Global Genetic Diversity, Phylogenetic Relationships and Codon Usage Bias of Stagonosporopsis Species Causing Gummy Stem Blight

Abstract Gummy stem blight (GSB) is a devastating fungal disease of cucurbit crops worldwide, caused by species of the genus Stagonosporopsis . Despite its global economic importance, comprehensive studies integrating phylogenetic relationships, population genetics, and codon usage bias across international isolates remain limited. This study aimed to investigate the global genetic diversity, evolutionary relationships, and codon usage patterns of Stagonosporopsis species using RNA polymerase II second largest subunit (RPB2) gene as a molecular marker. A total of 234 sequences from diverse geographical regions were retrieved from GenBank and analyzed using phylogenetic reconstruction, haplotype network analysis, and comprehensive codon usage bias indices including relative synonymous codon usage (RSCU), effective number of codons (ENC), codon adaptation index (CAI), neutrality plot, and parity rule 2 (PR2) analysis. Phylogenetic analysis revealed distinct species-level clades with high bootstrap support, indicating that RPB2 is a reliable marker for resolving evolutionary relationships within the genus. Haplotype analysis identified 43 distinct haplotypes with high haplotype diversity (Hd = 0.861), while analysis of molecular variance (AMOVA) indicated that most genetic variation (92.06%) occurs within populations, suggesting historical gene flow and global dispersal. Codon usage analysis revealed moderate bias shaped primarily by mutational pressure, with additional influences from natural selection, as evidenced by ENC-GC3 plots, neutrality plots, and PR2 bias. Optimal codons were identified, and correspondence analysis revealed distinct clustering patterns associated with nucleotide composition. These findings provide the integrated perspective on the global genetic structure and molecular evolution of Stagonosporopsis species. The observed genetic diversity and codon usage patterns have important implications for understanding pathogen adaptation, population dynamics, and evolutionary mechanisms, ultimately contributing to improved disease management strategies and resistance breeding programs in cucurbit crops.