Molecular and experimental evidence of multi-resistance of Cercospora beticola field populations to MBC, DMI and QoI fungicides

Abstract

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.

Fig. 1
Fig. 2

References

  1. 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.

    CAS  Article  PubMed  Google Scholar 

  2. 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.

    CAS  Article  PubMed  Google Scholar 

  3. 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.

    CAS  Article  PubMed  Google Scholar 

  4. 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.

    CAS  Article  Google Scholar 

  5. 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.

    CAS  Article  PubMed  Google Scholar 

  6. 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.

    CAS  Article  PubMed  Google Scholar 

  7. 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.

    CAS  Article  PubMed  Google Scholar 

  8. 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.

  9. 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.

    CAS  Article  PubMed  Google Scholar 

  10. 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.

    CAS  Article  Google Scholar 

  11. 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.

  12. 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.

    CAS  Article  Google Scholar 

  13. Clement, M., Posada, D., & Crandall, K. A. (2000). TCS: a computer program to estimate gene genealogies. Molecular Ecology, 9, 1657–1659.

    CAS  Article  PubMed  Google Scholar 

  14. 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.

    Google Scholar 

  15. Davidse, L. C. (1986). Benzimidazole fungicides: mechanism of action and biological impact. Annual Review of Phytopathology, 24, 43–65.

    CAS  Article  Google Scholar 

  16. 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.

    CAS  Article  Google Scholar 

  17. 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.

    CAS  Article  PubMed  Google Scholar 

  18. 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.

    Google Scholar 

  19. 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.

    CAS  Article  Google Scholar 

  20. 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.

    CAS  Article  PubMed  Google Scholar 

  21. 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.

    CAS  Article  PubMed  Google Scholar 

  22. FRAC Code List (2016). Fungicide sorted by mode of action (including FRAC Code numbering). www.frac.info.

  23. Georgopoulos, S. G., & Dovas, C. (1973). Occurrence of Cercospora beticola strains resistant to benzimidazole fungicides in northern Greece. Plant Disease Reporter, 57, 321–324.

    Google Scholar 

  24. 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.

    CAS  Article  PubMed  Google Scholar 

  25. Groenewald, M., Groenewald, J. Z., & Crous, P. W. (2005). Distinct species exist within the Cercospora apii morpho-type. Phytopathology, 95, 951–959.

    CAS  Article  PubMed  Google Scholar 

  26. 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.

  27. Jones, A. L., & Ehret, G. R. (1980). Resistance of Coccomyces hiemalis to benzimidazole fungicides. Plant Disease, 64, 767–769.

    Article  Google Scholar 

  28. 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.

    CAS  Article  Google Scholar 

  29. 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.

    Article  Google Scholar 

  30. 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.

    Google Scholar 

  31. 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.

    CAS  Article  Google Scholar 

  32. 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.

    CAS  Article  Google Scholar 

  33. 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.

    CAS  Article  Google Scholar 

  34. 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.

    Article  Google Scholar 

  35. 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.

    Article  Google Scholar 

  36. 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.

    Google Scholar 

  37. 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.

    Google Scholar 

  38. Luo, C. X., & Schnabel, G. (2008). Adaptation to fungicides in Monilinia fructicola isolates with different fungicide resistance phenotypes. Phytopathology, 98, 230–238.

    CAS  Article  PubMed  Google Scholar 

  39. 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.

    CAS  Article  Google Scholar 

  40. 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.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  41. 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.

    CAS  Article  PubMed  Google Scholar 

  42. 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.

    Article  Google Scholar 

  43. 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.

    Google Scholar 

  44. 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.

    Google Scholar 

  45. 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.

    CAS  Article  Google Scholar 

  46. Pearson, R. C., & Taschenberg, E. F. (1980). Benomyl-resistant strains of Uncinula necator on grapes. Plant Disease, 64, 677–680.

    CAS  Article  Google Scholar 

  47. Raicu, C., Tusa, C., Coicev, A., & Jon, S. (1964). Chemical control of Cercospora beticola Sacc. in sugar beets. Hemizacija poljoprivrede, 9, 613–623.

    Google Scholar 

  48. 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.

    CAS  Article  Google Scholar 

  49. 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.

    Google Scholar 

  50. Ruppel, E. G., & Scott, P. R. (1974). Strains of Cercospora beticola resistant to benomyl in the USA. Plant Disease Reporter, 58, 434–436.

    Google Scholar 

  51. 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.

    CAS  Article  PubMed  Google Scholar 

  52. 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.

    Google Scholar 

  53. 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.

    Article  Google Scholar 

  54. 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.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  55. 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.

    CAS  PubMed  PubMed Central  Google Scholar 

  56. 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.

  57. 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.

    Google Scholar 

  58. 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.

    CAS  Article  Google Scholar 

  59. 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.

    CAS  Article  Google Scholar 

  60. 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.

    Article  Google Scholar 

  61. 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.

  62. 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.

    CAS  Article  Google Scholar 

Download references

Acknowledgements

This research has been funded by the national projects III43001 and TR31018 financed by the Ministry of Education, Science and Technological development of Serbia.

Author information

Affiliations

Authors

Corresponding author

Correspondence to Nenad R. Trkulja.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

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

Download citation

Keywords

  • Multi-resistance
  • Cercospora beticola
  • Molecular characterization
  • CYP51
  • ß-tubulin
  • Cob