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.
Three Valencia-type clones (N7-11, B7R9T35 and B7R9T36) with excellent fruit quality were discovered in our collection (older trees) that have no HLB symptoms and are PCR-negative for Liberibacter. Trees of these clones have been propagated for further study. Juice quality, as assessed by a large citrus juice company in Florida, was found to be excellent. We continued to monitor performance, assess HLB severity, tree growth and yields of late maturing sweet oranges at different field sites throughout Florida. Also, we received seeds from collaborators in Sicily of rootstock candidates that showed good preliminary performance at St. Helena, as well as some new candidates of identical parentage for future trials. Seeds were planted to produce larger numbers of trees for extended trials. Gauntlet rootstock screening: Approximaly 75 HLB+ Valencia budstick-grafted hybrid rootstocks already passed through the ‘hot psyllid’ house were planted at Picos Farm in Ft. Pierce. Three parental combinations from 2013 crosses produced numerous hybrids that are growing off beautiful trees from grafted HLB+ Valencia budsticks: C2-5-12 pummelo x C. latipes (papeda); Amblycarpa+HBPummelo x Sour orange+rangpur; and Amblycarpa+HBPummelo x White #1. Stick-grafting and production of rooted cuttings from 2013 gauntlet crosses has been completed. The two early-maturing (December) Valencia somaclones identified, B7-70 and SF14W-65, have been cleaned up and a few pathogen-free trees were provided by the Parent Tree Program (DPI). We are following our plans for rapid pathogen-free budwood increase. We are moving quickly to have a substantial population of budwood increase trees available for nurseries at the time of the official release announcement. These clones have been put up for official release approval this summer, 2015.
Three Valencia-type clones (N7-11, B7R9T35 and B7R9T36) with excellent fruit quality were discovered in our collection (older trees) that have no HLB symptoms and are PCR-negative for Liberibacter. Trees of these clones have been propagated for further study. Juice quality, as assessed by a large citrus juice company in Florida, was found to be excellent. We continued to monitor performance, assess HLB severity, tree growth and yields of late maturing sweet oranges at different field sites throughout Florida. Also, we received seeds from collaborators in Sicily of rootstock candidates that showed good preliminary performance at St. Helena, as well as some new candidates of identical parentage for future trials. Seeds were planted to produce larger numbers of trees for extended trials. Gauntlet rootstock screening: Approximaly 75 HLB+ Valencia budstick-grafted hybrid rootstocks already passed through the ‘hot psyllid’ house were planted at Picos Farm in Ft. Pierce. Three parental combinations from 2013 crosses produced numerous hybrids that are growing off beautiful trees from grafted HLB+ Valencia budsticks: C2-5-12 pummelo x C. latipes (papeda); Amblycarpa+HBPummelo x Sour orange+rangpur; and Amblycarpa+HBPummelo x White #1. Stick-grafting and production of rooted cuttings from 2013 gauntlet crosses has been completed. The two early-maturing (December) Valencia somaclones identified, B7-70 and SF14W-65, have been cleaned up and a few pathogen-free trees were provided by the Parent Tree Program (DPI). We are following our plans for rapid pathogen-free budwood increase. We are moving quickly to have a substantial population of budwood increase trees available for nurseries at the time of the official release announcement. These clones have been put up for official release approval this summer, 2015.
The UF/CREC Citrus Breeding and Genetics Program is currently focused on 4 compelling and critical objectives: 1. Development of rootstocks that can impart HLB tolerance/resistance to grafted scions. 2. Breeding of HLB tolerant/resistant processing sweet orange-like hybrids. 3. Screening of the UF-CREC germplasm collection to identify and validate HLB tolerant or resistant selections. 4. Advanced field trials, release and commercialization of promising HLB tolerant/resistant scion and rootstock cultivars. This project is a continuation of previous citrus breeding and genetics efforts, with a heightened and renewed emphasis on developing cultivar options for Florida citrus growers that can enable a greater likelihood of sustained viability and profitability in the face of endemic HLB. The project began on 1 November 2015. In the late autumn and early winter, we collected data from several ongoing field trials of rootstock cultivars throughout the state. Trees were assessed for HLB incidence and severity through all plantings, and yields and fruit quality was determined in selected replicated trials. Hybrid families planted in the field were evaluated to identify selections producing fruit that resembled sweet orange in appearance, and exhibiting few or no HLB symptoms. Fruit from the best of these were tested for juice quality and a few were also analyzed for volatile components and compared with standard sweet orange. Fruit samples were provided to a major juice company for processing and assessment of the juice for flavor. The entire collection of raw material produced by the breeding program and growing in groves at several different locations throughout the growing areas of Florida were assessed for HLB symptoms, to identify a subset of individuals displaying few or no symptoms of HLB, regardless of parentage. A new large field trial with more than 20,000 trees on over 100 previously identified and new rootstock candidates was prepared and planted.
The goal of this project was to determine the feasibility of developing a high-throughput screen of citrus seedlings to identify naturally resistant plants based on optical sensing. Although the project initially focused on fluorescent imaging, the use of polarized light proved more advantageous. The buildup of starch in the leaves is a hallmark of the disease, and starch grains rotate reflected light having a wavelength around 595 nm by 90 . The polarized images were acquired using a conventional DLS camera. The strategy was to infect seedling by exposure to a population of psyllids that were positive for CLas (Candidatus Liberibacter asiaticus). Employing the natural method of inoculation has the advantage of detecting seedling resistance at all stages of the process, from alterations in psyllid feeding behavior to resistance to the bacterial pathogen. Seedlings of sweet orange were exposed to infected psyllids for eight weeks and allowed to recover for either two months or four months before image acquisition and analysis of HLB status by quantitative PCR. Image analysis was conducted by manually segmenting the images and analyzing pixel distributions. A total of seven different texture feature sets were identified and assessed in pairwise comparisons using a support vector machine (SVM) approach to predict the HLB infection status. In comparisons among seedlings that had all been exposed to infected psyllids, the accuracies ranged from 59.79% (moderately vs. strongly infected) to 98.06% (questionable vs. weakly infected) depending on the comparison. Previous studies employed clearly symptomatic leaves; whereas, the current study included pre-symptomatic leaves which is a more difficult task. The present study imaged leaves on seedlings where angles and distances to the camera are variable, which is challenging compared to studies using individually mounted leaves. In future studies, increased prediction accuracies seem likely if distances of the leaves to the camera lens are incorporated to normalize image texture. Also, our preliminary results with fluorescence imaging and recent literature suggest that prediction accuracies could be significantly improved by combining fluorescence and polarization imaging to create an index of infection.
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.
Citrus Huanglongbing (HLB) poses the greatest threat to the survival of the Florida and US citrus industry. Research to incorporate HLB resistance/tolerance into citrus has been recommended by the National Research Council as one of the top priority topics for addressing the HLB threat. A number of Poncirus and Citrus cultivars have been recently found to be tolerant to HLB. Identification, characterization and validation of candidate genes responsible for HLB-resistance are of the remarkable value to develop new HLR-resistant/tolerant citrus varieties. More than 70 different accessions of Citrus species and relatives have been observed in a field planting in Florida (with cooperation of Dr. E. Stover, USDA). Leaf samples were collected from about 450 trees. The titer of CLas in the HLB-infected plants was determined by TaqMan probe-based qPCR, and the HLB symptom severity on the plants was evaluated by experienced researchers. These accessions showed diverse responses (susceptible, tolerant and resistant) against HLB. Many HLB-tolerant citrus relatives were revealed, including Citrus latipes, Poncirus trifoliata, Severinia buxifolia and Microcitrus australis. Interestingly, S. buxifolia plants did not show severe HLB symptoms yet had relatively low Ct values when assayed for CLas titer. Two general trends observed so far were increased Ct values on the same trees in the summer than in the spring or fall and the inconsistent relationships between stem diameter and shoot growth and HLB severity across a diverse group of Citrus, Poncirus, and distant relatives. To identify sequence polymorphisms in candidate genes and assess their association with HLB tolerance/resistance, the genomes of 15 citrus accessions have been sequenced to about 25x coverage (9 to 11 Gb/genome) and the genome of one Poncirus trifoliata accession has been sequenced to about 50x coverage (~21 Gb). We have detected 614 nucleotide-binding site – leucine-rich repeat (NB-LRR) genes with more than 50% coverage in the re-sequenced genomes, and 149 NB-LRR genes with more than 95% coverage in re-sequenced genomes. No NB-LRR genes were lost in the re-sequenced genomes. This superfamiliy of genes represents the largest group of disease resistance genes in plants, conferring resistance to bacterial, fungal, viral and nematode diseases. Most of the NB-LRR genes confer disease resistance in other plants in a race-specific fashion, but some render broad-spectrum and durable quantitative disease resistance. Single nucleotide polymorphisms (SNPs, one of the most common sequence polymorphisms) were found in 330 NB-LRR genes of Poncirus trifoliata DPI-50-7, 98 NB-LRR genes of Poncirus trifoliata Flying Dragon, 156 NB-LRR genes of Poncirus hybrid US-897 and 107 NB-LRR genes of Poncirus US-812. We also detected 72 genes from another two gene families that have more than 90% coverage in the DPI-50-7, Flying Dragon, US-812, and US-897. Real-time PCR was performed with HLB-resistant and HLB-susceptible samples to detect the relative change in the expression of these candidate genes. Results so far showed that several candidate genes were up-regulated with higher expression values in HLB-tolerant Poncirus genotypes when compared with HLB-susceptible citrus genotypes.
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.
Objective 1. (Greenhouse experiment): seed was extracted, seed coats removed using a chemical-removal procedure and planted of the needed rootstocks: x639, Swingle, WGFT+50-7 and UFR-3. Seed germination is underway. Objective III: To evaluate the effect of complete, balanced and constant nutrition on HLB-affected mature trees (composition, delivery and economics). The trial sites have been confirmed at two locations: Peace River Packing groves, Fort Meade and Orange Co, Arcadia. The trial sites have been surveyed and experimental setup has been custom designed for two locations and both the trials (CRF+ Tiger Micronutrient and hybrid fertilizer). The control release fertilizer has been formulated for both the trials and has been acquired from Harrell s and H.J. Bakers. Currently, data is being collected for before treatment time point. Data collected includes tree canopy measurements, leaf area index, leaf and soil nutrient levels, tree health, and HLB qPCR test). First fertilizer application will be made in second week of February at both the locations. Ojective 5. (funded by Orie Lee, using donated fertilizer products): Alligator Vernia/Rough Lemon Enhanced Nutrition Experiment Treatments: 6 tree plots (randomized), 2 plots per treatment treatments 2 times per year. The first treatment was applied first week of October, 2015. 1. Control no extra nutrition 2. Harrells St. Helena mix (2lbs per tree) 3. Harrells St. Helena mix (2lbs.)+ 2x TigerSul manganese (90 gm) 4. Harrells St. Helena mix (2lbs.) + 2x Florikan polycoated sodium borate (32 gm) 5. Harrells St. Helena mix (2lbs.) + 2x TigerSul manganese (90 gm) + 2x FL sodium borate (32 gm) 6. 4x TigerSul manganese (180 gm) 7. 4x Florikan polycoated sodium borate (64 gm) 8. 4xTigerSul manganese (180 gm) + 4x Florikan polycoated sodium borate (64 gm) Baseline yield data was taken from each plot, just completed (January, 2016).
The general goal of this project is to rapidly propagate complex citrus rootstock material for field testing. The rootstock materials to be tested will be products of the Citrus Improvement Program at the UF-IFAS-CREC in Lake Alfred. Dr. Jude Grosser has assumed responsibility for completing the project, following the resignation of Dr. Barrett Gruber. Ms. Amy Dubois is the OPS assistant taking care of the trees at the IRREC, she will continue in this roll. The inventory of recovered liners that could make a good field tree are provided below. These will be grown to grafting size, grafted with selected scions (including a dark red grapefruit somaclone N11-15 showing possible tolerance/resistance to HLB) and planted in IR field trials. Additionally, we now plan to include selected new cybrid grapefruit clones (Flame, Ruby somaclone N11-11, and White Marsh) that contain cytoplasm from ‘Meiwa’ kumquat as scions. These new grapefruit clones have shown significantly improved canker tolerance in greenhouse assays as compared to traditional grapefruit clones, and they are not GMO. Having them in the field on improved rootstocks will expedite determining their true canker resistance, as well as their response to HLB. Dr. Ahmad Omar will assist with grafting. Viable cutting inventory: Rootstock # of liners recovered 1. A+HBPxCH+50-7-12-14 44 2. 46×31-00-S10x46x31-00-S11-S5 78 3. Orange 10 x Green 7-11-1 52 4. A+VolkxOrange19-11-5 90 5. A+HBJL2BxOrange14-09-7 71 6. A+HBJL2BxOrange19-09-31 14 7. A+HBJL1-09-14 25 8. A+FDxOrange19-11-11 50 Viable seedling inventory: 1. 46×20-04-S22 86 2. 46×20-04-42 94 3. 46×20-04-48 78 4. 46×20-04-S13 86
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.
Excellent progress was made in continuing the development of new hybrid rootstocks for the Florida citrus industry. As requested by CRDF, project 15-002 will place highest priority on hybrid rootstocks being considered for release to growers over the next six years, including about 400 Supersour-type rootstocks. It is expected that at least one of these SuperSour rootstocks will be released within three years, with more to be released in the following years as further information is collected from ongoing field trials. During this quarter, six new replicated rootstock field trials were planted, including trials on flatwoods and ridge sites. Emphasis in the new trials was on SuperSour selections, although standard rootstocks and previously released USDA rootstocks were included to allow good comparisons for relative assessment of field performance. The new plantings included two trials with Hamlin scion, two with Valencia scion, and two with specialty scion cultivars. Analysis of results from previously established trials indicates that HLB introduces additional variability to trials that requires more than the previously effective five or six statistical replicates to provide clear evidence of rootstock performance. Based on this observation, new USDA rootstock trials will usually include more than six statistical replications, regardless of the number of trees per replication. Using statistical comparisons is essential to develop reliable information about relative rootstock performance in field trials. During this quarter, trees in field trials were scored for health, HLB symptoms, and samples were collected from some groups for PCR detection of Las infection. Typically, trees in plantings with good ACP control remain nearly free of Las infection for the first 1-2 years, regardless of surrounding tree infection. Because of this, rootstock HLB tolerance can only be assessed when trees in field trials are more than 2-3 years old, or when special steps are taken to ensure trees become infected earlier. During this quarter, yield and fruit quality data were taken from 8 rootstock field trials with early maturing scion varieties. Analysis was completed on data from several established trials to assess relative rootstock performance, rootstock effects on yield, fruit quality, tree size, and HLB symptom development. A comprehensive presentation on standard and new rootstocks was made at the Florida Citrus Show. A new publication was prepared, providing a comprehensive comparison of the new USDA rootstocks with other standard rootstocks, and will be submitted for publication in the next quarter. Trees in the USDA nursery on a large number of advanced rootstock selections, especially SuperSour-type, were continued in propagation for field trials to be planted in 2016. Nursery experiments were conducted with promising new rootstocks to determine nursery-related traits important for commercial use. Cooperative work continued with commercial nurseries involved with micropropagation, to facilitate more rapid deployment of the best new rootstocks. Greenhouse experiments continued to assess rootstock tolerance to HLB, CTV, and high pH. Cooperative work continued with UF researchers and a commercial nursery to propagate trees for use in multiple rootstock field trials sponsored by the HLB MAC program. Trees from the commercial nursery are scheduled to be planted into six cooperative field trials in 2016, and six more field trials in 2017.
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