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Sensitivity of Cercospora beticola populations to fentin-acetate, benomyl and flutriafol in Greece
Crop Protection 22 (2003) 735–740
Sensitivity of Cercospora beticola populations to fentin-acetate, benomyl and flutriafol in Greece G.S. Karaoglanidisa, D.A. Karadimosb,*, P.M. Ioannidisa, P.I. Ioannidisb a
Plant Protection Department, Hellenic Sugar Industry SA, 59032, Platy Imathias, Greece b Plant Protection Department, Hellenic Sugar Industry SA, 41110 Larissa, Greece Received 7 August 2002; accepted 3 February 2003
Abstract Cercospora beticola isolates were collected, over 2 years (2000–2001), from sugar beet fields of 4 areas of Greece (Imathia, Larissa, Serres and Orestiada) with different histories of fungicide use. The isolates were used to determine their sensitivity to benomyl, fentin-acetate and flutriafol. In Orestiada and Larissa, where benomyl has been used in one spray application per year since 1996 or 2000, the resistance frequency was more than 50%. In contrast to Imathia and Serres, where benomyl has not been used since the detection of resistance in the early 1970s. Resistance to fentin-acetate, was significantly decreased since previous surveys in 1978. The resistance frequency ranged from 2–3% in the Serres’ population to 8–11% in the Orestiada’ population. In Larissa and Orestiada no population shift towards decreased sensitivity to flutriafol was detected but in Serres and Imathia, which were more heavily treated with sterol demethylation-inhibiting fungicides (DMIs), populations clearly showed decreased sensitivity, although to a smaller frequency than in earlier surveys when more DMI sprays were being applied. r 2003 Elsevier Science Ltd. All rights reserved. Keywords: Cercospora leaf-spot; Sugar beet; Fungicide resistance
1. Introduction Sugar beet leaf-spot, caused by Cercospora beticola, is the most important foliar disease of sugar beet in warm and humid areas worldwide (Byford, 1996, Windels et al., 1998). It can cause serious yield losses in the absence of treatments, ranging from 10% to 50% (Shane and Teng, 1992; Wolf and Verreet, 2002). Control of sugar beet leaf-spot, in Greece and in many other countries as well, has relied on an integrated approach involving use of tolerant cultivars, crop rotation and fungicide applications (Ioannidis, 1994). The benzimidazole derivatives were the first systemic fungicides that became available for C. beticola control. In Greece, applications of benomyl began in 1971 with excellent results but, within 2 years, the fungal populations developed resistance and fungicide efficacy was completely lost (Georgopoulos and Dovas, 1973). This has also been reported in several other countries (Ruppel and Scott, 1974; D’Ambra et al., 1974; Pal *Corresponding author. Tel.: +30-410-582-152. E-mail address: [email protected] (D.A. Karadimos).
and Mukhopadhyay, 1985; Weiland and Halloin, 2001). In Greece, after the emergence of benzimidazole resistance the use of benomyl was discontinued. However, in 1995 it was found that resistance frequency to benomyl had greatly decreased since 1972. Since then, benomyl has been used in one application early in the season in some areas of Greece (Karadimos et al., 2000). Fentin fungicides are generally very effective against C. beticola (Stallknecht and Calpouzos, 1968) and used worldwide for controlling the disease (Byford, 1996). Fentin derivatives were the only available fungicides providing satisfactory control of C. beticola after the emergence of benomyl resistance. However, during the 1976 and 1977 growing seasons, control of Cercospora leaf-spot by fentin fungicides, was unsatisfactory and laboratory tests showed that resistance had developed (Giannopolitis, 1978). Since then the use of fentin derivatives, in most areas of sugar beet cultivation, has been restricted to 2 or 3 applications in mixture with maneb, early in the season. Resistance of C. beticola to fentin derivatives has also been reported in USA (Bugbee, 1995; Campbell et al., 1998) and in Italy (Cerato and Grassi, 1983).
0261-2194/03/$ - see front matter r 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0261-2194(03)00036-X
G.S. Karaoglanidis et al. / Crop Protection 22 (2003) 735–740
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Sterol demethylation-inhibiting fungicides (DMIs) have been used in Greece, for the control of this disease, since 1979. These fungicides are used always in mixture with a protectant fungicide, either maneb or chlorothalonil. However, after 1990 a reduction in the performance of DMI fungicides was observed in field experiments for fungicide evaluation, on sugar beet, in Northern Greece and an extensive monitoring program showed that fungal populations had shifted towards decreased sensitivity (Karaoglanidis et al., 2000). The current study was conducted in order to determine the sensitivity of C. beticola populations to benomyl, fentin-acetate and flutriafol after the end of the 2000 and 2001 spraying seasons and to evaluate the effects of the applied fungicide spray programmes on the fungal population sensitivity to these fungicides. A preliminary report of this research has already been published (Ioannidis et al., 2001).
2.2. Pathogen isolation
2. Materials and methods
Sugar beet leaves with distinct sporulating lesions were collected, from about 10 widely distributed fields in each sampling area, after the end of the spraying period, during October 2000 and 2001. Single-lesion isolates of C. beticola were obtained by transferring conidia, with the aid of a fine glass needle, to Aspergillus Complete Medium (ACM), composed of 20 g agar (Oxoid, Unipath Ltd, Basingstoke, England), 10 g dextrose (Merck, Darmstadt, Germany) and 1 g yeast extract (Oxoid) per liter and acidified with 0.5 ml l1 lactic acid in order to suppress bacterial growth. Conidia from only one lesion per leaf were transferred to the culture medium. In total 150 isolates per sampling site were collected in 2000, while in 2001 250, 133, 130 and 122 isolates were collected from Orestiada, Serres, Larissa and Imathia areas, respectively. After 2 days of incubation at 25 C, in the dark, the fungal colonies were transferred to fresh ACM. In each petri dish 6 colonies were started and incubated for 10 days at 25 C.
2.1. Sampling sites
2.3. Sensitivity tests
Four different areas in northern and central Greece, Larissa, Imathia, Serres and Orestiada, with different histories of fungicide use, were sampled. During 2000 and 2001 in all the four areas, fentin-acetate mixed with maneb was used in the first 2–3 fungicide spray applications of the season. DMIs in mixture with maneb or chlorothalonil were applied later in the season in 3–4 spray applications (Table 1). In the past, Imathia and Serres were the areas which received the higher DMIs selection pressure since they were applied in a greater number of sprays. The use of benomyl was discontinued after the emergence of resistance strains in 1972 but was reintroduced into the spray programs in 1996 in Larissa and in 2000 in Orestiada, in mixture with maneb.
Fungicides used in this study were commercial formulations of benomyl (Benlate 50WP, DuPont Agro Hellas), fentin-acetate (Brestan 60WP, Agrevo Hellas S.A.) and flutriafol (Impact, 12.5SC, Zeneca Hellas, Greece). Autoclaved ACM was cooled to 50 C and amended with aqueous fungicide solutions at discriminatory concentrations. The discriminatory concentration for benomyl was 1 mg ml1, a concentration completely inhibitory for the sensitive isolates but not for the resistant isolates. For fentin-acetate the isolates were classified in three sensitivity groups, resistant, moderately resistant and sensitive, according to their vegetative growth at 0.25 mg ml1. For the measurement of isolates’ sensitivity to flutriafol, 1 mg ml1 was
Table 1 Fungicidea spray schedules applied in sugar beet fields in four sampled areas of Greece and application doses during a 2-year period (2000–2001) Spray application
Sampling site Larissa
1st 2nd 3rd 4th 5th 6th 7th a
Orestiada
Imathia
Serres
2000
2001
2000
2001
2000
2001
2000
2001
FA/MN FA/MN FA/MN FL/MN FL/MN DF/MN DF/MN
FA/MN FA/MN FA/MN DP/MN DP/MN DF/MN DF/MN
FA FA/MN BN/MN DF/FP/CH
FA FA/MN BN/MN DF/FP/CH
FA/MN FA/MN FA/MN DF/FP/CH FL/CH FL/CH
FA/MN FA/MN FA/MN DF/FP/CH DP/CH DP/CH DP/CH
FA/MN FA/MN FA/MN DF/FP/CH FL/MN FL/MN DF/MN
FA/MN FA/MN FA/MN DF/FP/CH DP/MN DP/FA DP/CH
Fungicides: FA: fentin acetate, Brestan 60WP, Agrevo Hellas S.A., 0.30 kg/ha a.i., MN: maneb, Dithane 80WP, Rohm and Haas Hellas, S.A., 1.92 kg/ha a.i., BN: benomyl, Benor 50WP, Alpha Agrochemicals S.A., 0.25 kg/ha a.i., CH: chlorothalonil, Daconil, 75WP, Zeneca Hellas S.A., 0.75 kg/ha a.i., FP: fenpropimorph, Corbel 75EC, Syngenta Hellas AEVE, 0.30 kg/ha a.i., DP: difenoconazole+propiconazole, Armure 15/15 EC, Syngenta Hellas S.A., 0.075 kg/ha a.i., FL: flutriafol, Impact 12.5 SC, Zeneca Hellas S.A. 0.05 Kg/ha a.i., DF: difenoconazole, Score 25EC, Syngenta Hellas S.A., 0.05 kg/ha a.i. or 0.075 kg/ha a.i.
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used as discriminatory concentration. Control petri dishes were not amended with fungicide. Tests for each isolate were replicated twice per concentration of each fungicide. Mycelial plugs of 5 mm diameter were removed from the colony margins and placed upside down on the fungicide-amended and fungicide-free petri dishes and incubated at 25 C, in the dark. Radial growth of each isolate was measured (minus the diameter of inoculation plug) after 7 days by calculating the mean of two perpendicular colony diameters. The mean diameters of colonies were expressed as percentages of the colony diameters in control treatments and the relative growth (RG) was estimated. Chi-square analysis was used to analyse the sensitivity distribution of the populations to flutriafol, based on the RG of the isolates from the four areas sampled. The statistical analysis was supported by Mstat-C statistical program (Mstat-C, version 2.10, Michigan State University).
3. Results
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3.2. Sensitivity to fentin-acetate In the current study, sensitivity of C. beticola populations to fentin-acetate was measured for the first time since the emergence of resistance was detected in Greece, in 1977. For the measurement of sensitivity was used the discriminatory concentration suggested by Giannopolitis (1978). The areas of Orestiada, Serres and Imathia were sampled both in 2000 and 2001, while in the area of Larissa sampling of the population was carried out only in 2001. Data are presented in Table 3. In all sampled areas, resistance frequency has decreased in comparison to the levels of resistance detected by Giannopolitis (1978). In all the 4 areas sampled, the percentage of resistant strains was lower than 11%, whereas 29% of the strains was resistant when resistance had been detected for first time in 1977 (Table 3). Among the areas sampled Orestiada showed the higher fentin-resistance frequency with values of 8.6% and 10.8% in 2000 and 2001, respectively, while the lowest resistance frequency was observed within the Serres population where only 2–3% of the isolates tested was resistant to fentin-acetate.
3.1. Sensitivity to benomyl 3.3. Sensitivity to flutriafol Since resistance to benomyl has a qualitative character (Georgopoulos and Skylakakis, 1986), the sensitivity of C. beticola was studied by using a discriminatory concentration. Data on the sensitivity to benomyl of fungal populations in the several regions are presented in Table 2. During both years of the study high resistance frequencies with values of 80% and 68% in 2000 and 2001, respectively, were observed within the population of Orestiada which has been treated with one benomyl application per year. The benomyl-resistance frequency within the population of Larissa was also high, with values of 68% and 50.7% in 2000 and 2001, respectively. The resistance frequency in the populations of the remaining two areas, Imathia and Serres, was lower with values of 22% and 15%, respectively, in 2000 and 25% and 11.2%, respectively in 2001.
Quantification of sensitivities of C. beticola to flutriafol was based on the degree of inhibition of mycelial growth. For each population sampled, a sensitivity distribution was constructed according to the RG of isolates at the dosage of 1 mg ml1 flutriafol. The sensitivity distribution was continuous, as expected for a DMI fungicide. Results showed that sensitivity distributions of the C. beticola populations in the areas of Imathia and Serres have clearly shifted towards decreased sensitivity while in the remaining two areas, Larissa and Orestiada, the populations are more sensitive (Fig. 1). Chi-squared analysis of frequency distribution data showed that growth of isolates of Serres and Imathia populations, at 1 mg ml1 flutriafol was similar (P > 0:05). More than 32.2% of the isolates
Table 2 Sensitivity of C. beticola to the benzimidazole fungicide benomyl, in four areas of Greece in 2000 and 2001 Region
Year
Number of isolates
Sensitivitya Resistant isolates
Larissa Orestiada Imathia Serres a b
2000 2001 2000 2001 2000 2001 2000 2001
150 130 150 250 150 122 150 133
b
102 (68) 66 (50.7) 120 (80) 170 (68) 33 (22) 31 (25.4) 23 (15.3) 15 (11.2)
Sensitive isolates showed no vegetative growth at 1 mg ml1 benomyl, while resistant isolates formed normal colonies. Percentage of the population.
Sensitive isolates 48 (32) 64 (49.3) 30 (20) 80 (32) 117 (78) 91 (74.6) 127 (84.7) 118 (88.8)
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Table 3 Sensitivity of C. beticola to fentin-acetate, in four areas of Greece in 2000 and 2001 Sampling area
Year
Sensitivitya
Number of isolates
Sensitive isolates Larissa Orestiada
2001 2000 2001 2000 2001 2000 2001
Imathia Serres
130 150 250 150 122 150 133
32 31 52 38 21 59 45
b
(24.6) (20.6) (20.8) (25.3) (17.2) (39.3) (33.8)
Moderately resistant isolates
Resistant isolates
89 (68.4) 106 (70.6) 171 (68.4) 106 (70.6) 94 (77.0) 88 (58.6) 84 (63.1)
9 (6.9) 13 (8.6) 27 (10.8) 6 (4.0) 7 (5.7) 3 (2.0) 4 (3.0)
Sensitive isolates were considered those showing no vegetative growth at 0.25 mg ml1 fentin-acetate, as moderately resistant isolates were considered those showing a vegetative growth of less than 2 mm per day at 0.25 mg ml1 fentin-acetate and as resistant isolates were considered those showing a vegetative growth of more than 2 mm per day at 0.25 mg ml1 fentin-acetate. b Percentage of population. a
100
45 40
80 RG fentin acetate
35 30 25 20 Frequency of isolates (%)
r = 0.26 P= 0.15 n= 100
2000
15 10 5
60 40 20
0 0
10
20
30
40
50
60
70
80
90
40
0 0
2001
35
20
40 60 RG flutriafol
80
100
Fig. 2. Linear regression among RG values of 100 C. beticola isolates at 1 mg ml1 flutriafol and 0.25 mg ml1 fentin-acetate.
30 25 20 15 10 5 0 0
10
20
30
40
50
60
70
80
90
Relative growth Fig. 1. Sensitivity distribution of C. beticola isolates, from Larissa area (closed bars), Orestiada area (hatched bars), Imathia area (opened bars) and Serres area (horizontal line bars), according to their RG at 1 mg ml1 flutriafol.
in both areas during both years of the study, had RG of 50% or more. At 1 mg ml1 flutriafol, conversely, only 20.5% and 21.2% of the Larissa and Orestiada populations, respectively, had RG of 50% or more during 2000, while the respective values of 2001 were even lower reaching only 19.4 and 13.6% for Larissa and Orestiada populations, respectively. Since fungicide sensitivity was determined using the same isolates, a cross-resistance analysis between flutriafol and fentin-acetate was carried out. The analysis was performed by correlating the RG values
of 100 randomly selected isolates at 1 mg ml1 flutriafol and 0.25 mg ml1 fentin-acetate. The isolates were arbitrarily selected from the Serres population. Based on this analysis there was no correlation (r ¼ 0:26; P > 0:05) between sensitivities to flutriafol and fentinacetate (Fig. 2).
4. Discussion The problem of fungicide resistance development in C. beticola is one of the most important factors limiting chemical control of Cercospora leaf-spot disease, in Greece and in many other countries (Ioannidis and Karaoglanidis, 2000). The favourable environmental conditions prevailing in most of the sugar beet cultivation areas in Greece and resulting in high disease incidence, the polycyclic nature of the disease, the abundant sporulation of the pathogen and the fact that large areas of sugar beet cultivation are treated with a common spray programme, are factors contributing to the development of fungicide resistance.
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Benomyl was the first fungicide in which resistance was developed by C. beticola with resistance frequency values reaching at 80–90% (Georgopoulos and Dovas, 1973). After detection of resistance the use of benomyl was discontinued. In a previous sensitivity monitoring study, carried out during 1995–1996, resistance frequency to benomyl had decreased to values of 20–25% (Karadimos et al., 2000), despite the predictions for a stable resistance frequency due to the equal competitive ability of resistant and sensitive strains (Dovas et al., 1976, Ruppel, 1975). A similar decrease of benomyl resistance frequency has also been reported for Mycosphaerella fijiensis in banana (Smith, 1988). Based on these findings benomyl was reintroduced into the spray programme in the area of Larissa during 1997–1998 and in the area of Orestiada during 2000–2001, by applying it once per season, at reduced dose in mixture with maneb. According to the findings of the current study in the two areas in which benomyl have been reintroduced into the spray programme, resistance frequency increased rapidly, even after only one or two spray applications and reached the levels of resistance frequency in the early 1970s, when for first time resistance to benomyl had been detected. Converserly, resistance frequency in the areas of Serres and Imathia, where benomyl had not been used since early 1970s, continued slowly dropping. Such results indicate that the reintroduction of benomyl into the spray programmes cannot be suggested for the control of sugar beet leaf-spot, since the resistant strains are selected rapidly even with only one benomyl spray application. In all the four areas sampled, fentin-resistance frequency was lower than that determined by Giannopolitis (1978). This lower resistance frequency is probably related to the fact that after the emergence of resistance, fentin fungicides are used only in two or three spray applications always early in the season, when disease incidence is relatively low (Ioannidis, 1994; Ioannidis and Karaoglanidis, 2000). Such a tactic allows the reduction, later in the season, of the resistant strains selected under the selection force of the fentin fungicides, since in the absence of fentin fungicides the resistance strains do not compete well with the sensitive strains (Giannopolitis and Chrysayi-Tokousbalides, 1980). Among the four sampled areas the higher resistance frequency was detected, during both years of the study, in Orestiada. This is probably related to the fact that in this region fentin-acetate was applied without any partner fungicide in the first spray application. Sensitivity to flutriafol was measured by using a discriminatory concentration despite the fact that resistance to DMIs has a quantitative character (Georgopoulos and Skylakakis, 1986). Such a procedure has also been accomplished for the measurement of sensitivity to DMIs in several other pathogens (Smith
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et al., 1991; Peever and Milgroom, 1992, Romero and Sutton, 1997) and also for the measurement of C. beticola sensitivity to bitertanol (Georgopoulos, 1987). The concentration of 1 mg ml1 flutriafol was selected as equal to or slightly higher than the mean EC50 of the wild-type population (Smith et al., 1991). Additionally, it has been found that RG values at this concentration have the higher correlation factor with the EC50 value of each individual isolate, and field experiments, carried out during 1999 and 2000, showed that such isolates cannot be efficiently controlled by the recommended dose of flutriafol (Karaoglanidis, unpublished data). The pathogen populations at Serres and Imathia were less sensitive to flutriafol than pathogen population at Orestiada and Larissa. Differences among the sampled areas are correlated to the DMI application history of each area. Imathia and particularly Serres areas were heavily treated with DMIs for about 20 years, while in the other two areas DMIs were used less often. However, resistance frequencies detected in the current study of Imathia and Serres populations were lower compared to those found in a previous monitoring program carried out for a 4-year period (1996–1999) in the same areas (Karaoglanidis et al., 2002). This lower resistance frequency could be correlated to the reduction of the number of DMIs spray applications from 5–6 to 3–4 per season, since field experimental data on the effects of various fungicide spray programmes on the sensitivity to DMIs showed that the number of DMIs treatments per season affects significantly the fungal population sensitivity to DMIs (Karaoglanidis et al., 2001). When isolates from the Serres population were analysed for their cross-sensitivity between the triazole fungicide flutriafol and the chemically unrelated fentinderivative fentin-acetate, no cross-resistance was observed. This absence of cross-resistance relationship reflects to the differences in the mode of action of these two fungicides since fentin derivatives inhibit the ATP formation (Stockdale et al., 1970), while triazole fungicides inhibit the oxidative sterol 14a-demethylation in the ergosterol biosynthesis pathway (Siegel, 1981). Severe failures in controlling Cercospora leaf-spot of sugar beet caused by resistance development are mainly restricted to benzimidazole fungicides and particularly in areas with high disease incidence requiring a high number of fungicide spray applications for successful disease control. Although field resistance has also developed to fentin and DMI fungicides, in these cases resistance is more easily managed due to its quantitative pattern. Conventional protective fungicides such as maneb and chlorothalonil could aid management of resistance to all these fungicide classes, by applying them in tank-mixtures. However, introduction into the spray programmes of novel compounds with higher disease control efficacy is necessary since conventional
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protective fungicides do not provide adequate disease control, particularly under conditions of high disease incidence. The introduction of strobilurin fungicides, which have shown excellent field performance, particularly in mixtures with triazole fungicides (Karadimos, unpublished data), could contribute towards this aim. Additionally, continuous and extensive monitoring programmes for fungicide sensitivity determination are required in order to indicate the need to change the applied spray programmes and thus, prevent failures of control.
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Sensitivity of Cercospora beticola populations to fentin-acetate, benomyl and flutriafol in Greece G.S. Karaoglanidisa, D.A. Karadimosb,*, P.M. Ioannidisa, P.I. Ioannidisb a
Plant Protection Department, Hellenic Sugar Industry SA, 59032, Platy Imathias, Greece b Plant Protection Department, Hellenic Sugar Industry SA, 41110 Larissa, Greece Received 7 August 2002; accepted 3 February 2003
Abstract Cercospora beticola isolates were collected, over 2 years (2000–2001), from sugar beet fields of 4 areas of Greece (Imathia, Larissa, Serres and Orestiada) with different histories of fungicide use. The isolates were used to determine their sensitivity to benomyl, fentin-acetate and flutriafol. In Orestiada and Larissa, where benomyl has been used in one spray application per year since 1996 or 2000, the resistance frequency was more than 50%. In contrast to Imathia and Serres, where benomyl has not been used since the detection of resistance in the early 1970s. Resistance to fentin-acetate, was significantly decreased since previous surveys in 1978. The resistance frequency ranged from 2–3% in the Serres’ population to 8–11% in the Orestiada’ population. In Larissa and Orestiada no population shift towards decreased sensitivity to flutriafol was detected but in Serres and Imathia, which were more heavily treated with sterol demethylation-inhibiting fungicides (DMIs), populations clearly showed decreased sensitivity, although to a smaller frequency than in earlier surveys when more DMI sprays were being applied. r 2003 Elsevier Science Ltd. All rights reserved. Keywords: Cercospora leaf-spot; Sugar beet; Fungicide resistance
1. Introduction Sugar beet leaf-spot, caused by Cercospora beticola, is the most important foliar disease of sugar beet in warm and humid areas worldwide (Byford, 1996, Windels et al., 1998). It can cause serious yield losses in the absence of treatments, ranging from 10% to 50% (Shane and Teng, 1992; Wolf and Verreet, 2002). Control of sugar beet leaf-spot, in Greece and in many other countries as well, has relied on an integrated approach involving use of tolerant cultivars, crop rotation and fungicide applications (Ioannidis, 1994). The benzimidazole derivatives were the first systemic fungicides that became available for C. beticola control. In Greece, applications of benomyl began in 1971 with excellent results but, within 2 years, the fungal populations developed resistance and fungicide efficacy was completely lost (Georgopoulos and Dovas, 1973). This has also been reported in several other countries (Ruppel and Scott, 1974; D’Ambra et al., 1974; Pal *Corresponding author. Tel.: +30-410-582-152. E-mail address: [email protected] (D.A. Karadimos).
and Mukhopadhyay, 1985; Weiland and Halloin, 2001). In Greece, after the emergence of benzimidazole resistance the use of benomyl was discontinued. However, in 1995 it was found that resistance frequency to benomyl had greatly decreased since 1972. Since then, benomyl has been used in one application early in the season in some areas of Greece (Karadimos et al., 2000). Fentin fungicides are generally very effective against C. beticola (Stallknecht and Calpouzos, 1968) and used worldwide for controlling the disease (Byford, 1996). Fentin derivatives were the only available fungicides providing satisfactory control of C. beticola after the emergence of benomyl resistance. However, during the 1976 and 1977 growing seasons, control of Cercospora leaf-spot by fentin fungicides, was unsatisfactory and laboratory tests showed that resistance had developed (Giannopolitis, 1978). Since then the use of fentin derivatives, in most areas of sugar beet cultivation, has been restricted to 2 or 3 applications in mixture with maneb, early in the season. Resistance of C. beticola to fentin derivatives has also been reported in USA (Bugbee, 1995; Campbell et al., 1998) and in Italy (Cerato and Grassi, 1983).
0261-2194/03/$ - see front matter r 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0261-2194(03)00036-X
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Sterol demethylation-inhibiting fungicides (DMIs) have been used in Greece, for the control of this disease, since 1979. These fungicides are used always in mixture with a protectant fungicide, either maneb or chlorothalonil. However, after 1990 a reduction in the performance of DMI fungicides was observed in field experiments for fungicide evaluation, on sugar beet, in Northern Greece and an extensive monitoring program showed that fungal populations had shifted towards decreased sensitivity (Karaoglanidis et al., 2000). The current study was conducted in order to determine the sensitivity of C. beticola populations to benomyl, fentin-acetate and flutriafol after the end of the 2000 and 2001 spraying seasons and to evaluate the effects of the applied fungicide spray programmes on the fungal population sensitivity to these fungicides. A preliminary report of this research has already been published (Ioannidis et al., 2001).
2.2. Pathogen isolation
2. Materials and methods
Sugar beet leaves with distinct sporulating lesions were collected, from about 10 widely distributed fields in each sampling area, after the end of the spraying period, during October 2000 and 2001. Single-lesion isolates of C. beticola were obtained by transferring conidia, with the aid of a fine glass needle, to Aspergillus Complete Medium (ACM), composed of 20 g agar (Oxoid, Unipath Ltd, Basingstoke, England), 10 g dextrose (Merck, Darmstadt, Germany) and 1 g yeast extract (Oxoid) per liter and acidified with 0.5 ml l1 lactic acid in order to suppress bacterial growth. Conidia from only one lesion per leaf were transferred to the culture medium. In total 150 isolates per sampling site were collected in 2000, while in 2001 250, 133, 130 and 122 isolates were collected from Orestiada, Serres, Larissa and Imathia areas, respectively. After 2 days of incubation at 25 C, in the dark, the fungal colonies were transferred to fresh ACM. In each petri dish 6 colonies were started and incubated for 10 days at 25 C.
2.1. Sampling sites
2.3. Sensitivity tests
Four different areas in northern and central Greece, Larissa, Imathia, Serres and Orestiada, with different histories of fungicide use, were sampled. During 2000 and 2001 in all the four areas, fentin-acetate mixed with maneb was used in the first 2–3 fungicide spray applications of the season. DMIs in mixture with maneb or chlorothalonil were applied later in the season in 3–4 spray applications (Table 1). In the past, Imathia and Serres were the areas which received the higher DMIs selection pressure since they were applied in a greater number of sprays. The use of benomyl was discontinued after the emergence of resistance strains in 1972 but was reintroduced into the spray programs in 1996 in Larissa and in 2000 in Orestiada, in mixture with maneb.
Fungicides used in this study were commercial formulations of benomyl (Benlate 50WP, DuPont Agro Hellas), fentin-acetate (Brestan 60WP, Agrevo Hellas S.A.) and flutriafol (Impact, 12.5SC, Zeneca Hellas, Greece). Autoclaved ACM was cooled to 50 C and amended with aqueous fungicide solutions at discriminatory concentrations. The discriminatory concentration for benomyl was 1 mg ml1, a concentration completely inhibitory for the sensitive isolates but not for the resistant isolates. For fentin-acetate the isolates were classified in three sensitivity groups, resistant, moderately resistant and sensitive, according to their vegetative growth at 0.25 mg ml1. For the measurement of isolates’ sensitivity to flutriafol, 1 mg ml1 was
Table 1 Fungicidea spray schedules applied in sugar beet fields in four sampled areas of Greece and application doses during a 2-year period (2000–2001) Spray application
Sampling site Larissa
1st 2nd 3rd 4th 5th 6th 7th a
Orestiada
Imathia
Serres
2000
2001
2000
2001
2000
2001
2000
2001
FA/MN FA/MN FA/MN FL/MN FL/MN DF/MN DF/MN
FA/MN FA/MN FA/MN DP/MN DP/MN DF/MN DF/MN
FA FA/MN BN/MN DF/FP/CH
FA FA/MN BN/MN DF/FP/CH
FA/MN FA/MN FA/MN DF/FP/CH FL/CH FL/CH
FA/MN FA/MN FA/MN DF/FP/CH DP/CH DP/CH DP/CH
FA/MN FA/MN FA/MN DF/FP/CH FL/MN FL/MN DF/MN
FA/MN FA/MN FA/MN DF/FP/CH DP/MN DP/FA DP/CH
Fungicides: FA: fentin acetate, Brestan 60WP, Agrevo Hellas S.A., 0.30 kg/ha a.i., MN: maneb, Dithane 80WP, Rohm and Haas Hellas, S.A., 1.92 kg/ha a.i., BN: benomyl, Benor 50WP, Alpha Agrochemicals S.A., 0.25 kg/ha a.i., CH: chlorothalonil, Daconil, 75WP, Zeneca Hellas S.A., 0.75 kg/ha a.i., FP: fenpropimorph, Corbel 75EC, Syngenta Hellas AEVE, 0.30 kg/ha a.i., DP: difenoconazole+propiconazole, Armure 15/15 EC, Syngenta Hellas S.A., 0.075 kg/ha a.i., FL: flutriafol, Impact 12.5 SC, Zeneca Hellas S.A. 0.05 Kg/ha a.i., DF: difenoconazole, Score 25EC, Syngenta Hellas S.A., 0.05 kg/ha a.i. or 0.075 kg/ha a.i.
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used as discriminatory concentration. Control petri dishes were not amended with fungicide. Tests for each isolate were replicated twice per concentration of each fungicide. Mycelial plugs of 5 mm diameter were removed from the colony margins and placed upside down on the fungicide-amended and fungicide-free petri dishes and incubated at 25 C, in the dark. Radial growth of each isolate was measured (minus the diameter of inoculation plug) after 7 days by calculating the mean of two perpendicular colony diameters. The mean diameters of colonies were expressed as percentages of the colony diameters in control treatments and the relative growth (RG) was estimated. Chi-square analysis was used to analyse the sensitivity distribution of the populations to flutriafol, based on the RG of the isolates from the four areas sampled. The statistical analysis was supported by Mstat-C statistical program (Mstat-C, version 2.10, Michigan State University).
3. Results
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3.2. Sensitivity to fentin-acetate In the current study, sensitivity of C. beticola populations to fentin-acetate was measured for the first time since the emergence of resistance was detected in Greece, in 1977. For the measurement of sensitivity was used the discriminatory concentration suggested by Giannopolitis (1978). The areas of Orestiada, Serres and Imathia were sampled both in 2000 and 2001, while in the area of Larissa sampling of the population was carried out only in 2001. Data are presented in Table 3. In all sampled areas, resistance frequency has decreased in comparison to the levels of resistance detected by Giannopolitis (1978). In all the 4 areas sampled, the percentage of resistant strains was lower than 11%, whereas 29% of the strains was resistant when resistance had been detected for first time in 1977 (Table 3). Among the areas sampled Orestiada showed the higher fentin-resistance frequency with values of 8.6% and 10.8% in 2000 and 2001, respectively, while the lowest resistance frequency was observed within the Serres population where only 2–3% of the isolates tested was resistant to fentin-acetate.
3.1. Sensitivity to benomyl 3.3. Sensitivity to flutriafol Since resistance to benomyl has a qualitative character (Georgopoulos and Skylakakis, 1986), the sensitivity of C. beticola was studied by using a discriminatory concentration. Data on the sensitivity to benomyl of fungal populations in the several regions are presented in Table 2. During both years of the study high resistance frequencies with values of 80% and 68% in 2000 and 2001, respectively, were observed within the population of Orestiada which has been treated with one benomyl application per year. The benomyl-resistance frequency within the population of Larissa was also high, with values of 68% and 50.7% in 2000 and 2001, respectively. The resistance frequency in the populations of the remaining two areas, Imathia and Serres, was lower with values of 22% and 15%, respectively, in 2000 and 25% and 11.2%, respectively in 2001.
Quantification of sensitivities of C. beticola to flutriafol was based on the degree of inhibition of mycelial growth. For each population sampled, a sensitivity distribution was constructed according to the RG of isolates at the dosage of 1 mg ml1 flutriafol. The sensitivity distribution was continuous, as expected for a DMI fungicide. Results showed that sensitivity distributions of the C. beticola populations in the areas of Imathia and Serres have clearly shifted towards decreased sensitivity while in the remaining two areas, Larissa and Orestiada, the populations are more sensitive (Fig. 1). Chi-squared analysis of frequency distribution data showed that growth of isolates of Serres and Imathia populations, at 1 mg ml1 flutriafol was similar (P > 0:05). More than 32.2% of the isolates
Table 2 Sensitivity of C. beticola to the benzimidazole fungicide benomyl, in four areas of Greece in 2000 and 2001 Region
Year
Number of isolates
Sensitivitya Resistant isolates
Larissa Orestiada Imathia Serres a b
2000 2001 2000 2001 2000 2001 2000 2001
150 130 150 250 150 122 150 133
b
102 (68) 66 (50.7) 120 (80) 170 (68) 33 (22) 31 (25.4) 23 (15.3) 15 (11.2)
Sensitive isolates showed no vegetative growth at 1 mg ml1 benomyl, while resistant isolates formed normal colonies. Percentage of the population.
Sensitive isolates 48 (32) 64 (49.3) 30 (20) 80 (32) 117 (78) 91 (74.6) 127 (84.7) 118 (88.8)
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Table 3 Sensitivity of C. beticola to fentin-acetate, in four areas of Greece in 2000 and 2001 Sampling area
Year
Sensitivitya
Number of isolates
Sensitive isolates Larissa Orestiada
2001 2000 2001 2000 2001 2000 2001
Imathia Serres
130 150 250 150 122 150 133
32 31 52 38 21 59 45
b
(24.6) (20.6) (20.8) (25.3) (17.2) (39.3) (33.8)
Moderately resistant isolates
Resistant isolates
89 (68.4) 106 (70.6) 171 (68.4) 106 (70.6) 94 (77.0) 88 (58.6) 84 (63.1)
9 (6.9) 13 (8.6) 27 (10.8) 6 (4.0) 7 (5.7) 3 (2.0) 4 (3.0)
Sensitive isolates were considered those showing no vegetative growth at 0.25 mg ml1 fentin-acetate, as moderately resistant isolates were considered those showing a vegetative growth of less than 2 mm per day at 0.25 mg ml1 fentin-acetate and as resistant isolates were considered those showing a vegetative growth of more than 2 mm per day at 0.25 mg ml1 fentin-acetate. b Percentage of population. a
100
45 40
80 RG fentin acetate
35 30 25 20 Frequency of isolates (%)
r = 0.26 P= 0.15 n= 100
2000
15 10 5
60 40 20
0 0
10
20
30
40
50
60
70
80
90
40
0 0
2001
35
20
40 60 RG flutriafol
80
100
Fig. 2. Linear regression among RG values of 100 C. beticola isolates at 1 mg ml1 flutriafol and 0.25 mg ml1 fentin-acetate.
30 25 20 15 10 5 0 0
10
20
30
40
50
60
70
80
90
Relative growth Fig. 1. Sensitivity distribution of C. beticola isolates, from Larissa area (closed bars), Orestiada area (hatched bars), Imathia area (opened bars) and Serres area (horizontal line bars), according to their RG at 1 mg ml1 flutriafol.
in both areas during both years of the study, had RG of 50% or more. At 1 mg ml1 flutriafol, conversely, only 20.5% and 21.2% of the Larissa and Orestiada populations, respectively, had RG of 50% or more during 2000, while the respective values of 2001 were even lower reaching only 19.4 and 13.6% for Larissa and Orestiada populations, respectively. Since fungicide sensitivity was determined using the same isolates, a cross-resistance analysis between flutriafol and fentin-acetate was carried out. The analysis was performed by correlating the RG values
of 100 randomly selected isolates at 1 mg ml1 flutriafol and 0.25 mg ml1 fentin-acetate. The isolates were arbitrarily selected from the Serres population. Based on this analysis there was no correlation (r ¼ 0:26; P > 0:05) between sensitivities to flutriafol and fentinacetate (Fig. 2).
4. Discussion The problem of fungicide resistance development in C. beticola is one of the most important factors limiting chemical control of Cercospora leaf-spot disease, in Greece and in many other countries (Ioannidis and Karaoglanidis, 2000). The favourable environmental conditions prevailing in most of the sugar beet cultivation areas in Greece and resulting in high disease incidence, the polycyclic nature of the disease, the abundant sporulation of the pathogen and the fact that large areas of sugar beet cultivation are treated with a common spray programme, are factors contributing to the development of fungicide resistance.
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Benomyl was the first fungicide in which resistance was developed by C. beticola with resistance frequency values reaching at 80–90% (Georgopoulos and Dovas, 1973). After detection of resistance the use of benomyl was discontinued. In a previous sensitivity monitoring study, carried out during 1995–1996, resistance frequency to benomyl had decreased to values of 20–25% (Karadimos et al., 2000), despite the predictions for a stable resistance frequency due to the equal competitive ability of resistant and sensitive strains (Dovas et al., 1976, Ruppel, 1975). A similar decrease of benomyl resistance frequency has also been reported for Mycosphaerella fijiensis in banana (Smith, 1988). Based on these findings benomyl was reintroduced into the spray programme in the area of Larissa during 1997–1998 and in the area of Orestiada during 2000–2001, by applying it once per season, at reduced dose in mixture with maneb. According to the findings of the current study in the two areas in which benomyl have been reintroduced into the spray programme, resistance frequency increased rapidly, even after only one or two spray applications and reached the levels of resistance frequency in the early 1970s, when for first time resistance to benomyl had been detected. Converserly, resistance frequency in the areas of Serres and Imathia, where benomyl had not been used since early 1970s, continued slowly dropping. Such results indicate that the reintroduction of benomyl into the spray programmes cannot be suggested for the control of sugar beet leaf-spot, since the resistant strains are selected rapidly even with only one benomyl spray application. In all the four areas sampled, fentin-resistance frequency was lower than that determined by Giannopolitis (1978). This lower resistance frequency is probably related to the fact that after the emergence of resistance, fentin fungicides are used only in two or three spray applications always early in the season, when disease incidence is relatively low (Ioannidis, 1994; Ioannidis and Karaoglanidis, 2000). Such a tactic allows the reduction, later in the season, of the resistant strains selected under the selection force of the fentin fungicides, since in the absence of fentin fungicides the resistance strains do not compete well with the sensitive strains (Giannopolitis and Chrysayi-Tokousbalides, 1980). Among the four sampled areas the higher resistance frequency was detected, during both years of the study, in Orestiada. This is probably related to the fact that in this region fentin-acetate was applied without any partner fungicide in the first spray application. Sensitivity to flutriafol was measured by using a discriminatory concentration despite the fact that resistance to DMIs has a quantitative character (Georgopoulos and Skylakakis, 1986). Such a procedure has also been accomplished for the measurement of sensitivity to DMIs in several other pathogens (Smith
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et al., 1991; Peever and Milgroom, 1992, Romero and Sutton, 1997) and also for the measurement of C. beticola sensitivity to bitertanol (Georgopoulos, 1987). The concentration of 1 mg ml1 flutriafol was selected as equal to or slightly higher than the mean EC50 of the wild-type population (Smith et al., 1991). Additionally, it has been found that RG values at this concentration have the higher correlation factor with the EC50 value of each individual isolate, and field experiments, carried out during 1999 and 2000, showed that such isolates cannot be efficiently controlled by the recommended dose of flutriafol (Karaoglanidis, unpublished data). The pathogen populations at Serres and Imathia were less sensitive to flutriafol than pathogen population at Orestiada and Larissa. Differences among the sampled areas are correlated to the DMI application history of each area. Imathia and particularly Serres areas were heavily treated with DMIs for about 20 years, while in the other two areas DMIs were used less often. However, resistance frequencies detected in the current study of Imathia and Serres populations were lower compared to those found in a previous monitoring program carried out for a 4-year period (1996–1999) in the same areas (Karaoglanidis et al., 2002). This lower resistance frequency could be correlated to the reduction of the number of DMIs spray applications from 5–6 to 3–4 per season, since field experimental data on the effects of various fungicide spray programmes on the sensitivity to DMIs showed that the number of DMIs treatments per season affects significantly the fungal population sensitivity to DMIs (Karaoglanidis et al., 2001). When isolates from the Serres population were analysed for their cross-sensitivity between the triazole fungicide flutriafol and the chemically unrelated fentinderivative fentin-acetate, no cross-resistance was observed. This absence of cross-resistance relationship reflects to the differences in the mode of action of these two fungicides since fentin derivatives inhibit the ATP formation (Stockdale et al., 1970), while triazole fungicides inhibit the oxidative sterol 14a-demethylation in the ergosterol biosynthesis pathway (Siegel, 1981). Severe failures in controlling Cercospora leaf-spot of sugar beet caused by resistance development are mainly restricted to benzimidazole fungicides and particularly in areas with high disease incidence requiring a high number of fungicide spray applications for successful disease control. Although field resistance has also developed to fentin and DMI fungicides, in these cases resistance is more easily managed due to its quantitative pattern. Conventional protective fungicides such as maneb and chlorothalonil could aid management of resistance to all these fungicide classes, by applying them in tank-mixtures. However, introduction into the spray programmes of novel compounds with higher disease control efficacy is necessary since conventional
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protective fungicides do not provide adequate disease control, particularly under conditions of high disease incidence. The introduction of strobilurin fungicides, which have shown excellent field performance, particularly in mixtures with triazole fungicides (Karadimos, unpublished data), could contribute towards this aim. Additionally, continuous and extensive monitoring programmes for fungicide sensitivity determination are required in order to indicate the need to change the applied spray programmes and thus, prevent failures of control.
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