W. Gams & Hol.-Jech.) Réblová & Hern.-Restr., Chloridium chloridioides (W. Gams & Hol.-Jech.) Réblová & Hern.-Restr., Chloridium subglobosum (W. Gams & Hol.-Jech.) Réblová & Hern.-Restr., Chloridium fuscum (Corda) Réblová & Hern.-Restr., Chloridium ypsilosporum (Hol.-Jech.) Réblová & Hern.-Restr., Chloridium costaricense (G. Weber et al.) Réblová & Hern.-Restr., Chloridium cuneatum (N.G. Liu et al.) Réblová & Hern.-Restr., Fusichloridium cylindrosporum (W. Gams & Hol.-Jech.) Réblová, Gongromeriza myriocarpa (Fr.) Réblová, Gongromeriza pygmaea (P. Karst.) Réblová, Gongromerizella lignicola (F. Mangenot) Réblová, Gongromerizella pachytrachela (W. Gams & Hol.-Jech) Réblová, Gongromerizella pini (Crous & Akulov) Réblová; New name: Chloridium pellucidum Réblová & Hern.-Restr.; Epitypifications (basionyms): Chaetopsis fusca Corda, Gonytrichum caesium var. subglobosum W. Gams & Hol.-Jech.; Lectotypification (basionym): Gonytrichum caesium Nees & T. Nees. Citation: Réblová M, Hernández-Restrepo M, Sklenár F, Nekvindová J, Réblová K, Kolarík M (2022). Consolidation of Chloridium: new classification into eight sections with 37 species and reinstatement of the genera Gongromeriza and Psilobotrys. Studies in Mycology 103: 87-212. doi: 10.3114/sim.2022.103.04.

Phylogenetic Reassessment, Taxonomy, and Biogeography of Codinaea and Similar Fungi.

The genus Codinaea is a phialidic, dematiaceous hyphomycete known for its intriguing morphology and turbulent taxonomic history. This polyphasic study represents a new, comprehensive view on the taxonomy, systematics, and biogeography of Codinaea and its relatives. Phylogenetic analyses of three nuclear loci confirmed that Codinaea is polyphyletic. The generic concept was emended; it includes four morphotypes that contribute to its morphological complexity. Ancestral inference showed that the evolution of some traits is correlated and that these traits previously used to delimit taxa at the generic level occur in species that were shown to be congeneric. Five lineages of Codinaea-like fungi were recognized and introduced as new genera: Codinaeella, Nimesporella, Stilbochaeta, Tainosphaeriella, and Xyladelphia. Dual DNA barcoding facilitated identification at the species level. Codinaea and its segregates thrive on decaying plants, rarely occurring as endophytes or plant pathogens. Environmental ITS sequences indicate that they are common in bulk soil. The geographic distribution found using GlobalFungi database was consistent with known data. Most species are distributed in either the Holarctic realm or tropical geographic regions. The ancestral climatic zone was temperate, followed by transitions to the tropics; these fungi evolved primarily in Eurasia and Americas, with subsequent transitions to Africa and Australasia.

Optimization of granular formulations of Metarhizium humberi microsclerotia with humectants.

Granular microsclerotial formulations of entomopathogenic fungi deserve attention because of their post-application, in situ production of new conidia that enhance and prolong mycoinsecticidal efficacy against a target pest insect. Because high ambient moisture is a crucial condition to induce fungal development and conidiogenesis on granules, we tested the impacts of the additions of three humectants-glycerin, propylene glycol, and polyethylene glycol 400-on water absorption by pellets incorporating microsclerotia of Metarhizium humberi IP 46 with microcrystalline cellulose or vermiculite carriers, and on the production of infective conidia of IP 46 microsclerotia in ambient humidities suboptimal for routine conidiogenesis. Glycerin facilitated greater and faster absorption of water than the other humectants. Microcrystalline cellulose absorbed low quantities of water without any added humectant whereas vermiculite did not. IP 46 did not grow or sporulate on pellets prepared with or without glycerin at 86% relative humidity (RH) or on control pellets without glycerin at 91% RH; conidial production on pellets prepared with vermiculite or microcrystalline cellulose and 10% glycerin reached 1.1 × 105 conidia/mg and 1 × 105 conidia/mg, respectively, after 20 days of exposure at 91% RH. Hence, these results strongly support glycerin as a suitable humectant for granular microsclerotial formulations of this fungus.

Phylogeny and taxonomy of Catenularia and similar fungi with catenate conidia.

The genus Catenularia (Chaetosphaeriaceae) was reviewed, and its relationships with morphologically similar fungi were evaluated using molecular and morphological data. Eleven species are accepted, four of which have been verified with molecular DNA data. The correct epithet 'cupulifera' is proposed for the type species C. cupulifera comb. nov. Four other combinations are proposed, namely C. catenulata comb. nov., C. elsikii comb. nov., C. minor comb. nov. and C. novae-zelandiae comb. nov. Catenularia is an uncommon fungus inhabiting mainly decaying bark, wood and bamboo culms of various hosts and shows a widespread geographical distribution. It is circumscribed for fungi with mononematous, macronematous, simple conidiophores with terminal monophialides, usually accompanied with capitate hyphae. The conidia are aseptate, brown, cuneiform to rounded-obconic with an angular outline, adhering in chains. The diagnostic values of taxonomic characteristics of capitate hyphae and conidia (i.e. colour, shape in transverse section, setulae and formation) at the generic level were evaluated. An account of morphology, taxonomy and phylogeny of species accepted in Catenularia is provided. Based on ribosomal DNA sequences, Chalarodes obpyramidata sp. nov., characterised by catenate, angular, hyaline conidia with apical setulae, is revealed as closely related to Catenularia. The new genus Fuscocatenula gen. nov. is proposed for catenularia-like fungi having pigmented conidia with protracted maturation and round outline, with two species accepted, F. submersa comb. nov. and F. variegata comb. nov. A new species Nawawia antennata sp. nov. is introduced and Nawawia is compared with morphologically similar taxa.

Reflections on Menisporopsis, Multiguttulispora and Tainosphaeria Using Molecular and Morphological Data.

The genera Menisporopsis, Multiguttulispora and Tainosphaeria (Chaetosphaeriaceae) are saprobes inhabiting decaying plant material. This study is based on an integrated morpho-molecular characterisation to assess their generic concepts and explore phylogenetic relationships. Menisporopsis is revealed as polyphyletic, and species with 1-septate conidia and synnemata growing unilaterally along the seta are placed in the new segregate genus Arcuatospora. Codinaea dimorpha and C. triseptata are shown to be congeneric with Multiguttulispora sympodialis, the type species. Two new combinations are proposed: M. sympodialis is found conspecific with M. dimorpha. The Tainosphaeria complex is resolved into three genera. We found that the morphological separation of three groups within the genus is consistent with phylogenetic relationships. Tainosphaeria s. str. is accepted with five species. Tainosphaeria aseptata and T. lunata are transferred to the newly erected Phialoturbella, whereas T. obclavata is revealed as conspecific with Phialogeniculata guadalcanalensis, reducing it to a synonym. A new genus Flectospora is erected for a chloridium-like fungus nested in the Tainosphaeria clade. Based on molecular evidence, we show that asymmetrical, scolecosporous ascospores are a unique teleomorphic characteristic among family members. Therefore, we propose new combinations for Chaetosphaeria hispida in Paragaeumannomyces and Ch. spinosa in the new genus Ericiosphaeria, both exhibiting this rare morphotype.

Phylogeny, Global Biogeography and Pleomorphism of Zanclospora.

Zanclospora (Chaetosphaeriaceae) is a neglected, phialidic dematiaceous hyphomycete with striking phenotypic heterogeneity among its species. Little is known about its global biogeography due to its extreme scarcity and lack of records verified by molecular data. Phylogenetic analyses of six nuclear loci, supported by phenotypic data, revealed Zanclospora as highly polyphyletic, with species distributed among three distantly related lineages in Sordariomycetes. Zanclospora is a pleomorphic genus with multiple anamorphic stages, of which phaeostalagmus-like and stanjehughesia-like are newly discovered. The associated teleomorphs were previously classified in Chaetosphaeria. The generic concept is emended, and 17 species are accepted, 12 of which have been verified with DNA sequence data. Zanclospora thrives on decaying plant matter, but it also occurs in soil or as root endophytes. Its global diversity is inferred from metabarcoding data and published records based on field observations. Phylogenies of the environmental ITS1 and ITS2 sequences derived from soil, dead wood and root samples revealed seven and 15 phylotypes. The field records verified by DNA data indicate two main diversity centres in Australasia and Caribbean/Central America. In addition, environmental ITS data have shown that Southeast Asia represents a third hotspot of Zanclospora diversity. Our data confirm that Zanclospora is a rare genus.

Delimitation and phylogeny of Dictyochaeta, and introduction of Achrochaeta and Tubulicolla, genera nova.

Dictyochaeta (Chaetosphaeriaceae) is a phialidic dematiaceous hyphomycete with teleomorphs classified in Chaetosphaeria. It is associated with significant variability of asexual morphological traits, which led to its broad delimitation. In the present study, six loci: nuc rDNA internal transcribed spacer region ITS1-5.8S-ITS2 (ITS barcode), nuc 18S rDNA (18S), nuc 28S rDNA (28S), DNA-directed RNA polymerase II second largest subunit gene (RPB2), translation elongation factor 1-α (TEF1-α), and ß-tubulin (TUB2), along with comparative morphological and cultivation studies, are used to reevaluate the concept of Dictyochaeta and establish species boundaries. Based on revised species, morphological characteristics of conidia (shape, septation, absence or presence of setulae), collarettes (shape), and setae (presence or absence) and an extension of the conidiogenous cell proved to be important at the generic level. The dual DNA barcoding using ITS and TEF1-α, together with TUB2, facilitated accurate identification of Dictyochaeta species. Thirteen species are accepted, of which seven are characterized in this study; an identification key is provided. It was revealed that D. fuegiana, the type species, is a complex of three distinct species including D. querna and the newly described D. stratosa. Besides, a new species, D. detriticola, and two new combinations, D. callimorpha and D. montana, are proposed. An epitype of D. montana is selected. Dictyochaeta includes saprobes on decaying wood, bark, woody fruits, and fallen leaves. Dictyochaeta is shown to be distantly related to the morphologically similar Codinaea, which is resolved as paraphyletic. Chaetosphaeria talbotii with a Dictyochaeta anamorph represents a novel lineage in the Chaetosphaeriaceae; it is segregated from Dictyochaeta, and a new genus Achrochaeta is proposed. Multigene phylogenetic analysis revealed that D. cylindrospora belongs to the Vermiculariopsiellales, and a new genus Tubulicolla is introduced.

Delimitation, new species and teleomorph-anamorph relationships in Codinaea, Dendrophoma, Paragaeumannomyces and Striatosphaeria (Chaetosphaeriaceae).

The Chaetosphaeriaceae are a diverse group of pigmented, predominantly phialidic hyphomycetes comprised of several holomorphic genera including Chaetosphaeria, the most prominent genus of the family. Although the morphology of the teleomorphs of the majority of Chaetosphaeria is rather uniform, their associated anamorphs primarily exhibit the variability and evolutionary change observed in the genus. An exception from the morphological monotony among Chaetosphaeria species is a group characterised by scolecosporous, hyaline to light pink, multiseptate, asymmetrical ascospores and a unique three-layered ascomatal wall. Paragaeumannomyces sphaerocellularis, the type species of the genus, exhibits these morphological traits and is compared with similar Chaetosphaeria with craspedodidymum- and chloridium-like synanamorphs. Morphological comparison and phylogenetic analyses of the combined ITS-28S sequences of 35 isolates and vouchers with these characteristics revealed a strongly-supported, morphologically well-delimited clade in the Chaetosphaeriaceae containing 16 species. The generic name Paragaeumannomyces is applied to this monophyletic clade; eight new combinations and five new species, i.e. P. abietinus sp. nov., P. elegans sp. nov., P. granulatus sp. nov., P. sabinianus sp. nov. and P. smokiensis sp. nov., are proposed. A key to Paragaeumannomyces is provided. Using morphology, cultivation studies and phylogenetic analyses of ITS and 28S rDNA, two additional new species from freshwater and terrestrial habitats, Codinaea paniculata sp. nov. and Striatosphaeria castanea sp. nov., are described in the family. A codinaea-like anamorph of S. castanea forms conidia with setulae at each end in axenic culture; this feature expands the known morphology of Striatosphaeria. A chaetosphaeria-like teleomorph is experimentally linked to Dendrophoma cytisporoides, a sporodochial hyphomycete and type species of Dendrophoma, for the first time.

A SET domain-containing protein involved in cell wall integrity signaling and peroxisome biogenesis is essential for appressorium formation and pathogenicity of Colletotrichum gloeosporioides.

The chromatin modulator Set5 plays important regulatory roles in both cell growth and stress responses of Saccharomyces cerevisiae. However, its function in filamentous fungi remains poorly understood. Here, we report the pathogenicity-related gene CgSET5 discovered in a T-DNA insertional mutant M285 of Colletotrichum gloeosporioides. Bioinformatic analysis revealed that CgSET5 encodes a SET domain-containing protein that is a homolog of the budding yeast S. cerevisiae Set5. CgSET5 is important for hyphae growth and conidiation and is necessary for appressorium formation and pathogenicity. CgSet5 regulates appressorium formation in a mitogen-activated protein kinase-independent manner. Inactivation of CgSET5 resulted in a significant reduction in chitin content within the cell wall, indicating CgSet5 plays a vital role in cell wall integrity. CgSet5 is involved in peroxisome biogenesis. We identified CgSet5 as the histone H4 methyltransferase, which methylates the critical H4 lysine residues 5 and 8 in C. gloeosporioides. We carried out a yeast two-hybrid screen to find CgSet5 interacting partners. We found CgSet5 putatively interacts with an inorganic pyrophosphatase named CgPpa1, which co-localized in the cytoplasm with CgSet5. Finally, CgPpa1 was found to strongly interact with CgSet5 in vivo during appressorium formation by bimolecular fluorescence complementation assays. These data corroborate a complex control function of CgSet5 acting as a core pathogenic regulator, which connects cell wall integrity and peroxisome biogenesis in C. gloeosporioides.

The brlA Gene Deletion Reveals That Patulin Biosynthesis Is Not Related to Conidiation in Penicillium expansum.

Dissemination and survival of ascomycetes is through asexual spores. The brlA gene encodes a C2H2-type zinc-finger transcription factor, which is essential for asexual development. Penicillium expansum causes blue mold disease and is the main source of patulin, a mycotoxin that contaminates apple-based food. A P. expansum PeΔbrlA deficient strain was generated by homologous recombination. In vivo, suppression of brlA completely blocked the development of conidiophores that takes place after the formation of coremia/synnemata, a required step for the perforation of the apple epicarp. Metabolome analysis displayed that patulin production was enhanced by brlA suppression, explaining a higher in vivo aggressiveness compared to the wild type (WT) strain. No patulin was detected in the synnemata, suggesting that patulin biosynthesis stopped when the fungus exited the apple. In vitro transcriptome analysis of PeΔbrlA unveiled an up-regulated biosynthetic gene cluster (PEXP_073960-PEXP_074060) that shares high similarity with the chaetoglobosin gene cluster of Chaetomium globosum. Metabolome analysis of PeΔbrlA confirmed these observations by unveiling a greater diversity of chaetoglobosin derivatives. We observed that chaetoglobosins A and C were found only in the synnemata, located outside of the apple, whereas other chaetoglobosins were detected in apple flesh, suggesting a spatial-temporal organization of the chaetoglobosin biosynthesis pathway.

New insights into the systematics of Bactrodesmium and its allies and introducing new genera, species and morphological patterns in the Pleurotheciales and Savoryellales (Sordariomycetes).

The newly discovered systematic placement of Bactrodesmium abruptum, the lectotype species of the genus, prompted a re-evaluation of the traditionally broadly conceived genus Bactrodesmium. Fresh material, axenic cultures and new DNA sequence data of five gene regions of six species, i.e. B. abruptum, B. diversum, B. leptopus, B. obovatum, B. pallidum and B. spilomeum, were studied. Bactrodesmium is a strongly resolved lineage in the Savoryellales (Sordariomycetes), supported by Bayesian and Maximum Likelihood methods. The genus Bactrodesmium is emended and delimited to hyphomycetes characterised by sporodochial conidiomata, mononematous often fasciculate conidiophores, holoblastic conidiogenesis and acrogenous, solitary, dry, pigmented, transversely or rarely longitudinally septate conidia. The conidia are seceding rhexolytically, exhibiting multiple secession patterns. An identification key to 35 species accepted in Bactrodesmium is given, providing the most important diagnostic characters. Novel DNA sequence data of B. longisporum and B. stilboideum confirmed their placement in the Sclerococcales (Eurotiomycetes). For other Bactrodesmium, molecular data are available for B. cubense and B. gabretae, which position them in the Dothideomycetes and Leotiomycetes, respectively. All four species are excluded from Bactrodesmium and segregated into new genera, Aphanodesmium, Gamsomyces and Kaseifertia. Classification of 20 other species and varieties not recognised in the genus is discussed. Based on new collections of Dematiosporium aquaticum, the type species of Dematiosporium, the genus is emended to accommodate monodictys-like freshwater lignicolous fungi of the Savoryellales characterised by effuse colonies, holoblastic conidiogenous cells and dictyosporous, pigmented conidia with a pore in each cell. Study of additional new collections, cultures and DNA sequence data revealed several unknown species, which are proposed as taxonomic novelties in the Savoryellales and closely related Pleurotheciales. Ascotaiwania latericolla, Helicoascotaiwania lacustris and Pleurotheciella erumpens are described from terrestrial, lentic and lotic habitats from New Zealand and France, respectively. New combinations are proposed for Helicoascotaiwania farinosa and Neoascotaiwania fusiformis. Relationships and systematics of the Savoryellales are discussed in the light of recent phylogenies and morphological patterns newly linked with the order through cultural studies.

FvatfA regulates growth, stress tolerance as well as mycotoxin and pigment productions in Fusarium verticillioides.

FvatfA from the maize pathogen Fusarium verticillioides putatively encodes the Aspergillus nidulans AtfA and Schizasaccharomyces pombe Atf1 orthologous bZIP-type transcription factor, FvAtfA. In this study, a ΔFvatfA deletion mutant was constructed and then genetically complemented with the fully functional FvatfA gene. Comparing phenotypic features of the wild-type parental, the deletion mutant and the restored strains shed light on the versatile regulatory functions played by FvAtfA in (i) the maintenance of vegetative growth on Czapek-Dox and Potato Dextrose agars and invasive growth on unwounded tomato fruits, (ii) the preservation of conidiospore yield and size, (iii) the orchestration of oxidative (H2O2, menadione sodium bisulphite) and cell wall integrity (Congo Red) stress defences and (iv) the regulation of mycotoxin (fumonisins) and pigment (bikaverin, carotenoid) productions. Expression of selected biosynthetic genes both in the fumonisin (fum1, fum8) and the carotenoid (carRA, carB) pathways were down-regulated in the ΔFvatfA strain resulting in defected fumonisin production and considerably decreased carotenoid yields. The expression of bik1, encoding the polyketide synthase needed in bikaverin biosynthesis, was not up-regulated by the deletion of FvatfA meanwhile the ΔFvatfA strain produced approximately ten times more bikaverin than the wild-type or the genetically complemented strains. The abolishment of fumonisin production of the ΔFvatfA strain may lead to the development of new-type, biology-based mycotoxin control strategies. The novel information gained on the regulation of pigment production by this fungus can be interesting for experts working on new, Fusarium-based biomass and pigment production technologies. Key points • FvatfA regulates vegetative and invasive growths of F. verticillioides. • FvatfA also orchestrates oxidative and cell wall integrity stress defenses. • The ΔFvatfA mutant was deficient in fumonisin production. • FvatfA deletion resulted in decreased carotenoid and increased bikaverin yields.