Z.W. de Beer

One fungus, which genes? Development and assessment of universal primers for potential secondary fungal DNA barcodes

The aim of this study was to assess potential candidate gene regions and corresponding universal primer pairs as secondary DNA barcodes for the fungal kingdom, additional to ITS rDNA as primary barcode. Amplification efficiencies of 14 (partially) universal primer pairs targeting eight genetic markers were tested across > 1 500 species (1 931 strains or specimens) and the outcomes of almost twenty thousand (19 577) polymerase chain reactions were evaluated. We tested several well-known primer pairs that amplify: i) sections of the nuclear ribosomal RNA gene large subunit (D1–D2 domains of 26/28S); ii) the complete internal transcribed spacer region (ITS1/2); iii) partial β -tubulin II (TUB2); iv) γ-actin (ACT); v) translation elongation factor 1-α (TEF1α); and vi) the second largest subunit of RNA-polymerase II (partial RPB2, section 5–6). Their PCR efficiencies were compared with novel candidate primers corresponding to: i) the fungal-specific translation elongation factor 3 (TEF3); ii) a small ribosomal protein necessary for t-RNA docking; iii) the 60S L10 (L1) RP; iv) DNA topoisomerase I (TOPI); v) phosphoglycerate kinase (PGK); vi) hypothetical protein LNS2; and vii) alternative sections of TEF1α. Results showed that several gene sections are accessible to universal primers (or primers universal for phyla) yielding a single PCR-product. Barcode gap and multi-dimensional scaling analyses revealed that some of the tested candidate markers have universal properties providing adequate infra- and inter-specific variation that make them attractive barcodes for species identification. Among these gene sections, a novel high fidelity primer pair for TEF1α, already widely used as a phylogenetic marker in mycology, has potential as a supplementary DNA barcode with superior resolution to ITS. Both TOPI and PGK show promise for the Ascomycota, while TOPI and LNS2 are attractive for the Pucciniomycotina, for which universal primers for ribosomal subunits often fail.

Redefining Ceratocystis and allied genera.

The genus Ceratocystis was established in 1890 and accommodates many important fungi. These include serious plant pathogens, significant insect symbionts and agents of timber degradation that result in substantial economic losses. Virtually since its type was described from sweet potatoes, the taxonomy of Ceratocystis has been confused and vigorously debated. In recent years, particulary during the last two decades, it has become very obvious that this genus includes a wide diversity of very different fungi. These have been roughly lumped together due to their similar morphological structures that have clearly evolved through convergent evolution linked to an insect-associated ecology. As has been true for many other groups of fungi, the emergence of DNA-based sequence data and associated phylogenetic inferences, have made it possible to robustly support very distinct boundaries defined by morphological characters and ecological differences. In this study, DNA-sequence data for three carefully selected gene regions (60S, LSU, MCM7) were generated for 79 species residing in the aggregate genus Ceratocystis sensu lato and these data were subjected to rigorous phylogenetic analyses. The results made it possible to distinguish seven major groups for which generic names have been chosen and descriptions either provided or emended. The emended genera included Ceratocystis sensu stricto, Chalaropsis, Endoconidiophora, Thielaviopsis, and Ambrosiella, while two new genera, Davidsoniella and Huntiella, were described. In total, 30 new combinations have been made. This major revision of the generic boundaries in the Ceratocystidaceae will simplify future treatments and work with an important group of fungi including distantly related species illogically aggregated under a single name.

Fungal Planet description sheets: 154–213

Novel species of microfungi described in the present study include the following from South Africa: Camarosporium aloes, Phaeococcomyces aloes and Phoma aloes from Aloe, C. psoraleae, Diaporthe psoraleae and D. psoraleae-pinnatae from Psoralea, Colletotrichum euphorbiae from Euphorbia, Coniothyrium prosopidis and Peyronellaea prosopidis from Prosopis, Diaporthe cassines from Cassine, D. diospyricola from Diospyros, Diaporthe maytenicola from Maytenus, Harknessia proteae from Protea, Neofusicoccum ursorum and N. cryptoaustrale from Eucalyptus, Ochrocladosporium adansoniae from Adansonia, Pilidium pseudoconcavum from Greyia radlkoferi, Stagonospora pseudopaludosa from Phragmites and Toxicocladosporium ficiniae from Ficinia. Several species were also described from Thailand, namely: Chaetopsina pini and C. pinicola from Pinus spp., Myrmecridium thailandicum from reed litter, Passalora pseudotithoniae from Tithonia, Pallidocercospora ventilago from Ventilago, Pyricularia bothriochloae from Bothriochloa and Sphaerulina rhododendricola from Rhododendron. Novelties from Spain include Cladophialophora multiseptata, Knufia tsunedae and Pleuroascus rectipilus from soil and Cyphellophora catalaunica from river sediments. Species from the USA include Bipolaris drechsleri from Microstegium, Calonectria blephiliae from Blephilia, Kellermania macrospora (epitype) and K. pseudoyuccigena from Yucca. Three new species are described from Mexico, namely Neophaeosphaeria agaves and K. agaves from Agave and Phytophthora ipomoeae from Ipomoea. Other African species include Calonectria mossambicensis from Eucalyptus (Mozambique), Harzia cameroonensis from an unknown creeper (Cameroon), Mastigosporella anisophylleae from Anisophyllea (Zambia) and Teratosphaeria terminaliae from Terminalia (Zimbabwe). Species from Europe include Auxarthron longisporum from forest soil (Portugal), Discosia pseudoartocreas from Tilia (Austria), Paraconiothyrium polonense and P. lycopodinum from Lycopodium (Poland) and Stachybotrys oleronensis from Iris (France). Two species of Chrysosporium are described from Antarctica, namely C. magnasporum and C. oceanitesii. Finally, Licea xanthospora is described from Australia, Hypochnicium huinayensis from Chile and Custingophora blanchettei from Uruguay. Novel genera of Ascomycetes include Neomycosphaerella from Pseudopentameris macrantha (South Africa), and Paramycosphaerella from Brachystegia sp. (Zimbabwe). Novel hyphomycete genera include Pseudocatenomycopsis from Rothmannia (Zambia), Neopseudocercospora from Terminalia (Zambia) and Neodeightoniella from Phragmites (South Africa), while Dimorphiopsis from Brachystegia (Zambia) represents a novel coelomycetous genus. Furthermore, Alanphillipsia is introduced as a new genus in the Botryosphaeriaceae with four species, A. aloes, A. aloeigena and A. aloetica from Aloe spp. and A. euphorbiae from Euphorbia sp. (South Africa). A new combination is also proposed for Brachysporium torulosum (Deightoniella black tip of banana) as Corynespora torulosa. Morphological and culture characteristics along with ITS DNA barcodes are provided for all taxa.

Ophiostoma spp. associated with pine- and spruce-infesting bark beetles in Finland and Russia

The timber and pulp industries of Finland rely heavily on importations from Russia as source of raw timber. These imports raise the risk of accidentally importing forest pests and pathogens, especially bark beetles and their associated fungi, into Finland. Although ophiostomatoid fungi have previously been reported from Finland and Russia, the risks of accidentally moving these fungi has prompted a first survey to compare the diversity of conifer-infesting bark beetles and associated fungi from boreal forests on both sides of the Finnish-Russian border. The aim of the present study was to identify and characterise Ophiostoma species isolated in association with 11 bark beetle species infesting Pinus sylvestris and Picea abies during this survey in the eastern parts of Finland and neighbouring Russia. Fungal isolates were grouped based on morphology and representatives of each morphological group were subjected to DNA sequence comparisons of the internal transcribed spaced region (ITS1, 5.8S, ITS2) and β-tubulin gene region. A total of 15 species of Ophiostoma were identified, including seven known species, five new species, and three species for which the identity remains uncertain. In the O. piceae-complex we identified O. canum, O. floccosum, O. karelicum and O. rachisporum sp. nov., and related to these, some isolates belonging to the European clade of O. minus in the O. minus-complex. Ophiostoma bicolor and O. fuscum sp. nov. were identified in the O. ips-complex, while O. ainoae, O. brunneo-ciliatum, O. tapionis sp. nov. and O. pallidulum sp. nov. were shown to group close to, but not in a strict monophyletic lineage with species of the O. ips-complex. Together with a single O. abietinum-like isolate, the only species that grouped close to the Sporothrix schenckii- O. stenoceras complex, was O. saponiodorum sp. nov.

Fungal Planet description sheets: 371-399

Novel species of fungi described in the present study include the following from Australia: Neoseptorioides eucalypti gen. & sp. nov. from Eucalyptus radiata leaves, Phytophthora gondwanensis from soil, Diaporthe tulliensis from rotted stem ends of Theobroma cacao fruit, Diaporthe vawdreyi from fruit rot of Psidium guajava, Magnaporthiopsis agrostidis from rotted roots of Agrostis stolonifera and Semifissispora natalis from Eucalyptus leaf litter. Furthermore, Neopestalotiopsis egyptiaca is described from Mangifera indica leaves (Egypt), Roussoella mexicana from Coffea arabica leaves (Mexico), Calonectria monticola from soil (Thailand), Hygrocybe jackmanii from littoral sand dunes (Canada), Lindgomyces madisonensis from submerged decorticated wood (USA), Neofabraea brasiliensis from Malus domestica (Brazil), Geastrum diosiae from litter (Argentina), Ganoderma wiiroense on angiosperms (Ghana), Arthrinium gutiae from the gut of a grasshopper (India), Pyrenochaeta telephoni from the screen of a mobile phone (India) and Xenoleptographium phialoconidium gen. & sp. nov. on exposed xylem tissues of Gmelina arborea (Indonesia). Several novelties are introduced from Spain, namely Psathyrella complutensis on loamy soil, Chlorophyllum lusitanicum on nitrified grasslands (incl. Chlorophyllum arizonicum comb. nov.), Aspergillus citocrescens from cave sediment and Lotinia verna gen. & sp. nov. from muddy soil. Novel foliicolous taxa from South Africa include Phyllosticta carissicola from Carissa macrocarpa, Pseudopyricularia hagahagae from Cyperaceae and Zeloasperisporium searsiae from Searsia chirindensis. Furthermore, Neophaeococcomyces is introduced as a novel genus, with two new combinations, N. aloes and N. catenatus. Several foliicolous novelties are recorded from La Réunion, France, namely Ochroconis pandanicola from Pandanus utilis, Neosulcatispora agaves gen. & sp. nov. from Agave vera-cruz, Pilidium eucalyptorum from Eucalyptus robusta, Strelitziana syzygii from Syzygium jambos (incl. Strelitzianaceae fam. nov.) and Pseudobeltrania ocoteae from Ocotea obtusata (Beltraniaceae emend.). Morphological and culture characteristics along with ITS DNA barcodes are provided for all taxa.

The divorce of Sporothrix and Ophiostoma: solution to a problematic relationship

One of the causal agents of human sporotrichosis, Sporothrix schenckii, is the type species of the genus Sporothrix. During the course of the last century the asexual morphs of many Ophiostoma spp. have also been treated in Sporothrix. More recently several DNA-based studies have suggested that species of Sporothrix and Ophiostoma converge in what has become known as Ophiostoma s. lat. Were the one fungus one name principles adopted in the Melbourne Code to be applied to Ophiostoma s. lat., Sporothrix would have priority over Ophiostoma, resulting in more than 100 new combinations. The consequence would be name changes for several economically important tree pathogens including O. novo-ulmi. Alternatively, Ophiostoma could be conserved against Sporothrix, but this would necessitate changing the names of the important human pathogens in the group. In this study, we sought to resolve the phylogenetic relationship between Ophiostoma and Sporothrix. DNA sequences were determined for the ribosomal large subunit and internal transcribed spacer regions, as well as the beta-tubulin and calmodulin genes in 65 isolates. The results revealed Sporothrix as a well-supported monophyletic lineage including 51 taxa, distinct from Ophiostoma s. str. To facilitate future studies exploring species level resolution within Sporothrix, we defined six species complexes in the genus. These include the Pathogenic Clade containing the four human pathogens, together with the S. pallida-, S. candida-, S. inflata-, S. gossypina- and S. stenoceras complexes, which include environmental species mostly from soil, hardwoods and Protea infructescences. The description of Sporothrix is emended to include sexual morphs, and 26 new combinations. Two new names are also provided for species previously treated as Ophiostoma.

Levels of specificity of Xylaria species associated with fungus-growing termites: a phylogenetic approach

Fungus‐growing termites live in obligate mutualistic symbiosis with species of the basidiomycete genus Termitomyces, which are cultivated on a substrate of dead plant material. When the termite colony dies, or when nest material is incubated without termites in the laboratory, fruiting bodies of the ascomycete genus Xylaria appear and rapidly cover the fungus garden. This raises the question whether certain Xylaria species are specialised in occupying termite nests or whether they are just occasional visitors. We tested Xylaria specificity at four levels: (1) fungus‐growing termites, (2) termite genera, (3) termite species, and (4) colonies. In South Africa, 108 colonies of eight termite species from three termite genera were sampled for Xylaria. Xylaria was isolated from 69% of the sampled nests and from 57% of the incubated fungus comb samples, confirming high prevalence. Phylogenetic analysis of the ITS region revealed 16 operational taxonomic units of Xylaria, indicating high levels of Xylaria species richness. Not much of this variation was explained by termite genus, species, or colony; thus, at level 2–4 the specificity is low. Analysis of the large subunit rDNA region, showed that all termite‐associated Xylaria belong to a single clade, together with only three of the 26 non‐termite‐associated strains. Termite‐associated Xylaria thus show specificity for fungus‐growing termites (level 1). We did not find evidence for geographic or temporal structuring in these Xylaria phylogenies. Based on our results, we conclude that termite‐associated Xylaria are specific for fungus‐growing termites, without having specificity for lower taxonomic levels.

Ophiostoma denticiliatum sp. nov. and other Ophiostoma species associated with the birch bark beetle in southern Norway.

Ophiostomatoid fungi were isolated from Scolytus ratzeburgi infesting Betula pendula and B. pubescens in Norway. Fungi were identified based on morphology, DNA sequence comparison for two gene regions and phylogenetic analyses. The most abundant fungus was Ophiostoma karelicum, suggesting a specific relationship between the fungus, the vector insect and the host tree. Our results suggest that O. karelicum occurs across the geographic range of S. ratzeburgi and its close relatedness to the Dutch elm disease fungi suggests that it could be important if introduced into other parts of the world. Other fungi, only occasionally isolated from S. ratzeburgi, were identified as O. quercus and a novel taxon, described here as O. denticiliatum sp. nov.

Two new Ophiostoma species from Protea caffra in Zambia

The genus Ophiostoma (Ophiostomatales) has a global distribution and species are best known for their association with bark beetles (Curculionidae: Scolytinae) on conifers. An unusual assemblage of these fungi is closely associated with the African endemic plant genus Protea (Proteaceae). Protea-associated Ophiostoma species are ecologically atypical as they colonise the fruiting structures of various serotinous Protea species. Seven species have been described from this niche in South Africa. It has been speculated that novel species may be present in other African countries where these host plants also occur. This view was corroborated by recent collections of two unknown species from Protea caffra trees in Zambia. In the present study we evaluate the species delineation of these isolates using morphological comparisons with other Protea-associated species, differential growth studies and analyses of DNA sequence data for the β-tubulin and internal transcribed spacer (ITS1, 5.8S, ITS2) regions. As a result, the species O. protea-sedis sp. nov., and O. zambiensis sp. nov. are described here as new. This study brings the number of Protea-associated Ophiostoma species to nine and highlights the need for more inclusive surveys, including additional African countries and hosts, to elucidate species diversity in this uncharacteristic niche.

Genera of phytopathogenic fungi: GOPHY 1

Genera of Phytopathogenic Fungi (GOPHY) is introduced as a new series of publications in order to provide a stable platform for the taxonomy of phytopathogenic fungi. This first paper focuses on 21 genera of phytopathogenic fungi: Bipolaris, Boeremia, Calonectria, Ceratocystis, Cladosporium, Colletotrichum, Coniella, Curvularia, Monilinia, Neofabraea, Neofusicoccum, Pilidium, Pleiochaeta, Plenodomus, Protostegia, Pseudopyricularia, Puccinia, Saccharata, Thyrostroma, Venturia and Wilsonomyces. For each genus, a morphological description and information about its pathology, distribution, hosts and disease symptoms are provided. In addition, this information is linked to primary and secondary DNA barcodes of the presently accepted species, and relevant literature. Moreover, several novelties are introduced, i.e. new genera, species and combinations, and neo-, lecto- and epitypes designated to provide a stable taxonomy. This first paper includes one new genus, 26 new species, ten new combinations, and four typifications of older names.