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Fungal taxonomy / Taxonomie fongique

Conidial morphology and ecological

characteristics as diagnostic tools for identifying
Claviceps purpurea from salt-marsh habitats
Alison J. Fisher, J.M. DiTomaso, and T.R. Gordon

Abstract: Claviceps purpurea associated with grass hosts in salt-marsh habitats, also known as G3 ergot, can be
differentiated from C. purpurea infecting grasses in other habitats, using genetic, chemical, and morphological criteria.
However, only morphological analysis can be preformed on herbarium specimens, which should not be destroyed, or
older samples that cannot be grown in culture. To determine if conidial characteristics could be employed to identify
the three infraspecific groups of C. purpurea (G1–G3), sclerotia from terrestrial grasses (G1), moist habitats (G2), and
salt-marsh habitats (G3) were examined. All G1 sclerotia sank in water. All G3 sclerotia floated in water, except those
from Washington State, which had variable buoyancy characteristics. Group 2 sclerotia did not float in water, except
those collected from Calamagrostis nutkaensis and Ammophila breviligulata. Based on the length of conidia derived
from a plant host, G1 samples are indistinguishable from G2 samples but are significantly different from G3. Conidia
produced in culture were on average smaller than conidia produced on plant hosts. These results indicate that
ecological and conidial characteristics can be used to distinguish G3 from G1, but cannot consistently separate either
from G2.
Key words: ergot, Claviceps purpurea, Spartina, cereal grains, conidial morphology, salt-marsh habitats.
Résumé : Aussi connu comme l’ergot G3, le Claviceps purpurea associé à des graminées hôtes dans des habitats de
marais salants peut être différencié du C. purpurea qui infecte des graminées dans d’autres habitats, en utilisant des
critères génétiques, chimiques et morphologiques. Cependant, seules des analyses morphologiques peuvent être faites
sur des spécimens d’herbier, qui ne doivent pas être détruits, ou sur de plus vieux échantillons qui ne peuvent pas être
cultivés. Afin de déterminer si les caractéristiques des conidies peuvent être utilisées pour identifier les trois groupes
infraspécifiques du C. purpurea (G1–G3), des sclérotes provenant de graminées terrestres (G1), d’habitats humides
(G2) et d’habitats de marais salants (G3) furent examinés. Tous les sclérotes G1 sombrèrent dans l’eau. Tous les
sclérotes G3 flottèrent sur l’eau, sauf ceux provenant de l’État de Washington, lesquels affichèrent des caractéristiques
de flottabilité variables. Les sclérotes G2 ne flottèrent pas sur l’eau, à l’exception de ceux obtenus du Calamagrostis
nutkaensis et de l’Ammophila breviligulata. Selon la longueur des conidies provenant d’une plante hôte, les
échantillons G1 ne peuvent pas être distingués des échantillons G2, mais ils sont significativement différents des G3.
Les conidies produites en culture étaient en moyenne plus petites que les conidies produites sur des plantes hôtes. Les
présents résultats indiquent que les caractéristiques écologiques et conidiennes peuvent être utilisées pour distinguer les
G3 des G1, mais ne sont pas suffisamment constantes pour les distinguer des G2.

Mots clés : ergot, Claviceps purpurea, Spartina, céréales, morphologie conidienne, habitats de marais salants.

et al.: ergot on grasses / Claviceps purpurea / salt-marsh habitats / conidial morphology 395
Introduction a worldwide distribution and a large host range within the
Poaceae. Grass florets become infected by ascospores re-
Claviceps purpurea (Fr.) Tul., commonly known as ergot, leased from perithecia or conidia from neighboring plants.
is a well known pathogen of cereal grains and forage, with

Accepted 27 April 2005.

Alison J. Fisher1,2 and J.M. DiTomaso. Department of Plant Sciences, One Shields Avenue, University of California, Davis, CA
95616-8755, USA.
T.R. Gordon. Department of Plant Pathology, One Shields Avenue, University of California, Davis, CA 95616-8755, USA.
Corresponding author (e-mail: afisher@pw.usda.gov).
Present address: Exotic and Invasive Weeds Research Unit, Agricultural Research Service, United States Department of
Agriculture, 800 Buchanan Street, Albany, CA 94710, USA.

Can. J. Plant Pathol. 27: 389–395 (2005)

390 Can. J. Plant Pathol. Vol. 27, 2005

After colonization of the flower, a sugary honeydew is pro- ples found in terrestrial (G1) and moist, freshwater (G2) en-
duced by the plant, which contains large numbers of conidia vironments, using genetic, chemical, and morphological
of C. purpurea. Conidia can be spread by rain splash, insect characteristics. Field-collected G2 and G3 sclerotia and
movement, or plant-to-plant contact. Later, sclerotia are salt-marsh floated in water while G1 sclerotia sank. In addi-
produced in place of seeds. Claviceps purpurea sclerotia tion, each group had a distinct conidial-size range. Lengths
grow from the rachis of the flower, beyond the bracts, and of conidia ranged from 5 to 8 µm for G1, from 7 to 10 µm
are easily visible in infected populations. Sclerotia fall to for G2, and from 9.5 to 12 µm for G3 (Pañoutová et al.
the ground when inflorescences shatter, usually in autumn. 2000, 2002). These studies provided an ecological context
With suitable environmental conditions, sclerotia germinate, for infraspecific variation in C. purpurea, much of which
produce stalked stromata containing perithecia, and forcibly was already documented (Loveless 1971; Stäger 1922).
release ascospores to start a new infection cycle (Luttrell Ecological and morphological characters are important tools
1980). Claviceps purpurea infects grasses in diverse habi- for assessing infraspecific differences within C. purpurea, es-
tats, including cultivated fields and unmanaged landscapes pecially for herbarium specimens where genetic or chemical
such as coastal salt marshes. analysis would require destruction of the preserved mate-
Sclerotia of C. purpurea obtained from grasses in salt- rial. Pañoutová et al. (2000, 2002) found consistent differ-
marsh habitats float in water. This adaptive trait allows for ences between C. purpurea from salt-marsh and other
aquatic dispersal and later aerial release and dissemination habitat types, however, only two geographic regions were
of spores that must reach the host inflorescence to establish sampled, New Jersey, United States and the United King-
infections. On the other hand, sclerotia from terrestrial dom. The objective of our study was to determine if flota-
grasses sink in water, leading to the suggestion that sclero- tion and conidial dimensions could function as definitive
tial buoyancy may serve to differentiate ecotypes within diagnostic tools for identification of G3 C. purpurea. To
C. purpurea (Duncan et al. 2002; Pañoutová et al. 2000). this end, G3 C. purpurea from nine geographic regions
Based on this characteristic and alkaloid profiles, Samuelson were compared with representative G1 and G2 samples col-
and Gjerstad (1966) proposed that C. purpurea from lected from terrestrial and moist, freshwater habitats, re-
Spartina spp. in coastal Argentina be designated as a sepa- spectively. In addition, we determined whether there were
rate species, Claviceps maritima Samuel. & Gjer., or significant differences in morphology of G3 conidia pro-
“feather ergot”. duced on a plant host compared with those produced in vi-
Historically, variation in conidial size among samples tro. Lastly, groupings based on size of conidia were overlaid
from different host plants has also been taken as evidence on a phenogram based on amplified fragment length poly-
morphisms (AFLPs), to determine if morphological and ge-
of host-specific groups within C. purpurea. Loveless (1971)
netic markers yielded similar indications of infraspecific
identified four groups based on conidial dimensions, with
mean lengths ranging from 5.4 to 8.4 µm. Shorter conidia
were measured from grasses in terrestrial or coastal habitats
such as Lolium perenne L., Arrhenatherum elatius (L.) Materials and methods
Beauv., Secale cereale L., Ammophila arenaria (L.) Link,
Festuca arundinacea Schreb., Triticum aestivum L., Agropyron Field collection and culturing
pungens (Pers.) Roem. & Schult., Agropyron repens (L.) The origins of samples used in this study are listed in Ta-
Beauv., Dactylis glomerata L., and Holcus lanatus L. The ble 1. Collections from the same location were taken from
longest conidia (8.4 µm) were measured from Nardus inflorescences at least 1 m apart. Sclerotia were refrigerated
stricta L., a grass associated with nutrient-poor, peat soils, at 4 °C for up to 6 months before processing. To reduce su-
and Spartina townsendii H. & J. Groves, a salt-marsh cord perficial contaminants, sclerotia were treated by immersion
grass. Spartina townsendii is a hybrid resulting from a cross in 70% ethanol for 30 s, followed by 1.5 min in 1.3% so-
between Spartina maritima (Curt.) Fern, a native from the dium hypochlorite (bleach) and three successive, 3-min
United Kingdom, and Spartina alterniflora Lois., intro- rinses in sterile deionized water. Sclerotia were sliced hori-
duced to the United Kingdom from the Atlantic Coast of the zontally, the rind was removed with a blade, and fungal tis-
United States. Comparing conidia collected from Lolium sue was plated onto potato dextrose agar (PDA) (Difco®).
perenne, Dactylis glomerata, Deschampsia caespitosa (L.) To ensure that each isolate represented a single genotype,
Beauv., and Nardus stricta, Loveless (1971) found no dif- cultures were streaked onto water agar, and a hyphal tip was
ference in size between samples taken from honeydew on subcultured. Pure cultures were grown on 42.5-mm filter
an infected host and isolates grown in culture, suggesting paper that were laid over 60-mm PDA plates; fully colo-
genetic control of spore size, an important discovery for di- nized papers were removed, dried, and refrigerated at 4 °C.
agnostics. Conidial size was also consistent whether plants
were collected from nature or spores were inoculated onto a Conidial size
common host, Triticum aestivum (Loveless and Peach Conidial dimensions were recorded for 6 G1 isolates, 7
1974). G2, and 22 G3 (Table 1), using both laboratory-grown cul-
Using a larger collection of isolates than in previous stud- tures, and conidia produced on plant hosts. Dried cultures
ies, Pañoutová et al. (2000) synthesized previous research on on filter paper (see above) were used to establish PDA cul-
C. purpurea ecology, morphology, alkaloid chemistry, and tures of each isolate, and these were incubated at 25 °C for
genetics and proposed three groups, each with a particular 10 to 14 days before conidia were collected in 0.5% KCl.
habitat association: Claviceps purpurea from salt-marsh To obtain conidia from an infected host, plants were inocu-
habitats, also known as G3, was distinguishable from sam- lated with spore suspensions. Spore suspensions were ob-
Fisher et al.: ergot on grasses / Claviceps purpurea / salt-marsh habitats / conidial morphology 391

Table 1. Origin and host plant of Claviceps purpurea isolates.

Isolate Location Year Host Group
CMD 1 MacDoel, California, USA 2001 Secale cereale G1
IA 1 Aberdeen, Idaho, USA 2000 Secale cereale G1
NGE 1 L’Anse-aux-Meadows, Newfoundland, Canada 2001 Leymus mollis G1
WFA 1 Nahcotta, Washington, USA 2002 Festuca arundinacea G1
WLL 1a Leadbetter, Washington, USA 2001 Leymus mollis (Trin.) Pilger G1
WLS 1 Nahcotta, Washington, USA 2002 Lolium spp. G1
WAB 1 Long Beach, Washington, USA 2002 Ammophila breviligulata G2
WAB 2 Long Beach, Washington, USA 2002 Ammophila breviligulata G2
WCN 2 Willapa Bay, Washington, USA 2002 Calamagrostis nutkaensis G2
WDG 1a Leadbetter, Washington, USA 2002 Dactylis glomerata G2
WDS 1a Willapa River, Washington, USA 2002 Deschampsia caespitosa G2
WHS 1 Long Beach, Washington, USA 2002 Holcus lanatus G2
WPA 1 Leadbetter, Washington, USA 2002 Calamagrostis nutkaensis G2
ADI 1 Dolphin Island, Alabama, USA 2000 Spartina alterniflora G3
ARG 1 Celpa Marsh, Argentina 2002 Spartina densiflora Brongn. G3
CDE 1a Point Reyes NS, California, USA 2002 Spartina foliosa G3
CDE a1 Point Reyes NS, California, USA 2002 Spartina alterniflora G3
CNB 21 Bolinas Lagoon, California, USA 2001 Spartina foliosa Trin. G3
CPE 10 Palo Alto, California, USA 2001 Spartina foliosa G3
CSL 1 Mountain View, California, USA 2002 Spartina foliosa G3
CSM 10 San Mateo, California, USA 2001 Spartina foliosa G3
FSA 1 St. Augustine, Florida, USA 2000 Spartina alterniflora G3
GML 1 Marsh Landing, Georgia, USA 2000 Spartina alterniflora G3
IRE1 2 Dublin, Ireland 2001 Spartina anglica Hubbard G3
NYF 1 Flax River, New York, USA 2001 Spartina alterniflora G3
RIH 1 Rhode Island, USA 2001 Spartina alterniflora G3
WDI 1a Willapa River, Washington, USA 2002 Distichlis spicata G3
WGP 1 Goose Point, Washington, USA 2002 Spartina alterniflora G3
WKL 1 Kaffe Louis, Washington, USA 2002 Spartina alterniflora G3
WLT 1 Leadbetter, Washington, USA 2001 Spartina alterniflora G3
WNC 1 North Cove, Washington, USA 2002 Spartina alterniflora G3
WNR 1 Naselle River, Washington, USA 2002 Spartina alterniflora G3
WPR 1 Palix River, Washington, USA 2002 Spartina alterniflora G3
WTA 1 Tarlatt Slough, Washington, USA 2002 Spartina alterniflora G3
WWR 1 Willapa River, Washington, USA 2002 Spartina alterniflora G3
Collection numbers are CMD 1 37641, WLL 1 37642, WDG 1 37643 WDS 1 37644, CDE 1 37645, and WDI 1 37646 from
NRRL (Northern Regional Research Laboratory, now the National Center For Agricultural Utilization Research), Agricultural
Research Service Culture Collection, Peoria, Illinois, United States.

tained by adding 10–15 mL of 0.5% KCl to plates, wiping respectively. Nine to 14 days after inoculation, conidia were
the surface with a glass rod, and pouring the liquid suspen- obtained by collecting honeydew. Honeydew was refriger-
sion into a vial; suspension concentrations were at least ated at 4 °C, and conidia suspended therein were measured
106 spores/mL. For each isolate, inoculations were accom- within 48 h. Sixty conidia were measured from each isolate,
plished by dipping 2- to 4-day-old inflorescences of 30 from culture and 30 from honeydew. Spores were
Spartina foliosa, into the spore suspension, just prior to added to a drop of 0.5% KCl and measured at a magnifica-
anthesis. These plants were grown in a greenhouse at the tion of × 1000, with phase contrast, using an ocular mi-
University of California, Davis, United States, and follow- crometer. Means ± standard errors were calculated for each
ing inoculation, their inflorescences were wrapped in poly- isolate. SAS (version 8.2) was used for all statistical analy-
vinyl chloride (plastic wrap). After inoculation, plants were ses (SAS Institute Inc. 1989).
placed for 24 h in a growth chamber, under the following
conditions: temperature, 17 °C (day) and 14 °C (night); Float test
photoperiod daylength, 15 h; light level (maximum photo- In 2000, 2001, and 2002, field-collected sclerotia were
synthetically activate radiation), 1100 µE m–2s–1; relative bagged on site and brought to University of California, Da-
humidity, 80%). After 24 h, the plastic wrap was removed, vis. Sclerotia were removed from the inflorescence and
and plants were returned to the greenhouse. Average daily stored at 4 °C for up to 3 months before processing. To test
greenhouse temperature and humidity were 24 °C and 77%, for flotation, sclerotia were placed in 20 mL of deionized
392 Can. J. Plant Pathol. Vol. 27, 2005

Fig. 1. Conidial size of G1 (䉱; terrestrial), G2 (䊏), and G3 (䊊; salt marsh) from culture of Claviceps purpurea isolates. Standard-
error bars are shown.

water for 10 min, stirring with a glass rod every 3 min. The The source of conidia (plant host or culture) had no effect
number of sclerotia on the surface of the water at the end of on conidial length for G1 (paired t test, P = 0.0992) and G2
this time period was recorded. (P = 0.4359). However, within G3, conidia from honeydew
To look more closely at the population in Willapa Bay, were significantly longer than those from PDA cultures
Washington, United States, in September 2003, sclerotia (paired t test, P = 0.0136). Mean length of conidia was 8.9
were collected from Spartina alterniflora at five locations ± 0.2 µm in vitro and 9.9 ± 0.3 µm in planta.
in the estuary. In October 2003, sclerotia were collected The range of conidial dimensions of G1, G2, and G3 iso-
from Spartina alterniflora at two sites, from Distichlis lates from PDA are shown in Fig. 1. The range of conidial
spicata (L.) Greene at one site, and from Lolium spp. at one dimensions of G1, G2, and G3 collected from inoculated
site and brought to University of California, Davis. In Octo- Spartina foliosa is shown in Fig. 2. Within salt-marsh G3
ber 2003, sclerotia were placed in deionized water for a lon- isolates, plant-produced conidia from the northeast United
ger duration, 2 h, and stirred 3 times after the first hour to States (New York and Rhode Island) were on average lon-
simulate natural conditions. In addition, an on-site flotation ger (11.6 ± 1.6 µm) than conidia from the southeast United
assay was performed on sclerotia collected from two sites States (Alabama, Florida, and Georgia) (9.9 ± 0.5 µm), Cal-
in Willapa Bay: Leadbetter and North Cove. Sclerotia were ifornia (8.7 ± 0.3 µm), and Washington (9.5 ± 0.2 µm)
collected in the field, removed from the inflorescence, and (Fig. 3). Variances of all isolates were similar.
later the same day, placed in 20 mL of deionized water for To compare morphological and molecular indicators of
10 min, stirring every 3 min. The number of sclerotia on the relationships, groupings based on conidial size within G3
surface of the water at the end of this time period was re- were overlaid on a cladogram according to AFLPs pre-
corded. sented by Fisher et al. (2005) (Fig. 4). Isolates from Cali-
fornia and Washington grouped together with 100%
Results bootstrap support, and these geographic regions are identi-
fied as one population, using morphological data as well
Average lengths and widths for conidia of all isolates (Figs. 3 and 4). Ireland and the northeast United States clus-
used in this study ranged from 6.1 to 13.6 µm and 3.0 to ter with 94% bootstrap support, and conidia from these iso-
5.2 µm, respectively, including those produced in vivo and lates are the longest in our collection.
in vitro. When produced on a common inoculated plant All G1 sclerotia collected from 2000 to 2003 sank in wa-
host, Spartina foliosa, G3 conidia were significantly longer ter. Of the G2 isolates, one of two sclerotia (WAB 1) col-
(9.92 ± 0.21 µm) than those of G1 (7.74 ± 0.49 µm) (P = lected from Ammophila breviligulata Fern. did float, and
0.0031), while G2 conidia (8.98 ± 0.24 µm) were not con- one of two sclerotia (WPA 1) collected from Calamagrostis
sistently distinct from either those of G1 or G3. Conidial nutkaensis (Presl.) Steud. floated; all other G2 sclerotia
lengths ranged from 6.0 to 8.0 µm for G1, from 8.0 to 10.0 sank in water. All G3 sclerotia floated in water, except those
µm for G2, and from 7.0 to 13.5 µm for G3. When har- from Washington State, which sank. When G3 sclerotia from
vested from culture, both G2 (8.83 ± 0.19 µm) and G3 Washington State were tested, immediately after collection
(9.4 ± 0.21 µm) conidia were significantly longer than G1 in 2002 and 2003, 86 of 93 from Leadbetter floated
conidia (7.26 ± 0.29 µm) (P < 0.0001). Conidial width did (92.5%), and 82 of 121 from North Cove floated (67.8%).
not differ among G1, G2, or G3 isolates (P < 0.3185). When sclerotia from Washington State were tested in larger
Fisher et al.: ergot on grasses / Claviceps purpurea / salt-marsh habitats / conidial morphology 393

Fig. 2. Conidial size of G1 (䉱; terrestrial), G2 (䊏), and G3 (䊊; salt marsh) from isolates of Claviceps purpurea inoculated on
Spartina foliosa.

Fig. 3. Length of conidia of G3 collected from Spartina foliosa inoculated with Claviceps purpurea isolates from six geographic
regions: Ireland, Argentina, northeast United States (NE USA, including New York and Rhode Island), southeast United States (SE
USA, including Alabama, Georgia, and Florida), Washington, United States (WA USA), and California, United States (CA USA).

numbers, 64.3% to 100% of the sclerotia from Spartina morphological and ecological characters can be used to dif-
alterniflora and Distichlis spicata floated (Table 2). ferentiate three infraspecific groups within C. purpurea.
However, characters are more variable in samples from
Discussion aquatic and salt-marsh habitats than has been reported
(Pañoutová et al. 2000, 2002). For example, sclerotia from
The results of this study confirm previous reports that grasses in most salt-marsh habitats floated in water, but those
394 Can. J. Plant Pathol. Vol. 27, 2005

Fig. 4. Unweighted pair-group method with arithmetic average (UPGMA) cladogram based on 221 amplified fragment length
polymorphism (AFLP) markers as described in Fisher et al. (2005). Included are 33 isolates, representative of three previously
described infraspecific groups within Claviceps purpurea, for which we have conidia measurements. Bootstrap support is shown only
where values are >90%. Following ANOVA, mean separation with the Student–Newman–Keuls (SNK) statistic was used to compare
the average conidia lengths of G3 isolates. Mean conidia length and isolate origins follow SNK values. Southeast United States
includes isolates from Alabama, Florida, and Georgia; northeast United States includes isolates from New York and Rhode Island.

from Washington State had variable characteristics, with We found a wider range for the length of G3 conidia,
some floating and others sinking. One G2 sclerotium from 7.0–13.5 µm, than previously reported, by Pañoutová et al.
Ammophila breviligulata (WAB 1) floated while another (2000, 2002), 9.5–12 µm. Our data show that conidia of G3
from the same host did not (WAB 2). The same was true C. purpurea, whether produced in culture or on a plant host
for two sclerotia collected from different Calamagrostis (i.e., those collected from honeydew), are consistently lon-
nutkaensis plants. The tendency to float on water has been ger than conidia produced by G1 C. purpurea, which is in
attributed to the presence of intercellular air spaces within agreement with results presented by Pañoutová et al. (2000).
the sclerotium rind (Duncan et al. 2002) and is clearly adap- However, some G3 conidia overlapped the range reported by
tive for survival and dispersal in aquatic environments. Pañoutová et al. (2000) for G2, 7–10 µm. Group 3 conidia
However, sclerotia lose their ability to float with age from California and Washington were smaller than average
(Stäger 1922) perhaps as a result of decay of the rind. In for the salt-marsh group, 8.6 µm and 9.1 µm, respectively;
2000–2002, sclerotia were collected in the field and trans- the longest conidia came from NYF 1 (13.0 µm) and IRE 12
ported to the laboratory to be tested. When sclerotia from (12.5 µm) samples from New York, United States, and Ire-
all locations were refrigerated before processing, only salt- land, respectively. These two regions were closest to the col-
marsh sclerotia from Washington State sank. Without a pe- lection sites for isolates previously reported (Pañoutová et al.
riod of storage, flotation rates were similar to flotation rates 2000, 2002), which may explain why previous papers re-
after refrigeration. Our data suggest that this trait is variable ported less of an overlap with the shorter, G2 spores. Our es-
in the Washington State population of G3 C. purpurea. timate of average conidial size is perhaps smaller than it
Fisher et al.: ergot on grasses / Claviceps purpurea / salt-marsh habitats / conidial morphology 395

Table 2. Buoyancy determinations for population samples of sclerotia of Claviceps purpurea

from grasses in Willapa Bay, Washington State, United States.
Year Site Host N Flotation (%)
2002 Cedar River Spartina alterniflora 2 100
2002 Kaffee Louis Spartina alterniflora 6 66.7
2002 Leadbetter Spartina alterniflora 28 64.3
2002 Upper Palix Spartina alterniflora 6 100
2002 Willapa River Spartina alterniflora 15 93.3
2003 Leadbetter Spartina alterniflora 93 92.5
2003 North Cove Distichlis spicata 121 67.8
2003 Palix River Spartina alterniflora 3 66.7
2003 Naselle River Lolium spp.a 6 0
Note: N, number of sclerotia.
Lolium spp. are the only grasses, in this analysis, presumed to host terrestrial C. purpurea (G1).

would be had we collected equally from western Europe and was supported by Jastro Shields and Humanities awards
the Pacific Coast of the United States. Regional populations from the University of California, Davis, and the National
produced conidia of different lengths, which suggest that Science Foundation, Biocomplexity, Division of Environ-
these populations may be isolated. Northeast United States mental Biology (No. 0083583) to A. Hastings.
isolates had larger conidia than isolates from the southeast
United States, California, and Washington (Fig. 4). Conidia
from Ireland and Argentina were also long, although low References
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We thank Brenna Aegerter, Sharon Kirkpatrick, and
Jeness Peterson for their technical assistance. This work