Research Project: Genetic Improvement of Citrus for Enhanced Resistance to Huanglongbing Disease and Other Stresses

Location: Subtropical Insects and Horticulture Research

2023 Annual Report

Objectives
1. Develop and release new citrus rootstock and scion cultivars with traits urgently needed for successful commercial fruit production, especially resistance or tolerance to Huanglongbing (HLB). 1.A. Develop and select promising candidates and assess performance in multiyear field trials, leading to release of outstanding new cultivars. 1.B. Select promising candidates and assess huanglongbing resistance and tolerance in the greenhouse. 2. Introduce new useful traits into USDA citrus breeding populations by sexual hybridization, mutation, and genetic transformation, and recombine traits as needed to obtain desired new types. 2.A. Use hybridization to create new germplasm from diverse parental types with useful horticultural characteristics. 2.B. Create new scions with useful traits through mutation. 2.C. Create scions and rootstocks resistant to huanglongbing and other citrus diseases via genetic modification technologies. 3. Improve the methodology to create, select, propagate, and evaluate new citrus rootstock and scion cultivars for valued traits, especially tolerance or resistance to huanglongbing, and employ these methods for the development and commercialization of new cultivars. 3.A. Refine and evaluate methods to assess huanglongbing tolerance/resistance, and apply appropriate methods to select superior individuals. 3.B. Evaluate rootstocks propagated by seed, cuttings, and micropropagation for horticultural performance and genetic fidelity suitable for large-scale nursery production and commercial field use. 4. Develop in vitro, molecular markers, model systems, and other technology that facilitates the project goals. 4.A. Develop rapid in-vitro methods to accelerate assessment of citrus huanglongbing resistance. 4.B. Develop populations to identify molecular markers associated with resistance or tolerance to huanglongbing.

Approach
This project will focus on development of new citrus rootstock and scion cultivars with traits urgently needed for commercial production, especially tolerance and resistance to huanglongbing (HLB). These new rootstock and scion cultivars will enhance the competitiveness of the U.S. citrus industry by improving production efficiency, plant health, and value of the U.S. citrus crop. Breeding citrus for resistance to HLB is particularly difficult because of the long life cycle of citrus, predominant apomixis among most important cultivars, and lack of significant HLB resistance in the important cultivated citrus scion cultivars. The primary objectives of this project are to generate new citrus genotypes through conventional breeding, mutation, and genetic modification technologies; evaluate them as candidate cultivars for commercial use; and release new superior cultivars. Due to the very long-term cycle for development of new citrus cultivars, a significant portion of the project is focused on the multiyear evaluation of hybrids created under previous projects, and creating new hybrids for future evaluation and based on the best available knowledge. These components of the project focus on creation and evaluation of germplasm and are not hypothesis-driven research. Under this project, improved methods will be developed for genetic modification of citrus, since current methods are notably inefficient and result in plants containing many foreign sequences and difficult to commercialize. Plant responses to HLB will be investigated, and increased knowledge will be used to design strategies for creation and more rapid selection of tolerant and resistant cultivars. Development of improved screening methods will increase the efficiency of new cultivar development with these traits. Among global efforts to develop new citrus cultivars with HLB resistance and tolerance, this program has the unique advantage of numerous advanced breeding lines, produced by more than a century of interbreeding and introgression, which contain alleles for resistance or tolerance to HLB from related species and genera, combined with other critical traits.

Progress Report
This is the fifth and final year of a project that continues the long-term goals of previous projects at the U.S. Horticultural Research Laboratory (USHRL) in Fort Pierce Florida to develop new citrus scion and rootstock cultivars with traits critical for successful commercial production and marketability. The current project involves new work especially focused on developing rootstocks and scions resistant or tolerant to the disease huanglongbing. Toward this goal, the project has created and is testing thousands of new hybrids with potential outstanding traits, including resistance to huanglongbing. In addition, under this and subordinate externally funded projects, research is being conducted to document horticultural, genetic, gene expression, metabolomic, physiological, and anatomical differences that are associated with disease sensitivity or tolerance, and to explore new avenues to develop huanglongbing resistant cultivars and methods to grow susceptible cultivars. A major new effort has begun to map important rootstock traits in the genome and integrate molecular markers and genomic selection into the breeding program. Two new HLB-tolerant rootstocks have been proposed for release and are awaiting final approval. Continued assessment of canopy health of grapefruit trees treated with different forms of iron, an essential plant nutrient. Micronutrient fertilization, including iron, is important in the management of huanglongbing disease. These grapefruit trees are infected with huanglongbing and are now bearing fruit. The initial study was completed but the cooperator is collecting fruit data, canopy size, and tree vigor measures. The initial experiment compared the effects of 7 forms of iron on various measures of grapefruit tree growth and health, but with the trees bearing fruit, the effects on fruit number, size, and quality can be determined. Research was continued to identify treatments to improve the health of citrus trees that have huanglongbing disease. These efforts included 1) maintaining multiple psyllid colonies that are required to produce greenhouse trees that have huanglongbing disease, 2) developing and using a 3D-printed infusion device that allows for trunk injection of seedling trees with trunk diameters as small as 4 mm, and 3) set up multiple field trials and currently testing >80 trunk injection treatments with the potential to manage huanglongbing disease. Direct trunk infusion of diseased trees in the greenhouse and field will be used to identify treatments that improve tree health. Psyllid inoculation of greenhouse trees will be used to both produce infected trees for management treatments, but also to assess the level of resistance of citrus selections from the citrus breeding program. The psyllid colonies are calibrated to verify that they carry the bacteria that causes huanglongbing disease. Eight thousand sweet orange and grapefruit seedlings have been established in the greenhouse. These are being used as the source trees for psyllid infection. About 1,500 citrus plants have been treated with psyllids carrying the huanglongbing disease. These infected plants have severe symptoms and have been treated with over 30 therapeutic molecules by direct trunk infusion. These treatments include infusion with oxytetracycline, now the standard trunk injection treatment for huanglongbing trees in Florida. Embryogenic cell lines from varieties important to the Florida and California citrus industries were started, including Donaldson and Tobias. Donaldson is being widely propagated for testing as an huanglongbing-tolerant sweet orange. Tobias flowers within two years making it a potential breeding parent to reduce juvenility. Embryogenic cells were provided to University of Maryland collaborators and are being used to develop CRISPR technology for citrus. The citrus scion breeding effort had a strong emphasis on the evaluation and selection of new varieties exhibiting superior agronomic traits, fruit quality, and huanglongbing tolerance. Extensive assessments were conducted, incorporating materials from the USDA germplasm repository, resulting in the identification of promising grapefruit selections, including 'Florida Red,' 'Henderson,' and 'Red Blush.' These selections may exhibit potential for early-onset huanglongbing-tolerance or improved tolerance through commercial management programs. Consequently, these grapefruit selections were submitted for further evaluation and clean-up, thus expanding the germplasm base and enabling more informed and promising crosses. Hybridization efforts in the 2022 season yielded thousands of unique hybrids, targeting HLB resistance/tolerance and seedless selections. The breeding focus encompassed the production of sweet orange-like, red grapefruit-like, and seedless mandarin hybrids, all with an emphasis on fruit quality combined with huanglongbing tolerance. A total of 776 flowers from 20 parental combinations were crossed, based on our growing understanding of huanglongbing resistance and tolerance. The selection process involved rigorous screening for fruit quality attributes, leading to the identification of 24 new citrus selections with commercial promise, including sweet oranges, grapefruits, and mandarins, all showing evidence of disease tolerance that will need to be validated with replicated field trials. Ongoing evaluations were carried out on existing hybrids to assess fruit quality attributes as they commenced bearing. These selections were made based on their potential as cultivars and parents for future breeding endeavors. The continuous evaluation and screening process will facilitate the release of notable cultivars, including sweet-orange-like hybrids, mandarins with excellent flavor, and seedless varieties. The breeding program's trajectory is focused towards evaluating materials from older crosses, collections, and segregating populations, which have demonstrated resilience in the challenging Florida environment and offer unique parental combinations. These materials are subjected to internal testing and comparison with industry standards, with grower feedback obtained through fruit displays and field tours, enabling the assessment of their success within specific operational contexts. In the past year, 27 new hybrids were selected from large populations for further evaluation as rootstocks in Florida field trials and for initiation of field testing in California and Texas, following quarantine clearance in California. Data was collected on tree size, health, cropping, and fruit quality from 30 established replicated rootstock field trials. Two new replicated rootstock field trials were planted, and trees were prepared for planting of two additional new replicated rootstock field trials in the coming year, including 25 new hybrid rootstocks. Work continued under material transfer agreements and externally-funded grants to cooperatively test new scions and rootstocks in field trials with university and private partners in Florida, and in other states. Information was collected to support the upcoming release of new hybrid rootstocks with field tolerance to huanglongbing and outstanding field performance in graft combination with commercial scions. Greenhouse studies were conducted to improve methods for growth and evaluation of citrus trees in the greenhouse during the winter months. The winter is typically a time of little growth for citrus even under greenhouse conditions, and our studies have demonstrated that careful manipulation of light spectra and intensity can dramatically improve citrus tree growth during the winter. Work continued under collaboration with funding from a NIFA grant to sequence 380 rootstock hybrids and map important rootstock traits for the development of selectable molecular markers. A large portion of the data from the rootstock mapping trials has been collected, and work to complete genetic map is underway. Important traits to be mapped include rootstock influence on fruit quantity, fruit quality, tree size, tree health, and tolerance of huanglongbing disease.

Accomplishments
1. Release of two new citrus rootstocks. ARS researchers in Fort Pierce, Florida, proposed and prepared release documentation for two new huanglongbing-tolerant rootstocks that exhibited outstanding multiyear field performance with sweet orange scion under severe disease conditions. The two new rootstocks demonstrated superior tree health, superior fruit cropping, and reduced preharvest fruit drop, as compared with two of the most commonly used commercial rootstocks over 3-5 crop seasons on both flatwoods and ridge environmental conditions. The two new rootstocks are expected to provide good new options to increase production and profitability of commercial sweet orange fruit under huanglongbing-endemic conditions.

2. New 3D printed trunk infusion device. ARS researchers in Fort Pierce, Florida, develop a 3D printed trunk infusion device for infusing chemicals into small diameter tree trunks as small as 4 mm. Conventional trunk injection devices typically require a trunk diameter of >4 inches (100 mm) and thereby limits their use in high-throughput testing using small greenhouse plants or trees. The infusion device can be inexpensively produced with standard 3D printers. This makes large scale testing of molecules and formulations possible. This technology is particularly useful for woody perennial and tree research where field testing is more costly than using greenhouses and/or growth chambers. The device is being used to screen and identify molecules that improve the health of citrus trees that have huanglongbing disease.

3. Field testing of 80 molecules. ARS researchers in Fort Pierce, Florida, are testing >80 molecules in the field to identify molecules that will improve the horticultural health of citrus trees that have huanglongbing disease. The molecules were selected based on their antimicrobial efficacy, systemic movement, and minimal EPA approval requirements. The molecules are delivered by trunk injection, using methods currently being used by the citrus industry to control HLB. Field testing of molecules on this scale has not been reported as most studies on molecule screening use lab assays. No lab assay can determine if a molecule will work in the field. The approach is based on the screening principles of design of experiments. The number of molecules being screened far exceeds the number of molecules field tested by the research community for managing huanglongbing. Molecules that are discovered to have large effects in rejuvenating diseased citrus trees can then be rapidly used by the citrus industry. The citrus industry has only one approved molecule for injection, oxytetracycline, and finding alternatives to oxytetracycline is critical for the long-term sustainability of the citrus industry.

4. Evaluation of promising grapefruit cultivars. ARS researchers in Fort Pierce, Florida have identified promising heirloom grapefruit selections from the USDA's former germplasm repository. The extensive evaluations have resulted in the identification of several promising heirloom grapefruit selections, including 'Florida Red,' 'Henderson,' and 'Red Blush.' While these selections may not exhibit exceptional HLB tolerance in the field long-term, they demonstrate potential for early onset HLB tolerance or improved tolerance through commercial management programs. These varieties have been sent to the state for cleanup and will be included in future replicated trials to validate our findings. These heirloom selections provide additional options for grapefruit cultivation in Florida and expand our understanding of HLB tolerance of underutilized heirloom varieties.

5. Selection of 24 promising citrus scion cultivars. ARS researchers in Fort Pierce, Florida, have selected 24 new citrus varieties with commercial promise. Through rigorous screening and evaluations, a total of 24 new citrus varieties were selected during the 2022-2023 seasons, exhibiting commercial promise and demonstrating evidence of huanglongbing tolerance. These selections encompass a wide range of citrus types, including sweet oranges, grapefruits, and mandarins. The breeding focus involved thousands of unique hybrids produced from targeted crosses aimed at HLB tolerance and seedless easy-peel selections. The extensive evaluation and selection process involved assessing fruit quality attributes and the performance of parental combinations. Notable achievements include the selection of seven totally seedless sweet-orange-like selections, the identification of sweet-orange-like hybrids with Poncirus in their pedigree, and mandarins showing good huanglongbing tolerance. These selections represent a step towards the development of citrus varieties with commercial traits and improved resilience to disease.

Review Publications
Mattia, M.R., Du, D., Yu, Q., Kahn, T.L., Roose, M.L., Hiraoka, Y., Wang, Y., Munoz, P.T., Gimitter, F.G. 2022. Genome-Wide association study of healthful flavonoids among diverse mandarin accessions. Plants. 11(3):317. https://doi.org/10.3390/plants11030317.
Zhang, Y., Cheng, Y., Fang, H., Roberts, N., Zhang, L., Vakulskas, C.A., Niedz, R.P., Culver, J.N., Qi, Y. 2022. Highly efficient genome editing in plant protoplasts by Ribonucleoprotein delivery of CRISPR-Cas12a nucleases. Frontiers in Genome Editing. 4: Article 780238. https://doi.org/10.3389/fgeed.2022.780238.
Krystel, J.A., Stover, E.W., Dominguez, J., Jayachandran, K., Shetty, K. 2023. Endophytes and plant extracts as potential antimicrobial agents against Candidatus Liberibacter asiaticus, causal agent of huanglongbing. Microorganisms. https://doi.org/10.3390/microorganisms11061529.
Stover, E.W., Mayo, S., Driggers, R.E., Adair, R.C. 2022. Planting in metallized reflective mulch did not significantly benefit development of new hybrid seedlings. HortScience. 57:2. https://doi.org/10.21273/HORTSCI16248-21.
Pokhrel, S., Meyering, B., Bowman, K.D., Albrecht, U. 2020. Horticultural attributes and root architectures of field-grown ‘Valencia’ trees grafted on different rootstocks propagated by seed, cuttings, and tissue culture. HortScience. 56(2):163–172. https://doi.org/10.21273/HORTSCI15507-20.
Bowman, K.D., Albrecht, U. 2020. Rootstock influences on health and growth following Candidatus Liberibacter asiaticus infection in young sweet orange trees. Agronomy. 10(12):1907. https://doi.org/10.3390/agronomy10121907.
Pence, V.C., Finke, L.R., Niedz, R.P. 2020. Evaluating a DOE screen to reduce hyperhydricity in the threatened plant, Cycladenia humilis var. jonesii. In Vitro Cellular and Developmental Biology - Plants. 56:215–229. https://doi.org/10.1007/s11627-019-10038-y.
Fong, H., Culver, J.N., Niedz, R.P., Qi, Y. 2023. Delivery of CRISPR-Cas12a ribonucleoprotein complex for genome editing in an embryogenic citrus cell line. Methods in Molecular Biology. https://doi.org/10.1007/978-1-0716-3131-7_10.
Bowman, K.D., Mccollum, G.T., Seymore, D. 2023. Genetic modulation of valencia sweet orange field performance by 50 rootstocks under huanglongbing-endemic conditions. Frontiers in Plant Science. https://doi.org/10.3389/fpls.2023.1061663.
Tardivo, C., Qureshi, J., Bowman, K.D., Albrecht, U. 2023. Relative influence of rootstock and scion on Asian citrus psyllid colonization and Candidatus Liberibacter asiaticus distribution. Scientia Horticulturae. 58(4):395-403. https://doi.org/10.21273/HORTSCI17039-22.