The objectives of this project are: 1. Evaluate psyllid populations, HLB incidence and intensity, gene expression, tree growth, soil moisture, soil nutrients, foliar nutrients, and eventually yield in newly planted citrus blocks, 2. Assess separate contributions of vector control and foliar nutritional to the above parameters, 3. Evaluate effectiveness of reflective mulch to repel ACP and reduce incidence of HLB, 4. Provide economic analysis of costs and projected benefits, and 5. Extend results to clientele. Management of the experimental block (‘Hamlin’ orange on Carrizo citrange planted 3-4 July on a 10-acre block at A. Duda & Sons, Inc. farm in Hendry County was turned over to the grower having completed the planned 3 years. Trees will still be harvested by plot this year and the data used to complete a planned paper. A field day was conducted in conjunction with the company on 19 June attended by about 25 citrus growers and 14 other participants to present the results of the 3-year study. Part of the study was included in a dissertation completed by now Dr. Scott Croxton who graduated this summer. Another trial planted 5 May 2013 at SWFREC consists of 24, 250 ft. rows of Ray Ruby grapefruit on smooth flat Seville divided into 8 main plots, half receiving organic amendments since 1993, including 12 tons/acre composted yard trimming waste (YTW) applied in a 6 ft swath to the plant drill prior to planting. The plot had been underlain with drain tile and is flat except for 6 in high beds 32 inches wide on 18 ft centers covered with polyethylene film mulch and irrigated through two drip tape lines. Each 3-row plot is divided into 2 subplots: whiteface or metalized mulch. Soil bed temperatures have been about the same under white or reflective mulch over the last 3 months. Trunk x-section area, canopy area and height have been consistently for trees on compost compared to trees with no compost whereas growth differences between white and reflective mulch have not been significant. Sticky card captures have been 5 times greater on white mulch, but twice as high on compost compared to no compost. Infested flush followed similar trends. Incidence of HLB was 4% on metalized mulch compared to 13% on white (over both compost treatments) and 11% on compost compared to 6% on no-compost over both mulch treatments. Flooding caused considerable foot rot this summer. Soil samples were taken for microbiota analysis in Sep 2015. Plastic mulch was removed in Oct to evaluate the planned transition to microjet irrigation. Fertigation is used at the Vero Beach experiment usingliquid 6-0-8 (with minors) fertilizer injected continuously with all irrigation events. Herbicides are applied via herbicide boom to both the composted Urban Plant Debris (UPD) and the Bare Ground (BG) treatments in a 9 ft band 2.5 ft from edges of the 4 ft wide Metallized Reflective Mulch (MRM) ~2.5 ft.) beds to create 9 ft. wide weed free zone. Tree opening ins MRM are treated with spot sprays of glyphosate. Belay 2.13 SC and Admire Pro) were applied by soil drench for ACP control and Ridomil Gold SL using a calibrated electric timed liquid applicator. Insects and mites are monitored weekly. ACP adults and nymphs, Diaprepes Root Weevil, and orange dog larvae have been half or less on MRM than other treatments whereas Sri Lankan weevils and citrus leafminer were unaffected. Soil moisture readings as percent Volumetric Water Content (%VMC) for each treatment are also recorded.
Our project aims to provide durable long term resistance to Diaprepes using a plant based insecticidal transgene approach. In this quarter, several transgenic lines expressing each of our test genes have been regenerated and many of them have rooted. The rooted material has been successfully acclimatized to the greenhouse for growth and further testing. We are in the process of confirming the gene expression levels in these transgenic lines to isolated lines that have excellent gene expression. These selected lines will be propagated for subsequent evaluation with Diaprepes neonates.
Seasonal root sampling continues in two field sites for root density and root growth. We are collecting a second year of root growth data from Hamlin/Swingle and have 1 year of root growth data on Valencia/Swingle. Results so far emphasize the need to use treatments that improve root longevity as the main method of managing HLB root loss. Root growth stimulation is unlikely to improve root density. Preliminary tests of root tubes are complete and easy observation of root growth and root dieback have been confirmed. This will allow for more rapid quantification of root growth and death using nondestructive sampling so that the same roots can be monitored over time. The root tubes have been installed in treated and untreated plots of a thermotherapy trial to combine efforts and provide both basic disease information and treatment effects on the root system. Sampling at a rootstock trial site continues. Only one rootstock tested to date has shown a significant difference in response to HLB. Initial sampling at a second rootstock trial site has begun. This will allow comparison of some of the rootstocks on Ridge and Flatwoods soils. We continue to monitor the most promising rootstocks identified in the field trial to HLB using rhizotrons in the greenhouse. A second set of rootstocks is ready for testing in rhizotrons as soon as greenhouse space is available from the breakdown of the first set. Changes in image acquisition and soil type have been tested to improve the data analysis steps in these followup experiments. The first experiment is ready for takedown, but is awaiting some final root collections for phytohormone and microscopic analysis. Method development to characterize the mechanism by which Liberibacter causes root death continues with a second round of samples to be collected shortly with improved tissue selection to avoid variability found in the first attempt at microscopy analysis of cell death.
Seasonal root sampling continues in two field sites for root density and root growth. We have completed a second year of root growth data from Hamlin/Swingle and are continuing to collect a second year of root growth data on Valencia/Swingle. Results continue to emphasize the need to use treatments that improve root longevity as the main method of managing HLB root loss. Additional root growth appears to occur at the expense of older roots and is unlikely to provide sustained improvement in root density. Root growth, lifespan, and death measurements are being made from root tubes installed in treated and untreated plots of a thermotherapy trial to combine efforts and provide both basic disease information and treatment effects on the root system. Visual observation shows that the method is working well, but data analysis has been delayed due to an unexpected software glitch that the company is currently working on fixing. Only one rootstock tested to date has shown a significant difference in response to HLB. It remains the only rootstock with significantly better root density (increased) when infected by HLB. Root loss has not been observed yet in this rootstock. We are planning to collect fruit drop data this year on the rootstocks that have been measured to see if the improved root density of this rootstock correlates with better fruit retention. We continue to monitor the most promising rootstocks identified in the field trial to HLB using rhizotrons in the greenhouse. A second set of rootstocks is ready for testing in rhizotrons as soon as greenhouse space is available from the breakdown of the first set. Changes in image acquisition and soil type have been tested to improve the data analysis steps in these followup experiments. The first experiment is ready for takedown, but is awaiting some final root collections for phytohormone and microscopic analysis. A second set of root samples was collected for phytohormone analysis. Based on initial tests we have collected multiple growth stages and ages of roots to get a larger picture of the effects of HLB on phytohormones. Results from the initial run suggested that contrary to our hypothesis it was not the primary phytohormones that were altered by Las infection. One expected phytohormone was not detected in any sample and we are working to resolve this lack of detection. More microscopy samples have been collected and we are awaiting processing before we perform the final breakdown.
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 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.
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