Our project is based on the biology of quorum sensing of Candidatus Liberibacter asiaticus. The system here the two components system LuxR-AHL. The Genome of Candidatus Liberibacter asiaticus (CLas) revealed the presence of luxR that encodes LuxR protein, one of the two components typical of bacterial “quorum sensing” or cell-to-cell communication systems. Interestingly, the genome lacks the second components; luxI that produce Acyl-Homoserine Lactones (AHLs) suggesting that CLas has a solo LuxR system. In the current project, we will test the effect of AHL-producing citrus plants on the pathogencity of CLas. We have selected different Lux-I genes from different bacteria expressing different AHLs. In order to isolate the genes, we will obtain bacteria from the American type culture collection including Agrobacterium tumefaciens, Aeromonas hydrophilia, Agrobacterium vitis, Burkholderia cepacia, Chromobacterium violaceum, Panteoa stewartii, Pectobacterium carotovorum, Pseudomonas aeruginosa, Pseudomonas aeruginosa, Pseudomonas fluorescens, Rhizobium leguminosarum bv. Viciae, Rhizobium leguminosarum bv. Viciae, Rhizobium leguminosarum bv. Viciae, Rhodobacter sphaeroides, Serratia liquifaciens, Sinorhizobium meliloti, Vibrio anguillarum, Once we receive the bacteria we will culture them prior to insolate the genes by PCR. We will insert genes in CTV-based vector prior to the infiltration inside citrus trees. Our aim is to interfere with CLas signaling by expressing AHL which will enhance the bacterial aggregation and attachment and results in localization of the bacterium in certain branches.
This overall 3 year project was focused on determining the optimum combination of chemotherapy, thermotherapy, and nutrient therapy that can be registered for use in field citrus and control HLB. In this quarter (Jan 2016 to March 2015), we continue to evaluate 1) the effect of Pen and SD on control of HLB disease by gravity bag infusion in the field; 2) the efficiency of effective chemical compounds (Pen, SDX, Pcy and Carv) against HLB disease by gravity bag infusion; 3) the effectiveness of a combination of chemotherapy, thermotherapy and nutrient therapy against HLB in the field trials; 4) the efficacy of the new adjuvants to improve the uptake of antimicrobials. The chemical compounds (Pen and EBI-602) and additional nutrients were applied to the heat-treated citrus for three times by foliage spray, using our optimized nano-delivery system. The preliminary results showed that Pen was the more effective to control Las bacterium than EBI-602. The disease severity index (SDI) decreased by 6% after applied with Pen. The integrated practices (antimicrobial treatment coupled with heat treatment and nutrition fertilization) could decrease the fruit drop by 10~20 %, increase the fruit and juice weight by 3~13 %, and decrease the ratio of brix to acid by 0.2~5.0 %. The preliminary results from the other five antimicrobials (SD, Pen, SDX, Pcy and Carv) applied by gravity bag infusion showed that there were not different in the Las bacterial titers among the treated antimicrobials. Compared to the untreated plants, all antimicrobials reduced the Las bacterial titers, especially PEN. Both SD and Pen reduced the DSI through two years application. In last quarter, we tried to evaluate two new adjuvants (Bio and MF200) for improving the effectiveness of Pen by foliar spray. The preliminary results indicated that Pens formulated in both Bio and MF200 decreased the Las bacterial titers a lots. Ten antimicrobials were prepared in two different concentrations of the nano formulations (0.1 % and 1.0 %) in the greenhouse test. The Ct values kept over 36.o in the PEN-treatment. In next quarter, we will keep our application. Pcy and Carv will be changed application from trunk-injection to foliar spray. One papers has been published in the Crop Protection.
OVERVIEW: The Budwood Certification Program continues to build and develop the Foundation and Increase tree collection. Demand for budwood continues to increase and is anticipated to exceed 250,000 this year. PROGRAM OPERATIONS Texas Citrus Budwood Certification Program: 1. Budwood sales during the period were 229,664. Rio Red Grapefruit, Olinda and Standard Valencia Oranges, and Improved Meyer Lemon were the major varieties requested. 2. Much needed maintenance has been conducted on the main screenhouses. The old double poly roof on Screenhouses 1 thru 4 was replaced in November. A new layer of insect screen followed by a new layer of poly followed by aluminet shade cloth was put in place. 3. New roll-up curtains were installed in January to help protect the trees in the screenhouses during inclimate weather. 4. The project to upgrade Phase I of the Screen Structures will begin in the early summer. All existing trees will be removed and the screen and wood frame will be replaced with new galvanized metal frame and new insect screen. New Foundation trees will be planted in the structure when completed. 5. The Budwood Program Database continues to expand and is a key component in the management of the Texas program. The database program contains all records and information for all budwood production and sales, all foundation and increase trees, test results, and chemical applications. This database has the ability for an unlimited range of reporting information on the Texas program, including budwood orders, supply management, sales, testing, pesticide and fertilizer applications, budget analysis, and records needed for all compliance with TDA and USDA regulations. Stephenville Foundation Screenhouse : 1. New, clean Foundation trees continue to be added to established Foundation trees at the Stephenville greenhouse location. There are currently 75 Foundation trees consisting of over 60 different citrus species. Additional trees will be added as they are propagated to bring the total capacity to 100. These trees are being maintained as a reserve source of clean material for the Texas citrus industry. Texas Germplasm Introduction Program 1. The quarantine facility for the Germplasm Introduction Program is nearing completion and will be ready for USDA and TDA certification as a quarantine facility this summer. 2. Mark VanNess (Program Manager) and Sonia Del Rio (Lab Technician) visited the California CCPP in the fall to observe and receive training in their shoot-tip grafting program. Mark and Sonia visited Florida’s Germplasm Introduction Program in January and March to receive training and observe the Florida program. The new Texas program will operate based on the practices learned from both the California and Florida programs. Diagnostic Lab Testing – Foundation/Increase Trees: 1. All Foundation and Increase trees located in Weslaco and the Stephenville greenhouse were tested for HLB in October, 2015, and will be tested again for HLB and CTV in April, 2016. All tests were negative. 2. All Foundation Trees underwent virus/viroid testing in the fall of 2015. All test on all viruses and viroids were negative.
The first quarter of 2016 was very strenuous for Core Citrus Transformation Facility (CCTF). Two out of five employees left the facility in January and were eventually replaced by the new members of staff. CREC Center Director informed CCTF of possible move of the lab to the new site in March. Although March 24th was anticipated date for the move, that did not happen and CCTF still operates from its present location. CCTF received unprecedented number of orders (26) within the last quarter. Such a high volume of incoming orders called for an additional increase in production capacity of CCTF. I have purchased necessary consumables and tools for this transition and have taken steps to gradually ramp-up the input of starting material for experiments. However, uncertainty associated with possible move to new location prevented search for additional employees. I expect new recruit to begin working in the month of April. Partial increase in work load in March was little overwhelming for the present labor force and resulted in loss of some transgenic shoots and plants. I hope that when all aspects of CCTF functioning stabilize, so will the level of our production. The newly announced date for the move to new location is June 17th and I will try to organize it in such a way so that it will affect productivity of CCTF to the least possible extent. Between January and April, CCTF produced 57 plants. These plants belong to newer orders placed within the last 12-15 months. Four of the produced plants were Valencia oranges, three were Pineapple sweet oranges, eight were Carrizo citrange, and the rest were Duncan grapefruit. Transgenic rootstock plants carrying NPR1 produced in our facility are still in our greenhouse. They are at the stage when they could easily be propagated by cuttings. I am awaiting further instructions on what to do with these plants.
This is the final report for Project 13-926.3C. Over the last two years, we have been able to confirm that Citrus Leafminer Mating Disruption is a viable control solution for Phyllocnistic citrella. Data from 734 Citrus Holdings, Indian River Exchange Packers and The Packers of Indian River, all distributed across the state, confirmed that the performance of DCEPT CLM is in fact tremendously beneficial to CLM control. Performance across all of the trials, including monitoring data and damage evaluations, was consistent. This evidences the consistent and predictable performance of pheromone based mating disruption. After evaluating all of the data from this project, we can confirm that DCEPT CLM will quickly reduce populations of CLM and maintain control for up to twelve weeks. For these twelve weeks, pheromone monitoring traps will capture little to no adult CLM males because the populations are being controlled by the pheromone being released by DCEPT CLM. Additionally, damage will follow the same trend as the traps and will be equal or lower than farms that utilize conventional spray programs that spray as often as every two weeks. We also believe that DCEPT CLM, as a sole control input, can control CLM using a single application without CLM targeted conventional insecticide sprays for up to twelve weeks. Going forward, for farmers that plan to proceed with a single application of DCEPT CLM, our recommendation to farmers will be to target DCEPT CLM applications at the most important ten to twelve weeks of the citrus growing season. This will allow the farmer to have flexibility and choice, one where they can mix and match control strategies. For example, a farmers can protect their citrus during the highest pressure portion of the season with a DCEPT CLM application and protect the rest of the season with conventional insecticide sprays. The farmer could also apply DCEPT CLM twice which will cover nearly the entire susceptible season of Florida citrus. Unlike insecticide treated blocks, DCEPT CLM applications will also maintain the major advantage of residual performance. Although DCEPT CLM will not be able to eliminate damage after twelve weeks in the field, it will still maintain residual performance that keeps CLM populations lower than blocks treated with conventional insecticides. For example, at The Packers of Indian River, blocks that were treated with DCEPT CLM were able to maintain monitoring trap captures three to four times lower than blocks treated with insecticides every two weeks for an additional four weeks. Lastly, these three trials confirmed that DCEPT CLM performance can be maintained at essentially the same levels with applications in farms with tree densities ranging from 125-175 trees per acre. This confirmation is important because it will allow Florida farmers with various tree densities to adopt CLM mating disruption. We will now recommend that all farmers proceed with a minimum application rate of 125 DCEPT CLM per acre.
This project (Hall-15-016) is an extension of a project that came to a close last summer (Hall-502). The driving force for this project is the need to evaluate citrus transformed to express proteins that might mitigate HLB, which requires citrus be inoculated with CLas. USDA-ARS-USHRL, Fort Pierce Florida is producing thousands of scion or rootstock plants transformed to express peptides that might mitigate HLB. The more rapidly this germplasm can be evaluated, the sooner we will be able to identify transgenic strategies for controlling HLB. The purpose of this project is to support a high-throughput facility to evaluate transgenic citrus for HLB-resistance. This screening program supports citrus breeding and transformation efforts by Drs. Stover and Bowman. Briefly, individual plants to be inoculated are caged with infected psyllids for two weeks, and then housed for six months in a greenhouse with an open infestation of infected psyllids. Plants are then moved into a psyllid-free greenhouse and evaluated for growth, HLB-symptoms and Las titer, and finally the plants are transplanted to the field where evaluations of resistance continue. CRDF funds for the inoculation program 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 technician is assigned ~50% to the program. USDA provides for the program two small air-conditioned greenhouses, two walk-in chambers, and a large conventional greenhouse. Currently 18 individual colonies of infected psyllids are maintained. Some of the individual colonies are maintained on CLas-infected lemon plants while others are maintained on CLas-infected Citron plants. Update: Two technicians funded by the grant have been fully trained in establishing and maintaining colonies of infected psyllids, conducting qPCR assays on plant and psyllid samples, and running the inoculations. As of March 17, 2016, a total of 8,169 plants have passed through inoculation process. A total of 160,395 psyllids from colonies of CLas-infected ACP have been used in no-choice inoculations. Not included in these counts of inoculated plants and psyllids used in inoculations are many plants inoculated over the past year to assess transmission rates, which has provided insight into the success of our inoculation methods and strategies for increasing success. Research concluded during September 2015 showed that seedling citrus with flush is significantly more prone to contracting the HLB pathogen than seedling citrus without flush: Hall, D. G., U. Albrecht, and K. D. Bowman. 2016. Transmission rates of ‘Ca. Liberibacter asiaticus’ by Asian citrus psyllid are enhanced by the presence and developmental stage of citrus flush. J. Econ. Entomol. doi: 10.1093/jee/tow009. Therefore, the program has been changed to ensure that plants to be inoculated have flush. Current research indicates that the no-choice inoculation step used in our program is successful 75 to 95% of the time when approximately 75% of ACP placed on a plant test positive for CLas and have CLas titers of around CT=26 to 29 (success contingent on flush being present on a plant).
We continue to produce transgenic, mature citrus trees and transfer them to scientists (Drs. Dutt, Louzada, McNellis, Mou, Wang) as soon the primary or secondary grafts heal. Mature scion transformation efficiencies have increased to 7.6%, and micrografting efficiencies have improved to 77%. Approximately 154 transgenics (primary transgenics and vegetative progeny) have been transferred to Dr. Dawson’s lab for additional testing, and another ~50 will be transferred next month. For out-of-state transport of transgenics, USDA APHIS permits were obtained by scientists prior to shipping. Shipping certification was also obtained through UF. Transgenic, mature citrus has been shipped to Dr. McNellis at Penn State. A manuscript was submitted to a scientific journal describing biolistic transformation of immature citrus rootstock, never previously reported in the literature. Biolistic transformation to produce transgenics will augment those produced with Agrobacterium. A rapid, high throughput, nondestructive MUG assay is being developed to screen whole putative transgenic citrus shoots for GUS expression. It is quantifiable and more sensitive than using X-Gluc as substrate. Fluorescence can be quantified on a plate reader, or visualized on a gel doc with known controls. It is anticipated that GUS expression will correlate to copy number similar to NPTII expression. It remains to be determined whether the shoots will survive immersion in the MUG substrate and subsequent micrografting, but minimal exposure to the substrate, followed by rinsing, might not be harmful. Data are being collected describing the method for potential publication. We are still optimizing the PMI selectable marker using biolistics and Agrobacterium transformations in immature and mature citrus transformation. The results so far look promising and shoot growth doesn’t appear to be negatively impacted like shoot growth on kanamycin medium. Sour orange and Volkameriana seed have been purchased for the growth room because seed of our preferred rootstock varieties have been sold-out. It remains to be determined if these varieties perform well in the growth room.
During the period of project 767 funding, we accomplished the following: 1) screened Liberibacter crescens against a wide variety of antimicrobials that we predicted to be phloem mobile (this was done independently of a contract from CRDF to assess compounds of CRDF’s choosing); 2) worked with Erik Mirkov of Texas A&M to determine the efficacy of oxytetracycline and streptomycin against tomato yellows disease caused by Ca. L. solanacearum; 3) encouraged and worked with NuFarm to test the efficacy of oxytetracycline in the field against HLB; 4) mutagenesis of L. crescens identified 314 genes that are essential for growth in culture, of those 238 have homologs in Ca. L. asiaticus and can be considered excellent candidates for antimicrobial development; 5) developed a list of seven antimicrobials that are excellent candidates as second generation compounds for treatment of HLB in the field. A paper describing our findings in activities one through three above is in preparation and we hope to submit it soon. A paper on activity four is in revision in Frontiers in Microbiology. The major findings from these activities are as follows: 1. Three classes of phloem-mobile antibiotics were found to be highly effective against L. crescens: the cephalosporins, the penicillins, and the tetracyclines. The cephalosporins are still used widely in medicine making regulatory approval for citrus use difficult. Penicillins often cause allergic reactions in humans making them difficult to use on a fruit tree crop. The tetracyclines appear to be a very viable option for HLB since oxytetracycline has already been approved for use on bacterial diseases of fruit tree crops. 2. Erik Mirkov of Texas A&M showed that oxytetracycline, but not streptomycin, is very effective against tomato yellows disease. Thus, we recommended that oxytetracycline be tested in the field for control of HLB. 3. NuFarm showed HLB symptom relief in field trials in 2014 and 2015 using oxytetracycline. Streptomycin was not effective in these trials. Since streptomycin is not predicted to be phloem-mobile, we were not surprised by the failure of streptomycin to relieve Liberibacter-induced disease symptoms on tomato and citrus. 4. Thanks to our work on the essential gene list of L. crescens, we now have 238 excellent targets in L. asiaticus for antimicrobial action. This list of genes will soon be in the public domain as a paper describing this work is expected to be published soon in the open access journal, Frontiers in Microbiology. 5. Based on all of the above and on the properties of many antimicrobials tested against L. crescens, we have proposed that a second generation of antimicrobials be tested in preparation for their use in HLB control. These include sulbactim and thiamphenicol and other compounds that require the involvement of other parties. We expect all of them to be effective but have varying degrees of regulatory hurdles. All need to be tested in the tomato yellows assay followed by testing in the field on infected citrus trees. Our view is that a second generation of antimicrobials needs to be available: a) to be used in rotation with oxytetracycline and streptomycin to slow the appearance of resistance to these compounds and b) to replace oxytetracycline and/or streptomycin when resistance dominates in the L. asiaticus population.
This proposal is aimed at following previous work in CRDF-710 and CRDF-818 with a series of precise experiments that will: 1. Elucidate the nature of the HLB signal(s) 2. Provide additional evidence on its transmission in terms of movement across tissues and between trees though underground organs. 3. Determine the progression of physical symptoms from its inception. 4. Examine the in-tree variation in CLas titer. All experiments were completed and final testing done in December. 1. To test for he unlikely, but increasing, possibility that HLB is transmitted by extracellular vectors, we isolated DNA from HLB leaves and inject these into 2 year old Valencia trees. The trees are being kept in a greenhouse and are under observation. Trees continue growing normally. Trees tested in September 11, one tree tested HLB+, though a high PCR value. Trees were retested again late December and all tested HLB-. 2. Experiments for objective 2 are well under way. Two trees (one healthy and one HLB+) were root grafted in three different locations and placed in special pots large enough to accommodate the 2 trees (5 pairs). The trees were placed in a greenhouse and kept under observation. PCR analyses were conducted once more in September 2016. At this time, 3 out of the 5 pairs of the initially healthy trees tested positive, although clear visible symptoms were not evident in all cases. By the end of December, 4 of originally healthy trees tested HLB+. One remains under observation. 3. Grafted trees with HLB material are being monitored weekly using Narrow-band imaging under polarized illumination. Although we continue to have issues with the background, we have established a standard curve and a correlation relationship between starch levels, PCR values, and polarized light readings. 4. Trees have been grafted for a substantial amount of time and some are showing HLB symptoms. However, given that analysis of this objective destroys the trees, only trees with clear symptoms are tested. PCR analyses was conducted in a total of three trees that showed symptoms and tested HLB+. In any of the trees, there was correlation between PCR values and leaf position. These possible results prompted the design of a new system for HLB determination with a much higher level of precision.
Our project aims to provide durable long term resistance to Diaprepes using a plant based insecticidal transgene approach. In this quarter, 30 additional transgenic lines were analyzed for gene expression using qPCR. Of them, 10 were determined to be high expressers while the rest were medium to low in expression. Most lines tested negative for the transgene in the aerial leaf and stem while there was gene expression in the roots. In addition, a number of additional lines have been transferred from in vitro medium and acclimated to greenhouse conditions. We expect to begin propagation of the larger plants in the next quarter for challenge with Diaprepes neonates.
Our goal to examine expression of transcripts in host Sinorhizobium meliloti (Sm) due to introduced Candidatus Liberibacter asiaticus (CLas) transcription factor genes. These data will lead us to identify targets to screen for novel anti-bacterials. Between September and December 2015, we have analyzed the first Affymetrix GeneChip experimental data to reveal targets for Clas RpoH in S. meliloti. We have also completed construction of clones and mutants for several more of our target genes. RpoH results: Using RNA from the S. meliloti rpoH1H2 mutant strain expressing CLas rpoH or from control strains, we prepared cDNA and probed a genomic AffyChip. We found 20 genes activated over 2-fold by CLas RpoH and 39 genes activated by Sm RpoH1. There were 14 genes shared between the two, that is, expressed by both types of RpoH1. Based on these results, we can identify strong candidates for CLas RpoH target gene fusions. Our top 3 candidate promoters for fluorescent fusions to use in high-throughput compound screening are: ibpA, clpB, and groES5. We already have uidA (GUS) and mCherry (red fluorescent protein) transcriptional fusions to these genes. We have also made progress in constructions for analyzing 6 transcription factors from CLas . We have constructed visRN, ldtR, and lsrB mutants in S. meliloti. Each of these is being systematically tested for growth and other functions with the cloned (synthetic) corresponding CLas gene. As previously reported, we found some function of CLas LsrB copmlementing slow growth of S. meliloti .lsrB in complementation of slow growth. We further discovered that S. meliloti .lsrB is sensitive to the cell envelope disrupting agent, deoxycholate (DOC); we demonstrated that overexpression of CLas lsrB partially restores growth of the Sm .lsrB mutant on DOC. In another case, we found that Sm .visNR has a severe motility defect in culture. Overexpressing CLas visNR restores motility, demonstrating that the synthetic CLas visNR is functional. We have successfully constructed phrR1 and phrR2 single mutants and are in the process of constructing the double mutant. As we described in the last report, construction of a ctrA deletion strain is complicated since ctrA is an essential gene. Our recent work revealed another surprise, which is that the cloned CLas ctrA gene is deleterious to survival of S. meliloti wild type cells when grown on standard rich medium (LB). We can maintain the plasmid in the cells despite this, because the CLas ctrA gene is on a regulatable promoter. Without IPTG inducer, no CLas CtrA protein is produced and the S. meliloti cells are viable; at high IPTG, the cells die. By using alternative media and low levels of inducer, we are currently testing whether the CLas ctrA gene can be modulated to supply appropriate function to S. meliloti. While this surprising result complicates our strategy, it opens up another possible avenue for chemical discovery. We might ask the question, are there any compounds in our chemical library that would interfere with the toxicity of CLas CtrA to S. meliloti? We will explore this possible design.
The objective of this is to use “new technologies” to accelerate the elimination of graft transmissible pathogens in germplasm accessions for use in citrus breeding in Florida. These “new technologies” include the application of cryotherapy (freezing the buds in liquid nitrogen followed by recovery of the treated buds by grafting onto seedling rootstocks) and the use of “mini-plant-indexing” which allows the biological indexing for graft transmissible pathogens using young seedling indicator plants, 60-75 days old seedlings. During the reporting period advanced citrus selections were passed through cryo treatment. Shoot tips of each selection have been recovered following treatment to determine viability. CLas-infected bud eyes were passed through cryo-treatment in the previous rating period. Plants recovered from these shoot tips are to be transfered to the USHRL where they will be tested for CLas and observed for development of HLB symptoms. Seven promising USDA promising new scion selections were propagated and readied for shipment to Ft. Collins. These selections were passed through cryo treatment and are in cryogenic storage. During the next quarter plants will be regenerated from cryo treated tissue to determine recovery and estimate % of viable shoots tips in storage. Regular shipments of additional selections from the USHRL to Ft. Collins will continue throughout the remaining duration of this project.
This quarter, using Cas9m4, with conjugated activation or repression domains, we intended to modify the expression of citrus proteins responsible for regulating flowering, namely TERMINAL FLOWER-1 (TFL), in order to reduce juvenility. TFL is a repressor of flowering and has been shown to inhibit flowering when overexpressed and to increase flowering when enhanced in Arabidopsis thaliana. We want to down-regulate TFL transiently, so we intend to decrease maturation times and do so without the use of transgenic insertion that is deemed unfavorable. For this quarter, we have run two different time course experiments, one with Agrobacterium and the other using cell penetrating peptides (CPPs). The time course experiments were performed on three different citrus varieties: ‘Duncan’ grapefruit for the Agrobacterium experiment and ‘Pineapple’ sweet orange and a trifoliate cultivar ‘812’ for the CPP experiment. For both experiments, they plants were microinjected with the Cas9 repressor of and a sgRNA construct target the 5′ UTR of TFL. Control solutions were included. Sample leaves that had been treated with the experimental or control treatment were removed for a period of up to five days. After which, the leaves were harvested for their RNA and cDNA preparations were made. The real-time analysis run on these samples has been performed and the results are being investigated for accuracy. For the next quarter, we hope to have good, clear results of the experiments above. While perfecting the real-time analysis of the data more experiments will also be run to further test our CRISPR/Cas9 transient expression system in citrus.
The objective of this is to use “new technologies” to accelerate the elimination of graft transmissible pathogens in germplasm accessions for use in citrus breeding in Florida. These “new technologies” include the application of cryotherapy (freezing the buds in liquid nitrogen followed by recovery of the treated buds by grafting onto seedling rootstocks) and the use of “mini-plant-indexing” which allows the biological indexing for graft transmissible pathogens using young seedling indicator plants, 60-75 days old seedlings. During the current reporting period, budwood from seven promising scion selections from the USDA citrus improvement project were sent to the germplasm preservation laboratory in Ft. Collins. Each of those selections have been passed through cryo treatment and are being held in cryo storage at the Ft. Collins facility. This brings the total number of USDA advanced scion selections in cryo storage to 16. Of all selections that are in cryo storage there has been and average success rate (based on number of successfully recovered buddlings / total buddlings) has been ca. 50%, although results do vary between selections with the least successful being 10% and the most successful 80%. Based on the percent success and the total number of shoot tips stored it is estimated that at least 10 viable shoot tips will remain viable for each selection. Some of the selections from previous cryo treatment have been returned to Ft. Pierce and are being grown in the greenhouse to evaluate trueness to type. Our permit to receive material in Florida from the germplasm preservation laboratory in Ft. Collins has expired so we are renewing the permit to allow movement of the cryo treated material back into Florida for evaluation. Additional selections have been rescued from the field since the last reporting period and these will be cryo treated and preserved. The cryo therapy and preservation approach has proven to be a viable method for storage of citrus germplasm. Although we have yet to determine if cryo therapy will eliminate graft transmissible pathogens, the treatment is effective for germplasm preservation. The advantages of cryo treatment include security of the material, the small footprint required for storage and elimination of the need to store selections as whole plants. Comparison of results with the mini-plant index protocol with the results of indexing using the traditional, 10-14 month old indicator plants on a total of 48 accessions has been conducted. The results have been the same except in one instance when symptoms of Vein enation virus was found in the mini-plant index but not in the traditional index. We will continue to evaluate plant material recovered from cryo treatment for the presence of CLas as well as other graft transmissible pathogens.
Previously our model for the appearance of symptoms was based on the assumption that inoculum accumulates at a rate proportional to the number of infected nymphs present in the citrus trees. Comparisons of these simulations with data from Southern Gardens showed that this model was not accurate. We have now implemented a model where the rate at which symptoms develop is proportional to the amount of inoculum in the tree. Furthermore the inoculum in the tree decays at some rate. We are continuing to refine this model for the appearance of symptoms and need to investigate what the carrying capacity for inoculum is. We will investigate the impact of a local carrying capacity at the flush level as well as a carrying capacity for the whole tree. This model will continue to be refined using data from Southern Gardens and through discussions with plant pathologists to understand the phloem system and its function in symptom development. We have had discussions with statisticians on the design of field trials to answer questions regarding how many constructs can be used in the field trial while still differentiating between constructs. Based on initial simulations from the previous model, it seems that at least four constructs can be used while still differentiating between constructs. As changes are made to the model for symptom appearance, we will need to run more simulations to determine how many constructs can be used.
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