Phylogenomic Characterization of Two Alternaria Lineages Associated with Cotton Leaf Spot in Tennessee

Alternaria leaf spot is an important foliar disease of cotton (Gossypium hirsutum L.) and has historically been associated primarily with Alternaria macrospora in the United States. In November 2025, symptomatic Upland cotton leaves from a research greenhouse at Tennessee Technological University's Oakley Farm in Livingston, Tennessee, yielded two Alternaria isolates, 2-2 and 9-1. Quantitative conidial characterization showed substantial overlap in dimensions, septation, and shape. Concatenated ITS-RPB2 phylogenetic analysis supported different broad affinities, placing isolate 2-2 within an A. alternata- dominated group and isolate 9-1 within a group associated with the A. arborescens species complex (AASC), although their immediate relationships were weakly resolved. Phylogenomic analysis of 1,501 single-copy BUSCO orthologs from 63 genomes placed isolate 2-2 within the A. alternata lineage, sister to BMP2343, and isolate 9-1 within the AASC, sister to MOD1-FUNGI6. In controlled pathogenicity assays, isolate 9-1 reproduced foliar lesions on cotton, whereas mock-inoculated plants remained symptomless, and molecularly confirmed re-isolation fulfilled Koch's postulates. Isolate 2-2 produced insufficient conidia under the culture conditions used to prepare a standardized inoculum, and its pathogenicity was therefore not determined. These results reveal phylogenetically distinct Alternaria lineages associated with symptomatic cotton and establish an AASC isolate as a causal agent of cotton leaf spot in Tennessee.