Objective 1: Transform citrus with constitutively active resistant proteins (R proteins) that will only be expressed in phloem cells. The rationale is that by constitutive expression of an R protein, the plant innate immunity response will be at a high state of alert and will be able to mount a robust defense against infection by phloem pathogens. Overexpression of R proteins often results in lethality or in severe stunting of growth. By restricting expression to phloem cells we hope to limit the negative impact on growth and development. Results: We sequenced all of the constructs introduced into Arabidopsis which consisted of the AtSUC2 promoter (940 upstream from ATG) driving expression of the mutated (constitutive) and wild type forms of SSI4 and SNC1 (R proteins). For SSI4, which is derived from the Nossen cultivar of arabidopsis, two closely related genes (MUF8.3 and 8.2) are present in the Columbia cultivar. We cloned both, and the original from the Nossen cultivar, and cloned them behind the AtSUC2 promoter. These wild type versions of SSI4 will be used as controls for non-active forms of the R protein (pathogen activated). The four AtSUC2/R protein constructs (mutant and wild type of each of the two R proteins) were transferred from the pCAMBIA1305.1 vector, which confers hygromycin resistance, into pCAMBIA2301 with kanamycin resistance since the former is detrimental to transformation into citrus. The four constructs were submitted to the UF Citrus Research Facility at Lake Alfred for transformation into citrus. Transformation of these constructs into the Duncan variety of grapefruit is currently in progress at the Lake Alfred Citrus Research and Education Center (Dr. Vladimir Orbovic). Conclusions: Our hypothesis was that phloem-restricted expression of the R protein constitutive mutants would limit potential negative impacts on growth. The Arabidopsis transgenic plants expressing R protein mutants did not seem to be significantly affected in the majority of cases. Approximately 8% of the snc1 transgenics exhibited a stunted phenotype, very similar to the snc1 mutant expressed from its native promoter (not phloem specific). The first series of ssi4 transgenics (construct 5-2) had a point mutation (C>T) in the coding region that generated a premature stop codon and shortened the protein by 78 aa in the C-terminal region, past the leucine rich repeat (LLR). Five percent of these truncated ssi4 transgenics showed phenotypic differences mostly in the rosette appearance and lighter green, splotchy coloring. However, this effect will be investigated again in the new full-length ssi4 transgenic plants.
This report covers the period April 1 through June 30, 2010. This project was funded July 1, 2009. A no-cost extension of this project was applied for in June 2010 and was granted during this reporting period. Two coordinating meetings were held during this period. The field personnel, in particular, closely coordinated their activities with the personnel analyzing data. Coordination with the aerial application program through Southeastern Air and the Indian River Citrus league continued as did the mapping of data for subsequent time and space analyses of Asian citrus psyllid populations. Due to the massive amount of data being collected in the field, four Garmin GPS units and five laptop computers were purchased during this reporting period. The Garmin units were delivered on June 25, 2010.. This program increased its monitoring activities by increasing the number of tap samples. There were 730 tap sample sites: Indian River 265, St. Lucie 315, and Martin 150. 1. 4040 traps were set and retrieved during this reporting period: Indian River 1,325, St. Lucie 1,575 and Martin 750. 2. Trapping locations by county: Indian River 205; St. Lucie, 225; Martin 75. 3. There were 9,845 psyllids caught in traps duirng this reporting period: Indian River 5,433, St. Lucie3,672 and Martin 740. Over 12,000 miles were driven by field personnel during this period. The data have been analyzed by time and space by Drs. Hall and Gottwald(USDA) and hot spots identified. In our previous report we stated that the data analyses from the first aerial application were inconclusive. The data from the second application in Feb-Mar 2010 also led to an inconclusive conclusion. The second application was made over a period of one month and the lengthy application period appears to be the reason for this result. The one month application period was the result of adverse weather and interference with harvesting.
1- The physical construction/renovation of the growth room did not start yet. A draft of the final layout of the lamps and benches was presented. Several scenarios about air filtration and distribution were discussed as well as safety, security and the irrigation system. Regarding irrigation, it seems like there are concerns about the amount of run-off water leaving the growth room. A holding tank was proposed as a way to contain the water that is coming out of the facility, however the existence of a retention pond south of the building might be the best solution. No decision has been reached at this point regarding the disposal of the water. We make clear to everyone that we need hose bibs in the growth room and there will approximately 2000 gallons/week of run off water coming from the growth room, this is inclusive of watering the plants and maintaining cleanliness. 2 – The first material to establish mother plants from Hamlin 1-4-1 was released from Dr. Peggy Sieburth lab. The clean shoot tips are approximately 4 weeks old and they will need to be grafted in approximately 4 more weeks on rootstocks that are currently growing inside the lab. These rootstocks are being maintained inside the lab for 6 months under laboratory conditions and they needed to be transferred to bigger pots 2 months ago. Under these conditions the grafting will be delay until the growth room is available. Since these rootstocks are already suffering, a new batch was started as a backup. We hope that the shoot tips can hold a few months more until the grafting can be performed. We will need to transfer them several times which in normal conditions is not required. Maintaining shoot tips for a long time on in vitro conditions might also induce juvenility, which we will want to avoid. 3 – Construction of the growth room will start on October 25th according to information provided by the CREC maintenance supervisor. 4 – A grower was selected and he will be at CREC in a few weeks.
In this quarter, the research was focused on the efficacy of the screened chemical compounds against citrus HLB in the field. A completely randomized design with three replicates was conducted at the USHRL farm in Fort Pierce. Seventy-two HLB-affected citrus trees were treated with PS (penicillin and streptomycin) by trunk-injection. Water was used as a control. The applications were repeated once after two months, and samples were taken for PCR assay. Potential phytotoxic effects of PS were also determined in the field. The primary results showed PS may eliminate the Las-bacteria in the treated citrus. When HLB-affected citrus were injected in the field with different dosages of PS, the resulting Ct values increased from 25.7 at pre-treatment up to 34.2 at 2 months after treatment with PS-5 (5.0 g penicillin+ 0.5g streptomycin in 100 ml water solution), which is an approximate 500-fold decrease in the Las bacterial population in the treated citrus plants. At 4 months after the second treatments, the Ct value was ‘ 40.0 (undetectable) indicating elimination of the Las bacterial population in the treated plants. Similar results were found with PS-10 (10.0 g penicillin+ 1.0 g streptomycin in 100 ml water solution). However, phytotoxic effects on citrus were found with PS-10. Penicillin residues in the fruit from the treated citrus were positive (more than 5 ppb) at one week after treatment, but negative (less than 5 ppb) after one month. A grafting system using HLB-affected citrus scions was optimized and used to screen the chemical compounds. HLB-affected citrus (Lime) cuttings (three to four buds) were collected and soaked overnight in solutions of MDL, PS, Kasu, AG, DBNPA, Chaptin, Oxy and KO, respectively, and grafted into Las-free grapefruit. Scions without any treatment were used as controls. A randomized block design was conducted with three replicates and 45 scions per treatment. New growth from the scions was observed in 1 to 2 weeks. Leaf samples were collected for PCR assay 3 month post-grafting. The results suggested that the scion treatment system may be used for screening anti-Las bacteria compounds to control citrus HLB. HLB-affected trees characteristically have a damaged root system and nutritional deficiencies because of interrupted transport of photosynthetic products from shoots to the roots and mineral nutrients from roots to the shoots. Greenhouse and field experiments were conducted to develop an integrated approach for keeping groves with HLB productive by means of disease-curing antibiotics combined with nutritional priming and root restoration. Key nutrients, plant growth regulators, and transporters were screened and several chemicals, called growth-priming substances, have been identified to be useful in keeping the HLB-affected citrus growing and promoting root restoration. Soil application once at two-week intervals with the growth-priming substances in combination with antibiotics was found to reduce the accumulation of starch in HLB-affected citrus leaves and enhance the emergence of new flushes from the severely diseased young trees. These growth-priming substance and antibiotic combinations are being evaluated in field trials.
Obj. 1,2. Construct cDNA libraries from (a) adult/immature psyllids, dissected gut, salivary glands and accessory salivary glands; Sequence random cDNA clones, assemble ESTs, and select unigene sets; Obj.3. Produce RNA-seq libraries and compare transcript levels in psyllids and digestive organs at key acquisition access periods (AAP) to determine the relative abundance of insect mRNAs affected by bacterial infection. We have received from NCGR the Illumina assembled sequences from the Diaphorina citri psyllid libraries: (1) adult gut liberi-infected, (2) immature whole body liberi-infected, (3) adult whole body, and (4) immature whole body. Each library resulted in over 24M sequences and the total set of ~100M sequences assembled into 121444 contigs. The distribution of lengths is very similar to that of our 454 assembled sequences. We have also downloaded the Hunter labs’ Sanger sequences from GenBank, which will be assembled with the 454 and Illumina consensus sequences for further analysis. The website software has been enhanced based on feedback from Dr. Brown. A web domain has been purchased for this project, where the results will be made public once analysis is complete. The first four libraries (above) have been assembled, organized in PAVE, and predictive annotation is nearly complete with links to the GenBank database. Data-mining, qPCR analysis of transcripts, FISH probe design and optimization using our epifluorescence microscope equipped with a filter for Cy5-labeled probes will commence Oct 1 with the hiring of a post-doc. The last two of six adult D. citri (from our P. Stansly/Roberts collaborators) EST libraries have been constructed from, and are undergoing preparation for, Illumina sequencing to improve coverage over initial 454 runs. Space and energy, devoted to rearing potato psyllids, have been ramped up to facilitate transmission studies, dissections, FISH, and SEM/TEM. Use of potato psyllids has accelerated this study because it is a far more tractable system. With it, we can dissect a detailed time-course study to produce data points for extrapolation to the more difficult-to-study D. citri-asiaticus system. Toward B. cockerelli library preparation, two of three collections of whole adults and immatures (all stages) have been made, as well as 1000 dissected, liberi-infected, adults guts. 1000 liberi-uninfected guts will be in hand in less than two weeks. RNA isolation and cDNA synthesis will proceed for these libraries. Transmission studies to determine % efficiency transmission by single B. cockerelli psyllids to tomato plants have been initiated using liberi-infected and -uninfected colonies and a range of AAPs. Two replicated experiments have been completed and a third is underway (0-24 hrs). Next, the more time consuming studies using Liberi-uninfected psyllids will be initiated to determine AAPs and IAPs over time-course, and to be correlated with FISH, qPCR, and TEM (project-34),and quantitative gene expression analysis (RNA-seq). Key time points will be identified and results investigated by RNA-seq analysis and/or qPCR to validate transcript involvement for the D. citri-Liberibacter system.
Obj. 1.DNA bar coding to establish the identity and diversity in south Florida (Stansly, Brown). new mtCOI primers were designed based on amplicons obtained using general homopteran primers, resulting in increased specificity. PCR primers were used to obtain a ~1200 bp product; sequence analysis is underway. Obj. 2. qPCR detection of Ca. Liberibacter presence (or absence) in the psyllid colony cohorts over different IAPs has been optimized and analysis is underway for the first two replicates. Obj 3. Define the gross association of Ca. Liberibacter in thick sections’develop a ‘gross anatomical road map’ of Ca. Liberibacter accumulation in key organs, tissues, and cells; Obj. 4 Using the resultant ‘thick section road map’, elucidate at the TEM level the specific organs, tissues and cells where Ca. Liberibacter accumulates in the vector. We have adopted the potato psyllid, Bactericera cockerelli, as a surrogate for the citrus psyllid because we can rear it on tomato easily, and set experimental parameters (‘acquisition access’, ‘inoculation access’ etc.) that have the tightest possible definition with regard to timespan of actual feeding and source infectivity at the locus of the stylet. Our rearing system is ramped up to broad, robust capacity, providing for multiple studies by several experimenters in the transmission context simultaneously. Guts of infective and uninfective specimens of all life cycle stages (1st thru 5th instars, metamorphosing adult, unpigmented emerging adult (‘tenerals’), and mature adult) have been processed, mounted on stubs, and are being studied under the scanning electron microscope (SEM). Graphic differences occur that correlate directly to host-vector relationships at the histological level. These comparisons assist us with interpretation of cross-sections, and demonstrate the abundance of bacteria in infected adults relative to indigenous flora, as well as the buildup of bacteria during each consecutive stage. The pre-oral organ complex, referred to as the ‘oral box’, is the 0.1mm3 chamber that houses the convergence of the salivary ducts and esophagus, and the transfer of their fluids to the stylets, which in turn, penetrate the plant for feeding. The potential for bacteria to access the stylets, through this box, during egestion events, is very high and cannot be overlooked. Along these lines, it is known that in Psyllidae, the unused length of the larval stylets is externally wound into a groove around the face, but the unused length of the adult stylets is held inside an internal sac (Pollard 1970, Ullman and McLean 1986). Our studies of the oral box have shown that the internalization process retains the cuticular barrier between stylets and blood. The basal sections inside the oral box, however, may undergo modification during metamorphosis that can admit bacteria, and must be given scrutiny as well. 10-15 infective and uninfective adults + source plant material were collocated to the same paraffin block face in four different runs. 4-micron thick sections were cut from these blocks and mounted in alternation on slides and stubs for, respectively, the light microscope and the SEM. Slides were treated with ‘in-situ hybridization’ techniques that attach a visible gold/silver label to bacteria, allowing them to be localized inside organs, and directly compared to sections magnified under the SEM. These methods give consistent label in the gut, as well as a more general distribution in the abdomen. Strong signal also occurs in the spaces between salivary gland cells and inside the oral box. These localizations are best pursued further under the transmission electron microscope (TEM). Strong signal occurs in new apical growth (‘flush’) of infected source plants, but in sharply delimited patches, or ‘compartments’.
Samples for phloem plugging determination were collected from HLB infected mature field grown trees and from young greenhouse grown trees. A total of 29 trees and 4 cultivars were sampled; 22 trees were field grown. Callose plugs were 2.7 and 2.4 times more prevalent than phloem protein 2 (PP2) plugs in the field and greenhouse samples, respectively. Three Murcott field trees has a ratio of 13 callose plugs per each PP2 plug. For grapefruit, Valencia and Hamlin orange samples, the ratio range was 1.8 to 4.2 callose/PP2 plug. This data suggests that blocking callose production may be more productive than blocking PP2 production. Transformation experiments were initiated in efforts to produce plants that over-express the citrus ‘-1,3-glucanase gene, both with the constitutive 35S promoter (p35S ‘ BG-35T) and the Suc 2 phloem specific promoter (pSuc2-BG-35T). Using a modified Agrobacterium-mediated transformation protocol, 26 transgenic sweet orange shoots were regenerated containing p35S ‘ BG-35T (Valencia and Vernia sweet oranges) and 24 containing pSuc2-BG-35T (Valencia and Vernia). All have been micro-grafted to Carrizo citrange to expedite their greenhouse evaluation. Twenty transgenic Carrizo shoots were also recovered containing p35S ‘ BG-35T. Construction of plasmids for protoplast transformation has also been completed for the ‘-1,3-glucanase gene, and protoplast transformation experiments using new robust sweet orange suspension lines are being initiated. Protoplast transformation generally results in higher transgene copy numbers than Agrobacterium-mediated transformation. Twenty-eight genes containing a signal peptide (identified by SignalP) were cloned in TMV30b GFP viral vector. Thirteen constructs were successfully assayed in tobacco plants for symptom expression. Two (AS7 and AS13) out of the 13 showed interesting symptoms, and hence were selected for further characterization. AS 7 produced wilting and death of the whole tobacco plant within 2-3 weeks. AS13 transformed plants developed phyllody, stunting and very clear growth defects. The symptoms were significantly different from the infection using the empty vector (TMV 30BGFP). The expression of these genes in planta was checked using RT-PCR. The leaf, petiole and roots of tobacco plants infected with AS7 were collected after ~3weeks for observation under the light microscope. AS7 caused very clear phloem collapse in the petiole, after 3 weeks of inoculation. These symptoms were not observed earlier, 10 days after infection.
A scFv library with activity against ‘Ca. Liberibacter asiaticus’ has been prepared at Beltsville. mRNA was purified from mouse spleens and converted into cDNA. The mice had been immunized with psyllid extracts confirmed to be carrying a high concentration of “Ca. Liberibacter asiaticus” A complete library of variable heavy chain (VH) and variable light chain (VL) genes were made by PCR amplification of the cDNA using a set of 44 primers. The (VH) and (VL) gene segments were then joined in a random combinatorial fashion by overlap extension PCR. The scFv genes were then ligated into the pKM19 phagemid vector which was used to infect Escherichia coli DH5. F’ cells with the aide of a helper phage. The resulting phage library is presently being screened to select phage clones expressing antibodies that bind to “Ca. Liberibacter asiaticus”. Our first attempts to select desired antibodies using extracts from HLB-infected rough lemon were not successful, probably because the concentration of the target bacteria in the rough lemon extracts was too low. We have therefore modified the screening procedure by incorporating magnetic microbeads. These microbeads bind to rabbit antibodies. To use them we raised standard polyclonal antisera in a rabbit against the outer membrane protein of “Ca. Liberibacter asiaticus”. These beads are added to plant and insect extracts to bind the “Ca. Liberibacter asiaticus” and then concentrated by magnetic separation. The phage libraries are then added to the “Ca. Liberibacter asiaticus” on the beads and screened in that manner. The outer membrane protein and antibody system for immunocapture of ‘Ca. Liberibacter asiaticus’ has however not proven to be successful. The polyclonal antibodies made in rabbits against purified outer membrane protein from ‘Ca. Liberibacter asiaticus’ work very well in detecting the purified outer membrane protein but are not effective in immunocapture. The most likely explanation is that the outer membrane protein is not well enough exposed on the surface of the ‘Ca. Liberibacter asiaticus’ cell. We have therefore cloned genes encoding the type IV pilus protein of ‘Ca. Liberibacter asiaticus’, since this protein should be well exposed on the cell surface and should be useful for immunocapture in place of the outer membrane protein. Correct cloning was confirmed by DNA sequencing and these genes have been expressed in E. coli and the encoded proteins and have beenpurified. Emphasis is on recovering the proteins in native, soluble form, which is difficult, but we have now been able to accomplish it. These proteins will be used as antigen to prepare polyclonal rabbit antiserum for the immunocapture portion of the antibody screening process. In related research, we have also cloned and expressed a dinucleotide polyphosphate hydolase, and a polysialic acid capsule expression protein. These proteins, as well as the type IV pilus protein will be biotinylated, combined with strepavidin coated magnetic beads and used to bind phage expressing antibodies that recognize these targets. Thus we will finish with uncharacterized scFv antibodies binding “Ca. Liberibacter asiaticus” as well as a set of antibodies that bind to specific pathologically relevant proteins of the pathogen.
In the first quarter, a greenhouse hydroponics experiment was established to investigate the relationships between boron nutrition and HLB pathogen viability and / or disease expression in citrus. Many of the symptoms of HLB, especially the earlier symptoms have remarkable similarity to boron deficiency. The most notable similarity is that of vein corking, which is associated with rapid unorganized replication of phloem tissue that results from plugging and collapse of the original phloem sieve tubes. It is uncertain why the phloem is especially sensitive to boron deficiency. Boron deficiency also has root symptoms similar to HLB. In the early stages of boron deficiency, the roots stop developing and the tips become disorganized. After extended boron deficiency the roots begin to die back. Determining the mechanism of this possible HLB symptom escape will provide important information about how Las causes disease and provide a possible strategy for HLB management by citrus growers.
A major objective of this project is to develop an understanding of how the HLB bacterium (Las) interacts with citrus genotypes to cause disease. After finding that different citrus genotypes respond differently to Las from extremely sensitive (sweet orange and grapefruit) to tolerance with minor symptoms, we have focused on the one citrus genotype that is most resistant to citrus. Las is restricted to very low levels in Poncirus trifoliata. Most plants remain PCR negative, but a few have barely detectable levels of Las. We have found that under some conditions Las appears not to be able to move through poncirus. We have plants with lower living inoculum that is highly infected with Las, but sensitive sweet orange shoots grafted on top of the poncirus plants have not become infected. We are examining the value of using Poncirus rootstocks and interstocks to reduce or prevent spread of the disease in sweet orange or grapefruit. We have developed a containment plant growth room to examine natural infection of citrus trees by psyllid inoculation. We have made several significant observations: First, we have found that the time period between when plants first become exposed to infected psyllids and the time that new psyllids can acquire Las for those plants can be as little as 6 weeks. We are examining this process in more detail now. Second, when we allowed the infected psyllids a choice of different citrus genotypes, there was a large difference in the time and number of plants that were inoculated by the psyllids: (Citrus macrophylla >> Swingle citrumelo >> Volkamer lemon = Duncan grapefruit > Madam Vinous sweet orange >> Carrizo citrange). Most of the Citrus macrophylla plants became infected with only 2 months of exposure in the epidemic room, whereas only a few of the sweet orange and grapefruit became infected after 4 months. Since there was such a clear preference, we are now investigating its cause ‘ whether the preference is related to genotype, growth habit, flushing, or other possible differences. It is clear that psyllids reproduce on new flush, but feed on older leaves. We are examining whether and how well the psyllid can transmit the disease in the absence of flush. We have developed methods to greatly speed up results of field tests for transgenic or other citrus trees or trees being protected by the CTV vector plus antibacterial or antipsyllid genes. In order to interpret results of a field test, most control trees need to become diseased. Under natural field pressure in areas in which USDA APHIS will allow field tests, this level of infection could take 2-3 years. By allowing the trees to become adequately inoculated by infected psyllids in a containment facility, we can create the level of inoculation that would naturally occur in the field within 2-3 years in 2-5 months in the containment room, after which the trees are moved to the field test site. Another large experiment is underway. Another objective is to provide knowledge and resources to support and foster research in other laboratories. A substantial number of funded projects in other labs are based on our research and reagents. We supply infected psyllids to Mike Davis’s lab for culturing of Las and Kirsten Pelz-Stelinski’s lab for psyllid transmission experiments. Among the plants being screened for resistance or tolerance to HLB for other labs are: 1) a series of elite lines for the citrus improvement group; 2) a series of transgenic plants designed to examine the relationship of pectin production to disease development for Jude Grosser, Gene Albrigo, and Nian Wang; 3) we are testing a series of transgenic plants that we made in collaboration with Zhonglin Mou to have increased disease resistance. The trees, which have high resistance to citrus canker, so far do not look like they have resistance to HLB.
This is a project to find an interim control measure to allow the citrus industry to survive until resistant or tolerant trees are available. We are approaching this problem in three ways. First, we are attempting to find products that will control the greening bacterium in citrus trees. We have chosen initially to focus on antibacterial peptides because they represent one of the few choices available for this time frame. We also are testing some possible anti-psyllid genes. Second, we are developing virus vectors based on CTV to effectively express the antibacterial genes in trees in the field as an interim measure until transgenic trees are available. With effective antibacterial or antipsyllid genes, this will allow protection of young trees for perhaps the first ten years with only pre-HLB control measures. Third, we are examining the possibility of using the CTV vector to express antibacterial peptides to treat trees in the field that are already infected with HLB. With effective anti-Las genes, the vector should be able to prevent further multiplication and spread of the bacterium in infected trees and allow them to recover. We have completed several large screenings of antibacterial peptides against Las in sweet orange trees. About 50 different antibacterial constructs have been tested in trees. We have found two peptides that appear to effectively protect sweet orange trees from HLB. However, we and other labs continue screening for better genes that more effectively control HLB and can be approved for use in a food crop. We also are improving the CTV-based vector to be able to produce multiple genes at the same time. This could allow expression of genes against HLB and canker or multiple of genes against HLB. Another major goal is to do a field test of the CTV vector with antibacterial peptides, which is an initial step in obtaining EPA and FDA approval for use in the field. After some delays, we have received permission for USDA APHIS and are now establishing the field test.
1- The first objective of the second year was to build a plant growth room at the Citrus Research and Education Center in Florida (CREC). The physical construction/renovation of the growth room has not been started so far. The construction is expected to start at the end of September. The rootstocks that are currently growing in the lab are big and we will need to start growing a new batch until the growth room is ready. They cannot be transferred to another greenhouse because it will defeat the purpose of growing under controlled conditions. 2- Training of the manager Dr. Zapata has been completed at the IVIA under the supervision of Dr. Pena. 3- Initial material to establish the mother plants are being produced at the Department of Agriculture with Dr. Peggy Sieburth. She will start releasing the in vitro plants in September. Ideally these plants should be used immediately for grafting on the rootstocks; plants will be kept in vitro until the growth room is ready. The plants cannot be kept for more than 4-5 months on in vitro conditions. 4- A search for a full growth room technician started. The final hiring process will be completed once the growth room construction starts. We expect this technician to go for 2 weeks of training in Spain. At this moment, construction/renovation of the growth room is a major bottleneck for the progress of the project. Clean materials for the most important scion varieties of Florida have been obtained with the help of Dr. Peggy Siebuth through shoot-tip grafting. Clean rootstocks (stored at the lab at this moment-the only clean area we count with) are already 6-month-old and will be ready to be grafted with the clean scions for next October. However, we do not know yet when the growth room will be finalized. Only after this facility is fully operative, we will be able to perform the grafts with the clean materials. If the construction of the growth room is delayed further (more than 3 months), we have the risk of losing both the scions and the rootstocks, and consequently lose months of work (that could not be repeated until next year due to Dr. Sieburth’s agenda) and a lot of money. These delays (no growth room after 1.5 years of project) are making impossible to fulfil our objective for the end of this 3-year project. This situation is out of my understanding- Leandro Pe’a
Concerning the Nested PCR one step we are working on improvement of sensitive of reactions but with few progress in the sensibility of NPCR one step comparing against double step nested PCR, as discussed in the annual report. On the other hand, we did great progress on development of antibodies against Ca. Liberibacter asiaticus. All the 9 peptides mentioned on the third report were injected in rabbits for the polyclonal antiserum (PA) production. Female New Zealand white rabbits weighing 2 kg were endovenously inoculated with 200 ug of each antigenic peptides. Afterwards, this procedure was repeated two times at 14 days intervals, using 300 ug and 400 ug of antigenic peptides, respectively. Seven days after the last injection, the blood was collected through cardiac puncture and the serum titer and specificity were determined by indirect enzyme-linked immunosorbent assay (ELISA) according to Clark et al. (Clark, M.F., R.M. Lister & M. Bar-Joseph. 1986. ELISA techinques, p.742-766. In H. Weissbach & A. Weissbach (eds.), Methods in enzymology, v.118. New York, Academic Press, 820p). Preimmune serum was used as a negative control. Antibody titres of nine PAs were checked by an enzyme- linked colorimetric assay (ELISA) using the Anti-Peptide Antibodies methodology (Table 1). Just one (T3699-3) of the nine PA resulted in low reactivity against the antigen (10’g/mL). For the nest steps we will test the eight PA against the Liberibacter asiatic infected and or symptomatic plants. Table 1. ELISA assays of Polyclonal antibodies against the antigens. Peptides name T3699-1 T3699-2 T3699-3 T3699-4 T3699-5 T3699-6 T3699-7 T3699-8 T3699-9 Dilution Antibody O.D. (405 nm) 1:500 2.100 1.936 0.875 1.455 2.032 2.117 2.153 1.819 2.042 1:1.000 2.037 1.695 0.500 1.311 2.001 2.026 2.132 1.548 1.928 1:2.000 1.904 1.286 0.264 1.129 1.917 1.793 1.993 1.332 1.625 1:4.000 1.601 1.408 0.126 0.758 1.884 1.569 1.825 1.109 1.560 1:8.000 1.336 1.077 0.038 0.573 1.706 1.297 1.584 0.750 1.346 1:16.000 1.110 0.761 0.000 0.284 1.495 1.112 1.388 0.509 1.220 1:32.000 0.832 0.550 0.000 0.164 1.252 0.998 1.060 0.287 1.013
Continued efforts to improve transformation efficiency: ‘ The effect of antioxidants lipoic acid, glycine betaine and glutathione are being evaluated for increased transformation efficiency. Some treatments are showing a significant increase in transformation efficiency across a range of citrus genotypes.’ We have successfully developed an efficient transformation system for embryogenic citrus callus, (publication in Plant Cell Reports in press). This system works well for polyembryonic mandarin types (i.e. W. Murcott, Ponkan) that are seedless or more recalcitrant using the common Agrobacterium-mediated citrus method. The method should also work well for lemons. Horticultural manipulations to reduce juvenility in commercial citrus: The RES (Rapid Evaluation System). ‘ Commercial sweet orange, grapefruit, and specialty mandarin cultivars were propagated and planted in the RES. Several juvenile hybrids from our breeding program flowered and set fruit after only one year – our goal is to force flowering and fruit set in juvenile sweet oranges and grapefruits in 1-2 years. If successful, the same approach could be applied to transgenics. A two-year old field trial using a juvenile Valencia budline on more than 70 rootstocks is showing significant rootstock affects on precocious bearing. Transformation of precocious but commercially important sweet orange clones: transgenic plants of precocious ‘Vernia’ sweet orange somaclones were regenerated and micrografted for further study of early flowering. Transgenic approaches to reduce juvenility: ‘ Whole plants generated from ciFT and empty vector control transformation experiments of Carrizo are being evaluated by both PCR amplification assays and a repeat of the screening histochemical GUS assay. No obvious phenotypes have yet been observed among the whole plants, however, flowers have occasionally been observed to occur on in vitro shoots. ‘ Putative transformed Duncan grapefruit whole plants in soil and shoots being rooted in vitro have been generated. We will be doing additional transformation experiments as soon as fresh seed becomes available. We will shortly have T1 seed from additional plants and will soon be able to proceed with assays to phenotype this generation and compare the effects that each of the ciFT genes has on expression and morphology. ‘Through a project being conducted by an HHMI-sponsored undergraduate student, Melanie Pajon, we will also be cloning the tomato FT ortholog and using it to obtain citrus transformed by a heterologous FT gene.Transformation of Samsun tobacco with the ciFT genes has resulted in a number of T0 plants of each of the 3 ciFT constructs, some of which have produced T1 seeds. We will shortly have T1 seed from additional plants and will soon be able to proceed with assays to phenotype this generation and compare the effects that each of the ciFT genes has on expression and morphology. Phenotypes of the T0 plants have ranged from early flowering, multi-branching, dwarfs to ones very similar in architectures in the wild type parent.
Weather towers are in place in a ridge grove location and collecting data for determining weather conditions to optimize low volume spray applications. Towers will be placed in a flatwoods grove location during September. As noted in earlier reports the ability to measure spray droplet size from low volume spray machines is not possible. Funding for that part of the research project has been returned to the granting agency.
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