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Sensitivity of Cercospora beticola isolates from Serbia to carbendazim and flutriafol
Crop Protection 66 (2014) 120e126
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Sensitivity of Cercospora beticola isolates from Serbia to carbendazim and flutriafol Dragana Budakov a, Nevena Nagl b, *, Vera Stojsin a, Ferenc Bagi a, Dario Danojevi c b, cb Oliver T. Neher c, Ksenija Taski-Ajdukovi a b c
Faculty of Agriculture, University of Novi Sad, Trg Dositeja Obradovica 8, 21000 Novi Sad, Serbia Institute of Field and Vegetable Crops, Maksima Gorkog 30, 21000 Novi Sad, Serbia The Amalgamated Sugar Company, 1951 S. Saturn Way, Suite 100, Boise, ID 83709, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 April 2014 Received in revised form 15 September 2014 Accepted 16 September 2014 Available online
Cercospora beticola, causal agent of Cercospora leaf spot (CLS) of sugar beet, is primarily controlled by fungicides. Benzimidazole and demethylation inhibiting fungicides, including carbendazim and flutriafol, have been widely used in Serbia. Since these fungicide groups have a site-specific mode of action, there is a high risk for developing resistance in target organisms, which is the most important limiting factor in Cercospora leaf spot chemical control. A rapid identification of flutriafol and carbendazim resistance can help researchers in examining the potential of different fungicide resistance management practices, as well as in selection of fungicides for use in the areas where resistance has occurred. One hundred singleconidia isolates were collected from 70 representative locations of the sugar beet production region in Serbia. Evaluation of the isolates' sensitivity was based on the reduction of mycelial growth on medium amended with 1.25 mg mL1 flutriafol and 5 mg mL1 carbendazim. Resistance to flutriafol and carbendazim was detected in 16% and 96% of the tested isolates, respectively. All isolates resistant to flutriafol were resistant to carbendazim as well, which is the first report of a double resistance to fungicides in C. beticola. Detection of the isolates resistant to flutriafol and carbendazim using Cleaved Amplified Polymorphic Sequence (CAPS) markers confirmed the results of the in vitro tests. The efficacy of carbendazim, flutriafol, azoxystrobin, and tetraconazole at commercially recommended doses was evaluated in field trials where sugar beet plants in plots were inoculated with a mixture of isolates either sensitive and/or resistant to flutriafol and carbendazim. Carbendazim and flutriafol efficacy was very low in plots inoculated with isolates resistant to these fungicides. Presented results will contribute to development of a pathogen population sensitivity monitoring strategy that could be used for an effective CLS management in the region. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Cercospora leaf spot (CLS) Fungicide resistance Carbendazim Flutriafol CAPS Fungicide efficacy
1. Introduction Cercospora leaf spot (CLS), caused by Cercospora beticola Sacc., is the most important foliar disease of sugar beet in warm and humid environmental conditions that often occur during summer months in the sugar beet growing regions in South-Eastern Europe. In the absence of appropriate control measures in the areas with a high disease incidence, severe epidemics of CLS can result in a significant reduction of root yield, recoverable sugar, sucrose concentration and an increase in impurities leading to higher processing costs
* Corresponding author. Tel.: þ381 (0)21 4898327. E-mail addresses: [email protected] (N. Nagl), [email protected] (K. Taski-Ajdukovi c). http://dx.doi.org/10.1016/j.cropro.2014.09.010 0261-2194/© 2014 Elsevier Ltd. All rights reserved.
(Windels et al., 1998; Jacobsen and Franc, 2009). CLS is primarily controlled by fungicide applications, although it can also be managed by planting disease-tolerant varieties, crop rotation and tillage. In conditions of a high disease pressure, frequent reapplications of the same fungicide are common. This can select for resistance in the target organisms, which is the most important limiting factor for CLS chemical control. Several classes of protective and systemic fungicides with various modes of action are available for use against this disease worldwide. In the West Balkans, benzimidazoles are among the fungicides registered for CLS control, while the sterol demethylation inhibiting (DMI) fungicides have the highest number of registered active ingredients available. Since these fungicide groups have a site-specific mode of action, there is a high risk for developing resistance (Brent and Hollomon, 2007).
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Benzimidazoles act primarily by binding to fungal tubulin and interfering with mitosis and the fungal cytoskeleton (Davidse, 1986). The mechanism of resistance to benzimidazole fungicides in C. beticola is based on a mutation in the b-tubulin gene, which reduces benzimidazole binding (Davidson et al., 2006). Benzimidazole fungicides have been used extensively in the management of CLS, but resistance to them has become a major problem in many sugar beet growing areas (Georgopoulos and Dovas, 1973; Briere et al., 2001; Weiland and Halloin, 2001). Once resistance has developed, the frequency of benzimidazole-resistant strains remains stable or only drops slowly even in the absence of selection pressure (Karaoglanidis and Ioannidis, 2010). Sterol demethylation inhibiting (DMI) fungicides are inhibitors of sterol C-14 alpha-demethylation during ergosterol biosynthesis, which is the main fungal sterol (Siegel, 1981). They represent the most important class of fungicides used to control CLS, providing an excellent protective and curative activity against Cercospora spp. (Dahmen and Staub, 1992). In the United States, a reduction in the performance of DMI fungicides has occurred over time as a result of the presence of C. beticola strains with a reduced sensitivity to DMIs (Secor et al., 2010). Population shift towards DMI resistance was step-wise, which indicates a polygenic control of the resistance (Brent and Hollomon, 2007). Resistance problems with the use of DMIs have been reported in several phytopathogenic fungi and two different resistance mechanisms have been identified: mutations in the C-14 alpha-demethylase (CYP51) gene and overexpression of the CYP51 gene (Ma and Michailides, 2005). It has been suggested that overexpression of the C-14 alpha-demethylase (Cyp51) gene is the mechanism of DMI resistance in C. beticola (Nikou et al., 2009; Bolton et al., 2013). Northern Serbia is a major sugar beet producing region with about 60,000 ha under this crop. Since a reduction in the performance of benzimidazole and some DMI fungicides has been observed (Mari c et al., 1976; Bala z et al., 1999; Budakov et al., 2010), the aims of this study were to: i) perform a sensitivity screening of C. beticola isolated from the sugar beet growing areas in Serbia to benzimidazole and DMI fungicides; ii) develop a CAPS (Cleaved Amplified Polymorphic Sequence) marker for detection of the resistant and sensitive isolates to benzimidazoles; iii) test a
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previously developed CAPS marker for detection of the DMI resistant isolates; iv) field-evaluate fungicide efficacy on CLS control, caused by the isolates with known sensitivity levels to benzimidazoles and DMIs; and v) evaluate how fungicide resistance determined by in vitro tests and CAPS markers correlates with a reduced disease control in the field. 2. Materials and methods 2.1. Sampling of leaves and collection of fungal isolates Sugar beet leaves with typical symptoms of CLS were collected during the growing season of 2007, from 70 locations in the main sugar beet growing region in Serbia (Fig. 1). A total of 100 isolates were collected, with the number of isolates per location being proportional to the acreage under sugar beet in that area. All sampling sites had a history of benzimidazole and DMI fungicide use, including carbendazim and flutriafol. Collected leaves were washed under tap water and the lesions were cut out and placed in Petri dishes on a wet filter paper. After 24e36 h of incubation at 25e26 C, monosporial isolations were performed after conidia had formed within lesions. Single conidia were lifted with a sterile entomological needle and placed on potato dextrose agar (PDA, Carl Roth GmbH). The cultures were transferred onto PDA slants and kept refrigerated at 4 C until further analysis. 2.2. In vitro sensitivity testing For in vitro fungicide sensitivity tests, we used two commercially available fungicide formulations containing flutriafol and carbendazim as active ingredients: Takt (125 g a.i. L1 flutriafol, Herbos, Croatia) and Galofungin (500 g a.i. L1 carbendazim, Galenika, Serbia). Baseline sensitivities for flutriafol and carbendazim were determined by testing five references C. beticola isolates (CB 581, CB 582, CB 586, CB 587 and CB 588) from beetroot and chard grown in fields in which none of the fungicides examined had been used. In order to test the sensitivity to flutriafol, the concentrations of 0.3, 0.6, 1.25, 2, 4, 8 and 16 mg mL1 were applied in the medium. An average EC50 was 0.8 mg mL1, hence the next higher concentration
Fig. 1. Map of Northern Serbia showing locations where Cercospora beticola isolates were collected.
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(1.25 mg mL1) was a discriminative concentration. The sensitivity to carbendazim was tested with a series of concentrations: 1, 2, 4, 5, 8, 10, 16, 20, 32 and 50 mg mL1. Since none of the tested isolates was suppressed with tested concentrations, a discriminative concentration of 5 mg mL1 was used (Weiland and Halloin, 2001). The autoclaved PDA was amended with fungicide aqueous solutions, while the control Petri dishes contained non-amended PDA. The tests were performed in 90 mm-diameter Petri dishes with 3 mycelial plugs per isolate (5 mm in diameter) turned upside down on the fungicide-amended PDA and the control Petri dishes. Each isolate-fungicide combination was replicated 3 times. A colony diameter was determined by averaging two perpendicular measurements of the diameter after 4 days of incubation at 25e26 C and subtracting the diameter of the mycelial plug. Obtained values were used to calculate the relative growth (RG). Based on the mean RG, the isolates were divided into four groups: i) sensitive (RG < 20%); ii) with a decreased sensitivity (RG ¼ 20e39.9%); iii) moderately resistant (RG ¼ 40e69.9%); and iv) resistant (RG 70%). 2.3. Molecular analysis After the fungicide sensitivity was determined in vitro, 27 isolates with varying degrees of resistance to flutriafol were chosen for further analysis of the C-14 alpha-demethylase gene, while 23 isolates were chosen for analysis of the b-tubulin gene, in order to identify the carbendazim resistance-causing mutation. Prior to DNA extraction, the isolates were grown on PDA for 10 days at 25e26 C, after which 500 mg of fungal tissue was harvested by scraping off mycelia from the agar surface. The DNA was extracted following the procedure described by Budakov et al. (2012). In order to detect a mutation indicating the DMI resistant isolates, specific primers CYP51RT-F (50 -AACTCCAAATTGATGGAGCA30 ) and CYP51RT-R (50 -CGGCTAGCAGTGTAAATGGT-30 ) were used for amplification of the C-14 alpha-demethylase gene fragment (Nikou et al., 2009). Amplification started with an initial denaturation at 94 C for 4 min, followed by 38 cycles of denaturation at 94 C for 40 s, annealing at 52 C for 35 s and extension at 72 C for 45 s. The final extension was at 72 C for 10 min. Primers Bt512F (50 CCAGCTTTTCCGCCCAGACAAC-30 ) and Bt922R (50 -ACGGCACCATG TTCACGGCAAGC-30 ) were used for amplification of the b-tubulin gene fragment (Davidson et al., 2006). The amplification profile was as follows: initial denaturation at 94 C for 4 min, followed by 38 cycles of denaturation at 94 C for 40 s, annealing at 62 C for 40 s and extension at 72 C for 1 min. The final extension lasted 10 min at 72 C. All PCR reactions were carried out in a total volume of 25 mL containing: 30 ng of the DNA template, 2.5 mL of buffer, 0.2 mM of each dNTP, 2.5 mM MgCl2, 2 units of Taq polymerase (Fermentas) and 0.25 mM of each primer (Metabion). The reactions were performed in TPersonal and T1 thermocyclers (Biometra). The PCR products were separated on a 1% agarose gels containing 0.005% ethidium bromide and visualized under UV light. Restriction enzyme Alw26I (BsmAI, Fermentas) was used to detect the mutation in C-14 alpha-demethylase gene (Nikou et al., 2009). After restriction mapping of the b-tubulin gene sequence (Davidson et al., 2006), a mutation responsible for resistance to benzimidazoles at position 198 was detected by digestion with Bsh1236I (BSTUI, Fermentas) restriction enzyme. The PCR products were digested in the manufacturer's reaction buffer and electrophoresed on a 1.7% agarose gel containing 50% high resolution agarose (Carl Roth GmbH). 2.4. Field trials Fungicides containing active ingredients carbendazim and flutriafol were used in field trials to test their efficacy in controlling
the selected C. beticola isolates. In order to test for alternative spraying options in areas with a reported decreased sensitivity to carbendazim and flutriafol, azoxystrobin (Quinone outside Inhibitors-QoIs)- and tetraconazole (DMIs)-based fungicides were also included. Azoxystrobin was selected as a representative of a novel group of fungicides in Serbia that has already been widely used in CLS control worldwide. Tetraconazole was chosen for several reasons: i) it belongs to the same chemical group as flutriafol, but has been reported to have higher efficacy; ii) no crossresistance with flutriafol has been reported yet; and iii) it is registered for use in Serbia as a single ingredient in pesticide formulations. The isolates with known sensitivity levels to flutriafol and carbendazim were selected previously in vitro and used as inoculum for field trials. The inoculum was prepared in the form of a conidial suspension with conidia produced on Sugar Beet Leaf extract Agar (SBLA) (150e200 g of fresh sugar beet leaves in 1 L of distilled water þ 20% Agar). Concentration of the suspension was 500 spores per ml, which was determined by counting spores using a haemocytometer. Field trials were conducted on sugar beet cv. LARA, during the growing seasons of 2010 and 2011, on an experimental field of the Institute of Field and Vegetable Crops, Novi Sad, Serbia, in a completely randomized factorial design with four replicates. In 2010 and 2011, the size of one plot was 12 m2 (3 rows of sugar beet, 8 m long) and 24 m2 (6 rows of sugar beet, 8 m long), respectively. Individual plots were inoculated on July 8th 2010 and July 4th 2011, when the weather conditions were favorable for disease development, using a back-pack sprayer (Solo 456) and 200 L of water per ha. The plots were inoculated with four isolate mixtures, containing the isolates with a similar sensitivity to tested fungicides: a) sensitive to flutriafol and carbendazim (FSBS); b) moderately resistant to flutriafol and sensitive to carbendazim (FRBS); c) resistant to flutriafol and carbendazim (FRBR); and d) sensitive to flutriafol and resistant to carbendazim (FSBR) (Appendix B). Each inoculated plot, except for the non-treated control, was treated throughout the vegetation period with one of the following fungicides: Galofungin (Galenika, Serbia, carbendazim 500 g a.i. L1), Takt (Herbos, Croatia, flutriafol 125 g a.i. L1), Eminent (Isagro, tetraconazole 125 g a.i. L1) and Quadris (Syngenta, azoxystrobin 125 g a.i. L1). The first fungicide treatment was applied seven days after inoculation, with subsequent treatments on August 1st, August 20th and September 4th in 2010 and July 20th, August 3rd and August 16th in 2011. The fungicides were applied using a Solo 456 back-pack sprayer at 400 L ha1 of fungicide mixture. Disease intensity was evaluated four times until harvest on the same dates when the fungicide treatments were applied. Evaluation was based on the percent of leaf infection using a scale from 0 to 9, where 0 represents a healthy plant and 9 is a plant with 100% necrotic leaves. These values were used to calculate the AUDPC (Area Under the Disease Progress Curve) (Wolf and Veerret, 2002). Approximately two weeks after the last treatment in each year, the plants were harvested and the root yield and sugar content were determined in a sugar beet automatic laboratory (Venema Automation BV, The Netherlands). Fungicide efficacy was calculated on the basis of an average penultimate disease intensity evaluation. 2.5. Data analysis Results of the field trials: AUDPC, root yield and sugar concentration were expressed as mean ± standard deviation. Statistical analysis was performed by analysis of variance (ANOVA) using Statistica 12 software (StatSoft, Tulsa, OK, USA). Comparisons between means were made with Fisher's least significant difference (LSD) test, P ¼ 0.05, among the isolates for each fungicide treatment.
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Table 1 Sensitivity of C. beticola isolates and composition of isolate mixtures used in field trials. Isolate
Flutriafol RG (%)
CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB
80 254 84 546 72 253 543 551 538 179 591 412 523 605 600 504 82 10 603 3 270 257 152 94 515 322 236 50 382 561 133 317 526 175 74 183 319 188 609 191 316 75 577 44 112 206 20 300 292 567 135 596 165 207 115 125 33 587 157 1 171 173 204 369 388 425 440
113.33 112.30 106.74 106.62 101.82 101.81 99.03 96.77 95.74 92.86 90.16 89.66 87.86 86.00 82.48 75.27 69.75 69.22 68.53 63.25 60.00 53.54 50.00 48.57 47.90 46.75 44.90 32.00 24.42 24.11 23.30 20.10 19.87 19.80 19.74 19.10 19.05 18.75 18.39 17.54 17.14 17.06 16.84 16.67 16.51 16.27 15.48 14.97 14.33 13.89 13.79 12.59 11.67 11.30 10.98 9.87 9.50 9.43 8.33 2.26 0.00 0.00 0.00 0.00 0.00 0.00 0.00
Isolates used in field trials
Carbendazim Sensitivity
RG %
In vitro
CAPS
R R R R R R R R R R R R R R R R MR MR MR MR MR MR MR MR MR MR MR DS DS DS DS DS S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S
R R e R e e R e R e R e e R e R R S S S S S S S S S S e S e S S e e e e e e e e S e e e e e e e e e e e e e e S e S S e e e e S e e e
121.90 102.98 101.00 104.83 98.58 102.72 105.07 106.81 97.59 99.31 106.03 99.43 91.62 86.00 98.60 98.92 105.24 103.26 2.80 96.14 97.50 100.00 112.38 137.14 10.86 94.08 123.88 102.83 93.02 104.56 98.92 97.82 95.96 99.42 97.99 94.24 127.27 102.74 101.53 100.78 2.86 102.78 92.63 104.66 149.21 100.02 77.42 113.26 102.81 102.07 99.30 128.67 91.27 100.33 97.65 0.00 99.01 84.18 94.02 113.56 99.81 93.43 104.65 95.27 98.99 97.05 98.78
Sensitivity In vitro
CAPS
FRBR
R R R R R R R R R R R R R R R R R R S R R R R R S R R R R R R R R R R R R R R R S R R R R R R R R R R R R R R S R R R R R R R R R R R
e R e R e e e e R e R e e R e R R e S R e R R R S R R e e e R R e e e e e e e e S e e e e e e e e e e e e e e S R R R e e e e e R e e
þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ
FRBS
FSBR
FSBS
þ
þ
þ þ
þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ
RG-relative growth; S-Sensitive; DS-Decreased sensitivity; MR-Moderately resistant; R-Resistant; FRBR-resistant to flutriafol and carbendazim; FRBS-moderately resistant to flutriafol and sensitive to carbendazim; FSBR-sensitive to flutriafol and resistant to carbendazim; FSBS-sensitive to flutriafol and carbendazim.
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3. Results 3.1. In vitro sensitivity testing A relative mycelial growth on the medium amended with flutriafol in comparison to the non-amended control ranged from 0% to 113.3% (Table 1). Thirty six percent of isolates were sensitive to flutriafol, 38% had a decreased sensitivity, 10% were moderately resistant and 16% of the tested isolates were resistant to flutriafol. When the medium was amended with carbendazim, 96 isolates had an equal growth on the fungicide-amended and non-amended medium (77.4e149.2%). In four isolates (CB125, CB316, CB515, and CB603), RG was suppressed and varied from 0 to 10.99% on carbendazim-amended medium. After in vitro sensitivity testing, 16 isolates resistant to flutriafol and carbendazim (FRBR), 2 moderately resistant to flutriafol and sensitive to carbendazim (FRBS), 36 isolates sensitive to flutriafol and resistant to carbendazim (FSBR) and 2 sensitive to flutriafol and carbendazim (FSBS) were used for further investigation. 3.2. CAPS markers Eight resistant (CB80, CB254, CB546, CB543, CB538, CB591, CB605, CB504), 11 moderately resistant (CB82, CB10, CB603, CB3, CB257, CB270, CB152, CB94, CB515, CB322, CB236) and 8 isolates sensitive to flutriafol (CB382, CB133, CB317, CB316, CB125, CB587, CB157, CB369) were chosen for further analysis of the C-14 alphademethylase gene using CAPS, while 19 resistant (CB254, CB538, CB546, CB591, CB82, CB152, CB257, CB322, CB33, CB504, CB157, CB388, CB587, CB3, CB133, CB317, CB236, CB605, CB94) and all isolates sensitive (CB125, CB316, CB515, CB603) to carbendazim were chosen for detection of the mutation in the b-tubulin gene responsible for resistance (Table 1). After PCR reactions with C-14 alpha-demethylase gene-specific primers, an amplification product of 200 bp in size was obtained in all isolates. After restriction with Alw26I enzyme, only one band of 200 bp was detected in the resistant isolates (RG 70%) and the moderately resistant isolate CB82 (RG 69.35%), whereas two bands of 80 and 120 bp were detected in the sensitive and other moderately resistant isolates (Fig. 2a). As a result of amplification with the b-tubulin specific
primers, a 570 bp-long PCR product was obtained in all isolates. As expected, digestion of the amplification product with Bsh1236I enabled differentiation between the resistant and the sensitive isolates: three bands were detected in the resistant isolates (250, 200 and 120 bp), whereas there were two bands in the sensitive isolates (450 and 120 bp) (Fig. 2b). 3.3. Field trials Based on the results of the in vitro sensitivity tests and the CAPS marker analysis, C. beticola isolates were used for sugar beet inoculations in field trials aimed at determining fungicide efficacy. In both 2010 and 2011, disease progressed quickly after inoculation due to favorable environmental conditions, i.e. high temperatures and humidity during July and August. The first symptoms were observed seven days after inoculation, at the moment of the first fungicide treatment. Disease symptoms developed uniformly in all plots, with the spots being formed mainly on middle leaves within the rosette. At the second disease evaluation, a black pseudostroma formed within the spots. The disease progressed fastest in nontreated control plots and in plots inoculated with the resistant isolates treated with the fungicides corresponding to the resistance. In those plots, the leaf canopy completely decayed and regrowth occurred by the middle and end of August. In both years, disease intensity (AUDPC) was highest in the non-treated control, while the lowest AUDPC occurred in all inoculated plots treated with azoxystrobin (Fig. 3a). Disease intensity was higher in plots inoculated with the isolates resistant to flutriafol and carbendazim and treated with the fungicides corresponding to the resistance. We did not observe a noticeable pattern in the effect of fungicide treatments on root yield in plots inoculated with the selected isolates (Fig. 3b). Sugar concentration was lowest in plots treated with carbendazim and flutriafol and inoculated with isolates resistant to both fungicides (FRBR), while in 2010 this was also the case in plots inoculated with FRBS isolates and treated with flutriafol (Fig. 3c). In 2010, there were no differences in sugar content in azoxystrobin and tetraconazole treatments, regardless of the sensitivity of the isolates used for inoculation. In carbendazim treatments, efficacy was very low in plots inoculated with isolates resistant to this fungicide (Fig. 3d). In flutriafol treatments, efficacy was very low in plots inoculated with
Fig. 2. Detection of flutriafol and carbendazim resistance in C. beticola isolates using CAPS. (a) Restriction of C-14 alpha-demethylase product with Alw26I restriction enzyme. Tested isolates were: (1) CB80, (2) CB254, (3) CB538, (4) CB543, (5) CB546, (6) CB591, (7) CB605, (8) CB82, (9) CB587, (10) CB603, (11) CB94, (12) CB152, (13) CB257, (14) CB207, (15) CB322, (16) CB33, (17) CB157, (18) CB369, (19) CB388. M ¼ 100 bp GeneRulerTM, Fermentas, M1 ¼ DNA Ladder, Low Range, GeneRulerTM, Fermentas; (b) Restriction of b-tubulin gene fragment with Bsh1236I. Tested isolates were: (1) CB504, (2) CB538, (3) CB543, (4) CB546, (5) CB605, (6) CB94, (7) CB33, (8) CB254, (9) CB591, (10) CB82, (11) CB257, (12) CB322, (13) CB157, (14) CB587, (15) CB603, (16) CB125. M ¼ 100 bp GeneRulerTM, Fermentas.
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Fig. 3. Effect of carbendazim (Carb), flutriafol (Flutr), azoxystrobin (Azoxy) and tetraconazole (Tetrac) treatments against C. beticola isolates. Tested parameters were (a) AUDPC, (b) sugar beet root yield, (c) sugar content, and (d) fungicide efficacy. Isolates: FRBR e flutriafol-resistant, carbendazim-resistant; FRBS e flutriafol-resistant, carbendazim-sensitive; FSBR e flutriafol-sensitive, carbendazim-resistant; FSBS e flutriafol-sensitive, carbendazim-sensitive. Error bar represents standard deviation. Values (bars) within a graph with the same letter are not significantly different according to Fisher's least significant difference (LSD) test at P ¼ 0.05.
isolates resistant to flutriafol. Azoxystrobin had the most consistent efficacy across all inoculated plots, given that it did not drop below 70% regardless of isolate sensitivity. 4. Discussion The emergence of resistance to carbendazim and flutriafol became a limitation to effective and sustained control of CLS in all sugar beet growing regions in Serbia. Since the early control of CLS is essential for successful disease management, it is necessary to determine whether fungicide resistance occurs and to what extent (Hanson, 2010). Rapid identification of flutriafol and carbendazim resistance can help researchers in examining the potential of different fungicide resistance management practices, as well as in selection of fungicides for use in areas where resistance has occurred. The frequency of isolates resistant to flutriafol detected in laboratory tests was considerable (16%), indicating that development of resistance to flutriafol and other DMIs with a positive crossresistance might soon become one of the major problems in CLS control. Since C. beticola is not known for inoculum movement across wide geographical areas (Bolton et al., 2013), it can be assumed that the resistance developed on several locations independently. All isolates resistant to flutriafol were resistant to carbendazim as well, which is the first report of a double resistance to
fungicides in C. beticola. Almost all tested isolates (96%) were resistant to carbendazim. In our opinion, this is probably due to the fact that benzimidazoles were never abandoned from fungicide spraying schedules, although their application has been significantly reduced after detection of resistance in the region (Mari c et al., 1976). It is very likely that as a result of such a disease management practice, the frequency of benzimidazole-resistant isolates was steadily increasing over time and that nowadays the resistant isolates have become predominant in C. beticola from this sugar beet growing region. This could also be a consequence of a high fitness of the resistant isolates for survival under various fungicide concentrations and temperature regimes (Trkulja et al., 2013). In order to assess a large number of fungal isolates for the occurrence of flutriafol and carbendazim resistance and to avoid pathogen sensitivity in in vitro analysis, CAPS markers were used to distinguish between the sensitive and the resistant isolates. The results confirmed the assumption of Nikou et al. (2009) that a silent mutation at position 169 of the C-14 alpha-demethylase gene, detected by CAPS, can be used as a marker for detection of resistant C. beticola isolates on medium amended with flutriafol. A broad range in sensitivity levels suggests quantitative control of the trait, as the relative mycelial growth ranged from complete inhibition to growth that exceeded the control. Regarding carbendazim
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resistance, mutation of the b-tubulin gene at position 198 was successfully detected by CAPS and enabled differentiation between the sensitive and resistant isolates, as confirmed with other fungal pathogens (Canas-Gutierrez et al., 2006; Chung et al., 2010). In selected isolates, sensitivity levels to carbendazim and, for highly resistant phenotypes, to flutriafol, were detected by CAPS markers. These data completely correlate with the results of in vitro screening, making CAPS markers a useful tool in monitoring C. beticola for fungicide resistance. During the field trials in 2010, fungicide efficacy was relatively low, even in fungicides with known high efficacy (flutriafol, azoxystrobin and tetraconazole), which can be explained by extremely favorable environmental conditions for disease development during July and August in that particular year. Azoxystrobin treatment was highly effective against CLS, probably due to its multiple effects on this pathogen, such as inhibition of spore germination, as well as eradicative and curative effect (Anesiadis et al., 2003; Karaoglanidis and Bardas, 2006). Tetraconazole provided very effective CLS control that resulted in high yield and high sucrose content, as previously described (Khan and Smith, 2005). Although it is known that a cross-resistance between the DMI fungicides exists, various DMIs can have different resistance mechanisms in the target organism (Karaoglanidis and Ioannidis, 2010). In this experiment, tetraconazole had a higher efficacy than flutriafol in controlling flutriafolresistant isolates, which can be explained by a relatively stable sensitivity of C. beticola to tetraconazole (Secor et al., 2010) and a differential fungicide efficacy between members of the DMI group (Karaoglanidis and Ioannidis, 2010). Flutriafol efficacy was very low in plots inoculated with flutriafol-resistant (FRBR) isolates and in plots inoculated with moderately resistant (FRBS) isolates. Efficacy of carbendazim was very low in plots inoculated with isolates resistant to this fungicide, which is in agreement with the qualitative nature of resistance to benzimidazoles. Detection of carbendazim resistance using CAPS and in vitro laboratory tests was also confirmed in field trials. According to Nikou et al. (2009), a mutation in the C-14 alpha-demethylase gene can be used for detection of C. beticola isolates highly resistant to flutriafol. This, however, is not in accordance with the findings from Bolton et al. (2012) that specific polymorphisms in or near the CbCyp51 gene are not associated with an enhanced resistance to DMI fungicides in C. beticola. In our current study, the use of this particular CAPS marker for detection of the abovementioned mutation resulted in a successful differentiation between the isolates with a high level of resistance. Although the CAPS marker for flutriafol resistance is not functionally connected to expression of the resistance itself, the results from our study indicate that it could be a useful tool for detecting resistance to flutriafol in C. beticola isolates from northern Serbia. In summary, the detection and measurement of C. beticola resistance to fungicides prior to actual control failures can help in developing strategies for the prevention of increased fungicide resistance and the successful management of CLS. Acknowledgments Funding for this research was provided by the Ministry of Education and Science, Republic of Serbia, project number TR 31015. The authors wish to thank Dr. Bojana Stanic for her assistance in English language editing. Appendix A. Supplementary data Supplementary data associated with this article can be found in the online version, at http://dx.doi.org/10.1016/j.cropro.2014.09. 010.
References Anesiadis, T., Karaoglanidis, G., Tzavella-Klonari, K., 2003. Protective, curative and eradicant activity of the Strobilurine fungicide azoxystrobin against Cercospora beticola and Erysiphe betae. J. Phytopathol. 151, 647e651. Bala z, F., Stojsin, V., Gavran-Starovi c, M., 1999. Sensitivity of Cercospora beticola Sacc. in Vojvodina to some fungicides. Pesticides 14, 5e13. Bolton, M., Birla, K., Rivera-Varas, V., Rudolf, K., Secor, G., 2012. Characterization of CbCyp51 from field isolated of Cercospora beticola. Phytopathology 102, 298e305. Bolton, M., Rivera, V., Secor, G., 2013. Identification of the G143A mutation associated with QoI resistance in Cercospora beticola field isolates from Michigan, United States. Pest Manag. Sci. 69, 35e39. Brent, K.J., Hollomon, D.J., 2007. Fungicide Resistance in Crop Pathogens: How can it be Managed. Fungicide Resistance Action Committee, Brussels, Belgium. Briere, S.C., Franc, G.D., Kerr, E.D., 2001. Fungicide sensitivity characteristics of Cercospora beticola isolates recovered from the high plains of Colorado, Montana, Nebraska and Wyoming. 1. Benzimidazole and Triphenyltin hydroxide. J. Sugar Beet Res. 38, 111e120. Budakov, D., Nagl, N., Taski-Ajdukovi c, K., Stojsin, V., Bagi, F., 2012. Evaluation of Rapid protocols for DNA isolation from Cercospora beticola Sacc. Field Veg. Crop Res. 49, 202e207. Budakov, D., Stojsin, V., Bagi, F., Jani cijevi c, M., 2010. Managing resistance of Cercospora beticola Sacc. as a basis for integral concept of disease control on sugar beet. In: Kastori, R., Kadar, I., Aleksi c, N., Kozi c, T., Grbi c, M., Ðuranovi c, S., Samsonov, M. (Eds.), Proceedings of XIV International Eco-conference, Novi Sad, Serbia, pp. 169e173. Canas-Gutierrez, G.P., Patino, L.F., Rodriguez-Arango, E., Arango, R., 2006. Molecular characterization of benomyl-resistant isolates of Mycosphaerella fijiensis, collected in Colombia. J. Phytopathol. 154, 403e409. Chung, W.H., Chung, W.C., Peng, M.T., Yang, H.R., Huang, J.W., 2010. Specific detection of benzimidazole resistance in Colletotrichum gloeosporoides from fruit crops by PCR-RFLP. New Biotechnol. 27, 17e24. Dahmen, H., Staub, T., 1992. Protective, curative and eradicant activity of difenoconazole against Venturia inaequalis, Cercospora arachidicola and Alternaria solani. Plant Dis. 76, 774e777. Davidse, L.C., 1986. Benzimidazole fungicides: mechanisms of action and biological impact. Annu. Rev. Phytopathol. 24, 43e65. Davidson, R.M., Hanson, L.E., Franc, G.D., Panella, L., 2006. Analysis of b-tubuline gene fragments from benzimidazole-sensitive and -tolerant Cercospora beticola. J. Phytopathol. 154, 321e328. Georgopoulos, S.G., Dovas, C., 1973. Occurrence of Cercospora beticola strains resistant to benzimidazole fungicides in northern Greece. Plant Dis. Rep. 62, 321e324. Hanson, L., 2010. Genetics of fungicide resistance in Cercospora and Mycosphaerella. In: Lartey, R., Weiland, J.J., Panella, L., Crous, P., Windels, C. (Eds.), Cercospora Leaf Spot of Sugar Beet and Related Species. APS Press, St. Paul, MN, USA, pp. 179e188. Jacobsen, B., Franc, G., 2009. Cercospora leaf spot. In: Harveson, R.M., Hanson, L.E., Hein, G.L. (Eds.), Compendium of Beet Diseases and Pests. APS Press, St. Paul, MN, USA, pp. 7e10. Karaoglanidis, G., Bardas, G., 2006. Control of benzimidazol- and DMI-resistant strains of Cercospora beticola with strobilurin fungicides. Plant Dis. 90, 419e424. Karaoglanidis, G.S., Ioannidis, P., 2010. Fungicide resistance of Cercospora beticola in Europe. In: Lartey, R., Weiland, J.J., Panella, L., Crous, P., Windels, C. (Eds.), Cercospora Leaf Spot of Sugar Beet and Related Species. APS Press, St. Paul, MN, USA, pp. 189e211. Khan, M., Smith, L., 2005. Evaluating Fungicides for controlling of Cercospora leaf spot on sugar beet. Crop Prot. 24, 79e86. Ma, Z., Michailides, T.J., 2005. Advances in understanding molecular mechanisms of fungicide resistance and molecular detection of resistant genotypes in phytopathogenic fungi. Crop Prot. 24, 853e863. Mari c, A., Petrov, M., Masirevi c, S., 1976. Occurrence of tolerance in Cercospora beticola Sacc to benomyl in Yugoslavia and management practice against this pathogen. Zastita bilja 27, 227e236. Nikou, D., Malandrakis, A., Konstantakaki, M., Vontas, J., Markoglou, A., Ziogas, B., 2009. Molecular characterization and detection of overexpressed C-14 alphademethylase-based DMI resistance in Cercospora beticola field isolates. Pestic. Biochem. Phys. 95, 18e27. Secor, G.A., Rivera-Varas, V.V., Gudmestad, N.C., Weiland, J.J., 2010. Sensitivity of Cercospora beticola to foliar fungicides in the Red River Valley of North Dakota and Minnesota. In: Lartey, R., Weiland, J.J., Panella, L., Crous, P., Windels, C. (Eds.), Cercospora Leaf Spot of Sugar Beet and Related Species. APS Press, St. Paul, MN, USA, pp. 213e222. Siegel, M.R., 1981. Sterol-inhibiting fungicides: effects on sterol biosynthesis and sites of action. Plant Dis. 65, 986e989. Pfaf-Dolovac, E., Dolovac, N., Mitrovi Trkulja, N., Ivanovi c, Z., c, P., Tosevski, I., Jovi c, J., 2013. Characterization of benzimidazole resistance of Cercospora beticola in Serbia using PCR-based detection of resistance-associated mutations of the btubulin gene. Eur. J. Plant Pathol. 135, 889e902. Weiland, J.J., Halloin, J.M., 2001. Benzimidazole resistance in Cercospora beticola sampled from sugarbeet fields in Michigan, USA. Can. J. Plant Pathol. 23, 78e82. Windels, C.E., Lamey, H.A., Hilde, D., Widner, J., Knudsen, T., 1998. Cercospora leaf spot model for sugar beet: in practice by the industry. Plant Dis. 82, 716e726. Wolf, P.F.J., Veerret, J.A., 2002. An Integrated Pest Management System in Germany for the control of fungal leaf diseases in sugar beet: the IPM sugar beet model. Plant Dis. 86, 336e344.
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Sensitivity of Cercospora beticola isolates from Serbia to carbendazim and flutriafol Dragana Budakov a, Nevena Nagl b, *, Vera Stojsin a, Ferenc Bagi a, Dario Danojevi c b, cb Oliver T. Neher c, Ksenija Taski-Ajdukovi a b c
Faculty of Agriculture, University of Novi Sad, Trg Dositeja Obradovica 8, 21000 Novi Sad, Serbia Institute of Field and Vegetable Crops, Maksima Gorkog 30, 21000 Novi Sad, Serbia The Amalgamated Sugar Company, 1951 S. Saturn Way, Suite 100, Boise, ID 83709, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 April 2014 Received in revised form 15 September 2014 Accepted 16 September 2014 Available online
Cercospora beticola, causal agent of Cercospora leaf spot (CLS) of sugar beet, is primarily controlled by fungicides. Benzimidazole and demethylation inhibiting fungicides, including carbendazim and flutriafol, have been widely used in Serbia. Since these fungicide groups have a site-specific mode of action, there is a high risk for developing resistance in target organisms, which is the most important limiting factor in Cercospora leaf spot chemical control. A rapid identification of flutriafol and carbendazim resistance can help researchers in examining the potential of different fungicide resistance management practices, as well as in selection of fungicides for use in the areas where resistance has occurred. One hundred singleconidia isolates were collected from 70 representative locations of the sugar beet production region in Serbia. Evaluation of the isolates' sensitivity was based on the reduction of mycelial growth on medium amended with 1.25 mg mL1 flutriafol and 5 mg mL1 carbendazim. Resistance to flutriafol and carbendazim was detected in 16% and 96% of the tested isolates, respectively. All isolates resistant to flutriafol were resistant to carbendazim as well, which is the first report of a double resistance to fungicides in C. beticola. Detection of the isolates resistant to flutriafol and carbendazim using Cleaved Amplified Polymorphic Sequence (CAPS) markers confirmed the results of the in vitro tests. The efficacy of carbendazim, flutriafol, azoxystrobin, and tetraconazole at commercially recommended doses was evaluated in field trials where sugar beet plants in plots were inoculated with a mixture of isolates either sensitive and/or resistant to flutriafol and carbendazim. Carbendazim and flutriafol efficacy was very low in plots inoculated with isolates resistant to these fungicides. Presented results will contribute to development of a pathogen population sensitivity monitoring strategy that could be used for an effective CLS management in the region. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Cercospora leaf spot (CLS) Fungicide resistance Carbendazim Flutriafol CAPS Fungicide efficacy
1. Introduction Cercospora leaf spot (CLS), caused by Cercospora beticola Sacc., is the most important foliar disease of sugar beet in warm and humid environmental conditions that often occur during summer months in the sugar beet growing regions in South-Eastern Europe. In the absence of appropriate control measures in the areas with a high disease incidence, severe epidemics of CLS can result in a significant reduction of root yield, recoverable sugar, sucrose concentration and an increase in impurities leading to higher processing costs
* Corresponding author. Tel.: þ381 (0)21 4898327. E-mail addresses: [email protected] (N. Nagl), [email protected] (K. Taski-Ajdukovi c). http://dx.doi.org/10.1016/j.cropro.2014.09.010 0261-2194/© 2014 Elsevier Ltd. All rights reserved.
(Windels et al., 1998; Jacobsen and Franc, 2009). CLS is primarily controlled by fungicide applications, although it can also be managed by planting disease-tolerant varieties, crop rotation and tillage. In conditions of a high disease pressure, frequent reapplications of the same fungicide are common. This can select for resistance in the target organisms, which is the most important limiting factor for CLS chemical control. Several classes of protective and systemic fungicides with various modes of action are available for use against this disease worldwide. In the West Balkans, benzimidazoles are among the fungicides registered for CLS control, while the sterol demethylation inhibiting (DMI) fungicides have the highest number of registered active ingredients available. Since these fungicide groups have a site-specific mode of action, there is a high risk for developing resistance (Brent and Hollomon, 2007).
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Benzimidazoles act primarily by binding to fungal tubulin and interfering with mitosis and the fungal cytoskeleton (Davidse, 1986). The mechanism of resistance to benzimidazole fungicides in C. beticola is based on a mutation in the b-tubulin gene, which reduces benzimidazole binding (Davidson et al., 2006). Benzimidazole fungicides have been used extensively in the management of CLS, but resistance to them has become a major problem in many sugar beet growing areas (Georgopoulos and Dovas, 1973; Briere et al., 2001; Weiland and Halloin, 2001). Once resistance has developed, the frequency of benzimidazole-resistant strains remains stable or only drops slowly even in the absence of selection pressure (Karaoglanidis and Ioannidis, 2010). Sterol demethylation inhibiting (DMI) fungicides are inhibitors of sterol C-14 alpha-demethylation during ergosterol biosynthesis, which is the main fungal sterol (Siegel, 1981). They represent the most important class of fungicides used to control CLS, providing an excellent protective and curative activity against Cercospora spp. (Dahmen and Staub, 1992). In the United States, a reduction in the performance of DMI fungicides has occurred over time as a result of the presence of C. beticola strains with a reduced sensitivity to DMIs (Secor et al., 2010). Population shift towards DMI resistance was step-wise, which indicates a polygenic control of the resistance (Brent and Hollomon, 2007). Resistance problems with the use of DMIs have been reported in several phytopathogenic fungi and two different resistance mechanisms have been identified: mutations in the C-14 alpha-demethylase (CYP51) gene and overexpression of the CYP51 gene (Ma and Michailides, 2005). It has been suggested that overexpression of the C-14 alpha-demethylase (Cyp51) gene is the mechanism of DMI resistance in C. beticola (Nikou et al., 2009; Bolton et al., 2013). Northern Serbia is a major sugar beet producing region with about 60,000 ha under this crop. Since a reduction in the performance of benzimidazole and some DMI fungicides has been observed (Mari c et al., 1976; Bala z et al., 1999; Budakov et al., 2010), the aims of this study were to: i) perform a sensitivity screening of C. beticola isolated from the sugar beet growing areas in Serbia to benzimidazole and DMI fungicides; ii) develop a CAPS (Cleaved Amplified Polymorphic Sequence) marker for detection of the resistant and sensitive isolates to benzimidazoles; iii) test a
121
previously developed CAPS marker for detection of the DMI resistant isolates; iv) field-evaluate fungicide efficacy on CLS control, caused by the isolates with known sensitivity levels to benzimidazoles and DMIs; and v) evaluate how fungicide resistance determined by in vitro tests and CAPS markers correlates with a reduced disease control in the field. 2. Materials and methods 2.1. Sampling of leaves and collection of fungal isolates Sugar beet leaves with typical symptoms of CLS were collected during the growing season of 2007, from 70 locations in the main sugar beet growing region in Serbia (Fig. 1). A total of 100 isolates were collected, with the number of isolates per location being proportional to the acreage under sugar beet in that area. All sampling sites had a history of benzimidazole and DMI fungicide use, including carbendazim and flutriafol. Collected leaves were washed under tap water and the lesions were cut out and placed in Petri dishes on a wet filter paper. After 24e36 h of incubation at 25e26 C, monosporial isolations were performed after conidia had formed within lesions. Single conidia were lifted with a sterile entomological needle and placed on potato dextrose agar (PDA, Carl Roth GmbH). The cultures were transferred onto PDA slants and kept refrigerated at 4 C until further analysis. 2.2. In vitro sensitivity testing For in vitro fungicide sensitivity tests, we used two commercially available fungicide formulations containing flutriafol and carbendazim as active ingredients: Takt (125 g a.i. L1 flutriafol, Herbos, Croatia) and Galofungin (500 g a.i. L1 carbendazim, Galenika, Serbia). Baseline sensitivities for flutriafol and carbendazim were determined by testing five references C. beticola isolates (CB 581, CB 582, CB 586, CB 587 and CB 588) from beetroot and chard grown in fields in which none of the fungicides examined had been used. In order to test the sensitivity to flutriafol, the concentrations of 0.3, 0.6, 1.25, 2, 4, 8 and 16 mg mL1 were applied in the medium. An average EC50 was 0.8 mg mL1, hence the next higher concentration
Fig. 1. Map of Northern Serbia showing locations where Cercospora beticola isolates were collected.
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(1.25 mg mL1) was a discriminative concentration. The sensitivity to carbendazim was tested with a series of concentrations: 1, 2, 4, 5, 8, 10, 16, 20, 32 and 50 mg mL1. Since none of the tested isolates was suppressed with tested concentrations, a discriminative concentration of 5 mg mL1 was used (Weiland and Halloin, 2001). The autoclaved PDA was amended with fungicide aqueous solutions, while the control Petri dishes contained non-amended PDA. The tests were performed in 90 mm-diameter Petri dishes with 3 mycelial plugs per isolate (5 mm in diameter) turned upside down on the fungicide-amended PDA and the control Petri dishes. Each isolate-fungicide combination was replicated 3 times. A colony diameter was determined by averaging two perpendicular measurements of the diameter after 4 days of incubation at 25e26 C and subtracting the diameter of the mycelial plug. Obtained values were used to calculate the relative growth (RG). Based on the mean RG, the isolates were divided into four groups: i) sensitive (RG < 20%); ii) with a decreased sensitivity (RG ¼ 20e39.9%); iii) moderately resistant (RG ¼ 40e69.9%); and iv) resistant (RG 70%). 2.3. Molecular analysis After the fungicide sensitivity was determined in vitro, 27 isolates with varying degrees of resistance to flutriafol were chosen for further analysis of the C-14 alpha-demethylase gene, while 23 isolates were chosen for analysis of the b-tubulin gene, in order to identify the carbendazim resistance-causing mutation. Prior to DNA extraction, the isolates were grown on PDA for 10 days at 25e26 C, after which 500 mg of fungal tissue was harvested by scraping off mycelia from the agar surface. The DNA was extracted following the procedure described by Budakov et al. (2012). In order to detect a mutation indicating the DMI resistant isolates, specific primers CYP51RT-F (50 -AACTCCAAATTGATGGAGCA30 ) and CYP51RT-R (50 -CGGCTAGCAGTGTAAATGGT-30 ) were used for amplification of the C-14 alpha-demethylase gene fragment (Nikou et al., 2009). Amplification started with an initial denaturation at 94 C for 4 min, followed by 38 cycles of denaturation at 94 C for 40 s, annealing at 52 C for 35 s and extension at 72 C for 45 s. The final extension was at 72 C for 10 min. Primers Bt512F (50 CCAGCTTTTCCGCCCAGACAAC-30 ) and Bt922R (50 -ACGGCACCATG TTCACGGCAAGC-30 ) were used for amplification of the b-tubulin gene fragment (Davidson et al., 2006). The amplification profile was as follows: initial denaturation at 94 C for 4 min, followed by 38 cycles of denaturation at 94 C for 40 s, annealing at 62 C for 40 s and extension at 72 C for 1 min. The final extension lasted 10 min at 72 C. All PCR reactions were carried out in a total volume of 25 mL containing: 30 ng of the DNA template, 2.5 mL of buffer, 0.2 mM of each dNTP, 2.5 mM MgCl2, 2 units of Taq polymerase (Fermentas) and 0.25 mM of each primer (Metabion). The reactions were performed in TPersonal and T1 thermocyclers (Biometra). The PCR products were separated on a 1% agarose gels containing 0.005% ethidium bromide and visualized under UV light. Restriction enzyme Alw26I (BsmAI, Fermentas) was used to detect the mutation in C-14 alpha-demethylase gene (Nikou et al., 2009). After restriction mapping of the b-tubulin gene sequence (Davidson et al., 2006), a mutation responsible for resistance to benzimidazoles at position 198 was detected by digestion with Bsh1236I (BSTUI, Fermentas) restriction enzyme. The PCR products were digested in the manufacturer's reaction buffer and electrophoresed on a 1.7% agarose gel containing 50% high resolution agarose (Carl Roth GmbH). 2.4. Field trials Fungicides containing active ingredients carbendazim and flutriafol were used in field trials to test their efficacy in controlling
the selected C. beticola isolates. In order to test for alternative spraying options in areas with a reported decreased sensitivity to carbendazim and flutriafol, azoxystrobin (Quinone outside Inhibitors-QoIs)- and tetraconazole (DMIs)-based fungicides were also included. Azoxystrobin was selected as a representative of a novel group of fungicides in Serbia that has already been widely used in CLS control worldwide. Tetraconazole was chosen for several reasons: i) it belongs to the same chemical group as flutriafol, but has been reported to have higher efficacy; ii) no crossresistance with flutriafol has been reported yet; and iii) it is registered for use in Serbia as a single ingredient in pesticide formulations. The isolates with known sensitivity levels to flutriafol and carbendazim were selected previously in vitro and used as inoculum for field trials. The inoculum was prepared in the form of a conidial suspension with conidia produced on Sugar Beet Leaf extract Agar (SBLA) (150e200 g of fresh sugar beet leaves in 1 L of distilled water þ 20% Agar). Concentration of the suspension was 500 spores per ml, which was determined by counting spores using a haemocytometer. Field trials were conducted on sugar beet cv. LARA, during the growing seasons of 2010 and 2011, on an experimental field of the Institute of Field and Vegetable Crops, Novi Sad, Serbia, in a completely randomized factorial design with four replicates. In 2010 and 2011, the size of one plot was 12 m2 (3 rows of sugar beet, 8 m long) and 24 m2 (6 rows of sugar beet, 8 m long), respectively. Individual plots were inoculated on July 8th 2010 and July 4th 2011, when the weather conditions were favorable for disease development, using a back-pack sprayer (Solo 456) and 200 L of water per ha. The plots were inoculated with four isolate mixtures, containing the isolates with a similar sensitivity to tested fungicides: a) sensitive to flutriafol and carbendazim (FSBS); b) moderately resistant to flutriafol and sensitive to carbendazim (FRBS); c) resistant to flutriafol and carbendazim (FRBR); and d) sensitive to flutriafol and resistant to carbendazim (FSBR) (Appendix B). Each inoculated plot, except for the non-treated control, was treated throughout the vegetation period with one of the following fungicides: Galofungin (Galenika, Serbia, carbendazim 500 g a.i. L1), Takt (Herbos, Croatia, flutriafol 125 g a.i. L1), Eminent (Isagro, tetraconazole 125 g a.i. L1) and Quadris (Syngenta, azoxystrobin 125 g a.i. L1). The first fungicide treatment was applied seven days after inoculation, with subsequent treatments on August 1st, August 20th and September 4th in 2010 and July 20th, August 3rd and August 16th in 2011. The fungicides were applied using a Solo 456 back-pack sprayer at 400 L ha1 of fungicide mixture. Disease intensity was evaluated four times until harvest on the same dates when the fungicide treatments were applied. Evaluation was based on the percent of leaf infection using a scale from 0 to 9, where 0 represents a healthy plant and 9 is a plant with 100% necrotic leaves. These values were used to calculate the AUDPC (Area Under the Disease Progress Curve) (Wolf and Veerret, 2002). Approximately two weeks after the last treatment in each year, the plants were harvested and the root yield and sugar content were determined in a sugar beet automatic laboratory (Venema Automation BV, The Netherlands). Fungicide efficacy was calculated on the basis of an average penultimate disease intensity evaluation. 2.5. Data analysis Results of the field trials: AUDPC, root yield and sugar concentration were expressed as mean ± standard deviation. Statistical analysis was performed by analysis of variance (ANOVA) using Statistica 12 software (StatSoft, Tulsa, OK, USA). Comparisons between means were made with Fisher's least significant difference (LSD) test, P ¼ 0.05, among the isolates for each fungicide treatment.
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Table 1 Sensitivity of C. beticola isolates and composition of isolate mixtures used in field trials. Isolate
Flutriafol RG (%)
CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB CB
80 254 84 546 72 253 543 551 538 179 591 412 523 605 600 504 82 10 603 3 270 257 152 94 515 322 236 50 382 561 133 317 526 175 74 183 319 188 609 191 316 75 577 44 112 206 20 300 292 567 135 596 165 207 115 125 33 587 157 1 171 173 204 369 388 425 440
113.33 112.30 106.74 106.62 101.82 101.81 99.03 96.77 95.74 92.86 90.16 89.66 87.86 86.00 82.48 75.27 69.75 69.22 68.53 63.25 60.00 53.54 50.00 48.57 47.90 46.75 44.90 32.00 24.42 24.11 23.30 20.10 19.87 19.80 19.74 19.10 19.05 18.75 18.39 17.54 17.14 17.06 16.84 16.67 16.51 16.27 15.48 14.97 14.33 13.89 13.79 12.59 11.67 11.30 10.98 9.87 9.50 9.43 8.33 2.26 0.00 0.00 0.00 0.00 0.00 0.00 0.00
Isolates used in field trials
Carbendazim Sensitivity
RG %
In vitro
CAPS
R R R R R R R R R R R R R R R R MR MR MR MR MR MR MR MR MR MR MR DS DS DS DS DS S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S
R R e R e e R e R e R e e R e R R S S S S S S S S S S e S e S S e e e e e e e e S e e e e e e e e e e e e e e S e S S e e e e S e e e
121.90 102.98 101.00 104.83 98.58 102.72 105.07 106.81 97.59 99.31 106.03 99.43 91.62 86.00 98.60 98.92 105.24 103.26 2.80 96.14 97.50 100.00 112.38 137.14 10.86 94.08 123.88 102.83 93.02 104.56 98.92 97.82 95.96 99.42 97.99 94.24 127.27 102.74 101.53 100.78 2.86 102.78 92.63 104.66 149.21 100.02 77.42 113.26 102.81 102.07 99.30 128.67 91.27 100.33 97.65 0.00 99.01 84.18 94.02 113.56 99.81 93.43 104.65 95.27 98.99 97.05 98.78
Sensitivity In vitro
CAPS
FRBR
R R R R R R R R R R R R R R R R R R S R R R R R S R R R R R R R R R R R R R R R S R R R R R R R R R R R R R R S R R R R R R R R R R R
e R e R e e e e R e R e e R e R R e S R e R R R S R R e e e R R e e e e e e e e S e e e e e e e e e e e e e e S R R R e e e e e R e e
þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ
FRBS
FSBR
FSBS
þ
þ
þ þ
þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ
RG-relative growth; S-Sensitive; DS-Decreased sensitivity; MR-Moderately resistant; R-Resistant; FRBR-resistant to flutriafol and carbendazim; FRBS-moderately resistant to flutriafol and sensitive to carbendazim; FSBR-sensitive to flutriafol and resistant to carbendazim; FSBS-sensitive to flutriafol and carbendazim.
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3. Results 3.1. In vitro sensitivity testing A relative mycelial growth on the medium amended with flutriafol in comparison to the non-amended control ranged from 0% to 113.3% (Table 1). Thirty six percent of isolates were sensitive to flutriafol, 38% had a decreased sensitivity, 10% were moderately resistant and 16% of the tested isolates were resistant to flutriafol. When the medium was amended with carbendazim, 96 isolates had an equal growth on the fungicide-amended and non-amended medium (77.4e149.2%). In four isolates (CB125, CB316, CB515, and CB603), RG was suppressed and varied from 0 to 10.99% on carbendazim-amended medium. After in vitro sensitivity testing, 16 isolates resistant to flutriafol and carbendazim (FRBR), 2 moderately resistant to flutriafol and sensitive to carbendazim (FRBS), 36 isolates sensitive to flutriafol and resistant to carbendazim (FSBR) and 2 sensitive to flutriafol and carbendazim (FSBS) were used for further investigation. 3.2. CAPS markers Eight resistant (CB80, CB254, CB546, CB543, CB538, CB591, CB605, CB504), 11 moderately resistant (CB82, CB10, CB603, CB3, CB257, CB270, CB152, CB94, CB515, CB322, CB236) and 8 isolates sensitive to flutriafol (CB382, CB133, CB317, CB316, CB125, CB587, CB157, CB369) were chosen for further analysis of the C-14 alphademethylase gene using CAPS, while 19 resistant (CB254, CB538, CB546, CB591, CB82, CB152, CB257, CB322, CB33, CB504, CB157, CB388, CB587, CB3, CB133, CB317, CB236, CB605, CB94) and all isolates sensitive (CB125, CB316, CB515, CB603) to carbendazim were chosen for detection of the mutation in the b-tubulin gene responsible for resistance (Table 1). After PCR reactions with C-14 alpha-demethylase gene-specific primers, an amplification product of 200 bp in size was obtained in all isolates. After restriction with Alw26I enzyme, only one band of 200 bp was detected in the resistant isolates (RG 70%) and the moderately resistant isolate CB82 (RG 69.35%), whereas two bands of 80 and 120 bp were detected in the sensitive and other moderately resistant isolates (Fig. 2a). As a result of amplification with the b-tubulin specific
primers, a 570 bp-long PCR product was obtained in all isolates. As expected, digestion of the amplification product with Bsh1236I enabled differentiation between the resistant and the sensitive isolates: three bands were detected in the resistant isolates (250, 200 and 120 bp), whereas there were two bands in the sensitive isolates (450 and 120 bp) (Fig. 2b). 3.3. Field trials Based on the results of the in vitro sensitivity tests and the CAPS marker analysis, C. beticola isolates were used for sugar beet inoculations in field trials aimed at determining fungicide efficacy. In both 2010 and 2011, disease progressed quickly after inoculation due to favorable environmental conditions, i.e. high temperatures and humidity during July and August. The first symptoms were observed seven days after inoculation, at the moment of the first fungicide treatment. Disease symptoms developed uniformly in all plots, with the spots being formed mainly on middle leaves within the rosette. At the second disease evaluation, a black pseudostroma formed within the spots. The disease progressed fastest in nontreated control plots and in plots inoculated with the resistant isolates treated with the fungicides corresponding to the resistance. In those plots, the leaf canopy completely decayed and regrowth occurred by the middle and end of August. In both years, disease intensity (AUDPC) was highest in the non-treated control, while the lowest AUDPC occurred in all inoculated plots treated with azoxystrobin (Fig. 3a). Disease intensity was higher in plots inoculated with the isolates resistant to flutriafol and carbendazim and treated with the fungicides corresponding to the resistance. We did not observe a noticeable pattern in the effect of fungicide treatments on root yield in plots inoculated with the selected isolates (Fig. 3b). Sugar concentration was lowest in plots treated with carbendazim and flutriafol and inoculated with isolates resistant to both fungicides (FRBR), while in 2010 this was also the case in plots inoculated with FRBS isolates and treated with flutriafol (Fig. 3c). In 2010, there were no differences in sugar content in azoxystrobin and tetraconazole treatments, regardless of the sensitivity of the isolates used for inoculation. In carbendazim treatments, efficacy was very low in plots inoculated with isolates resistant to this fungicide (Fig. 3d). In flutriafol treatments, efficacy was very low in plots inoculated with
Fig. 2. Detection of flutriafol and carbendazim resistance in C. beticola isolates using CAPS. (a) Restriction of C-14 alpha-demethylase product with Alw26I restriction enzyme. Tested isolates were: (1) CB80, (2) CB254, (3) CB538, (4) CB543, (5) CB546, (6) CB591, (7) CB605, (8) CB82, (9) CB587, (10) CB603, (11) CB94, (12) CB152, (13) CB257, (14) CB207, (15) CB322, (16) CB33, (17) CB157, (18) CB369, (19) CB388. M ¼ 100 bp GeneRulerTM, Fermentas, M1 ¼ DNA Ladder, Low Range, GeneRulerTM, Fermentas; (b) Restriction of b-tubulin gene fragment with Bsh1236I. Tested isolates were: (1) CB504, (2) CB538, (3) CB543, (4) CB546, (5) CB605, (6) CB94, (7) CB33, (8) CB254, (9) CB591, (10) CB82, (11) CB257, (12) CB322, (13) CB157, (14) CB587, (15) CB603, (16) CB125. M ¼ 100 bp GeneRulerTM, Fermentas.
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Fig. 3. Effect of carbendazim (Carb), flutriafol (Flutr), azoxystrobin (Azoxy) and tetraconazole (Tetrac) treatments against C. beticola isolates. Tested parameters were (a) AUDPC, (b) sugar beet root yield, (c) sugar content, and (d) fungicide efficacy. Isolates: FRBR e flutriafol-resistant, carbendazim-resistant; FRBS e flutriafol-resistant, carbendazim-sensitive; FSBR e flutriafol-sensitive, carbendazim-resistant; FSBS e flutriafol-sensitive, carbendazim-sensitive. Error bar represents standard deviation. Values (bars) within a graph with the same letter are not significantly different according to Fisher's least significant difference (LSD) test at P ¼ 0.05.
isolates resistant to flutriafol. Azoxystrobin had the most consistent efficacy across all inoculated plots, given that it did not drop below 70% regardless of isolate sensitivity. 4. Discussion The emergence of resistance to carbendazim and flutriafol became a limitation to effective and sustained control of CLS in all sugar beet growing regions in Serbia. Since the early control of CLS is essential for successful disease management, it is necessary to determine whether fungicide resistance occurs and to what extent (Hanson, 2010). Rapid identification of flutriafol and carbendazim resistance can help researchers in examining the potential of different fungicide resistance management practices, as well as in selection of fungicides for use in areas where resistance has occurred. The frequency of isolates resistant to flutriafol detected in laboratory tests was considerable (16%), indicating that development of resistance to flutriafol and other DMIs with a positive crossresistance might soon become one of the major problems in CLS control. Since C. beticola is not known for inoculum movement across wide geographical areas (Bolton et al., 2013), it can be assumed that the resistance developed on several locations independently. All isolates resistant to flutriafol were resistant to carbendazim as well, which is the first report of a double resistance to
fungicides in C. beticola. Almost all tested isolates (96%) were resistant to carbendazim. In our opinion, this is probably due to the fact that benzimidazoles were never abandoned from fungicide spraying schedules, although their application has been significantly reduced after detection of resistance in the region (Mari c et al., 1976). It is very likely that as a result of such a disease management practice, the frequency of benzimidazole-resistant isolates was steadily increasing over time and that nowadays the resistant isolates have become predominant in C. beticola from this sugar beet growing region. This could also be a consequence of a high fitness of the resistant isolates for survival under various fungicide concentrations and temperature regimes (Trkulja et al., 2013). In order to assess a large number of fungal isolates for the occurrence of flutriafol and carbendazim resistance and to avoid pathogen sensitivity in in vitro analysis, CAPS markers were used to distinguish between the sensitive and the resistant isolates. The results confirmed the assumption of Nikou et al. (2009) that a silent mutation at position 169 of the C-14 alpha-demethylase gene, detected by CAPS, can be used as a marker for detection of resistant C. beticola isolates on medium amended with flutriafol. A broad range in sensitivity levels suggests quantitative control of the trait, as the relative mycelial growth ranged from complete inhibition to growth that exceeded the control. Regarding carbendazim
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resistance, mutation of the b-tubulin gene at position 198 was successfully detected by CAPS and enabled differentiation between the sensitive and resistant isolates, as confirmed with other fungal pathogens (Canas-Gutierrez et al., 2006; Chung et al., 2010). In selected isolates, sensitivity levels to carbendazim and, for highly resistant phenotypes, to flutriafol, were detected by CAPS markers. These data completely correlate with the results of in vitro screening, making CAPS markers a useful tool in monitoring C. beticola for fungicide resistance. During the field trials in 2010, fungicide efficacy was relatively low, even in fungicides with known high efficacy (flutriafol, azoxystrobin and tetraconazole), which can be explained by extremely favorable environmental conditions for disease development during July and August in that particular year. Azoxystrobin treatment was highly effective against CLS, probably due to its multiple effects on this pathogen, such as inhibition of spore germination, as well as eradicative and curative effect (Anesiadis et al., 2003; Karaoglanidis and Bardas, 2006). Tetraconazole provided very effective CLS control that resulted in high yield and high sucrose content, as previously described (Khan and Smith, 2005). Although it is known that a cross-resistance between the DMI fungicides exists, various DMIs can have different resistance mechanisms in the target organism (Karaoglanidis and Ioannidis, 2010). In this experiment, tetraconazole had a higher efficacy than flutriafol in controlling flutriafolresistant isolates, which can be explained by a relatively stable sensitivity of C. beticola to tetraconazole (Secor et al., 2010) and a differential fungicide efficacy between members of the DMI group (Karaoglanidis and Ioannidis, 2010). Flutriafol efficacy was very low in plots inoculated with flutriafol-resistant (FRBR) isolates and in plots inoculated with moderately resistant (FRBS) isolates. Efficacy of carbendazim was very low in plots inoculated with isolates resistant to this fungicide, which is in agreement with the qualitative nature of resistance to benzimidazoles. Detection of carbendazim resistance using CAPS and in vitro laboratory tests was also confirmed in field trials. According to Nikou et al. (2009), a mutation in the C-14 alpha-demethylase gene can be used for detection of C. beticola isolates highly resistant to flutriafol. This, however, is not in accordance with the findings from Bolton et al. (2012) that specific polymorphisms in or near the CbCyp51 gene are not associated with an enhanced resistance to DMI fungicides in C. beticola. In our current study, the use of this particular CAPS marker for detection of the abovementioned mutation resulted in a successful differentiation between the isolates with a high level of resistance. Although the CAPS marker for flutriafol resistance is not functionally connected to expression of the resistance itself, the results from our study indicate that it could be a useful tool for detecting resistance to flutriafol in C. beticola isolates from northern Serbia. In summary, the detection and measurement of C. beticola resistance to fungicides prior to actual control failures can help in developing strategies for the prevention of increased fungicide resistance and the successful management of CLS. Acknowledgments Funding for this research was provided by the Ministry of Education and Science, Republic of Serbia, project number TR 31015. The authors wish to thank Dr. Bojana Stanic for her assistance in English language editing. Appendix A. Supplementary data Supplementary data associated with this article can be found in the online version, at http://dx.doi.org/10.1016/j.cropro.2014.09. 010.
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