Morphological and Molecular Characterization of Pyrenophora graminea Causing Stripe Disease on Barley Seedlings

Abstract

Stripe disease symptoms characterized by elongated chlorotic streaks, leaf necrosis, stunted growth, and reduced seedling vigor were frequently observed in barley fields cultivated under cool and humid environmental conditions. Diseased barley seedlings were collected from infected fields to identify and characterize the associated fungal pathogen through morphological and molecular approaches. Fungal isolates obtained from symptomatic leaf tissues consistently produced dark olivaceous to brown colonies with septate mycelia and cylindrical conidia typical of Pyrenophora graminea. Considerable variation in colony morphology, pigmentation, and sporulation intensity was observed among isolates recovered from different barley-growing regions. Microscopic examination confirmed the presence of characteristic conidial morphology and fungal structures corresponding to descriptions of the pathogen. Pathogenicity assays performed on healthy barley seedlings reproduced typical stripe disease symptoms, including chlorotic striping, tissue necrosis, and suppression of seedling growth. Re-isolation of the fungus from inoculated plants fulfilled Koch’s postulates and confirmed the pathogenic nature of the isolates. Molecular identification was conducted through amplification and sequencing of the internal transcribed spacer (ITS) region and additional conserved gene loci commonly used for fungal taxonomy. Sequence analysis demonstrated high similarity between the obtained isolates and authenticated P. graminea sequences available in international databases. Phylogenetic analysis grouped the isolates within the P. graminea clade with strong bootstrap support, confirming species identity and genetic relatedness among isolates. Environmental conditions characterized by low temperature and prolonged moisture favored disease development and pathogen transmission through infected seed material. The combined use of morphological and molecular characterization provided accurate identification of the pathogen and improved differentiation from closely related barley pathogens.