Molecular characterization and detection of overexpressed C-14 alpha-demethylase-based DMI resistance in Cercospora beticola field isolates D. Nikou, A. Malandrakis, M. Konstantakaki, J. Vontas, A. Markoglou, B. Ziogas * Laboratory of Pesticide Science, Agricultural University of Athens, Votanikos, 118 55 Athens, Greece article info Article history: Received 15 December 2008 Accepted 30 April 2009 Available online 8 May 2009 Keywords: CYP51 Resistance mutations Real time PCR PCR–RFLPs Diagnostics abstract Cercospora beticola strains were isolated from sugar beet fields in N. Greece heavily sprayed with tria- zoles. Two resistant phenotypes with resistance factors ranging from 18 to 25 and 65 to 115 were iden- tified. Epoxiconazole-resistant isolates were less sensitive to the triazole flutriafol but not to the benzimidazole benomyl and the carboxamide boscalid. No fitness penalties were associated with resis- tance mutations in the highly resistant phenotype and most isolates retained their resistance levels even after four generations on fungicide free medium. The C-14 alpha-demethylase gene (CYP51) was isolated for the first time in C. beticola. Sequence comparison between resistant and sensitive strains revealed three mutations in only two out of five of the resistant isolates. However, 3-dimensional model analysis showed that these mutations are unlikely to interfere with fungicide binding. Study of transcriptional levels of the C-14 alpha-demethylase gene showed that overexpression was strongly associated with the highly DMI-resistant phenotype. A neutral mutation observed only in highly resistant isolates permit- ted the development of a PCR–RFLP molecular resistance diagnostic. Ó 2009 Elsevier Inc. All rights reserved. 1. Introduction Cercospora beticola, causal agent of Cercospora leaf spot disease, is one of the most serious pathogens infecting sugar beets in Greece and other areas with warm–humid climatic conditions that favor the development of the disease. Control measures against the fungus include resistant sugar beet varieties and crop rotation but the disease is managed effectively only when combined with fre- quent fungicide applications. Different chemistries ranging from the organotin, dithiocarba- mate and benzimidazole fungicides to the mainly used today sterol biosynthesis inhibiting triazoles and the recently introduced QoIs, have been used over the years for the control of the disease. How- ever, fungicide resistance development has resulted in serious problems in the efficacy of chemical control [1]. Effectiveness of benzimidazoles, the first group of site-specific inhibitors against C. beticola was compromised by resistance development shortly after their introduction [2,3]. DMI fungicides replaced benzimidazoles when a number of fun- gicides from the triazole group were registered against Cercospora leaf spot disease [4]. DMI fungicides, the larger member of the chemical group of EBIs, inhibit the sterol C-14 demethylation dur- ing the process of ergosterol formation in higher fungi [5,6]. They have been introduced in agriculture to control a large number of plant pathogens since 1970 and have been used for the control of C. beticola for almost 3 decades. The step-wise population shift to- wards reduced sensitivity to DMIs has led to practical control prob- lems since the 1980s [7]. Studies on mechanisms involved in DMI resistance have re- ported target-site modifications in the C-14 alpha-demethylase (CYP51) gene [8–11], overexpression of the target gene [12–14] and increased expulsion mediated through increased activity of ABC transporters [15]. Triazole-resistant isolates of C. beticola have been reported in the last 10 years in Greece [1,4]. A study of sterol content in DMI-resistant and sensitive isolates did not reveal the mechanism by which resistance is achieved in the pathogen population in Greece [16]. In the present work, a number of C. beticola isolates resistant to triazoles from Greek sugar beet fields were identified, and subse- quently used to study the mechanism and molecular basis for resistance to DMIs by assessing the possible role of target gene (CYP51) modification and overexpression. 2. Materials and methods 2.1. Fungal strains and culture conditions Ten strains of C. beticola Sacc. isolated from sugar beet fields in Aminteo, Veria, Xanthi and Serres Greece and a wild type (222s) characterized previously [17] were used in this study. 0048-3575/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.pestbp.2009.04.014 * Corresponding author. Fax: +30 210 5294514. E-mail address: ziv@aua.gr (B. Ziogas). Pesticide Biochemistry and Physiology 95 (2009) 18–27 Contents lists available at ScienceDirect Pesticide Biochemistry and Physiology journal homepage: www.elsevier.com/locate/pest