Whole-Genome Analysis Reveals Biosynthetic Potential and Antifungal Activity of Nakaseomyces kungkrabaensis BRKS1-2 Isolated from Boesenbergia rotunda Rhizome

Yeasts associated with medicinal-plant tissues are potential sources of antifungal compounds for plant disease management. Microorganisms isolated from surface-sterilised rhizomes of Boesenbergia rotunda and Alpinia galanga were screened against Fusarium oxysporum f. sp. lycopersici (Fol), F. oxysporum f. sp. cubense (Foc), and Sclerotium rolfsii. In dual-culture assays, BRKS1-2 showed the highest mean inhibition across pathogens (35.5%; p < 0.001), including 42.2 ± 5.3% against Foc and 39.0 ± 0.9% against S. rolfsii, and was the only isolate active against S. rolfsii. Cellfree culture filtrates collected after 24–72 h inhibited all pathogens, with activity increasing over time (p < 0.001). ITS and LSU rRNA phylogenies placed BRKS1-2 within Nakaseomyces, although ITS did not resolve species identity. Whole-genome sequencing produced an 11.5-Mb draft assembly (37.2% GC) at 185.8× mean mapped depth, with 99.90% of filtered reads mapped. Genome-wide comparison showed 98.3% average nucleotide identity with the N. kungkrabaensis reference, supporting identification as N. kungkrabaensis. Gene prediction identified 5,112 protein-coding genes, 222 tRNA genes, and five rRNA genes. LC-MS/MS detected 74 fungal protein groups, mainly associated with central metabolism and stress responses. Fungal-mode antiSMASH predicted four terpene-related clusters and one putative beta-lactone cluster; all abutted contig boundaries and were therefore partial. These findings demonstrate the in vitro antifungal activity of BRKS1-2 and identify candidate biosynthetic loci for investigating the responsible compounds.