Horticultural & Management


Organic acids compared to conventional acidification for improved nutrient uptake and root physiology

Report Date: 04/10/2023   Project: 20-004   Year: 2023

Organic acids compared to conventional acidification for improved nutrient uptake and root physiology

Report Date: 04/10/2023
Project: 20-004   Year: 2023
Percentage Completion: 0.75
Category: Horticultural & Management
Author: Davie Kadyampakeni
Sponsor: Citrus Research and Development Foundation

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 under way on March 28, 2023 using HLB free trees due to challenges with CLas inculation and are evluating varying pH levels in rhizotrons. All protocols are developed and pretesting for the study is finalized. The portion CLas impacts will be conducted on selected and periodic field root samples from CLas infected trees.

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 collected soil and leaf tissue samples which show sufficiency in all treatments. We are now evaluating root density, and PCR of selected trees. We also applied all acids and elemental S in the appropriate treatments and are monitoring canopy changes and soil trends as described in the project deliverables. We also picked fruit and did fruit quality determinations in March for one site in Fort Meade.

Results on the objective were shared at the CREC Symposium omn March 24, 2023. The title of the poster was “Impacts of acidification and fertilization on soil quality and citrus tree production”.

2. Please state what work is anticipated for next quarter:
We will finalize harvesting and fruit quality determinations at the Lake Alfred site in April 2023.
The greenhouse and field work portions on objective 1 will continue in April through May 2023.
Early results of the greenhouse work will be shared at the Florida State Horticultural Society (FSHS) annual meeting.

3. Please state budget status (underspend or overspend, and why):
The budget is on track and meeting the project milestones.



Citrus Research and Field Trials (CRAFT) Program Year Four

Report Date: 04/03/2023   Project: 22-010   Year: 2023

Citrus Research and Field Trials (CRAFT) Program Year Four

Report Date: 04/03/2023
Project: 22-010   Year: 2023
Percentage Completion: 0.5
Category: Horticultural & Management
Author: Tamara Wood
Sponsor: Citrus Research and Development Foundation

1. Please state project objectives and what work was done this quarter to address them:

The online application window for CRAFT Cycle Four opened in October 2022 and closed December 31, 2022. CRAFT received 130 applications representing more than 6,300 acres of potential new Florida citrus plantings. Solid set proposals accounted for 2,561 acres and reset proposals accounted for 3,747 acres. The geographic distribution represented 13 counties and all citrus-producing regions in Florida. Projects focusing on oranges, grapefruit, hybrid and others were all proposed.

Following a staff review for eligibility, the CRAFT Technical Working Group began review and development of all proposals. Proposals were assigned to subgroups of similar projects and assigned a group leader to assist in the development of project design.

As of March 22, 2023 five projects have been withdrawn, 69 have been fully developed and 56 have been contacted and are in progress. The Technical Working Group continues to work with applicants to complete the development and review process.

2. Please state what work is anticipated for next quarter:

The Technical Working Group will finalize the development and review of all Cycle Four proposals within the next quarter and will present a final slate of recommended projects and waitlist to the Board of Directors for consideration. Following Board approval, CRAFT staff will draft participant contracts which will be finalized prior to the June 30, 2023 deadline.

Upon execution of participant contracts those projects which have completed planting will undergo a pre-audit by CRAFT contractors to ensure compliance with established protocols and agreements. Additionally, CRAFT-USDA Data Portal access will be established for each participant to allow for entry of project production data.

3. Please state budget status (underspend or overspend, and why):

As of March 31, 2023 no funds have been spent. Invoices will be submitted by CRAFT to CRDF following approval of projects and execution of participant contracts.



Integrated management of sting nematode in newly planted citrus trees

Report Date: 03/28/2023   Project: 21-013   Year: 2023

Integrated management of sting nematode in newly planted citrus trees

Report Date: 03/28/2023
Project: 21-013   Year: 2023
Percentage Completion: 0.33
Category: Horticultural & Management
Author: Larry Duncan
Sponsor: Citrus Research and Development Foundation

  The first 10 rootstocks from the USDA breeding program were evaluated for sting nematode tolerance during the first week of March, approximately 20 weeks after infesting the seedlings with sting nematodes.  Compared to unchallenged trees, the fibrous root damage was not readily evident and the stubby root symptoms were markedly less pronounced than in the previous trial. The morphology of all plants were documented photographically and the shoots, structural and fibrous roots were dried and weighed. The average fibrous root mass of the infested as a proportion of the average of the uninfested lines was marginally correlated (r=0.6; P=0.07) with the subjective visual damage rating assigned each seedling and was used to rank the genotypes for nematode tolerance. All USDA hybrid rootstocks comprised trifoliate orange crossed with either pumelo or mandarin parents. The tolerance ratio of the average fibrous root mass (infested/uninfested) for the seven hybrid rootstocks as well as that of Swingle citrumelo (also a trifoliate hybrid) was 0.99, indicating no root reduction.  That of the remaining two non-hybrid rootstocks (sweet and sour orange) averaged 0.71 indicating a 29% reduction of fibrous roots in plants exposed to the nematode.  It is unclear why the extent of stubby root symptoms caused by sting nematode differed between the two trials done to date. The most likely explanation is that the infestation in the first trial was done by transplanting infested Bermuda grass into the tanks prior to introducing the seedlings.  Due to competition between the grass and the citrus in that trial, we changed the methodology in the second trial and infested the seedlings directly following planting.  Nematode populations monitored in each trial after three months and at termination were consistently higher in the second trial; nevertheless, the initial population to which plants were exposed was highest in trial 1. This discrepancy should be overcome in future trials because all tanks are now heavily infested.  Regardless, the relationships between the tolerance ratio (infested/uninfested fibrous roots) and variables other than nematode damage which might have affected the ratio were the same in both trials. For example, the ratio might have been affected by the inherent size of the different lines. In both trials the smallest lines tended to have greater tolerance ratios (correlations were significant at P<0.05 and p<0.10, respectively).  However, when the tolerance ratio was regressed on two variables (inherent size (average weight of uninfested fibrous roots) and actual reduction in fibrous roots (infested  - noninfested fibrous roots), the inherent plant size was not significant (P=0.35 and P=0.55), whereas the net reduction of fibrous roots due to nematodes was highly significant in both trials (P<0.001). The two variables explained 78% of the variability of the tolerance ratio in the first trial and 88% in the second.  Validation of the results of both experiments to date will be attempted by comparing the most tolerant lines in both trials to widely used conventional rootstocks including sweet and sour oranges, Kuharske citrange, Cleopatra mandarin, Volkamer lemon and C-35. We have also begun trials to evaluate several of the most and least tolerant lines in trial one for sting nematode resistance (inability to reproduce) in addition to tolerance (ability to thrive despite nematode ability to reproduce).  These trials require individually potted plants grown in nematode infested and non-infested soil. We will expand these experiments to evaluate promising lines in trial two and conventional rootstock varieties for comparison.   The average number of sting nematodes in the surface soil (0-12 in depth) of the field trial remained low, just 6 nematodes per 250 cm3 soil (0-25 nematodes range). Unlike the summertime measurement, there were no treatment effects on the nematode numbers. Similarly, the fibrous root density was 3 times higher in January than the previous July, but there were no effects of treatment on the root weight or the change in root weight.    



Determine optimal timing for application of fertilizer to improve fruit quality and reduce preharvest drop

Report Date: 03/12/2023   Project: 21-024   Year: 2022

Determine optimal timing for application of fertilizer to improve fruit quality and reduce preharvest drop

Report Date: 03/12/2023
Project: 21-024   Year: 2022
Percentage Completion: 0.3
Category: Horticultural & Management
Author: Arnold Schumann
Sponsor: Citrus Research and Development Foundation

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 order to compare the effects of early versus late / sustained fertilization on health and fruit production of Hamlin and Valencia trees, the following completed treatments were evaluated in the fourth quarter of 2022:Treatments: Early fertilization as % completed of the recommended fertilization (RF; 160 lb N/acre) before June T1: 25% of RF   (T25%)T2: 50% of RF    (T50%)T3: 75% of RF    (T75%)T4: 100% of RF   (T100%)During the fourth quarter of 2022, the trees in the experiment were recovering from substantial damage caused by hurricane Ian in late September, and were impacted again by hurricane Nicole in October. The Hamlin trees lost most of their fruit due to the onset of maturity making them most vulnerable to drop. The Valencia trees retained more fruit, but losses were also significant. The Sugarbelle trees suffered considerable limb and tree breakage, as well as fruit drop. Fruit drop occurred during the storm events, and continued for weeks afterwards as the stress caused premature coloring and abscission. Soil lysimeter leachate sampling, leaf sampling, processing and analysis continued, and tree size and health was assessed after the third Aerobotics drone survey was completed in November. Since many of the Hamlin plots had no fruit remaining at all, we could only sample the few remaining fruit for analysis and observational comparison of fruit quality. In December we harvested Sugarbelle fruit from experiment plots to assess the efficacy of foliar nutrient sprays (P and K) and gibberellic acid (GA) on fruit peel firmness, color, and internal fruit quality. The peel strength measurements conducted with a digital penetrometer were NS and inconclusive, mainly due to the unsuitability of measuring only force (N) to puncture the peel. We plan to measure future fruit peel strength by quantifying the force required to distort the peel by an exact fixed distance. That will allow the calculation of work done, or energy expended (J), which is a more reasonable measure of peel strength / firmness. GA applied once at 10 ppm in August significantly reduced TSS relative to the two best treatments, dipotassium phosphate (DKP) sprayed in May, July and September, and potassium nitrate sprayed in July. The GA treatment had the lowest TSS overall. Titrateable acidity, TSS/acid ratio, and fruit weight were NS across treatments. 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):The growth and yield data associated with all different timed fertilizer events in year 1 is not complete yet and was severely impacted by hurricanes, and therefore we don’t have new results to report for this objective. B) New developments: NoneC) Issues: The severe damage caused by hurricanes in 2022 will likely cause longer-term setbacks to the trees in these experiments.2. Please state what work is anticipated for next quarter: The first 2023 season fertilizer applications will be made to designated treatment plots in February. Soil, lysimeter sampling, leaf sampling, processing and analysis will be ongoing, as will tree size and fruit measurement. We plan to assess fruit yield and quality for the Valencia experiment if enough fruit remains. In Feb/Mar the roots of the Hamlin and Valencia trials will be sampled at two soil depths by collecting and sieving soil samples taken around the driplines of trees. We plan to apply OTC by trunk injection to all the trees in Hamlin and Valencia trials, since this remedy is now labeled and available, and will likely be the new norm for growing citrus in HLB-endemic Florida. The OTC will be applied to all trees, similar to the way herbicide, pesticide, or irrigation is applied to all trees irrespective of treatment.  3. Please state budget status (underspend or overspend, and why): Spending rate is approximately on track.    



Citrus Research and Field Trials (CRAFT) Program Cycle III

Report Date: 02/23/2023   Project: 21-004C   Year: 2022

Citrus Research and Field Trials (CRAFT) Program Cycle III

Report Date: 02/23/2023
Project: 21-004C   Year: 2022
Percentage Completion: 1
Category: Horticultural & Management
Author: Tamara Wood
Sponsor: Citrus Research and Development Foundation

The contract executed between CRAFT and CRDF was effective September, 2021, however work began on Cycle III projects beginning in July 2021 and deliverables executed during that period will be reported herein. CRAFT PARTICIPATION APPLICATION PROCESS: The CRAFT Technical Working Group, USDA/ARS and CRAFT staff were all involved with preparing the CRAFT application content for the Cycle III online application experience. The application period was open to Florida citrus growers from July-October, 2021. CRAFT received 84 applications representing  more than 3,300 acres, including 2,470 acres for solid set plantings and 878 acres for reset plantings. The geographic distribution represented 10 counties throughout Florida’s citrus producing regions in.Based upon the contractual requirement stating 50 percent of the funding be allocated to small growers (less than 2,500 acres) and 50 percent be allocated to large growers, applications have been sorted into their respective categories. There were a total of 72 applications within the small grower category accounting for 2,399 acres and 12 applications within the large grower category accounting for 949 acres. PROJECT SELECTION & DEVELOPMENT:The Technical Working Group reviewed all projects submitted, worked with growers to establish proposed experimental designs, and evaluatied the relative value of each project. Based upon the projects presented, applications were organized within subgroups of similar projects (for purposes of replication) and assigned a group leader to help develop  the project design. The groups are as follows:1.         Rootstock/Scion2.         Soil/Tree Fertility3.         Pest Management4.         Biostimulants5.         ResetsAs of June 30, 2022 a total of 48 projects had been approved by the Board of Directors and contracted for inclusion in Cycle III. Small growers represented 25 of the projects totaling 773 acres and utilizing $2,500,750 of funding, while large growers represented 23 projects totaling 1,045 acres and utilizing $2,499,300 of funding. Remaining applications were placed on a waitlist in case of future funding availability due to withdrawal of contracted projects. Cycle III projects are now pending planting. Following completion of planting, pre-audits of each project are used to confirm the planting of trees and compliance with contract to date. Growers are expected to report production data in real time or at least quarterly in to the USDA-CRAFT Data Portal. 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.   In addition to execution of contracts, CRAFT staff continued communications and outreach efforts including, 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.  



Right Leaf Sampling-The first and most critical step to good nutrition program

Report Date: 02/15/2023   Project: 20-011   Year: 2022

Right Leaf Sampling-The first and most critical step to good nutrition program

Report Date: 02/15/2023
Project: 20-011   Year: 2022
Percentage Completion: 0.75
Category: Horticultural & Management
Author: Tripti Vashisth
Sponsor: Citrus Research and Development Foundation

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. In this quarter we continued collecting tagged leaf flush and analyse them for nutrient content.  In this quarter we also harvested the hamlin site in Fort Meade however, we were unable to harvest Arcadia Hamlin site as the grove was seriously damaged by hurricane Ian. The preliminary analysis shows that the mild trees are responding to the fertilizer treatments that based on summer flush nutrient analysis. Mild trees getting additional fertilizer are not lossing as much canopy density as the control trees. It is interesting that the response of fertilizer is not same in mild and severe trees. This observation indicates towards the variabilty that we see in groves with nutritional treatments.  Another interesting observation is that we are seeing significant differences between nutritional profile of fruiting and non-fruiting branches therefore, raising the question about value in using fruting branch leaf in plance of non fruiting branch when adjusting the fertilizer. We continue to collect data to answer this additional question. 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 results4. Harvesting Valencia at Fort Meade and Arcadia   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. 



Reducing fruit drop by altering hormonal responses within the tree through nutritional and hormonal therapies: a mechanistic affordable approach

Report Date: 02/14/2023   Project: 21-007   Year: 2023

Reducing fruit drop by altering hormonal responses within the tree through nutritional and hormonal therapies: a mechanistic affordable approach

Report Date: 02/14/2023
Project: 21-007   Year: 2023
Percentage Completion: 0.25
Category: Horticultural & Management
Author: Fernando Alferez
Sponsor: Citrus Research and Development Foundation

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.2) To determine effects of GA3 and 2,4D applications on fruit retention when applied at different times during fruit development.3) To develop a strong and proactive outreach program.  For objective 1 we harvested Hamlin on the first week of January, 2023. As a reminder, we performed treatments at three different dates: on early June 2022, a second set of treatments by the end of July, 2022, and a third set by early September 2022. Every set of treatments was performed in an independent block, so treatments are applied only once, to assess the best timing to achieve the best results. Results for this first season, seem to confirm our hypothesis that only one treatement of Zn and Zn+K, done at the right time, may increase even more the yield. these are the data for the first year:In Kg per 3 trees, control was 67,8, Zn+K applied in June was 70.3 (4% increase, not significant at p<0.05), Zn+K applied by the end of July was 83.9 (25% increase, significantly different), and Zn+K applied in early September was 105,4 Kg (57% increase, statistically significant at p<0.05).These are encouraging results, especially considering that  Ian hit in late September and we had massive fruit drop (around 75%) in the SW FL area. We expect that these data will improve next season. So far we see that only one treatment performed in September can be enough to have a susbtantial increae in yield, although we should confirm this next year, hopefully without the background noise that a hurricane imposes. Treatments only with Zn or K also yield statistically significant better returns, but not as remarkable as the combined Zn+K treatment.  For objective 2 we also found better yields with combined application of GA and 2,4 D. The repeated application of GA and 2,4 D yielded 180 lb/tree as compared to 142 lb/tree in control (p value =0.1). Due to shortge in supply of GA and 2,4 D, the first application of GA was made in July and in August for 2,4D. Altogether tree received 3 GA and 2 2,4 D application in entire season. No effect of GA alone or 2,4 D were seen.  Objective 3:- F. Alferez, ASHS invited webinar on fruit drop mecanisms. January 12, 2023. - F Alferez, Combining IPCs and brasinosteroids in citrus & increasing yield by using Zin. An update. Citrus Production School, February 9, 2023 in Arcadia.-Vashisth,.T. Use of PGRs to improve citrus proiduction, January 12, 2023 in Polk OJ Break 2. Please state what work is anticipated for next quarter: In next quarter,  we will continue treatments now in Valencia for objective 1 and 2  we will start assesing fruit drop in this variety. We will also collect samples for analysis and assess flushing, blooming, and fruit set in both varieties.    3. Please state budget status (underspend or overspend, and why):Spending is now on track.    



Determining best timing for Brassinosteroid (Brs) application to achieve maximum beneficial effects on citrus tree health and fruit yield and quality

Report Date: 02/14/2023   Project: 22-003   Year: 2023

Determining best timing for Brassinosteroid (Brs) application to achieve maximum beneficial effects on citrus tree health and fruit yield and quality

Report Date: 02/14/2023
Project: 22-003   Year: 2023
Percentage Completion: 0.1
Category: Horticultural & Management
Author: Fernando Alferez
Sponsor: Citrus Research and Development Foundation

1. Please state project objectives and what work was done this quarter to address them: Specific objectives are: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 have confirmed in field conditions that Br application activates SAR gene expression for at least 6 weeks in young citrus plants. We have also found that in  greenhouse-controlled conditions, trees maintained in cages and exposed to hot psyllids that were added, % of trees infected in non-treated controls is 60% after 4 months. Strikingly, no plants treated with Br are infected in the same conditions of heavy psyllid pressure, and eggs and nimphs are not present or significantly in lower numbers. There exists the possibility of an altered volatile profile in leaves after Br treatment taht may repel the insects, and we will explore this possibility in next quarters.2, To determine the best time of application (frequency) to achieve maximum protection against pests and disease in newly planted trees. We are ready to assay this both in the field and in controlled conditions in cages. For this we have pruned young plants in cages to allow new flushes before treatments and introduction of hot psyllids. We are waiting for the spring flush in the field to perform treatments in field conditions and to assess psyllid population dynamics after Br treatment.3, To determine the effect of Br application on advancing fruit maturation in both Valencia and Hamlin.We finished Br applications at Duda farm by the end of November in Hamlin. These applications started at the end of August 2022. In non-treated control trees we found a decrease in Brix between Oct 31 and Nov 15 (from 8.45 to 7.9) that was coincident with fruit set from off blooms in early October. Brix levels recovered later. Interestingly, that decrease was not found in Br treated trees, were the Brix remained stable at around 8.4-8.5. The most striking finding, though, was that acidity dropped faster after Br treatment as compared to controls and this led to increased ratios earlier in the season. This ratios were higher in Br treatment thereafter and at the end of the season, at harvest. In controls sugar/acid ratio was stable around 9 from September to December 1st. It only increased to 11 by the time of harvest in January 5. With Br treatment, values of 12 were achieved in early December, 1 month in advance, and by January 5th ratio was 14. We have collected juice samples throughout the maturation process and we will be analyzing them for organic acid composition and sugar profiles, to tdetect any possible differences. We are partnering with Prof Yu Wang at CREC for this. Outreach:- F Alferez, Combining IPCs and brasinosteroids in citrus & increasing yield by using Zinc. An update. Citrus Production School, February 9, 2023 in Arcadia.  2. Please state what work is anticipated for next quarter:For objectives 1 and 2, treatments and samplings will continue.For objective 3, we will continue treatments on Valencia, that started in early January. We will assess maturity periodically.     3. Please state budget status (underspend or overspend, and why):Spending is now on track.    



Improving the Systemic Uptake of Therapeutic Compounds by Trunk Injections

Report Date: 02/12/2023   Project: 22-017   Year: 2023

Improving the Systemic Uptake of Therapeutic Compounds by Trunk Injections

Report Date: 02/12/2023
Project: 22-017   Year: 2023
Percentage Completion: .17
Category: Horticultural & Management
Author: Amit Levy
Sponsor: Citrus Research and Development Foundation

1. Please state project objectives and what work was done this quarter to address them:Objective 1: Using callose inhibitors to improve systemic uptake and reduce HLB symptoms.A study was implemented incorporating 12 replicates of all proposed treatments.  We continued our field experiment on sweet orange ~8 years old (Citrus x sinensis) trees: (1) injected control (water), (2) Oxytetracycline (2 g per tree), (3) DDG (0.1 mM), (4) DDG (0.1mM) + Oxytetracycline (2 g per tree), (5) DDG (1mM), (6) 3AB (0.1mM), (7) 3AB (0.1mM) + Oxytetracycline (2 g per tree), (8) 3AB (1mM). Then we designed the appropriate map for the experiment. We measured baseline stomatal conductance, trunk diameter, canopy volume and canopy density. Injection treatments were accompanied with measurements of canopy dimensions and density, and followed by measurements of stomatal conductance.  No treatment reduced stomatal conductance, and we conclude that the treatments do not negatively impact tree physiology in the short term. Canopy growth will be measured at 6 months post-treatment. Furthermore, six mature leaf samples per tree were collected to measure the CLas titer and quantify calloselevel of each tree. Leaf samples were collected to measure the CLas titre and quantify the callose level of trees 1 and 2 weeks after treatments. Lastly, we measured the Oxytetractcline on the trees. Suprisingly we saw that there was no difference between the different samples, and they were all the same. We went back and evaluated all our procedures. Unfortunatly, we found that we made a mistake in the Oxytetracycline measurements. and injected only a small amount, a small percentange of what we had planned. We decided to repeat the injections.Objective 2: Maintaining water-saturated injection site: Sealed trunk injection ports that stay functional by avoiding woundresponses.Experments planned.Objective 3: Targeted root delivery.No progress. 2. Please state what work is anticipated for next quarter:We will reinject trees and evaluate the trees for callose level and Clas titre monthly. We will also measure the canopy volume and density. We will begin experiments for objectives 2 and 3. 3. Please state budget status (underspend or overspend, and why):Underspend- since it took some time to initiate the work 4. Please show all potential commercialization products resulting from this research, and the status of each:We are still evaluating the effect of the callose inhibitor in the field trials  



Improving the Systemic Uptake of Therapeutic Compounds by Trunk Injections

Report Date: 02/12/2023   Project: 22-017   Year: 2023

Improving the Systemic Uptake of Therapeutic Compounds by Trunk Injections

Report Date: 02/12/2023
Project: 22-017   Year: 2023
Percentage Completion: .17
Category: Horticultural & Management
Author: Amit Levy
Sponsor: Citrus Research and Development Foundation

1. Please state project objectives and what work was done this quarter to address them:Objective 1: Using callose inhibitors to improve systemic uptake and reduce HLB symptoms.A study was implemented incorporating 12 replicates of all proposed treatments.  We continued our field experiment on sweet orange ~8 years old (Citrus x sinensis) trees: (1) injected control (water), (2) Oxytetracycline (2 g per tree), (3) DDG (0.1 mM), (4) DDG (0.1mM) + Oxytetracycline (2 g per tree), (5) DDG (1mM), (6) 3AB (0.1mM), (7) 3AB (0.1mM) + Oxytetracycline (2 g per tree), (8) 3AB (1mM). Then we designed the appropriate map for the experiment. We measured baseline stomatal conductance, trunk diameter, canopy volume and canopy density. Injection treatments were accompanied with measurements of canopy dimensions and density, and followed by measurements of stomatal conductance.  No treatment reduced stomatal conductance, and we conclude that the treatments do not negatively impact tree physiology in the short term. Canopy growth will be measured at 6 months post-treatment. Furthermore, six mature leaf samples per tree were collected to measure the CLas titer and quantify calloselevel of each tree. Leaf samples were collected to measure the CLas titre and quantify the callose level of trees 1 and 2 weeks after treatments. Lastly, we measured the Oxytetractcline on the trees. Suprisingly we saw that there was no difference between the different samples, and they were all the same. We went back and evaluated all our procedures. Unfortunatly, we found that we made a mistake in the Oxytetracycline measurements. and injected only a small amount, a small percentange of what we had planned. We decided to repeat the injections.Objective 2: Maintaining water-saturated injection site: Sealed trunk injection ports that stay functional by avoiding woundresponses.Experments planned.Objective 3: Targeted root delivery.No progress. 2. Please state what work is anticipated for next quarter:We will reinject trees and evaluate the trees for callose level and Clas titre monthly. We will also measure the canopy volume and density. We will begin experiments for objectives 2 and 3. 3. Please state budget status (underspend or overspend, and why):Underspend- since it took some time to initiate the work 4. Please show all potential commercialization products resulting from this research, and the status of each:We are still evaluating the effect of the callose inhibitor in the field trials  



Improving the Systemic Uptake of Therapeutic Compounds by Trunk Injections

Report Date: 02/12/2023   Project: 22-017   Year: 2023

Improving the Systemic Uptake of Therapeutic Compounds by Trunk Injections

Report Date: 02/12/2023
Project: 22-017   Year: 2023
Percentage Completion: 0.17
Category: Horticultural & Management
Author: Amit Levy
Sponsor: Citrus Research and Development Foundation

1. Please state project objectives and what work was done this quarter to address them:Objective 1: Using callose inhibitors to improve systemic uptake and reduce HLB symptoms.A study was implemented incorporating 12 replicates of all proposed treatments.  We continued our field experiment on sweet orange ~8 years old (Citrus x sinensis) trees: (1) injected control (water), (2) Oxytetracycline (2 g per tree), (3) DDG (0.1 mM), (4) DDG (0.1mM) + Oxytetracycline (2 g per tree), (5) DDG (1mM), (6) 3AB (0.1mM), (7) 3AB (0.1mM) + Oxytetracycline (2 g per tree), (8) 3AB (1mM). Then we designed the appropriate map for the experiment. We measured baseline stomatal conductance, trunk diameter, canopy volume and canopy density. Injection treatments were accompanied with measurements of canopy dimensions and density, and followed by measurements of stomatal conductance.  No treatment reduced stomatal conductance, and we conclude that the treatments do not negatively impact tree physiology in the short term. Canopy growth will be measured at 6 months post-treatment. Furthermore, six mature leaf samples per tree were collected to measure the CLas titer and quantify calloselevel of each tree. Leaf samples were collected to measure the CLas titre and quantify the callose level of trees 1 and 2 weeks after treatments. Lastly, we measured the Oxytetractcline on the trees. Suprisingly we saw that there was no difference between the different samples, and they were all the same. We went back and evaluated all our procedures. Unfortunatly, we found that we made a mistake in the Oxytetracycline measurements. and injected only a small amount, a small percentange of what we had planned. We decided to repeat the injections.Objective 2: Maintaining water-saturated injection site: Sealed trunk injection ports that stay functional by avoiding woundresponses.Experments planned.Objective 3: Targeted root delivery.No progress. 2. Please state what work is anticipated for next quarter:We will reinject trees and evaluate the trees for callose level and Clas titre monthly. We will also measure the canopy volume and density. We will begin experiments for objectives 2 and 3. 3. Please state budget status (underspend or overspend, and why):Underspend- since it took some time to initiate the work 4. Please show all potential commercialization products resulting from this research, and the status of each:We are still evaluating the effect of the callose inhibitor in the field trials  



OTC Directed Research Solicitation: Use of CRDF Rootstock Trial Locations for Testing Bactericides Inserted into Trees Through Systemic Delivery Devices

Report Date: 01/15/2023   Project: 23-005   Year: 2023

OTC Directed Research Solicitation: Use of CRDF Rootstock Trial Locations for Testing Bactericides Inserted into Trees Through Systemic Delivery Devices

Report Date: 01/15/2023
Project: 23-005   Year: 2023
Percentage Completion: 0.25
Category: Horticultural & Management
Author: Ute Albrecht
Sponsor: Citrus Research and Development Foundation

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.  Leaves were collected for CLas analysis. Trunk injury/wound evaluations were conducted. PCR analyses were continued. Statistical data analyses continued. So far, data analysis showed the following: The uptake rate of the injected OTC solution (Rectify) did not differ whether injections were done into the rootstock or the scion trunk. However, there was a large variability during the day and among the trial sites – the uptake rate was anywhere from <30 minutes to >3 hours.Injections in the rootstock seemed to have caused slightly less visible injury than injections in the scion. Some rootstock differences were also noted.In one of the trials (Venus location), we measured lower CLas titer levels in injected trees.  2. Please state what work is anticipated for next quarter: Leaves will be collected for CLas and OTC analysis. Disease ratings will be conducted. We will prepare for the harvest. Harvest dates will depend on the grower collaborators. 3. Please state budget status (underspend or overspend, and why):The budget is as expected. 



OTC Directed Research Solicitation: Use of CRDF Rootstock Trial Locations for Testing Bactericides Inserted into Trees Through Systemic Delivery Devices

Report Date: 01/15/2023   Project: 23-005   Year: 2023

OTC Directed Research Solicitation: Use of CRDF Rootstock Trial Locations for Testing Bactericides Inserted into Trees Through Systemic Delivery Devices

Report Date: 01/15/2023
Project: 23-005   Year: 2023
Category: Horticultural & Management
Author: Ute Albrecht
Sponsor: Citrus Research and Development Foundation

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.  Leaves were collected for CLas analysis. Trunk injury/wound evaluations were conducted. PCR analyses were continued. Statistical data analyses continued. So far, data analysis showed the following: The uptake rate of the injected OTC solution (Rectify) did not differ whether injections were done into the rootstock or the scion trunk. However, there was a large variability during the day and among the trial sites – the uptake rate was anywhere from <30 minutes to >3 hours.Injections in the rootstock seemed to have caused slightly less visible injury than injections in the scion. Some rootstock differences were also noted.In one of the trials (Venus location), we measured lower CLas titer levels in injected trees.  2. Please state what work is anticipated for next quarter: Leaves will be collected for CLas and OTC analysis. Disease ratings will be conducted. We will prepare for the harvest. Harvest dates will depend on the grower collaborators. 3. Please state budget status (underspend or overspend, and why):The budget is as expected. 



OTC Directed Research Solicitation: Use of CRDF Rootstock Trial Locations for Testing Bactericides Inserted into Trees Through Systemic Delivery Devices

Report Date: 01/15/2023   Project: 23-005   Year: 2023

OTC Directed Research Solicitation: Use of CRDF Rootstock Trial Locations for Testing Bactericides Inserted into Trees Through Systemic Delivery Devices

Report Date: 01/15/2023
Project: 23-005   Year: 2023
Percentage Completion: 0.25
Category: Horticultural & Management
Author: Ute Albrecht
Sponsor: Citrus Research and Development Foundation

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.  Leaves were collected for CLas analysis. Trunk injury/wound evaluations were conducted. PCR analyses were continued. Statistical data analyses continued. So far, data analysis showed the following: The uptake rate of the injected OTC solution (Rectify) did not differ whether injections were done into the rootstock or the scion trunk. However, there was a large variability during the day and among the trial sites – the uptake rate was anywhere from <30 minutes to >3 hours.Injections in the rootstock seemed to have caused slightly less visible injury than injections in the scion. Some rootstock differences were also noted.In one of the trials (Venus location), we measured lower CLas titer levels in injected trees.  2. Please state what work is anticipated for next quarter: Leaves will be collected for CLas and OTC analysis. Disease ratings will be conducted. We will prepare for the harvest. Harvest dates will depend on the grower collaborators. 3. Please state budget status (underspend or overspend, and why):The budget is as expected. 



OTC Directed Research Solicitation: Use of CRDF Rootstock Trial Locations for Testing Bactericides Inserted into Trees Through Systemic Delivery Devices

Report Date: 01/15/2023   Project: 23-005   Year: 2023

OTC Directed Research Solicitation: Use of CRDF Rootstock Trial Locations for Testing Bactericides Inserted into Trees Through Systemic Delivery Devices

Report Date: 01/15/2023
Project: 23-005   Year: 2023
Percentage Completion: 0.25
Category: Horticultural & Management
Author: Ute Albrecht
Sponsor: Citrus Research and Development Foundation

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.  Leaves were collected for CLas analysis. Trunk injury/wound evaluations were conducted. PCR analyses were continued. Statistical data analyses continued. So far, data analysis showed the following: The uptake rate of the injected OTC solution (Rectify) did not differ whether injections were done into the rootstock or the scion trunk. However, there was a large variability during the day and among the trial sites – the uptake rate was anywhere from <30 minutes to >3 hours.Injections in the rootstock seemed to have caused slightly less visible injury than injections in the scion. Some rootstock differences were also noted.In one of the trials (Venus location), we measured lower CLas titer levels in injected trees.  2. Please state what work is anticipated for next quarter: Leaves will be collected for CLas and OTC analysis. Disease ratings will be conducted. We will prepare for the harvest. Harvest dates will depend on the grower collaborators. 3. Please state budget status (underspend or overspend, and why):The budget is as expected.