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Sensitivity to the fungicides thiophanate-methyl and ziram was characterized for newly identified clades in the sooty blotch and flyspeck (SBFS) disease complex. Isolates obtained from apple orchards in Illinois, Iowa, Missouri, and Wisconsin were previously grouped into clades based on parsimony analysis of the internal transcriber spacer (ITS) and large subunit (LSU) regions of rDNA. Two isolates from each of eight clades were cultured on water agar amended with a range of fungicide concentrations. Radial growth of colonies was measured after incubation for 21 days at 25°C. Fungicide sensitivity (ED50) did not differ significantly (P = 0.05) between isolates within a clade, but several clades differed significantly. There was a greater variability among clades for sensitivity to thiophanate-methyl than for ziram. Significant differences in fungicide sensitivity among, but not within, clades provide evidence that these clades differ physiologically. Understanding differences in fungicide sensitivity among SBFS clades could have practical implications for improved management of SBFS. Introduction Sooty blotch and flyspeck diseases (SBFS) are caused by a complex of saprophytic fungi that colonize the cuticle of pome fruits. Sooty blotch signs are variable, but generally appear as brown to black smudges on the apple peel, whereas flyspeck appears as groups of tiny brown or black spots (Fig. 1). The fungi blemish the fruit, downgrading its value from fresh-market to cull status, and thereby can cause severe economic losses for apple growers (24). The SBFS complex includes Peltaster fructicola, Geastrumia polystigmatis, and Leptodontium elatius, which cause sooty blotch, and Zygophiala jamaicensis, which causes flyspeck (16). Six mycelial types -punctate, fuliginous, rimate, ramose, flyspeck, and discrete speck -are currently used to describe the diverse morphology of members of the SBFS complex on apples (3,11). Fig. 1. Sooty blotch and flyspeck signs on Golden Delicious apple from Pella, IA. Plant Health Progress 9 December 2003 Batzer et al. (4) presented genetic evidence indicating that the SBFS complex in the Midwest could be separated into approximately 25 clades. After the large (28S) subunit (LSU) and internal transcribed spacer (ITS) regions of rDNA were amplified and sequenced, maximum parsimony analysis was used to separate isolates into clades. Each clade exhibited characteristic mycelial morphology on apples and in culture. In several instances, more than one clade shared the same mycelial type. Applying molecular genetic techniques has revealed that the SBFS disease complex in the Midwest encompasses far more genetic diversity than previously documented. Previous studies have shown a correlation between SBFS mycelial type and environmental response. Hickey (14) showed that morphologically distinct isolates from the SBFS complex differed in response to temperature and pH, and that the peak time of fruit colonization was earlier for flyspeck than sooty blotch. Batzer (5) found that mycelial type influenced efficacy of removal of SBFS fungi by postharvest sanitizing agents. A study comparing incidence and severity of sooty blotch mycelial types at four locations in North Carolina showed that environmental factors affect mycelial types differently (22). Additionally, Johnson and Sutton (18) found that P. fructicola and L. elatius, which exhibit punctate and fuliginous mycelial types, respectively, responded differently to temperature and relative humidity. Hickey (14) found differences in fungicide sensitivity between sooty blotch and flyspeck fungi. Dodine suppressed growth of sooty blotch mycelium, but not flyspeck mycelium, on fungicide-amended agar. Johnson (17) determined that EC50 for fluazinam was more than 100 times greater for L. elatius than P. fructicola, whereas EC50 for benomyl was 30 times greater for P. fructicola than L. elatius. To prevent economic losses from SBFS, a protectant fungicide spray program is implemented in apple orchards throughout the eastern half of the United States, as well as in other humid areas of apple production worldwide. Use of weather-based disease-warning systems to reduce the frequency of fungicide sprays in orchards has provided promising but inconsistent results (12,13,21; Gleason, unpublished data). In both conventional and integrated pest management (IPM) approaches, management regimes have been applied without regard for the species of SBFS present in each orchard. More effective management may require a better understanding of the environmental biology and fungicide sensitivity of members of the SBFS complex. Few fungicides are currently registered for control of SBFS. Among these, thiophanate-methyl and ziram are among the most affordable and widely used compounds in the U.S. The purpose of the present study was to characterize the sensitivity of newly discovered SBFS fungi to the fungicides thiophanate-methyl and ziram. A preliminary report has been published (2). Isolates and Fungicides Fourteen SBFS isolates from orchards in Illinois, Iowa, Missouri, North Carolina, and Wisconsin were chosen to represent eight clades that encompassed five mycelial types as they appeared on apples (Fig. 1;Table 1). Plant Health Progress 9 December 2003 Table 1. Characterization of clades in the SBFS complex, including recognized species assessed for fungicide sensitivity in this study. x These isolates were originally thought to be in a single clade due to close genetic similarity. When further study revealed that they differed significantly in several morphological characteristics, these isolates were split into two separate clades (23). y Isolate Pf002 has been identified as Peltaster fructicola. z Isolate Zj003 has been identified as Zygophiala jamaicensis. Two fungicides that are widely used against SBFS (10) were chosen for the study. Thiophanate-methyl is a systemic fungicide in the benzimidazole group, whereas ziram is a protectant fungicide in the dimethyldithiocarbamate group (25). For this study, commercial formulations of each fungicide were used: Topsin M 70WP (thiophanate-methyl; Cerexagri, King of Prussia, PA) and Ziram 76DF (ziram; Cerexagri). Characterization of Isolate Sensitivity to Thiophanate-methyl and Ziram Stock suspensions of each fungicide were prepared in distilled water using 1:200 serial dilutions. The most concentrated suspension corresponded to the lowest labeled rate for the fungicide on apples (10). Fungicide stock suspensions were added to autoclaved 2% water agar (after cooling to approximately 40°C). Three ml of the amended media were added to each well in a six-well (well diameter = 3.5 cm) tissue culture plate (Costar #3516, Corning Inc., New York, NY). Inoculum was prepared by streaking 30-day-old cultures onto potato dextrose agar (PDA) to achieve a uniform lawn of mycelium. After 17 days of incubation at 23°C, 3.5-mm-diameter fungal plugs were placed mycelium down onto the media in the six-well plates. The inoculated plates were incubated in the dark at 25°C (Fig. 2). Clade Isolate Mycelial Type Origin FG2 UIF1 fuliginous Urbana, IL FG2 UMF2 fuliginous Columbia, MO P4 GTE5 punctate Sparta, IL P4 CUE2 punctate Rockford, IL P1 GTE1 punctate Sparta, IL yP1 Pf002 punctate Moore County, NC xRL1 MWD2 rimate Indianola, IA xRL2 UID2 rimate Urbana, IL DS1 MSTB8 discrete speck New Munster, WI DS1 PEB1 discrete speck Pella, IA FS3 MWA8 flyspeck Indianola, IA FS3 GTA8 flyspeck Sparta, IL FS1 MSTA6 flyspeck New Munster, WI zFS1 Zj003 flyspeck Henderson County, NC Plant Health Progress 9 December 2003 Each experiment was arranged in a randomized complete block design with two factors: isolate and fungicide concentration (μg/ml). Thiophanate-methyl and ziram trials were treated as separate experiments. Fourteen fungal isolates and six fungicide concentrations were tested (Tables 1, 2, and 3). Each well contained one of the fungicide concentrations. The experimental unit thus consisted of a six-well plate in which a single fungal isolate was exposed to five concentrations of a fungicide and an unamended control. Each experimental unit was replicated six times, and each experiment was repeated once. In the second run of the experiment, fungicide concentrations were modified to improve the accuracy of ED50 determination (Tables 2 and 3). Table 2. Thiophanate-methyl concentrations used in the study. Isolates were tested in one of two concentration ranges, based on preliminary data indicating that the isolates were grouped into two distinct ranges of sensitivity. Concentrations were adjusted between Trials 1 and 2 to more closely encompass the response range within which ED50 occurred. a Group 1T included isolates GTE5, CUE2, GTE1, Pf002, MWD2, and UID2. b Group 2T included isolates UIF1, UMF2, MSTB8, PEB1, MWA8, GTA8, MSTA6, and Zj003. Table 3. Ziram concentrations used in the study. Isolates were tested in one of two concentration ranges, based on preliminary data indicating that the isolates were grouped into two distinct ranges of sensitivity. Concentrations were adjusted between Trials 1 and 2 to more closely encompass the response range within which ED50 occurred. c Group 1Z included isolates UIF1, UMF2, and MWD2. d Group 2Z included isolates GTE5, CUE2, GTE1, Pf002, UID2, MSTB8, PEB1, MWA8, GTA8, MSTA6, and Zj003. Fig. 2. Mycelial plugs were placed on fungicide-amended agar in wells of tissue culture plates. Isolate MWD2 is shown on thiophanate-methyl-amended agar. Fungicide concentrations (μg/ml) are shown below corresponding wells. Trial Group Concentrations (μg/ml) 1 1Ta 0.00 0.30 0.40 0.86 1.71 3.00 2Tb 0.00 0.10 0.12 0.20 0.30 0.60 2 1Ta 0.00 0.30 0.40 0.60 0.86 1.71 2Tb 0.00 0.10 0.12 0.16 0.20 0.30 Trial Group Concentrations (μg/ml) 1 1Zc 0.00 0.45 0.90 1.20 2.40 18.00 2Zd 0.00 0.23 0.45 0.90 1.20 3.60 2 1Zc 0.00 0.45 0.90 1.03 1.20 2.40 2Zd 0.00 0.23 0.45 0.60 0.90 1.20 Plant Health Progress 9 December 2003 After 21 days, two perpen

Sensitivity of Newly Identified Clades in the Sooty Blotch and Flyspeck Complex on Apple to Thiophanate-methyl and Ziram