The antibody developer, Creative Biolabs, Inc., has nearly completed screening for antibodies against the Candidatus Liberibacter asiaticus NodT protein. They have identified six high-affinity binding antibodies to the 30 amino acid peptide antigen used. The materials will be shipped to Dr. McNellis’ lab at Penn State University within the next few weeks. We anticipate beginning to screen the antibodies for their ability to detect the native NodT protein produced by Candidatus Liberibacter asiaticus starting in mid-August, working through the fall.
We have successfully made transgenic Arabidopsis plants for most of the constructs that we made so far. Some of the transformations were made in the corresponding mutant background while others were made in Col-0 background (due to the lack of corresponding mutants). The presence of the transgenes was confirmed by PCR with gene specific primers. We have been in the process of testing disease resistance of these plants to P. syringae. If a citrus SA gene confers broad disease resistance, we expect to see enhanced resistance to P. syringae when the citrus gene is overexpressed in Col-0 and/or to see the complementation of the phenotypes exhibited by its corresponding mutant. For ctNDR1, we have so far obtained 29 independently transformed US942 transgenic citrus plants carrying ctNDR1 overexpressing construct (US942::d35S::CtNDR1). The presence of the transgene in the plants was confirmed by transgene-specific primers. We performed disease resistance assays with Xanthomonas citri subsp (Xac), using excised leaf discs from 24 US942::d35S::CtNDR1 plants. Twelve leaf discs (that was all we could sacrifice for now) from each independently transformed line were used in the infection. Leaf disks, each from a single leaf, were tested in three infection groups and each infection was conducted independently on a separate day. Our preliminary results indicate that US-942::d35S::ctNDR1 transgenic clones had significantly reduced growth of Xac, as compared to untransformed controls. Therefore, these results suggest that overexpression of ctNDR confers enhanced resistance to citrus canker disease.
We have successfully made transgenic Arabidopsis plants for most of the constructs that we made so far. Some of the transformations were made in the corresponding mutant background while others were made in Col-0 background (due to the lack of corresponding mutants). The presence of the transgenes was confirmed by PCR with gene specific primers. We have been in the process of testing disease resistance of these plants to P. syringae. If a citrus SA gene confers broad disease resistance, we expect to see enhanced resistance to P. syringae when the citrus gene is overexpressed in Col-0 and/or to see the complementation of the phenotypes exhibited by its corresponding mutant. For ctNDR1, we have so far obtained 29 independently transformed US942 transgenic citrus plants carrying ctNDR1 overexpressing construct (US942::d35S::CtNDR1). The presence of the transgene in the plants was confirmed by transgene-specific primers. We performed disease resistance assays with Xanthomonas citri subsp (Xac), using excised leaf discs from 24 US942::d35S::CtNDR1 plants. Twelve leaf discs (that was all we could sacrifice for now) from each independently transformed line were used in the infection. Leaf disks, each from a single leaf, were tested in three infection groups and each infection was conducted independently on a separate day. Our preliminary results indicate that US-942::d35S::ctNDR1 transgenic clones had significantly reduced growth of Xac, as compared to untransformed controls. Therefore, these results suggest that overexpression of ctNDR confers enhanced resistance to citrus canker disease.
Objective 1. Develop a simple in-field system to raise citrus canopy temperature. This objective is driven by the need of such system to achieve objective 2 and test our central hypothesis. A moving greenhouse was developed to cover single trees during the summer of 2012. Four trees (~ 2.5.2.5.2.5 m) were treated, one tree per day, during the months of September (trees T1 through T3) and October (tree T4). From each tree, three symptomatic branches were sampled to determine microbial kill before (0 h) and 2, 3, 4, and 5 h during the treatment. Temperature distribution throughout the canopy and on the sampled branches was also recorded. Maximal temperatures in the ranges 50 to 53 ‘C were reached at the top (2.4 m) of the canopy whereas at the bottom of the canopy (i.e., 0.6 m) maximal temperatures ranged from 36 to 43 ‘C. Due to varied micro-meteorological conditions during the treatment, temperatures of the T1 through T4 sampled branches reached above 40.C for 217, 166, 35, 228 min, respectively. For T1, T2 and T4 trees, average temperatures of the sampled branches reached above 45 ‘C for 87, 35, and 49 min or more. Attempt to quantitatively determine microbial kill by determining percent live bacteria at selected time intervals during thermal treatment was unreliable due to the very uneven distribution of initial proportion of live-to-dead bacteria and analysis variability. However, overall, after thermal treatments, live microbial populations decreased. These findings indicate that practical application of heat will require supplementing solar heat with electrical heating elements and fans to produce adequate, fast, uniform thermal treatment of trees.
The objectives of this project are: 1. Evaluate psyllid populations, HLB incidence and intensity, gene expression, tree growth, soil moisture, soil nutrients, foliar nutrients, and eventually yield in newly planted citrus blocks, 2. Assess separate contributions of vector control and foliar nutritional applications to the above parameters, 3. Evaluate the effectiveness of reflective mulch to (repel) ACP, 4. Provide economic analysis of costs and projected benefits and 5. Extend results to clientele. Paperwork was not completed and funds made available by UF-IFAS until 13 March 2012. This quarter the experiment was established on a 10-acre block on the A. Duda & Sons, Inc. farm in Hendry County south of LaBelle at 26.64315 degrees S. -81.45456 degrees W and 26 ft elevation. The land is part of the old outside nursery that is now being converted to production grove. Already provided with 4 inch drain tile, it was releveled flat, bedded with 18 inches elevation between swale bottom and bed top with the swales having a 0.1% grade south to north. Irrigation lines were installed in April with a 20HP electric pump and two 48′ sand media filters which will provide water originating from the nearby Townsend canal. Each tree is provided with 2 Toro Turbo-Plus II Drip Emitter 2 Lph with pressure compensation. The experimental design of main plots is factorial RCB with 4 replicates and 4 treatments: insecticide alone, foliar nutrition alone, insecticide + nutrition, and untreated control. Each plot is split into two subplots, mulch and no mulch. Mulch is metalized (aluminized/reflective) polyethylene film shown in preliminary evaluations to repel Asian citrus psyllid and together with a drip irrigation/fertigation system increase citrus growth rate over the unmulched control. Planting density is 23 ft between rows and 9 feet within the row giving 210 trees per acre. There are 16 main plots each 5 rows wide and 26 trees long, each divided into two subplots of 5 rows x 13 trees. All plots were treated with Allion herbicide at a rate of 5 fl oz/ac. The metalized mulch specially prepared by the ImaFlex company was 6 ft wide, 2.5 or 3 mil thick protected on top. with a clear coat layer. Mulch was laid 11-12 June using a Kenco plastic mulch layer with 4.5 exposed above the soil and holes punched with sharpened post hole diggers. Trees, ‘Hamlin’ orange on ‘Carrizo’ citrange rootstock, were planted 2-3 July and Scott’s Suncote 15-8-11 12-14 month complete control release fertilizer (1 lb) added to each tree hole. Imidacloprid (Nuprid 2F) at a rate of 22.4 oz/ac was applied in designated plots on 9 July 2012 by spraying 8 oz of solution onto bare soil within 6 inches of all sides of the trunk of the tree using an EZ-Dose’ sprayer with a pressure of 45 PSI and a flow rate of 3.7 gpm.
The objectives of this project are: 1. Evaluate psyllid populations, HLB incidence and intensity, gene expression, tree growth, soil moisture, soil nutrients, foliar nutrients, and eventually yield in newly planted citrus blocks, 2. Assess separate contributions of vector control and foliar nutritional applications to the above parameters, 3. Evaluate the effectiveness of reflective mulch to (repel) ACP, 4. Provide economic analysis of costs and projected benefits and 5. Extend results to clientele. The experiment was planted 3-4 July on a 10-acre block on the A. Duda & Sons, Inc. farm in Hendry County south of LaBelle at 26.64315 degrees S. -81.45456 degrees W and 26 ft elevation. The land is part of the old outside nursery that is now being converted to production grove. The experimental design of main plots is factorial RCB with 4 replicates and 4 treatments: insecticide alone, foliar nutrition alone, insecticide + nutrition, and untreated control. Each plot is split into two subplots, mulch and no mulch. Mulch is metalized (aluminized/reflective) polyethylene film shown in preliminary evaluations to repel Asian citrus psyllid and together with a drip irrigation/fertigation system increase citrus growth rate over the unmulched control. Planting density is 23 ft between rows and 9 feet within the row giving 210 trees per acre. Already provided with 4 inch drain tile, it was leveled flat, bedded with 18 inches elevation between swale bottom and bed top with the swales having a 0.1% grade south to north. The experimental design of main plots is factorial RCB with 4 replicates and 4 treatments: insecticide alone, foliar nutrition alone, insecticide + nutrition, and untreated control. Each plot is split into two subplots, mulch and no mulch. Mulch is metalized (aluminized/reflective) polyethylene film shown in preliminary evaluations to repel Asian citrus psyllid and together with a drip irrigation/fertigation system increase citrus growth rate over the unmulched control. Planting density is 23 ft between rows and 9 feet within the row giving 210 trees per acre. Imidacloprid (Nuprid 2F) at a rate of 22.4 oz/ac was applied in designated plots on 9 July 2012 by spraying 8 oz of emulsion onto bare soil within 6 inches of all sides of the trunk of the tree using an EZ-Dose’ sprayer with a pressure of 45 PSI and a flow rate of 3.7 gpm. Flush inspection and sticky cards were placed in the block on 13 Aug. Sticky cards are observed and replaced every other week for ACP and other common citrus pests. About 10 psyllids have been found with no obvious patterns. Only one nymph has been found on flush. Ten trees per plot were selected and marked for trunk diameter measurement which was completed 21-22 Aug. Injection of liquid fertilizer (6-2-6) began on 5 September. Cyazypyr (Verimark) insecticide was applied at a rate of 15 oz/ac in designated plots on 11 October 2012 by spraying 8 oz of solution onto bare soil within 6 inches of all sides of the trunk of the tree using an EZ-Dose’ sprayer with a pressure of 45 PSI and a flow rate of 3.7 gpm. Normal grove care operations. These include two applications of Intrepid for leaf miner one application of glyphosate for weed control (Kocide) twice, to control canker and replanted 10 dead trees.
This report covers the first month of the project funding, and new data during this period is limited. Objective 1. Evaluate existing transformed lines: Experimental lines were generated during the first funding cycle, and one of our principal efforts is analysis and testing of candidate transformed lines of Duncan grapefruit to find stable transgenic lines that correctly express gene constructs. Objective 2. Expand stable transformations: This time of year seeds are poor, but we are planning how to best ramp up our efforts for transformation of Ruby Red grapefruit and sweet orange in September. Objective 3. Refine constructs: We have new genetic elements that we are incorporating into our expression constructs with the aim of improving resistance. Objective 4. Sequence more TAL effectors from additional canker accessions: We have identified several cankers strains that have particular phenotypes or geographies that may reflect differences in TAL effectors. We are working on isolating and sequencing these genes, with the particular goal of testing the effectiveness of our resistance strategy against these strains.
Objective 1. Develop a simple in-field system to raise citrus canopy temperature. This objective is driven by the need of such system to achieve objective 2 and test our central hypothesis. An adjustable aluminum frame was built and mounted on wheels in Dr. Eshani’s Workshop. In its current configuration (8 ft x 8 ft), the frame is being tested on three year-old trees. The frame can be adjusted to larger trees. Trees with twigs that displayed well developed HLB symptoms were selected. Experimental work began in July after trees were acclimated to summer high temperatures. The first set of experiments to determine the tarp material that provides the fastest rate of heating and to determine the temperature profiles within the canopy are underway under Drs. Reyes and Khot’s supervision. Maximal temperature reached at the surface (top) and center of the canopy were 62 ‘C and 56 ‘C respectively. Destruction of live CLas bacteria is being determined in Dr. Wang’s lab. Results are expected within next week. Experiments will continue throughout the summer. A Ph.D. student was identified to work in this project but declined the offer at the last minute. A second student that has applied to UF ABE program to work on this project and is expected to join in the spring 2012. A request to change student salary to OPS salary between August 2012 and December 2012 was submitted to ensure project progress continuity.
One of our specific objectives has been to determine how psyllid behavior is affected by Las-infection in healthy and infected citrus. In previous experiments we have determined that ACP adults initially prefer to settle on Las-infected plants. This attraction to Las-infected plants appears to be dependent upon the plant release of the volatile chemical methyl salicylate. Based on published data, it is known that Las-infected plants have nutrient deficiencies. Therefore, we hypothesized that while the Las-infected plants are initially attractive to psyllids, after prolonged feeding the psyllid experiences imbalanced nutrition and choose to seek a better host. To examine this hypothesis we planned to determine how psyllids settle on plants with known nutrient deficiencies, using the following choice tests: 1. Healthy vs. Nutrient Deficient Citrus 2. Healthy vs. HLB infected Citrus 3. Healthy vs. Nutrient Deficient Citrus + ‘HLB infected plant odor’ (HLB odor) 4. Healthy vs. Healthy + HLB odor However, in order to apply ‘HLB-infected plant odor’ to deficient and healthy plants, we needed to evaluate the volatile profile of our healthy and deficient trees, as well as HLB-infected trees. By using these data, we will be able to mimic the smell of HLB-infected plants and apply it to trees during our settling experiments. Previously, we found that our existing volatile collection bell jars were too small for use with 4-5 year old Valencia trees, so we had new bell jar custom made. We received these bell jars in this past quarter and have nearly completed volatile collections on 12 of 16 trees. The samples are currently being analyzed by GC and GC-MS to identify and quantify differences in the volatile blends. In preparation for settling experiments, we have constructed a 2′ x 3′ x 3′ mesh assay chamber that is sufficiently large to house two experimental trees simultaneously. We are currently evaluating odor release devices for applying ‘HLB odor’, by releasing psyllids in the cage and catching them on small yellow sticky traps baited with the release device. Preliminary data suggests that a 0.5mL polyethylene ‘beem vial’ may be an acceptable release device to affix to experimental trees. We will be quantifying release rates in order to obtain a release rate that is most similar to HLB-infected plants.
This is a 4-year project with 2 main objectives: (1) Over-express the Arabidopsis MAP kinase kinase 7 (AtMKK7) gene in citrus to increase disease resistance (Transgenic approach). (2) Select for citrus mutants with increased disease resistance (Non-transgenic approach). For objective 1, besides transgenic citrus plants overexpressing the Arabidopsis MKK7 (AtMKK7) and NPR1 genes, we have also generated transgenic citrus plants expressing the Arabidopsis NAC1, MOD1, and EDS5 genes. These three genes have been shown to confer disease resistance in Arabidopsis. The transgenic plants are growing and will be propagated for canker and greening resistance test. In addition, we recently established an Arabidopsis-Xanthomonas citri subsp. citri (Xcc) pathosystem. Using this pathosystem, we have found that several genes of the SA signaling pathway function in nonhost resistance to Xcc. We are using the pathosystem to identify novel genes conferring nonhost resistance against citrus canker. For objective 2, we focused on the direct genetic screen for citrus varieties with increased resistance to citrus greening. More seedlings from gamma ray-irradiated Ray Ruby grapefruit seeds were inoculated with psyllids carrying greening bacteria. The first batch of seedlings inoculated with psyllids carrying greening bacteria have been moved out, and we are monitoring the development of greening symptoms on the seedlings.
A series of transgenics scions and rootstocks, produced in the last several years, continue to move forward in the testing pipeline. It appears prudent to replicate plants of each transgenic event and conduct challenges that last 10-14 months. Most of these plants in our program have been transformed with AMPs driven by several constitutive and vascular specific promoters. Plants from the initial round of scion transformations are now replicated and are being exposed to HLB, using graft inoculations and CLas infected psyllids in greenhouse and field environments. Challenge with HLB through exposure to infected ACP (D. Hall collaboration) is being conducted on a replicated set of 33 independent Hamlin transformants, 5 Valencia transformants, 4 midseason transformants, and 3 non-transformed controls. Several events grew better than all controls at 14 months after initiating the challenge, with 35% greater trunk-cross-sectional area increase than the overall experimental average and 64% greater growth than the mean of the controls, but do not show immunity to CLas development. These will soon be placed in the field for further evaluation. Forty four AMPs were screened in-vitro, a number of which were synthetics specifically designed to enhance efficacy against alpha-proteobacters. There appeared to be a ceiling of activity which could not be exceeded. The most active AMPs included Tachyplesin 1 from horseshoe crab, SMAP-29 from sheep, D4E1 and D2A21. A series of promoters were tested with the GUS gene. The three vascular-specific promoters show expression only in phloem and xylem, while other promoters show broad expression in tested tissues. Sucrose synthase promoter from Arabidopsis drives high GUS expression more consistently than the other phloem-specific promoters citrus SS promoter or a phloem promoter from wheat dwarf virus. A ubiquitin promoter from potato drives unusually consistent and high GUS activity. D35S produces the highest level of expression but with great variability between events. Anthocyanin regulatory genes, give bright red shoots (UF Gray collaboration) and were tested as a visual marker for transformation, as a component of a citrus-only transgenic system. When antibiotics were left out of regeneration media, almost no red shoots were recovered. However, high anthocyanin apples are reported to have field resistance to bacterial fire-blight. Red citrus transgenics will be tested for HLB, ACP, and canker resistance. CLas sequence data target a transmembrane transporter (Duan collaboration),as a possible transgenic solution for HLB-resistance. In E. coli expressing the CLas translocase, two exterior epitope-specific peptides suppressed ATP uptake by 60+% and significantly suppressed CLas growth in culture. After verification these will be used to create transgenes. In our program, new constructs and resulting transgenics are in process, including hairpins to suppress PP-2 through RNAi (to test possible reduction in vascular blockage even when CLas is present), chimeral constructs that should enhance AMP effectiveness (designed by Goutam Gupta of Los Alamos National Lab), and a citrus promoter driving citrus defensins (designed by Bill Belknap of USDA/ARS, Albany, CA).
A transgenic test site has been prepared at the USDA/ARS USHRL Picos Farm in Ft. Pierce, to support HLB/ACP/Citrus Canker resistance screening for the citrus research community. There are numerous experiments in place at this site where HLB, ACP, and citrus canker are widespread. The first trees have been in place for more than two years. Dr. Jude Grosser of UF has provided 550 transgenic citrus plants expressing genes expected to provide HLB/canker resistance, which have been planted in the test site. Dr. Grosser planted an additional 89 trees including preinoculated trees of sweet orange on a complex tetraploid rootstock that appeared to confer HLB resistance in an earlier test. Dr. Kim Bowman has planted several hundred rootstock genotypes transformed with the antimicrobial peptide D4E1. Texas A&M Anti-ACP transgenics produced by Erik Mirkov and expressing the snow-drop Lectin (to suppress ACP) have been planted along with 150 sweet orange transgenics from USDA expressing the garlic lectin. Eliezer Louzada of Texas A&M has permission to plant his transgenics on this site, which have altered Ca metabolism to target canker, HLB and other diseases. More than 120 citranges, from a well-characterized mapping population, and other trifoliate hybrids (+ sweet orange standards) have been planted in a replicated trial in collaboration with Fred Gmitter of UF and Mikeal Roose of UCRiverside. Plants will be monitored for CLas development and HLB symptoms. Data from this trial should provide information on markers and perhaps genes associated with HLB resistance, for use in transgenic and conventional breeding. Dr. Roose has completed initial genotyping on a sample of the test material using a “genotyping by sequencing” approach. Additional plantings are welcome from the research community.
The goal of this proposal is to examine how the efficiency of HLB transmission by psyllids varies depending on the stage of infection and plant development. We are on schedule for the second year of the project. There was a delay for getting the funds for this year, however they are being released now. We have set trials using sweet orange and grapefruit plants that have young growing flushes and plants that have only matured flushes. These plants have been exposed to HLB-infected psyllids. Leaves on which psyllids fed were analyzed by PCR to see if the HLB bacterium could be detected soon after the exposure of leaves to infected psyllids. As a result in this experiment, we were able to detect presence of the bacterium fairly early after the initial exposure. Plants exposed to infected psyllids have been transferred to greenhouse and further monitored for the development of infection. We are in a process of analyzing and comparing infection rates of plants with young flushes versus plants with only matured flushes. Additional trials are in progress. To characterize potential inoculum sources of the bacterium available for psyllids within an infected tree we are examining presence of viable bacterium in different types of flushes that are produced during the development of the disease after psyllid-mediated inoculation of a tree and evaluating the proportion of psyllids that acquired the bacterium after their exposure to different types of flushes during infection development and their ability to transmit infection to new trees. We conducted several trials in which healthy psyllids were placed on either a young growing flush or an older symptomatic flush of an infected tree. Psyllids were secured on those flushes by using small traps made up of mesh material and after 21 days psyllids were analyzed by PCR with HLB-specific primers. Data from PCR analyses demonstrated that Las-positive psyllids were collected from both types of flushes. Psyllids that acquired bacteria from different flushes were next transferred onto healthy receptor plants. These plants are being monitored for the development of infection. Currently we are analyzing numbers of plants that became infected upon inoculation with psyllids fed on different types of flushes. Another goal is to understand whether the HLB bacterium can have different forms or can be present at different stages in different types of tissues. One of the approaches that are being undertaken is to compare levels of the HLB bacterium genes expression in old symptomatic and young pre-symptomatic flushes. This work is ongoing. The next objective is to examine psyllid transmission rates from and to citrus varieties that are highly tolerant to HLB. We have propagated 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. Currently these plants are being exposed to HLB-infected psyllids. After 1-month exposure, plants will be moved to greenhouse and monitored for the development of HLB infection. We are analyzing infection rates for these varieties. Later infected plants will be used as inoculum donors to examine psyllid transmission to new plants.
This is a proposal to find ways to optimally deploy the superinfecting vector to prevent existing field trees from development of the HLB disease and to treat trees that already established the disease. This is a new project and the funds for this project are currently being released. The research is in progress. We already have designated personal who would conduct the proposed research. Plant material that will be used in this project is being prepared. Using plant material and inoculum sources that are already available we are setting up initial experiments with the purpose to examine the levels of multiplication of the superinfecting CTV ector in trees infected with different field isolates of CTV to investigate how previous infection of trees with the virus affects the ability of the vector to infect and multiply in those trees and to examine the effect of various rootstock/scion combinations on the superinfecting ability of the vector in order to evaluate what combinations would support high levels of vector expression. These two objectives are our main focus for the first year of funding.
This project has been an administration challenge from the beginning. It was impossible to get funds to our Brazilian colleague and difficult to get funds to our USDA partners. Dr. Machado had only requested funding for the first two years of the project. Since he could not get funding in Brazil, he asked me to support a “sandwich” student in my lab, which Dr. Turpin approved. This student is returning to Brazil this month after one year in my lab. She worked on controlling tissue contamination, which is our main problem with mature transformation, but with only partial success. Drs. Grosser and Gmitter have had the same difficulties at the CREC and do not feel that they should continue on this project without improved facilities. The part of the project that has been successful is the work on small penetrating peptides (CPPs) in the Moore lab. This technology allows an alternative to Agrobacterium-based transformation of citrus. The Agrobacterium method produces a low rate of stable transformants (~9%), takes several months, and commercialization is potentially difficult because of the negative views of bacterial genetic modifications. We propose an alternative method of transformation using cell penetrating peptides (CPPs). CPPs are positively charged short amino acid sequences able to simultaneously bind proteins and nucleic acids and deliver them across cellular membranes and cell walls. Many applications are available using CPPs including transient expression assays and gene silencing. We have developed a standard method for the transient expression of reporter genes (GUS and GFP) in citrus. Our data indicate that up to 50% of treated explants express GUS when CPPs are used alone. Several optimization steps have been tested. For instance, the efficiency is increased to 100% when CPPs are used in conjunction with a lipid reagent. Our main goal is to use this method to improve stable citrus transformation efficiency compared to the Agrobacterium method. We have produced 84/163 segments which survived kanamycin selection and produced shoots. We will use PCR and reporter gene analysis to confirm their stable integration. Further experiments, comprising RNAi and protein expression, will also be performed.
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