HLB’s impacts have led to grower interest in advanced production and harvesting systems with the potential for early and sustainable yield, as well as ease of harvest and other management efficiencies. The goal of this project is to identify appropriate rootstocks among exiting field trials and those soon to be planted that are well suited to advanced citrus production and harvesting systems. Existing field trials previously planted with size-controlling rootstock candidates have continued to be observed, including the portion of the St. Helena project planted with dwarfing selections. Yield and fruit quality data were collected from the St. Helena trial (last week of January); overall yields increased 42% over last year (where yields were down 18%), as HLB incidence in the trial increased from 56% to 92%. This remarkable increase in yield was primarily attributed to the addition of TigerSul micronutrients to the CRF mix and elevating the boron and manganese in the CRF. Many of the tree-size controlling rootstocks in the trial produced fruit with higher lbs. solids and juice color than the more vigorous rootstocks; thus this information will be utilized in the economic analysis for the 2015 Field Day handout, and will help to identify the best HLB-tolerant rootstocks for ACPS along with recommended planting densities. Collection of botanical data on two high-performing tree-size controlling somatic hybrid rootstocks (sour orange+trifoliate orange 50-7; and White grapefruit+trifoliate orange 50-7) in the trial was completed as necessary for patenting and Fast Track release later in 2015. After 7 years, all 86 trees on the White grapefruit+50-7 rootstock in the St. Helena trial remain healthy and productive. New Trials: Planting of a major rootstock trial with Lykes is underway (>20,000 trees to be planted at high densities at two sites, Basinger and Camp Mack). This trial includes 19 tree-size controlling rootstocks from the CREC, as well as additional tree-size controlling rootstocks from the Forner Spanish breeding program and from the California breeding program (C-Series). Progress was also made with propagation of trees for the major rootstock trial with Cutrale, which will also include several new tree-size controlling rootstocks. ACPS Data collection: Yield and fruit quality data were collected in February from the ‘Hamlin’ section of a large ACPS rootstock trial in Indiantown being conducted in collaboration with Gardinier Florida Citrus (w/ Lee Jones). Collection of yield and fruit quality data from the ‘Valencia’ part of the trial began on March 30th, and was completed April 4th. Data is currently being analyzed. Several new potentially HLB-tolerant rootstocks that have capacity to dwarf trees for use in ACPS (diploid and tetraploid Flying Dragon hybrids) were identified that are amenable to seed propagation. Seed was planted for use in subsequent trials.
Little resistance to HLB, associated with infection by Candidatus Liberibacter asiaticus (CLas), is found within commercial citrus varieties, though Poncirus trifoliata has been described as resistant or tolerant. An intergeneric F1 population was developed from the crossing of Citrus sinensis and P. trifoliata. The objective of this study was to construct a high-density linkage map for further mapping HLB resistance QTLs. A composite of 182 intergeneric progenies derived from four crosses between C. sinensis cultivars. Sanford, Ridge Pineapple, Fiwicke, or Ruby as the maternal parents, and P. trifoliata cultivars Argentina or Flying Dragon as the paternal parents, were used for genotyping by the Illumina GoldenGate Assay. Totally 1536 SNP markers were developed from the sweet orange BAC library. Genotyping data were analyzed by the GenomeStudio (Illumina), and a linkage map was created by JoinMap 4.1 using the two-way pseudo-testcross mapping strategy and regression mapping algorithm. Individuals from this same population were replicated and planted in the field with no ACP control, to assess symptom development, as well as to use qPCR to track CLas levels, over the course of the study. A preselected subset was used as a monitoring population to quantify HLB symptom severity, relative vigor (stem diameter), and citrus canker severity. Combining all results of qPCR monitoring across the two years of the project, 5 F1 hybrids have been for the most part CLas-free, except for occasional low-level and inconsistent detections of CLas in a small number of the replicates. One F1 hybrid has always tested negative for CLas, among all the individual replicates across all sample dates. The variation of the population mean Ct value across all F1 progeny showed good normal distribution, on which the Ct values of susceptible and resistant parents were located at each end respectively. Analysis of genotyping data showed that 99% of the markers were heterozygous in maternal parents (C. sinensis), but only 6% in paternal parents (P. trifoliata), and 5% in both. Finally 698 high-quality SNP markers were mapped to nine linkage groups for sweet orange, but only 42 could be mapped to seven linkage groups for the paternal parents. A comparison of the genetic maps to the published sweet orange (C. sinensis) genome revealed both conservation and variations. The completed linkage map is still being utilized to associate with the phenotyping data for QTL mapping and cloning of HLB resistance genes in the future. At the current time we can only map QTLs for sensitivity from sweet orange; but with the availability of the Poncirus genome and/or access to GBS data from our collaborators, we will soon be able to map tolerance/resistance QTLs from the male parents. We have also conducted artificial inoculation experiments in the greenhouse with a subset of individuals from the mapping population, to compare natural inoculation in the field with the more severe test of graft inoculation. Although funding for the project has ceased, we intend to continue the work to its logical conclusions, as possible.
We routinely monitor previously identified candidate survivor trees at the CREC, the GCREC, and some Polk County commercial groves where we have planted out materials from the CREC breeding program, including those more recently identified. Some of the previously identified selections have succumb to HLB and we have removed these materials from the program. We continue to grow out and propagate recovered rootsprouts and scion materials collected, to produce groups of trees that can be tested for their responses to HLB. A recent decision by FDACS/DPI, that researchers may propagate and plant in the field trees grown from non-certified sources, will help us move some of the selections we have propagated into the field for longer-term evaluations. Materials collected from a grove in Lake County that appeared to be completely free of HLB symptoms were propagated and increased. PCR results have been negative; these plant materials were placed in a growth room with CLas+ ACP populations to attempt natural inoculation to assess their responses; however, as Dr. Brlansky retired and the growth room was transferred to another researcher, we were forced to terminate the experiment prematurely. Materials previously collected from a legacy Parson Brown tree found in Marion County, and apparently free of HLB, have been propagated, and are being grown off for field planting. We visited several newly identified candidate trees, but in most cases materials were not collected because the trees actually were more symptomatic than we had been led to believe. However, we have learned of two new locations with reportedly very healthy trees surrounded by rather severe HLB diseased trees, one in Osceola County and the other in St. Lucie County; we will visit these in January or February 2015. We plan to revisit trees from which samples were collected previously to determine their current status and performance; scion and rootstock materials will be collected, tested, and propagated, if they remain in very healthy condition.
In preliminary studies on ACP biology, it was observed that there was a notable suppression of successful development from eggs to adults, on Cleopatra mandarin (Citrus reshni; CM) trees both growing in the field and also in greenhouse experiments. Based on these results, we looked at ACP viability in a field-grown population of CM-derived hybrids from the UF-CREC breeding program, in Lake Alfred. The trees were 18 years old and growing on their own roots. A total of 91 trees in three families produced by crossing Cleopatra mandarin with three male parents were selected from field plantings for the project and for future evaluations. Routine insecticide applications were withheld for a period of 2 months prior to the experiment. Large branches on the south sides of the trees were severely pruned, to stimulate a synchronized and vigorous flush of new shoot growth to introduce ACP. Pairs of mated ACP were caged on each shoot and after three weeks, the number of adults that developed on each was counted. Of 30 individual hybrids that were used, only 9 of these supported the development of new adults; the remaining 21 had absolutely no surviving adults. This experiment was repeated with the same results. We propagated replicates from each individual for observations of psyllid reproduction in controlled greenhouse and laboratory conditions. Additional trees were propagated from additional numbers of the hybrids from the families to provide increased numbers of replicates for the ACP reproduction and feeding behavior studies. We conducted several rounds of experiments in the greenhouse. Previously, ACP were able to reproduce on only 3 of the 26 plants screened (ACP did not reproduce on 88% of the plants screened). Since then we have screened seventy-three additional individual plants for the ability of ACP to reproduce on them. Of these, ACP were unable to complete their life cycle on 52 of these plants (71% did not support ACP reproduction). Our results indicated that several of the replicated hybrids were unable to support adult psyllid development. These results were compared to results from previous experiments conducted in the field, and essentially demonstrated the consistency of the response of ACP to specific individual hybrids. In addition, new candidate plants have been identified from other studies of HLB impact on diverse citrus germplasm, which have shown either no impact or dramatically delayed infection, and new hybrid families have been produced from some of these for possible future studies. These include certain mandarins as well as other complex citrus hybrids. It appears that suppression of ACP by Cleopatra mandarin and hybrids derived from it is a heritable characteristic, thereby opening new possibilities to understand the genetic control and underlying mechanisms of the characteristic. It remains to be determined whether these new candidates likewise possess genetics that may be transmitted to offspring and whether the mechanisms underlying the phenomenon are the same or different. Such information might lead to new strategies to minimize the HLB-vectoring capacity of ACP.
This project is built on the legacy of materials produced and field trials planted across the past several years. The objectives are to evaluate existing families and created germplasm in the field and in greenhouses for their responses to HLB and citrus canker, to carefully observe and document rootstock effects on severity and rates of progression of HLB symptoms, and to maintain the facilities and activities involved in the state-wide assessment of new scion and rootstock performance with a focus on HLB responses. Assessments of HLB field tolerance are continuously carried out in the vast collection of raw germplasm that we maintain, and new selections have been identified, and several previously found continue to hold up to HLB; additional evidence is accumulating supporting what may be differential sensitivity to HLB among sweet orange clones from the CREC program. We monitored performance, assessed HLB severity, tree growth and yields at 15 different field sites throughout Florida. A new set of 25 HLB+ Valencia budstick-grafted hybrid rootstocks was rotated into the ‘hot psyllid’ house, following selection based on freedom from symptoms in the greenhouse test. Another set of approximately 50 hybrid rootstocks were removed from the ‘hot psyllid’ house and prepared for field planting (spring of 2015); another set of 50 was rotated into the ‘hot psyllid’ house. Several new clones were entered into the DPI Parent Tree Program, including a cybrid sweet orange with exceptional juice quality and preliminary evidence of HLB tolerance, two early maturing Valencia somaclones, and twelve new promising rootstock hybrids with preliminary evidence of HLB tolerance. The two Valencia somaclones identified, B7-70 and SF14W-65 had 16 ratio and 11.5 brix in mid-November (6 year old trees on rough lemon at St. Cloud), when Hamlins were still at 12 ratio. B7-70 appears to be slightly earlier than SF14W-65. Both clones have been significantly earlier than Valquarius the past two years. These Valencia clones have potential to replace Hamlin which would significantly increase the quality of our NFC and concentrate juices. Observations were made at a rootstock trial in Vero Beach from where several of the UF rootstocks already approved for release have been selected; a field day was held in October to highlight these and other promising rootstocks at this location. Another large field day was held at the St. Helena planting in December. Seedlings grown from seed collected from more than 3 dozen new candidate rootstocks were evaluated for trueness to type, and new seeds have just been collected from additional new candidates not evaluated previously. We recovered seed from 2014 diploid and tetraploid rootstock crosses (20 successful crosses), and these were planted in calcareous soil inoculated with two species of Phytophthora. Seed was extracted from 8 UFR FAST TRACK rootstocks and distributed to participating nurseries. Seed was also extracted from 16 additional new rootstocks showing potential for HLB tolerance and provided to nurseries for propagations of planned large-scale field trials.
HLB’s impacts have led to grower interest in advanced production and harvesting systems with the potential for early and sustainable yield, as well as ease of harvest and other management efficiencies. The goal of this project is to identify appropriate rootstocks among exiting field trials and those soon to be planted that are well suited to advanced citrus production and harvesting systems. Existing field trials previously planted with size-controlling rootstock candidates have continued to be observed, including the portion of the St. Helena project planted with dwarfing selections; a field day was held there in December to demonstrate the performance of the tree size controlling rootstocks. A 40-acre Hamlin/Valencia cooperative rootstock trial with trees planted between 300-500/acre has been monitored for tree growth and HLB responses, as was a high density planting of LB8-9 (Sugar Belle). Data are being compiled on rootstocks to identify new candidate rootstocks for larger’scale ACPS trials. Seedlings grown from previously untested Flying Dragon-derived hybrids, as well as from a range of other complex interspecific hybrids with tree size control potential, were grown to assess seedling vigor and other characteristics; selections were either discarded because of poor growth habits, excessive phenotypic variation, or poor germination. The best performers from this group were listed and substantial quantities of seed were harvested and made available to 3 different cooperators for new plantings and trials, hopefully to be planted in 2015 or spring 2016. Seeds were harvested from rootstock hybrids that are bearing their first fruit, and these have been planted in the greenhouse to assess germination rates, relative seedling vigor, and trueness to type. Additionally, large lots of seed from candidates for ACPS planting already identified from existing field trials were harvested and distributed to cooperating nurseries to produce trees for new trial opportunities in the 2015-16, with interested collaborative growers. Finally, a field day was also held to showcase some of the UFR rootstocks in a trial near Vero Beach, several of which are showing good performance even though they are affected by HLB, and with ACPS potential by virtue of smaller tree size combined with higher yield efficiencies.
The project has two objectives: (1) Increase citrus disease resistance by activating the NAD+-mediated defense-signaling pathway. (2) Engineer non-host resistance in citrus to control citrus canker and HLB. For objective 1, we continued optimizing the NAD+ treatment conditions. Both soil drench and foliar spraying of NAD+ were tested again. Foliar spraying did not provide significant protection against citrus canker, soil drench induced strong resistance against the pathogen. We previously observed strong systemic protection against canker by NAD+ one month after the treatment in upper new flushes and are repeating the experiment to confirm the systemic effects. We are also testing different recipes for stabilizing NAD+ on the surface of leaves or in soil, since this chemical is not stable under aqueous conditions. For objective 2, transgenic citrus plants expressing the Arabidopsis nonhost resistance genes are growing in greenhouse and will be tested for canker resistance. We are also propagating the transgenic plants to produce progenies for HLB resistance test. Citrus homologs of the Arabidopsis nonhost resistance genes have been cloned and sequenced. To test their functionality, we have cloned the citrus homologs into plant expression vector and have transformed into the corresponding Arabidopsis mutants. T1 plants are growing.
In this project, we have conducted transcriptome analysis on 25 samples of tolerant/susceptible citrus plants using RNA-Seq. We first performed bioinformatics analysis on RNA-seq data of three HLB-tolerant ‘Jackson’ grapefruit hybrid and three HLB-susceptible ‘Marsh’ grapefruit hybrid. We identified 686 differentially expressed (DE) genes between two groups using FDR threshold of 0.1. Among them, 247 genes were up-regulated and 439 were down-regulated in tolerant citrus trees. We have experimentally verified the expressions of 14 up-regulated genes and 20 down-regulated genes using real time PCR. 11 of 14 up-regulated genes and 18 of 20 down-regulated genes were validated. We performed Gene Ontology (GO) enrichment analysis of DE genes. Genes associated with beta-amyrin synthase, cycloartenol synthase and Camelliol C synthase were significantly up-regulated in the HLB tolerant citrus trees while terpene synthase genes (CiClev10014707, Ciclev10017785) were down-regulated in the tolerant citrus trees. Some PR-protein genes were significantly up-regulated in the tolerant citrus trees, including several TIR-NBS-LRR genes. Many cell wall degradation-related genes, such as cellulose synthase/transferase, cellulase and expansins were up-regulated in the susceptible citrus trees. Some glucan hydrolase genes were also up-regulated in the tolerant citrus trees. These genes may play important roles in symptom development. The DE genes were also enriched in two classes of RLKs, LRR-RLKs and DUF26-RLKs. We then applied the MapMan software to identify DE genes related to disease response. The MapMan annotated 155 DE genes related to disease response. We found multiple pathways have been suppressed or activated in HLB tolerant citrus tree, which lead to the promotion of the basal resistance or immunity in citrus tree. For examples, suppression of beta glucanases, DMR6-like genes, expansin and DET2 uphold the suppression of immunity of citrus tree and activation of NPR1-like genes also induces the immune responses to HLB. The mechanism how to trig the suppression and activation of these genes is still unclear. Further experimental studies on the NBS-LRR and RLK genes differentially expressed between HLB tolerant and HLB susceptible citrus trees may help understand the HLB related receptor genes. Meanwhile, our functional analysis also showed that the HLB development related genes were enriched in HLB susceptible citrus trees, such as viral life cycle and symbiosis related genes. We predicted a protein-protein interaction (PPI) network of citrus using the PPIs of Arabidopsis. There are 1259 proteins and 2298 interactions in our Citrus PPI network. Among 1259 proteins, 42 proteins are differentially expressed between the HLB resistance and susceptible citrus. An interested PPI sub-network includes 14 citrus NPR1-likes proteins and three TGA proteins. There were four NPR1-like genes were significantly up-regulated in HLB tolerant citrus trees and one NPR1-like gene up-regulated in HLB sensitive citrus trees. There is also one TGA gene up-regulated in HLB tolerant citrus trees. Another interested sub-network includes the RPS2 protein. There were two LRR kinase receptors significantly up-regulated in HLB tolerant citrus trees and four LRR kinase receptors significantly up-regulated in sensitive citrus trees. In conclusion, by profiling the transcriptome of HLB tolerant and HLB susceptible citrus trees, we found that multiple pathways have been suppressed or activated in HLB tolerant citrus tree, which lead to the promotion of the basal resistance or immunity of citrus trees. This study may help us understand HLB-tolerance and provide guidance for breeding HLB-tolerant citrus in the future. The down-regulation genes can be served as the candidate targets for RNAi to improve HLB tolerance in citrus trees. Two manuscripts were prepared: one published, and the other has been submitted.
In this project, we have conducted transcriptome analysis on 25 samples of tolerant/susceptible citrus plants using RNA-Seq. We first performed bioinformatics analysis on RNA-seq data of three HLB-tolerant ‘Jackson’ grapefruit hybrid and three HLB-susceptible ‘Marsh’ grapefruit hybrid. We identified 686 differentially expressed (DE) genes between two groups using FDR threshold of 0.1. Among them, 247 genes were up-regulated and 439 were down-regulated in tolerant citrus trees. We have experimentally verified the expressions of 14 up-regulated genes and 20 down-regulated genes using real time PCR. 11 of 14 up-regulated genes and 18 of 20 down-regulated genes were validated. We performed Gene Ontology (GO) enrichment analysis of DE genes. Genes associated with beta-amyrin synthase, cycloartenol synthase and Camelliol C synthase were significantly up-regulated in the HLB tolerant citrus trees while terpene synthase genes (CiClev10014707, Ciclev10017785) were down-regulated in the tolerant citrus trees. Some PR-protein genes were significantly up-regulated in the tolerant citrus trees, including several TIR-NBS-LRR genes. Many cell wall degradation-related genes, such as cellulose synthase/transferase, cellulase and expansins were up-regulated in the susceptible citrus trees. Some glucan hydrolase genes were also up-regulated in the tolerant citrus trees. These genes may play important roles in symptom development. The DE genes were also enriched in two classes of RLKs, LRR-RLKs and DUF26-RLKs. We then applied the MapMan software to identify DE genes related to disease response. The MapMan annotated 155 DE genes related to disease response. We found multiple pathways have been suppressed or activated in HLB tolerant citrus tree, which lead to the promotion of the basal resistance or immunity in citrus tree. For examples, suppression of beta glucanases, DMR6-like genes, expansin and DET2 uphold the suppression of immunity of citrus tree and activation of NPR1-like genes also induces the immune responses to HLB. The mechanism how to trig the suppression and activation of these genes is still unclear. Further experimental studies on the NBS-LRR and RLK genes differentially expressed between HLB tolerant and HLB susceptible citrus trees may help understand the HLB related receptor genes (PLoS ONE 10(3): e0121893). Meanwhile, our functional analysis also showed that the HLB development related genes were enriched in HLB susceptible citrus trees, such as viral life cycle and symbiosis related genes. We predicted a protein-protein interaction (PPI) network of citrus using the PPIs of Arabidopsis. There are 1259 proteins and 2298 interactions in our Citrus PPI network. Among 1259 proteins, 42 proteins are differentially expressed between the HLB resistance and susceptible citrus. An interested PPI sub-network includes 14 citrus NPR1-likes proteins and three TGA proteins. There were four NPR1-like genes were significantly up-regulated in HLB tolerant citrus trees and one NPR1-like gene up-regulated in HLB sensitive citrus trees. There is also one TGA gene up-regulated in HLB tolerant citrus trees. Another interested sub-network includes the RPS2 protein. There were two LRR kinase receptors significantly up-regulated in HLB tolerant citrus trees and four LRR kinase receptors significantly up-regulated in sensitive citrus trees. In conclusion, by profiling the transcriptome of HLB tolerant and HLB susceptible citrus trees, we found that multiple pathways have been suppressed or activated in HLB tolerant citrus tree, which lead to the promotion of the basal resistance or immunity of citrus trees. This study may help us understand HLB-tolerance and provide guidance for breeding HLB-tolerant citrus in the future. The down-regulation genes can be served as the candidate targets for RNAi to improve HLB tolerance in citrus trees.
Analysis and writing continue in preparation for the end of this project in June. One student is completing her MS degree, and with her data completed and analyzed, manuscripts are being developed. These may not be completed by the termination date of the project, but we hope to have at least first drafts done. The FT3 gene is also being put into the CTV vector and is being put into CRISPR/Cas systems in collaborations with others.
A chimeral construct that should enhance AMP effectiveness (designed by Goutam Gupta of Los Alamos National Lab) is being tested. Many transformed Carrizo with the chimera AMP were obtained. Exposure to canker inoculum showed remarkable resistance in chimera compared to control. RNA was isolated from 16 transgenic Hamlin containing Chimera. RT-qPCR showed 50% of them have relative high gene expression. One of them showed over hundred times higher expression compare to plant expressing the lowest level of chimera. These promising transgenic lines were replicated by grafting for HLB challenge. About 30 Hamlin transformed with thionin also were obtained. Twenty transgenic lines were confirmed containing thionin gene by PCR. RNA was isolated from 16 transgenic Hamlin containing thionin. Six of them have relative high gene expression by RT-qPCR. These transgenic lines will be replicated for HLB challenge. Two new chimeral peptide have been developed and is used to transform citrus. Many transformed Carrizo shoots with new chimera construct were obtained. Some were transferred to the rooting medium. Replicated trangenic lines expressing chimera, thionin and D4E1were grafted with HLB infected rough lemon. Las tilter will be checked by qPCR periodically. To explore broad spectrum resistance, a flagellin receptor gene FLS2 from tobacco was cloned into pBinARSplus vector Flagellins are frequently PAMPS (pathogenesis associated molecular patterns) in disease systems and CLas has a full flagellin gene despite having no flagella detected to date. The consensus FLS2 clone was obtained and used to transform Hamlin and Carrizo so that resistance transduction may be enhanced in citrus for HLB and other diseases. Many putative transformants were generated on the selective media. DNA was isolated from 80 of them: 38 Carrizo and 7 Hamlin are positive by PCR test. Reactive Oxygen Species (ROS) assay showed typical ROS reaction in three of transgenic Hamlin which suggest nbFLS is functional in citrus PAMP-triggered immunity. However, there is only slight canker resistance by infiltration test. Spray inoculation was tried and some of them show obvious canker resistance. To confirm that high ROS production was not due to variability in Hamlin, we examined 40 Hamlin seedlings and no or very low level ROS production was detected. In contrast, relatively higher ROS production was detected from wild-type Carrizo seedings compared to Hamlin seedlings. Two potential FLS2 orthologues were identified in Hamlin and their expression was shown much lower compare to nbFLS2. Replicated trangenic Carrizo lines expressing nbFLS2 were challenge with ACP. Las titer will be checked by qPCR periodically. To disrupt HLB development by manipulating Las pathogenesis, a luxI homolog potentially producing a ligand to bind LuxR in Las was cloned into binary vector and transformed citrus. Both transformed Carrizo and Hamlin were obtained. Further investigation are underway. A series of transgenics scions produced in the last several years continue to move forward in the testing pipeline. Several D35S::D4E1 sweet oranges show initial growth in the field which exceeds that of controls. A large number of ubiquitin::D4E1 and WDV::D4E1 plants and smaller numbers with other AMPs are replicated and in early stages of testing. In collaboration with Bill Belknap two new citrus-derived promoters have been tested using a GUS reporter gene and have been shown to have extraordinarily high levels of tissue-specific expression. The phloem-specific promoter is being used to create a construct for highly phloem specific expression of the chimeral peptide using citrus genes only.
Trees of seemingly HLB resistant/tolerant sweet orange-like hybrids and mandarin -types have been propagated on x639. Replicated trials with standards will be established. Six locations each of all sweet orange-like together and 4 with all mandarins will be established with 6-8 trees of each cultivar at each site. We have identified cooperators (in Ridge, IR and Gulf coast) for complete replicated block plantings at each site. In October 2013, 34 unique genotypes (USDA hybrids) some of which appear to have tolerance to HLB, and 16 standard commercial varieties were exposed to an ACP no-choice feeding trial and have been transferred to the field at Ft. Pierce Fl. Standard growth measurements and disease ratings were initiated in July 2014 and will continue on a monthly basis. As of December 2014, the first HLB symptoms are apparent. Evaluation of existing standard and non-standard cultivars (‘Hamlin’, ‘Temple’, ‘Fallglo’, ‘Sugar Belle’, ‘Tango’, and ‘Ruby Red’) for HLB resistance/tolerance is complete. In August 2010, the plants were established at Pico’s farm in Ft. Pierce Fl. Data on the growth rate, disease severity, and Candidatus Liberibacter asiaticus (CLas) titer levels have been collected since April 2012. During the 4-year period, there were significant differences in disease severity, stem diameter, and CLas levels among the varieties. All trees exhibited symptoms of HLB and tested positive for CLas, with similar titers measured at most recent sample dates.’Fallglo’ had the lowest incidence of HLB symptoms, whereas ‘Ruby Red’ had the highest incidence. ‘Ruby Red’ also appears to be in significant decline. Despite the high initial titer levels found in ‘SugarBelle’, it had the greatest overall increase in diameter and was the healthiest in overall appearance. In Nov. 2014 ‘Temple’ trees had significantly greater fruitload, with 26 fruit/tree, followed by ‘Tango’ with 10 fruit /tree, ‘Hamlin/Kinkoji’ with 5 fruit/tree and all others with 0-1.4 fruit/tree. Production was compromised in all varieties by the severe HLB pressure at this site, and commercial value of the observed tolerance remains uncertain. Progress has been made on the antibiotic treatment of HLB infected bud-wood to compare growth at different levels of CLas infection. Bud-wood of nine HLB symptomatic varieties, 3 fairly resistant (‘Temple’, GnarlyGlo’, and ‘Nova’) 3 tolerant (‘Jackson’, FF-5-51-2, and Ftp 6-17-48), and 3 susceptible (‘Flame’, ‘Valencia’, and ‘Murcott’). In November 2013 and May 2014, HLB positive bud-wood was treated with various concentrations of penicillin and streptomycin and grafted on sour orange rootstock. Standard growth measurements (stem diameter and height), disease severity were evaluated and leaves were sampled for qPCR analysis. Evaluations and sampling will continue on quarterly basis. Development of periclinal chimeras with resistant vascular tissue from Poncirus and remaining layers from sweet orange is currently underway. One hundred and fifty etiolated seedlings of the trifoliate ‘Rubidoux’ and the sweet orange ‘Hamlin’ have been approach grafted together. Generation of new chimeras has been difficult. Several adventitious buds have emerged from the treated graft region, with several appearing to be chimeral. The newly emerged plants will be tested using LC/MS to determine the origin of the three layers. To increase the success rate, additional plants will be grafted over the next twelve months. A method for the rapid identification of potential sources of HLB resistance is also being developed. This project involves the screening of citrus seedlings at the 3 to 5 leaf stage, or very small micrografted trees, that are exposed to HLB infect ACP feeding. CLas titer levels, using real time PCR, are evaluated at 3, 6, and 9 weeks Seedlings of Hamlin and Dancy show early CLas proliferation and systemic movement. Only very low levels of CLas have been observed in Carrizo.
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 five years. A number of successes have already been documented at the Picos Test Site funded through the CRDF. The UF Grosser transgenic effort has identified promising material, eliminated failures, continues to replant with new advanced material, with ~200 new trees in April 2015 (Grosser, personal comm.). The ARS Stover transgenic program has trees from many constructs at the test site and is seeing some modest differences so far, but new material is being planted this spring that has shown great promise in the greenhouse (unpublished). A trial of more than 85 seedling populations from accessions of Citrus and citrus relatives (provided as seeds from the US National Clonal Germplasm Repository in Riverside, CA) has been underway for 5 years in the Picos Test Site. P. trifoliata, Microcitrus, and Eremocitrus are among the few genotypes in the citrus gene pool that continue to show substantial resistance to HLB (Lee et al., in preparation), and P. trifoliata also displayed reduced colonization by ACP (Westbrook et al., 2011). A new UF-Gmitter led association mapping study has just been initiated using the same planting, to identify genes associated with HLB- and ACP-resistance. A collaboration between UF, UCRiverside and ARS is well-underway with more than 1000 Poncirus-hybrid trees (including 100 citranges replicated) being evaluated to map genes for HLB/ACP resistance. Marked differences in initial HLB symptoms and Las titer were presented at the 2015 International HLB conference (Gmitter et al., unpublished) and David Hall is now assessing ACP colonization. Several USDA citrus hybrids/genotypes with Poncirus in the pedigree have fruit that approach commercial quality, were planted within the citrange site. As of April 2014 at the Picos Test Site, several of these USDA hybrids had grown to a height of seven ft, with dense canopies and good fruit set, while sweet oranges are stunted (3 ft) with very low vigor (Stover et al., unpublished). A Fairchild x Fortune mapping population will be planted at the Picos Test Site this spring in an effort led by Mike Roose to identify genes associated with tolerance. This replicated planting will also include a number of related hybrids (among them our easy peeling remarkably HLB-tolerant 5-51-2) and released cultivars. Valencia on UF Grosser tertazyg rootstocks have been at the Picos Test Site for several years, having been Las-inoculated before planting, and several continue to show excellent growth compared to standard controls (Grosser, personal comm.).
We continue to conduct the weekly Agrobacterium-mediated transformations and screen putatively transformed mature scion and rootstock shoots for clients. The total number of transgenics produced and detected thus far (excluding the 157 reported earlier) is ~100, and ~ half of these survived primary and secondary grafting (excluding the 66 reported earlier). The transformation efficiency for a plasmid with no reporter gene and a weak promoter driving the npt11 selectable marker was 2.8% for mature scion and rootstock. As expected, mature rootstock gave a higher transformation efficiency than mature scion. As previously mentioned, the weaknesses of this protocol are the high number of escapes particularly with constructs having weak promoters driving the npt11 selectable marker, difficulties with micro-grafting, and for certain constructs with no reporter genes, the difficulties in screening by PCR. PCR is expensive, time-consuming, labor intensive and prone to error. After the standard tissue culture protocol, growth in selective liquid media has assisted in identifying 4 transgenic events, which indicates that better selection is important. GFP expressing transgenic shoots were regenerated from mature citrus after biolistics, but subsequently died after micro-grafting. We will consider purchasing mature scion from nurseries solely for biolistics because we cannot produce enough in the growth room to facilitate the weekly Agrobacterium transformations and optimize biolistics. This mature scion will not enter the growth facility unless it has been cleaned through shoot-tip grafting. Parameters still to be optimized include use of the hepta adapter, the number of bombardments per treatment, growth stages (days after culture initiation) of target explants, and osmotic treatment on medium containing sorbitol and mannitol (hours and concentrations). We have had to find an alternate California seed source because certain varieties that we regularly use in tissue culture are contaminated by a fungus. Spraying fungicide does not eliminate this seed borne fungus. A manuscript entitled, “Genetic Transformation of Commercially Important Mature Citrus Scions” authored by Hao Wu, Yosvanis Acanda, Alka Shankar, Michael Peeples, Calvin Hubbard, Vladimir Orbovi. and Janice Zale has been accepted for publication in Crop Science.
Objective 1: Generate functional EFR variants (EFR+) recognizing both elf18-Xac and elf18-CLas A number of strategies to engineer an EFR variant that recognized elf18-Clas were tested over the grant period, but none were successful. These strategies included PCR mutagenesis of EFR, screening of natural variants in an extensive Arabidopsis accession collection, creating targeted mutations based on the modeled interactions among elf18, EFR, and BAK1, and testing high-throughput selection strategies such as phage display and fluorescence activated cell sorting. Funding for this activity has ended and effort toward this objective has ceased. Objective 2. Generate functional XA21-EFR chimera (XA21-EFRchim) recognizing axYS22-Xac. This objective was completed and a manuscript describing the XA21-EFR chimera and the complementary EFR-XA21 chimera has been published (Holton et al., 2015, PLoS Pathogens 11, e1004602). Objective 3: Generate transgenic citrus plants expressing both EFR+ and XA21-EFRchim. Putative transgenic citrus plants have been generated for four constructs: EFR, EFR plus XA21, EFR plus XA21-EFRchim, and the empty vector pCAMBIA2201. To date, 174 Duncan grapefruit, 20 sweet orange, and 5 Carrizo citrange plants have been tranferred to soil, and PCR analysis is beginning to determine whether the intact transgenes are present.
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