Ji Won Lee, Jihye Choi and Jin-Won Kim* Triazole Fungicides Sensitivity of Sclerotinia homoeocarpa from Golf Courses in Korea Ji Won Lee, Jihye Choi and Jin-Won Kim* Department of Environmental Horticulture, The University of Seoul, Seoul 130-743, Korea Abstract Dollar spot caused by Sclerotinia homoeocarpa is a serious turfgrass disease in many countries such as North America, Canada, and Europe as well as in Korea. Chemical control is the most prevalent method to suppress dollar spot to an acceptable threshold. Among the fungicides, triazole fungicides belong to demethylation inhibiting (DMI) fungicides have been mainly used for turf from 1990’s in Korea. Repeated applications of triazole fungicides, however, lead to decreased sensitivity to triazole fungicides. In this study, 66 isolates from ten golf courses were evaluated the sensitivity to three triazole fungicides, hexaconazole, tebuconazole and propiconazole. Twelve isolates representing sensitive (S) and resistant (R) isolates were selected based on the EC50 value for three triazole, and had a distinct gap between relative mycelial growth in 0.1 a.i. μg ml-1 of S and R. Among the 12 isolates, S0-41, S10-2-1, and S5-13-1 were sensitive, and S5-11-1, S2-1-1, S0-7, S1-8-1 and S8-8-1 were resistant to all three fungicides. Coefficients of correlation between each fungicides were significantly high (r≥0.8), thus cross-resistance between three triazole fungicides was confirmed. S. homoeocarpa populations collected from golf courses with history of repeated treatment of triazole fungicides had 2.7 to 20 times higher mean EC50 value compared to isolates from golf course with no history of application of fungicides. Introduction S Dollar spot caused by Sclerotinia homoeocarpa is the major turfgrass disease in many countries. To control dollar spot disease, fungicides are mostly used, and among the fungicides, demethylation inhibiting (DMI) fungicide including triazole is the largest group registered for control of dollar spot. However, continuous use of triazole for turf leads to reduced sensitivity of S. homoeocarpa to the fungicides. There are numerous researches of triazole-resistant strains of S. homoeocarpa, however, in Korea, only a few reports were published (Shim et al., 2001; Kim et al., 2010). R Materials and Methods Isolates were collected from ten golf courses and Korea Turfgrass Research Institute (KTRI). Symptomatic leaf blades exhibiting straw-colored tan with dark brown borders were surface sterilized. Sterilized tissues were placed on potato dextrose agar (PDA, Difco, USA) and incubated at 25°C. Compared with characteristics of species-identified isolates, which have fluffy white mycelium, dark brown to olive colored stromata, and fast growth rate, collected isolates were identified as S. homoeocarpa. Pure cultures were obtained with at least twice times of transfers from the actively growing margin of colonies. All 66 isolates were tested for their sensitivity to three triazole fungicides (hexaconazole, tebuconazole and propiconazole), and fungicide-amended PDA was used for in vitro sensitivity assay. Sterile distilled water was used to prepare dilutions of the commercial formulations of the fungicides. Each diluted fungicides was added to cooled PDA (50-55°C). Agar plugs (5mm in diameter) were transferred from the edge of actively growing mycelium to each PDA plates amended with hexaconazole FL (a.i. cont. 2%), tebuconazole FL (a.i. cont. 20%), and propiconazole EC (a.i. cont. 25%) at 0, 0.001, 0.01, 0.1, 1.0 and 10.0 a.i. μg ml-1. Radial mycelial growth (RMG) was measured after 48h of incubation at 25°C (Fig. 1). The inhibition of relative mycelial growth was calculated as [1-(radial growth on fungicide-amended PDA/radial growth on non-amended PDA)]×100. EC50 values defined as the effective concentration of active ingredient (a. i.) that inhibits mycelium growth by 50% were obtained for each isolates using PROC PROBIT (SAS 9.4; SAS Institute, Cary, NC) (Hsiang et al., 1997; Miller et al., 2002). Correlation analysis between each fungicides were conducted with pairwise comparisons of the logEC50 values. Coefficients of correlation were evaluated with regression analysis for all 66 isolates. S R S R Control T 0.01 T 0.1 T 1.0 T 10.0 T 0.001 R (S5-11-1) Fig. 2. Inhibition of mycelial growth at 0, 0.001, 0.01, 0.1, 1.0, and 10.0 a.i. μg ml-1 of hexaconazole, propiconazole and tebuconazole using twelve selected isolates. Relative mycelial growth in 0.1 a.i. μg ml-1 of fungicides had a distinct gap between S and R. Fig. 3. Pairwise correlations of hexaconazole, tebuconazole, and propiconazole between Log EC50 values for 66 Sclerotinia homoeocarpa Isolates including eight isolates representing R (red dot) and five isolates representing S (blue dot). S (S10-2-1) Conclusion Fig. 1. Mycelium growth of tebuconazole-resistant (S5-11-1) and -sensitive (S10-2-1) isolates of Sclerotinia homoeocarpa on PDA plates amended with 10-fold dilutions of tebuconazole and non-amended PDA (control). Compared to S. homoeocarpa population from golf courses with no history of triazole fungicides, populations from golf courses with history of triazole fungicides had 2.7 to 20 times higher mean EC50 value (data not shown). Popko et al. (2012) suggested that, in the golf courses with high mean EC50 value, selection pressure of resistant isolates may have caused control failure of disease. Also, cross-resistance between three triazole fungicides was confirmed; therefore, it seems advisable to avoid using triazole fungicides with same mode of action. Results and Discussion In vitro fungicides sensitivity assay. Sensitivity of S. homoeocarpa isolates to triazole fungicides was determined with in vitro sensitivity assay. The EC50 values of each isolates to three fungicides were quantitatively dispersed from 0.001 to 1.1 a.i. μg ml-1. Three isolates for each fungicides were selected as sensitive isolates (S) based on EC50 values. Based on EC50 values of S10-2-1 isolated from golf courses with no history of fungicides, five isolates for each fungicides, which have more than 26 times higher EC50 for hexaconazole and tebuconazole, and more than 50 times higher for propiconazole, were selected as resistant isolates (R). Total twelve isolates representing S or R were selected, and had a distinct gap between relative mycelial growth in 0.1 a.i. μg ml-1 of S and R. Among them, S0-41, S10-2-1, and S5-13-1 were sensitive, and S5-11-1, S2-1-1, S0-7, S1-8-1 and S8-8-1 were resistant to all fungicides (Fig. 2). Cross-resistance between three triazole fungicides. Cross-resistance between three triazole fungicides was determined with correlation analysis of LogEC50 values for 66 isolates. Coefficients of correlation of hexaconazole-tebuconazole, hexacozole-propiconazole and propiconazole-tebuconazole combinations were 0.80, 0.82 and 0.82, respectively (Fig. 3). Coefficients of correlation between each fungicides were significantly high (P<0.001). Cross-resistance of four DMI fungicides except with triadimefon was confirmed by Miller et al.(2002) and Ok et al.(2011) determined high correlation (r ≥ 0.67) between six DMI fungicides including triadimefon. Similar to the result by Ok et al.(2011), propiconazole and tebuconazole were highly correlated in this study. Shim, G. Y., Min, G. Y., Shin, H. D. and Lee, H. J. 2001. Occurrence of chemical resistance and control of dollar spot caused by Sclerotinia homoeocarpa in turfgrass of golf course. Kor. Turfgrass Sci. 15:1-8. Kim, J. H., Choi, H. Y., Shim, G. Y. and Kim, Y. H. 2010. Chemical resistance and control of dollar spot caused by Sclerotinia homoeocarpa on turfgrass of golf courses in Korea. Kor. Turfgrass Sci. 24:170-175. Hsiang, T., Yang, L., and Barton, W. 1997. Baseline sensitivity and cross-resistance to demethylation-inhibiting fungicides in Ontario isolates of Sclerotinia homoeocarpa. Eur. J. Plant Pathol. 103: 409–416. Miller, G. L., Stevenson, K. L., and Burpee, L. L. 2002. Sensitivity of Sclerotinia homoeocarpa isolates to propiconazole and impact on control of dollar spot. Plant Dis. 86:1240-1246. Ok, C.-H., Popko, J. T., Jr., Campbell-Nelson, K., and Jung, G. 2011. In vitro assessment of Sclerotinia homoeocarpa resistance to fungicides and plant growth regulators. Plant Dis. 95:51-56. Popko, J. T., Jr., Ok, C.-H., Campbell-Nelson, K., and Jung, G. 2012. The association between in vitro propiconazole sensitivity and field efficacy of five New England Sclerotinia homoeocarpa populations. Plant Dis. 96:552-561. References ☏ 02) 6490-2690 E-mail: jwkim@uos.ac.kr