Effects are expected to be lower in populations with high recombination rates. M. graminicola populations display a high degree of sexual recombination both during and between growing seasons [18], [20] and the populations included in this study were at gametic equilibrium [21], [22]. Thus, even if there were close linkage between the genes 
encoding cyproconazole tolerance and virulence, the high recombination rate observed in populations of M. graminicola would lead to a rapid decay in disequilibrium. We hypothesize that the observed correlation is due to pleiotropic effects of genes that affect both virulence and cyproconazole tolerance. Host defense systems usually involve the production of compounds that have lethal or inhibitory effects on the penetration, survival and reproduction of pathogens [54]. These defense-related compounds may share some structural or functional characteristics with synthetic antimicrobials. Pathogen strains having the ability to detoxify the compounds produced by resistant hosts may also have the ability to detoxify synthetic antimicrobial compounds, leading to a simultaneous increase in virulence and antimicrobial resistance. This detoxification processEvolution of Virulence and Sudan I site fungicide ResistanceFigure 4. 
Correlation between variation in cyproconazole resistance and variation in two measures of virulence for five populations of Mycosphaerella graminicola. Cyproconazole resistance was determined by calculating the relative colony size of an isolate grown on Petri plates with and without the fungicide. Correlation was estimated at the 4EGI-1 biological activity population level: A) phenotypic variation in Percentage Leaf Area Covered by Pycnidia (PLACP); B) phenotypic variation in Percentage Leaf Area Covered by Lesions (PLACL); C) genetic variation in Percentage Leaf Area Covered by Pycnidia (PLACP); and D) genetic variation in Percentage Leaf Area Covered by Lesions (PLACL). doi:10.1371/journal.pone.0059568.gFigure 5. Correlation between population mean in cyproconazole resistance and two measures of virulence in Mycosphaerella graminicola. Cyproconazole resistance was determined by calculating the relative colony size of an isolate grown on Petri plates with and without the fungicide: A) Percentage Leaf Area Covered by Pycnidia (PLACP) on Toronit; B) Percentage Leaf Area Covered by Pycnidia (PLACP) on Greina; C) Percentage Leaf Area Covered by Lesions (PLACL) on Toronit; and D) Percentage Leaf Area Covered by Lesions (PLACL) on Greina. doi:10.1371/journal.pone.0059568.gEvolution of Virulence and Fungicide Resistancecould involve mechanisms such as reducing the entry of natural and synthetic compounds into pathogen cells through the action of efflux pumps located in the cytoplasmic membrane. It has been reported that some efflux pumps, such as ABC transporters and MgrA protein, have the ability to transport a broad range of structurally unrelated compounds during pathogen infection, therefore affecting both virulence and antimicrobial resistance, in many plant and human pathogens [55], [56], [57], [58], [59]. The positive correlation between virulence and cyproconazole tolerance could also be due to pathogen metabolites that can destroy or modify the structures and functions of both natural and synthetic antimicrobials. An example of such a defense metabolite is melanin. Melanin is composed of dark-brown or black pigments formed by the oxidative polymerization of phenolic compounds and can be produced by a broad array of 11967625 plant.Effects are expected to be lower in populations with high recombination rates. M. graminicola populations display a high degree of sexual recombination both during and between growing seasons [18], [20] and the populations included in this study were at gametic equilibrium [21], [22]. Thus, even if there were close linkage between the genes encoding cyproconazole tolerance and virulence, the high recombination rate observed in populations of M. graminicola would lead to a rapid decay in disequilibrium. We hypothesize that the observed correlation is due to pleiotropic effects of genes that affect both virulence and cyproconazole tolerance. Host defense systems usually involve the production of compounds that have lethal or inhibitory effects on the penetration, survival and reproduction of pathogens [54]. These defense-related compounds may share some structural or functional characteristics with synthetic antimicrobials. Pathogen strains having the ability to detoxify the compounds produced by resistant hosts may also have the ability to detoxify synthetic antimicrobial compounds, leading to a simultaneous increase in virulence and antimicrobial resistance. This detoxification processEvolution of Virulence and Fungicide ResistanceFigure 4. Correlation between variation in cyproconazole resistance and variation in two measures of virulence for five populations of Mycosphaerella graminicola. Cyproconazole resistance was determined by calculating the relative colony size of an isolate grown on Petri plates with and without the fungicide. Correlation was estimated at the population level: A) phenotypic variation in Percentage Leaf Area Covered by Pycnidia (PLACP); B) phenotypic variation in Percentage Leaf Area Covered by Lesions (PLACL); C) genetic variation in Percentage Leaf Area Covered by Pycnidia (PLACP); and D) genetic variation in Percentage Leaf Area Covered by Lesions (PLACL). doi:10.1371/journal.pone.0059568.gFigure 5. Correlation between population mean in cyproconazole resistance and two measures of virulence in Mycosphaerella graminicola. Cyproconazole resistance was determined by calculating the relative colony size of an isolate grown on Petri plates with and without the fungicide: A) Percentage Leaf Area Covered by Pycnidia (PLACP) on Toronit; B) Percentage Leaf Area Covered by Pycnidia (PLACP) on Greina; C) Percentage Leaf Area Covered by Lesions (PLACL) on Toronit; and D) Percentage Leaf Area Covered by Lesions (PLACL) on Greina. doi:10.1371/journal.pone.0059568.gEvolution of Virulence and Fungicide Resistancecould involve mechanisms such as reducing the entry of natural and synthetic compounds into pathogen cells through the action of efflux pumps located in the cytoplasmic membrane. It has been reported that some efflux pumps, such as ABC transporters and MgrA protein, have the ability to transport a broad range of structurally unrelated compounds during pathogen infection, therefore affecting both virulence and antimicrobial resistance, in many plant and human pathogens [55], [56], [57], [58], [59]. The positive correlation between virulence and cyproconazole tolerance could also be due to pathogen metabolites that can destroy or modify the structures and functions of both natural and synthetic antimicrobials. An example of such a defense metabolite is melanin. Melanin is composed of dark-brown or black pigments formed by the oxidative polymerization of phenolic compounds and can be produced by a broad array of 11967625 plant.