The main goal of this project is to assess how the efficiency of HLB transmission by psyllids varies depending on the stage of infection and plant development. 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 14 days after the beginning of the experiment) the bacteria could be already visualized in the initial sites of introduction. To characterize inoculum sources of the bacterium available for psyllids within an infected tree, we are 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 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. 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). 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. 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 were moved to greenhouse and monitored for the development of HLB infection. The first four varieties showed the highest infection rates (80-100% infection), while only about 10% of Carrizo citrange and Poncirus trifoliate became infected. 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.
In this project we are examining ways 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 are conducting initial experiments to examine the levels of multiplication of the superinfecting CTV vector in trees infected with different field isolates of CTV. We already prepared plant material that will be used in this project. Inoculum sources (different isolates of CTV propagated in the greenhouse as well as collected on the field) are also available. A series of 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 or T30 isolate of CTV, the isolates that were propagated in our greenhouse, as well as with CTV-infected material obtained from field. In different experiment sets we are using isolates that contain only single strains and isolates that contain mixtures of strains for primary inoculations. 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. For another objective, 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 now growing and later will be used for the experiments similar to the experiments described above.
HLB incidence is approaching 100%, especially in young groves. The 2012-2013 season was dry before July and after September to the present. Fruit drop statewide has led to five reductions in the USDA crop estimate (unprecedented). The concensus among researchers and growers is that most of the drop is due to HLB. Fruit drop is greater than in past seasons due to increased HLB incidence and disease effects which we defined from our recent greenhouse and field studies on root health of HLB-affected trees,i.e. 1) Candidatus Liberibacter asiaticus (Las) moves to the roots after initial infection /transmission in the shoots; 2) Las infects structural and fibrous roots; 3) Las colonizes the roots before the shoots, phloem is not plugged; 3) the infection causes a rapid fibrous root loss of 27-40% before symptoms in the canopy; 4) Phytophthora interacts to further reduce root health but the majority of the root loss is due to HLB; 5) Phytophthora populations of HLB trees in groves initially increase and then the populations decline rapidly as roots are lost due to Las infection, . Statewide drop in Phytophthora counts in 2012 may indicate more HLB-induced root loss which accelerates fruit drop. This Phytophthora population dynamic was confirmed in potting soil at 2, 8 and 14 mpi for bud-inoculated trees (HLB+) and mock-inoculated (HLB-) trees. We measured a 27-40% reduction in root density for presymptomatic and recently symptomatic HLB trees. Root loss equates with the ‘ 30% yield losses on early symptomatic trees in Florida treated with good irrigation and nutritional management. Recent population survey of a mefenoxam treated block showed a significantly lower population of Phyopthtora per root mass than in the adjacent non-treated half of the block. Hence, we are recommending that, when the Phytophthora count is >10-20 prop/cm3, the fungus should be managed aggressively to sustain root health. We are furthermore recommending alternation of fungicides as follows: after spring shoot flush – Aliette/phosphites; 45 days later ‘ mefenoxam (injection); 45 days later – Aliette/phosphites; After fall shoot flush ‘ mefenoxam (injection). The costs of root health management should be balanced with other resources for HLB, i.e. psyllid control, best management practices for irrigation and nutrition, as well as, control of other pests and diseases.
Objective 1 (To define the role of chemotaxis in the location and early attachment to the leaf and fruit surface). Multiple assays to determine the ability of canker strains to move in response to chemical stimuli have been concluded. Differences among 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 sensors. Cluster analysis of chemotaxis profiles and MCP sequences grouped narrow host range citrus bacterial canker (CBC) strains into the same clade. In addition an in silico study was performed to identify MCPs from complete genome sequence in databases. MCP sequences were clustered in twenty seven phylogenetic groups, fifteen of these groups included MCP sequences found in every strain and are considered conserved in Xanthomonas. Twelve groups are restricted to certain strains and of interest due to their possible link with the unique chemotactic profile for strains associated with host range. Moreover, differences among strains were detected mainly as amino acid modifications in the putative ligand binding domain.To confirm chemotaxis at an early stage of the infection and to determine possible differences due to host range, the bacterial strains were exposed to leaf fractions from different plant species. No response was elicited by leaf washings, revealing inability of absent or low concentrations compounds to stimulate a chemotaxis response on the intact leaf surface. In contrast, apoplastic fluids definitely produced chemoattractant and repellant responses, even more so than those produced by crude leaf extract. Therefore the natural chemotactic interaction is probably due to apoplastic fluids emanating from opened stomata or leaf damages. Moreover differences were shown between narrow and wide host range strains of CBC in their chemotaxis behavior. 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). Type IV pilus from Xanthomonas citri subsp. citri is being purified in order to obtain antibodies as a strategy to confirm such protein is a main component of the protein fraction of the biofilm matrix. In addition assays to detect cellulose and amyloid fiber production by CBC strains, using calcofluor and Congo red have been started. Differences in biofilm formation among the diverse CBC strains as compared to X. campestris and X. alfalfae subsp. citrumelonis were shown in minimal or nutritive culture media. In addition assays to evaluate presence of DNA in the biofilm matrix are under progress by varying DNAse concentration during the biofilm development or for biofilm removal.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.
A transgenic test site at the USDA/ARS USHRL Picos Farm in Ft. Pierce supports 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 over three years. Dr. Jude Grosser of UF has provided ~600 transgenic citrus plants expressing genes expected to provide HLB/canker resistance, which have been planted in the test site. Dr. Grosser planted an additional group of 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, and Ed Stover 50 sweet oranges (400 trees due to replication) 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 are being 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. Early in the next quarter Dr. Grosser is removing the unsuccessful trees from the first planting and planting additional transgenics among the promising trees still under trial. Additional plantings are welcome from the research community.
Citrus scions continue to advance which have been transformed with diverse constructs including AMPs, hairpins to suppress PP-2 through RNAi (to test possible reduction in vascular blockage even when CLas is present), a citrus promoter driving citrus defensins (citGRP1 and citGRP2) designed by Bill Belknap of USDA/ARS, Albany, CA), and genes which may induce deciduousness in citrus. Putative transgenic plants of several PP-2 hairpins and of PP-2 directly are grafted in the greenhouse and growing for transgene verification, replication and testing. Over 30 putative transgenic plants with citGRP1 were transferred to soil. They will soon be ready for RNA isolation and RT-PCR to check gene expression. About 10 transgenic Hamlin shoots with citGRP2 are in the rooting medium for rooting. Fifteen transgenic Hamlin shoots with peach dormancy related gene MADS6 are in the rooting medium for rooting. In addition numerous putative transformants are present on the selective media. A chimeral construct that should enhance AMP effectiveness (designed by Goutam Gupta of Los Alamos National Lab) is finally completed. Sequence information was confirmed and used to transform Hamlin. Some kanamycin-resistant shoots have already been obtained. To explore broad spectrum resistant plants, a flagellin receptor gene FLS2 from tobacco was amplified and cloned into pBinARSplus vector. Flagellins are frequently PAMPS (pathogenesis associated molecular patterns) in disease systems and CLas has a full flagellin gene despite having no flagella detected to date. The consensus FLS2 clone was obtained and will be use to transform Hamlin and Carrizo so that resistance transduction may be enhanced in citrus responding to HLB and other diseases. Other targets identified in genomic analyses are also being pursued. A series of transgenics scions produced in the last several years continue to move forward in the testing pipeline. Several D35S::D4E1 sweet oranges show initial growth in the field which exceeds that of controls. A large number of ubiquitin::D4E1 and WDV::D4E1 plants and smaller numbers with other AMPs are replicated and queued for testing with no-choice ACP and then free-flying ACP infection.
Oral uptake of dsRNA targeting specific Asian citrus psyllid genes can induce psyllid mortality and reduce Liberibacter titer in infected psyllids. Research has shown that Asian citrus psyllid (ACP) mortality can be induced when adults feed on leaves from citrus that have been infected with a Citrus tristeza virus (CTV) expression vector modified to produce dsRNAs targeting specific ACP genes. The ACP mortality was shown to be directly associated with dsRNA abundance within the leaves. Also, when ACP infected Candidatus Liberibacter asiaticus (CLas) are fed on these plants for 15 days, the remaining live psyllids do not contain detectable CLas. Furthermore, none of the emergent adults that developed on these plants, from eggs laid by the CLas carrying ACP, contained detectable CLas. This is in contrast to what was observed in adults obtained from plants producing dsRNAs targeting the jellyfish green fluorescent protein (GFP) used as a control. The Clas bacterium could still be detected in adult ACP that fed on GFP-dsRNA expressing citrus and also in some adults that developed and emerged on these plants. Studies continue on comparing the effect of multiple ACP gene targeted dsRNA molecules (individually and in combination) that are fed to psyllids either in diets or produced in citrus using the CTV vector. As part of this work, a method was developed that allows complete life cycle development of the ACP on excised citrus flush thus improving efficiency of studies of dsRNA feeding on ACP nymphal stages.
During this period we have analyzed the response of ‘Duncan’ grapefruit (considered susceptible to HLB) and ‘Sun Chu Sha’ mandarin (considered moderately tolerant to HLB) after inoculation with flagellin 22 (flg22) from Liberibacter using comparative Ct real time PCR. The expression of 16 defense-associated genes was analyzed. In ‘Sun Chu Sha’ we observed that PBS1, NDR1, RAR1, SGT1, EDS1, EDR1, PAL1, AZI1, NPR2, NPR3 and RdRp were significantly induced compared to water controls. However, in ‘Duncan’ only PR1 was significantly induced compared to water controls. These results were presented during the 3d International Research Conference on HLB held in Orlando, Florida on February 4-8 of 2013.
The long-term goal of this project is to develop a novel and robust measure to protect citrus from high-priority diseases that threaten the industry and economy. The novelty stems from the fact that we are enhancing the plant defense (called innate immunity) by introducing a protein chimera of two domains: one for recognizing the pathogen that causes the disease and the other for lysing the pathogen. The synergy of the pathogen recognition and lysis makes the chimera a robust therapeutic agent for clearing the pathogen before it can cause infection. In this three-year project, our goal is to demonstrate that a protein chimera shows broad-spectrum activity on three citrus pathogens, namely ‘Candidatus Liberibacter’, Xylella fastidiosa subsp. pauca, and Xanthomonas citri subsp. citri that respectively cause HLB, CVC, and canker. Our initial focus is to design a chimera that recognizes and kills Liberibacter from the citrus phloem thereby providing protection against HLB. Toward this end, we have chosen thionin from vascular plants such as citrus. Thionins are small antimicrobial peptides that can attach to the membrane LPS of gram-negative bacteria such as Liberibacter. Thionins can also lyse the membrane. We have attached another small helical antimicrobial peptide (discovered by our collaborator, Ed Stover, USDA-ARS, Ft. Pierce, FL) to thionin using a flexible linker. The idea is to exploit the synergy of membrane recognition and lysis to direct rapid clearance of Liberibacter by the chimera. We constructed a chimera gene with an upstream signal sequence and a constitutive promoter. The signal sequence is attached for phloem delivery whereas the constitutive promoter is attached to ensure high level of expression. We have successfully performed Agrobacterium mediated transformation of citrus carrizo. The full grown trees will be ready for Liberibacter challenge and efficacy testing in 9 months.
Our objective has been to determine how Asian Citrus Psyllid (ACP) behavior is affected by Ca. Liberibacter asiaticus (Las) infection by comparing ACP response to healthy versus infected citrus trees. In previous experiments, we have determined that ACP adults initially settle on Las-infected plants as compared with uninfected controls. We hypothesized that while the Las-infected plants are initially attractive to ACP, after prolonged feeding, the ACP experiences imbalanced nutrition and therefore leave infected plants to seek a better host (non-infected tree). We have finished conducting our settling experiments to determine how ACP settle in response to choice tests between: control vs. old infection, control vs. new infection, new vs. old infection, old infection vs. nutrient spray, new infection vs. nutrient spray, and control vs. nutrient spray. All plants used in settling experiments were approximately four-year old Hamlin sweet oranges. Control plants were uninfected (healthy) plants. Nutrient sprayed plants were old-HLB infected plants. Infected plants were either newly infected (<5 month since PCR detection) or old infected (>12 months since PCR detection). Our results show that newly infected plants are very attractive to ACP. ACP prefer to settle on these plants over controls and old infected plants. Old infected plants are not as attractive as newly infected plants when compared with controls. ACP initially settle evenly between control and old or nutrient sprayed plants, but then choose to move to the infected plants over seven days. When ACP are given a choice between newly infected plants and nutrient sprayed plants, the nutrient sprayed plants appear to regain some of their attractiveness and ACP settle more evenly between these two plant treatments. In addition, we have conducted a settling experiment between control and newly infected citrus in the presence of high amounts of methyl salicylate released from controlled-release devices. It appears from these experiments, that high levels of methyl salicylate may interfere with ACP’s ability to differentiate between infected and uninfected controls. We are currently exploring this hypothesis by measuring ACP preference to infected vs. uninfected citrus odor in olfactometer assays using methyl salicylate pre-exposed ACP. This may result in a commercial product for disrupting the ACP’s ability of finding infected citrus host plants. ISCA technologies is currently working on a initial proprietary product to disrupt ACP host finding behavior for beta-testing in the field. We are currently in the process of writing a manuscript that summarizes these experiments, which we plan to submit for scientific peer review by early summer.
In the first three months of 2013, Citrus Core Transformation Facility (CCTF) continued to operate at the steady rate without any interruptions in production of transgenic plants. Within this period, CCTF received highest number of orders ever-twelve. Five orders were placed for production of transgenic Duncan grapefruit (pW14, pHGJ1, pHGJ2, pHGJ3, and pHGJ4); four for production of transgenic Mexican lime plants (pOA1, pOA2, pOA3, and pGF1); and three for production of Valencia plants (pOA1, pOA2, and pOA3). All the binary vectors received from clients were already mobilized into appropriate Agrobacterium strains and initial co-incubation experiments were performed. Most of the work done in the CCTF revolved around the recently placed orders. Regarding the production of transgenic plants, CCTF has achieved following results. Transgenic Duncan plants carrying genes from these different vectors were produced: fifteen from the pX4, twenty six from the pX7, thirteen from the pX11, ten from the pX16, three from the pX19, five from the pX28, twelve from pNah, and five from pBI121 vector (to serve as control plants). There were also twelve Duncan plants produced carrying the gene from pMED14 vector and one carrying the gene from pMED16. Despite the high flux of workers in the facility, productivity remained high. Every effort will be made to keep production of transgenic material at satisfactory level considering high volume of incoming orders.
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 and reduce incidence of HLB, 4. Provide economic analysis of costs and projected benefits, and 5. Extend results to clientele. The experiment was planted 3-4 July 2012 on a 10-acre block planted on a 23 x 9 ft spacing at 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 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 of 16 plots is split into two subplots 5 rows wide and 13 trees long, mulch and no mulch. Mulch provided by Imaflex Inc. is metalized (aluminized/reflective) polyethylene film of 3 mils thickness covered with a clear protective polyethylene coat. Metalized mulch was shown in preliminary evaluations on single plots to repel Asian citrus psyllid and together with a drip irrigation/fertigation system increase citrus growth rate over the unmulched control. The block was planted 3-4 Jul 2012 and monitoring ACP with flush inspection and sticky cards commenced 13 Aug. Sticky cards are monitored for ACP and other common citrus pests and replaced every other week. About 106 psyllids have been found on sticky cards of which 80% are in no-mulch plots with the majority in plots with trees that do not have receive chemical control . Flush infested with ACP eggs and nymphs have become much more common. These are predominately in no-mulch plots with 85% of the total observed infested flush. Very few infested flush have been found in plots receiving insecticides while no infested flush have been found in plots with both mulch and insecticide applications. The third leaf samples were collected on July 9 for HLB. Tree growth measurements were taken on July 12 for the third time which consisted of trunk diameter and height measurements. Monthly foliar nutrition applications continue to be applied in the first week of each month. Leaf samples for the 3rd nutrient analysis were collected 29 Jan 2013. Normal grove care operations continued. These include one application of Agrmek on July 4 for leafminer, one application of glyphosate for weed control in mulched plots May 30, one application of glyphosate May 30 and Kocide once to control canker on May 30. All trees were desuckered and any low hanging limbs pruned July 2 to reduce the chance of herbicide damage.
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 and reduce incidence of HLB, 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 planted on a 23 x 9 ft spacing at 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 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 of 16 plots is split into two subplots 5 rows wide and 13 trees long, mulch and no mulch. Mulch provided by Imaflex Inc. is metalized (aluminized/reflective) polyethylene film of 3 mils thickness covered with a clear protective polyethylene coat. Metalized mulch was shown in preliminary evaluations on single plots to repel Asian citrus psyllid and together with a drip irrigation/fertigation system increase citrus growth rate over the unmulched control. The block was planted 3-4 Jul 2012 and monitoring ACP with flush inspection and sticky cards commenced 13 Aug. Sticky cards are monitored for ACP and other common citrus pests and replaced every other week . About 12 psyllids have been found on sticky card of which 3/4 are in no-mulch plots with more in plots that do not have chemical control than plots that receive insecticides. To date, several flush have been found infested with ACP eggs and nymphs in no-mulch receiving foliar nutrition without insecticides. The first leaf samples were collected on October 17 for HLB testing of which one tested positive but subsequently came up negative following multiple times retesting. A second general leaf sample on 19 Feb all tested negative. Monthly foliar nutrition applications were suspended after the Nov 7 spray in preparation for the winter dormant season and resumed on Mar 7. Leaf samples for the 2nd nutrient analysis were collected 29 Jan 2013. Tree growth measurements of height and trunk area were taken for a second time on 12 Feb 2013. These measurements showed trees grown on mulch were slightly taller than trees in unmulched plots they also had a larger trunk cross sectional area. Trees in the foliar nutrition and insecticide treatments were taller and had larger trunks followed by all other treatments which were similar. Normal grove care operations continued. These include one application of Vendex for spider mite control on Jan 15, one application of glyphosate for weed control in mulched plots Apr 3, one application of glyphosate and Solicam in unmulched plots Apr 3 and Kocide once to control canker on Apr 10. All trees were desuckered and any low hanging limbs pruned to reduce the chance of herbicide damage on Feb 7. Over the nights of Feb 16-18 and March 1-3 a freeze threat was experienced but the flood irrigation plan was executed so that no damage was incurred. New water sensor probes were tested and calibrated for soil moisture data collection.
Obj 1: The GOseq method was incorporated into the PAVE database to aid in identifying candidate effectors to take advantage of the ability to select statistically among significant differentially expressed genes. Predicted ‘effectors’ found in both the Asian citrus psyllid (ACP) and potato psyllid (PoP: study system) transcriptomes have been bioinformatically validated using automated BLAST annotation against the NCBI-GenBank database. PCR primers were designed around conserved nucleotides for each contig and RT-PCR was carried out using PoP cDNA to amplify the corresponding transcript and confirm (in vivo) expression of each transcript. To date, expression of 22 transcripts has been confirmed in PoP and confirmation of all genes in ACP by RT-PCR is in progress. Initially dsRNA screening will employ PoP to test for knockdown of transcripts. Obj 2: Yeast-2 hybrid studies were initiated to study protein-protein interactions important in psyllid-Liberibacter interactions involved in the circulative, propagative pathway. To accomplish this 500 guts from Las-infected ACP and 500 guts and 1000 salivary glands from uninfected ACP were extirpated and used to construct the libraries. Three cDNA libraries: uninfected gut, uninfected salivary gland and infected bacteria have been completed. Furthermore, three Mate & Plate ‘prey’ libraries (uninfected gut, uninfected salivary gland and infected bacteria) have also been completed, with high transformation efficiencies of > 2 million clones for each (50 ml x 10 tubes ea). The titers (cfu/ml) of the libraries are 4.0 x 107 cfu/ml for the uninfected gut, 2.6 x 107 cfu/ml for the uninfected salivary gland and 1.2 x 107 cfu/ml for the infected bacteria, respectively. Bacterial ‘bait’ primers were designed for 4 CLas-specific target genes, amplified each by PCR from infected ACP genomic DNA, and cloned into E. coli using pGBKT7. Transformation of selected candidate ‘baits’ into libraries is in progress. Obj 3: The second biological replicate for proteomic identification of putative effectors is underway. An additional 50 guts and 250 salivary glands of infected and uninfected potato psyllid adults were fractionated by SDS-PAGE (10’14% acrylamide) and digested with trypsin to yield peptides. The peptide mixture was analyzed by nano-LC-MS/MS using an EASY nano-LC (Proxeon, Thermo Fisher Scientific, San Jose, Ca) coupled to an Orbitrap LTQ Velos mass spectrometer. Proteins were identified at 99% confidence with XCorr score cut-offs determined by reversed database search. Mass spectra of peptides were analyzed with SEQUEST and XTandem software. Data were processed and organized using the Scaffold program. A probability threshold of 95% was adopted, and only proteins present in at least two replicates were considered expressed. The PAVE EST database was mined to select optimally sized open reading frames from among the annotated transcripts in the ACP and PoP adult psyllid, gut, and salivary gland databases, and the resultant translated proteins were used for protein identification. Obj 4: Several psyllid and Liberibacter genes were selected based on their potential for involvement as interactors, and tested for knockdown activity in an RNA-interference (RNAi) assay using oral delivery (parafilm membrane). Good quality dsRNA was synthesized (MEGAscript RNAi kit). This approach, together with microinjection of dsRNA, will form the basis for in vitro functional analysis of candidate effectors identified bioinformatically and/or in the yeast dihybrid assays.
Models relating yield to ground and foliar enhanced fertilization in the presence of HLB have been fully developed and explored. Although the results are promising, the amount of data available does not permit a general recommendation. Based on these initial results, several growers have volunteered to provide more data. Efforts are being made to obtain the data.
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