This project is a continuation of a previous project #95 “PREPARATION OF ANTIBODIES AGAINST CANDIDATUS LIBERIBACTER ASIATICUS”. Progress reports for the previous project are on file. The reimbursable agreement with CRDF was established on September 5, 2012. We continue to study the literature to identify vectors to use for a future scFv library made as part of this project. The goal is to find a suitable vector that is not encumbered by intellectual property and patent issues. I have written twice to a laboratory in Germany which has published results with a suitable vector but have had no reply. We are also optimizing the cloning strategies that will be used to move already selected scFv into transgenic plants. A visiting scientist arrived and began work 6-15-2013. We have obtained the vector, pUSHRL-26, to be used for plant transformation of the scFv constructs from Ed Stover at Fort Pierce and the plasmid has been purified. We have purchased the restriction enzymes and designed primers to be used for PCR to amplify the cloned scFv encoding inserts from vector pKM19. The cloned inserts will be sequenced to confirm that they are correct and then cloned into the transformation vector. Related research with the existing scFv is underway on project 551.
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, we are planning a microarray experiment to identify genes that are induced by NAD+ in citrus. Microarray chips have been ordered. The first batch of RNA samples we prepared did not pass the quality control (QC). We are making the second batch of RNA samples. Microarray will be performed soon. For objective 2, two non-host resistance genes against citrus canker have been cloned into the T-DNA vector pBI1.4T, a vector with good transformation efficiency in citrus. The plasmids have been mobilized into Agrobacterium and will be used for citrus transformation.
The efficiency of transmission process of Candidatus Liberibacter asciaticus (CLas) depends on the success of specific interactions between CLas and the insect vector Asian citrus psyllids (ACP). CLas is circulative propagative within ACP. The bacteria need to pass the intestinal barrier to reach the hemolymph where they multiply then they must invade the salivary glandes in order to be inoculated in a new plant host while insect feeding. Passing these biological barriers needs specific interactions between CLAS cells and the epithelial cells in the guts and the salivary glands cells. In the last few months, we were successful in identifying the receptors in the Asian citrus psyllids (ACP). The receptors are the proteins that CLas cells recognize and bind to before passing the gut to the hemolymph. Same thing, when cells invade the salivary glands to reach the food canal. the main technique we use in our study is the protein overlay assay. In this method, non CLas-carrying insect total proteins were separated by 2D-SDS-PAGE. After 2D-SDS-PAGE, the proteins were blotted onto PVDF membrane. the membrane was overlaid with extract from infect citrus then we detect the complex using antibodies against CLas membrane proteins. this method called (Far-western) Comparisons between the stained electrophoretic profiles of ACP proteins in the gel and results of far-Western blot experiments on the membrane allow to cut the protein spots from 2-DE gels for LC-MS/MS analysis. Some ACP proteins (receptors) were identified. The function of these proteins was analyzed with bioinformatics. These genes were cloned, proteins were expressed, antibody against these proteins were made. a publication described this work is in preparation. Recently, we started to identify the membrane proteins from CLas that interact and bind to the identified receptors. Since we have identified the receptors in ACP, we are planing to perform negative FAR-Western to identify the ligands in CLas. For this reason, we predicted the antigenic domains in the identified ACP-receptors to produce the antibodies. We already obtained antibodies against beta-actin. our aim for the next few months is to identify the proteins of Clas (ligands) that adhere to ACP cells using antibodies against identified ACP proteins. the yeast double hybrid system will be used to validate specific interaction for each couple (receptor-ligand) Overall, our research work is carried out according to milestone of the project.
This project is to assess how the efficiency of HLB transmission by psyllids varies depending on the stage of infection and plant development. Our main accomplishments on this project are listed below. 1) Electron microscopy examination of the sites on the leaves of citrus plants where HLB-positive psyllids fed for 7 days demonstrated that even at early stages of infection (starting from 3-4 weeks after the beginning of the experiment) the bacteria could be already visualized in the initial sites of introduction. 2) In order to characterize inoculum sources of the bacterium available for psyllids within an infected tree, we examined the proportion of psyllids that acquired the bacterium after their exposure to different types of flushes, young growing or matured symptomatic ones. Data from PCR analyses demonstrated that Las-positive psyllids were collected from both types of flushes. We also conducted a similar experiment that was slightly modified in a way that psyllids fed on old and young leaves that were detached from plants and kept in 50 ml tubes (‘detached leaf experiment’). Some differences in the bacterium acquisition were obtained from these two experiment series. On average 48.33% of psyllids fed on old symptomatic flushes tested positive and 58.33% of psyllids fed on young pre-symptomatic flushes were positive. In the ‘detached leaf’ experiment, an average acquisition from young pre-symptomatic tissue was significantly higher than from old symptomatic flushes: with average of 64.26% and 23.9%, respectively. Psyllids that acquired bacteria from different flushes were next transferred onto healthy receptor plants. Analysis of numbers of plants that became infected upon inoculation with psyllids fed on different types of flushes revealed that more receptor plants that were inoculated by psyllids kept on young flushes became infected (52% of Duncan grapefruit plants and 53% of Madam Vinous sweet orange plants) and less proportion of receptor plants inoculated with psyllids that fed on old mature flushes got infected (19 and 33% of the same varieties, respectively). 3) In order to assess what types of flushes are more susceptible to psyllid inoculation with the HLB bacteria, we exposed sweet orange and grapefruit plants that have young growing flushes and plants that have only matured flushes to HLB-infected psyllids (“no young flush” plants). According to our data, both young and mature flushes could be inoculated by psyllids, yet inoculation efficiency of mature flushes is significantly lower. 4) Overall, our results support the initial observation of young flushes being more likely crucial for the disease spread at both steps of the pathogen transmission, either acquisition and inoculation are higher when young flush are present. Nonetheless, transmission associated with old tissues, which occurs at a reduced level, should not be ignored also. 5) To examine psyllid transmission rates to different citrus genotypes, we analyzed psyllid inoculation of 6 different varieties of citrus: Valencia sweet orange, Duncan grapefruit, Persian lime, Eureka lemon, Carrizo citrange, and Poncirus trifoliata. Those varieties represent plants with different degrees of susceptibility to HLB. The first four varieties showed the highest infection rates (80-100% infection), while only about 10% of Carrizo citrange and Poncirus trifoliate became infected. Poncirus and poncirus hybrids have been shown to have much greater tolerance to HLB. The fact that they are also more tolerant to psyllid inoculation with the bacterium suggests that developing hybrids of such varieties that in addition to being more tolerant would also have acceptable horticultural characteristics and would produce fruit and juice of a sufficient quality could be a solution to battle HLB epidemics before more sustainable approaches are in place.
The general goal of this project is to rapidly propagate complex citrus rootstock material for field testing. The rootstock materials to be tested will be products of the Citrus Improvement Program at the UF-IFAS-CREC in Lake Alfred. Specifically, these materials will be selected based upon their performance in the ‘HLB gauntlet’: Promising rootstock genotypes will have already been evaluated in the greenhouse and field for their ability to grow-off citrus scions that have been exposed to CLas-positive budwood and CLas-positive Asian citrus psyllids. Once candidate rootstock materials have successfully passed through this gauntlet, they will be propagated via rooted cuttings en masse in a psyllid-free greenhouse at the UF-IFAS-IRREC in Fort Pierce. From there, rootstock materials will be budded with scion materials and planted in the field for further testing for their long-term performance. The start date for this project was April, 2013. To date, the progress of this project is as follows: – Misting chamber to propagate candidate, rootstock materials as rooted-cuttings has been constructed. – Propagation materials (containers, soilless media, and rooting hormones) have been purchased. – Competitive bids have been prepared to support the construction of a new, dedicated propagation greenhouse at the IRREC. Funds allocated to this project are also being used to retro-fit an existing greenhouse at the IRREC to meet FDACS-DPI standards. The existing greenhouse will also be employed as a propagation facility for this project. – The initial cohort of advanced, tetratzygous citrus rootstock materials for en masse propagation have been identified. They are currently emerging from the ‘HLB gauntlet’ and will soon be ready for en masse propagation.
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 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 and are known to regulate 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 will significantly expand 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 regulating citrus defense. We have three specific objectives in this proposal: 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. Currently we have cloned 10 full-length genes in these categories in the entry vector pJET. Five of the genes were further cloned to the binary vector pBIN19plusARS and transferred to Agrobacteria. The Agro strains were sent to our collaborator Dr. Bowman’s lab to initiate citrus transformation. In the mean time, we started the process of transforming Arabidopsis to overexpress these genes and to test their defense function. In addition, we are continuing to generate and/or characterize transgenic citrus plants expressing the SA positive regulators, as proposed in the previous project, although the support of this previous project has already been terminated. A paper describing the cloning and characterization of the citrus NDR1 ortholog was recently published in the journal Frontiers in Plant Science.
Field experiments with nutritional and other horticultural management impacts on HLB disease: Final PCR testing of subject trees. The 20 “Valencia” and 20 Hamlin trees being treated with a comprehensive and holistic HLB management program, including foliar micro and macro nutrients and psyllid control, were sampled to perform PCR analysis at the end of the fifth year since HLB was discovered in the grove and tree yields were recorded annually. Five years before, half of the trees (10) were initially symptomatic for HLB (PCR+) and the other half were non-symptomatic (PCR undetermined; healthy). Refer to the previous two reports for the 2012/13 season yields. Samples were collected on May 5, 2013. Samples consisted of 10 HLB asymptomatic Hamlins, 11 HLB symptomatic Hamlins, 12 HLB Valencia trees with greening symptoms and 10 asymptomatic Valencia trees. Approximately 20 leaves were collected from around each tree and from each sample 4 leaves were selected and prepared for real time PCR for the detection of Candidatus Liberibacter asiaticus. Only 7 of the 21 Valencia trees tested PCR positive. These trees were in both the symptomatic as well as the asymptomatic designated categories. All of the Hamlin symptomatic trees tested PCR positive and in addition two of the asymptomatic Hamlin trees tested PCR positive. Samples of all the trees were run in fluorescent dye tests to determine if the nutrient applications had an effect on phloem translocation. The 7 PCR positive Valencia trees translocated less dye than the PCR negative trees. The PCR positive and negative Hamlin trees translocated similar amounts of dye.
In cooperation with Bryan Belcher from Davis Citrus Management we identified that bicarbonate in irrigation water from deep wells impacts root health and exacerbates Huanglongbing (HLB) symptom expression for trees on Carrizo and Swingle rootstocks. We have since learned that there is a long history of management of high bicarbonate and pH of irrigation water for Carrizo rootstock groves in the Central Valley of California. In February, we took FL growers, including John Gose from Lykes, to CA to tour Paramount Citrus groves to learn about conditioning water and soils with high bicarbonates (>100ppm) and alkalinity (> pH 7.5). The primary means are sulfuric acid injection from storage tanks managed by contractors or use of sulfur burners to generate acidity from elemental sulfur. The target is to reduce pH of water used for irrigation to 6.5. In Florida, Bryan Belcher has acidified irrigation water with N-furic acid (a mixture of urea and sulfuric acid) by injection at the well in same way as fertigation is applied. N-furic has the advantage of being safe to handle but the disadvantage of higher cost of treatment (2-3X sulfuric acid). We are currently surveying root health in Davis Citrus Managment groves that have shallow and deep wells and varying liming histories. In addition, we have set up demonstration trials to observe whether bicarbonates in the well water and soil (as residual from dolomite application) are associated with greater HLB effects on root mass density and tree health. We are also in the process of developing a site to conduct a controlled study with a combination of acidification treatments of soil and water with the requisite conditions of elevated bicarbonates.
Huanglongbing (HLB), caused by Candidatus Liberibacter asiaticus (Las), is well known to be a destructive disease of citrus that results in losses of fruit quality and quantity, canopy dieback, and tree decline. Recently, early Las infection of roots and associated root loss has been identified. The role of root infection in disease development has focused attention on the potential for interactions of HLB with soil-borne pathogens and pests. Concurrent with the spread of HLB through Florida citrus groves an unexpected rise in populations of Phytophthora spp., especially P. nicotianae (Pn), was observed in a statewide survey during unfavorable environmental conditions (i.e., periods of exceptional drought). A greenhouse study demonstrated that HLB induces a rapid increase in Pn propagule counts in the soil, but that the interaction is observed only until HLB drastically reduces root mass, confirming most recent field population results. The presence of Pn at the time of inoculation caused a significant shift in Las colonization to the root system associated with a delay in foliar symptom development. Hence, damage to the root system caused by Las-Pn interactions may reduce stress tolerance of infected trees before appearance of HLB symptoms. Fourteen months after inoculation, HLB-Pn interaction reduced total leaf area which could limit the total photosynthetic capacity of the tree, exacerbating the disruption of carbohydrate supply. Although both HLB and Pn reduced fibrous root mass, no interaction was observed. Based on preliminary results, Las-Pn interactions are likely to increase the rate of decline of HLB-affected trees.
Two field sites have been identified and temporal root sampling has begun. Sampling has already revealed seasonal variation in root infections and shifts in the root flush cycle caused by Liberibacter. Root cages to monitor new root growth in healthy and HLB affected groves are under construction and will be deployed in the next 2 months. Sampling at a rootstock trial site is underway with 6 months of data on the effects of HLB on these new experimental rootstocks. This has already begun to demonstrate how these new rootstock lines respond to Liberibacter infection. Meanwhile seedlings of the most promising of these rootstocks along with conventional and recently released rootstocks have been planted and will be inoculated with HLB to begin greenhouse experiments as soon as they reach a sufficient caliper for graft inoculation.
Objective 1 (To define the role of chemotaxis in the location and early attachment to the leaf and fruit surface). Xanthomonas citri subsp. citri (Xcc) A strain types are causal agents of Citrus Bacterial Canker (CBC) on most Citrus sp. and close relatives. Two narrow host range strains of CBC, Aw and A*, from Florida and Southwest Asia, respectively, are only able to infect Mexican Lime. As for many phytopathogens in the early stage of infection, Xcc must ingress the leaf surface to colonize the apoplast. This process may be mediated by plant signals originating from the sites of entry, such as stomata or wounds. In this study, the chemotactic profile of wide and narrow host range strains of CBC was compared with other xanthomonads causing Citrus Bacterial Spot (CBS) and Crucifer Black Rot (CBR) and related to carbon source utilization evaluated by Biolog GN system. Differences among these species and types of xanthomonads were found for motility, chemotaxis, bacterial growth and the profile of chemicals that act as chemotaxis inducers. The diversity of chemotaxis profiles was related to the patterns of methyl-accepting chemotaxis proteins (MCPs) that act as chemotactic sensors. Cluster analysis based on chemotaxis profiles and MCPs grouped narrow host range CBC strains into the same clade. Chemotactic response of CBC and CBR strains towards leaf fractions from sweet orange, Mexican lime and Chinese cabbage were compared. Differential chemotaxis responses occurred for leaf washes and apoplastic fluids depending on the combination of xanthomonad and host plant. These results suggest that xanthomonads sense signals from the host which facilitate the location of leaf entry points for specific bacterium plant associations. Objective 2 (To investigate bifofilm formation and composition and its relationship with bacteria structures related with motility in different strains of Xcc and comparison to non-canker causing xanthomonads). Differences in biofilm formation were demonstrated among the diverse CBC strains and compared to X. campestris and X. alfalfae subsp. citrumelonis. Presently, type IV pilus from Xanthomonas citri subsp. citri strains is being purified in order to obtain antibodies to be used in a microscopy strategy to confirm such protein as a main component of the protein fraction of the biofilm matrix and determine possible differences among bacterial strains. In addition assays to detect cellulose and amyloid fiber production by CBC strains, using calcofluor and Congo red, respectively, are underway. Amyloid fiber but not cellulose has been detected in preliminary assays. Moreover, role of DNA in the biofilm matrix is being evaluated by applying DNAase at different concentrations during the biofilm formation or removal process. First results confirmed the positive effect of the DNAase and therefore the role of free DNA as a component of the biofilm matrix. More assays are underway to compare altering of DNA with that of other biofilm matrix elements. Finally, initial assays of gene expression revealed differences in the level of transcription between wide and narrow host range strains of CBC for genes related to biofilm and motility.
he objectives are 1) apply a protocol for sampling grapefruit for streptomycin resistance, 2) quantify the local systemic activity of streptomycin for control of Xcc inoculum in lesions of grapefruit; 3) evaluate the efficacy of mixing copper with streptomycin compared to streptomycin alone for reduction in risk of streptomycin resistance in Xcc. Treatments of streptomycin in three grove company locations as well as a trial with additional sprays this season will be monitored in September 2013 for incidence of resistance using a sampling protocol previously developed in our program. As soon as the results are available they will be forwarded to EPA section 18 for their information/comment.
The objectives of this proposal are 1) to conduct a statewide survey of tangerine and tangerine hybrid groves to determine the proportion of strobilurin resistant Alternaria alternata isolates along with the identification and characterization of resistance-causing mutations; 2) establish the baseline sensitivity of Alternaria alternata to the SDHI class fungicide, boscalid and characterize field or laboratory SDHI resistant mutants to determine the likelihood of SDHI resistance development in Florida tangerine production and 3) Develop an accurate and rapid assay to evaluate sensitivity to DMI fungicides. During this quarter we accomplished: ‘ The paper ‘Distribution of QoI resistance in population of tangerine-infecting Alternaria alternata in Florida’ was submitted to Plant Disease. This paper was accepted for publication. ‘ The book chapter ‘Fungicide resistance in citrus’ was submitted to be published in the book ‘Fungicide resistance in North America, 2do edition’. ‘ The tangerine non-pathogenic Alternaria populations were tested for azoxystrobin sensitivity. During this time 124 isolates were screening for sensitivity using the rezasurin-based microtiter assay. ‘ In summary: 90 isolates (72%) were resistant to azoxystrobin (EC50 values higher than 10 ‘g/ml), while 34 isolates (28%) were sensitive (EC50 values lower than 0.5 ‘g/ml). ‘ DNA of 20 tangerine non-pathogenic isolates was extracted to identify the G143 point mutation in the cytochrome b gene. ‘ In vitro fitness components of A. alternata tangerine pathotype were established using five QoI-resistant (R) and five QoI-sensitive (S) isolates. This experiment was performed twice. Fitness parameters evaluated were: mycelium growth, sporulation and conidium germination. Those isolates were added to the last 10 isolates tested previously. ‘ DNA extraction and RFLP-PCR analyses were performed using the cytochrome b gene amplification of the remaining 10 isolates (used in the In vitro fitness experiment) to identify the G143A mutation in resistant isolates. In summary: all five R isolates carried the G143A mutation. In contrast, the five S isolates did not have the G143A point mutation. ‘ The study of baseline sensitivity to boscalid was started using the rezasurin-based microtiter assay. During this trimester, 405 isolates (arbitrary selected from 2008-2010 survey) were tested. The mean Effective concentration needed to reduce the growth by 50% (EC50) was 0.57 ‘g/ml. ‘ The paper ‘Stability and fitness of Alternaria alternata tangerine pathotype’ was started.
The Mature Tissue Transformation Laboratory (MTTL) continued to increase its preparedness for the first incoming orders. Number of rootstock plants available for budding was increased significantly and supply is now at the level necessary for normal operation. In the last three months, seven co-incubation experiments were performed. In three experiments, 2212 explants of Valencia were used. Two co-incubations were done with 497 Hamlin explants. One experiment was done with 240 explants from Pineapple orange plants and one with 701 explant of Ray Ruby. The experiment with Ray Ruby was the first experiment done using the grapefruit explants. The data were analyzed from three experiment performed in the previous reporting period and from four experiments performed in this reporting period. For both binary vectors used pCAMBIA2301 and pTLAB21, transformation rate is about 3%. Because of the problems with the budding success rate, the decision was made to change provider of grafting services again. Within the last 12 months, major efforts were directed towards keeping the facility operational, employees retained, and number of rootstock plants increased to levels needed for performing 9-10 experiments per quarter. Those goals have been achieved. By doing multiple repetitions of transformation experiments with bacterial strains carrying two different binary vectors, proper estimation of transformation success rate was obtained. That rate is at satisfactory level for citrus mature tissue transformation. However, there is a possibly lingering problem that needs to be addressed. Many of the plants produced to be the source of explants in co-incubation experiments have thorns that are one of the major features of juvenility. As a result, at least a half of transgenic plants already produced in the MTTL also have thorns. Within next few months, the oldest transgenic plants in our inventory will reach the age where they should theoretically flower. If they do not flower, protocol used for production of transgenic plants will have to be re-evaluated. Also, we must make sure that the sources of our germ-free certified material for production of ‘mother’ plants are really mature trees.
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 two USHRL projects funded by CRDF for transforming citrus. Non-transgenic citrus can also be subjected to the screening program. CRDF funds are being used for the inoculation steps of the program. Briefly, individual plants 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. This report marks the end of the first year of the project, during which we have achieved large-scale production of CLas positive ACP. To date on this project, a technician dedicated to the project has been hired, a second career technician has been assigned part-time, two small air-conditioned greenhouses for rearing psyllids are in use, and 18 individual CLas-infected ACP colonies are being used for caged infestations. A total of 3,583 transgenic plants have passed through the screening program. A total of 71,760 psyllids have been used in no-choice inoculations. USDA-ARS is providing approximately $18,000 worth of PCR-testing annually to track CLas levels in psyllids and rearing plants. Additionally, steps to manage pest problems (spider mites, thrips and other unwanted insects) are costing an additional $1,400 annually for applications of M-Pede and Tetrasan and releases of beneficial insects.
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