Seedborne pathogen complex of cotton: morpho-molecular identification and PCR-based detection of Paramyrothecium roridum

Abstract

Abstract Seedborne pathogens represent a major constraint to cotton production by impairing seed quality, seedling establishment and early crop vigour, while also facilitating long-distance disease dissemination. The extensive cultivation of genetically uniform tetraploid cotton hybrids has made them vulnerable to seed- and soilborne diseases. The present investigation was aimed at exploring the diversity of major seedborne pathogens associated with cotton, to identify them using integrated morphological and molecular approaches, and to develop a sensitive PCR-based detection assay for Paramyrothecium roridum. Multiple seed health testing methods employed, resulted in the isolation of nine isolates representing eight predominant pathogens, including Fusarium spp., Lasiodiplodia theobromae, Alternaria spp., Paramyrothecium roridum and Xanthomonas citri pv. malvacearum. Cultural and microscopic characterisation revealed considerable morphological diversity among the isolates, and growth responses varied significantly across culture media. Molecular identification using ITS, β-tubulin, and 16S rRNA gene sequencing confirmed species-level identities. Pathogenicity assays on cotton validated the virulence of all isolates and successful reisolation confirmed Koch’s postulates. Furthermore, an ITS-based primer pair was designed and optimised for P. roridum, producing a specific 354 bp amplicon with high specificity and a detection limit of 1 pg DNA. The assay reliably detected the pathogen in artificially inoculated seeds. The findings of this study highlight the importance of integrating morphological and molecular approaches for comprehensive characterisation of cotton seedborne pathogens.