Phylogeny and systematics of the genus Clonostachys.

Introduction: Clonostachys, a genus with rich morphological and ecological diversity in Bionectriaceae, has a wide distribution among diverse habitats. Methods and Results: In the present study, a phylogenetic framework is reconstructed for the family Bionectriaceae focusing on Clonostachys through increased taxon-sampling using the nrLSU sequence. Through surveying Clonostachys in China, Vietnam, and Thailand over the past 3 years, seven Clonostachys spp. were found and identified. Two new species, C. chuyangsinensis and C. kunmingensis, are described and illustrated based on morphological characteristics and molecular data. The phylogenetic positions of the seven species were evaluated based on four genomic loci (ITS, nrLSU, TUB2, and TEF1). Discussion: Moreover, the genetic divergence comparisons of Clonostachys species for three markers (ITS, TUB2, and TEF1) are also provided. The results indicated that the TEF1 sequence data provided the best resolution for distinguishing species of Clonostachys, followed by sequence data for the TUB2 and ITS regions.

Assessing the Biocontrol Potential of Clonostachys Species Isolated as Endophytes from Coffea Species and as Mycoparasites of Hemileia Rusts of Coffee in Africa.

During surveys conducted in South America and Africa to identify natural fungal enemies of coffee leaf rust (CLR), Hemileia vastatrix, over 1500 strains were isolated, either as endophytes from healthy tissues of Coffea species or as mycoparasites growing on rust pustules. Based on morphological data, eight isolates-three isolated from wild or semiwild coffee and five from Hemileia species on coffee, all from Africa-were provisionally assigned to the genus Clonostachys. A polyphasic study of their morphological, cultural and molecular characteristics-including the Tef1 (translation elongation factor 1 alpha), RPB1 (largest subunit of RNA polymerase II), TUB (ß-tubulin) and ACL1 (ATP citrate lyase) regions-confirmed these isolates as belonging to three species of the genus Clonostachys: namely C. byssicola, C. rhizophaga and C. rosea f. rosea. Preliminary assays were also conducted to test the potential of the Clonostachys isolates to reduce CLR severity on coffee under greenhouse conditions. Foliar and soil applications indicated that seven of the isolates had a significant effect (p < 0.05) in reducing CLR severity. In parallel, in vitro tests that involved conidia suspensions of each of the isolates together with urediniospores of H. vastatrix resulted in high levels of inhibition of urediniospore germination. All eight isolates showed their ability to establish as endophytes in C. arabica during this study, and some proved to be mycoparasites of H. vastatrix. In addition to reporting the first records of Clonostachys associated with healthy coffee tissues and with Hemileia rusts of coffee, this work provides the first evidence that Clonostachys isolates have potential as biological control agents against CLR.

Revising Clonostachys and allied genera in Bionectriaceae.

Clonostachys (Bionectriaceae, Hypocreales) species are common soil-borne fungi, endophytes, epiphytes, and saprotrophs. Sexual morphs of Clonostachys spp. were placed in the genus Bionectria, which was further segregated into the six subgenera Astromata, Bionectria, Epiphloea, Myronectria, Uniparietina, and Zebrinella. However, with the end of dual nomenclature, Clonostachys became the single depository for sexual and asexual morph-typified species. Species of Clonostachys are typically characterised by penicillate, sporodochial, and, in many cases, dimorphic conidiophores (primary and secondary conidiophores). Primary conidiophores are mononematous, either verticillium-like or narrowly penicillate. The secondary conidiophores generally form imbricate conidial chains that can collapse to slimy masses, particularly on sporodochia. In the present study, we investigated the species diversity within a collection of 420 strains of Clonostachys from the culture collection of, and personal collections at, the Westerdijk Fungal Biodiversity Institute in Utrecht, the Netherlands. Strains were analysed based on their morphological characters and molecular phylogeny. The latter used DNA sequence data of the nuclear ribosomal internal transcribed spacer regions and intervening 5.8S nrDNA (ITS) and partial 28S large subunit (LSU) nrDNA and partial protein encoding genes including the RNA polymerase II second largest subunit (RPB2), translation elongation factor 1-alpha (TEF1) and ß-tubulin (TUB2). Based on these results, the subgenera Astromata, Bionectria, Myronectria and Zebrinella are supported within Clonostachys. Furthermore, the genus Sesquicillium is resurrected to accommodate the former subgenera Epiphloea and Uniparietina. The close relationship of Clonostachys and Sesquicillium is strongly supported as both are inferred phylogenetically as sister-genera. New taxa include 24 new species and 10 new combinations. Recognition of Sesquicillium distinguishes species typically forming a reduced perithecial stroma superficially on plant tissue from species in Clonostachys often forming well-developed, through bark erumpent stromata. The patterns of observed perithecial wall anatomies, perithecial wall and stroma interfaces, and asexual morph diversifications described in a previously compiled monograph are used for interpreting ancestral state reconstructions. It is inferred that the common ancestor of Clonostachys and Sesquicillium may have formed perithecia superficially on leaves, possessed a perithecial wall consisting of a single region, and formed intercalary phialides in penicilli of conidiophores. Character interpretation may also allow hypothesising that diversification of morphs occurred then in the two genera independently and that the frequently stroma-linked Clonostachys morphs evolved together with the occupation of woody host niches and mycoparasitism. Taxonomic novelties: New species: Clonostachys aurantiaca L. Zhao & Crous, Clonostachys australiana L. Zhao & Crous, Clonostachys bambusae L. Zhao & Crous, Clonostachys buxicola L. Zhao & Crous, Clonostachys cylindrica L. Zhao & Crous, Clonostachys ellipsoidea L. Zhao & Crous, Clonostachys flava L. Zhao, Crous & Schroers, Clonostachys fujianensis L. Zhao & Crous, Clonostachys fusca L. Zhao, Crous & Schroers, Clonostachys garysamuelsii L. Zhao & Crous, Clonostachys hongkongensis L. Zhao & Crous, Clonostachys longiphialidica L. Zhao, Crous & Schroers, Clonostachys obovatispora, L. Zhao & Crous, Clonostachys palmae L. Zhao, Crous & Schroers, Clonostachys parasporodochialis L. Zhao & Crous, Clonostachys penicillata L. Zhao, Crous & Schroers, Clonostachys reniformis L. Zhao & Crous, Clonostachys vacuolata L. Zhao, Crous & Schroers, Clonostachys venezuelae L. Zhao, Crous & Schroers, Mycocitrus synnematus L. Zhao & Crous, Nectriopsis didymii L. Zhao & Crous, Sesquicillium intermediophialidicum L. Zhao & Crous, Sesquicillium neerlandicum L. Zhao & Crous, Sesquicillium symmetricum L. Zhao & Crous. New combinations: Mycocitrus coccicola (J.A. Stev.) L. Zhao & Crous, Mycocitrus coxeniae (Y.P. Tan et al.) L. Zhao & Crous, Sesquicillium essexcoheniae (Y.P. Tan et al.) L. Zhao & Crous, Sesquicillium lasiacidis (Samuels) L. Zhao, Crous & Schroers, Sesquicillium phyllophilum (Schroers) L. Zhao, Crous & Schroers, Sesquicillium rossmaniae (Schroers) L. Zhao, Crous & Schroers, Sesquicillium saulense (Lechat & J. Fourn.) L. Zhao & Crous, Sesquicillium sesquicillii (Samuels) L. Zhao, Crous & Schroers, Sesquicillium spinulosisporum (Lechat & J. Fourn.) L. Zhao & Crous, Sesquicillium tornatum (Höhn.) Schroers. New synonyms: Clonostachys aranearum W.H. Chen et al., Clonostachys chuyangsinensis H. Yu & Y. Wang, Clonostachys eriocamporesiana R.H. Perera & K.D. Hyde, Clonostachys granuligera (Starbäck) Forin & Vizzini, Clonostachys indica Prasher & R. Chauhan, Clonostachys spinulosa R.H. Perera et al., Clonostachys squamuligera (Sacc.) Forin & Vizzini, Clonostachys wenpingii (J. Luo & W.Y. Zhuang) Z.Q. Zeng & W.Y. Zhuang. Epitypes (basionyms): Fusidium buxi J.C. Schmidt ex Link, Verticillium candelabrum Bonord. Citation: Zhao L, Groenewald JZ, Hernández-Restrepo M, Schroers H-J, Crous PW (2023). Revising Clonostachys and allied genera in Bionectriaceae. Studies in Mycology 105: 205-266. doi: 10.3114/sim.2023.105.03.

Redisposition of acremonium-like fungi in Hypocreales.

Acremonium is acknowledged as a highly ubiquitous genus including saprobic, parasitic, or endophytic fungi that inhabit a variety of environments. Species of this genus are extensively exploited in industrial, commercial, pharmaceutical, and biocontrol applications, and proved to be a rich source of novel and bioactive secondary metabolites. Acremonium has been recognised as a taxonomically difficult group of ascomycetes, due to the reduced and high plasticity of morphological characters, wide ecological distribution and substrate range. Recent advances in molecular phylogenies, revealed that Acremonium is highly polyphyletic and members of Acremonium s. lat. belong to at least three distinct orders of Sordariomycetes, of which numerous orders, families and genera with acremonium-like morphs remain undefined. To infer the phylogenetic relationships and establish a natural classification for acremonium-like taxa, systematic analyses were conducted based on a large number of cultures with a global distribution and varied substrates. A total of 633 cultures with acremonium-like morphology, including 261 ex-type cultures from 89 countries and a variety of substrates including soil, plants, fungi, humans, insects, air, and water were examined. An overview phylogenetic tree based on three loci (ITS, LSU, rpb2) was generated to delimit the orders and families. Separate trees based on a combined analysis of four loci (ITS, LSU, rpb2, tef-1α) were used to delimit species at generic and family levels. Combined with the morphological features, host associations and ecological analyses, acremonium-like species evaluated in the present study are currently assigned to 63 genera, and 14 families in Cephalothecales, Glomerellales and Hypocreales, mainly in the families Bionectriaceae, Plectosphaerellaceae and Sarocladiaceae and five new hypocrealean families, namely Chrysonectriaceae, Neoacremoniaceae, Nothoacremoniaceae, Pseudoniessliaceae and Valsonectriaceae. Among them, 17 new genera and 63 new combinations are proposed, with descriptions of 65 new species. Furthermore, one epitype and one neotype are designated to stabilise the taxonomy and use of older names. Results of this study demonstrated that most species of Acremonium s. lat. grouped in genera of Bionectriaceae, including the type A. alternatum. A phylogenetic backbone tree is provided for Bionectriaceae, in which 183 species are recognised and 39 well-supported genera are resolved, including 10 new genera. Additionally, rpb2 and tef-1α are proposed as potential DNA barcodes for the identification of taxa in Bionectriaceae. Taxonomic novelties: New families: Chrysonectriaceae L.W. Hou, L. Cai & Crous, Neoacremoniaceae L.W. Hou, L. Cai & Crous, Nothoacremoniaceae L.W. Hou, L. Cai & Crous, Pseudoniessliaceae L.W. Hou, L. Cai & Crous, Valsonectriaceae L.W. Hou, L. Cai & Crous. New genera: Bionectriaceae: Alloacremonium L.W. Hou, L. Cai & Crous, Gossypinidium L.W. Hou, L. Cai & Crous, Monohydropisphaera L.W. Hou, L. Cai & Crous, Musananaesporium L.W. Hou, L. Cai & Crous, Paragliomastix L.W. Hou, L. Cai & Crous, Proliferophialis L.W. Hou, L. Cai & Crous, Proxiovicillium L.W. Hou, L. Cai & Crous, Ramosiphorum L.W. Hou, L. Cai & Crous, Verruciconidia L.W. Hou, L. Cai & Crous, Waltergamsia L.W. Hou, L. Cai & Crous; Clavicipitaceae: Subuliphorum L.W. Hou, L. Cai & Crous; Neoacremoniaceae: Neoacremonium L.W. Hou, L. Cai & Crous; Nothoacremoniaceae: Nothoacremonium L.W. Hou, L. Cai & Crous; Plectosphaerellaceae: Allomusicillium L.W. Hou, L. Cai & Crous, Parafuscohypha L.W. Hou, L. Cai & Crous; Pseudoniessliaceae: Pseudoniesslia L.W. Hou, L. Cai & Crous; Sarocladiaceae: Polyphialocladium L.W. Hou, L. Cai & Crous. New species: Bionectriaceae: Alloacremonium ferrugineum L.W. Hou, L. Cai & Crous, Al. humicola L.W. Hou, L. Cai & Crous, Acremonium aerium L.W. Hou, L. Cai & Crous, A. brunneisporum L.W. Hou, L. Cai & Crous, A. chlamydosporium L.W. Hou, L. Cai & Crous, A. ellipsoideum L.W. Hou, Rämä, L. Cai & Crous, A. gamsianum L.W. Hou, L. Cai & Crous, A. longiphialidicum L.W. Hou, L. Cai & Crous, A. multiramosum L.W. Hou, Rämä, L. Cai & Crous, A. mycoparasiticum L.W. Hou, L. Cai & Crous, A. stroudii K. Fletcher, F.C. Küpper & P. van West, A. subulatum L.W. Hou, L. Cai & Crous, A. synnematoferum L.W. Hou, Rämä, L. Cai & Crous, Bulbithecium ammophilae L.W. Hou, L. Cai & Crous, B. ellipsoideum L.W. Hou, L. Cai & Crous, B. truncatum L.W. Hou, L. Cai & Crous, Emericellopsis brunneiguttula L.W. Hou, L. Cai & Crous, Gliomastix musae L.W. Hou, L. Cai & Crous, Gossypinidium sporodochiale L.W. Hou, L. Cai & Crous, Hapsidospora stercoraria L.W. Hou, L. Cai & Crous, H. variabilis L.W. Hou, L. Cai & Crous, Mycocitrus odorus L.W. Hou, L. Cai & Crous, Nectriopsis ellipsoidea L.W. Hou, L. Cai & Crous, Paracylindrocarpon aurantiacum L.W. Hou, L. Cai & Crous, Pn. foliicola Lechat & J. Fourn., Paragliomastix rosea L.W. Hou, L. Cai & Crous, Proliferophialis apiculata L.W. Hou, L. Cai & Crous, Protocreopsis finnmarkica L.W. Hou, L. Cai, Rämä & Crous, Proxiovicillium lepidopterorum L.W. Hou, L. Cai & Crous, Ramosiphorum echinoporiae L.W. Hou, L. Cai & Crous, R. polyporicola L.W. Hou, L. Cai & Crous, R. thailandicum L.W. Hou, L. Cai & Crous, Verruciconidia erythroxyli L.W. Hou, L. Cai & Crous, Ve. infuscata L.W. Hou, L. Cai & Crous, Ve. quercina L.W. Hou, L. Cai & Crous, Ve. siccicapita L.W. Hou, L. Cai & Crous, Ve. unguis L.W. Hou, L. Cai & Crous, Waltergamsia alkalina L.W. Hou, L. Cai & Crous, W. catenata L.W. Hou, L. Cai & Crous, W. moroccensis L.W. Hou, L. Cai & Crous, W. obpyriformis L.W. Hou, L. Cai & Crous; Chrysonectriaceae: Chrysonectria crystallifera L.W. Hou, L. Cai & Crous; Nectriaceae: Xenoacremonium allantoideum L.W. Hou, L. Cai & Crous; Neoacremoniaceae: Neoacremonium distortum L.W. Hou, L. Cai & Crous, N. flavum L.W. Hou, L. Cai & Crous; Nothoacremoniaceae: Nothoacremonium subcylindricum L.W. Hou, L. Cai & Crous, No. vesiculophorum L.W. Hou, L. Cai & Crous; Myrotheciomycetaceae: Trichothecium hongkongense L.W. Hou, L. Cai & Crous; Plectosphaerellaceae: Brunneomyces polyphialidus L.W. Hou, L. Cai & Crous, Parafuscohypha proliferata L.W. Hou, L. Cai & Crous; Sarocladiaceae: Chlamydocillium acaciae L.W. Hou, L. Cai & Crous, C. antarcticum L.W. Hou, L. Cai & Crous, C. guttulatum L.W. Hou, L. Cai & Crous, C. lolii L.W. Hou, L. Cai & Crous, C. soli L.W. Hou, L. Cai & Crous, C. terrestre L.W. Hou, L. Cai & Crous, Parasarocladium chondroidum L.W. Hou, L. Cai & Crous,Polyphialocladium fusisporum L.W. Hou, L. Cai & Crous, Sarocladium agarici L.W. Hou, L. Cai & Crous, S. citri L.W. Hou, L. Cai & Crous, S. ferrugineum L.W. Hou, L. Cai & Crous, S. fuscum L.W. Hou, L. Cai & Crous,S. theobromae L.W. Hou, L. Cai & Crous; Valsonectriaceae: Valsonectria crystalligena L.W. Hou, L. Cai & Crous, V. hilaris L.W. Hou, L. Cai & Crous. New combinations: Bionectriaceae: Acremonium purpurascens (Sukapure & Thirum.) L.W. Hou, L. Cai & Crous, Bulbithecium arxii (Malloch) L.W. Hou, L. Cai & Crous, Bu. borodinense (Tad. Ito et al.) L.W. Hou, L. Cai & Crous, Bu. pinkertoniae (W. Gams) L.W. Hou, L. Cai & Crous, Bu. spinosum (Negroni) L.W. Hou, L. Cai & Crous, Emericellopsis exuviara (Sigler et al.) L.W. Hou, L. Cai & Crous, E. fimetaria (Pers.) L.W. Hou, L. Cai & Crous, E. fuci (Summerb. et al.) L.W. Hou, L. Cai & Crous, E. moniliformis (A. Giraldo et al.) L.W. Hou, L. Cai & Crous, E. salmonea (W. Gams & Lodha) L.W. Hou, L. Cai & Crous, E. tubakii (Gams) L.W. Hou, L. Cai & Crous, Fusariella arenula (Berk. & Broome) L.W. Hou, L. Cai & Crous, Hapsidospora chrysogena (Thirum. & Sukapure) L.W. Hou, L. Cai & Crous, H. flava (W. Gams) L.W. Hou, L. Cai & Crous, H. globosa (Malloch & Cain) L.W. Hou, L. Cai & Crous, H. inversa (Malloch & Cain) L.W. Hou, L. Cai & Crous, Hydropisphaera aurantiaca (C.A. Jørg.) L.W. Hou, L. Cai & Crous, Lasionectria atrorubra (Lechat & J. Fourn.) L.W. Hou, L. Cai & Crous, L. bisepta (W. Gams) L.W. Hou, L. Cai & Crous, L. castaneicola (Lechat & Gardiennet) L.W. Hou, L. Cai & Crous, L. cerealis (P. Karst.) L.W. Hou, L. Cai & Crous, L. olida (W. Gams) L.W. Hou, L. Cai & Crous, Lasionectriopsis dentifera (Samuels) L.W. Hou, L. Cai & Crous, Lasionectriella arenuloides (Samuels) L.W. Hou, L. Cai & Crous, La. marigotensis (Lechat & J. Fourn.) L.W. Hou, L. Cai & Crous, Monohydropisphaera fusigera (Berk. & Broome) L.W. Hou, L. Cai & Crous, Musananaesporium tectonae (R.F. Castañeda) L.W. Hou, L. Cai & Crous, Mycocitrus zonatus (Sawada) L.W. Hou, L. Cai & Crous, Nectriopsis microspora (Jaap) L.W. Hou, L. Cai & Crous, Ovicillium asperulatum (A. Giraldo et al.) L.W. Hou, L. Cai & Crous, O. variecolor (A. Giraldo et al.) L.W. Hou, L. Cai & Crous, Paracylindrocarpon multiloculatum (Samuels) L.W. Hou, L. Cai & Crous, Pn. multiseptatum (Samuels)L.W. Hou, L. Cai & Crous, Paragliomastix chiangraiensis (J.F. Li et al.) L.W. Hou, L. Cai & Crous, Px. luzulae (Fuckel) L.W. Hou, L. Cai & Crous, Px. znieffensis (Lechat & J. Fourn.) L.W. Hou, L. Cai & Crous, Protocreopsis rutila (W. Gams) L.W. Hou, L. Cai & Crous, Proxiovicillium blochii (Matr.)L.W. Hou, L. Cai & Crous, Stanjemonium dichromosporum (Gams & Sivasith.) L.W. Hou, L. Cai & Crous, Verruciconidia persicina (Nicot) L.W. Hou, L. Cai & Crous, Ve. verruculosa (W. Gams & Veenb.-Rijks) L.W. Hou, L. Cai & Crous, Waltergamsia citrina (A. Giraldo et al.) L.W. Hou, L. Cai &Crous, W. dimorphospora (A. Giraldo et al.) L.W. Hou, L. Cai & Crous, W. epimycota (Samuels) L.W. Hou, L. Cai & Crous, W. fusidioides (Nicot) L.W. Hou, L. Cai & Crous, W. hennebertii (W. Gams) L.W. Hou, L. Cai & Crous, W. parva (A. Giraldo et al.) L.W. Hou, L. Cai & Crous, W. pilosa (A. Giraldo et al.) L.W. Hou, L. Cai & Crous, W. zeylanica (Petch) L.W. Hou, L. Cai & Crous; Cephalothecaceae: Phialemonium thermophilum (W. Gams & J. Lacey) L.W. Hou, L. Cai & Crous; Clavicipitaceae: Subuliphorum camptosporum (W. Gams) L.W. Hou, L. Cai & Crous; Coniochaetaceae: Coniochaeta psammospora (W. Gams) L.W. Hou, L. Cai & Crous; Nothoacremoniaceae: Nothoacremonium exiguum (W. Gams) L.W. Hou, L. Cai & Crous; Neoacremoniaceae: Neoacremonium minutisporum (Sukapure & Thirum.) L.W. Hou, L. Cai & Crous; Ne. taiwanense (K.L. Pang et al.) L.W. Hou, L. Cai & Crous; Ne. vitellinum (W. Gams) L.W. Hou, L. Cai & Crous; Plectosphaerellaceae: Allomusicillium domschii (W. Gams) L.W. Hou, L. Cai & Crous, Brunneomyces pseudozeylanicus (W. Gams) L.W. Hou, L. Cai & Crous; Pseudoniessliaceae: Pseudoniesslia minutispora (W. Gams et al.) L.W. Hou, L. Cai & Crous; Sarocladiaceae: Chlamydocillium curvulum (W. Gams) L.W. Hou, L. Cai & Crous, Parasarocladium funiculosum (Sukapure & Thirum.) L.W. Hou, L. Cai & Crous; Valsonectriaceae: Valsonectria inflata (C.H. Dickinson) L.W. Hou, L. Cai & Crous, V. roseola (G. Sm.) L.W. Hou, L. Cai & Crous. Epitype (basionym): Sphaeria violacea J.C. Schmidt ex Fr. Neotype (basionym): Mastigocladium blochii Matr. Citation: Hou LW, Giraldo A, Groenewald JZ, Rämä T, Summerbell RC, Zang P, Cai L, Crous PW (2023). Redisposition of acremonium-like fungi in Hypocreales. Studies in Mycology 105: 23-203. doi: 10.3114/sim.2023.105.02.

Three New Species of Clonostachys (Hypocreales, Ascomycota) from China.

Three new species of Clonostachys are introduced based on specimens collected from China. Clonostachys chongqingensis sp. nov. is distinguished by pale yellow to pale orange-yellow perithecia with a very low papilla, clavate to subcylindrical asci possessing ellipsoidal to elongate-ellipsoidal spinulose ascospores 13-16 × 4.5-5.5 µm; it has acremonium- to verticillium-like conidiophores and ellipsoidal to rod-shaped conidia. Clonostachys leptoderma sp. nov. has pinkish-white subglobose to globose perithecia on a well-developed stroma and with a thin perithecial wall, clavate to subcylindrical asci with ellipsoidal to elongate-ellipsoidal spinulose ascospores 7.5-11 × 2.5-3.5 µm; it produces verticillium-like conidiophores and ellipsoidal to subellipsoidal conidia. Clonostachys oligospora sp. nov. features solitary to gregarious perithecia with a papilla, clavate asci containing 6-8 smooth-walled ascospores 9-17 × 3-5.5 µm; it forms verticillium-like conidiophores and sparse, subfusiform conidia. The morphological characteristics and phylogenetic analyses of combined nuclear ribosomal DNA ITS1-5.8S-ITS2 and beta-tubulin sequences support their placement in Clonostachys and their classification as new to science. Distinctions between the novel taxa and their close relatives are compared herein.

Stilbocrea banihashemiana sp. nov. a New Fungal Pathogen Causing Stem Cankers and Twig Dieback of Fruit Trees.

Stem cankers and twig dieback were the most serious disease of fig (Ficus carica) and loquat (Eriobotrya japonica) noticed in a survey of fruit tree orchards in the Fars Province, Iran. Isolates of Bionectriaceae were consistently recovered from symptomatic fig and loquat trees. Phylogenetic analyses of multiple nuclear loci, internal transcribed spacer regions (ITS) of rDNA, RNA polymerase II subunit 2 (rpb2), and translation elongation factor 1-α (tef1), combined with morphological observations, revealed that isolates could be referred to a still unknown taxon, which was formally described as Stilbocrea banihashemiana sp. nov. Phylogenetically, isolates from fig and loquat trees clustered in a well-supported monophyletic group within the Stilbocrea clade of Bionectriaceae, closely related to S. walteri. Stilbocrea banihashemiana sp. nov. was characterized by the lack of stilbella-like asexual structure in both natural substrates and pure cultures and produced two morphologically distinct types of conidia, globose and cylindrical, formed on short and long simple phialides. In pathogenicity tests, S. banihashemiana sp. nov. induced stem cankers in both fig and loquat, wood discoloration in fig and twig dieback in loquat. Pathogenicity tests also showed that the potential host range of this novel pathogen includes other economically relevant horticultural trees.

Characterization and microsatellite marker development for a common bark and ambrosia beetle associate, Geosmithia obscura.

Symbioses between Geosmithia fungi and wood-boring and bark beetles seldom result in disease induction within the plant host. Yet, exceptions exist such as Geosmithia morbida, the causal agent of Thousand Cankers Disease (TCD) of walnuts and wingnuts, and Geosmithia sp. 41, the causal agent of Foamy Bark Canker disease of oaks. Isolates of G. obscura were recovered from black walnut trees in eastern Tennessee and at least one isolate induced cankers following artificial inoculation. Due to the putative pathogenicity and lack of recovery of G. obscura from natural lesions, a molecular diagnostic screening tool was developed using microsatellite markers mined from the G. obscura genome. A total of 3256 candidate microsatellite markers were identified (2236, 789, 137 di-, tri-, and tetranucleotide motifs, respectively), with 2011, 703, 101 di-, tri-, and tetranucleotide motifs, respectively, containing markers with primers. From these, 75 microsatellite markers were randomly selected, screened, and optimized, resulting in 28 polymorphic markers that yielded single, consistently recovered bands, which were used in downstream analyses. Five of these microsatellite markers were found to be specific to G. obscura and did not cross-amplify into other, closely related species. Although the remaining tested markers could be useful, they cross-amplified within different Geosmithia species, making them not reliable for G. obscura detection. Five novel microsatellite markers (GOBS9, GOBS10, GOBS41, GOBS43, and GOBS50) were developed based on the G. obscura genome. These species-specific microsatellite markers are available as a tool for use in molecular diagnostics and can assist future surveillance studies.

Clonostachys viticola sp. nov., a novel species isolated from Vitis vinifera.

A collection of fungal isolates obtained from crop plants, specifically grapevine and blueberry, in Peru were characterised through morphological and DNA sequence analyses of the nuclear ribosomal internal transcribed spacer (ITS), beta-tubulin (tub2) and translation elongation factor 1-alpha (tef-1α) regions. Isolates produced monomorphic and dimorphic conidiophores typical of members of the genus Clonostachys. Single- and multi-locus gene phylogenies confirmed the isolates as representing members of the genus Clonostachys, more closely related to species in the subgenus Bionectria. In phylogenetic analyses the isolates grouped in two separate clades, one corresponding to the species Clonostachys pseudochroleuca and the other one distinct from all known species of the genus Clonostachys. These isolates are recognized as representing a novel species species for which the name Clonostachys viticola is proposed.

The Protean Acremonium. A. sclerotigenum/egyptiacum: Revision, Food Contaminant, and Human Disease.

Acremonium is known to be regularly isolated from food and also to be a cause of human disease. Herein, we resolve some sources of confusion that have strongly hampered the accurate interpretation of these and other isolations. The recently designated type species of the genus Acremonium, A. alternatum, is known only from a single isolate, but it is the closest known relative of what may be one of the planet's most successful organisms, Acremonium sclerotigenum/egyptianum, shown herein to be best called by its earliest valid name, A. egyptiacum. The sequencing of ribosomal internal transcribed spacer (ITS) regions, actin genes, or both for 72 study isolates within this group allowed the full range of morphotypes and ITS barcode types to be elucidated, along with information on temperature tolerance and habitat. The results showed that nomenclatural confusion and frequent misidentifications facilitated by morphotaxonomy, along with misidentified early sequence deposits, have obscured the reality that this species is, in many ways, the definitive match of the historical concept of Acremonium: a pale orange or dull greenish-coloured monophialidic hyphomycete, forming cylindrical, ellipsoidal, or obovoid conidia in sticky heads or obovoid conidia in dry chains, and acting ecologically as a soil organism, marine organism, plant pathogen, plant endophyte, probable insect pathogen, human opportunistic pathogen, food contaminant, probable dermatological communicable disease agent, and heat-tolerant spoilage organism. Industrially, it is already in exploratory use as a producer of the antibiotic ascofuranone, active against trypanosomes, cryptosporidia, and microsporidia, and additional applications are in development. The genus-level clarification of the phylogeny of A. egyptiacum shows other historic acremonia belong to separate genera, and two are here described, Parasarocladium for the Acremonium radiatum complex and Kiflimonium for the Acremonium curvulum complex.

In situ mass spectrometry monitoring of fungal cultures led to the identification of four peptaibols with a rare threonine residue.

Peptaibols are an intriguing class of fungal metabolites due both to their wide range of reported bioactivities and to the structural variability that can be generated by the exchange of variable amino acid building blocks. In an effort to streamline the discovery of structurally diverse peptaibols, a mass spectrometry surface sampling technique was applied to screen the chemistry of fungal cultures in situ. Four previously undescribed peptaibols, all containing a rare threonine residue, were identified from a fungal culture (MSX53554), which was identified as Nectriopsis Maire (Bionectriaceae, Hypocreales, Ascomycota). These compounds not only increased the known threonine-containing peptaibols by nearly 20%, but also, the threonine residue was situated in a unique place compared to the other reported threonine-containing peptaibols. After the initial in situ detection and characterization, a large-scale solid fermentation culture was grown. The four peptaibols were isolated and characterized by mass spectrometry. In addition, one of the peptaibols was fully characterized by NMR and amino acid analysis using Marfey's reagent and exhibited moderate in vitro anticancer activity.

The complete mitochondrial genome of the important mycoparasite Clonostachys rosea (Hypocreales, Ascomycota).

The complete nucleotide sequence of mitochondrial genome of the important mycoparasitic fungus Clonostachys rosea was determined using the next-generation sequencing technology. The circular molecule is 40,921 bp long with a GC content of 27.90%. Gene prediction revealed 42 genes encoding 15 conserved proteins, 25 tRNAs, the large and small ribosomal RNAs. All genes are located on the same strand. It is found to be similar to the previously sequenced mitochondrial genomes of Acremonium chrysogenum and Nectria cinnabarina. The differences lie in the copy number of trnG-UCC and locations of trnN-GUU and cox2. The phylogenetic analysis confirmed C. rosea as a sister taxon of A. chrysogenumin (Bionectriaceae). The mitochondrial genome of C. rosea will contribute to the understanding of phylogeny and evolution of Hypocreales.

Two new Geosmithia species in G. pallida species complex from bark beetles in eastern USA.

Species of Geosmithia are cosmopolitan but understudied fungi, and most are associated with phloem-feeding bark beetles on various woody hosts. We surveyed 207 bark and ambrosia beetles from 37 species in the eastern USA for associated fungi. The community is dominated by species in the G. pallida species complex (GPSC) and included several Geosmithia isolates that appear to be new to science. The new Geosmithia isolates exhibited the characteristic brownish-colored colonies typical for the G. pallida species complex and were phylogenetically resolved as two genealogically exclusive lineages based on a concatenated multilocus data set based on the internal transcribed spacers (ITS) of the nuc rDNA (ITS1-5.8S-ITS2 = ITS), and the translation elongation factor 1-α (TEF1-α), ß-tubulin (TUB2), and RNA polymerase II second largest subunit (RPB2) genes. Two new Geosmithia species, G. brunnea and G. proliferans, are proposed, and their morphological traits and phylogenetic placements are presented.

Diversity of Clonostachys species assessed by molecular phylogenetics and MALDI-TOF mass spectrometry.

We assessed the species diversity among 45 strains of Clonostachys from different substrates and localities in Brazil using molecular phylogenetics, and compared the results with the phenotypic classification of strains obtained from matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Phylogenetic analyses were based on beta tubulin (Tub), ITS-LSU rDNA, and a combined Tub-ITS DNA dataset. MALDI-TOF MS analyses were performed using intact conidia and conidiophores of strains cultivated on oatmeal agar and 4% malt extract agar. Six known species were identified: Clonostachys byssicola, Clonostachys candelabrum, Clonostachys pseudochroleuca, Clonostachys rhizophaga, Clonostachys rogersoniana, and Clonostachys rosea. Two clades and two singleton lineages did not correspond to known species represented in the reference DNA dataset and were identified as Clonostachys sp. 1-4. Multivariate cluster analyses of MALDI-TOF MS data classified the strains into eight clusters and three singletons, corresponding to the ten identified species plus one additional cluster containing two strains of C. rogersoniana that split from the other co-specific strains. The consistent results of MALDI-TOF MS supported the identification of strains assigned to C. byssicola and C. pseudochroleuca, which did not form well supported clades in all phylogenetic analyses, but formed distinct clusters in the MALDI-TOF dendrograms.