Mycologia, 95(4), 2003, pp. 630–636. q 2003 by The Mycological Society of America, Lawrence, KS 66044-8897 Fusarium commune is a new species identified by morphological and molecular phylogenetic data Kerstin Skovgaard1 Søren Rosendahl renberg and O’Donnell 1998). These cultures shared several morphological characteristics typically found in F. oxysporum, but they differed in that they produced polyphialides as well as long, slender monophialides. Morphological characters frequently are homoplastic, and the circumscription of taxa, based on the size and shape of conidia and conidiophores and the color and texture of colonies, has resulted in an underestimation of species diversity within Fusarium Link (Brayford 1996, O’Donnell 1996). Phylogenetic species recognition, based on DNA sequence data from multiple loci, allows greater numbers of species to be distinguished than in the exclusive use of morphological features (Taylor et al 2000). Based on morphological characters alone, between two and 10 taxa have been recognized in section Liseola (Booth 1971, Gerlach and Nirenberg 1982, Nelson et al 1983), four species in section Dlaminia (Kwasna et al 1991) and two species in section Elegans (Gerlach and Nirenberg 1982). Using multigene genealogies, O’Donnell et al (1998) recognized 36 species within the Gibberella fujikuroi Saw. complex, represented by part or all species of sections Liseola, Elegans and Dlaminia. Over the past half-decade, combined molecular phylogenetic and morphological approaches have been shown to be invaluable in the diagnosis of fusaria (Aoki and O’Donnell 1999, Aoki et al 2001, Gams et al 1999, Geiser et al 2001, Nirenberg and O’Donnell 1998, O’Donnell et al 1998). Evolutionary relationships among and within the F. oxysporum species complex have been investigated with multilocus DNA sequence data (Baayen et al 2000, Skovgaard et al 2001). Fusarium hostae Geiser, a species causing root and crown rot of hosta, recently was discovered as the putative sister taxon of F. redolens Wollenw., based on the analysis of partial b-tubulin and translation elongation factor 1a sequences (Baayen et al 2001, Geiser et al 2001). In this study we describe a new Fusarium species based on morphology and phylogenetic analysis of partial (EF-1a) and the mitochondrial small subunit ribosomal DNA (mtSSU rDNA) sequences. Department of Mycology, University of Copenhagen, Oester Farimagsgade 2D, 1353 Copenhagen K, Denmark Kerry O’Donnell Microbial Properties Research Unit, National Center for Agricultural Utilization Research, U.S. Department of Agriculture, Agricultural Research Service, 1815 N. University Street, Peoria, Illinois Helgard I. Nirenberg Institut für Pflanzenvirologie, Mikrobiologie und biologische Sicherheit, Biologische Bundesanstalt für Land- und Forstwirtschaft, Koenigin-Luise-Strasse 19, 14195 Berlin, Germany Abstract: Fusarium commune sp. nov. was isolated from soil and Pisum sativum in Denmark and several widespread locations within the northern hemisphere from diverse substrates including white pine, Douglas fir, carnation, corn, carrot, barley and soil. Fusarium commune is characterized by and distinguished from its putative sister taxon, the F. oxysporum complex, in having long, slender monophialides and polyphialides when cultured in the dark. Based on the combined DNA sequence data from translation elongation factor 1a (EF-1a) and the mitochondrial small subunit ribosomal DNA (mtSSU rDNA), the 15 isolates of F. commune analyzed formed a strongly supported clade closely related to but independent of the F. oxysporum and Gibberella fujikuroi species complexes. Key words: gene genealogies, hyphomycetes, mitochondrial small subunit rDNA, phylogeny, soil fungi, translation elongation factor a INTRODUCTION During a study of Fusarium oxysporum Schlect. emend. Snyder & Hansen occurring in pea fields in Denmark, several fusaria were isolated that could not be identified as any described species (Gerlach and Nirenberg 1982, Booth 1971, Nelson et al 1983, Ni- MATERIALS AND METHODS Strains used in this study are listed in TABLE I together with substrate and geographic origin. All isolates are stored in Accepted for publication March 13, 2003. 1 Corresponding author. E-mail: kerstins@bot.ku.dk 630 TABLE I. Strains of Fusarium used in this study Sourcea Species NRRL 22172 (BBA 62264) New Caledonia B.C., Canada OR, USA Ontario, Canada Ontario, Canada Finland Finland Japan Austria Austria The Netherlands Denmark Denmark Denmark Denmark Denmark SC, USA Australia Sweden Denmark Denmark — Denmark Denmark USA Soil Pseudotsuga menziesii (Mirb.) Frane Pseudotsuga menziesii Pinus strobes L. Pinus strobes Barley root (Hordeum vulgare L.) Carrot root (Daucus carota L.) River sediment Zea mays L. leaf Zea mays leaf Dianthus caryophyllus L. Pisum sativum L. Soil Soil Soil Soil Hosta sp. Musa sp. Pisum sativum Pisum sativum Soil — Pisum sativum Pisum sativum Zea mays Germany Zea mays SP. NOV. 25174 (FRC M-1425 5 BBA 65829) 22900 (BCRI P4C2P17A) 22903b (BCRI 3139) 25043 (BBA 69585) 25049 (BBA 69586) 26897 (ARCF 93144) 26898d (ARCF 94193 5 BBA 71641) 28058 (FRC 0-1173) 28180 (MA 1208) 28182 (MA 1210) 28387d (PD 90/1377) 31076bcd (AAS 156 5 BBA 71639) 31077d (AAS 345) 31079d (AAS 362) 31080 (AAS 363 5 BBA 71640) 31081 (AAS 364) 29889 (FRC 0-2074) 25603 (HCK A2) 31073 (Bødker L5) 31074 (AAS 112) 31078 (AAS 350) 22057 ( JFL 4853) 31075 (AAS 127) 31255 (AAS 120) 22016 ( JFL 2192) FUSARIUM COMMUNE, NRRL NRRL NRRL NRRL NRRL NRRL NRRL NRRL NRRL NRRL NRRL NRRL NRRL NRRL NRRL NRRL NRRL NRRL NRRL NRRL NRRL NRRL NRRL NRRL NRRL ET AL: beomiforme commune commune commune commune commune commune commune commune commune commune commune commune commune commune commune hostae oxysporum oxysporum oxysporum oxysporum proliferatum (Matsushima) Nirenb. redolens redolens subglutinans (Wollenw. & Reinking) Nelson et al. F. verticillioides (Sacc.) Nirenb. Host/Substrate SKOVGAARD F. F. F. F. F. F. F. F. F. F. F. F. F. F. F. F. F. F. F. F. F. F. F. F. F. Geographic origin a AAS, K. Skovgaard, Botanical Institute, Department of Mycology, University of Copenhagen, Copenhagen, Denmark; ARCF, T. Yli-Mattila, Agricultural Research Centre of Finland, Jokioinen, Finland; BBA, Institut für Pflanzenvirologie, Mikrobiologi und biologische Sicherheit, Biologische Bundesanstalt für Land- und Forstwirtschaft, Berlin, Germany; BCRI, British Columbia Research Inc., Vancouver, Canada; Bødker, L. Bødker, Danmarks JordbrugsForskning, Flakkebjerg, Denmark; FRC, Fusarium Research Center, Penn State University, University Park, USA; HCK, C. Kistler, USDA, St. Paul, USA; JFL, J. F. Leslie, Department of Plant Pathology, Kansas State University, Manhattan, USA; MA, Vienna Institute of Applied Microbiology (VIAM), Vienna, Austria; NRRL, National Center for Agricultural Utilization Research, Peoria, USA; PD, Plantenziektenkundige Dienst, Wageningen, The Netherlands. b Pathogenicity tested at BBA. c Ex-type. d Isolates examined. 631 632 MYCOLOGIA liquid nitrogen at the National Center for Agricultural Utilization Research, Peoria, IL (NRRL). The ex-type culture of Fusarium commune NRRL 31076 also is stored in soil vials and as freeze-dried cultures at the Institut für Pflanzenvirologie, Mikrobiologie und biologische Sicherheit, Biologische Bundesanstalt für Land- und Forstwirtschaft culture collection (BBA), Berlin, Germany, as well as in glycerol at280 C at the Botanical Institute, Department of Mycology, University of Copenhagen, Copenhagen, Denmark (AAS). Morphological examination. Cultures were grown on potato-dextrose agar (PDA; Difco, Detroit, Michigan) at 20 C in the dark. Colony colors were determined using the Methuen Handbook of Colour (Kornerup and Wanscher 1978). Microscopic characters were studied after 10 to 14 days’ growth on synthetic low nutrient agar (SNA) overlain with a 1 3 2 cm piece of sterile filter paper (Nirenberg 1990). Characteristic morphological traits were photographed and measurements of conidia, chlamydospores and phialides were made after the cultures were incubated either in the dark or under continuous black light (Philips TLD 18w/ 08) (Nirenberg 1990). At least 30 randomly selected 1-, 3and 5-septate conidia were measured and their mean values and ranges (shown in brackets) were determined. Pathogenicity test. Two isolates of Fusarium commune, NRRL 22903 and 31076, were tested for their pathogenicity toward Pinus sylvestris L. and Picea rubens Sarg. These conifer species are hosts of F. blasticola Rostr., a morphologically similar species initially thought to be conspecific with F. commune (see below). From 14-day-old SNA cultures of these two isolates, five 10 mm2 pieces were transferred separately to each of seven Erlenmeyer flasks filled with a sterile peat-straw-sand mixture (2:2:1). Each culture was allowed to grow through the mixture at 20 C. After 19 d the inoculated soil was mixed with a commercial soil, TKS 1 (Flora Gard, Berlin, Germany), and sand (2:3:1). The inoculum of each fungus was used to fill 20 plastic pots (9 cm in diameter). Eight-week-old seedlings of each of the conifer species were planted individually in 10 pots. Negative controls treated the same way but lacked fungi. The pots were placed in a greenhouse at 15 C to 18 C. After three months the temperature was raised to 25 C for the next two months. Symptoms were evaluated after five months’ incubation. Sequencing and phylogenetic analysis. Isolates were grown as shake cultures (200 rpm) in a yeast-malt broth (0.3% yeast extract, 0.3% malt extract, 0.5% peptone, 2% glucose) 2–3 days at room temperature. Genomic DNA was extracted from lyophilized mycelium by the CTAB-method (O’Donnell and Cigelnik 1997). Amplification and sequencing of the mtSSU rDNA and EF-1a genes was carried out employing the primers and thermocycling parameters described by White et al (1990) and O’Donnell et al (2000). PCR products were purified with GeneClean II (Bio 101, La Jolla, CA). Cycle sequencing products were spun through Sephadex G-50 columns (Pharmacia, Piscataway, NJ) to remove unincorporated dye-labeled nucleotides and sequenced on an automated ABI 377 sequencer (Perkin-Elmer, Foster City, CA). Sequencher 3.0 (GeneCodes, Ann Arbor, MI) and Bioedit (Hall 1999) were used to edit and align the sequence data. The final alignment is available through TreeBASE. Sequences were deposited in GenBank under accessions numbers AF362261 to AF362292. Sequence data from EF1a and mtSSU rDNA were tested for combinability with the partition homogeneity test implemented in PAUP 4.0b2 (Swofford 1999). Branch and bound searches were performed with default options. Alignment gaps were treated as missing data and 1000 parsimony bootstrap replications were conducted to test clade support. RESULTS Fusarium commune Skovgaard, O’Donnell et Nirenberg, sp. nov. FIGS. 1–6 Coloniae in PDA in dies radium 4.6–5.6 mm expandentes, temperatura 20 C in obscuritate. Mycelium aerium album usque ad aurantiaco-album, lanosum usque ad byssaceum. Color coloniarum in reverso griseo-fulvidus, nonnumquam griseo-violaceus. Odor insensibilis. Sporulatio in mycelio aerio praecox in SNA, conidia capitulis aggregata. Sporodochia post 10 dies formata sub luce nigra continua. Conidiophora in mycelio aerio prostrata. Phialides cylindricae pro parte maxima monophialidicae nonnumquam polyphialidicae; monophialides seu breves (ca 17 mm) seu longiores (ad 60 mm), 3.5–4.0 mm latae; polyphialides not magis quam 30 mm longae. Conidia in mycelio aerio oblonge ovalia, recta vel curvata, plerumque non-septata, aliquando 1- vel 2septata, conidia non-septata: (4.0–) 5.5–7.7 (-8.0) 3 (2.0–) 2.5–3.5 (–4.0) mm; conidia sporodochialia plerumque falcata et 3-septata: (22–) 24–30 (–38) 3 3.8–4.1 mm obscuritate, (32–) 34–42 (–50) 3 3.8–4.2 mm sub luce nigra continua; chlamydosporae 8–12 mm diam, leves, singulares vel binae. Teleomorphosis ignota. Holotypi origo geographica in Dania, in humo. Ex holotypo culturae NRRL 31076 5 BBA 71639 5 AAS 156. HOLOTY PUS. Colonia sicca BBA 71639, deposita in herb. B. Colonies with a radial growth rate of 5.1 mm per day on PDA at 20 C in the dark. Aerial mycelium white to orange white, generally abundant, densely floccose to fluffy, later resupinate in degenerated cultures. Colony reverse grayish yellow with magenta to dull violet pigmentation, often in rings. The grayish magenta was dominant in older and degenerate cultures. Odor not detectable. Sporulation starting early in aerial mycelium, abundant after 10 days on SNA. 0-septate conidia produced in slimy droplets and sporodochia, typically formed after 10 days under continuous black light. Conidiophores consisting of short monophialides up to 17 mm long and 4.0 mm wide, or longer and more slender monophialides up SKOVGAARD ET AL: FUSARIUM COMMUNE, SP. NOV. 633 FIGS. 1–6. Fusarium commune (Scale bar 5 25 mm). 1. Polyphialidic conidiophore. 2. Polyphialidic and monophialidic conidiophores of the aerial mycelium. 3. Polyphialidic and long monophialidic conidiophores of the aerial mycelium. 4. Long sporodochial conidia. 5. Chlamydospores. 6. Sporodochial conidia. Figs. 1–3, 5 from cultures maintained in the dark, Figs. 4, 6 from cultures maintained under continuous black light. to 60 mm long and 3.5 mm wide. Polyphialides up to 30 mm long and 3.5 mm wide, appearing in cultures incubated in the dark. Aerial conidia mostly 0-septate, cylindrical, straight to slightly curved, measuring (4.0–) 5.5–7.7 (–8.0) 3 (2.0–) 2.5–3.5 (–4.0) mm. Conidia borne in sporodochia typically fusiform with a slightly curved apical cell and a foot-shaped basal cell, bending equally toward both ends. Three-sep- tate conidia (22–) 24–30 (–38) 3 3.8–4.1 mm formed in the dark and under continuous black light (32–) 34–42 (–50) 3 3.8–4.2 mm, five-septate conidia under continuous black light (52–) 56–60 (–64) 3 (3.8–) 3.9–4.6 (–5.0) mm. Chlamydospores 8–12 mm diam, smooth, intercalary or terminal, and produced singly or in pairs. Cardinal temperatures: 7.5 C, 27.5 C and 35.0 C. Teleomorph unknown. 634 MYCOLOGIA FIG. 7. Single most-parsimonious phylogram based on the combined analysis of EF-1a and mtSSU rDNA gene sequences. Bootstrap frequencies from 1000 replications are given above nodes. Consistency index 5 0.80, retention index 5 0.89, tree length 5 285.ol6 Sequence data. Two substitutions were found within the alignment of EF-1a (572 bp) and mtSSU rDNA (698 bp) from the 15 isolates of F. commune. Results of the partition homogeneity test indicated that the EF-1a and mtSSU rDNA partitions could be combined (P 5 0.267). A branch and bound search of the combined dataset, rooted with sequences of F. beomiforme Nelson et al, yielded a single most-parsi- monious tree of 285 steps (FIG. 7, consistency index 5 0.80, retention index 5 0.89). The 15 isolates of F. commune formed a strongly supported monophyletic group (bootstrap 5 100%). Pathogenicity test. Disease symptoms were not observed after five months incubation of Pinus sylvestris SKOVGAARD ET AL: FUSARIUM COMMUNE, and Picea rubens with strains of F. commune (NRRL 22903 and 31076). SP. NOV. 635 (Ueno et al 1977, Marasas et al 1984), studies are in progress to elucidate the mycotoxin potential of F. commune. DISCUSSION Fusarium commune and F. oxysporum are morphologically similar in that they both produce conidia on short monophialides in false heads on the aerial mycelium and chlamydospores singly or in pairs. Unique features of F. commune include long, slender monophialides in addition to the occasional production of polyphialides. Ten of the 15 strains of F. commune used in this study were identified originally as F. oxysporum. Four of the 15 isolates studied (NRRL 22900, 25043, 28058, 31080) produced longer sporodochial conidia than the type isolate, and the other isolates in the dark and under black light. In these four strains, 3-septate conidia measured (36–) 40–50 (–52) 3 3.7–4.0 mm in the dark and (36–) 44–56 (–60) 3 3.8–4.1 mm under continuous black light; 5-septate conidia were (44–) 52–58 (–60) 3 3.9–4.1 mm under continuous black light. Little variation in EF-1a and mtSSU rDNA sequences was obser ved within F. commune, even though the 15 strains were isolated from a wide range of substrates and geographic locations throughout the northern hemisphere. No relationship was observed between the minor sequence differences in the F. commune clade and differences in macroconidial morphology. The molecular phylogenetic analysis identified F. commune as a putative sister group to the F. oxysporum complex, a result consistent with the high morphological similarity of these taxa (FIG. 7). Fusarium redolens as well as the newly described F. hostae (Geiser et al 2001) formed a sister group to the rest of the ingroup taxa. Fusarium commune was considered to be conspecific with F. blasticola (Wollenweber and Reinking 1935), a species isolated originally from seedlings of Pinus montana Lamarck and described as Fusoma parasitica Tub. (Tubeuf 1895). Although conidia were not observed in the type material of F. blasticola, the description of this species as possessing slender (3.5 mm) 3-septate conidia that occur rarely in sporodochia (Hartig 1892, Wollenweber and Reinking 1935) is inconsistent with our observations of F. commune. In addition, results of the infection tests indicate that F. commune is not pathogenic to seedlings of Pinus sylvestris and Picea rubens, two of the hosts of F. blasticola. Collectively, these results support the separation of F. commune and F. blasticola. The phytopathological role of F. commune on other hosts, if any, is still unknown. 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