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Sensitivity of Verticillium dahliae to prochloraz and prochloraz–manganese complex and control of Verticillium wilt of cotton in the field
Crop Protection 22 (2003) 51–55
Sensitivity of Verticillium dahliae to prochloraz and prochloraz–manganese complex and control of Verticillium wilt of cotton in the field Sener Kurta,*, Sibel Dervisa, Suat Sahinlerb a
Department of Plant Protection, Faculty of Agriculture, Mustafa Kemal University, 31040 Hatay, Turkey b Department of Biometry, Faculty of Agriculture, Mustafa Kemal University, 31040 Hatay, Turkey Accepted 27 May 2002
Abstract Twenty-eight isolates of Verticillium dahliae from the cotton growing areas of Cukurova and Amik plains of Turkey were evaluated in vitro for their sensitivities to prochloraz and prochloraz–manganese complex. The fungicide sensitivity was determined by measuring mycelial growth on agar medium amended with prochloraz and prochloraz–manganese complex at dosages up to 100 mg ml1. The mean ED50 values for both locations ranged from 0.2590 to 0.3574 mg ml1 for prochloraz and 0.5239– 0.8409 mg ml1 for prochloraz–manganese complex. The resistance factors for both fungicides were greater for the Cukurova than the Amik isolates. In field trials, the higher concentration of prochloraz (506 g a.i. ha1) gave a slight reduction in disease severity. However, the higher concentration (1250 g a.i. ha1) of prochloraz–manganese complex resulted in a significant effect (P ¼ 0:05) on disease control. r 2002 Elsevier Science Ltd. All rights reserved. Keywords: Verticillium dahliae; Cotton; Prochloraz; Prochloraz–manganese complex; Sensitivity; Wilt disease control
1. Introduction Verticillium wilt of cotton (Gossypium hirsutum L.), caused by the soilborne pathogen fungus Verticillium dahliae Kleb., is one of the most important plant diseases in Turkey (Dolar, 1986; Esentepe et al., 1986) and in most cotton-growing areas of the world (Bell, 1992). Due to the highly virulent strains or pathotypes (SS-4 and T-1) of V. dahliae, the disease can result in great economic losses in cotton lint yields (Schnathorst and Mathre, 1966). Today, the major cotton producing areas of Turkey are infested with V. dahliae. In addition, the highly virulent pathotype (T-1) and mild virulent pathotype (SS-4) of V. dahliae were found in the Cukurova region of Turkey (Bicici and Kurt, 1998). The fungus is now recognized as one of the most widely distributed and destructive pathogens in agricultural soils cropped continuously to cotton (Butterfield and DeVay, 1977). The pathogen can persist in the soil *Corresponding author. Tel.: +90-326-2455-836; fax: +90-3262455-832. E-mail address: [email protected] (S. Kurt).
or decomposed plant material as microsclerotia for more than 20 years (Wilhelm, 1955; El-Zik, 1985). Infected plants show general yellowing, epinasty and defoliation of leaves and may be killed quickly or remain stunted (Anonymous, 1984). Diseased tissues become necrotic and turn brown, with uniformly distributed tan to brown flecks in the stem (Schnathorst, 1981; Bell, 1992). Verticillium wilt of cotton can be effectively controlled by using tolerant cultivars, crop rotation, plant density, fertilizer and water, solarization, systemic fungicides, induction of phytoalexing gossypol and applying the principles of integrated pest management (Bell, 1992; Canıhos et al., 1997; Kurt and Bicici, 1998). As a protectant, eradicant and translaminar fungicide, prochloraz has been used in Turkey since 1991 as a foliar spray for powdery mildew, Rhizoctonia root rot, Fusarium foot rot, and eyespot control on cereals. The objective of this study was to determine the sensitivity of the V. dahliae isolates against the fungicides prochloraz and prochloraz–manganese complex and the effectiveness of these fungicides to Verticillium wilt of cotton in the field.
0261-2194/02/$ - see front matter r 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 2 6 1 - 2 1 9 4 ( 0 2 ) 0 0 0 9 7 - 2
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S. Kurt et al. / Crop Protection 22 (2003) 51–55
2. Materials and methods 2.1. Selection of fungal isolates Fourteen isolates of V. dahliae were collected from 1994 to 1996 primarily in the naturally infected cotton growing areas of Cukurova region of Turkey. In addition, 14 isolates of V. dahliae were obtained from areas of the Hatay Amik plain in 1997, where upland cotton (Gossypium hirsutum L.), mostly cv. Sure Grow 125, has been grown for a long time. The infected stem tissues were aseptically cut into small pieces and surface-disinfested in 0.525% NaOCL solution for 2 min, rinsed twice in sterile distilled water, and placed on potato dextrose agar (PDA, Difco) and ethanol agar plates amended with streptomycin sulfate at 100 mg ml1 to inhibit bacterial growth and incubated at 231C to 241C in dark for 5 to 7 days. After incubation, hyphal plugs of the growing colonies of each isolate were transferred to a 6-cm diameter petri dish of PDA amended with the same concentrations of antibiotics and incubated in the dark at 231C for 10 days. For pathogenicity of V. dahliae isolates, 6–7 weekold cotton plants, Cukurova 1518, which is a susceptible cotton cultivar, were inoculated with 0.5 ml of a conidial suspension (2 106 conidia/ml) of V. dahliae using a hypodermic needle. Their pathogenicity was verified within 14 days after inoculation (Melouk, 1992).
2.2. Assessment of fungicide sensitivity in vitro Prochloraz (Sportak 45% EC, 450 g/l) and prochloraz–manganese complex (Sporgon 50 WP, 46% w/w) used in this trial were obtained from Aventis Crop Sciences Co., Adana, Turkey. The sensitivity of each isolate of V. dahliae against the fungicides prochloraz and prochloraz–manganese complex was determined by comparing the mycelial radial growth of each isolate on PDA medium containing fungicide with the growth of the same isolate on a medium without the fungicide. For fungicide sensitivity testing, a technical grade active ingredient of each fungicide was used, and all dilutions were made in sterile distilled water. The concentrations of prochloraz and prochloraz–manganese complex were 0.1, 0.5, 1, 5, 10, 25, 50, 75, and 100 mg ml1. Dilutions with or without fungicide were added to molten, cooled PDA (ca. 501C) before the medium was dispensed into petri plates. The same amount of medium was distributed to each petri plate. Hyphal plugs from the edge of actively growing colonies of V. dahliae were inverted and placed in the centre of petri dishes containing approximately 10 ml of PDA amended with each of the nine concentrations of fungicide or one nonamended control. Plates were incubated at 241C for 10 to 14 days in the dark. Colony diameters were measured periodi-
cally in millimeters on all plates when the control treatment was at least 5 cm diameter. Relative growth for each isolate was expressed as the mean adjusted colony diameter on each fungicide concentration as a percentage of the mean adjusted diameter on unamended PDA multipled by 100. ED50 values (the concentration of fungicide causing a 50% reduction in growth compared with nonamended controls) for each isolate were estimated by fitting the best regression model of relative growth on the log10transformed fungicide concentration (Reynolds et al., 1997; McManus et al., 1999). The range and mean ED50 values were calculated for both location and fungicides tested. For each fungicide and location, a resistance factor was determined. This was expressed as the ratio of the highest ED50 to the mean ED50 for each sample. All ED50 values were assigned to classes as follows: o0.1 mg ml1; 0.1 to o0.5 mg ml1; 0.5 to o1.0 mg ml1; 1.0 to o5.0 mg ml1; and X5.0 mg ml1. Correlation coefficients (r) were calculated to determine the correlation between log10-transformed ED50 for prochloraz and prochloraz–manganese complex. 2.3. Control of Verticillium wilt of cotton in naturally infested fields Field trials were conducted at two locations in the Hatay province where severe Verticillium wilt had been reported. Plots were established in naturally infested commercial cotton (cv. Sure Grow 125, susceptible cultivar against wilt pathogen) fields in early June. Standard commercial cotton seed was sown at each location, in late May, 1999 and 2000. The fungicides were applied as sprays at different dosages (Table 2) to the soil in 200 l ha1 water, using a manually operated knapsack sprayer with 1101 nozzles at 138 kPa pressure, to plots (3.2 20 m with 1.0 m border between plots). Areas were replicated four times in a randomized block design. The first spray was applied at flowering on 13 July 1999, and 29 June 2000, with two subsequent applications made at 21-day intervals. Verticillium wilt disease was assessed were made on 6 September 1999 and 13 September 2000 when disease severity in the unsprayed control treatment was highest. Thirty plants were selected at random from each of the plots at harvest. The main stem of each cotton plant was cut near the ground, and at a height of approximately 40 cm, and rated on a scale of 0–4 for intensity and pattern of vascular discoloration. The score of 0 indicated the absence of discoloration; (1) very slight streaking in the wood nearest the pith; (2) slight streaking distributed sporadically throughout the wood; (3) distinct dark discoloration throughout the wood; and (4) intense uniform discoloration and wood deterioration (Wilhelm et al., 1974). The effectiveness of fungicides was calculated by using Abbott formula
S. Kurt et al. / Crop Protection 22 (2003) 51–55
53
(% effectiveness=control-treatment/control 100). The ratings were subjected to a square root transformation prior to analysis of variance. Treatment means were separated using Duncan’s Multiple Range Test. All analysis was conducted by using the SPSS statistical computer software program.
The other isolates (39.3%) were between 0.5 and 5.0 mg ml1. There was a significant and positive correlation between the sensitivities to the prochloraz and the prochloraz–manganese complex for isolates from Cukurova and Amik. The highly significant correlation coefficients were 0.895 and 0.902 (po0:01) for Cukurova and Amik isolates, respectively.
3. Results
3.3. Control of Verticillium wilt of cotton in naturally infected fields
3.1. Fungal isolates All 24 isolates of V. dahliae from Cukurova and Amik plains grew well on PDA amended with streptomycin sulfate at the rate of 100 mg ml1.
Treatments with either prochloraz or prochloraz– manganese complex during the flowering stage generally reduced the incidence of Verticillium wilt but the greater effect was only obtained with the highest dosage applied, especially in 2000 (Table 2).
3.2. Fungicide sensitivity assay in vitro 4. Discussion In this study, both prochloraz and prochloraz– manganese complex demonstrated good activity in reducing in vitro growth of V. dahliae, so in vivo studies were conducted in naturally infested cotton fields. The
60 Percentage of isolates (%)
The ED50 values for prochloraz ranged from 0.00027 to 3.08 and 0.00108 to 1.41 mg ml1 for Cukurova and Amik, respectively (Table 1). The ED50 values for prochloraz–manganese complex ranged from 0.00043 to 3.11 and 0.0 to 3.57 mg ml1 for Cukurova and Amik, respectively. The mean ED50 value for prochloraz for the Cukurova isolates was 0.2590 mg ml1, compared with 0.3574 mg ml1 for the Amik isolates. The mean ED50 value for prochloraz–manganese complex for the Cukurova isolates was 0.5239 mg ml1, compared with 0.8409 mg ml1 for the Amik isolates. The resistance factors for both fungicides were greater for the Cukurova than the Amik isolates (Table 1). The mean was relatively greater for prochloraz–manganese complex than prochloraz. This difference indicated that resistance factor for prochloraz–manganese complex fungicide were higher than prochloraz. Based on the frequency distributions of ED50 values to prochloraz and prochloraz–manganese complex for isolates of V. dahliae (Fig. 1), 85.7% of the isolates were very sensitive (o0.5 mg ml1) to the prochloraz. The ED50 values of the other isolates were between 0.5 and 5.0 mg ml1. 60.7% of the isolates were very sensitive (o0.5 mg ml1) to the prochloraz–manganese complex.
50 40
Prochloraz Prochloraz-manganese complex
50 39.3
35.7
30 21.4
21.4
17.9
20
10.7 10
3.6
0 <0.1
0.1−0.5 0.5−1.0 −1 Fungicide concentration (mg ml )
1.0−5.0
Fig. 1. Frequency distributions of ED50 values to prochloraz and prochloraz–manganese complex for isolates of V. dahliae collected in 1995 and 1997 from cotton fields of Cukurova and Amik plains of Turkey, that had never been exposed to selected fungicides.
Table 1 Frequency distributions of ED50 values to prochloraz and prochloraz–manganese complex for isolates of V. dahliae collected in 1995 and 1997 from two locations in Turkey with no history of exposure to selected fungicides Fungicide
Prochloraz Prochloraz–manganese complex a
Isolate
14 14 14 14
Resistance factor=maximum ED50–mean ED50.
Location
Cukurova Amik Cukurova Amik
ED50 (mg ml1)
Resistance factora
Range
Mean
0.000266 to 3.08 0.001075 to 1.41 0.000429 to 3.11 0.0 to 3.57
0.2590 0.3574 0.5239 0.8409
11.9 3.9 5.9 4.2
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S. Kurt et al. / Crop Protection 22 (2003) 51–55
Table 2 Effect of different fungicides on control of V. dahliae in naturally infected cotton fields in 1999 and 2000 Treatment
Rate (a.i. ha1)
Disease severity index a
2000
1999
Effectiveness (%) 1999b
2000
Avg.
Prochloraz (g)
202 304 405 506 Control
2.73 2.16 1.56 1.47 3.04
b c d d a
2.74 2.35 1.77 1.31 3.43
b c d e a
9.5 28.3 48.8 51.8
c b a a
30.3 31.1 48.2 61.7
c b a a
19.9 29.7 48.5 56.7
c b a a
Prochloraz–manganese complex (g)
500 750 1000 1250 Control
2.85 2.46 1.46 1.43 3.31
b b c c a
2.66 2.28 1.66 0.94 3.62
b c d e a
13.9 24.8 56.0 56.6
b b a a
26.5 37.1 54.2 80.0
d c b a
20.2 30.9 55.1 68.2
d c b a
a Disease rating scale was 0=the absence of discoloration; 4=intense uniform discoloration and wood deterioration; Plots were randomized, based on four replication per fungicide treatment. Thirty plants in each plot were evaluated. b Means followed by the same letter are not significantly different according to Duncan’s Multiple Range Test at P ¼ 0:05: Effectiveness (%)=(control-treatment)/control 100.
significant difference in the sensitivity of isolates from Cukurova region compared to the sensitivity of isolates from Amik plain to prochloraz and prochloraz–manganese complex showed that the V.dahliae populations, where selected fungicides have not been used, have altered toward resistance to these fungicides. The response of a pathogen population to fungicide used depends on genetic variation of resistance being present in the population. In addition, the wide range of ED50 values and low mean ED50 values for that location indicate a wider range in sensitivity among isolates within that fungal population. Inevitably, during the first seasons of a new fungicide use, there will be some instances of poor control due to the environmental problems (Wolfe and Barrett, 1986). However, resistance in the laboratory does not mean that resistance will develop in the field. The nature of the disease and the biology of the fungus play significant roles in the development of fungicide resistance. Although it is not yet possible to predict the development of resistance to a new fungicide with certainty, as much information as possible should be obtained about the potential of plant pathogens to become resistant to such a fungicide (Dekker, 1986). Field tests conducted in naturally infested soils revealed that prochloraz gave only partial disease control when used at the highest concentration, although these fungicides reduced the damage from Verticillium wilt caused by V. dahliae. Our study indicated that prochloraz was the least active of the fungicides on isolates causing Verticillium wilt. In a similar study, benzimidazole fungicides were found to provide complete control of Verticillium wilt when applied as drenches at 2500 ppm to the leaves and the stems of infected cotton plants twice in 1–3 day intervals (Buchenauer and Erwin, 1971). Unfortunately the yield
data on our field trials was not available to correlate changes in disease control with yield. However, our data supports further fungicide studies based on the IPM program, in which these new fungicides are used in combination with other practices to reduce the incidence of the disease (resistant cultivars, crop rotation, etc.) will serve as an important part of the disease management program.
Acknowledgements This work was supported by the grant of the Research Fund of Mustafa Kemal University, Hatay.
References Anonymous, 1984. Integrated Pest Management for cotton in the western region of the United States. Publication No. 3305, University of California, Division of Agriculture and Natural Resources, USA, 144pp. Bell, A.A., 1992. Verticillium wilt. In: Watkins, G.M. (Ed.), Compendium of Cotton Diseases. APS, St. Paul, MN, USA, pp. 41–44. Bicici, M., Kurt, S., 1998. Etiology, incidence and prevalence of cotton wilt disease and strains of the wilt pathogen in Cukurova. In: Proceedings of the World Cotton Research Conference-2, New Frontiers in Cotton Research, Vol II, Athens, Greece, pp. 914–918. Buchenauer, H., Erwin, D.C., 1971. Control of Verticillium wilt of cotton by spraying foliage with benomyl and thiabendazole solubilized with hydrochloric acid. Phytopathology 61, 433–434. Butterfıeld, E.J., DeVay, J.E., 1977. The effect of Verticillium wilt on the growth and development of cotton. Proc. Am. Phytopathol. Soc. (Abstr. ) 4, 173. Canıhos, Y., Kurt, S., Bicici, M., Erkılıc, A., 1997. Induction of phytoalexing gossypol production of cotton plants treated with some herbicides and its antifungal activity. In: Fourth Balkan
S. Kurt et al. / Crop Protection 22 (2003) 51–55 Conference on Operational Research, Volume of Abstracts, Thessaloniki, Greece, 20–23 October, 102pp. Dekker, J., 1986. Preventing and managing fungicide resistance. In: Fungicide Resistance: Strategies and Tactics for Management. National Academy Press, Washington, DC, USA, pp. 347–354. Dolar, S., 1986. Studies on determination of susceptibility of some cotton cultivars against wilt disease (Verticillium dahliae Kleb.) in the Mediterranean region of Turkey. Plant Prot. Bull. 24 (5), 148–158. El-Zik, K.M., 1985. Integrated control of Verticillium wilt of cotton. Plant Dis. 69 (12), 1025–1032. Esentepe, M., Karcılıoglu, A., Sezgin, E., Onan, E., 1986. Investigations on relation between the severity of cotton wilt disease (verticillium dahliae kleb. ) and yield loss in Aegean region. J. Turkish Phytopathol. 15 (1), 27–17336. Kurt, S., Bicici, M., 1998. Development of Verticillium dahliae in cotton plants grown in Cukurova and reaction of the some cultivars to the wilt. In: Proceedings of the World Cotton Research Conference-2 (New Frontiers in Cotton Research), Vol II, Athens, Greece, pp. 919–922. McManus, P.S., Best, V.M., Voland, R.P., Leininger, B.L., 1999. Sensitivity of Monilinia oxycocci to fenbuconazole and propiconazole in vitro and control of cranberry cottonball in the field. Plant Dis 83, 445–450.
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Melouk, H.A., 1992. Verticillium. In: Singleton, L.L., Mihail, J.D., Rush, C.M. (Eds.), Methods for Research on Soilborne Phytopathogenic Fungi. APS Press, The American Phytopathological Society, St. Paul, MN, USA, pp. 175–178. Reynolds, K.L., Brenneman, T.B., Bertrand, P.F., 1997. Sensitivity of Cladosporium caryigenum to propiconazole and fenbuconazole. Plant Dis 81, 163–166. Schnathorst, W.C., 1981. Life cycle and epidemiology of Verticillium. In: Mace, M.E., Bell, A.A., Beckman, C.H. (Eds.), Fungal Wilt Diseases of Plants. Academic Press, New York, USA, pp. 81–111. Schnathorst, W.C., Mathre, D.E., 1966. Host range and differentiation of a severe form of Verticillium albo-atrum in cotton. Phytopathology 56, 1155–1161. Wilhelm, S., 1955. Longevity of the Verticillium wilt fungus in the laboratory and field. Phytopathology 45, 180–181. Wilhelm, S., Sagen, J.E., Tietz, H., 1974. Resistance to Verticillium wilt in cotton: sources, techniques of identification, inheritance trends, and the resistance potential of multiline cultivars. Phytopathology 64, 924–931. Wolfe, M.S., Barrett, J.A., 1986. Response of plant pathogens to fungicides. In: Fungicide Resistance: Strategies and Tactics for Management. National Academy Press, Washington, DC, pp. 245–256.
Sensitivity of Verticillium dahliae to prochloraz and prochloraz–manganese complex and control of Verticillium wilt of cotton in the field Sener Kurta,*, Sibel Dervisa, Suat Sahinlerb a
Department of Plant Protection, Faculty of Agriculture, Mustafa Kemal University, 31040 Hatay, Turkey b Department of Biometry, Faculty of Agriculture, Mustafa Kemal University, 31040 Hatay, Turkey Accepted 27 May 2002
Abstract Twenty-eight isolates of Verticillium dahliae from the cotton growing areas of Cukurova and Amik plains of Turkey were evaluated in vitro for their sensitivities to prochloraz and prochloraz–manganese complex. The fungicide sensitivity was determined by measuring mycelial growth on agar medium amended with prochloraz and prochloraz–manganese complex at dosages up to 100 mg ml1. The mean ED50 values for both locations ranged from 0.2590 to 0.3574 mg ml1 for prochloraz and 0.5239– 0.8409 mg ml1 for prochloraz–manganese complex. The resistance factors for both fungicides were greater for the Cukurova than the Amik isolates. In field trials, the higher concentration of prochloraz (506 g a.i. ha1) gave a slight reduction in disease severity. However, the higher concentration (1250 g a.i. ha1) of prochloraz–manganese complex resulted in a significant effect (P ¼ 0:05) on disease control. r 2002 Elsevier Science Ltd. All rights reserved. Keywords: Verticillium dahliae; Cotton; Prochloraz; Prochloraz–manganese complex; Sensitivity; Wilt disease control
1. Introduction Verticillium wilt of cotton (Gossypium hirsutum L.), caused by the soilborne pathogen fungus Verticillium dahliae Kleb., is one of the most important plant diseases in Turkey (Dolar, 1986; Esentepe et al., 1986) and in most cotton-growing areas of the world (Bell, 1992). Due to the highly virulent strains or pathotypes (SS-4 and T-1) of V. dahliae, the disease can result in great economic losses in cotton lint yields (Schnathorst and Mathre, 1966). Today, the major cotton producing areas of Turkey are infested with V. dahliae. In addition, the highly virulent pathotype (T-1) and mild virulent pathotype (SS-4) of V. dahliae were found in the Cukurova region of Turkey (Bicici and Kurt, 1998). The fungus is now recognized as one of the most widely distributed and destructive pathogens in agricultural soils cropped continuously to cotton (Butterfield and DeVay, 1977). The pathogen can persist in the soil *Corresponding author. Tel.: +90-326-2455-836; fax: +90-3262455-832. E-mail address: [email protected] (S. Kurt).
or decomposed plant material as microsclerotia for more than 20 years (Wilhelm, 1955; El-Zik, 1985). Infected plants show general yellowing, epinasty and defoliation of leaves and may be killed quickly or remain stunted (Anonymous, 1984). Diseased tissues become necrotic and turn brown, with uniformly distributed tan to brown flecks in the stem (Schnathorst, 1981; Bell, 1992). Verticillium wilt of cotton can be effectively controlled by using tolerant cultivars, crop rotation, plant density, fertilizer and water, solarization, systemic fungicides, induction of phytoalexing gossypol and applying the principles of integrated pest management (Bell, 1992; Canıhos et al., 1997; Kurt and Bicici, 1998). As a protectant, eradicant and translaminar fungicide, prochloraz has been used in Turkey since 1991 as a foliar spray for powdery mildew, Rhizoctonia root rot, Fusarium foot rot, and eyespot control on cereals. The objective of this study was to determine the sensitivity of the V. dahliae isolates against the fungicides prochloraz and prochloraz–manganese complex and the effectiveness of these fungicides to Verticillium wilt of cotton in the field.
0261-2194/02/$ - see front matter r 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 2 6 1 - 2 1 9 4 ( 0 2 ) 0 0 0 9 7 - 2
52
S. Kurt et al. / Crop Protection 22 (2003) 51–55
2. Materials and methods 2.1. Selection of fungal isolates Fourteen isolates of V. dahliae were collected from 1994 to 1996 primarily in the naturally infected cotton growing areas of Cukurova region of Turkey. In addition, 14 isolates of V. dahliae were obtained from areas of the Hatay Amik plain in 1997, where upland cotton (Gossypium hirsutum L.), mostly cv. Sure Grow 125, has been grown for a long time. The infected stem tissues were aseptically cut into small pieces and surface-disinfested in 0.525% NaOCL solution for 2 min, rinsed twice in sterile distilled water, and placed on potato dextrose agar (PDA, Difco) and ethanol agar plates amended with streptomycin sulfate at 100 mg ml1 to inhibit bacterial growth and incubated at 231C to 241C in dark for 5 to 7 days. After incubation, hyphal plugs of the growing colonies of each isolate were transferred to a 6-cm diameter petri dish of PDA amended with the same concentrations of antibiotics and incubated in the dark at 231C for 10 days. For pathogenicity of V. dahliae isolates, 6–7 weekold cotton plants, Cukurova 1518, which is a susceptible cotton cultivar, were inoculated with 0.5 ml of a conidial suspension (2 106 conidia/ml) of V. dahliae using a hypodermic needle. Their pathogenicity was verified within 14 days after inoculation (Melouk, 1992).
2.2. Assessment of fungicide sensitivity in vitro Prochloraz (Sportak 45% EC, 450 g/l) and prochloraz–manganese complex (Sporgon 50 WP, 46% w/w) used in this trial were obtained from Aventis Crop Sciences Co., Adana, Turkey. The sensitivity of each isolate of V. dahliae against the fungicides prochloraz and prochloraz–manganese complex was determined by comparing the mycelial radial growth of each isolate on PDA medium containing fungicide with the growth of the same isolate on a medium without the fungicide. For fungicide sensitivity testing, a technical grade active ingredient of each fungicide was used, and all dilutions were made in sterile distilled water. The concentrations of prochloraz and prochloraz–manganese complex were 0.1, 0.5, 1, 5, 10, 25, 50, 75, and 100 mg ml1. Dilutions with or without fungicide were added to molten, cooled PDA (ca. 501C) before the medium was dispensed into petri plates. The same amount of medium was distributed to each petri plate. Hyphal plugs from the edge of actively growing colonies of V. dahliae were inverted and placed in the centre of petri dishes containing approximately 10 ml of PDA amended with each of the nine concentrations of fungicide or one nonamended control. Plates were incubated at 241C for 10 to 14 days in the dark. Colony diameters were measured periodi-
cally in millimeters on all plates when the control treatment was at least 5 cm diameter. Relative growth for each isolate was expressed as the mean adjusted colony diameter on each fungicide concentration as a percentage of the mean adjusted diameter on unamended PDA multipled by 100. ED50 values (the concentration of fungicide causing a 50% reduction in growth compared with nonamended controls) for each isolate were estimated by fitting the best regression model of relative growth on the log10transformed fungicide concentration (Reynolds et al., 1997; McManus et al., 1999). The range and mean ED50 values were calculated for both location and fungicides tested. For each fungicide and location, a resistance factor was determined. This was expressed as the ratio of the highest ED50 to the mean ED50 for each sample. All ED50 values were assigned to classes as follows: o0.1 mg ml1; 0.1 to o0.5 mg ml1; 0.5 to o1.0 mg ml1; 1.0 to o5.0 mg ml1; and X5.0 mg ml1. Correlation coefficients (r) were calculated to determine the correlation between log10-transformed ED50 for prochloraz and prochloraz–manganese complex. 2.3. Control of Verticillium wilt of cotton in naturally infested fields Field trials were conducted at two locations in the Hatay province where severe Verticillium wilt had been reported. Plots were established in naturally infested commercial cotton (cv. Sure Grow 125, susceptible cultivar against wilt pathogen) fields in early June. Standard commercial cotton seed was sown at each location, in late May, 1999 and 2000. The fungicides were applied as sprays at different dosages (Table 2) to the soil in 200 l ha1 water, using a manually operated knapsack sprayer with 1101 nozzles at 138 kPa pressure, to plots (3.2 20 m with 1.0 m border between plots). Areas were replicated four times in a randomized block design. The first spray was applied at flowering on 13 July 1999, and 29 June 2000, with two subsequent applications made at 21-day intervals. Verticillium wilt disease was assessed were made on 6 September 1999 and 13 September 2000 when disease severity in the unsprayed control treatment was highest. Thirty plants were selected at random from each of the plots at harvest. The main stem of each cotton plant was cut near the ground, and at a height of approximately 40 cm, and rated on a scale of 0–4 for intensity and pattern of vascular discoloration. The score of 0 indicated the absence of discoloration; (1) very slight streaking in the wood nearest the pith; (2) slight streaking distributed sporadically throughout the wood; (3) distinct dark discoloration throughout the wood; and (4) intense uniform discoloration and wood deterioration (Wilhelm et al., 1974). The effectiveness of fungicides was calculated by using Abbott formula
S. Kurt et al. / Crop Protection 22 (2003) 51–55
53
(% effectiveness=control-treatment/control 100). The ratings were subjected to a square root transformation prior to analysis of variance. Treatment means were separated using Duncan’s Multiple Range Test. All analysis was conducted by using the SPSS statistical computer software program.
The other isolates (39.3%) were between 0.5 and 5.0 mg ml1. There was a significant and positive correlation between the sensitivities to the prochloraz and the prochloraz–manganese complex for isolates from Cukurova and Amik. The highly significant correlation coefficients were 0.895 and 0.902 (po0:01) for Cukurova and Amik isolates, respectively.
3. Results
3.3. Control of Verticillium wilt of cotton in naturally infected fields
3.1. Fungal isolates All 24 isolates of V. dahliae from Cukurova and Amik plains grew well on PDA amended with streptomycin sulfate at the rate of 100 mg ml1.
Treatments with either prochloraz or prochloraz– manganese complex during the flowering stage generally reduced the incidence of Verticillium wilt but the greater effect was only obtained with the highest dosage applied, especially in 2000 (Table 2).
3.2. Fungicide sensitivity assay in vitro 4. Discussion In this study, both prochloraz and prochloraz– manganese complex demonstrated good activity in reducing in vitro growth of V. dahliae, so in vivo studies were conducted in naturally infested cotton fields. The
60 Percentage of isolates (%)
The ED50 values for prochloraz ranged from 0.00027 to 3.08 and 0.00108 to 1.41 mg ml1 for Cukurova and Amik, respectively (Table 1). The ED50 values for prochloraz–manganese complex ranged from 0.00043 to 3.11 and 0.0 to 3.57 mg ml1 for Cukurova and Amik, respectively. The mean ED50 value for prochloraz for the Cukurova isolates was 0.2590 mg ml1, compared with 0.3574 mg ml1 for the Amik isolates. The mean ED50 value for prochloraz–manganese complex for the Cukurova isolates was 0.5239 mg ml1, compared with 0.8409 mg ml1 for the Amik isolates. The resistance factors for both fungicides were greater for the Cukurova than the Amik isolates (Table 1). The mean was relatively greater for prochloraz–manganese complex than prochloraz. This difference indicated that resistance factor for prochloraz–manganese complex fungicide were higher than prochloraz. Based on the frequency distributions of ED50 values to prochloraz and prochloraz–manganese complex for isolates of V. dahliae (Fig. 1), 85.7% of the isolates were very sensitive (o0.5 mg ml1) to the prochloraz. The ED50 values of the other isolates were between 0.5 and 5.0 mg ml1. 60.7% of the isolates were very sensitive (o0.5 mg ml1) to the prochloraz–manganese complex.
50 40
Prochloraz Prochloraz-manganese complex
50 39.3
35.7
30 21.4
21.4
17.9
20
10.7 10
3.6
0 <0.1
0.1−0.5 0.5−1.0 −1 Fungicide concentration (mg ml )
1.0−5.0
Fig. 1. Frequency distributions of ED50 values to prochloraz and prochloraz–manganese complex for isolates of V. dahliae collected in 1995 and 1997 from cotton fields of Cukurova and Amik plains of Turkey, that had never been exposed to selected fungicides.
Table 1 Frequency distributions of ED50 values to prochloraz and prochloraz–manganese complex for isolates of V. dahliae collected in 1995 and 1997 from two locations in Turkey with no history of exposure to selected fungicides Fungicide
Prochloraz Prochloraz–manganese complex a
Isolate
14 14 14 14
Resistance factor=maximum ED50–mean ED50.
Location
Cukurova Amik Cukurova Amik
ED50 (mg ml1)
Resistance factora
Range
Mean
0.000266 to 3.08 0.001075 to 1.41 0.000429 to 3.11 0.0 to 3.57
0.2590 0.3574 0.5239 0.8409
11.9 3.9 5.9 4.2
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S. Kurt et al. / Crop Protection 22 (2003) 51–55
Table 2 Effect of different fungicides on control of V. dahliae in naturally infected cotton fields in 1999 and 2000 Treatment
Rate (a.i. ha1)
Disease severity index a
2000
1999
Effectiveness (%) 1999b
2000
Avg.
Prochloraz (g)
202 304 405 506 Control
2.73 2.16 1.56 1.47 3.04
b c d d a
2.74 2.35 1.77 1.31 3.43
b c d e a
9.5 28.3 48.8 51.8
c b a a
30.3 31.1 48.2 61.7
c b a a
19.9 29.7 48.5 56.7
c b a a
Prochloraz–manganese complex (g)
500 750 1000 1250 Control
2.85 2.46 1.46 1.43 3.31
b b c c a
2.66 2.28 1.66 0.94 3.62
b c d e a
13.9 24.8 56.0 56.6
b b a a
26.5 37.1 54.2 80.0
d c b a
20.2 30.9 55.1 68.2
d c b a
a Disease rating scale was 0=the absence of discoloration; 4=intense uniform discoloration and wood deterioration; Plots were randomized, based on four replication per fungicide treatment. Thirty plants in each plot were evaluated. b Means followed by the same letter are not significantly different according to Duncan’s Multiple Range Test at P ¼ 0:05: Effectiveness (%)=(control-treatment)/control 100.
significant difference in the sensitivity of isolates from Cukurova region compared to the sensitivity of isolates from Amik plain to prochloraz and prochloraz–manganese complex showed that the V.dahliae populations, where selected fungicides have not been used, have altered toward resistance to these fungicides. The response of a pathogen population to fungicide used depends on genetic variation of resistance being present in the population. In addition, the wide range of ED50 values and low mean ED50 values for that location indicate a wider range in sensitivity among isolates within that fungal population. Inevitably, during the first seasons of a new fungicide use, there will be some instances of poor control due to the environmental problems (Wolfe and Barrett, 1986). However, resistance in the laboratory does not mean that resistance will develop in the field. The nature of the disease and the biology of the fungus play significant roles in the development of fungicide resistance. Although it is not yet possible to predict the development of resistance to a new fungicide with certainty, as much information as possible should be obtained about the potential of plant pathogens to become resistant to such a fungicide (Dekker, 1986). Field tests conducted in naturally infested soils revealed that prochloraz gave only partial disease control when used at the highest concentration, although these fungicides reduced the damage from Verticillium wilt caused by V. dahliae. Our study indicated that prochloraz was the least active of the fungicides on isolates causing Verticillium wilt. In a similar study, benzimidazole fungicides were found to provide complete control of Verticillium wilt when applied as drenches at 2500 ppm to the leaves and the stems of infected cotton plants twice in 1–3 day intervals (Buchenauer and Erwin, 1971). Unfortunately the yield
data on our field trials was not available to correlate changes in disease control with yield. However, our data supports further fungicide studies based on the IPM program, in which these new fungicides are used in combination with other practices to reduce the incidence of the disease (resistant cultivars, crop rotation, etc.) will serve as an important part of the disease management program.
Acknowledgements This work was supported by the grant of the Research Fund of Mustafa Kemal University, Hatay.
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