Inter-laboratory validation of high throughput sequencing for the detection of viruses and viroids in grapevine, apple and stone fruits

Authors: Abrahamian, Peter; CAI, WEILI - Animal And Plant Health Inspection Service (APHIS); NUNZIATA, SCHYLER - Animal And Plant Health Inspection Service (APHIS); STEVENS, KRISTIAN - University Of California, Davis; HU, XIAOJUN - Animal And Plant Health Inspection Service (APHIS); HWANG, MIN SOOK - University Of California, Davis; COSTA, LARISSA - Animal And Plant Health Inspection Service (APHIS); BELANGER, CHARLES - Animal And Plant Health Inspection Service (APHIS); ATHA, BENJAMIN - Animal And Plant Health Inspection Service (APHIS); YANG, YU - Animal And Plant Health Inspection Service (APHIS); NOUROLAH, SOLTANI - University Of California, Davis; HURTADO-GONZALES, OSCAR - Animal And Plant Health Inspection Service (APHIS); AL RWAHNIH, MAHER - University Of California, Davis; RIVERA, YAZMIN - Animal And Plant Health Inspection Service (APHIS)

Submitted to: PhytoFrontiers
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/25/2025
Publication Date: 6/25/2025
Citation: Abrahamian, P., Cai, W., Nunziata, S., Stevens, K., Hu, X., Hwang, M., Costa, L., Belanger, C., Atha, B., Yang, Y., Nourolah, S., Hurtado-Gonzales, O., Al Rwahnih, M., Rivera, Y. 2025. Inter-laboratory validation of high throughput sequencing for the detection of viruses and viroids in grapevine, apple and stone fruits. PhytoFrontiers. https://doi.org/10.1094/PHYTOFR-03-25-0025-R.
DOI: https://doi.org/10.1094/PHYTOFR-03-25-0025-R

Interpretive Summary: High-throughput sequencing (HTS) is emerging as a powerful tool for detecting plant pathogens, offering the ability to identify unknown viruses that conventional diagnostic methods often fail to detect. It represents a significant advancement for post-entry quarantine programs, but its broader use faces challenges such as high costs, risks of contamination, and the need for standardized validation. This study evaluated an RNA-seq method for detecting plant viruses and viroids in apple, grape, and stone fruits like peach and cherry across three U.S. laboratories. Samples included 33 viruses and 6 viroids, prepared with a standardized control virus and tested using a consistent protocol for nucleic acid extraction and library preparation. All three labs detected the expected pathogens athough detection rates declined with fewer reads. A low false-positive rate was observed, primarily in grapevine samples. HTS results were compared to PCR-based methods, with HTS showing strong reliability when data quality was sufficient.The study highlights HTS as a transformative diagnostic tool for supporting plant health, capable of detecting multiple pathogens simultaneously. However, its implementation depends on meeting specific criteria such as maintaining data quality, ensuring consistency in lab protocols, and requiring expert analysis for accurate interpretation. These findings provide a solid foundation for adopting HTS in plant pathogen detection, particularly in quarantine and certification programs.

Technical Abstract: High-throughput sequencing (HTS) is increasingly being used as a diagnostic tool for plant pathogen detection. HTS has demonstrated to be a paradigm shift for post-entry quarantine programs by increasing their ability to detect unknown plant viruses that have remained undetected by conventional diagnostic tools. However, wide adoption of HTS-based diagnostics faces several hurdles such as a relative high cost, increased risk of contamination, and a lack of validation. The use of HTS as a diagnostic test can be divided into eight different processes, all which influence its performance and the reported diagnoses. In this study, we validated the TruSeq stranded Total RNA with Ribo-depletion library preparation protocol for plant virus and viroid detection in three woody plant groups across three U.S.-based laboratories. The samples used here included apple, grape, and several Prunus spp. (peach, cherry, nectarine, apricot, and plum) that together represented 33 viruses and 6 viroids. Proficiency (blind) sample panels of each crop group were prepared from petiole and budwood tissue, spiked with a standardized quantity of Phaseolus vulgaris cv. Black turtle soup (BTS) endornavirus as exogeneous positive control, and distributed to each laboratory. In addition, composite samples of infected plant tissue with multiple viruses were diluted into healthy plant tissue in several proportions and evaluated via HTS. All three laboratories used a standardized set of guidelines for quality assessment, including the same nucleic acid extraction and library preparation protocols during the entire process. HTS-based detection of the expected viruses and viroids on each tested sample was achieved by all three laboratories. When normalizing all datasets, 100% virus detection rate was achieved in datasets containing between 10 M to 15 M reads but decreased with lower number of reads. A low false-positive detection rate was observed only on grapevine samples. HTS results were also compared against real-time PCR and conventional PCR-based detection. Overall, our study provides a robust analysis of suitability for HTS as a reliable tool for virus and viroid detection in the evaluated crops upon meeting the criteria of a minimum of 15 M reads and subject matter expert interpretation.