The Genome of Candidatus Liberibacter asiaticus (CLas) revealed the presence of luxR that encodes LuxR protein, one of the two components typical of bacterial “quorum sensing” or cell-to-cell communication systems. Interestingly, the genome lacks the second components; luxI that produce Acyl-Homoserine Lactones (AHLs) suggesting that CLas has a solo LuxR system. In the current project, we will test the effect of AHL-producing citrus plants on the pathogencity of CLas. Although the postdoctoral researcher has joined our lab two months later than the proposed starting date, we have selected different Lux-I genes from different bacteria expressing different AHLs. The selected genes included TraI (accession number: L22207.1) from Agrobacterium tumefaciens, AhyI (accession number: CAA61653) from Aeromonas hydrophilia, AYsI (accession number: DQ058009) from Agrobacterium vitis, CepI (accession number: AF019654.1) from Burkholderia cepacia, CviI (accession number: AY277257.1) from Chromobacterium violaceum, EsaI (accession number: L32183) from Panteoa stewartii, ExpI (Accession number: AY507108.1) from Pectobacterium carotovorum, LasI (protein ID: AHW72185.1) from Pseudomonas aeruginosa, RhlI (accession number: U11811.1) from Pseudomonas aeruginosa, PhzI (accession number: AAC18898.1) from Pseudomonas fluorescens, CinI (accession number: AF210630) from Rhizobium leguminosarum bv. Viciae, RhiI (protein ID: CAK10388) from Rhizobium leguminosarum bv. Viciae, RaiI (accession number: AJ427969) from Rhizobium leguminosarum bv. Viciae, CerI (accession number: AF016298) from Rhodobacter sphaeroides, SwrI (accession number: U22823.1) from Serratia liquifaciens, SinI (Protein ID: CAC46418.1) from Sinorhizobium meliloti, VanI (accession number: U69677) from Vibrio anguillarum, LuxI (protein ID: AAD48474.1) from Vibrio fischeri, LuxM (accesion number: L13940) from Vibrio harveyi, Yenl (accession number: X76082.1) from Yersinia enterocolitica, YpsI (accession number: AF079973.1) from Yersinia pseudotuberculosis, and YtbI (accession number: AF079136.2) from Yersinia pseudotuberculosis. Currently, we are synthesizing the genes in order to insert them in CTV-based vector prior to the infiltration inside citrus trees.
Citrus trees transformed with a chimera AMP and a thionin alone showed remarkable resistance in citrus canker compared to control. These promising transgenic lines were replicated by grafting for HLB challenge. Replicated transgenic Carrizo lines expressing thionin, chimera and control were grafted with HLB infected rough lemon buds. Las titer was checked from new flush rough lemon leaves at six month after grafting. Las titer from 18.6-36.5 was detected in 90% of transgenics expressing the chimera. Some transgenic lines expressing thonin had the lower Las tilter(most in 33.3-36.4 ranges). Transgenic root sample were further tested and most were detected with las titer from 30 to 35. All root samples will be checked at 9 month for Las titer. Two new chimeral peptides (second generation) were developed and used to produce many Carrizo plants and Hamlin shoots. Transgenic carrizo plants carrying second generation AMPs were transferred to soil cones. DNA was isolated from 46 plants and 40 of them are PCR positive. To explore broad spectrum resistance, a flagellin receptor gene FLS2 from tobacco was used to transform citrus. Flagellins are frequently PAMPS (pathogenesis associated molecular patterns) in disease systems and CLas has a full flagellin gene despite having no flagella detected to date. The consensus FLS2 clone was obtained and used to transform Hamlin and Carrizo so that resistance transduction may be enhanced in citrus for HLB and other diseases. Reactive Oxygen Species (ROS) assay showed typical ROS reaction in transgenic Hamlin indicating nbFLS is functional in citrus PAMP-triggered immunity. Trees showed significant canker resistance to spray inoculation. Replicated Carrizo and Hamlin were challenged with ACP feeding. Las titer will be tested periodically. To disrupt HLB development by manipulating Las pathogenesis, a luxI homolog potentially producing a ligand to bind LuxR in Las was cloned into binary vector and transformed citrus. Both transformed Carrizo and Hamlin were obtained. Replicated transgenic Carrizo plants were challenged by ACP feeding. Las tilter will be tested soon. Transgenic Hamlin were propagated by grafting for HLB challenge. In collaboration with Bill Belknap two new citrus-derived promoters have been tested using a GUS reporter gene and have been shown to have extraordinarily high levels of tissue-specific expression. The phloem-specific promoter was used to create a construct for highly phloem specific expression of the chimeral peptide using citrus genes only. A Las expressed gene with a nuclear-localization sequence has been identified and studied, including creating transgenic citrus that express this p235 gene. Carrizo transformed with this gene displays leaf yellowing similar to that seen in HLB-affected trees. Gene expression levels, determined by RT-qPCR amplification, correlated with HLB-like symptoms. P235 translational fusion with GFP shows the gene product binds to citrus chloroplasts. Antibodies (ScFv) to the Las invA and TolC genes, and constructs to overproduce them, were created by John Hartung under an earlier CRDF project. We have transgenic Carrizo reflecting almost 400 independent transgenic events and 17 different ScFv ready for testing. A series of AMP transgenics scions produced in the last several years continue to move forward in the testing pipeline. Many trees are in the field and some are growing well but are not immune to HLB. A large number of ubiquitin::D4E1 and WDV::D4E1 plants and smaller numbers with other AMPs are replicated and now in the field.
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 three 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. 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. 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. Three CRISPR constructs are being created in the Staskawicz lab: 1) A construct targeting two sites that will produce a deletion in Bs5 in both Carrizo and Duncan (the bs5 transgene will be added); 2) A construct targeting a site overlapping the two conserved leucines, containing a bs5 repair template for Carrizo that will not be cut; and 3) a construct targeting the same site, with a repair template for Duncan grapefruit.
Chimeral constructs that should enhance AMP effectiveness (designed by Goutam Gupta of Los Alamos National Lab) are being tested and are among the most promising transgenics we have created, along with thionin transgenics. Trees transformed with a chimera AMP showed remarkable resistance in citrus canker compared to control. These promising transgenic lines were replicated by grafting for HLB challenge. Transgenic Hamlin lines expressing thionin were grafted onto Carrizo for HLB challenge. Replicated transgenic Transgenic Carrizo lines expressing thionin, chimera and control were grafted with HLB infected rough lemon. Promising resistance to HLB was observed based on plant growth and phenotype. Las titer is being checked from root and new flush rough lemon leaves. Two new chimeral peptides from citrus genes only were developed and used to produce many Carrizo plants and Hamlin shoots which will be tested soon as part of the next generation of this project. To explore broad spectrum resistance, a flagellin receptor gene FLS2 from tobacco was used to transform citrus. Flagellins are frequently PAMPS (pathogenesis associated molecular patterns) in disease systems and CLas has a full flagellin gene despite having no flagella detected to date. The consensus FLS2 clone was obtained and used to transform Hamlin and Carrizo so that resistance transduction may be enhanced in citrus for HLB and other diseases. Reactive Oxygen Species (ROS) assay showed typical ROS reaction in transgenic Hamlin indicating nbFLS is functional in citrus PAMP-triggered immunity. Trees showed significant canker resistance to spray inoculation. To disrupt HLB development by manipulating Las pathogenesis, a luxI homolog potentially producing a ligand to bind LuxR in Las was cloned into binary vector and transformed citrus. Both transformed Carrizo and Hamlin were obtained. Further investigation are underway. In collaboration with Bill Belknap two new citrus-derived promoters have been tested using a GUS reporter gene and have been shown to have extraordinarily high levels of tissue-specific expression. The phloem-specific promoter was used to create a construct for highly phloem specific expression of the chimeral peptide using citrus genes only. Transgenic plants of PP-2 hairpins (for suppression of PP-2 through RNAi to test possible reduction in vascular blockage even when CLas is present) and of PP-2 directly are grafted in the greenhouse. 40 putative transgenic plants transformed with citGRP1 were tested by PCR and twenty two of them were confirmed with citGRP1 insertion. RNA was isolated from some and RT-PCR showed gene expression. Some transgenics with over-expression of citGRP1 had increased resistance to canker by detached leaf assay but do not appear as potent as some other AMPs. Transgenic Carrizo and Hamlin with peach dormancy genes show no evidence of enhanced or accelerated dormancy A Las expressed gene with a nuclear-localization sequence has been identified and studied, including creating transgenic citrus that express this p235 gene. Carrizo transformed with this gene displays leaf yellowing similar to that seen in HLB-affected trees. Gene expression levels, determined by RT-qPCR amplification, correlated with HLB-like symptoms. P235 translational fusion with GFP shows the gene product binds to citrus chloroplasts. Antibodies (ScFv) to the Las invA and TolC genes, and constructs to overproduce them, were created by John Hartung under an earlier CRDF project. We have transgenic Carrizo reflecting almost 400 independent transgenic events and 17 different ScFv ready for testing. A series of AMP transgenics scions produced in the last several years continue to move forward in the testing pipeline. Many trees are in the field and some are growing well but are not immune to HLB. A large number of ubiquitin::D4E1 and WDV::D4E1 plants and smaller numbers with other AMPs are replicated and now in the field.
In the July-September 2015 period of our work, we have continued to finish strain constructions and have begun Affymetrix GeneChip experiments in order to examine expression of transcripts in host Sinorhizobium meliloti due to introduced Candidatus Liberibacter asiaticus (CLas) transcription factor genes. Previously we showed that the cloned CLas rpoH gene can complement phenotypes of an S. meliloti double rpoH1 rpoH2 deletion mutant. We have now isolated total RNA from the S. meliloti rpoH1H2 mutant strain expressing CLas rpoH and prepared labeled cDNA. Controls for this experiment are: S. meliloti rpoH1H2 carrying the empty vector, pSRK-Gm; and S. meliloti rpoH1H2 carrying S. meliloti rpoH1, cloned in pSRK-Gm. All samples (3 biological replicates of each of 3 strains) have been submitted to our campus facility for hybridization to the S. meliloti genome chip, which will reveal the total transcriptome arising from CLas rpoH expression. Beyond rpoH, we intend to study 6 transcription factors from CLas . As described in the previous report, we are creating S. meliloti host strains with deletions for each of the S. meliloti genes corresponding to those CLas regulators, so we can optimally measure function of the introduced CLas genes. As of July report, we had constructed the visRN and ldtR mutants. We have since constructed an lsrB mutant. The S. meliloti lsrB deletion strain grows poorly. Using the cloned CLas lsrB gene carried on plasmid pSRK-Gm, we showed that induced expression of CLas lsrB partly rescues the poor growth phenotype of the S. meliloti mutant. We are encouraged that this shows at least partial function of CLas LsrB in S. meliloti. We are working on construction of a double phrR1 and phrR2 mutant; meanwhile we have introduced the cloned CLas phrR gene into wild type S. meliloti, in case we are unable to make the phrR1 phrR2 double mutant.. Construction of a ctrA deletion strain requires an extra step, since ctrA is essential for S. meliloti viability. Our progress to date includes successful cloning of the CLas and S. meliloti ctrA genes in vector pSRK-Gm. Each of these plasmids was introduced into S. meliloti containing a single crossover of the ctrA deletion construct. In parallel, we have constructed wild type S. meliloti strains containing the cloned CLas and Sm ctrA genes. We will screen these two strains (overexpressing either CLas ctrA or S. meliloti ctrA) in parallel on sucrose. If the optimized CLas ctrA gene does not allow for viability of a S. meliloti ctrA deletion strain, then we will study the CLas CtrA regulator in wild type S. meliloti.
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 almost 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). 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.).
For the last three months, Core Citrus Transformation Facility (CCTF) continued to provide its service for production of transgenic Citrus plants. Within this period of time, there were only two new orders placed but three clients requested work on three previously placed orders bringing total number to five. Probably as a result of recent Knowledge Mapping meeting organized by CRDF there is an increased interest for transgenic plants and CCTF was already informed of eight new incoming orders. That does not include additional orders from another client with whom CCTF manager communicated for the last three months. Forty plants were produced during the last quarter which represents lower productivity than usual. Partially, this is the result of work on group of three orders that have not yielded any plants. Another reason is the low quality of seeds used to obtain seedlings as starting material for experiments. High majority of produced plants belong to eight orders placed within the last 12-15 months. Five plants belong to three older orders. Most of the transgenic plants are Duncan grapefruit and Valencia orange and one of them is Carrizo citrange. The work has begun on determination of level of expression of AtNPR1 gene in rootstock plants produced for CRDF. Once all plants produced by CCTF and Mature tissue lab are processed, those with the highest expression levels will be selected for further use and propagated.
Mature plant production continues with various genetic constructs with reporter genes from Drs. Dutt, McNellis and Wang. Additional scientists have expressed interest in our services. Transformation efficiencies have significantly increased with reporter genes. We are also trying to further increase Agrobacterium transformation efficiencies of mature citrus by incorporating vacuum infiltration and sonication treatments. These treatments significantly increased transformation efficiencies in immature citrus. In addition to Agrobacterium, we can now supplement plant production with plants produced using biolistics. Scions and rootstocks have been micropropagated (budded scions and rooted cuttings) into three replicates for one location. It remains to be determined whether we will continue micropropagation for replicates at additional locations. We have almost concluded screening Dr. Orbovic’s putative transgenics with qPCR to identify high-expressing lines. This work should be finished by the end of this month. I am switching to citrus pots in the growth room, which can be planted to higher densities than current planting densities. Recently we discovered that mature scion grows significantly faster after budding if the rootstock is not cut-off after the buds break.
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 are being trained on how to establish and maintain colonies of infected psyllids, how to conduct qPCR assays on plant and psyllid samples, and how to run the inoculations. As of March 31, 2015, a total of 7,448 plants have passed through inoculation process. A total of 148,960 psyllids from colonies of CLas-infected ACP have been used in no-choice inoculations.
The objectives of this project are: 1. Evaluate psyllid populations, HLB incidence and intensity, gene expression, tree growth, soil moisture, soil nutrients, foliar nutrients, and eventually yield in newly planted citrus blocks, 2. Assess separate contributions of vector control and foliar nutritional to the above parameters, 3. Evaluate effectiveness of reflective mulch to repel ACP and reduce incidence of HLB, 4. Provide economic analysis of costs and projected benefits, and 5. Extend results to clientele. Management of the experimental block (‘Hamlin’ orange on Carrizo citrange planted 3-4 July on a 10-acre block at A. Duda & Sons, Inc. farm in Hendry County was turned over to the grower having completed the planned 3 years. Trees will still be harvested by plot this year and the data used to complete a planned paper. A field day was conducted in conjunction with the company on 19 June attended by about 25 citrus growers and 14 other participants to present the results of the 3-year study. Part of the study was included in a dissertation completed by Dr. Scott Croxton who graduated in summer 2015. Another trial planted 5 May 2013 at SWFREC consists of 24, 250 ft. rows of Ray Ruby grapefruit on smooth flat Seville divided into 8 main plots, half receiving organic amendments since 1993, including 12 tons/acre composted yard trimming waste (YTW) applied in a 6 ft swath to the plant drill prior to planting. All evaluated soil parameters parameters were significantly different between compost and no compost: 5.6 vs 6.9, organic matter (%) 1.29 vs 3.13, CEC (meq/100 g) 1.19 vs 5.73, P, K, Ca and MG (mg/kg) 32.05 vs 234.3, 8.73 vs 19.4, 414.6 vs 2071.8, 27.0 vs 113.5 respectively. The plot had been underlain with drain tile and is flat except for 6 in high beds 32 inches wide on 18 ft centers covered with polyethylene film mulch and irrigated through two drip tape lines. Each 3-row plot was divided into 2 subplots: whiteface or metalized mulch. Soil bed temperatures between Jul and Dec 2015 tended to be in non-compost than compost and greater under metalized mulch compared to white mulch: averaging 80.43 (metalized non compost) 78.85 (white non compost, 79.07 (metalized compost) and 77.73 (white compost) Sticky card captures have been 5 times greater on white mulch, but twice as high on compost compared to no compost. Infested flush followed similar trends. Incidence of HLB was 4% on metalized mulch compared to 13% on white (over both compost treatments) and 11% on compost compared to 6% on no-compost over both mulch treatments. Trunk x-section area, canopy area and height have increased significantly more for trees on compost compared to trees with no compost. From Jul 2013 through Jul 2015, non compost trunk area grew from 65 to 2407 mm compared to 61 to 3,333 mm2 with compost, canopy area (m2) from 0.43 to 3.69 m2 non compost vs 0.65-5.34 m2 compost, and tree height from 1.13 to 2.02 m non compost compared to 1.17 to 2.22 m compost. In contrast, growth differences between white and reflective mulch were not significant. Flooding and over irrigation caused considerable foot rot this summer. Soil samples were taken for microbiota analysis in Sep 2015. Plastic mulch was removed in Oct to evaluate the planned transition to microjet irrigation. Constraints on increased compost use lie with short supply rather than grower acceptance, in spite of 3 major suppliers in the local tri-county area including a huge facility run by Lee County. One grower even asked me to stop promoting use of compost in citrus because supply was not keeping up with demand.
The objectives of this project are: 1. Evaluate psyllid populations, HLB incidence and intensity, gene expression, tree growth, soil moisture, soil nutrients, foliar nutrients, and eventually yield in newly planted citrus blocks, 2. Assess separate contributions of vector control and foliar nutritional to the above parameters, 3. Evaluate effectiveness of reflective mulch to repel ACP and reduce incidence of HLB, 4. Provide economic analysis of costs and projected benefits, and 5. Extend results to clientele. Management of the experimental block (‘Hamlin’ orange on Carrizo citrange planted 3-4 July on a 10-acre block at A. Duda & Sons, Inc. farm in Hendry County was turned over to the grower having completed the planned 3 years. Trees will still be harvested by plot this year and the data used to complete a planned paper. A field day was conducted in conjunction with the company on 19 June attended by about 25 citrus growers and 14 other participants to present the results of the 3-year study. Part of the study was included in a dissertation completed by now Dr. Scott Croxton who graduated this summer. Another trial planted 5 May 2013 at SWFREC consists of 24, 250 ft. rows of Ray Ruby grapefruit on smooth flat Seville divided into 8 main plots, half receiving organic amendments since 1993, including 12 tons/acre composted yard trimming waste (YTW) applied in a 6 ft swath to the plant drill prior to planting. The plot had been underlain with drain tile and is flat except for 6 in high beds 32 inches wide on 18 ft centers covered with polyethylene film mulch and irrigated through two drip tape lines. Each 3-row plot is divided into 2 subplots: whiteface or metalized mulch. Soil bed temperatures have been about the same under white or reflective mulch over the last 3 months. Trunk x-section area, canopy area and height have been consistently for trees on compost compared to trees with no compost whereas growth differences between white and reflective mulch have not been significant. Sticky card captures have been 5 times greater on white mulch, but twice as high on compost compared to no compost. Infested flush followed similar trends. Incidence of HLB was 4% on metalized mulch compared to 13% on white (over both compost treatments) and 11% on compost compared to 6% on no-compost over both mulch treatments. Flooding caused considerable foot rot this summer. Soil samples were taken for microbiota analysis in Sep 2015. Plastic mulch was removed in Oct to evaluate the planned transition to microjet irrigation. Fertigation is used at the Vero Beach experiment usingliquid 6-0-8 (with minors) fertilizer injected continuously with all irrigation events. Herbicides are applied via herbicide boom to both the composted Urban Plant Debris (UPD) and the Bare Ground (BG) treatments in a 9 ft band 2.5 ft from edges of the 4 ft wide Metallized Reflective Mulch (MRM) ~2.5 ft.) beds to create 9 ft. wide weed free zone. Tree opening ins MRM are treated with spot sprays of glyphosate. Belay 2.13 SC and Admire Pro) were applied by soil drench for ACP control and Ridomil Gold SL using a calibrated electric timed liquid applicator. Insects and mites are monitored weekly. ACP adults and nymphs, Diaprepes Root Weevil, and orange dog larvae have been half or less on MRM than other treatments whereas Sri Lankan weevils and citrus leafminer were unaffected. Soil moisture readings as percent Volumetric Water Content (%VMC) for each treatment are also recorded.
Our project aims to provide durable long term resistance to Diaprepes using a plant based insecticidal transgene approach. In this quarter, several transgenic lines expressing each of our test genes have been regenerated and many of them have rooted. The rooted material has been successfully acclimatized to the greenhouse for growth and further testing. We are in the process of confirming the gene expression levels in these transgenic lines to isolated lines that have excellent gene expression. These selected lines will be propagated for subsequent evaluation with Diaprepes neonates.
Seasonal root sampling continues in two field sites for root density and root growth. We are collecting a second year of root growth data from Hamlin/Swingle and have 1 year of root growth data on Valencia/Swingle. Results so far emphasize the need to use treatments that improve root longevity as the main method of managing HLB root loss. Root growth stimulation is unlikely to improve root density. Preliminary tests of root tubes are complete and easy observation of root growth and root dieback have been confirmed. This will allow for more rapid quantification of root growth and death using nondestructive sampling so that the same roots can be monitored over time. The root tubes have been installed in treated and untreated plots of a thermotherapy trial to combine efforts and provide both basic disease information and treatment effects on the root system. Sampling at a rootstock trial site continues. Only one rootstock tested to date has shown a significant difference in response to HLB. Initial sampling at a second rootstock trial site has begun. This will allow comparison of some of the rootstocks on Ridge and Flatwoods soils. We continue to monitor the most promising rootstocks identified in the field trial to HLB using rhizotrons in the greenhouse. A second set of rootstocks is ready for testing in rhizotrons as soon as greenhouse space is available from the breakdown of the first set. Changes in image acquisition and soil type have been tested to improve the data analysis steps in these followup experiments. The first experiment is ready for takedown, but is awaiting some final root collections for phytohormone and microscopic analysis. Method development to characterize the mechanism by which Liberibacter causes root death continues with a second round of samples to be collected shortly with improved tissue selection to avoid variability found in the first attempt at microscopy analysis of cell death.
Seasonal root sampling continues in two field sites for root density and root growth. We have completed a second year of root growth data from Hamlin/Swingle and are continuing to collect a second year of root growth data on Valencia/Swingle. Results continue to emphasize the need to use treatments that improve root longevity as the main method of managing HLB root loss. Additional root growth appears to occur at the expense of older roots and is unlikely to provide sustained improvement in root density. Root growth, lifespan, and death measurements are being made from root tubes installed in treated and untreated plots of a thermotherapy trial to combine efforts and provide both basic disease information and treatment effects on the root system. Visual observation shows that the method is working well, but data analysis has been delayed due to an unexpected software glitch that the company is currently working on fixing. Only one rootstock tested to date has shown a significant difference in response to HLB. It remains the only rootstock with significantly better root density (increased) when infected by HLB. Root loss has not been observed yet in this rootstock. We are planning to collect fruit drop data this year on the rootstocks that have been measured to see if the improved root density of this rootstock correlates with better fruit retention. We continue to monitor the most promising rootstocks identified in the field trial to HLB using rhizotrons in the greenhouse. A second set of rootstocks is ready for testing in rhizotrons as soon as greenhouse space is available from the breakdown of the first set. Changes in image acquisition and soil type have been tested to improve the data analysis steps in these followup experiments. The first experiment is ready for takedown, but is awaiting some final root collections for phytohormone and microscopic analysis. A second set of root samples was collected for phytohormone analysis. Based on initial tests we have collected multiple growth stages and ages of roots to get a larger picture of the effects of HLB on phytohormones. Results from the initial run suggested that contrary to our hypothesis it was not the primary phytohormones that were altered by Las infection. One expected phytohormone was not detected in any sample and we are working to resolve this lack of detection. More microscopy samples have been collected and we are awaiting processing before we perform the final breakdown.
The Budwood Certification Program provided over 246,000 buds from 67 different varieties to the industry in FY 2014-2015. This was the largest amount of budwood provided in the last 10 years. All structures are certified by TDA and USDA with monthly inspections. All trees were tested every 6 months for HLB and annually for CTV in accordance with the USDA-APHIS regulations for intrastate movement of quarantined plant material. In addition, the Texas Germplasm Introduction Program is starting up, with a shoot-tip grafting and quarantine facility for introducing clean, pathogen free varieties specific to the Texas Industry. Increase Screenhouses 3 and 4 have over 4,000 potted increase trees. Screenhouse 3 contains newly budded Increase and Foundation trees awaiting transplanting. All trees were tested for HLB in October, 2014 and April, 2015 for HLB and CTV. A total 106,659 buds were cut from Screenhouses 3 and 4. Intensive inspections, pest and fertility management continue to be in place. A total of 117,001 buds were cut from Increase screen structures I, II, and III. As the trees in Screenhouses 3 and 4 develop, the number of buds cut from the screen structures has decreased. All trees were tested in October, 2014 for HLB and in April, 2015 for CTV and HLB. An intensive fertilization, health and pest management program continues to be in place. Screenhouse 1 and 2 are filled to 70% capacity with certified Foundation trees. Additional trees will be added in the fall and winter. All trees were sampled and tested for HLB in October, 2014 and again in April, 2015 for HLB and CTV. All trees have tested negative. All trees are currently undergoing PCR testing for Viruses and Viroids. There are currently 147 Foundation trees in Screenhouse 5. All trees were sampled and tested for HLB in October, 2014 and again in April, 2015 for HLB and CTV. All trees are currently undergoing PCR testing for Viruses and Viroids. The Foundation greenhouse in Stephenville, Texas is the remote site location for reserve Foundation varieties. Currently there are 70 Foundation trees at Stephenville. The greenhouse was certified by TDA in August. Mark VanNess and Sonia del Rio traveled to California s Rubidoux Citrus Quarantine Facility in Riverside California in August for shoot-tip grafting training and also to learn the procedures for establishing a germplasm introduction program in Texas. The TajMahal building has been designated as the facility for growing and indexing the quarantined varieties until they are approved for release by the USDA and TDA. The program will be approved and certified by USDA prior to the start of any shoot-tip grafting. Budwood sales for FY 2014-2015 were 246,075. This exceeds last year s total of 196,080. Rio Red buds totaled 144,139, (59%), down from last year s number of 155,521 Rio Red buds (79%). The number of Olinda Valencia buds cut was 43,200 (18%), the largest number of Olinda s ever cut. More Olinda Increase trees have been budded in anticipation of higher numbers in the future.
(863) 956-8817
(863) 956-5894
700 Experiment Station Road
Lake Alfred, FL 33850
Email Us
