All of the research described in the previous report is still ongoing or is still being analyzed. 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 is being analyzed. A study of CiFT3 transgenic tobacco plants treated with various growth regulators has been performed and all of the data have been collected except for the flowering dates of the nontransgenic control plants that have not yet flowered. 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. The complete construct is completed and is being tested in tobacco for a rapid test before citrus experiments are started. This research was presented at the ASHS national meeting.
An extensive survey of HLB-affected groves indicated that greater decline in fibrous root health and greater expression of HLB symptoms is observed where irrigation water is high in bicarbonates (> 100 ppm) and/or soil pH > 6.5. Affected groves employ micro-sprinkler irrigation that concentrates fibrous roots in the wetted zone and soils often have a history of excessive dolomite liming to manage high residual copper. Affected orchards have off-color foliage, thin canopies due to excessive leaf drop, twig dieback and more severe HLB symptoms in leaves and fruit. HLB symptom expression of trees on different rootstocks is ranked Swingle citrumelo > Carrizo citrange > sour orange > Cleopatra mandarin which follows rootstock intolerance of bicarbonate. To identify the relationship between HLB decline and bicarbonate stress, fibrous roots of 8-15 yr old Valencia orange trees on Swingle or Carrizo rootstock were sampled in 37 orchards with varying soil pH and irrigation water quality. Lower root density was correlated to irrigation water pH and soil pH > 6.2. Fruit production over three seasons (2009-2012) during which HLB incidence was accelerating revealed that groves under high bicarbonate stress declined 20% in yield compared to groves under low bicarbonate stress with a 6% increase in production. Yield loss under bicarbonate stress was correlated with reduced fibrous root density compared to the non-stress condition. To confirm that treatments with acidified irrigation water reduce the impact of bicarbonate stress on root health, we are surveying 8 grove ridge locations in Highlands county and 4 flatwoods locations in Desoto 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. This survey will continue 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.
Stress intolerance of HLB infected trees is a direct consequence of greater than 30 percent loss of fibrous root density compared to non-diseased trees. HLB-induced 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. To understand the possible interaction between these two pathogens and host tolerance, two rootstocks, Cleopatra mandarin (Citrus reticulata) and sour orange (Citrus aurantium) were inoculated with Las, Phytophthora nicotianae (P.n.), both or neither. P.n. infection, root loss, and carbohydrate content of fibrous roots were assayed. P.n. infection increased on both rootstocks indicating that Las reduced their tolerance to P.n. Differences in P.n. infection on Las positive seedlings at 5 and 11 weeks after inoculation suggest the interaction changed over time. Both pathogens caused significant root loss alone, but Las in combination with P.n. did not cause additional root loss compared to Las alone. Based on these results, we hypothesize that 1) early in disease development, Las increases susceptibility to P.n. infection by increasing zoospore attraction and/or facilitating penetration; 2) Las induces root loss resulting in a temporary drop in P.n. population by reducing available food supply; 3) As new root flushes occur, P.n. starts the infection cycle again and this cycle repeats until there is a complete loss of fibrous root system. To test the value of P. n. control in slowing tree decline in the presence of both pathogens, P.n.-infested nursery trees were inoculated or mock-inoculated with HLB-infected 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.n. 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.n. root infection and increased fibrous root mass compared to untreated controls for HLB(-) trees. Neither Ridomil or Aliette significantly reduced P.n. 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.n.
Seasonal root sampling continues in two field sites for root density and root growth. Sampling has been completed for a full year for Hamlin/Swingle and 6 months for Valencia/Swingle. Differences in root growth patterns for different scions are already becoming apparent. The role of HLB and Las in modifying quantity and timing of root growth between these two scions is being analyzed. Results so far emphasize the need to use treatments that promote root longevity as the main method of managing HLB root loss. Root growth stimulation is unlikely to improve root density. As more of the healthy trees in the field trial become PCR positive for Las it is increasingly difficult to find sufficient presumed healthy trees for comparison. This is especially difficult for trees of sufficient age for seasonal root sampling. While field sampling continues, the results will become more descriptive of the decline of HLB affected trees than a comparison to healthy trees. We will soon be purchasing equipment to allow non-destructive measurements of root density and root growth, so that young plantings can be sampled allowing for direct comparison of healthy and HLB affected trees in the field. Sampling at the UF-CREC St. Helena rootstock trial site has been underway for 1.5 years to evaluate the effects of HLB on new experimental rootstocks. These data demonstrate how the new rootstock lines respond to Las infection. The findings been presetned for presentation at recent grower meetings (Citrus Expo and OJ Breaks). The most promising rootstocks have been included in greenhouse trials to understand why they are responding differently with the objective of finding and validating parameters for rapid screening for new rootstocks. The two most promising rootstock breeding lines and Swingle as a standard rootstock control have been graft inoculated in the greenhouse and transplanted to rhizotrons to monitor root growth and death. Data analysis of root growth and root longevity in response to Las is currently underway. Dying roots are becoming apparent, so sampling for comparative physiological responses will begin soon.
FireWall 50WP (65.8% streptomycin sulfate; Agrosource, Inc.) has been granted a second year of 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 of FireWall registration, EPA requires monitoring of Xanthomonas citri subsp. citri (Xcc) for streptomycin resistance. This survey for 2014 season will be conducted in November and the report submitted to EPA by the end of the year. A recently completed greenhouse trial 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 aginst canker in that the penetration of the leaf insures that streptomycin is protected from wash-off by rainfall, weathering and degradation by UV light.
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 two USHRL projects funded by CRDF for transforming citrus. Non-transgenic citrus can also be subjected to the screening program. CRDF funds are being used for the inoculation steps of the program. Briefly, individual plants 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. USDA-ARS is providing approximately $18,000 worth of PCR-testing annually to track CLas levels in psyllids and rearing plants. Additionally, steps to manage pest problems (spider mites, thrips and other unwanted insects) are costing an additional $1,400 annually for applications of M-Pede and Tetrasan and releases of beneficial insects. To date on this project, it funds a technician dedicated to the project, a career technician has been assigned part-time (~50%) to oversee all aspects of the project, two small air-conditioned greenhouses for rearing psyllids are in use, and 18 individual CLas-infected ACP colonies located in these houses are being used for caged infestations. Additionally, we established new colonies in a walk-in chamber at USHRL to supplement production of hot ACP. Some of the individual colonies are maintained on CLas-infected lemon plants while others are maintained on CLas-infected Citron plants. As of September 2, 2014, a total of 6,208 transgenic plants have passed through inoculation process. A total of 122,855 bacteriliferous psyllids have been used in no-choice inoculations.
The overall aim of this project is to develop and evaluate soft nanoparticles (SNP) to deliver natural biocides to the phloem of HLB infected trees by foliar and/or bark application. In the last quarter (July ‘ Oct ’14) the efficacies of formulations of all three EOs employed (EO-A, EO-B and Thyme oil) were evaluated through multiple methods. Subsequent to a careful selection of formulations based on efficacy against L. crescens, phytotoxicity and EPA registration of surfactants used, preparation of selected formulations have been scaled up. Currently, tests to assess efficacy of the formulations in HLB infected citruses via a bud graft technique at undergoing at Indian River Cirrus and Education Center, Ft. Pierce. Normally about six months are required for the assay to give results. To investigate the penetration of formulations into citrus leaves, dye doped microemulsion formulations have been prepared and applied by foliar application. From a number of oil soluble dyes, formulations were initially developed with fluorescein and Nile Red dyes. However, upon application to plants and successive dilution in the leaf, significant background interference was observed in the form of auto-fluorescence of chlorophyll and other leaf components (red region). Presently, new dyes have been selected between blue and green fluorescence. Formulations have now been developed with the new dyes like Bodipy 505/515, Vybrant DiO etc. which show fluorescence in 475-525 and 550-650 nm range. Leaf penetration experiments will begin in the upcoming weeks. In another approach we are aiming to developing methods for detection of EO transport to the phloem. Duncan grapefruit and Valencia orange plants have been acquired in order to do so. As an additional impact, zebra chip disease on tomato and potato caused by Ca. Liberibacter solanacearum (CLso) a close relative of CLas was identified as a rapid disease surrogate. The assays were all done at Texas A&M University. Some SNP formulations were successful in relieving zebra chip symptoms on tomato plants in a period of 30 days. QPCR tests are currently being conducted to get a quantitative evaluation.
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. 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 remarkabe resistance in chimera compared to control. Canker infiltration showed greatly increased resistance in two chimera AMP and several thionin transgenics, at 107CFU/ml. RNA was isolated from transgenic plants containing chimera and thionin. RT-PCR showed gene expression in the transgenic plants. Further gene expression level was evaluated with RT-qPCR. Our results showed gene expression variation between different transgenic lines, from several fold to 35 fold. Transgenic lines containing D4E1 were evaluated with Xcc infiltration. All the transgenic lines with canker development at 105 CFU/ml while some transgenic lines show less canker development at 104 CFU/ml. Bacterial growth rate in transgenic lines containing D4E1, chimera and thionin was investigated by qPCR. Our results showed some transgenic lines containing chimera and thionin had low Xcc growth rate. More transformed Hamlin carrying chimera were generated and over 30 were confirmed positive by PCR. About 20 Hamlin transformed with thionin also were obtained. They will be tested by RT-PCR and replicated for HLB challenge. Putative 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 and growing for transgene verification, replication and testing. 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 and infiltration with Xanthomonas. Over 60 transgenic Carrizo with GRP2 were transferred to soil. DNA was isolated from 20 of them and 19 of them are PCR positive. Some of them showed canker resistance when infiltrated with Xcc at concentration of 105/CFU. Fifteen transgenic Carrizo and seven transgenic Hamlin with peach dormancy related gene MADS6 were planted in soil and they are ready for DNA isolation. 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 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.
This quarter we have continued to make progress on our transformation approaches: 1. Stable transformation in citrus using new vectors. We previously found that the original vector system used to create the Bs3 promoter constructs was contributing to low transformation efficiency in citrus, and switched to a pCAMBIA-based vector system. Two ProBs314EBE:avrGf2 transgenic plants created using the new vectors in citrus cultivar Carrizo have now been confirmed by PCR to contain the transgene, and these will be examined for appropriate gene expression by RT-PCR. An ongoing pipeline of transformants are being generated with the new vectors. To date a total of 2,056 putative transgenic shoots of grapefruit, sweet orange and Carrizo were screened for this period. Results show that no GUS positive has been observed for the sweet orange cultivar transformed with any of the constructs analyzed, however, 3 shoots were chimeric for the pCAMBIA2201:NosT:Bs3super::avrGF2 construct. In general, Grapefruit had a combined total of 12 and 41 shoots being GUS positive and chimeric for GUS, respectively for all constructs anlayzed while Carrizo citrange had 185 and 180 shoots being GUS positive and chimeric for GUS, respectively. These GUS and chimeric shoots will later be screened via PCR once rooted and transferred to soil for acclimatization. 2. Stable transformation in tomato test system: A tomato test system was previously designed and tested in which the 14 EBE promoter was fused to the avrBs4 gene capable of inducing a hypersensitive reaction in tomato. T1 generation of Bonny Best and Large Red Cherry transformed with ProBs3_14EBE:avrBs4 were screened for pathogenicity reaction with X. euvesicatoria strain (Race 9). Promising resistant transgenics have been selected for T2 generation analysis to confirm that this test system, in which resistance is induced by the effectors AvrBs3 and AvrHah1, is functional for conferring stably-transformed transgenic disease resistance.
We are still working to obtain stably transformed citrus containing the BS3 promoter with added TAL effector binding elements (4 or 14 EBE) fused to a defense response inducing gene. We have obtained three transformants with a 14 EBE construct driving either the AvrGf1 or AvrGf2 Xanthomonas effector gene in Carrizo citrange, a citrus variety more amenable to transformation. PCR-based analysis of gene expression demonstrated that these constructs were induced as expected upon infection with the virulent strain X. citri strain Xcc306, validating that the promoter works in stably-transformed citrus. Whereas expression of AvrGf1 or 2 genes in orange and grapefruit triggers a hypersensitive defense response, this reaction doesn’t occur in Carrizo and we are not able to assess resistance to X. citri in these plants. In Duncan grapefruit, our efforts to transform constructs with AvrGf1 and AvrGf2 transgenes, where an inducible hypersensitive response is expected, have led to the isolation of seven putative transformants. These were sequenced to determine whether the transgene construct was intact. We found that all seven had deletions in the area of the transgene comprising parts of the promoter region and Avr gene. We believe our difficulty in obtaining transgenic grapefruit is arising either because the construct may have a tendency to recombine in Agrobacterium or during the transformation process, or the promoter may be leaky at some point during the transformation process, even though we have shown that it is tightly regulated in transient assays in leaves. Therefore, we are taking further steps to assess the stability and background expression of the construct. To investigate construct stability, transgenic tobacco plants (Nicotiana tabacum) resulting from the constructs pCAMBIA2201, pCAMBIA2201:NosT:Bs3super::avrGF2, and pCAMBIA2201:NosT:Bs34box::avrGF2 have been generated to determine whether the constructs are stable in another system. If construct sequence optimization is necessary, it will be easier in the tobacco system. We are also carefully assessing the potential for background expression of the construct. Four Carrizo transgenics containing the 14EBE construct fused to GUS were obtained, and these will be verified for the presence of the intact transgene by PCR, followed by GUS staining to determine whether there is any unexpected expression in any tissues other than leaves. In addition, N. tabacum and N. benthamiana transgenic plants carrying the 14 EBE construct fused to GUS will be stained to determine whether GUS is expressed anywhere in the plant. If one of the added EBEs produces unwanted background expression, we will be better able to determine which one is problematic in this system. We will also attempt to transform Duncan grapefruit with a construct containing the Bs3 promoter without any added EBEs, fused to an Avr gene or to GUS, to assess whether the unaltered promoter produces any background expression. Work also continues in the tomato model system, where one transgenic line carrying the disease resistance construct showed a reduction in symptoms in initial tests. T2 plants are being generated for further study.
Objective 1: Generate functional EFR variants (EFR+) recognizing both elf18-Xac and elf18-CLas. In order to perform screening on complex EFR mutant libraries required to discover mutants which respond to elf18-CLas ,we have been developing a FACS-based screen. To this end we have generated a number of reporter lines (using GFP) in both suspension cultures and transgenic Arabidopsis plants. The reporter lines are driven by the FRK1, WRKY30 and PER4 promoters. We have tested two PER4p:GFP cell suspension lines for responsiveness to elf18, and both of these give clear induction of the reporter gene following treatment. However, when protoplasts were produced of these lines, elf18 responsiveness was no longer observed. We are currently retesting these lines to ensure the buffer conditions and EFR expression is correct. In addition, plant and cell suspension lines transformed with FRK1p:GFP and WRKY30p:GFP are also in the process of being tested. In addition to the mutagenesis approach, we screened the Nordborg collection of Arabidopsis ecotypes for sensitivity to elf18-CLas or reciprocal chimeric peptides of elf18-Ecoli and elf18-CLas. Of this collection, none show ROS in response to either elf18-CLas or the chimeric peptides. We did observed one line (Se-0) which had enhanced response to the CLas-Ecoli-elf18 chimera. This chimera has some activity in Col-0, but only at high concentrations. Further testing of this ecotype revealed that it also enhanced ROS to elf18-Ecoli and to flg22, indicating that it was not a variant of EFR which was causing the enhanced ROS. Indeed the sequence of EFR from Se-0 contains no non-synonymous SNPs. We have been also investigating the possibility of targeting other PAMPs. To this end we conducted a bioinformatic 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. We are currently waiting for delivery of the CLas-CSP22 peptide to test. Objective 2. Generate functional XA21-EFR chimera (XA21-EFRchim) recognizing axYS22-Xac. These constructs have been constructed and tested and a manuscript is under revision. Objective 3: Generate transgenic citrus plants expressing both EFR+ and XA21-EFRchim. Transformation experiments are ongoing; to date, a total of 5,781 ‘Duncan’ grapefruit and 956 sweet orange segments have been collectively transformed with the constructs EFR, EFR-XA21, EFR-XA21-EFRchim and pCAMBIA2201 (empty vector control). A total of 580 and 219 grapefruit and sweet orange shoots, respectively, were transferred to rooting media. These shoots were first analyzed histochemically for GUS expression. The results show that collectively 6 grapefruit shoots were GUS positive with the constructs EFR-XA21, EFR-XA21-EFRchim and pCAMBIA2201 and 1 sweet orange shoot GUS positive with the construct EFR-XA21-EFRchim. Other grapefruit shoots (47) collectively stained partially (chimeric) for GUS with the constructs EFR, EFR-XA21, EFR-XA21-EFRchim and pCAMBIA2201, while 5 sweet orange shoots stained chimeric for GUS with the constructs EFR, EFR-XA21 and EFR-XA21-EFRchim. Chimeric shoots were those segments with less than 85% of blue staining.
The measurement of petiole phloem area for all treatment was completed by December 2014. Data from all heat treated trees with dry air are being compiled. Physiological measurements including leaf water potential, stomatal conductance, and phloem area are being analyzed to evaluate possible effects of heat treatment on HLB-infected citrus trees. Chlorophyll fluorescence, fruit set, fruit diameter, leaf area, and PCR data are being analyzed to assess the effects of heat treatment on overall tree health and symptom reduction. According to the preliminary analysis of the physiological measurements, there is no evidence that heat treatment had an effect on these markers. Since the physiological measurements can heavily depend on weather, they might not be the best indicators of heat treatment performance. Chlorophyll fluorescence measurements were not significantly different among heat treated and control trees but further studies on this parameter need to be done in future experiments. Preliminary data analysis shows that trees heated to 55’C had significantly more and larger fruit when compared to control trees. Starting in January 2015, fruit drop measurements on trees heated with dry air will be collected. Fruit from under heated and control trees is counted on a weekly basis. This data will also be used to evaluate the effect of the heat treatment on symptom reduction. Fruit quality will also be analyzed. Fruit quality analysis was done on trees heated with steam in August 2014. Total acid, total brix, acid/brix ratio, and pounds solids per box were evaluated. There was no significant difference in total acid between heated and control trees. However, total brix, acid/brix ratio, and pounds solid per box were all significantly higher in heat treated trees when compared to untreated trees.
During summer 2014, 9- and 13-month post treatment physiological tests on stomatal conductance, water potential, and leaf anatomy samples were continued. Also, fruit set data, fruit diameter data, average leaf area data, and average leaf area index (LAI) was collected and analyzed. The results of the fruit set analysis show there is a significant difference between the amount of fruit on the trees that were heated to 50’C and 55’C as compared to the sick control trees. The fruit set on the trees heated to 55’C and 50’C are significantly higher compared to untreated trees. The fruit diameter data shows there is a significant difference between the average fruit diameter on the 55’C treated trees as compared to the control trees. There is also a significant difference between the 45’C treated trees and the 55’C treated trees. The fruit on the 55’C trees is also significantly larger than the fruit on the 50’C trees. There was no noticeable difference in the average leaf size for any heat-treated or control trees. However, there were some differences in the average LAI. This measurement was taken to help measure the leaf density of the tree canopy. The trees heated to 55’C and 45’C had a noticeably higher average LAI than the control trees. The trees heated to 55’C had a significantly higher LAI than the trees heated to 50’C. The trees heated to 50’C dropped more leaves than the trees heated to 45’C directly after treatment. Leaf anatomy samples were processed and analyzed. The leaf petioles from 7 days after treatment and 1, 3, and 6 months after treatment were cross-sectioned and stained to measure phloem area. Since the disease is phloem-limited, the phloem was analyzed for any changes due to heat treatment. Ideally, in heat-treated trees that are recovering, the phloem area should increase once the bacterium is no longer present. The results suggest there was an increase in phloem area directly after treatment in all treated trees, and it continued until 1 month after treatment. Then, the phloem area began to decrease again.
The work in the Core Citrus Transformation Facility (CCTF) continued without interruption. Co-incubation experiments using different type of explants and Agrobacterium strains are still being done on a weekly basis resulting in production of more transgenic plants. Three new orders were received during the last three months although most of facility’s productivity came as result of work on older orders. Significant effort was invested into production of rootstock plants carrying the NPR1 gene requested by the CRDF. About 850 shoots were tested in the primary PCR with 135 of them being positive. Forty eight of these shoots died either before or after grafting, and 27 were negative in the second PCR. The other 60 positive shoots were grafted. Out of those, 27 were moved to pots. Presently, there are 29 plants in the greenhouse that are 6-10 inches tall. In about six weeks many of those plants will reach the size when they could be cut into nodal explants for propagation. In the period covered by this report, CCTF produced plants for the following orders: pNPR1-30 plants, pNPR1-G-five plants, pX4- two plants, pELP3-G-one plant, pELP4-G- one plant, pMG105- eight plants, pPR2-one plant, pHGJ2- one plant, pHGJ8-one plant, pW14- two plants, pMED16-four plants, pN7-three plants, pN18- four plants. Out of total of 63 transgenic plants, 29 were Carrizo citrange, three were Swingle citrumelo, one was C. macrophylla, one was Mexican lime, one was Valencia orange, and 28 were Duncan grapefruit. Within previous year the efficiency of transformation and the ability of explants to regenerate shoots are little lower than they were in previous years. The reason for this is probably the low quality of seedling explants which are starting material in our experiments. Seeds used for production of Duncan grapefruit, Valencia orange, and Hamlin orange seedlings that are cut into explants are obtained from fruit harvested on CREC property where HLB is widespread. Fruit have unhealthy appearance; seeds are smaller and have altered color. Seed vivipary which occurs in older fruit of Duncan appears four months earlier than it used to. This is a strong indicator of disrupted hormonal balance within the fruit which may contribute to changes we noticed. For this reason, we intend to change the way fruit are acquired. Duncan grapefruit will be picked from CREC property only between September and January. Depending on the quality of fruit this season CCTF may start getting Duncan fruit from the outside source even before January. Fruits of sweet oranges will be acquired from the outside sources throughout the whole season and CCTF will require assistance for this.
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