Post on 16-May-2023
Short title: Fusarium dactylidis produces nivalenol wheat
Fusarium dactylidis sp. nov., a novel nivalenol toxin-producing species sister to F.
pseudograminearum isolated from orchard grass (Dactylis glomerata) in Oregon and
New Zealand
Takayuki Aoki1
Genetic Resources Center (MAFF), National Institute of Agrobiological Sciences, 2-1-2
Kannondai, Tsukuba, Ibaraki 305-8602, Japan
Martha M. Vaughan
Susan P. McCormick
Mark Busman,
Todd J. Ward
Amy Kelly
Kerry O'Donnell
Bacterial Foodborne Pathogens and Mycology Research Unit, National Center for
Agricultural Utilization Research, Agricultural Research Service, US Department of
Agriculture, Peoria, Illinois 60604-3999
Peter R. Johnston
Landcare Research Manaaki Whenua, Private Bag 92170, Auckland 1142, New Zealand
In Press at Mycologia, preliminary version published on December 30, 2014 as doi:10.3852/14-213
Copyright 2014 by The Mycological Society of America.
David M. Geiser
Department of Plant Pathology and Environmental Microbiology, Pennsylvania State
University, University Park, Pennsylvania 16802
Abstract: The B trichothecene toxin-producing clade (B clade) of Fusarium includes the
etiological agents of Fusarium head blight, crown rot of wheat and barley and stem and
ear rot of maize. B clade isolates also havebeen recovered from several wild and
cultivated grasses, including Dactylis glomerata (orchard grass or cock's foot), one of
the world's most important forage grasses. Two isolates from the latter host are formally
described here as F. dactylidis. Phenotypically F. dactylidis most closely resembles F.
ussurianum from the Russian Far East. Both species produce symmetrical sporodochial
conidia that are similar in size and curved toward both ends. However, conidia of F.
ussurianum typically end in a narrow apical beak while the apical cell of F. dactylidis is
acute. Fusarium dactylidis produced nivalenol mycotoxin in planta as well as low but
detectable amounts of the estrogenic mycotoxin zearalenone in vitro. Results of a
pathogenicity test revealed that F. dactylidis induced mild head blight on wheat.
Key words: cock's foot, crown rot, forage, Fusarium head blight, genotyping,
morphology, mycotoxins, pathogenicity, phylogenetics, RPB1, RPB2, trichothecene,
zearalenone
INTRODUCTION
The 21 phylogenetically distinct species within the B trichothecene toxin-producing
clade (hereafter B clade) of Fusarium comprises some of the world's economically most
destructive mycotoxigenic plant pathogens (Aoki et al. 2012). Nested within this clade
are the primary etiological agents of Fusarium head blight (FHB) and crown rot of small
grain cereals (Sarver et al. 2011, Gardiner et al. 2012), ear and stem rot of maize
(Boutigny et al. 2011) and pathogens of exotic forest plantations (Roux et al. 2001).
Eighteen of the genealogically exclusive B clade species, which were discovered
employing genealogical concordance phylogenetic species recognition (GCPSR, Taylor
et al. 2000), currently fit the morphological description of Fusarium graminearum
Schwabe sensu lato accepted by Gerlach and Nirenberg (1982) and Nelson et al. (1983).
The latter species also was known as Gibberella zeae (Schwein.) Petch before the
of dual nomenclature (Geiser et al. 2013). During global surveys of isolates identified as
F. graminearum, using DNA sequence data from multiple loci (O'Donnell et al. 2000,
2004; Starkey et al. 2007) and a multilocus genotyping assay (MLGT) for trichothecene
chemotype prediction and species determination within the B clade (Ward et al. 2008),
two isolates from Dactylis glomerata L. (commonly known as orchard grass and cock's
foot) were discovered (i.e. ICMP 5269, FRC R-7593). They represent a
distinct species that is sister to F. pseudograminearum O'Donnell & T. Aoki. Given that
glomerata is one of the world's most widely cultivated forages for livestock (Steward
Ellison 2011), the objectives of this study were to: (i) characterize the orchard grass
isolates phenotypically and formally describe them as Fusarium dactylidis T. Aoki et al.,
(ii) assess pathogenicity of this novel B clade species to wheat and (iii) determine, as
previously predicted by a MLGT assay (Ward et al. 2008), whether the F. dactylidis
isolates could elaborate the trichothecene mycotoxin nivalenol in vitro and in planta.
MATERIALS AND METHODS
Fungal isolates studied.—ICMP 5269 stored at the International Collection of Microorganisms from
(ICMP), Auckland, New Zealand, was accessioned in the ARS Culture Collection as G. zeae NRRL
(= CBS 119181) in Dec 1999. It originally was isolated from orchard grass in Palmerston North,
New Zealand, on 30 Oct 1958. After identifying it as F. graminearum, Joan M. Dingley placed it in her
private collection as JMD No. 5941. In Dec 1976 this isolate was accessioned in ICMP as F.
graminearum 5269 and it was deposited in the New Zealand Fungal and Plant Disease Collection,
Auckland, New Zealand (PDD, https://scd.landcareresearch.co.nz) as a dried subculture (= PDD 104196).
A second isolate of the novel B clade species from orchard grass was received from the Fusarium
Research Center (FRC) at Pennsylvania State University as F. culmorum FRC R-7593 in March 2000 and
accessioned in the ARS Culture Collection as NRRL 29380 (= CBS 123656). FRC R-7593 originally was
isolated from cultivated orchard grass in Oregon in 1983 by Ronald E. Welty as No. 2A-1, Forage Seed
and Cereal Research Unit, ARS-USDA, Corvallis, Oregon. After identifying the isolate as F. roseum Link
cv. Graminearum, Welty sent it to Paul E. Nelson at FRC in Mar 1984 who initially identified it as F.
graminearum but subsequently re-identified it as F. culmorum. Herein the two isolates of F. dactylidis
were compared phenotypically with other morphologically similar B clade species listed in Sarver et al.
(2011). The holotype specimen was prepared from a culture of NRRL 29298 and deposited in the US
National Fungus Collection (BPI), Beltsville, Maryland.
Morphological characterization.—Methods follow Aoki et al. (2001, 2005). Isolates were cultured on
potato dextrose agar (PDA, Difco, Detroit, Michigan) in the dark to characterize colony morphology,
odor and color using a standardized color atlas (Kornerup and Wanscher 1978). PDA cultures also were
used for determining mycelial growth rates in the dark at eight temperatures (5–40 C) at 5 C intervals
(Aoki et al. 2003). Culture plates were examined at 1 and 4 d post-inoculation, and radial growth was
calculated as arithmetic mean values per day by measuring 16 radii around the colony. Measurements of
growth rates at different temperatures were replicated twice, and the data averaged for each strain.
Conidia and conidiophores were examined in cultures on synthetic low nutrient agar (SNA, Nirenberg
1990) in the dark, under continuous black light (National FL8BL-B 8 W/08, Panasonic, Osaka, Japan) or
under an ambient daylight photoperiod and PDA. Conidia and conidiophores were described from water
mounts. Phenotypic characters were compared with data published previously (Aoki and O'Donnell 1999;
O'Donnell et al. 2004, 2008; Starkey et al. 2007; Yli-Mattila et al. 2009; Sarver et al. 2011).
Multilocus genotyping and molecular phylogenetics.—The MLGT assay for the simultaneous
determination of B clade species identity and trichothecene chemotype prediction was described by Ward
et al. (2008). Briefly, the multiplex reactions contained allele-specific primer extension (ASPE) probes
that targeted two different genes to identify each B clade species and genetic variation previously detected
within Tri3 and Tri12 (Ward et al. 2002) for determining trichothecene chemotype (i.e. nivalenol = NIV,
3-acetyldeoxynivalenol = 3ADON or 15-acetyldeoxynivalenol = 15ADON). As reported in Ward et al.
(2008), ASPE probes EFsp(31) and ATsp(33) were validated for the identification of F. dactylidis and
ASPE probes T12-N-37 and T3-N-2 were developed for identifying NIV-producing isolates.
The trichothecene biosynthetic gene cluster sequence of F. dactylidis strain NRRL 29380 (GenBank
accession number KP057243) was obtained from a draft genome sequence generated by BGI Americas
Corp. (Cambridge, Massachusetts) on an Illumina Hiseq 2000 platform (Illumina Inc., San Diego,
California), using 90 bp paired-end sequencing. The trichothecene biosynthetic gene cluster sequence of
Fusarium graminearum strain 88-1 (AF336365.2, Lee et al. 2002) was used to retrieve homologous
sequences from the F. dactylidis draft genome for manual annotation and in silico translation in
Sequencher 4.10.1 (Gene Codes Corp., Ann Arbor, Michigan). Given the close sister group relationship
between F. dactylidis and F. pseudograminearum, BLAST queries were conducted to determine whether
the novel virulence genes discovered in F. pseudograminearum, FpAH1 and FpDLH1 (EKJ74132.1 and
EKJ71690.1, Gardiner et al. 2012), were present in the genome of strain NRRL 29380.
Extraction of total genomic DNA, PCR amplification and DNA sequencing of portions of the
RPB1 and RPB2 subunits of RNA polymerase II followed O'Donnell et al. (2013). The maximum
parsimony (MP) analysis was conducted with PAUP* 4.0b10 (Swofford 2003), and the maximum
likelihood analysis was implemented with GARLI 1.0 (Zwickl 2006) on the CIPRES Science Gateway
TeraGrid employing the GTR + I + Γ model of evolution. Nucleotide sequence data generated in the
present study were deposited in GenBank (accession numbers KM361637–KM36167) and the NEXUS
file and the most-parsimonious tree were deposited in TreeBASE (accession number S16253, tree number
Tr77032).
Toxin production in liquid culture.—Mycotoxin production by F. dactylidis strains NRRL 29298 and
29380 was evaluated with a two-stage liquid culture protocol modified from Greenhalgh et al. (1986).
Cultures were initiated by inoculating 50 mL stage 1 media (3 g NH4Cl, 2 g MgSO4·7H2O, 0.2 g
FeSO4·7H2O, 2 g KH2PO4, 2 g peptone, 2 g yeast extract, 2 g malt extract and 20 g glucose per L water)
in a 250 mL Erlenmeyer flask with mycelia scraped from a V8 juice agar plate (20% V8 juice, 0.3%
CaCO3, 2% agar; Stevens 1974). After culturing 3 d in the dark at 28 C on a rotary shaker at 200 rpm, the
cultures were transferred aseptically to a 400 mL beaker, macerated with a stick blender (Cuisinart Smart
Stick, East Windsor, New Jersey), pelleted in a centrifuge, and 25 mL of the supernatant was discarded.
The remaining material was vortexed briefly to obtain a homogenous suspension and then 1.5 mL mixture
was used to inoculate 20 mL stage 2 media (10% sucrose-YEP: 100 g sucrose, 1 g yeast extract, 1 g
peptone per L water) in a 50 mL Erlenmeyer flask. After 7 d, a 5 mL aliquot was extracted with 2 mL
ethyl acetate and analyzed by GC/MS for trichothecenes using a Hewlett-Packard 5891 mass-selective
detector fitted with a HP-5MS column (30 m by 0.25 μm thick film). The column was held at 120 C at
injection, heated to 260 C at 25 C min−1 and held 6.4 min. A NIV standard was prepared by hydrolysis of
acetylated NIV with 0.1 N NaOH after it was purified from a liquid culture of F. cerealis NRRL 37851.
Toxin production in solid culture.—To prepare inoculum, conidia of F. dactylidis strains NRRL 29298 and
29380 were harvested after 5 d growth on V8 agar and suspended in water at 106 spores/mL. Cracked corn
(50 g cracked corn + 22 mL water) and rice (20 g rice + 9 mL water) were autoclaved in 300 mL
Erlenmeyer flasks for 30 min and allowed to cool to room temperature. Each experimental flask was
inoculated with 2 mL inoculant and shaken once a day for the first 3 d. Negative control flasks were
inoculated with 2 mL sterile water. After incubation for 28 d in the dark, mycotoxins were extracted 1 h
with continuous shaking with acetonitrile-water, 86:14 vol/vol (10 g culture material / 40 mL extraction
solvent) or acetonitrile-water, 1:1 vol/vol (10 g culture material/50 mL extraction solvent). After the
extraction solvent was removed by filtration (Whatman 2V pleated paper filter, Sigma-Aldrich, St Louis,
Missouri), extracts were analyzed with a liquid chromatograph (Dionex Model U3000, Thermo Scientific,
San Jose, California) coupled to a QTRAP 3200 tandem mass spectrometer (AB SCIEX, Thornhill,
Ontario, Canada) with electrospray or atmospheric pressure chemical ionization. For trichothecene and
zearalenone (ZON) mycotoxin analysis, acetonitrile-water extracts were separated on a Kinetex column
(C18, 2.0 µm particle size, 4.6 mm diam × 150 mm long; Phenomenex, Golden, Colorado) with a
gradient (15–90% acetonitrile over 12 min) flow (0.9 mL/min) of acetonitrile-water with 0.3%
ammonium acetate. Eluting mycotoxins were detected with atmospheric pressure chemical ionization in
negative mode with multiple reaction monitoring. Deoxynivalenol (DON), NIV, 3ADON and 15ADON
B-type trichothecenes and ZON were monitored by observing the 355–359, 371–359, 397–359, 397–307
and 317–175 transitions respectively. Toxins were identified with Analyst software (AB SCIEX, Thornhill,
Ontario, Canada).
Wheat head blight assays and toxin analysis.—Two wheat head blight assays of varying inoculum levels
were employed to evaluate virulence and toxin production of F. dactylidis strains NRRL 29298 and 29380
in planta. Susceptible cultivar Norm spring wheat was grown to anthesis in growth chambers controlled at
25 C day/20 C night, 500 μmol m−2 sec−1 photosynthetic photo flux density 12 h photoperiod and 50–60%
relative humidity as described by Desjardins et al. (2000). Wheat heads were inoculated by injecting 10
µL suspension containing approximately 103 conidia in mung bean broth (Gale et al. 2005) into a floret.
Negative control heads were injected with sterile mung bean broth, and for comparison a set of heads
were injected with F. graminearum NRRL 29169 (= Gz3639 = CBS 110271). After inoculation the heads
were covered with plastic bags for 3 d to ensure high humidity during the initial phase of pathogenesis. In
the first assay a single floret of a central spikelet of the head was inoculated for each of 10 heads
representing biological replicates. Disease spread was evaluated by calculating the percentage of
symptomatic spikelets with premature whitening or necrosis over 21 d post inoculation. Disease
progression for the different strains of Fusarium was compared by an analysis of covariance (ANCOVA).
As an alternate more severe approach all florets on one side of the head were inoculated by injecting each
floret with 10 µL conidial suspension. Mature heads from both experiments were harvested 35 d post
inoculation. A subset of 10 heads per treatment was individually threshed, and the total seed weight per
head and the percentage of diseased seed was determined. Variable means were compared using a
student's t-test. The remaining 10 heads were combined in pairs (n = 5), ground and toxin was extracted
with 20 mL extraction solvent (acetonitrile-water, 86:14 vol/vol) in a 50 mL polypropylene screw-cap
centrifuge tube with shaking for 1 h. Each sample was purified by forcing 5 mL extract through a
MycoSep 225 Trich cartridge (Romer Labs, Union, Missouri). Two milliliters of the purified extract were
transferred to a 1 dram vial and dried under a nitrogen stream. Trimethylsilyl (TMS) derivatives were
prepared by adding 100 µL Tri-Sil reagent (Pierce, Rockford, Illinois) to the dried extract. After 30 min
900 µL isooctane was added, followed by 1 mL water. The mixture was vortexed gently until the organic
(top) layer became translucent. The organic layer was analyzed by GC/MS as described above. TMS
derivatives were prepared with purified NIV in quantities of 2–20 µg, and these data were used to
construct a standard curve. Compound identifications were confirmed with NMR and mass spectral
analysis.
RESULTS
Molecular phylogenetics of F. dactylidis.—Maximum likelihood (ML) and maximum
parsimony (MP) analyses of 22 partial RPB1 and RPB2 sequences representing all 21 B
clade species strongly supported the genealogical exclusivity of F. dactylidis and its
sister group relationship with F. pseudograminearum (FIG. 1, 100% ML/MP bootstrap
support). In addition reanalysis of the 12-gene 72-taxa dataset published for the B clade
(Sarver et al. 2011), revealed that six of the seven gene partitions supported a F.
dactylidis-F. pseudograminearum sister group relationship (i.e. α-tubulin, β-tubulin,
EF-1α, histone H3, reductase, URA-Tri101-PHO; MP bootstrap support = 87–100%;
data not shown) and the highly conserved ITS rDNA + LSU rDNA domains D1/D1
(1133 bp alignment), supported F. dactylidis monophyly. Although analysis of MAT1-1
strongly supported F. dactylidis monophyly, the MAT phylogeny resolved it as sister to
a clade comprising F. pseudograminearum, F. culmorum, F. cerealis (Cooke) Sacc. and
F. lunulosporum Gerlach (data not shown).
Multilocus genotyping of the trichothecene biosynthetic genes.—MLGT analyses with
species and trichothecene chemotype-specific probes indicated that F. dactylidis isolates
NRRL 29298 and 29380 possess trichothecene biosynthetic gene clusters that might
enable them to produce nivalenol toxins in vitro and in vivo. Probe T12-N(37), one of
two NIV-specific ASPE probes, produced positive genotypes with NRRL 29298 and
29380 (Ward et al. 2008); however ASPE probe T3-N-2 produced negative genotypes
with these strains. Analysis of the Tri3 gene sequence obtained from the trichothecene
gene cluster of NRRL 29380 revealed that this strain harbored the NIV-specific
variation targeted by the T3-N-2 probe. However the probe failed due to an unrelated
point mutation under the probe sequence. This finding will enable us to redesign
T12-N(37) to accommodate this novel genetic variation. In addition the NRRL 29380
trichothecene gene cluster included intact open-reading frames for Tri13 (trichothecene
C-4 hydoxylase) and Tri7 (trichothecene C-4 acetyltransferase), which are responsible
for the NIV chemotype (Lee et al. 2002).
Toxin production and pathogenicity assays.—Both isolates of F. dactylidis produced
acetylated NIV in liquid culture, but the amount produced (NRRL 29298 = 295 ± 36
μg/mL, NRRL 29380 = 27 ± 12 μg/mL) were significantly different statistically
(student's t-test, P < 0.001). In contrast both isolates produced NIV in culture on solid
substrate at statistically indistinguishable levels (NRRL 29298 = 8.3 ± 10.1 μg/mL,
NRRL 29380 = 6.4 ± 2.3 μg/mL, student's t test, P > 0.2 on corn and NRRL 29298 =
20.8 ± 11.7 μg/mL and NRRL 29380 = 16.7 ± 19.7 μg/mL, student's t-test, P > 0.2 on
rice). Similarly both isolates produced ZON on culture on solid substrate at statistically
indistinguishable levels (NRRL 29298 = 2.5 ± 3.4 μg/mL, NRRL 29380 = 0.2 ± 0.2
μg/mL, student's t-test, P > 0.2 on corn and NRRL 29298 = 19.4 ± 13.2 μg/mL and
NRRL 29380 = 18.2 ± 16.1 μg/mL, student's t-test, P > 0.2 on rice). Moreover NIV was
detected in wheat heads of cultivar Norm that were infected with NRRL 29298 (43 ±
5.0 μg/mL), but this toxin was below detectible limits in plants infected with NRRL
29380 (statistical difference student's t-test, P < 0.001). Fusarium dactylidis strains
displayed moderate virulence on wheat. While disease progression was modest in
comparison to the highly virulent 15ADON-producing F. graminearum strain NRRL
29169, F. dactylidis NRRL 29298 and 29380 were able to spread beyond the inoculated
spikelet (FIG. 2A). Furthermore wheat that received a heavy inoculum of F. dactylidis
NRRL 29298 produced significantly less grain yield per head and an increased level of
diseased seed compared to strain NRRL 29380 (FIG. 2B, C).
TAXONOMY
Fusarium dactylidis T. Aoki, Geiser, P.R. Johnst. & O'Donnell, sp. nov. FIGS. 3–6
MycoBank MB809785
Typification: NEW ZEALAND, Manawatu, Palmerston North, isolated from Dactylis
glomerata L. (orchard grass or cock's foot), 30 Oct 1958, J.M. Dingley No. 5941
(holotype BPI 892886, dried culture of NRRL 29298; Isotype PDD 104196, a dried
culture of ICMP 5269. Ex-type cultures NRRL 29298 = CBS 119181 = ICMP 5269).
Etymology: The epithet refers to the associated plant host genus, Dactylis.
Colonies in darkness on PDA have fast average growth rates of 6.2–6.9 mm and
7.2–9.1 mm per d at 20 C and 25 C respectively. Colony color on PDA at first White,
becoming orange white (5–6A2), reddish white (9–10A2), pale red (7–9A3), pastel red
(7–9A4–5), dull red (8–9B3–4), then turning grayish red (8–10B5–6, 8–10C5), grayish
rose (11B6), red (9–11A6–8, 9–11B7–8) to brownish red (8–10C6, 9–11C7–8) or
reddish brown (9D–E7–8) to violet brown (10–11E–F7–8); reverse white at margin,
reddish pigmentation centrally, reddish white (9–10A2), pale red (7–9A3), pastel red
(7–9A4–5), grayish red (8–9B4–6, 8–9C4–5), brownish red (8–9C6, 9–11C7–8),
reddish brown (9D–E7–8), violet brown (10–11E–F7–8). Colony margin entire to
undulate. Odor absent or moldy. Aerial mycelium on PDA generally abundant,
sometimes sparsely developed, loose to densely floccose. Hyphae on SNA 1–10 µm
wide. Chlamydospores and sclerotia absent, but round intercalary or terminal cell
swellings sometimes present in hyphae or older conidia. Sporulation on SNA under
black light quick and abundant from conidiophores formed directly on substrate
mycelium or aggregated in sporodochia on the agar surface, in darkness less abundant
or arrested; sporodochia formed abundantly or sparsely. Conidiophores branched or
unbranched, forming monophialides on the apices, or as single monophialides formed
on substrate hyphae. Phialides subulate, ampulliform to subcylindrical, sometimes
doliiform, monophialidic, 12.5–24.5 × 3.5–5.5 μm, with slight periclinal thickening at
the tip. Short collarets sometimes are visible at the tip of phialides under higher
magnification. Conidia of a single type, typically falcate and curved, dorsiventral,
(1–)3–5(–7)-septate, usually widest at or slightly above the midregion of their length,
tapering and curving equally toward both ends, typically with an acute apical cell and
often with a distinct basal foot cell. Upper and lower halves of conidia nearly
symmetrical. Under black light, three-septate conidia: 30–65.5 × 3–6.5 μm in total range,
36.3–49.3 × 4.9–5.4 μm on average (ex-holotype strain: 34.5–49.3–65.5 × 4–4.9–5.5
μm); five-septate conidia: 34.5–68 × 4–6 μm in total range, 40.3–51.9 × 4.9–5.1 μm on
average (ex-holotype strain: 38.5–51.9–68 × 4–4.9–6 μm).
Other strains examined: NRRL 29380 = CBS 123656 = FRC R-7593, ex D. glomerata in Oregon,
USA, Ronald E. Welty 2A-1, 1983.
Distribution: New Zealand and Oregon, USA.
DISCUSSION
The reddish pigmentation produced by the sparsely to densely floccose colonies of F.
dactylidis on PDA is typical of other B clade species. Fusarium dactylidis only
produces multiseptate conidia from monophialides produced in sporodochia or directly
on substrate hyphae. The conidia average ca. 5 µm wide, similar to those in F.
aethiopicum O'Donnell et al. (O'Donnell et al. 2008), F. ussurianum T. Aoki et al.
(Yli-Mattila et al. 2009), F. vorosii B. Tóth et al. (Starkey et al. 2007), F. cerealis
(Nirenberg 1990) and F. culmorum (Aoki et al. 2012; FIGS. 5, 6). Conidia formed by F.
dactylidis on SNA under black light are usually widest at or slightly above the
midregion and taper and curve equally toward both ends (FIGS. 3, 6). Although F.
cerealis and F. culmorum also form symmetrical conidia, those of F. dactylidis are on
average longer than those of F. culmorum and shorter than those of F. cerealis (FIGS. 5,
6). In addition conidia of F. cerealis differ in that they possess a narrow apical beak. F.
aethiopicum and F. vorosii produce asymmetrical and predominantly straight conidia
(FIG. 6; Aoki et al. 2012), which are distinct from the conidia of F. dactylidis that were
almost symmetrical and curved toward both ends (FIGS. 5, 6). Conidial shape and size
of F. dactylidis is similar to that of F. ussurianum (Yli-Mattila et al. 2009), but conidia
of F. ussurianum (FIG. 6) typically possess a narrow apical beak that is rarely observed
in F. dactylidis, and the latter species typically produce conidia with an acute apical cell
that is rarely observed in F. ussurianum. While this comparison shows that F. dactylidis
can be distinguished phenotypically from other B clade species, most end users will
prefer to use DNA sequence data to obtain a definitive identification.
Our results on trichothecene production and pathogenicity of F. dactylidis are
consistent with the available data that indicate NIV-producing strains are generally less
virulent than DON-producing strains in wheat and that toxin production is a virulence
factor (Proctor et al. 1995, Jansen et al. 2005). Although NIV appears to be less
phytotoxic than DON, NIV has been reported to be more toxic than DON in human cell
lines (Abbas et al. 2013). The present study is the first to document ZON production by
F. dactylidis, which is well known for its toxicity to mammals (Creppy 2002). Thus
future studies are needed to assess whether livestock and other animals are exposed to
NIV and ZON produced by F. dactylidis, given that orchard grass is the fourth most
important forage grass worldwide (Stewart and Ellison 2011). Moreover, in light of the
fact that Ronald E. Welty informed Paul E. Nelson that his isolate No. 2A-1 (= NRRL
29380) was highly pathogenic, inducing crown rot and leaf blight symptoms on 6 wk
old orchard grass seedlings, plant breeders may benefit from testing genetically diverse
populations of D. glomerata for broad-based resistance to F. dactylidis.
Fusarium dactylidis isolates NRRL 29298 and 29380 both possess MAT1-1
idiomorphs that appear to be functional based on in silico translations (O'Donnell et al.
2004, Sarver et al. 2011), suggesting this species may possess a heterothallic or
self-sterile reproductive mode. To assess whether any of the perithecial collections
accessioned in the PDD herbarium as G. zeae on D. glomerata might be F. dactylidis,
attempts were made to PCR amplify and sequence the ITS rDNA region from DNA
extracts of perithecia from three New Zealand collections (PDD 12851, 24746, 26041
that were collected in 1954, 1965 and 1967 respectively). DNA sequence data was
obtained only from PDD 12851 collected in Central Otago District in 1954. While the
data clearly indicated the perithecia were not produced by F. dactylidis, a definitive
identification was not possible because the ITS rDNA allele is shared by several B clade
fusaria, including F. graminearum and F. cortaderiae O'Donnell et al., both of which
have been reported from New Zealand (Mounds et al. 2005).
In contrast to the trichothecene gene cluster of NRRL 29380, Tri13 and Tri7 are
either missing or are present as pseudogenes in the genomes of 3ADON and 15ADON
strains (Lee et al. 2002, Kimura et al. 2003). While F. dactylidis' sister F.
pseudograminearum was reported to have acquired the novel virulence genes FpAH1
and FpDLH1 horizontally from bacteria (EKJ74132.1, EKJ71690.1; Gardiner et al.
2012), BLAST queries suggest these genes are absent in the genome of F. dactylidis
NRRL 29380.
While the present study has advanced our understanding of the pathogenic and
toxigenic potential of F. dactylidis, it also has exposed significant gaps in our
knowledge of this novel NIV-producing species. To help fill this void, surveys are
needed for the presence of this pathogen in geographically and genetically diverse
populations of orchard grass, which is widely cultivated for forage in Europe, North
America, Asia and Oceania (Xie et al. 2010) and also rotated with corn, wheat and
soybean in organic farming (Teasdale et al. 2004). These surveys are needed to address
several basic aspects of its biology, including: (i) Is F. dactylidis segregating for
additional trichothecene chemotypes? (ii) Is it transmitted vertically in seed, which
might explain the transoceanic disjunction of the two known isolates? (iii) Does it
possess a heterothallic reproductive mode, and if so, where and on what host(s) does it
undergo sexual reproduction? and (iv) Is it distributed worldwide on wild and cultivated
orchard grass, and if so what is its global population genetic structure? Last,
comparative pathogenomic analyses and functional studies are needed to elucidate why
F. pseudograminearum (Gardiner et al. 2012) and F. dactylidis are excellent crown rot
but weak head blight pathogens of small grain cereals.
ACKNOWLEDGMENTS
We acknowledge Dr Joan M. Dingley, formerly of New Zealand Fungal and Plant Disease Collection, and
the late Dr Ronald E. Welty, of the Forage Seed and Cereal Research Unit, ARS-USDA, for depositing
the two isolates of F. dactylidis from orchard grass upon which this research was conducted. The authors
thank Nathane Orwig for running sequences in the NCAUR DNA Core Facility, Thomas R. Usgaard for
annotating the trichothecene gene cluster of F. dactylidis NRRL 29380, Stacy Sink for generating the
DNA sequence data reported in this study and Jennifer M. Teresi and Deborah S. Shane for skilled
assistance with the toxin analyses and pathogenicity experiments. The mention of conpany names or trade
products does not imply that they are endorsed or recommended by the US Department of Agriculture
over other companies or similar products not mentioned. The USDA is an equal opportunity provider and
employer.
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LEGENDS
FIG. 1. Molecular phylogeny of 21 B clade species inferred from combined analysis of partial RPB1 +
RPB2 sequence data. The phylogram is one of eight equally most-parsimonious trees inferred via
maximum parsimony implemented in PAUP* 4.0b10 (Swofford 2003). Maximum likelihood (ML,
Zwickl 2006) and maximum parsimony bootstrap (BS) values ≥ 70% are above nodes (ML-BS/MP-BS).
The five-digit number next to each strain represents the ARS Culture Collection (NRRL) accession
number. The two thickened internodes identify Fusarium dactylidis and F. pseudograminearum as
strongly supported sisters and the F. graminearum species complex (FGSC). CI = consistency index,
MPT = most-parsimonious tree, PIC = parsimony informative character, RI = retention index.
FIG. 2. Fusarium dactylidis virulence in wheat. A. Disease progression in wheat cultivar Norm tracked for
21 d after a single floret was inoculated with either a mung bean broth negative control, F. graminearum
NRRL 29169 (= Gz3639) or F. dactylidis strains NRRL 29298 and 29380. Regression lines were
compared with an analysis of covariance (ANCOVA). B. Yield of mature wheat heads inoculated at all of
the florets on one side of the head with the negative control and Fusarium strains NRRL 29169, 29298 or
29380 represented as the mean of total seed weight per head in grams. C. Mean percentage of diseased
seeds was estimated and comparisons were made with an analysis of variance (ANOVA) followed by a
Tukey-Kramer honestly significant difference. Values represent averages ± SEM (n = 10) and letters
indicate significant differences (P<0.001).
FIG. 3. Microscopic features of Fusarium dactylidis NRRL 29298 and 29380 on SNA under black light. A,
B. Sporodochia formed on agar surface. C. Sporodochial conidia formed directly from phialides on
substrate hypha. D. Sporodochial conidiophore forming conidia on monophialides. E. Four–five-septate
sporodochial conidia formed from monophialides. F, G. Symmetrical four–six-septate sporodochial
conidia, widest at or slightly above midregion, curved and tapering gradually toward both ends, with a
foot cell and an acute apex. H, I. Swollen intercalary cells in old conidia. A, F, G. NRRL 29380. B–E.
NRRL 29298 ex-holotype. Bars: A, B = 50 μm. C–I = 25 μm.
FIG. 4. Radial growth rate of Fusarium dactylidis NRRL 29298 and 29380 on PDA in dark at eight
temperatures 5–40 C at 5 C intervals.
FIG. 5. Scatter diagram showing mean length and width of five-septate conidia of Fusarium dactylidis
strains NRRL 29298 and 29380 cultured on SNA under continuous black light, together with 20 other B
clade species. Plots are based on mean size of 50 randomly selected five-septate conidia.
FIG. 6. Comparison of conidial morphology of Fusarium dactylidis (left: NRRL 29380; right: NRRL
29298) with seven closely related B clade fusaria. “S” indicates species that produce symmetrical conidia.
Note that F. graminearum, F. aethiopicum and F. vorosii produce asymmetrical conidia that typically are
widest above the midregion. However the other species, including F. dactylidis, produce conidia that
typically are widest at the midregion. Arrows identify conidia with a narrow apical beak. Bar = 25 μm.
FOOTNOTES
Submitted 20 Aug 2014; accepted for publication 18 Nov 2014
1Corresponding author: E-mail: taoki@nias.affrc.go.jp