This is the second year of a currently funded multi-investigator, multi-institution project, with the second year end time of 6/30/2011. A total of $224,000 are the current funds allocated to the second year of the project. We are requesting a 6 month no-cost-extension on this grant. There are two major reasons for this. First, there was a delay in dispersing the funds. It took an unusually long time for the funds to flow from agency to UF. Then, there was a further delay at UF establishing subaccounts at UF (CREC) and particularly with USDA, Ft. Pierce. It was October before all of the subaccounts were established. Finally,there is $24,000 in the second year budget for Dr. Machado in Brazil. He was never able to submit the necessary paperwork to receive these funds because of government restrictions. He has a student coming to the Moore lab in July for training and Dr. Machado has asked that the funds be used for her. The second reason for requesting the NCE is that there were a number of personnel changes this year. Because of this, we are requesting permission to adjust what funding is in specific categories and that we can adjust some funding between PIs. Randy Neidz (USDA) has had a post-doc working on this research until recently, and USDA post-docs are costly, so he has been funding much of the supplies used on the project from another source. He has hired a non-PhD person with tissue culture experience to continue the work on the project, but at a lower salary ($25,000 for the NCE). The rest of his funds would be primarily in materials. Jude Grosser (UF) has spent almost all of his current funding on this project. I am requesting that we be permitted to transfer $4,653 from Fred Gmitter’s subaccount to Jude and that I transfer $12,000 from the main account (originally alloted to Dr. Machado) to Dr. Grosser’s subaccount. This will give him adequate money to pay a post-doc and purchase supplies. I will use the other $12,000 originally allocated to Dr. Machado for fees and stipend for his student. I hope this is clear. If I can provide you with any further information or cost breakdown, please let me know.
As proposed, a transgenic test site has been prepared at the USDA/ARS USHRL Picos Farm in Ft. Pierce, where HLB and ACP are widespread. The first trees have been in place for more than fourteen months. Dr. Jude Grosser of UF has provided 300 transgenic citrus plants expressing genes expected to provide HLB/canker resistance, which have been planted in the test site. Dr. Grosser has just planted an additional 89 tress including preinoculated trees of sweet orange on a complex tetraploid rootstock that appeared to confer HLB resistance in an earlier test. USHRL has a permit approved from APHIS to conduct field trials of their transgenic plants at this site, with several hundred transgenic rootstocks in place. Dr. Kim Bowman has planted several hundred rootstock genotypes transformed with the antimicrobial peptide D4E1. An MTA is in place to permit planting of Texas A&M transgenics produced by Erik Mirkov. More than 120 citranges, from a well-characterized mapping population, and other trifoliate hybrids (+ sweet orange standards) have been propagated for a replicated trial in collaboration with Fred Gmitter of UF and are growing well in the greenhouse. These will be planted in July 2011, and monitored for CLas development and HLB symptoms. Data from this trial should provide information on markers and perhaps genes associated with HLB resistance, for use in transgenic and conventional breeding. An experimental attract/kill product, to disrupt citrus leaf miner (CLM) without disrupting ACP, was not effective last year. Our experience suggests CLM may significantly compromise tree growth where insecticides are avoided to permit ready transfer of Las by psyllids. CLM damage also compromises ability to view HLB symptoms. Several applications of Admire are being used to encourage an undamaged flush on transgenic trees. We are still learning how to grow trees for best assessment of HLB-resistance. In June the test site was visited by APHIS Biotechnology Regulatory Services, and we received notice that the site is in compliance with all relevant regulations.
The goal of the proposed research is to understand how Candidatus Liberibacter asiaticus causes Huanglongbing (HLB) disease on citrus. Citrus HLB is the most devastating disease on citrus. There are very few options for management of the disease due to the lack of understanding of the pathogen and citrus interaction. Understanding the citrus and citrus HLB pathogen interaction is needed in order to provide knowledge to develop sustainable and economically viable control measures. We are currently assessing citrus genes modulated by Las infection in 1) Comparison of citrus leaves, stems and roots to Las infection (completed, paper in writing), 2)Comparison of healthy vs. infected leaf samples in citrus grove (microarray data collected and QRT-PCR is underway),3) Comparison of different citrus varieties that are different in tolerance and susceptibility (in progress). The alteration of gene expressions by Las in leaf, stem and root tissues of Valencia sweet orange was investigated using Affymetrix microarray analysis. Out of 30,279 probe sets, a total of 2590 probes had significantly altered expression, out of which 1915 were in leaves, 884 in stems, and 111 in roots. Only 313 probes where shared among the three tissues, out of which 289 were regulated in both leaves and stems, 16 in leaves and roots, 6 in root and stems, while two transcripts were regulated in all the tissues. Further analysis identified genes whose expression is regulated in a tissue-specific manner. Genes involved in carbohydrate metabolism, cell wall biogenesis, lipid metabolism, hormone signaling, secondary metabolism, transportation, amino acid metabolism, pathogenesis, and signaling and transcriptional regulation were predominantly altered in leaves, and a relatively fewer number in stems but least in roots. Within these groups, tissue specific regulation was observed for several genes and gene groups including cell wall pectins, transcriptional factors with MADS box and PHOR1 domains, G-proteins, legume-lectin family proteins, pectatelyase, SUT4, SUC6, BAP12, protein kinase THESEUS1 and vacuolar invertase, which were regulated only in leaves; major intrinsic protein family proteins, crinkly 4-like protein, SUT1, ERF5, CPRD2, CNGC1, CSLD4, and FERONIA in stems; and NAT12, GLR4, DDM1, SCL14, APS kinase, resistance protein RGC2, CCR4-associated factor 1-related protein and arabidopsis response regulator 1 in roots. These differences shade light into the molecular diversity involved in the regulation of tissue response to Ca. Liberibacter infection. In another study, host response of Rangpur lime (Citrus . limonia Osbeck) which shows tolerance to the bacteria, to Ca. L. asiaticus infection, was examined using suppression subtractive hybridization (SSH). Quantitative real-time reverse transcription PCR is being used to confirm the SSH results.
The goal of this proposal is to continue our ongoing research to determine how infection of citrus with Candidatus Liberibacter asiaticus (Las) affects the behavioral response of the Asian citrus psyllid (ACP) to its host plants. Our results to date indicate that HLB-infected citrus plants are more attractive to ACP adults than uninfected plants initially but psyllids subsequently disperse to make uninfected plants their final settling choice. The chemical profile of infected and uninfected plants indicated that infection with the Las pathogen significant changes the volatile release profile of citrus trees as compared with uninfected plants. Specifically, HLB-infected plants release less methyl anthranilate (MA) but more methyl salicylate (MeSA) than uninfected control plants. Similarly, infected plants release lower quantities of D-limonene than uninfected plants. The current objectives of this project are to determine which chemicals mediate initial attraction by psyllids to infected plants. We also working to determine why psyllids leave infected plants after initial attraction to make uninfected plants their final settling choice. Our goal is to determine if these chemicals can be exploited for ACP management. Based on head space volatiles of HLB-infected and uninfected plants, we have obtained synthetic versions of the majority of the chemicals that appear to mediate psyllid behavior in response to uninfected and HLB-infected citrus plants. Psyllid behavioral bioassays with these chemicals are in progress. The second aspect of this research has been to determine how HLB infection affects the nutritional status of host and plants and correlate these changes with possible effects on psyllid behavior. For this objective, we have been cultivating plants with various nutrient deficiencies. These plants are almost ready for behavioral experiments with psyllids. They will be used to determine the role of plant nutritional status on psyllid behavior.
Candidatus Liberibacter asiaticus (Las) is a fastidious phloem-inhabiting gram-negative bacterium transmitted by Asian citrus psyllid (ACP). Transmission mechanisms of Las have been intensely investigated recently, yet key information gaps still exist. In this research, we investigated whether Las is transmitted between infected and uninfected psyllid adults during courtship. Forced mating between Las-infected males and uninfected females showed that Las is sexually transmitted from Las-infected male psyllids to uninfected females in low proportions but not from infected females to uninfected males or among psyllids of the same sex. Las was detected in the ovaries and eggs of infected females. A latent period of 7 days or more was required to detect the bacterium in the recipient females. Given the evidence for sexual transmission of Las during courtship, we investigated whether the bacterium was present in the reproductive organs of ACP. Long rod shaped as well as spherical structures resembling Las were observed in female ovaries using a transmission electron microscope but were not observed in the ovaries of uninfected ACP females. The size of rod shaped structures varied from 0.39-0.67 ‘m long and 0.19 to 0.39 ‘m wide. The spherical structures measured from 0.61 to 0.80 ‘m in diameter. This investigation proved that Las is sexually transmitted by ACP during courtship and established evidence that the bacteria colonize reproductive organs. Moreover, these findings provide an alternative explanation for Las spread among psyllids even in absence of infected host trees.
In this third quarter, 208 plants of Valencia orange on Kuharske Carrizo rootstocks were inoculated with HLB on the 3rd week of April of 2011. The HLB inoculation was done using a standard inverted ‘T’ budding technique as describe by Ferguson (2007), two HLB infected buds per tree were used. Infected and non-infected HLB trees were kept in the greenhouse under natural light conditions at 17-25’C. The HLB infected buds were obtained from greenhouse-grown trees that were inoculated in March 2008 with HLB infected buds from field trees in a commercial citrus grove in Lake Placid, Florida. This source was tested in 2008 and found to be CTV-free, thus there are no interacting factors between HLB and CTV. Four to six mature leaves per tree were harvest randomly from the greenhouse-grown inoculum source trees to verify HLB infection by qRT-PCR prior to inoculation. qRT- PCR analysis for DNA extracts was done following the standard method for HLB detection (Li, et al., 2006), four primer and one Taqman probe empirically designed based on 16S sequences of Ca Liberibacter species were used. The reverse primer (HLBr) used is specific to the genus Liberibacter and recognize all three species (Las, Lam, Laf) in the genus. The reverse primer (HLBr) was used in combination with the primers HLBas, HLBaf and HLBam specific to Las, Laf and Lam, respectively HLB infection for the HLB inoculated plants will be verified by qRT-PCR, starting on July 11, 2012 and continuing sampling and looking for HLB infection monthly. After HLB infection is detected, we will apply therapeutic compounds to HLB and healthy plants that will enhance the Systematic Acquired Resistance (SAR) response and counteract ethylene in early infected tissues, study the regulation of glucose transport on HLB infected and non infected plants and performed a bioassay using sucrose + atrazine at two different rates. Leaves form infected and non-infected trees will be also sampled for RNA extraction to study gene expression patterns on HLB early infected plants. Literature cited: Ferguson J. 2007. Your Florida dooryard citrus guide. Horticultural Sciences Department, Florida Cooperative Extension Service, Institute of Food and Agricultural Sciences, University of Florida. Document HS 884. Li, W., Hartung J.S. Levy L. 2006. Quantitative real time PCR for detection and identification of Candidatus Liberobacter species associated with citrus huanglongbing. J. Microbiol. Methods 66: 104-115.
We have transferred the initial indexed mature material obtained from Dr. Peggy Sieburth’s lab to the growth room. As we mentioned in previous reports the material was not in excellent condition in vitro because of excess storage time and only 20 % of the material prepared during the last quarter of 2010 survived the in vitro conditions. Usually these in vitro grafted plants are grafted in rootstocks on greenhouse conditions, but since the rootstocks were not ready because they were growing in lab conditions, they were transferred to soil directly. The rootstocks produced in lab conditions were small, and they were not actively growing and they are still recovering from the stress suffered in the laboratory and only a small portion of them will be used for grafting. We started preparing new source of material from the same 3 orange types: Valencia SPB 1-14-10, Hamlin 1-4-1 and Pineapple F-60-3. The mature budsticks are coming from indexed plants from the Department of Agriculture. New rootstocks from Swingle citrumelo and C. macrophylla were planted once the growth room was ready and they are currently growing. We will need a few more months to use this material. The Growth Room was finished and we passed the local inspection, however we operated for many weeks without being able to control the growth room due to lack of computer access to the program, the greenhouse technician does not have still access to the program. There were also several inconsistencies among the different companies involved in the construction that were solved during this post construction period to be able to operate the growth room. We are still dealing with some changes in the humidifier in the small room and finalizing the drainage of water from the growth room to the outside field. Fortunately the facility is under warranty for one year and the contractors are addressing most of the problems. We addressed the need of an emergency stand by generator for the facility as well as some improvements that were not in the construction plan, that were requested initially. These improvements are still necessary to make production in the growth room reliable. We experienced already a few electricity failures in the facility due to lightening and thunderstorms that affect the electrical service, and it took several hours to fix the situation on site and put the growth room back up and running again. The temperature increases considerably during the time of the electrical outage and there was no “clean water available” since the UV light system will not work under these conditions. It will compromise the whole process if we have more than 2 days with no water and electricity.
In previous informs we have already mentioned that mature tissues from the three sweet orange genotypes we pretended to transform through this project, namely Hamlin, Valencia and Pineapple, are readily transformable, as demonstrated by positive results from molecular analyses of marker transgenes incorporated into plants established in the greenhouse from the three sweet orange types. The procedures have been transferred in detail to our lab at the CREC. We are not working any more with Hamlin at our IVIA’s lab, but Valencia and Pineapple are being routinely transformed with several transgenes of interest not related to this project, meaning that, at least for these two sweet orange types, we can efficiently insert other transgenes able to modify plant phenotype and likely provide new improvement traits. For Carrizo citrange, we first established a procedure for transformation of mature tissues and then used the system to incorporate a hairpin construct aimed to induce RNA interference to silence and endogenous GA20-oxidase gene and them reducing gibberellin biosynthesis into actively growing tissues. These transgenic plants would be semidwarf and could be used as rootstocks to provide semi-dwarfing characteristics to non-transgenic scions grafted into them. A field trial assay initiated 5 years ago in Moncada (Valencia, Spain) indicates that GA20-oxidase antisense lines provide semi-dwarfing architecture (a one-third reduction in height) to mandarin scions. In theory, RNAi-inducing constructs should work better than antisense ones. We already have PCR-positive shoots for the hairpin construct in the growth room. We have initiated experiments to attempt transformation of mature tissues from Swingle citrumelo and Star Ruby grapefruit. We are also preparing new source material of Ray Ruby grapefruit to initiate transformation experiments by the end of the year. These objectives were not contemplated in the original project. For improving citrus tree management, we proposed to over-express flowering-time genes in both the Carrizo citrange rootstock and the Pineapple sweet orange scion. We have now at least ten independent transgenic lines of Pineapple sweet orange and Carrizo citrange over-expressing either FT or AP1 flowering-time genes already established in the greenhouse. We continue characterizing them in detail. In Florida, construction of the growth room has been finalized, but several important issues need to be set up to make production of healthy source plant material reliable. We are helping the manager and her team to establish a calendar for production of rootstock, clean scions, mother plants, propagations, etc. to get maximum occupation of the available space in the growth room and being able to make the maximum number of mature transformation experiments per year. We are also helping to establish substrate, fertirrigation and phytosanitary treatments.
The main objective of this project is to provide comprehensive 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. In order to achieve this goal, 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. For the funding period since March 2011 we received nucleic acid extracts of 20 California isolates of CTV that we selected for our study. Those isolates have been earlier collected from main citrus growing regions and California and then propagated in CTV isolates collections (CCTEA, CCPP). Previously some biological characterization has been done for those isolates. Most of the isolates are severe stem-pitters, however two or three isolates among those 20 appear to be mild. Thus, these isolates may potentially represent good candidates as protecting isolates. We conducted examination of the genotype (strain) composition of those isolates using conventional reverse transcription PCR and quantitative real-time reverse transcription PCR analysis with genotype-specific primers. Most of the isolates have a mixture of the T30 plus VT genotypes. Few isolates have T3 or T36 genotypes. The mild isolates appear to contain VT genotype as well. Thus, the latter isolates could potentially provide protection against aggressive VT isolates. Dr. Folimonova is planning a trip to California to examine a possibility of setting up in-greenhouse cross-protection trials using those mild isolates to examine their ability to protect against stem pitting isolates containing a similar genotype composition. We are also planning to conduct additional bio-indexing experiments to repeat evaluation of those mild isolates to eliminate any possibility that they might demonstrate more severe phenotype in some particular citrus varieties. In order to obtain an understanding what CTV genotypes are currently present in the southern part of California where no previous eradication occurred, we are planning to conduct additional sampling of the new material from this region (Vidalakis lab). Those samples will be then used for inoculation of greenhouse trees for bioindexing characterization (Vidalakis lab) as well as sent (as nucleic acid extracts) to Folimonova lab for further molecular characterization.
This is a 4-year project with 2 main objectives: (1) Over-express the Arabidopsis MAP kinase kinase 7 (AtMKK7) gene in citrus to increase disease resistance (Transgenic approach). (2) Select for citrus mutants with increased disease resistance (Non-transgenic approach). For objective 1, 20 transgenic Duncan grapefruit plants have been generated, and these plants have been growing in greenhouse for 6 months and ready for canker resistance test. Since canker resistance test is straightforward, we will test all 20 transgenic plants. However, for greening resistance test, the transgenic plants will need to be propagated. We will first extract total RNA from each of the 20 plants and determine the expression levels of AtMKK7. We will choose 4 to 6 lines that highly express the transgene AtMKK7 for propagation. Six plants from each line will be used for greening resistance test. For objective 2, we have been using hypocotyls as explants for gamma irradiation mutagenesis. A total of about 75,000 hypocotyl cuttings have been irradiated in three batches with a irradiation dosage of 40 Gy. Shoots formed on the irradiated cuttings were transferred onto selective medium containing 0.2 mM of sodium iodoacetate. Several shoots are currently growing on the selective medium. To increase the screening efficiency, we have also been using seeds for this objective. A large quantity of Ray Ruby grapefruit was obtained in the Spring of 2011 from the Indian River area. Seeds were obtained from the fruits and cleaned with Pectinase. Seeds were then treated with 8-hydroxyquinoline as a preservative to allow long-term storage of seeds at 4’C. Moisture content of the seeds was determined for future reference. Two quarts of seeds have been treated with gamma irradiation. One quart was irradiated at 50 Gy, the other at 100 Gy. Both untreated and irradiated seeds were plated into large glass Petri dishes as well as Magenta boxes containing water agar. Shoots have been formed on the seeds and will be transferred onto selective medium containing 0.2 mM of sodium iodoacetate. Based on the result of this batch of seeds, we will treat other seeds with either the same condition or a modified dosage. Shoots formed on these gamma irradiated seeds will be screened on the selective medium. Those shoots that are resistant to sodium iodoacetate will be grafted onto rootstocks to generate plants for resistance test.
1. mtCOI haplotyping. Additional samples are gradually received for mtCOI haplotyping. Samples from a center of diversity in Asia and elsewhere are being collected to explore baseline diversities in different locations, in an attempt to relate populations from the US and elsewhere in the Western Hemisphere to haplotypes from a primary region of endemism. 2. Time-course and transmission studies are underway using bioassay to assess transmission frequency in relation to qPCR and/or dot blot hybridization detection of Ca. Liberibacter solanacearum (AZ) and in Ca. L. asiaticus (FL) in individual psyllids reared on infected plant material. This will be followed by acquisition access feeds over time course in relation to qPCR and SEM and TEM correlations. Single psyllids reared on infected tomato and given a range of inoculation access feeds on tomato from 30 min, and 1,2, 4hr-transmitted Ca. L. solanacearum 20, 35, 30, and 75% of the time (20 plants per rep). When five psyllids were used instead transmission frequency was 5, 35, 25, and 70%, respectively. Studies suggest that once acquired psyllids transmit at relatively high frequency (70%); tests are underway for potato psyllid at 8,12,and 24 hrs. The goal is to select individuals for light microscopy, and SEM-TEM ultrastructural observations to pin point localization following a range of different AAP and IAPs. IAP studies are underway for Ca. L. asiaticus using seedlings for bioassay, together with qPCR and/or dot blot monitoring, all difficult undertakings owing to the apparent uneven distribution of bacteria in plants, which confounds reliable detection and transmission bioassay to further localization efforts. Extrapolations from the more tractable potato psyllid study system are expected to help pinpoint key AAP and IAP time-points for localization studies. 3. Transmission and scanning electron microscope studies focused on large monocultures [interpreted as thick biofilms] of rod-shaped, fastidious bacteria that are consistently associated with the alimentary canal, from the oral box (including salivary glands) to the posterior midgut, of infected but not uninfected psyllids, and most consistently observed in 3rd instar and older psyllids. In addition, studies have revealed the dynamic behavior in the positions that the intestine assumes within the abdomen of live psyllids. As with the causal, bacterial agent for Pierce’s disease, attempts to reliably implement FISH for determining the time-course proliferation and gross anatomical affinities of the target pathogen within the psyllid vector body remain tentative. We are continuing to try to optimize FISH for light microscopy, and are implementing colloidal gold – DNA hybridization for ultrastructural studies. 100nm thick, plastic Z-section libraries of the oral box have been made from infected and non-infected potato psyllids to map the tissue/organ organization where bacteria have been observed to reside. The net accomplishments of this objective are visualization of Ca. Liberibacter in identified anatomical structures, and constructing a model for the mode and pathway of transmission.
Diagnostic service for growers for detection of Huanglongbing to aid in management decisions, June 2011. As we did in previous reports, this update covers the entire period that Huanglongbing Diagnostic Laboratory has been in service because one of the objectives for the funding for continued, uninterrupted diagnostic services to growers while expanding our ability to provide diagnostics quickly and assist with research efforts. The HLB Diagnostic Laboratory has been operational at UF-IFAS-SWFREC since February 2008. Since the opening of the lab, there has been continued development of techniques, protocols and efficiency. The lab has been in operation for fifteen months, and as the first week of June, 2011, we have processed more than 25,700 grower samples, with nearly 1200 of that number arriving since March 2011. Additionally, approximately 15,000 total samples have been received for research, of which more than 2100 were within that same time frame. Additionally, approximately 15,000 total samples have been received for research, which was approximately twice the number processed in 2100 within that same time frame. Techniques, Protocols and Research For DNA extractions, we continue to use the magnetic particle based system, which has proved both reliable and fast. Current methods of sample processing have become streamlined and therefore seen little change. We have recently introduced the use of TaqMan Fast Advanced MasterMix for real-time-PCR reactions as this is more economical and has shown comparable-to-superior amplification and detection of gene(s)-of-interest when compared to the TaqMan Fast Universal PCR MasterMix. Protocol for the detection of HLB in Asian Citrus Psyllid has been validated, including quantification of HLB in both plant and psyllid samples, with the primary goal of serving research projects within the entomology and plant pathology department that also contribute funds from their reserach grants to support the labor and supply costs for research samples. The protocol that was established in 2010 was the quantification of the HLB bacteria in both the psyllid and host tissue using a standardize curve. The basic service remains the available to growers, researchers, extension faculty and dooryard citrus growers. However, we are also expanding the data analysis of PCR processed data from individual groves that consented to have their data used. In conjuction with an epidemiologists and computer mathematician, the spread of the disease will be modeled. This studies are not supported by lab funds but are an offshoot of the database collection. The intent is to have additional tools for looking at the spread of HLB in sites where incidence is still relatively low.
Diagnostic service for growers for detection of Huanglongbing to aid in management decisions, June 2011. As we did in previous reports, this update covers the entire period that Huanglongbing Diagnostic Laboratory has been in service because one of the objectives for the funding for continued, uninterrupted diagnostic services to growers while expanding our ability to provide diagnostics quickly and assist with research efforts. The HLB Diagnostic Laboratory has been operational at UF-IFAS-SWFREC since February 2008. Since the opening of the lab, there has been continued development of techniques, protocols and efficiency. The lab has been in operation for fifteen months, and as the first week of June, 2011, we have processed more than 25,700 grower samples, with nearly 1200 of that number arriving since March 2011. Additionally, approximately 15,000 total samples have been received for research, of which more than 2100 were within that same time frame. Additionally, approximately 15,000 total samples have been received for research, which was approximately twice the number processed in 2100 within that same time frame. Techniques, Protocols and Research For DNA extractions, we continue to use the magnetic particle based system, which has proved both reliable and fast. Current methods of sample processing have become streamlined and therefore seen little change. We have recently introduced the use of TaqMan Fast Advanced MasterMix for real-time-PCR reactions as this is more economical and has shown comparable-to-superior amplification and detection of gene(s)-of-interest when compared to the TaqMan Fast Universal PCR MasterMix. Protocol for the detection of HLB in Asian Citrus Psyllid has been validated, including quantification of HLB in both plant and psyllid samples, with the primary goal of serving research projects within the entomology and plant pathology department that also contribute funds from their reserach grants to support the labor and supply costs for research samples. The protocol that was established in 2010 was the quantification of the HLB bacteria in both the psyllid and host tissue using a standardize curve. The basic service remains the available to growers, researchers, extension faculty and dooryard citrus growers. However, we are also expanding the data analysis of PCR processed data from individual groves that consented to have their data used. In conjuction with an epidemiologists and computer mathematician, the spread of the disease will be modeled. This studies are not supported by lab funds but are an offshoot of the database collection. The intent is to have additional tools for looking at the spread of HLB in sites where incidence is still relatively low.
The Core Citrus Transformation Facility (CCTF) continues to serve the community of researchers exploring ways to improve Citrus plants and make them tolerant/resistant to diseases. CCTF does its service by producing transgenic material. Within the last quarter, the CCTF facility produced the following transgenic citrus plants (transgene in parenthesis): three Mexican lime plants (pHK vector); nine Duncan plants (ELP3 gene); one Duncan plant (MKK7 gene); four Duncan plants (p7 gene); nine Duncan plants (p10 gene); one Mexican lime and two Hamlin plants (p33 gene); six Duncan plants (SUC-CitNPR1 gene); two Duncan plants (pWG19-5 vector); two Duncan plants (pWG20-7 vector); 11 Duncan plants (pWG21-1 vector); seven Duncan plants (pWG22-1 vector); two Duncan plants (pWG24-13 vector); and three Duncan plants (pWG25-13 vector). There are additional 30 plants in soil that need to be tested for the presence of the transgene of interest. Within last three months, the CCTF facility also sustained a loss of 30 soil-adapted transgenic plants due to unknown contamination coming from the rootstock plants (probably Phytophthora). Steps have been taken to prevent this from happening again in the future, including improved greenhouse sanitation and use of more resistant rootstocks in micrografting.
-RNA isolation/treatments: A Trizol/RNeasy hybrid RNA extraction protocol was adapted for RNA isolation from the tissues of ACP and the potato psyllid. 1 ug total RNA or more was isolated for 454 or Illumina sequencing library construction: 40-50 adults; 60-70 larvae; 90-100 guts; 200-250 salivary glands. For ACP two 454 Titanium libraries were constructed (uninfected/infected adults); six Illumina Paired-end libraries (uninfected guts, uninfected adults, uninfected larvae, infected guts, infected adults, and infected larvae) were made for Illumina sequencing. The 454 uninfected gut library generated 69,066,955 bps and 236,095 clean reads (296 bps av. read length) and assembled 429,995 ESTs. For the 454 AdtLib 124,458,137 bps and 433,870 clean reads were generated (286 bp aver length), and assembled 233,891 ESTs. To improve coverage the initial 454 runs (429,995 ESTs uninfected guts/ 233,891 ESTs infected adults), we constructed two Illumina libraries and obtained ~65M sequences. Taken together with ~100M sequences from the other 4 libraries we have a deep coverage of the ACP whole transcriptome. 36 ACP genes were selected as potential targets for RNAi construction, full length cloning, expression profiling, and function identification. For potato psyllid, we constructed 6 Illumina paired end libraries from 6 potato psyllid samples (uninfected; infected guts, uninfected; infected adults, uninfected;infected larvae). Libraries were sequenced using an Illumina PE 2×54 sequencing format, yielding >24M sequences each, and a total set of ~160M sequences. -Data management/Annotation: redesigned the PAVE web interface to simplify it. Second have updated the annotation of the Sanger EST and 454 assemblies with the latest taxonomic UniProt databases (annoDBs). The top 25 hits are stored and displayed for each annoDB. The new interface makes it easy to filter on the annoDB and species, e.g. show all the unitrans with hits from SwissProt Virus, or show all unitrans hits. The interface provides a more versatile way to query the expression level data; (1) it shows the data as absolute, normalized or percentage, (2) it allows the user to view up-regulated or down-regulated unitrans by fold change, difference in percentage or difference in absolute value. Query results can be viewed by unitrans, grouped by annoDB IDs, or grouped by GO IDs. The new annotation and interface will be posted on the www.sohomoptera.org site by July, 2011. -Data mining for ACP has been initiated and is underway for annotated transcriptomes (phase I) of the whole adult and gut ACP to identify candidate genes for functional analysis, with an emphasis on gut and bacterial targets for siRNA-mediated silencing. Primers for PCR and RT-PCR have been designed for 36 candidate psyllid gut genes with potential involvement in pathogenesis (iron metabolism, transport, endocytosis). Presently 28 candidate genes have been amplified by PCR and RT-PCR, cloned (partial or coding region), and sequenced, thereby, demonstrating, the quality of EST sequences, and that these gene targets are expressed mRNA in the Asian citrus psyllid. Additional targets are in process. -FISH probes were designed for Liberibacter FISH localization: 16S rRNA for Ca. Liberibacter solanacearum and asiaticus, 16S rRNA for Carsonella ruddii (primary endosymbiont, potato psyllid) as an internal control, and three other specific probes for in situ localization in potato psyllid. Studies are underway to validate the FISH protocol.
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