Objective 1. To define the role of chemotaxis in the location and early attachment to the leaf and fruit surface. The importance of motility and chemotaxis in the infection process for citrus Xanthomonas strain varying in host range is under investigation. Membrane protein receptors (MCP) profiles were predicted from complete genome sequence analysis and PCR amplification and nucleotide sequencing for wide (Xcc62, Xcc306) and narrow (XccIran2, XccIran10, Xcc12879) Xanthomonas citri subsp. citri (Xcc) strains compared to B and C canker strains (Xfa69, Xfa341), X. alfalfae subsp. citrumelonis (Xac) and X. campestris pv. campestris (Xc). All Xcc strains showed similar MCP content except Xcc306 which lacked some of the MCP sequences. To confirm differences in MCP profiles among wide and narrow host range Xcc, the evaluation will include 40 addtional strains. These results should confirm whether there is a relationship between MCP profile and chemotaxis differences among Xcc strains. Objective 2. To investigate biofilm formation, composition and structure in relation to motility for different Xcc strains and non-canker xanthomonads. Previously, we detected extracellular DNA (eDNA) in the initial stages of the biofilm formation. Differences between wide and narrow host range strains of Xcc were found when the biofilms were treated with DNAse which reduced biofilm formation and disrupted preformed biofilms. To evaluate the potential direct effect of eDNA on bacterial aggregation, cultures of Xcc, Xac and Xc were amended with genomic DNA (gDNA) from each of the strains. In most cases no differences were found in biofilm formation between cultures treated and non-treated with gDNA. Therefore, the role of the DNA in biofilm formation is more complex. The next objective was to determine the origin and nature of the DNA detected in biofilm structure. To characterize such DNA, gDNA and eDNA were analyzed by BOX and ERIC PCR and DNA profiles compared but no differences were found. Futures assays will use RAPDs and several primer sets to detect any differences in DNA. In addition to characterizing biofilm, exopolysaccharide (EPS) production was evaluated in the different strains after growth in nutrient broth medium amended with 2% sucrose. No quantitative differences in EPS were detected among strains but narrow host range strains showed qualitative differences which may be due their high DNA content. Further assays are planned to clarify this difference.
FireWall 50WP (50% streptomycin; Agrosource, Inc.) was granted by an EPA section 18 registration for control of citrus canker in Florida grapefruit. The label for FireWall restricts use to no more than two applications per season. As a condition for FireWall registration, EPA requires monitoring of Xanthomonas citri subsp. citri (Xcc) for streptomycin resistance in treated groves. The objective of this survey was to apply our published protocol (Behlau et al., 2012) for sampling canker-infected grapefruit leaves for isolation and detection of streptomycin resistant Xcc. The survey for 2015 season applications was conducted in January 2016. After sprays with FireWall, no Xcc colonies resistant to streptomycin were detected from two grove locations that received two sprays of FireWall in spring- summer 2015. Xcc in one of the locations has been exposed to streptomycin for three seasons without development of resistance. A report of our findings will be submitted to FDACS.
To confirm that treatments with acidified irrigation water reduce the impact of bicarbonate stress on root health, we surveyed 8 ridge groves in Highlands county and 4 flatwoods groves in Hardee county with high bicarbonate stress as detected in our 2013 survey. All the blocks are less than 10 year old Valencia trees on bicarbonate sensitive rootstocks, Swingle and Carrizo. The survey is bimonthly to follow the recovery of these blocks and at harvest to compare 2014 season block yields 1.0 to 1.5 years after acid treatments began. From May to November root density fluctuated but was maintained at about the same level throughout the season. Root density was about twice as high in the ridge as in the flatwoods locations. Phytophthora populations were low or non-detectable in ridge groves but were always present and about 2-3x higher in the flatwoods groves. Soil pH ranged from 5.0-6.0 in the ridge after 1-1.5 years of acidification. pH in th flatwoods groves was initially in this range but rose above 6.5 and remained at that level. Flatwoods groves with higher bicarbonates in irrigation wells and soil require higher rates of acidification treatment to reduce below 6.5 as recommended for Swingle and Carrizo rootstock groves.
Recent evaluation of root production on HLB-affected trees compared to presumed healthy trees confirms that root loss is due to reduced root longevity. This root loss is exacerbated by biotic and abiotic stresses in the rhizosphere. Increased susceptibility of Las’infected roots to Phytophthora spp. is evidenced by statewide populations that have fluctuated from unprecedented highs in the 2011 season to an unprecedented low in 2013 compared to 25 years of pre-HLB soil populations. Phytophthora propagules per soil volume and per root resurged in 2014 in response to a more than doubling of root density based on intensive (i.e., local repeated measures) and extensive (i.e., statewide survey) sampling compared to 2013. Substantially higher root density in Florida groves in spring and summer 2014 is predicted to result in less fruit drop than in the previous two seasons. Both field surveys and greenhouse experiments show that Huanglongbing (HLB) increases the susceptibility of the citrus root system to the damaging root pathogen Phytophthora nicotianae. This increased P. nicotianae infection suggests that root system damage may occur more quickly in the presence of both pathogens. Chemical control of P. nicotianae may slow infection of the root system and subsequent yield decline in HLB-affected trees in Phytophthora-infested groves. To test the value of P. nicotianae control in slowing tree decline in the presence of both pathogens, 100 P. nicotianae-infested nursery trees were inoculated or mock-inoculated with HLB-affected budwood. After HLB inoculation, trees were treated bimonthly with Ridomil Gold SL (mefenoxam) or Aliette WDG (Aluminum tris O-ethyl phosphonate) compared to untreated controls. HLB status, P. nicotianae infection and propagule counts, and visible symptoms were assessed bimonthly. After 12 months, the trees were harvested and biomass of roots and shoots was measured. As expected Ridomil and Aliette significantly reduced P. nicotianae root infection and increased fibrous root mass compared to untreated controls for HLB(-) trees. Neither Ridomil or Aliette significantly reduced P. nicotianae root infection or increased fibrous root density for HLB(+) trees, although both measures quantitatively improved with Ridomil performing slightly better than Aliette. These data suggest that HLB reduces the effectiveness of fungicide control of Phytophthora root rot as a consequence of increased susceptibility to P. nicotianae.
FireWall 50WP (50% streptomycin; Agrosource, Inc.) was granted by an EPA section 18 registration for control of citrus canker in Florida grapefruit. The label for FireWall restricts use to no more than two applications per season. As a condition for FireWall registration, EPA requires monitoring of Xanthomonas citri subsp. citri (Xcc) for streptomycin resistance in treated groves. The objective of this survey was to apply our published protocol (Behlau et al., 2012) for sampling canker-infected grapefruit leaves for isolation and detection of streptomycin resistant Xcc. The survey for 2014 season was conducted in November and the report recently submitted to FDACS. After sprays with FireWall, no Xcc colonies resistant to streptomycin were detected from two grove locations that received one or two sprays of FireWall in summer 2014. Thus far, Xcc in the Felda location has been exposed to streptomycin for two seasons and the Vero Beach location for one season without development of resistance. Two greenhouse trials measured the residual systemic activity of streptomycin against Xcc in leaves to be 10 weeks after foliar spray application. Trans-cuticular movement of streptomycin explains the consistent performance in field trials against canker in that the penetration of the leaf insures that streptomycin is protected from wash-off by rainfall, weathering and degradation by UV light.
The Core Citrus Transformation Facility (CCTF) continued to provide service for production of transgenic citrus plants. The work load that included: co-incubation experiments, explants incubation, shoot harvesting and inspection, PCR testing, micro-grafting, and care of plants in the greenhouse was kept at maximum level allowed by the number of holidays within the last quarter of the year. The work done by the CCTF from October through December was concentrated on older orders as five new orders were placed to CCTF from two different clients in late December. Production of transgenic rootstock plants ordered by the CRDF continued at slower pace. One out of 29 plants that were previously growing in the greenhouse died. Nineteen more ‘pot-adapted’ plants were moved from the laboratory to the greenhouse bringing the total number up to 47. The growth of first plants moved to the greenhouse was not as vigorous as expected and they were not cut into explants for propagation. That may be done in the middle of February. In the period covered by this report, CCTF produced plants for the following orders: pNPR1-17 plants, pNPR1-G-five plants, pELP3-G-12 plants, pELP4-G-five plants, pMG36-eight plants, pX7-2- five plants, pHGJ27- one plant. Three orders that are being serviced at this time require detection of transgenes in transgenic plants by microscopy. The methodology for this detection is still being worked out with the EM lab personnel. If this was solved at earlier date, the output of the facility would have been higher. Plants produced in this quarter were mostly Duncan grapefruit and Carrizo citrange with the exception of one Valencia sweet orange plant. As it was anticipated in the previous report, the quality of Duncan fruits and seeds from the CREC’s grove has deteriorated further. Before the end of January, CCTF will contact local grower to get supply of better quality Duncan fruit.
All of the research described in the previous report is being analyzed or active research is being transferred to a new grant from CRB. The one year study of the in vivo tracking of FT1, FT2, and FT3 in various citrus trees differing in age and phenotype is concluded and analyzed. There were some surprises. RNA levels of FT3, the FT homologue from citrus that other research by us and others indicates is most closely associated with flowering, were low in leaves at bloom time, whereas we expected high levels of expression. This may indicate that high levels of FT protein are produced at that time and sent to apices and mRNA is depleted. A study of CiFT3 transgenic tobacco plants treated with various growth regulators has been performed and the data is now being analyzed. The growth hormones produced striking and individually different phenotypes in each treatment. The data includes plant height and leaf number, size, and area. The endogenous ciFT3 promoter from sweet orange was successfully cloned to be used in the transcription activator-like (TAL) effector system inducible by methoxyfenozide that will hopefully activate the naturally present FT3 gene in citrus. Large numbers of citrus seeds are being germinated for transformation studies with this construct.
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 almost 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. We have installed tubes for the preliminary tests of a new root scanner to nondestructively measure root growth and root death. Preliminary tests are being used to determine the optimum depth of tube needed for future experiments. This will allow for more rapid quantification of root growth and death with less damage to observed trees. These will be used in future experiments of treatments used to increase root longevity and for observing the effects of HLB on young plantings that are too easily damaged by extensive soil core sampling. Sampling at a rootstock trial site continues. As some of the rootstocks initially chosen for observation have not performed better than Swingle, these rootstocks will no longer be sampled and others that show positive canopy responses are under consideration for root sampling. The response the root system of the most promising rootstocks identified in the field trial to HLB is currently being observed in rhizotrons in the greenhouse. The first experiment is nearing completion and data analysis of root growth and root longevity in response to Las is currently underway. Additional rootstocks are being considered for the second round of rhizotron experiments. Method development to characterize the mechanism by which Liberibacter causes root death is underway.
The overall aim of this project is to develop and evaluate oil-in-water (o/w) microemulsion (ME) formulations to deliver antimicrobial essential oils (EOs) to the phloem of HLB-infected trees. In the last quarter leaf uptake and phloem transport studies of the EO formulations were initiated using two approaches: (i) fluorescence microscopy with dye doped formulations and (ii) GC/MS spectrometry. Previously we have reported the development of o/w formulations with maximum oil loadings of 7% for thyme oil and EO-A and 5% (w/w) for EO-B showing high inhibition of L. crescens. Further modifications in the interfacial properties of the formulations, by varying the surfactants, co-surfactant and the ionic strength have led to increased oil loadings from 7 to 16% for thyme oil and from 5 to 10% for other oils. This will make the formulations more economically viable and reduce the amount of surfactant applied on the plants. Experiments to determine the change in the droplet size and stability of the formulations due to increased oil loading are underway. Method development experiments were performed to estimate EO uptake and transport in citrus plants by using GC/MS and LC/MS. Initial GC/MS analysis of thyme oil revealed its major components to be thymol, p-cymene, .-terpinene with carvacrol in small amounts. Macerated leaves from Valencia orange and Duncan grapefruit plants were also analyzed by GC/MS for headspace analysis to establish the background and interference from molecules with similar emission as EO components. Additionally the detection limit of the EO (or component) was determined by GC/ MS headspace analysis by mixing known amount of EO to macerated citrus leaves. Preliminary experiments with thyme oil formulation show that thymol component can be detected in the headspace of leaf samples upto 1mg/ L thyme oil. Future studies include estimation of EO transport in the citrus plants. To investigate the penetration of formulations into citrus leaves, dye doped formulations were prepared with the lipid soluble dyes e.g. Bodipy 505/515, Vybrant DiO etc. having fluorescence emission in 475-525 and 550-650 nm range. Initial studies done by foliar application of the dye doped formulations showed non-uniform uptake of the microemulsions and photo-bleaching during imaging. Further studies are underway to study the uptake of EOs.
A transgenic 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 over four years. Dr. Jude Grosser of UF has provided ~600 transgenic citrus plants expressing genes expected to provide HLB/canker resistance, which have been planted in the test site. Dr. Grosser planted an additional group of trees including preinoculated trees of sweet orange on a complex tetraploid rootstock that appeared to confer HLB resistance in an earlier test. Dr. Kim Bowman has planted several hundred rootstock genotypes, and Ed Stover 50 sweet oranges (400 trees due to replication) transformed with the antimicrobial peptide D4E1. Texas A&M Anti-ACP transgenics produced by Erik Mirkov and expressing the snow-drop Lectin (to suppress ACP) have been planted along with 150 sweet orange transgenics from USDA expressing the garlic lectin. More than 120 citranges, from a well-characterized mapping population, and other trifoliate hybrids (+ sweet orange standards) have been planted in a replicated trial in collaboration with Fred Gmitter of UF and Mikeal Roose of UCRiverside. Plants are being monitored for CLas development and HLB symptoms. Data from this trial should provide information on markers and perhaps genes associated with HLB resistance, for use in transgenic and conventional breeding. Dr. Roose has completed initial genotyping on a sample of the test material using a “genotyping by sequencing” approach. So far, the 1/8th poncirus hybrid nicknamed Gnarlyglo is growing extraordinarily well. It is being used aggressively as a parent in conventional breeding. In a project led by Richard Lee, an array of seedlings from the Germplasm Repository are in place, with half preinoculated with Liberibacter. Data and tissue samples were collected from almost every tree in the test site during the last quarter. Additional plantings are welcome from the research community.
A chimeral construct that should enhance AMP effectiveness (designed by Goutam Gupta of Los Alamos National Lab) is being tested. Many transformed Carrizo with the chimera AMP were obtained. Exposure to canker inoculum showed remarkable resistance in chimera compared to control. RNA was isolated from 16 transgenic Hamlin containing Chimera. RT-qPCR showed 50% of them have relative high gene expression. One of them showed over hundred times higher expression compare to plant expressing the lowest level of chimera. These promising transgenic lines were replicated by grafting for HLB challenge. About 30 Hamlin transformed with thionin also were obtained. Twenty transgenic lines were confirmed containing thionin gene by PCR. They will be tested by RT-PCR and replicated for HLB challenge. A new chimeral peptide from citrus genes only has been developed and is being used to transform citrus. To explore broad spectrum resistance, a flagellin receptor gene FLS2 from tobacco was cloned into pBinARSplus vector 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. Many putative transformants were generated on the selective media. DNA was isolated from 80 of them: 38 Carrizo and 7 Hamlin are positive by PCR test. Reactive Oxygen Species (ROS) assay showed typical ROS reaction in three of transgenic Hamlin which suggest nbFLS is functional in citrus PAMP-triggered immunity. However, there is only slight canker resistance by infiltration test. Spray inoculation was tried and some of them show obvious canker resistance. To confirm that high ROS production was not due to variability in Hamlin, we examined l 40 Hamlin seedlings and no or very low level ROS production was detected. In contrast, relative higher ROS production was detected from wild-type Carrizo seeding compared to Hamlin seedlings. Two potential FLS2 orthologues were identified in Hamlin and their expression was shown much lower compare to nbFLS2. 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. A series of transgenics scions produced in the last several years continue to move forward in the testing pipeline. Several D35S::D4E1 sweet oranges show initial growth in the field which exceeds that of controls. A large number of ubiquitin::D4E1 and WDV::D4E1 plants and smaller numbers with other AMPs are replicated and in early stages of testing. 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 is being used to create a construct for highly phloem specific expression of the chimeral peptide using citrus genes only.
We continue to work to obtain stably transformed citrus containing the BS3 promoter with added TAL effector binding elements (4 or 14 EBE) fused to a marker or defense response inducing gene. A large transformation experiment has been carried out in Duncan grapefruit and sweet orange with an expanded set of constructs, and 75 putative transgenic grapefruit plants and 79 putative transgenic sweet orange plants have been transferred to soil. These plants will be screened by PCR for the presence of the intact transgenes. A model system has been developed in tomato varieties Bonny Best and Large Red Cherry. Transgenic plants have been generated that carry the BS3 promoter with 14 added TAL effector binding sites fused to the effector AvrBs4 (14EBE:avrBs4) which is known to trigger resistance in tomato. We previously reported that one transgenic line carrying this disease resistance construct showed a reduction in symptoms in response to Xanthomonas euvesicatoria strain 85-10 carrying AvrBs3 in initial tests. Further testing has now shown that a hypersensitive reaction was observed in several T2 lines derived from multiple transformation events upon activation with X. euvesicatoria 85-10 carrying AvrBs3. 85-10 carrying empty vector produced a susceptible reaction (disease symptoms) in all transgenic plants, demonstrating that this resistance cassette and pathogen triggered resistance is working successfully in tomato. Homozygous 14EBE:avrBs4 plants will be generated and tested for disease resistance. Experiments continue to determine whether there is cryptic regulation of the 14EBE construct in plants that may contribute to our low transformation efficiency in citrus. We are testing this through marker gene or resistance gene cassettes in model systems – tobacco, tomato and Carrizo. Transgenic tobacco plants carrying the 14EBE:GUS construct have been generated, and whole seedlings were stained to examine whether the construct is expressed anywhere in the plant. No GUS expression was observed in 48 of 50 seedlings tested, demonstrating that this construct is not expressed in tobacco in the absence of pathogen induction, except in low frequency. The recovery of transgenic tomato plants carrying the 14EBE:avrBs4 construct demonstrates that background expression is not an issue in tomato either. In citrus, four transgenic Carrizo lines have been confirmed to contain an intact 14EBE:GUS construct, and these will be used to examine the regulation of the 14EBE promoter in citrus. We expect to obtain further 14EBE:GUS lines in Duncan grapefruit and sweet orange from the current transformation batch, and these will be useful for examining promoter activity in the absence and presence of pathogen triggers.
Objective 1: Generate functional EFR variants (EFR+) recognizing both elf18-Xac and elf18-CLas In order to screen a large EFR mutant population for the gain of responsiveness to elf18-CLas we have been developing reporter lines with GFP under the control of the various PAMP-inducible promoters (FRK1, WRKY30 and PER4) which could be used to screen with FACS. Of these lines PER4p:GFP produces the lowest background and clearest PAMP-induced expression. We have generated lines in both Arabidopsis cell suspension and plants. The cell suspension lines fail to respond after protoplast mediated transformation. We suspect that this is a consequence of the protoplasting procedure and are investigating alternative buffers which may enable detection of the reporter following elf18 treatment. We are currently bulking seed to test the stable transgenic plant lines to determine if protoplasts derived from plant tissue will be more reproducible. These lines have the additional advantage that some have been generated in an efr-1 background so any weak basal induction by elf18-CLas would be eliminated. In addition, we have been also investigating the possibility of targeting other PAMPs. To this end we conducted bioinformatics comparison of known PAMPs with those in C. Liberibacter asiaticus. From these search we identified CSP22 (Felix & Boller, JBC 2003, 278:6201) as a potential candidate, since it is conserved in the sequence required for recognition. After a long delay in production we have now received the CSP22-CLas peptide and are growing plants to test their activity. Objective 2. Generate functional XA21-EFR chimera (XA21-EFRchim) recognizing axYS22-Xac. The manuscript relating to the generation of chimeric XA21:EFR receptors has now been accepted in PLOS Pathogens and will be online shortly. Objective 3: Generate transgenic citrus plants expressing both EFR+ and XA21-EFRchim. Transformation experiments are ongoing; to date, a total of 10,556 ‘Duncan’ grapefruit, 2,025 sweet orange and 191 Carrizo segments have been collectively transformed with the constructs EFR, EFR-XA21, EFR-XA21-EFRchim and pCAMBIA2201 (empty vector control). Regenerated shoots from transformed segments are being screened for GUS expression, and GUS positive plants are transferred to soil. So far, grapefruit plantlets (110) from all 4 constructs and sweet orange plantlets (17) from the 3 constructs EFR, EFR-XA21 and EFR-XA21-EFRchim have been transferred to soil.
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 applications to the above parameters, 3. Evaluate the 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. The experiment was planted 3-4 July on a 10-acre block planted on a 23 x 9 ft spacing at the A. Duda & Sons, Inc. farm in Hendry County south of LaBelle at 26.64315 degrees S. -81.45456 degrees W and 26 ft elevation. The experimental design of main plots is factorial RCB with 4 replicates and 4 treatments: insecticide alone, foliar nutrition alone, insecticide + nutrition, and untreated control. Each of 16 plots is split into two subplots 5 rows wide and 13 trees long, mulch and no mulch. Mulch provided by Imaflex Inc. is metalized (aluminized/reflective) polyethylene film of 3.5 mils thickness covered with a clear protective polyethylene coat. Metalized mulch was shown in preliminary evaluations on single plots to repel Asian citrus psyllid and together with a drip irrigation/fertigation system increase citrus growth rate over the unmulched control. The block was planted 3-4 July 2012 and monitoring ACP with flush inspection and sticky cards commenced 13 August. Sticky cards are monitored for ACP and other common citrus pests and replaced every other week. 4011 psyllids have been found on sticky cards of which greater than 67% are in no-mulch plots while only 15% have been found in plots that receive insecticides and 4% in plots with both mulch and insecticides. All of 79,944 shoots have been examined so far of which 12,316 were infested with ACP and 1759 were infested with Aphids, both predominately in no-mulch plots with more than 65% infestation. In contrast, 1396 ACP and 569 aphid-infested flush were found on insecticide-treated trees and 463, and192 on trees on mulch and receiving insecticide drenches. Leaf samples for HLB testing were collected July 2014 of which there were 55 positive samples: 12 from mulched treatments and 10 from insecticide plots. Only 1 tree was positive in plots with both insecticide and mulch. Trunk area cross section measurements collected on July 1, 2014 showed different results from previous reports. Trees receiving mulch and foliar nutrition are now 20% larger than trees receiving nutrition. Also the no-treatment control with mulch is still larger than without mulch. However, the same is no longer true for the treatments reviving insecticide applications. Treatments receiving insecticide drenches either with and without foliar nutrition and no mulch now have larger trunks than the same treatments with mulch. This may be a result of an irrigation system failure during 13 days in Feb and 15 in May which caused more drought stress to trees on mulch while the unmulched trees benefited more from rainfall (1.53 inches at FAWN SWFREC). HLB samples and growth measurements are scheduled to be collected again late January 2015. The first fruit harvest was completed 9 Dec with 19% more fruit by weight was collected from plots with mulch when compared to plots without mulch. Insecticides increased yield by 17%, foliar nutrition increased yield by 7% compared to the check. Juice analysis was conducted January 14. Normal grove care operations continued which included Kocide sprayed monthly for control of canker, and one application of Intrepid for leafminer control. Delegate was sprayed in all insecticide plots late on October 15 as well as an application of Baythroid to all trees as a dorment season spray January 8. A manuscript entitled “Role of Metalized Reflective Mulch with Nutritional and Insecticidal Treatments in Mitigation of HLB in New Citrus Plantings is in preparation.
A manuscript has been submitted, which summarizes scion (Hamlin, Valencia, Pineapple, and Ray Ruby) introduction into the growth facility by shoot-tip grafting (STG) from Dec, 2011 to July, 2013 (20 mo). Sixty-six mother plants out of 171 STGs introduced from FDACS were determined to be disease-free after micropropagation, budding, and disease indexing. A total of 157 putative transgenics were generated, 66 survived micro-grafting, and 42 expressed the NPTII protein. NPTII immunostrips, ELISAs and Southern blots were used to characterize the transgenics. Similar to previous reports in immature and mature citrus, there were a large number of escapes using kanamycin as the selection agent. The biggest loss during this period was due to micro-grafting as only 66 out of 157 (42%) GUS or GFP positive shoots survived. We have determined that micro-grafting success is dependent on the transformation batch and shoot age; it must occur early after shoot development. Alternately, rooting mature citrus must be established. Several of these transgenics flowered after the T5 fluorescent bulbs were replaced with LED lights, night temperatures decreased, drought stress applied, or if the trees were moved to natural light. Because our facility has no natural lighting, a greenhouse with natural and supplemental lighting is necessary to obtain early flowering and fruiting of desirable events. We continue to produce transgenic mature citrus scion and rootstock using plasmids with disease resistance genes obtained from various scientists. Since most of these constructs have no GUS or GFP markers, we micro-graft all shoots and screen with PCR, which is a more rigorous process than with reporters. Transgenics are double and triple checked with PCR and NPTII immunostrips to ensure they are stable, not chimeric, and expressing the NPTII protein. We have successfully micrografted at least as many putative transgenics. There are currently 3,600 putative transgenics in the pipeline to be screened. For replicated disease screening, the number of transgenics will be increased at least threefold by budding, and expression in vegetative progeny can be determined. For one particular genetic construct, budding with transgenic immature rootstock can begin at any time to facilitate experiments to determine the contribution of each genotype in imparting tolerance. One mature Swingle rootstock tree, transgenic for a disease resistance gene, is over four feet tall and should flower soon for seed production. We continue to optimize biolistics in order to increase our productivity. Thus far, the results are promising and we have recorded 200-300 transient GUS and GFP foci per shot in mature scion and rootstock shoots. If 0.01% of these foci develop into plants, 2 to 3 transgenics might be produced after each shot. During the optimization process, we have determined optimum stage height, gold particle size, and helium pressure. The primary advantage of using biolistics is that it avoids all of the antibiotics used to suppress growth of Agrobacterium, which also suppress shoot growth in scion and rootstock. The growth facility is being certified as a nursery. This will hasten the pace of providing plants to scientists, growers, and industry. Routine disease testing in April will be conducted by FDACS.
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