Development of Two SYBR Green Quantitative Real-Time PCR Assays for the Detection and Quantification of...

Several fungal species in the Botryosphaeriaceae family are associated with trunk diseases of major agricultural crops, such as almond and grapevine. These diseases can lead to considerable yield losses in vineyards and orchards. Botryosphaeriaceae can also infect plant material and seedlings during nursery operations. Latent infections in contaminated propagation materials play an important role in the spread of these fungal pathogens. Accurate identification and early detection of these pathogens are crucial for improving disease management. Molecular tools, such as real-time PCR, are widely used as rapid and sensitive approaches to improve disease diagnostics, allowing early detection and quantification of target pathogens. In this study, we developed two SYBR Green qPCR assays for the specific detection and quantification of Botryosphaeria dothidea and species within the Neofusicoccum parvum-ribis complex. Assays were validated using artificially inoculated and naturally infected grapevine and almond plants. We assessed the specificity of the assays on 79 isolates, including various Botryosphaeriaceae and off-target species, such as other pathogens associated with grapevine trunk diseases. Target pathogens were successfully detected in all artificially inoculated plants, as well as in most naturally infected field samples tested. These results demonstrate that both SYBR Green qPCR assays are specific, selective, and sensitive, validating their use for detecting B. dothidea and N. parvum-ribis species in wood samples. This cost-effective tool can be applied for accurate detection of these pathogens from both symptomatic and asymptomatic plants, enabling large-scale analyses and early screening to promote the production of healthy planting material.