Determining Practical Fungicide Resistance Development and Drift in the Control of Cucurbit Powdery Mildew in Pumpkin

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1 2010 Plant Management Network. Accepted for publication 11 October Published. Determining Practical Fungicide Resistance Development and Drift in the Control of Cucurbit Powdery Mildew in Pumpkin Christian A. Wyenandt, Extension Specialist in Vegetable Pathology, Daniel L. Ward, Extension Specialist in Pomology, and Nancy L. Maxwell, Former Field Researcher IV, Department of Plant Biology and Pathology, Rutgers University, Rutgers Agricultural Research and Extension Center, 121 Northville Road, Bridgeton, NJ Corresponding author: Christian A. Wyenandt. Wyenandt, C. A., Ward, D. L., and Maxwell, N. L Determining practical fungicide resistance development and drift in the control of cucurbit powdery mildew in pumpkin. Online. Plant Health Progress doi: /php rs. Abstract In 2006 and 2007, nine fungicides were evaluated to determine if practical fungicide resistance could be identified and if fungicide resistance drift occurred in cucurbit powdery mildew of pumpkin. The fungicides and/or tank mixes whose active ingredient(s) were evaluated included: sulfur (FRAC code M1), chlorothalonil (M5), myclobutanil (3), pyraclostrobin (11), azoxystrobin (11), quinoxyfen (13), chlorothalonil + myclobutanil (M5 + 3), famoxadone + cymoxanil ( ), pyraclostrobin + boscalid (11 + 7), and water only (control). Based on visual ratings of upper and lower leaf surfaces, a FRAC code 11 resistance cucurbit powdery mildew population was present in both years. Practical resistance and cross resistance were identified where a FRAC code 11 fungicide had not been applied season-long as well as where a FRAC code 11 fungicide was applied weekly or in rotation with another fungicide chemistry. Resistance to a FRAC code 3 fungicide was not identified where a FRAC code 3 fungicide had been applied season-long, or in rotation, or where no FRAC code 3 fungicide was applied. This study demonstrates that cucurbit powdery mildew populations resistant and/or cross resistant to FRAC code 11 fungicides can develop and have the potential to disseminate into and be detected in areas where no FRAC code 11 fungicides have been applied. Introduction In the United States, pumpkins (Cucurbita pepo) are grown primarily for wholesale food processing or for ornamental use during holidays such as Halloween and/or Thanksgiving. In 2007, 1000 ha of pumpkin were harvested in New Jersey, accounting for approximately 5% of United States production (3,38). Although considered a minor crop based on the number of acres produced in New Jersey, pumpkin and other cucurbit crops play an important role in keeping small roadside farm markets operational during the fall season through events such as autumn festivals and educational grade school tours. Some roadside farm markets grow pumpkins and other cucurbits crops specifically for u-pick customers. Powdery mildew, caused by Podosphaera (sect. Sphaerotheca) xanthii (Castagne) U. Braun & N. Shishkoff [also known as Sphaerotheca fusca (Fr.) S. Blumer and S. fuliginea (Schlechtend.:Fr.) Pollacci] or Golovinomyces cichoracearum DC (formerly Erisiphe cichoracearum DC), is an important disease of cucurbit crops throughout the United States (40). Powdery mildew may overwinter as cleistothecia on crop debris; however, in most years, conidia of the pathogen are wind dispersed into northern regions from southern states each production season (40). Older, mature leaves are usually infected first leading to premature defoliation resulting in yield reduction (28,40). Premature defoliation can also lead to sunscald injury resulting in unmarketable fruit. Stems infected by powdery mildew before harvest will prematurely turn brown and reduce the post-harvest longevity of marketable fruit. Control of cucurbit

2 powdery mildew begins with preventing nutritional stress, planting tolerant or resistant cultivars such as Gladiator, Magician, or Gold Bullion, and preventative fungicide applications (1,9,21,27, 33,37). Pumpkin breeding lines and cultivars have been consistently evaluated for cucurbit powdery mildew resistance, and in recent years, a number of new cultivars with powdery mildewtolerance or resistance have been released commercially (5,9,12,13,14,15,17,24,29,30). Fungicide resistance has been researched and well documented (10,11,16,20,29,31,43,36). In many crops, including cucurbits, fungicides which are translaminar or systemic are able to suppress powdery mildew on the abaxial (i.e., underside or bottom) sides of leaves where conditions are more favorable for development (22). Effective control of cucurbit powdery mildew depends on the presence of the pathogen, the timing of fungicide applications, the fungicide(s) used to control the disease, the age of the leaves, and pumpkin cultivar (6,7,8,9,18,19,23). Some newer fungicide chemistries for cucurbit powdery mildew control have modes-of-action that target fungal growth and development at specific metabolic site(s) (22,26). The first case of fungicide resistance development in the United States was reported in cucurbit powdery mildew to the FRAC (Fungicide Resistance Action Committee) code 1 fungicide, benomyl (36). In the late 1990s a new class of fungicide chemistry, known as the strobilurin fungicides (FRAC code 11), were commercially-released for the control of cucurbit powdery mildew (10). Cucurbit powdery mildew resistance to a strobilurin fungicide was first reported in the United States in 2002 (25). Management of fungicide resistance in cucurbit powdery mildew has been studied and techniques developed to directly or indirectly detect it under field conditions (2,4,24,25,39). When such indirect techniques or methods are used to measure fungicide resistance development, terms such as field or practical resistance are used to describe the observable loss of chemical control. Indirect observations of fungicide resistance development are extremely important and useful for disease management because these observations can be used to make or adjust current fungicide recommendations in real-time in specific states and/or geographic regions. In 2006 and 2007, a study using fungicide-treated potted pumpkin plants was overlaid onto a larger field study to assess nine fungicides to determine if practical fungicide resistance and/or cross resistance development and drift could be identified in cucurbit powdery mildew following five different seasonlong fungicide programs. Field Study for Season-Long Fungicide Programs for Cucurbit Powdery Mildew Control From 2005 to 2007, studies were conducted in a field (Aura sandy loam, ph = 6.4) at the Rutgers Agricultural Research and Extension Center (RAREC) in Bridgeton, NJ, to evaluate the effects of five different fungicide programs for cucurbit powdery mildew control in pumpkin production (39). Five weekly fungicide programs were done season-long (10 total applications per program) in five separate blocks each 24 m wide by 46 m long (Table 1). Between each block, 4.6-m-wide windbreaks were established by seeding sorghum-sudangrass hybrid Green Grazer V (Seedway Inc., Hall, NY) at 67.2 kg/ha on 20 April 2005, 3 May 2006, and 25 May 2007 to prevent drift between fungicide programs. The same seeding rate was used to create a 9.1-m-wide windbreak on the southwest edge of the field to reduce wind speed during fungicide applications. Within each strip for each season-long fungicide program, sub-sub plots consisted of five treated (fungicide application) and five untreated plots (no fungicide application). Each strip consisted of two rows of pumpkin cultivar Howden seeded on a 3.1-m center with 0.6 m between each hill (2 seeds/hill). The first sub-subplot in each strip was randomly assigned and the succeeding sub-subplots alternated between fungicide and no fungicide application. Each fungicide treatment sub-subplot was 7.6 m long with a 3.1-m in-row space between each sub-subplot. The five season-long fungicide programs and the active ingredients of the nine fungicides used in the potted plant bioassay are presented in Table 1.

3 Table 1. Active ingredients, FRAC code, and percent cucurbit powdery mildew severity on adaxial and abaxial leaf surfaces of pumpkin cultivar Howden treated with nine selected fungicides following five different season-long fungicide programs in 2006 and 2007 at the Rutgers Agricultural Research and Extension Center, Bridgeton, NJ season-long fungicide programs and leaf surface z Active ingredients applied to bioassay plants w FRAC code Protectants only alt. 11 alt weekly Group 11 weekly Ad x Ab x Ad Ab Ad Ab Ad Ab Ad Ab sulfur M1 0 y myclobutanil + chlorothalonil 3 + M famoxadone + cymoxanil myclobutanil azoxystrobin pyraclostrobin + boscalid pyraclostrobin quinoxyfen chlorothalonil M control (water only) Active ingredients applied to bioassay plants w FRAC code 2007 season-long fungicide programs and leaf surface z Protectants only alt. 11 alt weekly Group 11 weekly Ad x Ab x Ad Ab Ad Ab Ad Ab Ad Ab sulfur M1 9 y myclobutanil + chlorothalonil 3 + M famoxadone + cymoxanil myclobutanil azoxystrobin pyraclostrobin + boscalid pyraclostrobin quinoxyfen chlorothalonil M control (water only) w Active ingredient rates applied to run-off on bioassay plants were: sulfur at 2.24 kg/ha; myclobutanil at 0.35 kg/ha; chlorothalonil at 3.5 liters/ha; famoxadone + cymoxanil at 0.56 kg/ha; myclobutanil at 0.35 kg/ha; azoxystrobin at 0.35 kg/ha; pyraclostrobun + boscalid at 1.30 kg/ha; pyraclostrobin at 0.84 kg/ha; quinoxyfen at 0.29 liters/ha; or control (distilled water only). x Ad = adaxial, Ab = abaxial. y Mean powdery mildew severity on the adaxial and abaxial surfaces of three leaves from each fungicide treated potted plant. Powdery mildew severity was rated on a scale of 0.0 to (where 0.0 = none, 100 = 100%) (i.e., percentage of adaxial or abaxial of leaf surfaces with symptoms of powdery mildew). z The five season-long fungicide programs were: protectants only (2.24 kg/ha manzate kg/ha sulfur alternated weekly with 2.24 kg/ha maneb liters/ha copper hydroxide); alt. 11 (3.5 liters/ha chlorothalonil kg/ha myclobutanil alternated weekly with 0.35 kg/ha azoxystrobin); alt. weekly with (2.24 kg/ha maneb kg/ha myclobutanil alternated weekly with 0.56 kg/ha famoxadone + cymoxanil); weekly (3.5 liters/ha chlorothalonil kg/ha myclobutanil alternated weekly with 0.35 kg/ha myclobutanil); Group 11 weekly (3.5 liters/ha chlorothalonil kg/ha azoxystrobin alternated weekly with 0.35 kg/ha azoxystrobin).

4 Seedling Bioassays to Determine Fungicide Resistance Development Following Five Different Season-Long Fungicide Programs Whole plots within the field study were used as the blocks for a randomized complete block design containing treatments that were allocated to potted pumpkin plants. In 2006 and 2007, three seeds of powdery mildew-susceptible pumpkin cultivar Howden were sown in a 393-cm³ pot containing Metro-Mix 360 potting soil (Scotts-Sierra Horticultural Products Company, Marysville, OH). All pots were placed in a greenhouse under mist irrigation until germination, then thinned to one plant per pot and placed on greenhouse benches with drip tubes until each plant had three to five true leaves. Each leaf to be evaluated was labeled using black magic marker. On 1 October 2006 and 23 September 2007, each pot was labeled according to fungicide treatment and a squirt bottle containing the equivalent rate per hectare of product (Table 1) was used to apply each fungicide to the adaxial and abaxial sides of each marked leaf (3 per plant) until run-off. After applying each fungicide treatment, plants were placed back into the greenhouse and allowed to dry for 24 h before being placed into the field. On 2 October 2006 and 24 September 2007 each pot containing one of the nine fungicide treated and a control plant were placed in the center of an untreated sub-plot in each of the five season-long fungicide program plots for 48 h to allow for infection by cucurbit powdery mildew. After 48 h all pots were placed back into the greenhouse with drip tube irrigation so leaves would remain dry during the evaluation period. After 7 days, powdery mildew severity on the adaxial and abaxial surfaces of three leaves from each treated plant were rated on a scale of 0.0 to (where 0.0 = none, 100 = 100%) (i.e., percentage of adaxial or abaxial of leaf surfaces with symptoms of powdery mildew). Powdery mildew severity on the adaxial or abaxial surfaces of the leaves were analyzed separately using ANOVA to test the main effects of fungicide treatment and year as well as their interaction. The MIXED procedure of the SAS system (ver. 9.2 SAS Institute, Cary, NC) was used to perform the analysis on the means of the three leaf surfaces (subsamples) from each plant. Departures from model assumptions were investigated using graphs of the standardized residuals. The year by fungicide treatment interaction means were further analyzed using Tukey's HSD (α = 0.05) to compare all pairs of fungicide treatment means within each year (35). Year had a significant interaction with fungicide treatment on powdery mildew severity on the adaxial leaf surfaces of potted plants (P = ) with severity on the potted plants higher in 2006 than 2007 (P = ) (Fig. 1). In both years, severity was higher on adaxial leaf surfaces (Fig. 1). Resistance to the FRAC 11 fungicide, azoxystrobin, was observed in both years of the study (Fig. 1). Strobilurin efficacy was poor on plants placed in locations where a FRAC code 11 fungicide had not been applied season-long, suggesting that a resistant FRAC code 11 cucurbit powdery mildew population had developed and drifted during each production season (Table 1). The efficacy of another FRAC 11 fungicide, pyraclostrobin (Cabrio, BASF Corporation, Research Triangle Park, NC), was also poor and comparable to the control in both years even though it had not been applied during each production season, suggesting that crossresistance was present to FRAC 11 fungicides (Table 1 and Fig. 1). Control of powdery mildew with pyraclostrobin + boscalid was better compared to famoxadone + cymoxanil which was comparable to the FRAC 11 fungicides, azoxystronin and pyraclostrobin, in both years (Fig. 1). This suggests that the lack of control with famoxadone + cymoxanil was due to the failure of the famoxadone (FRAC code 11), because cymoxanil (FRAC code 27) has no activity against cucurbit powdery mildew (Table 2). In the pyraclostrobin + boscalid treatment in both years, control was likely achieved due to the efficacy of boscalid (FRAC code 7), and not pyraclostrobin (FRAC code 11) (Fig. 1). Other studies have reported boscalid efficacy against cucurbit powdery mildew, although it is not labeled for use as a stand-alone product by the manufacturer. More recently, resistance in cucurbit powdery mildew to boscalid has been detected (McGrath, unpublished). Myclobutanil provided good to excellent

5 control in both years on plants placed in blocks where no myclobutanil had been applied season-long, or where it had been applied weekly or in rotations with a different mode-of-action fungicides (Table 1 and Fig. 1). This suggests that resistance in the powdery mildew population to the FRAC code 3 fungicide was not present in both years. The fungicide containing quinoxyfen (FRAC 13) provided a high level of control of cucurbit powdery mildew in both years of this study (Table 1 and Fig. 1).

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7 Fig. 1. Percent powdery mildew development on the adaxial (B,D) or abaxial (A,C) leaf surfaces of pumpkin cultivar Howden in response to nine fungicide treatments at the end of the production season following five different fungicide programs in 2006 (A,B) and 2007 (C,D). Fungicide treatments are: Control = Water only; Q = Quintec; N + B = Nova + Bravo; N = Nova; P = Pristine; S = Sulfur; B = Bravo; T = Tanos; C = Cabrio; and A = Amistar. Bars with no lower case letters in common are significantly different (α = 0.05) according to Tukey's HSD.

8 Conclusions and Recommendations This study demonstrates that cucurbit powdery mildew can develop resistance, as well as cross resistance, to important high-risk fungicide chemistries in the mid-atlantic region of the United States. Applications of high-risk fungicides, such as those in FRAC code 11 (or other high-risk FRAC groups) for cucurbit powdery mildew control in the mid-atlantic and Northeast region needs to be done judiciously because the threats for resistance and cross resistance selection are high. This study also helps demonstrate that fungicide resistant cucurbit powdery mildew populations have the potential to disseminate (i.e., drift) into areas (i.e., other fields or nearby farms) where no high-risk fungicides had been applied. This may also apply to larger geographic areas where one particular high-risk fungicide or group of fungicides have been overused resulting in a resistant pathogen population that can disseminate over large geographic distances. For example, a FRAC code 11 resistant cucurbit powdery mildew population could move into the mid-atlantic or Northeast regions from southeastern states each year if those high-risk fungicides are routinely used on southern vegetable farms. Therefore, the successful management of fungicide resistance development in highly mobile pathogens such as cucurbit powdery mildew or downy mildew, caused by Pseudoperonospora cubensis (Berk. & M.A. Curtis) Rostovzev, may require more regional cooperation between states and/or regions when developing vegetable fungicide recommendations. Otherwise, growers using high-risk fungicides exclusively may select resistant strains and thereby reduce the efforts of growers using a resistance management program (22). Parnell et al. (32) suggested that in-field strategies, such as the alternation and/or tank mixing of fungicides used to combat fungicide resistance may be more useful through the restricted deployment of fungicides over large areas. Increased restrictions on fungicide use (i.e., such as those in FRAC 11 or other high-risk fungicides) in this manner may be extremely beneficial in managing fungicide resistance development in an important disease such as cucurbit powdery mildew that has the potential to disseminate over vast geographic areas (i.e., the east coast of the United States) each year, while at the same time preserving the efficacy and lifespan of the active ingredient(s). The use of broad-spectrum protectant fungicides, such as chlorothalonil, as well as, the more specific use of an elemental fungicide, such as sulfur, for cucurbit powdery mildew control remains critically important for managing potential resistance development. A protectant fungicide should be tank mixed or rotated with high risk fungicides as much as possible during the production season for cucurbit powdery mildew control and resistance management. Literature Cited 1. Alexander, S. A., and Waldenmaier, C. M Evaluation of fungicides for control of powdery mildew in pumpkin, Fungic. Nematic. Tests. 54: Cohen, R., Burger, Y., and Katzir, N Monitoring physiological races of Podosphaera xanthii (syn. Sphaerotheca fuliginea), the causal agent of powdery mildew in cucurbits: factors affecting race identification and the importance for research and commerce. Phytoparasitica 32: Ingerson-Mahar, J., Rabin, J., and Wyenandt, A Crop profile for pumpkins in New Jersey. Online. New Jersey Agric. Exp. Stn., Rutgers Univ., New Brunswick, NJ. 4. Cushman, K. E., Evans, W. B., Ingram, D. M., Gerard, P. D., Straw, R. A., Canaday, C. H., Wyatt, J. E., and Kenty, M. M Reduced foliar disease and increased yield of pumpkin regardless of management approach or fungicide combinations. HortTechnology 17: Epinat, C., Pitrat, M., and Bertrand, F Genetic analysis of resistance of five melon lines to powdery mildews. Euphytica 65: Everts, K. L Integrated pumpkin disease management using a cover crop, host resistance and reduced fungicide application. (Abstr.) Phytopathology 89:S Everts, K. L First report of benomyl resistance in Didymella bryoniae in Delaware and Maryland. Plant Dis. 83:304.

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