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

Location: Subtropical Insects and Horticulture Research

2019 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
Good progress is being made to develop and field test new hybrid citrus rootstocks with outstanding horticultural traits and superior tolerance to huanglongbing. Over 300 new hybrid rootstocks are now under evaluation in 30 multiyear replicated field trials with commercial scions. About 100 new promising hybrids are currently being propagated in preparation for planting new replicated field trials. Other new hybrids with unique huanglongbing-tolerant parentage have been produced and after preliminary evaluation, will be entered into additional replicated field trials. Field performance data being collected from field trials seasonally on yield, fruit quality, tree size, and tree health indicates that some of the new hybrid rootstocks exhibit outstanding combinations of traits. Three new hybrid rootstocks, SuperSour 1, SuperSour 2, and SuperSour 3, exhibiting outstanding field performance were released in 2018, and other superior new rootstocks are expected to be released over the next two years. Focused research on rapid greenhouse testing for tolerance to huanglongbing is paying dividends in the development of broadly applicable methods and evaluation criteria that can be readily employed by the various research laboratories involved in related research. Meaningful differences in the tolerance of scion and rootstock cultivars to huanglongbing are readily observed and measured under controlled conditions within the greenhouse in less than 12 months. Coordinated research on gene expression and metabolomics differences associated with huanglongbing tolerance has identified several plant genetic/metabolic pathways that may be used as markers in breeding and as guides in developing therapeutic treatments. Conventional citrus nursery management has relied upon apomictic seeds for propagation of citrus rootstocks, which has become increasingly problematic in both breeding and commercial use, as some of the parental material with the most tolerance to huanglongbing does not exhibit apomixis. Even the seed supply for the best of the new huanglongbing-tolerant apomictic rootstocks lags far behind the demand. ARS research has contributed to the development of effective methods for rootstock propagation by stem cuttings and tissue culture, and has been leading efforts to ensure that rootstocks propagated by the alternative methods will maintain optimum field performance. Several new trials with USDA hybrid rootstocks are a major part of the work to broaden the germplasm base (to include species without apomixis) and remove the limitations imposed on rate of propagation by obligate seed use. Advances continue in developing improved conventional citrus scions. Over 20,000 new scion hybrids have been planted in the last ten years, with 6,000 more in the greenhouse for subsequent planting. More than 130 selections were made for use as parents or potential cultivars. Outstanding fruit quality and potentially useful huanglongbing-tolerance is evident in many populations, including the first progeny derived from our huanglongbing -tolerant release US SunDragon. Very late maturing selections were made this year with both purely conventional and Poncirus-containing genetics, providing the promise of a substantial extension of the citrus maturity window for fresh and processing fruit. As our understanding of huanglongbing-tolerance advances we make better crosses each year. Ten cultivars have been released in the last six years. In the last ten years, three thousand trees have been planted and evaluated following budwood irradiation for reduced seed count or improved color. Eight low-seeded variants have been selected, and most are in replicated trials. A large replicated trial of 50 advanced selections and cultivars is in the sixth year of severe huanglongbing challenge and several of our selections, with both purely conventional and Poncirus-containing genetics, are showing excellent growth despite huanglongbing pathogen-infection. Large replicated plantings continue to be phenotyped, in an effort to identify genes associated with huanglongbing-tolerance and huanglongbing-resistance. In a multi-institution collaboration led by ARS researchers, the Florida orange juice standards are being re-assessed to potentially include new huanglongbing-tolerant scion types. Progress continues in developing transgenic citrus with huanglongbing-resistance. Initial replicated trees expressing a modified plant thionin (Mthionin) which greatly reduces Candidatus Liberibacter asiaticus when transgenic vs. wild-type rootstocks are grafted with infected scions (1800X reduction at 12 months) have been placed in a field planting, and many additional Mthionin Carrizo and Hamlin have been propagated. Transgenics expressing chimeral peptides (containing separate lytic and gram negative membrane recognition sequences from citrus) have been created and show greatly suppressed huanglongbing pathogen in detached leaf psyllid inoculations. To develop the data package to deregulate Mthionin transgenics, ARS researchers have met with regulators and participated in the Specialty Crops Regulatory Assistance workshop. Grapefruit has been grafted onto replicated rootstocks transgenically expressing antibodies to two huanglongbing-pathogen proteins. Earlier, in the rootstock itself, ARS researchers have shown pathogen suppression of 125 times. A phloem-specific promoter from citrus is very successful, with expression of a marker gene 400 times greater in midribs than in laminar leaf area. This promoter is being used in creating transgenics targeting the huanglongbing pathogen and vector, and is showing excellent activity. New transgenics have also been created expressing peptides or double-stranded ribonucleic acids identified by ARS researchers to prevent huanglongbing pathogen acquisition by Asian citrus psyllid. Two of these peptides markedly increased psyllid mortality in detached leaf feeding assays. Genome editing is underway, to knock-out a few key targets where loss-of-function may produce desired traits. In work the previous year ARS researchers followed expression of 28 candidate effectors which are produced by the huanglongbing pathogen to colonize its citrus host. The 6 effectors which were most expressed during early infection are now being transgenically expressed with a Flag-tag. This will permit pull-down of the citrus gene products to which the effectors bind, allowing identification and CRISPR knock-down of gene products which may be critical for disease development. In collaboration with university colleagues, ARS researchers recently perfected early-flowering transgenics (flowering within 6-8 months of seed germination) and are using these flowers as pollen-sources in hybridizations. ARS researchers are also assessing the value of immediately using this construct to transform seedling tissues from priority hybrids, primarily intended as a tool to expedite breeding for the next generation of hybrids. ARS researchers are also testing an exogenous application, we hypothesize will promote flowering in juvenile citrus, which should be applicable to all priority parents. Finally and serendipitously, ARS researchers have a population of plants that have Poncirus in the pedigree, but are otherwise conventional, which flowered within three years of seed germination. This early flowering trait is being tested for heritability, and may provide a substantial acceleration in using our most advanced material as parents. A research plan was modified to develop systems to rapidly identify huanglongbing-tolerant phenotypes, and develop systems to rapidly test strategies for controlling/curing huanglongbing. This included both in vitro and ex vitro systems. The rationale for this shift away from potential long-term solutions (i.e., breeding, genetic transformation and gene editing) was because of the severe impact of huanglongbing on the Florida citrus economy and the urgency of quick solutions. Most of the progress was in reducing the concepts to researchable questions with some experiments now underway. In vitro research was focused on identifying in vitro screens useful for assessing certain aspects of huanglongbing management. For example, an attempt was made to establish a stage 2 in vitro huanglongbing-infected proliferating shoot culture. Such a culture might be useful as 1) a clean source of Liberibacter for culture efforts, 2) a source of tissue for genetic transformation where transformants generated in tissue culture could be assayed directly for the degree of resistance conferred by the candidate gene, and 3) a culture system to screen management strategies (e.g., mineral nutrition, test molecules that are only available in small amounts). The experiment to develop a stage 2 huanglongbing-infected culture was not successful, though the results were important as it showed that the in vitro environment was not sufficient to overcome the effects of the disease. Work on citrus stage 2 cultures continues as it relates directly to micropropagation, and citrus conservation (germplasm can be rescued, and huanglongbing-free types propagated). Ex vitro research focused on 1) developing a greenhouse-level system for rapidly screening disease management and control strategies; 2) using ferrous iron as a huanglongbing control strategy; 3) evaluating several water modification technologies (nanobubble, corona discharge, and cold plasma); and 4) developing a machine learning/artificial intelligence system for efficient image analysis and data collection at all scales – in vitro, greenhouse, and field. This research includes hypothesis testing of potential control strategies (e.g., ferrous iron project), and developing and building screening systems that span from single leaf assays to field plot level testing. Scalable screening systems match the research question to the appropriate screen.

Accomplishments
1. Release of three new huanglongbing-tolerant citrus rootstocks. Huanglongbing disease has proven devastating to the Florida citrus industry and severely threatens citrus production in other parts of the United States, but the use of tolerant rootstocks has been shown to be effective in ameliorating disease effects. ARS researchers in Ft. Pierce, Florida, have released three new huanglongbing-tolerant citrus rootstocks that produced sweet orange trees with improved health, fruit yield, and fruit quality over multiple years in a huanglongbing-endemic environment. The new tolerant rootstocks will allow continued profitable production of sweet orange in the presence of huanglongbing, where trees on many other rootstocks will fail.

2. Release of one new huanglongbing-tolerant citrus scion cultivar. Huanglongbing disease has proven devastating to most citrus, but some hybrid scions have been shown to have field tolerance. ARS researchers in Ft. Pierce, Florida, have released the first citrus scion cultivar with good fruit quality and with tolerance to huanglongbing. US SunDragon is being widely tested in Florida and continues to show outstanding tolerance to huanglongbing. It is being promoted as a breeding parent, niche fruit, and making it possible to once again grow citrus in Florida home-owner plantings. Initial tests of juice quality, by a major processor and in a scientific study, show considerable promise for inclusion of US Sundragon in orange juice blends. The use of the new tolerant scion will allow profitable production of orange-like fruit in the presence of huanglongbing, where trees of conventional sweet orange will fail.

3. Release of one new citrus scion cultivar with outstandingly good flavor. The retail market for citrus fruit is highly competitive for United States producers, with seedless mandarins now available nearly year-round from global mix of production. There is a need for new citrus scion cultivars to provide unique and distinctive quality to garner a portion of the market under these conditions. ARS researchers in Ft. Pierce, Florida, have released a citrus scion cultivar, US Superna, which stands out in this regard, because of its extraordinary flavor. An article about this cultivar in the Los Angeles Times was entitled “The USDA 88-2 Super Nova may be your favorite new fruit.” It has been described as the “best-tasting citrus” ever produced. About 100 acres of this cultivar are already planted in California.

4. Development of standardized methods for testing citrus scion and rootstock cultivars for huanglongbing-tolerance. Huanglongbing disease has proven devastating to most citrus, but some scion and rootstock cultivars have been shown to have field tolerance. Prior to our work, methodologies for evaluating cultivar tolerance to huanglongbing was difficult, controversial, and took several years to obtain reliable results. ARS researchers in Ft. Pierce, Florida, in collaboration with university researchers have developed methods for standardized greenhouse testing for huanglongbing tolerance. Those methods, and their applications in comparisons of tolerance in scion and rootstock cultivars were published, and used as the basis for testing guidelines used by the granting agencies. This standardization of methods will greatly improve the process of creating and testing new citrus cultivars.

5. Development of supplemental lighting methods to accelerate citrus nursery production. The rate of plant growth in the greenhouse citrus nursery is dramatically slowed during the short days of winter, which significantly reduces potential nursery profitability and rate at which trees on new scion and rootstock cultivars can be prepared for planting into the field. ARS researchers in Ft. Pierce, Florida, have developed supplemental lighting methods to extend daylength during the winter and consequently accelerate tree growth in the nursery up to 50 percent. The methods were demonstrated to be effective with either high pressure sodium or low-power light emitting diode sources of illumination. The use of supplemental illumination to extend daylength in the citrus nursery will improve growth of nursery trees, and enable more rapid deployment of the newest huanglongbing-tolerant citrus scion and rootstock cultivars into commercial field production.

6. Development of early flowering tool to accelerate citrus breeding. Citrus has a very long life-cycle, which greatly slows the breeding of improved cultivars with needed traits under natural conditions. ARS researchers in Ft. Pierce, Florida, have achieved early-flowering in seedling citrus using a transgenic approach, in collaboration with university colleagues. Transgenic plants first produced flowers in 5 months after initial seed generation and have flowered continuously for the subsequent year. Pollen has been shown to germinate and appears to have been used successfully to make new hybrids. This approach, when combined with DNA markers for priority traits, should permit what would ordinarily be 100 years of breeding progress in about twelve years. This method, combined with transformation and a proper selection system, should permit transgene-free genome-edited plants within three years.

7. Development of method to use shoot cultures for screening huanglongbing control strategies. Huanglongbing disease is devastating to citrus production, and work to evaluate different control strategies in the greenhouse or field are very laborious and difficult to obtain accurate information. ARS researchers in Ft. Pierce, Florida, have developed methods to establish shoot cultures infected with the disease and that express symptoms, which may be used to evaluate small amounts of therapeutic agents or other compounds under controlled conditions in the laboratory. If the materials were demonstrated effective against the disease in shoot cultures, then the much more expensive evaluation of the control strategy in the greenhouse or field can be more reasonably justified.

8. Development of method to use shoot cultures to remove huanglongbing from germplasm. Although possible, removal of huanglongbing disease from clonal cultivars by shoot tip grafting is difficult, and alternative methods for cultivar cleanup would be valuable. ARS researchers in Ft. Pierce, Florida, have developed methods to develop disease-free shoot cultures from clonal citrus cultivars infected with huanglongbing disease. Refinement of these methods may be useful in rescue of unique germplasm that is infected with the disease. These methods could also be used in production of huanglongbing-negative stock plants for commercial micropropagation.

9. Development of method to use nanobubble treated irrigation water to improve health of huanglongbing-infected trees. Huanglongbing disease is devastating to citrus production, and treatments that improve tree health in the field are of great interest and value. ARS researchers in Ft. Pierce, Florida, have developed methods to generate oxygen-treated nanobubble water with much higher dissolved oxygen than normal water. Treatment of huanglongbing-infected trees with the nanobubble water was observed to increase root growth after only 4 months. This appears of tremendous importance, since one of the most damaging aspects of the disease is to reduce root growth and root health on infected trees. Use of nanobubble treated water may be used therapeutically to improve health and productivity of disease-infected trees in the field.

Review Publications
Shi, Q., Pitino, M., Zhang, S., Krystel, J.A., Cano, L.M., Shatters, R.G., Hall, D.G., Stover, E.W. 2019. Temporal and spatial detection of Candidatus Liberibacter asiaticus putative effector transcripts during interaction with Huanglongbing-susceptible, -tolerant, and -resistant citrus hosts. Biomed Central (BMC) Plant Biology. 19:122. https://doi.org/10.1186/s12870-019-1703-4.
George, J., Shi, Q., Stelinski, L., Stover, E.W., Lapointe, S.L. 2019. Host selection, oviposition and feeding by a phytopathogen vector, Diaphorina citri (Hemiptera: Liviidae), modulated by plant exposure to formic acid. FRONTIERS IN ECOLOGY AND EVOLUTION. 7:78. https://doi.org/10.3389/fevo.2019.00078.
Albrecht, U., Bowman, K. 2019. Reciprocal influences of rootstock and scion citrus cultivars challenged with Ca. Liberibacter asiaticus. Scientia Horticulturae. 254:133-142. https://doi.org/10.1016/j.scienta.2019.05.010
Stover, E.W., Wright, G. 2019. Walter Tennyson Swingle, a relentless intellect that transformed American pomology. Journal of American Pomological Society. 73:129-138.
Niedz, R.P., Hert, M.M. 2018. A filter paper-based liquid culture system for citrus shoot organogenesis - a mixture-amount plant growth regular experiment. In Vitro Cellular & Developmental Biology - Plants. 54(6):658-671.
Huang, M., Roose, M., Yu, Q., Du, D., Yuan, Y., Zhang, Y., Deng, Z., Stover, E., Gmitter, F. 2018. Construction of high-density genetic maps and detection of QTLs associated with huanglongbing tolerance in citrus. Frontiers in Plant Science. https://doi.org/10.3389/fpls.2018.01694.
Stover, E., Driggers, R., Hearn, C.J., Bai, J., Baldwin, E., McCollum, T.G., Hall, D.G. 2016. Breeding "sweet oranges" at the USDA U.S. Horticultural Research Laboratory. Acta Horticulturae. 1127:41-44. https://doi.org/10.17660/ActaHortic.2016.1127.7.
Shi, Q., George, J., Krystel, J.A., Zhang, S., Lapointe, S.L., Stelinski, L.L., Stover, E.W. 2019. Hexaacetyl-chitohexaose, a chitin-derived oligosaccharides, transiently activiates citrus defenses and alters the feeding behavior of asian citrus psyllid. Horticulture Research. 6(76):1-10. https://doi.org/10.1038/s41438-019-0158-y.
Stover, E.W., Hall, D.G., Grosser, J., Gruber, B., Moore, G. 2018. Huanglongbing-related responses of 'Valencia' sweet orange on eight citrus rootstocks during greenhouse trials. HortTechnology. 28(6):776-782. https://doi.org/10.21273/HORTTECH04137-18.
Shi, Q., Febres, V.J., Zhang, S., Yu, F., McCollum, T.G., Hall, D.G., Moore, G.A., Stover, E. 2018. Identification of huanglongbing tolerance-associated genes using Candidatus Liberibacter asiaticus flagellin 22 as a proxy to challenge citrus. Molecular Plant-Microbe Interactions. 31:200-211.
Stover, E., Aradhya, M., Gozlecki, S., Crane, J., Matsumoto Brower, T., Mayo- Riley, C., Zee, F., Gottwald, T., Hall, D. 2018. Guava SSR analysis: diversity assessment in US and similarity to accessions associated with reducing citrus huanglongbing in Vietnam. Journal of American Pomological Society. 72:242-250.
Hao, G., Ammar, D., Duan, Y., Stover, E.W. 2019. The 'Candidatus Liberibacter asiaticus' effector LasP235 targeting the citrus chloroplast induces Huanglongbing-like symptoms in transgenic citrus. AGRI GENE. 12:100085. https://doi.org/10.1016/j.aggene.2019.100085.
Albrecht, U., Bordas, M., Lamb, B., Meyering, B., Bowman, K.D. 2017. Influence of propagation method on root architecture and other traits of young citrus rootstock plants. HortScience. 52(11):1569-1576. https://doi.org/10.21273/HORTSCI12320-17.
Albrecht, U., Tripathi, I., Kim, H., Bowman, K.D. 2018. Rootstock effects on metabolite composition in leaves and roots of young navel orange (Citrus sinensis L. Osbeck) and pummelo (C. grandis L. Osbeck) trees. Trees. 33(1):243-265. https://doi.org/10.1007/s00468-018-1773-1.
Bowman, K.D. 2018. Weeping dragon, a unique ornamental citrus. HortScience. 53(11):1708-1710. https://doi.org/10.21273/HORTSCI11607-18.
Deng, Z., Rawat, N., Kumar, B., Albrecht, U., Du, D., Huang, M., Yu, Q., Zhang, Y., Duan, Y., Bowman, K.D., Gmitter, F. 2017. Genome resequencing and transcriptome profiling reveal structural diversity and expression patterns of constitutive disease resistance genes in Huanglongbing-tolerant Poncirus trifoliata and its hybrids. Horticulture Research. 4:17064. https://doi.org/10.1038/hortes.2017.64.
Man, H.P., Hibbard, B.E., Lapointe, S.L., Niedz, R.P., French, B.W., Pereira, A.E., Finke, D.L., Shelby, K., Coudron, T.A. 2019. Multidimensional approach to formulating a specialized diet for northern corn rootworm larvae. Scientific Reports. 9:3709. https://doi.org/10.1038/s41598-019-39709-x.
Patt, J.M., Meikle, W.G., Niedz, R.P., Woods, D. 2018. Synthetic ligands of olfactory binding proteins modulate aggregation response of Asian citrus psyllid in the presence of host-plant volatiles. Frontiers in Plant Science. 9:1891. https://doi.org/10.3389/fpls.2018.01891.
Xin, X., He, Z., Hill, M.R., Niedz, R.P., Jiiang, X., Sumerlin, B.S. 2018. Efficiency of biodegradable and pH-responsive polysuccinimide nanoparticles (PSI-NPs) as smart nanodelivery systems in grapefruit: in vitro cellular investigation. In Vitro Cellular and Developmental Biology. 18(7):e1800159. https://doi.org/10.1002/mabi.201800159.