The haploid Clementine and sweet orange sequences have been assembled, annotated, and are available to the research community at Phytozome and at citrusgenomedb.org. The new Clementine v. 1.0, will soon be publicly available. New citrus sequences were generated by the Machado lab in Brazil (Ponkan mandarin, 454 and Illumina), the Gmitter lab and UF-ICBR (low-acid pummelo, Illumina), and Illumina datasets for Willowleaf/Avana mandarin, W. Murcott, Chandler pummelo, and Seville sour orange have been provided (Morgante, IGA-Italy; Talon, IVIA-Spain; and M. Roose-UCR). Comparative analysis has elucidated the phylogeny of sweet orange, Clementine, Ponkan and Willowleaf, and sour orange; all are admixtures of C. reticulata and C. maxima, in varying degrees. The fine-scale characterization of citrus genotypes opens the possibility that ancient C. reticulata/C. maxima admixtures (such as sweet and sour orange) can be recreated by conventional breeding guided by a set of genome-wide markers, enabling incorporation of specific, limited genomic regions from other citrus or relatives to confer disease resistance, yet retaining the essence of marketable fruit phenotypes. A manuscript based on these results has been expanded and prepared for submission. Work proceeds on the other objectives of this project. We identified miRNAs induced in citrus-pathogen interactions, presumably regulating target genes involved in signaling pathways and metabolic events important for plant resistance. In order to set up protocols to validate microRNA expression in plant-pathogen interactions and identify target genes, we performed a comprehensive analysis of the expression of 7 different citrus miRNAs in the context of 4 different Xanthomonas citri subsp. citri (XC) ‘ Citrus limon interactions, and certain miRNAs appear to be XC strain specific in their responses. We have used the GoldenGate assay platform for hi-throughput genotyping of DNA from >150 individuals of a large mapping family; we have produced a preliminary linkage map that shows excellent coverage and distribution of markers. The genotyping by sequencing (GBS) project has proceeded and currently it appears that it may generate as many as 2000 high-quality SNP markers for mapping a large segregating Citrus x Poncirus family. A large scale RNA-seq project to uncover differences in gene expression over time between HLB-sensitive and tolerant citrus, that weren’t seen previously in microarray studies, or to validate those already seen, has progressed. RNA samples have been prepared from appropriate times in the disease process, and libraries have been recreated to test, prior to the full sequencing effort.
This project is assessing a range of citrus germplasm and relatives for tolerance or resistance to HLB, through greenhouse assays and field tests; these germplasm resources were selected on the basis of research and observations in Asia and Florida. We have produced seedlings from 7 pummelo accessions (10-15 each), Citrus latipes (13 seedlings) and some hybrids of this species with trifoliate orange, 4 natural pummelo-mandarin introgression hybrids (9-16 each), 6 other miscellaneous wild citrus types (4-12 each), and various sweet orange lines for which there is anecdotal evidence of differential sensitivity to HLB. Subsets of these families have been inoculated with HLB-infected, PCR positive budwood of Carrizo citrange to ensure freedom from CTV cross-contamination, are being grown in a climate controlled, DPI-certified greenhouse and monitored for symptom development. Currently symptoms are being noted among some of the accessions, and we are collecting information on disease symptom progression. We have increased the numbers of individuals of most accessions, and have begun re-inoculations of previously inoculated seedlings with the same HLB source. We have extracted nucleic acids from most individuals, run RT-PCR on these, and we continue to find very few PCR+ plants. We will continue to monitor these individuals in the greenhouse. The Core Citrus Mapping Population, a genetically well-characterized collection of more than 250 citranges that we proposed to test at the Picos Road Farm near Ft. Pierce have been budded and growing off, prior to field planting. This population is of significant interest as the trifoliate orange and some of its hybrids are very HLB-tolerant, and this experiment is an opportunity to explore potential tolerance from these sources. Currently, there are at least 8 propagations of a total of 102 individuals from the original CCMP, and we plan to plant these this summer 2011. To conclude, a wide range of genetic materials have been produced and prepared for greenhouse and field testing for their tolerance or susceptibility to HLB. We have been unable to find a cooperator to plant the same experimental materials in the field as we have been testing in our greenhouse. The exception to this is the CCMP, soon to be planted.
This project is assessing a range of citrus germplasm and relatives for tolerance or resistance to HLB, through greenhouse assays and field tests; these germplasm resources were selected on the basis of research and observations in Asia and Florida. We have produced seedlings from 7 pummelo accessions (10-15 each), Citrus latipes (13 seedlings) and some hybrids of this species with trifoliate orange, 4 natural pummelo-mandarin introgression hybrids (9-16 each), 6 other miscellaneous wild citrus types (4-12 each), and various sweet orange lines for which there is anecdotal evidence of differential sensitivity to HLB. Subsets of these families have been inoculated with HLB-infected, PCR positive budwood of Carrizo citrange to ensure freedom from CTV cross-contamination, are being grown in a climate controlled, DPI-certified greenhouse and monitored for symptom development. Currently symptoms are being noted among some of the accessions, and we are collecting information on disease symptom progression. Additional seedlings that have reached sufficient size have now also been inoculated with the same HLB source. We have extracted nucleic acids from most individuals, run RT-PCR on these, and have found very few PCR+ plants. We will continue to monitor these individuals in the greenhouse. The Core Citrus Mapping Population, a genetically well-characterized collection of more than 250 citranges that we proposed to test at the Picos Road Farm near Ft. Pierce have been budded and growing off, prior to field planting. This population is of significant interest as the trifoliate orange and some of its hybrids are very HLB-tolerant, and this experiment is an opportunity to explore potential tolerance from these sources. We continue to seek additional germplasm resources, to expand the breadth and depth of the material categories we described in our proposal; a source for new C. latipes hybrids has been identified. We are still exploring other options within Florida for a field trial, but no secure, long-term commitments have been forthcoming, despite multiple discussions with growers throughout the state. To conclude, a wide range of genetic materials have been produced and prepared for greenhouse and field testing for their tolerance or susceptibility to HLB. We have expanded, and are continuing to expand, the number of types we wish to challenge. We will be developing new information about potentially tolerant/resistant germplasm that can lead to expanded efforts to capture and exploit the genetic basis for this phenomenon.
This study addresses two general questions: 1) Will our constructs disrupt normal growth and development in citrus, and 2) Will these constructs confer a degree of resistance to infection by Liberibacter asiaticus? We have answers for the first question and seek a one-year extension to address the second. Objective 1. Express R proteins in a phloem-specific manner in Arabidopsis and citrus. It was evident very early from the results of our experiments, and of others, that the Arabidopsis SUC2 promoter was phloem-specific in citrus and, thus, efforts were directed towards the generation of transformed citrus containing wild type and constitutive mutants of the two R genes, SSI4 and SNC1. Our rationale was that by restricting expression to phloem tissues (or to the wounding response) potential negative effects on growth and development would be minimized. From 30-60 transformants of each R gene variant were obtained in Arabidopsis and at least 10 in citrus (Duncan grapefruit). In Arabidopsis, some stunting was observed when transformed with the constitutive ssi4, but not with wild type SSI4, or with wild type or mutant SNC1. A similar result was obtained with citrus; however, the stunted growth (or seedling death) phenotype was much more pronounced. However, as with Arabidopsis, no abnormal phenotype was observed with either variant of SNC1. e triggered by psyllid feeding. This objective is a variation of the first, except the restriction in expression of the potentially harmful R genes was imposed by the wound-inducible PAD4 promoter, a promoter known to be activated by aphid feeding in Arabidopsis. Our rationale was that in case AtSUC2-directed expression resulted in a stunted growth phenotype, the use of an inducible promoter, such as PAD4, would provide a way to evaluate the effectiveness of R protein expression in inhibiting Liberibacter infection. Our expectation was that the Pad4 promoter would not be active, except upon deliberate wounding under controlled conditions. As with the AtSUC2 promoter, the expression pattern of PAD4 was more variable in Arabidopsis as determined using a GUS reporter; however, PAD4/GUS expression in transformed citrus appeared to be strictly wound-inducible. Expression of the R gene variants using the PAD4 promoter gave a result similar to that obtained in Arabidopsis: expression of the SSI4 constitutive mutant was sometimes harmful to the plant; whereas, expression of the constitutive mutant of SNC1 was not. These experiments can be summarized as follows: 1-Restricted expression of the wild type SSI4 and SNC1 genes using either the AtSUC2 or AtPAD4 promoter had no negative impact on growth and development in citrus. 2-Similarily, expression of the constitutive mutant of SNC1 had minimal effect on growth and development. In contrast, expression of the constitutive mutant of SSI4 is sometimes harmful to normal growth and development in both Arabidopsis and in citrus. Additionally, preliminary tests indicate that none of the constructs effected psyllid feeding preferences. In preparation for assessing disease resistance of the transformed citrus, a single leaf assay to monitor the early events in the transfer of Liberibacter from the psyllid to the plant has been developed as outlined in the proposal pending with the CRDF (Nov 2012). In brief, the real time PCR protocol was refined by developing calibration curves for the Las and various plant and psyllid control amplicons so that detection is now reproducible down to 12 copies. In addition, significant improvements have been made in single-leaf cage design that will enable feeding to be restricted to a 6 mm area of leaf.
For the last three months of 2012, the Mature Tissue Transformation Laboratory (MTTL) continued to operate in the ‘maintenance’ capacity mode. Level of operation was determined by the amount of plant material available and its quality. The process of increasing the number of rootstock plants is slow and it has been hindered by low germination rate of seeds that are old. Although new seeds were ordered in December, they will not be available until late January/mid February when Swingle citrumelo and C. macrophylla fruit are available. One of the batches of Hamlin buds grafted in early October had low percentage ‘take/success’ rate. The outside provider of grafting services claimed that the buds coming from mother plants were not of the highest quality. In the meantime, this person has left the business and the facility contracted other provider. In couple of experiments, a high percentage of explants that were used in co-incubations with Agrobacterium got contaminated. We are investigating whether those incidences were the result of human error in the steps of transformation taking place in the laboratory, or if plants that served as starting material for explants were infected while in the growth chamber. During these three months, six co-incubation experiments were performed. Four of those experiments were done with Valencia explants and two with Hamlin explants. For the Valencia experiments, we cut 2170 explants and 1030 explants were cut for Hamlin experiments. In order to be able to assess the ability of the lab to process different orders at the same time, two additional Agrobacterium strains were used for co-incubation experiments. One of those harbored a binary vector with the gene for green fluorescent protein (GFP) as a reporter gene. In one of the Hamlin experiments, out of 16 shoots inspected for GFP fluorescence two were positive. Those two shoots were micro-grafted on Carrizo rootstock plants. Some GUS assays were done on shoots obtained from experiments done earlier. Out of 19 shoots, one was positive. Two additional Ray Ruby plants were cleaned of microorganisms and are ready to become source of budding material. One more Hamlin plant was also cleaned.
The four most promising anti-NodT scFv antibodies have been selected for further development. Anti-NodT antibody #1 has been successfully expressed in E. coli. This means that we can generate as much of the antibody as needed. The antibodies are being augmented with two 6xHis epitope tags – one at the amino terminus, and one at the carboxy terminus. The protein can be detedcted with anti-His antibodies. The anti-NodT scFv antibody is soluble, and should be usable for protein immunoblotting and other applications. We experienced some delays in cloning the scFv antibody DNA into the appropriate citrus transformation vector. However, these difficulties have now been solved and we now expect to have the scFv citrus transformation construct completed within a few weeks, and we will commence transformation immediately.
McTeer trial – (3-year old SugarBelle trees on 15 rootstocks, nearly 100% HLB infected as of September (2011)- remediation program initiated in January by application of southern pine biochar and Harrell’s UF mix slow release fertilizer): Continued evaluation of this trial shows significant differences in tree health among rootstocks, with Orange #19 showing the healthiest trees. However, HLB has significantly impacted the quality of fruit trees on all rootstocks, even from very healthy looking trees. We will look at these trees one more season to see if continued remediation will improve fruit quality next season. St. Helena trial (20 acre trial of more than 70 rootstocks, Vernia and Valquarius sweet orange scions, 12 acres of 4.5 year old trees, Harrell’s UF mix slow release fertilizer and daily irrigation). Full data (yield, fruit quality and HLB infection rates) was presented in our Field Day handout. Big differences have shown up in HLB infection rates per rootstock. Control commercial rootstocks have the highest infection rates, with most >70% infected. The rate of infection on tetraploid rootstocks was half that of diploids, with tetrazyg Orange #15 showing the lowest rate (just 7%). Disease severity of infected trees is also being impacted by rootstock. Good candidate rootstocks for ACPS are emerging. Greenhouse Experiments – Rootstock liners have been grown off for the nutrition and rootstock comparison studies, and have been moved to the HLB house for graft inoculation, to begin this quarter. Protection of seed source trees: The release of new and improved rootstocks to the Florida Industry will require a large and stable source of viable nucellar seeds for our nurseries. Since seed source trees will be growing in the HLB environment, such trees should be protected from HLB. Transgenic tetraploid lines containing an insecticidal Snowdrop lectin gene were regenerated from the tetrazyg selections Green #7 and Orange #4. 12 transgenic lines of Orange #4 and 3 of Green #7 have been successfully micrografted, acclimatized and transferred to the greenhouse. A construct containing the Snowdrop Lectin insecticidal gene combined with the antimicrobial gene CEMA was completed. Transformations are underway. We have codon optimized the Snowdrop Lectin insecticidal gene (GNA) for optimal expression in citrus. Two vectors containing this optimized gene have been produced; a) codon optimized GNA fused with a Tobacco PR1b signal peptide for improved extracellular secretion of the GNA protein by plant cells; b) codon optimized GNA fused with a HDEL C-terminal extension for retention of the GNA protein in in the endoplasmic reticulum. Dual protection against psyllids and Liberibacter: A construct containing the native Snowdrop Lectin insecticidal gene with the antimicrobial gene CEMA have been constructed. Transformation using this vector are being carried out.
Progress with the rapid flowering system (pvc pipe scaffolding system) in the greenhouse: Selected transgenic plants produced from juvenile explant, budded to precocious tetraploid rootstocks in airpots are growing well in our RES system, with some plants reaching 8 feet in height. Additional transgenics were propagated onto additional new rootstocks expected to reduce juvenility, including the somatic hybrid Amblycarpa + Flying Dragon. The goal is to reduce juvenility by several years to accelerate flowering and fruiting of the transgenic plants. Experiments to efficiently stack promising transgenes are underway. Experiments to efficiently stack promising transgenes are underway. The first transformation experiments using the two-transgene Gateway based cloned construct combining our best transgene for HLB resistance (NPR-1 from Arabidopsis) with our best transgene against canker that also has some affect on HLB (the synthetic CEME lytic peptide gene) were initiated, and so far 30 putative transgenic lines of the sweet orange cultivars Hamlin and Valencia have been regenerated. These plantlets have been micrografted to Carrizo rootstock. The goal is to provide stable resistance to both HLB and canker, with transgene backup to prevent Liberibacter from overcoming single transgene resistance.A construct containing CEMA gene stacked with the NPR1 gene has been constructed. Also, another vector containing a AttacinE gene stacked with the NPR1 gene is also under construction. Correlation of transgene expression with disease resistance response: More than 150 transgenic lines with different genes have been analyzed using ELISA by either C-myc or LIMA antibody (which also works for CEME) to measure transgene expression. As expected, significant differences were observed in our transgenic plants. Correlations between the data obtained from ELISA and other molecular data with HLB challenge response data are underway. Transgenic lines examined by ELISA include 40 lines with NPR-1, 50 lines with LIMA, and 9 lines with CEME. Improved transformation methodology (for seedless or recalcitrant cultivars, and eventually marker-free consumer-friendly transformation): We have finished construction of several parts of the T-DNA region of a pCAMBIA0390 derived binary vector for cre-lox based marker-free selection. A fusion codA-hptII gene driven by the d35S promoter have been constructed and a cre gene driven by a glucocorticoid-responsive elements promoter have also been constructed and cloned into a pUC based vector. We are experiencing problems cloning the glucocorticoid receptor gene driven by a constitutive mirabilis mosaic virus promoter as all sequenced clones have mutations and/or deletions in them. Work is underway to rectify this.
Work has been continuing on the development of a construct using the FT3 cDNA insert and an FMV promoter. This construct will eventually be used to test the efficacy of the FT3 cDNA as compared to the genomic DNA construct currently being used. Over the past several months, extensive testing was conducted to establish a more effective disinfestation technique for use on seeds and other explant tissue. This technique should allow for the continuous use of seed for transformation, even many months after their initial collection. Transformation of Carrizo has picked up following the most recent harvest of seed. These transformants will be used in the experiments examining the effects of GA and day length on FT phenotype. This is month 7 of the in vivo tracking of FT1, FT2, and FT3 and samples are continuing to be collected and processed. These data will be evaluated at the end of the year-long trial to compare month-to-month variations in gene expression. The FT3 protein that was commercially synthesized has finally arrived and experiments with direct application of the protein will be commencing shortly.
This is a continuing project to find economical approaches to citrus production in the presence of Huanglongbing (HLB). We are developing trees to be resistant or tolerant to the disease or to effectively repel the psyllid. First, we are attempting to identify genes that when expressed in citrus will control the greening bacterium or the psyllid. Secondly, we will express those genes in citrus. We are using two approaches. For the long term, these genes are being expressed in transgenic trees. However, because transgenic trees likely will not be available soon enough, we have developed the CTV vector as an interim approach to allow the industry to survive until resistant or tolerant trees are available. A major goal is to develop approaches that will allow young trees in the presence of HLB inoculum to grow to profitability. We also are using the CTV vector to express anti-HLB genes to treat trees in the field already infected with HLB. At this time we are continuing to screen possible peptide candidates in our psyllid containment room. We are now screening about 60 different peptides for activity against HLB. We are also working with other groups to screen possible compounds against psyllids on citrus. Several of these constructs use RNAi approaches to control psyllids.
The objective of this project is to find poncirus hybrids that exist now that are sufficiently tolerant and of sufficient horticultural and juice quality to be used now for new planting in the presence of high levels of Huanglongbing (HLB) inoculum. We believe there is a good chance that there mature budwood exists with these properties that could be available immediately for new plantings. Although these trees are not likely to be equal in juice and horticultural qualities of the susceptible varieties of sweet oranges grown in Florida, with their tolerance to HLB they could be an acceptable crutch until better trees are developed. We surveyed the trees at the Whitney field station and found 5 lines that we thought could be acceptable for juice. Those have been propagated and are beginning to be tested for tolerance and horticultural properties.
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 three years. Dr. Jude Grosser of UF has provided 550 transgenic citrus plants expressing genes expected to provide HLB/canker resistance, which have been planted in the test site. Dr. Grosser planted an additional 89 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 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. Eliezer Louzada of Texas A&M has permission to plant his transgenics on this site, which have altered Ca metabolism to target canker, HLB and other diseases. 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. Additional plantings are welcome from the research community.
Evaluation of existing standard cultivars (‘Temple’, ‘Fallglo’, ‘Sunburst’, ‘Sugar Belle’, ‘Tango’, ‘Hamlin’, and ‘Ruby’) for HLB tolerance/resistance is underway . Trees were planted in 2010, using a randomized complete block design, at Picos Farm, Ft. Pierce, Fl. HLB symptom development and tree growth (diameter and height) are being monitored on a monthly basis. All of the cultivars in this trial exhibit symptoms of HLB and have tested positive for Candidatus Liberibacter asiaticus (CLas). Preliminary results indicate that there are a range of host responses with ‘Temple’ in the most tolerant group. A second project involves the treatment of various resistant/tolerant citrus accessions and susceptible standards with various concentrations of antibiotics to generate a range of CLas titer levels. In February 2013, budwood with various concentrations of CLas, derived from the antibiotic treated plants, will be evaluated for their potential to result in HLB symptoms in disease free material. The budded plants will be evaluated for growth and HLB symptoms development over a 2-year period. Temporal progression and systemic movement of the bacteria in the inoculated plants will be determined along with HLB symptom development, and growth of the plants.. Development of periclinal chimera using resistant geneotypes and standard varieties is in progress. In vitro shoots have been established from nodal and internodal explants excised from mature, certified disease free plants of Red Carrizo, Temple, Hamlin, and Valencia. After root formation, chimeras will be generated using a procedure developed by Ohtsu (1994). After successfully generating the chimeras with HLB resistant vascular system and good fruit using the previously mentioned cultivars, additional cultivars such as ‘Sweet Orange’ and grapefruit will be added to this study. An additional study has been added to the project. Screening and evaluating new scion materials is a lengthy process and require multiple testing locations. Due to the urgency to develop tolerant/resistant material, a shorter evaluation cycle procedure is being investigated. If this screening method is successfully, it may be useful to quickly identify new sources of resistance varieties that may enhance and improve citrus production in Florida.
Dr. Guixia Hao, who has extensive experience in plant transformation and molecular biology, began working on this project 9/23/2012. New constructs have been used to transform citrus scions including hairpins to suppress PP-2 through RNAi (to test possible reduction in vascular blockage even when CLas is present), a citrus promoter driving citrus defensins (designed by Bill Belknap of USDA/ARS, Albany, CA), and genes which may induce deciduousness in citrus. Numerous putative transformants are present on the selective media. A chimeral construct that should enhance AMP effectiveness (designed by Goutam Gupta of Los Alamos National Lab) is finally completed and will be used in transformations next quarter. A series of transgenics scions produced in the last several years, continue to move forward in the testing pipeline.
Development continued on new rootstocks with outstanding attributes for Florida production, including tolerance to HLB. Yield, fruit quality, and tree size data were collected from ten rootstock trials with early ripening scions. Propagation continued to prepare trees for four more field trials to plant this year. Propagations of supersour selections were prepared for budding of trees for field trials, and another group was prepared for field planting. Cooperative work was continued with a commercial nursery to multiply 250 advanced supersour selections for placement of trees into cooperative field trials with growers at multiple locations. Work continued to assess supersour tolerance of CTV, Phytophthora, and Diaprepes using carefully controlled tests in the greenhouse and the field. Preparations were made for controlled testing of supersour selections for tolerance to high pH. Specialized testing of the supersour hybrids and concurrent field trials will effectively identify specific supersour selections that are equal or superior to sour orange in horticultural attributes and effects on fruit quality, as well as provide disease resistance or tolerance. Experiments were initiated to study the most important components of the tolerance to HLB exhibited by some citrus rootstocks. The final stage of a study of metabolic changes in HLB infected germplasm is being completed to supplement the gene expression study completed last year, including HLB susceptible and tolerant cultivars. Detailed evaluation of specific defense-related genes continued, including CtCDR1 and CtPDF2, identified by microarray as being responsive to HLB in tolerant rootstocks. Constructs are being built using this knowledge and that will allow the creation of new cultivars with increased HLB tolerance using only citrus origin genes. Knowledge gained about specific citrus resistance genes will also help guide crosses for the creation of conventional hybrids with improved HLB tolerance or resistance. A study to define the interaction of rootstock tolerance with scion tolerance/susceptibility is nearly complete, and is expected to be published by mid-year. Additional trees were propagated to examine the effect of rootstock tolerance to HLB in trees with the scaffold composed of the tolerant variety. Trees were also propagated for a field planting that will examine the same high grafting technique. Collaborative work continued to assess rootstock interaction with scion, nutrition, and management factors in determining tree tolerance to HLB. A manuscript was published on collaborative work demonstrating the association of particular small RNAs and nutrition, with HLB infection. Collaborative work continued on the relationship of small RNA to the HLB tolerance of selected citrus genotypes. Collaborative work began to compare the early response of trees infected by HLB to those infected by CTV, both phloem-limited pathogens. Selected citrus plant resistance genes were inserted by genetic transformation into outstanding rootstock and scion cultivars to develop new varieties with increased resistance to HLB. More than 300 new transgenic rootstock selections with potential resistance to HLB were produced, targeting increased expression of the citrus resistance genes CtNPR1, CtEDS1, CtMOD1, CtEDS5, CtPAD4, CtNDR1, or CtACD1. Twenty-five new transgenic rootstocks with selected antimicrobial genes were propagated and entered into a replicated greenhouse test with ACP inoculation to assess tolerance to HLB. A field trial continued with selected transgenic rootstocks to evaluate performance under natural field infection with HLB. The field trees are nearly 100% infected with HLB, but differ widely in the severity of symptoms and the effect of HLB on plant growth. Three presentations were made at the International Citrus Congress on research progress in the USDA rootstock program, including descriptions of new USDA citrus rootstocks, rootstocks for tree size control, and methods to test citrus selections for HLB resistance.
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