After just a year of support for this project, we have not yet completed any of our objectives. However, we have set up all of the experiments in the greenhouse and field needed to accomplish those objectives.
A greenhouse trial over the winter months taught us the level of foliarly-applied potassium phosphate needed to maintain proper P nutrition in citrus. A second greenhouse trial was established to determine whether foliarly applied potassium phosphate would decrease citrus levels in phloem compared to the application of calcium phosphate (i.e. rock phosphate). By September of this year, it was clear that just one month of treatment with foliar potassiumphosphate decreased organic acids level in phloem (citrate, malate, and alpha-ketoglutarate) by more than half compared to trees whose roots were treated with calcium phosphate.
The first greenhouse trial 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. 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. 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.
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.
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 expect our first results from this in six months.
Given the speed with which foliar potassium phosphate can reduce organic 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.
Our team (Triplett, Vincent, Killiny, and Wang) are working very well together and meet to discuss the project every two weeks.major
Update for this quarter:
No new plantings. Canker was assessed in a number of the transgenic trees from UF and USDA and in the trifoliate and trifoliate hybrid planting of UCRiverside. Growth and CLas titer data were collected on Stover lab transgenics. The Stover BRS permit was renewed and associated plantings were reported to be in compliance. The McNellis trees will be planted next quarter.
Previous quarter
A number of trials are underway at the Picos Test Site funded through the CRDF. A detailed current status is outlined below this paragraph. In the last quarter, the most significant advances have been: 1) Planting of USDA Mthionin transgenics with 108 transgenic Hamlin grafted on wild type Carrizo (7 events represented), 81 wild type Hamlin grafted on transgenic Carrizo (16 events represented) and 16 non-transgenic controls. 2) Planting was made of transgenics from Zhonglin Mou of UF under Stover permit, with 19 trees of Duncan, each expressing one of four resistance genes from Arabidopsis, and 30 Hamlin expressing one of the genes, along with ten non-transgenic controls of each scion type. 3) Renewal and approval for BRS permit effective 9/1/19 through 8/31/20. 4) Continuation of an experiment on pollen flow from transgenic trees. FF-5-51-2 trees are slightly more than 1000 ft from the US-802, and are self-incompatible and mono-embryonic. If pollen from transgenic trees is not detected from open-pollination, it should reduce isolation distances required by BRS. 5) Early-flowering transgenic Carrizo (flowered ex-vitro within five months of seed sowing, and used at 12 months) was used to pollinate some of the same FF-5-51-2 and some fruit appear to have set. 6) What should be the final samples from the C. Ramadugu-led Poncirus trial (#3 below) completed preparation and were shipped in ethanol to UC Riverside.
Previously established at the site:
1) The UF Grosser, Dutt and Gmitter transgenic effort has a substantial planting of diverse transgenics. These are on an independent permit, while all other transgenics on the site are under the Stover permit.
2) Under the Stover permit a replicated planting of 32 transgenic trees and controls produced by Dr. Jeff Jones at UF were planted. These trees include two very different constructs, each quite specific in attacking the citrus canker pathogen.
3) A broad cross-section of Poncirus derived material is being tested by USDA-ARS-Riverside and UCRiverside, and led by Chandrika Ramadugu. These are seedlings of 82 seed source trees from the Riverside genebank and include pure trifoliate accessions, hybrids of Poncirus with diverse parents, and more advanced accessions with Poncirus in the pedigree. Plants are replicated and each accession includes both graft-inoculated trees and trees uninfected at planting. Likely 2019 will be the last year for data collection.
4) More than 100 citranges, from a well-characterized mapping population, and other trifoliate hybrids (+ sweet orange standards) were planted in a replicated trial in collaboration with Fred Gmitter of UF and Mikeal Roose of UCRiverside. Plants were monitored for CLas titer development and HLB symptoms. Data from this trial should provide information on markers and perhaps genes associated with HLB resistance, for use in transgenic and conventional breeding. Manuscripts have been published reporting HLB tolerance associated QTLs and differences in ACP colonization. Trees continue to be useful for documenting tolerance in a new NIFA project.
5) A replicated Fairchild x Fortune mapping population was planted at the Picos Test Site in an effort led by Mike Roose to identify loci/genes associated with tolerance. This planting also includes a number of related hybrids (including our easy peeling remarkably HLB-tolerant 5-51-2) and released cultivars. Genotyping, HLB phenotyping and growth data have been collected and will continue to be conducted under a new NIFA grant.
6) Valencia on UF Grosser tertazyg rootstocks have been at the Picos Test Site for several years, having been CLas-inoculated before planting, and several continue to show excellent growth compared to standard controls (Grosser, personal comm.).
7) In a project led by Fred Gmitter there is a planting of 1132 hybrids of C. reticulata x C. latipes. C. latipes is among the few members of genus Citrus reported to have HLB resistance, and it is expected that there will be segregation for such resistance. The resulting plants may be used in further breeding and may permit mapping for resistance genes.
8) Seedlings with a range of pedigree contributions from Microcitrus are planted in a replicated trial, in a collaboration between Malcolm Smith (Queensland Dept. of Agriculture and Fisheries) and Ed Stover. Microcitrus is reported to have HLB resistance, and it is expected that there will be segregation for such resistance. The resulting plants may be used in further breeding and may permit mapping for resistance genes.
9) Conventional scions on Mthionin-producing transgenic Carrizo are planted from the Stover team and are displaying superior growth to trees on control Carrizo.
10) Numerous promising transgenics identified by the Stover lab in the last two years have been propagated and will be planted in the test site. New transgenics from Tim McNellis of PSU will be planted in the next quarter.
11) Availability of the test site for planting continues to be announced to researchers.
Project rationale and focus:
The driving force for this three-year project is the need to evaluate citrus germplasm for tolerance to HLB, including germplasm transformed to express proteins that might mitigate HLB, which requires citrus be inoculated with CLas. Citrus can be bud-inoculated, but since the disease is naturally spread by the Asian citrus psyllid, the use of psyllids for inoculations more closely resembles “natural infection”, while bud-inoculations might overwhelm some defense responses. CRDF funds supported high-throughput inoculations to evaluate HLB resistance in citrus germplasm developed by Drs. Ed Stover and Kim Bowman. The funds cover the costs associated with establishing and maintaining colonies of infected psyllids; equipment such as insect cages; PCR supplies for assays on psyllid and plant samples from infected colonies; and two GS-7 USDA technicians. A career base-funded USDA technician is also assigned ~50% to the program. USDA provides greenhouses, walk-in chambers and laboratory space to accommodate rearing and inoculations.
Most recent quarter:
Stover lab: 11,560 ACP used for 1388 leaves inoculated by DLA ( detached leaf assay), 108 small trees inoculated by no choice caged assay, and 13 assays run. A total of 14 different experiments, most testing Liberibacter killing transgenics.
Bowman lab: Continued grow out for first group of budded trees that were inoculated in July. First PCR and scoring will be done in November, 2019. Rootstocks were budded with Valencia for test groups 2, 3, and 4, to be inoculated beginning in early 2020. Once these Valencia budded trees are to size, there will be a regular ACP inoculation of a new group of trees every 1-2 months.
Previous quarter:
Over 7000 infected ACP were used in the last quarter, in part to screen 450 trees, but also for other related uses. The Stover lab used 1700 ACP in no-choice inoculation of transgenic citrus. 2700 ACP were used for detached leaf assays in which leaves of putative CLas killing transgenics and related controls are exposed to CLas-infected ACP for 4 days, allowed 3 days for ACP-free metabolism and then assessed for CLas titer in leaves and the ACP. One thousand ACP were used in an assay in which CLas+ ACP are used to develop a uniform homogenate for rapid testing of putative CLas-disrupting peptides
The Bowman lab has transitioned to use of grafted trees with a commercial scion in 2.5 liter pots. The first group of test plants will be removed from ACP inoculation the second week of July to begin post-inoculation evaluation. Subsequent groups of test plants for rootstock evaluation are being prepared.
Previously achieved in this project:
The 35 day federal government shutdown, and the threat of a possible shutdown on Feb 15, directly disrupted our ability to initiate and conduct experiments using the CLas+ ACP colonies. In addition, considerable rehibilitation of colonies and supporting plants was necessary due to the minimal care that could be provided during the shutdown. Only 2400 CLas+ ACP were used for experiments in this quarter and were used for detached leaf assessments of plants expressing three different transgenic constructs. We anticipate a normal demand in the current quarter.
As of December 21, 2018, a total of 14,111 plants had passed through the inoculation process. A total of 361,255 psyllids from colonies of CLas-infected ACP had been used in inoculations. Not included in these counts of inoculated plants and psyllids used in inoculations were many used to refine inoculation procedures, which provided insight into the success of our inoculation methods and strategies for increasing success. After inoculations, plants were returned to the breeders and subsequently subjected to further inoculations when they are transplanted to the field.
In addition to inoculating germplasm, infected psyllids were supplied to other researchers for other purposes. This side of the project grew over time, and detailed records were not maintained on how many were given out until 2018. More than 10,000 infected psyllids were supplied to the research community for an array of experiments during 2018. Recipients included researchers with USDA in Fort Pierce, Ithaca and Beltsville, UF in Gainesville, Cornell in Ithaca, University of California, and University of Nevada.
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 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. We have also identified two other possible transgenics from plants received from Dr. Vladimir Orbovic. Both responded to infiltration with a high concentration of bacterial cells by exhibiting a hypersensitive reaction within 4 days of infiltratin. One of the transgenics appeared to have a growth defect, but recently has developed normal shoots. Both transgenic trees contain the avrGf2 gene (based on PCR for detection of avrGf2). These trangenics will be grafted onto rootstock within the next two weeks. 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 ihas developed 45 putative grapefruit and sweet orange transformants. We are screening these currently via PCR. We have also grafted our lone transgenic plant onto two rootstocks (812 and Sour Orange) and planted these 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 June. At that point there was not a significant amount of disease to show any possible differences.
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 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. We have identified additional transgenics from plants received from Dr. Vladimir Orbovicthat that will be grafted onto rootstocks once the rootstock and transgenic trees are of adequate size.
In this study, we conducted the following specific objectives:1) GFP labeling of Candidatus Liberibacter. 2) Elucidation of plant- Candidatus Liberibacter asiaticus (Las) interaction through real-time monitoring of Las movement and multiplication in planta. 3) Investigate the effect of different control approaches on the dynamic population of Las in planta.
Objective 1. We constructed pDH3::PgyrA-GFP which has a wide bacterial host range replicon, repW, but cannot be inserted into a genome. Transformants and the GFP expression in Liberibacter crescens BT-1 were confirmed.
Objective 2. Our data showed that Las moves with phloem sap from source to sink tissues and remains in the young flush after ACP transmission and before the young flush matures. This observation prompted us to develop a method for early diagnosis of HLB, which allows inoculum removal to prevent ACP acquisition and transmission of Las. We conducted targeted early detection of Las in cultivar Valencia sweet orange (Citrus sinensis) before HLB symptom expression. ACPs secrete salivary sheaths at their feeding sites, which can be visualized using Coomassie brilliant blue staining owing to the presence of salivary sheaths secreted by ACP. Epifluorescence and confocal microscopy indicate the presence of salivary sheaths beneath the blue spots on ACP-fed leaves. Quantitative real-time polymerase chain reaction (PCR) and conventional PCR assays are able to detect Las in the ACP feeding surrounding areas as early as 2 to 20 days after ACP feeding. This finding lays a foundation to develop much-needed tools for early diagnosis of HLB before symptom expression, thus assisting Las inoculum removal and preventing HLB from spreading.
Objective 3. We evaluated the spatiotemporal dynamics of oxytetracycline in planta and its control effect against HLB via trunk injection. Las-infected ‘Hamlin’ sweet orange trees on ‘Swingle’ citrumelo rootstock at the early stage of decline were treated with oxytetracycline hydrochloride (OTC) using trunk injection with varying number of injection ports. Spatiotemporal distribution of OTC and dynamics of Las populations were monitored by high-performance liquid chromatography method and qPCR assay, respectively. Uniform distribution of OTC throughout tree canopies and root system was achieved 2 days postinjection. High levels of OTC (>850 µg/kg) were maintained in leaf and root for at least 1 month and moderate OTC (>500 µg/kg) persisted for more than 9 months. Reduction of Las populations in root system and leaves of OTC-treated trees were over 95% and 99% (i.e., 1.76 and 2.19 log reduction) between 2 and 28 days postinjection. Conditions of trees receiving OTC treatment were improved, fruit yield was increased, and juice acidity was lowered than water-injected control even though their differences were not statistically significant during the test period. Our study demonstrated that trunk injection of OTC could be used as an effective measure for integrated management of citrus HLB.
We tested HLB control via trunk injection of plant defense activators and antibiotics. In this study, eight plant defense activators and three antibiotics were evaluated in three field trials for their effect to control HLB by trunk injection of young and mature sweet orange trees. Results showed that four trunk injections of several activators, including salicylic acid, oxalic acid, acibenzolar-S-methyl, and potassium phosphate, provided significant control of HLB by suppressing Las titer and disease progress. Trunk injection of penicillin, streptomycin, and oxytetracycline hydrochloride resulted in excellent control of HLB. In general, antibiotics were more effective in reduction of Las titer and HLB symptom expressions than plant defense activators. These treatments also resulted in increased yield and better fruit quality. Injection of both salicylic acid and acibenzolar-S-methyl led to significant induction of pathogenesis-related (PR) genes PR-1 and PR-2 genes. Meanwhile, injection of either potassium phosphate or oxalic acid resulted in significant induction of PR-2 or PR-15 gene expression, respectively. These results suggested that HLB diseased trees remained inducible for systemic acquired resistance under field conditions. In summary, this study presents information regarding controlling HLB via trunk injection of plant defense activators and antibiotics, which helps citrus growers in decision making regarding developing an effective HLB management program.
We evaluated the effect of the combinations of plant defense elicitors, nitrogen (N) fertilizer, and compost to control HLB. After four applications over two consecutive growing seasons we found that the combination of compost, urea, and plant defense elicitors β-aminobutyric acid, plus ascorbic acid and potassium phosphite with or without salicylic acid, slowed down the progression of HLB and reduced disease severity by approximately 18%, compared with the untreated control. Our data showed no decline in fruit yield, indeed treatment resulted in a higher yield compared with the untreated control.
The present reporting period runs from June 16 – September 15, 2019. Mr. Chad Vosburg is the M.S. degree student in the Penn State Department of Plant Pathology graduate program who is working on the project. Chad took a trip to Fort Pierce, FL, September 12 – 15, 2019. During this trip, he set up plant propagations sufficient for 1-2 runs of an HLB resistance test for all the FT-scFv grapefruit lines. Existing propagated plants were cut back to induce a new flush of growth, which is essential for a successful HLB infection test. Personnel at the USHRL will initiate the HLB screenhouse test when the plants have reached the optimal stage of re-growth. Chad also worked with personnel at the USHRL to set up a field test of two of the FT-scFv lines. Chad is now working on mastering PCR skills for CLas detection that will be part of the HLB infection tests in the lab at Penn State. We anticipate that the first run of an HLB resistance test will beinitiated during the next reporting period. Mr. Jeremy Held, a Ph.D. student in the Intercollege Graduate Program in Plant Biology at Penn State, continued his studies of the stabilty and expression of the FT-scFv protein in the grapefruit lines. He optimized western blotting and antibody selection and plans to test for movement of FT-scFv across the graft union using immunoprecipitation during the next reporting period. Finally, we initiated tests to check whether FT-scFv plants are fertile. Initial fruits obtained from the FT-scFv plants were seedless, possibly because they are being grown in greenhouses or growth chambers. Jeremy performed some cross-pollinations of FT-scFv flowers in the spring of 2019, and one of these fruits was cut open during the reporting period and found to have normal seed set. This is good news if the FT-scFv construct were ever to be used as a citrus breeding acceleration tool, since precocity is one of the phenotypes of FT-scFv plants. Jeremy also tested pollen viability from FT-scFv plants and found the pollen to be viable and able to germinate in the laboratory in pollen germination medium.
Between the July and October, Citrus transformation Facility continued its operation without any interruptions. Within this period, we accepted three new orders. All orders required production of transgenic Duncan grapefruit plants.
In the last three months, CTF produced 74 plants. Among the produced plants there were: six Duncan plants (BB3), 18 Duncan plants (BB4), seven Duncan plants (HGJ34), three Duncan plants (HGJ74), two Duncan plants (HGJ87), three Duncan plants (HGJ88), ten Duncan plants ZM14, four Mexican lime plants (M2SF), one Mexican lime plant (contSF), one Murraya (BB1), one Pomelo plants (HGJ68), five Valencia plants (BB3), five Valencia plants (BB4), eight Valencia plants (JJ7). Codes in parentheses represent names of different binary vectors (genes) used in transformation experiments.
One of the CTF employees working on the USDA grant left the lab in the first week of September. After securing additional funding from other participants of that USDA grant, I have submitted position description to HR department and expect to hire new employee before the end of the year. Currently, there are five employees in the CTF.
Most of the fruit of Duncan grapefruit and Valencia orange that was stored in the cold room at CREC rotted and started growing mold. Only six bins of Valencia and ten bins of grapefruit were kept as a source of seeds. The crew working in A. Schumann’s CUPS harvested half of the yield from Duncan grapefruit trees we have there. These fruits will be used for seed extraction in a matter of weeks. We will ask A. Schumann’s crew to harvest the rest of Duncan grapefruit from CUPS for us in January.
To decrease our dependence on CRDF funding, two research proposals were submitted to two federal agencies: NSF and NIFA. It should be ~Christmas before we hear whether the NSF was successful, but NIFA will likely be delayed because of its move to Kansas City. I am working on a third proposal that will be submitted to the USDA SCRI Citrus whenever the RFP is announced. This RFP will also be delayed because of USDA’s move to Kansas City. CRDF funding obligations would be decreased if these proposals were funded. I have contacted scientists at UF, the USDA in Fort Pierce, Texas A&M, University of Arizona, Standford, UC Davis & UC Riverside to increase our clientele. In the near future, our prices will increase as suggested by the CREC Director, the CRDF, and the External Review committee. The actual dollar amount will be determined in consultation with the CREC Director.
It was a productive quarter for the transgenic event production. Forty-four transgenic shoots were identified, 18 surived micrografting, 6 died, and 10 are still healing. Use of the GFP reporter partially accounts for this increased productivity. In spring & summer, our productivity also naturally inceases because citrus is more vigorous. In addition, a new staff member was screening for transgenics & this might have contributed to increased productivity. We received two new vectors from Dr. Mou. Dr. Bonning contacted me about potential work in the future. Dr. Wang’s group is interested in transforming mature grapefruit for commercialization, but we had to introduce grapefruit cultivars first (see below) & then determine which cultivars are amenable to transformation. Dr. Dutt has provided three vectors for our collaborative CRDF project together.
We discovered that Dr. Grosser’s OLL4 and Valquarius cultivars have relatively high Agrobacterium transformation efficiencies. UF15 rootstock is recalcitrant to Agrobacterium transformation with no shoots forming in tissue culture. We introduced new budwood cultivars from FDACS. The budwood introductions were Marsh, Flame, Ray Ruby, and Duncan grapefruits, Temple sweet orange (zygotic embryos), and US-942. We will introduce OLL20 in the future because it is important to the juice industry.
We continue to conduct biolistic transformation of mature scions with citrus reporter genes & selectable markers with the ultimate goal being cis/intragenic production. Next week, we will test whether Dr. Dutt’s reporter can be used without a selectable marker. A new plant – derived selectable marker has been tested with excellent results. Replication of these experiments is currently underway. A similar vector with a citrus-derived selectable marker will be tested in the future. This system is an alternative to E. coli genes for antibiotic resistance & selection. Using citrus-derived sequences should also be more consumer friendly. A manuscript should be produced from this research. PEG-mediated DNA precipitation is being tested to increase biolistic transformation efficiency rather than protamine sulfate or spermidine.
Dr. Wu performed Southern blots for another scientist to show copy number of the transgenes for a scientific publication.
RC Webview, the electronic system that runs the growth facility, has been offline for ~1 week & the reasons are unclear. UF Gainesville is supposedly in the process of connecting a new server. With no other choice available, we have to water with city water which might contain slime mold spores, does not contain enough chlorine, and hasn’t passed through 2 UV filters.
All significant results remain the same as in the previous quarter.
The long term field trial continues with weekly psyllid counts and quarterly CLas infection testing. Treatments continue to have similar effects on ACP counts. Plants in both of the kaolin treatments continue to show higher growth rates than the other two treatments. The red treatment has the highest growth rate, trunk cross-sectional area, and canopy volume. Kaolin treated trees that are infected grow more than untreated-infected trees, but less than treated uninfected trees. The field trial will continue until the project ends, when we expect to have the first economic yield.
We are now performing follow-up repetitions of the MS student’s thesis work. We anticipate publication submission of this work in the Fall.
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.
These treatments have been initiated at all sites in spring of 2019. The pretreatment data and first and second round of fertilizer application have been already made. The leaf and soil nutrient analysis has been completed and currently we are interpreting the data. Overall, the trees are looking good and they have a good crop load.
The yield and consumer panel results from Objective 1 have been presented at Citrus Expo 2019. These results will be additionally presented at SWFREC and CREC on October 29th, 8th, respectively.’
This a fertilizer evaluation trial and the progress on it is timely and as per expectations
Objective 1: Investigate the efficacy of bactericides treatments for preventing new infections for young citrus trees protection. Hypothesis: Bactericidal treatment will protect young trees from CLas colonization.Initial leaf samples were collected before treatments to evaluate CLas titers in the uninfected trees. Antibiotic treatments were applied from May 2019 through September of 2020. From early June through September, 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 and ACP from May 2019 through June 2020 are described. Currently, citrus leaves tissue samples from July through September 2020are being processed to analyze the CLas-infection rate. Results: CLas titers were highest during the past quarter compared to previous quarters across all treatments. The average tree CT value prior to the beginning og the study was 37.5-39.5. Trees are considered CLas-infected (positives) when CT values are below 35. Trees receiving monthly or quarterly applications of Firewall/Fireline tested CLas-positive beginning January 2020 and December 2019, respectively. Trees covered by Tree Defender bags or treated with insecticide only tested CLas-positive beginning December 2019 and February 2020, respectively.During May-June 2020, CLas values reached the higest level across all treatments since the study was initiated. CLas titers were lowest in the insecticide only treatment during this period, followed by monthly applications of Firewall/Fireline rotation. Trees receiving quarterly applications of Firewall/Fireline had the second highest titer of CLas, compared to other treatments. The highest titers of CLas during May-June 2020 were observed in trees covered with Tree Defender bags. Objective 2. Determine the effect of bactericides application frequency on Las infection of citrus. Hypothesis: Bactericidal treatment will reduce CLas infection in mature trees. Antibiotic treatments were applied from May 2019 through September of 2020. From early May through June, CLas titer was monitored in leaf tissue and Asian citrus psyllid adults in response to antibiotic treatments. Currently, citrus leaf samples from July through September are being processed to analyze the CLas-infection rate. Results: The average CT value of citrus trees at the onset of the study was 28. From May 2019-June 2020, trees receiving monthly applications of Firewall/Fireline had the lowest CT values, as compared with trees receiving quarterly antibiotic applications or insecticide only, with the exception of January 2020 when mean CLas titers across all treatments were not significantly different.
The purpose of this project is to reveal the mechanisms of bactericide uptake and transport in citrus plant and establish a theoretical basis for developing technologies to improve the efficacy of bactericides, which is helpful to provide potential solution to the development of effective chemotherapeutic tools for HLB management. Achieving this outcome will require progress in the following three tasks: (1) to compare the delivery efficacy of bactericides with three application methods (foliar spraying, truck injection, and root administration) based on the uptake and dynamic movement/distribution of the bactericide within the tree; (2) to clarify the systemic movement and transportation mechanisms of bactericides within the phloem of tree; and (3) to investigate the effects of citrus variety and age on the delivery efficacy of bactericides. This project requires a combination of greenhouse studies and field trials, and a reliable and accurate method for quantifying bactericides (oxytetracycline and streptomycin) in citrus tissues. This project officially started on December 1, 2018. This is the 3rd quarterly progress report covering 6/01 to 08/31, 2019. During this period we have started and/or completed the following work/research tasks:
1) The extraction method for oxytetracycline and streptomycin in citrus samples was further improved. Now we have a simpler and faster extraction method with a small SPE cartridge (Waters Oasis HLB 1 cc, 30 mg) to replace the previously used big one (Waters Oasis HLB 6 cc, 500 mg). Besides, the newly developed method was validated in terms of specificity, linearity, precision, accuracy and recovery with reference to the guideline established by the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use Validation of analytical procedures.
2) We finished the planned citrus foliar spraying experiment. Plant samples (leaves, branches, and roots) were collected from the treated citrus trees at 0 (control), 7, 15 and 30 days after treatment. The samples were extracted and analyzed for bactericides using the above newly developed method. Preliminary results showed that both oxytetracycline and streptomycin could be detected in all the plant samples even at day 30 after spraying except for streptomycin in root. More biological replicates will be analyzed to verify the results.
3) For the trunk injection experiment, work was performed to develop some specific apparatus for trunk injection, and some trial tests were conducted to optimize flow speed of bactericide solution and ensure steady absorption of antibiotics into citrus trees under field conditions.
The work planned for the next quarter:
The major goals of research for the next three months are to obtain the final results of foliar spraying experiment and evaluate the delivery efficacy of bactericides within citrus trees based on the three different application methods (foliage spraying, trunk injection, and root administration). The following research tasks will be conducted in the 4th quarter: 1) More biological replicates of plant samples from the foliar spraying experiment will be analyzed to verify the present result; 2) The planned trunk injection experiment will be conducted; 3) Prepare for root administration of bactericide experiment.
Objective 1: Investigate the efficacy of bactericides treatments for preventing new infections for young citrus trees protection.
Hypothesis: 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.
We applied bactericide treatments from May through September. CLas titer was monitored in leaf tissue in response to antibiotic treatments using quantitative real-time PCR analysis. In this report, the results of CLas-infection rate in citrus leaves from May and June is described. Currently, citrus leaves tissue samples from July through September are being processed to analyze the CLas-infection rate.
*Trees were considered CLas-infected (positives) when CT values were below 35.
1. Antibiotics (monthly rotation): Prior to bactericide application (May), 15% of trees (20 trees/treatment) were CLas positive (Ct<35). After the bactericide application (June), 35% of trees were CLas positive (Ct<35).
2. Antibiotics (quarterly rotation): Prior to bactericide application (May), 100% of trees were CLas negative (Ct>35). After bactericide application (June), 40% trees were CLas positive (Ct<35).
3. Negative Control (insecticide + Tree defender exclusion netting): Prior to bactericide application (May), 100% of trees were CLas negative (Ct>35). After the bactericide application (June), 45% trees were CLas positive (Ct<35).
4. Positive Control (insecticide only): Prior to bactericide application (May), 100% of trees were CLas negative (Ct>35). After the bactericide application (June), 5% trees were CLas positive (Ct<35).
Counting of ACP adults using taps was conducted bi-weekly from May through September, 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 farm manager. As 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 September. Also, to determine the effect of citrus vegetative growth (flush-like structures) in CLas-infection rate, 1 ft.3 was used to count the number of flush-like structures per tree. Results showed that the presence of flush-like structures incremented from May to July and decreased in September.
Objective 2. Determine the effect of bactericides application frequency on Las infection of citrus.
Hypothesis: Bactericidal treatment will reduce CLas infection in mature trees.
We applied bactericide treatments from May through September. CLas titer was monitored in leaf tissue in response to antibiotic treatments using quantitative real-time PCR analysis. In this report, the results of CLas-infection rate in citrus leaves from May and June is described. Currently, citrus leaves tissue samples from July through September are being processed to analyze the CLas-infection rate.
*Trees were considered CLas-infected (positives) when CT values were below 35.
1. Antibiotics (monthly rotation): Prior to bactericide application (May), 100% of trees (20 trees/treatment) were CLas positive (Ct<35). After the bactericide application (June), 100% of trees were CLas positive (Ct<35). Although positive, bacterial titers declined in trees receiving antimicrobial treatments.
2. Antibiotics (quarterly rotation): Prior to bactericide application (May), 100% of trees were CLas positive (Ct<35). After the bactericide application (June), 100% of trees were CLas positive (Ct<35).
3. Positive Control (insecticide only): Prior to bactericide application (May), 100% of trees were CLas positive (Ct<35). After the bactericide application (June), 100% of trees were CLas positive (Ct<35).
Counting of ACP adults using taps was conducted bi-weekly from May through September, presence of other life stages such as eggs and nymphs were scouted visually. Preliminary results showed high ACP populations in treatments from May to August, excepting for June. The number of eggs and nymphs were not collected during May and first collection of June. However, populations increased from late June to August and reached high population levels. Currently, ACP adults that were collected bi-weekly are being processed to analyze the CLas-infection rate using quantitative real-time PCR analysis. Also, to determine the effect of citrus vegetative growth (flush-like structures) in CLas-infection rate, 1 ft3 was used to count the number of flush-like structures per tree. Flush was not collected during May and June. However, results showed that the presence of flush was high in July and August.
August 31, 2019 – In this quarter, we have continued to work on objectives outlined in our chronogram.
Objective 1. We have completed assessment of trees planted in our pilot study (planted 22 months ago) for CLas infection and HLB symptoms. All the non-covered trees are PCR-positive for CLas whereas all trees covered with IPC have tested negative. We are continuing with quantification of leaf drop and comparing leaf drop in both treatments; 6-month cumulative data show no significant differences in leaf drop in IPC-covered trees compared with non-covered trees. Interestingly, when counted seasonally, in spring leaf drop was significantly higher in non-covered trees as compared to IPC trees, whereas in summer, it was slightly higher inside IPCs. This fact points out a seasonal component that we will investigate as the project progresses.
In August, we have replaced the old 4-ft IPCs with new 8-ft covers, donated by The Tree Defender, Inc, because the trees had filled the volume of the cover completely. This also has opened the possibility of studying the dynamics of branch unfolding, which we are doing visually (photography documentation) and by measuring canopy growth and leaf area index. We have also assessed other pest and disease incidences inside the IPCs. We have found less incidences of canker inside IPCs and approximately equal incidences of greasy spot. However, greasy spot severity is higher inside the IPCs. We have found more incidence of other pests such as mites, armyworms, and leafrollers inside the IPCs, and a total absence of predators (beneficials). This suggests that relying only on IPC for insect control is not sufficient, and insect management must still be conducted. No psyillid have been found inside the IPCs.
Objective 2. To study the edge effect in different IPC layouts, we are now preparing to plant 700 trees of SugarBelle, Tango and Early Pride mandarins and using 3 different arrangements (targeted, alternated and patterned, as described in the proposal) of IPC. We have performed initial measurements of the tree parameters (trunk diameter, and leaf sampling, for CLas, cholorophyll and sugar analysis).
Objectives 3 and 4. We are continuing to measure fruit set and development inside the IPCs and comparing this with our CUPS planting. We are taking fruitlet and fruit samples regularly for biochemical analysis.
Outreach, Professional Presentations and Extension Activities for this quarter :
-Grower Presentation: “Growing Young Citrus Trees Under Individual Protective Covers (IPCs): What We Know After 18 Months” Citrus Expo 2019, August 15, Fort Myers, Fl.
-Industry Magazine Article: “Individual Protective Covers for Psyllid Exclusion and HLB Disease Prevention in Young Trees”. Article submitted to Citrus Industry Magazine in July to be published in October issue.
-Our Project was also noted in the September’s issue of Citrus Industry Mag’s UF/IFAS. The Citrus State Opinion Column by Jack Payne highlighted this work as an example of collaboration between growers, extension agents, and scientists in Florida. The column was entitled “Collaboration breeds solutions”.
August 2019 The objectives for this proposal are 1) Conduct ground and aerial applications of fungicides to determine the efficacy and economics of fungicide treatments; 2) Determine if Luna Sensation has enough systemic activity to protect flowers from before they fully develop and open; 3) Determine if the period flowering of trees affected by huanglongbing can be narrowed to eliminate the offseason bloom that contributes to the PFD inoculum increase in groves. Field trials were conducted each year to test new compounds and try new programs with existing compounds. Unfortunately, in all years but 2016, there was minimal PFD which did not allow for great confidence in the trial results We tried three different methods to investigate the question of Luna Sensation and did not come to a satisfactory answer. The first year, the experimental approach was flawed and the remaining years, there was insufficient disease to be able to generate an answer. We tried to induce flowering in the greenhouse but failed. Three years of PGR field trials were undertaken and it was found that GA does compress the flowering period but did not reduce yeild when applied once a month from September to December. It also appears to increase the amount of force needed to remove fruit from trees and reduce fruit drop. The Citrus Advisory Scheduler was developed based on a Brazilian model. It was modified and optimized for the Floridian situation. It brings data in from the FAWN system and will allow growers to monitor their risk for PFD. It also has a log in so growers can get alerts on their phones or e-mail systems. We did 4 years of validation trials for the Citrus Advisory Scheduler. We determined that the system is very reliable when indicating there is low risk for a PFD infection event. Unfortunately, there was limited disease during this program and we were unable to evaluate how well it worked when predicting moderate or high risk of PFD infection.
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