Journal of Plant Pathology (2017), 99 (1), 177-184 Edizioni ETS Pisa, 2017 177

STABILITY OF TETRACONAZOLE-RESISTANT ISOLATES OF BETICOLA AFTER EXPOSURE TO DIFFERENT TEMPERATURE AND TIME TREATMENTS

S. Arabiat1, M.F.R. Khan2, M. Bolton3 and G. Secor1

1 Department of Plant Pathology, North Dakota State University, Fargo, ND, 58108 2 Department of Plant Pathology, North Dakota State University and University of Minnesota, Fargo, ND, 58108 3 United States Department of Agriculture–Agricultural Research Service, Northern Crop Science Laboratory, Fargo, ND, 58102

SUMMARY INTRODUCTION

Cercospora leaf spot caused by Cercospora beticola is Cercospora leaf spot is one of the most destructive fo- one of the most damaging foliar diseases of sugar beet. liar diseases affecting sugar beet ( vulgaris L.) world- The sterol demethylation inhibitor (DMI) fungicide tetra- wide (Holtschulte, 2000; Weiland and Koch, 2004). This conazole is widely-used to manage Cercospora leaf spot. disease is caused by the hemibiotrophic Cercospora However, there has been an increase in prevalence of beticola Sacc. (Saccardo, 1876). Cercospora beticola belongs tetraconzole-resistant isolates in recent years. Knowledge to the phylum , class , order about the stability of tetraconazole resistance in tetracon- , and family . It over- zole-resistant isolates after exposure to cold temperatures winters as pseudostromata and reproduces asexually by in the absence of the selection pressure imposed by tetra- producing conidia (Asher and Hanson, 2006). Favorable conazole application would be important information for conditions for disease development are temperatures from fungicide resistance management. To explore this, we in- 25ºC to 35ºC during the day and above 18ºC at night and oculated sugar beet plants with two known tetraconazole- in high relative humidity (Pool and Mckay, 1916; Weiland sensitive and two known tetraconazole-resistant isolates and Koch, 2004; Khan et al., 2009) Cercospora leaf spot is of C. beticola. Four weeks after inoculation, symptomatic a polycyclic disease and significant crop losses can occur leaves were harvested and subsequently exposed to six dif- under favorable conditions if not managed properly (Shane ferent temperature/time treatments: −20ºC (4 weeks), 4ºC and Teng, 1992). For example, the American Crystal Sugar (4 weeks), 20ºC (4 weeks), −20ºC (2 weeks)/4ºC (2 weeks), Company estimated a loss to Cercospora leaf spot in 1998 −20ºC (1 week)/4ºC (1 week)/−20ºC (1 week)/4ºC (1 week), at $40 million (Ellington et al., 2001). and −20ºC (1 week)/20ºC (1 week)/−20ºC (1 week)/20ºC Disease management practices of Cercospora leaf spot (1 week). Subsequently, spore production, spore germina- include crop rotation, using of resistant cultivars, and tion, radial mycelial growth, sensitivity to tetraconazole, fungicide applications (Jacobsen, 2010; Secor et al., 2010a; and disease severity were evaluated for each isolate and Skaracis et al., 2010). Several fungicide applications may be compared to the control (the same fungal isolates used in needed during the growing season to manage the patho- the inoculation series and maintained on CV8 media). Af- gen in areas where the disease is endemic. In the north ter exposure to all temperature/time treatments, all tested central USA, three to four applications may be needed isolates were found sTable for the parameters evaluated. during the growing season (Secor et al., 2010a). Several However, the tetraconazole-resistant isolate 09-347 after fungicides belonging to different Fungicide Resistant Ac- exposure to −20ºC and −20ºC/4ºC/−20ºC/4ºC treatments tion Committee (FRAC) groups have been registered for became 38.6 and 32.8 times more sensitive to tetracon- use for Cercospora leaf spot management on sugar beet azole, respectively. Taken together, cold temperatures do including members of the dithiocarbamate (FRAC M3), not appear to impart a significant fitness penalty in C. benzimidazole (FRAC 1), triphenyltin hydroxide (TPTH; beticola. FRAC 30), demethylation inhibitor (DMI; FRAC 3), and quinone outside inhibitor (QoI; FRAC 11) (Friskop et al., Keywords: , DMI fungicide, Tetraconazole 2014). Continuous or prolonged usage of fungicides from resistance. the same FRAC group increases the risk of resistance de- velopment. FRAC recommends rotating or mixing fungi- cides from different FRAC groups to manage fungicide resistance (Brent and Hollomon, 2007). Over the past 40 years, C. beticola has developed re- sistance to several fungicide classes, including benzimid- Corresponding author: M.F.R. Khan E-mail: [email protected] azoles (Briere et al., 2001; Campbell et al., 1998; Davidson et al., 2006; Giannopolitis and Chrysayi-Tokousbalides, 178 Tetraconazole resistance of C. beticola Journal of Plant Pathology (2017), 99 (1), 177-184

1980; Ruppel and Scott, 1974), TPTH (Briere et al., 2001; conditions found in sugar beet growing regions in the Bugbee, 1995, 1996; Giannopolitis and Chrysayi-Tokous- northern United States and northern Europe. balides, 1980), DMIs (Karaoglanidis et al., 2000, 2002; Secor et al., 2010b), and QoI fungicides (Birla et al., 2012; Bolton et al., 2013). In other pathosystems, resistance to MATERIALS AND METHODS DMIs was shown to be due to single-site mutations (Wy- and and Brown, 2005), overexpression of the Cyp51gene Cercospora beticola isolates. Four known isolates of (Schnabel and Jones, 2001) or energy-dependent drug ef- C. beticola (collected from North Dakota and Minnesota) flux mechanisms (Nakaune et al., 1998; Palani and Lalitha- were chosen based on sensitivity to tetraconazole (Bolton kumari, 1999). In C. beticola, tetraconazole resistance was et al., 2012a). The isolates were obtained by culturing shown to be associated with overexpression of the Cyp51 single germinated spore on clarified V8-medium (CV8; gene (Bolton et al., 2012a). Indeed, a recent RNA-seq study Bolton et al., 2012). Two isolates, 07-230 and 08-640, had identified most genes in the ergosterol pathway were sig- tetraconzole EC50 (effective concentration that inhibits ra- nificantly induced in both tetraconazole-sensitive and -re- dial mycelial growth by 50%) values of 0.006 μg ml−1 and sistant isolates of C. beticola in response to tetraconazole, 0.008 μg ml−1, respectively and were considered sensitive to but Cyp51 was induced several-fold higher in the resistant tetraconazole. Two isolates, (07-981 and 09-347), had EC50 isolate than the sensitive isolate (Bolton et al., 2016). Cross- values greater than 1 μg ml−1 and were considered resistant resistance is known to occur among the DMIs fungicides to tetraconazole (Bolton et al., 2012a). The isolates were (Karaoglanidis and Thanassoulopoulos, 2002; Karaoglani- confirmed as tetraconazole-sensitive and -resistant based dis et al., 2000, 2002). on radial mycelial growth assay and on Cyp51 gene expres- Fitness can be defined as the survival and reproductive sion (Bolton et al., 2012a). success of an allele, individual, or group (Pringle and Tay- lor, 2002). Fitness of fungicide-resistant isolates plays an Sugar beet plants. Three seeds of C. beticola-suscepti- important role in developing stable resistance to fungicides ble sugar beet variety BTS 89RR10 (Niehaus, 2011) were within a fungal population (Peever and Milgroom, 1994). planted per 15-cm diameter plastic pot (T. O. Plastic Inc., Several studies have been conducted to evaluate the fitness Clearwater, MN, USA) filled with potting mix LC1 (Sun of DMI-resistant C. beticola isolates. For example, DMI- Gro Horticulture Distribution Inc., Agawam, MA, USA). resistant C. beticola isolates have been shown to exhibit After emergence, seedlings were thinned to one seedling reduced virulence and spore production (Karaoglanidis per pot. Plants were fertilized once using Osmocote 15-9- et al., 2001; Moretti et al., 2003), as well as reduced ra- 12 (Everris NA Inc., Dublin, OH, USA). Pots were placed dial mycelial growth (Moretti et al., 2003; Nikou et al., in the greenhouse with a 16-hour photoperiod and an aver- 2009). Other studies have shown that resistant and sensi- age day and night temperature of 24ºC and 16ºC, respec- tive isolates were similar in spore germination (Moretti tively. Plants were watered for optimal plant growth. et al., 2003; Karaoglanidis et al., 2001), mycelial growth, competitive ability, incubation period, germ tube length Inoculum and inoculation. Spores of C. beticola were (Karaoglanidis et al., 2001), spore production (Nikou et produced following the method reported by Secor and al., 2009; Moretti et al., 2003), and disease severity (Bolton Rivera (2012). Suspensions of 10,000 spore ml−1 were pre- et al., 2012b; Nikou et al., 2009). Cold temperature (≤ 5°C) pared for each isolate in water with the aid of a hemocy- have been shown to adversely affect DMI-resistant isolates tometer. Sugar beet plants were inoculated at the 4-leaf of several fungi, including C. beticola (Karaoglanidis and growth stage using a preval spray gun (Preval, Coal City, Thanassoulopoulos, 2002), M. fructicola (Cox et al., 2007; IL, USA). To ensure consistency of inoculation, the first Zhu et al., 2012), and V. inaequalis (Köller et al., 1991). three true leaves were sprayed with the spore suspension Cercospora beticola overwinters in plant debris either until runoff. After inoculation, the pots were placed in incorporated in the soil or on the soil surface (Khan et al., humid chambers with a misting controller (1626D, Phyto- 2008). In some sugar beet production areas such as Fargo, tronics Inc., Earth City, MO, USA) for 10 days. The plants North Dakota, sugar beet infected debris are exposed were misted for 20 seconds every 2 minutes for the first to cold conditions during the winter where the average day and then for 10 seconds every 2 minutes for 9 days, air temperature during winter of 2014 was −13°C with a after which they were moved to the greenhouse and kept maximum air temperature of 6°C and the minimum air at the same conditions described above. temperature was −28°C (https://ndawn.ndsu.nodak.edu). To evaluate if tetraconazole-resistant isolates of C. beticola Temperature/time treatments. Four weeks after inocu- are affected by these cold temperatures, studies were con- lation, the three inoculated leaves from each plant were ducted to evaluate spore production, spore germination, excised, placed in paper bags, and kept at one of six dif- radial mycelial growth, disease severity and sensitivity to ferent temperature/time treatments: −20ºC (4 weeks); 4ºC tetraconazole after exposure to different temperature/ (4 weeks); 20ºC (4 weeks); −20ºC (2 weeks)/4ºC (2 weeks); time treatments that were similar to the environmental −20ºC (1 week)/4ºC (1 week)/−20ºC (1 week)/4ºC (1 week); Journal of Plant Pathology (2017), 99 (1), 177-184 Arabiat et al. 179 and −20ºC (1 week)/ 20ºC (1 week)/−20ºC (1 week)/20ºC (SAS Institute Inc., Cary, NC, USA). The design for all (1 week). Those tested conditions were chosen to mimic experiments was a randomized complete design within the winter weather conditions of North Dakota and Min- each isolate; the factors were temperature/time treat- nesota. After 4 weeks of exposure to temperature/time ment replicates (three sub-isolates per temperature/time treatments, leaves were placed in humid chambers for treatment) and the temperature/time treatments. Two 24 h to induce sporulation. Spores were then collected by replicates were used for each treatment. The experiment adding sterilized Tween-water [0.06% Tween 20 (Sigma- was conducted twice. Aldrich, St. Louis, MO) and 0.02% filter sterilized ampi- To measure disease severity, 4-leaf stage sugar beet cillin (Sigma-Aldrich)] to the leaf surface with a pipette to plants were inoculated using spores harvested from six- liberate the spores from the lesions, which were then trans- day-old cultures as described above. Disease severity was ferred with the same pipette to 2 ml tube. Subsequently, evaluated by counting the number of lesions on the three spores were transferred to water agar (WA) (1.5% wt vol−1) inoculated leaves after 4 weeks. The number of lesions was plates. After 24 h, three germinated spores from each tem- transformed to a category from 1 to 10 using the rating perature/time treatment were transferred to CV8 media scale published by Jones and Windels (1991) and revised and kept at 20 ± 2ºC for 14 days (Secor and Rivera, 2012). by Bolton et al. (2012b). To confirm the causal agent of This resulted in a total of 72 sub-isolates (four C. beticola the symptoms on sugar beet plants, spores were collected isolates X six temperature/time treatments X three sub- from representative lesions, cultured on CV8 media and isolates per temperature/time treatment) that were tem- confirmed as C. beticola based on morphological charac- perature treated and an additional 4 isolates that were teristics (Asher and Hanson, 2006). maintained as their respective controls. For controls, the same fungal isolates used in the inoculation series were Data analysis. The two trials for each experiment were maintained on CV8 (Bolton et al., 2012) at 21ºC in the combined based on a non-significant F-test (Proc ttest, dark). SAS 9.3). Tukey’s test was used to separate between means at α = 0.05 using SAS version 9.3. The non-parametric Stability of tetraconazole-resistant and -sensitive iso- Kruskal-Wallis test was used to analyze the disease severity lates of C. beticola after temperature/time treatments. data. The disease severity median for each pot was calcu- For all 76 isolates, agar plugs (5 mm) from 14-day-old cul- lated, and Proc Rank was used to calculate mean rank us- tures were transferred to CV8 plates and kept in the dark ing SAS 9.3. Using the ranked disease severities, standard at 20 ± 2ºC for 14 days for radial mycelial growth mea- errors and the confidence intervals were calculated for surement. For spore production, agar plugs (5 mm) from each treatment using LD-CI macro to compare between 14-day-old cultures were transferred to CV8 media and different treatments (Shah and Madden, 2004). kept under constant fluorescent light at 20 ± 2ºC. After 14 days, spores were dislodged from the culture surface by adding 2 ml of sterilized Tween-water. Spore concen- RESULTS trations were estimated using a hemocytometer. The ger- mination percentage was determined by placing 100 μl of For each combination of isolate and temperature/time spore suspension on WA media. After 24 h, the number of treatment, three sub-isolates were tested to determine if germinated spores per 100 observed spores was recorded. there was variation within the isolate. Since there was no The germination percentage was calculated using % ger- significant difference (P > 0.05) among the three sub-iso- mination = ((germinated spores) / (germinated spores + lates for each isolate and temperature/time treatment, the non-germinated spores)) × 100. The spores were considered average was reported. germinated if the germ tube length was at least equal to No significant difference (P > 0.05) was found for spore the spore length. production between the mean of control tetraconazole- Tetraconazole sensitivity of C. beticola isolates from sensitive (28,084 spore ml−1) and control tetraconazole-re- different temperature/time treatments in addition to the sistant isolate (29,084 spore ml−1). Spore production from control isolates were tested following the radial mycelial all temperature/time treatments was not significantly dif- growth assay method of Secor and Rivera (2012). Briefly, ferent (P > 0.05) from the control for each isolate. the mean of two perpendicular diameters for each plate For spore germination, no significant difference was calculated. The percentage of mycelium growth reduc- (P > 0.05) was found between mean of tetraconazole-sen- tion relative to the growth in the non-amended media was sitive isolate (99.96%) and tetraconazole-resistant isolate calculated using the following: [100 - (growth diameter in (99.92%). For each isolate, spore germination from all amended media / growth diameter in non-amended me- temperature/time treatments was found not significantly dia) × 100)], and regressed against the fungicide concen- different (P > 0.05) from the control. trations logarithm. The concentration that causes 50% The radial mycelial growth varied significantly among mycelium inhibition was determined by interpolation of C. beticola sub-isolates exposed to different temperature/ the 50% intercept (Russell, 2004) using SAS version 9.3 time treatments. Resistant sub-isolates had similar or 180 Tetraconazole resistance of C. beticola Journal of Plant Pathology (2017), 99 (1), 177-184

−1 Table 1. Effect of six different temperature/time treatments of the control isolate was 8.72 μg ml . The EC50 values de- on radial mycelial growth of two tetraconazole-resistant and creased significantly in two temperature/time treatments, two tetraconazole-sensitive Cercospora beticola isolates. Clari- the −20ºC and −20ºC/4ºC/−20ºC/4ºC, which exhibited fied V8 medium was used. The cultures were kept for 14 days EC values of 0.22 μg ml−1 and 0.26 μg ml−1, respectively. in dark at 20 ± 2ºC. 50 The factor of change (EC50 value for the control/EC50 Radial mycelial growth (cm) value for temperature-time treatment) was 38.6 for the −20ºC treatment and 32.8 for the −20ºC/4ºC/−20ºC/4ºC Temperature/ Tetraconazole-sensitiveb Tetraconazole-resistantc time treatmenta isolate isolate treatment. The control tetraconazole-resistant isolate 07- −1 07-230 08-640 09-347 07-981 981 had an EC50 value of 16.22 μg ml . For 07-981 isolate, 4 4.14bc* 4.63bc 3.19b 3.76ab all sub-isolates from all temperature/time treatments had 20 4.18bc 4.71b 3.27b 3.53bc a factor of change of 1 to 1.3, except for the sub-isolate −20 4.10c 4.80b 4.33a 3.75ab from −20ºC/20ºC/−20ºC/20ºC treatment which had a −20/4 4.47a 4.43c 3.13b 3.88a factor of change of 0.85. The lowest EC50 values were bc bc b c −20/20/−20/20 4.25 4.58 3.22 3.49 12.2 μg ml−1 for −20ºC/4ºC/−20ºC/4ºC and 13.56 μg ml−1 −20/4/−20/4 4.28b 4.63bc 4.43a 3.82a for −20ºC/4ºC, which were significantly different from the Controld 3.65d 5.18a 3.24b 3.65abc EC50 value of the control (Table 2). Control meane 4.41A 3.45B In general, there were variations among disease se- * Means followed by same lowercase letter are not significantly differ- verities for all the sub-isolates exposed to different tem- ent within the column at P ≤ 0.05 according to Tukey’s test. a 4ºC (4 weeks), 20ºC (4 weeks), −20ºC (4 weeks), −20ºC (2 weeks)/4ºC (2 weeks), −20ºC perature/time treatments. Sensitive isolates (07-230 and (1 week)/20ºC (1 week)/−20ºC (1 week)/20ºC (1 week), and −20ºC (1 08-640) exposed to the −20ºC/4ºC/−20ºC/4ºC treatment week)/4ºC (1 week)/−20ºC (1 week)/4ºC (1 week). b tetraconazole-sensitive caused a significant increase in disease severity compared −1 −1 isolates had EC50 values of 0.007 μg ml and 0.008 μg ml for 07-230 and c to the disease severity for the control (Table 3). For sen- 08-640 isolates, respectively. tetraconazole-resistant isolates had EC50 value greater than 1 μg ml−1. d same fungal isolates used in the inoculation sitive isolate 07-230, −20ºC/4ºC treatment resulted in series and maintained on clarified V8-medium.e mean for control isolates significantly higher disease severity than the control followed by the same uppercase letter are not significantly different at (Table 3). For resistant isolate 09-347, all temperature/ P ≤ 0.05. time treatments had disease severity that were not signifi- cantly different from the control except for 20ºC which greater radial mycelial growth after exposure to different had disease severity significantly lower than the control temperature/time treatments compared to their respec- (Table 3). For resistant isolate 07-981, disease severity for tive controls (Table 1). For resistant isolate 09-347, two all treatments were not significantly different from the treatments, −20ºC and −20ºC/4ºC/−20ºC/4ºC, resulted in control (Table 3). significantly higher radial mycelial growth (4.33 cm and 4.43 cm, respectively) compared to the other four tested treatments. For the other temperature/time treatments, DISCUSSION the radial mycelial growth was not significantly different from each other and from the control (Table 1). For tetra- Resistant isolates resulted in similar spore production, conazole-resistant 07-981 isolate, no significant difference spore germination, and disease severity after exposure was found between the control and all temperature/time to six different temperature/time treatments compared treatments (Table 1). The control tetraconazole-sensitive to the control. However, one tetraconazole-resistant iso- isolate 07-230 showed significantly lower radial mycelial late became more sensitive to tetraconazole and showed growth (3.65 cm) than when exposed to different tem- higher radial mycelial growth than the control after cold perature/time treatments, and the radial mycelial growth treatments. from the −20ºC/4ºC treatment was significantly higher There was no significant difference in mean spore pro- than the other temperature/time treatments (Table 1). In duction and mean spore germination between control contrast, the other tetraconazole-sensitive isolate (08-640) tetraconazole-resistant and control tetraconazole-sensi- exhibited significantly higher radial mycelial growth in tive isolates. Similar results were reported for C. beticola the control compared to all temperature/time treatments (Moretti et al., 2003; Nikou et al., 2009) and other patho- (Table 1). The mean radial mycelial growth of the control gens including Monilia fructicola (Cox et al., 2007) and tetraconazole-resistant isolates was 3.45 cm which was sig- Pyrenophora teres (Peever and Milgroom, 1994). Karao- nificantly lower than the control tetraconazole-sensitive glanidis et al. (2001) found that flutriafol-sensitive C. isolates which was 4.41 cm (Table 1). beticola isolates had significantly higher spore production Two C. beticola isolates sensitive to tetraconazole compared to flutriafol-resistant isolates. Different results (07-230 and 08-640) remained sensitive after all tempera- might be due to their evaluation of sporulation in vivo ture/time treatments and their EC50 values were not sig- and not in vitro as was done in this study or because they nificantly different from the control EC50 values (Table 2). used C. beticola flutriafol-resistant isolates and we used For tetraconazole-resistant isolate 09-347, the EC50 value tetraconazole-resistant isolates. Journal of Plant Pathology (2017), 99 (1), 177-184 Arabiat et al. 181

Table 2. Effect of six different temperature/time treatments on tetraconazole sensitivity (EC50; effective concentration that inhibit radial mycelial growth by 50%) of four Cercospora beticola isolates. Clarified V8 medium was used. The cultures were kept for 14 days in dark at 20 ± 2ºC.

−1 EC50 (µg ml ) Temperature/time treatmenta Tetraconazole-sensitive isolate Tetraconazole-resistant isolate 07-230 FCc 08-640 FC 09-347 FC 07-981 FC 4 0.008a* 0.87 0.007b 1.15 9.10b 0.95 14.04bcd 1.16 20 0.008a 0.86 0.007ab 1.11 10.26a 0.84 15.36bc 1.06 −20 0.008a 0.83 0.009a 0.94 0.22d 38.64 15.43bc 1.05 −20/4 0.008a 0.84 0.008ab 0.96 7.83c 1.10 13.59cd 1.19 −20/20/−20/20 0.008a 0.86 0.008ab 1.04 8.61bc 1.00 19.15a 0.85 −20/4/−20/4 0.008a 0.89 0.007ab 1.11 0.26d 32.84 12.20d 1.33 Controlb 0.007a 0.008ab 8.64bc 16.22b * numbers followed by same letter are not significantly different within a column at P ≤ 0.05 according to Tukey’s test. a 4ºC (4 weeks), 20ºC (4 weeks), −20ºC (4 weeks), −20ºC (2 weeks)/4ºC (2 weeks), −20ºC (1 week)/20ºC (1 week)/−20ºC (1 week)/20ºC (1 week), and −20ºC (1 week)/4ºC (1 week)/−20ºC (1 b c week)/4ºC (1 week). same fungal isolates used in the inoculation series and maintained on clarified V8-medium. factor of change= EC50 value of control isolate / EC50 value of isolates from different temperature treatments.

Radial mycelial growth varied among isolates; control weeks at different temperature/time treatments which may tetraconazole-resistant isolates had the same or lower ra- not have been enough to cause a pronounced decrease in dial mycelial growth compared to control tetraconazole- the EC50 values as occurred in other studies where dif- sensitive isolates. Karaoglanidis et al. (2001) found similar ferent fungal pathogens were incubated for 3 months variation in radial mycelial growth of C. beticola. However, and longer (Karaoglanidis and Thanassoulopoulos, 2002; for the mean radial mycelial growth of the control isolates, Köller et al., 1991). Cox et al. (2007) found that in M. fructi- the tetraconazole-resistant isolates had significantly lower cola the percentage of growth inhibition at the discrimina- radial mycelial growth compared to tetraconazole-sensitive tory dose of 0.3 μg ml−1 increased by 165% after 8 months isolates, which was also found by Moretti et al. (2003). In of incubation at 5ºC and by 273% after 34 months at the contrast, Nikou et al. (2009) and Karaoglanidis et al. (2001) same temperature. The instability of sensitivity to tetracon- found that mean radial mycelial growth was not signifi- azole after 4 weeks of continuous exposure to −20ºC and cantly different between flutriafol-resistant and flutriafol- weekly exposure to −20ºC followed by 4ºC for a total of 4 sensitive isolates which could be a result of the difference weeks in C. beticola could be important because in areas in C. beticola strains evaluated. Also the variation could such as North Dakota and Minnesota with low tempera- be due to difference in research methodologies. In this ture winters could possibly result in tetraconazole-resistant study we used 5 mm plugs, CV8 media, and 14-day-old population reverting to sensitive again which could then cultures while Nikou et al. (2009) used 2 mm mycelium be effectively controlled with fungicides, including DMI plugs, Aspergillus complete medium and 5-day-old cul- fungicides. To study if this phenomenon occurs in nature, tures and Karaoglanidis et al. (2001) used liquid malt me- samples of C. beticola should be collected from areas with dium. Temperature/time treatments had different effects known tetraconazole resistance (Secor et al., 2010b) at on radial mycelial growth of tetraconazole-resistant and the end of one growing season and early and late in the -sensitive isolates. The most pronounced effect was on the subsequent growing season and evaluated to determine if resistant isolate 09-346 where two treatments [−20ºC and overwintering low temperatures impact the frequency of −20ºC /4ºC/−20ºC/4ºC] resulted in significantly higher tetraconazole-resistant isolates. radial mycelial growth than the control. Instability of isolates to maintain fungicide resistance Resistance to DMIs was found unsTable after exposing has been reported for other fungicides beside DMIs. For resistant isolates to cold treatments in pathogens such as example, resistance of C. beticola to triphenlytin hydroxide V. ineaqualis (Köller et al., 1991), and M. fructicola (Cox (TPTH) was found unstable in North Dakota and Min- et al., 2007; Zhu et al., 2012). The instability of resistance nesota, and the resistant population reverted to sensitive was also found in the current study where two treatments again (Secor et al., 2010b). The instability was explained (−20ºC and −20ºC/4ºC/−20ºC/4ºC) impacted the 09-347 by the reduction in TPTH use and concomitant expo- isolate and resulted in an increase in sensitivity to tetracon- sure to fungicides with different mode of action, includ- azole. For the other resistant isolate (07-981), −20ºC/4ºC ing QoI and DMI, which were used in most areas instead and −20ºC/4ºC/−20ºC/4ºC treaments also caused increase of TPTH, the lack of fitness of resistant isolates, and the in tetraconazole sensitivity. Instability of DMI-resistant iso- inability of resistant isolates to survive the adverse win- lates was also reported for C. beticola from Greece (Karao- ter conditions (Secor et al., 2010b). However, no study glanidis et al., 2002; Karaoglanidis and Thanassoulopou- has been conducted to determine if the reversion back los, 2002). In the current study, isolates were kept for 4 to sensitivity to TPTH was a result of cold conditions. 182 Tetraconazole resistance of C. beticola Journal of Plant Pathology (2017), 99 (1), 177-184

Table 3. Effect of temperature/time treatments on disease severity caused by four known Cercospora beticola isolates. Inoculated sugar beet (BTS 89RR10) plants were kept in the greenhouse with 16-hour photoperiod, 24ºC day temperature, and 16ºC night temperature for 4 weeks. Non-parametric analysis was used to analyze the data.

a Temperature/ 95% CI of the disease severity Isolate b Disease Median Disease Severity Rank time treatment Lower limit Upper limit 07−230 Controlc 4.0 0.32 0.21 0.45 4 4.7 0.52 0.40 0.64 20 4.8 0.50 0.40 0.61 −20 4.8 0.52 0.39 0.64 −20/4 4.9 0.56 0.47 0.64 −20/20/−20/20 4.4 0.41 0.30 0.54 −20/4/−20/4 5.0 0.67 0.58 0.75 08−640 Control 4.0 0.48 0.39 0.57 4 4.5 0.38 0.32 0.44 20 5.0 0.62 0.50 0.72 −20 5.0 0.34 0.27 0.41 −20/4 5.0 0.47 0.34 0.61 −20/20/−20/20 4.0 0.49 0.38 0.60 −20/4/−20/4 5.0 0.73 0.63 0.80 07−981 Control 6.0 0.52 0.42 0.62 4 5.5 0.39 0.28 0.52 20 6.0 0.52 0.38 0.66 −20 6.0 0.55 0.45 0.64 −20/4 6.0 0.47 0.37 0.58 −20/20/−20/20 6.0 0.47 0.37 0.58 −20/4/−20/4 6.0 0.57 0.48 0.66 09−347 Control 4.5 0.59 0.47 0.70 4 5.0 0.66 0.51 0.77 20 4.0 0.33 0.23 0.46 −20 4.0 0.47 0.39 0.57 −20/4 4.0 0.38 0.29 0.49 −20/20/−20/20 4.0 0.52 0.43 0.61 −20/4/−20/4 4.5 0.54 0.45 0.63 a 95% confidence intervals of disease severity rank.b 4ºC (4 weeks), 20ºC (4 weeks), −20ºC (4 weeks), −20ºC (2 weeks)/4ºC (2 weeks), −20ºC (1 week)/20ºC (1 week)/−20ºC (1 week)/20ºC (1 week), and −20ºC (1 week)/4ºC (1 week)/−20ºC (1 week)/4ºC (1 week). c same fungal isolates used in the inoculation series and maintained on clarified V8-medium.

Metalaxyl-resistant isolates of the oomycete pathogen Phy- mycelial growth and disease severity of tetraconazole-re- tophthora infestans were found less frequently than sensi- sistant isolates between before and after exposing them tive isolates at the beginning of the growing season, which to different temperature/time treatments for any fungal was explained by the adverse effect of overwintering on pathogen. the survival of the pathogen (Kadish and Cohen, 1992). Because of space limitation in the greenhouse and the This adverse effect of overwintering on the P. infestans number of isolates we had after temperature/time treat- survival was confirmed in the laboratory by exposing resis- ments (72 sub-isolates and 4 isolates) we were able to study tant and sensitive isolates to cold temperature and looking only four C. beticola isolates. Future research with more to fitness parameters which showed that resistant isolates isolates will be necessary to draw a stronger conclusion, did not survive the cold temperature (Kadish and Cohen, but none the less we did see unsTable resistance in a single 1992). In contrast, our experiment showed that C. beticola isolate among four, suggesting that it may be a common resistant isolates had the same level of survivability as sen- phenomenon. sitive isolates, but cold treatment may affect the stability Cercospora beticola isolates resistant to tetraconazole of DMI resistance. had similar or greater spore production, spore germina- In the greenhouse, all C. beticola isolates were able to tion, radial mycelial growth, and disease severity after cause disease symptoms on sugar beet plants after expo- exposure to the tested cold temperatures. However, re- sure to different temperature/time treatments. The tem- sistance to tetraconazole was unstable, and low tempera- perature/time treatments effects varied among the isolates, ture during winter in sugar beet production areas such and no significant difference was found in resistant isolates as North Dakota and Minnesota may significantly impact after exposure to different temperature/time treatments. DMI-resistant population which could have an important To our knowledge, no previous studies have been reported role in fungicide resistance management. Even though comparing the spore production, spore germination, radial one tetraconazole-resistant isolate became sensitive to Journal of Plant Pathology (2017), 99 (1), 177-184 Arabiat et al. 183 tetraconazole after exposure to cold temperatures, some Campbell L.G., Smith G.A., Lamey H.A., Cattanach A.W., resistant isolates may still survive. Based on these results, 1998. Cercospora beticola tolerant to triphenyltin hydroxide if this phenomenon occurs in the field, it may be prudent and resistant to thiophanate methyl in North Dakota and to not use DMI fungicides in the early applications but Minnesota. Journal of Sugar Beet Research 35: 29-41. instead use other chemistries with the aim of significantly Cox K.D., Bryson P.K., Schnabel G., 2007. Instability of propi- reducing any surviving overwintering DMI-resistant popu- conazole resistance and fitness in Monilinia fructicola. Phy- lation so as to prolong the usefulness of DMI fungicides topathology 97: 448-453. for controlling C. beticola. Crous P.W., Kang J.C., Braun U., 2001. A phylogenetic redefini- tion of anamorph genera in Mycosphaerella based on ITS rDNA sequence and morphology. Mycologia 93: 1081-1101. Davidson R.M., Hanson L.E., Franc G.D., Panella L., 2006. ACKNOWLEDGEMENTS Analysis of β-tubulin gene fragments from benzimidazole- sensitive and -tolerant Cercospora beticola. Journal of Phyto- Thanks to the Sugarbeet Research and Education pathology 154: 321-328. Board of Minnesota and North Dakota and the Western Ellington R.L., Cattanach A.W., Weiland J.J., 2001. A Cerco- Sugar Cooperative for partial funding of this research, spora leaf spot management program for American Crystal Vivianna Rivera-Varas for providing Cercospora beticola Sugar Company growers in 1999-2000. Resource document. isolates, and Yangxi Liu for his technical support. American Society of Sugar Beet Technologists 31: 100-112. Friskop A., Markell S., Khan M., 2014. 2014 North Dakota Field Crop Plant Disease Management Guide. North Da- REFERENCES kota State University Extension Service, Fargo, North Da- kota. Asher M.J.C., Hanson L.E., 2006. Fungal and bacterial dis- Giannopolitis C.N., Chrysayi-Tokousbalides M., 1980. Biology eases. In: Draycott A.P. (ed.). Sugar Beet, pp. 286-315. 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Received July 25, 2016 Accepted September 19, 2016