The third mass screen of germplasm for sting nematode tolerance in large sand-filled tanks was completed in January 2024. Sixteen rootstocks were compared by planting four replicates of each in each of 4 tanks (256 seedlings total) – two tanks infested with sting nematode and 2 that were not infested. The trial began in May and ran for 7 months, two months longer than the previous trials, to enhance the nematode pressure. The trial is noteworthy for comparing rootstocks created at USDA and UF to each other and to historical industry standards. Most of the rootstocks have been available for propagation for a number of years; however, only the historical industry standards were available for comparison the last time sting nematode tolerance was evaluated 4 decades ago. An initial trial in this project evaluated UF rootstocks, whereas USDA rootstocks were screened in the second trial. Here we look for differences among the rootstocks and for consistency with the previous trials. Nematode populations were evaluated, prior to plant recovery from tanks, finding more than 200 sting nematodes of all stages per 250 cm3 soil in each infested tank, and no nematodes were detected in non-infested tanks. Plants were carefully dug from tanks and photographed after soil was rinsed from roots. They were then separated (top, tap roots, fibrous roots), dried for 72 h at 70oC, and weighed. Roots appeared abnormally thickened when damaged heavily by the nematode which may mask differences if measured exclusively by mass (Fig 1). A rating system (1-10) was used to assign a score to each plant within a rootstock, relative to the best plant(s) (given 10) in the non-treated group of that rootstock. Each rootstock was assigned a tolerance index T, where T= mean rank of 8 infested plants/mean rank of 8 non-infested plants. Three investigators rated the plants independently, the T rankings were highly significantly correlated, and were averaged. Fig. 2 shows these subjective ratings (Tsubj)plotted against tolerance estimated as the mean fibrous root weight from infested tanks/mean fibrous root weight from non-infested tanks (Twt). Tolerance of the rootstocks was surprisingly similar within 1) those used historically, or 2) produced by USDA or 3) by UF. The four historical standard rootstocks had low T values measured visually or by mass suggesting that most newer releases confer some tolerance to the nematode. While USDA rootstocks tended to have higher Tsubj values than historical standards, this was not the case for Twt. UF rootstocks tended to have the most tolerant lines according to both measures. The results of the trial were consistent with previous results and field observations. Kuharske which is widely used for burrowing nematode control was heavily damaged by sting nematode in a 3-year trial to evaluate cover crops and nematicides. Swingle citrumelo was rated as susceptible to sting nematode in previous surveys. In this and our previous tank trial using USDA rootstocks, US812 and US942 and US1516 outperformed sweet orange or sour orange. Similarly, in this and the previous trial using UF rootstocks, UF1, UF5 and Orange 16 had the highest Tsubj and Twt scores. There were discrepencies, however, such as LB8-9xS13-15-16 which performed poorly in the first compared to this trial. The very small plant size of LB8-9xS13-15-16 compared to the better performing rootstocks may make relative differences between infested and non-infested plants more difficult to measure accurately. The rootstock screening trials have provided consistent evidence that newer rootstocks are somewhat more tolerant under these conditions than the previous generation of commonly used rootstocks. The widespread occurrence of sting nematode in Florida may be a factor driving grower adoption of newer rootstocks. This is somewhat encouraging; however, there is no evidence in these trials that an acceptibly tolerant citrus cultivar has been identified. We shall continue screening new cultivars with Kuharske and UF5 as standards for intolerance and tolerance, respectively.
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:Trunk diameter, fruit growth, fruit drop measurement was done in Hamlin and Valencia during October to December. Additionally, routine leaf sampling was accomplished during October and December for leaf nutrient analysis. In the 2nd week of November, treatment-wise fruit was harvested from the Hamlin variety for yield data, fruit drop % calculation, and fruit quality measurement. Temporal treatment responses on different variables and results recorded during October to December were presented and interpreted below.1. Trunk Growth: There were no significant treatment differences on relative growth rate of trunk diameter (mm/cm/month) in both Hamlin and Valencia varieties during October to December 2023. However, overall trunk growth rate in approximately in two years of the experiment was significantly higher in Valencia variety in treatment T8 and T9 (fertilizer applied 125% RDF (recommended dose of fertilizers) with 100% in spring and 25% fall season).2. Fruit relative growth rate (RGR): Fruit diameter of tagged fruit was measured every two weeks to calculate the relative growth rate of fruit after physiological fruit drop in May in both varieties. In both years, fruit RGR was not significant after August in Hamlin and Valencia. Overall fruit RGR (mm/cm/month) was also statistically non-significant between treatments in both varieties in both years.3. Fruit drop: Fruit drop was extremely high, and started after physiological fruit drop in May. We counted dropped fruit every two-week intervals in both varieties. Treatment 6 (50% fertilization in August), Treatment 8 (125% fertilization in spring and 25% in August) and Treatment 2 ( linear fertilization with 25% in August) resulted in a higher numbers of cumulative number of fruit drop per tree in Hamlin in 2023. However, only Treatment 2 showed higher fruit drop in Valencia. Fruit drop percentage was calculated based on number of fruit harvested and number of fruit drops before fruit harvesting. In 2023, only 3 replications were used for calculation in Hamlin because the 4th replication was heavily affected by citrus canker in addition to HLB disease. The fruit drop rate was higher than 70% in all fertilization treatments which were not statistically significant between treatments in Hamlin variety.4. Fruit quality at harvest:a. Fruit size (diameter and weight): After harvesting, 10 fruits were selected to measure the fruit diameter and fruit wt. There was no treatment effects on fruit diameter and single fruit weight in Hamlin variety in 2023. b. Juice quality: The fertilization treatment did not affect the juice quality of Hamlin fruit. Total soluble solids (TSS), titratable acidity (TA), and TSS:TA ratio were NS. c. Fruit yield over time: Yield was very low in Hamlin due to perhaps severe HLB infestation and excessive fruit drop. The projected potential yield of these trees was only 153 boxes/acre if no fruit drops had occurred. However cumulative excessive fruit drop resulted in very low fruit retention and less fruit yield. Treatment 10 resulted in a higher yield (~58 boxes/acre) than other treatments which was still not likely profitable. 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):Due to the absence of significant differences to date, we have not made progress with this objective. The hurricanes of 2022 caused damage that takes years to repair, and the constant high pressure of citrus canker on the Hamlin trees in particular has been an additional source of noise that masks any treatment response.B) New developments: NoneC) Issues: None, except those mentioned above that are beyond our control.2. 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 January to March:1. Sampling for leaf nutrient analysis2. Fruit diameter measurement (Valencia)3. Collection of leachates for nitrate N loss and analysis4. Scanning of the Minirhizotron for root growth5. Fruit drop count in every two weeks until harvesting in Valencia6. Harvesting Valencia variety for yield and fruit quality measurement 3. Please state budget status (underspend or overspend, and why):Spending rate is slightly below the target by about 10%. This is considered within the normal deviations of budgeting.
1. Please state project objectives and what work was done this quarter to address them:In the first quarter 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. Participation Agreements were sent to growers in December and are to be returned by January 31, 2024 unless otherwise approved by CRAFT. Participants from both Rounds One and Two have begun submitting their three-year historical data to CRAFT. This data will be added to the CRAFT Data Portal moving forward. To-date, 97 percent of Round One projects and 36 percent of Round Two projects have submitted their historical data. 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 will be 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. As of January 2024 CRAFT’s Scientific Coordinator has reached out to all growers and has finalized design proposals for the majority of projects. All projects will be presented to the CRAFT Technical Working Group at their February meeting for review and discussion with the expectation of finalizing a slate of recommendations for the Board of Directors during the March TWG meeting. The final slate of projects approved by the CRAFT Board of Directors for Cycle Five will join the 226 projects representing more than 9,000 acres in Cycles One – Four. In addition to the development of projects and 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. 2. Please state what work is anticipated for next quarter:The development and approval of Cycle V projects will continue, as well as the execution of ETT Round Two contracts and submittal of historical data. ETT Round One participants will also begin entering data for the harvest year 2023-24. Additionally, Cycle Four projects continue to be planted. 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. 3. Please state budget status (underspend or overspend, and why):As of January 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 Fruit drop, disease ratings, and tree measurements were conducted. We are continuing with PCR/CLas analyses.Selected information from these trials was presented at the MISA conference in november at UCF. The grower collaborator for trial 1 has unfortunately stopped tree care after our year 2 injections, and trees have been declining rapidly. We have therefore terminated our evaluations in this trial. 2. Please state what work is anticipated for next quarter: Fruit drop assements will continue. Tree size measurements will be conducted. Leaf samples will be collected for PCR/CLas analysis. We are preparing for the harvest. Harvest dates will be coordinated with the grower collaborators. 3. Please state budget status (underspend or overspend, and why):Although our activities are on track, a large portion of funds intended to be used for OTC analysis at the USDA National Science Lab in Gastonia, NC, and for fruit quality analysis in 2024 remains unspent because the start/end date of the grant coincides with the harves time. We have asked for a no cost extension to complete these research activities.
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.
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: 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 did a new set of treatments by the end of September in Hamlin as we did last year. This date of treatment was the one that got best results last season in terms of yield, despite the effects of hurricane Ian. As before, every set of treatments is performed in an independent replicated block, so treatments are applied only once, to assess the best timing to achieve the best results. Trees under Zn and Zn+K treatments continue having greener canopies. This is consistent with effects we saw last year. Also, we are performing canopy analysis by using the ImageJ program. In Hamlin, trees treated with Zn continue to have significantly more dense canopies and more canopy volume than controls. These measurements have been done also for objective 2, and we are analyzing data. For objectives 1 and 2 we are also analizyng foliar nutrient concentrations.We see less oxidative stress levels in trees treated with Zn and Zn+K.Fruit drop is being now monitoired weekly. We still don’t see differences in treatments but this is usually seen in October-November. Objective 3:-Optimizing foliar sprays of zinc and potassium to prevent fruit drop, by Fernando Alferez, Daniel Boakye, Murillo De Sousa and Pablo Orozco. Citrus Industry, Tip of the Week, October 2023. 2. Please state what work is anticipated for next quarter: Treatments for objectives 1 and 2 will continue as programmed in Hamlin. We will continue fruit drop assesments weekly. Sampling will continue as well and we will finisnh data analysis of samples analyzed in the last quarter for objectives 1 and 2. Harvesting of Hamlin will be done in December-January. Treatments in Valencia will start. 3. Please state budget status (underspend or overspend, and why): Budget is 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 have continued treatments and samplings. After 8 months (with a monthly application), most of the trees (around 60%) are still HLB-negative. Denser foliage has consolidated in a denser canopy. Fall flush was highly syncronized, following the trend we observed during Summer flush. 90% of the trees treated with Brs achjieved fully expanded leaf status at the same time. This happened only in 20% of non-treated trees. This indicates that flushing occurred at the same time in Br-treated trees, and will allow better planification of insecticide sprays. 2, To determine the best time of application (frequency) to achieve maximum protection against pests and disease in newly planted trees. Immunity is still greatly increased after 45d of treatment. We have now started plots applying Brs every 45d or 60d. We will monitor the same parameters, icluding Ct values and tree health. 3, To determine the effect of Br application on advancing fruit maturation in both Valencia and Hamlin. Treatments started in September on Hamlin. We performed two different sets of treatments: biweekly treatment and only once treatment. We are following quality every 15 days, including internal quality and external color development. So far, it is remarkable that internal maturation and quality are in general superior than last year. At the beginning of the treatments, control fruit was already at 9.38 Brix. This value was about 8.2 last season. So far we do not see a significant increase in Brix with Br treatment. This is coincident with what we saw last year, as differences started to be significant in November.Values for early November are 9 for control and 9.43 for Br-treated. However, ratio is greatly increased, 13.32 in Br-treated as compared to 11.5 in controls. External color is also increased. 2. Please state what work is anticipated for next quarter:For objectives 1 and 2 we are continuing sampling and molecular analysis.In objective 3, treatments in mature Hamlin trees are already running. We will be harvesting fruit in December-January, depending on the commercial farm schedule. Last season, harvesting was performed by January 5th. We will determine yield and fruit quality. We will start treatnments in Valencia in early January.For objectives 1 and 2 we will continue treatments, samplings and molecular analysis to determine the best timing for treatments to achieve the most protection against psyllid infestation. For objective 3, we will start in August treatments on Hamlin. We will also continue analyzing juice samples from Hamlin and start analysis on juice samples from Valencia. We have included also an injection treatment of Brs in trees, This was not in the proposal, but we believe it may have interest. We have started in October. We will be injecting once per month until harvest and measuring the same parameters as in objectives 1, 2 and 3. 3. Please state budget status (underspend or overspend, and why): Spending is 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.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. Trees were monitored. No phytotocicity was obserevd after the spring injections. Fall injections were conducted in September in the Valencia trial. No phytotoxicity was noted. 2. Please state what work is anticipated for next quarter: We will conduct tree measurements. Leaves will be collected for CLas assessment. In the Hamlin trial fruit will be collected for fruit quality assessment, and trees will be harvested. Tentative harvest time is middle of December. 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: 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:Routine measurements of tree health and performance continued, including leaf nutrient analyses, chlorophyll index, canopy and trunk size, fruit growth rates and incidental fruit drop (not pre-harvest). The study comprehensively analyzed the nutrient dynamics of leaves, revealing notable variations in nitrogen (N) concentrations. During the cell-division stage / physiological fruit drop in May to the cell-enlargement stage starting June, both Hamlin and Valencia varieties exhibited higher leaf N concentrations in most fertilization treatments, except for two linear supply treatments (T1 and T2). This increase in leaf nitrogen could contribute to enhanced crop health and performance. In contrast, potassium (K) concentrations in leaves remained consistent across various fertilization treatments throughout the growing period. However, a spike in leaf K concentration was observed in both Hamlin and Valencia during the fruit maturation phase (September-), particularly in treatments with linear nutrient supply and late fertilization. This is significant given that numerous studies indicate that lower potassium levels are preferable for improved fruit quality and peel coloration. Conversely, the temporal fluctuations of other nutrients did not show significant variations across different fertilization 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):In the 2023 growing season, an increase in fruit drop was observed in both Hamlin and Valencia varieties as the season progressed. Specifically, the lowest cumulative fruit drop per tree in Hamlin was recorded under treatment 3 (100% sigmoidal fertilizer supply). On the other hand, Valencia showed a higher cumulative fruit drop per tree, particularly in the linear supply with late fertilization treatment (T2). The overall percentage of fruit drop will be determined after the harvest.Regarding fruit growth, no significant differences were observed in the relative fruit growth rate among the various fertilization 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 October to December:1. Sampling for leaf nutrient analysis2. Fruit diameter measurement3. Collection of leachates for nitrate N loss and analysis4. Scanning of the Minirhizotron for root growth5. Fruit drop count in every two weeks till harvesting in Hamlin and Valencia6. Harvesting Hamlin variety for yield and fruit quality measurement7. Soil sample collection from Hamlin variety blockIn the Sugar Belle peel thickness experiment, all treatment allocation was completed for this year’s experiment and final harvesting will be done during December for fruit quality and peel thickness measurements. Leaf sample collection will be done after harvesting of fruits and processed for leaf nutrient concentration. 3. Please state budget status (underspend or overspend, and why):Spending rate is approximately on track.
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.
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. We request 6-month extension because will need to collect fruit yield and juice quality data in March/April 2024.
2. Please state what work is anticipated for next quarter:
A poster on Objective 1 will be presented at the Soil Science Society of America annual meeting in St. Louis, MO.
3. Please state budget status (underspend or overspend, and why):
The budget is on track and meeting the project milestones. However, we plan to request a no-cost extension so as to collect a fourth harvest in 2024 and also owing to some delays we had in executing the greenhouse portion of the project due to changes in project leadership.
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 Leaf CLas detection analyses are in progress. Leaves were collected for nutrient analysis (in progress). Trunk injury/wounding was assessed in each trial. August and September injections were conducted in trials 4 and 5. September injections were conducted in trial 1. Selected information from these trials was presented at several industry events and conferences, including the Citrus Expo, Packinghouse Day, ASHS, and the International Citrus and Beverage Conference. Research findings were also disseminated in Citrus Industry magazine and through the UF/IFAS citrus podcast. 2. Please state what work is anticipated for next quarter:We will conduct tree size measurements. CLas titer analysis will continue. Fruit drop and tree health will be monitored. 3. Please state budget status (underspend or overspend, and why):The budget is on track although it may appear to be underspent as a large portion of funds are intended to be used for fruit OTC analysis at the USDA National Science Lab in Gastonia, NC, and for fruit quality analysis in 2024.
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. In all three trials: Trunk diameters were measured. Leaves were collected for CLas and OTC detection; laboratory analysis is ongoing. Leaves were collected for nutrient analysis; analysis is in progress. Roots were collected to assess root density. So far we did not detect any significant differences between OTC-injected and non-injected trees for the root densities but we found differences among rootstocks.Selected information from these trials was presented at the Citrus Expo and in Citus Industry magazine. 2. Please state what work is anticipated for next quarter: We will continue with leaf collections for CLas and OTC analysis. Tree health will be monitored. Trunk/wound damage will be assessed. CLas and OTC analysis will continue. 3. Please state budget status (underspend or overspend, and why):The budget is as expected.
On January 5, 2023 18 plants each of LB8-9xS13-15-16 and UFR-5 were potted in 10x10x30 cm containers with autoclaved Astatula sand:ProMix (3:1). Half of the pots of each rootstock were inoculated with 210 sting nematodes in 10 ml water and the remaining half with 10 ml of nematode suspension filtrate from which nematodes were removed by passing repeatedly through a 25µ seive. The plants were maintained in a greenhouse until July 5 when they were removed from pots, separated into tops, tap-pioneer roots, fibrous roots, dried (70o) and weighed. Sting nematodes in 250 cm3 soil from each pot were recovered by sucrose centrifugation. The trial was designed to compare the resistance (nematode reproduction) and tolerance (of nematode damage) between two UF rootstocks that performed best and worst in the mass-screening-tolerance assays reported previously. It is not possible to attribute nematode reproduction to any rootstock in the latter assay, because all lines are grown together in the same large tank. The top weights of UFR-5, and LB8-9xS13-15-16 plants were reduced by 18% and 36% (P=0.05), respectively, in the pots with nematodes compared to the non-nematode pots. Fibrous root density was reduced by nematodes by 10% and 48% (NS due to high variability in the damage) in the respective rootstocks. Two of the LB8-9xS13-15-16 plants died during the trial. The average nematode density in pots with UFR-5 was 535 (all stages) and in pots with LB8-9xS13-15-16 was 378. The trial demonstrated that while UFR-5 exhibited greater tolerance to sting nematodes compared to LB8-9xS13-15-16 in the tank-tolerance assay, the line is not resistant to nematode reproduction. UFR-5 also showed somewhat greater tolerance to the nematode in the pot study than did LB8-9xS13-15-16; however, more pronounced damage in pots compared to that in the tank (Figure 1 in accompanying MS Word document) suggests that the relative rootstock tolerance under more stringent conditions such as the field, where the nematode is not presented with a choice of rootstocks (as in the tank study), remains unresolved. Additional resistance trials with these and additional rootstocks are scheduled to run in tandem with the ongoing and remaining tolerance studies.
The third oxamyl application of the trial occurred on March 24 and soil samples were taken on 1 May. As reported previously, the sting nematode populations remained low in this grove (<10 nematodes/250 cm3 soil), and it was not possible to distinguish significant treatment differences, although average population density during the past year was 47% lower in plots treated with oxamyl in the uncovered trees and 23% lower in the plots with IPCs. Fibrous root density in May was unaffected by nematide application in either covered or uncovered trees. It is noteworthy that prior to the trial, in January 2022, when the orchard was replanted, 20 trees throughout the block were pulled to examine the root systems (Fig. 1 in the attached word document). Every tree had an extensive surface root system with varying amounts of fibrous roots; however no tree had roots deeper than approximately 12 inches and every tree exhibited extensive sting nematode damage at about the 12 inch depth. In other words, sting nematode damage was severely limiting the depth of the root systems. Moreover, the measured sting population density throughout the block was an order of magnitude larger at that time than since the block was replanted and this trial began. Rootstock may be affecting sting nematode density in the trial. The rootstock US 812 was introduced in the grove when it was replanted in 2022. A CRAFT roostock trial in an adjacent grove includes a number of USDA and UF rootstocks, and sting nematode populations measured in that trial several years ago showed US 812 to have the lowest number of nematodes of the rootstocks in the trial. US 812 was also among the top two performing rootstocks in the ongoing greenhouse tolerance trials and will be evaluated a second time in combination with UF and other USDA rootstocks a trial that will terminate in October 2023. In an ancillary project, initiated in this grove at the same time as the current IPC-nematicide trial, we are evaluating an unregistered nematicide produced by Syngenta. The trial is being conducted in the same manner as the IPC-nematicide trial (spring and autumn applications followed by summer and winter assessments), but without any protection from HLB. The Syngenta material was the best-performing of four new nematicides tested in a previous CRDF trial to control sting nematode. The new chemistries are much safer to use than traditional nematicides, have high toxicity to nematodes but very low toxicity to mamals birds and fish. Moreover, the recent availablity of several new nematicides will allow rotation of chemistries with different modes of action to help prevent loss of efficacy due to nematode resistance development or the development of accelerated microbial degradation. The new product is being tested at low and high rates (L and H), with and without an adjuvent (S), and with applicatons either once a year in spring (S) or twice annually in spring and fall (F). To date the new nematicide has performed consistently as well as or better than oxamyl against sting nematode (Fig. 2 in the attached Word document), albeit under conditions of relatively low population density in this grove. All treatments have measured efficacy ranging from 57% to 90% against the nematode. On May 14 the following rootstocks were planted into the tanks infested or not with sting nematode and will be harvested in 20 weeks to comprise the third tolerance trial: UFR 1, UFR 5, Orange 16, MG 11, CB8-9xS13-15-16, S10x639-12-32, US 812, US 942, US 802, US 1284, US 1283, US 1516, Swingle, Kuharske, Sour orange, Sweet orange.
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