The Role of Oxalic Acid in <i>Clarireedia jacksonii</i> Virulence and Development on Creeping Bentgrass

Dollar spot is a destructive foliar disease of amenity turfgrass caused by <i>Clarireedia</i> spp. fungi, mainly <i>C. jacksonii</i>, on the Northern United States region's cool-season grass. Oxalic acid (OA) is an important pathogenicity factor in related fungal plant pathogens such as <i>Sclerotinia sclerotiorum</i>; however, the role of OA in the pathogenic development of <i>C. jacksonii</i> remains unclear due to its recalcitrance to genetic manipulation. To overcome these challenges, a CRISPR/Cas9-mediated homologous recombination approach was developed. Using this novel approach, the oxaloacetate acetylhydrolase (<i>oah</i>) gene that is required for the biosynthesis of OA was deleted from a <i>C. jacksonii</i> wild-type (WT) strain. Two independent knockout mutants, Δ<i>Cjoah-1</i> and Δ<i>Cjoah-2</i>, were generated and inoculated on potted creeping bentgrass along with a WT isolate and a genome sequenced isolate LWC-10. After 12 days, bentgrass inoculated with the mutants Δ<i>Cjoah-1</i> and Δ<i>Cjoah-2</i> exhibited 59.41% lower dollar spot severity compared with the WT and LWC-10 isolates. OA production and environmental acidification were significantly reduced in both mutants when compared with the WT and LWC-10. Surprisingly, stromal formation was also severely undermined in the mutants in vitro, suggesting a critical developmental role of OA independent of plant infection. These results demonstrate that OA plays a significant role in <i>C. jacksonii</i> virulence and provide novel directions for future management of dollar spot. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.