Our focus in the last quarter was to develop a greater diversity of candidates that could serve as replacements for the CecB lytic peptide domain of our CAP previously described (Dandekar et al., 2012 PNAS 109(10): 3721-3725). We had successfully used the structural motif Lys10, Lys11, Lys16, and Lys29 a unique feature of CecB. We were able to identify 52 aa CsHAT protein that is highly conserved among citrus. Since the protein is too large to syntheize we have begun making constructs to express these in plants. We designed 3 constructs 1) CsP14a with a secretion sequence and Flag tag, 2) CsP14a ‘ CecB (this is construct 1 expressed as a CAP with the original CecB and 3) CsP14a-CsHAT52 (here construct 1 is linked to the 52 aa CsHAT protein from Citrus). These three constructs are being incorporated into CTV vectors so that they can be used for challenging HLB, this work is in progress. We have made Construct 1 and 3 in binary vectors and incorporated these into Agrobacterium strains and these are being used to transform tobacco, construct 2 is in the process of being made. In order to understand better the functionality of the alpha helical domains of CecB we have developed two new computational tools PAGAL and SCALPEL to better predict the antimicrobial activities in portions of existing proteins. PAGAL (Properties and corresponding graphics of alpha helical structures in proteins) implements previously known and established methods of evaluating the properties of alpha helical structures, providing very useful information of the hydrophobicity and charge moments. We have successfully used PAGAL to search 4000 plant proteins in the PDB database and we now have a database that contains a listing of the properties of each and every alpha helical structure present in all of these proteins and the properties of all of the alpha helical structures present in these 4000 plant proteins. We then developed the second program SCALPEL (Search characteristic alpha helical peptides in the PDB database and locate it in the genome) to search for alpha helical structures of a particular type. Once we obtain a particular hit that has the right properties that we are interested in investigating we then use BLAST to find the corresponding citrus protein. Using these two programs we have focused on our phase 1 search which is currently underway where we are examining all small proteins and will compare them to the N-terminal 21 aa domain of CecB that we call CBNT21 (+ve charge). We determined using these two programs that this domain to be the most active of the two alpha helical domains of CecB. We would predict would be the most active in terms of lytic properties. We identified three small proteins in citrus PPC20 (+ve charge very similar to CBNT21 in its structural properties); CsHAT22 (+ve charge smaller version of the 52 aa CsHAT that is currently being tested in plants), ISS15 (-ve charge, very small protein). We also will test CATH12 smallest definsin (12 aa) to define how small a peptide we can use successfully against our pathogens of interest. All 5 proteins have been synthesized and are currently being tested for their lytic activity against E.coli, Xylella, Xanthomonas and Agrobacterium to determine their range of clearance of these pathogens.
The main accomplishments during this quarter: 1) We repeated the transformation experiments with juvenile explants of succari sweet orange and carrizo citrange and we have confirmed drastic increases in transformation efficiency using the K or I genes. 2) We have confirmed most shoots of succari sweet orange and carrizo citrange hosting the K or I genes are transgenic. 3) We grafted some transgenic shoots onto rootstock and they have survived well, and in tissue culture vessels, we have observed little morphological changes if the K gene was used while we have observed a bushy phenotype were observed if the I gene was used. 4) Some grafted transgenic shoots were transferred to soil and grown in a greenhouse. These plants also exhibited normal growth characteristics compared to the control plants. These results demonstrate that the K and I genes can drastically increase transformation efficiency of juvenile tissues of succari sweet orange and carrizo citrange, suggesting they should be useful for adult tissue transformation. Further, these results also suggest that the K gene may not need to be eliminated in transgenic citrus plants. 5) We have put much of our time and effort on adult tissue transformation but nothing is significant to report at this time.
In May 2014 we initiated a bimonthly survey of 4 flatwoods groves and 8 ridge sites in Hardee, Desoto and Highlands counties where soil acidification or irrigation water was implemented 1-1.5 years ago. All the blocks are Valenicia orange on Swingle or Carrizo, roostocks sensitive to excess bicarbonates. The survey assesses seasonal changes in soil pH, root mass density and Phytophthora populations to quantify the response to bicarbonate management, as well as, root growth dynamics of HLB-affected trees. It is expected that seasonal root flushes have been considerably altered from the root growth cycle of healthy tree. The results so far indicate that acidification has dropped rhizosphere pH about 1 unit from the baseline measured last year, and the root mass density and tree health has responded positively. As expected with a resurgence of root production, Phytophthora populations have increased in all 4 flatwoods locations and 3 of the 8 ridge sites.
The goal of this project is to understand the biology of HLB by identifying key components and processes involved in disease development. We identified four secreted proteins (also called effectors) from the causative agent, Candidatus Liberibacter asiaticus (CLas), which are consistently, and in some cases, highly expressed in infected citrus trees. We have generated antibodies against these CLas-specific proteins and developed serological detection methods for HLB. This method is currently under extensive evaluation using field and greenhouse trees. We are also using these effectors as molecular probes to identify their targets in citrus because effectors have been shown to perform key virulence functions during bacterial infection. This project will reveal important information of HLB pathogenesis, therefore providing guidance for disease management. A major approach that we are using to find the effector targets is yeast two hybrid (Y2H) screen. In the first year of this project, we cloned the four CLas effector genes into an Y2H bait vector, transformed them into the yeast strain AH109, and confirmed that the effectors are highly expressed in the yeast without self activation activities. Therefore, these constructs are appropriate for Y2H screens. In the past quarter (April to June, 2014), our main progress include: 1) Finished the construction of a citrus cDNA library, which is currently used for Y2H screening. We collected RNA samples from asymptomatic and symptomatic tissues of HLB-infected sweet orange leaves. These RNA samples were mixed with RNA extracted from healthy tissues to ensure that we would be able to cover as many genes as possible. A cDNA library was constructed and further normalized to reduce the level of cDNAs representing frequent transcripts using double strand specific nuclease (Evrogen). The cDNA was also size-fractionated to achieve an average insert size of >1 kb, with additional enrichment for long cDNAs that tend to represent full length transcripts. The cDNA library was transformed to a target complexity of about 3 millions of primary clones. Normalization of the library was confirmed with sequencing of 50 randomly picked colonies, which exhibited increase in complexity measured by diversity of transcripts. We are now in the process of Y2H screening using this cDNA library and the four CLas effectors as the baits. 2) Determined the subcellular localization of the effectors in plant cells. We have made gene expression constructs that produce fusion proteins with each CLas effector gene tagged to two consecutive genes encoding the yellow fluorescence protein (YFP). We used two YFP tags because these CLas effectors are small in size and may therefore diffuse to certain sub-cellular localizations non-specifically. We expressed these fusion proteins in plant cells and examined their localizations using confocal microscope. Our data showed that three of the four proteins can enter plant nucleus. This is interesting because they may manipulate plant physiology by associating with specific plant targets in the nucleus. These results will provide insight into the biological function of the effectors and also provide guidance when we interpret the Y2H data.
Seasonal root sampling continues in two field sites for root density. Root cages have been installed at the second field site (Valencia) to compare the root growth dynamics in healthy and HLB affected trees with two sweet orange scions that have large differences in seasonal carbohydrate dynamics. Sampling has already revealed seasonal variation in root infections and apparent shifts in the root flush cycle caused by Liberibacter. The interaction between root growth and root density in Las infected trees is becoming clearer with continued sampling of root cages in the Hamlin block. The introduction of a Valencia block will provide insight if these dynamics are differ based on differing seasonal carbohydrate demands for fruit development. As more of our healthy trees become PCR positive for Las it is increasingly difficult to find sufficient presumed healthy trees in the field. It is especially hard to find trees of sufficient age for seasonal root sampling. While field sampling continues, it will become more of a description of HLB affected tree decline than a comparison to healthy trees. Therefore, more emphasis will be placed on greenhouse experiments for direct healthy v. HLB comparisons as the project continues. Sampling at a rootstock trial site is underway with a full year of data on the effects of HLB on these new experimental rootstocks. This has already begun to demonstrate how these new rootstock lines respond to Liberibacter infection. The most promising lines have been graft inoculated in the greenhouse and will be transplanted to rhizotrons to monitor root growth and death as soon as the graft success can be confirmed.
The general goal of this project is to rapidly propagate complex citrus rootstock material for field testing. The rootstock materials to be tested will be products of the Citrus Improvement Program at the UF-IFAS-CREC in Lake Alfred. Specifically, these materials will be selected based upon their performance in the ‘HLB gauntlet’: Promising rootstock genotypes will have already been evaluated in the greenhouse and field for their ability to grow-off citrus scions that have been exposed to CLas-positive budwood and CLas-positive Asian citrus psyllids. Once candidate rootstock materials have successfully passed through this gauntlet, they will be propagated via rooted cuttings en masse in a psyllid-free greenhouse at the UF-IFAS-IRREC in Fort Pierce. From there, rootstock materials will be budded with scion materials and planted in the field for further testing for their long-term performance. The start date for this project was April, 2013. To date, the progress of this project is as follows: – Two (2) misting chambers to propagate candidate, rootstock materials as rooted-cuttings have been constructed. – Propagation materials (containers, soilless media, and rooting hormones) have been purchased. – Funds from this project were used to support the construction of a new greenhouse at the IRREC. This greenhouse is completed and operational. – The first cohort of advanced, tetratzygous citrus rootstock materials for en masse propagation are currently being propagated. – The second cohort of advanced, tetrazygous citrus rootstock materials for en masse propagation have been identified and are being prepared to have cuttings taken from them. – In addition to the 1st & 2nd cohorts of tetrazygous rootstocks, promosing diploid rootstocks have also been identified and are being prepared to have cuttings taken from them.
In this exploratory study soft nano-particle (SNP) formulations were developed for delivery of anti-bacterial essential oils to plant phloem where Liberibacter asiaticus resides. Two essential oils (EO-A and EO-B which are single component) as identified by InnoCentiveTM assay were selected for developing SNP formulations. Both oils are naturally occurring terpenoid essential oils, generally regarded as safe (GRAS) with a number of botanical sources, including oregano and thyme. SNPs with Thyme Oil have were also formed and characterized. Microemulsion formulations were developed using agriculturally approved surfactants with different hydrophilic – lipophilic balance and charge. Formulations containing EO-A loading up to 7% (w/w), 35% (w/w) for EO-B and 20% for Thyme Oil, having droplet size ranging from ca.3 to 30 nm were developed. The stability of the formulations was tested by dilution with water, dilution with phosphate buffer saline (PBS) and in the presence of commonly used spreaders sticker adjuvants such as Cohere and Cling. While most formulations were stable, some made with ionic surfactant displayed instability when diluted with PBS. The developed EO formulations and their respective controls were tested for the anti-bacterial activity against the surrogate bacteria, Liberibacter crescens (Department of Microbiology and Cell Science, UF). The antibacterial efficacies of the SNPs were tested at three different dilutions 1, 5 and 10% (v/v). All SNP formulations showed > 90% inhibition at 1, 5 and 10 % (v/v) dilution. Some of the control solutions containing surfactants also showed high bacterial inhibition. High (> 90%) inhibition was observed with most formulations at concentrations as low as 100 ppm of EOs. While initially the SNPs were developed to achieve high EO loadings for maximum bacterial inhibition, it led to SNP formulations having relatively high surfactant concentration. The high concentration of the surfactants used is likely contributing to the antibacterial efficacy of the EO SNPs. Phytotoxicity of select formulations was tested at 1:1, 1:10 and 1:20 dilutions (formulations EO-A and EO-B) at Lake Alfred facilities (UF). All SNP formulations showed low phytotoxicity when applied at 1:20 dilutions. SNP formulations at these and lower dilutions showed high inhibition of L. crescens. Antibacterial and phytotoxicity results indicate that with suitable dilutions the SNP formulations can be used to for performing foliar applications tests on citrus crops. Future experiments are being planned for testing efficacy of SNP formulations in HLB infected plants.
We hypothesized that groves with high bicarbonate stress are suffering from HLB because they support lower fibrous root density compared to groves with lower bicarbonates (less than 100 ppm) in irrigation water and/or soil pH (less than 6.5). To confirm this relationship, we surveyed 37 grove locations in Highlands and Desoto counties with varying liming history and deep vs. shallow wells mostly on Swingle and Carrizo. Lower root density is significantly related to well water pH greater than 6.5 and to soil pH greater than 6.2. Yield records from these blocks reveal that groves under high bicarbonate stress production have declined 20 percent over the last three seasons (2009-2012) in contrast to Ridge groves with low bicarbonate stress which have increased 6 percent in production even though HLB incidence has accelerated. The yield losses are correlated with less fibrous root density, which reduces root system capacity for water and nutrient uptake. Evidence from research on other crops indicates that bicarbonate impairs the root’s ability to take up important nutritional cations including Ca, Mg and K, as well as micronutrients, especially Mn and Fe. In Florida, Bryan Belcher of Davis Citrus Management has acidified irrigation water with sulfuric or N-furic acid (a mixture of urea and sulfuric acid) by injection at the well in the same way as fertigation. N-furic has the advantages of being safer to handle and providing some additional N due to the urea component, but the disadvantage is higher cost of treatment compared to sulfuric acid. Since irrigation is not necessary when it rains, acidification treatment only occurs during the dry season when the bicarbonates are loading into the wetted area under the tree. When the rain begins, these bicarbonates are flushed from the rhizosphere. Our labs and grower cooperators are also evaluating acidification of soil by amendment with elemental sulfur applied in prilled or finely ground form. Sulfur (S) releases acid when it interacts with Thiobacillus bacteria in soil to form acid (H+) ions. This process of acidification with S is slower than treatment of the water but provides for longer lasting reduction in soil pH. Sulfur can be applied in prilled form with a fertilizer spreader or with a herbicide boom as a slurry. Sulfur can also be added to dry and fertigation formulations to lower the pH by as much as one unit after repeated ground applications. This spring we detected a 20% in crease in root mass density of Swingle trees associated with a drop in root zone pH from 6.4 to 5.9 at 9 months after soil amendment with prilled sulfur (Tiger 90). Although the soil levels were high, the drop in pH did not result in evelvarion of leaf copper concentration into the toxicity range. Root mass density and well as copper levels in leaves in groves under acidification management will be monitored in 8 ridge and 4 flatwoods sites for changes in pH of irrigated root zone, root mass density and and nutrient levels in leaves.
We hypothesized that groves with high bicarbonate stress are suffering from HLB because they support lower fibrous root density compared to groves with lower bicarbonates (less than 100 ppm) in irrigation water and/or soil pH (less than 6.5). To confirm this relationship, we surveyed 37 grove locations in Highlands and Desoto counties with varying liming history and deep vs. shallow wells mostly on Swingle and Carrizo. Lower root density is significantly related to well water pH greater than 6.5 and to soil pH greater than 6.2. Yield records from these blocks reveal that groves under high bicarbonate stress production have declined 20 percent over the last three seasons (2009-2012) in contrast to Ridge groves with low bicarbonate stress which have increased 6 percent in production even though HLB incidence has accelerated. The yield losses are correlated with less fibrous root density, which reduces root system capacity for water and nutrient uptake. Evidence from research on other crops indicates that bicarbonate impairs the root’s ability to take up important nutritional cations including Ca, Mg and K, as well as micronutrients, especially Mn and Fe. In Florida, Bryan Belcher of Davis Citrus Management has acidified irrigation water with sulfuric or N-furic acid (a mixture of urea and sulfuric acid) by injection at the well in the same way as fertigation. N-furic has the advantages of being safer to handle and providing some additional N due to the urea component, but the disadvantage is higher cost of treatment compared to sulfuric acid. Since irrigation is not necessary when it rains, acidification treatment only occurs during the dry season when the bicarbonates are loading into the wetted area under the tree. When the rain begins, these bicarbonates are flushed from the rhizosphere. Our labs and grower cooperators are also evaluating acidification of soil by amendment with elemental sulfur applied in prilled or finely ground form. Sulfur (S) releases acid when it interacts with Thiobacillus bacteria in soil to form acid (H+) ions. This process of acidification with S is slower than treatment of the water but provides for longer lasting reduction in soil pH. Sulfur can be applied in prilled form with a fertilizer spreader or with a herbicide boom as a slurry. Sulfur can also be added to dry and fertigation formulations to lower the pH by as much as one unit after repeated ground applications. This spring we detected a 20% in crease in root mass density of Swingle trees associated with a drop in root zone pH from 6.4 to 5.9 at 9 months after soil amendment with prilled sulfur (Tiger 90). Although the soil levels were high, the drop in pH did not result in evelvarion of leaf copper concentration into the toxicity range. Root mass density and well as copper levels in leaves in groves under acidification management will be monitored in 8 ridge and 4 flatwoods sites for changes in pH of irrigated root zone, root mass density and and nutrient levels in leaves.
Stress intolerance of HLB trees is a direct consequence of more than 30 percent loss of fibrous root density compared to non-diseased trees. This root loss may be compounded an additional 20% by the interaction with bicarbonates in the rhizosphere as a result of irrigation with well water high in bicarbonates and/or over-liming with dolomite to avoid copper toxicity. HLB and bicarbonate stress also favor infection by root pathogens such as Phytophthora spp. In field surveys and greenhouse trials with trees with and without Liberibacter asiaticus (Las) inoculation, populations of Phytophthora spp. are elevated in the rhizosphere of HLB trees. When Las interacts with Phytophthora, fibrous root loss is greater than that caused by HLB alone depending on the stage of Las infection and time of year. Also, HLB damage of fibrous roots alters the concentration of soluble sugars by increasing leakage that attracts and accelerates infection by root pathogens such as P. nicotianae. Although HLB associated root loss has been attributed to carbohydrate depletion by HLB and Phytopththora this relationship may vary with rootstock. Rootstock tolerance to Phytophthora is broken by the interaction with Las such that resistance to biotic or abiotic stress is greatly reduced. This results in premature root loss and reduction in root system capacity for water and nutrient uptake. The normal annual cycle of root death and replacement of fibrous roots appears to be disrupted. To confirm this, bimonthly changes in fibrous root mass density and rhizosphere population Phytophthora are currently under study in 8 ridge and 4 flatwoods sites in south central Florida.
FireWall 50WP (65.8% streptomycin sulfate; Agrosource, Inc.) has been granted by an EPA section 18 registration for control of citrus canker in Florida grapefruit. The label for FireWall restricts use to no more than two applications per season. As a condition for FireWall registration, EPA requires monitoring of Xanthomonas citri subsp. citri (Xcc) for streptomycin resistance in treated groves. The objective of this survey is to apply our published protocol for sampling canker-infected grapefruit leaves for isolation and detection of streptomycin resistant Xcc.
Objective 1. To define the role of chemotaxis in the location and early attachment to the leaf and fruit surface. As reported previously, methyl accepting chemotaxis proteins (MCPs) from Xanthomonas strains were deduced based on whole genome sequence data and PCR analysis. MCPs from citrus pathogenic strains (Xcc) were closely related to one another and distinct from X. campestris pv. campestris (Xc). MCPs from Aw strains were more related to X. fuscans pv. aurantifolia (Xfa) and X.alfalfae subsp. citrumelonis (Xac) than to strain 306 of Xcc. Another analysis has been conducted to convert whole sequence data to binary form according to the absence or presence of each MCP. New specific PCR primers have been designed to detect the presence or absence of certain MCPs. Gene expression analysis confirmed the higher expression of fimA (XAC3241) the major subunit of Pilus type IV, at early stage of the biofilm formation in A and Aw when bacteria grown in LB or XVM2 minimal media; another fimA gene (XAC3240), however, showed a different expression pattern in Aw, showing the highest expression in mature biofilm. Expression of fimA gene (XAC3240) in Xcc A strain is higher in mature biofilm when this strain was grown in LB medium, while it is higher in early stage of the biofilm formation when grown in XVM2 medium. Differences in gene expression between Xcc A and Aw strains in the two nutrient environments are in accord with the structural changes observed during biofilm formation. Objective 2. To investigate biofilm formation and composition and its relationship with bacteria structures related with motility in different strains of Xcc and comparison to non-canker causing xanthomonads. DNAse treatments were performed to confirm DNA content of aggregates and elucidate the role of DNA in biofilm formation. DNAse treatment of Xcc A, A*, Aw, Xac and Xc for different incubation times reduced biofilm formation for all citrus pathogenic strains but had little or no effect of biofilm formation by Xc and Xac. Biofilm formation by wide host range strains of Xcc was more affected at the beginning of the incubation period (0 to 24 h) while formation by restricted host range strains which were affected after a longer incubation of 72h. Xac biofilm was only affected at early stages of incubation. DNAse treatment of preformed biofilms reduced biofilm 40 to 50% confirming the high DNA content of mature aggregates. By comparison preformed biofilm of Xac and Xc was reduced only 20%. Differences in development of DNA structures of the biofilm between wide and narrow host range strains is related to the rate of biofilm formation by these strains analyzed by microscopic observations.
This is a continuing project to find economical approaches to citrus production in the presence of Huanglongbing (HLB). We are developing trees to be resistant or tolerant to the disease or to effectively repel the psyllid. First, we are attempting to identify genes that when expressed in citrus will control the greening bacterium or the psyllid. Secondly, we will express those genes in citrus. We are using two approaches. For the long term, these genes are being expressed in transgenic trees. However, because transgenic trees likely will not be available soon enough, we have developed the CTV vector as an interim approach to allow the industry to survive until resistant or tolerant trees are available. A major goal is to develop approaches that will allow young trees in the presence of HLB inoculum to grow to profitability. We also are using the CTV vector to express anti-HLB genes to treat trees in the field already infected with HLB. We have modified the CTV vector to produce higher levels of gene products to be screened. At this time we are continuing to screen possible peptide candidates in our psyllid containment room. We are now screening about 80 different genes or sequences for activity against HLB. We are starting to test the effect of two peptides or sequences in combination. We are attempting to develop methods to be able to screen genes faster. We are also working with other groups to screen possible compounds against psyllids on citrus. Several of these constructs use RNAi approaches to control psyllids. Preliminary results suggest that the RNAi approach against psyllids will work. We are screening a large number of transgenic plants for other labs. We are beginning to work with a team of researchers from the University of California Davis and Riverside campuses to express bacterial genes thought to possibly control Las. We are testing about 80 genes for induction of resistance or tolerance to HLB in citrus, but are eliminating many that are not effective and are focusing on about 20 that still are under test and about half of a dozen that have some activity. We recently examined all of the peptides constructs for stability. The earliest constructs have been in plants for about nine years. Almost all of the constructs still retain the peptide sequences. One of the peptides in the field test remained stable for four years. A recent advance is that has greatly speeded up our screen is that we now can estimate when plants become infected with HLB and can tell whether a peptide is working more quickly.
The goal of this project is to examine conditions for optimal deployment of a superinfecting Citrus tristeza virus (CTV)-based vector as a tool to be used in the field to prevent existing field trees from the development of the HLB disease and to treat trees that already established the disease. In order to provide protection against HLB, the superinfecting CTV vector will be carrying an anti-HLB gene (i.e. a gene of an effective antimicrobial peptide).The majority of trees in Florida are already infected with some CTV isolates. The main question for us is how these pre-existing isolates would affect the establishment of infection with the superinfecting vector and, thus, expression and the production level of an anti-HLB polyprotein. We are examining how preexisting infection with different CTV strains affects the ability of the superinfecting CTV vector to infect and get established in the same trees. We are assaying the levels of multiplication of the superinfecting CTV vector in trees infected with different field isolates of CTV. We first graft-inoculated sweet orange trees with the T36,T30 and/or T68 isolate of CTV, singly or in mixtures (these isolates were propagated in our greenhouse) as well as with CTV-infected material obtained from the field trees (FS series isolates). In addition to wild type isolates, we also included several CTV constructs that could be used as vectors for expression of genes of interest in trees to see how they compete with wild type isolates. Real time PCR analysis protocol is being optimized for quantification of multiplication of CTV genotypes in the inoculated trees. 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. Real time PCR quantification is also being employed to these tests. In these experiments we are using different citrus rootstock/scion combinations in order to find combinations that would support the highest levels of superinfecting vector multiplication and thus, highest levels of expression of the anti-HLB protein of interest from this vector. These combinations include trees of Valencia and Hamlin sweet oranges and Duncan and Ruby Red grapefruit on three different rootstocks: Swingle citrumelo, Carrizo citrange, and Citrus macrophylla. Evaluation of results is ongoing.
The objective of this enhancement project is to characterize the chemical composition for different citrus cultivars that show different degrees of the susceptibility and tolerance. Previously we developed a centrifugation based method to collection the pure phloem sap in order to be able to quantify the compounds. The phloem sap of fourteen different citrus cultivars (listed in the end of report) was collected from one-year-old greenhouse plants. The collected phloem samples were derivatized either by methychloroformate (MCF) that specific for organic and amino acids or trimethylsilyl (TMS) that is specific for sugars. The derivatized samples were analyzed by GC-MS. Thirty-two compounds were identified in the phloem sap after MCF derivatization. These compounds were classified to three groups: amino acids, organic acids, and fatty acids. Over than fifty compounds were detected in the citrus phloem sap after TMS derivatization. The compounds detected after TMS derivatization included sugars, sugar alcohols, organic acids, fatty acids, and amino acids. Our MCF results showed a significant correlation between the concentration of phenylalanine, tryptophan, tyrosine, serine, leucine, valine, and histidine with citrus resistance to Candidatus Liberibacter asiaticus (CLas). Phenylalanine, tryptophan, and tyrosine are precursors for phenylpropanoid biosynthesis phenylpropanoid biosynthetic pathway which is induced in response to biotic and abiotic stress. At this point we are processing the GC-MS data obtained after TMS derivatization. Once we process all the data, we will analyze it to identify other potential phloem sap metabolites that are linked to citrus resistance to CLas. Citrus cultivars included: Valencia, Pineapple and Madam Vinous sweet orange (C. sinensis (L.) Osbeck), Duncan grapefruit (C. paradisi MacFadyen), Sour orange (C. aurantium L.), Volkamer lemon (C. limonia Osbeck ‘Volkameriana’), C. macrophylla Wester, Palestine Sweet lime (C. aurantifolia), Mexican lime (C. aurantifolia), Carrizo citrange (X Citroncirus webberi J. Ingram & H. E. Moore), Severinia buxifolia (Poiret) Ten, Poncirus trifoliata (L.) Raf. and Citrus latipes (Swingle),
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