Source: The Regents of University of California submitted to NRP
NOVEL, NON-TRANSGENIC, HYBRID CITRUS VARIETIES WITH RESISTANCE TO HUANGLONGBING: EVALUATION AND CULTIVAR DEVELOPMENT.
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
OTHER GRANTS
Reporting Frequency
Annual
Accession No.
1024588
Grant No.
2020-70029-33201
Cumulative Award Amt.
$4,670,000.00
Proposal No.
2020-08470
Multistate No.
(N/A)
Project Start Date
Sep 1, 2020
Project End Date
Aug 31, 2025
Grant Year
2020
Program Code
[ECDRE]- Emergency Citrus Disease Research and Extension Program
Recipient Organization
The Regents of University of California
200 University Office Building
Riverside,CA 92521
Performing Department
College of Nat & Agr Sciences
Non Technical Summary
Citrus huanglongbing (HLB) has negatively impacted all the major citrus growing areas of the world. The financial losses incurred due to HLB have been estimated to be several billions of dollars in the US alone. Citrus is an important food crop and consumed widely in the USA and other parts of the world. The fruits are considered to be nutrient-dense and are rich in beneficial phytochemicals like flavonoids, carotenoids, coumarins, terpenes, and limonoids. The medicinal properties associated with citrus fruits protect against several chronic diseases. Citrus is also an essential component of the diet for most people. Juice derived from various types of citrus is consumed widely. Cultivating citrus provides revenue that supports multi-billion dollar industries in the USA.The devastation caused by HLB has now threatened the citrus industry in Florida. Texas and California, the other two prominent citrus-producing states, are likely to be in a similar situation if the disease is not controlled. Despite research conducted on HLB for the past fifteen years, tangible, practical solutions are not available to the growers. HLB is aptly described as the greatest threat to citrus cultivation in the USA. The disease complex is complicated because of the uncultivable nature of the pathogen (leading to lack of understanding of the disease organism), late symptom expression (resulting in the spread of the pathogen before an infected plant can be identified), and, unequal distribution of the pathogen in the host plants (resulting in sampling issues and false negatives in HLB surveys). In addition, the widespread distribution of the rapidly proliferating psyllid vector and difficulties associated with controlling of psyllid populations contribute to the challenges related to developing practical solutions. Short-term disease management can be achieved by controlling psyllids, preventing spread by adhering to phytosanitary practices, and implementing strict regulations. Environmentally friendly and financially sustainable solutions will be needed to continue citrus cultivation in the presence of the HLB associated pathogen and the psyllid vector. HLB has now established in the three major citrus growing states of the USA. HLB disease management practices that are in vogue are useful for a limited time only; long-term control of HLB will require genetic solutions. The most promising solution is to develop disease-resistant cultivars that prevent the establishment of the associated pathogen. Since there is no appreciable HLB resistance in the genus Citrus, utilizing genetic resources of wild relatives is a viable option to introgress resistance traits into cultivated citrus. We have previously characterized HLB sensitivity across a broad spectrum of phylogenetically diverse citrus and relative genera under field conditions. We have reported that Australian limes in the groups Microcitrus and Eremocitrus, that diverged from the mandarin lineage approximately 6 million years ago, have HLB resistance trait that is superior to citrus. Building on this insight, we created citrus x Microcitrus hybrids that possess both good fruit quality and strong HLB resistance; these materials are the building blocks of new HLB solutions that we exploit here todevelop commercially acceptable, non-transgenic, HLB-resistant cultivars.Our project builds on previous work where we identified natural sources of HLB resistance, bred for new HLB resistant varieties, and developed the genomic resources required for marker-assisted selection of HLB resistance. Here, we propose to evaluate the commercial potential of these by conducting multi-site evaluations of a limited number of hybrids in Florida, Texas, and California. Our project aims to develop an integrated pipeline that will accelerate the farm-level implementation of our research and hasten the time-lines for transitioning these materials from the lab to the grower. Our pipeline will enable the rapid identification of valuable hybrids by combining multiple strategies including the use of precocious fruiting to accelerate fruit evaluation, metabolomics and genomics to quickly eliminate undesirable hybrids, and multi- location field trials to assess HLB resistance, horticultural performance, and consumer acceptance in real-world contexts. Our approach is to incorporate resistance-associated genomic segments from Australian limes to cultivated citrus types. The process of selecting promising hybrids from breeding populations is generally time consuming and involves greenhouse evaluations, preliminary horticultural data, field performance evaluations of single trees and finally, by multi-location field trials. We are proposing a fast-track approach to deliver new cultivars where multiple evaluations will be carried out in parallel to hasten the process of variety development. Molecular data (genomic and metabolomic) developed in a complementary project and phytopathological data proposed in the current project will be used to select advanced hybrids that may have commercial potential.Through this project, we will be able to evaluate the field performance of four F1 hybrids in six locations and identify advanced hybrids with resistance traits incorporated. The research conducted will be beneficial to accelerate citrus breeding and develop tools for selection of useful hybrids with putative commercial value.
Animal Health Component
35%
Research Effort Categories
Basic
35%
Applied
35%
Developmental
30%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2010930104030%
2020999108120%
2122499108030%
2064010106020%
Knowledge Area
212 - Pathogens and Nematodes Affecting Plants; 206 - Basic Plant Biology; 202 - Plant Genetic Resources; 201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
0999 - Citrus, general/other; 4010 - Bacteria; 0930 - Lemon; 2499 - Plant research, general;

Field Of Science
1040 - Molecular biology; 1060 - Biology (whole systems); 1081 - Breeding; 1080 - Genetics;
Goals / Objectives
The over-arching goal of this project is to identify potentially valuable HLB resistant/tolerant novel hybrids generated in our breeding program for future cultivar development. We determined earlier that the Australian citrus relative genera have good resistance to HLB. Through traditional breeding, we have already generated novel hybrids of citrus and Microcitrus. For development of disease resistant cultivars that produce acceptable fruits deemed useful to the industry, several evaluation criteria are required. We propose to develop methods and processes for efficient selection and evaluation of future hybrids from breeding programs. Field trials in different agro climatic citrus growing regions, are necessary for assessing horticultural and resistance traits in real life situations. One of the major problems associated with citrus breeding is the long juvenility. Development of methods to induce early flowering and fruiting will help expedite the process of preselection of hybrids. Third, evaluating disease resistance using different isolates of the pathogen is essential to ensure durable resistance in diverse environments. Transportation of citrus budwood across state borders is highly regulated since citrus has several graft-transmissible pathogens that can get introduced to new areas. To facilitate movement of research material, we will develop a pathogen detection array that can be used to test for 20 pathogens. Validation of the array by multiple budwood certification agencies is essential. Thorough evaluation of newly generated hybrids for HLB resistance is required. We propose to conduct HLB challenges through grafts and psyllids in greenhouses to ensure reliable resistance. Acquiring permits for the budwood transportation, handling of intellectual property, multiplication of budwood and maintenance of mother trees in different locations are needed. Finally, educating the public, gathering information on public perception of newly created hybrids, feedback from stakeholders and through fruit taste panels will benefit the research team since the project can be configured to fit industry needs. Economic analysis of the novel hybrids and comparison of their performance with other standard cultivars will assess the financial viability of the new hybrids. The eight objectives are:1: Establish multi-state field trials for four F1 hybrids (mandarin X Australian lime) already selected for HLB resistance and fruit quality. (Albrecht, Kunta, Mauk, Roose, Ramadugu). Four lemon-like HLB resistant F1 hybrids were pre-selected based on HLB disease response in contained research facilities in CA, limited horticultural evaluations in field and through fruit taste panels in CA. We propose to conduct field trials in FL, TX and CA with a total of 600 trees for three years.2: Induce precocious fruiting in advanced hybrids. (Cutler and Ramadugu). Early flowering expedites breeding; availability of fruits will be helpful for pre-selection of hybrids from the population. We propose to accelerate the selection process by early phenotyping of fruits induced using a newly developed inducible FT technology. Fruit metabolome data (generated in a parallel project) will be used to identify lines with undesirable profiles. Three approaches for precocious flowering. 2.1: transgenic induction of FT via Agrobacterium-mediated transformation; 2.2: citrus leaf blotch virus-mediated FT expression; 2.3: induction of flowers and fruits in the field by cultural practices.3: Evaluate advanced hybrids for HLB resistance in controlled greenhouse conditions in CA, FL and TX. (Duan, Kunta, Ramadugu, Main). Tasks identified are: 3.1: Develop Illumina and PACBIO sequence for Microcitrus australis, a resistant citrus relative used to generate several hybrids. This data will complement sequence information being developed in a parallel project for three other Australian limes. Main will analyze sequence data, curate and make the sequence available to browse, visualize, search and download through various query and visualization tools in a public genome database. 3.2: Advanced hybrids will be selected based on SNP genotyping, haplotype data and fruit metabolomics information. Representative resistant hybrids with different parentages will be selected. 3.3: Multi-locational testing for HLB resistance: Budwood from 50 advanced hybrids will be shipped to FL and TX under permits for greenhouse challenge inoculations. Local CLas isolates will be utilized in FL, TX and CA. Selected advanced hybrids will be used for transcriptome analysis to predict a list of HLB resistance related genes. 4: Development of rapid methods for detection of multiple pathogens to facilitate rapid release of quarantined plants by budwood certification programs in CA, FL and TX. (Kunta, Duan, and Ramadugu). Rapid testing for multiple citrus pathogens would facilitate safe interstate movement of budwood. We will develop and evaluate a reverse transcription-qPCR based assay for simultaneous detection citrus pathogens. Using this platform, it will be possible to evaluate two accessions for 20 pathogens in under 2 hours (starting from RNA).5: Transfer of budwood of selected hybrids to collaborating nurseries in all three states for propagation. (Albrecht, Kunta, Roose, Ramadugu). The bud sticks of selected hybrids will be provided to budwood certification programs in all three states. Coordination of further tasks will be carried out by co-PDs.6: Obtain permits for interstate movement of hybrids and for the release of five selected hybrids by working with regulatory agencies. (Albrecht, Kunta, Ramadugu, Roose). Permits will be obtained for interstate movement of budwood of advanced hybrids (through CCPP).7: Propagate and release selected varieties for further field trials and plan for future commercialization. (Albrecht, Kunta, Ramadugu and Roose). Participating nurseries in FL, TX and CA will help in propagation. Patent development, licensing and commercialization is supported with the guidance of the Univ. of California Riverside (UCR) through patenting and licensing of intellectual property, inter-institutional material transfer, and confidential disclosure agreements. The information, products, methodologies, technologies, and results, patented and unpatented, will be made available. UCR will work with co-PIs from other institutions through an IP management plan and/or inter-institutional agreements to allow access to all pertinent IP and expedite future commercialization process.8. Extension to familiarize the industry with novel hybrids, conduct taste panels and obtain feedback. Surveys to measure social and economic impact. Albrecht, Kunta, Mauk, Ramadugu, Main (extension) Guan (social and economic analysis). The team will disseminate information to growers, industry personnel and scientists by making presentations in scientific and grower meetings, conduct taste panels to assess organoleptic properties of fruits, and generate a project-specific website. Main will make information on genomic sequences of four HLB resistant Australian limes (three from a previous project and one from the current project) publicly available in a separate browser.The economic viability of varieties to be developed in this project will be critical for grower adoption of the varieties. We will assess the economic and social benefits accounting for increased yield, reduced chemical use, and reduced cost of production, to inform the decision making of growers, regulators and other stakeholders. The economic performance of the traditional varieties will be compared to the resistant varieties to calculate the grower-level benefit. Data on production costs and revenues of different varieties will be collected and the most profitable variety will be identified. The environmental benefits of reductions in pesticide use will be further estimated in the project.
Project Methods
Obj.1. Multi-state field trial: Four selected F1 hybrids that were pre-selected based on HLB resistance phenotype observed in greenhouse evaluation experiments in CA and limited horticultural (including taste panel) evaluations. Field trials will have a total of 600 trees (200 per state). Trees will be propagated on US-942 & US-812 rootstocks in FL, on sour orange & Bitters C22 in TX, and Carrizo citrange & Alemow in CA. Standard varieties serve as control plants. In FL, the trees will be planted in two commercial groves [Arcadia, Desoto county (FL1) & Fort Meade, Polk county (FL2)]. In TX, trees will be in TAMU-KCC (TX1) and a private farm in the Lower Rio Grande Valley (TX2). In CA, trees will be in the Coachella Valley Agricultural Research station in Thermal (CA1) & UCR Citrus Research Center in Riverside (CA2), both in the Riverside county. Planting will be in a randomized split-plot design with rootstock cultivar as the main plot factor and scion cultivar as the sub plot factor with 10 single tree replications per rootstock, per site. Field trees will be managed using standard practices followed by the cooperating grower/research facility. Tall stakes will support the trees, promote vertical growth, and encourage early flowering.Horticultural evaluations will document tree height, canopy volume, density and color, trunk diameters at six months initially and annually later on. HLB symptoms will be assessed biannually as per standard guidelines (NCBCM, 2018). CLas testing will be done every fall by qPCR and for multiple pathogens in year 3 using NGS. In year three, once the trees are productive, fruit yield, size, color, percent juice, BRIX, percent acid, and BRIX/acid ratios will be measured. Fruit quality analysis will be done locally and juice metabolomic analysis will be at the UCR metabolomics core facility in Riverside. Tasting panels will assess the juice quality.Obj.2. Precocious flowering of advanced hybrids. We will use constructs of FTgene cloned under an inducible promoter responsive to the agrochemical Mandipropamid. i). Juvenile internode segments of selected advanced hybrids will be transformed with the iFT construct using Agrobacterium-mediated transformation. An RFP marker will quickly identify transformed shoots. These will be propagated on rootstocks, and floral induction will be done using Mandipropamid. ii). Citrus leaf blotch virus containing the FT gene will be used to graft inoculate hybrids to induce flowering as per published protocols. Permits will be obtained as needed. iii). Advanced hybrids will be grown in the field in Riverside - trained as single stems up to 5-6 feet to promote flowering. With these three approaches, we expect to obtain precocious flowering and fruiting. Evaluation will be by measuring fruit parameters, taste panels where appropriate, and metabolome analysis.Obj.3. Multiple tasks: i) Develop genomic sequence information for Microcitrus australis, a highly HLB resistant accession used as a parent in breeding. Illumina and PACBIO sequences will be generated, assembled, curated, and validated. The data will complement genomic sequences that are being generated for three other resistant parental lines in a parallel project (Eremocitrus and two other Microcitrus species). Co-PI Main will make the sequence data available to browse, visualize, search, and download through the various query and visualization tools on a public database.5X sequences of selected advanced hybrids will be generated, and data analyzed to identify resistance-related genes in advanced hybrids.ii). Advanced hybrids will have about 16-38% of Microcitrus genome and 62-84% citrus genome (from pummelos, grapefruit, mandarin, tangor, etc, depending on parents chosen for individual crosses) and are expected to have citrus-like fruits. Hybrids chosen for evaluations will be pre-selected to represent diverse parentages; selections will also be based on SNP genotyping and haplotyping data. Emphasis will be given to hybrids with increased genomic content from the citrus parent.iii). For screening of backcross hybrids, budwood from 50 advanced hybrids will be evaluated for HLB resistance in controlled greenhouses in FL, TX and CA (in BSL3 facility) using either graft or psyllid challenges. CLas isolates from FL, TX, and CA will be used for greenhouse inoculations. From a limited number of selected advanced hybrids, samples will be collected at 0, 15, 30, and 45 days after HLB challenge for RNAseq analysis. Bioinformatics and functional genomic analysis will predict HLB resistance-associated genes in advanced hybrids.Advanced hybrids with resistance and suitable fruit quality will be identified for future field trials (separate from the current project).Obj.4. Development of qPCR array for accurate and rapid detection of multiple pathogens to facilitate safe release and movement of quarantined plants by budwood certification programs. A reverse-transcription-qPCR based assay will be developed for simultaneous detection of about 20 citrus pathogens in about 2 hours' time (starting from an RNA prep). The array will be validated by other budwood certification programs.Obj. 5. Transfer of budwood of selected hybrids from APHIS-certified greenhouses in CA to collaborating nurseries in FL, TX, and CA for propagation. Under valid permits and material transfer agreements (as needed), budwood will be sent to FL and TX certification programs. After pathogen testing, it will be provided to collaborating nurseries for field trials. The nurseries will propagate 40 plants of each of the four hybrids on two different rootstocks selected locally and provide trees to participating grower collaborators. A total of 200 plants will be produced in each state for field trials (40 plants each from 4 hybrids and a control variety).Obj. 6. Obtain permits for interstate movement of hybrids for HLB resistance evaluations in greenhouses. Budwood will be shipped through Citrus Clonal Protection Program in CA to the respective budwood certification agencies (in FL and TX) and then to individual PIs (Duan and Kunta) for evaluation of advanced hybrids in greenhouse conditions.Obj. 7. Propagate and release selected varieties for further field trials and plan for future commercialization. Participating nurseries in FL, TX, and CA will maintain mother trees as needed and propagate selected hybrids for greenhouse and field evaluations. Patent development, licensing & possible commercialization will be supported by the UCR office of technology partnerships through patenting and licensing of intellectual property, inter-institutional material transfer, and confidential disclosure agreements. UCR will work with collaborating organizations through an IP management plan and inter-institutional agreements to allow access to all pertinent IP and expedite future commercialization processes.Obj. 8. Extension, economic, and social analysis. The project team will disseminate information to growers, industry personnel, and scientists by making presentations in scientific and grower meetings, conduct taste panels to obtain public perception about organoleptic properties, generate project-specific website for making information available. Every year, fruit tasting events will be organized in CA (only in year 3 in FL and TX) to assess the fruit quality of the recently generated hybrids used in the current project. Co-PI Main will post curated genomic sequences on a public database available to the community. Economic and social impacts will be measured. Data on yield, chemical use, cost of production etc. will be used for analysis. The performance of the traditional varieties will be compared to the resistant varieties to calculate the grower level benefit using the partial budgeting approach and the net present value approach. Economic impact analysis of resistant varieties will also be conducted at the industry level to evaluate sustainability.

Progress 09/01/23 to 08/31/24

Outputs
Target Audience:The target audience for this citrus project consists of citrus researchers, citrus growers, industry personnel, breeders, students, geneticists, horticulturists, bud wood testing facilities, molecular biologists, and bioinformatics specialists. We have interacted with grower cooperators, regulators, other breeders, citrus enthusiasts, and pathologists. We have developed tools for testing budwood that needs to be transported to other states in the United States. It is imperative that the material transported is free of citrus pathogens (mainly graft-transmitted pathogens). In the United States, the movement of citrus is highly regulated. Research done in our project facilitates the testing process and is useful for several regulatory agencies and research laboratories. The field trials we are conducting in different locations are helpful to growers looking for alternative varieties if the current cultivars cannot be grown because of huanglongbing-associated disease issues. The genomic tools developed in the project are crucial for researchers developing disease-resistant citrus varieties. The novel hybrids evaluated in the current project may be valuable as substitute varieties if deemed necessary. However, we want to emphasize that our program constantly improves and leads to better varieties every year. Changes/Problems:Freeze in Texas durign January 2024 killed hybrid plants located in the field trial at the commercial orchard. Few plants representing each hybrid at South Research Farm of the Citrus Center survived although several hybrid plants were killed. Freeze (Winter Storm Uri) occurred in February 2021 devastated citrus industry in South Texas with an estimated crop loss of at least $230 million (Texas A&M AgriLife Extension Service). A large number of the trees were dead or severely damaged. Growers encountered a total loss of Valencia orange crop and losses of over 60% grapefruit crop. It also created a severe shortage of rootstock and availability of plants for replanting. This has created disruption in the priorities of the researchers and budwood program mainly to focus on meeting immediate industry needs. What opportunities for training and professional development has the project provided?In this project, we provided many project personnel with training and professional development opportunities. The project objectives consist of field trials, genome sequencing, transcriptome studies, genome comparisons, identification of QTLs, genotyping studies, pathogenicity experiments to determine disease response in novel hybrids in both greenhouse and field situations, development of methods to screen for pathogens in budwood material that needs to be transported across state borders, facilitating budwood transport and evaluation of novel hybrids in different agroclimates, etc. The undergraduate students who work part-time in the laboratories are trained in plant care, grafting, evaluating field plants for horticultural traits, setting up field trials, etc. We have trained at least ten individuals in these areas of plant research. The graduate students in cooperators labs were involved in genome assemblies, identification of structural variants in selected individuals of the breeding population, identification of haploblocks in hybrids (to compare the selected disease-resistant and susceptible plants), conducting genome-based annotations using specific software packages, etc. We have also interacted with service providers to learn the various methods involved in genomic studies. We believe that the project-specific training provided in the various participating laboratories has contributed significantly to professional development for individuals at multiple levels, including undergraduate students, graduate students, laboratory technicians, postdoctoral fellows, project scientists, and professional researchers. How have the results been disseminated to communities of interest?We have communicated our research thorugh various avenues including presentations in scientific and grower meetings, delivering invited lecutres to educate the industry about on-going research that is of interest, discussions with industry members, conducting meetings, etc. The following list of activities outlines the extension activities. Presentation at the Citrus Day event, March 2024, Riverside,CA. 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. 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. Ramadugu presented a talk on "Citrus huanglongbing - Can Indian Germplasm provide solutions?". International Webinar of "Citrus Horizons: Genomics Tales, Germplasm Utilization and Cultivar Chronicles". Indian Council of Agricultural Research - Citrus Research Institute, Nagpur, Maharashtra, India. December 2023. Chandrika Ramadugu, 2023. Presented an invited key note lecture on: "Citrus Huanglognbing: strategies to develop disease resistant varieties through breeding". Asian Citrus Congress 2023. Organized jointly by Indian Society of Citriculture, Nagpur, India; Indian Council of Agricultural research - Central Citrus Research Institute, Nagpur, India and Asia-Pacific Association of Agricultural Research Institutions, Bangkok, Thailand. At Nagpur, Maharashtra, India, October 2023. Presented at the Asian Citrus Congress 2023, October 28-30th, 2023 at Nagpur, India. Event was attended by citrus scientists and industry personnel from India and 15 Asian countries. Ten scientists from the USA attended the event. Invited keynote speaker. Chandrika chaired a technical session: on Current approaches in Citrus health management, insect-pest and disease surveillance and diagnostic approaches on 29.10.23. Chandrika Ramadugu "Developing HLB resistant varieties". Oral presentation at the Santa Barbara County Citrus Industry Workshop. June 15, 2023. Santa Barbara County Citrus Industry Workshop. Citrus Pest and Disease Prevention Program. June 2023. Ramadugu was invited to make an oral presentation to the citrus growers in Santa Barbara. Presentation was made to a group of about 25 citrus growers from Santa Barbara, Ventura and San Luis Obispo counties (California). Website: Huanglongbingresistantcitrus. https://huanglongbingresistantcitrus.ucr.edu/ 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. Larissa Nunes da Silva, Anas Fadli, Chandrika Ramadugu, Ute Albrecht. Cultivating Novel Microcitrus Hybrids as Scion for HLB Management in Disease-Endemic Growing Conditions. Presented at the International Research Conference on Citrus Huanglongbing organized by Citrus research board, March 2024. 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. Manjunath L. Keremane, Chandrika Ramadugu, Khushwant Singh, and Robert Krueger. New Pathogen Testing Tools for Safe Movement of Citrus Germplasm in the Huanglongbing (HLB) Era. Presented at the International Research Conference on Citrus Huanglongbing. Organized by Citrus Research Board, Riverside, CA. 03/2024. Jong-Won Park, Khushwant Singh, Tara Rickman, Erik Burchard, Manjunath Keremane, Robert Krueger, Chris Dardick, Margaret Staton, Mikeal Roose, Chandrika Ramadugu, and Madhurababu Kunta. Generating de novo transcriptome assemblies of Australian native lime species using various tissue types and its use as a reference for transcriptome data analysis. Presented at the International Research Conference on Citrus Huanglongbing. Organized by Citrus Research Board, Riverside, CA. 03/2024. 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. 03/2024. What do you plan to do during the next reporting period to accomplish the goals?We will complete the field trials in Florida, California, and Texas (one site only since the trees in one of the sites died during January 2024 because of extreme cold weather). We will observe growth parameters and other horticultural traits, including rootstock/scion compatibility, and determine the CLas titer. We will also collect samples to determine if other pathogens are detected in these hybrids and evaluate the general resistance pattern to different citrus pathogens in the field trial trees. We will utilize the pathogen array developed in this project to conduct these evaluations. We will continue evaluation of advanced hybrids under greenhouse conditions in Florida and Texas when exposed to the CLas pathogen in the two states and compare the reulsts with the observations made in California. We are in the process of comparing hybrid sequences with genome sequences of the parents - the three Australian limes and the three Mandarin genotypes. This analysis will indicate if the hybrids showing resistance phenotypes have certain genomic regions of the Australian limes constantly represented in their genomes. The extension aspect is an integral part of the study since communicating the results to interested parties is essential for discussing the progress of the project. We plan to conduct a project-specific meeting in Riverside in 2025 January so that the scientists can present their research to the group and the advisory board members and other interested individuals are aware of the direction of our research for this project.

Impacts
What was accomplished under these goals? Obj.1: Establish multi-state field trials for four F1 hybrids (mandarin X Australian lime) already selected for HLB resistance and fruit quality. For this objective, we conducted field trials in two locations in the three states - Florida, California, and Texas. The field trial in California is for performing the horticultural evaluation only since HLB is not reported in the areas chosen for the field trial. In Florida, we are evaluating the tree health ratings, tree size assessments, and CLas titer analyses. The hurricane in 2022 and other unfavorable growing conditions, along with tree characteristics including a weeping, brittle growth phenotype prone to trunk breakage (hybrids with Microcitrus inodora parentage) and poor adaptation to Florida conditions, have resulted in the decline of some trees. Promising hybrids with very low CLas titer and relatively good growth characters are identified and are under observation. In California, horticultural traits were documented; the fruit yield is excellent (about 3-500 fruits per tree), and the tree health appears to be satisfactory. Hybrids on 'Alemow' rootstock performed better than the trees on the Carrizo. We will conclude the analysis during the winter of 2024/2025. In Texas, the planting was delayed initially because of Hurricane Uri. During January 2024, there were freezing temperatures in Texas, and in one of the locations, all the field trial trees were compromised due to unusually low temperatures. So, we could not obtain any field data regarding fruit yield. However, the field trees in Texas did not show any titer for CLas. The hybrid plants in the second field are exposed to natural infections through psyllids. HLB-positive trees surround the field trial trees in the second location, indicating the presence of inoculum. The surviving trees are being monitored in Texas. Obj.2: Induce precocious fruiting in advanced hybrids. We were not successful in transforming Microcitrus hybrids with the FT gene. Although callus initiation was recorded in a number of genotypes, there was no differentiation. Co-PI Cutler is continuing the experiments with novel approaches to enable specific hybrids to flower. The cultural practices used to induce flowering were successful only in certain genotypes. Inducing early flowering is an essential component of developing and evaluating new hybrids. Hence, we will pursue our research in this area. Obj.3. Evaluate advanced hybrids for HLB resistance in controlled greenhouse conditions in CA, FL, and TX. We have developed long-read PACBIO sequences for Microcitrus australis, a resistant citrus relative used to generate hybrids in the breeding program. The haplotype sequences were assembled de novo using HiC analysis (Phase genomics) information. This work complements our published research on genome assemblies of three other Australian limes - Microcitrus australasica, M. inodora and Eremocitrus glauca. We have also sequenced three mandarin genotypes used to generate disease-resistant hybrids. Co-PI Main has facilitated the analysis by creating a private database for the team to visualize the data and compare it with the PACBIO sequences of selected HLB-resistant and susceptible F1 hybrids generated in our breeding program. SNP genotyping of advanced hybrids is in process. We have transported budsticks from 75 advanced hybrids to Florida and Texas under valid permits for greenhouse evaluations. The hybrids were selected based on preliminary genotyping results, field performance in California (for fruit evaluation), and pathogenicity tests conducted at the Contained research facilities in Riverside to document disease response when exposed to CLas pathogen. The challenge tests done in Florida using psyllids to challenge the plants showed promising results. We are completing the tests done in Texas to confirm the results. Obj.4: Development of rapid methods for detecting multiple pathogens to facilitate rapid release of quarantined plants by budwood certification programs in CA, FL, and TX. We have already developed a pathogen testing platform in a 96-well format. The citrus pathogen array can test for 20 common pathogens and will be very useful in testing budwood to be transported across state borders. During this year, we have standardized the storage conditions (of the pathogen array) and improved the sensitivity of the assays by modifying specific probe sequences and other assay parameters. Obj. 5,6,7: Include transfer of budwood of selected hybrids to all three states for propagation, obtain permits for interstate movement of hybrids and for the release of five hybrids chosen by working with regulatory agencies, and propagate and release selected varieties for further field trials. We have fulfilled these requirements. However, since we are now generating superior hybrid lines (compared to the hybrid genotypes used for the field trial in the current project), we will focus on developing other selections as future cultivars. Obj. 8: Extension to familiarize the industry with novel hybrids, conduct taste panels and obtain feedback. Surveys to measure social and economic impact. PI Ramadugu has conducted several extension activities in California, as outlined in the following sections. Co-PI Albrecht has presented the project results to interested individuals and growers in Florida. In Texas, co-PI Kunta has presented the project information to the students from South Texas Community College, students and administrators of Texas A&M University-Kingsville, Citrus Center Advisory Board Meeting, and Citrus Center visiting students under the USDA AgDiscovery Summer Youth Program organized by the University of Texas Rio Grande Valley. The co-PI communicated about the project periodically with the growers and the stakeholders. The project team has routinely shared the results of the performance to the growers. Co-PI Zhengfei Guan has made significant contributions to understanding the challenges and potential solutions facing the U.S. citrus industry in light of the ongoing HLB crisis. Economic impact of HLB cure: The publication titled "Curing Citrus Greening: Implications along the Supply Chain" offers valuable insights into the potential financial benefits of a cure for HLB. By predicting a total welfare surplus of $1.457 billion if a cure restores lost acreage, this research comprehensively analyzes the distributional effects of technology shocks within the orange supply chain. Innovative Disease Management Framework: The paper "How Resistant Cultivars Reshape Disease Management: A Theoretical Model with an Application to Citrus Greening Disease" introduces a theoretical framework for assessing the value and impact of resistant cultivars in perennial agricultural systems. This study highlights the potential of disease-resistant cultivars as a long-term solution to agricultural challenges. The findings underscore the importance of coordination among growers to mitigate adverse impacts during the transition to resistant cultivars. Supply Chain Challenges in the Orange Juice Industry: The research on "The Squeeze on Orange Juice Industry: A Deep Dive into Supply Chain Challenges and the Impact of Citrus Greening," fills a gap in the literature by exploring the orange juice industry's challenges from a supply chain perspective. The detailed mapping of the supply chain from production to retailing reveals each player's multifaceted difficulties, offering insights crucial for both policymaking and strategic business decisions. Industry Insights on Declining Production and Rising Imports: The publication "The United States Orange Industry: Declining Production and Climbing Imports," provides an overview of the ongoing decline in U.S. orange production due to HLB and other challenges. This research informs industry stakeholders and policymakers of the current threats to the domestic citrus industry.

Publications

  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Danda, T., J.-W. Park, K. L. Timmons, M. S�tamou, E. S. Louzada, and M. Kunta. 2023. A field deployable real-time loop-mediated isothermal amplification targeting five Copy nrdB gene for the detection of Candidatus Liberibacter asiaticus in citrus. Plant Pathology. J. 39(4): 309-318.
  • Type: Other 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.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Larissa Nunes da Silva, Anas Fadli, Chandrika Ramadugu, Ute Albrecht. Cultivating Novel Microcitrus Hybrids as Scion for HLB Management in Disease-Endemic Growing Conditions. 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: 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: Manjunath L. Keremane, Chandrika Ramadugu, Khushwant Singh, and Robert Krueger. New Pathogen Testing Tools for Safe Movement of Citrus Germplasm in the Huanglongbing (HLB) Era. 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: Jong-Won Park, Khushwant Singh, Tara Rickman, Erik Burchard, Manjunath Keremane, Robert Krueger, Chris Dardick, Margaret Staton, Mikeal Roose, Chandrika Ramadugu, and Madhurababu Kunta. Generating de novo transcriptome assemblies of Australian native lime species using various tissue types and its use as a reference for transcriptome data analysis. 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. 03/2024.
  • Type: Journal Articles Status: Published Year Published: 2024 Citation: Manjunath Keremane, Khushwant Singh, Chandrika Ramadugu, Robert R. Krueger and Todd H. Skaggs. 2024. NextGen Sequencing, and Development of a pipeline as a tool for the detection and discovery of citrus pathogens to facilitate safer germplasm exchange. Plants 2024, 13, 411. https://doi.org/10.3390/plants13030411
  • 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: Journal Articles Status: Published Year Published: 2023 Citation: Park, J.-W., W.E. Braswell and M. Kunta. 2023. Co-occurrence analysis of citrus root bacterial microbiota under citrus greening disease. Plants 13:80. https://doi.org/10.3390/plants13010080


Progress 09/01/22 to 08/31/23

Outputs
Target Audience:Our target audience consists of citrus growers, researchers, industry personnel, regulators, and other interested individuals. For obtaining regulatory approvals, we routinely interact with state-level and federal-level regulatory agencies like the California Department of Food and Agriculture, Division of Plant Industry, Texas Budwood Program, and Animal and Plant Health Inspection Services. The pathogen testing array we developed is of interest to other budwood testing organizations. We interact with growers to seek help in conducting field trials. The research conducted is routinely presented at various venues (scientific conferences and grower meetings - listed in the section 'how have the results been disseminated to communities of interest') and results in many useful discussions with growers, industry leaders, researchers, and students. After COVID-19-related restrictions were lifted, we were able to conduct fruit-tasting events and obtained feedback from the citrus industry representatives regarding the acceptability of the fruits generated from hybrids in our program. Changes/Problems:The field trial in Texas was delayed significantly because of the winter storm 'Uri' that occurred in February 2021. Due to the severe conditions caused by the storm, the citrus industry in South Texas was severely affected with an estimated loss for citrus growers of at least $230 million (as per Texas Comptroller of Public Accounts). The effect on our project was due to the unavailability of rootstocks needed to propagate the field trial hybrid plants. The shortage of labor and materials needed resulted in delaying our field planting. We do not expect fruits from the Texas field plants during 2023/24 winter. It will probably require at least one more year to obtain this crucial data essential to evaluate the hybrids in the field. In addition, one of the four hybrids has not flowered or fruited both in Florida and Riverside fields and hence it requires more time for complete horticultural field evaluation. Our efforts to induce the flowering of novel hybrids using the inducible FT gene are only partially successful. In other citrus systems, we can generate transgenic plants and induce flowering after induction with Mandipropamid®. In hybrids with Microcitrus parentage, there is transformation as evidenced by the presence of red calli (expressing the marker gene) but regeneration of the tissue has been challenging. Since precocious flowering is a very important trait for the early evaluation of novel hybrid plants, we are pursuing this objective by modifying our approach based on other published information. Other COVID-related problems during the early stages of the project (2020-2022) included delays in obtaining the necessary permits for budwood transfer from Riverside to the US Horticultural Laboratory in Fort Pierce, Florida where greenhouse evaluations are now in progress. Fruit evaluations were not possible in the initial years of the project (2020-2022) due to COVID-19 associated restrictions imposed by the University of California Riverside. What opportunities for training and professional development has the project provided?In Texas, co-PI Madhurababu Kunta's team has trained several underrepresented Hispanic students in STEM fields. Both laboratory techniques and skills needed for plant research such as propagation techniques to successfully grow and maintain citrus plants under greenhouse conditions were taught. Three Hispanic candidates who were previously trained in the laboratory of Kunta are now pursuing Master's degrees in Biology, and, Plant and Soil science. Two of these individuals are also working as full-time research assistants with Kunta. The mentorship they received from Kunta in the STEM field made them interested in science and pursue further education and training. In Riverside, CA, Ramadugu worked with four undergraduate students who came from socially, economically, or educationally disadvantaged backgrounds and trained them in basic laboratory skills and greenhouse techniques. Technicians and postdoctoral researchers working on the project got opportunities to learn new skills because they participated in the project and obtained needed training through project collaborators. The professional development of project personnel was made possible through the collaborating co-PIs who are extremely qualified in their fields of study. How have the results been disseminated to communities of interest?A.Co-PI Madhu Kunta arranged an educational, "Ag Discovery" lab tour to the students from the University of Texas Rio Grande Valley (UTRGV) and associated high school teacher participants from 15 school districts (24 high school teacher participants, 4 co-mentor high school teachers). The team is associated with the USDA grant-funded UTRGV Agro-Ecology professional group. Internships and assistantship opportunities were provided to the students. B. Ramadugu arranged two fruit-tasting sessions: 1). A group of 37 participants consisting of project participants from NIFA projects and industry members from California, Florida, and Texas were in the first fruit-tasting session conducted in January 2023. The team gathered for a project meeting in Riverside (mentioned in detail in section C below). Four F1 hybrids and one advanced hybrid were used for tasting sessions. The F1 hybrids with lemon-like flavors were provided as juice and the advanced hybrid with mandarin-like flavor was provided as fruit sections for taste evaluation. Extracted juice from a standard lemon and a standard mandarin were used for comparison. Cloud-based sensory software Compusense® was utilized to analyze the data. 2). Fruit tasting was conducted with a group of 75 participants at the UCR Citrus Day event held in March 2023 in Riverside. Juice from four F1 hybrids and a standard lemon were utilized for taste evaluations and the Compusense® software was used for analysis. C. Ramadugu arranged a program-specific meeting in Riverside in January 2023. Forty-seven people attended the meeting (33 in person and 14 by Zoom). Collaborators from two NIFA grants, their postdocs, technicians from the Riverside team, industry participants from Florida, Texas, and California (Advisory board members for the projects), and a few local citrus enthusiasts attended the meeting. Sixteen scientists presented their research associated with the projects. Six industry representatives talked about the importance of HLB, and the impact on the different citrus industries in the USA. The discussions that followed during the in-person meeting were very valuable to all the participants. The research being conducted was conveyed to the scientists and industry personnel. There were new collaborations made and useful discussions because of this event. D. Ramadugu and Singh made an oral presentation to highlight their research on "Finding genetic solutions for a serious citrus disease - huanglongbing" at the Plant and Animal Genomics meeting held in San Diego, CA in January 2023. At the same meeting, our USDA collaborator, Keremane, made an oral presentation about methods to detect citrus pathogens in budwood designated for interstate movement so that safe germplasm exchange can be achieved (objective four). E. Kunta and Park presented a talk on "Molecular genetics approach for the improvement of citrus tolerance to HLB" at the 77th annual meeting of Subtropical Agriculture and Environments" in Feb 2023 at South Padre, TX. The meeting was attended by local growers, students, scientists, and citrus industry personnel from Texas. F. Ramadugu gave an oral presentation on "Novel genomes of three Australian limes: towards the identification of the source of resistance against huanglongbing (HLB) disease" at the second meeting of the International Society for Citrus huanglongbing and phloem-colonizing bacterial pathosystems (IS-CHPP), Clearwater, FL in Oct 2022. Scientists from the USA and some International labs were present at the meeting. A few growers and industry representatives also attended this meeting. G. Ramadugu presented a poster at the Visalia meeting describing progress in citrus breeding and metabolomics analysis of fruit juice from novel hybrids generated in our breeding program. October 2022. H. Kunta and Park presented the results of our research at the XIV International Citrus Congress in Mersin, Turkey in November 2022. The title was "Comparison of gene expression profiles of HLB-tolerant and - sensitive F1 hybrids derived from a cross between mandarins and an Australian lime". I. Ramadugu gave an invited talk on "Citrus Huanglongbing: Finding solutions for disease management and long-term cultivation" at the University of California Riverside on the Citrus Day event organized in March 2023. About 140 growers, scientists, students, and others attended the meeting. Taste evaluations of fruit juice from hybrids generated in Ramadugu's breeding program were done during this event. J. Ramadugu presented a talk for citrus growers in Santa Barbara, CA. The event was organized by the Santa Barbara County Citrus Industry workshop and CPDPP in collaboration with UC ANR (University of California Agriculture and Natural Resources) in June 2023. K. Co-PI Albrecht presented the results of a field trial conducted in Florida with F1 hybrids generated by breeding citrus with Microcitrus. The event was the Annual Conference of the American Society for Horticultural Sciences, Orlando, FL, August 2023. L. Ramadugu was invited to give a talk at the Joint Congress of California Citrus Nursery Society and the International Society of Citrus Nurserymen. Oct 2022. The title of the talk was "Disease resistant, novel citrus cultivars generated through breeding citrus with Australian limes". What do you plan to do during the next reporting period to accomplish the goals?Objective 1: In the next year we plan to obtain data for the field trial plants in Florida and California. Certain genotypes have fruited and will be utilized for assessing horticultural performance. One genotype (hybrid 2) is slow in flowering and fruiting and it may take some more time to evaluate fruit traits. In Texas, the field planting could not be done in time due to winter storm "Uri" in 2021 (described in the changes/problems section). Hence we may not obtain fruits during 2023/24 winter. Resistance/susceptibility to HLB under field conditions will be monitored in the next year in both Florida and Texas (in California there is no HLB in the field and hence only horticultural evaluations will be possible). Objective 2: We will pursue the flowering objective by co-transforming the iFT construct in combination with the morphogenetic regulator, PLT 5 to obtain early flowering that is essential for the evaluation of horticultural traits of the new hybrids. We have now planted over 1300 hybrids in the fields in Riverside, CA and some genotypes can flower and fruit if certain practices are followed. We will encourage flowering in the field trees through these cultural methods. The resulting fruits will provide an opportunity to assess the usefulness of the advanced hybrids. Objective 3: We have almost completed the genome sequencing of Microcitrus australis genotype utilized in the breeding program. We will upload the information generated to the Citrus Genome Database and other Genbank sites so that other researchers can access the information. We will continue the analysis of the genotyping data generated so far with other traits like HLB resistance, tolerance, fruit traits, and horticultural performance. We will continue the evaluation of the greenhouse challenge plants transported earlier and identify promising individuals for further study. We will continue the analysis of the transcriptome data generated to identify promising genes associated with disease resistance. Objective 4: We have made significant progress in the development of a qPCR assay in a 96-well PCR plate arrayed with required primers and Taqman probes for the detection of 26 citrus pathogens (viruses, bacteria, including HLB-associated CLas). A total of 48 genomic targets can be detected in under 90 minutes in a qPCR assay. We will validate the array in the laboratories of collaborators in different states. Objectives 5, 6, and 7: Source plants are maintained in Florida, Texas, and California. However, we anticipate superior hybrids in the advanced hybrid population now available in Riverside, CA. The advanced hybrids will have fruit quality similar to citrus and hence may be more appropriate for cultivar development. Rigorous testing of the advanced hybrids needs to be completed for the HLB resistance phenotype. Objective 8: We will conduct extension - similar to what was done in the September 2022-August 2023 period. Economic analysis will be conducted by the economist on the team.

Impacts
What was accomplished under these goals? Objective 1. The goal for this objective is to conduct field evaluations of four F1 (first filial generation) hybrids generated by crossing various mandarin types with Australian limes - Microcitrus inodora and M. australasica. We planned to have about 240 trees per state and a total of 720 trees in six locations (2 per state). The field trees in Florida and Texas can be evaluated for HLB tolerance/resistance since the disease is prevalent in both these states. In California, although about 6,000 positive trees have been identified (and removed, for disease mitigation), the fields where the plants are being evaluated do not have the HLB pathogen; hence the hybrid evaluation in California is only for horticultural traits. In FL and TX, both horticultural and disease resistance evaluations are feasible. The field trials in Florida and California were planted as per our original strategy. Evaluations of plant horticultural characters including growth parameters, compatibility with the rootstocks being used, canopy characters, number of fruits per tree, juice characteristics, etc. are being recorded. In addition, in Florida, disease response evaluations are in progress since the pathogen is present in the fields. The results are not fully conclusive yet. Certain hybrids appear to be disliked by the psyllids and these exhibit much better growth parameters and lower CLas titers. Further evaluations will confirm the usefulness of these hybrids. All the hybrids have not flowered in FL and CA. Hybrid no. 2 would require some more time before flowers and fruits can be obtained. In Texas, due to the delay in planting (unavailability of required rootstock caused by the severe winter storm 'Uri' in February 2021), the field plants are still small and are yet to be evaluated. Objective 2. Obtaining flowers and fruits promptly is essential for horticultural evaluation of the hybrids. Citrus has a long juvenility - a shortage caused by the severe winter storm 'Uri' in February 2021 that prolongs the breeding approach. We are trying to induce precocious flowering using FT, a flower-inducing gene described in other systems. Efforts have focused on transforming stem explants obtained from advanced hybrids with Microcitrus parentage using Agrobacterium-mediated transformation of stem tissues. Nearly half of the hybrids tested (22 of 58 hybrids) produced calli with subsets of cells expressing Red Fluorescent Protein (RFP), a marker gene on the iFT (inducible flowering gene FT) construct used in the transformation experiments. This observation suggested that the transformation of hybrid stems is possible; however, efforts to regenerate shoots from these transformed calli have repeatedly failed, and subsequent transformation experiments have produced similar results (RFP+ cell clusters with poor shoot regeneration). Shoot induction from stem explants remains a key bottleneck with this approach. Recent reports have shown that the expression of shoot meristem developmental regulators can improve regeneration in recalcitrant species and varieties. We examined whether co-transformation using previously described shoot-inducing constructs. Our results demonstrated the successful isolation of emerging shoot meristems expressing an RFP-harboring construct in material co-transformed with morphogenetic regulator PLT5; however, the shoots were morphologically abnormal and not rescuable by grafting. Development and characterization of chemically regulated PLT6 expression construct is ongoing. In another approach to induce precocious flowering, we have planted about 1300 advanced hybrid trees in the field in Riverside, California. Certain genotypes can be induced to produce flowers precociously if grown as tall, single-stemmed plants. Until we standardize the conditions for producing flowers early through molecular approaches, the cultural practices that promote early flowering can be utilized for fruit evaluation of novel hybrids. Obj 3. We have completed the genomic sequencing of Microcitrus australis using material from the exact genotype that was used in our breeding trials to generate HLB-resistant progeny. We utilized the PacBio long-read CCS sequencing approach to generate extra-long scaffolds (N50 over 28 MB). In combination with HiC methodology, we have assembled the chromosome length scaffolds and generated a phased genome. Annotations using programs Braker and GenSAS are in progress. Another sub-objective in this main objective is to evaluate hybrids selected based on genotyping information, and fruit metabolome data (generated in a different project) and evaluate the selected hybrids for HLB resistance in different environments using multiple CLas isolates known to exist in Texas and Florida. Genotyping information generated from the hybrids is used as an aid in the selection of the hybrids for other evaluations. Hybrids shipped (under permits) to the US Horticultural Laboratory in Florida and to Texas A&M Citrus Center are being evaluated in the greenhouse. Seventy-two hybrid selections were sent to Florida and grafted onto 8 rootstock plants of US-942 and R7T6-SO. 87 selections were sent to TX. In the Florida greenhouse, about 50 individual trees are without detectable CLas several months after the graft challenge. We will continue monitoring the challenged plants to confirm disease tolerance/resistance. In California, a limited number of hybrids are challenged in the contained research facility in Riverside to get preliminary information about disease tolerance. We have many promising candidates that are being challenged multiple times with two isolates of the CLas pathogen (Psy62 from Florida and Hacienda Heights from CA). Objective 4. We have successfully developed pathogen arrays that have the capability of testing citrus plant tissue for 26 citrus pathogens by qPCR-based assays. Designed in a 96-well format, the array wells are prefilled with primers, Taqman probes, and qPCR master mix, and the assays are run using cDNA (total RNA extractions made from plant tissue are first reverse transcribed and the cDNA is utilized for the assay). We are in the process of publishing these results. Budwood certification programs in different states will benefit immensely from this citrus pathogen array since multiple pathogens can be tested simultaneously. Since the array is designed as a modular test, reagents for any single well can be replaced with suitable primers and probes to detect a pathogen that is more relevant to the geographic region where testing is performed. Objectives 5, 6, and 7. Budwood transfer to different collaborating groups has been achieved. We have permits required for the interstate transfer of hybrid germplasm. Commercialization aspects will be considered later if the current set of hybrids is deemed to be of interest. Objective 8. The extension and outreach performed for the project have been described in detail in the section - 'how have the results been disseminated to communities of interest'.

Publications

  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Jessica Trinh, Tianrun Li, Jessica Franco, Tania Toruno, Danielle M. Stevens, Shree P. Thapa, Justin Wong, Rebeca Pineda, Emmanuel Avila de Dios, Tracy Kahn, Danelle K. Seymour, Chandrika Ramadugu and Gitta Coaker. 2023. Variation in microbial feature perception in the Rutaceae family with immune receptor conservation in citrus. Plant Physiology, kiad263, https://doi.org/10.1093/plphys/kiad263.
  • Type: Book Chapters Status: Published Year Published: 2023 Citation: Chandrika Ramadugu and David Karp. 2023. Citron Genomics. In 'The Citron Compendium' edited by Eliezer Goldschmidt and Moshe Barr-Joseph. Springer. Pages 41-62.
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Muzi Li, Kelsey Galimba, Yuwei Xiao, Chris Dardick, Stephen M Mount, Ann Callahan, Zhongchi Liu, 2022. Comparative transcriptomic analysis of apple and peach fruits: insights into fruit type specification. The Plant Journal. 109(6): 1614-1629.
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Danda, T., J.-W. Park, K. L. Timmons, M. S�tamou, E. S. Louzada, and M. Kunta. 2023. A field deployable real-time loop-mediated isothermal amplification targeting five Copy nrdB gene for the detection of Candidatus Liberibacter asiaticus in citrus. Plant Pathology. J. 39(4): 309-318. https://doi.org/10.5423/PPJ.OA.02.2023.0030
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: B Wu, Q Yu, Z Deng, Y Duan, F Luo, F Gmitter Jr. 2023. A chromosome-level phased genome enabling allele-level studies in sweet orange: a case study on citrus Huanglongbing tolerance.
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: L Archer, U Albrecht. 2023. Wound reaction to trunk injection of oxytetracycline or water in huanglongbing-affected sweet orange (Citrus sinensis). Trees. Pages 1-15.


Progress 09/01/21 to 08/31/22

Outputs
Target Audience:The target audience consists of citrus researchers, molecular biologists, breeders, producers, and regulatory personnel involved in citrus research, improvement, and management in the US. For the current project, we have several citrus growers/nursery workers as collaborators and seek their input since it is valuable for our overall goal of generating novel hybrids and evaluating them in different parts of the country. We collaborate with regulators for permit concerns. Our target audience also consists of budwood certification and testing laboratories for the movement of experimental material; they are also valuable partners in our efforts to validate novel technologies for improved citrus pathogen detection platforms developed in the project. We collaborate with citrus nursery personnel, growers, and other industry members to arrange the field trials in three states since their help and opinion are valuable for the project. When we have opportunities, we present our research to citrus industry members in Florida, Texas, and California. Due to Covid-related constraints, the interactions were not at an optimal level in the past year. However, we anticipate that this situation will change in 2022-2023. We have presented our research project to students and teachers in Texas to educate the community on different research types. We encourage the student population to choose STEM fields during their studies. In California, we present our research to the industry and share fruits from novel hybrids with the local citrus industry personnel. This helps us to obtain feedback and incorporate suggestions into our plans. Changes/Problems:In Texas, due to winter storm 'Uri' during February 2021, it was not possible to obtain rootstock plants essential for the propagation of field trial plants in the greenhouses. 'Uri' caused a lot of destruction that delayed several aspects of the current project. The field trial is now scheduled to be initiated in September of 2022 and will require additional time for obtaining field data from the experimental hybrids. Due to restrictions associated with the Covid pandemic, the US Horticultural Research laboratory in Fort Pierce was not able to maintain essential services and instruments. These were needed for obtaining APHIS permits for transfer of budwood from Riverside facility to Fort Pierce. This caused a substantial delay in conducting greenhouse related disease evaluations for hybrid plants. A major turnover of personnel in co-PI Guan's team coupled with Covid restrictions delayed hiring of individuals for conducting the economic analysis proposed in the project. Access to contained research facilities in California were restricted because of Covid and resulted in delay of hybrid evaluations after HLB challenge. Evaluation of fruits of novel hybrids was delayed since the research team was not able to contact local growers and other citrus enthusiasts. We hope that during 2022-2023, Covid restrictions will be lifted completely so that we can make significant progress in our project. What opportunities for training and professional development has the project provided?We have provided opportunities several under-graduate and graduate students and technicians to get training in various aspects of the project including: greenhouse maintenance of citrus plants, grafting to propagate citrus, field maintenance of plants, promoting early flowering in the field by following certain cultural practices, genome sequence analysis, using gene annotation software, conducting HLB resistance evaluations in greenhouse and field, conducting DNA extractions, qPCR and analysis of data, plant transformations, extraction of RNA for transcriptome analysis, etc. Co-PI Kunta has presented the project to high school science and Ag science teachers from Agroecology professional development Institute, University of Texas - Rio Grande valley. Periodic communication of the project to citrus growers was made in Florida (by Albrecht), in Texas (by Kunta) and in California (by Ramadugu). How have the results been disseminated to communities of interest?We have been very active in communicating with other citrus scientists in scientific conferences and also in grower meetings like: Plant and Animal Genome meeting in San Diego, ASHS (American Society of Horticultural sciences) annual meeting in Chicago and California citrus conference in Visalia, CA. A web seminar was presented for ASHS professional interest group (Citrus) describing the current project and the overall goal of the breeding program. The presentations were virtual when in-person meetings were not allowed due to Covid. We have described our research in trade journals like Citrus industry and Citrograph. Made presentation of project summary at the Citrus Expo in Fort Myers, FL. We interacted with about 60 citrus growers in Riverside CA during the annual Citrus day event organized by University of California Riverside during March 2022. A list of the activities is included in other sections of the current report. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, we plan to do the following activities: Field planting of already propagated F1 hybrids (240 trees on two rootstocks) in two locations in Texas. Continuation of horticultural and disease evaluations in Florida and in California (horticultural evaluation only in CA). We will monitor growth parameters, conduct soil/leaf analysis to determine the nutritional status of the field plants. Some weak field trees in California field plantings will be replaced with duplicate plants in September. qPCR analysis will be done for field plants in Florida and Texas only (for determining CLas titer). PIs involved in this objective (Ramadugu, Kunta and Ute) will travel to other states to visit the field plantings and exchange notes. Due to Covid, such activities were not possible in the previous year. For inducing flowers using transformation technologies, the newly constructed vectors described earlier will be utilized for Agrobacteriaum-mediated transformations and shoot regenerations. Field planting of back cross hybrids will continue. We plan to grow the field plants as tall, single-stemmed plants and prune all side branches. This cultural practice was observed to induce flower and fruit production in at least certain genotypes. We will complete sequencing of HLB-resistant accession, Microcitrus australis. We will conduct in-depth bio-informatic analysis of other sequences that are already generated and use the sequence information for data mining. We will try to organize fruit tasting events in Riverside so that feedback can be obtained from the industry regarding suitability of the novel hybrid fruits for human consumption. Also, this will help us to determine if certain hybrids develop off-flavors that are passed on to the breeding progeny due to linkage drag. We hope to attend scientific meetings in person to foster collaborations and exchange ideas with other scientific personnel. We will also plan more extension meetings with citrus industry people interested in our project.

Impacts
What was accomplished under these goals? Developing novel hybrids with disease resistance will provide long-term solutions to the citrus HLB problem. Towards this goal, we have generated hybrids by crossing citrus with HLB resistant Australian limes. Research performed here will help in evaluating four F1 hybrids in three states under field conditions, test HLB tolerance of advanced hybrids in greenhouses, identify promising genotypes based on molecular data generated, and eventually lead to cultivar development. We are developing genome sequence information for Microcitrus australis, a resistant accession, in this project. To facilitate rapid testing and movement of citrus budwood across states, we are developing additional tools like citrus pathogen testing array. Novel hybrids generated by traditional breeding will be readily accepted by the public once we establish the disease resistance in the field and obtain acceptable fruit quality. Obj1. In Florida field trials were initiated in the Fall of 2021. The four previously selected F1 hybrids were propagated on Carrizo and US 942 rootstocks and planted in Fort Meade and Arcadia. Valencia and Lisbon lemon were included as controls. The experimental design is a RCBD with 12 and 9 replications. Field trees were monitored for growth, health and psyllid colonization. Monthly measurements of plant height, trunk diameter and characterization of the new flush was documented. Disease evaluation included monthly psyllid counts (adults, nymphs, eggs), HLB symptom rating and determination of CLas titers at six month intervals. Climate data and soil moisture was checked monthly. Leaf and soil samples were collected and sent to a private lab for nutritional analysis. In Florida, the four hybrids established well; some hybrids have produced flowers and fruits. Psyllid feeding on hybrid2 was significantly low. Hybrid2 produces seedless fruits with mandarin/orange-like characteristics. On hybrids 1, 3 and 4, psyllid feeding and reproduction activity varied depending on the location. Valencia, hybrid1 and hybrid3 were most attractive to the psyllids while hybrid2 was the most repellent. There is no correlation yet between the flushing data and psyllid colonization. Hybrids 1,2 and 3 appeared to be more resistant than hybrid4 and Lisbon lemon controls. Hybrid2 had a continuous and intensive flushing behavior while maintaining a healthy green foliage with low CLas titers (Ct close to 40) indicating its superior ability to resist HLB or avoid its transmission by repelling psyllids. Rootstock or rootstock/scion effects on CLas titers were not detected. We continue to monitor the disease response and horticultural performance in the sub-tropical, HLB-endemic environment of Florida. In the field situation in Florida, there is continuous reinoculation by ACP. Next CLas titer analysis in October 2022 will confirm if resistance trends observed in hybrids 1, 2 and 3 will continue. In California the field trials were initiated in Riverside and South Coast Research Station (Irvine) in September 2021. The plants are monitored for horticultural traits and presence of other pests but not for HLB since psyllids are not present and HLB is not expected. We evaluated leaves of hybrid field trees for symptoms of Citrus leaf miner (CLM), Asian Citrus Psyllid (ACP), and thrips. Tree health and total fruit per tree were recorded. Most trees had CLM damage and populations developing in the new flush leaves. Thrip damage was observed on the leaves and fruit. Some hybrids produced flowers and fruits in 2022. In Texas, due to freeze issues, field planting was delayed. Budwood received from Riverside was tested for several pathogens/diseases including HLB, exocortis viroid, hopstunt viroid, Citrus viroid III, canker, citrus variegated chlorosis, citrus leprosis virus, citrus tristeza virus, citrus tatterleaf virus, citrus leaf blotch virus, psorosis virus, citrus black spot, and sweet orange scab. Propagations on sour orange and C22 are ready for field planting in Sep 2022. Obj.2. Co-PI Cutler has developed genetic constructs that allow for chemically inducible flowering (iFT) to potentially assist in the pre-selection of desirable hybrids from breeding populations. The strategy is to transform stem segments from juvenile seedling with the iFT construct so that fruit phenotype data can be acquired rapidly. Many challenges exist in working with hybrid material, including difficulties with shoot regeneration and lower transformation levels. Recent reports have shown that the expression of shoot meristem developmental regulators can improve regeneration in recalcitrant species. Systematic comparisons of different regulatory transcription factors in Solanum (PLT5, ESR1, WUS-P2A-BBM, WUS, and WIND1) showed that Plethora-like5 (PLT5) had improved properties for transformation and shoot regeneration (Lian et al. 2022). We have now designed constructs for both transient and stable co-expression of PLT5 in citrus. Preliminary results show successful isolation of emerging shoot meristems expressing an RFP-harboring construct in material co-transformed with PLT5 and in lines where the expression cassette was on the same construct as the RFP marker. These tests showed shoot emergence from 50% of the explants. We will conduct a larger-scale test of the effects of the PLT5 construct alongside other developmental regulators (WUS and GRF) in advanced hybrids. We have some success with modifying cultural practices in the field and inducing early flowering in some novel hybrids. Obj.3. a). Genome sequencing of Microcitrus australis, the Australian Round lime, is in progress. We are generating long reads using PacBio coupled with the scaffolding of the contigs using proximity ligation and HiC methods. Genome sequence analysis is in progress with collaboration from co-PI Main. b). We have generated SNP genotyping data for 56,000 loci using 1050 hybrids belonging to F1, and back cross hybrids. Genotyping correlates resistance traits in the breeding population with specific genomic fragments of Australian limes. c). We are transporting budwood of 200 advanced hybrids to TX and FL for greenhouse HLB challenge with the local CLas isolates, and disease evaluation after psyllid feeding or grafting with diseased budwood. Transcriptome studies are in progress. Obj.4. To streamline the budwood testing process and facilitate faster and safer germplasm exchange between different states, we have developed a citrus pathogen testing array consisting of pre-loaded DNA primers and Taqman probes for 26 citrus pathogens in a 96 well format. Starting from citrus RNA, using the array, we can test for the presence of 26 pathogens (a total of 48 targets) in 90 minutes in a qPCR assay. We have made significant progress toward this objective. We are now trying to generate array plates that can be shared with labs in FL and TX to benefit from citrus budwood testing programs all over the USA. Obj.5. Source trees of the four F1 hybrids are currently maintained in Florida (by Alico), California (at the UCR greenhouses), and Texas (at the citrus center). Objective fulfilled. Obj.6. We have APHIS permits for interstate movement of experimental backcross hybrids. Selection of the hybrids for release is not yet done since rigorous testing needs to be completed. Obj.7. About 1000 back cross hybrids are now under observation in California field plantings to evaluate horticultural performance. Obj.8. We presented posters describing our HLB projects and displayed hybrid fruits on UCR citrus day held in March 2022. Taste panels could not be conducted for this project and hopefully will be possible during the 2022 winter season. Abstracts describing field trails were presented to growers at Citrus Expo held in Fort Myers, FL, in August 2021. Extension work was done in Texas involving high school teachers. Oral presentations were given to various citrus groups.

Publications

  • Type: Journal Articles Status: Published Year Published: 2022 Citation: " da Gra�a, J. V. & M. Kunta. 2022. A review of citrus diseases and pathogens in Texas. Subtropical Agriculture and Environments 73: 17-28.
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: " Louzada, E., O. Vazquez, S. Chavez, M. S�tamou & M. Kunta. 2022. Optimization of vqPCR for reliable detection of viable Candidatus Liberibacter asiaticus in citrus. HortScience 57: 692-697. doi:1021273/HORTSCI16600-22.
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: " Park, J.-W., J. V. da Gra�a, M. Gonzalez, E. S. Louzada, O. J. Alabi & M. Kunta. 2022. First report of citrus virus A in Texas associated with oak leaf patterns in Citrus sinensis. Plant Disease 106: 2005. doi.org/10.1094/PDIS-03-21-0628-PDN.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: " Park, J.-W., J. V. da Gra�a, M. S�tamou & M. Kunta. 2021. Diversity of Citrus tristeza virus strains in the Upper Gulf Coast of Texas. Plant Disease 105: 592-598.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: " Perez, E., M. Kunta, V. Ancona, J. V. da Gra�a, C. Ayin, G. Santilla & V. Mavrodieva. 2021. The return of Asiatic citrus canker to Texas: Surveys and eradication efforts. Plant Health Progress 22: 143-148.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Keremane, M. L., McCollum, T. G., Roose, M. L., Lee, R. F., & Ramadugu, C. (2021). An Improved Reference Gene for Detection of Candidatus Liberibacter asiaticus Associated with Citrus Huanglongbing by qPCR and Digital Droplet PCR Assays. Plants 2021. 10(10);2111. https://doi.org/10.3390/plants10102111
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Snyder, J., Dickens, K.L., Halbert, S.E., Dowling, S., Russell, D., Henderson, R., Rohrig, E. and Ramadugu, C. 2022. The Development and Evaluation of insect traps for the Asian Citrus Psyllid, Diaphorina citri (Hemiptera: Psyllidae), vector of Citrus Huanglongbing. MDPI Insects 13:295. https://doi.org/10.3390/insects13030295


Progress 09/01/20 to 08/31/21

Outputs
Target Audience:Target audience consisted of citrus producers (nursery owners, growers), citrus regulatory personnel,citrus researchers, breeders and the public. We are in constant communication with citrus growers/nurserymen interested in collaborating with the project team in conducting field trials in Florida, Texas, and California. Arrangements for budwood propagation and preparation of land for field trials involved several research personnel, growers, nurserymen and workers in the citrus fields on a continuous basis. We interacted with regulatory agencies in Florida, Texas and California to arrange budwood testing through various organizations (Citrus Clonal Protection Program in Riverside, CA; The Division of Plant Industry in Gainesville, Florida; Texas Department of Agriculture and Texas budwood testing program in Weslaco, TX). We communicated with grower collaborators in the three states to arrange field trials in six locations. The pathogen array we developed will be evaluated by the budwood testing and regulatory agencies in California, Florida, and Texas. Arrangements are now being made to ship the ready-made array plates, supplies required for the qPCR runs and detailed protocols to collaborating scientists in all three states. We presented an abstract at the Citrus Expo held in Fort Myers during August 2021 and discussed project details with interested growers. In California, we interact with growers interested in our novel hybrids. Due to Covid-related restrictions, during the current year, this interaction was minimum. Changes/Problems:The field trial in CA was planned in two locations - University of California Riverside and Coachella Valley Agricultursl Research Station which is located about 70 miles from Riverside. Due to HLB quarantine, it will be difficult to transport propagated hybrid plants from Riverside to Thermal. Hence we changed the field trial location to South Coast Research and Extension Center, located in Irvine, CA. Riverside and Irvine are under HLB quarantine. Thermal is not under HLB quarantine. Due to Covid related complicaitons, we were not able to get permits for sending budwood material of advanced hybrids to co-PI Duan in Fort Pierce, Florida. The USDA laboratory where Dr. Duan works has strict regulations regarding entry of non-USDA personnel into the facility during Covid. The autoclave and biological safety cabinet located in the facility need to be certified by outside agencies that are not able to get access to the facility. Hence the delay in obtaining permits. We hope that in the next few months this problem will be resolved. The Texas field trial will start in Spring of 2022 (instead of September 2021). The freeze caused by winter storm Uri during February 2021 devastated the local citrus industry with an estimated crop loss of $230 million. It was not possible for us to obtain the necessry plant material required for the plant propagations. There was also shortage of labor and delays in obtaining permits that resulted in delays. What opportunities for training and professional development has the project provided?We have trained several Hispanic students that are underrepresented in STEM fields in both laboratory techniques and skills needed for citrus research such as propagaiton techniques to successfully grow and maintain the plants under greenhouse conditions. Many of these students are socially, economically, or educationally disadvantaged. Fresh graduates with BS degrees were trained in doing basic molecular biology techniques, exposed to bioinformatic procedures, trained in plant molecular biology and microbiology techniques. A postdoc hired in the project got trained in conducting advanced analysis essential for the project. How have the results been disseminated to communities of interest?Due to Covid19-related restrictions in several regions, we were not able to conduct extension work as usual. When possible, the members of our project team have communicated with growers. Kunta presented project progress report to the citrus community in Weslaco. Albrecht was able to present research work to members of the community in Florida that attended the Citrus Expo event at Fort Myers in Florida during August 2021. Development of pathogen array was communicated with the regulatory agencies in all three states and arrangements are now made for collaborative work to validate the arrays so that improvements can be done. The goal is to make the pathogen array useful for all budwood testing units in the country so that teting for multiple pathognes can occur in a reliable and rapid manner. This will facilitate germplasm transfer which is crucial for condcuting greenhouse and field experiments in different agronomic situations. For the field trial experiments, Ramadugu, Mauk, Albrecht and Kunta have communicated with several research personnel, growers, nursery managers and workers about the project goals and the nature of the novel hybrids generated for developing HLB resistance. What do you plan to do during the next reporting period to accomplish the goals?Specifically, we plan to accomplish the following tasks: Objective 1: For the multi-state field trials, we will have 240 trees planted in the field in each of the three states. Each of the six sites selected will have 120 field trees. If covid regulaitons are lifted, PI Ramadugu will travel to Florida and Texas to visit the field plantings. Co-PIs Albrecht and Kunta will travel to California for observing the hybrid plants in the field and to exchange ideas. Objective 2: We will pursue our efforts to transform more back cross hybrids using the iFT technology described earlier. We will focus on selected hybrids that appear to be amenable for transformaion process (based on parentage and genotype, certain hybrids may be recalcitrant). We will plant another 500 back cross hybrids in the fields in Riverside and follow cultural management practices to promote flowering and fruiting in the field. Objective 3: We hope that Covid restrictions will be lifted in florida and permits can be obtained to transport budwood to Fort Pierce research station. We will transport 100 back cross hybrids to US Horicultural Research lab in Fort Pierce for greenhouse evaluation of hybrid material. We are in the process of generating PACBIO long read sequences for Microcitrus australis. We will perform Hi-C scaffolding and develop reliable genome assembly for Microcitrus australis. The assembled genome will be annotated both by using in-silico analysis of the genome and supporting data from RNAseq. SNP genotyping of 1500 back cross hybrids will be done using an Affymetrix chip with 56,000 SNPs and data analyzed. Objective 4: Pathogen arrays developed in Riverside will be shared with research collborators interesed in budwood esing in Florida and Texas. Validation by multiple laboratories (using different instruments and operators) is required for fine-tuning the system and making it useful to the citrus industries in other parts of the country. If other citrus industries require testing for different pathognes, the modular system developed can be altered to incorporate additional targets. Objective 5: Budwood transfer to different collaborating groups has been acheived. PI will travel to the required sites and ensure that the selected hybrids are established in the different locations. Objective 6: We have already obtained permits for interstate movement of budwood required for field trials. Objective 7: Commercialization aspects will be considered after field performance data is collected from different growing regions. Objective 8: We envisage that covid restrictions will be lifted soon in the cirus growing states involved in this project. We plan tot attend scientific meetings, arrange extension talks in grower meetings and conduct taste panels to assess the organoleptic properties of novel hybirds. Data required for assessing the economic viabiliy of the novel hybrids will be collected by the economist on the team.

Impacts
What was accomplished under these goals? Citrus huanglongbing (HLB) has proved to be a major challenge for citrus cultivation in the USA. Substantial financial resources are required for vector control and disease management. Financially and environmentally sustainable solutions are needed for citrus cultivation. Development of genetic resistance is useful for HLB management. Citrus breeding is generally not favored since it is time consuming and difficult. We have already conducted crosses for 8 years and have advanced hybrids on hand. Research performed in the current grant will evaluate four hybrids in three states under field conditions, identify advanced hybrids that are HLB resistant and lead to cultivar development. We have designed pathogen arrays to facilitate rapid testing and movement of citrus budwood across the country. The research conducted in this project will identify disease resistant hybrids and develop additional tools for selection of useful hybrids. Novel hybrids generated by traditional breeding will be readily accepted by the public - once we establish the disease tolerance in the field and obtain acceptable fruit quality. Obj.1: We are ready to conduct field trials in FL, TX and CA for four selected F1 hybrids. Budwood was sent to various regulatory agencies [Citrus Clonal Protection Program in CA, Division of Plant Industry in FL, Texas budwood certification program in TX] for pathogen testing and passed on to the nurseries/co-PIs for propagations. The budwood was propagated by Alico Inc., FL on US 942 and Carrizo rootstocks. Trees will be planted in two citrus groves (Alico Inc. and Peace River Packing Co.) in Sep2021. In CA, the four hybrids were propagated on Carrizo and Alemow rootstocks and will be planted in the Univ. of CA Riverside fields and in South Coast Research and Education center, Irvine, CA. during Sep2021. For the TX field trial, budwood was propagated on sour orange and Bitters C22 rootstocks. Control plants were Valenica and Lisbon lemon (FL), Washington navel and Lisbon lemon (CA), Rio Red grapefruit and Meyer lemon (TX). The total number of trees for the field trial in the three states will be 720. Obj.2: The goal is to obtain flowering from advanced hybrids in a short period of time. The advanced or back cross hybrids are generated by crossing promising F1 hybrids (Citrus X Microcitrus) with other types of citrus cultivars. Cutler's lab has developed genetic constructs for chemically inducible flowering (iFT). Stem segments from hybrids are transformed with the iFT construct so that fruit phenotype data can be acquired rapidly. In standard Carrizo, flowering and fruiting can be obtained in a year. Transformation of hybrids is technically more challenging. Stem segments from 58 hybrid progeny were evaluated in Agrobacterium-mediated transformations; 22 hybrids have produced calli with a variable subset of cells that express Red Fluorescent Protein (RFP), a marker gene. One of the stem explants has produced an RFP+ shoot, which we will rescue. Shoot induction remains a bottleneck with this approach and we will test whether the co-transformation of shoot-inducing constructs can be used to increase transformation frequencies. Hybrids that are transformable will be targeted in subsequent experiments. Microcitrus parents used to generate the hybrid progeny show precocious flowering and hence are amenable to early flowering if certain cultural practices are followed. We have planted 350 advanced hybrids in the fields in Riverside, CA. We expect flowering and fruiting from a portion of these in the next few years. This would provide fruit information that can be used for hybrid selection. Obj.3: We are developing PACBIO long read (CCS) sequences for HLB-resistant parent accession, Microcitrus australis. SNP genotyping of 1500 advanced hybrids using Affymetrix citrus array (for 56,000 SNPS) is in progress. Fruit metabolomics data generated in parallel projects will be utilized for selection of advanced hybrids for further evaluation. Multi-locational field testing for HLB resistance: budwood from 100 advanced hybrids was selected for shipping to Texas and FL (total 200). We are currently awaiting permits for Florida and we are ready to ship material to TX. Obj.4: Movement and release of germplasm requires testing for multiple citrus pathogens at different times. We have developed an array for rapid, efficient detection of 26 citrus pathogens (both DNA and RNA) by real time PCR. The array includes primers and probes for detection of seven citrus viroids, twelve viruses, seven bacterial pathogens including Ca.Liberibacter asiaticus, and others. The array was prepared by loading primers and probes, lyophilizing the plates and storing sealed, 96-well PCR plates at -20 °C. Synthetic gene-blocks conaining target sequences for 48 targets were used for standardizing the assays. The gene blocks, primers and probes for most targets were developed in this study. The array was evaluated by testing known positive samples. The presence of various pathogens in twenty samples were analyzed by transcriptome analysis and blast searches. Raw sequences and many individual pathogen genome sequences have been deposited in NCBI. Using one array plate, RNA extracts from three plants can be tested for 48 targets each within 90 minutes. Obj.5: We have transferred budwood of selected hybrids for propagation. Budwood sent to Florida was tested by Division of Plant Industry and are now maintained at Alico Inc. nursery for further propagation as needed. Budwood sent to Texas was tested first by CCPP in CA and then by the Texas budwood certification program in Weslaco and are now maintained at Citrus center greenhouses for future propagations. In CA, the budwood is maintained in an APHIS-certified greenhouse facility at Univ. of CA Riverside. Obj.6. We have obtained permits needed for field trial and greenhouse testing of advance hybrids for the Texas group. For the Florida group, permits for field trial were obtained. Permits for greenhouse work in Fort Pierce are still pending due to Covid-related delays. Obj.7. Propagation of hybrids has been done in the participating nurseries in all three states. Patenting and commercialization will take place in future based on field performance in all three states. Obj.8: Extension. Due to Covid-related restrictions, only very limited extension work could be done. In FL, we presented an abstract of our field trial aspect of the project in Citrus Expo held in Fort Myers, FL during Aug 2021. In TX, we provided a tour of the lab and explained project to high school teachers. Albrecht, Mauk, Ramadugu and Kunta discussed details of the project with growers, nurserymen and researchers from FL, CA and TX. Meetings were held in person or by phone conversations.

Publications

  • Type: Other Status: Published Year Published: 2021 Citation: Cutler, S., Roose, M. and Park, S.Y. 2021. Accelerated citrus breeding by genetic engineering. Citrograph Summer 2021, 52-55. https://citrus-research-board-static.sfo2.digitaloceanspaces.com/citrograph/pdf/CRB-Citrograph-Mag-Q3-Summer-2021-Web.pdf
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Lee, R.F., Keremane, M.L. and Ramadugu, C. 2021. Use of young plants for biological indexing of graft transmissible pathogens of citrus. Crop Protection 143: 105524. https://www.sciencedirect.com/science/article/pii/S0261219420304579
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Stover, E., Ramadugu, C, Roose, M., Krystel, J., Lee, R.F. and Keremane, M. 2021. Incidence of Asiatic citrus canker on trifoliate orange and its hybrid accessions in a Florida field planting. HortScience 56(5): 525-531. https://journals.ashs.org/hortsci/view/journals/hortsci/56/5/article-p525.xml
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Louzada, E.S. and Ramadugu, C. 2021. Grapefruit: History, use and breeding. HortTechnology 1: 1-16. https://doi.org/10.21273/HORTTECH04679-20
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Huang, C.Y., Niu, D., Kund, G., Jones, M., Albrecht, U., Nguyen, L., Bui, C., Ramadugu, C., Bowman, K, Trumble, J. and Jin, H. 2021. Identification of citrus immune regulators involved in defence against huanglongbing using a new functional screening system. Plant Biotechnology Journal 19(4): 757-766. https://doi.org/10.1111/pbi.13502
  • Type: Other Status: Published Year Published: 2021 Citation: Albrecht, U., Alferez, F. and Zekri, M. 2021. Journal EDIS. 2021-2022 Florida Citrus production guide: rootstock and scion selection: HS1308. https://edis.ifas.ufl.edu/publication/hs1308
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Albrecht, U., Tripathi, I. Bowman, K.D. 2020. Rootstock influences the metabolic response to Candidatus Liberibacter asiaticus in grafted sweet orange trees. Trees 34(2): 405-431. https://doi.org/10.1007/s00468-019-01925-3
  • Type: Book Chapters Status: Published Year Published: 2021 Citation: Kunta, M., J.-W. Park, W. E. Braswell, J. V. da Graca & P. Edwards. 2021. Modern tools for detection and diagnosis of plant pathogens. Pages 63-96. In: Plant Pathology: Emerging Trends and Opportunities (K. P. Singh, S. Jahagirdar & B. K. Sarma, Eds.). Springer Nature Int. Publishing
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Braswell, W., J.-W. Park, P. A. Stansly, B. C. Kostyk, E. S. Louzada, J.V. da Gra�a, and M. Kunta. 2020. Root samples provide early and improved detection of Candidatus Liberibacter asiaticus in citrus. Nature Scientific Reports 10, Article number: 16982. https://doi.org/10.1038/s41598-020-74093-x