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.
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.
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.
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