This three-year project is to continue the search and evaluation of citrus tree survivors found under high pressure of HLB and its pathogen, on the basis of additional visits to groves in severely HLB-affected production areas, primarily in Florida, but also in areas of southern China that we have visited previously. Past exploration in China has identified three such trees and at least one of these remains free of HLB after several years. The field visits will be conducted by our collaborators and ourselves, when possible; we expect to visit Florida groves seasonally every year, and southern China once within the 3 years. Funding for this project became available on 18 May, 2012, so no new activities have been initiated at this point. We have previously propagated a few trees from Pineapple budwood collected in Martin County. Two of 5 original source trees were found to be qPCR negative, while > 90% of the other trees in the block were dead. These trees are maintained in a greenhouse at the CREC and are being grown off to sufficient size for use in future experiments.
This three-year project is to continue the search and evaluation of citrus tree survivors found under high pressure of HLB and its pathogen, on the basis of additional visits to groves in severely HLB-affected production areas, primarily in Florida, but also in areas of southern China that we have visited previously. Past exploration in China has identified three such trees and at least one of these remains free of HLB after several years. The field visits will be conducted by our collaborators and ourselves, when possible; we expect to visit Florida groves seasonally every year, and southern China once within the 3 years. We have previously propagated a few trees from Pineapple budwood collected in Martin County. Two of 5 original source trees were found to be qPCR negative, while > 90% of the other trees in the block were dead. These trees are maintained in a greenhouse at the CREC and are being grown off to sufficient size for use in future experiments. This summer, we have focused visits to properties at the CREC, the GCREC, and some Polk County commercial groves where we have planted out materials from the CREC breeding program, with the express purpose of identifying particularly healthy appearing trees, that can be found in blocks as HLB symptoms are becoming more widespread and obvious. These trees have been noted and marked on maps, for future observations, as the season progresses and symptom expression increases in the autumn. They include commercial varieties on typical rootstocks, as well as on experimental rootstocks from our program, and some other citrus accessions.
We have reported previously on the escape trees that have been identified and assessed in collaboration with colleagues in Guangdong and Guangxi provinces in China. There has been no change in the status of the trees and no additional testing has been carried out. The trees propagated remain in their respective locations. The two trees free of HLB symptoms from Guangdong had been propagated in the greenhouse at the Guangdong Institute of Fruit Tree Research facilities. Some were grafted with HLB-infected budwood in the greenhouse, and others were planted in their research field to assess their reaction to natural inoculation with HLB. Under observations for several months, the propagated trees in the field, surrounded by severe HLB disease and intense inoculum and vector pressure in Guangdong, appeared not to show any HLB symptoms and no pathogen was detected by qPCR. Most of the trees propagated from the individual symptom-free tree found in Guangxi, that were inoculated in a protected greenhouse, were confirmed to be infected and all qPCR positive trees displayed HLB symptoms. Though they apparently were not resistant to inoculation, the question remains as to why the original source tree was not infected and symptom-free; the possibility of vector resistance in the host could be explored further. Unfortunately, the two scientists assigned to this project in Guangdong have moved to new positions; we are in discussions with the GIFTR director regarding the future status of the materials and the project.
Two trees were found growing in HLB-ravaged orchards in Guangdong and one in Guangxi province, which were free of HLB symptoms, while other trees planted at the same time were either dead or declining. The trees in Guangdong were propagated at the Guangdong Institute of Fruit Tree Research (GIFTR), and were repeatedly tested by inoculation with HLB-affected budwood . The original source trees were tested multiple times by qPCR and remained negative for HLB over time, as did the propagations inoculated in the greenhouse. One original source tree was destroyed during highway expansion. Several propagations of one selection were replanted in an infected orchard location at the GIFTR, under high inoculum and vector pressure, but they remained HLB-negative throughout the project. Their current status is unknown as the scientists working on the project with us have moved to other places. The HLB-escape tree found in Guangxi was transplanted to a protected location at the Guangxi Citrus Research Institute (GCRI). Several propagations were made from this tree and inoculated with HLB-infected budwood; most of the re-propagated trees exhibited HLB symptoms and were found to be qPCR positive. In conclusion, HLB-survivor trees were found through our collaborators in China, they were inoculated in greenhouses, and some remained symptom free and qPCR negative; one selection remained so in a field planting, throughout the duration of the project. To expand further our search for survivors, and to continue to learn about Chinese citrus industry adjustments in response to HLB, we established contact with a citrus extension specialist in the Fujian Provincial Academy of Agricultural Sciences. A valuable side benefit of the project was the opportunity to survey regions where HLB is severe and widespread; in doing so we visited groves that appeared to be unaffected by HLB though surrounded by severely declining groves. These surprising locations were seen both in Guangxi and Guangdong; these were mostly mandarin plantings but we also saw healthy sweet oranges in Guangxi. We investigated the management programs which enabled them to survive > 8-10 years in good health. We interviewed growers, pathologists, horticulturists, and entomologists associated with these healthy groves. We visited the Pinghe County pummelo production area in Fujian province, where we saw HLB managed through good psyllid control, good nutrition and subsequent tree health, and natural tolerance of the pummelo variety. We revisited specific orchards we saw previously in Guangdong and Guangxi in healthy condition in 2008, despite widespread infection of neighboring plots. One orchard in Guangdong was devastated with HLB in 2010, and were informed that fruit prices for this variety were very low and the grower had stopped taking care of his trees. Orchards revisited in Guangxi were a different matter. These orchards not only maintained their healthy condition, but were substantially more productive and healthier in appearance than two years earlier. These observations confirm the utility and effectiveness of the management strategy that was developed and employed under direction of the extension entomologist at the GCRI. Although located in different provinces several hundred miles apart, the key elements outlined to us were the same. These include critically timed and thorough pesticide applications, use of pathogen-free planting materials, and maintenance of tree health through good nutrition; when tree removal was in order, it was performed only once a year and after intensive psyllid eradication measures were taken. We have reported on our experiences and observations throughout China in several articles in The Citrus Industry, at many grower group meetings throughout Florida, as well as in California and Brazil. We learned that there may be potential to find mutants of commercial cultivars that are not affected by HLB in the field, though there remain many questions about underlying mechanisms. Nonetheless, there is justification to continue such efforts in China, as well as now in Florida. And also importantly, we learned many of the approaches that are employed to manage HLB in regions of China where the disease has been endemic for more than 100 years, and provided some degree of hope for the same in Florida.
Two trees have been found growing in HLB-ravaged orchards in Guangdong and one other in Guangxi province, that appeared to be free of HLB symptoms, while all other trees planted at the same time were either dead or declining, and replants likewise were afflicted. The trees from Guangdong were propagated at the Guangdong Institute of Fruit Tree Research facilities, the original source trees have been tested three times after propagation using standard RT-PCR protocols, and they remain PCR negative for HLB. Unfortunately we have learned now that one of the original source trees was destroyed as the orchard was removed for highway expansion, but fortunately the propagated trees remain alive. Repeated qPCR tests on the propagated trees have shown them to be HLB-negative. These greenhouse grown trees were inoculated with HLB-infected budwood in early spring 2010, but until now symptoms have been observed. The symptom-free tree found in Guangxi, which was transplanted to the Guangxi CRC, is housed under screen and pushed to produce additional propagation materials. Previously produced test trees that were inoculated remain symptom free and qPCR negative, according to reports from our collaborator.
Objective 1: Generate functional EFR variants (EFR+) recognizing both elf18-Xac and elf18-CLas. Mutagenesis of Arabidopsis EFR Random mutagenesis was performed on the extracellular domain of EFR, and a library containing approximately 10^6 clones with an average mutation frequency of 0.4% was produced. From this library 13,000 clones were screened for ROS induction in response to elf18CLas. Unfortunately, No elf18CLas responsive clones were found. It was observed that there was a high frequency of non-functional clones in this library (as assessed by ROS production induced by wild-type elf18), so a further library was produced with a lower mutation rate (0.1%). A further 6,000 clones were screened from this library without isolating any elf18CLas responsive clones. Given the lack of positive results arising from these screens it has been decided to use different approaches to engineer elf18CLas responsiveness to EFR. Firstly, target mutations were produced in EFR at sites which are known to be important for elf18 binding and responsiveness. However none of these produced a response to elf18CLas. Secondly, we have shown that elf18CLas fails to compete well with elf18, in ROS and growth inhibition assays, suggesting that binding of elf18CLas EFR is not occurring. Therefore, a first step toward engineering an EFR variant capable of responding to elf18CLas is to evolve an EFR variant that gains binding to elf18CLas. In order to engineer EFR capable of binding elf18CLas, experiments have been initiated to determine the feasibility of performing a phage display screen to identify mutants of EFR. Initial data indicates that fragments of the EFR extracellular domain can be expressed in E. coli and can bind to biotin-labeled wild-type elf18. Further experiments are underway to determine the minimal region of EFR necessary for binding and the specificity of binding, which will later enable mutagenesis of this region. Objective 2: Generate functional XA21-EFR chimera (XA21-EFRchim) recognizing axYS22-Xac. Transgenic Arabidopsis plants are being produced with XA21 or XA21-EFR to assess their resistance to Xanthomonas campestris pv. campestris 8004. This work is required to test unambiguously the functionality of XA21 in conferring anti-bacterial disease resistance in dicots. In addition, tomato plants are being transformed with XA21 to determine functionality in this species. These plants will also be crossed with tomato EFR lines to determine the effectiveness of the presence of both genes in bacterial resistance.
Transgenic studies have proceeded, with several hundred plants containing various combinations of natural or synthetic genes and promoters produced, many currently in greenhouse testing and field trial locations. Additional transgenic plants were propagated for new hot psyllid greenhouse tests, and for field planting. The sweet orange citrus genome sequence was mined to identify genes controlling anthocyanin expression, in an effort to develop visual and citrus-derived markers for genetic transformation; several candidates have been identified for further experiments. More than 875 transgenic plants have now been planted with a collaborator in Martin County, and these are being monitored regularly, along with a second site in Indian River County. The plant materials growing out include sweet oranges, grapefruit and mandarin hybrids. Several new rootstock trials with more than 15,000 trees were planted throughout Florida in the last year, to assess their adaptation to evolving advanced citrus production systems; these trials have been monitored regularly, and data has been collected on their early performance. We have made significant progress on new rootstock candidate HLB response screening in greenhouse tests; rootstock hybrids are showing diverse responses when grafted with HLB-infected Valencia, ranging from extreme sensitivity to high levels of tolerance; four complex tetraploid rootstocks have shown some repression of HLB in greenhouse tests (one was symptom-free up to 22 months ). We initiated a program to rotate new germplasm (rootstock and transgenic) through a ‘hot psyllid’ house (in collaboration with Dr. Stelinski) to ensure HLB inoculation prior to approved field planting; two groups of 50 trees have been rotated through so far, scheduled for planting at a collaborators field site, under permit from DPI. Rootstock candidates that produce nucellar seedlings have been identified using SSR markers; these rootstocks were preselected for potential tree size control and some for tolerance of Diaprepes/Phytophthora. Hybrid plants for rootstock improvement from the previous season were planted, and new crosses made 2010 were just planted in the field. Previous work to develop rootstocks against other maladies (CTV, blight, Phytophthora, Diaprepes, etc.) continues, as we collected data from replicated trials and plantings. Final data have been collected from a field trial of various Valencia somaclones and seedless Midsweet selections, and following final analysis the most consistently high yielding clones from each will be moved forward for release; most candidates have already moved through the DPI-Parent Tree Program. New pummelo-grapefruit seedless hybrids have been selected, some showing field tolerance to canker; their fruit have been assayed for furanocoumarin content and several with good fruit quality have been found FC-free, potentially producing grapefruit cultivars that alleviate drug interaction concerns. Patents have been issued by the US-PTO for Valquarius (SF14W-62) and Valenfresh (N7-3), very early- and late- maturing Valencia selections respectively, and licensing is in process. Patent applications and documents for release were developed for 7 new cultivars, and these were approved for release and commercialization in January 2011 by the UF-IFAS Cultivar Release Committee.
More than two thousand transgenic lines we have produced thus far, and are planted in field trials (2 locations under permit) or are in greenhouse tests at the CREC and with grower-collaborators; we continue to monitor the HLB and canker resistance or tolerance of these lines over time. These transgenic lines contain various combinations of natural or synthetic genes and promoters. New candidate genes continue to be identified by genome mining as well as from other disease resistance plant research. Citrus-specific promoters, transcription factors, and other genetic elements are being identified and incorporated into some of the new constructs to produce more consumer friendly transgenic plants, by limiting foreign genetic elements or controlling their expression in specific tissues. Canker-tolerant transgenic grapefruit lines have been found in field and greenhouse tests, including some containing a broad spectrum, ancient disease resistance gene from rice; the latter are being propagated for HLB challenge. Data are being collected on the early performance of new advanced selections in trials planted to assess adaptation to advanced citrus production systems. We have made significant progress on new rootstock candidate HLB response screening in greenhouse tests. Diverse responses of rootstocks are being noted when grafted with HLB-infected Valencia, ranging from extreme sensitivity to high levels of tolerance. Greenhouse experiments are being continued examining interactions of rootstock and nutrients in severity of HLB symptom expression. Hot psyllid greenhouse facilities are now being used routinely to assess performance of transgenic citrus (representing our most advanced constructs with phloem-limited promoters and previously proven genes), as well as hybrids between Citrus and Poncirus, for responses to psyllid feeding and HLB development. More than 150 new rootstock candidates preselected for potential tree size control and some for tolerance of Diaprepes/Phytophthora, and have been used to produce new trees that were planted into new rootstock trials, or held for pending trials. Rootstocks developed for resistance to other maladies (CTV, blight, Phytophthora, Diaprepes, etc.) are evaluated, as we collected data from replicated trials and plantings. Additional seedless pummelo-grapefruit hybrids have been identified during the 2011-12 season, some showing field tolerance to canker, good fruit quality, and FC-free, potentially producing grapefruit cultivars that address canker and marketing issues of ordinary grapefruit. Trees were propagated onto 30 new sour orange-like hybrid rootstocks, some already shown to be tolerant of CTV quick decline, and planted in a new field trial. A second demonstration planting of advanced sweet orange selections and newly-released cultivars, selected for high yields and superior juice quality, was established to assess to demonstrate their performance and utility in commercial processing, in collaboration with a major juice processor; these trials allow comparisons to be made between different production regions with the same sweet orange candidate selections. A well-attended field day was held in mid-November at the large CREC field experiment at the St. Helena block in Dundee; this featured Vernia and Valquarius orange trees grown on a number advanced rootstock selections, and highlighted early performance (yield and HLB effects).
We continue to monitor HLB and canker resistance or tolerance among the more than two thousand transgenic lines we have produced thus far, in field trials (2 locations under permit) and in greenhouse tests at the CREC and with grower-collaborators. These transgenic lines contain various combinations of natural or synthetic genes and promoters. Currently, 54 independent transgenic events represented by 162 individual plants have shown resistance to HLB after 10 months in a ‘hot psyllid’ greenhouse structure, showing no symptoms and negative results following qPCR diagnostics; these results are based on all 3 replicates of each transgenic line showing the same results. Among these plants are commercial cultivars of orange, grapefruit, as well as new sweet oranges and promising new rootstocks from our breeding program. Genetic constructs include both antimicrobial peptides and native citrus defense genes. We continue genome mining to identify and test new candidate genes, citrus-specific promoters, transcription factors, and other genetic elements. More than 875 transgenic plants in a collaborators field site in Martin County are being monitored regularly, along with a second site in Indian River County; apparently healthy trees in these trials are being tested by qPCR for the presence of Liberibacter. Canker-tolerant transgenic grapefruit lines have been found in field and greenhouse tests, including some containing a broad spectrum, ancient disease resistance gene from rice; the latter have been tested tree times for canker responses, and continue to exhibit phenotypes that are much less severe than standard control. Data collection continues on the early performance of new advanced selections in trials planted to assess adaptation to advanced citrus production systems. We have made significant progress on new rootstock candidate HLB response screening in greenhouse tests, and identified several that appear to overcome infection, with PCR positive results coming to PCR negative; these are being propagated for new field trials to be planted in 2013. Greenhouse experiments examining interactions of rootstock and nutrients in severity of symptom expression have shown clear differences among rootstocks in response to various nutrient regimes and symptom expression. More than 150 new rootstock candidates preselected for potential tree size control and some for tolerance of Diaprepes/Phytophthora have been used to produce new trees that were planted into new rootstock trials, or held for pending trials. Rootstocks developed for resistance to other maladies (CTV, blight, Phytophthora, Diaprepes, etc.) are evaluated, as we collected data from replicated trials and plantings. Several field trials are revealing very obvious differences among experimental rootstocks for their ability to tolerate HLB infection as well as the rate at which they are becoming infected, suggesting that rootstocks may provide an important protective benefit even in infected trees; we are focusing significant efforts now to carefully monitor disease ingress and severity in the dozens of rootstock trials we have statewide Two demonstration planting of advanced sweet orange selections and newly-released cultivars, selected for high yields and superior juice quality, have been carefully monitored for HLB and canker resistance; these trials are grown using ACPS techniques, and trees are growing off very well. Finally, genetic mechanisms underlying HLB tolerance lemon have been determined. Microscopy and fluorescent compound uptake have shown that phloem is regenerated and is functional in lemon, even in symptomatic leaves. Expression of genes controlling cell wall biosynthesis is upregulated in lemon, in support on phloem regeneration. Further, defense associated genes are slightly upregulated early in disease in lemon, but are highly upregulated later in orange, utilizing all reserves rapidly and accelerating disease progression and decline.
The haploid Clementine and sweet orange sequences have been assembled, annotated, and are available to the research community at Phytozome and at citrusgenomedb.org. The new Clementine v. 1.0, has finally been validated and made publicly available in USDOE’s JGI Phytozome v 9.0 in December 2012. This assembly is a vast improvement over the first version (v 0.9) that was made available to the research community in January 2011. The genome is organized into 9 super-scaffolds, representing the basic nine chromosomes of citrus. This was made possible by the ICGC collaborative genetic mapping effort, in which are lab was a primary contributor. (See Ollitrault et al. BMC Genomics, 2012, 13:593 DOI:10.1186/1471-2164-13-593). New citrus sequences were generated by the Machado lab in Brazil (Ponkan mandarin, 454 and Illumina), the Gmitter lab and UF-ICBR (low-acid pummelo, Illumina), and Illumina datasets for Willowleaf/Avana mandarin, W. Murcott, Chandler pummelo, and Seville sour orange have been provided (Morgante, IGA-Italy; Talon, IVIA-Spain; and M. Roose-UCR). These have been further analyzed to the phylogeny of sweet orange, Clementine, Ponkan and Willowleaf, and sour orange; all are admixtures of C. reticulata and C. maxima, in varying degrees. Surprisingly, we have identified the male parent that gave rise to sweet orange. The fine-scale characterization of citrus genotypes opens the possibility that ancient C. reticulata/C. maxima admixtures (such as sweet and sour orange) can be recreated by conventional breeding guided by a set of genome-wide markers, enabling incorporation of specific, limited genomic regions from other citrus or relatives to confer disease resistance, yet retaining the essence of marketable fruit phenotypes. Additionally, we have been able to understand better the evolutionary relationships of ancestral citrus species, and their diversification over time. A manuscript based on these results has been expanded and prepared for submission. Work proceeds on the other objectives of this project. New experiments with new vectors have been initiated to attempt transient gene silencing of HLB and citrus canker-associated genetic targets identified from our microarray studies. We have also initiated preliminary yeast-2-hybrid experiments, as an alternative approach to identifying the effects of target genes on plant phenotypes and disease resposnse. We have used the GoldenGate assay platform for hi-throughput genotyping of DNA from >150 individuals of a large mapping family, and a second family is currently being mapped. Plans have been made for collaboration with the Dvorak lab to anchor the sweet orange genome sequence to the linkage map, thus substantially improving the quality and utility of the previously produced assembly. The genotyping by sequencing (GBS) project is proceeding, once proof of concept was provided, and mapping is underway in a large segregating Citrus x Poncirus family. The RNA-seq project to uncover differences in gene expression over time between HLB-sensitive and tolerant citrus has proceeded. RNA samples have been prepared from appropriate times in the disease process, and libraries have been recreated to test, prior to the full sequencing effort. Preliminary runs have enabled us to multiplex libraries and these are ready for sequencing once we have lanes available on the instrument.
The haploid Clementine and sweet orange sequences have been assembled, annotated, and are available to the research community at Phytozome and at citrusgenomedb.org. The new Clementine v. 1.0, will soon be publicly available. New citrus sequences were generated by the Machado lab in Brazil (Ponkan mandarin, 454 and Illumina), the Gmitter lab and UF-ICBR (low-acid pummelo, Illumina), and Illumina datasets for Willowleaf/Avana mandarin, W. Murcott, Chandler pummelo, and Seville sour orange have been provided (Morgante, IGA-Italy; Talon, IVIA-Spain; and M. Roose-UCR). Comparative analysis has elucidated the phylogeny of sweet orange, Clementine, Ponkan and Willowleaf, and sour orange; all are admixtures of C. reticulata and C. maxima, in varying degrees. The fine-scale characterization of citrus genotypes opens the possibility that ancient C. reticulata/C. maxima admixtures (such as sweet and sour orange) can be recreated by conventional breeding guided by a set of genome-wide markers, enabling incorporation of specific, limited genomic regions from other citrus or relatives to confer disease resistance, yet retaining the essence of marketable fruit phenotypes. A manuscript based on these results has been expanded and prepared for submission. Work proceeds on the other objectives of this project. We identified miRNAs induced in citrus-pathogen interactions, presumably regulating target genes involved in signaling pathways and metabolic events important for plant resistance. In order to set up protocols to validate microRNA expression in plant-pathogen interactions and identify target genes, we performed a comprehensive analysis of the expression of 7 different citrus miRNAs in the context of 4 different Xanthomonas citri subsp. citri (XC) ‘ Citrus limon interactions, and certain miRNAs appear to be XC strain specific in their responses. We have used the GoldenGate assay platform for hi-throughput genotyping of DNA from >150 individuals of a large mapping family; we have produced a preliminary linkage map that shows excellent coverage and distribution of markers. The genotyping by sequencing (GBS) project has proceeded and currently it appears that it may generate as many as 2000 high-quality SNP markers for mapping a large segregating Citrus x Poncirus family. A large scale RNA-seq project to uncover differences in gene expression over time between HLB-sensitive and tolerant citrus, that weren’t seen previously in microarray studies, or to validate those already seen, has progressed. RNA samples have been prepared from appropriate times in the disease process, and libraries have been recreated to test, prior to the full sequencing effort.
This project is assessing a range of citrus germplasm and relatives for tolerance or resistance to HLB, through greenhouse assays and field tests; these germplasm resources were selected on the basis of research and observations in Asia and Florida. We have produced seedlings from 7 pummelo accessions (10-15 each), Citrus latipes (13 seedlings) and some hybrids of this species with trifoliate orange, 4 natural pummelo-mandarin introgression hybrids (9-16 each), 6 other miscellaneous wild citrus types (4-12 each), and various sweet orange lines for which there is anecdotal evidence of differential sensitivity to HLB. Subsets of these families have been inoculated with HLB-infected, PCR positive budwood of Carrizo citrange to ensure freedom from CTV cross-contamination, are being grown in a climate controlled, DPI-certified greenhouse and monitored for symptom development. Currently symptoms are being noted among some of the accessions, and we are collecting information on disease symptom progression. We have increased the numbers of individuals of most accessions, and have begun re-inoculations of previously inoculated seedlings with the same HLB source. We have extracted nucleic acids from most individuals, run RT-PCR on these, and we continue to find very few PCR+ plants. We will continue to monitor these individuals in the greenhouse. The Core Citrus Mapping Population, a genetically well-characterized collection of more than 250 citranges that we proposed to test at the Picos Road Farm near Ft. Pierce have been budded and growing off, prior to field planting. This population is of significant interest as the trifoliate orange and some of its hybrids are very HLB-tolerant, and this experiment is an opportunity to explore potential tolerance from these sources. Currently, there are at least 8 propagations of a total of 102 individuals from the original CCMP, and we plan to plant these this summer 2011. To conclude, a wide range of genetic materials have been produced and prepared for greenhouse and field testing for their tolerance or susceptibility to HLB. We have been unable to find a cooperator to plant the same experimental materials in the field as we have been testing in our greenhouse. The exception to this is the CCMP, soon to be planted.
This project is assessing a range of citrus germplasm and relatives for tolerance or resistance to HLB, through greenhouse assays and field tests; these germplasm resources were selected on the basis of research and observations in Asia and Florida. We have produced seedlings from 7 pummelo accessions (10-15 each), Citrus latipes (13 seedlings) and some hybrids of this species with trifoliate orange, 4 natural pummelo-mandarin introgression hybrids (9-16 each), 6 other miscellaneous wild citrus types (4-12 each), and various sweet orange lines for which there is anecdotal evidence of differential sensitivity to HLB. Subsets of these families have been inoculated with HLB-infected, PCR positive budwood of Carrizo citrange to ensure freedom from CTV cross-contamination, are being grown in a climate controlled, DPI-certified greenhouse and monitored for symptom development. Currently symptoms are being noted among some of the accessions, and we are collecting information on disease symptom progression. Additional seedlings that have reached sufficient size have now also been inoculated with the same HLB source. We have extracted nucleic acids from most individuals, run RT-PCR on these, and have found very few PCR+ plants. We will continue to monitor these individuals in the greenhouse. The Core Citrus Mapping Population, a genetically well-characterized collection of more than 250 citranges that we proposed to test at the Picos Road Farm near Ft. Pierce have been budded and growing off, prior to field planting. This population is of significant interest as the trifoliate orange and some of its hybrids are very HLB-tolerant, and this experiment is an opportunity to explore potential tolerance from these sources. We continue to seek additional germplasm resources, to expand the breadth and depth of the material categories we described in our proposal; a source for new C. latipes hybrids has been identified. We are still exploring other options within Florida for a field trial, but no secure, long-term commitments have been forthcoming, despite multiple discussions with growers throughout the state. To conclude, a wide range of genetic materials have been produced and prepared for greenhouse and field testing for their tolerance or susceptibility to HLB. We have expanded, and are continuing to expand, the number of types we wish to challenge. We will be developing new information about potentially tolerant/resistant germplasm that can lead to expanded efforts to capture and exploit the genetic basis for this phenomenon.
This study addresses two general questions: 1) Will our constructs disrupt normal growth and development in citrus, and 2) Will these constructs confer a degree of resistance to infection by Liberibacter asiaticus? We have answers for the first question and seek a one-year extension to address the second. Objective 1. Express R proteins in a phloem-specific manner in Arabidopsis and citrus. It was evident very early from the results of our experiments, and of others, that the Arabidopsis SUC2 promoter was phloem-specific in citrus and, thus, efforts were directed towards the generation of transformed citrus containing wild type and constitutive mutants of the two R genes, SSI4 and SNC1. Our rationale was that by restricting expression to phloem tissues (or to the wounding response) potential negative effects on growth and development would be minimized. From 30-60 transformants of each R gene variant were obtained in Arabidopsis and at least 10 in citrus (Duncan grapefruit). In Arabidopsis, some stunting was observed when transformed with the constitutive ssi4, but not with wild type SSI4, or with wild type or mutant SNC1. A similar result was obtained with citrus; however, the stunted growth (or seedling death) phenotype was much more pronounced. However, as with Arabidopsis, no abnormal phenotype was observed with either variant of SNC1. e triggered by psyllid feeding. This objective is a variation of the first, except the restriction in expression of the potentially harmful R genes was imposed by the wound-inducible PAD4 promoter, a promoter known to be activated by aphid feeding in Arabidopsis. Our rationale was that in case AtSUC2-directed expression resulted in a stunted growth phenotype, the use of an inducible promoter, such as PAD4, would provide a way to evaluate the effectiveness of R protein expression in inhibiting Liberibacter infection. Our expectation was that the Pad4 promoter would not be active, except upon deliberate wounding under controlled conditions. As with the AtSUC2 promoter, the expression pattern of PAD4 was more variable in Arabidopsis as determined using a GUS reporter; however, PAD4/GUS expression in transformed citrus appeared to be strictly wound-inducible. Expression of the R gene variants using the PAD4 promoter gave a result similar to that obtained in Arabidopsis: expression of the SSI4 constitutive mutant was sometimes harmful to the plant; whereas, expression of the constitutive mutant of SNC1 was not. These experiments can be summarized as follows: 1-Restricted expression of the wild type SSI4 and SNC1 genes using either the AtSUC2 or AtPAD4 promoter had no negative impact on growth and development in citrus. 2-Similarily, expression of the constitutive mutant of SNC1 had minimal effect on growth and development. In contrast, expression of the constitutive mutant of SSI4 is sometimes harmful to normal growth and development in both Arabidopsis and in citrus. Additionally, preliminary tests indicate that none of the constructs effected psyllid feeding preferences. In preparation for assessing disease resistance of the transformed citrus, a single leaf assay to monitor the early events in the transfer of Liberibacter from the psyllid to the plant has been developed as outlined in the proposal pending with the CRDF (Nov 2012). In brief, the real time PCR protocol was refined by developing calibration curves for the Las and various plant and psyllid control amplicons so that detection is now reproducible down to 12 copies. In addition, significant improvements have been made in single-leaf cage design that will enable feeding to be restricted to a 6 mm area of leaf.
For the last three months of 2012, the Mature Tissue Transformation Laboratory (MTTL) continued to operate in the ‘maintenance’ capacity mode. Level of operation was determined by the amount of plant material available and its quality. The process of increasing the number of rootstock plants is slow and it has been hindered by low germination rate of seeds that are old. Although new seeds were ordered in December, they will not be available until late January/mid February when Swingle citrumelo and C. macrophylla fruit are available. One of the batches of Hamlin buds grafted in early October had low percentage ‘take/success’ rate. The outside provider of grafting services claimed that the buds coming from mother plants were not of the highest quality. In the meantime, this person has left the business and the facility contracted other provider. In couple of experiments, a high percentage of explants that were used in co-incubations with Agrobacterium got contaminated. We are investigating whether those incidences were the result of human error in the steps of transformation taking place in the laboratory, or if plants that served as starting material for explants were infected while in the growth chamber. During these three months, six co-incubation experiments were performed. Four of those experiments were done with Valencia explants and two with Hamlin explants. For the Valencia experiments, we cut 2170 explants and 1030 explants were cut for Hamlin experiments. In order to be able to assess the ability of the lab to process different orders at the same time, two additional Agrobacterium strains were used for co-incubation experiments. One of those harbored a binary vector with the gene for green fluorescent protein (GFP) as a reporter gene. In one of the Hamlin experiments, out of 16 shoots inspected for GFP fluorescence two were positive. Those two shoots were micro-grafted on Carrizo rootstock plants. Some GUS assays were done on shoots obtained from experiments done earlier. Out of 19 shoots, one was positive. Two additional Ray Ruby plants were cleaned of microorganisms and are ready to become source of budding material. One more Hamlin plant was also cleaned.
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