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.
Leaf and soil nutrient analysis from a survey of 20-22 blocks in two flatwoods locations in Hendry and Collier County were compared for changes from the 2014 to 2015 season. The blocks were treated with 150 lb Tiger sulfur & 3.0 gal N-phuric acid/ton liquid fertilizer per acre (50% dry and 50% liquid) for three seasons following our recommendations for acidification since 2013. Acidification reduced soil pH by 0.6 units to 6.4 in the Hendry Co. location and by 0.3 units to 6.7 in the Collier Co. location. Soil Ca was increased in both locations; soil Mg as well as leaf Mg was unaffected by the pH drop. The increase in Ca availability and uptake was due to release of soil Ca as no fertilizer Ca was supplied. These results confirm the major benefit of soil acidification is to increase availability of Ca. Also following our recommendation increase in leaf Ca was achieved in flatwoods groves in Hardee Co. with low pH (<6.0) and soil Ca (< 500 lb/acre) by applying Ca sulfate (gypsum) at 1000 lb/acre as a soil amendment. Program on Management of soil and water bicarbonates, pH and nutrient availability was presented on December 2, 2015 as part of Indian River Citrus School at UF-Indian River Research and Education Center (IRREC) in Ft. Pierce. A recent analysis of groves management costs for acidification treatments reported a range from $50-75/acre (Singerman, A. and Muraro, R. 2015. Summary of 2014/15 Production Costs for Indian River Fresh Market Grapefruit and Southwest Florida Juice Oranges. UF-IFAS EDIS FE968, 10 pp)
During this reporting period (October, November, and December, 2015), the transgenic plants being produced for this project continued to grow at two different locations in secure greenhouses and growth chambers. Seven independently-transformed citrus plants carrying the FLT-antiNodT fusion protein expression construct are growing in Dr. McNellis’ lab at the Pennsylvania State University at University Park, PA, and an additional eight independently-transformed citrus plants carrying the FLT-antiNodT fusion protein expression construct are growing at Dr. Tim Gottwald’s lab at the United States Horticultural Laboratory in Fort Pierce, Florida. These plants are continuing to be propagated at both Ft. Pierce and Penn State. We now have propagated each line at Penn State with about 10 propagated trees rooted per transgenic line. In addition, we used genomic DNA analysis (Southern blotting) to confirm the presence of the anti-HLB gene in the genome of the grapefruit trees. Control plants that have been through the transformation process were also generated during the reporting period. These plants are the best comparison to the FLT-antiNodT plants in terms of plant behavior and disease resistance. These control plants will be sent to Penn State from Lake Alfred during the next reporting period. We call these the “transformation control” trees. Our collaboration with Dr. Janice Zale (University of Florida Mature Citrus Transformation Facility, Lake Alfred) to transform varieties important to the Florida citrus industry, including the ‘Valencia’ and ‘Hamlin’ sweet orange varieties and the ‘Citrumello’ and ‘Carrizo’ rootstocks with the FLT-antiNodT expression construct, continued during the reporting period. Hamlin and Carrizo transformants are now growing at Lake Alfred. Dr. Zale will maintain the original transformants, and will send propagated cuttings to Penn State soon. During the reporting period, Dr. McNellis applied for and was granted USDA permits to move sweet orange, rootstock, and “transformation control” trees to Penn State. This will set us up well for tests on these new trees.
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 gotten our early real-time PCR results. Using an activator construct pCAMBIA-2201-Cas9m4-VP16-EcR along with a sgRNA construct, pCAMBIA-1302-TFL-sgRNA-968 for one experiment, we have generated data from two different experiments. Statistical analysis awaits, but the early data suggest that instead of up-regulating TFL as predicted, we have slightly down-regulated the gene, suggesting that targeting the 5 UTR of the TFL gene does not allow the transient CRISPR machinery to work. Our follow up experiment is using a repressor, pCAMBIA-2201-Cas9m4-KRAB, to verify the extent to which we can down-regulate the gene and hopefully cause early flowering.
The project has two objectives: (1) Increase citrus disease resistance by activating the natural SAR inducer-mediated defense-signaling pathway. (2) Engineer non-host resistance in citrus to control citrus canker and HLB. For objective 1, we performed concentration gradient experiments to determine the lowest concentration of the natural SAR inducer, which is sufficient for canker resistance. We used 0, 0.25, 0.5, 0.75, and 1 mM of the SAR inducer, as we have found that 1 mM of the SAR inducer was able to induce strong canker resistance. We used both infiltration and soil drench to treat citrus plants with the SAR inducer. For infiltration, treated leaves were inoculated 24 hours later and for soil drench, leaves on the treated plants were inoculated 7 days later. As in the previous experiments, 5 plants were used for each treatment; three leaves on each plant were inoculated; 6 inoculations on each leaf were carried out, and a total of 90 inoculations were used for each treatment. Results showed that, for both infiltration and soil drench, the strength of canker resistance is concentration dependent in the range between 0 to 1 mM. Therefore, 1 mM is likely the concentration that should be used for inducing canker resistance. We will confirm this result in the coming season. Moreover, we found that treatment of citrus plants with the SAR inducer produced systemic residual resistance to canker. We cut back previously treated plants and tested canker resistance on leaves on the new flushes. Canker disease symptom development was significantly attenuated on the leaves on previously treated plants. We will confirm this interesting observation in the coming season. Meanwhile, we are designing experiment to determine whether the systemic residual resistance is conferred by the SAR inducer residue or products induced by the inducer. For objective 2, among 49 independent transgenic lines expressing the Arabidopsis nonhost resistance genes, 20 lines showed increased resistance to citrus canker. We have propagated 10 lines that exhibited good canker resistance in the first test. The progeny plants are growing in the greenhouse and will be tested when they are ready.
This project (Hall-15-016) is an extension of a project that recently came to a close (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 were hired during August and have been trained to establish and maintain colonies of infected psyllids, conduct qPCR assays on plant and psyllid samples, and run the inoculations. As of December 31, 2015, a total of 7,853 plants have passed through inoculation process. A total of 154,595 psyllids from colonies of CLas-infected ACP have been used in no-choice inoculations. 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. (in press)
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