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Peer Reviewed

2003 Plant Management Network.
Accepted for publication 12 November 2003. Published 9 December 2003.

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

Tara Tarnowski, Jean Batzer, Mark Gleason, and Sara Helland, Department of Plant Pathology, Phillip Dixon, Department of Statistics, Iowa State University, Ames, IA, 50011

Corresponding author: Mark Gleason.

Tarnowski, T., Batzer, J., Gleason, M., Helland, S., and Dixon, P. 2003. Sensitivity of newly identified clades in the sooty blotch and flyspeck complex on apple to thiophanate-methyl and ziram. Online. Plant Health Progress doi:10.1094/PHP-2003-1209-01-RS.


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 25C. 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.


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.


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).

Table 1. Characterization of clades in the SBFS complex, including
recognized species assessed for fungicide sensitivity in this study.

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

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 40C). 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 23C, 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 25C (Fig. 2).


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 (g/ml) are shown below corresponding wells.


Each experiment was arranged in a randomized complete block design with two factors: isolate and fungicide concentration (g/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.

Trial Group Concentrations (g/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

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.

Trial Group Concentrations (g/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

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.

After 21 days, two perpendicular measurements of colony diameter were made using a ruler. Colony diameter was determined by subtracting the initial plug. To calculate the ED50 for each isolate, the growth response was fitted to a non-linear logistic model using the log of the concentration values as the predictor variable. Non-linear regression was performed using PROC NLIN in SAS (SAS, Version 8.2, SAS Institute Inc., Cary, NC). The fungicide concentration value at which the colony diameter was 50% of the colony diameter on non-amended agar (ED50) was then used as the response variable for each experimental unit. The log of this value was then analyzed using ANOVA to check for treatment differences while treating the two runs of the experiment as blocking factors. Tukeys adjustment was used in obtaining the p-values from testing differences in the least squares means of the treatments (PROC GLM, SAS).

Results & Management Implications

Preliminary experiment. Isolates were tested for sensitivity to thiophanate-methyl and ziram over a wide range of fungicide concentrations. The isolates segregated into two general ranges of sensitivity in response to each fungicide. Based on these results, appropriate fungicide concentrations were selected to determine the ED50 of each isolate with each fungicide.

Main experiment. Several clades differed significantly (P < 0.05) in sensitivity to thiophanate-methyl (Fig. 3A). There was more than a 10-fold difference between the most and least sensitive clades, DS1 and P4, respectively. Clades P1 and P4 were least sensitive, whereas DS1 showed little growth at any fungicide concentration tested. Clade RL2 (isolate UID2) was twice as sensitive as clade RL1 (isolate MWD2). This provides further justification for the separation of these isolates into different clades. While there was wide variation in sensitivity among clades, isolates within a clade did not differ significantly.


Fig. 3. Estimated ED50 of isolates for thiophanate-methyl (A) and ziram (B). Bars followed by the same letters do not differ significantly (P < 0.05) in fungicide sensitivity, based on Tukeys HSD test.

Fewer significant differences in sensitivity were evident among clades in response to ziram than to thiophanate-methyl (Fig. 3B). However, clades FG2 and RL1 were significantly less sensitive than clade DS1 and several individual isolates (Pf002, UID2, and Zj003). RL2 was significantly more sensitive to ziram than RL1.

This is the first published report that newly discovered clades in the SBFS complex differ significantly in their sensitivity to fungicides. These differences in fungicide sensitivity are also the first evidence that the clades are physiologically distinct, as well as genetically and morphologically distinct (4,24). The absence of significant differences between isolates within clades further emphasizes the uniqueness of each clade. Our findings thus support the view that these clades comprise taxonomically distinct units, perhaps at the species or genus level.

Two isolates, MWD2 and UID2, that showed significant differences in sensitivity to both fungicides were initially assumed to be in the same clade because they differ by only three base pairs in the ITS region of rDNA (4). However, Van deVoort et al. (23) found that UID2 grew significantly faster than MWD2 on three agar media. The two isolates also differ in appearance on agar. On PDA, MWD2 exhibited flat, black mycelia with white tufts, whereas UID2 exhibited green, fluffy growth. The spore shapes also differ. On malt extract agar (MEA), MWD2 had olive-green mycelia with aerial hyphae, whereas UID2 was tan-colored and sometimes exhibited callus-like growth with no conidia. These morphological and physiological differences warranted splitting UID2 and MWD2 into different clades.

Because solubility in water of both thiophanate-methyl and ziram is low, our results may not accurately determine inherent toxicity of these fungicides to the fungi we studied. Nevertheless, whereas absolute values of ED50 might have changed had the fungicides been solubilized rather than suspended, relative sensitivity of each clade would probably remain the same. We used water suspensions and commercial fungicide formulations, rather than pure technical grade material, in order to simulate fungicide preparation by growers as closely as possible.

Differences in fungicide sensitivity among species of the same genus have been characterized for Pythium in wheat (20), Fusarium in pumpkin (7), Colletotrichum in almond, peach, and other fruits (1,9), and Phytophthora in pepper (19). There have been fewer investigations of intraspecific differences in fungicide sensitivity. Quantitative differences in sensitivity to imazalil, thiabendazole, and o-phenylphenyl were characterized for 18 isolates of Penicillium digitatum from citrus, and the effects of metalaxyl on Pythium ultimum var. sporangiiform (76 isolates) and P. aphanidermatum (21 isolates) have been determined (6,15).

Our results have the potential to lead to more effective SBFS management strategies. Species composition within the SBFS complex varies among geographic locations. Furthermore, the SBFS assemblage within an orchard may be dominated by one or a few species. In a preliminary survey, the prevalence and incidence of SBFS mycelial types varied sharply among nine apple orchards in four states (IA, MO, IL, and WI) (4). L. elatius, a common SBFS species found in North Carolina, was not isolated from apples in the Midwest. In a North Carolina study, the prevalence and incidence of SBFS mycelial types differed among four orchards (22). Although in vitro fungicide sensitivity may not correlate precisely to SBFS control in the field (8), knowledge of the composition of the disease complex within a specific orchard and the fungicide sensitivities of the predominant clades could allow a grower or consultant to better select fungicides, rates, and spray timing to suppress SBFS.

The discovery of sensitivity differences to commonly used fungicides in the SBFS complex on apples indicates that members of these newly identified clades have significant functional and physiological differences. Thus, members of the SBFS complex may respond differently to various fungicide-based management strategies. With further understanding of fungicide sensitivity differences and ecological interactions within the SBFS complex, more effective SBFS management strategies may be developed.

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