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Plant Disease / December 2002 1363
Detection and Management of Downy Mildew in Rose Rootstock
B. J. Aegerter, Department of Plant Pathology, University of California, Davis 95616; J. J. Nuñez, University of
California Cooperative Extension, Kern County, Bakersfield 93307; and R. M. Davis, Department of Plant Pathol-
ogy, University of California, Davis
More than 50% of the bare-root roses
produced in the United States are grown
near Wasco, in Kern County, CA. In 2000,
over 23 million rose plants were harvested
at a farm-gate value of $46 million (10).
Since the early 1990s, this nursery produc-
tion system has had a sporadic but continu-
ing problem with rose downy mildew,
caused by the oomycete Peronospora
sparsa. Infected leaves quickly abscise,
often resulting in severe defoliation and
reduced plant vigor. During the first 2
months after budbreak, rootstock cuttings
that are defoliated by downy mildew may
fail to root, resulting in reduced stands. On
older bushes, the reduced vigor can result
in failure of the canes to reach the diameter
required for the top grade and such plants
often are never harvested. Dieback of
young canes has been observed when the
stems are severely diseased, presumably
due to invasion by secondary pathogens
such as Botrytis spp.
Downy mildew currently is managed
with frequent preventative applications of
foliar fungicides after the plants begin to
grow in January or February. Such applica-
tions are costly in terms of materials and
labor, and can be difficult to undertake
during rainy weather. Even when fungi-
cides are applied, growers find the cur-
rently registered materials only marginally
effective. Difficulties in disease control are
compounded because the source of initial
inoculum is unknown. Production fields
are fumigated with methyl bromide prior to
planting; therefore, soilborne inoculum
seems an improbable source. Caneberries
(Rubus spp.), which are alternative hosts of
P. sparsa, are not cultivated in this produc-
tion area, and infected wild hosts have not
been observed in Kern County.
Because roses are vegetatively propa-
gated, it has been suggested that mycelium
of P. sparsa overwinters in rose propaga-
tion stock (5), but such transmission has
never been demonstrated. In the bare-root
rose industry in Kern County, rootstock is
propagated by cuttings taken from blocks
of perennial mother plants and the scion is
propagated by buds collected from produc-
tion fields. To avoid viruses, growers peri-
odically return to material from Foundation
Plant Material Services at the University of
California (UC) at Davis. Rootstock cut-
tings are obtained from mother plants in
the fall and typically planted in November.
These cuttings root in the field and begin
growing in January or February, depending
on the planting date and seasonal tempera-
tures. In some years, downy mildew ap-
pears on these cuttings just as they emerge
from dormancy and this is typically the
first downy mildew in a season. In the past,
this was a particular problem with the root-
stock Manetti’s rose (Rosa × noisettiana
Thory cv. Manetti). These observations led
us to suspect that at least one source of
initial inoculum may be within the root-
stock cuttings.
In addition to concerns about sources of
the pathogen within propagation materials,
there is also the potential of exportation of
infected plants to other areas. In the ab-
sence of symptoms, however, there is cur-
rently no means by which growers can
ascertain if plants might be latently in-
fected. Microscopic examination, while
useful, is limited to a small volume of
plant material and lacks specificity because
the mycelial morphology of various fungi
is similar. DNA-based methods, such as
the polymerase chain reaction (PCR), are
particularly suitable for the detection of
obligate parasites like downy mildews.
Toward development of an integrated
control program for rose downy mildew,
we initiated investigations into the poten-
tial for infected stems to serve as sources
of initial inoculum. The specific objectives
of this research were to (i) develop and
implement a PCR-based method for the
detection of P. sparsa in woody rose tis-
sues, (ii) examine survival structures
within rose tissues by epifluorescent mi-
croscopy, and (iii) document the efficacy
of fungicidal or hot-water dips of rootstock
propagation wood in the control of epidem-
ics of downy mildew in rose nurseries.
MATERIALS AND METHODS
Development of PCR-based detection
assay. The internal transcribed spacer
(ITS) region of the nuclear ribosomal DNA
(rDNA) was chosen as a target for amplifi-
cation of P. sparsa DNA. The ITS has
sequence variation at the interspecific level
but low levels of intraspecific variation
(11).
To obtain DNA for amplification and se-
quencing, symptomatic leaves collected
from commercial fields in Kern County,
CA were placed in a plastic box overnight
at room temperature and 100% relative
humidity to induce sporulation. Under a
dissecting microscope, roughly 200 spo-
rangiophores bearing sporangia were re-
moved from the leaves with sterile jew-
eler’s forceps and placed into a 1.5-ml
microcentrifuge tube containing 100 µl of
PCR buffer (undiluted GeneAmp10X PCR
ABSTRACT
Aegerter, B. J., Nuñez, J. J., and Davis, R. M. 2002. Detection and management of downy mil-
dew in rose rootstock. Plant Dis. 86:1363-1368.
A technique utilizing the polymerase chain reaction (PCR) was developed to investigate the
occurrence and location of Peronospora sparsa in dormant, woody rose tissues. PCR primers
were designed to amplify the internal transcribed spacer region of the ribosomal DNA of the
pathogen. Inhibition of the reaction by plant compounds was minimized by optimizing the re-
agents used in the extraction of DNA from roses and in the amplification reaction. The PCR
assay was capable of detecting as little as 2 pg of DNA from P. sparsa against a background o
f
4 ng of DNA from rose cane cortex. With this method, DNA of P. sparsa was detected in the
cortex of stem and root tissues of symptomatic plants. Pathogen DNA also was detected in the
cortex of crown tissues of asymptomatic mother plants used as a source of propagation materi-
als. Epifluorescent and differential interference contrast microscopy were used to confirm the
presence of abundant hyphae and oospores within the stem cortex of infected canes. Preplant
treatments of dormant rootstock cuttings in fungicides or hot water were evaluated during natu-
ral outbreaks of the disease in commercial rose nurseries. In three trials conducted over 2 years,
a 10-min preplant dip in the systemic fungicides metalaxyl or mefenoxam at rates of 100 to
10,000 mg a.i./liter reduced the area under the disease progress curve by 63 to 76% relative to
nontreated plots. The evidence from PCR assays, microscopy, and fungicide trials all support
the occurrence of perennating infections of P. sparsa within rose.
Corresponding author: B. J. Aegerter
E-mail: bjaegerter@ucdavis.edu
This research was supported, in part, by contribu-
tions from the Garden Rose Council.
Accepted for publication 7 August 2002.
Publication no. D-2002-1010-01R
© 2002 The American Phytopathological Society
1364 Plant Disease / Vol. 86 No. 12
buffer II; Applied Biosystems, Foster City,
CA). The suspension was sonicated with a
Vibra Cell sonicator (Model VC50; Sonic
& Materials Inc., Danbury, CT) using a
pulse of 2 s duration delivering 39 W at the
probe tip. The tubes then were centrifuged
at maximum speed (16,000 × g) for 5 s to
pellet the cellular material. DNA was
stored at –20°C prior to use in PCR.
PCR amplification of ITS1 and ITS2
and the 5.8S RNA gene of the rDNA was
accomplished with the oligonucleotide
primers ITS4 and ITS5 (24). The PCR was
performed in 50-µl reactions containing 2.5
mM MgCl2, 200 µM of each deoxynu-
cleoside triphosphate, 0.1 µM of each oli-
gonucleotide primer, and 1 unit of Taq
polymerase in 1× GeneAmp PCR buffer II
(Applied Biosystems). To this mixture, 1 to
3 µl of sonicated sporangial suspension was
added. The reaction was carried out in a
thermal cycler (EasyCycler, Ericomp Inc.,
San Diego, CA) programmed for 40 cycles,
each consisting of denaturation at 94°C for 2
min, annealing at 55°C for 2 min, and exten-
sion at 72°C for 2.5 min, with a single final
extension at 72°C for 10 min.
Due to difficulties in obtaining satisfac-
tory sequence data from the PCR product,
the amplification fragment from four dif-
ferent isolates from Kern County was
cloned into a plasmid in Escherichia coli
with the TA cloning system (TOPO TA,
Invitrogen Corp., Carlsbad, CA) using the
manufacturer’s recommendations. Success-
ful transformants transferred to 10 ml of
Luria-Bertani broth (13) were grown for 24
h on a shaker at 37°C. Plasmid DNA was
extracted from E. coli with the QIAprep
Spin Mini-prep Kit (Qiagen Inc., Valencia,
CA). The amplification fragment was cut
from the plasmid vector with the restriction
enzyme EcoR1. These cloned fragments
were submitted for sequencing to the DBS
Automated Sequencing Facility at UC
Davis. Sequences were determined with
ABI PRISM dye terminator cycle sequenc-
ing with dRhodamine terminator chemis-
try. The reactions were electrophoresed on
an ABI PRISM 377 DNA sequencer (Ap-
plied Biosystems) using a 5% Long Ranger
gel. The data were analyzed using ABI
Prism Á Sequencing 2.1.1 software (Ap-
plied Biosystems).
Primer design. The GenBank database
was searched for sequences with high simi-
larity to the DNA fragments. As there were
no Peronospora sequences in the database
at that time, the closest matches were ITS
sequences from eleven species of Phy-
tophthora (2). These were aligned with the
Peronospora sparsa sequence using the
software of the Genetics Computer Group,
University of Wisconsin, Madison. A num-
ber of regions that were unique to the Per-
onospora sequence were identified. Unique
sequences near the beginning of the ITS1
were used to design several forward prim-
ers. Other unique sequences, near the end
of the ITS2, were used to design reverse
primers. Candidate primer sequences were
entered into the program Primer Designer
(ver 2.0; Scientific and Educational Soft-
ware, Durham, NC) to evaluate the po-
tential for problems due to formation of
hairpin loops or primer dimers. Primers
were synthesized by the Molecular Struc-
ture Facility at UC Davis. Various primer
pairs were evaluated for specificity by
including them in reaction mixtures with
DNA from other fungal pathogens of rose,
including Phytophthora cactorum, Botrytis
cinerea, and a Phragmidium sp. These
fungi were all isolated from rose, and my-
celium or spores were sonicated in PCR
buffer as described above.
Preparation of rose DNA. Preliminary
results indicated that DNA extracted from
rose tissues using standard protocols for
plants (4,15) was not suitable for amplifi-
cation, presumably due to inhibition of Taq
polymerase by plant compounds. Success-
ful results were obtained with either a phe-
nol-chloroform extraction protocol opti-
mized for another rosaceous plant (17) or
with a modification of the manufacturer’s
protocol for the DNeasy Plant Mini Kit
(Qiagen, Inc.). The DNeasy kit was se-
lected for use in all tissue-assay extractions
due to its ease of use and quick processing
time. The procedure is based on the selec-
tive binding of the DNA to a silica gel
membrane in the presence of high con-
centrations of chaotropic salt and then
elution of the DNA off of the membrane by
a low-salt buffer. Fresh rose tissue (100 to
200 mg) was ground to a powder in a mor-
tar and pestle with liquid nitrogen. These
powdered samples were stored in cryo-
genic vials at –80°C until further proc-
essing. The major modifications to the
manufacturer’s extraction protocol were
the replacement of the supplied lysis buffer
and elution buffer. For the lysis step, the
buffer contained 2.5% cetyltrimethyl-
ammonium bromide (CTAB), 1% polyvi-
nyl pyrrolidone (PVP-40), 1.4 M NaCl, 50
mM EDTA, and 100 mM Tris-HCl (pH
8.0). Just prior to use, 0.5% 2-mercap-
toethanol was added to the CTAB lysis
buffer. For the elution step, the elution
buffer provided was replaced with 10 mM
Tris-HCl at pH 9.0. The elution step was
conducted twice with 100 µl of buffer each
time for a final elution volume of 200 µl.
Reactions including the specific primers
were carried out as described above for
ITS-PCR. To determine the number and
size of amplified DNA fragments, 10 µl of
each reaction was examined by electropho-
resis in 1.5% agarose gels in 0.5% Tris-
borate EDTA buffer. Gels were stained
with ethidium bromide and DNA was visu-
alized with UV light. Any samples that
tested negative with the specific primers
were retested with the conserved primers
to determine if the DNA was suitable for
amplification.
PCR reactions sometimes were inhibited
by rose tissue; therefore, optimization of
the protocol was conducted with various
concentrations (0.1, 0.2, 0.3, 0.4, and
0.5%) of nonfat dry milk (Difco Laborato-
ries, Detroit) added to the reaction mixture
to neutralize inhibitory compunds (3). The
sensitivity of the PCR assay was evaluated
by spiking DNA extracted from healthy
rose cortex tissue (4 ng of DNA per reac-
tion) with various dilutions of P. sparsa
DNA (ranging from 0.4 to 200 pg of DNA
per reaction). For this purpose, concen-
trated suspensions of P. sparsa sporangia
were processed with the DNeasy Plant
Mini Kit (Qiagen Inc.). The DNA was
quantified visually by staining with
ethidium bromide and comparing the in-
tensity with DNA standards of known con-
centration.
PCR assay of field samples. In January
and February 1999, rootstock cuttings of
cvs. Manetti and Dr. Huey were selected
randomly from eight recently planted com-
mercial fields. Fifteeen cuttings per field
were selected and the emerging vegetative
buds were removed and processed sepa-
rately from the cane. Each cane was proc-
essed through an electric pencil sharpener
which was cleaned between samples. For
each cane, the resulting fragments were
mixed and two 200-mg samples each were
ground further in liquid nitrogen for
extraction of DNA as described above.
DNA also was extracted from buds (typi-
cally two per cutting) ground in liquid
nitrogen. Therefore, for each cutting, three
assays were performed; one on DNA from
buds and two on DNA from the cane.
In December 1999, samples of crown
tissue were collected from 20 mother
plants. These plants were selected due to
their location in a section of a block that
had severe foliar downy mildew symptoms
in the spring of 1998. Cortex discs (col-
lected with a 1-cm cork borer) from two
plants were combined and chilled over-
night on ice. These 10 samples subse-
quently were ground in liquid nitrogen and
DNA was extracted as above. Samples
from 16 additional crowns were obtained
in February 2000 and processed individu-
ally.
Root samples also were collected from
miniature roses from a commercial nursery
that had a downy mildew outbreak. From
each of four plants sampled, 200 mg of
crown tissue and two 200-mg samples of
root tissue were collected and processed as
described above.
Preparation of samples for micros-
copy. Rose stem tissues were collected
from symptomatic field-grown plants and
inoculated greenhouse plants and stored in
a solution of formaldehyde-alcohol-acetic
acid immediately after collection (8). Tis-
sue was prepared for embedding with par-
affin using a Leica TP 1020 Automatic
Tissue Processor (Leica, Deerfield, IL).
The material was dehydrated in a series of
ethanol solutions at 10% increments from
50 to 80% for 1 h each. Samples then were
Plant Disease / December 2002 1365
transferred to 1% Eosin in 95% ethanol for
1 h followed by 2 h in 100% ethanol. The
ethanol series was followed by 2 h in
Hemo-De (Fisher Scientific Co., Pitts-
burgh, PA), 4 h in Hemo-De with paraffin
chips, and 11 h in melted paraffin at 60°C.
All steps were performed under vacuum
with vertical agitation to assist infiltration.
The material was embedded in paraffin
blocks using a Leica EG1160 Histo Em-
bedder, sectioned with a Microm HM340E
rotary microtome (Carl Zeiss, Inc., Thorn-
wood, NY) at 10 and 12 µm, and placed
onto glass slides by floating them in a
42°C water bath. The slides then were
placed on a slide warmer at 45°C for 24 h.
The staining procedure was initiated by
removing the paraffin by immersion in two
washes of 100% Hemo-De and one wash
of 50% Hemo-De in ethanol. Sections then
were hydrated in a four-step ethanol series
from 100 to 50% ethanol. Hydrated sec-
tions then were stained in 1% Aniline Blue
(C.I. 42755) in 0.067 M K2HPO4 (7). Sec-
tions were either mounted in water and
examined immediately or dehydrated
through an ethanol series and mounted in
Permount (Fisher Scientific). Specimens
were examined with a Microphot-SA mi-
croscope equipped for epifluorescence
microscopy and differential interference
contrast microscopy (Nikon Inc., Melville,
NY). Colonization of tissues was diag-
nosed by the observation of oospores or the
characteristic haustoria (6,25).
Evaluation of preplant treatments of
propagation materials. Trials were con-
ducted to evaluate fungicidal or hot-water
dips of dormant cuttings for the control of
downy mildew. Four trials were conducted
in commercial rose nurseries in the vicinity
of Wasco, CA and, in 1999 to 2000, one
trial was replicated on the UC Davis cam-
pus. In commercial fields, normal grower
practices were used with the exception that
standard fungicide sprays were not applied.
All trials were conducted with the root-
stock cv. Manetti’s Rose due to its high
susceptibility to downy mildew. The trial
fields were established with cuttings that
had been treated with bleach (5 min in
0.5% sodium hypochlorite) and rooting
hormone (0.3% indole 3-butyric acid),
which are standard industry practices.
Hardwood rootstock cuttings measured
about 30 cm in length with all but two buds
per cutting removed. These cuttings subse-
quently were stored at 4°C for up to 4
weeks before planting in November. The
dormant cuttings were treated immediately
prior to planting by submersion in fungi-
cide solutions for 10 min. Hot-water treat-
ments varied in temperature and duration
(Tables 1 and 2). Spacing between the
planted cuttings was 15 cm within the row
and 103 cm between rows (except in 1997,
when the cuttings were planted in double
rows with 103 cm between bed centers).
The soil in the Wasco area was a sandy
loam, while the soil in Davis was a fine-
silty loam. An experimental unit (plot) was
a single bed 6.1 m long. Nontreated buffer
rows were included between treated rows.
Trials were conducted five times over 3
years, although not all treatments were
included in all trials. Except where noted,
all trials contained five replications ar-
ranged in a randomized complete block
design.
The following fungicides were used in
these studies: azoxystrobin (Quadris
2.08SC, 249.3 g a.i. liter–1; Syngenta Crop
Protection, Greensboro, NC), dimetho-
morph (Acrobat 50WP, 50% a.i.; BASF,
Research Triangle Park, NC), fosetyl-Al
(Aliette 80WDG, 80% a.i.; Aventis Crop-
Science, Research Triangle Park, NC),
mefenoxam (Ridomil Gold WP, 44% a.i.;
Syngenta), and metalaxyl (Ridomil 2E,
239.7 g a.i. liter–1, Syngenta). Rates are
provided in Tables 1 and 2.
In all trials, disease assessments were
made two times per week through the
spring until all plants were infected or until
symptoms stopped appearing. Disease
incidence was defined as the proportion of
plants with downy mildew symptoms.
From these measurements, the area under
the disease progress curve (AUDPC) was
calculated as described by Shaner and
Finney (18). The AUDPC calculations
were based on disease incidence over a 75-
day period in 1998 (27 January to 10 April)
and a 29-day period in 2000 (6 March to 3
April). In 1999 and 2000, a subset of the
plants within each plot was harvested at
budding and oven dried for weight meas-
urements.
Statistical analyses. Analysis of vari-
ance (ANOVA) was used to evaluate dif-
ferences in disease incidence, dry weight,
and shoot length between treatments.
Analyses were conducted with the general
linear models (GLM) procedure of SAS
(version 6.12; SAS Institute Inc., Cary,
NC). Treatment means were compared by
Fisher’s least significant difference (LSD)
test at P = 0.05. Due to differences in
treatments and experimental design be-
tween trials, data from each trial were
analyzed separately.
RESULTS
PCR with conserved primers. The
primers ITS4 and ITS5 directed amplifica-
tion of a roughly 820-bp fragment from
DNA extracted from sporangia of P.
sparsa. Two products were detected from
DNA from symptomatic rose tissues, one
the same size as that from sporangial
DNA, and a second, smaller product. This
smaller fragment was the same size as that
produced by amplification of DNA ex-
tracted from healthy rose tissue, and is
presumably due to amplification of rose
Tab l e 1 . Effect of preplant fungicidal and hot-water dips on incidence of downy mildew in rose
nurseries, Kern County, CA
AUDPCy
Treatmentz 1998 trial 2000 trial 1 2000 trial 2
Control 2,217.2 a 111.5 a 48.1 a
Azoxystrobin (10,000 mg a.i./liter) 2,038.3 a … …
Azoxystrobin (1,000 mg a.i./liter) … 45.4 c …
Dimethomorph (1,000 mg
a.i./liter)
… 55.4 bc …
Fosetyl-Al (10,000 mg a.i./liter) 1,987.9 a 55.7 bc …
Hot water (44°C for 15 min) … 44.2 c …
Hot water (46°C for 10 min) … 107.9 ab …
Mefenoxam (100 mg a.i./liter) … 36.5 c …
Mefenoxam (500 mg a.i./liter) … 40.0 c 17.7 b
Mefenoxam (1,000 mg a.i./liter) … 26.3 c …
Metalaxyl (10,000 mg a.i./liter) 783.0 b … …
Metalaxyl (5,000 mg a.i./liter) 662.6 b … …
LSD, P = 0.05 501.0 54.6 24.5
y Area under the disease progress curve calculated from twice-weekly assessments of the percentage
of plants with downy mildew symptoms during the periods 27 January to 10 April 1998, and 6
March to 3 April 2000. Means in the same column followed by a common letter are not statistically
different (P = 0.05) according to Fisher’s least significant difference (LSD) test. Values are means
of four replications (1998 trial) or five replications (2000 trials).
z Cuttings of rootstock ‘Manetti’ were submerged for 10 min (unless otherwise noted) immediately
prior to planting.
Tab l e 2 . Effect of preplant fungicidal or hot-
water dips on rose shoot dry weight at four
months after bud break, 1999
Treatmentx
Rate
(mg a.i./liter)
Dry weighty
(g)
Control 0 164.0 a
Fosetyl-Al 10,000 164.0 a
Azoxystrobin 1,000 148.0 ab
Metalaxyl 100 136.0 ab
Dimethomorph 1,000 128.0 bc
Metalaxyl 1,000 128.0 bc
Metalaxyl 10,000 104.0 cd
Hot waterz … 92.0 d
LSD, P = 0.05 … 30.1
x Cuttings of rootstock ‘Manetti’ were sub-
merged for 10 min (unless otherwise noted)
immediately prior to planting.
y Means followed by a common letter are not
statistically different (P = 0.05) according to
Fisher’s least significant difference (LSD)
test. Values are means of five replications.
z Temperature of 46 to 47°C for 15 min.
1366 Plant Disease / Vol. 86 No. 12
rDNA. Primers ITS4 and ITS5 also di-
rected amplification of a roughly 820-bp
fragment from DNA of Phytophthora cac-
torum. In reactions containing DNA of
Botrytis cinerea and Phragmidium sp., the
amplified fragments were smaller and
larger, respectively, than those amplified
from the oomycetes Peronospora and Phy-
tophthora. Reliable amplification of DNA
extracted from rose tissue required the
inclusion of 0.4% nonfat milk in the PCR
mix.
PCR with specific primers. From the
ITS sequences of four isolates of P.
sparsa, a forward primer (PS3, 5
ATTTTGTGCTGGCTGGC 3) and a re-
verse primer (PS1, 5 TGCCACACGAC-
CGAAGC 3) were designed. These prim-
ers reproducibly directed amplification of a
660-bp fragment from extracts from spo-
rangia of all 15 tested isolates of P. sparsa.
These isolates included 10 from field-
grown roses in Kern County; 1 from a rose
greenhouse in Contra Costa County, CA; 3
from wholesale ornamental nurseries in
Solano County, CA, and in Oregon; and 1
from commercial caneberries in Santa Cruz
County. These primers also directed ampli-
fication of the same size fragment from
100% of DNA extracts from symptomatic
rose leaf and stem cortex samples (n = 64).
No PCR products were detected from reac-
tions containing DNA extracted from
healthy rose tissue (n = 11) or from reac-
tions with DNA from other rose pathogens,
including Phytophthora cactorum, B. cine-
rea, and Phragmidium sp. (two or three
isolates of each species). In reactions con-
taining 4 ng of DNA extracted from
healthy rose cortex, the detection limit was
2 pg of P. sparsa DNA.
PCR assay of field samples. Positive
PCR results never were obtained from
DNA extracted from rootstock cuttings
collected from recently established com-
mercial production fields (n = 92 cuttings).
However, the Peronospora-specific PCR
product was detected in 17% of the sam-
ples (n = 26) of crown of asymptomatic
rootstock mother plants which exhibited
foliar symptoms the previous season. In
addition, 73% of root samples from symp-
tomatic miniature rose plants were positive
(n = 22) for P. sparsa and all four crowns
tested positive.
The specific primers always directed am-
plification of the 660-bp Peronospora frag-
ment from DNA extracts of stem epidermal
and cortex tissues from inoculated, sympto-
matic canes (n = 14), but produced PCR
products less consistently from extracts of
pith plus vascular tissues (5 out of 15).
Microscopy. The aniline-blue staining
method allowed clear differentiation of
pathogen structures from plant tissue when
viewed with epifluorescent microscopy
(Fig. 1). Mycelia and diagnostic haustoria
were always visible within symptomatic
canes. The mycelium was located intercel-
lularly between epidermal and cortex pa-
renchyma cells. The pathogen never was
observed by microscopy within vegetative
buds collected from rootstock cuttings (n =
27 buds).
Oospores formed profusely in the meso-
phyll of symptomatic leaves and the cortex
parenchyma of stems from both field and
artificially inoculated plants (Fig. 1). Inter-
estingly, abundant oospores formed in
leaves inoculated with either of two single-
lesion isolates, suggesting that P. sparsa
may be homothallic (B. Aegerter and R.
Davis, unpublished).
Attempts to examine crown and root tis-
sues by microscopy were not successful
due to the failure of the paraffin to fully
penetrate these lignified tissues.
1998 field trial. Bud break occurred in
January and downy mildew appeared im-
mediately thereafter and was severe in
many fields in Kern County through mid-
May. There was a positive correlation be-
tween the variances and the means of the
AUDPC of the treatments; thus, the data
were log transformed prior to analysis.
However, the results of the statistical tests
were comparable between transformed and
nontransformed data; therefore, the non-
transformed data and analysis are pre-
sented (Table 1). There were significant
differences in the AUDPC between treat-
ments (P < 0.0001). Compared with the
nontreated control, metalaxyl applied at
5,000 and 10,000 mg a.i./liter reduced the
AUDPC by 70 and 65%, respectively,
whereas azoxystrobin and fosetyl-Al did
not significantly affect disease incidence
(Table 1). No phytotoxicity symptoms or
growth reductions were observed.
1999 field trials. Downy mildew did not
occur in any commercial rose fields in
Kern County during the spring of 1999;
therefore, disease severity data were not
collected. However, 4 months after bud
break, a subset of plants from each plot
was harvested to evaluate the visually ob-
servable variation in growth between plots.
Subsequent analysis of dry weight meas-
urements indicated that this variation was
attributable to a treatment effect (P =
0.0002). The hot-water dip, dimethomorph,
and the two higher rates of metalaxyl
(1,000 and 10,000 mg a.i./liter) reduced
dry weight over the control treatment (Ta-
ble 2). The hot-water dip also reduced the
stand establishment by 65% compared with
the control plots (P = 0.0001), whereas no
other treatments had a significant effect on
stand (data not presented). Azoxystrobin,
fosetyl-Al, and metalaxyl at 100 mg
a.i./liter had no effect on dry weight (Table
2). Shoot length at 2 months after bud
break also varied between treatments (P =
0.01); fosetyl-Al significantly increased
shoot length, whereas the other treatments
had no effect (data not presented).
2000 field trials. In Kern County,
downy mildew did not occur at bud break
in February but did appear from the second
week of March through the first week of
April. There were significant differences in
AUDPC between treatments (P = 0.025).
Mefenoxam (the active enantiomer con-
tained in metalaxyl), hot water (44°C for
15 min), azoxystrobin, dimethomorph, and
fosetyl-Al reduced disease incidence rela-
tive to the control (Table 1). The shorter
hot-water dip (46°C for 10 min) had no
effect on disease (Table 1). Shoot weights
were not significantly different between
treatments (P = 0.258), but trends were
similar to what was observed in 1999 (i.e.,
fosetyl-Al resulted in the most growth and
mefenoxam at 1,000 mg a.i./liter resulted in
the least growth; data not presented). In a
replication of this trial conducted at UC
Davis during the same time period, no
disease occurred and there were no differ-
ences in dry weights between treatments (P
= 0.93, data not presented). In a third trial
conducted in Wasco, which included a
variety of control measures, a preplant dip
with mefenoxam at 500 mg a.i./liter re-
duced disease by 63% (P = 0.026, Table 1).
Fig. 1. Peronospora sparsa within rose stem cortex tissue; haustorium (left) viewed with epifluores-
cent microscopy (×600 magnification) and oospores (right) viewed with differential interference con-
trast microscopy (×200 magnification).
Plant Disease / December 2002 1367
DISCUSSION
The PCR-based technique developed
here for the detection of P. sparsa has
commercial application in the detection of
the pathogen in propagation materials or
harvested plants. In this study, the tech-
nique was used to reveal the pathogen
within rose stems, crowns, and roots, in-
cluding the crowns of symptomless root-
stock mother plants that were highly ligni-
fied. A major challenge in the development
of a reliable PCR assay for roses was the
inhibition of the reaction by plant com-
pounds. This was presumably due to in-
hibitors of Taq polymerase activity such as
tannins, polyphenols, and polysaccharides
that tend to cause problems of this nature
when working with plants in the family
Rosaceae (17). In our experiments, reliable
DNA amplification was obtained through
use of a silica-gel membrane extraction
method with a CTAB extraction buffer
containing high salt concentrations and
PVP. Finally, addition of nonfat milk to the
reaction mixture improved amplification.
The mechanism by which the milk attenu-
ates the inhibition is not known (3).
The results of the PCR assay and micro-
scopic observations confirm previous re-
ports that P. sparsa colonizes stem cortex
tissue (14). However, this is the first report
of P. sparsa occurring in crown and root
tissues of rose. The less consistent occur-
rence of P. sparsa in vascular tissues sug-
gests that the pathogen must be colonizing
the crown by mycelial growth through the
stem cortex.
The role of oospores in the disease cycle
of rose downy mildew is unknown. Baker
(1) observed oospores in rose leaves but
found their occurrence only sporadic. Al-
though we found numerous oospores in
both leaves and stems, epidemiological
importance does not necessarily follow.
Mycelium is formed abundantly in the
stem and the host is perennial and vegeta-
tively propagated; therefore, the pathogen
likely need not rely upon survival of its
oospores. As a sexual spore, they might be
the product of out-crossing and hence a
source of genetic variation. However, pre-
liminary results from inoculations with two
single-lesion isolates suggest that P. sparsa
may be homothallic.
Perennating crown infections previously
have been demonstrated in downy mildews
of other crops. The hop downy mildew
pathogen, Pseudoperonospora humuli,
overwinters as mycelium in the perennial
crown of hop plants (19). Crowns become
infected by mycelial growth of the patho-
gen downward from systemically infected
spikes or by zoospores that infect stem
bases (19). Such infections have been
demonstrated to be a source of inoculum
for epidemics the following season (9). In
onion, mycelium of Peronospora destruc-
tor is reported to survive within bulbs (16).
Using microscopy, Tate (20) demonstrated
what he called “systemic” cortex infection
of boysenberry (Rubus loganobaccus) by
P. sparsa. In this host, the pathogen seems
to overwinter in the crown tissue and
grows along with the emerging canes the
following season. Tate (20) suggested that
the practice of taking cuttings from pro-
duction blocks ensured disease transmis-
sion into new plants and production areas.
In arctic bramble (R. arcticus) cultivated in
Finland, downy mildew caused by P.
sparsa was observed on new growth from
roots collected from commercial fields and
planted in a growth chamber (12). In roses,
it is possible that transmission of the
pathogen to new growth from infected
crowns or canes may likewise occur.
The use of systemic fungicides to de-
crease survival of downy mildews in per-
ennial, woody plant parts previously has
been demonstrated in hops (9) and cane-
berries (21). In hops, controlling downy
mildew with metalaxyl in one year reduced
the number of systemically infected shoots
(primary infections) the following spring,
while spraying metalaxyl over new shoots
emerging from systemically infected
crowns reduced secondary infections (9).
In caneberries, metalaxyl soil drenches
conducted at transplanting reduced inci-
dence of downy mildew on suckers arising
from infected crowns (21). Preplant dips of
infected crowns in metalaxyl were not
effective in this system (21).
In our studies, preplant dips of dormant
rose cuttings in metalaxyl effectively re-
duced incidence of downy mildew on
shoots emerging from these cuttings. Al-
though the higher concentrations of
metalaxyl (1,000 mg a.i./liter or higher)
reduced subsequent growth from the cut-
tings, lower concentrations (100 and 500
mg a.i./liter mefenoxam) were not phyto-
toxic and effectively controlled downy
mildew. Metalaxyl and mefenoxam are
closely related; the former is a racemic
mixture of active and inactive enantiomers
while the later contains only the active
enantiomer. Fosetyl-Al was not effective
under high disease pressure but was effec-
tive under moderately disease-conducive
conditions; further testing is needed. The
efficacy of dimethomorph, azoxystrobin,
and hot-water dips should be evaluated in
future trials. A hot-water dip, which could
avoid the pesticide registration process,
would be particularly useful if the tempera-
ture and duration could be optimized to
maximize downy mildew control and
minimize damage to the plant. In studies
on the control of nematodes with hot-water
treatment, damage to rose roots was re-
duced by prior storage of the dormant
plants at 38°C for 24 h to induce heat-
hardening (22).
Success in controlling downy mildew by
dipping rose cuttings in fungicides or hot
water supports our conclusion that P.
sparsa overwinters in woody tissue. Pre-
plant dip treatments may delay the start of
a mildew outbreak or reduce its severity,
presumably by reducing the number of
perennating infections serving as initial
inoculum (23). A more economical solu-
tion, in the long term, may be to reduce
incidence of the pathogen in propagation
materials by chemically treating or remov-
ing infected rootstock mother plants. Plants
could be tested by PCR and eliminated if
positive. This approach, which has worked
well in reducing the transmission of vi-
ruses in roses, would be successful if sec-
ondary spread of the pathogen was limited.
ACKNOWLEDGMENTS
We thank the Garden Rose Council for finan-
cial support, M. Rademacher, S. Riley, and M.
Williams for technical assistance, and P. Guzman
for helpful advice.
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