The contract with Compac Sorting Equipment LTD and University of Florida was developed and signed by both parties for the optical sizing/grading unit. The equipment has been serviced by Compaq, including updating the operating system to the latest software, computers, and electronic communication. They replaced the electronics because the chips used in the dedicated PLC’s are no longer available. Their engineers designed a new system to replace the legacy systems. This is a key to limiting the possibility of expensive repairs down the road. The payment from UF should be sent soon and the equipment delivered shortly after that. We anticipated receiving the equipment by the end of February at the very latest.
This single-lane machine has extensive capabilities with both visible spectrum and near infrared (NIR) cameras to measure fruit color, dimensions, weight, and internal constituents. Computing algorithms can be utilized the calculate fruit volume, density, internal Brix concentrations, defects, and the manufacturer believes it can accurately detect and separate HLB-affected fruit. The unit has three drops for separating different classifications of fruit.
Sept 2015
Objective 1: Evaluate the optimal spray timing for Florida and investigate if tree skirting or alternative products improves fungicidal control of citrus black spot.
Objective 3: A MAT-1-1 isolate may enter Florida and allow for the production of ascospores. The industry needs to know if this happens, as it will affect management practices. Additionally, the existing asexual population may be more diverse than currently measured. If multiple clonal linages exist, then there may be different sensitivities to fungicides or other phenotypic traits. We also need to determine whether P. paracitricarpa or P. paracapitalensis are present in Florida for regulatory concerns due to misidentification. We plan to survey for the MAT-1-1 mating type, unique clonal lineages, and two closely related Phyllosticta spp.
We are still waiting for symptom experssion in the fungicide and skirting trials. As explained in the previous quarterly report, we expect symptom expression in early 2020 as Valencia, a late harvested cultivar, start to ripen. We have marked off products that should not be harvested and have occassionally visited the trials to make sure that no symptoms have started to express unexpected. None have been observed so far.
The subcontract with CRI is finally complete. Despite this, 23 isolates were sequenced (6 from South Africa and 17 from the USA) using the Ion Torrent System. The genomes of all the isolates have been successfully assembled and analysed using a customised bioinformatics pipeline. Previous genotypes obtained with SSR primers were confirmed and new SSR primers were developed in silico. To date, mapping and SNP variant statistics as well as in silico genotyping data revealed significantly less variation between the USA isolates than between the isolates from South Africa.
To investigate the fine-scale genetic differences within the USA P. citricarpa population, the assembled genomes were annotated by mapping the reference genes to the assembled contig sets, using GMAP. The variant calling results together with the annotations were further analysed using SNPeff, to detect putative variable genes. In silico detection of mating types were also performed, and confirmed that only one mating type is present in the USA.
Five more South African isolates is in the process of being sequenced. For some of the isolates selected for the next round of sequencing, the available DNA are not of high enough quality, and new cultures of these isolates are grown and DNA will be extracted.
New fungicide products are being tested for the management of CBS in South Africa. Products being evaluated include Enable (copper hydroxide) sprayed in alternation with Amistar Top, Luna Sensation or Headline, which is also tested in alternation with copper hydroxide. Fungicides are applied every 4 weeks from October 2019 until March 2020. Additional fungicides including Miravis, Miravis Top, Ph-D, Priaxor and Luna experience could not be tested due to their unavailability in South Africa. The fungicides are being tested in a ‘Valencia’ orange orchard with a history of CBS.
Within Objective 3 (Survey for the MAT-1-1 mating type and two closely related Phyllosticta spp.), the culture collection of P. citricarpa isolates from Florida which contains more than 125 single-speed isolates has been plated and individual isolates cultured for DNA extraction. Thirty-six isolates have been extracted and high quality DNA purified. PCR conditions for amplifying the tef1 sequence from Phylosticta spp. are currently under evaluation and optimization. Amplicons will be sequenced and compared to determine if the cryptic P. paracitricarpa and P. paracapitalensis species exist within our collections. In regards to screening for matting type, all isolates from our current collection have been screened and determined to be of the MAT1-2 mating type. Screens are ongoing at the Division of Plant Industry to evaluate mating types of new isolates coming from outside of the current quarantine zone and we are coordinating with Dr. Hector Urbina and colleagues to monitor the findings. Beyond Florida, permits have been approved from both the US and Cuba to receive extracted DNA samples from the Cuban collection of P. citricarpa isolates. DNA representing these isolates will be screened for mating type and used later for analysis of global P. citricarpa population structure. Additional efforts have been focused on determining the role of fruit developmental etiology on susceptibility to CBS. A quarantine greenhouse experiment has been established with fruit-bearing Myer lemon trees. Eighty-six fruit of varying developmental stages as well as controls have been inoculated. We are currently monitoring for symptom development and collecting data on environmental parameters and fruit development.
The purpose of this project is to reveal the mechanisms of bactericide uptake and transport in citrus plant and establish a theoretical basis for developing technologies to improve the efficacy of bactericides, which is helpful to provide potential solution to the development of effective chemotherapeutic tools for HLB management. Achieving this outcome will require progress in the following three tasks: (1) to compare the delivery efficacy of bactericides with three application methods (foliar spraying, truck injection, and root administration) based on the uptake and dynamic movement/distribution of the bactericide within the tree; (2) to clarify the systemic movement and transportation mechanisms of bactericides within the phloem of tree; and (3) to investigate the effects of citrus variety and age on the delivery efficacy of bactericides. This project requires a combination of greenhouse studies and field trials. Prior to conducting these experiments, a sensitive and accurate quantifying method of bactericides (oxytetracycline and streptomycin) in citrus tissues is needed. This project officially started on December 1, 2018. This is the 4th quarterly progress report covering 9/01 to 11/31, 2019. During this period, we have conducted and/or completed the following work/research tasks:
1) The procedures of sample preparation method are further improved. Based on the test results of leaves, stems and root samples from foliar spraying, the recovery of both bactericides (streptomycin, oxytetracycline) is approximately 70%, which is within the acceptable range.
2) The greenhouse experiments of citrus tree trunk injection were completed. All the samples were collected and are being prepared with the above-mentioned newly modified method for the analysis of bactericides using a LC-MS. The results are expected to be available at the end of December 2019.
3) The root administration experiments were initiated in greenhouse, and the treatments included two bactericides (streptomycin, oxytetracycline) and three different concentrations for each bactericide on young citrus trees. So far, two batches of samples (leaves, stems and root) were collected and are being prepared for the analysis of bactericides using the LC-MS, and the last batch of samples will be collected on December 20.
The work planned for the next quarter:
The major goals of research for the next three months are to analyze the obtained data and to evaluate the delivery efficacy of three different application methods for bactericides in field. To accomplish the goals, field experiments will be carried out and the following research tasks will be performed during the next quarter (December 1, 2019 to February 29, 2020): 1) Collection and analysis of the obtained data; 2) Selection of desired sites and citrus trees for field experiments; 3) Conducting foliar spraying treatments of both bactericides for the field experiments.
This report is for the first quarter of year 1 of project 19-001C. For the first quarter, 5,866 samples were run which is 241 samples over the budgeted amount for the period. Of these, 97% were plant samples and 3% were psyllid samples. This total also includes approximatley 1000 samples that were run using alternate assays as requested by the customer. Beginning with the 19-001C funding period, the Southern Gardens Diagnostic Laboratory (SGDL) said it would run alternate assays as requested by the customer provided that the customer provides the primer sequences and testing details.
October 2019
The objectives for this proposal are 1) Conduct field trials of new products and fungicide programs for PFD management as well as validation trials for the Citrus Advisory System (CAS); 2) Investigate the reasons for the movement of Postbloom fruit drop (PFD) to new areas and recent major outbreaks; 3) Evaluate methods for initial inoculum reduction on leaves so that early fungicide applications could be more effective and identify the constituents of the flower extracts using “omics” techniques.
The spray trial and CAS validation trial data were collected and analyzed. The data were reported as part of the comprehensive final report for project 16-010C in September. Planning for the next season will be underway in the next quarter.
The wind tunnel experiments continued throughout this quarter. The majority of the data for spores from leaves has been completed. Leaves were inoculated in planta with Colletotrichum acutatum and allowed to form appressoria. The approssoria were stimulated to germinate and for secondary conidia. The trees were then exposed to three windspeeds: 5, 10, and 20 m/s, with and without rain within the wind tunnel at the USDA Tropical Research Unit in Fort Pierce. Petri dishes with selective media were placed a different distances from the inoculated plants prior to the treatments. The number of CFU were counted to assess how well the inoculum moved. The data are in the process of being modelled to select the best fitting models. Even though the data are still being analyzed, it is evident that some inoculum can move up to 15m with just wind and even more inoculum moves when there is wind and rain. This has not been reported for C. acutatum before which is considered to be an exclusively splash dispersed organism. The secondary conidiation inoculum has not been studies in detail previously but is the primary PFD inoculum and responsible for initial outbreaks of disease. We plan to study the movement of the primary inoculum produced on flowers when they become available in the spring.
Little progress has been made with the modeling to better predict PFD outcomes because of the focus on the inoculum movement. The movement was a priority because of the limited access to the USDA facilities.
There was also little work on the germination stimulation with the flower extracts because of the reasons stated above. The experiments have been restarted and progress with the different extracts are expected in time to inform our efforts at flowering this spring.
Dr. Yu Wang’s lab has been busy analysing the constituents of the water extracts of the flowers at pin head, button bloom, and open flowers. All the standards and chemical techniques were obtained and methods optimized. The sugars and sugar alcohols in the flower stages were identified. In agreement with previous studies that sucrose stimulated the germination of appressoria, glucose and fructose concentrations increase with flower maturity. Some sugars and sugar alcohols did not change signficantly among flower maturity stages while some increased at button stage but decreased at open bloom.
Sept 2019
Objective 1: Evaluate the optimal spray timing for Florida and investigate if tree skirting or alternative products improves fungicidal control of citrus black spot.
Objective 3: A MAT-1-1 isolate may enter Florida and allow for the production of ascospores. The industry needs to know if this happens, as it will affect management practices. Additionally, the existing asexual population may be more diverse than currently measured. If multiple clonal linages exist, then there may be different sensitivities to fungicides or other phenotypic traits. We also need to determine whether P. paracitricarpa or P. paracapitalensis are present in Florida for regulatory concerns due to misidentification. We plan to survey for the MAT-1-1 mating type, unique clonal lineages, and two closely related Phyllosticta spp.
In the fungicide and skirting control trial, seven applications of fungicides have been completed. They were applied approximately every 28 days. Skirting was done on June 4, 2019. We plan to have one more application for one treatment. Activity on this part of the project will be minimal in the next few months as we will need to wait for enough symptom development to rate which is expected no earlier than February based on previous years.
Applications were continued at an approximate 28 day interval for the fungicide screening trial throughout the summer for a total of 6 applications. Again, this part of the project will have minimal activity because we will need to wait for symptom development. Since again the cultivar is Valencia, symptom expression is expected in early 2020.
The subcontracting process with CRI has been slow although some progress has been made and should be complete soon. Despite this, 23 isolates were sequenced (6 from South Africa and 17 from the USA) using the Ion Torrent System. The genomes of all the isolates have been successfully assembled and analysed using a customised bioinformatics pipeline. Previous genotypes obtained with SSR primers were confirmed and new SSR primers were developed in silico. To date, mapping and SNP variant statistics as well as in silico genotyping data revealed significantly less variation between the USA isolates than between the isolates from South Africa.
Jeff Rollins is still working on travel to Cuba because of changes to Federal rules and regualtions. Trees and fruit have been selected for on tree experiments and inoculum production is underway.
We continued our tree evaluations at the three trial locations (1) SWFREC, (2) Hendry County – Duda & Sons, and (3) Polk County – Peace River Packing Co. during the third quarter of 2019. Tree evaluations included monthly (SWFREC) or bimonthly (Hendry County and Polk County) root growth measurements using rhizotrons and leaf flush ratings. In addition to rhizotron measurements, root cores were collected at each location to determine root densities and other fibrous root properties. Leaves were collected from all trials for nutrient analysis. Statistical data analysis is still in progress.
Two-year tree growth data are currently being collected for trees located at SWFREC, and preparations at SWFREC are in progress for whole tree excavation and detailed root architectural analysis, plant biomass determination, hydraulic conductivity measurements, and measurement of other tree parameters.
Updates on project progress were presented during the Citrus Expo seminar presentation in August (“Bigger is not always better – large-scale rootstock evaluations on a Ridge and flatwoods site”) and poster display “Rootstock propagation methods” with printed educational material. Additional information was presented during a seminar at SWFREC in September titled “Which rootstock to plant in an HLB-endemic environment”. A new EDIS publication “Citrus nursery production guide, chapter 6: Citrus rootstock propagation: traditional techniques and recent advances” was completed and is available at https://edis.ifas.ufl.edu/hs1329.
We are awaiting to plant the fourth trial at a site in Indian River County (FLARES Inc.) once the grower has finalized his decision on the grove location. Trees are field-ready and in the holding stage at the USDA Horticultural Research Laboratory DPI-certified greenhouses in Ft. Pierce.
Progress on objectives 1, 2, 6, and 8 were made as follows:
Objective 1 – The ratio effect of Fe2+ and citric acid concentration and proportion on hydroxyl radical production and stability will be quantified and validated by response surface methods with accompanying mathematical models and ANOVA.
An assay to quantify hydroxyl radical production via ferrous iron induced Fenton reaction was setup and calibrated (i.e., standard curves). The hydroxyl radicals produced by the Fenton reaction degrade the dye methylene blue, and is measured by spectrophotometry. This assay is required to determine the Fe2+/citric acid ratio effects.
Objective 2 – Phytotoxic effects of Fe2+ and citric acid concentration and proportion will be quantified and validated by response surface methods with accompanying mathematical models and ANOVA.
Citron plants for this experiment were established from bud cuttings.
Objective 6 and 8 (Note: there is no objective 7) – Effects of Fe2+ and citric acid treatment on HLB status and horticultural measures of young newly planted trees AND mature HLB-infected field trees are in progress. Prior to treatment applications, the caliper and height for each of the newly trees was measured. Tree condition for the mature trees was visually determined by disease indexing (DI) according to the methods of T. R. Gottwald, B. Aubert and Z. Xue-Yuan. Trees with a similar DI were selected, flagged, randomized and mapped for treatment.
Aerial images were acquired (drone) for the young and mature field planting. Images will provide canopy size, density, and shape data. This data, in combination with ground data, will form a baseline measure for treatment comparisons over time.
The summer cover crop mix was planted in late May/early June at both locations. This mix included buckwheat, brown top millet, and dove millet. Sunnhemp was included in the mixes for the nitrogen-fixation treatments (1/2 of the rows).
Dataloggers and soil moisture probes continued to record soil moisture every hour. Root growth measurements using the mini-rhizotron tubes installed in both groves were performed in March and July 2019. Data on these measurements are currently being analyzed and will continue in Fall 2019.
Weed density and identification measurements were made in March and Aug 2019. Initial analysis of data collected from March 2019 revealed a general (72-84%) reduction in density of weeds in citrus row middles that received cover crop treatments when compared to controls with no cover crop in the row-middles. The next data collection is scheduled towards the end of this year.
Collection of samples for the annual assessment of leaf and soil nutrient concentrations, trunk diameter, canopy size, and soil microbial community composition have begun and will be completed for all treatments at both locations by the end of August. DNA will be extracted from soil samples and sent for sequencing analysis within the next quarter.
The postdoctoral research associate, Dr. Antonio Castellano-Hinojosa, joined the project on July 1. The graduate student under the supervision of Drs. Kadyampakeni and Kanissery, Miurel Brewer, began working in May 2019.
The purpose of the project is to develop new guidelines for restoring root health and improving overall tree nutrition in 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 implemented at the 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 concentration, and root growth. Data collection will continue, 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 CREC, a second graduate student started working on the project this summer at the IRREC. The project has also recruited agricultural assistants to support data collection and other measurements. Recruiting at IRREC is taking longer than expected due to the lack of interested and/or qualified applicants. A pool of applicants from an advertisement at Craiglist is under review. One candidate has been identified and is in the process of being hired. Drs. Ferrarezi, Rossi and Wright also decided on conducting the trial at the IRREC Research Grove since we were not able to identify a grower collaborator to partner up due to the challenges of implementing a fertigation system in a commercial grove. Trees planted in double row, high-density staggered in diamond set (392 trees/acre) were cut down to one row (196 trees/acre) for the trial. Plans for Next QuarterThe team will continue with data collection and reporting on the progress of the project.
The purpose of this project is to optimize the CRISPR technology for citrus genome editing. This study is related to the CRDF RMC-18 Research Priorities 4AB. We are optimizing the CRISPR-Cas9 technology in citrus genome editing by conducting the following three objectives: Objective 1. Expanding the toolbox of citrus genome editing. In this study, we will adapt StCas9, NmCas9, AsCpf1, FnCpf1 and LbCpf1 on genome modification of citrus. Lately, we have shown CRISPR-Cpf1 can be readily used as a powerful tool for citrus genome editing. One manuscript entitled CRISPR-LbCas12a-mediated modification of citrus has been published on Plant Biotechnol J.Objective 2. Optimization of the CRISPR-Cas mediated genome editing of citrus. In this study, we will first test different promoters in driving expression of Cas9 and Cpf1. We have identified one optimized promoter which showed higher efficacy in driving gene expression in citrus than 35S promoter and Arabidopsis U6 promoter. We are further characterizing the promoter and test its efficacy in driving sgRNA and in genome editing of citrus. We have also developed a method to increase the transient expression efficiency. Objective 3. Optimization of the CRISPR technology to generate foreign DNA free genome editing in citrus. We have conducted transient expression of Cas9/sgRNA plasmid and Cas9 protein/sgRNA ribonucleoprotein complex in citrus protoplast. The plasmid-transformed protoplast has 1.7% editing efficiency, and the RNP-transformed samples have approximately 3.4% efficiency. The genome modified protoplast cells are undergoing regeneration. We aim to increase the efficacy to over 20% and eventually generate non-transgenic genome modified citrus. One manuscript is under preparation. One patent has been filed on the CRISPR-Cas mediated genome editing of citrus.
During the second quarter of 2019, we continued our horticultural field trial evaluations as outlined in the proposal. Trees in all three trials are composed of Valencia scion on eight different rootstocks (US-802, US-812, US-897, US-942, US-1516, X-639, Swingle, Cleopatra). The SWFREC trial (Collier County) was established in October 2017. The two commercial trials were established in April 2018 on a typical Ridge site in Polk County and on a flatwoods type-site in southwest FL (Hendry County). Rhizotron root growth measurements at the SWFREC location were continued in monthly intervals. Results showed some differences in root growth among propagation methods within a rootstock cultivar during the first year of growth in the field. But, when averaged across all rootstock cultivars, no differences associated with propagation method were observed. Statistical analyses are still ongoing. After one year of growth, significant differences in tree height were found among trees on different rootstocks independent of the propagation method. The largest tree size was measured for US-1516 and the lowest for US-897. No statistically significant differences in tree size were attributed to the method by which rootstocks were propagated. Other horticultural parameters such as trunk diameter and the ratio of rootstock to scion trunk diameter were significantly influenced by rootstock and by propagation method. Nutrient analysis of leaves showed no significant differences associated with the propagation method, but differences were found among trees on different rootstocks for all nutrients except nitrogen and potassium. The SWFREC trial was demonstrated during a field day in April 2019.During the first year of growth in the commercial locations, no dieback of trees was observed for trees at the Polk County location. A small percentage of trees died within several weeks after transplant at the Hendry County location, but dieback was not related to the propagation method or rootstock cultivar. After one year of growth, no significant differences in tree height, canopy spread, and trunk diameter among trees were observed based on the method by which rootstocks were propagated in both trials. In contrast, significant differences were observed for trees on different rootstocks independent of the propagation method. Data analysis is still ongoing. Rhizotrons for root observations were installed during the previous quarter, and the first measurements were conducted.Tree propagations for the additional field trial to be conducted in Indian River County were completed and trees are maintained at the US Horticultural Research Lab in Ft. Pierce until ready for planting. Trees have Valencia scion and are grafted on four different rootstocks (US-812, US-897, US-942, and US-1516) propagated by seed, cuttings, and tissue culture.
Systemic characterization of three Mg-Zn gel based multi-metal formulations containing Cu A.I. [Cu:Mg:Zn (10%:45%:45%, MM10C45M45Z), Cu:Mg:Zn (15%:42.5%: 42.5%, MM15C42.5M42.5Z) and Cu:Mg:Zn (20%:40%: 40%, MM20C40M40Z)] is in progress. The hydrodynamic size of as-prepared materials was measured through dynamic light scattering (DLS). MM10C45M45Z, MM15C42.5M42.5Z, and MM20C40M40Z showed increased hydrodynamic size with higher Cu content (290.2 nm, 334.7 nm, and 354.2 nm respectively), suggesting Cu could induce the aggregation of primary particles. The primary particle size of multi-metal formulations (MM25C75M, MM25C75Z, MM10C45M45Z, MM15C42.5M42.5Z, and MM20C40M40Z) will be evaluated through electron microscopy. The electron microscopy results for the as-prepared multi-metal formations will be included in the next report.The antimicrobial efficacy of as-prepared materials was tested in vitro against Pseudomonas syringae. To determine the minimum inhibitory concentration (MIC) of the as-prepared materials and selected controls, a macrodilution assay was performed (Nature Protocols, 2008, 3, 163-175). The MIC value was determined by measuring the optical density at 600 nm after 48 h incubation at 27 °C under shaking (150 rpm). The MIC value of MM25C75M, MM15C42.5M42.5Z, and MM20C40M40Z was 80 ppm of metallic Cu, which was similar to copper controls (copper nitrate and Kocide 3000). The MIC value of MM10C45M45Z was 40 ppm of metallic Cu. The enhance antimicrobial efficacy may due to the smaller particle size. The MIC value of MM25C75Z was 5 ppm of metallic Cu. This result suggests that MM25C75Z formulation might contain ultra-small size particles. To determine the minimum bactericidal concentration (MBC) of as-prepared materials and selected controls, a colony-forming unit (CFU) assay was performed (Nature Protocols, 2008, 3, 163-175). The MBC value of MM25C75M, MM25C75Z and MM10C45M45Z was 80, 10 and 40 ppm metallic Cu respectively. All multi-metal formulations showed enhanced bactericidal efficacy when compare with commercial copper bactericide control (Kocide 3000, MBC: 320 ppm metallic Cu).To evaluate the systemic mobility of the Cu A.I., a plant uptake study was performed using Citrus reshni (Cleopatra mandarin) seeding in a growth chamber (Panasonic Environmental Test Chamber, Model MLR-325H, Kadoma, Japan). Temperature and relative humidity were maintained at 35 ºC and 85%. The citrus seeding was foliar-sprayed using a hand-pump sprayer with 300 ml of MM25C75M, MM25C75Z, MM10C45M45Z (500 and 1000 µg/ml metallic Zn concentration) and deionized water as a control. After 48 h incubation in the plant growth chamber, citrus seedlings were removed from the soil and gently washed with 1% cleaning detergent (Alconnox ®, Alconnox Inc.) and 0.1% HCl. Leaves, roots and stem sections were separated after washing and left in an oven for 48 h (45 ºC). Then, the dried leaves, roots, and stems were acid digested (EPA method 3050 B Acid Digestion of sediments, Sludge, and Soil). Cu, Zn and Mg contentions in leaves, stems, and roots will be quantified with Atomic Absorption Spectroscopy (AAS). The AAS results will be included in the next report.
The purpose of this project is to optimize the CRISPR technology for citrus genome editing. This study is related to the CRDF RMC-18 Research Priorities 4AB. We are optimizing the CRISPR-Cas9 technology in citrus genome editing by conducting the following three objectives: Objective 1. Expanding the toolbox of citrus genome editing. In this study, we will adapt StCas9, NmCas9, AsCpf1, FnCpf1 and LbCpf1 on genome modification of citrus. As a proof of concept, CsPDS and/or CsLOB1 are chosen for targeting through transient expression of Cas9-sgRNA or Cpf1-crRNA via Xcc-facilitated agroinfiltration. Recently, we employed CRISPR-LbCpf1, derived from Lachnospiraceae bacterium ND2006, to edit the citrus genome. First, LbCpf1 was successfully used to modify Duncan CsPDS via Xcc-facilitated agroinfiltration. Next, GFP-p1380N-35S-LbCpf1-crRNA-lobp and GFP-p1380N-Yao-LbCpf1-crRNA-lobp were constructed to edit the PthA4 effector binding elements in the CsLOB1 promoter (EBEPthA4-CsLOBP) in transgenic Duncan grapefruit. Totally, seven GFP-p1380N-35S-LbCpf1-crRNA-lobp-transformed Duncan plants were generated, designated as #D35s1 to #D35s7, and ten GFP-p1380N-Yao-LbCpf1-crRNA-lobp-transformed Duncan plants were created, designated as #DYao1 to #DYao10. LbCpf1-directed EBEPthA4-CsLOBP modification was observed in three 35S-LbCpf1-transformed Duncan (#D35s1, #D35s4 and #D35s7). However, no LbCpf1-mediated indels were observed in the Yao-LbCpf1-transformed plants. Importantly, transgenic line #D35s4, containing the highest mutation rate, alleviates Xcc.pthA4:dCsLOB1.4 infection. Therefore, CRISPR-LbCpf1 can be readily used as a powerful tool for citrus genome editing. We have also made progress regarding use other Cas proteins. Objective 2. Optimization of the CRISPR-Cas mediated genome editing of citrus. In this study, we will first test different promoters in driving expression of Cas9 and Cpf1. We have identified one optimized promotors which showed higher efficacy in driving gene expression in citrus than 35S promoter and Arabidopsis U6 promoter. We are further characterizing the promoter and test its efficacy in driving sgRNA and in genome editing of citrus. We have also developed a method to increase the transient expression efficiency. Objective 3. Optimization of the CRISPR technology to generate foreign DNA free genome editing in citrus. We have conducted transient expression of Cas9/sgRNA plasmid and Cas9 protein/sgRNA ribonucleoprotein complex in citrus protoplast. The plasmid-transformed protoplast has 1.7% editing efficiency, and the RNP-transformed samples have approximately 3.4% efficiency. The genome modified protoplast cells are undergoing regeneration. We aim to increase the efficacy to over 20% and eventually generate non-transgenic genome modified citrus. One manuscript is under preparation. We are also filing patent for this technology.
The spring cover crop mix was planted in late February and early March in both locations. This mix included Daikon radish, white clover, crimson clover, buckwheat, oats, and sunflower. Daikon radish germination was particularly good in both locations.
Dataloggers and soil moisture probes continued to record soil moisture every hour. A rain gauge will be installed at each site to record rainfall data. Preparations are underway for the next assessment of root growth via the mini-rhizotron tubes installed in both groves. Weed emergence in row-middles and tree-rows were collected in mid-March from North grove location. Weed emergence data in South grove location will be collected in mid-summer.
Yield and juice quality data was collected for the North grove location March 28-April 4 and for the South grove April 11-18. As treatments had only just begun, this yield and juice data will serve as baseline data for comparison with subsequent years of the project.
The summer cover crop mix is being planted at both locations in the next two weeks. This mix will include dove millet, buckwheat, brown top millet, and sunnhemp.
A postdoctoral research associate will be joining the project in July and a graduate student for Drs. Kadyampakeni and Kanissery will begin this summer. Dr. Wades graduate student will begin this fall.
The next collection of samples for soil and leaf measurements will occur this summer.
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