sudden-death syndrome of soybean is caused by two

660

Mycologia, 95(4), 2003, pp. 660–684.q 2003 by The Mycological Society of America, Lawrence, KS 66044-8897

Sudden-death syndrome of soybean is caused by two morphologically andphylogenetically distinct species within the Fusarium solani species complex—

F. virguliforme in North America and F. tucumaniae in South America

Takayuki Aoki1,2

National Institute of Agrobiological Sciences, GeneticDiversity Department, 2-1-2 Kannondai, Tsukuba,Ibaraki 305-8602 Japan

Kerry O’DonnellMicrobial Genomics and Bioprocessing Research Unit,National Center for Agricultural Utilization Research,United States Department of Agriculture, AgriculturalResearch Service, Peoria, Illinois 61604-3999

Yoshihisa Homma1

Japan International Research Center for AgriculturalSciences, Biological Resources Division, 1-2 Ohwashi,Tsukuba, Ibaraki 305-8686 Japan

Alfredo R. Lattanzi1

Instituto Nacional de Tecnologıa Agropecuaria,Estacion Experimental Agropecuaria (INTA-EEA)Marcos Juarez, Casilla de Correo 21, 2580 MarcosJuarez, Cordoba, Argentina

Abstract: Soybean sudden-death syndrome has be-come a serious constraint to commercial productionof this crop in North and South America during thepast decade. To assess whether the primary etiologi-cal agent is panmictic in both hemispheres, morpho-logical and molecular phylogenetic analyses wereconducted on strains selected to represent the knownpathogenic and genetic diversity of this pathogen.Maximum-parsimony analysis of DNA sequencesfrom the nuclear ribosomal intergenic spacer regionand the single copy nuclear gene translation elon-gation factor 1-a, together with detailed morpholog-ical comparisons of conidial features, indicate thatSDS of soybean in North and South America iscaused by two phylogenetically and morphologicallydistinct species. Fusarium virguliforme sp. nov., for-mally known as F. solani f. sp. glycines, is describedand illustrated for the SDS pathogen in North Amer-ica, and F. tucumaniae sp. nov. is proposed for theSouth American pathogen. The molecular phyloge-netic results challenge the forma specialis naming sys-tem because pathogenicity to soybean might haveevolved convergently in F. tucumaniae and F. virguli-

Accepted for publication February 2, 2003.1 Collaborators via the JIRCAS research project, ‘‘Soybean improve-ment, production and utilization in South America’’2 Corresponding author. E-mail: [email protected]

forme. Phylogenetic evidence indicates the two SDSpathogens do not share a most recent common an-cestor, since F. tucumaniae was resolved as a sister toa pathogen of Phaseolus vulgaris, F. phaseoli comb.nov. All three pathogens appear to have evolutionaryorigins in the southern hemisphere since they aredeeply nested within a South American clade of theF. solani species complex.

Key words: Argentina, conidiogenesis, Fusariumphaseoli, Glycine max, Phaseolus vulgaris, SDS

INTRODUCTION

During the past decade, sudden-death syndrome(SDS) of soybean (Glycine max (L.) Merr.) hasreached epidemic proportions in North and SouthAmerica (Roy et al 1997, Wrather et al 1997). Firstdiscovered in Arkansas in 1972, this disease has be-come widespread in soybean-growing regions in theUnited States (Rupe et al 2001), Argentina (Ploper1993) and Brazil (Nakajima et al 1993). The etiolog-ical agent first was reported as Fusarium solani(Mart.) Sacc. (Rupe 1989, Roy et al 1989), but morerecently it has been described as F. solani f. sp. gly-cines (Roy 1997a) to emphasize its putative host spe-cialization. Although some recent taxonomic treat-ments of Fusarium Link (Nelson et al 1983, Burgesset al 1998) follow Snyder and Hansen (1941) in rec-ognizing F. solani as the only species within the infra-generic section Martiella Wollenw., van Etten and Kis-tler (1988) have emphasized that the seven matingpopulations (MPs) of Nectria haematococca Berk. &Broome (Sakurai and Matuo 1960, Matuo 1972, Ma-tuo and Snyder 1973) represent biologically distinctspecies. This latter view was supported by recent mo-lecular phylogenetic analyses on DNA sequences thatindicate the Martiella fusaria (i.e., the F. solani spe-cies complex) comprise at least 26 phylogeneticallydistinct species (O’Donnell 2000), most of whichhave not been described formally. Taxonomy of thisclade is further complicated because Neocosmospora isthe nomenclaturally and phylogenetically correct te-leomorph name for the F. solani complex (for a dis-cussion, see O’Donnell 2000).

Previous molecular and morphological analyseshave included only North American isolates of theSDS pathogen (O’Donnell and Gray 1995, Achen-

661AOKI ET AL: FUSARIUM CAUSE SOYBEAN SDS

bach et al 1996, O’Donnell 2000, Li et al 2000, Rupeet al 2001). All of the molecular data, based on DNAsequences (O’Donnell and Gray 1995, O’Donnell2000, Li et al 2000), random amplified polymorphicDNA (Achenbach et al 1996) and restriction frag-ment-length polymorphisms (Rupe et al 2001), indi-cate that North American isolates of the SDS patho-gen are genetically homogeneous and closely relatedto a root-rot pathogen of Phaseolus vulgaris L., F. so-lani f. sp. phaseoli (Burkh.) W.C. Snyder & H.N. Han-sen.

The objective of this study was to compare mor-phologically and molecularly North and South Amer-ican isolates of the soybean SDS pathogen using ge-nealogical concordance phylogenetic species recog-nition (Taylor et al 2000). Precise knowledge of aspecies’ limits and phylogeographic structure provideessential genetic data for tracking the intercontinen-tal movement of foreign pathogens associated withworld trade. Toward this end, South American iso-lates of the SDS pathogen were recovered during a2001 field survey in Argentina, where the disease hasbeen reported (Ivancovich et al 1992, Ploper 1993,Botta et al 1993). Based on detailed morphologicalcomparisons and molecular phylogenetic analyses onDNA sequences from multiple loci, the Argentineanand North American isolates of the SDS pathogenand the bean root-rot pathogen were resolved asthree distinct species within a South American cladeof the F. solani species complex.

MATERIALS AND METHODS

Strains used in this study were isolated from soybean plantsexhibiting symptoms of SDS or supplied from culture col-lection, and are listed in TABLE I. Infected soybean plantswere collected at fields in three different regions of Argen-tina in February and March 2001: General Rocca (state ofCordoba, 15 Feb 2001), Las Rosas (state of Santa Fe, 19 Feb2001) and San Agustin (state of Tucuman, 1 Mar 2001).Isolation of the causal fungus was performed at INTA-EEAat Marcos Juarez. The stem base and roots were cut fromdiseased soybean plants, washed under running tap waterfor 10 min and cut into pieces 1–5 cm in length with orwithout removing the cortex. The pieces were surface ster-ilized with 70% ethanol for 30 s and then in a solution of10% ethanol with 0.5% sodium hypochlorite for 5 min, fol-lowed by a rinse in sterilized distilled water for 10 min. Sam-ples were placed on sterilized filter paper in 9-cm Petri dish-es to remove excess water and were dried overnight. Driedplant pieces were placed on synthetic low nutrient agar(SNA) (Nirenberg 1976, 1990) and incubated at 25 C. Six-teen isolates of the Argentinean soybean SDS pathogenwere obtained and studied together with two SDS strainsisolated at General Rocca, Cordoba, in 2000. Pathogenicitytests of the Argentinean isolates on various soybean cultivars

were performed separately from this study and their path-ogenic ability has been demonstrated.

Eight strains of F. solani f. sp. glycines causing soybeanSDS in the United States (Abney et al 1993, O’Donnell andGray 1995, Roy 1997a, Li et al 1998) together with twostrains of F. solani f. sp. phaseoli isolated from root rot ofbean (P. vulgaris) were examined to evaluate their taxo-nomic and phylogenetic relationships with the Argentineanisolates. Strain NRRL 22276, F. solani f. sp. phaseoli, hasbeen studied as a typical bean root-rot pathogen (Roy1997a). These strains were compared with strains of F. so-lani f. sp. cucurbitae race-1 (MP-I), f. sp. mori (MP-III), f. sp.xanthoxyli (MP-IV), f. sp. cucurbitae race-2 (MP-V), f. sp. pisi(MP-VI), f. sp. robiniae (MP-VII). All strains are stored bylyophilization or in liquid nitrogen vapors at 2175 C in theAgriculture Research Service Culture Collection (NRRL),National Center for Agricultural Utilization Research, Pe-oria, Illinois, U.S.A., and at the MAFF Genebank System,National Institute of Agrobiological Sciences, Tsukuba, Ibar-aki, Japan.

Examination of morphological characters. Fusarium strainswere grown on potato-dextrose agar (PDA; Difco, Detroit,Michigan), SNA and steamed rice (Burkholder 1919) in 9-cm plastic Petri dishes. Cultures were incubated at 20 C inthe dark, under continuous fluorescent light (MitsubishiFL40S-W) or under daylight. Average and standard devia-tion (SD) in the size of individual conidial types were de-rived from the measurement of 50 conidia, randomly cho-sen according to the number of septa from cultures grownunder each of the cultural conditions. Colony morphology,color and odor were based primarily on cultures grown onPDA. Colors cited are given according to Kornerup andWanscher (1978). Dried cultures were deposited as holo-types of the new taxa in the herbarium of the U.S. NationalFungus Collection (BPI), USDA/ARS, Beltsville, Maryland,U.S.A. Descriptive terms for anamorph morphology followNirenberg and O’Donnell (1998).

Assessment of growth rate at different temperatures. For com-parison of mycelial growth rates at various temperatures,agar blocks ca 5 3 5 mm were cut from the margins of 2-wk old cultures on SNA and transferred onto PDA. Theseculture plates were incubated under eight different tem-peratures between 5 and 40 C at 5 C intervals in the dark.Cultures were examined after 1 d and 5 d under a dissectingmicroscope, and colony margins were marked with perma-nent ink on the reverse side of the Petri dishes. Radial my-celial growth rates were calculated as mean values per dayby measuring the difference in colony size in 16 directionsaround the colony during the four days of incubation. Mea-surements were repeated at least twice and averaged.

Molecular biology. Total genomic DNA was prepared as de-scribed in O’Donnell (2000). Domains D1 and D2 of thenuclear large subunit rDNA (28S), the nuclear ribosomalinternal transcribed spacer (ITS) region and a portion ofthe translation elongation factor 1-a gene (EF-1a) were am-plified and sequenced with primers and reagents describedin White et al (1990) and O’Donnell (2000). The nuclearribosomal intergenic spacer (IGS) was amplified with the

662 MYCOLOGIAT

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664 MYCOLOGIA

primer pair NL11 (59-CTGAACGCCTCTAAGTCAG) andCNS1 (59-GAGACAAGCATATGACTAC). In addition, thesefour internal primers were used to sequence the entire IGSregion: SCNS3 (59-GGTCTGAAAGATCAGGTACG), SCNS5(59-TACCCTATACCTCCGCCAAC) and SCNS7 (59-TACCCT-ATACCACCTAGTAGC). Sequencing reactions were puri-fied by gel filtration and run on either an Applied Biosys-tems model 377 or 3100 automated sequencer, as previouslydescribed (O’Donnell 2000).

Molecular phylogenetic analysis. DNA sequences were ed-ited and aligned visually using Sequencher 4.1.2 (GeneCodes Corporation, Ann Arbor, Michigan). Sequences ofFusarium illudens C. Booth and Nectria plagianthi Dingleywere selected as outgroup taxa based on a previous phylo-genetic analysis (O’Donnell 2000). PAUP*4.0b4a (Swofford2002) was used to conduct unweighted parsimony analyseson the aligned 28S rDNA, ITS rDNA and EF-1a sequencesas separate and combined datasets for the 43 taxon matrix.Sequences from the IGS region for the three species de-scribed in this study were analyzed separately. For all anal-yses the heuristic search option was used with 1000 randomaddition sequences with MULPARS on and TBR branchswapping. Phylogenetically informative indels were coded asa fifth character state. Clade stability was assessed by 1000parsimony bootstrap replications. Combinability of the in-dividual partitions was assessed with the nonparametricTempleton Wilcoxon signed-ranks (WS-R) test implement-ed in PAUP* using 70% bootstrap consensus trees as con-straints. Sequences have been deposited in GenBank asAY220150-AY220239, and the alignments have been depos-ited in TreeBASE as M1370 and M1371.

TAXONOMY

Fusarium tucumaniae T. Aoki, O’Donnell, Yos. Hom-ma et Lattanzi, sp. nov. FIGS. 1–18Coloniae in agaro PDA dicto 20 C obscuritate tarde ex-

pandentes, albae, luteolo-albae vel coeruleo-griseae, in par-te sporulante pustulis dilute flavis vel viridi-albis, sub lucefluorescente vel diurna pustulis dilute flavis, viridi-albis, gri-seo-luteis, griseo-viridibus, griseo-glaucis, viridibus, obscureviridibus vel caerulescentibus. Mycelium aerium vulgo par-cum, pionnoti simile, nonnumquam copiosum, laxum veldense floccosum, album, flavo-album vel coeruleo-griseum.Reversum saepe incoloratum, griseo-luteum vel dilute brun-neum. Chlamydosporae in hyphis et in conidiis frequentes,plerumque subglobosae, terminales vel intercalares, singu-lae, raro catenatae, hyalinae vel pallide pigmentatae, levesvel asperatae. Sclerotia absentia. Sporodochia plerumquecopiosa in agaris SNA et PDA, parca in fasciculis hypharumin PDA. Conidiophora aeria copiosa in SNA, nonnumquamin PDA, simplicia vel parce ramosa, brevia vel ad 270 mmlonga, 2–6.5 mm lata, monophialides terminales integratasformantia. Phialides aeriae simplices, subulatae vel subcylin-dricae. Conidia aeria dimorpha: (1) cylindrica curvata velfalcata, (2–)3(–5)-septata, basi distincta pediformi, in coni-diophoris majoribus formata; (2) minuta, oblongo-ellipso-idea vel breviter clavata vel ovalia, in conidiophoris ad 50mm longis et 1.5–3 mm latis formata, 0(–1)-septata, 3.5–18.5

3 1.5–4.5 mm. Conidiophora sporodochialia verticillata velraro simplicia; monophialides simplices, subulatae, ampul-liformes vel subcylindricae. Conidia plerumque cylindricavel modice curvata, nonnumquam falcata, circumscriptioneinterna et externa quasi parallelis, sursum modice expan-dentia, cellulae apicali acutae, basilari pediformi, (2–)3–4(–7)-septata; ubi 3-septata in SNA 35.5–85.5 3 3.5–6 mm, ubi4-septata in SNA 49.5–95 3 4–5.5 mm, ubi 5-septata in SNA49–105.5 3 3.5–6 mm.

Colonies on PDA showing radial mycelial growthrates of 1.0–2.2 mm per d at 20 C in the dark. Colonycolor on PDA white (1A1) to yellowish-white (2-4A2),sometimes with bluish-gray (20-21B2-3) to grayish-blue (22-23D6) tint, conidial pustules pale yellow (3-4A3), light yellow (4A4-5) or greenish-white (28-30A2) in the dark and light yellow (4A4-5), greenish-white (28-30A2), grayish-yellow (1-3B3-5), grayish-green (27-30B-C3-5, 25-30D-E4-6, 25-26E7), darkgreen (25-26F6-8) to dark turquoise (24F6-8) underfluorescent or daylight. Aerial mycelium generallysparse with pionnotal colony appearance, some de-veloped abundantly, then loose to dense floccose,white (1A1), yellowish-white (2-4A2), sometimes blu-ish-gray (22-23B3) to grayish-blue (22-23D6). Colonymargin entire to undulate. Reverse pigmentation of-ten absent, sometimes grayish-yellow (4C4-6) to lightbrown (5D4-5). Odor absent or sometimes putrid ormoldy. Chlamydospores formed frequently in hyphaeand in conidia, mostly subglobose, often terminal, oc-casionally intercalary, single, rarely in chains, hyaline,pale to yellowish-gray or pale-yellow, smooth torough-walled, sometimes verruculose, 9-13 3 8.5-12.5mm. Sclerotia absent. Sporulation generally rapidand abundant; on PDA often light-colored in thedark, greenish-to-bluish under fluorescent light ordaylight; sporodochia normally formed abundantlyon SNA and PDA, but sparsely in mycelial strains onPDA. Aerial conidiophores formed abundantly onSNA, sometimes on PDA, generally unbranched, oc-casionally sparsely branched from their base or mid-dle, up to 270 mm long, 2-6.5 mm wide, forming mon-ophialides integrated in the apices. Aerial phialidessimple, subulate to subcylindrical. Aerial conidia oftwo types; (1) curved cylindrical to falcate, (2-)3(-5)-septate, with a foot cell, morphologically indistin-guishable from falcate sporodochial conidia, formedmainly on taller conidiophores; (2) minute, oblong-ellipsoidal, short-clavate to oval, 0(–1)-septate, 3.5–(6.5–8.2)–18.5 3 1.5–(2.4–2.8)–4 mm (ranges of theaverages for individual isolates in parentheses) [extype: 5.5–(7.3 6 0.89)–9.5 3 2–(2.5 6 0.25)–3 mm(averages 6 SDs in parentheses)] formed on shortconidiophores up to 50 mm long, 1.5–3 mm wide.Sporodochial conidiophores branched verticillately,or rarely unbranched, forming apical monophialides.


FIG. 1. Fusarium tucumaniae (NRRL 31096, ex holotype) cultured in the dark. A–E: cultured on SNA, F–H: cultured onPDA. A. Septate, falcate conidia with a foot cell formed on tall, slender aerial conidiophores. B. Aseptate, short-clavate conidiaformed on a short aerial conidiophore. C, F. Septate, falcate to curved cylindrical conidia with a foot cell formed on branchedsporodochial conidiophores. D. Chlamydospores in conidia. E, H. Smooth to rough-walled, terminal or intercalary chla-mydospores formed in hyphae. G. Aseptate, minute ovate conidia observed in a culture on PDA. Scale bar: 25 mm.

Sporodochial phialides simple, subulate, ampulli-form to subcylindric, often with a conspicuous col-larette at the tip. Sporodochial conidia generally cy-lindrical and gently curved, sometimes falcate, withdorsal and ventral lines nearly parallel or graduallywider upward, with an acuate apical cell and a dis-tinct basal foot cell, (2–)3–4(–7)-septate; 3-septate onSNA: 35.5–(52.4–71.4)–85.5 3 3.5–(4.5–4.9)–6 mm[ex type: 38.5–(63.9 6 12.39)–83.5 3 4–(4.8 6 0.27)–5.5 mm], on PDA: 38–(57.5–67.4)–81 3 3.5–(4.4–4.9)–5.5 mm [ex type: 45–(66.4 6 5.89)–81 3 4–(4.76 0.29)–5.5 mm]; 4-septate on SNA: 49.5–(61.3–

77.3)–95 3 4–(4.7–5.0)–5.5 mm [ex type: 49.5–(64.06 7.62)–84 3 4–(4.7 6 0.25)–5.5 mm], on PDA: 48–(60.3–76.0)–88 3 3.5–(4.4–5.0)–5.5 mm [ex type: 66–(72.1 6 3.21)–81 3 4–(4.8 6 0.27)–5.5 mm]; 5-sep-tate on SNA: 49–(67.9–86.4)–105.5 3 3.5–(4.6–4.9)–6 mm [ex type: 76–(84.9 6 4.45)–94 3 4–(4.9 60.21)–5.5 mm].

Type specimen. ARGENTINA. TUCUMAN: San Agustin,dried culture isolated from Glycine max, 1 Mar 2001, T. Aoki& Y. Homma (HOLOTY PE, BPI 841955).

Ex holotype culture. NRRL 31096 5 MAFF 2384185 MJ-172.

666 MYCOLOGIA

FIGS. 2–10. Aerial conidia and conidiophores of Fusarium tucumaniae cultured on SNA in the dark (2–6: aerial view, 7–10: water mounted). 2–4. Falcate aerial conidia formed on slender conidiophores arising from hyphae on the agar surface.5, 6. Minute conidia formed on short aerial conidiophores arising from hyphae on the agar surface. 7–9. Slender aerialconidiophores and falcate conidia with a foot cell formed on the agar surface. 10. Aseptate, short clavate to oblong conidiaformed on a short aerial conidiophore. 2–6, 8, 9 from NRRL 31096, 7 from NRRL 31099, and 10 from NRRL 31092. Scalebars: 2–7 5 50 mm, 8–10 5 20 mm.

Additional cultures studied. ARGENTINA. CORDOBA:General Rocca, from G. max, 3 Mar 2000, Y. Homma (NRRL31085, NRRL 31086), 15 Feb 2001, T. Aoki (NRRL 31087,NRRL 31088, NRRL 31089, NRRL 31099); SANTA FE: LasRosas, from G. max, 19 Feb 2001, T. Aoki (NRRL 31100);TUCUMAN: San Agustin, from G. max, 1 Mar 2001, T. Aoki(NRRL 31090, NRRL 31091, NRRL 31092, NRRL 31093,NRRL 31094, NRRL 31095, NRRL 31097, NRRL 31098).Additional data are presented in TABLE I.

Etymology. tucumaniae; based on the Latin namefor Argentina, Tucumania.

Notes. Key morphological characters that distin-guish F. tucumaniae from other species within the F.solani complex include the production of septate, fal-cate conidia with a foot cell formed on tall and slen-der, aerial conidiophores (FIGS. 1A, 2–4, 7–9) togeth-er with long, slender sporodochial conidia with most-ly pointed apices (FIGS. 1C, F, 11–15). In contrast toF. virguliforme and F. phaseoli n. spp. described in thisstudy, the second type of sporodochial conidia arenever formed in F. tucumaniae.


FIGS. 11–18. Sporodochial conidia, conidiophores and chlamydospores of Fusarium tucumaniae cultured in the dark(water mounted). 11, 12. Branched sporodochial conidiophores forming falcate to curved cylindrical conidia. 13–15. Spo-rodochial conidia observed on SNA (13) and PDA (14, 15); septation is obscured in immature conidia on PDA (14), butsepta become clearer as the conidia mature (15). 16, 17. Chlamydospores in hyphae. 18. Chlamydospore in a conidium. 11–13, 16–18 cultured on SNA, and 14, 15 on PDA. 11 from NRRL 31086, 12, 13, 16–18 from NRRL 31096, 14 from NRRL31098, and 15 from NRRL 31085. Scale bars: 11 5 50 mm, 12–18 5 20 mm.

Fusarium virguliforme O’Donnell & T. Aoki, sp. nov.5 Fusarium solani (Mart.) Sacc. f. sp. glycines K. Roy s.

str., Plant Dis. 81: 259–266. 1997.? 5 Fusarium martii Appel & Wollenw. var. viride Sherb.,

Mem. Cornell Univ. Agric. Exp. Stat. 6: 247–249, 1915.FIGS. 19–46

Coloniae in agaro PDA dicto ut in Fusarium tucumaniaesimiles. Sclerotia absentia. Chlamydosporae in hyphis etconidiis frequentes, ut in F. tucumaniae similes. Conidi-ophora aeria et sporodochialia ut in F. tucumaniae similia.Conidia aeria dimorpha: (1) curvata cylindrica vel falcata,(2–)3(–4)-septata, basi pediformi, praecipue in conidi-ophoris majoribus formata; (2) minuta, oblongo-ellipsoideavel breviter clavata, in parte minore coloniae in conidi-ophoris ad 60 mm longis et 2–2.5 mm latis formata, 0(–1)-septata, 3–18.5 3 1.5–3.5 mm. Conidia sporodochialia di-morpha: (1) falcata, dorsiventralia, plerumque latissima in

parte media, utrinque aequaliter angustatae, cellulis apical-ibus et basilaribus vix distinctis, (2–)3–4(–5)-septata, in PDA et SNA formata; ubi 3-septata inSNA 33–65.5 3 3.5–6 mm, ubi 4-septata in SNA 43–67 34.5–6 mm, ubi 5-septata in SNA 46–79.5 3 4–7 mm; (2) vir-guliformia vel nonnumquam breviter clavata, ad apicem in-flata et rotundata, ad basim angustata et curvata, in PDAsaepe obscuritate formata, 0–1(–2)-septata, 12–33.5 3 4–7.5mm.

Colonies on PDA showing radial mycelial growthrates of 1.3–1.7 mm per d at 20 C in the dark. Colonycolor on PDA white (1A1) to yellowish-white (2-4A2)or pale yellow (2-4A3), sometimes with bluish-gray(20-21B2-3) tint, conidial pustules pale yellow (3-4A3) to light yellow (3-4A4-5) in the dark and lightyellow (3-4A4-5), grayish-yellow (1-4B3-7), grayish-or-ange (5B3-5), greenish-white (26-29A2), grayish-tur-

668 MYCOLOGIA

FIG. 19. Fusarium virguliforme (NRRL 31041, ex holotype) cultured in the dark. A–E: cultured on SNA, F–J: cultured onPDA. A. Septate, falcate conidia with a foot cell formed on tall, slender aerial conidiophores. B. Minute, aseptate and short-clavate conidia formed on a short aerial conidiophore. C. Compactly branched sporodochial conidiophores forming septate,falcate conidia with a foot cell from bottle-shaped phialides. D, I. Chlamydospores formed in conidia. E, J. Smooth to rough-walled, terminal or intercalary chlamydospores formed in hyphae. F. Sporodochial conidiophore forming septate, falcateconidia with a foot cell and comma-shaped conidia from phialides. G. Minute, aseptate and short-clavate conidia observedin a culture on PDA. H. Zero–1-septate comma-shaped conidia formed in culture on PDA. Scale bar: 25 mm.

quoise (24B-D3-6), pastel green (28-30A4), grayish-green (25-30B-C3-5, 25-30D-E4-7), dark green (25-30F5-8) to dark turquoise (24F7-8) under fluorescentor daylight. Aerial mycelium sparse with pionnotalcolony appearance, or sometimes developed abun-dantly, then loose to dense floccose, white (1A1), yel-lowish-white (2-4A2), sometimes bluish-gray (21-23B-C2-3). Colony margin entire to often undulate. Re-verse pigmentation often absent, sometimes grayish-

yellow (4B-C4-6), grayish-orange (5B4-6) tobrownish-orange (5C3-6), or olive brown (4D-F5-6) toyellowish-brown (5D-F5-6). Yellowish exudate some-times present. Odor absent or sometimes putrid ormoldy. Chlamydospores formed abundantly in my-celium and in conidia, mostly subglobose, intercalaryor terminal, mostly single, rarely in chains, hyaline topale or pale-yellow, smooth to rough-walled, 4–15 mmdiam. Typical sclerotia absent. Sporulation generally


FIGS. 20–27. Aerial conidia and conidiophores of Fusarium virguliforme cultured on SNA in the dark (20–24, 26: aerialview, 25, 27: water mounted). 20–24. Falcate aerial conidia formed on slender conidiophores arising from hyphae on theagar surface. 25. Slender, unbranched aerial conidiophores. 26. Minute conidia formed on short aerial conidiophores infalse heads. 27. Aseptate, short-clavate to oblong aerial conidia. 20, 21, 23, 25, 26 from NRRL 22490, 22 from NRRL 22823,and 24, 27 from NRRL 31041. Scale bars: 20–24, 26 5 50 mm, 25, 27 5 20 mm.

rapid and abundant; on PDA often light-colored inthe dark, greenish-to-bluish under fluorescent lightor daylight; sporodochia normally formed abundant-ly on SNA and PDA but sparsely in mycelial strainson PDA. Aerial conidiophores formed abundantly onSNA, rarely on PDA, unbranched or sparselybranched, up to 290 mm long, 2–7 mm wide, formingmonophialides integrated in the apices. Aerial phial-ides simple, subulate to subcylindrical, often with aconspicuous collarette at the tip. Aerial conidia oftwo types; (1) curved cylindrical to falcate, (2–)3(–4)-septate, with a foot cell, morphologically indistin-guishable from falcate sporodochial conidia, formedmainly on taller conidiophores; (2) minute, oblong-ellipsoidal to short-clavate, 0(–1)-septate, 3–(5.5–10.8)–18.5 3 1.5–(2.3–2.7)–3.5 mm [ex type: 6–(9.06 1.10)–11.5 3 2–(2.5 6 0.28)–3.5 mm] formed in asmall portion of the colony and on short conidio-phores up to 60 mm long, 2–2.5 mm wide. Sporodo-chial conidiophores branched verticillately, or rarelyunbranched, forming apical monophialides. Sporo-dochial phialides simple, subulate, ampulliform tosubcylindric, with a conspicuous collarette at the tip.Sporodochial conidia of two types; (1) typically fal-

cate, dorsiventral, most frequently widest at the mid-region of their length, often tapering and curvingequally toward both ends, with the apex and foot celltypically similarly pointed and often indistinguish-able, (2–)3–4(–5)-septate, formed on PDA and onSNA; apical and basal halves often morphologicallysymmetrical; 3-septate on SNA: 33–(47.3–54.8)–65.53 3.5–(4.8–5.4)–6 mm [ex type: 37.5–(48.8 6 4.33)–58 3 4.5–(5.2 6 0.26)–5.5 mm], on PDA: 27–(45.0–52.2)–60.5 3 4–(4.7–5.2)–6 mm [ex type: 27–(45.5 65.21)–56 3 4.5–(5.0 6 0.20)–5.5 mm]; 4-septate onSNA: 43–(53.3–56.9)–67 3 4.5–(5.2–5.4)–6 mm [extype: 47–(54.2 6 3.73)–64 3 4.5–(5.3 6 0.26)–6 mm],on PDA: 45.5–(54.3–57.2) –66 3 4.5–(5.0–5.2)–6 mm[ex type: 50.5–(55.6 6 2.87)–63 3 4.5–(5.0 6 0.13)–5.5 mm]; 5-septate on SNA: 46–(57.8–63.5)–79.5 3 4–(5.0–5.4)–7 mm [ex type: 47.5–(57.8 6 3.50)–67.5 35–(5.4 6 0.29)–6.5 mm]; (2) comma-shaped to some-times short-clavate, with a swollen apex often round-ed but rarely pointed and with a tapering and curv-ing base, formed only on PDA often in the dark, 0–1(–2)-septate, 12–(18.2–22.4)–33.5 3 4–(5.1–6.4)–7.5mm [ex type: 15–(21.4 6 2.74)–26 3 5–(6.2 6 0.62)–7.5 mm].

670 MYCOLOGIA


←

FIGS. 28–46. Sporodochial conidia, conidiophores and chlamydospores of Fusarium virguliforme cultured in the dark (28:aerial view, 29–46: water mounted). 28–30. Branched sporodochial conidiophores forming falcate to curved cylindrical co-nidia; comma-shaped conidia were formed on the same conidiophores (arrowheads in 30). 31–34. Sporodochial conidiaobserved in culture on SNA (31) and PDA (32–34); septation is obscured in young conidia on PDA (32) but becomes clearas vacuoles form in the conidia (33, 34). 35–41. Zero–1-septate comma-shaped conidia formed only on PDA. 42–45. Terminalor intercalary chlamydospores in hyphae. 46. Chlamydospore in a conidium. 28, 29, 31, 42–45 cultured on SNA, and 30, 32–41, 46 on PDA. 28, 34, 42 from NRRL 22823, 29–31, 35–37, 43–46 from NRRL 31041, 32 from NRRL 31039, 33, 41 fromNRRL 22292, 38, 39 from NRRL 22489 and 40 from NRRL 22490. Scale bars: 28–30 5 50 mm, 31–46 5 20 mm.

Type specimen. UNITED STATES. ILLINOIS: dried cul-ture isolated from Glycine max, 1998, Shuxian Li (HOLO-TY PE, BPI 841956).

Ex holotype culture. NRRL 31041 5 MAFF 2385535 Shuxian Li # 95.

Additional cultures studied. UNITED STATES: from G.max, K. W. Roy (NRRL 22489, NRRL 22490); UNITEDSTATES. ILLINOIS: from G. max, P. Stevens (NRRL 22292),1994, S. Li (NRRL 31039), S. Li (NRRL 31040); INDIANA:from G. max, T. S. Abney (NRRL 22823), 1989, T. S. Abney(NRRL 22825). Additional data are presented in TABLE I.

Etymology. virguliformis (Lat. comma-shaped);based on the morphology of the second type of spo-rodochial conidia.

Notes. Fusarium virguliforme is distinguished fromother species within the F. solani complex by the pro-duction of comma-shaped sporodochial conidia onPDA (FIGS. 19H, 35–41) together with septate, falcateaerial conidia with a foot cell on SNA (FIGS. 19A, 20–25). Minute, oblong-ellipsoidal to short-clavate conid-ia were formed from short conidiophores up to 60mm long (FIGS. 19B, 26, 27), but they were observedonly in a small portion of the entire colony. Fusariumvirguliforme resembles F. martii var. viride isolatedfrom potato in the dimensions of its sporodochialconidia. Sherbakoff (1915) illustrated a comma-shaped conidium from a culture on a potato stemplug (p. 245, FIG. 44G) but did not include a descrip-tion of sporodochial conidia of this shape. Conidialmasses of this fungus on potato agar rich in glucose(nearly equivalent to PDA) were described as palesmoke-gray and without dark blue coloration (Sher-bakoff 1915), while those of F. virguliforme on PDAare variable but often greenish-to-bluish when cul-tured under fluorescent light or daylight. Further-more, the host plants of these taxa are different. Un-fortunately, no authentic material of F. martii var. vir-ide was left by Sherbakoff which makes it difficult toascertain whether the variety and F. virguliforme areconspecific.

Fusarium phaseoli (Burkh.) T. Aoki & O’Donnell,comb. nov.Fusarium martii Appel & Wollenw. f. phaseoli Burkh.,Mem. Cornell Univ. Agric. Exp. Stat. 26: 1007–1012, 1919.(designated in trinomial as F. martii phaseoli)

5 Fusarium solani (Mart.) Sacc. f. phaseoli (Burkh.) W.C.Snyder & H.N. Hansen s. str., Amer. J. Bot. 28: 740,1941. (presently considered as F. solani f. sp. phaseoli)

5 Fusarium martii Appel & Wollenw. var. minus Sherb.,Mem. Cornell Univ. Agric. Exp. Stat. 6: 249–250, 1915.

FIGS. 47–68Colonies on PDA showing radial mycelial growth

rates of 1.5–1.6 mm per d at 20 C in the dark. Colonycolor on PDA white (1A1) to yellowish white (3-4A2),orange white (3-4A2) or grayish-orange (5B3-4) tobrownish-orange (5-6C3-4), sometimes with grayishtint (1B-C1), conidial pustules sometimes present,pale yellow (3-4A3) to light yellow (3-4A4-5) in thedark and light yellow (3-4A4-5), grayish-yellow (1-3B-C3-5), pale green (28-30A3), or grayish-green (26-29B-C3-5, 26-30D-E4-6, 28-30E7) to dark green (28-30F6-8) under fluorescent or daylight. Aerial myce-lium generally abundant, loose to dense floccose,white (1A1), yellowish-white (3-4A2), sometimes gray-ish-white (1B1) to pastel gray (1C1), sometimessparse with pionnotal colony appearance. Colonymargin entire to undulate. Reverse pigmentation of-ten grayish-yellow (4C4-6), grayish-orange (5B4-6) tobrownish-orange (5C4-6), sometimes absent. Brown-ish exudate sometimes present. Odor absent or some-times putrid or moldy. Colony color on steamed ricesimilar to that on PDA but with a more pinkish tint,sometimes with conidial pustules of coral red (9B7),brownish-red (9C7) to reddish-brown (9D-E7) onsporodochia. Chlamydospores formed frequently inmycelium and in conidia, mostly subglobose, some-times oblong, often terminal, occasionally intercala-ry, single, sometimes in chains, hyaline, pale to yel-lowish-gray or pale-yellow, smooth to rough-walled,6.5–13 3 4–12 mm. Sclerotia absent, but forming scat-tered plectenchymatic, wart-like, sporodochial stro-mata of coral red (9B7), brownish-red (9C7) to red-dish-brown (9D-E7) on steamed rice. Sporulationgenerally rapid and abundant on SNA and in pion-notal strains on PDA, sparse in mycelial strains onPDA; on PDA often light-colored in darkness, alsogreenish-to-bluish under fluorescent light or day-light; sporodochia formed abundantly on SNA andin pionnotal strains on PDA, but generally less fre-quently on PDA. Aerial conidiophores formed on

672 MYCOLOGIA

FIG. 47. Fusarium phaseoli cultured on SNA in the dark. A, D. Septate, falcate conidia with a foot cell formed on tall,slender aerial conidiophores. B, G. Septate, falcate conidia with a foot cell formed on branched sporodochial conidiophores.C, H. Chlamydospores formed in hyphae and in a conidium. E. Obovate to ellipsoidal, large conidia formed on aerialconidiophores. F. Aseptate, short-clavate conidia formed on short aerial conidiophores. A–C from NRRL 22276, and D–Hfrom NRRL 31156. Scale bar: 25 mm.

SNA, unbranched or sparsely branched, up to 210mm long, 2–4.5 mm wide, forming monophialides in-tegrated in the apices. Aerial phialides simple, subu-late to subcylindrical. Aerial conidia of three types;(1) curved cylindrical to falcate, (2–)3(–4)-septate,with a foot cell, mostly morphologically indistinguish-able from sporodochial conidia but sometimes short-er and thicker, formed mainly on taller conidio-phores; (2) minute, short-clavate to ellipsoidal, 0(–1)-septate, 5–10.5 3 1.5–3 mm formed in a minorportion of a colony on SNA on short conidiophoresup to 45 mm long, 1.5–3 mm wide; (3) large, obovate,short-clavate to ellipsoidal, with a rounded apex anda truncate to rounded base, 0–2(–3)-septate, 13–34.53 5.5–7.5 mm, formed sometimes on taller conidio-phores. Sporodochial conidiophores branched verti-cillately, or rarely unbranched, forming apical mon-

ophialides. Sporodochial phialides simple, subulate,ampulliform to subcylindric, sometimes with a con-spicuous collarette at the tip. Sporodochial conidiaof two types; (1) typically falcate, dorsiventral, withdorsal and ventral lines nearly parallel or often grad-ually and slightly widen upwards, with an acuate api-cal cell and a rounded but protruding basal foot cell,(2–)3–4(–5)-septate; apical and basal parts oftencurved ventrally but asymmetrically; 3-septate onSNA: 32.5–(42.1–45.8)–55 3 4–(4.9–5.2)–6.5 mm, onPDA: 32–(41.8–47.9)–58 3 4.5–(5.1–5.2)–6 mm; 4-septate on SNA: 42.5–(50.9–51.4)–59 3 4.5–(5.1–5.3)–6 mm, on PDA: 44.5–(51.6–54.7)–63.5 3 4.5–(5.2–5.4)–6 mm; 5-septate on SNA: 42.5–(54.9–58.3)–73.5 3 4.5–(5.1–5.3)–6.5 mm; (2) short-clavate to el-lipsoidal or naviculate straight or slightly curved, witha rounded apex and a truncate base, sometimes


FIG. 48. Fusarium phaseoli cultured in the dark. A, D. Septate, falcate conidia with a foot cell formed on sporodochialconidiophores. B, F. Chlamydospores formed in conidia. C, G. Terminal or intercalary chlamydospores formed in hyphae.E. Short clavate to ellipsoidal or naviculate 0–1-septate sporodochial conidia formed in cultures on PDA. H. Sporodochialconidia formed in cultures on steamed rice. A–C, H from NRRL 22276, and D–G from NRRL 31156. A–G cultured on PDA,and H on steamed rice. Scale bar: 25 mm.

formed on PDA and on steamed rice, 0–1(–2)-sep-tate, 13.5–32.5 3 3.5–6 mm.

Cultures studied. UNITED STATES: from Phaseolusvulgaris, H. VanEtten (NRRL 22276); UNITEDSTATES. MICHIGAN: from P. vulgaris (NRRL31156). Additional data are presented in TABLE I.

Notes. Morphological and cultural features of thestrains examined agreed well with the original de-scription of F. martii f. phaseoli given by Burkholder(1919) especially in the dimensions and morphologyof septate sporodochial conidia formed on PDA(FIGS. 47B, G, 48A, D, 61–64). Fusarium phaseoli canbe differentiated from other members of the F. solanicomplex by the production of septate, falcate aerialconidia with a foot cell (FIGS. 47A, D, 49–53) togeth-er with falcate sporodochial conidia with asymmetricends. In F. phaseoli, minute, short-clavate to ellipsoi-dal conidia were only produced on short aerial co-nidiophores up to 45 mm long and in a minor por-tion of a colony. Fusarium phaseoli is similar to F. vir-guliforme in its conidial dimensions (FIGS. 69, 70).However, F. phaseoli does not produce comma-shapedconidia in sporodochia on PDA. Falcate sporodochial

conidia of F. phaseoli also differ morphologically fromthose of F. virguliforme in that those of F. phaseolipossess an acuate apical cell and a protruding foot-like basal cell, which frequently is curved ventrally(FIGS. 47B, G, 48A, D, 61–64). The midregion of thedorsal and ventral lines of the sporodochial conidiaof F. phaseoli are nearly parallel and often are grad-ually and slightly wider toward the apex. Therefore,the apical and basal parts of the sporodochial conidiaof F. phaseoli are asymmetrical, in most cases, anddiagnostic. In contrast, the apical and basal parts ofthe sporodochial conidia of F. virguliforme often aresymmetrical (FIGS. 19C, F, 31–34). Fusarium phaseolialso formed short clavate to ellipsoidal or sometimesnaviculate, straight or slightly curved sporodochialconidia with a rounded apex and a truncate base onPDA and on steamed rice (FIGS. 48E, H, 65). Themorphology of these conidia corresponds well withthose described and illustrated by Burkholder (1919;p. 1009, Fig. 134C) for F. martii f. phaseoli on steamedrice. In addition, large obovate to ellipsoidal conidiaalso were observed on elongate aerial conidiophoresof F. phaseoli on SNA (FIGS. 47E, 54–55).

674 MYCOLOGIA

FIGS. 49–56. Aerial conidia and conidiophores of Fusarium phaseoli cultured on SNA in the dark (49–52: aerial view, 53–56: water-mounted). 49–53. Falcate aerial conidia formed on slender conidiophores arising from hyphae on the agar surface;shorter and thicker conidia with a blunt base were observed occasionally (51, 52). 54, 55. Slender aerial conidiophoresforming ellipsoidal, obovate or naviculate conidia. 56. Minute conidia formed on short aerial conidiophores in false heads.49–56 from NRRL 31156. Scale bars: 49–52, 54 5 50 mm, 53, 55, 56 5 20 mm.

Fusarium phaseoli is similar morphologically to F.martii (Appel and Wollenweber 1910) and its variety,var. minus (Sherbakoff 1915), both of which were syn-onymized with F. solani in several taxonomies (Sny-der and Hansen 1941, Booth 1971, Gerlach and Ni-renberg 1982, Nelson et al 1983). Appel and Wollen-weber (1910) were the first to describe and illustrateseptate aerial conidia with a foot cell formed on longand slender conidiophores for F. martii. Sporodochi-al conidia of F. phaseoli and F. martii have dorsal andventral lines that are nearly parallel and a curved api-cal and a distinct basal cell (Appel and Wollenweber1910, Sherbakoff 1915). However, the protruding

basal cells in the conidia of F. martii are long andstraight in contrast to those of F. phaseoli. In F. phas-eoli, the basal cells in the sporodochial conidia areoften distinct (FIGS. 47B, G, 48A, D, 61–64) but with-out a long and straight protrusion. Measurementsprovided for 3–4-septate sporodochial conidia of F.martii by Appel and Wollenweber (1910) were 44–603 4.75–5.5 mm on average (39–71 3 4.5–6 mm intotal range) on various sterilized natural substrates.These values differ from those of F. phaseoli but theyare similar to F. virguliforme (FIGS. 69, 70). Fusariummartii var. minus, in contrast, was described as havingshorter and narrower sporodochial conidia (Sherbak-


FIGS. 57–68. Sporodochial conidia, conidiophores and chlamydospores of Fusarium phaseoli cultured in the dark (57:aerial view, 58–68: water mounted). 57–60. Branched sporodochial conidiophores forming falcate conidia. 61–64. Sporodo-chial conidia observed in culture on SNA (61) and PDA (62–64); septation is obscured in young conidia on PDA (62) butbecomes conspicuous as the conidia mature (63, 64). 65. Naviculate sporodochial conidium. 66–68. Terminal or intercalarychlamydospores in hyphae. 57–61, 66–68 cultured on SNA, and 62–65 on PDA. 57–62, 65–68 from NRRL 31156, and 63, 64from NRRL 22276. Scale bars: 57, 58 5 50 mm, 59–68 5 20 mm.

off 1915), which are more similar to those of F. phas-eoli. Fusarium phaseoli cultured on steamed riceformed reddish, plectenchymatic, wart-like stromata,which were scattered in the aerial mycelium and cov-ered by large sporodochia. Identical structures were

reported for F. martii var. minus by Sherbakoff(1915). Sherbakoff (1915) also illustrated slightlycurved, ellipsoidal to naviculate, 0–2-septate sporo-dochial conidia of this variety on a potato tuber plug,as well as minute, short-clavate to ellipsoidal, 0-sep-

676 MYCOLOGIA

FIG. 69. Plots of mean values of length and width of 3-and 4-septate sporodochial conidia in Fusarium tucumaniae,F. virguliforme and F. phaseoli grown on SNA in the dark at20 C.

FIG. 70. Plots of mean values of length and width of 3-and 4-septate sporodochial conidia in Fusarium tucumaniae,F. virguliforme and F. phaseoli grown on PDA in the dark at20 C.

tate aerial conidia on a red raspberry cane plug, thatresemble the conidia produced by F. phaseoli on PDAand SNA, respectively. Colony color of F. martii var.minus was described as light gray, drab and dark ol-ive-buff on potato agar rich in glucose, suggesting agreenish pigmentation. Based on these comparisons,F. martii var. minus is considered synonymous to F.phaseoli, although their original hosts are differentand colony color appeared more greenish or bluishin F. phaseoli cultured under light.

Another root-rot pathogen of bean, F. aduncispo-rum Weimer & Harter (5 F. solani var. aduncisporum(Weimer & Harter) Wollenw.), was described as hav-ing different morphological and physiological fea-tures from F. martii f. phaseoli (Weimer and Harter1926). Although F. aduncisporum was placed in syn-onymy with F. solani f. phaseoli (5 F. phaseoli) by Sny-der and Hansen (1941) and in F. solani by Gerlachand Nirenberg (1982), it possesses distinctly curvedto hook-shaped sporodochial conidia and the endsof its conidia are described as rounded with a basalcell that was either not foot-like or only slightly so.Because no living culture of F. aduncisporum wasavailable, the taxon tentatively was not included inthe synonymy of F. phaseoli.

Morphological comparison of the three species and F. so-lani. Features shared by SDS isolates from Argenti-na (5 F. tucumaniae), from the United States (5 F.virguliforme) and in the bean root-rot isolates fromthe United States (5 F. phaseoli) include the forma-tion of plural types of conidia, especially on the aerialconidiophores. Three different types of aerial conid-ia were found: (A-1) falcate, multiseptate aerial co-nidia with a foot cell formed mainly on tall and slen-der conidiophores observed in all three species onlyon SNA; (A-2) minute, short-clavate to ellipsoidal ae-rial conidia formed on short conidiophores in a mi-nor portion of an entire colony in all three species;(A-3) large, obovate, short-clavate to ellipsoidal co-nidia formed on taller conidiophores only in F. phas-eoli on SNA. Three types of sporodochial conidia alsowere found: (S-1) falcate, multiseptate conidia with afoot cell formed in all three species; (S-2) comma-shaped conidia only in F. virguliforme on PDA; (S-3)short clavate to ellipsoidal or naviculate conidia in F.phaseoli on PDA and on steamed rice. Therefore, F.virguliforme and F. phaseoli formed two conidial typesin sporodochia.

A distinctive morphological character common tothe three new species, but not recognized in the con-


cept of F. solani, is the production of the curved cy-lindrical to falcate, multiseptate aerial conidia with afoot cell (A-1). These conidia were formed by allstrains of the three species cultured on SNA withoutexception from phialides on generally tall and slen-der conidiophores. These conidiophores were sep-tate, simple or sparsely branched, often more than100 mm long, and bore slender phialides integratedin their apices. Sporodochial conidiophores, in con-trast, branched repeatedly and compactly and theirphialides were distinct and often bottle-shaped (FIGS.1C, F, 11, 12, 19C, F, 28–31, 47B, G, 48A, D, 57–60).Septate aerial conidia (A-1) mostly were indistin-guishable morphologically from the septate sporo-dochial conidia (S-1) because both conidial typeshave a foot cell at the base. Some shorter and thickermultiseptate conidia of this type, but with a roundedbase, were occasionally observed in F. phaseoli (FIGS.51, 52). In the typical F. solani complex strains (MP-I, III–VII) examined in this study, oval, ellipsoidal tosubcylindrical, 0–1-septate conidia (so-called ‘‘micro-conidia’’) were formed abundantly on tall and slen-der aerial conidiophores on both SNA and PDA. Inolder SNA culture, these conidiophores occasionallyformed slightly curved, subcylindrical multiseptateconidia with tapering ends, together with a mass ofthe oval to ellipsoidal ‘‘microconidia’’. Minute, shortclavate to ellipsoidal, mostly 0-septate conidia (A-2)were observed in the three new species in a minorportion of an entire colony on SNA and rarely onPDA. However, they were formed separately on shortaerial conidiophores, often less than 50 mm long, butnot on the same tall and slender aerial conidiophoresforming the falcate, multiseptate conidia (A-1) (FIGS.1B, G, 5, 6, 10, 19B, G, 26, 27, 47F, 56). In addition,their morphology clearly was different from ‘‘micro-conidia’’ formed by the typical F. solani complexstrains, where aerial ellipsoidal conidia were largerand formed on tall, slender conidiophores that wereoften more than 200 mm in length.

The morphology of septate sporodochial conidiawas evaluated on SNA and on PDA. Falcate sporo-dochial conidia (S-1) were most frequently 3- to 4-septate in the three species described in this study.Septation of the sporodochial conidia observed onPDA often was difficult to score in younger conidiabecause the cytoplasmic contents were granular.However, septa became distinct as the conidia be-came vacuolated in age (FIGS. 14, 15, 32–34, 62–64).Conidial septation was clearly observed in cultures onSNA (FIGS. 11–13, 29, 31, 61). Sizes of the septatesporodochial conidia were compared from cultureson SNA and PDA and average values of conidiallength and width for individual strains were plottedin FIGS. 69 and 70 according to the number of septa.

Measurements of 3- and 4-septate conidia of thethree species yielded similar results on SNA (FIG. 69)and on PDA (FIG. 70). Conidia of F. tucumaniae werelonger and narrower than those of the other two spe-cies, especially in the size of 4-septate conidia on SNA(FIG. 69). In cultures on PDA, F. tucumaniae clearlywas distinguishable from the other species based onthe dimensions of 3- or 4-septate conidia (FIG. 70).The sharply pointed ends of conidia in F. tucumaniaeprovided an additional diagnostic character (FIGS.1C, F, 11–15). There was a tendency for conidia of F.virguliforme, especially those that were 4-septate, tobe longer than those of F. phaseoli, but the ranges ofconidial sizes often overlapped such that this char-acter lacked diagnostic value (FIGS. 19F, 69, 70). Co-nidia of F. virguliforme and those of F. phaseoli, how-ever, showed other morphological differences. Co-nidia of F. virguliforme most frequently were widest atthe midregion and tapered and curved equally to-wards both ends. The apical and foot cells were sym-metrical and often indistinguishable (FIGS. 31–34). Incontrast, the dorsal and ventral lines of conidia in F.phaseoli were nearly parallel or often gradually wid-ened upward, and their apical and basal parts fre-quently were curved ventrally. Therefore, differencesbetween the apical and foot cell were conspicuous inF. phaseoli (FIGS. 61–64).

Diagnostic morphological features were discoveredfor F. virguliforme and F. phaseoli by the comparisonof cultures on SNA and PDA. All strains of F. virgu-liforme cultured on PDA formed comma-shaped co-nidia in sporodochia (S-2; FIGS. 19F, 30) which werefrequently 0–1(–2)-septate, curved, and wider andswollen upwards (FIGS. 19H, 35–41), especially fromcultures incubated in the dark. Although comma-shaped conidia were not observed in cultures onSNA, septate aerial conidia with a foot cell were con-stantly formed on tall conidiophores. Strains of F.phaseoli did not form comma-shaped conidia on PDAor SNA, although short-clavate to naviculate conidiawere sometimes formed in sporodochia on PDA andon steamed rice (S-3; FIGS. 48E, H, 65). On SNA,relatively large, ellipsoidal to obovate, 0–2(–3)-sep-tate conidia were formed by strains of F. phaseoli ontall and slender aerial conidiophores (A-3; FIGS. 47E,54, 55). This morphology was unique to strains of F.phaseoli.

Terminal or intercalary chlamydospores wereformed commonly and often abundantly by strains ofthe three species (FIGS. 1E, H, 16, 17, 19E, J, 42–45,47C, H, 48C, G, 66–68). Some conidial chlamydo-spores also were observed (FIGS. 1D, 18, 19D, I, 46,47H, 48B, F), especially in old cultures. They weresmooth- to rough-walled and occasionally possessed ayellowish pigment.

678 MYCOLOGIA

FIG. 71. Comparison of radial growth rates per day on PDA of Fusarium tucumaniae, F. virguliforme and F. phaseoli, withstrains of the representative MPs of the F. solani complex under different temperature from 5 to 40 C. The thick horizontaland vertical bars indicate means and total ranges, respectively, among the strains of each species (number of strains examinedin parentheses).

Colony characteristics were compared on PDA in9-cm plastic Petri dishes at 20 C, but no clear differ-ence among the three species was observed. Mycelialand pionnotal strains were found in each of the threespecies and greater numbers of conidia were formedon the colonies of the pionnotal strains. Pionnotalsectors sometimes were observed in colonies of my-celial strains, where conidial production was higherthan in the mycelial parts of the colonies. These factsmight suggest the possible occurrence of mutation,but intraspecific variation observed among strains ap-peared to be related to the light conditions em-ployed. When cultures were grown in the dark, col-ony color often remained whitish to yellowish, al-though greenish or bluish coloration sometimes wasobserved. When cultured under fluorescent light orunder daylight, colonies frequently became moregreenish to bluish and dark green, and a dark tur-quoise coloration also was observed as an extreme inone-month-old cultures. However, differences in col-ony morphology or coloration were not useful forspecies delimitation.

Difference in radial mycelial growth rates. Average ra-dial mycelial growth rates on PDA in the dark at eightdifferent temperatures between 5 to 40 C were cal-

culated for 16 strains of F. tucumaniae, eight strainsof F. virguliforme and two strains of F. phaseoli, andare summarized in FIG. 71. Eleven representativestrains of the F. solani complex (MP-I, III–VII) wereexamined for comparison. Optimal temperature formycelial growth was 25 C for all strains: 3.5–5.0 mm/day for F. solani of MP-I, III–VII; 1.5–3.0 mm/day forF. tucumaniae; 1.7–2.1 mm/day for F. virguliforme;1.5–1.6 mm/day for F. phaseoli. Average growth ratesof the latter three species were nearly half that of therepresentative F. solani complex strains. This ratiowas nearly the same for the other temperatures ex-amined.

Molecular phylogenetic relationships among strains.Aligned sequences of the 28S rDNA, nuclear ribo-somal ITS region and translation elongation factor(EF-1a) gene were analyzed as a combined datasetbased on the results of the Templeton WS-R test,which indicated that the partitions could be com-bined. Fusarium tucumaniae, F. phaseoli and F. virgu-liforme shared identical 28S rDNA and ITS haplo-types, except for a single base-pair indel within theITS2 that distinguishes F. virguliforme from the othertwo species. As suggested by the ITS2 indel, F. tucu-maniae and F. phaseoli were resolved as sister taxa in


FIG. 72. The single most-parsimonious phylogram inferred from the combined nuclear 28S rDNA, ribosomal ITS regionand EF-1a gene for the Fusarium solani species complex. Bootstrap replication frequencies above 70% are indicated abovenodes. Sequences of Fusarium illudens and Nectria plagianthi were used as outgroup taxa. Note that the SDS pathogen withinNorth America, F. virguliforme, is a sister to F. phaseoli and F. tucumaniae within a South American clade of this speciescomplex. CI, consistency index; RI, retention index; MP, mating populations or biological species.

the EF-1a gene tree (not shown), in the combinedanalysis (FIG. 72), and in the nuclear ribosomal IGSphylogeny (FIG. 73). Of these three species, intraspe-cific variation was detected only within the IGS re-gion in F. tucumaniae (FIG. 73), where two mono-phyletic subclades (designated A 5 86% bootstrapand B 5 98% bootstrap, respectively) of similar sizewere resolved, comprising four haplotypes. All six iso-lates from the northern pampean subregion of Cor-doba shared an identical IGS haplotype within sub-clade A. However, the nine isolates from the north-western state of Tucuman comprised 3 IGS haplo-types, one within subclade A and two within subcladeB (FIG. 73). The single isolate from the northernpampean state of Santa Fe (NRRL 31100) shared anidentical IGS haplotype with four isolates from Tu-cuman within subclade B.

DISCUSSION

Two morphologically and phylogenetically distinctspecies within the F. solani species complex are re-sponsible for SDS of soybean, F. tucumaniae in Ar-

gentina and F. virguliforme in the United States (Roy1997a, Rupe et al 2001). The species responsible forroot rot of Phaseolus vulgaris in the United States, F.phaseoli, based on F. martii f. phaseoli (5 F. solani f.phaseoli) (Burkholder 1919, Snyder and Hansen1941) was resolved cladistically as the sister of F. tuc-umaniae, indicating that host specificity might nothave a single evolutionary origin [i.e., ‘‘F. solani f. sp.glycines,’’ the name currently used to describe theSDS pathogen is non-monophyletic, see FIGS. 72 and73]. These new taxa differ morphologically fromeach other and from representative strains of themating populations of the F. solani complex exam-ined in this study. Fusarium tucumaniae is differenti-ated from F. virguliforme and F. phaseoli, based on itslonger and more slender sporodochial conidia, es-pecially by the mean sizes of its 4-septate conidia,which are more than 60 mm long. Fusarium virguli-forme can be distinguished from F. phaseoli based onpresence or absence of comma-shaped conidia andon differences in the morphology of falcate sporo-dochial conidia. Fusarium virguliforme forms comma-shaped conidia as the second type of sporodochial

680 MYCOLOGIA

FIG. 73. Single most-parsimonious tree inferred from the nuclear ribosomal intergenic region (IGS). Pathogenicity andconidial phenotypes are mapped onto the phylogram. Sequences of an alternate, more distant outgroup species, NRRLFusarium sp. 22387 (see FIG. 72 and O’Donnell 2000) supports a F. phaseoli—F. tucumaniae sister group relationship. CI,consistency index; RI, retention index.

conidia on PDA, but F. phaseoli does not. Falcate spo-rodochial conidia of F. virguliforme most frequentlyare widest at the mid-region of their length and taperand curve equally toward both ends, such that theapical and basal halves often are symmetrical mor-phologically. In contrast, falcate sporodochial conidiaof F. phaseoli possess nearly parallel dorsal and ventrallines and often are slightly wider toward the apex.Sporodochial conidia of F. phaseoli possess an acuateand curved apical cell and usually a distinct foot-likebasal cell, which differentiates it from F. virguliforme.

The most important morphological characterfound in this study is the exclusive production ofmultiseptate phialidic conidia from tall and slenderaerial conidiophores (A-1) by all three new speciescultured on SNA. Morphologically, these septate ae-rial conidia mostly are indistinguishable from theseptate sporodochial conidia of each species becauseboth conidial types are falcate and have a foot cell atthe base. Fusarium phaseoli, however, occasionallyformed shorter and thicker aerial conidia with arounded base. In typical F. solani complex strains

(MP-I, III–VII) examined in this study (Clade 3 sensuO’Donnell 2000; FIG. 72), oval, ellipsoidal to subcy-lindrical, 0–1-septate conidia were formed abundant-ly on tall and slender aerial conidiophores, occasion-ally with slightly curved, subcylindrical multiseptateconidia with tapering ends originating from the samephialides. Although production of these aerial conid-ia is common to the three new species described, itnever has been recognized as a part of the speciesconcept of F. solani. Appel and Wollenweber (1910),however, illustrated identical aerial conidia and co-nidiophores for F. martii Appel & Wollenw. and de-scribed them as ‘‘conidiophores isolated in the air’’from a culture on potato tuber. Aerial conidiophoresof F. martii were illustrated as tall and slender, un-branched or sparsely branched, in comparison withthe thick sporodochial conidiophores, which borecompact, dense branches. The species concept of F.martii, used by Sherbakoff (1915) for isolates frompotatoes, did not include aerial conidial structures.Because the species was reduced to F. solani var. mar-tii (Appel & Wollenw.) Wollenw. (Wollenweber


1931), these unique features of the aerial conidia andconidiophores apparently have never been reported.

Because of successive discoveries of additional co-nidial types within Fusarium (Pascoe 1990, Nirenbergand Aoki 1997, Aoki and Nirenberg 1999, Aoki et al2001), the traditional terms ‘‘macroconidia’’ and‘‘microconidia’’ have proven to be inadequate for de-scribing the full range of anamorph morphology ex-hibited by the fusaria. The three species described inthis study formed plural conidial types and, as an ex-treme, F. phaseoli produced three and two differenttypes of aerial and sporodochial conidia, respectively.For this reason, we have adopted the anatomical ter-minology given by Nirenberg and O’Donnell (1998)to describe anamorph morphology. Although F. vir-guliforme (5 F. solani f. sp. glycines) has been report-ed to form ‘‘microconidia’’ rarely (Roy 1997a, Roy etal 1997, Rupe and Hartman 1999), in this study, mi-nute, oblong-ellipsoidal to short-clavate, aerial conid-ia (A-2) corresponding to ‘‘microconidia’’ were ob-served in all strains of F. virguliforme cultured onSNA. Failure to observe this conidial type could bedue to their infrequent production in only a smallportion of each colony. By comparison, aerial falcateand multiseptate conidia on the tall conidiophores(A-1) were scattered on the entire surface of SNAcultures.

In this study, the close morphological and molec-ular phylogenetic relationship of F. virguliforme andF. phaseoli was resolved. However, the Argentine soy-bean SDS pathogen F. tucumaniae appears to bemore closely related to F. phaseoli than it is to F. vir-guliforme. These three species share a morphologicalcharacter with F. martii, i.e., the septate aerial conidiawith a foot cell. Fusarium phaseoli, F. tucumaniae andF. virguliforme all are nested within a putative SouthAmerican clade (Clade 2) together with several un-identified Fusarium species, while representatives ofF. solani complex MPs I–VII are nested within Clade3 (O’Donnell 2000) together with F. ambrosium(Gadd & Loos) Agnihothrudo & Nirenberg, F. solanif. sp. piperis Albuquerque and Neocosmospora vasin-fecta E.F. Smith (FIG. 72). Fusarium solani f. sp. pisi(Snyder and Hansen 1941), once classified as F. mar-tii var. pisi F.R. Jones (1923), is nested within Clade3. Although only one strain of F. solani f. sp. pisi wasincluded in this study, its morphological featuresshould be examined to determine whether it pro-duces conidiophores that form only septate aerial co-nidia with a foot cell. This feature is uniquely sharedby the three new species and F. martii described andillustrated by Appel and Wollenweber (1910).

DNA sequences from three of the five loci sampledresolved the three species described in this study asreciprocally monophyletic clades within a South

American clade of the F. solani species complex, andfour of the loci support a ((F. tucumaniae, F. phaseoli)(F. virguliforme)) relationship. As previously shown(O’Donnell 2000), sequences of the nuclear 28S andITS rDNA regions are too conserved to resolve thespecies limits of F. virguliforme and F. phaseoli, al-though a single base-pair indel within the ITS2 re-gion differentiates these taxa. Due to the high con-servation of these nuclear ribosomal loci, O’Donnelland Gray (1995) incorrectly concluded that thesespecies are conspecific. Sequences of the EF-1a gene(O’Donnell 2000), and especially those of the nucle-ar ribosomal IGS region, possess enough phyloge-netic signal to resolve these three closely related spe-cies. Although partial sequences of the IGS regionhave been used to investigate phylogenetic relation-ships within the F. oxysporum complex where putativeparalogs were discovered (Appel and Gordon 1996),the current study represents the first time that ho-mology assessment has not been an issue in the useof the IGS for low-level phylogenetics within Fusari-um. Sequences of the IGS also work well for phylog-eny reconstruction within the Gibberella fujikuroi spe-cies complex (O’Donnell unpubl).

In EF-1a, ITS and IGS gene trees, the two SDSpathogens did not form a monophyletic group,which suggests that either their pathogenicity to soy-bean has evolved convergently, as demonstrated forformae speciales within the F. solani (O’Donnell 2000)and F. oxysporum species complexes (O’Donnell et al1998, Baayen et al 2001), or else the most recentcommon ancestor of F. phaseoli might have lost itspathogenicity to this host. Pathogenicity studies arein progress to determine whether F. phaseoli can in-duce typical SDS symptoms on soybean together withstrains of closely related species. Rupe et al (2001)reported that one of these species, represented bystrain NRRL 22743 from Brazil (as F. solani f. sp. phas-eoli), could induce SDS-like symptoms on soybean.

Since it was first reported in Arkansas in 1972, SDSof soybean has been reported in the United Statesfrom at least 13 soybean-growing states (Hershman etal 1990, Jardine and Rupe 1993, Yang and Rizvi 1994,Hartman et al 1995, Roy 1997b, Roy et al 1997, Rupeand Hartman 1999, Pennypacker 1999) and fromOntario, Canada (Anderson and Tenuta 1998). Thedisease also has been reported in Argentina and Bra-zil (Ivancovich et al 1992, Botta et al 1993, Ploper1993, Nakajima et al 1993, Wrather et al 1997, Rupeand Hartman 1999). Although the causal pathogenof soybean SDS recently has been described as F. so-lani f. sp. glycines, based primarily on its pathogenic-ity to this host (Roy 1997a, Roy et al 1997), results ofour study clearly document the existence of two mor-phological and phylogenetic species corresponding

682 MYCOLOGIA

to this formae speciales, F. virguliforme currently re-sponsible for SDS within North America and F. tuc-umaniae in South America. Given that these twopathogens are deeply nested within a South Ameri-can clade of the F. solani complex, it seems likely thatthey switched to soybean sometime within the past100 years, after this crop was introduced to SouthAmerica from Asia. What remains unclear is the iden-tity the original hosts of these pathogens and theircurrent geographic distribution. F. virguliforme sur-prisingly has not been found in South America, al-though an explicit biogeographic hypothesis suggeststhat it is endemic to this region (O’Donnell 2000).Thus far F. tucumaniae is responsible for the knownoutbreaks of SDS of soybean in South America. Thehigh genetic similarity of strains of F. virguliforme iso-lated from soybean within the United States(O’Donnell and Gray 1995, Achenbach and Patrick1996, Li et al 2000, Rupe et al 2001) suggests that itmight represent a single introduction (Goodwin et al1994) or that this species is predominately or exclu-sively clonal in North America.

Results of this study provide a phylogenetic frame-work for understanding the species limits of severaleconomically important soybean and green or drybean pathogens. Because precise knowledge of thegenetic diversity of plant pathogens is crucial to thesuccess of disease-control efforts and breeding pro-grams (Taylor et al 1999), our results further high-light the failure of the forma specialis naming systembecause strains classified as f. sp. glycines and f. sp.phaseoli are not reciprocally monophyletic(O’Donnell unpubl). Clearly, plant breeding effortsmight benefit by including representatives of eachspecies when testing new varieties so as to increasethe likelihood of achieving broad-based resistance tothis pathogen complex. Finally, a multiplex polymer-ase chain reaction assay is being developed to rapidlydetect and identify the three pathogens reported inthis study based on informative variation within thealigned IGS sequences. In addition to the use of thisunique molecular diagnostic tool in better under-standing the phylogeographic structure and patho-biology of the soybean SDS pathogens, it has poten-tial use in tracking the global movement of thesepathogens.

ACKNOWLEDGMENTS

The authors thank Prof. Walter Gams for preparing the Lat-in diagnoses of F. tucumaniae and F. virguliforme. Specialthanks are due to all members of the soybean researchgroup at INTA-EEA, Marcos Juarez, Sr. Francisco H. Fuen-tes, INTA-EEA, Famailla, and members of the Phytopathol-ogy section, EEAOC, Tucuman, Argentina, for their assis-

tance in obtaining fresh soybeans exhibiting typical SDSsymptoms and for providing invaluable information on theoccurrence of SDS in Argentina.

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sudden-death syndrome of soybean is caused by two

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