Transgenic plants containing our stacked transgenes are being clonally propagated for disease resistance evaluation and the first trees will be challenged for HLB resistance in spring 2015. Improving Consumer Acceptance: Following the successful demonstration of the inducible cre-lox gene system, the plant transformation vector has been modified to contain our NPR1 gene and Agrobacterium mediated citrus transformation is underway to incorporate this gene. Induction of early flowering to reduce juvenility (Carrizo transformed with the FT gene): After numerous attempts, we have finally produced transgenic Carrizo citrange plants expressing the clementine-derived CFT3 gene. Several of the plants have flowered once in the greenhouse in the juvenile state. These plants have been micrografted to standard rootstock and are in the greenhouse for further evaluation and observation. The new transgenic field site at the Southwest Research and Education Center (working with Dr. Phil Stansly) was successfully established, and approximately 320 transgenic citrus plants were planted as follows: Constructs: pCIT 107O (35s-CEMA) Line/Events: 15 Constructs: pCIT 109 (35s-SABP2) Line/Events: 24 (SABP2 is a SAR-inducing gene showing great promise) Constructs: pCIT 109A (AtSUC2-SABP2) Line/Events: 57 Constructs: pCIT105 (35s-CEME) Line/Events: 34 Constructs: pLC 216 (35s-LIMA) Line/Events: 190. Note: most of these are transgenic LIMA rootstocks (Carrizo/Orange 16) with non-transgenic Valencia scion. Plants in our Indoor RES structure have not flowered this year. It is possible greenhouse temperature may have played a role in the flowering process. We will attempt to keep the greenhouse unheated this fall in hopes of initiating flowering in spring 2015. We have achieved rapidly growing transgenic sweet orange trees through thorniness. We are also planning an outdoor RES type structure for transgenics.
St. Helena trial (20 acre trial of more than 70 rootstocks, Vernia and Valquarius sweet orange scions, 12 acres of 5.5 year old trees, Harrell’s UF mix slow release fertilizer and daily irrigation). The 2nd annual application of CRF was applied, this was the 2nd application that contained TigerSul micronutrients. The formula was modified according to our greenhouse results, and included increased concentrations of manganese and boron. Trees appear to be responding well, as even some control trees on Swingle and Volk were showing a significant improvement in health. Approximately 120 trees were reset; we added trees on rootstocks US-897 and x639 for comparison. Rootstock trial at the GREC (Balm): a 3-year old trial of Vernia on 15 rootstocks (nearly 100% infected with HLB) was treated with Compo CRF (donated) and a blend of TigerSul micronucrients and poly-coated sodium borate (Florikan) – in an effort to see if the positive results in the greenhouse study could be extended to the field. This treatment will be repeated in January. Greenhouse Experiments – Nutritional study: highly symptomatic trees on various rootstocks were treated with the 3x overdose of TigerSul manganese and polymer-coated sodium borate (Florikan); several trees are showing recovery (new healthy growth). Protecting Seed Source Trees: Transgenic Orange #4 (UFR-2)plants containing the GNA transgene have been clonally multiplied as rooted cuttings and are being sized up for evaluation. Transgenic Orange #16 and Orange #19 (UFR-4) tetrazyg plants transformed with GNA have being clonally multiplied in a mistbed to provide replicated plants for evaluation.
Our project aims to provide durable long term resistance to Diaprepes using a plant based insecticidal transgene approach. In this quarter, we have cloned all the components necessary for this study. The plant transformation vectors containing the GNA, APA and ASAL genes driven by either the root specific RB7 promoter or the citrus derived C1867 promoter have been constructed. Vectors containing the CpTI gene driven by a SLREO promoter that targets the transgene to the mature root cortex have also been produced. In addition, plant transformation vectors containing the gus gene driven by these root specific promoters have also been produced to demonstrate proof of functionality of the root specific promoters. Transformation experiments to produce genetically modified rootstocks with each of this promoters will be initiated in the next quarter as seeds become available.
HLB’s impacts have led to grower interest in advanced production and harvesting systems with the potential for early and sustainable yield, as well as ease of harvest and other management efficiencies. The goal of this project is to identify appropriate rootstocks among exiting field trials and those soon to be planted that are well suited to advanced citrus production and harvesting systems. Existing field trials previously planted with size-controlling rootstock candidates have continued to be observed, including the portion of the St. Helena project planted with dwarfing selections, a 40-acre Hamlin/Valencia cooperative rootstock trial with trees planted between 300-500/acre, and a high density planting of LB8-9 (Sugar Belle). Data are being compiled on rootstocks to identify new candidate rootstocks for larger’scale ACPS trials. Seedlings of some Flying Dragon hybrids that have come through the ‘HLB gauntlet’ screening process (grafted with CLas-infected Valencia budsticks, and then cycled through a hot psyllid house, ending with no obvious HLB symptoms) were planted in the field, under a DPI permit for further observation. Seed were collected from previously untested Flying Dragon-derived hybrids, as well as from a range of other complex interspecific hybrids with tree size control potential; these seed were characterized for polyembryony and have been planted to assess relative trueness to type and seedling vigor; observations are in progress to identify the best candidates for new trials. A large scale, ACPS trial was planned in collaboration with a major citrus outfit, and seeds collected in 2013 have been provided to the propagating nursery.
HLB’s impacts have led to grower interest in advanced production and harvesting systems with the potential for early and sustainable yield, as well as ease of harvest and other management efficiencies. The goal of this project is to identify appropriate rootstocks among exiting field trials and those soon to be planted that are well suited to advanced citrus production and harvesting systems. Existing field trials previously planted with size-controlling rootstock candidates have continued to be observed, including the portion of the St. Helena project planted with dwarfing selections, a 40-acre Hamlin/Valencia cooperative rootstock trial with trees planted between 300-500/acre, and a high density planting of LB8-9 (Sugar Belle). Data are being compiled on rootstocks to identify new candidate rootstocks for larger’scale ACPS trials. Visits were made this summer to three different trials featuring tree size controlling rootstocks by team members, and assessments of tree conditions were made. Seedlings grown from previously untested Flying Dragon-derived hybrids, as well as from a range of other complex interspecific hybrids with tree size control potential, were grown to assess seedling vigor and other characteristics; selections were either discarded because of poor growth habits, excessive phenotypic variation, or poor germination. The best performers from this group were listed and substantial quantities of seed have been or are being harvested currently, to be available for new plantings and trials in the next year. Seeds are being harvested from rootstock hybrids that are bearing their first fruit, to be evaluated for potential in greenhouse tests in the coming year. Additionally, large lots of seed from candidates for ACPS planting already identified from existing field trials are being harvested to prepare for new trial opportunities in the next year. Finally, a field day has been planned to showcase some of the CRDF Matrix rootstocks in a trial near Vero Beach, several of which are showing good performance even though they are affected by HLB, and with ACPS potential by virtue of smaller tree size combined with higher yield efficiencies.
The transgenic plants to be developed for this project are now growing in two different locations in secure greenhouses and growth chambers. Eight independently-transformed citrus plants carrying the FLT-antiNodT fusion protein expression construct were shipped from the Citrus Transformation Facility at the University of Florida Citrus Research and Education Center at Lake Alfred, FL, to Dr. McNellis’ lab at the Pennsylvania State University at University Park, PA, in early October, 2014. An additional seven independently-transformed citrus plants carrying the FLT-antiNodT fusion protein expression construct were shipped to Dr. Tim Gottwald’s lab at the United States Horticultural Laboratory in Fort Pierce, Florida. The plants at both locations are growing well. In summary, a total of 15 independent transgenic lines now exist for the FLT-antiNodT fusion protein expression construct. These plants will need to be grown for some time to produce larger plants, which will be used to vegetative propagation of multiple individual plants for each independent transgenic line. Interestingly, at least one of the transgenic lines at University Park is blooming, despite being less than 8 inches tall. This is an expected phenotype of the transgene, since overexpression of the FLT domain of the engineered FLT-antiNodT fusion protein is expected to trigger precocious flowering due to the “florigen” blooming promotion activity of the Flowering Locus T (FLT) protein. This bodes well for the successful expression of the FLT-antiNodT fusion protein from the transgene in these plants. Plants at University Park will be used for analysis of the transgene expression, while plants at Fort Pierce will be tested for resistance to citrus greening, in collaboration with Dr. Gottwald.
Leaf samples were collected from 895 of the original 912 plants that covered the whole field population (116 individuals in total) in Fort Pierce this May; 17 plants were no longer living from the original planting. Real-time PCR was used on these samples to quantify CLas levels, as a measure of hist responses to HLB disease. A total of 16 individuals were found to be basically PCR-negative for CLas (average Ct value >35); these included 6 Poncirus parents and 10 F1 hybrids, of which 4 F1 progenies were completely PCR-negative to for CLas (each repeat’s Ct value >35). Compared with the results from last October 2013 on the same population, the PCR-based measures of CLas were more serious; the average Ct value of the whole population in October 2013 was 32.6 but is now 31.2 for this May 2014). Last October, 6 F1 hybrids were completely PCR-negative for CLas (i.e., no CLas was detected in any of the replicates of each hybrid); however, in our May assessments, two of these F1 hybrids now had some individual replicates wherein CLas was detected although at rather low titer. Notably the other 4 F1 hybrids with all replicates remaining PCR-negative are exactly the same individuals in October 2013 and May 2014. Besides, HLB symptoms ratings, canker symptoms were ranked and stem diameters were measured for the preselected monitor population (20 individuals comprising 164 plants); this information was recorded in June 2014. The results showed that the average rating (on a scale of 0-3, 0 being none and 3 being severe) for HLB symptoms is 1.4, and for canker symptoms is 1.8. Finally, we finished the propagations of the greenhouse backup population for the field trial accessions that could be propagated; at last we obtained a 257-plant F1 population derived from 73 progenies in total. These plants were transferred to another protected greenhouse structure waiting for the inoculation Candidatus Liberibacter asiaticus. The results from the natural infection assessments in the field will be compared with the artificially inoculated plants in the greenhouse.
This July 2014, leaf samples were collected from 887 of the original 912 plants that covered the entire field population again to quantify the CLas levels by real-time PCR. At this sampling time, 6 F1 progenies remain basically PCR-negative for CLas (i.e., most replicates remain with undetectable levels of CLas), of which 3 are completely HLB free among all replicates. The average Ct value of the whole population declined further to 29.9. Combining all results of PCR monitoring over time, 4 F1 hybrids generally have been for the most part CLas-free, except for occasional low-level and inconsistent detections of CLas by PCR in a small number of the replicates. Two F1 hybrids have always tested negative for CLas, among all the individual replicates across all sample dates. In addition, we collected samples from the selected monitoring population again, and we took an additional step to have greater confidence in our results; we quantified CLas using double reference genes for both the plant and the bacterium. The results verified the accuracy of using a single reference gene for the whole population (correlation R2=0.91). Further, this step also verified the 2 constantly and completely CLas-negative F1 hybrid progenies in all replicates of each. Finally, HLB symptoms, canker symptoms, and stem diameters were assessed again for the pre-selected monitoring population. The average rating across the entire population for HLB was 1.6 and for canker was 2.1. We have begun artificial inoculation experiments in the protected greenhouse structure, by testing a recently developed inoculation method recommended to us by a colleague, using sap extracts from PCR+ plants rather than grafting the infected budsticks. We have started experiments to determine the effectiveness of this approach in our hands using 3 year-old Valencia orange trees. Finally, we have produced a sequence-based marker genetic linkage map for this population, to support subsequent QTL mapping efforts.
We have continued to monitor previously identified candidate survivor trees at the CREC, the GCREC, and some Polk County commercial groves where we have planted out materials from the CREC breeding program. Most of the trees now display symptoms after 2.5 years, and the number of unaffected has decreased. Further assessments of other populations of trees has revealed new candidates among younger trees that had not previously been surveyed. Trees identified at several locations through out Florida have been sampled for both budwood and root tissues. We have propagated rootsprouts, by budding and by rooted cuttings. Groups of small trees are growing of for further propagations to test their responses to HLB. We extracted DNA from these new rootsrpouts from other survivor trees and compared their DNA fingerprints with what we produced from feeder roots collected previously, as expected, the fingerprints were identical. The use of SSR DNA fingerprinting was supported by the enhancement supplement (now ended) and we are continuing this approach so at the least we can confirm whether the rootsystems sampled are nucellar or of zygotic origin. New rootstock samples collected from trees were used to confirm nucellar embryony. Thus far, all except one of the rootstocks collected has been shown to be a nucellar seedling of the presumed rootstock. Routine fingerprinting of all scion varieties sampled has shown them all to be true to type; this does not discount the possibility of mutations for HLB tolerance/resistance. We have gathered information on several other possible survivors identified and visited some groves to assess the condition of the reported trees. In several instances, the reported survivors actually exhibited more symptoms than would qualify as a ‘healthy’ survivor. Two hybrids of unknown origin that we have been observing for several years in CREC groves, and were found to be HLB-free have begun to display symptoms; one of these is declining quickly while the other is remaining healthy overall, despite symptom development in a small sector. New reports of survivors have been decreasing, though there have been some. Materials have been collected from an unusual grove in Lake County that appears to be completely free of HLB symptoms.
We routinely monitor previously identified candidate survivor trees at the CREC, the GCREC, and some Polk County commercial groves where we have planted out materials from the CREC breeding program, including those more recently identified. We continue to grow out and propagate recovered rootsprouts and scion materials collected, to produce groups of trees that can be tested for their responses to HLB. We have been informed of some additional newly suspected survivors, and have cataloged the information for visits in the autumn and winter, when HLB symptoms become more obvious. The materials collected from a grove in Lake County that appears to be completely free of HLB symptoms have been propagated and increased. PCR results have been negative; these plant materials now have placed into a growth room with CLas+ ACP populations to attempt natural inoculation to assess their responses. Materials were collected from a legacy Parson Brown tree found in Marion County, and apparently free of HLB. Plans are in place to visit several newly identified candidate trees in the coming months, as well as to revisit trees from which samples were collected previously to determine their current status and performance; scion and rootstock materials will be collected, tested, and propagated, as seen fit.
This project is built on the legacy of materials produced and field trials planted across the past several years. The objectives are to evaluate existing families and created germplasm in the field and in greenhouses for their responses to HLB and citrus canker, to carefully observe and document rootstock effects on severity and rates of progression of HLB symptoms, and to maintain the facilities and activities involved in the state-wide assessment of new scion and rootstock performance with a focus on HLB responses. Assessments of HLB field tolerance are continuously carried out in the vast collection of raw germplasm that we maintain, and new selections have been identified, and several previously found continue to hold up to HLB; additional evidence is accumulating supporting what may be differential sensitivity to HLB among sweet orange clones from the CREC program. We have essentially completed observations for the season on fifteen different individual rootstock trials planted throughout the citrus production regions of FL. Additional materials from the CRDF Rootstock Matrix list have been sent to the DPI PTP for STG and indexing, so certified materials can be made available to nurseries and TC companies for rapid increase of those trees for subsequent field trials and demonstrations. Individual Valencia trees on 70 rootstock hybrids (grown from grafted budsticks of HLB-infected Valencia) successfully made it through the greenhouse and ‘hot psyllid’ house phases of the HLB gauntlet and were planted at Picos Farm including 45 complex tetraploids, 15 robust Flying Dragon hybrids (ACPS potential), and 10 diploid sour orange-like hybrids. The first phase of an ‘interstock’ experiment was completed by budstick grafting interstock candidates (10 per selection) onto Swingle citrumelo rootstock liners. Candidates included HLB tolerant pummelos, polyploid pummelo/mandarin hybrids, a wide intergeneric hybrid, and sweet orange as a control. Six new rootstock trials were planted; five of these were with rootstocks introduced from outside of Florida and all showing some evidence of tolerance of HLB and one of our UF scion cultivars, Valquarius. The 6th trial was a major replanting of the grove at the PSREU in Citra, to introduce new mandarin cultivars, along with better tree size-controlling rootstocks for mechanical harvesting. Finally, a new cybrid sweet orange showing HLB tolerance and with exceptional juice quality (Rhode Red Valencia nucleus + Hamlin cytoplasm – Brix: 16; Ratio: 18.44; Juice %: 54.7; Lbs. solids/box: 8.7) was discovered.
This project is built on the legacy of materials produced and field trials planted across the past several years. The objectives are to evaluate existing families and created germplasm in the field and in greenhouses for their responses to HLB and citrus canker, to carefully observe and document rootstock effects on severity and rates of progression of HLB symptoms, and to maintain the facilities and activities involved in the state-wide assessment of new scion and rootstock performance with a focus on HLB responses. Assessments of HLB field tolerance are continuously carried out in the vast collection of raw germplasm that we maintain, and new selections have been identified, and several previously found continue to hold up to HLB; additional evidence is accumulating supporting what may be differential sensitivity to HLB among sweet orange clones from the CREC program. A new set of 25 HLB+ Valencia budstick-grafted hybrid rootstocks was rotated into the ‘hot psyllid’ house, following selection based on freedom from symptoms in the greenhouse test. Several new clones were entered into the DPI Parent Tree Program, including a cybrid sweet orange with exceptional juice quality and preliminary evidence of HLB tolerance, two early maturing Valencia somaclones, and twelve new promising rootstock hybrids with preliminary evidence of HLB tolerance. A total of 383 new transgenic citrus trees with potential HLB tolerance/resistance were planted at the USDA Picos Farm site under APHIS permit. Careful observations were made at a rootstock trial in Vero Beach from where several of the UF rootstocks already approved for release have been selected; a field day is being planned to highlight these and other promising rootstocks at this location. Hybrid rootstocks grown out from the previous season’s crosses have been prepared for field planting. Seedlings grown from seed collected from more than 3 dozen new candidate rootstocks were evaluated for trueness to type, and new seeds have just been collected from additional new candidates not evaluated previously.
We aim in this project to genetically manipulate defense signaling networks to produce citrus cultivars with enhanced disease resistance. Defense signaling networks have been well elucidated in the model plant Arabidopsis but not yet in citrus. Salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) are key hubs on the defense networks that are important for broad-spectrum disease resistance. With a previous CRDF support, the PI’s laboratory has identified ten citrus genes with potential roles as positive SA regulators. Characterization of these genes indicate that Arabidopsis can be used not only as an excellent reference to guide the discovery of citrus defense genes and but also as a powerful tool to test function of citrus genes. This new project significantly expanded the scope of defense genes to be studied by examining the roles of negative SA regulators and genes affecting JA and ET-mediated pathways in citrus defense control. We originally proposed a three-year research with three specific objectives: 1) identify SA negative regulators and genes affecting JA- and ET-mediated defense in citrus; 2) test function of citrus genes for their disease resistance by overexpression in Arabidopsis; and 3) produce and evaluate transgenic citrus with altered expression of defense genes for resistance to HLB and other diseases. However only one-year funding was provided to support our research. With this support, our effort has been focusing on Aim 1. We have cloned 15 citrus defense genes affecting SA, ET, and/or JA pathways from citrus cDNA libraries and made overexpression constructs with the full-length cDNA clones of these genes in the binary vector pBINplusARS. While our focus is on gene cloning with the one-year support, we also initiated Arabidopsis transformation and testing disease resistance of the transgenic plants for these cloned genes (Aim 2). So far we obtained T1 seeds for 10 gene overexpression constructs of the cloned citrus genes and preliminary testing of transgenic plants overexpressing CsJAR1 or CsACD1 revealed promising results for future studies. In addition, citrus transformation with 10 citrus gene overexpression constructs has been initiated (Aim 3). In addition, we continue to characterize transgenic citrus plants expressing the SA positive regulators, as proposed in the previous project (#129), although the support of the project was already terminated. Our recent analysis of transgenic citrus overexpressing CsNDR1 (five out of ten transgenic plants) showed that these plants had lower rates of HLB positive three months after inoculation psyllids carrying Liberibacter asiaticus, suggesting that CsNDR1 overexpression confers enhanced HLB resistance. This result is consistent with CsNDR1 overexpression in conferring enhanced disease resistance in Arabidopsis (Lu et al., 2013). We will follow up with these plants for a further test of HLB resistance in the next few months. Lu, H., Zhang, C., Albrecht, U., Shimizu, R., Wang, G., and Bowman, K.D. (2013). Overexpression of a citrus NDR1 orthodox increases disease resistance in Arabidopsis. Front Plant Sci 4, 157.
Huanglongbing (HLB) is the most serious threat to the U.S. citrus industry. Several transcriptome studies on citrus-HLB interactions have been published in recent years that provide a large pool of HLB-responsive candidate genes. These studies have identified many genes with respect to HLB infection but there is only a modest overlap in results which could be due to multiple factors such as lab-specific or genotype-specific effects. Interrogating all the transcriptome studies through meta-analysis can provide a better understanding that cannot be gained by analyzing single studies. A combined list of 7,412 differentially expressed gene probes was generated by using a Teradata in-house SQL script. Weighted gene co-expression network analysis was conducted and 21 modules with major hub genes were identified that show the greatest number of interconnections and contribute most to citrus-HLB interaction. We have shortlisted 2,000 candidate genes based on several criteria such as their expression patterns among different datasets, putative function and position as hub genes in the co-expression gene network. These candidate genes will be validated using high throughput target capture and massively parallel sequencing of targeted gene regions among susceptible and tolerant citrus genotypes. Toward this objective, we have designed a target capture system based on the Agilent SureSelect system. Different citrus accessions and relatives were collected and tested for their responses to HLB; they revealed a wide range of responses from susceptible to tolerant and resistant. DNA from these selected accessions will be isolated and used for the SureSelect library preparation. The bait of the library will be custom designed 120mer probes at every 60bp interval specific to the shortlisted candidates. The library will be sequenced using Illumina HiSeq 2000 to rapidly identify sequence variations in the candidate genes in selected genotypes. Phenotyping of these selected accessions is continuing in the field of Florida, using parameters such as the titer of CLas, and HLB symptom severity ratings. The titer of CLas in the HLB-infected plants was determined by TaqMan probe based realtime PCR, and the HLB severity on the plants was evaluated by experienced researchers. These phenotype data continue to be collected several times every year, and correlations among these data will be assessed before use in association analysis. From the preliminary investigation, we can see a great difference in HLB tolerance among these citrus species and relatives. Many HLB-tolerant citrus relatives were revealed from the preliminary investigation, including Citrus latipes, Poncirus trifoliata, and Severinia buxifolia. This study will lead to the identification of the genes most likely associated with HLB tolerance.
The project has two objectives: (1) Increase citrus disease resistance by activating the NAD+-mediated defense-signaling pathway. (2) Engineer non-host resistance in citrus to control citrus canker and HLB. For objective 1, both soil drench and foliar spraying of NAD+ have been performed. In the side-by-side experiment with the plant defense activator Actogard, soil drench provided good protection against citrus canker, whereas foliar spraying had limited effects. We are repeating the experiment and trying to find the best approach for NAD+ application. We have also been testing NAD+ analogs to identify potential chemicals for citrus disease control. For objective 2, newly generated transgenic plants are growing in greenhouse. Presence and expression of the transgenes have been tested. All transgenic plants are growing in the greenhouse and will be tested for canker resistance. Citrus homologs of the defense genes have been cloned and sequenced. The will be used for functionality test through complementation experiment.
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