Cercospora leaf spot (CLS) caused by Cercospora beticola occurs annually in Serbia causing severe yield losses of sugar beet, which requires intensive use of fungicides. In recent years we have observed unsatisfactory control of CLS originating from northwestern Serbia. Frequency of C. beticola populations resistant to Quinone outside inhibitors (QoI) was 81% (51/63 isolates), 98% (62/63) to sterol-demethylation inbibitors (DMI) and 54% (34/63) to methyl-2-benzimidazole carbamate fungicides (MBC). The genetic basis underlying the resistance was tested by characterizing the cob, CYP51 and ß-tubulin genes, associated with resistance to QoI, DMI and MBC fungicides, respectively. Isolates that were resistant to QoI fungicides had the G143A mutation in the cob gene. Characterization of the CYP51 gene revealed seven diverse haplotypes; however, no correlation with sensitivity or resistance to DMI fungicides could be identified. Resistance to MBC fungicides was associated with the presence of the E198A mutation in the ß-tubulin gene of all resistant isolates. From a total of 63 isolates originating from sugar beet fields of northwestern Serbia, 62 isolates showed resistance to multiple modes of action. Three multi-resistant phenotypes were identified: MR1 (N = 29) - resistant to QoI and DMI fungicides (QoI-R and DMI-R) but sensitive to MBC fungicides (MBC-S); MR2 (N = 11, QoI-S, DMI-R and MBC-R); and MR3 (N = 22), resistant to all three groups of fungicides (QoI-R, DMI-R and MBC-R). This is the first report of C. beticola resistance to QoI fungicides in Serbia. This study revealed development of multi-resistance of C. beticola isolates to MBC, DMI and QoI fungicides, which represents the first record of this phenomenon in C. beticola populations.
This is a preview of subscription content, log in to check access.
Buy single article
Instant access to the full article PDF.
Price includes VAT for USA
Subscribe to journal
Immediate online access to all issues from 2019. Subscription will auto renew annually.
This is the net price. Taxes to be calculated in checkout.
Avila-Adame, C., & Köller, W. (2003). Impact of alternative respiration and target-site mutations on responses of germinating conidia of Magnaporthe grisea to Qo-inhibiting fungicides. Pest Management Science, 59, 303–309.
Bardas, G. A., Veloukas, T., Koutita, O., & Karaoglanidis, G. S. (2010). Multiple resistance of Botrytis cinerea from kiwifruit to SDHIs, QoIs and fungicides of other chemical groups. Pest Management Science, 66, 967–973.
Bartlett, D. W., Clough, J. M., Godwin, J. R., Hall, A. A., Hamer, M., & Parr-Dobrzanski, B. (2002). The strobilurin fungicides. Pest Management Science, 58, 649–662.
Birla, K., Rivera-Varas, V., Secor, G. A., Khan, M. F. R., & Bolton, M. D. (2012). Characterization of cytochrome b from European field isolates of Cercospora beticola with quinone outside inhibitor resistance. European Journal of Plant Pathology, 134, 475–488.
Bolton, M. D., Birla, K., Rivera-Varas, V., Rudolph, K., & Secor, G. A. (2012). Characterization of CbCyp51 from field isolates of Cercospora beticola. Phytopathology, 102, 298–305.
Bolton, M. D., Rivera, V., & Secor, G. (2013). Identification of the G143A mutation associated with QoI resistance in Cercospora beticola field isolates from Michigan, United States. Pest Management Science, 69, 35–39.
Bolton, M. D., Ebert, M. K., Faino, L., Rivera-Varas, V., de Jonge, R., Van de Peer, Y., Thomma, B. P. H. J., & Secor, G. A. (2016). RNA-sequencing of Cercospora beticola DMI-sensitive and –resistant isolates after treatment with tetraconazole identifies common and contrasting pathway induction. Fungal Genetics and Biology, 92, 1–13.
Brown, M. C., Waller, C. D., Charlet, C, Palmer, R. (1986). The use of flutriafol based fungicides for the control of sugar beet diseases in Europe. In Proc. BCPC – pests and diseases, BCPC, Farnham, Surrey, UK.
Brunner, P. C., Stefanato, F. L., & McDonald, B. A. (2008). Evolution of the CYP51 gene in Mycosphaerella graminicola evidence for intragenic and selective replacement. Molecular Plant Pathology, 9, 305–316.
Budakov, D., Nagl, N., Stojšin, V., Bagi, F., Danojević, D., Neher, T. N., & Taski-Ajduković, K. (2014). Sensitivity of Cercospora beticola isolates from Serbia to carbendazim and flutriafol. Crop Protection, 66, 120–126.
Byford, W. J. (1996). A survey of foliar diseases of sugar beet and their control in Europe. In 59th International Institute of Beet Research (IIRB) Conference.
Chapman, K. S., Sundin, G. W., & Beckerman, J. L. (2011). Identification of resistance to multiple fungicides in field populations of Venturia inaequalis. Plant Disease, 95, 921–926.
Clement, M., Posada, D., & Crandall, K. A. (2000). TCS: a computer program to estimate gene genealogies. Molecular Ecology, 9, 1657–1659.
Cools, H., & Fraaije, B. (2012). Resistance to azole fungicides in Mycosphaerella graminicola: Mechanisms and management. In T. S. Thind (Ed.), Fungicide resistance in Crop protection risk and management. Wallingford: CAB International.
Davidse, L. C. (1986). Benzimidazole fungicides: mechanism of action and biological impact. Annual Review of Phytopathology, 24, 43–65.
Davidson, R. M., Hanson, L. E., Franc, G. D., & Panella, L. (2006). Analysis of ß-tubulin gene fragments from benzimidazole sensitive and tolerant Cercospora beticola. Journal of Phytopathology, 154, 321–328.
De Miccolis Angelini, R. M., Rotolo, C., Masiello, M., Gerin, D., Pollastro, S., & Faretra, F. (2014). Occurrence of fungicide resistance in populations of Botryotinia fuckeliana (Botrytis cinerea) on table grape and strawberry in southern Italy. Pest Management Science, 70, 1785–1796.
Edin, E., & Torriani, S. (2012). The role of intraspecific parallel genetic adaptation to QoIs in Europe. In T. S. Thind (Ed.), Fungicide resistance in Crop protection risk and management. Wallingford: CAB International.
Estep, L. K., Torriani, S. F. F., Zala, M., Anderson, N. P., Flowers, M. D., McDonald, B. A., Mundt, C. C., & Brunner, P. C. (2015). Emergence and early evolution of fungicide resistance in North American populations of Zymoseptoria tritici. Plant Pathology, 64, 961–971.
Fiaccadori, R., Cicognani, E., Alberoni, G., Collina, M., & Brunelli, A. (2011). Sensitivity to strobilurin fungicides of Italian Venturia inaequalis populations with different origin and scab control. Pest Management Science, 67, 535–540.
Fontaine, S., Remuson, F., Fraissinet-Tachet, L., Micoud, A., Marmeisse, R., & Melayah, D. (2009). Monitoring of Venturia inaequalis harbouring the QoI resistance G143A mutation in French orchards as revealed by PCR assays. Pest Management Science, 65, 74–81.
FRAC Code List (2016). Fungicide sorted by mode of action (including FRAC Code numbering). www.frac.info.
Georgopoulos, S. G., & Dovas, C. (1973). Occurrence of Cercospora beticola strains resistant to benzimidazole fungicides in northern Greece. Plant Disease Reporter, 57, 321–324.
Gisi, U., Sierotzki, H., Cook, A., & McCaffery, A. (2002). Mechanisms influencing the evolution of resistance to Qo inhibitor fungicides. Pest Management Science, 58, 859–867.
Groenewald, M., Groenewald, J. Z., & Crous, P. W. (2005). Distinct species exist within the Cercospora apii morpho-type. Phytopathology, 95, 951–959.
Holtschulte, B. (2000). Cercospora beticola – Worldwide distribution and incidence, in advances in sugar beet research. Vol. 2, Cercospora beticola Sacc. Biology, agronomic influence and control measures in sugar beet 2000. Int. Inst. Beet Res. Brussels.
Jones, A. L., & Ehret, G. R. (1980). Resistance of Coccomyces hiemalis to benzimidazole fungicides. Plant Disease, 64, 767–769.
Joseph-Horne, T., & Hollomon, D. W. (2000). Functional diversity within the mitochondrial electron transport chain of plant pathogenic fungi. Pest Management Science, 56, 24–30.
Karadimos, D. A., & Karaoglanidis, G. S. (2006). Comparative efficacy, selection of effective partners and application time of strobilurin fungicides for control of Cercospora leaf-spot of sugar beet. Plant Disease, 90, 820–825.
Karaoglanidis, G. S., & Ioannidis, P. M. (2010). Fungicide resistance of Cercospora beticola in Europe. In R. T. Lartey, J. J. Weiland, L. Panella, P. W. Crous, & C. E. Windels (Eds.), Cercospora leaf spot of sugar Beet and related species. American Phytopathological Society: St. Paul.
Karaoglanidis, G. S., & Thanassoulopoulos, C. C. (2002). Phenotypic instability of resistance to sterol demethylation inhibiting fungicides (DMIs) in Cercospora beticola. Journal of Phytopathology, 150, 692–696.
Karaoglanidis, G. S., Ioannidis, P. M., & Thanassoulopoulos, C. C. (2000). Reduced sensitivity of Cercospora beticola isolates to sterol demethylation inhibiting fungicides. Plant Pathology, 49, 567–572.
Karaoglanidis, G. S., Loannidis, P. M., & Thanassoulopoulos, C. C. (2002). Changes in sensitivity of Cercospora beticola populations to sterol-demethylation-inhibiting fungicides during a 4-year period in northern Greece. Plant Pathology, 51, 55–62.
Kirk, W., Hanson, L., Franc, G., Stump, W., Gachango, E., Clark, G., & Stewart, J. (2012). First report of strobilurin resistance in Cercospora beticola in sugarbeet Beta vulgaris in Michigan and Nebraska, USA. New Disease Reports, 26, 3.
Koenraadt, H., Somerville, S. C., & Jones, A. L. (1992). Characterization of mutations in the beta-tubulin gene of benomyl-resistant field strains of Venturia inaequalis and other plant pathogenic fungi. Phytopathology, 82, 1348–1354.
Köller, W. (1991). Fungicide resistance in plant pathogens. In D. Pimentel & A. A. Hanson (Eds.), Handbook of Pest Management in Agriculture (Vol. II). Boca Raton: CRC Press.
Kristek, A., Glavaš–Tokić, R., Kristek, S., & Antunović, M. (2006). Importance of sort selection and fungicides application in Cercospora beticola sacc. Sugar beet leaf spot prevention and quality root high yield achievement. Poljoprivreda, 1, 27–34.
Luo, C. X., & Schnabel, G. (2008). Adaptation to fungicides in Monilinia fructicola isolates with different fungicide resistance phenotypes. Phytopathology, 98, 230–238.
Ma, Z., & Michailides, T. J. (2005). Advances in understanding molecular mechanisms of fungicide resistance and molecular detection of resistant genotypes in phytopathogenic fungi. Crop Protection, 24, 853–863.
Ma, Z., Yoshimura, M., & Michailides, T. J. (2003). Identification and characterization of benzimidazole resistance in Monilinia fructicola from stone fruit orchards in California. Applied and Environmental Microbiology, 69, 7145–7152.
Ma, Z. H., Yoshimura, M. A., Holtz, B. A., & Michailides, T. J. (2005). Characterization and PCR-based detection of benzimidazole-resistant isolates of Monilinia laxa in California. Pest Management Science, 61, 449–457.
Malandrakis, A., Markoglou, A., Nikou, D., Vontas, J., & Ziogas, B. (2006). Biological and molecular characterization of laboratory mutants of Cercospora beticola resistant to Qo inhibitors. European Journal of Plant Pathology, 116, 155–166.
Marić, A., Petrov, M., & Maširević, S. (1976). Pojava tolerantnosti kod Cercospora beticola Sacc. prema benomilu u Jugoslaviji i mogućnosti suzbijanja ovog parazita. Zaštita Bilja, 27, 227–236.
Marić, A., Maširević, S., & Jercović, Z. (1984). Increase in resistance of Cercospora beticola to benomyl and first occurrence of strains tolerant to fentin acetate in Vojvodini. Zaštita Bilja, 35, 207–215.
Nikou, D., Malandrakis, A., Konstantakaki, M., Vontas, J., Markoglou, A., & Ziogas, B. (2009). Molecular characterization and detection of overexpressed C-14 alpha-demethylase-based DMI resistance in Cercospora beticola field isolates. Pesticide Biochemistry and Physiology, 95, 18–27.
Pearson, R. C., & Taschenberg, E. F. (1980). Benomyl-resistant strains of Uncinula necator on grapes. Plant Disease, 64, 677–680.
Raicu, C., Tusa, C., Coicev, A., & Jon, S. (1964). Chemical control of Cercospora beticola Sacc. in sugar beets. Hemizacija poljoprivrede, 9, 613–623.
Rosenzweig, N., Hanson, L. E., Clark, G., Jiang, Q. W., Franc, G. D., Stewart, J., & Kirk, W. W. (2015). Use of PCR-RFLP analysis to monitor fungicide resistance in Cercospora beticola populations from sugarbeet (Beta vulgaris) in Michigan, United States. Plant Disease, 99, 355–362.
Rossi, V., Racca, P., & Giosue, S. (1995). Geophytopathological analysis of Cercospora leaf spot of sugar beet in the Mediterranean area. Phytopathologia Mediterranea, 34, 69–82.
Ruppel, E. G., & Scott, P. R. (1974). Strains of Cercospora beticola resistant to benomyl in the USA. Plant Disease Reporter, 58, 434–436.
Samuel, S., Papayiannis, L. C., Leroch, M., Veloukas, T., Hahn, M., & Karaoglanidis, G. S. (2011). Evaluation of the incidence of the G143A mutation and cyt b intron presence in the cytochrome bc-1 gene conferring QoI resistance in Botrytis cinerea populations from several hosts. Pest Management Science, 67, 1029–1036.
Schnabel, G., Amiri, A., & Brannen, P. M. (2012). Field kit-and internet-supported fungicide resistance Monitoring. In T. S. Thind (Ed.), Fungicide resistance in Crop protection risk and management. Wallingford: CAB International.
Secor, G. A., Rivera, V. V., Khan, M. F. R., & Gudmestad, N. C. (2010). Monitoring fungicide sensitivity of Cercospora beticola of sugar beet for disease management decisions. Plant Disease, 94, 1272–1282.
Tamura, K., Stecher, G., Peterson, D., Filipski, A., & Kumar, S. (2013). MEGA6: Molecular evolutionary genetics analysis version 6.0. Molecular Biology and Evolution, 30, 2725–2729.
Templeton, A. R., Crandall, K. A., & Sing, C. F. (1992). A cladistic analysis of phenotypic associations with haplotypes inferred from restriction endonuclease mapping and DNA-sequence data. III. Cladogram estimation. Genetics, 132, 619–633.
Trkulja, N. (2013). Resistance of Cercospora beticola Sacc. to fungicides from benzimidazoles and triazoles groups and molecular characterization of the resistance mechanisms. PhD Thesis, Megatrend University, Faculty of Biofarming, Bačka Topola, Serbia.
Trkulja, N., Aleksić, G., Starović, M., Dolovac, N., Ivanović, Ž., & Živković, S. (2009). Osetljivost izolata Cercospora beticola prema karbendazimu i flutriafolu u Srbiji. Zaštita Bilja, 270, 237–245.
Trkulja, N., Ivanović, Ž., Pfaf-Dolovac, E., Dolovac, N., Mitrović, M., Toševski, I., & Jović, J. (2013). Characterisation of benzimidazole resistance of Cercospora beticola in Serbia using PCR-based detection of resistance-associated mutations of the β-tubulin gene. European Journal of Plant Pathology, 135, 889–902.
Trkulja, N., Milosavljević, A., Stanisavljević, R., Mitrović, M., Jović, J., Toševski, I., & Bošković, J. (2015). Occurrence of Cercospora beticola populations resistant to benzimidazoles and demethylation-inhibiting fungicides in Serbia and their impact on disease management. Crop Protection, 75, 80–87.
Trueman, C. L., Hanson, L. E., Rosenzweig, N., Jiang, Q. W., & Kirk, W. W. (2013). First report of QoI insensitive Cercospora beticola on sugar beet in Ontario, Canada. Plant Disease, 97, 1255.
Ziogas, B. N., & Malandrakis, A. A. (2015). Sterol biosynthesis inhibitors: C14 demethylation (DMIs). In H. Ishii & W. D. Hollomon (Eds.), Fungicide resistance in plant pathogens: Principles and a guide to practical management. Tokyo: Springer.
Ziogas, B. N., Nikou, D., Markoglou, A. N., Malandrakis, A. A., & Vontas, J. (2009). Identification of a novel point mutation in the β-tubulin gene of Botrytis cinerea and detection of benzimidazole resistance by a diagnostic PCR-RFLP assay. European Journal of Plant Pathology, 125, 97–107.
This research has been funded by the national projects III43001 and TR31018 financed by the Ministry of Education, Science and Technological development of Serbia.
About this article
Cite this article
Trkulja, N.R., Milosavljević, A.G., Mitrović, M.S. et al. Molecular and experimental evidence of multi-resistance of Cercospora beticola field populations to MBC, DMI and QoI fungicides. Eur J Plant Pathol 149, 895–910 (2017). https://doi.org/10.1007/s10658-017-1239-0
- Cercospora beticola
- Molecular characterization