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Department of Plant Pathology, Throckmorton Plant Sciences Center, Kansas State University, Manhattan, Kansas 66506-5502
Brett A. Summerell
Suzanne Bullock
Royal Botanic Gardens, Mrs. Macquaries Road, Sydney, New South Wales 2000, Australia
John F. Leslie 1
Department of Plant Pathology, Throckmorton Plant Sciences Center, Kansas State University, Manhattan, Kansas 66506-5502
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSTAXONOMYRESULTSDISCUSSIONLITERATURE CITED |
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The Gibberella fujikuroi species complex (Fusarium section Liseola and allied taxa) is composed of an increasingly large number of morphological, biological and phylogenetic species. Most of the known species in this group have been isolated from agricultural ecosystems or have been described from a small number of isolates. We sampled Fusarium communities from native prairie grasses in Kansas and recovered a large number of isolates that superficially resemble F. anthophilum. We used a combination of morphological, biological and molecular characters to describe a new species, Gibberella konza (Gibberella fujikuroi mating population I [MP-I]), from native prairie grasses in Kansas. Although female fertility for field isolates of this species appears to be low, G. konza is heterothallic, and we developed reliably female fertile mating population tester strains for this species. The F. konzum anamorph is differentiated from F. anthophilum and from other Fusarium species in section Liseola by mating compatibility, morphology, AFLP fingerprint profile and differences in ß-tubulin DNA sequence.
Key words: AFLP, biological species, DNA sequence, Fusarium anthophilum, mating population, ß-tubulin
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSTAXONOMYRESULTSDISCUSSIONLITERATURE CITED |
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recognized seven species within this section, while Nelson et al (1983) recognized only four. Since that time, cross-fertility data and data from molecular markers have been used to analyze section Liseola and have revealed an abundance of cryptic biological species and phylogenetic lineages.
Within the Gibberella fujikuroi (Sawada) Wollenw. species complex (primarily Fusarium section Liseola), at least eight biological species have been defined with sexual cross-fertility criteria, reviewed in Samuels et al (2001). Additional Fusarium species with no known sexual stage, but whose morphology allies them with Fusarium section Liseola, also have been described (e.g., Marasas et al 1985, 1987, 2001; Nelson et al 1987; Rheeder et al 1996). At least 25 additional phylogenetic lineages have been identified within the G. fujikuroi complex by using molecular characters (e.g., O'Donnell et al 1998, Steenkamp et al 1999, Marasas et al 2001). Together these findings indicate that the G. fujikuroi complex encompasses a large and incompletely documented set of species.
In addition to morphology, several types of molecular and biochemical methods have been used to differentiate species within Fusarium section Liseola. Huss et al (1996) described an isozyme methodology that could be used to distinguish the (then seven) described biological species in G. fujikuroi. Voigt et al (1995) and Amoah et al (1996) used RAPD (random amplified polymorphic DNA) markers to distinguish these same biological species, while Möller et al (1999) developed PCR primers for species-specific RAPD fragments to differentiate them. DNA sequence data also have been used to aid in distinguishing probable species and phylogenetic subgroupings within section Liseola (e.g., O'Donnell et al 1998, 2000, Steenkamp et al 1999, 2000), as has amplified fragment length polymorphism (AFLP, Vos et al 1995); e.g., Marasas et al (2001).
We have been evaluating Fusarium communities associated with prairie grasses in Kansas and have recovered isolates that are superficially similar in morphology to Fusarium anthophilum (A Braun) Wollenw. (Nelson et al 1983) from greater than 50% of the sampled grasses. AFLP fingerprint data from the majority of these isolates indicate that these isolates all are members of the same phylogenetic lineage but are different from the ex-type strains of F. anthophilum and from a number of other potentially closely related Fusarium species. The prairie isolates are cross-fertile with one another but are infertile with tester strains from the described biological species (G. fujikuroi mating populations A–H). This result indicates that these isolates represent a new, distinct biological species within the G. fujikuroi species complex (mating population I [MP-I]). Here we formally describe this new Gibberella species, Gibberella konza, with Fusarium konzum as its anamorph.
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MATERIALS AND METHODS |
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We isolated Fusarium from asymptomatic prairie grasses by surface sterilizing stem and seed head segments in 95% ethanol for 2 min and then plating them onto semiselective peptone-PCNB media (Nash and Snyder 1962). After 4–7 d incubation at 25 C, we isolated Fusarium colonies that emerged from these materials. We purified cultures by subculturing single conidia separated by micromanipulation. Isolates routinely were cultured on carnation leaf agar (CLA) (Fisher et al 1982) or on modified Czapek's complete medium (Correll et al 1987).
We measured growth rates of strains on plates of potato-dextrose agar (PDA; Difco, Detroit, Michigan) at both 25 and 30 C after 3 d on three replicate cultures per temperature for each of four representative strains (KSU 08373, 10595, 10653 and 10689) on two separate occasions (Burgess et al 1994).
All strains are maintained as spore suspensions in 15% glycerol at -70 C (KSU). We deposited a dried culture of isolate KSU 10653, as the holotype of F. konzum, with the New South Wales Plant Pathology Herbarium, Orange, NSW, (Australia) as DAR 76034. A preserved heterothallic cross between strains KSU 11615 and KSU 11616 on carrot-agar media was deposited as the holotype of Gibberella konza as DAR 76033. Isolates KSU 10595 (=FGSC 8912, ATCC MYA-2882) and 10653 (=FGSC 8913, ATCC MYA-2883), and the female fertile mating-type tester strains (strain numbers—KSU 11615 (=FGSC 8911, ATCC MYA-2884) and KSU 11616 (=FGSC 8910, ATCC MYA-2885) have been deposited with the Fungal Genetics Stock Center, University of Kansas Medical Center, Kansas City, Kansas (FGSC numbers), and the American Type Culture Collection, Manassas, Virginia (ATCC numbers). Strains with FRC numbers also are deposited at the Fusarium Research Center, Department of Plant Pathology, Pennsylvania State University, University Park, Pennsylvania. Strains with MRC numbers also are deposited at PROMEC, Medical Research Council, Tygerberg, South Africa. Strains with BBA numbers also are deposited at Biologische Bundesanstalt für Land- und Forstwirtschaft, Berlin, Germany. Strains with ITEM numbers also are deposited at Istituto Tossine e Micotossine da Parassati Vegetale du CNR (Bari, Italy). Strains with NRRL numbers also are deposited at the National Center for Agricultural Utilization Research (NCAUR), USDA-ARS, Peoria, Illinois.
We used these standard Gibberella fujikuroi mating population mating-type tester strains in all mating tests: FGSC 7600 (MATA-1), FGSC 7603 (MATA-2), FGSC 7611 (MATB-1), FGSC 7610 (MATB-2), FGSC 8931 (MATC-1), FGSC 8932 (MATC-2), FGSC 7615 (MATD-1), FGSC 7614 (MATD-2), FGSC 7616 (MATE-1), FGSC 7617 (MATE-2), FGSC 7057 (MATF-1), FGSC 7056 (MATF-2), FGSC 8934 (MATG-1), FGSC 8933 (MATG-2), KSU H-10847 (MATH-1) and KSU H-10850 (MATH-2).
We estimated AFLP diversity and genetic distances among F. konzum isolates and among other Fusarium species and phylogenetic lineages. Preliminary isozyme data (not shown) indicated that the putative F. konzum isolates had affinity to isolates identified as belonging to the "American" clade identified by O'Donnell et al (1998), so we focused most of our molecular comparisons to Fusarium species within that grouping. We compared F. konzum isolates to representative isolates previously characterized as F. anthophilum [KSU 03811, (FRC M-847, BBA 63435); KSU 03817, (FRC M-853, BBA 63633); KSU 03818, (FRC M-854, BBA 63270, NRRL 22943); KSU 05007, (FRC M-1208); KSU 05008, (FRC M-1238)] and to the morphologically similar F. succisae (Schröt.) Sacc. [KSU 03832 (FRC M-1221, BBA 63627, NRRL 13613) and KSU 03853 (FRC M-1222, BBA 63162, NRRL 25215)] to determine whether these isolates belonged to the same or to different phylogenetic lineages. We also compared AFLP profiles of F. konzum, F. anthophilum and F. succisae isolates to these Fusarium isolates to assess interspecies AFLP similarity: F. begoniae Nirenberg & O'Donnell [KSU 10767 (MRC-7542, BBA 67781, NRRL 25300)], F. bulbicola Nirenberg & O'Donnell [KSU 03808 (FRC M-844, BBA 62274), KSU 10759 (MRC-7534, BBA 63628, NRRL 13618), KSU 03814 (FRC M-850, BBA 63620, NRRL 13600)], F. circinatum Nirenberg & O'Donnell (teleomorph Gibberella circinata Nirenberg & O'Donnell) [KSU 10766 (MRC-7541, BBA 69720, NRRL 25331), KSU 10847 (MRC 7488), KSU 10850 (MRC 6213), KSU 00861 (ITEM 2969), KSU 00867 (ITEM 2975), KSU 00869 (ITEM 2977) and KSU 00870 (ITEM 2978)], F. guttiforme Nirenberg & O'Donnell [KSU 05035 and KSU 10764 (MRC-7539, BBA 69661, NRRL 25295)], F. napiforme Marasas, Rabie, Lübben, Nelson, Toussoun, & van Wyk [KSU 05015 (FRC 1646)], F. sacchari (Butl.) W. Gams (Gibberella fujikuroi mating population B) [KSU 03852 (ATCC 201264, FRC M-6865, FGSC 7610, NRRL 22043), KSU 04797 (ATCC 38480, MRC 2292), and KSU 04799 (ATCC 38478, MRC 2381)], F. subglutinans (Wollenw. & Reinking) Nelson, Toussoun & Marasas (teleomorph G. subglutinans Nelson, Toussoun & Marasas [KSU 02192, (FGSC 7617, FRC M-3693, ATCC 201271, NRRL 22016)] and F. sterilihyphosum Britz, Couthino, Wingfield & Marasas [KSU 03874 (MRC-2802, ITEM 2987)].
Amplified fragment length polymorphisms (AFLP) –
We generated AFLPs (Vos et al 1995) as described by Zeller et al (2000). After preamplification, AFLP fingerprints were generated with the two base-pair extension primer combinations EcoRI+TT/MseI+AC, and EcoRI+GG/MseI+CT. To analyze AFLP profiles, bands between 100 and 400 base pairs in size were scored manually as presence or absence. We assumed that bands of the same molecular size in different individuals were identical (homologous characters). Each band was treated as a single independent locus with two alleles, and unresolved bands or missing data were scored as ambiguous. The Dice coefficient (Nei 1972) was used to calculate pairwise UPGMA genetic distances among isolates with the CLUSTER procedure of SAS (version 6.11, SAS Institute, Cary, North Carolina).
We also examined the utility of these AFLP data to describe phylogenetic relationships among the isolates. We used the neighbor-joining (NJ) subroutine with PAUP* version 4.08b (Swofford 1999) to construct a network of relatedness among these isolates and species. For NJ analyses, pairwise genetic distances were calculated with the distance measure = mean character difference and 1000 bootstrap resamplings of the data were conducted to assess the degree of support for the resulting clusters.
DNA sequencing and phylogenetic analyses –
We obtained ß-tubulin DNA sequence data for these isolates from GenBank [species (isolate number(s); GenBank accession number)]: F. anthophilum (NRRL 13602; U61541), F. begoniae (NRRL 25300, = KSU 10767; U61543), F. bulbicola (NRRL 13618, = KSU 10759; U61546), F. dlamini Marasas, Nelson & Toussoun (NRRL 13164; U34430), F. guttiforme (NRRL 22945; U34420), F. napiforme (NRRL 13604; U34428), F. sacchari (NRRL 13999; U34414), F. subglutinans (NRRL 22016, = KSU 02192; U34417), F. succisae (NRRL 13613, = KSU 03832; U34419), Gibberella circinata (NRRL 25331, = KSU 10766; U61547). We used PCR primers T1 and T2 and the PCR reaction conditions described in O'Donnell and Cigelnik (1997), except that the annealing temperature was increased to 58 C to amplify a homologous region of the ß-tubulin gene from these Fusarium isolates used in AFLP analyses: KSU 03808, 03811, 03814 (=NRRL 13600), 03818, 03874, 05007, 05035, 10578 and 10653.
PCR products were purified and desalted with the Wizard® PCR Preps DNA Purification System (Promega, Madison, Wisconsin). DNA sequences were obtained by direct sequencing of these PCR products by using PCR primers T1 and T2 with an ABI 3700 automated sequencer at the Kansas State University sequencing facility. Sequences from both strands were aligned to minimize ambiguous nucleotide positions, and each sequence then aligned with ClustalW (Thompson et al 1994), as implemented in the program BioEdit version 4.7.8 (Hall 1999). Preliminarily aligned sequences were aligned and edited manually with BioEdit, as necessary. We exported these aligned DNA sequence data, conducted NJ analyses, with distances calculated as mean character difference and with gaps treated as missing data, and assessed support for branching patterns with bootstrap analyses (1000 resamplings) with PAUP* version 4.08b (Swofford 1999). Final sequence alignments are available as TreeBASE accession number SN1142 <http://www.treebase.org/treebase/index.html>. The 10 unpublished DNA sequences were deposited in GenBank under accession numbers AY222286–AY222295.
Mating-type specific PCR and crossing procedures –
We identified mating-type idiomorphs (MAT-1 or MAT-2) for the F. konzum isolates with PCR-based assays, as described in either Kerényi et al (1999) or Steenkamp et al (2000). We confirmed all mating-types assigned by PCR with crosses to female-fertile tester isolates (KSU 11615 MAT-2 or KSU 11616 MAT-1) developed as a part of this study. We conducted crosses as described in Klittich and Leslie (1988), except that after fertilization we maintained cultures at 22 C with a 12:12 h light:dark cycle. We tested all isolates for both male and female fertility with KSU 11615 and KSU 11616. Crosses were made initially with KSU 11615 and 11616 as the female parent and then again with each field isolate as the female parent but fertilized with either KSU 11615 or KSU 11616. We conducted each cross at least twice.
We estimated Ne (effective population size) based on mating-type ratios and on the relative frequency of female-fertile isolates from the field populations by using the equations derived in Leslie and Klein (1996). We used methods described by Brown et al (1980) and by Smouse and Neel (1977) on data from clone-censored samples to test whether these AFLP data for populations of G. konza differ from expectations of random mating.
Morphology methods –
The description of Gibberella konza was based on crosses (KSU 11615 x KSU 11616) produced on carrot agar. Perithecia were treated with 3% KOH and 100% lactic acid to observe any color reaction and measured in situ. Asci and ascospores were mounted in water for measurement and photography. Measurements were taken of 20 each of perithecia, asci and ascospores. Whole perithecia were fixed in 6.5% glutaraldehyde in 100 mM sodium cacodylate buffer at pH 7.6 for 4 h at room temperature (Bullock et al 1980), dehydrated in a graded ethanol series, and finally infiltrated and embedded in LR White resin. Sections, 1.5 µm thick, were cut with a Reichert ultramicrotome, dried onto poly-L-lysine-coated glass slides, and stained with 0.5% Toluidine Blue O for 10 s (Feder and O'Brien 1968). Sections were mounted in immersion oil.
The description of Fusarium konzum is based on cultures on PDA, CLA, soil agar (SA) (Klotz et al 1988) and SNA (Nirenberg 1976). Cultures were incubated under a light bank of three fluorescent and one black (near uv) light with a 12 h photoperiod at 22 C. Macroconidia and microconidia were mounted in water, and measurements were taken of the length and width of 30 macroconidia and 30 of each of the microconidial types. Macroconidia used for measurements were from sporodochia from carnation leaf pieces on CLA, and microconidia were from aerial mycelium on SNA. The size, presence and nature of monophialides, polyphialides, microconidia, macroconidia and sporodochia were recorded and photographed. The colony morphology also was recorded with descriptions of pigmentation according to Rayner (1970).
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TAXONOMY |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSTAXONOMYRESULTSDISCUSSIONLITERATURE CITED |
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[Perithecia superficialia, livida, 360–840 µm alta x 360–780 µm lata. Asci fusiformes 58–64 µm alta x 8–9 µm lata, dehiscentes, octospori. Ascosporae exudatae in cirrhis, laeves, hyalinae, ellipsoidae vel obovoidae, 0–1 septatae, plerumque 1 septatae et ad septum leviter constrictae, 12–18 x 4–37 µm. Anamorphosis: Fusarium konzum Zeller, Summerell et Leslie.]
HOLOTYPUS. Cultura exsiccata in agaro ex KSU 11615 x KSU 11616 (DAR 76033).
Etymology. Konza (English) = in reference to the fact that first recognized isolates of this species were isolated from native prairie grasses at the Konza Prairie Biological Station, Manhattan, Kansas. The word Konza is one of many English derivations of the Native American tribal name Kansa.
Teleomorph. Perithecia superficial, solitary to aggregated in groups of a few and seated on a minute stromatic base, obovoidal, and warty; 360–840 (mean = 636) µm x 360–780 (mean = 628) µm diam; blue-black, color not changing in 3% KOH, turning red in 100% lactic acid. Perithecial wall 83.0–138.6 (mean = 99.2) µm wide laterally, formed of two obvious regions. Outer region 47.4–98.2 (mean = 82.2) µm wide; cells ± angular to elliptic, 11.0–33.0 (mean = 20.0) µm length x 5.6–15.4 (mean = 10.2) µm width, with the largest cells at the exterior and the smallest cells toward the interior of the wall, walls of cells 2.4–5.8 (mean = 3.8) µm wide and pigmented. Inner region 10.4–26.2 (mean = 17.0) µm wide; cells ± angular to elliptic, 9.8–59.6 (mean = 26.0) µm length x 1.2–5.6 (mean = 3.4) µm width. Cells fusiform, thin-walled and irregular, walls of cells 0.6–2.4 (mean = 1.2) µm wide and pigmented. Outer and the inner wall regions merging imperceptibly; cells of the outer region more pigmented. Perithecial apex continuous with the outer and inner wall layers; cells at the apex smaller appearing as a reticulum; cells forming the ostiolar opening ± clavate and thick-walled at the cell tips, nonpigmented merging periphyses. Cells of the apex attaining the same length to form an apical disk.
Asci fusiform, dimensions 58–64 x 8–9 µm, regularly dehiscing upon examination under the microscope with eight spores. Ascospores exuded in a cirrhus, ellipsoidal to obovoid with both ends rounded, 0–1 septate and slightly constricted at the septum, 12–18 (mean = 15.2) x 4–7 (mean = 5.9) µm (Figs. 1–2). Heterothallic species reproductively isolated from previously described species of Gibberella.
Fusarium konzum Zeller, Summerell et Leslie sp. nov. Figs. 6–17
[Coloniae in agaro PDA 72 horus apud 25 C 2.1–3.4 cm diam, apud 30 C 2.1–3.8 cm diam, floccosae vel pulveraceae, primo candidae, diende violaceae. Coloniae retrosum incolor ad violaceus, subinde miniatus. Sporodochia in agaro CLA rare, pallida aurentiaca. Macroconidia sparsa, hyalina, 3–5 septata crassitunicata, plerumque 3-septata, recta vel falcata, cellula basali pedicellata, cellula apicali curvata, 30–52 x 4–6 µm. Microconidia in capitulis falsis, hyalina, ellipsoidea (2 x 5 µm), napiformia (6 x 6 µm) et pyriformia (4 x 5 µm) producentes in monophialides et polyphialides. Microconidia numquam in catenis. Chlamydosporae absentiae.]
HOLOTYPUS. Cultura exsiccata in agaro ex KSU 10653, sejuncta a ramentis plantarum (Sorghastrum nuttans) in humo, Konza Prairie Biological Station, Manhattan, Kansas, U.S.A. (DAR 76034).
Etymology. Refers to the teleomorph.
Anamorph.
Colonies on PDA produce abundant floccose mycelium that are initially white and become violet. Colony color (bottom) on PDA after 10 d at 25 C with a 12 h photoperiod ranged from colorless to violet [pigment code 9'D-53''''K in Rayner (1970)]; occasionally some cultures produce a brick red-pigment [pigment code 5 in Rayner (1970)]. Colony diam on PDA after 72 h incubation in the dark was 2.1–3.4 (mean = 2.9) cm at 25 C and 2.1–3.8 (mean = 2.8) cm at 30 C.
Sporodochia are rare; sporodochia from leaf pieces on CLA are pale orange. Macroconidia are not common. Typically macroconidia are hyaline, 3–5 septate, mostly 3 septate, falcate, with a pedicellate foot cell and a slightly curved apical cell, 30–52 x 4–6 µm (mean 37 x 4.5) µm (Figs. 6–7). Microconidia are produced on monophialides and polyphialides produced laterally in the aerial hyphae on the water-agar surface of the CLA and, more abundantly, on SNA (Figs. 10–17). Polyphialides are generally simple with only 2–3 polyphialidic openings per cell (Fig. 11). Phialidic apertures are elongate and typical of species in section Liseola (Figs. 11–17). The phialidic cells are 3–15 µm long x 2–4 µm in diam. Microconidia are produced either singly or in small false heads consisting of 2–4 microconidia per phialide (Figs. 12–15). Three types of microconidia are produced: oval, hyaline 0–1 septate microconidia (7–10 x 2–5 µm) (Figs. 8, 12, 13); pyriform, 0-septate (occasionally 1-septate) microconidia (3–6 x 3–8 µm) (Figs. 9, 10); and larger napiform to globose, 0-septate microconidia (5–6 x 5–6 µm) (Figs. 10, 11). The pyriform and napiform microconidia are more abundant on SNA than on CLA (Figs. 16, 17). Microconidia were not produced in chains, although false chains of the napiform microconidia were observed as spores initially adhere in short chains of 2–3 conidia before collapsing into irregular, dry heads on SNA (Figs. 16, 17). No chlamydospores have been observed.
Isolates examined. U.S.A., Kansas: Lyon County, culture isolated from an Andropogon gerardii plant by K. A. Zeller, 1995, KSU 08373 (ITEM-3106), MAT-1, Riley County, cultures isolated from A. gerardii plants by K. A. Zeller, 1997, KSU 10578 (ITEM-3132), MAT-2; KSU 10595 (FGSC 8912, ATCC MYA-2882, ITEM-3141), MAT-2, culture isolated from an Andropogon scoparius plant by K. A. Zeller, 1997, KSU 10638 (ITEM-3162), MAT-2, cultures isolated from Sorghastrum nuttans plants by K. A. Zeller, 1997, KSU 10653 EX-HOLOTYPE (DAR 76034, HOLOTYPE; ISOTYPES, FGSC 8913, ITEM 3168, ATCC MYA- 2883), MAT-1; KSU 10663 (ITEM-3173), MAT-1; KSU 10676 (ITEM-3177), MAT-1; KSU 10678 (ITEM-3179), MAT-2; KSU 10681 (ITEM-3180), MAT-2; KSU 10689 (ITEM-3183), MAT-1, cultures isolated from A. gerardii plants by K. A. Zeller, 1998, (KSU 11656, MAT-1; KSU 11704, MAT-1; KSU 11799, MAT-1; KSU 11810, MAT-2; KSU 11815, MAT-2; KSU 11819, MAT-2; KSU 11827, MAT-1; KSU 11830, MAT-1; KSU 11838, MAT-1; KSU 11839, MAT-2; KSU 11840, MAT-1; KSU 11844, MAT-1; KSU 11850, MAT-1; KSU 11853, MAT-1; KSU 11856, MAT-1; KSU 11868, MAT-1; KSU 11870, MAT-1; KSU 11873, MAT-1; KSU 11886, MAT-2; KSU 11899, MAT-2; KSU 11900, MAT-2; KSU 11908, MAT-1; KSU 11911, MAT-1; KSU 11923, MAT-1; 24 isolates from 22 plants), cultures isolated from A. scoparius plants by K. A. Zeller, 1998, (KSU 11804, MAT-1; KSU 11893, MAT-1; KSU 11925, MAT-1; three isolates from three plants), culture isolated from a Bouteloua curtipendula [Michx.] Torr. plant by K. A. Zeller, 1998, (KSU 11875, MAT-1), cultures isolated from S. nuttans plants by K. A. Zeller, 1998, (KSU 11639, MAT-1; KSU 11652, MAT-1; KSU 11822, MAT-1; KSU 11824, MAT-1; KSU 11834, MAT-1; KSU 11835, MAT-1; KSU 11841, MAT-1; KSU 11843, MAT-1; KSU 11858, MAT-2; KSU 11859, MAT-1; KSU 11914, MAT-1; KSU 11919, MAT-1; KSU 11920, MAT-1; 13 isolates from eight plants), cultures isolated from a Sporobolis hetrolepis [Gray] plant by K. A. Zeller, 1998, (KSU 11916, MAT-1; KSU 11918, MAT-1; two isolates from one plant), USA, Female-Fertile Laboratory Strains, generated by crosses and back-crosses between parental isolates 10595 and 10653 by K. A. Zeller, 2000, KSU 11615 (FGSC 8911, ATCC MYA-2884), MAT-2; KSU 11616 (FGSC 8910, ATCC MYA-2885), MAT-1.
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RESULTS |
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Assessments of phylogenetic relatedness –
The AFLP fingerprint profiles from the F. anthophilum-like isolates can be used to group the isolates into three related, but distinct, phylogenetic lineages (Fig. 18). Bootstrap support for the unity of the F. konzum group, after excluding isolate KSU 10578, is 100%. Bootstrap support for unity of a second cluster that includes an isolate previously identified as F. anthophilum (KSU 03818) by comparisons of DNA sequence data (O'Donnell et al 1998) also was 100% (Fig. 18). Thus, F. konzum, F. succisae and F. circinatum clearly are different from F. anthophilum if AFLP polymorphisms are used as the distinguishing character. Other groups that received 100% support were F. sacchari, F. circinatum and a pair of F. succisae isolates, KSU 03832 and KSU 03833. These two F. succisae isolates had identical AFLP fingerprints. Fusarium succisae grouped with an isolate (KSU 05007) received as F. anthophilum in 96% of the replications. A group that included F. begoniae (KSU 10767), two isolates from pineapples (KSU 05035 and KSU 10764), and F. sterilihyphosum (KSU 03874) was supported in 93% of the replications. High (>90%) bootstrap support commonly is observed when grouping isolates within species or when grouping closely related species. Bootstrap support for specific branching orders at higher taxonomic levels is much lower, and other data are needed to determine these phylogenetic relationships.
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2, p < 0.001). All isolates with the same AFLP fingerprint also had the same MAT allele. The mating-type ratio (26:11) for just the 37 clonal haplotypes also was significantly different from 1:1 (2, p < 0.02). Interspecies and intraspecies mating compatibility testing and estimates of Ne – We observed no fertile combinations when isolates KSU 08373, 10595, 10638, 10653, 10663, 10676, 10678, 10681 and 10689 were crossed as males with tester strains for the described mating populations of G. fujikuroi (MPs A-H). These isolates also were not fertile when crossed to isolates from the other clades, including F. anthophilum.
After determining the mating-type idiomorph for each isolate with PCR assays, we intercrossed isolates KSU 08373, 10595, 10638, 10653, 10663, 10676, 10678, 10681 and 10689 in all possible compatible (MAT-1 x MAT-2) pairs. A single isolate (KSU 10653, MAT-1) was female fertile when crossed to isolates KSU 10595 (MAT-2) and 10689 (MAT-2). To develop reliable, female-fertile testers of both mating-types, we collected 15 progeny from the cross of KSU 10653 x 10595 and used these progeny as females in back crosses to KSU 10653. We identified one female-fertile MAT-2 strain (KSU 11615) in the F1 progeny and backcrossed it to KSU 10653 (B1 generation). We identified a female-fertile MAT-1 strain (KSU 11616) among the progeny of the B1 cross. We used KSU 11615 and KSU 11616 as mating-type testers in the crosses with the remaining F. konzum isolates, but only 45/52 strains were fertile as males in these crosses, despite high AFLP similarity and known MAT compatibility. Of these infertile isolates, four were MAT-1 and three were MAT-2, based on PCR assays. Based on these data, we estimate that Ne is approximately 75% of the count, due to the skewed mating-type ratio.
Female fertility was low, with only 2/52 field isolates capable of functioning as the female parent under the conditions used. After excluding the seven strains that were not fertile as males from the calculation, Ne based on the frequency of hermaphrodite strains is about 16% of the count. Ne based on hermaphrodite frequency remains low (22% of the count), even after censoring the clonal strains and male-sterile isolates.
Despite these data suggesting low incidence of female fertility, we are not able to exclude the hypothesis that this population of G. konza differs significantly from the expectation of random mating. Using the approach of Brown et al (1980), the observed heterozygosity (42.06) was higher than expected (13.47) but the resulting s2k value (variance) was only 2.12 and was considerably less than the 95% cut-off value of 20.04 (data not shown). We obtained a similar result when using the multivariate approach of Smouse and Neel (1977). In this case, we estimated an R-value of 0.057 and a probability of 1.0 (2 value 56.82, 465 df). Both analyses indicate that multilocus associations in these samples cannot be differentiated from expectations of random mating.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSTAXONOMYRESULTSDISCUSSIONLITERATURE CITED |
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). Agricultural ecosystems are characterized by host (crop) populations that are relatively uniform genetically, numerically and spatially (reviewed in Burdon et al 1989) and that experience regular, human-mediated disturbance and migration. The existing data for populations of fungal plant pathogens suggest that widespread populations of agricultural pathogens may be relatively uniform across a broad range, while their parallels from nonagricultural ecosystems will have a less-uniform distribution, may be endemic and/or more locally diverse, e.g., Gordon et al (1992) and Kohn (1995). Some of the changes in Fusarium communities or species in a native ecosystem relative to those observed in agriculture might include differences in fertility (either higher or lower), less migration, higher local species diversity, better-defined spatial structure due to a lower level of physical disturbance, long-term associations of individual isolates with individual perennial plants, changes in species composition, and/or a higher frequency of species perceived as "generalists."
Leslie (1995) included F. anthophilum on a list of Fusarium species within sections Liseola or Elegans with no known sexual stage but suggested that additional biological species remained to be found and might be associated with such taxonomic entities. Numerous studies since have increased the number of recognized biological species, e.g., Klassen and Nelson (1996) and Britz et al (1999) and have greatly expanded the number of proposed phylogenetic species (O'Donnell et al, 1998, 2000, Nirenberg et al 1998, Steenkamp et al 1999, Marasas et al 2001). As with the other morphological species recognized within Fusarium section Liseola, the F. anthophilum morphological type might contain more than a single biological species or phylogenetic lineage (Figs. 18–19).
Analyses of AFLP fingerprints and ß-tubulin sequence data could not conclusively resolve the phylogenetic relatedness of F. anthophilum, F. succisae, G. circinata and G. konza isolates. In all cases, the AFLP similarity between clusters of these species was 50–60%, although bootstrap support separating these species clusters was high (96–100%). We have observed a comparable level of AFLP profile similarity (50–55%) between some isolates of G. fujikuroi mating populations C (G. fujikuroi) and D (G. intermedia) (Leslie et al unpublished) and between some of the cross-fertile phylogenetic lineages of G. zeae (Schwein.) Petch (Jurgenson et al 2002). The conclusion based on ß-tubulin sequence data is similar; genetic distances between F. anthophilum, F. bulbicola, F. succisae, G. circinata and G. konza are similar and low, and no strong phylogenetic statement can be made about their evolutionary interrelatedness.
Ne values based on both mating-type ratios and hermaphrodite frequencies are the lowest reported to date for this group of fungi. The reduction in Ne due to skewed mating-type ratio could be an artifact of our relatively small sample of strains for this species. Gibberella konza also has a lower percentage female-fertile isolates than has been observed in most populations of G. moniliformis, G. thapsina or G. intermedia (Kuhlman) Samuels (Leslie and Klein 1996, Mansuetus et al 1997, Chulze et al 2000), but it is comparable to that found in some populations of G. thapsinum or G. circinata (Klittich et al 1997, Britz et al 1998). The low estimate for Ne suggests that sexual reproduction is not common in G. konza. However, this estimate of Ne might be artificially low if the conditions used to assess female fertility are not optimal, a problem that has resulted in the re-evaluation of Ne data for G. circinata (see Covert et al 1999). In fact, our multilocus association analyses of G. konza cannot exclude the hypotheses of random mating and recombination for this population. More explicit resolution of these apparent contradictions will require further testing of G. konza populations for indications of gametic disequilibrium (Milgroom 1996).
Taxonomy –
Gibberella konza has been isolated only as the anamorph F. konzum, from stems and inflorescences of the perennial grass species Andropogon gerardii, A. scoparius, Bouteloua curtipendula, Sorghastrum nuttans and Sporobolis hetrolepis sampled from tallgrass prairie ecosystems in Kansas. This species appears to have a generalist distribution across these prairie grasses. We have preliminary data (K. A. Zeller et al unpubl) that indicate that F. konzum may be more commonly isolated from plants in lowland, mesic sites. However, we need to examine samples from shortgrass prairies, mountain meadows and other grassland ecosystems to determine the range of this species. The cultures in this study were isolated from surface-sterilized, asymptomatic plant materials, so this species is most likely to exist as an endophyte or as a latent plant pathogen (e.g., Sinclair and Cerkauskas 1996) in these grass hosts, rather than as an epiphyte.
Fusarium konzum shares a number of characteristics with species in the section Liseola, especially with F. anthophilum, particularly the presence of pyriform and napiforme to globose microconidia formed on monophialides and polyphialides. The shape and size of the macroconidia and all three types of microconidia fall within those reported for F. anthophilum and the colony diameter at 25 and 30 C after 3 d falls within the range reported by Burgess et al (1994). This species can be differentiated from F. anthophilum on the basis of characteristics of the phialides. The monophialides of F. konzum generally are longer than the monophialides typically found in F. anthophilum (Figs. 11–17), and the polyphialides of F. konzum are somewhat more swollen than those found in F. anthophilum (Figs. 11, 12). The globose microconidia observed in F. konzum have not been reported for F. anthophilum. Because the teleomorph of F. anthophilum has not been reported, it is not possible to compare the teleomorphs of these two species.
Gibberella konza does not differ significantly from several of the described species of Gibberella found within G. fujikuroi. The size and nature of the perithecia of G. konza falls within the range of many of the described species of Gibberella. The ascospores of G. konza are smaller than those of G. moniliformis, G. intermedia and G. thapsina (Samuels et al 2001) but cannot be clearly differentiated from ascospores of G. fujikuroi. These characters must be viewed with some caution because the conditions under which perithecia are produced can affect the size of both the perithecia and ascospores (Summerell et al 1998).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Accepted for publication March 3, 2003.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSTAXONOMYRESULTSDISCUSSIONLITERATURE CITED |
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