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)
The main accomplishments during this quarter: We have confirmed the K gene overexpression-mediated improvement on transformation efficiency of a lemon cultivar we used. We have tested the effects of the K gene on genetic transformation efficiencies of 6 citrus cultivars and we observed 3-15 fold increases if compared to our control vector, and 3-11 fold increases if compared to the highest transformation efficiencies of the same cultivars previously reported by others. We have observed and confirmed the stimulatory effects of one non-conventional regulator of gene expression on shoot regeneration efficiencies of some citrus cultivars. We have been testing the effects of that factor and other factors in combinations of the K gene on transformation efficiencies of both mature and juvenile citrus explants and our preliminary results suggest that there are significant improvements in transformation efficiency for both juvenile and mature tissues. We are also repeating the effects of endogenous auxin and the auxin transport on efficiencies of shoot regeneration and Agrobacterium-mediated infection of mature tissues of citrus. One manuscript reporting the drastically improvement of six citrus cultivars including a lemon cultivar has been accepted for publication in “Plant Cell, Tissue and Organ Culture”. The article is currently in the production stage and should be out in either February or March issue.
Objective 1: Assess canker resistance conferred by the PAMP receptors EFR and XA21 Three constructs were used for genetic transformation of Duncan grapefruit and sweet orange as part of a previous grant: EFR, EFR coexpressed with XA21, and EFR coexpressed with an XA21:EFR chimera. Putative transgenics are currently being verified by PCR in the Jones lab, and five PCR positive plants have been identified so far. To ensure that there will be sufficient events to analyze to come to a conclusion about the effectiveness of these genes, we will initiate more transformations in Duncan grapefruit at the Core Citrus Transformation Facility at UF Lake Alfred. EFR, XA21, and XA21 + EFR constructs have been re-created with the inclusion of a GFP marker for identification of transformants. Objective 2: Introduction of the pepper Bs2 disease resistance gene into citrus Constructs are being created in the Staskawicz lab to express Bs2 under the 35S promoter and under a resistance gene promoter from tomato. Constructs are also being created in which Bs2 is co-expressed with other R genes that may serve as accessory factors for Bs2. Constructs with tagged Bs2 have been confirmed to function in transient assays, and have been transformed into Arabidopsis. Protein expression will be confirmed by immunoblot. GFP is currently being added to the constructs to facilitate selection of transformants in citrus. Objective 3: Development of genome editing technologies (Cas9/CRISPR) for citrus improvement The initial target for gene editing is the citrus homolog of Bs5 of pepper. The recessive bs5 resistance allele contains a deletion of two conserved leucines. The citrus Bs5 homolog was sequenced from both Carrizo citrange and Duncan grapefruit, and conserved CRISPR targets were identified. Four CRISPR constructs are being created in the Staskawicz lab: C1) A construct targeting two sites that will produce a 100 bp deletion in Bs5 in both Carrizo and Duncan (the bs5 transgene will be added); C2) A construct targeting a site overlapping the two conserved leucines; C3) C2 with the addition of a bs5 repair template for Carrizo that will not be cut; and C4) C2 with a similar repair template for Duncan grapefruit. C1 and C2 have been tested by co-delivery into Nicotiana benthamiana leaves with another construct carrying the targeted DNA from Carrizo or Duncan varieties. “C1” clearly cut the target sites of both varieties, causing 100-bp deletions. Sequence analysis confirms that “C2” cuts the target site in Carrizo. Considering this site is identical in both Duncan alleles, we expect it to cut Duncan as well. And, considering “C3” and “C4” are built from “C2,” we expect them to target the cut site as well. Sequence analysis is underway to confirm these expectations. In addition, to aid in the selection of positive transgenics, we are currently adding a GFP reporter into each CRISPR construct.
A test site at the USDA/ARS USHRL Picos Farm in Ft. Pierce supports HLB/ACP/Citrus Canker resistance screening for the citrus research community. There are numerous experiments in place at this site where HLB, ACP, and citrus canker are widespread. The first trees have been in place for six years. A number of successes have already been documented at the Picos Test Site funded through the CRDF. The UF Grosser transgenic effort has identified promising material, eliminated failures, continues to replant with new advanced material, with ~200 new trees in April 2015 (Grosser, personal comm.). The ARS Stover transgenic program has trees from many constructs at the test site and is seeing some modest differences so far, but new material has been planted that has shown great promise in the greenhouse and the permit has been updated to plant many new transgenics. A trial of more than 85 seedling populations from accessions of Citrus and citrus relatives (provided as seeds from the US National Clonal Germplasm Repository in Riverside, CA) has been underway for 6 years in the Picos Test Site. P. trifoliata, Microcitrus, and Eremocitrus are among the few genotypes in the citrus gene pool that continue to show substantial resistance to HLB (Lee et al., in preparation, with the last samples collected this week), and P. trifoliata also displayed reduced colonization by ACP (Westbrook et al., 2011). Marked tolerance to HLB is apparent in many accessions with citron in their pedigree. All replicates of one alleged “standard sour orange” looks remarkably healthy and may permit comparison of more susceptible and tolerant near-isogenic variants. A new UF-Gmitter led association mapping study has just been initiated using the same planting, to identify genes associated with HLB- and ACP-resistance. A broader cross-section of Poncirus-derived genotypes are on the site in a project led by UC Riverside/USDA-ARS Riverside, in which half of the trees of each seed source were graft-inoculated prior to planting. A collaboration between UF, UCRiverside and ARS is well-underway with more than 1000 Poncirus-hybrid trees (including 100 citranges replicated) being evaluated to map genes for HLB/ACP resistance. Marked differences in initial HLB symptoms and Las titer were presented at the 2015 International HLB conference (Gmitter et al., unpublished). In July 2015 David Hall led assessment of ACP colonization across the entire planting, and the Gmitter lab will map markers associated with reduced colonization. Several USDA citrus hybrids/genotypes with Poncirus in the pedigree have fruit that approach commercial quality, were planted within the citrange site. Several of these USDA hybrids have grown well, with dense canopies and good fruit set but copious mottle, while sweet oranges are stunted with very low vigor (Stover et al., unpublished). A Fairchild x Fortune mapping population was just planted at the Picos Test Site in an effort led by Mike Roose to identify genes associated with tolerance. This replicated planting includes a number of related hybrids (among them our easy peeling remarkably HLB-tolerant 5-51-2) and released related cultivars. Valencia on UF Grosser tetrazyg rootstocks have been at the Picos Test Site for several years, having been Las-inoculated before planting, and several continue to show excellent growth compared to standard controls (Grosser, personal comm.).
Background information The objective of this project is to quantify the relative effect of copper (Cu), windbreak (Wb) and leafminer control (Lc) on the spatial and temporal progress of Asiatic citrus canker (ACC) under conducive conditions for epidemics and disease loss. The experiment is set up in a 10 ha plot planted with Valencia sweet orange grafted on Rangpur lime located in the municipality of Xambre, Paran , Brazil. The different treatments are the combination of up to three control measures (Cu, Wb, Lc) or none. The presence or absence of windbreak represents a plot. The presence or absence of copper sprays and leafminer control represents a subplot. Each subplot is composed of 112 trees. Each of the eight treatments has three replicates. Cu treated plots are being sprayed with Kocide (35% metallic copper) at 1 kg metallic copper/ha every 21 days. Lc is being performed with application of abamectin at 150 ml/ha every 21 days. Casuarina is used as a natural Wb around the plots. Disease evaluations started in December 2013 and include percentage of ACC symptomatic trees, proportion of the canopy affected by the disease, incidence of symptomatic fruits at harvest, and yield. CRDF funding will cover the period of November 2015 to October 2016. Pre-funding progress results Nineteen months since the onset of the epidemics (July 2015), the incidences of diseased trees in the plots with complete management (Cu, Wb, Lc) and no management reached 45.2 and 97.6%, respectively. ACC diseased trees under complete management showed a minimal citrus canker severity of 0.8% as opposed to 31.4% observed on trees without any control. At first harvest, the incidences of symptomatic fruits from trees treated with the tree measures and none were 3.8 and 58.5%, respectively. Finally, production of trees in the first harvest revealed the same trend observed for other assessments. Fruit yield of trees under complete management (40 kg/tree) was 186% higher than control trees (14 kg/tree). The combination of Cu and Wb is showing the greatest importance for disease control. Post-funding progress results Trees continued to be assessed monthly as previously described. By the end of this report, disease assessments of December 2015 had been performed but not processed. The assessments of January 2016 had not been concluded. Thus, up to November 2015, disease has not progressed since July 2015 as conducive conditions for the pathogen in the trial started in December. In November 2015, the incidences of diseased trees in the plots with complete management and no management reached 43.8 and 97.9%, respectively. Severity of citrus canker in the canopy dropped for all treatments and reached 0.6 and 2.5%. The same trend was observed in the previous year and it was mainly due to drop of ACC-affected leaves, production of spring flushes and lack of favorable weather conditions for disease outbreaks, which are expected to occur in the upcoming months.
A test site at the USDA/ARS USHRL Picos Farm in Ft. Pierce supports HLB/ACP/Citrus Canker resistance screening for the citrus research community. There are numerous experiments in place at this site where HLB, ACP, and citrus canker are widespread. The first trees have been in place for six years. A number of successes have already been documented at the Picos Test Site funded through the CRDF. The UF Grosser transgenic effort has identified promising material, eliminated failures, continues to replant with new advanced material, with ~200 new trees in April 2015 (Grosser, personal comm.). The ARS Stover transgenic program has trees from many constructs at the test site and is seeing some modest differences so far, but new material has been planted that has shown great promise in the greenhouse and the permit has been updated to plant many new transgenics. A trial of more than 85 seedling populations from accessions of Citrus and citrus relatives (provided as seeds from the US National Clonal Germplasm Repository in Riverside, CA) has been underway for 6 years in the Picos Test Site. P. trifoliata, Microcitrus, and Eremocitrus are among the few genotypes in the citrus gene pool that continue to show substantial resistance to HLB (Lee et al., in preparation, with the last samples collected this week), and P. trifoliata also displayed reduced colonization by ACP (Westbrook et al., 2011). Marked tolerance to HLB is apparent in many accessions with citron in their pedigree. All replicates of one alleged “standard sour orange” looks remarkably healthy and may permit comparison of more susceptible and tolerant near-isogenic variants. A new UF-Gmitter led association mapping study has just been initiated using the same planting, to identify genes associated with HLB- and ACP-resistance. A broader cross-section of Poncirus-derived genotypes are on the site in a project led by UC Riverside/USDA-ARS Riverside, in which half of the trees of each seed source were graft-inoculated prior to planting. A collaboration between UF, UCRiverside and ARS is well-underway with more than 1000 Poncirus-hybrid trees (including 100 citranges replicated) being evaluated to map genes for HLB/ACP resistance. Marked differences in initial HLB symptoms and Las titer were presented at the 2015 International HLB conference (Gmitter et al., unpublished). In July 2015 David Hall led assessment of ACP colonization across the entire planting, and the Gmitter lab will map markers associated with reduced colonization. Several USDA citrus hybrids/genotypes with Poncirus in the pedigree have fruit that approach commercial quality, were planted within the citrange site. Several of these USDA hybrids have grown well, with dense canopies and good fruit set but copious mottle, while sweet oranges are stunted with very low vigor (Stover et al., unpublished). A Fairchild x Fortune mapping population was just planted at the Picos Test Site in an effort led by Mike Roose to identify genes associated with tolerance. This replicated planting includes a number of related hybrids (among them our easy peeling remarkably HLB-tolerant 5-51-2) and released related cultivars. Valencia on UF Grosser tetrazyg rootstocks have been at the Picos Test Site for several years, having been Las-inoculated before planting, and several continue to show excellent growth compared to standard controls (Grosser, personal comm.).
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