We aim in this project to genetically manipulate defense signaling networks to produce citrus cultivars with enhanced disease resistance. Defense signaling networks have been well elucidated in the model plant Arabidopsis but not yet in citrus. Salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) are key hubs on the defense networks and are known to regulate broad-spectrum disease resistance. With a previous CRDF support, the PI’s laboratory has identified ten citrus genes with potential roles as positive SA regulators. Characterization of these genes indicate that Arabidopsis can be used not only as an excellent reference to guide the discovery of citrus defense genes and but also as a powerful tool to test function of citrus genes. This new project will significantly expand the scope of defense genes to be studied by examining the roles of negative SA regulators and genes affecting JA and ET-mediated pathways in regulating citrus defense. We have three specific objectives in this proposal: 1) identify SA negative regulators and genes affecting JA- and ET-mediated defense in citrus; 2) test function of citrus genes for their disease resistance by overexpression in Arabidopsis; and 3) produce and evaluate transgenic citrus with altered expression of defense genes for resistance to HLB and other diseases. Currently we have cloned 8 full-length genes in these categories in the entry vector pJET and two of the genes were further cloned to the binary vector pBIN19plusARS. Transformation of Arabidopsis and citrus plants will be simultaneously performed to obtained transgenic plants over-expressing the constructs for further analysis of plant disease resistance. In addition, we are continuing to generate and/or characterize transgenic citrus plants expressing the SA positive regulators, as proposed in the previous project, although the support of this previous project has already been terminated. A manuscript describing the cloning and characterization of the citrus NDR1 ortholog was recently under revision in the journal Frontiers in Plant Science.
Huanglongbing (HLB) caused by Candidatus Liberibacter asiaticus (Las) infection has emerged as a major threat to citrus production worldwide owing to the fact that it can take up to two years following infection before outward symptoms become apparent. The goal of this project was to find metabolic indicators for Las infection in citrus that is complementary to PCR, and may be useful in areas where Las remains an exotic threat such as California. Our work began looking at the effect of this pathogen on citrus fruit metabolism. Through measuring metabolites in healthy, asymptomatic, and symptomatic fruit, we were able to detect distinct differences that provided some potential information on the mechanism of Las attack. Specifically, we determined that the bacteria may be suppressing plant defense mechanisms, such as preventing generation of hydrogen peroxide, nitric oxide, and cinnamic acid, all compounds that can aid in killing potential pathogens. This work was published in the Journal of Proteome Research in 2012, and has provided valuable preliminary information for understanding the mechanism of Las infection. At the same time, we began to study the effect of Las infection on citrus leaves. Through collaboration with the USDA in Fort Pierce (Mark Hilf), we obtained leaf samples from a longitudinal study in which asian citrus psyllids (ACP) carrying Las were released into a controlled greenhouse and allowed to infect the citrus plants, including varieties of sweet orange. At the end of the experiment, none of the trees were symptomatic for Las infection, and approximately 8-9 months after infection, sweet orange varieties of citrus tested positive for Las. Our work indicated that we could detect obvious plant metabolic changes, related to stress and plant defense, 3-5 months prior to detection by PCR-based methods. These results provided the necessary information for us to propose extending our project by an additional 2 years. The work that we started this year continues from our first year of funding. We have obtained additional samples from other longitudinal experiments performed by Dr. Hilf that were started at different times to rule out any changes in plant metabolism caused by weather. These new experiments appear to confirm our previous findings. We have also coordinated to collect leaf samples from citrus infected with various pathogens such as citrus tristeza virus (CTV) or citrus canker. In collaboration with Dr. Cynthia LeVesque and Dr. MaryLou Polek, we have received citrus leaf samples that have been damaged by Las negative ACP. We have determined that the metabolic signature from insect damage is different than the signature from Las pathogen infection further confirming that our results in the longitudinal experiment were due to the pathogen rather than damage from the insect. We are currently in the process of writing the results from the Las negative ACP experiment for publication.
In the past few months, the most significant progress made on the FT project was the completion of the new FMVcDNA27 construct, which contains an FT3 cDNA insert in the pCAMBIA2201 vector with a constitutive FMV promoter. This construct was created as a first step towards the development of a new FT3 construct with an inducible promoter. In order to ensure that the cDNA was as effective as the genomic, this FMVcDNA27 construct will be compared to the original p27 construct which contains a genomic FT3 insert in the pCAMBIA2201 vector with the FMV promoter. Transformation of Carrizo and tobacco tissue is already underway in order to compare the action of these two constructs. Additionally, we have arranged for the materials transfer of two inducible promoter systems from the Danforth Foundation. Both of these promoters are inducible by the chemical methoxyfenozide, a widely-available pesticide, approved for field use. One system is driven by the CsMV constitutive promoter, and the other by the RTBV vascular-specific promoter. Once we have verified that the smaller and more manageable cDNA is as effective as the original genomic version of the FT3 gene, we will begin development of the inducible promoter constructs.
USDA-ARS-USHRL, Fort Pierce Florida has thus-far produced over 3,000 scion or rootstock plants transformed to express peptides that might mitigate HLB, and many additional plants are being produced. The more rapidly this germplasm can be evaluated, the sooner we will be able to identify transgenic strategies for controlling HLB. The purpose of this project is to support a high-throughput facility to evaluate transgenic citrus for HLB-resistance. This screening program supports two USHRL projects funded by CRDF for transforming citrus. Non-transgenic citrus can also be subjected to the screening program. CRDF funds are being used for the inoculation steps of the program. Briefly, individual plants are caged with infected psyllids for two weeks, and then housed for six months in a greenhouse with an open infestation of infected psyllids. Plants are then moved into a psyllid-free greenhouse and evaluated for growth, HLB-symptoms and Las titer. This report marks the end of the third quarter of the project, during which we have began large-scale production of CLas positive ACP. To date on this project, a technician dedicated to the project has been hired, a second career technician has been assigned part-time, two small air-conditioned greenhouses for rearing psyllids are completed and are functioning well, and 18 individually caged CLas-infected plants are being used to rear ACP for infestations. A total of 2,124 transgenic plants have passed through the screening program. A total of 43,680 psyllids have been used in no-choice inoculations. USDA-ARS is providing approximately $18,000 worth of PCR-testing annually to track CLas levels in psyllids and rearing plants. Maintaining an open infestation of infected psyllids (phase 2 of the inoculation process) was challenging this past quarter because of surprise pest problems, notably western flower thrips which invaded the greenhouse initially attacking new flush and then first instar psyllids (facultative predation). Tamarixia radiata invaded the house during January, killing a majority of psyllid nymphs. Measures have been taken to limit these unwanted pests, but these measures are costing an additional $1,400 annually for applications of M-Pede and Tetrasan and releases of beneficial insects for the control of spider mites and thrips.
The objectives of this project are: 1) to generate transcriptome profiles of both susceptible and resistant citrus responding to HLB infection using RNA-Seq technology; 2) to identify key resistant genes from differentially expressed genes and gene clusters between the HLB-susceptible and HLB-resistant plants via intensive bioinformatics and other experimental verifications such as RT-PCR; and 3) to create transgenic citrus cultivars with new constructs containing the resistant gene(s). First group of samples for RNA-Seq were selected at Picos Farm at Fort Pierce, including three Jackson grapefruit plants (resistant/tolerant) and three Marsh grapefruit plants (susceptible). Five pioneer Illumina paired-end libraries were constructed and sequenced by Hiseq-2000 at BGI. Each library has about 10 M 2.90 bp paired-end reads with more than 10 G data. We maped the RNA-Seq data to reference genome, C. clementina using the computer program STAR. About 85% of the raw reads could be uniquely mapped. The transfrags of each library were assembled with cufflinks and merged with cuffmerg. 24275 genes of original predicted genes had been found with expressions. And a total of 10539 novel transfrags were identified with cufflinks, which were missing from the original reference genome annotation. Some of the NBS genes were found to have an expression. For C. clementine and C. sinensis, there were 118,381 and 214,858 mRNAs or ESTs deposited in GenBank and 93 out of 607 and 221 out of 484 NBS related genes match one or more ESTs respectively. The number of EST varied from 1 to 25. The expression abundance of each gene was measured by FPKM. The distribution curves of density of FPKM of 5 samples are very similar, indicating that the gene expression is similar and the quality of sequencing is high. We also performed the principal component analysis (PCA) study on the expressions of five samples. The plot of first PC against second PC showed that R2017 and R20T18, were grouped to the one group (resistance group) and R19T23, R19T24 and R20T24 were grouped to another group (susceptible group). This result showed that the gene expressions were significantly different in resistant and susceptible citrus. Using cuffdiff, a total of 821 genes were identified as difference expressed genes (DE genes) between the two groups using both p-value and FDR threshold of 0.01. Among them, 306 genes are up-regulated expression genes and 515 are down-regulated in resistant citrus. Using program iAssembler, a total of 53981 uni-transfrags were obtained. Most of the assembled uni-transfrags should be novel genes, comparing with the citurs reference genome. To reveal the differences in resistance, we also identified the exon variations (SNP/INDEL). A total of 612618 SNP/INDELs were identified using mpileup method employed in samtools. We focused on two types of mutations that could contribute to the resistance difference. The first type mutation is the mutation in the genes of susceptible citrus leading to pseudogenes (Type1) and the other type of mutation is the mutations in resistant citrus genes that may gain a new function (Type2). Type1 mutation should have a homozygote mutation genotype in the susceptible citrus. We identified 146 candidate genes having Type1 mutations, which had high impact variations, such as frame shift, splice site acceptor, splice site donor, start lost, stop gain or stop lost and 3578 genes with Type2 mutations. We expect that with more libraries being sequencing, the candidates should be reduced to a reasonable number for further validation.
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.
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