Morphological and Molecular Characterization of Alternaria brassicae Causing Dark Leaf Spot on Brassica Oilseeds

Abstract

Dark leaf spot symptoms observed on Brassica oilseed crops were investigated to determine the causal pathogen and characterize its morphological and molecular features. Field surveys in major oilseed-growing regions revealed circular to irregular necrotic lesions with concentric rings and chlorotic halos on leaves, leading to premature defoliation and reduced photosynthetic efficiency under conducive environmental conditions. Fungal isolates obtained from infected leaf tissues produced dark, septate mycelium with rapid growth on potato dextrose agar and abundant multicellular, muriform conidia borne singly or in short chains, consistent with Alternaria brassicae. Pathogenicity tests conducted on healthy Brassica seedlings under controlled conditions successfully reproduced dark leaf spot symptoms, confirming virulence and fulfilling Koch’s postulates. Molecular identification using ITS rDNA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and Alternaria major allergen (Alt a 1) gene sequencing confirmed isolates as A. brassicae, showing high similarity with reference sequences from global databases. Phylogenetic analysis placed all isolates within a well-supported A. brassicae clade, indicating limited intraspecific variation among populations from different locations. Minor sequence polymorphisms suggested possible local adaptation and evolutionary divergence under varying agro-climatic conditions. Disease severity was strongly associated with high humidity, moderate temperatures, and extended leaf wetness duration, which favored sporulation and secondary infection cycles. The study highlights the significance of A. brassicae as a major foliar pathogen of Brassica oilseeds and emphasizes the importance of integrated disease management strategies. Recommended approaches include crop rotation, resistant cultivars, optimized planting density, and timely fungicide applications to reduce disease incidence and improve oilseed productivity.