In the first year of this project (4th quarter), we evaluated the performance of a ground penetrating radar (GPR) to map root architecture of HLB-infected citrus trees, investigated the influence of several factors on the accuracy of the GPR system and developed an enclosure unit for the GPR for remote control of the system. The use of GPR system was a non-destructive approach to map tree roots without having any effect on the roots and the root-soil envronment. It can provide a rapid technique for root mapping. The GPR system consisted of two ground penetrating radar units with frequencies of 900 MHz and 1,600 MHz (TRU Model, Tree Radar Inc., USA) connected to a mobile scanning cart that had a control unit mounted on it which collected the data being sent from the radar and generated root morphology and root density maps. Additionally, a commercial software (TreeWin roots) was used to generate 3D root maps. In the initial evaluation of the system, we found that the GPR unit with 1,600 MHz frequency was more accurate in detecting root location and depth than the GPR unit with 900 MHz frequency. We have also found that the GPR system can distinguish between live and dead roots which is a very important aspect for studying the effects of diseases on citrus tree root systems.
In the first phase, we have investigated the influence of several factors on the accuracy of the GPR system. These factors were: (i) GPR Frequency; (ii) root diameter; (iii) root moisture level; (iv) root depth; (v) root spacing; (vi) survey angle; and (vii) soil moisture level. Experiments were conducted at the citrus research grove of the University of Florida Southwest Florida Research and Education Center (SWFREC) in Immokalee, USA. Upon conducting these experiments, we have found that the GPR can detect a root successfully when the root diameter is more than 6 mm. If two or more roots are in close proximity, the GPR system can only distinguish between them when the distance is more than 10 cm. We summarized all the results of these experiments and prepared a manuscript which we submitted for peer-review to the Agronomy Journal, Special Issue of Precision Agriculture. It was accepted and published:
“Zhang X., Derival M., Albrecht U., and Ampatzidis Y., 2019. Evaluation of a Ground Penetrating Radar to Map Root Architecture of HLB-infected Citrus Trees. Agronomy (Special Issue: Precision Agr.), 9(7), 354. https://doi.org/10.3390/agronomy9070354.”
Received: 3 May 2019 / Revised: 23 June 2019 / Accepted: 1 July 2019 / Published: 3 July 2019.
We have acknowledged CRDF: “Funding: This research was funded by the University of Florida Citrus Initiative and the Citrus Research and Development Foundation.”
In the second phase, we started developing an automated platform for the GPR system. In the current conditions, the operator has to manually go around the trees making very accurate 360-degree peripheral scans while maintaining a constant distance from the tree trunk. This process is time consuming and prone to manual measurement errors. The automated platform will help in eliminating this manual measurement errors thereby increasing the accuracy of the system and also reducing the amount of time taken for data collection. For the first prototype, we designed and built an enclosure unit for the radar unit which will allow the operator to control it remotely. We used 3D printed wheels which were connected to dual channel motor drivers; these motor drivers are remotely controlled by an 8-channel transmitter. Although the enclosure unit worked well on a regular terrain, it failed to make smoother movements of the soil. Hence, we worked on building a different enclosure unit; this time with an agile tracked chassis that is capable of efficient movement on even an irregular terrain. The new enclosure unit works well and can be efficiently used for the remote control of the GPR. We are currently working on developing the adjustable arm that can be used to connect the GPR to the trees which will help it make almost perfect 360-degree peripheral scans around the tree.
Our next objectives are:
(i) To complete the development of adjustable arm connection for GPR to citrus trees.
(ii) Evaluate the remote-controlled prototype in the field and compare it to the manual operation of the GPR system.
Objective 1 – Ongoing.
Objective 2 – Determine the phytotoxic levels of Fe + organic acid solutions on citrus.
Preliminary range experiments were started using F11 to determine the concentration where leaf burn occurs. F11 is the Japanese Fe + citric acid product developed from the patent. In discussions with the Japanese inventors (who visited Florida to see the project), we just learned that another product, F11-C, is the product that was developed from this patent specifically for citrus. Therefore, we have modified all experiments, greenhouse and field, to use F11-C rather than F11. Aichi Steel, the manufacturer of F11 and F11-C, have provided us with all the product required for the project.
Objective 3 – Determine the effect of Fe2+ + organic acid solutions on HLB titer using a rapid greenhouse, HLB-infected citron, rooted shoot bud assay.
Being modified. Originally, we planned on using HLB-infected citron bud cuttings because they show clear HLB symptoms, but are still able to grow. This is unlike HLB-infected grapefruit that can barely be kept alive. However, in our experiments to characterize the “citron system” we have been able to detect very little growth differences between HLB-infected vs healthy citron plants. This means that treatment effects on HLB-infected plants cannot be determined, only the general effects on plant growth. We are working on another system based on very young sweet orange plants and psyllid inoculation where the preliminary experiment looked very promising. The HLB effect was very strong, the requirement for a good system to test treatments. The validation experiment is now being rerun at a larger scale. If it validates, then we will use this system on the objective 3 (and objectives 4 and 5) experiments where it should work quite well.
Objective 4 – See objective 3 discussion.
Objective 5 – See objective 3 discussion.
Objectives 6 and 7 – These are the field tests on mature and young trees. The trees have been planted, baseline ground and aerial data taken, and treatments initiated.
Progress report for the fourth quarter of the 2018/2019 project year
The purpose of the project is to develop new guidelines for restoring root health and improving overall tree nutrition for Florida oranges and grapefruit. The objectives of the project are to:
1. Determine optimal nutrient concentrations in roots and leaves for multiple grapefruit and orange varieties.
2. Compare and contrast fertigation, soil, and foliar fertilization to identify best application method for uptake of nutrients into both underground and aboveground components.
3. Investigate the relationship between root and leaf nutrient contents to tree health, yield, and fruit quality as well as bacteria titer.
4. Generate updated and new guidelines for optimal nutrient contents for roots and leaves for HLB-affected trees.
Progress to date:
The project is being conducted at three sites: Citrus Research and Education Center (CREC), Southern Gardens Citrus near Clewiston, FL and Indian River Research and Education Center (IRREC). Data collection continued in the third quarter particularly on canopy size, soil and leaf nutrient concentrations, HLB disease ratings and root growth and longevity. Data collection continues, and analyses will be done as needed. Updates and data will be presented in future extension meetings after about a year or two of data collection and validation of results to get feedback from growers and the citrus industry.
In addition to one graduate student working on the project at CREC, a second graduate student started working on the project this summer at the IRREC.
At the start of fall 2019, an agricultural assistant was recruited at IRREC to help with data collection, and application of treatments.
In terms of outreach, some of the project co-PIs, Dr. Rossi and Dr. Johnson participated in the “International Root Workshop” in August 2019 that involved international root experts and several graduate students. Also, Drs. Ferrarezi and Dr. Kadyampakeni facilitated 4 statewide workshops on Citrus Nutrition conducted at CREC (October 8, 2019), IRREC (October 23, 2019), Southwest Florida Research and education Center (SWFREC, October 8, 2019) and Sebring (October 8, 2019). Finally, Drs. Kadyampakeni, Ferrarezi and Johnson also presented portions of their work on citrus nutrition and root health at the Materials, Innovation and Sciences in Agriculture (MISA) Conference (October 24-25, 2019) in Orlando, Florida. Two graduate students working on the project presented their proposed work at the South Florida Graduate Student Symposium.
Plans for Next Quarter
The team will continue with data collection including yield and reporting on the progress of the project.
The purpose is to evaluate the control effect of bactericides via trunk injection.
Objective 1.
1.1. Determination of the in planta minimum bactericidal concentrations (MBCs) of bactericides against Las
We developed a new method for evaluating the effects of oxytetracycline (OTC) treatment on Las titers in planta, and determined the relationship between OTC residue levels and control levels achieved for Las using mathematical modeling in greenhouse and field experiments. In greenhouse, OTC injection at 0.05 g/tree decreased Las titers to an undetectable level (Ct value ≥ 36.0) from 7 to 30 DPA, and produced a residue level of OTC at 0.68-0.73 µg/g fresh tissue over this period. In the field, OTC injection at 0.50 g/tree resulted in the decline of Las titers by 1.52 log reduction from 14 to 60 DPA, with residue levels of OTC at 0.27-0.33 µg/g fresh tissue. In both trials, a first-order compart model of OTC residue dynamics in leaves of trunk-injected trees was specified for estimating the retention of effective concentrations. Furthermore, nonlinear modeling revealed significant positive correlations between OTC residue levels in leaves and the control levels for Las achieved. The results suggested that the minimum concentration of OTC required to suppress Las populations in planta to below the detection limit is 0.68 and 0.86 µg/g, and the minimum concentration of OTC required for initial inhibition of Las growth in planta is approximately 0.17 and 0.215 µg/g in leaf tissues under greenhouse and field conditions, respectively. This finding highlights that a minimum concentration of OTC should be guaranteed to be delivered to target Las in planta for effective control of citrus HLB. This study has been published by Phytopathology in a manuscript entitled: The in planta effective concentration of oxytetracycline against Candidatus Liberibacter asiaticus for suppression of citrus Huanglongbing.
In addition, we evaluated the inhibitory activity of streptomycin (STR) against Las in a greenhouse experiment. Citrus trees were trunk-injected with STR, and leaves were inspected for Las populations and STR residues using qPCR and HPLC assays respectively, at various times after STR injection. Assays for Las titers and STR concentration in leaf samples from field trials are also ongoing.
1.2. Effect of bactericides via trunk injection on citrus HLB disease progression, tree health, yield and fruit quality in different aged trees with a different disease severity
The field experiments were performed at four different groves on different aged trees with a different disease severity. They are one located in Avon Park, FL, 3-year old Valencia trees (planted in 04/2016); one in Bartow, FL, 2-year old W. Murrcot trees (planted in 01/2017, “CUPS”); and one in Auburndale, FL, 7-year old Hamlin trees (planted in 02/2012). The last one is in CREC-, Lake Alfred, FL, 20-year old Hamlin trees (planted in 03/1999). The HLB disease severity and tree size (canopy volume and trunk diameter) in the four groves were estimated immediately prior to treatment application. For the field tests, the experiment design is a randomized complete block design (RCBD) for 9 treatments, including 6 injection treatments (3 different doses for OTC or STR), 2 spray treatments (OTC or STR spraying), and one No treatment as a negative control. Each injection treatment consisted of 9 or 15 trees divided into 3 blocks of 3 or 5 trees each. Each spray treatment consisted of 30 trees divided into 3 blocks of 10 trees each. For all the four field trials, the injection treatment applications were completed by the end of April 2019. The first application of spray treatments were completed during spring flushing in February or March 2019, and the second applications were conducted in late June to early July 2019. Leaf samples have been collected from the treated trees at the following time points: 0 (pre- injection), 7, 14, 28 days, 2, 4, and 6 months after treatment. The estimation of Las titers in these leaf samples are ongoing with qPCR assays. The first estimation of HLB disease severity and growth performance (height, trunk diameter, and canopy volume) of immature trees after treatment were performed on May, 2019 (three months after the injection) and continued in a 3-months interval.
Objective 2.
2.1. Examination of dynamics and residues of bactericide injected into citrus and systemic movement within the vascular system
Leaf and root samples have been collected from OTC or STR treated trees in the Avon Park grove at the following time points:0 (pre- injection), 7, 14, 28 days, 2, 4, and 6 months after injection. The samples are being processed for OTC or STR extraction, and the concentrations of OTC and STR in these samples will be determined by HPLC assays. Then, the distribution of bactericides in different plant tissues will be compared in terms of translocation time and dosage.
2.2. Determination of the residue contents of bactericides in fruit and juice in each harvest
This study will be initiated during fruit harvest.
2.3. Analysis of degradation metabolites of bactericides injected into citrus trees
Leaf samples were collected from OTC or STR injected trees in the Avon Park grove at two and four months after treatment for the analysis of the degradation metabolites of the bactericides. The samples are being processed for the extraction of the degradation metabolites.
Objective 3.
3.1. Greenhouse assays of the effect of bactericides via trunk injection on Las acquisition by ACP
This assay will be initiated in the spring of 2020.
3.2. Field assays of the effect of bactericides via trunk injection on Las acquisition by ACP
This assay will be initiated in the spring of 2020.
Objective 4.
4.1. Monitoring resistance development in Las against bactericides
Leaf samples for this test have been collected from 5 trees injected with OTC and 5 trees injected with STR at the highest doses in each of the three groves at six months after the injection. DNA extraction from these samples is ongoing.
4.2. Evaluation of potential side effects of trunk injection of bactericides
We evaluated possible phytotoxity caused by OTC or STR in immature trees (3-year old Valencia) in the Avon Park grove from one week to one month after injection. The trees were be examined for the following symptoms: fruitlet drop, fruit drop, quantity of leaf drop, non-insect related leaf rolling, and leaf discoloration. There was no significant difference in fruitlet drop, fruit drop, quantity of leaf drop, or non-insect related leaf rolling between OTC or STR treatment and untreated control. About 20% (3 out of 15) trees injected with OTC or STR at the highest dose (2.0 g/tree) showed leaf discoloration (yellowing) on some young shoots. These phytotoxicity-like symptoms disappeared at 6 months post injection. There was no infection symptom by Phytophthora in the area surrounding drilling sites (injection holes), probably due to the application of the fungicide Ridomil gold immediately after drilling.
In 7-year old Hamlin trees in the Auburndale grove, three trees injected with STR at the highest dose (3.0 g/tree) showed leaf discoloration (yellowing) on some young shoots, a possible phytotoxitic effect. These phytotoxicity-like symptoms disappeared at 6 months post injection. Other treated trees all showed normal growth.
We will continue the surveys for potential side effects.
Trunk growth between February and June of trees in the nematicide trial did not differ significantly. However, growth rates for all nematicide treatments were nominally higher than for the untreated trees. Growth for a Syngenta experimetal nematicide was 40% greater than the control, and the oxamyl, aldicarb, fluopyram and, fluazaindolizine were between 17-20% greater than the control. There was a weak inverse relationship (P = -0.09) between the trunk growth and the May sting nematode populations, measured following the nematicide treatments. The root weights ranged between 0.28 and 0.37 mg dw/g soil fw and did not differ between treatments. Sting nematode populations were significantly lower (P = 0.04) in perennial peanut plots (6 nematodes/250 cc soil) than in control plots (23 nematodes), 5 months after laying the peanut sod. Fall 2019 nematicide treatments were initiated in September and will continue throughout October. The materials are being injected for two hours, begining 30 minutes after irrigation begins. The plots are irrigated for a further 30 minutes following the nematicide applications.
After just a year of support for this project, we have not yet completed any of our objectives. However, we have set up all of the experiments in the greenhouse and field needed to accomplish those objectives.
A greenhouse trial over the winter months taught us the level of foliarly-applied potassium phosphate needed to maintain proper P nutrition in citrus. A second greenhouse trial was established to determine whether foliarly applied potassium phosphate would decrease citrus levels in phloem compared to the application of calcium phosphate (i.e. rock phosphate). By September of this year, it was clear that just one month of treatment with foliar potassiumphosphate decreased organic acids level in phloem (citrate, malate, and alpha-ketoglutarate) by more than half compared to trees whose roots were treated with calcium phosphate.
The first greenhouse trial taught us the appropriate levels of potassium phosphate to spray on citrus trees in the field. In April 2019, a field trial commenced in a grove o 20-year-old infected trees. There were 10 replicates and four treatments in a randomized complete block design. We spray the trees with 0, 1x, 3x, and 9x the optimal level observed in the greenhouse. Even at 1x, the plants are receiving enough P for flushing and fruit development. The plants are sprayed six times a year including after each flush.
A second field trial was established in August 2019 in the Immokalee area. That trial is the same design as that in Polk County. The idea was to have a trial on the ridge (Polk County) and flatwoods (Collier County) regions of citrus production. In both trials, the outcomes being measured are CLas titer in leaf midribs and leaf area index. Baseline samples were taken prior to the first spray and subsequent sampling will be done at 6, 12, and 18 months after the first spray.
As we wait for field results, we are now testing the effect of a foliar potassium phosphate spray (compared to root-applied calcium phosphate) on CLas titer in graft-infected trees in the greenhouse. We expect our first results from this in six months.
Given the speed with which foliar potassium phosphate can reduce organic acids levels in citrus phloem, we expect to see positive results in the field in reducing CLas titer in the first quarter of 2020. I am pleased to report that this project is working as planned so far. Foliar potassium phosphate does reduce citrate levels and the levels of other organic acids in phloem. As organic acids, particularly citrate, are the preferred carbon source for Liberibacter crescens, we expect this treatment to starve the pathogen.
Our team (Triplett, Vincent, Killiny, and Wang) are working very well together and meet to discuss the project every two weeks.major
Update for this quarter:
No new plantings. Canker was assessed in a number of the transgenic trees from UF and USDA and in the trifoliate and trifoliate hybrid planting of UCRiverside. Growth and CLas titer data were collected on Stover lab transgenics. The Stover BRS permit was renewed and associated plantings were reported to be in compliance. The McNellis trees will be planted next quarter.
Previous quarter
A number of trials are underway at the Picos Test Site funded through the CRDF. A detailed current status is outlined below this paragraph. In the last quarter, the most significant advances have been: 1) Planting of USDA Mthionin transgenics with 108 transgenic Hamlin grafted on wild type Carrizo (7 events represented), 81 wild type Hamlin grafted on transgenic Carrizo (16 events represented) and 16 non-transgenic controls. 2) Planting was made of transgenics from Zhonglin Mou of UF under Stover permit, with 19 trees of Duncan, each expressing one of four resistance genes from Arabidopsis, and 30 Hamlin expressing one of the genes, along with ten non-transgenic controls of each scion type. 3) Renewal and approval for BRS permit effective 9/1/19 through 8/31/20. 4) Continuation of an experiment on pollen flow from transgenic trees. FF-5-51-2 trees are slightly more than 1000 ft from the US-802, and are self-incompatible and mono-embryonic. If pollen from transgenic trees is not detected from open-pollination, it should reduce isolation distances required by BRS. 5) Early-flowering transgenic Carrizo (flowered ex-vitro within five months of seed sowing, and used at 12 months) was used to pollinate some of the same FF-5-51-2 and some fruit appear to have set. 6) What should be the final samples from the C. Ramadugu-led Poncirus trial (#3 below) completed preparation and were shipped in ethanol to UC Riverside.
Previously established at the site:
1) The UF Grosser, Dutt and Gmitter transgenic effort has a substantial planting of diverse transgenics. These are on an independent permit, while all other transgenics on the site are under the Stover permit.
2) Under the Stover permit a replicated planting of 32 transgenic trees and controls produced by Dr. Jeff Jones at UF were planted. These trees include two very different constructs, each quite specific in attacking the citrus canker pathogen.
3) A broad cross-section of Poncirus derived material is being tested by USDA-ARS-Riverside and UCRiverside, and led by Chandrika Ramadugu. These are seedlings of 82 seed source trees from the Riverside genebank and include pure trifoliate accessions, hybrids of Poncirus with diverse parents, and more advanced accessions with Poncirus in the pedigree. Plants are replicated and each accession includes both graft-inoculated trees and trees uninfected at planting. Likely 2019 will be the last year for data collection.
4) More than 100 citranges, from a well-characterized mapping population, and other trifoliate hybrids (+ sweet orange standards) were planted in a replicated trial in collaboration with Fred Gmitter of UF and Mikeal Roose of UCRiverside. Plants were monitored for CLas titer 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. Manuscripts have been published reporting HLB tolerance associated QTLs and differences in ACP colonization. Trees continue to be useful for documenting tolerance in a new NIFA project.
5) A replicated Fairchild x Fortune mapping population was planted at the Picos Test Site in an effort led by Mike Roose to identify loci/genes associated with tolerance. This planting also includes a number of related hybrids (including our easy peeling remarkably HLB-tolerant 5-51-2) and released cultivars. Genotyping, HLB phenotyping and growth data have been collected and will continue to be conducted under a new NIFA grant.
6) Valencia on UF Grosser tertazyg rootstocks have been at the Picos Test Site for several years, having been CLas-inoculated before planting, and several continue to show excellent growth compared to standard controls (Grosser, personal comm.).
7) In a project led by Fred Gmitter there is a planting of 1132 hybrids of C. reticulata x C. latipes. C. latipes is among the few members of genus Citrus reported to have HLB resistance, and it is expected that there will be segregation for such resistance. The resulting plants may be used in further breeding and may permit mapping for resistance genes.
8) Seedlings with a range of pedigree contributions from Microcitrus are planted in a replicated trial, in a collaboration between Malcolm Smith (Queensland Dept. of Agriculture and Fisheries) and Ed Stover. Microcitrus is reported to have HLB resistance, and it is expected that there will be segregation for such resistance. The resulting plants may be used in further breeding and may permit mapping for resistance genes.
9) Conventional scions on Mthionin-producing transgenic Carrizo are planted from the Stover team and are displaying superior growth to trees on control Carrizo.
10) Numerous promising transgenics identified by the Stover lab in the last two years have been propagated and will be planted in the test site. New transgenics from Tim McNellis of PSU will be planted in the next quarter.
11) Availability of the test site for planting continues to be announced to researchers.
Project rationale and focus:
The driving force for this three-year project is the need to evaluate citrus germplasm for tolerance to HLB, including germplasm transformed to express proteins that might mitigate HLB, which requires citrus be inoculated with CLas. Citrus can be bud-inoculated, but since the disease is naturally spread by the Asian citrus psyllid, the use of psyllids for inoculations more closely resembles “natural infection”, while bud-inoculations might overwhelm some defense responses. CRDF funds supported high-throughput inoculations to evaluate HLB resistance in citrus germplasm developed by Drs. Ed Stover and Kim Bowman. The funds 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 base-funded USDA technician is also assigned ~50% to the program. USDA provides greenhouses, walk-in chambers and laboratory space to accommodate rearing and inoculations.
Most recent quarter:
Stover lab: 11,560 ACP used for 1388 leaves inoculated by DLA ( detached leaf assay), 108 small trees inoculated by no choice caged assay, and 13 assays run. A total of 14 different experiments, most testing Liberibacter killing transgenics.
Bowman lab: Continued grow out for first group of budded trees that were inoculated in July. First PCR and scoring will be done in November, 2019. Rootstocks were budded with Valencia for test groups 2, 3, and 4, to be inoculated beginning in early 2020. Once these Valencia budded trees are to size, there will be a regular ACP inoculation of a new group of trees every 1-2 months.
Previous quarter:
Over 7000 infected ACP were used in the last quarter, in part to screen 450 trees, but also for other related uses. The Stover lab used 1700 ACP in no-choice inoculation of transgenic citrus. 2700 ACP were used for detached leaf assays in which leaves of putative CLas killing transgenics and related controls are exposed to CLas-infected ACP for 4 days, allowed 3 days for ACP-free metabolism and then assessed for CLas titer in leaves and the ACP. One thousand ACP were used in an assay in which CLas+ ACP are used to develop a uniform homogenate for rapid testing of putative CLas-disrupting peptides
The Bowman lab has transitioned to use of grafted trees with a commercial scion in 2.5 liter pots. The first group of test plants will be removed from ACP inoculation the second week of July to begin post-inoculation evaluation. Subsequent groups of test plants for rootstock evaluation are being prepared.
Previously achieved in this project:
The 35 day federal government shutdown, and the threat of a possible shutdown on Feb 15, directly disrupted our ability to initiate and conduct experiments using the CLas+ ACP colonies. In addition, considerable rehibilitation of colonies and supporting plants was necessary due to the minimal care that could be provided during the shutdown. Only 2400 CLas+ ACP were used for experiments in this quarter and were used for detached leaf assessments of plants expressing three different transgenic constructs. We anticipate a normal demand in the current quarter.
As of December 21, 2018, a total of 14,111 plants had passed through the inoculation process. A total of 361,255 psyllids from colonies of CLas-infected ACP had been used in inoculations. Not included in these counts of inoculated plants and psyllids used in inoculations were many used to refine inoculation procedures, which provided insight into the success of our inoculation methods and strategies for increasing success. After inoculations, plants were returned to the breeders and subsequently subjected to further inoculations when they are transplanted to the field.
In addition to inoculating germplasm, infected psyllids were supplied to other researchers for other purposes. This side of the project grew over time, and detailed records were not maintained on how many were given out until 2018. More than 10,000 infected psyllids were supplied to the research community for an array of experiments during 2018. Recipients included researchers with USDA in Fort Pierce, Ithaca and Beltsville, UF in Gainesville, Cornell in Ithaca, University of California, and University of Nevada.
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 within the next two weeks. 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 ihas developed 45 putative grapefruit and sweet orange transformants. We are screening these currently via PCR. 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. Citrus canker has developed on plants in the field and the trees were rated for disease in June. At that point there was not a significant amount of disease to show any possible differences.
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 two most promising EFR transgenic plants (based on ROS activity) onto two rootstocks (812 and Sour Orange) and planted them in the field at Fort Pierce in collaboration Dr. Ed Stover. They were planted in the field in late March. There was some citrus canker on the trees, although they were not uniformly infected. We have identified additional transgenics from plants received from Dr. Vladimir Orbovicthat that will be grafted onto rootstocks once the rootstock and transgenic trees are of adequate size.
In this study, we conducted the following specific objectives:1) GFP labeling of Candidatus Liberibacter. 2) Elucidation of plant- Candidatus Liberibacter asiaticus (Las) interaction through real-time monitoring of Las movement and multiplication in planta. 3) Investigate the effect of different control approaches on the dynamic population of Las in planta.
Objective 1. We constructed pDH3::PgyrA-GFP which has a wide bacterial host range replicon, repW, but cannot be inserted into a genome. Transformants and the GFP expression in Liberibacter crescens BT-1 were confirmed.
Objective 2. Our data showed that Las moves with phloem sap from source to sink tissues and remains in the young flush after ACP transmission and before the young flush matures. This observation prompted us to develop a method for early diagnosis of HLB, which allows inoculum removal to prevent ACP acquisition and transmission of Las. We conducted targeted early detection of Las in cultivar Valencia sweet orange (Citrus sinensis) before HLB symptom expression. ACPs secrete salivary sheaths at their feeding sites, which can be visualized using Coomassie brilliant blue staining owing to the presence of salivary sheaths secreted by ACP. Epifluorescence and confocal microscopy indicate the presence of salivary sheaths beneath the blue spots on ACP-fed leaves. Quantitative real-time polymerase chain reaction (PCR) and conventional PCR assays are able to detect Las in the ACP feeding surrounding areas as early as 2 to 20 days after ACP feeding. This finding lays a foundation to develop much-needed tools for early diagnosis of HLB before symptom expression, thus assisting Las inoculum removal and preventing HLB from spreading.
Objective 3. We evaluated the spatiotemporal dynamics of oxytetracycline in planta and its control effect against HLB via trunk injection. Las-infected ‘Hamlin’ sweet orange trees on ‘Swingle’ citrumelo rootstock at the early stage of decline were treated with oxytetracycline hydrochloride (OTC) using trunk injection with varying number of injection ports. Spatiotemporal distribution of OTC and dynamics of Las populations were monitored by high-performance liquid chromatography method and qPCR assay, respectively. Uniform distribution of OTC throughout tree canopies and root system was achieved 2 days postinjection. High levels of OTC (>850 µg/kg) were maintained in leaf and root for at least 1 month and moderate OTC (>500 µg/kg) persisted for more than 9 months. Reduction of Las populations in root system and leaves of OTC-treated trees were over 95% and 99% (i.e., 1.76 and 2.19 log reduction) between 2 and 28 days postinjection. Conditions of trees receiving OTC treatment were improved, fruit yield was increased, and juice acidity was lowered than water-injected control even though their differences were not statistically significant during the test period. Our study demonstrated that trunk injection of OTC could be used as an effective measure for integrated management of citrus HLB.
We tested HLB control via trunk injection of plant defense activators and antibiotics. In this study, eight plant defense activators and three antibiotics were evaluated in three field trials for their effect to control HLB by trunk injection of young and mature sweet orange trees. Results showed that four trunk injections of several activators, including salicylic acid, oxalic acid, acibenzolar-S-methyl, and potassium phosphate, provided significant control of HLB by suppressing Las titer and disease progress. Trunk injection of penicillin, streptomycin, and oxytetracycline hydrochloride resulted in excellent control of HLB. In general, antibiotics were more effective in reduction of Las titer and HLB symptom expressions than plant defense activators. These treatments also resulted in increased yield and better fruit quality. Injection of both salicylic acid and acibenzolar-S-methyl led to significant induction of pathogenesis-related (PR) genes PR-1 and PR-2 genes. Meanwhile, injection of either potassium phosphate or oxalic acid resulted in significant induction of PR-2 or PR-15 gene expression, respectively. These results suggested that HLB diseased trees remained inducible for systemic acquired resistance under field conditions. In summary, this study presents information regarding controlling HLB via trunk injection of plant defense activators and antibiotics, which helps citrus growers in decision making regarding developing an effective HLB management program.
We evaluated the effect of the combinations of plant defense elicitors, nitrogen (N) fertilizer, and compost to control HLB. After four applications over two consecutive growing seasons we found that the combination of compost, urea, and plant defense elicitors β-aminobutyric acid, plus ascorbic acid and potassium phosphite with or without salicylic acid, slowed down the progression of HLB and reduced disease severity by approximately 18%, compared with the untreated control. Our data showed no decline in fruit yield, indeed treatment resulted in a higher yield compared with the untreated control.
The present reporting period runs from June 16 – September 15, 2019. Mr. Chad Vosburg is the M.S. degree student in the Penn State Department of Plant Pathology graduate program who is working on the project. Chad took a trip to Fort Pierce, FL, September 12 – 15, 2019. During this trip, he set up plant propagations sufficient for 1-2 runs of an HLB resistance test for all the FT-scFv grapefruit lines. Existing propagated plants were cut back to induce a new flush of growth, which is essential for a successful HLB infection test. Personnel at the USHRL will initiate the HLB screenhouse test when the plants have reached the optimal stage of re-growth. Chad also worked with personnel at the USHRL to set up a field test of two of the FT-scFv lines. Chad is now working on mastering PCR skills for CLas detection that will be part of the HLB infection tests in the lab at Penn State. We anticipate that the first run of an HLB resistance test will beinitiated during the next reporting period. Mr. Jeremy Held, a Ph.D. student in the Intercollege Graduate Program in Plant Biology at Penn State, continued his studies of the stabilty and expression of the FT-scFv protein in the grapefruit lines. He optimized western blotting and antibody selection and plans to test for movement of FT-scFv across the graft union using immunoprecipitation during the next reporting period. Finally, we initiated tests to check whether FT-scFv plants are fertile. Initial fruits obtained from the FT-scFv plants were seedless, possibly because they are being grown in greenhouses or growth chambers. Jeremy performed some cross-pollinations of FT-scFv flowers in the spring of 2019, and one of these fruits was cut open during the reporting period and found to have normal seed set. This is good news if the FT-scFv construct were ever to be used as a citrus breeding acceleration tool, since precocity is one of the phenotypes of FT-scFv plants. Jeremy also tested pollen viability from FT-scFv plants and found the pollen to be viable and able to germinate in the laboratory in pollen germination medium.
Between the July and October, Citrus transformation Facility continued its operation without any interruptions. Within this period, we accepted three new orders. All orders required production of transgenic Duncan grapefruit plants.
In the last three months, CTF produced 74 plants. Among the produced plants there were: six Duncan plants (BB3), 18 Duncan plants (BB4), seven Duncan plants (HGJ34), three Duncan plants (HGJ74), two Duncan plants (HGJ87), three Duncan plants (HGJ88), ten Duncan plants ZM14, four Mexican lime plants (M2SF), one Mexican lime plant (contSF), one Murraya (BB1), one Pomelo plants (HGJ68), five Valencia plants (BB3), five Valencia plants (BB4), eight Valencia plants (JJ7). Codes in parentheses represent names of different binary vectors (genes) used in transformation experiments.
One of the CTF employees working on the USDA grant left the lab in the first week of September. After securing additional funding from other participants of that USDA grant, I have submitted position description to HR department and expect to hire new employee before the end of the year. Currently, there are five employees in the CTF.
Most of the fruit of Duncan grapefruit and Valencia orange that was stored in the cold room at CREC rotted and started growing mold. Only six bins of Valencia and ten bins of grapefruit were kept as a source of seeds. The crew working in A. Schumann’s CUPS harvested half of the yield from Duncan grapefruit trees we have there. These fruits will be used for seed extraction in a matter of weeks. We will ask A. Schumann’s crew to harvest the rest of Duncan grapefruit from CUPS for us in January.
To decrease our dependence on CRDF funding, two research proposals were submitted to two federal agencies: NSF and NIFA. It should be ~Christmas before we hear whether the NSF was successful, but NIFA will likely be delayed because of its move to Kansas City. I am working on a third proposal that will be submitted to the USDA SCRI Citrus whenever the RFP is announced. This RFP will also be delayed because of USDA’s move to Kansas City. CRDF funding obligations would be decreased if these proposals were funded. I have contacted scientists at UF, the USDA in Fort Pierce, Texas A&M, University of Arizona, Standford, UC Davis & UC Riverside to increase our clientele. In the near future, our prices will increase as suggested by the CREC Director, the CRDF, and the External Review committee. The actual dollar amount will be determined in consultation with the CREC Director.
It was a productive quarter for the transgenic event production. Forty-four transgenic shoots were identified, 18 surived micrografting, 6 died, and 10 are still healing. Use of the GFP reporter partially accounts for this increased productivity. In spring & summer, our productivity also naturally inceases because citrus is more vigorous. In addition, a new staff member was screening for transgenics & this might have contributed to increased productivity. We received two new vectors from Dr. Mou. Dr. Bonning contacted me about potential work in the future. Dr. Wang’s group is interested in transforming mature grapefruit for commercialization, but we had to introduce grapefruit cultivars first (see below) & then determine which cultivars are amenable to transformation. Dr. Dutt has provided three vectors for our collaborative CRDF project together.
We discovered that Dr. Grosser’s OLL4 and Valquarius cultivars have relatively high Agrobacterium transformation efficiencies. UF15 rootstock is recalcitrant to Agrobacterium transformation with no shoots forming in tissue culture. We introduced new budwood cultivars from FDACS. The budwood introductions were Marsh, Flame, Ray Ruby, and Duncan grapefruits, Temple sweet orange (zygotic embryos), and US-942. We will introduce OLL20 in the future because it is important to the juice industry.
We continue to conduct biolistic transformation of mature scions with citrus reporter genes & selectable markers with the ultimate goal being cis/intragenic production. Next week, we will test whether Dr. Dutt’s reporter can be used without a selectable marker. A new plant – derived selectable marker has been tested with excellent results. Replication of these experiments is currently underway. A similar vector with a citrus-derived selectable marker will be tested in the future. This system is an alternative to E. coli genes for antibiotic resistance & selection. Using citrus-derived sequences should also be more consumer friendly. A manuscript should be produced from this research. PEG-mediated DNA precipitation is being tested to increase biolistic transformation efficiency rather than protamine sulfate or spermidine.
Dr. Wu performed Southern blots for another scientist to show copy number of the transgenes for a scientific publication.
RC Webview, the electronic system that runs the growth facility, has been offline for ~1 week & the reasons are unclear. UF Gainesville is supposedly in the process of connecting a new server. With no other choice available, we have to water with city water which might contain slime mold spores, does not contain enough chlorine, and hasn’t passed through 2 UV filters.
All significant results remain the same as in the previous quarter.
The long term field trial continues with weekly psyllid counts and quarterly CLas infection testing. Treatments continue to have similar effects on ACP counts. Plants in both of the kaolin treatments continue to show higher growth rates than the other two treatments. The red treatment has the highest growth rate, trunk cross-sectional area, and canopy volume. Kaolin treated trees that are infected grow more than untreated-infected trees, but less than treated uninfected trees. The field trial will continue until the project ends, when we expect to have the first economic yield.
We are now performing follow-up repetitions of the MS student’s thesis work. We anticipate publication submission of this work in the Fall.
This project is an continuation of an objective of existing CRDF funded project (# 00124558 ; ended in March 2019, final report submited to CRDF) with some added treatments to be evaluated in comparison to control (dry conventional fertilizer with foliar micronutrients). Objective 1 which is the continuation of # 00124558 included 10 treatments.
The added treatments from objective 2
1. CRF + Tiger Micronutrients+ Mn 50%
2. CRF + Tiger Micronutrients+ Zn 50%
3. CRF + Tiger Micronutrients+ Fe 50%
4. CRF + Tiger Micronutrients+ B 50%
5. CRF + Tiger Micronutrients+ Mn +Zn 20%
6. CRF + Tiger Micronutrients+ Mn +Fe 20%
7. CRF + Tiger Micronutrients+ Zn +Fe 20%
8. CRF + Tiger Micronutrients+ Zn +B 20%
9. CRF + Tiger Micronutrients+ Fe + B 20%
10. CRF + Tiger Micronutrients+ Mn +Zn 50%
11. CRF + Tiger Micronutrients+ Mn +Fe 50%
12. CRF + Tiger Micronutrients+ Zn +Fe 50%
13. CRF + Tiger Micronutrients+ Zn +B 50%
14. CRF + Tiger Micronutrients+ Fe + B 50%
The treatment for objective 3:
1.CRF + Foliar Micronutrients + Tiger 90;
2.CRF + Tiger Micronutrients
So altogether currently there are 25 treatments of citrus nutrition that are being compared to control.
These treatments have been initiated at all sites in spring of 2019. The pretreatment data and first and second round of fertilizer application have been already made. The leaf and soil nutrient analysis has been completed and currently we are interpreting the data. Overall, the trees are looking good and they have a good crop load.
The yield and consumer panel results from Objective 1 have been presented at Citrus Expo 2019. These results will be additionally presented at SWFREC and CREC on October 29th, 8th, respectively.’
This a fertilizer evaluation trial and the progress on it is timely and as per expectations
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