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Influence of fungicide spray schedules on the sensitivity of Cercospora beticola to the sterol demethylation-inhibiting fungicide flutriafol
Crop Protection 20 (2001) 941–947
Influence of fungicide spray schedules on the sensitivity of Cercospora beticola to the sterol demethylation-inhibiting fungicide flutriafol G.S. Karaoglanidisa,*, P.M. Ioannidisb, C.C. Thanassoulopoulosa a
Faculty of Agriculture, Plant Pathology Laboratory, Aristotelian University of Thessaloniki, POB 269, 54006 Thessaloniki, Greece b Plant Protection Department, Hellenic Sugar Industry S.A., Plati Imathias 59032, Greece Received 23 January 2001; received in revised form 5 March 2001; accepted 22 March 2001
Abstract A field experiment was conducted over a three-year period (1997–1999) to study the efficacy of several fungicide spray-schedules and their effects on the sensitivity of Cercospora beticola populations to the DMI fungicide flutriafol. Spray applications of flutriafol, either alone at the recommended dose, or at a reduced dose in mixtures with maneb, or in alternation with tank mixed fentin-acetate and maneb, were included in the spray programs. Applications of flutriafol at the recommended dose showed a significantly greater control efficacy in comparison with the other treatments, while applications of flutriafol alternated with tank mixed fentin-acetate and maneb showed lower efficacy in comparison with the remaining flutriafol treatments. Fungal populations from plots continuously treated with flutriafol, either alone at the full dose or at reduced dose with maneb, had lower sensitivity to flutriafol in comparison with populations from plots treated alternatively with flutriafol and tank mixed fentin-acetate and maneb. Repeated applications of flutriafol, at full or reduced doses, favoured the selection of highly resistant strains. Since applications of flutriafol in alternation with tank mixed fentin-acetate and maneb do not maintain a high level of disease control, the only available antiresistance strategy would be the restriction of the number of flutriafol treatments, by applying them only when environmental conditions are favourable for disease development and by using alternative fungicides during the rest of the growing season. r 2001 Elsevier Science Ltd. All rights reserved. Keywords: Fungicide resistance; Sterol demethylation inhibitors; Sugar beet leaf-spot
1. Introduction Sterol demethylation-inhibiting fungicides (DMIs) constitute the most important fungicide group used for the control of sugar beet leaf-spot caused by Cercospora beticola (Byford, 1996). In Greece they have been used since 1979, in mixtures with protective fungicides such as maneb or chlorothalonil (Ioannidis, 1994). During the last decade, the DMIs used have been almost exclusively the triazole fungicides flutriafol and difenoconazole (Ioannidis and Doulias, 1998). The use of DMIs at reduced dose in tank mixtures with protectant fungicides has been adopted in order to avoid or delay resistance development, since the control efficacy of these spray programs was similar to those obtained by applications of the DMIs alone (Ioannidis, 1994; Ioannidis and Doulias, 1998). However, the extensive use of DMIs has led to a reduction of fungal sensitivity in some areas of *Corresponding author. Tel.:+30-31-998831; fax: +30-31-998854. E-mail addresses: [email protected] (G.S. Karaoglanidis).
northern Greece (Karaoglanidis et al., 2000). Despite this sensitivity shift, fungicide field performance was not significantly affected until 1998. However, during 1999 the first signs of reduced disease control were detected in an area of northern Greece (Karaoglanidis, 2000). Suggesting appropriate anti-resistance strategies for DMI fungicides is a difficult task since their efficacy largely depends on several parameters such as the region, the infection pressure, the crop and the pathogen (Scheinpflug, 1988; Kuck, 1994). Data on the effects of various DMI anti-resistance strategies are largely restricted to cereal pathogens, apple scab and grape powdery mildew. However, some general recommendations were made by the FRAC-DMIs Working Group. According to Brent (1995) DMIs should not be used in repeated applications alone, or at reduced dose levels. They can be used in alternation or in mixtures with non cross-resistance fungicides but should be reserved for the critical part of the spraying period . Maintenance of the field performance of DMIs is important for sugar beet leaf-spot control since alter-
0261-2194/01/$ - see front matter r 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 2 6 1 - 2 1 9 4 ( 0 1 ) 0 0 0 4 9 - 7
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native fungicides are unsatisfactory, particularly under climatic conditions favourble for the development of the disease. Therefore a 3-year field experiment, in sugar beet fields of northern Greece, was conducted during 1997– 1999 to assess several fungicide spray schedules, based on flutriafol in controlling Cercospora leaf-spot. The sensitivity of C. beticola isolates was also determined.
2. Materials and methods 2.1. Experimental design Field trials were conducted in a different sugar beet field each year in the region of Imathia, northern Greece, during a three-year period from 1997 to 1999. In this area disease incidence is rather high and 6–8 fungicide applications per growing season are required for satisfactory disease control. The prolonged use of DMI fungicides in this area has led to a slight shift towards decreased sensitivity of the pathogen population (Karaoglanidis et al., 2000). The sugar beet cultivar used was ‘‘Rizor’’, which is sensitive to Cercospora leaf-spot disease. In order to minimize, the risk of interplot-interference, the plots were separated from each other by a 4 m buffer zone which remained free of sugar beet plants. Experimental field plots were of 6 12 m2 in size, arranged in a randomized block design. The experiment consisted of seven fungicide spray schedules with four replicate plots per treatment. The treatments used were: (I) flutriafol at the full recommended dose, (II) tank mixture of flutriafol at reduced dose and maneb, (III) tank mixture of flutriafol at reduced dose plus fenpropimorph and chlorothalonil, (IV) flutriafol at the full recommended dose in alternation with a tank mixture of fentin-acetate and maneb, (V) tank mixture of flutriafol at reduced dose and maneb in alternation with a tank mixture of fentin-acetate and maneb, (VI) tank mixture of fentin-acetate and maneb and (VII) control, untreated (Table 1). All the
‘‘companion’’ fungicides were protectants applied at the full dose, except fenpropimorph which is an ergosterol biosynthesis inhibitor (EBIs) belonging to the morpholine group. The triple mixture of flutriafol, fenpropimorph and chlorothalonil was included in the experimental design since it simultaneously controls Cercospora leaf-spot and powdery mildew. Fungicides were applied preventively. Spray applications were initiated before the appearance of any disease symptoms on the plants, just after the ‘‘closing’’ of the rows and repeated at intervals of 15–18 days. In total six spray applications were applied per plot. Fungicide solutions were applied through a D3 cone nozzle using an AZO precision sprayer, at a volume of 2.8 l per plot (400 l/ha) and pressure 4 bar. 2.2. Disease assessment Assessments of the disease were carried out at 15–20 day intervals after the appearance of the first symptoms in the untreated plots. Assessments were based on foliage damage, measured using the 12-degree disease index rating scale of Horsfall and Barrat (1945). 2.3. Pathogen isolations The first isolations were made just after the appearance of disease symptoms on sugar beet leaves in the control plots, in order to measure the initial sensitivity of C. beticola population within the experimental field. The second set of isolations was carried out about 20 days after the 6th spray application, when isolates were obtained from all the plots for all the treatments. Fifteen leaves with sporulating lesions were sampled from each experimental plot and in total 60 leaves per treatment were obtained. Only plants from the two central rows of each plot were selected, and only one leaf was cut off from each plant. The leaves were, transferred into the laboratory to isolate the pathogen. Isolations, were made from one lesion on each leaf, on 2% water agar (Oxoid, Unipath Ltd, Basingstoke,
Table 1 Fungicide spray application schedules and application doses Treatmenta
Application dose (kg or l ha@1)
I. Flutriafol II. Flutriafol–maneb III. Flutriafol-fenpropimorph-chlorothalonil IV. Flutriafol / fentin-acetate–manebb V. Flutriafol–maneb / fentin-acetate–manebb VI. Fentin-acetate–maneb VII. Control
0.125 0.062+1.92 0.025+0.375+0.75 [email protected] [email protected]+1.92 0.30+1.92 F
a Flutriafol: Impact, 12.5 SC, Zeneca Hellas S.A., maneb: Dithane 80WP, Rohm and Haas Hellas S.A., fenpropimorph: Corbel, 75EC, Novartis Hellas S.A., chlorothalonil: Daconil, 75 WP, Zeneca Hellas S.A., fentin-acetate: Brestan, 60 WP, Agrevo Hellas S.A. b Treatments in alternation schemes. Spray applications: 1st, 3rd, 5th fentin-acetate/maneb2nd, 4th, 6th flutriafol or flutriafol/maneb.
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England) by transferring conidia onto the medium with a needle. In total 50 isolates were obtained per treatment. Colonies were visible after two days and they were then transferred to new petri dishes containing aspergillus complete medium (ACM), composed of 20 g agar (Oxoid), 10 g dextrose (Merck, Darmstadt, Germany) and 1 g yeast extract (Oxoid) per litre of medium. The isolates were then incubated for 8–10 days at 251C, in the dark. 2.4. Sensitivity determination The fungicide used in this study was pure technical grade of flutriafol generously supplied by the manufacturer Zeneca Hellas S.A., Athens, Greece. Stock solutions were prepared by dissolving the pure technical grade of flutriafol in methanol. Autoclaved ACM was amended with 0.10, 0.25, 0.50, 1.00, 2.00, 4.00, 8.00 and 16.00 mg ml@1, by adding appropriate volumes of the fungicide solutions to the medium. Control medium was not amended with fungicide. In all cases, including the control dishes, the final methanol concentration was not greater than 0.1% (v/v). Tests for each isolate were replicated three times for each concentration of each fungicide. Sensitivity to flutriafol was determined in terms of EC50 values (Effective Concentration that causes 50% inhibition of mycelial growth). The sensitivity assay technique, described by Karaoglanidis et al. (2000), involved mycelial plugs cut with a 5-mm-diameter cork borer, from the colony margins, placed inverted on the fungicide-amended or unamended culture medium and incubated at 251C in the dark for seven days. The mean colony diameter was then measured and expressed as percentage of the mean diameter of the untreated control. This relative growth index was then used to calculate the EC50 value of each isolate by regressing it against the Log10 fungicide concentration. 2.5. Data analyses Disease incidence in the experimental plots was determined by the Horsfall and Barrat disease index scale. Mean values of disease incidence were subjected to an analysis of variance and compared by Duncan’s multiple range test. Since DMI EC50 values typically follow a log-normal distribution, the mean EC50 values of the pathogen populations were compared by a t-test carried out on log-transformed EC50 values of the individual isolates and then converted to original scale values. Additionally, fungal isolates were classified as either resistant or sensitive according to their relative growth at 0.5 flutriafol with resistant strains being considered to be those that had relative growth values >80% at that concentration of fungicide. This discriminatory concen-
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tration was selected as being equal to or slightly higher than the mean EC50 of the wild-type population, which in a previous study has been determined to have a value of 0.45 mg ml@1 (Karaoglanidis et al., 2000). Field experiments carried out during 1999 showed that such isolates cannot be efficiently controlled by the recommended dose of flutriafol (Karaoglanidis, 2000). Frequencies of resistant strains within treatments were compared by Chi-square analysis. All the statistical analyses were performed using the MStat-C statistical program (MStat-C, version 2.10, Michigan State University).
3. Results 3.1. Efficacy of fungicide spray schedules Disease incidence and control by the several fungicide spray schedules were not significantly different during the first stages of disease development in all the three years of the study. In 1997, no significant differences were detected even at the end of the spraying period, with the exception of the mixture fentin-acetate-maneb (Table 2). In that year the plots treated with flutriafol either alone, or in mixture with maneb, or in alternation with the mixture fentin-acetate-maneb, had relatively low disease incidence ranging from 2.0 to 3.0. Differences between treatments were not significant (P>0.05). Climatic conditions were favourable during 1998 for disease development, allowing better discrimination between the fungicide treatments. Foliage in control plots and in plots treated with the tank mix fentinacetate-maneb had been completely destroyed by the end of the spraying period. The lowest value of disease incidence was observed in the plots treated with six successive spray applications of flutriafol at the fullrecommended dose, as well as in the plots treated with the triple mixture of flutriafol, fenpropimorph and chlorothalonil (Table 2). Environmental conditions in 1999 were extremely favourable for the development of the disease. The lowest foliage damage was observed again in the plots treated with six successive spray applications of flutriafol at the full-recommended dose (Po0.05). In the plots treated with flutriafol, in an alternation scheme with the mixture of fentin-acetate and maneb, disease incidence was rather high with values of 6.7 and 6.2 , which were not significantly different (P>0.05) from the disease incidence value of 7.7 in plots treated only with the mixture of fentin-acetate and maneb. The disease incidence in plots treated successively with flutriafol in mixtures with maneb or with fenpropimorph and chlorothalonil, was significantly lower (Po0.05).
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Table 2 Effect of several fungicide spray application programs on incidence of sugar beet leaf-spot caused by Cercospora beticola, determined after the termination of fungicide sprays, in experimental sugar beet field plots during 1997–1999 Treatment
1997
1998
Disease incidencea I. Flutriafol II. Flutriafol–maneb III. Flutriafol–fenpropimorph-chlorothalonil IV. Flutriafol / fentin-acetate-manebd V. Flutriafol-maneb / fentin-acetate-manebd VI. Fentin-acetate-maneb VII. Control
2.0 2.5 3.1 3.0 3.0 6.0 11.0
ab (0.0)c a (0.5) a (0.3) a (0.5) a (0.0) b (0.7) c (0.0)
1999
Control (%)
Disease incidence
Control (%)
Disease incidence
Control (%)
95.3 93.6 89.8 80.6 80.6 37.5 0.0
4.5 5.2 4.5 4.8 5.0 11.0 11.0
63.0 58.0 63.0 66.7 62.5 0.0 0.0
2.7 5.0 4.9 6.7 6.2 7.7 11.0
92.1 62.5 64.6 23.0 33.3 12.4 0.0
a (0.2) b (0.6) a (0.7) b (0.5) b (0.8) c (0.0) c (0.0)
a (0.4) b (0.0) b (0.2) c (0.3) bc (0.5) c (0.8) d (0.0)
a
Foliage damage was scored according to the Horsfall and Barrat 12-degree (0–11) disease index scale. Mean values followed by different letter in the column are significantly different at P=0.05 according to Duncan’s Multiple Range Test. c Standard Deviation. d Spray applications in alternation schemes. b
Table 3 Sensitivity to flutriafol, in terms of EC50 values, of Cercospora beticola isolates collected from untreated plots and plots treated with several fungicide spray schedules during a three-year period (1997–1999), in experimental field trials conducted in northern Greece Treatment
Year 1997
Control–initial I. Flutriafol II. Flutriafol–maneb III. Flutriafol–fenpropimorph-chlorothalonil IV. Flutriafol / fentin-acetate-manebd V. Flutriafol–maneb / fentin-acetate-manebd VI. Fentin-acetate-maneb VII. Control
1998
Range
Mean EC50
0.07–1.05 0.12–4.86 0.05–2.61 0.10–2.68 0.12–1.20 0.09–1.02 0.10–1.41 0.10–1.33
0.51 0.76 0.74 0.63 0.58 0.55 0.56 0.56
a
b (0.19) ab (0.65)c a (0.56) ab (0.41) b (0.20) b (0.25) b (0.22) b (0.33)
1999
Range
Mean EC50
Range
Mean EC50
0.09–1.35 0.30–10.40 0.23–8.53 0.10–11.30 0.15–7.56 0.26–7.87 0.07–1.36 0.10–2.45
0.50 2.24 1.50 1.35 1.06 0.96 0.51 0.52
0.12-1.25 0.28-9.42 0.15-10.56 0.20-8.63 0.15-5.31 0.09-4.28 0.07-1.45 0.10-1.51
0.48 c (0.22) 1.87 a (2.54) 1.23 b (3.11) 1.17 b (2.31) 0.98 bc (1.20) 1.02 bc (1.25) 0.53 c (0.31) 0.52 c (0.26)
d (0.21) a (3.20) b (2.04) b (2.09) c (1.19) c (1.12) d (0.20) d (0.40)
Mean EC50 values of isolates in mg ml@1 flutriafol. Mean values followed by different letter in the column are significantly different according to t-test at P=0.05. c Standard deviation d Spray applications in alternation schemes. a
b
3.2. Fungicide resistance The sensitivity of the fungal population to flutriafol was determined twice each year (Table 3). During the three years of the study the initial mean sensitivity of the pathogen’s population ranged from 0.48 to 0.51 mg ml@1 flutriafol and in the untreated plots it had not changed significantly (P>0.05) at the end of the spraying period. Moreover, the mean sensitivity of isolates obtained from plots that had been treated with six successive applications of the tank mix fentin-acetate-maneb was not significantly different (P>0.05) from the mean sensitivity of the isolates obtained from the untreated plots. In all the three years of the study isolates obtained from plots treated continuously with flutriafol showed the lowest (Po0.05) sensitivity to flutriafol with mean EC50 values of 0.76, 2.24 and 1.87 mg ml@1 for 1997, 1998 and 1999, respectively. A significant sensitivity
shift, in comparison with the initial population sensitivity, was also observed in the isolates obtained from plots treated with the tank mixes of flutriafol with either maneb or with fenpropimorph and chlorothalonil. The lowest sensitivities were observed in isolates obtained from plots treated with flutriafol either alone or in tank mix with maneb at reduced dose in alternation with the mixture of fentin-acetate-maneb. In the last two treatments the mean population sensitivity was not significantly different (P>0.05) from the respective sensitivity of isolates obtained from the untreated plots or the plots treated with the mixture of fentin-acetate and maneb during the years 1997 and 1999, while a shift towards decreased sensitivity was observed during 1998. Population sensitivity shifts in the flutriafol-treated plots have resulted from the increased frequency of resistant strains (Fig. 1). Initial resistance frequency within the population was relatively low with only 2–4%
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Fig. 1. Frequency (%) of flutriafol-resistant Cercospora beticola isolates obtained from experimental sugar beet field plots treated with several fungicide spray application schedules in 1997 (solid bars), 1998 (open bars) and 1999 (hatched bars).
of isolates showing resistance. These frequencies of resistance remained stable in the populations from untreated plots or from plots treated with the mixture of fentin-acetate-maneb. In 1997 the proportions of resistant isolates in plots treated with repeated applications of flutriafol at full dose or in mixtures at reduced dose were similar (P>0.05), while no resistant strains were detected in plots treated with flutriafol in alternation schemes. During that year the resistance frequency did not exceed the value of 8% in any of the treatments. However, in 1998 and 1999 higher (Po0.05) resistance frequencies were observed in plots treated with repeated applications of flutriafol at the full dose, with the proportion of resistant isolates reaching values of 28% and 32% respectively. In plots treated with flutriafol at reduced dose plus maneb resistance frequencies were 16% in 1998 and 26% in 1999, while in plots treated with repeated applications of flutriafol-fenpropimorphchlorothalonil the respective resistance frequency values were 14% and 22%. The differences between the two treatments during both the years were not significant (P>0.05). The resistance frequencies in plots treated with flutriafol in alternation with the mixture of fentinacetate and maneb were significantly lower and did not exceed 8%.
4. Discussion The measurement of pathogen sensitivity to fungicides is, to some degree, limited by two important
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factors, the initial population sensitivity within the experimental field and the probability of inter-plot interference (Skylakakis, 1984). The initial resistance frequency is a determinative factor since high resistance tends to dilute the delaying effect of mixtures or alternations. The initial resistance frequency in the fields selected for the current research, as determined early in the season was relatively low and did not exceed a value of 2%–4% over the three-year period. The second limiting factor also dilutes the delaying effects of spray schedules applied in the field plots. In order to minimize the risk of inter-plot interference due to conidia movement between adjacent plots, a 4 m buffer zone free of sugar beet and weed plants was established between plots. Moreover, pathogen isolates were only taken from plants within the two central rows of each plot. Indirect evidence of limited inter-plot interference was the fact that the population sensitivity remained stable or only slightly decreased, compared to the initial population sensitivity, in all the three years of the study, in both untreated plots and plots treated with the tank mix of fentin-acetate and maneb. The fungicide sprays were applied preventively, starting early in the summer before the appearance of disease symptoms. In 1997 the weather conditions were not favourable for disease development and disease incidence was low. All the treatments in which flutriafol was used showed satisfactory disease control. In the following two years, disease incidence was significantly higher allowing a better discrimination among the different treatments. Applications of flutriafol at the normal dose showed the best field performance, followed by the treatments of flutriafol at reduced dose, in tank mixture with either maneb or fenpropimorph and chlorothalonil. Applications of flutriafol alternated with the tank mix fentin-acetate-maneb did not provide a satisfactory disease control. Flutriafol applied at the full dose in six successive treatments showed an excellent field performance. Conversely, when flutriafol was applied in tank mixtures at a reduced dose, disease control efficacy was lower perhaps because strains with mean sensitivity to flutriafol survived. Undoubtedly, the use of the protective fungicides maneb and chlorothalonil contributed to the elimination of the whole population, independent of its sensitivity to flutriafol. However, their control efficacy is restricted since some fractions of the population can escape their action. Reduced performance of EBI fungicides applied in reduced dose has also been observed in cereal powdery mildew (Hardwick et al., 1994; J rgensen and rgensen, 1994; Engels et al., 1996), apple scab (Ko. ller, 1996) and Septoria leaf-spot of wheat (Shaw and Pijls, 1994). However, in other reports no difference in performance was observed between full and reduced doses of EBIs (Schulz, 1994; Zziwa and Burnett, 1994), whereas according to another report,
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application of the triazole fungicide triadimenol at reduced dose in mixture with the protective fungicide dinocap showed better field performance in controlling grape powdery mildew, as compared to applications at the full dose (Steva, 1994). Applications of flutriafol in alternation schemes showed lower field performance compared to the continuous flutriafol treatments, due to the low efficacy of fentin-acetate and maneb, particularly under favourable environmental conditions such as those usually prevailing in Greece during August. Evidence for this is provided by the low disease control achieved in plots successively treated with six spray applications of the above-described mixture during 1998 and 1999. In all the three years of the study, the initial population sensitivity to flutriafol was similar and fluctuated around 0.5 mg ml@1. Results showed that the levels of sensitivity remained stable in the control treatments throughout the three years, as well as in plots treated with fentin-acetate and maneb. Conversely, in plots treated with flutriafol, either in successive applications or in alternation schemes, a 2–3-fold shift towards reduced sensitivity was observed. The sensitivity shift was more pronounced in 1998 and 1999. This is probably related to the high disease incidence during 1998 and 1999, since the selection of resistant strains is faster when disease incidence is high (Georgopoulos and Skylakakis, 1986). During the three years of the experiment the most significant shift toward reduced sensitivity was observed in plots treated with six spray applications of flutriafol at the full dose. Obviously, applications of flutriafol at high dose controlled efficiently the major part of the pathogen’s population while allowed the survival of only a small part of population, the most resistant, which was sampled at the end of the growing season. A significant shift towards higher EC50 values was also found in C. beticola isolates obtained from plots treated with six successive applications of reduced doses of flutriafol in mixtures with either maneb or fenpropimorph and chlorothalonil. The reduction of application dose favours polygenically controlled resistance by allowing the survival of both highly resistant strains and strains with moderate sensitivity (Georgopoulos, 1994; Brent, 1995). Similar patterns of sensitivity shift were observed in cereal powdery mildew (Hunter et al., 1984; Porras et al., 1990). Conversely, in other studies it was found that applications of the pyrimidine fungicide fenarimol at reduced dose resulted in faster selection of resistant isolates in Venturia inaequalis, in comparison to applications at the full-recommended dose (Ko. ller, 1996; Ko. ller and Wilcox, 1999). However, no differences were observed in sensitivity shifts when EBIs were applied at full or reduced dose to control pathogens such as Pseudocercosporella herpotrichoides (Hunter et al., 1993), Septoria tritici (Shaw and Pijls, 1994) or
Erysiphe graminis (Zziwa and Burnett, 1994; Hau and Pons, 1996). The shift towards higher EC50 values was less significant in isolates obtained from plots treated with flutriafol in alternation with tank mix of fentin-acetate and maneb. The use of this specific mixture reduced the flutriafol selection pressure on the population and delayed the evolution of resistance. Additionally, the interpolation of this mixture between flutriafol treatments favoured the selection of more sensitive strains when the use of flutriafol was discontinued, since sensitive strains are more fit than the resistant strains in the absence of DMIs (Ioannidis and Karaoglanidis, 2000). A similar delaying effect of alternations on the evolution of resistance to the triazole fungicide triadimenol has been previously reported in Uncinula necator (Steva, 1994). A successful anti-resistance strategy should fit two requirements simultaneously. Firstly, it should contribute to the overall reduction of the selection pressure exercised on the pathogen’s population by the ‘‘at risk’’ fungicide. Secondly, it should provide satisfactory disease control. However, sometimes these two requirements do not result from a single strategy, as has been shown by the results of the current study. Although, applications of flutriafol in alternation with tank mix fentin-acetate and maneb could be suggested to provide such a strategy, this spray application program did not provide satisfactory disease control, particularly under favourable environmental conditions for disease development. The alternation strategy could be of high value if alternative fungicides existed. Fungicides of the strobilurin group may play such a role in the future, since they have shown excellent field performance in experimental field trials (Ioannidis and Doulias, 1998). On the other hand, neither successive spray applications of flutriafol at full dose, nor successive applications in reduced dose, could be suggested as efficient antiresistance strategies, despite their good disease control performance. Such strategies favoured the selection of highly resistant strains and their prolonged use over large areas will probably lead to an increase of resistance frequency, resulting in inefficient disease control in the field. It is concluded that the selection of highly resistant strains of C. beticola is dependent firstly on the number of DMIs treatments, and secondly, on the dose applied. Consequently, the reduction in the number of treatments is a prerequisite for successful management of DMIs resistance in C. beticola. The restriction of the number of DMIs treatments cannot be achieved by using them in alternation schemes with tank mix fentinacetate and maneb, due to its low field performance under favourable environmental conditions for disease development. Consequently, the use of DMIs is recommended when it is strictly necessary, whereas
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alternative fungicides can be used during the rest of the spraying period. The development of models predicting the disease development and determining the critical period for the initiation of the epidemic (Skarakis et al., 1996) could contribute to the determination of the critical time for DMIs applications.
Acknowledgements The authors acknowledge Zeneca Hellas S.A. for supplying the technical material of flutriafol used in sensitivity determinations. We also thank S. Daucopoulos and G. Akrivos for maintaining field experiments. Miss M. Thanassoulopoulos, teacher of English language is acknowledged for language corrections. The first of the authors was financially supported by the Greek Scholarship Foundation.
References Brent, J.K., 1995. Fungicide resistance in crop pathogens: how it can be managed? FRAC Monograph No. 1, GIFAP, Brussels. Byford, W.J., 1996. A survey of foliar diseases of sugar beet and their control in Europe. Proceedings of the 59th IIRB Congress, February 1996, pp. 1–10. Engels, A.J.G., Mantell, B.C., de Waard, M.A., 1996. Effect of split applications of fenpropimorph-containining fungicides on sensitivity of Erysiphe gramminis f. sp. tritici. Plant Pathology. 45, 636–643. Georgopoulos, S.G., 1994. Early evaluation of fungicide resistance risk. In: Heaney, S., Slawson, D., Hollomon, D.W., Smith, M., Russell, P.E., Parry, D.W. (Eds.), Fungicide Resistance, BCPC Monograph No. 60. Farnham, UK, pp. 389–396. Georgopoulos, S.G., Skylakakis, G., 1986. Genetic variability in the fungi and the problem of fungicide resistance. Crop Protection 5, 299–305. Hardwick, N.V., Jenkins, J.E.E., Collins, B., Groves, S.G., 1994. Powdery mildew (Erysiphe gramminis) on winter wheat: control with fungicides and the effects on yield. Crop Protection 13, 93–98. Hau, B., Pons, J., 1996. Selection of populations of barley powdery mildew influenced by fungicide strategies. In: Lyr, H., Russell, P.E., Sisler, H.D. (Eds.), Modern Fungicides and Antifungal Compounds. Intercept Ltd., Andover, UK, pp. 357–364. Horsfall, J.G., Barrat, R.W., 1945. An improved grating system for measuring plant diseases. Phytopathology 35, 655. Hunter, T., Brent, K.J., Carter, G.A., 1984. Effects of fungicide spray regimes on sensitivity and control of barley powdery mildew. Proceedings of the British Crop Protection ConferenceFPests and Diseases, pp. 471–476. Hunter, T., Arnold, G., Jordan, V.W.L., Brent, K.J., 1993. Effects of different fungicide programs on infection of wheat crops by Pseudocercosporella herpotrichoides and on build-up of fungicide resistance. In: Lyr, H., Polter, C. (Eds.), Proceedings of 10th International Symposium on Systemic Fungicides and Antifungal Compounds, pp. 165–174. Ioannidis, P.M., 1994. Fungicide chemicals and techniques in controlling Cercospora beticola Sacc. in Greece. Proceedings of
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IIRBMediterranean Committee Meeting, Thessaloniki, Greece, pp. 139–151. Ioannidis, P.M., Doulias, K., 1998. Cercospora beticola and powdery mildew control under Greek conditions. Proceedings of IIRB. Mediterranean Committee Meeting, Brussels, pp. 89–99. Ioannidis, P.M., Karaoglanidis, G.S., 2000. Competition between DMIs-sensitive and -resistant strains of Cercospora beticola on untreated sugar beet crop. Proceedings of the 63rd IIRB Congress, Interlaken, Switzerland, pp. 489–496. J rgensen, L.N., 1994. Duration of effect of EBI fungicides when using reduced rates in cereals. Proceedings of the British Crop Protection Conference-Pests and Diseases, Vol. 2, pp. 702–710. Karaoglanidis, G.S., 2000. Resistance of Cercospora beticola to triazole fungicides. Ph.D Thesis, Aristotelian University, Thessaloniki, Greece (in Greek with an English abstract). Karaoglanidis, G.S., Ioannidis, P.M., Thanassoulopoulos, C.C., 2000. Reduced sensitivity of Cercospora beticola isolates to sterol demethylation inhibiting fungicides. Plant Pathology. 49, 567–572. Ko. ller, W., 1996. Recent developments in DMI resistance. In: Lyr, H., Russell, P.E., Sisler, H.D. (Eds.), Modern Fungicides and Antifungal Compounds. Intercept Ltd., Andover, UK, pp. 301–311. Ko. ller, W., Wilcox, W.F., 1999. Evaluation of tactics for managing resistance of Venturia inaequalis to sterol demethylation inhibitors. Plant Diseases. 83, 857–863. Kuck, K.H., 1994. Evaluation of antiresistance strategies. In: Heaney, S., Slawson, D., Hollomon, D.W., Smith, M., Russell, P.E., Parry, D.W. (Eds.), Fungicide Resistance, BCPC Monograph No. 60. British Crop Protection Council, Farnham, UK, pp. 43–46. Porras, L., Gisi, U., St.ahle-Csech, U., 1990. Selection dynamics in triazole treated populations of Erysiphe gramminis on barley. Proceedings of the British Crop Protection ConferenceFPests and Diseases, Vol. 3, pp. 1163–1168. Scheinpflug, H., 1988. Resistance management strategies for using DMI fungicides. In: Delp, C.J. (Ed.), Fungicide Resistance in North America. APS Press, Minnesota, pp. 93–94. Schulz, U., 1994. Evaluating antiresistance strategies for control of Erysiphe gramminis. In: Heaney, S., Slawson, D., Hollomon, D.W., Smith, M., Russell, P.E., Parry, D.W. (Eds.), Fungicide Resistance. BCPC Monograph No 60, Farnham, UK, pp. 55–58. Shaw, M.W., Pijls, C.F.N., 1994. The effect of reduced dose on the evolution of fungicide resistance in Septoria tritici. In: Heaney, S., Slawson, D., Hollomon, D.W., Smith, M., Russell, P.E., Parry, D.W. (Eds.), Fungicide Resistance, BCPC Monograph No. 60. Farnham, UK, pp. 47–54. Skarakis, G.N., Ioannidis, P.M., Ioannidis, P.I., 1996. Integrated management systems against sugar beet leaf-spot disease. Proceedings of the 59th IIRB Congress, pp. 45–53. Skylakakis, G., 1984. Quantitative evaluation of strategies to delay fungicide resistance. Proceedings of the British Crop Protection ConferenceFPests and Diseases, Vol. 2, pp. 565–572. Steva, H., 1994. Evaluating anti-resistance strategies for control of Uncinula necator. In: Heaney, S., Slawson, D., Hollomon, D.W., Smith, M., Russell, P.E., Parry, D.W. (Eds.), Fungicide Resistance, BCPC Monograph No. 60. British Crop Protection Council, Farnham, UK, pp. 59–66. Zziwa, M.C.N., Burnett, F.G., 1994. The effect of reduced doses on the sensitivity of powdery mildew to fenpropimorph in barley field trials. In: Heaney, S., Slawson, D., Hollomon, D.W., Smith, M., Russell, P.E., Parry, D.W. (Eds.), Fungicide Resistance, BCPC Monograph No. 60. British Crop Protection Council, Farnham, UK, pp. 303–308.
Influence of fungicide spray schedules on the sensitivity of Cercospora beticola to the sterol demethylation-inhibiting fungicide flutriafol G.S. Karaoglanidisa,*, P.M. Ioannidisb, C.C. Thanassoulopoulosa a
Faculty of Agriculture, Plant Pathology Laboratory, Aristotelian University of Thessaloniki, POB 269, 54006 Thessaloniki, Greece b Plant Protection Department, Hellenic Sugar Industry S.A., Plati Imathias 59032, Greece Received 23 January 2001; received in revised form 5 March 2001; accepted 22 March 2001
Abstract A field experiment was conducted over a three-year period (1997–1999) to study the efficacy of several fungicide spray-schedules and their effects on the sensitivity of Cercospora beticola populations to the DMI fungicide flutriafol. Spray applications of flutriafol, either alone at the recommended dose, or at a reduced dose in mixtures with maneb, or in alternation with tank mixed fentin-acetate and maneb, were included in the spray programs. Applications of flutriafol at the recommended dose showed a significantly greater control efficacy in comparison with the other treatments, while applications of flutriafol alternated with tank mixed fentin-acetate and maneb showed lower efficacy in comparison with the remaining flutriafol treatments. Fungal populations from plots continuously treated with flutriafol, either alone at the full dose or at reduced dose with maneb, had lower sensitivity to flutriafol in comparison with populations from plots treated alternatively with flutriafol and tank mixed fentin-acetate and maneb. Repeated applications of flutriafol, at full or reduced doses, favoured the selection of highly resistant strains. Since applications of flutriafol in alternation with tank mixed fentin-acetate and maneb do not maintain a high level of disease control, the only available antiresistance strategy would be the restriction of the number of flutriafol treatments, by applying them only when environmental conditions are favourable for disease development and by using alternative fungicides during the rest of the growing season. r 2001 Elsevier Science Ltd. All rights reserved. Keywords: Fungicide resistance; Sterol demethylation inhibitors; Sugar beet leaf-spot
1. Introduction Sterol demethylation-inhibiting fungicides (DMIs) constitute the most important fungicide group used for the control of sugar beet leaf-spot caused by Cercospora beticola (Byford, 1996). In Greece they have been used since 1979, in mixtures with protective fungicides such as maneb or chlorothalonil (Ioannidis, 1994). During the last decade, the DMIs used have been almost exclusively the triazole fungicides flutriafol and difenoconazole (Ioannidis and Doulias, 1998). The use of DMIs at reduced dose in tank mixtures with protectant fungicides has been adopted in order to avoid or delay resistance development, since the control efficacy of these spray programs was similar to those obtained by applications of the DMIs alone (Ioannidis, 1994; Ioannidis and Doulias, 1998). However, the extensive use of DMIs has led to a reduction of fungal sensitivity in some areas of *Corresponding author. Tel.:+30-31-998831; fax: +30-31-998854. E-mail addresses: [email protected] (G.S. Karaoglanidis).
northern Greece (Karaoglanidis et al., 2000). Despite this sensitivity shift, fungicide field performance was not significantly affected until 1998. However, during 1999 the first signs of reduced disease control were detected in an area of northern Greece (Karaoglanidis, 2000). Suggesting appropriate anti-resistance strategies for DMI fungicides is a difficult task since their efficacy largely depends on several parameters such as the region, the infection pressure, the crop and the pathogen (Scheinpflug, 1988; Kuck, 1994). Data on the effects of various DMI anti-resistance strategies are largely restricted to cereal pathogens, apple scab and grape powdery mildew. However, some general recommendations were made by the FRAC-DMIs Working Group. According to Brent (1995) DMIs should not be used in repeated applications alone, or at reduced dose levels. They can be used in alternation or in mixtures with non cross-resistance fungicides but should be reserved for the critical part of the spraying period . Maintenance of the field performance of DMIs is important for sugar beet leaf-spot control since alter-
0261-2194/01/$ - see front matter r 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 2 6 1 - 2 1 9 4 ( 0 1 ) 0 0 0 4 9 - 7
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native fungicides are unsatisfactory, particularly under climatic conditions favourble for the development of the disease. Therefore a 3-year field experiment, in sugar beet fields of northern Greece, was conducted during 1997– 1999 to assess several fungicide spray schedules, based on flutriafol in controlling Cercospora leaf-spot. The sensitivity of C. beticola isolates was also determined.
2. Materials and methods 2.1. Experimental design Field trials were conducted in a different sugar beet field each year in the region of Imathia, northern Greece, during a three-year period from 1997 to 1999. In this area disease incidence is rather high and 6–8 fungicide applications per growing season are required for satisfactory disease control. The prolonged use of DMI fungicides in this area has led to a slight shift towards decreased sensitivity of the pathogen population (Karaoglanidis et al., 2000). The sugar beet cultivar used was ‘‘Rizor’’, which is sensitive to Cercospora leaf-spot disease. In order to minimize, the risk of interplot-interference, the plots were separated from each other by a 4 m buffer zone which remained free of sugar beet plants. Experimental field plots were of 6 12 m2 in size, arranged in a randomized block design. The experiment consisted of seven fungicide spray schedules with four replicate plots per treatment. The treatments used were: (I) flutriafol at the full recommended dose, (II) tank mixture of flutriafol at reduced dose and maneb, (III) tank mixture of flutriafol at reduced dose plus fenpropimorph and chlorothalonil, (IV) flutriafol at the full recommended dose in alternation with a tank mixture of fentin-acetate and maneb, (V) tank mixture of flutriafol at reduced dose and maneb in alternation with a tank mixture of fentin-acetate and maneb, (VI) tank mixture of fentin-acetate and maneb and (VII) control, untreated (Table 1). All the
‘‘companion’’ fungicides were protectants applied at the full dose, except fenpropimorph which is an ergosterol biosynthesis inhibitor (EBIs) belonging to the morpholine group. The triple mixture of flutriafol, fenpropimorph and chlorothalonil was included in the experimental design since it simultaneously controls Cercospora leaf-spot and powdery mildew. Fungicides were applied preventively. Spray applications were initiated before the appearance of any disease symptoms on the plants, just after the ‘‘closing’’ of the rows and repeated at intervals of 15–18 days. In total six spray applications were applied per plot. Fungicide solutions were applied through a D3 cone nozzle using an AZO precision sprayer, at a volume of 2.8 l per plot (400 l/ha) and pressure 4 bar. 2.2. Disease assessment Assessments of the disease were carried out at 15–20 day intervals after the appearance of the first symptoms in the untreated plots. Assessments were based on foliage damage, measured using the 12-degree disease index rating scale of Horsfall and Barrat (1945). 2.3. Pathogen isolations The first isolations were made just after the appearance of disease symptoms on sugar beet leaves in the control plots, in order to measure the initial sensitivity of C. beticola population within the experimental field. The second set of isolations was carried out about 20 days after the 6th spray application, when isolates were obtained from all the plots for all the treatments. Fifteen leaves with sporulating lesions were sampled from each experimental plot and in total 60 leaves per treatment were obtained. Only plants from the two central rows of each plot were selected, and only one leaf was cut off from each plant. The leaves were, transferred into the laboratory to isolate the pathogen. Isolations, were made from one lesion on each leaf, on 2% water agar (Oxoid, Unipath Ltd, Basingstoke,
Table 1 Fungicide spray application schedules and application doses Treatmenta
Application dose (kg or l ha@1)
I. Flutriafol II. Flutriafol–maneb III. Flutriafol-fenpropimorph-chlorothalonil IV. Flutriafol / fentin-acetate–manebb V. Flutriafol–maneb / fentin-acetate–manebb VI. Fentin-acetate–maneb VII. Control
0.125 0.062+1.92 0.025+0.375+0.75 [email protected] [email protected]+1.92 0.30+1.92 F
a Flutriafol: Impact, 12.5 SC, Zeneca Hellas S.A., maneb: Dithane 80WP, Rohm and Haas Hellas S.A., fenpropimorph: Corbel, 75EC, Novartis Hellas S.A., chlorothalonil: Daconil, 75 WP, Zeneca Hellas S.A., fentin-acetate: Brestan, 60 WP, Agrevo Hellas S.A. b Treatments in alternation schemes. Spray applications: 1st, 3rd, 5th fentin-acetate/maneb2nd, 4th, 6th flutriafol or flutriafol/maneb.
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England) by transferring conidia onto the medium with a needle. In total 50 isolates were obtained per treatment. Colonies were visible after two days and they were then transferred to new petri dishes containing aspergillus complete medium (ACM), composed of 20 g agar (Oxoid), 10 g dextrose (Merck, Darmstadt, Germany) and 1 g yeast extract (Oxoid) per litre of medium. The isolates were then incubated for 8–10 days at 251C, in the dark. 2.4. Sensitivity determination The fungicide used in this study was pure technical grade of flutriafol generously supplied by the manufacturer Zeneca Hellas S.A., Athens, Greece. Stock solutions were prepared by dissolving the pure technical grade of flutriafol in methanol. Autoclaved ACM was amended with 0.10, 0.25, 0.50, 1.00, 2.00, 4.00, 8.00 and 16.00 mg ml@1, by adding appropriate volumes of the fungicide solutions to the medium. Control medium was not amended with fungicide. In all cases, including the control dishes, the final methanol concentration was not greater than 0.1% (v/v). Tests for each isolate were replicated three times for each concentration of each fungicide. Sensitivity to flutriafol was determined in terms of EC50 values (Effective Concentration that causes 50% inhibition of mycelial growth). The sensitivity assay technique, described by Karaoglanidis et al. (2000), involved mycelial plugs cut with a 5-mm-diameter cork borer, from the colony margins, placed inverted on the fungicide-amended or unamended culture medium and incubated at 251C in the dark for seven days. The mean colony diameter was then measured and expressed as percentage of the mean diameter of the untreated control. This relative growth index was then used to calculate the EC50 value of each isolate by regressing it against the Log10 fungicide concentration. 2.5. Data analyses Disease incidence in the experimental plots was determined by the Horsfall and Barrat disease index scale. Mean values of disease incidence were subjected to an analysis of variance and compared by Duncan’s multiple range test. Since DMI EC50 values typically follow a log-normal distribution, the mean EC50 values of the pathogen populations were compared by a t-test carried out on log-transformed EC50 values of the individual isolates and then converted to original scale values. Additionally, fungal isolates were classified as either resistant or sensitive according to their relative growth at 0.5 flutriafol with resistant strains being considered to be those that had relative growth values >80% at that concentration of fungicide. This discriminatory concen-
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tration was selected as being equal to or slightly higher than the mean EC50 of the wild-type population, which in a previous study has been determined to have a value of 0.45 mg ml@1 (Karaoglanidis et al., 2000). Field experiments carried out during 1999 showed that such isolates cannot be efficiently controlled by the recommended dose of flutriafol (Karaoglanidis, 2000). Frequencies of resistant strains within treatments were compared by Chi-square analysis. All the statistical analyses were performed using the MStat-C statistical program (MStat-C, version 2.10, Michigan State University).
3. Results 3.1. Efficacy of fungicide spray schedules Disease incidence and control by the several fungicide spray schedules were not significantly different during the first stages of disease development in all the three years of the study. In 1997, no significant differences were detected even at the end of the spraying period, with the exception of the mixture fentin-acetate-maneb (Table 2). In that year the plots treated with flutriafol either alone, or in mixture with maneb, or in alternation with the mixture fentin-acetate-maneb, had relatively low disease incidence ranging from 2.0 to 3.0. Differences between treatments were not significant (P>0.05). Climatic conditions were favourable during 1998 for disease development, allowing better discrimination between the fungicide treatments. Foliage in control plots and in plots treated with the tank mix fentinacetate-maneb had been completely destroyed by the end of the spraying period. The lowest value of disease incidence was observed in the plots treated with six successive spray applications of flutriafol at the fullrecommended dose, as well as in the plots treated with the triple mixture of flutriafol, fenpropimorph and chlorothalonil (Table 2). Environmental conditions in 1999 were extremely favourable for the development of the disease. The lowest foliage damage was observed again in the plots treated with six successive spray applications of flutriafol at the full-recommended dose (Po0.05). In the plots treated with flutriafol, in an alternation scheme with the mixture of fentin-acetate and maneb, disease incidence was rather high with values of 6.7 and 6.2 , which were not significantly different (P>0.05) from the disease incidence value of 7.7 in plots treated only with the mixture of fentin-acetate and maneb. The disease incidence in plots treated successively with flutriafol in mixtures with maneb or with fenpropimorph and chlorothalonil, was significantly lower (Po0.05).
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Table 2 Effect of several fungicide spray application programs on incidence of sugar beet leaf-spot caused by Cercospora beticola, determined after the termination of fungicide sprays, in experimental sugar beet field plots during 1997–1999 Treatment
1997
1998
Disease incidencea I. Flutriafol II. Flutriafol–maneb III. Flutriafol–fenpropimorph-chlorothalonil IV. Flutriafol / fentin-acetate-manebd V. Flutriafol-maneb / fentin-acetate-manebd VI. Fentin-acetate-maneb VII. Control
2.0 2.5 3.1 3.0 3.0 6.0 11.0
ab (0.0)c a (0.5) a (0.3) a (0.5) a (0.0) b (0.7) c (0.0)
1999
Control (%)
Disease incidence
Control (%)
Disease incidence
Control (%)
95.3 93.6 89.8 80.6 80.6 37.5 0.0
4.5 5.2 4.5 4.8 5.0 11.0 11.0
63.0 58.0 63.0 66.7 62.5 0.0 0.0
2.7 5.0 4.9 6.7 6.2 7.7 11.0
92.1 62.5 64.6 23.0 33.3 12.4 0.0
a (0.2) b (0.6) a (0.7) b (0.5) b (0.8) c (0.0) c (0.0)
a (0.4) b (0.0) b (0.2) c (0.3) bc (0.5) c (0.8) d (0.0)
a
Foliage damage was scored according to the Horsfall and Barrat 12-degree (0–11) disease index scale. Mean values followed by different letter in the column are significantly different at P=0.05 according to Duncan’s Multiple Range Test. c Standard Deviation. d Spray applications in alternation schemes. b
Table 3 Sensitivity to flutriafol, in terms of EC50 values, of Cercospora beticola isolates collected from untreated plots and plots treated with several fungicide spray schedules during a three-year period (1997–1999), in experimental field trials conducted in northern Greece Treatment
Year 1997
Control–initial I. Flutriafol II. Flutriafol–maneb III. Flutriafol–fenpropimorph-chlorothalonil IV. Flutriafol / fentin-acetate-manebd V. Flutriafol–maneb / fentin-acetate-manebd VI. Fentin-acetate-maneb VII. Control
1998
Range
Mean EC50
0.07–1.05 0.12–4.86 0.05–2.61 0.10–2.68 0.12–1.20 0.09–1.02 0.10–1.41 0.10–1.33
0.51 0.76 0.74 0.63 0.58 0.55 0.56 0.56
a
b (0.19) ab (0.65)c a (0.56) ab (0.41) b (0.20) b (0.25) b (0.22) b (0.33)
1999
Range
Mean EC50
Range
Mean EC50
0.09–1.35 0.30–10.40 0.23–8.53 0.10–11.30 0.15–7.56 0.26–7.87 0.07–1.36 0.10–2.45
0.50 2.24 1.50 1.35 1.06 0.96 0.51 0.52
0.12-1.25 0.28-9.42 0.15-10.56 0.20-8.63 0.15-5.31 0.09-4.28 0.07-1.45 0.10-1.51
0.48 c (0.22) 1.87 a (2.54) 1.23 b (3.11) 1.17 b (2.31) 0.98 bc (1.20) 1.02 bc (1.25) 0.53 c (0.31) 0.52 c (0.26)
d (0.21) a (3.20) b (2.04) b (2.09) c (1.19) c (1.12) d (0.20) d (0.40)
Mean EC50 values of isolates in mg ml@1 flutriafol. Mean values followed by different letter in the column are significantly different according to t-test at P=0.05. c Standard deviation d Spray applications in alternation schemes. a
b
3.2. Fungicide resistance The sensitivity of the fungal population to flutriafol was determined twice each year (Table 3). During the three years of the study the initial mean sensitivity of the pathogen’s population ranged from 0.48 to 0.51 mg ml@1 flutriafol and in the untreated plots it had not changed significantly (P>0.05) at the end of the spraying period. Moreover, the mean sensitivity of isolates obtained from plots that had been treated with six successive applications of the tank mix fentin-acetate-maneb was not significantly different (P>0.05) from the mean sensitivity of the isolates obtained from the untreated plots. In all the three years of the study isolates obtained from plots treated continuously with flutriafol showed the lowest (Po0.05) sensitivity to flutriafol with mean EC50 values of 0.76, 2.24 and 1.87 mg ml@1 for 1997, 1998 and 1999, respectively. A significant sensitivity
shift, in comparison with the initial population sensitivity, was also observed in the isolates obtained from plots treated with the tank mixes of flutriafol with either maneb or with fenpropimorph and chlorothalonil. The lowest sensitivities were observed in isolates obtained from plots treated with flutriafol either alone or in tank mix with maneb at reduced dose in alternation with the mixture of fentin-acetate-maneb. In the last two treatments the mean population sensitivity was not significantly different (P>0.05) from the respective sensitivity of isolates obtained from the untreated plots or the plots treated with the mixture of fentin-acetate and maneb during the years 1997 and 1999, while a shift towards decreased sensitivity was observed during 1998. Population sensitivity shifts in the flutriafol-treated plots have resulted from the increased frequency of resistant strains (Fig. 1). Initial resistance frequency within the population was relatively low with only 2–4%
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Fig. 1. Frequency (%) of flutriafol-resistant Cercospora beticola isolates obtained from experimental sugar beet field plots treated with several fungicide spray application schedules in 1997 (solid bars), 1998 (open bars) and 1999 (hatched bars).
of isolates showing resistance. These frequencies of resistance remained stable in the populations from untreated plots or from plots treated with the mixture of fentin-acetate-maneb. In 1997 the proportions of resistant isolates in plots treated with repeated applications of flutriafol at full dose or in mixtures at reduced dose were similar (P>0.05), while no resistant strains were detected in plots treated with flutriafol in alternation schemes. During that year the resistance frequency did not exceed the value of 8% in any of the treatments. However, in 1998 and 1999 higher (Po0.05) resistance frequencies were observed in plots treated with repeated applications of flutriafol at the full dose, with the proportion of resistant isolates reaching values of 28% and 32% respectively. In plots treated with flutriafol at reduced dose plus maneb resistance frequencies were 16% in 1998 and 26% in 1999, while in plots treated with repeated applications of flutriafol-fenpropimorphchlorothalonil the respective resistance frequency values were 14% and 22%. The differences between the two treatments during both the years were not significant (P>0.05). The resistance frequencies in plots treated with flutriafol in alternation with the mixture of fentinacetate and maneb were significantly lower and did not exceed 8%.
4. Discussion The measurement of pathogen sensitivity to fungicides is, to some degree, limited by two important
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factors, the initial population sensitivity within the experimental field and the probability of inter-plot interference (Skylakakis, 1984). The initial resistance frequency is a determinative factor since high resistance tends to dilute the delaying effect of mixtures or alternations. The initial resistance frequency in the fields selected for the current research, as determined early in the season was relatively low and did not exceed a value of 2%–4% over the three-year period. The second limiting factor also dilutes the delaying effects of spray schedules applied in the field plots. In order to minimize the risk of inter-plot interference due to conidia movement between adjacent plots, a 4 m buffer zone free of sugar beet and weed plants was established between plots. Moreover, pathogen isolates were only taken from plants within the two central rows of each plot. Indirect evidence of limited inter-plot interference was the fact that the population sensitivity remained stable or only slightly decreased, compared to the initial population sensitivity, in all the three years of the study, in both untreated plots and plots treated with the tank mix of fentin-acetate and maneb. The fungicide sprays were applied preventively, starting early in the summer before the appearance of disease symptoms. In 1997 the weather conditions were not favourable for disease development and disease incidence was low. All the treatments in which flutriafol was used showed satisfactory disease control. In the following two years, disease incidence was significantly higher allowing a better discrimination among the different treatments. Applications of flutriafol at the normal dose showed the best field performance, followed by the treatments of flutriafol at reduced dose, in tank mixture with either maneb or fenpropimorph and chlorothalonil. Applications of flutriafol alternated with the tank mix fentin-acetate-maneb did not provide a satisfactory disease control. Flutriafol applied at the full dose in six successive treatments showed an excellent field performance. Conversely, when flutriafol was applied in tank mixtures at a reduced dose, disease control efficacy was lower perhaps because strains with mean sensitivity to flutriafol survived. Undoubtedly, the use of the protective fungicides maneb and chlorothalonil contributed to the elimination of the whole population, independent of its sensitivity to flutriafol. However, their control efficacy is restricted since some fractions of the population can escape their action. Reduced performance of EBI fungicides applied in reduced dose has also been observed in cereal powdery mildew (Hardwick et al., 1994; J rgensen and rgensen, 1994; Engels et al., 1996), apple scab (Ko. ller, 1996) and Septoria leaf-spot of wheat (Shaw and Pijls, 1994). However, in other reports no difference in performance was observed between full and reduced doses of EBIs (Schulz, 1994; Zziwa and Burnett, 1994), whereas according to another report,
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application of the triazole fungicide triadimenol at reduced dose in mixture with the protective fungicide dinocap showed better field performance in controlling grape powdery mildew, as compared to applications at the full dose (Steva, 1994). Applications of flutriafol in alternation schemes showed lower field performance compared to the continuous flutriafol treatments, due to the low efficacy of fentin-acetate and maneb, particularly under favourable environmental conditions such as those usually prevailing in Greece during August. Evidence for this is provided by the low disease control achieved in plots successively treated with six spray applications of the above-described mixture during 1998 and 1999. In all the three years of the study, the initial population sensitivity to flutriafol was similar and fluctuated around 0.5 mg ml@1. Results showed that the levels of sensitivity remained stable in the control treatments throughout the three years, as well as in plots treated with fentin-acetate and maneb. Conversely, in plots treated with flutriafol, either in successive applications or in alternation schemes, a 2–3-fold shift towards reduced sensitivity was observed. The sensitivity shift was more pronounced in 1998 and 1999. This is probably related to the high disease incidence during 1998 and 1999, since the selection of resistant strains is faster when disease incidence is high (Georgopoulos and Skylakakis, 1986). During the three years of the experiment the most significant shift toward reduced sensitivity was observed in plots treated with six spray applications of flutriafol at the full dose. Obviously, applications of flutriafol at high dose controlled efficiently the major part of the pathogen’s population while allowed the survival of only a small part of population, the most resistant, which was sampled at the end of the growing season. A significant shift towards higher EC50 values was also found in C. beticola isolates obtained from plots treated with six successive applications of reduced doses of flutriafol in mixtures with either maneb or fenpropimorph and chlorothalonil. The reduction of application dose favours polygenically controlled resistance by allowing the survival of both highly resistant strains and strains with moderate sensitivity (Georgopoulos, 1994; Brent, 1995). Similar patterns of sensitivity shift were observed in cereal powdery mildew (Hunter et al., 1984; Porras et al., 1990). Conversely, in other studies it was found that applications of the pyrimidine fungicide fenarimol at reduced dose resulted in faster selection of resistant isolates in Venturia inaequalis, in comparison to applications at the full-recommended dose (Ko. ller, 1996; Ko. ller and Wilcox, 1999). However, no differences were observed in sensitivity shifts when EBIs were applied at full or reduced dose to control pathogens such as Pseudocercosporella herpotrichoides (Hunter et al., 1993), Septoria tritici (Shaw and Pijls, 1994) or
Erysiphe graminis (Zziwa and Burnett, 1994; Hau and Pons, 1996). The shift towards higher EC50 values was less significant in isolates obtained from plots treated with flutriafol in alternation with tank mix of fentin-acetate and maneb. The use of this specific mixture reduced the flutriafol selection pressure on the population and delayed the evolution of resistance. Additionally, the interpolation of this mixture between flutriafol treatments favoured the selection of more sensitive strains when the use of flutriafol was discontinued, since sensitive strains are more fit than the resistant strains in the absence of DMIs (Ioannidis and Karaoglanidis, 2000). A similar delaying effect of alternations on the evolution of resistance to the triazole fungicide triadimenol has been previously reported in Uncinula necator (Steva, 1994). A successful anti-resistance strategy should fit two requirements simultaneously. Firstly, it should contribute to the overall reduction of the selection pressure exercised on the pathogen’s population by the ‘‘at risk’’ fungicide. Secondly, it should provide satisfactory disease control. However, sometimes these two requirements do not result from a single strategy, as has been shown by the results of the current study. Although, applications of flutriafol in alternation with tank mix fentin-acetate and maneb could be suggested to provide such a strategy, this spray application program did not provide satisfactory disease control, particularly under favourable environmental conditions for disease development. The alternation strategy could be of high value if alternative fungicides existed. Fungicides of the strobilurin group may play such a role in the future, since they have shown excellent field performance in experimental field trials (Ioannidis and Doulias, 1998). On the other hand, neither successive spray applications of flutriafol at full dose, nor successive applications in reduced dose, could be suggested as efficient antiresistance strategies, despite their good disease control performance. Such strategies favoured the selection of highly resistant strains and their prolonged use over large areas will probably lead to an increase of resistance frequency, resulting in inefficient disease control in the field. It is concluded that the selection of highly resistant strains of C. beticola is dependent firstly on the number of DMIs treatments, and secondly, on the dose applied. Consequently, the reduction in the number of treatments is a prerequisite for successful management of DMIs resistance in C. beticola. The restriction of the number of DMIs treatments cannot be achieved by using them in alternation schemes with tank mix fentinacetate and maneb, due to its low field performance under favourable environmental conditions for disease development. Consequently, the use of DMIs is recommended when it is strictly necessary, whereas
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alternative fungicides can be used during the rest of the spraying period. The development of models predicting the disease development and determining the critical period for the initiation of the epidemic (Skarakis et al., 1996) could contribute to the determination of the critical time for DMIs applications.
Acknowledgements The authors acknowledge Zeneca Hellas S.A. for supplying the technical material of flutriafol used in sensitivity determinations. We also thank S. Daucopoulos and G. Akrivos for maintaining field experiments. Miss M. Thanassoulopoulos, teacher of English language is acknowledged for language corrections. The first of the authors was financially supported by the Greek Scholarship Foundation.
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