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© 2010 Plant Management Network.
Accepted for publication 27 April 2010. Published 14 June 2010.


Resistance to Dodine in Populations of Venturia inaequalis in Quebec, Canada


Odile Carisse and Tristan Jobin, Agriculture and Agri-Food Canada, 430 Gouin Boulevard, St-Jean-sur-Richelieu, Quebec, J3B 3E6, Canada


Corresponding author: Odile Carisse. carisseo@agr.gc.ca


Carisse, O., and Jobin, T. 2010. Resistance to dodine in populations of Venturia inaequalis in Quebec, Canada. Online. Plant Health Progress doi:10.1094/PHP-2010-0614-01-RS.


Abstract

Dodine was introduced in the USA and Canada in the early 1960s for the control of apple scab. Following control failure, growers stopped using dodine in the mid-1970s. Despite the curtailment of dodine use more than 30 years ago, persistent resistance to the fungicide was suspected in V. inaequalis populations. The dodine sensitivity was determined for two populations that were not exposed to dodine for at least 30 years − a wild type population (25 monoconidial isolates) and a population constructed with isolates collected in orchards managed for apple scab (156 isolates). The sensitivity to dodine was determined by monitoring growth of these isolates on agar Petri dishes amended with 0, 0.01, 0.1, 1.0, or 10.0 µg/ml of dodine. Sensitivity to fungicide was evaluated based on ED50 values. Both populations showed a lognormal distribution of ED50 values. The ED50 means were 0.525 μg/ml and 1.735 μg/ml for the wild type and managed orchards populations, respectively. In managed orchard, 31.4% of the isolates were resistant to dodine (ED50 > 1.0 μg/ml). Cross-resistance with myclobutanil and with kresoxim-methyl was tested and found not to be significant. The results of this study suggest that resistance to dodine is still present in the populations of V. inaequalis from Quebec and that reintroduction of dodine should only be done along with an appropriate resistance management strategy.


Introduction

Dodine (n-dodecylguanidine acetate) was introduced in the USA and Canada in the 1960s as a protective and eradicant fungicide for the control of apple scab, caused by Venturia inaequalis (Cke) Wint. Dodine was intensively used and provided satisfactory control for about nine years. In 1968, reduced control of apple scab with dodine was reported, and failure in controlling apple scab was reported 1 year later (22,23). Initially, resistance to dodine was reported for the Lake Ontario fruit belt counties and in the Geneva area of New York State. Resistance to dodine was then reported for other apple production areas of the USA (4,7,11,25), Canada (15,17,19), New Zealand (1), and Poland (16).

In apple-growing areas of Eastern Canada, apple scab is the key disease for scheduling fungicide sprays. Most current strategies for managing apple scab are based on controlling primary infections caused by ascospores, in order to avoid epidemic build-up caused by secondary infections and, thus, reduce or eliminate the need for subsequent fungicide applications during the summer months. To achieve this goal, fungicide applications generally begin in the spring at the green tip phenological stage and continue throughout the spring to early summer, for a total of approximately 8 to 10 applications (13). The number of fungicide sprays made each year in individual orchards is related to the cultivar susceptibility and to the intended market (fresh or processed fruits). In some orchards, fungicides are applied routinely following predetermined fixed-interval; in other orchards, fungicides are applied based on risk estimated from various forecasting systems (14). In Quebec, sterol demethylation inhibitor fungicides (DMIs) and strobilurin-based fungicides (QoIs) are the two main classes of fungicides used in post infection management of apple scab (6). These fungicides are prone to selecting for resistance in V. inaequalis, and resistance to these compounds has been reported for most major apple production areas (2,3,5,6,8,12,18).

In Quebec, the current strategy for managing resistance to DMIs consists of applying DMIs in mixture with protective fungicide. This strategy allows for control of apple scab, but as the frequency of DMI-resistant isolates in target populations increases control increasingly relies on the protective fungicide (8,9,21). Therefore, programs initially designed to take advantage of the post infection properties of DMIs will need to be adjusted to provide fungicide timing that relies mostly on the pre-infection effect of the fungicide. In this context, dodine was evaluated as an alternative to conventional protective fungicides in a mixture with a DMI fungicide (9). As opposed to conventional protective fungicide, dodine provides some post infection activity which may allow for maintaining current scab management programs. However, the frequency and distribution of dodine resistance in populations of V. inaequalis is important for developing innovative and informed approaches to managing apple scab in a context of increasing fungicide resistance. The objective of this study was to evaluate the status of dodine resistance in two populations of V. inaequalis: a wild type population constructed from isolates collected in never-treated trees and a population from managed orchards that were not treated with dodine for at least 30 years.


Testing Dodine Sensitivity in V. inaequalis Populations

Commercial formulations of n-dodecylguanidine acetate (Equal 65WP, Norac Concepts Inc., Guelph, Ontario, Canada), myclobutanil (Nova 40W, Dow AgroSciences, Indianapolis, IN), and kresoxim-methyl (Sovran 50, BASF, Port Arthur, TX) were obtained from Centre Agricole Bienvenue Inc. (Rougemont, QC). Potato dextrose agar (PDA) was obtained from Difco Laboratories (Detroit, MI). Disposable petri dishes (150 mm × 15 mm) were obtained from Fisher Scientific (Whitby, ON). All other chemicals were obtained from Sigma Chemical Company (St. Louis, MO), unless otherwise stated.

Origin of V. inaequalis isolates. The populations described in this paper were collected from 51 orchards located in five different apple-producing regions of Quebec (Laurentides, Montérégie, Quebec City region, Missisquoi-Estrie and Southwest Quebec). Details on the distribution of the orchards and isolates are provided in Figure 1. Sampling was done in the summer of 2003, between 2 June and 1 August. The sampled orchards were selected to include various incidences of apple scab and diverse fungicide spray schedules. The isolates used to construct the wild type population were collected in 2003 from trees located in the Montérégie and Missisquoi-Estrie apple-producing regions. The isolates were collected from never-treated backyard or abandoned trees separated from any commercial orchards by at least 10 km.


 

Fig. 1. Geographical location of apple orchards surveyed in 2003 and in parenthesis the number of isolates tested for sensitivity of Venturia inaequalis to dodine.

 

Collection of apple scab lesions and culturing of V. inaequalis. Each isolate originated from a distinct leaf and from a distinct lesion. Methods previously described were used to obtain monoconidial cultures of V. inaequalis (6,10,20). In brief, leaves with distinct sporulating scab lesions were stored in brown paper bags in the freezer for a maximum of six months or individual lesions were cut out and placed in 2-ml Eppendorf tubes and kept at room temperature for a maximum of 3 mo. Individual lesions were immersed in 1 ml water, and the suspension was spread onto PDA medium. After 24 h, a single germinated conidium was transferred to PDA containing antibiotics (tetracycline, 15 mg/liter, and novobiocin, 100 mg/liter). After further growth, the isolate was ready for the subsequent tests.

Assays for fungicide resistance. Commercial formulations of dodine were dissolved in water and incorporated into the autoclaved medium, which had been cooled down to 55°C. Mycelial plugs measuring 5 mm in diameter were removed with a cork borer from the margin of an actively growing colony of V. inaequalis and placed on PDA medium amended with increasing doses of fungicides and on unamended PDA medium. Ten different isolates were placed per Petri dish (150 mm in diameter) (Fig. 2), and the procedure was repeated twice. The fungicide concentrations used were 0, 0.01, 0.1, 1.0, and 10 μg/ml active ingredient (ai). After 4 weeks of incubation at 18°C, radial growth was measured with a digital caliper (CE, model 111-411) as the average of two diameters perpendicular to each other, minus the initial plug diameter. Relative growth values were defined as: (mean mycelial expansion on amended medium) / (mean mycelial expansion on control) × 100.  ED50 values were defined as the fungicide concentration, in μg/ml ai, at which 50% of the radial growth was inhibited, as calculated by regressing mycelial expansion against the log10 of fungicide concentration. Cross-sensitivity was investigated by comparing the ED50 values for 156 isolates tested for dodine and myclobutanil and for dodine and kresoxim-methyl (6).


 

Fig. 2. Growth of Venturia inaequalis isolates on PDA medium amended with dodine.

 

Data analysis. The means of the ED50 values from the two populations were compared based on the Kolmogorov Smirnov test, and the frequency distribution of log-transformed ED50 values for each population was analyzed using univariate procedures. Classes of ED50 values were determined by measuring the range factor (highest ED50 / lowest ED50) from repeated in vitro tests with a subsample of 20 isolates (24). The correlations between the ED50 values for dodine and myclobutanil and between the ED50 values for dodine and kresoxim-methyl were analyzed using the Spearman correlation coefficient. Log-linear regression analysis was used to compare the wild type population and the population from managed orchards and populations from different geographical regions based on isolates classified as dodine-sensitive (ED50 < 1.0 μg/ml) and dodine-resistant (ED50 ≥ 1.0 μg/ml). This discriminatory concentration was selected based on previous reports in the scientific literature (4,11,16,17) and to facilitate comparison with the previous results. To compare dodine sensitivity of isolates collected in different apple-producing regions, the isolates classified as sensitive were further subdivided into sensitive (ED50 < 0.2 μg/ml) and moderately resistant (ED50 ≥ 0.2- < 1.0 μg/ml) isolates. All analyses were performed using the SYSTAT 11 software program (Systat Software, Richmond, CA).


Persistent Resistance to Dodine

The findings of this study demonstrate that isolates of V. inaequalis resistant to dodine are still present in the Quebec population. The wild type population (n = 25) constructed with isolates from never-treated backyard or abandoned trees yielded a mean and a median ED50 of 0.525 and 0.263 μg/ml, respectively, with values ranging from 0.057 to 3.424 μg/ml (Fig. 3). These represent a range factor (highest ED50/lowest ED50) of 60.07 and a ratio of highest ED50 / mean ED50 of 6.51. The frequency distribution of ED50 values was lognormal (P = 0.257). To construct a population representative of the whole apple-producing area, 51 managed orchards from five different regions in the province of Quebec (Canada) were sampled for scab lesions. This population (n = 156) yielded a mean and median ED50 of 1.735 and 0.419 μg/ml, respectively, and values ranging from 0.009 to 8.889 μg/ml (Fig. 3). These represent a range factor (highest ED50 / lowest ED50) of 987.67 and a ratio of highest ED50 / mean ED50 of 5.12. The frequency distribution of ED50 values was lognormal (P = 0.772). The mean ED50 values for the population of V. inaequalis from managed orchards (1.735 μg/ml) was significantly (P < 0.05) higher than the mean ED50 values of the wild-type population (0.525 μg/ml) according to the Kolmogorov-Smirnov test. The sensitivities to dodine observed in this study are comparable to some other reports. Gilpatrick and Blowers (4) reported ED50 values for isolates originating from orchard with good scab control using dodine ranging from 0.3 to 0.65, while in orchards with poor scab control, ED50 ranged from 0.75 to 1.25 μg/ml. Similarly, McKay and MacNeill (15) reported mean ED50 of 0.262, 0.293, and 1.198 μg/ml, for isolates collected in orchards never, occasionally, and frequently treated with dodine, respectively. On the other hand, ED50 values obtained in this study are higher than values reported by Koller et al. (11) who reported, for a baseline population, values ranging from 0.27 to 1.2 μg/ml (mean of 0.17 μg/ml). Nevertheless, these authors concluded that isolates with ED50 > 1.0 μg/ml should be considered resistant to dodine, which is consistent with our observations of mean ED50 values of 0.525 and 1.735 for wild type and managed orchards populations.


 

Fig. 3. Frequency distributions of Venturia inaequalis isolate sensitivities to dodine for (A) isolates from never treated trees sampled in Quebec during the summer of 2003; (B) isolates from commercial orchards sampled in Quebec during the summer of 2003. ED50 values (in μg/ml are based on mycelial radial growth on PDA amended with different doses of the fungicides for four weeks.

 

A total of 156 isolates were tested for their sensitivity to both dodine and myclobutanil and to dodine and kresoxim-methyl. The Pearson correlation coefficients were low with r = -0.10 and r = 0.06 with P = 0.214 and P = 0.445 for myclobutanil and kresoxim-methyl, respectively, indicating that there was no significant correlation between sensitivity to dodine and myclobutanil or between sensitivity to dodine and kresoxim-methyl.

Based on logistic linear regression analysis of the binary data (sensitive or resistant), there was no significant population effect (wild vs managed orchards populations) on the proportion of resistant isolates. Overall, the proportion of sensitive, moderately resistant, and resistant isolates was similar in the wild type population and in the population constructed from isolates originating from managed orchards with 25.6, 42.9, and 31.4% of the isolates classified as sensitive, moderately resistant, and resistant, respectively (Table 1). However, these proportions varied with the geographical origin of the isolates (Table 2). Missisquoi-Estrie was the region with the highest proportion of moderately resistant and resistant isolates, while Quebec City was the region with the highest proportion of sensitive isolates.


Table 1. Classification of fungicide resistance of 181 isolates of Venturia inaequalis based on ED50 values.

Classificationx Percentage of isolates in:
Managed orchardsy
(n = 156)
Wild type treesz
(n = 25)
Both populations
(n = 181)
Sensitive 25.6 32.0 26.5
Moderately resistant 43.0 56.0 44.8
Resistant 31.4 12.0 28.7

 x Isolates were classified as sensitive if the ED50 value was less than 0.2 μg/ml, moderately resistant if the ED50 value was more than 0.2 μg/ml and less than 1.0 μg/ml, and resistant if the ED50 value was more than 1.0 μg/ml.

 y Population constructed from 156 isolates collected in 51 managed orchards from five different regions in the province of Quebec (Canada).

 z The wild type population (n = 26) constructed with isolates from backyard apple trees and untreated abandoned trees.


Table 2. Classification of fungicide resistance of 187 isolates of Venturia inaequalis originating from different apple growing region of the province of Quebec.

Classificationx Percentage of isolates from the region of:
Laurentides Montérégie Missisquoi
-Estrie
Quebec
City region
Southwest
Quebec
Sensitive 30.5 21.9 15.6 53.8 42.8
Moderately resistant 38.9 49.4 40.6 38.5 42.9
Resistant 30.6 28.7 43.8 7.7 14.3

 x Isolates were classified as sensitive if the ED50 value was less than 0.2 μg/ml, moderately resistant if the ED50 value was more than 0.2 μg/ml and less than 1.0 μg/ml, and resistant if the ED50 value was more than 1.0 μg/ml.


In this study, detailed information on historical use of dodine was not available. However, it is known that dodine has not been used for at least 30 years, and that most orchards in the region surveyed were replaced in the early 1980s following winter injuries. Hence we assumed that most orchards with dodine resistance in the 1970s do not exist anymore or were replanted with new trees. Nevertheless, for both wild and managed orchard populations, the range of ED50 values observed in this study was similar to values reported in the literature (4,15) with the exception of Jones and Walker (7) and Yoder and Klos (25) who reported growth of V. inaequalis on paper disk dipped in solution containing 300 μg/ml of dodine. Despite the relatively high proportion of resistant isolates observed in this study, it is unlikely that data on laboratory sensitivity to dodine can be used to determine if dodine will fail to control V. inaequalis in the Quebec’s orchards. Resistance to dodine in wild type populations was reported in Eastern Canada (15) and Yoder and Klos (25) observed resistant isolates in orchard with good scab control with dodine. Furthermore, it is difficult to compare sensitivities to dodine reported in the literature mainly because of the large variation reported and of the overlap in the different ranges of ED50 values reported for non-exposed and exposed to dodine populations (1,4,11,16,17). Ross and Newbery (17), reported a lot of variation in dodine resistance and a poor correlation with usage of this fungicide. In this study, the relatively high level of resistance to dodine observed and the overlap in the range of ED50 values observed for the wild-type and managed V. inaequalis populations, could be explained at least in part by the absence of treatments with dodine for a period of at least 30 years. Hence, the comparison made in this study is different than in most studies where a never-exposed population is compared with an exposed population. There are only few studies on the persistence of fungicide resistance, McKay and MacNeill (15), reported a high level of resistance to dodine after nine year of not using it. This observation is supported by our observations of high level of resistance after 30 years without application of dodine. The relative high level of resistance observed in the population originating from never treated trees could be explained by a possible mixing of sensitive and resistant populations over the years.

Following the introduction of DMI fungicides in the 1980s, the management of apple scab moved from protective to post infection fungicide applications. Overall, this approach provided adequate control with, in some cases, reduced number of fungicide applications. The increasing frequency of DMI resistant isolates stimulated a more intensive use of protective fungicides generally less expensive and not or less prone to fungicide resistance. Increasing use of protective fungicides will impact fungicide timing hence it was proposed to use dodine as a partner fungicide for DMI fungicides (9). However, the results of this study suggest that the risk of dodine resistance development and of control failure is still present in the orchards of the province of Quebec. Field efficacy trials and resistance monitoring will be required before the reintroduction of dodine into apple scab management programs.


Acknowledgments

The authors are grateful to Julie Roy and Genevičve Legault for their technical assistance. This work was financially supported by a Matching Investment Initiative between Agriculture and Agri-Food Canada and the Fédération des producteurs de pommes du Quebec.


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