Sensitivity of Cercospora beticola to fungicides in Slovakia

Sugar beet belongs to traditional crops in Europe. In Slovakia, it is mainly grown as a technical crop for the sugar industry. Sugar beet is currently grown on about 22 000 hectares in Slovakia. The farmers accepted the offer of sugar companies to grown more sugar beets, which helped to stabilize sugar production to satisfy consumption in Slovakia (Černý et al., 2019). An important factor in growing of sugar beet is the control of diseases and pests (Almquist et al., 2016; Černý et al., 2018). The already emerging plants may be threatened by pests such as wireworms (Elateridae), mangold flea beatle or brassy flea beatle (Chaetocnema concinna, Ch. tibialis), beet tortoise beetle (Cassida nebulosi) (Hajyieva and Soroka, 2008) and diseases such as damping-off sedlings (Phoma sp., Pythium sp., Aphanomyces sp., Fusarium sp., Rhizoctonia sp.). Damage by foliar pathogens lead to reduction of assimilative leaves surface. The significant leaf pathogens are Cercospora beticola, Erysiphe betae, Uromyces betae and Alternaria alternata (Mahmound, 2016; Mahlein et al., 2012; Hudec and Roháčik, 2002). Cercospora beticola causes Cercospora leaf spot disease, which is the most important disease worldwide (Tedford et al., 2017). Cercospora beticola is a necrotrophic fungus that uses C. beticola toxin (CBT) to kill infected plants. CBT causes the typical symptoms of leaf spots and prevents root formation. Cercospora leaf spot is economy problem for growers (Khan and Khan, 2009), because it causes decrease of assimilate transport to root. The result of disease damage is lower yield and sugar quality and high storage rots (Harveson and Bolton, 2013).


Introduction
Sugar beet belongs to traditional crops in Europe. In Slovakia, it is mainly grown as a technical crop for the sugar industry. Sugar beet is currently grown on about 22 000 hectares in Slovakia. The farmers accepted the offer of sugar companies to grown more sugar beets, which helped to stabilize sugar production to satisfy consumption in Slovakia (Černý et al., 2019). An important factor in growing of sugar beet is the control of diseases and pests (Almquist et al., 2016;Černý et al., 2018). The already emerging plants may be threatened by pests such as wireworms (Elateridae), mangold flea beatle or brassy flea beatle (Chaetocnema concinna, Ch. tibialis), beet tortoise beetle (Cassida nebulosi) (Hajyieva and Soroka, 2008) and diseases such as damping-off sedlings (Phoma sp., Pythium sp., Aphanomyces sp., Fusarium sp., Rhizoctonia sp.). Damage by foliar pathogens lead to reduction of assimilative leaves surface. The significant leaf pathogens are Cercospora beticola, Erysiphe betae, Uromyces betae and Alternaria alternata (Mahmound, 2016;Mahlein et al., 2012;Hudec and Roháčik, 2002). Cercospora beticola causes Cercospora leaf spot disease, which is the most important disease worldwide (Tedford et al., 2017). Cercospora beticola is a necrotrophic fungus that uses C. beticola toxin (CBT) to kill infected plants. CBT causes the typical symptoms of leaf spots and prevents root formation. Cercospora leaf spot is economy problem for growers (Khan and Khan, 2009), because it causes decrease of assimilate transport to root. The result of disease damage is lower yield and sugar quality and high storage rots (Harveson and Bolton, 2013).
The term "resistance of pathogens to fungicides" is a phenomenon in chemical control in recent years. The term is used by Fungicide resistance action committee; it means gained and heritable reduction in susceptibility of a fungus to fungicide (FRAC, 2017). Fungal resistance represents a serious problem for farmers and may causes significant damage to crops (Setiawan et al., 2000). The terms "reduced sensitivity" or "tolerance" refer for lower fungicide efficacy, while term "resistance" indicates total loss of fungicidal efficacy against pathogens (FRAC, 2016). Resistance of C. beticola in sugar beet seems to be controlled by 4-5 pairs of genes with an additive action (Smith and Gaskill, 1970); their expression strongly interacts (66%) with the environment (Van den Bosch et al., 2011).
Fungicidal resistance seems to be actual problem in agricultural practice (Budakov et al., 2014). High level of resistance of C. beticola isolates to benzimidazoles (93.3-98.6%) was reported in Serbia, whereas 6.2-42.4% of isolates were resistant to demethylation inhibitors (DMI) fungicides (Trkulja et al., 2015). Pathogen strain CbCyp51 induced several-fold higher in C. beticola DMi-resistant strain than C. beticola wild type (Bolton et al., 2016). Resistance to azoxystrobin (QoI) was found out in 41% isolates of C. beticola, with higher EC 50 value than 0.2 μg ml -1 in New York. The mutation of G143A was identified in these isolates, which is known as a cause of resistance to QoI fungicides (Vaghefi et al., 2016).
This study is focused on monitoring of Cercospora beticola sensitivity to the most frequently used fungicides, authorised against Cercospora leaf spot in Slovakia. The work is based on hypothesis of possible different fungicide sensitivity in C. beticola population, which could results to diverse risk of fungicide resistance in certain localities of Slovakia.

Material and methods
Cercospora beticola isolates were obtained from infected sugar beet leaves collected at the end of vegetation (Shrestha et al., 2017) from several localities of Slovakia. The localities and their characteristics are shown in Table 1. The fragments of leaves with pathogen sporulation spots were cut and put into petri dishes with potato dextrose agar (PDA) (Aggarwal et al., 2014). The petri dishes were incubated for 1-2 days in laboratory conditions. Isolates from spores were obtained by transferring the individual germinating conidia by a sterile needle to a nutrient medium. The isolates were determined microscopically according producing of typical conidia (Trkulja et al., 2013). Isolates were incubated at 25 °C and photoperiods 12/12, which are optimal conditions for growing of Cercospora beticola (Forsyth, 1963). After 14 days of incubation, each isolate was inoculated into three petri dishes containing PDA and incubated under the same temperature and light conditions.

Test of C. beticola sensitivity to fungicides
Sensitivity test of C. beticola to fungicides was performed with several concentrations for each fungicide to determine the EC 50 (EC 50 = half maximal effective concentration refers to the concentration of fungicide, which induces a response halfway the baseline and maximum) (Karaoglanidis and Thanassoulopoulos, 2003). Concentration of each active ingredient was based on recommended dose per hectare by the fungicide manufacturers, diluted in 200, 400 and 1000 liters of (spraying) water per hectare. The fungicides represented commercial formulations of those authorised against Cercospora leaf spot in Slovakia: trifloxystrobin + cyproconazole (Sfera 525 SC), kresoxym-methyl + epoxiconazole (Juwel), azoxystrobin + cyproconazole (Amistar Xtra), thiophanate-methyl + tetraconazole (Yamato), thiophanate-methyl (Topsin 500 SC), prochloraz + propiconazole (Bumper Super), picoxystrobin (Acanto), tetraconazole (Eminent 125ME), and difenoconazole (Score). Fungicides were aseptically added to the sterile medium prior to inoculation until the agar was still liquid. Sensitivity to fungicides was tested by inoculating 5 mm fragment of pathogens strain, removed from the mycelium edge of 14 days old culture. The fragment was upside down transformed into Petri Dishes with PDA (Karaoglanidis et al, 2002;Russell, 2004). The effect of fungicides and concentrations on mycelial growth was determined by measuring the diameter of colonies mycelium after 14 days (Malandrakis et al., 2006). The percent inhibition (PI) of each fungicide was calculated by following formula (Tumbek et al., 2011): where: PI -percentage of inhibition a -average diameter of the nontreated (check) sample colony b -average diameter of the treated sample PI of the least 3 concentrations for each fungicide and each strain were subjected to regression analysis against the decadic logarithm of the fungicide dose to determination the EC 50 value using by MS Excel. Differences between isolates and regions were determined by analysing the PI value for all doses by analysis of variance (ANOVA), at P = 0.05 to expressed statistically significant differences between fungicides and localities (Gaurilčikienė et al., 2006). Fungi-toxic curve was created from the results as the relationship between relative growth (RR = average of the treated sample colony/average of the control colony × 100) and fungicide concentration. Petri dishes with an equivalent amount of agar without fungicide were used as control (check) samples (Malandrakis et al., 2006). The concentrations of the active ingredients used in the tests are given in Table 2.

Results and discussion
Results of sensitivity test of Cercospora beticola population based on the mean of PI values showed that the mean PI value of thiophanate-methyl for each concentration achieved less than 27% for 6 of 10 localities. All the isolates from Mojmírovce, Hronovce, Senec, and Oslany localities were not able to grow on thiophanate-methyl concentration from 350 to 1750 µg ml -1 (Table 3). Thiophanate-methyl as a single fungicide failed to protect against C. beticola, because the combination of thiophanate-methyl +  (Table 4). The fungicide achieved great results, IP ranged from 83.83 to 100% at all isolates (Table 5). The high sensitivity of C. beticola isolates was observed also in single-site fungicide with the active ingredient tetraconazole, PI ranged from 81.18 to 93.63% (Table 4). PI values of tetraconazole were only  * concentration of active ingredient in the PDA (µg ml -1 ); ** PIpercentage of inhibition (%); *** the differences between the values marked with the same letters in the column are not statistically significant, LSD test, P = 0.05 * concentration of active ingredient in the PDA (µg ml -1 ); ** PIpercentage of inhibition (%); *** The differences between the values marked with the same letters in the column are not statistically significant, LSD test, P = 0.05 slightly lower than for multi-site fungicide prochloraz + propiconazole.
According to work of Karaoglanidis and Thanassoulopoulos (2003), isolates was categorized to 3 groups based on growth rate. These categories of results are presented in table 6. Percentage of categorized isolates presents table 7. The most numbers of sensitive isolates were observed to single-site fungicide tetraconazole (100%) and to multi-site fungicide prochloraz + propiconazole (100%). Resistant strains of C. beticola were recorded to all single-site fungicides, except tetraconazole. High percentage of susceptible strains was observed by using of thiophanate-methyl + tetraconazole (90% sensitive, 10% reduced sensitivity). No resistant strains were confirmed to multi-site fungicides. The most numbers of resistant isolates were observed by using of thiophanate methyl (60%), slightly lower numbers of resistant isolates were observed by picoxystrobin (40%). For difenoconazole, it was 80% of isolates determined as "resistant" and 10% as "reduced sensitivity".
EC 50 values for the isolates were calculated by regression analysis from fungicide PI values. Application of multisite fungicides kresoxim-methyl + epoxiconazole provided excellent inhibitory effect for all of isolates with average EC 50 value 6.10E-05 ppm. All multi-site fungicides (trifloxystrobin + cyproconazole, kresoximmethyl + epoxiconazole, azoxystrobin + cyproconazole, thiophanate methyl + tetraconazole, prochloraz + propiconazole) achieved average EC 50 value <5 ppm. The highest average EC 50 value was achieved by picoxystrobin (3,622.70 ppm). Average EC 50 value for the singlesite fungicides (thiophanate methyl, picoxystrobin, tetraconazole, difenoconazole), except tetraconazole, varied from 618.71 (difenoconazole) to 3,622.7 ppm (picoxystrobin). Average EC 50 for tetraconazole achieved 14.03 ppm ( Table 6). Ppm of recommended dose of active ingredients in field conditions is compared with EC 50 values of active ingredients established by laboratory assay (Table 7). Laboratory EC 50 values of picoxystrobin, thiophanate-methyl and difenoconazole were several times higher than ppm of recommended concentration for field conditions. Based on EC 50 values, the isolates of C. beticola were sorted into three categories -sensitive, medium sensitive and resistant (Giannopolitis, 1978). All the tested isolates were categorized as sensitive to multi-site fungicides. 20% of isolates were resistant to picoxystrobin according EC 50 , while according growth rate, it was 40% of isolates.
Among tested sites, isolates from the Oslany site were categorized as very susceptible to all active ingredient, all isolates achieved EC 50 value ≤0.06 µg ml -1 . Sensitivity of Cercospora beticola isolates to thiophanate-methyl from localities Dolné Saliby, Bolešov, Nižná, Horné Chlebany, Senica, and Nové Zámky was different significantly compared with isolates from localities Oslany, Mojmírovce, Hronovce and Senec. The highest EC 50 value to thiophanate-methyl was observed on isolates from Horné Chlebany (2,804.82 µg ml -1 ). EC 50 value to picoxystrobin varied from <0.01 to 16.71 µg ml -1 . Sensitivity of isolates to fungicides from each locality had compared each other on base EC 50 values, statistically significant difference was found out to three active ingredients -thiophanatemethyl, picoxystrobin and difenoconazole ( Table 6).
Assessment of resistance to fungicides, based on the growth rate of isolates is considered as a good indicator (Karaoglanidis and Thanassoulopoulos, 2003). Results

Table 6
Numbers of the Cercospora beticola isolates sorted into 3 categories according different criteria * percentage categories based on daily growth, sensitive (S) -no growth; reduced sensitivity (RS) = <2 mm growth per day; resistant (R) = >2 mm per day ; ** average of EC 50 value of isolates to active ingredients; *** concentration of active ingredients in application dose in field conditions; **** percentage categories based on EC 50 value, S -sensitive (<0.5), MS -medium sensitive (0.5-5.0), R -resistant (>5.0) (Giannopolitis, 1978) of this work showed, that 60% of tested isolates was resistant to thiophanate-methyl, based on growth rate. Resistance to thiophanate-methyl was not confirmed in all of the tested localities. Isolates from Mojmírovce, Senec, Hronovce and Oslany were categorized as very sensitive to thiophanate-methyl. This could be caused by agrotechnical measures, anti-resistance strategy or by low frequency of using thiophanate-methyl. According Karaoglanidis and Thanassoulopoulos (2003), the level of resistant strain can be low without using benzimidazole, but frequency of occurrence of resistant strains to benzimidazoles is increasing with use of benzimidazoles on field conditions. Results of Trkulja et al. (2013) study showed that there was no difference in occurrence or frequency of resistant isolates between use and non-use of benzimidazoles fungicides (Trkulja et al., 2013). In our results, the significant difference was found between isolates from localities, where the thiophanate-methyl was used for several years ago (Horné Chlebany, Senica, Bolešov) and those with a very low thiophanate-methyl use (Oslany, Hronovce, and Mojmírovce).
Development of resistant strains could by a consequence of higher using of benzimidazole fungicides. Consumption of benzimidazoles in Slovakia was 35425 litres in 2015, which is 10% more than in 2014 (UKSUP, 2016). Resistance to benzimidazole is caused by mutation at codon 198 in the β-tubulin (Davidson et al., 2006). Results of Groenewald's (2008) study showed high genetic variability of Cercospora beticola isolates. According to FRAC (2017), thiophanate-methyl belongs to the group of "high risk for resistance". One of the reasons of high risk for resistance could be a fact that thiophanate-methyl was first registered in 1971 (general information) yet. On the other side, the combination of thiophanate-methyl + tetraconazole achieved excellent results. It could be caused by mixture by other active ingredient -tetraconazole, because it recorded 100% of sensitive isolates to single tetraconazole in our study.
Laboratory results in this study confirmed the presence of resistant C. beticola strains to active ingredient picoxystrobin. Picoxystrobin is classified in "high risk for resistance" group, because of their specific mode of action (Grasso et al., 2006;FRAC, 2017). According to Karaoglanidis and Thanassoulopoulos (2003) classification, 40% of tested isolates in our work were categorized as resistant, but according to Giannopolitis (1978) classification, only 20% of our tested isolates were resistant. Diameter of untreated colony for picoxystrobin was slightly smaller than other check colonies. That could be reason of difference in the results. Lower EC 50 value than 0.01 µg ml -1 to picoxystrobin was observed on 3 isolates only (Bolešov, H. Chlebany and Senica). It had the lowest number of isolates with EC 50 value lower than 0.01 µg ml -1 among all tested fungicides.
Quinone outside inhibitors (QoI) fungicides is good combinable with demethylation inhibitors (DMi) fungicides in anti-resistant strategy against Cercospora leaf spot (CLS) (Karadimos and Karaoglanidis 2006). Increase of resistance C. beticola strain to QoI fungicides in Poland was delayed (Brila et al., 2012). It could be result of low frequent using of chemical control in their climate (Piszczek et al., 2017). DMi fungicides are known for their broad-spectral fungicide, curative and protective effect (Bolton et al., 2012), and have been using more than 20 years and still have a sufficient effect (Nikou et al., 2009).  Karaoglanidis and Thanassoulopoulos (2003), based on EC 50 was slight stricter. From Trkulja study (2015) followed, that 20% of resistance isolates to DMI were also resistance to methyl benzimidazole carbamates (MBC). In this study, the cross resistance between DMi and MBC fungicides was not confirmed.
The highest inhibitory effect against C. beticola isolates was demonstrated by using of prochloraz + propiconazole.
Average PI value of prochloraz + propiconazole varied from 90.42 to 96.02%. EC 50 value achieved <0.01 µg.ml -1 on all of tested localities. Both active ingredients belong to the group of DMi fungicides, and by FRAC were classified as "medium risk". According to Karaoglanidis and Karadimos (2003), all single-site fungicides had reduced efficacy.
Monitoring of sensitivity of C. beticola to fungicides can be an excellent tool to determine the development of C. beticola resistance and effective recommendations for sugar beet production areas (Kirk et al., 2012). In this work, the hypothesis of possible different fungicide sensitivity in C. beticola population was confirmed. According to the results, the risk of fungicide resistance is different in certain localities of Slovakia.

Conclusions
Cercospora beticola is a pathogen with high risk of developing of resistance. Occurrence of fungicide resistance in C. beticola population was confirmed in Slovakia. Resistant strains were confirmed for three (thiophanate-methyl, picoxystrobin and difenoconazole) from nine tested fungicides. Different criteria of assessment of fungicide resistance (based on EC 50 and on growth rate -inhibition percentage) showed slightly different results, but both of the criteria confirmed occurrence of resistant C. beticola strains to thiophanatemethyl, picoxystrobin and difenoconazole in Slovakia. Fields with higher frequency of application of these fungicides significantly supported the development of resistant strains. Their use should be reconsidered on critical areas. It is very important to focus on anti-resistant strategy and reduce of using of risk fungicides on localities, where it was confirmed occurrence of resistant Cercospora beticola strains. The highest frequency of fungicide resistant strains was confirmed in localities Dolné Saliby (thiophanate-methyl and picoxystrobin) and Senec (picoxystrobin and difenoconazole). The lowest level of risk of fungicide resistance was confirmed in the locality Oslany. Assessment of any C. beticola strains have not confirmed reduced sensitivity to active ingredients tetraconazole and prochloraz + propiconazole. These active ingredients achieved the highest efficacy against C. beticola isolates from all of the tested localities. The serious risk of fungicide resistance in some localities in Slovakia was confirmed in this work. For farmers, avoid of risk fungicides application in certain localities is recommended.