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1306 PHYTOPATHOLOGY
Biological Control
Identification of the Infection Route of a Fusarium Seed Pathogen
into Nondormant Bromus tectorum Seeds
JanaLynn Franke, Brad Geary, and Susan E. Meyer
First and second authors: Department of Plant and Wildlife Sciences, Brigham Young University, Provo, UT 84602; and third author: U.S.
Department of Agriculture Forest Service Rocky Mountain Research Station, Shrub Sciences Laboratory, Provo, UT 84606.
Accepted for publication 6 June 2014.
ABSTRACT
Franke, J., Geary, B., and Meyer, S. E. 2014. Identification of the
infection route of a Fusarium seed pathogen into nondormant Bromus
tectorum seeds. Phytopathology 104:1306-1313.
The genus Fusarium has a wide host range and causes many different
forms of plant disease. These include seed rot and seedling blight diseases
of cultivated plants. The diseases caused by Fusarium on wild plants are
less well-known. In this study, we examined disease development caused
by Fusarium sp. n on nondormant seeds of the important rangeland weed
Bromus tectorum as part of broader studies of the phenomenon of stand
failure or “die-off” in this annual grass. We previously isolated an un-
described species in the F. tricinctum species complex from die-off soils
and showed that it is pathogenic on seeds. It can cause high mortality of
nondormant B. tectorum seeds, especially under conditions of water
stress, but rarely attacks dormant seeds. In this study, we used scanning
electron microscopy (SEM) to investigate the mode of attack used by this
pathogen. Nondormant B. tectorum seeds (i.e., florets containing caryop-
ses) were inoculated with isolate Skull C1 macroconidia. Seeds were then
exposed to water stress conditions (–1.5 MPa) for 7 days and then trans-
ferred to free water. Time lapse SEM photographs of healthy versus
infected seeds revealed that hyphae under water stress conditions grew
toward and culminated their attack at the abscission layer of the floret
attachment scar. A prominent infection cushion, apparent macroscopically
as a white tuft of mycelium at the radicle end of the seed, developed
within 48 h after inoculation. Seeds that lacked an infection cushion
completed germination upon transfer to free water, whereas seeds with an
infection cushion were almost always killed. In addition, hyphae on seeds
that did not initiate germination lacked directional growth and did not
develop the infection cushion. This strongly suggests that the fungal
attack is triggered by seed exudates released through the floret attachment
scar at the initiation of germination. Images of cross sections of infected
seeds showed that the fungal hyphae first penetrated the caryposis wall,
then entered the embryo, and later ramified throughout the endosperm,
completely destroying the seed.
The invasive winter annual grass Bromus tectorum (cheatgrass,
downy brome) forms extensive near-monocultures over hundreds
of thousands of hectares in semiarid regions of western North
America. A common but poorly understood phenomenon in B.
tectorum stands is the occurrence of stand failure or “die-off”
over sometimes large areas. Stand failure in B. tectorum is
thought to be caused by soilborne pathogens, possibly related to
those that cause similar stand failure in winter cereal crops (1). In
a recent study, Fusarium sp. n isolates representing an un-
described species in the Fusarium tricinctum species complex
were obtained from killed nondormant (readily germinable) B.
tectorum seeds that had been planted into die-off soils (26). These
Fusarium isolates were found to be highly pathogenic on non-
ormant B. tectorum seeds, particularly under water-stress condi-
tions, simulating field conditions following early autumn storms.
In the pathogenicity test, nondormant seeds were inoculated with
Fusarium sp. n macroconidia and kept at –1.5 MPa for 1 week.
The seeds were subsequently exposed to a free-water environment
at which point 25 to 83% of the seeds were killed. Even without
the water-stress treatment, mortality as high as 43% was ob-
served. These results suggested that Fusarium sp. n might be an
important causal organism implicated in the die-off phenomenon
and prompted further studies of its pathogenesis on B. tectorum
seeds.
Members of the genus Fusarium are important crop pathogens
worldwide and have been the subject of intensive study, particu-
larly those species that impact winter cereal crops (11). Several
Fusarium species are reported to cause seed “rot” diseases, often
as part of a complex of diseases that affect different stages of the
host, such as seeds, seedlings, and the crowns of developing plants
(e.g., diseases caused by F. g r a m ine a r u m and related species) (29).
When seed rot and seedling blight are caused by the same
organism, preemergence mortality may be due to pathogen attack
either before or after germination. There are apparently few
studies on the mode of attack by Fusarium directly on un-
germinated seeds. The most extensive work has been with Fu-
sarium species that infect maize seed (5,6,21). More recently,
work has been done on species pathogenic on the seeds of root-
parasitic plants in the context of biological control. Heiko et al.
(15) showed that infection by F. oxysporum f. sp. orthoceras
resulted in the destruction of the germ tube of the seed and
reduced the number of parasitic attachments of Orobanche
cumana to its host plant (sunflower). In a more recent study (30),
a F. oxysporum isolate from Germany was noted to have the
capability to reduce O. ramosa seed germination by 40%. In a
more specialized study regarding the mode of infection for
F. nygamai into Striga hermonthica seeds, Sauerborn et al. (34)
showed that this pathogen penetrated the seed coat along the cell
walls, thereby requiring less energy, and that penetration culmi-
nated in the disintegration of the embryo and endosperm.
Baughman and Meyer (1) determined that dormant (not readily
germinable) B. tectorum seeds occurred at similar densities in the
persistent seed banks of die-off and adjacent non-die-off soils.
This suggested that the pathogen responsible for seed death and
stand failure only impacted nondormant seeds in the process of
Corresponding author: J. Franke; E-mail address: janalynn.franke@gmail.com
*The e-Xtra lo
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Vol. 104, No. 12, 2014 1307
initiating germination and had little or no effect on dormant seeds.
Most studies of the interactions between seeds and their patho-
gens in the “spermosphere” have involved crop species whose
seeds are nondormant at planting, so that the impact of dormancy
status on pathogenesis has not been considered (31). Studies of
losses to potentially pathogen-caused decay in weed seed banks
have also not explicitly considered dormancy status, even though
seed dormancy in weeds of arable lands is common (10). The
relationship between seed dormancy and pathogen-caused mor-
tality has recently received theoretical consideration, but there are
few empirical studies available to test model predictions (4).
Although we have isolated Fusarium sp. n strains from die-off
soils and demonstrated their pathogenicity on B. tectorum seeds,
the actual mechanism used by the fungus to infect and kill rapidly
germinating, nondormant B. tectorum seeds is poorly understood.
Therefore, the objective of this study was to visually examine the
mode of attack that is implemented by this pathogen to cause
mortality of nondormant B. tectorum seeds and to gain insight
into why it may be less able to attack dormant seeds.
MATERIALS AND METHODS
Genetic identification. The Fusarium sp. n isolate Skull C1
used in the current study was obtained from the strain collection
used by Meyer et al. (26) in the pathogenicity test previously
described. Amplification of a portion of the TEF (translation
elongation factor) gene was conducted using primers ef1 (forward
primer; 5′-ATGGGTAAGGA(A/G)GACAAGAC-3′) and ef2
(reverse primer; 5′-GGA(G/A)GTACCAGT(G/C)ATCATGTT-3′)
(9). Sequencing results were used in a BLAST search of the
FUSARIUM-ID (9) and NCBI GenBank databases.
Inoculum production, seed inoculation, and incubation.
Skull C1 was cultured onto SNA (Spezieller Nahrstoffaremer
agar) lined with one sterile filter paper and grown for 2 to 3 weeks
to induce sporulation (22). Once macroconidia were observed,
spores were suspended in sterile ddH2O (double deionized water)
by pipetting 10 ml of ddH2O into the petri dish and gently
knocking the spores off of the mycelium with the back of a sterile
spatula. Spore concentration was determined with a hemacytom-
eter and diluted to 250,000 spores ml–1. Nondormant B. tectorum
seeds (Brigham Young University Farm, Spanish Fork, UT; 2011)
were immersed in the spore suspension and shaken for 1 min;
excess inoculum was then discarded. Inoculated seeds were
placed in a sterile petri dish lined with a PEG-8000 (polyethylene
glycol)-soaked blue blotter (Anchor Paper Company, St. Paul,
MN). The PEG concentration was calculated to create a water
potential of –1.5 MPa (27) and was checked with the Aqualab
dew point water activity meter 4TE (Decagon, Pullman, WA).
Seeds were incubated at 25°C in the PEG solution for 7 days and
then transferred to free water and incubated at 25°C for up to 14
additional days.
Sample selection, preparation, and viewing. Inoculated seeds
were randomly sampled at 2, 24, 48, 72, and 96 h after
inoculation. Selected samples were then fixed by gently placing
Fig. 1. Scanning electron microscopic images of uninoculated Bromus tectorum seeds. A, Abscission layer (Ab) of the floret attachment scar (Fs). B, Vertical cross
section showing the unimbibed caryopsis surrounded by the floret bracts (Fb). The vascular tissue (Vt) begins at the abscission layer (Ab) of the attachment scar
(Fs) and ends at the caryopsis wall. Inside are the embryo (Em) and the endosperm (En). C, A close up of the cross section of the unimbibed endosperm with
amyloplast (Am) filled starch cells (St). It is protected by the floret bracts (Fb) on the outside and the caryopsis wall (C) on the immediate exterior. D, A close up
of imbibed B. tectorum endosperm with starch cells (St) which have begun to digest the amyloplasts (Am). The endosperm is protected by the caryopsis wall (C).
1308 PHYTOPATHOLOGY
samples into a 2% gluteraldehyde mixture for 48 h. The fixed
samples were placed into a sodium cacodylate working buffer at a
pH of 7.2 to 7.4 to wash the samples. The buffer solution was
replaced five times at 15-min intervals using the same buffer.
Ethanol dehydration steps were then used with ethanol
concentrations of 30, 50, 75 (five times, each for 15 min), 90, and
100% (five times, each for 30 min). Finally, dehydrated samples
were dried in a critical-point dryer (Tousimis Autosamdri 931.GL,
Rockville, MD) to ensure sample preservation by first replacing
100% ethanol with liquid carbon dioxide and then carbon dioxide
gas.
Each sample was mounted to a metal stub using epoxy.
Samples were then coated with gold palladium after drying for
24 h. Coated samples were viewed using the FEI XL30 ESEM
FEG and the FEI Helios Nanolab 600 electron microscope (FEI,
Hillsboro, OR) at a voltage of 5.0 Kv.
Vertical cross-section SEM sample preparation and view-
ing. Cross-section samples were selected from uninfected and
severely infected seeds. The uninfected samples were randomly
selected from a group of uninoculated nondormant seeds and
were photographed both unimbibed and after imbibition. The
severely infected samples were visually selected from a dish of
nondormant seeds that had been imbibed and inoculated 14 days
prior (7 days in PEG followed by 7 days in free water). The
severely infected samples were chosen based on the appearance
of a white tuft at the radicle end of the seed. This ensured that the
location of the fungal attack could be visualized within the seed.
Selected samples were fixed and dehydrated using the previ-
ously stated protocol for dehydration through the 75% ethanol
solution. Samples were removed from the 75% ethanol solution
and slowly submerged in liquid nitrogen with tweezers, and held
down until the bubbling had ceased. The sample was removed
from liquid nitrogen and immediately fractured with a frozen
razor blade. The fractured sample was immediately placed back
into the 75% ethanol solution to continue dehydration steps. Seed
preservation, mounting, coating, and viewing were completed
following the previously stated protocol.
RESULTS
Sequencing results. A nucleotide BLAST query of NCBI
GenBank revealed that the TEF region of the Skull C1 isolate was
a 100% match to accession JX397848, which was obtained from
BBA71608 from corn in Serbia. The sequence from this strain
was deposited as Fusarium cf. reticulatum, i.e., as similar but not
identical to F. reticulatum, a member of the F. tricinctum species
complex. In the associated publication (32), the authors present a
phylogenetic tree based on maximum likelihood analysis that
includes BBA71608 along with 60 other strains belonging to
F. tricinctum and other species within the complex. This tree
shows that strain BBA71608 falls clearly within the F. tricinctum
species complex but that it is not identical to any known species.
This indicates that the taxon represented by Skull C1 is an
undescribed species within the F. tricinctum species complex.
B. tectorum seed anatomy. In B. tectorum, as in most grasses,
the dispersal unit is comprised of floret bracts (lemma, palea) that
enclose a one-seeded fruit (caryopsis) in which the fruit wall
(pericarp) and seed coat (testa) are fused into a single multi-
layered structure, the caryopsis wall (25; Fig. 1A). Within the
caryopsis are the embryo and the endosperm. The attachment scar
is located at the embryo end of the floret, near the point of radicle
emergence. It represents the vascular tissue through which the
Fig. 2. Scanning electron microscopic images of Skull C1 macroconidia on imbibed Bromus tectorum seeds. A, Macroconidia (Mc) on the floret bracts (Fb) of the
seed immediately after inoculation. B, Macroconidial development of an adhesion pad (Ap) on the floret bract (Fb) 2 h after inoculation. C, Macroconidial
development of an adhesion pad (Ap) on bract hair (Bh) 2 h after inoculation. D, A close up of macroconidial adhesion pad (Ap) as shown in C. E, Macroconidial
(Mc) development of a germ tube (Gt) on the surface of the seed 2 h after inoculation.
Vol. 104, No. 12, 2014 1309
seed was provisioned by the maternal plant. The attachment scar
remains after dispersal as a porous, highly vascularized area that
represents a direct pathway toward the now-mature caryopsis
(Fig. 1B), beginning at the abscission layer and ending at the
exterior of the caryopsis wall. Seeds in our research were initially
not imbibed; therefore, the endosperm had not begun diges-
tion (Fig. 1C). However, once a nondormant seed becomes
imbibed, the seed germination process is initiated. This begins
with the digestion of starch cells within the endosperm (Fig. 1D)
and presumably results in the diffusion of products of endo-
sperm digestion through the weakening zone of radicle emergence
in the caryopsis wall and then through the porous abscission
layer.
Fusarium infection process on nondormant B. tectorum
seeds. Nondormant B. tectorum seeds were inoculated with Skull
C1 macroconidia and allowed to imbibe while under water stress
as described earlier (Fig. 2A). The macroconidia developed adhe-
sion pads only 2 h after contact with the surface of the floret
bracts (Fig. 2B to D). Some macroconidia did not produce adhe-
sion pads but instead immediately produced germ tubes (Fig. 2E).
Within 24 h, the majority of the macroconidia had germinated and
begun to focus their hyphal growth toward the abscission layer of
the floret attachment scar (Fig. 3A and B). Within 48 h, the
hyphae grew preferentially toward and culminated at the abscis-
sion layer regardless of macroconidial germination location. The
hyphae prepared for penetration down the vascular tissue of the
floret attachment scar by developing a large infection cushion
(Fig. 3C). Little additional hyphal growth occurred between 48
and 96 h in PEG (Fig. 3D). During this time, growth was tem-
porarily halted until the water potential was increased by trans-
ferring the seeds to free water on day 7 (Fig. 4A), at which point
mycelial proliferation recommenced (Fig. 4B). Occasionally a
B. tectorum seed was able to germinate quickly enough to
outgrow the infection cushion (Fig. 5A). On even rarer occasions,
a viable seed was apparently still functionally dormant and failed
to initiate germination. This resulted in wandering mycelial
growth on the surface of the floret bracts in place of a well-
developed infection cushion at the floret attachment scar, and was
similar to the mycelial growth observed on dormant seeds in
earlier experiments (Fig. 5B).
Fig. 3. Scanning electron microscopic time-lapse images of Skull C1 hyphal growth on imbibed Bromus tectorum seeds. A, Hyphal growth (Hg) towards the floret
attachment scar (Fs) 24 h after inoculation. B, Close-up of hyphal growth (Hg) at floret attachment scar (Fs) 24 h after inoculation. C, An infection cushion (Ic) is
developed at the abscission layer of the floret attachment scar 48 h after inoculation. D, Infection cushion (Ic) 96 h after inoculation.
1310 PHYTOPATHOLOGY
Vertical cross section of infected seed. Because the infection
of the embryo takes place within the floret bracts, a vertical cross
section was necessary to identify the location of the mycelial
attack (Fig. 6). Fourteen days after macroconidial inoculation
(7 days after transfer to free water), hyphae had successfully
colonized the vascular tissue of the seed attachment scar (Fig.
7A). During the 7 days in water, penetration hyphae successfully
breached the caryopsis wall and came in contact with the nutrient-
rich embryo. Colonization began in the intercellular region and
eventually spread to intracellular growth (Fig. 7B and C). Once
colonization in the embryo had occurred, hyphae grew into the
endosperm where they began intercellular growth (Fig. 7D). The
end result was the complete destruction of the nondormant
B. tectorum seed.
DISCUSSION
The regulation of Fusarium pathogenesis on seeds in an
artificial inoculation experiment may be quite different from the
process as it occurs in soil. In semiarid ecosystems, soils are
rarely at saturation, and nutrients available to microorganisms are
sporadically available at best, resulting in nutrient competition.
This can in turn result in the suppression of pathogen spore
germination, a phenomenon known as fungistasis (8,23). Fungi-
stasis may be overcome by an increase in nutrient status, which
could be provided by a change in the status of organic matter in
the soil (3) or directly by exudates from germinating seeds (31).
In the experiment described here, Fusarium spores were im-
mediately exposed to an environment conducive to germination,
either because of an absence of competing soil microorganisms or
possibly because of the presence of seed exudates. It is not known
whether the spores in this study are capable of germination in the
absence of an exogenous nutrient source, or whether the imbibing
seeds provided this nutrient source. In either case, spore attach-
ment and germination occurred essentially simultaneously within
24 h of inoculation.
In previous studies with plant-pathogenic fungi such as Coch-
liobolus heterostrophus, conidia were capable of forming a firm
Fig. 5. Scanning electron microscopic images of Skull C1-inoculated Bromus tectorum seeds that escaped infection. A, A weak infection cushion (Ic) on a
B
romus
tectorum floret 7 days after inoculation, allowing seedling growth. B, Hyphal growth on an inoculated B. tectorum seed that failed to germinate.
Fig. 4. Images taken of infected seeds (florets). A, Infected Bromus tectorum seed in polyethylene glycol (PEG) 7 days after Skull C1 macroconidial inoculation
with a well-developed infection cushion. B, Well-colonized B. tectorum seed in free water 21 days (7 days in PEG and 14 days in free water) after macroconidial
inoculation.
Vol. 104, No. 12, 2014 1311
attachment to plant and artificial surfaces within 60 min after
inoculation (2). According to Jones and Epstein (17), many plant-
pathogenic fungi depend on spore attachment as the first step in
host infection. Their results showed that mutants incapable of
attaching to the host had a dramatic decrease in virulence on
nonwounded host tissue (fruit). The extracellular matrix material
produced prior to germ tube production proved to be the key to
infection and was not dependent on nutrient availability. In this
study system, this initial step most likely happens in the field in
the first wetting event after spore dissemination.
As mentioned earlier, there is evidence that the Fusarium sp. n
strains implicated in B. tectorum die-offs only effectively attack
germinating (nondormant) seeds (1). Evidence gathered from the
vertical cross-sectional view of an uninoculated nondormant B.
tectorum seed suggests that the vascular tissue of the floret
attachment scar could be the exit route for exudates released by
the germinating seed. This would result in the directional growth
toward the abscission layer of the floret attachment scar as
exhibited by the Skull C1 isolate in this study. Spores were able to
germinate on seeds that did not initiate germination, but hyphal
growth meandered with no obvious direction. The directional
pattern of response to an apparent nutrient gradient emanating
from the radicle end of a germinating seed permitted the pathogen
to target the most vulnerable point on the seed and to rapidly
complete penetration before radicle protrusion could occur
following transfer to free water.
The development of hyphae dramatically increases fungal nu-
trient acquisition. Numerous studies have examined the mecha-
nisms behind hyphal growth. Riquelme et al. (33) showed that in
Neurospora crassa the spitzen korper position located on the tip
of hyphae directly affects the direction and morphology of hyphal
growth. Their results showed directional fungal growth and the
hyphal ability to adaptively change direction. Grow (12) dis-
cussed the different mechanisms used by fungi for directional
orientation, such as thigmotropism (which was not apparent in
this study). A second mechanism is directional growth in response
to chemical gradients. In the present study, it appears that the
recognition of a gradient of seed exudates emanating from the
floret attachment scar allowed hyphae to sense the most direct
path to the source of nutrients. This resulted in a conspicuous
white infection cushion at the abscission layer of the floret
attachment scar.
There was no evidence suggesting that Skull C1 used advanced
morphological structures to penetrate the exterior of the floret
bracts or the caryopsis wall. Studies have shown that some fungal
species have forgone penetration structure development and
completely bypassed the plant cuticle by taking advantage of
features of plant or seed anatomy to infect the host (e.g., through
the stomates; 19). According to Mendgen et al. (24), some
Fusarium species enter their host with little cell differentiation or,
in other words, a very underdeveloped appressorium. These Fu-
sarium species use a method in which they produce a net-like mat
Fig. 6. Scanning electron microscopic images of a vertical cross section taken from Skull C1-inoculated Bromus tectorum seeds. Vertical cross section of a
severely infected seed 14 days (7 days in polyethylene glycol [PEG] and 7 days in H2O) after macroconidial inoculation. The hyphae have visibly grown down the
vascular tissue (Vt) of the floret attachment scar and penetrated the caryopsis wall (C), colonizing the embryo (Em).
1312 PHYTOPATHOLOGY
of mycelium where the penetration hyphae are produced. This
method is very similar to the pattern observed in the present
study. By taking advantage of the porous floret attachment scar
and the weakening caryopsis wall at the point of radicle emer-
gence, Skull C1 was able to penetrate host tissues and cause seed
death without appressorial development.
According to our results, water stress at –1.5 MPa prevented
seeds from completing germination while fungal growth could
still occur, a result similar to that observed with Pyrenophora
semeniperda, another B. tectorum seed pathogen (7). However,
we observed that for Skull C1 there was a growth lag between 48 h
in PEG and exposure to free water. It appears that this Fusarium
species can germinate, grow directionally, and produce an infec-
tion cushion at reduced water potential. However, the actual
penetration of the caryopsis wall, and subsequent access to the
abundant resources within the seed, did not take place until after
transfer to free water. Seeds are also allowed to complete germi-
nation normally once exposed to free water. However, by this
time, Skull C1 had already penetrated the floret bracts and was
ready to penetrate the caryopsis wall.
Money (28) showed that hyphae require a substantial amount of
turgor pressure to penetrate solid media. This ultimately is caused
by the inability of hyphae to generate enough pressure inside
while the exterior pressure is so low. According to Howard et al.
(16), penetration of rice by Magnaporthe grisea was significantly
reduced after an incubation period of 48 h in PEG. Mechanical
penetration occurred after sufficient turgor pressure was estab-
lished. In contrast, Harold et al. (14) showed that exposure to low
turgor pressure had little effect on hyphal morphogenesis and
growth while exposed to water stress. Kaminskyj et al. (18) first
noted that hyphal extension and diameter increased when sub-
jected to water stress. This was reaffirmed by Money (28). Our
research agrees with these previous studies in that Skull C1
growth recommenced after exposure to free water, suggesting that
free water is crucial to the production of the turgor pressure
required for penetration of the caryopsis wall.
Other confounding variables may decrease hyphal penetration
of the caryopsis wall or seed coat. Polyphenolic and phenolic
compounds in seed coats and their interactions with pathogens
have been studied extensively. Results suggest that hyphae may
grow substantially on seed surfaces lower in polyphenolic com-
pounds before penetrating polyphenolic-rich surfaces (such as the
seed coat), delaying penetration (13,20). Because such a clear
relationship exists in our study between water potential and the
growth potential of the hyphae, it may be argued that poly-
phenolic and phenolic compounds probably played a minor role
in protecting the seeds from attack.
This study has demonstrated that Fusarium sp. n pathogenesis
on nondormant B. tectorum seeds is focused on the porous tissue
of the floret attachment scar, close to the point of potential radicle
emergence. This represents the most vulnerable location for
pathogen attack on a rapidly germinating seed. A nutrient gradient
produced by seed exudates apparently directs hyphal growth to-
ward this attachment scar, where an infection cushion is produced
even at water potentials that suppress seed germination. Upon
subsequent transfer to free water, the fungus is able to quickly
breach the caryopsis wall. This results in rapid colonization of the
embryo, seed death, and a major increase in pathogen mycelial
production. Knowledge of this mechanism of pathogenesis will
enable us to determine how the pathogen operates to cause seed
mortality in the field, and will help to clarify its role in the
B. tectorum die-off phenomenon.
Fig. 7. Scanning electron microscopic images of close-ups of the vertical cross section shown in Figure 6. A, Hyphae (Hp) have grown down and colonized the
vascular tissue (Vt) of the floret attachment scar. B and C, Hyphae (Hp) colonizing the embryo. D, Hyphae (Hp) growing in the intercellular region of the
endosperm starch cells (St).
Vol. 104, No. 12, 2014 1313
ACKNOWLEDGMENTS
This research was funded in part by a grant from the USDI Bureau of
Land Management Idaho State Office in support of the Integrated
Cheatgrass Die-Off Project. We thank H. Finch-Boekweg and M. Stand-
ings for microscope assistance in obtaining the images for this paper and
S. Sink and K. O’Donnell (NCAUR, ARS, USDA, Peoria, IL) for
assistance with the molecular phylogenetic identification of Skull C1.
LITERATURE CITED
1. Baughman, O. W., and Meyer, S. E. 2013. Is Pyrenophora semeniperda
the cause of downy brome (Bromus tectorum) die-offs? Invasive Plant Sci.
Manag. 6:105-111.
2. Braun, E. J., and Howard, R. J. 1994. Adhesion of Cochliobolus
heterostrophus conidia and germlings to leaves and artificial surfaces.
Exp. Mycol. 18:211-220.
3. Bonanomi, G., Gaglione, S. A., Incerti, G., and Zonia, A. 2013.
Biochemical quality of organic amendments affects soil fungistasis. Appl.
Soil Ecol. 72:135-142.
4. Dalling, J. W., Davis, A. S., Schutte, B. J., and Arnold, A. E. 2011. Seed
survival in soil: Interacting effects of predation, dormancy and the soil
microbial community. J. Ecol. 99:89-95.
5. Duncan, K. E., and Howard, R. J. 2010. Biology of maize kernel infection
by Fusarium verticillioides. Mol. Plant-Microbe Interact. 23:6-16.
6. Fandohan, P., Hell, K., Marasas, W. F. O., and Wingfield, M. J. 2003.
Infection of maize Fusarium species and contamination with fumonisin in
Africa. Afr. J. Biotechnol. 12:570-579.
7. Finch, H., Allen, P. S., and Meyer, S. E. 2013. Environmental factors
influencing Pyrenophora semeniperda-caused seed mortality in Bromus
tectorum. Seed Sci. Res. 23:57-66.
8. Garbeva, P., Hol, W. H., Termorshuizen, A. J., Kowalchuk, G. A., and De
Boer, W. 2011. Fungistasis and general soil biostasis–a new synthesis.
Soil Biol. Biochem. 43:469-477.
9. Geiser, D. M., Jimenez-Gasco, M. D. M., Kang, S., Makalowska, I.,
Veeraraghavan, N., Ward, T. J., Zhang, N., Kuldau, G. A., and O’Donnell,
K. 2004. FUSARIUM-ID v. 1.0: A DNA sequence database for identi-
fying Fusarium. Eur. J. Plant Pathol. 110:473-479.
10. Gomez, R., Liebman, M., and Munkvold, G. 2014. Weed seed decay in
conventional and diversified cropping systems. Weed Res. 54:13-25.
11. Goswami, R. S., and Kistler, H. C. 2004. Heading for disaster: Fusarium
graminearum on cereal crops. Mol. Plant Pathol. 5:515-525.
12. Grow, N. A. R. 1994. Growth and guidance of the fungal hyphae.
Microbiology 140:3193-3205.
13. Halloin, J. M. 1983. Deterioration resistance mechanisms in seeds.
Phytopathology 73:335-339.
14. Harold, F. M., Harold, R. L., and Money, N. P. 1995. What forces drive
cell wall expansion? Can. J. Bot. 73(S1):379-383.
15. Heiko, T., Sauerborn, J., Muller-Stover, D., Ziegler, A., Singh-Bedi, J.,
and Kroschel, J. 1998. The potential of Fusarium oxysporum f. sp.
orthoceras as a biological control agent for Orobanche cumana in
sunflower. Biol. Control 1:41-48.
16. Howard, R. J., Ferrari, M. A., Roach, D. H., and Money, N. P. 1991.
Penetration of hard substrates by a fungus employing enormous turgor
pressures. Microbiology 88:11281-11284.
17. Jones, M. J., and Epstein, L. 1990. Adhesion of macroconidia to the plant
surface and virulence of Nectria haematococca. Appl. Environ.
Microbiol. 56:3772-3778.
18. Kaminskyj, S. G. W., Garrill, A., and Heath, I. B. 1992. The relation
between turgor and tip growth in Saprolegnia ferax: Turgor is necessary,
but not sufficient to explain apical extension rates. Exp. Mycol. 16:64-75.
19. Knogge, W. 1996. Fungal infection of plants. Plant Cell 8:1711-1722.
20. Lattanzio, V., Lattanzio, V. M. T., and Cardinali, A. 2006. Role of
phenolics in resistance mechanisms of plant against fungal pathogens and
insects. Phytochem. Adv. Res. 661:23-67.
21. Lawrence, E. B., Nelson, P. E., and Ayers, J. E. 1981. Histopathology of
sweet corn seed and plants infected with Fusarium monliforme and F.
oxysporum. Phytopathology 71:379-386.
22. Leslie, J. F., and Summerell, B. A. 2006. The Fusarium Laboratory
Manual. Blackwell Publishing, Ames, IA.
23. Lockwood, J. L. 1977. Fungistasis in soils. Biol. Rev. 52:1-43.
24. Mendgen, K., Hahn, M., and Deising, H. 1996. Morphogenesis and
mechanisms of penetration by plant pathogenic fungi. Annu. Rev.
Phytopathol. 34:364-386.
25. Metcalfe, C. R. 1960. Anatomy of the Monocotyledons. I. Gramineae.
Clarendon Press, Oxford, UK.
26. Meyer, S. E., Franke, J.-L., Baughman, O., Beckstead, J., and Geary,
B. D. 2014. Does Fusarium-caused seed mortality contribute to Bromus
tectorum stand failure in the Great Basin? Weed Res. 54:511-519.
27. Michel, B. E. 1983. Evaluation of the water potentials of polyethylene
glycol 8000 both in the absence and presence of other solutes. Plant
Physiol. 72:66-70.
28. Money, N. P. 1995. Turgor pressure and the mechanics of fungal
penetration. Can. J. Bot. 73(Suppl. 1):S96-S102.
29. Moya-Elizando, E. M. 2013. Fusarium crown rot disease: Biology,
interactions, management and function as a possible sensor of global
climate change. Cien. Inv. Agric. 40:235-252.
30. Muller-Stover, D., Kohlschmid, E., and Sauerborn, J. 2009. A novel strain
of Fusarium oxysporum from Germany and its potential for biocontrol of
Orobanche ramosa. Weed Res. 2:175-182.
31. Nelson, E. B. 2004. Microbial dynamics and interactions in the
spermosphere. Annu. Rev. Phytopathol. 42:271-309.
32. Niessen, L., Gräfenhan, T., and Vogel, R. F. 2012. ATP citrate lyase 1
(acl1) gene-based loop-mediated amplification assay for the detection of
the Fusarium tricinctum species complex in pure cultures and in cereal
samples. Internat. J. Food Microbiol. 158:171-185.
33. Riquelme, M., Reynaga-Pena, C. G., Gierz, G., and Bartnicki-Garcia, S.
1998. What determines growth direction in fungal hyphae? Fungal Genet.
Biol. 24:101-109.
34. Sauerborn, J., Dorr, I., Abbasher, A., Thomas, H., and Kroschel, J.
1996. Electron microscopic analysis of the penetration process of
Fusarium nygamai, a hyperparasite of Striga hermonthica. Biol. Control
7:53-59.
