Abstract
We investigated the molecular basis of resistance of the obligate biotrophic grape powdery mildew fungus Uncinula necator to sterol demethylation-inhibiting fungicides (DMIs). The sensitivity of 91 single-spore field isolates of U. necator to triadimenol was assessed by using a leaf disc assay. Resistance factors (RF) ranged from 1.8 to 26.0. The gene encoding the target of DMIs (eburicol 14 alpha-demethylase) from five sensitive and seven resistant isolates was cloned and sequenced. A single mutation, leading to the substitution of a phenylalanine residue for a tyrosine residue at position 136, was found in all isolates exhibiting an RF higher than 5. No mutation was found in sensitive or weakly resistant (RF, < 5) isolates. An allele-specific PCR assay was developed to detect the mutation. Among the 91 isolates tested, only isolates with RF higher than 5 carried the mutation. Three of the 19 resistant isolates and all sensitive and weakly resistant isolates did not possess the mutation. The mutation at codon 136 is thus clearly associated with high levels of resistance to triadimenol.
Full Text
The Full Text of this article is available as a PDF (218.9 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Aoyama Y., Funae Y., Noshiro M., Horiuchi T., Yoshida Y. Occurrence of a P450 showing high homology to yeast lanosterol 14-demethylase (P450(14DM)) in the rat liver. Biochem Biophys Res Commun. 1994 Jun 30;201(3):1320–1326. doi: 10.1006/bbrc.1994.1848. [DOI] [PubMed] [Google Scholar]
- Aoyama Y., Noshiro M., Gotoh O., Imaoka S., Funae Y., Kurosawa N., Horiuchi T., Yoshida Y. Sterol 14-demethylase P450 (P45014DM*) is one of the most ancient and conserved P450 species. J Biochem. 1996 May;119(5):926–933. doi: 10.1093/oxfordjournals.jbchem.a021331. [DOI] [PubMed] [Google Scholar]
- Chen C., Kalb V. F., Turi T. G., Loper J. C. Primary structure of the cytochrome P450 lanosterol 14 alpha-demethylase gene from Candida tropicalis. DNA. 1988 Nov;7(9):617–626. doi: 10.1089/dna.1988.7.617. [DOI] [PubMed] [Google Scholar]
- Geber A., Hitchcock C. A., Swartz J. E., Pullen F. S., Marsden K. E., Kwon-Chung K. J., Bennett J. E. Deletion of the Candida glabrata ERG3 and ERG11 genes: effect on cell viability, cell growth, sterol composition, and antifungal susceptibility. Antimicrob Agents Chemother. 1995 Dec;39(12):2708–2717. doi: 10.1128/aac.39.12.2708. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hargreaves J. A., Keon J. P. Isolation of an Ustilago maydis ERG11 gene and its expression in a mutant deficient in sterol 14 alpha-demethylase activity. FEMS Microbiol Lett. 1996 Jun 1;139(2-3):203–207. doi: 10.1111/j.1574-6968.1996.tb08203.x. [DOI] [PubMed] [Google Scholar]
- Hitchcock C. A., Barrett-Bee K. J., Russell N. J. Inhibition of 14 alpha-sterol demethylase activity in Candida albicans Darlington does not correlate with resistance to azole. J Med Vet Mycol. 1987 Oct;25(5):329–333. [PubMed] [Google Scholar]
- Hitchcock C. A. Resistance of Candida albicans to azole antifungal agents. Biochem Soc Trans. 1993 Nov;21(4):1039–1047. doi: 10.1042/bst0211039. [DOI] [PubMed] [Google Scholar]
- Kalb V. F., Woods C. W., Turi T. G., Dey C. R., Sutter T. R., Loper J. C. Primary structure of the P450 lanosterol demethylase gene from Saccharomyces cerevisiae. DNA. 1987 Dec;6(6):529–537. doi: 10.1089/dna.1987.6.529. [DOI] [PubMed] [Google Scholar]
- Kirsch D. R., Lai M. H., O'Sullivan J. Isolation of the gene for cytochrome P450L1A1 (lanosterol 14 alpha-demethylase) from Candida albicans. Gene. 1988 Sep 7;68(2):229–237. doi: 10.1016/0378-1119(88)90025-x. [DOI] [PubMed] [Google Scholar]
- Lamb D. C., Corran A., Baldwin B. C., Kwon-Chung J., Kelly S. L. Resistant P45051A1 activity in azole antifungal tolerant Cryptococcus neoformans from AIDS patients. FEBS Lett. 1995 Jul 17;368(2):326–330. doi: 10.1016/0014-5793(95)00684-2. [DOI] [PubMed] [Google Scholar]
- Sloane D. L., So O. Y., Leung R., Scarafia L. E., Saldou N., Jarnagin K., Swinney D. C. Cloning and functional expression of the cDNA encoding rat lanosterol 14-alpha demethylase. Gene. 1995 Aug 19;161(2):243–248. doi: 10.1016/0378-1119(95)00211-n. [DOI] [PubMed] [Google Scholar]
- Sommer R., Tautz D. Minimal homology requirements for PCR primers. Nucleic Acids Res. 1989 Aug 25;17(16):6749–6749. doi: 10.1093/nar/17.16.6749. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sommer S. S., Groszbach A. R., Bottema C. D. PCR amplification of specific alleles (PASA) is a general method for rapidly detecting known single-base changes. Biotechniques. 1992 Jan;12(1):82–87. [PubMed] [Google Scholar]
- Strömstedt M., Rozman D., Waterman M. R. The ubiquitously expressed human CYP51 encodes lanosterol 14 alpha-demethylase, a cytochrome P450 whose expression is regulated by oxysterols. Arch Biochem Biophys. 1996 May 1;329(1):73–81. doi: 10.1006/abbi.1996.0193. [DOI] [PubMed] [Google Scholar]
- van Nistelrooy J. G., van den Brink J. M., van Kan J. A., van Gorcom R. F., de Waard M. A. Isolation and molecular characterisation of the gene encoding eburicol 14 alpha-demethylase (cYP51) from Penicillium italicum. Mol Gen Genet. 1996 Apr 10;250(6):725–733. doi: 10.1007/BF02172984. [DOI] [PubMed] [Google Scholar]