Characterization of the Molecular Mechanisms of Resistance against DMI Fungicides in Cercospora beticola Populations from the Czech Republic

Abstract

Cercospora leaf spot (CLS), caused by the fungal pathogen Cercospora beticola, is the most important foliar pathogen of sugar beet worldwide. Extensive reliance on fungicides to manage CLS has resulted in the evolution of fungicide resistance in C. beticola worldwide, including populations in the Czech Republic. One important class of fungicides used to manage CLS is the sterol demethylation inhibitors (DMI). The aim of our study was to assess DMI resistance in C. beticola from the Czech Republic and elucidate the molecular basis of DMI resistance in this population. A total of 50 isolates were collected in 2018 and 2019 from the major sugar beet growing regions of the Czech Republic and assessed for in vitro sensitivity to the DMI fungicides propiconazole, prochloraz, and epoxiconazole. These analyses identified three strains that exhibited 50% effective concentration (EC50) values > 1.0 μg mL–1 against respective fungicides, which were therefore considered resistant. In contrast, strains that exhibited lowest EC50 values were considered sensitive. To explore the molecular basis of resistance in these three strains, the cytochrome P450-dependent sterol 14α-demethylase (Cyp51) gene was sequenced. Sequence analysis identified a Y464S mutation in all three resistant strains. To assess whether Cyp51 gene expression may play a role in DMI resistance, selected strains were grown in vitro with and without fungicide treatment. These analyses indicated that Cyp51 gene expression was significantly induced after fungicide treatment. Thus, we conclude that Y464S point mutation along with induced Cyp51 gene overexpression is likely responsible for resistance against DMI fungicides in C. beticola from the Czech Republic.

In Vitro Evaluation of Azoxystrobin, Boscalid, Fentin-Hydroxide, Propiconazole, Pyraclostrobin Fungicides against Alternaria alternata Pathogen Isolated from Carya illinoinensis in South Africa

“…Ergosterol is the most common sterol in fungi because it is an important component of the fungal cell membrane and is essential for fungal growth [ 51 ]. However, the resistance to DMIs has been identified in some phytopathogenic fungal species of Aspergillus , Fusarium , Rhynchosporium, Penicillium [ 52 , 53 , 54 , 55 ], Venturia nashicola [ 56 ], Villosiclava virens [ 57 ], and Cercospora beticola [ 58 ] for their ability to substitute some amino acids, such as CYP51, in the target protein. Future preliminary studies on the molecular characterization of mutational genes, such as CYP51A , CYP51B , and CYP51C , found in filamentous fungi resulting in amino acid substitutions that alter the structure of the CYP51 protein [ 59 , 60 , 61 ] are worth investigating as a tool to better elucidate the resistance mechanism of A. alternata .…”

Inactivation of Cercospora lactucae-sativa through Application of Non-Thermal Atmospheric Pressure Gliding Arc, Tesla Coil and Dielectric Barrier Discharge Plasmas

“…lactucae-sativa is an airborne plant pathogen that can infect dozens of plants, and brassicaceae plants are one of its main targets [4,5]. The fungus is difficult to eliminate and can be resistant to fungicides due to its extensive reliance on fungicides [28]. Moreover, in organic farming, the incidence and severity of leaf spot disease on lettuce are regularly observed to result in a reduction in product quality and quantity [2].…”

Alterations in CYP51 of Cercospora beticola and their effects on DMI sensitivity

“…After 6 days, isolates were transferred onto V8 agar (2% agar, 15% vegetable juice, 0,3% CaCO3) for conidial production. Isolates from 2021-2022 (all = 406) were from Austria (18), Czech Republic (24), France (106), Germany (51), Lithuania (24), Poland (115), Romania (20), Switzerland (18), Türkiye (9) and UK (21).…”

“…A minor increase in EC 50 values in European populations was reported by Muellender et al (2021), who identi ed several point mutations in the DMI target gene sterol 14α-demethylase (CYP51). In addition to the detection of point mutations in the target gene that lead to an amino acid exchange (Kumar et al 2021; Muellender et al 2021), CYP51 overexpression (Nikou et al 2009;Muellender et al 2021), variable codon usage, and a silent mutation (Spanner et al 2021) have also been discussed as mechanisms for DMI adaptation in C. beticola. In their studies with isolates from the United States, Spanner et al (2021) reported that the phenylalanine of mutation L144F can be encoded by two different base triplets, TTT or TTC, both of which are associated with increased EC 50 values for tetraconazole.…”