Evaluation of ITS copy number variation among Clarireedia jacksonii isolates using single-copy genetic markers cam and hsp60

Molecular tools are widely used for plant pathogen detection and quantification. Recent quantitative PCR applications for the turfgrass disease causal agent Clarireedia jacksonii were developed targeting the ITS region and showed potential utility in improved disease management. However, accurate and consistent pathogen quantification is contingent on stable copy numbers of the target genes across populations. In this study, we evaluated the genomic copy number of molecular markers in C. jacksonii using in silico analyses, quantitative PCR, and digital PCR. Calmodulin ( cam ), elongation factor 1-alpha ( ef1α ), and minichromosome maintenance protein 7 ( mcm7 ) were consistently identified as single-copy loci, whereas ITS exhibited multiple copies in two out of nine assemblies. Digital PCR confirmed that cam and heat shock protein 60 ( hsp60 ) closely followed theoretical single-copy expectations across a genomic DNA dilution series of C. jacksonii isolate HRS10. In the analysis of 27 C. jacksonii isolates, ITS was consistently detected as a multicopy locus, with copy number estimates ranging from 33 to 48 copies per genome when normalized using cam and from 34 to 52 copies per genome when normalized using hsp60 . These results demonstrate that ITS copy number variation among C. jacksonii isolates was consistently observed, independent of the single-copy reference locus used for normalization. Therefore, single-copy genetic markers, such as cam and hsp60 , may represent superior choices for accurate and stable estimation of fungal genome equivalents in C. jacksonii .