Stemphylium Leaf Spot of Parsley in California

Caused by Stemphylium vesicarium

Steven T. Koike, University of California Cooperative Extension, Salinas 93901; Nichole O’Neill and Julie Wolf, United States
Department of Agriculture–Agricultural Research Service (USDA-ARS), Systematic Mycology and Microbiology, and Peter Van
Berkum, USDA-ARS, Soybean Genomics and Improvement Laboratory, Beltsville, MD 20705; and Oleg Daugovish, University of
California Cooperative Extension, Ventura 93003

Abstract
Koike, S. T., O’Neill, N., Wolf, J., Van Berkum, P., and Daugovish, O. 2013. Stemphylium leaf spot of parsley in California caused by Stemphylium
vesicarium. Plant Dis. 97:315-322.

From 2009 through 2011, a previously undescribed disease occurred rot and celery) but not on leek, onion, spinach, and tomato. Isolates
on commercial parsley in coastal (Ventura County) California. Symp- caused brown lesions to form when inoculated onto pear fruit but only
toms of the disease consisted of circular to oval, tan to brown leaf spots when the fruit tissue was wounded. Using a freeze-blotter seedborne
and resulted in loss of crop quality and, hence, reduced yields. A fun- pathogen assay, parsley seed was found to have a low incidence
gus was consistently isolated from symptomatic parsley. Morphologi- (0.25%) of S. vesicarium. When inoculated onto parsley leaves, three
cal and molecular data identified the fungus as Stemphylium vesicar- of four isolates from seed caused the same leaf spot disease. This is the
ium. When inoculated onto parsley leaves, the isolates caused first documentation of a foliar parsley disease caused by S. vesicarium.
symptoms that were identical to those seen in the field; the same fun- The occurrence of S. vesicarium on parsley seed indicates that infested
gus was recovered from test plants, thus completing Koch’s postulates. seed may be one source of initial inoculum. Based on the negative
Additional inoculation experiments demonstrated that 10 of 11 tested results in the host range experiments, it appears that this parsley patho-
flat leaf and curly parsley cultivars were susceptible. The parsley iso- gen differs from the S. vesicarium that causes disease on leek, garlic,
lates also caused small leaf spots on other Apiaceae family plants (car- onion, and pear fruit.

Parsley (Petroselinum crispum) is a familiar leafy plant in the plantings were affected. Initial symptoms consisted of leaf spots
Apiaceae family that is grown as both a fresh market commodity that were 2 to 3 mm in diameter, circular to oval in shape, and
for use as a vegetable, herb, and garnish and as a dehydrated prod- chlorotic. As disease progressed, the spots enlarged to 5 to 8 mm,
uct for various culinary uses. California is the number one pro- retained a circular to oval shape, and turned tan to light brown in
ducer of parsley in the United States, with over 1,043 ha grown in color, with chlorotic borders. In some cases, leaf spots exhibited a
2010 (6), representing approximately half of the country’s parsley ringspot or target appearance due to alternating lighter- and darker-
(50). Two coastal counties, Monterey and Ventura, grow 49% of colored tissue (Fig. 1). When the disease was severe, the leaf spots
California’s parsley; in 2010, these counties accounted for 509 ha coalesced and the leaves became prematurely chlorotic and senes-
of parsley that was valued at $13.5 million (6). The California cent, eventually drying up and resulting in leaf dieback (Fig. 1).
parsley crop consists of both curly and flat leaf cultivars. Parsley Leaf petioles were also diseased and had narrow, elongated, brown
crops are direct seeded, grown to harvestable size, hand harvested lesions. Spots occurred mostly but not exclusively on older foliage.
or mechanically mowed, then allowed to regrow for additional When the parsley was harvested, the remaining lower, older leaves
harvests. This harvest-and-regrowth practice can be done up to still attached to the plants often exhibited the most severe symp-
three times. toms; the disease also reappeared on the subsequent regrowth fol-
Parsley production in California involves high seeding rates that lowing a harvest. Overall maximum disease incidence was esti-
result in dense plant populations and thick canopies. This planting mated at 30%. Sparse fungal growth was sometimes observed on
practice, combined with the fact that all parsley crops are irrigated adaxial leaf spot surfaces but obvious fungal fruiting bodies were
with overhead sprinklers, creates conditions in which foliar dis- absent from the spots. Because of the high quality standards for
eases can be problematic. In California, the most damaging foliar this leafy commodity, the problem resulted in yield loss because
disease has historically been Septoria late blight caused by Septo- diseased sections in some fields were not harvested. The purposes
ria petroselini (23). Septoria late blight has been managed by using of this study were to determine the cause of this leaf spot problem,
pathogen-free seed and by applying foliar fungicides (24,32). characterize the pathogen, and investigate possible sources of pri-
Other, less important foliar concerns in California are powdery mary inoculum.
mildew caused by Erysiphe orontii (26), bacterial leaf spot caused
by two pathovars of Pseudomonas syringae (5), and a virus disease Materials and Methods
caused by Apium virus Y (25,49). Isolation of the causal agent. Symptomatic leaves were surface
From 2009 through 2011, unfamiliar foliar symptoms were ob- sterilized by soaking leaf pieces in 1% bleach (0.525% sodium
served on commercial flat leaf parsley grown for the fresh market hypochlorite) solutions for 3 min. Small (3-by-3-mm) sections of
in Ventura County, CA. Both conventional and organic parsley tissue were aseptically excised from leaf spot margins and placed
into petri plates containing corn meal agar (CMA; Difco Laborato-
ries) that was acidified (25% lactic acid at 2 ml/liter). Plates were
Corresponding author: S. T. Koike, E-mail: [email protected] incubated in light from a combination of cool white and Vita-Lite
fluorescent tubes on a cycle of 12 h of light and 12 h of darkness at
Accepted for publication 4 September 2012.
22°C and examined after 5 to 7 days for fungal growth. Single-
spored cultures of isolated fungi were subsequently stored on po-
http://dx.doi.org/10.1094 / PDIS-06-12-0611-RE tato dextrose agar (PDA; Difco Laboratories) slants at 5°C. To test
© 2013 The American Phytopathological Society for bacterial pathogens, small pieces of the surface-sterilized leaf

Plant Disease / March 2013 315

spots were aseptically excised from leaf spot margins and macer- GGG CCG TCA ACG ACC TTC-3′ designed with the software
ated in 40 µl of sterile distilled water (SDW). The resulting suspen- package Oligo Primer Analysis Software (version 6.65; Molecular
sions were streaked onto sucrose peptone agar plates and incubated Biology Insights, Inc.) using sequences of Stemphylium spp. de-
at 22°C. After 3 to 5 days, these plates were examined for bacterial posited in GenBank. The polymerase chain reaction (PCR) was
growth. Nonsterilized leaf spot pieces were also excised and placed optimized using a commercially available kit (Invitrogen). The
in a drop of water on glass slides to examine for bacterial stream- reaction conditions that were used were 10 µl of 5× buffer C (60
ing with a compound microscope. mM Tris-HCL, 15 mM (NH4)2SO4, and 2.5 mM MgCl2 at a final
Morphological characterization of Stemphylium isolates. pH of 8.5); 1.25 µl each (10 mM) of dATP, dCTP, dGTP, and
Seven isolates taken from diseased plants were single spored and dTTP; 5 µl each (10 pmol/µl) of the primers gpd f and gpd r (gpd
grown on half-strength V8 agar medium incubated in light on a region); 2 µl of Perkin Elmer Taq polymerase; and 22 µl of sterile
cycle of 12 h of light and 12 h of darkness at 22°C. Morphological water. Amplifications were performed with an ERICOMP Delta
examination was conducted for all isolates after 7 days and mea- Cycler II system using the following program parameters: 35 cy-
surements taken for two representative isolates after 14 days. At cles of 94°C for 30 s, 57°C for 1 min, and 72°C for 1.5 min; and a
least 50 conidia and 20 conidiophores were measured per isolate. final extension at 72°C for 3 min. The presence of PCR products
Photomicrographs were taken using a Zeiss Axioplan II Imaging was verified by UV illumination of horizontal agarose (0.7%
Microscope, Axiocam digital camera, and Axiovision imaging [wt/vol]) gels after electrophoresis in the presence of ethidium
software (Carl Zeiss Microimaging Inc.). Semi-permanent mounts bromide (EtBr) (0.08 µg/ml).
were prepared in Shear’s solution (300 ml of 2% aqueous potassium The presence of a single PCR product of the expected molecular
acetate, 120 ml of glycerin, and 120 ml of 95% ethyl alcohol). size using each primer pair with each of the different templates was
Molecular characterization of Stemphylium isolates. For ex- verified before sequence analysis. Each PCR product was then
tracting DNA, isolates were grown in shake cultures at 22°C for 14 purified to remove the PCR primers by using the Ampure PCR
days in 100 ml of potato dextrose broth (24 g/liter). The resulting purification system (Agincourt Bioscience Corporation). The puri-
mycelial mats were collected by filtration and then lyophilized. fied PCR products were used in each of two forward and two re-
The lyophilized mycelium (50 mg/isolate) was ground in liquid verse sequence reactions with the nested sequence primers gpd-ef
nitrogen using a sterile micropestle. DNA was extracted using the 5′-CGG CTT CGG TCG CAT G-3′, gpd-if 5′-CAC GGC CAG
Qiagen DNeasy Plant Mini Kit according to the manufacturer’s TTC AAG-3′, gpd-er 5′-GCC AAG CAG TTG GTT GTG-3′, and
instructions. Samples were stored at –20°C. gpd-intU 5′-CGC GGC GGT TGG AGG ACA TTT-3′. An Applied
A partial section of the gpd gene was amplified by using the pri- Biosystems 3130 DNA Analyzer in combination with a Dye Deoxy
mers gpd f5′-GCA CCG ACC ACA AAA ATC-3′ and gpd r5′- Terminator Cycle Sequencing Kit (Applied Biosystems) was used

Fig. 1. Symptoms of Stemphylium leaf spot of parsley caused by Stemphylium vesicarium. A, Initial symptoms are small, circular, chlorotic spots. B, As disease develops, the
spots enlarge, turn tan to brown, and have chlorotic borders. C and D, Spots may exhibit concentric rings and result in necrosis and early senescence of the leaf.

316 Plant Disease / Vol. 97 No. 3

for sequencing the purified PCR products as described previously unwounded and wounded green and yellow pear fruit. Fruit were
(51). Sequences were aligned with GeneDoc (version 2.6.001; examined for up to 10 days for lesion development. The experi-
K. B. Nicholas and H. B. Nicholas; http://www.nrbsc.org/gfx/gene ment was completed twice.
doc/index.html). Accuracy of the alignments was checked and Assaying for seedborne inoculum. Because Stemphylium
adjustments made using GeneDoc. A neighbor-joining tree of pathogens on crops such as onion (1,35), radish (Raphanus sativus)
Jukes-Cantor distances of the partial glyceraldehyde-3-phosphate (4), and spinach (19) are known to be seedborne, parsley seed was
dehydrogenase DNA sequences was generated with the Molecular tested for Stemphylium vesicarium using a freeze-blotter assay
Evolutionary Genetics Analysis software, version 4.0 (48), with the (10,11). The grower who first reported this disease provided seed
sequence of Alternaria alternata (GenBank accession AF 081400) (‘Italian Flat Leaf’) from the original seed lot used to grow the af-
as an outgroup. Confidence values for the branches were obtained fected crop. For each experiment, four replications of 100 seeds
by bootstrap analysis using 2,000 permutations of the dataset. The each were placed in a stainless steel tea strainer that was agitated
sequences for gpd were deposited in GenBank under accession for 1 min in a 1.2% bleach (0.525% sodium hypochlorite) solution.
number JX139753. Seed were triple-rinsed in SDW, placed on sterile paper towels in a
Pathogenicity of Stemphylium vesicarium isolates to parsley. laminar flow hood to dry, and finally arranged singly onto steri-
To document pathogenicity to parsley, six representative isolates lized steel-blue germination blotter paper (Anchor Paper Com-
(STM 09-12B1, 09-13B1, 09-14B1, 09-15B1, 09-17B1, and 09- pany) lining plastic incubation boxes. The boxes were 10-by-10-
18B2) of Stemphylium vesicarium recovered from diseased parsley cm, clear acrylic containers with tight-fitting, clear lids (Hoffman
were grown for 4 weeks on V8 juice agar medium under a cycle of Manufacturing, Inc.). The blotter paper was moistened with ap-
12 h of light and 12 h of darkness at 22°C. Conidial suspensions proximately 15 ml of SDW.
(1.5 × 105 conidia/ml) were prepared, Tween 20 was added (1 drop The parsley seed were arranged on the blotters in six rows of 6
per 500 ml), and the mixture was sprayed with a hand-held mister seeds/box (maximum of 36 seeds/box) using sterilized forceps;
until runoff onto 24 parsley (‘Italian Flat Leaf’) plants, growing in therefore, three boxes were used for each replication. The lid was
six-pack containers, each having 6 to 10 true leaves. Plants were then tightly secured on each box. The boxes were incubated in the
subsequently placed in clear plastic bags for 48 h, removed from dark for 24 h while the seed imbibed water, placed in a freezer
bags, and then incubated in a greenhouse (20 to 24°C). Control (–20°C) for 24 h to freeze-kill the imbibed seed, and then placed
plants were sprayed with SDW plus Tween 20 and handled in the under lights (near-UV light and cool white fluorescent light) on a
same way. When symptoms developed, isolations were conducted cycle of 12 h of light and 12 h of darkness at 22°C. If not subject to
as described above. The inoculation experiment was conducted two freezing, imbibed seed will germinate, making it difficult to ob-
times. serve fungal growth on the seed coats. Seed were examined for
To evaluate the susceptibility of various parsley cultivars to the Stemphylium spp. and other dematiaceous fungal species with a
pathogen, separate conidial suspensions of four isolates (STM 09- dissecting stereomicroscope at 5, 10, and 14 days after being re-
12B1, 09-13B1, 09-14B1, and 09-15B1) were prepared as de- moved from the freezer. Suspected Stemphylium fungi were iso-
scribed above and sprayed onto 12 plants each of five flat and six lated and single-spored cultures of these isolates were subsequently
curly leaf parsley cultivars, growing in six-pack containers, each stored on PDA slants at 5°C. This experiment was completed four
having 6 to 10 true leaves. An SDW plus Tween 20 control was times.
included for each cultivar. Plants were incubated as described Pathogenicity of seedborne isolates to parsley. To determine
above. Results were evaluated at 24 days post inoculation (dpi) whether Stemphylium isolates obtained from parsley seed were
when 10 symptomatic leaves were randomly collected per cultivar. pathogenic to parsley, isolates were grown for 4 weeks on V8 juice
For all leaflets, spots were measured and categorized as being agar medium under a cycle of 12 h of light and 12 h of darkness at
either smaller or larger than 5 mm in diameter. Isolations were 22°C. Conidial suspensions (1 × 105 conidia/ml) were prepared
conducted from these collected leaves. The inoculation experiment and sprayed onto 12 parsley (‘Dark Green Italian’) plants as de-
was conducted two times. scribed above. Inoculated plants were incubated as per the previous
Pathogenicity of S. vesicarium parsley isolates to other pathogenicity tests. Control plants were sprayed with SDW plus
plants. Host range was investigated with the same four isolates Tween 20 and handled in the same way. Plants were evaluated at
used in the parsley cultivar inoculations. Separate conidial suspen- 24 dpi to determine presence or absence of leaf spots. If symptoms
sions of these four isolates were prepared as before and sprayed developed, isolations were conducted and resulting Stemphylium
onto 12 plants each of following plants: carrot (Daucus carota isolates identified to species. This inoculation experiment was
subsp. sativus ‘Scarlet Nantes’ and ‘Tendersweet’), celery (Apium conducted three times.
graveolens ‘Conquistador’), leek (Allium porrum ‘Lancelot’), on-
ion (A. cepa, ‘California Red’), spinach (Spinacia oleracea ‘Bo- Results
lero’ and ‘Lazio’), and tomato (Solanum lycopersicum ‘Cham- Isolation of the causal agent. Cut leaf spot edges lacked bacte-
pagne’). Prior to inoculating the leek and onion plants, leaves were rial streaming when examined with a compound microscope, and
lightly rubbed with cheesecloth so as to partially remove the waxy no bacteria were recovered from the leaf section suspensions
cuticle (43). Parsley (‘Dark Green Italian’) plants were inoculated streaked onto sucrose peptone agar. Leaf spot surfaces generally
as a positive control. An SDW plus Tween 20 control was included did not have visible fungal growth and no distinct fruiting bodies
for each cultivar. Plants were incubated as described above. Plants were present. However, a fungus was consistently isolated from
were evaluated at 24 dpi for presence or absence of leaf spots, and tissues removed from the margins of leaf spots and plated onto
the experiment was completed two times. acidified CMA. Colonies on acidified CMA were dark green to
In addition, organically grown pear (Pyrus communis ‘Bartlett’) gray in color, with whitish aerial mycelium, and sporulated
fruit that were never treated with fungicides were inoculated with sparsely. To obtain greater sporulation, isolates were transferred to
the same spore suspensions used on plants. Fruit were placed on V8 juice agar medium and incubated under lights for all experi-
wire racks inside plastic crisper boxes and water was added to the ments. Monoconidial cultures of representative isolates were ob-
bottom of the boxes. Two unripe green and two ripe yellow pear tained from these V8 juice agar cultures and stored on PDA slants
fruit were used for each inoculation. One drop of inoculum was at 5°C. No other fungi were isolated from the leaf spots.
placed on each of six unwounded and six wounded spots on each Morphological and molecular characterization of Stem-
fruit. Wounding was done by pushing a sterilized dissecting needle phylium isolates. The seven isolates of Stemphylium recovered
approximately 2 mm deep into the fruit. The boxes were closed from symptomatic parsley leaves were morphologically similar.
with their lids and incubated at 22°C in the dark for 48 h (22). Af- Dictyoconidia were brown colored, broadly ellipsoidal to oblong,
ter 48 h, the pear fruit were placed in a dry, open crisper and main- and borne singly (Fig. 2). Conidia often had three main transverse
tained at 22°C. An SDW plus Tween 20 control was used on both septa, at which points there were conspicuous constrictions. Outer

Plant Disease / March 2013 317

conidial walls had verrucose ornamentation. Conidia dimensions (<5 mm in diameter) for the second experiment that was conducted
were mostly 22 to 28 (mean of 26) µm long and 13 to 16 (mean of during the warmer, drier month of June (Table 2). None of the
15) µm wide; the mean length/width ratio was 1.8 (Table 1). control plants treated with SDW developed symptoms.
Conidiophores were mostly unbranched, 6 to 8 µm wide, with dis- For curly leaf parsley, five of the six cultivars (‘Dark Moss
tinctly swollen apical cells (8 to 11 µm wide) having darkly pig- Curled’, ‘Evergreen’, ‘Jane’, ‘Krausa’, and ‘Moss Curled’) devel-
mented bands (Table 1). These morphological features fit the oped leaf spots that were consistently less than 5 mm in diameter
descriptions for S. vesicarium (46). A Pleospora sp. teleomorph (Table 2). Curly leaf ‘Triple Curled’ generally did not develop leaf
stage was not observed on diseased parsley collected from the field spots following inoculations, except with isolates STM 09-13B1
and did not develop on inoculated plants or in culture. From se- (second inoculation only) and STM 09-14B1 (first inoculation
quence analysis of the partial gpd gene, the parsley isolates were only) (Table 2). The pathogen was consistently reisolated from leaf
identified within the genus Stemphylium. The partial gpd sequences spots on all curly leaf parsley. None of the control plants treated
of the parsley isolates S. vesicarium (EGS 37-067), S. herbarum with SDW developed symptoms.
(EGS 30-181.1 and EGS 36-138.2), S. alfalfae (EGS 36-088, EGS Pathogenicity of S. vesicarium isolates from parsley to other
40-038, and EGS 39-127), and a Stemphylium sp. (EGS 44-149) plants. After 24 days, positive control flat leaf parsley (‘Dark
were identical (Fig. 3). Based on the morphological and molecular Green Italian’) developed typical leaf spots resembling symptoms
data, the parsley isolates were identified as Stemphylium vesicar- seen in the field. Celery and both carrot cultivars developed small
ium (Wallr.) E. G. Simmons (7,45,46). (<5 mm in diameter) leaf spots that were circular to oval, tan to
Pathogenicity of S. vesicarium to parsley. For all six isolates, light brown in color, and sometimes exhibited a ring spot
leaf spots developed on inoculated parsley (‘Italian Flat Leaf’) appearance (Table 2). The S. vesicarium pathogen was consistently
plants between 20 and 24 dpi and closely resembled symptoms ob- reisolated from parsley, celery, and two carrot cultivars. Leaf spots
served on the originally infected field plants. Fungal fruiting did not develop on inoculated leek, onion, spinach, or tomato
bodies and structures did not form on leaf spot surfaces. Stem- (Table 2). For inoculated pear fruit, circular, dark-brown lesions
phylium fungi morphologically similar to the original isolates were developed at wounded sites on both green immature and yellow
consistently isolated from the leaf spots and, after morphological mature fruit. S. vesicarium was reisolated from these fruit lesions.
and molecular characterization, confirmed to be the same as the No lesions formed at the unwounded sites on green immature or
original isolates. None of the control plants treated with SDW plus yellow mature fruit. None of the control plants or fruit treated with
Tween 20 developed symptoms. Results from the two experiments SDW plus Tween 20 developed symptoms. Results from the two
were the same. experiments were the same (Table 2).
Susceptibility of parsley cultivars. Leaf spots developed on all Assaying for seedborne inoculum. Parsley seed tested with a
five flat leaf parsley cultivars tested for susceptibility to S. vesicar- freeze-blotter assay were evaluated for dematiaceous fungi at 5, 10,
ium (Table 2) and the pathogen was reisolated from symptomatic and 14 days after removal from the freezer. The recorded numbers
tissue. Slight differences in disease severity were observed among of seed supporting growth of dematiaceous fungi did not increase
the flat leaf cultivars. ‘Dark Green Italian’ and ‘Italian Flat Leaf’ from 10 to 14 days; however, in some cases, by day 14, white,
consistently had larger lesions (≥5 mm in diameter) than the other aerial, floccose fungal growth obscured the seed. Therefore, num-
cultivars in both the first and second experiments (Table 2). For bers recorded at day 10 were considered as the final counts (Table
‘Forest Green’, ‘Rialto’, and ‘Titan’, the leaf spots were smaller 3). With the seed assay repeated four times (400 seeds per experi-

Fig. 2. Conidia of Stemphylium vesicarium isolated from parsley and grown on V8 agar medium under lights (×400).

318 Plant Disease / Vol. 97 No. 3

ment), the mean percentage of seed supporting growth of Stem- ley seed (16,36) and with a parsley seedling disease that involved a
phylium was 0.25%. A Ulocladium fungus was present on 0.63% number of fungi (34,37).
of the seed, while an Alternaria sp. having small conidia borne in S. vesicarium is reported on a number of other agronomic crops
long chains appeared at 2.9%. The Stemphylium isolates were mor- and causes purple leaf blotch or leaf blight of garlic (A. sativum),
phologically similar to S. vesciarium isolated from leaf spots and, leek (A. ampeloprasum), and onion (A. cepa) (39,40,43,47); Stem-
therefore, were single spored and stored in PDA slants. phylium leaf spot or purple spot of asparagus (Asparagus offici-
Pathogenicity of seedborne isolates to parsley. Four S. ves- nalis) (8,13,27); and foliar diseases of alfalfa (28,29,44) and soy-
icarium isolates were recovered from the parsley seed assays (Ta- bean (9). S. vesicarium also causes a stem and root rot on radish
ble 3), stored as single-spored cultures, and used to inoculate pars- sprouts (4) and brown spot of European pear (P. communis) leaves
ley plants. Three of the isolates caused leaf spots 20 to 24 dpi when and fruit (22,31).
inoculated onto parsley (‘Dark Green Italian’); the fourth isolate The parsley S. vesicarium isolates caused similar leaf spots
failed to cause disease after repeated inoculations. All Stemphylium when inoculated onto carrot and celery. Therefore, this pathogen is
fungi reisolated from leaf spots of inoculated plants were morpho- not host specific to parsley but can infect other plants in the
logically similar to S. vesicarium from field plantings. None of the Apiaceae family. However, the host ranges and possible host speci-
control plants treated with SDW plus Tween 20 developed symp- ficities of S. vesicarium pathogens from various crops remain un-
toms. Results from the three inoculation experiments were the same. clear due to differing reports. Some previous reports indicate that
S. vesicarium can infect a broad range of plants, with onion devel-
Discussion oping typical purple blotch symptoms when inoculated with an
This is the first documentation of Stemphylium leaf spot of pars- asparagus isolate (43) and asparagus, garlic, and onion each being
ley caused by S. vesicarium in California and the first characteriza- susceptible to S. vesicarium isolates from all three of these plants
tion of a Stemphylium foliar pathogen on this crop (15). Morpho- (3). However, a different isolate of S. vesicarium from onion failed
logical and molecular data supported the identification of the to infect asparagus (13). Kohl et al. (22) found that asparagus and
parsley isolates as S. vesicarium, which belongs to the S. vesicar- onion isolates did not infect pear leaves or fruit. In this study, parsley
ium species group (46). Species within this Stemphylium clade are isolates of S. vesicarium did not infect onion when inoculated under
indistinguishable based on DNA sequences using different loci controlled conditions. Additional studies using larger numbers of
(20) and include S. vesicarium, S. herbarum, and S. alfalfa. In isolates from various hosts would be needed to further address the
multiple inoculation experiments, the flat leaf parsley cultivars question of cross-pathogenicity of S. vesicarium pathogens.
were affected more severely than curly leaf types. The pathogen Using a freeze-blotter seed assay and inoculations onto parsley,
does not appear to be extremely aggressive, and the disease devel- we confirmed that pathogenic isolates of S. vesicarium were seed-
oped slowly under experimental conditions. The ability of S. vesi- borne on parsley at an incidence of 0.25%. Three of four isolates
carium to infect parsley appears sensitive to environmental condi- from seed caused leaf spot symptoms when inoculated onto parsley
tions, because experiments conducted during warmer, drier plants. Seedborne S. vesicarium has been reported for alfalfa (29),
ambient conditions (in June) resulted in less severe symptoms and, onion (1,35), and radish (4). Therefore, one possible inoculum
in some cases, lack of leaf spot development. source for this disease appears to be parsley seed. Although the
To our knowledge, the only other report of a Stemphylium foliar incidence of infested parsley seed was low, such an infestation rate
pathogen was a 1962 abstract describing a Stemphylium leaf spot could be significant for the farmer. For typical parsley production
occurring on parsley in New Jersey (30); however, the pathogen in California, the seeding rate is approximately 17 kg/ha or 4.41
species was not identified or characterized. The Lewis report (30) million seed/ha. Given the 0.25% infestation rate, potentially
indicated that, upon inoculating multiple crops in the Apiaceae there could be 18,750 infested seed/ha. Because all parsley is
family, the Stemphylium isolate from parsley infected parsley, cel- irrigated with overhead sprinkler systems, this low level of
ery, celeriac, fennel, dill, and parsnip; however, carrot, caraway, infested seed could result in significant disease due to a favorable
and anise did not develop disease. In our host range experiments, environment consisting of high-density plantings and splashing
both carrot and celery were susceptible to S. vesicarium isolates irrigation water.
from parsley. Given the lack of details in the 1962 report, it is not If freeze-blotter assays for detecting S. vesicarium on parsley
possible to draw conclusions regarding any possible relatedness of seed were to be used commercially, seed and lab technicians would
the California and New Jersey outbreaks. In other reports concern- need to exercise caution in examining the seed. The Alternaria spp.
ing Stemphylium spp., S. botryosum has been associated with pars- found on parsley seed should be readily recognized morpho-

Table 1. Dimensions of conidia and conidiophores of Stemphylium vesicarium isolates from parsley
Isolate measurements
Structurea 09-14B1 09-18B2 Means for two isolates
Conidia
Length (µm)
Range (18.4–) 19.9–28.0 (–41.3) (16.4–) 22.3–33.5 (–41.1) 25.9
SD 4.4 5.6 …
Width (µm)
Range (10.9–) 13.0–16.4 (–18.5) (10.3–) 13.8–18.2 (–23.2) 15.4
SD 1.7 2.2 …
Length/width ratio
Range 1.2–2.2 1.2–2.4 …
Mean 1.7 1.8 1.8
Conidiophores
Width (µm)
Range 5.5–8.3 5.5–8.3 6.9
SD 0.5 0.6 …
Apical cell width (µm)
Range 8.3–11.1 8.3–11.1 9.7
SD 0.8 0.9 …
a Fungal structures were taken from 4-week-old cultures on V8 juice agar plates that were incubated in light on a cycle of 12 h of light and 12 h of darkness
at 22°C. For each isolate, 50 spores and 20 conidiophores were measured. SD = standard deviation.

Plant Disease / March 2013 319

logically due to the beaked conidia that are borne in chains. senescent leaves of orchard floor cover crop species support the
However, when examining seed with a dissecting microscope, development of both P. allii pseudothecia and S. vesicarium co-
Ulocladium spp. appear very similar morphologically to Stem- nidia (31,41,42).
phylium; therefore, caution is required in differentiating these two In California, although parsley is grown as an annual crop, each
genera (45). In addition, because previous studies indicate that S. planting is harvested multiple times. Each time a flush of growth is
botryosum can be present as a contaminant on parsley seed, the cut and harvested, many loose leaves and stem pieces remain in the
seed technician will need to differentiate between the pathogenic S. field and become embedded in the parsley crowns. The cut ends of
vesicarium that causes leaf spot and S. botryosum that is not known stems still attached to the plant crowns dry out and die. Such de-
to cause a foliar disease on parsley. Such a differentiation based on tached parsley debris and senescing cut stem ends could function
morphology will necessitate examining spores using glass-slide- as substrates for either the conidial or possibly the pseudothecial
prepared specimens and a compound microscope. stage of this foliar pathogen. Also, after cutting and removing a
A thorough examination of the epidemiology of this disease is flush of foliage, the older, lower leaves remain attached to the
beyond the scope of this report. However, it is interesting to note plant; these older leaves were often found to have leaf spots. Infec-
that some epidemiological aspects of Stemphylium diseases of tion of subsequent regrowth in the field suggests that in-field
asparagus, garlic, and pear may also pertain to parsley. For these inoculum is coming from the crop debris or remaining diseased
other diseases, inoculum is associated with senescing and dead foliage. Splashing water from overhead sprinkler irrigation would
host tissues and debris. Pseudothecia of the Pleospora herbarum provide the means and environment for subsequent inoculum
perfect stage develop on senescent and dead asparagus fern debris dispersal, infection, and disease development. The nature of this
and are a key inoculum source for purple spot (12,13,18,21). P. inoculum (conidia versus ascospores) is not known for parsley.
allii pseudothecia also are prevalent on crop residues, senescent Future studies could examine such field situations for the presence
leaves, and old flower stalks of garlic (13,38). Pear leaf litter and of pseudothecia or conidia of this pathogen.

Fig. 3. Jukes-Cantor distance tree generated using the neighbor-joining method for the aligned partial DNA sequences of glyceraldehyde-3-phosphate dehydrogenase. The
analysis was done using the software MEGA version 4.0 (48) using an alignment length of 562 bp. Numbers at the branch nodes indicate the confidence values obtained
from bootstrap analysis using 2,000 permutations of the data set.

320 Plant Disease / Vol. 97 No. 3

 Although Septoria late blight is typically the most destructive spots and, therefore, could be mistaken for bacterial leaf spot.
disease of parsley in California, the addition of Stemphylium leaf Bacterial leaf spot causes angular, tan to brown leaf spots that lack
spot adds yet another challenge that California growers must deal any mycelial or fungal structures. Stemphylium leaf spot also lacks
with while producing large volumes of high-quality, defect-free fungal structures in the spots but is characterized by oval to round
parsley. Information is lacking regarding which fungicides might spots that often contain concentric rings of light and dark tissues.
be effective for controlling S. vesicarium on parsley. Field studies
involving other crops have demonstrated that chlorothalonil, pro- Acknowledgments
cymidone, and tebuconazole can manage Stemphylium spp. in We thank P. Elia (United States Department of Agriculture–Agricultural Re-
search Service, Beltsville, MD) and P. Ayala and K. Kammeijer (University of
asparagus, garlic, or onion (2,14,17); thiram is used to control California Cooperative Extension, Salinas) for assisting with this study; the
Stemphylium spp. on pear (33). None of these fungicides, however,
is registered for use on parsley in California. The cultivar experi-
ment indicates that at least one flat leaf parsley cultivar might be Table 3. Stemphylium and other dematiaceous fungi observed on parsley
resistant; therefore, a search for other resistant parsley cultivars seed following a freeze-blotter seed assay
could assist growers. Curly leaf cultivars consistently developed Number of seed colonized by a
less severe symptoms than flat leaf varieties. dematiaceous species/400 total testeda
Stemphylium leaf spot is not the only new foliar problem of Exp.b Stemphylium Ulocladium Alternaria
parsley that has been recently documented in California. A new
bacterial leaf spot disease caused by two different pathovars of 1 0 3 3
2 1 3 26
Pseudomonas syringae (pvs. apii and coriandricola) was found in
3 1 3 14
commercial fields (5). Therefore, growers and field personnel must 4 2 1 3
distinguish between four foliar diseases of parsley (23) (Table 4). Mean 1 2.5 11.5
Powdery mildew can easily be identified because of the typical Seedborne (%) 0.25 0.63 2.9
white, powdery, superficial mycelium and sporulation on leaves. a For each experiment, the seed assay was completed using 100 seeds in
Septoria late blight is distinctive because of the obvious brown to each of four replications. Final counts are based on seed examined at the
black pycnidia that form in most of the angular leaf spots; however, 10-day period. The experiment was conducted four times.
early stages of late blight will not have pycnidia present in leaf b Experiment number. Mean = mean number/experiment.

Table 2. Pathogenicity of Stemphylium vesicarium isolates from parsley to parsley cultivars and other commercial crops
Stemphylium isolates and control (first test/second test)a
Plant, cultivarb 09-12B1 09-13B1 09-14B1 09-15B1 Water
Parsley cultivars
Parsley, flat leaf
‘Dark Green Italian’ ++/++ ++/++ ++/++ ++/++ –/–
‘Forest Green’ ++/+ ++/++ ++/+ ++/+ –/–
‘Italian Flat Leaf’ ++/++ ++/++ ++/++ ++/++ –/–
‘Rialto’ +/+ ++/+ ++/+ ++/+ –/–
‘Titan’ ++/+ ++/+ +/++ ++/+ –/–
Parsley, curly
‘Dark Moss Curled’ +/+ +/+ +/+ +/+ –/–
‘Evergreen’ +/+ +/+ +/+ +/+ –/–
‘Jade’ +/+ +/– +/+ +/+ –/–
‘Krausa’ +/– +/+ +/+ +/+ –/–
‘Moss Curled’ +/– +/+ +/+ +/+ –/–
‘Triple Curled’ –/– –/+ +/– –/– –/–
Other crops
Carrot
‘Scarlet Nantes’ +/+ +/+ +/– +/+ –/–
‘Tendersweet’ +/+ +/+ +/+ +/+ –/–
Celery, ‘Conquistador’ +/+ +/+ +/+ +/– –/–
Leek, ‘Lancelot’ –/– –/– –/– –/– –/–
Onion, ‘California red’ –/– –/– –/– –/– –/–
Spinach
‘Bolero’ –/– –/– –/– –/– –/–
‘Lasio’ –/– –/– –/– –/– –/–
Tomato, ‘Champagne’ –/– –/– –/– –/– –/–
Pear fruit, unripec
Unwounded –/– –/– –/– –/– –/–
Wounded +/+ +/+ +/+ +/+ –/–
Pear fruit, ripec
Unwounded –/– –/– –/– –/– –/–
Wounded +/+ +/+ +/+ +/+ –/–
Positive controld +/++ +/+ ++/+ ++/+ –/–
a For plants, 10 leaves per cultivar were evaluated for the development of leaf spots at 24 days post inoculation using the following scale: – = no leaf spots;
+ = minor leaf spots (spot size <5 mm in diameter); ++ = moderate leaf spots (spot size ≥5 mm in diameter). For pear fruit, results were evaluated for the
development of lesions for up to 10 days post inoculation using the following scale: – = no lesions and + = lesions present.
b All plants had 6 to 10 true leaves when spray inoculated with conidial suspensions (105 conidia/ml). Controls were treated with sterile distilled water plus

Tween 20. Plants were placed in clear plastic bags for 48 h, unbagged, then maintained in a greenhouse (20 to 24°C). Results from the two pathogenicity
tests are presented as first test/second test.
c Pear fruit (‘Bartlett’) were inoculated with the same conidial suspensions. Both green unripe and yellow ripe fruit were used and inoculum was placed on

both unwounded and wounded spots. Controls were treated with sterile distilled water plus Tween 20. Pear fruit were placed in an enclosed humid chamber
(lidded plastic box) at 22°C for 48 h and later incubated in a dry plastic box in the open air at room temperature. Results from the two pathogenicity tests
are presented as first test/second test.
d Control was Dark Green Italian flat leaf parsley.

Plant Disease / March 2013 321

Table 4. Diagnostic features of foliar diseases of parsley in California
Symptoms and signs
Tan to brown Leaf spots Dark brown to black Mycelial growth on
Disease, pathogen leaf spots circular, oval fungal structures leaf surface
Septoria late blight, Septoria petroselini Yes No (usually angular) Yes (pycnidia) No
Stemphylium leaf spot, Stemphylium vesicarium Yes Yes No Yes (sparse)
Powdery mildew, Erysiphe orontii No No No Yes (profuse, white)
Bacterial leaf spot, Pseudomonas syringae Yes No (always angular) No No

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322 Plant Disease / Vol. 97 No. 3