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 breakdown of the 2nd experiment testing different irrigation pH’s on each half of the split root system (to mimic irrigated and non-irrigated root zones) is complete. Data is being analyzed and samples for RNAseq are being prepared. The 2nd repetition of the 1st experiment is underway to confirm the results that US942 becomes highly sensitive to low pH (5.5) when infected with HLB (root damage and excessive root leakage causing irrigation leachate pH to increase. We also expect to partially confirm this finding in the ongoing data analysis of the 2nd experiment.
Propagation of experimental trees proved more difficult than anticipated. We have transplanted the trees for the first repitition into split root rhizotrons and are preparing for HLB inoculation and sampling. The postdocs in Drs. Johnson and Wang’s labs have coordinated and done test runs of sampling procedures for RNA and metabolite samples, which will begin 2 weeks after inoculations. 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. Although we thought we had enough plant material for both experiments vigorously growing, we have had some of the combinations struggle recently. We are contacting nurseries to see if they can provide us with grafted trees for the 2nd rep by next spring.
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. Yield has been collected from Hamlin sites and the Valencia harvest is being planned currently. Phytophthora samples have been taken and this years treatments will begin in April. We have built plot by plot microjet irrigation systems that connect to our trailer mounted handgun sprayer to mimic irrigation injection of materials for ease of application. We are currently adding the split plot factor to our plot design to begin foliar brown rot sprays in midsummer through October. We are preparing to inoculate seedlings with HLB for an additional greenhouse experiment to determine if the newly labeled chemistries have the same limitation on HLB-affected plants as fosetyl-Al and mefanoxam have shown. 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. We are waiting for enough CLas+ budwood for graft inoculations or a large enough field population of psyllids after the spring flush to do a psyllid inoculation, whichever comes first.
1. Please state project objectives and what work was done this quarter to address them:Project objectives:Objective 1: Determine the effect of systemic acquired resistance (SAR) in enhanced tolerance to HLB Objective 2: Monitor tree health and HLB levels Objective 3: Conduct Juice analysis and evaluate quality parameters from selected Parson Brown trees. We have identified and collected leaf samples in the spring from 8 Parson Brown groves. Groves are spread all around the major citrus growing regions. We sampled from 3 groves in polk county, 2 groves in Highlands county, 1 grove in St. Lucie county, 1 grove in glades county and 1 grove in Collier county. Samples were collected for both RNA and DNA extractions. Most of the trees are from the PB 56-2 clone but some groves had trees of the older PB 1-2-3 clone. All growers reported lower to no fruit drop on the parson brown irrespective of rootstock used. This is in comparison with Hamlin which had severe fruit drop this year. Hamlin leaf samples from the same block or neighboring blocks were also collected for analysis. 2. Please state what work is anticipated for next quarter:In the next quarter, we will again sample the same groves and collect similar leaf samples. HLB levels will be evaluated using qPCR and we will evaluate the genetic differences between the samples. 3. Please state budget status (underspend or overspend, and why):We are on target with the spending for this project.
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 (Oct. 1 – Dec. 31) 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. There were three cold fronts that impacted the trial location in Indian River County during late Nov. through Dec. that were documented to be below 40. F. Significant leaf drop occurred 5-7 days after the cold weather but there was no treatment effect observed based on visual assessment of extent of abscission. Also, HLB leaf symptoms were prominent throughout the trial in late Dec. and will be visually assessed next quarter. A ground application of herbicide was made, and plant material was removed in preparation for a UAV flight for aerial imagery data collection. Aerial images were taken with a UAV equipped with a 20 MP digital camera on 12/31/2020. A set of overlapping images were taken at two altitudes – 100 and 150 feet. Images taken at 100 feet are for photo-documentation (0.36 inch/pixel) of the experiment and will be made available at the completion of the project. Images taken at 150 feet (0.53 inch/pixel) were processed by segmenting the image into trees and background using machine learning image analysis. Once the trees were segmented (identified and separated from the background), various measures were made/calculated including canopy area, density, perimeter, convex hull area and perimeter, and minimum and maximum caliper diameters. These measures, as well as the ground measures, will be correlated (statistical analysis) with the iron treatments to determine their effects. One additional set of images, including a photo-documentation set, will be collected at the end of the project.
This project is a continuation of previously funded CRDF grants to TWO BLADES focused on utilizing multiple strategies to produce canker-resistant citrus plants plus the addition of a new strategy using gene editing. 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 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, the transgenic material was planted. Citrus canker has developed on plants in the field and the trees were rated for disease in November, 2020 and there was considerable defoliation on all trees including JJ5. We also observed disease on all susceptible Duncan trees, but no evidence on the transgenic JJ5. The disease at this point was difficult to rate on Duncan given the defoliation. I am planning to evaluate in June. As for developing a different transgenic with ProBs314EBE:avrGf2, 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. 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 is in the process of creating additional transformants. 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. They and the original EFR transgenic plants were tested for susceptibility by being inoculated in October by pinprick inoculation. Leaves from all EFR positive grapefruit leaves were susceptble based on the pin-prick inoculatons with typical pustule formation simlar to the wild-type Duncan control. Lesions on leaves of one of the EFR positive sweet orange trees were smaller.We have also tested for EFR gene expression and determined that all EFR grapefruit and orange had similar gene expression levels. We have inoculated young leaves from representative trees of each transgenic event by spray inoculation in February and will rate the leaves for susceptibility. 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. The latter was to serve as the negative control as it had undergone the transformation process. We have grafted the buds and several have developed into branches. We are currently growing these. We have recently inoculated leaves from the two CRISPR events, C1 and C2, by pinprock inoculation and assessed the pustule size in December. Pustules on C2 leaves were similar in size to non-edited leaves. Interestingly we observed reduced pustule formation on C1 leaves. We are waiting for new leaves to develop for conducting further testing this spring.
Nematicide effects on nematode populations were measured in December, as previously described. The average number of sting nematodes in aldicarb plots was 10% higher (NS) than that in untreated plots, whereas oxamyl, Syngenta product and Nimitz plots had 34%-42% fewer sting nematodes (P<0.05) than in untreated plots. Nematode numbers in the plots treated with the other nematicides ranged from 89%-93% of that in untreated plots, none of which were significant differences. The average cumulative numbers of sting nematodes measured 60 days following each of the four seasonal applications thus far during the project (areas under the curve) were 55%, 54%, 37%, 21%, and 8% lower than those in untreated plots for the Syngenta compound (P=0.05), oxamyl (P=0.05), Nimitz, (P=0.05) Salibro, and Velum Prime, respectively. Cumulative sting nematodes in aldicarb treated plots were 16% more numerous than in untreated plots.Average fibrous root mass density for all nematicide treatments ranged between 7%-47% greater than for non-treated plots. Unlike during the previous June, no treatment resulted in significant root increase during the fall period. The growth of the tree trunks during 2 years between February 2019 and January 2021 was 26% greater for trees treated with oxamyl than that of untreated trees (Dunnett, P<0.05). Growth rates of trees treated with other nematicides were between 2-13% greater than the untreated trees, but the differences were not significant. There remained a strong inverse linear relationship (r= -0.75, n=7, P=0.05) between trunk growth during 2 years and the average sting nematode population density (log-transformed) during that time. The harvested fruit in were counted. The number of oranges per tree in both 2020 and 2021 were very highly correlated (r=0.68 and r=0.64, P=0.001) with trunk cross-sectional area measured at the beginning of the trial (2/2019). Larger trees initially produced more fruit, irrespective of treatment. The average trunk area differed greatly between treatments at the beginning of the trial, therefore we determined the %increase in number of fruit per treatment between2020 and 2021. The yield/tree declined by 22% in untreated trees between 2020 and 2021, but increased in all of the nematicide treated plots. Aldicarb and oxamyl plots increased (P=0.05) by 166% and 126%, respectively while plots treated with combinations of Velum Prime and Nimitz increased by 44% and those treated with combinations of Salibro and the Syngenta product increased by 23%.In a second trial comparing untreated trees to trees treated with aldicarb in April 2019 and 2020, there were no differences in the trunk cross-sectional areas of either treatment at 20 months after the first treatment (untreated=2403 mm2 vs aldicarb=2409 mm2). Nor was there a significant increase in growth since summer 2020. There was no treatment effect on the very small number of fruit per tree (10.2 untreated vs 6.2 aldicarb). Although young tree growth in this grove is very uneven, the cause(s) are other than sting nematodes which were below detectable levels in most plots.In the perennial peanut trial, sting nematodes in the native vegetation middles increased to 85 nematodes/250 cc soil from an average of 51 nematodes during the previous 6 months. However, the populations remained below 10 nematodes in the plots planted with peanut regardless of whether there was some incursion of weeds in some of the peanut plots. Indeed, during the monthly measurement the past 6 months, sting nematodes in peanut middles averaged just 7.7% ± 2.02% (mean and standard error) of those in native vegetation middles. Unlike the previous spring, oxamyl did not significantly reduce the sting nematodes in the plots during the autumn (just 25% reduction). Citrus fibrous root density in plots without peanut middles or oxamyl treatment was 0.21 mg/g soil, compared to 28 mg/g in plots treated with oxamyl, 29 mg/g in plots with peanut middles, and 30 mg/g in plots with both peanut and oxamyl. There was an interaction (P=0.01) between oxymyl and peanut effects on trunk size. Trees were 14% larger (P<0.1) when peanut rather than native plants were in the row middles. Trees treated with oxymyl in peanut plots were 26% larger than those not treated (P=0.03), but were not different than untreated trees in plots with weed middles. Trunk size was highly correlated with number of fruit per tree, but there were no significant effects or interactions between fruit number and either treatment.
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 acollected data on tree health, leaf and soil nutrition. We also applied fertilzer for spring at both sites. Due to low soil pH, we have skipped Tiger sul application and instead applied regular micronutirents. We are continuously monitopring soil pH and will resume Tiger Sul application when pH is high enough.In addition we recently harvested Arcadia site. The data on fruit yield, fruit size, huice quality has been collected. nfortunately, due to Covid-19 related restriction we could not perform sensory analysis, however we collected juice for flavor analysis in order to asses the effect of nutritional treatments on sensory aspect of juice. Currently, we are in process of data organization and analysis of field data and flavor analysis of juice samples.We are also getting ready to harvest second trial site. We anticipate to harvest it in next 2-3 weeks. This quarter the results of field trial were pesented at virtual OJ break.
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 1st quarter of the third year of our grant, the Stover lab has focused on producing additional transgenics of the couple lines that are still needed. They tried a new shoot elongation medium with good results and have a large number of transformants for expression testing. The Rogers lab has continued small-scale 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 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.
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 accepted for publication by Phytopathology.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 2021. 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. We have analyzed the residues of OTC and STR for fruit samples harvested in January 2021. 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. We have determined the time points to test OTC and STR treatment on ACP acquisition of Las. 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. We have collected another set of samples to monitor Las resistance against OTC and STR.
Progress report for the first quarter of the 2020/2021 project yearThe purpose of the project is to develop new guidelines for restoring root health and improving overall tree nutrition for Florida oranges and grapefruits. 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 samplings for the first quarter of the third year of the project are complete. However, one key lesson emerging in this project is that there is no benefit in doubling macronutrients such as Ca, K and Mg, but rather keeping them at the current recommendation but elevating micronutrient levels (2x to 4x) such as Mn, B, Zn and Fe. 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. Yield and fruit quality data were collected in February and are being analyzed. 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. Graduate student Tanyaradzwa Chinyukwi will defend her MS thesis in March 2021. Portions of her work will be published in refereed journals and IFAS/EDIS extension bulletins and trade journal articles this summer/fall. Orange fruit yield and juice quality parameters will be determined in March/April 2021 on the Ridge and Flatwoods sites. Lukas Hallman will defend his MS Thesis in May 2021. Portions of his work will be published in refereed journals and IFAS/EDIS extension bulletins and trade journal articles this summer/fall, as well as presented at FSHS and ASHS planned for June and August 2021.
Progress report for the second quarter of the 2020/2021 project yearThe purpose of the project is to develop new guidelines for restoring root health and improving overall tree nutrition for Florida oranges and grapefruits. 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. Fruit yields and juice quality were evaluated at both the Ridge and Flatwoods sites in March 2021 and no significant differences were observed between treatments. All samplings for the second quarter of the third year of the project are 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. Yield and fruit quality data were collected in February and are being analyzed. 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. Graduate student Tanyaradzwa Chinyukwi working with Dr. Kadyampakeni graduated on May 3, 2021. Portions of her work will be published in two Citrus Industry Magazine issue of May 2021 and Florida State Horticultural Society Conference Proceedings of 2020 and 2021. Addition outputs from Tanyaradzwa’s work will be published in refereed journals. Lukas Hallman will defend his MS Thesis in July 2021. Portions of his work will be published in refereed journals and IFAS/EDIS extension bulletins and trade journal articles this summer/fall, as well as presented at ASHS and FSHS planned for August and September 2021. Please state budget status (underspend or overspend, and why): We are on track with our planned budget spending but would need a no cost extension to get a fourth harvest from our experimental blocks.
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. The 2nd annual compost application was conducted. All one-year horticultural measurements were completed according to the standardized guidelines. No significant differences among compost and no-compost plots were found yet, but there was a trend for trees in the compost plots to be larger than trees in the no-compost plots. Significant rootstock effects were found with US-802 generally producing the largest and healthiest trees among the four rootstocks in this trial. The soil moisture content was measured and significant differences were found between compost plots and no-compost plots with the soil in the compost plots having a 40% higher volumetric water holding capacity than non-treated soils. There was no significant rootstock effect on soil volumetric water content. Rhizospere sample processing and data organization and analysis continued. 2. Please state what work is anticipated for next quarter:We will continue with sample processing and data analysis. 3. Please state budget status (underspend or overspend, and why): Approximately 40% of funds have been spent, which is in accordance with the timeline.
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 November, 2020 and there was considerable defoliation on all trees including JJ5. We also observed disease on all susceptible Duncan trees a, but no evidence on the transgenic JJ5. The disease at this point was difficult to rate on Duncan given the defoliation. As for developing a different transgenic with ProBs314EBE:avrGf2, 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. They are currently being tested for disease severity having been inoculated in October by pinprick inoculation. We are also testing 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. We have recently inoculated leaves from the two CRISPR events, C1 and C2 by pinprock inoculation and should have results in early December.
1. Please state project objectives and what work was done this quarter to address them: The overall goal of the proposed research is to test the effectiveness of Frateuria defendens (Frd) in reducing HLB damage in FL citrus. The original proposal was revised for a 1-year feasibility study. Prior to the start of the funding period, we prepared our facility and cleared all regulatory hurdles to allow import of and work with the bacterium in FL under a valid APHIS permit. Due to the regulated nature of the organism, all experiments are being conducted in a greenhouse.Objective 1. Determine the optimal application method of Frd to citrus trees.To optimize the application process, the Israel team conducted a series of in-lab and in-field experiments to optimize the culturing process and determine optimal spray concentrations and time of survival of Frd. During several virtual meetings, methods and outcomes were discussed among the Israel and FL team, and protocols were established. Upon start of the funding period, culturing methods were successfully implemented in the Albrecht lab. We also established real-time polymerase chain reaction methods to detect Frd in planta. At the same time, methods were established and successfully implemented to detect viable bacteria in plants after application. Objective 2. Determine if Frd pre-treatment can prevent trees from getting infected with Candidatus Liberibacter asiaticus (CLas).Due to the short duration of this grant and the complications associated with creating a homogenous population of infected plants, we are first testing efficacy of Frd in infected symptomatic plants (curative). Should Frd show efficacy in restoring health of infected plants, we will initiate additional longer-term pre-treatment (preventative) experiments.Prior to the start of the grant, we prepared plant material to be used for greenhouse testing. For this, we grafted buds from HLB-symptomatic field plants (Valencia sweet orange) onto UFR-5 rootstock liners to create a population of CLas -infected grafted Valencia trees for experimentation. Six months after grafting, all plants were tested by PCR for CLas presence and titer levels. Plants were divided into different populations based on titer levels, disease severity, and size to be used for either spray experiments (smaller plants) and larger plants (injection experiments). Plants that were PCR negative for CLas were not included.In December, the first experiment was initiated to test efficacy of spray applications of Frd in restoring plant health and reducing CLas titers. Frd is being was sprayed in biweekly and monthly intervals at the concentrations determined optimal during the pre-grant stage. Control plants are receiving culturing medium only. Leaves are collected regularly to assess CLas and Frd titers and horticultural variables (plant height, stem diameters, leaf number, etc.) are being measured to assess efficacy of treatments on plant health. 2. Please state what work is anticipated for next quarter: A second experiment using the larger set of infected plants is in preparation to examine efficacy of Frd by trunk injection; we will continue optimizing the injection method before commencing with the experiment. We will continue with the spray experiment, collecting horticultural information and determining CLas and Frd titers. 3. Please state budget status (underspend or overspend, and why): Approximately 25% of funds have been spent.
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