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Background and Aims: Ramularia collo-cygni is an ascomycete fungus that colonizes barley primarily as a benign endophyte, although this interaction can become pathogenic, causing the disease Ramularia leaf spot (RLS). Factors, particularly reactive oxygen species, that resulted in the transition of the fungus from endophyte to necrotrophic parasite and the development of disease symptoms were investigated., Methods: Disease development in artificially inoculated seedlings of barley varieties varying in partial resistance to RLS was related to exposure to abiotic stress prior to inoculation. Histochemical and molecular analysis determined the effect of R. collo-cygni colonization on accumulation of reactive oxygen species and antioxidant gene expression. Development of RLS on barley lines defective in antioxidant enzymes and with altered redox status or non-functional chloroplasts was compared with the accumulation of fungal biomass to determine how these factors affect disease symptom expression., Key Results: Exposure to abiotic stress increased symptom development in all susceptible and most partially resistant barley varieties, in association with greater hydrogen peroxide (H2O2) levels in leaves. Decreased activity of the antioxidant enzymes superoxide dismutase and catalase in transgenic and mutant plants had no effect on the disease transition, whereas manipulation of H2O2 levels during asymptomatic growth of the fungus increased disease symptoms in most susceptible varieties but not in partially resistant plants. Barley mutants that undergo rapid loss of green leaf area when infected by R. collo-cygni or albino mutants with non-functional chloroplasts showed reduced development of RLS symptoms., Conclusions: These results imply that in seedlings the pathogenic transition of the normally endophytic fungus R. collo-cygni does not result from senescence as such, but rather is promoted by factors that result in changes to host reactive oxygen species. Barley varieties vary in the extent to which these factors promote RLS disease., (© The Authors 2017. Published by Oxford University Press on behalf of the Annals of Botany Company.)