Citrus transformation Facility continued its operation during the second quarter of 2019. Within this period, we accepted seven orders. Six of the orders required production of transgenic Duncan grapefruit plants and one was for production of transgenic Mexican lime plants.Altogether, CTF produced 67 plants in this quarter. They belong to following orders: eight Duncan plants BB3, seven Duncan plants BB4, seven Duncan plants HGJ74, six Duncan plants ZM3, five Duncan plants ZM6, two Duncan plants ZM12, 11 Duncan plants JJ8, three Mexican lime plants LM2, five Mexican lime plants M2SF, three Pineapple orange plants AL2, nine Pomelo plants HGJ68, one Valencia plant BB4. A new employee was hired in April to work for the next six months on the CRDF-funded project lead by Jeff Jones. With the departure of one of the employees at the end of June, there are presently six members of staff in CTF.The fruit of Duncan grapefruit and Valencia oranges that we stored in the cold room at CREC, provide steady source of seeds. The other seeds were acquired from the Lyn Citrus nursery in California.
During the second quarter of 2019, we continued our horticultural assessments according to the objectives outlined in the proposal. In April 2019, we collected fruit samples from the Valencia trees at both the Lake Wales and the Basinger location. Because of the high cost, only fruit on all rootstocks that were fully replicated were analyzed for the following quality parameters: fruit weight, % juice, % solids, % acid, brix/acid ratio, and juice color. Fruit quality analyses were conducted at the CREC Processing Pilot Plant. Fruits were harvested in both locations shortly thereafter and yield data were collected from trees on all rootstocks, even if not fully replicated, as they may provide important information to the breeders.At the Basinger location, the average yield across all trees and rootstocks was 33 lbs. fruit/tree for Valencia and 13.6 lbs. fruit/tree for Hamlin. Hamlin yields were low because of citrus canker, which spread through this grove after hurricane Irma and caused many fruits to drop. Although adjacent to the Hamlins, Valencia fruit were not as affected. The average tree size at the Basinger location was 5.4 ft for Valencia and 5.6 ft for Hamlin. At the Lake Wales location, the average yield across all trees and rootstocks was 26 lbs. fruit/tree for both Valencia and Hamlin. The average tree size was 5.3 ft for Valencia and 5.6 ft for Hamlin. The largest trees across all trials were those on X-639 and C-54 with an average height of 6.7 ft and 6.3 ft, respectively. The highest yielding trees across all trials were those on C-22 and FA-517 with an average of 32-33 lbs. fruit/tree, followed by C-146, US-897, and UFR-5 with an average of 29-30 lbs. fruit/tree. Significant differences among trees on different rootstocks were also found for most fruit quality parameters (Valencia). The largest fruit were produced on trees with the Spanish rootstocks. The highest percentage of solids was imparted by FA-517 and several of the UF rootstocks including UFR-5 and UFR-6. The brix/acid ratio was highest in fruit on FA-517 and another Spanish rootstock.To conduct proper statistical analyses, we only included fully replicated rootstocks, which resulted in approximately 30 rootstocks depending on the scion and the trial location. Our analyses showed some statistically significant trends among rootstocks although results varied with scion and location. Overall, the most prominent differences were found for tree size and yield efficiency imparted by different rootstocks. For example, trees on some rootstocks (e.g. X-639) were large and exceptionally healthy looking but yielded few and lower-quality fruit (low yield efficiency). In contrast, trees on other rootstocks that were not remarkable in appearance yielded significantly more fruit than trees on X-639.
This new project is a continuation of work that was ongoing under CRDF 15-010; the official start date for the project was 1 February 2019. We report below on the activities in the first two months of the project by objectives. 1. Develop new rootstocks that impart HLB-tolerance to scion cultivars. Trees that were selected from the Gauntlet screen from 2018 crosses were stick grafted with CLas-infected Valencia budwood for further selection of tolerant types and are now under observation. Several crosses were made in spring 2019 for rootstock improvement at the diploid and tetraploid levels, using parents that have previously demonstrated good genetic combining ability, to generate new families for selection that combine tolerance of high pH calcareous soil and Phytophthora, with appreciable tolerance of HLB in grafted scions. Previous good parental combinations have included A+HBP x Sour Orange + Rangpur, among others at the tetraploid level. Recently we have used LB8-9 Sugar Belle as a seed parent with various pollen parents and have selected several strong survivors in the Gauntlet. We have repeated some of these crosses to increase the likelihood of finding superior performers. We are using DNA fingerprinting techniques to verify the origins of the Super-Root Mutants that have been selected from in vitro propagations in a commercial nursery. 2. Develop new, HLB-tolerant scion cultivars from sweet orange germplasm, as well as other important fruit types such as grapefruit, mandarins, and acid fruit. A large number of crosses were made in the spring 2019, to produce families for selection of individuals that may meet this objective and be valuable for commercialization following advanced testing, as well as to continue the process of parent development to improve future outcomes from breeding. The former will meet the needs of the industry in the near-term while the latter will lay the basis for a future of quantitatively enhanced tolerance of HLB. Over 25 crosses were made for grapefruit development, using at least one parent that has been shown to be highly tolerant of HLB and citrus canker; 10 crosses were made to create families of sweet orange like hybrids, including 2 using Parson Brown as pollen parent at the recommendation of some citrus growers; and 40 crosses for mandarin hybrid development and parent building. Some of the crosses for mandarin improvement may yield also sweet orange-like hybrids. 3. Screen our ever-growing germplasm collection for more tolerant types and evaluate fruit quality of candidate selections. We evaluated the fruit quality attributes of several very tolerant types previously identified, but we have not carried out whole collection assessments of tree health vis a vis HLB in these two months.4. Conduct studies to unravel host responses to CLas and select targets for genetic manipulations leading to consumer-friendly new scion and rootstock cultivars. We have completed anatomical studies of HLB-tolerant LB8-9 and Bearss lemon, and demonstrated that phloem regeneration is an obvious physical mechanism of their apparent tolerance.
Two students were successfully recruited to work on the project during the present reporting period, March 15 – June 15, 2019. Mr. Chad Vosburg has accepted an offer of admission as a M.S. degree student in the Penn State Department of Plant Pathology graduate program. He has experience working with HLB and is currently involved in HLB research as a technician. Chad will begin his studies in August, 2019 and will be working on the citrus infection and HLB disease measurement parts of the project. Mr. Jeremy Held, who was rotating in the PI’s lab starting in Februrary, 2019, has elected to stay in the PI’s lab as a Ph.D. student in the Intercollege Graduate Program in Plant Biology at Penn State. Jeremy will be working on the tree molecular characterization objectives of the project. Jeremy has already successfully detected the FT-scFv protein by protein gel immunoblotting and verified the pollen viability of trees producing the FT-scFv protein. Personnel to complete this project are now in place. The PI visited the lab of collaborator Dr. Tim Gottwald at the United States Horticultural Research Laboratory (USHRL) in Fort Pierce, Florida, on June 10 – 11, 2019. Chad Vosburg, who is from the nearby area, was also able to be present for part of these meetings. The current status of existing trees was determined, and a plan was developed by Drs. McNellis and Gottwald and Mr. Vosburg and Mr. Earl Taylor (Gottwald Lab research assistant) for tree propagations that were needed and setting up a series of HLB resistance tests. It was determined that at least 4 of the FT-scFv lines had sufficient numbers of trees available to set up a test immediately. It was determined that Chad would best work at USHRL as a “guest” to expedite his starting work on the project at the USHRL. We plan to initiate plant propagations and one HLB resistance experiment run during the next reporting period, prior to Chad’s formal beginning of studies. Chad will be able to participate in this work in part due to his close proximity to the USHRL this summer.
Objective 1, Mthionin Constructs: Assessment of the Mthionin transgenic lines is ongoing. Detached leaf assays, with CLas+ ACP feeding, have been conducted and lines with the most promising results have begun greenhouse studies. These studies (With 9 Carrizo lines and 4 Hamlin lines, 98 total plants with controls) include graft inoculation of Carrizo rooted cuttings with CLas+ rough lemon, no-choice caged ACP inoculation of Carrizo rooted cuttings, and no-choice caged ACP inoculation of Hamlin grafted on Carrizo with all combinations of WT and transgenic.Data collection from the first round of field plantings with Mthionin transgenic Carrizo (45 plants) rootstock with non-transgenic rough lemon continues. Initial results show transgenics maintaining higher average CLas CT, significantly decreased leaf mottle and significantly increased health values after 6 months. A large group of Mthionin plants went into the ground in April, including transgenic Carrizo with WT Hamlin scions (81 plants) and transgenic Hamlin on non-transgenic Carrizo rootstock (108 plants) with WT/WT controls (16 plants). Additional grafts of WT Ray Ruby (118 plants) and WT Valencia (118 plants) onto transgenic rootstock are being propagated for future plantings. Mthionin construct transformations have also been completed on 250 Valencia explants to provide sufficient events for this critical variety. Objective 2, Citrus Chimera Constructs: Detached leaf assays, with CLas+ ACP feeding, were conducted on lines representing chimera constructs TPK, PKT, CT-CII, TBL, LBP/’74’, `73′, and `188′. Multiple lines from several constructs were moved forward into greenhouse studies based on these results as noted below. Definitive results for TPK, PKT, CII, and TBL were hindered by low inoculation rates. Assays for these constructs are being repeated to identify which lines of each are best suited for greenhouse studies. Detached leaf feeding assay protocols have also been adjusted to improve sensitivity (See section 4)No-choice caged ACP inoculation has been conducted on 8 lines of citrus Thionin-lipid binding protein chimeras (`73′, and ’74’). Early data from CLas+ plants showed a statistically significant reduction (13x) in CLas titer for transgenics vs WT in the CLas+ plants. However, many plants shown little to no amplification of CLas DNA at 3 and 6 months post inoculation. Amplification by this time would be expected from a successful inoculation, indicating low inoculation efficiency. All plants in this experiment will be re-inoculated by bud grafting with HLB+ rough lemon to allow for continued greenhouse studies. Moving forward, we will be emphasizing parallel field trials for phenotyping efforts and modify the ACP inoculation in greenhouse studies to increase CLas pressure. 475 rooted cuttings were previously made from Hamlin and Carrizo lines expressing constructs `74′ and `188′ and are now of a size appropriate for CLas exposure. 360 of these plants will be grafted with WT scions (for Carrizo lines) or rootstocks (For Hamlin lines) for field trials. The remaining 115 plants will be ACP inoculated for greenhouse studies, with the caged no-choice feeding time period extended from 7 days to 14.Seven new transformations, totaling over 2000 explants, have been completed to generate Valencia, Hamlin and Ray Ruby lines expressing constructs `74′, `188′, and TPK.Objective 3, ScFv Constructs: Greenhouse studies on the 5 scFv lines in the 1st round of ACP-inoculation has been completed with the best performing lines showing significantly reduced CLas titer over the 12 month period (up to 250x reduction) and a much higher incidence of no CLas rDNA amplification in all tissue types. The best lines have been used as rootstock for WT Ray Ruby scions and will be moved to the field after necessary permit approvals. An additional 129 rooted cuttings are propagated for follow up plantings.Like the `74′ results discussed for objective 2, the 2nd round of ACP-inoculations of scFv plants (150 plants, 12 lines) had a poor infection rate. The plants are to be re-inoculated by budding with HLB+ rough lemon. The 370 scFv rooted cuttings already propagated for a 3rd round of ACP-inoculations will use the higher pressure 14 day feeding protocol described above. Objective 4, Screening Development and Validation: Details of the high throughput ACP homogenate assay, and its use for selecting lytic peptides for activity against CLas, has been submitted for publication and remains in use for early screening of therapeutics in the lab. The detached leaf ACP-feeding assay has undergone several small revisions to improve sensitivity and maintain consistent inoculation; increasing from 10 to 20 ACP per leaf, decreasing the feeding period (7 days to 3) and adding a 4 day incubation period between feeding and tissue collection.An array of phloem specific citrus genes has been selected for investigation as potential reference genes to improve detached tissue and plant sampling techniques. The use of a phloem specific endogene would allow for samples to be normalized to phloem cells instead of total citrus cells, more accurately evaluating bacterial titer and potential therapeutic effects with the phloem limited CLas. Objective 5, Transgene Characterization: Transgenic Carrizo lines expressing His6 tagged variants of chimeric proteins TBL (15 lines), BLT (15 lines), TPK (17 lines), and PKT (20 lines) have been generated and confirmed for transgene expression by RT-qPCR. These plants will be used for generating data on the movement and distribution of transgene products in parallel to antibody based approaches.
The first steps in this project have taken place. As we will be using the very latest DNA sequencing technologies, and we are attempting to produce nearly full-length genome sequences, it is important to begin with the highest quality DNA preparation as starting material for library construction for sequencing. We have collected young leaves and young flush from the five cultivars selected for sequencing. This required multiple samplings because we were not always able to get the ideal tissue types; just slightly more mature vegetative tissue than the optimum did not give us the purity and the quantity of high-molecular weight (HMW) DNA required. We also optimized previous protocols we have used to be able to produce quantity and quality required. Finally, we have been successful and samples have been sent to UC Berkeley Genome Sequencing Laboratory for library construction.
In this quarter, the propagated transgenic lines have been budded up with non-transgenic HLB free Valencia budwood. It has been observed that the bud take frequency is higher on cutting made from juvenile transgenic lines when compared to the mature lines. Seed source trees, budded onto US802 rootstock have been produced for field evaluation and are being sized up. Transgenic NPR1 lines that have been observed to be tolerant to HLB under field conditions are also being propagated to use as interstocks in this study. ELISA protocols are being developed to rapidly test for transgene products in the fruit and juice.A USDA-APHIS permit has been applied for evaluation of these transgenic lines.
Huanglongbing (HLB) and Citrus Bacterial Canker (CBC) present serious threats to the future success of citrus production in the US. Insertion of transgenes conferring resistance to these diseases or the HLB insect vector is a promising solution. Genes for antimicrobial peptides (AMPs) with diverse promoters have been used to generate numerous transformants of rootstock and scion genotypes. New promoters and/or transgenes are being regularly introduced with more than a thousand new transformation attempts on citrus epicotyl sections each week. Plants have progressed from the initial round of scion transformations and are now replicated and ready for exposure to HLB, using CLas infected psyllids in a greenhouse environment. Transformed rootstock varieties, with two AMPs (D4E1 and Pyrrhocoricin) and 170 transgenic plants, are being challenged using graft inoculations in two new replicated experiments. A wide series of promoters driving a reporter gene are being tested in transformed citrus and show very different levels of expression with some being expressed in all tissues and some only in vascular tissue. Liberibacter sequence data were used to target a transmembrane transporter,as a possible transgenic solution for HLB-resistance. Collaboration with a USDA team in Albany, CA is providing constructs with enhanced promoter activity, minimal IP conflicts, and reduced regulatory and consumer concerns. Genes are being identified from citrus genomic data, from Carrizo citrange sequence generated using USDA funds, to permit transformation and resistance using citrus-only sequences. Citrus-derived T-DNA border analogues have been shown to be effective in producing transgenic tobacco and will be tested in citrus in next quarter. Genes for anthocyanin production are being tested as a visual marker for transformation, as a component of a citrus-only transgenic system. Transgenes are being developed to suppress (using an RNAi strategy) a lectin-like protein produced in the phloem of HLB-infected citrus. It is possible that suppression of this protein may significantly reduce disease symptoms. High throughput evaluation of HLB resistance will require the ability to efficiently assess resistance in numerous plants. Graft-inoculation, controlled psyllid-inoculation, and ‘natural’ psyllid inoculation in the field are being compared. The first trial has been in the field for 21 months and a repeated trial has been in the field for 9 months. Leaf samples have been collected monthly and PCR analysis of CLas conducted. These data will be analyzed over the next quarter.
As proposed, a transgenic test site has been prepared at the USDA/ARS USHRL Picos Farm in Ft. Pierce. The first trees have been in place for more than eight months. Answers have been provided to numerous questions from regulators to facilitate field testing approval. Cooperators have been made aware that the site is ready for planting. Dr. Jude Grosser of UF has provided 300 transgenic citrus plants expressing genes expected to provide HLB/canker resistance, which have been planted in the test site. USHRL has a permit approved from APHIS to conduct field trials of their transgenic plants at this site. An MTA is in place to permit planting of Texas A&M transgenics produced by Erik Mirkov. Alphascents provided an experimental pheromone attract/kill product Malex to disrupt citrus leaf miner (CLM). Our experience suggests CLM may significantly compromise tree growth where insecticides are avoided to permit ready transfer of Las by psyllids. CLM damage also compromises ability to view HLB symptoms. Unfortunately, this product had little effect on leaf miner. The decision has been made to apply Admire this fall to encourage an undamaged flush on transgenic trees. We are still learning how to grow trees for best assessment of HLB-resistance. More than 120 citranges, from a well-characterized mapping population, and other trifoliate hybrids (+ sweet orange standards) have been propagated for a replicated trial in collaboration with Fred Gmitter of UF. These will be planted in the spring of 2011, and monitored for CLas development and HLB symptoms. Data from this trial should provide information on markers and perhaps genes associated with HLB resistance, for use in transgenic and conventional breeding.
Huanglongbing (HLB) and Citrus Bacterial Canker (CBC) present serious threats to the future success of citrus production in the US. Insertion of transgenes conferring resistance to these diseases or the HLB insect vector is a promising solution. Genes for antimicrobial peptides (AMPs) with diverse promoters have been used to generate numerous transformants of rootstock and scion genotypes. New promoters and/or transgenes are being regularly introduced with more than a thousand new transformation attempts on citrus epicotyl sections each week. Plants have progressed from the initial round of scion transformations and are now replicated and ready for exposure to HLB, using graft inoculations and CLas infected psyllids in a greenhouse environment. A detailed set of AMP activity assays will be initiated next quarter on a replicated set (8 plants of each) of 39 independent Hamlin transformants. Transformed rootstock varieties, with two AMPs (D4E1 and Pyrrhocoricin) and 170 transgenic plants, are being challenged using graft inoculations in two new replicated experiments. A series of promoters are being tested with the GUS gene to see how effective they are. As expected, the three vascular-specific promoters show expression only in phloem and xylem, while other promoters show broad expression in tested tissues. Sucrose synthase promoter from Arabidopsis drives high GUS expression more consistently than citrus SS promoter or a phloem promoter from wheat dwarf virus. A ubiquitin promoter from potato drives consistent and very high GUS activity, even though the mRNA levels are similar to D35S promoter. use of this promoter may reduce the number of independent transformants needed. Liberibacter sequence data were used to target a transmembrane transporter,as a possible transgenic solution for HLB-resistance. Radioisotope permits are finally in place to assess effect of identified peptides on preventing ATP uptake in E. coli expressing the Liberibacter translocase. Collaboration with a USDA team in Albany, CA is providing constructs with enhanced promoter activity, minimal IP conflicts, and reduced regulatory and consumer concerns. Genes are being identified from citrus genomic data, from Carrizo citrange sequence generated using USDA funds, to permit transformation and resistance using citrus-only sequences. Citrus-derived T-DNA border analogues have been shown to be effective in producing transgenic Carrizo and tobacco and will be tested in citrus scions soon. Genes for anthocyanin production are being tested as a visual marker for transformation, as a component of a citrus-only transgenic system. Transgenes are being developed to suppress (using an RNAi strategy) a lectin-like protein produced in the phloem of HLB-infected citrus. It is possible that suppression of this protein may significantly reduce disease symptoms. High throughput evaluation of HLB resistance will require the ability to efficiently assess resistance in numerous plants. Graft-inoculation, controlled psyllid-inoculation, and ‘natural’ psyllid inoculation in the field are being compared. The first trial has been in the field for 24 months and a repeated trial has been in the field for 12 months. Leaf samples have been collected monthly and PCR analysis of CLas conducted.
As proposed, a transgenic test site has been prepared at the USDA/ARS USHRL Picos Farm in Ft. Pierce. The first trees have been in place for more than eleven months. Answers have been provided to numerous questions from regulators to facilitate field testing approval. Cooperators have been made aware that the site is ready for planting. Dr. Jude Grosser of UF has provided 300 transgenic citrus plants expressing genes expected to provide HLB/canker resistance, which have been planted in the test site. USHRL has a permit approved from APHIS to conduct field trials of their transgenic plants at this site, with several hundred transgenic rootstocks in place. An MTA is in place to permit planting of Texas A&M transgenics produced by Erik Mirkov. More than 120 citranges, from a well-characterized mapping population, and other trifoliate hybrids (+ sweet orange standards) have been propagated for a replicated trial in collaboration with Fred Gmitter of UF and are growing well in the greenhouse. These will be planted in the spring of 2011, and monitored for CLas development and HLB symptoms. Data from this trial should provide information on markers and perhaps genes associated with HLB resistance, for use in transgenic and conventional breeding. A proposal is being prepared that would provide high density mapping of this material to provide tighter mapping of linked resistance loci. Alphascents provided an experimental pheromone attract/kill product Malex to disrupt citrus leaf miner (CLM). Our experience suggests CLM may significantly compromise tree growth where insecticides are avoided to permit ready transfer of Las by psyllids. CLM damage also compromises ability to view HLB symptoms. Unfortunately, this product had little effect on leaf miner. The decision has been made to apply Admire this fall to encourage an undamaged flush on transgenic trees. We are still learning how to grow trees for best assessment of HLB-resistance.
Huanglongbing (HLB) and Citrus Bacterial Canker (CBC) present serious threats to the future success of citrus production in the US. Insertion of transgenes conferring resistance to these diseases or the HLB insect vector is a promising solution. Genes for antimicrobial peptides (AMPs) with diverse promoters have been used to generate numerous transformants of rootstock and scion genotypes. New promoters and/or transgenes are being regularly introduced with more than a thousand new transformation attempts on citrus epicotyl sections each week. Plants have progressed from the initial round of scion transformations and are now replicated and ready for exposure to HLB, using CLas infected psyllids in a greenhouse environment. Transformed rootstock varieties, with two AMPs (D4E1 and Pyrrhocoricin) and 170 transgenic plants, are being challenged using graft inoculations in two new replicated experiments. A wide series of promoters driving a reporter gene are being tested in transformed citrus and show very different levels of expression with some being expressed in all tissues and some only in vascular tissue. Liberibacter sequence data were used to target a transmembrane transporter,as a possible transgenic solution for HLB-resistance. Collaboration with a USDA team in Albany, CA is providing constructs with enhanced promoter activity, minimal IP conflicts, and reduced regulatory and consumer concerns. Genes are being identified from citrus genomic data, from Carrizo citrange sequence generated using USDA funds, to permit transformation and resistance using citrus-only sequences. Citrus-derived T-DNA border analogues have been shown to be effective in producing transgenic tobacco and will be tested in citrus in next quarter. Genes for anthocyanin production are being tested as a visual marker for transformation, as a component of a citrus-only transgenic system. Transgenes are being developed to suppress (using an RNAi strategy) a lectin-like protein produced in the phloem of HLB-infected citrus. It is possible that suppression of this protein may significantly reduce disease symptoms. High throughput evaluation of HLB resistance will require the ability to efficiently assess resistance in numerous plants. Graft-inoculation, controlled psyllid-inoculation, and ‘natural’ psyllid inoculation in the field are being compared. The first trial has been in the field for 21 months and a repeated trial has been in the field for 9 months. Leaf samples have been collected monthly and PCR analysis of CLas conducted. These data will be analyzed over the next quarter.
1. Development of rootstocks that can impart HLB tolerance/resistance to grafted scions. Seedlings grown from over one dozen unreleased rootstocks, selected on the basis of their abilities to control tree size, size support good fruit loads, and to have minimal HLB symptom expression, are being budded with sweet orange scions for field planting at the St. Helena site next season. As part of the `gauntlet’ screening, we stick-grafted approximately 75 new candidate rootstock hybrids produced from HLB-tolerant parents in 2017 with HLB+ Valencia sweet orange for HLB screening; these are currently under evaluation, as are approximately 100 gauntlet candidate rootstock hybrids (including 47 hybrids crossing HLB-tolerant `LB8-9′ Sugar Belle® with complementary rootstock germplasm such as salt tolerant pummelo/mandarin hybrids and trifoliate orange 50-7). `Super Root Mutants’ of 10 selections of UFR and other rootstocks, including 3 mutants of UFR-1, 3 of UFR-3, one of UFR-4, one of UFR-17 and one of SO+50-7, are being used to produce whole trees for further evaluations of HLB tolerance. One of these from UFR-1 has been found to be a zygotic triploid, and not a mutant from the original line.2. Breeding of HLB tolerant/resistant processing sweet oranges and orange-like hybrids. New hybrids produced have been potted up to grow until field planting next season. Fruit and juice samples of existing UF releases and new oranges under consideration for release were presented to industry at Fruit Display days in November and December. Blends of sweet orange like-selections also were presented and ranked higher than most sweet orange juice samples. New selections were identified from field plantings of orange-like hybrids and likewise were well received. The newer selections have been entered into the DPI PTP for cleanup and certification.3. Screening of the UF-CREC germplasm collection to identify and validate HLB tolerant or resistant selections. Another season of observations was begun in fall-winter 2018 and the new data, combined with previous seasons’ data, are being reanalyzed to more accurately identify and characterize tolerant individuals. This information is also being used to target specific genome regions that may harbor genes for tolerance, to be used in other projects.4. Advanced field trials, release and commercialization of promising HLB tolerant/resistant scion and rootstock cultivars. We continued focused effort on field trial data management, analysis and interpretation. Files from more than 80 sites have been opened, conditions of the trials have been noted, and based on this information plans for 2018-19 field data collection were developed, prioritized, and implemented. Efforts to review and summarize data have continued, and information was organized for inclusion in our website, which was launched in late 2018. This website can be accessed through this link: https://citrusresearch.ifas.ufl.edu/rootstock-field-data/. We visited 18 different field trial sites in fall 2018 and evaluated trees fir HLB responses and overall tree health, and we harvested fruit from several for juice quality analysis. Yield data were also collected from some of these trials and those data are being entered into our database. Finally, a very substantial effort was undertaken this year to rescue promising individual trees of diverse scion and rootstock germplasm from our 50-acre research block at the GCREC in Balm. These blocks have not been irrigated since early fall of 2017, and we were forced to leave the site. All trees in this block were subjectively assessed for potential HLB tolerance, as well as general overall health and appearance, using a 0-4 scale (0=dead; 4=completely heathy appearing tree). We harvested budwood from ~2300 individuals with scores = 3 and propagations for field planting in another location. Between June and end of September 2018, we had to revisit the block and collect additional budwood from trees that were not successfully propagated. We are growing off these trees for planting in 2019 at a new location; it appears that we were successful in recovering ~1800 of the selected individuals.
Significant progress has been made establishing a website, presenting data from our field trials. Ten trials have been entered initially, and several more will be added during the current quarter. Website access: https://citrusresearch.ifas.ufl.edu/rootstock-trials/ The first quarter of this grant was a very busy time for data collection. Summary of data-collection activities this quarter:Trial Name and Data Type Collected Banack (Vero) Juice Quality 2018-19 Cody Estes (Vero Beach) Tree Health Ratings 2018-19 CPI Ranch 1 Tree Health Ratings 2018-19 Doe Hill Grove (Bryan Paul) Tree Health Ratings, yield and juice quality 2018-19 Duda (Immokalee) Vernia/rootstock yield & Juice Quality, tree health ratings 2018-19Duda (Immokalee) APS-OHS Valencia/rootstock Yield and Juice quality, tree health ratings 2018-2019 Ed English Tree Height and Tree Health Ratings 2018-19 Hammond Tree Health Ratings, Yield and juice quality 2018-19 IMG Yield and fruit quality 2018-2019 Lee Groves – 13E Frank’s Block (St. Cloud): OLL seedling/rootstock; fruit quality, PCR 2018-2019 Lee Groves (St. Cloud) – Alligator – Mathew Block: tree health and Juice Quality 2018-19 Lee Groves – 13W Karen’s Block (St. Cloud): OLL/rootstock; yield, fruit quality and PCR 2018-2019 Heller Brothers (Fort Pierce): Vernia/rootstock Yield & Juice Quality, tree health ratings 2018-19 Hidden Golf Course (CREC Variety Trial): Tree Size and Health Ratings 2018-19 Peace River (Larry Black) – Tree size and Health, Fruit Count 2018-19 POST OFFICE (Haines City) OLL/rootstock; Tree Size and Health Ratings 2018-19 Raley Tree (Dundee): Tree Health & Size Ratings, yield and fruit quality 2018-19 Saint Helena (Lee Groves, Dundee): Vernia and Valencia Yield & Juice QualityData, Tree Size and PCR 2018-19 Serenoa (Immokalee): HLB Rating 2018-19 Smoak (Lake Placid): Yield Data, fruit quality, tree size and health 2018-19 Steve Brewer (Land-O-Lakes): fresh mandarin trial – Tree Health & Size Ratings 2018-19 Teaching block (CREC): Berna /Salustiana Yield, tree health ratings, fruit drop 2018-19 Hammond (Fort Pierce): Minneola Fresh Fruit Yield 2018-19 Jackson Citrus (LaBelle): OLL/Valencia B9-65/rootstock reset Tree Size and Health Ratings 2018-19 Wayne Simmons (LaBelle): OLL/Valencia B9-65/rootstock reset Tree Height and Tree Health Ratings 2018-19 Wheeler Bros. (Waverly): Yield 2018-19 Additional trial data will be added to the website as data is analyzed and formatted.
1. Development of rootstocks that can impart HLB tolerance/resistance to grafted scions. Seedlings grown from over one dozen unreleased rootstocks, selected on the basis of their abilities to control tree size, size support good fruit loads, and to have minimal HLB symptom expression, are being budded with sweet orange scions for field planting at the St. Helena site next season. As part of the `gauntlet’ screening, we stick-grafted approximately 75 new candidate rootstock hybrids produced from HLB-tolerant parents in 2017 with HLB+ Valencia sweet orange for HLB screening; these are currently under evaluation, as are approximately 100 gauntlet candidate rootstock hybrids (including 47 hybrids crossing HLB-tolerant `LB8-9′ Sugar Belle® with complementary rootstock germplasm such as salt tolerant pummelo/mandarin hybrids and trifoliate orange 50-7). `Super Root Mutants’ of 10 selections of UFR and other rootstocks, including 3 mutants of UFR-1, 3 of UFR-3, one of UFR-4, one of UFR-17 and one of SO+50-7, are being used to produce whole trees for further evaluations of HLB tolerance. One of these from UFR-1 has been found to be a zygotic triploid, and not a mutant from the original line.2. Breeding of HLB tolerant/resistant processing sweet oranges and orange-like hybrids. New hybrids produced have been potted up to grow until field planting next season. Fruit and juice samples of existing UF releases and new oranges under consideration for release were presented to industry at Fruit Display days in November and December. Blends of sweet orange like-selections also were presented and ranked higher than most sweet orange juice samples. New selections were identified from field plantings of orange-like hybrids and likewise were well received. The newer selections have been entered into the DPI PTP for cleanup and certification.3. Screening of the UF-CREC germplasm collection to identify and validate HLB tolerant or resistant selections. Another season of observations was begun in fall-winter 2018 and the new data, combined with previous seasons’ data, are being reanalyzed to more accurately identify and characterize tolerant individuals. This information is also being used to target specific genome regions that may harbor genes for tolerance, to be used in other projects.4. Advanced field trials, release and commercialization of promising HLB tolerant/resistant scion and rootstock cultivars. We continued focused effort on field trial data management, analysis and interpretation. Files from more than 80 sites have been opened, conditions of the trials have been noted, and based on this information plans for 2018-19 field data collection were developed, prioritized, and implemented. Efforts to review and summarize data have continued, and information was organized for inclusion in our website, which was launched in late 2018. This website can be accessed through this link: https://citrusresearch.ifas.ufl.edu/rootstock-field-data/. We visited 18 different field trial sites in fall 2018 and evaluated trees fir HLB responses and overall tree health, and we harvested fruit from several for juice quality analysis. Yield data were also collected from some of these trials and those data are being entered into our database. Finally, a very substantial effort was undertaken this year to rescue promising individual trees of diverse scion and rootstock germplasm from our 50-acre research block at the GCREC in Balm. These blocks have not been irrigated since early fall of 2017, and we were forced to leave the site. All trees in this block were subjectively assessed for potential HLB tolerance, as well as general overall health and appearance, using a 0-4 scale (0=dead; 4=completely heathy appearing tree). We harvested budwood from ~2300 individuals with scores = 3 and propagations for field planting in another location. Between June and end of September 2018, we had to revisit the block and collect additional budwood from trees that were not successfully propagated. We are growing off these trees for planting in 2019 at a new location; it appears that we were successful in recovering ~1800 of the selected individuals.
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