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.
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.
Starting with the 2021, Juvenile Tissue Citrus Transformation Facility (JTCTF) started operating as an EBA unit. As a result of this transition, relationship between the clients and JTCTF is redefined in a sense that clients will be paying for the work JTCTF has done and not for the produced plants. Towards this goal, JTCTF has created a price list for the services it offers. In accordance with changed status of JTCTF, administration at CREC has established an account that will be used for financial transaction associated with facility’s operation. In the first three months of 2021, Juvenile Tissue Citrus Transformation Facility (JTCTF) accepted five new orders. Four of those orders were for transgenic sweet orange plants and one for Duncan grapefruit plants. One of the orders is associated with the existing USDA grant, while four other orders will be paid for. Certain number of orders that were expected because JTCTF was designated subcontractor have not materialized because those clients have not started working on projects for which they received funding as they had difficulties in hiring personnel.At this time, JTCTF has four employees but one has not worked since December of 2020 because of unresolved immigration status. Another employee is working during the night which has allowed us to return to the level of attendance of pre-COVID period. As a result, the facility operated at about 75% capacity. Between January and April, JTCTF produced 10 plants. Those include five Duncan grapefruit plants transformed with HGJ87 and HGJ88 plasmids. The rest are sweet orange plants transformed with pXH-H1 and pXH-H2 plasmids.
Starting with the 2021, Juvenile Tissue Citrus Transformation Facility (JTCTF) started operating as an EBA unit. As a result of this transition, relationship between the clients and JTCTF is redefined in a sense that clients will be paying for the work JTCTF has done and not for the produced plants. Towards this goal, JTCTF has created a price list for the services it offers. In accordance with changed status of JTCTF, administration at CREC has established an account that will be used for financial transaction associated with facility’s operation. In the first three months of 2021, Juvenile Tissue Citrus Transformation Facility (JTCTF) accepted five new orders. Four of those orders were for transgenic sweet orange plants and one for Duncan grapefruit plants. One of the orders is associated with the existing USDA grant, while four other orders will be paid for. Certain number of orders that were expected because JTCTF was designated subcontractor have not materialized because those clients have not started working on projects for which they received funding as they had difficulties in hiring personnel.At this time, JTCTF has four employees but one has not worked since December of 2020 because of unresolved immigration status. Another employee is working during the night which has allowed us to return to the level of attendance of pre-COVID period. As a result, the facility operated at about 75% capacity. Between January and April, JTCTF produced 10 plants. Those include five Duncan grapefruit plants transformed with HGJ87 and HGJ88 plasmids. The rest are sweet orange plants transformed with pXH-H1 and pXH-H2 plasmids.
1. Please state project objectives and what work was done this quarter to address them: Objective 1. Investigate effects of rootstock propagation method and the interaction with rootstock on root structure, root growth, and tree performance during the first 3 years of growth in the field.Bimonthly root growth measurements with the minirhizotron imaging system continued in all trials. Leaves were collected in trials 2 and 3 for CLas detection; samples are being analyzed. Trials 2 and 3 third-year horticultural data collection (tree size, health ratings, etc.) was completed. We also counted and collected fruits for fruit quality analysis and yield determination in trials 2 and 3. There were very few fruit in trial 2, but fruit quality analysis was completed for trial 3. We are working on completing the analysis of the year 3 data of trials 2 and 3 to be included in a publication of all three years of data. Objective 2. Investigate if trees on rootstocks propagated by tissue culture or cuttings differ in susceptibility to Phytophthora-induced decline or wind-induced blow-over compared with trees on rootstocks propagated by seed.Monthly root growth measurements with the rhizotron imaging system continued. We are still working on the PCR and ELISA assays. 2. Please state what work is anticipated for next quarter: We will continue with our minirhizotron root imaging and other data collection. We will continue with the statistical analysis and interpretation of all data collected. A Citrus Industry Magazine article will be prepared sumarizing findings of this project. 3. Please state budget status (underspend or overspend, and why): Approximately 65% of funds have been spent, which is somewhat underspent due to Covid-19 related complications that affected research, travel, and hiring of personnel.
1. Please state project objectives and what work was done this quarter to address them: Objective 1. Investigate rootstock effects on horticultural performance of Valencia and Hamlin trees commercially grown under HLB-endemic conditions using standardized field data collection procedures.We conducted additional HLB foliar disease symptom and canopy color ratings at both Valencia trials for seasonal comparison. We also collected leaves for additional nutrient analysis and CLas detection.Valencia fruit samples were collected at the Basinger location for fruit quality analysis in advance of the harvesting. Fruit quality analysis was completed for these samples at the CREC pilot plant.We are working on the leaf CLas analysis.We are continuing with the statistical analysis of all new data and are working on a manuscript for publication of three years of Valencia data. Objective 2. Develop outreach to transfer information to growers and other industry clientele.Nothing to report in this quarter. 2. Please state what work is anticipated for next quarter: Fruits will be collected from the Valencia trees at the Lake Wales (Camp Mack) location for fruit quality analysis, and yield will be assessed. We will continue with the data analysis. A rootstock seminar including trial updates will be given in June at SWFREC (or virtual, depending on the Covid-19 situation). 3. Please state budget status (underspend or overspend, and why): Approximately 64% of funds have been spent, which is somewhat underspent due to Covid-19 related complications that affected research, travel, and hiring of personnel.
1. Please state project objectives and what work was done this quarter to address them: Objective 1. Investigate effects of rootstock propagation method and the interaction with rootstock on root structure, root growth, and tree performance during the first 3 years of growth in the field.Bimonthly root growth measurements with the minirhizotron imaging system continued in all trials. Leaves were collected in trials 2 and 3 for CLas detection; samples are being analyzed. Trials 2 and 3 third-year horticultural data collection (tree size, health ratings, etc.) was completed. We also counted and collected fruits for fruit quality analysis and yield determination in trials 2 and 3. There were very few fruit in trial 2, but fruit quality analysis was completed for trial 3. We are working on completing the analysis of the year 3 data of trials 2 and 3 to be included in a publication of all three years of data. Objective 2. Investigate if trees on rootstocks propagated by tissue culture or cuttings differ in susceptibility to Phytophthora-induced decline or wind-induced blow-over compared with trees on rootstocks propagated by seed.Monthly root growth measurements with the rhizotron imaging system continued. We are still working on the PCR and ELISA assays. 2. Please state what work is anticipated for next quarter: We will continue with our minirhizotron root imaging and other data collection. We will continue with the statistical analysis and interpretation of all data collected. A Citrus Industry Magazine article will be prepared sumarizing findings of this project. 3. Please state budget status (underspend or overspend, and why): Approximately 65% of funds have been spent, which is somewhat underspent due to Covid-19 related complications that affected research, travel, and hiring of personnel.
1. Please state project objectives and what work was done this quarter to address them: Objective 1. Investigate rootstock effects on horticultural performance of Valencia and Hamlin trees commercially grown under HLB-endemic conditions using standardized field data collection procedures.We conducted additional HLB foliar disease symptom and canopy color ratings at both Valencia trials for seasonal comparison. We also collected leaves for additional nutrient analysis and CLas detection.Valencia fruit samples were collected at the Basinger location for fruit quality analysis in advance of the harvesting. Fruit quality analysis was completed for these samples at the CREC pilot plant.We are working on the leaf CLas analysis.We are continuing with the statistical analysis of all new data and are working on a manuscript for publication of three years of Valencia data. Objective 2. Develop outreach to transfer information to growers and other industry clientele.Nothing to report in this quarter. 2. Please state what work is anticipated for next quarter: Fruits will be collected from the Valencia trees at the Lake Wales (Camp Mack) location for fruit quality analysis, and yield will be assessed. We will continue with the data analysis. A rootstock seminar including trial updates will be given in June at SWFREC (or virtual, depending on the Covid-19 situation). 3. Please state budget status (underspend or overspend, and why): Approximately 64% of funds have been spent, which is somewhat underspent due to Covid-19 related complications that affected research, travel, and hiring of personnel.
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