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.
1) Trees have been in the ground for 4 years in a trial of 50 selections and cultivars on US-802 following no-choice ACP inoculation and several months in an ACP house. Standard growth measurements and disease ratings were initiated in July 2014 and will continue on a semi-annual basis. HLB is now widespread and the trees looking healthiest include a full sib of our best mandarin selection (unfortunately not in this trial). Several of our best grapefruit-like hybrids looked good last year but are now declining. The one true grapefruit is the least healthy selection in the trial. 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. These trees are cropping this year, and fruit will be used in a complementary project exploring synthesis of orange-like juice from HLB-tolerant types. The 5 selections with the highest percent of full canopy volume ratings had 46% to 57% increase in canopy volume over the last year while the 7 with the lowest percent of full canopy volume ranged from 0 to 23% increase. Several selections that had looked good last year suddenly dropped to a very low canopy increase, and it appears this may be a relatively early indicator of HLB tolerance. 2) In June 2015 a field planting was established of: seedling trees of 133 Fortune x Fairchild hybrids from an earlier mapping study, seedlings of 27 Ponkan-like accessions, budded trees of 10 advanced ARS selections that are predominately mandarin, and budded trees of Fortune, Fairchild and Valencia. Data collection is underway. A genotyping project was initiated with 688 seedlings trees of Fortune x Fairchild and 235 Ponkan type seedlings. This samples will be collected at picos farm and determined with zygosity KASP markers designed by Roose group at UCR. Monoembryonic hybids will be identified and used for HLB-tolerance mapping analysis as part of a new NIFA grant. 3) Replicated trials in multiple locations are established of our best sweet-orange-like cultivars and mandarin-types. Volatiles from sweet-orange-like hybrids are so similar to sweet orange that likely can be legally named sweet orange. 4) RNA-seq compared transcriptome responses in HLB moderately 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. In addition, several genes were similarly regulated by CLas-flg22 and CLas treatments. Genes identified were recently published and are valuable for studying HLB tolerance mechanisms and potential for screening for HLB-tolerant citrus using CLas-flg22 as a pathogen proxy. A population of mandarin hybrids from a 2015 cross was used to test for CLas-flg22 responsiveness. Leaf injections of CLas-flg22 and Xcc-flg22 were conduct on about 60 seedlings, and 8 randomly selected plants were subject to expression analysis of marker genes identified by transcriptomics previously. CLas-flg22 injection induced marker genes in several plants, although the number of markers induced was inconsistent. The analysis of the remaining 50 plus plants is underway. 5) A group of CLas effector (28) was predicted using bioinformatics tools. The study of transcriptional level of these effectors in infected citrus samples was initiated. Many of the effectors were successfully detected by RT-qPCR in citrus RNA, suggesting their roles in pathogen virulence and host response modulation. The number of effectors detected has a positive linear relationship with bacterial titer, but less association with infection stage within the first week of CLas inoculation. The effectors expression were compared in citrus hosts with various levels of HLB tolerance, including citron, Duncan grapefruit, Cleopatra mandarin, Pomeroy trifoliate, Carrizo citrange and Washington navel orange, and indicated relatively high expression of CLIBASIA_03695, CLIBASIA_00460, CLIBASIA_00420, CLIBASIA_04580, CLIBASIA_05320, CLIBASIA_04425, CLIBASIA_00525 and CLIBASIA_05315 in either a host-specific or -nonspecific manners. In another study, the expression of effectors was compared between leaves and roots of citrus that has been Ca. L. asiaticus-infected for more than a year. Results indicated relative high expression of CLIBASIA_03875, CLIBASIA_04800 and CLIBASIA_05640 in all leaf and certain root tissues of citron, Duncan and Cleopatra. Some of the highly expressed effector candidates will be further tested for the correlation with host tolerance/susceptibility, and the potential used as biomarkers for resistance screening of breeding materials. 6) Seedlings with a range of pedigree contributions from Microcitrus have been received in a collaboration with M. Smith, Queensland Aus. citrus breeder, are being grown, and will be planted next quarter for field testing of HLB resistance. 7) Our putative chimeras have not proven to be successful. We identified a chimera (Satsuma and Poncirus) from the Citrus genebank, arranged its importation, and we finally got permission to accept this material and maintain it in a quarantine death house. Cuttings of the chimera and each separate component (Owari and Poncirus) were rooted and were challenged by hot ACP feeding and data will be collected in the next quarters.
1) Assessed use of isolated leaf inoculation and small plant destructive sampling: Isolated leaf inoculations with ACP do not readily distinguish between resistant and susceptible citrus selections, but prove useful in assessing CLas-killing transgenics. Within a week, such assays have shown marked reductions in CLas in leaves and in ACP. Small plant destructive inoculation assays now permit us to distinguish between susceptible Valencia and resistant Carrizo after 12 weeks. 2) Data collection continues on transgenics. Transgenic plants expressing a modified thionin (Mthionin) are promising for HLB resistance and they have been extensively propagated for testing in the greenhouse and the field. Non-grafted Mthionin are in the field. A propagated group of 200 Mthionin transgenic Carrizo are potted up and grafted with wild type Hamlin to be planted in field next year. More Mthionin Carrizo are propagated and will be used to graft with Valencia and Ray ruby scions for field planting. In greenhouse a group of grafted plants with wt/transgenic and transgenic/wt rookstock and scion combinations were created and subjected to ACP inoculation. The 3-month post inoculation samples were recently collected for titer analysis. About 100 small rooted cuttings were grafted with CLas+ rough lemon for identification of the most resistant lines. The 3-month sampling was just completed. Tissue specific constructs of the very promising Mthionin gene have been developed, with 10 Carrizo confirmed. The root specific variant was transformed only into Carrizo, and 17 plantlets were regenerated. A CTV-expression vector for Mthionin was created by Dr. W. Dawson, has been verified, and will be graft inoculated into a range of plants (both infected and healthy for challenging) next quarter. 3) Transgenics expressing LuxI from Agrobacterium, and an array of ScFv transgenics (more in 4 below) have been propagated and are in replicated testing. New chimeral peptides (citrus only genes) have been used to produce many Carrizo plants and shoots of Hamlin, Valencia and Ray Ruby. A total of 35 lines of Carrizo with citrus thionin V2-LBP construct, and 20 lines with citrus thionin V1-LBP construct have been generated. A total of 18 independent Carrizo lines, each expressing citrus thionin-EDS and citrus thionin D2A21 chimeras respectively, were produced with confirmation of high level transgene expressions. A group of Carrizo transgenics expressing two variants of citrus thionin chimera (code 73 and 74) are being tested side-by-side with modified thionin transgenics. These plants were inoculated by ACP no-choice inoculation and reached 3 month mark for the first disease test in July. Using the detached leaf ACP-inoculation assay, it was shown that transgenic Carrizo expressing citrus thionin V1-LBP chimera has significantly less CLas titer after 1 week of ACP feeding than the wild type controls. Psyllid fed on detached leaves of the citrus thionin V1-LBP chimera Carrizo had lower CLas titer in bodies compared to the ones fed on the wide type leaves.. Comparison among individual lines from modified thionin transgenics were conducted using detached leaves and discovered variations in antimicrobial ability between them, suggesting this protocol allows us to screen better performing lines for further tests. His-6 affinity tagged variants of citrus thionin-BPI/LBP expressing constructs have been created with C-terminal and N-terminal thionin orientations. These constructs have been transformed into benthamiana for efficient in plantae production and purification of protein for use in detached tissue assays with multiple lines for each construct confirmed as transgenic and currently undergoing analysis for expression levels. 4) Antibodies (ScFv) to the CLas invA and TolC genes, and constructs to overproduce them, were created by John Hartung under an earlier CRDF project. Two representative constructs, one targeting each gene, have been challenged by CLas + ACP. At all time points measured after inoculation, transgenic plants are showing consistent and statistically significant decreases in bacterial titer (as much as 400x) when measured by qPCR and a much higher incidence of plants with no measurable bacterial DNA amplification. Additional plants representing 21 independent events from all 7 constructs have been replicated as rooted cuttings for ACP challenge of whole plants. A second round of ACP inoculations has been conducted on 150 plants replicated from twelve independent transformation events representing three different ScFv constructs. Additional lines will be inoculated once sufficient mature transgenic material becomes available. Thirty ScFv-transgenic Carrizo plants (10 each from the 3 best performing constructs in greenhouse studies) are being grafted with Ray Ruby scions in parallel with non-transgenic controls for initial field studies. Approximately 120 additional rooted cuttings for a follow up trial are being propagated. 5) Arabidopsis DMR6 (downy mildew resistance 6)-like genes were previously shown to be downregulated in more tolerant Jackson compared to susceptible Marsh grapefruit. DMR6 acts as a suppressor of plant immunity and it is upregulated during pathogen infection. In a gene expression survey of DMR6 orthologs in Hamlin , Clementine , Carrizo , rough lemon, sour orange and citron, expression levels were significantly higher in all CLas-infected trees compared with healthy trees in each citrus genotype. We developed 2 RNA silencing (hairpinRNA) constructs targeting citrus DMR6 and DLO1 respectively. Citrus DMR6 is silenced in hairpin transgenic plants and with an average silencing efficiency of 41.4%. DMR6 silenced Carrizo plants (28 independent so far) exhibit moderate to strong activation of plant defense response genes. Determination of silencing efficiency of DLO1 in transgenic plants (20 plants so far) are ongoing. Carrizo plants carrying these constructs with multiple events each were transferred into larger pots to stimulate growth in early 2018 and subsequent propagations. CRISPR constructs with guide RNA targeting DMR6 and DLO1 were made and transformed into Carrizo, generating 3 and 1 confirmed transgenic line respectively. Mutation at the target locus has been confirmed in the first of these plants after heat shock treatment. The remaining plants are being sequenced to detect mutations and new transformations into Hamlin are in preparation. 6) Budwood from our best performing Mthionin, citrus gene chimeras and ScFv antigen binding fragment expressing transgenics have been sent to DPI for cleanup and then broad field testing.
1) Trees have been in the ground for 4 years in a trial of 50 selections and cultivars on US-802 following no-choice ACP inoculation and several months in an ACP house. Standard growth measurements and disease ratings were initiated in July 2014 and will continue on a semi-annual basis. HLB is now widespread and the trees looking healthiest include a full sib of our best mandarin selection (unfortunately not in this trial). Several of our best grapefruit-like hybrids looked good last year but are now declining. The one true grapefruit is the least healthy selection in the trial. 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. These trees are cropping this year, and fruit will be used in a complementary project exploring synthesis of orange-like juice from HLB-tolerant types. The 5 selections with the highest percent of full canopy volume ratings had 46% to 57% increase in canopy volume over the last year while the 7 with the lowest percent of full canopy volume ranged from 0 to 23% increase. Several selections that had looked good last year suddenly dropped to a very low canopy increase, and it appears this may be a relatively early indicator of HLB tolerance. 2) In June 2015 a field planting was established of: seedling trees of 133 Fortune x Fairchild hybrids from an earlier mapping study, seedlings of 27 Ponkan-like accessions, budded trees of 10 advanced ARS selections that are predominately mandarin, and budded trees of Fortune, Fairchild and Valencia. Data collection is underway. A genotyping project was initiated with 688 seedlings trees of Fortune x Fairchild and 235 Ponkan type seedlings. This samples will be collected at picos farm and determined with zygosity KASP markers designed by Roose group at UCR. Monoembryonic hybids will be identified and used for HLB-tolerance mapping analysis as part of a new NIFA grant. 3) Replicated trials in multiple locations are established of our best sweet-orange-like cultivars and mandarin-types. Volatiles from sweet-orange-like hybrids are so similar to sweet orange that likely can be legally named sweet orange. 4) RNA-seq compared transcriptome responses in HLB moderately 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. In addition, several genes were similarly regulated by CLas-flg22 and CLas treatments. Genes identified were recently published and are valuable for studying HLB tolerance mechanisms and potential for screening for HLB-tolerant citrus using CLas-flg22 as a pathogen proxy. A population of mandarin hybrids from a 2015 cross was used to test for CLas-flg22 responsiveness. Leaf injections of CLas-flg22 and Xcc-flg22 were conduct on about 60 seedlings, and 8 randomly selected plants were subject to expression analysis of marker genes identified by transcriptomics previously. CLas-flg22 injection induced marker genes in several plants, although the number of markers induced was inconsistent. The analysis of the remaining 50 plus plants is underway. 5) A group of CLas effector (28) was predicted using bioinformatics tools. The study of transcriptional level of these effectors in infected citrus samples was initiated. Many of the effectors were successfully detected by RT-qPCR in citrus RNA, suggesting their roles in pathogen virulence and host response modulation. The number of effectors detected has a positive linear relationship with bacterial titer, but less association with infection stage within the first week of CLas inoculation. The effectors expression were compared in citrus hosts with various levels of HLB tolerance, including citron, Duncan grapefruit, Cleopatra mandarin, Pomeroy trifoliate, Carrizo citrange and Washington navel orange, and indicated relatively high expression of CLIBASIA_03695, CLIBASIA_00460, CLIBASIA_00420, CLIBASIA_04580, CLIBASIA_05320, CLIBASIA_04425, CLIBASIA_00525 and CLIBASIA_05315 in either a host-specific or -nonspecific manners. In another study, the expression of effectors was compared between leaves and roots of citrus that has been Ca. L. asiaticus-infected for more than a year. Results indicated relative high expression of CLIBASIA_03875, CLIBASIA_04800 and CLIBASIA_05640 in all leaf and certain root tissues of citron, Duncan and Cleopatra. Some of the highly expressed effector candidates will be further tested for the correlation with host tolerance/susceptibility, and the potential used as biomarkers for resistance screening of breeding materials. 6) Seedlings with a range of pedigree contributions from Microcitrus have been received in a collaboration with M. Smith, Queensland Aus. citrus breeder, are being grown, and will be planted next quarter for field testing of HLB resistance. 7) Our putative chimeras have not proven to be successful. We identified a chimera (Satsuma and Poncirus) from the Citrus genebank, arranged its importation, and we finally got permission to accept this material and maintain it in a quarantine death house. Cuttings of the chimera and each separate component (Owari and Poncirus) were rooted and were challenged by hot ACP feeding and data will be collected in the next quarters.
1) Assessed use of isolated leaf inoculation and small plant destructive sampling: Isolated leaf inoculations with ACP do not readily distinguish between resistant and susceptible citrus selections, but prove useful in assessing CLas-killing transgenics. Within a week, such assays have shown marked reductions in CLas in leaves and in ACP. Small plant destructive inoculation assays now permit us to distinguish between susceptible Valencia and resistant Carrizo after 12 weeks. 2) Data collection continues on transgenics. Transgenic plants expressing a modified thionin (Mthionin) are promising for HLB resistance and they have been extensively propagated for testing in the greenhouse and the field. Non-grafted Mthionin are in the field. A propagated group of 200 Mthionin transgenic Carrizo are potted up and grafted with wild type Hamlin to be planted in field next year. More Mthionin Carrizo are propagated and will be used to graft with Valencia and Ray ruby scions for field planting. In greenhouse a group of grafted plants with wt/transgenic and transgenic/wt rookstock and scion combinations were created and subjected to ACP inoculation. The 3-month post inoculation samples were recently collected for titer analysis. About 100 small rooted cuttings were grafted with CLas+ rough lemon for identification of the most resistant lines. The 3-month sampling was just completed. Tissue specific constructs of the very promising Mthionin gene have been developed, with 10 Carrizo confirmed. The root specific variant was transformed only into Carrizo, and 17 plantlets were regenerated. A CTV-expression vector for Mthionin was created by Dr. W. Dawson, has been verified, and will be graft inoculated into a range of plants (both infected and healthy for challenging) next quarter. 3) Transgenics expressing LuxI from Agrobacterium, and an array of ScFv transgenics (more in 4 below) have been propagated and are in replicated testing. New chimeral peptides (citrus only genes) have been used to produce many Carrizo plants and shoots of Hamlin, Valencia and Ray Ruby. A total of 35 lines of Carrizo with citrus thionin V2-LBP construct, and 20 lines with citrus thionin V1-LBP construct have been generated. A total of 18 independent Carrizo lines, each expressing citrus thionin-EDS and citrus thionin D2A21 chimeras respectively, were produced with confirmation of high level transgene expressions. A group of Carrizo transgenics expressing two variants of citrus thionin chimera (code 73 and 74) are being tested side-by-side with modified thionin transgenics. These plants were inoculated by ACP no-choice inoculation and reached 3 month mark for the first disease test in July. Using the detached leaf ACP-inoculation assay, it was shown that transgenic Carrizo expressing citrus thionin V1-LBP chimera has significantly less CLas titer after 1 week of ACP feeding than the wild type controls. Psyllid fed on detached leaves of the citrus thionin V1-LBP chimera Carrizo had lower CLas titer in bodies compared to the ones fed on the wide type leaves.. Comparison among individual lines from modified thionin transgenics were conducted using detached leaves and discovered variations in antimicrobial ability between them, suggesting this protocol allows us to screen better performing lines for further tests. His-6 affinity tagged variants of citrus thionin-BPI/LBP expressing constructs have been created with C-terminal and N-terminal thionin orientations. These constructs have been transformed into benthamiana for efficient in plantae production and purification of protein for use in detached tissue assays with multiple lines for each construct confirmed as transgenic and currently undergoing analysis for expression levels. 4) Antibodies (ScFv) to the CLas invA and TolC genes, and constructs to overproduce them, were created by John Hartung under an earlier CRDF project. Two representative constructs, one targeting each gene, have been challenged by CLas + ACP. At all time points measured after inoculation, transgenic plants are showing consistent and statistically significant decreases in bacterial titer (as much as 400x) when measured by qPCR and a much higher incidence of plants with no measurable bacterial DNA amplification. Additional plants representing 21 independent events from all 7 constructs have been replicated as rooted cuttings for ACP challenge of whole plants. A second round of ACP inoculations has been conducted on 150 plants replicated from twelve independent transformation events representing three different ScFv constructs. Additional lines will be inoculated once sufficient mature transgenic material becomes available. Thirty ScFv-transgenic Carrizo plants (10 each from the 3 best performing constructs in greenhouse studies) are being grafted with Ray Ruby scions in parallel with non-transgenic controls for initial field studies. Approximately 120 additional rooted cuttings for a follow up trial are being propagated. 5) Arabidopsis DMR6 (downy mildew resistance 6)-like genes were previously shown to be downregulated in more tolerant Jackson compared to susceptible Marsh grapefruit. DMR6 acts as a suppressor of plant immunity and it is upregulated during pathogen infection. In a gene expression survey of DMR6 orthologs in Hamlin , Clementine , Carrizo , rough lemon, sour orange and citron, expression levels were significantly higher in all CLas-infected trees compared with healthy trees in each citrus genotype. We developed 2 RNA silencing (hairpinRNA) constructs targeting citrus DMR6 and DLO1 respectively. Citrus DMR6 is silenced in hairpin transgenic plants and with an average silencing efficiency of 41.4%. DMR6 silenced Carrizo plants (28 independent so far) exhibit moderate to strong activation of plant defense response genes. Determination of silencing efficiency of DLO1 in transgenic plants (20 plants so far) are ongoing. Carrizo plants carrying these constructs with multiple events each were transferred into larger pots to stimulate growth in early 2018 and subsequent propagations. CRISPR constructs with guide RNA targeting DMR6 and DLO1 were made and transformed into Carrizo, generating 3 and 1 confirmed transgenic line respectively. Mutation at the target locus has been confirmed in the first of these plants after heat shock treatment. The remaining plants are being sequenced to detect mutations and new transformations into Hamlin are in preparation. 6) Budwood from our best performing Mthionin, citrus gene chimeras and ScFv antigen binding fragment expressing transgenics have been sent to DPI for cleanup and then broad field testing.
The objectives of this proposal are: 1) conduct a field trial using the selected grapefruit seedlings to ensure the productivity of the trees in Florida where HLB is endemic; and 2) evaluate the quality of the fruit produced. Achievement of these goals will produce a more resistant/tolerant variety that could be available in the near future since its use would not require the regulatory approval. Based on two year’s graft-inoculation assays in greenhouse and the performance of individual seedlings in the field, four lines of the seedlings (with greater HLB resistance/tolerance) were selected for further propagation on three different rootstock (commercial sour orange, newly selected USDA-sour orange and 942). All these propagates (ca. 250 trees) are growing well in greenhouse, and expected to go to field next month. These propagates will be tested by qPCR before going to fields. The fruit quality (Brix, sucrose, glucose and fructose, soluble solids, pH, % TA and total ascorbic acid) of the four selected seedlings showed no significant difference from their maternal trees. New propagations of the selections from the the Scott grove seedlings, including 245 Red Ruby and 51 white Marsh plants on three different rootstock and 38 Red Ruby cuttings were planted in our research farm on Nov.15, 2017. Before going to field, samples were taken from these plants, and processed for qPCR detection. We will monitor the disease incidence and Las titers if there is an infection. Meanwhile we will go second round of selection and propagations on Scott seedlings, and then plant them in Scott grove for field evaluation. Because of the limitation of our greenhouse, and the new development of bud sport selection with several commercial grapefruit varieties such as Flame and Star Ruby. The propagation of the selected seedlings for Scott grove trial was delayed. However, another 80 trees generated from the cutting of the seedlings will be planted next week at USHRL Picos Research Farm for further evaluation. It is worth noting that some of the bud sport grapefruit plants showed less than 10% HLB affection rate after two years at USHRL Picos Farm with high disease pressure.
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