Growth Sensitivity of Corynespora cassiicola to Thiophanate-methyl, Iprodione, and Fludioxonil

An African violet production facility has relied solely on thiophanate-methyl, a sitespecific fungicide that possesses a high risk for pathogens to develop resistance, to manage a devastating Corynespora leaf spot problem. During a disease outbreak in September 2007, 325 isolates of C. cassiicola were collected and 40 isolates were randomly selected to determine the pathogen's sensitivity for mycelium growth on agar amended with various concentrations of thiophanatemethyl, iprodione, or fludioxonil. EC values, concentration resulting in a 50% reduction in mycelium growth, were determined and indicate a population that currently is sensitive to all three fungicides. Due to the high risk of the pathogen developing resistance to thiophanate-methyl and iprodione, a moderate risk for cross-sensitivity between iprodione and fludioxonil, and phytotoxicity and visual residue problems with protective fungicides such as chlorothalonil, a fungicide rotation is recommended with fludioxonil as the main chemical selection. Introduction Corynespora leaf spot, caused by Corynespora cassiicola (Berk. & M.A. Curtis) C.T. Wei, can cause devastating epidemics on African violets (Saintpaulia ionantha H. Wendl.). The disease is characterized by water soaked lesions that expand rapidly on leaf surfaces and petioles (21). A large greenhouse facility in Tennessee that produces more than 10 million African violets annually has experienced epidemics of Corynespora leaf spot for more than five years. The facility receives cuttings from several overseas operations and distributes to a large number of businesses. As a result, the pathogen’s population likely includes an influx and efflux of isolates, typical of many greenhouse and nursery commodities. Outbreaks at the TN facility occur mostly from May through September (31). Symptoms occur on plants in all stages of production and can be severe enough that plants are discarded. In an effort to eliminate inoculum, thousands of plants have been discarded during a single episodic event. Common cultural and sanitation practices alone have not provided adequate control. To date, fungicides are the only tool that has restricted disease development. Thiophanate-methyl (Cleary 3336; W.A. Cleary Corp., Somerset, NJ) and chlorothalonil (Daconil; Syngenta Crop Protection, Greensboro, NC) were effective in reducing incidence of Corynespora leaf spot in experiments conducted at the facility (31). Due to phytotoxicity and visible residue issues resulting from application of chlorothalonil, thiophanate-methyl has been solely relied upon for control of Corynespora leaf spot. 50 26 September 2011 Plant Health Progress Thiophanate-methyl is a methyl benzimidazole carbamate (MBC) fungicide [FRAC code 1 (7)]. MBC chemicals inhibit nuclear division that consequently stops hyphal growth. MBC fungicides have been classified as being high risk for selecting resistance in pathogen populations, and resistance has been documented for a number of pathogens in other crops (11,12,26,29,33). Continued use of this single fungicide product at a greenhouse operation increases the probability of chemical control failure, and could be a factor in the Tennessee facility. One objective of this research was to establish a thiophanatemethyl sensitivity profile for the C. cassiicola population, since it has already been subjected to continuous selection pressure at this production facility. An additional objective was to establish sensitivity profiles for iprodione and fludioxonil, which were selected because these products have not been used on this crop, but are registered for use on many greenhouse and field crops that serve as hosts for C. cassiicola. Iprodione is a dicarboximide fungicide (FRAC code 2) that inhibits spore germination and mycelium growth by affecting lipid synthesis and metabolism (28). Fludioxonil is a phenylpyrrole fungicide (FRAC code 12) that inhibits spore germination and mycelium growth by affecting the osmotic signal transduction pathway (8,10,13,18). By establishing sensitivity profiles of these three fungicides for the C. cassiicola population, the influence of previous exposure to thiophanate-methyl can be assessed, the similarity of iprodione and fludioxonil efficacy to baseline data of other organisms can be determined, and recommendations for product use patterns can be developed. Fungal Isolate Collection Leaves of African violets symptomatic for Corynespora leaf spot were collected at a large African violet production facility during a disease outbreak in September 2007. Symptomatic leaves were individually placed into sterile polyethylene bags and transported to the lab. Excised lesions were submersed in a solution of 10% sodium hypochlorite and 5% ethanol for 30 sec, and then rinsed in sterile deionized water for 10 sec (4) and set on potato dextrose agar (PDA; Difco Laboratories, Detroit, MI) amended with chlorotetracycline hydrochloride (8 μg/ml) and streptomycin sulfate (8 μg/ml). Cultures were grown in an incubator at 23 ± 1°C with a diurnal 12-h light and dark cycle for 48 h before transferring the leading edge of mycelium to unamended PDA. Isolates of C. cassiicola was identified based on colony and conidial morphology (25). Each of 325 collected isolates were grown on sterile cotton stems embedded in 75 ml PDA in GA-7 culture vessels (Magenta Corp., Chicago, IL) at 23 ± 1°C with 12-h light and dark cycles. Isolate Sensitivity to Fungicides Forty isolates were selected randomly to assess their sensitivity to the selected fungicides. Technical grade thiophanate-methyl (97% a.i.; W.A. Cleary Corp., Somerset, NJ), iprodione (99% a.i.; Bayer Environmental Science, Research Triangle, NC), and fludioxonil (98% a.i.; Syngenta Crop Protection, Greensboro, NC) were dissolved in acetone to provide stock solutions of 20 mg/ml that were added to partially cooled PDA to obtain concentrations of 0, 0.01, 0.1, 1, 10, and 100 μg/ml. Acetone was added to achieve equal concentrations in all dilutions. Plugs of the leading mycelium edge from twelve-day-old cultures were placed upside down on PDA amended with fungicide or unamended media (25). Each fungicide concentration and isolate combination was replicated three times. Cultures were incubated for 11 days at 23 ± 1°C in complete darkness. Mean colony diameter was obtained for each culture by taking two perpendicular measurements, with the original 5-mm plug diameter subtracted from each measurement (16,17). The experiment was repeated once. Preliminary linear mixed model analysis indicated no significant difference (P > 0.1) between the two runs; therefore, runs were combined for each isolate and fungicide concentration to determine EC (effective concentration that reduces mycelial growth by 50%) values. Lack-of-fit tests were also conducted to compare fit of nonlinear and simple linear regression models to the data for each fungicide and isolate. In this study, simple linear regression models 50 26 September 2011 Plant Health Progress provided a poor fit (P ≤ 0.05), whereas the nonlinear models (log-logistic doseresponse curve) provided an acceptable fit (P > 0.05). Fungicide concentrations were logarithmically (log ) transformed and data were analyzed using nonlinear regression analysis with the NLIN procedure of SAS (version 9.2; SAS Institute Inc., Cary, NC). The following log-logistic doseresponse function was used, where y is the mean colony diameter growth response, x is the fungicide dose, C is the lower growth limit, D is the upper growth limit, b is the slope, and EC is the dose giving the 50% response (23,30). The log-logistic dose-response curve is used in studies where the dose response ranges from no effect to complete inhibition (23,30). A resistance factor (RF) for each fungicide was defined as the ratio of the least sensitive isolate’s EC value to the mean EC for that fungicide (19). Cross-sensitivity relationships between fungicides were calculated using the EC values for each isolate to generate Pearson correlation coefficients (r) for each pair of fungicides. Sensitivity to Thiophanate-methyl A true baseline distribution is not represented within this study for thiophanate-methyl because of the previous reliance on this chemical for control of C. cassiicola at the production facility. The range of thiophanatemethyl EC values was 0.0157 to 0.1539 μg/ml (Fig. 1) and the mean value was 0.0553 μg/ml. Despite the past exposure to thiophanate-methyl, the EC range falls within the sensitive grouping for several pathogens (24,26,32,33,34), and overlaps but extends slightly above the sensitive range for a few pathogens (28). The upper EC value (0.1539 μg/ml) is well below the discriminatory doses of 1 μg/ml (24) and 10 μg/ml (32,34) previously used to identify fungal isolates resistant to thiophanate-methyl. The RF value of 2.8 was similar to the RF value for a sensitive population of Fusicladium effusum sensitive to thiophanatemethyl (24). 10