Fusarium metavorans sp. nov.: The frequent opportunist ‘FSSC6’

Fusarium metavorans sp. nov.: The frequent opportunist ‘FSSC6’ Abstract The Fusarium solani species complex (FSSC) is the most common group of fusaria associated with superficial and life-threatening infections in humans. Here we formally introduce Fusarium metavorans sp. nov., widely known as FSSC6 (Fusarium solani species complex lineage 6), one of the most frequent agents of human opportunistic infections. The species is described with multilocus molecular data including sequences of internal transcribed spacer region (ITS), portions of the translation elongation factor 1-a gene (TEF1), and the partial RNA polymerase II gene (rPB2). A phylogenetic approach was used to evaluate species delimitation. Topologies of the trees were concordant. Phylogenetic analyses suggest that the FSSC consists of three major clades encompassing a large number of phylogenetic species; Fusarium metavorans corresponds to phylogenetic species 6 within FSSC clade 3. The species has a global distribution and a wide ecological amplitude, also including strains from soil and agents of opportunistic plant disease; it was also isolated from the gut of the wood-boring cerambycid beetle Anoplophora glabripennis. taxonomy, molecular phylogenetics, morphology, RPB2, TEF1, fusariosis, Fusarium metavorans Introduction Fusarium is a large genus of the Ascomycota (Nectriaceae, Hypocreales). Currently the genus comprises over 300 species, mostly common endophytic and plant-pathogenic species, isolated from a wide range of hosts and with global distribution.1,2 Some species produce mycotoxins in crops that can affect human and animal health when entering the food chain.3 The main Fusarium toxins are fumonisins and trichothecenes.3 Strains of Fusarium also are encountered as opportunistic pathogens on humans causing eye, skin, nail, and occasionally disseminated infections.4,5 However, the role of fusaria in human pathology is not well understood. In plants, weather conditions such as temperature and humidity may predispose individuals to Fusarium infection, the cutaneous microenvironment, or host defense mechanisms. Modern polyphasic Fusarium taxonomy has led to narrow species concepts with numerous micro-species. Based on partial translation elongation factor-1 alpha (TEF1), the RNA polymerase II gene (rPB1) and RNA polymerase (rPB2), 20 species complexes were recognized in the genus.6,7Fusarium solani species complex (FSSC) currently contains about 60 phylogenetic distinct entities.8–13 Based on the biological species concept, Matue and Syner14 described seven mating populations within F. solani known as Nectria haematococca mating populations I–VII. Each group was correlated to one of the known formae speciales defined by pathogenicity on specific hosts.15–18 Members of the FSSC represent a diverse set of self-fertile (homothallic) and self-sterile (heterothallic) species, although sexual cycles are only known for approximately one-third of the taxa.8,10 Molecular phylogenetic relationships recognize three major clades in FSSC, with clade 3 accommodating several clinically relevant phylogenetic entities. Several of the lineages in clade 3 have been described with formal names,12,19,20 while others remained unnamed. Thus far, six entities are named and are listed as such in the Atlas of Clinical Fungi:21F. falciforme (FSSC3+4), F. keratoplasticum (FSSC2), F. lichenicola (FSSC16), F. petroliphilum (FSSC1), F. pseudensiforme, and F. solani sensu stricto (FSSC5). FSSC6 is currently labeled according to its sequence type (ST) and remains to be described. Another lineage associated with opportunistic infections in this clade that has been named is FSSC 27 (Phialophora cyanescens = Cylindrocarpon cyanescens), which was recently recombined as Neocosmospora cyanescens, MB 813864.22 In a recent study from Japan it was reported that also other STs, that is, FSSC9 and FSSC18, are associated with opportunistic infections and with mycotic keratitis.23 Both haplotypes are phylogenetically distinct from described species but remain unnamed yet. The ISHAM Working Group on Fusarium aims to collect clinical strains on a global basis, store and identify these with state-of-the-art technology, in order to obtain insight into species diversity involved in human infections, and to develop subsequent diagnostics and management recommendations. The main objectives of the study, for which part of the material was collected by network members are to (1) to describe phylogenetic and morphological features that correlate with FSSC6 as circumscribed by molecular analyses; (2) to provide a valid Latin binomial to FSSC6 in order to facilitate studies comparing the etiology and epidemiology of the entity and to improve communication among public health and agricultural scientists. Methods Phenotypic studies A Fusarium strain was isolated from human pleura of lung cancer patient in Greece and submitted to the Centraalbureau voor Schimmelcultures (CBS) reference collection housed at Westerdijk Fungal Biodiversity Institute, the Netherlands under accession number CBS 135789 (Table 1). The strain was cultured on plates of malt extract agar (MEA; Oxoid, UK), oatmeal agar (OA; home-made at CBS), potato dextrose agar (PDA; Oxoid), synthetic nutrient agar (SNA; CBS),24 and carnation leaf agar (CLA; CBS).25 Cultures were grown under 12 h light-dark (l/d) cycles with UV and daylight color fluorescent lights at 24°C. Morphological characters examined included the shape and size of macroconidia produced in sporodochia on CLA,25 the shape and mode of formation of microconidia on CLA and SNA,25 the production of chlamydospores on CLA, and pigmentation of the agar on PDA, SNA, and MEA. Microscopic slides of CBS 135789 strain were prepared from cultures grown on a CLA after 5 days of incubation at 24°C by mounting structures in lactic acid. Slides were examined with a Nikon Eclipse 80i light microscope, and pictures were taken using a camera attached to the microscope (Nikon; digital-sight DS-5 M). A minimum of 10 measurements per structure were taken after processing in Adobe Photoshop CS3, and the average was calculated. Table 1. GenBank accession numbers of Fusarium spp. of the F. solani species complex used in phylogenetic analysis of F. metavorans. Species  Collection  RPB2  TEF 1α  ITS  F. solani (FSSC5)  NRRL 44903  GU170585.1  GU170620.1  GU170640.1  F. solani (FSSC5)  CBS 140079ET  KT313623  KT313611  KT313633  F. solani (FSSC5)  NRRL 32810  EU329624.1  DQ247118.1  DQ094577.1  F. keratoplasticum (FSSC2)  NRRL 43433  DQ790561.1  DQ790473.1  DQ790517.1  Fusarium sp. (FSSC8)  NRRL 43467  EF469979.1  EF452940.1  EF453092.1  F. lichenicola (FSSC16)  NRRL 28030  EF470146.1  KR673968.1  DQ094355.1  Fusarium sp. (FSSC44)  NRRL 34617  KT313616.1  EF428716.1  KT313627.1  Fusarium sp. (FSSC24a)  NRRL 32751  EU329611.1  DQ247070.1  DQ094531.1  Fusarium sp. (FSSC9)  NRRL 32755  EU329613.1  DQ247073.1  DQ094534.1  F. petroliphilum (FSSC1)  NRRL 28546  EU329544.1  DQ246887.1  DQ094361.1  Fusarium sp. (FSSC7)  NRRL 43507  DQ790578.1  DQ790490.1  DQ790534.1  Fusarium sp. (FSSC18)  NRRL 31158  EU329559.1  DQ246916.1  DQ094389.1  Fusarium sp. (FSSC15)  NRRL 28009  EF470136.1  DQ246869.1  DQ094351.1  Fusarium sp. (FSSC29)  NRRL 28008  EF470135.1  DQ246868  DQ094350.1  Fusarium sp. (FSSC35)  NRRL 46707  EU329665.1  HM347127.1  EU329716.1  Fusarium sp. (FSSC25)  NRRL 31169  KR673999.1  DQ246923.1  DQ094396.1  Fusarium sp. (FSSC26)  NRRL 28541  EU329542.1  DQ246882.1  EU329674.1  Fusarium sp. = N. cyanescens (FSSC27)  NRRL 37625  EU329637.1  FJ240353.1  EU329684.1  Fusarium sp. (FSSC28)  NRRL 32437  EU329581.1  DQ246979.1  DQ094446.1  Fusarium sp. (FSSC12)  NRRL 22642  EU329522.1  DQ246844.1  DQ094329.1  Fusarium sp. (FSSC14)  NRRL 22611  EU329518.1  DQ246841.1  DQ094326.1  Fusarium sp. (FSSC33)  NRRL 22354  EU329504.1  AF178338.1  AF178402.1  Fusarium sp. (FSSC6)  NRRL 43489  DQ790572.1  DQ790484.1  DQ790528.1  Fusarium sp. (FSSC6)  NRRL 44892  GU170601.1  GU170618.1  GU170638.1  Fusarium sp. (FSSC6)  F201135  KM520374.1  KM527108.1  KM527100.1  Fusarium sp. (FSSC6)  F201334  KM520376.1  KM527110.1  KM527102.1  Fusarium sp. (FSSC6)  NRRL 43717  EF470233.1  FJ240356.1  EU329688.1  Fusarium sp. (FSSC6)  NRRL 44904  GU170586.1  GU170621.1  GU170641.1  Fusarium sp. (FSSC6)  CBS 135789  KU604374.1  KU711773.1  KR071699.1  F. falciforme (FSSC 3+4)  NRRL 43441  DQ790566.1  DQ790478.1  DQ790522.1  Fusarium sp. (FSSC20)  NRRL 22608  EU329517.1  DQ246838.1  DQ094323.1  Fusarium sp. (FSSC24)  NRRL 22389  EU329506.1  AF178340.1  DQ094314.1  F. solani f.sp. pisi MPVI (FSSC1)  NRRL 22278  EU329501.1  AF178337.1  DQ094309.1  F. solani f.sp. robiniae MPVII (FSSC13)  NRRL 22161  EU329494.1  AF178330.1  DQ094311.1  F. solani f.sp. mori MPIII (FSSC17)  NRRL 22157  EU329493.1  AF178359.1  DQ094306.1  F. ambrosium (FSSC19)  NRRL 20438  JX171584.1  AF178332.1  DQ094315.1  F. solani f.sp. xanthoxyli  NRRL 22163  EU329496.1  AF178328.1  AF178394.1  F. solani f.sp. batata  NRRL 22400  EU329509.1  AF178343.1  DQ094303.1  F. pseudensiforme  NRRL 46517  KC691674.1  KC691555.1  KC691584.1  F. striatum (FSSC21)  NRRL 22101  EU329490.1  AF178333.1  AF178398.1  Fusarium sp. (FSSC34)  NRRL 46703  EU329661.1  HM347126.1  EU329712.1  Fusarium sp. (FSSC30)  NRRL 22579  EU329515.1  AF178352.1  AF178415.1  Fusarium sp. (FSSC31)  NRRL 22570  EU329513.1  AF178360.1  AF178422.1  Fusarium sp. (FSSC32)  NRRL 22178  EU329498.1  AF178334.1  DQ094313.1  F. solani’ f.sp. cucurbitae MPI (FSSC10)  NRRL 22153  EU329492.1  AF178346.1  DQ094302.1  F. brasiliense (FSSC) clade2  NRRL 22743  EU329507.1  AF178341.1  AF178405.1  F. illudens (FSSC) clade1  NRRL 22090  JX171601.1  AF178326.1  AF178393.1  F. staphyleae (Outgroup)  NRRL 22316  JX171609.1  AF178361.1  AF178423.1  N. croci  CBS 142423  LT746329  LT746216  LT746264  N. macrospora  CBS 142424  LT746331  LT746218  LT746266  Species  Collection  RPB2  TEF 1α  ITS  F. solani (FSSC5)  NRRL 44903  GU170585.1  GU170620.1  GU170640.1  F. solani (FSSC5)  CBS 140079ET  KT313623  KT313611  KT313633  F. solani (FSSC5)  NRRL 32810  EU329624.1  DQ247118.1  DQ094577.1  F. keratoplasticum (FSSC2)  NRRL 43433  DQ790561.1  DQ790473.1  DQ790517.1  Fusarium sp. (FSSC8)  NRRL 43467  EF469979.1  EF452940.1  EF453092.1  F. lichenicola (FSSC16)  NRRL 28030  EF470146.1  KR673968.1  DQ094355.1  Fusarium sp. (FSSC44)  NRRL 34617  KT313616.1  EF428716.1  KT313627.1  Fusarium sp. (FSSC24a)  NRRL 32751  EU329611.1  DQ247070.1  DQ094531.1  Fusarium sp. (FSSC9)  NRRL 32755  EU329613.1  DQ247073.1  DQ094534.1  F. petroliphilum (FSSC1)  NRRL 28546  EU329544.1  DQ246887.1  DQ094361.1  Fusarium sp. (FSSC7)  NRRL 43507  DQ790578.1  DQ790490.1  DQ790534.1  Fusarium sp. (FSSC18)  NRRL 31158  EU329559.1  DQ246916.1  DQ094389.1  Fusarium sp. (FSSC15)  NRRL 28009  EF470136.1  DQ246869.1  DQ094351.1  Fusarium sp. (FSSC29)  NRRL 28008  EF470135.1  DQ246868  DQ094350.1  Fusarium sp. (FSSC35)  NRRL 46707  EU329665.1  HM347127.1  EU329716.1  Fusarium sp. (FSSC25)  NRRL 31169  KR673999.1  DQ246923.1  DQ094396.1  Fusarium sp. (FSSC26)  NRRL 28541  EU329542.1  DQ246882.1  EU329674.1  Fusarium sp. = N. cyanescens (FSSC27)  NRRL 37625  EU329637.1  FJ240353.1  EU329684.1  Fusarium sp. (FSSC28)  NRRL 32437  EU329581.1  DQ246979.1  DQ094446.1  Fusarium sp. (FSSC12)  NRRL 22642  EU329522.1  DQ246844.1  DQ094329.1  Fusarium sp. (FSSC14)  NRRL 22611  EU329518.1  DQ246841.1  DQ094326.1  Fusarium sp. (FSSC33)  NRRL 22354  EU329504.1  AF178338.1  AF178402.1  Fusarium sp. (FSSC6)  NRRL 43489  DQ790572.1  DQ790484.1  DQ790528.1  Fusarium sp. (FSSC6)  NRRL 44892  GU170601.1  GU170618.1  GU170638.1  Fusarium sp. (FSSC6)  F201135  KM520374.1  KM527108.1  KM527100.1  Fusarium sp. (FSSC6)  F201334  KM520376.1  KM527110.1  KM527102.1  Fusarium sp. (FSSC6)  NRRL 43717  EF470233.1  FJ240356.1  EU329688.1  Fusarium sp. (FSSC6)  NRRL 44904  GU170586.1  GU170621.1  GU170641.1  Fusarium sp. (FSSC6)  CBS 135789  KU604374.1  KU711773.1  KR071699.1  F. falciforme (FSSC 3+4)  NRRL 43441  DQ790566.1  DQ790478.1  DQ790522.1  Fusarium sp. (FSSC20)  NRRL 22608  EU329517.1  DQ246838.1  DQ094323.1  Fusarium sp. (FSSC24)  NRRL 22389  EU329506.1  AF178340.1  DQ094314.1  F. solani f.sp. pisi MPVI (FSSC1)  NRRL 22278  EU329501.1  AF178337.1  DQ094309.1  F. solani f.sp. robiniae MPVII (FSSC13)  NRRL 22161  EU329494.1  AF178330.1  DQ094311.1  F. solani f.sp. mori MPIII (FSSC17)  NRRL 22157  EU329493.1  AF178359.1  DQ094306.1  F. ambrosium (FSSC19)  NRRL 20438  JX171584.1  AF178332.1  DQ094315.1  F. solani f.sp. xanthoxyli  NRRL 22163  EU329496.1  AF178328.1  AF178394.1  F. solani f.sp. batata  NRRL 22400  EU329509.1  AF178343.1  DQ094303.1  F. pseudensiforme  NRRL 46517  KC691674.1  KC691555.1  KC691584.1  F. striatum (FSSC21)  NRRL 22101  EU329490.1  AF178333.1  AF178398.1  Fusarium sp. (FSSC34)  NRRL 46703  EU329661.1  HM347126.1  EU329712.1  Fusarium sp. (FSSC30)  NRRL 22579  EU329515.1  AF178352.1  AF178415.1  Fusarium sp. (FSSC31)  NRRL 22570  EU329513.1  AF178360.1  AF178422.1  Fusarium sp. (FSSC32)  NRRL 22178  EU329498.1  AF178334.1  DQ094313.1  F. solani’ f.sp. cucurbitae MPI (FSSC10)  NRRL 22153  EU329492.1  AF178346.1  DQ094302.1  F. brasiliense (FSSC) clade2  NRRL 22743  EU329507.1  AF178341.1  AF178405.1  F. illudens (FSSC) clade1  NRRL 22090  JX171601.1  AF178326.1  AF178393.1  F. staphyleae (Outgroup)  NRRL 22316  JX171609.1  AF178361.1  AF178423.1  N. croci  CBS 142423  LT746329  LT746216  LT746264  N. macrospora  CBS 142424  LT746331  LT746218  LT746266  View Large Physiology Cardinal growth temperatures were determined on 2% MEA for CBS 135789. Plates were incubated in the dark for 1 week at temperatures of 21–36°C at intervals of 3°C; growth was also recorded at 37°C and at 40°C. DNA amplification and sequencing The following genes were amplified directly from the genomic DNA for multilocus sequence typing, using internal transcribed spacer region (ITS),26 elongation factor 1 alpha (TEF1),27 and the second largest subunit of RNA polymerase (rPB2).28 Polymerase chain reaction (PCR) amplification and sequencing were performed according to a protocol applied by Al-Hatmi et al.29 Phylogenetic inference To confirm the identity of our new Fusarium species, we evaluated its position in Bayesian phylogenetic and RAxML trees of the following individual gene markers (ITS, TEF1, and rPB2). Comparative sequences were retrieved from GenBank (Table 1) were analyzed together. Sequences were aligned with the program MAFFT (www.ebi.ac.uk/Tools/msa/mafft/), followed by manual adjustments with MEGA v6.2 and BioEdit v7.0.5.2. A single alignment was constructed for ITS, TEF1, and rPB2. The analysis included 48 sequences for ITS, TEF1 and rPB2. The best-fit model of evolution was determined by ModelTest v0.1.1. The trees were constructed by the outgroup method using the CIPRES Science Gateway online version30 and edited in MEGA v6.2. Sequences included in this study were retrieved from GenBank NRRL 22316 F. staphyleae was used as outgroup. Genetic relationships were investigated by phylogenetic analysis using Bayesian inference (BI) and maximum likelihood (ML) methods. Antifungal susceptibility Antifungal susceptibility testing (AFST) of CBS 135789 was performed by the CLSI broth microdilution as described in the CLSI document M38-A2 (Clinical and Laboratory Standards Institute 2008) with modification as described by Al-Hatmi et al.31 The following drugs were used, amphotericin B (Sigma-Aldrich), fluconazole (Pfizer, Groton, CT, USA), itraconazole (Janssen Pharmaceutica, Tilburg, The Netherlands), voriconazole (Pfizer), posaconazole (Merck), isavuconazole (Basilea Pharmaceutica, Basel, Switzerland), micafungin (Astellas, Ibaraki, Japan), and anidulafungin (Pfizer). Three reference strains (Paecilomyces variotii ATCC 22319, Candida krusei ATCC 6258, and Candida parapsilosis ATCC 22019) were included as quality controls. Results BLAST results of the TEF1 and rPB2 sequences in GenBank revealed that CBS 135789 matched FSSC6 strain (NRRL 43489, F201135, F201334, NRRL 44892, NRRL 44904, and NRRL 43717) with 99% similarity. For further understanding of relations between species, a phylogenetic tree was constructed for each locus separately, that is, ITS, TEF1, and rPB2. Each gene was analyzed separately prior to multilocus analysis. The cladistic analysis was based on sequences of the ITS, TEF1, and rPB2 regions. In gene trees of ITS, TEF1, and rPB2 separately, strain CBS 135789 was found as a member of a monophyletic clade supported by high bootstrap values (data not shown). No major topological variations were detected between trees derived from ML and BI phylogenetic inferences. Trees with identical overall topologies and resolving a monophyletic FSSC6 as shown in the Bayesian inference (BI) and maximum likelihood (ML) trees were encountered in all of the 100 ML inferences and in the BI and ML bootstrap consensus trees. All clades had statistical support between 70 and 100%, and all species were well separated. Intraspecific polymorphism within FSSC6 clusters was observed with ITS, TEF1, and rPB2. The FSSC6 clade received highest possible support in a multilocus study including strains CBS 135789, NRRL 43489, F201135, F201334, NRRL 44892, NRRL 44904, and NRRL 43717 (100% bs/ 1 pp) (Fig. 1). Analysis of individual gene ITS, TEF1, and rPB2 genealogies showed that CBS 135789 was nested within FSSC6 in clade 3, which is known to accommodate opportunistic human or animal pathogens. The final combined analysis of the three mentioned loci data sets encompassed 150 sequences representing 42 taxa and sequence type (STs) and comprised 2061 bp (ITS 497 bp, TEF1 701 bp, and rBP2 863 bp) (Fig. 1) and with F. staphyleae (NRRL 22316) as the outgroup. Figure 1. View largeDownload slide Maximum-likelihood phylogeny of Fusarium solani complex inferred from combined data set (ITS, TEF1, rPB2). Values in the nodes are Maximum-likelihood bootstrap supports (right) and Bayesian posterior probabilities (left). Numbers on the nodes are RaxML bootstrap values above 70% and Bayesian posterior probability values above 0.7. The tree is rooted to NRRL 22316, F. staphyleae. The colored clades are the species that are reported so far from human infection. This Figure is reproduced in color in the online version of Medical Mycology. Figure 1. View largeDownload slide Maximum-likelihood phylogeny of Fusarium solani complex inferred from combined data set (ITS, TEF1, rPB2). Values in the nodes are Maximum-likelihood bootstrap supports (right) and Bayesian posterior probabilities (left). Numbers on the nodes are RaxML bootstrap values above 70% and Bayesian posterior probability values above 0.7. The tree is rooted to NRRL 22316, F. staphyleae. The colored clades are the species that are reported so far from human infection. This Figure is reproduced in color in the online version of Medical Mycology. Taxonomy   Fusarium metavorans Al-Hatmi, S.A. Ahmed and de Hoog, sp. nov. – Fig. 2. MycoBank MB 821742. Figure 2. View largeDownload slide Morphological description of Fusarium metavorans CBS 135789. (A) Growth on MEA agar, obverse, reverse white, reverse yellowish to light orange; (B) Colony characteristics on PDA white to yellowish-white; (C–D). The production of microconidia in false heads on phialides formed on the hyphae single; (E–I) Monophialides with false head and microconidia; (J) Microconidia, abundant and ovoidal; (K) Chlamydospores within hyphae (intercalary) or at hyphal tips (terminal). Scale bar = 10 μm. This Figure is reproduced in color in the online version of Medical Mycology. Figure 2. View largeDownload slide Morphological description of Fusarium metavorans CBS 135789. (A) Growth on MEA agar, obverse, reverse white, reverse yellowish to light orange; (B) Colony characteristics on PDA white to yellowish-white; (C–D). The production of microconidia in false heads on phialides formed on the hyphae single; (E–I) Monophialides with false head and microconidia; (J) Microconidia, abundant and ovoidal; (K) Chlamydospores within hyphae (intercalary) or at hyphal tips (terminal). Scale bar = 10 μm. This Figure is reproduced in color in the online version of Medical Mycology. Etymology: named after the unspecific feeding pattern involving plants, insects and humans. Holotype: CBS 135789 preserved in metabolically inactive condition under liquid nitrogen; living derived from type CBS 135789, pleural effusion of patient with lung cancer, Athens, Greece. Description based on colonies of CBS 135789 grown for 10 d at 25°C on PDA, MEA, and CLA under 12h/12h alternating cool white fluorescent light / darkness. Colonies on MEA reaching 65 mm diam, growing rapidly with average daily growth rates of 5.3–6.2 mm. Obverse with cottony aerial mycelium, reverse yellowish to light orange (Fig. 2A). Colonies on PDA reaching 60 mm diam, average daily growth rate 4.9–5.5 mm. Obverse white to reddish-white at the edge (Fig. 2B), reverse pigmentation in a gradient from pale yellow at the margin to dark brown to red at the center. Conidia formed at colony surface and in the aerial mycelium, the latter in clear, translucent drops of liquid at the tips of long, unbranched conidiophores. Colonies on SNA reaching 70–80 mm diam. Obverse aerial mycelium cottony, white, dense near the center, nearly absent toward the margin. Conidia formed abundantly from erect, typically unbranched conidiophores at the agar surface. Microconidia in false heads on phialides formed on hyphae (Fig. 2C, D). Conidiophores 21.5–118.5 μm (mean 68.9 μm), 3.3 μm wide at the base, unbranched or branched up to three times, straight, thin-walled, smooth; each branch terminating in a single phialide. Phialidic collarettes thickened, flared (Fig. 2E–I). Conidia 0–1 septate, ellipsoidal with rounded apex and truncate base, 3.9–11.1 × 2.3–3.1 μm (Fig. 2I). Chlamydospores appearing after 4 weeks of incubation, single or in chains, consisting of enlarged, thick-walled vegetative cells, within hyphae (intercalary) or at hyphal tips (terminal), 6.0–13.4 μm diam (Fig. 2J). Notes.—Fusarium metavorans was isolated from human pleura of lung cancer patient in Greece. However, this fungus is also associated with plants and ants, which might suggest some level of diversity and geographically widely distributed species. It was considered as a human opportunist, causing superficial and disseminated infections. Fusarium metavorans is morphologically similar to F. solani sensu stricto (FSSC5).13 However, the new species differs mainly by its coloured brown to red pigmentations. Also shows similar micro-morphological characteristics to F. solani sensu stricto (FSSC5) but differs in the absence of macroconidia and the longer conidial chains with up to 10–20 micro-conidia in the terminal branched and unbranched with light and increase size of its conidiophores and phialides. The consensus morphology associated with F. metavorans corresponds well with other species in F. solani species complex. In addition, genetically very well differentiated as trees with identical overall topologies and resolving a monophyletic F. metavorans as shown in the Maximum likelihood and MrBayes consensus tree (Fig. 1) were encountered in all (100% bs/ 1 pp). Physiology Cardinal growth temperatures of F. metavorans type strain CBS 135789: optimal development at (27−)33°C, growth observed in the entire range between 21 and 37°C. Maximum growth temperature of the strain analyzed was 37°C. No growth observed at 40°C. Antifungal susceptibility Antifungal susceptibility testing performed with broth microdilution according to CLSI M38A resulted in the following MICs: amphotericin B 2 μg/ml; fluconazole >64 μg/ml; both posaconazole and voriconazole >4 μg/ml; itraconazole and isavuconazole >16 μg/ml; anidulafungin and micafungin >8 μg/ml. Discussion The taxonomy of Fusarium is evolving. Unlike many other filamentous fungi, only few phenotypic characteristics are available to differentiate Fusarium species. Many of the morphological characteristics described to differentiating the 300 known species overlap, interfering with identification but also species delimitation in Fusarium. Cryptic, molecularly defined species exist, which are morphologically identical.32 Therefore, sequence-based characteristics are essential to confirm the species identities.7,25 Also the genus level is debated. Lombard et al.33 reclassified the F. solani complex into the genus Neocosmospora, a decision opposing the consensus paper of Geiser et al.,9 supported by many authors from clinical and phytopathological backgrounds, with a plea for nomenclatural stability and preservation of the name Fusarium. Lombard's paper has been followed by only few authors; with the result that now confusingly two names exist for the F. solani complex.34 Recently, Sandoval-Denis et al.,35 described two new species within “FSSC,” named as Neocosmospora macrospora and Neocosmospora croci. However, to preserve the nomenclature stability, in the present article we adhere to Fusarium as the best-known descriptor for the etiological agents and the clinical entities. Members of the Fusarium solani species complex (FSSC) are the most commonly encountered fusaria in human infections. The clinical spectrum encompasses skin, nail, eye, bone, nasal cavities, contaminated wounds, and disseminated infection including in patients with cancer.11,25,36 The species complex accounts for approximately 60% of the total Fusarium infections, judging from literature supported by multilocus sequence analysis.37 Phylogenetically FSSC has been suggested to include 60 narrowly defined phylogenetic species, associated in three main clades: Clade 1, Clade 2, and Clade 3.8,9 Distinct morphological traits are minimal or absent between these clades. Clade 3 is known to accommodate opportunistic pathogens on humans, animals, and plants.8 Clade 3 also contains the type of F. solani sensu stricto (FSSC5).13 Seven of the most common species which occur on human hosts in Clade 3 have been formally named: F. falciforme (FSSC 3+4), F. keratoplasticum (FSSC2), F. lichenicola (FSSC16), F. petroliphilum (FSSC1), F. pseudensiforme, F. solani sensu stricto (FSSC5) and F. metavorans (FSSC6) is officially introduced in this study.12,13,19,20,36 In addition, FSSC27, (Phialophora cyanescens = Cylindrocarpon cyanescens), was recently recombined as Neocosmospora cyanescens.22 Previous studies have consistently revealed the monophyletic group described in this paper within FSSC with strains from the United States,8,11 Colombia,32 Japan,23 Turkey,38 and The Netherlands (unpublished data), all papers demonstrating an association with human infection in lineage FSSC6 (Fusarium metavorans). Sequences of ITS, TEF1, and rBP2 derived from strain CBS 135789, originating from a disseminated infection, matched with STs known as FSSC6 in public DNA sequence repositories. In a comparative analysis of 50 sequences representing 42 Fusarium species and STs of FSSC, it was shown that CBS 135789 was different from all these but showed 100% identity with strains NRRL 43489, F201135, F201334, NRRL 44892, NRRL 44904, and NRRL 43717 of FSSC6; the group formed a strongly supported monophyletic group (100% bs/ 1 pp) (Fig. 1). It was sister to F. solani f. sp. mori clade, a host specific pathogen on mulberry.39 Phylogenetic analysis of the conserved gene region ITS placed F. metavorans unambiguously in the FSSC, but ITS does not provide sufficient resolution at the species level, as holds true for many Fusarium species.6 Using partial coding genes TEF1 and RPB2, F. metavorans was easily distinguished from all described Fusarium species within FSSC. Fusarium metavorans is morphologically similar to other Fusarium species of FSSC that occur on human patients.21 It produces microconidia from false heads on phialides formed on hyphal conidiophores (Fig. 2C, D). A number of aberrant morphological variants were observed in isolate CBS 135789 of F. metavorans concerning the production of yellowish-white pigments on PDA. Fusarium metavorans does not produce macroconidia but instead produces septate or non-septate conidia from long mycelial conidiophores, which is typical for species in FSSC. The monophialides and the production of ovoidal microconidia, presence of chlamydospores and false microconidial head are observed in all species within FSSC. Fusarium metavorans (FSSC6) has been encountered in areas of research different from human infection, including quarantine, plant, soil, and the gut of the wood-boring cerambycid beetle Anoplophora glabripennis in the United States.40 This suggests that the species likely is a saprobe on virgin, watery substrates. In other fungal genera with plant-pathogenic members, such as Alternaria, it was noted that the human opportunists are just the species with predominantly saprobic life styles.41 Chemical analyses have demonstrated that F. metavorans is able to degrade lignocellulose,42 synthesize amino acids,43 and contribute to the synthesis of sterols.44 We find that our new species is capable of growth at 37°C and can thus survive at human body temperature. Antifungal susceptibility profiles obtained from F. metavorans showed remarkable similarity to other Fusarium species4,5 with regard to high degrees of resistance to all antifungals tested. In this study, the description of a novel species involved in opportunistic plant, animal and human infections has enlarged the spectrum of species of potential clinical interest in the genus Fusarium. The intrinsic broad spectrum antifungal resistance of Fusarium species has also been confirmed in F. metavorans. Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and the writing of the paper. References 1. O’Donnell K, Ward TJ, Robert VARG, Crous PW, Geiser DM, Seogchan K. DNA sequence-based identification of Fusarium: current status and future directions. Phytoparasitica . 2015; 43: 583– 595. Google Scholar CrossRef Search ADS   2. Moussa TAA, Al-Zahrani HS, Kadasa NMS, Ahmed SA, de Hoog GS, Al-Hatmi AMS. Two new species of the Fusarium fujikuroi species complex isolated from the natural environment. Antonie van Leeuwenhoek . 2017; 110: 819– 832. Google Scholar CrossRef Search ADS PubMed  3. Milicevic D, Skrinjar M, Baltic T. Real and perceived risks for mycotoxin contamination in foods and feeds: challenges for food safety control. Toxins . 2010; 2: 572– 592. Google Scholar CrossRef Search ADS PubMed  4. Al-Hatmi AMS, Meis JF, de Hoog GS. Fusarium: molecular diversity and intrinsic drug resistance. PLoS Pathog . 2016; 12: e1005464. Google Scholar CrossRef Search ADS PubMed  5. Al-Hatmi AM, Bonifaz A, Ranque R, de Hoog GS, Verweij PE, Meis JF. Current antifungal treatment of fusariosis. Int J Antimicrob Agents . 2017; http://dx.doi.org/doi:10.1016/j.ijantimicag.2017.06.017. 6. Geiser DM, Jimenez-Gasco MD, Kang SC et al.   FUSARIUM-ID v. 1.0: a DNA sequence database for identifying Fusarium. Eur J Plant Pathol.  2004; 110: 473– 479. Google Scholar CrossRef Search ADS   7. O’Donnell K, Rooney AP, Proctor RH et al.   Phylogenetic analyses of RPB1 and RPB2 support a middle cretaceous origin for a clade comprising all agriculturally and medically important fusaria. Fungal Genet Biol . 2013; 52: 20– 31. Google Scholar CrossRef Search ADS PubMed  8. O’Donnell K, Sutton DA, Fothergill A et al.   Molecular phylogenetic diversity, multilocus haplotype nomenclature, and in vitro antifungal resistance within the Fusarium solani species complex. J Clin Microbiol . 2008; 46: 2477– 2490. Google Scholar CrossRef Search ADS PubMed  9. Geiser DM, Aoki T, Bacon CW et al.   One fungus, one name: defining the genus Fusarium in a scientifically robust way that preserves longstanding use. Phytopathology . 2013; 103: 400– 408. Google Scholar CrossRef Search ADS PubMed  10. O’Donnell K, Nirenberg HI, Aoki T, Cigelnik E. A multigene phylogeny of the Gibberella fujikuroi species complex: detection of additional phylogenetically distinct species. Mycoscience . 2000; 1: 61– 78. Google Scholar CrossRef Search ADS   11. Zhang N, O’Donnell K, Sutton DA et al.   Members of the Fusarium solani species complex that cause infections in both humans and plants are common in the environment. J Clin Microbiol  2006; 44: 2186– 2190. Google Scholar CrossRef Search ADS PubMed  12. Nalim FA, Samuels GJ, Wijesundera RL, Geiser DM. New species from the Fusarium solani species complex derived from perithecia and soil in the Old World tropics. Mycologia . 2011; 103: 1302– 1330. Google Scholar CrossRef Search ADS PubMed  13. Schroers HJ, Samuels GJ, Zhang N, Short DP, Juba J, Geiser DM. Epitypification of Fusisporium (Fusarium) solani and its assignment to a common phylogenetic species in the Fusarium solani species complex. Mycologia . 2016; 108: 806– 819. Google Scholar CrossRef Search ADS PubMed  14. Matuo T, Snyder WC. Use of morphology and mating populations in the identification of formae speciales in Fusarium solani. Phytopathology . 1973; 63: 562– 565. Google Scholar CrossRef Search ADS   15. Toussoun TA, Snyder WC. The pathogenicity, distribution and control of two races of Fusarium (Hypomyces) solani f.sp. cucurbitae. Phytopathology.  1961; 51: 17– 22. 16. Suga HT, Hasegawa H, Mitsui KK, Hyakumachi M. Phylogenetic analysis of the phytopathogenic fungus Fusarium solani based on the rDNA-ITS region. Mycol Res . 2000; 104: 1175– 1183. Google Scholar CrossRef Search ADS   17. Chung WC, Chen LW, Huang JH, Chung H. A new “forma specialis” of Fusarium solani causing leaf yellowing of Phalaenopsis: Leaf yellowing of Phalaenopsis. Plant Pathol . 2011; 60: 244– 252. Google Scholar CrossRef Search ADS   18. Bueno CJ, Fischer IH, Rosa DD et al.   Fusarium solani f. sp. passiflorae: a new forma specialis causing collar rot in yellow passion fruit. Plant Pathol . 2014; 63: 382– 389. Google Scholar CrossRef Search ADS   19. Summerbell R, Schroers H-J. Analysis of phylogenetic relationship of Cylindrocarpon lichenicola and Acremonium falciforme to the Fusarium solani species complex and a review of similarities in the spectrum of opportunistic infections caused by these fungi. J Clin Microbiol . 2002; 40: 2866– 2875. Google Scholar CrossRef Search ADS PubMed  20. Short DPG, O’Donnell K, Thrane U et al.   Phylogenetic relationships among members of the Fusarium solani species complex in human infections and the descriptions of F. keratoplasticum sp. nov. and F. petroliphilum stat. nov. Fungal Genet Biol . 2013; 53: 59– 70. Google Scholar CrossRef Search ADS PubMed  21. de Hoog GS, Guarro J, Gené J, Figueras MJ. Atlas of Clinical Fungi , 3rd ed. Utrecht, Netherlands: Centraalbureau voor Schimmelcultures, 2011. 22. Summerbell RC, Scott JA. Conidiogenesis: its evolutionary aspects in the context of a philosophy of opportunity (lectics). In: Li DW, eds. Biology of Microfungi . London: Springer Science, 2016: 169– 195. Google Scholar CrossRef Search ADS   23. Muraosa Y, Oguchi M, Yahiro M, Watanabe A, Yaguchi T, Kamei K. Epidemiological study of Fusarium species causing invasive and superficial fusariosis in Japan. Med Mycol J . 2017; 58: E5– E13. doi: 10.3314/mmj.16-00024. Google Scholar CrossRef Search ADS PubMed  24. Nirenberg HI. Studies on the morphologic and biologic differentiation in Fusarium section Liseola. Mitt Biol Bundesanst Land- Forstw Berlin-Dahlem . 1976; 169: 1– 117. 25. Leslie JF, Summerell BA. The Fusarium Laboratory Manual . Oxford: Blackwell, 2007. 26. White TJ, Bruns T, Lee S, Taylor J. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Gelfand Innis D., Sninsky J., White T, eds., PCR Protocols: A Guide to Methods and Applications . New York: Academic Press, 1990: 315– 322. Google Scholar CrossRef Search ADS   27. O’Donnell K, Sutton DA, Rinaldi MG et al.   Internet-accessible DNA sequence database for identifying Fusaria from human and animal infections. J Clin Microbiol . 2010; 48: 3708– 3718. Google Scholar CrossRef Search ADS PubMed  28. Reeb V, Lutzoni F, Roux C. Contribution of RPB2 to multilocus phylogenetic studies of the euascomycetes (Pezizomycotina, fungi) with special emphasis on the lichen-forming Acarosporaceae and evolution of polyspory. Mol Phylogenet Evol . 2004; 32: 1036– 1060. Google Scholar CrossRef Search ADS PubMed  29. Al-Hatmi AMS, Mirabolfathy M, Hagen F et al.   DNA barcoding, MALDI-TOF and AFLP data support Fusarium ficicrescens as a distinct species within the F. fujikuroi species complex. Fungal Biol . 2016; 120: 265– 278. Google Scholar CrossRef Search ADS PubMed  30. Miller MA, Pfeiffer W, Schwartz T. The CIPRES science gateway: a community resource for phylogenetic analyses. In Proceedings of the 2011 TeraGrid Conference: Extreme Digital Discovery . Salt Lake City, UT, USA: ACM 2011: 1– 8. 31. Al-Hatmi AM, van Diepeningen AD, Curfs-Breuker I, de Hoog GS, Meis JF. Specific antifungal susceptibility profiles of opportunists in the Fusarium fujikuroi complex. J Antimicrob Chemother . 2015; 70: 1068– 1071. Google Scholar PubMed  32. Guevara-Suarez M, Cano-Lira JF, de Garcia MCC et al.   A genotyping of Fusarium isolates from onychomycosis in Colombia: detection of two new species within the Fusarium solani species complex and in vitro antifungal susceptibility testing. Mycopathologia . 2016; 181: 165– 174. Google Scholar CrossRef Search ADS PubMed  33. Lombard L, van der Merwe NA, Groenewald JZ, Crous PW. Generic concepts in Nectriaceae. Stud Mycol . 2015; 80: 189– 245. Google Scholar CrossRef Search ADS PubMed  34. Aoki T, O’Donnell K, Geiser DM. Systematics of key phytopathogenic Fusarium species: current status and future challenges. Plant Pathol . 2014; 80: 189– 201. 35. Sandoval-Denis M, Guarnaccia V, Polizzi G, Crous PW. Symptomatic citrus trees reveal a new pathogenic lineage in Fusarium and two new Neocosmospora species. Persoonia . 2018; 40: 1– 25. 36. Al-Hatmi AMS, Hagen F, Menken SBJ, Meis JF, de Hoog GS. Global molecular epidemiology and genetic diversity of Fusarium, a significant emerging human opportunist from 1958–2015. Emerg Microbes Infect . 2016; 5: e33; doi:10.1038/emi.2016.126. Google Scholar CrossRef Search ADS PubMed  37. Alfonso EC, Rosa RH. Fungal Keratitis. In: Krachmer JH, Mannis MJ, Holland EJ, eds. Cornea and External Diseases: Clinical Diagnosis and Management . St Louis: Mosby, 1997: 1253– 1266. Google Scholar CrossRef Search ADS   38. Dalyan Cilo B, Al-Hatmi AM, Seyedmousavi S et al.   Emergence of fusarioses in a university hospital in Turkey during a 20-year period. Eur J Clin Microbiol Infect Dis.  2015; 34: 1683– 1691. Google Scholar CrossRef Search ADS PubMed  39. Sakurai Y, Matuo T. On the form name and race of Hypomyces solani (Rke. et Berth.) Snyd. et Hans. which is pathogenic to the mulberry trees. Ann Pytopathol Soc Japan . 1959; 24: 219– 223. Google Scholar CrossRef Search ADS   40. Herr JR, Scully ED, Geib SM, Hoover K, Carlson JE, Geiser DM. Genome sequence of the fungus FSSC6, a Fusarium species (MYA-4552) isolated from the midgut of Anoplophora glabripennis, an invasive, wood-boring beetle. Genome Announc . 2016; 4: e00544– 16. Google Scholar CrossRef Search ADS PubMed  41. Van Baarlen P, Van Belkum A, Summerbell RC, Crous PW, Thomma BPHJ. Molecular mechanisms of pathogenicity: how do pathogenic microorganisms develop cross-kingdom host jumps? FEMS Microbiol Rev . 2007; 31: 239– 277. Google Scholar CrossRef Search ADS PubMed  42. Scully ED, Geib SM, Hoover K et al.   Metagenomic profiling reveals lignocellulose degrading system in a microbial community associated with a wood-feeding beetle. PLoS One . 2013; 8: e73827. doi:10.1371/journal.pone.0073827 Google Scholar CrossRef Search ADS PubMed  43. Ayayee PA, Larsen T, Rosa C, Felton GW, Ferry JG, Hoover K. Essential amino acid supplementation by gut microbes of a wood-feeding cerambycid. Environ Entomol . 2016; 45: 66– 73. doi:10.1093/ee/nvv153. Google Scholar CrossRef Search ADS PubMed  44. Scully ED, Geib SM, Carlson JE. Functional genomics and microbiome profiling of the Asian longhorned beetle (Anoplophora glabripennis) reveal insights into the digestive physiology and nutritional ecology of wood feeding beetles. BMC Genomics . 2014; 15: 1096. Google Scholar CrossRef Search ADS PubMed  © The Author 2017. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Medical Mycology Oxford University Press

Loading next page...
 
/lp/ou_press/fusarium-metavorans-sp-nov-the-frequent-opportunist-fssc6-odL80FN10Q
Publisher
Taylor & Francis
Copyright
© The Author 2017. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com
ISSN
1369-3786
eISSN
1460-2709
D.O.I.
10.1093/mmy/myx107
Publisher site
See Article on Publisher Site

Abstract

Abstract The Fusarium solani species complex (FSSC) is the most common group of fusaria associated with superficial and life-threatening infections in humans. Here we formally introduce Fusarium metavorans sp. nov., widely known as FSSC6 (Fusarium solani species complex lineage 6), one of the most frequent agents of human opportunistic infections. The species is described with multilocus molecular data including sequences of internal transcribed spacer region (ITS), portions of the translation elongation factor 1-a gene (TEF1), and the partial RNA polymerase II gene (rPB2). A phylogenetic approach was used to evaluate species delimitation. Topologies of the trees were concordant. Phylogenetic analyses suggest that the FSSC consists of three major clades encompassing a large number of phylogenetic species; Fusarium metavorans corresponds to phylogenetic species 6 within FSSC clade 3. The species has a global distribution and a wide ecological amplitude, also including strains from soil and agents of opportunistic plant disease; it was also isolated from the gut of the wood-boring cerambycid beetle Anoplophora glabripennis. taxonomy, molecular phylogenetics, morphology, RPB2, TEF1, fusariosis, Fusarium metavorans Introduction Fusarium is a large genus of the Ascomycota (Nectriaceae, Hypocreales). Currently the genus comprises over 300 species, mostly common endophytic and plant-pathogenic species, isolated from a wide range of hosts and with global distribution.1,2 Some species produce mycotoxins in crops that can affect human and animal health when entering the food chain.3 The main Fusarium toxins are fumonisins and trichothecenes.3 Strains of Fusarium also are encountered as opportunistic pathogens on humans causing eye, skin, nail, and occasionally disseminated infections.4,5 However, the role of fusaria in human pathology is not well understood. In plants, weather conditions such as temperature and humidity may predispose individuals to Fusarium infection, the cutaneous microenvironment, or host defense mechanisms. Modern polyphasic Fusarium taxonomy has led to narrow species concepts with numerous micro-species. Based on partial translation elongation factor-1 alpha (TEF1), the RNA polymerase II gene (rPB1) and RNA polymerase (rPB2), 20 species complexes were recognized in the genus.6,7Fusarium solani species complex (FSSC) currently contains about 60 phylogenetic distinct entities.8–13 Based on the biological species concept, Matue and Syner14 described seven mating populations within F. solani known as Nectria haematococca mating populations I–VII. Each group was correlated to one of the known formae speciales defined by pathogenicity on specific hosts.15–18 Members of the FSSC represent a diverse set of self-fertile (homothallic) and self-sterile (heterothallic) species, although sexual cycles are only known for approximately one-third of the taxa.8,10 Molecular phylogenetic relationships recognize three major clades in FSSC, with clade 3 accommodating several clinically relevant phylogenetic entities. Several of the lineages in clade 3 have been described with formal names,12,19,20 while others remained unnamed. Thus far, six entities are named and are listed as such in the Atlas of Clinical Fungi:21F. falciforme (FSSC3+4), F. keratoplasticum (FSSC2), F. lichenicola (FSSC16), F. petroliphilum (FSSC1), F. pseudensiforme, and F. solani sensu stricto (FSSC5). FSSC6 is currently labeled according to its sequence type (ST) and remains to be described. Another lineage associated with opportunistic infections in this clade that has been named is FSSC 27 (Phialophora cyanescens = Cylindrocarpon cyanescens), which was recently recombined as Neocosmospora cyanescens, MB 813864.22 In a recent study from Japan it was reported that also other STs, that is, FSSC9 and FSSC18, are associated with opportunistic infections and with mycotic keratitis.23 Both haplotypes are phylogenetically distinct from described species but remain unnamed yet. The ISHAM Working Group on Fusarium aims to collect clinical strains on a global basis, store and identify these with state-of-the-art technology, in order to obtain insight into species diversity involved in human infections, and to develop subsequent diagnostics and management recommendations. The main objectives of the study, for which part of the material was collected by network members are to (1) to describe phylogenetic and morphological features that correlate with FSSC6 as circumscribed by molecular analyses; (2) to provide a valid Latin binomial to FSSC6 in order to facilitate studies comparing the etiology and epidemiology of the entity and to improve communication among public health and agricultural scientists. Methods Phenotypic studies A Fusarium strain was isolated from human pleura of lung cancer patient in Greece and submitted to the Centraalbureau voor Schimmelcultures (CBS) reference collection housed at Westerdijk Fungal Biodiversity Institute, the Netherlands under accession number CBS 135789 (Table 1). The strain was cultured on plates of malt extract agar (MEA; Oxoid, UK), oatmeal agar (OA; home-made at CBS), potato dextrose agar (PDA; Oxoid), synthetic nutrient agar (SNA; CBS),24 and carnation leaf agar (CLA; CBS).25 Cultures were grown under 12 h light-dark (l/d) cycles with UV and daylight color fluorescent lights at 24°C. Morphological characters examined included the shape and size of macroconidia produced in sporodochia on CLA,25 the shape and mode of formation of microconidia on CLA and SNA,25 the production of chlamydospores on CLA, and pigmentation of the agar on PDA, SNA, and MEA. Microscopic slides of CBS 135789 strain were prepared from cultures grown on a CLA after 5 days of incubation at 24°C by mounting structures in lactic acid. Slides were examined with a Nikon Eclipse 80i light microscope, and pictures were taken using a camera attached to the microscope (Nikon; digital-sight DS-5 M). A minimum of 10 measurements per structure were taken after processing in Adobe Photoshop CS3, and the average was calculated. Table 1. GenBank accession numbers of Fusarium spp. of the F. solani species complex used in phylogenetic analysis of F. metavorans. Species  Collection  RPB2  TEF 1α  ITS  F. solani (FSSC5)  NRRL 44903  GU170585.1  GU170620.1  GU170640.1  F. solani (FSSC5)  CBS 140079ET  KT313623  KT313611  KT313633  F. solani (FSSC5)  NRRL 32810  EU329624.1  DQ247118.1  DQ094577.1  F. keratoplasticum (FSSC2)  NRRL 43433  DQ790561.1  DQ790473.1  DQ790517.1  Fusarium sp. (FSSC8)  NRRL 43467  EF469979.1  EF452940.1  EF453092.1  F. lichenicola (FSSC16)  NRRL 28030  EF470146.1  KR673968.1  DQ094355.1  Fusarium sp. (FSSC44)  NRRL 34617  KT313616.1  EF428716.1  KT313627.1  Fusarium sp. (FSSC24a)  NRRL 32751  EU329611.1  DQ247070.1  DQ094531.1  Fusarium sp. (FSSC9)  NRRL 32755  EU329613.1  DQ247073.1  DQ094534.1  F. petroliphilum (FSSC1)  NRRL 28546  EU329544.1  DQ246887.1  DQ094361.1  Fusarium sp. (FSSC7)  NRRL 43507  DQ790578.1  DQ790490.1  DQ790534.1  Fusarium sp. (FSSC18)  NRRL 31158  EU329559.1  DQ246916.1  DQ094389.1  Fusarium sp. (FSSC15)  NRRL 28009  EF470136.1  DQ246869.1  DQ094351.1  Fusarium sp. (FSSC29)  NRRL 28008  EF470135.1  DQ246868  DQ094350.1  Fusarium sp. (FSSC35)  NRRL 46707  EU329665.1  HM347127.1  EU329716.1  Fusarium sp. (FSSC25)  NRRL 31169  KR673999.1  DQ246923.1  DQ094396.1  Fusarium sp. (FSSC26)  NRRL 28541  EU329542.1  DQ246882.1  EU329674.1  Fusarium sp. = N. cyanescens (FSSC27)  NRRL 37625  EU329637.1  FJ240353.1  EU329684.1  Fusarium sp. (FSSC28)  NRRL 32437  EU329581.1  DQ246979.1  DQ094446.1  Fusarium sp. (FSSC12)  NRRL 22642  EU329522.1  DQ246844.1  DQ094329.1  Fusarium sp. (FSSC14)  NRRL 22611  EU329518.1  DQ246841.1  DQ094326.1  Fusarium sp. (FSSC33)  NRRL 22354  EU329504.1  AF178338.1  AF178402.1  Fusarium sp. (FSSC6)  NRRL 43489  DQ790572.1  DQ790484.1  DQ790528.1  Fusarium sp. (FSSC6)  NRRL 44892  GU170601.1  GU170618.1  GU170638.1  Fusarium sp. (FSSC6)  F201135  KM520374.1  KM527108.1  KM527100.1  Fusarium sp. (FSSC6)  F201334  KM520376.1  KM527110.1  KM527102.1  Fusarium sp. (FSSC6)  NRRL 43717  EF470233.1  FJ240356.1  EU329688.1  Fusarium sp. (FSSC6)  NRRL 44904  GU170586.1  GU170621.1  GU170641.1  Fusarium sp. (FSSC6)  CBS 135789  KU604374.1  KU711773.1  KR071699.1  F. falciforme (FSSC 3+4)  NRRL 43441  DQ790566.1  DQ790478.1  DQ790522.1  Fusarium sp. (FSSC20)  NRRL 22608  EU329517.1  DQ246838.1  DQ094323.1  Fusarium sp. (FSSC24)  NRRL 22389  EU329506.1  AF178340.1  DQ094314.1  F. solani f.sp. pisi MPVI (FSSC1)  NRRL 22278  EU329501.1  AF178337.1  DQ094309.1  F. solani f.sp. robiniae MPVII (FSSC13)  NRRL 22161  EU329494.1  AF178330.1  DQ094311.1  F. solani f.sp. mori MPIII (FSSC17)  NRRL 22157  EU329493.1  AF178359.1  DQ094306.1  F. ambrosium (FSSC19)  NRRL 20438  JX171584.1  AF178332.1  DQ094315.1  F. solani f.sp. xanthoxyli  NRRL 22163  EU329496.1  AF178328.1  AF178394.1  F. solani f.sp. batata  NRRL 22400  EU329509.1  AF178343.1  DQ094303.1  F. pseudensiforme  NRRL 46517  KC691674.1  KC691555.1  KC691584.1  F. striatum (FSSC21)  NRRL 22101  EU329490.1  AF178333.1  AF178398.1  Fusarium sp. (FSSC34)  NRRL 46703  EU329661.1  HM347126.1  EU329712.1  Fusarium sp. (FSSC30)  NRRL 22579  EU329515.1  AF178352.1  AF178415.1  Fusarium sp. (FSSC31)  NRRL 22570  EU329513.1  AF178360.1  AF178422.1  Fusarium sp. (FSSC32)  NRRL 22178  EU329498.1  AF178334.1  DQ094313.1  F. solani’ f.sp. cucurbitae MPI (FSSC10)  NRRL 22153  EU329492.1  AF178346.1  DQ094302.1  F. brasiliense (FSSC) clade2  NRRL 22743  EU329507.1  AF178341.1  AF178405.1  F. illudens (FSSC) clade1  NRRL 22090  JX171601.1  AF178326.1  AF178393.1  F. staphyleae (Outgroup)  NRRL 22316  JX171609.1  AF178361.1  AF178423.1  N. croci  CBS 142423  LT746329  LT746216  LT746264  N. macrospora  CBS 142424  LT746331  LT746218  LT746266  Species  Collection  RPB2  TEF 1α  ITS  F. solani (FSSC5)  NRRL 44903  GU170585.1  GU170620.1  GU170640.1  F. solani (FSSC5)  CBS 140079ET  KT313623  KT313611  KT313633  F. solani (FSSC5)  NRRL 32810  EU329624.1  DQ247118.1  DQ094577.1  F. keratoplasticum (FSSC2)  NRRL 43433  DQ790561.1  DQ790473.1  DQ790517.1  Fusarium sp. (FSSC8)  NRRL 43467  EF469979.1  EF452940.1  EF453092.1  F. lichenicola (FSSC16)  NRRL 28030  EF470146.1  KR673968.1  DQ094355.1  Fusarium sp. (FSSC44)  NRRL 34617  KT313616.1  EF428716.1  KT313627.1  Fusarium sp. (FSSC24a)  NRRL 32751  EU329611.1  DQ247070.1  DQ094531.1  Fusarium sp. (FSSC9)  NRRL 32755  EU329613.1  DQ247073.1  DQ094534.1  F. petroliphilum (FSSC1)  NRRL 28546  EU329544.1  DQ246887.1  DQ094361.1  Fusarium sp. (FSSC7)  NRRL 43507  DQ790578.1  DQ790490.1  DQ790534.1  Fusarium sp. (FSSC18)  NRRL 31158  EU329559.1  DQ246916.1  DQ094389.1  Fusarium sp. (FSSC15)  NRRL 28009  EF470136.1  DQ246869.1  DQ094351.1  Fusarium sp. (FSSC29)  NRRL 28008  EF470135.1  DQ246868  DQ094350.1  Fusarium sp. (FSSC35)  NRRL 46707  EU329665.1  HM347127.1  EU329716.1  Fusarium sp. (FSSC25)  NRRL 31169  KR673999.1  DQ246923.1  DQ094396.1  Fusarium sp. (FSSC26)  NRRL 28541  EU329542.1  DQ246882.1  EU329674.1  Fusarium sp. = N. cyanescens (FSSC27)  NRRL 37625  EU329637.1  FJ240353.1  EU329684.1  Fusarium sp. (FSSC28)  NRRL 32437  EU329581.1  DQ246979.1  DQ094446.1  Fusarium sp. (FSSC12)  NRRL 22642  EU329522.1  DQ246844.1  DQ094329.1  Fusarium sp. (FSSC14)  NRRL 22611  EU329518.1  DQ246841.1  DQ094326.1  Fusarium sp. (FSSC33)  NRRL 22354  EU329504.1  AF178338.1  AF178402.1  Fusarium sp. (FSSC6)  NRRL 43489  DQ790572.1  DQ790484.1  DQ790528.1  Fusarium sp. (FSSC6)  NRRL 44892  GU170601.1  GU170618.1  GU170638.1  Fusarium sp. (FSSC6)  F201135  KM520374.1  KM527108.1  KM527100.1  Fusarium sp. (FSSC6)  F201334  KM520376.1  KM527110.1  KM527102.1  Fusarium sp. (FSSC6)  NRRL 43717  EF470233.1  FJ240356.1  EU329688.1  Fusarium sp. (FSSC6)  NRRL 44904  GU170586.1  GU170621.1  GU170641.1  Fusarium sp. (FSSC6)  CBS 135789  KU604374.1  KU711773.1  KR071699.1  F. falciforme (FSSC 3+4)  NRRL 43441  DQ790566.1  DQ790478.1  DQ790522.1  Fusarium sp. (FSSC20)  NRRL 22608  EU329517.1  DQ246838.1  DQ094323.1  Fusarium sp. (FSSC24)  NRRL 22389  EU329506.1  AF178340.1  DQ094314.1  F. solani f.sp. pisi MPVI (FSSC1)  NRRL 22278  EU329501.1  AF178337.1  DQ094309.1  F. solani f.sp. robiniae MPVII (FSSC13)  NRRL 22161  EU329494.1  AF178330.1  DQ094311.1  F. solani f.sp. mori MPIII (FSSC17)  NRRL 22157  EU329493.1  AF178359.1  DQ094306.1  F. ambrosium (FSSC19)  NRRL 20438  JX171584.1  AF178332.1  DQ094315.1  F. solani f.sp. xanthoxyli  NRRL 22163  EU329496.1  AF178328.1  AF178394.1  F. solani f.sp. batata  NRRL 22400  EU329509.1  AF178343.1  DQ094303.1  F. pseudensiforme  NRRL 46517  KC691674.1  KC691555.1  KC691584.1  F. striatum (FSSC21)  NRRL 22101  EU329490.1  AF178333.1  AF178398.1  Fusarium sp. (FSSC34)  NRRL 46703  EU329661.1  HM347126.1  EU329712.1  Fusarium sp. (FSSC30)  NRRL 22579  EU329515.1  AF178352.1  AF178415.1  Fusarium sp. (FSSC31)  NRRL 22570  EU329513.1  AF178360.1  AF178422.1  Fusarium sp. (FSSC32)  NRRL 22178  EU329498.1  AF178334.1  DQ094313.1  F. solani’ f.sp. cucurbitae MPI (FSSC10)  NRRL 22153  EU329492.1  AF178346.1  DQ094302.1  F. brasiliense (FSSC) clade2  NRRL 22743  EU329507.1  AF178341.1  AF178405.1  F. illudens (FSSC) clade1  NRRL 22090  JX171601.1  AF178326.1  AF178393.1  F. staphyleae (Outgroup)  NRRL 22316  JX171609.1  AF178361.1  AF178423.1  N. croci  CBS 142423  LT746329  LT746216  LT746264  N. macrospora  CBS 142424  LT746331  LT746218  LT746266  View Large Physiology Cardinal growth temperatures were determined on 2% MEA for CBS 135789. Plates were incubated in the dark for 1 week at temperatures of 21–36°C at intervals of 3°C; growth was also recorded at 37°C and at 40°C. DNA amplification and sequencing The following genes were amplified directly from the genomic DNA for multilocus sequence typing, using internal transcribed spacer region (ITS),26 elongation factor 1 alpha (TEF1),27 and the second largest subunit of RNA polymerase (rPB2).28 Polymerase chain reaction (PCR) amplification and sequencing were performed according to a protocol applied by Al-Hatmi et al.29 Phylogenetic inference To confirm the identity of our new Fusarium species, we evaluated its position in Bayesian phylogenetic and RAxML trees of the following individual gene markers (ITS, TEF1, and rPB2). Comparative sequences were retrieved from GenBank (Table 1) were analyzed together. Sequences were aligned with the program MAFFT (www.ebi.ac.uk/Tools/msa/mafft/), followed by manual adjustments with MEGA v6.2 and BioEdit v7.0.5.2. A single alignment was constructed for ITS, TEF1, and rPB2. The analysis included 48 sequences for ITS, TEF1 and rPB2. The best-fit model of evolution was determined by ModelTest v0.1.1. The trees were constructed by the outgroup method using the CIPRES Science Gateway online version30 and edited in MEGA v6.2. Sequences included in this study were retrieved from GenBank NRRL 22316 F. staphyleae was used as outgroup. Genetic relationships were investigated by phylogenetic analysis using Bayesian inference (BI) and maximum likelihood (ML) methods. Antifungal susceptibility Antifungal susceptibility testing (AFST) of CBS 135789 was performed by the CLSI broth microdilution as described in the CLSI document M38-A2 (Clinical and Laboratory Standards Institute 2008) with modification as described by Al-Hatmi et al.31 The following drugs were used, amphotericin B (Sigma-Aldrich), fluconazole (Pfizer, Groton, CT, USA), itraconazole (Janssen Pharmaceutica, Tilburg, The Netherlands), voriconazole (Pfizer), posaconazole (Merck), isavuconazole (Basilea Pharmaceutica, Basel, Switzerland), micafungin (Astellas, Ibaraki, Japan), and anidulafungin (Pfizer). Three reference strains (Paecilomyces variotii ATCC 22319, Candida krusei ATCC 6258, and Candida parapsilosis ATCC 22019) were included as quality controls. Results BLAST results of the TEF1 and rPB2 sequences in GenBank revealed that CBS 135789 matched FSSC6 strain (NRRL 43489, F201135, F201334, NRRL 44892, NRRL 44904, and NRRL 43717) with 99% similarity. For further understanding of relations between species, a phylogenetic tree was constructed for each locus separately, that is, ITS, TEF1, and rPB2. Each gene was analyzed separately prior to multilocus analysis. The cladistic analysis was based on sequences of the ITS, TEF1, and rPB2 regions. In gene trees of ITS, TEF1, and rPB2 separately, strain CBS 135789 was found as a member of a monophyletic clade supported by high bootstrap values (data not shown). No major topological variations were detected between trees derived from ML and BI phylogenetic inferences. Trees with identical overall topologies and resolving a monophyletic FSSC6 as shown in the Bayesian inference (BI) and maximum likelihood (ML) trees were encountered in all of the 100 ML inferences and in the BI and ML bootstrap consensus trees. All clades had statistical support between 70 and 100%, and all species were well separated. Intraspecific polymorphism within FSSC6 clusters was observed with ITS, TEF1, and rPB2. The FSSC6 clade received highest possible support in a multilocus study including strains CBS 135789, NRRL 43489, F201135, F201334, NRRL 44892, NRRL 44904, and NRRL 43717 (100% bs/ 1 pp) (Fig. 1). Analysis of individual gene ITS, TEF1, and rPB2 genealogies showed that CBS 135789 was nested within FSSC6 in clade 3, which is known to accommodate opportunistic human or animal pathogens. The final combined analysis of the three mentioned loci data sets encompassed 150 sequences representing 42 taxa and sequence type (STs) and comprised 2061 bp (ITS 497 bp, TEF1 701 bp, and rBP2 863 bp) (Fig. 1) and with F. staphyleae (NRRL 22316) as the outgroup. Figure 1. View largeDownload slide Maximum-likelihood phylogeny of Fusarium solani complex inferred from combined data set (ITS, TEF1, rPB2). Values in the nodes are Maximum-likelihood bootstrap supports (right) and Bayesian posterior probabilities (left). Numbers on the nodes are RaxML bootstrap values above 70% and Bayesian posterior probability values above 0.7. The tree is rooted to NRRL 22316, F. staphyleae. The colored clades are the species that are reported so far from human infection. This Figure is reproduced in color in the online version of Medical Mycology. Figure 1. View largeDownload slide Maximum-likelihood phylogeny of Fusarium solani complex inferred from combined data set (ITS, TEF1, rPB2). Values in the nodes are Maximum-likelihood bootstrap supports (right) and Bayesian posterior probabilities (left). Numbers on the nodes are RaxML bootstrap values above 70% and Bayesian posterior probability values above 0.7. The tree is rooted to NRRL 22316, F. staphyleae. The colored clades are the species that are reported so far from human infection. This Figure is reproduced in color in the online version of Medical Mycology. Taxonomy   Fusarium metavorans Al-Hatmi, S.A. Ahmed and de Hoog, sp. nov. – Fig. 2. MycoBank MB 821742. Figure 2. View largeDownload slide Morphological description of Fusarium metavorans CBS 135789. (A) Growth on MEA agar, obverse, reverse white, reverse yellowish to light orange; (B) Colony characteristics on PDA white to yellowish-white; (C–D). The production of microconidia in false heads on phialides formed on the hyphae single; (E–I) Monophialides with false head and microconidia; (J) Microconidia, abundant and ovoidal; (K) Chlamydospores within hyphae (intercalary) or at hyphal tips (terminal). Scale bar = 10 μm. This Figure is reproduced in color in the online version of Medical Mycology. Figure 2. View largeDownload slide Morphological description of Fusarium metavorans CBS 135789. (A) Growth on MEA agar, obverse, reverse white, reverse yellowish to light orange; (B) Colony characteristics on PDA white to yellowish-white; (C–D). The production of microconidia in false heads on phialides formed on the hyphae single; (E–I) Monophialides with false head and microconidia; (J) Microconidia, abundant and ovoidal; (K) Chlamydospores within hyphae (intercalary) or at hyphal tips (terminal). Scale bar = 10 μm. This Figure is reproduced in color in the online version of Medical Mycology. Etymology: named after the unspecific feeding pattern involving plants, insects and humans. Holotype: CBS 135789 preserved in metabolically inactive condition under liquid nitrogen; living derived from type CBS 135789, pleural effusion of patient with lung cancer, Athens, Greece. Description based on colonies of CBS 135789 grown for 10 d at 25°C on PDA, MEA, and CLA under 12h/12h alternating cool white fluorescent light / darkness. Colonies on MEA reaching 65 mm diam, growing rapidly with average daily growth rates of 5.3–6.2 mm. Obverse with cottony aerial mycelium, reverse yellowish to light orange (Fig. 2A). Colonies on PDA reaching 60 mm diam, average daily growth rate 4.9–5.5 mm. Obverse white to reddish-white at the edge (Fig. 2B), reverse pigmentation in a gradient from pale yellow at the margin to dark brown to red at the center. Conidia formed at colony surface and in the aerial mycelium, the latter in clear, translucent drops of liquid at the tips of long, unbranched conidiophores. Colonies on SNA reaching 70–80 mm diam. Obverse aerial mycelium cottony, white, dense near the center, nearly absent toward the margin. Conidia formed abundantly from erect, typically unbranched conidiophores at the agar surface. Microconidia in false heads on phialides formed on hyphae (Fig. 2C, D). Conidiophores 21.5–118.5 μm (mean 68.9 μm), 3.3 μm wide at the base, unbranched or branched up to three times, straight, thin-walled, smooth; each branch terminating in a single phialide. Phialidic collarettes thickened, flared (Fig. 2E–I). Conidia 0–1 septate, ellipsoidal with rounded apex and truncate base, 3.9–11.1 × 2.3–3.1 μm (Fig. 2I). Chlamydospores appearing after 4 weeks of incubation, single or in chains, consisting of enlarged, thick-walled vegetative cells, within hyphae (intercalary) or at hyphal tips (terminal), 6.0–13.4 μm diam (Fig. 2J). Notes.—Fusarium metavorans was isolated from human pleura of lung cancer patient in Greece. However, this fungus is also associated with plants and ants, which might suggest some level of diversity and geographically widely distributed species. It was considered as a human opportunist, causing superficial and disseminated infections. Fusarium metavorans is morphologically similar to F. solani sensu stricto (FSSC5).13 However, the new species differs mainly by its coloured brown to red pigmentations. Also shows similar micro-morphological characteristics to F. solani sensu stricto (FSSC5) but differs in the absence of macroconidia and the longer conidial chains with up to 10–20 micro-conidia in the terminal branched and unbranched with light and increase size of its conidiophores and phialides. The consensus morphology associated with F. metavorans corresponds well with other species in F. solani species complex. In addition, genetically very well differentiated as trees with identical overall topologies and resolving a monophyletic F. metavorans as shown in the Maximum likelihood and MrBayes consensus tree (Fig. 1) were encountered in all (100% bs/ 1 pp). Physiology Cardinal growth temperatures of F. metavorans type strain CBS 135789: optimal development at (27−)33°C, growth observed in the entire range between 21 and 37°C. Maximum growth temperature of the strain analyzed was 37°C. No growth observed at 40°C. Antifungal susceptibility Antifungal susceptibility testing performed with broth microdilution according to CLSI M38A resulted in the following MICs: amphotericin B 2 μg/ml; fluconazole >64 μg/ml; both posaconazole and voriconazole >4 μg/ml; itraconazole and isavuconazole >16 μg/ml; anidulafungin and micafungin >8 μg/ml. Discussion The taxonomy of Fusarium is evolving. Unlike many other filamentous fungi, only few phenotypic characteristics are available to differentiate Fusarium species. Many of the morphological characteristics described to differentiating the 300 known species overlap, interfering with identification but also species delimitation in Fusarium. Cryptic, molecularly defined species exist, which are morphologically identical.32 Therefore, sequence-based characteristics are essential to confirm the species identities.7,25 Also the genus level is debated. Lombard et al.33 reclassified the F. solani complex into the genus Neocosmospora, a decision opposing the consensus paper of Geiser et al.,9 supported by many authors from clinical and phytopathological backgrounds, with a plea for nomenclatural stability and preservation of the name Fusarium. Lombard's paper has been followed by only few authors; with the result that now confusingly two names exist for the F. solani complex.34 Recently, Sandoval-Denis et al.,35 described two new species within “FSSC,” named as Neocosmospora macrospora and Neocosmospora croci. However, to preserve the nomenclature stability, in the present article we adhere to Fusarium as the best-known descriptor for the etiological agents and the clinical entities. Members of the Fusarium solani species complex (FSSC) are the most commonly encountered fusaria in human infections. The clinical spectrum encompasses skin, nail, eye, bone, nasal cavities, contaminated wounds, and disseminated infection including in patients with cancer.11,25,36 The species complex accounts for approximately 60% of the total Fusarium infections, judging from literature supported by multilocus sequence analysis.37 Phylogenetically FSSC has been suggested to include 60 narrowly defined phylogenetic species, associated in three main clades: Clade 1, Clade 2, and Clade 3.8,9 Distinct morphological traits are minimal or absent between these clades. Clade 3 is known to accommodate opportunistic pathogens on humans, animals, and plants.8 Clade 3 also contains the type of F. solani sensu stricto (FSSC5).13 Seven of the most common species which occur on human hosts in Clade 3 have been formally named: F. falciforme (FSSC 3+4), F. keratoplasticum (FSSC2), F. lichenicola (FSSC16), F. petroliphilum (FSSC1), F. pseudensiforme, F. solani sensu stricto (FSSC5) and F. metavorans (FSSC6) is officially introduced in this study.12,13,19,20,36 In addition, FSSC27, (Phialophora cyanescens = Cylindrocarpon cyanescens), was recently recombined as Neocosmospora cyanescens.22 Previous studies have consistently revealed the monophyletic group described in this paper within FSSC with strains from the United States,8,11 Colombia,32 Japan,23 Turkey,38 and The Netherlands (unpublished data), all papers demonstrating an association with human infection in lineage FSSC6 (Fusarium metavorans). Sequences of ITS, TEF1, and rBP2 derived from strain CBS 135789, originating from a disseminated infection, matched with STs known as FSSC6 in public DNA sequence repositories. In a comparative analysis of 50 sequences representing 42 Fusarium species and STs of FSSC, it was shown that CBS 135789 was different from all these but showed 100% identity with strains NRRL 43489, F201135, F201334, NRRL 44892, NRRL 44904, and NRRL 43717 of FSSC6; the group formed a strongly supported monophyletic group (100% bs/ 1 pp) (Fig. 1). It was sister to F. solani f. sp. mori clade, a host specific pathogen on mulberry.39 Phylogenetic analysis of the conserved gene region ITS placed F. metavorans unambiguously in the FSSC, but ITS does not provide sufficient resolution at the species level, as holds true for many Fusarium species.6 Using partial coding genes TEF1 and RPB2, F. metavorans was easily distinguished from all described Fusarium species within FSSC. Fusarium metavorans is morphologically similar to other Fusarium species of FSSC that occur on human patients.21 It produces microconidia from false heads on phialides formed on hyphal conidiophores (Fig. 2C, D). A number of aberrant morphological variants were observed in isolate CBS 135789 of F. metavorans concerning the production of yellowish-white pigments on PDA. Fusarium metavorans does not produce macroconidia but instead produces septate or non-septate conidia from long mycelial conidiophores, which is typical for species in FSSC. The monophialides and the production of ovoidal microconidia, presence of chlamydospores and false microconidial head are observed in all species within FSSC. Fusarium metavorans (FSSC6) has been encountered in areas of research different from human infection, including quarantine, plant, soil, and the gut of the wood-boring cerambycid beetle Anoplophora glabripennis in the United States.40 This suggests that the species likely is a saprobe on virgin, watery substrates. In other fungal genera with plant-pathogenic members, such as Alternaria, it was noted that the human opportunists are just the species with predominantly saprobic life styles.41 Chemical analyses have demonstrated that F. metavorans is able to degrade lignocellulose,42 synthesize amino acids,43 and contribute to the synthesis of sterols.44 We find that our new species is capable of growth at 37°C and can thus survive at human body temperature. Antifungal susceptibility profiles obtained from F. metavorans showed remarkable similarity to other Fusarium species4,5 with regard to high degrees of resistance to all antifungals tested. In this study, the description of a novel species involved in opportunistic plant, animal and human infections has enlarged the spectrum of species of potential clinical interest in the genus Fusarium. The intrinsic broad spectrum antifungal resistance of Fusarium species has also been confirmed in F. metavorans. Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and the writing of the paper. References 1. O’Donnell K, Ward TJ, Robert VARG, Crous PW, Geiser DM, Seogchan K. DNA sequence-based identification of Fusarium: current status and future directions. Phytoparasitica . 2015; 43: 583– 595. Google Scholar CrossRef Search ADS   2. Moussa TAA, Al-Zahrani HS, Kadasa NMS, Ahmed SA, de Hoog GS, Al-Hatmi AMS. Two new species of the Fusarium fujikuroi species complex isolated from the natural environment. Antonie van Leeuwenhoek . 2017; 110: 819– 832. Google Scholar CrossRef Search ADS PubMed  3. Milicevic D, Skrinjar M, Baltic T. Real and perceived risks for mycotoxin contamination in foods and feeds: challenges for food safety control. Toxins . 2010; 2: 572– 592. Google Scholar CrossRef Search ADS PubMed  4. Al-Hatmi AMS, Meis JF, de Hoog GS. Fusarium: molecular diversity and intrinsic drug resistance. PLoS Pathog . 2016; 12: e1005464. Google Scholar CrossRef Search ADS PubMed  5. Al-Hatmi AM, Bonifaz A, Ranque R, de Hoog GS, Verweij PE, Meis JF. Current antifungal treatment of fusariosis. Int J Antimicrob Agents . 2017; http://dx.doi.org/doi:10.1016/j.ijantimicag.2017.06.017. 6. Geiser DM, Jimenez-Gasco MD, Kang SC et al.   FUSARIUM-ID v. 1.0: a DNA sequence database for identifying Fusarium. Eur J Plant Pathol.  2004; 110: 473– 479. Google Scholar CrossRef Search ADS   7. O’Donnell K, Rooney AP, Proctor RH et al.   Phylogenetic analyses of RPB1 and RPB2 support a middle cretaceous origin for a clade comprising all agriculturally and medically important fusaria. Fungal Genet Biol . 2013; 52: 20– 31. Google Scholar CrossRef Search ADS PubMed  8. O’Donnell K, Sutton DA, Fothergill A et al.   Molecular phylogenetic diversity, multilocus haplotype nomenclature, and in vitro antifungal resistance within the Fusarium solani species complex. J Clin Microbiol . 2008; 46: 2477– 2490. Google Scholar CrossRef Search ADS PubMed  9. Geiser DM, Aoki T, Bacon CW et al.   One fungus, one name: defining the genus Fusarium in a scientifically robust way that preserves longstanding use. Phytopathology . 2013; 103: 400– 408. Google Scholar CrossRef Search ADS PubMed  10. O’Donnell K, Nirenberg HI, Aoki T, Cigelnik E. A multigene phylogeny of the Gibberella fujikuroi species complex: detection of additional phylogenetically distinct species. Mycoscience . 2000; 1: 61– 78. Google Scholar CrossRef Search ADS   11. Zhang N, O’Donnell K, Sutton DA et al.   Members of the Fusarium solani species complex that cause infections in both humans and plants are common in the environment. J Clin Microbiol  2006; 44: 2186– 2190. Google Scholar CrossRef Search ADS PubMed  12. Nalim FA, Samuels GJ, Wijesundera RL, Geiser DM. New species from the Fusarium solani species complex derived from perithecia and soil in the Old World tropics. Mycologia . 2011; 103: 1302– 1330. Google Scholar CrossRef Search ADS PubMed  13. Schroers HJ, Samuels GJ, Zhang N, Short DP, Juba J, Geiser DM. Epitypification of Fusisporium (Fusarium) solani and its assignment to a common phylogenetic species in the Fusarium solani species complex. Mycologia . 2016; 108: 806– 819. Google Scholar CrossRef Search ADS PubMed  14. Matuo T, Snyder WC. Use of morphology and mating populations in the identification of formae speciales in Fusarium solani. Phytopathology . 1973; 63: 562– 565. Google Scholar CrossRef Search ADS   15. Toussoun TA, Snyder WC. The pathogenicity, distribution and control of two races of Fusarium (Hypomyces) solani f.sp. cucurbitae. Phytopathology.  1961; 51: 17– 22. 16. Suga HT, Hasegawa H, Mitsui KK, Hyakumachi M. Phylogenetic analysis of the phytopathogenic fungus Fusarium solani based on the rDNA-ITS region. Mycol Res . 2000; 104: 1175– 1183. Google Scholar CrossRef Search ADS   17. Chung WC, Chen LW, Huang JH, Chung H. A new “forma specialis” of Fusarium solani causing leaf yellowing of Phalaenopsis: Leaf yellowing of Phalaenopsis. Plant Pathol . 2011; 60: 244– 252. Google Scholar CrossRef Search ADS   18. Bueno CJ, Fischer IH, Rosa DD et al.   Fusarium solani f. sp. passiflorae: a new forma specialis causing collar rot in yellow passion fruit. Plant Pathol . 2014; 63: 382– 389. Google Scholar CrossRef Search ADS   19. Summerbell R, Schroers H-J. Analysis of phylogenetic relationship of Cylindrocarpon lichenicola and Acremonium falciforme to the Fusarium solani species complex and a review of similarities in the spectrum of opportunistic infections caused by these fungi. J Clin Microbiol . 2002; 40: 2866– 2875. Google Scholar CrossRef Search ADS PubMed  20. Short DPG, O’Donnell K, Thrane U et al.   Phylogenetic relationships among members of the Fusarium solani species complex in human infections and the descriptions of F. keratoplasticum sp. nov. and F. petroliphilum stat. nov. Fungal Genet Biol . 2013; 53: 59– 70. Google Scholar CrossRef Search ADS PubMed  21. de Hoog GS, Guarro J, Gené J, Figueras MJ. Atlas of Clinical Fungi , 3rd ed. Utrecht, Netherlands: Centraalbureau voor Schimmelcultures, 2011. 22. Summerbell RC, Scott JA. Conidiogenesis: its evolutionary aspects in the context of a philosophy of opportunity (lectics). In: Li DW, eds. Biology of Microfungi . London: Springer Science, 2016: 169– 195. Google Scholar CrossRef Search ADS   23. Muraosa Y, Oguchi M, Yahiro M, Watanabe A, Yaguchi T, Kamei K. Epidemiological study of Fusarium species causing invasive and superficial fusariosis in Japan. Med Mycol J . 2017; 58: E5– E13. doi: 10.3314/mmj.16-00024. Google Scholar CrossRef Search ADS PubMed  24. Nirenberg HI. Studies on the morphologic and biologic differentiation in Fusarium section Liseola. Mitt Biol Bundesanst Land- Forstw Berlin-Dahlem . 1976; 169: 1– 117. 25. Leslie JF, Summerell BA. The Fusarium Laboratory Manual . Oxford: Blackwell, 2007. 26. White TJ, Bruns T, Lee S, Taylor J. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Gelfand Innis D., Sninsky J., White T, eds., PCR Protocols: A Guide to Methods and Applications . New York: Academic Press, 1990: 315– 322. Google Scholar CrossRef Search ADS   27. O’Donnell K, Sutton DA, Rinaldi MG et al.   Internet-accessible DNA sequence database for identifying Fusaria from human and animal infections. J Clin Microbiol . 2010; 48: 3708– 3718. Google Scholar CrossRef Search ADS PubMed  28. Reeb V, Lutzoni F, Roux C. Contribution of RPB2 to multilocus phylogenetic studies of the euascomycetes (Pezizomycotina, fungi) with special emphasis on the lichen-forming Acarosporaceae and evolution of polyspory. Mol Phylogenet Evol . 2004; 32: 1036– 1060. Google Scholar CrossRef Search ADS PubMed  29. Al-Hatmi AMS, Mirabolfathy M, Hagen F et al.   DNA barcoding, MALDI-TOF and AFLP data support Fusarium ficicrescens as a distinct species within the F. fujikuroi species complex. Fungal Biol . 2016; 120: 265– 278. Google Scholar CrossRef Search ADS PubMed  30. Miller MA, Pfeiffer W, Schwartz T. The CIPRES science gateway: a community resource for phylogenetic analyses. In Proceedings of the 2011 TeraGrid Conference: Extreme Digital Discovery . Salt Lake City, UT, USA: ACM 2011: 1– 8. 31. Al-Hatmi AM, van Diepeningen AD, Curfs-Breuker I, de Hoog GS, Meis JF. Specific antifungal susceptibility profiles of opportunists in the Fusarium fujikuroi complex. J Antimicrob Chemother . 2015; 70: 1068– 1071. Google Scholar PubMed  32. Guevara-Suarez M, Cano-Lira JF, de Garcia MCC et al.   A genotyping of Fusarium isolates from onychomycosis in Colombia: detection of two new species within the Fusarium solani species complex and in vitro antifungal susceptibility testing. Mycopathologia . 2016; 181: 165– 174. Google Scholar CrossRef Search ADS PubMed  33. Lombard L, van der Merwe NA, Groenewald JZ, Crous PW. Generic concepts in Nectriaceae. Stud Mycol . 2015; 80: 189– 245. Google Scholar CrossRef Search ADS PubMed  34. Aoki T, O’Donnell K, Geiser DM. Systematics of key phytopathogenic Fusarium species: current status and future challenges. Plant Pathol . 2014; 80: 189– 201. 35. Sandoval-Denis M, Guarnaccia V, Polizzi G, Crous PW. Symptomatic citrus trees reveal a new pathogenic lineage in Fusarium and two new Neocosmospora species. Persoonia . 2018; 40: 1– 25. 36. Al-Hatmi AMS, Hagen F, Menken SBJ, Meis JF, de Hoog GS. Global molecular epidemiology and genetic diversity of Fusarium, a significant emerging human opportunist from 1958–2015. Emerg Microbes Infect . 2016; 5: e33; doi:10.1038/emi.2016.126. Google Scholar CrossRef Search ADS PubMed  37. Alfonso EC, Rosa RH. Fungal Keratitis. In: Krachmer JH, Mannis MJ, Holland EJ, eds. Cornea and External Diseases: Clinical Diagnosis and Management . St Louis: Mosby, 1997: 1253– 1266. Google Scholar CrossRef Search ADS   38. Dalyan Cilo B, Al-Hatmi AM, Seyedmousavi S et al.   Emergence of fusarioses in a university hospital in Turkey during a 20-year period. Eur J Clin Microbiol Infect Dis.  2015; 34: 1683– 1691. Google Scholar CrossRef Search ADS PubMed  39. Sakurai Y, Matuo T. On the form name and race of Hypomyces solani (Rke. et Berth.) Snyd. et Hans. which is pathogenic to the mulberry trees. Ann Pytopathol Soc Japan . 1959; 24: 219– 223. Google Scholar CrossRef Search ADS   40. Herr JR, Scully ED, Geib SM, Hoover K, Carlson JE, Geiser DM. Genome sequence of the fungus FSSC6, a Fusarium species (MYA-4552) isolated from the midgut of Anoplophora glabripennis, an invasive, wood-boring beetle. Genome Announc . 2016; 4: e00544– 16. Google Scholar CrossRef Search ADS PubMed  41. Van Baarlen P, Van Belkum A, Summerbell RC, Crous PW, Thomma BPHJ. Molecular mechanisms of pathogenicity: how do pathogenic microorganisms develop cross-kingdom host jumps? FEMS Microbiol Rev . 2007; 31: 239– 277. Google Scholar CrossRef Search ADS PubMed  42. Scully ED, Geib SM, Hoover K et al.   Metagenomic profiling reveals lignocellulose degrading system in a microbial community associated with a wood-feeding beetle. PLoS One . 2013; 8: e73827. doi:10.1371/journal.pone.0073827 Google Scholar CrossRef Search ADS PubMed  43. Ayayee PA, Larsen T, Rosa C, Felton GW, Ferry JG, Hoover K. Essential amino acid supplementation by gut microbes of a wood-feeding cerambycid. Environ Entomol . 2016; 45: 66– 73. doi:10.1093/ee/nvv153. Google Scholar CrossRef Search ADS PubMed  44. Scully ED, Geib SM, Carlson JE. Functional genomics and microbiome profiling of the Asian longhorned beetle (Anoplophora glabripennis) reveal insights into the digestive physiology and nutritional ecology of wood feeding beetles. BMC Genomics . 2014; 15: 1096. Google Scholar CrossRef Search ADS PubMed  © The Author 2017. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

Journal

Medical MycologyOxford University Press

Published: Apr 1, 2018

There are no references for this article.

You’re reading a free preview. Subscribe to read the entire article.


DeepDyve is your
personal research library

It’s your single place to instantly
discover and read the research
that matters to you.

Enjoy affordable access to
over 18 million articles from more than
15,000 peer-reviewed journals.

All for just $49/month

Explore the DeepDyve Library

Search

Query the DeepDyve database, plus search all of PubMed and Google Scholar seamlessly

Organize

Save any article or search result from DeepDyve, PubMed, and Google Scholar... all in one place.

Access

Get unlimited, online access to over 18 million full-text articles from more than 15,000 scientific journals.

Your journals are on DeepDyve

Read from thousands of the leading scholarly journals from SpringerNature, Elsevier, Wiley-Blackwell, Oxford University Press and more.

All the latest content is available, no embargo periods.

See the journals in your area

DeepDyve

Freelancer

DeepDyve

Pro

Price

FREE

$49/month
$360/year

Save searches from
Google Scholar,
PubMed

Create lists to
organize your research

Export lists, citations

Read DeepDyve articles

Abstract access only

Unlimited access to over
18 million full-text articles

Print

20 pages / month

PDF Discount

20% off