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.

ABSTRACTWe investigated genetic factors that govern the reduced propiconazole sensitivity ofSclerotinia homoeocarpafield isolates collected during a 2-year field efficacy study on dollar spot disease of turf in five New England sites. These isolates displayed a >50-fold range ofin vitrosensitivity to a sterol demethylation inhibitor (DMI) fungicide, propiconazole, making them ideal for investigations of genetic mechanisms of reduced DMI sensitivity. TheCYP51gene homolog inS. homoeocarpa(ShCYP51B), encoding the enzyme target of DMIs, is likely a minor genetic factor for reduced propiconazole sensitivity, since there were no differences in constitutive relative expression (RE) values and only 2-fold-higher induced RE values for insensitive than for sensitive isolate groups. Next, we mined RNA-Seq transcriptome data for additional genetic factors and found evidence for the overexpression of a homolog ofBotrytis cinerea atrD(BcatrD),ShatrD, a known efflux transporter of DMI fungicides. TheShatrDgene showed much higher constitutive and induced RE values for insensitive isolates. Several polymorphisms were found upstream ofShatrDbut were not definitively linked to overexpression. The screening of constitutive RE values ofShCYP51BandShatrDin isolates from two golf courses that exhibited practical field resistance to propiconazole uncovered evidence for significant population-specific overexpression of both genes. However, linear regression demonstrated that the RE ofShatrDdisplays a more significant relationship with propiconazole sensitivity than that ofShCYP51B. In summary, our results suggest that efflux is a major determinant of the reduced DMI sensitivity ofS. homoeocarpagenotypes in New England, which may have implications for the emergence of practical field resistance in this important turfgrass pathogen.

Fungi are known to utilize transcriptional regulation of genes that encode efflux transporters to detoxify xenobiotics; however, to date it is unknown how fungi transcriptionally regulate and coordinate different phases of detoxification system (phase I, modification; phase II, conjugation; and phase III, secretion). Here we present evidence of an evolutionary convergence between the fungal and mammalian lineages, whereby xenobiotic detoxification genes (phase I coding for cytochrome P450 monooxygenases [CYP450s] and phase III coding for ATP-binding cassette [ABC] efflux transporters) are transcriptionally regulated by structurally unrelated proteins. Following next-generation RNA sequencing (RNA-seq) analyses of a filamentous fungus, Sclerotinia homoeocarpa, the causal agent of dollar spot on turfgrasses, a multidrug resistant (MDR) field strain was found to overexpress phase I and III genes, coding for CYP450s and ABC transporters for xenobiotic detoxification. Furthermore, there was confirmation of a gain-of-function mutation of the fungus-specific transcription factor S. homoeocarpa XDR1 (ShXDR1), which is responsible for constitutive and induced overexpression of the phase I and III genes, resulting in resistance to multiple classes of fungicidal chemicals. This fungal pathogen detoxifies xenobiotics through coordinated transcriptional control of CYP450s, biotransforming xenobiotics with different substrate specificities and ABC transporters, excreting a broad spectrum of xenobiotics or biotransformed metabolites. A Botrytis cinerea strain harboring the mutated ShXDR1 showed increased expression of phase I (BcCYP65) and III (BcatrD) genes, resulting in resistance to fungicides. This indicates the regulatory system is conserved in filamentous fungi. This molecular genetic mechanism for xenobiotic detoxification in fungi holds potential for facilitating discovery of new antifungal drugs and further studies of convergent and divergent evolution of xenobiotic detoxification in eukaryote lineages.

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Dollar spot, caused by Sclerotinia homoeocarpa, is a prevalent turfgrass disease, and the fungus exhibits widespread fungicide resistance in North America. In a previous study, an ABC-G transporter, ShatrD, was associated with practical field resistance to demethylation inhibitor (DMI) fungicides. Mining of ABC-G transporters, also known as pleiotropic drug resistance (PDR) transporters, from RNA-Seq data gave an assortment of transcripts, several with high sequence similarity to functionally characterized transporters from Botrytis cinerea, and others with closest blastx hits from Aspergillus and Monilinia. In addition to ShatrD, another PDR transporter showed significant over-expression in replicated RNA-Seq data, and in a collection of field-resistant isolates, as measured by quantitative polymerase chain reaction. These isolates also showed reduced sensitivity to unrelated fungicide classes. Using a yeast complementation system, we sought to test the hypothesis that this PDR transporter effluxes DMI as well as chemically unrelated fungicides. The transporter (ShPDR1) was cloned into the Gal1 expression vector and transformed into a yeast PDR transporter deletion mutant, AD12345678. Complementation assays indicated that ShPDR1 complemented the mutant in the presence of propiconazole (DMI), iprodione (dicarboximide) and boscalid (SDHI, succinate dehydrogenase inhibitor). Our results indicate that the over-expression of ShPDR1 is correlated with practical field resistance to DMI fungicides and reduced sensitivity to dicarboximide and SDHI fungicides. These findings highlight the potential for the eventual development of a multidrug resistance phenotype in this pathogen. In addition, this study presents a pipeline for the discovery and validation of fungicide resistance genes using de novo next-generation sequencing and molecular biology techniques in an unsequenced plant pathogenic fungus.

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The succinate dehydrogenase inhibitor (SDHI) fungicide, fluopyram, is used as a soybean seed treatment to manage Fusarium virguliforme, the casual agent of sudden death syndrome (SDS). More recently, other species within clade 2 of the Fusarium solani species, F. tucumaniae in South America and F. brasiliense in America and Africa, have been recognized as additional agents capable of causing SDS. To determine if fluopyram could be used for management of SDS caused by these species, in vitro sensitivity tests of the three Fusarium species to fluopyram were conducted. The mean EC50 values of F. brasiliense and F. virguliforme strains to fluopyram were 1.96 and 2.21 μg ml-1, respectively, but interestingly F. tucumaniae strains were highly sensitive (mean EC50 = 0.25 μg ml-1) to fluopyram compared to strains of the other two species. A sequence analysis of Sdh genes of Fusarium strains revealed that the F. tucumaniae strains contain an arginine at codon 277 in the SdhB gene instead of a glycine as in other Fusarium species. Replacement of glycine to arginine in SdhB-277 in a F. virguliforme wild-type strain Mont-1 through genetic transformation resulted in increased sensitivity to two SDHI fungicides, fluopyram and boscalid. Similar to a F. tucumaniae strain, the Mont-1 (SdhBG277R) mutant caused less SDS and root rot disease than Mont-1 on soybean seedlings with the fluopyram seed treatment. Our study suggests the amino acid difference in the SdhB in F. tucumaniae results in fluopyram being efficacious if used as a seed treatment for management of F. tucumaniae, which is the most abundant SDS causing species in South America. The establishment of baseline sensitivity of Fusarium species to fluopyram will contribute to effective strategies for managing Fusarium diseases in soybean and other pathosystems such as dry bean.

Fungicides have extensively been used to effectively combat fungal diseases on a range of plant species, but resistance to multiple active ingredients has developed in pathogens such as Sclerotinia homoeocarpa, the causal agent of dollar spot on cool-season turfgrasses. Recently, ZnO and Ag nanoparticles (NPs) have received increased attention due to their antimicrobial activities. In this study, the NPs' toxicity and mechanisms of action were investigated as alternative antifungal agents against S. homoeocarpa isolates that varied in their resistance to demethylation inhibitor (DMI) fungicides. S. homoeocarpa isolates were treated with ZnO NPs and ZnCl (25-400 μg ml) and Ag NPs and AgNO (5-100 μg ml) to test antifungal activity of the NPs and ions. The mycelial growth of S. homoeocarpa isolates regardless of their DMI sensitivity was significantly inhibited on ZnO NPs (≥200 μg ml), Ag NPs (≥25 μg ml), Zn ions (≥200 μg ml), and Ag ions (≥10 μg ml) amended media. Expression of stress response genes, glutathione S-transferase (Shgst1) and superoxide dismutase 2 (ShSOD2), was significantly induced in the isolates by exposure to the NPs and ions. In addition, a significant increase in the nucleic acid contents of fungal hyphae, which may be due to stress response, was observed upon treatment with Ag NPs using Raman spectroscopy. We further observed that a zinc transporter (Shzrt1) might play an important role in accumulating ZnO and Ag NPs into the cells of S. homoeocarpa due to overexpression of Shzrt1 significantly induced by ZnO or Ag NPs within 3 h of exposure. Yeast mutants complemented with Shzrt1 became more sensitive to ZnO and Ag NPs as well as Zn and Ag ions than the control strain and resulted in increased Zn or Ag content after exposure. This is the first report of involvement of the zinc transporter in the accumulation of Zn and Ag from NP exposure in filamentous plant pathogenic fungi. Understanding the molecular mechanisms of NPs' antifungal activities will be useful in developing effective management strategies to control important pathogenic fungal diseases.