The purpose is to evaluate the control effect of bactericides via trunk injection.Objective 1. 1.1. Determination of the in planta minimum bactericidal concentrations (MBCs) of bactericides against Las We developed a new method for evaluating the effects of oxytetracycline (OTC) treatment on Las titers in planta, and determined the relationship between OTC residue levels and control levels achieved for Las using mathematical modeling in greenhouse and field experiments. In greenhouse, OTC injection at 0.05 g/tree decreased Las titers to an undetectable level (Ct value = 36.0) from 7 to 30 DPA, and produced a residue level of OTC at 0.68-0.73 µg/g fresh tissue over this period. In the field, OTC injection at 0.50 g/tree resulted in the decline of Las titers by 1.52 log reduction from 14 to 60 DPA, with residue levels of OTC at 0.27-0.33 µg/g 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 Las achieved. The results suggested that the minimum concentration of OTC required to suppress Las populations in planta to below the detection limit is 0.68 and 0.86 µg/g, and the minimum concentration of OTC required for initial inhibition of Las growth in planta is approximately 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 Las in planta for effective control of citrus HLB. This study has been published by Phytopathology in a manuscript entitled: The in planta effective concentration of oxytetracycline against Candidatus Liberibacter asiaticus for suppression of citrus Huanglongbing. In addition, we evaluated the inhibitory activity of streptomycin (STR) against Las in a greenhouse experiment. Citrus trees were trunk-injected with STR, and leaves were inspected for Las populations and STR residues using qPCR and HPLC assays respectively, at various times after STR injection. Assays for Las titers and STR concentration in leaf samples from field trials are also ongoing. We are summarizing the data for publication and presenting the information to citrus growers. 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 (2, 3, 7 and 20 years old) with a different disease severity. For all the four field trials, the injection treatment applications were completed by the end of April 2019. The first application of spray treatments were completed during spring flushing in February or March 2019, and the second applications were conducted in late June to early July 2019. Leaf samples have been collected from the treated trees at the following time points: 0 (pre- injection), 7, 14, 28 days, 2, 4, and 6 months after treatment. 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 on May, 2019 (three months after the injection) and continued in a 3-months interval. Fruit yield and quality data were collected in January 2020.Objective 2. 2.1. Examination of dynamics and residues of bactericide injected into citrus and systemic movement within the vascular systemLeaf 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), 7, 14, 28 days, 2, 4, 6 and 8 or 9 months after injection. The samples are being processed for OTC or STR extraction, and the concentrations of OTC and STR in these samples were determined by HPLC assays. 2.2. Determination of the residue contents of bactericides in fruit and juice in each harvestFruit samples were collected during harvest in January 2020. The samples are being processed for OTC or STR extraction, and the concentrations of OTC and STR in these samples are being determined by HPLC assays.2.3. Analysis of degradation metabolites of bactericides injected into citrus treesLeaf 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 samples are being processed for the extraction of the degradation metabolites.Objective 3. 3.1. Greenhouse assays of the effect of bactericides via trunk injection on Las acquisition by ACPThis assay will be initiated in the spring of 2020.3.2. Field assays of the effect of bactericides via trunk injection on Las acquisition by ACPThis assay will be initiated in the spring of 2020.Objective 4. 4.1. Monitoring resistance development in Las against bactericidesLeaf 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 six and nine months after the injection. PCR-sequencing analysis on Las 16SrRNA gene showed there was no mutation compared with the reported sequence at the 6-month samples. 4.2. Evaluation of potential side effects of trunk injection of bactericides We evaluated possible phytotoxity caused by OTC or STR in immature trees (3-year old Valencia) from one week to one month after injection. The trees were be examined for the following symptoms: fruitlet drop, fruit drop, quantity of leaf drop, non-insect related leaf rolling, and leaf discoloration. There was no significant difference in fruitlet drop, fruit drop, quantity of leaf drop, or non-insect related leaf rolling between OTC or STR treatment and untreated control. About 20% (3 out of 15) trees injected with OTC or STR at the highest dose (2.0 g/tree) showed leaf discoloration (yellowing) on some young shoots. These phytotoxicity-like symptoms disappeared at 6 months post injection. There was no infection symptom by Phytophthora in the area surrounding drilling sites (injection holes), probably due to the application of the fungicide Ridomil gold immediately after drilling.In 7-year old Hamlin trees in the Auburndale grove, three trees injected with STR at the highest dose (3.0 g/tree) showed leaf discoloration (yellowing) on some young shoots, a possible phytotoxitic effect. These phytotoxicity-like symptoms disappeared at 6 months post injection. Other treated trees all showed normal growth.We are continuing the surveys for potential side effects.
Progress report for the first quarter of the 2019/2020 project yearThe purpose of the project is to develop new guidelines for restoring root health and improving overall tree nutrition for Florida oranges and grapefruit. The objectives of the project are to:1. Determine optimal nutrient concentrations in roots and leaves for multiple grapefruit and orange varieties.2. Compare and contrast fertigation, soil, and foliar fertilization to identify best application method for uptake of nutrients into both underground and aboveground components.3. Investigate the relationship between root and leaf nutrient contents to tree health, yield, and fruit quality as well as bacteria titer.4. Generate updated and new guidelines for optimal nutrient contents for roots and leaves for HLB-affected trees. Progress to date:The project is being conducted at three sites: Citrus Research and Education Center (CREC), Southern Gardens Citrus near Clewiston, FL and Indian River Research and Education Center (IRREC). Data collection continued in the first quarter of 2019/2020 particularly on soil, root and leaf nutrient concentrations, HLB disease ratings, tree physiological characteristics, and root growth and longevity. Data collection continues, and analyses will be done as more data is collected. First harvest of grapefruit was completed in Fort Pierce in January 2020 to determine fruit yield and juice quality. Root measurements, soil characterization, and canopy size determinations are completed every 6 months. Data analysis and comparison of early trends is under way. Early results show no significant differences in soil and tissue nutrient contents at the CREC and Clewiston sites for baseline samples collected in May 2019 but differences were observed in November 2019, where treatments providing 2x macronutrients and 2 to 4x micronutrients showed greater tissue and soil concentrations than standard practices but canopy size and yield did not differ across treatments in the first year. We will continue to monitor the trends over the coming months and report any observations accordingly. In terms of outreach, some of the project co-PIs, Dr. Rossi, Dr. Johnson, Dr. Ferrarezi and Dr. Kadyampakeni facilitated seminars at the Florida Citrus Show on January 22-23, 2020 in Fort Pierce, FL. Plans for Next QuarterThe team will continue with fertilizer treatments and data collection including yield and juice quality (at Lake Alfred and Clewiston sites), HLB rating assessments and root growth measurements and reporting on the progress of the project. Students working on the project will submit abstracts for presentation at the Florida State Horticultural Society Meeting in Sarasota, FL and the American Society of Horticultural Science in Orlando, FL.
This project is a continuation of previously funded CRDF grants to TWO BLADES focused on utilizing multiple This project is a continuation of previously funded CRDF grants to TWO BLADES focused on utilizing multiple strategies to produce canker-resistant citrus plants. The project has focused on transforming Duncan grapefruit with genes that express EFR or a gene construct designated ProBs314EBE:avrGf2 that is activated by citrus canker bacteria virulence factors. This project is a continuation of previously funded CRDF grants to TWO BLADES focused on utilizing multiple strategies to produce canker-resistant citrus plants. The project has focused on transforming Duncan grapefruit with genes that express EFR or a gene construct designated ProBs314EBE:avrGf2 that is activated by citrus canker bacteria virulence factors. Objective 1. To determine if Bs3-generated transgenic grapefruit plants are resistant to diverse strains of the citrus canker bacterium or to alternate target susceptibility genes in greenhouse experiments and to the citrus canker bacterium in field experiments in Fort Pierce. As stated in a previous report, the transgenic Duncan grapefruit containing the Bs3-executor transgene, designated JJ5, shows a high level of resistance to an array of strains representing a worldwide collection. Furthermore, using real time PCR, we have validated that the gene is activated by one or more TAL effectors and that there is minimal activation without these genes. Buds from the original transgenic tree were grafted onto two rootstocks (812 and Sour Orange) and planted in late March in the field at Fort Pierce in collaboration Dr. Ed Stover. Citrus canker has developed on plants in the field and the trees were rated for disease in September and there was considerable disease on all susceptible Duncan trees, but no evidence on the transgenic, JJ5. We have also identified two other possible transgenics from plants received from Dr. Vladimir Orbovic. Both, and an additional one which has since been identified, responded to infiltration with a high concentration of bacterial cells by exhibiting a hypersensitive reaction within 4 days of infiltration. One of the transgenics appeared to have a growth defect, but recently has developed normal shoots. All three transgenic trees contain the avrGf2 gene (based on PCR for detection of avrGf2). These transgenics were grafted onto rootstock and are in various stages (i.e, some of buds have broken and the shoots are developing while others are still dormant). During the past three months we have placed our constuct in a different vector that is acceptable for future transgenic purposes. The previous constructs contain an additional selectable marker that allowed for identifying putative transgenics with a higher success rate that contained the targeted construct. Given that there was concern about the additional marker, the new construct contains only NPT as a selectable marker. The construct was sent to Vladimir Orbovic, who has developed 45 putative grapefruit and sweet orange transformants. We have screened these via PCR and there was one weak positive. The transgenic was grown and inoculated with Xcc and found to be susceptible. Objective 2. To determine if EFR-generated transgenic grapefruit plants are resistant to the citrus canker bacterium in field experiments in Fort Pierce. We have grafted our two most promising EFR transgenic plants (based on ROS activity) onto two rootstocks (812 and Sour Orange) and planted them in the field at Fort Pierce in collaboration Dr. Ed Stover. They were planted in the field in late March. There was some citrus canker on the trees, although they were not uniformly infected. In September the trees were rated for disease severity and the transgenics carrying EFR had considerably more disease than the susceptible wild-type Duncan grapefruit. We have identified additional transgenics from plants received from Dr. Vladimir Orbovic that that have been grafted onto rootstocks. These will be tested for ROS activity and for EFR gene expression. Objective 3. To determine if bs5-generated transgenic Carrizo plants are resistant to X. citri and to generate transgenic grapefruit carrying the pepper bs5. We have recently received budwood from UC Berkeley. The budwood was from two transgenic events and a third was from a tree that was run through the transformation process but that was negative for the gene, serves as budwood that had undergone the transformation process but that was negative for the transgene. This will serve as a negative control.
The objective of this project is to compare commercial adjuvants effects on systemic delivery of oxytetracycline (OTC) and streptomycin (Strep). Implementing this objective involves two experiments: One testing effects on delivery of OTC and one testing effects on delivery of Strep. Because the compound is expected to behave differently (based on preliminary experiments and based on recommendations of the company marketing both ingredients), we will use a different set of adjuvants for each, with some overlap. We have made three modifications to the original plan: 1.) We included injection as a positive control, because effective delivery has been reported through trunk injection. 2.) We included two experimental adjuvants in addition to all of the adjuvants recommended by AgroSource for the OTC study. 3.) We chose to implement this experiment on CLas infected trees in the field, because results using uninfected trees could be questioned on the basis that delivery might be affected by infection status. All of these changes expand, rather than reducing, the results and potential impact.
We have acquired all the adjuvants and antimicrobials materials needed and designed both experiments. We have implemented the first experiment, addressing delivery in December 2019 and have collected the materials for testing, and quantification of OTC in the samples is in process. We will use the results from the first experiment to assess experimental design for the Strep study. We expect to have complete results from the OTC study in Q2. We rate our % objective completion at 25% because we have completed approximately ½ of the objective to address OTC, which could be considered ½ of the complete objective.
Fall 2019 nematicide treatments were initiated in September and continued throughout October. Effects on nematode populations were measured (as previously described) in December. Randomly assigned pairs of the new materials were rotated (one of each pair in spring, the other in autumn) to comply with annual rate limits and to reduce the occurrence of pesticide resistance in the sting nematode populations. Oxamyl and aldicarb were not rotated with other materials. Oxamyl was applied twice in each of spring and fall. Aldicarb was applied just once in April, per label requirements prior to deregistration. All nematicides except oxamyl (which was sprayed by hand during the final third of the irrigation cycle) were injected for two hours, beginning 30 minutes after irrigation began and ending a half hour before the irrigation run ended. This injection period was double that used in the spring applications and it appears to have produced more favorable results. One pair of new products reduced sting nematodes by 67%-84% (depending on the order of treatment) and the other pair by 19%-47%. Oxamyl reduced the nematodes by 62% and aldicarb had no effect on the sting nematode population. Growth of the trees as measured by trunk cross sectional area between February and December was greatest (P=0.05) for the oxamyl treatment. Growth of trunks in no other treatment was significantly different from the control; however there was an inverse linear relationship (P=0.02) between trunk growth and the average sting nematode population density (log-transformed) for each treatment measured in June and December 2019. Numbers of fruit on the young trees and dropped fruit were counted in November. Although fruit count was positively related to trunk diameter (r= 0.63, P=0.000), there was an pronounced inverse relationship between fruit number and trunk growth during the 2019 season (n=224, r= -0.63, P=0.000). Fruit count was unrelated to nematode population density.
In the perennial peanut trial the trunk girth in November was inversely related to the sting nematode population density measured the previous summer in the row middles. However, ANOVA detected no effect of cover crop on trunk girth. Although root mass density was four-fold in peanut compared to native vegetation in both July and December, the sting nematode population in peanut was just a third of that in native vegetation during 2019 (P=0.04). Unlike in the nematicide comparison trial above, oxamyl did not reduce the sting nematodes measured in December in the tree row. Possibly, the lower population density (by two thirds) in the peanut trial compared to the nematicide trial obscures effects of management on population density. Fruit count was related (r=0.93) to the trunk girth, but was not affected by cover crop or oxamyl.
This project is an continuation of an objective of existing CRDF funded project (# 00124558 ; ended in March 2019, final report submited to CRDF) with some added treatments to be evaluated in comparison to control (dry conventional fertilizer with foliar micronutrients). Objective 1 which is the continuation of # 00124558 included 10 treatments.
The added treatments from objective 2
1. CRF + Tiger Micronutrients+ Mn 50%
2. CRF + Tiger Micronutrients+ Zn 50%
3. CRF + Tiger Micronutrients+ Fe 50%
4. CRF + Tiger Micronutrients+ B 50%
5. CRF + Tiger Micronutrients+ Mn +Zn 20%
6. CRF + Tiger Micronutrients+ Mn +Fe 20%
7. CRF + Tiger Micronutrients+ Zn +Fe 20%
8. CRF + Tiger Micronutrients+ Zn +B 20%
9. CRF + Tiger Micronutrients+ Fe + B 20%
10. CRF + Tiger Micronutrients+ Mn +Zn 50%
11. CRF + Tiger Micronutrients+ Mn +Fe 50%
12. CRF + Tiger Micronutrients+ Zn +Fe 50%
13. CRF + Tiger Micronutrients+ Zn +B 50%
14. CRF + Tiger Micronutrients+ Fe + B 50%
The treatment for objective 3:
1.CRF + Foliar Micronutrients + Tiger 90;
2.CRF + Tiger Micronutrients
So altogether currently there are 25 treatments of citrus nutrition that are being compared to control.
Within this quater the fertilizer application was made for the third round and final data collection for the year 2019 was done.
The results of this trial were presented at 4 nutrition events in month of October and November. In addition a citrus industry article highlighting the finding of the first tial was submitted for Febraury.
Currently, we are getting prepared for the harvest in March-April of 2020.
This a fertilizer evaluation trial and the progress on it is timely and as per expectations.
The proposed field trial was established in a commercial citrus grove on 22 acres of a typical SW Florida flatwoods-type site in Hendry County (Lat/Lon: 26° 34′ 28.3518″ N, 81° 30′ 34.3772″ W). A total of 3232 trees were planted in 32 rows on 16 beds, each separated by furrows, at a spacing of 12 ft within rows and 25 feet between rows (145 trees per acre). Each row contained 101 trees. Trees were composed of Valencia scion (clone 1-14-19) on four different rootstocks: 1) X-639, a high-vigor inducing cultivar; 2) US-802, a high-vigor inducing cultivar; 3) US-812, a moderate-vigor inducing cultivar; and 4) US-897, a low-vigor inducing cultivar. Except for US-802, which is a pummelo × trifoliate hybrid, rootstocks are mandarin × trifoliate hybrids. Trees were arranged in a randomized split-plot design with treatment (compost or no compost) as the main plot and rootstock (X-639, US-802, US-812, US-897) as the subplot. Plots were arranged in eight blocks (16 beds) across the experimental site with each block containing two beds either treated with compost or without compost. Each bed contains 200 experimental trees, 100 per row, arranged in sets of 50 trees of each rootstock cultivar and separated by a non-experimental tree in the center of each row. OMRI certified compost (Green Care Recycling, Ft Myers, FL) was applied at a rate of 5 tons per acre and tilled into the soil. A subset of trees in each experimental unit was selected and tree heights and trunk diameters (above and below the graft union) were measured. Soil samples were collected from each experimental unit and divided for nutrient analysis and soil microbial analysis.
Objective 1: We hypothesized that bactericidal treatment will protect young trees from CLas colonization.
Initial leaf samples were collected prior to treatments to evaluate CLas titers in the uninfected trees. Bactericidal treatments were applied from May through December. CLas titer was monitored in leaf tissue in response to antibiotic treatments using quantitative real-time PCR analysis. In this report, the results of the CLas-infection rate in citrus leaves from May to July are described. Currently, citrus leaves tissue samples from August through December are being processed to analyze the CLas-infection rate.Trees were considered CLas-infected (positives) when CT values were below 35 (CN= Copy number).
1. Bactericides (monthly rotation): Prior to bactericide application (May), 15% of trees (20 trees/treatment) were CLas positive (Ct<35) and the overall CT mean of the treatment was 34.5. After the bactericide application (June), 35% of trees were CLas positive (Ct<35) and the overall CT mean of the treatment was 35.1. After the second application (July), 65% of trees were CLas positive (CT<35) and the overall CT mean of the treatment was 33.6. However, CLas titers decreased 5.94-fold from May (CP = 4096) to July (CP = 690).
2. Bactericides (quarterly rotation): Prior to bactericide application (May), 100% of trees were CLas negative (Ct>35) and the overall CT mean of the treatment was 39.2. After the bactericide application (June), 40% of trees were CLas positive (Ct<35) and the overall CT mean of the treatment was 34. After the second application (July), 80% of trees were CLas positive (CT<35) and the overall CT mean of the treatment was 34.1. However, CLas titers decreased 9.05-fold from June (CN = 1277) to July (CN = 141).
3. Negative Control (insecticide + Tree defender exclusion netting): Prior to bactericide application (May), 100% of trees were CLas negative (Ct>35) and the overall CT mean of the treatment was 38.6. After the bactericide application (June), 45% of trees were CLas positive (Ct<35) and the overall CT mean of the treatment was 34.7. After the second application (July), 85% of trees were CLas positive (CT<35) and the overall CT mean of the treatment was 33.7. Additionally, CLas titers increased 1.06-fold from June (CN = 205) to July (CN = 218).
4. Positive Control (insecticide only): Prior to bactericide application (May), 100% of trees were CLas negative (Ct>35) and the overall CT mean of the treatment was 39.1. After the bactericide application (June), 5% of trees were CLas positive (Ct<35) and the overall CT mean of the treatment was 36.1. After the second application (July), 55% of trees were CLas positive (CT<35) and the overall CT mean of the treatment was 34.5. Additionally, CLas titers increased 12-fold from June (CN = 14) to July (CN = 168).
Enumeration of ACP adults using taps was conducted bi-weekly from May through December, the presence of other life stages such as eggs and nymphs were scouted visually. Preliminary results showed a low ACP population in citrus locations due to the active vector management performed by the farm manager. As a consequence, no ACP adults were collected to analyze the CLas-infection rate using quantitative real-time PCR analysis. The overall number of eggs and nymphs were low or undetectable in citrus trees from May to December.
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 1, 2, 4, 8, and 12 weeks 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 4th quarter of the first year of our grant, the post-doctoral researcher, Tami Collum, continues to optimize nucleic acid extraction protocols for citrus. She traveled to the Stover lab to learn their citrus propagation and infection protocols. The Stover lab continues Agrobacterium-mediated transformation of seedling epicotyls from all three citrus genotypes (Carrizo, Poncirus and Hamlin sweet orange) with the His-FLAG tagged RPL18 (ribosomal protein L18) under the 35S promoter and all three phloem promoters pSUC2, pSUL and p396ss. Carrizo transgenic plants with three promoters (p35S::HF-RPL18, pSUL::HF-RPL18, and p396ss::HF-RPL18) have been shown to be expressing the transgenic RPL18 by qRT-PCR and transferred to Ft. Detrick. Carrizo with pSUC2 and Poncirus transformants are close behind with multiple lines in soil and ready to ship to Ft. Detrick in January. Hamlin transformation was intensified in last quarter and now many putative transformants are in rooting media. They will be transferred to soil early next quarter before expression testing and transfer to Ft. Detrick. For objective 6 (Additional Approach: Phloem limited citrus tristeza virus vectors will be used to express the His-FLAG-tagged ribosomal protein in healthy and CLas infected citrus) Dr. Dawson’s lab has moved all necessary constructs into citrus. CTV-infected plants will be shipped to Maryland in January.
The objectives of this study are to identify optimal pH range for root function and minimize root turnover on HLB-affected rootstocks and how uneven pH levels in the root zone (e.g. irrigated vs. row middle portions of root system) affect the overall health of the tree. This is being done in a split root system in the greenhouse where pH of different parts of the root system can be controlled an maintained.
Trees for the first repetition of the experiment have been inoculated and regular pictures of roots in the rhizotrons are being taken and root tracings are being done on the controls and trees that have tested qPCR postive. A new individual side drip irrigation system has been developed to make irrigation and leachate collection less prone to error and more efficient. Currently about 20% of inoculated trees are qPCR positive for Las, so not quite enough are positive yet for statistical comparisons of treatments.
Because of the difficulty in developing a buffer system across the entire desired pH range that does not alter plant physiology, we have changed to using CREC well water and adjusting it’s pH with sulfuric acid. The well water provides good buffering capacity from pH 7.5 to 5.5 and this more accurately represents what occurs in grower groves.
We are currently testing methods to collect and quantify root leakage non-destructively in the rhizotron system that should provide additional information on root health with HLB and soil/irrigation water pH.
In the first year of this project (4th quarter), we evaluated the performance of a ground penetrating radar (GPR) to map root architecture of HLB-infected citrus trees, investigated the influence of several factors on the accuracy of the GPR system and developed an enclosure unit for the GPR for remote control of the system. The use of GPR system was a non-destructive approach to map tree roots without having any effect on the roots and the root-soil envronment. It can provide a rapid technique for root mapping. The GPR system consisted of two ground penetrating radar units with frequencies of 900 MHz and 1,600 MHz (TRU Model, Tree Radar Inc., USA) connected to a mobile scanning cart that had a control unit mounted on it which collected the data being sent from the radar and generated root morphology and root density maps. Additionally, a commercial software (TreeWin roots) was used to generate 3D root maps. In the initial evaluation of the system, we found that the GPR unit with 1,600 MHz frequency was more accurate in detecting root location and depth than the GPR unit with 900 MHz frequency. We have also found that the GPR system can distinguish between live and dead roots which is a very important aspect for studying the effects of diseases on citrus tree root systems.
In the first phase, we have investigated the influence of several factors on the accuracy of the GPR system. These factors were: (i) GPR Frequency; (ii) root diameter; (iii) root moisture level; (iv) root depth; (v) root spacing; (vi) survey angle; and (vii) soil moisture level. Experiments were conducted at the citrus research grove of the University of Florida Southwest Florida Research and Education Center (SWFREC) in Immokalee, USA. Upon conducting these experiments, we have found that the GPR can detect a root successfully when the root diameter is more than 6 mm. If two or more roots are in close proximity, the GPR system can only distinguish between them when the distance is more than 10 cm. We summarized all the results of these experiments and prepared a manuscript which we submitted for peer-review to the Agronomy Journal, Special Issue of Precision Agriculture. It was accepted and published:
“Zhang X., Derival M., Albrecht U., and Ampatzidis Y., 2019. Evaluation of a Ground Penetrating Radar to Map Root Architecture of HLB-infected Citrus Trees. Agronomy (Special Issue: Precision Agr.), 9(7), 354. https://doi.org/10.3390/agronomy9070354.”
Received: 3 May 2019 / Revised: 23 June 2019 / Accepted: 1 July 2019 / Published: 3 July 2019.
We have acknowledged CRDF: “Funding: This research was funded by the University of Florida Citrus Initiative and the Citrus Research and Development Foundation.”
In the second phase, we started developing an automated platform for the GPR system. In the current conditions, the operator has to manually go around the trees making very accurate 360-degree peripheral scans while maintaining a constant distance from the tree trunk. This process is time consuming and prone to manual measurement errors. The automated platform will help in eliminating this manual measurement errors thereby increasing the accuracy of the system and also reducing the amount of time taken for data collection. For the first prototype, we designed and built an enclosure unit for the radar unit which will allow the operator to control it remotely. We used 3D printed wheels which were connected to dual channel motor drivers; these motor drivers are remotely controlled by an 8-channel transmitter. Although the enclosure unit worked well on a regular terrain, it failed to make smoother movements of the soil. Hence, we worked on building a different enclosure unit; this time with an agile tracked chassis that is capable of efficient movement on even an irregular terrain. The new enclosure unit works well and can be efficiently used for the remote control of the GPR. We are currently working on developing the adjustable arm that can be used to connect the GPR to the trees which will help it make almost perfect 360-degree peripheral scans around the tree.
Our next objectives are:
(i) To complete the development of adjustable arm connection for GPR to citrus trees.
(ii) Evaluate the remote-controlled prototype in the field and compare it to the manual operation of the GPR system.
Objective 1 – Ongoing.
Objective 2 – Determine the phytotoxic levels of Fe + organic acid solutions on citrus.
Preliminary range experiments were started using F11 to determine the concentration where leaf burn occurs. F11 is the Japanese Fe + citric acid product developed from the patent. In discussions with the Japanese inventors (who visited Florida to see the project), we just learned that another product, F11-C, is the product that was developed from this patent specifically for citrus. Therefore, we have modified all experiments, greenhouse and field, to use F11-C rather than F11. Aichi Steel, the manufacturer of F11 and F11-C, have provided us with all the product required for the project.
Objective 3 – Determine the effect of Fe2+ + organic acid solutions on HLB titer using a rapid greenhouse, HLB-infected citron, rooted shoot bud assay.
Being modified. Originally, we planned on using HLB-infected citron bud cuttings because they show clear HLB symptoms, but are still able to grow. This is unlike HLB-infected grapefruit that can barely be kept alive. However, in our experiments to characterize the “citron system” we have been able to detect very little growth differences between HLB-infected vs healthy citron plants. This means that treatment effects on HLB-infected plants cannot be determined, only the general effects on plant growth. We are working on another system based on very young sweet orange plants and psyllid inoculation where the preliminary experiment looked very promising. The HLB effect was very strong, the requirement for a good system to test treatments. The validation experiment is now being rerun at a larger scale. If it validates, then we will use this system on the objective 3 (and objectives 4 and 5) experiments where it should work quite well.
Objective 4 – See objective 3 discussion.
Objective 5 – See objective 3 discussion.
Objectives 6 and 7 – These are the field tests on mature and young trees. The trees have been planted, baseline ground and aerial data taken, and treatments initiated.
Progress report for the fourth quarter of the 2018/2019 project year
The purpose of the project is to develop new guidelines for restoring root health and improving overall tree nutrition for Florida oranges and grapefruit. The objectives of the project are to:
1. Determine optimal nutrient concentrations in roots and leaves for multiple grapefruit and orange varieties.
2. Compare and contrast fertigation, soil, and foliar fertilization to identify best application method for uptake of nutrients into both underground and aboveground components.
3. Investigate the relationship between root and leaf nutrient contents to tree health, yield, and fruit quality as well as bacteria titer.
4. Generate updated and new guidelines for optimal nutrient contents for roots and leaves for HLB-affected trees.
Progress to date:
The project is being conducted at three sites: Citrus Research and Education Center (CREC), Southern Gardens Citrus near Clewiston, FL and Indian River Research and Education Center (IRREC). Data collection continued in the third quarter particularly on canopy size, soil and leaf nutrient concentrations, HLB disease ratings and root growth and longevity. Data collection continues, and analyses will be done as needed. Updates and data will be presented in future extension meetings after about a year or two of data collection and validation of results to get feedback from growers and the citrus industry.
In addition to one graduate student working on the project at CREC, a second graduate student started working on the project this summer at the IRREC.
At the start of fall 2019, an agricultural assistant was recruited at IRREC to help with data collection, and application of treatments.
In terms of outreach, some of the project co-PIs, Dr. Rossi and Dr. Johnson participated in the “International Root Workshop” in August 2019 that involved international root experts and several graduate students. Also, Drs. Ferrarezi and Dr. Kadyampakeni facilitated 4 statewide workshops on Citrus Nutrition conducted at CREC (October 8, 2019), IRREC (October 23, 2019), Southwest Florida Research and education Center (SWFREC, October 8, 2019) and Sebring (October 8, 2019). Finally, Drs. Kadyampakeni, Ferrarezi and Johnson also presented portions of their work on citrus nutrition and root health at the Materials, Innovation and Sciences in Agriculture (MISA) Conference (October 24-25, 2019) in Orlando, Florida. Two graduate students working on the project presented their proposed work at the South Florida Graduate Student Symposium.
Plans for Next Quarter
The team will continue with data collection including yield and reporting on the progress of the project.
The purpose is to evaluate the control effect of bactericides via trunk injection.
Objective 1.
1.1. Determination of the in planta minimum bactericidal concentrations (MBCs) of bactericides against Las
We developed a new method for evaluating the effects of oxytetracycline (OTC) treatment on Las titers in planta, and determined the relationship between OTC residue levels and control levels achieved for Las using mathematical modeling in greenhouse and field experiments. In greenhouse, OTC injection at 0.05 g/tree decreased Las titers to an undetectable level (Ct value ≥ 36.0) from 7 to 30 DPA, and produced a residue level of OTC at 0.68-0.73 µg/g fresh tissue over this period. In the field, OTC injection at 0.50 g/tree resulted in the decline of Las titers by 1.52 log reduction from 14 to 60 DPA, with residue levels of OTC at 0.27-0.33 µg/g 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 Las achieved. The results suggested that the minimum concentration of OTC required to suppress Las populations in planta to below the detection limit is 0.68 and 0.86 µg/g, and the minimum concentration of OTC required for initial inhibition of Las growth in planta is approximately 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 Las in planta for effective control of citrus HLB. This study has been published by Phytopathology in a manuscript entitled: The in planta effective concentration of oxytetracycline against Candidatus Liberibacter asiaticus for suppression of citrus Huanglongbing.
In addition, we evaluated the inhibitory activity of streptomycin (STR) against Las in a greenhouse experiment. Citrus trees were trunk-injected with STR, and leaves were inspected for Las populations and STR residues using qPCR and HPLC assays respectively, at various times after STR injection. Assays for Las titers and STR concentration in leaf samples from field trials are also ongoing.
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 severity
The 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 (planted in 04/2016); one in Bartow, FL, 2-year old W. Murrcot trees (planted in 01/2017, “CUPS”); 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 (planted in 03/1999). 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 first application of spray treatments were completed during spring flushing in February or March 2019, and the second applications were conducted in late June to early July 2019. Leaf samples have been collected from the treated trees at the following time points: 0 (pre- injection), 7, 14, 28 days, 2, 4, and 6 months after treatment. 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 on May, 2019 (three months after the injection) and continued in a 3-months interval.
Objective 2.
2.1. Examination of dynamics and residues of bactericide injected into citrus and systemic movement within the vascular system
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), 7, 14, 28 days, 2, 4, and 6 months after injection. The samples are being processed for OTC or STR extraction, and the concentrations of OTC and STR in these samples will be determined by HPLC assays. Then, the distribution of bactericides in different plant tissues will be compared in terms of translocation time and dosage.
2.2. Determination of the residue contents of bactericides in fruit and juice in each harvest
This study will be initiated during fruit harvest.
2.3. Analysis of degradation metabolites of bactericides injected into citrus trees
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 samples are being processed for the extraction of the degradation metabolites.
Objective 3.
3.1. Greenhouse assays of the effect of bactericides via trunk injection on Las acquisition by ACP
This assay will be initiated in the spring of 2020.
3.2. Field assays of the effect of bactericides via trunk injection on Las acquisition by ACP
This assay will be initiated in the spring of 2020.
Objective 4.
4.1. 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 six months after the injection. DNA extraction from these samples is ongoing.
4.2. Evaluation of potential side effects of trunk injection of bactericides
We evaluated possible phytotoxity caused by OTC or STR in immature trees (3-year old Valencia) in the Avon Park grove from one week to one month after injection. The trees were be examined for the following symptoms: fruitlet drop, fruit drop, quantity of leaf drop, non-insect related leaf rolling, and leaf discoloration. There was no significant difference in fruitlet drop, fruit drop, quantity of leaf drop, or non-insect related leaf rolling between OTC or STR treatment and untreated control. About 20% (3 out of 15) trees injected with OTC or STR at the highest dose (2.0 g/tree) showed leaf discoloration (yellowing) on some young shoots. These phytotoxicity-like symptoms disappeared at 6 months post injection. There was no infection symptom by Phytophthora in the area surrounding drilling sites (injection holes), probably due to the application of the fungicide Ridomil gold immediately after drilling.
In 7-year old Hamlin trees in the Auburndale grove, three trees injected with STR at the highest dose (3.0 g/tree) showed leaf discoloration (yellowing) on some young shoots, a possible phytotoxitic effect. These phytotoxicity-like symptoms disappeared at 6 months post injection. Other treated trees all showed normal growth.
We will continue the surveys for potential side effects.
Trunk growth between February and June of trees in the nematicide trial did not differ significantly. However, growth rates for all nematicide treatments were nominally higher than for the untreated trees. Growth for a Syngenta experimetal nematicide was 40% greater than the control, and the oxamyl, aldicarb, fluopyram and, fluazaindolizine were between 17-20% greater than the control. There was a weak inverse relationship (P = -0.09) between the trunk growth and the May sting nematode populations, measured following the nematicide treatments. The root weights ranged between 0.28 and 0.37 mg dw/g soil fw and did not differ between treatments. Sting nematode populations were significantly lower (P = 0.04) in perennial peanut plots (6 nematodes/250 cc soil) than in control plots (23 nematodes), 5 months after laying the peanut sod. Fall 2019 nematicide treatments were initiated in September and will continue throughout October. The materials are being injected for two hours, begining 30 minutes after irrigation begins. The plots are irrigated for a further 30 minutes following the nematicide applications.
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