1. Please state project objectives and what work was done this quarter to address them: This report covers the period of March 1, 2021 – May 31, 2021. The objective of this project is to test transgenic ‘Ducan’ grapefruit trees expressing an anti-HLB antibody fused to the FT (Flowering Locus T) protein (FT-scFv protein). Several accomplishments were made during this reporting period. Graduate student Mr. Chad Vosburg was able to take a trip to Florida to assess trees again, take samples, and send samples for qPCR analysis at Southern Gardens. Chad visited Fort Pierce for several days in May, 2021. The samples taken and/or processed, and then sent to Southern Garderns included those from a field trial natural inoculation, an Asian citrus psyllid (ACP) infection in the greenhouse, and a repeate experiment of graft challenge of FT-scFv scions grafted to HLB-infected rough lemon rootstocks. These samples were prepared and sent to Southern Gardens for qPCR detection and quantification of ‘Candidatus Liberibacter asiaticus’ (CLas) within the tissues. Data are anticipated to be received from these samples in July-August 2021. In addition, growth data were taken from FT-scFv scions on infected rough lemon. The transgenic scions appeared to be consistently growing faster and more robustly than the control non-transgenic scions. However, this difference did not meet a standard cutoff for statistical significance. The FT-scFv protein appears to provide a mild benefit to the grapefruit trees when inoculated by grafting, even if not statistically significant. The replicate grafting inoculation experiment begun in January, 2021, has additional replicates and may provide more statistically robust results. In March, 2021, we applied for a no-cost extension through November of 2021, and it was granted in May, 2021. 2. Please state what work is anticipated for next quarter:Additional qPCR data from the field, graft, and psyllid-transmission HLB challenge tests will be received from Southern Gardens in the summer of 2021. These data will be analyzed during the next reporting period. Additional sampling of field grown trees will be performed with help from collaborators at the USDA USHRL. These will be sent to Southern Gardens for qPCR CLas quantification for an additional infection time point. 3. Please state budget status (underspend or overspend, and why):This project is running under budget thanks for wage support from Penn State for Chad Vosburg’s work on the project, which resulted in some salary funds not being expended.
1. Please state project objectives and what work was done this quarter to address them:The project objectives are: 1.Evaluate existing transgenic Carrizo and Swingle AtNPR1 overexpressing rootstocks in the laboratory and greenhouse. 2. Conduct a replicated field trial with the best transgenic rootstocks budded with non-transgenic `Valencia’ and test for GMO gene products in the fruit or juice. 3. Produce additional transgenic rootstock lines and stack other gene(s) responsible for SAR using mature transformation. 4. Evaluate transgene segregation analyses of the rootstock progeny and large-scale propagation of select lines.We have continued on the evaluation of transgenic trees in the greenhouse and field. Trees in the greenhouse and field have not been been tested against HLB in this quarter as sampling is done bi-annually. We have produced several trees with each gene as outlined in the proposal and objective 3 is almost complete. RNA has been isolated from all the new lines and we are preparing to propagate selected lines that have enhanced gene expression. Western blot analysis of some prior propagated lines have been done. All previously identified lines with high gene expression have been clonally propagated by budding onto rootstock for field planting to provide more seed source trees. Additionally seedlings from several existing seed source trees have been produced and budded with Valencia sweet orange. We are also exploring the ability of some of the high expressing lines as interstocks to protect the non-transgenic scion against HLB. White grapefruit is being used in this study due to its extreme susceptibility to HLB. 2. Please state what work is anticipated for next quarter: We will continue on our evaluation of the transgenic lines, propagate additional lines for evaluation and test the interstock trees against HLB. 3. Please state budget status (underspend or overspend, and why): We are on track with the budget and have not overspent.
1. Please state project objectives and what work was done this quarter to address them: Since this CRDF proposal was written in 2018 & the external review occurred, the status of our lab has changed at UF, & in the future, we will conduct less research & focus more on service. In any event, the objectives of this project were to produce disease resistant, commercially & agronomically desirable, mature citrus transgenics, cisgenics, & intragenics that will flower & fruit naturally using Agrobacterium & biolistics as a service for research & commercialization. The research focus of this project is to improve Agrobacterium & biolistic transformation efficiency of mature citrus, so that the mature citrus protocols become more productive, decrease prices for scientists, & contribute more to financial self-sufficiency of our lab. We made great strides in increasing Agro-mediated transformation efficiency of some cultivars in Year 2 (after testing virB5 & plant hormones) & found a completely unrelated method that improves transformation efficiency. A manuscript was submitted for review to my supervisor. An all-citrus selectable marker that functions well in transformation, as an alternative to nptII, was also elucidated in Year 2. A manuscript is being written describing the new selection system. The objective of research into DNA-free gene editing will be continued only in collaboration with a customer(s). From one customer, we received four vectors, performed five transformations, none of his vectors worked & no transgenics were produced. This scientist is using a new vector backbone that doesn’t function in mature citrus scions or rootstocks. We are in the process of assisting him troubleshoot this issue. In addition, the Agro strain he is using, EHA105, tends to rearrange the vectors & the genes of interest can be lost. We transformed some of his vectors into the AGL1 strain, which is RecA- & doesn’t rearrange as often. From now on, we will always include a control transformation with a reliable vector that consistently yields results. Another scientist gave us three vectors to transform into mature citrus & we produced 50 transgenic shoots. He did not want these shoots grafted onto rootstock, but we took high quality photographs for him. The scientist wrote a manuscript with the photographs of the transgenics & included us as co-authors. For another order, we produced ~82 transgenic shoots in one rootstock transformation, an excess of 40 were micrografted, & it is too soon to tell how many will survive. 2. Please state what work is anticipated for next quarter: A number of vectors have been transformed into mature citrus & we are awaiting results. In addition, we have more orders for Agro-mediated transgenics, so we are budding mature citrus for these transformations. In addition, two potential orders for biolistics are pending, however the efficiency of biolistics is still being increased by testing different osmotic treatments & cultivars. Unfortunately the lab member responsible for biolistics is away on sick leave, so we must conduct the optimizations for him. Budding of Kuharske, Carrizo, US942, Hamlin, Valencia, & EV1 was performed for biolistic transformation in order to collect stem tissue explants more appropriate for bombardments. In addition, Dr. Dutt is currently making genetic constructs that will be tested for one biolistic order. We might get additional orders once the outcomes of grant competitions are announced. 3. Please state budget status (underspend or overspend, and why): CRDF funding is sufficient, however we will probably overspend the Director’s account since we lost time & money doing five transformations that didn’t work, unless we can make up for it in later quarters.
Previously, we generated raw sequence data for Valencia orange (S, sensitive), Ruby Red grapefruit (S), Clementine mandarin (S), LB8-9 Sugar Belle® mandarin hybrid (T, tolerant), and Lisbon lemon (T) and preliminary assemblies and analyses were carried out. Because of reduced sequencing costs, we were able to enter additional important genomes into the pipeline beyond those originally proposed, including Carrizo citrange, sour orange, and Shekwasha (an important breeding parent for HLB tolerance); these also have now been sequenced and assembled. Now, we have completed Hi-C sequencing of two genomes, and by incorporating these data with PacBio sequence assembly from two of our target genomes we have produced improved chromosome scale assemblies and have phased the two parental chromosomes of the target genomes using Illumina short reads from citrons, pummelos and mandarins. Minor assembly errors in repetitive DNA regions have been repaired, by genome alignment and comparison to the Poncirus assembly we recently published, resulting in polished assemblies of these two accessions. Initial preliminary characterizations of the presence/absence variations among the four phased haploid genomes have been made. Notable variations among these four involve transposable elements including MITES with characteristic sizes. A genome graph-based approach is being pursued, with the goal of building a genome graph containing the different types of structural variations that are now being identified. The availability of high-quality assemblies for the 3 basic species (C. medica, reticulata, and maxima) will allow thorough and complete characterization of large-scale structural variations (SVs: deletions, insertions, etc.) in our target genomes of commercial interest. SVs are a driving force for phenotypic diversity especially among somatic mutants (e.g., different oranges, grapefruits, etc.), and this type of information will become more important as we test different sweet orange mutants exhibiting enhanced tolerance of HLB and attempt to determine the underlying causes of such tolerance. Currently, we are developing this approach using 27 previously published whole genome shotgun sequences of a particular citrus species and aligning them to our high-quality assembly of the same species. We have found several distinct basic genome types, with varying admixture patterns; a detailed characterization is in progress. We now have the transcriptome data for two of our target genomes, using both Illumina and PacBio sequencing platforms, and genome annotation (i.e., identify all the genes within the genome) is in progress. Samples have been prepared and collected, and plans are in place to proceed with Hi-C sequencing of the target genomes not yet completed and polished, as well as to generate the transcriptome data required for genome annotation, and further characterization of large-scale structural variations within and among the genomes upon which we are focused.
1. Develop new rootstocks that impart HLB-tolerance to scion cultivars. Seeds were extracted from 27 candidate rootstock seed source trees, including several UFRs and other experimental rootstocks, that were fund to be free of seed transmissible pathogens by DPI testing. We are authorized to distribute seeds to licensed nurseries for the UFRs, and to collaborative nurseries and other organizations within Florida, in the US, and globally. New trials will be established with these rootstocks in Australia (New South Wales and Queensland) and South Africa in the next year. A rootstock trial with previously exported rootstocks was propagated in Sicily this spring. These trials will provide information relating to performance under diverse soil and environmental conditions and can help development of recommendations for Florida conditions. More than 3000 flowers were pollinated in twenty-one new rootstock cross combinations of 13 different parents, to recombine multiple useful traits for rootstock improvement. We updated and added new data to existing rootstock trial files and added new files to our website (https://crec.ifas.ufl.edu/citrus-research/rootstock-trials/); currently there is information from 26 locations. Seedlings are being selected from 2020 diploid and tetraploid rootstock crosses in calcareous, high pH soil inoculated with two species of Phytophthora, the first step of the `gauntlet’ screening. More than 50 gauntlet rootstock candidates in citripots are growing off for grafting of replicate trees as necessary to meet the new CRDF guidelines for Stage 1 rootstock evaluations (approximately 500 liners). 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. Shoots generated from 37 interploid crosses made using selected HLB tolerant plants in 2020, including 10 crosses for red grapefruit improvement, 10 for sweet orange improvement, and 10 for mandarin improvement are in the process of micrografting on to 3 different rootstocks, and some have been already moved to the greenhouse. Seedlings from somaclone seedling-derived populations of early maturing (January), high soluble solids OLL selections were planted, to screen for even earlier maturing OLL types; seedling populations of a nearly seedless HLB-tolerant mandarin were also planted to get a completely seedless selection, and to extend the maturity season. A September-maturing pink grapefruit hybrid C2-53, with canker and HLB tolerance exceeding standard grapefruit, was approved for release by the IFAS cultivar release committee. 3. Screen our ever-growing germplasm collection for more tolerant types and evaluate fruit quality of candidate selections. We have continued monitoring a unique hybrid family of more than 400 individuals (with many of these planted as 3 tree replicates) from the cross of Clementine mandarin with a wild species reported to be nearly resistant to CaLas attacks, by collecting detailed HLB phenotypic data, including Ct values and other tree health measures as described previously. The frequency of CaLas-negative trees remains unexpectedly high. We have revisited a replicated planting of sweet orange-Poncirus hybrids that was mapped for HLB tolerance QTLs (Huang et al. 2018) and are collecting new phenotypic data to study long term performance of these trees.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 selected ~ 450 mandarin hybrids for GWAS studies, using the data referred to in Obj 3 above. DNA samples were prepared, each individual was genotyped using the citrus Axiom SNP array, and GWAS analysis is proceeding. This work will validate previously identified, or identify new genomic regions, associated with HLB tolerance or sensitivity. We continued seedling and callus line transformation experiments using the BG gene, and have plants coming from 3 orange, 1 grapefruit, and 2 rootstock lines.
Objective 1, Mthionin Constructs: Assessment of the Mthionin transgenic lines is ongoing. As the most proven of our transgenics, we continue to use them as a reference in detached leaf assays, as well as studying them in established greenhouse and field trials. The first MThionin field trial (45 plants, WT or transgenic Carrizo on rough lemon scions) has shown transgenics maintaining higher average CLas CT values (2.5 CT higher @ 18 months), but with a high degree of variability. The larger second MThionin planting (205 total grafted plants of transgenic Hamlin scions, transgenic Carrizo rootstock, or WT/WT controls) is producing encouraging results; with the transgenic Hamlin on WT Carrizo having statistically better trunk diameter, tree height and canopy volume compared to controls. Leaf samples have also been collected and are being processed for CLas quantification. The Mthionin construct has also been extensively transformed into additional varieties; with 10 confirmed transgenic lines of US-942 and 44 putative lines of Valencia and Ray Ruby now undergoing expression analysis. Objective 2, Citrus Chimera Constructs: Detached leaf assays, with CLas+ ACP feeding, have been conducted to screen citrus lines expressing chimera constructs TPK, PKT, CT-CII, TBL, BLT, LBP/’74’, ’73’, and ‘188’ (as well as scFv-InvA, scFv-TolC, Topaz and Onyx). Testing of all 35s driven Carrizo lines is complete and the analysis of phloem specific and scion-types is well underway. This work has already identified numerous lines with significant effects on CLas transmission and increased ACP mortality (up to 95% from TBL and >70% from TPK). Endosymbiont analysis of high mortality causing transgenics (outlined in Objective 4) has also shown an accompanying reduction in Profftella titer that may indicate a mode of action. The best performing of these lines have been moved forward to greenhouse trials. Initial ACP-inoculated greenhouse trials on 8 lines of citrus Thionin-LBP chimeras (’73’, and ’74’) showed a statistically significant reduction (13x) in CLas titer for ’74’ transgenics vs WT. However, many plants of both treatments remained CLas negative due to low inoculation efficiency. In June, 150 plants representing the best performing 6 lines of `74′ and 7 lines of `188′ were no-choice caged ACP inoculated using a new protocol to improve transmission rates. At 3 months, control plants tested positive at twice the rate of the earlier inoculation; 6-12 month tissue samples are now collected, processed and ready for qPCR. A larger greenhouse study is also underway to directly compare the best performing 3rd generation chimera (TPK and TBL) with the earlier 1st (Mthionin) and 2nd (`74′ and `188′) lines. A total of 420 grafted plants (all on WT Carrizo rootstock for uniformity) were made and bud inoculated with CLas+ RL to ensure high transmission. The first (3 month) growth assessment and leaf collection is complete, with samples awaiting qPCR analysis. An additional ~1200 rooted cuttings have been made from those same lines for paired ACP-inoculated greenhouse and field trials. Field trials of 2nd generation chimeras (`74′, and `188′) with included MThionin plants are ongoing; with 165 plants (WT Hamlin and Ray Ruby on transgenic Carrizo) and 70 plants (WT Valencia on transgenic Carrizo) moved to the field in August 2020 and May 2021 respectively. An additional 200 plants (transgenic Hamlin on WT rootstocks) are being produced complete the planting. Eighteen new transformations, totaling over 6200 explants, have been completed to generate sufficient events of Valencia, Ray Ruby, US-942, and Hamlin lines expressing `74′, `188′, TBL, TPK and other advanced chimera constructs. From this effort, over 325 new lines from 74-Valencia, 74-Ray Ruby, 74-US-942, 74-Hamlin, 188-Ray Ruby, 188-Valencia, 188-US-942, TBL-US-942, TBL-Hamlin, TBL-Ray Ruby, TPK-Ray Ruby, TPK-US-942 and TPK-Hamlin are now in soil. Transgene expression analysis has confirmed the first 29 of these lines as positive with the remainder still being tested. In addition to the use of the Mthionin and its chimeric variants, new strategies have been implemented in our Laboratory to develop HLB resistant citrus. These efforts include the expression of insecticidal peptides to control ACP (CLas vector) and the downregulation of the DMR6 genes to enhance defense responses against HLB disease. 54 independent transgenic lines of Carrizo, Hamlin and Ray Ruby expressing the insecticide peptide Topaz (a code name to protect IP), under constitutive and phloem specific (SCAmpP-3) promoters were evaluated by detached leaf assay. From these, 12 lines (4 event of each genotype) showed significant ACP mortality and were selected to move up in the screening pipeline for HLB/ACP tolerance. Also, 24 Carrizo transgenic events highly expressing Onyx (a code name to protect IP), a peptide with antimicrobial and insecticide activity, were evaluate by DLA. The 5 Onyx lines showing high ability to kill ACP (to 83% mortality) were selected for further evaluation. These strongly performing lines were replicated as rooted cuttings (103 Onyx and 189 Topaz plants) that will soon enter greenhouse trials. The available Onyx transgenic material is being expanded through production of additional constitutive (13 Valencia and 6 Ray Ruby) and phloem specific lines (25 Carrizo, 5 Hamlin, 5 Valencia, and 13 Ray Ruby). Down regulated DMR6 Carrizo, either by stable expression of specific hairpin RNA or by specific Cas9-sgRNA were generated, cloned, and are being assessed. Since DMR6 is a broad immune suppressor, downregulated plants were first evaluated for Canker resistance as a quicker assay. After Xanthomonas challenge, both transgenic and gene edited DMR6 lines showed reduced bacterial titers and statistically significant reductions in Canker symptoms compared to controls; including some lines that developed no symptoms whatsoever. As an effort to accelerate development of non-transgenic HLB resistant plants through gene editing, we transformed early flower transgenic plants (carrying FT-SCFV gene) with the DMR6 targeting CRISPR construct. The early flowering trait will greatly decrease the time needed to produce an edited but non-transgenic offspring. A set of 30 plants resulted from this gene stacking effort will be evaluated for the presence of both genes. Objective 3, ScFv Constructs: ACP inoculated greenhouse studies on 5 scFv lines have been completed with transgenics showing significantly reduced CLas titer (up to 250x reduction) and a significantly higher incidence of no CLas rDNA amplification in roots and leaves compared to WT. These lines have been grafted with WT Ray Ruby scions and are undergoing field trials at Picos farm. The first assessment was completed in March with leaf tissue collected and awaiting CLas quantification. An additional 129 rooted cuttings are propagated for follow up plantings with grafted Hamlin scions. A second greenhouse trial testing new lines (150 plants from 12 lines) have been bud inoculated with HLB+ RL. A group of 370 plants for a third greenhouse trial has been propagated with the first 54 plants to reach a suitable size ACP-inoculated using the improved protocol. Plant tissue from both second and third (partial) greenhouse trials has been collected and processed; now awaiting qPCR analysis for CLas quantification. Objective 4, Screening Development and Validation: A protocol using a high throughput ACP homogenate assay for selecting lytic peptides for activity against CLas is now in use. A manuscript on the protocol has been published in Plant Methods (DOI: 10.1186/s13007-019-0465-1) to make it available to the HLB research community. Transgenic Nicotiana benthamiana plants expressing His-6 tagged variants of the chimeras TBL, TPK, PKT and LBP have also been generated to produce sufficient protein extracts for use in exogenous applications. The detached leaf ACP-feeding assay (DLA) has undergone several small revisions to improve sensitivity and maintain consistent inoculation; adjusting feeding period and ACP numbers. We have also expanded the analysis of ACP bodies to include quantification of other major endosymbionts (Wolbachia, Carsonella and Profftella) to better investigate the activity of peptides causing CLas mortality. An array of phloem specific citrus genes has been selected for investigation as potential reference genes to improve detached tissue and plant sampling techniques. Multiple sets of sequence specific qPCR primers for each gene have been synthesized and tested for efficiency. Six varieties of citrus have been propagated for endogene stability testing. A phloem specific endogene would allow normalizing to phloem cells, more accurately evaluating CLas titer relative to Citrus DNA and potential therapeutic effects. The best performing lines of Mthionin, chimeras `74′,`188′, TPK, TBL and scFv transgenics have been submitted to Florida Department of Plant Industry for shoot-tip graft cleanup in preparation for future field studies. Hamlin/Mthionin transgenics (3 lines), Carrizo/Mthionin (2 lines) and Carrizo/’74’ (1 line) have been returned certified clean. Objective 5, Transgenic Product Characterization: Experiments are also underway track the movement and distribution of transgene products using antibodies and affinity tagged protein variants. CLas+ RL have been grafted as scions onto MThionin expressing Carrizo as a platform to test peptide movement and effects across the graft union. Transgenic Carrizo lines expressing His6 and/or Flag tagged variants of chimeric proteins TBL (15 lines), BLT (15 lines), TPK (17 lines), PKT (20 lines), scFv-InvA (22 lines) and scFv-TolC (18 lines) have been generated and expression confirmed by RT-qPCR. Total protein samples have been extracted from His-tagged transgenic lines and sent to our CRADA partner for testing.
Field variety trials are a simple but effective tool to test plant horticultural performance under different environmental conditions and enhance the commercial adoption of new cultivars. Large-scale, rapid implementation of HLB-tolerant cultivars depends on reliable data, and the Millennium Block is addressing the need of establishing field plantings to generate regional, updated information for the Indian River IIR) Citrus District. The project has two objectives: (i) Assess performance of new grapefruit cultivars with certain rootstocks under HLB endemic conditions in the IR district and (ii) Evaluate the influence of UFR and other recent rootstocks on grapefruit, navel, and mandarin in the IR in comparison to legacy/standard rootstocks.
Trials tested: T1) grapefruit cultivars on three rootstocks, T2) 36 rootstocks with Ray Ruby grapefruit as the scion, T3) 30 rootstocks with Glenn 56-11 navel orange, and T4) 30 rootstocks with UF-950 mandarin.
We planted 3,400 trees in Sep/2019, 1,100 trees in Aug/2020, and 400 trees on Jun/2021. Only grapefruit trees on UFR-8 are going to be planted in Fall since not ready yet. Controlled-release poly coated fertilizer was applied in Sep/2019, Jan, May and Sep/2020, Jan and May/2021. Irrigation controller, sand media filtration system and water flow meter were installed. The group met with the certified crop advisor to develop a spray program schedule based on time of year and conditions to be applied as determined by IPM scouting. Hoop boom was modified to spray young trees with higher accuracy, reducing the waste of agrochemical products. We created a location map and began production and distribution of QR tags to be used with scanner codes during data collection in the field.
Tree height, tree width in two positions (E-W/N-S), and trunk diameter were measured in three central trees from each experimental plot in Feb, July and Nov/2020, and Feb/2021, and canopy volume calculated. have been presented at the 2020 and 2021 ASHS annual conferences. The study is on schedule, and the following summary data reflect the best and worst treatments in each trial during the first 18 months of growth.
Tree size data from Feb/2021 show some scion-rootstock combinations are exhibiting significant differences in canopy volume. In Trial 1, Pummelette UF-5-1-99-2 grapefruit on US-942 was 6x larger (4.7 m3) than US 1-83-179 grapefruit hybrid on Sour Orange (0.8 m3) (P<0.0001). In Trial 2, Ray Ruby grapefruit on US-812 was ~3x larger (4.1 m3) than on Orange 16 (1.3 m3) (P<0.0001). In Trial 3, Glenn F-56-11 navel orange on C-22 was ~2x larger (2.9 m3) than on Willits (1.2 m3) (P<0.0001). In Trial 4, UF-950 mandarin on US-942 was ~2x larger (2.5 m3) than on WGFT+50-7 (1.2 m3) (P<0.0001). Cultivars grafted on different rootstocks have no differences in tree phenological stage. HLB is spreading in the field and individual trees are getting infected as visual HLB symptoms are seen on some treatments in each trial. Leaf samples were taken in Mar/21 for HLB diagnostic and bacterial titer quantification. Asian citrus psyllids, Diaprepes root weevils, whiteflies, and other insects are less abundant in the field, except for leaf miners, which caused severe damage due to the excessive rainfall and wind gusts that made pesticide application challenging during this quarter. Nonetheless, tree growth has not been significantly affected by these pests. Leaf samples for determining HLB incidence were taken from a pool of trees from each experimental plot in May and Sept/2020, and Feb/2021 and sent to the Southern Gardens lab for analysis; on average, all samples tested negative (no trees with Ct values <32) but there are several positive trees with visible symptoms. Fruit phenology, pests and diseases have been monitored monthly. Canopy thickness, canopy color and HLB incidence have been measured quarterly in all experimental plots. The Ferrarezi Lab organized a very successful drive-thru field day to showcase the results to growers on 10/09/2020 with 49 attendees (limited by covid-19 regulations). An estimated 24,000 acres of citrus were represented at the event (70% of the current grapefruit industry acreage, highlighting the importance of the event and my program engagement with the industry). Attendees came from local and neighboring counties including St. Lucie, Charlotte, and Okeechobee. Another field day took place on 12/10/2020 with 4 large growers and industry leaders. Overall, trees are showing morphological differences among scions/rootstocks. Although HLB is spreading in the field, symptomatic trees are building up vigorous canopies. Longer-term evaluation is required to identify the most promising scions and rootstocks to determine their profitability and capability of meeting grower and market needs.
Create new candidate hybrids. This spring focused on growing out previous crosses, selecting among hybrids, establishing source trees, propagating trees for additional trials, and collecting data from existing trials. Additional crosses among the best parental material are planned for next spring, as resources are available. Propagate and plant new field trials. Budwood increase trees of selected scions were grown, in preparation for budding trees for new rootstock trials. Trees for stage 2 rootstock trials with Valencia and Hamlin on selected released rootstocks and the best of the next generation hybrids are being grown in the greenhouse in preparation for field planting in 2021. Nursery trees for two new Stage 1 trials with 60 new rootstocks and Valencia orange were propagated in the greenhouse in preparation for field planting in fall 2021. Some planned propagation for new trials was delayed because of USDA institutional Coronavirus shutdown. Collect data from field trials. Extensive information on tree performance is collected from established field trials, and includes measurement of tree size, fruit crop, fruit quality, and pathogen titer, HLB symptoms, and assessments of tree health. Cropping data is collected during the time of scion harvest, and during this quarter data on yield and fruit quality from nine replicated trials with Valencia scion. Early fruit drop data was also collected from selected Valencia scion trials. Assessments of tree health and measurements of tree size were completed on 23 trials during this quarter. Progress continued in working through the backlog of brix, acid, and color for the fruit quality analysis of last season fruit quality assessments caused by institutional Coronavirus restrictions.Evaluate effectiveness for seed propagation of new rootstocks and develop seed sources. Some of the newest hybrid rootstocks can be uniformly propagated by seed, but others cannot. As the best rootstocks are identified through testing, seed sources are established and used to determine trueness-to-type from seed. Studies were continued this quarter to evaluate seed propagation for 25 of the most promising SuperSour hybrid rootstocks. SSR analysis of progeny is progressing more slowly than planned because of institutional Coronavirus shutdown and restrictions. Cooperative work continues to compare field performance of rootstocks propagated by seed, cuttings, and tissue culture.Posting field trial results for grower access. The USDA rootstock trials produce large amounts of information that is useful to identify the most promising of the new hybrids, as well as comparative information on the relative performance of many commercially available rootstocks. During this quarter, updated trial summaries were prepared for uploading to the website https://www.citrusrootstocks.org/.Release of superior new rootstocks for commercial use. Release of new USDA rootstocks is based on robust data from multiple trees in replicated field trials over multiple years, including information on tree survival and health, canopy size, fruit yield and fruit quality, and observations on tolerance of disease and other biotic and abiotic threats. Several of the 350 advanced Supersour rootstock hybrids in field trials are exhibiting outstanding performance in comparison with the commercial standard rootstocks. Information on some of the most promising of the next-generation USDA hybrid rootstocks was provided to CRDF for consideration as candidates for the next set of CRDF Stage 3 trials. Performance data continues to be collected, but it is anticipated that 2-3 of the most outstanding of these will be officially released in 2022-23.
1. Please state project objectives and what work was done this quarter to address them:
Objective 1: Determine how different cover crop mixtures impact soil and root health and weed cover in established commercial citrus groves.
Cover crops were last planted at the end of Nov 2020 and included sunnhemp, Austrian winter pea, daikon radish, oats, and winter rye. While the harvest in March did terminate some of the cover crops, there is still growth in the row middles. Further analysis of the soil nutrient and microbial samples collected in August 2020 found that the types of microbes performing denitrification (part of the nitrogen cycle) are completely different under the legume+non-legume cover crops compared to the other treatments. Soil organic matter has also significantly increased under cover crop treatments. Analysis of the weed data indicates cover crop planting impacts will have a significantly longer-term effect on weed suppression, which could be potentially attributed to the substantial reduction in the addition of weed seeds to the soil seed bank in the cover-cropped areas. The germination and establishment of cover crops in the second location appear to be improving over time, as noted from the comparisons of cover crop density in planted row-middles between 2019 and 2020.
Objective 2: Examine the impact of eco-mowing in conjunction with cover crops on soil and root health and weed cover in established commercial citrus groves.
Eco-mowing occurred at the end of November 2020 with the planting of the next round of cover crops. Data from Year 2 (collected in Aug 2020) is still being analyzed. Visual root growth assessments show continued root growth under cover cropping and eco-mowing, but analysis is ongoing. Soil moisture appears to be similar across all treatments, possibly due to the presence of a high water table at both sites. Quantitative data on root growth (volume, length and area), root dieback, and recent soil moisture dynamics by treatments will be presented in the next quarter and is being finalized now. Preliminary evaluation shows that conducting eco-mowing in the row-middles has unremarkable effects on weed emergence and coverage in the tree-rows.
Objective 3: Quantify the effect of cover crops and eco-mowing on tree growth and production.
Yield data for the second year of cover crops was collected in March 2021. Preliminary analysis of yield data indicates little change in with treatments in one location, and a slight increase with cover crops at the second site. Analysis of fruit quality, canopy volume, and trunk size is ongoing. Canopy and trunk size measurements, and leaf nutrient status will continue, and quantitative differences will be reported in the next quarter. However, the marginal changes are not unexpected, as trees of this age could take at least three years to show responses to treatments. We will continue to assess canopy volume and trunk size.
Objective 4: Identify the economic benefits of using cover crops
The cover crop survey is being administered via Qualtrics. Low response rates in Florida necessitated including other citrus producing states (Texas and California). To date, we identified about 60 usable surveys with respondents from each state. We attended the Citrus Show in May 2021 to encourage more Florida citrus growers to take the survey. Preliminary responses suggest that cover crops are more widely used by CA citrus growers than FL growers.
Objective 5: Communicate results to growers using field days and extension materials
Preliminary results are being presented at the Soil Science Society of America/American Society of Agronomy meeting in November. Observations on the impacts of cover cropping on weed control were presented at Weed Science Society meetings during Feb/March 2021. Information on cover crops was provided as a Tip of the Weed for the Citrus Industry magazine in May 2021. Cover crop information and preliminary data were also presented at two regional grower extension talks in March and April 2021. Discussions are underway about how to host a field day, or a virtual field day, in the summer or fall of 2021.
2. Please state what work is anticipated for next quarter:
Analysis of soil inorganic N fractions (ammonium and nitrate) for samples collected in August 2020 continues and updates on other soil/leaf nutrient variables will be provided in the next quarter. Half-yearly weed data analysis will be scheduled in the upcoming quarter. Canopy and trunk size measurements and leaf nutrient status along with root image collections and soil moisture monitoring will continue. Some soil moisture data loggers were disconnected due to wild animals and/or field equipment and data will be recovered this month and reported in the next quarter. The next set of cover crops are scheduled to be planted in June 2021. The annual soil sample collection will occur in August 2021. The composition of the mixes is still being discussed. The economics team will begin survey data analysis. They will also construct the framework for partial budgeting and assessing the cost of cover crop use and continue to collect data. Partial budgeting analysis will continue, and a report will be drafted.
3. Please state budget status (underspend or overspend, and why):
We are mostly on track with our planned budget spending, however delays in hiring and limited travel resulted in the economics team underspending.
The project has five objectives:(1) Remove the flowering-promoting CTV and the HLB bacterial pathogen in the transgenic plants(2) Graft CTV- and HLB-free buds onto rootstocks(3) Generate a large number of vigorous and healthy citrus trees(4) Plant the citrus trees in the site secured for testing transgenic citrus for HLB responses(5) Collect the field trial data In this quarter, the following activities have been conducted: (1) Transgenic citrus plants for field trials were maintained in the greenhouse. These plants will be transplanted into the field on May 20, 2021. (2) Cloning the citrus gene encoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). We have constructed a vector based on citrus DNA sequences for generating cisgenic or intragenic citrus plants. However, the transformation efficiency of the vector is extremely low. We plan to develop a transformation selection method based on citrus DNA sequences to facilitate this process. It has been shown in other plant species that an EPSPS variant is able to provide tolerance to glyphosate. We thus cloned the citrus EPSPS gene full-length coding sequence from sweet orange total cDNA. This gene will be mutated to create a similiar citrus EPSPS variant. (3) Optimizing conditions for analyzing nicotinamide adenine dinucleotide-binding activities of a group (10) of citrus lectin receptor kinase proteins using Monolith NT.115. Four different buffers with a pH value in the range of 5-8 have been tested. Although binding activity was detected for some of the proteins using the binding test model, a reliable Kd value has not been achieved. Our goal is to find the functional citrus nicotinamide adenine dinucleotide-binding recptor for generation of intragenic or cisgenic citrus plants. We are testing the transgenic citrus plants expressing the Arabidopsis nicotinamide adenine dinucleotide-binding receptor for HLB resistance/tolerance.
The purpose of this project is to optimize the CRISPR technology for citrus genome editing. This study is related to the CRDF RMC-18 Research Priorities 4AB. Objective 1. Expanding the toolbox of citrus genome editing. In this study, we will adapt StCas9, NmCas9, AsCpf1 (from Acidaminococcus), FnCpf1 (from Francisella novicida) and LbCpf1 (from Lachnospiraceae) on genome modification of citrus. Lately, we have shown CRISPR-Cpf1 can be readily used as a powerful tool for citrus genome editing. In our recent study, we employed CRISPR-LbCas12a (LbCpf1), which is derived from Lachnospiraceae bacterium ND2006, to edit a citrus genome for the first time. Our study showed that CRISPR-LbCas12a can readily be used as a powerful tool for citrus genome editing. One manuscript entitled CRISPR-LbCas12a-mediated modification of citrus has been published on Plant Biotechnol J. We are currently further optimizing LbCas12a-crRNA-mediated genome editing to make homologous biallelic mutations. We are also testing AsCpf1 and FnCpf1 for their application in citrus genome editing and generating homologous biallelic mutations. We have successfully generated both homozygous and biallelic mutations in the EBE region of LOB1 gene in pumlo. This work has been submitted for publication. We are in the process of generating homozygous and biallelic lines of other citrus varieties.Recently, we have developed multiplex genome editing toolkits for citrus including a PEG mediated protoplast transformation, a GFP reporter system that allows rapid assessment of the CRISPR constructs, citrus U6 promoters with improved efficacy, tRNA-mediated or Csy4-mediated multiplex genome editing. Using the toolkits, we have successfully conducted genome modification of embryogenic protoplast cells and epicotyl tissues. We have achieved a biallelic mutation rate of 44.4% and a homozygous mutation rate of 11.1%, indicating that the CRISPR-mediated citrus genome editing technology is mature and could be implemented in citrus genetic improvement as a viable approach. In addition, our study lay the foundation for non-transgenic genome editing of citrus. One manuscript entitled Development of multiplex genome editing toolkits for citrus with high efficacy in biallelic and homozygous mutations has been published on Plant Molecular Biology.We have successfully developed base editing tools for citrus genome editing. We have further optimized LbCas12a. Objective 2. Optimization of the CRISPR-Cas mediated genome editing of citrus. In this study, we are testing different promoters including INCURVATA2 promoter, the cell division-specific YAO promoter, and the germ-line-specific SPOROCYTELESS promoter, and ubiquitin promoter in driving the expression of Cas9 and Cpf1 orthologs. To optimize the expression of sgRNA and crRNA, we have identified multiple citrus U6 promoters and two of the citrus U6 promoters showed higher efficacy in driving gene expression in citrus than 35S promoter and Arabidopsis U6 promoter. We have further increased the mutation efficacy to 50%. We have further optimized the CRISPR/Cas9 system. Now, the biallelic mutation rate reaches 89% for Carrizo citrange and 79% for Hamlin sweet orange. We have generated one homozygous line in the promoter region of canker susceptibility genes of Hamlin. We have successfully generated one biallelic mutant of grapefruit that is canker resistant. We also successfully generated multiple biallelic and homozygous mutant lines of sweet orange that are canker resistant. Objective 3. Optimization of the CRISPR technology to generate foreign DNA free genome editing in citrus. We have conducted transient expression of Cas9/sgRNA plasmid and Cas9 protein/sgRNA ribonucleoprotein complex in citrus protoplast. We are also conducting citrus genome editing using Cpf1/crRNA plasmids and ribonucleoprotein complex in citrus protoplast. The plasmid-transformed protoplast has 1.7% editing efficiency, and the RNP-transformed samples have approximately 3.4% efficiency. The genome modified protoplast cells are undergoing regeneration. We aim to increase the efficacy to over 20% and eventually generate non-transgenic genome modified citrus. One patent has been filed on the CRISPR-Cas mediated genome editing of citrus. We have lately optimized the citrus protoplast isolation and manipulation, our data showed that more than 98% of the isolated protoplasts were alive. We regularly obtained a transfection efficiency of approximately 66% or above. Genome modified lines in canker are being regenerated. We are editing 6 putative HLB susceptibility genes for sweet orange.
True sweet oranges: New OLL sweet orange candidates repeated for early-mid season maturity (consecutive years), including OLL-FB-4-13 (mentioned in previous report) and OLL-FB-9-33. OLL-FB-4-13, OLL-FB-9-33, and OLL-FB-1-22 were selected for inclusion in the CRDF scion trial, and were entered into the Parent Tree Program (using special slots provided by Ben Rosson and Kristen Aslan). A new selection OLL-FB-4-08 was identified with January 1 maturity (first time fruiting). Potential HLB resistance from ‘gauntlet’ rootstock candidates: qPCR was run on leaves sampled from more than 625 gauntlet rootstock trees at the USDA Picos Farm, and 54% tested questionable or negative for active infection. Among these, qPCR was run on roots on 60 of the best performing trees. Among these, 13 trees had root ct values of 40, meaning no bacteria was detected in the roots. Two of these also had leaves that tested negative and 4 others questionable. We also identified 19 gauntlet rootstocks in this group that were negative for active root infections, and among these 4 had no active infections in the leaves, and 9 had questionable leaf infections. Considering the severe fruit quality problems, especially from young trees the past 2 seasons in Florida, we also focused on identifying the best of these producing high brix fruit right off the bat. 3 potentially resistant rootstocks were identified producing high brix fruit: N+HBPxOrange19-12-3 (a backcross of UFR-4 onto it’s mom), B11-R5-T25-11-3 (a Flying Dragon hybrid with potential for high density plantings), and A+HBPxCH+50-7-12-11 (also tree-size controlling, with UFR-6 pollen parent). Several gauntlet rootstocks are also showing an apparent tolerance mechanism, as they show consistently good tree health, but had higher CLas titers in the roots than the trees above; these include a few hybrids made with SugarBelle. Identification of probable zygotic rootstocks at St. Helena showing good HLB tolerance: 5 trees on apparent zygotic rootstocks were identified producing 3 – 3-5 boxes of Vernia/Valquarius fruit per tree with 12 – 12.5 brix. One is a tetraploid from Orange 12 (Nova+HBP-derived), two from HBPummelo x Shekwasha, and two from HBPummelo (open pollination). One rootstock genotype has been recovered, and scaffold roots of the other selections have been cut in efforts to retreive the rootstock germplasm. Fruit quality data was collected from the Trailer Park trial – two combinations of 3.75 year-old trees made 6.0 and 5.9 lbs solids per box: Valencia B9-65/UFR-4 and OLL-4/UFR-4, respectively. Yield data was collected from the St. Helena trial. There was no issue with fruit drop, and lbs. solids across more than 50 rootstocks averaged around 6.0 lbs. solids per box. Overall yield increased slightly. These positive results were attributed to the continued year-round use of CRF containing an enhanced micronutrient package. Yield data was collected from the Peace River Valencia/rootstock trial, and the Duda trials. Data analysis and entry onto the Rootstock Data Website: annual updates included: Duda Valencia APS rootstock trial, Duda Vernia rootstock trial, Smoak Valencia rootstock trial, Bryan Paul Doe Hill multi-scion rootstock trial, and the IMG navel/grapefruit rootstock trial. Data analyses for subsequent trial updates was performed for (in progress): Banack multi-scion rootstock trial, Hidden Golf Trailer Park trial, Wheeler Bros. scion/rootstock trial, Orie Lee OLL clone/rootstock demonstration trial, Teaching Block scion/rootstock trial, Bryan Paul Doe Hill Grove multi-scion/rootstock trial, Citra (PSREU) scion/rootstock trial, Hammond IR Minneola rootstock trial, IR Marsh grapefruit/rootstock trial, the St. Helena rootstock survey trial, and trial and the Peace River Valencia/rootstock trial.
Update for this quarter:A substantial infrastructure renewal project is underway at the Picos location on USDA base funding. A full renovation of the water management system has been completed and will provide improved storm protection. Drainage channels were re-dug and cleared of vegetation. Culverts were inspected and replaced if damaged. The road surfaces have also improved to provide better vehicle access. Tree maintenance and field trials have received priority support under current conditions. Full assessments have been conducted on USDA transgenic plantings by onsite personnel. UF collaborators have been permitted into the test site; samples and data have been collected. A manuscript detailing results from the canker resistance assessment of replicated trifoliate and trifoliate hybrids planted in collaboration with NCGR-Citrus/Dates and UCRiverside was accepted for publication in Hortscience as Incidence of Asiatic Citrus Canker on Trifoliate Orange and its Hybrid Accessions in a Florida Field Planting. This site is also participating in a trial program to use drone based aerial photography for mapping and HLB assessment. The primary BRS permit, which covers the transgenic materials planted by Z. Mou, J. Jones, T. McNellis as well as USDA scientists has been renewed (AUTH – 0000043619 effective 1/27/2021). Recent quarters:An additional permit has been approved (AUTH – 0000043620 effective 12/17/2020) for material with “Confidential Business Information” for a project led by R. Shatters. Stover analyzed data on canker incidence for as Incidence of Asiatic Citrus Canker on Trifoliate Orange and its Hybrid Accessions in a Florida Field Planting. Most notably: Almost all accessions with lower ACC lesion incidence were hybrids vs. pure trifoliate, though a few pure Poncirus had lower ACC than most. Based on chloroplast genome data from 57K Affymetrix SNP chip, provided by M. Roose, 11 of 33 reported seed parentage for hybrids was inaccurate, convention of female first was not followed. Of 34 hybrids validated, similar numbers had Poncirus, grapefruit, and sweet orange chloroplasts. Chloroplast type did not affect ACC incidence, but in each year accessions with grapefruit chloroplasts had small but statistically higher ACC severity than those with Poncirus chloroplasts. Hybrids of Citrus with Poncirus have markedly reduced ACC sensitivity compared to Poncirus, indicating that this trait is readily overcome in breeding. A manuscript has been prepared and submitted from this data. Previously established at the site:A number of trials are underway at the Picos Test Site funded through the CRDF. A detailed current status is outlined below this paragraph. Continuation of an experiment on pollen flow from transgenic trees. FF-5-51-2 trees are slightly more than 1000 ft from the US-802, and are self-incompatible and mono-embryonic. If pollen from transgenic trees is not detected from open-pollination, it should reduce isolation distances required by BRS. Early-flowering transgenic Carrizo (flowered ex-vitro within five months of seed sowing, and used at 12 months) was used to pollinate some of the same FF-5-51-2 What should be the final samples from the C. Ramadugu-led Poncirus trial (#3 below) completed preparation and were shipped in ethanol to UC Riverside. Availability of the test site for planting continues to be announced to researchers. Plantings:1) The UF Grosser, Dutt and Gmitter transgenic effort has a substantial planting of diverse transgenics. These are on an independent permit, while all other transgenics on the site are under the USDA permits.2) Under the Stover permit, a replicated planting of 32 transgenic trees and controls produced by Dr. Jeff Jones at UF were planted. These trees include two very different constructs, each quite specific in attacking the citrus canker pathogen. 3) A broad cross-section of Poncirus derived material is being tested by USDA-ARS-Riverside and UCRiverside, and led by Chandrika Ramadugu. These are seedlings of 82 seed source trees from the Riverside genebank and include pure trifoliate accessions, hybrids of Poncirus with diverse parents, and more advanced accessions with Poncirus in the pedigree. Plants are replicated and each accession includes both graft-inoculated trees and trees uninfected at planting. 4) More than 100 citranges, from a well-characterized mapping population, and other trifoliate hybrids (+ sweet orange standards) were planted in a replicated trial in collaboration with Fred Gmitter of UF and Mikeal Roose of UCRiverside. Plants were monitored for CLas titer 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. Manuscripts have been published reporting HLB tolerance associated QTLs and differences in ACP colonization. Trees continue to be useful for documenting tolerance in a new NIFA project.5) A replicated Fairchild x Fortune mapping population was planted at the Picos Test Site in an effort led by Mike Roose to identify loci/genes associated with tolerance. This planting also includes a number of related hybrids (including our easy peeling remarkably HLB-tolerant 5-51-2) and released cultivars. Genotyping, HLB phenotyping and growth data have been collected and will continue to be conducted under a new NIFA grant.6) Valencia on UF Grosser tertazyg rootstocks have been at the Picos Test Site for several years, having been CLas-inoculated before planting, and several continue to show excellent growth compared to standard controls (Grosser, personal comm.).7) In a project led by Fred Gmitter, there is a planting of 1132 hybrids of C. reticulata x C. latipes. C. latipes is among the few members of genus Citrus reported to have HLB resistance, and it is expected that there will be segregation for such resistance. The resulting plants may be used in further breeding and may permit mapping for resistance genes. 8) Seedlings with a range of pedigree contributions from Microcitrus are planted in a replicated trial, in a collaboration between Malcolm Smith (Queensland Dept. of Agriculture and Fisheries) and Ed Stover. Microcitrus is reported to have HLB resistance, and it is expected that there will be segregation for such resistance. The resulting plants may be used in further breeding and may permit mapping for resistance genes. 9) Conventional scions on Mthionin-producing transgenic Carrizo are planted from the Stover team and are displaying superior growth to trees on control Carrizo.10) Planting of USDA Mthionin transgenics with 108 transgenic Hamlin grafted on wild type Carrizo (7 events represented), 81 wild type Hamlin grafted on transgenic Carrizo (16 events represented) and 16 non-transgenic controls. 11) Grafted trees of conventional sweet orange and grapefruit scions on transgenic rootstock expressing antimicrobial citrus-thionin and bacterial recognition domain fusion proteins (165 trees with controls) as a collaboration between USDA and Innate Immunity.12) Planting was made of transgenics from Zhonglin Mou of UF under Stover permit, with 19 trees of Duncan, each expressing one of four resistance genes from Arabidopsis, and 30 Hamlin expressing one of the genes, along with ten non-transgenic controls of each scion type. 13) Transgenic trees expressing FT-ScFv (12 transgenic and 12 control) to target CLas from Tim McNellis of Penn State14)Numerous promising transgenics identified by the Stover lab in the last two years have been propagated and will be planted in the test site.
This quarter:The Bowman lab has established three new experiments with grafted Valencia trees on groups of rootstocks which were inoculated with ACP during the quarter (Janyuary to March 2021). Each experiment compares replications on nine different rootstocks. For that, 189 trees were inoculated with 3,780 ACP from the positive ACP colonies. Periodic evaluation of tree health, growth, and CLas titer via PCR were conducted on trees from rootstock experiments that were inoculated during the previous months following a set schedule. Periodic colony checks were conducted by PCR to maintain CLas positive colonies.The Stover lab conducted weekly detached leaf assays (DLAs) challenging transgenic citrus with CLas inoculated by infected ACP in the lab, which is used to identify best performing transgenic events (transgenics varying by position of transgene insertion etc.). Six detached leaf assays experiments, envolving individual 290 leaves, were inoculated using 2,900 Clas infected ACPs in this quarter. Transgenic material tested in DLAs were of three constructs (ONYX and two Chimerical Thionin), and a total of 46 independent events were tested alongside WT controls.The balance of Covid-19 restricted lab hours was spent processing the leaf and ACP samples in preparation for CLas qPCR. We continue to see substantial ACP mortality from feeding on CLas-killing transgenic leavesIn addition 600 CLas+ ACP were provided to Dr. YongPing Duan of USDA.Project rationale and focus:The driving force for this three-year project is the need to evaluate citrus germplasm for tolerance to HLB, including germplasm transformed to express proteins that might mitigate HLB, which requires citrus be inoculated with CLas. Citrus can be bud-inoculated, but since the disease is naturally spread by the Asian citrus psyllid, the use of psyllids for inoculations more closely resembles “natural infection”, while bud-inoculations might overwhelm some defense responses. CRDF funds supported high-throughput inoculations to evaluate HLB resistance in citrus germplasm developed by Drs. Ed Stover and Kim Bowman. The funds cover the costs associated with establishing and maintaining colonies of infected psyllids; equipment such as insect cages; PCR supplies for assays on psyllid and plant samples from infected colonies; and two GS-7 USDA technicians. A career base-funded USDA technician is also assigned ~50% to the program. USDA provides greenhouses, walk-in chambers and laboratory space to accommodate rearing and inoculations. Previous quarter:Dean Gabriel of UF, and USDA scientists Kim Bowman, Ed Stover and G avin Poole have all run experiments totalling ~7,300 ACP. Samples have all been collected on-time from ongoing experiments. All samples collected, that have not been analyzed, have been processed for qPCR.
Update for this quarter:A substantial infrastructure renewal project is underway at the Picos location on USDA base funding. A full renovation of the water management system has been completed and will provide improved storm protection. Drainage channels were re-dug and cleared of vegetation. Culverts were inspected and replaced if damaged. The road surfaces have also improved to provide better vehicle access. Tree maintenance and field trials have received priority support under current conditions. Full assessments have been conducted on USDA transgenic plantings by onsite personnel. UF collaborators have been permitted into the test site; samples and data have been collected. A manuscript detailing results from the canker resistance assessment of replicated trifoliate and trifoliate hybrids planted in collaboration with NCGR-Citrus/Dates and UCRiverside was accepted for publication in Hortscience as Incidence of Asiatic Citrus Canker on Trifoliate Orange and its Hybrid Accessions in a Florida Field Planting. This site is also participating in a trial program to use drone based aerial photography for mapping and HLB assessment. The primary BRS permit, which covers the transgenic materials planted by Z. Mou, J. Jones, T. McNellis as well as USDA scientists has been renewed (AUTH – 0000043619 effective 1/27/2021). Recent quarters:An additional permit has been approved (AUTH – 0000043620 effective 12/17/2020) for material with “Confidential Business Information” for a project led by R. Shatters. Stover analyzed data on canker incidence for as Incidence of Asiatic Citrus Canker on Trifoliate Orange and its Hybrid Accessions in a Florida Field Planting. Most notably: Almost all accessions with lower ACC lesion incidence were hybrids vs. pure trifoliate, though a few pure Poncirus had lower ACC than most. Based on chloroplast genome data from 57K Affymetrix SNP chip, provided by M. Roose, 11 of 33 reported seed parentage for hybrids was inaccurate, convention of female first was not followed. Of 34 hybrids validated, similar numbers had Poncirus, grapefruit, and sweet orange chloroplasts. Chloroplast type did not affect ACC incidence, but in each year accessions with grapefruit chloroplasts had small but statistically higher ACC severity than those with Poncirus chloroplasts. Hybrids of Citrus with Poncirus have markedly reduced ACC sensitivity compared to Poncirus, indicating that this trait is readily overcome in breeding. A manuscript has been prepared and submitted from this data. Previously established at the site:A number of trials are underway at the Picos Test Site funded through the CRDF. A detailed current status is outlined below this paragraph. Continuation of an experiment on pollen flow from transgenic trees. FF-5-51-2 trees are slightly more than 1000 ft from the US-802, and are self-incompatible and mono-embryonic. If pollen from transgenic trees is not detected from open-pollination, it should reduce isolation distances required by BRS. Early-flowering transgenic Carrizo (flowered ex-vitro within five months of seed sowing, and used at 12 months) was used to pollinate some of the same FF-5-51-2 What should be the final samples from the C. Ramadugu-led Poncirus trial (#3 below) completed preparation and were shipped in ethanol to UC Riverside. Availability of the test site for planting continues to be announced to researchers. Plantings:1) The UF Grosser, Dutt and Gmitter transgenic effort has a substantial planting of diverse transgenics. These are on an independent permit, while all other transgenics on the site are under the USDA permits.2) Under the Stover permit, a replicated planting of 32 transgenic trees and controls produced by Dr. Jeff Jones at UF were planted. These trees include two very different constructs, each quite specific in attacking the citrus canker pathogen. 3) A broad cross-section of Poncirus derived material is being tested by USDA-ARS-Riverside and UCRiverside, and led by Chandrika Ramadugu. These are seedlings of 82 seed source trees from the Riverside genebank and include pure trifoliate accessions, hybrids of Poncirus with diverse parents, and more advanced accessions with Poncirus in the pedigree. Plants are replicated and each accession includes both graft-inoculated trees and trees uninfected at planting. 4) More than 100 citranges, from a well-characterized mapping population, and other trifoliate hybrids (+ sweet orange standards) were planted in a replicated trial in collaboration with Fred Gmitter of UF and Mikeal Roose of UCRiverside. Plants were monitored for CLas titer 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. Manuscripts have been published reporting HLB tolerance associated QTLs and differences in ACP colonization. Trees continue to be useful for documenting tolerance in a new NIFA project.5) A replicated Fairchild x Fortune mapping population was planted at the Picos Test Site in an effort led by Mike Roose to identify loci/genes associated with tolerance. This planting also includes a number of related hybrids (including our easy peeling remarkably HLB-tolerant 5-51-2) and released cultivars. Genotyping, HLB phenotyping and growth data have been collected and will continue to be conducted under a new NIFA grant.6) Valencia on UF Grosser tertazyg rootstocks have been at the Picos Test Site for several years, having been CLas-inoculated before planting, and several continue to show excellent growth compared to standard controls (Grosser, personal comm.).7) In a project led by Fred Gmitter, there is a planting of 1132 hybrids of C. reticulata x C. latipes. C. latipes is among the few members of genus Citrus reported to have HLB resistance, and it is expected that there will be segregation for such resistance. The resulting plants may be used in further breeding and may permit mapping for resistance genes. 8) Seedlings with a range of pedigree contributions from Microcitrus are planted in a replicated trial, in a collaboration between Malcolm Smith (Queensland Dept. of Agriculture and Fisheries) and Ed Stover. Microcitrus is reported to have HLB resistance, and it is expected that there will be segregation for such resistance. The resulting plants may be used in further breeding and may permit mapping for resistance genes. 9) Conventional scions on Mthionin-producing transgenic Carrizo are planted from the Stover team and are displaying superior growth to trees on control Carrizo.10) Planting of USDA Mthionin transgenics with 108 transgenic Hamlin grafted on wild type Carrizo (7 events represented), 81 wild type Hamlin grafted on transgenic Carrizo (16 events represented) and 16 non-transgenic controls. 11) Grafted trees of conventional sweet orange and grapefruit scions on transgenic rootstock expressing antimicrobial citrus-thionin and bacterial recognition domain fusion proteins (165 trees with controls) as a collaboration between USDA and Innate Immunity.12) Planting was made of transgenics from Zhonglin Mou of UF under Stover permit, with 19 trees of Duncan, each expressing one of four resistance genes from Arabidopsis, and 30 Hamlin expressing one of the genes, along with ten non-transgenic controls of each scion type. 13) Transgenic trees expressing FT-ScFv (12 transgenic and 12 control) to target CLas from Tim McNellis of Penn State14)Numerous promising transgenics identified by the Stover lab in the last two years have been propagated and will be planted in the test site.
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