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.
Two trials, (1) factorial combinations of perennial peanut in row middles with nematicide in irrigated zone and (2) efficacy against sting nematode of oxamyl, aldicarb, fluensulfone, fluopyram, fluazaindolizine, and an experimental compound, were treated with recommended rates of each nematicide. All nematicides except aldicarb and oxamyl were applied in dedicated irrigation lines by irrigating (microjet) for 30 minutes, injecting the nematicide for 60 minutes, followed by 30 additional minutes of irrigation. Prior to treatments, dye was injected sequentially into the manifold controlling each treatment to ensure the plumbing was treating all of the experimental trees. On May 30 and 31, two months after treatments were initiated, soil samples were taken from all trials and processed (sucrose centrifugation) for nematode determination. Two nematicides reduced sting nematode populations by more than 70%, with no significant effect yet by four compounds. Because there is evidence that some of the new compounds move in the soil very litte after application, the injection period will be extended to 2 hours in an effort to increase efficacy. Effects on fibrous root density are being processed. Treatments of most products will be resumed in the autumn. Perennial peanut is establishing reasonably well in all plots since the onset of the rainy season. The large effort of watering and weed management required of the sod laid during the spring dry season argues for starting such plots during the summer. Similarly, the sunn hemp crop did not establish well during the dry season and is will be replanted in early July. A second nematicide trial was initiated at a second young orchard by treating with aldicarb on 26 April, with all remaining treatments to be initiated in autumn after the plots are plumbed.
This project is an continuation of an objective of existing CRDF funded project (# 00124558 ; ended in March 2019) with some added treatments to be evaluated. The added treatments are: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 These treatments have been initiated at all sites. The pretreatmentr data and first round of fertilizer application have been already made. The leaf and soil samples have been sent tout of nutrient analysis. Overally, the trees are looking good and they have a good crop loead. The trees seem uniform in symptom levels .The second application of the fertilizer will be made in late June-early July. A consumer taste panel was performed in April on 4 of the best performing treatments based on the yield.
The federal agency, USDA APHIS, will issue new guidance about the deregulation & environmental release of transgenics produced with plant pest sequences (35S promoter, Nos terminator, T-DNA borders) in the near future, which will significantly decrease the time & cost to release transgenics to the growers. This is extremely important for the citrus industry because Agrobacterium-mediated transgenics with plant pest sequences that are being field tested now could theoretically be commercialized, relatively soon. The FDA & EPA will still examine these plants. The final report of the external review of the transformation labs was received & it concluded that both transformation labs are necessary & do not have to be merged. This is encouraging since our objectives are completely different. However, now we are faced with what might be a formal split between the CRDF & UF over the role of direct support organizations. The Mature Citrus Facility produced ~85 transgenics this quarter, which is a significant increase in productivity & we are not sure what caused this increase. Use of the gfp reporter in some transformations, which makes transgenics easier to identify likely contributed somewhat to this increase, & the fact that it was spring when our productivity naturally increases probably contributed. We always seem to produce more transgenics during spring. However, not all will grow large enough for micrografting & only 57 transgenics have been micrografted thus far. Of these 57 shoots, 30 will definitely survive. I submitted a research proposal to the National Science Foundation (NSF). It will take ~6 months to review & ~7% of proposals are funded. In collaboration with another scientist, a letter of intent was submitted to USDA AFRI for another research proposal. I contacted many scientists to advertise our services but have heard back from few. In the future, after transgenics have been made for current customers, our prices will increase to better cover operating costs. Our biolistic transformation manuscript in In Vitro Cellular & Developmental Biology is finally in press. It took a long time for this journal to review & publish the manuscript. Currently we are working on other manuscripts with other UF CREC scientists. We have introduced additional new cultivars from the UF Plant Improvement Team & other breeders. EV2, WP Murcott, Valquarius, OLL4 scions have been introduced. OLL4 & Valquarius have very high Agrobacterium transformation efficiency. UF 15, UF 17, X639, US 942 rootstocks were introduced & are being tested in Agrobacterium transformations. We also recently introduced several grapefruit cultivars (Flame, Marsh, Ray Ruby & Duncan). In the past, grapefruit (Ray Ruby, Grosser’s Red Grapefruit, & Ruby Red Grapefruit) were recalcitrant using the standard Agrobacterium protocol.
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.
The goal of this project is to investigate the effect of bactericides in new citrus plantings, and to evaluate the effects of repeated ACP inoculation access in infected groves treated with bactericides Objective 1: Investigate efficacy of bactericide treatments for preventing new infections. This objective is necessary to determine the potential role of bactericides in an integrated pest management program for young tree protection. Objective 2. Determine the effect of bactericide application frequency on Las infection of citrus. This experiment will determine the most effective application frequency for bactericides to maximize tree health. This will inform revised recommendations for the use of antimicrobials in commercial citrus groves.Objective 3: Quantify the effect of repeated inoculation of the efficacy of bactericides. This experiment will determine whether bactericides are sufficient to overcome the pressure of repeated Las inoculation by ACP. This will inform revised recommendations for integrated pest management programs to improve use of insecticides in conjunction with bactericides in commercial citrus groves. Bactericidal treatments were applied from May 2019 through June 2020 for each objective. However, the UF/IFAS Citrus Research and Education Center (CREC) closed on March 23rd due to COVID-19, limiting our access to the center and equipment. We were able to conduct field work as of April 20th. Samples were collected from early March, May, and June in order to quantify CLas titer in leaf tissue in response to antibiotic treatments using quantitative real-time PCR analysis. Currently, citrus leaves tissue samples from January through June are being processed to analyze the CLas-infection rate for trees in objectives 1-3.ACP adult populations were assessed by tap sampling bi-weekly from May through June 2020. Preliminary results showed a low ACP population in citrus locations due to the active vector management performed by the farm manager. Therefore, no ACP adults were collected to analyze the CLas-infection rate using quantitative real-time PCR analysis. Treatments and sampling will continue through late fall 2020 for each objective. PCR analysis will resume in June. Data will be analyzed during the 3rd quarter of 2020 in order to provide an updated preliminary assessement of bactericidal effects .
This project aims at investigating efficacy and feasibility of individual tree covers such as the tree defender to protect the tree from psyllids in the first 2-3 years after planting, and higher capacity nets during productive years. Little is known on the physiology and tree health parameters of trees grown under individual protective covering (IPC). There are no reports in the scientific literature regarding the use of these IPC systems in citrus.The project started in December 2018. We are following the chronology developed in our proposal. Our Project has 4 main objectives: Objective 1. Assessing tree growth and absence of psyllids and HLB disease symptoms (including CLas bacteria titer) under protective covering. Objective 2. Assessment of alternative netting approaches involved in `targeted’, `alternated’ or `patterned’ setup of IPC in groves for more cost-effective protection. Objective 3. Monitoring the transition from vegetative to reproductive stage in the covered trees as compared to the uncovered. Objective 4. Comparing individual tree protection with CUPS-like systems. Objective 1: We continued assessing the trees (Valencia on Swingle) planted in our pilot study 18 months ago for incidence of CLas. Today, virtually all of the uncovered trees in the trial (80-100% depending on the treatment) are already PCR-positive for CLas whereas all trees covered with IPC have tested negative. We have started quantification of leaf drop and compared leaf drop in both treatments; cumulative data of April through June show no significant increase in leaf drop in IPC-covered trees. Objective 2: We have identified a collaborator in southwest Florida to evaluate different layouts of IPCs and compare with the grove that we are setting up in Polk County.Objective 3: We have measured this season’s blooming and are now monitoring fruit set and development. We saw more blooming in uncovered than in covered trees. There are two possible reasons for these observations: 1) we measured more vegetative growth in IPC trees, which is known to delay transition to the reproductive stage, or 2) uncovered trees are more stressed by HLB than IPC trees, which may result in more blooming.Objective 4: We finished planting the trees in our CUPS facility and we started to perform different treatments to study fruit set in ‘Tango’, `SugarBelle’, and `US Early Pride’ trees. These treatments consist of different applications of gibberellic acid for overcoming absence of external pollinators. We will be assessing fruit set and fruit development in the coming months.Outreach, Professional Presentations and Extension Activities:-Citrus Industry, January 2019. “HLB reduction strategies”.-IRCHLB VI meeting, California, March 2019. “Individual Protective Covers (IPCs) prevent young citrus trees from psyllids and infection with CLas, and promote vegetative growth”.-Spring Citrus Field Day at SWFREC April 2019. Demonstration of the SWFREC IPC experimental grove with an attendance of over 60 growers and industry representatives.-FSHS ANNUAL CONFERENCE, Orlando, FL, June 2019. “Effect of Individual Protective Cover (IPC) on Physiology and Growth of Citrus Trees”.-Florida Citrus Industry Annual Conference. Educational Session, June 2019. “Protective Covers on Young Trees”.
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. Prior to conducting these experiments, a sensitive and accurate quantifying method of bactericides (oxytetracycline and streptomycin) in citrus tissues is needed. This project officially started on December 1, 2019. This is the 2nd quarterly progress report covering 3/01 to 05/31, 2019. During this period we have started and/or completed the following work/research tasks: 1) We conducted a literature search for methods that can be used for analyses of oxytetracycline and streptomycin in plant samples and the methods we found in literature are adequate for analysis of oxytetracycline or streptomycin only in processed food such as fruit gel. They are not reliable for determination of oxytetracycline or streptomycin in plant samples, because of matrix effects that result in poor extraction efficiency. 2) In this project, we need a reliable method for simultaneous determination of both oxytetracycline and streptomycin in citrus plant tissue samples, but it is not available in literature. Therefore, we designed a method based on information in literature and our previous experience in the extraction of other antibiotics in plant samples for simultaneous analysis of oxytetracycline and streptomycin in citrus plants, especially we replaced methanol with acetonitrile and used lower concentration of formic acid for the extraction of antibiotics in citrus plant tissue samples. 3) Laboratory tests demonstrated the potential and feasibility of the newly designed method for simultaneous analysis of oxytetracycline and streptomycin in citrus plants, but matrix interference with antibiotic analysis was still significant. 4) To further improve this method, a solid-phase extraction (SPE) method was developed to clean up sample matrix, and analytical conditions of instrumentation were modified by changing the gradient profile of LC and lowering the operation temperature of MS/MS. These modifications substantially improved instrumental sensitivity for detecting oxytetracycline and streptomycin plant samples. 5) This newly developed method has now been successfully applied to the simultaneous detection of oxytetracycline and streptomycin in citrus leaf samples. The work planned for the next quarter:The major goals of research for the next six months are to evaluate the delivery efficacy of bactericides within citrus trees based on three different application methods (foliage spraying, trunk injection, and root administration). The following research will be conducted in the 3rd quarter: 1) acquisition and culture of citrus seedlings in greenhouse; 2) foliar spraying of streptomycin or oxytetracycline and periodic sampling; 3) sample extraction and instrumental analyses; and 4) preparation of materials for experiments with application methods of trunk injection and/or root administration.
June 2019Objective 1: Evaluate the optimal spray timing for Florida and investigate if tree skirting or alternative products improves fungicidal control of citrus black spot.Objective 3: A MAT-1-1 isolate may enter Florida and allow for the production of ascospores. The industry needs to know if this happens, as it will affect management practices. Additionally, the existing asexual population may be more diverse than currently measured. If multiple clonal linages exist, then there may be different sensitivities to fungicides or other phenotypic traits. We also need to determine whether P. paracitricarpa or P. paracapitalensis are present in Florida for regulatory concerns due to misidentification. We plan to survey for the MAT-1-1 mating type, unique clonal lineages, and two closely related Phyllosticta spp. In the fungicide and skirting control trial, three applications of fungicides have been completed. They are applied approximately every 28 days. Skirting was planned for early June. We expect to continue with the applications through the summer and into the fall. The two potential groves for the fungicide spray trial were scouted for CBS incidence and symptom severities in in March 2019. The selected grovewas chosen for the trial based on the higher incidence of CBS. To keep treatments consistent, only mature trees were rated for CBS. Twenty-fve fruit per tree were rated and three trees were grouped based on the average rating values into a replication. There are five replications per treatment and each treatment consists of at least one replicate at each CBS incidence level. The trial is spread out over eight rows, each consisting of 117 trees, plus guard rows on both sides of the rows containing the treatments. A total of 10 treatments were selected and treatments are applied with a handgun at 200 psi. The first application was done over a two-day period starting on May 16th, this was a two-week delay from the original plan due to unforeseen grove activities. The second application was done over a two-day period beginning on June 3rd. The first two applications were completed as planned without any weather delays; although there was significant rainfall five hours after the last treatment of the second application. The third application is planned to begin on June 24th. Whilst the CRDF-funded project has already officially started, the sub-contract between UF and CRI must still be concluded. Funding has therefore not been allocated yet. Nonetheless, a postdoctoral researcher has been appointed from 1 March 2019, and preparations for genotyping-by-sequencing of a collection of Phyllosticta citricarpa isolates from USA has been initiated. Eighteen isolates (4 from South Africa and 14 from USA) were sequenced (600nt read length) using the Ion Torrent System. Thus far, the genomes of eight isolates ( 3 from SA and 5 from the USA) have been successfully assembled and analysed using a customised bioinformatics pipeline. The following available reference sequences were used: GC12: 5748 contigs (Florida, USA) and GCMC3: 6716 contigs (Zhejiang, China). The rest of the isolates will be sequenced before comparative analysis commences. Jeff Rollins has been able to secure the most of the necessary permits for him to be able to travel to Cuba and collect isolates to determine the mating type and species identifications. As the travel rules change based on Federal government policy, a few more details need to be worked out and final trip arrangement. We recieved MAT 1-1 DNA from South Africa to use as positive controls for our experiments. Trees are fruiting for some on tree experiments.
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.This is the first year, 2nd quarter progress report; our grant started December 1, 2018. In the last three months, we have brought on board our post-doctoral researcher, Dr. Tamara D. Collum. Objective 1 (development of transgenic constructs) has been completed. 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 the necessary constructs and has begun moving them into citrus. The majority of our efforts in the 2nd quarter were focused on objective 2 (production of transgenic citrus lines). The Stover lab has performed Agrobacterium-mediated transformation of seedling epicotyls from all three citrus genotypes indicated in the grant (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 putative transgenic plants with three promoters are already rooted and established in the greenhouse; transformation was initiated with the fourth construct. Putative transgenic plants of Poncirus are also in hand, with only one construct in soil and the other three in rooting and/or shoot induction and selection media. Hamlin transformation was initiated with three constructs; explants are still in shoot induction/selection media. shoots are already evident on the shoot induction/selection medium for p35S::HF-RPL18 (35 shoots). More in vitro germinated seedlings are growing for additional Hamlin transformations. Likely the Carrizo and Poncirus transformations already created will be sufficient for this project. Since Hamlin has a much lower transformation efficiency, some transformations will likely need to be repeated in the next quarter. Gene insertion and expression will be verified for many plants in the coming months. Plans are underway for transfer of the first plants to Ft. Detrick.
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 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 once they reach the appropriate size. 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 is in the process of screening putative grapefruit and sweet orange transformants. We hope to have additional transgenics later this year. 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. 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 tow most promising EFR transgenic plants (based on ROS activity) onto two rootstocks (812 and Sour Orange) and are in the process of planting in the field at Fort Pierce in collaboration Dr. Ed Stover. They were planted in the field in late March. We have identified additional transgenics from plants received from Dr. Vladimir Orbovicthat that will be graftered onto rootstocks in the near future.
Objective 1: Investigate efficacy of bactericide treatments for preventing new infections. This objective is necessary to determine the potential role of bactericides in an integrated pest management program for young tree protection. Experiment were initiated in new tree plantings in a Wauchula grove.Initial leaf samples were collected prior to treatments to evaluate CLas titers in the uninfected trees. The following treatments were applied this second quarter of 2019: Treatments were applied to trees as follows:1. Antibiotics (monthly rotation) 2. Antibiotics (quarterly rotation)3. Negative Control (insecticide + Tree defender exclusion netting)4. Positive Control (insecticide only)Tree height and circumference were quantified. ACP populations were monitored biweekly (eggs, nymphs, adults). No flush, eggs, or nymphs were detected after treatment applications. Tap sampling of adults was conducted, and insects are currently being processed to determine the infection rate. Objective 2. Determine the effect of bactericide application frequency on Las infection of citrus.This experiment will determine the most effective application frequency for bactericides to maximize tree health. This will inform revised recommendations for the use of antimicrobials in commercial citrus groves. Experiment were initiated in new tree plantings in a Lake Alfred grove. Initial leaf samples were collected prior to treatments to evaluate CLas titers in the uninfected trees, then one week after treatment applications. The following treatments were applied this second quarter of 2019: 1. Antibiotics (monthly rotation) 2. Antibiotics (quarterly rotation)3. Positive Control (insecticide only) Tree height and circumference were quantified. ACP populations were monitored biweekly (eggs, nymphs, adults). No flush, eggs, or nymphs were detected after treatment applications. Tap sampling of adults was conducted, and insects are currently being processed to determine the infection rate. Objective 3: Quantify the effect of repeated inoculation of the efficacy of bactericides. This experiment will determine whether bactericides are sufficient to overcome the pressure of repeated Las inoculation by ACP. This will inform revised recommendations for integrated pest management programs to improve use of insecticides in conjunction with bactericides in commercial citrus groves. We have procured trees and will initiate experiments next quarter.
Huanglongbing (HLB) and Citrus Bacterial Canker (CBC) present serious threats to the future success of citrus production in the US. Insertion of transgenes conferring resistance to these diseases or the HLB insect vector is a promising solution. Genes for antimicrobial peptides (AMPs) with diverse promoters have been used to generate numerous transformants of rootstock and scion genotypes. New promoters and/or transgenes are being regularly introduced with more than a thousand new transformation attempts on citrus epicotyl sections each week. Plants have progressed from the initial round of scion transformations and are now replicated and ready for exposure to HLB, using CLas infected psyllids in a greenhouse environment. Transformed rootstock varieties, with two AMPs (D4E1 and Pyrrhocoricin) and 170 transgenic plants, are being challenged using graft inoculations in two new replicated experiments. A wide series of promoters driving a reporter gene are being tested in transformed citrus and show very different levels of expression with some being expressed in all tissues and some only in vascular tissue. Liberibacter sequence data were used to target a transmembrane transporter,as a possible transgenic solution for HLB-resistance. Collaboration with a USDA team in Albany, CA is providing constructs with enhanced promoter activity, minimal IP conflicts, and reduced regulatory and consumer concerns. Genes are being identified from citrus genomic data, from Carrizo citrange sequence generated using USDA funds, to permit transformation and resistance using citrus-only sequences. Citrus-derived T-DNA border analogues have been shown to be effective in producing transgenic tobacco and will be tested in citrus in next quarter. Genes for anthocyanin production are being tested as a visual marker for transformation, as a component of a citrus-only transgenic system. Transgenes are being developed to suppress (using an RNAi strategy) a lectin-like protein produced in the phloem of HLB-infected citrus. It is possible that suppression of this protein may significantly reduce disease symptoms. High throughput evaluation of HLB resistance will require the ability to efficiently assess resistance in numerous plants. Graft-inoculation, controlled psyllid-inoculation, and ‘natural’ psyllid inoculation in the field are being compared. The first trial has been in the field for 21 months and a repeated trial has been in the field for 9 months. Leaf samples have been collected monthly and PCR analysis of CLas conducted. These data will be analyzed over the next quarter.
As proposed, a transgenic test site has been prepared at the USDA/ARS USHRL Picos Farm in Ft. Pierce. The first trees have been in place for more than eight months. Answers have been provided to numerous questions from regulators to facilitate field testing approval. Cooperators have been made aware that the site is ready for planting. Dr. Jude Grosser of UF has provided 300 transgenic citrus plants expressing genes expected to provide HLB/canker resistance, which have been planted in the test site. USHRL has a permit approved from APHIS to conduct field trials of their transgenic plants at this site. An MTA is in place to permit planting of Texas A&M transgenics produced by Erik Mirkov. Alphascents provided an experimental pheromone attract/kill product Malex to disrupt citrus leaf miner (CLM). Our experience suggests CLM may significantly compromise tree growth where insecticides are avoided to permit ready transfer of Las by psyllids. CLM damage also compromises ability to view HLB symptoms. Unfortunately, this product had little effect on leaf miner. The decision has been made to apply Admire this fall to encourage an undamaged flush on transgenic trees. We are still learning how to grow trees for best assessment of HLB-resistance. More than 120 citranges, from a well-characterized mapping population, and other trifoliate hybrids (+ sweet orange standards) have been propagated for a replicated trial in collaboration with Fred Gmitter of UF. These will be planted in the spring of 2011, and monitored for CLas 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.
Huanglongbing (HLB) and Citrus Bacterial Canker (CBC) present serious threats to the future success of citrus production in the US. Insertion of transgenes conferring resistance to these diseases or the HLB insect vector is a promising solution. Genes for antimicrobial peptides (AMPs) with diverse promoters have been used to generate numerous transformants of rootstock and scion genotypes. New promoters and/or transgenes are being regularly introduced with more than a thousand new transformation attempts on citrus epicotyl sections each week. Plants have progressed from the initial round of scion transformations and are now replicated and ready for exposure to HLB, using graft inoculations and CLas infected psyllids in a greenhouse environment. A detailed set of AMP activity assays will be initiated next quarter on a replicated set (8 plants of each) of 39 independent Hamlin transformants. Transformed rootstock varieties, with two AMPs (D4E1 and Pyrrhocoricin) and 170 transgenic plants, are being challenged using graft inoculations in two new replicated experiments. A series of promoters are being tested with the GUS gene to see how effective they are. As expected, the three vascular-specific promoters show expression only in phloem and xylem, while other promoters show broad expression in tested tissues. Sucrose synthase promoter from Arabidopsis drives high GUS expression more consistently than citrus SS promoter or a phloem promoter from wheat dwarf virus. A ubiquitin promoter from potato drives consistent and very high GUS activity, even though the mRNA levels are similar to D35S promoter. use of this promoter may reduce the number of independent transformants needed. Liberibacter sequence data were used to target a transmembrane transporter,as a possible transgenic solution for HLB-resistance. Radioisotope permits are finally in place to assess effect of identified peptides on preventing ATP uptake in E. coli expressing the Liberibacter translocase. Collaboration with a USDA team in Albany, CA is providing constructs with enhanced promoter activity, minimal IP conflicts, and reduced regulatory and consumer concerns. Genes are being identified from citrus genomic data, from Carrizo citrange sequence generated using USDA funds, to permit transformation and resistance using citrus-only sequences. Citrus-derived T-DNA border analogues have been shown to be effective in producing transgenic Carrizo and tobacco and will be tested in citrus scions soon. Genes for anthocyanin production are being tested as a visual marker for transformation, as a component of a citrus-only transgenic system. Transgenes are being developed to suppress (using an RNAi strategy) a lectin-like protein produced in the phloem of HLB-infected citrus. It is possible that suppression of this protein may significantly reduce disease symptoms. High throughput evaluation of HLB resistance will require the ability to efficiently assess resistance in numerous plants. Graft-inoculation, controlled psyllid-inoculation, and ‘natural’ psyllid inoculation in the field are being compared. The first trial has been in the field for 24 months and a repeated trial has been in the field for 12 months. Leaf samples have been collected monthly and PCR analysis of CLas conducted.
(863) 956-8817
(863) 956-5894
700 Experiment Station Road
Lake Alfred, FL 33850
Email Us
