In the third and final year of funding, Core Citrus Transformation Facility (CCTF) maintained its level of performance and produced transgenic citrus plants for many satisfied customers. Considering that the major goals of proposed project to increase the capacity of the transformation lab were met within the first year of funding, during the second and third year of project duration CCTF had a task to maintain the achieved level of operation. The number of experiments and the quantity of produced transgenic material were at the level projected in the grant proposal. By accomplishing these tasks, CCTF assisted other researchers in efforts to improve different citrus cultivars by increasing their resistance and/or tolerance to diseases. Newly placed orders for transgenic plants stayed at high level. Altogether, thirteen new orders were received for processing during the third year of funding: pY46-Carrizo; pY102-Carrizo; pY141-Carrizo; pY150-Carrizo; pCitIntra-Duncan; pAZI-Duncan; pAtBI-Duncan; pBCR2-Duncan; pDPR1-Duncan; pLP1-Hamlin; pLP1-C-mac; pLP2-Hamlin; pLP2-C-mac. During the last quarter of this funding year, work was mostly concentrated on recent orders. Fourteen Duncan plants were produced carrying a gene of interest from the p35S-TRX vector and 23 more Duncan plants were produced carrying a gene from the pSucTRX vector. Multiple Duncan plants were produced toward satisfaction of ‘WG’ group of orders: eight-pWG22-1 plants, three-pWG21-1, and four pWG25-13 plant. Also, following Carrizo plants were produced for the ‘Yale’ order: nine plants with the gene from the pY46 vector and 11 plants with the gene from the pY102 vector. Eighteen Duncan plants were produced after treatment with bacteria harboring pBCR2 vector. Three more Duncan plants were produced with the EDS5 gene and six Mexican limes with the P35 gene. Four additional Duncan plants carrying a gene from pSUC-CitNPR1 were produced. In the previous three quarters of this year, small number of plants was produced for completion of older orders including: pNAC1 (1 plant); pMKK7 (1 plant); p33 gene (3 plants); pSUC-CitNPR1 (10 plants); p7+ p10 gene (32 plants). Most of the work was done on orders received in the last quarter of the second year and those placed in the third year of funding. The latter include: 12 Duncan plants (pWG19-5 vector); 11 Duncan plants (pWG20-7 vector); 18 Duncan plants (pWG21-1 vector); 22 Duncan plants (pWG22-1 vector); seven Duncan plants (pWG24-13 vector); 17 Duncan plants (pWG25-13 vector); 15 Duncan plants (pWG27-3 vector); 36 Duncan plants (ELP3 gene); 23 Duncan plants (ELP4 gene); nine Duncan plants (EDS5 gene); 10 Hamlin plants (pLC220 vector); 26 Duncan plants (p35 gene); 11 Duncan plants (35S-TRX vector), two Duncan plants (SUC-TRX vector). Joint efforts of Citrus industry and academic institutions to find solutions against huanglongbing (HLB), canker, and other citrus diseases are getting stronger with some positive results already being published. Continued funding for CCTF which is an integral part of this community and contributes greatly towards common goal will allow for the progress to go on by keeping production of transgenic material un-interrupted and at high levels.
In the 3rd and final year of the project, significant progress was made on several fronts. Juvenile Explant Transformation Protocol R&D: Key components of our transformation system were investigated in order to improve transformation and regeneration efficiency. The best medium for citrus transformation was determined to be the MS medium. Optimum hormonal levels in tissue culture medium was determined to be 3 mgL-1 BAP supplemented with 0.5 mgL-1 NAA for trifoliate rootstocks, Mexican lime and recalcitrant citrus cultivars like Volkamer lemon and mandarin / tangerines, and 1 mgL-1 BAP for sweet oranges and grapefruits. It was determined that a 3 hour soak in an auxin rich medium containing 1 mgL-1 2,4-D, and 0.5 mgL-1 NAA with 3 mgL-1 BAP significantly improved the transformation efficiency in a number of cultivars evaluated. Optical density of the bacteria was a determining factor in the genetic transformation of citrus. Trifoliate rootstock explants could tolerate a higher OD (0.6) while optimum transformation was observed at a lower OD for sweet oranges (0.15 or 0.3). We also observed co-cultivation duration was observed to be cultivar dependent. 3 days co-cultivation duration was observed to be optimum for cultivars with a thicker epicotyl such as trifoliate rootstocks or tetraploid selections. The optimum period for co-cultivation of sweet oranges was observed to be 2 days. Addition of 1mgL-1 GA3 resulted in rapid elongation of shoots, allowing in vitro micrografting within a month of initial selection of shoots. We determined that addition of Lipoic acid ‘ an antioxidant to shoot regeneration medium following transformation dramatically enhanced the transformation efficiency. This addition has resulted in improved transformation efficiency in otherwise recalcitrant cultivars. Transgenic plants from precocious sweet orange somaclones including OLL8, B4-79, Vernia 2-1, and a precocious mandarin W. Murcott, containing the LIMA antimicrobial construct, were produced and were grafted to precocious rootstocks (Amblycarpa+ Benton and Changsha + Benton somatic hybrids) for continued early flowering-induction experiments. Seasonal effects of on regeneration potential and transformation success rate were also evaluated, and confirmed that each cultivar behaved differently based on the time of fruit harvest and seed germination. A rapid ex vitro micrografting technique suitable for propagating in vitro and young ex vitro transgenic stem pieces was developed. Combining all of these advances is expected to cut in half the time from initiating an experiment to flowering and fruiting transgenic trees, thus making juvenile transformation more competitive with mature tissue transformation. In addition we have developed an efficient protocol using cell suspension cultures that has enabled us to transform seedless or recalcitrant cultivars such as the precocious mandarin cultivar W. Murcott and the seedless cultivar Okitsu Wase satsuma. This protocol has created an avenue for insertion of useful traits into any polyembryonic citrus cultivar that can be established as an embryogenic cell suspension culture. This research supported 8 journal publications. Transformation with Early Flowering Genes: We have regenerated many transgenic citrus plants that include: poplar FT gene behind either the 35S or heat shock promoter; co-transformed Carrizo plants with two cassettes, one containing 35S-cft1 and the other containing AtSUC2 ‘ gus; and numerous transgenic plantlets of Hamlin and Carrizo containing P27, P28, P29, PATFT and pPTFT. All of these plants are at various stages of evaluation.
Over the first year of this project, we have established the methodologies to aseptically acquire LAS inoculum from fruits from infected trees, detect LAS via qPCR, and determine LAS cell viability via qPCR-EMA. Then, we applied these methods in 10 replicated experiments to monitor LAS viability in different culture media over time. We are currently finishing the analysis of the data from the experiments and expect to publish this data within the year. For the replicated experiments, LAS suspension obtained from seed of infected pomelo fruits has been used to inoculate replicate flasks containing four different types of media: 1/3 King’s B media (control), J50 media (50% juice from the infected fruit), G50 media (50% store-bought grapefruit juice), and G media (100% store-bought grapefruit juice). Samples were then collected every other day for 18 days. These samples were assessed via EMA-qPCR for viable cells. Results show that LAS viability is maintained longer in a juice-based media than in 1/3 King’s B media. Total DNA and viable DNA quickly approach 0 detectable genomic equivalents after a few days in the 1/3 King’s B media, while viable cells are detectable until close to the end of the experiments in the other culture media. However, LAS cell viability changes over this time in a reproducible oscillating pattern that may be an indication of cryptic growth. Two complete experiments and three partial experiments have been analyzed at this time, with the remaining analysis currently being conducted. Culture media samples were collected at both the beginning and end of each of these experiments. We are currently working to use the analytical technique ICP-OES to analyze the concentrations of various nutrients in both the LAS cells and the media to determine what nutrients, if any, change in concentration in each fraction during the course of the experiments. This could provide an indication of nutrients utilized by LAS. It will also provide a comparison between nutrient concentrations between the four different media used here. We hope to send culture samples to a colleague in Florida for similar analyses of amino acids soon. Flasks used during the experiments showed evidence of biofilm formation at the liquid-air interface. The biofilm was almost nonexistent in flasks containing 1/3 KB media, while it was most pronounced in flasks containing G50 or G media. We have recently been working to reproduce this biofilm on glass slides, but the LAS inoculum concentration in seeds is much lower this time of year so it has been difficult to obtain sufficient biofilm formation. We do see that LAS cells adhere more easily to a special charged-surface glass slide called PlusGold. Since in our previous experiments using LAS inoculum in microfluidic chambers showed the cells would not adhere much, we will try to make chambers using these glass slides instead. Once this year’s new crop of pomelo fruit is available, we will be able to test these new microfluidic chambers for LAS cell attachment.
Challenge with Ca. Liberibacter spp ‘ Transcriptome experiments by microarrays were performed using plants of sweet orange (Citrus sinensis L. Osb. cv Pera) grafted onto Rangpur lime (C. limonia L. Osb.). The first challenge with Ca. Liberibacter americanus was carried out in 25 plants of four months old plants by grafting infected (PCR positive) budwoods. They were monitored bimonthly by end-point PCR for detection of the bacterium. PCR positive plants were challenged again by new grafting with infected budwoods, and pruned. Full expanded leaves of three plants displaying initial symptoms of HLB and PCR positive for the bacterium, and leaves of healthy plants in the conditions were collected and stored at -80’ C. To validate specific gene expression, eight plants of sweet orange (cv Hamlin) grafted onto Rangpur lime were challenged with infected budwoods, either with Ca. Liberibacter americanus or Ca. Liberibacter asiaticus. Genome wide transcription approach – Global transcriptional levels of diseased and healthy plants were evaluated with a Roche Nimblegen Systems customized 385K chip containing 32,000 unigenes of sweet orange. Total RNA concentration and purity were determined from the ratio of absorbance readings at 260 and 280 nm using a Nanodrop ND8000 spectrophotometer (Nanodrop Technologies), and RNA integrity was tested in a denaturing agarose gel. Raw data were imported to NimbleScan 2.5v software, which employs three steps of preprocessing: convolution background correction, quantile normalization, and a summarization of expression measures in a probe-level with a robust multiarray model fit (RMA) using the median polish algorithm. Unigene transcripts with p-values ‘0.05, fold change |FC| ‘ 2.0 and odds probability ‘ 0.95 were considered as differentially expressed genes (DEG). To identify relevant molecular mechanisms potentially associated with sweet orange response against Ca.L.americanus infection gene set enrichment analysis (GSEA) method, that evaluates microarray data at level of gene sets was carried out. Specific gene expression analysis – To obtain reliable of gene expression measurements, we performed a screening of candidate reference genes of our microarray analyses, adopting the following cutoff: absolute logFC ‘0.5, average expression (AveExp ‘7.0) and standard deviation (stdev ‘0.5). Besides that, we decided to evaluate the expression stability of 18S ribosomal and GAPDH that were usually used to normalize transcript levels. Primer efficiencies, Cq values and normalized relative quantities (NRQ) were calculated as described. The most stable reference genes were identified using the geNorm 3.5v (medgen.ugent.be/~jvdesomp/geNorm/) algorithm. Stepwise exclusion of the reference genes with the lowest stability of expression (the highest M) allows ranking the reference genes according to their expression stability. Based on the predicted function, retrieved from TAIR, 24 genes were selected to be validated by real time quantitative PCR (RT-qPCR). Amplicon specificity was checked by 2% (w/v) agarose gel electrophoresis and by melting-curve analysis. Sequence identity was confirmed by direct sequencing of PCR products using an Applied Biosystems Model 3730 DNA sequencer. Relative quantification was carried out in a 96-well optical plate with an ABI PRISM 7500 FAST sequence detection system (Applied Biosystems) using the Fast SYBR green PCR master mix (Applied Biosystems). All assays were performed using three technical replicates and a non-template control, as well as three biological replicates. To analyse dissociation curve profile program was run after the 40 cycles of PCR: 95’C for 15 sec followed by a constant increase in temperature between 60 and 95’C. Raw data of fluorescence accumulation for each individual assay were imported into R statistical package version 2.922 (R Development Core Team). The complete manuscript is been submitted to an international journal for evaluation and, if accepted, to be published. As soon as the manuscript is accepted I will send the final report of the project. If requested I can submit the preliminary version of the manuscript in confidence.
Under objective 1: Biofilm from A and Aw strains of Xcc were evaluated in time course experiments on fruit and leaf surface using scanning electronic microscopy (SEM) and confocal laser scanning microscopy (CLSM). Differences among wide and limit host range strains were shown, confirming previous results for in vitro biofilms. Initially, biofilm formation by the wide host range strains showed greater ability for aggregation than the narrow host range strains. Wide host range bacteria were aggregates comprised of a well-developed biofilm matrix whereas, narrow host range bacteria formed a labyrinth with a fiber of unknown composition. These structures were detected both by CLSM and SEM. Ongoing experiments are evaluating the differences in the matrix between the two host range strains and whether those differences are related to their motility. Under objective 2: Inefficacy of several bactericide treatments previously reported were evaluated by treating the bacteria in preformed biofilms and measuring their viability after the treatments. Viable and culturable bacteria were isolated after the treatments confirming the resistance of the aggregates to bactericidal treatments.
Under Objective 1: In order to confirm biofilm formation is a bacterial survival strategy and not just a physical aggregation, biofilm was induced in vitro bacterial suspensions of the wide host range strain of Xanthomonas citri susbp. Citri (Xcc). RNA extractions were performed from bacteria from the culture suspension (planktonic state) as well as those deposited on a glass slide surface (biofilm state). Real Time RT-PCR using GumD primers previously demonstrated as valid for viability determination of Xcc was used to assay the same concentration of RNA from planktonic or biofilm bacteria. Higher level of viability was detected for those bacteria in aggregated (biofilm) form compared to those in planktonic form. Greater viability of the bacterium in aggregates was also confirmed using the labile GFP probe to detect viable cells with confocal microscopy, and the use of a bacterial respiration assay. Differences in viability between the aggregated and planktonic bacterial states increased through the time based on GumD RNA detection. No viable cells were detected in the planktonic form after 72 hours while viability was maintained in the biofilm form. RNA detection of Xcc viability was also tested on plant material. To do so RNA extraction was performed from canker lesions after Xcc inoculation of sweet orange leaves. Positive results were obtained from lesions after no treatment but not after a bactericidal treatment of the extract by heating. Under Objective 2: In preliminary assays of bacterial viability for aggregates treated with bactericide treatments was evaluated by the methodology described above. Complete elimination of viable bacteria was not achieved with any of the bactericide treatments evaluated. New assays are currently in course with different bactericide concentrations.
The antibody developer, Creative Biolabs, Inc., now has the purchase order from Penn State, which was required to initiate the project. The target peptide for NodT is now being synthesized. Screening of antibody libraries for high-quality antibodies is expected to take approximately 6 weeks, and was initiated on April 24, 2012. They plan to isolate about 5-20 antibodies with ability to bind the NodT protein.
For the Asian citrus psyllid (ACP), we constructed and sequenced two Illumina Paired-end libraries for ACP uninfected/infected salivary glands (SGs) and generated 818 M reads, totaling 83 Gb raw data. We are presently combining the ACP SG with the alimentary canal (guts) data generated previously to make the assembly DcGs. In addition, combining the data that we generated from two 454 Titanium libraries and six Illumina Paired-end libraries of Asian citrus psyllid (ACP) and the Sanger sequence data from Genbank for this species, we have made a hybrid assembly ‘ACPisp’ to yield 98,265 Unitrans from 140 M Illumina reads, 664 K 454 reads and 19 K Sanger reads, respectively. Of the 98,265 total unitrans, 22,543 unitrans (23%) were annotated with an average unitrans length of 1,848 bp. This dataset will provide deeper insights into the whole trancriptome of ACP. To translate peptide sequences from transcript (coding) sequences: (1) the ESTScan program was installed, (2) the hemiptera sequences were downloaded from Genbank, (3) these were used to train the ESTScan matrix, and (4) ESTScan was used with the hemiptera matrix to extract the peptide sequences from the ACPisp transcript sequences. An additional matrix was created using all the Genbank insecta sequences, and a second set of peptides were generated using the insecta sequences. The hemiptera matrix performed better than the insecta matrix, creating slightly more and longer sequences. ESTScan was also used to extract peptides from our WFB assembly using the same matrix. The two peptide files were used as input into orthoMCL, and the resulting clusters were used as input into cmpPAVE in order to investigate functionally similar transcripts between these two genomes. Secondly, we have made an extensive comparative transcriptome analysis of ACP and whitefly (also important agricultural sucking pest and invasive species), the potato psyllid, and pea aphid. Results indicated the presence of a suite of immunity and stress-related genes. Thirdly, for potato psyllid, we constructed and sequenced a suite of Illumina RNA-seq libraries to investigate infected and uninfected potato psyllid gene expression changes reared at different temperatures. We have identified a branch of genes that were differentially expressed under different temperatures in the Ca. Liberibacter-infected and uninfected potato psyllid. This work also will provide a good reference for the study of temperature affects for ACP in the future. We continue work on several manuscripts that are expected to be submitted during May-July.
The final step in completing the general transmission pathway is determination of the origin of Lib individuals that are able to access the stylets (adult, larval, or both) for reinoculation. Once acquired by adult PoP, the potential for dispersal of Lib by flight relies on its ability to exit the adult, or pass transovarially to the next generation larvae. Exit from either is, potentially, multidirectional. Our methodology is therefore scenario driven. Scenario #1) [Inoculum comes from primarily from larvae]. Our, and other lab’s, feeding experiments show that adults can transmit Lib during feeding, but our TEM shows no transepithelial, nor transcuticular, movement (n hot=20, n cold = 10), which would explain the origin of their midgut biofilms. If transmission by the adult is very inefficient, it must be transovarially transmitted to the next generation larvae after flight. SEM of all instars (n=333, 195) shows that 3rd and older larvae have biofilms on the midgut exterior also, but they have yet to be examined by TEM. It may be that larvae are the primary source of inoculum under field conditions. FISH of dissected guts assisted in identification of Lib, but does not distinguish between internal and external bacteria as needed for this scenario (pub. #1). #2) [Transepithelial movement to the adult midgut exterior may occur at point loci missed in our sampling.] FISH with thick paraffin sections of infra-hour time course AAP is required to see if Lib leaks thru the epithelium at point loci to establish blood-side biofilms. #3) [Access to the adult midgut exterior by chitinolysis through foregut/hindgut intima.] Alternatively, Lib may chitinolyse through weak points in the intima, and sprawl to the midgut exterior. #4) [Adult midgut biofilms as a source of inoculum.] Although the origin of adult midgut biofilms is unknown, we demonstrated that they sprawl anteriorly, proliferate in the oral region, burrow into the salivary glands, and reach their core. We have not found them inside the ducts yet, but this anatomy may get clogged or disabled by the biofilm’s intrusive movements. Lib are ca. half the diameter of the duct lumen. Alternatively, Lib may also access the mouthparts by chitinolysis through the oral region’s endoskeleton (purported for sharpshooters, Brlansky et al. 1983). Our study of the oral region (pubs #2, 3) elucidated its anatomy, but did not show any compromises of cuticle with Lib exiting through them. Views (n = 5) of the oral lumina, available in our TEM libraries, do not show fastidious bacteria, as occurs with sharpshooters (Alves et al. 2008). Our time-course experiments are underway, and when minimum AAP for adults is determined, FISH and colloidal gold may reveal where leakage into the interior occurs. Publications. We are preparing a third manuscript on the topic of: Stylet biogenesis and installation into their functional positions during the pharate adult stage of the potato psyllid, Bactericera cockerelli (Sulc). J.M. Cicero and J.K. Brown.
At this stage, we have completed Aim 1 (Identify genes positively regulating SA-mediated defense in citrus) and most work described in Aim 2 (Complement Arabidopsis SA mutants with corresponding citrus homologs). We have so far cloned more than ten citrus SA genes, all of which are at various stages of gene transformation and analysis of transgenic plants. We are actively working on Aim 3 (Assess the roles of SA regulators in controlling disease resistance in citrus) to make citrus transgenic plants over-expressing the SA genes and to assay the plants for resistance to HLB and citrus canker diseases. So far we have confirmed transgenic citrus expressing ctNDR1, ctPAD4, and ctEDS5. Additional constructs are in the pipeline of citrus transformation. While the cloned citrus SA genes are at various stages of analysis, the most advancement that has been made so far is with ctNDR1. We have obtained data to support that manipulating the level of ctNDR1 could lead to enhanced disease resistance. The main results on ctNDR1 are summarized below. We are in the middle of preparing a manuscript for publication. 1. Overexpression of CsNDR1 could complement the Arabidopsis mutant ndr1-1 for its disease susceptibility to and the lack of hypersensitive response to Pseudomonas syrinage avrRpt2 infection. ctNDR1 conferred resistance is largely dependent on the expression of ctNDR1 (dosage dependency) in Arabidopsis 2. The Arabidopsis NDR1 was previously shown to act downstream of a subset of resistance genes (i.e. RPS2 that recognizes avrRpt2) but not required by other resistance genes, such as RPS4. However, we found that ctNDR1 overexpression increases resistance to both P. syringae strains expressing avrRpt2 or avrRps4, suggesting the activation of general defense mechanism in the ctNDR1 overexpression plants. 3. Consistent with enhanced disease resistance to different pathogens, we found that higher expression of ctNDR1 also led to increased accumulation of salicylic acid, a key signaling molecule that activates broad disease resistance. 4. We performed quantitative RT-PCR analysis of NDR1 in mock-inoculated and Ca. L. asiaticus-inoculated ‘Cleopatra’ mandarin seedlings. We found that expression of ctNDR1 was inducible by HLB in two independent experiments (experiment 1 with 32 week-old seedlings and experiment 2 with 30 week-old seedlings). These results suggest a potential role of ctNDR1 in HLB resistance. 5. We have so far obtained 29 independently transformed transgenic citrus plants carrying ctNDR1 overexpressing construct. The presence of the transgene was confirmed by transgene-specific primers. We should begin to test these plants for resistance to citrus canker disease in the next few months.
In this research, we have been developing a repellent formulation for Asian citrus psyllid (ACP). A DMDS-based SPLAT formulation has been developed. It is currently formulated and produced by ISCA technologies. Our recent results have produced inconsistent results; however, there have been cases where the formulation has effectively reduced psyllid populations both in Florida and in trials in another state. In the final year of the project, we have tested new active ingredients that may be more effective than DMDS for several reasons. First, these newly identified active ingredients are less noxious than DMDS and simply do not smell as bad to humans. This makes them much easier to work with. Also, these chemicals are less phytotoxic and easier to formulate. Therefore, these new formulations may not have the drawbacks that are associated with DMDS-SPLAT. In this previous quarter, we have been working to evaluate these new formulations and we have determined that two of the newly identified active ingredients are effective in repelling psyllids when deployed in SPLAT. We are currently finishing up these tests to hopefully determine whether a more practical SPLAT-based repellent formulation can be developed than our current DMDS-based standard.
Our objective was to determine how psyllid behavior is affected by Las-infection of citrus. In previous experiments we have determined that ACP adults initially prefer to settle on Las-infected plants. This attraction to Las-infected plants appears to be dependent upon the plant release of the volatile chemical, methyl salicylate. Based on published data, it is known that Las-infected plants have nutrient deficiencies. Therefore, we hypothesized that while the Las-infected plants are initially attractive to psyllids, after prolonged feeding the psyllids experience imbalanced nutrition and choose to seek a better host. To examine this hypothesis we planned to determine how psyllids settle on plants with known nutrient deficiencies. Our previous experimental design was limited to settling behavior of psyllids on nutrient deficient and healthy plants. To obtain more robust conclusions, we have modified our experimental design to incorporate HLB-infected plants. In these experiments we plan to test psyllid settling preference in several scenarios: 1. Healthy vs. Nutrient Deficient Citrus 2. Healthy vs. HLB infected Citrus 3. Healthy vs. Nutrient Deficient Citrus + ‘HLB infected plant odor’ (HLB odor) 4. Healthy vs. Healthy + HLB odor If psyllids initially settle on HLB infected plants because of attractive odors and subsequently move due to the poor nutrient quality of the host, we expect the following results: In scenario 1, psyllids should settle and stay on healthy plants. In scenario 2, psyllids should initially settle on HLB-infected plants and then move to healthy plants (this has been previously shown in experiments). In scenario 3, psyllids should settle on nutrient deficient plants with HLB odor and then move to healthy plants. In the last scenario psyllids should settle and stay on healthy plants with HLB odor. In the last quarter, we have been successful in making nutrient deficient uninfected plants to use in these experiments. We sent leaf tissue samples from ten healthy, ten unfertilized (Nutrient Deficient), and ten HLB-infected Valencia plants to a private lab for nutrient evaluation. The results show that our unfertilized plants contain significantly less nitrogen, potassium, magnesium, calcium, boron, zinc, manganese, and iron when compared to Healthy plants. There were no significant differences in sulfur, phosphorus, or copper content between unfertilized and control plants. The HLB-infected plants have been fertilized regularly and are contain levels of nutrients that are not significantly less than healthy plants with the exception of zinc. We are in the process of assessing the volatile profile of the three plant types: Healthy, Nutrient Deficient, and HLB infected citrus. In preliminary trials we found that our existing volatile collection bell jars were too small to use on the plants selected, resulting in physical damage to the plants during handling. We have since ordered larger bell jars and are prepping for volatile collections. Upon analysis of Healthy, Nutrient Deficient and HLB infected plants, we will determine the characteristic components of HLB infected plant odor. These odors will then be applied to the plants according to the design outlined above.
Our progress for the current quarter is as follows: 1. Transgenic Duncan grapefruit lines expressing avrGf1 driven by the Bs3- PIP14 box promoter are currently being tested for resistance by spray inoculating X. citri subsp citri 306 at 10^8 cfu per ml suspended in sterile tap water on young citrus leaves. Resistance confirmed by transgenic lines will be assessed by evaluating canker lesions between transgenic and non-transgenic control plants. 2. Compared to AvrGf1, the type 3 effector AvrGf2, identified from X. fuscans subsp aurantifolii strain C, seems to provoke a faster hypersensitive reaction on Duncan grapefruit. The avrGf2 coding sequence was fused with the Bs3- PIP14 box promoter into the pK7FWG2 binary vector and transformed into A. tumefaciens strain Agl-1. In planta transient expression in Duncan grapefruit was assessed for induction by X. citri subsp citri 306 TALEs using the pathogen inducible promoter (Bs3- PIP14 box):avrGf2 construct for citrus canker resistance. Upon activation of the construct by Xcc TALEs, the expressed AvrGf2 elicited a resistance response that was faster and more effective than that of a Bs3-PIP14 promoter construct driving AvrGf1. 3. Transgenic Duncan grapefruit carrying the AvrGf2 construct are in the development pipeline to produce stable transformants. Transfomation was performed on 2,049 grapefruit epicotyl segments. 4. Germination and epicotyl transformation experiments with Duncan grapefruit are continuing. Molecular characterization involving PCR is ongoing and used to confirm transgenic lines from the putative transgenic plants regenerated. To date, a total of 190 putative transgenic plants have been screened and 23%, 20% and 6% of the plants tested positive for the Bs3 promoter, nptII and the avrGf1 genes, respectively.
Cytoplasmic citrus leprosis infected samples previously sent from our cooperator in Colombia to quarantine facilities at the USDA, APHIS, PPQ, CPHST, Beltsville, MD. continued to be negative in PCR and antibody tests for cytoplasmic citrus leprosis virus. Additional sets of samples were sent during this funding period. All samples were prepared for electron microscopy to verify the PCR results. Viral particles similar to those previously published for cytoplasmic citrus leprosis were discovered in all the samples. Sizes and locations of the virions were verified. Sequencing was done and a new virus was identified by our lab in conjunction with another funded project. New primers are now available for detection and we plan to perform mites transmissions on both types of the virus at the USDA, ARS, FDWSRU in Fort Detrick. Mr.Leon in Colombia as earlier reported did mite transmission experiments with the previously PCR negative isolates from Colombia. With our new primers we have found that he has transmitted this new virus with Brevipalpus mites from Colombia. Symptoms of the new virus are identical to citrus leprosis.
All data analyses and writing for this research was completed, and a publication entitled “Viability of ‘Candidatus Liberibacter asiaticus’ prolonged by addition of citrus juice to culture medium” has been submitted to a peer-reviewed journal and is currently under review. Data finalized and discussed in this publication include: qPCR data from total and viable LAS cells over time in different culture media and next-generation sequencing data of the microbial communities associated with LAS in cultures, among several other aspects of the research conducted for the purpose of this grant. This data was also presented at the 3rd International Conference on Huanglongbing, and comments from other researchers in this field were incorporated into the manuscript. While this publication does not provide a defined method for the culture of LAS, it does provide many useful clues for prolonging LAS viability which we expect to be instrumental in future development of a culture medium via collaborations among the scientific community.
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