The Budwood Certification Program provided over 246,000 buds from 67 different varieties to the industry in FY 2014-2015. This was the largest amount of budwood provided in the last 10 years. All structures are certified by TDA and USDA with monthly inspections. All trees were tested every 6 months for HLB and annually for CTV in accordance with the USDA-APHIS regulations for intrastate movement of quarantined plant material. In addition, the Texas Germplasm Introduction Program is starting up, with a shoot-tip grafting and quarantine facility for introducing clean, pathogen free varieties specific to the Texas Industry. Increase Screenhouses 3 and 4 have over 4,000 potted increase trees. Screenhouse 3 contains newly budded Increase and Foundation trees awaiting transplanting. All trees were tested for HLB in October, 2014 and April, 2015 for HLB and CTV. A total 106,659 buds were cut from Screenhouses 3 and 4. Intensive inspections, pest and fertility management continue to be in place. A total of 117,001 buds were cut from Increase screen structures I, II, and III. As the trees in Screenhouses 3 and 4 develop, the number of buds cut from the screen structures has decreased. All trees were tested in October, 2014 for HLB and in April, 2015 for CTV and HLB. An intensive fertilization, health and pest management program continues to be in place. Screenhouse 1 and 2 are filled to 70% capacity with certified Foundation trees. Additional trees will be added in the fall and winter. All trees were sampled and tested for HLB in October, 2014 and again in April, 2015 for HLB and CTV. All trees have tested negative. All trees are currently undergoing PCR testing for Viruses and Viroids. There are currently 147 Foundation trees in Screenhouse 5. All trees were sampled and tested for HLB in October, 2014 and again in April, 2015 for HLB and CTV. All trees are currently undergoing PCR testing for Viruses and Viroids. The Foundation greenhouse in Stephenville, Texas is the remote site location for reserve Foundation varieties. Currently there are 70 Foundation trees at Stephenville. The greenhouse was certified by TDA in August. Mark VanNess and Sonia del Rio traveled to California s Rubidoux Citrus Quarantine Facility in Riverside California in August for shoot-tip grafting training and also to learn the procedures for establishing a germplasm introduction program in Texas. The TajMahal building has been designated as the facility for growing and indexing the quarantined varieties until they are approved for release by the USDA and TDA. The program will be approved and certified by USDA prior to the start of any shoot-tip grafting. Budwood sales for FY 2014-2015 were 246,075. This exceeds last year s total of 196,080. Rio Red buds totaled 144,139, (59%), down from last year s number of 155,521 Rio Red buds (79%). The number of Olinda Valencia buds cut was 43,200 (18%), the largest number of Olinda s ever cut. More Olinda Increase trees have been budded in anticipation of higher numbers in the future.
The main accomplishments during this quarter: We repeated and confirmed the effects of K and I genes on genetic transformation for cultivars Valencia and Washington oranges and observed drastic increases in transformation efficiencies if compared to a conventional Ti-plasmid vector containing no K or I gene. We have confirmed that the K and I genes can drastically enhance transformation efficiencies of juvenile explants of 5 different citrus cultivars. We started test the effects of the K gene on transformation efficiency of a lemon cultivar. Lemon is difficult for genetic transformation. Our major efforts have been in testing the effects of the K and I genes and other factors on mature tissues. We used K and I genes to do genetic transformation of mature Pineapple orange. The K gene resulted in about two fold increases in transformation efficiency while the I gene produced about three fold increases in efficiency compared to control vector. We have also started testing effects of other factors on transformation of mature tissues in combination with the K gene. We have made some significant progress but microbial contaminations of adult tissues harvested from greenhouse grown trees have sometimes caused problems for us. One example is that we have repeated and confirmed the effects of the transport of an endogenous plant hormone in explants on shoot regeneration efficiency. We observed that manipulating that process improves shoot regeneration and transformation efficiency of juvenile citrus explants. We are testing the effects of the same manipulation on transformation efficiency of adult tissues of citrus.
The overall objective of this project was to use the PAMP receptors EFR and XA21 to engineer citrus plants resistant to both HLB (causal agent Candidatus Liberibacter asiaticus, CLas) and citrus canker (Xanthomonas axonopodis pv citri, Xac). Since neither receptor recognizes a PAMP from CLas, the first objective was to engineer a variant of EFR (EFR+) to recognize the elf18 peptide from CLas. This novel receptor would then be combined with XA21 or an XA21-EFR chimera that recognizes a PAMP from Xac. A number of strategies to engineer an EFR+ variant that recognized elf18-Clas were tested, but none were successful. These included PCR mutagenesis, screening of natural variants in an extensive Arabidopsis accession collection, creating targeted mutations based on the modeled interactions among elf18, EFR, and BAK1, and testing high-throughput strategies such as phage display and FACS. However, we were able to successfully create a functional XA21-EFR chimera. Although we did not generate an EFR+ variant that recognized elf18-CLas, expression of EFR and XA21 may still provide significant protection against citrus canker. Therefore, we transformed three constructs into citrus: EFR alone, EFR with XA21, and EFR with the XA21-EFR chimera described above. The latter two constructs have the potential of providing stronger and more durable resistance than EFR alone. Some transgenic events have been obtained in Duncan grapefruit and sweet orange, and these have been transferred to Dr. Jeff Jones lab at the University of Florida for testing with citrus canker.
For 2015 season, FireWall 50WP (50% streptomycin; Agrosource, Inc.) has been granted an EPA section 18 registration for control of citrus canker in Florida grapefruit. The label for FireWall restricts use to no more than two applications per season. As a condition for FireWall registration, EPA requires monitoring of Xanthomonas citri subsp. citri (Xcc) for streptomycin resistance in treated groves. The objective of this survey is to apply our published protocol (Behlau et al., 2012) for sampling canker-infected grapefruit leaves for isolation and detection of streptomycin resistant Xcc. The survey for 2015 season will be conducted in November 2015 and the report of results submitted to FDACS. Greenhouse trials to measure the residual systemic activity of streptomycin against Xcc in leaves after foliar spray confirm trans-cuticular and upward movement of streptomycin into new foliage via the xylem.
Our survey of 8 ridge groves in Highlands County and 4 flatwoods groves in Hardee Co. with high bicarbonate stress continues. The survey is bimonthly to follow the recovery of these blocks and at harvest to compare 2015 season block yields 2.0 to 2.5 years after acid treatments began. Soil pH continues to range from 5.0-6.0 in the ridge and in the flatwoods groves has dropped after treatments resumed fall 2014. The grower reports that overall yields have improved 10% in flatwoods groves but were down 4% in ridge groves. The additional survey of 5 grove locations initiated January 2015 in the flatwoods with relatively low bicarbonate stress continues. The root zone pH of these groves is less than 6.5 and fibrous root density higher than in high stress flatwoods groves surveyed in 2014. Phytophthora populations are damaging in 3 of the 5 groves in the new survey. To lower the pH or to increase the calcium status, the grower is strongly considering Tiger Sulfur or gypsum amendments according to soil and leaf test.
Our survey of 8 ridge groves in Highlands County and 4 flatwoods groves in Hardee Co. with high bicarbonate stress continues. The survey is bimonthly to follow the recovery of these blocks and at harvest to compare 2015 season block yields 2.0 to 2.5 years after acid treatments began. Soil pH continues to range from 5.0-6.0 in the ridge and in the flatwoods groves has dropped after acidification treatments resumed in fall 2014. The additional survey of 5 grove locations initiated January 2015 in the flatwoods with relatively low bicarbonate stress continues. The root zone pH of these groves is less than 6.5 and fibrous root density higher than in high stress flatwoods groves surveyed in 2014. Phytophthora populations are damaging in 3 of the 5 groves in the new survey. To lower the pH or to increase the calcium status, the grower is considering Tiger Sulfur or gypsum amendments according to soil and leaf test.
For 2015 season, FireWall 50WP (50% streptomycin; Agrosource, Inc.) has been granted an EPA section 18 registration for control of citrus canker in Florida grapefruit. The label for FireWall restricts use to no more than two applications per season. As a condition for FireWall registration, EPA requires monitoring of Xanthomonas citri subsp. citri (Xcc) for streptomycin resistance in treated groves. The objective of this survey is to apply our published protocol (Behlau et al., 2012) for sampling canker-infected grapefruit leaves for isolation and detection of streptomycin resistant Xcc. The survey for 2015 season will be conducted in November 2015 and the report of results submitted to FDACS. Greenhouse trials to measure the residual systemic activity of streptomycin against Xcc in leaves after foliar spray confirm trans-cuticular and upward movement of streptomycin into new foliage via the xylem.
The transgenic plants to be developed for this project are now growing in 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. The plants at both locations are growing well. At Penn State, all the transgenic lines have been successfully propagated as vegetative cuttings. All of the lines growing at Penn State have been found to express the FLT-antiNodT fusion protein, with five of the seven lines expressing very high levels of the protein. We must continue to let these plants grow a bit more before starting the HLB resistance tests. We have initiated a collaboration with Dr. Janice Zale (University of Florida Mature Citrus Transformation Facility, Lake Alfred) to transform varieties important to the Florida citrus industry, including the ‘Valencia’ and ‘Hamlin’ sweet orange varieties and the ‘Citrumello’ rootstock with the FLT-antiNodT expression construct. We have immediately started transformations with the available transformation construct used to transform ‘Duncan’ grapefruit, in plasmid pTLab21. In addition, we are developing an FLB-antiNodT expression cassette in the transformation construct pBI121, which has a history of successful approval for transgenic plant development. In June, Dr. McNellis submitted a Stakeholder Relevance Statement to the USDA Specialty Crop Citrus Disease Research and Extension program to further develop this project. However, a full proposal was not invited. Dr. McNellis will present a poster describing the results of this project to date at the American Phytopathological Society conference in Pasadena, California, August 1-5, 2015.
Currently there are 9 total trails evaluating thermotherapy in the state. 8 of the trials are underway with the 9th yet to begin. 2 of the trials were treated by Dr. Eshani while the remaining 3 trials used private machines. Site 1 Cliff Whitaker – Lake County. This trial was done in conjunction with Dr. Ehsani. There are 20 trees being evaluated in 4 classification: steam, thyme oil, steam and thyme combined, and control. Each group has 5 trees. Measurements being taken are DI, trunk circumference, height, canopy diameter, PCR, and fruit drop counts. Site 2 Larry Davis – Hardee County. I contacted Mr. Davis on 4/3/15 and set up the trial on 4/6/15. There are 24 trees being evaluated. All of the trees are steam treated, larry didn t want to leave any trees untreated. Measurements being taken are DI, trunk circumference, height, canopy diameter, PCR, and fruit drop counts. Site 3 Premier Energy Raley grove in Dundee this trial was set up 5/21/15 and treatment was 5/28/15. There are 40 valencia on Carrizo trees being evaluated, 10 control and 30 treated. Measurements being taken are DI, trunk circumference, height, canopy diameter, PCR, % leaf drop, and fruit drop counts. Wheeler grove in Lake Wales This trial has 15 control trees and 15 treated trees. The trail was set up on 6/9/15 the treatment was done on 6/12/15. Measurements being taken are DI, trunk circumference, height, canopy diameter, PCR, % leaf drop, and fruit drop counts. Dunson grove in Haines City – this trial was set up on 6/8/15. Treatment has yet to be done. This grove is under a less intense management strategy. Measurements being taken are DI, trunk circumference, height, canopy diameter, PCR, % leaf drop, and fruit drop counts. Site 4 Daniel Scott St. Lucie County Trial A – 36 trees (Red grapefruit on Sour) this trial was a comparison of 3 different temps applied at different durations. The grower was trying to determine what combination of time and temperature worked best. Measurements being taken are DI, height, and PCR. Trial B 54 (Red grapefruit on Sour) this trial was a comparison of 3 different temps applied at different durations. The grower was trying to determine what combination of time and temperature worked best. Measurements being taken are DI, height, and PCR. Trail C 35 trees (ray ruby on sour) 20 treated trees and 15 control trees. We will take post treatment PMA samples on 7/23/15. Measurements being taken are DI, trunk circumference, height, canopy diameter, PCR, % leaf drop, and fruit drop counts. Site 5 Uncle Matt s Eddy Block in Winter Garden. This is the only trial that is being conducted in an organic grove. Most of the trees are in poor condition. If thermotherapy works here, I ll have hope for the whole industry. There are 10 control trees and 20 treated trees. The study trees are split into 4 groups: Large treated trees, Small treated trees, Large control trees, and Small control trees. Measurements being taken are DI, trunk circumference, height, canopy diameter, PCR, % leaf drop, and fruit drop counts.
The project has two objectives: (1) Increase citrus disease resistance by activating the natural SAR inducer-mediated defense-signaling pathway. (2) Engineer non-host resistance in citrus to control citrus canker and HLB. For objective 1, we treated citrus plants with the natural SAR inducer using three different approaches: leaf infiltration, foliar spray, and soil drench. Three concentrations were tested: 1, 5, and 10 mM. For leaf infiltration, the infiltrated leaves were inoculated 1 day later with the canker bacterial pathogen; for foliar spray, treated leaves were inoculated 3 days later; and for soil drench, leaves on treated plants were inoculated 7 days later. For each treatment, 5 plants were used. Three leaves on each plant were inoculated and 6 inoculations on each leaf were conducted. A total of 90 inoculations were used for each treatment. Fourteen days after inoculation, numbers of lesions formed on the inoculated leaves were counted. Results showed that all concentrations of the SAR inducer induced strong resistance to citrus canker. The inoculated plants have been cut back. Systemic residual resistance will be tested on the new flushes. We are repeating this comprehensive testing experiment and will identify the most efficient treatment method. For objective 2, transgenic citrus plants expressing the Arabidopsis nonhost resistance genes have been propagated. The progenies are growing in greenhouse and will be tested for disease resistance.
Report for period ending 3/31/15 During the period of 1/1/15 to 3/31/15, Mr. Page assisted with the daily activities associated with CRDF funded field trials. During this reporting period, Mr. Page provided assistance on a daily basis through activities as described in the following: Stayed in constant contact with contracted crop consultants (CC’s) to make sure trials were on schedule, reports were to be submitted on time, and trouble shoot any potential obstacles that might arise in advance. As needed, supplies were ordered and delivered to the CC’s to ensure samples were collected and processed in a timely manner. During this period which coincided with fruit harvest season, Mr. Page worked with the CREC packinghouse manager to coordinate the delivery and analysis of fruit samples from field trials for fruit quality assessments. This involved making sure the proper analyses were planned and fruit was delivered on schedule. For trials where quantification of live vs. dead HLB bacterium assessment was required, coordination was made with the Wang lab (CREC) to ensure the proper protocols were being followed to ensure samples were collected and processed in the appropriate manner. During this time, the large-scale evaluation of new rootstock plantings was underway. Mr. Page worked with the grower-cooperators to make sure plots were planted and flagged to make sure data could be collected in a timely and efficient manner. This involved continued communication with the nurseries and growers to make sure trees were delivered and planted according to schedule. Multiple on-site visits were made to these locations with photo documentation of the entire process. For other ongoing CPDC projects, work continued collecting leaf/root samples, coordination of laboratory testing and fruit analysis was conducted as required. Because of the increasing number of trials and on-site visits required for monitoring and data collection, two new employees were hired to help conducted field trail evaluations.
Report for period ending 6/30/15 (Final Report) During the period of 4/1/15 to 6/30/15, Mr. Page assisted with the daily activities associated with CRDF funded field trials. During this reporting period, Mr. Page provided assistance on a daily basis through activities as described in the following: The major focus of field trials underway at this time were the new large-scale plantings being established. Numerous on-site visits were made as these plantings were being established to ensure plots were appropriately established and marked, tree measurements were made and PCR samples were taken. In one situation, a problem was noted with insufficient tree counts being received from the nursery. Mr Page worked to correct this short-fall in trees delivered to make sure planting stayed on schedule. With the assistance of his newly hired employees, Mr. Page has kept up to date with all the ongoing field trials, collecting tree growth data, PCR analyses, and root and fruit samples required by the established monitoring protocols. Additional trials are being established evaluating GRAS compounds and other treatments for which evaluations have been contracted through the CRDF CPDC. In the coming year, Mr. Page will continue to provide oversight to these ongoing projects to ensure that all required sample analyses, data collection and reports are submitted in a timely manner.
Report for period ending 12/31/14 During the period of 10/1/14 to 12/31/14, Mr. Page assisted with the daily activities associated with CRDF funded field trials. The general duties provided by Mr. Page on this project include: attending routine meetings with Drs. Browning and Syvertsen to provide updates on the status of trials, maintaining constant communication with crop consultants (CC’s) to ensure projects are moving forward, setup contracts with soil testing laboratory, sort out results from testing labs, submission of invoices, contact product reps for material samples, organization of spreadsheets for data analysis, scheduling submission dates for the CC s field trial data and on-site visits to field sites to make sure trials are on schedule and up-to-date. During this reporting period, Mr. Page provided oversight on the numerous ongoing CRDF-CPDC projects. Activities included organizing submission dates for CC’s to submit data, collecting root samples for analysis from CC’s and getting those samples to the appropriate laboratory for testing in a timely manner, working with researchers (in particular Nian Wang) to streamline sample submission for PCR testing, work with researchers to implement the proper canker evaluation protocols for ongoing trial work, travel to meet CC’s to pick up plant samples for testing as needed, on-site visits to field trials to photo-document ongoing trials, take tree growth data and collect leaf samples for PCR analysis.
Report for period ending 12/31/14 During the period of 10/1/14 to 12/31/14, Mr. Page assisted with the daily activities associated with CRDF funded field trials. The general duties provided by Mr. Page on this project include: attending routine meetings with Drs. Browning and Syvertsen to provide updates on the status of trials, maintaining constant communication with crop consultants (CC’s) to ensure projects are moving forward, setup contracts with soil testing laboratory, sort out results from testing labs, submission of invoices, contact product reps for material samples, organization of spreadsheets for data analysis, scheduling submission dates for the CC s field trial data and on-site visits to field sites to make sure trials are on schedule and up-to-date. During this reporting period, Mr. Page provided oversight on the numerous ongoing CRDF-CPDC projects. Activities included organizing submission dates for CC’s to submit data, collecting root samples for analysis from CC’s and getting those samples to the appropriate laboratory for testing in a timely manner, working with researchers (in particular Nian Wang) to streamline sample submission for PCR testing, work with researchers to implement the proper canker evaluation protocols for ongoing trial work, travel to meet CC’s to pick up plant samples for testing as needed, on-site visits to field trials to photo-document ongoing trials, take tree growth data and collect leaf samples for PCR analysis.
Our project aims to analyze gene regulators from Ca. Liberibacter asiaticus by expressing these in the related bacterium, Sinorhizobium meliloti. In previous reports we described initial success in cloning the CLas homolog of RpoH, and in expressing this to examine one phenotype. In our work from January-April 2015, we worked on optimizing expression. In the following section, the abbreviation Sm rpoH1 means the native S. meliloti rpoH gene, specifically the rpoH1 copy which is the most important rpoH gene for the S. meliloti stress response; La rpoH designates the CLas homolog with codon usage optimized for expression in S. meliloti; .rpoH1H2 is a host strain of S. meliloti in which both of the native copies of rpoH have been deleted. The pSRK-Gm vector expressing Sm rpoH1 and La rpoH has a lac promoter (inducible by IPTG) to control rpoH expression. 1. We have strains in which RpoH-controlled genes (targets) are fused with the uidA gene (GUS). We tested the Sm rpoH1 plasmid and optimized La rpoH plasmids in strains with rpoH target gene-uidA fusions in the .rpoH1H2 strain. a. Under inducing conditions (+IPTG), expression of the target gene fusions is much higher when the native S. meliloti rpoH genes are deleted. b. In addition, expression levels are higher with the La rpoH plasmid than with the Sm rpoH1 plasmid (we discussed possible reasons why in Dec 2014 report). c. We determined empirically that the optimum IPTG concentration for induction is 0.5 mM. 2. Another way to optimize expression is to test various ribosome binding site (RBS) sequences. We re-made Sm rpoH1 and codon-optimized La rpoH constructs in the same pSRK-Gm vector, but utilizing the vector RBS instead of the RBS inferred from S. meliloti genes predicted to be highly expressed (published by Schroeder et al., 2005, Appl.Env.Microbiol 71:5858). The goal of this test was to determine whether using a different RBS/cloning site would decrease the leakiness of the lac promoter (as discussed in Sept 2014 report). 3. The S. meliloti .rpoH1H2 mutant does not grow at 37 C. We tested the ability of constructs in (2) to restore growth of the .rpoH1H2 mutant at 37 C IPTG, and to carry out regulation of target genes with target gene-uidA fusion assays. a. In the heat stress assay (complementation of .rpoH1H2), non-induced controls (minus IPTG) show much less growth than induced (plus IPTG) strains. b. Basal (minus IPTG) GUS activity of rpoH target-uidA fusions is lower. c. Therefore, these new constructs are more tightly regulatable than the constructs using the S. meliloti RBS consensus. Several important lessons come from these results. First, we now know that native genes (such as Sm rpoH1 and rpoH2) can confound observation of introduced CLas genes. This may have implications for experimental design in the next group of experiments. Second, we know that the RBS can make a difference in whether we get tight off/on control with IPTG induction of the RpoH. These results will inform our next round of constructs and complementation tests.
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