AbstractAsexual urediniospore infection of primary cereal hosts by Puccinia graminis f. sp. tritici (Pgt), the wheat stem rust pathogen, was considered biphasic. The first phase, spore germination and appressoria formation, requires a dark period and moisture. The second phase, host entry by the penetration peg originating from the appressoria formed over the guard cells, was thought to require light to induce natural stomata opening. Previous studies concluded that inhibition of colonization by the dark was due to lack of penetration through closed stomata. A sensitive WGA-Alexa Fluor 488 fungal staining, surface creation and biovolume analysis method was developed enabling visualization and quantification of fungal growth in planta at early infection stages surpassing visualization barriers using previous methods. The improved method was used to investigate infection processes of Pgt during stomata penetration and colonization in barley and wheat showing that penetration is light independent. Based on the visual growth and fungal biovolume analysis it was concluded that the differences in pathogen growth dynamics in both resistant and susceptible genotypes was due to light induced pathogen growth after penetration into the substomatal space. Thus, light induced plant or pathogen cues triggers pathogen growth in-planta post penetration.