This project is a continuation of previously funded CRDF grants to TWO BLADES focused on utilizing multiple This project is a continuation of previously funded CRDF grants to TWO BLADES focused on utilizing multiple strategies to produce canker-resistant citrus plants. The project has focused on transforming Duncan grapefruit with genes that express EFR or a gene construct designated ProBs314EBE:avrGf2 that is activated by citrus canker bacteria virulence factors. This project is a continuation of previously funded CRDF grants to TWO BLADES focused on utilizing multiple strategies to produce canker-resistant citrus plants. The project has focused on transforming Duncan grapefruit with genes that express EFR or a gene construct designated ProBs314EBE:avrGf2 that is activated by citrus canker bacteria virulence factors. Objective 1. To determine if Bs3-generated transgenic grapefruit plants are resistant to diverse strains of the citrus canker bacterium or to alternate target susceptibility genes in greenhouse experiments and to the citrus canker bacterium in field experiments in Fort Pierce. As stated in a previous report, the transgenic Duncan grapefruit containing the Bs3-executor transgene shows a high level of resistance to an array of strains representing a worldwide collection. Furthermore, using real time PCR, we have validated that the gene is activated by one or more TAL effectors and that there is minimal activation without these genes. We have also identified two other possible transgenics from plants received from Dr. Vladimir Orbovic. Both responded to infiltration with a high concentration of bacterial cells by exhibiting a hypersensitive reaction within 4 days of infiltratin. One of the transgenics appeared to have a growth defect, but recently has developed normal shoots. Both transgenic trees contain the avrGf2 gene (based on PCR for detection of avrGf2). These trangenics will be grafted onto rootstock once they reach the appropriate size. During the past three months we have placed our constuct in a different vector that is acceptable for future transgenic purposes. The previous constructs contain an additional selectable marker that allowed for identifying putative transgenics with a higher success rate that contained the targeted construct. Given that there was concern about the additional marker, the new construct contains only NPT as a selectable marker. The construct was sent to Vladimir Orbovic, who is in the process of screening putative grapefruit and sweet orange transformants. We hope to have additional transgenics later this year. We have also grafted our lone transgenic plant onto two rootstocks (812 and Sour Orange) and planted these in late March in the field at Fort Pierce in collaboration Dr. Ed Stover. Objective 2. To determine if EFR-generated transgenic grapefruit plants are resistant to the citrus canker bacterium in field experiments in Fort Pierce. We have grafted our our tow most promising EFR transgenic plants (based on ROS activity) onto two rootstocks (812 and Sour Orange) and are in the process of planting in the field at Fort Pierce in collaboration Dr. Ed Stover. They were planted in the field in late March. We have identified additional transgenics from plants received from Dr. Vladimir Orbovicthat that will be graftered onto rootstocks in the near future.
Objective 1: Investigate efficacy of bactericide treatments for preventing new infections. This objective is necessary to determine the potential role of bactericides in an integrated pest management program for young tree protection. Experiment were initiated in new tree plantings in a Wauchula grove.Initial leaf samples were collected prior to treatments to evaluate CLas titers in the uninfected trees. The following treatments were applied this second quarter of 2019: Treatments were applied to trees as follows:1. Antibiotics (monthly rotation) 2. Antibiotics (quarterly rotation)3. Negative Control (insecticide + Tree defender exclusion netting)4. Positive Control (insecticide only)Tree height and circumference were quantified. ACP populations were monitored biweekly (eggs, nymphs, adults). No flush, eggs, or nymphs were detected after treatment applications. Tap sampling of adults was conducted, and insects are currently being processed to determine the infection rate. Objective 2. Determine the effect of bactericide application frequency on Las infection of citrus.This experiment will determine the most effective application frequency for bactericides to maximize tree health. This will inform revised recommendations for the use of antimicrobials in commercial citrus groves. Experiment were initiated in new tree plantings in a Lake Alfred grove. Initial leaf samples were collected prior to treatments to evaluate CLas titers in the uninfected trees, then one week after treatment applications. The following treatments were applied this second quarter of 2019: 1. Antibiotics (monthly rotation) 2. Antibiotics (quarterly rotation)3. Positive Control (insecticide only) Tree height and circumference were quantified. ACP populations were monitored biweekly (eggs, nymphs, adults). No flush, eggs, or nymphs were detected after treatment applications. Tap sampling of adults was conducted, and insects are currently being processed to determine the infection rate. Objective 3: Quantify the effect of repeated inoculation of the efficacy of bactericides. This experiment will determine whether bactericides are sufficient to overcome the pressure of repeated Las inoculation by ACP. This will inform revised recommendations for integrated pest management programs to improve use of insecticides in conjunction with bactericides in commercial citrus groves. We have procured trees and will initiate experiments next quarter.
Huanglongbing (HLB) and Citrus Bacterial Canker (CBC) present serious threats to the future success of citrus production in the US. Insertion of transgenes conferring resistance to these diseases or the HLB insect vector is a promising solution. Genes for antimicrobial peptides (AMPs) with diverse promoters have been used to generate numerous transformants of rootstock and scion genotypes. New promoters and/or transgenes are being regularly introduced with more than a thousand new transformation attempts on citrus epicotyl sections each week. Plants have progressed from the initial round of scion transformations and are now replicated and ready for exposure to HLB, using CLas infected psyllids in a greenhouse environment. Transformed rootstock varieties, with two AMPs (D4E1 and Pyrrhocoricin) and 170 transgenic plants, are being challenged using graft inoculations in two new replicated experiments. A wide series of promoters driving a reporter gene are being tested in transformed citrus and show very different levels of expression with some being expressed in all tissues and some only in vascular tissue. Liberibacter sequence data were used to target a transmembrane transporter,as a possible transgenic solution for HLB-resistance. Collaboration with a USDA team in Albany, CA is providing constructs with enhanced promoter activity, minimal IP conflicts, and reduced regulatory and consumer concerns. Genes are being identified from citrus genomic data, from Carrizo citrange sequence generated using USDA funds, to permit transformation and resistance using citrus-only sequences. Citrus-derived T-DNA border analogues have been shown to be effective in producing transgenic tobacco and will be tested in citrus in next quarter. Genes for anthocyanin production are being tested as a visual marker for transformation, as a component of a citrus-only transgenic system. Transgenes are being developed to suppress (using an RNAi strategy) a lectin-like protein produced in the phloem of HLB-infected citrus. It is possible that suppression of this protein may significantly reduce disease symptoms. High throughput evaluation of HLB resistance will require the ability to efficiently assess resistance in numerous plants. Graft-inoculation, controlled psyllid-inoculation, and ‘natural’ psyllid inoculation in the field are being compared. The first trial has been in the field for 21 months and a repeated trial has been in the field for 9 months. Leaf samples have been collected monthly and PCR analysis of CLas conducted. These data will be analyzed over the next quarter.
As proposed, a transgenic test site has been prepared at the USDA/ARS USHRL Picos Farm in Ft. Pierce. The first trees have been in place for more than eight months. Answers have been provided to numerous questions from regulators to facilitate field testing approval. Cooperators have been made aware that the site is ready for planting. Dr. Jude Grosser of UF has provided 300 transgenic citrus plants expressing genes expected to provide HLB/canker resistance, which have been planted in the test site. USHRL has a permit approved from APHIS to conduct field trials of their transgenic plants at this site. An MTA is in place to permit planting of Texas A&M transgenics produced by Erik Mirkov. Alphascents provided an experimental pheromone attract/kill product Malex to disrupt citrus leaf miner (CLM). Our experience suggests CLM may significantly compromise tree growth where insecticides are avoided to permit ready transfer of Las by psyllids. CLM damage also compromises ability to view HLB symptoms. Unfortunately, this product had little effect on leaf miner. The decision has been made to apply Admire this fall to encourage an undamaged flush on transgenic trees. We are still learning how to grow trees for best assessment of HLB-resistance. More than 120 citranges, from a well-characterized mapping population, and other trifoliate hybrids (+ sweet orange standards) have been propagated for a replicated trial in collaboration with Fred Gmitter of UF. These will be planted in the spring of 2011, and monitored for CLas development and HLB symptoms. Data from this trial should provide information on markers and perhaps genes associated with HLB resistance, for use in transgenic and conventional breeding.
Huanglongbing (HLB) and Citrus Bacterial Canker (CBC) present serious threats to the future success of citrus production in the US. Insertion of transgenes conferring resistance to these diseases or the HLB insect vector is a promising solution. Genes for antimicrobial peptides (AMPs) with diverse promoters have been used to generate numerous transformants of rootstock and scion genotypes. New promoters and/or transgenes are being regularly introduced with more than a thousand new transformation attempts on citrus epicotyl sections each week. Plants have progressed from the initial round of scion transformations and are now replicated and ready for exposure to HLB, using graft inoculations and CLas infected psyllids in a greenhouse environment. A detailed set of AMP activity assays will be initiated next quarter on a replicated set (8 plants of each) of 39 independent Hamlin transformants. Transformed rootstock varieties, with two AMPs (D4E1 and Pyrrhocoricin) and 170 transgenic plants, are being challenged using graft inoculations in two new replicated experiments. A series of promoters are being tested with the GUS gene to see how effective they are. As expected, the three vascular-specific promoters show expression only in phloem and xylem, while other promoters show broad expression in tested tissues. Sucrose synthase promoter from Arabidopsis drives high GUS expression more consistently than citrus SS promoter or a phloem promoter from wheat dwarf virus. A ubiquitin promoter from potato drives consistent and very high GUS activity, even though the mRNA levels are similar to D35S promoter. use of this promoter may reduce the number of independent transformants needed. Liberibacter sequence data were used to target a transmembrane transporter,as a possible transgenic solution for HLB-resistance. Radioisotope permits are finally in place to assess effect of identified peptides on preventing ATP uptake in E. coli expressing the Liberibacter translocase. Collaboration with a USDA team in Albany, CA is providing constructs with enhanced promoter activity, minimal IP conflicts, and reduced regulatory and consumer concerns. Genes are being identified from citrus genomic data, from Carrizo citrange sequence generated using USDA funds, to permit transformation and resistance using citrus-only sequences. Citrus-derived T-DNA border analogues have been shown to be effective in producing transgenic Carrizo and tobacco and will be tested in citrus scions soon. Genes for anthocyanin production are being tested as a visual marker for transformation, as a component of a citrus-only transgenic system. Transgenes are being developed to suppress (using an RNAi strategy) a lectin-like protein produced in the phloem of HLB-infected citrus. It is possible that suppression of this protein may significantly reduce disease symptoms. High throughput evaluation of HLB resistance will require the ability to efficiently assess resistance in numerous plants. Graft-inoculation, controlled psyllid-inoculation, and ‘natural’ psyllid inoculation in the field are being compared. The first trial has been in the field for 24 months and a repeated trial has been in the field for 12 months. Leaf samples have been collected monthly and PCR analysis of CLas conducted.
As proposed, a transgenic test site has been prepared at the USDA/ARS USHRL Picos Farm in Ft. Pierce. The first trees have been in place for more than eleven months. Answers have been provided to numerous questions from regulators to facilitate field testing approval. Cooperators have been made aware that the site is ready for planting. Dr. Jude Grosser of UF has provided 300 transgenic citrus plants expressing genes expected to provide HLB/canker resistance, which have been planted in the test site. USHRL has a permit approved from APHIS to conduct field trials of their transgenic plants at this site, with several hundred transgenic rootstocks in place. An MTA is in place to permit planting of Texas A&M transgenics produced by Erik Mirkov. More than 120 citranges, from a well-characterized mapping population, and other trifoliate hybrids (+ sweet orange standards) have been propagated for a replicated trial in collaboration with Fred Gmitter of UF and are growing well in the greenhouse. These will be planted in the spring of 2011, and monitored for CLas development and HLB symptoms. Data from this trial should provide information on markers and perhaps genes associated with HLB resistance, for use in transgenic and conventional breeding. A proposal is being prepared that would provide high density mapping of this material to provide tighter mapping of linked resistance loci. Alphascents provided an experimental pheromone attract/kill product Malex to disrupt citrus leaf miner (CLM). Our experience suggests CLM may significantly compromise tree growth where insecticides are avoided to permit ready transfer of Las by psyllids. CLM damage also compromises ability to view HLB symptoms. Unfortunately, this product had little effect on leaf miner. The decision has been made to apply Admire this fall to encourage an undamaged flush on transgenic trees. We are still learning how to grow trees for best assessment of HLB-resistance.
Huanglongbing (HLB) and Citrus Bacterial Canker (CBC) present serious threats to the future success of citrus production in the US. Insertion of transgenes conferring resistance to these diseases or the HLB insect vector is a promising solution. Genes for antimicrobial peptides (AMPs) with diverse promoters have been used to generate numerous transformants of rootstock and scion genotypes. New promoters and/or transgenes are being regularly introduced with more than a thousand new transformation attempts on citrus epicotyl sections each week. Plants have progressed from the initial round of scion transformations and are now replicated and ready for exposure to HLB, using CLas infected psyllids in a greenhouse environment. Transformed rootstock varieties, with two AMPs (D4E1 and Pyrrhocoricin) and 170 transgenic plants, are being challenged using graft inoculations in two new replicated experiments. A wide series of promoters driving a reporter gene are being tested in transformed citrus and show very different levels of expression with some being expressed in all tissues and some only in vascular tissue. Liberibacter sequence data were used to target a transmembrane transporter,as a possible transgenic solution for HLB-resistance. Collaboration with a USDA team in Albany, CA is providing constructs with enhanced promoter activity, minimal IP conflicts, and reduced regulatory and consumer concerns. Genes are being identified from citrus genomic data, from Carrizo citrange sequence generated using USDA funds, to permit transformation and resistance using citrus-only sequences. Citrus-derived T-DNA border analogues have been shown to be effective in producing transgenic tobacco and will be tested in citrus in next quarter. Genes for anthocyanin production are being tested as a visual marker for transformation, as a component of a citrus-only transgenic system. Transgenes are being developed to suppress (using an RNAi strategy) a lectin-like protein produced in the phloem of HLB-infected citrus. It is possible that suppression of this protein may significantly reduce disease symptoms. High throughput evaluation of HLB resistance will require the ability to efficiently assess resistance in numerous plants. Graft-inoculation, controlled psyllid-inoculation, and ‘natural’ psyllid inoculation in the field are being compared. The first trial has been in the field for 21 months and a repeated trial has been in the field for 9 months. Leaf samples have been collected monthly and PCR analysis of CLas conducted. These data will be analyzed over the next quarter.
The experimental design of the Polk County field trial is completed. Christopher Vincent has designed this trial carefully to include sufficient replication. He also planned the trial to reduce the effect of disease severity variablity on the results of the experiment. There will be ten replications for each of the four treatments and three trees per experimental unit. Only the middle tree will be sampled for CLas titer. The canopy denisty of each tree will also be measured.
The trees in the Poly County trial will be sampled next Tuesday prior to the first phosphate applications. This will give us baseline numbers on disease severity in each tree. The first phosphate spray applications will occur later that same week. Trees will be spray with one of four phosphate concentrations, including one treatment with no phosphate.
Another trial is being planned with Southern Gardens in Hendry County. With the two sites, we will have field trials in the two major soil types for citrus in Florida. We are delayed in the establishment of ths site as Southern Gardens continues its search for plots that would be suitable I do not expect this to take very long. Southern Gardens is committed to doing this.
The current plan is for Nian Wang’s lab at the CREC to determine CLas titer in samples from the Polk County trial. The Hendry County samples will be processed in Eric Triplett’s lab in Gainesville.
Meanwhile, the Triplett and Killiny labs have greenhouse experiments underway. In the Triplett lab, the optimal level of phosphate comcentration for foliar fertilization has been determined. Also, a new experiment is underway to compare calcium phosphate fertilizaiton on roots versus foliar phosphate fertilization. The former is expected to mimic field conditions where the plant is forced to reduce the pH to solubilize phosphate. The leaf sprays will mimic the intervention we have planned for the control of HLB.
In the Killiny lab, similar experiments are underway in the greenhouse as described in the paragraph above. In all of these greenhouse experiments, Killiny will measure citrate levels in leaf midribs from all treatments.
We will sample eight leaves per tree, four times each year for CLas titer. Canopy density will be used to measure disease severity. We plan to spray the plants four times a year.
The experimental design of the Polk County field trial is completed. Christopher Vincent has designed this trial carefully to include sufficient replication. He also planned the trial to reduce the effect of disease severity variablity on the results of the experiment. There will be ten replications for each of the four treatments and three trees per experimental unit. Only the middle tree will be sampled for CLas titer. The canopy denisty of each tree will also be measured.
The trees in the Poly County trial will be sampled next Tuesday prior to the first phosphate applications. This will give us baseline numbers on disease severity in each tree. The first phosphate spray applications will occur later that same week. Trees will be spray with one of four phosphate concentrations, including one treatment with no phosphate.
Another trial is being planned with Southern Gardens in Hendry County. With the two sites, we will have field trials in the two major soil types for citrus in Florida. We are delayed in the establishment of ths site as Southern Gardens continues its search for plots that would be suitable I do not expect this to take very long. Southern Gardens is committed to doing this.
The current plan is for Nian Wang’s lab at the CREC to determine CLas titer in samples from the Polk County trial. The Hendry County samples will be processed in Eric Triplett’s lab in Gainesville.
Meanwhile, the Triplett and Killiny labs have greenhouse experiments underway. In the Triplett lab, the optimal level of phosphate comcentration for foliar fertilization has been determined. Also, a new experiment is underway to compare calcium phosphate fertilizaiton on roots versus foliar phosphate fertilization. The former is expected to mimic field conditions where the plant is forced to reduce the pH to solubilize phosphate. The leaf sprays will mimic the intervention we have planned for the control of HLB.
In the Killiny lab, similar experiments are underway in the greenhouse as described in the paragraph above. In all of these greenhouse experiments, Killiny will measure citrate levels in leaf midribs from all treatments.
We will sample eight leaves per tree, four times each year for CLas titer. Canopy density will be used to measure disease severity. We plan to spray the plants four times a year.
The experimental design of the Polk County field trial is completed. Christopher Vincent has designed this trial carefully to include sufficient replication. He also planned the trial to reduce the effect of disease severity variablity on the results of the experiment. There will be ten replications for each of the four treatments and three trees per experimental unit. Only the middle tree will be sampled for CLas titer. The canopy denisty of each tree will also be measured.
The trees in the Poly County trial will be sampled next Tuesday prior to the first phosphate applications. This will give us baseline numbers on disease severity in each tree. The first phosphate spray applications will occur later that same week. Trees will be spray with one of four phosphate concentrations, including one treatment with no phosphate.
Another trial is being planned with Southern Gardens in Hendry County. With the two sites, we will have field trials in the two major soil types for citrus in Florida. We are delayed in the establishment of ths site as Southern Gardens continues its search for plots that would be suitable I do not expect this to take very long. Southern Gardens is committed to doing this.
The current plan is for Nian Wang’s lab at the CREC to determine CLas titer in samples from the Polk County trial. The Hendry County samples will be processed in Eric Triplett’s lab in Gainesville.
Meanwhile, the Triplett and Killiny labs have greenhouse experiments underway. In the Triplett lab, the optimal level of phosphate comcentration for foliar fertilization has been determined. Also, a new experiment is underway to compare calcium phosphate fertilizaiton on roots versus foliar phosphate fertilization. The former is expected to mimic field conditions where the plant is forced to reduce the pH to solubilize phosphate. The leaf sprays will mimic the intervention we have planned for the control of HLB.
In the Killiny lab, similar experiments are underway in the greenhouse as described in the paragraph above. In all of these greenhouse experiments, Killiny will measure citrate levels in leaf midribs from all treatments.
We will sample eight leaves per tree, four times each year for CLas titer. Canopy density will be used to measure disease severity. We plan to spray the plants four times a year.
The objectives of this proposal are: 1) conduct a field trial using the selected grapefruit seedlings to ensure the productivity of the trees in Florida where HLB is endemic; and 2) evaluate the quality of the fruit produced. Achievement of these goals will produce a more resistant/tolerant variety that could be available in the near future since its use would not require the regulatory approval. Based on two year’s graft-inoculation assays in greenhouse with two HLB isolates and the performance of individual seedlings in the field, four lines of the seedlings (with greater HLB resistance/tolerance) were selected for further propagation on three different rootstock (commercial sour orange, newly selected USDA-sour orange and 942). The fruit quality (Brix, sucrose, glucose and fructose, soluble solids, pH, % TA and total ascorbic acid) of the four selected seedlings showed no significant difference from their maternal trees. First group of the propagates on three different rootstock from the selections of Scott Grove’s seedling variants were grown in our research farm, Picos farm, where the plants are under extreme high HLB disease pressure with very aggressive HLB pathogens. These new plantings (July, 2017; Nov, 2017; and May, 2018) showed different disease index, the longer the planting was, the higher the disease index, which was also highly correlated with the titers of Ca. Liberibacter asiaticus (Las) in infectd plants. It is worth noting that the new HLB isolate from Picos farm caused severe HLB disease on most of grapefruit selections of seedlings and bud sports in our latest greenhouse evaluation. Those selections were either resistant or tolerant to the previous HLB isolates we maintained in greenhouse. By the end of this year, we will expect to draw a better conclusion if the selected seedling variants display better resistance/tolerance to HLB pathogens at the Picos Research Farm. Second group of the propagates on three different root stocks mentioned above (Ca. 750 plants) have been budded and grown in our greenhouse, and are expected to be planted in Scott grove this month. All the propagates have been tested for the presence of Las. The propagates that carry a low titer of Las will be palnted separately and serve as the experiment for “Cross-protection”.
The objectives of this proposal are: 1) conduct a field trial using the selected grapefruit seedlings to ensure the productivity of the trees in Florida where HLB is endemic; and 2) evaluate the quality of the fruit produced. Achievement of these goals will produce a more resistant/tolerant variety that could be available in the near future since its use would not require the regulatory approval. Based on two year’s graft-inoculation assays in greenhouse with two HLB isolates and the performance of individual seedlings in the field, four lines of the seedlings (with greater HLB resistance/tolerance) were selected for further propagation on three different rootstock (commercial sour orange, newly selected USDA-sour orange and 942). The fruit quality (Brix, sucrose, glucose and fructose, soluble solids, pH, % TA and total ascorbic acid) of the four selected seedlings showed no significant difference from their maternal trees. First group of the propagates on three different rootstock from the selections of Scott Grove’s seedling variants were grown in our research farm, Picos farm, where the plants are under extreme high HLB disease pressure with very aggressive HLB pathogens. These new plantings (July, 2017; Nov, 2017; and May, 2018) showed different disease index, the longer the planting was, the higher the disease index, which was also highly correlated with the titers of Ca. Liberibacter asiaticus (Las) in infectd plants. It is worth noting that the new HLB isolate from Picos farm caused severe HLB disease on most of grapefruit selections of seedlings and bud sports in our latest greenhouse evaluation. Those selections were either resistant or tolerant to the previous HLB isolates we maintained in greenhouse. By the end of this year, we will expect to draw a better conclusion if the selected seedling variants display better resistance/tolerance to HLB pathogens at the Picos Research Farm. Second group of the propagates on three different root stocks mentioned above (Ca. 750 plants) have been budded and grown in our greenhouse, and are expected to be planted in Scott grove this month. All the propagates have been tested for the presence of Las. The propagates that carry a low titer of Las will be palnted separately and serve as the experiment for “Cross-protection”.
The objectives of this proposal are: 1) conduct a field trial using the selected grapefruit seedlings to ensure the productivity of the trees in Florida where HLB is endemic; and 2) evaluate the quality of the fruit produced. Achievement of these goals will produce a more resistant/tolerant variety that could be available in the near future since its use would not require the regulatory approval. Based on two year’s graft-inoculation assays in greenhouse with two HLB isolates and the performance of individual seedlings in the field, four lines of the seedlings (with greater HLB resistance/tolerance) were selected for further propagation on three different rootstock (commercial sour orange, newly selected USDA-sour orange and 942). The fruit quality (Brix, sucrose, glucose and fructose, soluble solids, pH, % TA and total ascorbic acid) of the four selected seedlings showed no significant difference from their maternal trees. First group of the propagates on three different rootstock from the selections of Scott Grove’s seedling variants were grown in our research farm, Picos farm, where the plants are under extreme high HLB disease pressure with very aggressive HLB pathogens. These new plantings (July, 2017; Nov, 2017; and May, 2018) showed different disease index, the longer the planting was, the higher the disease index, which was also highly correlated with the titers of Ca. Liberibacter asiaticus (Las) in infectd plants. It is worth noting that the new HLB isolate from Picos farm caused severe HLB disease on most of grapefruit selections of seedlings and bud sports in our latest greenhouse evaluation. Those selections were either resistant or tolerant to the previous HLB isolates we maintained in greenhouse. By the end of this year, we will expect to draw a better conclusion if the selected seedling variants display better resistance/tolerance to HLB pathogens at the Picos Research Farm. Second group of the propagates on three different root stocks mentioned above (Ca. 750 plants) have been budded and grown in our greenhouse, and are expected to be planted in Scott grove this month. All the propagates have been tested for the presence of Las. The propagates that carry a low titer of Las will be palnted separately and serve as the experiment for “Cross-protection”.
In the first six months of this project, we investigated the influence of several limiting factors on the performance of a ground penetrating radar (GPR) to accurately detect HLB-affected citrus roots and determine their main structural characteristics. First, single-factor experiments were conducted to evaluate GPR performance. Factors evaluated were: (i) root diameter; (ii) root moisture level; (iii) root depth; (iv) root spacing; (v) survey angle; and (vi) soil moisture level. Second, two multi-factor field experiments were conducted to evaluate the performance of the GPR in complex orchard environments. Experiments were conducted at the citrus research grove of the University of Florida Southwest Florida Research and Education Center (SWFREC) in Immokalee, FL, USA.According to the GPR work principle, three subsystems must be investigated at the outset for accurate root detection: (I) the tree root system; (II) the soil system; and (III) the GPR system. In this study, three types of limiting factors were studied: (1) root property (root diameter and root water content); (2) root position (root depth, horizontal and vertical distance between roots) and scan direction; (3) soil property (soil type and moisture). Four output parameters were evaluated: (i) the shape of the hyperbola; (ii) the signal strength; (iii) the signal overlap and interference, and (iv) the signal noise.Upon root detection, the GPR generated a hyperbola in the radar profile; from the width of the hyperbola, the diameter of the root was successfully determined when roots were larger than 6 mm in diameter. The GPR also distinguished live from dead roots, which is indispensable for studying the effects of soil-borne and other diseases on the citrus tree root system. If two or more roots are located in close proximity, the GPR can distinguish the roots only if their horizontal distance is larger than 10 cm and their vertical distance is larger than 5 cm. The results demonstrated that GPR is useful for studying citrus tree root systems under southwest Florida growing conditions.Furthermore, we started developing an automated (remote-controlled) platform for the GPR technology. The current GPR technology requires an operator to manually complete 360-degree peripheral scans around the tree trunk (per tree) to cover the entire rooting system. A wheel encoder measures and records the distance covered by the GPR. This manual procedure generates many errors ( noise ), mainly because it is difficult for the operator to follow a perfect circular path (360-degree peripheral scan) around the tree (for more information please check our preliminary work at Derival et al., 2018). This automated system will increase data collection accuracy and decrease data collection time. The first prototype is ready for field trials. In the next quarters, we will:1) A GPR evaluation manuscript is under preparation. We will submit it to a high-quality peer-reviewed journal. 2) Evaluate the remote-controlled prototype in the field. Based on the evaluation metrics, we will develop further and improve the first prototype.In this activity, we will compare the automated GPR collected data and the developed 3D maps with the actual root systems of citrus trees. First, we will generate the 3D maps using the automated GPR; then, we will excavate the trees to compare the developed maps with the actual root system. This activity will be limited to non-commercial experimental trials in place at SWFREC.
During this reporting period, we have conducted the following studies. We have continued to work on method development to produce gene-edited and transgene-free plants derived from mature citrus tissues. For the approach to create a chemical resistance to reduce chimeras, we have not observed resistance. We are trying to determine what are possible reasons. We also started a different strategy to create chemical resistance. For that purpose, we have made some gene constructs and will test their efficiencies.
We have completed experiments to repeat some previously conducted experiments in which we observed inconsistent results using the proposed genes regarding their effects when used in mature citrus tissues. We have also completed to test the effects of several chemicals on transient and stable expression activities and also regeneration efficiencies in mature citrus tissues. Our results suggest that there are some improvement of shoot regeneration from calli derived from mature tissues of citrus. We will write a manuscript to report these results.
We have observed that fresh young shoot tissues propagated from mature citrus shoots through vegetative propagation can be regenerated but the efficiencies are somewhat low. The Agrobacterium-mediated transformation efficiency of young fresh tissues generated from mature tissues is very low. To circumvent these problems, we have constructed additional genes. We are starting to test their effectivities using both juvenile and mature citrus tissues.
To develop an in-planta transformation method for mature citrus tissues, we have done additional experiments from the last report. We observed low efficiencies for the Agrobacterium infection based on our transient or stable transformation assays. We have also observed low de novo regeneration efficiencies using mature citrus plants in our in-planta transformation experiments. Thus, we believe that new tools have to be incorporated into the in-planta transformation method in order to successfully use it in mature citrus plants. We are preparing tissues for test whether the new genes that can enhance shoot regeneration we have recently constructed can enhance regeneration efficiencies from mature plants in our in-planta transformation experiments.
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