Submitted to: Phytopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/12/2011
Publication Date: 2/6/2012
Citation: Bolton, M.D., Birla, K., Rivera-Varas, V., Rudolph, K.D., Secor, G.A. 2012. Characterization of CbCyp51 from field isolates of Cercospora beticola. Phytopathology. 102(3):298-305. Interpretive Summary: Leaf spot disease of sugarbeet is caused by the fungus Cercospora beticola. To control disease, growers apply fungicides called sterol demethylation inhibitors or DMIs. However, loss of disease control from DMIs has been reported in the Red River Valley sugarbeet growing region. DMIs target a protein called Cyp51 in fungi. Some fungi gain resistance to DMIs through mutations in the Cyp51 gene. To investigate whether this happens in C. beticola, we cloned Cyp51 and sequenced the gene from isolates with varying degrees of resistance to DMI fungicides. However, there were no mutations associated with fungicide resistance. The gene was over-expressed in fungicide resistant isolates and was turned on even further when tetraconazole was added to the growth medium. The results of this study offer important insight into how fungicide resistance develops in C. beticola. This information is useful for plant pathologists that wish to utilize molecular tools to monitor fungicide resistance in C. beticola.
Technical Abstract: The hemi-biotrophic fungus Cercospora beticola causes leaf spot of sugarbeet. Leaf spot control measures include the application of sterol demethylation inhibitor (DMI) fungicides, but reduced sensitivity to DMIs has been reported recently in the Red River Valley sugarbeet growing region. Here, we report the cloning and molecular characterization of CbCyp51, which encodes the DMI target enzyme sterol P450 14a-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 81% of the sequenced isolates, followed by protein variant 2 that composed 16% 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 over 2,440 nucleotides encompassing CbCyp51 coding and flanking regions from 32 isolates with varying EC50 values 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 over-expressed in isolates with high EC50 values compared to isolates with low EC50 values. After exposure to tetraconazole, isolate with high EC50 values responded with further induction of CbCyp51, with a positive correlation of CbCyp51 expression and tetraconazole concentration up to 2.5 µg ml-1.