Grant Information
| Knowledge Area | Subject of Investigation | Field of Science | Percent |
|---|---|---|---|
| 202 - Plant Genetic Resources | 999 - Citrus, general/other | 1081 - Breeding | 50% |
| 201 - Plant Genome, Genetics, and Genetic Mechanisms | 999 - Citrus, general/other | 1080 - Genetics (excludes breeding) | 40% |
| 212 - Pathogens and Nematodes Affecting Plants | 999 - Citrus, general/other | 1160 - Pathology | 10% |
Huanglongbing (HLB) and its causal agent Candidatus Liberibacter asiaticus (CLas) are a serious problem for the US citrus industry, with Florida and Texas already heavily affected, and California at an early stage. Rootstock cultivars with genetic tolerance to HLB improve tree health, fruit production, and fruit quality in HLB-affected orchards, but even the best rootstock available suffers large yield losses when infected. Further gains in tolerance or resistance to HLB are urgently needed to sustain the industry. Field trees in Florida are now all infected with CLas, providing an opportunity to screen thousands of trees for overall performance in environments with high disease pressure. Previous work to create and evaluate over 300 new rootstock hybrids across three HLB- endemic regions in Florida in replicated sweet orange field trials will be leveraged to deliver the best performing HLB-tolerant rootstocks for commercial release, including expanded collection of performance information over the next two years (Objective 1). Top performing rootstocks will be further screened for resistance to Phytophthora and other relevant soil-borne pathogens to ensure adoption in HLB-endemic and HLB-threatened regions. This extensive set of phenotypic data will be integrated with genetic information to identify the genetic control of HLB-tolerance and other important traits, enabling rapid selection of superior tolerant hybrid rootstocks in future breeding cycles (Objective 2). Commercial testing and release of rootstocks will occur in close consultation with industry members and will be disseminated to stakeholders at all levels through virtual and in- person seminars and large industry events.
Objective 1Methods. Beginning in 2006, Bowman initiated sexual hybridization targeting the creation of superior new rootstocks with tolerance to HLB from parentage that included several selections of mandarin and pummelo (the progenitor species of sour orange), and hybrids of those species with trifoliate orange (P. trifoliata) that have combined good rootstock traits and HLB-tolerance. Trees of the new hybrid rootstocks were propagated for the trials in the USDA certified citrus nursery in Ft. Pierce, beginning in 2013, using stem cuttings for rootstock propagation and standard budding practice for grafting of the scions. From 2014 to 2020, 15 rootstock trials were planted with 'Valencia 1-14-19' or 'Hamlin 1-4-1' scion among three locations.In addition to the new hybrid rootstocks, four standard rootstocks with a long history of use, diverse genetic backgrounds, and known characteristics were also planted in each trial to compare the new rootstocks with those in commercial use.Beginning in the year after planting, Bowman has collected metrics on tree survival, tree and canopy characteristics, fruit yield and quality, and yield efficiency across the 15 replicated trials. Rootstock hybrids with performance superior to the current best HLB-tolerant commercial rootstocks will be identified to select a minimum of three new rootstock cultivars for release to growers before the end of 2023. For the current project proposal, Bowman and Albrecht will continue periodic measurements of tree performance similar to prior published studies and based on the guidelines outlined in the National Citrus Breeding Collaboration Report.In addition to tree size and canopy measurements, we will assess tree health by visual rating of foliar HLB symptoms and canopy density. Fruit yield and fruit quality analysis will be conducted annually during harvest times typical for the location.Rolshausen will also evaluate selected new rootstocks that showed good performance under Florida conditions for their level of tolerance against some of the main relevant soil-borne pests and diseases for citriculture production globally, including Phytophthora and Fusarium species and nematodes. Root and soil samples will be collected from the top ten performing rootstocks grown in the trials currently being evaluated by Bowman and Albrecht. Known susceptible (Ridge sweet orange) and resistant (Swingle) rootstocksto Phytophthora species and nematodes will also be included for comparison. Data will be collected in Florida and tolerance to nematodes and Phytphothora will be determined as a measure of total female nematodes per gram of fresh roots and fungal propagules per cm3 of soil, respectively. Total DNA will also be extracted from roots and sent to UC Riverside for further analysis. Phytophthora and Fusarium load in roots will also be measured by quantitative PCR. Rolshausen will also evaluate rootstock performance in environmental growth chambers using citrus seedlings. Resistance to the most frequent pathogens Tylenchulus semipenetrans, Fusarium solani, Phytophthora citrophthora and P. nicotianae for California will be measured using established protocols. The commercial roostocks to be tested include six HLB-tolerant commercial cultivars (US-802, US-812, US-897, and US-942, US-1283 and US-1284), and four of the best performing new hybrids to be selected from the 15 Florida field trials. Resistant and susceptible roostocks to both nematodes and Phytophthora will be included.Objective 2Methods.Genome-wide association mapping will be performed to identify alleles underlying the contribution of rootstock to HLB tolerance and other important horticultural traits in trees with a grafted sweet orange scion. Genetic data will first be collected from all rootstock hybrids and their parents using standard practices. DNA sequencing libraries will be pooled and sequenced to a coverage depth of about 20X per sample with 150 bp paired-end sequencing reads on the Illumina NovaSeq S4. High quality genetic variants segregating in the population of rootstock hybrids will be discovered using the Genome Analysis Toolkit (GATK) following best variant calling practices.Missing genotype calls will be imputed with AlphaFamImpute.Variants will then be LD pruned (r2
Target Audience
During this reporting period we have interacted with citrus industry stakeholders through regional, state in-person events and seminars and through the preparation of extension materials. We are particularly focused on raising awareness of the ongoing research supported through this proposal to growers interested in the adoption of new HLB-tolerant rootstocks in CA, TX and FL. We delivered at least 9 presentations at events attended by citrus industry stakeholders.
Changes / Problems
Nothing Reported
Training & Professional Development
One postdoctoral research associates and two graduate students were trained on research of citrus production and rootstocks and/or genetic analysis of citrus germplasm. Graduate students and postdoctoral researchers receive mentorship in research and also career advancement. This occurs through training focused one-on-one and group meetings, encouragement to establish an individualized development plan to identify and accomplish long-term career goals, and access to career and networking events at the relevant universities. ?
Dissemination Streams
We have presented information on the data collected from these trials to grower and other stakeholder communities and will continue to do so in the upcoming reporting period, including at least 9 presentations on this project to audiences composed mostly of citrus industry stakeholders. The goal is to ensure there is interest and awareness of these new HLB-tolerant rootstock hybrids to facilitate rapid adoption in relevant regions upon release. ?
Next Reporting Steps
This project is in no cost extension. We expect the remainder of the funds to be spent in the next year and project objectives will transitions to those outlined in 2023-70029-41305.
Target Audience
During this reporting period we have interacted with citrus industry stakeholders through regional and state in-person events and seminars and through the preparation of extension articles and other associated material. We are particularly focused on raising awareness of the ongoing research supported through this proposal to growers interested in the adoption of new HLB-tolerant rootstocks, both in CA and FL. We delivered at least 7 presentations at citrus industry events. To reach a broader audience an extension article summarizing the ongoing research supported by this proposal was posted on 'Science for Citrus Health' (https://ucanr.edu/sites/scienceforcitrushealth/). These efforts highlighted the future project goals as well as information on specific rootstocks and their future availability.
Changes / Problems
There are no major changes in the workplan. Research funds from this proposal were not available to support the collection of field data until January 2022 and spending reflects this challenge.
Training & Professional Development
Two postdoctoral research associates and two graduate students were trained on research on citrus production and rootstocks and/or genetic analysis of citrus germplasm. Graduate students and postdoctoral researchers receive mentorship in research and also career advancement. This occurs through training focused one-on-one and group meetings, encouragement to establish an individualized development plan to identify and accomplish long-term career goals, and access to career and networking events at the relevant universities.
Dissemination Streams
We have presented information on the data collected from these trials to grower and other stakeholder communities and will continue to do so in the upcoming reporting period, including at least 7 presentations on this project to audiences comprised mostly of citrus industry stakeholders. The goal is to ensure there is interest and awareness of these new HLB-tolerant rootstock hybrids to facilitate rapid adoption in relevant regions upon release. ?
Next Reporting Steps
Collection of field trial data for 2023/2024 season is ongoing as anticipated and will continue for the duration of the project. Hybrids will be continually evaluated as data becomes available to identify top performing rootstock hybrids with enhanced tolerance to HLB. Testing of released rootstocks from Bowman's USDA-ARS program will continue in CA to evaluate their response to other soileborne pathogens. Whole-genome genetic variation data will be integrated with phenotypic data to facilitate genome-wide dissection of genetic control of tree performance and disease progression. New selections will continue to be submitted to clean budwood programs in Florida and California in anticipation of future release. All PIs will continue to disseminate research results through extension activities at the local, regional, and national level. <br><br>
<br>What was accomplished under these goals? Objective 1: Data on field tree performance was collected during 2022-23 from 14 established replicated trials in Florida with sweet orange scion and containing about 350 different rootstocks. Data collected from trials included tree survival, tree size, canopy health, fruit yield, fruit quality, and CLas titer in leaf tissue. Tree care in the trials was continued using methods as is standard in commercial production in Florida. Comprehensive datasets through 2023 are being assembled for all trials to use for statistical and genetic analysis. Based on phenotypic and genetic analysis of the first set of 46 hybrids (doi: 10.3389/fpls.2023.1061663), as well as performance in additional trials, two selections were made in 2023 and are pending release by the USDA-ARS. Material for these hybrids as well as at least 30 other potentially promising hybrids is available as clean budwood from the FDACS program. Hybrid rootstocks from the USDA-ARS in Florida that are tolerant to HLB were also screened for their tolerance/resistance against four major California citrus root pathogens including Phytophthora nicotiana, Phytophthora citrophthora, Fusarium solani and the nematode Tylenchulus semipenetrans. These Florida citrus hybrid rootstocks included US-802, US-812, US-897, US-942, US-1283, US-1284, US-1516. Levels of resistance of these rootstocks were compared to the industry standard rootstocks Sour orange, Swingle citrumelo, Ridge and Cleopatra mandarin. The seeds of all citrus rootstocks were grown to seedlings under controlled conditions in growth chamber and challenged with the soilborne pathogens for 6 months (Aphis Permit# P526P-22-06877). Rootstock resistance was assessed as a measure of plant health phenotype (e.g., biomass, disease severity ratings) and abundance of pathogens. Fungi were quantified by qPCR following DNA extraction from roots. Nematodes were quantified by counting population from roots and soils. Sample processing and data analyses are currently being finalized. Resistance to these rootstocks to soilborne pathogens was also assessed by collecting root and soil samples from field trials in Florida. Nematode count from root and soil samples was done at University of Florida following sampling. Root and soil samples were also frozen and shipped to UC Riverside (Aphis Permit# 525-23-143-94712). Incidence and severity of Phytophthora and Fusarium pathogens will be evaluated by qPCR from root samples. A greenhouse study of salinity tolerance has also been initiated including the released rootstocks described. Objective 2: The genetic composition of the 350 rootstock hybrids combined with measurements of tree performance and fruit quality will enable genome-wide association mapping to dissect the genetic basis of traits associated with tolerance to HLB in the field. To initiate work on this objective and prepare for DNA extraction and sequencing, source plant material was identified for the 350 hybrids and parents and shipped from Ft. Pierce, Florida to Riverside, CA (APHIS permit #: P526P-22-03899). DNA extraction and sequencing for both parents and hybrids has been completed. Parents were sequenced to an average depth of 56X coverage and hybrids to a lower depth of ~15X coverage. SNP calling for parents has been completed with over 10 million genetic variants detected. SNP calling of hybrids is ongoing and upon completion will be integrated with phenotypic data to identify alleles associated with HLB tolerance across environments. Deng has tested the TELL-seq sequencing technology on two citrus rootstock cultivars and begun testing single marker assay technologies for eventual development of markers linked to HLB tolerance, including characterization of genetic variation in accessions of interest, including Poncirus trifoliata which is the most important source of HLB tolerance in citrus rootstocks. To identify sequence polymorphism and develop specific molecular markers, we re-sequenced the genomes of four additional accessions of P. trifoliata (Flying Dragon, Little Leaf, Pomeroy, and Rubideau) and its only related evergreen species (P. polyandra) and the genomes of seven citrus rootstock cultivars (US-802, US-812, US-897, US-942, Carrizo, Swingle, and X639) and three of their parents in the genus Citrus (Citrus grandis 'Siamese', C. paradisi 'Duncan' grapefruit, and C. reticulata 'Cleopatra'). Some of these varieties include parents of the 350 hybrids that form the basis of this proposal. <br><br><b>Publications</b><br>
Target Audience
During this reporting period we have interacted with citrus industry stakeholders through regional, state in-person events and seminars. We are particularly focused on raising awareness of the ongoing research supported through this proposal to growers interested in the adoption of new HLB-tolerant rootstocks, both in CA and FL. Seymour presented information on new HLB tolerant rootstocks at the California Citrus Conference in October, 2021 and at UCR Citrus Day, an field-day at UCR for growers and other stakeholders. These presentations highlighted the future goal and timeline for future availability of HLB-tolerant rootstocks in both regions.
Changes / Problems
There are no major changes in the workplan. Research funds from this proposal were not available to support the collection of field data until January 2022 and spending reflects this challenge.
Training & Professional Development
Two postdoctoral research associates were trained to analyze citrus genome sequencing data. Graduate students and postdoctoral researchers receive mentorship in research and also career advancement. This occurs through training focused one-on-one and group meetings, encouragement to establish an individualized development plan to identify and accomplish long-term career goals, and access to career and networking events at the relevant universities. ?
Dissemination Streams
We have presented information on the data collected from these trials to grower and other stakeholder communities and will continue to do so in the upcoming reporting period, including a presentation by Bowman on this topic in October 2022. The goal is to ensure there is interest and awareness of these new HLB-tolerant rootstock hybrids to facilitate rapid adoption in relevant regions upon release. ?
Next Reporting Steps
Collection of field trial data for 2022/2023 season is ongoing as anticipated and will continue for the duration of the project. Hybrids will be continually evaluated as data becomes available to identify top performing rootstock hybrids with enhanced tolerance to HLB. Permits have been obtained to move seed and leaf material from Florida to California for greenhouse studies at UCR to start assessing under controlled conditions resistance levels of citrus hybrid rootstocks to Phytophthora and nematodes. Rolshausen lab will visit Florida to begin assessing under field conditions resistance levels of citrus hybrid rootstocks to Phytophthora and nematodes. Whole-genome sequence data will be produced and genetic variants identified in both parents and hybrids and will facilitate genome-wide dissection of genetic control of tree performance and disease progression. New selections will continue to be submitted to clean budwood programs in Florida and California in anticipation of future release. All PIs will continue to disseminate research results through extension activities at the local, regional, and national level. <br><br>
<br>What was accomplished under these goals? Objective 1: Field performance data was collected from October 2021 to April 2022 from the 16 sweet orange rootstock field trials to be used for the ECDRE project. Data collected from the trials included tree survival, tree size, canopy health, amount of fruit crop, and several fruit quality factors that are important for sweet orange. Some data has been collected monthly since October 2021, but research funds from this proposal were not available to support these activities until January 2022, despite the award date in September 2021. Nine rootstock hybrids with excellent field performance in early planted trials associated with this project have been submitted to the California Citrus Clonal Protection program for introduction into California, as planned. Material for these hybrids is available as clean budwood from the FDACS program. Three of the nine have been released by the USDA already and would be freely available in CA once they have completed quarantine and clean-up. To initiate testing of soilborne pathogens in the greenhouse in California Rolshausen has an APHIS permit (#P526-211124-004) to move citrus seeds from the USDA-ARS in Florida (Dr Bowman) to UC Riverside (Dr. Rolshausen). A manuscript summarizing the performance of 46 rootstocks in the first of the 16 field trials planted is near submission. The phenotypic data presented in this manuscript forms the basis of the first selections to be made from this material with tentative release in early 2023. Objective 2: The genetic composition of the 350 rootstock hybrids combined with measurements of tree performance and fruit quality will enable genome-wide association mapping to dissect the genetic basis of traits associated with tolerance to HLB in the field. To initiate work on this objective and prepare for DNA extraction and sequencing, source plant material was identified for the 350 hybrids and parents and shipped from Ft. Pierce, Florida to Riverside, CA (APHIS permit #: P526P-22-03899). DNA extraction and sequencing is in progress. Initial estimates of heritability were obtained for approximately 30 traits related to tree performance and disease progression using pedigree-based analysis of the first trial of 46 new hybrids. The heritability for traits related to overall yield and tree size (a proxy for health) was substantial (>0.4), indicating there is rootstock-mediated genetic control of HLB-tolerance in the common scion. We will work to dissect the specific variants underlying these differences with the whole-genome sequence data generated for this project. Deng has tested the TELL-seq sequencing technology on two citrus rootstock cultivars and begun testing single marker assay technologies for eventual development of markers linked to HLB tolerance. <br><br><b>Publications</b><br>