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Peer Reviewed

© 2013 Plant Management Network.
Accepted for publication 23 April 2012. Published 29 July 2013.

Emergence of Single Point Mutation in PvCesA3, Conferring Resistance to CAA Fungicides, in Plasmopara viticola Populations in Japan

Yoshinao Aoki, Maki Hashimoto, and Shunji Suzuki, Laboratory of Fruit Genetic Engineering, The Institute of Enology and Viticulture, University of Yamanashi, Kofu, Yamanashi 400-0005

Corresponding author: Shunji Suzuki.

Aoki, Y., Hashimoto, M., and Suzuki, S. 2013. Emergence of single point mutation in PvCesA3, conferring resistance to CAA fungicides, in Plasmopara viticola populations in Japan. Online. Plant Health Progress doi:10.1094/PHP-2013-0729-01-BR.

Mandipropamid, one of the carboxylic acid amide (CAA) fungicides, was registered for control of grapevine downy mildew disease (caused by Plasmopara viticola) in June 2009 in Japan and its use was started in the 2010 growing season. Almost simultaneously with the registration of mandipropamid in Japan, the increasing occurrence of quinone outside inhibitor fungicide resistance in P. viticola populations became a serious problem in Japan (4). Because of this, mandipropamid was widely used in Japanese vineyards. Dimethomorph and benthiavalicarb-isopropyl, other CAA fungicides, were registered in 1999 and 2000 in Japan, respectively. However, dimethomorph has been hardly used for control of grapevine downy mildew disease.

In Europe, mandipropamid-resistant P. viticola isolates were detected as early as in 2004 (5). A single point mutation from GGC to AGC in codon 1105 of PvCesA3, which resulted in a single amino acid substitution of serine for glycine, confers mandipropamid resistance to P. viticola (3). Mandipropamid fungicide resistance is recessively inherited (5). Heterozygotes harboring resistant and susceptible PvCesA3 alleles are susceptible to mandipropamid fungicide, whereas homozygotes harboring the mutation in both alleles only express resistance (3). Therefore, to inhibit the spread of CAA fungicide-resistant P. viticola, we have to recognize the emergence of the mutation in P. viticola populations.

Downy mildew infected leaves were collected from two vineyards located in Yamanashi Prefecture, the major grape-growing region in Japan, in September 2012. Mandipropamid was first applied to both vineyards in the 2011 growing season (Table 1), and then applied to vineyard A in the 2012 growing season. Benthiavalicarb-isopropyl, another CAA fungicide, was also applied to vineyard A in both years. As control samples, downy mildew infected leaves were collected from three vineyards to which CAA fungicides had never been applied. The detection of mandipropamid-resistant PvCesA3 was performed by the PCR-restriction fragment length polymorphism (PCR-RFLP) assay (1) and the allele-specific primer PCR (ASP-PCR) assay (2).

Table 1. Fungicide management programs in vineyards sampled.

Year Vineyard A Vineyard B
Date Chemical Date Chemical
2011 13 Apr Lime sulfur
14 Apr Lime sulfur
21 May Amisulbrom
31 May Cymoxanil/
13 May Fosetyl-Al
10 Jun Cyazofamid/
24 May Cymoxanil/
22 Jun Mandipropamidx
1 Jun Manzeb
9 Jun Mandipropamidx
3 Jul Bordeaux mixture
21 Jun Cyazofamid/
1 Aug Bordeaux mixture
29 Jun Cyprodinil/
13 Jul Bordeaux mixture
25 Jul Bordeaux mixture
27 Aug Kresoxim-methyl


13 Apr Lime sulfur
5 Apr Lime sulfur
21 May Cymoxanil/
31 May Cyazofamid/
16 May Fosetyl-Al
10 Jun Mandipropamidx
8 Jun Cyazofamid/
18 Jun Amisulbrom
18 Jun Amisulbrom
28 Jun Bordeaux mixture
17 Jul Bordeaux mixture
23 Jul Bordeaux mixture
25 Jul Bordeaux mixture


9 Aug Bordeaux mixture

 x CAA fungicide.

Consequently, we detected the mandipropamid-resistant PvCesA3 from one of the 35 single-lesion samples collected from vineyard A (Fig. 1a and 1b). DNA sequencing demonstrated that a single point mutation from GGC to AGC in codon 1105 occurred in the PvCesA3 of the sample (Fig. 1c). In contrast, the resistant allele was not detected in any of the 21 samples collected from vineyard B. Furthermore, no mutation in codon 1105 of the PvCesA3 allele was detected in 164 samples collected from three vineyards to which mandipropamid had never been applied. As DNA sequencing also detected a non-mutated codon 1105 in PvCesA3 clones amplified from the sample by PCR, we judged that the sample might have been a susceptible heterozygote to mandipropamid or that it might have been a heterogeneous population among susceptible and resistant P. viticola isolates. This is the first report that the resistant PvCesA3 allele has been emerged in P. viticola populations in Japan.


Fig. 1. Emergence of mandipropamid-resistant PvCesA3 inPlasmopara viticola. (a) PCR-RFLP assay. White arrowhead indicates non-mutated PvCesA3 and black arrowheads indicate resistant PvCesA3. Numbers at the left indicate positions of DNA size. (b) ASP-PCR assay. White arrow indicates ribosomal RNA gene as internal control, and black arrow indicates resistant PvCesA3. Numbers at the bottom indicate positions of DNA size. (c) DNA sequence analysis. Arrows indicate a single point mutation in the PvCesA3. R, mandipropamid-resistant PvCesA3. S, susceptible PvCesA3.


The risk for CAA fungicide resistance in P. viticola is evaluated as ‘moderate’ (5). To data, the grape grower in vineyard A has not experienced a loss of efficiency of CAA fungicides against downy mildew. We cannot rule out the possibility that the use of benthiavalicarb-isopropyl promoted the emergence of G1105S mutation in PvCesA3, although benthiavalicarb-isopropyl had never been applied to the vineyards sampled prior to 2011.

Literature Cited

1. Aoki, Y., Furuya, S., and Suzuki, S. 2011. Method for rapid detection of PvCesA3 gene allele conferring resistance to mandipropamid, a carboxylic acid amide fungicide, in Plasmopara viticola populations. Pest Manag. Sci. 67:1557-1561

2. Aoki, Y., Hada, Y., and Suzuki, S. 2013. Development of multiplex allele-specific primer PCR assay for simultaneous detection of QoI and CAA fungicide resistance alleles in Plasmopara viticola populations. Pest Manag. Sci. 69:268-273

3. Blum, M., Waldner, M., and Gisi, U. 2010. A single point mutation in the novel PvCesA3 gene confers resistance to the carboxylic acid amide fungicide mandipropamid in Plasmopara viticola. Fung. Gen. Biol. 47:199-510

4. Furuya, S., Mochizuki, M., Saito, S., Kobayashi, H., Takayanagi, T., and Suzuki, S. 2010. Monitoring of QoI fungicide resistance in Plasmopara viticola populations in Japan. Pest Manag. Sci. 66:1268-1272

5. Gisi, U., Waldner, M., Kraus, N., Dubuis, P. H., Sierotzki, H. 2007. Inheritance of resistance to carboxylic acid amide (CAA) fungicides in Plasmopara viticola. Plant Pathol. 56:199-208.