Characterisation of benzimidazole resistance of Cercospora beticola in Serbia using PCR-based detection of resistance-associated mutations of the beta-tubulin gene
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2013
Authors
Trkulja, NenadIvanović, Žarko
Pfaf-Dolovac, Erika
Dolovac, Nenad
Mitrović, Milana
Toševski, Ivo
Jović, Jelena
Article (Published version)
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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 beta-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 beta-tubulin gene sequence revealed two amino acid replacements in the benzimidazole-resistant isolates of C. beticola. One was a glutamic acid to alanine chang...e 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 beta-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.
Keywords:
Cercospora leaf spot / Novel point mutation Phe167Tyr / PCR-RFLP diagnostics / PCR mutation-specific diagnostic assay / Sugar beet / Temperature sensitivitySource:
European Journal of Plant Pathology, 2013, 135, 4, 889-902Publisher:
- Springer, Dordrecht
Funding / projects:
- Development of integrated approach in plant protection for control harmful organisms (RS-31018)
- Agrobiodiversity and land-use change in Serbia: an integrated biodiversity assessment of key functional groups of arthropods and plant pathogens (RS-43001)
DOI: 10.1007/s10658-012-0135-x
ISSN: 0929-1873
WoS: 000315601500025
Scopus: 2-s2.0-84874569380
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IZBISTY - JOUR AU - Trkulja, Nenad AU - Ivanović, Žarko AU - Pfaf-Dolovac, Erika AU - Dolovac, Nenad AU - Mitrović, Milana AU - Toševski, Ivo AU - Jović, Jelena PY - 2013 UR - https://plantarum.izbis.bg.ac.rs/handle/123456789/272 AB - 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 beta-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 beta-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 beta-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. PB - Springer, Dordrecht T2 - European Journal of Plant Pathology T1 - Characterisation of benzimidazole resistance of Cercospora beticola in Serbia using PCR-based detection of resistance-associated mutations of the beta-tubulin gene EP - 902 IS - 4 SP - 889 VL - 135 DO - 10.1007/s10658-012-0135-x ER -
@article{ author = "Trkulja, Nenad and Ivanović, Žarko and Pfaf-Dolovac, Erika and Dolovac, Nenad and Mitrović, Milana and Toševski, Ivo and Jović, Jelena", year = "2013", 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 beta-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 beta-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 beta-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.", publisher = "Springer, Dordrecht", journal = "European Journal of Plant Pathology", title = "Characterisation of benzimidazole resistance of Cercospora beticola in Serbia using PCR-based detection of resistance-associated mutations of the beta-tubulin gene", pages = "902-889", number = "4", volume = "135", doi = "10.1007/s10658-012-0135-x" }
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 beta-tubulin gene. in European Journal of Plant Pathology Springer, Dordrecht., 135(4), 889-902. https://doi.org/10.1007/s10658-012-0135-x
Trkulja N, Ivanović Ž, Pfaf-Dolovac E, Dolovac N, Mitrović M, Toševski I, Jović J. Characterisation of benzimidazole resistance of Cercospora beticola in Serbia using PCR-based detection of resistance-associated mutations of the beta-tubulin gene. in European Journal of Plant Pathology. 2013;135(4):889-902. doi:10.1007/s10658-012-0135-x .
Trkulja, Nenad, Ivanović, Žarko, Pfaf-Dolovac, Erika, Dolovac, Nenad, Mitrović, Milana, Toševski, Ivo, Jović, Jelena, "Characterisation of benzimidazole resistance of Cercospora beticola in Serbia using PCR-based detection of resistance-associated mutations of the beta-tubulin gene" in European Journal of Plant Pathology, 135, no. 4 (2013):889-902, https://doi.org/10.1007/s10658-012-0135-x . .