Characterization of CbCyp51 from Field Isolates of Cercospora beticola

Abstract

The hemibiotrophic fungus Cercospora beticola causes leaf spot of sugar beet. Leaf spot control measures include the application of sterol demethylation inhibitor (DMI) fungicides. However, reduced sensitivity to DMIs has been reported recently in the Red River Valley sugar beet-growing region of North Dakota and Minnesota. Here, we report the cloning and molecular characterization of CbCyp51, which encodes the DMI target enzyme sterol P450 14α-demethylase in C. beticola. CbCyp51 is a 1,632-bp intron-free gene with obvious homology to other fungal Cyp51 genes and is present as a single copy in the C. beticola genome. Five nucleotide haplotypes were identified which encoded three amino acid sequences. Protein variant 1 composed 79% of the sequenced isolates, followed by protein variant 2 that composed 18% of the sequences and a single isolate representative of protein variant 3. Because resistance to DMIs can be related to polymorphism in promoter or coding sequences, sequence diversity was assessed by sequencing >2,440 nucleotides encompassing CbCyp51 coding and flanking regions from isolates with varying EC(50) values (effective concentration to reduce growth by 50%) to DMI fungicides. However, no mutations or haplotypes were associated with DMI resistance or sensitivity. No evidence for alternative splicing or differential methylation of CbCyp51 was found that might explain reduced sensitivity to DMIs. However, CbCyp51 was overexpressed in isolates with high EC(50) values compared with isolates with low EC(50) values. After exposure to tetraconazole, isolates with high EC(50) values responded with further induction of CbCyp51, with a positive correlation of CbCyp51 expression and tetraconazole concentration up to 2.5 μg ml(-1).

Challenges and Prospects for Building Resilient Disease Management Strategies and Tactics for the New York Table Beet Industry

“…This finding indicates a shift towards resistance in the population typical of quantitative resistance demonstrated in other plant pathogens to demethylation inhibitors [67]. Resistance to demethylation inhibitor fungicides has also been reported in C. beticola populations found on sugar beet [68][69][70] including on sugar beet in Ontario, Canada [71], which shares high similarity to the C. beticola population in New York [59]. This suggests that long-term use and dependence on fungicides in this class as the central tenet of a disease management program is unsustainable.…”

Evaluation of the potential for sexual reproduction in field populations of Cercospora beticola from USA

“…Mutation is an important source of variation and does not require physical interaction. Although this source of variation cannot be ignored, the localized (per-field) ability to become resistant to fungicides (Secor et al 2010;Bolton et al 2012) in conjunction with relatively small population sizes associated with C. beticola suggests that mutation alone is unable to explain the genetic diversity found in this species.…”

“…Field isolates of C. beticola are well-known for high levels of variability, particularly with regard to fungicide resistance (Ruppel 1972;Karaoglanidis et al 2000Karaoglanidis et al , 2001Moretti et al 2004;Secor et al 2010;Bolton et al 2012). Studies of the genetic structure of C. beticola populations have shown high degrees of genetic variation (Moretti et al , 2006Groenewald et al 2008), likely underpinning the high level of phenotypic variability associated with this species.…”

Efficacy of Variable Tetraconazole Rates Against Cercospora beticola Isolates with Differing In Vitro Sensitivities to DMI Fungicides

“…DMI-resistant isolates initially were thought to cause less disease than DMIsensitive isolates, suggesting that DMI resistance would not become widespread in field populations (11,13). However, established resistance to DMIs has been reported in many fungal pathogens (30,32,43), including C. beticola (1,23). DMIs are inhibitors of sterol P450 14a-demethylase (CYP51), a protein required for ergosterol biosynthesis in fungi (17).…”