Performing Department
(N/A)
Non Technical Summary
California is the undisputed champion of avocado growing in the US, producing 95% of the country's crop worth around $400 million annually. Avocado industry has been impacted byAvocado Sunblotch Viroid Disease (ASBVd) for over a century. While ASBVd doesn't kill the trees, it stunts their growth and slashes fruit production by a massive 95%, impacting the fruit's appearance and marketability. Detecting ASBVd is currently difficultdue to the uneven spread of the disease within trees and the existence of infected but symptomless trees. One of the major challenges to preventing the spread of ASBVd is the lack of ideal rapid, accurate, and sensitive detection of pathogen in trees and nursery materials. This proposalfocuses on developing a fast, accurate, and high throughput method, referred to as ANC-NANO, to detect ASBVd in avocado nurseries and avocado orchards in California to prevent the spread of the disease; and to detect the disease in honeybees (avocado pollinators) to help early detection of ASBVd in orchards. While the technical details involve recombinase polymerase amplification, DNA to nanoparticle conversion, and nanopore array nanoparticle obstruction, the key takeaway is that ANC-NANO can find even the tiniest traces of viroid down to 1 copy/mL in hundreds of samples simultaneously. This method can also be used for sensitive and multiplexed detection of other fungal and viral avocado diseases. This revolutionary technology has the potential to transform not just avocado disease diagnosis, but plant disease detection as a whole, potentially leading to breakthroughs in plant pathology field.
Animal Health Component
0%
Research Effort Categories
Basic
(N/A)
Applied
100%
Developmental
(N/A)
Goals / Objectives
This project focuses on developing a fast, accurate, and high throughput method to detect avocado sunblotch viroid disease (ASBVd) in avocado nurseries and avocado orchards in California to enable intervention in disease epidemiology and prevent the spread of the disease; and to combine the developed method with the ability of honeybees to serve as a surveillance tool to enable early diagnosis in symptomatic and asymptomatic trees. This method is based on recombinase polymerase amplification (RPA), DNA to nanoparticle conversion and nanopore array nanoparticle obstruction referred to as ANC-NANO system. ANC-NANO is inspired by existing challenges and the latest research efforts (and their drawbacks) in pathogenic viroid/viral detection. This proposed detection scheme combines advantages of RPA, DNA/RNA to nanoparticle conversion and nanoparticle obstruction-based detection andallows for high-throughput multiplexed detection.Since ASBVd spreads through pollen, insects acting as pollinators can carry the viroid's RNA from tree to tree. In Southern California avocado groves, a diverse community of around 60 insect species visit avocado flowers for pollination. To facilitate this process, avocado growers often collaborate with beekeepers to place beehives within the orchards during flowering season. This proposed method, ANC-NANO, leverages the unique ability of honeybees to collect pollen from numerous avocado trees, effectively creating a large-scale sample. This, combined with the high sensitivity of ANC-NANO, allows for early detection of ASBVd in honeybees. Early detection serves as a reliable surveillance tool for the avocado industry, enabling growers to take swift action and limit disease spread within their orchards. New varieties or rootstocks of nurseries can introduce sunblotch into avocado industry, so nursery propagation techniques and grove management strategies exert influence on the severity of the disease in California. To exercise careful propagation of nursery stock and to reduce sunblotch to a relatively minor disease, an ongoing monitoring, detection, and diagnosis approach is required in nurseries and established groves. We will use our developed platform to perform high-throughput detection of avocado sunblotch viroid in leaves, fruits, and propagative plant materials in nurseries and avocado orchards in California.Our specific objectives are as following:Developing and establishing the ANC-NANO system for rapid and ultrasensitive RNA detection of ASBV and comparing the system to the established ddPCR method. Surveillance for Avocado Sunblotch Viroid using the European honeybee (Apis mellifera)and tree pollen.High-throughput detection of Avocado Sunblotch Viroid in nurseries and avocado orchards in California with the ANC-NANO system.
Project Methods
Aim 1: Developing and establishing the ANC-NANO protocol for rapid and ultrasensitive RNA detection of viroids and comparative analysis with ddPCR.First step is to develop RNA to nanoparticle conversion protocol. The success of this technique hinges on the specificity of two key steps: the RPA reaction and the cDNA-to-nanoparticle conversion. The conversion process must reliably generate a sufficient number of DNA-bound charged nanoparticles that can be readily separated and detected by electrophoresis-driven nanoparticle obstruction. To optimize this conversion protocol, we will use avocado sunblotch viroid RNA as our target RNA. To achieve efficient, one-step RNA to cDNA conversion within the RPA temperature range, we will incorporate a compatible reverse transcriptase. Additionally, we will optimize the reaction conditions by fine-tuning the ionic components, concentrations, and primer length to ensure rapid and specific cDNA amplification. Furthermore, we will optimize the amplified DNA fragment length and PNA nanoparticle size. This optimization ensures sufficient DNA copies are generated during the 20-minute RPA reaction, while the resulting DNA-bound nanoparticles possess sufficient charge for effective separation from unbound nanoparticles using electrophoresis. Concurrent with optimizing the DNA-nanoparticle conversion protocol, we will refine the detection scheme based on nanopore array nanoparticle obstruction using a dual approach; fluorescence microscopy and electrical measurement. Then, we will validate the correlation between the initial linear slope of the nanoparticle obstruction profiles and the concentration of nanoparticles. Through these investigations, we will be able to identify the maximum number of nanopores in a single nanopore array.Following the establishment of conversion and detection protocols, we will validate the system's ability to detect ASBVd RNA using controlled samples. We will target a specific viroid genome segment and prepare a series of cDNA solutions in buffer solutions with concentrations ranging from 0 to 100,000 copies/mL. Utilizing ANC-NANO for RNA detection, we will establish a correlation between the target RNA concentration and the measured nanoparticle obstruction. Finally, to make a comparative analysis with ANC-NANO, we will develop a reverse transcription droplet digital PCR assay for sensitive detection of ASBVd.Aim 2: Bee and Pollen Sample Collection and ASBVd detectionDuring avocado flowering season,beehives will bestrategically placed within and around (at distances of 100m, 200m, and 400m) confirmed ASBVd-infected trees in at least four avocado orchards.To test if bee-collected pollen harbors detectable ASBVd, we will collect honeybees will be collected from both infected trees and the hives themselves. We will collect approximately 100 worker bees from up to six hives per orchard using established methods (opening hives and rolling an open tube over a frame of bees). Additionally, a handheld insect vacuum will be used to collect at least 40 bees directly from flowers of diseased trees. All bee samples will be transported to the lab for further analysis.Following established protocols, we will collect pollen samples from 10 randomly chosen cells within each hive across the tested orchards. Using a spatula, pollen will be transferred to microcentrifuge tubes and stored at -20°C until further processing.Optimizing RNA extraction methods is crucial for both pollen and bee samples. We will develop protocols for extracting RNA from individual bees and from pooled samples of approximately 50 bees per hive. Adult bee samples (individual or pooled) will be homogenized in a phosphate-buffered saline (PBS) buffer followed by RNA extraction using a manual method or commercially available RNA extraction kits. Extracted RNA from both pollen and bee samples will then undergo the optimized RT-RPA reaction, DNA-nanoparticle conversion, and subsequent analysis using the nanopore-array device for viroid detection through nanoparticle blockage.Aim 3: Surveillance for ASBVd in avocado orchards in Southern California3-1: Development of the ASBV disease detection survey for avocado growersTo identify potential ASBVd infection zones within California avocado-producing regions, we will develop an anonymous paper or email survey targeting avocado growers. The survey will inquire about the presence of typical ASBVd symptoms within their orchards.Survey Distribution:Collaboration:We will collaborate with Pest Control Advisers (PCAs), farm advisors, and cooperative extension agents to ensure the survey reaches a broad audience.Target Audience:The survey will be distributed to avocado growers, California Avocado Commission members, orchard and nursery managers, and other relevant stakeholders.Data Analysis:Data Collection:Survey responses will be collected and analyzed statistically to determine the incidence rate of ASBVd across California.3-2: Sample collectionOnce we received the survey results and analyzed data to locate infected trees, prior to doing any diagnostic tests for ASBVd, we will inspect the trees for symptoms and when recognized, we will sample and test the diseased trees individually along with neighboring symptomatic or asymptomatic trees either in the same or adjacent row, since there is indication for transmission of the viroid by natural root grafting. Given the uneven distribution of viroid in the tree, a proper sampling approach is of importance, thus, while we will work on developing an effective sampling strategy, to economize, we will use both individual samples and pool leaves from within and amongst different trees into batches for detection testing.3-3: High-throughput detection of avocado samples from avocado orchards and nurseries in Southern California using ANC-NANOTo demonstrate the super capability of high-throughput ASBV detection of ANC-NANO, we will create a 12x8 nanopore-array array device that is compatible with commercially available automatic pipetting system and can simultaneously handle up to 96 samples. We will first optimize an effective and high throughput RNA extraction from various avocado tissues. RNA solution extracted from each individual or batch of avocado samples collected from avocado orchards or nurseries, will then be detected by the ANC-NANO strategy using the new 12x8 nanopore-array array platform.This project will significantly impact the overall knowledge of the avocado industry on ASBV disease by sharing our findings with nurserymen, growers, farm advisors and extension agents. We will achieve this through various outreach activities, including presentations at industry meetings (national, regional, and local), workshops, field days, and publications. Collaborating with University of California Cooperative Extension, we will publish in existingnewsletters, bulletins, and factsheets to educate growers and nursery managers on disease diagnosis, pathogen control, and ultimately, avocado production free of sunblotch viroid disease. Additionally, our research will be published in peer-reviewed and trade journals, reaching a wider audience, and contributing to the scientific community.By developing our nanopore array for sensitive detection of plant diseases, screening pollen and honeybee samples, and enabling on-site detection in avocado nurseries and orchards, this project ensures its sustainability and ushers in a new era for ASBVd detection in California.