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Applied and Environmental Microbiology, September 1999, p. 3950-3954, Vol. 65, No. 9
Unité de Recherches en Santé
Végétale,1 and Laboratoire
de Biologie Cellulaire et
Moléculaire,2 Institut National de la
Recherche Agronomique, Domaine de la Grande Ferrade, 33883 Villenave d'Ornon cédex, France
Received 22 March 1999/Accepted 29 June 1999
Isolates of the obligately biotrophic fungus Uncinula
necator cluster in three distinct genetic groups (groups I, II,
and III). We designed PCR primers specific for these groups in order to
monitor field populations of U. necator. We used the
nucleotide sequences of the gene that encodes eburicol
14 Powdery mildew is the most
economically important fungal disease of grapes (Vitis
vinifera L.) throughout the world. The causal agent is the
haploid, heterothallic ascomycete Uncinula necator (Schw.)
Burr. This obligately biotrophic fungus develops only on green organs
of plants belonging to the family of Vitaceae (3). In
Europe, India, and Australia, the only member of the family Vitaceae is
the cultivated grapevine, V. vinifera L.). In temperate
climates, the asexual mycelium of U. necator may overwinter
on leaf primordia inside dormant buds (17, 18). The fungus
begins growing again shortly after bud break, which results in the
appearance of shoots covered with white, sporulating mycelium; these
shoots are called "flagshoots" (3). U. necator also may overwinter as cleistothecia (the ascigerous stage
of the fungus) on the bark of grapevines (11, 16). In this
case, primary infections are caused by ascospores released during the spring (12). Although both flagshoots and cleistothecia are observed in Europe, cleistothecia are considered the most important way
that the fungus overwinters in Europe (23).
Recent randomly amplified polymorphic DNA (RAPD) (22)
studies showed that U. necator isolates collected from
plants with typical flagshoot symptoms (group I isolates) had RAPD
patterns very different than the RAPD patterns of isolates that
presumably arose from ascospores (group III isolates) (5,
7). A similar situation seems to exist in Australia
(10), but the population structure of U. necator
in India seems to be more complex; in India a third genetic group
(group II) has been identified (7). The genetic variation
between groups is considerably greater than the genetic variation
within groups, suggesting genetic isolation (7).
In European vineyards in which U. necator isolates belonging
to group I and group III are both present, the fungal population may
shift from predominantly group I isolates to predominantly group III
isolates during the grapevine growing season (5, 7). For
large-scale field studies, molecular markers that allow workers to
detect isolates belonging to each genetic group are needed. As U. necator is an obligate biotroph, large-scale field studies in
which RAPDs are used are extremely time- and labor-intensive. Specific
PCR primers that distinguish the genetic groups of U. necator are needed for such field studies.
Our primary objective was to develop PCR primers specific for groups I
and III. A secondary objective was to obtain preliminary data on
changes in the populations of U. necator in vineyards in
which isolates belonging to both of these genetic groups are present.
We cloned and sequenced the single-copy gene encoding eburicol
14 Isolates of U. necator.
We used 132 single-spore
isolates, including 90 isolates from a previous study (7)
and 42 new isolates collected from 1992 to 1998 in Australia (30 isolates), Tunisia (8 isolates), Europe (3 isolates), and Israel (1 isolate) (Table 1). Fungal material (conidia and mycelium) from all of the isolates from Europe, India, Tunisia, Israel and from the 22 Australian isolates collected in 1998 was produced and collected as previously described (5). Freeze-dried conidia of the eight remaining Australian isolates (isolates 920103, 930202, 930304b, 931201, 931302, 931502, 940301, and
940402) were obtained from B. E. Stummer (University of Adelaide, Adelaide, Australia).
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Nested Allele-Specific PCR Primers Distinguish
Genetic Groups of Uncinula necator
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
-demethylase (CYP51) and of the ribosomal DNA internal
transcribed spacer 1 (ITS1), ITS2, and 5.8S regions. We identified four
point mutations (three in CYP51 and one in ITS1) that
distinguished groups I and II from group III based on a sample of 132 single-spore isolates originating from Europe, Tunisia, Israel, India,
and Australia. We developed a nested allele-specific PCR assay in which
the CYP51 point mutations were used to detect and
distinguish groups I and II from group III in crude mildewed samples
from vineyards. In a preliminary study performed with samples from
French vineyards in which isolates belonging to genetic groups I and
III were present, we found that a shift from a population composed
primarily of group I isolates to a population composed primarily of
group III isolates occurred during the grapevine growing season.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
-demethylase (CYP51), a highly conserved cytochrome P-450 enzyme
essential for sterol biosynthesis (2; for a review see reference 24). CYP51 is the only
U. necator gene whose sequence is known. We previously found
a point mutation in CYP51 that could be used to identify
U. necator isolates that are resistant to a CYP51-inhibiting
fungicide (8). In other studies (1, 15, 19), the
ribosomal DNA (rDNA) region encompassing internal transcribed spacers
(ITS) (21) and 5.8S rDNA has been used to distinguish fungal
species, subspecies, or isolates. We cloned and sequenced both
CYP51 and the ITS-5.8S rDNA region from U. necator isolates belonging to all three genetic groups in order to
identify mutations that distinguish the genetic groups.
MATERIALS AND METHODS
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
TABLE 1.
Assignment of 42 single-spore U. necator
isolates to genetic groups I through III on the basis of their
RAPD patterns
Powdery mildew field samples.
U. necator
populations were monitored in three vineyards in France (Manosque,
Nîmes, and Montirat) in which both flagshoots and cleistothecia
had been observed for at least 5 years. During the 1998 grape growing
season, these vineyards were not sprayed with fungicides. Mildewed
tissues were collected from grapevines at different times during the
grape growing season (Table 2), packed in
healthy grapevine leaves and newspapers, and mailed to the laboratory.
To minimize the effect of sampling, only small amounts of mildewed
tissues were collected from each grapevine. Three successive samples
were obtained from two grapevines in Nîmes and from two
grapevines in Manosque. Four successive samples were obtained from five
grapevines in Montirat. The first samples consisted exclusively of
flagshoots, since they were the only early powdery mildew symptoms
observed.
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20°C until
nucleic acids were extracted.
DNA extraction and RAPD assay. Total genomic DNA extraction and a RAPD analysis were performed as described previously (6). The uncharacterized isolates (Table 1) were assigned to genetic groups I, II, and III by using band patterns obtained with 46 primers, as described previously (7).
CYP51 cloning and sequencing. We considered the CYP51 sequence of a group III, fungicide-sensitive isolate (GenBank accession no. U72657) the reference sequence for U. necator CYP51. A 1,756-bp DNA fragment encompassing the entire CYP51 coding sequence was amplified by PCR by using primers C14 and C14R (8). For each isolate, both strands of three DNA inserts were sequenced by using specific primers (9).
ITS and 5.8S rDNA cloning and sequencing. Primers ITS1 (21) and UncITS4 (5'-AATGATTCGAGGTCAACCTGTCAATCC; based on a partial sequence of U. necator rDNA kindly provided by J. Mugnier [Rhône-Poulenc Agro, Lyon, France]) were used for PCR amplification of an expected approximately 550-bp DNA fragment encompassing ITS region 1 (ITS1), 5.8S rDNA, and most of ITS2 from one isolate per genetic group. The cloning and sequencing procedures used have been described previously (9).
Allele-specific PCR assay.
Allele-specific PCR primers were
designed by using the fact that a 3' mismatch does not prime in a PCR
at a specific annealing temperature (20). Primers
MUT2(I-II), MUT3(I-II), and MUT4(I-II) (Table
3) were designed to specifically prime
CYP51 sequences containing a T at nucleotide 110, a C at
nucleotide 575, and a T at nucleotide 1587, respectively. Primers
MUT2(III), MUT3(III), and MUT4(III) (Table 3) were designed to
specifically prime CYP51 sequences containing a G at
nucleotide 110, a T at nucleotide 575, and a C at nucleotide 1587, respectively. Primers U14DM, M1I, and M1 were based on CYP51
sequences that were identical in all of the U. necator CYP51
sequences studied. Primer MUT2(I-II) or MUT2(III) was used with primer
U14DM, primer MUT3(I-II) or MUT3(III) was used with primer M1I, and
primer MUT4(I-II) or MUT4(III) was used with primer M1 (Table 3); all
of the primers were used at a final concentration of 0.1 µM.
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NAS-PCR. We developed a nested allele-specific PCR (NAS-PCR) assay targeting the point mutations found in CYP51. Nested PCR (4) involves two rounds of amplification by PCR. The first-round PCR was performed by using primers C14 and C14R (8). Subsequently, 1-µl aliquots of the first-round PCR mixture were subjected to a second round of PCR amplification, in which we used each of the six primer pairs designed for allele-specific PCR amplification of CYP51. In the second-round PCR the amount of Goldstar DNA polymerase (Eurogentec S. A., Seraing, Belgium) used was 0.2 U per sample, and the allele-specific PCR primers (Table 3) were used at a final concentration of 0.05 µM each. Otherwise, the PCR conditions were as described previously (8). For each combination of allele-specific second-round PCR primer pair and mildewed field sample, two independent NAS-PCR amplifications were performed by using two different DNA solutions extracted independently. Amplified DNA fragments were visualized under UV light after electrophoresis at 100 V on ethidium bromide-stained (0.4 µg/ml) 1% agarose gels electrophoresed in 0.5× Tris-borate-EDTA buffer.
DNA extracted from grapevines and from 34 filamentous fungi and yeasts collected in the field from grapevines and identified by D. Blancard and P. Lecomte (Institut National de la Recherche Agronomique, Bordeaux, France) were tested for amplification by using NAS-PCR. The yeasts and fungi tested were Acremonium sp., Alternaria alternata, Armillaria mellea, Botryotinia fuckeliana, Cephalosporium sp., Chaetomium sp., Coniella diplodiella, Coniothyrium sp., Diplodia natalensis, Elsinoe ampelina, Epicoccum sp., Eutypa lata, Fusarium roseum, Fusarium sp., Gliocladium sp., Greeneria uvicola, Guignardia bidwellii, Kloeckera apiculata, Pestalotia sp., Phellinus ignarius, Phialophora parasitica, Phomopsis viticola, Plasmopara viticola, Pseudopeziza tracheiphila, Rhizopus nigricans, Rosellinia necatrix, Saccharomyces sp., Saccharomycopsis ludwigii, Sphaeropsis sp., Stereum hirsutum, Trichoderma sp., Ulocladium sp., Verticillium dahliae, and Verticillium lecanii. To confirm the specificity of allele-specific PCR primers for genetic groups, 13 single-spore isolates of U. necator were taken from powdery mildew colonies obtained from grapevines from each of the three vineyards and independently characterized by using allele-specific PCR and RAPD analysis.| |
RESULTS |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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RAPD amplifications. European isolates collected in 1998 and Tunisian, Israeli, and Australian isolates were assigned either to group I or to group III on the basis of their RAPD patterns (Table 1). Australian isolates 920103 and 930202, which were assigned to PCR-restriction fragment length polymorphism group A (10), had RAPD patterns typical of group I, and the six Australian isolates assigned to PCR-restriction fragment length polymorphism group B (10) had RAPD patterns typical of group III. Thus, genetic groups A and B described previously for Australian isolates corresponded to RAPD groups I and III, respectively. The 132 isolates which we studied included 16 group I isolates, 12 group II isolates, and 104 group III isolates (7) (Table 1).
CYP51 cloning and sequencing and allele-specific PCR detection of mutations. The CYP51 sequences of 12 group III isolates, including 7 isolates from France, 2 isolates from Portugal, 1 isolate from Switzerland, 1 isolate from Germany, and 1 isolate from Israel, were determined previously (8). In this study, we determined the CYP51 sequences of 10 additional isolates, including 3 group I isolates (isolates FCP1.1 [France] [7], GNE1.1 [Germany] [7], and 930202 [Australia] [Table 1]), 1 group II isolate (isolate IHY1.1 [India] [7]), and 6 group III isolates (isolates SLE1.1 and SNO2.1 [Switzerland] [7], IBA1.2 and IPA1.1 [India] [7], PVA2.1 [Portugal] [7], and 931502 [Australia] [Table 1]).
Of the 18 CYP51 sequences determined for group III isolates, 5 had a point mutation at nucleotide 462 that was associated with high levels of resistance to a fungicide that inhibits CYP51 (8). The CYP51 sequences of the 13 other group III isolates studied were identical to the sequences of fungicide-sensitive European isolates (reference sequences). Three point mutations were found in the CYP51 sequences of the three group I isolates and one group II isolate studied (GenBank accession no. AF042067). The sequence changes were a G-T transversion at nucleotide position 110 corresponding to a Gly-37-Val substitution, a T-C transition at nucleotide position 575 resulting in a Ile-156-Thr substitution, and a C-T transversion at nucleotide position 1587 (Arg-493) that is silent. Amplifications performed with allele-specific PCR primers U14DM and MUT2(I-II), primers M1I and MUT3(I-II), and primers M1 and MUT4(I-II) yielded fragments of the expected sizes (125, 447, and 613 bp, respectively) for all 16 group I isolates and all 12 group II isolates (Fig. 1). No amplification was obtained with DNA extracted from the 104 group III isolates, while a fragment of the expected size (1,756 bp) was obtained from these DNA samples when we used primers C14 and C14R as a control for PCR efficiency. Conversely, amplifications performed with primers U14DM and MUT2(III), primers M1I and MUT3(III), and primers M1 and MUT4(III) yielded fragments of the expected sizes for all 104 group III isolates. No amplification was obtained with DNA extracted from all 16 group I isolates and all 12 group II isolates, while a fragment of the expected size was obtained when primers C14 and C14R were used (data not shown).
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ITS and 5.8S rDNA cloning and sequencing and allele-specific PCR detection of a mutation. A 559-bp DNA fragment encompassing ITS1, most of ITS2, and 5.8S rDNA was cloned from group I isolate FCP1.1, group II isolate ISA1.1, and group III isolate PVA2.1 (7) and sequenced. The 5.8S rDNA sequence was identical to the 5.8S rDNA sequence of Uncinula adunca, the organism that is most closely related to U. necator for which this sequence is available (14). Group I and II isolates had identical ITS and rDNA sequences (GenBank accession no. AF049332). The ITS1 sequences of these isolates differed from the ITS1 sequence of the group III isolate (GenBank accession no. AF049331) by a single C-to-T substitution at nucleotide 70.
Amplifications with primers UITS and MIT1(I-II) and primers UITS and MIT1(III) were performed with the same 132 DNA samples used for detection of mutations in CYP51. Amplification of a DNA fragment of the expected size (84 bp) was obtained with all of the group I and II isolates when we used primers UITS and MIT1(I-II) but not with any of the group III isolates. Conversely, amplification of a DNA fragment of the expected size was obtained with all group III isolates when we used primers UITS and MIT1(III) but not with any of the group I and II isolates (data not shown).NAS-PCR analysis. NAS-PCR amplifications performed with DNA extracted from grapevines and from 34 yeast and fungal species associated with grapevines in vineyards yielded no amplified fragments. All NAS-PCR amplifications performed with DNA extracted from samples containing U. necator yielded three major fragments (Fig. 2), one of which was polymorphic.
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DISCUSSION |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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A comparison of the CYP51 sequences of three group I U. necator isolates, one group II U. necator isolate, and 18 group III U. necator isolates identified three point mutations that distinguished groups I and II from group III. The mutation at codon 37 causes a Gly-Val substitution in the hydrophobic, N-terminal, putative transmembrane region of CYP51. The mutation at codon 156 causes an Ile-Thr substitution 15 amino acids from the end of the CR2 domain, a highly conserved region that is believed to be involved in enzyme substrate recognition (2). The mutation at codon 493 is silent. The occurrence of three point mutations in CYP51 was surprising, since this gene is highly conserved and is essential for the survival of the fungus (2, 24). Thus, mutations in CYP51 are likely to be either neutral or adaptive but probably not deleterious. The silent mutation at codon 493 is probably neutral, but the biological significance of the mutations at codons 37 and 156 in group I and II CYP51 sequences is not known.
Although ITS rDNA regions have been reported to be highly variable, even in closely related species (14, 15), we found only one point mutation that distinguished group I and II isolates from group III isolates. Thus, we could develop no molecular tool derived from the rDNA sequence to distinguish groups I and II.
The results of allele-specific PCR amplification experiments performed with DNA from 145 single-spore isolates of U. necator (132 isolates listed in reference 7 and Table 1 and 13 isolates obtained from our 1998 population survey) demonstrated that the four point mutations in CYP51 and ITS1 always distinguished groups I and II (34 isolates studied) from group III (111 isolates studied). Based on our sample, we are 95% certain that groups I and II are positively identified at least 91% of the time, and that isolates from group III are positively identified at least 99% of the time.
Using NAS-PCR, we found that in May, flagshoot symptoms were associated with group I isolates (Table 2). During June, group III isolates were first detected, but group I isolates were still detected in two vineyards (Nîmes and Montirat). Only group III isolates were detected in July and September, suggesting that this group represented the majority of U. necator isolates present at the end of the growing season. Our findings are consistent with the hypothesis that there are genetically isolated groups of isolates within U. necator and that these isolates occupy distinct, specialized niches (5, 7), as was shown for other phytopathogenic fungi (13).
The molecular tools which we developed can be used to reliably and efficiently distinguish groups I and II from group III. PCR primers that distinguish group I from group II might be derived from specific RAPD fragments. The NAS-PCR technique is much more suitable than RAPD analysis for field studies, however, since crude samples from the field can be directly processed. Because only 5% of the first-round PCR mixture is required for second-round, allele-specific PCR amplification, numerous second-round PCR amplifications can be performed with a single field sample. We plan to use NAS-PCR to monitor U. necator populations in vineyards in various geographic locations. More generally, NAS-PCR may be very useful for conducting large-scale population genetic studies with diverse organisms. When resistance of an organism to chemical control results from point mutations in the gene encoding the target enzyme, NAS-PCR also may be used in resistance monitoring programs.
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ACKNOWLEDGMENTS |
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This work was supported by grant 970 307 002 from the Conseil Régional d'Aquitaine.
We thank G. Brarda (Chambre d'Agriculture de l'Aude, Carcassonne, France), H. Guillemont (Chambre d'Agriculture du Roussillon, Perpignan, France), M. Blanc (Institut Technique de la Vigne de Manosque, Manosque, France), and B. Molot (Institut Technique de la Vigne de Nîmes, Nîmes, France) for collecting and mailing grapevine mildewed samples. We thank D. Blancard and P. Lecomte (Station de Pathologie Végétale, Institut National de la Recherche Agronomique, Bordeaux, France) for providing DNA samples from yeasts and fungi associated with grapevines.
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FOOTNOTES |
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* Corresponding author. Present address: Laboratoire de Malherbologie et Agronomie, Institut National de la Recherche Agronomique, BV 1540, 21034 Dijon Cédex, France. Fax: 33 3 80 69 32 62. E-mail: delye{at}bordeaux.inra.fr.
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Applied and Environmental Microbiology, September 1999, p. 3950-3954, Vol. 65, No. 9
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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