The first nematicide treatment occurred in the IPC trial in late April 2022 and the second will occur in late May. The effects of these treatments will be determined in early July when soil samples will be taken to obtain the first nematode and root mass density measurements. The site is on a hilltop and is extremely windy. We spent several days following planting installing grade stakes inside the IPCs to better support the PVC frames. To date they are secure.As noted previously, maintenance and new construction on our greenhouse that started at the beginning of January was still ongoing in April, precluding further research on the rootstock tolerance trials until completed. Plants and cultures had to be moved to a laboratory facility to protect the citrus from psyllid infestations and to protect all of the cultures from excessively high temperatures when the evaporative cooling systems were being replaced. Plants were reinstalled in the greenhouse during the second week of April and pruned to reinitiate growth in preparation for innoculations to occur in June. Cultures were sampled and found to contain fewer nematodes than previously, but were still in good condition and will recover. The sting nematode populations in the large tanks that remained in the greenhouse were in good condition. Presumably, the depth of soil allowed the nematodes to persist despite the high ambient temperatures they experienced for a week. The first rootstock lines are ready and will be installed in the tanks during the first week of June.
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. During this initial quarter, for objectives 1 and 2 we have identified the trees from Hamlin and Valencia in Central and Southwest Florida that we will be using in our studies, flagged them and organized them in a completely randomized block design. We have also taken baseline initial data on HLB severity ratings, tree height, canopy volume, leaf area index, and scion and rootstock diameters. Also, leaf samples for nutrient analysis and determination of initial Ct values for HLB have been taken.For objective 2, GA3 and 2,4D treatments have already started. For objective 1, we are starting first set of treatments by the end of May.Objective 3: Outreach-F. Alferez, Foliar applications of Zinc and Potassium to increase yield by reducing fruit drop in Hamlin oranges. 2022 Florida Citrus Growers’ Institute, Avom Park April 5, 2022.-T. Vashisth, Update on Gibberellic acid studies.2022 Florida Citrus Growers’ Institute, Avom Park April 5, 2022.-T, Vashisth and F. Alferez., wokshop by zoom on “gibberellic acid research update and other PGRs for Florida citrus”. 217 registered participants. 2. Please state what work is anticipated for next quarter: In the second quarter, we will perform first set of treatments for objective 1 in both Valencia and Hamlin trees and continue with data collection. Treatments for objective 2 will continue as planned (applications every 45 days). Importantly, a new PhD student, Dyvia Aryal, Ms graduated from University of Hannover, Germany, will join the project in June-July, to conduct studies on this project under Dr Alferez supervision 3. Please state budget status (underspend or overspend, and why): There has been underspending on student salary and tuition, because student could not join in January as expected, due to a delay in proposal approval. As stated above, Dyvia will join in Summer, as soon as she gets her visa approved (in process).
Objective 1. To illustrate whether application of bactericides via trunk injection could efficiently manage citrus HLB and how bactericides via trunk injection affects Las and HLB diseased trees. 1.1. Determination of the in planta minimum bactericidal concentrations (MBCs) of bactericides against LasWe developed a new method for evaluating the effects of oxytetracycline (OTC) treatment on CLas titers in infected plants and determined the relationship between OTC residue levels and control levels achieved for CLas using mathematical modeling in greenhouse and field experiments. In both greenhouse and field, OTC spray did not reduce the titers of CLas, and it produced undetectable or mild levels of OTC residue in leaves within 7 days post-application (DPA). In greenhouse, OTC injection at 0.05 g per tree decreased CLas titers to an undetectable level (cycle threshold value = 36.0) from 7 to 30 DPA and produced a residue level of OTC at 0.68 to 0.73 µg/g of fresh tissue over this period. In the field, OTC injection at 0.50 g per tree resulted in the decline of CLas titers by 1.52 log reduction from 14 to 60 DPA, with residue levels of OTC at 0.27 to 0.33 µg/g of fresh tissue. In both trials, a first-order compart model of OTC residue dynamics in leaves of trunk-injected trees was specified for estimating the retention of effective concentrations. Furthermore, nonlinear modeling revealed significant positive correlations between OTC residue levels in leaves and the control levels for CLas achieved. The results suggested that the minimum concentrations of OTC required to suppress CLas populations in planta to below the detection limit are 0.68 and 0.86 µg/g and that the minimum concentrations of OTC required for initial inhibition of CLas growth in planta are ~0.17 and ~0.215 µg/g in leaf tissues under greenhouse and field conditions, respectively. This finding highlights that a minimum concentration of OTC should be guaranteed to be delivered to target CLas in infected plants for effective control of citrus HLB.We investigated the in planta minimum bactericidal concentrations of streptomycin (STR) and its effect on CLas titers in planta applied by foliar spray and trunk injection of 3-year-old citrus trees that were naturally infected by CLas in the field. After foliar spray, STR levels in leaves peaked at 2 to 7 days postapplication (dpa) and gradually declined thereafter. The STR spray did not significantly affect CLas titers in leaves of treated plants as determined by quantitative PCR. After trunk injection, peak levels of STR were observed 7 to 14 dpa in the leaf and root tissues, and near-peak levels were sustained for another 14 days before significantly declining. At 12 months after injection, moderate to low or undetectable levels of STR were observed in the leaf, root, and fruit, depending on the doses of STR injected, with a residue level of 0.28 µg/g in harvested fruit at the highest injection concentration of 2.0 µg/tree. CLas titers in leaves were significantly reduced by trunk injection of STR at 1.0 or 2.0 g/tree, starting from 7 dpa and throughout the experimental period. The reduction of CLas titers was positively correlated with STR residue levels in leaves. The in planta minimum effective concentration of STR needed to suppress the CLas titer to an undetectable level (cycle threshold =36.0) was 1.92 µg/g fresh weight. Determination of the in planta minimum effective concentration of STR against CLas and its spatiotemporal residue levels in planta provides the guidance to use STR for HLB management. 1.2. Effect of bactericides via trunk injection on citrus HLB disease progression, tree health, yield and fruit quality in different aged trees with a different disease severityThe field experiments were performed at four different groves on different aged trees with a different disease severity. They are one located in Avon Park, FL, 3-year old Valencia trees; one in Bartow, FL, 2-year old W. Murrcot trees; and one in Auburndale, FL, 7-year old Hamlin trees (planted in 02/2012). The last one is in CREC-, Lake Alfred, FL, 20-year old Hamlin trees. The HLB disease severity and tree size (canopy volume and trunk diameter) in the four groves were estimated immediately prior to treatment application. For the field tests, the experiment design is a randomized complete block design (RCBD) for 9 treatments, including 6 injection treatments (3 different doses for OTC or STR), 2 spray treatments (OTC or STR spraying), and one No treatment as a negative control. Each injection treatment consisted of 9 or 15 trees divided into 3 blocks of 3 or 5 trees each. Each spray treatment consisted of 30 trees divided into 3 blocks of 10 trees each. For all the four field trials, the injection treatment applications were completed by the end of April 2019. The 1st application of spray treatments were completed during spring flushing in February or March 2019, the 2nd applications were conducted in late June to early July 2019, and the 3rd applications were conducted in early to middle October 2019. Leaf samples have been collected from the treated trees at the following time points: 0 (pre- injection), 7, 14, 28 days, 2, 4, 6, 8, 10 and 12 months after treatment (MPT). Our data demonstrate that for 6-year-old trees, 2.0 g OTC/tree, but not 1.0 g OTC /tree via trunk injection significantly reduced CLas titers. However, neither concentrations reduced CLas titers for 22-year-old trees. Spray had no effect on CLas titers. STC at 1 g or 2 g/tree via trunk injection had no effect on CLas titers. OTC (2.0 g/tree) and STR (2.0 g/tree) but not at lower concentrations significantly increased fruit yield, but had no effect on quality. Objective 2. To examine the dynamics and residues of bactericide injected into citrus and systemic movement within the vascular system of trees and characterize the degradation metabolites of bactericides in citrus. Residues of OTC or STR in fruit harvested from 22-year old Hamlin sweet orange trees injected with OTC or STR at different doses were determined. Injections were conducted in February-March 2021 and fruit was harvested in January 2022. Neither OTC or STR was detected at 1 g or 2 g/tree via trunk injection.Residue of OTC or STR in fruit harvested from 6-year old Hamlin sweet orange trees injected with OTC or STR at different doses were determined. Injections were conducted in March 2021 and fruit was harvested in January 2022. The following residues were observed: OTC (1.0 g/tree): 0.08 ppm ± 0.03; OTC (2.0 g/tree): 0.19 ppm ± 0.07, STR (1.0 g/tree): 0.09 ppm ± 0.03; STR (2.0 g/tree): 0.17 ppm ± 0.08. Objective 3. To determine whether trunk injection of bactericides could decrease Las acquisition by Asian citrus psyllids (ACP)Twenty 1.5-year old citrus (Valencia sweet orange) plants were graft-inoculated by Las carrying buds in February 2020. These plants are being tested for Las infection and 4 plants were confirmed with Las infection (Ct values are between 34.0 and 35.0) at 4 months after grafting. They will be subjected to OTC or STR treatment by trunk injection and ACP acquisition access for 7 to 14 days. We have determined the time points to test OTC and STR treatment on ACP acquisition of Las. We have conducted the test regarding how trunk injection of OTC affect ACP acquisition of Las. OTC significantly reduced ACP acquisition of Las. Objective 4. To monitor resistance development in Las against bactericides and evaluate potential side effects of trunk injection of bactericides Monitoring resistance development in Las against bactericides. Leaf samples for this test have been collected from 5 trees injected with OTC and 5 trees injected with STR at the highest doses in each of the three groves at 6 and 9 months after the injection, respectively. PCR-sequencing analysis on Las 16SrRNA gene showed there was no mutation compared with the reported sequence. However, accurate evaluation needs to be conducted once CLas is cultured. No obvious side effects were observed at low OTC or STR concentrations. However, at some high concentrations, phytotoxicity was observed on leaves.
HLB is known to make citrus roots more susceptible to Phytophthora root rot. It also reduces the efficacy of chemical management of Phytophthora root rot, creating a difficult management scenario. Current Phytophthora management recommendations are based on pre-HLB work done in the 1980s. These three conditions raise the question of whether yield improvement from Phytophthora management is enough to pay for the management costs themselves. The goal of this project is to develop new soil propagule density managment thresholds and recommendations for chemical management of Phytophthora root rot based on ecomonic analysis of yield responses in different soil conditions. Objective 1) Determine if labelled Phytophthora management maintains efficacy in the field on HLB-affected trees for reducing fibrous root loss and improving yield.During this quarter, soil samples for root density and phythophthora counts were collected from both sites. The Hamlin block was harvested. The quality and sizing data were generated in the packinghouse facility.In preliminarily reviewing the data, there were significant differences among the root densities for Hamlin but not the Valencia. The range for Hamlin was 0.096 to 0.314 roots/ml of soil and for Valencia it was 0.032 to 0.076 roots/ml soil. The treatment with the greatest number of roots was the Ridomil, Presidio, Phosphite, and Orondis regimen and it had significantly more than the UTC. There were no significant differences among the total phytophthora propagule count for the Valencia which ranged from 2.8 to 9.6 propagules/cm3 of soil. When separated for the species there was also no difference. In the Hamlin trees, there was a significant difference among treatments. The Ridomil, Presidio, Phosphite, and Orondis treatment still had no propagules and a statitically equivalent number were observed in the Phosphite alternated with Orondis tretatment. The treatment with the most propagules was Phosphite alternated with Ridomil treatment which had 56.0 propagules/cm3 of soil, significantly higher than other treatments. In the fall propagules counts at both sites, the block effect was much weaker but additional statistical analysis will still be needed to take this factor into account in a better manner. When looking at the Hamlin fruit weights, there was no treatment effect but there was a significant block effect. In looking at the pounds solid per box from each plot, there was a weakly significant effect (P < 0.1) for treatments which ranged from 5.40 to 5.02 lb solids/box. The best treatment was the Phosphite treatment and block was highly significant. The brix and acid had no signficant differences but the brix acid ratio with the highest ratio is for the UTC (16.36) and lowest was Phosphites rotated with Presidio (15.24). Objective 2) Determine benefit-cost thresholds for Phytophthora treatment on HLB-affected treesAs planned in the proposal, this objective awaits this years yield results to begin calculating benefit-cost based on a combination of change in yield from the previous year and comparison among treatments within blocks.
HLB is known to make citrus roots more susceptible to Phytophthora root rot. It also reduces the efficacy of chemical management of Phytophthora root rot, creating a difficult management scenario. Current Phytophthora management recommendations are based on pre-HLB work done in the 1980s. These three conditions raise the question of whether yield improvement from Phytophthora management is enough to pay for the management costs themselves. The goal of this project is to develop new soil propagule density managment thresholds and recommendations for chemical management of Phytophthora root rot based on ecomonic analysis of yield responses in different soil conditions. Objective 1) Determine if labelled Phytophthora management maintains efficacy in the field on HLB-affected trees for reducing fibrous root loss and improving yield.During this quarter, Prophyte treatments were applied to the Hamlin and Valencia sites in July and October. The root density and phytophthora counts for all treatments were done in August. Soil applied treatments were undertaken in late September and early October.In preliminarily reviewing the data, there were no significant differences among the root densities for Hamlin and Valencia. The range for Hamlin was 0.046 to 0.092 roots/ml of soil and for Valencia it was 0.02 to 0.072 roots/ml soil. There were no significant differences among the total phytophthora propagule count for the Valencia which ranged from 12.8 to 23.2 propagules/cm3 of soil. When separated for the species there was alss no difference. In the Hamlin trees, there was a significant difference among treatments. The Ridomil, Presidio, Phosphite, and Orondis treatment had no propagules and this was signficantly fewer than the Phosphite alternated with Ridomil treatment which had 19.2 propagules/cm3 of soil. In both sites, there was a significant block effect and a new statistical analysis will be needed to take this factor into account. No brown rot measurements were taken this year.Objective 2) Determine benefit-cost thresholds for Phytophthora treatment on HLB-affected treesAs planned in the proposal, this objective awaits this years yield results to begin calculating benefit-cost based on a combination of change in yield from the previous year and comparison among treatments within blocks.
1. Please state project objectives and what work was done this quarter to address them:
The final data analyses were done for this paper during this quarter. This included the qPCR of CLas titer in all trees for the last two samplings of the project. Analysis of CLas titer after the six month mark did not show a decline. Yields and canopy cover also did not improve.
CLas titer was determined in two labs using two different primer sets. This was done to give us more confidence in the results. Both labs gave essentially the same results. This will be described in detail in the manuscript.
All samplings are now complete.
Our work is being completed with a draft of a manuscript that describes the results of all of our greenhouse and field work on this project. This is expected by May 2022.
2. Please state what work is anticipated for next quarter:
The paper is expected to be submitted in May 2022 to the APS Journal, Plant Health Reports
3. Please state budget status (underspend or overspend, and why):
We did not overspend on this project.
1. Please state project objectives and what work was done this quarter to address them:
During this period a manuscript describing this work is nearly completed. The current draft is attached along with this report. Our sense of this work is that this apporach still has promise. Greenhouse results were very encouraging. Foliarly applied phosphate did resude citrate levels in phloem. CLas titers did decline in graft-infected citrus saplings in the greenhouse upon foliar phosphate application. Citrus was also found to get all of its phosphate needs through foliar fertilization.
However, the field experiments were harder. We were not able to identify plots with low infection rates at the beginning of the work. The heavily infected plants may have been too weak to have the full benefits of foliar phosphate fertilization. In the first 3-6 months of the field experiments, declines in CLas titer were observed with the lowest level of foliar phosphate application but the plants did not recover in subsequent quarters.
More work on this would require larger groves with more experimentation on the frequency of foliar phosphate application. We choose application every two months and used that frequency throughout. More frequent application at lower doses may improve the efficay of foliar phosphate treatments. This treatment is also more likely to work on younger trees with less infection. Younger trees were used at the Imokalee site but those trees were highly infected. The trees in Hamilton were more than 25 years old.
I remain confident that nutritional approaches can help this problem. It will just require the correct nutrients applied at the right time. The upcoming manuscript is entitled A systems biology approach suggests a simple strategy to alleviate citrus greening disease.
Abstract of manuscript:
Citrus greening disease is now completely endemic to Florida citrus trees, having spread rapidly across all counties in the past 20 years and causing devastating economic losses. The diseases etiological agent is the unculturable bacterium, Candidatus Liberibacter asiaticus (CLas). Liberibacter crescens, the closest culturable relative, was discovered to prefer citrate as its most effective carbon and energy source. Plants load citrate in response to phosphorus deficiency. In Floridas calcareous soils, supplemental phosphate fertilization is very low due to the assumption that it is readily available for plants through the soil. It is likely that citrus trees are loading citrate to mine phosphorus from the soil, which could inadvertently exacerbate CLas infection. In greenhouse experiments, foliarly applied phosphate is easily taken up by the plants, resulting in decreased free-citrate levels in the phloem, and delaying the onset of CLas infection. Our field experiments show improvements in mature trees where CLas infection is well established, and infected psyllids maintain infection.
2. Please state what work is anticipated for next quarter:
None. This report and the attached manuscript respresent our final contributions to CRDF.
3. Please state budget status (underspend or overspend, and why):
We did not overspend on this project.
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 were able to fertilize the trees for spring based on spring and summer flush nutrient analysis. In addition we have tageed newly emereged spring flush for nutrient analysis for this year. We were able to perform another set of nutriet analysis in this quarter as well. In addition we have been collecting leaf samples from fruiting an non fruting branches for comparison. The prelimnary analysis shows that spring flush are deficient in immobile nutrients as compared to random leaves therefore, suggesting that method of sampling can sway the results signifcantly. In addition we are seeing that fruting branches show low levels of consistent acculumation of micronutrients in the leaves wherease the non fruiting branch decrease in micronutrients from summer to fall thus the suggesting a higher metabolism of nutrients. 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.
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: We established two replicated field trials with 10 fertilizer rate and timing combinations to evaluate the growth, fruit development, maturity, yields and quality of 5-year old Valencia and Hamlin trees on X-639 rootstock. The first fertilizer of about 20% annual N was applied in the first week of March. Soil samples were collected from each plot for nutrient analysis, vacuum lysimeters were installed under trees to sample nutrient leachates, and minirhizotrons were installed in the field to measure root growth. Soil P tests in the site were hight to very high, so we omitted P fertilizer from the blended fertilizer in order to deplete soil P reserves down to nominal levels. The third field trial was established on 5-year old Sugarbelle trees, and sub-objectives were modified to focus on improving fresh-fruit characteristics, particularly peel thickness, fruit size, color break, juice content, and internal quality (brix, acid). The basis for this refocusing was in response to the Sugarbelle “soft fruit” and quality reports of the 2021/22 season. 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): We are using the existing Citrus Diagnosis smartphone AI app as a baseline, and exploring improved methods for classifying foliar nutrient deficiency symptoms such as magnesium, iron, manganese and zinc so that the intensity of deficiency can be quantified from the severity and extent of chlorotic tissue on an area basis. Semantic segmentation models were trained on leaf images to quantify chlorosis and generative adversarial networks were tested to generate hypothetical synthetic nutrient symptoms to include in a comprehensive fertilizer management and nutrient modeling app. B) New developments: sub-objectives concerning the Sugarbelle trial were refocused on solving the current fruit quality issues, but temporal fertilization strategies are still at the core of the trial. C) Issues: We are concerned about the low fruit set in the city block. The trees flowered in January, and the bloom was apparently damaged by the January 30 freeze. The trees produced some recovery bloom about a month later, but it was small. 2. Please state what work is anticipated for next quarter: The second of four fertilizer applications will be made in the first week of April, and the third will be in mid May. Some treatments in the 10-treatment structure will not receive fertilizer at every date, or receive variable amounts of fertilizer in order to construct 10 different fertilization strategies (supply curves) over time. Soil, lysimeter and leaf sampling, processing and analysis will be ongoing, as will tree size and fruit measurement. The first Aerobotics drone survey will fly in early May, to evaluate tree health and size indicators. 3. Please state budget status (underspend or overspend, and why): Grove chargebacks for 3 acres of field experiments were initiated, partial salaries of 3 personnel were allocated for the new efforts of this project, and supplies for installing minirhizotrons were purchased. There may be a temporary underspend due to system lag in reporting the modified salary allocations to this project, which was done recently.
1. Please state project objectives and what work was done this quarter to address them: Objective 1: Determine effects of lowered soil pH on CLas populations and root physiology including internal root apoplast and vascular tissue pH. Due to a collapse in our inoculum trees, we are psyllid inoculating trees for these experiments and will start the greenhouse portion of the study in Spring 2022. All CLas inoculated and non-CLas inoculated trees are ready for the experiment slated for April 29, 2022. 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, PCR and of selected trees. 2. Please state what work is anticipated for next quarter: Fruit harvest and fruit quality evaluation will also be accomplished this quarter on April 19 and April 21, 2022. Injection of acids and S application were delayed until harvest to avoid any impacts on fruit drop. 3. Please state budget status (underspend or overspend, and why): The budget is on track and meeting the project milestones.
During October 2021-Janury 2022 we surveyed sting nematode populations in several Polk and Highlands County orchards before focusing on 12 quadrats (all moderately-highly infested) across 30 acres of a declining grove near Babson Park destined for replanting in spring 2022. The nine-year-old trees in the grove were clipped to preserve the current irrigation system; however, the grower uprooted 20 trees throughout the grove, revealing uniformly severe root damage on all trees immediately below 30 cm depth. There were relatively fewer symptoms on the surface layer of fibrous roots supporting the need for deep soil sampling to accurately assess sting nematode abundance in citrus. The extensive and comprehensive damage to root systems by sting nematode suggested that the grove would be suitable for the trial.Three hundred trees (Valquarius on 812) were planted on 3/22/2022 and half the trees were covered immediately with individual protective covers (IPC). The design of the experiment is a completely randomized block with 15 blocks, each with 4 factorial treatments (all combinations of IPCs and nematicide application), each applied to 4-tree plots. Plots are separated by single buffer trees. Thus, we will evaluate the effect of sting nematodes on trees exposed to or protected from Asian citrus psyllids that are treated or not treated for nematode control. We dissassembled the fittings and poly-tubing used to deliver nematicides to 32, 4-tree plots in a previous experiment and reconfigured and installed the equipment in these 15 blocks. The first nematicide treatment will occur in April 2022 and will occur in each autumn and spring thereafter. In November 2020 we collected sting nematode-infested soil from a citrus orchard and extracted and handpicked 30-50 mixed-stage individuals per 8 pots each of St. Augustine grass and sorghum sudangrass to establish cultures to serve as inoculum for trials to evaluate tolerance of rootstocks to sting nematode. Within two months, sampled pots contained as many as 40 nematodes per cm3 of soil. In December 2022 we autoclaved Candler sandy soil and filled 4, 150 gallon tanks in a greenhouse. St. Augustine grass stolons and soil from the nematode cultures were transplanted into two of the tanks in January. the other two tanks were planted with non-infested St. Augustine. Construction on the greenhouse that began in early January was still ongoing in April, precluding further research until completed.
Objective 1. To illustrate whether application of bactericides via trunk injection could efficiently manage citrus HLB and how bactericides via trunk injection affects Las and HLB diseased trees. 1.1. Determination of the in planta minimum bactericidal concentrations (MBCs) of bactericides against LasThis has been completed for both streptomycin and oxytetracycline against Las. A manuscript entitled: “Residue dynamics of streptomycin in citrus delivered by foliar spray and trunk injection and effect on Candidatus Liberibacter asiaticus titer” was accepted for publication by Phytopathology.1.2. Effect of bactericides via trunk injection on citrus HLB disease progression, tree health, yield and fruit quality in different aged trees with a different disease severityThe field experiments were performed at four different groves on different aged trees with a different disease severity. They are one located in Avon Park, FL, 3-year old Valencia trees; one in Bartow, FL, 2-year old W. Murrcot trees; and one in Auburndale, FL, 7-year old Hamlin trees (planted in 02/2012). The last one is in CREC-, Lake Alfred, FL, 20-year old Hamlin trees. The HLB disease severity and tree size (canopy volume and trunk diameter) in the four groves were estimated immediately prior to treatment application. For the field tests, the experiment design is a randomized complete block design (RCBD) for 9 treatments, including 6 injection treatments (3 different doses for OTC or STR), 2 spray treatments (OTC or STR spraying), and one No treatment as a negative control. Each injection treatment consisted of 9 or 15 trees divided into 3 blocks of 3 or 5 trees each. Each spray treatment consisted of 30 trees divided into 3 blocks of 10 trees each. For all the four field trials, the injection treatment applications were completed by the end of April 2019. The 1st application of spray treatments were completed during spring flushing in February or March 2019, the 2nd applications were conducted in late June to early July 2019, and the 3rd applications were conducted in early to middle October 2019. Leaf samples have been collected from the treated trees at the following time points: 0 (pre- injection), 7, 14, 28 days, 2, 4, 6, 8, 10 and 12 months after treatment (MPT). The estimation of Las titers in these leaf samples are ongoing with qPCR assays. The first estimation of HLB disease severity and growth performance (height, trunk diameter, and canopy volume) of immature trees after treatment were performed in May 2019 (three months after the injection) and continued in a 3-months interval. Fruit yield and quality data were collected for the Bartow trial (W. Murrcot), Auburndale trial (Hamlin), and CREC trial (Hamlin) in January 2021. We investigated the effect of 10 adjuvants on oxytetracycline absorption via foliar spray. Four adjuvants including Flame slightly increased the antimicrobial effect of OTC on Las.We have collected data for yield and quality. Objective 2. To examine the dynamics and residues of bactericide injected into citrus and systemic movement within the vascular system of trees and characterize the degradation metabolites of bactericides in citrus. Leaf and root samples have been collected from OTC or STR treated trees in the Avon Park grove at the following time points:0 (pre- injection), 2, 4, 7, 14, 28 days, 2, 4, 6, 8, 10, and 12 months after injection. The samples have been processed for OTC or STR extraction, and the concentrations of OTC and STR in these samples were determined by HPLC assays. Fruit samples were collected for the Bartow trial (W. Murrcot), Auburndale trial (Hamlin), and CREC trial (Hamlin) during harvest in January 2020, and for the Avon Park trial (Valencia) in April 2020. The samples were processed for OTC or STR extraction, and the concentrations of OTC and STR in these samples were determined by HPLC assays. We have collected data for 60 and 360 days post treatment. We have analyzed the residues of OTC and STR for fruit samples harvested in January 2022. Objective 3. To determine whether trunk injection of bactericides could decrease Las acquisition by Asian citrus psyllids (ACP)Twenty 1.5-year old citrus (Valencia sweet orange) plants were graft-inoculated by Las carrying buds in February 2020. These plants are being tested for Las infection and 4 plants were confirmed with Las infection (Ct values are between 34.0 and 35.0) at 4 months after grafting. They will be subjected to OTC or STR treatment by trunk injection and ACP acquisition access for 7 to 14 days. We have determined the time points to test OTC and STR treatment on ACP acquisition of Las. We have conducted the test regarding how trunk injection of OTC affect ACP acquisition of Las. OTC signficantly reduces ACP acquisition of Las. Objective 4. To monitor resistance development in Las against bactericides and evaluate potential side effects of trunk injection of bactericides Monitoring resistance development in Las against bactericides. Leaf samples for this test have been collected from 5 trees injected with OTC and 5 trees injected with STR at the highest doses in each of the three groves at 6 and 9 months after the injection, respectively. PCR-sequencing analysis on Las 16SrRNA gene showed there was no mutation compared with the reported sequence. We are further confirming the results. Evaluation of potential side effects of trunk injection of bactericides have been completed. We have collected another set of samples to monitor Las resistance against OTC and STR. PCR-sequencing analysis on Las 16SrRNA gene showed there was no mutation compared to the reported sequence, indicating no major changes in bacterial resistance against antimicrobials. Leaf samples were collected from OTC or STR injected trees in the Avon Park grove at two and four months after treatment for the analysis of the degradation metabolites of the bactericides. The extraction of the degradation metabolites were completed and will be subjected to HPLC assays. We are in the process of analyzing data related to how adjuvants affect bactericide delivery via foliar spray, bactericide residues in plants, and effect of bactericides on HLB positive citrus tress of different ages. We also investigated how trunk injection of OTC and STR affects phloem tissues. We are analyzing data for final report.
Conclusions of the one year preliminary study: `Parson Brown’ trees, irrespective of location, clone or rootstock had enhanced SAR activity as indicated by enhanced PR1 and PR2 gene expression. Statistically significant differences were always observed in the different locations during the March and December samplings. PP2 gene downregulation indirectly suggests an active protection mechanism in the phloem of the `Parson Brown’. Targeted RNA seq and metabolomics of the phloem would confirm these results and provide more insights into the exact mechanism of HLB tolerance. All trees (`Parson Brown’ and `Hamlin’) evaluated in this study were HLB positive with similar CaLas titer. `Parson Brown’ trees however, maintained a good canopy and held on to fruit much better than comparable `Hamlin’ trees. Oil content % ranged from 0.007 to 0.0011 in `Hamlin’ juice whereas, it ranged from 0.020 to 0.042 in `Parson Brown’ juice, depending on the location the fruit was harvested from. This could be due to clonal differences between trees grown in different locations. Lbs. Solids Per Box were comparable between `Parson Brown’ and `Hamlin.’ However, `Hamlin’ has recorded good Lbs. Solids Per Box in 2021, and samples will need be evaluated for multiple seasons to understand differences between the two early season sweet oranges. Limonin levels were less than 5 ppm in both `Parson Brown’ and `Hamlin’ and within acceptable levels. It was not possible to identify specific clones based on the data generated. Whole genome sequencing can shed insights into clonal differences by looking at the overall pattern of SNPs and INDELS among the specific selections. `Parson Brown’ trees at the Premier Citrus location in Lorida had the best growth, canopy coverage, followed by trees at the groves managed by Pat and Marty McKenna and the Premier Citrus location in Ft. Pierce. There were no significant differences in the oil content from fruit from these locations. It may be worthwhile to reintroduce a couple of these superior lines into the DPI’s parent tree program for the benefit of the citrus stakeholders.
Conclusions of the one year preliminary study: · Trees were evaluated on a quarterly basis from 8 different locations. Tree aged ranged from 20 to 40 years in these locations. Most locations had changed management several times in these years and there was no clonal identification records.· `Parson Brown’ trees, irrespective of location, clone or rootstock had enhanced SAR activity as indicated by enhanced PR1 and PR2 gene expression. Statistically significant differences were always observed in the different locations during the March and December samplings. · PP2 gene downregulation was observed only in the `Parson Brown’ trees. This indirectly suggests an active protection mechanism in the phloem of the `Parson Brown’. Targeted RNA seq and metabolomics of the phloem would confirm these results and provide more insights into the exact mechanism of HLB tolerance. · All trees (`Parson Brown’ and `Hamlin’) evaluated in this study were HLB positive with similar CaLas titer. `Parson Brown’ trees however, maintained a good canopy and held on to fruit much better than comparable `Hamlin’ trees. · Oil content % ranged from 0.007 to 0.0011 in `Hamlin’ juice whereas, it ranged from 0.020 to 0.042 in `Parson Brown’ juice, depending on the location the fruit was harvested from. This could be due to clonal differences between trees grown in different locations and could help to differentiate between the different clones. · Lbs. Solids Per Box were comparable between `Parson Brown’ and `Hamlin.’ However, `Hamlin’ has recorded good Lbs. Solids Per Box in 2021, and samples will need be evaluated for multiple seasons to understand differences between the two early season sweet oranges. · Limonin levels were less than 5 ppm in both `Parson Brown’ and `Hamlin’ and within acceptable levels.· It was not possible to identify specific clones based on the data generated. Whole genome sequencing can shed insights into clonal differences by looking at the overall pattern of SNPs and INDELS among the specific selections. · `Parson Brown’ trees at a grower location in Lorida had the best growth, canopy coverage, followed by trees in Lake Wales, Sebring and Ft. Pierce. There were no significant differences in the oil content from fruit from these locations. It may be worthwhile to reintroduce a couple of these superior lines into the DPI’s parent tree program for the benefit of the citrus stakeholders.
Our project is examining phloem gene expression changes in response to CLas infection in HLB-susceptible sweet orange and HLB-resistant Poncirus and Carrizo (a sweet orange – Poncirus cross). We are using a recently developed methodology for woody crops that allows gene expression profiling of phloem tissues. The method leverages a translating ribosome affinity purification strategy (called TRAP) to isolate and characterize translating mRNAs from phloem specific tissues. Our approach is unlike other gene expression profiling methods in that it only samples gene transcripts that are actively being transcribed into proteins and is thus a better representation of active cellular processes than total cellular mRNA. Sweet orange, and HLB-resistant Poncirus and Carrizo (sweet orange x Poncirus) will be transformed to express the tagged ribosomal proteins under the control of characterized phloem-specific promoters; tagged ribosomal proteins under control of the nearly ubiquitous CaMV 35S promoter will be used as a control. Transgenic plants will be exposed to CLas+ or CLas- ACP and leaves sampled 30, 60, 90, and 120 days later. Ribosome-associated mRNA will be sequenced and analyzed to identify differentially regulated genes at each time point and between each citrus cultivar. Comparisons of susceptible and resistant phloem cell responses to CLas will identify those genes that are differentially regulated during these host responses. Identified genes will represent unique phloem specific targets for CRISPR knockout or overexpression, permitting the generation of HLB-resistant variants of major citrus cultivars.During the 1st quarter of our 1st 6 month no cost extension (the original end date was 11/30/2021; the current end date is 05/31/2022), the Stover lab has finally identified several high expressing lines of the p396 phloem-specific promoter in Poncirus. These will be shipped to the Rogers lab shortly, as soon as their BRS transgenic movement permit is renewed. APHIS has changed the on-line interface for applying for BRS movement permits and renewals are much delayed. At least 4 high expressing lines for each of the other 8 promoter/genotype combinations are in the containment greenhouse facility at Ft. Detrick and are being prepared for no-choice psyllid inoculation experiments.The Rogers lab has continued no-choice psyllid inoculation experiments on the many lines they have received from the Stover lab and continued ribosome pull-downs from the tissue collected. ARS facilities are still at a maximum of 25% occupancy due to the COVID-19 pandemic; we are teleworking the remaining time. This 25% occupancy cap will be lifted on Monday, March 28th, as long as the CDC community COVID metrics are in the ‘low’ category (which they are currently for Frederick County, MD). This will allow more rapid progress on grant milestones. Additionally, we are hopeful that the relaxed restrictions on in-person work will make our facility a more attractive place to work for potential post-doc candidates. Hiring a post-doc to help on the project will also allow more rapid progress on grant milestones.
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