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Sudden-death syndrome of soybean is caused by two morphologically and phylogenetically distinct species within the Fusarium solani species complex—F. virguliforme in North America and F. tucumaniae in South America

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

Kerry O'Donnell

     Microbial Genomics and Bioprocessing Research Unit, National Center for Agricultural Utilization Research, United States Department of Agriculture, Agricultural Research Service, Peoria, Illinois 61604-3999

Yoshihisa Homma ¹

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

Alfredo R. Lattanzi ¹

     Instituto Nacional de Tecnología Agropecuaria, Estación Experimental Agropecuaria (INTA-EEA) Marcos Juárez, Casilla de Correo 21, 2580 Marcos Juárez, Córdoba, Argentina

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS TAXONOMY DISCUSSION LITERATURE CITED

 

Soybean sudden-death syndrome has become a serious constraint to commercial production of this crop in North and South America during the past decade. To assess whether the primary etiological agent is panmictic in both hemispheres, morphological and molecular phylogenetic analyses were conducted on strains selected to represent the known pathogenic and genetic diversity of this pathogen. Maximum-parsimony analysis of DNA sequences from the nuclear ribosomal intergenic spacer region and the single copy nuclear gene translation elongation factor 1-, together with detailed morphological comparisons of conidial features, indicate that SDS of soybean in North and South America is caused by two phylogenetically and morphologically distinct species. Fusarium virguliforme sp. nov., formally known as F. solani f. sp. glycines, is described and illustrated for the SDS pathogen in North America, and F. tucumaniae sp. nov. is proposed for the South American pathogen. The molecular phylogenetic results challenge the forma specialis naming system because pathogenicity to soybean might have evolved convergently in F. tucumaniae and F. virguliforme. Phylogenetic evidence indicates the two SDS pathogens do not share a most recent common ancestor, since F. tucumaniae was resolved as a sister to a pathogen of Phaseolus vulgaris, F. phaseoli comb. nov. All three pathogens appear to have evolutionary origins in the southern hemisphere since they are deeply nested within a South American clade of the F. solani species complex.

Key words: Argentina, conidiogenesis, Fusarium phaseoli, Glycine max, Phaseolus vulgaris, SDS

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS TAXONOMY DISCUSSION LITERATURE CITED

 
During the past decade, sudden-death syndrome (SDS) of soybean (Glycine max (L.) Merr.) has reached epidemic proportions in North and South America (Roy et al 1997, Wrather et al 1997). First discovered in Arkansas in 1972, this disease has become widespread in soybean-growing regions in the United States (Rupe et al 2001), Argentina (Ploper 1993) and Brazil (Nakajima et al 1993). The etiological agent first was reported as Fusarium solani (Mart.) Sacc. (Rupe 1989, Roy et al 1989), but more recently it has been described as F. solani f. sp. glycines (Roy 1997a) to emphasize its putative host specialization. Although some recent taxonomic treatments of Fusarium Link (Nelson et al 1983, Burgess et al 1998) follow Snyder and Hansen (1941) in recognizing F. solani as the only species within the infrageneric section Martiella Wollenw., van Etten and Kistler (1988) have emphasized that the seven mating populations (MPs) of Nectria haematococca Berk. & Broome (Sakurai and Matuo 1960, Matuo 1972, Matuo and Snyder 1973) represent biologically distinct species. This latter view was supported by recent molecular phylogenetic analyses on DNA sequences that indicate the Martiella fusaria (i.e., the F. solani species complex) comprise at least 26 phylogenetically distinct species (O'Donnell 2000), most of which have not been described formally. Taxonomy of this clade is further complicated because Neocosmospora is the nomenclaturally and phylogenetically correct teleomorph name for the F. solani complex (for a discussion, see O'Donnell 2000).

Previous molecular and morphological analyses have included only North American isolates of the SDS pathogen (O'Donnell and Gray 1995, Achenbach et al 1996, O'Donnell 2000, Li et al 2000, Rupe et al 2001). All of the molecular data, based on DNA sequences (O'Donnell and Gray 1995, O'Donnell 2000, Li et al 2000), random amplified polymorphic DNA (Achenbach et al 1996) and restriction fragment-length polymorphisms (Rupe et al 2001), indicate that North American isolates of the SDS pathogen are genetically homogeneous and closely related to a root-rot pathogen of Phaseolus vulgaris L., F. solani f. sp. phaseoli (Burkh.) W.C. Snyder & H.N. Hansen.

The objective of this study was to compare morphologically and molecularly North and South American isolates of the soybean SDS pathogen using genealogical concordance phylogenetic species recognition (Taylor et al 2000). Precise knowledge of a species' limits and phylogeographic structure provide essential genetic data for tracking the intercontinental movement of foreign pathogens associated with world trade. Toward this end, South American isolates of the SDS pathogen were recovered during a 2001 field survey in Argentina, where the disease has been reported (Ivancovich et al 1992, Ploper 1993, Botta et al 1993). Based on detailed morphological comparisons and molecular phylogenetic analyses on DNA sequences from multiple loci, the Argentinean and North American isolates of the SDS pathogen and the bean root-rot pathogen were resolved as three distinct species within a South American clade of the F. solani species complex.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS TAXONOMY DISCUSSION LITERATURE CITED

 
Strains used in this study were isolated from soybean plants exhibiting symptoms of SDS or supplied from culture collection, and are listed in Table I. Infected soybean plants were collected at fields in three different regions of Argentina in February and March 2001: General Rocca (state of Córdoba, 15 Feb 2001), Las Rosas (state of Santa Fe, 19 Feb 2001) and San Agustin (state of Tucumán, 1 Mar 2001). Isolation of the causal fungus was performed at INTA-EEA at Marcos Juárez. The stem base and roots were cut from diseased soybean plants, washed under running tap water for 10 min and cut into pieces 1–5 cm in length with or without removing the cortex. The pieces were surface sterilized with 70% ethanol for 30 s and then in a solution of 10% ethanol with 0.5% sodium hypochlorite for 5 min, followed by a rinse in sterilized distilled water for 10 min. Samples were placed on sterilized filter paper in 9-cm Petri dishes to remove excess water and were dried overnight. Dried plant pieces were placed on synthetic low nutrient agar (SNA) (Nirenberg 1976, 1990) and incubated at 25 C. Sixteen isolates of the Argentinean soybean SDS pathogen were obtained and studied together with two SDS strains isolated at General Rocca, Córdoba, in 2000. Pathogenicity tests of the Argentinean isolates on various soybean cultivars were performed separately from this study and their pathogenic ability has been demonstrated.

Examination of morphological characters – Fusarium strains were 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 in the dark, under continuous fluorescent light (Mitsubishi FL40S-W) or under daylight. Average and standard deviation (SD) in the size of individual conidial types were derived from the measurement of 50 conidia, randomly chosen according to the number of septa from cultures grown under each of the cultural conditions. Colony morphology, color and odor were based primarily on cultures grown on PDA. Colors cited are given according to Kornerup and Wanscher (1978). Dried cultures were deposited as holotypes of the new taxa in the herbarium of the U.S. National Fungus Collection (BPI), USDA/ARS, Beltsville, Maryland, U.S.A. Descriptive terms for anamorph morphology follow Nirenberg and O'Donnell (1998).

Assessment of growth rate at different temperatures – For comparison of mycelial growth rates at various temperatures, agar blocks ca 5 x 5 mm were cut from the margins of 2-wk old cultures on SNA and transferred onto PDA. These culture plates were incubated under eight different temperatures between 5 and 40 C at 5 C intervals in the dark. Cultures were examined after 1 d and 5 d under a dissecting microscope, and colony margins were marked with permanent ink on the reverse side of the Petri dishes. Radial mycelial growth rates were calculated as mean values per day by measuring the difference in colony size in 16 directions around the colony during the four days of incubation. Measurements were repeated at least twice and averaged.

Molecular biology – Total genomic DNA was prepared as described in O'Donnell (2000). Domains D1 and D2 of the nuclear large subunit rDNA (28S), the nuclear ribosomal internal transcribed spacer (ITS) region and a portion of the translation elongation factor 1- gene (EF-1) were amplified and sequenced with primers and reagents described in White et al (1990) and O'Donnell (2000). The nuclear ribosomal intergenic spacer (IGS) was amplified with the primer pair NL11 (5'-CTGAACGCCTCTAAGTCAG) and CNS1 (5'-GAGACAAGCATATGACTAC). In addition, these four internal primers were used to sequence the entire IGS region: SCNS3 (5'-GGTCTGAAAGATCAGGTACG), SCNS5 (5'-TACCCTATACCTCCGCCAAC) and SCNS7 (5'-TACCCTATACCACCTAGTAGC). Sequencing reactions were purified by gel filtration and run on either an Applied Biosystems model 377 or 3100 automated sequencer, as previously described (O'Donnell 2000).

Molecular phylogenetic analysis – DNA sequences were edited and aligned visually using Sequencher 4.1.2 (Gene Codes Corporation, Ann Arbor, Michigan). Sequences of Fusarium illudens C. Booth and Nectria plagianthi Dingley were selected as outgroup taxa based on a previous phylogenetic analysis (O'Donnell 2000). PAUP*4.0b4a (Swofford 2002) was used to conduct unweighted parsimony analyses on the aligned 28S rDNA, ITS rDNA and EF-1 sequences as separate and combined datasets for the 43 taxon matrix. Sequences from the IGS region for the three species described in this study were analyzed separately. For all analyses the heuristic search option was used with 1000 random addition sequences with MULPARS on and TBR branch swapping. Phylogenetically informative indels were coded as a fifth character state. Clade stability was assessed by 1000 parsimony bootstrap replications. Combinability of the individual partitions was assessed with the nonparametric Templeton Wilcoxon signed-ranks (WS-R) test implemented in PAUP* using 70% bootstrap consensus trees as constraints. Sequences have been deposited in GenBank as AY220150-AY220239, and the alignments have been deposited in TreeBASE as M1370 and M1371.

	TAXONOMY

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS TAXONOMY DISCUSSION LITERATURE CITED

 
Fusarium tucumaniae T. Aoki, O'Donnell, Yos. Homma et Lattanzi, sp. nov. Figs. 1–18

View larger version (63K): [in this window] [in a new window]   FIG. 1. Fusarium tucumaniae (NRRL 31096, ex holotype) cultured in the dark. A–E: cultured on SNA, F–H: cultured on PDA. A. Septate, falcate conidia with a foot cell formed on tall, slender aerial conidiophores. B. Aseptate, short-clavate conidia formed on a short aerial conidiophore. C, F. Septate, falcate to curved cylindrical conidia with a foot cell formed on branched sporodochial conidiophores. D. Chlamydospores in conidia. E, H. Smooth to rough-walled, terminal or intercalary chlamydospores formed in hyphae. G. Aseptate, minute ovate conidia observed in a culture on PDA. Scale bar: 25 µm

Coloniae in agaro PDA dicto 20 C obscuritate tarde expandentes, albae, luteolo-albae vel coeruleo-griseae, in parte sporulante pustulis dilute flavis vel viridi-albis, sub luce fluorescente vel diurna pustulis dilute flavis, viridi-albis, griseo-luteis, griseo-viridibus, griseo-glaucis, viridibus, obscure viridibus vel caerulescentibus. Mycelium aerium vulgo parcum, pionnoti simile, nonnumquam copiosum, laxum vel dense floccosum, album, flavo-album vel coeruleo-griseum. Reversum saepe incoloratum, griseo-luteum vel dilute brunneum. Chlamydosporae in hyphis et in conidiis frequentes, plerumque subglobosae, terminales vel intercalares, singulae, raro catenatae, hyalinae vel pallide pigmentatae, leves vel asperatae. Sclerotia absentia. Sporodochia plerumque copiosa in agaris SNA et PDA, parca in fasciculis hypharum in PDA. Conidiophora aeria copiosa in SNA, nonnumquam in PDA, simplicia vel parce ramosa, brevia vel ad 270 µm longa, 2–6.5 µm lata, monophialides terminales integratas formantia. Phialides aeriae simplices, subulatae vel subcylindricae. Conidia aeria dimorpha: (1) cylindrica curvata vel falcata, (2–)3(–5)-septata, basi distincta pediformi, in conidiophoris majoribus formata; (2) minuta, oblongo-ellipsoidea vel breviter clavata vel ovalia, in conidiophoris ad 50 µm longis et 1.5–3 µm latis formata, 0(–1)-septata, 3.5–18.5 x 1.5–4.5 µm. Conidiophora sporodochialia verticillata vel raro simplicia; monophialides simplices, subulatae, ampulliformes vel subcylindricae. Conidia plerumque cylindrica vel modice curvata, nonnumquam falcata, circumscriptione interna et externa quasi parallelis, sursum modice expandentia, cellulae apicali acutae, basilari pediformi, (2–)3–4(–7)-septata; ubi 3-septata in SNA 35.5–85.5 x 3.5–6 µm, ubi 4-septata in SNA 49.5–95 x 4–5.5 µm, ubi 5-septata in SNA 49–105.5 x 3.5–6 µm.

Colonies on PDA showing radial mycelial growth rates of 1.0–2.2 mm per d at 20 C in the dark. Colony color 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), dark green (25-26F6-8) to dark turquoise (24F6-8) under fluorescent or daylight. Aerial mycelium generally sparse with pionnotal colony appearance, some developed abundantly, then loose to dense floccose, white (1A1), yellowish-white (2-4A2), sometimes bluish-gray (22-23B3) to grayish-blue (22-23D6). Colony margin entire to undulate. Reverse pigmentation often absent, sometimes grayish-yellow (4C4-6) to light brown (5D4-5). Odor absent or sometimes putrid or moldy. Chlamydospores formed frequently in hyphae and in conidia, mostly subglobose, often terminal, occasionally intercalary, single, rarely in chains, hyaline, pale to yellowish-gray or pale-yellow, smooth to rough-walled, sometimes verruculose, 9-13 x 8.5-12.5 µm. Sclerotia absent. Sporulation generally rapid and abundant; on PDA often light-colored in the dark, greenish-to-bluish under fluorescent light or daylight; sporodochia normally formed abundantly on SNA and PDA, but sparsely in mycelial strains on PDA. Aerial conidiophores formed abundantly on SNA, sometimes on PDA, generally unbranched, occasionally sparsely branched from their base or middle, up to 270 µm long, 2-6.5 µm wide, forming monophialides integrated in the apices. Aerial phialides simple, subulate to subcylindrical. Aerial conidia of two types; (1) curved cylindrical to falcate, (2-)3(-5)-septate, with a foot cell, morphologically indistinguishable from falcate sporodochial conidia, formed mainly on taller conidiophores; (2) minute, oblong-ellipsoidal, short-clavate to oval, 0(–1)-septate, 3.5–(6.5–8.2)–18.5 x 1.5–(2.4–2.8)–4 µm (ranges of the averages for individual isolates in parentheses) [ex type: 5.5–(7.3 ± 0.89)–9.5 x 2–(2.5 ± 0.25)–3 µm (averages ± SDs in parentheses)] formed on short conidiophores up to 50 µm long, 1.5–3 µm wide. Sporodochial conidiophores branched verticillately, or rarely unbranched, forming apical monophialides. Sporodochial phialides simple, subulate, ampulliform to subcylindric, often with a conspicuous collarette at the tip. Sporodochial conidia generally cylindrical and gently curved, sometimes falcate, with dorsal and ventral lines nearly parallel or gradually wider upward, with an acuate apical cell and a distinct basal foot cell, (2–)3–4(–7)-septate; 3-septate on SNA: 35.5–(52.4–71.4)–85.5 x 3.5–(4.5–4.9)–6 µm [ex type: 38.5–(63.9 ± 12.39)–83.5 x 4–(4.8 ± 0.27)–5.5 µm], on PDA: 38–(57.5–67.4)–81 x 3.5–(4.4–4.9)–5.5 µm [ex type: 45–(66.4 ± 5.89)–81 x 4–(4.7 ± 0.29)–5.5 µm]; 4-septate on SNA: 49.5–(61.3–77.3)–95 x 4–(4.7–5.0)–5.5 µm [ex type: 49.5–(64.0 ± 7.62)–84 x 4–(4.7 ± 0.25)–5.5 µm], on PDA: 48–(60.3–76.0)–88 x 3.5–(4.4–5.0)–5.5 µm [ex type: 66–(72.1 ± 3.21)–81 x 4–(4.8 ± 0.27)–5.5 µm]; 5-septate on SNA: 49–(67.9–86.4)–105.5 x 3.5–(4.6–4.9)–6 µm [ex type: 76–(84.9 ± 4.45)–94 x 4–(4.9 ± 0.21)–5.5 µm].

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

Ex holotype culture. NRRL 31096 = MAFF 238418 = MJ-172.

Additional cultures studied. ARGENTINA. CÓRDOBA: General Rocca, from G. max, 3 Mar 2000, Y. Homma (NRRL 31085, NRRL 31086), 15 Feb 2001, T. Aoki (NRRL 31087, NRRL 31088, NRRL 31089, NRRL 31099); SANTA FE: Las Rosas, from G. max, 19 Feb 2001, T. Aoki (NRRL 31100); TUCUMÁN: 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 name for Argentina, Tucumania.

Notes. Key morphological characters that distinguish F. tucumaniae from other species within the F. solani complex include the production of septate, falcate conidia with a foot cell formed on tall and slender, aerial conidiophores (Figs. 1A, 2–4, 7–9) together with long, slender sporodochial conidia with mostly pointed apices (Figs. 1C, F, 11–15). In contrast to F. virguliforme and F. phaseoli n. spp. described in this study, the second type of sporodochial conidia are never formed in F. tucumaniae.

View larger version (172K): [in this window] [in a new window]   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 aerial conidiophores and falcate conidia with a foot cell formed on the agar surface. 10. Aseptate, short clavate to oblong conidia formed on a short aerial conidiophore. 2–6, 8, 9 from NRRL 31096, 7 from NRRL 31099, and 10 from NRRL 31092. Scale bars: 2–7 = 50 µm, 8–10 = 20 µm

View larger version (153K): [in this window] [in a new window]   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. Sporodochial conidia observed on SNA (13) and PDA (14, 15); septation is obscured in immature conidia on PDA (14), but septa 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 NRRL 31098, and 15 from NRRL 31085. Scale bars: 11 = 50 µm, 12–18 = 20 µm

= Fusarium solani (Mart.) Sacc. f. sp. glycines K. Roy s. str., Plant Dis. 81: 259–266. 1997.

? = Fusarium martii Appel & Wollenw. var. viride Sherb., Mem. Cornell Univ. Agric. Exp. Stat. 6: 247–249, 1915. Figs. 19–46

View larger version (72K): [in this window] [in a new window]   FIG. 19. Fusarium virguliforme (NRRL 31041, ex holotype) cultured in the dark. A–E: cultured on SNA, F–J: cultured on PDA. 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, falcate conidia with a foot cell and comma-shaped conidia from phialides. G. Minute, aseptate and short-clavate conidia observed in a culture on PDA. H. Zero–1-septate comma-shaped conidia formed in culture on PDA. Scale bar: 25 µm

Colonies on PDA showing radial mycelial growth rates of 1.3–1.7 mm per d at 20 C in the dark. Colony color 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 light yellow (3-4A4-5), grayish-yellow (1-4B3-7), grayish-orange (5B3-5), greenish-white (26-29A2), grayish-turquoise (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 fluorescent or daylight. Aerial mycelium sparse with pionnotal colony appearance, or sometimes developed abundantly, then loose to dense floccose, white (1A1), yellowish-white (2-4A2), sometimes bluish-gray (21-23B-C2-3). Colony margin entire to often undulate. Reverse pigmentation often absent, sometimes grayish-yellow (4B-C4-6), grayish-orange (5B4-6) to brownish-orange (5C3-6), or olive brown (4D-F5-6) to yellowish-brown (5D-F5-6). Yellowish exudate sometimes present. Odor absent or sometimes putrid or moldy. Chlamydospores formed abundantly in mycelium and in conidia, mostly subglobose, intercalary or terminal, mostly single, rarely in chains, hyaline to pale or pale-yellow, smooth to rough-walled, 4–15 µm diam. Typical sclerotia absent. Sporulation generally rapid and abundant; on PDA often light-colored in the dark, greenish-to-bluish under fluorescent light or daylight; sporodochia normally formed abundantly on SNA and PDA but sparsely in mycelial strains on PDA. Aerial conidiophores formed abundantly on SNA, rarely on PDA, unbranched or sparsely branched, up to 290 µm long, 2–7 µm wide, forming monophialides integrated in the apices. Aerial phialides simple, subulate to subcylindrical, often with a conspicuous collarette at the tip. Aerial conidia of two types; (1) curved cylindrical to falcate, (2–)3(–4)-septate, with a foot cell, morphologically indistinguishable from falcate sporodochial conidia, formed mainly on taller conidiophores; (2) minute, oblong-ellipsoidal to short-clavate, 0(–1)-septate, 3–(5.5–10.8)–18.5 x 1.5–(2.3–2.7)–3.5 µm [ex type: 6–(9.0 ± 1.10)–11.5 x 2–(2.5 ± 0.28)–3.5 µm] formed in a small portion of the colony and on short conidiophores up to 60 µm long, 2–2.5 µm wide. Sporodochial conidiophores branched verticillately, or rarely unbranched, forming apical monophialides. Sporodochial phialides simple, subulate, ampulliform to subcylindric, with a conspicuous collarette at the tip. Sporodochial conidia of two types; (1) typically falcate, dorsiventral, most frequently widest at the midregion of their length, often tapering and curving equally toward both ends, with the apex and foot cell typically similarly pointed and often indistinguishable, (2–)3–4(–5)-septate, formed on PDA and on SNA; apical and basal halves often morphologically symmetrical; 3-septate on SNA: 33–(47.3–54.8)–65.5 x 3.5–(4.8–5.4)–6 µm [ex type: 37.5–(48.8 ± 4.33)–58 x 4.5–(5.2 ± 0.26)–5.5 µm], on PDA: 27–(45.0–52.2)–60.5 x 4–(4.7–5.2)–6 µm [ex type: 27–(45.5 ± 5.21)–56 x 4.5–(5.0 ± 0.20)–5.5 µm]; 4-septate on SNA: 43–(53.3–56.9)–67 x 4.5–(5.2–5.4)–6 µm [ex type: 47–(54.2 ± 3.73)–64 x 4.5–(5.3 ± 0.26)–6 µm], on PDA: 45.5–(54.3–57.2) –66 x 4.5–(5.0–5.2)–6 µm [ex type: 50.5–(55.6 ± 2.87)–63 x 4.5–(5.0 ± 0.13)–5.5 µm]; 5-septate on SNA: 46–(57.8–63.5)–79.5 x 4–(5.0–5.4)–7 µm [ex type: 47.5–(57.8 ± 3.50)–67.5 x 5–(5.4 ± 0.29)–6.5 µm]; (2) comma-shaped to sometimes short-clavate, with a swollen apex often rounded but rarely pointed and with a tapering and curving base, formed only on PDA often in the dark, 0–1(–2)-septate, 12–(18.2–22.4)–33.5 x 4–(5.1–6.4)–7.5 µm [ex type: 15–(21.4 ± 2.74)–26 x 5–(6.2 ± 0.62)–7.5 µm].

Type specimen. UNITED STATES. ILLINOIS: dried culture isolated from Glycine max, 1998, Shuxian Li (HOLOTYPE, BPI 841956).

Ex holotype culture. NRRL 31041 = MAFF 238553 = Shuxian Li # 95.

Additional cultures studied. UNITED STATES: from G. max, K. W. Roy (NRRL 22489, NRRL 22490); UNITED STATES. 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 sporodochial conidia.

Notes. Fusarium virguliforme is distinguished from other species within the F. solani complex by the production of comma-shaped sporodochial conidia on PDA (Figs. 19H, 35–41) together with septate, falcate aerial conidia with a foot cell on SNA (Figs. 19A, 20–25). Minute, oblong-ellipsoidal to short-clavate conidia were formed from short conidiophores up to 60 µm long (Figs. 19B, 26, 27), but they were observed only in a small portion of the entire colony. Fusarium virguliforme resembles F. martii var. viride isolated from potato in the dimensions of its sporodochial conidia. Sherbakoff (1915) illustrated a comma-shaped conidium from a culture on a potato stem plug (p. 245, Fig. 44G) but did not include a description of sporodochial conidia of this shape. Conidial masses of this fungus on potato agar rich in glucose (nearly equivalent to PDA) were described as pale smoke-gray and without dark blue coloration (Sherbakoff 1915), while those of F. virguliforme on PDA are variable but often greenish-to-bluish when cultured under fluorescent light or daylight. Furthermore, the host plants of these taxa are different. Unfortunately, no authentic material of F. martii var. viride was left by Sherbakoff which makes it difficult to ascertain whether the variety and F. virguliforme are conspecific.

View larger version (166K): [in this window] [in a new window]   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 conidia; comma-shaped conidia were formed on the same conidiophores (arrowheads in 30). 31–34. Sporodochial conidia observed in culture on SNA (31) and PDA (32–34); septation is obscured in young conidia on PDA (32) but becomes clear as vacuoles form in the conidia (33, 34). 35–41. Zero–1-septate comma-shaped conidia formed only on PDA. 42–45. Terminal or 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 from NRRL 22292, 38, 39 from NRRL 22489 and 40 from NRRL 22490. Scale bars: 28–30 = 50 µm, 31–46 = 20 µm

View larger version (112K): [in this window] [in a new window]   FIGS. 20–27. Aerial conidia and conidiophores of Fusarium virguliforme cultured on SNA in the dark (20–24, 26: aerial view, 25, 27: water mounted). 20–24. Falcate aerial conidia formed on slender conidiophores arising from hyphae on the agar surface. 25. Slender, unbranched aerial conidiophores. 26. Minute conidia formed on short aerial conidiophores in false 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 = 50 µm, 25, 27 = 20 µm

Fusarium martii Appel & Wollenw. f. phaseoli Burkh., Mem. Cornell Univ. Agric. Exp. Stat. 26: 1007–1012, 1919. (designated in trinomial as F. martii phaseoli)

= 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)

= Fusarium martii Appel & Wollenw. var. minus Sherb., Mem. Cornell Univ. Agric. Exp. Stat. 6: 249–250, 1915. Figs. 47–68

View larger version (61K): [in this window] [in a new window]   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 aerial conidiophores. F. Aseptate, short-clavate conidia formed on short aerial conidiophores. A–C from NRRL 22276, and D–H from NRRL 31156. Scale bar: 25 µm

Cultures studied. UNITED STATES: from Phaseolus vulgaris, H. VanEtten (NRRL 22276); UNITED STATES. MICHIGAN: from P. vulgaris (NRRL 31156). Additional data are presented in Table I.

Notes. Morphological and cultural features of the strains examined agreed well with the original description of F. martii f. phaseoli given by Burkholder (1919) especially in the dimensions and morphology of septate sporodochial conidia formed on PDA (Figs. 47B, G, 48A, D, 61–64). Fusarium phaseoli can be differentiated from other members of the F. solani complex by the production of septate, falcate aerial conidia with a foot cell (Figs. 47A, D, 49–53) together with falcate sporodochial conidia with asymmetric ends. In F. phaseoli, minute, short-clavate to ellipsoidal conidia were only produced on short aerial conidiophores up to 45 µm long and in a minor portion of a colony. Fusarium phaseoli is similar to F. virguliforme in its conidial dimensions (Figs. 69, 70). However, F. phaseoli does not produce comma-shaped conidia in sporodochia on PDA. Falcate sporodochial conidia of F. phaseoli also differ morphologically from those of F. virguliforme in that those of F. phaseoli possess 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 the dorsal and ventral lines of the sporodochial conidia of F. phaseoli are nearly parallel and often are gradually and slightly wider toward the apex. Therefore, the apical and basal parts of the sporodochial conidia of F. phaseoli are asymmetrical, in most cases, and diagnostic. In contrast, the apical and basal parts of the sporodochial conidia of F. virguliforme often are symmetrical (Figs. 19C, F, 31–34). Fusarium phaseoli also formed short clavate to ellipsoidal or sometimes naviculate, straight or slightly curved sporodochial conidia with a rounded apex and a truncate base on PDA and on steamed rice (Figs. 48E, H, 65). The morphology of these conidia corresponds well with those described and illustrated by Burkholder (1919; p. 1009, Fig. 134C) for F. martii f. phaseoli on steamed rice. In addition, large obovate to ellipsoidal conidia also were observed on elongate aerial conidiophores of F. phaseoli on SNA (Figs. 47E, 54–55).

View larger version (51K): [in this window] [in a new window]   FIG. 48. Fusarium phaseoli cultured in the dark. A, D. Septate, falcate conidia with a foot cell formed on sporodochial conidiophores. 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. Sporodochial conidia 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 µm

View larger version (183K): [in this window] [in a new window]   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. Sporodochial conidia observed in culture on SNA (61) and PDA (62–64); septation is obscured in young conidia on PDA (62) but becomes conspicuous as the conidia mature (63, 64). 65. Naviculate sporodochial conidium. 66–68. Terminal or intercalary chlamydospores in hyphae. 57–61, 66–68 cultured on SNA, and 62–65 on PDA. 57–62, 65–68 from NRRL 31156, and 63, 64 from NRRL 22276. Scale bars: 57, 58 = 50 µm, 59–68 = 20 µm

View larger version (132K): [in this window] [in a new window]   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 conidiophores forming 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 = 50 µm, 53, 55, 56 = 20 µm.

View larger version (28K): [in this window] [in a new window]   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 at 20 C.

View larger version (29K): [in this window] [in a new window]   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 at 20 C.

Another root-rot pathogen of bean, F. aduncisporum Weimer & Harter (= F. solani var. aduncisporum (Weimer & Harter) Wollenw.), was described as having different morphological and physiological features from F. martii f. phaseoli (Weimer and Harter 1926). Although F. aduncisporum was placed in synonymy with F. solani f. phaseoli (= F. phaseoli) by Snyder and Hansen (1941) and in F. solani by Gerlach and Nirenberg (1982), it possesses distinctly curved to hook-shaped sporodochial conidia and the ends of its conidia are described as rounded with a basal cell that was either not foot-like or only slightly so. Because no living culture of F. aduncisporum was available, the taxon tentatively was not included in the synonymy of F. phaseoli.

Morphological comparison of the three species and F. solani – Features shared by SDS isolates from Argentina (= F. tucumaniae), from the United States (= F. virguliforme) and in the bean root-rot isolates from the United States (= F. phaseoli) include the formation of plural types of conidia, especially on the aerial conidiophores. Three different types of aerial conidia were found: (A-1) falcate, multiseptate aerial conidia with a foot cell formed mainly on tall and slender conidiophores observed in all three species only on SNA; (A-2) minute, short-clavate to ellipsoidal aerial conidia formed on short conidiophores in a minor portion of an entire colony in all three species; (A-3) large, obovate, short-clavate to ellipsoidal conidia formed on taller conidiophores only in F. phaseoli on SNA. Three types of sporodochial conidia also were found: (S-1) falcate, multiseptate conidia with a foot 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 types in sporodochia.

A distinctive morphological character common to the three new species, but not recognized in the concept of F. solani, is the production of the curved cylindrical to falcate, multiseptate aerial conidia with a foot cell (A-1). These conidia were formed by all strains of the three species cultured on SNA without exception from phialides on generally tall and slender conidiophores. These conidiophores were septate, simple or sparsely branched, often more than 100 µm long, and bore slender phialides integrated in their apices. Sporodochial conidiophores, in contrast, branched repeatedly and compactly and their phialides 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 indistinguishable morphologically from the septate sporodochial conidia (S-1) because both conidial types have a foot cell at the base. Some shorter and thicker multiseptate conidia of this type, but with a rounded base, 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 to subcylindrical, 0–1-septate conidia (so-called "microconidia") were formed abundantly on tall and slender aerial conidiophores on both SNA and PDA. In older SNA culture, these conidiophores occasionally formed slightly curved, subcylindrical multiseptate conidia with tapering ends, together with a mass of the oval to ellipsoidal "microconidia". Minute, short clavate to ellipsoidal, mostly 0-septate conidia (A-2) were observed in the three new species in a minor portion of an entire colony on SNA and rarely on PDA. However, they were formed separately on short aerial conidiophores, often less than 50 µm long, but not on the same tall and slender aerial conidiophores forming 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 "microconidia" formed by the typical F. solani complex strains, where aerial ellipsoidal conidia were larger and formed on tall, slender conidiophores that were often more than 200 µm in length.

The morphology of septate sporodochial conidia was evaluated on SNA and on PDA. Falcate sporodochial conidia (S-1) were most frequently 3- to 4-septate in the three species described in this study. Septation of the sporodochial conidia observed on PDA often was difficult to score in younger conidia because the cytoplasmic contents were granular. However, septa became distinct as the conidia became vacuolated in age (Figs. 14, 15, 32–34, 62–64). Conidial septation was clearly observed in cultures on SNA (Figs. 11–13, 29, 31, 61). Sizes of the septate sporodochial conidia were compared from cultures on SNA and PDA and average values of conidial length and width for individual strains were plotted in Figs. 69 and 70 according to the number of septa. Measurements of 3- and 4-septate conidia of the three species yielded similar results on SNA (Fig. 69) and on PDA (Fig. 70). Conidia of F. tucumaniae were longer and narrower than those of the other two species, especially in the size of 4-septate conidia on SNA (Fig. 69). In cultures on PDA, F. tucumaniae clearly was distinguishable from the other species based on the dimensions of 3- or 4-septate conidia (Fig. 70). The sharply pointed ends of conidia in F. tucumaniae provided an additional diagnostic character (Figs. 1C, F, 11–15). There was a tendency for conidia of F. virguliforme, especially those that were 4-septate, to be longer than those of F. phaseoli, but the ranges of conidial sizes often overlapped such that this character lacked diagnostic value (Figs. 19F, 69, 70). Conidia of F. virguliforme and those of F. phaseoli, however, showed other morphological differences. Conidia of F. virguliforme most frequently were widest at the midregion and tapered and curved equally towards both ends. The apical and foot cells were symmetrical and often indistinguishable (Figs. 31–34). In contrast, the dorsal and ventral lines of conidia in F. phaseoli were nearly parallel or often gradually widened upward, and their apical and basal parts frequently were curved ventrally. Therefore, differences between the apical and foot cell were conspicuous in F. phaseoli (Figs. 61–64).

Diagnostic morphological features were discovered for F. virguliforme and F. phaseoli by the comparison of cultures on SNA and PDA. All strains of F. virguliforme cultured on PDA formed comma-shaped conidia in sporodochia (S-2; Figs. 19F, 30) which were frequently 0–1(–2)-septate, curved, and wider and swollen upwards (Figs. 19H, 35–41), especially from cultures incubated in the dark. Although comma-shaped conidia were not observed in cultures on SNA, septate aerial conidia with a foot cell were constantly formed on tall conidiophores. Strains of F. phaseoli did not form comma-shaped conidia on PDA or SNA, although short-clavate to naviculate conidia were sometimes formed in sporodochia on PDA and on steamed rice (S-3; Figs. 48E, H, 65). On SNA, relatively large, ellipsoidal to obovate, 0–2(–3)-septate conidia were formed by strains of F. phaseoli on tall and slender aerial conidiophores (A-3; Figs. 47E, 54, 55). This morphology was unique to strains of F. phaseoli.

Terminal or intercalary chlamydospores were formed commonly and often abundantly by strains of the three species (Figs. 1E, H, 16, 17, 19E, J, 42–45, 47C, H, 48C, G, 66–68). Some conidial chlamydospores also were observed (Figs. 1D, 18, 19D, I, 46, 47H, 48B, F), especially in old cultures. They were smooth- to rough-walled and occasionally possessed a yellowish pigment.

Colony characteristics were compared on PDA in 9-cm plastic Petri dishes at 20 C, but no clear difference among the three species was observed. Mycelial and pionnotal strains were found in each of the three species and greater numbers of conidia were formed on the colonies of the pionnotal strains. Pionnotal sectors sometimes were observed in colonies of mycelial strains, where conidial production was higher than in the mycelial parts of the colonies. These facts might suggest the possible occurrence of mutation, but intraspecific variation observed among strains appeared to be related to the light conditions employed. When cultures were grown in the dark, colony color often remained whitish to yellowish, although greenish or bluish coloration sometimes was observed. When cultured under fluorescent light or under daylight, colonies frequently became more greenish to bluish and dark green, and a dark turquoise coloration also was observed as an extreme in one-month-old cultures. However, differences in colony morphology or coloration were not useful for species delimitation.

Difference in radial mycelial growth rates – Average radial mycelial growth rates on PDA in the dark at eight different temperatures between 5 to 40 C were calculated for 16 strains of F. tucumaniae, eight strains of F. virguliforme and two strains of F. phaseoli, and are summarized in Fig. 71. Eleven representative strains of the F. solani complex (MP-I, III–VII) were examined for comparison. Optimal temperature for mycelial 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 for F. tucumaniae; 1.7–2.1 mm/day for F. virguliforme; 1.5–1.6 mm/day for F. phaseoli. Average growth rates of the latter three species were nearly half that of the representative F. solani complex strains. This ratio was nearly the same for the other temperatures examined.

View larger version (25K): [in this window] [in a new window]   FIG. 71. Comparison of radial growth rates per day on PDA of Fusarium tucumaniae, F. virguliforme and F. phaseoli, with strains of the representative MPs of the F. solani complex under different temperature from 5 to 40 C. The thick horizontal and vertical bars indicate means and total ranges, respectively, among the strains of each species (number of strains examined in parentheses).

View larger version (42K): [in this window] [in a new window]   FIG. 72. The single most-parsimonious phylogram inferred from the combined nuclear 28S rDNA, ribosomal ITS region and EF-1 gene for the Fusarium solani species complex. Bootstrap replication frequencies above 70% are indicated above nodes. Sequences of Fusarium illudens and Nectria plagianthi were used as outgroup taxa. Note that the SDS pathogen within North America, F. virguliforme, is a sister to F. phaseoli and F. tucumaniae within a South American clade of this species complex. CI, consistency index; RI, retention index; MP, mating populations or biological species.

View larger version (39K): [in this window] [in a new window]   FIG. 73. Single most-parsimonious tree inferred from the nuclear ribosomal intergenic region (IGS). Pathogenicity and conidial phenotypes are mapped onto the phylogram. Sequences of an alternate, more distant outgroup species, NRRL Fusarium sp. 22387 (see Fig. 72 and O'Donnell 2000) supports a F. phaseoli—F. tucumaniae sister group relationship. CI, consistency index; RI, retention index.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS TAXONOMY DISCUSSION LITERATURE CITED

The most important morphological character found in this study is the exclusive production of multiseptate phialidic conidia from tall and slender aerial conidiophores (A-1) by all three new species cultured on SNA. Morphologically, these septate aerial conidia mostly are indistinguishable from the septate sporodochial conidia of each species because both conidial types are falcate and have a foot cell at the base. Fusarium phaseoli, however, occasionally formed shorter and thicker aerial conidia with a rounded base. In typical F. solani complex strains (MP-I, III–VII) examined in this study (Clade 3 sensu O'Donnell 2000; Fig. 72), oval, ellipsoidal to subcylindrical, 0–1-septate conidia were formed abundantly on tall and slender aerial conidiophores, occasionally with slightly curved, subcylindrical multiseptate conidia with tapering ends originating from the same phialides. Although production of these aerial conidia is common to the three new species described, it never has been recognized as a part of the species concept of F. solani. Appel and Wollenweber (1910), however, illustrated identical aerial conidia and conidiophores for F. martii Appel & Wollenw. and described them as "conidiophores isolated in the air" from a culture on potato tuber. Aerial conidiophores of F. martii were illustrated as tall and slender, unbranched or sparsely branched, in comparison with the thick sporodochial conidiophores, which bore compact, dense branches. The species concept of F. martii, used by Sherbakoff (1915) for isolates from potatoes, did not include aerial conidial structures. Because the species was reduced to F. solani var. martii (Appel & Wollenw.) Wollenw. (Wollenweber 1931), these unique features of the aerial conidia and conidiophores apparently have never been reported.

Because of successive discoveries of additional conidial types within Fusarium (Pascoe 1990, Nirenberg and Aoki 1997, Aoki and Nirenberg 1999, Aoki et al 2001), the traditional terms "macroconidia" and "microconidia" have proven to be inadequate for describing the full range of anamorph morphology exhibited by the fusaria. The three species described in this study formed plural conidial types and, as an extreme, F. phaseoli produced three and two different types of aerial and sporodochial conidia, respectively. For this reason, we have adopted the anatomical terminology given by Nirenberg and O'Donnell (1998) to describe anamorph morphology. Although F. virguliforme (= F. solani f. sp. glycines) has been reported to form "microconidia" rarely (Roy 1997a, Roy et al 1997, Rupe and Hartman 1999), in this study, minute, oblong-ellipsoidal to short-clavate, aerial conidia (A-2) corresponding to "microconidia" were observed in all strains of F. virguliforme cultured on SNA. Failure to observe this conidial type could be due to their infrequent production in only a small portion of each colony. By comparison, aerial falcate and multiseptate conidia on the tall conidiophores (A-1) were scattered on the entire surface of SNA cultures.

In this study, the close morphological and molecular phylogenetic relationship of F. virguliforme and F. phaseoli was resolved. However, the Argentine soybean SDS pathogen F. tucumaniae appears to be more closely related to F. phaseoli than it is to F. virguliforme. These three species share a morphological character with F. martii, i.e., the septate aerial conidia with a foot cell. Fusarium phaseoli, F. tucumaniae and F. virguliforme all are nested within a putative South American clade (Clade 2) together with several unidentified Fusarium species, while representatives of F. solani complex MPs I–VII are nested within Clade 3 (O'Donnell 2000) together with F. ambrosium (Gadd & Loos) Agnihothrudo & Nirenberg, F. solani f. sp. piperis Albuquerque and Neocosmospora vasinfecta E.F. Smith (Fig. 72). Fusarium solani f. sp. pisi (Snyder and Hansen 1941), once classified as F. martii var. pisi F.R. Jones (1923), is nested within Clade 3. Although only one strain of F. solani f. sp. pisi was included in this study, its morphological features should be examined to determine whether it produces conidiophores that form only septate aerial conidia with a foot cell. This feature is uniquely shared by the three new species and F. martii described and illustrated by Appel and Wollenweber (1910).

DNA sequences from three of the five loci sampled resolved the three species described in this study as reciprocally monophyletic clades within a South American clade of the F. solani species complex, and four of the loci support a ((F. tucumaniae, F. phaseoli) (F. virguliforme)) relationship. As previously shown (O'Donnell 2000), sequences of the nuclear 28S and ITS rDNA regions are too conserved to resolve the species limits of F. virguliforme and F. phaseoli, although a single base-pair indel within the ITS2 region differentiates these taxa. Due to the high conservation of these nuclear ribosomal loci, O'Donnell and Gray (1995) incorrectly concluded that these species are conspecific. Sequences of the EF-1 gene (O'Donnell 2000), and especially those of the nuclear ribosomal IGS region, possess enough phylogenetic signal to resolve these three closely related species. Although partial sequences of the IGS region have been used to investigate phylogenetic relationships within the F. oxysporum complex where putative paralogs were discovered (Appel and Gordon 1996), the current study represents the first time that homology assessment has not been an issue in the use of the IGS for low-level phylogenetics within Fusarium. Sequences of the IGS also work well for phylogeny reconstruction within the Gibberella fujikuroi species complex (O'Donnell unpubl).

In EF-1, ITS and IGS gene trees, the two SDS pathogens did not form a monophyletic group, which suggests that either their pathogenicity to soybean has evolved convergently, as demonstrated for formae speciales within the F. solani (O'Donnell 2000) and F. oxysporum species complexes (O'Donnell et al 1998, Baayen et al 2001), or else the most recent common ancestor of F. phaseoli might have lost its pathogenicity to this host. Pathogenicity studies are in progress to determine whether F. phaseoli can induce typical SDS symptoms on soybean together with strains of closely related species. Rupe et al (2001) reported that one of these species, represented by strain NRRL 22743 from Brazil (as F. solani f. sp. phaseoli), could induce SDS-like symptoms on soybean.

Since it was first reported in Arkansas in 1972, SDS of soybean has been reported in the United States from at least 13 soybean-growing states (Hershman et al 1990, Jardine and Rupe 1993, Yang and Rizvi 1994, Hartman et al 1995, Roy 1997b, Roy et al 1997, Rupe and Hartman 1999, Pennypacker 1999) and from Ontario, Canada (Anderson and Tenuta 1998). The disease also has been reported in Argentina and Brazil (Ivancovich et al 1992, Botta et al 1993, Ploper 1993, Nakajima et al 1993, Wrather et al 1997, Rupe and Hartman 1999). Although the causal pathogen of soybean SDS recently has been described as F. solani f. sp. glycines, based primarily on its pathogenicity to this host (Roy 1997a, Roy et al 1997), results of our study clearly document the existence of two morphological and phylogenetic species corresponding to this formae speciales, F. virguliforme currently responsible for SDS within North America and F. tucumaniae in South America. Given that these two pathogens are deeply nested within a South American clade of the F. solani complex, it seems likely that they switched to soybean sometime within the past 100 years, after this crop was introduced to South America from Asia. What remains unclear is the identity the original hosts of these pathogens and their current geographic distribution. F. virguliforme surprisingly has not been found in South America, although an explicit biogeographic hypothesis suggests that it is endemic to this region (O'Donnell 2000). Thus far F. tucumaniae is responsible for the known outbreaks of SDS of soybean in South America. The high genetic similarity of strains of F. virguliforme isolated from soybean within the United States (O'Donnell and Gray 1995, Achenbach and Patrick 1996, Li et al 2000, Rupe et al 2001) suggests that it might represent a single introduction (Goodwin et al 1994) or that this species is predominately or exclusively clonal in North America.

Results of this study provide a phylogenetic framework for understanding the species limits of several economically important soybean and green or dry bean pathogens. Because precise knowledge of the genetic diversity of plant pathogens is crucial to the success of disease-control efforts and breeding programs (Taylor et al 1999), our results further highlight the failure of the forma specialis naming system because strains classified as f. sp. glycines and f. sp. phaseoli are not reciprocally monophyletic (O'Donnell unpubl). Clearly, plant breeding efforts might benefit by including representatives of each species when testing new varieties so as to increase the likelihood of achieving broad-based resistance to this pathogen complex. Finally, a multiplex polymerase chain reaction assay is being developed to rapidly detect and identify the three pathogens reported in this study based on informative variation within the aligned IGS sequences. In addition to the use of this unique molecular diagnostic tool in better understanding the phylogeographic structure and pathobiology of the soybean SDS pathogens, it has potential use in tracking the global movement of these pathogens.

	ACKNOWLEDGMENTS

	FOOTNOTES

 
¹ Collaborators via the JIRCAS research project, "Soybean improvement, production and utilization in South America"

² Corresponding author. E-Mail: taoki{at}nias.affrc.go.jp

Accepted for publication February 2, 2003.

	LITERATURE CITED

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS TAXONOMY DISCUSSION LITERATURE CITED

 
Abney TS, Richards TL, Roy KW., 1993 Fusarium solani from ascospores of Nectria haematococca causes sudden-death syndrome of soybean. Mycologia 85:801-806

Achenbach LA, Patrick J., 1996 Use of RAPD markers as a diagnostic tool for the identification of Fusarium solani isolates that cause soybean sudden-death syndrome. Pl Dis 80:1228-1232

Anderson TR, Tenuta A., 1998 First report of Fusarium solani f. sp. glycines causing sudden-death syndrome of soybean in Canada. Pl Dis 82:448.

Aoki T, Nirenberg HI., 1999 Fusarium globosum from subtropical Japan and the effect of different light conditions on its conidiogenesis. Mycoscience 40:1-9

———, O'Donnell K, Ichikawa K., 2001 Fusarium fractiflexum sp. nov. and two other species within the Gibberella fujikuroi species complex recently discovered in Japan that from aerial conidia in false heads. Mycoscience 42:461-478

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