In the first three months of 2013, Citrus Core Transformation Facility (CCTF) continued to operate at the steady rate without any interruptions in production of transgenic plants. Within this period, CCTF received highest number of orders ever-twelve. Five orders were placed for production of transgenic Duncan grapefruit (pW14, pHGJ1, pHGJ2, pHGJ3, and pHGJ4); four for production of transgenic Mexican lime plants (pOA1, pOA2, pOA3, and pGF1); and three for production of Valencia plants (pOA1, pOA2, and pOA3). All the binary vectors received from clients were already mobilized into appropriate Agrobacterium strains and initial co-incubation experiments were performed. Most of the work done in the CCTF revolved around the recently placed orders. Regarding the production of transgenic plants, CCTF has achieved following results. Transgenic Duncan plants carrying genes from these different vectors were produced: fifteen from the pX4, twenty six from the pX7, thirteen from the pX11, ten from the pX16, three from the pX19, five from the pX28, twelve from pNah, and five from pBI121 vector (to serve as control plants). There were also twelve Duncan plants produced carrying the gene from pMED14 vector and one carrying the gene from pMED16. Despite the high flux of workers in the facility, productivity remained high. Every effort will be made to keep production of transgenic material at satisfactory level considering high volume of incoming orders.
The objectives of this project are: 1. Evaluate psyllid populations, HLB incidence and intensity, gene expression, tree growth, soil moisture, soil nutrients, foliar nutrients, and eventually yield in newly planted citrus blocks, 2. Assess separate contributions of vector control and foliar nutritional applications to the above parameters, 3. Evaluate the effectiveness of reflective mulch to repel ACP and reduce incidence of HLB, 4. Provide economic analysis of costs and projected benefits, and 5. Extend results to clientele. The experiment was planted 3-4 July 2012 on a 10-acre block planted on a 23 x 9 ft spacing at the A. Duda & Sons, Inc. farm in Hendry County south of LaBelle at 26.64315 degrees S. -81.45456 degrees W and 26 ft elevation. The experimental design of main plots is factorial RCB with 4 replicates and 4 treatments: insecticide alone, foliar nutrition alone, insecticide + nutrition, and untreated control. Each of 16 plots is split into two subplots 5 rows wide and 13 trees long, mulch and no mulch. Mulch provided by Imaflex Inc. is metalized (aluminized/reflective) polyethylene film of 3 mils thickness covered with a clear protective polyethylene coat. Metalized mulch was shown in preliminary evaluations on single plots to repel Asian citrus psyllid and together with a drip irrigation/fertigation system increase citrus growth rate over the unmulched control. The block was planted 3-4 Jul 2012 and monitoring ACP with flush inspection and sticky cards commenced 13 Aug. Sticky cards are monitored for ACP and other common citrus pests and replaced every other week. About 106 psyllids have been found on sticky cards of which 80% are in no-mulch plots with the majority in plots with trees that do not have receive chemical control . Flush infested with ACP eggs and nymphs have become much more common. These are predominately in no-mulch plots with 85% of the total observed infested flush. Very few infested flush have been found in plots receiving insecticides while no infested flush have been found in plots with both mulch and insecticide applications. The third leaf samples were collected on July 9 for HLB. Tree growth measurements were taken on July 12 for the third time which consisted of trunk diameter and height measurements. Monthly foliar nutrition applications continue to be applied in the first week of each month. Leaf samples for the 3rd nutrient analysis were collected 29 Jan 2013. Normal grove care operations continued. These include one application of Agrmek on July 4 for leafminer, one application of glyphosate for weed control in mulched plots May 30, one application of glyphosate May 30 and Kocide once to control canker on May 30. All trees were desuckered and any low hanging limbs pruned July 2 to reduce the chance of herbicide damage.
The objectives of this project are: 1. Evaluate psyllid populations, HLB incidence and intensity, gene expression, tree growth, soil moisture, soil nutrients, foliar nutrients, and eventually yield in newly planted citrus blocks, 2. Assess separate contributions of vector control and foliar nutritional applications to the above parameters, 3. Evaluate the effectiveness of reflective mulch to repel ACP and reduce incidence of HLB, 4. Provide economic analysis of costs and projected benefits, and 5. Extend results to clientele. The experiment was planted 3-4 July on a 10-acre block planted on a 23 x 9 ft spacing at the A. Duda & Sons, Inc. farm in Hendry County south of LaBelle at 26.64315 degrees S. -81.45456 degrees W and 26 ft elevation. The experimental design of main plots is factorial RCB with 4 replicates and 4 treatments: insecticide alone, foliar nutrition alone, insecticide + nutrition, and untreated control. Each of 16 plots is split into two subplots 5 rows wide and 13 trees long, mulch and no mulch. Mulch provided by Imaflex Inc. is metalized (aluminized/reflective) polyethylene film of 3 mils thickness covered with a clear protective polyethylene coat. Metalized mulch was shown in preliminary evaluations on single plots to repel Asian citrus psyllid and together with a drip irrigation/fertigation system increase citrus growth rate over the unmulched control. The block was planted 3-4 Jul 2012 and monitoring ACP with flush inspection and sticky cards commenced 13 Aug. Sticky cards are monitored for ACP and other common citrus pests and replaced every other week . About 12 psyllids have been found on sticky card of which 3/4 are in no-mulch plots with more in plots that do not have chemical control than plots that receive insecticides. To date, several flush have been found infested with ACP eggs and nymphs in no-mulch receiving foliar nutrition without insecticides. The first leaf samples were collected on October 17 for HLB testing of which one tested positive but subsequently came up negative following multiple times retesting. A second general leaf sample on 19 Feb all tested negative. Monthly foliar nutrition applications were suspended after the Nov 7 spray in preparation for the winter dormant season and resumed on Mar 7. Leaf samples for the 2nd nutrient analysis were collected 29 Jan 2013. Tree growth measurements of height and trunk area were taken for a second time on 12 Feb 2013. These measurements showed trees grown on mulch were slightly taller than trees in unmulched plots they also had a larger trunk cross sectional area. Trees in the foliar nutrition and insecticide treatments were taller and had larger trunks followed by all other treatments which were similar. Normal grove care operations continued. These include one application of Vendex for spider mite control on Jan 15, one application of glyphosate for weed control in mulched plots Apr 3, one application of glyphosate and Solicam in unmulched plots Apr 3 and Kocide once to control canker on Apr 10. All trees were desuckered and any low hanging limbs pruned to reduce the chance of herbicide damage on Feb 7. Over the nights of Feb 16-18 and March 1-3 a freeze threat was experienced but the flood irrigation plan was executed so that no damage was incurred. New water sensor probes were tested and calibrated for soil moisture data collection.
Obj 1: The GOseq method was incorporated into the PAVE database to aid in identifying candidate effectors to take advantage of the ability to select statistically among significant differentially expressed genes. Predicted ‘effectors’ found in both the Asian citrus psyllid (ACP) and potato psyllid (PoP: study system) transcriptomes have been bioinformatically validated using automated BLAST annotation against the NCBI-GenBank database. PCR primers were designed around conserved nucleotides for each contig and RT-PCR was carried out using PoP cDNA to amplify the corresponding transcript and confirm (in vivo) expression of each transcript. To date, expression of 22 transcripts has been confirmed in PoP and confirmation of all genes in ACP by RT-PCR is in progress. Initially dsRNA screening will employ PoP to test for knockdown of transcripts. Obj 2: Yeast-2 hybrid studies were initiated to study protein-protein interactions important in psyllid-Liberibacter interactions involved in the circulative, propagative pathway. To accomplish this 500 guts from Las-infected ACP and 500 guts and 1000 salivary glands from uninfected ACP were extirpated and used to construct the libraries. Three cDNA libraries: uninfected gut, uninfected salivary gland and infected bacteria have been completed. Furthermore, three Mate & Plate ‘prey’ libraries (uninfected gut, uninfected salivary gland and infected bacteria) have also been completed, with high transformation efficiencies of > 2 million clones for each (50 ml x 10 tubes ea). The titers (cfu/ml) of the libraries are 4.0 x 107 cfu/ml for the uninfected gut, 2.6 x 107 cfu/ml for the uninfected salivary gland and 1.2 x 107 cfu/ml for the infected bacteria, respectively. Bacterial ‘bait’ primers were designed for 4 CLas-specific target genes, amplified each by PCR from infected ACP genomic DNA, and cloned into E. coli using pGBKT7. Transformation of selected candidate ‘baits’ into libraries is in progress. Obj 3: The second biological replicate for proteomic identification of putative effectors is underway. An additional 50 guts and 250 salivary glands of infected and uninfected potato psyllid adults were fractionated by SDS-PAGE (10’14% acrylamide) and digested with trypsin to yield peptides. The peptide mixture was analyzed by nano-LC-MS/MS using an EASY nano-LC (Proxeon, Thermo Fisher Scientific, San Jose, Ca) coupled to an Orbitrap LTQ Velos mass spectrometer. Proteins were identified at 99% confidence with XCorr score cut-offs determined by reversed database search. Mass spectra of peptides were analyzed with SEQUEST and XTandem software. Data were processed and organized using the Scaffold program. A probability threshold of 95% was adopted, and only proteins present in at least two replicates were considered expressed. The PAVE EST database was mined to select optimally sized open reading frames from among the annotated transcripts in the ACP and PoP adult psyllid, gut, and salivary gland databases, and the resultant translated proteins were used for protein identification. Obj 4: Several psyllid and Liberibacter genes were selected based on their potential for involvement as interactors, and tested for knockdown activity in an RNA-interference (RNAi) assay using oral delivery (parafilm membrane). Good quality dsRNA was synthesized (MEGAscript RNAi kit). This approach, together with microinjection of dsRNA, will form the basis for in vitro functional analysis of candidate effectors identified bioinformatically and/or in the yeast dihybrid assays.
Models relating yield to ground and foliar enhanced fertilization in the presence of HLB have been fully developed and explored. Although the results are promising, the amount of data available does not permit a general recommendation. Based on these initial results, several growers have volunteered to provide more data. Efforts are being made to obtain the data.
Objective 1: Generate functional EFR variants (EFR+) recognizing both elf18-Xac and elf18-CLas. Mutagenesis of Arabidopsis EFR Random mutagenesis was performed on the extracellular domain of EFR, and a library containing approximately 10^6 clones with an average mutation frequency of 0.4% was produced. From this library 13,000 clones were screened for ROS induction in response to elf18CLas. Unfortunately, No elf18CLas responsive clones were found. It was observed that there was a high frequency of non-functional clones in this library (as assessed by ROS production induced by wild-type elf18), so a further library was produced with a lower mutation rate (0.1%). A further 6,000 clones were screened from this library without isolating any elf18CLas responsive clones. Given the lack of positive results arising from these screens it has been decided to use different approaches to engineer elf18CLas responsiveness to EFR. Firstly, target mutations were produced in EFR at sites which are known to be important for elf18 binding and responsiveness. However none of these produced a response to elf18CLas. Secondly, we have shown that elf18CLas fails to compete well with elf18, in ROS and growth inhibition assays, suggesting that binding of elf18CLas EFR is not occurring. Therefore, a first step toward engineering an EFR variant capable of responding to elf18CLas is to evolve an EFR variant that gains binding to elf18CLas. In order to engineer EFR capable of binding elf18CLas, experiments have been initiated to determine the feasibility of performing a phage display screen to identify mutants of EFR. Initial data indicates that fragments of the EFR extracellular domain can be expressed in E. coli and can bind to biotin-labeled wild-type elf18. Further experiments are underway to determine the minimal region of EFR necessary for binding and the specificity of binding, which will later enable mutagenesis of this region. Objective 2: Generate functional XA21-EFR chimera (XA21-EFRchim) recognizing axYS22-Xac. Transgenic Arabidopsis plants are being produced with XA21 or XA21-EFR to assess their resistance to Xanthomonas campestris pv. campestris 8004. This work is required to test unambiguously the functionality of XA21 in conferring anti-bacterial disease resistance in dicots. In addition, tomato plants are being transformed with XA21 to determine functionality in this species. These plants will also be crossed with tomato EFR lines to determine the effectiveness of the presence of both genes in bacterial resistance.
This study addresses two general questions: 1) Will our constructs disrupt normal growth and development in citrus, and 2) Will these constructs confer a degree of resistance to infection by Liberibacter asiaticus? We have answers for the first question and seek a one-year extension to address the second. Objective 1. Express R proteins in a phloem-specific manner in Arabidopsis and citrus. It was evident very early from the results of our experiments, and of others, that the Arabidopsis SUC2 promoter was phloem-specific in citrus and, thus, efforts were directed towards the generation of transformed citrus containing wild type and constitutive mutants of the two R genes, SSI4 and SNC1. Our rationale was that by restricting expression to phloem tissues (or to the wounding response) potential negative effects on growth and development would be minimized. From 30-60 transformants of each R gene variant were obtained in Arabidopsis and at least 10 in citrus (Duncan grapefruit). In Arabidopsis, some stunting was observed when transformed with the constitutive ssi4, but not with wild type SSI4, or with wild type or mutant SNC1. A similar result was obtained with citrus; however, the stunted growth (or seedling death) phenotype was much more pronounced. However, as with Arabidopsis, no abnormal phenotype was observed with either variant of SNC1. e triggered by psyllid feeding. This objective is a variation of the first, except the restriction in expression of the potentially harmful R genes was imposed by the wound-inducible PAD4 promoter, a promoter known to be activated by aphid feeding in Arabidopsis. Our rationale was that in case AtSUC2-directed expression resulted in a stunted growth phenotype, the use of an inducible promoter, such as PAD4, would provide a way to evaluate the effectiveness of R protein expression in inhibiting Liberibacter infection. Our expectation was that the Pad4 promoter would not be active, except upon deliberate wounding under controlled conditions. As with the AtSUC2 promoter, the expression pattern of PAD4 was more variable in Arabidopsis as determined using a GUS reporter; however, PAD4/GUS expression in transformed citrus appeared to be strictly wound-inducible. Expression of the R gene variants using the PAD4 promoter gave a result similar to that obtained in Arabidopsis: expression of the SSI4 constitutive mutant was sometimes harmful to the plant; whereas, expression of the constitutive mutant of SNC1 was not. These experiments can be summarized as follows: 1-Restricted expression of the wild type SSI4 and SNC1 genes using either the AtSUC2 or AtPAD4 promoter had no negative impact on growth and development in citrus. 2-Similarily, expression of the constitutive mutant of SNC1 had minimal effect on growth and development. In contrast, expression of the constitutive mutant of SSI4 is sometimes harmful to normal growth and development in both Arabidopsis and in citrus. Additionally, preliminary tests indicate that none of the constructs effected psyllid feeding preferences. In preparation for assessing disease resistance of the transformed citrus, a single leaf assay to monitor the early events in the transfer of Liberibacter from the psyllid to the plant has been developed as outlined in the proposal pending with the CRDF (Nov 2012). In brief, the real time PCR protocol was refined by developing calibration curves for the Las and various plant and psyllid control amplicons so that detection is now reproducible down to 12 copies. In addition, significant improvements have been made in single-leaf cage design that will enable feeding to be restricted to a 6 mm area of leaf.
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 new greenhouse is compliant with DPI regulations for propagation facilities. – The initial cohort of advanced, tetratzygous citrus rootstock materials for en masse propagation have been identified and are currently being propagated.
For the last three months of 2012, the Mature Tissue Transformation Laboratory (MTTL) continued to operate in the ‘maintenance’ capacity mode. Level of operation was determined by the amount of plant material available and its quality. The process of increasing the number of rootstock plants is slow and it has been hindered by low germination rate of seeds that are old. Although new seeds were ordered in December, they will not be available until late January/mid February when Swingle citrumelo and C. macrophylla fruit are available. One of the batches of Hamlin buds grafted in early October had low percentage ‘take/success’ rate. The outside provider of grafting services claimed that the buds coming from mother plants were not of the highest quality. In the meantime, this person has left the business and the facility contracted other provider. In couple of experiments, a high percentage of explants that were used in co-incubations with Agrobacterium got contaminated. We are investigating whether those incidences were the result of human error in the steps of transformation taking place in the laboratory, or if plants that served as starting material for explants were infected while in the growth chamber. During these three months, six co-incubation experiments were performed. Four of those experiments were done with Valencia explants and two with Hamlin explants. For the Valencia experiments, we cut 2170 explants and 1030 explants were cut for Hamlin experiments. In order to be able to assess the ability of the lab to process different orders at the same time, two additional Agrobacterium strains were used for co-incubation experiments. One of those harbored a binary vector with the gene for green fluorescent protein (GFP) as a reporter gene. In one of the Hamlin experiments, out of 16 shoots inspected for GFP fluorescence two were positive. Those two shoots were micro-grafted on Carrizo rootstock plants. Some GUS assays were done on shoots obtained from experiments done earlier. Out of 19 shoots, one was positive. Two additional Ray Ruby plants were cleaned of microorganisms and are ready to become source of budding material. One more Hamlin plant was also cleaned.
The four most promising anti-NodT scFv antibodies have been selected for further development. Anti-NodT antibody #1 has been successfully expressed in E. coli. This means that we can generate as much of the antibody as needed. The antibodies are being augmented with two 6xHis epitope tags – one at the amino terminus, and one at the carboxy terminus. The protein can be detedcted with anti-His antibodies. The anti-NodT scFv antibody is soluble, and should be usable for protein immunoblotting and other applications. We experienced some delays in cloning the scFv antibody DNA into the appropriate citrus transformation vector. However, these difficulties have now been solved and we now expect to have the scFv citrus transformation construct completed within a few weeks, and we will commence transformation immediately.
Progress with the rapid flowering system (pvc pipe scaffolding system) in the greenhouse: Selected transgenic plants produced from juvenile explant, budded to precocious tetraploid rootstocks in airpots are growing well in our RES system, with some plants reaching 8 feet in height. Additional transgenics were propagated onto additional new rootstocks expected to reduce juvenility, including the somatic hybrid Amblycarpa + Flying Dragon. The goal is to reduce juvenility by several years to accelerate flowering and fruiting of the transgenic plants. Experiments to efficiently stack promising transgenes are underway. Experiments to efficiently stack promising transgenes are underway. The first transformation experiments using the two-transgene Gateway based cloned construct combining our best transgene for HLB resistance (NPR-1 from Arabidopsis) with our best transgene against canker that also has some affect on HLB (the synthetic CEME lytic peptide gene) were initiated, and so far 30 putative transgenic lines of the sweet orange cultivars Hamlin and Valencia have been regenerated. These plantlets have been micrografted to Carrizo rootstock. The goal is to provide stable resistance to both HLB and canker, with transgene backup to prevent Liberibacter from overcoming single transgene resistance.A construct containing CEMA gene stacked with the NPR1 gene has been constructed. Also, another vector containing a AttacinE gene stacked with the NPR1 gene is also under construction. Correlation of transgene expression with disease resistance response: More than 150 transgenic lines with different genes have been analyzed using ELISA by either C-myc or LIMA antibody (which also works for CEME) to measure transgene expression. As expected, significant differences were observed in our transgenic plants. Correlations between the data obtained from ELISA and other molecular data with HLB challenge response data are underway. Transgenic lines examined by ELISA include 40 lines with NPR-1, 50 lines with LIMA, and 9 lines with CEME. Improved transformation methodology (for seedless or recalcitrant cultivars, and eventually marker-free consumer-friendly transformation): We have finished construction of several parts of the T-DNA region of a pCAMBIA0390 derived binary vector for cre-lox based marker-free selection. A fusion codA-hptII gene driven by the d35S promoter have been constructed and a cre gene driven by a glucocorticoid-responsive elements promoter have also been constructed and cloned into a pUC based vector. We are experiencing problems cloning the glucocorticoid receptor gene driven by a constitutive mirabilis mosaic virus promoter as all sequenced clones have mutations and/or deletions in them. Work is underway to rectify this.
Monthly monitoring of all the alternative host plants continued this quarter. Transmission rate data were the same as found in last quarter on the 6 citrus relatives tested. Severinia buxifolia (Sb), Calamondin (Cal), Zanthoxylum fagara (Zf), Citropsis gilletiana (Cg), Choisya spps (Ch), and Esenbeckia runyonii (Er), were all found to be alternative hosts of Candidatus Liberibacter asiaticus. Bacterial populations in Zanthozylum fagara, Citropsis gillentiana, Choisa spp. and Esenbeckia runyonii maintained for a relatively short period of time, or maintained at a low population level (Cq value around 35). This followed the same trend found with Murraya paniculata. Las persistency in the plant, percentage of psyllids with Las and Cq values in the psyllids was summarized. Monthly monitoring of the live bacterial population dynamics in Severinia buxifolia and Valencia sweet orange by PMA-qPCR ended in April 2012. The data of the last month showed that Severinia and Valencia both contained high levels of live bacteria around 10^7, and 3×10^7 bacteria/gram tissue, respectively. The total population was similar to the these levels in both Severinia and Valencia.
Three different psyllid transmission tests using all of the alternative host plants were completed except for tests using Amyris since all transmission attempts from HLB infected citrus to Amyris was negative (no PCR positives or symptomatic plants) Monthly monitoring of the bacterial population dynamics in Severinia buxifolia by PMA-qPCR showed that the live bacterial population level dropped in January 2012 to about 10^6 bacteria/gram tissue, and then came back to the high level in February and March (about 10^7 bacteria/gram tissue), while total population fluctuated from 10^7 to 1.2×10^8 bacteria/gram tissue. Monthly monitoring of bacterial population dynamics in Valencia sweet orange showed that the live bacterial population also dropped in January 2012 to about 10^7 bacteria/gram tissue, and then came back to the high level in February and March (about 4×10^7 bacteria/gram tissue). The total population fluctuated from 10^7 to 10^8 bacteria/gram tissue.
In alternative hosts tested in psyllid transmission experiments, positive PCR transmission results from citrus to alternative host were obtained for the following species: Severinia buxifolia (Sb), Calamondin (Cal), Zanthoxylum fagara (Zf), Citropsis gilletiana (Cg), Choisya spps (Ch), and Esenbeckia runyonii (Er). No eggs or nymphs were found on any of these alternative hosts (except for Sb and Cal), and further transmission tests (three reps were done for each) are still under investigation. The psyllid feeding behavior and living status on those plants was recorded since the psyllids seemed not to readily feed on some of the plants even when force fed. Monthly monitoring of the live bacterial population dynamics in Severinia buxifolia by PMA-qPCR showed that the live bacterial population remained high (about 10^7 bacteria/gram tissue) through this time period, while total population fluctuated from 10^7 to 10^8 bacteria/gram tissue. Monthly monitoring of live bacterial population dynamics in Valencia sweet orange by PMA-qPCR showed that the live bacterial population remained on the high level (about 3×10^7 bacteria/gram tissue) through these three months, while total population was about the same as live ones in these three months, 3.2×10^7 bacteria/gram tissue.
HLB in Florida is associated with bacterial pathogen Candidatus Liberibacter asiaticus (Las) and transmitted by Asian citrus psyllid (Diaphorina citri) to citrus. Some plant species have been listed as hosts of the psyllid and/or the associated bacterium based on field samples, but their status as alternative hosts has never been throughly studied. In this work the following rutaceous plants were investigated: Murraya paniculata (orange jasamine), Murraya (Bergera) koenegii, Murraya exotica, Severinia buxifolia, Citrofortunella microcarpa (Calomondin), Citropsis gilletiana, Esenbeckia runyonii, Zanthoxylum fagara, Choisya aztec ‘Pearl’, Choisya ternata ‘Sundance’, Helietta parvifolia, and Amyris texana, were studied to investigate their alternative host status. Possible transmission pathways for each plant were tested with repeated psyllid transmission experiments as well as grafting where compatible. After inoculation, plants were monitored for symptom development and tested by real-time PCR (qPCR). At the USDA, ARS, FDWSRU, Ft. Detrick, MD, psyllid transmission tests, Asian citrus psyllids transmitted Las to M. paniculata and M. exotica, but not M. koneigii. Disease symptoms did not develop in Murraya plants, and positive infections were determined by PCR. Back-inoculations from M. paniculata to sweet orange was successful however there was some variability in infection rates, titer, and the Las bacterium did not persist in M. paniculata (published). At the Texas A&M Citrus Center, Weslaco a collection of eight rutaceous species were established and included two Amyris species (torch wood), Zanthozylum fagara (lime pricklyash), Helietta parvifolia (baretta), Esenbeckia berlandia, Casimiroa tetrameria and two Choisya species (Mexican orange) and were tested as hosts for the psyllid. Esenbeckia berlandieri (jopoy), Amyris madrensis (torchwood), Choisya ternata and C. arizonica all were found to be feeding hosts for the psyllid. Egg laying was found on torchwood and egg laying and nymphal development were found on C. ternata. At CREC we experimentally demonstrated the alternative host status of several plant species, of which 6 plants (Calomondin, C. gilletiana, E. runyonii, Z. fagara, C. aztec and C. ternata) were 1st time reported as hosts of Las. S. buxifolia, Calomondin, C. gilletiana, E. runyonii, Z. fagara, C. aztec and C. ternata were infected by Las although the transmissibility varied and was based on bacterial persistency and psyllid activities. At the USDA, ARS, Beltsville quarantine work with dodder as an alternative host to study the plant infection process and for its use with plants that are not graft compatible with citrus was done and published. qPCR has limitations since it does not differentiate live bacterial genomes (LBG)and dead bacterial cells. Propidium monoazide (PMA), a novel DNA-binding dye, has been used with many bacterial pathogens to effectively remove DNA from dead cells, but no applications on uncultured bacteria like Las have been reported. In our work we devised PMA-qPCR protocols and optmized them to work with plant and psyllid materials. They were then used to determine LBG in various studies, such as establishing correlation between LBG and microscopic counting, checking the reactions of different citrus plants to Las infection, and checking the connection between LBG and leaf symptom expression. Lastly, the LBG dynamics inside HLB positive citrus and non-citrus hosts was monitored monthly through a 20-month period, and a seasonal development pattern was observed in both hosts. The optimized PMA-qPCR developed provides an accurate way to determine LBG in plant hosts of Las, which should benefit various HLB research and serve as a crucial component in HLB management. This work has been accepted for publication.
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