1. Please state project objectives and what work was done this quarter to address them:
This project aims to test SAMP efficacy tests in the field and greenhouse on important citrus varieties in Florida and California. Dr. Megan Dewdney is in charge of all the field trials in FL. A large scale field test on 240 young Hamlin trees with 3 different treatments and 1 control set was laid out in a completely randomized design. A second field test on infected 4-year old Hamlin sweet orange bearing trees was also initiated. In addition, we also performed new greenhouse tests on important citrus varieties in collaboration with Dr. Kris Godfrey (UCD). Fruthermore, we also have surprising findings that SAMP may also suppress CLas in ACP in collaboration with entomologists Dr. Kris Godfrey and Dr. Kerry Mauck (UCR). Thus, SAMP is the only antimicrobial peptide that we know of that has three biological functions: 1) bactericidal activity, 2) inducing host immunity activity and 3) inhibiting ACP activity.
2. Please state what work is anticipated for next quarter:
Test I: Field Test I- SAMP efficacy test on 240 newly planted Hamlin sweet orange trees
Dr. Megan Dewdney started a field trial with a total 240 young Hamlin sweet orange trees to determine the SAMP efficacy on newly planted trees against HLB. The trial will last for at least 2-3 years. The experiment was laid out in a completely randomized design with three treatments and sixty trees per treatment. The treatments are 1. Untreated control, 2. Treatment in the nursery 1 week prior to planting and field applications (10 µM in 158 ml/tree) every 2 months, 3. No nursery treatment with field applications every 2 months starting 10 days post-planting, 4. The injection treatment with Invaio simple injection device. It is expected that the treatment volume will increase as the trees grow over the next two years (Figure 1). We will monitor the tree growth, disease rating, and CLas titer in trees and ACP after this summer.
Test II: Field Test II- SAMP efficacy test on 125 infected 4-year old Hamlin sweet orange trees.
The second field trial was set up to test if SAMP can remediate an HLB-affected young citrus grove. This experiment is ongoing in the Ridge region of Florida on the deep sandy soils. We have located a four-year-old commercial Hamlin sweet orange grove in the Lake Wales region. The trial involves total of 125 bearing trees and was laid out as a randomized complete block design with five blocks and five replicates per block. The treatments in this trial were an untreated control and bimonthly foliar application. Three additional treatments from an additional funding source are to look at the peptide as various injection treatments. These treatments are 2 injections in spring and summer, 3 injections in spring, summer, and fall, and 3 injections per year starting at the same time as the foliar spray (Figure 2). The treatment started from August 2021. We expect to monitor the trial at least for 2 years. We will monitor the tree growth, disease rating, and CLas titer in trees and ACP.
Test III: Three greenhouse trials with important varieties of California which were conducted in BSL3-UC Davis :
A. Tests of SAMP treatment using trunk injection and foliar spray on the early infected tree with Washington Navel orange and Tengo Mandarin trees (Table 1, Figure 3 and 4).Total of 30 Navel trees and 22 Tengo trees
B. Test of using SAMP foliar spray to protect trees from CLas infection with 16 Washington Navel orange.
SAMP has clear disease control and plant protection effect on all the three trials.
3. Please state budget status (underspend or overspend, and why): The budget was spent in full to purchase 100g SAMP.
Updates for this quarter:Site management and field trials are progressing well. The site remains available for access to all researchers and all regulatory protocols for the care and disposal of transgenic material are being observed. The trees have been hedged and topped to promote growth, open canopies and access to nutritional sprays. The foliar spray program, applied every two weeks, includes: 435 horticultural oil, Magna-Bon CS2005 copper, 20-10-20 and/or 15-5-15 CA-MG, N-Sure 28-0-0, Keyplex 1400 DP, and Ocean Organics 0.2-0-6. Wedgeworth’s 12-13-15, 2Mg slow release nitrogen and potassium with greening guard was also applied above the root zone in March. Discussions have begun with APHIS-BRS to set conditions for new or expanded transgenic release permits to allow field trials of a novel system using engineered tissue to delivery therapeutic peptides to non-transgenic trees. This effort is in support of NIFA project 2020-70029-33176, Therapeutic Molecule Evaluation and Field Delivery Pipeline for Solutions to HLB, with field trials expected to begin later this year once all regulatory requirements are met. The testing site is also being actively utilized this breeding season. Crosses have been made with transgenic pollen to help elucidate if sexual embryos can be rescued from polyembryonic females, making use of the transgenic markers to determine if sexual hybridization is successful. This could greatly impact both transgenic and conventional breeding efforts by allowing the use of polyembryonic citrus accessions as females. A third year of crossings has also been made with the early flowering (FT) transgenics, continuing the work described below. Recent quarters:A significant USDA-funded infrastructure project has been completed, fully renovating the water management systems and significantly improving storm and flood protection. USDA has also acquired a topper hedger to facilitate canopy management and reflect the best practices of commercial farms. An additional BRS transgenic release permit was approved (AUTH – 0000043620) for material with confidential business information (CBI) for a project led by R. Shatters. The primary BRS permit has also been renewed and amended to include a new construct from UF (Now AUTH – 0000206702). The annual site review from APHIS/BRS has been conducted successfully. Four new plantings from UF expressing resistance genes and two new plantings from USDA-CRADA partners expressing antimicrobial peptides and anti-CLas plantibodies have been made. With recent plantings, the transgenic site is operating at full capacity. Fall 2021 assessments were completed for USDA plantings as described below for trials #8, #9, #10, #11 and #15. Fruit have been harvested from the second year of controlled crosses using pollen from early flowering (FT) transgenics on traditional varieties maintained in the testing site. Seeds from these fruit and those of future crossings will be used to assess inheritability of the phenotype and for CRISPR gene stacking to combine genome editing with accelerated breeding traits. The UCRiverside-led trifoliate and trifoliate hybrid trial has concluded, a manuscript regarding identified HLB-tolerance is in preparation; and these trees can be removed as needed to make space available for future plantings. Dr. Stover analyzed data on canker incidence for this trial and a manuscript detailing these results has been published in HortScience DOI: 10.21273/HORTSCI15684-20. Previously established at the site:A number of trials are underway at the CRDF funded Picos Test Site. A detailed current status is outlined below this paragraph. We continue investigation of potential pollen flow from transgenic trees to assess the possibility of reducing the isolation distances. Availability of the test site for planting continues to be announced to researchers. Supplemental: Full details on trial 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 analysis 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) Multiple plantings with grafted trees of l Hamlin, Valencia and grapefruit scions on transgenic rootstock expressing antimicrobial citrus-thionin and bacterial recognition domain fusion proteins (219 trees with controls) as a collaboration between USDA and the New Mexico Consortium.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) Planting from Zhonglin Mou of UF that includes transgenic grapefruit (31 plants) and sweet orange (60 plants) scions expressing two different resistance genes and grafted on WT swingle rootstocks; as well as non-transgenic controls. 14) Transgenic trees expressing FT-ScFv (12 transgenic and 12 control) to target CLas from Tim McNellis of Penn State15)Numerous promising transgenics identified by the Stover lab in the last two years have been propagated and will be planted in the test site.
1. Please state project objectives and what work was done this quarter to address them: This report covers the period of December 1, 2021 – February 28, 2022. 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). Chad Vosburg graduated in December, 2021 with his M.S. degree in Plant Pathology and his M.S. Thesis was approved. The PI reviewed the M.S. thesis elements related the HLB resistance tests that we completed in the greenhouse and an ongoing test in the field at Fort Pierce. An initial draft of a manuscript describing these results was prepared by the PI. 2. Please state what work is anticipated for next quarter:In the next quarter, the PI will finalized the manuscript in collaboration with Chad Vosburg and other co-workers. The manuscript will be submitted by the PI to a peer-reviewed journal for review in the next quarter. We anticipate requesting another no-cost extension to allow us to use grant funds to pay publication costs for the manuscript. Review, acceptance and publication could take several additional months, at least. 3. Please state budget status (underspend or overspend, and why):The project remains underspent in part due to support for graduate stipends and tuition waviers from the Plant Pathology & Environmental Microbiology Department of Penn State and the Penn State Graudate School. We have sufficient funds remaining for publication costs for the upcoming manuscript.
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. This method has been succefully used to generate non-transgenic biallelic mutants of sweet orange. 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. ErCas12a has been succes for non-transgenic gene editing of embryogenic Hamlin sweet orange protoplast cells. We are editing 6 putative HLB susceptibility genes for sweet orange. One biallelic mutant line has been generated for ACD2. We have successfully adapted the adenine base editors (ABE) to modify the TATA box in the promoter region of the canker susceptibility gene LOB1 from TATA to CACA in grapefruit and Hamlin sweet orange. Inoculation of the TATA-edited plants with the canker pathogen Xanthomonas citri subsp. Citri (Xcc) demonstrated that the TATA-edited plants were resistant to Xcc. In addition, cytosine base editors (CBE) was successfully used to edit the acetolactate synthase (ALS) gene of Carrizo citrange, a hybrid of Citrus sinensis `Washington’ sweet orange X Poncirus trifoliata. Editing the ALS genes conferred resistance of Carrizo to the herbicide chlorsulfuron. Two ALS-edited Carrizo plants did not show green florescence although the starting construct for transformation contains a GFP expression cassette. We performed PCR amplification for Cas9 gene in the mutant plants and found that Cas9 gene was undetectable in the herbicide resistant citrus plants. This indicates that the ALS edited plants are transgene-free, representing the first transgene-free gene-edited citrus using the CRISPR technology. In summary, we have successfully adapted the base editors for precise citrus gene editing. The CBE base editor has been used to generate transgene-free citrus via transient expression. This work has been been published by Frontiers in genome editing. In addition, we are further developing non-transgenic genome editing technology using ALS gene as a selection marker.
True sweet oranges: This was a very exciting quarter for identifying true sweet orange candidates to replace Hamlin. Multiple Vernia somaclone-derived seedling clones repeated for early maturity (first week of December) for the 3rd consecutive year. The best of these was MB-R25-T7, which had a 16.17 ratio and the highest brix (11.16), and significantly higher soluble solids than the Hamlin control. The Vernia somaclone-seedling population, planted in the Mathew Block at the Orie Lee Alligator Grove, has never had psyllid control. We began supplementing trees with CRF about 2 years ago to prevent decline. Trees were commercially harvested last week, and there was still no fruit drop observed. At this time, the brix in the MB-R25-T7 was over 13. This new Vernia clone was entered into the PTP in January. We also discovered a new early-maturing OLL clone in the somaclone-derived seedling block of OLLs (located in the Lee Family Home Grove in St. Cloud). Clone FB-R7-T35 had a 14.12 ratio in mid-January with 12 brix, 5.7 lbs. solids/box (exceptionally high for a juvenile tree) with a 37.3 juice color score. This clone had an extended juvenility and did not have fruit last year. This new OLL clone will be entered into the PTP the first week of April, when new slots become available. Both of these clones are being propagated at the CREC to expedite further evaluation (with permission from DPI). Promising orange-like hybrid: Triploid hybrid C7-11-7 [Sugar Belle x (Murcott + Succari sweet orange)] produces fruit morphologically indistinguishable from a true sweet orange, but with better external color. The original tree is exceptionally productive and appears to have HLB tolerance slightly better than standard sweet oranges. Juice from fruit of the original tree (with HLB for many years) had 13.87 brix, 15.1 ratio, 39.3 color score and 8.31 lbs. solids per box (double the state average for Hamlin!) in mid-January. This selection will also be entered into the PTP in early April. Potential HLB tolerance/resistance from ‘gauntlet’ rootstock candidates: Rootstock sprouts recovered from 12 superior gauntlet trees that had the tops cut off to induce sprouting for rootstock recovery, all successfully grafted, were analyzed for CLas by PCR. Most interestingly, multiple grafted trees from two of these rootstock candidates had ct values above 36, indicating no CLas bacteria replication; and both hybrids were from the same cross (A+HBPxOrange 3-12-12 and A+HBPxOrange 3-12-10). Both hybrids appear to be more vigorous than either parent. Orange 3 is UFR-1, and both tetraploid parents in this cross produce fruit with high soluble solids (good rootstock pedigree). Further experiments are being designed to determine the value of strong rootstocks that do not support CLas replication in the root systems. Since recovered lines of these two rootstock candidates are pathogen free, TC propagation was initiated. A 3rd hybrid from this cross looks excpetional in the ‘gauntlet’ this spring, and it will also be cut to recover the rootstock. CREC Trailer Park Trial: A good candidate for juice blending was identified: 18A-10-11. This selection is a low-seeded cybrid of ‘Furr’ that produces a large firm fruit, with exceptional quality juice (12.5 brix, 19.84 ratio and 46.1 color in mid-January). We used this selection in juice blends at both the CREC display and Gator Day at the State Capitol, and all were very well received. The exceptional juice flavor holds up well to pasteurization. Rootstock candidate identified from Strang/Gapway trial. We were able to recover the x639 mutant (apparent deletion mutant) showing the exceptional HLB tolerance. One successfully grafted tree and 12 rooted cuttings have been obtained. PCR analysis showed a ct value of 40, indicating no CLas bacteria in the recovered rootstock. Additional propagation is underway as needed for a more robust field trial. (Also note that the GapWay property has been sold, so the trial has been lost; however, the rootstock tree was dug up and successfully moved back to the CREC). Field Trial Data Collection, etc.: various data (tree health, fruit quality, yield, etc.) was collected from the following trials: Tom Hammond, Greene River Citrus, IMG, Peace River Growers, Bryan Paul Citrus, Lee Family Groves, and Duda. Data analysis and entry onto the Rootstock Data Website: new data posted on website included: Jackson Citrus, Lykes, GFC Citrus, Lee Family Groves, Banack Citrus and Duda. Trial data worked on, not yet added to website: Greene River Citrus, Peace River Growers, IMG, Bryan Paul Doe Hill, Citra trial, Cutrale, and the Serenoa trial. The Access Data Plan was also implemented on the website.
In the current project we are establishing the volatile and non-volatile polar metabolite profiles of new scions and rootstocks and evaluating them for their tolerance to HLB by challenges with psyllids and HLB.Progress on Objectives: This reporting period we focused on data from the study involving a new mandarin hybrid we call Lucky (Sugar Belle x Nova×Osceola) and the two parental varieties by challenging with ACP and taking leaf samples after infestation.Objective 1.1. To understand the mechanism behind the tolerance of new varieties toward HLB. The comparison between the varietal responses will allow us to determine the response to CLas-infected ACP infestation. Subsequent PCR testing will help determine susceptibility to HLB.a. For the hybrid Lucky (its parents are Sugar Belle and Nova × Osceola), we successfully repeated the challenge with CLas-infected ACPs ending in September. Leaf samples were taken initially (before infestation with ACP) and then 3 days, 1 month, 2 months and 3 months after ACPs were introduced into the cages. We will determine the biochemical response of Lucky to ACP infestation and its relative tolerance to CLas over time, with PCR confirmation of HLB status scheduled for Mar 2022 (9 months). The chromatograms have now been integrated and a summary is provided here:b. VOC profile summary: At this time we have finished the preliminary integration and have a general idea of the VOC profile of each of the three varieties. Overall, we detected 54 volatile compounds among the leaf extracts of the three varieties. `Lucky’ and Nova produced sabinene and beta-ocimene as their major monoterpenes, whereas in Sugar Belle (SB) the major monoterpenes are beta-ocimene and gamma-terpinene. Linalool appears at similar levels in all three, and limonene is relatively low in all three (compared to sweet orange varieties, where it is the dominant monoterpene). The dominant sesquiterpenes are caryophyllene and beta- and gamma-elemene.c. The majority of the compounds detected were in common between the three varieties, but a few were unique to each variety. As reported previously, the characteristic VOCs for SB included thymol and thymol methyl ester. Neither `Lucky’ nor Nova produced thymol, but Nova produced traces of the thymol methyl ester. In addition, we detected the monoterpenes para-cymenene and para-methatriene in SB, which was not found in the hybrid `Lucky’ offspring or Nova. Overall, the VOC profile of `Lucky’ showed contributions from both parents but did not express all of the possible compounds (44/54). There are many that are found uniquely or in two of the three varieties. These include para-cymene, para-menthatriene, and thymol (in SB only), thymol methyl ester (SB, Nova), germacrene C (Nova only), beta- and gamma-elemene (SB, Nova), 7-epi-thujene (Nova, Lucky), beta-cubebene (Lucky), 4-epicubedol (new, in Nova), and beta-sesquiphellandrene (Nova, Lucky).d. We will continue the data analysis to determine the effect of the infestation on the VOC profile of the three varieties over time. We expect to see changes in the VOC profile with increasing time after the infestation. e. Most importantly, survival of ACPs on the three varieties was not good again, as was found previously in our January 2021 experiment, which was attributed to cold weather. The second biology experiment was carried out in a protected growth room between July and August, 2021 and showed that ACP survives and reproduces poorly on the two parents. The ACP count was the highest on the hybrid Lucky. Therefore, we can suggest a correlation may exist between the lack of inheriting thymol and several other compounds from its parents and the higher ACP counts on Lucky, so maybe it is not very Lucky after all.Objective 2.To support objective 2 (understanding the role of rootstocks in citrus tolerance to HLB), we have two remaining experiments. 1. USDA rootstocks. We collected leaves from all of the USDA rootstocks grown from seeds since last year (US-802, 812, 897, 942, 1283, 1284, 1516) for volatile metabolite profiling. These samples are in the freezer and await processing and analysis. In addition, we have scheduled challenges with ACP and HLB graft-inoculations in the early spring of 2022 to determine if any of these are tolerant toward HLB.2. Grapefruit varieties on different rootstocks from the CUPS. The volatile data was reported in the previous reporting period. However, the data for the non-volatile metabolites are still being analyzed at this time.
A large-scale trial of HLB-tolerant citrus cultivars is addressing the need of Indian River growers to identify the best rootstocks and scions for growing fresh fruit. The project has two objectives: (i) Assess the performance of new grapefruit cultivars with selected rootstocks; and (ii) Evaluate the influence of UFR and other rootstocks on grapefruit scion development in comparison to legacy/standard rootstocks. There are two trials: Trial 1) 18 grapefruit cultivars on three rootstocks; Trial 2) 32 rootstocks with `Ray Ruby’ grapefruit as the scion. The final 90 grapefruit trees on UFR-8 rootstock are scheduled to be planted in March-April 2022, which will bring the total number of trees with grapefruit scions to 2,741. Control-release polycoated fertilizer was applied appropriately in February 2022. All trees were treated as needed with appropriate agrochemicals to manage canker, Asian citrus psyllids, mites, and citrus leafminers. One night of subzero temperatures for a short period caused slight freeze damage to many trees, but the trees are expected to recover fully. No trees died due to the subzero temperatures. Trunk diameter and canopy volume were measured on the three middle trees in each experimental plot in October 2021 to assess tree size. At the time, there were significant differences among scion/rootstock combinations. In Trial 1, `Pummelette UF-5-1-99-2′ grapefruit on US-942 was 6X larger (395.5 ft3) than `US 1-83-179′ grapefruit hybrid on US-942 (70.6 ft3). In Trial 2, grapefruit on UFR-15 was 3X larger (314.3 ft3) than on UFR-17 (123.6 ft3). Many trees produced fruit. Size, color and taste of fruit varied among rootstock/scion combinations. No fruit data were collected because a tree’s first crop is not indicative of future production. Long-term evaluation is needed to identify the most promising scions and rootstocks to determine their profitability and capability of meeting grower and market needs. HLB is spreading in the study grove and visual blotchy mottle symptoms in the canopy are increasing. Leaf samples for quantifying CLas titer were collected in September 2021 and sent to Southern Gardens for analysis. Trees that are CLas-free (ct values >38) and CLas-infected and symptomatic (ct values of 26-32) can be found in the same plots, but many symptomatic trees are developing vigorous canopies. The incidences of Asian citrus psyllids (ACP), Diaprepes root weevils, whiteflies, leafminers, and citrus canker were low during the reporting period, probably due a decrease in temperatures and relative humidity, which reduces flush emergence and slows pest activity. Canker damage is noticeable but not uniform; it is especially significant on `Ray Ruby’ grapefruit trees. Nevertheless, tree growth has not been significantly affected by these pests due the timely applications of agrochemicals. Results of the study were presented at the annual Florida Citrus Show in January 2022 in Fort Pierce, FL by graduate student Martin Zapien.
Create new candidate hybrids. Planning is in progress for new hybrids to be created during next quarter (Spring 2022), based on parental combinations yielding the best progeny in previous trials. Hybrids from previous crosses were selected and propagated this quarter. New seed source trees for advanced selections were prepared for field planting in Spring 2022.Propagate and plant new field trials. One new Stage 1 field trial with Valencia and one new Stage 2 field trial with Valencia were field planted this quarter. Trees for a Stage 2 Hamlin trial with selected released rootstocks and the best of the next generation hybrids are being grown in the greenhouse in preparation for field planting in Spring 2022. Nursery trees for one new Valencia Stage 1 trial with 60 new rootstocks are being prepared for field planting in 2022. Budwood increase trees were grown in preparation for budding trees for new rootstock trials. Trees with Valencia scion and HLB-tolerant interstocks were propagated as a preliminary analysis of interstock feasibility.Collect data from field trials. Measurements of fruit crop and fruit quality data were collected on 6 rootstock trials with Hamlin and other early season scions. The USDA researcher assisting with the analysis of fruit quality from USDA rootstock trials retired this quarter, so responsibility for this aspect of the work shifted entirely onto the Bowman program. Because of the Bowman program emphasis on rootstock trials, it is anticipated that this change will allow for an increase in the pace of fruit quality analysis associated with the rootstock trials. Evaluate effectiveness for seed propagation of new rootstocks and develop seed sources. Studies continued to evaluate seed propagation for promising SuperSour hybrid rootstocks, using morphological and SSR analyses of seedling progeny for trueness-to-type. Additional effort was initiated on this subject as a graduate student project. As the best rootstocks are identified through field trials, seed sources are established. Cooperative work continues to compare field performance of rootstocks propagated by seed, cuttings, and tissue culture. Evidence indicates that performance of rootstocks is primarily determined by rootstock cultivar, with only small influences by propagation method.Field trial results for grower access. The USDA rootstock trials produce large amounts of information that is useful to help growers make informed decisions about rootstock choice for new plantings. A manuscript is in preparation that has a detailed comparison of field performance for Valencia on 50 SuperSour rootstocks and other commercial rootstocks, and selected excerpts from this were prepared for presentation at the 2022 Florida Citrus Show and the January 2022 meeting of the Florida Citrus Research Foundation. During this quarter, updated trial summaries were prepared for uploading to the USDA citrus rootstock program website https://www.citrusrootstocks.org/, and information was provided to individual growers and groups, as requested.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. Assessments of trees in trials includes 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 commercial standard rootstocks. Performance data continues to be collected and is being used to critically compare the new hybrids with each other and existing rootstocks. It is anticipated that 2-3 of the most outstanding of the new SuperSour hybrids will be officially released in 2022-23.
This project addressed CPDC priority 3A. Collect standardized data on existing field trials to evaluate citrus scion and rootstock response to HLB. The purpose of the project was to conduct evaluations of rootstocks in existing replicated field trials regarding their horticultural performance and ability to mitigate HLB-induced decline. The cooperative trials were planted in 2015 and contain more than 20,000 Valencia and Hamlin trees on more than 30 different replicated rootstock cultivars and other non-replicated ones. Trials were under commercial management (Lykes Bros.) and planted near Basinger (Highlands County) and Lake Wales (Polk County). For annual evaluations, only replicated rootstocks were included using 6 replications. The complete set of rootstocks was included in year 3 evaluations. The rootstocks included UF/IFAS (CREC) released and experimental selections, California selections (available in FL), and selections from Spain. The Spanish selections are proprietary and not available for use in the USA currently but may be available soon. The UF selections are tetraploid cultivars, meaning that they were created by somatic hybridization or from crosses at the tetraploid level. All other rootstocks were diploids and created by sexual hybridization.All replicated rootstocks were evaluated annually using the methods established in the Denver protocol and outlined in the proposal (also see appendix). Data collected include tree size (height, canopy volume, trunk diameter), fruit yield, fruit quality (total soluble sugars, acid, brix/acid ratio, etc.), leaf nutrients, canopy health (canopy color, canopy density, foliar HLB disease symptoms), and wind resistance/tree leaning (after hurricane Irma). A comprehensive data was assembled for each scion/rootstock combination and trial location and is attached to this report as 4 Excel files: 1) Albrecht_18-029C_VAL_Replicated_Yrs1-3.xlsx; 2) Albrecht_18-029C_VAL_All_Yr3.xlsx; 3) Albrecht_18-029C_HAM_Replicated_Yrs1-3.xlsx; 4) Albrecht_18-029C_HAM_All_Yr3.xlsx.
This project addressed CPDC priority 3A. Collect standardized data on existing field trials to evaluate citrus scion and rootstock response to HLB. The purpose of the project was to conduct evaluations of rootstocks in existing replicated field trials regarding their horticultural performance and ability to mitigate HLB-induced decline. The cooperative trials were planted in 2015 and contain more than 20,000 Valencia and Hamlin trees on more than 30 different replicated rootstock cultivars and other non-replicated ones. Trials were under commercial management (Lykes Bros.) and planted near Basinger (Highlands County) and Lake Wales (Polk County). For annual evaluations, only replicated rootstocks were included using 6 replications. The complete set of rootstocks was included in year 3 evaluations. The rootstocks included UF/IFAS (CREC) released and experimental selections, California selections (available in FL), and selections from Spain. The Spanish selections are proprietary and not available for use in the USA currently but may be available soon. The UF selections are tetraploid cultivars, meaning that they were created by somatic hybridization or from crosses at the tetraploid level. All other rootstocks were diploids and created by sexual hybridization.All replicated rootstocks were evaluated annually using the methods established in the Denver protocol and outlined in the proposal (also see appendix). Data collected include tree size (height, canopy volume, trunk diameter), fruit yield, fruit quality (total soluble sugars, acid, brix/acid ratio, etc.), leaf nutrients, canopy health (canopy color, canopy density, foliar HLB disease symptoms), and wind resistance/tree leaning (after hurricane Irma). A comprehensive data was assembled for each scion/rootstock combination and trial location and is attached to this report as 4 Excel files: 1) Albrecht_18-029C_VAL_Replicated_Yrs1-3.xlsx; 2) Albrecht_18-029C_VAL_All_Yr3.xlsx; 3) Albrecht_18-029C_HAM_Replicated_Yrs1-3.xlsx; 4) Albrecht_18-029C_HAM_All_Yr3.xlsx.
1. Please state project objectives and what work was done this quarter to address them: This report covers the period of September 1, 2021 – November 30, 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). We have inoculation tests completed using graft transmission and Asian citrus psyllid (ACP) transmission of hte pathogen (Candidatus Liberibacter asiaticus, or CLas), and a natural infection trial in a grove at USHRL. Graduate student Chad Vosburg finalized his data analysis of the field, psyllid, and grafting mediated CLas infections. His committee approved the final version of his thesis, and Chad is set to graduate in December, 2021. During the reporting period, he revised and finalized his M.S. thesis document as a draft of a publication of the methods and infection results. 2. Please state what work is anticipated for next quarter:In the next quarter, graduate student Chad Vosberg will graduate (December, 2021). The PI will start to revise the draft documents created by Chad for submission to a peer-reviewed journal for publication. 3. Please state budget status (underspend or overspend, and why):This project is underspend partially due to support by the Plant Pathology and Environmental Microbiology Department for Chad’s stipend and tuition. In the next several months, the PI will draw part of his salary support from the grant because he is now the primary person revising the manuscript for submission to a peer-reviewed journal for publication in the next quarter.
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
Four of the proposed objectives have been accomplished. Objective (5) is delayed due to the Covid-19 pandemic. Activites conducted for each of the objectives are described below.
(1) We previously generated three HLB-tolerent transgenic lines, ‘Hamlin’ 13-3, 13-29, and Duncan ’57-28′. The transgenic plants carried both the HLB-causing bacterium CLas and the flowering-promoting CTV vector. To remove CTV and CLas, the plants were treated in a growth chamber with alternating temperatures of 25 and 42 degree C every 4 hours for a total of 60 to 90 days. New shoots formed on the treated plants were tested for the presence of CLas and CTV by quantitative PCR (qPCR) with specific primers. The alternating temperature treatment is known to be highly efficient for removing CTV. We found that it is also effective for eliminating CLas. The new shoots obtained were free of both CTV and CLas.
(2) & (3) Budwoods from the new shoots were grafted onto ‘Swingle’ rootstocks. A total of 83 transgenic plants were produced using CTV- and CLas-free budwoods of the above mentioned transgenic lines (28 for 13-3, 31 for 13-29, and 28 for 57-28). Moreover, 13 plants propagated from a new NPR1 transgenic line generated through mature tissue transformation, 17 plants from an EDS5 transgenic line, and 7 plants from an ELP3 transgenic line were produced. All these transgenic lines showed HLB tolerance in the greenhouse. In addition, a total of 27 transgenic rootstock plants were produced. These transgenic plants include eight transgenic ‘Carrizo’ lines that express three different disease resistance genes. The transgenic rootstocks were replicated and grafted with ‘Valencia’. All plants were grown and maintained in the greenhouse for two to three years.
(4) The transgenic plants were planted in The Picos Farm in 2019 and 2021 (no plants were planted in 2020 due to Covid-19). A total of 69, 98, and 27 plants were planted on May 9, 2019, May 20, 2021, and October 8, 2021.
(5) This objective has not been finished. The transgenic plants transplanted on May 9, 2019 and May 20, 2021 were examed. The plants grow well in the field and one plant from the 2019 planting has shown HLB symptoms. Field trial data will be collected in the coming years.
Besides the proposed objectives, we continue working on development of techniques that are able to produce consumer friendly citrus products. These techniques include CTV-delivered gene silencing, transgene-free CRISPR, and cisgenesis or intragenesis (cis/intragenesis). Our CTV and CRISPR projects are supported by USDA. The cis/intragenesis project is partially supported with the CRDF funds. We have previously created an intragenic vector. Unfortunately, the efficiency of the vector is extremely low. Our goal is to develop a strategy to significantly improve the efficiency. We are using the citrus 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) mutant (called EPSPS TIPS) that provides tolerance to glyphosate as a selective marker to increase the intragenic vector efficiency. A strong citrus promoter is needed to drive the EPSPS TIPS gene, we are building a system to identify such citrus promoters.
Once the efficiency of the intragenic vector is improved, it can be used to either silence or overexpress a target gene. We are using CTV-delivered gene silencing to identify targets for silencing, which is supported by USDA. We are also screen for genes for overexpression through cis/intragenesis. We recently discovered two nicotinamide adenine dinucleotide (NAD)-binding receptors in the model plant Arabidopsis, which when overexpressed, increase resistance to bacterial pathogens. With the support of the CRDF funds, we generated transgenic citrus expressing the Arabidopsis NAD receptors. The transgenic citrus plants were inoculated with CLas-infected psyllids and are maintained in the greenhouse for HLB symptom development. We are grafting the transgenic scions onto sweet orange rootstocks for easier detection of HLB resistance or tolerance. Furthermore, the citrus genome encodes several putative NAD-binding receptors. NAD-binding activities of two of the putative receptors were tested. These citrus receptors will be overexpressed using the intragenic vector to create HLB tolerance.
In summary, we have accomplished four of the proposed five objective (~70% of the proposed work) and will finsih field data collection in the coming years.
ObjectiveThis project had only one objective which was to support the operation of the facility that would provide service for production of transgenic citrus plants to researchers working towards solution against huanglongbing (HLB) and citrus canker. The Citrus Transformation Facility (CTF) has been a part of the multipronged approach to fight citrus diseases by producing genetically transformed citrus plants that were used to gain important knowledge about possible introduction of tolerance/resistance traits. This knowledge represented a foundation for understanding of effects that introduced gene(s) could have on the growth of transgenic plants and on their ability to sustain and survive diseases. Within the scientific community working in this field there are laboratories without transformation capabilities, and for those research groups CTF was the place where the ideas from projects came to life because of the production of transgenic plants. Major accomplishments per objectiveThe major accomplishment of this project is that it has facilitated the existence of the CTF throughout the last three years. The CTF has undergone the transition into the Educational Business Activity (EBA) unit within the University of Florida that overhauled the way it provides service and operates as business. This transition together with the wide-ranging effects of COVID-19 pandemic have negatively affected operation of the CTF and funding that came from this project together with the funds provided by the University of Florida have kept the CTF open. Within the last three years, the CTF has produced 388 transgenic plants (Table 1). Number of plants produced in 2019 was 246, in 2020 it was 108, and in 2021 it was 36. Without exception, all the plants we created were associated with the research directed towards finding a solution to HLB and citrus canker diseases. Some of our transgenic plants including some Duncan grapefruits and all Pomelos exhibited increased resistance to citrus canker. The other plants we produced have the potential to either be tolerant or resistant to HLB, or in the case of Indian curry leaf plants (Murraya koeinigii), they produce chemicals that can kill Asian Citrus psyllids. Besides Duncan grapefruits and Pomelos, we also produced transgenic Mexican lime, Valencia sweet orange, Kumquats, Pineapple sweet oranges, and Indian curry leaf plants. All of these plants stayed in the state of Florida and were used in tests to determine the efficiency of introduced transgenes on desired traits.Table 1. The plants produced by CTF in the three-year period (2019-2021).Cultivar Number of plants produced Duncan grapefruit 257 Mexican lime 32 Valencia sweet orange 51 Pomelo 22 Kumquat 4 Pineapple sweet orange 10 M. koenigii 12 Total 388 The number of orders placed at the CTF was 57. In 2019, there were 25 orders, in 2020 there were seven, and in 2021 there were 25 orders. The work done on these orders was performed within 266 experiments that included 358 co-incubations of seedling explants with appropriate bacterial strains. Altogether, 314,000 explants were processed in these experiments. Approximately, 170,000 explants were inspected under the microscope equipped with the blue light source for the presence of GFP-fluorescing shoots and buds. Close to 3,000 PCR reactions were done in search of shoots either carrying or expressing a transgene of interest. Also, 1,750 GUS histochemical assays were performed to confirm the transgenic nature of shoot and plants. In average, the transformation rate was about 1% and that number included both chimeric and fully transformed shoots. The percentage of shoots that were successfully grafted was low in the range of 50%.With the help of staff from CREC’s Business office, during the year 2021 I was able to organize the system of payments for the work performed by the CTF as an EBA and get first payments processed.
Fourth quarter 2021- 100% done Project rationale and focus: The driving force for this three-year project is the need to evaluate citrus germplasm for resistance to HLB, including germplasm transformed to produce 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 for the last 3 years. 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 also assigned ~30% of her time to the program in order to maintain colonies (including watering, setting up new cages, terminating old cages, cleaning growth chambers and cages). USDA provides greenhouses, walk-in chambers and laboratory space to accommodate rearing and inoculations. This quarter: Colonies of CLas infected psyllids supplied a total of 3,770 ACPs used for (1) transgenic events evaluation, (2) applied research for CLas control in citrus performed by USDA and University researchers; and (3) monitoring the colony quality by qPCR.The Stover lab conducted 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.) expressing antimicrobial peptides and defensive proteins targeting CLas, as well as natural insecticide peptides to control ACP. Five detached leaf assays experiments, involving individual 228 leaves, were inoculated using 2,280 CLas infected ACPs in this quarter. Transgenic material tested in DLAs were Carrizo plants expressing ONYX peptide and chimeric AMP TS, both under SCAmp-P3 phloem specific promoter. A total of 38 independent events were tested alongside WT controls. The leaves (midribs) and ACPs are being processed and submitted to qPCR for CLas titer after each DLA to better understand the effect of the transgenic peptide in bacteria control and transmission. These trials have being very useful in terms of providing information that allow to select the best transgenic events (ones causing high ACP mortality and/or low CLas transmission to plant) for propagation and further evaluation at greenhouse environment. We continue to see substantial ACP mortality from feeding on CLas-killing transgenic leaves, with some ONYX events killing around 80% of the psyllids with reproducible results. Research involving evaluation of the microbiome of ACPs fed on transgenic causing high insect mortality was conducted this quarter using 440 ACPs fed in a set of 44 transgenic leaves. A research paper has been prepared (Rapid in vivo screening for huanglongbing resistance in genetically modified citrus by detached leaf assay- J.Krystel, M. Grando, Q. Shi, E. Cochrane, E. Stover) in order to report important modifications implemented into the DLAs using Clas+ ACPs to evaluate transgenic plants and investigate the mode of actions of peptide in controlling the psyllids. In addition, 1,080 CLas+ ACP were provided to researcher collaborators:780 for Florida International University, for Jessica Dominguez, a Ph.D. student, who is developing a thesis in alternative compounds to control CLas bacteria and 300 ACP were furnished to Dr. Randy Niedz (USDA Fort Pierce) for activities in a HLB NIFA project. Periodic colony checks were conducted by PCR to maintain CLas positive colonies. This quarter 410 ACPs were used for Clas detection by qPCR to monitor colony quality. Also, six new colony cages were set up this quarter to renew and support the demand of the hot ACPs. For that 24 HLB positive plants were infested with 2.500 ACPs. Previous quarter: United States Department of Agriculture scientists Kim Bowman, Ed Stover, Michelle Heck, Randy Niedz, and researcher collaborators have all run experiments totaling 6,840 ACPs. Samples have all been collected on-time from ongoing experiments. ACP mortality were computed and statistically analyzed. Surviving ACPs and leaf midribs were collected and processed for future qPCR analysis to access the bacteria CLas titer).
1. Please state project objectives and what work was done this quarter to address them: The objectives of this project were to produce disease resistant, commercially & agronomically acceptable, mature citrus transgenics, intragenics, & GMO/non-GMO edited trees using Agrobacterium as a service for research & commercialization. The research focus of this project was to improve transformation efficiencies, so that the mature citrus protocols become more productive, decrease prices for scientists, & contribute more to financial self-sufficiency of our lab. A biolistic transformation protocol was also developed to overexpress all-citrus intragenic sequences using a citrus gene for selection, which is useful for food crops. A final comprehensive report will be submitted in the near future to CRDF. The efficiency of the mature citrus Agrobacterium transformation protocol was increased significantly, primarily due to the introduction of new cultivars developed by the Plant Improvement Team & determining which ones were not recalcitrant to Agrobacterium. The mature citrus Agrobacterium protocol was originally developed for Pineapple sweet orange but Pineapple is not commercially important to the Florida juice industry. Instead we have found several new sweet orange cultivars that are readily transformed with Agrobacterium. Currently we are focusing on producing non-GMO edited mature citrus & have seen some encouraging results. In addition, we continue to produce all-citrus intragenics using biolistic & Agrobacterium transformation. The plants produced using these technologies might not be as strictly regulated compared to plants produced by transformation with genes from foreign organisms. The USDA APHIS, EPA & FDA will work with each scientist on a case by case basis to deregulate superior intragenic trees. Intragenic trees can likely be provided to the growers relatively rapidly. During the last quarter, ~82 transgenics were produced using Agrobacterium transformation of mature rootstocks & ~87 Cas9 trangenic edited scions were produced for another scientist. We have two new potential projects involving scientists from other states & countries. A manuscript describing a protocol for a new liquid selection system in mature rootstock was published, which showed significantly higher Agrobacterium transformation efficiency in liquid than on solid medium. In addition, Agrobacterium transformation efficiency also increased in mature scions using liquid selection & another manuscript is in preparation. Currently, a manuscript is in review about the new citrus, intragenic selectable marker. It is the responsibility of the scientists to field test their trees & to my knowledge, at least ~ two to three projects are being field tested in the Picos Farm during this last granting cycle. The lab transitioned to an Educational Business Account for earning salaries & operating funds, however it is difficult to earn enough money for salaries by selling plants. Prices are being increased again in 2022 since we will no longer receive CRDF funding.
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