The goal of this project is to obtain knowledge of the genetic and biological diversity of CTV isolates in CA and to identify and put in place mild isolates of the virus that can provide sustained protection against severe SP isolates. We are conducting molecular characterization of CTV isolates (Folimonova lab) collected from various regions in California. This approach will provide important information regarding genetic diversity of CTV populations in this region required for further selection of mild protective isolates. We have developed approaches for molecular characterization of CTV isolates by designing several sets of oligonucleotide primers that specifically amplify sequences of particular CTV genotypes (strains), including T30, T36, T3, VT, T68 and RB (Poncirus resistance braking strain) genotypes and further tested and optimized protocols for conventional reverse transcription PCR and quantitative real-time reverse transcription PCR amplification using the selected primers coupled with further sequencing analysis. We have analyzed the genotype composition of nearly 90 CTV isolates that have been collected from main citrus growing regions in California and then propagated in CTV isolates collections (CCTEA, CCPP). Most of the isolates had a mixture of the T30 plus VT genotypes. Few isolates have T3 or T36 genotypes. We also got amplification of PCR products with primers specific to the T68 and RB genotypes for a few isolates. The latter genotypes are not very well characterized, so follow up sequencing will be needed to determine whether some of the tested isolates contain those two genotypes. As the next step, we are setting up cross-protection trials using selected mild isolates along with isolates that cause severe stem pitting phenotype. Our first trial set has been already set up using T519 (mild isolate) and SY 568 (severe isolate) in Ray Yokomi lab at the USDA facility in Parlier. Currently plants that were inoculated first with the mild isolate and sequentially challenged with the severe one are being monitored for symptoms development. From the results of the molecular characterization of a large number of CTV isolates, we made a list of isolates (mild and severe) that will be used for additional cross-protection trials. We are now in a process of setting up those trials in Ray Yokomi and Georgios Vidalakis labs. In order to further understand how CTV cross-protection works in the field, we studied the cross-protection system developed by Klaus Bederski in Peru. This system represents an example of successful in-field selection of mild protecting isolates that were able to provide sustained protection of sweet oranges against severe stem pitting isolates that were introduced into the country. Molecular characterization of the Peruvian protecting isolates and isolates that caused severe stem pitting disease demonstrated that both protecting and severe isolates shared the same genotype composition: they all contained the VT genotype. This result very well correlates with the data that we obtained from our studies on CTV cross-protection mechanism and confirms that sustained protection could be provided only by mild isolates that have the same genotype as that of the severe isolate.
Eight experiments to estimate the viability of LAS over time, each conducted using a separate LAS-infected pomelo, have recently been completed. Data analyses from these experiments are ongoing. These are expected to be the final experiments of this type, and data from these experiments will be part of a publication. Time-series experiments to monitor LAS viability were conducted in four different types of media. These were: K (1/3 King’s B), J50 (50% juice from the infected pomelo), G50 (50% store-bought grapefruit juice), and G ( 100% storebought grapefruit juice). Replicate culture flasks for each media type were inoculated with LAS inoculum from seeds of the infected pomelo fruit. Starting immediately after inoculation and continuing every other day for a total of 10 time points (18 days), samples were collected from the culture flasks. Triplicate samples were frozen unmodified from the culture flasks, while another triplicate set was treated with ethidium monoazide (EMA) and then frozen. Additional samples of the media before and after each experiment were collected for later analyses, and pH and salinity of these samples were measured. Also during the course of the time-series, samples were collected and cultured on both 1/3 KB solid media and G50 solid media to see if any colonies would develop. As 1/3 KB media served as the control media, and previous experiments showed a quick loss of LAS viability in this medium, growth on these plates served as an estimate of contamination. Experiments with significant contamination were excluded from further analyses. No growth of any kind was seen on G50 solid media. Currently, data analysis of the frozen samples is being conducted. DNA extractions are being performed, and the DNA is being analyzed by qPCR to determine both total LAS DNA and viable LAS DNA (EMA-treated samples). So far, results agree with previous findings showing that LAS viability is maintained longer in media containing fruit juice. Also, plans are being made to analyze media sample nutrient concentrations before and after the experiments via inductively coupled plasma-optical emission spectroscopy. This will allow media composition comparisons and may help identify nutrients that LAS requires for growth. While conducting these experiments, it was noticed that a white biofilm forms at the air-liquid interface of many of the culture flasks. This biofilm was more frequent and clearly larger in flasks containing juice in the media. In an effort to reproduce this biofilm for further study, 50 ml conicals containing a glass slide have recently been inoculated with LAS to monitor biofilm formation. Any growth will be observed under a microscope, with the intention of gaining more information on the conditions needed to produce this biofilm in microfluidic chambers in the future. We would like to determine how this biofilm forms and if it contains any LAS cells or not.
The first objective of the project was to hire a Florida-based faculty scientist that could be trained under Dr. Leandro Pena in Spain, for the purpose of learning the mature tissue transformation technique and transferring the technology to Florida. The scientist (Dr. Cecilia Zapata) was hired, at the end of the first year of the three year project, and traveled to Dr. Pena’s lab at the IVIA, Spain, where she was trained in all tissue culture techniques associated with citrus mature transformation, starting with preparation of the source of material at the greenhouse and ending with the acclimatization of transformants in the greenhouse. It was emphasized that the preparation of plant material needed for mature transformation is the key to successfully and consistently obtaining mature transformants, and this can only be achieved by producing budsticks in a highly controlled and clean environment. The second objective of the project was to build a greenhouse at the Citrus Research and Education Center in Florida for the purpose of creating and growing citrus for mature transformation and to establish a Mature Transformation Laboratory. Unfortunately, the greenhouse was unable to be constructed due to miscalculations in the planning budget in the original proposal. Instead a growth room was constructed adding a clean head house structure that could be used as support of the growing area. It took approximately 7 months to construct the growth room. Up to date, almost a year after finishing the construction, we are still correcting a few problems with humidity, computer sensors and the water filtration system. Also, a generator needs to be purchased; without it, any prolonged electricity failure could jeopardize the whole project. The laboratory is fully operational. The third objective of the project was to obtain mature transgenic plants from the most important Florida citrus cultivars. We started using the growth room and plant the rootstocks at the beginning of April 2011. At the same time three (3) sweet orange varieties were indexed in vitro and micrografted; the cultivars introduced were Hamlin 1-4-1, Valencia SPB 1-14-19 and Pineapple F-60-3. A calendar was established in October 2011 and firsts mature transformation experiments were performed in November 2011, all the protocols developed at the IVIA are adjusting to our specific environmental conditions and clone specificities. In our conditions, mature Valencia was very responsive to organogenic regeneration. We obtained positive plants. Hamlin was also transformed but was less responsive to organogenic regeneration, we have a few positive plants, but we are still adjusting the protocol to improve the efficiency. Our control cultivar, Pineapple, has some quality problems with the starting material and new introductions are needed in the future, however since we don’t have too much space in the growth room, we are introducing another important cultivar for Florida. The Valencia positive plants are already growing in the growth room to be tested by PCR and Southern Blot.
The overall objective of this project is to develop and use a high-throughput system to screen for chemicals that disrupt interactions in a model of the ACP/HLB/Citrus system that uses the related bacterium Candidatus Liberibacter psyllaurous (CLps) which causes psyllid yellows of tomato. During year 1, most work focused on development of a system for the model plant Arabidopsis thaliana which has the best developed genetics of any plant and has been used in previous chemical genomics experiments. We tested 19 diverse Arabidopsis lines for susceptibility to infection by psyllids and found significant differences among lines in the percentage of plants that became infected, and in the amount of bacteria present in tissue samples from each plant as judged from the Ct value of qPCR detection. No lines were completely resistant, the most resistant having only 1 of 5 plants infected. We did not detect any visible disease symptoms on leaves or stems of infected plants, and later experiments with a susceptible line showed no differences in root weight, number of fruits, number of seeds or other traits. However, seeds produced by most inoculated plants had much lower germination than those of non-inoculated plants. Potato psyllid nymphs can mature into adults on Arabidopsis, but the these adults soon die and do not lay eggs. We repeated screening of soil-grown plants of 10 Arabidopsis ecotypes for susceptibility or partial resistance using a larger number of Arabidopsis plants per ecotype to obtain better discrimination among ecotypes. Results of these experiments have not yet been analyzed. For chemical screening experiments, we must be able to grow plants in a system that facilitates introduction of small volumes of chemicals through the root system, and it must be possible to inoculate such plants with potato psyllids raised on infected tomato plants. Repeated attempts to infect Arabidopsis plants grown in solid culture media, liquid culture media, or hydroponics were not successful. Only plants grown in soil were infected by psyllid nymphs. This does not appear related to the size of the plant, but the culture condition may induce stress responses that make the plants immune to infection and/or reduce psyllid feeding. Late in the project year we began to focus on developing a system for chemical genomics assays in tomato. Small side shoots from tomato plants were placed in 50 ml culture tubes with the cut stem end immersed in water in a microfuge tube. This design was adapted from one shown on a poster by Ammar et al. at the Citrus Health Research Forum in Denver in October 2011. Adult psyllids are placed in the culture tubes and within 7 days most tomato shoots were qPCR positive for CLps. After two weeks Ct values ranged from 22 to 29. This system appears promising since chemicals can be introduced into the water for plant uptake. We have not yet demonstrated chemical uptake, but the system has all of the other essential characteristics necessary for a chemical genomics experiment and we plan to initiate these experiments early in year 2. The project received a no-cost extension to January 31, 2012 due to delays in initial funding, so there are 5 quarterly reports this year.
An Excel model that uses the income approach to asset valuation as the methodology has previously been developed by UF/IFAS to evaluate citrus investments in Florida. This model was modified to develop an economic model to evaluate emerging solutions to citrus greening. The model can be divided into three components: grove characteristics, disease characteristics, and economic variables. Each component enables the user to enter values for a number of variables that, when combined with variables from the other components, represent the planting and operation of a new grove, or the operation of an existing grove, as a business unit. Each variable can be changed to determine its effect on costs, production, revenues and cash flows. An economic evaluation of the tradeoffs among current greening management alternatives was then done that included the following: (1) removing infected trees and spraying to control psyllids; (2) not managing greening; (3) resetting removed trees; (4) switching to an enhanced foliar nutrient program instead of removing diseased trees and (5) replanting the entire grove when cumulative tree losses make it economically unproductive, and replanting to traditional tree densities versus higher tree densities. The results of this analysis were published on EDIS and in a citrus trade magazine, as well as presented at two industry citrus conferences, at two International Research Conferences on HLB, and at three grower meetings. An economic model was also developed that evaluates the grove replanting decision. The general principles of asset replacement were applied to orange groves, where the existing typical grove (defender) was compared to self-replacement with another typical grove and to replacement with a grove planted at high tree densities (advanced production systems, or APS). This was called the challenger. Two types of groves were analyzed: A traditional grove (defender) planted with 145 trees per acre and a grove planted with 270 trees per acre (challenger). The results showed that once the grove was replanted with an APS grove, if trees lost were reset, the grove never needed replanting. If trees lost were not reset, the grove had an economic life of 28 years, far beyond the typical planning horizon of 15-20 years. Thus, groves planted at tree densities similar to APS groves have either infinite or much longer economic lives. Data for both groves were from commercial groves in Florida planted at these densities. When data from an APS grove become available from this project, this model will be used to see if APS groves potentially have infinite economic lives. If they do, that suggests that APS technology offers a way for the Florida citrus industry to survive greening until a cure is discovered. This optimal grove replanting analysis was published in a leading Florida citrus trade magazine, and presented at both a scientific conference and at grower meetings. It is also in review for publication in the Journal of Agricultural and Applied Economics as ‘Optimal Asset Replacement under Conditions of Technological Change: The Use of Advanced Citrus Production Systems to Mitigate Endemic Citrus Greening.’
Preliminary examination of limb, scaffold and trunk phloem of healthy and HLB infected trees indicated that in diseased trees development of callose plugging was evident at all levels and started behind the newer phloem, was present in low amounts in healthy plants, could be seen in early infection stages and was readily seen by light microscopy with aniline blue or by TEM with normal fixation. Further evaluations by phloem age are underway. Huanglongbing (HLB) or citrus greening disease, caused by Candidatus Liberibacter asiaticus, is a phloem-limited fastidious pathogen transmitted by the Asian citrus psyllid, Diaphorina citri, and appears to be an intracellular pathogen that maintains an intimate association with the psyllid or the plant throughout its life cycle. The molecular basis of the interaction of this pathogen with its hosts is not well understood. We hypothesized that during infection, Ca. L. asiaticus differentially expresses the genes critical for its survival and pathogenicity in either host. To test this hypothesis, quantitative reverse transcription PCR was utilized to compare the gene expression of Ca. L. asiaticus in planta and in psyllid. Overall, 362 genes were analyzed for their gene expression in planta and in psyllid. Among them, 263 genes were up-regulated in planta compared with in psyllid, 18 genes were overexpressed in the psyllid, and 81 genes showed similar levels of expression in both plant and psyllid. Our study indicates that Ca. L. asiaticus adjusts its expression of genes involved in transport systems, secretion system, flagella, LPS, heme biosynthesis, stress resistance, hemolysin and serralysin in a host specific manner to adapt to the distinct environment of plant and insect. To our knowledge, this is the first large-scale study to evaluate the differential expression of Ca. L. asiaticus genes in a plant host and its insect vector. Efforts to propagate transgenic plants with the beta-glucanase gene continued, resulting in more than 150 Duncan and/or Valencia plants with this transgene, controlled by either or 35S promoter, or the phloem-specific Suc2 promoter. PCR analysis on a subset of these revealed that 90% are showing the specific band for the BG gene. Additional transgenic plants are being regenerated from in vitro cultures developed by Dr. Abdullah ‘ about 23 transgenic lines with the BG gene in either OLL#20 or Jin Cheng sweet oranges. These plantlets will be micrografted to rootstocks in 2012. Transgenic plants developed earlier are being moved to the Southern Gardens ‘hot psyllid’ greenhouse for inoculation.
Continued efforts to improve transformation efficiency: ‘ Evaluation of transgene expression of transgenic citrus plants with different phloem specific promoters. Several transgenic ‘Mexican lime’ lines containing the d35s promoter and the 4 phloem specific promoters were evaluated for transgene activity. Transgene analysis was carried out using PCR, RT-PCR, q-PCR and Southern Blot analyses. Publicataion: Dutt M., Ananthakrishnan G, Jaromin MK, Brlansky RH, & Grosser JW (2012) Evaluation of four phloem-specific promoters in vegetative tissues of transgenic citrus plants. Tree Physiology 32(1):83-93. ‘ q-PCR approach to evaluate copy number and gene expression levels. Copy number of several transgenic lines has being evaluated using gene specific TaQMAN probes. Most transgenic lines had 1 ‘ 4 copies of the transgene stably incorporated into the genome. In addition a qPR-PCR approach is being used to evaluate gene expression levels in all transgenic lines. Horticultural manipulations to reduce juvenility in commercial citrus: ‘ Continued to grow selected precocious rootstock seedlings for subsequent budding with transgenic precocious sweet oranges (Vernia and OLL series). Several transgenic lines of our precious sweet orange and mandarin transgenic lines (B4-79, W. Murcott, B10-68 and OLL8) have been grafted onto precocious rootstock including Amblycarpa + Benton and Changsha + Benton. Grafted trees have been transplanted into airpots in a heated greenhouse for evaluation (Fig 1.), with plans to grow the trees in a RES (Rapid Evaluation System horticultural manipulation) type system in the greenhouse. We are now planning to establish a transgenic site on CREC property, and hope to be able to include a small structure to apply the RES technology (horticultural manipulation to reduce the time of juvenility) to actual transgenic plants. Transformation with early-flowering genes: ‘ We have regenerated many transgenic plants with the poplar FT behind either the 35S or heat shock promoter. Some of them are quite large now and the ones with HS promoter were maintained in a growth chamber at high temperature for several months, but none have bloomed yet. We are growing T1 tobacco with all 3 citrus FTs in order to determine phenotypes. We developed a co-transformation strategy to transform Carrizo citrange with two cassettes, one containing 35S-cft1 and the other containing AtSUC2 ‘ gus. We generated 122 transgenic Carrizo plants using the two vectors. PCR analysis revealed that 16 lines contained both cassettes. Plants have not flowered 12 months after transformation. Plants are currently being evaluated in an unheated greenhouse for cold stress in order to initiate flowering in spring 2012. Numerous transgenic plantlets of Hamlin and Carrizo were regenerated containing P27, P28, P29, PATFT and pPTFT.
For Asian citrus psyllid, we have constructed and sequenced two 454 Titanium libraries (uninfected guts and infected adults), and have generated 663,886 ESTs. We constructed and sequenced six Illumina Paired-end libraries (uninfected guts, uninfected adults, uninfected nymphs, infected guts, infected adults, and infected nymphs), obtaining 209 M sequence reads. These high-quality reads were assembled with different combinations. The assembly from 4 adult/nymph (no guts) Illumina libraries yielded a total of 45,799 unique transcripts (unitrans). The mixed assembly (six Illumina and two 454 libraries) resulted in a total of 151,307 unitrans. With unitrans annotation and data mining, we investigated gene/unitrans expression levels based on read numbers and identified genes of interest that are misexpressed among treatments. A suite of candidate genes are under investigation as potential targets for RNAi, full length cloning, and further functional analysis. We functionally analyzed the ACP and PP Illumina unitrans using Gene Ontology (GO) classification. Both ACP and PP annotated unitrans were categorized into 59 functional groups, with the majority assigned to the Biological Process category. Significant hits for response to stimulus and nutrient reservoir activity highlights dependency on signalling molecules to sense food and a lifecycle revolving around plants. We found many predicted GO functions like organelle, extracellular region, and biological adhesion interesting because of their putative role in supporting parasites. We further analyzed individual libraries to determine the overall gene regulation pattern. Results show that Liberibacter infection had a greater impact on psyllid nymphs than adults. The majority (99%) of PP nymph unitrans were down- regulated in all GO categories and the majority (56%) of PP adult unitrans remained unchanged in response to Liberibacter infection. Although not as dramatic, ACP data shows a similar phenomenon of differences in functional categories being more pronounced between infected and uninfected nymphs, compared to infected and uninfected adults. Data suggest the two developmental stages may contribute in different ways to support the pathogen; some pathogenesis genes may differ between stages. Predicted functions of PP unitrans unique to Liberibacter-infected adult PP were mostly of bacterial origins with functions related to motility. Predicted functions of unitrans specific to Liberibacter-infected PP nymphs were related to antimicrobial transport and inactivation of MAPK activities. A similar analysis of ACP unitrans was performed and data shows that ACP has more unique functional classifications for unitrans in the uninfected libraries compared to PP. Our database is designed to utilize the OrthoMCL program, which identifies orthologous proteins. Of the roughly 128,000 annotated PP and ACP Illumina unitrans, 10% were orthologous to known or predicted invertebrate proteins. We are currently using this program to identify protein/gene families common between the genomes in hopes of identifying important psyllid genes, which can be used in RNAi and other functional bioassays. We are optimizing microinjection using a hand-held microinjector to prepare for dsRNA-RNAi knockout assays for functional genomic characterization of a suite of target psyllid genes. Presentation of dsRNAs by artificial feeding and qPCR quantification of gene expression levels for target genes is already underway. Manuscripts: 1. Fisher TW, Vyas M, He R, Cicero J, Nelson W, Crowe J, Soderlund C, Gang DR, Brown JK. 2012. Characterization of the potato psyllid transcriptome and comparison with the Asian Citrus Psyllid transcriptome in response to Candidatus Liberibacter infections. BMC Gen. (In prep). 2. Vyas M, Yin G, Fisher TW, He R, Cicero J, Nelson W, Crowe J, Soderlund C, Gang DR, Brown JK. 2012. Unravelling the Asian Citrus Psy transcriptome. BMC Gen.(in prep).
During the last quarter the goal has been to bring together accumulated data and discoveries into two publications. Our final step toward closure and submission of these manuscripts was to perfect a method that increased the rigor of the FISH assay for localization in psyllid organs. We have tested and confirmed the specificity of three Ca. Liberibacter probes, and have used a Carsonella ruddii-specific probe as an internal control. FISH probe localization was highly successful for Liberibacter solanacearum localization in dissected guts using the 16S rRNA, outer membrane protein, and abc transporter gene sequences as probes under modified fixation conditions and using the new fluorescent camera. This approach distinguishes bacteria on the inside of the gut from those on the outside, which is extremely important for time-course studies of proliferation inside the body and advancement along the transmission pathway to its terminus at the stylets. We have successfully dissected the following organs from psyllids: gut, malpighian tubules, salivary gland, ovary, testis, fat body, hemolymph and bacteriocytes,to study the differential localization of specific Ca. Liberibacter genes in the different tissues using the standardized FISH protocol. Experiments are in progress to determine APP and IAP parameters and link them with transmission frequency, virus load using qPCR to quantify Ca. Liberibacter load in individual psyllids, and the FISH technique developed here to track the bacterium progress in associating with guts and salivary glands/oral box. Experiments will be completed next quarter. TEM Liberibacter localization in sectioned whole psyllids is underway using a cocktail DIG- labeled probe with the focus being on gut, blood, salivary glands, and the oral box. Time course points of study are guided by the above experiments. Pathogen localization cannot be resolved without understanding the mouthpart complex (oral box), and our studies (soon to be published) have increased our understanding in morphology from its state of progress some 50 to 80 years ago. Manuscripts: 1. ‘Strongarm’ movement of Ca. Liberibacter solanacearum through the anatomical transmission pathway in the potato psyllid vector, Bactericera cockerelli (Sulc) (Hemiptera: Triozidae). J.M. Cicero, T.W.Fisher, J.A. Qureshi, P.A. Stansly and J.K.Brown. Ann. Entomol. Soc. Am. (In preparation). 2. Elucidation of the oral region of the general pathogen transmission pathway and scrutiny for potential occupation by Ca. Liberibacter solanacearum in the potato psyllid vector, Bactericera cockerelli (Sulc) (Hemiptera: Triozidae). J.M. Cicero, J.A. Qureshi, P.A. Stansly, and J.K. Brown. Ann. Entomol. Soc. Am. (In preparation).
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. Recently, we have been testing a newer formulation of the SPLAT with a different active ingredient, which cannot be disclosed here. This new formulation with the new active ingredient is working more consistently than DMDS to repell psyllids. Also, it is much less noxious than DMDS and does not produce a foul smell. We are working to determine if this new repellent formulation will produce more consistent results than the DMDS formulation. Also, we have been investigating blends of compounds in the SPLAT formulation as an active ingredient to determine if blends may be more effective than single components, such as DMDS. Finally, we have been working to develop a release device that can be applied to foliage, such that the repellent SPLAT formulation can be deployed without adhering to leaves or fruit. We have found that this is necessary, because some of the repellent active ingredients, like DMDS, are phyto-toxic.
Our objective was to determine how psyllid host seeking behavior is affected by Las-infection. In previous experiments, we have determined that ACP adults initially prefer to settle on Las-infected plants than non-infected counterparts. This attraction to Las-infected plants appears to be dependent upon the plant’s release of one specific volatile chemical that attracts psyllids. After settling on infected plants for a short period of time sufficient to acquire the Las pathogen, these potentially infected psyllids move from infected plants to healthy plants. Thus, the Las-pathogen is affecting the behavior of psyllids in way that promotes its own spread. 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. In the last quarter, we have worked towards creating nutrient deficient, yet uninfected plants. We have successfully achieved a low level of nutrient deficiency in some plants and conducted initial settling trials. In these trials, psyllids were initially caged on healthy citrus. Subsequently, the cage was removed and the psyllids were allowed to either stay on the healthy plant or move to a nutritionally deficient plant. In this experiment, an average of 69.5% of psyllids chose to remain on the healthy plants while on average 26.7% moved from healthy to nutrient deficient plants. In concurrent experiments, psyllids were initially caged on nutrient deficient plants and then allowed to move to healthy plants. In these experiments, an average of 72.4% of psyllids chose to move to the healthy plants, whereas an average of 24.7% of psyllids remained on the nutrient deficient plants. We have continued to restrict application of fertilizer to these nutrient deficient plants in order to achieve further deficiencies. Samples of plant tissues have been sent for nutrient analysis, and upon receiving that data, another replication of these experiments will be conducted. In future experiments we will rescue these plants with each nutrient to determine which nutrients may be involved in affecting psyllid settling behavior. These data may be useful in predicting psyllid movement between citrus groves depending on nutritional status of trees. Also, this will help explain movement of pathogen by psyllids from tree to tree as a result of pathogen-altered psyllid host seeking behavior.
Analysis of transgenic citrus lines: We confirmed the presence of transgenes in transformed citrus plants using standard PCR techniques. In one line, SUC2-snc1 mutant (20-7), it was unusually difficult to establish that the desired construct was present. Serial dilutions of genomic templates were employed to reduce interference of inhibitory substances present in plant genomic DNA extracts. This interference seemed to be correlated with this specific construct. It is possible, therefore, that constitutively expressed snc1 mutant may affect the production of phenolic or other interfering compounds. In order to evaluate the survival of Liberibacter asiaticus (Las) in our transformed citrus lines, we first focused on the development of an assay to detect the presence of the bacteria in heavily infected, symptomatic citrus leaves. Infected leaves were obtained from the UF Lake Alfred laboratory of Dr. William Dawson. These were sectioned into midveins and blades in order to determine the distribution of the infecting pathogen. Original quantities of tested materials (leaf samples) were in the range of 40 mg. PCR primers for detection of the Liberibacter asiaticus were based on 16S ribosomal DNA, and as plant controls, the cytochrome oxidase COX gene was used (Pelz-Stelinski et al., 2010, J. Econ. Entomol. 103, 1531-1541). We were able to detect Las and Cox amplicons in genomic DNA isolates from these relatively small quantities of transgenic citrus leaf material: either in green (asymptomatic) or yellow symptomatic Las-infected leaves. Setting up a calibrated curve for real time quantitative PCR: Next, we generated Las and Cox PCR amplicons to be used in standard curves in real-time PCR reactions for copy number determinations. The Wingless (Wg) gene that serves as the psyllid control was obtained from the genomic DNA isolated from 10 uninfected psyllids. Real-time PCR reactions required testing multiple variables in order to fine-tune the Las-detection assay, some being the primer and amplicon concentrations. We tested a range of amplicon concentrations from 10 ng to 1 pg (=12,190,283 copies), and in later experiments, down to 12 copies of Las and 14 copies of Cox and Wg. As a starting point, we tested the expression of AtPAD4-GUSplus transgenic plants responsive to wounding to correlate the wounding event itself with the actual psyllid feeding. Preliminary citrus wounding experiments by slit-cutting, or needle-puncturing determined that the AtPAD4 promoter was very specifically induced by wounding. We performed numerous histochemical studies, including aniline blue, acid fuchsin, toluidine blue, Evans blue as individual and with combined staining, and fluorescence antibody labeling techniques to detect psyllid stylet sheaths. These were performed on cross-sections identified by GUS staining spots generated in response to psyllid-feeding (=wounding). After numerous attempts we were unable to establish this technology as a useful tool to meet our early Liberibacter detection requirements in citrus plants.
This is the end of the second year plus a 6 month NCE for a currently funded multi-investigator, multi-institution project. Although many parts of this research were successful, it cannot be continued in its present form. The USDA group, who was receiving almost 50% of the funding, does not want to continue for a third year. The post-doc who was working on the project has left and it is difficult to get a new post-doc, particularly in Ft. Pierce, when only a year is left on the project. The group has published a paper on their successful efforts (Marutani-Hert, Mizuri, Evens, Terence,McCollum, Gregory, and Niedz, Randall. 2011. Bud emergence and shoot growth from mature citrus nodal stem segments. Plant Cell, Tissue and Organ Culture 106:81-91). The Moore laboratory is also making good progress on the use of cell penetrating peptides to get molecules into citrus without having to use Agrobacterium. In the past 6 months, we have been doing successful experiments with DNA as well as proteins. This is far from a mature technology but shows. promise.
This project sought the development of in vitro regeneration techniques for Murraya paniculata, a presumed host plant citrus relative highly favored by psyllids; these regeneration methods were then to be used to attempt first the genetic transformation of Murraya with marker genes, to optimize the transformation protocol. If successful, then insecticidal or psyllid-suppressive gene construct could be introduced. The ultimate objective was to attempt the development of a deadly trap plant for psyllids that could be deployed in citrus groves to potentially decrease psyllid populations and consequent inoculum potential. Further, such deadly trap plants could be used in the urban landscape to decrease the reservoir of uncontrolled CLas inoculum from commercial or residential areas impacting nearby citrus production areas. We were successful in developing a reasonably efficient regeneration protocol for Murraya via organogenesis, with defined levels of hormone and growth regulator supplementation as well as appropriate plant tissue management and handling techniques; a manuscript on this work is under preparation, the first ever report of in vitro regeneration of this citrus relative. We struggled, however, with the objective of achieving successful genetic transformation. One bottleneck was the unavailability of a reliable source of abundant and viable seed sources necessary to initiate the large-scale experiments that we wanted to conduct. Despite this, we explored various parameters for genetic transformation of Murraya, including assessments of shoot sensitivity to the selection agent kanamycin using untransformed shoots, determinations of bacterial growth curves, and appropriate and effective antibiotic concentrations for bacterial selection. Using the optimized protocol for organogenic shoot regeneration from appropriate seedling tissues, transformation experiments were conducted after testing various plasmids and Agrobacterium strains. Various factors, including a range of OD values (cell density or concentration in liquid culture) of Agrobacterium cultures, the duration of explant incubation in bacterial cultures, duration of co-cultivation period, and the composition of co-cultivation and regeneration media were likewise tested, and we established a standardized transformation protocol. Optimal conditions for transformation using shoot tips and lateral buds, to develop an alternative method using a different tissue source should the organogenic approach prove too difficult or inefficient for transformation, were also explored. Regeneration of buds and some shoots occurred from organogenic cultures of longitudinally cut seedling epicotyl segments, following these transformation experiments. Observations of the regenerating cultures revealed several buds and shoots displaying green fluorescence, indicating successful genetic transformation. Their growth was monitored, as well as the stability and uniformity of GFP expression over time. Nearly all of these transformation events proved to be either chimeric or transient, so further production of new transgenic events was pursued. Though the project has ended, we have shared our results with ctrus transformation experts, and the work is continuing in collaboration now with the Core Citrus Transformation Facility at the UF-CREC, to attempt to further refine and improve our abilities to transform Murraya, and perhaps ultimately to produce, test, and deploy the deadly trap plants we aimed to develop, to test their value and utility as part of integrated approaches to manage HLB disease in Florida citrus.
Funds for this project have now been received. The antibody service provider has provided a quote for anti-NodT antibody production. Antibody production and screening will be initiated soon and is expected to take approximately 3 months.
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