He two USA populations (ORER and ORES) were collected on the same day in 1990 from a field planted to the partially resistant cultivar Madsen and the highly susceptible cultivar Stephens, respectively. The fungal isolates were stored in silica gel at 280uC after they were isolated from infected leaves. Isolates in each population were genotyped using restriction fragment length polymorphisms (RFLPs) and DNA fingerprints.Evolution of Virulence and Fungicide Resistanceremoved at three day intervals. At 22 days after the inoculation an average of 8?0 leaves was collected from each isolate-cultivar combination. These leaves were mounted onto blue paper sheets and their images were digitized. Percentage Leaf Area Covered by Pycnidia (PLACP) and Percentage Leaf Area Covered by Lesions (PLACL) were measured with the image analysis software Assess 2.0 [37]. All inoculations and all virulence assessments were made during a single day to minimize environmental variance among treatments.Data analysisFrequencies of cyproconazole resistance (natural logarithm transformed) and both PLACL and PLACP (square root transformed) in the fungal isolates were grouped using a binning approach and each group was labelled with the mid-point value of the lower and upper boundaries of the corresponding bins. Analyses of variance for cyproconazole tolerance, PLACL and PLACP were performed using the general linear model procedure implemented in SAS [39]. The raw data were leftward skewed. After applying a natural logarithm transformation to cyproconazole tolerance and a square root transformation to the PLACL and PLACP datasets, the transformed data showed a more even distribution than the non-transformed data, so the transformed data were used in the ANOVA. Least significant differences [40] were used to compare cyproconazole tolerance, PLACL and PLACP among populations sampled from different regions and hosts. The environmental variance of cyproconazole tolerance, PLACL and PLACP in each population was estimated using the among-replicate variance [41], [42]. In Anlotinib site common garden experiments with C.I. 19140 web asexually reproducing species, any variance among replicates can be attributed to environmental effects because individuals in different replicates have the same genotype (i.e. they are identical clones). Therefore, variance among replicates in this case is equivalent to the environmental variance of cyproconazole tolerance, PLACL and PLACP (for details see [24], [41], [42]). Our earlier analyses [24] indicated that the environmental variance estimated using the large number of isolates with two replicates was not significantly different from the variance estimated using the limited number of isolates with 10 replicates. Therefore, we included all isolates in the analysis of environmental variance. Genetic variance in each M. graminicola population was estimated by subtracting the environmental variance from the phenotypic variance in the corresponding population. The association between virulence and fungicide tolerance in the pathogen populations was evaluated by simple 16574785 linear correlation using transformed data [39].analyses revealed that these bin allocations yielded the optimum distribution to display the trait frequency, with enough strains occupying each bin to calculate and display a frequency with equal spacing between bin means. The square root transformed distributions of PLACL were unimodal and symmetric, peaking at the post-transformation level of 6 (Fig. 1A, 1B) f.He two USA populations (ORER and ORES) were collected on the same day in 1990 from a field planted to the partially resistant cultivar Madsen and the highly susceptible cultivar Stephens, respectively. The fungal isolates were stored in silica gel at 280uC after they were isolated from infected leaves. Isolates in each population were genotyped using restriction fragment length polymorphisms (RFLPs) and DNA fingerprints.Evolution of Virulence and Fungicide Resistanceremoved at three day intervals. At 22 days after the inoculation an average of 8?0 leaves was collected from each isolate-cultivar combination. These leaves were mounted onto blue paper sheets and their images were digitized. Percentage Leaf Area Covered by Pycnidia (PLACP) and Percentage Leaf Area Covered by Lesions (PLACL) were measured with the image analysis software Assess 2.0 [37]. All inoculations and all virulence assessments were made during a single day to minimize environmental variance among treatments.Data analysisFrequencies of cyproconazole resistance (natural logarithm transformed) and both PLACL and PLACP (square root transformed) in the fungal isolates were grouped using a binning approach and each group was labelled with the mid-point value of the lower and upper boundaries of the corresponding bins. Analyses of variance for cyproconazole tolerance, PLACL and PLACP were performed using the general linear model procedure implemented in SAS [39]. The raw data were leftward skewed. After applying a natural logarithm transformation to cyproconazole tolerance and a square root transformation to the PLACL and PLACP datasets, the transformed data showed a more even distribution than the non-transformed data, so the transformed data were used in the ANOVA. Least significant differences [40] were used to compare cyproconazole tolerance, PLACL and PLACP among populations sampled from different regions and hosts. The environmental variance of cyproconazole tolerance, PLACL and PLACP in each population was estimated using the among-replicate variance [41], [42]. In common garden experiments with asexually reproducing species, any variance among replicates can be attributed to environmental effects because individuals in different replicates have the same genotype (i.e. they are identical clones). Therefore, variance among replicates in this case is equivalent to the environmental variance of cyproconazole tolerance, PLACL and PLACP (for details see [24], [41], [42]). Our earlier analyses [24] indicated that the environmental variance estimated using the large number of isolates with two replicates was not significantly different from the variance estimated using the limited number of isolates with 10 replicates. Therefore, we included all isolates in the analysis of environmental variance. Genetic variance in each M. graminicola population was estimated by subtracting the environmental variance from the phenotypic variance in the corresponding population. The association between virulence and fungicide tolerance in the pathogen populations was evaluated by simple 16574785 linear correlation using transformed data [39].analyses revealed that these bin allocations yielded the optimum distribution to display the trait frequency, with enough strains occupying each bin to calculate and display a frequency with equal spacing between bin means. The square root transformed distributions of PLACL were unimodal and symmetric, peaking at the post-transformation level of 6 (Fig. 1A, 1B) f.