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.
We got our experiment in rhizotrons completed in September 2023. Sample processing and data analysis is under way. Root tracing and quantification of root traits will be completed in the next quarter. We should get the data processed and published in the next quarter or immediately following project completion.
Objective 2: Field test multiple acidification materials including organic acids for tree response CLas suppression, nutrient uptake, and root and vascular pH changes. In this quarter, we continued root sampling and soil pH determinations and applied final round of the treatments. We also finalized canopy determinatiions for trees used for the study.
2. Please state what work is anticipated for next quarter:
We will collect fruit yield and juice quality data in March/April 2024 to finalize objective 2 milestones.
We will get one article prepared and published in a refereed journal.
3. Please state budget status (underspend or overspend, and why):
The budget is on track and meeting the project milestones.
Recent work demonstrated that irrigation and soil acidification can improve the health and productivity of huanglongbing (HLB)-affected citrus; however, the best long-term methods to achieve this response have not been investigated. This work was done on highly pH-sensitive rootstocks, so the mechanism and broader applicability to other rootstocks is not fully understood. pH adjustment with sulfuric acid or sulfur treatment does temporarily modify the pH, but other acidifiers such as organic acids could provide additional benefits while acidifying the soil. The purpose of this project was to understand the physiological response of HLB-affected roots to soil acidification and to determine if organic acids that also provide improvement in soil health can effectively acidify the root zone and improve nutrient uptake and tree productivity through two main objectives.
Objective 1. Determine effects of lowered soil pH on Candidatus Liberibacter asiaticus (CLas) populations and root physiology including internal root apoplast and vascular tissue pH. Large-scale field trials of acidification on different rootstocks would require long-term experiments with carefully laid out plantings of multiple rootstocks at multiple sites with an overlaid acidification treatment. Understanding how acidification affects HLB-affected root physiology and CLas populations in pH-sensitive and tolerant rootstocks could speed up recommendations for acidification on new rootstocks.
Objective 2. Field test multiple acidification materials including organic acids for tree response, CLas suppression, nutrient uptake, and root and vascular pH changes. Organic acids provide multiple benefits to soil and root health that sulfur-based acidification cannot provide. If these acidifiers can provide the same acidification benefit, it would increase growers’ toolset of recommended acidification treatments and provide additional benefits to grove management in nutrient uptake and soil health.
This project addressed priority 2 of the RFP, specifically whether lower soil pH lowers internal root pH and subsequently lowers bacteria levels in the tree and if using organic acids for acidification can increase root nutrient uptake. Key findings from the greenhouse study were that acidic soils stimulate root growth, particularly around pH 5.5 conditions; citrus roots exhibit remarkable resilience and internal compensation mechanisms in response to pH variations. Optimizing soil pH and nutrient management can mitigate the impacts of HLB and promote the resilience of citrus trees. The field study showed a positive correlation between soil pH and soil calcium (Ca), magnesium (Mg0, zinc (Zn), manganese (Mn), and boron (B), possibly suggesting that the availability of these soil nutrients increased as pH increased. A negative correlation was observed between soil pH and soil iron (Fe), sulfur (S), potassium (K), and phosphorus (P), indicating decreased nutrients in soil solution as pH increased. There was no significant canopy difference between treatments but a difference between time points due to trees deteriorating due to the bacteria or loss caused by natural hazards, especially in hurricane-prone Florida. Results show no significant difference in fruit yield and fruit quality parameters in all treatments. In both sites, soil pH positively correlated to soluble solids and soluble solids to acid ratio but negatively to titratable acidity.
Project Outputs and Presentations
Journal manuscripts in preparation or accepted
1) Sambani, D., T. Vashisth, D. Bright, D. Kadyampakeni. In preparation. Physiological responses of huanglongbing (HLB)-affected citrus tree roots to soil acidification. Journal of Environmental Quality.
2) Sambani, D., T. Vashisth, D. Bright, H. Ghoveisi, L. Rossi, T. Ayankojo, A. Atta, D. Kadyampakeni. The impact of organic acids on soil health and acidification for enhanced nutrient uptake and tree productivity in HLB-affected citrus trees. HortScience (In review)
3) Sambani, D. and Kadyampakeni, D. Exploring the Nexus of Citrus HLB Dynamics and Interactive Influences of Soil Nutrient Availability: A Comprehensive Review. Journal of Plant Nutrition (accepted).
4) Kadyampakeni, D., T. Vashisth, D. Sambani. 2024. Soil fertility and soil pH interaction. Citrus Industry Tip of the Week article, Aug. 2024.
5) Kadyampakeni, D. and T. Vashisth. In preparation. Interaction of soil pH and soil fertility. EDIS Publication.
Conference Proceedings/Presentations
1) Sambani, D., T. Vashisth, L. Rossi, and D. Kadyampakeni. 2024. The physiological responses of citrus tree roots to soil acidification. SSSA 2024 Bouyoucos Summer Conference, June 10-12, 2024, San Juan, Puerto Rico.
2) Sambani, D., T. Vashisth and D. Kadyampakeni. 2023. The physiological responses of citrus tree roots to soil acidification. Poster presentation ASA/CSSA/SSSA Tri-Society Meetings, St. Louis, Missouri. Oct. 29-Nov. 1, 2023.
3) Sambani, D., T. Vashisth and D. Kadyampakeni. 2023. The physiological responses of citrus tree roots to soil acidification. ASHS Poster Presentation, Orlando, FL. July 31-Aug. 4, 2023.
4) Sambani, D., T. Vashisth, L. Rossi and D. Kadyampakeni. 2023. The physiological responses of HLB-affected citrus tree roots to soil acidification. Southern Fruitworkers Conference, Quincy, FL. November 14-16, 2023.
5) Sambani, D., T. Vashisth, L. Rossi and D. Kadyampakeni. 2024. The physiological responses of HLB-affected citrus tree roots to soil acidification. 9th UF Water Institute Biennial Symposium, Gainesville, FL. Feb. 20-22, 2024.
6) Sambani, D., T. Vashisth and D. Kadyampakeni. 2023. The physiological responses of citrus tree roots to soil acidification. FSHS Oral Presentation, Daytona Beach, FL. June 11-13, 2023.
Acknowledgments
The researchers are grateful to the support of the Citrus Research and Development Foundation for funding this research.
1. Please state project objectives and what work was done this quarter to address them: Objective 1: To compare different rates and annual use patterns of trunk-injected OTC on late-season (Valencia) and early-season (Hamlin) sweet orange tree and their effects on health, yield, and fruit quality over a period of 3 years in a commercial citrus production environment.Year-2 injections were completed in May for the Hamlins and in June for the Valencias. Tree size and canopy volume data were collected. Leaf samples were collected for CLas analysis. Obective 2: To compare spring with late summer injections in Valencia orange trees to determine if OTC effects on yield can be enhanced, and the harvest window extended, through timing of injections.Valencia OTC fruit residue analyses were completed. Fruit from trees injected in June 2023 had residues below the allowed 10 ppb regardless of the rate. Fruit from trees injected in September had higher residues, suggesting that trees should not be injected in September. This year’s late summer injections will therefore be conducted in August instead of September. 2. Please state what work is anticipated for next quarter: We are preparing for the Valencia August injections. Leaf sampling will be conducted for CLas analysis. Phytopthora trunk rot has been observed in this location. The rot does not seem to be associated with OTC injections; we will conduct measurements/ratings to document this. 3. Please state budget status (underspend or overspend, and why):The budget is as expected.
1. Please state project objectives and what work was done this quarter to address them: Objective: To test OTC alone and in combination with Streptomycin and Zn at low and neutral pH (using a stabilizing adjuvant) in a commercial citrus grove. The trial was initiated at a commercial location in SW Florida. Trees are Valencia /X-639 planted in 2017. Baseline data were collected (tree size, canopy volume, canopy health). Leaves were collected for CLas analysis. Trees were injected in June. There were 10 treatments: 1. Low pH control (acidified water) 2. Neutral pH control (adjuvant) 3. Neutral pH+Zn 4. Low pH OTC 5. Low pH STM (streptomycin) 6. Low pH OTC+ STM 7. Neutral pH OTC 8. Neutral pH OTC+Zn 9. Neutral pH STM 10. Neutral pH OTC+STMLeaves were collected again 3 days and 1 month after injection.Trees experienced severe phytotoxicity with the neutral pH adjuvant alone (and in combination with OTC and STM). Low pH OTC injections also resulted in phytotoxicity, but less severe than the neutral pH treatments. Addition of Zn to the neutral pH adjuvant reduced phytotoxicity to the level of low pH OTC. No phytotoxicity was observed for low pH STM and the low pH control. Phytotoxicity ratings were conducted for all trees to quantify effects. We also started a small-tree experiment at the SWFREC farm, injecting low pH water and neutral pH adjuvant with and without OTC for destructively sampling after harvest to study the internal injury. The same phytotoxicity was observed after injection (more for the neutral pH treatments and less for the low pH treatments). Therefore, additional research is being done reformulating the neutral pH adjuvant for scalability and to minimize plant toxicity. For this, several industry-grade ingredients (chloride-free) have been tested for compatibility and performance of dissolving OTC at near-neutral pH. 2. Please state what work is anticipated for next quarter: Trunk wound sizes will be measured. Leaves will be collected for CLas and OTC detection. New adjuvant optimizations will be tested for toxicity on young citrus trees using the chemjet injection system. Formulation uptake, visual toxicity, and SPAD measurements will be used to determine the best candidate. 3. Please state budget status (underspend or overspend, and why):The subcontract was finally set up allowing us to start spending funds.
1. Please state project objectives and what work was done this quarter to address them: Specific objectives are: 1) To determine the right timing for Zn and K treatments to minimize fruit drop.We started analyzing auxin content in Valencia and Hamlin. For Hamlin, we see a 3-fold increase in IAA content in both abscission zone and flavedo around October and November, when treatments of Zn+K are performed in late september-early October. This is transient but is coincident with a decrease in fruit drop. In Valencia results are less clear and we are still analyzing samples, but we see this IAA increase around late February following Zn+K treatment.2) To determine effects of GA3 and 2,4D applications on fruit retention when applied at different times during fruit development.After harvesting was finished we have started planning the new set of treatments and selected the plots to start treatments in the coming quarter. 3) To develop a strong and proactive outreach program. -Alferez F. New tools for growing citrus under endemic HLB, Davie UF Extension, Master Gardener Talk Series, June 4, 2024.-Otavio de Sousa, M., Alferez F. 2024. Fine-tuning zinc and potassium treatments to increase yield in sweet orange FSHS annual meeting, June 2024, Orlando. 2. Please state what work is anticipated for next quarter: We will resume treatments in objectives 1 and 2, characterize tree health, and continue auxin analysis. 3. Please state budget status (underspend or overspend, and why): Budget continues on track
1. Please state project objectives and what work was done this quarter to address them:The objectives of this project are:1, To study the effect of Brs on priming immunity on young, newly planted trees. This will allow to know for how long immune response will last after Br application, so we can adjust timing (number of applications).We monitored summer flushes and they were more synchronized than in controls. Also it is apparent more root growth in trees treated with Brs. The canopies continue being denser than in controls and more fruit is growing in Br treated trees. In general tree health continues to be improved significantly. Since we are getting more synchronized flushes, we started concentrating Br sprays only at the time of flushing in a set of trees and follow psyllid population, with the idea of limiting Br application only to these moments of flushing and maximum psyllid pressure, as described in our previous report.2, To determine the best time of application (frequency) to achieve maximum protection against pests and disease in newly planted trees. After we determined that gene expression of genes related to SA pathway is maximum at 30 days, but still significant by 60 days, we have started treatments every other month in young trees to see if this rate is enough for protection.3, To determine the effect of Br application on advancing fruit maturation in both Valencia and Hamlin.We have contacted with CPI in Felda and we have started plans to use trees that have been injected with OTC and show symptoms of canopy recovery but not significant improvement in fruit quality to test if Br application can boost internal quality in the next season. As a reminder, this idea came after seeing this year that we get most of the Br effect on increasing Brix on trees with the best canopies (in our plots that are not treated with OTCs).Outreach:-Susmita Gaire, Ute Albrecht, Ozgur Batuman, Mongi Zekri, Fernando Alferez (2024).Individual Protective Covers (IPCs) Improve Yield and Quality of Citrus Fruit under Endemic Huanglongbing (HLB). Preprints (accepted in Plants, in press) https://doi.org/10.20944/preprints202407.2288.v1 CRDF project 22-003 is aknowledged in the publication, since the use of Brs as a strategy to prolong tree health and improve quality is discussed in the paper.2. Please state what work is anticipated for next quarter:-We will continue analyzing juice quality and determine gene expression as related not only to plant immunity, but also we are interested in sugar and acid metabolism in response to Br.-We will select trees with different response to OTC in terms of fruit quality, we will design the trial plots and will start to perform the treatments during this coming months in collaboration with CPI.-Finally, for objectives 1 and 2 we are starting a new set of trials: i) concentrate Br treatments in young trees only at the time of flushing and ii) apply Br every other month.-Outreach: September 19. Alferez, F. Evaluating Brassinosteroids to improve citrus fruit quality and tree protection under HLB. On zoom.3. Please state budget status (underspend or overspend, and why):This project was approved for 3rd year funding in July. We have reduced the budget in order to be more cost-efficient in this last year of the project.
1. Please state project objectives and what work was done this quarter to address them:
Overall goal and specific objectives. The overall goal in this proposal is to evaluate the utility of trunk-injected OTC for reducing CLas infection and transmission in non-bearing and bearing citrus. The specific objectives are to:
Objective 1) Quantify CLas reduction and tree health in response to OTC.
Experiment 1.1. Mature Trees. Field trials were established with grower collaborators in groves at Wauchula and Frostproof, Florida.
For the Wauchula grove, treatments were applied to 10-year-old, CLas-infected Valencia trees of a standard size and CLas titer. Four treatments under evaluation are: 1) insecticide control of ACP on productive trees, 2) insecticides plus addition of trunk injection of OTC on productive trees, 3) insecticides control of ACP on non-productive trees, and 4) insecticides plus addition of trunk injection of OTC on non-productive trees. The insecticide treatment consists of a program that is intended to represent what certain growers are doing currently4-6 annual sprays of formulations labeled for ACP that are rotated between the following active ingredients: (thiamethoxam, imidacloprid, spinosyn, fenpropathrin, cyantraniliprole, methoxyfenozide, clothianidin, and diflubenzuron). Rectify was injected into mature trees at 50-100mL/tree (8250 ppm solution), according to the product label, in April 2023 using tree injectors. Each treatment was applied to four replicate groups of 0.022 ha plots that consisted of four rows, with five trees/row comprising 20 trees per plot.
Treatments at the Frostproof site were applied to 10-year-old, CLas-infected Valencia or Hamlin trees of a standard size and CLas titer. Four treatments are being evaluated: 1) insecticide control of ACP on Valencia trees, 2) insecticides + addition of trunk injection of OTC (Rectify) on Valencia trees, 3) insecticide control of ACP Hamlin trees, and 4) insecticides + addition of trunk injection of OTC (ReMedium) on Hamlin trees. The insecticide treatment consisted of a program that is intended to represent what many growers are doing currently4-6 annual sprays of formulations labeled for ACP and were rotated between the following active ingredients: (thiamethoxam, imidacloprid, spinosyn, fenpropathrin, cyantraniliprole, methoxyfenozide, clothianidin, and diflubenzuron). Rectify was injected into mature trees at 50-100mL/tree (8250 ppm solution), according to the product label, in March 2023 using tree injectors. Each treatment was applied to four replicate groups of 0.022 ha plots consisting of four rows, with five trees/row comprising 20 trees per plot. According to the product label, using tree injectors, Oxytetracycline (ReMedium) was injected into mature trees at 50-100 mL/tree (5,500 ppm solution) in March 2023. Each treatment was applied to four replicate groups of 0.022 ha plots that consisted of four rows, with five trees/row comprising 20 trees per plot.
Tree Infection Sampling. Monthly leaf samples are harvested from all treatment plots in both experiments. Four mature leaves are randomly selected from five trees in the second row of each replicate plot at each sample date to assess CLas infection. Collected leaves were placed into plastic bags in the field and transported to the laboratory in coolers with ice, where they were stored at -20oC for subsequent CLas detection, as described below.
Detection of CLas in plants and psyllids. Dual-labeled probes were used to detect CLas in ACP and citrus plants using an ABI 7500 qPCR system (Applied Biosystems, Foster City, CA) in a multiplex TaqMan qPCR assay described in (Li et al. 2006). DNA from insect and plant samples was isolated using the DNeasy blood and tissue or DNeasy plant kits (Qiagen Inc, Valencia, CA), respectively. Las-specific 16S rDNA from psyllid and plant extracts were amplified using probe-primer sets targeting internal control sequences specific to ACP [insect wingless] or plant [cytochrome oxidase] gene regions (Li et al. 2006). DNA amplifications were conducted in 96-well MicroAmp reaction plates (Applied Biosystems). Quantitative PCR reactions consisted of an initial denaturation step of 95°C for 10 min followed by 40 cycles of 95°C for 15 s and 60 °C for 60s. Each 96-well plate containing ACP samples included a no template control, a positive control (Las DNA in DNA extractions from ACP), and a negative control (no Las DNA in DNA extractions from ACP).
Tree health. Tree growth was assessed at the beginning of experiments to determine the effect of OTC injection over time at both locations. Tree size measurements (height, canopy, width, and trunk diameter at tree base) are taken from each tree in each treatment replicate. The measurements have been made at the initiation and mid-way through the experiment, and will also be made at the end of study. New leaf growth (flush) is being assessed monthly during the growing season by quantifying the number of flushes in an open 0.3m3 cube placed into three random positions per five trees of each replicate (Hall and Albrigo 2007).
Recent activities:
Leaf samples corresponding to May through December 2023 were successfully collected at both locations and are currently being processed for CLas detection. Additionally, flush numbers were successfully collected. Tree health measurements were done at time zero and in the middle of the experiment at both locations. This report presents results regarding CLas infections obtained from May to August 2023 in both locations.
Wauchula site. At this field site, we are investigating whether application of Rectify could stop tree decline and improve the productivity of tree in severe decline in commercial groves. In May 2023, a month after injections were applied to trees, a decrease in CLas abundance occurred in injected unproductive and productive trees compared to their respective control treatments. In June 2023, injected productive trees exhibited significantly lower CLas abundance than the control. In July and August 2023, significantly lower CLas abundance was observed on injected unproductive trees, followed by injected productive trees as compared with their respective controls. Overall, trees injected with Rectify showed lower CLas abundance than their respective controls, with statistically significant reductions in bacterial titer observed 60 days after injection.
Frostproof site. At this field site, we are comparing the efficacy of two OTC formulations (Rectify and Remedium) in reducing CLas infection in commercial groves. In May 2023, two months after injections were applied, trees injected with Rectify had lower CLas abundance than those injected with Remedium. This trend continued in June and July. However, CLas abundance was statistically lower in Rectify-treated trees during these two comnths compared with their respective controls, which pathogen titer was not statistically reduced in Remedium-treated trees compared with controls. In August 2023, there was lower CLas abundance observed in Rectify or Remedium-treated trees than in controls. Overall, Rectify-treated trees had the lowest CLas abundance among treatments 60 days after injection and this result remained consistent throughout the summer of 2023. In general with both formulations, OTC-injected trees had lower CLas abundance in all cases than not-injected trees.
Objective 2) Determine the effect of OTC injection on psyllid populations.
Psyllid Population Sampling. On each sampling date and each location, Asian citrus psyllid (ACP) adults were sampled in all plots by placing a 22 × 28 cm white plastic sheet horizontally and 30 cm underneath a randomly chosen branch. Each branch was struck three times with a 40 cm length of PVC pipe. Adult ACPs falling onto the sheet were quickly counted (Monzo et al. 2015). All trees in the replicate were sampled in this manner each month.
Recent activities:
ACP adult populations have been monitored monthly from May through December 2023 at both locations. During the next reporting period, we will include results regarding the effect of OTC on ACP adult populations.
Objective 3) Determine the effect of OTC injection on CLas transmission.
Field trials were established at a collaborator grove at Babson Park, Florida. In this grove, treatments were applied to 10-year-old, CLas-infected Valencia trees of a standard size and CLas titer. There were four treatments evaluated: 1) Rectify (8,220 ppm), 2) Remedium (5,500 ppm), 3) Fireline (70,000 ppm) (positive control), and 4) Insecticides only (negative control). For all antibiotics, trunk injections were performed using Chemjets. In this location, CLas infection in trees was evaluated by randomly collecting four leaves at time zero, 2, 7, 30, 45, 60, and 90 days after injection. Additionally, 40 leaves per tree (20 top and 20 bottom) were collected from 2 trees in each replicate plot to measure the translocation of treatments at the times mentioned above.
Experiment 3.1. Acquisition assays. Psyllid nymphs, which develop on immature leaf tissue, acquire CLas more efficiently than adults; therefore, acquisition of CLas from OTC-treated infected citrus trees was compared with acquisition from untreated infected trees, using the abovementioned treatments. ACP adults (five female and five male) from uninfected laboratory cultures were on young leaf growth (flush) of treated or control infected trees for oviposition. Each treatment was replicated three times on individual trees. Following oviposition, adults were collected and preserved for CLas detection. Egg clutches were left on trees enclosed in mesh sleeves. After nymphs reached adulthood, psyllids and leaves from test plants were collected. Transmission assays were repeated on the same trees every four months following treatments to determine the influence of treatments on pathogen acquisition over time. The effect of OTC injection on the acquisition of CLas was assessed by comparing the CLas titer in ACP caged on citrus trees before and after treatments and across time.
Experiment 3.2. Inoculation assays. A subsample of 10 ACP per treatment collected from the above trees was transferred to uninfected citrus seedlings in an insect-proof greenhouse. ACP was enclosed on plants for inoculation feeding for 7d. After that, ACP adults were collected for CLas detection using RT-PCR. Furthermore, leaves will be collected every 30, 45, 60, 90, 120, 150, and 180 days for CLas detection.
Recent activities:
.Leaves were collected to measure both CLas infection and OTC translocation and are currently being processed. The first replication of sampling for experiments 3.1 and 3.2 was successfully completed in June 2023. A second replication was performed in August for all experiments. The upcoming report will include results regarding the effectiveness of OTC formulations on CLas transmission (CLas acquisition and inoculation) and OTC translocation. This report presents results regarding the effects of OTC treatment on CLas transmission by ACP during the priod from initiation of the experiment (day 0) to 90 days after injections.
Babson Park field site. At time zero, CLas infection levels were similar across treatments. Two and seven days after treatments were applied, CLas abundance fluctuated across treatments, and no statistical differences were observed. At 30 days after treatments were applied, trees injected with Fireline exhibited the lowest CLas abundance, followed by Rectify and Remedium treated trees and then the control. At 30 days after treatment, CLas abundance was statistically lower in Fireline or Rectify-treated trees than that observed in control trees. After 45 days, Fireline-treated trees showed significantly lower CLas abundance than the other treatments tested, and no statistical differences were observed between pathogen load in trees treated with Rectify and Remedium as compared with control trees. At 60 days after treatments, the lowest CLas abundance was observed in Remedium-treated trees, followed by Fireline and then Rectify-treated trees. However, these reductions in CLas abundance were not statistically different from the control. A similar pattern was observed at 90 days after treatments were applied, but the highest CLas abundance among treatments was observed in Rectify treated trees. Overall, Fireline-treated trees exhibited the lowest CLas abundance across time, followed by Remedium-treated trees.
2. Please state what work is anticipated for next quarter:
This investigation is ongoing. TWe are currently wrapping up the initial field season for the project and have harvested fruit for analysis. Fruit/juice quality data will be reported next quarter.
3. Please state budget status (underspend or overspend, and why):
The budget spending is on track as anticipated.
4. Please show all potential commercialization products resulting from this research, and the status of each:
Not applicable at this time. This project is evaluating registered and available products.
1. Please state project objectives and what work was done this quarter to address them:The overall goal of the project is to develop fertilization strategies to best match nutrient supply and demand, and develop recommendations for optimal nutrient application timing as compared to a simple constant supply, which will improve fruit yield, quality, and reduce fruit drop. A)Objective 1) Test if a reduced N-P-K nutrient supply in the fall is safe for sustaining HLB-affected citrus, and whether it can improve fruit quality to facilitate earlier maturity / harvesting and reduce fruit drop:In this quarter, fertilization treatments were applied (2nd and 3rd application) in the third year of the experiment. Routine leaf sampling was done in May and processed for nutrient analysis. Nutrient data from leaf sampling in March 2024 is described below, and leaf data from May 2024 samples is still pending. Fruit diameter measurements were started with fruit tagging, and dropped fruits counts began after the physiological drop for the 2024 season, and then will be continued to measure every 2 weeks until harvesting. Destructive root sampling in the Hamlin experiment was done in January and scanned using a flatbed scanner to estimate total root length and volume (presented below). Additionally, total root biomass per unit soil area was also calculated. Fruit growth and drop: These measurements were started after the physiological drop in May. Eight fruits were tagged in each experimental unit to measure the fruit growth recording over time, measured at every 2-week interval until November. Similarly, fruit drop counting from each plot was started after removing physiological drop fruits. Fruit dropped in both Hamlin and Valencia varieties were less in June than May, and fertilizer treatments had no significant effect on fruit drops to date. Fruit drop counting data will be used to calculate the cumulative fruit drop per tree and fruit drop rate. Leaf nutrient status in March 2024: Leaf sampling was done one month after the first 2024 season fertilization in the experiment. Most of the macronutrients and micronutrients in leaf samples were in the optimum concentration range in the Hamlin trial except Mn and Zn, which were in the low range. The status of leaf nutrients was not significantly different between fertilizer treatments. This might be due to an almost equal proportion of fertilizer applications in February and efficient nutrient uptake by the Hamlin trees during leaf flushing and bloom growth stages. However, leaf nutrients N, K, Zn, and Fe were deficient in the Valencia trees. Contrasting results might be due to the later ripening stages of the 2023 crop for the Valencia variety, where internal physiology controls nutrient uptake and distribution. Root dynamics: Root growth measurements for the 2023 season root samples were calculated using Rhizovision® root analysis software, and the total root biomass at two soil depth intervals (topsoil 0-20 cm and subsoil 20-40 cm) was measured. Total root length, total root volume, and root dry biomass per unit soil volume of fibrous (<2mm size) and coarse (>2 mm size) roots were calculated.Total root length, volume, and biomass were significantly higher in topsoil than in subsoil. The measured parameters of fibrous roots were significantly higher than those of coarse roots in surface topsoil except total length of fibrous roots which was higher in subsoil. In contrast, root biomass and volume of coarse roots were measured higher in subsoil (20-40 cm). There were no significant differences in root parameters in response to sigmoidal fertilizer management compared to the control linear fertilizer management treatments in 2023. However, overall root parameters were higher in treatment 3 (100% sigmoidal supply with 25% fall application on August) in subsoil. Objective 2) Develop an optimized, practical fertilizer timing management profile to boost fruit quality and reduce fruit drop for HLB-affected citrus based in part on the sigmoidal nutrient demand curve defined by four physiological growth phases (0=bloom/fruit set; 1=cell division; 2=cell enlargement; 3=maturation):Fruit growth measurement and drop counting of year 3 were started in June and will be reported in the next quarter. Initial data on fruit drop showed no significant difference in fertilizer management treatments. Leaf nutrient status during March was also not significantly different from fertilizer management treatments; however, suboptimal or deficient leaf nutrients N, K, Fe, and Zn were recorded in Valencia. Sigmoidal fertilizer management treatments were not significantly affecting root parameters like total root volume, root length, and root biomass compared to the linear fertilizer management (control) treatments. B) New developments: NoneC) Issues: None2. Please state what work is anticipated for next quarter:Routine lysimeter sampling, leaf sampling, processing and analysis will be ongoing, as will tree size, root growth and fruit drop and growth measurement.Plan for July to September 2024 Fertilization treatment applications (4th or 5th application) Routine leaf sampling every 2-month interval (July 30, September 30) Root scanning via Minirhizotron Trunk size measurement Routine lysimeter leachate collection Schedule Aerobatics flight for tree health, vigor, canopy volume, and area estimation 3. Please state budget status (underspend or overspend, and why):Spending rate is slightly below the target by about 15%. This is considered within the normal deviations of budgeting. The departure of a TEAMS employee from our lab has caused unanticipated reduction in salary expenses. We will request a 6-month no cost extension in September in order to complete the collection and analysis of the third year field data (Hamlin harvest in December, Valencia in February / March 2025).
1. Please state project objectives and what work was done this quarter to address them:In the first three quarters of the agreement, the CRAFT Foundation accepted applications for Round Two of the Existing Tree Therapies program as well as Cycle Five of the tradtional CRAFT new planting program. After review by CRAFT staff to validate the eligibility of applications, the CRAFT Board of Directors voted to approve all eligible Existing Tree Therapies applications submitted in Round Two, including those rolled over from the Round One waitlist. A total of approximately 920 projects were approved. Approximations are due to consolidation of applications, which may require some revision prior to final execution of agreements. These projects include acreage from all major citrus-growing regions in the state, as well as varieties including oranges, grapefruit, hybrids and others. While the majority of projects are focusing on the injection of oxytetracycline, treatments of giberellic acid, 2,4-D and brassinosteroids are also included in the approved projects. The Round Two approved projects combine with those previously approved in Round One for a total of more than 1,100 Existing Tree Therapies projects representing approximately 74,000 acres. Participants from both Rounds One and Two have submitted their three-year historical data, first-year treatment data, and 2023-24 harvest data to CRAFT. Many participants have also submitted their second-year treatment data. This data will be added to the CRAFT Data Portal moving forward. Cycle Five of the traditional new planting program received 175 new applications. An additional 46 applications rolled over from the Cycle Four waitlist for consideration in this cycle as well. A total of 211 applications representing more than 14,000 acres were reviewed and considered for approval by the CRAFT Technical Working Group and Board of Directors. Based upon the projects presented, applications were organized by solid-set and reset and further within subgroups of similar projects for replication and evaluation purposes. The Board of Directors approved a total of 71 new planting projects under CRDF funding for a total of 3,282 acres, including 360 acres utilizing Program for Expedited Propagation (PEP) Materials. These projects, along with approximately 60 others representing more than 6,000 additional acres approved by the CRAFT Board of Directors for Cycle Five under other, federal funding agreements will join the 226 projects representing more than 9,000 acres in Cycles One – Four. All Participation Agreements were fully executed and returned to CRAFT prior to the June 30, 2024 deadline and planting of the Cycle Five projects has begun. In addition to the development of projects and execution of contracts, CRAFT staff continued communications and outreach efforts including, a featured presentation at the Florida Citrus Industry Annual Conference, attendance at industry tradeshows and Expos, articles in multiple industry and general publications; updating of the CRAFT website (craftfdn.org); public meetings of the Technical Working Group and Board of Directors; and more. A full list of communications efforts is available upon request. 2. Please state what work is anticipated for next quarter:Existing Tree Therapies projects will continue to report treatments and harvest data for a term of three years to allow for full analysis of the impacts of approved therapies. Following completion of planting, pre-audits of each Cycle Five project will be used to confirm the planting of trees and compliance with contract. Growers will be expected to report production data in real time or at least quarterly in to the USDA-CRAFT Data Portal for a term of six years. The data portal will include a separate entry point for data/ measurements from third party partners such as FDACS and Aerobotics. Data entered by participants and third parties for each project will be visible initially by the growers for their individual projects, and at a later date, on public dashboards developed by CRAFT and USDA-ARS. 3. Please state budget status (underspend or overspend, and why):As of June 30, 2024 contracted grower payments were in line with budget and all invoices had been submitted to CRDF.
1. Please state project objectives and what work was done this quarter to address them: Objectives: 1) Test the efficacy of different injection devices, 2) Determine the most effective formulation of OTC, 3) Determine the best month of injection and most appropriate OTC concentration based on tree size. Trial 1: SW FL(Duda) – 8-year-old Valencia/Carrizo.** Trial 2: SW FL (Graves Bros) – 8-year-old Valencia/Kuharske. Trial 3: East coast (Graves Bros) – 9-year-old Valencia/sour orange. Trial 4: East coast (Graves Bros) – 4-year-old Valencia/x639. Trial 5: Central ridge (King Ranch) – 4 year-old OLL8/x630**The grower collaborator for trial 1 stopped tree care after our year 2 injections, and trees have been declining rapidly. We have therefore terminated our evaluations in this trial. Harvests and fruit quality analyses were completed for trials 3 and 4. In trial 3, we found significant increases in yield and juice quality after OTC injection. Depending on the OTC dose and the method by which it was administerd, yields were increased by 74-131% (control: 31 lbs. fruit/tree, injected: 54-72 lbs fruit /tree) and Brix was increased by 11-20% (control: 9.7, injected: 10.8-11.6). Also improved by OTC injections were the juice color, acid, and the Brix/acid ratio. Both methods (Chemjet injection+Arbor-OTC and Flexinject injection+ReMedium) produced similar results. The best juice quality was obtained injecting ReMedium with the Flexinject injector at the high rate (1.1 g OTC/tree), but at the lower rate (0.55 g OTC/tree) two-sided injection using the chemjets produced slightly higher yields than one-sided Flexinject injections. In trial 4 we found significant differences among the different treatments. Overall, most treatments improved fruit yield and juice quality, with higher OTC doses producing better results. The highest dose (0.75 g/tree) resulted in 52 lbs of fruit/tree compared to 20-22 lbs for the non-injected and OTC-foliar spray controls. OTC was delivered double-sided either using the Treecise system (dose: 0.18g and 0.38 g/tree) or the chemjet injectors (dose: 0.38 g and 0.75 g/tree). Best results for the juice quality were obtained when OTC was injected in August and at the high rate (0.75 g/tree) – Brix was 11.3 compared to 8.7 for the controls.The same rate injected in May produced a Brix of only 9.6, but May injection resulted in larger fruit than August injection. The same trend was observed in the previous year. Some of the data from this project were presented at the Citrus Institute, the Citrus OJ Break, the Florida Citrus Industry Annual Conference, and other venues. 2. Please state what work is anticipated for next quarter: We are still waiting for some of the results from the OTC residue analyses (performed by the NSL lab). PCR analyses will still need to be completed as will wounding evaluations. Data will be included for presentation at the Citrus Expo and in an upcoming Citrus Industry magazine article. 3. Please state budget status (underspend or overspend, and why):Everything is on track.
1. Please state project objectives and what work was done this quarter to address them: Main objective: To determine the large-scale efficacy of trunk injection of OTC on citrus tree health, fruit quality, and yield in existing rootstock trials in three different commercial growing environments. Sub-objectives: a) test the efficacy of OTC injection in the rootstock trunk compared to injection into the scion, and b) determine whether OTC injection increases/restores root densities and if there are interactions with the rootstock. Harvests and fruit quality analyses were completed for the trial in SW Florida. Tree care was not as desired in this location as the collaborator (Duda) has ceased citrus production in this area. Overall, trees were still relative productive in this trial with an average of 102 boxes per acre. The best performing rootstock in this location in terms of production was UFR-2 (144 boxes/acre), and the worst was Swingle (46 boxes/acre). Across all rootstocks OTC injection increased yield by 21% but differences were not statistically significant. The yield for trees on US-812, UFR-2, and Swingle improved the most (28-29%) while there was little to moderate improvement for trees on UFR-4 (5%) and US-942 (14%). OTC injection increased juice quality significantly across all rootstocks.There was no significant difference between rootstock and scion injection for yield and juice quality. Wound closure was the same on scion and rootstock. However, bark cracks were longer after scion injection than after rootstock injection. There were significant differences among rootstocks for both wound closure and bark crack length. Year-2 injections were completed. This year we measured significant differences in the uptake rate between rootstock and scion injection: trees injected into the scion took OTC up significantly faster than trees injected in the rootstock. 2. Please state what work is anticipated for next quarter: We will monitor the trees and continue to collect leaf samples for CLas detection. Fibrous root density collections will be performed. 3. Please state budget status (underspend or overspend, and why):The budget is mostly as expected.
1. Please state project objectives and what work was done this quarter to address them:
Valencia fruit was harvested by the end of April in trial site 1.
Similar to the last year, 400 ppm of both Fertizink and Nuzink (treatments applied in October 2023, January 2024 and February 2024) increased yield as compared to control (180 Kg of fruit per 3 trees replicate as compared to 120 kg per three-tree replicate, 33% increase) with no significant differences between both compounds and by 17% as compared to trees treated on the same dates with Zn sulfate, that was incorporated as a grower standard control. Internal quality was not significantly affected this year. Additionally, the fruit juice and rinds have been digested for Zn content (analysis pending). We continued analyzing auxin content in fruit flavedo. After three applications of both compounds, NuZn and FertiZn, auxin increased as in last year by similar levels. Noticeably, trees this year are in worst shape, with very thin canopies, which may explain the lower yields, even though physiologically trees responded to the treatments by increasing endogenous auxin content. Next year study will be very important as we will have trees with better health, for instance trees that have received 2 years of OTC injections, these treatments could serve as a bridge for the gap year.
The greening severity was evaluated in trial site 2 (ridge) for both Valencia and Hamlins (400 ppm and 800 ppm Zn). In this site, we observed a dose dependent effect on the greening severity for both Hamlin and Valencia. Over 80% trees treated with 800 ppm of NuZn and FertiZn are rated as healthy compared to those treated with 400 ppm, the later rate displayed some symptoms of greening. Nevertheless, NuZn and FertiZn at these rates significantly increased the percentage of healthy rated trees compared to Zn sulfate. The greening evaluation correlates with the yield results. Valencia plot treated with NuZn and FertiZn (800 ppm), demonstrated a yield increase of 25% and 45%, respectively, compared to the untreated control and an increase of 8% and 25% compared to Zn sulfate.
Fruit and juice quality was assessed for Hamlin and Valencia (trial site 2) harvested in Fall 2023. FertiZn at 800ppm performed the best in terms of fruit size and weight in comparison to all other treatments including controls at both rates. None of the treatments have any significant impact on juice quality.
The raintfastness, Zn absorption and in planta mobility were evaluated using swingle liners. On average NuZn and FertiZn demonstrated a 20% and 130% increase of bound Zn, respectively, over Zn Sulfate. These findings correlate with the percentage of ion to insoluble Zn found in the formulations. A week after treatment, the Zn content in the plants was analyzed. It was found that FertiZn increased the Zn content in roots by 160% compared to Zn Sulfate. This might explain the reduction in greening severity and the impact on overall physiological response.
2. Please state what work is anticipated for next quarter:
In the next quarter, we will quantify the Zinc content in juice of fruit harvested trial site 1. Additionally, fruit shelf-life data will be analyzed to determine if the treatments possess other benefits. In year 3 trial, we will analyze Zn leaf residue on field samples. We will also communicate our research findings through peer-reviewed publications.
3. Please state budget status (underspend or overspend, and why):
on track
1. Please state project objectives and what work was done this quarter to address them: Specific objectives are: 1) To determine the right timing for Zn and K treatments to minimize fruit drop.Harvest in Valencia was done in late April. We performed treatments at four different dates: on early September 2023, a October 2023, December 2023, and February 2024. Yields from plots treated with Zn and Zn+K in December were significantly higher than non-treated controls. Our yield numbers were 16 pounds of fruit per tree in nontreated controls, 24 pounds of fruit per tree in Zn-treated trees, and 27 pounds of fruit per tree in Zn+K treated trees. These differences were statistically significant at p<0.05. We found that cummulative fruit drop declined when treatments were performed in December and February, which may explain why we had more yield with the December treatments. Yield in trees treated in February was higher in Zn+K although not siginificant at p<0.05. We have also measured canopy volume and we have seen an increase with Zn+K treatment in December: 2.88 m3 as compared to 2.28 m3 in controls. This is a 26% increase in canopy that may explain the increase in yield with this treatment performed at this time.2) To determine effects of GA3 and 2,4D applications on fruit retention when applied at different times during fruit development.We harvested the Valencia and analyzed the harvest data of both varieties. GA+2,4 D treatment together significantly reduced the fruit drop by 18% in Valencia. No such effect was seen in Hamlin as the trees were harvested significantly early before the drop began. 3) To develop a strong and proactive outreach program. -Alferez F. New tools for growing citrus under endemic HLB, Collier Fruit Growers Association, Naples, April 15, 2024.-1 communication was submitted and accepted for the FSHS annual meeting in Orlando this year. 2. Please state what work is anticipated for next quarter: We are analyzing auxins in all samples collected in objective 1 and we are planning next season's treatments in objectives 1 and 2 3. Please state budget status (underspend or overspend, and why): Budget continues on track
1. Please state project objectives and what work was done this quarter to address them: The objectives of this project are: 1, To study the effect of Brs on priming immunity on young, newly planted trees. This will allow to know for how long immune response will last after Br application, so we can adjust timing (number of applications).Spring flush that started in early February was highly syncronized, following the trend we observed during Summer and Fall flushes. We are counting less psyllids per flush after Br application. The canopies are denser than in controls and there is a significantly higher fruit set (30% more fruit) in Br treated trees. In general tree health is improved significantly. Since we are getting more synchronized flushes, we are planning to concentrate Br sprays only at the time of flushing in a set of trees and follow psyllid population, with the idea of limiting Br application only to these moments of flushing and maximum psyllid pressure. 2, To determine the best time of application (frequency) to achieve maximum protection against pests and disease in newly planted trees. Gene expression of genes related to SA pathway, and PR5 and NPR1 is maximum at 30 days, but still significant by 60 days. This confirms past year’s results and indicates that treatments may be performed every other month in young trees. We plan to do a trial with application every other month in the next season.3, To determine the effect of Br application on advancing fruit maturation in both Valencia and Hamlin. We started treatments in Valencia mature fruit-bearing trees on January.We performed two different sets of treatments: biweekly treatment and only once treatment, as in Hamlin. We have noticed this year a great variability in tree health and canopy density in these trees. In general the grove is in decline and most of the trees were categorized as 4 in the disease indexing developed by Jim Syvertsen and Brandon Page for CRDF. Interestingly we saw only a significant effect of Br application on trees that were 2 and 3 in the scale (there were neither 0 nor 1 trees). Fruit was harvested in late April and in the healthiest trees Brix was 9.6 in Br-treated in Mid March as compared to 8.8 in controls. Ratio was also greatly improved. These differences were not significant in trees categorized as 4 in the disease indexing (8.8 for controls as compared to 9.2 in Br-treated). Outreach: -Alferez, F. New tools for growing citrus under endemic HLB. Collier Co Fruit Growers Association, Naples, April 15.-Pérez-Hedo, M.; Urbaneja, A.; Alférez, F. Homobrassinolide Delays Huanglongbing Progression in Newly Planted Citrus (Citrus sinensis) Trees. Plants 2024, 13, 1229. https:// doi.org/10.3390/plants13091229 CRDF project 22-003 is aknowledged in the publication. 2. Please state what work is anticipated for next quarter: We will finish analyzing juice quality and determine gene expression as related not only to plant immunity, but also we are interested in sugar and acid metabolism in response to Br.We are planning to use trees that have been injected with OTC and show symptoms of canopy recovery but not significant improvement in fruit quality to test if Br application can boost internal quality in the next season. This idea came after seeing this year that we get most of the Br effect on increasing Brix on trees with the best canopies (in our plots that are not treated with OTCs). We will focus this quarter on identifying those trees in commercial groves and select trees with different response to OTC in terms of fruit quality to perform the treatments during next season. Finally, for objectives 1 and 2 we will perform a new set of trials: i) concentrate Br treatments in young trees only at the time of flushing and ii) apply Br every other month. 3. Please state budget status (underspend or overspend, and why): Spending continues on track.
1. Please state project objectives and what work was done this quarter to address them: Objective 1: To compare different rates and annual use patterns of trunk-injected OTC on late-season (Valencia) and early-season (Hamlin) sweet orange tree and their effects on health, yield, and fruit quality over a period of 3 years in a commercial citrus production environment.Trees were harvested and fruit quality was assed in December 2023. We measured less fruit drop and increased yields (18-32%) for injected trees compared with non-injected trees, but yield differences were only significant at p = 0.12. In general, the highest OTC rate (1.65 g/tree) and the 75% rate (0.825 g/tree) resulted in the highest yield (32% increase). The highest pound solids and percent juice were measured for the trees that received the highest rate (p < 0.001). Please note that the highest rate (1.65 g/tree) was administerd by split-injections (0.825 g each on two opposite sides of the trunk). Fruit size and juice color were also improved by the injections. A subset of fruits was collected and processed for OTC residue analysis by the National Science Lab in Gastonia.Leaves were collected for CLas analysis (in progress) and tree ratings were performed. Trees that received the highest OTC rate are the densest and show the least foliar HLB symptoms compared with the controls. Obective 2: To compare spring with late summer injections in Valencia orange trees to determine if OTC effects on yield can be enhanced, and the harvest window extended, through timing of injections.Canopy ratings were performed and fruit drop was monitored. Injected trees look denser; trees that received the highest OTC rate are the densest and healthiest looking trees. 2. Please state what work is anticipated for next quarter: The Valencia trial will be harvested, fruit will be collected for fruit quality assessment and OTC residue analysis. Tentative harvest time is March 2024. 3. Please state budget status (underspend or overspend, and why):The budget is as expected.
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