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.
Highest priority is being placed on advanced stage work to develop hybrid rootstocks that can be made available for release to growers in the next two years, especially selections from the SuperSour project. Based on outstanding performance in field trials, three Supersour rootstocks will be released in 2018, tentatively identified as US SuperSour 1, 2, and 3. I am working with FDACS-DPI to get clean plant material of these rootstocks to nurseries now, to ensure that trees will be available to growers soon after release. During the quarter, performance data was collected from established field trials, including crop size, fruit quality, tree size, health, and infection status by qPCR. New information on rootstocks and field performance was presented to growers at the Florida Citrus Show in January and the Peace River Packers Field Day on March 7. A summary of performance from one of the trials is given below. Trees in the USHRL nursery are being prepared for planting three additional field trials with the most promising SuperSour rootstocks later in 2018. Additional information is available on the USDA rootstock breeding project, on request.
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), and several of our best grapefruit-like hybrids. 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 or pummelo. It may take 2-3 more years to clearly distinguish tolerant material. These trees are cropping this year, though Irma dropped a lot of fruit, 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. The r2 for percent of full canopy volume vs. last year s % growth was 0.37 across the entire planting. 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 NIFA grant proposal was submitted and funded to mobilize tolerance for industry use, and this planting will contribute. It is a collaboration between ARS, UF and UC Riverside. 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 initial detection was achieved from 2 weeks to 10 weeks after inoculation in multiple citrus genotypes. A comparison between root and leaf CLas expression was carried out. Results indicated different deployment of effectors between above- and underground CLas infections. Using a detached leaf assay for ACP-mediated inoculation, we also detected the expression the effector as early as 6h after ACP exposure, and observed continuous expression of effectors by CLas from 6h to 7 days. In addition, expression patterns of effectors were different between HLB susceptible and tolerant/resistant citrus hosts, which was validated by testing, although only a few genotypes were tested. Correlation between effector expressione and host tolerance/susceptibility is underway analyzing citrus types with various genetic backgrounds. 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. Consistently detected effectors may have important roles in pathogen virulence. Citrus transformation for expression of CLas effectors will permit investigation of host modulation and discovery of binding targets in citrus. 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 soon 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) have been rooted and will be challenged by hot ACP feeding 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 are promising for HLB resistance and they have been extensively propagated for testing in the greenhouse and the field. 67 plants representing 13 independent events and wild types (4-5 replicates each) were transferred into larger pots and are getting ready for field planting and resistance evaluation. The remaining plants were used for bud-inoculation tests as transgenic root stock and/or scion. About 100 small rooted cuttings were grafted with CLas+ rough lemon for identification of the most resistant lines. Another 805 rooted cuttings were created at the end of 2017 from 18 events for future experimentations. Tissue specific constructs of the very promising Mthionin gene have been developed. The phloem-specific variant has been transformed into Carrizo and is currently being transformed into other citrus types including Hamlin and Ray Ruby. The root specific variant is being transformed only into Carrizo. 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. Using the detached leaf ACP-inoculation assay, it was shown than transgenic Carrizo expressing citrus thionin V1-LBP chimera have significantly less CLas titer after 1 week of ACP feeding than the wild type controls. 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 six and nine months post inoculation, transgenic plants are showing consistent and significant decreases in bacterial titer (300x) as 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. 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 6) Budwood from our best performing Mthionin transgenics and citrus gene chimeras have been sent to DPI for cleanup and then broad field testing.
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. No transformants were obtained for CSPR, and this construct has been abandoned. 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 transformants are currently being analyzed at UC Berkeley. To date, we have identified one plant 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. More plants remain to be analyzed. Any plants confirmed to have biallelic loss of function mutations in Bs5 plus the introduced bs5 resistance allele will be propagated and shipped to Dr. Jeff Jones’ lab at UF Gainesville for canker testing.
The driving force for this project (Hall-15-016) is the need to evaluate citrus germplasm for tolerance to HLB, including germplasm transformed to express proteins that might mitigate HLB, which requires citrus be inoculated with CLas. Citrus breeders at USDA-ARS-USHRL, Fort Pierce Florida continue producing germplasm that needs to be evaluated. The more rapidly germplasm can be evaluated, the sooner breeders can identify HLB-tolerant germplasm for the Florida citrus industry. The purpose of this project is to support a high-throughput facility to evaluate citrus germplasm for HLB resistance. This screening program supports citrus breeding and transformation efforts by Drs. Stover and Bowman. The original inoculation program called for individual plants to be caged with 20 infected psyllids for a two-week infestation, and then housed for six months in a greenhouse with an open infestation of infected psyllids. As indicated below, the open infestation step was abandoned. After the caged inoculation step, plants are moved into a psyllid-free greenhouse and evaluated for growth, HLB symptoms and CLas titer, and finally the plants are transplanted to the field where evaluations of resistance continue and additional inoculations by field psyllids occurs. CRDF funds for the inoculation program cover the costs associated with establishing and maintaining colonies of infected psyllids; equipment such as insect cages; PCR supplies for assays on psyllid and plant samples from infected colonies; and two GS-7 USDA technicians. A career technician is assigned ~50% to the program. USDA provides for the program two small air-conditioned greenhouses, two walk-in chambers, and a large conventional greenhouse. Currently 18 individual colonies of infected psyllids are maintained. Some of the individual colonies are maintained on CLas-infected lemon plants while others are maintained on CLas-infected citron plants. Update: As of March 15, 2018, a total of 11,888 plants have passed through inoculation process. A total of 326,295 psyllids from colonies of CLas-infected ACP have been used in no-choice inoculations. Not included in these counts of inoculated plants and psyllids used in inoculations are many plants inoculated over the past year to assess transmission rates, which has provided insight into the success of our inoculation methods and strategies for increasing success. We have abandoned the greenhouse open-infestation step because of continual problems mainly with invasive pests such as thrips, scales and parasitoids. Research indicated that the no-choice inoculation step should usually average around 74% effective and gets plants back to the breeders faster. The plants are subjected to further inoculations in the field. The no-choice inoculation procedure was evaluated monthly for 12 months, and success in getting seedlings infected was evaluated six months after each monthly infestation. The results indicated a 74% average success rate in getting seedlings infected when flush (immature leaves) was present, with success ranging from 40 to 100%. Success was significantly related to how many ACP on a seedling tested positive for CLas, thus greater success rates would be expected using more than 20 ACP per seedling. Based on the results of the research, an infestation shorter than two weeks would be as effective, which would be advantageous for guarding against excessive ACP damage to seedlings if more than 20 per seedling were used. It remains possible that modifications to the no-choice inoculation procedure would increase success rates and reduce variability, for example 25 to 30 ACP per seedling for a 1 week period, perhaps with larger seedlings in larger cages.
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