During this reporting period (January, February, and March, 2016), control plants that have been through the transformation process, but not containing the transgene, were generated and sent to Penn State, and they are growing well at the Penn State location. These plants are the best comparison to the FLT-antiNodT plants in terms of plant behavior and disease resistance. We call these the “transformation control” trees. The transgenic plants being produced for this project continued to grow at two different locations in secure greenhouses and growth chambers. Seven independently-transformed citrus plants carrying the FLT-antiNodT fusion protein expression construct are growing in Dr. McNellis’ lab at the Pennsylvania State University at University Park, PA, and an additional eight independently-transformed citrus plants carrying the FLT-antiNodT fusion protein expression construct are growing at Dr. Tim Gottwald’s lab at the United States Horticultural Laboratory in Fort Pierce, Florida. Dr. McNellis has applied for and been granted an APHIS BRS permit to send propagated FLT-antiNodT plants to Florida for replicated testing for HLB resistance in Dr. Gottwald’s lab. However, before sending the plants, we must obtain the needed Florida state permit (FDACS 08084), and this is in progress. Dr. Janice Zale (University of Florida Mature Citrus Transformation Facility, Lake Alfred) transformed ‘Hamlin’ sweet orange and the ‘Carrizo’ rootstock with the FLT-antiNodT expression construct, and we received these plants at Penn State in early April, 2016. During the next reporting period, we will test these plants for expression of the FLT-antiNodT anti-HLB protein. Dr. McNellis will also produce rooted cuttings of all these lines for later testing for HLB resistance in Florida.
During this reporting period (January, February, and March, 2016), control plants that have been through the transformation process, but not containing the transgene, were generated and sent to Penn State, and they are growing well at the Penn State location. These plants are the best comparison to the FLT-antiNodT plants in terms of plant behavior and disease resistance. We call these the “transformation control” trees. The transgenic plants being produced for this project continued to grow at two different locations in secure greenhouses and growth chambers. Seven independently-transformed citrus plants carrying the FLT-antiNodT fusion protein expression construct are growing in Dr. McNellis’ lab at the Pennsylvania State University at University Park, PA, and an additional eight independently-transformed citrus plants carrying the FLT-antiNodT fusion protein expression construct are growing at Dr. Tim Gottwald’s lab at the United States Horticultural Laboratory in Fort Pierce, Florida. Dr. McNellis has applied for and been granted an APHIS BRS permit to send propagated FLT-antiNodT plants to Florida for replicated testing for HLB resistance in Dr. Gottwald’s lab. However, before sending the plants, we must obtain the needed Florida state permit (FDACS 08084), and this is in progress. Dr. Janice Zale (University of Florida Mature Citrus Transformation Facility, Lake Alfred) transformed ‘Hamlin’ sweet orange and the ‘Carrizo’ rootstock with the FLT-antiNodT expression construct, and we received these plants at Penn State in early April, 2016. During the next reporting period, we will test these plants for expression of the FLT-antiNodT anti-HLB protein. Dr. McNellis will also produce rooted cuttings of all these lines for later testing for HLB resistance in Florida.
A 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 six years and new trees are being added every few months. A number of successes have already been documented at the Picos Test Site funded through the CRDF. The UF Grosser transgenic effort has identified promising material, eliminated failures, continues to replant with new advanced material, with ~200 new trees in April 2015. The ARS Stover transgenic program has trees from many constructs at the test site and is seeing some modest differences so far, but new material has been planted that has shown great promise in the greenhouse and the permit has been updated to plant many new transgenics. A trial of more than 85 seedling populations from accessions of Citrus and citrus relatives (provided as seeds from the US National Clonal Germplasm Repository in Riverside, CA) has been underway for 6 years in the Picos Test Site. P. trifoliata, Microcitrus, and Eremocitrus are among the few genotypes in the citrus gene pool that continue to show substantial resistance to HLB (Ramadugu et al, Plant Disease, 2016), and P. trifoliata also displayed reduced colonization by ACP (Westbrook et al., 2011). Marked tolerance to HLB is apparent in many accessions with citron in their pedigree (Miles et al., 2016). All replicates of one alleged “standard sour orange” looks remarkably healthy and may permit comparison of more susceptible and tolerant near-isogenic variants. A new UF-Gmitter led association mapping study has just been initiated using the same planting, to identify genes associated with HLB- and ACP-resistance. A broader cross-section of Poncirus-derived genotypes are on the site in a project led by UC Riverside/USDA-ARS Riverside, in which half of the trees of each seed source were graft-inoculated prior to planting. A collaboration between UF, UCRiverside and ARS is well-underway with more than 1000 Poncirus-hybrid trees (including 100 citranges replicated) being evaluated to map genes for HLB/ACP resistance. Marked differences in initial HLB symptoms and Las titer were presented at the 2015 International HLB conference (Gmitter et al., unpublished). In July 2015 David Hall led assessment of ACP colonization across the entire planting, and the Gmitter lab will map markers associated with reduced colonization. Several USDA citrus hybrids/genotypes with Poncirus in the pedigree have fruit that approach commercial quality, were planted within the citrange site. Several of these USDA hybrids have grown well, with dense canopies and good fruit set but copious mottle, while sweet oranges are stunted with very low vigor (Stover et al., unpublished). A Fairchild x Fortune mapping population was just planted at the Picos Test Site in an effort led by Mike Roose to identify genes associated with tolerance. This replicated planting includes a number of related hybrids (among them our easy peeling remarkably HLB-tolerant 5-51-2) and released related cultivars. Valencia on UF Grosser tetrazyg rootstocks have been at the Picos Test Site for several years, having been Las-inoculated before planting, and several continue to show excellent growth compared to standard controls (Grosser, personal comm.).
Our significant progresses during this reporting time period are: 1) Using mature shoot segments of Valencia and Washington navel, we have demonstrated that the Kn1 gene can improve transformation efficiencies by approximately 2-fold compared to the control vector, which is much lower than those observed in juvenile citrus transformation. 2) We used an epigenetic modulator in our transformation experiments and observed about a 2- to 3-fold increase in overall transformation efficiency in mature tissues of Valencia and Washington navel oranges. We further demonstrated that the epigenetic modulator produced a 10-fold increase in shoot regeneration efficiency of mature citrus with no transformation when compared to the controls. 3) With expression of a 35S::GUS gene containing an intron as an indicator, we examined Agrobacterium infection and T-DNA integration activities in mature citrus using tobacco leaf discs and juvenile citrus tissues as references. Consistent with the fact that tobacco leaf discs can be efficiently transformed with Agrobacterium, we observed very high levels of transient and stable expression of GUS in the cut edges of tobacco discs. When juvenile citrus tissues were used for Agrobacterium infection, we observed reasonable levels of both transient and stable GUS gene expression. Using mature explants of Valencia, however, we observed extremely low levels of transient and stable expression of the GUS gene. As we have shown that although both the Kn1 and Ipt gene dramatically enhanced transformation efficiencies of juvenile citrus via increased shoot regeneration, they were far less effective at improving transformation on mature citrus tissue. Also, in mature Valencia and Washington navel oranges, we found that using an epigenetic modulator led to about 10-fold increase in shoot regeneration, but only a 2- to 3-fold increase in transformation efficiency (i.e., transgenic shoot production). We hypothesized that after improved shoot regeneration, Agrobacterium-mediated T-DNA integration remained the major challenges to improving mature citrus transformation. We are now working to enhance efficiencies of Agrobacterium-mediated stable T-DNA integration. Combining the various molecular tools we have, we would like to develop a ‘vector’ that is highly efficient and genotype-independent for mature citrus transformation. One manuscript reporting the drastically improvement of six citrus cultivars including a lemon cultivar has been published: Hu et al (2016): Kn1 gene overexpression drastically improves genetic transformation efficiencies of citrus cultivars. Plant cell, Tissue and Organ Culture. 125: 81-91. Two manuscripts are under preparation, reporting some of the results summarized above.
Objective 1: Assess canker resistance conferred by the PAMP receptors EFR and XA21 Three constructs were used for genetic transformation of Duncan grapefruit and sweet orange as part of a previous grant: EFR, EFR coexpressed with XA21, and EFR coexpressed with an XA21:EFR chimera. Putative transgenics are currently being verified by PCR in the Jones lab, and six PCR positive plants have been identified so far. To ensure that there will be sufficient events to analyze to come to a conclusion about the effectiveness of these genes, we have initiated more transformations in Duncan grapefruit at the Core Citrus Transformation Facility at UF Lake Alfred. EFR, XA21, and XA21 + EFR constructs have been re-created with the inclusion of a GFP marker for confirmation of transformants; selection is underway. In addition, we will add the recently-identified Cold Shock Protein Receptor (CSPR) to the transformation queue. Objective 2: Introduction of the pepper Bs2 disease resistance gene into citrus Constructs have been created in the Staskawicz lab to express Bs2 under the 35S promoter and under a resistance gene promoter from tomato. Constructs have also been created in which Bs2 is co-expressed with other R genes that may serve as accessory factors for Bs2. Constructs with tagged Bs2 have been confirmed to function in transient assays, and protein expression has been confirmed by immunoblot. These constructs have also been transformed into Arabidopsis for analysis, and two constructs have been provided to the Lake Alfred transformation facility, Objective 3: Development of genome editing technologies (Cas9/CRISPR) for citrus improvement The initial target for gene editing is the citrus homolog of Bs5 of pepper. The recessive bs5 resistance allele contains a deletion of two conserved leucines. The citrus Bs5 homolog was sequenced from both Carrizo citrange and Duncan grapefruit, and conserved CRISPR targets were identified. Four CRISPR constructs are being created in the Staskawicz lab: C1) A construct targeting two sites that will produce a 100 bp deletion in Bs5 in both Carrizo and Duncan (the bs5 transgene will be added); C2) A construct targeting a site overlapping the two conserved leucines; C3) C2 with the addition of a bs5 repair template for Carrizo that will not be cut; and C4) C2 with a similar repair template for Duncan grapefruit. The constructs have been tested by co-delivery into Nicotiana benthamiana leaves with another construct carrying the targeted DNA from Carrizo or Duncan varieties, and verified to function. To aid in the selection of positive transgenics, we are currently adding a GFP reporter into each CRISPR construct.
Citrus trees transformed with a chimera AMP (thionin-D4E1) and the thionin alone showed remarkable resistance in citrus canker compared to control. These promising transgenic lines were replicated for HLB challenge. Replicated transgenic Carrizo lines expressing thionin, chimera and control were grafted with HLB infected rough lemon buds. Las titer was checked from new flush rough lemon leaves at six month after grafting. Las titer from 18.6-36.5 was detected in 90% of transgenics expressing the chimera. Some transgenic lines expressing thonin had lower Las titer(most in 33.3-36.4 ranges). Transgenic root sample were further tested and most were detected with las titer from 30 to 35. Root samples from control plants and transgenic Carrizo expressing chimera and thionin were taken nine months after grating inoculation. Our results showed transgenic Carrizo expressing thionin significantly inhibited Las growth (0.5% of control level) compared to control and transgenic Carrizo expressing chimera. Antibody against thionin will be produced for Western detection. Two new chimeral peptides (second generation) were developed and used to produce many Carrizo plants and Hamlin shoots. Transgenic Carrizo plants carrying second generation AMPs were obtained. DNA was isolated from 46 plants and 40 of them are PCR positive. To explore broad spectrum resistance, a flagellin receptor gene FLS2 from tobacco was used to transform citrus. The consensus FLS2 clone was obtained and used to transform Hamlin and Carrizo so that resistance transduction may be enhanced in citrus for HLB and other diseases. Reactive Oxygen Species (ROS) assay showed typical ROS reaction in transgenic Hamlin indicating nbFLS is functional in citrus PAMP-triggered immunity. Trees showed significant canker resistance to spray inoculation. Replicated Carrizo and Hamlin were challenged with ACP feeding. Leaves were taken six months after ACP feeding inoculation. DNA will be isolated and Las titer will be tested. To disrupt HLB development by manipulating Las pathogenesis, a luxI homolog potentially producing AHLs to bind LuxR in Las was cloned into binary vector and transformed citrus. Both transformed Carrizo and Hamlin were obtained. Replicated transgenic Carrizo plants were challenged by ACP feeding. Las titer will be tested soon. Transgenic Hamlin were propagated by grafting for HLB challenge. In collaboration with Bill Belknap two new citrus-derived promoters have been tested using a GUS reporter gene and have been shown to have extraordinarily high levels of tissue-specific expression. The phloem-specific promoter was used to create a construct for highly phloem specific expression of the chimeral peptide using citrus genes only. A Las protein p235 with a nuclear-localization sequence has been identified and studied. Carrizo transformed with this gene displays leaf yellowing similar to that seen in HLB-affected trees. Gene expression levels, determined by RT-qPCR , correlated with HLB-like symptoms. P235 translational fusion with GFP shows the gene product targets to citrus chloroplasts. Transcription data were obtained by RNA-Seq. Data analysis and comparison are underway. Antibodies (ScFv) to the Las invA and TolC genes, and constructs to overproduce them, were created by John Hartung under an earlier CRDF project. We have transgenic Carrizo reflecting almost 400 independent transgenic events and 17 different ScFv ready for testing. A series of AMP transgenics scions produced in the last several years continue to move forward in the testing pipeline. Many trees are in the field and some are growing well but are not immune to HLB. A large number of ubiquitin::D4E1 and WDV::D4E1 plants and smaller numbers with other AMPs are replicated and now in the field.
The Huanglongbing Diagnostic Lab at UF-IFAS-SWFREC has now been in operation for 8 years. As of March 2016, we have processed more than 38,000 grower samples. For the 2016 calendar year to date, we’ve received 917 samples from growers, which is on track towards a calendar year total very close to 2015 levels. The 3,995 growers samples processed during 2015 represented a 46% increase in the number of grower’s samples over the previous calendar year, which in itself had seen a 37% increase over 2013 numbers. These increases are likely due to the increased efforts to mitigate the HLB-associated tree stresses. Growers in this area, and most other regions, currently have one or more HLB mitigation program that they are evaluating. These growers are using the HLB lab to evaluate the effectiveness of their efforts. Another evidence of increased grower usage of the lab is seen in the fact that 60% of the individuals who submitted samples during 2015 were new clients who had not previously submitted samples. So far, new clients comprise 46% of submitters in 2016. Additionally, nearly 43,800 samples have been received for research for the entire period of diagnostic service, supported by grant funding of individual researchers. This brings the grand total to more than 81,800 plant samples processed. Grower samples are typically processed and reports returned within a two to four week time period. For this report, focusing on the quarter from March 2016, there were 855 growers samples processed and 1,071 research. Since the start of the current grant in July 2015, the lab has received 3,093 growers samples, which is even higher than the expected increases in sample volume. The HLB Diagnostic Lab continues to offer the service of detection of CLas in psyllids as funded in this grant. Current methods of sample processing have become streamlined and therefore seen no change in procedure.
Liberibacter crescens strain BT-1, has recently been cultured under laboratory conditions and is the model system for our studies. We had previously reported that we had initiated studies using the well-studied R2 tailocin to design fusions between N-terminal tail fiber region of the R2 tailocin and C-terminal portions of tail spike from BT-1 prophages. Tailocins are protein assemblages that function like the tails of phages, by adsorbing to the bacterial cell and then puncturing the cell envelope. Unlike phages, tailocins do not have a capsid and thus inject no DNA, instead relying on the membrane puncturing activity to kill the cell. Like phages, tailocins use tail fibers to recognize specific receptors on the target cell surface. Tailocins are thus potent and specific lethal nanoparticles. The assembly of active tailocin requires chaperones specific to the C-terminal and the availability of chaperone can limit tailocin production. In order to understand the protein folding necessary to assemble functional hybrid tailocins, we are fusing different N-terminal region-encoding portions of the tail fiber gene of the well-studied tailocin R2 to a series of C-terminal regions of the P2 tail fiber. This is being accomplished in- trans using a broad host vector with a tac promoter. This series of experiments will allow us to understand the fusion(s) points necessary to construct active tailocins. Using the developed overlay system that incorporates several modifications of medium BM7, we are testing broad host phages, tailocins and environmental samples to identify phages active against L. crescens.
The goal of this project is to find non-copper treatment options to control citrus canker, caused by Xanthomonas citri ssp. citri (Xcc). The hypothesis of the proposed research is that we can control citrus canker by manipulating the effector binding element (EBE) of citrus susceptibility gene CsLOB1, which is indispensable for citrus canker development upon Xcc infection. We have previously identified that CsLOB1 is the citrus susceptibility gene to Xcc. The dominant pathogenicity gene pthA4 of Xcc encodes a transcription activator-like (TAL) effector which recognizes the EBE in the promoter of CsLOB1 gene, induces gene expression of CsLOB1 and causes citrus canker symptoms. To test whether we can successfully modify the EBE in the promoter region of CsLOB1 gene, we first used Xcc-facilitated agroinfiltration to modify the PthA4-binding site in CsLOB1 promoter via Cas9/sgRNA system. Positive results have been obtained from the Cas9/sgRNA construct, which was introduced into Duncan grapefruit. We analyzed the Cas9/sgRNA-transformed Duncan grapefruit. The PthA4-binding site in CsLOB1 promoter was modified as expected. Currently we are using both Cas9/sgRNA and TALEN methods to modify EBE in sweet orange using transgenic approach. Transgenic Duncan and Valencia transformed by Cas9/sgRNA has been established. Totally four transgenic Duncan grapefruit lines have been acquired and confirmed. Mutation rate for the type I CsLOB1 promoter is up to 82%. GUS reporter assay indicated mutation of the EBE of type I CsLOB1 promoter reduces its induction by Xac. The transgenic lines are being grafted to be used for test against citrus canker. In the presence of wild type Xcc, transgenic Duncan grapefruit developed canker symptoms 5 days post inoculation similarly as wild type. An artificially designed dTALE dCsLOB1.3, which specifically recognizes Type I CsLOBP, but not mutated Type I CsLOBP and Type II CsLOBP, was developed to evaluate whether canker symptoms, elicited by Xcc.pthA4:dCsLOB1.3, could be alleviated on Duncan transformants. Both #D18 and #D22 could resist against Xcc.pthA4:dCsLOB1.3, but not wild type Xcc. Our data suggest that activation of a single allele of susceptibility gene CsLOB1 by Xcc-derived PthA4 is enough to induce citrus canker disease and mutation of both alleles of CsLOB1, given that they could not be recognized by PthA4, is required to generate citrus canker resistant plants. The data has been published by Plant Biotechnology Journal Transgenic Valencia transformed by Cas9/sgRNA has been established in our lab. Three transformants have been verified by PCR. The PthA4-binding site in CsLOB1 promoter was modified as expected, only one transgenic line seems to be bi-allelic mutant. The EBE modifed transgenic line is being evaluated for resistance against Xac. One Cas9/sgRNA binary vector, which is designed to target CsLOB1 open reading frame, designated as GFP-Cas9/sgRNA:cslob1, was used to transform Duncan grapefruit epicotyls by Agrobacterium-mediated method. Several transgenic citrus lines were created, verified by PCR analysis and GFP detection. Cas9/sgRNA:cslob1-directed modification was verified on the targeted site, based on the direct sequencing of PCR products and the chromatograms of individual colony. Upon Xcc infection, some transgenic lines showed delayed canker symptom development. We are currently analyzing the genome modified plants using transgenic approaches including off-targets. To generate non-transgenic DNA free canker resistant citrus, Cas9 containing nucleus localization signal was overexpressed and purified. The purified Cas9 showed activity in cutting target sequence and will be used to generate canker resistant plants.
Previously, we have shown that the Genome of Candidatus Liberibacter asiaticus (CLas) possess luxR gene that encodes LuxR protein, one of the two components typical of bacterial “quorum sensing”. However, the genome lacks the second component; luxI that produce Acyl-Homoserine Lactones (AHLs) suggesting that CLas has a solo LuxR system. In the current project, we have only one objective: study the effect of AHL-producing citrus plants on the pathogenicity of CLas. We have selected different Lux-I genes from different bacteria expressing structurally different AHLs. Due to the difficulties we faced in growing different bacteria to extract DNA for LuxI isolation, we changed our strategy to synthesizing these genes (G-Blocks). Synthesized genes include 1- Agrobacterium tumefaciens N-(B-oxo-octan-1-oyl)-L-homoserine lactone(Hwang et al., 1994) TraI Accession number#L22207.1 2- Pseudomonas fluorescens Six acyl-HSLs, including the 3-hydroxy forms, N-(3-hydroxy-hexanoyl)-L-homoserine lactone(3-OH-C6-HSL), N-(3-hydroxy-octanoyl)-L-homoserine lactone (3-OH-C8-HSL), and N-(3-hydroxy-decanoyl)-L-homoserine lactone (3-OH-C10-HSL); the alkanoyl forms hexanoyl-homoserine lactone (C6-HSL) and octanoyl-homoserine lactone (C8-HSL)(Khan et al., 2005) PhzI Accession number#AAC18898.1 3-Rhizobium leguminosarum bv. viciae 3-OH-C14:1-HSL (Wilkinson et al., 2002, Edwards et al., 2009) CinI Accession number#AF210630 4-Serratia liquifaciens N-(butyryl)-L- homoserine lactone (BHL) (Oulmassov et al., 2005) SwrI Accession number#U22823.1 5- Vibrio fischeri OHHL (Schaefer et al., 1996) LuxI Accession number#AAD48474.1 We already started the first step of expressing these genes by inserting them in CTV-based vector prior to the infiltration inside citrus trees. Our aim is to interfere with CLas signaling by expressing AHL which will enhance the bacterial aggregation and attachment and results in localization of the bacterium in certain branches.
Our project is based on the biology of quorum sensing of Candidatus Liberibacter asiaticus. The system here the two components system LuxR-AHL. The Genome of Candidatus Liberibacter asiaticus (CLas) revealed the presence of luxR that encodes LuxR protein, one of the two components typical of bacterial “quorum sensing” or cell-to-cell communication systems. Interestingly, the genome lacks the second components; luxI that produce Acyl-Homoserine Lactones (AHLs) suggesting that CLas has a solo LuxR system. In the current project, we will test the effect of AHL-producing citrus plants on the pathogencity of CLas. We have selected different Lux-I genes from different bacteria expressing different AHLs. In order to isolate the genes, we will obtain bacteria from the American type culture collection including Agrobacterium tumefaciens, Aeromonas hydrophilia, Agrobacterium vitis, Burkholderia cepacia, Chromobacterium violaceum, Panteoa stewartii, Pectobacterium carotovorum, Pseudomonas aeruginosa, Pseudomonas aeruginosa, Pseudomonas fluorescens, Rhizobium leguminosarum bv. Viciae, Rhizobium leguminosarum bv. Viciae, Rhizobium leguminosarum bv. Viciae, Rhodobacter sphaeroides, Serratia liquifaciens, Sinorhizobium meliloti, Vibrio anguillarum, Once we receive the bacteria we will culture them prior to insolate the genes by PCR. We will insert genes in CTV-based vector prior to the infiltration inside citrus trees. Our aim is to interfere with CLas signaling by expressing AHL which will enhance the bacterial aggregation and attachment and results in localization of the bacterium in certain branches.
This overall 3 year project was focused on determining the optimum combination of chemotherapy, thermotherapy, and nutrient therapy that can be registered for use in field citrus and control HLB. In this quarter (Jan 2016 to March 2015), we continue to evaluate 1) the effect of Pen and SD on control of HLB disease by gravity bag infusion in the field; 2) the efficiency of effective chemical compounds (Pen, SDX, Pcy and Carv) against HLB disease by gravity bag infusion; 3) the effectiveness of a combination of chemotherapy, thermotherapy and nutrient therapy against HLB in the field trials; 4) the efficacy of the new adjuvants to improve the uptake of antimicrobials. The chemical compounds (Pen and EBI-602) and additional nutrients were applied to the heat-treated citrus for three times by foliage spray, using our optimized nano-delivery system. The preliminary results showed that Pen was the more effective to control Las bacterium than EBI-602. The disease severity index (SDI) decreased by 6% after applied with Pen. The integrated practices (antimicrobial treatment coupled with heat treatment and nutrition fertilization) could decrease the fruit drop by 10~20 %, increase the fruit and juice weight by 3~13 %, and decrease the ratio of brix to acid by 0.2~5.0 %. The preliminary results from the other five antimicrobials (SD, Pen, SDX, Pcy and Carv) applied by gravity bag infusion showed that there were not different in the Las bacterial titers among the treated antimicrobials. Compared to the untreated plants, all antimicrobials reduced the Las bacterial titers, especially PEN. Both SD and Pen reduced the DSI through two years application. In last quarter, we tried to evaluate two new adjuvants (Bio and MF200) for improving the effectiveness of Pen by foliar spray. The preliminary results indicated that Pens formulated in both Bio and MF200 decreased the Las bacterial titers a lots. Ten antimicrobials were prepared in two different concentrations of the nano formulations (0.1 % and 1.0 %) in the greenhouse test. The Ct values kept over 36.o in the PEN-treatment. In next quarter, we will keep our application. Pcy and Carv will be changed application from trunk-injection to foliar spray. One papers has been published in the Crop Protection.
OVERVIEW: The Budwood Certification Program continues to build and develop the Foundation and Increase tree collection. Demand for budwood continues to increase and is anticipated to exceed 250,000 this year. PROGRAM OPERATIONS Texas Citrus Budwood Certification Program: 1. Budwood sales during the period were 229,664. Rio Red Grapefruit, Olinda and Standard Valencia Oranges, and Improved Meyer Lemon were the major varieties requested. 2. Much needed maintenance has been conducted on the main screenhouses. The old double poly roof on Screenhouses 1 thru 4 was replaced in November. A new layer of insect screen followed by a new layer of poly followed by aluminet shade cloth was put in place. 3. New roll-up curtains were installed in January to help protect the trees in the screenhouses during inclimate weather. 4. The project to upgrade Phase I of the Screen Structures will begin in the early summer. All existing trees will be removed and the screen and wood frame will be replaced with new galvanized metal frame and new insect screen. New Foundation trees will be planted in the structure when completed. 5. The Budwood Program Database continues to expand and is a key component in the management of the Texas program. The database program contains all records and information for all budwood production and sales, all foundation and increase trees, test results, and chemical applications. This database has the ability for an unlimited range of reporting information on the Texas program, including budwood orders, supply management, sales, testing, pesticide and fertilizer applications, budget analysis, and records needed for all compliance with TDA and USDA regulations. Stephenville Foundation Screenhouse : 1. New, clean Foundation trees continue to be added to established Foundation trees at the Stephenville greenhouse location. There are currently 75 Foundation trees consisting of over 60 different citrus species. Additional trees will be added as they are propagated to bring the total capacity to 100. These trees are being maintained as a reserve source of clean material for the Texas citrus industry. Texas Germplasm Introduction Program 1. The quarantine facility for the Germplasm Introduction Program is nearing completion and will be ready for USDA and TDA certification as a quarantine facility this summer. 2. Mark VanNess (Program Manager) and Sonia Del Rio (Lab Technician) visited the California CCPP in the fall to observe and receive training in their shoot-tip grafting program. Mark and Sonia visited Florida’s Germplasm Introduction Program in January and March to receive training and observe the Florida program. The new Texas program will operate based on the practices learned from both the California and Florida programs. Diagnostic Lab Testing – Foundation/Increase Trees: 1. All Foundation and Increase trees located in Weslaco and the Stephenville greenhouse were tested for HLB in October, 2015, and will be tested again for HLB and CTV in April, 2016. All tests were negative. 2. All Foundation Trees underwent virus/viroid testing in the fall of 2015. All test on all viruses and viroids were negative.
The first quarter of 2016 was very strenuous for Core Citrus Transformation Facility (CCTF). Two out of five employees left the facility in January and were eventually replaced by the new members of staff. CREC Center Director informed CCTF of possible move of the lab to the new site in March. Although March 24th was anticipated date for the move, that did not happen and CCTF still operates from its present location. CCTF received unprecedented number of orders (26) within the last quarter. Such a high volume of incoming orders called for an additional increase in production capacity of CCTF. I have purchased necessary consumables and tools for this transition and have taken steps to gradually ramp-up the input of starting material for experiments. However, uncertainty associated with possible move to new location prevented search for additional employees. I expect new recruit to begin working in the month of April. Partial increase in work load in March was little overwhelming for the present labor force and resulted in loss of some transgenic shoots and plants. I hope that when all aspects of CCTF functioning stabilize, so will the level of our production. The newly announced date for the move to new location is June 17th and I will try to organize it in such a way so that it will affect productivity of CCTF to the least possible extent. Between January and April, CCTF produced 57 plants. These plants belong to newer orders placed within the last 12-15 months. Four of the produced plants were Valencia oranges, three were Pineapple sweet oranges, eight were Carrizo citrange, and the rest were Duncan grapefruit. Transgenic rootstock plants carrying NPR1 produced in our facility are still in our greenhouse. They are at the stage when they could easily be propagated by cuttings. I am awaiting further instructions on what to do with these plants.
This is the final report for Project 13-926.3C. Over the last two years, we have been able to confirm that Citrus Leafminer Mating Disruption is a viable control solution for Phyllocnistic citrella. Data from 734 Citrus Holdings, Indian River Exchange Packers and The Packers of Indian River, all distributed across the state, confirmed that the performance of DCEPT CLM is in fact tremendously beneficial to CLM control. Performance across all of the trials, including monitoring data and damage evaluations, was consistent. This evidences the consistent and predictable performance of pheromone based mating disruption. After evaluating all of the data from this project, we can confirm that DCEPT CLM will quickly reduce populations of CLM and maintain control for up to twelve weeks. For these twelve weeks, pheromone monitoring traps will capture little to no adult CLM males because the populations are being controlled by the pheromone being released by DCEPT CLM. Additionally, damage will follow the same trend as the traps and will be equal or lower than farms that utilize conventional spray programs that spray as often as every two weeks. We also believe that DCEPT CLM, as a sole control input, can control CLM using a single application without CLM targeted conventional insecticide sprays for up to twelve weeks. Going forward, for farmers that plan to proceed with a single application of DCEPT CLM, our recommendation to farmers will be to target DCEPT CLM applications at the most important ten to twelve weeks of the citrus growing season. This will allow the farmer to have flexibility and choice, one where they can mix and match control strategies. For example, a farmers can protect their citrus during the highest pressure portion of the season with a DCEPT CLM application and protect the rest of the season with conventional insecticide sprays. The farmer could also apply DCEPT CLM twice which will cover nearly the entire susceptible season of Florida citrus. Unlike insecticide treated blocks, DCEPT CLM applications will also maintain the major advantage of residual performance. Although DCEPT CLM will not be able to eliminate damage after twelve weeks in the field, it will still maintain residual performance that keeps CLM populations lower than blocks treated with conventional insecticides. For example, at The Packers of Indian River, blocks that were treated with DCEPT CLM were able to maintain monitoring trap captures three to four times lower than blocks treated with insecticides every two weeks for an additional four weeks. Lastly, these three trials confirmed that DCEPT CLM performance can be maintained at essentially the same levels with applications in farms with tree densities ranging from 125-175 trees per acre. This confirmation is important because it will allow Florida farmers with various tree densities to adopt CLM mating disruption. We will now recommend that all farmers proceed with a minimum application rate of 125 DCEPT CLM per acre.
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