Source: UNIVERSITY OF CALIFORNIA, RIVERSIDE submitted to NRP
CAP: LARGE-SCALE BREEDING AND EVALUATION OF THE NEXT GENERATION OF SUPERIOR ROOTSTOCKS FOR CITRUS PRODUCTION IN REGIONS WITH HUANGLONGBING
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
OTHER GRANTS
Reporting Frequency
Annual
Accession No.
1031585
Grant No.
2023-70029-41305
Cumulative Award Amt.
$6,854,123.64
Proposal No.
2023-06850
Multistate No.
(N/A)
Project Start Date
Sep 15, 2023
Project End Date
Sep 14, 2028
Grant Year
2023
Program Code
[ECDRE]- Emergency Citrus Disease Research and Extension Program
Recipient Organization
UNIVERSITY OF CALIFORNIA, RIVERSIDE
(N/A)
RIVERSIDE,CA 92521
Performing Department
(N/A)
Non Technical Summary
. ?
Animal Health Component
70%
Research Effort Categories
Basic
30%
Applied
70%
Developmental
0%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
20209991081100%
Knowledge Area
202 - Plant Genetic Resources;

Subject Of Investigation
0999 - Citrus, general/other;

Field Of Science
1081 - Breeding;
Goals / Objectives
. ?
Project Methods
Methods are described for each objective.Objective 1:Field trials: Twenty-one replicated rootstock field trials with sweet orange scion will be used for data collection and rootstock evaluation. This includes nineteen trials that were planted in Florida between 2014 and 2022 and two additional trials have been planned for planting in 2024. Beginning the year after planting, data collection from the trials includes tree survival and canopy health rated three times per year and tree canopy size measured once per year. Once cropping begins, fruit data will also be collected once per year, and includes fruit count and fruit quality using a fruit sample to measure total soluble solids, total acids, juice color, percent juice, and fruit weight. Other traits are calculated from these values including fruit yield per tree, yield efficiency, fruit yield per acre, and pounds solids per acre. Evaluations of trunk diameter, HLB symptoms, canopy thickness, canopy color, CLas and CTV infection frequency are measured every four years after harvest begins.Plant therapy: All citrus germplasm is introduced to the CCPP as budwood under quarantine following the conditions of federal and state permits. Upon receipt, budwood is surface sterilized, and then placed in tissue culture media to initiate vegetative meristem production for shoot-tip grafting (STG) following established protocols. A series of pre-index laboratory tests are used to identify the pathogen status of the material upon arrival. After 6-9 months of growth, the therapied plant undergoes a series of 80 laboratory and bioindexing tests over the next 8-12 months as part of the comprehensive variety index (VI) process. If all pre-index and VI index tests are negative, the accession is released from quarantine and can be used for research and field trials, for commercial propagation and planting, and in any U.S. state via the NCPN.Objective 2: All rootstock releases are currently available for field and greenhouse studies in Florida. First generation rootstocks are available for field and greenhouse studies in California and Texas, and next generation rootstocks will be available in year three. Field studies: Two demonstration blocks will be established in Florida for 20 next generation rootstock selections, including 5 recent SuperSour releases, and industry standards. Trial sites will include a flatwoods-type environment (east coast or southwest) with poorly drained soils and prone to infestation with Phytophthora and a well-drained environment in central Florida. Selected rootstocks will be propagated by cuttings and grafted with 'Valencia 1-14-19'. Trees will be planted in commercial citrus groves using 7 or more replications of each rootstock. To mimic current commercial production practices, half of the trees will be covered with Individual Protective Covers (IPCs) to protect new plantings from the insect vector. This practice is effective at preventing HLB infection and has been widely adopted by Florida growers (and is also investigated in Texas). The effect of IPC on rootstock performance will reveal the economic potential of HLB-infected and HLB-free rootstocks during the first years of establishment. Two demonstration trials will also be planted in both California and Texas, following similar experimental designs. The primary difference between trials will be selection of regionally important scions. In California, rootstocks will be grafted with 'Washington navel' sweet orange and 'Tango' mandarin. Texas field trials will evaluate rootstock performance with 'Marrs' sweet orange and 'Rio Red' grapefruit. Propagations for the first set of field trials in both states will begin in year one and include first generation HLB-tolerant rootstocks (US-802, US-812, US-897, US-942) alongside regional commercial standards. These hybrids have been extensively evaluated in Florida, with some widely adopted for commercial production, but they have never been systematically tested outside of Florida. The second set of field trials in both states will include a minimum of 5 next generation SuperSour rootstocks along with industry standards. Demonstration trials in California and Texas will follow the Florida design and planting density typical for sweet oranges, mandarins, and grapefruit.Greenhouse studies: Field performance of rootstock hybrids is the best indication of rootstock adaptability and an important guide for growers making decisions about new plantings. Focused greenhouse testing of rootstocks for their response to abiotic and biotic stresses will supplement field evaluations. In California, resistance to Tylenchulus semipenetrans, Fusarium solani, Phytophthora citrophthora and P. nicotianae, will be evaluated in environmental growth chambers on citrus seedlings using established protocols.Rootstock response to abiotic stresses will also be evaluated, including poor water quality, reduced water availability, and salinity for first generation rootstocks in year one and next generation rootstocks in year three. Rootstocks will be grafted with 'Washington navel' and grown in 10L pots with sandy loam soils from central California. The experimental design will be a split-plot design including 10 replications with rootstock as the main factor and salinity level (or soil water content level) as the subplot.Evaluations of water use efficiency and capacity to withstand poor water quality will be made using physiological and biochemical measurements.Objective 3:Methods. The following genomic and genetic resources and biotechnological tools will be generated. We have previously generated an average of 56X sequence coverage for each of the 50 parental cultivars with Illumina 150 bp paired-end sequence reads. Sequence libraries for the 378 hybrids have been constructed and pooled and are currently awaiting sequencing to 30X coverage. Additional whole-genome sequence (WGS): Generation of WGS for progeny of next generation rootstocks will support genomic prediction and fine-scale genetic analysis. DNA will be extracted from leaf tissue for each individual using a Qiagen DNEasy 96 Plant kit and barcoded sequence libraries produced with an Illumina DNA Prep kit. Libraries will be pooled and sequenced with 150 bp paired-end reads on the Illumina NovaSeq S4. Genetic marker development: Targeted marker assays will be implemented for genomic loci strongly associated with HLB tolerance following standard practices. To ensure performance across germplasm groups, all assays will be tested against each parental cultivar by high-resolution melting curve analysis. Genome assemblies: High molecular weight (HMW) DNA (>20 Kb) will be extracted from new flush from each parental cultivar. An additional high salt and low ethanol precipitation is typically required for the purification of HMW DNA from citrus. Next, PacBio HiFi library preparation and sequencing to 50X coverage with the PacBio Revio instrument will be performed. Citrus with on-demand flowering: To engineer transgenic lines for accelerated breeding, we will transform our inducible FT (iFT) construct into epicotyl segments using well-established Agrobacterium-mediated citrus transformation methods; we have used this protocol to create 36 transgenic citrus lines for 13 cultivars. The iFT construct harbors a visible mCHERRY marker, which allows facile identification of transgenic shoots, which are rescued by grafting onto 'Carrizo' rootstocks. The rescued lines will be tested for floral induction after inducer (mandipropamid) applications; three validated, morphologically normal lines will move forward for use in crosses.

Progress 09/15/23 to 09/14/24

Outputs
Target Audience:During this reporting period we have interacted with citrus industry stakeholders through regional, state in-person events and seminars and through the preparation of extension materials. We are particularly focused on raising awareness of the ongoing research supported through this proposal to growers interested in the adoption of new HLB-tolerant rootstocks in CA, TX and FL. We delivered at least 9 presentations at events attended by citrus industry stakeholders. We have held our first annual advisory meeting in March 2024. Our advisory board is comprised of citrus industry stakeholders from each region, including nursery and grower representatives. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two postdoctoral research associates and one graduate student were trained on research of citrus production and rootstocks and/or genetic analysis of citrus germplasm. Graduate students and postdoctoral researchers receive mentorship in research and also career advancement. This occurs through training focused one-on-one and group meetings, encouragement to establish an individualized development plan to identify and accomplish long-term career goals, and access to career and networking events at the relevant universities. How have the results been disseminated to communities of interest?We have presented information on the data collected from these trials to grower and other stakeholder communities and will continue to do so in the upcoming reporting period, including at least 9 presentations on this project to audiences composed mostly of citrus industry stakeholders. The goal is to ensure there is interest and awareness of these new HLB-tolerant rootstock hybrids to facilitate rapid adoption in relevant regions upon release. Below is a list of presentations where research related to this project was shared: Bowman, K.D. Southeast Fruit and Vegetable Growers Association Annual Conference, 11-14 January 2024, Savannah, Georgia. Invited presentation: "USDA Citrus Rootstock Development Research". Bowman, K.D. International Research Conference on Huanglongbing, 25-29 March 2024, Riverside, California. Invited presentation: "New HLB-Tolerant Citrus Rootstocks and the SuperSour Breeding Strategy". Bowman, K.D. International Rootstocks Symposium, 8-9 May 2024, Rimini, Italy. Invited presentation: "Aiming the USDA SuperSour breeding strategy at HLB-tolerant rootstocks". Bowman, K.D. Citrus Research International Research Symposium, 18-22 August 2024, Kwazulu-Natal, South Africa. Invited Keynote address: "Breeding Superior New Citrus Rootstocks: USDA Success, and Remaining Challenges from Diseases and other Stresses". Rolshausen, P. Botany and Plant Sciences Seminar, UC Riverside, CA 'Choosing a Career in Cooperative Extension'. 2024 Rolshausen, P. Annual Conference on Soilborne Plant Pathogens - APS Pacific Division Meeting. Corvallis, OR: 'Assessment of Huanglongbing-tolerant citrus hybrid rootstocks for resistance against citrus nematode (Tylenchulus semipenetrans Cobb)' 2024 Rolshausen, P. Organic Avocado and Citrus Workshop - UC ANR. Ventura, CA 'Citrus wood and root diseases'. 2023 Seymour, D.K. Southern California Plant Biology Symposium. San Diego, CA. 'Integrating scales to traverse the genotype-phenotype divide in citrus" October 2023. Seymour, D.K. International Research Conference on Huanglongbing. Riverside, CA. 'Dissecting the genetic basis of HLB tolerance in large-scale breeding and evaluation trials' March 2024. Seymour, D.K. Invited seminar University of British Columbia. Vancouver, Canada. 'Integrating scales to traverse the genotype-phenotype divide in citrus" January 2024. Seymour, D.K. UC Davis Plant Sciences Symposium. Davis, CA. 'Integrating scales to traverse the genotype-phenotype divide in citrus" January 2024. Seymour, D.K. Invited Seminar Institute of Subtropical and Mediterranean Horticulture. Malaga, Spain. 'Integrating scales to traverse the genotype-phenotype divide in citrus" January 2024. What do you plan to do during the next reporting period to accomplish the goals?Objective 1: Collection of field trial data for 2024/2025 season is ongoing as anticipated and will continue for the duration of the project. Hybrids will be continually evaluated as data becomes available to identify top performing rootstock hybrids with enhanced tolerance to HLB. Hybrids will continue to evaluated for their potential for seed propagation and sampled for molecular evaluations. In Florida, two new field trials of the most promising new rootstocks in Florida will be planted in the upcoming year. Therapy and quarantine for 23 new rootstock hybrids will continue at CCPP and new selections will continue to be submitted to clean budwood programs in Florida and California in anticipation of future release. Objective 2: Testing of released rootstocks from Bowman's USDA-ARS program will continue in CA to evaluate their response to soilborne pathogens Phytophthora nicotiana, Phytophthora citrophthora, Fusarium solani and Tylenchulus semipenetrans. In California, the first field trial of HLB-tolerant rootstocks will be planted at Lindcove REC. The goal of this trial is to evaluate the horticultural performance of two scion varieties Tango and Washington Navel grafted on the HLB-tolerant citrus hybrid rootstocks US-897, US-942, US-1516, US-812, Super Sour2 and compare them to California standards Carrizo and Rich. Greenhouse evaluation of HLB-tolerant rootstocks for their response to salt stress will begin. In Texas, 8 HLB-tolerant rootstocks will be grafted with orange and grapefruit and planted in a field trial for evaluation. Objective 3: Genome sequencing and assembly will be completed for parents of the SuperSour breeding program following the bioinformatic workflow established for the assembly and annotation of US-897. As additional trial data is collected we will incorporate it into genomic prediction models. Zygotic seedlings from next-generation rootstock hybrids are growing at the USDA-ARS in Ft. Pierce. Around 300 hybrid progeny will be selected for sequencing and genomic prediction used to identify the top 10% of performers which will be tested in future field trials for their performance in HLB-endemic conditions. Transgenic Cleopatra with inducible flowering will be used as a mother in crosses with US-1688 to create a "backcross" population that can be used to evaluate the genetic basis of HLB-tolerance in this family. All PIs will continue to disseminate research results through extension activities at the local, regional, and national level. A second advisory board meeting will be held in the coming year.

Impacts
What was accomplished under these goals? Objective 1. Select next generation rootstocks from large-scale field evaluations in HLB-endemic regions. Select and release new HLB-tolerant rootstocks: Continued evaluation of 378 next generation rootstock hybrids (grafted with a common sweet orange scion) in a series of 21 field trials will support the selection and release of rootstock hybrids with superior performance. Two rootstocks were selected from these trials released by the USDA in 2024 (SuperSour 4 and SuperSour 5). In the last year phenotypic data related to tree health, tree size, fruit crop size, and fruit quality was collected from 21 sweet orange rootstock field trials in Florida. Preliminary selections among these hybrid rootstocks have been made based on multiyear performance data. Develop source of certified disease-free material: Currency, 30 next generations are available as clean budwood from the FDACS program and 23 of those have been transferred to the Citrus Clonal Protection Program (CCPP) and therapy has begun to develop disease-free sources of rootstocks for commercial use in California. Material transfer agreements were developed and approved for transfer the preliminary 23 most promising selections from USDA Florida to California CCPP to complete quarantine testing that will allow broad testing and commercial use of these rootstocks in California and Texas. Three of these have been released by the USDA already and would be freely available in CA once they have completed therapy and quarantine. Objective 2. Determine commercial potential of new rootstocks selections in major citrus growing regions. To promote adoption of these new rootstocks in Florida, and other citrus growing regions including Texas and California, further characterization of rootstock characteristics is essential. Their potential to benefit regional market sectors depends on compatibility with multiple scion types, including sweet orange, mandarin, and grapefruit. In addition to scion compatibility, priority rootstock traits include seedling uniformity, tolerance to soil salinity and soil moisture, sensitivity to temperature extremes, and resistance to pests and diseases. In Florida: Trees were propagated with the most promising of the hybrid rootstocks for additional semi-commercial evaluation. Planned field planting is fall 2024. Selected new promising rootstocks that can be propagated by seed were transferred under permit as seed to California and Texas for preliminary evaluation and testing. Nucellar embryony of new HLB-tolerant rootstocks was evaluated using visual/SSR marker comparisons to determine whether they produce zygotic or nucellar embryos. In California: Seeds of citrus hybrid rootstocks US-897, US-942, US-1516, US-812, Super Sour2 were received and planted at the Lindcove Research and Extension Center in California under APHIS permit# PPQ 526-24-85-43993. These plants will be included in the initial trial to HLB-tolerant rootstocks to be planted in California. In addition, disease resistance evaluations of the hybrid rootstocks US-802, US-812, US-897, US-942, US-1283, US-1284, US-1516 to Phytophthora nicotiana, Phytophthora citrophthora, Fusarium solani and the nematode Tylenchulus semipenetrans were completed. In Texas: Seed for 8 hybrid rootstocks (including US-897, US-942, US-1516, US-812, Super Sour2) were planted and grafting with selected scions is planned in the coming year. These plants will be included in the initial trial to HLB-tolerant rootstocks to be planted in Texas. Objective 3: Optimize selection for HLB tolerance/resistance through implementation of genomics-assisted breeding to boost efficiency of future rootstock breeding cycles. New genomic resources, genetic analysis, and biotechnological tools will be integrated to overcome current challenges in citrus rootstock breeding, including the long generation time of citrus and need to evaluate many inferior progeny in the field. Genomic-assisted breeding: Initial genomic predictions for rootstock-mediated influence on performance of the grafted scion are promising. With 150 hybrids across 5 (of the 21) field trials, decent prediction accuracies can be obtained (r>0.6), including for traits related to yield. To further supplement genomic prediction efforts, we are assembling the complete genomes of the 50 parents. Leaf samples have been collected for these 50 lines. To start, we have completed a long-read assembly for US-897, including scaffolding and annotation. Sequencing and assembly of the remaining lines is in progress. Biotechnological tools: Cleopatra is one of the 50 parents in the SuperSour breeding program and the parent of important HLB-tolerant hybrids. Transgenic Cleopatra with an inducible flowering construct have been developed. Sequencing to confirm the transgene and uniform genetic background are in progress.

Publications

  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Bowman KD. 2023. Four new SuperSour rootstocks for improved production of sweet orange in a huanglongbing environment. HortScience 58(12):1622-1625.