The overall objective of this proposal 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). Most work during the first quarter of the project was focused on development of a chemical screen using potato psyllid/CLps/Arabidopsis as the model. As a first step toward this objective, we tested 19 diverse Arabidopsis lines for susceptibility to infection by psyllids. We found significant differences among lines in the percentage of plants that became infected, and in the amount of bacteria present in tissue samples from each plant as judged from the Ct value of qPCR detection. No lines were completely resistant, the most resistant having only 1 of 5 plants infected. The most resistant and susceptible lines will be tested again to confirm differences. We did not detect any visible disease symptoms on leaves or stems of infected plants, but have not yet evaluated seed production or root growth. We also found that about 92% of psyllids from our colony test positive for bacteria, a level adequate to obtain a high frequency of infected plants from inoculation with 3 psyllids per plant. Methods to grow Aradidopsis seedlings in 24-well plates were developed. However, when psyllid nymphs were transferred to the seedlings, they appeared to feed but we did not obtain any infected plants. The reason for this is being investigated but it may relate to the composition of the growth medium. We have planted seedlings to measure gene expression responses to CLps. We have not yet initiated chemical testing because the system to produce suitable plants for screening has not yet been successful.
Researchers at the USDA Ft. Pierce: 1. Source of mature tissue. Four populations of adult phase trees were maintained in the greenhouse including Valencia sweet orange/Sun Chu Sha (73 trees), Ruby Red grapefruit/US812 (62 trees), US-942 citrange rootstock/Cleo (32 trees), Calamondin (31 trees), and Etrog Arizona 861-S1 citron (67 trees). In vitro bud emergence and growth manuscript accepted for publication. A manuscript was submitted to the journal Plant Cell, Tissue and Organ Culture that documents the system developed for producing in vitro adult phase shoots from cultured nodes of greenhouse trees. Shoot regeneration from mature tissue explants. A system was developed for the production of shoots from cultured internodes from greenhouse trees. The system results in shoot and bud formation in 70-90% of the explants. A manuscript is in preparation that documents this research. Agrobacterium-mediated transformation of mature tissue explants. Transformation of mature internode explants from greenhouse trees has been demonstrated in grapefruit (1 plant), US-942 (4 plants), and Etrog citron (4 plants) using the beta-glucuronidase reporter gene. Current efforts are now directed toward identifying the factors important for a system of sufficient efficiency for routine transgenic plant production. New tissue culture method of Agrobacterium-mediated transformation of tissue explants. Preliminary results using alternative culture methods suggest improved transformation efficiencies. These approaches will be further explored. At the CREC in the Gmitter lab, work is continuing on the use of Thin Cell Layers (TCLs) as explants for mature tissue transformation. Experiments have been done to induce regeneration in the TCLs by manipulating the amount of growth regulators, carbon source and also by pre-treating the TCLs with BA but regeneration is still problematic from these explants. In the Grosser lab, research confirmed the work of others showing that the flush used to generate transformation explants was critical. In the Machado laboratory in Brazil, research on the regeneration ability of various sweet oranges continues. These differences are present in both mature and juvenile tissues. In the Moore laboratory in Gainesville, experiments still are focused on using small peptides as vehicles to deliver cargos to plant tissues. If these techniques could be worked out they would have a number of applications for citrus transformation, perhaps even eventually allowing the transfer of genes or gene products to existing trees. A transient transformation expression system has also been developed using citrus leaves.
Objective 2: Develop a method to elicit a robust plant defense response triggered by psyllid feeding. This objective is proposed as an alternative strategy in case constitutive expression of the mutant R proteins proves to be detrimental to the growth or vigor of the plant. By restricting expression of the R protein to the single cell that is pierced by the insect stylet, a defense can be mounted without endangering the overall health of the plant. In the long run, this may be the most effective strategy in fighting Liberibacter infection since the response can engineered to be quite robust. Results: To further insure more specific expression of the R proteins in the phloem, we identified a wound-inducible, phloem-specific promoter that triggers expression upon wounding resulting from aphid feeding. We substituted the AtSUC2-940 promoter (phloem-specific) with the AtPAD4-1002 promoter (PAD4) in SNC1, snc1, SSI4, and ssi4 R gene constructs in the pCAMBIA1305.1 vector. Additionally, we created a PAD4/GUSplus construct to verify phloem- and wound-specific activation. The PAD4 constructs were introduced to Arabidopsis plants, grown to seeds and harvested this week (Jan 2011). The transgenic seeds will be subjected to selective screening for R gene transformants. Due to the cloning limitations (restriction site configuration), all PAD4/R constructs had to be first assembled in the 1305.1 pCAMBIA vector prior to transfer into the 2301 pCAMBIA for transformation into citrus. We selected two constitutive R protein mutants and PAD4/GUSplus constructs, and transferred them to the Citrus Research and Education Facility at Lake Alfred (Dr. Vladimir Orbovic). Preliminary reports indicated that transformants are proving difficult to obtain, potentially due to the lethal nature of at least one R protein mutant. Conclusions: Our hypothesis is that phloem-restricted expression of the R protein constitutive mutants would limit the potential negative impacts on plant growth. Use of the PAD4 promoter to express the R protein constructs should further increase the stringency of transcriptional control over expression of the potentially harmful R proteins. Our goal is have only the single phloem cell that is penetrated by the stylet express the constitutively active R protein, hence limiting potentially detrimental effects and focusing a robust defense at the source of Liberibacter introduction. Additionally, these constructs with the PAD4 promoter may reduce spurious expression in callus cells during the transformation protocol into citrus.
A scFv library with activity against ‘Ca. Liberibacter asiaticus’ has been prepared at Beltsville. mRNA was purified from mouse spleens and converted into cDNA. The mice had been immunized with psyllid extracts confirmed to be carrying a high concentration of “Ca. Liberibacter asiaticus” A complete library of variable heavy chain (VH) and variable light chain (VL) genes were made by PCR amplification of the cDNA using a set of 44 primers. The (VH) and (VL) gene segments were then joined in a random combinatorial fashion by overlap extension PCR. The scFv genes were then ligated into the pKM19 phagemid vector which was used to infect Escherichia coli DH5. F’ cells with the aide of a helper phage. The resulting phage library is presently being screened to select phage clones expressing antibodies that bind to “Ca. Liberibacter asiaticus”. Our library is estimated to contain 2.1 x 10 7th primary, unique antibody clones. Because our antigen was individual psyllids from Florida infected with high concentrations of ‘Ca. Liberibacter asiaticus’, the library contains antibodies for both the pathogen and the vector insect. Our first attempts to select desired antibodies using extracts from HLB-infected rough lemon were not successful, probably because the concentration of the target bacteria in the rough lemon extracts was too low. We therefore modified the screening procedure by incorporating magnetic microbeads. These microbeads bind to rabbit antibodies. To use them we raised standard polyclonal antisera in a rabbit against the outer membrane protein of “Ca. Liberibacter asiaticus”. These beads are added to plant and insect extracts to bind the “Ca. Liberibacter asiaticus” and then concentrated by magnetic separation. The phage libraries are then added to the “Ca. Liberibacter asiaticus” on the beads and screened in that manner. The outer membrane protein and antibody system for immunocapture of ‘Ca. Liberibacter asiaticus’ has however not proven to be successful. The polyclonal antibodies made in rabbits against purified outer membrane protein from ‘Ca. Liberibacter asiaticus’ work very well in detecting the purified outer membrane protein but are not effective in immunocapture. The most likely explanation is that the outer membrane protein is not well enough exposed on the surface of the ‘Ca. Liberibacter asiaticus’ cell. We have therefore cloned genes encoding the type IV pilus protein of ‘Ca. Liberibacter asiaticus’, and the polysialic acid capsule protein since these proteins should be well exposed on the cell surface and should be useful for labeling ‘Ca. Liberibacter asiaticus in insect or plant tissues. Correct cloning was confirmed by DNA sequencing and these genes have been expressed in E. coli and the encoded proteins and have been purified. Emphasis is on recovering the proteins in native, soluble form, which was difficult, but we have now been able to accomplish it. The Type 4 pilus proteins and the polysialic acid capsule protein have been biotinylated and combined with strepavidin coated magnetic beads and used successfully to bind recombinant phage expressing antibodies that recognize these targets. Thus we have developed and demonstrated a protocol that will allow us to isolate scFv antibodies that, in principle, will recognize any proteins from “Ca. Liberibacter asiaticus” or the insect vector, Diaphorina citri. We will select scFv antibodies next against purified Outer Membrane Protein (OMP), using protein already purified. After our presentation at the 2nd IHRC in Orlando, several researchers expressed interest in our offer to select scFv antibodies upon request for any protein of interest to their research programs. scFv antibodies may be useful as labels for ultrastructural studies of infected plants and insects, advanced detection assays, and possible even for HLB control through a ‘plantibody-based’ approach.
For EDS1 cloning, we currently confirmed by sequencing the cloning of the full-length ctEDS1 and already moved the sequence from the pGEM T-easy vector to the binary vector pBINplusARS for plant transformation. With Carrizo sequence database (http://citrus.pw.usda.gov/) recently available, we designed primes to clone the 3′ end sequence of ctSID2, which we were previously unable to obtain with several methods. We performed the RACE reaction and have now obtained this missing sequence. We will subsequently design primers to amplify the full-length sequence of ctSID2. In addition, we did bioinformatics analysis and identified additional 9 citrus homologs of Arabidopsis defense genes that have available sequences in the database. We designed primers for these citrus genes and have been conducting RACE in order to amplify the genes. So far we have obtained the 3′ end sequences for ctNHL1, ctSFD1, and ctFAD7. Further cloning of 5′ end and/or 3′ end sequences of these citrus defense genes are currently underway. We continue to characterize the transgenic plants expressing ctNDR1/pBINplusARS. We obtained 5 homozygous ndr1 + ctNDR1/pBINplusARS. Through HR test and disease resistance assay with infection of the avirulent strain P. syringae avrRpt2, we further confirmed that over-expression of ctNDR1 could complement Arabidopsis ndr1 mutant. We are going to further characterize the defense phenotypes of these transgenic plants.
This is a 3-year project with 2 specific aims: (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 AtMKK7 gene, we also cloned the Arabidopsis MOD1 and NAC1 genes, which have been shown to provide resistance to bacterial pathogens in Arabidopsis. The T-DNA vectors have been transformed into Agrobacteria and plant transformation is underway. We have generated transgenic citrus plants overexpressing the Arabidopsis NPR1 gene and showed that they are more resistant to citrus canker. We are currently testing the resistance of these transgenic plants to HLB. We are also generating citrus plants that can accumulate more salicylic acid. For AtMKK7, about 1700 explants were incubated and regenerated shoots were tested with PCR. Twenty of these shoots were positive in the screen and all of them were grafted onto Carrizo. The twenty transgenic lines are being propagated. The presence of the AtMKK7 gene in the transgenic plants will be confirmed by PCR and the expression levels of AtMKK7 in each transgenic line will be analyzed using qRT-PCR. Resistance of the transgenic lines to citrus canker and greening (HLB) will be characterized when more transgenic plants are available. For objective 2, a total of 100 plates of hypocotyl cuttings (each plate with 40-50 stem pieces) was irradiated with a dosage of 30G gama ray. The irradiated stem pieces were placed on non-selective shooting medium. The plates are kept under 14 hour photoperiod. Shoots generated from the irradiated hypocotyls were transferred onto selective medium with 0.2 mM of sodium iodoacetate. Another batch of explants (90 tubes of ‘Duncan’ grapefruit seedlings) has been prepared for irradiation. For each irradiation, ten plates of hypocotyl cuttings were kept as non-irradiated controls for comparison. We will prepare more hypocotyls for irradiation.
Over the past quarter, we have continued to develop all aspects of our project. In particular we have progressed in the following areas: 1. Building and testing additional TAL effector and promoter constructs. We have synthetically assembled a number of TAL effector genes matching X. citri TAL effectors and showed that they transcriptionally activate our broad recognition or “super” promoter in a Nicotiana benthamiana system. We have also assembled promoters with individual TAL effector binding sites to test activity and specificity. 2. Testing activation of gene constructs against a diverse world wide collection of X. citri isolates. Using the transient transformation method that we have developed, we have tested the reaction of thirty X. citri isolates on grapefruit leaves. We see a very high correlation between isolates which are capable of inducing disease in standard susceptible germplasm and recognition by our promoter constructs, indicating that the resistance constructs we have created will be able to confer broad resistance to diverse strains of citrus canker. Additionally, we are preparing and testing X. citri strains with single or multiple disruptions in their TAL effector complement to test the role of specific TAL effector proteins in the disease and resistance process. 3. Stable transformations. The transformed lines generated last Fall and Winter have progressed through selection, shoot formation and rooting, and are now growing in soil. These lines are tested by PCR as they reach adequate size, and positively scored lines have been subjected to pathogen testing by pin-prick assay with X. citri. We have identified several canker resistant transgenic lines. We are currently setting up additional transformations to generate more transgenic material for line testing and with new promoter constructs. 4. Manuscript preparation We are in the process of drafting a manuscript of our results.
The goal of the proposed research is to understand how Candidatus Liberibacter asiaticus causes Huanglongbing (HLB) disease on citrus. Citrus HLB is the most devastating disease on citrus. There are very few options for management of the disease due to the lack of understanding of the pathogen and citrus interaction. Understanding the citrus and citrus HLB pathogen interaction is needed in order to provide knowledge to develop sustainable and economically viable control measures. Here are the major achievements: 1. Microarray analysis of host response of sweet orange to Las infection in greenhouse. The results were published in the following paper: Kim, J., Sagaram, U.S., Burns, J. K., and Wang N*. 2009 Response of sweet orange (Citrus sinensis) to Candidatus Liberibacter asiaticus infection: microscopy and microarray analyses. Phytopathology 2009 99:50-7. 2. We are currently assessing citrus genes modulated by Las infection in 1) Comparison of citrus leaves, stems and roots to Las infection (completed, paper in writing), 2)Comparison of healthy vs. infected leaf samples in citrus grove (microarray data collected and QRT-PCR is underway),3) Comparison of different citrus varieties that are different in tolerance and susceptibility (in progress). The alteration of gene expressions by Las in leaf, stem and root tissues of Valencia sweet orange was investigated using Affymetrix microarray analysis. Out of 30,279 probe sets, a total of 8667, 2795 and 1142 showed significantly altered (p< 0.05) expression in leaves, stems and roots, respectively. Using 2 fold change as cut-off value, 1008, 580 and 58 transcripts were significantly up-regulated in leaf, stem and root tissues, respectively, whereas, 1109, 350 and 58 were correspondingly down-regulated in Las infected plants. Differences were observed for genes involved in cell wall synthesis and remodeling, lipid metabolism, photosynthesis, secondary metabolism, and starch and sucrose metabolism. Biotic stress induced signaling and transcription factors, PR-proteins, heat shock proteins, hormones and genes involved in protein modification, redox reactions and secondary metabolism were affected more in leaves and stems than in roots. PR genes were mainly repressed in roots but showed both patterns in leaves and stems; JA genes were up-regulated in stems, down-regulated in roots but up- and down-regulated in leaves; Calvin cycle genes were mainly altered in roots; SA and heat shock proteins were not significantly altered in roots. Transcriptional factors with WRKY, AP2/EREBP, MYB, bZIP, bHLH and Zinc finger domains were differentially regulated in all tissues, but least in roots. Differences were shown by C2C2(Zn) DOF zinc finger family proteins, affected in leaves and stems only; homologs of MEE47, nuclear factor PBF-2 and ATRR1 were regulated in roots only while MADS box transcription factor family and several unclassified transcriptional factors were down-regulated only in leaf tissues. We are further analyzing the data to understand how Las affects leaves, stems, and roots since they have distinct roles and function and how they contribute to the HLB disease development.
The goal of this project is to transform the citrus and Arabidopsis NPR1 genes (CtNPR1 and AtNPR1), and the rice XIN31 gene into citrus, and to evaluate their resistance to both citrus canker (caused by Xanthomonas axonopodis pv. citri (Xac)) and greening diseases. The first year objectives include: (1) Molecular characterization of the transgenic plants; (2) Inoculation of the transgenic plants with Xac; (3) Inoculation of the transgenic plants with the HLB pathogen, and monitoring of the bacterium in planta with quantitative PCR; (4) Transformation of SUC2::NPR1 into citrus; (5) Plant maintenance. We have identified three transgenic lines overexpressing CtNPR1. These NPR1 overexpression lines were inoculated with 105 cfu/ml of Xac306 and the results showed high levels of resistance from the NPR1 overexpression lines, but not from the control plants. We also conducted growth curve analyses. Nineteen days after inoculation, the bacterial population in one of the NPR1 overexpression lines is 10,000 fold lower than that in the control plants. These results demonstrate that overexpression of CtNPR1 confers resistance to canker disease. We also graft-inoculated the NPR1 overexpression lines with greening to determine whether NPR1 is functional in greening resistance. We are in the process of monitoring Candidatus Liberibacter asiaticus populations in the inoculated plants using quantitative PCR. Finally, transformation of the SUC2::CtNPR1 construct, in which CtNPR1 is driven by a phloem-specific promoter from the Arabidopsis SUC2 gene, is in progress.
In this first quarter, we have investigated the experimental design and set up of plant materials and growth conditions for small molecule therapeutic treatments. We have conducted a bioinformatics and literature analysis of the transcriptome data obtained in our previous project. We have identified three different therapeutic strategies that will be tested to evaluate if they enhance citrus response to the disease, prolong life of HLB-infected plants and reduce the bacterial titre and counteract the detrimental effects on production. We have identified that a possible cause of the induced disorder in infected plants is related to an unbalanced hormone-mediated response and crosstalk. In young leaves, where the pathogen is usually transmitted by the psyllids, Systemic Acquired Resistance (SAR) was unexpectedly not induced while several genes involved in jasmonic acid and ethylene signaling and response were activated. The inductions of few SAR genes (i.e. PR1 and DIR1) in mature leaves might be too weak a response to counteract pathogen colonization/virulence. Interestingly several genes typically involved in plant responses to biotic stress were induced in the fruits such as WRKY transcription factors. Induction of ethylene biosynthesis and signaling is another detrimental response for fruits. Based on these results we defined a therapeutic strategy that will enhance SAR response and counteract ethylene in early infected tissues. An important aspect of the disease is related to the imbalance of the source-sink homeostasis due to the differential regulation and induced of several key genes involved in glucose intracellular transport, sucrose and starch metabolism. Based on these results, we have identified from public databases (i.e. Genevestigator) possible molecules that can be applied on leaves regulating these genes to correct the sugar gradient between sink and source tissues. A differential regulation of genes involved in photosynthetic light reactions was observed in fruit and leaf tissues in HLB infected (dowregulation in leaves and upregulation in fruits). This could possibly be related to the feedback regulation of sugar signaling response. Based on literature analysis this is related xenobiotic stress responses, we have also considered a possible strategy based on sugar signaling. We have constructed a solid experimental design that will clearly identify the effects of each small molecule applications on specific host responses to HLB infection. Plant categories were composed by four categories: HLB-infected and Control (healthy, uninfected) plants. Both types of plants were divided in treated and untreated groups to dissect treatment effects due to the direct enhancement of the host responses to pathogen infection and those that can have indirect effects and involved in other plant functions. The following parameters were defined: methods of HLB-infection through grafting, physiological conditions, plant age and growth conditions, timing of small molecule applications, molecular and morphological analysis, environmental conditions in greenhouse. Plants will be grown in pots and infections have yet to be started. Infected bark pieces will be obtained from citrus infected trees from commercial orchards near Lake Alfred (Florida) and confirmed positive for pathogen presence using qPCR. Plants will be randomly arranged in the greenhouse and kept under natural light conditions at 17’25 ‘C.
As previously reported Severinia buxifolia (box orange) has been determined to be a host for the Ca. L. asiaticus and transmission to citrus was obtained. Transmissions of Candidatus Liberibacter asiaticus were successfully done from S. buxifolia to both healthy sweet orange and to healthy S. buxifolia in two separate tests and a third test was performed but not yet sampled. To date we have a 50% transmission rate from S. buxifolia to sweet orange and a 72% rate from S. buxifolia to S. buxifolia. As earlier reported some plants that tested positive using real time PCR (qPCR) later tested negative. These plants were tested each month during this period and continued to test negative. This may be similar to results found with Murraya paniculata (orange jasamine) that the bacterium may be transmitted to the plants but doesn’t survive if not reinoculated. We have developed a method to quantify live and dead bacteria in plants and are using it to determine the population of live Candidatus Liberibacter asiaticus in citrus and S. buxifolia. As reported the various rutaceae (Esenbeckia berlandieri (jopoy), Amyris madrensis (torchwood), Choisya ternata and C. arizonica) were subjected to psyllid inoculation with Candidatus Liberibacter asiaticus since all be found to be graft incompatible with citrus. As reported all of these hosts were new ones for the psyllid and in transmission experiments the psyllid did survive and in some instances layed eggs on these hosts. To date only Choisya ternata again was found positive for Liberibacter asiaticus. Positive psyllid transmission tests with Candidatus L. africanus in quarantine with Choisya ternata were obtained. Results with Choisya arizonica are still pending. Calamondin (Citrus madurensis) was infected via grafting with Candidatus L. asiaticus and psyllid transmissions were done to sweet orange with the results pending. Surveys of wooded riparian areas near orange groves that have an edge effect of what appears to be the first entrance of HLB have been sampled for the presence of alternative hosts of HLB. We have run over 100 plants of various types including citrus and to date only citrus species have been positive for Candidatus Liberibacter asiaticus and that number is very low. Sampling will again be done this spring when psyllids are active. Primers to a different region of Candidatus Liberibacter asiaticus were produced and tested. The new primer set and probe generically detects all Candidatus Liberibacter species including the potato zebra chip bacterium (Candidatus Liberibacter solanacearum). These primers and probe were tested by two other labs for confirmation and our results were confirmed. They will be sent for testing to more labs working on Liberibacter species. We have developed these primers and probe to further check the qPCR using the 16s primers. Work to further test the susceptibility or resistance of the cultivar IAPAR73 continued. Numerous propagations of this cultivar were made on four different rootstocks and were subjected to further psyllid inoculation with Candidatus Liberibacter asiaticus. IAPAR 73 plants were prepared for quarantine for testing with Candidatus Liberibacter americanus and africanus.
The project has two specific aims. We outlined below the progress made for each of them in the final quarter. Specific Aim 1: Identify sweet orange responses to Huanglongbing disease (HLB) through deep transcriptome profiling using new DNA sequencing technologies. We successfully completed the analysis of transcriptome data from immature fruits, the final developmental stage considered. We have mapped the entire deep sequencing data obtained from the four types of samples (young and mature leaves, immature and mature fruits) using a customized reference composed of NCBI unigenes from two citrus species (C. sinensis and C. clementina) and the Affymetrix sequence dataset. This analysis has increased the number of annotated HLB-regulated genes. In total, 12-36 million 85 pair-ended reads were obtained. We identified 6383-11407 genes as HLB-differentially regulated (log Fold Ratio ><0.5) and functionally categorized using Mapman knowledgebase. We grouped them based on the regulation at different stage of disease identifying those that are specifically or commonly regulated at asymptomatic and symptomatic stages. We performed a gene set enrichment analysis to identify pathways that are highly affected by the disease such as sucrose and starch metabolism, glycolysis, pentose-phosphate and phenylpropanoids. In leaves, we observed a clear down regulation of photosynthesis and the up regulation of genes involved in sucrose and starch metabolism. These altered transcriptional regulation might induce an increase in glucose and a decrease in sucrose accumulation resulting in an alteration in source-sink homeostasis. In HLB infected fruits, photosynthesis was up regulated while starch metabolism was repressed. Specific Aim 2: Define and validate gene networks and identify host (sweet orange) response biomarkers regulated by HLB at different stages of infection. The major causes of the detrimental effects of HLB disease might be related to an inappropriate hormone-mediated response which disrupts source-sink homeostasis between leaves and fruits. The innate immune system is down regulated. SAR was not induced in infected young leaves and the crosstalk between JA, SA and ethylene seems to play a key role in pathogen success. Other hormones such as gibberellins and cytokinins were differentially regulated in both tissues. We dissected the different hormone signaling and their crosstalk choosing important key genes for qRT-PCR validation. Our analysis identified heat shock proteins as major hubs in the protein-protein network and qRT-PCR confirmed transcript down regulation at all stages of the disease. In addition, five-ten early regulated transcripts were identified as good host biomarkers of asymptomatic infections in different developmental stages and tissues. These genes belong to photosynthesis, sucrose metabolism, volatile pathways, transcription factors. These genes may be used to distinguish infections before symptoms appear and as host response complement to qPCR pathogen detection.
Three new formulations of SPLAT-DMDS were developed and evaluated. The formulations release the DMDS active ingredient for up to 3 months, which is approximately 3 times longer than previous formualtions. However, results in the field have still been inconsistent. In certain field trials, were have been able to measure reduced ACP populations but in other experiments there appears to be no effect. The most promising results are obtained when the repellent is deployed after the populations of ACP are initially killed off with an insecticide. Subsequently, psyllids appear to re-colonize DMDS-treated plots slower than untreated plots. However, even these experiments have been inconsistent. An experiment was conducted during the spring and summer that tested the effect of an olfactory psyllid repellent under standard psyllid management practices. The experiment has concluded. The formulation tested was the initial SPLAT-ACP Repel. Throughout this period, three applications of the formulation were made (April 29th and May 29th and June 29th). Applications were made to 3-4 acre replicated blocks of citrus and identical adjacent blocks were used as controls. The experiment was arranged as a randomized complete block with four replicates. All blocks received the same standard psyllid management with pesticides. Psyllid populations were monitored once or twice per week March 16th through August 18th. Yellow sticky monitoring traps were used as well as the tap sampling method. Throughout the course of the experiment, psyllid populations were extremely low in the experimental plots (Below 0.25 psyllids per ten traps per block on average). We observed no significant additional reduction of psyllid populations in the plots that received the olfactory repellent as compared with control blocks without the repellent. This result was likely due to the insecticide use practiced in this grove resulting in psyllid population densities that were likely too low to allow measurement of a treatment effect. During the initial part of the season, we observed that the DMDS active ingredient was highly phyto-toxic when the SPLAT formulation was applied to peripheral (pencil-thin) tree branches. However, application to larger branches (3-5 inch diameter or greater) did not cause phyto-toxic symptoms. We have also developed a method to measure DMDS concentrations in the field and we are using this method to determine how much of the DMDS or other repellents occurs when we deploy it from SPLAT or other release devices. Our intent is to determine exactly how much DMDS we need deployed in the field to achive desirable effects, so that a more consistent release device can be developed.
The goal of this proposal has been to investigate whether Ca. Las is transmitted between infected and uninfected ACP adults in a sex-related manner to understand its role in disease spread in field. We found that Ca. Las was sexually transmitted from Ca. Las infected male psyllids to healthy females but not from infected females to healthy males or among psyllids of the same sex. The experiments were repeated this season to confirm the findings. Our findings were consistent with our previous findings and we were able to confirm that Ca. Las is sexually transmitted between the adult psyllids during routine mating. We were able to detect bacteria in ACP ovaries of recipient females with PCR. However, we were unable to detect the presence of bacteria in genital parts of male and female psyllids with scanning and transmission electron microscopy probably due to washing of bacteria during sample preparation procedures. However, we found some other unidentified bacteria like structures in ovaries of recipient females. In addition studies are in progress to determine if Ca. Las recipient females are able to new citrus plants. Our ongoing efforts focus on identification of unidentified structures and to exploit florescent in situ hybridization analysis to detect presence of bacteria in genital parts of recipient and of the donor psyllids. The procedures for this are being standardized in collaboration with microbiologists.
In the last few months, we have continued working on genetic transformation of mature material from the three sweet orange genotypes (Valencia, Hamlin and Pineapple) with the aim of improving transgenic regeneration efficiency and having a reliable mature transformation procedure for each type that could be reproduced in Florida. After some last refining, Valencia sweet orange is routinely transformed at IVIA now. The transgenic nature of the first plants acclimated to the greenhouse has been confirmed through Southern blot analysis. Hamlin is more difficult to transform but with appropriate modifications of the tissue culture media and the source material used we have been able to produce already many transgenic plants, as confirmed also by Southern blot. Pineapple is routinely transformed at IVIA since the 90’s and is being used as control. Transformation of mature Carrizo citrange was initiated later, simply because we had not enough space and personnel to work with all the genotypes at the same time. During the last quarter, we have been more focused on developing a reliable transformation system for this genotype. More than 100 mature transformants (PCR-positive shoots) have been produced so far. The key in this case is using proper source material. We have preparing new source material to attempt transformation of mature citrumelo and grapefruit in the coming months. Regarding our second objective, at least ten independent transgenic lines of Pineapple sweet orange and Carrizo citrange expressing either FT or AP1 flowering-time genes are established in the greenhouse and we are now characterizing them in detail (genetic and phenotypically). Additionally, a hairpin construct aimed to induce RNA interference to silence and endogenous GA20-oxidase gene and them reducing gibberellin biosynthesis has been synthesized and incorporated into Agrobacterium tumefaciens. It will be used to transform Carrizo citrange. In Florida, construction of the growth room has been finally initialed and according to the schedule it will be finalized before the end of February. The PI and his greenhouse manager are planning to travel to Florida next March to supervise and setting up plant growth conditions, to set up the healthy citrus mother materials, and to establish substrate, fertirrigation and phytosanitary treatments.
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