1. Please state project objectives and what work was done this quarter to address them: To determine how many leaf nutrient sampling per year are required to effectively capture the tree nutritional statusand adjust fertilizer accordingly.2. To establish the relationship of leaf nutrient concentration with yield, fruit drop, and canopy density3. To determine how the leaf nutrient (all 14 nutrient) levels change in the tree throughout the year.4. To evaluate how the leaf age affects the leaf nutrient status. In this quarter alot of the activities were similar to previous quarter. We were able to fertilize the trees for spring based on spring and summer flush nutrient analysis. We were able to perform another set of nutriet analysis in this quarter as well. In addition we have been collecting leaf samples from fruiting an non fruting branches for comparison. Our analysis shows that spring flush are deficient in immobile nutrients as compared to random leaves therefore, suggesting that method of sampling can sway the results signifcantly. In addition we are seeing that fruting branches show low levels of consistent acculumation of micronutrients in the leaves wherease the non fruiting branch decrease in micronutrients from summer to fall thus the suggesting a higher metabolism of nutrients. We did write a ‘Tip of the week’ in June based ont he frutiting versus nonfruiting branch nutrient analysis data generated from this project. 2. Please state what work is anticipated for next quarter: 1. Data analysis and interpretation2. Collecting samples for nutrient analysis3. Applying fertilizer treatments based on leaf nutrient analysis results 3. Please state budget status (underspend or overspend, and why): The budget is being spent as per the plan where major funds have been used for nutrient anlaysis.
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.No further phytophthora counts have been taken from the southwest Florida site this quarter. Treatment applications were made in early May in both the Hamlins and Valencia.We initiated a second location for two new trials, early oranges and Valencia in the Wachula area. The sites have a history of phytophthora. The preliminary counts from the blocks varied between 40 and 128. The per plot data in the early cultivars are high, varying between 15 and 120 CFU/plot with a fewplots with counts at 10 or below CFU/plot. In our Valencia block, we were suprised to find very low counts in many plots after our composite samples had at least 40 CFU in the block. We are not sure if there was a difference in trees sampled or if there was some sort of seasonal decline of the population because of the local block conditions. Objective 2) Determine benefit-cost thresholds for Phytophthora treatment on HLB-affected treesWe are waiting for more data
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.No further phytophthora counts have been taken from the southwest Florida site this quarter. Treatment applications were made in early May in both the Hamlins and Valencia.We initiated a second location for two new trials, early oranges and Valencia in the Wachula area. The sites have a history of phytophthora. The preliminary counts from the blocks varied between 40 and 128. The per plot data in the early cultivars are high, varying between 15 and 120 CFU/plot with a fewplots with counts at 10 or below CFU/plot. In our Valencia block, we were suprised to find very low counts in many plots after our composite samples had at least 40 CFU in the block. We are not sure if there was a difference in trees sampled or if there was some sort of seasonal decline of the population because of the local block conditions. Objective 2) Determine benefit-cost thresholds for Phytophthora treatment on HLB-affected treesWe are waiting for more data
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. Five field trials were established to test these objectives. The trials had to be modified from the original proposal to accomodate the requests by the industry collaborators TJ Biotech and Invaio. Trials are as follows:Trial 1: Located in SW Florida (Duda) – 8-year-old Valencia/Carrizo trees. This trial examines two different oxytetracycline (OTC) rates and timing of injection using our established procedures.Trial 2: Located in SW Florida (Graves Bros) – 8-year-old Valencia/Kuharske trees. This trial examines different OTC rates and different volumes in which OTC is delivered. We used a different OTC formulation which we were told is the same as ArborOTC (which we have been using in the past) but marketed under a different name.Trial 3: Located on the east coast (Graves Bros) – 9 year-old Valencia/sour orange trees. This trial is conducted together with TJ Biotech and examines different OTC rates, different OTC formulations, and different injection procedures. The trial compares TJ Biotech’s methodology with ours as well as some combinations of both methodologies.Trial 4: Located on the east coast (Graves Bros) – 4-year-old Valencia/x639 trees. This trial is conducted together with Invaio and examines different OTC rates, timing of injection, and different injection procedures. The trial compares Invaio’s methodology with ours as well as some combinations of both methodologies. Invaio’s OTC formulation is the same as ours. We also included a comparison of scion vs. rootstock trunk injection. Per Invaio request, spray application of OTC (Fireline) is included as one treatment as an additional control. Trial 5: Located on the central ridge (King Ranch) – 4-year-old OLL-8/x639 trees. This trial is conducted together with Invaio and is a repeat of trial 4 (per Invaio request). The trial site was chosen by Invaio.Prior to trial establishment several meetings with TJ Biotech and Invaio took place and numerous emails were exchanged to determine locations, tree types, experimental designs, and objectives of trials 3-5. The resulting trials are the result of compromises between both companies needs and our/industry needs.Baseline measurements for all trials were conducted, leaves were collected prior to injection for CLas detection, and May injections were completed. 2. Please state what work is anticipated for next quarter:We will conduct August injections in the trials where injection timing is one of the objectives. 3. Please state budget status (underspend or overspend, and why):Budget status is as anticipated.
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 first set of treatments on the first week of June, and the second set of treatments, by the end of July. 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. We have observed and recorded that trees under Zn and Zn+K treatments have greener canopies. We have sampled leaves for chlorophyll analysis, after noticing this clear difference. Control trees are presenting some off-blooms in August. These off-blooms have not been observed in the treated trees. Off bloom trees have been marked to record fruit drop.In objective 2, GA3 and 2,4D treatments have continued as planned.Objective 3: Outreach. -Boakye, D, Alferez, F. The interplay between Zn, K, and IAA biosynthesis and signalling during the abscission process on Huanglongbing (HLB)-affected Hamlin trees. ASHS 2022 Annual Conference. July, 2022. Chicago. Oral presentation. 2. Please state what work is anticipated for next quarter: In next quarter, we will continue treatments for both objectives and we will start assesing fruit drop in Hamlin, we do not anticipate any fruit drop in Valencia. We are starting also analysis of samples collected to date. 3. Please state budget status (underspend or overspend, and why): Underspending on student salary and tuition is still happening as the student Divya Aryal did not join yet. We expect this to be corrected during this month, as she is already accepted in the HOS doctoral program and visa gets finally approved.
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 first set of treatments on the first week of June, and the second set of treatments, by the end of July. 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. We have observed and recorded that trees under Zn and Zn+K treatments have greener canopies. We have sampled leaves for chlorophyll analysis, after noticing this clear difference. Control trees are presenting some off-blooms in August. These off-blooms have not been observed in the treated trees. Off bloom trees have been marked to record fruit drop.In objective 2, GA3 and 2,4D treatments have continued as planned.Objective 3: Outreach. -Boakye, D, Alferez, F. The interplay between Zn, K, and IAA biosynthesis and signalling during the abscission process on Huanglongbing (HLB)-affected Hamlin trees. ASHS 2022 Annual Conference. July, 2022. Chicago. Oral presentation. 2. Please state what work is anticipated for next quarter: In next quarter, we will continue treatments for both objectives and we will start assesing fruit drop in Hamlin, we do not anticipate any fruit drop in Valencia. We are starting also analysis of samples collected to date. 3. Please state budget status (underspend or overspend, and why): Underspending on student salary and tuition is still happening as the student Divya Aryal did not join yet. We expect this to be corrected during this month, as she is already accepted in the HOS doctoral program and visa gets finally approved.
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: We completed the second split fertilizer application for designated treatments in early April, allowing a comparison of leaf nutrients sampled on May 2 at the 25% and 50% annual fertilizer allocations. For the Hamlin experiment, leaf N concentrations were significantly higher in the 50% complete treatment (2.53% N) than the 25% complete treatment (2.38% N). Leaf K concentrations were also significantly higher (1.41% K) in the 50% complete treatment than the 25% complete treatment (1.23% K). For the Valencia experiment, only leaf N was significantly higher for the 50% treatment (2.55% N) than the 25% treatment (2.33%). There were no other significant differences in foliar nutrients between treatments at that time. There were however deficiencies of Mn, and low levels of Ca, Mg, Zn, Fe and B in the two experiments according to the leaf analysis of May 2. Leaf samples collected on May 29 were analyzed and indicated improved nutrient status, with only Ca and Zn deficiencies in the experiments. Leaf N concentrations for Hamlin and Valencia experiments remained significantly different for the 50% and 25% completed fertilizer treatments (Hamlin: 2.69%, 2.81% N for 25%, 50% complete; Valencia: 2.49%, 2.66% N for 25%, 50% complete). The third split fertilizer application was in early June but the leaf samples were not analyzed yet. Dry and hot spring weather extending well into late June seemed detrimental to the trees despite daily irrigation. Fruit diameter was measured at every two weeks starting from phase 2 of fruit development (end of May), at 8 fruits per plot. Although fertilizer was applied 3 times, there was not any significant increase in fruit growth between treatments, but there was significant different in fruit growth between Hamlin and Valencia. The second Aerobotics drone survey flew at the end of May to collect data on tree size and health. There were no significant differences among treatments yet.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):Our investigations to use the intensity of leaf symptom expression to help determine foliar nutrition in the field with smartphone apps is focusing on Mn first. We showed that we could visually classify 10 classes of Mn deficiency severity (all in the deficient range of <18 ppm). The corresponding leaf Mn concentrations correlated linearly with the 10 classes. The challenge will be to detect not yet easily visible Mn deficiency symptoms so that early diagnoses can be made. We plan to repeat the process with N, Mg, Zn, and Fe deficiency symptoms.B) New developments: sub-objectives concerning the Sugarbelle trial focusing on solving the current fruit quality issues are progressing well. We applied the first replicated foliar spray treatments to Sugarbelle trees at the city block consisting of KNO3 and KH2PO4. Two more applications will go out this summer. 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: We are still concerned about the low fruit set in the city block compared to previous years. The early bloom in January followed by a dry hot spring has stressed the trees considerably. 2. Please state what work is anticipated for next quarter:The third and fourth fertilizer applications will be made to designated treatment plots in July and September. Soil, lysimeter sampling, leaf sampling, processing and analysis will be ongoing, as will tree size and fruit measurement. The third Aerobotics drone survey will fly in late September or early October, to evaluate tree health and size indicators. 3. Please state budget status (underspend or overspend, and why):Spending rate is approximately on track, slightly less than expected due to startup lag in Q1.
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: We completed the second split fertilizer application for designated treatments in early April, allowing a comparison of leaf nutrients sampled on May 2 at the 25% and 50% annual fertilizer allocations. For the Hamlin experiment, leaf N concentrations were significantly higher in the 50% complete treatment (2.53% N) than the 25% complete treatment (2.38% N). Leaf K concentrations were also significantly higher (1.41% K) in the 50% complete treatment than the 25% complete treatment (1.23% K). For the Valencia experiment, only leaf N was significantly higher for the 50% treatment (2.55% N) than the 25% treatment (2.33%). There were no other significant differences in foliar nutrients between treatments at that time. There were however deficiencies of Mn, and low levels of Ca, Mg, Zn, Fe and B in the two experiments according to the leaf analysis of May 2. Leaf samples collected on May 29 were analyzed and indicated improved nutrient status, with only Ca and Zn deficiencies in the experiments. Leaf N concentrations for Hamlin and Valencia experiments remained significantly different for the 50% and 25% completed fertilizer treatments (Hamlin: 2.69%, 2.81% N for 25%, 50% complete; Valencia: 2.49%, 2.66% N for 25%, 50% complete). The third split fertilizer application was in early June but the leaf samples were not analyzed yet. Dry and hot spring weather extending well into late June seemed detrimental to the trees despite daily irrigation. Fruit diameter was measured at every two weeks starting from phase 2 of fruit development (end of May), at 8 fruits per plot. Although fertilizer was applied 3 times, there was not any significant increase in fruit growth between treatments, but there was significant different in fruit growth between Hamlin and Valencia. The second Aerobotics drone survey flew at the end of May to collect data on tree size and health. There were no significant differences among treatments yet.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):Our investigations to use the intensity of leaf symptom expression to help determine foliar nutrition in the field with smartphone apps is focusing on Mn first. We showed that we could visually classify 10 classes of Mn deficiency severity (all in the deficient range of <18 ppm). The corresponding leaf Mn concentrations correlated linearly with the 10 classes. The challenge will be to detect not yet easily visible Mn deficiency symptoms so that early diagnoses can be made. We plan to repeat the process with N, Mg, Zn, and Fe deficiency symptoms.B) New developments: sub-objectives concerning the Sugarbelle trial focusing on solving the current fruit quality issues are progressing well. We applied the first replicated foliar spray treatments to Sugarbelle trees at the city block consisting of KNO3 and KH2PO4. Two more applications will go out this summer. 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: We are still concerned about the low fruit set in the city block compared to previous years. The early bloom in January followed by a dry hot spring has stressed the trees considerably. 2. Please state what work is anticipated for next quarter:The third and fourth fertilizer applications will be made to designated treatment plots in July and September. Soil, lysimeter sampling, leaf sampling, processing and analysis will be ongoing, as will tree size and fruit measurement. The third Aerobotics drone survey will fly in late September or early October, to evaluate tree health and size indicators. 3. Please state budget status (underspend or overspend, and why):Spending rate is approximately on track, slightly less than expected due to startup lag in Q1.
1. Please state project objectives and what work was done this quarter to address them: Objective. To determine the influence of compost during the first three years of tree establishment on growth, productivity, and root and soil health of citrus trees on rootstocks with different vigor-inducing capacity. Another round of compost was applied at the usual rate. Flush ratings and soil moisture measurements continued. The soil moisture in the compost treated plots continues to be significantly higher than in the control plots and we are seeing significantly more (two to three-fold more) weed pressure in the compost treated plots. Soil and leaf samples were collected for macro- and micronutrient analysis. Many of the soil nutrients continue to be present in higher concentrations in the compost treated plots than in the control plots except for copper which was reduced by the compost. However, higher nutrient concentrations in the soil did not always translate to higher concentrations in the leaves. The cation exchange capacity and the soil pH were also significantly higher in compost plots than control plots.The fibrous root respiration was measured and roots from the compost plots have a higher respiration rate than roots from the control plots. Significant differences were also found among rootstocks with the citrandarins having higher rates than US-802. This was also reflected in the specific root length which was lowest for US-802 and highest for US-897. The specific root length appears to be moderately correlated with pounds soluble solids. A manuscript for publication was submitted and is under review. A Citrus Industry magazine article on this study was published in the June 2022 issue. 2. Please state what work is anticipated for next quarter:Flush ratings and soil moisture measurements will continue. Root respiration and specific root length will be measured. We will conduct tree ratings and tree size measurements. Leaves will be collected for CLas determination. The weed biomass will be determined.Data analyses will continue. 3. Please state budget status (underspend or overspend, and why): Most of the funds have been spent.
1. Please state project objectives and what work was done this quarter to address them: Objective. To determine the influence of compost during the first three years of tree establishment on growth, productivity, and root and soil health of citrus trees on rootstocks with different vigor-inducing capacity. Another round of compost was applied at the usual rate. Flush ratings and soil moisture measurements continued. The soil moisture in the compost treated plots continues to be significantly higher than in the control plots and we are seeing significantly more (two to three-fold more) weed pressure in the compost treated plots. Soil and leaf samples were collected for macro- and micronutrient analysis. Many of the soil nutrients continue to be present in higher concentrations in the compost treated plots than in the control plots except for copper which was reduced by the compost. However, higher nutrient concentrations in the soil did not always translate to higher concentrations in the leaves. The cation exchange capacity and the soil pH were also significantly higher in compost plots than control plots.The fibrous root respiration was measured and roots from the compost plots have a higher respiration rate than roots from the control plots. Significant differences were also found among rootstocks with the citrandarins having higher rates than US-802. This was also reflected in the specific root length which was lowest for US-802 and highest for US-897. The specific root length appears to be moderately correlated with pounds soluble solids. A manuscript for publication was submitted and is under review. A Citrus Industry magazine article on this study was published in the June 2022 issue. 2. Please state what work is anticipated for next quarter:Flush ratings and soil moisture measurements will continue. Root respiration and specific root length will be measured. We will conduct tree ratings and tree size measurements. Leaves will be collected for CLas determination. The weed biomass will be determined.Data analyses will continue. 3. Please state budget status (underspend or overspend, and why): Most of the funds have been spent.
The first oxamyl nematicide treatment occurred in the IPC trial on 25 April 2022 and the second on 24 May. The effects of these treatments on sting nematode populations were measured on 11 July, 48 days after the final treatment. Two soil cores (30 cm deep x 2.5 cm dia) per tree were composited in each 4-tree plot. Samples were weighed and nematodes were separated from subsamples (250 cm3) by sucrose centrifugation. Fibrous roots in the entire sample were collected, air dried and are currently being separated from weed roots for measurement. The sting nematode populations were low, averaging just 5 nematodes per 250 cm3 soil. Nevertheless, oxamyl was shown to reduce the (log-transformed ) sting nematodes (P=0.015) by 53% and there was a suggestion of an interaction (P=0.07) with the IPC treatment in which nematodes were reduced by 19% compared to 88% in the non-covered trees. The first rootstock lines were planted in the four large Rubbermaid tanks on 17 June (UFR-1, UFR-4, UFR-5, UFR-6, UFR-15, UFR-17, MG-11, FG#2, Orange 14, Orange 16, A+VolkxOrange19-11-8, LB8-9xS10-15-18, LB8-9xS13-15-16, S10xx639-12-32, S11xSO+50-7-16-12, LB8-9×50-7-16-4, Blue 1). Two tanks are infested with sting nematode and two are non-infested controls. Each tank contains 4 plants of each rootstock. The greenhouse renovation improved the ambient conditions, especially the temperature control. The plants will be evaluated for root and shoot mass and nematode induced stubby root and tip gall symptoms.
The first oxamyl nematicide treatment occurred in the IPC trial on 25 April 2022 and the second on 24 May. The effects of these treatments on sting nematode populations were measured on 11 July, 48 days after the final treatment. Two soil cores (30 cm deep x 2.5 cm dia) per tree were composited in each 4-tree plot. Samples were weighed and nematodes were separated from subsamples (250 cm3) by sucrose centrifugation. Fibrous roots in the entire sample were collected, air dried and are currently being separated from weed roots for measurement. The sting nematode populations were low, averaging just 5 nematodes per 250 cm3 soil. Nevertheless, oxamyl was shown to reduce the (log-transformed ) sting nematodes (P=0.015) by 53% and there was a suggestion of an interaction (P=0.07) with the IPC treatment in which nematodes were reduced by 19% compared to 88% in the non-covered trees. The first rootstock lines were planted in the four large Rubbermaid tanks on 17 June (UFR-1, UFR-4, UFR-5, UFR-6, UFR-15, UFR-17, MG-11, FG#2, Orange 14, Orange 16, A+VolkxOrange19-11-8, LB8-9xS10-15-18, LB8-9xS13-15-16, S10xx639-12-32, S11xSO+50-7-16-12, LB8-9×50-7-16-4, Blue 1). Two tanks are infested with sting nematode and two are non-infested controls. Each tank contains 4 plants of each rootstock. The greenhouse renovation improved the ambient conditions, especially the temperature control. The plants will be evaluated for root and shoot mass and nematode induced stubby root and tip gall symptoms.
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 will 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. 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 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 will 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. 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 the next quarter. 3. Please state budget status (underspend or overspend, and why): The budget is on track and meeting the project milestones.
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) will be transformed to express the tagged ribosomal proteins under the control of characterized phloem-specific promoters; tagged ribosomal proteins under control of the nearly ubiquitous CaMV 35S promoter will be used as a control. Transgenic plants will be exposed to CLas+ or CLas- ACP and leaves sampled 30, 60, 90, and 120 days later. Ribosome-associated mRNA will be 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.During this most recent quarter, the 2nd quarter of our 1st 6 month no cost extension (the original end date was 11/30/2021; we were granted one 6 month no cost extension and one 3 month one; the current end date is 08/31/2022), the Stover lab sent the last of the transgenic lines needed for the project to the Rogers lab. Now at least 4 high expressing lines for each of the 9 promoter/genotype combinations are in the containment greenhouse facility at Ft. Detrick and are being prepared for no-choice psyllid inoculation experiments.The Rogers lab has continued no-choice psyllid inoculation experiments on the rooted cuttings available and ribosome pull-downs from the tissue collected. Work has progressed more quickly since the ARS 25% occupancy cap was lifted on Monday, March 28th. We have still not been able to identify a qualified and interested post-doc candidate and with less than 3 month left on the grant, now do not have time to hire someone. As many translatome RNA samples as possible will be sent for sequencing in July, leaving the month of August for data analysis.
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