Location: Subtropical Insects and Horticulture Research
2020 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 second year of a project that continues the long-term goals of previous projects 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 new rootstocks and scions resistant or tolerant to the disease huanglongbing. Toward this goal, the project has created and begun testing on 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 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.
In FY2020, fifty new hybrids were selected from large populations for further evaluation as rootstocks in field trials. Data was collected on tree size, health, and cropping from twelve established replicated rootstock and field trials. Three new replicated rootstock field trials were planted, to evaluate 45 new rootstock hybrids. Trees were prepared for planting of four new replicated rootstock field trials, including about 60 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 of Florida, the Citrus Research and Development Foundation, the Florida Citrus Research Foundation, and Peace River Packing Company in Florida, and with University of California and University of Georgia. Information was collected to support the upcoming 2021-22 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 evaluate scion and rootstock tolerance to biotic and abiotic stress, including focused studies of shoot and root responses to controlled infection with the bacteria causing huanglongbing, and measuring seasonal titer of the bacteria as affected by tolerant cultivars.
Advances continue in developing improved conventional citrus scions. Over 26,000 new scion hybrids have been produced in the last ten years, from more than 600 unique parental selections. More than 150 selections were made for use as parents or potential cultivars. Outstanding fruit quality and potentially useful huanglongbing-tolerance is evident in many populations, including progeny derived from our Huanglongbing (HLB)-tolerant release US SunDragon, which is 1/8th Citrus trifoliata. A high proportion of our crosses include C. trifoliata in their pedigree, many show potentially useful tolerance to huanglongbing, and USDA has been at the forefront of developing scions that include C. trifoliata in the pedigree. Selections made this year include: sweet-orange-like hybrids displaying early evidence of huanglongbing-tolerance, some of which are seedless; very grapefruit-like selections, with C. trifoliata in their pedigree, and displaying apparent huanglongbing tolerance; and seedless, easy-peel, high quality mandarins. As our understanding of HLB-tolerance advances we make “better” crosses each year.
In the last year, we released US Superna, which is a large mandarin with good evidence of huanglongbing tolerance in field performance. US Superna has exceptional flavor, acceptable peelability, and is cropping well in plantings in Florida and California. Another selection, a very sweet orange-like hybrid had previously displayed disappointing sensitivity to huanglongbing, but this year was statistically and markedly better than the commercial standard Hamlin in a substantial trial. 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, and the industry is using our work to petition for changed standards. Thousands of trees have been planted and evaluated following budwood irradiation for reduced seed count or improved color. Fifteen low-seeded variants have been selected, subjected to clean-up and most are replicated and in trials.
In a collaboration with the Florida citrus industry, and University of Florida and University of California, a replicated trial of 200 diverse accessions was established in a grower’s field to generate data for Genomic Selection. ARS scientists were key participants leading and implementing this effort.
Progress continues in developing transgenic citrus with huanglongbing-resistance using a range of transgenes. Several routinely display suppression of Candidatus Liberibacter asiaticus (CLas) and Asian citrus psyllid (ACP) when tested using CLas-infected ACP feeding on detached transgenic leaves. Many selections have been placed in field plantings, including wild-type grapefruit grafted onto replicated rootstocks transgenically expressing antibodies to two huanglongbing-pathogen proteins.
In collaboration with University of Florida, we recently perfected early-flowering transgenics (flowering within 6-8 months of seed germination) used these flowers as pollen-sources in hybridizations, and documented earlier flowering from progeny. It should be noted that resulting early-flowering plants likely will not be directly useful as cultivars, and this is primarily intended as a tool to expedite breeding for the next generation of hybrids. We are also testing a novel delivery system we hypothesize will promote flowering in juvenile citrus, which should be applicable to all priority parents. Also, we have a population of plants that have Poncirus in the pedigree, but are otherwise conventional, which flowered within three years of seed germination. Interestingly, this occurs only when specific seed parents are used, but not in reciprocal crosses. This early flowering trait is being tested for heritability, and may provide a substantial acceleration in using our most advanced material as parents.
Stage 2 shoot cultures of some commercially produced citrus rootstocks continue to be maintained and developed. These cultures are suitable for mass propagation and for testing as source tissue for genetic transformation experiments. Stage 2 citrus shoot cultures take over a year to develop. This relates to Objective 4 – Develop in vitro, molecular markers, model systems, and other technology that facilitates the project goals.
Aerial imagery is being developed as a tool for field data collection from trials. Images are taken with a drone, processed, analyzed by machine learning, and various measures obtained including canopy diameter, density, size, and an array of shape parameters. Initial experiments suggest that aerial imagery provides equivalent information as obtained from ground measures but with higher resolution.
Nanobubble technology is being tested to increase the dissolved oxygen levels of irrigation water and to evaluate its usefulness to improve health and productivity of citrus, including amelioration of huanglongbing (HLB) disease. Oxygen nanobubbles can result in dissolved oxygen levels >30 parts per million in irrigation water. Dissolved oxygen levels in untreated greenhouse water at our lab is approximately 6.5 parts per million dissolved oxygen. The results from the first experiment using HLB-infected citrus there were no differences in shoot mass but a 28% increase in the root mass. Experiments are in progress to confirm these results. Corona-discharge plasma being field tested as a method for increasing oxygen levels in field production. A corona-discharge plasma unit was installed in the field for treating irrigation water. Air enters the unit, is ionized, and then injected via a venturi into the irrigation stream. The result is that dissolved oxygen is increased from 2.5 parts per million to 8.5 parts per million, which is high quality irrigation water. Ground and aerial measures of tree growth are being evaluated twice per year.
A rapid customizable whole-plant huanglongbing management screening pipeline is being developed. It is not economical to use field trees to screen various management strategies (such as mineral nutrition), potential therapeutic molecules, and the various factors that affect their delivery and uptake. Assays are required to rapidly determine a molecule’s effectiveness against the bacteria that causes huanglongbing, its mobility in the plant, prophylactic and/or curative properties, and on psyllid mortality. A small whole-plant system is being developed that can produce in a few months large numbers of huanglongbing-infected citrus for testing.
Accomplishments
1. New website provides current information about citrus rootstock performance in field trials. Productive citrus trees of all fruiting types rely on good rootstock cultivars to develop strong root systems that can tolerate disease (including huanglongbing), environmental stresses, and support large crops of high quality fruit. With hundreds of citrus rootstocks already available, and numerous new improved rootstocks released regularly, growers and nurseries need better and current information that compares how the rootstocks perform with different fruit cultivars and under different conditions. ARS researchers in Ft. Pierce, Florida, have developed a new website that provides current and regularly updated information on comparative information among rootstocks, including results from more than 35 replicated field trials and more than 300 rootstocks. This website helps growers and nurseries make good decisions about rootstocks to be used for the millions of new citrus trees planted each year in Florida, Georgia, California, Arizona, and Texas.
2. Orange juice standards to be redefined to compensate for huanglongbing disease effects. Current orange juice standards in the United States restrict fruit used to a single genotype. This single sweet orange genotype is especially sensitive to the disease huanglongbing, which is ravaging the Florida and Texas industries, and threatening other citrus producing areas of the U.S. Other closely related genotypes provide very high quality juice and are much more tolerant to huanglongbing. ARS researchers in Ft. Pierce, Florida have led a multi-institution collaboration providing the rationale for re-assessing the Florida orange juice standards to include new huanglongbing-tolerant scion types. This was published in Horticulture Research- Nature, and the industry is using this work to petition for changed standards in the U.S. and internationally. This will result in an orange juice industry that is much better able to deal with production problems created by huanglongbing disease.
3. Corona-discharge plasma increases dissolved oxygen in field irrigation water. The amount of dissolved oxygen in water is considered the single most important factor to determine water quality. Ecologically, dissolved oxygen levels below 5 parts per million (ppm) are considered harmful to most life forms. High quality water for agricultural irrigation should have oxygen values greater than 8. It is not uncommon in Florida for the oxygen levels of the water used to irrigate citrus to be less than 2. ARS researchers at Ft. Pierce, Florida, led a study to evaluate the use of a corona-discharge plasma unit to increase dissolved oxygen levels in the irrigation water applied to field citrus trees, and to evaluate the effects on the growth of the trees. Air enters the unit, is ionized by the corona plasma, and then injected via a venturi into the irrigation stream. The result is that the dissolved oxygen is increased from 2.5 ppm to 8.5 ppm in the irrigation water, which is high quality irrigation water. Studies are continuing to measure the long-term positive effects on tree growth.
Review Publications
Stover, E., Gmitter, F.G., Grosser, J., Baldwin, E., Wu, G.A., Bai, J., Wang, Y., Chaires, P., Motamayor, J.C. 2020. Rationale for reconsidering current regulations restricting use of hybrids in orange juice. Horticulture Research. 7:38. https://doi.org/10.1038/s41438-020-0277-5.
Hall, D.G., Ramadugu, C., Hentz, M.G., Gmitter, F.G., Stover, E.W. 2019. Survey of Poncirus trifoliata hybrids for resistance to colonization by Asian citrus psyllid. Florida Entomologist.102(3):635-637.
Ali, S., Plotto, A., Scully, B.T., Wood, D.R., Stover, E.W., Owens, N.D., Pisani, C., Ritenour, M., Anjum, M., Nawaz, A., Naz, A., Bai, J. 2020. Fatty acid and volatile organic compound profiling of avocado germplasm grown under East-Central Florida conditions. Scientia Horticulturae. 261. https://doi.org/10.1016/j.scienta.2019.109008.
Krystel, J.A., Shi, Q., Shaw, J.J., Goutam, G., Hall, D.G., Stover, E.W. 2019. An in vitro protocol for rapidly assessing the effects of antimicrobial compounds on the unculturable bacterial plant pathogen, Candidatus Liberibacter asiaticus. Plant Methods. 15:85. https://doi.org/10.1186/s13007-019-0465-1
Bowman, K.D., McCollum, T.G., Plotto, A., Bai, J. 2019. Minnie finger lime: A new novelty citrus cultivar. HortScience. 54(8):1425-1428. https://doi.org/10.21273/HORTSCI13622-18.
