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.
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. In Objective 1 we have performed the applications of Zn and K and their combination in early June and in July, as we did last year. Also, we have performed canopy analysis by using the ImageJ program after the first year of tretments. In Hamlin, trees treated with Zn had significantly more dense canopies and more canopy volume than controls. This effect was less evident with K and in the combined treatment. In Valencia trees all treatments increased canopy volume and density significantly. These measurements have been done also for objective 2, and we are analyzing data.We have started to analyze samples to determine oxidative stress levels. In objective 3, 7 presentations from Dr Vashisth’s lab and 4 from Dr Alferez’s were delivered at the ASHS annual meeting in Orlando, July, 2023. 2. Please state what work is anticipated for next quarter: Treatments for objectives 1 and 2 will continue. We are starting now 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. 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 to confirm what we have previously reported. We do see again a very strong induction of immunity-related gene expression around 5 days after brassinosteroid treatment. After 9 months (with a monthly application), 60% of these trees are still HLB-negative and with a denser foliage, in part due to a more abundant flushing in May than the control-infected trees. Interestingly, we do see more synchronous flushing in trees treated with Brs as compared to controls. Trees treated with Brs had a massive flushing in June, whereas control trees had a more spread flushing in June and July. We are following this flushing dynamics in fall to confirm that Brs can synchronize flushing.2, To determine the best time of application (frequency) to achieve maximum protection against pests and disease in newly planted trees. We started the assesment of treatments after May flush, and we collected data on psyllid population, egg deposition and bacteria titer. We do see less psyllids and eggs per tree. We are now analyzing bacteria titer in samples collected through the first year. 3, To determine the effect of Br application on advancing fruit maturation in both Valencia and Hamlin. We have continued juice analysis of samples collected through the year. Treatments are planned to start in September for Hamlin and January in Valencia. We have prepared the experimental area in Duda, flagged the trees and collected baseline data (canopy, fruit set, tree height) as we are using the same trees than last year, so we can also assess any cummulative effects of treatment over time. Outreach:-Alferez, F., Shahid, M. (2023). Can brassinosteroids help citrus to cope with disease and extreme weather events? Citrus Industry, 104, 4 April 2023.-Alferez, F. Use of Auxins and Brassinosteroid in citrus to improve fruit growth and development. In Service Training Use of plant growth regulators in Florida horticulture industry. Daytona Beach, FL. June 13, 2023. 2. Please state what work is anticipated for next quarter: Treatments will start in Hamlin. 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: 1. 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. Major activities in this quarter were leaf sampling of tagged flush, nutrient analysis, leaf characteristics documentation. A signifcant portion of time was spent on data analysis as we have a massive data set. The preliminary analysis is showing that the fertilization based on spring flush yielded the highest fruit. It is possible because spring flush is monitored throughout the year and therefore, fertilizer can be adjusted atleast three times. We are also learning about the leas charateristic differences between spring and summer flush where summer flush seems to be larger and more productive. Lastly, fruiting and non fruiting branch comparison are showing distinct nutrient profile differences between the two branches. These results will be presented at ASHS 2023 as an oral presentation. 2. Please state what work is anticipated for next quarter: 1. Data analysis and interpretation 2. Final leaf collection for phenotyping and nutrient analysis. 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. 4. Please show all potential commercialization products resulting from this research, and the status of each: Changes in leaf sampling recommedation will happen as a result of this research. We anticipate to have recommendations by december 2023.
1. Please state project objectives and what work was done this quarter to address them: 1. 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 final leaf sampling was done for nutrient analysis and leaf characteristics documentation. Other activities included data analysis as we have a massive data set. The preliminary analysis is showing that the fertilization based on spring flush yielded the highest fruit. It is possible because spring flush is monitored throughout the year and therefore, fertilizer can be adjusted atleast three times. The results from nutrient profile comparison of fruting and non fruiting branch were presented at ASHS 2023 as an oral presentation. The major finding of this objective are that the fruting branch show severe deficiency of macro nutients as the fruit matures whereas the micronutrient depletes in non fruiting branch as the season progresses. 2. Please state what work is anticipated for next quarter: 1. Data analysis and interpretation 2. Developing recomendations for nutrient analysis 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. 4. Please show all potential commercialization products resulting from this research, and the status of each: Changes in leaf sampling recommedation will happen as a result of this research. We anticipate to have recommendations by december 2023.
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. Two field trials were established in collaboration with Peace River Packing Company, one with Valencia (Varner grove – trial 1) and one with Hamlin (Kurish south grove – trial 2) trees. Trees in both groves were planted in 2005. Trial 1 was established in a split-plot design. The main plot factor is the season of injection at 2 levels: (i) spring injections; and (ii) late summer/fall injections. The sub-plot factor is the injection rate in combination with different annual use patterns as outlined below: Year 1 Year 2 Year 3 1 utc utc utc 2 150% 0 tbd 3 100% 100% 0 4 100% 0 100% 5 75% 75% 0 6 75% 0 75% 7 50% 50% 0 8 50% 0 50% The trial was established in 16 rows of trees, with the main plot factor arranged by row. Eight replications were used, with each replication consisting of 4 trees. The total number of experimental trees is 512 (8 treatments x 4 trees x 8 reps x 2 times). Spring injections were conducted on June 15 using ReMedium and the Flexinject injectors. The OTC solution was prepared directly in the field to prevent degradation. Trees were injected between 9-12 am. Trial 2 was established in a complete randomized block design using the same rate and annual use pattern as in the Valencia trial. The trial was established in 10 rows, each comprising one block, for 10 replications. Each replicate consist of 3 trees. The total number of experimental trees is 240 (8 treatments x 3 trees x 10 reps). Injections were conducted on June 1 using ReMedium and the Flexinject injectors. The OTC solution was prepared directly in the field to prevent degradation. Trees were injected between 9-12 am. 2. Please state what work is anticipated for next quarter: We will complete the September injections in trial 1. 3. Please state budget status (underspend or overspend, and why):The budget is as expected.
1. Please state project objectives and what work was done this quarter to address them: Objective 1: Study the changes in the reactive oxygen species (ROS) and investigate the correlation between ROS accumulation, sucrose levels and callose deposition in early season sweet oranges Budded trees from Hamlin, Parson Brown and Roble sweet oranges have been infected with CaLas through stick grafting. Initial qPCR results indicate that 80% of the trees infected have tested positive for Clas. Samples will be obtained from trees during November for further investigations. We will also sample trees from stakeholder plots for comparison with the greenhouse evaluation. Objective 2: Quantify fruit drop and canopy yield efficiency in Parson Brown as compared to Hamlin. Understand the underlying mechanism of better fruit retention and yield efficiency in Parson Brown as compared to Hamlin. This experiment has been initiated and trees have been tagged at a grower – cooperator plot. Objective 3: Determine the mechanism underpinning the tolerance of Parson Brown to HLB by leaf volatiles and non-volatile metabolite analysis using GC-MS. The metabolite and leaf voliatile assay has been completed and the results are being analysed. Objective 4: Evaluate other early season sweet oranges We have sampled our experimental trees once and subsequent leaf samples will be collected in the Fall (November) for this objective. 2. Please state what work is anticipated for next quarter: Fruit drop studies will be intiated on field trees. Also, results obtained in objective 3 will be analysed in detail. 3. Please state budget status (underspend or overspend, and why): We are on track with the budget now. A postdoc has been hired to work on several of the parameters in this project from the second quarter.
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:Fertilization treatments for the year were only partially complete in June and too early for fruit quality and preharvest drop analyses. 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). Leaf nutrient concentration in the citrus leaves was affected by fertilizer application timing. Initially, fertilizer application of 20-25% of total recommended dose during February recorded low concentration of N,Ca, Mg, S and Zn. However the nutrients P, K, Cu, Fe and B were observed in optimum range in Hamlin and Valencia. After 50% fertilization of total recommended N (180lbs/ac, year 2 rate) by March, optimum to higher ranges of N concentrations were observed. In contrast, N concentration in Hamlin was observed below optimum ranges if only applied 25% N fertilizer was applied by March. This result suggests that 50% of recommended N application by March improves the N uptake in Hamlin variety and could be beneficial for tree growth and fruit development. Fruit drop was noticeably increased with time in Hamlin variety after natural fruit drop (June) but with no significant difference among treatments (fertilization timing and rate). See Issues, below. Routine lysimeter leachate sample collection continued in this quarter but sample analysis was not completed yet.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):There were no new fruit quality of drop data for 2023 to report in June, but so far trunk diameter, fruit growth and fruit drop were not significantly different with respect to fertilizer treatments.B) New developments: NoneC) Issues: The Hamlin trees dropped some low-hanging fruit after a routine glyphosate spray applied by the grove care crew. The Valencia trees were mostly unaffected, in part due to their fruit not hanging as low as the larger Hamlin fruit.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.The fourth fertilizer application will be applied in late August, phase 2 fruit growth.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) 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 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, on 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. There are four treatments 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 growers many are doing currently4-6 annual sprays of formulations labelled 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. In experiments at locations, leaves are being sampled monthly. 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. 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 in both locations. Tree size measurements (height, canopy, width, and trunk diameter at tree base) were at time zero, middle, and end of the from all trees in the replicate. New leaf growth (flush) was 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 July 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 of the experiment in both locations. The following report will include results regarding the effectiveness of OTC on CLas infection and tree health.
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 have been 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 ACP 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 July 2023 at both locations. During the next reporting period, we will include results regarding the effectiveness of OTC on ACP adult populations.
Objective 3) Determine the effect of OTC injection on CLas transmission.
Field trials were establsihed 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, 60, and 90 days for CLas detection.
Recent activities:
.Leaves for CLas infection and OTC translocation have been successfully collected for the first replication and are currently being processed. The first replication of experiments 3.1 and 3.2 was successfully done in June 2023. A second replication will be 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), CLas infection, and OTC translocation.
2. Please state what work is anticipated for next quarter:
This investigation is long term. The above described experiments will run throughout this year and next. We anticipate having progress updates on all three objectives as described above.
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:
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 inoculation and are evluating varying pH levels from 5.5, 6.5, 7.5 and 8.5 in rhizotrons under greenhoiuse conditions. All protocols are developed and pretesting for the study is finalized.
This quarter we measured physiological responses, root growth variables and apoplastic pH changes and are still completing micropscopy work and desdtrctive sampling through the next quarter. The portion CLas impacts will be conducted on selected and periodic field root samples from CLas infected trees. Early results of the greenhouse work were shared at the Florida State Horticultural Society (FSHS) annual meeting and a scientific note was published.
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 also completed fruit harvesting and juice quality determinations in the Lake Alfred site in April 2023.
2. Please state what work is anticipated for next quarter:
The greenhouse and field work portions on objective 1 will be completed in August 2023. Once the greenhouse and field work portion of objective one is done, we will prepare a journal manuscript and possibly a cittrus industry magazine article to share the results with other researchers and the citrus industry.
A poster on Objective 1 will be presented at the American Society of Horticultural Science in Orlando, in August 2023.
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: Located in SW Florida (Duda) – 8-year-old Valencia/Carrizo trees. HLB ratings were conducted. Wounds from year 1 injections were assessed. Year-2 April injections were completed. Leaves were collected for CLas analysis prior to injections. Trial 2: Located in SW Florida (Graves Bros) – 8-year-old Valencia/Kuharske trees. Trees were harvested and fruit quality analysis was completed. HLB ratings were conducted. Wounds from year 1 injections were assessed. Year-2 injections were completed. Leaves were collected for CLas analysis prior to injections. Trial 3: Located on the east coast (Graves Bros) – 9 year-old Valencia/sour orange trees. Trees were harvested and fruit quality analysis was completed. HLB ratings were conducted. Wounds from year 1 injections were assessed. Year-2 injections were completed. Leaves were collected for CLas analysis prior to injections. Trial 4: Located on the east coast (Graves Bros) – 4-year-old Valencia/x639 trees. Year-2 May injections were completed. HLB ratings were conducted. Wounds from year 1 injections were assessed. Leaves were collected for CLas analysis prior to injections. Trial 5: Located on the central ridge (King Ranch) – 4-year-old OLL-8/x639 trees. Year-2 May injections were completed. HLB ratings were conducted. Wounds from year 1 injections were assessed. Leaves were collected for CLas analysis prior to injections. Presentations including research findings from this project:- Citrus Growers Institute, Avon Park- Citrus Grower meeting, Tavares- USDA PPQ CHRP meeting, Gainesville- Citrus Annual Conference, Bonita Springs 2. Please state what work is anticipated for next quarter:A subset of trees in trials 1, 4, and 5 will be injected in September. Leaves will be collected for nutrient analysis. 3. Please state budget status (underspend or overspend, and why):The budget is slightly underspent as we are still waiting for the USDA National Science Lab in Gastonia, NC to complete the OTC residue analysis, which will be around $24,000.
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. Three trials were evaluated (rootstocks UFR-3 and UFR-16 were not included due to poor performance): Trial 1 (near Babson Park, Polk County): 6 rootstocks (US-812, US-942, US-897, UFR-2, UFR-4, Carrizo). Trial 2 (near Venus, Highlands County): 5 rootstocks (US-812, US-942, UFR-2, UFR-4, sour orange). Trial 3 (near Felda, Hendry County): 5 rootstocks (US-812, US-942, UFR-2, UFR-4, Swingle). The experimental design for OTC injections was established in all trials and trees were flagged accordingly. All trials were injected by a commercial crew guided by our team. One set of trees within each rootstock plot was injected in the scion. Another set was injected into the rootstock, and another set remained non-injected to serve as a control. The rate was 1.1 g of OTC (Rectify) and was delivered in a volume of 100 ml (11,000 ppm) using the FlexInject injectors.During injection, a subset of trees from 3 selected rootstocks in each trial were monitored to compare the speed of uptake between rootstock injections and scion injections. We found that the uptake rate was mostly affected by the time of day and the environmental conditions on the day of injection. The uptake rate varied from less than 20 minutes to several hours.Leaves from selected subsets of trees were collected 3 days, 1 week, and 1 month after injection to determine CLas titers and OTC concentrations. 2. Please state what work is anticipated for next quarter: We will continue with leaf collections for CLas and OTC analysis. We will also conduct root density measurements. 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: 1. 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. Major activities in this quarter were leaf sampling of tagged flush, nutrient analysis, leaf characteristics documentation and collecting harvest data from Valencia sites. Unfortunately, harvest data from Arcadia Valencia could not be collectred as the tree experienced significant fruit drop after the Hurricane Ian The preliminary analysus is showing that the fertilization based on spring flush yielded the highest fruit. It is possible because spring flush is monitored throughout the year and therefore, fertilizer can be adjusted atleast three times. We are also learning about the leas charateristic differences between spring and summer flush where summer flush seems to be larger and more productive. Lastly, fruiting and non fruiting branch comparison are showing distinct nutrient profile differences between the two branches. 2. Please state what work is anticipated for next quarter: 1. Data analysis and interpretation2. Collecting samples for nutrient analysis3. Applying fertilizer treatments based on leaf nutrient analysis results 3. Please state budget status (underspend or overspend, and why): The budget is being spent as per the plan where major funds have been used for nutrient anlaysis. 4. Please show all potential commercialization products resulting from this research, and the status of each: Changes in leaf sampling recommedation will happen as a result of this research.
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