Evaluation of existing cultivar/rootstock combinations for HLB resistance/tolerance has revealed potentially valuable tolerance but indicates that early HLB symptoms and earlier CLas titer are unrelated to growth and cropping. This suggests that tolerance verification requires 5+ years of field evaluation. Discovery and utilization of molecular markers may help to accelerate the selection of resistance/tolerance materials by screening young plants and avoiding pathogen inoculation. Plant defense elicited by pathogen-associated molecular patterns (PAMPs) is an important component of disease resistance. In previous studies, we have shown that canker resistance in citrus correlates with responsiveness to Xcc-flg22, the 22 amino acid active region from the flagellin of Xanthomonas citri ssp. citri, the causal agent of citrus canker (Shi, Febres et al. 2015). To study the association between HLB resistance/tolerance and citrus response to flg22 of HLB causal bacterium Candidatus Liberibacter asiaticus (CLas), we designed an RNA-seq experiment comparing transcriptome responses in HLB moderately tolerant Sun Chu Sha mandarin and susceptible Duncan grapefruit, to Xcc-flg22 and CLas-flg22 (project initiated with Gloria Moore at University of Florida). Recently data analysis revealed that a group of 86 genes were differentially regulated by CLas-flg22 in Sun Chu Sha mandarin but not by Duncan grapefruit and not associated with differential expression from Xcc-flg22, suggesting they may have roles in HLB tolerance. The 16 genes with highest differential expression were selected for RT-qPCR validation, and 10 genes were consistent with the RNA-seq results. To evaluate if these genes were associated with HLB tolerance, Cleopatra mandarin (similar to Sun Chu Sha ) and Duncan grapefruit plants were inoculated with CLas using psyllid infestation. CLas titer and gene expression were monitored biweekly for 10 weeks after inoculation. High bacterial titer (Ct<30) was observed at 2 weeks in Duncan but not until 6 weeks in Cleopatra . RT-qPCR results indicated that 5 of the studied genes were differentially expressed between the Cleopatra HLB-infected and the uninfected control plants, but not in Duncan . It is worth noting that the induction of these genes was detected before bacterial infection was detected. Although not fully annotated in the citrus genomic databases, the function of some of these genes include a peroxidase, gibberellin 2-beta-dioxygenase, glucan endo-1,3-beta-D-glucosidase and an F-box domain containing protein. We will continue to characterize the expression of these genes and their association to HLB tolerance in other citrus genotypes, and determine if they may serve as marker genes for selection of tolerant citrus material. Trees of seemingly HLB resistant/tolerant sweet orange-like hybrids and mandarin -types were propagated and replicated trials with standards and have been established in growers' fields, in cooperation with G. McCollum. A mapping population of Fortune x Fairchild has been planted (collaborating Roose and Gmitter) along with related material, in an effort to identify genes associated with tolerance in the mandarin phenotypic group. 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 in the spring for field testing of HLB resistance. In October 2013, 34 unique genotypes (USDA hybrids) some of which appear to have tolerance to HLB, and 16 standard commercial varieties were exposed to an ACP no-choice feeding trial and transferred to the field at Ft. Pierce FL. Standard growth measurements and disease ratings were initiated in July 2014 and will continue on a quarterly basis. HLB is now widespread and trees of more vigorous scion types are generally the healthiest at this point in time. At three years after planting, there continues to be great variation between selections and it may take 2-3 more years to clearly distinguish tolerant material.
Transgenic evaluation of antimicrobial peptides (AMPs) has so far has identified a modified thionin (Mthionin) that conferred resistance against HLB and canker when over-expressed in Carrizo plants (Hao et al, 2016 Front Plant Sci.). From the same transgenic Carrizo population, we screened another 37 positive plants by PCR validation of gene insertion and documented Mthionin transcript level of each plant by RT-qPCR. Multiple high, intermediate and low transgene expressing plants were selected and propagated using stem cuttings (a total of 250 cuttings). Once established these plants will be tested as rootstocks with sweet orange and grapefruit scions. compared with wild type Carrizo as a rootstock. An antibody has been developed for specific detection of Mthionin and a second to detect both Mthionin and citrus native thionin. Currently, we are evaluating binding capacity and sensitivity of these antibodies to antigen peptides and to full-length proteins (expressed by E.Coli). Later the antibodies will be used for access expression level and mobility of thionin protein between root stock and scion. Two additional Mthionin chimera genes (2nd generation of AMPs), Mthionin-D2A21 and Mthionin-lipid binding protein (LBP), have been used to produce transgenic Carrizo and Hamlin. So far we have obtained a number of transgenic Carrizo plants and made a propagation of about 100 for each transgene. These plants will be used for initial evaluation of HLB resistance through no choice ACP feeding inoculation and promising lines will be further propagated for field tests. The 3rd generation of AMPs includes two variants of modified citrus thionin genes combined with citrus LBP. Transformation of these two chimeras into Carrizo and Hamlin is underway. So far we have obtained a number of Carrizo regenerations. We are also in the process of the evaluation of HLB resistance in several Hamlin transgenic lines: we graft propagated transgenic Hamlin expressing Mthionin, D4E1 linker Mthionin, and LuxI, with wild type scion as the controls. These plants are established and inoculated by no choice feeding from 9 to 12 month ago. The bacterial titer tests showed that only very few plants were HLB positive, indicating the inoculation procedure was not successful. These plants were uniformly trimmed for new flush growth and will soon be re-inoculated with our improved protocols. The previous generations of transgenics remain in testing in the greenhouse and field.
Our productivity significantly decreased after the move to the packing house while the AC in our lab was being fixed. There was bacterial contamination of our cultures, presumably due to autoclave issues, unsealed windows, or poor temperature control. Bacterial and fungal clean tests of mature citrus budwood from the growth facility in LB & LW broth, respectively, showed that all mother trees were clean, even the new cultivar introductions (B770, OLL8, Vernia, red grapefruit). We anticipate having to do two Agrobacterium transformations per week to make-up for lost time. Needless to say our efficiencies declined because of the move. Due to the aforementioned difficulties, Agrobacterium transformations with disease resistant genes was slowed. Only ~10 transgenics were produced and one did not survive micrografting. The results of the remainder are pending. Ten immature Swingle transgenics for Dr. Wang and Vladimir were micrografted because Vladimir had micrografting issues in the packing house. One shoot died & the results for the others are pending. We have found a cDNA that dramatically increases our mature scion transformation efficiencies and we are investigating whether it will increase efficiencies in all cultivars. An invention disclosure entitled, A method to increase organogenesis and transformation efficiencies in recalcitrant woody species such as mature citrus, was submitted to UF/IFAS Tech Transfer. There have been significant unanticipated growth room repair & maintenance expenditures during this last quarter. The water softener had to be rebuilt & a new one must be purchased next fiscal year. Without the water softener, hard water clogs the humidifiers & a white residue is deposited on plants making them unsuitable for transformation. The AC ducts in both growth rooms must be replaced because of filth deposited inside them over the years. The sprayer broke down & was repaired again. In the future, a new sprayer must be purchased to alleviate costly repairs. Lights & ballasts are an ongoing significant expenditure. We had to replace some of the shelving in the laboratory because our shelving disappeared after the move. The use of the PMI selectable marker after biolistics of immature and mature citrus continues. Different sucrose concentrations are required for shoot regeneration in mature vs immature citrus. Similarly, more sucrose is necessary for shoot development in scion than rootstock. Mannose concentrations must be manipulated accordingly. A manuscript (25% funded CRDF & 75% funded CRB) was submitted to PCTOC & is in review: Y. Acanda, M. Canton, H. Wu and J. Zale (XXXX) Kanamycin selection in bioreactors allows visual selection of transgenic citrus shoots, PCTOC.
Our project is focused on the following objectives: 1. Development of rootstocks that can impart HLB tolerance/resistance to grafted scions. 2. Breeding of HLB tolerant/resistant processing sweet orange-like hybrids. 3. Screening of the UF-CREC germplasm collection to identify and validate HLB tolerant or resistant selections. 4. Advanced field trials, release and commercialization of promising HLB tolerant/resistant scion and rootstock cultivars. DPI Parent Tree Entries: Three gauntlet rootstock selections, plus two others already showing evidence of HLB tolerance in field trials. Several scions selections, submitted primarily because of high levels of tolerance to HLB in the field, including Cybrid Dancy (reduced seed); 1 Red pummelo, 1 nearly seedless pink pummelo, and seedless Pummelette ; 3 seedless mandarins; the so-called McTeer Murcott (low seeded and showing HLB tolerance under heavy field pressure); a late maturing true sweet orange exhibiting HLB tolerance in multiple replicates; two HLB tolerant sweet orange-like hybrids; one seedy but extremely high quality mandarin. Gauntlet activities: Several new candidates have been entered into the program, grafted with hot budsticks and their tops propagated. Seventy five candidates were moved from the greenhouse to a psyllid hot house, and another 145 trees were planted in Picos Farm for final field screening, in collaboration with USDA researchers. Transgenic plantings: 198 sweet orange trees and 27 W. Murcott trees, containing a total of 7 different constructs for possible resistance to HLB, were also planted at Picos Farm in collaboration with USDA researchers. Field trials: Meetings were held with our collaborators from Lykes and Cutrale Citrus to review plans and activities in large scale field trials with these companies. Replacement trees were replanted, and seeds for a large trial with Hamlin orange were collected and transferred to the relevant nurseries. Other activities: Meetings were held with CRDF staff to facilitate communications and mutual understanding of our current research agenda. Several interactions took place of our team with recently hired Dr. Catherine Hatcher of CRDF. We have made several field visits to some of our locations in an effort to help her understand the nature and landscape of our citrus breeding program. We feel we have established a good working relationship and anticipate that this will facilitate the more rapid deployment of potential genetic solutions to HLB in the industry. Our field research manager, Dr. Paul Ling, resigned during this time frame and we have begun a search for his replacement. Several meetings and teleconferences were held with collaborators from the USDA, UCR, DPI, and Rucks Nursery to develop and implement plans for two MAC projects aiming to plant several large scale trials in Florida of tolerant rootstock and scion cultivars.
Objective 1. (Greenhouse experiment): seedlings of the required rootstocks: x639, Swingle, WGFT+50-7, UFR-3 and UFR-15 potted in round citripots were trained to a single stem for subsequent stick grafting (in our HLB approved air-conditioned greenhouse). Liners are now stick-grafting size, grafting will begin in February, when conditions support a good graft-take. Objective III: To evaluate the effect of complete, balanced and constant nutrition on HLB-affected mature trees (composition, delivery and economics). All the pretreatment and mid-year data on tree health, canopy volume, leaf nutrients analysis has been collected and is currently being analyzed. We have applied the fertilizer for year 1 at both the locations. The trees have received full dosage of fertilizer for macro and micronutrients. Data collection on fruit drop has started at both locations. Next fertilizer application will be made in February. April is anticipated harvest time. Ojective 5. (funded by Orie Lee, 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. The third treatment was applied as follows (using all donated products from TigerSul, Harrell’s and Florikan). Permanent field plot signs were installed (w/ assistance from Frank Rogers). 1. Control no extra nutrition 2. Harrells St. Helena mix (2lbs per tree) 3. Harrells St. Helena mix (2lbs.)+ 2x TigerSul manganese (90 gm) 4. Harrells St. Helena mix (2lbs.) + 2x Florikan polycoated sodium borate (32 gm) 5. Harrells St. Helena mix (2lbs.) + 2x TigerSul manganese (90 gm) + 2x FL sodium borate (32 gm) 6. 4x TigerSul manganese (180 gm) 7. 4x Florikan polycoated sodium borate (64 gm) 8. 4xTigerSul manganese (180 gm) + 4x Florikan polycoated sodium borate (64 gm) Donated micronutrient treatments were also applied at the Hughes Post Office block (Haines City) – where there were yield increases ranging from a half-box to 3/4’s box per tree this past season on 100% HLB-infected trees, enhanced by specific treatments, especially those containing both boron and manganese. Trees look exceptional at present, and we expect to see a yield increase for the 2nd year in a row since we began the study.
The project has two objectives: (1) Increase citrus disease resistance by activating the natural SAR inducer-mediated defense-signaling pathway. (2) Engineer non-host resistance in citrus to control citrus canker and HLB. We repeated the concentration gradient experiments. A series of concentrations of the SAR inducer, including 0, 0.25, 0.5, 0.75, and 1 mM, were used to treat citrus plants by infiltration and soil drench. The infiltrated leaves and soil drenched plants were inoculated with canker bacterial pathogens 24 hours and 7 days later, respectively. Again, 5 plants were used for each treatment; three leaves on each plant were inoculated; 6 inoculations on each leaf were carried out, and a total of 90 inoculations were used for each treatment. Results confirmed that the strength of canker resistance is concentration dependent in the range between 0 to 1 mM. We also confirmed the systemic residual resistance activated by the SAR inducer. The SAR inducer-treated plants were cut back and leaves on the new flushes were tested for resistance to canker. As observed previously, canker disease symptom development was significantly delayed on the leaves on the new flushes. This result indicated that the SAR inducer not only activates resistance in the treated leaf tissues, but also in new flush leaves not treated with SAR inducer. In addition, experiments determining if the systemic residual resistance is conferred by the SAR inducer residue or products induced by the inducer are still ongoing.
The project has two objectives: (1) Increase citrus disease resistance by activating the natural SAR inducer-mediated defense-signaling pathway. (2) Engineer non-host resistance in citrus to control citrus canker and HLB. In order to understand how the SAR inducer activates disease resistance, we tested defense gene induction in the treated plants. Citrus leaves were infiltrated with 0. 0.25, 0.5, 1, 5, and 10 mM SAR inducer and the treated leaf tissues were collected at 0, 4, and 24 hours. Expression of a group of defense genes were analyzed by qPCR. These genes include PAL1,NPR1, PR5, CM1, ICS1, CM1, CM2, and PLDg. Results showed that the SAR inducer activated the expression of several defense genes such as NPR1, PR5, and CM1. We also tested if the SAR inducer treatment enhances pathogen induced defense gene expression. Citrus leaves were infiltrated with different concentrations of the SAR inducer. Thirty six hours later, the infiltrated leaves were inoculated with citrus canker bacterial pathogens. The inoculated leaf tissues were collected at 0, 4, 8, and 24 hours later. Expression of the above defense genes were analyzed by qPCR. We found that the bacterial pathogen induced expression of PAL1, NPR1, PR5, CM1, and ICS1 was significantly enhanced by the SAR inducer pretreatment. This results indicate that the SAR inducer can prime citrus plants for resistance to citrus canker. We have also started to test if the SAR inducer can elevate resistance or tolerance to HLB. We are currently testing the treatment conditions.
The project has three objectives: (1) Confirm HLB resistance/tolerance in transgenic citrus lines. (2) Determine the chimerism of the HLB-resistant/tolerant transgenic lines. (3) Confirm HLB resistance in citrus putative mutants (nontransgenic lines). The following work has been carried out in this quarter: (1) Inoculated the promising candidate transgenic plants with CTV carrying the flower-promoting gene FT3. (2) Propagated the transgenic line HAM 13-3, DUN 57-25, DUN205-25c, and DUN 207-8. (3) Infected progenies of transgenic plants with Las-carrying psyllids. We have generated more transgenic plants expressing various defense genes. These transgenic plants are growing in greenhouse and will be tested once they are ready.
During this reporting period July, August, and September, 2016), Dr. McNellis continued to work with USDA APHIS to obtain permitting to transfer a set of ‘Duncan’ grapefruit plants expressing the FLT-antiNodT fusion protein from Penn State University to Fort Detrick in Frederick, Maryland. These plants are to be tested for resistance to HLB using a psyllid-vectored inoculation system in a secure greenhouse. We anticipate approval during the next reporting period. In addition, Dr. McNellis’ team at Penn State continued to evaluate the solubility and stability of the FLT-antiNodT fusion protein in citrus extracts and presence of the FLT-antiNodT fusion protein in various plant tissues by protein gel immunoblotting. The FLT-antiNodT fusion protein appears to be produced and present in all tissues examined to date, although these tests are ongoing and will continue into the next reporting period. Dr. McNellis presented a poster describing the results of the project to date at the annual conference of the American Phytopathological Society in Tampa, FL, July 30 – August 4, 2016. A poster viewer at the conference had some suggestions as to how to determine whether the FLT-antiNodT fusion protein indeed binds to its target in vivo, and Dr. McNellis has developed an experimental plan for doing this, which will be initiated during the next reporting period.
Objective 1: Assess canker resistance conferred by the PAMP receptors EFR and XA21 Three constructs were used for genetic transformation of Duncan grapefruit and sweet orange as part of a previous grant: EFR, EFR coexpressed with XA21, and EFR coexpressed with an XA21:EFR chimera. Seven transgenics have survived and passed a PCR screen, and these have been grafted onto rootstocks. To ensure that there will be sufficient events to analyze to come to a conclusion about the effectiveness of these genes, we have initiated more transformations in Duncan grapefruit at the Core Citrus Transformation Facility at UF Lake Alfred. In addition, we have added the recently-identified Cold Shock Protein Receptor (CSPR) to the transformation queue. Selection is underway, but the GFP marker is not expressed in citrus, and the protocol may need to be optimized. Objective 2: Introduction of the pepper Bs2 disease resistance gene into citrus Two constructs were created to co-express Bs2 with other R genes that may serve as accessory factors for Bs2. These constructs have been provided to the Lake Alfred transformation facility, and selection of transformants in Duncan grapefruit is underway. Objective 3: Development of genome editing technologies (Cas9/CRISPR) for citrus improvement The initial target for gene editing is the citrus homolog of Bs5 of pepper. The recessive bs5 resistance allele contains a deletion of two conserved leucines. The citrus Bs5 homolog was sequenced from both Carrizo citrange and Duncan grapefruit, and conserved CRISPR targets were identified. A construct targeting a site overlapping the two conserved leucines has been tested by co-delivery into Nicotiana benthamiana leaves with another construct carrying the targeted DNA from Carrizo or Duncan varieties, and verified to function. A replacement recessive bs5 allele will be added, and this construct will be prioritized for transformation into Carrizo citrange for proof of concept. Resulting plants with biallelic mutations in Bs5 that contain the replacement bs5 allele will be selected and tested for canker resistance.
Objective 1: Assess canker resistance conferred by the PAMP receptors EFR and XA21 Three constructs were used for genetic transformation of Duncan grapefruit and sweet orange as part of a previous grant: EFR, EFR coexpressed with XA21, and EFR coexpressed with an XA21:EFR chimera. Seven transgenics have survived and passed a PCR screen, and these have been grafted onto rootstocks. To ensure that there will be sufficient events to analyze to come to a conclusion about the effectiveness of these genes, we have initiated more transformations in Duncan grapefruit at the Core Citrus Transformation Facility at UF Lake Alfred. In addition, we have added the recently-identified Cold Shock Protein Receptor (CSPR) to the transformation queue. Selection is underway, but the GFP marker is not expressed in citrus, and the protocol may need to be optimized. Objective 2: Introduction of the pepper Bs2 disease resistance gene into citrus Two constructs were created to co-express Bs2 with other R genes that may serve as accessory factors for Bs2. These constructs have been provided to the Lake Alfred transformation facility, and selection of transformants in Duncan grapefruit is underway. Objective 3: Development of genome editing technologies (Cas9/CRISPR) for citrus improvement The initial target for gene editing is the citrus homolog of Bs5 of pepper. The recessive bs5 resistance allele contains a deletion of two conserved leucines. The citrus Bs5 homolog was sequenced from both Carrizo citrange and Duncan grapefruit, and conserved CRISPR targets were identified. A construct targeting a site overlapping the two conserved leucines has been tested by co-delivery into Nicotiana benthamiana leaves with another construct carrying the targeted DNA from Carrizo or Duncan varieties, and verified to function. A replacement recessive bs5 allele will be added, and this construct will be prioritized for transformation into Carrizo citrange for proof of concept. Resulting plants with biallelic mutations in Bs5 that contain the replacement bs5 allele will be selected and tested for canker resistance.
New rootstocks are appropriate for large-scale grower use when outstanding performance and yield have been documented by multiple statistically replicated trials over multiple years. Outstanding performance has been documented for US-802 and US-942 rootstocks over multiple years in trials affected by HLB, and these rootstocks are available in large numbers through commercial nurseries. Other released new USDA rootstocks with outstanding performance documented at fewer sites and harvests are also commercially available, and should be used for smaller scale plantings until there is more experience with those varieties. It is anticipated that at least one of the best new SuperSour rootstocks will be released for commercial use within 3 years, based on outstanding performance. Field performance information is being collected on more than 400 new rootstocks in 17 different replicated field trials, include tree growth, tree health, fruit yield, fruit quality, and tolerance or resistance to HLB and other diseases. During this quarter, data collection was focused on tree size, health, and PCR evaluation of infection by Las. Data was collected from a replicated greenhouse trial to compare Valencia tree performance on the most HLB-tolerant rootstocks under optimum management conditions. The study will also follow field performance of trees on the most HLB-tolerant rootstocks in the first 1-3 years after they become infected with Las. Focused study in this trial will help to more clearly measure the ways in which tree performance is affected by HLB and estimate the economic viability of commercial production on the most tolerant rootstocks. Trees in the USDA nursery on a large number of advanced rootstock selections, especially SuperSour-type, were continued in propagation for field trials to be planted in 2017. New trials in propagation continue to focus on sweet orange scion, but include some plantings to assess performance with new scions that have better tolerance to HLB. Nursery experiments were conducted with promising new rootstocks to determine nursery-related traits important for commercial use. Cooperative work continued with commercial nurseries involved with micropropagation, to facilitate more rapid deployment of the best new rootstocks. A cooperative project is underway with Dr. Ute Albrecht (UF, Immolakee), Agromillora and Rucks Citrus Nursery to compare trees on rootstocks propagated by seed, cuttings, and micropropagation, so that growers can have confidence that rootstocks propagated by the different methods will have equivalent performance. A multi-year collaborative grant proposal was developed with the UF citrus breeding team and other UF and University of California researchers, and submitted to USDA NIFA to help fund expanded rootstock research and development efforts. Cooperative grant-funded work continued with UF researchers and a commercial nursery to propagate trees for use in multiple rootstock field trials sponsored by the HLB MAC program. Extensive information was provided, and cooperative planning for continuing rootstock testing and trials was begun with Catherine Hatcher. An invited presentation on tolerance to HLB in rootstocks was made at the International Citrus Congress in Brazil. A new paper that was developed to provide a comprehensive comparison of field performance and nursery characteristics for USDA rootstocks with other standard rootstocks was accepted, and should appear in the October issue of the journal HortScience. This publication will be a valuable reference for use by growers and nurseries in planning for which rootstocks to use in new plantings.
Good progress was made in the validation of the effectiveness of metabolite profiles for selection of HLB tolerant rootstocks. Focused studies were continued using plant material established in the USHRL greenhouse and field during the previous year. Trees that were inoculated with Las for controlled greenhouse and field metabolic studies were sampled and tested by PCR for Las infection, to identify those suitable for the next group of metabolic samples. Metabolomics data was received from the first set of grafted and non-grafted rootstocks that was sent to the West Coast Metabolomics Center (WCMC) at UC Davis earlier this year for GC-TOF-MS analysis. The research team at the Southwest Florida Research and Education Center (SWFREC)/UF began working with this first data set, in which over 500 metabolites were detected (30% of known chemical structure and 70% of unknown chemical structure) in four standard rootstocks (Cleopatra mandarin, Swingle citrumelo, Ridge Pineapple, and Sour orange), which were grown as seedlings and as grafted plants under greenhouse and under field conditions. Analysis of this first data set is currently being conducted to 1) identify metabolites associated with rootstock traits, 2) decipher tissue-specific (roots and leaves) metabolite profiles and their value for rootstock characterization, 3) identify rootstock effects on scion, and 4) identify significance and impact of environmental factors on metabolite profiles. Preparation of a manuscript for publication of preliminary data is in progress. The results from the second data set, for which samples were submitted to WCMC in June and July, and which include rootstocks with well-characterized responses to HLB, are required to focus in detail on the discovery of metabolites associated with tolerance to HLB and other positive rootstock attributes. The team is in regular contact with WCMC and was informed that results from this data set will be available in the coming weeks. Research results from a previous data set on leaf metabolite profiles of HLB tolerant and susceptible rootstocks were presented at the International Citrus Congress in Brazil in September 2016. A presentation of results from metabolomic studies is anticipated at the 5th International Research Conference on Huanglongbing, to be held in Orlando next spring.
The citrus relatives planting (85 seed source genotypes from the gene bank) has been assessed for growth and apparent HLB tolerance. Within the genus Citrus, measures of tolerance msuch as canopy density, health, and tree size, correlate positively with % citron in pedigree, with r2 of 0.3-0.6. A manuscript describing apparent tolerance to HLB in citrus and citrus-related germplasm has been submitted. Chemical, morphological and transcriptome characteristics are being assessed to determine what factors are associated with observed tolerance (three distinct projects), so they can be used in early screening and possibly directed transgenesis. A paper describing HLB resistance in this population has just been published in Plant Disease. These data suggest that citron-derived germplasm should be used as part of a portfolio of citrus cultivar improvement efforts for Florida production. In October 2013, 34 unique genotypes (USDA hybrids) some of which appear to have tolerance to HLB, and 16 standard commercial varieties were exposed to an ACP no-choice feeding trial and have been transferred to the field at Ft. Pierce FL. Standard growth measurements and disease ratings were initiated in July 2014 and will continue on a quarterly basis. HLB is now widespread and trees of more vigorous scion types are generally the healthiest at this point in time. Data taken in the next quarter should show marked distinctions between genotypes in HLB-tolerance. Development of periclinal chimeras with resistant vascular tissue from Poncirus and remaining layers from sweet orange is underway. Generation of new chimeras has been difficult. An existing periclinal chimera (Satsuma and Poncirus) has been imported and is now being grown at USHRL for testing. A method for the rapid identification of potential sources of HLB resistance is being developed. This project involves the screening of citrus seedlings at the 3 to 5 leaf stage, or very small micrografted trees, that are exposed to HLB infect ACP feeding. CLas titer levels, using real time PCR, are easily detectable in most plants at 3 weeks By nine weeks after exposure, susceptible genotypes can be clearly distinguished from reported resistant material by higher CLas levels in roots. Averaged across genotypes and tissues, total CLas per tree was 5 billion in week 3 after ACP exposure and doubled every 3 weeks through week 12. This should be especially useful for screening anti-Las transgenics. Trees of seemingly HLB resistant/tolerant sweet orange-like hybrids and mandarin -types were propagated on x639. Replicated trials with standards have been established, in cooperation with G. McCollum. Six locations each of all sweet orange-like together and 4 with all mandarins were established in replicated block plantings with 6-8 trees of each cultivar at each site (in Ridge, IR and Gulf coast). Evaluation of existing cultivar/rootstock combinations for HLB resistance/tolerance has revealed potentially valuable tolerance and indicates that early HLB symptoms and earlier CLas titer are unrelated to growth and cropping. In August 2010, the plants were established at Pico s farm in Ft. Pierce FL. Despite the high incidence of mottle in SugarBelle / SourOrange, it had the greatest overall increase in diameter. ‘SugarBelle’ and ‘Tango’ (which were not on the same stock as ‘Hamlin’ and so results should be viewed as comparing cultivar/rootstock combinations) were the healthiest in overall appearance in 10/15 and had the most fruit (88 per tree). A mapping population of Fortune x Fairchild has been planted (collaborating Roose and Gmitter) along with related material, in an effort to identify genes associated with tolerance in the mandarin phenotypic group. Seedlings with a range of pedigree contributions from Microcitrus have been received in a collaboration with M. Smith, Queensland Aus. citrus breeder, and are being grown for field testing of HLB resistance.
Citrus trees transformed with a chimera AMP and a thionin alone showed remarkable resistance in citrus canker compared to control. These promising transgenic lines were replicated for HLB challenge. Propagated transgenic Carrizo lines expressing thionin, chimera and control were grafted with HLB infected rough lemon buds. Twelve months after graft inoculation, Las titer was examined and compared in old leaves (most with HLB symptom), young expanded leaves (with or without HLB symptom) and fibrous roots of transgenic and control plants. Our results showed again that transgenic citrus expressing Mthionin has lower Las titer (200-1800X lower) compared to control and transgenic plant expressing chimera. These data suggest transgenic plants expressing thionin are promising for HLB resistance ( published in Frontiers in Plant Biology). Antibody against thionin has been produced for investigating the correlation of thionin expression and HLB resistance. Two new chimeral peptides (second generation) were developed and used to produce many Carrizo plants and Hamlin shoots. Transgenic carrizo plants carrying second generation AMPs were obtained. DNA was isolated from 46 plants and 40 of them are PCR positive. Furthermore, the third generation chimeral peptides were designed based on citrus thionins, the vector construction were finished and citrus transformation are underway. To explore broad spectrum resistance, a flagellin receptor gene FLS2 from tobacco was used to transform citrus. Trees expressing NbFLS2 showed significant canker resistance to spray inoculation. Replicated Carrizo and Hamlin were challenged with ACP feeding. Leaves were taken six months after ACP feeding inoculation. DNA was isolated and Las titer was tested. Our preliminary results showed that transgenic trees expressing NbFLS2 can reduced Las titer. To disrupt HLB development by manipulating Las pathogenesis, a luxI homolog potentially producing AHLs to bind LuxR in Las was cloned into binary vector and transformed citrus. Both transformed Carrizo and Hamlin were obtained. Replicated transgenic Carrizo plants were challenged by ACP feeding. Las tilter will be tested soon. Transgenic Hamlin were propagated by grafting for HLB challenge. In collaboration with Bill Belknap two new citrus-derived promoters have been tested using a GUS reporter gene and have been shown to have extraordinarily high levels of tissue-specific expression. The phloem-specific promoter was used to create a construct for highly phloem specific expression of the chimeral peptide using citrus genes only. A Las protein p235 with a nuclear-localization sequence has been identified and studied. Carrizo transformed with this gene displays leaf yellowing similar to that seen in HLB-affected trees. Gene expression levels, determined by RT-qPCR , correlated with HLB-like symptoms. P235 translational fusion with GFP shows the gene product targets to citrus chloroplasts. Transcription data were obtained by RNA-Seq. Data analysis and comparison are underway. Antibodies (ScFv) to the Las invA and TolC genes, and constructs to overproduce them, were created by John Hartung under an earlier CRDF project. We have transgenic Carrizo reflecting almost 400 independent transgenic events and 17 different ScFv ready for testing. A series of AMP transgenics scions produced in the last several years continue to move forward in the testing pipeline. Many trees are in the field and some are growing well but are not immune to HLB. A large number of ubiquitin::D4E1 and WDV::D4E1 plants and smaller numbers with other AMPs are replicated and now in the field.
(863) 956-8817
(863) 956-5894
700 Experiment Station Road
Lake Alfred, FL 33850
Email Us
