Objective 1 – Determine the effect of the ratio and concentration of Fe2+ + organic acid on hydroxyl radical production and stability. Objective complete. Objective 2 – Determine the phytotoxic levels of Fe2+ + organic acid solutions on citrus. Objective complete. Objective 3 – Determine the effect of Fe2+ + organic acid solutions on HLB titer using a rapid greenhouse, HLB-infected citron, rooted shoot bud assay. Continuing work to develop a rapid greenhouse screening system. Current systems are not yet ready for screening methods to cure or manage HLB. No CRDF funds being used for this research. Objective 4 – Requires screening system – see objective 3 discussion.Objective 5 – Requires screening system – see objective 3 discussion. Objectives 6 and 8 (Note: there is no 7) – These are the field tests for the various ferrous iron (Fe2+) and citric acid treatments on HLB status and horticultural measures for both mature (HLB symptomatic) and nonbearing (non-symptomatic) trees. Throughout this quarter (July 15 – Oct 15) as well as the project timeline, conventional pesticide spray applications were applied to all the treatments in the trial and were based on scouting and were in accordance with IFAS guidelines. Similarly, irrigation events were made based on tree and field conditions as determined by soil feel and appearance, tensiometer readings, water table observation well measurements and visual assessment of tree canopy. Fertilizer applications were made via fertigation and were `spoon fed’ with frequent small applications bi-weekly. No dry fertilizer was used. All 7 experimental treatments were applied to the trial block as per the protocol. However, there were some delayed applications for the weekly iron sprays for the F11-C treatment due to periods of daily rainfall, but these applications were made up when the rain ceased. All the trees in the trial were deemed to be in excellent condition nutritionally and health.
Our project is examining phloem gene expression changes in response to CLas infection in HLB-susceptible sweet orange and HLB-resistant Poncirus and Carrizo (a sweet orange – Poncirus cross). We are using a recently developed methodology for woody crops that allows gene expression profiling of phloem tissues. The method leverages a translating ribosome affinity purification strategy (called TRAP) to isolate and characterize translating mRNAs from phloem specific tissues. Our approach is unlike other gene expression profiling methods in that it only samples gene transcripts that are actively being transcribed into proteins and is thus a better representation of active cellular processes than total cellular mRNA. Sweet orange, and HLB-resistant Poncirus and Carrizo (sweet orange x Poncirus) will be transformed to express the tagged ribosomal proteins under the control of characterized phloem-specific promoters; tagged ribosomal proteins under control of the nearly ubiquitous CaMV 35S promoter will be used as a control. Transgenic plants will be exposed to CLas+ or CLas- ACP and leaves sampled 1, 2, 4, 8, and 12 weeks later. Ribosome-associated mRNA will be sequenced and analyzed to identify differentially regulated genes at each time point and between each citrus cultivar. Comparisons of susceptible and resistant phloem cell responses to CLas will identify those genes that are differentially regulated during these host responses. Identified genes will represent unique phloem specific targets for CRISPR knockout or overexpression, permitting the generation of HLB-resistant variants of major citrus cultivars.During the 4rd quarter of the second year of our grant, the Stover lab sent a large shipment of transgenic plants to the Rogers lab, comprising almost all the lines needed for the proposed experiments. Additionally, the Rogers lab conducted their first no-choice psyllid inoculation experiments. ARS employees are still been ordered to maximize telework due to the COVID-19 pandemic. This continues to slow down progress on the grant milestones. We are very much hoping to be allowed to move to the next phases of reopening soon, which will allow for much more rapid progress towards grant milestones.
The integrity of plumbing in all plots of the nematicide comparison trial (the midway point in the trial) was confirmed in early October with blue dye injections. Between October 21 and November 20 the nematicides, with the exception of aldicarb, were applied either once or twice, according to manufacturer. Efficacy and tree response following 2 years of nematode management will be determined during the final two weeks of December and first week of the new year. There was no activity in the second trial evaluating aldicarb (which is treated only in spring). Tree and nematode response at the end of the second year of treatment will be evaluated as above.Vydate was applied in the perennial peanut trial on November 4. Sting nematodes monitored monthly in the row middles during September-November declined by more than 2/3, with numbers in the peanut middles remaining at approximately 5% (P=0.001) as numerous as those in the middles with native vegetation. Hand cultivation of weeds in the 3 of 8 peanut plots containing weeds was discontined in October when the peanuts closed in the bare patches. Tree and nematode response at the end of the second year of treatment will be evaluated as above.
This project is an continuation of an objective of existing CRDF funded project (# 00124558 ; ended in March 2019, final report submited to CRDF) with some added treatments to be evaluated in comparison to control (dry conventional fertilizer with foliar micronutrients). Objective 1 which is the continuation of # 00124558 included 10 treatments.Altogether currently there are 25 treatments of citrus nutrition that are being compared to control. Within this quater the we successfully analyzed data collected from 25 treatments. In addition, we have applied fertilizer for summer. In a detailed soil and leaf nutrient analysis we found that due to continuous application Tiger Sul fertilizer for almost 5 years, the soil pH in the wetted zone of the tree has dropped signifcantly. The lowering of soil pH is more pronounced in Arcadia site than Fort Meade. Therefore, in order to rebpound the soil pH to range of 5.8-6.5, we have applied dolomite and for rest of the year we are continuing with non-tiger sul micronutirent. The rate od micronutirents is still kept the same and are soil applied with bulk fertilizer. We are continuously montiroing soil pH and making adjumstments as needed. Based on this information about soil pH we are getting ready to start an initiative to educate growers on usefulness of continuously monitoring soil pH.We will be conducting workshop, webinar as well as writing trade journal article.
This project is an continuation of an objective of existing CRDF funded project (# 00124558 ; ended in March 2019, final report submited to CRDF) with some added treatments to be evaluated in comparison to control (dry conventional fertilizer with foliar micronutrients). Objective 1 which is the continuation of # 00124558 included 10 treatments.Altogether currently there are 25 treatments of citrus nutrition that are being compared to control. Within this quater the we successfully applied Fall fertilizer, in order to avoid Tiger Sul fertilizer due to low soil pH concern we soil applied micronutiorents in sulfate form. In addition, in this quater detailed statistical analysis was conducted and the juice smaples from spring were being analyzed for analytical flavor profile, this work is still underway.
The objectives of this study are to identify optimal pH range for root function and minimize root turnover on HLB-affected rootstocks and how uneven pH levels in the root zone (e.g. irrigated vs. row middle portions of root system) affect the overall health of the tree. This is being done in a split root system in the greenhouse where pH of different parts of the root system can be controlled an maintained. The following progress reported is based on a October 31st report date. The 2nd experiment testing different irrigation pH’s on each half of the split root system (to mimic irrigated and non-irrigated root zones) was transplanted to rhizotrons and is underway. It is expected to complete in February or March of 2021. We continue to analyze data from the first experiment and trees for a 2nd repetition of the first experiment will be purchased when NCE funds are available. As we continue analyzing the results we have found that HLB infected roots appear to have caused an increase in pH in the leachate from the pH 5.5 irrigated US942 compared to the healthy and uninfected roots of the inoculated plant. This is based on limited data due to COVID restrictions limiting sampling in April and May. If repeated this suggests that the soil-root interaction is dramatically changed in CLas infected roots, whether due to leakage or alterations in citrus root physiology.
Propagation of experimental trees proved more difficult than anticipated. While we had very high success rate for Valencia on Swingle, all other combinations that included either Sugarbelle or UFR-4 had lower than expected success rates and the trees continue to underperform compared to the Val/Swingle. That said we have propagated enough and have vigorous growth for 2 repetitions of the experiment. We are doing the final preparations for planting the first rep in the rhizotron, but are waiting until after the dormant period. In the meantime we are doing the final planning for the intense sample collection and processing that will be done in the first few weeks after transplant and inoculation. We have also used the time from the delayed plant establishment to develop faster root tracing and analysis techniques to speed up later steps in the process.
HLB is known to make citrus roots more susceptible to Phytophthora root rot. It also reduces the efficacy of chemical management of Phytophthora root rot, creating a difficult management scenario. Current Phytophthora management recommendations are based on pre-HLB work done in the 1980s. These three conditions raise the question of whether yield improvement from Phytophthora management is enough to pay for the management costs themselves. The goal of this project is to develop new soil propagule density managment thresholds and recommendations for chemical management of Phytophthora root rot based on ecomonic analysis of yield responses in different soil conditions. We have identified multiple field sites with heavy and moderate Phytophthora infection and have begun plotting field maps to get the yield data where possible and begin treatments as the weather warms. Due to an observance of substantial brown rot in some of the groves we have been scouting without highly conducive weather and only with the severe (for brown rot) P. nicotianae, we will also include brown rot ratings and compare this in Valencia and Hamlin groves to include this in our economic analysis. We are considering adding a split plot factor of foliar brown rot sprays. Meanwhile we are planning an additional greenhouse Phytophthora and HLB experiment to determine if the newly labeled chemistries have the same limitation on HLB-affected plants as fosetyl-Al and mefanoxam have shown. Seedlings for these trials have been started and will begin when the weather warms in the spring. Many of the new chemistries are directly effective against Phytophthora in the soil rather than acting after uptake by the roots, so this is likely to reduce the limitations of Phytophthora management in HLB-affected groves and test the hypothesis
Progress report for the fourth quarter of the 2019/2020 project yearThe purpose of the project is to develop new guidelines for restoring root health and improving overall tree nutrition for Florida oranges and grapefruit. The objectives of the project are to:1. Determine optimal nutrient concentrations in roots and leaves for multiple grapefruit and orange varieties.2. Compare and contrast fertigation, soil, and foliar fertilization to identify best application method for uptake of nutrients into both underground and aboveground components.3. Investigate the relationship between root and leaf nutrient contents to tree health, yield, and fruit quality as well as bacteria titer.4. Generate updated and new guidelines for optimal nutrient contents for roots and leaves for HLB-affected trees. Progress to date:The project is being conducted at three sites: Citrus Research and Education Center (CREC), Southern Gardens Citrus near Clewiston, FL and Indian River Research and Education Center (IRREC). Data collection continued during this quarter on root scanning, canopy size determinations and soil sampling on the central Ridge and southwest Flatwoods along with fertilizer treatment applications. Trends on soil and root nutrient levels in Valencia orange, for example, continue to follow the pattern of treatments e.g. 4x>2x>1x, for soil applied treatments in summer and fall 2020. Similarly, for foliar applied treatments, the pattern in leaf nutrient concentration follows 1x<2x<4x. All sampling for the fourth quarter is complete. The aim of the study at the UF/IFAS IRREC in Fort Pierce, FL was to relate nutrient concentrations in grapefruit leaves and roots to indicators of tree health and root growth. The research was conducted on flatwoods soils in a randomized complete block design field study on `Ruby Red' grapefruit. Micronutrients (B, Fe, Mn and Zn) were applied using three different concentrations (1x, 2x, and 4x current UF/IFAS guidelines) in the form of either dry granular water-soluble fertilizer, controlled-release fertilizer, or liquid fertilizer. A total of 600 trees divided in 40 experimental units were employed. We collected leaf and root nutrient concentrations, canopy volume and tree height twice a year. Mini-rhizotrons were installed at the beginning of the experiment and root images were taken four times a year. Results showed increased micronutrient concentrations in the leaves among all treatments. There were no significant differences in tree height, canopy volume, root length, and root diameter. Graduate student Lukas Hallman presented his research at the American Society of Horticultural Science virtual Annual Meeting on August 10-13, 2020: Hallman, L.; Ferrarezi, R. S.; Kadyampakeni, D.; Wright, A. L.; Lange, J.; Johnson, E.; Rossi, L. 2020. Micronutrient uptake and root growth and development in HLB-affected grapefruit on Florida Flatwoods soils. HortScience 55(9): S3 (Abstr.). Graduate student Tanyaradzwa Chinyukwi presented her research at the American Society of Horticultural Science virtual Annual Meeting on August 10-13, 2020: Chinyukwi, T., S. Kwakye, and D. Kadyampakeni. 2020. Response of HLB-Affected trees to differential foliar, and Soil Macro- and Micronutrient Applications. HortScience 55(9) (Abstract) Graduate student Tanyaradzwa Chinyukwi presented portions of her research at the Florida State Horticultural Society virtual Annual Meeting on October 19, 2020. She has submitted a conference proceedings paper that will be in print soon. Chinyukwi, T., S. Kwakye, and D. Kadyampakeni. 2020. Performance of HLB-affected trees to soil macro- and micronutrient applications. Proceedings of the Florida State Horticultural Society 133:xx-xx. Plans for Next QuarterThe team will continue with fertilizer treatments and data collection including HLB rating assessments, canopy size and root growth measurements and reporting on the progress of the project.
1. Please state project objectives and what work was done this quarter to address them: Objective. To determine the influence of compost during the first three years of tree establishment on growth, productivity, and root and soil health of citrus trees on rootstocks with different vigor-inducing capacity. Leaf samples were collected for nutrient analysis. Leaf and soil nutrient data analysis was completed. Significant differences between compost plots and no-compost (control) plots were found for several variables. Leaf K concentrations were higher in trees on compost plots than control plots whereas the reverse was found for leaf Ca and B. None of the other nutrients were affected. A rootstock effect was measured only for leaf Mg concentrations which were higher in trees on US-812 compared with the other rootstocks. Soil K, Mg, Ca, and B concentrations were significantly higher in the compost plots than in control plots; the reverse was found for Mn and copper. Compost plots also had a higher organic matter content, a higher CEC, a higher pH, and a higher base saturation for Mg and Ca than control plots. Root cores were collected for root physiological (root respiration) and root structural analyses. Root length and structure was not significantly affected by the compost treatment, but some structural differences were found among rootstock cultivars. Root respiration was neither affected by the compost nor the rootstock cultivar. 2. Please state what work is anticipated for next quarter:We will conduct the one-year horticultural measurements.We will continue with the rhizospere sample processing.The 2nd annual compost application will be conducted. 3. Please state budget status (underspend or overspend, and why): Approximately 30% of funds have been spent, which is in accordance with the timeline.
Objective 1. To illustrate whether application of bactericides via trunk injection could efficiently manage citrus HLB and how bactericides via trunk injection affects Las and HLB diseased trees. 1.1. Determination of the in planta minimum bactericidal concentrations (MBCs) of bactericides against LasThis has been completed for both streptomycin and oxytetracycline against Las. A manuscript entitled: “Residue dynamics of streptomycin in citrus delivered by foliar spray and trunk injection and effect on Candidatus Liberibacter asiaticus titer” was submitted to Phytopathology for publication.1.2. Effect of bactericides via trunk injection on citrus HLB disease progression, tree health, yield and fruit quality in different aged trees with a different disease severityThe field experiments were performed at four different groves on different aged trees with a different disease severity. They are one located in Avon Park, FL, 3-year old Valencia trees; one in Bartow, FL, 2-year old W. Murrcot trees; and one in Auburndale, FL, 7-year old Hamlin trees (planted in 02/2012). The last one is in CREC-, Lake Alfred, FL, 20-year old Hamlin trees. The HLB disease severity and tree size (canopy volume and trunk diameter) in the four groves were estimated immediately prior to treatment application. For the field tests, the experiment design is a randomized complete block design (RCBD) for 9 treatments, including 6 injection treatments (3 different doses for OTC or STR), 2 spray treatments (OTC or STR spraying), and one No treatment as a negative control. Each injection treatment consisted of 9 or 15 trees divided into 3 blocks of 3 or 5 trees each. Each spray treatment consisted of 30 trees divided into 3 blocks of 10 trees each. For all the four field trials, the injection treatment applications were completed by the end of April 2019. The 1st application of spray treatments were completed during spring flushing in February or March 2019, the 2nd applications were conducted in late June to early July 2019, and the 3rd applications were conducted in early to middle October 2019. Leaf samples have been collected from the treated trees at the following time points: 0 (pre- injection), 7, 14, 28 days, 2, 4, 6, 8, 10 and 12 months after treatment (MPT). The estimation of Las titers in these leaf samples are ongoing with qPCR assays. The first estimation of HLB disease severity and growth performance (height, trunk diameter, and canopy volume) of immature trees after treatment were performed in May 2019 (three months after the injection) and continued in a 3-months interval. Fruit yield and quality data were collected for the Bartow trial (W. Murrcot), Auburndale trial (Hamlin), and CREC trial (Hamlin) in January 2020. Fruit yield was estimated for the Avon Park trial (Valencia) in April 2020. Leaf samples were collected for Las population and antibiotic residue assays for those trials. Objective 2. To examine the dynamics and residues of bactericide injected into citrus and systemic movement within the vascular system of trees and characterize the degradation metabolites of bactericides in citrus. Leaf and root samples have been collected from OTC or STR treated trees in the Avon Park grove at the following time points:0 (pre- injection), 2, 4, 7, 14, 28 days, 2, 4, 6, 8, 10, and 12 months after injection. The samples have been processed for OTC or STR extraction, and the concentrations of OTC and STR in these samples were determined by HPLC assays. Fruit samples were collected for the Bartow trial (W. Murrcot), Auburndale trial (Hamlin), and CREC trial (Hamlin) during harvest in January 2020, and for the Avon Park trial (Valencia) in April 2020. The samples were processed for OTC or STR extraction, and the concentrations of OTC and STR in these samples were determined by HPLC assays. We have collected data for 60 and 360 days post treatment. Objective 3. To determine whether trunk injection of bactericides could decrease Las acquisition by Asian citrus psyllids (ACP)Twenty 1.5-year old citrus (Valencia sweet orange) plants were graft-inoculated by Las carrying buds in February 2020. These plants are being tested for Las infection and 4 plants were confirmed with Las infection (Ct values are between 34.0 and 35.0) at 4 months after grafting. They will be subjected to OTC or STR treatment by trunk injection and ACP acquisition access for 7 to 14 days. This experiment is ongoing. Objective 4. To monitor resistance development in Las against bactericides and evaluate potential side effects of trunk injection of bactericides Monitoring resistance development in Las against bactericides. Leaf samples for this test have been collected from 5 trees injected with OTC and 5 trees injected with STR at the highest doses in each of the three groves at 6 and 9 months after the injection, respectively. PCR-sequencing analysis on Las 16SrRNA gene showed there was no mutation compared with the reported sequence. We are further confirming the results. Evaluation of potential side effects of trunk injection of bactericides have been completed.
Objective 1: Investigate the efficacy of bactericides treatments for preventing new infections for young citrus trees protection. Hypothesis: Bactericidal treatment will protect young trees from CLas colonization.Initial leaf samples were collected before treatments to evaluate CLas titers in the uninfected trees. Antibiotic treatments were applied from May 2019 through September of 2020. From early June through September, CLas titer was monitored in leaf tissue in response to antibiotic treatments using quantitative real-time PCR analysis. In this report, the results of the CLas-infection rate in citrus leaves and ACP from May 2019 through June 2020 are described. Currently, citrus leaves tissue samples from July through September 2020are being processed to analyze the CLas-infection rate. Results: CLas titers were highest during the past quarter compared to previous quarters across all treatments. The average tree CT value prior to the beginning og the study was 37.5-39.5. Trees are considered CLas-infected (positives) when CT values are below 35. Trees receiving monthly or quarterly applications of Firewall/Fireline tested CLas-positive beginning January 2020 and December 2019, respectively. Trees covered by Tree Defender bags or treated with insecticide only tested CLas-positive beginning December 2019 and February 2020, respectively.During May-June 2020, CLas values reached the higest level across all treatments since the study was initiated. CLas titers were lowest in the insecticide only treatment during this period, followed by monthly applications of Firewall/Fireline rotation. Trees receiving quarterly applications of Firewall/Fireline had the second highest titer of CLas, compared to other treatments. The highest titers of CLas during May-June 2020 were observed in trees covered with Tree Defender bags. Objective 2. Determine the effect of bactericides application frequency on Las infection of citrus. Hypothesis: Bactericidal treatment will reduce CLas infection in mature trees. Antibiotic treatments were applied from May 2019 through September of 2020. From early May through June, CLas titer was monitored in leaf tissue and Asian citrus psyllid adults in response to antibiotic treatments. Currently, citrus leaf samples from July through September are being processed to analyze the CLas-infection rate. Results: The average CT value of citrus trees at the onset of the study was 28. From May 2019-June 2020, trees receiving monthly applications of Firewall/Fireline had the lowest CT values, as compared with trees receiving quarterly antibiotic applications or insecticide only, with the exception of January 2020 when mean CLas titers across all treatments were not significantly different.
Our project is examining phloem gene expression changes in response to CLas infection in HLB-susceptible sweet orange and HLB-resistant Poncirus and Carrizo (a sweet orange – Poncirus cross). We are using a recently developed methodology for woody crops that allows gene expression profiling of phloem tissues. The method leverages a translating ribosome affinity purification strategy (called TRAP) to isolate and characterize translating mRNAs from phloem specific tissues. Our approach is unlike other gene expression profiling methods in that it only samples gene transcripts that are actively being transcribed into proteins and is thus a better representation of active cellular processes than total cellular mRNA. Sweet orange, and HLB-resistant Poncirus and Carrizo (sweet orange x Poncirus) will be transformed to express the tagged ribosomal proteins under the control of characterized phloem-specific promoters; tagged ribosomal proteins under control of the nearly ubiquitous CaMV 35S promoter will be used as a control. Transgenic plants will be exposed to CLas+ or CLas- ACP and leaves sampled 1, 2, 4, 8, and 12 weeks later. Ribosome-associated mRNA will be sequenced and analyzed to identify differentially regulated genes at each time point and between each citrus cultivar. Comparisons of susceptible and resistant phloem cell responses to CLas will identify those genes that are differentially regulated during these host responses. Identified genes will represent unique phloem specific targets for CRISPR knockout or overexpression, permitting the generation of HLB-resistant variants of major citrus cultivars.During the 3rd quarter of the second year of our grant, the Stover lab continues producing transgenic plants and the Rogers lab continues to propagate the transgenic lines. However, currently ARS employees have been ordered to maximize telework due to the COVID-19 pandemic. This has continued to slow down progress on the grant milestones. Still, this quarter 72 new transgenic plants were evaluated for transgene expression levels in the Stover lab. A number of high expressor were identified. There are now 20 Hamlin transgenic lines, 2 Poncirus lines, and 7 Carrizo lines ready for shipment to the Rogers lab once both labs are ready to reopen. The Rogers lab now has a CLas infected colony of Asian citrus psyllid and is ready to start exposing transgenic lines. We are very much hoping to be allowed to move to the next phase of reopening soon, which will allow for citrus shipments and CLas infections of transgenic lines.
This project is a continuation of previously funded CRDF grants to TWO BLADES focused on utilizing multiple This project is a continuation of previously funded CRDF grants to TWO BLADES focused on utilizing multiple strategies to produce canker-resistant citrus plants. The project has focused on transforming Duncan grapefruit with genes that express EFR or a gene construct designated ProBs314EBE:avrGf2 that is activated by citrus canker bacteria virulence factors. This project is a continuation of previously funded CRDF grants to TWO BLADES focused on utilizing multiple strategies to produce canker-resistant citrus plants. The project has focused on transforming Duncan grapefruit with genes that express EFR or a gene construct designated ProBs314EBE:avrGf2 that is activated by citrus canker bacteria virulence factors. We also are in the process of testing citrus that has been transformed to modify the bs5 gene to enhance resistance to the citrus canker bacterium. Objective 1. To determine if Bs3-generated transgenic grapefruit plants are resistant to diverse strains of the citrus canker bacterium or to alternate target susceptibility genes in greenhouse experiments and to the citrus canker bacterium in field experiments in Fort Pierce. In late March, 2019, in the field at Fort Pierce in collaboration Dr. Ed Stover thetransgenic material was planted. Citrus canker has developed on plants in the field and the trees were rated for disease in December and there was considerable disease on all susceptible Duncan trees, but no evidence on the transgenic, JJ5. Statistical analysis revealed that there was a significant difference in disease both at the second and third ratings. Because of Covid-19 I was unable to rate the plants until late on July 28, 2020 but have not had the time to analyze the data. We have two additional transgenics from plants received from Dr. Vladimir Orbovic. Both transgenic trees contain the avrGf2 gene (based on PCR for detection of avrGf2). These transgenics are reaching a stage where they can be assayed more thoroughly for resistance. We have placed our constuct in a different vector that is acceptable for future transgenic purposes. The previous constructs contain an additional selectable marker that allowed for identifying putative transgenics with a higher success rate that contained the targeted construct. Given that there was concern about the additional marker, the new construct contains only NPT as a selectable marker. The construct is with Vladimir Orbovic, who has additional transformants. We have screened these via inoculations and so far the ones we have received are susceptibe. More will be available in the future. Objective 2. To determine if EFR-generated transgenic grapefruit plants are resistant to the citrus canker bacterium in field experiments in Fort Pierce. We have grafted our two most promising EFR transgenic plants (based on ROS activity) onto two rootstocks (812 and Sour Orange) and planted them in the field at Fort Pierce in collaboration Dr. Ed Stover. They were planted in the field in late March and were recently rated in late July. I am in the process of analyzing the data. We have identified additional transgenics from plants received from Dr. Vladimir Orbovic that have been grafted onto rootstocks. The are in the process of testing this quarter for ROS activity and for EFR gene expression . Objective 3. To determine if bs5-generated transgenic Carrizo plants are resistant to X. citri and to generate transgenic grapefruit carrying the pepper bs5. We have recently received budwood from UC Berkeley. The budwood was from two transgenic events and a third was from a tree that was run through the transformation process but that was negative for the gene, serves as budwood that had undergone the transformation process but that was negative for the transgene. This will serve as a negative control. We have grafted the buds and several have developed into branches. We are currently growing these. Unfortunately the control grafts have not developed any branches as of now. Once they are of an appropriate size we will send DNA to Berkeley. Of course this will only occur once we are allowed to conduct research. We have grown the two CRISPR created bs5 Carrizo trees by pinprick inoculation and spray inoculation. For both inoculation methods we saw no clear-cut difference in susceptibility compare to Carrizo rootstock leaves. We have also compared bacterial growth of one of the CRISPR events with Carrizo rootstock leaves and saw no differences in populations. We plan to test the other CRISPR event when the leaves grow outl.
This project is a continuation of previously funded CRDF grants to TWO BLADES focused on utilizing multiple This project is a continuation of previously funded CRDF grants to TWO BLADES focused on utilizing multiple strategies to produce canker-resistant citrus plants. The project has focused on transforming Duncan grapefruit with genes that express EFR or a gene construct designated ProBs314EBE:avrGf2 that is activated by citrus canker bacteria virulence factors. This project is a continuation of previously funded CRDF grants to TWO BLADES focused on utilizing multiple strategies to produce canker-resistant citrus plants. The project has focused on transforming Duncan grapefruit with genes that express EFR or a gene construct designated ProBs314EBE:avrGf2 that is activated by citrus canker bacteria virulence factors. We also are in the process of testing citrus that has been transformed to modify the bs5 gene to enhance resistance to the citrus canker bacterium. Objective 1. To determine if Bs3-generated transgenic grapefruit plants are resistant to diverse strains of the citrus canker bacterium or to alternate target susceptibility genes in greenhouse experiments and to the citrus canker bacterium in field experiments in Fort Pierce. In late March, 2019, in the field at Fort Pierce in collaboration Dr. Ed Stover thetransgenic material was planted. Citrus canker has developed on plants in the field and the trees were rated for disease in December and there was considerable disease on all susceptible Duncan trees, but no evidence on the transgenic, JJ5. Statistical analysis revealed that there was a significant difference in disease both at the second and third ratings. Because of Covid-19 I was unable to rate the plants until late on July 28, 2020 but have not had the time to analyze the data. We have two additional transgenics from plants received from Dr. Vladimir Orbovic. Both transgenic trees contain the avrGf2 gene (based on PCR for detection of avrGf2). These transgenics are reaching a stage where they can be assayed more thoroughly for resistance. We have placed our constuct in a different vector that is acceptable for future transgenic purposes. The previous constructs contain an additional selectable marker that allowed for identifying putative transgenics with a higher success rate that contained the targeted construct. Given that there was concern about the additional marker, the new construct contains only NPT as a selectable marker. The construct is with Vladimir Orbovic, who has additional transformants. We have screened these via inoculations and so far the ones we have received are susceptibe. More will be available in the future. Objective 2. To determine if EFR-generated transgenic grapefruit plants are resistant to the citrus canker bacterium in field experiments in Fort Pierce. We have grafted our two most promising EFR transgenic plants (based on ROS activity) onto two rootstocks (812 and Sour Orange) and planted them in the field at Fort Pierce in collaboration Dr. Ed Stover. They were planted in the field in late March and were recently rated in late July. I am in the process of analyzing the data. We have identified additional transgenics from plants received from Dr. Vladimir Orbovic that have been grafted onto rootstocks. The are in the process of testing this quarter for ROS activity and for EFR gene expression . Objective 3. To determine if bs5-generated transgenic Carrizo plants are resistant to X. citri and to generate transgenic grapefruit carrying the pepper bs5. We have recently received budwood from UC Berkeley. The budwood was from two transgenic events and a third was from a tree that was run through the transformation process but that was negative for the gene, serves as budwood that had undergone the transformation process but that was negative for the transgene. This will serve as a negative control. We have grafted the buds and several have developed into branches. We are currently growing these. Unfortunately the control grafts have not developed any branches as of now. Once they are of an appropriate size we will send DNA to Berkeley. Of course this will only occur once we are allowed to conduct research. We have grown the two CRISPR created bs5 Carrizo trees and inoculated by pinprick inoculation and spray inoculation. For both inoculation methods we saw no clear-cut difference in susceptibility compare to Carrizo rootstock leaves. We have also compared bacterial growth of one of the CRISPR events with Carrizo rootstock leaves and saw no differences in populations. We plan to test the other CRISPR event when the leaves grow out.
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