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Applied and Environmental Microbiology, August 2000, p. 3277-3282, Vol. 66, No. 8
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Production and Characterization of Two Monoclonal
Antibodies Specific for Plasmopara halstedii
S.
Bouterige,1
R.
Robert,1,*
J. P.
Bouchara,1
A.
Marot-Leblond,1
V.
Molinero,2 and
J.
M.
Senet1
Groupe d'Etude des Interactions
Hôte-Parasite, Laboratoire de Parasitologie-Mycologie,
Faculté de Pharmacie, 49100 Angers,1
and Groupe d'Etude des Variétés et des Semences,
49071 Beaucouzé Cedex,2 France
Received 29 November 1999/Accepted 20 May 2000
 |
ABSTRACT |
Sunflower downy mildew, caused by the fungus Plasmopara
halstedii, is a potentially devastating disease. We produced two
monoclonal antibodies (MAbs) (12C9 and 18E2) by immunizing mice with a
partially purified extract of P. halstedii race 1. Both
MAbs detected in enzyme-linked immunosorbent assay (ELISA) all races of
P. halstedii present in France. No cross-reactions were
observed with Plasmopara viticola or with other fungi
commonly associated with sunflowers. Both MAbs recognized the same
three fungal antigens with molecular masses of 68, 140, and 192 kDa.
However, the epitopes on the fungal antigens were distinct and
repetitive. Seed homogenates from infected plants were incubated in
wells coated with MAb 18E2. This resulted in the trapping of P. halstedii antigens that were identified with biotinylated MAb
12C9. No reactions were seen with seed homogenates from healthy plants.
Thus, our results suggest that these MAbs might be used to develop a
sandwich ELISA detection system for P. halstedii in
infected seeds.
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INTRODUCTION |
Downy mildew caused by
Plasmopara halstedii (Farlow) Berlese et De Tony is one of
the economically most important diseases of sunflowers,
Helianthus annuus L. The fungus, which is an obligate parasite of sunflowers, occurs in all areas where sunflowers are cultivated extensively except Australia, South Africa, and possibly parts of North Africa (21, 23). Systemic downy mildew
infection alters the development of vegetative and generative parts of
the plant, as well as its metabolism (26, 32). Inoculation
of sunflower plants with P. halstedii at the two-leaf stage
through apical buds greatly inhibits stem elongation. The yield of
infected plants is usually less than 25% that of uninfected plants.
There is no fungicide to control this disease after infection has occurred.
Little information is available on the epidemiology and biochemistry of
P. halstedii or its relationship with its sunflower host.
Seven physiological races of P. halstedii have been
identified (10, 11, 24), and they are capable of attacking a
wide range of sunflower genotypes. In France, in addition to the three
races
1, A (equivalent to American race 4), and B (equivalent to
American race 3)classically encountered (19), two new
races, designated C and D, have been detected recently (13).
In the Red River Valley of North Dakota, Minnesota, and Manitoba, all
but race 1 have been identified (24). Some sunflower
varieties carry resistance genes (Pl) against P. halstedii races present in France and in the United States
(15, 17, 18, 20, 28). Genetic variation in the pathogen
appears limited, since no random amplification of polymorphic DNA
variation was found among isolates from races 1, A, and B or between
isolates of the same race, and very few (89% similarity) polymorphisms
were identified among all races of P. halstedii present in
France (22).
Downy mildew of sunflowers may result from oospores in the soil
(6). Contamination of seeds by P. halstedii has
also been implicated in the establishment of the disease
(6). The only effective control technique for
mildew-sensitive sunflower varieties is to treat seeds with metalaxyl
(Apron 35SD) (1). However, metalaxyl-resistant isolates of
P. halstedii have been described (1), and
laboratory tests of the effectiveness of fungicide treatment of the
seeds showed a decreased sensitivity of the fungus to the drug
(13). In this context, there is a greater need for the
development of efficient methods to detect P. halstedii. Our objective in this study was to develop a monoclonal antibody (MAb) that
recognized all of the races of P. halstedii present in France.
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MATERIALS AND METHODS |
Microorganisms and culture conditions.
We used one isolate
of each of the five races of P. halstedii present in France.
Isolates were maintained by Groupe d'Etude des Variétés et
des Semences (Angers, France) or Institut de Recherche Agronomique
(Clermont-Ferrand, France). Races 1, C, and D were maintained on
sunflower line HA89 (Peredovick variety), and races A and B were
maintained on hybrid GH RHA266 (Pharaon variety), which contains the
gene Pl1 and is resistant to race 1. Artificial infections
were made by immersing whole seedlings as described by Cohen and
Sackston (3).
Preparation of crude fungal extracts.
Zoosporangia and
hyphae of P. halstedii were collected by scraping 150 contaminated cotyledons with a paintbrush in 50 ml of distilled water.
The fungal suspension was sonicated for three periods of 5 min each
using a Vibra Cell sonicator (Bioblock Scientific, Illkirch, France)
(power, 24 W) and centrifuged at 12,000 × g for 10 min. The resulting supernatant, which corresponds to the crude fungal
extract, was stored as 2-ml aliquots at 
20°C until use.
For the preparation of MAbs, the crude extract of P. halstedii race 1 was partially purified by fractionated ammonium
sulfate precipitation. Saturated ammonium sulfate was added to the
crude extract to a final concentration of 0.65 M. The solution was
incubated for 15 min at room temperature before being centrifuged
(12,000 × g; 10 min). Ammonium sulfate was added to
the resulting supernatant to a final concentration of 2 M. After
incubation for 15 min and centrifugation as described above, the pellet
was resuspended in distilled water and dialyzed for 24 h at 4°C
against 200 volumes of distilled water.
Crude extracts of
Plasmopara viticola, cultivated on vine
leaves, and of other fungi (Table
1)
potentially encountered on
sunflower seeds as saprophytes or pathogens,
cultivated on malt
agar medium, were also prepared by sonication.
The total protein content of the extracts was determined by the method
of Bradford (
2), using bovine serum albumin (BSA)
as a
standard.
Germination of zoosporangia.
Zoosporangia were isolated by
scraping 10 contaminated cotyledons with a paintbrush in 50 ml of a 1%
saccharose solution. Germination (release of zoospores and germ tube
formation) followed incubation of the suspension of zoosporangia (about
5 × 106 per ml) at 16 to 18°C with gentle shaking.
The first zoospores released under these conditions appeared 2 h
after the beginning of the incubation, and most had initiated germ tube
formation after 5 h (4).
Immunization of mice.
MAbs were prepared from 8-week-old
female BALB/c mice (Iffa-Credo, L'Arbresle, France) which received
either three weekly subcutaneous injections or three intraperitoneal
injections. The first injection consisted of 200 µl of a 1:1 mix of
partially purified P. halstedii extract (450 µg of protein
per ml) and complete Freund's adjuvant (Sigma Chemical Co., St. Louis,
Mo.). Incomplete Freund's adjuvant was used for the two subsequent
boosters. Twelve days after the last booster, blood samples were tested
by immunoblotting. An additional injection of antigen was given
intravenously 3 days before the mice were euthanized for removal of
their spleens.
Hybridoma production.
Murine plasmocytoma cells, X63/Ag
8.653, were grown in RPMI 1640 medium (Gibco Laboratories, Grand
Island, N.Y.) containing 15% fetal calf serum, 2 mM glutamine, 1 mg of
ampicillin/ml, and 0.1 mg of gentamicin/ml. Cell fusion and selection
of hybrids were performed essentially as described by Dippold et al.
(5) with minor modifications. Ten days after fusion, culture
supernatants from wells with growing hybridomas were screened by
enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence,
and immunoblotting for the production of antibodies directed against P. halstedii race 1. Positive hybrids were subcloned twice
by limiting dilution and stored in liquid nitrogen. MAbs were obtained either from hybridoma cultures or from ascites fluid. Isotypes were
determined by ELISA with anti-isotype antibodies (Caltag Laboratories,
Burlingame, Calif.).
Purification and labeling of MAbs.
MAbs were purified by
ion-exchange chromatography on a DEAE-Sepharose (Pharmacia-Biotech, St.
Quentin, France) column in 25 mM Tris buffer (pH 8.8) containing 35 mM
NaCl. Ascites fluids previously equilibrated in the same buffer were
applied to the column at a flow rate of 2 ml/min. The MAbs were eluted
using 25 mM Tris buffer (pH 8.8) containing 75 mM NaCl, and the eluate was collected in 4-ml fractions. Labeling of the purified MAbs was performed essentially as described by Guesdon et al.
(8).
ELISA.
Ninety-six-well flat-bottom microtiter plates were
coated with crude fungal extract diluted in 50 mM carbonate buffer (pH 9.6) (25 µg of protein per ml; 100 µl per well). After a 1-h
incubation at 37°C, followed by three washes in 0.15 M
phosphate-buffered saline (PBS) (pH 7.2) containing 0.05% (vol/vol)
Tween 20 (PBST), 200 µl of a 3% BSA solution in PBS were added to
each well. The plates were incubated for 2 h at 37°C or
overnight at 4°C, washed three times in PBST, and incubated for
1 h at 37°C with the pooled mouse immune sera diluted 1:400 or
with undiluted culture supernatants (100 µl per well). After three
additional washes in PBST, 100 µl of alkaline phosphatase-conjugated
goat anti-mouse immunoglobulin G (IgG) 
chain antibodies (Caltag
Laboratories) diluted 1:2,000 in PBS was added to each well. The plates
were incubated for 1 h at 37°C and washed three times in PBST.
p-Nitrophenyl phosphate (1 mg/ml in 1 M diethanolamine
buffer [pH 9.8]) was used as a chromogen. The reaction was stopped
after a 30-min incubation at room temperature by the addition of 3 N
NaOH, and the absorbance at 405 nm was determined on a Titertek
multiscan (Labsystem, Les Ulis, France). All tests were performed in
triplicate. The controls were uncoated wells and the incubation of
coated wells with PBS instead of the immune sera or culture supernatants.
Indirect immunofluorescence.
Zoosporangia and hyphae
obtained by scraping cotyledons in distilled water and zoospores and
germ tubes initiated by germination of zoosporangia were pelleted by
centrifugation for 10 min at 180 × g, and the pellets
were resuspended in 200 µl of undiluted hybridoma culture
supernatants. After incubation for 1 h at 37°C, followed by
washing in PBS, the fungal elements were incubated for 1 h at
37°C in 200 µl of a 1:100 dilution in PBS of fluorescein isothiocyanate-conjugated goat anti-mouse IgG chain antibodies (Caltag Laboratories). After being washed, 20 µl of the fungal suspensions was dropped on glass slides, and the preparations were
examined with a Nikon microscope equipped with epifluorescence. The
specificity of the labeling was assessed by incubating the fungal
elements in PBS instead of the culture supernatants.
Characterization of the antigens.
Titration curves of the
MAbs were established by ELISA on wells coated with crude extract of
P. halstedii race 1. A concentration of the biotinylated MAb
which gives about 50% binding (2 to 4 µg/ml for MAb 18E2 and 0.5 to
1 µg/ml for MAb 12C9) was used for competition experiments in
combination with an equal concentration or a 2-, 10-, or 100-fold
excess of the unbiotinylated MAb.
To demonstrate that the epitopes were repetitive, we used a procedure
derived from that of Theolis and Breckenridge (
29).
Plates
were coated with purified MAbs (20 µg/ml for MAb 12C9 or
10 µg/ml
for MAb 18E2) and incubated successively with (i) crude
extract of
P. halstedii race 1, (ii) an appropriate dilution of
the
same biotinylated MAb corresponding to the minimum concentration
that
gives 100% binding (6 µg/ml for MAb 12C9 and 24 µg/ml for
MAb
18E2), and (iii) streptavidin-labeled alkaline phosphatase
polymer
(Sigma) diluted 1:500 in distilled water. All incubation
steps were
carried out at 37°C for 1 h and followed by three washes
in
PBST.
p-Nitrophenyl phosphate was used for color development
as described
above.
The antigen molecular mass was determined by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western
blotting. Samples were dissolved in 62.5 mM Tris-HCl buffer (pH
6.8)
containing 2% SDS, loaded on a 5 to 15% gradient polyacrylamide
slab
gel, and electrophoresed according to the method of Laemmli
(
12). After electrophoresis, the gels were stained with
Coomassie
brilliant blue or transferred electrophoretically to
0.45-µm-pore-size
Immobilon membranes (Millipore Corp., Bedford,
Mass.) as described
by Towbin et al. (
30). The blots were
saturated overnight at
4°C in 10% nonfat dry milk in PBS, washed in
PBS, and incubated
for 1 h with a 1:50 dilution of the mouse
immune sera in PBS or
with undiluted culture supernatants. After being
washed, the membranes
were incubated for 1 h with alkaline
phosphatase-conjugated goat
anti-mouse IgG
chain antibodies diluted
1:300 in PBS. Finally,
bands were detected by using the nitroblue
tetrazolium and 5-bromo-4-chloro-3-indolylphosphate
substrate system.
The molecular masses of the antigens were calculated
from the migration
of molecular mass standards (Sigma): myosin,
205 kDa;
-galactosidase, 116 kDa; phosphorylase b, 97.4 kDa;
BSA, 67 kDa;
ovalbumin, 45 kDa; and carbonic anhydrase, 29
kDa.
Antigen sensitivity to proteolytic enzymes or chemical agents was also
investigated by Western blotting and ELISA. The effects
of
2-mercaptoethanol were determined by the addition of 5%
2-mercaptoethanol
to the fungal extract prior to electrophoresis and
Western blotting.
Antigenic extract of
P. halstedii race 1 immobilized on Immobilon
sheets or microtiter plates was also incubated
at 37°C for 1 h
with either pronase E (2.5 mg/ml) or proteinase
K (0.16 mg/ml)
diluted in PBS (pH 7.6). After the plates or sheets were
washed
in PBS and incubated with nonfat dry milk, immunoreactivity with
the MAbs was determined. Periodate oxidation was performed by
incubating the immobilized antigens at room temperature in the
dark for
1 h with a 20 mM periodate solution in 50 mM acetate
buffer (pH
4.0) (
33). After the antigens were washed in PBST,
the
reaction was stopped by the addition of 1% glycine and incubation
for
30 min. The immobilized antigens were treated with the MAbs
as
described above. The control was treatment of the immobilized
antigens
with PBS or acetate buffer alone. ELISA results, which
correspond to
the means of triplicate determinations, are expressed
as the binding
(percent) relative to the control performed in
the absence of any
treatment of the
antigens.
ELISA detection of the fungus in seeds from infected plants.
Samples of seeds (2 g) collected from healthy or infected plants grown
at nine different locations in France were homogenized in liquid
nitrogen, sonicated in distilled water for three periods of 5 min each,
and centrifuged at 12,000 × g for 10 min. ELISAs were
performed by coating the plates with purified MAb 18E2 (10 µg/ml).
After saturation with BSA, the centrifugation supernatant from seed
homogenates was added to the coated wells (100 µl per well). The
presence of the fungus was detected with biotinylated purified MAb 12C9
(8 µg/ml; 100 µl per well). The control was incubation with PBS
instead of seed homogenates. The nonparametric Mann-Whitney test was
used for statistical evaluation of comparisons between infected and
noninfected seeds. A P value of 
0.01 was considered
statistically significant. To determine sensitivity, this ELISA was
performed with various concentrations of the crude fungal extract
instead of seed homogenates.
| |
RESULTS |
MAb isolation.
The fusions between X63/Ag 8.653 myeloma cells
and lymphocytes of BALB/c mice that had been immunized with the
partially purified extract of P. halstedii race 1 resulted
in 4,000 hybridomas. Of these, 313 produced antibodies directed towards
the fungus. However, only 16 remained positive after a second screening
performed by Western blotting. These 16 included 12C9 and 18E2, which
were IgG1 and IgG2b, respectively.
Immunofluorescence studies.
Immunofluorescence studies
performed on different morphological stages of the fungus revealed
similar binding patterns for the MAbs. As soon as zoosporangia began
differentiating, a faint fluorescence of their granular contents
appeared with both MAbs, but their surfaces were not labeled (Fig. 1a
and b). Extending the incubation time to
5 h resulted in production of germ tubes by almost all of the
zoospores. When immunofluorescence was performed with these later
developmental stages, the surfaces of the zoospores (Fig. 1a) and of
the mother cells of the germ tubes (Fig. 1c) were intensely labeled.
The hyphal part of the germ tubes did not stain (Fig. 1d), nor did
hyphae (data not shown).
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FIG. 1.
Visualization by indirect immunofluorescence of the
binding of MAb 12C9 to P. halstedii race 1 zoosporangia and
zoospores (a) or germ tubes (c). Note the faint fluorescence of the
granular contents of zoosporangia (a) and the intense staining of the
surfaces of zoospores (a; arrowhead) and of mother cells of germ tubes
(c). The surfaces of zoosporangia (b) and the hyphal walls of germ
tubes (d) visualized on the same fields by phase-contrast microscopy
were not labeled. Bars, 10 µm.
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|
Specificity of MAbs 12C9 and 18E2 for P. halstedii.
We
coated the plates with crude extracts from the different races of
P. halstedii present in France, P. viticola, a
related fungus that causes downy mildew of grapevine, and other molds potentially pathogenic to sunflowers (e.g., Alternaria
tenuis or Phomopsis sp.) or encountered on seeds of
sunflowers. All races of the fungus tested were recognized by both MAbs
(Table 1). The controls were negative, and no cross-reactions were
observed with P. viticola or with the other fungi tested.
Characterization of the antigens recognized by MAbs 12C9 and
18E2.
Titration curves (Fig. 2)
demonstrated the saturability of the binding and permitted the
determination of the minimal concentrations required for 50 and 100%
binding. For example, when plates were coated with crude extract of
P. halstedii race 1, a 0.5- to 1-µg/ml solution of
biotinylated MAb 12C9 was required for about 50% binding, whereas the
minimum concentration required to reach saturation was 6 µg/ml. These
concentrations were four times greater for MAb 18E2, suggesting that
the two MAbs differ in their affinity or their binding sites for the
crude fungal extract.
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FIG. 2.
Titration curve of biotinylated MAbs 12C9 and 18E2 by
ELISA. Crude extract of P. halstedii race 1 was used for
coating. The dotted lines correspond to the minimal concentrations of
the MAbs which give 100% binding, and the hatched areas indicate the
concentrations which give 50% binding.
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|
Competition experiments suggested that distinct epitopes were detected
by these MAbs on
P. halstedii antigens (Table
2).
Moreover, plates coated with an
unbiotinylated MAb trapped
P. halstedii antigens that could
be detected by the same, but biotinylated,
MAb, suggesting the presence
of repetitive epitopes for each MAb
on the fungal antigens (data not
shown).
From SDS-PAGE analysis of the crude extract of
P. halstedii
race 1, we identified about 15 polypeptides with molecular masses
ranging from 20 to 250 kDa (Fig.
3, lane
1). Three proteins, with
apparent
molecular masses of 68, 140, and 192 kDa, were identified
in Western
blots by using MAbs 12C9 or 18E2 as the probes (Fig.
3, lanes 2 and 3).
No bands were detected for either MAb when
electrophoresis was
performed under reducing conditions. Treatment
of blots of the crude
fungal extract with proteinase K or pronase
E resulted in complete loss
of immunoreactivity with MAb 12C9
or 18E2. Reduction of
immunoreactivity with MAb 12C9 was also
seen on blots treated with 20 mM periodate (Fig.
3, lane 5). Pretreatment
of blots with acetate
buffer alone (Fig.
3, lanes 6 and 8) or
with PBS had no detectable
effects. ELISA confirmed differences
in the susceptibilities of the
respective epitopes, since periodate
oxidation resulted in a 43%
reduction of the binding of MAb 12C9
while the binding of MAb 18E2 was
only slightly modified (Table
3).
Complete loss of binding was observed for both MAbs after
treatment of
the coated plates with proteolytic enzymes.
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FIG. 3.
Characterization of the antigens detected by MAbs 12C9
and 18E2. Lane 1, Coomassie blue staining of an SDS-PAGE gel of crude
extract of P. halstedii race 1 (80 µg of protein per
lane). Lanes 2 to 8, Western blot detection of the antigens recognized
by MAb 12C9 (lanes 2, 5, and 6) or 18E2 (lanes 3, 7, and 8) without
prior treatment of the crude extract (lanes 2 and 3) or after treatment
with 20 mM periodate (lanes 5 and 7). Controls consisted of omission of
the MAbs (lane 4) and incubation of the blots with acetate buffer
(lanes 6 and 8) before the addition of the MAb. The molecular masses
(in kilodaltons) of the protein standards are indicated on the left.
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TABLE 3.
Sensitivities of antigens detected by MAbs 12C9 and 18E2
to enzyme or chemical treatments as determined by ELISA
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|
Detection of the fungus in seeds from infected plants.
Since
both MAbs recognized distinct and repetitive epitopes on the same
antigens of P. halstedii, a sandwich ELISA system was
developed to detect the fungus in infected seeds. P. halstedii antigens were trapped with MAb 18E2 and detected with
biotinylated MAb 12C9 (Table 4). An
optical density (OD) value higher than 0.250 was obtained with all lots
of seeds from infected plants, but it did not exceed the control value
(0.115) when seed homogenates from healthy plants were used. The
difference between the results with infected and noninfected seeds was
highly significant (P < 0.01). The sensitivity of this
assay, evaluated with the crude fungal extract, was estimated to be 1 ng/ml.
| |
DISCUSSION |
P. halstedii causes extensive damage to sunflowers, and
the absence of efficient antifungal treatments has led to a search for
new varieties of sunflower resistant to the fungus. Several races of
P. halstedii have been identified (10, 11, 13, 19,
24), and some resistant sunflower hybrids have been produced (9, 16, 25), but the resistance is not durable. Moreover, metalaxyl is the only commercial antifungal agent available, and resistant strains of P. halstedii have been described
(1).
An alternative control strategy is a screen to detect infected seeds
and break the transmission of downy mildew by seeds by removing them.
ELISAs have been developed for downy mildew of peas caused by
Peronospora viciae f. sp. pisi (7),
Verticillium wilt of potato (27), and blight of
soybeans (31), as well as for downy mildew of sunflowers
(14). These tests rely on rabbit polyclonal antisera
specific for the causative agent of the disease. The target antigens in
these tests have not been identified. The reproducibility of such
ELISAs has been questioned, since the immunization of rabbits with
crude or partially purified fungal extracts may result in qualitative
or quantitative differences in the sera obtained. Such variation may be
overcome by the use of MAbs.
In the present study, we produced two MAbs that recognized antigens
found in all morphological stages of the fungus except the hyphae and
the hyphal portion of the germ tubes. These MAbs were specific for
P. halstedii. They recognized all races of the fungus that
are present in France and did not cross-react with P. viticola or with other fungi commonly recovered from sunflower plants. All of the races tested have been reported in other parts of
the world where sunflowers are grown commercially (19, 24). However, only a single isolate was available for each race of P. halstedii, and our results remain to be confirmed with other isolates. Both MAbs bound to the same three glycoproteins in the crude
extract. The relationships among these glycoproteins remain to be
defined. For example, they may be degradation products of the 192-kDa
or a larger glycoprotein, different degrees of polymerization of the
68-kDa molecule, or three distinct glycoproteins sharing common
epitopes. Both MAbs also bound distinct, repetitive epitopes. The
reduction of immunoreactivity with MAb 12C9 after periodate treatment
suggests that these epitopes are carbohydrates. The loss of recognition
after reduction with 2-mercaptoethanol suggests that the epitopes are conformational.
We used the specificities of these antibodies for P. halstedii and their recognition of distinct and repetitive
epitopes to develop a sandwich ELISA system for the detection of the
fungus. Thus, we could detect the fungus in seeds from infected plants from two geographically distinct areas of France, and no reaction was
observed with any lot of seeds from healthy plants. Although our study
was conducted exclusively with isolates from France, these races have a
global distribution. Therefore, our ELISA detection system can
potentially be used in all countries where sunflowers are grown
commercially. In conclusion, these MAbs may constitute valuable tools
for the diagnosis of seed-borne downy mildew of sunflowers and help
reduce the incidence of seed-borne disease transmission. As importing
and exporting agencies in most countries require a phytosanitary
certificate attesting the absence of downy mildew in the production
area, this test could make this regulation easier to enforce by
limiting movement of infected lots of seeds.
| |
ACKNOWLEDGMENTS |
This work was supported by a grant from Contrat de Plan
Etat-Région Pays de la Loire 1994-1998
"IIIAppréciation et contrôle de la qualité
sanitaire des semences."
We thank Sophie Aligon and Christine Giroult from GEVES for the
production of contaminated seedlings for P. halstedii races 1, A, and B; Pascal Walser and Denis Tourvieille from INRA for races C
and D; Marie-Pascale Latorse from Rhône-Poulenc (Lyon, France)
for the production of contaminated vine leaves; and Cipta Meliala and
Denis Tourvieille (Clermont-Ferrand, France) and Thomas Henry (Service
Régional de Protection des Végétaux de Beaune, Beaune, France) who gave us seeds from infected plants.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Groupe d'Etude
des Interactions Hôte-Parasite, Laboratoire de
Parasitologie-Mycologie, Faculté de Pharmacie, 16 Bd Daviers,
49100 Angers, France. Phone: (33) 02 41 22 66 62. Fax: (33) 02 41 48 67 33. E-mail: Raymond.Robert{at}univ-angers.fr.
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REFERENCES |
| 1.
|
Albourie, J. M.,
J. Tourvieille, and D. Tourvieille de Labrouhe.
1998.
Resistance to metalaxyl in isolates of the sunflower pathogen Plasmopara halstedii.
Eur. J. Plant Pathol.
104:235-242[CrossRef].
|
| 2.
|
Bradford, M. M.
1976.
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.
Anal. Biochem.
72:248-254[CrossRef][Medline].
|
| 3.
|
Cohen, Y., and W. E. Sackston.
1973.
Factors affecting infection of sunflowers by Plasmopara halstedii.
Can. J. Bot.
5:15-22.
|
| 4.
|
Delanoe, D.
1972.
Biologie et épidémiologie du mildiou du tournesol (Plasmopara helianthi Novot.).
Informations Tech. CETIOM
29:1-49.
|
| 5.
|
Dippold, W. G.,
K. O. Lloyd,
L. T. C. Li,
H. Ikeda,
H. F. Oettgen, and L. J. Old.
1980.
Cell surface antigens of human malignant melanoma; definition of six antigenic systems with mouse monoclonal antibodies.
Proc. Natl. Acad. Sci. USA
77:6114-6118[Abstract/Free Full Text].
|
| 6.
|
Doken, M. T.
1989.
Plasmopara halstedii (Farl.) Berl. et de Toni in sunflower seeds and the role of infected seeds in producing plants with systemic symptoms.
J. Phytopathol.
124:23-26.
|
| 7.
|
Fegies, N. C.,
R. Corbière,
V. Molinéro,
M. T. Bethenod,
J. Bertrandy,
R. Champion, and D. Spire.
1992.
Détection de Peronospora viciae dans les graines de pois par méthode sérologique, p. 315-316.
In
Proceedings of the 1st European Conference on Proteaginous. Assoc. Eur. Protéagineax, Paris, France.
|
| 8.
|
Guesdon, J. L.,
T. Ternynck, and S. Avrameas.
1979.
The use of avidin-biotin interaction of immunoenzymatic techniques.
J. Histochem. Cytochem.
27:1137-1139.
|
| 9.
|
Gulya, T. J., and J. F. Miller.
1985.
Registration of DM-I sunflower germplasm composite resistant to race 3 downy mildew.
Crop Sci.
25:719[Free Full Text].
|
| 10.
|
Gulya, T. J.,
J. F. Miller,
F. Viranyi, and W. E. Sackston.
1991.
Proposed internationally standardized methods for race identification of Plasmopara halstedii.
Helia
14:11-20.
|
| 11.
|
Gulya, T. J.,
W. E. Sackston,
F. Viranyi,
S. Masirevic, and K. Y. Rashid.
1991.
New races of the sunflower downy mildew pathogen (Plasmopara halstedii) in Europe and North and South America.
J. Phytopathol.
132:303-311.
|
| 12.
|
Laemmli, U. K.
1970.
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature (London)
227:680-685[CrossRef][Medline].
|
| 13.
|
Lafon, S.,
A. Penaud,
P. Walser,
M. C. De Guenin,
V. Molinero,
R. Mestre, and D. Tourvieille de Labrouhe.
1996.
Le mildiou du tournesol toujours sous surveillance.
Phytoma Défense Végétaux
484:35-37.
|
| 14.
|
Liese, A. R.,
A. R. Gotlieb, and W. E. Sackston.
1982.
Use of enzyme-linked immunosorbent assay (ELISA) for the detection of downy mildew (Plasmopara halstedii) in sunflower, p. 173-175.
In
Proceedings of the 10th International Sunflower Conference. International Sunflower Association, Queensland, Australia.
|
| 15.
|
Miller, J. F., and T. J. Gulya.
1987.
Inheritance of resistance to race 3 downy mildew in sunflower.
Crop Sci.
27:210-212[Abstract/Free Full Text].
|
| 16.
|
Miller, J. F., and T. J. Gulya.
1988.
Registration of six downy mildew resistant sunflower germplasm lines.
Crop Sci.
25:719.
|
| 17.
|
Miller, J. F., and T. J. Gulya.
1991.
Inheritance of resistance to race 4 of downy mildew derived from interspecific crosses in sunflower.
Crop Sci.
31:40-43[Abstract/Free Full Text].
|
| 18.
|
Mouzeyar, S.,
J. Philippon,
F. Vear, and D. Tourvieille de Labrouhe.
1992.
Genetical studies of resistance to downy mildew (Plasmopara helianthii Novot.) in sunflowers, p. 1162-1167.
In
Proceedings of the 13th International Sunflower Conference. Cetior, Paris, France.
|
| 19.
|
Mouzeyar, S.,
J. Philippon,
P. Walser,
F. Vear, and D. Tourvieille de Labrouhe.
1994.
Sunflower resistance to French races of downy mildew (Plasmopara halstedii).
Agronomie
14:335-336.
|
| 20.
|
Mouzeyar, S.,
P. Roeckel-Drevet,
L. Gentzbittel,
J. Philippon,
D. Tourvieille de Labrouhe,
F. Vear, and P. Nicolas.
1995.
RFLP and RAPD mapping of the sunflower Pl1 locus for resistance to Plasmopara halstedii race 1.
Theor. Appl. Genet.
91:733-737.
|
| 21.
|
Rashid, K. Y.
1993.
Incidence and virulence of Plasmopara halstedii on sunflower in western Canada during 1988-1991.
Can. J. Plant Pathol.
15:206-210.
|
| 22.
|
Roeckel-Drevet, P.,
V. Coelho,
J. Tourvieille,
P. Nicolas, and D. Tourvieille de Labrouhe.
1997.
Lack of genetic variability in French identified races of Plasmopara halstedii, the cause of downy mildew in sunflower Helianthus annuus.
Can. J. Microbiol.
43:260-263.
|
| 23.
|
Sackston, W. E.
1981.
Downy mildew of sunflower, p. 545-575.
In
D. M. Spencer (ed.), The downy mildews. Academic Press, London, United Kingdom.
|
| 24.
|
Sackston, W. E.
1990.
A proposed international system for designating races of Plasmopara halstedii.
Plant Dis.
74:721-723.
|
| 25.
|
Sackston, W. E.
1992.
On a treadmill: breeding sunflowers for resistance to disease.
Annu. Rev. Phytopathol.
30:529-551.
|
| 26.
|
Spring, O.,
A. Benz, and V. Faust.
1991.
Impact of downy mildew (Plasmopara halstedii) infection on the development and metabolism of sunflower.
J. Plant Dis. Protect.
98:597-604.
|
| 27.
|
Sundaram, S.,
J. Plasencia, and E. E. Bantarri.
1991.
Enzyme-linked immunosorbent assay for detection of Verticillium spp. using antisera produced to V. dahliae from potato.
Phytopathology
81:1485-1489.
|
| 28.
|
Tan, A. S.,
C. C. Jan, and T. J. Gulya.
1992.
Inheritance of resistance to race 4 of sunflower downy mildew in wild sunflower accessions.
Crop Sci.
32:949-952[Abstract/Free Full Text].
|
| 29.
|
Theolis, R., Jr., and W. C. Breckenridge.
1994.
Characterization of monoclonal antibodies to apolipoprotein (a) and development of a chemiluminescent assay for phenotyping apolipoprotein (a) isomorphs.
J. Immunol. Methods
172:43-58[CrossRef][Medline].
|
| 30.
|
Towbin, H.,
T. Staehelin, and J. Gordon.
1979.
Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and application.
Proc. Natl. Acad. Sci. USA
76:4350-4354[Abstract/Free Full Text].
|
| 31.
|
Velicheti, R. K.
1993.
Immunodetection of Phomopsis species in asymptomatic soybean plants.
Plant Dis.
77:70-73.
|
| 32.
|
Viranyi, F., and G. Oros.
1981.
Changes in the development and metabolism of sunflowers infected by Plasmopara halstedii.
Acta Phytopathol.
16:273-279.
|
| 33.
|
Woodward, M. R.,
W. W. Young, Jr., and R. A. Bloodgood.
1985.
Detection of monoclonal antibodies specific for carbohydrate epitopes using periodate oxidation.
J. Immunol. Methods
78:143-153[CrossRef][Medline].
|
Applied and Environmental Microbiology, August 2000, p. 3277-3282, Vol. 66, No. 8
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
