Genetic Diversity of Cotton Leafroll Dwarf Virus from the Southwestern United States and Its Implications for the Multi-Introduction Event Hypothesis and Future Evolution.

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Cotton leafroll dwarf virus (CLRDV) is a viral agent recently identified in the United States in 2017 in Alabama. Since its identification, CLRDV has spread to every cotton-growing state east of New Mexico. Oklahoma, Kansas, and Texas make up the westernmost border of reported CLRDV incidence, making monitoring of these states vital for proper control. Additionally, as the virus evolves, mutations that alter symptomology, such as mutations in the F-box-like motif in ORF0/P0, may occur and need to be monitored thoroughly during the growing seasons. Using high-throughput sequencing and PCR-derived Sanger sequencing, 4 CLRDV genomes and 21 P0 gene isolates were sequenced from Oklahoma, Kansas, and Texas from 2019 to 2021 to determine the genetic diversity among CLRDV isolates. Phylogenetic analyses of the complete genomes revealed seven clades, whereas ORF0 gene analyses resulted in large polytomic clusters. BEAST analyses of the 114 total P0 sequences from GenBank, downloaded before 2024, revealed a lower mean substitution rate than previously reported as well as an earlier root year (1914). In addition, using all available CLRDV genome sequences, 11 likely recombination events were determined. Examination of the P0 amino acid sequences revealed 13 mutations unique to the isolates collected in this study. Based on the phylogenetic and amino acid analyses, the CLRDV isolates from Texas (TX clade) may represent evidence for the multi-introduction event hypothesis into the United States. Additionally, based on our analyses in this study, we propose the Asian CLRDV isolates should be constituted as a potentially separate strain of CLRDV.