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Characterisation of benzimidazole resistance of Cercospora beticola in Serbia using PCR-based detection of resistance-associated mutations of the β-tubulin gene

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Abstract

A survey to detect and characterise benzimidazole resistance within populations of Cercospora beticola in Serbia was performed. From 52 field isolates collected from sugar beet and beet root, only eight were found to be benzimidazole-sensitive based on the inhibition of mycelial growth by discriminatory concentrations of carbendazim and thiophanate-methyl. Sensitivity tests revealed the presence of three resistant phenotypes among the tested isolates: high-resistance (HR), low-resistance (LR) and moderate-resistance (MR). The benzimidazole resistant isolates were characterised based on the DNA sequence of the β-tubulin gene and temperature sensitivity. The HR isolates showed no temperature sensitivity regardless of carbendazim concentration, whereas the LR and MR isolates were sensitive at lower temperatures. Analysis of the β-tubulin gene sequence revealed two amino acid replacements in the benzimidazole-resistant isolates of C. beticola. One was a glutamic acid to alanine change at position 198 (codon GAG to GCG) that was identified in HR isolates; this mutation has previously been reported to be associated with the development of benzimidazole resistance in C. beticola. The second replacement was a novel point mutation of phenylalanine (TTC) to tyrosine (TAC) at position 167, identified in low and moderate benzimidazole-resistant isolates, sharing a single LR/MR β-tubulin genotype. A diagnostic PCR-RFLP assay utilising a BsaI restriction site present in the benzimidazole sensitive and LR/MR genotypes but absent in the HR genotype was developed for the routine detection of high resistance. A mutation-specific PCR assay was developed for the diagnosis of LR/MR genotype based on a mutation from T to A at codon 167, which is unique to this genotype.

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References

  • Albertini, C., Gredt, M., & Leroux, P. (1999). Mutations of the β-tubulin gene associated with different phenotypes of benzimidazole resistance in the cereal eyespot fungi Tapesia yallundae and Tapesia acuformis. Pesticide Biochemistry Physiology, 64, 17–31.

    Article  CAS  Google Scholar 

  • Baraldi, E., Mari, M., Chierici, E., Pondrelli, M., Bertolini, P., & Pratella, G. C. (2003). Studies on thiabendazole resistance of Penicillium expansum of pears: pathogenic fitness and genetic characterization. Plant Pathology, 52, 362–370.

    Article  CAS  Google Scholar 

  • Canas-Gutierrez, G. P., Patino, L. F., Rodriguez-Arango, E., & Arango, R. (2006). Molecular characterization of benomyl-resistant isolates of Mycosphaerella fijiensis, collected in Colombia. Journal of Phytopathology, 154, 403–409.

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  • Davidse, L., & Ishii, T. (1995). Biochemical and molecular aspects of benzimidazoles, N-phenylcarbamates and N-phenylformamidoxines and the mechanisms of resistance to these compounds in fungi. In H. Lyr (Ed.), Modern selective fungicides (pp. 305–322). Jena: Gustav Fisher.

    Google Scholar 

  • 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.

    Article  CAS  Google Scholar 

  • Dovas, C., Skylakakis, G., & Georgopoulos, S. G. (1976). The adaptability of benomyl-resistant population of Cercospora beticola in northern Greece. Phytopathology, 66, 1452–1456.

    Article  CAS  Google Scholar 

  • Gafur, A., Tanaka, C., Shimizu, K., Ouchi, S., & Tsuda, M. (1998). Molecular analysis and characterization of the Cochliobolus heterostrophus β-tubulin gene and its possible role in conferring resistance to benomyl. The Journal of General and Applied Microbiology, 44(3), 217–223.

    Article  PubMed  CAS  Google Scholar 

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

    Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  • 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.

    Article  CAS  Google Scholar 

  • Karaoglanidis, G. S., Ioannidis, 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.

    Article  CAS  Google Scholar 

  • Karaoglanidis, G. S., Karadimos, D. A., Ioannidis, P. M., & Ioannidis, P. I. (2003). Sensitivity of Cercospora beticola populations to fentin-acetate, benomyl and flutriafol in Greece. Crop Protection, 22, 735–740.

    Article  CAS  Google Scholar 

  • 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 

  • Luo, Y., Ma, Z., & Michailides, T. (2007). Quantification of allele E198A in beta-tubulin conferring benzimidazole resistance in Monilinia fructicola using real-time PCR. Pest Management Science, 63, 1178–1184.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    Article  CAS  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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 

  • Maymon, M., Zveibil, A., Pivonia, S., Minz, D., & Freeman, S. (2006). Identification and characterization of benomyl resistant and sensitive populations of Colletotrichum gloeosporioides from Statice (Limonium spp.). Phytopathology, 96, 542–548.

    Article  PubMed  CAS  Google Scholar 

  • McKay, G. J., Egan, D., Morris, E., & Brown, A. E. (1998). Identification of benzimidazole resistance in Cladobotryum dendroides using a PCR-based method. Mycological Research, 102, 671–676.

    Article  CAS  Google Scholar 

  • Orbach, M. J., Porro, E. B., & Yanofsky, C. (1986). Cloning and characterization of the gene for fl-tubulin from a benomyl-resistant mutant of Neurospora crassa and its use as a dominant selectable marker. Molecular and Cellular Biology, 6, 2452–2461.

    PubMed  CAS  Google Scholar 

  • Qiu, J., Xu, J., Yu, J., Bi, C., Chen, C., & Zhou, M. (2011). Localisation of the benzimidazole fungicide binding site of Gibberella zeae β2-tubulin studied by site-directed mutagenesis. Pest Management Science, 67, 191–198.

    Article  PubMed  CAS  Google Scholar 

  • 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 

  • Russell, P. E. (2004). Sensitivity baselines in fungicide resistance research and management. FRAC Monograph No. 3. Brussels: Crop Life International.

    Google Scholar 

  • Saito, S., Suzuki, S., & Takayanagi, T. (2009). Nested PCR-RFLP is a high-speed method to detect fungicide-resistant Botrytis cinerea at an early growth stage of grapes. Pest Management Science, 65, 197–204.

    Article  PubMed  CAS  Google Scholar 

  • Shane, W. W., & Teng, P. S. (1992). Impact of Cercospora leaf spot on root weight, sugar yieldand purity of Beta vulgaris. Plant Disease, 76, 812–820.

    Article  CAS  Google Scholar 

  • Sisler, H. D. (1988). Fungicidal action and fungal resistance mechanisms. In C. J. Delp (Ed.), Fungicide resistance in North America (pp. 6–8). Minnesota: APS Pres.

    Google Scholar 

  • Solel, Z. (1970). The systemic fungicidal effect of benzimidazole derivatives and thiophanate against Cercospora leaf spot of sugar beet. Phytopathology, 60, 1186–1190.

    Article  CAS  Google Scholar 

  • Stallkercht, H. F., & Crane, G. L. (1969). The therapeutic effect of thiabendazole, 2- (4′-thiazolyl) benzimidazole, against Cercospora leaf spot on sugar beets when applied as a soil drench. Phytopathology, 59, 393.

    Google Scholar 

  • StatSoft, Inc. (2007). STATISTICA (data analysis software system), version 8.0. www.statsoft.com.

  • Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., & Kumar, S. (2011). MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology and Evolution, 28, 2731–2739.

    Article  PubMed  CAS  Google Scholar 

  • 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 

  • Weiland, J. J., & Halloin, J. M. (2001). Benzimidazole resistance in Cercospora beticola sampled from sugarbeet fields in Michigan, USA. Canadian Journal of Plant Pathology, 23, 78–82.

    Article  CAS  Google Scholar 

  • Yarden, O., & Katan, T. (1993). Mutations leading to substitutions at amino acids 198 and 200 of beta-tubulin that correlates with benomyl resistance phenotypes of field strains of Botrytis cinerea. Phytopathology, 83, 1478–1483.

    Article  CAS  Google Scholar 

  • Zhang, C. Q., Liu, Y. H., & Zhu, G. N. (2010). Detection and characterization of benzimidazole resistance of Botrytis cinerea in greenhouse vegetables. European Journal of Plant Pathology, 126, 509–515.

    Article  CAS  Google Scholar 

  • 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.

    Article  CAS  Google Scholar 

Download references

Acknowledgments

We thank the anonymous reviewers for their constructive comments. This research was supported by the grants number TR31018 and III43001 from the Ministry of Education, Science and Technological Development of the Republic of Serbia.

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Authors and Affiliations

  1. Department of Plant Disease, Institute for Plant Protection and Environment, Teodora Drajzera 9, 11000, Belgrade, Serbia

    Nenad Trkulja, Žarko Ivanović, Erika Pfaf-Dolovac & Nenad Dolovac

  2. Department of Plant Pests, Institute for Plant Protection and Environment, Banatska 33, 11080, Zemun, Serbia

    Milana Mitrović, Ivo Toševski & Jelena Jović

  3. CABI Europe—Switzerland, 1 Rue des Grillons, 2800, Delémont, Switzerland

    Ivo Toševski

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  1. Nenad Trkulja
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  2. Žarko Ivanović
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  3. Erika Pfaf-Dolovac
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Correspondence to Jelena Jović.

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Trkulja, N., Ivanović, Ž., Pfaf-Dolovac, E. et al. Characterisation of benzimidazole resistance of Cercospora beticola in Serbia using PCR-based detection of resistance-associated mutations of the β-tubulin gene. Eur J Plant Pathol 135, 889–902 (2013). https://doi.org/10.1007/s10658-012-0135-x

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  • DOI: https://doi.org/10.1007/s10658-012-0135-x

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