The objectives of this proposal are: 1) conduct a field trial using the selected grapefruit seedlings to ensure the productivity of the trees in Florida where HLB is endemic; and 2) evaluate the quality of the fruit produced. Achievement of these goals will produce a more resistant/tolerant variety that could be available in the near future since its use would not require regulatory approval. Based on two year’s graft-inoculation assays in greenhouse with two HLB bacterial isolates and the performance of individual seedlings in the field, four lines of the seedlings (with greater HLB resistance/tolerance) were selected for further propagation on three different rootstock (commercial sour orange, newly selected USDA-sour orange and 942). The fruit quality (Brix, sucrose, glucose and fructose, soluble solids, pH, % TA and total ascorbic acid) of the four selected seedlings showed no significant difference from their maternal trees. The first group of the propagates on three different rootstock from the selections of Scott Grove’s seedling variants were grown at our research farm, Picos Farm, where the plants are under extreme high HLB disease pressure with very aggressive HLB pathogens. These new plantings (July, 2017; Nov, 2017; and May, 2018) showed different disease index, the longer the planting was, the higher the disease index, which was also highly correlated with the titers of Ca. Liberibacter asiaticus (Las) in infected plants. It is worth noting that the new HLB isolate from Picos Farm caused severe HLB disease on most of grapefruit selections of seedlings and bud sports in our latest greenhouse evaluation. Those selections were either resistant or tolerant to the previous HLB isolates we maintained in greenhouse. Prelimilary data showed some of the selections are better than the others with either lower disease index or better canopy growth. In general, the average infection rate was 20.85% for one group of grapefruit plants (470) 20 months after planting, and 5.46% for the other group of grapefruit plants (366) 15 months after planting. The second group of the propagates on three different root stocks mentioned above (Ca. 750 plants) have been budded and grown in our greenhouse, and over 400 plants in 12 combinations have been planted in Scott Grove, and the remainding plants will be planted in September. All of the propagates have been tested for the presence of Las. Propagates carring a low titer of Las will be planted separately and serve as the experiment for “cross-protection”.
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. We have completed construction of the rhizotrons and split the roots of and transplanted trees into the rhizotrons. Part of the delay was because we constructed new Aluminum framed rhizotrons with hollow framing to allow easy collection of irrigation leachate for nutrient analysis. This will also allow them to be sterilized and reused in the second rep without the risk of the frames rotting before the end of the experiment, which became a problem with the wood frames used in prior experiments. The trees for the first rep of the experiment will be inoculated within the week and irrigation water treatments will begin.
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: 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. Previous quarter: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. Previously achieved in this project: 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.
In this study, we will pursue the following specific objectives:1) GFP labeling of Candidatus Liberibacter asiaticus. 2) Elucidation of plant-Las interaction through real-time monitoring of Las movement and multiplication in planta using GFP labeled Las. 3) Investigate the effect of different control approaches on the dynamic population of Las in planta using GFP labeled Las. Previously, the reporter plasmid, pBAM1::R-PgyrA-GFP, composed of Tn5 and narrow host-range origin was constructed and therefore the GFP gene can be inserted into the genome of bacteria. However, it was only successfully transferred into a genome of Pseudomonas fluorescence with low transformation efficiency and failed with other bacteria including Escherichia coli DH5a, Sinorhizobium meliloti Rm1021, and Liberibacter crescens BT-1. Recently, pDH3::PgyrA-GFP was constructed which has a wide bacterial host range replicon, repW, but cannot be inserted into a genome. Transformation of E. coli by PEG mediated method with pDH3::PgyrA-GFP showed high transformation efficiency (~2 x 104 CFU/µg of DNA) than with previous reporter plasmid (failed). Following application with L. crescens BT-1 by electroporation was also successful (1.9 x 103 CFU/µg of DNA). Transformants and the GFP expression in L. crescens BT-1 were confirmed by PCR and fluorescent microscopic analysis, respectively. As L. crescens is a phylogenetically closest species to Ca. L. asiaticus, there is a possibility that pDH3::PgyrA-GFP would be useful for GFP labeling of Ca. L. asiaticus. We have further confirmed the Lcr-GFP using western blot. The GFP plasmid is being used to transform Las. To facilitate Las transformation, we have tested multiple novel methods of culturing. Las population was observed to decrease at the beginning, and increase slowly. Repeated experiments show similar pattern which suggest we might be able to acquire enough Las cells for transformation after further optimization. We are testing new methods for culturing Las. Especially, we are testing co-culturing Las with citrus tissue culture and psyllid tissue culture. Currently, we are in the process of establishing a pure psyllid cell culture. We have used two approaches to label L. crescens. Preliminary data showed one approach works for Las in vitro. We are testing whether we can label Las in vivo and observe its movement. 2) We have conducted Las movement and multiplication in planta based on qPCR method. We have tested approaches to prevent Las movement in planta. In addition, based on the movement of Las in planta, we have developed a method for targeted early detection of Las before symptom expression. 3) We have been testing the effect of different control approaches including application with bactericides. One manuscript entitled: “Control of Citrus Huanglongbing via Trunk Injection of Plant Defense Activators and Antibiotics” has been published by Phytopathology. In addition, based on the movement of Las in planta, we have developed a method for targeted early detection of Las before symptom expression. A manuscript entitled Targeted Early Detection of Citrus Huanglongbing Causal Agent ‘Candidatus Liberibacter asiaticus’ Before Symptom Expression has been published by Phytopathology. We determined the in planta minimum inhibitory concentration of oxytetracycline against Candidatus Liberibacter asiaticus effective for control of citrus Huanglongbing which has been accepted with revision by Phytopathology. In addition, we further characterized the movement of Las in planta and the treatment effect of antibiotics using trunk injection and foliar spray. Two manuscripts are being prepared.
July 2019 The objectives for this proposal are 1) Conduct field trials of new products and fungicide programs for PFD management as well as validation trials for the Citrus Advisory System (CAS); 2) Investigate the reasons for the movement of Postbloom fruit drop (PFD) to new areas and recent major outbreaks; 3) Evaluate methods for initial inoculum reduction on leaves so that early fungicide applications could be more effective and identify the constituents of the flower extracts using omics techniques. Objective 1 was mostly covered by project 16-010C and activities are also reported there. In 2019, a field trial was set up and treatments were applied in a Valencia grove in Ft. Meade. There were few predicted infection events this year and only one application was made based on the PFD-FAD or CAS model predictions. Button counts were collected. There were also two CAS model validation trials and button data were also collected for both of these. Fruit data should be collected by the end of July for all the trials. The experiments with the wind tunnel located at the ARS facilities in Ft. Pierce have commenced although access is still limited as progress is made for the USDA screening process. We hope to finish the screening process in the very near future for greater access. Preliminary trials with Colletotrichum acutatum in the wind tunnel indicates that the spores are able to move considerable distances in the wind alone. The modelling to indicate possible distances and gradient are very preliminary but indicate that some modifications to the experimental set up may be needed including increasing the number of repetitions since the variability was high. Work on modelling of leaf wetness to better predict PFD outbreaks in Florida is on-going. Five FAWN weather stations were selected for this work and are equiped with leaf wetness sensors. The recorded data was compared to the output of four leaf wetness estimation models, singly and in combination. The most accurate models were considered for modifying the citrus advisory system (CAS). Analyses to look at the number of recommended sprays and model accuracy are underway. Experiments to assess the effect of available fungicides on the secondary conidiation of C. acutatum on citrus leaves. In the intial experiment, pyraclostrobin was used and no difference was seen in the secondary conidiation regardless if sugar or water were used for stimulation of conidia. In subsequent experiments, ferbam was used instead. To stimulate sporulation, leaves were exposed to sterile water or 2.5% sucrose solutions with or without ferbam. Once the treatments were complete, the leaves were coated with nail polish and the conidia and appressoria stripped from the leaf surface and counted. Ferbam affected the viability of the spores but not the number produced. It also signficantly reduced the number of appressoria. These techniques are being used to assess the effect of the water, methanol, and ethyl acetate extracts of the flowers. Preliminary trials with the extracts are underway to make sure that the experimental procedures developed for the fungicides will be adequate or if modifications will be necessary. Flowers were collected and extracted using water, methanol and ethyl acetate. The yields of all the extracts have been calculated for future reference. All the extracts have been dried for testing their antifungal effects. Once we receive the antifungal effects of the extracts, we will start the composition analyses. Preliminary metabolomics analyses are planned once some data from the fungal tests are available.
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.
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