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.

LITERATURE CITED

Abbas HK, Yoshizawa T, Shier WT. 2013. Cytotoxicity and phytotoxicity of

trichothecene mycotoxins produced by Fusarium spp. Toxicon 74:68–75.

Aoki T, O'Donnell K. 1999. Morphological and molecular characterization of Fusarium

pseudograminearum sp. nov., formerly recognized as the Group 1 population of F.

graminearum and PCR primers for its identification. Mycologia 91:597–609.

———, ———, Homma Y, Alfredo R, Lattanzi AR. 2003. 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. Mycologia

95:660–684.

———, ———, Ichikawa K. 2001. Fusarium fractiflexum sp. nov. and two other

species within the Gibberella fujikuroi species complex recently discovered in Japan that form aerial

conidia in false heads. Mycoscience 42:461–478.

———, ———, Scandiani MM. 2005. Sudden-death syndrome of soybean in

South America is caused by four species of Fusarium: Fusarium brasiliense sp. nov., F. cuneirostrum sp.

nov., F. tucumaniae and F. virguliforme. Mycoscience 46:162–183.

———, Ward TJ, Kistler HC, O'Donnell K. 2012. Systematics, phylogeny and

trichothecene mycotoxin potential of Fusarium head blight cereal pathogens. Mycotoxins 62:91–102.

Boutigny A-L, Ward TJ, van Coller GJ, Flett B, Lamprech SC, O'Donnell K, Viljoen A. 2011. Analysis

of the Fusarium graminearum species complex from wheat, barley and maize in South Africa provides

evidence of species-specific differences in host preference. Fungal Genet Biol 48:914–920.

Creppy EE. 2002. Update of survey, regulation and toxic effects of mycotoxins in Europe. Toxicol Lett

127:19–28.

Desjardins AE, Bai G, Plattner RD, Proctor RH. 2000. Analysis of aberrant virulence of Gibberella zeae

following transformation-mediated complementation of a trichothecene-deficient (Tri5) mutant.

Microbiology 146:2059–2068.

Gale LR, Bryant JD, Calvo S, Giese H, Katan T, O'Donnell K, Suga H, Taga M, Usgaard TR, Ward TJ,

Kistler HC. 2005. Chromosome complement of the fungal plant pathogen Fusarium graminearum

based on genetic and physical mapping and cytological observations. Genetics 171:985–1001.

Gardiner DM, McDonald MC, Covarelli L, Solomon PS, Rusu AG, Marshall M,

Kazan K, Chakraborty S, McDonald BA, Manners JM. 2012. Comparative pathogenomics reveals

horizontally acquired novel virulence genes in fungi infecting cereal hosts. PLoS Pathog 8:e1002952, doi:

10.1371/journal.ppat.1002952.

Geiser DM, Aoki T, Bacon CW, Baker SE, Bhattacharyya MK, Brandt ME, Brown DW,

Burgess LW, Chulze S, Coleman JJ, Correll JC, Covert SF, Crous PW, Cuomo CA, De Hoog GS, Di

Pietro A, Elmer WH, Epstein L, Frandsen RJ, Freeman S, Gagkaeva T, Glenn AE, Gordon TR, Gregory

NF, Hammond-Kosack KE, Hanson LE, Jímenez-Gasco Mdel M, Kang S, Kistler HC, Kuldau GA, Leslie

JF, Logrieco A, Lu G, Lysøe E, Ma LJ, McCormick SP, Migheli Q, Moretti A, Munaut F, O'Donnell K,

Pfenning L, Ploetz RC, Proctor RH, Rehner SA, Robert VA, Rooney AP, Bin Salleh B, Scandiani MM,

Scauflaire J, Short DP, Steenkamp E, Suga H, Summerell BA, Sutton DA, Thrane U, Trail F, van

Diepeningen A, Vanetten HD, Viljoen A, Waalwijk C, Ward TJ, Wingfield MJ, Xu JR, Yang XB,

Yli-Mattila T, Zhang N. 2013. One fungus, one name: defining the genus Fusarium in a scientifically

robust way that preserves longstanding use. Phytopathology 103:400–408.

Gerlach W, Nirenberg H. 1982. The genus Fusarium—a pictorial atlas. Mitt Biol

Bundesanst. Land- Forstwirtsch 209:1–406.

Greenhalgh R, Levandier D, Adams W, Miller JD, Blackwell BA, McAlees AJ, Taylor A. 1986.

Production and characterization of deoxynivalenol and other secondary metabolites of Fusarium

culmorum (CMI 14764, HLX 1503). J Agric Food Chem 34:98–102.

Jansen C, von Wettstein D, Schäfer W, Kogel K-H, Felk A, Maier FJ. 2005. Infection patterns in

barley and wheat spikes inoculated with wild-type and trichodiene synthase gene disrupted Fusarium

graminearum. Proc Natl Acad Sci USA 102:16892–16897.

Kimura M, Tokai T, O'Donnell K., Ward TJ, Fujimura M, Hamamoto H, Shibata T, Yamaguchi I. 2003.

The trichothecene biosynthesis gene cluster of Fusarium graminearum F15 contains a limited

number of essential pathway genes and expressed non-essential genes. FEBS Lett 539:105–110.

Kornerup A, Wanscher JH. 1978. Methuen handbook of color, 3rd ed. London: Methuen. 252 p.

Lee T, Han Y-K, Kim K-H, Yun S-H, Lee Y-W. 2002. Tri13 and Tri7 determine deoxynivalenol- and

nivalenol-producing chemotypes of Gibberella zeae. Appl Environ Microbiol 68:2148–2154.

Mounds RD, Cromey MG, Lauren DR, di Menna M, Marshall J. 2005. Fusarium graminearum, F.

cortaderiae and F. pseudograminearum in New Zealand: molecular phylogenetic analysis,

mycotoxin chemotypes and co-existence of species. Mycol Res 109:410–420.

Nelson PE, Toussoun TA, Marasas WFO. 1983. Fusarium species: an illustrated manual for identification.

University Park: Pennsylvania State Univ. Press. 193 p.

Nirenberg HI. 1990. Recent advance in the taxonomy of Fusarium. Stud Mycol 32:91– 101.

O'Donnell K, Rooney AP, Proctor RH, Brown DW, McCormick SP, Ward TJ, Randsen

RJN, Lysøe E, Rehner SA, Aoki T, Robert VARG, Crous PW, Groenewald JZ, Kang S, Geiser DM. 2013.

Phylogenetic analyses of RPB1 and RPB2 support a middle Cretaceous origin for a clade comprising all

agriculturally and medically important fusaria. Fungal Genet Biol 52:20–31.

———, Ward, TJ, Aberra D, Kistler HC, Aoki T, Orwig N, Kimura M, Bjørnstad Å,

Klemsdal SS. 2008. Multilocus genotyping and molecular phylogenetics resolve a novel head blight

pathogen within the Fusarium graminearum species complex from Ethiopia. Fungal Genet Biol

45:1514–1522.

———, ———, Geiser DM, Kistler HC, Aoki T. 2004. Genealogical concordance

between the mating type locus and seven other nuclear genes supports formal recognition of nine

phylogenetically distinct species within the Fusarium graminearum clade. Fungal Genet Biol

41:600–623.

Proctor RH, Hohn TM, McCormick SP. 1995. Reduced virulence of Gibberellazeae caused by

disruption of a trichothecene toxin biosynthetic gene. Mol Plant Microbe Interact 8:593–601.

Roux J, Steenkamp ET, Marasas WFO, Wingfield MJ, Wingfield BD. 2001.

Characterization of Fusarium graminearum from Acacia and Eucalyptus using β-tubulin and histone gene

sequences. Mycologia 93:704–711.

Sarver BAJ, Ward TJ, Gale LR, Broz K, Kistler HC, Aoki T, Nicholson P, Carter J, O'Donnell K. 2011.

Novel Fusarium head blight pathogens from Nepal and Louisiana revealed by multilocus genealogical

concordance. Fungal Genet Biol 48:1096–1107.

Starkey DE, Ward TJ, Aoki T, Gale LR, Kistler HC, Geiser DM, Suga H, Toth B, Varga J, O'Donnell K.

2007. Global molecular surveillance reveals novel Fusarium head blight species and trichothecene toxin

diversity. Fungal Genet Biol 44:1191–1204.

Stevens RR. 1974. Mycology guidebook. Seattle: Univ. Washington Press. 703 p.

Stewart AV, Ellison NW. 2011. Dacytlis. In: Kole C, ed. Wild crop relatives: Genomic and breeding

resources, millets and grasses. New York: Springer. p 73–87.

Swofford DL. 2003. PAUP*: phylogenetic analysis using parsimony (*and other methods). 4.0b10.

Sunderland, Massachusetts: Sinauer Associates.

Taylor JW, Jacobson DJ, Kroken S, Kasuga T, Geiser DM, Hibbett DS, Fischer MC. 2000. Phylogenetic

species recognition and species concepts in fungi. Fungal Genet Biol 31: 21–32.

Teasdale JR, Mangum RW, Radhakrishnan J, Cavigelli MA. 2004. Crop rotation, weed seedbank

dynamics in three organic framing crop rotations. Agron J 96:1429–1435.

Ward TJ, Bielawski JP, Kistler HC, Sullivan E, O'Donnell K. 2002. Ancestral

polymorphism and adaptive evolution in the trichothecene mycotoxin gene cluster of phytopathogenic

Fusarium. Proc Natl Acad Sci USA 99:9278–9283.

———, Clear RM, Rooney AP, O'Donnell K, Gaba D, Patrick S, Starkey DE, Gilbert J, Geiser DM,

Nowicki TW. 2008. An adaptive evolutionary shift in Fusarium head blight pathogen populations is

driving the rapid spread of more toxigenic Fusarium graminearum in North America. Fungal Genet Biol

45:473–484.

Xie W-G, Zhang X-Q, Cai H-W, Liu H-W, Peng Y. 2010. Genetic diversity and transferability of cereal

EST-SSR markers to orchard grass (Dactylis glomerata L.). Biochem Syst Ecol 38:740–749.

Yli-Mattila T, Gagkaeva T, Ward TJ, Aoki T, Kistler HC, O'Donnell K. 2009. A novel Asian clade within

the Fusarium graminearum species complex includes a newly discovered cereal head blight pathogen

from the Russian Far East. Mycologia 101:841–852.

Zwickl DJ. 2006. Genetic algorithm approaches for the phylogenetic analysis of large biological sequence

datasets under the maximum likelihood criterion [doctoral dissertation]. Austin: Univ. Texas Press.

125 p.

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