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.
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.
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.
Objective 1. Create hybrid rootstocks which combine germplasm from parental material with good rootstock traits and HLB tolerance, propagate the most promising of these hybrids, and establish replicated field trials with commercial scions. During this time period, hybrid seed from 2018 rootstock crosses were planted, including SuperSour type parentage of unique HLB tolerant types. One new rootstock trial with Star Ruby grapefruit was field planted in the East coast region in April, using a replicated randomized experimental design, and included several advanced hybrid rootstocks that have demonstrated outstanding field performance in other trials, along with several commercial standard rootstocks for comparison.Multiple trees of about 150 selected new rootstock hybrids were budded and grown in the nursery, in preparation for field planting other new rootstock field trials later in 2019. These nursery trees included many of the most promising SuperSour hybrids identified in ongoing trials established in previous years, as well as several commercial standard rootstocks. These nursery trees also include other new and different hybrids chosen because of newly available information about parentage and characteristics best associated with outstanding traits. Three new field trials with sweet orange scion will be planted from these trees in 2019, including one trial in the East coast region, one in the Central ridge region, and one in the Southwest region.Objective 2. Collect field performance data from early-stage replicated rootstock field trials and release new rootstock cultivars as justified by superior performance in multiyear field trials. Seventeen rootstock trials planted prior to summer 2018 (as described in the Proposal Appendix ii) were monitored and used for data collection on field performance, as appropriate during this quarter for the scion involved. Yield and fruit quality data were collected from multiple Valencia rootstock trials, including cooperative trials with Larry Black in Polk County and Duda in Hendry County. Fruit yield, fruit quality, tree size, and other metrics from the USDA cooperative Valencia trial in Polk County were summarized in a four page handout that was distributed at the CRDF Rootstock Field Day on April 25, 2019. Three SuperSour rootstocks identified in the trials as particularly outstanding in potential yield per acre and yield efficiency are US-2122, US-2101, and US-2134, and these rootstocks will be established in additional trials and examined more carefully for potential upcoming commercial release. Seed trees of all the most promising new SuperSour hybrid rootstocks have been planted at the USDA research farm in Ft. Pierce and the Whitmore Farm to facilitate availability of seed for new rootstocks at the time of release.The three new USDA rootstocks released in November 2018, identified as US SuperSour 1 (SS1), US SuperSour 2 (SS2), and US SuperSour 3 (SS3), are being propagated in nurseries for numerous planned commercial plantings. An informational sheet with performance data on the three rootstocks was prepared and distributed widely to industry.
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