Location: Subtropical Insects and Horticulture Research
2021 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 third year of a project that continues the long-term goals of previous projects at the USHRL in Ft. 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 by ARS researchers in Ft. Pierce, Florida, 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.
Advances continue in developing improved conventional citrus scions. Over 30,000 new scion hybrids have been planted in the last eleven years, with 6,000 more in the greenhouse for subsequent planting. 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 -tolerant release US SunDragon. The two most outstanding selections this year are both very sweet-orange-like, with one being totally seedless and one extremely sweet-orange-like and a cross between our two most huanglongbing-tolerant parents. As our understanding of tolerance advances ARS researchers in Ft. Pierce, Florida, make better crosses each year. Ten cultivars have been released in the last six years. Two processors have assessed US SunDragon and found that its juice markedly improves Hamlin juice at additions as low as 10%.
Extensive efforts were made by ARS researchers in Ft. Pierce,Florida, to enhance utilization of new USDA scion selections, through discussions, presentations, and drafts to consider expansion of the Orange Juice standards to include other hybrids, and consideration of the value of the “Juice Orange Group” cultinomic designation, and administration through a Citrus International Cultivar Registry Authority.
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, subjected to clean-up and most are replicated and in trials. A large replicated trial of 50 advanced selections and cultivars is in the seventh 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, and US Superna performed especially well again, with exceptional flavor, acceptable peelability, and good evidence of huanglongbing-tolerance.
In the past year, fifty new hybrids were selected from large populations for further evaluation as rootstocks in field trials. Data was collected by ARS researchers in Ft. Pierce, Florida, on tree size, health, cropping, and fruit quality from 21 established replicated rootstock field trials. Four new replicated rootstock field trials were planted by ARS researchers in Ft. Pierce, Florida, and trees were prepared for planting of additional new replicated rootstock field trials, including 45 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 by ARS researchers in Ft. Pierce, Florida, 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 by ARS researchers in Ft. Pierce, Florida, 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. Methods were developed by ARS researchers in Ft. Pierce, Florida, that allow effective evaluation of cultivar tolerance to huanglongbing disease in a one-year greenhouse test.
Studies by ARS researchers in Ft. Pierce, Florida, began to evaluate the use of huanglongbing-resistant microcitrus species germplasm as interstocks with common rootstock and scion cultivars. It is hypothesized that these graft combinations may provide greater tree tolerance to the disease, while still producing the same strong root systems and high fruit quality.
Progress continues in developing transgenic citrus with huanglongbing-resistance. 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, greatly increase psyllid mortality, suppression of huanglongbong in the greenhouse, and are replicated for more extensive tests including field trials already planted including new plantings in 2021. Grapefruit has been grafted onto replicated rootstocks transgenically expressing antibodies to two huanglongbing-pathogen proteins and field planted. A phloem-specific promoter from citrus, identified by ARS researchers in Ft. Pierce, Florida, is very successful, with expression of a marker gene 400X 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 in Ft. Pierce, Florida, to prevent huanglongbing pathogen acquisition by Asian citrus psyllid. Two of these peptides markedly increased psyllid mortality in detached leaf feeding assays. The Florida Department of Agriculture has received buds of the most promising transgenics for cleanup, and ARS researchers in Ft. Pierce, Florida, have received some clean budwood which has been established for source plants.
Genome editing is underway, to knock-down a few key targets where loss-of-function may produce desired traits. Initial tests show that knock-down of the gene Downy Mildew Resistance 6 markedly reduced sensitivity to asian citrus canker. In previous work, ARS researchers in Ft. Pierce, Florida, 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 and have been sent to an ARS collaborator to pull-down the citrus gene products to which the effectors bind, allowing identification and genome editing knock-down of gene products which may be critical for disease development. Initiated development of a gene editing system for citrus using protoplasts embryogenic cell lines in collaboration with an ARS partner. This gene editing system will be used to produce huanglongbing-resistant citrus.
ARS researchers in Ft. Pierce, Florida, completed a preliminary field trial that determined the effects of iron chelates of certain types on canopy health of field grapefruit trees that have huanglongbing disease. Micronutrient fertilization is important in the management of disease. The experiment compared the effects of 7 forms of iron and measured their effect on various measures of grapefruit tree growth and health.
New research was initiated by ARS researchers in Ft. Pierce, Florida, at the request of the nursery industry to investigate the manipulation of citrus nursery conditions with artificial light from light emitting diodes that will improve nursery production of the commercially important citrus scion and rootstock cultivars during winter months. The nursery supply of new trees to replace field trees declining or dead is a critical component of citrus industry survival, especially with the widespread tree loss from huanglongbing disease.
Accomplishments
1. ARS researchers in Ft. Pierce, Florida, assembled multiyear phenotype data on 350 new hybrid rootstock selections in 15 replicated field trials with sweet orange scion. The phenotype data is being used to map important rootstock traits in the citrus genome to allow better choice of parents for future crosses and accelerate identification of new hybrids with the best combinations of rootstock traits. Until important rootstock traits are mapped on the genome, identification of superior new hybrid rootstocks relies on large multiyear field trials that require intense investment of resources. Advance selection of superior parents and hybrids with genetic maps and molecular markers will accelerate rootstock breeding progress and reduce expenditure of resources on field testing of selections with low fitness.
2. ARS researchers in Ft.Pierce, Florida, led effort in establishing a large field trial of 200 citrus scions selections with stakeholders and university citrus breeders. This will serve as a foundation for future genomic selection. Accessions have been 25x sequenced and plans are being developed for deep phenotyping and development of a computational model to predict key phenotypes using genotype data. When this model is validated, it will greatly accelerate: selection of new hybrids, their entry into advanced trials, and their release as cultivars to the industry. The model will also be used to optimize parent selection for future crosses.
3. ARS Researchers in Ft. Pierce, Florida, evaluated incidence and severity of Asian citrus canker in progenies of 84 accessions of trifoliate orange and their hybrids. Asian citrus canker remains an important disease which compromises plant health and fruit marketability. Trifoliate orange is an important source of resistance to the disease huanglongbing, but displays great sensitivity to Asian citrus canker. Most of the 49 trifoliate orange accessions were in the highest sensitivity group, while most of the trifoliate orange hybrids were less sensitive, indicating that trifoliate orange sensitivity to Asian citrus canker is readily overcome through breeding. For all 3 years of the study, one trifoliate orange accession that was represented by only one tree was much less susceptible to Asian citrus canker than other accessions of trifoliate orange, which merits further investigation.
4. ARS researchers in Ft. Pierce, Florida, developed improved methods for rapid evaluation of tolerance to huanglongbing disease under greenhouse conditions. Evaluating tolerance to this disease under field conditions takes at least 5 years and large investments of resources. The new greenhouse methods provide clear comparisons of rootstock tolerance to huanglongbing in combination with sweet orange scion in one year, and the results of the greenhouse testing provide similar relative values for tolerance to what is shown from field testing of more than 5 years in a huanglongbing-endemic environment.
5. ARS researchers in Ft. Pierce, Florida, determined that certain forms of iron improved the canopy size and health of grapefruit trees. In nature, iron consists of a soluble form, ferrous (Fe2+) iron, and an insoluble form, ferric (Fe3+) iron. Fe2+ is the form of iron that plants and animals utilize. Fe2+ is the form taken up by the plant. Iron supplied as a synthetic chelate such as iron-EDTA is in the Fe3+ ionization state and must be converted to Fe2+ before the iron can be taken up and used by the plant. Huanglongbing-infected citrus trees have reduced iron levels and a reduced ability to take up iron. This project was based on the idea that huanglongbing disrupts iron uptake and/or metabolism in the tree, and/or the initiation of the Fenton Reaction by Fe2+ to increase a citrus tree’s natural resistance to huanglongbing. Therefore, this project tested the idea that the form of iron used in citrus nutrition, and particularly organic acid-stabilized ferrous iron (Fe2+), can rejuvenate and possibly cure citrus trees infected with huanglongbing. Seven forms of iron were applied to both bearing and nonbearing age grapefruit trees. The bearing grapefruit trees were abandoned on 03/01/2020 due to severe decline caused by irreversible root damage, primarily due to the Phytophthora/Diaprepes complex exacerbated by huanglongbing. Certain forms of iron were identified that improved the canopy size and health of the nonbearing grapefruit trees. The effects on fruit yield and quality will be determined over the next 2-3 years.
6. ARS researchers in Ft. Pierce, Florida, developed improved methods for winter citrus nursery production with power-efficient lighting. Replacing citrus trees lost in the field from huanglongbing disease and environmental problems is critical to helping the US citrus industry survive the current challenges from disease and increasingly harsh environmental conditions. The citrus nursery faces many challenges to provide sufficient young trees to replant trees lost in the field at a pace equal to that of field tree loss. The methods of extending winter daylength by four hours with light emitting diode lighting of the proper spectrum was shown to greatly increase nursery bud break and tree growth, with low power use. This will significantly improve productivity of the citrus nursery during the winter months.
