Development of an LSU rRNA-targeted qPCR assay and transcriptional profiling of defense-related genes to elucidate barley resistance to Bipolaris sorokiniana

Spot blotch, caused by Bipolaris sorokiniana, severely limits global barley production. This study characterized five isolates from Bolu, Türkiye, and screened 95 barley cultivars for resistance to the most aggressive isolate (B_BS01) using a 1–9 disease severity scale. Cultivars Gazda, Dara, Hilal, Nonius, and Bravo exhibited the highest resistance (Disease severity index: 35.00 %–36.67 %), forming a statistically distinct group. A quantitative PCR assay targeting the LSU rRNA locus of B. sorokiniana was developed, detecting pathogen DNA down to 0.1 pg with high specificity. This assay quantified starkly different colonization dynamics: pathogen DNA was effectively suppressed in resistant cultivars (Dara, Gazda), while it proliferated rapidly in susceptible ones (Meriç, Bülbül), resulting in up to 15-fold higher pathogen loads by 4 days post-inoculation. Temporal expression profiling of defense-related genes (PR1, PR2, PR3, PR5, PR10, CSD, LOX, PAL) was conducted in Dara and Meriç. Notably, PR1 and PR10 were more strongly induced in Meriç (17.06- and 10.56-fold at 72 h post-inoculation), whereas PR3 was preferentially upregulated in Dara. PR5 and LOX were downregulated in both cultivars; CSD showed moderate induction, and PR2 remained relatively stable. The combination of a sensitive qPCR assay and gene expression profiling provides robust tools for resistance screening and supports targeted breeding for spot blotch resistance in barley.