Will Triazoles Still Be of Importance in Disease Control of Zymoseptoria tritici in the Future?

: Septoria tritici blotch (STB), caused by Zymoseptoria tritici , is one of the most important foliar wheat diseases worldwide. Current control strategies of STB rely mainly on fungicides, whereby triazoles (demethylation inhibitors; DMIs) have been the backbone in the control of Z. tritici in the last decades. However, in recent years a gradual loss of sensitivity of Z. tritici to several active ingredients of the triazole group has been reported in several European wheat-growing areas. Nevertheless, a new triazole fungicide, namely, mefentrifluconazole, has recently become available in disease management of STB, which belongs to a completely new triazole subclass, the so-called isopropanol triazoles. In this study, the trend in sensitivity development of Z. tritici towards older triazoles (tebuconazole, prothioconazole, and propiconazole) and the new isopropanol triazole mefentrifluconazole was determined in microtiter assays using Z. tritici field populations isolated in 1999, 2009, 2014, and 2020 in a high-disease-pressure and high-fungicide-input area in northern Germany in order to investigate whether the loss of sensitivity of Z. tritici to older triazoles also applies to mefentrifluconazole. For the three triazole fungicides tebuconazole, prothioconazole and propiconazole, a significant shift towards decreasing sensitivity of Z. tritici field populations was observed from 1999 to 2020, whereas the efficacy of mefentrifluconazole in reducing the in vitro fungal growth by 50% (EC 50 ) remained unchanged over the investigated period, demonstrating a stable sensitivity of Z. tritici towards mefentrifluconazole. Although older triazoles are suffering from a loss of sensitivity of Z. tritici field populations due to the selection and spread of less triazole sensitive strains within the Z. tritici population, the efficacy of the new triazole mefentrifluconazole with its unique isopropanol unit was not affected by these changes within the Z. tritici population. Thus, the introduction of such new molecular units could also represent an important contribution for older groups of active ingredients, which previously suffered from a loss of sensitivity. within the triazole group is to be demonstrated. In our present study we investigate the efﬁcacy of mefentriﬂuconazole with its ﬂexible isopropanol unit in comparison to older triazoles in microtiter assays by using Z. tritici ﬁeld populations isolated between 1999 and 2020 in a high-disease-pressure and high-fungicide-input area in northern Germany. The aim of our present study was to clarify the role of triazoles, which previously suffered from a loss of sensitivity, in future control strategies against Z. tritici due to innovations in their chemical structure.


Introduction
Fungal disease control in cereals relies upon the presence of effective fungicides [1]. These agrochemical compounds are also intensively used in wheat to reduce yield losses caused by Z. tritici, the causal agent of Septoria tritici blotch (STB). STB is one of the most important foliar wheat diseases worldwide [2]. Especially in maritime climatic regions of Europe, STB is the most yield-reducing wheat disease [3,4]. In Europe, 70% of the annual fungicide usage in wheat is linked to disease control of Z. tritici [4]. However, the frequent use of fungicides in disease management of Z. tritici gives rise to selection of fungicide resistance. One example is the emergence of resistance to quinone-outside inhibitors (strobilurins; QoIs), which no longer provide reliable field control of Z. tritici in most European countries [5][6][7]. Currently, the control of STB depends on the application of

Sampling Area and Isolation of Z. tritici
Wheat plants of the cultivar "Ritmo", characterized as moderately to highly susceptible to STB [40], were sampled in 1999,2009,2014, and 2020 in the northernmost federal state of Germany, Schleswig-Holstein. The region between the North and Baltic Sea is characterized by large acreages of winter wheat, accounting for 28.9% of arable land [41], and maritime weather conditions, with an average annual temperature of 8.9 • C and an annual precipitation of 823 L/m 2 [42], which are conducive for Z. tritici infections in wheat [43,44]. In each sampling year, 30 wheat plants with Z. tritici lesions were collected at growth stage 51 (begin of ear emergence) [45] from two reference locations, namely Kluvensiek (Coordinates: x = 1092115, y = 7234219; EPSG-Code: 3857) and Futterkamp (Coordinates: x = 1183896, y = 7225601), which were part of a regional monitoring for leaf pathogens (IPM wheat model) [43] and Fusarium head blight (FHB) in wheat [46,47]. Wheat plants were taken from three fungicide untreated control plots (10 plants per plot; plot size 2 × 5 m) per location and year within the IPM and FHB monitoring and were stored at −20 • C until Z. tritici isolation.
For the preparation of Z. tritici field populations [19], leaf pieces (3 cm in length) from the upper three leaves with typical Z. tritici necroses containing pycnidia were washed in running tap water, surface sterilized (2 min in 2% sodium hypochlorite), triple rinsed with sterile deionized water, and dried on tissue paper. During incubation of leaf pieces on water agar (15 g in 1 L water; Carl Roth, Karlsruhe, Germany) for 24 h in the dark at 20 • C, cirrhi were discharged from pycnidia. Cirrhi from individual pycnidia were transferred to a Petri dish containing malt yeast agar (MYA; 4 g of yeast extract, malt extract, glucose plus 15 g of agar in 1 L water; all obtained from Carl Roth) as well as penicillin and streptomycin (each 50 mg/L; both obtained from Carl Roth) to prevent bacterial growth. In total, 15 Petri dishes, originating from 15 individual pycnidia, were prepared for each field plot per location and year, resulting in 45 plates per location and year. Spores produced on MYA plates were washed off using 10 mL sterile skim milk (100 g of skim milk powder in 1 L of water; Sigma-Aldrich, Schnelldorf, Germany) per plate. Spores of each plate, representing individual pycnidia isolates, were stored separately at −70 • C (stock solution) until further use. For the fungicide sensitivity tests, spores from the stock solution were transferred to MYA plates, distributed over the medium surface, and grown for 4 days at 20 • C. Subsequently, spores were washed off using sterile deionized water and spore concentrations of the solutions originating from individual pycnidia isolates were determined using a Fuchs Rosenthal haemocytometer and a light microscope. Spore solutions were diluted with sterile deionized water to approximately 2.5 × 10 5 spores/mL. Mixtures of 15 spore solutions originating from 15 isolated individual pycnidia isolates per field plot (replication) within the same location and year were prepared, representing Z. tritici field populations, which were used for the in vitro fungicide sensitivity bioassays described below.

Fungicide Sensitivity Testing
The in vitro fungicide sensitivity of Z. tritici field populations isolated in Northern Germany in 1999, 2009, 2014, and 2020 was determined by using microtiter assays as described by Birr et al. [19]. Analytical grade compounds (>98% purity) of tebuconazole, propiconazole, prothioconazole (all purchased from Sigma-Aldrich), and mefentrifluconazole (HPC Standards GmbH, Cunnersdorf, Germany) were used in six test concentrations of 125, 12.5, 1.25, 0.125, 0.0125, and 0 mg/L, respectively, which were obtained via serial dilutions with ethanol (HPLC grade). For each fungicide, triplicates of each test concentration were added to clear, flat-bottomed 96-well microtiter plates (Sarstedt, Nümbrecht, Germany) with 100 µL per well for each year of Z. tritici isolation. After evaporation of solvents overnight, 100 µL of culture medium (4 g of yeast extract, 4 g of malt extract, and 4 g of glucose in 1 L of deionized water; all obtained from Carl Roth) and 100 µL of spore solution with a dilution of 2.5 × 10 5 spores/mL (medium-spore solution) were transferred to each well containing the abovementioned fungicide dilutions. Each 96-well plate was also loaded with medium-water controls containing the abovementioned fungicide concentrations in triplicate, but without spore solution consisting of 100 µL medium and 100 µL sterile deionized water. Finally, three microtiter plates were created for each fungicide and each location according to the three replications of field plots per location used for Z. tritici isolation. Microtiter plates were incubated at 22 • C on an orbital shaker at 120 rpm with an 8 h photoperiod. After 5 days, the optical density (absorbance) was measured at 595 nm with a shaking period of 5 s prior to measurement using a Multiskan FC microtitration plate photometer (Thermo Fisher Scientific, Schwerte, Germany).

Data Analysis
Optical densities of the six tested fungicide concentrations (medium-spore solution) were corrected for the medium, the absorbance of the microtiter plate, and the intrinsic colour of the fungicide (medium-water controls) for each concentration separately within the same plate. The corrected optical density data were then normalized by dividing by the optical density of the fungicide untreated control (medium-spore solution with fungicide concentration of 0 mg/L). For these relative optical density data, means and standard deviations (±SD) were calculated for each year of Z. tritici isolation and each concentration including both locations and the three replications per location, split for the different fungicides. Using the sigmoid regression model of the software package Sigmaplot 13.0 (Systat Software, Erkrath, Germany), optical density data were plotted against the several fungicide concentrations for each year of Z. tritici isolation in order to determine the fungicide concentration at which the optical densities of the spore cultures were half that of the cultures with no fungicide. Those fungicide concentrations reducing the in vitro fungal growth by 50% (EC 50 ) were determined for each fungicide and year of Z. tritici isolation as well as for each of the six replications originating from the two locations. From these values EC 50 means (±SD) were calculated for each year of fungal isolation, split for the different fungicides.

Statistical Analysis
Statistical analysis was done for EC 50 values by use of the statistical software R, version 4.0.1 (R Foundation for Statistical Computing, Vienna, Austria) [48]. The data evaluation started with the definition of an appropriate statistical mixed model [49,50]. The statistical model included the fungicide (tebuconazole, propiconazole, prothioconazole, mefentrifluconazole) and year of Z. tritici isolation (1999,2009,2014, and 2020), as well as their interaction term as fixed factors. Replications (plates), nested in location, were regarded as random factor. The residuals were assumed to be approximately normally distributed and to be heteroscedastic. These assumptions are based on a graphical residual analysis. Based on this model, a Pseudo R 2 was calculated [51] and an analysis of variance (ANOVA) was conducted, followed by multiple contrast tests [52] in order to compare the several years (2009, 2014, and 2020) versus 1999, split for the different fungicides.

Results
Changes in fungicide sensitivity of Z. tritici field populations isolated in 1999, 2009, 2014, and 2020 were tested for the four DMI fungicides tebuconazole, propiconazole, prothioconazole, and mefentrifluconazole. Averaged over all tested fungicides and years of Z. tritici isolation, ANOVA results showed that EC 50 values were significantly affected by the interaction of fungicide and year (p < 0.0001; Table 1), indicating that the four tested DMIs expressed a different change in sensitivity of Z. tritici over time. Both single factors had a significant effect on EC 50 values (p < 0.0001). Table 1. Analysis of variance (ANOVA) for the effect of fungicide, year and their interaction on EC 50 values (mg/L) of Zymoseptoria tritici field populations summarized for all tested demethylation inhibitors (DMIs; tebuconazole, propiconazole, prothioconazole, mefentrifluconazole) and years of Z. tritici isolation (1999,2009,2014,2020). In each year, Z. tritici field populations were isolated from wheat crops (cultivar "Ritmo") at two locations in Northern Germany. For the three DMI fungicides tebuconazole, propiconazole, and prothioconazole a shift towards decreasing sensitivity of Z. tritici field populations was observed over the last 21 years from 1999 to 2020 ( Figure 1A-C and Figure 2A-C).

Effect
Agronomy 2021, 11, x FOR PEER REVIEW 5 of 12 For the three DMI fungicides tebuconazole, propiconazole, and prothioconazole a shift towards decreasing sensitivity of Z. tritici field populations was observed over the last 21 years from 1999 to 2020 (Figures 1A-C and 2A-C).  years of Z. tritici isolation (Figures 1D and 2D). A stable relationship between fungicide concentration and optical density of spore suspension was observed between 1999, 2009, 2014, and 2020, respectively ( Figure 1D). Compared to 1999 (1.956 mg/L), insignificant differences in EC50 values were determined for the remaining years 2009 (2.642 mg/L), 2014 (2.337 mg/L), and 2020 (2.716 mg/L) (Figure 2D), demonstrating a stable sensitivity of Z. tritici towards mefentrifluconazole.

Discussion
Septoria tritici blotch (STB), caused by the wheat pathogen Z. tritici, is mainly controlled by the intensive use of fungicides during crop growth, whereby triazoles have been used for STB control for more than 30 years. However, the frequent use of fungicides in disease management gives rise to selection of fungicide resistance. In fact, in recent years a gradual loss of sensitivity of Z. tritici field populations to several active ingredients of the triazole group has been documented in many wheat growing areas [11,13,16,18,20]. Although triazoles have suffered from a general loss of sensitivity due to the selection and spread of less triazole sensitive strains within the Z. tritici population, the efficacy of the new isopropanol triazole mefentrifluconazole is to be demonstrated. In our present study, the trend in sensitivity development of Z. tritici towards older triazoles (tebuconazole,  Figure 2B). From 1999 to 2020 the EC 50 values increased by 3.615 mg/L for tebuconazole, 12.733 mg/L for propiconazole, and 6.694 mg/L for prothioconazole (Figure 2A-C).
In contrast to the abovementioned older triazoles, the efficacy of mefentrifluconazole in reducing the in vitro fungal growth of Z. tritici remained unchanged for the four tested years of Z. tritici isolation (Figures 1D and 2D). A stable relationship between fungicide concentration and optical density of spore suspension was observed between 1999, 2009, 2014, and 2020, respectively ( Figure 1D). Compared to 1999 (1.956 mg/L), insignificant differences in EC 50 values were determined for the remaining years 2009 (2.642 mg/L), 2014 (2.337 mg/L), and 2020 (2.716 mg/L) (Figure 2D), demonstrating a stable sensitivity of Z. tritici towards mefentrifluconazole.

Discussion
Septoria tritici blotch (STB), caused by the wheat pathogen Z. tritici, is mainly controlled by the intensive use of fungicides during crop growth, whereby triazoles have been used for STB control for more than 30 years. However, the frequent use of fungicides in disease management gives rise to selection of fungicide resistance. In fact, in recent years a gradual loss of sensitivity of Z. tritici field populations to several active ingredients of the triazole group has been documented in many wheat growing areas [11,13,16,18,20]. Although triazoles have suffered from a general loss of sensitivity due to the selection and spread of less triazole sensitive strains within the Z. tritici population, the efficacy of the new isopropanol triazole mefentrifluconazole is to be demonstrated. In our present study, the trend in sensitivity development of Z. tritici towards older triazoles (tebuconazole, prothioconazole, and propiconazole) and mefentrifluconazole was investigated in microtiter assays using Z. tritici field populations isolated from the same reference locations and a uniform cultivar between 1999 and 2020 in northern Germany in order to check whether the general loss of sensitivity of Z. tritici to triazoles also applies to mefentrifluconazole.
The triazoles belong to the sterol biosynthesis inhibitors (SBIs). They act in the sterol biosynthesis of the fungal metabolism. In the metabolic process of sterol biosynthesis, the conversion of lanosterol to ergosterol takes place. Based on different targets, the SBIs can be divided into different classes [53]. The most important class of SBIs are the demethylation inhibitors (DMIs). Their representatives are the nitrogen heterocycles, the so-called azoles. The azoles are subdivided in triazoles, imidazoles, and pyrimidines. Triazoles got their name due to their chemical structure, which consists of a five-membered ring system with three nitrogen atoms. Triazoles are among the azole group most commonly used in disease control of STB [24]. Therefore, the omission of this important group of active ingredients due to resistance development would be dramatic for practical agriculture.
DMIs act by inhibiting the enzyme C 14 -demethylase, which is encoded by the CYP51 gene [53]. This enzyme is required for the biosynthesis of ergosterol. Due to the inhibition of the C 14 -demethylase, the conversion of lanosterol to ergosterol is prevented at position C 14 of lanosterol. An interaction occurs between the uncharged N atom of the azole ring and the central Fe 3+ of the haem group by binding to the active side of the cysteine pocket of the CYP51 enzyme [54][55][56]. This leads to an accumulation of methylsterols in the fungal cell, which are toxic for the cell and lead to an undersupply of ergosterol. Ergosterol is an essential component of the fungal cell membrane and is required for the growth of fungal structures. A decrease in the concentration of ergosterol leads to a restructuring of the membrane, resulting in increased permeability and susceptibility of the fungal cell [57][58][59].
Several mechanisms mediate reduced sensitivity of Z. tritici to DMIs [25]. The molecular mechanisms of fungal resistance to DMI fungicides are mostly known [60]. In addition to alterations in the CYP51 gene leading to amino acid changes of the CYP51 enzyme [20][21][22][23], there are two further molecular mechanisms, which can contribute to the decline in sensitivity towards DMIs. One is the overexpression of the target gene CYP51, the other one is the enhanced efflux reducing the accumulation of DMIs in the fungal cell [20,22,[25][26][27][28].
To date, all of these mechanisms have conferred a gradual loss of sensitivity of Z. tritici field populations towards DMIs. However, higher doses are now required to achieve effective disease control due to the reduced field efficacy of DMIs in controlling STB [10,17,32].
However, the amino acid substitutions in the CYP51 enzyme, which directly affect the structure of the active site of the enzyme, are by far most important for decreasing DMI sensitivities in European Z. tritici populations [20,21,33]. Using a model of the CYP51 enzyme, Cools et al. [21] described that amino acid substitutions in the CYP51 enzyme cause alterations of the binding site leading to a reduced interaction between DMIs and their binding site within the CYP51 enzyme, resulting in a reduced sensitivity of the fungus towards these active ingredients. As a result, an increased amount of active ingredient is required at the binding site to increase the binding strength of the active ingredient by increasing their concentration [61]. Several alterations in the CYP51 enzyme gene have been extensively determined in recent years by comparing the amino acid sequences of the Z. tritici wild type with isolates found after long-term DMI use [26].
The CYP51 enzyme is a functional protein composed of many different amino acids. These amino acids can have completely different side chains and thus completely differ-ent chemical properties (acidic or basic, hydrophilic or hydrophobic). The size, shape, and position result in the specific chemical behaviour of the enzyme [62].
The binding and conversion of the substrate by the enzyme takes place in the so-called active site. The active site gets its properties from the amino acids of which it consists. This is a special spatial structure, which is formed by the folding of polypeptide chains. The catalytic reaction in the metabolic process also takes place in this active site [63]. As a rule, only the spatial structure of the substrate to be converted matches the spatial structure of the active site of the enzyme. As a result, very specific enzymes are required for each metabolic pathway, and often they only fit there.
Due to the high specificity of the active site, the enzyme can only bind a certain target molecule, the substrate, and support the chemical reaction [62]. Due to this specificity, however, every mutation in the CYP51 gene and therefore every change in the amino acid sequence of the enzyme has considerable consequences for the substrate, and thus for the metabolism of the fungus itself. At the same time, it also has consequences for the fungicidal active ingredient that is supposed to bind at the same active site. The active ingredient acts here as a substrate substitute and blocks the active site of the enzyme for further catalytic reactions. Due to the high binding affinity of azoles to CYP51 enzymes in the fungal organism, they are considered as effective active ingredients in disease management of fungal diseases in many crops. However, this effectiveness of azoles has been significantly reduced due to various mutations in the CYP51 gene of Z. tritici [20][21][22][23].
Unfortunately, positive cross-resistance relationships were found between several triazoles [16,20,22,24,26,30]. Hence, the loss of sensitivity to a single active ingredient of this group affects also the sensitivity of most remaining triazoles. In our study, a reduced sensitivity was also observed for the older triazoles, namely, tebuconazole, prothioconazole, and propiconazole. In contrast, mefentrifluconazole, the first active ingredient of the isopropanol subclass, apparently occupies a special position within the triazole group. In fact, the investigated older triazoles have suffered from a loss of sensitivity of Z. tritici field populations between 1999 and 2020, whereas the efficacy of mefentrifluconazole with its unique isopropanol unit in reducing the fungal growth by 50% (EC 50 ) was not affected. Furthermore, the variance of efficacy was extraordinarily low compared to the older triazoles. This indicates that high efficacy levels can be expected regardless of location and population. The high efficacy in the field was also confirmed by field studies of Jørgensen et al. [38] in comparison to older triazoles commonly used in STB control. According to Strobel et al. [39], the efficacy of various triazoles against highly adapted strains of Z. tritici (glasshouse trials) was poor, whereas mefentrifluconazole was able to show full control. Thus, it could be demonstrated that mefentrifluconazole has a unique selling point in its behaviour towards less sensitive populations in the field. This is remarkable, since it appears that the novel flexible isopropanol unit, which is located in front of the ring structure of the triazole [39], copes much better with alterations of the active site of the CYP51 enzyme caused by mutations in the CYP51 gene. Due to this isopropanol unit, a high degree of structural flexibility of the molecule is possible, which influences the binding at the active site of the enzyme. Especially in populations with mutations in the CYP51 gene and therefore altered spatial structures of the binding pocket of the target enzyme, positive effects result. It seems that significantly higher binding forces act at the active site of the CYP51 enzyme, which in turn leads to an improved efficacy in less sensitive populations.

Conclusions
Mefentrifluconazole is a new triazole with a novel isopropanol unit. In contrast to older triazoles, the mefentrifluconazole molecule is more flexible in its structure and is able to bind even if the active site of the molecular target (CYP51 enzyme) is altered. Due to this isopropanol unit, no changes in sensitivity of Z. tritici field populations was observed between 1999 and 2020, whereas a significant shift towards decreasing sensitivity was determined for the older triazole fungicides. These results are of great importance for the sustainable use of triazoles and can secure this important fungicide group for further disease management of STB in a long-term.
Two important approaches can be derived from our presented results. Firstly, our findings open up new aspects in the long-term evaluation of several groups of active ingredients. Due to innovations in the chemical structure also older groups of active ingredients can be reactivated, which have previously suffered from a loss of sensitivity. This approach is very encouraging, as the preservation of as many groups of active ingredients as possible with different modes of action in the fungal metabolism is the basic prerequisite for sustainable resistance management. The higher the diversity of active ingredients with different modes of action available for disease control, the more difficult is the adaptation of the various pathogens to these fungicidal active ingredients. However, resistance management means not just managing active ingredients. Moreover, agronomic practices (e.g., sowing date, cultivar susceptibility, crop rotation, and tillage) must be included in anti-resistance strategies. Strengthening and maintaining one group of active ingredients simultaneously reduces the risk of the development and spread of resistance to other groups of active ingredients. Furthermore, flexible units as the isopropanol unit could represent the answer to further resistance development caused by selective alterations within the target enzymes.