Fungicide sensitivity assay

Isolates for cyp51 sequencing came from cultures that had been maintained in the laboratory on fungicide-free grape leaves for over two years by sequential transfer to new leaves every two to three weeks. Pure cultures for each isolate were prepared as described in the previous section. Fungicide sensitivity assays were conducted as close as possible to the time of DNA extraction for cyp51 sequencing. Sensitivities to myclobutanil, tebuconazole and fenarimol were determined by detached-leaf bioassays. Stock solutions of tebuconazole (Bayer Crop Science LP, Research Triangle Park, NC), myclobutanil (Dow Agrosciences LLC, Indianapolis, IN) and fenarimol (Gowan Company, Yuma, AZ) were prepared in acetone from technical grade fungicides and stored at -10 to -12°C. A 3x-serial dilution scheme was used as follows depending on the isolate and DMI fungicide: 0.001–0.1, 0.003–0.3, 0.01–1, 0.03–0.3, 0.1–1, 0.3–3, 1–10, or 3–30 mg liter^-1. Bioassays were done by a point-inoculation method where six spore chains from each culture were deposited at equal distance from each other on the adaxial side of a leaf on 0.7% water agar (two leaves per fungicide treatment). Leaves were shaken in fungicide solution as described in Colcol et al. [15], then incubated at 25°C under a 12-hr light regime for 6 to 8 days. Relative growth was calculated from the mean colony diameters as a percentage of the mean diameter of the control (no fungicide treatment). The relative diameter at different treatments was regressed on natural log-transformed fungicide concentration. From the linear section of the regression, the effective concentration to inhibit growth by 50% (EC₅₀) was calculated in Microsoft Excel as described in Colcol et al. [15]. The resistance factor (RF) was calculated for each isolate as EC₅₀ for the fungicide divided by the corresponding mean EC₅₀ of the sensitive sub-group consisting of three isolates (BLP1, BLP4, MVP9) taken from unexposed populations (locations with no known fungicide application). These isolates were part of the larger sensitive sub-group reported by Colcol et al. [15]. Mean EC₅₀ for our sensitive sub-group was calculated as 0.03 mg liter^-1 for tebuconazole, 0.18 mg liter^-1 for myclobutanil and 0.02 mg liter^-1 for fenarimol. Isolates with RF<3 were regarded as sensitive; 3<RF<10 as weakly resistant; and RF ≥10 as resistant.