A test site at the USDA/ARS USHRL Picos Farm in Ft. Pierce supports HLB/ACP/Citrus Canker resistance screening for the citrus research community. There are numerous experiments in place at this site where HLB, ACP, and citrus canker are widespread. The first trees have been in place for almost six years. A number of successes have already been documented at the Picos Test Site funded through the CRDF. The UF Grosser transgenic effort has identified promising material, eliminated failures, continues to replant with new advanced material, with ~200 new trees in April 2015 (Grosser, personal comm.). The ARS Stover transgenic program has trees from many constructs at the test site and is seeing some modest differences so far, but new material has been planted that has shown great promise in the greenhouse and the permit has been updated to plant many new transgenics. A trial of more than 85 seedling populations from accessions of Citrus and citrus relatives (provided as seeds from the US National Clonal Germplasm Repository in Riverside, CA) has been underway for 6 years in the Picos Test Site. P. trifoliata, Microcitrus, and Eremocitrus are among the few genotypes in the citrus gene pool that continue to show substantial resistance to HLB (Lee et al., in preparation, with the last samples collected this week), and P. trifoliata also displayed reduced colonization by ACP (Westbrook et al., 2011). A new UF-Gmitter led association mapping study has just been initiated using the same planting, to identify genes associated with HLB- and ACP-resistance. A broader cross-section of Poncirus-derived genotypes are on the site in a project led by UC Riverside/USDA-ARS Riverside, in which half of the trees of each seed source were graft-inoculated prior to planting. A collaboration between UF, UCRiverside and ARS is well-underway with more than 1000 Poncirus-hybrid trees (including 100 citranges replicated) being evaluated to map genes for HLB/ACP resistance. Marked differences in initial HLB symptoms and Las titer were presented at the 2015 International HLB conference (Gmitter et al., unpublished). In July 2015 David Hall led assessment of ACP colonization across the entire planting, and the Gmitter lab will map markers associated with reduced colonization. Several USDA citrus hybrids/genotypes with Poncirus in the pedigree have fruit that approach commercial quality, were planted within the citrange site. Several of these USDA hybrids have grown well, with dense canopies and good fruit set but copious mottle, while sweet oranges are stunted with very low vigor (Stover et al., unpublished). A Fairchild x Fortune mapping population was just planted at the Picos Test Site in an effort led by Mike Roose to identify genes associated with tolerance. This replicated planting includes a number of related hybrids (among them our easy peeling remarkably HLB-tolerant 5-51-2) and released related cultivars. Valencia on UF Grosser tetrazyg rootstocks have been at the Picos Test Site for several years, having been Las-inoculated before planting, and several continue to show excellent growth compared to standard controls (Grosser, personal comm.).
For the last three months, Core Citrus Transformation Facility (CCTF) continued to provide its service for production of transgenic Citrus plants. Within this period of time, there were only two new orders placed but three clients requested work on three previously placed orders bringing total number to five. Probably as a result of recent Knowledge Mapping meeting organized by CRDF there is an increased interest for transgenic plants and CCTF was already informed of eight new incoming orders. That does not include additional orders from another client with whom CCTF manager communicated for the last three months. Forty plants were produced during the last quarter which represents lower productivity than usual. Partially, this is the result of work on group of three orders that have not yielded any plants. Another reason is the low quality of seeds used to obtain seedlings as starting material for experiments. High majority of produced plants belong to eight orders placed within the last 12-15 months. Five plants belong to three older orders. Most of the transgenic plants are Duncan grapefruit and Valencia orange and one of them is Carrizo citrange. The work has begun on determination of level of expression of AtNPR1 gene in rootstock plants produced for CRDF. Once all plants produced by CCTF and Mature tissue lab are processed, those with the highest expression levels will be selected for further use and propagated.
Mature plant production continues with various genetic constructs with reporter genes from Drs. Dutt, McNellis and Wang. Additional scientists have expressed interest in our services. Transformation efficiencies have significantly increased with reporter genes. We are also trying to further increase Agrobacterium transformation efficiencies of mature citrus by incorporating vacuum infiltration and sonication treatments. These treatments significantly increased transformation efficiencies in immature citrus. In addition to Agrobacterium, we can now supplement plant production with plants produced using biolistics. Scions and rootstocks have been micropropagated (budded scions and rooted cuttings) into three replicates for one location. It remains to be determined whether we will continue micropropagation for replicates at additional locations. We have almost concluded screening Dr. Orbovic’s putative transgenics with qPCR to identify high-expressing lines. This work should be finished by the end of this month. I am switching to citrus pots in the growth room, which can be planted to higher densities than current planting densities. Recently we discovered that mature scion grows significantly faster after budding if the rootstock is not cut-off after the buds break.
Citrus Huanglongbing (HLB) poses the greatest threat to the survival of the Florida and US citrus industry. Research to incorporate HLB resistance/tolerance into citrus has been recommended by the National Research Council as one of the top priority topics for addressing the HLB threat. A number of Poncirus and Citrus cultivars have been recently found to be tolerant to HLB. Identification, characterization and validation of candidate genes responsible for HLB-resistance are of the remarkable value to develop new HLR-resistant/tolerant citrus varieties. More than 70 different accessions of Citrus species and relatives have been observed in a field planting in Florida (with cooperation of Dr. E. Stover, USDA). Leaf samples were collected from about 450 trees. The titer of CLas in the HLB-infected plants was determined by TaqMan probe-based qPCR, and the HLB symptom severity on the plants was evaluated by experienced researchers. These accessions showed diverse responses (susceptible, tolerant and resistant) against HLB. Many HLB-tolerant citrus relatives were revealed, including Citrus latipes, Poncirus trifoliata, Severinia buxifolia and Microcitrus australis. The genomes of 15 citrus accessions have been sequenced to about 25x coverage (9 to 11 Gb/genome) and the genome of one Poncirus trifoliata accession has been sequenced to about 50x coverage (~21 Gb). Initial analysis indicated good quality of these sequence reads. We have begun a more detailed analysis of sequence polymorphisms and structural variations within candidate genes. We are also validating the expression of our shortlisted candidate genes by real time PCR among susceptible and resistant citrus accessions.
This project (Hall-15-016) is an extension of a project that recently came to a close (Hall-502). The driving force for this project is the need to evaluate citrus transformed to express proteins that might mitigate HLB, which requires citrus be inoculated with CLas. USDA-ARS-USHRL, Fort Pierce Florida is producing thousands of scion or rootstock plants transformed to express peptides that might mitigate HLB. The more rapidly this germplasm can be evaluated, the sooner we will be able to identify transgenic strategies for controlling HLB. The purpose of this project is to support a high-throughput facility to evaluate transgenic citrus for HLB-resistance. This screening program supports citrus breeding and transformation efforts by Drs. Stover and Bowman. Briefly, individual plants to be inoculated are caged with infected psyllids for two weeks, and then housed for six months in a greenhouse with an open infestation of infected psyllids. Plants are then moved into a psyllid-free greenhouse and evaluated for growth, HLB-symptoms and Las titer, and finally the plants are transplanted to the field where evaluations of resistance continue. 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: Two technicians funded by the grant were hired during August and are being trained on how to establish and maintain colonies of infected psyllids, how to conduct qPCR assays on plant and psyllid samples, and how to run the inoculations. As of March 31, 2015, a total of 7,448 plants have passed through inoculation process. A total of 148,960 psyllids from colonies of CLas-infected ACP have been used in no-choice inoculations.
Most commonly grown citrus cultivars are sensitive to Huanglongbing (HLB). However, some citrus species and related genera are substantially more tolerant, such as Citrus jambhiri (rough lemon), Poncirus trifoliata and Microcitrus australis. Genome analysis will improve our understanding of the HLB tolerance mechanisms. Nuclear DNA from Citrus jambhiri was used to generate more than 235 million paired-end reads (2 X 100 nt) of the rough lemon genome. A reference-guided method was used to assemble the rough lemon genome. Based on analysis of the SNPs identified, rough lemon was found to have originated from the interspecific hybridization of mandarin, citron and pummelo, and its chloroplast is probably derived from mandarin. RNA-sequencing data was used for gene annotation, and some differentially expressed (DE) genes were identified. Most of DE genes were up-regulated in HLB affected trees, compared with non-affected trees. These DE genes were mainly involved in response to stress, carbohydrate metabolic process, response to abiotic stimulus, cell wall organization or biogenesis, ion transport and signaling. Based on our meta-analysis and co-expression network analysis of previously published gene expression data, we have shortlisted 2,000 HLB-responsive candidate genes in citrus (Du et al., 2015; Rawat et al., 2015). To identify sequence polymorphisms and validate the expression patterns of candidate genes, we have isolated genomic DNA and RNA from 20 citrus accessions. The DNA and RNA preps are being evaluated for quality and are to be sent to a commercial DNA sequencing company for large-scale sequencing on the HiSeq 2500. The sequencing data of these samples will be analyzed with the focus on 2000 candidate genes. The genomic indel variations and SNPs will be identified within these sequenced accessions for the selected candidate genes.
Our project aims to provide durable long term resistance to Diaprepes using a plant based insecticidal transgene approach. In this quarter, several transgenic lines expressing each of our test genes have been regenerated and many of them have rooted. The rooted material has been successfully acclimatized to the greenhouse for growth and further testing. We are in the process of confirming the gene expression levels in these transgenic lines to isolated lines that have excellent gene expression. These selected lines will be propagated for subsequent evaluation with Diaprepes neonates.
The main accomplishments during this quarter: We repeated and confirmed the effects of K and I genes on genetic transformation for cultivars Valencia and Washington oranges and observed drastic increases in transformation efficiencies if compared to a conventional Ti-plasmid vector containing no K or I gene. We have confirmed that the K and I genes can drastically enhance transformation efficiencies of juvenile explants of 5 different citrus cultivars. We started test the effects of the K gene on transformation efficiency of a lemon cultivar. Lemon is difficult for genetic transformation. Our major efforts have been in testing the effects of the K and I genes and other factors on mature tissues. We used K and I genes to do genetic transformation of mature Pineapple orange. The K gene resulted in about two fold increases in transformation efficiency while the I gene produced about three fold increases in efficiency compared to control vector. We have also started testing effects of other factors on transformation of mature tissues in combination with the K gene. We have made some significant progress but microbial contaminations of adult tissues harvested from greenhouse grown trees have sometimes caused problems for us. One example is that we have repeated and confirmed the effects of the transport of an endogenous plant hormone in explants on shoot regeneration efficiency. We observed that manipulating that process improves shoot regeneration and transformation efficiency of juvenile citrus explants. We are testing the effects of the same manipulation on transformation efficiency of adult tissues of citrus.
The overall objective of this project was to use the PAMP receptors EFR and XA21 to engineer citrus plants resistant to both HLB (causal agent Candidatus Liberibacter asiaticus, CLas) and citrus canker (Xanthomonas axonopodis pv citri, Xac). Since neither receptor recognizes a PAMP from CLas, the first objective was to engineer a variant of EFR (EFR+) to recognize the elf18 peptide from CLas. This novel receptor would then be combined with XA21 or an XA21-EFR chimera that recognizes a PAMP from Xac. A number of strategies to engineer an EFR+ variant that recognized elf18-Clas were tested, but none were successful. These included PCR mutagenesis, screening of natural variants in an extensive Arabidopsis accession collection, creating targeted mutations based on the modeled interactions among elf18, EFR, and BAK1, and testing high-throughput strategies such as phage display and FACS. However, we were able to successfully create a functional XA21-EFR chimera. Although we did not generate an EFR+ variant that recognized elf18-CLas, expression of EFR and XA21 may still provide significant protection against citrus canker. Therefore, we transformed three constructs into citrus: EFR alone, EFR with XA21, and EFR with the XA21-EFR chimera described above. The latter two constructs have the potential of providing stronger and more durable resistance than EFR alone. Some transgenic events have been obtained in Duncan grapefruit and sweet orange, and these have been transferred to Dr. Jeff Jones lab at the University of Florida for testing with citrus canker.
The transgenic plants to be developed for this project are now growing in two different locations in secure greenhouses and growth chambers. Seven independently-transformed citrus plants carrying the FLT-antiNodT fusion protein expression construct are growing in Dr. McNellis’ lab at the Pennsylvania State University at University Park, PA, and an additional eight independently-transformed citrus plants carrying the FLT-antiNodT fusion protein expression construct are growing at Dr. Tim Gottwald’s lab at the United States Horticultural Laboratory in Fort Pierce, Florida. The plants at both locations are growing well. At Penn State, all the transgenic lines have been successfully propagated as vegetative cuttings. All of the lines growing at Penn State have been found to express the FLT-antiNodT fusion protein, with five of the seven lines expressing very high levels of the protein. We must continue to let these plants grow a bit more before starting the HLB resistance tests. We have initiated a collaboration with Dr. Janice Zale (University of Florida Mature Citrus Transformation Facility, Lake Alfred) to transform varieties important to the Florida citrus industry, including the ‘Valencia’ and ‘Hamlin’ sweet orange varieties and the ‘Citrumello’ rootstock with the FLT-antiNodT expression construct. We have immediately started transformations with the available transformation construct used to transform ‘Duncan’ grapefruit, in plasmid pTLab21. In addition, we are developing an FLB-antiNodT expression cassette in the transformation construct pBI121, which has a history of successful approval for transgenic plant development. In June, Dr. McNellis submitted a Stakeholder Relevance Statement to the USDA Specialty Crop Citrus Disease Research and Extension program to further develop this project. However, a full proposal was not invited. Dr. McNellis will present a poster describing the results of this project to date at the American Phytopathological Society conference in Pasadena, California, August 1-5, 2015.
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. For objective 1, we treated citrus plants with the natural SAR inducer using three different approaches: leaf infiltration, foliar spray, and soil drench. Three concentrations were tested: 1, 5, and 10 mM. For leaf infiltration, the infiltrated leaves were inoculated 1 day later with the canker bacterial pathogen; for foliar spray, treated leaves were inoculated 3 days later; and for soil drench, leaves on treated plants were inoculated 7 days later. For each treatment, 5 plants were used. Three leaves on each plant were inoculated and 6 inoculations on each leaf were conducted. A total of 90 inoculations were used for each treatment. Fourteen days after inoculation, numbers of lesions formed on the inoculated leaves were counted. Results showed that all concentrations of the SAR inducer induced strong resistance to citrus canker. The inoculated plants have been cut back. Systemic residual resistance will be tested on the new flushes. We are repeating this comprehensive testing experiment and will identify the most efficient treatment method. For objective 2, transgenic citrus plants expressing the Arabidopsis nonhost resistance genes have been propagated. The progenies are growing in greenhouse and will be tested for disease resistance.
We completed computational comparative analysis of the three genome categories: Liberibacter, Citrus and Psyllid. The results are available at the website
The HLB-tolerant rootstocks US-1279, US-1281, US-1282, US-1283, and US-1284 were released by USDA in 2014, and are available from FDACS-DPI as clean sources for vegetative propagation and establishment of seed trees. Plant material was also provided to commercial nurseries for micropropagation. Fruit yield of Hamlin trees infected by HLB on these rootstocks was 2-4 times the yield of trees on Swingle, and the trees on these rootstocks also had fruit that is larger in size and higher in sugar content. The previously released USDA rootstocks, US-942, US-802, and US-897 were demonstrated in greenhouse testing and field trials to exhibit better tolerance to HLB than many of rootstocks commonly used in Florida. The promising new USDA rootstocks, along with other new HLB-tolerant rootstocks from USDA and Univ. of Florida will be used in a series of grower-cooperator field trials with funding provided by the HLB-MAC program in 2016-17. An updated rootstock selection guide including information about these and other new rootstocks was developed cooperatively with University of Florida and released in 2015 as an extension publication, “Florida Citrus Rootstock Selection Guide, 3rd Edition”. Yield, fruit quality, tree health, and other performance information was collected from 10-20 established rootstock field trials each year, and used to assess new rootstock performance at different sites. Thousands of budded nursery trees were prepared with Supersour and other new rootstocks, and numerous new field trials were planted in 2013-2015. About fifteen thousand new propagations of Supersour and other rootstocks were prepared for greenhouse testing for disease tolerance and budding for additional field trials in 2016. Greenhouse and field studies were used to evaluate the phytophthora tolerance of 70 new Supersour and other rootstocks. Greenhouse tests were used to assess Supersour tolerance of CTV. Trees were planted into the field to establish seed sources for the most promising Supersour selections. Studies of defense gene and metabolomic profiles of hybrid rootstocks that are highly tolerant to HLB were studied, so as to improve our ability to create and select conventional hybrid and transgenic rootstocks that possess a high level of tolerance. Additional studies were conducted on defense-related gene expression and small RNAs associated with HLB infection, in collaboration with University of Maryland and University of California research groups. Several research studies were published to document the new important information. New grant proposals were submitted to NIFA-SCRI and other funding agencies to follow up on new research opportunities in these areas. Thousands of new transgenic rootstocks were produced, including the novel genes for antimicrobial peptides and constructs that would optimize expression of natural citrus defense genes. Optimizing of citrus defense genes was directed in significant part by results from other research under this grant to identify defense gene expression and metabolic profiles associated with tolerance to HLB. Fifteen transgenic rootstock selections showing increased resistance to HLB have been identified from preliminary test groups with transgenes, and trees are being propagated for additional testing. Several hundred additional transgenic citrus have been produced and are awaiting screening with HLB, including transgenics with optimized expression of the citrus defense genes CtSID2, CtSFD1, CtPAD3, CtCDR1, CtMPK4, CtTGA7, CtDIR1, CtERF1, CtFAD7, CtFMO1, CtAZL1, CtRDR1, CtRAP4, CtCSD1, CtNHL3, CtNHO1, and CtNHL25. Work is continuing under the new CRDF-funded screening project to test these transgenic citrus for tolerance to HLB.
Progress has been made in identifying conventional citrus that is quite tolerant to HLB. Evaluation of existing standard and non-standard cultivars ( Hamlin , Temple , Fallglo , Sugar Belle , Tango , and Ruby Red ) for HLB resistance/tolerance is complete. In August 2010, the plants were established at Pico s farm in Ft. Pierce Fl. Data on the growth rate, disease severity, and Candidatus Liberibacter asiaticus (CLas) titer levels have been collected since April 2012. During the 4-year period, there were significant differences in disease severity, stem diameter, and CLas levels among the varieties. All trees exhibited symptoms of HLB and tested positive for CLas, with similar titers measured at most recent sample dates. Fallglo had the lowest incidence of HLB symptoms, whereas Ruby Red had the highest incidence. Ruby Red also appears to be in significant decline. Despite the high initial titer levels found in SugarBelle , it had the greatest overall increase in diameter and was the healthiest in overall appearance. In Nov. 2014 Temple trees had significantly greater fruitload, with 26 fruit/tree, followed by Tango with 10 fruit /tree, Hamlin/Kinkoji with 5 fruit/tree and all others with 0-1.4 fruit/tree. All cultivars except sweet oranges and grapefruit are progressing in production, but production was compromised in all varieties by the severe HLB pressure at this site, and commercial value of the observed tolerance remains uncertain. In October 2013, 34 unique genotypes (USDA hybrids) some of which appear to have tolerance to HLB, and 16 standard commercial varieties were exposed to an ACP no-choice feeding trial and have been transferred to the field at Ft. Pierce Fl. Standard growth measurements and disease ratings were initiated in July 2014 and will continue on a monthly basis. As of December 2014, the first HLB symptoms are apparent. Progress has been made on the antibiotic treatment of HLB infected bud-wood to compare growth at different levels of CLas infection. HLB-infected budwood was treated with various concentrations of antibiotics and grafted on sour orange rootstock using 3 fairly HLB-resistant ( Temple , GnarlyGlo , and Nova ) 3 tolerant ( Jackson , FF-5-51-2, and Ftp 6-17-48), and 3 susceptible ( Flame , Valencia , and Murcott ) genotypes. Standard growth measurements (stem diameter and height), disease severity were evaluated and leaves were sampled for qPCR analysis. Evaluations and sampling will continue on quarterly basis. Trees will be field planted July 2015. Development of periclinal chimeras with resistant vascular tissue from Poncirus and remaining layers from sweet orange is underway. One hundred and fifty etiolated seedlings of the trifoliate Rubidoux and the sweet orange Hamlin have been approach grafted together. Generation of new chimeras has been difficult. Several adventitious buds have emerged from the treated graft region, with several appearing to be chimeral. The newly emerged plants will be tested using LC/MS to determine the origin of the three layers. To increase the success rate, additional plants will be grafted over the next twelve months. An existing periclinal chimera has been imported and will be released from DPI. 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 evaluated at 3, 6, and 9 weeks Seedlings of Hamlin and Dancy show early CLas proliferation and systemic movement. Only low levels of CLas have been observed in Carrizo. Trees of seemingly HLB resistant/tolerant sweet orange-like hybrids and mandarin -types have been 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 will be established with 6-8 trees of each cultivar at each site. The first trials have been established with cooperators (in Ridge, IR and Gulf coast) for replicated block plantings at each site.
A transgenic test site at the USDA/ARS USHRL Picos Farm in Ft. Pierce supports HLB/ACP/Citrus Canker resistance screening for the citrus research community. There are numerous experiments in place at this site where HLB, ACP, and citrus canker are widespread. The first trees have been in place for over five years. A number of successes have already been documented at the Picos Test Site funded through the CRDF. The UF Grosser transgenic effort has identified promising material, eliminated failures, continues to replant with new advanced material, with ~200 new trees in April 2015 (Grosser, personal comm.). The ARS Stover transgenic program has trees from many constructs at the test site and is seeing some modest differences so far, but new material is being planted this spring that has shown great promise in the greenhouse (unpublished). A trial of more than 85 seedling populations from accessions of Citrus and citrus relatives (provided as seeds from the US National Clonal Germplasm Repository in Riverside, CA) has been underway for 5 years in the Picos Test Site. P. trifoliata, Microcitrus, and Eremocitrus are among the few genotypes in the citrus gene pool that continue to show substantial resistance to HLB (Lee et al., in preparation), and P. trifoliata also displayed reduced colonization by ACP (Westbrook et al., 2011). A new UF-Gmitter led association mapping study has just been initiated using the same planting, to identify genes associated with HLB- and ACP-resistance. A broader cross-section of Poncirus-derived genotypes are on the sire in a project led by UC Riverside/USDA-ARS Riverside, in which half of the trees of each seed source were graft-inoculated prior to planting. A collaboration between UF, UCRiverside and ARS is well-underway with more than 1000 Poncirus-hybrid trees (including 100 citranges replicated) being evaluated to map genes for HLB/ACP resistance. Marked differences in initial HLB symptoms and Las titer were presented at the 2015 International HLB conference (Gmitter et al., unpublished). In July 2015 David Hall will be leading assessment of ACP colonization across the entire planting, and the Gmitter lab will map markers associated with reduced colonization. Several USDA citrus hybrids/genotypes with Poncirus in the pedigree have fruit that approach commercial quality, were planted within the citrange site. As of April 2014 at the Picos Test Site, several of these USDA hybrids had grown to a height of seven ft, with dense canopies and good fruit set, while sweet oranges are stunted (3 ft) with very low vigor (Stover et al., unpublished). A Fairchild x Fortune mapping population will be planted at the Picos Test Site in July in an effort led by Mike Roose to identify genes associated with tolerance. This replicated planting will also include a number of related hybrids (among them our easy peeling remarkably HLB-tolerant 5-51-2) and released cultivars. Valencia on UF Grosser tertazyg rootstocks have been at the Picos Test Site for several years, having been Las-inoculated before planting, and several continue to show excellent growth compared to standard controls (Grosser, personal comm.).
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