Integrated Morphological And Molecular Approaches For Characterizing Fusarium Isolates Causing Wilt In Black Gram

Abstract

Fusarium species rank among the most harmful fungal pathogens impacting cereals and legumes, leading to significant yield reductions and posing a threat to global food security. Precise identification of these pathogens is crucial for formulating effective disease management strategies. This research focused on characterizing Fusarium isolates from farmers' fields across five different agroclimatic zones. Thirty isolates were collected from diseased, wilted black gram plants, surface-sterilized, and cultured on Potato Dextrose Agar (PDA) at a temperature of 28 ± 2 °C. Genus-level identification was achieved through colony morphology, growth patterns, and microscopic examination of conidial structures using lactophenol cotton blue staining. Malachite Green Agar 2.5 ppm (MGA 2.5) was employed for the selective recovery and enumeration of Fusarium spp. This medium was effective in both pure and mixed cultures and contaminated samples, with infected seedlings showing mortality within five days, confirming pathogenicity. Molecular characterization was conducted for species identification. Genomic DNA was extracted from pure isolates, and the internal transcribed spacer (ITS) region was amplified using universal primers ITS1 and ITS4. The resulting ~600 bp amplicons were sequenced, and BLASTn analysis against NCBI GenBank identified three predominant species: Fusarium oxysporum, Fusarium solani, and Fusarium proliferatum. This method, which combines morphological characteristics with ITS-based molecular identification, highlights the diversity of Fusarium spp. in black gram cropping systems. The results provide foundational data for pathogen monitoring, disease management, and the development of resistant varieties.