Design and Validation of Specific qPCR Primers for Soil-Borne and Apple Tree-Associated Phytopathogenic Fungi.

Soil-borne pathogenic fungi cause substantial economic losses worldwide by infecting the underground parts of plants. In fruit trees, infections are especially damaging, as they often result in the death of the entire plant. Therefore, early detection is essential for effective disease management caused by soil-borne pathogens. In this study, we designed and validated real-time PCR primers targeting eight soil-borne and apple tree-associated phytopathogenic fungi. Each primer set successfully detected 20 ng of target genomic DNA (gDNA) within 25 cycles, while the same amount of non-target gDNA mixture was detected only after 35 cycles of amplification. Moreover, target DNA amplification remained unaffected in the presence of mixed non-target gDNA background, confirming the high specificity of the primers. Sensitivity test showed that 1 fg of plasmid DNA, corresponding to about 290 copies, was detectable around 30 cycles with all primer sets. These primers support accurate pathogen detection and early diagnosis in various environmental samples.