Baseline sensitivities to fludioxonil and pyrimethanil in Penicillium expansum populations from apple in Washington State

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Postharvest Biology and Technology 47 (2008) 239–245

Baseline sensitivities to fludioxonil and pyrimethanil in Penicillium expansum populations from apple in Washington State H.X. Li 1 , C.L. Xiao ∗ Department of Plant Pathology, Washington State University, Tree Fruit Research and Extension Center, 1100 North Western Avenue, Wenatchee, WA 98801, United States Received 8 May 2007; accepted 30 June 2007

Abstract Penicillium expansum is the primary cause of blue mold, a common postharvest fruit rot disease of apple. In 2004, two new fungicides, fludioxonil and pyrimethanil, were registered for postharvest use on pome fruits in the U.S. To establish distribution of baseline sensitivity of P. expansum to fludioxonil and pyrimethanil before their commercial use, 120 isolates recovered from apple orchards and fruit packinghouses across the apple growing areas in central Washington were selected and tested in vitro for sensitivity to these two fungicides using mycelial growth assays. Baseline EC50 values ranged from 0.011 to 0.068 (average = 0.020) mg/L for fludioxonil and from 0.519 to 2.054 (average = 1.340) mg/L for pyrimethanil. One isolate showed reduced sensitivity to fludioxonil with an EC50 of 0.068 mg/L, which was significantly higher (P < 0.0001) than those of remaining isolates tested. Fludioxonil at 0.5 mg/L completely inhibited mycelial growth of all isolates tested except for the isolate with reduced sensitivity. Conidial germination and germ-tube elongation were completely inhibited by pyrimethanil at 0.5 mg/L and by fludioxonil at 0.1 mg/L except for the isolate with reduced sensitivity based on the mycelial growth assay. Discriminatory concentrations of 0.5 mg/L fludioxonil for mycelial growth and 0.5 mg/L pyrimethanil for germ-tube elongation were recommended for phenotyping isolates of P. expansum for resistance to these two fungicides. No cross-sensitivity correlation in P. expansum was observed among thiabendazole, fludioxonil, and pyrimethanil. Fludioxonil and pyrimethanil applied at label rates were effective in controlling blue mold on apple fruit inoculated with isolates exhibiting different degrees of sensitivity to these two fungicides. The results indicate that the current population of P. expansum can be effectively controlled by these two new postharvest fungicides. The information generated in this study on baseline sensitivity distribution is useful in monitoring future shifts in sensitivities to these two new fungicides in P. expansum populations from apples in the region. © 2007 Elsevier B.V. All rights reserved. Keywords: Blue mold; Fungicide resistance; Penbotec; Postharvest disease; Scholar; Thiabendazole

1. Introduction Penicillium expansum (Link) Thom. is the primary causal agent of blue mold, a common postharvest disease of apple (Rosenberger, 1990). P. expansum is essentially a wound mediated pathogen. Wounds on the fruit peel such as punctures and bruises that are created at harvest and during postharvest handling are the major avenues for infection by the fungus (Rosenberger, 1990). Control of blue mold and other postharvest diseases has been largely dependent on fungicides (Eckert and Ogawa, 1988; Errampalli, 2004). Thiabendazole (TBZ) has been

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Corresponding author. Tel.: +1 509 663 8181x229; fax: +1 509 662 8714. E-mail address: [email protected] (C.L. Xiao). 1 Present address: Department of Plant Pathology, Nanjing Agricultural University, Nanjing, China. 0925-5214/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.postharvbio.2007.06.022

commonly used as either a drench treatment prior to storage or an online treatment at packing for control of postharvest diseases of apple, but resistance to benzimidazole fungicides (benomyl and TBZ) in P. expansum often results in the failure of blue mold control (Bertrand and Saulie-Carter, 1978; Rosenberger and Meyer, 1979). Diphenylamine (DPA) is an anti-scald agent and is often used in combination with fungicides as a pre-storage drench treatment for control of scald and decay. Previous reports indicated that when DPA and TBZ are combined as a drench treatment, DPA inhibits benzimidazole-resistant strains of P. expansum while the benzimidazole continues to control sensitive isolates (Rosenberger and Meyer, 1985). Presently, most isolates of P. expansum are resistant to the DPA/benzimidazole combination due to repeated exposures to the combination (Sholberg et al., 2005b; Errampalli et al., 2006). In 2004, two new fungicides, fludioxonil and pyrimethanil, were registered for postharvest use on pome fruits in the U.S.

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Both fungicides are effective in controlling blue mold, including TBZ-resistant isolates (Errampalli and Crnko, 2004; Sholberg et al., 2005a). Fludioxonil belongs to the chemical class of phenylpyrrole. It inhibits conidial germination and mycelial growth of P. expansum (Errampalli, 2004). Field resistance to fludioxonil in Alternaria spp. from crucifers and P. digitatum from lemon has been reported (Iacomi-Vasilescu et al., 2004; Kanetis et al., 2006). Pyrimethanil belongs to the anilinopyrimidine class, whose in vitro antifungal activity could result from a block in the excretion of hydrolytic enzymes involved in the pathogenic process in a site-specific manner and/or from an inhibition of methionine biosynthesis in fungal cells (Leroux and Gredt, 1996; Takagaki et al., 2004). Resistance of Botrytis cinerea to the anilinopyrimidine fungicides has been reported, indicating a high risk for build-up of resistance to this class of fungicides in fungal pathogens (Chapeland et al., 1999; Leroux et al., 1999; Latorre et al., 2002; Baroffio et al., 2003; Moyano et al., 2004). Experience with TBZ resistance in P. expansum and occurrence of resistance to pyrimethanil and fludioxonil in other fungal pathogens suggests that it is necessary to establish a monitoring program for early detection of reduced sensitivity to the two new postharvest fungicides in P. expansum and to implement resistance management practices. Establishment of baseline sensitivity distribution is the first step in developing a fungicide resistance monitoring and management program (Russell, 2004). Sensitivities to fludioxonil and pyrimethanil in P. expansum from apple in Canada have been reported (Errampalli, 2004; Sholberg et al., 2005a), but the tests were based only on a limited number of isolates and not intended to establish the distribution of baseline sensitivity to the fungicides. In addition, only the sensitivity of the fungal mycelial growth to pyrimethanil was tested (Sholberg et al., 2005a). The objectives of the present study were to (1) establish distribution of baseline sensitivity to fludioxonil and pyrimethanil in P. expansum populations from apple in Washington State; (2) compare sensitivity of mycelial growth, conidial germination and germ-tube elongation to the two fungicides; (3) determine whether cross-sensitivity correlation in P. expansum occurs among thiabendazole, fludioxonil and pyrimethanil; and (4) determine whether fludioxonil and pyrimethanil applied at label rates are effective to control isolates with reduced sensitivity on apple fruit. 2. Materials and methods 2.1. Sources of Penicillium expansum isolates During April to August in 2004 and 2005, decayed apple fruit were sampled from seven packinghouses in central Washington State, which is the primary region for apple production in the state. The fruit were either drenched with TBZ or not treated prior to storage. Decayed fruit were collected during packing operations. To isolate Penicillium spp. from blue mold or blue mold-like decayed fruit, the peel at the margin of decayed area was removed and small pieces of decayed tissue were placed on acidified potato dextrose agar plate (APDA, 4 mL of a 25% (v/v)

solution of lactic acid per liter of medium). Cultures were transferred onto fresh APDA plates. Isolates of Penicillium spp. were identified to P. expansum following Pitt’s protocol and description of the fungus (Pitt, 2002). Isolates of P. expansum used in this study were single-spore cultured and stored as conidial suspensions in 15% glycerol (1:1, v/v) at −80 ◦ C. One hundred and ninety three isolates of P. expansum were obtained from decayed fruit in 2004 and 2005, respectively. Isolates of P. expansum also were collected from apple orchards. In November 2004, fallen apple fruit on the orchard floor with blue mold or blue mold-like symptoms were collected from 16 apple orchards across the apple-production areas in central Washington State. At least 20 decayed fruit were collected from each orchard. Isolations were performed as described above. A total of 303 P. expansum isolates was obtained from apple orchards. 2.2. Screening for resistance to thiabendazole Isolates of P. expansum were screened for resistance to TBZ. Technical-grade TBZ (99% a.i.; Sigma, St. Louis) was dissolved in methanol to make a 5 mg/mL stock solution. One milliliter of the stock solution was added into 1 L of potato dextrose agar (PDA) to make 5 mg/L TBZ-amended PDA. A sterile 4-mmdiameter plastic loop was used to transfer dry conidia from a 7-day-old PDA culture of each isolate to a 5-mL screw-cap tube containing 1 mL of sterile distilled water with 0.01% Tween 20 (Rosenberger et al., 1991). The resulting concentration was approximately 106 conidia/mL. The conidial suspension was poured into 16 mL of molten PDA at 50 ◦ C, and the medium was then poured immediately into a plate. Cultures were incubated at 20 ◦ C for 24 h. Agar plugs were cut from the cultures with a 6-mm-diameter cork borer. Three such plugs were placed upside-down on TBZ-amended PDA in each plate. Three replicate plates were used for each isolate. Colony diameters were measured after 5 days at 20 ◦ C. Isolates that were able to grow on the amended media with 5 mg/L TBZ were considered resistant to TBZ. 2.3. Sensitivity of P. expansum mycelial growth to fludioxonil and pyrimethanil Sixty isolates (8 TBZ-R and 52 TBZ-S isolates of P. expansum) recovered from apple orchards and 60 isolates (30 isolates each of TBZ-R and TBZ-S) from decayed apple fruit from packinghouses were tested for sensitivity of mycelial growth to fludioxonil and pyrimethanil. Technical-grade fludioxonil (93% a.i.; Syngenta Crop Protection, Greensboro, NC) and pyrimethanil (96% a.i.; Janssen Pharmaceutica, Belgium) were dissolved in acetone, each adjusted to a concentration of 10 mg/mL stock solution, and added to produce the following concentrations: 0, 0.01, 0.015, 0.03, 0.05, 0.1, 0.15, 0.2 and 0.5 mg/L of fludioxonil in PDA and 0, 0.5, 0.75, 1, 1.5, 2.5 and 10 mg/L of pyrimethanil in a l-asparagine-based agar medium (ASP-agar) (Hiber and Sch¨uepp, 1996) containing per liter: 1 g K2 HPO4 , 1 g MgSO4 ·7H2 O, 0.5 g KCl, 0.01 g FeSO4 ·7H2 O, 2 g l-asparagine, 22 g glucose, 15 g agar. The con-

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centration of acetone in all media, including the media without fungicides, was 0.1 mL/L. Inoculum was prepared as described above. A 6-mm-diameter agar plug was placed on the center of each plate containing a fungicide-amended medium. For each isolate, three replicates per concentration were used. The experiment was performed twice. The radial growth (colony diameter) of each isolate was measured after incubation for 7 days at 20 ◦ C in the dark. For each plate, the average colony diameter measured in two perpendicular directions was used for calculation of EC50 , which is the effective concentration of a fungicide that results in 50% inhibition of mycelial growth. 2.4. Sensitivity of conidial germination and germ-tube elongation Ten TBZ-sensitive and 10 TBZ-resistant P. expansum isolates selected from the 120 isolates were tested for the sensitivity of conidial germination and germ-tube elongation to fludioxonil and pyrimethanil. Sensitivity to fludioxonil was tested on PDA amended with 0, 0.01, 0.015, 0.02, 0.035, 0.05 and 0.1 mg/L of fludioxonil. Sensitivity to pyrimethanil was tested on an agar medium (10 g glucose, 2 g K2 HPO4 , 2 g KH2 PO4 and 12.5 g agar) (Leroux and Gredt, 1996) amended with 0, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35 and 0.5 mg/L of pyrimethanil. Conidial suspensions (approximately 105 conidia/mL) were made from 7- to 10-day-old PDA cultures grown at 20 ◦ C in the dark. The reverses of fungicide-amended PDA plates were marked with 4–5 parallel lines. A loop of the conidial suspension was streaked along each line. Cultures were incubated at 20 ◦ C for 20 and 36 h for fludioxonil- and pyrimethanil-amended treatments, respectively. The percentage of conidial germination (100–200 conidia from each treatment were examined) and the lengths of germ tubes (50–100 germinated conidia from each treatment were examined) were assessed under a microscope, using a micrometer. Only conidia with a germ tube at least twice its length was considered germinated (Baraldi et al., 2003). 2.5. Cross sensitivity to postharvest fungicides To determine cross sensitivity to the three postharvest fungicides available for pome fruits, the 20 P. expansum isolates that were used for testing sensitivity of conidial germination and germ-tube elongation to fludioxonil and pyrimethanil also were tested to establish EC50 of TBZ on mycelial growth. TBZ concentrations in amended PDA were 0, 0.2, 0.4, 0.6, 1, 2.5, 5 mg/L and 0, 100, 250, 500, 750, 1000 mg/L for TBZ-S and TBZ-R isolates, respectively. Technical-grade TBZ (99% a.i.; Sigma, St. Louis) was used to make TBZ-amended PDA for TBZ-S isolates. Because high concentrations of TBZ (>100 mg/L) were needed for estimating EC50 for TBZ-R isolates, a formulated TBZ (42.3% a.i.; Syngenta Crop Protection, Greensboro, NC) was used to make TBZ-amended PDA for TBZ-R isolates. Inoculum preparation and inoculation of fungicide-amended plates were the same as described above. Colony diameters were measured after 7 days at 20 ◦ C.

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2.6. Efficacy of fludioxonil and pyrimethanil for apple blue mold control Organic ‘Red Delicious’ apples were harvested from a commercial orchard and stored at 0 ◦ C for approximately 8 weeks before the experiment was initiated. Fruit were surfacedisinfested for 5 min in 0.6% sodium hypochlorite solution, rinsed three times with sterile water, and air-dried. Each apple fruit was wounded with a 4-mm-diameter finish-nail head to 3 mm in depth. Two P. expansum TBZ-R isolates representing different degrees of sensitivity to fludioxonil, including the isolate that was much less sensitive to fludioxonil than the remaining isolates in the population, and two other P. expansum TBZ-R isolates representing different degrees of sensitivity to pyrimethanil were included in the test. Conidial suspensions (approximately 1 × 104 conidia/mL) made from 7-day-old PDA cultures grown at 20 ◦ C were used as inoculum for fruit inoculation. Each apple was inoculated by delivering 20 ␮L of the conidial suspension with a pipette into the wound. At approximately 60 min after inoculation, apples were dipped for 1 min in either sterile water as controls or in one of the three following fungicide solutions: TBZ applied as Mertect 340F (42.3% a.i.; Syngenta Crop Protection, Greensboro, NC) at 1.25 mL/L, fludioxonil applied as Scholar (50% a.i.; Syngenta Crop Protection, Greensboro, NC) at 0.6 g/L (the lowest label rate) and 1.2 g/L (the highest label rate), and pyrimethanil applied as Penbotec 400SC (37.14% a.i.; Janssen Pharmaceutica, Belgium) at 1.25 mL/L (the lowest label rate) and 2.5 mL/L (the highest label rate). Fruit were air-dried for 15 min after dipping and then placed on fruit trays and stored in cardboard boxes at 0 ◦ C for 8 weeks. Each treatment had three replicate trays and each tray with 20 fruit. Fruit were considered decayed when a lesion developed on the fruit. Lesion diameters were measured and percent fruit with decay symptoms was calculated. The experiment was performed twice. 2.7. Statistical analysis For each P. expansum isolate, a linear regression of the percent inhibition of mycelial growth, conidial germination or germ-tube elongation relative to the control versus the logarithmic transformation for concentrations of fludioxonil and pyrimethanil were obtained using the procedure REG of SAS (Version 9.1, SAS institute, Cary, NC). The EC50 value (50% inhibition relative to the control) for each isolate was calculated based on the model. Frequencies of EC50 of fludioxonil and pyrimethanil were calculated. The average EC50 values from two experiments for each isolate were pooled for data analysis because the data of the two experiments did not differ statistically (P > 0.05). A t-test was performed to compare mean EC50 values between TBZ-R and TBZ-S isolates using SAS. The differences between EC50 of fludioxonil and pyrimethanil on mycelial growth, conidial germination and germ-tube elongation were examined by analysis of variance (ANOVA) and means were compared by the Fisher’s protected LSD at P ≤ 0.05. Using the procedure REG of SAS, simple linear correlation coefficients were calculated to evaluate potential correlation between

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sensitivity to TBZ and sensitivity to fludioxonil or pyrimethanil. For the decay-control experiment, the incidence of blue mold was arcsine-square-root transformed prior to analysis. The differences among treatments were examined by ANOVA and the means were separated by the Waller–Duncan K-ratio t-test. 3. Results 3.1. Sensitivity of Penicillium expansum population to fludioxonil Based on the 120 single-spore isolates of P. expansum collected from packinghouses and commercial orchards, EC50 values of fludioxonil on the fungal mycelial growth ranged from 0.011 to 0.068 mg/L, with a mean of 0.020 mg/L (Fig. 1A). Eighty percent of the isolates had EC50 values between 0.016 and 0.025 mg/L. Of the 120 isolates, one exhibited a reduced sensitivity to fludioxonil with an EC50 value of 0.068 mg/L that was significantly higher (P < 0.0001) than those of the remaining isolates, which ranged from 0.011 to 0.028 mg/L. This isolate also was highly resistant to TBZ, with EC50 > 200 mg/L. Fludioxonil at 0.5 mg/L completely inhibited mycelial growth of all isolates tested except the isolate with reduced sensitivity. There was no significant difference in sensitivity (mean EC50 value) to fludioxonil between the 82 TBZ-S and 38 TBZ-R isolates of P. expansum (P > 0.1494, data not shown). There was no significant difference (P > 0.05) in the mean EC50 values of fludioxonil between mycelial growth and germ-tube elongation for the 20 isolates of P. expansum (Table 1). The mean

EC50 of fludioxonil on conidial germination was significantly higher than that on mycelial growth and germ-tube elongation (P < 0.0001). Conidial germination and germ-tube elongation were completely inhibited by fludioxonil at 0.1 mg/L except the isolate with reduced sensitivity based on the mycelia growth assay. 3.2. Sensitivity of P. expansum population to pyrimethanil Among the 120 isolates of P. expansum tested, the mean EC50 values of pyrimethanil on mycelial growth ranged from 0.519 to 2.054 mg/L, with a mean of 1.340 mg/L (Fig. 1B). The mean EC50 value (1.384 mg/L) of the 82 TBZ-S isolates was higher (P > 0.0023) than the mean EC50 (1.247 mg/L) of the 38 TBZ-R isolates. The mean EC50 value of pyrimethanil on the mycelial growth of P. expansum was significantly higher than that on germtube elongation and conidial germination (P < 0.0001) (Table 1). There was no significant difference in EC50 values (P > 0.05) between conidial germination and germ-tube elongation. Conidial germination and germ-tube elongation of P. expansum isolates were completely inhibited by pyrimethanil at 0.5 mg/L. 3.3. Cross-sensitivity analysis There was no evidence of cross-sensitivity correlation either between fludioxonil and TBZ (R2 = 0.0084, P = 0.7003, Fig. 2A) or between pyrimethanil and TBZ (R2 = 0.0016, P = 0.8680, Fig. 2B) based on the P. expansum isolates tested. No cross-sensitivity correlation was detected between fludioxonil and pyrimethanil based on the EC50 values on conidial germination (R2 = 0.0118, P = 0.6480, Fig. 3A) and germ-tube elongation (R2 = 0.0399, P = 0.3982, Fig. 3B) of the 20 isolates tested. However, based on the mycelial growth assay, there was a weak correlation between fludioxonil and pyrimethanil (R2 = 0.2176, P = 0.0381, Fig. 3C). 3.4. Efficacy of fludioxonil and pyrimethanil for apple blue mold control Over 99% of P. expansum-inoculated fruit (four TBZ-R isolates) either treated with TBZ or in the controls developed blue mold symptoms, whereas no decay developed on the fruit treated with either fludioxonil or pyrimethanil at both the lowest and highest label rates (data not shown) under the test conditions. 4. Discussion

Fig. 1. (A and B) Distribution of EC50 of fludioxonil and pyrimethanil based on mycelial growth assay for 120 isolates of Penicillium expansum from apple.

In this study, we observed that fludioxonil significantly inhibited germination of conidia, germ-tube elongation and mycelial growth of both TBZ-R and TBZ-S isolates of P. expansum from apples. The range of EC50 of fludioxonil against mycelial growth was similar to those previously reported in P. expansum (Errampalli, 2004; Olaya et al., 2005) and in P. digitatum (Kanetis et al., 2004). In our study, the pathogen population exhibited a great range of sensitivity to fludioxonil, with an up to 6-fold difference in EC50 between the least and the most sen-

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Table 1 Mean and range of EC50 (mg/L) of fludioxonil and pyrimethanil against Penicillium expansum with three different assays Assay

Fludioxonil Mean

Mycelial growth Germ-tube elongation Conidial germination

ba

0.022 0.021 b 0.034 a

Pyrimethanil Range

Mean

Range

0.013–0.068 0.018–0.024 0.026–0.057

1.227 a 0.158 b 0.188 b

0.519–1.669 0.128–0.217 0.161–0.231

a Values represent means from two separate runs of the experiment. Values followed by the same letter in the column are not significantly different (P > 0.05) according to the Fisher’s protected LSD.

sitive isolates. One isolate derived from a decayed apple fruit sampled from a packinghouse was much less sensitive to fludioxonil compared with remaining isolates. The existence of a great range of sensitivity reflects that at the time of sampling, the P. expansum population from apple consisted of a mixture of phenotypes varying in sensitivity to fludioxonil. Reliable and suitable methods for testing the sensitivity of B. cinerea to anilinopyrimidines have been previously reported (Birchmore and Forster, 1996; Hiber and Sch¨uepp, 1996; Takagaki et al., 2004). Recommendations to improve reliability of in vitro tests for sensitivity to this class of fungicides include standardizing incubation time, using the optimum temperature for growth of the pathogen, and using synthetic media without free amino acids. In testing sensitivity of B. cinerea to anilinopyrimidines, Birchmore and Forster (1996) reported

Fig. 2. Cross-sensitivity analysis between thiabendazole and fludioxonil or pyrimethanil in Penicillium expansum isolates from apples. (A) Fludioxonil vs. thiabendazole and (B) pyrimethanil vs. thiabendazole.

that only conidia or freshly germinated conidia as inoculum should be used in in vitro sensitivity tests and aged mycelia should be excluded because they are less sensitive and could give rise to false positive results. In the present study, we used 24-h-old germinated spores as inoculum for testing sensitivity of mycelial growth to pyrimethanil on ASP-agar (Hiber and Sch¨uepp, 1996), a synthetic medium. This method avoided using a mixture of conidia and mycelia as inoculum for sensitivity tests and provided consistent results. Sholberg et al. (2005a) used agar plugs containing conidia and mycelia from the margin of 10- to 14-day-old PDA cultures as inoculum to test sensitivity of 17 P. expansum isolates to pyrimethanil. The EC50 on mycelial growth ranged from 0.054 to 0.566 mg/L, with a mean EC50 value of 0.314 mg/L, which was lower than that we reported in the present study. The discrepancy in EC50 may result from different media and inoculum used in these two studies. Studies to examine the effects of anilinopyrimidines on B. cinerea revealed a weak effect on conidial germination in vitro, but a strong inhibition of germ-tube elongation and initial mycelial growth (Neumann et al., 1992; Rosslenbroich and Stuebler, 2000; Moyano et al., 2004). However, in our study pyrimethanil effectively impaired conidial germination and germ-tube elongation of P. expansum, which were completely inhibited at 0.5 mg/L pyrimethanil. Pyrimethanil inhibited conidial germination of P. expansum in vitro at 0.13–0.22 mg/L, which was similar to that of P. digitatum with 0.2–0.4 mg/L (Smilanick et al., 2006). These results suggest that conidial germination of P. expansum may respond to pyrimethanil differently than that of B. cinerea. In our study, mycelia of P. expansum were not completely inhibited on ASP-agar medium with 10 mg/L of pyrimethanil, and even had very limited growth at 100 mg/L of pyrimethanil after incubation for 7 days (data not shown). This may, at least partially, result from aged mycelium on the agar plugs that was less sensitive to the fungicide than younger mycelium as observed on B. cinerea (Birchmore and Forster, 1996). Discriminatory concentration is often used for determining whether or not isolates are resistant to a fungicide (Chapeland et al., 1999; Baroffio et al., 2003; Moyano et al., 2004; Russell, 2004). Our results suggest that the mycelial growth assay may not be suitable for phenotyping isolates for resistance to pyrimethanil because there was no definitive minimal inhibitory concentration associated with this assay. However, the mycelial growth assay is relatively easy to perform and less time consuming than conidial germination and germ-tube elongation assays,

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this concentration, except for the isolate with reduced sensitivity to fludioxonil. No significant cross-sensitivity correlation was observed between TBZ and either fludioxonil or pyrimethanil. This also is consistent with reports in B. cinerea (Forster and Staub, 1996; Petsikos-Panayotarou et al., 2003; Kanetis et al., 2004) and supports observations by Errampalli and Crnko (2004) and Sholberg et al. (2005a) that fludioxonil and pyrimethanil are effective in controlling blue mold caused by TBZ-resistant isolates of P. expansum. Although one isolate was much less sensitive to fludioxonil, fludioxonil applied as Scholar at the label rate completely controlled blue mold on apple fruit inoculated with this isolate. Similarly, two isolates representing different degrees of sensitivity to pyrimethanil were completely controlled on apple fruit with pyrimethanil applied as Penbotec. All these isolates also were resistant to TBZ. Thus, our results indicate that the current population of P. expansum from apple in Washington can be effectively controlled by the fungicides and that these two fungicides can serve as alternatives to TBZ for controlling TBZ-resistant P. expansum. In conclusion, the baseline sensitivities of P. expansum to fludioxonil and pyrimethanil were established based on 120 wild-type isolates collected from apple packinghouses and orchards without a history of exposure to these two new fungicides. The discriminatory concentrations, based on the mycelial growth and germ-tube elongation of P. expansum for fludioxonil and pyrimethanil, respectively, were established for screening or phenotyping isolates for resistance to the two fungicides. The established baseline sensitivities and discriminatory concentrations of P. expansum to fludioxonil and pyrimethanil are essential for monitoring future shifts in the sensitivity or development of resistance to phenylpyrroles and anilinopyrimidines in P. expansum. Acknowledgements Plant Pathology New Series 0458, Department of Plant Pathology, College of Agricultural, Human, and Natural Resource Sciences Agricultural Research Center, Project No. WNP0367, Washington State University, Pullman, WA 991646430, USA. We thank R.J. Boal and Y.K. Kim for assistance with collecting decayed fruit samples from orchards and fruit packinghouses. This research was supported in part by the Washington Tree Fruit Research Commission. Fig. 3. Cross-sensitivity analysis between fludioxonil and pyrimethanil in Penicillium expansum isolates from apples based on (A) conidial germination assay, (B) germ-tube elongation assay, and (C) mycelial growth assay.

and it is suitable for estimating the distribution of EC50 in the baseline population as we demonstrated in this study. Based on the results in this study, we recommended using the germ-tube elongation assay with 0.5 mg/L of pyrimethanil as a discriminatory concentration for phenotyping isolates of P. expansum for resistance to pyrimethanil. For phenotyping isolates for resistance to fludioxonil, a mycelial growth assay with 0.5 mg/L fludioxonil is recommended since we demonstrated in this study that mycelial growth of P. expansum was completely inhibited at

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