Progress 07/01/24 to 02/28/25

Outputs
Target Audience:The primary target audiences for this project arecommercial growers, nurseries, and plant breeders managing high-value horticultural crops. These stakeholders often work with plant material that is proprietary, heritage, or otherwise irreplaceable. Once infected with viruses or viroids, this material cannot be easily replaced, making virus elimination a critical step in maintaining crop viability. Stakeholders were engaged throughout the project through direct communication and collaboration, particularly those who contributed virus-infected plant material for testing and protocol development. These partnerships ensured that the research addressed real-world infection scenarios and informed the development of crop-specific cleaning protocols. Additional outreach efforts targeted growers and industry partners who have expressed concern about the limited availability of virus cleaning services and long wait times at existing public programs (NCPN centers). Several stakeholders described frustration with multi-year delays, which informed the development of a scalable, commercial virus cleaning platform. By increasing the availability of virus elimination services, this work responds to grower demand andhelps relieve pressure on overburdened public infrastructure. A secondary audience for this work included Qualterra's internal technical staff, who applied project results to refine protocols and develop standardized workflows for virus elimination. Training and knowledge transfer efforts were carried out throughout the project to support internal implementation and capacity building. Changes/Problems:Several challenges that we encountered, and successfully navegated, during the project are described below. Addressing these challengesled to adjustments that improved workflows and informed planning for Phase II. The first involved sourcing appropriate virus-infected plant material. Although we had strong support from collaborators, additional coordination was needed to determine the optimal timing and developmental stage for collecting explants suitable for tissue culture initiation. This resulted in a delayed start while we identified material that was both infected and suitablefor culture. In response, we've already begun working with partners to pre-identify suitable material ahead of Phase II to avoid similar delays. Due to the slightly delayed start, we prioritized completing all treatments for cherry, which had the most severe infection (high-titer Cherry Virus A). This allowed us to fully test individual and combination therapies in what we expected to be the most challenging case. Cherry results showed that combination treatments, particularly chemo + thermo, were highly effective where single treatments were less effective. While not all combination therapies were completed for every crop during Phase I, we successfully completed the full set of individual therapies--including chemotherapy, thermotherapy, and cryotherapy--for all four crops. These treatments were sufficient to produce clean plants in each case. Infections in apple, pear, and grape, while sometimes complex, were generally more responsive than cherry, with several individual treatments clearing all detectable viruses. Combination therapies in these crops will be further explored in Phase II to determine whether they can further improve clearance rates or reduce treatment duration. Cryotherapy in cherry initially did not result in any clean plants. As we progressed, we identified a likely issue in the timing of key steps in the cryotherapy protocol. This led to adjustments, which were implemented in subsequent applications for other crops. The improved protocol was successful in producing clean plants in those crops. Although we did not have time to reapply the updated cryotherapy protocol to cherry before the end of Phase I, the success observed in other crops strongly supports continuing cryotherapy trials in cherry during Phase II using the revised method. A final challenge was addressed through revision of our molecular testing approach. The original plan included testing at regular intervals using RT-qPCR for intermediate testing and sequencing for endpoint testing. However, due to the nature of the treatments and the quantity of plant material available for sampling, intermediate sampling was not feasible (it would have required destructive sampling). We therefore performed onlyendpoint sampling and screening using high-throughput sequencing, which provides high sensitivity for detecting viruses. For chemotherapy, we also compared two durations (3-week and 6-week), allowing us to observe changes in titer and treatment impact over time. These adjustments strengthened our virus cleaning platform and improved our readiness for Phase II. Lessons learned during this phase are already being applied to improve planning, execution, and scalability moving forward. What opportunities for training and professional development has the project provided?This project supported both hands-on training and formal professional development for research and technical staff engaged in virus elimination, tissue culture, and sequencing workflows. Two members of the project team participated in a multi-day cryopreservation workshop held in Colorado. This specialized training focused on droplet vitrification methods for horticultural crops and included instruction on the preparation of cryoprotectant solutions, shoot tip handling, exposure to liquid nitrogen, and post-treatment recovery. Participants successfully applied these methods to apple material during the workshop and later used the training to implement in-house cryotherapy protocols at Qualterra, including the acquisition and setup of all necessary equipment. This workshop served as a key professional development opportunity, directly influencing the expansion of treatment capacity within the project. In addition to the cryotherapy workshop, two project team members attended the American Phytopathological Society (APS) annual meeting. Participation in this national conference provided exposure to current research in plant pathology, opportunities for networking with peers and collaborators, and insights into emerging challenges and innovations related to virus management in horticultural systems. The primary focus of technical skill development during the project was on implementing and refining virus elimination treatments. Although staff had prior experience in tissue culture, internal knowledge-sharing helped adapt those skills to the specific requirements of virus elimination. Senior R&D scientists provided guidance to plant technicians on best practices for meristem excision, shoot tip selection, and post-treatment recovery. These informal training efforts were shaped by ongoing observations from the trials and were critical to maintaining consistency and viability across treatments. In addition to treatment-related training, a new scientist was trained in the post-sequencing analysis workflow used to assess treatment efficacy. This included quality control of long-read sequencing data, alignment of reads to a curated viral reference database, and normalization of viral read counts by genome size and sequencing effort. These skills enabled accurate comparison of viral titers across samples and supported data-driven protocol refinement. How have the results been disseminated to communities of interest?Preliminary results from this project have been shared through ongoing conversations with growers, nurseries, and industry partners--particularly those who contributed virus-infected plant material for testing. These discussions have focused on treatment outcomes, recovery of clean plant material, and priorities for expanding access to virus elimination services. Stakeholder engagement has also informed planning for the Phase II application, ensuring alignment between project goals and the needs of the horticulture industry. Participation in the American Phytopathological Society (APS) annual meeting supported technical exchange and relationship-building with researchers and professionals working in plant pathology, virus management, and clean plant systems. This engagement contributed to awareness of emerging challenges and innovations relevant to the project. Internally, results have been disseminated through collaborative discussions and protocol refinement. Findings from virus elimination trials directly informed improvements to tissue culture practices, recovery workflows, and sequencing-based detection methods. These outcomes were integrated into training efforts and contributed to consistent implementation of protocols across the team. In addition to technical engagement, the team has been conducting ongoing market research to identify prospective clients, assess demand across crop sectors, and explore potential partnerships. These efforts have contributed to strategic outreach and informed early-stage planning for future service delivery. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Impact Statement This project developed and tested methods for eliminating viruses from high-value horticultural crops, including cherry, apple, pear, and grape. Viral infections in these crops can cause significant losses, and because the infected plants are often genetically unique or proprietary, they cannot be replaced. We successfully applied and evaluated multiple virus-cleaning methods, generating protocols that can be used to restore valuable plant material to a healthy, virus-free state. The outcomes of this work help growers retain the genetic integrity of their crops, reduce dependency on limited public cleaning services, and ensure continued productivity and access to specialty varieties. As a result, growers will have access to better tools to manage viral disease and maintain a reliable, diverse, and resilient plant supply for the horticulture industry. Objective 1:Test and assess efficacy of multiple methods for virus elimination in multiple crops; identify best treatment regimen for each crop and develop internal protocols for virus elimination in each crop. Sub-objectives: 1.1 Conduct chemotherapy, thermotherapy, cryotherapy, and combination therapies 1.2 Monitor virus levels pre- and post-cleaning via high-throughput sequencing 1.3 Develop crop-specific virus cleaning protocols based on outcomes Major activities completed/experiments conducted: Virus-infected plant material was obtained from commercial partners for four crops: cherry, apple, pear, and grape. Infected explants were initiated into tissue culture and propagated to ensure genetically uniform material for parallel treatment. Treatments included chemotherapy (3 or 6 weeks), thermotherapy (step-up and sustained heat exposure), cryotherapy (droplet vitrification method), and combinations of these techniques. Treated shoot tips were excised and transferred to recovery media. Protocols for meristem excision and post-treatment culture were refined throughout the project. High-throughput RNA sequencing was used before and after treatment to evaluate viral infection status. Sequencing reads were mapped to a curated viral reference database, and normalized viral titers were calculated based on viral genome length, aligned read counts, and sequencing effort per sample and per run. Data collected: A minimum of 40 plants per treatment per crop were used for each virus cleaning experiment. For each crop-treatment combination, the following data were collected: survival rates were recorded following treatment, material from treated plants was sampled and sequenced (pre- and post-treatment), viral titer metrics were calculated, cleaned plants were identified based on absence of viral reads, protocol variables (e.g., chemotherapy duration, temperature cycles) were logged, long-read sequencing data was obtained, and normalized viral titer was calculated for each sample. Summary statistics and discussion of results: Survival rates were recorded for all treatments and considered alongside viral elimination efficacy. Across all crops and treatment types, survival met or exceeded the minimum practical threshold of 25% typically required for successful recovery and propagation of cleaned plant material. In most cases, survival ranged between 40-85%, depending on the crop and treatment intensity. Combination therapies, particularly those involving extended chemotherapy or cryotherapy, were associated with the lowest survival rates (as low as 28% in some cases), while single treatments--especially thermotherapy and three-week chemotherapy--generally showed higher survival. Despite differences in survival across treatments, all experiments yielded sufficient viable explants for downstream recovery, sequencing, and assessment of virus status. Survival outcomes were used alongside viral titer results to guide protocol prioritization for future scale-up. For each crop, one or more treatments produced clean plants (i.e., with no detectable viral reads). Most treatments resulted in a measurable reduction in mean viral titer across all sampled individuals, and several treatments fully eliminated infection in 100% of tested samples. While not every treatment resulted in universal clearance, most yielded a subset of clean individuals, confirming that clean material can be recovered across a range of conditions. Cherry (Gisela 5): Infected with high-titer Cherry Virus A (CVA). All tested therapies led to titer reduction. Chemo + thermo (both 3- and 6-week versions) eliminated CVA in 100% of samples. Thermotherapy alone eliminated viruses in 79% of surviving plants. Six-week chemotherapy eliminated viruses in 52% of samples; three-week chemo eliminated viruses in 8% of samples. Cryotherapy reduced titer but yielded no clean samples; the cryo protocol will be repeated in Phase II following timing adjustments. Apple (Nic29): Infected with moderate-titer Apple Chlorotic Leaf Spot Virus (ACLSV). All individual therapies (3- and 6-week chemo, thermo, and cryo) eliminated the virus in 100% of tested plants. Combo therapies were not tested due to efficacy of individual therapies. Pear (proprietary variety): Infected with a mix of medium-, low-, and very low-titer viruses (ASGV, ASPV, AGCAV, ACLSV). Both chemotherapy andcryotherapy individual treatments eliminated viruses in 100% of tested plants. Individual thermotherapy reduced mean viral titer overall and achieved full elimination of viruses in 53% of samples, and when used in combination with cryotherapy (thermo + cryo), viruses were eliminated in 100% of samples. Grape (proprietary variety): Infected with high-titer viroid (GYSVd1) and low-titer virus (GBNIV). Six-week chemotherapy and thermotherapy eliminated GBNIV in all treated samples. GYSVd1 titer was significantly reduced, and complete viroid elimination was achieved in 75% of chemo-treated and 54% of thermo-treated plants. Across all crops, clean plants were recovered from at least one treatment, and all major therapies produced virus-free material in one or more crops. Treatments with higher survival and virus elimination rates were prioritized for future optimization. Combination therapies were especially effective in challenging high-titer infection(like that seen in cherry), while single treatments were generally sufficient in crops with lower viral loads. Key outcomes or other accomplishments realized: Development of crop-specific virus cleaning protocols for cherry, apple, pear, and grape, including both individual and combination therapy regimens. Demonstration of successful virus elimination in all tested crops and infection types, with clean plants recovered under at least one treatment for each crop. Demonstration of the effectiveness of high-throughput sequencing (HTS) for measuring viral titer and confirming post-treatment virus status across diverse pathogens. Establishment of in-house thermotherapy and cryotherapy systems, including optimized protocols and supporting infrastructure for treatment and recovery. Implementation of refined tissue culture methods for meristem excision and post-treatment plant recovery, improving survival rates across treatments. Generation of clean plant material and physical collections for follow-up testing and long-term validation of clean status.

Publications