Genetic variations for rust pre-infection structures on Brachypodium inbred accessions. Detached leaves of accessions Bd3-1, Bd18-1, Bd21, Bd21-3, Bd29-1 and Bd30-1 were spray-inoculated with P. emaculata urediniospores followed by staining with WGA-Alexa-fluor-488, 72 h after inoculation. Stained fungal structures were visualized and quantified by epifluorescence microscope. Percent of P. emaculata urediniospore germination, germ tubes showing oriented growth, appressoria formation on stomatal apertures and infection foci were measured on six Brachypodium accessions (a). One way Analysis of variance (ANOVA) indicated differences among accessions for all parameters (p-values 0.0015, 0.0000000008, 0.0000001 and 0.0001 for germination, oriented growth, appressoria and HR, respectively). (b) Bd3-1 and Bd30-1 pre-infection structures, including microscopic evaluation of hypersensitive response (HR). Two tailed Student’s t-test (p-values 0.010, 0.0007, 0.0005 and 0.0002 for germination, oriented growth, appressoria and HR, respectively) indicate significant differences among both accessions for all tested parameters. It is important to note that P. emaculata failed to sporulate on any of the accessions tested. For statistical analysis, data from three biological replications was used. Error bars indicate standard deviation from mean.