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.
New outbreaks of invasive fruit flies (Diptera: Tephritidae) continue to threaten agriculture world-wide. Establishment of these pests often results in serious economic and environmental consequences associated with quarantine, control, and eradication programs. Early fruit fly detection and eradication in the United States requires deployment of large numbers of traps baited with the highly attractive male specific parapheromone lures trimedlure (TML), cue-lure (C-L), and methyl eugenol (ME) to detect such pests as Mediterranean fruit fly, Ceratitis capitata (Wiedemann), melon fly, Bactrocera cucurbitae (Coquillett), and oriental fruit fly, B. dorsalis (Hendel), respectively. The current study compared the performance of solid single lure cones and plugs in conjunction with DDVP insecticidal strips; liquid lure with naled formulations; and single, double, and triple solid lure wafers impregnated with insecticide. Treatments were placed in AWPM and Jackson traps under Hawaiian climatic conditions in habitats where B. dorsalis, C. capitata, and B. cucurbitae occur together. The overall goal of this study was to develop a more convenient, effective, and safer means to use male lures and insecticides for improved detection and male annihilation of invasive fruit flies. In survey trials near Kona, HI captures of C. capitata, B. cucurbitae, and B. dorsalis with Mallet TMR wafers were equal to those for the standard TML, ME, and C-L traps used in Florida and California. A solid Mallet TMR wafer is more convenient to handle, safer, and may be used in place of several individual lure and trap systems, potentially reducing costs of large survey and detection programs in Florida and California, and male annihilation programs in Hawaii. With confirmatory trials completed in Hawaii, further testing will be conducted in citrus orchards under California weather conditions. Through Dr. Joseph Morse of the University of California, Riverside, we will conduct weathering trials of the novel TMR dispensers in California (Riverside, Lindcove, Bakersfield, Ventura, and Costa Mesa, CA) beginning in July 2012. Climate data will be obtained from Hobo weather recorders maintained at each location. Weathered dispensers will be sent to Hawaii and Washington for bioassays and chemical analyses, respectively. Roger Vargas of US PBARC will oversee bioassays in Hawaii. Peter Cook of Farmatech and John Stark of Washington State University will collaborate on chemical analysis of wafers in North Bend, WA. Currently, approximately 30,000 sets of TML, ME, and C-L traps are maintained throughout the state. From a worker safety, convenience, and economic standpoint, Farma Tech TMR Mallet solid wafers with DDVP may be more cost effective, convenient, and safer to handle than current liquid lure and insecticide formulations (e.g. naled) used for detection programs for TML, ME and C-L responding flies in California. Cost/benefit analyses of Mallet TMR vs. standard trapping systems will be done.
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. Money was just released in last week so no results to report.
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. The field survey of tangerine hybrid blocks is nearly finished with approximately 1000 isolates collected. Pathogenicity testing and collecting monoconidial isolates is underway. Analysis of data from 2008-2011 is under way and a manuscript is in preparation and is waiting for the 2012 results. Plants have been prepared for a series of experiments to look at the fitness characteristics among sensitive and resistant isolates.
The first objective of this project was to hire a Florida-based faculty scientist that could be trained under Dr. Leandro Pena in Spain, for the purpose of learning the mature tissue transformation technique and transferring the technology to Florida. The scientist (Dr. Cecilia Zapata) was hired, at the end of the first year of the three year project, and traveled to Dr. Pena’s lab at the IVIA, Spain, where she was trained in all tissue culture techniques associated with citrus mature transformation, starting with preparation of the source of material at the greenhouse and ending with the acclimatization of transformants in the greenhouse. It was emphasized that the preparation of plant material needed for mature transformation is the key to successfully and consistently obtaining mature transformants, and this can only be achieved by producing budsticks in a highly controlled and clean environment. The second objective of the project was to build a greenhouse at the Citrus Research and Education Center in Florida for the purpose of creating and growing citrus for mature transformation and to establish a Mature Transformation Laboratory. A growth room was constructed instead of a greenhouse due to budget constraints. It took approximately 7 months to construct the growth room. It is currently operational after more than a year of troubleshooting. The water filtration system still needs some adjustments to be able to obtain a better water quality. The water quality is affecting the plant growth and the humidifiers. A generator needs to be purchased; without it, any prolonged electricity failure could jeopardize the whole project. The laboratory is fully operational. The third objective of the project was to obtain mature transgenic plants from the most important Florida citrus cultivars. We started using the growth room and planted the rootstocks at the beginning of April 2011. Three (3) sweet orange varieties were indexed in vitro and micrografted. The cultivars introduced were Hamlin 1-4-1, Valencia SPB 1-14-19 and Pineapple F-60-3. A calendar was established in October 2011 and firsts mature transformation experiments were performed in November 2011, all the protocols developed at the IVIA were adjusted to our specific environmental conditions and clone specificities. Mature Valencia, in our conditions, was very responsive to organogenic regeneration. We obtained positive plants, checked by PCR, and they are currently growing in the growth room. Hamlin was also transformed but was less responsive to organogenic regeneration, but we were able to obtain a plant currently growing in the growth room. Pineapple did not respond and we discarded the cultivar after the last batch in the production calendar was used. We are still waiting from results on this last experiment. We are currently introducing another clone of the same Hamlin cultivar 1-4-1 to improve its quality. A few of the initial mother plants were not as cleaned as previous determined and we introduced more plants using antimicrobial to guarantee cleanliness. It seems like yearly introductions may be necessary to maintain the quality of the material desired in the experiments. We are also cleaning the rootstocks Swingle Citrumelo and Carrizo to be transformed in future experiments.
The overall objective of this project is to develop and use a high-throughput system to screen for chemicals that disrupt interactions in a model of the ACP/HLB/Citrus system that uses the related bacterium Candidatus Liberibacter psyllaurous (CLps) which causes psyllid yellows of tomato. Previous work focused on development of a system for the model plant Arabidopsis thaliana which has the best developed genetics of any plant and has been used in previous chemical genomics experiments. However, repeated attempts to infect Arabidopsis plants grown in solid culture media, liquid culture media, or hydroponics were not successful. Only plants grown in soil were infected by psyllid nymphs. The most recent experiments gave a low percentage of infected plants (about 15%), and differences between Arabidopsis lines observed previously were not repeatable. We believe that it will still be of interest to analyze the tolerance (lack of symptoms) seen in CLps infected Arabidopsis plants and are accumulating samples from CLps-positive plants for gene expression analysis. Because of the problems with Arabidopsis system, in late 2011 we began to develop a system for tomato which, as a natural host of CLps, is more easily infected. Compound leaves along with petioles from tomato plants were placed in 50 ml culture tubes with the cut end immersed in water in a microfuge tube. This design was adapted from one shown on a poster by Ammar et al. at the Citrus Health Research Forum in Denver in October. Adult psyllids are placed in the culture tubes and within 7 days most tomato leaf petioles are qPCR positive for CLps. After two weeks Ct values are typically less than 25 and about 80% of plants are infected. This system appears promising since chemicals can be introduced into the water for plant uptake. We will investigate methods to induce rapid rooting of these leaf petioles since this may increase chemical uptake. Experiments to test chemical uptake were initiated. We also began testing a transgenic tomato carrying a marker gene (GUS) driven by a pathogen responsive promoter (CaBP22). This system has facilitated detection of chemicals that activate defense pathways in Arabidopsis. Sweet orange seedlings were grown to test chemical application methods during the next quarter. This work is supported by a separate funding source, but little progress on the chemical genomics project is expected while this work is carried out.
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