During October 2021-Janury 2022 we surveyed sting nematode populations in several Polk and Highlands County orchards before focusing on 12 quadrats (all moderately-highly infested) across 30 acres of a declining grove near Babson Park destined for replanting in spring 2022. The nine-year-old trees in the grove were clipped to preserve the current irrigation system; however, the grower uprooted 20 trees throughout the grove, revealing uniformly severe root damage on all trees immediately below 30 cm depth. There were relatively fewer symptoms on the surface layer of fibrous roots supporting the need for deep soil sampling to accurately assess sting nematode abundance in citrus. The extensive and comprehensive damage to root systems by sting nematode suggested that the grove would be suitable for the trial.Three hundred trees (Valquarius on 812) were planted on 3/22/2022 and half the trees were covered immediately with individual protective covers (IPC). The design of the experiment is a completely randomized block with 15 blocks, each with 4 factorial treatments (all combinations of IPCs and nematicide application), each applied to 4-tree plots. Plots are separated by single buffer trees. Thus, we will evaluate the effect of sting nematodes on trees exposed to or protected from Asian citrus psyllids that are treated or not treated for nematode control. We dissassembled the fittings and poly-tubing used to deliver nematicides to 32, 4-tree plots in a previous experiment and reconfigured and installed the equipment in these 15 blocks. The first nematicide treatment will occur in April 2022 and will occur in each autumn and spring thereafter. In November 2020 we collected sting nematode-infested soil from a citrus orchard and extracted and handpicked 30-50 mixed-stage individuals per 8 pots each of St. Augustine grass and sorghum sudangrass to establish cultures to serve as inoculum for trials to evaluate tolerance of rootstocks to sting nematode. Within two months, sampled pots contained as many as 40 nematodes per cm3 of soil. In December 2022 we autoclaved Candler sandy soil and filled 4, 150 gallon tanks in a greenhouse. St. Augustine grass stolons and soil from the nematode cultures were transplanted into two of the tanks in January. the other two tanks were planted with non-infested St. Augustine. Construction on the greenhouse that began in early January was still ongoing in April, precluding further research until completed.
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. We investigated the effect of 10 adjuvants on oxytetracycline absorption via foliar spray. Four adjuvants including Flame slightly increased the antimicrobial effect of OTC on Las.We have collected data for yield and quality. 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 2022. 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. We have conducted the test regarding how trunk injection of OTC affect ACP acquisition of Las. OTC signficantly reduces 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. PCR-sequencing analysis on Las 16SrRNA gene showed there was no mutation compared to the reported sequence, indicating no major changes in bacterial resistance against antimicrobials. Leaf samples were collected from OTC or STR injected trees in the Avon Park grove at two and four months after treatment for the analysis of the degradation metabolites of the bactericides. The extraction of the degradation metabolites were completed and will be subjected to HPLC assays. We are in the process of analyzing data related to how adjuvants affect bactericide delivery via foliar spray, bactericide residues in plants, and effect of bactericides on HLB positive citrus tress of different ages. We also investigated how trunk injection of OTC and STR affects phloem tissues. We are analyzing data for final report.
Conclusions of the one year preliminary study: `Parson Brown’ trees, irrespective of location, clone or rootstock had enhanced SAR activity as indicated by enhanced PR1 and PR2 gene expression. Statistically significant differences were always observed in the different locations during the March and December samplings. PP2 gene downregulation indirectly suggests an active protection mechanism in the phloem of the `Parson Brown’. Targeted RNA seq and metabolomics of the phloem would confirm these results and provide more insights into the exact mechanism of HLB tolerance. All trees (`Parson Brown’ and `Hamlin’) evaluated in this study were HLB positive with similar CaLas titer. `Parson Brown’ trees however, maintained a good canopy and held on to fruit much better than comparable `Hamlin’ trees. Oil content % ranged from 0.007 to 0.0011 in `Hamlin’ juice whereas, it ranged from 0.020 to 0.042 in `Parson Brown’ juice, depending on the location the fruit was harvested from. This could be due to clonal differences between trees grown in different locations. Lbs. Solids Per Box were comparable between `Parson Brown’ and `Hamlin.’ However, `Hamlin’ has recorded good Lbs. Solids Per Box in 2021, and samples will need be evaluated for multiple seasons to understand differences between the two early season sweet oranges. Limonin levels were less than 5 ppm in both `Parson Brown’ and `Hamlin’ and within acceptable levels. It was not possible to identify specific clones based on the data generated. Whole genome sequencing can shed insights into clonal differences by looking at the overall pattern of SNPs and INDELS among the specific selections. `Parson Brown’ trees at the Premier Citrus location in Lorida had the best growth, canopy coverage, followed by trees at the groves managed by Pat and Marty McKenna and the Premier Citrus location in Ft. Pierce. There were no significant differences in the oil content from fruit from these locations. It may be worthwhile to reintroduce a couple of these superior lines into the DPI’s parent tree program for the benefit of the citrus stakeholders.
Conclusions of the one year preliminary study: · Trees were evaluated on a quarterly basis from 8 different locations. Tree aged ranged from 20 to 40 years in these locations. Most locations had changed management several times in these years and there was no clonal identification records.· `Parson Brown’ trees, irrespective of location, clone or rootstock had enhanced SAR activity as indicated by enhanced PR1 and PR2 gene expression. Statistically significant differences were always observed in the different locations during the March and December samplings. · PP2 gene downregulation was observed only in the `Parson Brown’ trees. This indirectly suggests an active protection mechanism in the phloem of the `Parson Brown’. Targeted RNA seq and metabolomics of the phloem would confirm these results and provide more insights into the exact mechanism of HLB tolerance. · All trees (`Parson Brown’ and `Hamlin’) evaluated in this study were HLB positive with similar CaLas titer. `Parson Brown’ trees however, maintained a good canopy and held on to fruit much better than comparable `Hamlin’ trees. · Oil content % ranged from 0.007 to 0.0011 in `Hamlin’ juice whereas, it ranged from 0.020 to 0.042 in `Parson Brown’ juice, depending on the location the fruit was harvested from. This could be due to clonal differences between trees grown in different locations and could help to differentiate between the different clones. · Lbs. Solids Per Box were comparable between `Parson Brown’ and `Hamlin.’ However, `Hamlin’ has recorded good Lbs. Solids Per Box in 2021, and samples will need be evaluated for multiple seasons to understand differences between the two early season sweet oranges. · Limonin levels were less than 5 ppm in both `Parson Brown’ and `Hamlin’ and within acceptable levels.· It was not possible to identify specific clones based on the data generated. Whole genome sequencing can shed insights into clonal differences by looking at the overall pattern of SNPs and INDELS among the specific selections. · `Parson Brown’ trees at a grower location in Lorida had the best growth, canopy coverage, followed by trees in Lake Wales, Sebring and Ft. Pierce. There were no significant differences in the oil content from fruit from these locations. It may be worthwhile to reintroduce a couple of these superior lines into the DPI’s parent tree program for the benefit of the citrus stakeholders.
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 30, 60, 90, and 120 days 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 our 1st 6 month no cost extension (the original end date was 11/30/2021; the current end date is 05/31/2022), the Stover lab has finally identified several high expressing lines of the p396 phloem-specific promoter in Poncirus. These will be shipped to the Rogers lab shortly, as soon as their BRS transgenic movement permit is renewed. APHIS has changed the on-line interface for applying for BRS movement permits and renewals are much delayed. At least 4 high expressing lines for each of the other 8 promoter/genotype combinations are in the containment greenhouse facility at Ft. Detrick and are being prepared for no-choice psyllid inoculation experiments.The Rogers lab has continued no-choice psyllid inoculation experiments on the many lines they have received from the Stover lab and continued ribosome pull-downs from the tissue collected. ARS facilities are still at a maximum of 25% occupancy due to the COVID-19 pandemic; we are teleworking the remaining time. This 25% occupancy cap will be lifted on Monday, March 28th, as long as the CDC community COVID metrics are in the ‘low’ category (which they are currently for Frederick County, MD). This will allow more rapid progress on grant milestones. Additionally, we are hopeful that the relaxed restrictions on in-person work will make our facility a more attractive place to work for potential post-doc candidates. Hiring a post-doc to help on the project will also allow more rapid progress on grant milestones.
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 second of the biannual compost applications for year 2 was conducted in November. Monthly leaf flush ratings and soil moisture measurements were conducted; we continue seeing the same trends for higher soil moisture content in the compost plots compared with non-compost plots. Fibrous root samples were collected for physiological (root respiration) and structural analyses. Preliminary analysis of soil microbial data indicates some differences in the rhizosphere bacterial composition between rootstocks. In addition, early analysis points to possible differences in the response of the soil microbial community to compost application based on the rootstock. 2. Please state what work is anticipated for next quarter:Flush ratings and soil moisture determination will continue.Fruit yield and fruit quality will be determined.Data analyses will continue.Soil microbial sequence data analysis will continue. 3. Please state budget status (underspend or overspend, and why): Approximately 61% of funds have been spent, which is mostly in accordance with the timeline.
This project was initially awarded to Dr. Ferrarezi. Following Dr. Ferrarezi departure from UF/IFAS the project was transferred to Dr. Rossi. Finally, the project was early terminated in December 2021. The purpose of this project was to assess nitrogen (N) fertilization rates for high-density plantings of different scion and rootstock combinations in four commercial citrus growers. The overall approach was to 1) determine the appropriate N rates needed to support tree growth and productivity, 2) compare data obtained with existing UF/IFAS N recommendations, and 3) revise best management practices (BMPs) for N application in young trees planted in high-density plantings affected by Huanglongbing (HLB). The Ph.D. student and Research Assistant dedicated to the project were hired in Spring 2021. Grower collaborators participating in the project were contacted in Spring 2021. The 4 growers comprised: 1) IMG Citrus (Mr. Brian Randolph), 2) Peace River Citrus (Mr. Larry Black Jr.), 3) Agromillora Florida/ Lost Lake Groves (Mr. Clay Pedersen), 4) Graves Brothers (Mr. David Howard). The graduate student and the research assistant scheduled and performed site visits to all locations in Spring 2021 and Summer 2021. The planting densities and variety/scion combinations were chosen, field maps generated, experimental units identified with permanent signs, and the current information regarding N fertilization were requested to growers in Spring 2021. Dr. Ferrarezi contacted Yara (Bill Easterwood) and received fertilizer donations for the first year (20 tons or 40k lbs). Unfortunately, the delivery was delayed due to COVID-19 logistics finding a local distributor, blending the four different formulations, and delivering in Fort Pierce. We were able to receive both calcium nitrate and ammonium nitrate despite the challenges of finding this fertilizer. That was only possible because of the partnership with Yara. Treatments were applied all locations in Summer 2021 and initial tree size and leaf area index data collection was performed as expected in Summer 2021. The project was early terminated by the CRDF Board at the end of the first year and money were returned to CRDF in January 2022.
1. Please state project objectives and what work was done this quarter to address them:
Objective 1: Determine effects of lowered soil pH on CLas populations and root physiology including internal root apoplast and vascular tissue pH.
Due to a collapse in our inoculum trees, we are psyllid inoculating trees for these experiments and will start the greenhouse portion of the study in Spring 2022.
Objective 2: Field test multiple acidification materials including organic acids for tree response CLas suppression, nutrient uptake, and root and vascular pH changes
We have marked out plots for our experiments at two sites on 15-year old and 14-year-old trees. we collected soil and leaf tissue samples for preliminary analysis. We have also applied elemental sulfur, sulfuric acid, fulvic acid and humic acid in August and September 2021. We determined canopy and trunk size and PCR measurements to establish baseline. We will determine root density responses and root PCR every 6 months.
We have also recruited two postdoctoral associates to help with data collection and documentation of the results.
2. Please state what work is anticipated for next quarter:
In the next quarter we will wait for psyllid or bud inoculated trees to develop initial symptoms before testing for CLas for objective 1. We will apply more acidifying products in January 2022.
3. Please state budget status (underspend or overspend, and why):
Now the budget spending is on track.
1. Please state project objectives and what work was done this quarter to address them: To determine how many leaf nutrient sampling per year are required to effectively capture the tree nutritional statusand adjust fertilizer accordingly.2. To establish the relationship of leaf nutrient concentration with yield, fruit drop, and canopy density3. To determine how the leaf nutrient (all 14 nutrient) levels change in the tree throughout the year.4. To evaluate how the leaf age affects the leaf nutrient status.TThe project was initiated in January 2021. In this quarter we were able to tag summer flush on mild and severely HLB-symptomatic trees as well as sample the spring flush for leaf nutrient analysis. We also collected the data on leaf size, weight, chlorophyll index, and starch along with nutrient profile. The preliminary findings were presented at annual meeting of ASHS, Denver, Colardo. 2. Please state what work is anticipated for next quarter: 1. Data analysis and interpretation2. Processing samples for starch3. Collecting samples for nutrient analysis4. Applying fertilizer treatments based on leaf results 3. Please state budget status (underspend or overspend, and why): Currently, the project is in early phase.The budget is being spent as per the plan where major funds have been used for nutrient anlaysis. We have found dedicated personnels to work on this project starting this quarter.
1. Please state project objectives and what work was done this quarter to address them: To determine how many leaf nutrient sampling per year are required to effectively capture the tree nutritional statusand adjust fertilizer accordingly.2. To establish the relationship of leaf nutrient concentration with yield, fruit drop, and canopy density3. To determine how the leaf nutrient (all 14 nutrient) levels change in the tree throughout the year.4. To evaluate how the leaf age affects the leaf nutrient status. The project was initiated in January 2021. In this quarter we were able to fertilize the trees based on spring flusg nutrient analysis. In addition we have been able to sample spring flush for the second time and are able to make comparisons between randomly picked mature leaves versus spring tagged mature leaf for nutrient analysis. The prelimnary analysis shows that spring flush are deficient in immobile nutrients as compared to random leaves therefore, suggesting that method of sampling can sway the results signifcantly. In this quarter we also sampled summer flusgh for the leaf nutrient analysis for the first time. 2. Please state what work is anticipated for next quarter: 1. Data analysis and interpretation2. Collecting samples for nutrient analysis3. Applying fertilizer treatments based on leaf nutrient analysis results 3. Please state budget status (underspend or overspend, and why): The budget is being spent as per the plan where major funds have been used for nutrient anlaysis.
Project Objectives:
The purpose of the project was to develop new guidelines for restoring root health and improving overall tree nutrition for Florida oranges and grapefruits. The objectives of the project were 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.
Summary Description of the Project:
The project was 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 on root, leaf and soil nutrient evaluations, root scanning, canopy size determinations and soil sampling on the central Ridge and southwest Flatwoods along with fertilizer treatment applications were done throughout the project. Mini-rhizotrons were installed at the beginning of the experiment and root images were taken monthly and root density data were measured half-yearly. Fruit yield and juice quality and bacteria titer data were measured yearly. We compared standard fertilization with elevated (1x and 2x of current recommendations) macronutrients (potassium, magnesium and calcium) along with elevated (1x, 2x and 4x of current recommendations) micronutrient blends (iron, zinc, boron and manganese). At the UF/IFAS IRREC in Fort Pierce, FL 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.
Major accomplishments of the project:
One key finding in HLB-affected oranges is that it is not beneficial to double macronutrients such as Ca, Mg and K, but rather increasing micronutrients such as Fe, Mn, B and Zn by 1x to 4x because fruit yield, root health and canopy volume were optimized where micronutrients were elevated compared to current guidelines. For HLB-affected grapefruit, the results of this study may show evidence that rehabilitation of HLB-affected trees may be limited by the age of the tree and number of years it has been HLB-affected and should start early at the establishment of a grove. Two graduate students completed graduate degrees in Soil and Water Sciences and Horticultural Sciences. We made 8 grower presentations, published two MS theses, 5 citrus industry articles and 3 refereed journal articles. Next steps Additional 5 articles will be published in refereed journals in the next few months upon completion of data analysis. This will also be followed by extension bulletins based on our current results and follow-up studies to follow on the inconsistent results on yield, canopy size, root growth and root density patterns and bacteria titers. In the next phase of the research, we plan to use other tools we have learned from growers and other projects such as using real-time artificial intelligence to measure nutrient deficiency, soil amendments and using variable rate fertilization tools, besides fertigation and controlled release fertilizers.
Please state budget status (underspend or overspend, and why):
We completed the project milestones and spent about 99.5% of the budget. We are grateful to the Florida Citrus Growers and CRDF for their financial support.
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. We investigated the effect of 10 adjuvants on oxytetracycline absorption via foliar spray. Four adjuvants including Flame slightly increased the antimicrobial effect of OTC on Las.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. We have conducted the test regarding how trunk injection of OTC affect ACP acquisition of Las. OTC signficantly reduces 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. PCR-sequencing analysis on Las 16SrRNA gene showed there was no mutation compared to the reported sequence, indicating no major changes in bacterial resistance against antimicrobials. Leaf samples were collected from OTC or STR injected trees in the Avon Park grove at two and four months after treatment for the analysis of the degradation metabolites of the bactericides. The extraction of the degradation metabolites were completed and will be subjected to HPLC assays. We are in the process of analyzing data related to how adjuvants affect bactericide delivery via foliar spray, bactericide residues in plants, and effect of bactericides on HLB positive citrus tress of different ages.
The purpose of this project is to assess nitrogen (N) fertilization rates for high-density plantings of different scion androotstock combinations in four commercial citrus growers. The overall approach is to 1) determine the appropriate N ratesneeded to support tree growth and productivity, 2) compare data obtained with existing UF/IFAS N recommendations, and3) revise best management practices (BMPs) for N application in young trees planted in high-density plantings affected byHuanglongbing (HLB). The Ph.D. student and Research Assistant dedicated to the project worked with the three growercollaborators participating in the project: IMG Citrus (Brian Randolph), Peace River Citrus (Larry Black Jr.), AgromilloraFlorida/ Lost Lake Groves (Clay Pedersen), Graves Brothers (David Howard), and performed the first applications of thefertilizers. Fertilizers were donated by Yara (Bill Easterwood) for the first year (20 tons or 40k lbs). Treatments were applied in all locations and the 2nd fertilization for the season was completed on 07/16/2021. The first tree size and leaf area index data collection was performed and analyzed as expected. After being informed by CRDF that the project will be prematurely concluded in December 2021, cooperating growers were informed that we will not proceed with further applications and data collections.
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 20, 2021and there was moderate disease on Duncan grapefruit trees but none on JJ5. We rated the plots again on July 28 (not June as mentioned in May report) 2021 and there were similar trends as in November 2020 although disease was lower given significant defoliation in the plots. We have also continued analyzing JJ5 for response to strains from Nian Wang to determine if those strains could overcome the resistance. Interestingly we noted unique phenotypes in plants inoculated with these strains although they were not typical disease reactions but more of a watersoaked appearance. We also used a 5′ Race kit to determine the transcription start in our JJ5 construct and observed that all strains tested activated trasncription of our 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. We have created a second construct for Vladimir Orbovic given the first attempt was not successful.The construct is with Vladimir Orbovic, who is in the process of creating additional transformants. He currently has identified one putative transgenic that has been potted and has several others that will be potted in the near 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. The trees were rated for disease in November, 2020 and there was considerable disease on all EFR plants with disease being more severe than on susceptible Duncan control. We rated the plots again on July 28 (not June as mentioned in May report) 2021 and there were similar trends as in November 2020 although disease was lower given significant defoliation in the plots. 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 have cut back the material and will analyze for susceptibility and bacterial growth once the leaves grow out again.
Frateuria defendens (Frd) is a recently discovered endophytic bacterium belonging to the family of Rhodanobacteraceae. The application of Frd by foliar spraying and soil drenching was documented to reduce the symptoms associated with phytoplasmas and liberibacters in various crops, including grapevine and carrots. It is thought that Frd has multiple modes of action including secretion of antimicrobials and priming of host defenses. This project was part of a collaborative effort between US (UF/IFAS SWFREC) and Israel (ARO) to develop an environmentally safe, affordable, and effective solution for the management of citrus HLB. It aimed to test whether Frd can effectively reduce CLas titers and HLB disease symptoms in citrus trees and can be developed as a commercial biocontrol agent. The project was originally intended as a 3-year study but was revised to conduct a 1-year feasability study. The overall goal of the project was to test the efficacy of Frd in reducing HLB disease effects in Florida citrus. The specific objectives were 1) to test the curative effects of Frd against HLB under greenhouse conditions, and 2) to optimize delivery of Frd and determine suitability for large-scale control of HLB in field conditions. Objectives had been modified slightly from the original proposal to make the study feasible within the reduced time frame of the funding period.Our studies showed that, under the conditions of our study, Frd had difficulties establishing in citrus leaves. This effect may have been associated with Frd-unfavorable biochemicals in the leaves and seemed to have been exacerbated by CLas infection.There was no clear evidence that Frd interacted with CLas directly (i.e., Frd applications did not reduce CLas titers) and/or had a curative effect against HLB disease. However, Frd applications had positive effects on plant growth and seemed to be able to mitigate some of the damage caused by HLB or to retard disease progression of severly affected plants under greenhouse conditions.The direct delivery of Frd via leaf infiltration or trunk injection was more effective than foliar sprays.
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