Horticultural & Management


A secure site for testing transgenic and conventional citrus for HLB and psyllid resistance

Report Date: 10/14/2015   Project: 607   Year: 2015

A secure site for testing transgenic and conventional citrus for HLB and psyllid resistance

Report Date: 10/14/2015
Project: 607   Year: 2015
Category: Horticultural & Management
Author: Ed Stover
Sponsor: Citrus Research and Development Foundation

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 almost six years. 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 (Grosser, personal comm.). 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 (Lee et al., in preparation, with the last samples collected this week), and P. trifoliata also displayed reduced colonization by ACP (Westbrook et al., 2011). 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.).



Support role of the Citrus Core Transformation Facility remains crucial for research leading to production of Citrus plants that may be tolerant or resistant to diseases

Report Date: 10/12/2015   Project: 15-033C   Year: 2015

Support role of the Citrus Core Transformation Facility remains crucial for research leading to production of Citrus plants that may be tolerant or resistant to diseases

Report Date: 10/12/2015
Project: 15-033C   Year: 2015
Category: Horticultural & Management
Author: Vladimir Orbovic
Sponsor: Citrus Research and Development Foundation

For the last three months, Core Citrus Transformation Facility (CCTF) continued to provide its service for production of transgenic Citrus plants. Within this period of time, there were only two new orders placed but three clients requested work on three previously placed orders bringing total number to five. Probably as a result of recent Knowledge Mapping meeting organized by CRDF there is an increased interest for transgenic plants and CCTF was already informed of eight new incoming orders. That does not include additional orders from another client with whom CCTF manager communicated for the last three months. Forty plants were produced during the last quarter which represents lower productivity than usual. Partially, this is the result of work on group of three orders that have not yielded any plants. Another reason is the low quality of seeds used to obtain seedlings as starting material for experiments. High majority of produced plants belong to eight orders placed within the last 12-15 months. Five plants belong to three older orders. Most of the transgenic plants are Duncan grapefruit and Valencia orange and one of them is Carrizo citrange. The work has begun on determination of level of expression of AtNPR1 gene in rootstock plants produced for CRDF. Once all plants produced by CCTF and Mature tissue lab are processed, those with the highest expression levels will be selected for further use and propagated.



Contined funding for the mature citrus facility to produce disease tolerant, transgenic citrus.

Report Date: 10/07/2015   Project: 15-045C   Year: 2015

Contined funding for the mature citrus facility to produce disease tolerant, transgenic citrus.

Report Date: 10/07/2015
Project: 15-045C   Year: 2015
Category: Horticultural & Management
Author: Janice Zale
Sponsor: Citrus Research and Development Foundation

Mature plant production continues with various genetic constructs with reporter genes from Drs. Dutt, McNellis and Wang. Additional scientists have expressed interest in our services. Transformation efficiencies have significantly increased with reporter genes. We are also trying to further increase Agrobacterium transformation efficiencies of mature citrus by incorporating vacuum infiltration and sonication treatments. These treatments significantly increased transformation efficiencies in immature citrus. In addition to Agrobacterium, we can now supplement plant production with plants produced using biolistics. Scions and rootstocks have been micropropagated (budded scions and rooted cuttings) into three replicates for one location. It remains to be determined whether we will continue micropropagation for replicates at additional locations. We have almost concluded screening Dr. Orbovic’s putative transgenics with qPCR to identify high-expressing lines. This work should be finished by the end of this month. I am switching to citrus pots in the growth room, which can be planted to higher densities than current planting densities. Recently we discovered that mature scion grows significantly faster after budding if the rootstock is not cut-off after the buds break.



High-Throughput Inoculation of Transgenic Citrus for HLB Resistance

Report Date: 10/05/2015   Project: 15-016   Year: 2015

High-Throughput Inoculation of Transgenic Citrus for HLB Resistance

Report Date: 10/05/2015
Project: 15-016   Year: 2015
Category: Horticultural & Management
Author: David Hall
Sponsor: Citrus Research and Development Foundation

This project (Hall-15-016) is an extension of a project that recently came to a close (Hall-502). The driving force for this project is the need to evaluate citrus transformed to express proteins that might mitigate HLB, which requires citrus be inoculated with CLas. USDA-ARS-USHRL, Fort Pierce Florida is producing thousands of scion or rootstock plants transformed to express peptides that might mitigate HLB. The more rapidly this germplasm can be evaluated, the sooner we will be able to identify transgenic strategies for controlling HLB. The purpose of this project is to support a high-throughput facility to evaluate transgenic citrus for HLB-resistance. This screening program supports citrus breeding and transformation efforts by Drs. Stover and Bowman. Briefly, individual plants to be inoculated are caged with infected psyllids for two weeks, and then housed for six months in a greenhouse with an open infestation of infected psyllids. Plants are then moved into a psyllid-free greenhouse and evaluated for growth, HLB-symptoms and Las titer, and finally the plants are transplanted to the field where evaluations of resistance continue. CRDF funds for the inoculation program cover the costs associated with establishing and maintaining colonies of infected psyllids; equipment such as insect cages; PCR supplies for assays on psyllid and plant samples from infected colonies; and two GS-7 USDA technicians. A career technician is assigned ~50% to the program. USDA provides for the program two small air-conditioned greenhouses, two walk-in chambers, and a large conventional greenhouse. Currently 18 individual colonies of infected psyllids are maintained. Some of the individual colonies are maintained on CLas-infected lemon plants while others are maintained on CLas-infected Citron plants. Update: Two technicians funded by the grant were hired during August and are being trained on how to establish and maintain colonies of infected psyllids, how to conduct qPCR assays on plant and psyllid samples, and how to run the inoculations. As of March 31, 2015, a total of 7,448 plants have passed through inoculation process. A total of 148,960 psyllids from colonies of CLas-infected ACP have been used in no-choice inoculations.



Role of Vector Management and Foliar Nutrition in Prevention and Mitigation of HLB in New Citrus Plantings

Report Date: 10/01/2015   Project: 447   Year: 2015

Role of Vector Management and Foliar Nutrition in Prevention and Mitigation of HLB in New Citrus Plantings

Report Date: 10/01/2015
Project: 447   Year: 2015
Category: Horticultural & Management
Author: Phil Stansly
Sponsor: Citrus Research and Development Foundation

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 Dr. Scott Croxton who graduated in summer 2015. 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. All evaluated soil parameters parameters were significantly different between compost and no compost: 5.6 vs 6.9, organic matter (%) 1.29 vs 3.13, CEC (meq/100 g) 1.19 vs 5.73, P, K, Ca and MG (mg/kg) 32.05 vs 234.3, 8.73 vs 19.4, 414.6 vs 2071.8, 27.0 vs 113.5 respectively. 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 was divided into 2 subplots: whiteface or metalized mulch. Soil bed temperatures between Jul and Dec 2015 tended to be in non-compost than compost and greater under metalized mulch compared to white mulch: averaging 80.43 (metalized non compost) 78.85 (white non compost, 79.07 (metalized compost) and 77.73 (white compost) 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. Trunk x-section area, canopy area and height have increased significantly more for trees on compost compared to trees with no compost. From Jul 2013 through Jul 2015, non compost trunk area grew from 65 to 2407 mm compared to 61 to 3,333 mm2 with compost, canopy area (m2) from 0.43 to 3.69 m2 non compost vs 0.65-5.34 m2 compost, and tree height from 1.13 to 2.02 m non compost compared to 1.17 to 2.22 m compost. In contrast, growth differences between white and reflective mulch were not significant. Flooding and over irrigation 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. Constraints on increased compost use lie with short supply rather than grower acceptance, in spite of 3 major suppliers in the local tri-county area including a huge facility run by Lee County. One grower even asked me to stop promoting use of compost in citrus because supply was not keeping up with demand.



Role of Vector Management and Foliar Nutrition in Prevention and Mitigation of HLB in New Citrus Plantings

Report Date: 10/01/2015   Project: 447   Year: 2015

Role of Vector Management and Foliar Nutrition in Prevention and Mitigation of HLB in New Citrus Plantings

Report Date: 10/01/2015
Project: 447   Year: 2015
Category: Horticultural & Management
Author: Phil Stansly
Sponsor: Citrus Research and Development Foundation

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.



Diaprepes control using a plant based insecticidal transgene approach

Report Date: 09/29/2015   Project: 925   Year: 2015

Diaprepes control using a plant based insecticidal transgene approach

Report Date: 09/29/2015
Project: 925   Year: 2015
Category: Horticultural & Management
Author: Manjul Dutt
Sponsor: Citrus Research and Development Foundation

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.



Understanding and reducing early root loss in HLB affected trees

Report Date: 09/28/2015   Project: 732   Year: 2015

Understanding and reducing early root loss in HLB affected trees

Report Date: 09/28/2015
Project: 732   Year: 2015
Category: Horticultural & Management
Author: James Graham
Sponsor: Citrus Research and Development Foundation

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.



Understanding and reducing early root loss in HLB affected trees

Report Date: 09/23/2015   Project: 732   Year: 2015

Understanding and reducing early root loss in HLB affected trees

Report Date: 09/23/2015
Project: 732   Year: 2015
Category: Horticultural & Management
Author: James Graham
Sponsor: Citrus Research and Development Foundation

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 Texas Citrus Budwood Certification Program 2014-2015 - FINAL REPORT

Report Date: 09/13/2015  

The Texas Citrus Budwood Certification Program 2014-2015 - FINAL REPORT

Report Date: 09/13/2015
Category: Horticultural & Management
Author: John da Gra�a
Sponsor: Citrus Research and Development Foundation

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 Texas Citrus Budwood Certification Program 2014-2015 - FINAL REPORT

Report Date: 09/13/2015  

The Texas Citrus Budwood Certification Program 2014-2015 - FINAL REPORT

Report Date: 09/13/2015
Category: Horticultural & Management
Author: John da Graca
Sponsor: Citrus Research and Development Foundation

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.



Development of Technologies Important for Creation and Commercialization of Transgenic HLB Resistant Citrus

Report Date: 08/30/2015   Project: 749   Year: 2015

Development of Technologies Important for Creation and Commercialization of Transgenic HLB Resistant Citrus

Report Date: 08/30/2015
Project: 749   Year: 2015
Category: Horticultural & Management
Author: Yi Li
Sponsor: Citrus Research and Development Foundation

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.



Engineering PAMP-receptor mediated broad spectrum resistance to HLB and canker

Report Date: 07/31/2015   Project: 556   Year: 2015

Engineering PAMP-receptor mediated broad spectrum resistance to HLB and canker

Report Date: 07/31/2015
Project: 556   Year: 2015
Category: Horticultural & Management
Author: Diana Horvath
Sponsor: Citrus Research and Development Foundation

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.



Monitoring streptomycin resistance in Xanthomonas citri in support of FireWall registration for canker

Report Date: 07/29/2015   Project: 730   Year: 2015

Monitoring streptomycin resistance in Xanthomonas citri in support of FireWall registration for canker

Report Date: 07/29/2015
Project: 730   Year: 2015
Category: Horticultural & Management
Author: James Graham
Sponsor: Citrus Research and Development Foundation

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.



Calcium carbonate may reduce root health and exacerbate HLB expression

Report Date: 07/29/2015   Project: 731   Year: 2015

Calcium carbonate may reduce root health and exacerbate HLB expression

Report Date: 07/29/2015
Project: 731   Year: 2015
Category: Horticultural & Management
Author: James Graham
Sponsor: Citrus Research and Development Foundation

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.