1. Please state project objectives and what work was done this quarter to address them:
Objective 1: Determine effects of lowered soil pH on CLas populations and root physiology including internal root apoplast and vascular tissue pH. Due to a collapse in our inoculum trees, we are re-inoculating trees for these experiments and planned to start the greenhouse portion of the study at the end of July 2022 because some trees did not turn out positive for CLas in April 2022. However, we still had few trees turning out positive in July and had to reinoculate the trees with CLas. A good number of trees are now postive for CLas and will now be subjected to varying pH levels in rhizotrons. All protocols are developed and pretesting for the study is finalized.
Objective 2: Field test multiple acidification materials including organic acids for tree response CLas suppression, nutrient uptake, and root and vascular pH changes. In this quarter, we collected soil and leaf tissue samples which show sufficiency in all treatments. We are now evaluating root density, and PCR of selected trees. We also applied all acids and elemental S in the appropriate treatments and are monitoring canopy changes and soil trends as described in the project deliverables,
2. Please state what work is anticipated for next quarter:
Fruit harvests and juice quality evaluations wll be completed in the next quarter.
3. Please state budget status (underspend or overspend, and why):
The budget is on track and meeting the project milestones.
1. Please state project objectives and what work was done this quarter to address them:
Objective 1: Determine effects of lowered soil pH on CLas populations and root physiology including internal root apoplast and vascular tissue pH. Due to a collapse in our inoculum trees, we are re-inoculating trees for these experiments and planned to start the greenhouse portion of the study at the end of July 2022 because some trees did not turn out positive for CLas in April 2022. However, we still had few trees turning out positive in July and had to reinoculate the trees with CLas. A good number of trees are now postive for CLas and will now be subjected to varying pH levels in rhizotrons. All protocols are developed and pretesting for the study is finalized.
Objective 2: Field test multiple acidification materials including organic acids for tree response CLas suppression, nutrient uptake, and root and vascular pH changes. In this quarter, we collected soil and leaf tissue samples which show sufficiency in all treatments. We are now evaluating root density, and PCR of selected trees. We also applied all acids and elemental S in the appropriate treatments and are monitoring canopy changes and soil trends as described in the project deliverables,
2. Please state what work is anticipated for next quarter:
Fruit harvests and juice quality evaluations wll be completed in the next quarter.
3. Please state budget status (underspend or overspend, and why):
The budget is on track and meeting the project milestones.
1. Please state project objectives and what work was done this quarter to address them: Objectives: 1) Test the efficacy of different injection devices, 2) Determine the most effective formulation of OTC, 3) Determine the best month of injection and most appropriate OTC concentration based on tree size. Trial 1: Located in SW Florida (Duda) – 8-year-old Valencia/Carrizo trees. – We conducted tree health ratings, fruit size assessments, monthly fruit drop measurements, and tree size measurements. Trial 2: Located in SW Florida (Graves Bros) – 8-year-old Valencia/Kuharske trees. – We conducted tree health ratings, fruit size assessments, monthly fruit drop measurements, and tree size measurements. Trial 3: Located on the east coast (Graves Bros) – 9 year-old Valencia/sour orange trees. – We conducted tree health ratings, tree size measurements, monthly fruit drop measurements, and collected leaves for CLas detection. Trial 4: Located on the east coast (Graves Bros) – 4-year-old Valencia/x639 trees. – We conducted tree health ratings and monthly fruit drop measurements, and collected leaves for CLas detection. Trial 5: Located on the central ridge (King Ranch) – 4-year-old OLL-8/x639 trees. – We conducted tree health ratings and monthly fruit drop measurements, and collected leaves for CLas detection. 2. Please state what work is anticipated for next quarter:Fruit drop countings will continue. We will prepare for fruit quality and yield assessment (pending the desired harvest time of the grower collaborator).3. Please state budget status (underspend or overspend, and why): Budget status is as anticipated (the majority of the budget will be for fruit quality and residue analyses at the end of the first year).
1. Please state project objectives and what work was done this quarter to address them: HLB is known to make citrus roots more susceptible to Phytophthora root rot. It also reduces the efficacy of chemical management of Phytophthora root rot, creating a difficult management scenario. Current Phytophthora management recommendations are based on pre-HLB work done in the 1980s. These three conditions raise the question of whether yield improvement from Phytophthora management is enough to pay for the management costs themselves. The goal of this project is to develop new soil propagule density managment thresholds and recommendations for chemical management of Phytophthora root rot based on ecomonic analysis of yield responses in different soil conditions. Objective 1) Determine if labelled Phytophthora management maintains efficacy in the field on HLB-affected trees for reducing fibrous root loss and improving yield. In this quarter, we collected root samples from the Felda area grove. The root density was measured and the raw data appear to show that some treatments have greater density than the control for the Hamlin oranges. There is much more variation in the Valencia plots and the trends are unclear. Trends are also unclear from the populations of the two phytophthora species. We have been working on the analysis of these plots and are reworking the analysis to use time as a factor. We have harvested one of the hamlin sites and have the second site scheduled with the grower for the first week of January. The fruit quality and size data have been gathered but not yet entered into the computer. Objective 2) Determine benefit-cost thresholds for Phytophthora treatment on HLB-affected treesThe co-PI responsible for the economic analysis of this project, asked to have their funds returned. I am not sure whether they will provide an analysis of this project. 2. Please state what work is anticipated for next quarter: The second Hamlin harvest. The Valencia harvests and spring applications of the treatments. Data analysis will continue to confirm whether there any treatment differences in yeild, fruit quality, root density, or inoculum levels of phytophthora. 3. Please state budget status (underspend or overspend, and why): The budget is underspent because when I took over the project, there had not been staff appointed to the project for several months. This caused funds not to be spent in a timely manner. Also I had been planning to ask for the no cost extension so that we could harvest the Valencia plots so I needed to make sure there were funds available for that purpose.
1. Please state project objectives and what work was done this quarter to address them: HLB is known to make citrus roots more susceptible to Phytophthora root rot. It also reduces the efficacy of chemical management of Phytophthora root rot, creating a difficult management scenario. Current Phytophthora management recommendations are based on pre-HLB work done in the 1980s. These three conditions raise the question of whether yield improvement from Phytophthora management is enough to pay for the management costs themselves. The goal of this project is to develop new soil propagule density managment thresholds and recommendations for chemical management of Phytophthora root rot based on ecomonic analysis of yield responses in different soil conditions. Objective 1) Determine if labelled Phytophthora management maintains efficacy in the field on HLB-affected trees for reducing fibrous root loss and improving yield. In this quarter, we collected root samples from the Felda area grove. The root density was measured and the raw data appear to show that some treatments have greater density than the control for the Hamlin oranges. There is much more variation in the Valencia plots and the trends are unclear. Trends are also unclear from the populations of the two phytophthora species. We have been working on the analysis of these plots and are reworking the analysis to use time as a factor. We have harvested one of the hamlin sites and have the second site scheduled with the grower for the first week of January. The fruit quality and size data have been gathered but not yet entered into the computer. Objective 2) Determine benefit-cost thresholds for Phytophthora treatment on HLB-affected treesThe co-PI responsible for the economic analysis of this project, asked to have their funds returned. I am not sure whether they will provide an analysis of this project. 2. Please state what work is anticipated for next quarter: The second Hamlin harvest. The Valencia harvests and spring applications of the treatments. Data analysis will continue to confirm whether there any treatment differences in yeild, fruit quality, root density, or inoculum levels of phytophthora. 3. Please state budget status (underspend or overspend, and why): The budget is underspent because when I took over the project, there had not been staff appointed to the project for several months. This caused funds not to be spent in a timely manner. Also I had been planning to ask for the no cost extension so that we could harvest the Valencia plots so I needed to make sure there were funds available for that purpose.
1. Please state project objectives and what work was done this quarter to address them: HLB is known to make citrus roots more susceptible to Phytophthora root rot. It also reduces the efficacy of chemical management of Phytophthora root rot, creating a difficult management scenario. Current Phytophthora management recommendations are based on pre-HLB work done in the 1980s. These three conditions raise the question of whether yield improvement from Phytophthora management is enough to pay for the management costs themselves. The goal of this project is to develop new soil propagule density managment thresholds and recommendations for chemical management of Phytophthora root rot based on ecomonic analysis of yield responses in different soil conditions. Objective 1) Determine if labelled Phytophthora management maintains efficacy in the field on HLB-affected trees for reducing fibrous root loss and improving yield. In this quarter, we collected root samples from the Felda area grove. The root density was measured and the raw data appear to show that some treatments have greater density than the control for the Hamlin oranges. There is much more variation in the Valencia plots and the trends are unclear. Trends are also unclear from the populations of the two phytophthora species. We have been working on the analysis of these plots and are reworking the analysis to use time as a factor. We have harvested one of the hamlin sites and have the second site scheduled with the grower for the first week of January. The fruit quality and size data have been gathered but not yet entered into the computer. Objective 2) Determine benefit-cost thresholds for Phytophthora treatment on HLB-affected treesThe co-PI responsible for the economic analysis of this project, asked to have their funds returned. I am not sure whether they will provide an analysis of this project. 2. Please state what work is anticipated for next quarter: The second Hamlin harvest. The Valencia harvests and spring applications of the treatments. Data analysis will continue to confirm whether there any treatment differences in yeild, fruit quality, root density, or inoculum levels of phytophthora. 3. Please state budget status (underspend or overspend, and why): The budget is underspent because when I took over the project, there had not been staff appointed to the project for several months. This caused funds not to be spent in a timely manner. Also I had been planning to ask for the no cost extension so that we could harvest the Valencia plots so I needed to make sure there were funds available for that purpose.
Trees in the field trial were measured on 22 November, eight months after planting and more than 6 months following the first nematicide application. The heights of the IPC trees were 14% greater than those of uncovered trees (P=0.000) and trunk girth one inch above the bud union was 13% greater on covered than uncovered trees (NS). Nematicide treatment had no effect on the tree size. As noted previously, by summer the nematicides reduced the sting nematodes (P=0.02) by 87% on the uncovered trees and 13% on the covered trees. The apparent interaction for the nematicide effect was marginally significant (P=0.08). The final nematicide treatment of 2022 occurred in November and the effects on the winter population density will be measured in January. The first 16 rootstocks from the UF breeding program were evaluated for sting nematode tolerance during the first week of November, approximately 18 weeks after planting in nematode infested tanks. Compared to unchallenged trees, the root damage was readily evident on the most susceptible lines and was documented photographically for each plant in the trial. The average fibrous root mass of the infested compared to the the uninfested lines was highly correlated (0.80) with the subjective visual damage rating assigned each seedling and was used to rank the genotypes. The mean ratio of the average fibrous root mass infested/uninfested for the four most ‘tolerant’ lines was five-fold that of the four least tolerant lines. Three of the most tolerant lines were derived from crosses between the same tetrazygotic parents and the fourth had 3 of the 4 ancestral genotypes. Two additional tanks were established in July and a 2nd trial was initiated using USDA rootstocks and a number of conventional rootstocks. The second trial will be completed in February 2023.
This three-year project was designed to study the effects of regular applications of compost on Valencia orange tree growth and productivity during the first three years of establishment. Trees were planted on 22 acres of land in a commercial citrus grove in southwest Florida near Felda. The trial was established in 2019 on eight raised beds at a planting density of 15 x 22 feet. The experimental design was a split-plot design with eight replications and a total of 3200 trees (145/acre). The main plot was treatment at two levels: compost or no-compost. For the compost treatment compost was applied at a rate of 5 tons per acre twice annually (May and November). To study the interaction with rootstock, four different rootstocks were included as the sub-plot: US-802, US-897, US-812, and X-639. Trees were arranged in two rows per bed, each row containing 100 trees with each rootstock sub-plot containing 50 trees. As root health is a major concern in the HLB era, this project complemented standard horticultural evaluations with evaluations of root health indicators, including anatomical and metabolic root health traits as well as the rhizosphere microbial community composition. We tested the hypothesis that increased nutrient retention through application of compost during the early phase of tree growth will result in better tree establishment and health and therefore higher productivity once trees become mature. We found that the biannual applications of compost increased several soil physicochemical properties such as CEC, pH, organic matter, and water content. Despite some variations across sampling times, soil potassium, magnesium, calcium, and boron content were generally increased after compost applications, while soil copper content was decreased. Increases in soil nutrient content translated into increases in leaf nutrient content in some instances such as for potassium. In addition to changes in soil properties and leaf nutrient status, compost amendments resulted in an increase in the fibrous root respiration rate, indicating a higher metabolic activity, but fibrous root length was not affected. Despite improvements in soil physicochemical properties and nutrient status, we did not measure any increases in growth or productivity after three years of field study. However, the total soluble solids content of fruits was decreased by the compost amendments. There were also differences associated with the rootstock, with US-897 producing the most and best-quality fruits. US-897 also had the longest and finest roots, which may be one reason for the higher fruit quality commonly associated with this rootstock. Changes in the rhizosphere microbiome were also measured in response to the compost amendments, but the impact was only correlated with specific changes in root nutrients for US-812 and US-897. One concern regarding the compost amendments was that they provided a favorable environment for weed growth, evidenced by a higher weed biomass measured in compost-treated vs. non-treated plots in some instances. The competition with weeds may have contributed to the lower amount of soluble solids measured in trees from compost-amended plots. Taken together, biannual compost applications at the rate and with the methodology used in our study did not result in any measurable effect on tree growth and productivity during the first three years after tree establishment despite some measurable improvements in soil and root health parameters.
1. Please state project objectives and what work was done this quarter to address them: The overall goal of the project is to develop fertilization strategies to best match nutrient supply and demand, and develop recommendations for optimal nutrient application timing as compared to a simple constant supply, which will improve fruit yield, quality, and reduce fruit drop. Objective 1) Test if a reduced N-P-K nutrient supply in the fall is safe for sustaining HLB-affected citrus, and whether it can improve fruit quality to facilitate earlier maturity / harvesting and reduce fruit drop: In order to compare the effects of early versus late / sustained fertilization on health and fruit production of Hamlin and Valencia trees, the following completed treatments were evaluated in the third quarter of 2022:Treatments: Early fertilization as % completed of the recommended fertilization (RF; 160 lb N/acre) before June T1: 25% of RF (T25%)T2: 50% of RF (T50%)T3: 75% of RF (T75%)T4: 100% of RF (T100%)The remaining mid- to late-season fertilizer treatments were applied during the third quarter and the results will be reported in the following quarter. Leaf samples were collected on monthly intervals and analyzed to determine the leaf nutrient concentrations. The preliminary results showed that if 50% of total nutrient requirement was applied before May, there was improvement in tree health (NDVE) and vigor (NDVI) indices, and canopy volume. The same treatment resulted in higher leaf nutrient concentrations but only nitrogen was statistically significant. The fruit growth (diameter) was not significantly difference between early fertilization treatment even applying 75% or 100% of total fertilizer. Early fertilization with higher rates increased fruit size but growth increments diminished over time in all treatments, as expected when fruit development enters growth stage 2 in summer.Preliminary results showed that there was a positive trend of crop health, fruit growth and leaf nutrient concentration with early higher fertilization but didn’t observe statistically significant differences.Objective 2) Develop an optimized, practical fertilizer timing management profile to boost fruit quality and reduce fruit drop for HLB-affected citrus based in part on the sigmoidal nutrient demand curve defined by four physiological growth phases (0=bloom/fruit set; 1=cell division; 2=cell enlargement; 3=maturation):The growth and yield data associated with all different timed fertilizer events in year 1 is not complete yet and therefore we don’t have new results to report for this objective. Our investigations to use the intensity of leaf symptom expression throughout the season to help determine foliar nutrition in the field with smartphone apps is continuing.B) New developments: The Sugarbelle trials focusing on solving the soft fruit quality issues are progressing well. We have now applied three replicated foliar spray treatments to Sugarbelle trees at the city block and on healthy trees in the CUPS, consisting of KNO3 and KH2PO4. The purpose of the sprays is to increase peel thickness and strength to avoid soft fruit, as well as to improve fruit size. C) Issues: The fruit drop and tree lodging/ branch breaking damage caused by hurricane Ian to the outdoor Hamlin, Valencia and Sugarbelle experiments is considerable. The Sugarbelle experiment in the CUPS sustained no damage. We expect that the reduced yield data will not be useful for calibrating the fertilizer timing models, but we will collect and use fruit quality data where possible. 2. Please state what work is anticipated for next quarter:The final late fertilizer applications will be made to designated treatment plots in November. Soil, lysimeter sampling, leaf sampling, processing and analysis will be ongoing, as will tree size and fruit measurement. We plan to assess fruit yield and quality for the Hamlin and Sugarbelle experiments in December / January. The third Aerobotics drone survey will now fly in late November due to delays from Hurricanes Ian and Nicole. 3. Please state budget status (underspend or overspend, and why):Spending rate is approximately on track.
1. Please state project objectives and what work was done this quarter to address them: Specific objectives are:1) To determine the right timing for Zn and K treatments to minimize fruit drop.2) To determine effects of GA3 and 2,4D applications on fruit retention when applied at different times during fruit development.3) To develop a strong and proactive outreach program. For objective 1 we did the new set of treatments by the end of September in Hamlin. As before, every set of treatments is performed in an independent block, so treatments are applied only once, to assess the best timing to achieve the best results. Trees under Zn and Zn+K treatments continue having greener canopies. Control trees are presenting some off-blooms still in September and October with new fruit set clearly present. These off-blooms have not been observed in the treated trees. Fruit drop is being recorded in Hamlin. Unfortunately, hurricane Ian has increased fruit drop but since we are collecting data weekly, we are going to be able to reduce the fruit drop background noise and in any case, see if our treatments had a beneficial impact even under hurricane conditions in alleviating fruit drop. We have lost also some trees due to the high wind. Interestingly, all of the trees were lost in a control plot. We do not have a clear explanation for this, but fortunately, treatments were not compromised and other trees in untreated plots are being used as new control replacements. In objective 2, GA3 and 2,4D treatments have continued as planned. Objective 3. Outreach-Citrus Grower Forum. Citrus Fix. Guest speakers Vashisth and Alferez. Invited by AMVAC. SWFREC, August 30.-Citrus Grower Forum. Citrus Fix. Guest speakers Vashisth and Alferez. Invited by AMVAC. Stuart Conference Center, Bartow, August 31. 2. Please state what work is anticipated for next quarter: In next quarter, we will perform the December treatment in Valencia for objective 1 and continue treatments for objective 2, and we will continue assesing fruit drop in Hamlin. 3. Please state budget status (underspend or overspend, and why): We are still underspending as the student joined late. As we start our analysis we will be spending more funds this quarter.
1. Please state project objectives and what work was done this quarter to address them:The objectives of this project are: 1, To study the effect of Brs on priming immunity on young, newly planted trees. This will allow to know for how long immune response will last after Br application, so we can adjust timing (number of applications). We planted 40 Valencia trees on carrizo and 40 trees on X639 in a complete randomized block design. Br application or water (as control)has been performed every 15 days. We have been monitoring psyllid infestation and collecting leaves for bacteria titer analysis.Tree growth is being monitored. It is still early to have any significant data, but psyllids are clearly less abundant in Br-treated plants. To better undesrtand the effects, we started an experiment before August 1st in controlled conditions in the greenhouse with the same setup of trees, that were enclosed in individual cages and 4 pairs of psyllids per cage were introduced. We have seen differences in oviposition and activation of SAR genes with treatment and we found that this activation lasts for at least 6 weeks. This opens the possibility of reduce the number of Br applications. We are still monitoring this in the open field.2, To determine the best time of application (frequency) to achieve maximum protection against pests and disease in newly planted trees. This objective is related to the first one. In discussions with some growers at our Citrus advisory committee, they raised the posibility of concentrating the Br application when psyllids are more abundant, durnig or at the beginning of flushes. I think this idea has good potential, so we are going to assay this as well. 3, To determine the effect of Br application on advancing fruit maturation in both Valencia and Hamlin. We started Br applications at Duda farm by the end of August in Hamlin. Treatments have been performed since then twice per month. The treatments are being performed in commercial conditions. While we did not see a statistically significant increase in Brix in September, during October we have started to see a slight increase: 8,6 as compared to 8,1 in average. Interestingly, acidity has been consistently lower in fruit from Br-treated trees, making the ratio significantly higher. In view of this fact, we have started to collect juice samples for further HPLC analysis of sugars and organic acids. Fruit drop has started for Hamlin and we are monitoring it weekly. Unfortunately, hurricane Ian has accelerated fruit drop and we have also lost some trees. However, our close monitoring of fruit drop, that we started before Ian is allowing us to actually assess the real impact of the hurricane on fruit drop. Outreach:-Invited talk. Brassinosteroids and fruit quality. Packinghouse day. August 27, 2022, Lake Alfred FL.-Invited talk. Combining IPCs and Brs to prolong young tree health. Updates and future steps. Citrus Expo 2022, August 18, Fort Myers. 2. Please state what work is anticipated for next quarter: For objective 3, we will harvest Hamlin when the commercial time is right, we will assess yield and final quality. We will also start in December treatments in Valencia.For objectives 1 and 2, treatments and samplings will continue as in this quarter. 3. Please state budget status (underspend or overspend, and why):Spending is on track. Student is on board and we will start spending more this quarter as we start in depth molecular analysis
The purpose of this project is to generate non-transgenic HLB resistant Valencia and Hamlin sweet orange plants using CRISPR-Cas technology. Objective 1. Generate non-transgenic HLB resistant/tolerant Valencia and Hamlin sweet orange plants by mutation of HLB susceptibility genes. In total, six putative S genes will be edited. Constructs needed for CRISPR genome editing are being made. Multiple edited lines were generated for ACD2 gene. However, further confirmation demonstrated none of them was biallelic/homozygous mutant. Objective 2. Generate cisgenic genome modified Valencia and Hamlin sweet orange plants by knock-in the gene encoding MaSAMP from Microcitrus. We are optimizing the knock-in method using the CRISPR technology.
1. Please state project objectives and what work was done this quarter to address them: Objective 1: Determine effects of lowered soil pH on CLas populations and root physiology including internal root apoplast and vascular tissue pH. Due to a collapse in our inoculum trees, we are re-inoculating trees for these experiments and planned to start the greenhouse portion of the study at the end of July 2022 because some trees did not turn out positive for CLas in April 2022. However, we still had few trees turning out positive in July and had to reinoculate the trees with CLas. A good number of trees are now postive for CLas and will now be subjected to varying pH levels in rhizotrons. All protocols are developed and pretesting for the study is finalized. Objective 2: Field test multiple acidification materials including organic acids for tree response CLas suppression, nutrient uptake, and root and vascular pH changes. In this quarter, we collected soil and leaf tissue samples which show sufficiency in all treatments. We are now evaluating root density, and PCR of selected trees. We also applied all acids and elemental S in the appropriate treatments and are monitoring canopy changes and soil trends as described in the project deliverables, Fruit harvest and fruit quality evaluations were completed in April 2022. 2. Please state what work is anticipated for next quarter: We will apply acids and elemental S in the next quarter. The greenhouse study on effects of pH on root physiology and CLas populations will also be conducted and completed in this and next quarter due to delays in getting CLas incoluation confirmed in trees. 3. Please state budget status (underspend or overspend, and why): The budget is on track and meeting the project milestones.
1. Please state project objectives and what work was done this quarter to address them: Objectives: 1) Test the efficacy of different injection devices, 2) Determine the most effective formulation of OTC, 3) Determine the best month of injection and most appropriate OTC concentration based on tree size. Trial 1: Located in SW Florida (Duda) – 8-year-old Valencia/Carrizo trees. – 2022 injections are completed. Fruit size evaluations were conducted trees were rated for HLB disease symptoms and canopy health.Trial 2: Located in SW Florida (Graves Bros) – 8-year-old Valencia/Kuharske trees. – 2022 injections are completed. Fruit size evaluations were conducted and trees were rated for HLB disease symptoms and canopy health.Trial 3: Located on the east coast (Graves Bros) – 9 year-old Valencia/sour orange trees. – 2022 injections are completed. Trees were monitored.Trial 4: Located on the east coast (Graves Bros) – 4-year-old Valencia/x639 trees. – August injections were conducted. Trees were rated for HLB disease symptoms and canopy health.Trial 5: Located on the central ridge (King Ranch) – 4-year-old OLL-8/x639 trees. – August injections were conducted. Trees were rated for HLB disease symptoms and canopy health. 2. Please state what work is anticipated for next quarter:Fruit drop countings and tree size measurements will be conducted. Fruit size evaluations in trial 1 will continue. Additional tree ratings and size measurements will be conducted. 3. Please state budget status (underspend or overspend, and why): Budget status is as anticipated (the majority of the budget will be for fruit analyses).
METHODOLOGY: Our project is examining phloem gene expression changes in response to CLas infection in HLB-susceptible sweet orange and HLB-resistant Poncirus and Carrizo (a sweet orange – Poncirus cross). We are using a recently developed methodology for woody crops that allows gene expression profiling of phloem tissues. The method leverages a translating ribosome affinity purification strategy (called TRAP) to isolate and characterize translating mRNAs from phloem specific tissues. Our approach is unlike other gene expression profiling methods in that it only samples gene transcripts that are actively being transcribed into proteins and is thus a better representation of active cellular processes than total cellular mRNA. Sweet orange, and HLB-resistant Poncirus and Carrizo (sweet orange x Poncirus) have been transformed to express the tagged ribosomal proteins under the control of characterized phloem-specific promoters; transformants expressing the tagged ribosomal proteins under control of the nearly ubiquitous CaMV 35S promoter were also constructed as a control. We are in the process of exposing transgenic plants to CLas+ or CLas- ACP and leaves sampled 30, 60, 90, and 120 days later. Ribosome-associated mRNA is being sequenced and analyzed to identify differentially regulated genes at each time point and between each citrus cultivar. Comparisons of susceptible and resistant phloem cell responses to CLas will identify those genes that are differentially regulated during these host responses. Identified genes will represent unique phloem specific targets for CRISPR knockout or overexpression, permitting the generation of HLB-resistant variants of major citrus cultivars. PROGRESS: During the three year and nine month grant period, our group has made at least four high-expressing lines for each of the nine promoter/genotype combinations and shipped them from the Stover lab to the Rogers lab. More than 150 rooted cuttings from these lines were exposed to CLas+ or CLas- ACP in no-choice psyllid inoculation experiments. Each rooted cutting was sampled four times, yielding more than 600 leaf samples for ribosome affinity purification and mRNA isolation. Approximately one-third of the samples have been processed and almost 60 have been sequenced. In a parallel approach, the tagged ribosomal proteins were cloned into a citrus tristeza viral expression vector. This vector and a control empty vector were moved into both Nicotiana benthamiana and Citrus macrophylla. Proof of concept experiments testing whether CTV can yield mRNA samples enriched for phloem genes appear promising. There were several issues that hindered our progress on project milestones. First, laboratory closures and occupancy caps due to the COVID-19 pandemic drastically limited personnel time in the lab and greenhouse. The Poncirus genotype is very slow-growing and the last Poncirus transgenic lines were not available until the end of year 3. The post-doc moved on to a permanent job after about 2 1/2 years on the project. In spite of extensive advertising, no suitable replacement post-doc candidate was identified. Additionally, mRNA yields from both of the citrus specific promoters were low and an ultracentrifugation step to concentrate ribosomes will be added prior to affinity purification. In summary, significant progress was made towards our goal of identifying phloem gene expression changes in response to CLas infection even though the final studies are not complete.
This project is a continuation of previously funded CRDF grants to TWO BLADES focused on utilizing multiple strategies to produce canker-resistant citrus plants plus the addition of a new strategy using gene editing. 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. We also are in the process of testing citrus that has been transformed to modify the bs5 gene to enhance resistance to the citrus canker bacterium. Objective 1. To determine if Bs3-generated transgenic grapefruit plants are resistant to diverse strains of the citrus canker bacterium in greenhouse experiments and to the citrus canker bacterium in field experiments in Fort Pierce. In late March, 2019, in the field at Fort Pierce in collaboration Dr. Ed Stover, the transgenic material was planted. Citrus canker has developed on plants in the field and the trees were rated for disease in November 20, 2021and there was moderate disease on Duncan grapefruit trees but none on JJ5. We rated the plots again on July 28 (not June as mentioned in May report) 2021 and there were similar trends as in November 2020 although disease was lower given significant defoliation in the plots. We have also analyzed JJ5 for response to strains from Dr. Nian Wang to determine if those strains with unusual characteristics in terms of targeting the susceptibility gene could overcome the JJ5 resistance. Interestingly we noted unique phenotypes in plants inoculated with these strains although they were not typical disease reactions but more of a watersoaked appearance. We noted that theygrew to higher populations in inoculated tissue, although the disease phenotype was very weak and not typical of canker. We also used a 5′ Race kit to determine the transcription start in our JJ5 construct and observed that all strains tested activated trasncription of our As for developing a different transgenic with ProBs314EBE:avrGf2, 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. Given that there was concern about the additional marker, the new construct contains only NPT as a selectable marker. We have created a second construct for Vladimir Orbovic given the first attempt was not successful.The construct was transferred to Vladimir Orbovic, who was tasked with creating additional transformants in sweet orange. He identified several putative transgenic trees that weregrown and tested for disease reaction. Three of the putative transgenics were shown to elicit a hypersensitive reaction when infiltrated with a bacterial suspension of a Xanthomonas citri strain. Currently, we are maintaining the transgenic plants in our greenhouse. We are hopeful that these transgenic trees will be of use in the near future given that these particular ones do not express GFP. We are in the process of publishing this work. 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 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 and were recently rated in late July. The trees were rated for disease in November, 2020 and there was considerable disease on all EFR plants with disease being more severe than on susceptible Duncan control. We rated the plots again on July 28 (not June as mentioned in May report) 2021 and there were similar trends as in November 2020 although disease was lower given significant defoliation in the plots. This research project was complted in 2021. We also conducted greenhouse experiments in which we inoculated by spray inoculation and by pin-prick inoculation. We observed no differences in disease reactions of any of the transgenic trees from different transformation events, when compared to reactions in wild-type Duncan Grapefruit. We are in the process of writing a paper on these results. Objective 3. To determine if bs5-generated transgenic Carrizo plants are resistant to X. citri and to generate transgenic grapefruit carrying the pepper bs5. We budwood from UC Berkeley. The budwood was from two transgenic events and a third was from a tree that was run through the transformation process, but that was negative for the gene. The latter served as the negative control as it had undergone the transformation process. We grafted the buds and several have developed into branches. In our initial tests, one of the transgenic trees when inoculated resulted in reduced canker symptoms, but no differences in bacterial populations when compared to the wild-type Carrizo plamts. The other transgenic tree reacted similarly as the wild-type Duncan grapefruit in symptom development as well as in bacterial populations. We cut back the material several times to stimulate plant growth, but for reasons unknow we were not able to get significant regrowth to confirm earlier results and therefore were unable to make enough progress prior to the grant being terminated to make substantive assessment as to the responses of the transgenic events.
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