The goal of this experiment is twofold, first to determine the effects of plant growth regulators on addressing vascular degeneration and fruit drop, and second to determine the effects of HLB and ACPS citriculture on drought tolerance. A field experiment was installed in April 2013 to test the efficacy of the synthetic auxin 2,4-D and a micro-emulsion ‘based surfactant to reduce HLB symptom severity in a mature ‘Hamlin’ orange block. The HLB incidence in the block is currently more than 50% and consequently the fruit yield losses due to pre-harvest fruit drop from symptomatic trees were devastating in the 2012/13 season. The experimental design is a 4×4 Latin Square with four replications and four factorial foliar spray treatments consisting of 2,4-D, Eco-Agra’ surfactant, 2,4-D + Eco-Agra’, and untreated control. Each whole plot is split into two sub-plots containing Swingle and Carrizo rootstocks. A basal foliar nutrient spray treatment applied to the whole experiment consists of a comprehensive, balanced fertilization program of micronutrients, macronutrients and potassium phosphite products timed to coincide with the major leaf flushes. The basal ground-applied fertilizer program consists of a dry granular bulk-blended N-P-K +Ca +Mg + Fe + Mn +Zn +B +S product applied four times in the growing season. The automated micro-sprinkler irrigation system is used to apply water to the trees according to seasonal evapotranspiration demand as needed, up to twice per day. Measurements of the trees in this experiment will begin in the summer, and are designed to determine if the PGR and surfactant treatments can reduce the debilitating damage caused by the Liberibacter pathogen to the phloem tissue, and to the whole-tree physiology. They will include photosynthetic rates, phloem transport functions, HLB symptom severity, leaf nutrient concentrations, leaf drop and fruit drop. Preliminary hydraulics data suggests that trees grown under conventional microsprinkler irrigation systems, and watered 2-3 times per week are more resistant to drought induced declines in hydraulic conductivity compared to trees in the ACPS system. We are now sampling the trees again for hydraulics measurements to confirm the preliminary results and are also investigating the anatomical components of the resistance.
Understanding the transmission of CLas within the citrus tree remains one of the principal obstacles in the global efforts to undermine the pathogenicity of HLB (citrus greening). The movement of CLas has been assumed to follow the photoassimilate stream through the phloem. However, many observations based on our knowledge of the bacteria and general phloem anatomy have exposed inconsistencies with the accepted beliefs. The brevity of available information on the ultrastructural properties of citrus phloem sieve elements has hindered efforts to understand the spread of the disease within a tree. For example, lateral movement of CLas around an infected stem appears improbable given the size of cytoplasmic plasmodesmata connections between adjacent sieve elements and the isolated nature of phloem cells. Furthermore, spreading of CLas from the roots to uninfected aerial tree parts through the phloem seems highly unlikely given the direction of phloem sap. To date we lack a thorough investigation into the ultrastructure of citrus phloem and the surrounding tissue, the potential pathways that CLas could utilize to move long distances through citrus trees, and the location of CLas habitat within different citrus tissue. Using a variety of grafting and girdling experiments, SEM, TEM, confocal, high resolution computed tomography, and PCR tissue analysis we aim to gain a better understanding of the anatomical traits that facilitate the spread of CLas through citrus. These data will allow us to develop new screening tools that breeders can use to select for resistant scion/rootstock combinations to confer resistance or tolerance to HLB. As of this progress report the Valencia/Swingle trees are in the process of being grafted together. Once the grafting heals the trees will have HLB infected buds and branches grafted onto the scion or rootstock depending on the experiment. Trees will be dissected prior to grafting on the HLB infected material, and then trees will be sampled for anatomical studies and PCR testing at regular intervals for the next year. We have hired personnel to maintain the plants in the greehouse during the duration of the experiments.
The main goal of this project is to optimally deploy the superinfecting Citrus tristeza virus (CTV)-based vector to prevent existing field trees from development of the HLB disease and to treat trees that already established the disease. We have several sets of the experiments in which we are examining how prior infection with different CTV strains affects the ability of the superinfecting CTV vector to infect and get established in the same trees as well as examining the levels of multiplication of the superinfecting CTV vector in trees infected with different field isolates of CTV. Plant material that is being used in this project and CTV inoculum sources (different isolates of CTV propagated in the greenhouse as well as collected on the field) have been prepared. The experiments to assess the effect of preexisting CTV infections on multiplication of the superinfecting vector in inoculated citrus trees are ongoing. We first graft-inoculated sweet orange trees with the T36,T30 or T68 isolate of CTV, the isolates that were propagated in our greenhouse, as well as with CTV-infected material obtained from field (FS series isolates). We are using isolates that contain only single strains and isolates that contain mixtures of strains for primary inoculations. Real time PCR analysis protocol is being optimized for quantification of multiplication of CTV genotypes in the inoculated trees. Trees with developed CTV infection along with uninfected control trees were challenged by graft-inoculation with the superinfecting vector carrying a GFP gene. The latter protein is used as a marker protein in this assay, which production represents a measure of vector multiplication. The trees are now being examined to evaluate level of replication of superinfecting virus. Tissue samples from the challenged trees are observed under the fluorescence microscope to evaluate the ability of the vector to superinfect trees that were earlier infected with the other isolates of the virus. Levels of GFP fluorescence are monitored and compared between samples from trees with and without preexisting CTV infection. Additionally, real time PCR quantification is also being employed to these tests. To select rootstock/scion combinations that would support the highest levels of superinfecting vector multiplication and thus, highest levels of expression of the foreign protein of interest from this vector, we are preparing trees of Valencia and Hamlin sweet oranges and Duncan and Ruby Red grapefruit on three different rootstocks: Swingle citrumelo, Carrizo citrange, and Citrus macrophylla. The plants are used for the experiments similar to the experiments described above.
Work continues on the construction and characterization of new FT constructs using cDNA clones. The experiments are underway to compare the new FMVcDNA27 construct, which contains an FT3 cDNA insert in the pCAMBIA2201 vector with a constitutive FMV promoter, with a corresponding genomic clone, which we have been using up to this time. Transformation of Carrizo and tobacco tissue is underway in order to compare the action of these two constructs. The new construct was created as a first step towards the development of a new FT3 construct with an inducible promoter. We have arranged for the materials transfer of two inducible promoter systems from the Danforth Foundation. Both of these promoters are inducible by the chemical methoxyfenozide, a widely-available pesticide, approved for field use on citrus. However, we have not yet received the inducible promotors. One system is driven by the CsMV constitutive promoter, and the other by the RTBV vascular-specific promoter. Once we have verified that the smaller and more manageable cDNA is as effective as the original genomic version of the FT3 gene, we hope to begin development of the inducible promoter constructs. Experiments to determine the behavior of the three genomic clones from citrus when overexpressed in tobacco have been completed and a manuscript is being written. Expression of the genes in mature nonstransgenic citrus plants is being recorded monthly.
Two naturally occurring terpenoid essential oils have been selected as the key components for the pesticide formulations. They are known to have antimicrobial properties which allow us to believe that they will be potent in combating the targeted bacteria. Agriculturally approved surfactants of selected HLB values have produced formulations that permit for encapsulation of the oil. Both essential oils have successfully formed stable micro-emulsions. Large changes in temperature cause some instability with higher oil loading. However, at room temperature, system is re-equilibrated. Essential oil A requires additional input of energy (sonication), where as essential oil B is more readily encapsulated. With the addition of co-surfactants, the oil loading percentages greatly increase in the respective microemulsions. For essential oil A, loading is increased from 1% to 7% (w/w). Essential oil B increases from 8% to 14% (w/w). Particle sizing measurements have been conducted and show mean particle sizes by number distribution of ~3 nm for essential oil A and ~7 nm for essential oil B. The droplet size of Essential oil B microemulsion was increased to about ~30 nm with increase in oil loading. These results have been compared using two particle-sizing instruments, Microtrac Nanotrac and Malvern Zetasizer Nano ZS. Further investigations are being carried out to determine the correlation between the particle size and the amount of oil in the system. Currently, surfactant weight percentages are ~ 20% for both Essential oil microemulsion systems. Additional experiments are being performed to increase the Essential oil loading percentages in both microemulsion systems.
A transgenic test site at the USDA/ARS USHRL Picos Farm in Ft. Pierce supports HLB/ACP/Citrus Canker resistance screening for the citrus research community. There are numerous experiments in place at this site where HLB, ACP, and citrus canker are widespread. The first trees have been in place for over three years. Dr. Jude Grosser of UF has provided ~600 transgenic citrus plants expressing genes expected to provide HLB/canker resistance, which have been planted in the test site. Dr. Grosser planted an additional group of trees including preinoculated trees of sweet orange on a complex tetraploid rootstock that appeared to confer HLB resistance in an earlier test. Dr. Kim Bowman has planted several hundred rootstock genotypes, and Ed Stover 50 sweet oranges (400 trees due to replication) transformed with the antimicrobial peptide D4E1. Texas A&M Anti-ACP transgenics produced by Erik Mirkov and expressing the snow-drop Lectin (to suppress ACP) have been planted along with 150 sweet orange transgenics from USDA expressing the garlic lectin. Eliezer Louzada of Texas A&M has permission to plant his transgenics on this site, which have altered Ca metabolism to target canker, HLB and other diseases. More than 120 citranges, from a well-characterized mapping population, and other trifoliate hybrids (+ sweet orange standards) have been planted in a replicated trial in collaboration with Fred Gmitter of UF and Mikeal Roose of UCRiverside. Plants are being 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. Dr. Roose has completed initial genotyping on a sample of the test material using a “genotyping by sequencing” approach. So far, the 1/16th poncirus hybrid nicknamed Gnarlyglo is growing extraordinarily well. It is being used aggressively as a parent in conventional breeding. Dr. Grosser removed the unsuccessful trees from the first planting and planted additional transgenics among the promising trees still under trial. Additional plantings are welcome from the research community.
Citrus scions continue to advance which have been transformed with diverse constructs including AMPs, hairpins to suppress PP-2 through RNAi (to test possible reduction in vascular blockage even when CLas is present), a citrus promoter driving citrus defensins (citGRP1 and citGRP2) designed by Bill Belknap of USDA/ARS, Albany, CA), and genes which may induce deciduousness in citrus. Putative transgenic plants of several PP-2 hairpins and of PP-2 directly are grafted in the greenhouse and growing for transgene verification, replication and testing. Over 40 putative transgenic plants with citGRP1 were transferred to soil. Nineteen of them were test by PCR and ten of them are transgenic plants with citGRP1 insertion. They will soon be ready for RNA isolation and RT-PCR to check gene expression. More than thirty kan resistant shoots were obtained from citGRP1 transformed Hamilin. About 10 transgenic Hamlin shoots with citGRP2 were rooted in the medium and nine of them were planted in soil. Belknap reports that potatoes transformed with citGRP2 are displaying considerable resistance to Zebra Chip in Washington state. Fifteen transgenic Hamlin shoots with peach dormancy related gene MADS6 are in the rooting medium for rooting. Seven transgenic Hamlin with MADS6 were planted in soil. In addition, numerous putative transformants are present on the selective media transformed with different constructs. A chimeral construct that should enhance AMP effectiveness (designed by Goutam Gupta of Los Alamos National Lab) is being tested. Many kan resistant transformants were generated on the selective media. About twenty kan resistant shoot are rooted in rooting medium and one of Hamlin transformatn was planted in soil. To explore broad spectrum resistant plants, a flagellin receptor gene FLS2 from tobacco was amplified and cloned into pBinARSplus vector. Flagellins are frequently PAMPS (pathogenesis associated molecular patterns) in disease systems and CLas has a full flagellin gene despite having no flagella detected to date. The consensus FLS2 clone was obtained and used to transform Hamlin and Carrizo so that resistance transduction may be enhanced in citrus responding to HLB and other diseases. The construct pBinARSplus:nbFLS2 was used to transform Hamlin and Carrizo. Many putative transformants were generated on the selective media. About forty resistant shoots were rooted in rooting medium and ten Hamlin transformats were plant in soil. Other targets identified in genomic analyses are also being pursued. A series of transgenics scions produced in the last several years continue to move forward in the testing pipeline. Several D35S::D4E1 sweet oranges show initial growth in the field which exceeds that of controls. A large number of ubiquitin::D4E1 and WDV::D4E1 plants and smaller numbers with other AMPs are replicated and in early stages of testing.
Our recent progress towards proposed research goals: Objective 1: Generate functional EFR variants (EFR+) recognizing both elf18-Xac and elf18-CLas. A] Mutagenesis of EFR to produce elf18-CLas responsiveness: Our initial approach of random mutagenesis and screening in tomato was unsuccessful, indicating the necessity to generate multiple mutations for elf18-CLas recognition. Presently we are evaluating phage display for this purpose. To this end, we have defined suitable conditions for specific binding of ectodomain fragments of EFR to biotinylated elf24. Elf24 has been used for these experiments to allow linkage of the biotin group to Lysine 24; this peptide is fully functional in the elicitation of ROS. We are in the process of evaluating binding of biotinylated elf24-CLas to different regions of EFR. Once optimal regions are determined, mutagenesis will be performed on these regions and cloned into phage display vectors for screening. B] Screening for natural variants of EFR: A small selection of Brassicae has been screened for elf18-CLas response, however none of these were positive. Pending the outcome of mutagenic approaches, the screen for elf18-CLas response will be expanded to a large number of Brassicae. Objective 2: Generate functional XA21-EFR chimera (XA21-EFRchim) recognizing axYS22-Xac. Assessing XA21 function in dicots: Transgenic XA21-EFR, XA21 and EFR lines have been generated in Arabidopsis and are now ready to assess their effectiveness in pathogen defence. We plan to test these lines against Xanthomonas, Pseudomonas and Argobacterium. In addition, we have generated transgenic tomato lines expressing XA21. These plants will be crossed with EFR tomato lines to determine the pathogen resistance conferred by these two genes in a heterologous system. Objective 3: Generate transgenic citrus plants expressing both EFR+ and XA21-EFRchim. We will initiate the construction of appropriate expression vectors of genes for citrus transformation and expression.
The objectives of this project are to characterize the molecular interactions between the effectors and the host mitochondrial proteins; to screen for molecules that inhibit the effector functions; and to control HLB using the inhibitor(s) and/or other related molecules. To understand the function(s) of LasA1 and LasA2, we have made several constructs in Gateway’ pDONR’ Vector, and pGWB expression vectors, which contain different versions of the LasA1 gene, the N-terminal region (LasA1-A), two version for the repeat region with different number of the repeat sequences (LasA1-B0 and LasA1-B1), the C-terminal region (LasA1-C), and the full LasA1 gene. We are analyzing these constructs for their transient expression in Nicotiana benthamana and stable expression in transgenic Arabidopsis thaliana. The transgenic lines were obtained by floral-dip transformation of Arabidopsis Col-0 plants and we are currently verifying the gene insertion and mRNA expression level on T2 Arabidopsis. Three transgenic T3 lines expressing the gene are selected for analyzing phenotypes and protein localization using GFP pGWB2 vector. We are testing the expression level of the gene constructs that were transiently expressed in N. benthamiana with 35S, PFLAG and GFP pGWB2, 6 and 12 vectors. Localization of different constructs of LasA1 and LasA2 proteins using GFP vectors and pull-downs for protein-protein interactions using the PFLAG vector are in progress.
Cytoplasmic (CiLV-C and CiLV-C2) and nuclear (CiLV-N) citrus leprosis virus cause citrus leprosis disease in North and South American. All types of the CiLV are reportedly transmitted by Brevipalpus mite species. We continued mite transmission experiments at the USDA, ARS, Foreign Disease and Weed Science Research Unit, Ft. Detrick, MD utilizing endemic healthy Brevivalpus yothersii (syn. phoenicis) mites from Florida. We completed mite transmission experiments after receiving the citrus leprosis affected samples from Mexico (CiLV-N) & Colombia (CiLV-C2). Six to seven weeks after completion of the transmission experiments none of the citrus seedlings showed any leprosis symptoms. For confirmation of the negative test results asymptomatic leaf tissue from the experiments was analyzed by reverse transcription polymerase chain reaction (RT-PCR) using CiLV type-specific primers but all plant samples were negative. Recently, nuclear CiLV was reported from Mexico but no prior sequence information was available. In work on another funded project we have successfully determined the entire genome sequence of nuclear CiLV and deposited the sequence in the NCBI Genbank. A manuscript also has been accepted for publication in the journal Genome Announcement (‘Genome assembly of citrus leprosis virus nuclear type reveals a close association with orchid fleck virus’. Contacts have been made with collaborators in Mexico, Colombia and Panama for further shipment of infected leprosis samples to continue the transmission experiments with all 3 types of CiLV. Using newly developed PCR primers we will determine the viruliferous status of the B. yothersii after acquisition from infected leprosis tissue. Based on these results we will determine if Florida endemic healthy Brevivalpus Florida mites are able to acquire the various citrus leprosis viruses. In addition the utilized mites will be sent to USDA cooperators to continue to compare their taxonomic status with those that do transmit in Colombia elsewhere. Mite transmission work in Colombia continued with work by our collaborator Guillermo Leon. In acquisition experiments it was found that B. yothersii (phoenicis) mites effectively acquired CiLV-C2 after 30 minutes feeding on leprosis symptomatic leaves of Valencia orange. In transmission experiments with mites that were allowed an acquisition period of 24 h, positive virus transmissions were accomplished after only 10 minutes of feeding. Transmission rates increased incrementally up to a maximum after 24 hours feeding. However in using PCR to detect CiLV-C2 in the mites variable results were found. It appears that a minimal level of virus must be present in the mite for positive detection however PCR negative mites may still acquire and transmit the virus.
‘Sun Chu Sha’ and ‘Nagami’ kumquat plants were pre-treated with Candidatus Liberibacter asiaticus flagellin 22 (CLas-flg22) peptide 24 hours prior to inoculation with Xanthomonas citri pv citri (Xcc) in an attempt to determine whether CLas-flg22 was capable of inducing PAMP-triggered immunity (PTI). The Xcc population/concentration in the inoculated leaves was determine via colony forming units (cfu) by a standard procedure in a time course of up to 2 days post inoculation (dpi). Bacterial growth in the CLas-flg22 pretreated leaves was lower than in those treated with water in both Nagami and Sun Chu Sha, indicating that PTI was triggered. We used Xcc instead of CLas because of the impossibility of infiltrating the latter pathogen. Similar experiments using Xcc-flg22 also triggered PTI but to much higher levels than CLas-flg22. In other words, bacterial growth was slowed much more.
This is a new project that only just was funded, so there is not yet much to report. However, we do have some preliminary results. Cell penetrating peptides (CPPs) are small protein fragments that have been shown to be able to pass through the cell membrane that surrounds mammalian cells. More significantly, when the CPPs translocate in this manner they can also escort ‘cargoes’ across the membrane. Cargoes include proteins, plasmid or linear DNA, RNA, and antibodies that cannot enter the cell or blood-brain barrier without the presence of CPPs. CPPs have also been shown by others to work to introduce cargoes into plant cells. We have determined what CPPs work effectively in citrus for the import of proteins and nucleic acids. Imported DNA clones transiently express marker proteins; experiments on stable transformation have begun.
The transformation construct for expressing the FLT-antiNodT fusion protein in citrus is nearing completion. We encountered major difficulties cloning the FLT-antiNodT expression cassette into the pTLAB21 citrus transformation vector. The FLT-antiNodT cassette DNA appeared to be unstable in E. coli when cloned into pTLAB21, which stymied our cloning efforts for several months. The instability of the FLT-antiNodT cassette in pTLAB21 was surprising, since the FLT-antiNodT cassette was stable in E. coli when cloned into non-transformation vectors such as pBluescript. For reasons unknown, the FLT-antiNodT cassette was specifically unstable in pTLAB21. However, we serendipitously discovered that the inclusion of an additional segment of DNA next to the FLT-antiNodT cassette in pTLAB21 actually stabilized the FLT-antiNodT cassette in pTLAB21. This piece of DNA was derived from the original FLT-antiNodT cassette cloning vector, pBluescript. This additional piece of DNA should not cause a problem for transformation of citrus or expression of the FLT-antiNodT antibody in transgenic citrus. We are now in the process of sequencing and verifying the pTLAB21-FLT-antiNodT transformation vector. Once this process is complete, we will commence citrus transformations. We anticipate that transformations will begin within the next reporting period.
In the period between April and July of 2013, Citrus Core Transformation Facility (CCTF) experienced significant changes in personnel. These changes reflected negatively on the productivity and as result, the goal of 100 transgenic plants per quarter was not reached. The experimental efforts were directed towards orders that were placed within previous six months. Produced transgenic plants were all Duncan grapefruit. They belong to following orders: MED16-11 plants; X20-four plants; X16-three plants; X19-one plant; X4-one plant; Bi121-six plants; N5-31 plants; N7-nine plants; HGJ3-one plant; and W14-one plant. As opposed to a few previous quarters when the number of placed orders was unusually high, facility received three orders during this time. The ‘genes’ of interest in those vectors were: gMOD1, ELP3-CIV, and ELP4-CIV. All three orders require transformation of Duncan grapefruit plants. Actually, two more orders were placed but after initial attempts to mobilize binary vectors into appropriate Agrobacterium strains the quality of cultures was reviewed and client decided to withdraw them. Within the first year (14 months) of funding of this project, CCTF received 35 orders for production of transgenic plants. Altogether, about 430 plants were produced. They belong to 23 orders received within this funding period and eight orders from previous period. Transgenic plants that were requested from clients in new orders were mostly Duncan grapefruit, but there were some requests for Valencia oranges, Carrizo citranges, and Mexican limes. Ratio of produced plants reflects well the ratio of requested plants in placed orders. CCTF remained a reliable producer of transgenic material. High influx of orders speaks of the important role that CCTF plays in efforts directed towards disease tolerance improvement of Citrus cultivars. Researchers that have well developed ideas or initial encouraging data about the beneficial activity of certain genes know that CCTF can help them test their hypotheses by producing citrus plants that carry those genes in them. While waiting for transgenic material from CCTF, those research groups can direct their efforts towards development of appropriate challenge tests that will be performed on transgenic plants or work on other aspects of protection against citrus diseases.
The project was extended with no cost for an additional nine months in order to conduct the final destructive sampling during the dismantling of the hydroponics greenhouse experiment, and to collect the final year of 2012/13 fruit yields from the ‘Hamlin’ and ‘Valencia’ field trials at Orange Hammock.
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