Recipient Organization
UNIVERSITY OF WISCONSIN SYSTEM
3203 N DOWNER AVE STE 273
MILWAUKEE,WI 532113153
Performing Department
(N/A)
Non Technical Summary
Non-Technical SummaryThe citrus industry is facing a severe threat from a disease known as Huanglongbing (HLB). Since its discovery in the U.S. in 2005, HLB has caused significant economic harm to citrus growers, resulting in billions of dollars in losses and threatening the viability of citrus farming. Existing treatments, such as antibiotics and pesticides, have had limited success and pose environmental risks. Given these challenges, the citrus industry urgently needs new, sustainable solutions to combat HLB. Addressing this issue is critical not only for protecting the livelihoods of citrus farmers but also for ensuring the continued availability of citrus products, reducing the environmental impact of current disease management strategies, and maintaining economic stability in rural communities that depend on citrus farming.To tackle this problem, this project aims to develop and implement a natural biopesticide, RejuAgro A (RAA), which is produced by the bacterium Pseudomonas soli. The project will test the effectiveness of RAA in managing HLB through trunk injection, where the biopesticide is delivered directly into the tree's vascular system. This method is expected to be efficient, targeting the disease site directly and reducing the risk of environmental contamination. Alongside improving the treatment method, the project will focus on scaling up RAA production to ensure it can be manufactured and applied at a commercial scale by citrus growers. Researchers will conduct large-scale field trials to evaluate RAA's effectiveness in reducing bacterial load in infected trees, improving fruit yield, and maintaining tree health.Thegoal of the project is to create a biopesticide that can be widely adopted by citrus growers, protect the environment from further degradation, and support the long-term economic health of citrus-growing communities. The success of this project would mark a significant advancement in agricultural practices and offer a sustainable solution for managing HLB.
Animal Health Component
40%
Research Effort Categories
Basic
30%
Applied
40%
Developmental
30%
Goals / Objectives
The overarching goal of this project is to develop and commercialize a novel biopesticide, RejuAgro A (RAA), for the management of Huanglongbing (HLB), a devastating disease affecting the citrus industry. This research focuses on optimizing the liquid formulation of RAA for trunk injection in citrus trees to control HLB and sustain citrus tree health and productivity over the tree's lifespan. By leveraging the unique properties of RAA, including its classification as a biopesticide and its dual action as an antimicrobial agent and a defense activator in citrus, this project aims to offer a sustainable, effective, and environmentally friendly alternative to existing treatments like oxytetracycline (OTC) and streptomycin (STR).ObjectivesEnhance RAA production through metabolic engineering of Pseudomonas soli T307:This objective seeks to maximize the yield of RAA by optimizing the biosynthetic pathway and employing genetic and chemical inducers to make production scalable and cost-effective. We will investigate the effects of temperature and chemical inducers (e.g., L-methionine, L-glutathione) and use metabolic rewiring techniques to upregulate the RAA biosynthetic gene cluster (ras operon) for increased production.Develop a strategic scale-up and robust formulation for commercial readiness:This objective involves scaling up RAA production from laboratory to industrial levels. We will conduct pilot studies to refine fermentation processes and develop a cost-effective liquid formulation for trunk injection in citrus trees. The goal is to create a commercially viable, EPA-recognized end product that can be deployed in large-scale agricultural settings.Evaluate the long-term efficacy of RAA in managing HLB through large-scale field trials:We will conduct extensive field trials in Florida to test different dosages and frequencies of RAA application via trunk injection. The field trials aim to demonstrate the ability of RAA to reduce CLas titers, maintain tree health, and improve fruit yield and quality over multiple years of treatment. Data on tree growth, fruit production, and defense marker gene activation will also be collected.Advance stakeholder engagement and educational outreach:This objective focuses on extending the findings to the citrus industry and educating growers on the use of RAA for HLB management. We will develop outreach materials, participate in industry events, and ensure direct communication with stakeholders. Surveys will be used to assess the adoption rate of RAA and gather feedback on the treatment's effectiveness in real-world conditions.By addressing these objectives, the project will help combat the spread of HLB, reduce reliance on clinical antibiotics in agriculture, and promote the adoption of sustainable disease management practices in the citrus industry.
Project Methods
This project focuses on the development, optimization, and dissemination of the biopesticide RejuAgro A (RAA) to combat HLB in citrus trees. The methods employed throughout the project involve a combination of field trials, metabolic engineering, and extension efforts aimed at reaching the intended audiences--citrus growers, researchers, regulatory agencies, and industry stakeholders. The following sections detail the scientific methods, educational efforts, and evaluation plans that will be used to achieve the project's goals.1. Scientific MethodsThe project utilizes a comprehensive, multidisciplinary approach that includes the following scientific methods:A. Metabolic Engineering of Pseudomonas soli T307To increase the production of RAA, the project employs metabolic engineering techniques. The steps include:Gene Cluster Identification and Manipulation: Using bioinformatics tools like AntiSMASH, the project identified the biosynthetic gene cluster responsible for RAA production. Targeted gene mutations were introduced to enhance or disrupt specific genes, followed by metabolic rewiring to maximize RAA output.Overexpression of Biosynthetic Pathways: Plasmids containing the biosynthetic gene cluster will be introduced to increase expression and optimize RAA production. This method will be tested at small (flask) and larger (fermenter) scales.Optimization of Culture Conditions: Different temperatures, nutrient conditions, and chemical inducers (e.g., L-methionine and L-glutathione) will be tested to identify the best conditions for maximizing RAA yield.B. Formulation and Scale-UpThe project employs pilot studies to scale up RAA production from laboratory fermenters (30L) to larger industrial fermenters (500L and 1,000L). The process involves:Centrifugation and Concentration: Disc stack centrifugation and falling film evaporation will be used to concentrate RAA from fermentation broth, improving efficiency for large-scale production.Product Development: The final liquid formulation will be optimized for trunk injection, a more effective delivery method compared to foliar sprays.C. Field TrialsLarge-scale field trials were conducted in Florida to evaluate the efficacy of RAA in managing HLB. Methods included:Trunk Injection: RAA was applied through trunk injections at various dosages and frequencies to determine the optimal conditions for reducing the bacterial population (CLas) and improving tree health.Sampling and Analysis: Leaf samples were collected before and after treatment, and DNA was extracted for qPCR to measure changes in CLas titers. Phytotoxicity, yield, and fruit quality were also monitored.Data Collection: Data was gathered on changes in CLas titers, tree health metrics, and fruit yields, as well as the effectiveness of trunk injection compared to other treatments like oxytetracycline.2. Efforts to Cause Change in Knowledge, Actions, and ConditionsTo ensure the project's findings reach the intended audiences and effect changes in knowledge, actions, and conditions, several key educational and outreach efforts will be employed:A. Workshops and Field DemonstrationsWorkshops and field demonstrations will be organized for citrus growers, agricultural extension agents, and other stakeholders. These events provide training on RAA application via trunk injection and help translate research findings into practical tools for managing HLB in the field.B. Extension and OutreachThe project will partner with local agricultural extension offices to distribute science-based knowledge to growers and industry stakeholders. Educational materials, newsletters, and reports were shared through extension channels.C. Formal and Informal EducationGraduate students participated in internships, lab work, and field trials, providing them with practical experience in biopesticide research and HLB management. Additionally, researchers will present their findings at scientific conferences and publish papers in peer-reviewed journals to advance scientific knowledge.D. Collaboration with IndustryThe project works closely with agricultural companies to scale up RAA production and make it commercially viable. Presentations at industry trade shows, such as the Citrus Expo, help promote the adoption of RAA in commercial citrus production.3. Types of Data Collected for EvaluationSeveral types of quantitative and qualitative data will be collected to evaluate the project's outcomes:Scientific Data: qPCR data on CLas titer reduction, fruit yield measurements, and phytotoxicity evaluations will provide quantitative evidence of RAA's efficacy.Survey Data: Surveys of citrus growers and extension agents will collect feedback on their knowledge, practices, and the likelihood of adopting RAA treatments.Economic Data: Data on the costs of RAA production, the cost savings for growers, and the economic benefits of increased fruit yields will be collected and analyzed.Regulatory Data: Information on the regulatory approval process, including timelines and feedback from the EPA and USDA, will be documented.By integrating these methods, efforts, and evaluations, this project aims to achieve its goals of providing a sustainable, effective solution for managing HLB and promoting long-term citrus industry resilience.