Bazı CAA ve QoI fungisitlerinin Plasmopara viticola’nın hastalık gelişimi, zoospor salınımı ve ε-viniferin birikimi üzerine etkileri

Öz

Bu çalışma, azoxystrobin + difenoconazole ve mandipropamid + zoxamide aktif maddelerini içeren iki fungisitin asma yaprakları üzerindeki Plasmopara viticola’ya karşı etkisini değerlendirmiştir. Farklı fungisit dozlarının, farklı üzüm çeşitlerinin yapraklarından toplanan P. viticola popülasyonu üzerindeki etkisi; koparılmış yapraklarda sporulasyon alanının analizi, zoospor salınımı, zoospor hareketliliği ve ε-viniferin birikimi araştırılmıştır. Mandipropamid + zoxamide karışımı için EC50 değeri 0.031 µg ml⁻¹ olarak belirlenmiş ve 1 µg ml⁻¹ dozunda sporulasyon alanının azaltılmasında %97.73 etkinlik elde edilmiştir. Azoxystrobin + difenoconazole karışımı için EC50 değeri 40 µg ml⁻¹ olarak belirlenmiş ve 300 µg ml⁻¹ dozunda sporulasyon alanı için %92.82 engelleme oranına ulaşılmıştır. Fungisit dayanıklılığı için G143A mutasyonu, allel-özgül polimeraz zincir reaksiyon (ASPCR) testi kullanılarak tespit edilmiştir. Ayrıca, mandipropamid + zoxamide karışımının düşük konsantrasyonda (0.01 µg ml⁻¹) uygulanmasının ε-viniferin birikimini teşvik ettiği, aynı zamanda zoospor hareketliliğini ve salınımını önemli ölçüde engellediği belirtilmiştir. Azoxystrobin + difenoconazole karışımının 10 µg ml⁻¹ dozunda hareketli zoospor sayısını %84 oranında azaltması, bu fungisitin tarla koşullarında enfeksiyon riskini azaltabileceğini göstermektedir. Bu hastalığın kontrolünde fungisit etkinliğinin sürdürülebilirliğinin değerlendirilmesinde, zoospor hareketliliğinin enfeksiyon döngüsünde temel bir basamak olması nedeniyle fungisitlerin zoospor hareketliliği üzerindeki etkilerinin de dikkate alınması gerektiği önerilmiştir.

Anahtar Kelimeler

• Plasmopara viticola
• fungisit duyarlılığı
• sporangium salınımı
• zoospor hareketliliği
• ε-viniferin

The effects of some CAA and QoI fungicides on disease development by Plasmopara viticola, zoospore release and ε-viniferin accumulation

Öz

This study evaluated the efficacy of two fungicide mixtures, azoxystrobin + difenoconazole and mandipropamid + zoxamide, in protecting grapevine leaves against Plasmopara viticola. The effects of different fungicide doses on the P. viticola population collected from various grapevine cultivars were investigated by assessing sporulation area on detached leaves, zoospore release, zoospore mobility, and ε-viniferin accumulation. The EC50 value for mandipropamid + zoxamide was 0.031 µg ml-1, yielding 97.73% inhibition of sporulation area at 1 µg ml-1. In contrast, the azoxystrobin + difenoconazole mixture exhibited an EC50 value of 40 µg ml-1 and achieved 92.82% inhibition of sporulation area at 300 µg ml-1. The G143A mutation, a known marker for fungicide resistance, was found using allele-specific polymerase chain reaction (ASPCR). Furthermore, the mandipropamid + zoxamide mixture promoted ε-viniferin accumulation at a low concentration (0.01 µg ml⁻¹) while significantly inhibiting zoospore mobility and release. The azoxystrobin + difenoconazole mixture reduced the number of motile zoospores by 84% at 10 µg ml⁻¹, suggesting its potential to lower infection risk under field conditions. These findings suggest that, in addition to their direct effects on pathogen development, fungicides should also be evaluated for their impact on zoospore mobility, a critical stage in the infection cycle, to enhance sustainable disease management strategies.

Anahtar Kelimeler

• Plasmopara viticola
• Fungicide sensitivity
• Sporangium release
• Zoospore mobility
• ε-Viniferin

Kaynakça

1. Alonso-Villaverde V., Voinesco F., Viret O., Spring J.L., Gindro K., 2011. The effectiveness of stilbenes in resistant Vitaceae: ultrastructural and biochemical events during Plasmopara viticola infection process. Plant Physiology and Biochemistry, 49 (3), 265–274. https://doi.org/10.1016/j.plaphy.2010.12.010
2. Aoki Y., Hada Y., Suzuki S., 2013. Development of a multiplex allele-specific primer PCR assay for simultaneous detection of QoI and CAA fungicide resistance alleles in Plasmopara viticola populations. Pest Management Science, 69, 268–273. https://doi.org/10.1002/ps.3384
3. Aziz A., Trotel-Aziz P., Dhuicq L., Jeandet P., Couderchet M., Vernet G., 2006. Chitosan oligomers and copper sulfate induce grapevine defense reactions and resistance to gray mold and downy mildew. Phytopathology, 96 (11), 1188–1194. https://doi.org/10.1094/PHYTO-96-1188
4. Barchenger D.W., Clark J.R., Threlfall R.T., Howard L.R., Brownmiller C.R., 2014. Effect of field fungicide applications on storability, physicochemical, and nutraceutical content of muscadine grape (Vitis rotundifolia Michx.) genotypes. HortScience, 49 (10),1315–1323. https://doi.org/10.21273/HORTSCI.49.10.1315
5. Blum M., Waldner M., Gisi U., 2010. A single point mutation in the novel PvCesA3 gene confers resistance to the carboxylic acid amide fungicide mandipropamid in Plasmopara viticola. Fungal Genetics and Biology, 47, 499–510. https://doi.org/10.1016/j.fgb.2010.02.009
6. Boso S., Alonso-Villaverde V., Gago P., Santiago J.L., Martínez M.C., 2014. Susceptibility to downy mildew (Plasmopara viticola) of different Vitis varieties. Crop Protection, 63, 26–35.
7. Buonassisi D., Colombo M., Migliaro D., Dolzani C., Peressotti E., Mizzotti C., Velasso R., Masiero S., Perazzillo M., Vezzulli S., 2017. Breeding for grapevine downy mildew resistance: a review of ‘omics’ approaches. Euphytica, 213, 103. https://doi.org/10.1007/s10681-017-1900-9
8. Cadle-Davidson L., 2008. Variation within and between Vitis spp. for foliar resistance to the downy mildew pathogen Plasmopara viticola. Plant Disease, 92, 1577–1584. https://apsjournals.apsnet.org/doi/pdf/10.1094/PDIS-92-11-1577

9. Campbell S.E., Brannen P.M., Scherm H., Brewer M.T., 2020. Fungicide sensitivity survey of Plasmopara viticola populations in Georgia vineyards. Plant Health Progress, 21 (4), 256–261. https://doi.org/10.1094/PHP-05-20-0039-RS
10. Chitarrini G., Soini E., Riccadonna S., Franceschi P., Zulini L., Masuero D., Vrhovsek U., 2017. Identification of biomarkers for defense response to Plasmopara viticola in a resistant grape variety. Frontiers in Plant Science, 8, 1524. https://doi.org/10.3389/fpls.2017.01524
11. Delmas C.E.L., Dussert Y., Delière L., Couture C., Mazet I.D., Richart Cervera S., Delmotte F., 2017. Soft selective sweeps in fungicide resistance evolution: recurrent mutations without fitness costs in grapevine downy mildew. Molecular Ecology, 26, 1936–1951. https://doi.org/10.1111/mec.14006
12. Delmotte F., Mestre P., Schneider C., Kassemeyer H.H., Kozma P., Richart-Cervera S., Delière L., 2014. Rapid and multiregional adaptation to host partial resistance in plant pathogenic oomycete: evidence from European populations of Plasmopara viticola, the causal agent of grapevine downy mildew. Infection, Genetics and Evolution, 27, 500–508. https://doi.org/10.1016/j.meegid.2013.10.017
13. Diez-Navajas A.M., Wiedemann-Merdinoglu S., Greif C., Merdinoglu D., 2008. Nonhost versus host resistance to the grapevine downy mildew, Plasmopara viticola, studied at the tissue level. Phytopathology, 98 (7), 776–780. https://doi.org/10.1094/PHYTO-98-7-0776
14. FRAC (Fungicide Resistance Action Committee) 2019. FRAC pathogen risk list. Available at: https://www.frac.info/docs/default-source/publications/pathogen-risk/frac-pathogen-list-2019.pdf
15. FRAC (Fungicide Resistance Action Committee) 2024. FRAC pathogen risk list 2024. Available at: https://www.frac.info/publications/downloads.
16. Francesca S., Simona G., Francesco Nicola T., Andrea R., Vittorio R., Federico S., Cynthia R., Maria Lodovica G., 2006. Downy mildew (Plasmopara viticola) epidemics on grapevine under climate change. Global Change Biology, 12, 1299–1307. https://doi.org/10.1111/j.1365-2486.2006.01175.x
17. Fontaine S., Remuson F., Caddoux L., Barrès B., 2019. Investigation of the sensitivity of Plasmopara viticola to amisulbrom and ametoctradin in French vineyards using bioassays and molecular tools. Pest Management Science, 75 (8), 2115–2123. https://doi.org/10.1002/ps.5461
18. Furuya S., Mochizuki M., Saito S., Kobayashi H., Takayanagi T., Suzuki S., 2010. Monitoring of QoI fungicide resistance in Plasmopara viticola populations in Japan. Pest Management Science, 66, 1268–1272. https://doi.org/10.1002/ps.2012
19. Genet J.L., Vincent O., 1999. Sensitivity to famoxadone of European Plasmopara viticola populations. Med Fac Landbouww Univ Gent, 64, 559–564.
20. Genet J.L., Jaworska G., Deparis F., 2006. Effect of dose rate and mixtures of fungicides on selection for QoI resistance in populations of Plasmopara viticola. Pest Management Science, 62, 188–194. https://doi.org/10.1002/ps.1146
21. Gessler C., Pertot I., Perazzolli M., 2011. Plasmopara viticola: a review of knowledge on downy mildew of grapevine and effective disease management. Phytopathologia Mediterranea, 50, 3–44. https://doi.org/10.14601/Phytopathol_Mediterr-9360
22. Gindro K., Pezet R., Viret O., 2003. Histological study of the responses of two Vitis vinifera varieties (resistant and susceptible) to Plasmopara viticola infections. Plant Physiology and Biochemistry, 41 (9), 846–853. https://doi.org/10.1016/S0981-9428(03)00124-4
23. Gindro K., Spring J.L., Pezet R., Richter H., Viret O., 2006. Histological and biochemical criteria for objective and early selection of grapevine varieties resistant to Plasmopara viticola. Vitis 45, 191–196. https://doi.org/10.5073/vitis.2006.45.191-196
24. Gindro K., Alonso-Villaverde V., Viret O., Spring J.L., Marti G., Wolfender J., Pezet L., 2012a. Stilbenes: biomarkers of grapevine resistance to disease of high relevance for agronomy, oenology and human health. In: plant defence: biological control, progress in biological control. Mérillon J.M., Ramawat K.G., (Eds.). Springer, Dordrecht, pp 25–54. https://doi.org/10.1007/978-94-007-1933-0_2
25. Gindro K., Alonso-Villaverde V., Voinesco F., Spring J.L., Viret O., Dubuis P.H., 2012b. Susceptibility to downy mildew in grape clusters: new microscopical and biochemical insights. Plant Physiology and Biochemistry, 52, 140–146. https://doi.org/10.1016/j.plaphy.2011.12.009
26. Gisi U., Sierotzki H., 2015. Oomycete fungicides: phenylamides, quinone outside inhibitors, and carboxylic acid amides. In: Fungicide resistance in plant pathogens: principles and a guide to practical management. Ishii H., Hollomon D.W., (Eds.). Springer, Tokyo, pp 145–174. https://doi.org/10.1007/978-4-431-55642-8_8
27. Gisi U., Waldner M., Kraus N., Dubuis P.H., Sierotzki H., 2007. Inheritance of resistance to carboxylic acid amide fungicides in Plasmopara viticola. Molecular Plant Pathology, 56, 199–208. https://doi.org/10.1111/j.1365-3059.2006.01512.x
28. Godard S., Slacanin I., Viret O., Gindro K., 2009. Induction of defence mechanisms in grapevine leaves by emodin- and anthraquinone-rich plant extracts and their conferred resistance to downy mildew. Plant Physiology and Biochemistry, 47 (9), 827–837. https://doi.org/10.1016/j.plaphy.2009.04.003
29. Ismail I., Taylor A.S., Van Den Heuvel S., Borneman A., Sosnowski M.R., 2024. Sensitivity of Plasmopara viticola to selected fungicide groups and the occurrence of the G143A mutant in Australian grapevine isolates. Pest Management Science, 80 (8), 3861–3872. https://doi.org/10.1002/ps.8089
30. Kiefer B., Riemann M., Kassemeyer H.H., Nick P., 2002. The host guides morphogenesis and stomatal targeting in grapevine pathogen Plasmopara viticola. Planta, 215, 387–393. https://doi.org/10.1007/s00425-002-0760-2
31. Kumaşoğlu M.F., Özer N., Aktürk B., Uzun H.İ., 2022. Plasmopara viticola infection process and accumulation of important stilbenes in some grapevine varieties grown in Turkey and in individuals for brined leaves. Journal of Plant Diseases and Protection 129, 967–979. https://doi.org/10.1007/s41348-021-00561-9
32. Langcake P., 1981. Disease resistance of Vitis spp. and the production of the stress metabolites resveratrol, ε-viniferin, α-viniferin and pterostilbene. Physiological Plant Pathology, 18 (2), 213–226. https://doi.org/10.1016/S0048-4059(81)80043-4
33. Liu R., Wang L., Zhu J., Chen T., Wang Y., Xu Y., 2015. Histological responses to downy mildew in resistant and susceptible grapevines. Protoplasma, 252, 259–270. https://doi.org/10.1007/s00709-014-0677-1
34. Massi F., Torriani S., Borghi L., Toffolatti S.L., 2021. Fungicide resistance evolution and detection in plant pathogens: Plasmopara viticola as a case study. Microorganisms, 9 (1), 119. https://doi.org/10.3390/microorganisms9010119
35. Matasci C.L., Gobbin D, Schärer H.J., Tamm L., Gessler C., 2008. Selection for fungicide resistance throughout a growing season in populations of Plasmopara viticola. European Journal of Plant Pathology, 120, 79–83. https://link.springer.com/article/10.1007/s10658-007-9190-0
36. Nanni I.M., Pirondi A., Contaldo N., Collina M., 2016. Screening of sensitivity to mandipropamid of Plasmopara viticola populations from Italian vineyards by molecular and biological methods. Letters in Applied Microbiology, 63 (4), 268–273. https://doi.org/10.1111/lam.12613
37. Nanni I.M., Oggiano I., Laricchia M., Cempini M., Collina M., 2024. Monitoring and tracking changes in sensitivity to zoxamide fungicide in Plasmopara viticola in Italy. Journal of Plant Diseases and Protection, 131, :1211–1216. https://doi.org/10.1007/s41348-024-00947-5
38. Olivier V., Spring J.L., Gindro K., 2018. Stilbenes: biomarkers of grapevine resistance to fungal diseases. OENO One, 52, 3. https://doi.org/10.20870/oeno-one.2018.52.3.2033
39. Özer N., Uzun H.İ., Aktürk B., Özer C., Akkurt M., Aydın S., 2021. Resistance assessment of grapevine leaves to downy mildew with sporulation area scoring. European Journal of Plant Pathology, 160, 337–348. https://doi.org/10.1007/s10658-021-02247-2
40. Paolocci M., Muganu M., Alonso-Villaverde V., Gindro K., 2014. Leaf morphological characteristics and stilbene production differently affect downy mildew resistance of Vitis vinifera varieties grown in Italy. Vitis, 53, 155–161. https://doi.org/10.5073/vitis.2014.53.155-161
41. Pezet R., Perret C., Jean-Denis J.B., Tabacchi R., Gindro K., Viret O., 2003. δ-Viniferin, a resveratrol dehydrodimer: one of the major stilbenes synthesized by stressed grapevine leaves. Journal of Agricultural Food Chemistry, 51, 5488–5492. https://doi.org/10.1021/jf030227o
42. Pezet R., Gindro K., Viret O., Richter H., 2004a. Effects of resveratrol, viniferins and pterostilbene on Plasmopara viticola zoospore mobility and disease development. Vitis, 43, 145–148. https://ojs.openagrar.de/index.php/VITIS/article/view/4364
43. Pezet R., Gindro K., Viret O., Spring J.L., 2004b. Glycosylation and oxidative dimerization of resveratrol are respectively associated to sensitivity and resistance of grapevine cultivars to downy mildew. Physiological and Molecular Plant Pathology, 65, 297–303. https://doi.org/10.1016/j.pmpp.2005.03.002
44. Reuveni M., Arroyo C.J., Ovadia S., 2023. An effective hybrid fungicide containing tea tree oil and difenoconazole for grape powdery mildew management. Agriculture, 13 (5), 979. https://doi.org/10.3390/agriculture13050979
45. Sambrook J., Fritsch E.F., Maniatis T., 1989. Molecular cloning: a laboratory manual, 2nd Edition. Cold Spring Harbor Laboratory Press, New York, USA, 1659 p.
46. Santos R.F., Fraaije B.A., Garrido L.D.R., Monteiro-Vitorello C.B., Amorim L., 2020. Multiple resistance of Plasmopara viticola to QoI and CAA fungicides in Brazil. Plant Pathology, 69, 1708–1720. https://doi.org/10.1111/ppa.13254
47. Smith B.J., 2013. Fruit quality, phytochemical content, and disease severity of muscadine grapes affected by fungicide applications. Pharmaceutical Crops, 4, 21–37. https://doi.org/10.2174/2210290601304010021
48. Toffolatti S.L., Russo G., Campia P., Bianco P.A., Borsa P., Coatti M., Sierotzki H., 2018. A time-course investigation of resistance to the carboxylic acid amide mandipropamid in field populations of Plasmopara viticola treated with anti-resistance strategies. Pest Management Science, 74 (12), 2822–2834. https://doi.org/10.1002/ps.5072
49. Toffolatti S.L., Lecchi B., Maddalena G., Marcianò D., Stuknytė M., Arioli S., Torriani S.F., 2024. The management of grapevine downy mildew: from anti-resistance strategies to innovative approaches for fungicide resistance monitoring. Journal of Plant Diseases and Protection, 131, 1225–1232. https://doi.org/10.1007/s41348-024-00867-4
50. Wang C., Wu J., Zhang Y., Lu J., 2018. Muscadinia rotundifolia ‘Noble’ defense response to Plasmopara viticola inoculation by inducing phytohormone-mediated stilbene accumulation. Protoplasma, 255, 95–107. https://doi.org/10.1007/s00709-017-1118-8
51. Wang Y., Cao X., Han Y., Han X., Wang Z., Xue T., Li H., 2022. Kaolin particle film protects grapevine cv. ‘Cabernet Sauvignon’ against downy mildew by forming particle film at the leaf surface, directly acting on sporangia and inducing the defense of the plant. Frontiers in Plant Science, 12, 796545. https://doi.org/10.3389/fpls.2021.796545