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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.

Species identification lies at the heart of biodiversity studies that has in recent years favoured DNA-based approaches. Microbial Biological Resource Centres are a rich source for diverse and high-quality reference materials in microbiology, and yet the strains preserved in these biobanks have been exploited only on a limited scale to generate DNA barcodes. As part of a project funded in the Netherlands to barcode specimens of major national biobanks, sequences of two nuclear ribosomal genetic markers, the Internal Transcribed Spaces and 5.8S gene (ITS) and the D1/D2 domain of the 26S Large Subunit (LSU), were generated as DNA barcode data for ca. 100 000 fungal strains originally assigned to ca. 17 000 species in the CBS fungal biobank maintained at the Westerdijk Fungal Biodiversity Institute, Utrecht. Using more than 24 000 DNA barcode sequences of 12 000 ex-type and manually validated filamentous fungal strains of 7 300 accepted species, the optimal identity thresholds to discriminate filamentous fungal species were predicted as 99.6 % for ITS and 99.8 % for LSU. We showed that 17 % and 18 % of the species could not be discriminated by the ITS and LSU genetic markers, respectively. Among them, ∼8 % were indistinguishable using both genetic markers. ITS has been shown to outperform LSU in filamentous fungal species discrimination with a probability of correct identification of 82 % vs. 77.6 %, and a clustering quality value of 84 % vs. 77.7 %. At higher taxonomic classifications, LSU has been shown to have a better discriminatory power than ITS. With a clustering quality value of 80 %, LSU outperformed ITS in identifying filamentous fungi at the ordinal level. At the generic level, the clustering quality values produced by both genetic markers were low, indicating the necessity for taxonomic revisions at genus level and, likely, for applying more conserved genetic markers or even whole genomes. The taxonomic thresholds predicted for filamentous fungal identification at the genus, family, order and class levels were 94.3 %, 88.5 %, 81.2 % and 80.9 % based on ITS barcodes, and 98.2 %, 96.2 %, 94.7 % and 92.7 % based on LSU barcodes. The DNA barcodes used in this study have been deposited to GenBank and will also be publicly available at the Westerdijk Institute's website as reference sequences for fungal identification, marking an unprecedented data release event in global fungal barcoding efforts to date.

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Scientific communication is facilitated by a data-driven, scientifically sound taxonomy that considers the end-user's needs and established successful practice. Previously (Geiser et al. 2013; Phytopathology 103:400-408. 2013), the Fusarium community voiced near unanimous support for a concept of Fusarium that represented a clade comprising all agriculturally and clinically important Fusarium species, including the F. solani Species Complex (FSSC). Subsequently, this concept was challenged by one research group (Lombard et al. 2015 Studies in Mycology 80: 189-245) who proposed dividing Fusarium into seven genera, including the FSSC as the genus Neocosmospora, with subsequent justification based on claims that the Geiser et al. (2013) concept of Fusarium is polyphyletic (Sandoval-Denis et al. 2018; Persoonia 41:109-129). Here we test this claim, and provide a phylogeny based on exonic nucleotide sequences of 19 orthologous protein-coding genes that strongly support the monophyly of Fusarium including the FSSC. We reassert the practical and scientific argument in support of a Fusarium that includes the FSSC and several other basal lineages, consistent with the longstanding use of this name among plant pathologists, medical mycologists, quarantine officials, regulatory agencies, students and researchers with a stake in its taxonomy. In recognition of this monophyly, 40 species recently described as Neocosmospora were recombined in Fusarium, and nine others were renamed Fusarium. Here the global Fusarium community voices strong support for the inclusion of the FSSC in Fusarium, as it remains the best scientific, nomenclatural and practical taxonomic option available.

Fungi of the genus Fusarium are well known as major plant pathogens and soil inhabitants, but also cause a broad spectrum of human infections. Fusariosis is the second most common mould infection after aspergillosis, and keratitis is the most encountered implantation infection in immunocompetent individuals. Natamycin is active against Fusarium species both in vitro and in vivo, and is used along with voriconazole as the mainstay of treatment for Fusarium keratitis. Onychomycosis is treated with terbinafine, voriconazole and sometimes itraconazole. Cure is possible despite high in vitro minimum inhibitory concentrations (MICs). Recently, disseminated infections have increased dramatically, mainly affecting severely immunocompromised patients. The remarkable intrinsic resistance of Fusarium species to most antifungal agents results in high mortality rates in this patient population. Recovery of neutropenia is essential for patient survival and treatment should include voriconazole or amphotericin B as first-line and posaconazole as salvage therapy.

Resistance patterns in the F. fujikuroi species complex are species specific and therefore identification down to species level is important for the choice of antifungal treatment.