Source: UNIVERSITY OF FLORIDA submitted to NRP
AN INTEGRATED APPROACH TO THE ACCELERATED DEVELOPMENT OF ROOTSTOCKS THAT IMPART HLB TOLERANCE TO TREES GRAFTED WITH COMMERCIAL SCIONS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
OTHER GRANTS
Reporting Frequency
Annual
Accession No.
1012148
Grant No.
2017-70016-26328
Cumulative Award Amt.
$4,714,286.00
Proposal No.
2016-10982
Multistate No.
(N/A)
Project Start Date
Mar 15, 2017
Project End Date
Mar 14, 2023
Grant Year
2017
Program Code
[CDRE]- Citrus Disease Research and Extension Program
Recipient Organization
UNIVERSITY OF FLORIDA
G022 MCCARTY HALL
GAINESVILLE,FL 32611
Performing Department
Horticultural Sci
Non Technical Summary
The goal of this project is to develop and deploy new HLB tolerant or resistant rootstocks to ensure the future vitality of the US citrus industry. This will be accomplished through partnership of two long-term, successful comprehensive citrus rootstock breeding programs, at the UF-CREC and the USDA-ARS-USHRL, in combination with a team of accomplished specialists to identify genetic, transcript, metabolic and physiological differences between HLB-tolerant and -susceptible rootstocks to uncover tolerance mechanisms and to develop efficient selectable markers that will facilitate the breeding process. The project will utilize extensive collections and existing field trials with novel and diverse citrus germplasm planted throughout Florida that have seen increasingly intense pressure from HLB, and thus have undergone a natural screening process. The germplasm resources and the team's collective experience in its evaluation provide a very solid basis for advanced breeding, directly targeting the industry's need for HLB tolerant trees through rootstock effects on scion performance. New rootstock candidates will be created using complementary HLB-tolerant parents already shown to transmit this characteristic to offspring. Candidate rootstocks will be identified in greenhouse and field studies, and then planted in replicated field trials for evaluation of tree survival and overall performance, fruit yield, and quality. This information will be made widely available through interaction with industry partners and project outreach, to support HLB-tolerant rootstock selection decisions for new plantings. Finally, candidates selected for release will be channeled through state and national agencies responsible for certified nursery production programs, and extensively propagated to ensure wide availability.
Animal Health Component
60%
Research Effort Categories
Basic
30%
Applied
60%
Developmental
10%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
20109991081100%
Knowledge Area
201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
0999 - Citrus, general/other;

Field Of Science
1081 - Breeding;
Goals / Objectives
The primary goal of this project is to develop and release rootstocks that impart adequate HLB tolerance/resistance to grafted commercial scions as necessary to provide commercially trees that can remain healthy and productive in the face of this devestating disease.The 2nd primary objective is to determine and better understand the mechanisms underlying any developed tolerance/resistance.The 3rd goal is to use the knowledge gained in the objectives above to develop molecular markers for selection of HLB tolerant individuals that will facilitate future rootstock breeding.The ultimate goal is to save our national citrus industry.
Project Methods
Objective 1. Creation of Hybrids: Rootstock breeding will be conducted at both the diploid and tetraploid levels by standard methods for cross hybridization within citrus. Each year, information learned during the previous year from field trials, along with molecular genetics, metabolomics and physiology studies will be used to refine parentage of the new hybrids created. Objective 2. Rootstock Screening (testing and selection): Integration of the different approaches being employed by the USDA and UF/CREC rootstock breeding programs offers a unique opportunity for synergism. We will conduct reciprocal testing of the best available materials (using rooted cutting populations) from the two groups using these two approaches. Correlation of expressed traits with the high performance of HLB-challenged hybrids would validate both approaches. Objective 3. Tolerance/Resistance Characterization: During the first two years, there will be extensive focus on the top rootstock candidates showing the greatest level of HLB tolerance at the time of project initiation. Once the best candidates are chosen, small RNA, transcriptome, and metabolite analyses (3a-c) will be conducted to select tolerant phenotypesfor the meta-analysis and marker development. Objective 4. Establishment and monitoring of field trials. Beginning in year 3 of this project, 3 new field trials will be planted each year, using the best candidate rootstocks selected by greenhouse/field performance in combination with the results of all the molecular, metabolomics and physiological testing associated with Objective 3. The three trials will include one in each of the major citrus production regions in Florida (Indian River, Central Ridge, and Gulf Coast). Trials will be primarily conducted cooperatively on grower farms, with the necessary material transfer agreements, and the details of the trial negotiated well in advance of field planting.Each trial will contain 12-20 selected new hybrid rootstocks and four to six standard commercial rootstocks. Experimental design of field trials will follow standard practice, with randomized complete block design and seven replicates of three trees for each rootstock. Trees will be managed following conventional practice, according the methods commonly used by the grower-cooperator. Typically, trials will be irrigated by micro sprinklers three times a week in the absence of adequate rain fall. Fertilizer and pesticides will be applied seasonally or as needed, and will include enhanced nutritional applications and aggressive psyllid control as currently practiced in most HLB-affected commercial citrus groves in Florida. Evaluation of rootstock field performance. Tree size in trials will be assessed annually by measures of canopy height and width. Tree health in trials will be assessed annually by visual ratings of canopy health and HLB symptoms. Periodic testing of trees for Las infection by PCR will be used to assess rate of Las spread and levels of infection within each trial according to methods described below. Once cropping begins, yields will be measured in standard 90 pound (40.8 kg) boxes at the time of commercial harvest. Quality of fruit will be determined on fruit samples taken at harvest and consisting of 50 randomly collected fruit per rootstock replication. Fruit quality parameters measured will vary depending upon the scion used in the trial, but typically will include fruit size, percent juice, total soluble solids (TSS), total acids, TSS:acid ratio, and juice color. Monitoring disease development (Cano, UF/IRREC; Albrecht, UF/SWFREC). Early detection methods using 16S rDNA-based real time PCR assays with specific primers can now be routinely used for detecting and monitoring CLas populations in both the scion (via leaf samples) and roots (via root samples including fibrous roots) from selected candidate trees in field trials (Li et al., 2006, Rouse et al., 2010 and Stansly et al., 2014; Bowman et al., 2016). Samples will be processed using the protocol as described in and real-time polymerase chain reaction (PCR) as described by Li et al. (2006) with a few modifications. Ct values will be considered positive if 32 or below, negative if 36 or higher, and inconclusive if between these two values as reported by Li et al. (2006) and Rouse et al. (2010). The positive control will be DNA from previously tested HLB positive citrus rootstocks and negative controls will be obtained from citrus grown under psyllid-free screen-house conditions at USDA Fort Pierce. Visual rating of trees: In addition to the proposed molecular diagnosis, we will monitor foliar disease symptoms through visual disease index ratings based on the CRDF-recommended method.Objective 5. Release of HLB tolerant rootstocks. New rootstocks selected for inclusion in each annual group of field trials will be provided to the Florida Department of Agriculture and Consumer Services, Bureau of Citrus Budwood Registration for development of a certified clean budwood source, suitable for commercial distribution and propagation. Trees will also be planted into the field sites on the USHRL and UF/CREC research farms to develop seed sources for future seed propagation. It is anticipated that one or more new rootstock selections will demonstrate superior HLB tolerance and field performance by December 2019, and will be proposed for official release, according to each institution's policies. New rootstocks from this project will be provided to the USDA Citrus Germplasm Repository in Riverside, California for certification and distribution to other States. This project is proposed as a long-term project and will request an additional five years of funding to leverage the gains in the second five years. It can be expected that knowledge gained during the first five years of the project will enable the project to create and select better rootstocks with a higher level of HLB-tolerance during the second five years.

Progress 03/15/17 to 03/14/23

Outputs
Target Audience:Citrus growers, packers and processors across the United States, impacted by HLB. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Post-docs, students and other project personnel attended and madepresentations at local growers' and national society meetings. How have the results been disseminated to communities of interest?Data generated by this projecthas been communicated through oral and poster presentations (trade shows including the annual Expo and Florida Citrus Show; and national scientific meetings), publication of papers and articles, and direct communications. The UF-CREC and USDA data websites continue to be akey delivery method of relevant information to citrus growers: UF: https://citrusresearch.ifas.ufl.edu/rootstock-field-data/; USDA: https://citrusrootstocks.org What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Objective 1. 900+genetically diverse hybrid rootstocks were selected from complementary combinations of tolerant parents. Rooted cuttings of these hybrids are being used to establish new field trials.Some hybrids are from two parents with excellent rootstock pedigree, and others from crosses using new HLB-resistant genetics. A putative deletion mutant of x639 rootstock continues to show excellent HLB tolerance in grafted scions that maintain high CLas titers, suggesting a suppression of the auto-immune response.(Bowman, Grosser & Gmitter). ?Objective 2. Replicated greenhouse testing of selected rootstocks with HLB was conducted (USDA-ARS)and used to evaluatetolerance and guide continuing breeding (Bowman and Albrech). Robust high-throughput 'gauntlet' screening of rootstock hybrids continued (UF-CREC). Leaves of 600+ individual rootstock hybrids (with HLB+ Valencia scion) were screened by PCR, and root PCR screening was expanded to include 150+ individuals. Several hybrid rootstocks repeated (2 consecutive years) for no replication of CLas in the roots, and a few also showed some ability to suppress CLas replication in the grafted scion (Cano, Grosser). A new ECDRE grant was funded to study these effects on long-term commercial tree performance. Objective 3. The influence of rootstocks on gene expression, metabolism, and physiological conditions were evaluated with different scions and rootstocks, and as affected by HLB. Patterns of gene expression and metabolism associated with HLB tolerance and superior health were identified. (Albrecht and Bowman) Multiple Omics Platforms: we mainly focused onhow rootstocks would affect the HLB tolerance from the metabolite level and how interaction between rootstocks and scions would affectfruit quality. Firstly, we developed a flavoromics platform to identify effects of scion/rootstock on fruit quality. Secondly,we developed an untargeted and targeted metabolomics combining with machine learning to understand the key metabolite changes in the orange juice.We developed an AI driven methodology to collect data online to analyze consumer preference to provide information for breeding consumer liked HLB-tolerant cultivars. (Wang and Niu) Root Metabolomics: Samples for metabolomics (including root samples from field trials) were sent to the West Coast Metabolomics Center (WCMC) at UC Davis, CA.,results pending. (Albrecht). We used iTRAQ proteomic profiling of sweet orange juice produced from fruit on trees growing on an HLB-sensitive rootstock (Swingle citrumelo) and an experimental HLB-tolerant rootstock ('46x20-04-48'), to identify differentially expressed proteins in healthy or HLB-symptomatic fruit coming from the sensitive or tolerant rootstocks. Many defense-associated proteins were down-regulated in the symptomatic fruit on 'Swingle' rootstock that were seldom detected in the symptomatic fruit on the '46x20-04-48' rootstock, especially the proteins involved in the jasmonate biosynthesis and signaling, protein hydrolysis, and vesicle trafficking pathways. This suggests these pathways are related to CLas sensitivity. Certain transcription factors also were negatively regulated in symptomatic fruit. It is noteworthy that genetic differences between the rootstocks tested can interact differently with a common scion to enhance scion tolerance to CLas. (Gmitter & Grosser) Seedlings of two citrus rootstocks (HLB-sensitive Swingle citrumelo and an HLB-tolerant tetraploid) and Valencia orange grafted on the two rootstocks were used to investigate scion-rootstock interactions. Valencia grafted on the candidate rootstock showed strong tolerance to HLB, while the Valencia/Swingle plants were HLB-sensitive with high titer and obvious decline. Analysis of differentially expressed small RNAs from phloem tissues was conducted by comparison of their expression levels between the infected and non-infected plants. Three novel miRNAs and their 5 target genes were selected to investigate the expression profiles. Expressions of the miRNAs were lower in the tolerant than the sensitive combination demonstrating that miRNA expression in the scion was affected by rootstock. The expression of Cs5g06500, a sieve element occlusion (SEO) gene, was up-regulated in the leaf midribs of HLB tolerant Swingle seedlings and the tolerant Valencia/candidate combination, suggesting that SEO genes may potentially affect sieve pore plugging after HLB infection. This work revealed that interactions between scion and rootstock could affect the tolerance of the plant to HLB via small RNA-regulated gene expression. (Gmitter & Grosser) An integrative analysis of microRNA and mRNA expression profiles in three grafted combinations grown in the field: HLB-sensitive sweet orange (Valquarius) grafted onto an HLB-sensitive rootstock (B) and two HLB-tolerant rootstocks (F and O). Through DEG and WGCNA analysis, three pairs of negatively expressed DE miRNAs/mRNAs were identified that were co-expressed in scion leaves on F and one pair that was co-expressed in O. We also found that csi-miRN19, a novel miRNA, was upregulated in both tolerant rootstocks and two downregulated mRNAs, DWSO3A02668.to1 (transcription elongation factor) and MSTRG.12612.9 (translation initiation factor). These two downregulated mRNAs reprogrammed the auxin biosynthesis and signaling pathway in the scion grafted onto the tolerant rootstocks, leading to more efficient defense responses and cell wall strengthening, resulting in greater disease tolerance. (Gmitter, Wang & Grosser) ?Rootstocks/Nutrient Management:A100% hydroponic greenhouse study was conducted with six rootstocks with a range of tolerance to HLB. Several physiological and molecular tools were applied to evaluate the rootstocks for their nutrient uptake potential. A+Volk × O-19 (HLB-tolerant) rootstock had greater nutrient uptake efficiency, whereas US-896 (HLB-susceptible) had lesser nutrient uptake efficiency. Swingle, one of the most popular pre-HLB rootstocks, had poor zinc uptake and the least expression ofZINC TRANSPORTER, suggesting that zinc applications should be emphasized in Swingle plantings. US-896 rootstock expressed the least level of nutrient transporter genes, such asIRON TRANSPORTER. UFR-4 (a good performer under HLB conditions) had a large root biomass, but the uptake efficiency for nutrients was poor, suggesting that the nutrient uptake potential is a complex process that is not solely dependent on root biomass. (Vashisth) Objective 4. Eighteen new replicated rootstock field trials were established under the USDA-ARS component of the grant between 2018-22, containing about 300 different rootstocks and 4400 trees. This included eight trials with sweet orange scion, three trials with lemon scion, two trials with grapefruit scion, and two trials with mandarin scion (Bowman). Field performance data was collected each season under the USDA-ARS component of the grant from 34 replicated rootstock field trials, including about 9000 total replications. Information collected included tree survival, tree size, canopy health, fruit yield and fruit quality (Bowman). Nine HLB-tolerant gauntlet rootstocks are included in a replicated trial with multiple scions to be planted this summer (DeLuca Trial/IFAS ). (Grosser). Three new hybrid citrus rootstocks were released in 2018 under the USDA-ARS component of the grant: US SuperSour 1, US SuperSour 2, and US SuperSour 3. FDACS data indicates that the three new USDA-ARS HLB-tolerant rootstocks were used for propagation of 65,600 trees in Florida from 2019-22 (Bowman). Three superior HLB-tolerant gauntlet rootstock selections and two promising deletion mutant rootstock candidates were sent to CCPP for introduction into CA (Grosser). Twenty-two (21 superior gauntlet selections and the x639 mutant) were introduced for TC micropropagation at Agromillora, FL., to be included in new CRAFT projects.(Grosser & Gmitter).

Publications

  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Albrecht, U., and Bowman, K. D. (2019). Reciprocal influence of rootstock and scion citrus cultivars challenged with Ca. Liberibacter asiaticus. Sci. Hortic. 254: 133144. https://doi.org/10.1016/j.scienta.2019.05.010
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Huang, L., Grosser, J., Gmitter Jr., F.G., Sims, C.A. and Wang, Y. Effects of scion/rootstock combination on flavor quality of orange juice from Huanglongbing (HLB)-affected trees: a two-year study of the targeted metabolomics. Journal of Agricultural and Food Chemistry, 2020, 68: 3286-3296. https://doi.org/10.1021/acs.jafc.9b07934.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Albrecht, U., Tripathi, I., Kim, H., Bowman, K. D. (2019). Rootstock effects on metabolite composition in leaves and roots of young navel orange (Citrus sinensis L. Osbeck) and pummelo (C. grandis L. Osbeck) trees. Trees 33:243265. https://doi.org/10.1007/s00468-018-1773-1
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Reuss, L., Feng, S., Hung, W-L., Yu, Q., Gmitter Jr., F.G., and Wang, Y. Analysis of flavor and other metabolites in lemon juice (Citrus limon) from Huanglongbing-affected trees grafted on different rootstocks. Journal of Food and Drug Analysis, 2020, 28:2,7. https://doi.org/10.38212/2224-6614.1060.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Yao, L., Yu, Q., Huang, M., Song, Z., Grosser, J., Chen, S., Wang, Y., Gmitter Jr., F.G. Comparative iTRAQ proteomic profiling of sweet orange fruit on sensitive and tolerant rootstocks infected by Candidatus Liberibacter asiaticus. 2020, PLoS ONE 15(2): e0228876. https://doi.org/10.1371/journal.pone.0228876
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Ghimire, L., Kadyampakeni, D., & Vashisth, T. (2020). Effect of Irrigation Water pH on the Performance of Healthy and Huanglongbing-affected Citrus, Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci., 145(5), 318-327. Retrieved Apr 27, 2023, from https://doi.org/10.21273/JASHS04925-20
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Bowman, K. D., and Albrecht, U. (2020). Rootstock influences on health and growth following Candidatus Liberibacter asiaticus infection in young sweet orange trees. Agronomy 10:1907. https://doi: 10.3390/agronomy10121907
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Bowman, K. D., McCollum, G., and Albrecht, U. (2021). SuperSour: A New Strategy for Breeding Superior Citrus Rootstocks. Front. Plant Sci. 12:741009. https://doi: 10.3389/ fpls.2021.741009
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Aparicio-Duran, L., Gmitter Jr., F.G., Arjona-Lopez, J.M., Calero-Velazquez, R., Hervalejo, A., and Arenas-Arenas, F.J. Water-stress influences on three new promising HLB-tolerant citrus rootstocks. Horticulturae, 2021, 7(10), 336; https://doi.org/10.3390/horticulturae7100336.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Aparicio-Dur�n, L., Gmitter, F. G., Arjona-L�pez, J. M., Grosser, J. W., Calero-Vel�zquez, R., Hervalejo, �., and Arenas-Arenas, F. J. Evaluation of three new citrus rootstocks under boron toxicity conditions. Agronomy, 2021, 11 (12), 2490. https://doi.org/10.3390/agronomy11122490
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Kunwar, S., Grosser, J., Gmitter Jr., F.G., Castle, W.S., and Albrecht, U. Field performance of Hamlin orange trees grown on various rootstocks in Huanglongbing-endemic conditions. HortScience, 2021, 56:244-253. https://doi.org/10.21273/HORTSCI15550-20.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Mahmoud, L.M., Huyck, P.J., Vincent, C.I., Gmitter, F.G., Grosser, J.W., Dutt, M., 2021. Physiological Responses and Gene Expression Patterns in Open-Pollinated Seedlings of a Pummelo-Mandarin Hybrid Rootstock Exposed to Salt Stress and Huanglongbing. Plants 10, 1439.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Arjona-L�pez, J.M., Gmitter Jr., F.G., Romero?Rodr�guez, E., Grosser, J.W., Hervalejo, A., L�pez?Herrera, C.J., and Arenas-Arenas, F.J. Susceptibility of novel promising citrus rootstocks to white root rot. Plants, 2022, 11(23): 3338. doi.org/10.3390/plants11233388.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Zapien-Macias, J.M., Ferrarezi, R.S., Spyke, P.D., Castle, W.S., Gmitter Jr., F.G., Grosser, J.W., and Rossi, L. Early performance of recently released rootstocks with grapefruit, navel orange, and mandarin scions under endemic Huanglongbing conditions in Florida. Horticulturae, 2022, 8, 1027. https://doi.org/10.3390/ horticulturae8111027.
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Bodaghi, S., Meyering, B., Bowman, K. D., Albrecht, U. (2022a). Different Sweet OrangeRootstock Combinations Infected by Candidatus Liberibacter asiaticus under Greenhouse Conditions: Effects on the Scion. HortScience 57(1):144-153. https://doi.org/10.21273/HORTSCI16205-21
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Bodaghi, S., Pugina, G., Meyering, B., Bowman, K. D., Albrecht, U. (2022b). Different Sweet Orange?Rootstock Combinations Infected by Candidatus Liberibacter asiaticus under Greenhouse Conditions: Effects on the Roots. HortScience 57(1):56-64. https://doi.org/10.21273/HORTSCI16206-21
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Arjona-L�pez, J.M., Aparicio-Dur�n, L., Gmitter Jr., F.G., Romero-Rodr�guez, E., Grosser, J.W., Hervalejo, A. and Arenas-Arenas, F.J. Physiological influence of water stress conditions on novel HLB-tolerant citrus rootstocks. Agronomy 2023, 13(1), 63; https://doi.org/10.3390/agronomy13010063.
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Kunwar, S., Meyering, B., Grosser, J., Gmitter Jr., F.G., Castle, W.S., and Albrecht, U. Field performance of Valencia orange trees on diploid and tetraploid rootstocks in different huanglongbing-endemic growing environments. Scientia Horticulturae, 2023, 309, https://doi.org/10.1016/j.scienta.2022.111635.
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Liu, X., Gmitter, F.G., Grosser, J.W., and Wang, Y. Effects of rootstocks on the flavor quality of huanglongbing-affected sweet orange juices using targeted flavoromics strategy. RSC Advances, 2023, 13(8):5590-9. doi: 10.1039/D2RA08182B.
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Liu, X., Wang, Z., Gmitter, F.G., Jr., Grosser, J.W., and Wang, Y. Effects of different rootstocks on the metabolites of Huanglongbing-affected sweet orange juices using a novel combined strategy of untargeted metabolomics and machine learning. Journal of Agricultural and Food Chemistry, 2023, 71(2):1246-57. doi: 10.1021/acs.jafc.2c07456.
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Ghimire, L., Grosser, J., & Vashisth, T. (2023). Differences in Nutrient Uptake Can Influence the Performance of Citrus Rootstocks under Huanglongbing Conditions, HortScience, 58(1), 40-46. 2023, from https://doi.org/10.21273/HORTSCI16753-22
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Bowman, K.D., McCollum, G., and Seymour, D. K. (2023). Genetic modulation of Valencia sweet orange field performance by 50 rootstocks under huanglongbing -endemic conditions. Front. Plant Sci. 14:1061663. https://doi: 10.3389/fpls.2023.1061663
  • Type: Journal Articles Status: Under Review Year Published: 2023 Citation: Zhu, S., Zhao, C., Yu, Q., Grosser, J.W., Gmitter Jr., F.G. Scion-rootstock interaction via novel small RNAs in phloem tissues affect citrus Huanglongbing tolerance.
  • Type: Journal Articles Status: Under Review Year Published: 2023 Citation: Wei, X., Zhuo, X., Yu, Q., Grosser, J. W., Gmitter Jr., F.G. Altered auxin biosynthesis and signaling pathway by a novel microRNA played a central role in the HLB tolerance in different citrus scion and rootstock combinations.
  • Type: Journal Articles Status: Other Year Published: 2023 Citation: Wang, Z., Wei, Xu., Gmitter, F., Grosser, J., Wang, Y. Mahcine learning-enhance multi-omics toward understanding tolerance mechanisms of particular citrus rootstocks to Huanglongbing


Progress 03/15/21 to 03/14/22

Outputs
Target Audience:Citrus growers affected by HLB Changes/Problems:A one-year NCE was obtained to deal with Covid-related delays and personnel issues. What opportunities for training and professional development has the project provided?Post-docs, students and other project personnel attend and make presentations at local growers' and national society meetings. How have the results been disseminated to communities of interest?So far, data has been communicated through oral and poster presentations (trade shows and national meetings), publication of papers and articles, and direct communications. The UF-CREC and USDA data websites are a key delivery method: UF: https://citrusresearch.ifas.ufl.edu/rootstock-field-data/; USDA: https://citrusrootstocks.org/ What do you plan to do during the next reporting period to accomplish the goals?Efforts will be concentrated on establishing advanced trials of the most HLB-tolerant rootstock candidates, and delivering the best rootstock candidates to clientele. We have also established a conduit with CCPP in CA to move promising new rootstock germplasm as efficiently as possible.

Impacts
What was accomplished under these goals? Objective 1. UF and USDA programs produced new genetically diverse new rootstock hybrids (Bowman, Grosser, Gmitter) ; these include the first successful crosses of US-942 and US-897 with salt-tolerant pummelo/mandarin hybrids (Grosser). Objective 2. Robust high-throughput 'gauntlet' screening of rootstock hybrids continued. Leaves of 593 individual rootstock hybrids were screened by PCR, and root PCR screening was expanded to include 131 individuals. Several hybrids had zero replication of CLas in the roots, and the S11x50-7-12-6 hybrid roostock showed no replication in the roots and suppressed CLas replication in the leaves two consecutive years, suggesting possible resistance(Cano, Grosser). Objective 3. For these studies, Valquarius sweet orange on 6different rootstocks have been evaluated, including two HLB-tolerant rootstocks Orange 1804 and FG1733, two commercial rootstocks (intermediate in response to HLB) Swingle and Volk, and two susceptible rootstocks Blue 3 and Changsha+Benton (four replicates). Effects of rootstocks on the flavor quality of orange juice using targeted flavoromics: Targeted flavoromics was implemented by the combined applications of GC-MS and LC-MS instrumental analysis and sensory evaluation. The sensory overall liking and flavor liking ratings of six different orange juices were ranked in the following manner: CH > 1804 > SW > Blue > FG > Volk. Orange juice from rootstock CH showed the best flavor quality compared with the other samples. In addition, our results indicated that raffinose, xylose, rhamnose, glucose, sorbitol, and myo-inositol showed strong contribution to sweetness, and alanine, glutamic acid, proline, arginine, serine, asparagine, and aspartic acid were also responsible for positive flavor quality. These results suggested that rootstock CH could enhance accumulation of some specific amino acids, sugars, and flavonoids, resulting in better sensory quality. These results indicate that fruit quality in trees with HLB is under genetic control independentfrom tree growth and health when trees are grown with optimized nutrition (Wang, Niu, Vashisth). Effects of rootstocks on the juice metabolites: Metabolites including amino acids, amines, flavonoids, coumarins, fatty acids, and glycosides were identified as the differential markers. Interestingly, Notably, the presence of key amino acids and amines especially polyamines indicated that different rootstocks migh affect metabolites by regulating the defense response against HLBassociated with amino acid or nitrogen metabolism. The possible metabolic pathways were studied (Wang, Niu). Omics to understand the tolerance of different rootstocks: Metabolites from the 24 leaf samples, root and soil samples have been analyzed using the untargeted metabolomics platform. Biomarkers have been identified using different statistic regressions, and these biomarkers are now in the process of confirmation and quantification. 16S rDNA extraction, followed by V5-V7 amplicon sequencing for leaf and root samples to identify the endogenous bacteria community, and V3-V4 amplicon sequencing for the environmental bacteria community. We are now in the process of integrating the results from the metabolomics and microbiome studies and verifying the differential impact and mechanism of different rootstocks on Huanglongbing tolerance (Wang and Niu). Fine root traits of selected rootstocks were examined and roots extracted roots for metabolomics analysis. We have expanded our studies to investigate some graft incompatibility reactions. Data analysis is underway (Albrecht). MicroRNA Analyses: MicroRNAs (miRNAs) are important gene expression regulators in plants immune system, but information on the function of miRNAs in citrus-HLB is still lacking. In this study, to understand the molecular mechanism of the interaction between citrus host and CLas bacteria, next-generation sequencing was employed to profile mRNAs and miRNAs that are differentially expressed under the infection in four scion/rootstock combinations, including two tolerant rootstocks (FG and 1804), and two sensitive rootstocks (Blue3 and CH). Phenotype observations of the grafting combinations showed that the growth of the two sensitive rootstock grafting combinations was significantly inhibited, though the typical blotchy mottled appearance was found on the leaves of all grafting combinations. Phloem collapse with cell wall distortion and thickening was observed in the leaf and root samples of sensitive grafting combinations, and a significant increase in callose deposition was observed in the leaf samples of the sensitive grafting combinations compared to the tolerant combinations, while they did not exhibit a significant callose accumulation in the root samples. The roots of the sensitive rootstocks had some dieback. Atotal of 80,323 known and 74,829 new transcripts were detected. The grafting combinations with the two tolerant rootstocks (FG & 1804) were set as control for DEG analysis, and eight pairwise comparisons of leaf and root samples were conducted for DEG analysis. Functional analysis of the DEGs by MapMan software showed a large number of bins with downregulated genes related to cell and cell wall metabolism, while many bins with upregulated genes related to signaling and nucleotide metabolism were enriched in leaves of the two sensitive grafting combinations compared to those two tolerant grafting combinations. In root samples of different grafting combinations, several bins with upregulated and downregulated bins related to signaling, hormone and secondary metabolism were enriched in the two sensitive rootstocks compared to those two tolerant rootstocks. The result of miRNA sequencing showed that 25 known and 35 novel miRNAs were obtained, and 45 miRNAs were differentially expressed in the eight pairwise comparison groups. Furthermore, the targets of DEMs were predicted by psRNATarget, a total of 1516 miRNA target genes were identified, of which 97 were found in the identified DEGs. Expression profiles of miRNAs/targets pairs showed several of them had one-to-one corresponding negatively, and they were involved in hormone, defense, and photosynthesis pathways. The corresponding miRNAs of these targets may play important roles in the interaction of citrus with CLas (Gmitter). Objective 4. Twenty two promising new HLB-tolerant rootstock hybrids were introduced into tissue culture for micropropagation at Agromillora Florida, Inc., an international company that has established a citrus propagation facility in Wildwood Fl. They are cooperating with our team on R&D on their own dime. Once a rootstock is successfully established in TC, it is entered into planned advanced trials (Grosser, Gmitter). Propagation of trees for four additional smaller trials is underway with the same rootstock selections. Leverage: Funding for a 10-acre replicated trial featuring (De Luca Preserve Trial) was obtained from UF (Forest Systems Jumpstart Program) to plant 9 new HLB-tolerant rootstocks identified in this project (and UFR-4 as a control), combined with 5 promising new scions with both processing and fresh fruit potential. We expect to plant the trial in December, 2022 (Grosser, Gmitter). Planted three stage-1 rootstock trials (new genetically diverse selections, Bowman, Grosser) and three new stage-2 replicated rootstock field trials including rootstocks which were identified in previous trials as exhibiting HLB-tolerance (Bowman). From previously established trials (more than 40 field trials, some USDA/UF collaborations), we collected information on tree health, tree size, crop yield, fruit quality, and CLas infection and titer(Bowman, Grosser, Gmitter). Objective 5. Submitted 19 new hybrid rootstock selections (12 USDA and 7 UF-CREC) with superior field performance to the FDACS-DPI Parent Tree Program for STG and indexing, making preparations for new release(Bowman, Grosser).

Publications

  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Bowman, KD, G McCollum and U Albrecht. 2021. SuperSour: A new strategy for breeding superior citrus rootstocks. Frontiers in Plant Science. Doi: 10.3389/fpls 2021.741009
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: 3. Mahmoud, L.M., Huyck, P.J., Vincent, C.I., Gmitter, F.G., Grosser, J.W., Dutt, M., 2021. Physiological Responses and Gene Expression Patterns in Open-Pollinated Seedlings of a Pummelo-Mandarin Hybrid Rootstock Exposed to Salt Stress and Huanglongbing. Plants 10, 1439.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: 4. Aparicio-Duran, F.G. Gmitter, J.M. Arjona-Lopez, J.W. Grosser, R. Calero-Velazquez, A. Hervalejo and F.J. Arenas-Arenas. 2021. Evaluation of three new Citrus Rootstocks under Boron Toxicity Conditions. Agronomy 11(12), 2490; https://doi.org/10.3390/agronomy11122490


Progress 03/15/20 to 03/14/21

Outputs
Target Audience:Citrus growers affected by HLB. Changes/Problems:Propagation of promising rootstocks is still a limitation - we are employing tissue culture micropropagation and rooted cutting procedures as appropriate. Broad Covid restrictions have reduced work associated with this grant, especially at the USDA-ARS; however, significant progress was still achieved. What opportunities for training and professional development has the project provided?Post-docs, students and other project personnel attend and make presentations at local growers' and national society meetings - all done via zoom for this period. ? How have the results been disseminated to communities of interest?So far, data has been communicated through oral and poster presentations, publications in refereed journals and trade journals, and direct communications. The mentioned new data websites are a key delivery method. What do you plan to do during the next reporting period to accomplish the goals?Efforts will be concentrated on establishing advanced trials of the most HLB-tolerant rootstock candidates, and delivering the best rootstock candidates to clientele.

Impacts
What was accomplished under these goals? Objective 1. UF (Grosser and Gmitter) and USDA (Bowman) programs continued producing new rootstock hybrids combining superior combinations of mandarin, pummelo and trifoliate orange. Numerous robust seedlings were recovered from crosses with the highly HLB-tolerant SugarBelle. 2021 crosses focused on combining HLB tolerance and ability for precocious bearing of high soluble solids fruit. Objective 2. Multiple rootstock field trials were evaluated to select the most promising rootstock cultivars for metabolomics and root physiological studies. Based on tree health and productivity, a subset was selected for root physiological and metabolomics analyses. Root physiological analyses are in progress, with sample extracts shipped to the West Coast Metabolomics Center for GC-MS TOF metabolite identification (Albrecht). All the fruits used for the sensory study in years 2018 and 2019 were also analyzed using the metabolomics techniques. A PLS-DA model combined with KEGG pathway enrichment analysis was conducted. Rootstocks had an impact on the biosynthetic pathways of secondary metabolites. Sugars and organic acids were not closely correlated with the overall liking and sensory perception of orange juice. Rather, flavonoids, terpenoids and volatile aromas played important roles in improving consumer overall liking (Wang and Niu). Continued evaluation of 'Gauntlet' rootstock candidates: CLas detection in leaves via qPCR: We collected citrus leaves from a total of 629 plants on individual rootstock candidates at USDA Picos Farm. CLas detection from leaf samples were carried using tree foliage was divided into 4 quadrants. 54% of the 629 'gauntlet' rootstock trees tested negative for an active infection. CLas detection in roots: Based on visual scores PCR we selected 85 trees qPCR analysis (in progress). Brix measurement on selected rootstock candidates: To quantify brix, we measured 3 medium size fruits using a refractometer. Four rootstock candidates with high health indices and negative PCR results produced fruit with high brix: A+HBPxCH+50-7-12-11, A+HBPxCH+50-7-12-24, N+HBPxOrange19-12-3 and A+HBPxWhite-1-13-31 (Cano, Grosser). Objective 3. Study 1. Omics to understand the tolerance of different rootstocks Grafted citrus trees (Valquarius sweet orange) of six different rootstocks were selected for Studies 1,2 &3, including two susceptible rootstocks, two commercial rootstocks, and two tolerant experimental rootstocks. For each rootstock, four biological replicates were set up (12-year old trees from same grove). Leaf, fibrous root, and rhizosphere soil samples were collected for metabolite analysis and microorganism amplicon sequencing. Metabolites were extracted from the 24 leaf samples, 24 root samples, and 24 soil samples, followed by the untargeted metabolomics study using UHPLC/Orbitrap-MS profiling loaded with RPLC column and HILIC column, respectively. Biomarkers will be identified using different statistic regressions, and these biomarkers will be used for the furthering confirmation and quantification by targeted metabolomics. 16S rDNA were extracted from those same samples, followed by V5-V7 amplicon sequencing for leaf and root samples to identify the endogenous bacteria community, and V3-V4 amplicon sequencing for soil samples to identify the environmental bacteria community. The different microorganism species will be analyzed. At last, all the results from the metabolomics and microbiome studies will be integrated to explain the differential impact and mechanism of different rootstocks on Huanglongbing tolerance (underway,Yang & Niu). ?Study 2. Transcriptomic and gene expression analyses: Leaf and root samples were collected for transcriptomic and gene expression analyses. In brief, DNA was extracted from 24 leaf and 24 root samples. qPCR was used to detect and confirm CaLas infection in all the samples. RNA-seq and siRNA-seq have been completed, and currently bioinformatic analysis on the raw data has been initiated. Besides the molecular study, plant phenotype data have been collected, including the measurement of trunk diameter, leaf fresh and dry weight, leaf area, leaf photosynthesis, and root microscopy (Gmitter). Study 3. Rootstock/HLB effects on juice quality: For each scion/rootstock combination, fruits were collected and used to prepare juice in replicate. In brief, sensory test was conducted by a panel to evaluate the overall liking and attributes of six different orange juices. The panel consisted of 72 consumers between age of 20 and 70 with a percentage of female (55%) and male (45%). Brix and titratable acidity (TA) of different orange juices was determined. The volatile compounds were extracted using headspace solid-phase microextraction (HS-SPME), and identified and semi-quantified using GC-MS. Furthermore, targeted metabolomics including sugars, sugar alcohols, organic acids, amino acids, flavonoids, coumarins, nucleotides, and plant hormones analysis was conducted on LC-MS to discriminate these six different orange juices. Untargeted metabolomics was also carried out to reveal more differentiation in these samples. Currently, the raw data of metabolite profiles mentioned above has been analyzed (Chemistry - Wang; statistics - Niu; standard juice quality - Grosser). Evaluations of rootstocks selected from the large Central ridge field trial with sweet orange scion were continued to assess horticultural traits and HLB tolerance, and the association with rootstock physiological traits. The rootstocks studied included different genetically diverse hybrids. In addition to assembling important horticultural data (tree size, yield, fruit quality, etc.) leaves and roots were collected at two different time points (seasons) to assess leaf and root CLas titers, fibrous root anatomy, fibrous root respiration, and fibrous root metabolites. These analyses are in progress. Preliminary data revealed significant differences in fibrous root traits among the rootstocks, specifically between those with sour orange-like parentage and those without (Albrecht). Greenhouse studies were initiated to study different types of graft incompatibilities that were observed in some scion?rootstock combinations to identify physiological markers that may be used for identifying incompatibility responses prior to long-term field evaluation (Albrecht, Bowman). Objective 4. Trial data websites are now on-line with extensive information about citrus rootstocks, including results from the field trials included for evaluation under this NIFA grant: USDA Trials (Bowman): https://www.citrusrootstocks.org/ and UF/CREC Trials (Grosser & Gmitter): https://crec.ifas.ufl.edu/citrus-research/rootstock-field-data/ Four new replicated stage 2 rootstock field trials of USDA selections were planted (Bowman). A stage 2 rootstock trial with multiple new red grapefruit scions on UF rootstock selections was planted, and two stage 1 trials of UF selections including some exhibiting HLB tolerance were planted (Grosser). Objective 5. Botanical descriptions were developed for 5 new UF rootstock candidates showing HLB tolerance, as needed for patenting and release, all are in the PTP (FDACS-DPI Parent Tree Program) (Grosser). Two new rootstock candidates were entered into the PTP, including the first rootstock hybrid containing the highly HLB-tolerant 'SugarBelle' as a parent, and another superior 'gauntlet' hybrid made from two salt tolerant pummelo x mandarin hybrids. 12 new USDA hybrid rootstock selections with superior field performance were submitted to the PTP, making preparations for new rootstock cultivar release in 3-4 years (Bowman). USDA greenhouse nursery propagations included 60 new rootstocks for Stage 1 replicated filed trials and 18 identified potentially HLB-tolerant rootstocks for Stage 2 trials (Bowman). UF propagations included 100 + rootstocks for Stage 1 trials and 9 rootstocks for Stage 2 trials (Grosser).

Publications

  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Yao, L., Yu, Q., Huang, M., Song, Z., Grosser, J.W., Chen, S., Wang, Y. and Gmitter, F.G. 2020. Comparative iTRAQ proteomic profiling of sweet orange fruit on sensitive and tolerant rootstocks infected by Candidatus Liberibacter asiaticus. PLoS ONE, doi.org/10.1371/journal.pone.0228876.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Mahmoud LM, Dutt M, Vincent CI & Grosser JW (2020) Salinity-Induced Physiological Responses of Three Putative Salt Tolerant Citrus Rootstocks. Horticulturae 6(4):90
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Kunwar, S, JW Grosser, FG Gmitter, WS Castle and U Albrecht. 2021. Field Performance of Hamlin Orange Trees Grown on Various Rootstocks in Huanglongbing-endemic Conditions. HortScience 56:244-253
  • Type: Other Status: Published Year Published: 2020 Citation: Grosser, J, F Gmitter and K Bowman (2020) New rootstocks in the citrus breeding pipeline. July issue of Citrus Industry Magazine, Vol. 101 No. 7.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Wang, Y, F Gmitter and J Grosser (2020). Rootstock and scion affect orange juice flavor. July issue of Citrus Industry Magazine, Vol. 101 No. 7.
  • Type: Other Status: Published Year Published: 2020 Citation: Ferrarezi, R, WS Castle, KD Bowman, JW Grosser, SH Futch, S Rogers and A Gonzalez (2020). How to use the Citrus Rootstock Selection Guide. July issue of Citrus Industry Magazine, Vol. 101 No. 7.
  • Type: Other Status: Published Year Published: 2020 Citation: Albrecht, A, S Kumar and J Grosser (2020). Rootstock effects on Valencia and Hamlin in large-scale commercial plantings. July issue of Citrus Industry Magazine, Vol. 101 No. 7.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Ghimire, L, D Kadyampakeni and T Vashisth (2020) Effect of irrigation water on the performance of healthy and Huanglongbing-affected Citrus. JASHS 145:318-327.


Progress 03/15/19 to 03/14/20

Outputs
Target Audience:Citrus growers affected by HLB. Changes/Problems:Propagation of promising rootstocks is still a limitation, as seed trees are not available for most new genotypes - we are employing tissue culture micropropagation and rooted cutting procedures as appropriate. What opportunities for training and professional development has the project provided?Post-docs, students and other project personnel attend and make presentations at local growers' and national society meetings. How have the results been disseminated to communities of interest?So far, data has been communicated through oral and poster presentations, publication of papers and articles, and direct communications. The mentioned new data websites are a key delivery method. What do you plan to do during the next reporting period to accomplish the goals?Efforts will be concentrated on establishing advanced trials of the most HLB-tolerant rootstock candidates, and delivering the best rootstock candidates to clientele.

Impacts
What was accomplished under these goals? IMPACT: Progress has been made to develop rootstocks that impart higher levels of HLB tolerance/resistance to grafted commercial scions. This research already is providing growers with better choices for current plantings, and will lead to future choices that will minimize or eliminate HLB as a threat to sustainable and profitable citriculture throughout the US citrus industry. Objective 1. UF (Grosser and Gmitter) and USDA (Bowman) programs produced new rootstock hybrids combining superior combinations of mandarin, pummelo and trifoliate orange. Crosses were conducted to combine SugarBelle with other good widely adapted rootstocks stacked with abiotic stress tolerance genes. Superior open-pollinated zygotic mandarin/pummelo hybrids were identified from existing field trials (Grosser), and zygotic seed was also planted in the soil/Phytophthora screen from selected superior Chinotto sour orange hybrids (Gmitter). Objective 2. A. Multiple rootstock field trials including mature productive sweet orange trees were evaluated to select the most promising rootstock cultivars for metabolomics and root physiological studies. These field trials included a large number of commercial and experimental varieties. Based on tree health and productivity of trees on these rootstocks in different locations that vary in soil type and other environmental factors, a subset was selected for root physiological and metabolomics analyses. Root physiological analyses are in progress. Leaves and roots were collected, processed, and metabolites were extracted. Additional rootstocks from other trials have been identified, but due to the covid-19 imposed halt of UF research activities, the final selection was not completed (Albrecht). B. All the fruits used for the sensory study in years 2018 and 2019 (mentioned below under Objective 4) were also analyzed using the metabolomics techniques. A PLS-DA model combined with KEGG pathway enrichment analysis was conducted. Rootstocks had an impact on the biosynthetic pathways of secondary metabolites. Sugars and organic acids were not closely correlated with the overall liking and sensory perception of orange juice. Rather, flavonoids, terpenoids and volatile aromas played important roles in improving consumer overall liking (Wang and Niu). C. Drs. Grosser and Gmitter continued to feed new rootstock hybrids into the 'gauntlet' screening process, and the cuttings are being challenged in the greenhouse with HLB+ scions. The first 'gauntlet' hybrid made with HLB-tolerant SugarBelle as a parent (LB8-9 X S13-15-16) continued to show exceptional growth with no HLB symptoms, and the Valencia scion ct value went from 24 (December) to 36 in (March), indicating a rootstock effect in suppressing the CLas population. Approximately 150 new rootstock hybrids grafted with CLas+ Valencia were planted in the 'gauntlet' screen at the USDA Picos Farm, including new SugarBelle hybrids from multiple crosses. D. We have identified several 'gauntlet' rootstock trees where both the roots and leaves have Ct values indicating no active infection; suggesting potential resistance coming from the rootstock. This evidence for rootstock-provided resistance is strong, as 6 trees tested negative for CLas 4 consecutive months. Eleven other trees tested negative 3 out of 4 months, and included two hybrids with salt tolerant S10 as the mother (Cano and Grosser). Objective 3. We compared the proteomes of Valencia fruit from trees grown on a tolerant experimental and a commercial sensitive rootstock (1). Many defense-associated proteins were down-regulated in the symptomatic fruit on the sensitive rootstock that were seldom detected in the symptomatic fruit on the tolerant one, especially the proteins involved in the jasmonate biosynthesis, jasmonate signaling, protein hydrolysis, and vesicle trafficking pathways. Therefore, we predict that the down-regulated proteins involved in the jasmonate signaling pathway and vesicle trafficking are likely to be related to citrus sensitivity to the CLas pathogen (Gmitter). E. Proteomic and metabolic analyses using GC-MS were likewise conducted on Valencia fruit from trees grown on a sensitive rootstock, to measure the proteins, sugars, organic acids, amino acids, and volatile terpenoids (2). The results showed that most of the differentially expressed proteins involved in glycolysis, the tricarboxylic acid (TCA) cycle and amino-acid biosynthesis were degraded, and terpenoid metabolism was significantly downregulated in the symptomatic fruit. Our study indicated that off-flavor of symptomatic fruit was associated with a reduction in the levels of terpenoid products, and the downregulation of proteins in glycolysis, the TCA cycle, and the terpenoid biosynthesis pathway (Gmitter). F. Nutrient uptake attributes of potentially HLB-tolerant rootstocks (Vashisth): HLB-tolerant 'gauntlet' rootstock, A+Volk X Orange-19-11-8 rootstock showed high nutrient uptake efficiency whereas US-896 showed least. UFR-4 had a large root biomass, but the uptake efficiency for nutrients was poor, suggesting that the nutrient uptake potential is a complex process and is not solely dependent on root biomass. Differences among the rootstocks in their morphology and density of the roots in the growing media could explain the variations in the nutrient absorption capacity of roots. Objective 4. A. Planted four new replicated rootstock field trials, including rootstocks which were identified in previous trials as exhibiting HLB-tolerance (Bowman). B. Additional propagations of top HLB-tolerant rootstocks and the major citrus rootstocks are being prepared for phase 2 field trials with five different scions and to be planted in 2021. These trials will help identify the best-of-the-best rootstocks for commercial use (Bowman). C. From previously established trials (more than 40 field trials, some USDA/UF collaborations), we collected information on tree health, tree size, crop yield, fruit quality, and CLas infection and titer, as appropriate for the tree age and condition (Bowman, Grosser, Gmitter). D. Continued ongoing evaluation of field performance and propagation information from 26 field trials to identify selections for use in phase 1 and 2 trials. Numerous advanced hybrid rootstock selections appear superior to the standard rootstocks currently heavily used in commercial HLB-endemic environments, and the best of these will be prepared for commercial release in 2021-23 (Bowman). E. A sensory and consumer preference study was conducted in 2019 using the OLL sweet orange/rootstock trial in St. Cloud. Results showed that scion/rootstock combination significantly affected the overall consumer liking of juice from the HLB-affected trees. HLB-tolerant rootstocks showed significant effects on the consumer liking and overall flavor, while scions had significant effects on the freshness and overall orange flavor intensity of the juice (Wang and Niu). F. Replications of trees on 6 rootstocks (two HLB-tolerant; two HLB-susceptible, and two commercial controls) from an established 11-year old field trial were used to initiate studies of the effects of HLB on juice consumer likeness and flavor quality (Wang and Niu), nutrition (Vashisth), metabolomics (Wang and Niu), CLas titers (Grosser) and transcriptomics (Gmitter). Objective 5. A. Botanical descriptions were developed for 4 new UF rootstock candidates as needed for patenting and release; 6 promising candidate rootstocks were entered into tissue culture micropropagation (Grosser). B. Submitted 12 new USDA hybrid rootstock selections with superior field performance to FDACS-DPI for STG and indexing, making preparations for new rootstock cultivar release in 3-4 years (Bowman). C. New websites are now online providing growers data from mentioned rootstock trials: UF: https://citrusresearch.ifas.ufl.edu/rootstock-field-data/; USDA: https://citrusrootstocks.org/

Publications

  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Huang, L, J.W. Grosser, F.G. Gmitter, C.A. Sims and Y. Wang. 2020. Effects of Scion/Rootstock Combination on Flavor Quality of Orange Juice from Huanglongbing (HLB)-Affected Trees: A Two-Year Study of the Targeted Metabolomics. J. Agric. Food Chem. 2020, 68, 10, 3286
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Lixiao Yao, Qibin Yu, Ming Huang, Zhen Song, Jude Grosser, Shanchun Chen, Yu Wang, Frederick G. Gmitter Jr. 2020. Comparative iTRAQ proteomic profiling of sweet orange fruit on sensitive and tolerant rootstocks infected by Candidatus Liberibacter asiaticus. PLoS ONE, doi.org/10.1371/journal.pone.0228876.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Lixiao Yao, Qibin Yu, Ming Huang, Weilun Hung, Jude Grosser, Shanchun Chen, Yu Wang, and Frederick G. Gmitter, Jr. 2019. Proteomic and metabolomic analyses provide insight into the off-flavour of fruits from citrus trees infected with Candidatus Liberibacter asiaticus. Hortic Res. 6: 31. doi: 10.1038/s41438-018-0109-z
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Albrecht, U, I Tripathi, KD Bowman. 2020. Rootstock influences the metabolic response to Candidatus Liberibacter asiaticus in grafted sweet orange trees. Trees 34:405-431.
  • Type: Journal Articles Status: Awaiting Publication Year Published: 2020 Citation: Grosser, Jude, Fred Gmitter and Kim Bowman. 2020. New Rootstocks in the Citrus Breeding Pipeline. Citrus Industry Magazine (in press).


Progress 03/15/18 to 03/14/19

Outputs
Target Audience: Nothing Reported Changes/Problems:We have encountered some difficulty in recovering some elite rootstock selections mother tree material as needed for propagation; more radical techniques to accomplish this are being tried What opportunities for training and professional development has the project provided?Post-docs and other project personnel attended local growers' meetings. In addition, post-docs have been presenting results at meetings. New skills and developments are shared through regular lab meetings with other lab members, including fellow post-docs, visiting scientists and research scholars, graduate students and technical staff. How have the results been disseminated to communities of interest?Results thus far are preliminary; this activity will become more important later in the project. So far, data has been communicated through oral and poster presentations, publication of papers and articles, and direct communications. Many of the more recently identified elite rootstock candidates have been included in recently planted MAC-funded replicated field trials. Field days will be held in subsequent years. What do you plan to do during the next reporting period to accomplish the goals?Research activities will be performed as previously proposed, and adjusted as necessary to deliver rootstocks that have the best chance to solve the HLB problem.

Impacts
What was accomplished under these goals? Objective 1. In an effort to capture the exceptional HLB tolerance of Sugar Belle (which cannot be used as a rootstock due to its susceptibility to Phytophthora and its zygotic seed production), Dr. Grosser and Dr. Gmitter created 47 hybrids with complementary rootstock germplasm and propagated via rooted cuttings. Some are already showing great promise in the 'gauntlet' screening. Objective 2. USDA Rootstock trial - 2014, USDA Picos Farm, Fort Pierce, FL (Albrecht, Bowman) Horticultural data (tree measurements and tree health indexes) collection was conducted in June 2018 and November 2018 and incuded: Leaf and root samples were collected for CLas (and CTV) detection in June 2018. DNA/RNA was extracted and CLas titers were determined by PCR according to standard procedures. Leaf and root samples were collected for metabolite analysis in June 2018. Samples were stored at -80°C. Metabolite analysis was not yet conducted as we were waiting for yield data information to determine the most suitable rootstocks to be included in the analysis. Horticultural performance data and tree health data differed from the previous year's data; therefore, metabolite analysis for tress from this trial was put on hold. USDA Rootstock trial - 2017, USDA Picos Farm, Fort Pierce, FL (Albrecht, Bowman) Horticultural data collection was conducted in November 2018 Lykes Rootstock trials - 2015, Lake Wales/Basinger, FL (Albrecht, Grosser, Gmitter) Data collected: yield, fruit quality, tree height, trunk diameter, canopy size, tree health, yield efficiency. Metabolite analysis and root phenological analysis will commence in 2019-2020. To facilitate rootstock selection process from available germplasm, Dr. Cano is carrying out qPCR work to monitor CLas titers. Results showed 25 rootstocks with potential HLB tolerance. All of Dr. Grosser and Dr. Gmitter's hybrids have been included in the 'gauntlet' screening process, and the cuttings are being challenged in the greenhouse with HLB+ scions. One hybrid, LB8-9 X S10-15-16, is already showing exceptional performance in the 'gauntlet' screen. Some rootstocks showing superior HLB tolerance appear to be reducing or eliminating CLas titers from the grafted Valencia scion and the roots - an exciting result that suggests the possible transfer of CLas resistance to grafted sweet orange scion. Among older 'gauntlet' trees, 52 promising rootstock selections were identified for potential HLB tolerance, and 14 exhibiting possible CLas resistance. Genetic patterns regarding HLB tolerance are clearly being identified. Among the newer, potentially HLB-tolerant trees identified, several hybrids made with the somatic hybrid sour orange + Rangpur (SORP) are doing exceptionally well. This parent is exceptionally tolerant of HLB, but was not a superior rootstock by itself due to lack of vigor, and lack of nucellar seed production. Combining it with more vigorous parents of good rootstock pedigree appear to be solving these problems. We have identified several 'gauntlet' trees where both the roots and leaves have Ct values (Cano) indicating no active infection; suggesting potential resistance coming from the rootstock. Of particular interest is the large number of hybrids in this group that have the S10 hybrid as a mother, previously selected for salinity tolerance. The performance of the hybrids made with parents containing abiotic stress genes is remarkable, suggesting that there is a considerable overlap in the mechanisms for HLB tolerance with abiotic stress tolerance. Objective 3. Dr. Vashisth conducted greenhouse experiments to evaluate the effect of irrigation water pH on performance of HLB-affected and healthy plants. Results showed that HLB-affected plants show higher levels of defoliation and decline at an alkaline pH of irrigation water as compared to healthy plants. Ability to tolerate high soil pH can confer some degree of HLB tolerance to rootstocks, which seems to be correlated to the improved ability to mine and translocate nutrients under such conditions. All three pH treatments had resulted in similar leaf fresh weight in Swingle, US-802, UFR-16, 46X20-04-06, and UFR 4 rootstocks. Objective 4. Dr. Bowman collected information on tree health, crop yield, fruit quality, and CLas infection and titer, as appropriate for the tree age and condition, from 23 established trials planted between 2011 and 2019 Dr. Grosser and Dr. Gmitter have also initiated propagation of several other promising 'gauntlet' selections. Propagations from these selections will be entered into replicated field trials subsequently. Promising 'gauntlet' rootstock genotypes recovered by grafting from induced sprouts from below the 'gauntlet' tree graft union. Trees on 18 rootstocks propagated with 6 selected scions for the Tri-State project reported last year are now ready for planting, and the field site located about 10 miles from the CREC (Eagle Lake) is being prepared. The 2nd round of 24 selected rootstocks were propagated and sent to a commercial nursery for grafting. Dr. Wang examined scion/rootstock combination trials conducted at various locations to screen the most HLB-tolerant combinations with good tree health, yield and fruit quality. In this study, the rootstocks, namely Orange 3 (UFR-1), Orange 14 and Orange 16, are sibling complex allotetraploidhybrids from a cross of somatic hybrids:[(Citrus clementinax× (C. paradisi x C. reticulata)) + C. grandis]× [(C. reticulata + Poncirus trifoliata).Rootstock "C" (UFR-6) is a somatic hybrid of Changsha mandarin + 50-7 trifoliate orange. The Orie Lee Late (OLL) somaclone sweet orange scions. In the past year, some trees including various OLL's on Orange 3 (UFR-1) and Orange 16, and in particular OLL 20 on the Changsha+50-7 (UFR-6) rootstock, grown with no psyllid control, showed good health and productivity PCR Ct values negative for and active CLas infection. Sensory analysis and consumer preference and overall flavor were best in OLL 20 on the UFR-6 rootstock. Dr. Bowman is preparing additional propagations of new rootstocks in the USDA greenhouse nursery now for field planting in 2019-20, and will include about 150 new rootstocks to be used in four new replicated field trials. Dr. Bowman also planted six new replicated rootstock field trials with four different scions, including rootstocks, which were identified in previous trials as exhibiting HLB-tolerance. Objective 5. Dr. Grosser's complex tetrazyg A+Volk+Orange 19-11-8 has continued to excel in the 'gauntlet' screen, and the seed tree has proven to be fruitful, producing abundant nucellar seed. Dr. Bowman released three new rootstocks that have exhibited field tolerance to HLB in November 2018, identified as US SuperSour 1, US SuperSour 2, and US SuperSour 3. Dr. Bowman also submitted 12 new USDA hybrid rootstock selections with superior field performance to FDACS-DPI for STG and indexing, and subsequent release. Laura Reuss has successfully completed the T4 webserver training required to manage the projects' informational webpage designed to disseminate research findings, as well as provide a platform to upload images, graphs and other data so that it can be made available to the public.

Publications


    Progress 03/15/17 to 03/14/18

    Outputs
    Target Audience: Nothing Reported Changes/Problems:Due to hurricane Irma, which caused extensive flooding, plant evaluations and sample collection had to be postponed in order for roots and trees to recover. Standard horticultural measurements were conducted in December 2017. What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

    Impacts
    What was accomplished under these goals? Objective 1. Dr. Grosser and Dr. Gmitter propagated cuttings of promising new rootstock hybrids produced from crosses of HLB tolerant parents, including hybrids containing the highly tolerant 'SugarBelle' as a parent. Many of these also have HLB tolerant trifoliate orange 50-7 parentage. Expanded crosses were conducted at both the diploid and tetraploid levels using HLB tolerant parents with good rootstock pedigree. Superior hybrids from 2016 crosses selected and propagated (via seed), including two additional sour-orange type rootstocks showing promise in the Orie Lee Alligator trial for more thorough study: 46x20-04-S15 and 46x20-04-6 (both Hirado Buntan pummelo x Cleopatra mandarin hybrids). Dr. Grosser and Dr. Gmitter also propagated the advanced rootstock selections, in Table 1 below, for grafting with 6 selected commercial scions representative of the US citrus industry - for robust HLB challenge field experiment. Grafting will be initiated in May, 2018. Rootstock Source UFR-1 clone# 55 TC super-root mutant UFR-1 clone# 28 TC super-root mutant UFR-1 clone# 02 TC super-root mutant UFR-1 original clone control (tetrazyg) UFR-6 clone# 1 TC super-root mutant 2247x6070-02-2 tetrazyg, controlled cross SG-2-P tetrazyg, open pollination SG-6-50 SFS x Swingle A+Volk x Orange 19-11-31 gauntlet tetrazyg A+Volk x Orange 19-11-8 gauntlet tetrazyg Afghan sour orange external donor 46x20-04-6 diploid, controlled cross 5-1-99-5xUS812-15-20 diploid, controlled cross 2247x2075-02-26 tetrazyg, controlled cross Milam+HBPxOr.14-9-10 gauntlet tetrazyg Table 1. Grosser/Gmitter: potentially HLB-tolerant rootstocks selected for initial extensive evaluation During 2017-2018, Dr. Bowman planted four new replicated rootstock field trials, which included 183 new hybrid rootstocks combining germplasm from HLB-tolerant parents In addition, Dr. Bowman cooperated with Dr. Grosser in planting five new rootstock field trials under the HLB MAC project, planned for use in this NIFA project. Additional propagations of new rootstocks in the USDA greenhouse nursery now are being prepared for field planting in 2018-19, and will include about 140 new rootstocks to be used in three new replicated field trials. Objective 2. The first phase of Dr. Albrecht's evaluation period focused on a replicated rootstock field trial at the USDA Picos Farm in Fort Pierce, FL, which was planted in 2014. The trial consisted of Valencia orange scion on different rootstock selections. Except for 4 known "standard" rootstocks (Cleopatra mandarin, sour orange, Swingle citrumelo, and Ridge pineapple), rootstocks are new and unreleased selections. Dr. Albrecht selected trees on 20 different rootstocks for analysis. Selection was based on cropping information at 16 months after planting and included high yielding as well as lower yielding rootstocks in addition to the 4 rootstock standards. Data were provided by Dr. Kim Bowman (USDA, ARS). The experimental design was completely randomized with 9-12 biological replicates (each replicate consisting of one tree) per rootstock. Due to hurricane Irma, which caused extensive flooding, plant evaluations and sample collection had to be postponed in order for roots and trees to recover. Standard horticultural measurements were conducted in December 2017 and included Scion trunk diameter Rootstock trunk diameter Tree height Canopy Volume Dr. Albrecht's tree health ratings were conducted on a scale of 1 to 4 with 1 = unhealthy tree of small size with much reduced canopy/die-back, and 4 = healthy tree with dense canopy and no die-back. Dead trees were assigned a value of 0. Horticultural measurements and tree evaluations will be adapted according to new standardized guidelines anticipated to be available during the new funding cycle. Leaves were collected from each tree to assess CLas titer levels by PCR (Lili Cano, UF/IRREC). Dr. Albrecht measured root production by the ingrowth core method, custom-made cylindrical root core cages, measuring 7.5 cm in diameter and 28 cm in length and a mesh size of 3mm, were designed and constructed. One cage each was placed below the canopy dripline of each trees at the side of the tree facing the bed. Before placing the cages, holes of the same dimensions as the cages were drilled with an auger. Cages were inserted, filled with root-free soil to approximate the original soil bulk density, capped, and covered with a thin layer of additional soil. This method has proven very suitable in previous field trial. The first assessment of root growth, health, and architecture will be conducted in June 2018 (6 months after root cage placement). Additional root samples will be collected at the same time to be used for root metabolite analysis. Studies will be extended to additional rootstock trials which were established in November 2017. From previously established trials (mention in Objective 1), Dr. Bowman collected information on tree health, crop yield, fruit quality, and CLas infection and titer, as appropriate for the tree age and condition. A summary of performance information from one of these trials (Valencia scion planted in 2014) was prepared and presented at a grower field day in March 2018. This summary included data on 24 rootstocks, 23 of which having 100% tree survival. Rootstock US-2122 reported with the best yield/tree (lbs) and yield efficiency (kg/m3). There was much grower interest and excitement to see outstanding field performance from some of the new rootstocks in trials. Dr. Bowman also collaborated with co-PIs Albrecht and Cano, to facilitate collection of field samples and data from previously established trials for further use in this project. Objective 3. Dr. Wang sampled OLL Sweet Oranges harvested from six putative HLB-tolerant tetrazyg rootstocks from 4-year old trees in the Orie Lee 13-W Trial, St. Cloud, FL. Two trees of each rootstock were randomly selected and two bushels of oranges were picked from each tree. Briefly, each rootstock had four replicates. About 100 panelists were invited to participate in the sensory testing of orange juices from each of these different rootstocks/scions. Results indicated the juice colors of sweet oranges OLL 4 and OLL 8 on rootstock Orange-16 were darker than those of other rootstocks. The sensory evaluation of flavor properties including sweetness, freshness, overall orange flavor intensity and overall flavor presented a similar trend to overall liking, indicating these properties may play an important role in consumer preference. Juice from OLL 4 showed a better flavor profile than juice from OLL 8 across the rootstock population.More tests are needed to confirm consistency for the next fruit season. Dr. Vashisth used replicate control and selected putative HLB-tolerant rootstocks for propagation and then potted for micrografting, which will begin in the next few weeks. Dr. Gmitter conducted the initial RNA-seq experiment, with sRNAs is still being analyzed, and elite rootstock selected candidates are being validated now. Objective 4. Dr. Grosser and Dr. Gmitter worked with Dr. Cano to identify superior performing rootstocks in the field component of their 'gauntlet' screen for both leaf and root PCR analysis (complete results pending). Some rootstocks showing superior HLB tolerance appear to be reducing or eliminating CLas titers from the grafted Valencia scion. Dr. Bowman submitted 12 new USDA hybrid rootstock selections with superior field performance to FDACS-DPI for STG and indexing, making preparations for new rootstock cultivar release in 3-4 years.

    Publications