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 1st quarter of the second year of our grant, the Stover lab continues producing transgenic plants and shipping the ones that are ready to Ft. Detrick. CTV-infected plants were shipped to Ft. Detrick in February and are expressing nicely. This has allowed the post-doctoral researcher, Tami Collum, to finish optimizing citrus nucleic acid extraction protocols and perform immunoprecipitation and extraction of high quality translatome RNA from p35S::HF-RPL18 and pSUL::HF-RPL18. Another shipment of transgenic plants is anticipated in the 2nd quarter.
During this 5th quarter of the Project our work continued without problems. Objective 1: We continued monitoring parameters including tree trunk diameter (rootstock and scion) and canopy areas. Some differences are starting to emerge in trunk diameter, although not statistically significant yet: Covered trees are starting to show bigger diameters in both rootstock and scion. All IPC-covered trees are still HLB-negative. We are continuing documenting canopy area expansion by photography and also by leaf are index measurements after replacing the old 4-ft IPCs with new 8-ft covers, donated by The Tree Defender, Inc. Objective 2. We have finished planting the 700 trees of SugarBelle, Tango and Early Pride mandarins, and we started to monitor regularly tree parameters (trunk diameter, and leaf sampling, for CLas, chlorophyll and sugar analysis). Objectives 3 and 4. We set up blooming and fruit development experiments this season inside the IPCs and in our CUPS facility, which is now fully operative. We have performed deficit irrigation to induce blooming in both IPC and CUPS this season. We are currently collecting data on these experiments. Outreach, Professional Presentations and Extension Activities for this quarter : – A CUPS Day.CUPS, mini-CUPS and other strategies to manage HLB. Talk on “Individual Protective Covers” . SWFREC, was delivered on Dec 17. 45 people registered, 44 attended. Impact: 100% of the attendants found the presentation useful and manifested a gain in knowledge. Half of the attendants will improve their management practices, which, in this case, may mean to adopt IPCs. In addition, 32 CEUs and 10 CCAs were certified. -As a follow up of the International invited seminar at IVIA, Valencia, Spain entitled “Living with HLB. The new reality of Florida Citriculture” that was presented by Fernando Alferez last December 4, 2019, the spanish professional magazine Phytoma published an article showcasing, among others, our IPC work. It can be seen here:https://www.phytoma.com/noticias/noticias-de-actualidad/florida-conviviendo-con-el-hlb-y-los-huracanes -Citrus Extension Agents presentation on “The Citrus Horticulture Program at SWFREC”. Immokalee, January 28, 2020. As a result of this presentation, I have been invited to deliver these presentations in the next months: -“Individual Protective Covers”. O.J Break Series, Highlands Co. Extension Office. March 20, 2020. -“Individual Protective Covers” In Service Training invited speaker. CREC, Lake Alfred, April 8-9, 2020.
In the fifth quarter of this project, we started developing a new calibration system to get a better estimation of the depth of the roots when compared to the existing calibration methods. The existing calibration methods use dielectric constant measured using soil type and soil moisture level to get the depth of the roots. This new method combines dielectric constant calibration in the field with the post-processed data that is extracted in the lab. This method uses two different dielectric constant values at different depths, one at a deep level and another at a shallow level. These both values will be used to correct the GPR estimated depth of the root and increase the accuracy of root depth estimation. For evaluating this calibration method, we conducted field experiments at SWFREC citrus grove to determine: (i) effect of roots diameter on GPR depth measurement, (ii) effect of dielectric constant on GPR depth measurement and (iii) effect of root depth on GPR depth measurement. Upon performing these experiments, it is determined that the accuracy of depth of shallow roots was better than the depth of roots present at a deeper level and also, the new calibration method can be used to improve the accuracy of depth estimation for roots present at a level closer to the ground. We have found that this is due to the dielectric constant value being constant at shallow levels, because there is almost no change in the soil moisture content. However, as the depth increases, the soil moisture content rises and the dielectric constant increases, leading to the use of average value of all the changing dielectric constant values at deeper levels. All the results and discussion of these experiments are being prepared into a manuscript and will be reviewed further.We are also working on the development of an adjustable arm connection for the GPR to citrus trees. Several 3D modelled arms have been designed and are being tested to improve the functionality of the arm. The important functionality points we are considering implementing in the arm development is that (i) the arm fits all different tree sizes varying from young to old trees. (ii) the arm contains a measurement scale that can adjusted and fixed so that the 360 degree rotation around the tree remains constant at all time. In the coming quarters, we will:1) Develop further the new calibration method to improve the accuracy of depth estimation.2) Review the above mentioned manuscript containing results and discussion of the new calibration method and submit it for publication.3) Continue the development and testing of the automated GPR system to increase the efficiency of the system.
We have set up all of the experiments in the greenhouse and field needed to accomplish those objectives. We have also set up experiments in paralell with the field experiments. Given that citrate is the preferred carbon source for Liberibacter and that plants load citrate into phloem in order to acid mine insoluble phosphate from the soil, we expect foliar phosphate fertilization to reduce citrate in phloem so drastically, that it starves CLas.
We now know that foliarly-applied potassium phosphate will decrease phloem citrate levels in half after just one month of treatment compared to citrus saplings that were fed calcium phosphate to the roots. We are now analyzing samples to see whether citrate levels continue to decline after three months. We will keep this experiment going for the length of the project and will sample phloem citrate levels every three months.
The first greenhouse experiments on needed phosphate levels taught us the appropriate levels of potassium phosphate to spray on citrus trees in the field. In April 2019, a field trial commenced in a grove o 20-year-old infected trees near Lake Hamilton in Polk County. There were 10 replicates and four treatments in a randomized complete block design. We spray the trees with 0, 1x, 3x, and 9x the optimal level observed in the greenhouse every two months. Even at 1x, the plants are receiving enough P for flushing and fruit development. The plants are sprayed six times a year including after each flush. We sample these trees for phloem CLas levels every three months. The samples from the first nine months are now being analyzed. We expect to see CLas titer declines in the foliar phosphate-treated plants after one year of treatment.
A second field trial was established in August 2019 in the Immokalee area. That trial is the same design as that in Polk County. The idea was to have a trial on the ridge (Polk County) and flatwoods (Collier County) regions of citrus production. In both trials, the outcomes being measured are CLas titer in leaf midribs and leaf area index. Baseline samples were taken prior to the first spray and subsequent sampling will be done at 6, 12, and 18 months after the first spray. We have now sampled these trees for the first six months after treatment.
As we wait for field results, we are now testing the effect of a foliar potassium phosphate spray (compared to root-applied calcium phosphate) on CLas titer in graft-infected trees in the greenhouse. We are assaying those plants for CLas titer now and expect to have results in a week. This experiment will tell us whether foliar phosphate treatments can prevent CLas infection.
Given the speed with which foliar potassium phosphate can reduce organic hose plants acids levels in citrus phloem, we expect to see positive results in the field in reducing CLas titer in the first quarter of 2020. I am pleased to report that this project is working as planned so far. Foliar potassium phosphate does reduce citrate levels and the levels of other organic acids in phloem. As organic acids, particularly citrate, are the preferred carbon source for Liberibacter crescens, we expect this treatment to starve the pathogen.
We expect to observe CLas titer declines in these experiments over the next two months. If we do, a manuscript will be prepared very quickly in order to disseminate our results as quickly as possible. We will also ask CRDF for help in identifying more field locations for this work.
An extension article will follow shortly thereafter.
Our team (Triplett, Vincent, Killiny, and Wang) are working very well together and meet to discuss the project every two weeks.
In this quarter, we received permission to continue much our work on this project during the pandemic shutdown. All experiments in the greenhouse and field continue to be maintained.
We also obtained exciting results from our greenhouse experiment where Nabil Killiny graft-inoculated citrus saplings with CLas. We then applied the nutritional treatments to this plants to test the notion that leaf fertilization of potassium phosphate would reduce CLas titer in the leaf midribs compared to root fertilization with calcium phosphate.
Attached with this report are the data. With potassium phosphate fertilization of leaves, the propotion of unfected plants after 3 and 6 months is 80% and 86.7%, respectively. Plants fed calcium phosphate to the roots (analogous to field plants receiving no phosphate fertilizer) had only 40% and 38.5% of unifected plants at 3 and 6 months, respectively. Control plants given neither fertilizer were 26.7% and 38.5% uninfected at 3 and 6 months.
In contrast, after 6 months, 23.1%, 13.3%, and 0% of the highly infected plants were given leaf, root, or no fertilizaiton. Highly infected plants did improve over time with the foliar potassium phosphate fertilization.
So the greenhouse experiments have confirmed our hypothesis, foliar phosphate fertilization prevents and alleviates CLas infection. Treating plants with the equivalent of rock phosphate that is found in Florida citrus groves (calcium phosphate) encourages infection and it gets worse over time.
Now we need the field confirmation of these results. We were in the process of doing qPCR measurements of CLas leaf titer from our two field experiments, when we were ordered to stop data collection and reduce staff in the lab. This happened about the same time as the state-wide stay-at-home orders.
We expect to be able to get back to the qPCR measurements by mid-May.
We are also continuing with our greenhouse experiment on the effects of foliar phosphate treatments on citrate levels in phloem. Again, our efforts on this were curtailed on this with the shutdown. But we will be back at it soon.
All field and greenhouse experiments are on-going. Nothing was lost during the shutdown.
I submitted a preproposal to the USDA to expand our phosphate trials to 20 field experiments over the current two. That preproposal also includes a new disease model. I am eager to share those results with CRDF when the experiments are done.
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.
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