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.
The goal of this experiment is twofold, first to determine the effects of plant growth regulators on addressing vascular degeneration and fruit drop, and second to determine the effects of HLB and ACPS citriculture on drought tolerance. A field experiment was installed in April 2013 to test the efficacy of the synthetic auxin 2,4-D and a micro-emulsion ‘based surfactant to reduce HLB symptom severity in a mature ‘Hamlin’ orange block. The HLB incidence in the block is currently more than 50% and consequently the fruit yield losses due to pre-harvest fruit drop from symptomatic trees were devastating in the 2012/13 season. The experimental design is a 4×4 Latin Square with four replications and four factorial foliar spray treatments consisting of 2,4-D, Eco-Agra’ surfactant, 2,4-D + Eco-Agra’, and untreated control. Each whole plot is split into two sub-plots containing Swingle and Carrizo rootstocks. A basal foliar nutrient spray treatment applied to the whole experiment consists of a comprehensive, balanced fertilization program of micronutrients, macronutrients and potassium phosphite products timed to coincide with the major leaf flushes. The basal ground-applied fertilizer program consists of a dry granular bulk-blended N-P-K +Ca +Mg + Fe + Mn +Zn +B +S product applied four times in the growing season. The automated micro-sprinkler irrigation system is used to apply water to the trees according to seasonal evapotranspiration demand as needed, up to twice per day. Measurements of the trees in this experiment will begin in the summer, and are designed to determine if the PGR and surfactant treatments can reduce the debilitating damage caused by the Liberibacter pathogen to the phloem tissue, and to the whole-tree physiology. They will include photosynthetic rates, phloem transport functions, HLB symptom severity, leaf nutrient concentrations, leaf drop and fruit drop. Preliminary hydraulics data suggests that trees grown under conventional microsprinkler irrigation systems, and watered 2-3 times per week are more resistant to drought induced declines in hydraulic conductivity compared to trees in the ACPS system. We are now sampling the trees again for hydraulics measurements to confirm the preliminary results and are also investigating the anatomical components of the resistance.
Understanding the transmission of CLas within the citrus tree remains one of the principal obstacles in the global efforts to undermine the pathogenicity of HLB (citrus greening). The movement of CLas has been assumed to follow the photoassimilate stream through the phloem. However, many observations based on our knowledge of the bacteria and general phloem anatomy have exposed inconsistencies with the accepted beliefs. The brevity of available information on the ultrastructural properties of citrus phloem sieve elements has hindered efforts to understand the spread of the disease within a tree. For example, lateral movement of CLas around an infected stem appears improbable given the size of cytoplasmic plasmodesmata connections between adjacent sieve elements and the isolated nature of phloem cells. Furthermore, spreading of CLas from the roots to uninfected aerial tree parts through the phloem seems highly unlikely given the direction of phloem sap. To date we lack a thorough investigation into the ultrastructure of citrus phloem and the surrounding tissue, the potential pathways that CLas could utilize to move long distances through citrus trees, and the location of CLas habitat within different citrus tissue. Using a variety of grafting and girdling experiments, SEM, TEM, confocal, high resolution computed tomography, and PCR tissue analysis we aim to gain a better understanding of the anatomical traits that facilitate the spread of CLas through citrus. These data will allow us to develop new screening tools that breeders can use to select for resistant scion/rootstock combinations to confer resistance or tolerance to HLB. As of this progress report the Valencia/Swingle trees are in the process of being grafted together. Once the grafting heals the trees will have HLB infected buds and branches grafted onto the scion or rootstock depending on the experiment. Trees will be dissected prior to grafting on the HLB infected material, and then trees will be sampled for anatomical studies and PCR testing at regular intervals for the next year. We have hired personnel to maintain the plants in the greehouse during the duration of the experiments.
The main goal of this project is to optimally deploy the superinfecting Citrus tristeza virus (CTV)-based vector to prevent existing field trees from development of the HLB disease and to treat trees that already established the disease. We have several sets of the experiments in which we are examining how prior infection with different CTV strains affects the ability of the superinfecting CTV vector to infect and get established in the same trees as well as examining the levels of multiplication of the superinfecting CTV vector in trees infected with different field isolates of CTV. Plant material that is being used in this project and CTV inoculum sources (different isolates of CTV propagated in the greenhouse as well as collected on the field) have been prepared. The experiments to assess the effect of preexisting CTV infections on multiplication of the superinfecting vector in inoculated citrus trees are ongoing. We first graft-inoculated sweet orange trees with the T36,T30 or T68 isolate of CTV, the isolates that were propagated in our greenhouse, as well as with CTV-infected material obtained from field (FS series isolates). We are using isolates that contain only single strains and isolates that contain mixtures of strains for primary inoculations. Real time PCR analysis protocol is being optimized for quantification of multiplication of CTV genotypes in the inoculated trees. Trees with developed CTV infection along with uninfected control trees were challenged by graft-inoculation with the superinfecting vector carrying a GFP gene. The latter protein is used as a marker protein in this assay, which production represents a measure of vector multiplication. The trees are now being examined to evaluate level of replication of superinfecting virus. Tissue samples from the challenged trees are observed under the fluorescence microscope to evaluate the ability of the vector to superinfect trees that were earlier infected with the other isolates of the virus. Levels of GFP fluorescence are monitored and compared between samples from trees with and without preexisting CTV infection. Additionally, real time PCR quantification is also being employed to these tests. To select rootstock/scion combinations that would support the highest levels of superinfecting vector multiplication and thus, highest levels of expression of the foreign protein of interest from this vector, we are preparing trees of Valencia and Hamlin sweet oranges and Duncan and Ruby Red grapefruit on three different rootstocks: Swingle citrumelo, Carrizo citrange, and Citrus macrophylla. The plants are used for the experiments similar to the experiments described above.
Work continues on the construction and characterization of new FT constructs using cDNA clones. The experiments are underway to compare the new FMVcDNA27 construct, which contains an FT3 cDNA insert in the pCAMBIA2201 vector with a constitutive FMV promoter, with a corresponding genomic clone, which we have been using up to this time. Transformation of Carrizo and tobacco tissue is underway in order to compare the action of these two constructs. The new construct was created as a first step towards the development of a new FT3 construct with an inducible promoter. We have arranged for the materials transfer of two inducible promoter systems from the Danforth Foundation. Both of these promoters are inducible by the chemical methoxyfenozide, a widely-available pesticide, approved for field use on citrus. However, we have not yet received the inducible promotors. One system is driven by the CsMV constitutive promoter, and the other by the RTBV vascular-specific promoter. Once we have verified that the smaller and more manageable cDNA is as effective as the original genomic version of the FT3 gene, we hope to begin development of the inducible promoter constructs. Experiments to determine the behavior of the three genomic clones from citrus when overexpressed in tobacco have been completed and a manuscript is being written. Expression of the genes in mature nonstransgenic citrus plants is being recorded monthly.
Two naturally occurring terpenoid essential oils have been selected as the key components for the pesticide formulations. They are known to have antimicrobial properties which allow us to believe that they will be potent in combating the targeted bacteria. Agriculturally approved surfactants of selected HLB values have produced formulations that permit for encapsulation of the oil. Both essential oils have successfully formed stable micro-emulsions. Large changes in temperature cause some instability with higher oil loading. However, at room temperature, system is re-equilibrated. Essential oil A requires additional input of energy (sonication), where as essential oil B is more readily encapsulated. With the addition of co-surfactants, the oil loading percentages greatly increase in the respective microemulsions. For essential oil A, loading is increased from 1% to 7% (w/w). Essential oil B increases from 8% to 14% (w/w). Particle sizing measurements have been conducted and show mean particle sizes by number distribution of ~3 nm for essential oil A and ~7 nm for essential oil B. The droplet size of Essential oil B microemulsion was increased to about ~30 nm with increase in oil loading. These results have been compared using two particle-sizing instruments, Microtrac Nanotrac and Malvern Zetasizer Nano ZS. Further investigations are being carried out to determine the correlation between the particle size and the amount of oil in the system. Currently, surfactant weight percentages are ~ 20% for both Essential oil microemulsion systems. Additional experiments are being performed to increase the Essential oil loading percentages in both microemulsion systems.
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