Hybrids were selected from populations of seedlings from crosses made the previous year. Selected SuperSour hybrids were identified by morphology, horticultural characteristics, and molecular markers for further study. Cuttings were made from 300 different SuperSour rootstock hybrids in preparation for disease testing and field trials. Cuttings made the previous time-period were grown-up in preparation for budding. A greenhouse system was developed to evaluate SuperSour hybrids for tolerance of high pH/calcareous soils. A project continued to compare graft inoculation and Asian citrus psyllid inoculation as methods for evaluating cultivar resistance or tolerance to HLB in the greenhouse. The results of this study will have important implications in focusing resources to be used for evaluating the HLB-resistance/tolerance of new conventional and transgenic cultivars. A study using the Affymetrix GeneChip citrus genome microarray, and that compared transcriptional response of susceptible citrus to the tolerant selection, US-897, was accepted for publication in the journal ‘Plant Science’. The study was focused on identification of genes associated with the HLB tolerance of US-897. The analysis identified several pathogenesis-related genes that were induced by Liberibacter infection of susceptible citrus, such as thaumatin, chitinase, phenylalanine ammonia-lyase, peroxidase, and a cytosolic copper/zinc superoxide dismutase. Microarray analysis identified 326 genes which were significantly up-regulated by at least 4-fold in the susceptible genotype, compared with only 17 genes in US-897. Notably up-regulated in susceptible citrus, but not US-897, were a Myb-like transcriptional regulator protein (generally associated with a salicylic acid-mediated hypersensitive response to bacteria) and phloem protein 2-B15 (PP2). Exclusively up-regulated in US-897 was a gene for a 2-oxoglutarate and Fe(II)-dependant oxygenase, an important enzyme involved in the biosynthesis of plant secondary metabolites associated with antimicrobial activity and plant defense. More than eight hundred genes were expressed at much higher levels in US-897 independent of infection with Liberibacter, some of them clearly also associated with disease resistance. Among these genes, a constitutive disease resistance protein (CDR1) were notable. One form of CDR1 was found to be expressed more than 200-fold higher in US-897 than in susceptible citrus. The involvement of these and other detected genes in tolerance to HLB and their possible use for biotechnology were discussed in the paper. Results of a study on early RNA and metabolic changes in HLB infected citrus, both tolerant US-897 and susceptible Cleopatra, were presented at the CHRP meeting in Denver, and provided significant insights into key metabolic pathways that may be targets for engineering HLB resistance. Metabolic changes were analyzed using gas chromatography coupled mass spectrometry. There were large differences between the metabolic constituents of non-infected and HLB-infected Cleopatra, with many compounds notably up-regulated in response to infection, including ornithine, proline, lysine, asparagine, and saccharic acid. Corresponding to the nearly non-existent symptom development of US-897 in response to HLB infection, few compounds were significantly different between non-infected and infected US-897. Also presented at the the CHRP meeting were the results of a study of volatile organic compounds produced by non-infected and infected Valencia trees. Samples were collected on polydimethylsiloxane coated magnetic glass twisters and analyzed by gas chromatography-mass spectrometry. Changes in volatile organic compound profiles were detected, but were not strong or consistent across time following infection.
The initial indexed mature material that was maintained in vitro for a long time did not adapt properly after planting in soil. The plants have already 3 months and they have very long leaves and are growing stunted, most of them are not behaving like normal plants. We are going to continue monitoring them the next couple of months to see if they recover and come out of this stage. The new batches of indexed mature material introduced from May to July 2011 are being grafted on Swingle citrumelo, Macrophylla and C. Volkameriana that were growing originally inside the laboratory. This material will also be used to establish mother plants and to establish the first batch of plants that will give us the material for mature transformation experiments. The rootstocks that we started planting when the growth room was finished are not ready for grafting yet. Only a few plants coming from this material were available to graft and the whole group will be ready in October 2011. The Growth Room is still under “modification”. It took almost 5 months to be able to have completely access to the computer program, however it is still an issue to get access for users that come and go which is the case with the personnel we currently have. The Citrus Research and Education Center does not have enough IT help to assist in a timely fashion with the several needs required for this project. The IT people usually respond within weeks instead of hours to any problem we may have. The humidifier in the small room is still not working properly. The company responsible for the job was not able to coordinate the different subcontractors to finalize the job. The warranty in this case will not work and we will need to pay for them to finish this task. Another problem that we are currently facing is related with filtrations among the different areas where the floor meets the walls but also window sealing. The water is moving between growth rooms and between the growth room and the office. The caulking applied to close the gaps and to provide a seal between the concrete floor and the panel walls is not working. The subcontractor came and applied a new coat of caulking but it was not enough to stop the filtration. It is still happening as of today. Water leaking through the window has not been addressed yet. We also experience problems with tripped breakers and bad electrical connections of some lamps. We are still monitoring the situation.
The main objective of this project is to manipulate calcium signals by over-expressing calcium signal modifier genes (CSM) from citrus to develop broad spectrum disease resistance. During the fiscal year 2010-2011 we produced 17 transgenic C-22 rootstocks, three Valencia and six Hamlin oranges over-expressing the CSM-1 gene. Previous grapefruit plants produced with this gene Were tested for disease resistance and shown to be resistant to citrus canker, Phytophthora nicotianae and Alternaria alternate and to the toxin tentoxin. Furthermore we engineer a new Agrobacterium binary vector with a citrus lectin gene to be used for genetic transformation during the fiscal year 2011-2012. As part of the transformation procedure, we are trying to accomplish genetic transformation without the use of antibiotic, and using only citrus genes. We were able to find a substance that increase the regeneration capacity of citrus stem segments used for genetic transformation and at the same time have the potential to be used for selecting transgenic plants and replace the antibiotic selection system. We are currently optimizing the system. We performed several crosses of Rio Red X Hirado Buntan pummelo and Rio Red X Wilking tangor. Hybrids will be recovered in November 2011.
In our initial schedule, the mature transformation facility (lab and greenhouse) at the CREC in Lake Alfred had to be implemented during the first year of the project. An existing laboratory was modified to fulfill the requirements of a tissue culture facility. The laboratory is now fully operative. Regarding the greenhouse, it became impossible to accommodate the budget to our plans for constructing the outstanding facility we requested. Alternatively, a growth room was designed profiting an existing structure at the CREC. The growth room construction was initiated in October 22nd 2010. The projected date of completion was February 11th 2011. Technically it was finalized by mid-April, however there were several technical/operational problems that came out during the following 4-5 months, especially regarding the refrigeration system (environmental conditions were not estable; some air handlers were not producing the air the manufacturer claims, in some cases the thermal expansion valve was changed because it was defective, air filters were not the ones we requested and they were changed, the humidifier in the small room is not located in the appropriate place for working properly), computer program (growth room technician still doesn’t have access to the program though this is currently being solved), water elimination after irrigation is defective, soil sterilizer (it needs a special accommodation to work ‘safely’). A generator should be purchased; without it, any prolonged electricity cut could jeopardize the whole project. The manager from Florida (Dr. Cecilia Zapata) completed her training in Spain during the first year, moved to Florida and has been working hard to set up the mature transformation facility at the CREC during the whole second year. Two part time OP technicians were hired to work on tissue culture and on plant preparation and a third OP technician was hired to work care at the growth room, under the supervision of the manager (and the PI at the initial stage). Another technician has been recently hired to help in the growth room and the lab because one of the OP technicians is leaving soon due to personal reasons. The Spanish lab has been monitoring the progress of the Florida facility. The PI and his manager at the IVIA greenhouses traveled to Florida last March 2011 to supervise the growth room construction and to set up healthy citrus germplasm bank establishment. The PI and his greenhouse manager will travel again to Florida next October 2011. An IVIA scientist with experience in mature citrus transformation will travel to Florida to help setting up the facility tentatively next November 2011. It is programmed that the IVIA scientist will spend three months in Florida (November 2011-February 2012). In Spain, mature tissues from the three sweet orange types (Hamlin, Pineapple and Valencia) plus Carrizo citrange were readily transformed. For our second objective, improving citrus tree management, we proposed to over-express flowering-time genes in both the Carrizo citrange rootstock and the Pineapple sweet orange scion. We have now at least ten independent transgenic lines of Pineapple sweet orange and Carrizo citrange over-expressing either CsFT or CsAP1 flowering-time genes already established in the greenhouse. We have characterized these transformants at the molecular level and continue characterizing them phenotypically in detail in the greenhouse. Moreover, for generating a dwarf-dwarfing rootstock, we have incorporated a construct aimed to induce RNA interference to downregulate the expression of a crucial gene in gibberellin biosynthesis, CcGA20ox1, in mature Carrizo citrange.
For the construct containing the ctEDS1 gene in the binary vector pBINplusARS, we selected the T0 seeds and obtained so far 15 independent T1 transgenic plants. The T2 plants will be planted to select for homozygous lines and also for an initial disease resistance test. For ctNDR1 transformation of the ndr1-1 mutant, we currently obtained 11 homozygous lines. We reported earlier that some of the lines showed enhanced disease resistance. Now we are at a stage to systematically analyze the defense phenotypes of the ctNDR1 overexpressing transgenic plants, using the homozygous lines that we have. For ctPAD4 + pad4-1 and ctEDS5 + eds5-1 plants, we have obtained over 10 and 3 independent T1 transformants, respectively. We have planted the T2 seeds and will test the segregating T2 plants for disease resistance and harvest seeds from multiple individual plants for selection of homozygous lines. For ctEDS5 + eds5-1, we are also selecting more T0 seeds in order obtain additional transformants. Additional newly cloned genes include ctSID2, encoding the major biosynthetic enzyme for salicylic acid biosynthesis, and ctNHL1, which is a homolog of NDR1. These two genes were obtained from RACE followed by RT-PCR. We have moved the ctNHL1 cDNA fragment from the pGEM T-easy vector to the binary vector pBINplusARS for plant transformation. For ctSID2, we only obtained the cDNA clone in the pGEM T-easy vector. However, we have had some trouble in moving this fragment into pBINplusARS. We are currently trying a few different approaches to address this problem. Since the recent release of the Citrus sinensis (sweet orange) and clementine genome sequence, we have conducted extensive bioinformatics analysis on defense related genes in citrus based on published literature. Such analysis confirmed citrus defense genes that have already been cloned in my laboratory with this support. In addition, we found that most published defense genes are present in citrus with full-length sequences available. Therefore, we anticipate that our further cloning and functional characterization of citrus defense genes should be greatly expedited. We have so far selected additional 10 candidate citrus defense genes. The cloning of some of these genes is underway.
Objective 1: Transform citrus with constitutively active resistant proteins (R proteins) that will only be expressed in phloem cells. The rationale is that by constitutive expression of an R protein, the plant innate immunity response will be at a high state of alert and will be able to mount a robust defense against infection by phloem pathogens. Overexpression of R proteins often results in lethality or in severe stunting of growth. By restricting expression to phloem cells we hope to limit the negative impact on growth and development. Results: The transgenic plants containing AtSUC2/snc1 and AtSUC2/ssi4 mutants, as well transgenic control plants are growing in the laboratory of Dr. Orbovic at the UF Citrus Research Facility (Lake Alfred) until they are ready for the next level experiments. Objective 2: Develop a method to elicit a robust plant defense response triggered by psyllid feeding. By further restricting expression of the R protein to a single cell that is pierced by the insect stylet, we anticipate that a defense can be mounted without a manifestation of a dwarf phenotype. Results: The vast majority of T1 and T2 transgenic Arabidopsis plants expressing snc1 and ssi4 mutant coding sequences under the control of the AtSUC2-940 promoter have wild type phenotypes. Although the AtSUC2 promoter has been reported to be phloem-specific, we have found that it often does not maintain this tissue-specific pattern of expression in transformed Arabidopsis. However, despite the likelihood of expression in tissues other than phloem, only a few transformants showed any negative developmental or growth abnormalities. This lack of a negative phenotype in Arabidopsis provides a basis for optimism for similar results in transformed citrus. Our working hypothesis is that expression of the constitutive R proteins (mutants) in the phloem will active components of the innate immunity response to provide enhanced protection from Liberibacter infection in phloem cells. In order to monitor the activation state on the innate immunity system, we will cross the R protein transformants with transformed Arabidopsis lines containing pathogen-inducible promoters driving GUS reporter genes. We cloned the PR2 (also known as BGL2), and PR5 pathogen-inducible promoters in front of the GUSplus gene in pCAMBIA 2301. They were sequenced, transformed via electroporation into Agrobacterium tumefaciens strain GV3101 and introduced into Arabidopsis (strain GV3101) through the floral dip protocol in order to generate stable transgenic lines. We currently await the T-1 seeds from these transformations. In parallel, we acquired BGL2-GUS (in pBI101 vector; from Dr. Xinnian Dong from the Duke University) stable transgenic line to use as an alternative donor. The introduction of our R protein constructs into reporter lines by crosspollination will be faster and more efficient than transformation by agrobacterium. Being able to monitor constitutive activation of the innate immunity system by GUS will provide a test of the hypothesis that our constructs will activate pathogen-inducible promoters and will allow us to select lines that have strict phloem-specific expression for further study.
This is a project to find an interim control measure to allow the citrus industry to survive until resistant or tolerant trees are available. We are approaching this problem in three ways. First, we are attempting to find products that will control the greening bacterium in citrus trees. We have chosen initially to focus on antibacterial peptides because they represent one of the few choices available for this time frame. We also are testing some possible anti-psyllid genes. Second, we are developing virus vectors based on CTV to effectively express the antibacterial genes in trees in the field as an interim measure until transgenic trees are available. With effective antibacterial or anti-psyllid genes, this will allow protection of young trees for perhaps the first ten years with only pre-HLB control measures. Third, we are examining the possibility of using the CTV vector to express antibacterial peptides to treat trees in the field that are already infected with HLB. With effective anti-Las genes, the vector should be able to prevent further multiplication and spread of the bacterium in infected trees and allow them to recover. We now are making good progress: ‘ We continue to screen potential genes for HLB control and are finding peptides that reduce disease symptoms and allow continued growth of infected trees. We have about 30 new peptides that are now being screened. ‘ We have greatly improved our efficiency of screening . ‘ We have greatly improved the CTV vector. ‘ We have modified the vector to allow addition of a second anti-HLB gene. ‘ We have obtained permission and established a field test to determine whether the CTV vector and antimicrobial peptides can protect trees under field conditions. ‘ We continue to supply infected and healthy psyllids to the research community. ‘ We are testing numerous genes against greening or the psyllid for other labs.
Based on lesion number, phenotype, bacterial growth curve and cellular reaction, two cybrids of canker susceptible Red grapefruit (RG) with field tolerant Valencia orange (VO) as the cytoplasm donor,CY#3 and CY#10 have been characterized as tolerant of Xanthomonas citri subsp. citri (Xcc). The influence of mitochondrial exchange in the cybrids on the expression of nuclear genes was evaluated in an assay of the two cybrids and the parental lines (VO and RG) in the greenhouse. After inoculation of young leaves with Xcc at 108cfu/ml, tissue was collected at 4 and 24 hr post inoculation, mRNA was extracted, and the level of gene expression was determined by RT-qPCR. Several primers sets were tested to determine the differential expression among the parents (RG and VO) and the two cybrids. For the 15 nuclear genes tested, there was differential expression of several genes compared with parents. Among the changes detected were higher levels of expression of pathogenesis related proteins, enzymes related to the salicylic acid and jasmonate pathways, detoxifying enzymes and genes involved in oxidative stress response. Nuclear genes predicted to be involved in mitochondrial retrograde signaling were also affected. Several mitochondria and plastid related genes were assayed and the level of expression of genes in the cybrids differed from the parental lines including: ribulose bisphosphate carboxylase, aconitase-iron-regulated protein, ferritin-3, chloroplast precursor and ascorbate peroxidase. These results suggest that nuclear gene expression is modulated with respect to the interaction with the heterologous organelles in the cybrids. At present further characterization of the mt genome from kumquat, VO, RGF, Rough Lemon (RL), and previously obtained cybrids (RL+VO) and (RG+VO) is underway. Eleven citrus mitochondrial (mt) genes and introns were amplified with specific primers. The PCR products have been sent for sequencing. The sequence data obtained will be used for the design of specific TaqMan probes and used as gene markers. This method will allow us to monitor the mt genes transferred in the cybridization process. Production of new cybrids lines with susceptible Red grapefruit using a callus line of Meiwa kumquat as the cytoplasmic donor will be evaluated for inheritance of HR resistance using the same approaches as with the system described above.
Over the past quarter, we have made progress in the following areas: 1. We have continued testing TAL effector and promoter constructs in a Nicotiana benthamiana system to examine effector specificity for induction. 2. We have isolated TAL effectors from new citrus canker strains. Newly isolated variants will be sequenced, compared to known citrus TAL effectors, and tested for their ability to trigger our engineered resistance approach. 3. We have been continuing the molecular characterization of transgenic lines and testing of response to bacterial infiltration. 4. We have undertaken transformation of additional citrus varieties important to the Florida citrus industry, specifically sweet orange and red grapefruit. We continue to test additional promoter constructs in Duncan grapefruit. 5. We have begun planning for field trials of transgenic material.
The content of this quarterly report is similar to that of the annual report submitted in June, 2011. This is a 4-year project with 2 main objectives: (1) Over-express the Arabidopsis MAP kinase kinase 7 (AtMKK7) gene in citrus to increase disease resistance (Transgenic approach). (2) Select for citrus mutants with increased disease resistance (Non-transgenic approach). For objective 1, 20 transgenic Duncan grapefruit plants have been generated, and these plants have been growing in greenhouse for 6 months and ready for canker resistance test. Since canker resistance test is straightforward, we will test all 20 transgenic plants. However, for greening resistance test, the transgenic plants will need to be propagated. We will first extract total RNA from each of the 20 plants and determine the expression levels of AtMKK7. We will choose 4 to 6 lines that highly express the transgene AtMKK7 for propagation. Six plants from each line will be used for greening resistance test. For objective 2, we have been using hypocotyls as explants for gamma irradiation mutagenesis. A total of about 75,000 hypocotyl cuttings have been irradiated in three batches with a irradiation dosage of 40 Gy. Shoots formed on the irradiated cuttings were transferred onto selective medium containing 0.2 mM of sodium iodoacetate. Several shoots are currently growing on the selective medium. To increase the screening efficiency, we have also been using seeds for this objective. A large quantity of Ray Ruby grapefruit was obtained in the Spring of 2011 from the Indian River area. Seeds were obtained from the fruits and cleaned with Pectinase. Seeds were then treated with 8-hydroxyquinoline as a preservative to allow long-term storage of seeds at 4’C. Moisture content of the seeds was determined for future reference. Two quarts of seeds have been treated with gamma irradiation. One quart was irradiated at 50 Gy, the other at 100 Gy. Both untreated and irradiated seeds were plated into large glass Petri dishes as well as Magenta boxes containing water agar. Shoots have been formed on the seeds and will be transferred onto selective medium containing 0.2 mM of sodium iodoacetate. Based on the result of this batch of seeds, we will treat other seeds with either the same condition or a modified dosage. Shoots formed on these gamma irradiated seeds will be screened on the selective medium. Those shoots that are resistant to sodium iodoacetate will be grafted onto rootstocks to generate plants for resistance test.
The objectives of this project include: (1) Characterization of the transgenic citrus plants for resistance to canker and greening; (2) Examination of changes in host gene expression in the NPR1 overexpression lines in response to canker or greening inoculations; (3) Examination of changes of hormones in the NPR1 overexpression lines in response to canker or greening inoculations; (4) Overexpression of AtNPR1 and CtNPR1 in citrus by using a phloem-specific promoter. We have transformed the cloned CtNPR1 (also named CtNH1) into the susceptible citrus cultivar ‘Duncan’ grapefruit. After survey on transgene expression, we now focus on the three lines, CtNH1-1, CtNH1-3, and CtNH1-5, which showed normal growth phenotypes, but high levels of CtNH1 transcripts. The three lines were inoculated with Xac306. They all developed significantly less severe canker symptoms as compared with the ‘Duncan’ grapefruit plants. To confirm resistance, we carried out growth curve analysis. Consistent with the lesion development data, as early as 7 days after inoculation (DAI), there is a differential Xac population in the infiltrated leaves between CtNH1-1 and ‘Duncan’ grapefruit. At 19 DAI, the level of Xac in CtNH1-1 plants is 104 fold lower than that in ‘Duncan’ grapefruit. These results indicate that overexpression of CtNH1 results in a high level of resistance to citrus canker. We are planning to propagate the CtNH1 line by grafting. We are in the process of inoculating the CtNH1 lines with Candidatus Liberibacter asiaticus (Las). We have completed the SUC2::CtNH1 construct, in which CtNH1 is driven by a phloem-specific promoter from the Arabidopsis SUC2 gene. The construct were transformed into ‘Duncan’ grapefruit. To date, five transgenic lines have been obtained.
We have completed Carrizo genomic DNA isolation and purification. The source plant material was obtained from the University of California Riverside Citrus Variety Collection. Libraries for 454 sequencing (for random and paired-end reads) are currently being assembled. The 454 GS FLX sequencer at the Albany location was recently updated by Roche personnel to allow longer reads (averaging >700 bp, approximately doubling read length), with final testing and training to be completed by July 20, 2011. These longer reads should result in significantly improved genome coverage relative to the 10x coverage indicated in the original proposal. The extended reads will also facilitate both contig assembly and identification/separation of the sweet orange and Poncirus genomes within Carrizo. Sequence acquisition is expected to be initiated within the next three weeks, with completion within three months. While the 454 modifications have delayed the sequencing, the resulting improvement in read length can be expected to expedite the assembly and physical map alignment phases of the project.
Huanglongbing (HLB) is a serious and devastating disease of citrus caused by Candidatus Liberibacter spp. and vectored by the Asian citrus psyllid (ACP), Diaphorina citri Kuwayama (Hemiptera: Psyllidae). The disease has the potential to greatly limit the production of citrus in Florida and other citrus growing regions worldwide. Current chemical and classical biological control of ACP and HLB is inadequate, but identifying and incorporating traits from Citrus spp. and Citrus relatives that confer resistance to ACP is a potential strategy to manage the disease. In a study by USDA-ARS, 87 genotypes primarily in the Rutaceae (orange subfamily Aurantioideae), were assessed in the field in South Florida for resistance to natural populations of ACP. The majority of genotypes hosted all three life stages of ACP, however there were differences among genotypes in the mean ranks for eggs (F = 3.13, df = 86, P < 0.001), nymphs (F = 9.01, df = 86, P < 0.001), and adults (F = 4.21, df = 86, P < 0.001). Very low levels of ACP were found on two genotypes of Poncirus trifoliata, 'Simmon's trifoliate' and 'little-leaf'. Poncirus trifoliata, the trifoliate orange, readily forms hybrids with Citrus spp. and is commonly incorporated into rootstock varieties, so it may be useful in breeding programs as a potential source of genes that confer resistance to insects. The field experiment was followed by no-choice tests in which female ACP had the opportunity to lay eggs for six days on 46 genotypes of P. trifoliata, 35 genotypes of xCitroncirus sp. (hybrids of P. trifoliata and another parent species), three genotypes from the Citrus genera that were not represented in the field, and a control (Citrus macrophylla) to determine whether any genotypes were resistant to ACP. All genotypes of Poncirus trifoliata, except for one, and 14 of the genotypes of xCitroncirus sp. were resistant to oviposition by ACP. Currently we are testing whether development, weight, and lifespan of ACP nymphs and adults are negatively influenced by the resistant genotypes. Studies also have been initiated to compare plant volatiles of the resistant genotypes to those of susceptible genotypes to ascertain whether that is the mechanism conferring resistance of plants. Collaborators at the Fujian Academy of Agricultural Sciences in Fuzhou, China, initiated several experiments on resistance to ACP within the Rutaceae. Seventy-one genotypes of grafted pants were evaluated in a free-choice experiment conducted in a screen house. The majority of genotypes hosted all three life stages of ACP, however, eight genotypes had low levels of all life stages of ACP: Citrus sinensis (two cultivars), C. reticulata, C. unshiu (three cultivars), and C. mitis. Two additional free-choice experiments in a screen house and the field with seedling and grafted plants, respectively, with 102 genotypes yielded no differences in abundance of ACP. Six morphological structures of the leaf were also measured for five genotypes of Citrus that varied in abundance of ACP to determine whether morphology was correlated to infestation levels of ACP. Morphology was significantly different among the genotypes, but there was no correlation with abundance of ACP. Current work is investigating whether the eight genotypes with low abundance of ACP are resistant in no-choice tests.
This is the second year of a currently funded multi-investigator, multi-institution project, with the second year end time of 6/30/2011. A total of $224,000 are the current funds allocated to the second year of the project. We are requesting a 6 month no-cost-extension on this grant. There are two major reasons for this. First, there was a delay in dispersing the funds. It took an unusually long time for the funds to flow from agency to UF. Then, there was a further delay at UF establishing subaccounts at UF (CREC) and particularly with USDA, Ft. Pierce. It was October before all of the subaccounts were established. Finally,there is $24,000 in the second year budget for Dr. Machado in Brazil. He was never able to submit the necessary paperwork to receive these funds because of government restrictions. He has a student coming to the Moore lab in July for training and Dr. Machado has asked that the funds be used for her. The second reason for requesting the NCE is that there were a number of personnel changes this year. Because of this, we are requesting permission to adjust what funding is in specific categories and that we can adjust some funding between PIs. Randy Neidz (USDA) has had a post-doc working on this research until recently, and USDA post-docs are costly, so he has been funding much of the supplies used on the project from another source. He has hired a non-PhD person with tissue culture experience to continue the work on the project, but at a lower salary ($25,000 for the NCE). The rest of his funds would be primarily in materials. Jude Grosser (UF) has spent almost all of his current funding on this project. I am requesting that we be permitted to transfer $4,653 from Fred Gmitter’s subaccount to Jude and that I transfer $12,000 from the main account (originally alloted to Dr. Machado) to Dr. Grosser’s subaccount. This will give him adequate money to pay a post-doc and purchase supplies. I will use the other $12,000 originally allocated to Dr. Machado for fees and stipend for his student. I hope this is clear. If I can provide you with any further information or cost breakdown, please let me know.
As proposed, a transgenic test site has been prepared at the USDA/ARS USHRL Picos Farm in Ft. Pierce, where HLB and ACP are widespread. The first trees have been in place for more than fourteen months. Dr. Jude Grosser of UF has provided 300 transgenic citrus plants expressing genes expected to provide HLB/canker resistance, which have been planted in the test site. Dr. Grosser has just planted an additional 89 tress including preinoculated trees of sweet orange on a complex tetraploid rootstock that appeared to confer HLB resistance in an earlier test. USHRL has a permit approved from APHIS to conduct field trials of their transgenic plants at this site, with several hundred transgenic rootstocks in place. Dr. Kim Bowman has planted several hundred rootstock genotypes transformed with the antimicrobial peptide D4E1. An MTA is in place to permit planting of Texas A&M transgenics produced by Erik Mirkov. More than 120 citranges, from a well-characterized mapping population, and other trifoliate hybrids (+ sweet orange standards) have been propagated for a replicated trial in collaboration with Fred Gmitter of UF and are growing well in the greenhouse. These will be planted in July 2011, and 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. An experimental attract/kill product, to disrupt citrus leaf miner (CLM) without disrupting ACP, was not effective last year. Our experience suggests CLM may significantly compromise tree growth where insecticides are avoided to permit ready transfer of Las by psyllids. CLM damage also compromises ability to view HLB symptoms. Several applications of Admire are being used to encourage an undamaged flush on transgenic trees. We are still learning how to grow trees for best assessment of HLB-resistance. In June the test site was visited by APHIS Biotechnology Regulatory Services, and we received notice that the site is in compliance with all relevant regulations.
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