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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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.
KeywordsCercospora leaf spot Novel point mutation Phe167Tyr PCR-RFLP diagnostics PCR mutation-specific diagnostic assay Sugar beet Temperature sensitivity
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.
- 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
- 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.PubMedCrossRefGoogle 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
- 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
- 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
- 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
- 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.
- 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
- 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.CrossRefGoogle Scholar