Mehdi Kamali, PhD (he/him/his)
Assistant Specialist
Department of Microbiology & Plant Pathology, University of California, Riverside
Riverside, California, United States
Li Liu
Department of Chemical and Environmental Engineering, University of California, Riverside
Riverside, California, United States
Zahra Pegahrad, Undergrad
University of California, Riverside
Riverside, California, United States
Valentina Valencia Bernal
Graduate Student
University of California, Riverside
Riverside, California, United States
Ke Du
Department of Chemical and Environmental Engineering, University of California, Riverside
Riverside, California, United States
Fatemeh Khodadadi, PhD
Department of Microbiology & Plant Pathology, University of California, Riverside
Riverside, California, United States
Avocado sunblotch viroid (ASBVd) presents a considerable challenge to the California avocado industry. Given the limitations in sensitivity of conventional detection methods, the development and implementation of advanced molecular diagnostics are essential for effective disease management. A novel Digital Loop-Mediated Amplification (dLAMP) assay was developed and validated for sensitive ASBVd detection. dLAMP performance was compared to droplet digital PCR (ddPCR) using 64 leaf, fruit, and flower samples collected from eight California orchards (Ventura, San Diego, Riverside) during the 2023-2024 season. Samples included symptomatic trees (n=6), surrounding asymptomatic trees (n=33), and individual/pooled samples. Although LAMP yielded inconsistent results, both dLAMP and ddPCR, effectively detected ASBVd. Using serially diluted leaf and fruit samples in various pooling strategies, dLAMP achieved a limit of detection (LOD) of 1 x 10⁻³ ng/µL of RNA. Pooling positive samples with negative controls increased the LOD, showing the effect of high-rate pooling on assay sensitivity. ddPCR demonstrated high sensitivity, detecting ASBVd down to 0.31 copies/µL (6.20 copies/reaction), with higher copy numbers in fruits than leaves or flowers. Both assays showed comparable ASBVd detection rates in 64 orchard samples, with approximately 31-33% positivity. This study demonstrates the potential of dLAMP and ddPCR for the sensitive and specific detection and quantification of ASBVd in avocado orchards, offering valuable tools for disease surveillance and management within California.