Citrus trees transformed with a chimera AMP and a thionin alone showed remarkable resistance in citrus canker compared to control. These promising transgenic lines were replicated for HLB challenge. Propagated transgenic Carrizo lines expressing thionin, chimera and control were grafted with HLB infected rough lemon buds. Twelve months after graft inoculation, Las titer was examined and compared in old leaves (most with HLB symptom), young expanded leaves (with or without HLB symptom) and fibrous roots of transgenic and control plants. Our results showed again that transgenic citrus expressing Mthionin has lower Las titer compared to control and transgenic plant expressing chimera. These data suggest transgenic plants expressing thionin are promising for HLB resistance (The manuscript for this research will be published in Frontiers in Plant Biology). Antibody against thionin will be produced for investigating the correlation of thionin expression and HLB resistance. 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. Furthermore, the third generation chimeral peptides were designed based on citrus thionins, the vector construction were finished and citrus transformation are underway. To explore broad spectrum resistance, a flagellin receptor gene FLS2 from tobacco was used to transform citrus. Trees expressing NbFLS2 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 was isolated and Las titer was tested. Our preliminary results showed that transgenic trees expressing NbFLS2 can reduced Las titer. 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 tilter 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.
No new plantings in last quarter, but land is prepared for additional UF trees. 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.).
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 are being used to generate canker resistant plants. We have conducted multiple tries of genome editing using protoplast. Currently, we are optimizing the condition to conduct genome editing using protoplast. We also tested different sgRNAs to generate deletion in the coding region of CsLOB1.
This overall 3 year project was focused on determining the optimum combination of chemotherapy, thermo-therapy, and nutrient therapy that can be registered for use in field citrus and control HLB. In this quarter (April 2016 to June 2016), three foliar sprays of the antimicrobial chemicals (Pen, Pcy, Carv and EBI-602) were applied at a two week interval for all treated trees from March to May, 2016. For gravity bag infusion, two refill applications of Pen, SD and SDX were conducted at two week interval in April, 2016. According to the Field Trial Tree Evaluation Methods developed by CRDF, we investigated tree canopy, tree health, fruit drop and fruit quality and Las bacterial titers by real-time PCR. Fruit quality tests were done on the field trail of combination of chemotherapy, thermo-therapy and nutrient therapy. A total of 50 fruit were harvested from the 3 trees in each trial replicate. Tests were run on 20 fruit from each sample for size, peel color, puncture resistance, fruit weight, juice weight, brix and acid. The tree canopy decline index (DI) was compared between the treated and control plants. Eight mature leaves with petioles from each of the treated and control trees were sampled around the canopy for PCR test. The preliminary results indicated that:1) the integrated practices (antimicrobial treatment coupled with heat treatment and nutrition fertilization) could decrease the fruit drop, increase the fruit and juice weight, and decrease the ratio of brix to acid; 2) compared to the control plants, all antimicrobials reduced the Las bacterial titers, especially PEN. 3) Both SD and Pen reduced the DSI through two years application; 4) two new adjuvants (Bio and MF200) improved the effectiveness of Pen by foliar spray; 5) 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 on our application and prepare the final reports. One papers has been published in
In the second quarter of 2016 Core Citrus Transformation Facility (CCTF) continued to operate without interruption although prospective moving date for the lab was June 17th. Eventually the date was pushed back to July 21st so facility is still in its old location. Due to the very high number of orders placed in the last quarter and increased work load, I have hired one more employee who was trained in the lab during the month of April. This new employee is working full time. However, another employee was taken back from 1.0 FTE to 0.4 FTE at her own request. The number of orders placed at the CCTF remained high. We have received 12 orders within the last 3 months. Seven of those orders were paid in advance although no material associated with transformation was received. Customer just wanted to secure the place in our work schedule for time when they are ready to send us plasmid constructs. The plants produced within the last quarter are almost all from the experiments associated with orders placed within last 9-12 months. We produced 67 plants: nine Carrizo citranges, six Swingle citrumelos, and 52 Duncan grapefruits. 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.
Research has documented how HLB damages the root system before symptoms appear in the canopy. We have revised our model to include symptom development. The model now tracks the inoculum from when it enters the tree by means of Diaphorina citri. The inoculum then travels through the phloem towards the roots when the flushing period is over. Some of the bacteria make it to the roots causing damage to the root system. We have run simulations comparing this new model with previous models that we developed. We have run simulations using the new model to determine the question of how many RNAi constructs can be simultaneously tsted in a 10 acre block. We tentatively conclude that eight constructs can be simultaneously tested. Our model does not include how CLas distributes in a tree. However, recent unpublished work indicates that it may not be important to understand this. According to the experiment, once pathogen has entered the tree, the disease progresses in a way that cannot be stopped by any current countermeasures. The only protection is to prevent infection. The model that we have developed is key to preventing the infection from spreading to a tree. In Brazil, effective management has been put in place that prevents spread of HLB to an extent that the participating groves are profitable.
This project (Hall-15-016) is an extension of a project that came to a close last summer (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 have been fully trained in establishing and maintaining colonies of infected psyllids, conducting qPCR assays on plant and psyllid samples, and running the inoculations. As of June 7, 2016, a total of 8,694 plants have passed through inoculation process. A total of 170,895 psyllids from colonies of CLas-infected ACP have been used in no-choice inoculations. Not included in these counts of inoculated plants and psyllids used in inoculations are many plants inoculated over the past year to assess transmission rates, which has provided insight into the success of our inoculation methods and strategies for increasing success. As reported in the last progress report and reiterated here, research recently showed that seedling citrus with flush is significantly more prone to contracting the HLB pathogen than seedling citrus without flush: Hall, D. G., U. Albrecht, and K. D. Bowman. 2016. Transmission rates of Ca. Liberibacter asiaticus by Asian citrus psyllid are enhanced by the presence and developmental stage of citrus flush. J. Econ. Entomol. 109: 558-563. doi: 10.1093/jee/tow009. Therefore, the program has been changed to ensure that plants to be inoculated have flush. Current research indicates that the no-choice inoculation step used in our program is successful an average of 79% of the time when approximately 70% of ACP placed on a plant test positive for CLas (Ct <36) and have CLas titers of around CT=26 to 29 (success contingent on flush being present on a plant).
This project (Hall-15-016) is an extension of a project that came to a close last summer (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 have been fully trained in establishing and maintaining colonies of infected psyllids, conducting qPCR assays on plant and psyllid samples, and running the inoculations. As of June 7, 2016, a total of 8,694 plants have passed through inoculation process. A total of 170,895 psyllids from colonies of CLas-infected ACP have been used in no-choice inoculations. Not included in these counts of inoculated plants and psyllids used in inoculations are many plants inoculated over the past year to assess transmission rates, which has provided insight into the success of our inoculation methods and strategies for increasing success. As reported in the last progress report and reiterated here, research recently showed that seedling citrus with flush is significantly more prone to contracting the HLB pathogen than seedling citrus without flush: Hall, D. G., U. Albrecht, and K. D. Bowman. 2016. Transmission rates of Ca. Liberibacter asiaticus by Asian citrus psyllid are enhanced by the presence and developmental stage of citrus flush. J. Econ. Entomol. 109: 558-563. doi: 10.1093/jee/tow009. Therefore, the program has been changed to ensure that plants to be inoculated have flush. Current research indicates that the no-choice inoculation step used in our program is successful an average of 79% of the time when approximately 70% of ACP placed on a plant test positive for CLas (Ct <36) and have CLas titers of around CT=26 to 29 (success contingent on flush being present on a plant).
The Mature Citrus Facility has made significant progress producing transgenics for clients as a service although we are attempting to make even greater progress. Since July 1, 2015, ~100 transgenics were produced with Agrobacterium, which exceeds previous production. The increase in productivity is primarily due to superior vectors with reporter genes, stronger promoters driving expression of the nptII selectable marker, and an increase in our micrografting efficiencies to 75 -77%. Our clients include Drs. Grosser, Dutt, Louzada, McNellis, Wang, and Mou. After optimizations for biolistic transformation of mature citrus have concluded, these transgenics will augment those generated with Agrobacterium. Our project objectives of increasing micrografting efficiencies, propagating transgenic events into replicates, applying for external funding, and service work have been met. Service work will continue for the same clients in the next funding cycle. A manuscript describing the biolistic transformation of immature citrus has been published, and another manuscript on the selection of transgenics without reporter genes in temporary immersion bioreactors is being submitted. An additional manuscript is in preparation describing the development of a quantitative in situ 4-MUG assay for transgenic, mature citrus shoots. The Mature Citrus Facility protocols have changed in an effort to speed the growth of mature scions. There is a tremendous growth advantage if rootstocks are not removed. After budding mature buds, rootstocks are left attached for the two flushes of stem growth. Mature buds will break and stems can be used in transformations within 6-8 weeks rather than 12-16 weeks specified in the earlier protocol. We continue to optimize for the PMI selectable marker using biolistics and Agrobacterium transformations. The number of nontransformed, escaped shoots appears to be significantly lower than with nptII as a selectable marker. Various treatments (cold treatments and hormone applications) were tried to in an effort to increase regeneration rates and transformation efficiencies in recalcitrant mature citrus scions, but none were satisfactory. However, a citrus DNA sequence drastically increases the number of transgenics in recalcitrant scions. An expression vector is being prepared to test in co-transformations. New breeder lines (3 sweet orange and 1 grapefruit) were introduced through shoot-tip grafting and are being budded for transformations. Protocols will initially follow those used for Hamlin and Valencia, but might still have to be optimized for these new cultivars. Some clients have asked for each transgenic event to be budded onto immature rootstock into replicates, and then flowering seems to be delayed. Every time mature citrus is budded onto immature rootstock, it is reinvigorated and this may potentially delay flowering. An experiment is being conducted to determine how many months flowering is delayed by grafting flowering tissue onto immature rootstock. This result will influence our recommendations to clients. Our lab will be moving to the packinghouse in July, 2016 in order to fix the AC in our current lab. This move will cause disturbances to plant production, but we will do everything within our power to minimize disturbances to the mature citrus transformation pipeline.
The project has three objectives: (1) Confirm HLB resistance/tolerance in transgenic citrus lines. (2) Determine the chimerism of the HLB-resistant/tolerant transgenic lines. (3) Confirm HLB resistance in citrus putative mutants (nontransgenic lines). For objective 1, we continued propagating the transgenic lines that overexpress Arabidopsis defense genes and inoculated the previously generated progenies. The new progeny plants are growing in the greenhouse. The progenies obtained in the last quarter have been inoculated with Las-infected psyllids for two months and moved back to the greenhouse for symptom development. HLB symptoms on the plants have been carefully monitored and recorded. For objective 2, we performed the second round of real-time quantitative PCR (qPCR) to determine the chimerism of the HLB-resitant/tolerant transgenic lines. The results indicated that several lines of the HLB-resitant/tolerant transgenic lines are not chimeric. If these lines are confirmed to be HLB-resitant/tolerant in objective 1, they will be able to be propagated by grafting for industry use. For objective 3, we continued propagating the gamma ray-mutagenized mutant lines that are likely resistant/tolerant to HLB and inoculated previously generated progenies. The new progeny plants are growing in the greenhouse. As for the transgenic progenies, those obtained earlier were inoculated with Las-infected psyllids and are currently in the greenhouse for symptom development.
The project has three objectives: (1) Confirm HLB resistance/tolerance in transgenic citrus lines. (2) Determine the chimerism of the HLB-resistant/tolerant transgenic lines. (3) Confirm HLB resistance in citrus putative mutants (nontransgenic lines). For objective 1, we continued propagating the transgenic lines that overexpress Arabidopsis defense genes and inoculated the previously generated progenies. The new progeny plants are growing in the greenhouse. The progenies obtained in the last quarter have been inoculated with Las-infected psyllids for two months and moved back to the greenhouse for symptom development. HLB symptoms on the plants have been carefully monitored and recorded. For objective 2, we performed the second round of real-time quantitative PCR (qPCR) to determine the chimerism of the HLB-resitant/tolerant transgenic lines. The results indicated that several lines of the HLB-resitant/tolerant transgenic lines are not chimeric. If these lines are confirmed to be HLB-resitant/tolerant in objective 1, they will be able to be propagated by grafting for industry use. For objective 3, we continued propagating the gamma ray-mutagenized mutant lines that are likely resistant/tolerant to HLB and inoculated previously generated progenies. The new progeny plants are growing in the greenhouse. As for the transgenic progenies, those obtained earlier were inoculated with Las-infected psyllids and are currently in the greenhouse for symptom development.
Cell-penetrating peptides (CPPs) are a class of peptides (short stretchers of amino acids, the building blocks of proteins) known to translocate across most organic membranes. CPPs are currently revolutionizing the pharmaceutical and medical industries where CPPs are being investigated as vehicles for the delivery of therapeutic compounds and other cargos (proteins, RNA, DNA, antibiotics). Surprisingly, CPPs have also been found to function in plant cells to ferry cargos across cell membranes. The goals of this project were 1) To determine if CPPs could be systemically transported in citrus. 2) To develop a CPP transformation protocol without Agrobacterium in citrus. 3) To evaluate the use of CPPs as delivery tools for disease therapies and study the role of defense genes. Experiments with CPPs alone were unsuccessful with DNA, although protein could be transported. The addition of CRISPR/Cas to the systems appears to be successful. Importantly, plants altered with this system are not being regulated as transgenic.
Our project aims to provide durable long term resistance to Diaprepes using a plant based insecticidal transgene approach. In this quarter,most of the transgenic lines produced have been confirmed for gene integration by conventional PCR and analyzed for gene expression using qPCR. 40% of the lines tested have been determined to be high expressers while the rest were medium to low in expression. Cuttings from the larger lines have been made and are being rooted in the mist bed for future challange with Diaprepes. A number of other potential root specific promoters have been identified from the phytozome database. qPCR gene expression analyses on non-transgenic leaves, flowers, fruit, phloem, seeds and roots have identified some that can potentially be used in the future for root specific gene expression. Results from some of these studies will be presented in the World Congress on In vitro Biology in the summer.
We hypothesized that groves suffering most from HLB were exhibiting stress associated with high soil concentrations of bicarbonate since they support lower fibrous root density compared to groves with lower bicarbonates (< 100 ppm) in irrigation water and/or soil pH (< 6.5). To confirm this relationship, we surveyed 41 grove locations in Highlands and Desoto Counties with varying liming history and deep vs. shallow wells mostly on Swingle and Carrizo. Lower root density was significantly related to well water pH > 6.5 and to soil pH > 6.2. Records from these blocks revealed that yields from groves under high bicarbonate stress declined 20% from 2011 to 2013, in contrast to ridge groves with low bicarbonate stress that increased 6% in production even though HLB incidence had accelerated. Yield losses were correlated with less fibrous root density which reduces root system capacity for water and nutrient uptake. Evidence from research on other crops indicated that bicarbonate impairs the root s ability to take up important nutritional cations including Ca, Mg and K as well as micronutrients, especially Mn and Fe. Similar data for citrus were lacking; hence there was a need for more specific information for the effects of HLB, bicarbonates and their interaction for trees on the commonly grown rootstocks in Florida (i.e. Swingle and Carrizo) under field conditions. For evaluation in replicated plot trials, acidification of irrigation water and soil treatments that reduce bicarbonate were applied to quantify their effects on root health. Irrigation water acidification was targeted to soil pHs of 7.5, 6.0, 5.0 and 4.0 without or without sulfur application to soil to maintain the pH targets. In response to acidification leaf nutrient concentrations have been maintained in the optimum or high range. Greater leaf concentrations of Mg, Ca, Fe, Mn and Zn in plots were measured at target soil pH of 4.0 and 5.0, but no differences were found with or without sulfur amendment. Lower irrigation applications and few fertigation applications resulted in reduced soil acidification in the irrigated zone. Survey for root density, tree nutrient status and yields was implemented in groves that received water/soil acidification treatments to reduce soil pH and bicarbonate concentrations in comparison to unmanaged groves that were either untreated or experienced little or no bicarbonate stress based on the status of irrigation water and/or liming history. Acidification of irrigation water in central ridge and south central flatwoods Valencia orange groves on Swingle rootstock maintained soil pH below 6.5 on the flatwoods and 6.0 on ridge. Over the last 2.5 seasons of survey, root density as an index of root heath was sustained. Phytophthora populations remained below the damaging level in ridge groves and in flatwoods increases to damaging levels were coincident with the fall root flush dropped back to non-damaging levels for remainder of the season. Compared to 2014-15, yields in the ridge blocks have increased up to 4% and on the flatwoods increased up to 20%. Soil and tree responses to acidification required several seasons to become completely manifested. The final goal is to perform an economic analysis of bicarbonate management in terms of irrigation water and soil acidification costs versus benefit from gains in tree health and productivity so that growers may prioritize their expenditure on practices that mitigate losses of tree productivity due to HLB. With a complete sets of 2015-2016 yield data from acidified grove blocks, a cost benefit analysis will be performed.
In September 2012, FireWall (Agrosource, Inc.) was granted an EPA Section 18 registration for control of citrus canker in Florida grapefruit. The FireWall label restricted use to no more than two applications per season. As a condition for FireWall registration, EPA required monitoring of Xanthomonas citri subsp. citri (Xcc) for streptomycin resistance in commercially treated groves. The objective of the survey was 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 was conducted in 3 or 4 grove locations each season in November-January. A report of the survey results were submitted each January to FDACS to be forwarded to EPA. No Xcc resistant to streptomycin were recovered from commercial groves during the 3 year survey. Greenhouse assays of sweet orange seedlings injection-infiltrated with Xcc consistently demonstrated that streptomycin, the active ingredient in Firewall, moves trans-laminar and controls Xcc infection in expanding leaves. Furthermore, this assay demonstrated that streptomycin moves upward into the next leaf flush where there is detectable activity against Xcc inoculated 10 weeks after spray application to initial flush. These assay results confirm streptomycin moves upward and explain the consistent performance of streptomycin against canker in field trials. Penetration of leaves by streptomycin insures that it is protected from wash-off by rainfall, weathering and degradation by UV light.
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