The project has three objectives: (1) Obtain mature tissues of the best transgenic lines. (2) Determine whether transgenics prevent psyllids from being infected. (3) Continue testing generations of vegetative propagation from the best transgenic lines. The following work has been conducted in this quarter: (1) We have started to treat the three independent transgenic lines ( Duncan 57-28, Hamlin 13-3, and Hamlin 13-29), which have gone through the long-term HLB test and exhibited robust tolerance to HLB disease. The first batch of plants, including two replicates of the transgenic line 57-28, three replicates of the line 13-3, and one replicate of the line 13-29, have been treated under the alternating temperature conditions (25 C for 4 hours and 42 C for 4 hours) for two months. These plants have generated some new shoots during the treatment. We have tested if heat treatment is able to remove CTV and the CLas bacteria. So far the new shoots are negative for both CTV and CLas, indicating that the treatment is effective. The new shoots will be used for generating citrus trees for field trials. (2) We have screened 28 new transgenic lines against HLB-infected psyllids. These lines were generated by the mature transformation laboratory. The following lines still look great and haven’t shown any HLB symptoms: #82-6 Hamlin, #70-4 Hamlin, #26 Hamlin, #65 Hamlin, #82 Hamlin, #73-5 Hamlin, #11 Pineapple, #33 Pineapple, #73-5 Pineapple, #78 Pineapple. Based on the nymph production phenotype, these plants should have been infected by HLB. We have tested bacterial titers in these plants by qPCR, and indeed, the majority of these plants are CLas positive. The bacterium-free plants (with low CLas titers) will be inoculated again. (3) The eight new transgenic lines (A99, A100, A102, A101, A72, A73, A97, and A98) were irrigated and fertilized regularly. After they reach appropriate size, they will be screened against HLB-infected psyllids. (4) The manuscript titled Overexpression of the Arabidopsis NPR1 protein in citrus confers tolerance to Huanglongbing has been revised and published in the Journal of Citrus Pathology in this quarter.
The project has three objectives: (1) Obtain mature tissues of the best transgenic lines. (2) Determine whether transgenics prevent psyllids from being infected. (3) Continue testing generations of vegetative propagation from the best transgenic lines. Major accomplishments per objective (1) Obtain mature tissues of the best transgenic lines: successfully achieved. The citrus flower-promoting gene FT3 was previously cloned into the CTV vector by the Dawson lab. The CTV-FT3 construct was introduced into Agrobacterium. Tobacco leaves were infiltrated with the resulting Agrobacterium. CTV-FT3 recombinant virions were purified from systemically infected tobacco leaves and bark flap inoculated into C. macrophylla seedlings, which began blooming in about five months. Buds from the matured C. macrophylla were grafted onto the original plants of all transgenic lines (EDS5-Dun-205-9, ELP3-Dun-207-8, ZMSN-Ham-73-1, ZMSN-Dun-137-2, NPR1-Ham-13-3, NPR1-Ham-13-29, and NPR1-Dun-57-25). All plants began blooming in 6-18 months. We have successfully achieved this objective and also demonstrated that CTV-FT3 is efficient for converting juvenile tissues to mature tissues. The CTV-FT3-infected C. macrophylla plants that have bloomed can be used as bud source for promoting maturation. The three independent transgenic lines (NPR1-Ham-13-3, NPR1-Ham-13-29, and NPR1-Dun-57-25) that have shown robust tolerance to HLB have been treated under the alternating temperature conditions (25 C for 4 hours and 42 C for 4 hours) to remove CTV and the CLas bacteria. The resulting clean germplasms will be used to generate trees for field trials. (2) Determine whether transgenics prevent psyllids from being infected: accomplished with negative results. CLas-infected psyllids can transfer the CLas bacteria to the next generation of psyllids by inoculating the flush area in which the nymphs develop, which allows the next generation of psyllids to continue spreading HLB without the need for another source plant. In our experiments, we noticed that several of the transgenic lines exhibit delayed or reduced levels of CLas after infection. We started to test if the delayed or reduced production of CLas is sufficient to prevent or reduce the infection of the progeny psyllids. CLas bacteria-carrying transgenic plants were placed in cages, and clean psyllids (not infected by CLas) were moved into the cages. The progenies of the psyllids were collected and tested for CLas titers. A total of six rounds of cage experiments with vegetatively propagated plants from the transgenic lines ELP3-Dun-207-8, NPR1-Ham-13-3, NPR1-Ham-13-29, and NPR1-Dun-57-25 were conducted. Results showed that none of the transgenes was able to pre-vent psyllids from being infected by CLas. (3) Continue testing generations of vegetative propagation from the best transgenic lines: successfully achieved. Three independent transgenic lines, NPR1-Ham-13-3, NPR1-Ham-13-29, and NPR1-Dun-57-25, have gone through at least six rounds of HLB inoculation. Three generations of progenies (18 replicates for NPR1-Ham-13-3, 31 replicates for NPR1-Ham-13-29, and 25 replicates for NPR1-Dun-57-25) were inoculated with CLas-infected psyllids. The inoculation was repeated until all plants were CLas positive based on qPCR. All progeny plants have shown no or minor HLB symptoms. The three transgenic lines are thus highly tolerant to HLB and will be put into the field for field trials.
Activities are reported by project objectives below. 1. Development of rootstocks that can impart HLB tolerance/resistance to grafted scions. Seedlings are being grown of over one dozen unreleased rootstocks already shown to control tree size support good fruit loads and to have minimal HLB symptom expression. These will be propagated with sweet orange scions for field planting at the St. Helena site next season. As part of the gauntlet screening, we stick-grafted approximately 75 new candidate rootstock hybrids produced from HLB-tolerant parents in 2017 with HLB+ Valencia sweet orange for HLB screening. We produced rooted cuttings of approximately 100 gauntlet candidate rootstock hybrids including 47 hybrids combining HLB-tolerant LB8-9 Sugar Belle with complementary rootstock germplasm (including salt tolerant pummelo/mandarin hybrids and trifoliate orange 50-7). Replicated cuttings will be used for further HLB-tolerance assessment. Super Root Mutants of 10 selections of UFR and other rootstocks discovered by Beth Lamb at the Phillip Rucks Nursery Tissue Culture Lab were potted up; this group includes 3 mutants of UFR-1, 3 of UFR-3, one of UFR-4, one of UFR-17 and one of SO+50-7. These lines are producing feeder roots at a much higher density than the standard clones, and we will screen these selections for potentially enhanced HLB tolerance. 2. Breeding of HLB tolerant/resistant processing sweet oranges and orange-like hybrids. New hybrids produced have been potted up to grow until field planting next season. 3. Screening of the UF-CREC germplasm collection to identify and validate HLB tolerant or resistant selections. The data from this continued effort are being analyzed cumulatively across multiple seasons to more accurately identify and characterize tolerant individuals. 4. Advanced field trials, release and commercialization of promising HLB tolerant/resistant scion and rootstock cultivars. We continued focused effort on field trial data management, analysis and interpretation. Files from more than 80 sites have been opened, conditions of the trials have been noted, and plans for 2018-19 field data collection have been developed and prioritized, based on this information. Efforts to review and summarize data have continued, and information is being organized for inclusion in our soon-to-be available website. We completed data analysis from evaluation of young resets of new sweet orange selections on multiple rootstocks in LaBelle/Immokalee trials, for tree size, health and yield: UFR-17, UFR-15 and 46×20-04-42 (pummelo x Cleo) were outstanding performers here. UFR-4 also did well. The best combinations for tree health and early fruit production were Valencia B9-65/UFR-17 and OLL-20/UFR-17. Our field team visited 10 field trial sites this summer, assessed trees for condition, and contacted grove management personnel, to make them aware of our continued interest in the trials. We planted one new rootstock trial, an additional 400+ trees in a fresh fruit scion trial, and 250 grapefruit hybrids at the IRREC.We collected detailed botanical and morphological information to support IP protection for release of 2 new rootstocks, one sweet orange and a deep red grapefruit.Finally, a very substantial effort was undertaken this year to rescue promising individual trees of diverse scion and rootstock germplasm from our 50-acre research block at the GCREC in Balm. These blocks have not been irrigated since early fall of 2017, and we were forced to leave the site. All trees in this block were subjectively assessed for potential HLB tolerance, as well as general overall health and appearance, using a 0-4 scale (0=dead; 4=completely heathy appearing tree). We harvested budwood from ~2300 individuals with scores = 3 and propagations for field planting in another location. Between June and end of September 2018, we had to revisit the block and collect additional budwood from trees that were not successfully propagated. We are growing off these trees for planting in 2019 at a new location.
Objective 1. (Greenhouse experiment): qPCR analysis was conducted on all trees to determine CLas titers. The combination of Valencia on WGFT+50-7 rootstock showed significantly reduced Clas populations, especially in Treatment #5 (Harrell s 12-3-9- St. Helena mix). Many plants of this combination in Treatment #5, and in a few other treatments showed no active infections (trees previously showing active infections). Lower numbers of trees of other combinations also showed reduced CLas titers; but all the data was just received, so the complete analysis is still underway. Evidence accumulating that nutrition can indeed suppress CLas titers. Objective 3: Field trials of CRF/TigerSul blends: Use of CRF with tiger micronutrient seems promising – data recently presented to CRDF board at Arcadia. 20-50% higher rates of micronutrients seem to improve the fruit yields Soil applied fertilizer program seems to be beneficial for HLB-affected trees Nutrition does have an effect on fruit quality! Focus should be on constant supply of nutrients Either frequent application of conventional fertilizer or adding some amount of CRF is beneficial Uninterrupted fertigation is supported by data Objective 5. (funded by Lee Groves, using donated fertilizer products): Alligator Vernia/Rough Lemon Enhanced Nutrition Experiment Treatments: 6 tree plots (randomized), 2 plots per treatment treatments 2 times per year. Positive results showing a therapeutic affect from overdoses of manganese against HLB is being presented at the annual ASHS meeting in Washington DC, and a manuscript has now been submitted to HortScience. Another round of treatments was applied to the trees, although this round we substituted Florikan polycoated Mn for TigerSul Mn to get longer distribution.
Our transgenic efforts have evolved greatly in the three years of this project. As data accumulated and new ideas came to the forefront, efforts were focused on those aspects of the research proving most valuable, while several of the initial objectives were deemphasized. A new project was approved by CRDF (18-022) that is the next series of steps following up on the successes of project 15-026. To accelerate screening for CLas-killing transgenics, a detached leaf inoculation method was developed via CLas+ ACP no-choice infestation. This high throughput lab based method can test plants at small seedling stage, is non-destructive, and provides guiding information on assessment 6-12 month earlier than greenhouse based tests. To evaluate AMPs for potential use in CLas-killing transgenes, we have developed an in vitro procedure for directly measuring their ability to disrupt CLas cells. A homogeneous CLas suspension is recovered by macerating CLas+ ACP in a specially developed extraction buffer and removing cellular debris through spin filter centrifugation. Homogenates are exposed to AMPs vs controls for 4 hours. CLas cell integrity is determined by use of a photo-reactive DNA binding dye. Small plant destructive inoculation assays, where all plant tissues are weighed and sampled after no-choice CLas+ ACP feeding, now permit us to distinguish between susceptible Valencia and resistant Carrizo after 12 weeks. This method is being used in our transgenic efforts to validate the detached leaf assay results. A modified plant Thionin (Mthionin), was designed by G. Gupta using biophysical modeling. Transgenic expression of this peptide in Carrizo citrange showed a marked and highly statistically significant decrease in symptoms when challenged with Xanthomonas citri, the causal agent of citrus canker. When transgenic plants were challenged through graft inoculation with HLB infected material, both transgenic Carrizo tissues and non-transgenic scion (Rough Lemon) tissue showed reduced CLas titer up to 12 month (latest time-point) post graft when compared to control plants, with root CLas titer 1800 times greater in wild-type Carrizo. We are testing Mthionin transgenics in the field. Several of our best lines are at DPI for cleanup and broader field trialing, ideally head-to-head with transgenics from other programs Newer generations of AMPs, categorized as 2nd and 3rd generation, were designed (also by G. Gupta) using citrus native thionin as the foundation combined with other citrus genes with high affinity for CLas membranes. Numerous lines and events of 2nd and 3rd gen AMP transgenic citrus have been subject to the detached leaf assessments. Some showed promising CLas clearance; indicated by transgenic Carrizo leaves showing significantly lower CLas titer compared to wild type controls after a 7-day ACP infestation. Interestingly, bacterial quantity in the ACP bodies was also lower after feeding on the transgenic leaves, suggesting an uptake of AMPs into the insect body and disruption of gut bacterial cells. Several of our best lines are also at DPI for cleanup and broader field trialing. ScFv sequences targeting CLas outer membrane proteins were developed by J. Hartung and used for the creation of transgenes disrupting the CLas infection process. Transgenic plants are showing a consistent and statistically significant decrease in Clas titer twelve months after no choice CLas+ ACP inoculation (up to 250x reduction, measured by qPCR) and have a much higher incidence of plants with no measurable bacterial DNA amplification. Approximately 120 additional rooted cuttings were propagated for field trials, with the primary focus being rootstocks to protect conventional scions. We sought (with W. Belknap and J. Thomson) to identify highly expressed genes in the citrus phloem, reasoning that their promoters might be useful for transgenics combatting HLB. Through this work, a citrus gene family was identified and characterized encoding a group of Small Cyclic Amphipathic Peptides (SCAmpPs) with highly conserved gene structure, but considerable variation in the ultimate gene products. Variants of a tissue-specific SCAmpP promoter were tested using GUS as a reporter gene and resulted in excellent phloem-specific expression: 500x greater expression in leaf midribs/petioles compared to laminar area and visibly greater GUS gene product activity in midribs and vascular tissue compared to GUS driven by D35S. These citrus promoters are being used in all new transgenics from our program, usually with a parallel set of transgenics driven by D35S, to combat gram-negative pathogens in other citrus tissues.
Identifying HLB-tolerance and -resistance has greatly evolved in the three years of this project. Several ambitious objectives failed, and efforts were focused on promising project areas. Progress was made and resulted in supporting data and new objectives in a new NIFA-CRDE project (Baldwin lead). Due to this NIFA funding we did not submit a new proposal to CRDF for this project. Evaluation of existing cultivar/rootstock combinations for HLB resistance/tolerance was completed and revealed potentially valuable tolerance. It further indicated that early HLB symptoms and CLas titer are unrelated to growth and cropping. ‘SugarBelle’ and ‘Tango’ were the largest, healthiest in overall appearance, and had the most fruit. Grapefruit was by far the worst performing scion in this trial. A replicated trial is underway of 50 selections and cultivars following no-choice ACP, months in an ACP house, and 5 years in the field. Several of our best grapefruit-like hybrids looked good last year but are now declining. The one true grapefruit is among the least healthy selection in the trial. Jackson grapefruit continues to look quite good. The best performers include hybrids containing Poncirus, and conventional hybrids which are predominately mandarin. It may take 2-3 more years to clearly distinguish tolerant material. Several selections that had looked good last year suddenly dropped to very low canopy growth, and it appears this may be a relatively early indicator of HLB tolerance. Replicated trials in multiple locations were established of our best sweet-orange-like cultivars and mandarin-types. These trials are in the field with data collection managed by Greg McCollum of USHRL. The easy peeling sweet-orange-like hybrid 1-76-55 continues to look better than sweet orange at most sites. The 1/16th Poncirus hybrid we have been studying under the name Gnarlyglo (released as US SunDragon) continues to look very healthy with no evidence of HLB compromised fruit. Most of the mandarin-types in this trial continue to display marked HLB-tolerance. A replicated field planting was established of 133 Fortune x Fairchild hybrids, 27 Ponkan-like accessions, 10 advanced ARS selections that are predominately mandarin, and trees of Fortune, Fairchild and Valencia. Data collection is underway and this planting will be critical for the new NIFA project identifying HLB-tolerance in mandarins. A replicated citrus relatives planting (85 seed source genotypes) was assessed for apparent HLB tolerance. Within the genus Citrus, measures of tolerance (canopy density, health, and tree size) correlated positively with % citron in pedigree, with r2 of 0.3-0.6. This has encouraged use of citron hybrids in breeding acid-citrus and introgression of citron genes into phenotypes like sweet oranges, grapefruit, and mandarin hybrids Seedlings with a range of pedigree contributions from Microcitrus have been grown in collaboration with M. Smith, Queensland Aus. citrus breeder, and will be planted this fall for field testing of HLB resistance. Trees of 54 scion pairs were planted in 2000-04 on both mandarin and trifoliate hybrid rootstocks. There was no significant effect of rootstock on CLas titer in the foliage or the roots. Scions which included Poncirus in the pedigree (no pure Poncirus), had one third the level of foliar CLas compared to other scions and less severe HLB symptoms, but scion type had no significant effect on root CLas titer. This provided further evidence that scions derived from Poncirus can contribute to HLB-tolerance. RNA-seq compared transcriptome responses in HLB- tolerant `Sun Chu Sha’ mandarin and susceptible `Duncan’ grapefruit, to Xcc-flg22 and CLas-flg22 (most active epitope from the pathogen flagella; project initiated with Gloria Moore at University of Florida). Differential expression of a number of genes occurred between tolerant and susceptible citrus infected with CLas, suggesting their involvement in HLB tolerance. Genes identified may be valuable for studying HLB tolerance using CLas-flg22 as a pathogen proxy. 28 CLas effectors were predicted and transcriptional levels determined in infected citrus. Many were detected suggesting roles in pathogen virulence and host response modulation. Highly expressed effectors will be further tested for potential as biomarkers for screening breeding materials. Small citrus seedlings or very small micrografted trees were exposed to CLas-infected ACP. CLas titer levels were detectable in most plants at 3 weeks. By nine weeks after exposure, susceptible genotypes can be clearly distinguished from resistant material (Carrizo and Poncirus) by higher CLas levels in roots. There was no evidence that HLB-tolerant genotypes differed from -susceptible material in the development of CLas titers, which is consistent with field observations. However, this methodology should be especially useful for screening anti-Las transgenics, with recent transition to using detached leaves for a much shorter assay.
Activities are reported by project objectives below. 1. Development of rootstocks that can impart HLB tolerance/resistance to grafted scions. Seedlings are being grown of over one dozen unreleased rootstocks already shown to control tree size support good fruit loads and to have minimal HLB symptom expression. These will be propagated with sweet orange scions for field planting at the St. Helena site next season. As part of the gauntlet screening, approximately 150 new rootstock candidates from 2016 crosses stick-grafted with HLB-affected budwood; these included hybrids using Sugar Belle, pummelo, and trifoliate orange 50-7. We planted 100 new gauntlet trees (HLB+ Valencia on individual new rootstock candidates) at the USDA Picos Farm in Fort Pierce. This planting includes numerous tetraploid hybrids of a recently characterized HLB-tolerant tetraploid sour orange-like seed parent with HLB-tolerant hybrids of trifoliate orange 50-7. Finally, a small preliminary screening trial using left-over cuttings from gauntlet rootstock candidates (34 trees on 22 new rootstock hybrids), grafted with dark red grapefruit clone N11-7, was planted in the Indian River area. 2. Breeding of HLB tolerant/resistant processing sweet oranges and orange-like hybrids. Sensory and chemical analyses of fruit of tolerant sweet oranges and sweet orange-like hybrids were conducted, to understand the potential value of such fruit to the OJ business. Also, we planted approximately 500 triploid scion hybrids in CREC research blocks, with emphasis on hybrids designed to produce sweet orange-like fruit from HLB-tolerant parents. Finally, 140 new transgenic trees were planted at an APHIS-permitted site for field testing. These trees were mostly oranges containing either BG or another new AMP gene Lima B, under control of either constitutive or phloem-limited promoters. 3. Screening of the UF-CREC germplasm collection to identify and validate HLB tolerant or resistant selections. We continued to monitor our germplasm collection and breeding families for performance against HLB, and we contine our genomic selection efforts based on HLB-phenotype and SNP genotyping. Genomic regions identified contain putative disease resistance/defense genes. The data from this continued effort are being analyzed cumulatively across multiple seasons to more accurately identify and characterize tolerant individuals. 4. Advanced field trials, release and commercialization of promising HLB tolerant/resistant scion and rootstock cultivars. We finished off a very busy fruit season with collecting yield data from 2 field trial sites and determined fruit juice quality from 5 field trials; in these cases, data were collected on tree performance in the face of HLB. At least 15 grove sites throughout the state were visited to check the status of existing field trials, and to make certain that appropriate personnel within the cooperators organizations were aware of our continued interest and commitment to their field trials with us. We have continued our efforts to develop cumulative reports over time of certain selected individual trials, to highlight the better performing combinations for yields and HLB tolerance. We are moving forward with plans to share these with the collaborative growers first, and then to make them available to the industry at large. Finally, a very substantial effort was undertaken to rescue promising individual trees of diverse scion and rootstock germplasm from our 50-acre research block at the GCREC in Balm. These blocks have not been irrigated since early fall of 2017, and we are being forced to leave the site. All trees in this block were subjectively assessed for potential HLB tolerance, as well as general overall health and appearance, using a 0-4 scale (0=dead; 4=completely heathy appearing tree). We harvested budwood from ~2300 individuals with scores = 3 and propagations for field planting in another location.
This project was based on the idea that blocking the function of the NodT outer membrane transporter of ‘Candidatus Liberibacter asiaticus’ (CLas) would block pathogenicity or survival of the bacterium within citrus plants. Single-chain, mini-antibodies (scFvs) recognizing a peptide corresponding to the major, predicted extracellular loop of CLas were isolated. The scFv with the strongest binding in a qualitative assay was selected and fused to the C-terminal end of the citrus Flowering Locus T (FT) protein as a gene fusion, encoding an FT-scFv protein. The antibody was fused to FT in order to promoter stability, mobility, and expression of the protein in the phloem. The FT-scFv coding region was placed under the control of the constitutive Cauliflower Mosaic Virus (CaMV) 35S promoter and introduced into ‘Duncan’ grapefruit (Citrus paradisi) using Agrobacterium-mediated transformation. Fifteen (15) independent transgenic lines were obtained, most of them expressing high levels of the FT-scFv protein, as determined by protein gel immunoblot analysis. Eight lines are maintained in Florida at the United States Horticultural Laboratory (USHRL) and seven lines are maintained at Penn State. Many of the FT-scFv lines have a precocious blooming phenotype, which could be useful for accelerated citrus breeding purposes. Prior attempts to overproduce FT in citrus have encountered problems with lack of plant survival, while FT-scFv plants survive and can produce fruit. All lines have been propagated vegetatively, and they continue to express FT-scFv after propagation. The HLB resistance or tolerance phenotype of the FT-scFv lines has not yet been tested, however. Graft-transmission of the FT-scFv protein has also not yet been tested. However, the materials need to accomplish these last two goals have been produced and this represents an opportunity for future analysis.
This project was based on the idea that blocking the function of the NodT outer membrane transporter of ‘Candidatus Liberibacter asiaticus’ (CLas) would block pathogenicity or survival of the bacterium within citrus plants. Single-chain, mini-antibodies (scFvs) recognizing a peptide corresponding to the major, predicted extracellular loop of CLas were isolated. The scFv with the strongest binding in a qualitative assay was selected and fused to the C-terminal end of the citrus Flowering Locus T (FT) protein as a gene fusion, encoding an FT-scFv protein. The antibody was fused to FT in order to promoter stability, mobility, and expression of the protein in the phloem. The FT-scFv coding region was placed under the control of the constitutive Cauliflower Mosaic Virus (CaMV) 35S promoter and introduced into ‘Duncan’ grapefruit (Citrus paradisi) using Agrobacterium-mediated transformation. Fifteen (15) independent transgenic lines were obtained, most of them expressing high levels of the FT-scFv protein, as determined by protein gel immunoblot analysis. Eight lines are maintained in Florida at the United States Horticultural Laboratory (USHRL) and seven lines are maintained at Penn State. Many of the FT-scFv lines have a precocious blooming phenotype, which could be useful for accelerated citrus breeding purposes. Prior attempts to overproduce FT in citrus have encountered problems with lack of plant survival, while FT-scFv plants survive and can produce fruit. All lines have been propagated vegetatively, and they continue to express FT-scFv after propagation. The HLB resistance or tolerance phenotype of the FT-scFv lines has not yet been tested, however. Graft-transmission of the FT-scFv protein has also not yet been tested. However, the materials need to accomplish these last two goals have been produced and this represents an opportunity for future analysis.
The availability of rootstocks highly tolerant or resistant to HLB, and with good horticultural traits, would eliminate this disease as a threat and permit higher crop production at reduced cost. The main goal of this project was to investigate whether we can identify metabolites (small molecules) in citrus rootstocks that are associated with specific rootstock traits, particularly tolerance to HLB, but also tolerance to other stresses and diseases. Identification of metabolites that are associated with specific rootstock traits would aid in the early selection of promising candidate rootstocks prior to long-term field testing and accelerate the release of trees for commercial use. Specific objectives of this project were 1) identify key metabolites that are associated with rootstock traits, 2) investigate the effect of grafting on metabolite profiles, and 3) establish metabolite profiles of trees on different rootstocks in response to HLB.The plant material used in this project consisted of a wide array of rootstocks grown as seedlings or as grafted trees in the greenhouse or in the natural field environment. Metabolite detection and identification was conducted by a commercial service at the West Coast Metabolomics Center, UC Davis, CA, using untargeted gas chromatography and mass spectrometry. Greenhouse and field experiments, sample extraction, and data analyses were conducted by PIs Bowman (USDA) and Albrecht (UF). Accomplishments: Objective 1. For proof-of-concept, four standard rootstocks (Cleopatra, Swingle, Ridge, and sour orange) were included in preliminary and all other studies. Many hundred metabolites were detected in leaves and roots of rootstock seedlings. The majority were present in higher concentrations in the leaves than in the roots, indicating that leaves are metabolically more active. Only one third of all detected metabolites were of known chemical identity; most were unknown. Metabolite profiles corresponded well with the taxonomic relationship of rootstocks. In the roots, several metabolites were identified with differences in concentrations that correspond well with field tolerance of rootstocks to unfavorable soil conditions. Several metabolites were also identified that may be associated with rootstock tolerance to different pathogens and diseases. Results from this study have been submitted for publication. In addition to the four standard rootstocks, metabolite profiles of seedlings of other greenhouse-grown rootstock cultivars (Carrizo, US-802, US-812, US-896, US-897, US-942, US-1516) were investigated. This large set of data is still being analyzed.Objective 2. The results from this objective were similar to those from objective 1. Many hundred known and unknown metabolites were identified in grafted greenhouse and field grown citrus trees. Large differences were detected based on rootstock variety that corresponded well with taxonomic relationships. Many root metabolites that varied most significantly among rootstock varieties were the same, whether plants were young and grown in the greenhouse, or older and grown in the field. This proves the consistency of the methodology used in our project, and broad applicability of the approach. The comparison of two different scion cultivars showed large metabolic differences and indicated a considerable influence of the scion on the rootstock. But importantly, metabolite profiles of leaves from the same scion differed based on the rootstock on which it was grafted. This clearly demonstrates that rootstocks can influence the scion metabolically. We also found that many of the root metabolites that showed the largest differences among rootstocks did not show the same differences in the leaves of a grafted scion. This indicates that some metabolites are restricted to the tissue in which they are synthesized or are unable to move across the graft union. Results from a part of this study have been submitted for publication.Objective 3. For this objective we conducted a greenhouse experiment with Valencia trees on 10 different rootstocks (the same mentioned above) and compared the metabolite profiles of leaves and roots from HLB infected and non-infected plants. We detected nearly 600 known and unknown metabolites. In general, concentrations of most leaf metabolites were reduced in infected plants compared with healthy plants. The reverse was observed in roots, where most were increased by infections. The type and extend of metabolic changes were clearly influenced by rootstock. This large set of data is still being analyzed.In conclusion, rootstocks can be differentiated by their metabolic composition and influence on the scion. Several of the most discriminating metabolites may be suitable markers for stress and disease tolerance. Metabolites with the most dramatic differences and changes among rootstocks in healthy and HLB infected plants were in the group of chemically unidentified compounds. This indicates that they are biologically highly significant and provides future opportunities for discovery of new molecules.
Excellent progress was made under this project to develop promising new citrus rootstocks, and three new SuperSour rootstocks are scheduled for release during 2018. Among the rootstocks currently available for commercial use in large quantities, our previously released rootstock US-942 continues as consistently the overall best performer in field trials. Other rootstocks recently released, including US-1516, appear promising, but nursery material and field performance data is still limited. Field data is being collected from numerous established USDA rootstock field trials, to help determine which of a large group of SuperSour hybrid rootstock selections will best provide outstanding tree performance in the face of HLB for the major citrus producing areas of Florida. Results from existing replicated field trials suggest that some of the new USDA SuperSour hybrid rootstocks can be expected to make sweet orange trees that produce more than twice as much good quality fruit as trees on standard sour orange or Swingle rootstocks. Additional rootstock field trials to be planted in 2018, as well as focused rootstock development work under a new NIFA-SCRI grant, will further expand the new information that can help Florida growers make good rootstock choices in the future. Numerous presentations by Dr. Bowman to Florida citrus growers during 2015-18 have helped to transmit updated knowledge about relative rootstock performance, information about new rootstocks under development, and future plans to growers.The overall goal of the project was to develop and release new hybrid rootstocks with outstanding field performance and field tolerance to HLB. The specific objectives with major accomplishments were as follows:Objective 1. Collect tree health, size, yield, and fruit quality data from existing rootstock trials. Rootstock trials established prior to the initiation of this project were the focus of field trial data collection during 2015-2018. Eight field trials planted between 2011 and 2014 received special emphasis for performance information that was collected, and contained standard rootstocks as well as new SuperSour hybrids. Summaries of information from several of these trials were presented at grower meetings and field days, and are being prepared for publication.Objective 2. Test and select the most promising new rootstocks from SuperSour and other progeny groups based on laboratory studies, greenhouse testing, and short term field evaluations. As indicated in previous reports, work conducted under this objective was minimized at the direction of CRDF at the time the grant was awarded. Resources that would have been devoted to this objective were re-allocated to the other four objectives instead.Objective 3. Propagate trees and plant additional rootstock field trials. Nine new replicated rootstock field trials were field planted from 2015-18 with trees propagated in the USDA rootstock breeding nursery in Ft. Pierce. Trees for eight additional field trials are growing in the USDA rootstock breeding nursery, and will be planted 2018-19. These trials include standard rootstocks, as well as many new SuperSour hybrid rootstocks.Objective 4. Release new rootstock cultivars. The rootstock US-1516 was released in 2015, based on outstanding field performance with HLB. The three rootstocks US SuperSour 1, US SuperSour 2, and US SuperSour 3 are being released in 2018, based on outstanding field performance with HLB. Objective 5. Present information about new and existing rootstocks to Florida growers at meetings and field days, and through grower-targeted and extension publications. Dr. Bowman made presentations to large groups of Florida growers during at least 14 different shows, meetings, or field days from 2016-18. Dr. Bowman distributed summary information about field performance of new rootstocks to large groups of Florida growers in at least five separate emails. Dr. Bowman authored four separate refereed publications from 2015-18, presenting detailed performance information on new rootstocks in multiyear replicated field trials. Dr. Bowman was a co-author of the Florida Citrus Rootstock Selection Guide, 3rd edition, EDIS publication SP248/HS1260.
The overall goal of the project was to test three complementary molecular genetic approaches for canker resistance to determine which can contribute to a stacked resistance approach. Objective 1: Assess canker resistance conferred by the PAMP receptors EFR and XA21 Transgenic Duncan grapefruit and sweet orange lines carrying either EFR alone or EFR plus an XA21-EFR chimera were tested for canker resistance in the greenhouse. The two most promising Duncan grapefruit lines carrying EFR were selected for further testing in the field in collaboration with Dr. Ed Stover at the USDA ARS. Some new Duncan grapefruit transformants carrying EFR, XA21, or both genes have been produced at the Core Citrus Transformation Facility at UF Lake Alfred, and any that survive will be analyzed for canker resistance. Objective 2: Introduction of the pepper Bs2 disease resistance gene into citrus Work on these constructs has been discontinued due to negative effects of the constructs in citrus. Objective 3: Development of genome editing technologies (Cas9/CRISPR) for citrus improvement Our gene editing target is the citrus homolog of Bs5 of pepper. The recessive bs5 resistance allele contains a deletion of two conserved leucines. The citrus Bs5 homologs were sequenced from both Carrizo citrange and Duncan grapefruit, and conserved CRISPR targets were identified. For proof of concept, we chose to mutate the native citrus Bs5 alleles while simultaneously introducing the effective resistance allele as a transgene, rather than to attempt precise gene editing. Two editing constructs were created, one targeting the two conserved leucines, and one targeting two sites in the second exon to create a deletion in Bs5. The constructs were transformed into Carrizo citrange, and the Bs5 gene was sequenced from twenty-six transformants. We have identified two plants with mutations knocking out both alleles of the native Bs5 gene and several other candidate plants that may also have a loss of function of both alleles. Promising lines will be propagated and shipped to Dr. Jeff Jones’ lab at UF Gainesville for canker testing.
The overall goal of the project was to test three complementary molecular genetic approaches for canker resistance to determine which can contribute to a stacked resistance approach. Objective 1: Assess canker resistance conferred by the PAMP receptors EFR and XA21 Transgenic Duncan grapefruit and sweet orange lines carrying either EFR alone or EFR plus an XA21-EFR chimera were tested for canker resistance in the greenhouse. The two most promising Duncan grapefruit lines carrying EFR were selected for further testing in the field in collaboration with Dr. Ed Stover at the USDA ARS. Some new Duncan grapefruit transformants carrying EFR, XA21, or both genes have been produced at the Core Citrus Transformation Facility at UF Lake Alfred, and any that survive will be analyzed for canker resistance. Objective 2: Introduction of the pepper Bs2 disease resistance gene into citrus Work on these constructs has been discontinued due to negative effects of the constructs in citrus. Objective 3: Development of genome editing technologies (Cas9/CRISPR) for citrus improvement Our gene editing target is the citrus homolog of Bs5 of pepper. The recessive bs5 resistance allele contains a deletion of two conserved leucines. The citrus Bs5 homologs were sequenced from both Carrizo citrange and Duncan grapefruit, and conserved CRISPR targets were identified. For proof of concept, we chose to mutate the native citrus Bs5 alleles while simultaneously introducing the effective resistance allele as a transgene, rather than to attempt precise gene editing. Two editing constructs were created, one targeting the two conserved leucines, and one targeting two sites in the second exon to create a deletion in Bs5. The constructs were transformed into Carrizo citrange, and the Bs5 gene was sequenced from twenty-six transformants. We have identified two plants with mutations knocking out both alleles of the native Bs5 gene and several other candidate plants that may also have a loss of function of both alleles. Promising lines will be propagated and shipped to Dr. Jeff Jones’ lab at UF Gainesville for canker testing.
Objective 1. (Greenhouse experiment): Leaf nutrition results: There was no significant differences in the N,P,K, Mg and Ca levels in any of the rootstock / fertilizer combinations. Plants growing in the Harrell s nursery mix treatment had boron levels 5-6 folder higher in all rootstocks when compared to Swingle. Additionally, Mn levels were double in all rootstocks when compared to Swingle. When both Mn and Bo was applied simultaneously to supplement the Harrell s nursery mix, their levels were doubled in all rootstocks except the GFT+50-7 and X639. Harrell’s St Helena Mix as well as the Florikote IFAS blend resulted in enhanced levels of Bo and Mn in all rootstocks. These levels were twice that observed in plants growing in the Harrell s nursery mix. Most of the rootstocks stick grafted with HLB + budwood were infected with no significant differences in the rate of infection between the different treatments / rootstock combinations. However, WGFT+ trifoliate orange 50-7 rootstocks on the St Helena mix exhibited higher cT values (lower bacterial titers) when compared to the other rootstocks. Additionally, this rootstock also performed better across the board (generally higher cT values) in all the other fertilizer combinations (note that this rootstock is also showing good tolerance in several field plantings, and is being considered for commercial release). Most trees, even though infected, continue to grow exceptionally well, except for treatment #3, where trees are not showing much growth and somewhat yellow. Objective 3: Report for April-June 2018. To evaluate the effect of complete, balanced and constant nutrition on HLB-affected mature trees (composition, delivery and economics).In March fruit were harvested from both locations, Arcadia and Fort Meade. The initial analysis shows that there has been significant improvement in yield and fruit quality with the use of CRF and Tiger micronutrients (20%-50% higher rates on Manganese, Boron, and Iron) over the control treatment (IFAS recommendation). The yields at Arcadia site were compromised due to Hurricane Irma therefore, the two sites show some amount of variability in the data. Overall, the yields seems to have improved with use of CRF and tiger micronutrients in combination. With the collection of year 3 data on yield and quality, we will have more confidence in results and be able to develop some guidelines for growers. The yield data was presented at OJ Break on May 15th, 2018 and at SWFREC on May 16th, 2018. Detailed results of year 1and 2 of this experiment will be presented at Citrus Expo 2018. The third year application of fertilizer has been initiated, March and June applications were completed. The year-3 tree health data has been collected. Everything is now going according to schedule. Objective 5. (funded by Lee Groves, using donated fertilizer products): Alligator Vernia/Rough Lemon Enhanced Nutrition Experiment Treatments: 6 tree plots (randomized), 2 plots per treatment treatments 2 times per year. Positive results showing a therapeutic affect from overdoses of manganese against HLB is being presented at the annual ASHS meeting in Washington DC (manuscript also being prepared.
Objective 1. (Greenhouse experiment): Leaf nutrition results: There was no significant differences in the N,P,K, Mg and Ca levels in any of the rootstock / fertilizer combinations. Plants growing in the Harrell s nursery mix treatment had boron levels 5-6 folder higher in all rootstocks when compared to Swingle. Additionally, Mn levels were double in all rootstocks when compared to Swingle. When both Mn and Bo was applied simultaneously to supplement the Harrell s nursery mix, their levels were doubled in all rootstocks except the GFT+50-7 and X639. Harrell’s St Helena Mix as well as the Florikote IFAS blend resulted in enhanced levels of Bo and Mn in all rootstocks. These levels were twice that observed in plants growing in the Harrell s nursery mix. Most of the rootstocks stick grafted with HLB + budwood were infected with no significant differences in the rate of infection between the different treatments / rootstock combinations. However, WGFT+ trifoliate orange 50-7 rootstocks on the St Helena mix exhibited higher cT values (lower bacterial titers) when compared to the other rootstocks. Additionally, this rootstock also performed better across the board (generally higher cT values) in all the other fertilizer combinations (note that this rootstock is also showing good tolerance in several field plantings, and is being considered for commercial release). Most trees, even though infected, continue to grow exceptionally well, except for treatment #3, where trees are not showing much growth and somewhat yellow. Objective 3: Report for April-June 2018. To evaluate the effect of complete, balanced and constant nutrition on HLB-affected mature trees (composition, delivery and economics).In March fruit were harvested from both locations, Arcadia and Fort Meade. The initial analysis shows that there has been significant improvement in yield and fruit quality with the use of CRF and Tiger micronutrients (20%-50% higher rates on Manganese, Boron, and Iron) over the control treatment (IFAS recommendation). The yields at Arcadia site were compromised due to Hurricane Irma therefore, the two sites show some amount of variability in the data. Overall, the yields seems to have improved with use of CRF and tiger micronutrients in combination. With the collection of year 3 data on yield and quality, we will have more confidence in results and be able to develop some guidelines for growers. The yield data was presented at OJ Break on May 15th, 2018 and at SWFREC on May 16th, 2018. Detailed results of year 1and 2 of this experiment will be presented at Citrus Expo 2018. The third year application of fertilizer has been initiated, March and June applications were completed. The year-3 tree health data has been collected. Everything is now going according to schedule. Objective 5. (funded by Lee Groves, using donated fertilizer products): Alligator Vernia/Rough Lemon Enhanced Nutrition Experiment Treatments: 6 tree plots (randomized), 2 plots per treatment treatments 2 times per year. Positive results showing a therapeutic affect from overdoses of manganese against HLB is being presented at the annual ASHS meeting in Washington DC (manuscript also being prepared.
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