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Title:   Author  item
                                            FRENKEL, OMER - Volcani Center (ARO)item
                                            Cadle-Davidson, Lanceitem
                                            WILCOX, WAYNE - Cornell Universityitem
                                            MILGROOM, MICHAEL - Cornell University
  Submitted to: Phytopathology
                                    Publication Type: Peer Reviewed Journal
                                    Publication Acceptance Date: 1/17/2014
                                    Publication  Date: 9/16/2014
                                    Citation: Frenkel, O., Cadle Davidson, L.E., Wilcox, W., Milgroom, M. 2014. Mechanisms of resistance to an azole fungicide in the grapevine powdery mildew fungus, Erysiphe necator. Phytopathology. 105(3):370-377.Interpretive Summary: Fungicides are the primary tool used to manage grape powdery mildew.  To thrive in the presence of fungicide applications, the fungus must evolve genetic resistance to fungicides. Grape powdery mildew has evolved resistance to the azole class of fungicides by a point mutation (A495T) in the sterol 14a-demethylase gene, known as CYP51.  This CYP51(A495T) mutation changes the protein sequence and explains the major differences in sensitivity among powdery mildew clones.  To further characterize the mechanisms of azole resistance, we collected 65 clones of grape powdery mildew from the eastern U.S. and 12 from Chile and quantified their sensitivity to the azole fungicide myclobutanil. From each clone, we sequenced the CYP51 gene, and we measured the gene expression of CYP51 and four other candidate genes. Sequence variation in CYP51 was relatively low, with sequences of 40 U.S. clones identical to the sensitive reference sequence. Nine U.S. clones and five from Chile carried the known CYP51(A495T) mutation and had high levels of azole resistance, as expected. We also found a new point mutation CYP51(A1119C) in 15 U.S. clones, whose resistance was equivalent to that for the CYP51(A495T) clones. The new CYP51(A1119C) mutation does not change the protein sequence, but clones carrying mutation had significantly greater CYP51 expression. The other 4 candidate genes had no significant effects on azole sensitivity. Both increased CYP51 expression and the CYP51(A495T) mutation affecting protein sequence are apparently responsible for azole resistance in eastern U.S. populations of grape powdery mildew.
                                Technical Abstract: We studied the mechanisms of azole resistance in the grapevine powdery mildew fungus, Erysiphe necator, by quantifying the sensitivity to myclobutanil (EC50) in 65 isolates from the eastern U.S. and 12 from Chile. From each isolate, we sequenced the gene for sterol 14a-demethylase (CYP51), and measured the expression of CYP51 and homologs of four putative efflux transporter genes, which we identified in the E. necator transcriptome. Sequence variation in CYP51 was relatively low, with sequences of 40 U.S. isolates identical to the reference sequence. Nine U.S. isolates and five from Chile carried a previously identified A to T nucleotide substitution in position 495 (A495T), which results in an amino acid substitution in codon 136 (Y136F) and correlates with high levels of azole resistance. We also found a nucleotide substitution in position 1119 (A1119C) in 15 U.S. isolates, whose mean EC50 value was equivalent to that for the Y136F isolates. Isolates carrying mutation A1119C had significantly greater CYP51 expression, even though A1119C does not affect the CYP51 amino acid sequence. Regression analysis showed no significant effects of the expression of efflux transporter genes on EC50. Both the Y136F mutation in CYP51 and increased CYP51 expression apparently are responsible for azole resistance in eastern U.S. populations of E. necator.


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