Preharvest Applications of Fungicides for Control of Sphaeropsis Rot in Stored Apples

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1 2013 Plant Management Network. Accepted for publication 18 July Published. Preharvest Applications of Fungicides for Control of Sphaeropsis Rot in Stored Apples Y. K. Kim, Pace International, Wapato, WA 98951; and C. L. Xiao, United States Department of Agriculture-Agricultural Research Service, San Joaquin Valley Agricultural Sciences Center, Parlier, CA Corresponding author: C. L. Xiao. Kim, Y. K., and Xiao, C. L Preharvest applications of fungicides for control of Sphaeropsis rot in stored apples. Online. Plant Health Progress doi: /php rs. Abstract Sphaeropsis rot caused by Sphaeropsis pyriputrescens is a recently reported postharvest fruit rot disease of apple in Washington State and causes significant economic losses. Infection of apple fruit by the fungus occurs in the orchard, but decay symptoms develop during storage or in the market. The objective of this study was to evaluate preharvest fungicide applications to control Sphaeropsis rot. Thirty isolates of the fungus collected from various sources were tested for sensitivity to the registered fungicides Pristine, Topsin M, and Ziram using an in vitro mycelial growth assay. In the orchard, Golden Delicious apple fruit were inoculated with the conidial suspension of the fungus at 2 or 5 weeks before harvest, sprayed with fungicides within 2 weeks before harvest, and harvested and stored at 0 C for disease evaluation. All three fungicides effectively inhibited mycelial growth of the fungus in the in vitro tests. On apple fruit in four seasons, Pristine applied 1 week and Ziram applied 2 weeks before harvest significantly reduced incidence of Sphaeropsis rot compared to the nontreated control by 43 to 80% and 42 to 83%, respectively. In 4 years of testing, the performance of Topsin M was less consistent than that of Pristine and Ziram. Introduction Postharvest fruit rot diseases, left uncontrolled, can result in significant economic losses of apples during the postharvest storage period which can last for 12 months. Sphaeropsis rot, caused by Sphaeropsis pyriputrescens Xiao & J.D. Rogers, is a recently described postharvest fruit rot disease of apple (11,13). A survey for postharvest diseases of apples conducted on cultivars of Red Delicious, Fuji, and Golden Delicious during in Washington State indicated that in addition to gray mold caused by Botrytis cinerea and blue mold caused by Penicillium expansum, Sphaeropsis rot was a major postharvest disease of apples, accounting for approximately 17% of the total rots and that S. pyriputrescens was present in all major apple production counties in central Washington State (1). Sphaeropsis rot has the potential to cause significant losses of fruit during storage. In one instance where no fungicides had been applied to Red Delicious fruit, 24% of the fruit were decayed by S. pyriputrescens in storage (13). It is believed that infections of apple fruit by S. pyriputrescens occur in the orchard but remain latent, and Sphaeropsis rot symptoms develop in storage (2,13). Control of postharvest diseases has been largely dependent on the postharvest use of fungicides. In Washington State, postharvest fungicide treatments are usually applied to bins of fruits via recirculating drench solutions, and a postharvest drench with the fungicides Penbotec (pyrimethanil), Scholar (fludioxonil), or Mertect (thiabendazole) applied to the fruit on the day of harvest is commonly used by packers to control blue mold, gray mold, Sphaeropsis rot, and other diseases in stored apples (10). However, there are concerns about the accumulation of decay-causing pathogens in the recirculating drenches (5). Preharvest fungicides applied near harvest provide an alternative

2 to postharvest fungicides for decay control. For example, Pristine (a premixed formulation of pyraclostrobin and boscalid) applied within two weeks before harvest is effective in reducing postharvest losses resulting from gray mold and blue mold in stored apples (8). In the present study, we evaluated the effectiveness of Pristine, Topsin M, and Ziram as preharvest sprays for control of Sphaeropsis rot in stored apples. Laboratory Tests In total, 30 single-spore isolates of S. pyriputrescens used in a previous study (12) were selected for testing in vitro sensitivity to preharvest fungicides. Twenty of the isolates were recovered from decayed apple fruit and 10 were recovered from twig dieback or cankers of apple and crabapple trees. Fungal isolation was performed following the procedures of Kim and Xiao (3) and identification was based upon the descriptions of the fungus by Xiao and Rogers (11). The cultures were stored in 15% glycerol at -80 C prior to use. Technical grade pyraclostrobin (98% a.i.; BASF Corporation, Research Triangle Park, NC), boscalid (99% a.i.; BASF Corporation), Zinc dimethyldithiocarbamate (97.1% a.i.; Cerexagri, Inc., King of Prussia, PA), and thiophanate-methyl (98.2% a.i.; Cerexagri, Inc.) were used for in vitro sensitivity tests. Stock solutions of the fungicides were made in dimethyl sulfoxide (DMSO) and added into potato dextrose agar (PDA; Difco Laboratories, Detroit, MI) to produce the following concentrations: 0, 0.001, 0.01, 0.1, 0.5, 1, and 10 µg/ml of pyraclostrobin; 0, 0.01, 0.1, 0.5, 1, 5, and 10 µg/ml of boscalid; 0, 0.025, 0.05, 0.1, 0.5, 1, and 10 µg/ml of Zinc dimethyldithiocarbamate; and 0, 0.1, 0.25, 0.5, 1, 2.5, 5, and 10 µg/ml of thiophanate-methyl. To make a Pristine stock solution at a desired concentration, one part of pyraclostrobin and two parts of boscalid solution at the same concentration as the desired concentration for Pristine were mixed by volume. A stock solution of salicylhydroxamic acid (SHAM, 99% a.i.; Sigma-Aldrich Inc., St. Louis) was prepared at a concentration of 100 mg/ml in methanol and added into pyraclostrobin- and Pristine-amended media to inhibit the alternative oxidase respiration (4). Since the highest concentration of boscalid stock solution with the technical grade could not inhibit mycelia growth of the fungus by 50% relative to the control, additional concentrations were prepared with a commercial formulation (Endura, BASF Corporation) and the fungus was tested at 25, 50, 100, and 200 µg/ml of boscalid. The same amount of solvent was added into the control plates. For the mycelial growth assay, single-spore isolates were grown on PDA for 3 days at 20 C in the dark. A 5-mm-diameter plug of mycelium was removed from the margin of actively growing colonies with a sterile cork borer, placed inversely at the center of each plate and incubated at 20 C in the dark for 3 days. Colony diameters were measured at two perpendicular directions. There were two replicate plates for each fungicide concentration for each isolate. The experiment was conducted twice for all isolates. EC 50 value, which is the effective concentration of a fungicide that inhibits mycelial growth by 50% relative to the control, was calculated for each isolate and each fungicide by regression analysis of the percent inhibition versus the log of the fungicide concentration using SAS PROC REG (version 9.1; SAS Institute Inc., Cary, NC). A t test was performed to compare EC values between Pristine and Pyraclostrobin using SAS. 50 Preharvest Fungicide Efficacy Trials Fungicide efficacy trials were conducted in each season from 2005 to 2008 in a research orchard of Golden Delicious trees planted in 1985 at the Washington State University Tree Fruit Research and Extension Center, Wenatchee, WA. Insects and weeds were controlled following the recommendations commonly used for commercial apple production in the region (6). In each year, Flint 50W (trifloxystrobin) was applied to the trees in late April (pink stage) followed by another application in early June (4 weeks after petal fall stage, i.e. the second cover spray after the petal fall) to control powdery mildew on the foliage and no fungicides were applied in the orchard thereafter.

3 A single-spore isolate (CLX2380) recovered from a decayed apple was used in this study. To produce conidial inoculum for inoculation of fruit in the field, the isolate was grown on oatmeal agar [OMA, 60 g of finely ground iron- and zinc-fortified oatmeal (Gerber, Fremont, MI), 15 g agar in 1 liter of deionized water, autoclaved at 121 C for 90 min], sealed with Parafilm and incubated at 20 C under a 12-h light/12-h dark cycle (10 W/m²) for 3 weeks (1). Plates were flooded with sterile water, conidia were scraped off from the surface of the medium, and the resulting conidial suspensions were filtered through two layers of cheesecloth. The final concentration was adjusted to conidia/ml with a hemacytometer and Tween 20 (0.01%) was added into the final suspension. Fruit of Golden Delicious were inoculated with the conidial suspension at 5 or 2 weeks before harvest (16 August or 7 September in 2005 and 2006, 17 August or 6 September in 2007, and 21 August or 11 September in 2008). At each inoculation time, fruit per replicate were randomly selected from two trees for each treatment. Fruit were inoculated with the conidial suspension using a hand sprayer until runoff. The treatments were arranged as a randomized complete block design with four replicates per treatment on each inoculation date. The fruit were then covered with moistened plastic bags to maintain high humidity and white paper bags to prevent heat from morning sunlight reaching the fruit. Inoculations were performed near sunset to avoid high temperature affecting viability of the fungal conidia. After approximately 15 h of incubation, the bags were removed. Pristine at oz/gal, Topsin M at oz/gal, and Ziram at oz/gal were applied to the fruit until runoff using a backpack sprayer. Ziram was applied only to the fruit that were inoculated 5 weeks before harvest as its preharvest interval is 14 days for apple. The fruit were harvested at maturity stages acceptable for commercial harvest (21 September 2005, 20 September 2006 and 2007, and 24 September 2008), placed on sterilized fiberboard apple-trays wrapped in perforated polyethylene bags, and stored in cardboard apple boxes at 0 C. Decay development on the fruit, infection sites (stem or calyx) on the fruit and percentage of fruit with the symptoms of Sphaeropsis rot were recorded monthly after harvest for up to 9 months. Analysis of variance was performed with SAS PROC GLM (Version 9.1, SAS Institute) to compare the efficacy of the fungicide treatments. Mean separation was conducted using a Fisher s least significant difference (LSD, P = 0.05). All percentage data were arcsine-transformed prior to analysis. In vitro Sensitivity to Selected Fungicides Pyraclostrobin, thiophanate-methyl and Ziram were effective in inhibiting mycelial growth of the fungus in the in vitro tests (Table 1). Boscalid was much less effective than other three fungicides. The EC 50 of Pristine was lower than that of either pyraclostrobin or boscalid alone. Table 1. Mean and range of EC50 (µg/ml) values of pyraclostrobin, boscalid, Pristine, ziram, and thiophanate-methyl for mycelial growth of Sphaeropsis pyriputrescens isolated from different host. Hosts Apple fruit Apple fruit Postharvest treatment Nondrenched No. of isolates 10 Pyraclostrobin Boscalid Pristine Ziram x z Values are the means of pooled data from the two runs of the experiments. Mean Range Mean Mean Range Mean Range Mean Range z Thiophanatemethyl TBZdrenched Apple tree Crabapple tree > > > >

4 Efficacy of Fungicides Applied before Harvest Pristine applied 1 week before harvest and Ziram applied 2 weeks before harvest significantly reduced incidence of Sphaeropsis rot on stored apple fruit in all 4 seasons (Figs. 1 and 2). On the fruit inoculated 2 weeks before harvest, Pristine reduced the incidence of Sphaeropsis rot by 56.7%, 80%, 56.9%, and 57.7% compared to the non-treated control at 9 months after harvest in the , , , and seasons, respectively. On the fruit inoculated 5 weeks before harvest, Pristine reduced the incidence of Sphaeropsis rot by 70.4%, 79.9%, 38.3%, and 43.2% compared to the nontreated control 9 months after harvest in the respective seasons. Ziram significantly reduced incidence of Sphaeropsis rot compared to the non-treated control by 83.3, 73.9, 41.7, and 55.1% in the respective seasons. Topsin M significantly reduced Sphaeropsis rot incidence except on the fruit inoculated 2 weeks before harvest in and and on the fruit inoculated 5 weeks before harvest in (Figs. 1 and 2). Over the four seasons of this study, the performance of Topsin M was less consistent than that of Pristine and Ziram. Fig. 1. Efficacy of Pristine and Topsin M applied 1 week before harvest for the control of Sphaeropsis rot in stored Golden Delicious apple fruit in the , , , and seasons. Fruit were inoculated 2 weeks before harvest in the orchard with the conidial suspension of Sphaeropsis pyriputrescens, harvested, stored at 0 C and monitored for decay development for up to 9 months after harvest. Columns marked with the same letter are not significantly different at P = 0.05 according to Fisher s protected least significant difference.

5 Fig. 2. Efficacy of Pristine and Topsin M applied 1 week before harvest and Ziram applied 2 weeks before harvest for the control of Sphaeropsis rot in stored Golden Delicious apple fruit in the , , , and seasons. Fruit were inoculated 5 weeks before harvest in the orchard with the conidial suspension of Sphaeropsis pyriputrescens, harvested, stored at 0 C, and monitored for decay development for up to 9 months after harvest. Columns marked with the same letter are not significantly different at P = 0.05 according to Fisher s protected least significant difference. Implications for Control of Postharvest Fruit Rots One of the challenges for control of postharvest fruit rot diseases of apple is that multiple diseases need to be targeted. Fungicides that have broad-spectrum activities against major pathogens would have the potential as a preharvest treatment for control of postharvest diseases of apples. Pristine is considered a reduced-risk fungicide, which poses a low risk to humans and the environment, according to the U.S. Environmental Protection Agency classification (7). Pristine applied within 2 weeks before harvest also was effective in controlling gray mold and blue mold (8). Our results suggest that Pristine can be an effective alternative to postharvest fungicides for control of major postharvest diseases of apples given that there are continuing concerns about postharvest use of synthetic pesticides leading to residues on fresh produce as postharvest fungicide treatments are significant contributors to detectable residues on apples (9) and that there are potential food safety risks associated with using recirculating solutions on fresh produces. However, it should also be noted that preharvest application of fungicides may not be as effective as postharvest fungicides registered for apple such as Scholar (fludioxonil), Penbotec (pyrimethanil), and Mertect (thiabendazole) for control of Sphaeropsis rot (12).

6 When these postharvest fungicides are used as a drench treatment on the day of harvest, all three of the postharvest fungicides are equally effective for control of Sphaeropsis rot in stored apples, reducing decay incidence by 92 to 100% compared to the nontreated control (12). Nonetheless, preharvest fungicides can be practical alternatives for postharvest disease control when postharvest use of fungicides is not desirable. Acknowledgments We thank R. J. Boal for technical assistance. This research was supported in part by the Washington Tree Fruit Research Commission. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendations or endorsement by the United States Department of Agriculture. USDA is an equal opportunity provider and employer. Literature Cited 1. Kim, Y. K., Xiao, C. L., and Rogers, J. D Influence of culture media and environmental factors on mycelial growth and pycnidial production of Sphaeropsis pyriputrescens. Mycologia 97: Kim, Y. K., and Xiao, C. L Infection courts and timing of infection of apple fruit by Sphaeropsis pyriputrescens in the orchard in relation to Sphaeropsis rot in storage. Phytopathology 97:S Kim, Y. K., and Xiao, C. L Distribution and incidence of Sphaeropsis rot in apple in Washington State. Plant Dis. 92: Olaya, G., and Koller, W Diversity of kresoxim-methyl sensitivities in baseline populations of Venturia inaequalis. Pestic. Sci. 55: Rosenberger, D. A Blue mold. Pages in: Compendium of Apple and Pear Diseases. A. L. Jones, and H. S. Aldwinkle, eds. American Phytopathological Society, St. Paul, MN. 6. Smith, T. J., Dunley, J., Beers, E. H., Brunner, J. F., Grove, G. G., Xiao, C. L., Elfving, D. C., Peryea, F., Parker, R., Mayer, D. F., Woodruff, R., Daniels, C., Maxwell, T., and Roberts, S Crop Protection Guide for Tree Fruits in Washington. Ext. Bull. (EB 0419), Washington State University, Pullman, WA. 7. US-EPA Accomplishments under the Food Quality Protection Act (FQPA)- 10th Anniversary of the Food Quality Protection Act. US-EPA, Washington, DC. 8. Xiao, C. L., and Boal, R. J Preharvest application of a boscalid and pyraclostrobin mixture to control postharvest gray mold and blue mold in apples. Plant Dis. 93: Xiao, C. L., and Boal, R. J Residual activity of fludioxonil and pyrimethanil against Penicillium expansum on apple fruit. Plant Dis. 93: Xiao, C. L., and Kim, Y. K Control of postharvest diseases in apples with reduced-risk fungicides. Stewart Postharvest Review doi: /spr Xiao, C. L., and Rogers, J. D A postharvest fruit rot in d Anjou pears caused by Sphaeropsis pyriputrescens sp. nov. Plant Dis. 88: Xiao, C. L., Kim, Y. K., and Boal, R. J Control of Sphaeropsis rot in stored apple fruit caused by Sphaeropsis pyriputrescens with postharvest fungicides. Plant Dis. 95: Xiao, C. L., Rogers, J. D., and Boal, R. J First report of a new postharvest fruit rot on apple caused by Sphaeropsis pyriputrescens. Plant Dis. 88:223.