Recipient Organization
UNIVERSITY OF FLORIDA
G022 MCCARTY HALL
GAINESVILLE,FL 32611
Performing Department
(N/A)
Non Technical Summary
The project is focused on accelerating the delivery of conventionally developed HLB tolerant citrus scions and rootstocks as pathogen-free budlines for replicated multi-site testing by adopting an alternating temperature treatment. This simple technology will reduce the current clean-up time by 1.5-2 years and significantly increase the number of selections that can be processed. The project will utilize extensive collections of novel HLB tolerant/resistant citrus germplasm generated by the UF-CREC breeding team, including many that have the potential to create new niche/specialty marketing opportunities for national citrus growers. For example, new cold-hardy selections would be available for north Florida and other states like Georgia. Future plantings will be less susceptible to tree decline and crop losses, creating a more stable and expanded citrus industry. Using pathogen-free HLB tolerant scion/rootstock combinations reduces fruit production costs, including pesticide and fertilizer applications, which would positively impact human health and the environment. Commercial use of the high-quality and unique HLB-tolerant scions and rootstocks will provide Florida growers with more sustainable and profitable alternatives to replace the hundreds of acres currently going out of production due to HLB. Growers, processors, packers, and marketers will all benefit, and the project will also accelerate the delivery of new unusual and lucrative niche-market varieties. In short, developing a reliable rapid protocol for generating pathogen-free material with no rejuvenation is necessary for the timely delivery of these new scions and rootstocks to our ravaged Florida citrus industry.
Animal Health Component
100%
Research Effort Categories
Basic
(N/A)
Applied
100%
Developmental
(N/A)
Goals / Objectives
Florida field-tested Huanglongbing (HLB) resistant/tolerant scions and rootstocks by using an alternating temperature treatment. The project is an elevated risk/high reward that would enable HLB tolerant/resistant germplasms from different citrus genetic backgrounds, which have been developed by conventional breeding (not genetically modified or CRISPR manipulated genomes), to serve as resources for breeding programs and for dissecting the genetic basis of resistance to HLB. The results of this project would expedite large multi-site field testing in Florida necessary to determine the actual value of HLB resistant/tolerant scions and rootstocks.The four objectives planned to accomplish the goals of the project are:Identify and Propagate elite conventionally bred field-tested HLB tolerant citrusApply alternating temperature treatments to induce healthy growth from diseased plantsConfirm HLB and CTV-free cultivars after alternating temperature treatmentsPropagate pathogen-free cultivars to be validated by the Florida PTP battery of tests
Project Methods
?The four objectives are strongly interrelated. The project team members will assemble an outstanding group of scientists with expertise in different technology areas. PD will identify the most promising scions and rootstock to be cleaned up based on his 30+ years of citrus breeding (Objective 1). qPCR and ELISA will be used to confirm the infection of propagated materials before treatment (Co-PIs - Objective 1). Pre-treatment and post-treatment qPCR and ELISA to verify that the reproduced materials are pathogen-free (Co-PIs - Objective 2, 3, and 4).The citrus breeding program at the UF/CREC conducts several fruit/juice displays every year and invites different stakeholders to evaluate the new citrus cultivars in all the mentioned categories. New selections identified with potential for specialty crop niche markets, the fresh market industry, or the processing industry are identified by input provided by participating citrus growers and stakeholders, but unfortunately, there is no pathogen-free budwood for the immediate release of these cultivars. Besides, many of these new selections have been standing well in the field under intense HLB pressure. Since HLB became a problem, the CREC Citrus Improvement Program began a new 'gauntlet' screening program to quickly identify new rootstock hybrids with the best potential to mitigate or eliminate HLB from any grafted scions (Grosser et al. 2020). So far, more than 15,000 hybrid seedlings encompassing tremendous genetic diversity have been screened, and promising selections are now being identified. These selections require immediate large-scale testing to help save all types of citriculture in Florida, and pathogen-free material is needed to make this happen. Thus, developing a reliable rapid protocol for generating pathogen-free material with no rejuvenation is necessary for the timely delivery of these new scions and rootstocks to all phases of our citrus industry.The selection of the scion cultivars that will be included in this project for the generation of pathogen-free budlines will depend on the stakeholder's evaluations to meet their demand or that fill potential new specialty niches. All the outcomes from this project expedite the potential benefits of new varieties to the citrus industry at both the local and the national levels. Since most of the target citrus germplasm in this project are not commercially available yet, collaboration with different stakeholders (DPI, Florida PTP, FFSP, FNGLA and growers) will facilitate introducing these new cultivars to the citrus industry. Work will occur on all the project objectives each year.Objective 1: Identify and Propagate elite conventionally bred field-tested HLB tolerant citrus.Budwoods from new varieties but not commercially available yet will be collected from the source tree and propagated by grafting onto selected rootstocks that push vigorous scion growth. This will include any promising scions (sweet orange, mandarin, grapefruit, pummelo, tangerine, and unique acid-fruit types) and rootstocks. The selected cultivars for cleanup will be chosen based on their fruit quality evaluation, specialty crop potential, and consumer acceptance as well as grower interest and the most important HLB performance in the field. Grafted plants will grow in the greenhouse for three months to reach at least 30" before moving to the alternating temperatures chamber.HLB diagnosis of mature leaves for Candidatus Liberibacter asiaticus (CLas) titer and activity will be performed by using the quantitative polymerase chain reaction (q-PCR) according to Li et al. (2006)on the propagated plants before treatment.CTV diagnostics is dependent on double antibody sandwiched enzyme-linked immunosorbent assay (DAS-ELISA) and reverse transcription-polymerase chain reaction (RT-PCR). ELISA is easy to perform and suitable for large scale processing of samples. However, RT-PCR which is more laborious is more sensitive. ELISA is used to semi-quantify the amount of CTV in a sample by running a serial dilution analysis. DAS-ELISA protocol (Garnsey and Cambra 1991) is established and routinely run in EL Mohtar lab. ELISA positive post-treatment plants will be discarded whereas ELISA negative plants will be subject to RT-PCR using CTV coat protein primers (Hilf et al. 2005) to confirm the ELISA negative tests. Template RNA for RT-PCR will be extracted from the midribs of treated plants using a Trizol reagent (Ambion) or RNeasy® Plant Mini kit (Qiagen). Proper positive and negative controls will be included in all assays.Objective 2: Apply alternating temperature treatments to induce healthy growth from diseased plants.Exposing actively growing infected citrus plants (HLB and CTV) to the alternating temperature regimes does not cure the tree but prevents the establishment of the pathogens in the newly growing tissue. The temperature regimes will oscillate between 4 hours at 45°C and 4 hours at 25°C for three months. The oscillation between the temperatures will minimally affect plant growth but degrades viral ssRNA and the continuous heat shock will prevent bacteria from establishing in the new tissue. To the best of our knowledge, there is no non-destructive effective rapid protocol to clean up infected citrus plants from HLB. The established meristem-tip culture protocol generally takes about 2 years. Thus, the proposed new technique will offer an alternative method to produce propagative pathogen-free materials and still maintain the genetic integrity of the original plant in significantly less time.Objective 3: Confirm HLB and CTV-free cultivars after alternating temperature treatments.On a monthly basis, the plants inside the chamber will be screened for HLB using qPCR and CTV using ELISA test as described in objective 1. This will allow us to monitor the titer reduction of HLB and CTV in the new growth to make sure that the collected budwoods for the next objective are HLB and CTV-free.Our preliminary data showed that CLas and CTV could be eliminated from citrus materials after alternate temperature regime treatment (45°C for 4 hours and 25°C for 4 hours) for 3-4 months.Objective 4: Propagate pathogen-free cultivars to be validated by the Florida PTP battery of testsPlants confirmed to be pathogen-free from the previous objective will be used as a source for budwoods for micrografting onto the most promising rootstocks. After establishing the grafted plants, another round of qPCR and ELISA will be performed to make sure that they are still pathogen-free. At this stage, plants confirmed pathogen-free will be provided to the Florida Department of Agriculture and Consumer Services, Bureau of Citrus Budwood Registration for validation by their standard battery of PCR-based tests for all the important pathogens, the same testing that is performed on selections coming from meristem-tip culture. It should be noted that this procedure does not work for the elimination of viroids, but most new selections in the breeding program have not been in the field long enough to acquire any viroids; thus, this should not be a problem, but demonstrates the importance of thorough validation by DPI. Any selections validated to be pathogen free could be made available for commercial use in Florida, and also made available to the USDA Citrus Germplasm Repository in Riverside, California, for certification and distribution to other States. New pathogen-free scions and rootstocks developed by this project must be available to the industry as quickly as possible to maintain a sustainable, profitable US citrus industry.