Morphological and Molecular Characterization of Cercospora kikuchii Causing Purple Seed Stain on Soybean

Abstract

Purple seed stain symptoms characterized by reddish-purple discoloration on seed coats, foliar lesions, chlorosis, and premature defoliation were observed in soybean (Glycine max) fields, leading to reduced seed quality and market value. Diseased leaf and seed samples collected from affected fields were used for pathogen isolation and characterization through morphological, pathogenicity, and molecular approaches. Fungal isolates consistently produced slow-growing gray to dark colonies with septate hyphae and conidiophores bearing long, hyaline, multiseptate conidia typical of Cercospora kikuchii. Considerable variation in colony morphology, sporulation intensity, and lesion severity was observed among isolates obtained from different soybean-growing regions. Pathogenicity assays conducted on healthy soybean plants reproduced characteristic symptoms, including foliar spots and seed discoloration, whereas control plants remained symptom-free. Re-isolation of the fungus from inoculated tissues fulfilled Koch’s postulates, confirming its pathogenic role. Molecular identification was performed using amplification and sequencing of the internal transcribed spacer (ITS) region along with additional conserved loci including translation elongation factor 1-α (TEF1-α). Sequence analysis revealed high similarity with authenticated C. kikuchii isolates available in global databases. Phylogenetic analysis grouped field isolates within the C. kikuchii clade with strong bootstrap support, confirming species identity and revealing limited genetic variability among regional populations. Disease development was favored by warm temperatures, high humidity, and prolonged leaf wetness, which enhanced spore germination and infection efficiency. Severe infections resulted in reduced photosynthetic area, seed quality deterioration, and significant yield losses. The integration of morphological and molecular tools provided accurate identification and improved understanding of pathogen variability and epidemiology.