Ithout the fungicide. Data were natural logarithm transformed and labelled using the mid-point values of the corresponding bins. doi:10.1371/journal.pone.0059568.gORE. S AUS1 2Percentage Leaf Area Covered by Lesions. Percentage Leaf Area Covered by Pycnidia. Values followed by different letters are significantly different at P#0.05. doi:10.1371/journal.pone.0059568.tEvolution of Virulence and Fungicide ResistanceFigure 3. Correlations between cyproconazole 223488-57-1 resistance and two measures of virulence in 141 isolates of Mycosphaerella graminicola evaluated on two Swiss wheat cultivars. 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.gcorrelation coefficients. The process was repeated 10000 times. Results from the randomization analysis revealed that the observation of a positive association between the two traits could not be attributed to local adaptation or population differentiation (data not shown). We also found significantly higher PLACL, PLACP and cyproconazole tolerance in the pathogen population sampled from the resistant wheat cultivar Madsen than the susceptible cultivar Stephens (Table 1). These two pathogen populations were sampled from the same field at the same point in time and therefore most likely originated from the same source population. Because no triazole fungicides were applied to this field, and fungicide use was rare in this region, we do not believe that the difference in triazole tolerance between the M. graminicola populations from the resistant and susceptible hosts is due to selection for fungicide tolerance. This interpretation is supported by the lack of CYP51 sequence variation among isolates from the two hosts [25]. Instead, we hypothesize that the resistant host selected for higher pathogen virulence, which in turn was linked to or had secondary functions related to triazole tolerance (see below for details). Resistant hosts selecting for higher pathogen virulence has 3397-23-7 biological activity already been predicted theoretically [47], [48] and reported from experiments [49], [50]. Because quantitative host resistance decreases pathogen growth rate, pathogens can compensate for lower growth rates by evolving towards an increasing competitive ability, which in turn can result in increased virulence [46]. Both linkage (i.e. hitch-hiking) and pleiotropic effects could lead to a positive association between virulence and fungicide tolerance in pathogens, but hitch-hiking is unlikely to be the cause in thiscase. First, hitch-hiking refers to the process through which an allele increases in frequency because it is linked to an allele that is under positive selection [51], [52]. Cyproconazole tolerance, PLACL and PLACP are quantitative traits that display 
continuous variation within populations (Figs. 1?). It is possible that each of these traits is affected by many minor genes, but it is unlikely that all or most of the genes contributing to the increase of cyproconazole tolerance are closely linked to the genes governing the increase of PLACL or PLACP. Second, recombination rate plays a key role in determining the degree of hitch-hiking [53]. Hitch-hiking.Ithout the fungicide. Data were natural logarithm transformed and labelled using the mid-point values of the corresponding bins. doi:10.1371/journal.pone.0059568.gORE. S AUS1 2Percentage Leaf Area Covered by Lesions. Percentage Leaf Area Covered by Pycnidia. Values followed by different letters are significantly different at P#0.05. doi:10.1371/journal.pone.0059568.tEvolution of Virulence and Fungicide ResistanceFigure 3. Correlations between cyproconazole resistance and two measures of virulence in 141 isolates of Mycosphaerella graminicola evaluated on two Swiss wheat cultivars. 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.gcorrelation coefficients. The process was repeated 10000 times. Results from the randomization analysis revealed that the observation of a positive association between the two traits could not be attributed to local adaptation or population differentiation (data not shown). We also found significantly higher PLACL, PLACP and cyproconazole tolerance in the pathogen population sampled from the resistant wheat cultivar Madsen than the susceptible cultivar Stephens (Table 1). These two pathogen populations were sampled from the same field at the same point in time and therefore most likely originated from the same source population. Because no triazole fungicides were applied to this field, and fungicide use was rare in this region, we do not believe that the difference in triazole tolerance between the M. graminicola populations from the resistant and susceptible hosts is due to selection for fungicide tolerance. This interpretation is supported by the lack of CYP51 sequence variation among isolates from the two hosts [25]. Instead, we hypothesize that the resistant host selected for higher pathogen virulence, which in turn was linked to or had secondary functions related to triazole tolerance (see below for details). Resistant hosts selecting for higher pathogen virulence has already been predicted theoretically [47], [48] and reported from experiments [49], [50]. Because quantitative host resistance decreases pathogen growth rate, pathogens can compensate for lower growth rates by evolving towards an increasing competitive ability, which in turn can result in increased virulence [46]. Both linkage (i.e. hitch-hiking) and pleiotropic effects could lead to a positive association between virulence and fungicide tolerance in pathogens, but hitch-hiking is unlikely to be the cause in thiscase. First, hitch-hiking refers to the process through which an allele increases in frequency because it is linked to an allele that is under positive selection [51], [52]. Cyproconazole tolerance, PLACL and PLACP are quantitative traits that display continuous variation within populations (Figs. 1?). It is possible that each of these traits is affected by many minor genes, but it is unlikely that all or most of the genes contributing to the increase of cyproconazole tolerance are closely linked to the genes governing the increase of PLACL or PLACP. Second, recombination rate plays a key role in determining the degree of hitch-hiking [53]. Hitch-hiking.
