Correlation of transgene expression with disease resistance response: Western blot analysis for plants containing LIMA and GNA was completed, and as expected, data shows a strong correlation between transgene expression and desired phenotype. This supports the dogma that fairly large populations of transgenic plants are necessary (for each transgene/cultivar) to obtain adequate transgene expression while maintaining cultivar integrity. Improved transformation methodology: 1. In efforts to reduce transgene mediated metabolic load on the plant, we have transformed Hamlin suspension cultures with constructs containing our reporter gene driven by either an embryo specific Carrot DC3 promoter or an embryo specific Arabidopsis At2S2 promoter. Transgenic plants expressed anthocyanin only in the embryogenic cells. Cotyledonary cells did not express anthocyanin indicating tissue specific activity of the promoter. Plants germinated from these embryos have been observed to lack anthocyanin production and have been micro grafted for growth. RT-PCR will be carried out on these plants to confirm inactivity of the visual selectable marker. 2. A binary vector containing Dual T-DNA borders for gene segregation and marker free transformation of citrus suspension cell transformation and selection have been constructed. Hamlin and W Murcott cells have been transformed with this construct. Preliminary data indicates that the transformation vector is functional and able to incorporate both T-DNAs into the plant genome. Somatic embryos containing this dual T-DNA cassette have been isolated and have been placed in embryo maturation medium for growth and development. Plants will be tested by once the plants are large enough. Subsequent experiments will confirm if negative selection pressure can differentiate between cells that contain the marker free T-DNA from the T-DNA containing the selectable positive/negative fusion marker cassette and if it can be removed from the citrus genome. 3. The binary vector for an inducible cre-lox based marker free selection has been constructed containing a heat inducible excision system containing the cre gene driven by a Soybean heat shock gene promoter. Tobacco tissue has been transformed with the construct and we are waiting for the plants to grow to a suitable size for heat shock treatment and excision. Renewal of transgenic field permit with APHIS: following completion and review of paperwork, our permit was renewed for 3 years; we now have permission to allow transgenic trees to flower and fruit; the Southern Gardens transgenic site was added to our permit. Transgenic testing: Nearly 200 new transgenic citrus trees were planted at the Picos USDA Farm site (resets and new tree positions). Paperwork is nearly complete to move HLB resistant transgenic trees from the SG hot psyllid house to field sites (petition approved by DPI). Two large sets of transgenic plants were propagated for testing in the SG hot psyllid house; they include new transgenes LIMA-B and SABP2 (the latter being a plant gene showing promise). We expect to move these plants to the SG psyllid challenge house within the next few weeks.
This is a continuing project to find economical approaches to citrus production in the presence of Huanglongbing (HLB). We are developing trees to be resistant or tolerant to the disease or to effectively repel the psyllid. First, we are attempting to identify genes that when expressed in citrus will control the greening bacterium or the psyllid. Secondly, we will express those genes in citrus. We are using two approaches. For the long term, these genes are being expressed in transgenic trees. However, because transgenic trees likely will not be available soon enough, we have developed the CTV vector as an interim approach to allow the industry to survive until resistant or tolerant trees are available. A major goal is to develop approaches that will allow young trees in the presence of HLB inoculum to grow to profitability. We also are using the CTV vector to express anti-HLB genes to treat trees in the field already infected with HLB. We have modified the CTV vector to produce higher levels of gene products to be screened. At this time we are continuing to screen possible peptide candidates in our psyllid containment room. We are now screening about 80 different genes or sequences for activity against HLB. We are starting to test the effect of two peptides or sequences in combination. We are attempting to develop methods to be able to screen genes faster. We are also working with other groups to screen possible compounds against psyllids on citrus. Several of these constructs use RNAi approaches to control psyllids. Preliminary results suggest that the RNAi approach against psyllids will work. We are screening a large number of transgenic plants for other labs. We are beginning to work with a team of researchers from the University of California Davis and Riverside campuses to express bacterial genes thought to possibly control Las. Since we are testing about 80 genes for induction of resistance or tolerance to HLB in citrus, we changing our focus to controlling psyllids until we have more conclusion from the peptides under screen.
The potential for intellectual property protection for the proposed prototype and technology was discussed. Protection will reconsidered upon collecting more field data. PI Ehsani visited with research team at the USDA-ARS in Ft. Pierce who are doing similar research to explore potential collaboration and effort leveraging. Expanding the research scope through this collaboration is being discussed. The results of experiments where trees where treated in the summer of 2012 and fruit harvested and analyzed in the spring of 2013 were presented at the 2013 Annual Meeting of the American Society of Agricultural and Biological Engineering. Experiments with the new prototype were initiated in May to treat the canopies at three different temperatures with varied heating periods ranged from 20 to 180 min. The 2012 field-trials confirmed the limitation of QPCR technique in quantifying HLB kill due to varied live and dead bacterial load in the leaves from same branch and within branches of the untreated tree. Therefore, the tree physiological measurements such as number of leaves are being conducted to quantify the effect of treatment on tree health. It was found that despite supplemental heating, reaching temperature above ___ was difficult. It was hypothesized that ground water evaporation is responsible for producing evaporative cooling and delaying the rate of heating. An additional heating element will be added to the system and the ground will be covered with a tarp to prevent evaporation.
The potential for intellectual property protection for the proposed prototype and technology was discussed. Protection will reconsidered upon collecting more field data. PI Ehsani visited with research team at the USDA-ARS in Ft. Pierce who are doing similar research to explore potential collaboration and effort leveraging. Expanding the research scope through this collaboration is being discussed. The results of experiments where trees where treated in the summer of 2012 and fruit harvested and analyzed in the spring of 2013 were presented at the 2013 Annual Meeting of the American Society of Agricultural and Biological Engineering. Experiments with the new prototype were initiated in May to treat the canopies at three different temperatures with varied heating periods ranged from 20 to 180 min. The 2012 field-trials confirmed the limitation of QPCR technique in quantifying HLB kill due to varied live and dead bacterial load in the leaves from same branch and within branches of the untreated tree. Therefore, the tree physiological measurements such as number of leaves are being conducted to quantify the effect of treatment on tree health. It was found that despite supplemental heating, reaching temperature above ___ was difficult. It was hypothesized that ground water evaporation is responsible for producing evaporative cooling and delaying the rate of heating. An additional heating element will be added to the system and the ground will be covered with a tarp to prevent evaporation.
Function of individual X. citri transcription activator like effectors (TALEs): We previously reported the development of an assay using stable transgenic Nicotiana benthiamiana plants containing a 4 EBE promoter:GUS construct to be tested for activation by a number of PthA homologs we have cloned from various X. citri strains. The PthA homolog-EBE specificity was were tested by creating transconjugants of X. campestris pv campestris 8004 transconjugants carrying the various PthAs and infiltrating into the stable transgenic Nicotiana benthiamiana plants. X. campestris pv campestris was used in lieu of X. citri for screening in Nicotiana because X. citri doesn’t infect Nicotiana. We observed strong gus activity in transconjugants containing pthA1(21.5 repeats), pthA2 (15.5 repeats) and pthA4 (17.5 repeats) from strain A44; pthA1- 17.5 repeats from Etrog; but not PthA4 homolog from Miami strain. Transformation and production of stable citrus lines: Because we have had difficulty recovering intact and functioning stable transgenic citrus lines, we have undertaken a number of efforts to overcome this bottleneck. Changes included testing a different transformation vector, different promoter and construct components, adjusting the transformation methodology, and adding Carrizo citrange, given it’s greater transformation efficiency, for comparison with ‘Duncan’ grapefruit and ‘Pineapple’ sweet orange. So far we have regenerated 312, 22 and 261 putative transgenic shoots from grapefruit, sweet orange and Carrizo, respectively and transferred to rooting media. Ongoing transformation experiments with previously used constructs have resulted in a number of putative transgenic shoots that have been rooted and transferred to soil in trays or 4′ pots. PCR screening of putative transgenic grapefruit and sweet orange plants regenerated from segments transformed with various constructs were analysed for 3 genes, 2 of which are present in the transgene of the constructs used (avrGF2 and nptII). A total of 54 plants were tested, but none of the plants tested contained the avrGF2 gene while 36 of the plants contained the nptII gene. We also tested for the presence of the virC gene which indicates bacterial contamination. One plant was positive for virC. Histochemical GUS screening was also carried out on putative transgenic grapefruit, sweet orange and Carrizo. Segments were scored based on the extent of the blue staining observed on the segments. GUS positive shoots were considered those segments that stained entirely blue while chimeric GUS shoots were those segments that stained less than 80%. No GUS positive shoots were observed for grapefruit or sweet orange cultivars for any of the constructs analyzed. Some grapefruit shoots were observed to be chimeric. Carrizo citrange showed the best results with several plants being GUS positive and a large number of plants chimeric for GUS. Another effort we have undertaken was to have an external contract transformation lab test our constructs along side their standard transformation control. In these experiments we provided a 14 EBE promoter construct in the original vector driving either AvrGf1 or GUS, for stable transformation of tobacco and Carrizo. The results so far indicate that our construct was considerable less successful in their hands for transformation compared to their control. These results suggest that our difficulties arise from the original vector used. Now that we have made new constructs we expect to have greater success in the production of stable transgenic lines
CLas colonizes its insect vector and is transmitted in circulative propagative manner. The bacteria multiply within the insect vector hemolymph. We noticed that the bacteria also form biofilm on the gut surface. In general bacteria need a cell-to-cell signaling system (Quorum sensing) in order to form a biofilm. Genome of Candidatus Liberibacter asiaticus (CLas) reveals the presence of luxR that encodes LuxR protein, one of the two components cell-to-cell communication systems. But the genome lacks the second components; luxI that produce Acyl-Homoserine Lactone (AHL) suggesting that CLas has a solo LuxR system. We confirmed the functionality of LuxR by expressing in E. coli and the acquisition of different AHLs We detect AHLs in the insect vector (psyllid) healthy or infected with CLas but not in citrus plant meaning that Insect is the source of AHL. Main findings: 1-Using different bacterial biosensor, we partly identify these AHLs (number of Carbon). CLas biofilm formation on the surface of insect Gut confirms the presence of cell-to-cell communication in insect while the planktonic state of CLas in plant indicate the absence of this communication. 2- In plant, we found molecules that bind to LuxR but inactive its function (plant defense). We try now to characterize these molecule and study their effect on biofilm formation inside insect. We use purified molecule to feed infected insect through artificial diet system. 3-We produced citrus plants that express LuxR protein in the phloem sap in order to test I- If the acquired LuxR proteins in insect interfere with the biofilm formation in insect (cure the insect from CLas) II- if the expression of LuxR in plant induce biofilm formation (localize the infection in plant) We found that feeding infected ACP with CLas on the LUXR expressing plants reduce the bacterial populations in insect and reduced the infection rate significantly. This result strongly indicates that we can target this system to interfere with the insect transmission and the spread of Disease. In last few months we focused on identifying the AHL- like molecules in plant Phloem sap. for that were analyzed the phloem sap chemical composition from suspectable and resistent varities of citrus. We found some candidates and we start testing them. The main aim of this project is to express molecules in plant that interfere the growth of CLas in insect by feeding.
We have made progress with the scFv library made with the earlier grant from CRDF. We had previously used the scFv when expressed as part of the M13 phage vector particle in ELISA and dot blot formats. Our efforts in the past quarter have built on that work, and now we are using the scFv alone in tissue print assays of citrus plants to detect ‘Ca. Liberibacter asiaticus’. scFv are expressed and purified from from E. coli cells using a 6X His tag incorporated in the scFv protein. Study of the literature showed that the media used to grow the E. coli and other details of the culture conditions greatly influence the yield of scFv obtained from culture lysates. We have found that a very rich and buffered medium, (2X yeast extract Tryptone broth with phosphate buffer) works best. The medium is supplemented with glycine, sucrose and IPTG at various stages of the expression protocol. With this protocol we have produced purified scFv at concentrations in the mg/ml range. Differences are observed among different scFv clones. Results from SDS-PAGE gels are consistent with post translational folding being problematic for some scFv as has been reported in the literature. The tissue print assays continue on nitrocellulose membranes. Cross reactions with healthy plant tissue can be a problem, especially if the concentration of scFv is too high. However, color development is observed in the vascular cylinder (phloem) of HLB infected petioles but not in comparable petioles from healthy trees. In some tissue prints, color development is observed in discrete spots outside of the phloem cylinder. Similar results are obtained with all scFv that were selected to bind to proteins expressed on the surface of ‘Ca. Liberibacter asiaticus’. These targets include an ATPase associated with the type IV pilus, a pilus assembly protein, two flagellar proteins, the major outer membrane protein OmpA, and the efflux protein TolC. Previous work has used a secondary monoclonal antibody directed at the 6X His Tag of the scFv molecules conjugated with alkaline phosphatase and blocking of the membranes with skim milk. We have recently made tissue prints with a modified technique, using ‘Super block’, a commercial product used in Northern and Southern blotting on nitrocellulose membranes and detection with a monoclonal antibody directed at the FLAG epitope on the scFv. This protocol produces remarkably sharper tissue prints with dramatically reduced background, and color tightly focussed in the vascular cylinder of HLB infected, but not healthy petioles. We will carry out further experiments with the scFv using the FLAG epitope for detection of the scFv. The scFv will also be used to label sections for examination by fluorescence and electron microscopy. We have provided several scFv to cooperating researchers at USDA ARS Ft. Pierce.
This project is a continuation of a previous project #95 “PREPARATION OF ANTIBODIES AGAINST CANDIDATUS LIBERIBACTER ASIATICUS”. Progress reports for the previous project are on file. The reimbursable agreement with CRDF was established on September 5, 2012. We continue to study the literature to identify vectors to use for a future scFv library made as part of this project. The goal is to find a suitable vector that is not encumbered by intellectual property and patent issues. I have written twice to a laboratory in Germany which has published results with a suitable vector but have had no reply. We are also optimizing the cloning strategies that will be used to move already selected scFv into transgenic plants. A visiting scientist arrived and began work 6-15-2013. We have obtained the vector, pUSHRL-26, to be used for plant transformation of the scFv constructs from Ed Stover at Fort Pierce and the plasmid has been purified. We have purchased the restriction enzymes and designed primers to be used for PCR to amplify the cloned scFv encoding inserts from vector pKM19. The cloned inserts will be sequenced to confirm that they are correct and then cloned into the transformation vector. Related research with the existing scFv is underway on project 551.
The project has two objectives: (1) Increase citrus disease resistance by activating the NAD+-mediated defense-signaling pathway. (2) Engineer non-host resistance in citrus to control citrus canker and HLB. For objective 1, we are planning a microarray experiment to identify genes that are induced by NAD+ in citrus. Microarray chips have been ordered. The first batch of RNA samples we prepared did not pass the quality control (QC). We are making the second batch of RNA samples. Microarray will be performed soon. For objective 2, two non-host resistance genes against citrus canker have been cloned into the T-DNA vector pBI1.4T, a vector with good transformation efficiency in citrus. The plasmids have been mobilized into Agrobacterium and will be used for citrus transformation.
The efficiency of transmission process of Candidatus Liberibacter asciaticus (CLas) depends on the success of specific interactions between CLas and the insect vector Asian citrus psyllids (ACP). CLas is circulative propagative within ACP. The bacteria need to pass the intestinal barrier to reach the hemolymph where they multiply then they must invade the salivary glandes in order to be inoculated in a new plant host while insect feeding. Passing these biological barriers needs specific interactions between CLAS cells and the epithelial cells in the guts and the salivary glands cells. In the last few months, we were successful in identifying the receptors in the Asian citrus psyllids (ACP). The receptors are the proteins that CLas cells recognize and bind to before passing the gut to the hemolymph. Same thing, when cells invade the salivary glands to reach the food canal. the main technique we use in our study is the protein overlay assay. In this method, non CLas-carrying insect total proteins were separated by 2D-SDS-PAGE. After 2D-SDS-PAGE, the proteins were blotted onto PVDF membrane. the membrane was overlaid with extract from infect citrus then we detect the complex using antibodies against CLas membrane proteins. this method called (Far-western) Comparisons between the stained electrophoretic profiles of ACP proteins in the gel and results of far-Western blot experiments on the membrane allow to cut the protein spots from 2-DE gels for LC-MS/MS analysis. Some ACP proteins (receptors) were identified. The function of these proteins was analyzed with bioinformatics. These genes were cloned, proteins were expressed, antibody against these proteins were made. a publication described this work is in preparation. Recently, we started to identify the membrane proteins from CLas that interact and bind to the identified receptors. Since we have identified the receptors in ACP, we are planing to perform negative FAR-Western to identify the ligands in CLas. For this reason, we predicted the antigenic domains in the identified ACP-receptors to produce the antibodies. We already obtained antibodies against beta-actin. our aim for the next few months is to identify the proteins of Clas (ligands) that adhere to ACP cells using antibodies against identified ACP proteins. the yeast double hybrid system will be used to validate specific interaction for each couple (receptor-ligand) Overall, our research work is carried out according to milestone of the project.
This project is to assess how the efficiency of HLB transmission by psyllids varies depending on the stage of infection and plant development. Our main accomplishments on this project are listed below. 1) Electron microscopy examination of the sites on the leaves of citrus plants where HLB-positive psyllids fed for 7 days demonstrated that even at early stages of infection (starting from 3-4 weeks after the beginning of the experiment) the bacteria could be already visualized in the initial sites of introduction. 2) In order to characterize inoculum sources of the bacterium available for psyllids within an infected tree, we examined the proportion of psyllids that acquired the bacterium after their exposure to different types of flushes, young growing or matured symptomatic ones. Data from PCR analyses demonstrated that Las-positive psyllids were collected from both types of flushes. We also conducted a similar experiment that was slightly modified in a way that psyllids fed on old and young leaves that were detached from plants and kept in 50 ml tubes (‘detached leaf experiment’). Some differences in the bacterium acquisition were obtained from these two experiment series. On average 48.33% of psyllids fed on old symptomatic flushes tested positive and 58.33% of psyllids fed on young pre-symptomatic flushes were positive. In the ‘detached leaf’ experiment, an average acquisition from young pre-symptomatic tissue was significantly higher than from old symptomatic flushes: with average of 64.26% and 23.9%, respectively. Psyllids that acquired bacteria from different flushes were next transferred onto healthy receptor plants. Analysis of numbers of plants that became infected upon inoculation with psyllids fed on different types of flushes revealed that more receptor plants that were inoculated by psyllids kept on young flushes became infected (52% of Duncan grapefruit plants and 53% of Madam Vinous sweet orange plants) and less proportion of receptor plants inoculated with psyllids that fed on old mature flushes got infected (19 and 33% of the same varieties, respectively). 3) In order to assess what types of flushes are more susceptible to psyllid inoculation with the HLB bacteria, we exposed sweet orange and grapefruit plants that have young growing flushes and plants that have only matured flushes to HLB-infected psyllids (“no young flush” plants). According to our data, both young and mature flushes could be inoculated by psyllids, yet inoculation efficiency of mature flushes is significantly lower. 4) Overall, our results support the initial observation of young flushes being more likely crucial for the disease spread at both steps of the pathogen transmission, either acquisition and inoculation are higher when young flush are present. Nonetheless, transmission associated with old tissues, which occurs at a reduced level, should not be ignored also. 5) To examine psyllid transmission rates to different citrus genotypes, we analyzed psyllid inoculation of 6 different varieties of citrus: Valencia sweet orange, Duncan grapefruit, Persian lime, Eureka lemon, Carrizo citrange, and Poncirus trifoliata. Those varieties represent plants with different degrees of susceptibility to HLB. The first four varieties showed the highest infection rates (80-100% infection), while only about 10% of Carrizo citrange and Poncirus trifoliate became infected. Poncirus and poncirus hybrids have been shown to have much greater tolerance to HLB. The fact that they are also more tolerant to psyllid inoculation with the bacterium suggests that developing hybrids of such varieties that in addition to being more tolerant would also have acceptable horticultural characteristics and would produce fruit and juice of a sufficient quality could be a solution to battle HLB epidemics before more sustainable approaches are in place.
The general goal of this project is to rapidly propagate complex citrus rootstock material for field testing. The rootstock materials to be tested will be products of the Citrus Improvement Program at the UF-IFAS-CREC in Lake Alfred. Specifically, these materials will be selected based upon their performance in the ‘HLB gauntlet’: Promising rootstock genotypes will have already been evaluated in the greenhouse and field for their ability to grow-off citrus scions that have been exposed to CLas-positive budwood and CLas-positive Asian citrus psyllids. Once candidate rootstock materials have successfully passed through this gauntlet, they will be propagated via rooted cuttings en masse in a psyllid-free greenhouse at the UF-IFAS-IRREC in Fort Pierce. From there, rootstock materials will be budded with scion materials and planted in the field for further testing for their long-term performance. The start date for this project was April, 2013. To date, the progress of this project is as follows: – Misting chamber to propagate candidate, rootstock materials as rooted-cuttings has been constructed. – Propagation materials (containers, soilless media, and rooting hormones) have been purchased. – Competitive bids have been prepared to support the construction of a new, dedicated propagation greenhouse at the IRREC. Funds allocated to this project are also being used to retro-fit an existing greenhouse at the IRREC to meet FDACS-DPI standards. The existing greenhouse will also be employed as a propagation facility for this project. – The initial cohort of advanced, tetratzygous citrus rootstock materials for en masse propagation have been identified. They are currently emerging from the ‘HLB gauntlet’ and will soon be ready for en masse propagation.
We aim in this project to genetically manipulate defense signaling networks to produce citrus cultivars with enhanced disease resistance. Defense signaling networks have been well elucidated in the model plant Arabidopsis but not yet in We aim in this project to genetically manipulate defense signaling networks to produce citrus cultivars with enhanced disease resistance. Defense signaling networks have been well elucidated in the model plant Arabidopsis but not yet in citrus. Salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) are key hubs on the defense networks and are known to regulate broad-spectrum disease resistance. With a previous CRDF support, the PI’s laboratory has identified ten citrus genes with potential roles as positive SA regulators. Characterization of these genes indicate that Arabidopsis can be used not only as an excellent reference to guide the discovery of citrus defense genes and but also as a powerful tool to test function of citrus genes. This new project will significantly expand the scope of defense genes to be studied by examining the roles of negative SA regulators and genes affecting JA and ET-mediated pathways in regulating citrus defense. We have three specific objectives in this proposal: 1) identify SA negative regulators and genes affecting JA- and ET-mediated defense in citrus; 2) test function of citrus genes for their disease resistance by overexpression in Arabidopsis; and 3) produce and evaluate transgenic citrus with altered expression of defense genes for resistance to HLB and other diseases. Currently we have cloned 10 full-length genes in these categories in the entry vector pJET. Five of the genes were further cloned to the binary vector pBIN19plusARS and transferred to Agrobacteria. The Agro strains were sent to our collaborator Dr. Bowman’s lab to initiate citrus transformation. In the mean time, we started the process of transforming Arabidopsis to overexpress these genes and to test their defense function. In addition, we are continuing to generate and/or characterize transgenic citrus plants expressing the SA positive regulators, as proposed in the previous project, although the support of this previous project has already been terminated. A paper describing the cloning and characterization of the citrus NDR1 ortholog was recently published in the journal Frontiers in Plant Science.
Field experiments with nutritional and other horticultural management impacts on HLB disease: Final PCR testing of subject trees. The 20 “Valencia” and 20 Hamlin trees being treated with a comprehensive and holistic HLB management program, including foliar micro and macro nutrients and psyllid control, were sampled to perform PCR analysis at the end of the fifth year since HLB was discovered in the grove and tree yields were recorded annually. Five years before, half of the trees (10) were initially symptomatic for HLB (PCR+) and the other half were non-symptomatic (PCR undetermined; healthy). Refer to the previous two reports for the 2012/13 season yields. Samples were collected on May 5, 2013. Samples consisted of 10 HLB asymptomatic Hamlins, 11 HLB symptomatic Hamlins, 12 HLB Valencia trees with greening symptoms and 10 asymptomatic Valencia trees. Approximately 20 leaves were collected from around each tree and from each sample 4 leaves were selected and prepared for real time PCR for the detection of Candidatus Liberibacter asiaticus. Only 7 of the 21 Valencia trees tested PCR positive. These trees were in both the symptomatic as well as the asymptomatic designated categories. All of the Hamlin symptomatic trees tested PCR positive and in addition two of the asymptomatic Hamlin trees tested PCR positive. Samples of all the trees were run in fluorescent dye tests to determine if the nutrient applications had an effect on phloem translocation. The 7 PCR positive Valencia trees translocated less dye than the PCR negative trees. The PCR positive and negative Hamlin trees translocated similar amounts of dye.
In cooperation with Bryan Belcher from Davis Citrus Management we identified that bicarbonate in irrigation water from deep wells impacts root health and exacerbates Huanglongbing (HLB) symptom expression for trees on Carrizo and Swingle rootstocks. We have since learned that there is a long history of management of high bicarbonate and pH of irrigation water for Carrizo rootstock groves in the Central Valley of California. In February, we took FL growers, including John Gose from Lykes, to CA to tour Paramount Citrus groves to learn about conditioning water and soils with high bicarbonates (>100ppm) and alkalinity (> pH 7.5). The primary means are sulfuric acid injection from storage tanks managed by contractors or use of sulfur burners to generate acidity from elemental sulfur. The target is to reduce pH of water used for irrigation to 6.5. In Florida, Bryan Belcher has acidified irrigation water with N-furic acid (a mixture of urea and sulfuric acid) by injection at the well in same way as fertigation is applied. N-furic has the advantage of being safe to handle but the disadvantage of higher cost of treatment (2-3X sulfuric acid). We are currently surveying root health in Davis Citrus Managment groves that have shallow and deep wells and varying liming histories. In addition, we have set up demonstration trials to observe whether bicarbonates in the well water and soil (as residual from dolomite application) are associated with greater HLB effects on root mass density and tree health. We are also in the process of developing a site to conduct a controlled study with a combination of acidification treatments of soil and water with the requisite conditions of elevated bicarbonates.
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