Detection and Quantification of Avocado Sunblotch Viroid in California Avocado Orchards Using Digital Loop-Mediated Amplification and Droplet Digital PCR

Avocado sunblotch viroid (ASBVd) poses a significant threat to California's avocado industry. Early and accurate detection is crucial for disease management, but traditional detection methods lack sensitivity, highlighting the need for advanced molecular techniques. This study developed and validated a novel digital LAMP (dLAMP) assay for sensitive ASBVd detection, comparing its performance with droplet digital PCR (ddPCR) in California avocado orchards during the 2023 to 2024 season. Sixty-four samples of leaves, fruits, and flowers were collected from eight orchards across major California avocado-producing counties (Ventura, San Diego, Riverside), including symptomatic and surrounding asymptomatic trees (<i>n</i> = 33), as well as individual and pooled samples. Whereas RT-LAMP assays encountered inconsistencies, dLAMP and ddPCR proved effective for ASBVd detection. dLAMP assays, using various pooling strategies with serially diluted leaf and fruit samples, demonstrated a limit of detection (LOD) of 1 × 10^-3 ng/µl for RNA template. Pooling positive samples with negative ones increased the LOD, highlighting the potential impact of high-rate pooling on assay sensitivity. ddPCR demonstrated high sensitivity, detecting ASBVd down to 0.31 copies/µl, with varying copy numbers across tissues (fruits > leaves > flowers). dLAMP and ddPCR assays showed comparable ASBVd detection rates in orchard samples, with approximately 31 to 33% positivity (20/64 for dLAMP and 21/64 for ddPCR). These findings demonstrate the potential of dLAMP and ddPCR for sensitive and specific ASBVd detection and quantification in avocado orchards, providing valuable tools for disease surveillance, management, and insights into viroid distribution within California.