Morphological and Molecular Characterization of Plenodomus lingam Causing Blackleg Disease on Canola

Abstract

Blackleg disease symptoms characterized by leaf lesions, stem cankers, internal vascular discoloration, and premature plant senescence were observed in canola (Brassica napus) fields, resulting in significant yield reduction under conducive environmental conditions. Diseased plant samples collected from affected fields were subjected to pathogen isolation and characterization using morphological, pathogenicity, and molecular approaches. Fungal isolates consistently produced slow-growing gray to dark colonies with abundant pycnidia embedded in host tissues and culture media, typical of Plenodomus lingam. Microscopic examination revealed hyaline, cylindrical conidia consistent with descriptions of the pathogen’s asexual stage. Considerable variation in colony morphology, pycnidial density, and sporulation intensity was observed among isolates from different canola-growing regions. Pathogenicity assays conducted on healthy canola seedlings reproduced characteristic blackleg symptoms, including leaf spotting, stem lesions, and crown cankers, whereas control plants remained symptom-free. Re-isolation of the fungus from inoculated tissues fulfilled Koch’s postulates, confirming its pathogenic role. Molecular characterization was performed through amplification and sequencing of the internal transcribed spacer (ITS) region along with additional conserved loci including β-tubulin and actin genes. Sequence analysis revealed high similarity with authenticated P. lingam isolates in international databases. Phylogenetic analysis grouped the isolates within the P. lingam clade with strong bootstrap support, confirming species identity and revealing moderate genetic diversity among regional populations. Disease development was favored by cool temperatures and prolonged moisture conditions typical of canola-growing environments, which enhanced infection efficiency and pathogen spread. Infected plants exhibited reduced vigor, poor pod formation, and significant yield losses.