Location: Subtropical Insects and Horticulture Research
2022 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 fourth year of a project at the U.S. Horticultural Research Laboratory 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 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 scientists at Fort 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.
In the past year, 20 new hybrids were selected from large populations for further evaluation as rootstocks in field trials. Data was collected on tree size, health, cropping, and fruit quality from 25 established replicated rootstock field trials. Three new replicated rootstock field trials were planted, 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 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 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 that allow effective evaluation of cultivar tolerance to huanglongbing disease in an 18-month greenhouse test.
Work began under collaboration with funding from a new NIFA grant to sequence 400 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, with additional data to be collected in the coming year. Important traits to be mapped include rootstock influence on fruit quantity, fruit quality, tree size, tree health, and tolerance of huanglongbing disease.
The scion breeding program continued the development of citrus scions with improved quality and disease tolerance traits for use in the juice industry and the fresh market. New conventional crosses were made with individuals that have shown superior tolerance to huanglongbing in the field. Many of the new crosses incorporated new material that had not been used previously in the breeding program, and have the potential to produce a wider variety of commercial traits. This season, an increased effort was made to evaluate some of the oldest collections in the breeding program, which has led to the rediscovery of varieties that are interest to the commercial industry such as the sweet oranges ‘Donaldson’ and ‘Tobias’ and three grapefruit cultivars. Additional trees of these cultivars are being propagated to assess the tolerance of these individuals in replicated trials.
Several hundred irradiated mutants of ‘Bower’ and ‘Dancy’ were planted by ARS scientists at Fort Pierce, Florida, in the field at the Whitmore foundation farm in Groveland, Florida. An observation was made that ‘Bower’ and ‘Dancy’ appeared more tolerant to huanglongbing in a replicated trial of 50 advanced stage accessions and Florida varieties. ‘Dancy and ‘Bower’ were irradiated to reduce seed count and improve other organoleptic fruit qualities. There were also 700 pumelo (grapefruit- like) hybrids that were screened for low seed count and four tentative selections were made that will require an additional season of screening to validate low seed count.
Advanced phenotyping methodologies are being tested in collaboration with Breeding Insights and other USDA researchers. The testing of advanced technologies such as image analysis and machine learning are being evaluated for use in the breeding program on advanced stage replicated trials with the goal of validation of new technologies and deployment of successful technologies on the majority of the breeding populations.
Greenhouse studies are underway for molecular marker development of differentially expressed genes that are associated with huanglongbing tolerance in citrus scions. In this study ARS scientists at Fort Pierce, Florida, are deploying RNA sequencing for tolerant and susceptible genotypes to discover candidate genes for huanglongbing tolerance that may lead to the development of molecular markers that can be used to screen citrus scion trees for huanglongbing tolerance.
Assessment of canopy health continued for 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 have started to bear fruit. The initial study was completed but the cooperator will be collecting fruit data and requested additional canopy size and health measures. The initial experiment compared the effects of seven forms of iron on various measures of grapefruit tree growth and health, but with the trees starting to fruit, the effects on fruit number, size, and quality can also be determined.
Research was initiated to identify treatments to improve the health of citrus trees that have huanglongbing disease. These efforts included 1) reinitiating multiple psyllid colonies that are required to produce greenhouse trees that have huanglongbing disease, 2) developing a rapid laboratory assay that uses detached leaves, and 3) setting up a field trial to test a treatment with purported benefits to manage huanglongbing disease. The psyllid inoculation and detached leaf assay will be used to identify treatments that improve the health of treated trees or assess the level of resistance of citrus selections from the citrus breeding program. The psyllid colonies were calibrated to verify that they carry the bacteria that causes huanglongbing disease. Several thousand sweet orange and grapefruit seedlings have been established in the greenhouse. These are being used as the source trees for psyllid infection. About 300 citrus plants have been treated with the huanglongbing disease psyllids, with the oldest set being about 3 months old. It takes 3-5 months for symptoms to emerge. The detached leaf assay uses leaves from huanglongbing-infected citrus trees. Each leaf is placed into a solution containing a test molecule, a compound with putative antibacterial properties. The amount of bacteria are measured at the start and 7 days later to determine if the test molecule reduces the number of bacteria. The detached leaf assay has been set up and is now being calibrated using molecules known to affect this bacteria – e.g., the antibiotic streptomycin. A field trial was setup to determine if a foliar/soil treatment improves the health of huanglongbing infected trees as purported by some citrus growers.
Research funded by the nursery industry to investigate the manipulation of citrus nursery conditions with artificial light from light emitting diodes was continued. These studies 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. Completed an eight year replicated rootstock field trial with Valencia scion and 46 new hybrid rootstocks. The overall trial was severely damaged by infection from huanglongbing disease, and several new rootstocks were identified with outstanding performance in comparison with the most common citrus rootstocks. Cross comparisons of performance of these rootstocks in other trials is being used by ARS scientists at Fort Pierce, Florida, to determine those most promising for widespread commercial use and targeted for official release in the coming year.
2. Selected new citrus scion cultivars that can be used for juice blends and as parents. A broad range of huanglongbing tolerant citrus that can be used directly or as parents have received interest from growers and processors. Specifically, some of the advanced mandarin and sweet orange-like hybrids have high brix levels, which are desirable to processors, despite repeated infection in an HLB endemic environment. Due to the low brix levels of late season commercial sweet oranges, processors are now considering the inclusion of USDA mandarins and orange-like hybrids in quantities of 10%, as permitted by juice regulations to improve juice quality. As a result, there has been renewed interest in some mandarin and orange-like hybrids that are in collaborative trials. In addition, there are individuals that we have selected as breeding parents for use in the introgression and breeding of improved huanglongbing tolerant citrus cultivars. The breeding parents have useful tolerance to huanglongbing, but may have certain flaws that prevent direct commercial use. The use of these as breeding parents will result in the production of new hybrids with increased huanglongbing tolerance and more commercially desired fruit quality traits.
3. Identified inoculation techniques for evaluating huanglongbing tolerance in greenhouse. This is part of an ongoing study where groups of huanglongbing tolerant and susceptible material were selected from field trials, and used to determine if differences in tolerance can be observed following infection in the greenhouse. We were able to show clear differences in the greenhouse performance of tolerant and susceptible materials. We plan to refine this process and establish correlation between our short-term greenhouse trial and longer-term field tolerance. In this case, we worked backwards from field tolerance to demonstrating the same effects were achieved in the greenhouse. This will allow us to develop candidate genes and markers associated with HLB tolerance, through the differential expression of genes when inoculated in the greenhouse. For the next step, we will identify promising selections by inoculation in the greenhouse, and then test those selections for long-term tolerance in the field. Comparative assays in the greenhouse will allow for the creation of genomic tools that will aid in the accelerated development of citrus scions.
4. Developed transgenic clones that are highly resistant to bacterial disease. ARS scientists at Fort Pierce, Florida, developed transgenics that are highly resistant to bacterial diseases such as citrus canker when inoculated in the greenhouse by reducing the expression of Downey Mildew Resistance 6 genes using CRISPR and RNA interference. Newly developed non-transgenic methods are currently being adapted to produce plants with the same resistance to bacterial disease that may be more accepted by consumers.
5. Established a field trial to evaluate a therapeutic treatment for huanglongbing disease. Established a field trial of 122 trees composed of Valencia and Hamlin sweet oranges, Ruby Red grapefruit, and Minneola tangelo to determine the effects on canopy health and fruit yield and quality of a treatment claimed by some citrus growers to substantially improve the health of citrus trees that have huanglongbing disease. The treatment is applied to the foliage and soil and is environmentally benign.
Review Publications
Bodaghi, S., Pugina, G., Meyering, B., Bowman, K.D., Albrecht, U. 2022. Different sweet orange-rootstock combinations infected by Candidatus Liberibacter asiaticus under greenhouse conditions: Effects on the roots. HortScience. 57(1), 56-64. https://doi.org/10.21273/HORTSCI16206-21.
Bodaghi, S., Meyering, B., Bowman, K.D., Albrecht, U. 2021. Different sweet orange-rootstock combinations infected by Candidatus Liberibacter asiaticus under greenhouse conditions: Effects on the scion. HortScience. 57(1), 144-153. https://doi.org/10.21273/HORTSCI16205-21.
Bowman, K.D., Mccollum, T.G., Albrecht, U. 2021. SuperSour: A new strategy for breeding superior citrus rootstocks. Frontiers in Plant Science. 12, 741009. https://doi.org/10.3389/fpls.2021.741009.
