Location: Subtropical Plant Pathology Research
Project Number: 6034-22000-045-028-I
Project Type: Interagency Reimbursable Agreement
Start Date: Jan 1, 2025
End Date: Dec 31, 2027
Objective:
Objective 1. Characterizing the NMV-M citrus isolate, including its host range and transmission. Objective 2. Determining the molecular mechanisms underlying its HLB-suppressing effects. Objective 3. Using NMV-infected citrus to prevent or suppress HLB in greenhouse and field trials. Objective 4. Developing an expression vector of NMV-M/CFL with antimicrobial peptides and/or siRNA; Objective 5. Conducting extension and outreach activities to inform stakeholders of project results and obtaining permits for commercialization.
Approach:
Objective 1. 1a. Our preliminary data indicates that the virus can be detected in ACP at relatively high titers. We will focus our transmission study in part on major citrus-feeding insects, such as ACP and leafhopper, and determine the virus’ host range by graft and dodder transmission. We will use electron microscopy to illustrate how both the virus and Las bacteria co-exist in a phloem cell and how the virus attenuates the phloem cell destruction caused by Las; 1b. Surveys in the major citrus producing regions will be performed to assess the natural distribution and incidence of NMV-M in different citrus cultivars. Objective 2. The completed genome and transcriptome data of Red Ruby grapefruit and Valencia sweet orange derived from bud-sport selections, will be used to conduct a time course gene-expression profiling to reveal potential citrus target gene(s) that interact with the virus and Las in the HLB complex network. We will use other molecular tools for protein-protein interactions such as a pull-down assay to screen potential targets using the NMV-M/CFL coat protein and its antibody. Objective 3. The systemic distribution of NMV-M/CFL will be evaluated among different varieties. We have identified two HLB resistant/tolerant scions and two rootstocks as the best combinations of virus and host citrus for enhancing HLB resistance. We will expand the screening to all important commercial citrus varieties to provide more virus and host combinations for field trials. We will further conduct a small-scale field trial of new planting or grafting NMV-M to HLB-affected plants at the USHRL Picos farm to evaluate the prevention and/or revitalization of HLB-affected plants. Objective 4. 4a. We will synthesize the cDNA of this virus based on its complete genome sequence and generate an infectious cDNA clone of the virus and an expression vector following similar cloning strategies as described in Matsumura et al., 2018 and 2022; 4b. We will generate the expression vector with the antimicrobial peptide, such as the stable antimicrobial peptide (SAMP) identified from Australian finger lime and siRNA; 4c. We will initiate a permit application for commercializing the developed technology in Florida. Objective 5. 5a. We will organize a field demonstration event in Florida at USHRL Picos Farm to showcase NMV-M/CFL-induced prevention and/or suppression of HLB to stakeholders and citrus growers. We will present our progress at major grower meetings, outreach activities, and regular trade journal articles. News, results, and progress will be published on conventional and/or social media platforms. Project updates will be disseminated to grower associations in U.S. citrus-producing states through extension agents/cooperators. Project results will be shared via scientific meetings, such as the American Phytopathological Society conference and the International Research Conference on HLB.
