Source: CLEMSON UNIVERSITY submitted to NRP
CAP: FAST SELECTION, ASSESSMENT, AND DELIVERY OF HLB-RESISTANT/TOLERANT CITRUS VARIANTS AS NEW SCION AND ROOTSTOCK CULTIVARS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
OTHER GRANTS
Reporting Frequency
Annual
Accession No.
1031577
Grant No.
2023-70029-41309
Cumulative Award Amt.
$4,115,736.00
Proposal No.
2023-06841
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
CLEMSON UNIVERSITY
(N/A)
CLEMSON,SC 29634
Performing Department
(N/A)
Non Technical Summary
Huanglongbing (HLB) is the most devastating disease of citrus in the U.S. Economic losses over the past five years in FL alone amount to over $1 billion per year, with nearly 5,000 jobs lost annually. The U.S. citrus industry is in dire need of new citrus cultivars with stable and broad-spectrum resistance/tolerance to HLB as well as desirable horticultural characteristics, crop yield, and fruit quality. The other major citrus states are at high risk for a similar disastrous situation if suitable remedies are not forthcoming soon. Neither a simple cure nor successful management strategies have been identified even after 15+years of the disease' presence in Florida.Previous efforts to create new HLB resistant/tolerant cultivars were focused on conventional hybrid breeding or the development of transgenic plants. Unfortunately, the approaches used in those projects are inadequate to meet the current crisis because of the lack of sources of resistance in citrus, time constraints associated with citrus breeding and genetic engineering, and regulatory obstacles for transgenic citrus. Meanwhile, more than 15 years of HLB epidemics and tens of millions of infected trees in groves across Florida provide an unprecedented opportunity and high probability for selecting new mutant scions that are resistant or at least highly tolerant. Over the past years, our group has selected multiple citrus varieties with field resistance or tolerance to HLB from Florida groves. A recent study showed that HLB is a pathogen-triggered immune disease. These natural variants may have HLB resistance/tolerance through evasion of the immune response or loss of susceptibility genes. Although the exploitation of natural resistance/tolerance provides a key solution to developing new HLB-resistant/tolerant citrus scions and rootstocks, it still requires a significant amount of time to confirm stable resistance of the selected varieties. It is critical to develop rapid methods to shorten the selection process, to provide solutions within a shorter time frame to ensure the survival of the citrus industry, especially in Florida.Our previous research shows that transposon activities are one of the important drivers of diversification in sweet oranges. By monitoring the transposon activities in selected varieties, we may be able to determine their genetic stability, and then facilitate selection. Furthermore, there are many available sequences of citrus genomes, including several HLB-resistant/tolerant citrus plants and their counterparts that our group and others have sequenced. It is critical that we can integrate all genome-based information to understand the HLB-resistant/tolerant or susceptibility-related genes. With more sequences of HLB-resistant/tolerant citrus plants and their counterparts in this project, we can build a pangenome that includes all HLB-resistant/tolerant related variants. This should help identify HLB-resistant/tolerant or susceptibility-related genes.This project addresses the "Development of commercial citrus varieties (rootstocks and scions) for both fresh and processed markets with genetic tolerance and resistance to HLB using traditional breeding techniques and/or gene editing", which is one of the ECDRE focus areas.In this proposal, we first present a systematic approach with emphasis on the rapid selection, assessment, and delivery of new HLB-resistant/tolerant citrus variants as new scion and rootstock cultivars. Second, we will develop a novel pangenome to identify HLB resistance/tolerance or susceptibility-related genes. Moreover, with the HLB susceptibility-related genes identified in our previous study, we will develop new varieties via gene editing using CRISPR-Cas and test their resistance to HLB. We will conduct multistate field trails of our previous selected varieties and evaluate their horticultural performance and economic impacts. Meanwhile, we will conduct active outreach and extension to disseminate our project results to growers, stakeholders, and the public.Our practical methods for rapidly selecting, assessing, and delivering stable and broad-spectrum HLB-resistant/tolerant citrus variants will allow HLB-resistant/tolerant scion and rootstock cultivars to be developed and deployed much quicker without regulatory constraints. The integrated citrus pangenome can help identify the genetic elements associated with HLB resistance/tolerance or susceptibility. The identified HLB resistance/ tolerance or susceptibility related genes will provide a foundation for understanding the molecular mechanisms of Las infection and HLB symptom progression. Precise gene editing based on the CRISPR system will enable targeted disruption of citrus genes that are essential to Las infection, multiplication, and pathogenesis, production of loss-of-function mutants, and development of new HLB resistant/tolerant citrus cultivars that are free of GMO fingerprints and potentially free of regulatory obstacles to the market. Horticulture and socio-economics-based support tools will be developed for the industry to adopt new cultivars with confidence. The selected citrus varieties with HLB resistance/tolerance are expected to be the most durable, eco-friendly, and cost-effective solution to this devastating disease.The successful achievement of our goals and objectives will broadly impact the entire US citrus industry, by maintaining the viability of industries not yet fully impacted as in Florida, and by providing the plant materials that will encourage Florida growers to replant with confidence. The combined economic impacts of our US citrus industries will be measured in billions of dollars. The many communities throughout the country having economies built around viable citrus production will avoid the major disturbances that will come from the economic disasters that HLB incurs. The success of this project will set up a model for the citrus community across the globe to follow and solve the HLB problems in the not-too-distant future.
Animal Health Component
40%
Research Effort Categories
Basic
30%
Applied
40%
Developmental
30%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2010999108180%
2010999108020%
Knowledge Area
201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
0999 - Citrus, general/other;

Field Of Science
1081 - Breeding; 1080 - Genetics;
Goals / Objectives
The goal of this proposal is to develop a new generation of citrus cultivars that not only survive but are also profitable in the presence of HLB. Over the past years, we have successfully demonstrated that HLB-resistant/tolerant citrus plants can be selected in commercial groves and breeding orchards. The first generation of HLB-resistant/tolerant bud-sports and seedling variants that have been cleaned up by the Division of Plant Industry (DPI), Florida, which includes grapefruit, Valencia sweet orange, pummelo, and sour orange (as rootstock), will be subjected to advanced field trials. However, the selection and evaluation process still requires a long time. In this proposal, taking advantage of the success of our previous projects, we will develop practical methods for rapidly selecting, assessing, and delivering stable and broad-spectrum HLB-resistant/tolerant citrus variants as new scion and rootstock cultivars. We will pursue five interrelated research objectives in this project.Objective 1: Establish and optimize methods for rapid selection, assessment, and delivery of HLB-resistant/tolerant citrus bud-sports and seedling variants. Currently, transforming a selected citrus mutant into cultivars still takes a long time to make sure that it is stabilized, uniform, and resistant to HLB variants. Here, we will develop two methods to accelerate this process to allow fast selection and evaluation of citrus HLB-resistant/tolerant mutants. First, uniformity of propagations from individual scions/rootstocks is critical for their HLB resistance/tolerance performance. We will develop an enzyme-linked immunoassay (ELISA)-based method to assess performance uniformity. Second, we demonstrated that stick-grafting is much more successful than bud-grafting for inoculation, leading to the recovery of a higher percentage of HLB-resistant/tolerant mutants. Therefore, we will develop an assay to evaluate the resistance of variants using a simultaneous multi-strain inoculation strategy to ensure broad-spectrum HLB resistance/tolerance. Finally, in addition to current selections of grapefruit, sweet orange, and pummelo, we will expand our selection to mandarins, lemons, and other important citrus plants. Co-PI Duan and Gmitter each will lead two postdocs (1.5 FTE/year) and one technician (0.5 FTE/year) to develop the fast selection and evaluation methods. Co-PI Mattia and Grosser each will lead one technician (0.25 FTE/year) to select mutant citrus.Objective 2: Identify genetic variants and genes related to HLB resistance/tolerance.Understanding the genetic mechanism of selected HLB-resistant/tolerant mutants is critical for HLB-resistant/tolerant citrus breeding. We will re-sequence five resistant/tolerant and five susceptible siblings of representative plants and their parents. Bioinformatics and comparative genomics analysis will be used to identify HLB resistance/tolerance or susceptibility-related segment deletions, copy number variations (CNVs), structural variations (SVs), SNPs, and indels in each citrus line. There are many available sequences of citrus genomes, including several HLB-resistant/tolerant citrus plants and their counterparts that our group and others have sequenced. It is critical that we integrate all genome-based information to understand the HLB-resistant/tolerant or susceptibility-related genes. We will build a citrus pangenome by integrating our previous with newly sequenced genomes and applying the pangenome to identify genes with moderate/high impact variants. The identified candidate genes will be experimentally validated in a wide range of citrus species and relatives that display either resistant/tolerant or susceptible phenotypes. Co-PIs Duan, Gmitter, Mattia, and Grosser will provide the RNA samples of the citrus trees. PI Luo (0.06 FTE/year) and one postdoc (1 FTE/year) will perform bioinformatics and functional genomic analysis.Objective 3: Validate gene-edited citrus mutants for HLB resistance. Previously we edited two genes in two cultivars ('Duncan' grapefruit and Carrizo citrange) and produced more than a dozen novel dmr6 and sweet1 mutants. Recently, 173 novel mutant candidate plants (140 from 'Duncan' grapefruit and 33 from Kuharske citrange) were generated to target four other S genes singly or in pairs. These new mutants will be grown, propagated, inoculated, and evaluated for HLB resistance, potential changes in horticultural characteristics, potential side effects, and potential off-target mutations. Completing these evaluations will enable us to select the best target genes to edit in commercial citrus cultivars using mature citrus tissues to produce non-juvenile, non-transgenic HLB-resistant mutants as the next-generation citrus cultivars that can grow in HLB-endemic regions. Co-PI Gmitter, Deng, and Chater will lead one graduate student (0.5 FTE/year) to work on evaluating the gene-edited trees.Objective 4: Multistate field evaluations of HLB-resistant/tolerant citrus scion and rootstock cultivars. Based on preliminary selections of scion and rootstock cultivars with superior HLB resistance/tolerance attributes and fruit quality, a systematic evaluation of these novel varieties in multi-state field trials will be beneficial to the citrus industries. We propose to conduct field evaluations of 6 genotypes in Florida and Texas to monitor HLB tolerance and horticultural performance. In California, we will evaluate 3 of these selections that are available through the Citrus Clonal Protection Program (CCPP) in Riverside. Gene-edited selections were generated by the team and may be very valuable to the industry based on the preliminary data obtained from the field trees in Florida. The success of developed cultivars will ultimately be determined by their economic performance. We will conduct an economic assessment using scenario analysis and simulation. Co-PI Chater, Ramadugu, and Alabi will each lead one technician (0.15-0.25 FTE) on field evaluation. Co-PI Guan (0.1 FTE) will lead one student (0.2 FTE) to perform the economic analysis and report information on economic aspects in the fifth year.Objective 5: Outreach and dissemination of project results to stakeholders and the public. Outreach and dissemination of project results will keep stakeholders and the public informed and engaged with our research, which is critical to the success of the project. We will assemble a stakeholder advisory committee. Extension demonstration and informational activities will be held at the UF-CREC, the Citrus Expo, the Florida Citrus Show, and relevant grower organization meetings in CA or TX. The availability of these resistant/tolerant cultivars will be communicated during these extension events. We will establish replicated stage 2 field trials with these rootstocks across the US citrus production regions, during and after the project. A stakeholder advisory committee will be established with membership from FL, TX, and CA. A website will provide updates from this project to the public. Progress updates will be published in industry and peer-reviewed journals. Co-PI Chater, Ritenour, Ramadugu, and Alabi will lead the outreach in FL, CA, and TX.
Project Methods
Methods1, Methods for Objective 11.1 Developing an assay to evaluate the resistance of citrus variants using a simultaneous multi-strain inoculation: We will graft the selected bud-sticks onto severely HLB-affected plants of different genetic backgrounds, and measure how quickly Las bacteria invade, multiply, and cause HLB in the new growth from these bud-sticks. We will propagate the selected citrus bud-sports on different rootstocks and stick-graft inoculate them with multiple Las strains simultaneously. Inoculated plants will be scored by symptom development periodically and Las titer detection and quantification will be conducted at 3, 6, 9 and 12 months after inoculation using 16S rDNA and rRNA based RT-qPCR to measure Las bacterial titers and their relative cellular activities.1.2 Developing enzyme-linked immunoassay (ELISA)-based method to screen the uniformity of progeny of selected scions/rootstocks: We will collect newly expanded leaves of similar maturity, and extract in PBS buffer (1:2 [w/v]) in a FastPrep machine (MP Biomedical, Irvine, CA). The supernatants are the sources of total soluble proteins. The ELISA assay will be conducted by using a femtoELISA HRP kit with 1:1000 dilution of primary antibody and 1:3000 dilution of the secondary antibody. Absorbance of each reaction will be read at 450 nm on a FLUO Star Optima microplate reader. All tests will be replicated three times.1.3. Bud sport selection and evaluation: We propose more extensive searches of bud sport and controlled inoculation. in addition to current selections of grapefruit, sweet orange, and pummelo, we will expand our selection to mandarins, lemons, and other important citrus plants.2. Methods for Objective 22.1 Resequencing experiments and analysis:We will sequence the genomes by a service provider. We will assemble the genomes using PECAT or Hifiasm, selecting one of the parent genomes as the reference. For large SV detection, we will align re-sequenced and reference genomes using Mummer. Genetic variant annotation and functional effect prediction for the small variants will be predicted by SnpEff, and genes overlapping the SVs analyzed with BEDTools.2.2 Constructing Citrus Pangenome for identifying HLB-resistance related genetic variants: We will use Minigraph-Cactus to create the haplotype-aware citrus pangenome and develop statistical methods to predict the impact of each variant, and identify genes with moderate or high impacts.2.3 Experimental validation of selected genes and genetic variations: Candidate SNPs, indels, SVs, CNVs and segment deletions will be validated using PCR followed by sequencing.3. Methods for Objective 33.1 Determine types and frequencies of mutations: The targeted regions will be amplified from each of the 173 plants in high-fidelity PCR and subjected to deep amplicon sequencing. Amplicon sequence reads will be analyzed, to determine the types and frequencies of mutations.3.2 Propagate mutants and produce clonal plants and evaluate their HLB resistance: Plants with high frequencies of mutations will be propagated by grafting and cuttings to produce at least 20 clonal plants required for each selected mutant, and eight clonal plants will be inoculated with Las. Inoculated plants will be evaluated in eight randomized complete blocks in the approved/secured greenhouses and fields. Levels of HLB resistance will be determined and assessed every 3 months.3.3 Evaluate horticultural characteristics and potential side effects: Clonal plants will be planted in a secure field in Florida, in eight randomized complete blocks, and exposed to natural inoculation of Las by ACPs. Data on tree, shoot, leaf, flower, and fruit characteristics will be taken 2-3 times a year over 3 years.3.4 Identify potential off-target mutations: Mutants with strong HLB resistance will be subjected to genome re-sequencing. The genome of each selected mutant will be sequenced to 100x coverage or more and compared to the genome of wildtype citrus to identify off-target mutations.4 Methods for Objective 44.1 Field Trials: First, four selected scion selections that produce fruits of grapefruit, mandarin, and sweet orange and two rootstock varieties will be included in the field trial in all three states using randomized complete block design and 8 replicates per genotype/rootstock combination. These six genotypes were previously sent to CCPP in CA. Trials will be managed using standard irrigation, insecticide applications, fertilization and weed management practices. In CA, the scion selections will be grafted on Carrizo and field trial will be in the UC Riverside fields; in TX, we will propagate the scion selections on sour orange and plant in the TAMU Citrus Center experimental fields; in FL, the scions will be grafted on US942 and planted in Lake Alfred. The rootstock selections will be evaluated by grafting three scion cultivars (Washington navel orange, Tango mandarin and Lisbon lemon) on each of the two rootstock varieties. At least 8 replicates per rootstock/scion combination will be planted.4.2 Horticultural and disease evaluations: We will score tree height, canopy volume, appearance, density, trunk diameters using standard procedures - at planting, after six months and then annually. For disease evaluations, we will collect leaf samples every six months and conduct qPCRs to quantify Las titers. We will document disease symptoms. When the field trees are fruiting, we will collect data on fruit yield, size, external and internal coloration, percent juice, Brix, acid percent, and Brix/acid ratio.4.3 Economic analysis: We will conduct economic assessment using scenario analysis and simulation based on yield scenarios of the cultivar and price data from USDA and other sources.?5 Methods for Objective 55.1 Organization and engagement of advisory committee: An advisory committee will be organized to provide oversight on the relevancy of progress made by the research team. This panel will include key stakeholders, and research and extension scientists, who will meet at least once a year. The team will report progress made during the year and challenges encountered, and the panel will evaluate progress against the proposed milestones of each objective.5.2 Extension program and stakeholder panel: We will have grower visits to established trials and greenhouse experiments. We will conduct surveys to collect baseline data for project metric evaluation mechanisms. We will include pre- and post-workshop questionnaires to determine the program's effectiveness in disseminating knowledge and determine stakeholder engagement and opinion of project activities and progress. After each activity, the group will meet with project personnel to discuss survey and questionnaire results and to review the activities' success. The stakeholder group will meet at least once per year, to allow for meetings that focus on project progress on research activities. Stakeholder groups will include the historical citrus grower meetings in FL and CA.5.3 Dissemination of project results: Results will be communicated to stakeholders, researchers, and the public through multiple channels, including presentations at trade/professional/stakeholder advisory meetings, scholarly journals and conferences, project websites and social media accounts, and on-site visits (field days and workshops).

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

Outputs
Target Audience:Target audiences: citrus industry stakeholders across the U.S. and members of the public; citrus growers and nurserymen in each of the citrus-producing states; Citrus Research and Development Foundation, Florida Citrus Mutual, California Citrus Mutual, California Citrus Research Board, California Citrus Nursery Society, Texas Citrus Mutual, Texas Citrus Pest and Disease Management Corporation, and the state department of agriculture in CA, FL and TX.? Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This is an interdisciplinary project that involves expertise in bioinformatics, citrus breeding, genetics, plant pathology, and extension service. The project has provided researchers in multiple disciplines an unprecedented opportunity to work together to conduct this challenging research. The PIs maintain regular communication/meetings to discuss the progress of their individual projects. The postdoc research associates, graduate students, technicians and students in the different groups can gain knowledge and expertise from members in other groups. The project supports postdocs, graduate students and undergraduates to attend citrus HLB meetings and other professional meetings. How have the results been disseminated to communities of interest?We have disseminated our results to communities of interest through social media, grower meetings, invited talks and conference presentations. The details of dissemination activities are listed in the accomplishment of Objective 5. What do you plan to do during the next reporting period to accomplish the goals?Based on our accomplishments in the first year, our goals for the next reporting period will primarily focus on the following aspects: Continue developing an enzyme-linked immunoassay (ELISA)-based method to assess performance uniformity. Continue developing an assay to evaluate the resistance of variants using a simultaneous multi-strain inoculation strategy to ensure broad-spectrum HLB resistance/tolerance. Continue our selection to mandarins, lemons, and other important citrusplants. Continue working on annotating SVs in mutant genome and plan to experimental verify them in the future. Continue evaluation and validation of gene-edited mutants for HLB resistance. Continue field trials in Florida, Texas and California Have a Stakeholder Advisory Committee meeting to obtain feedback on our research progress. Continue dissemination of results to communities of interest through social media, grower meeting, conference presentation and the project website.

Impacts
What was accomplished under these goals? The project has been fruitful first year. Below are summaries of accomplishments for each objective. Objective 1: New evaluation and selection using heavily HLB-affected trees as recipients in the graft-based assay demonstrated the effectiveness of the assay, which yielded a highly HLB resistant Valencia line and one grapefruit line, and one highly tolerant pumelo lines. The evaluation procedure shortened the time required for graft-based assay more than a year. After multiple inoculations in the muti-year-evaluation in the USHRL greenhouse, the 'Ca. Liberibacter asiaticus' (Las)-resistant seedlings of pumelo revealed Las alone may not be able to cause full spectrum of HLB disease. This is another aspect of supporting evidence as our previous finding from in vitro Las culture and inoculation https://doi.org/10.1093/hr/uhae011. Fruits from new lines of grapefruit and pumelo with improved HLB resistance/tolerance are being analyzed for fruit qualities. It is worth noting that one grapefruit mutant had the leaves and canopy as mandarin. Progress with Development of Variant Germplasm: OLL sweet oranges - 25 clones selected for HLB tolerance and high juice quality (replicates planted in the field); 2 other OLL clones previously selected for enhanced HLB tolerance were stepped up for large-scale field trials and made available for CRAFT. Vernia sweet oranges - 10 clones selected for early maturity (December); two cybrid clones with kumquat cytoplasm selected for enhanced HLB tolerance and higher brix in juice. Red grapefruit - 2 cybrid clones of Ruby somaclone N11-11 (with mt and cp genomes from 'Meiwa' kumquat) selected for both HLB and citrus canker tolerance. Trees were propagated for larger field trial, and topworked to expedite fruiting. X639 citrus rootstock - discovered variant STR-4-1 with enhanced HLB tolerance, entered into TC micropropagation, and large field trial planted with OLL-20 sweet orange scion. This variant clone was sequenced, multiple mutations found. Objective 2: Five grapefruit (Citrus paradisi) tissue samples were sequenced using the PacBio HiFi Sequencing technique. One of these samples, named White-Marsh, is a mutant with white fruit color, derived from Ruby Red through a volunteering seedling (VS) and exhibiting improved resistance to Citrus Huanglongbing (HLB). The other four samples serve as controls: RG3 (Ruby Red from a nursery), PT 7-4 (Ruby Red from a VS with enhanced HLB tolerance), YT 6-4 (Ruby Red, a bud sport from a mature tree with improved HLB tolerance and a distinct canopy phenotype), and YT 6-9 (Ruby Red, a bud sport from a mature tree with improved HLB tolerance). We assembled five genomes using Hifiasm v0.19.9 with default parameters and generated phased assemblies. We annotated RG3 genome, and predicted 32,601 and 31,672 genes in the haplotype 1 and haplotype 2 scaffold assemblies, respectively. We first detected the structural variations (SVs) by performing whole-genome alignments (WGA) between the genome assembly of mutants and the RG3 sample (reference) using the 'nucmer' and 'dnadiff' programs from MUMmer v4.0.0. To further reduce false positives, we employed a mapping-based SV detection approach by aligning the PacBio long reads from each sample to the RG3 scaffold genome using Minimap v2.28. After intersecting SVs from both detection strategies, we detected 30 unique SVs in White-Marsh genome, consisting of 18 deletions and 12 insertions; 24 unique SVs in PT 7-4 genome, encompassing 20 deletions and 4 insertions; only 2 unique SVs in YT 6-4 genome, comprising 1 deletion and 1 insertion; only 3 unique deletions in YT 6-9 genome. Objective 3: We isolated genomic DNA from nearly 280 'Duncan' and Kuharske citrange plants that were generated from a previous gene editing experiment, amplified the targeted genomic region(s) in these plants, and conducted amplicon sequencing. This led to the identification and confirmation of new mutants. Objective 4: We continue to send our selections to Department of Plant Industry (DPI), Florida for cleaning-up, and have received more than a dozen of cleaned-up lines of grapefruit, Valencia, navel orange and pumelo etc. All these lines with improved HLB resistance/tolerance are subjected to further propagations for field trials in USHRL Pico's Research Farm. A few lines showed promising performance even under extremely high HLB disease pressure for different periods of challenging time. A trial of Donaldson sweet orange, a new HLB-tolerant variant, was established at the Pico farm in Ft. Pierce, Florida, alongside commercial standards. This trial aimed to test the HLB tolerance of Donaldson and other varieties under field conditions. The research team in California is involved in field testing of four previously selected citrus cultivars for horticultural evaluation. Since HLB is not widespread in California, the field assessment will be restricted to horticultural performance aspect only. The four genotypes selected for the field trial are: 'Sugar Belle', 'Marathon', 'UF-950' and 'OLL-8'. Two standard genotypes will be included as controls; these are: 'Washington Navel Orange' and 'Tango mandarin'. The genotypes are maintained at the Citrus Clonal Protection Agency located in Riverside, CA. In preparation for the field trial, we treated an area at the University of California Riverside Agricultural Research Station and designated it for the experimental field trial. The field was treated to minimize nematode-associated problems and the field is now ready for planting. Two rootstocks will be used for the field trial - 'Carrizo' and Citrus macrophylla ('Alemow'). Sixteen replicates for each of the six genotypes will be propagated on each of the rootstock varieties and a total of 192 trees will be planted in the field in a randomized complete block design (RBCD) and periodically assessed for growth parameters, rootstock compatibility, yield, canopy, and overall horticultural performance. Objective 5: Dr. Chandrika Ramadugu gave a presentation at the Citrus Day event, March 2024, UCR. Organized by University of California Riverside and CAPCA. "Generating novel hybrids with citrus huanglongbing resistance" Presented a poster and displayed fruits generated in the breeding program to an audience of 105 people including growers, citrus industry personnel, scientists, students and research collaborators. Offered hybrid fruits for tasting. Ramadugu gave the keynote address of the 78th Annual Conference of Subtropical Agriculture and Environments Society, South Padre Island, Texas. Organized by the Subtropical Agriculture and Environments Society. Feb 9, 2024. "Developing science-based solutions for a citrus disease". Meeting attended by about 150 people. During the reporting year, Dr. Alabi gave an invited talk titled "Huanglongbing (citrus greening disease): current situation and epidemiological insights" during the virtually held Syngenta-UK Biological Science Seminar Series on January 24, 2024. He mentioned the project's goals and progress during his presentation. He also provided updates on the efforts of the team and progress made at the bimonthly business meetings of the Texas Citrus Pest and Disease Management Corporation and mentioned the project during talks at County Master Gardener programs. Dr. Alabi participated in a project meeting held on the sidelines of the International Research Conference on Huanglongbing VII, UC-Riverside, Riverside, CA, 03/26-30/2024. He attended the Plant Health 2024 Annual American Phytopathological Society Meeting. We have our first advisory group meeting on September 6th. Each team member gave a presentation about research progress. The advisory group was satisfied with the progress of the team and gave us valuable suggestions for the future research.

Publications

  • Type: Journal Articles Status: Published Year Published: 2024 Citation: Fan Nie, Peng Ni, Neng Huang, Jun Zhang, Zhenyu Wang, Chuanle Xiao, Feng Luo#, Jianxin Wang. De novo diploid genome assembly using long noisy reads, Nature Communications 15, 2964, 2024.
  • Type: Journal Articles Status: Published Year Published: 2024 Citation: Desen Zheng, Cheryl Armstrong, Wei Yao, Bo Wu*, Weiqi Luo, Charles Powell, Wayne Hunter, Feng Luo, Dean Gabriel, Yongping Duan. Towards the completion of Kochs postulates for the citrus huanglongbing bacterium, Candidatus Liberibacter asiaticus, Horticulture Research, uhae011, 2024.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Parajuli, S., Tapia, R.R., Fetouh, M., Gmitter Jr., F.G., Deng, Z. 2024. CRISPR/Cas9-mediated editing of DMR6 and SWEET1 genes for resistance to citrus canker and Huanglongbing. Oral presentation to IRCHLB (International Research Conference on Huanglongbing) VII, Riverside, CA, USA, March 26-29, 2024.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Ashfaq, H., Deng, Z. 2024. Increasing citrus resistance against citrus canker and Huanglongbing through genome editing. Poster presentation to IRCHLB (International Research Conference on Huanglongbing) VII, Riverside, CA, USA, March 26-29, 2024.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Deng, Z., Gmitter Jr., F.G., Chater, J. 2024. Objective 3: Validate gene-edited citrus mutants for HLB resistance and generate novel non-transgenic HLB-resistant selections. Oral presentation to the project advisory group meeting, Fort Pierce, FL, September 5, 2024.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Jianyang Liu, Khushwant Singh, Matthew Huff, Jong-Wong Park, Tara Rickman, Manjunath Keremane, Robert Krueger, Ping Zheng, Jodi Humann, Madhurababu Kunta, Mikeal Roose, Dorrie Main, Margaret Staton, Chandrika Ramadugu, Chris Dardick. "Analysis of R-genes in Australian Limes in Comparison to Commercial Citrus Cultivars ". Presented at the International Research Conference on Citrus Huanglongbing organized by Citrus research board, March 2024.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Matthew Huff, Khushwant Singh, Beant Kapoor, Ryan Kuster, Madhura Babu Kunta, Jong Won Park, Jianyang Liu, Chris Dardick, Manjunath Keremane, Robert Krueger, Chandrika Ramadugu, Margaret Staton. Citrus Pan-Genome: The Search for Genes of Resistance in Australian Limes. Presented at the International Research Conference on Citrus Huanglongbing. Organized by Citrus Research Board, Riverside, CA. 03/2024.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Chandrika Ramadugu and Mikeal L. Roose. Genome-assisted breeding to incorporate huanglongbing resistance in citrus. Presented at the International Research Conference on Citrus Huanglongbing. Organized by Citrus Research Board, Riverside, CA.
  • Type: Journal Articles Status: Published Year Published: 2024 Citation: Khushwant Singh, Matthew Huff, Jianyang Liu, Jong-Won Park, Tara Rickman, Manjunath Keremane, Robert R. Krueger, Madhurababu Kunta, Mikeal L. Roose, Chris Dardick, Margaret Staton and Chandrika Ramadugu. 2024. Chromosome-scale, de novo, phased genome assemblies of three Australian Limes: Citrus australasica, C. inodora, and C. glauca. Plants 2024, 13, 1460. https://doi.org/10.3390/plants13111460
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Chandrika Ramadugu and Mikeal Roose, 2024. Breeding HLB-resistant citrus and field evaluation of novel hybrids. Citrograph Summer 2024. Volume 15, Number 3. pages 52-56.