A beta-tubulin-based qPCR assay for monitoring of Peronospora variabilis and disease progression in quinoa.

Peronospora variabilis, the causal agent of downy mildew disease, is an important pathogen affecting quinoa production worldwide. Conventionally its detection relies primarily on visual scoring of chlorosis, at times complemented by PCR-based assays targeting the cytochrome oxidase subunit 2 (cox2) or internal transcribed spacer (ITS) regions. In this study, we evaluated three candidate qPCR markers (cox2), NADH dehydrogenase subunit 1 (nad1), and β-tubulin (btub) and developed a btub-based qPCR assay for quantifying P. variabilis during infection. Among the three markers, the btub assay showed the best overall performance, with amplification efficiencies of 99-111% and coefficients of determination (R²) above 0.99 and was therefore selected for pathogen quantification. The btub-based qPCR assay enabled quantification of P. variabilis throughout disease progression and revealed three distinct phases of pathogen accumulation. Pathogen DNA accumulation was compared with chlorophyll, carotenoids, phenolics, betalains, and a rapid Red, Green, and Blue (RGB) colorimetric analysis. The pathogen accumulation curves revealed similar infection dynamics among cultivars previously classified as tolerant or susceptible, suggesting that differences in previously reported cultivar tolerance are not readily explained by pathogen accumulation in the sampled leaf tissue alone. This work introduces a qPCR assay targeting btub gene for quantifying P. variabilis in host plants, addressing the current incompleteness of validated molecular tools for tracking pathogen DNA accumulation during disease development and providing a valuable tool for linking pathogen growth with host physiological responses in future studies on plant-pathogen interactions.