ASHS 2025 Conference

There is an urgent need to diversify high-value fruit crops in low-chill areas, especially as climate change decreases the number of chill hours in cropping areas. Apple is largely considered a temperate crop, yet a subset of germplasm exhibits strong flowering responses even after minimal chill. These genotypes originate from Israeli breeding programs, as well as heritage commercial varieties and backyard discoveries. Future breeding efforts will benefit from characterization of the genetic mechanisms that govern flowering in response to limited chill. In this study we hypothesized that examination of sequence polymorphisms and flowering associated gene sequence differences may permit grouping of low chill materials based on common mechanisms. Relatedness was examined using a series of simple sequence repeat markers (SSRs). In addition, genomic sequence from a number of low chill accessions, including Dorsett Golden, Shell of Alabama, and an early-flowering accession from Mississippi, was compared to publicly available reads from ‘Anna’ (low chill), moderate chill (‘Fuji’, ‘Gala’) and high chill (‘Honeycrisp’; ‘Antonovka’) varieties. Genomes were aligned to Golden delicious reference genome, and shared and unique variants were identified. The data show that all low and moderately low chill cultivars share common sequence polymorphisms not found in high-chill germplasm. Examination of flowering and dormancy-related genes shows common sequence polymorphisms shared within low chill materials that contrast against high chill genotypes. These include members of MADS-boxes family, Frigida family, Early bud break (AP2/ERF) family, auxin responsive factors, transcription factors DELLA family, FRIGIDA INTERACTING PROTEIN, and others. This study illuminates potential mechanisms of low-chill responses, opening opportunities for marker-assisted breeding and increased genetic diversity in development of low chill apple cultivars.

Prior to the formal breeding programsof the 19th century, farmers contributed to apple (Malus× domestica Borkh.) breeding by selecting trees based on the desirable characteristics. The transcontinental seed spread was common, and modern breeding programs identified elite commercial trees and high quality fruits. There is a rekindling interest in low-chill apples, both as a high value crop in the USA Southeast, as well as development of new varieties to confront climate change. But the genetic record is poor. Dorsett Golden (DG) is low chill apple cultivar believed to have been discovered in The Bahamas by a Mrs. Dorsett in 1950. As legend has it, she was a jet-setting traveler that loved apples, and planted a set of trees when she relocated to the Bahamas. This allegedly led to the identification of ‘Dorsett Golden’. While this quickly became the accepted story, some questioned the claim. Observations of phenological data in DG trees next to other Israeli germplasm (e.g. ‘Anna’, ‘Ein Shemer’) led Dr. Wayne Sherman to posit in 1980 that DG’s origin was likely from the Israeli breeding program. To test this hypothesis, whole genome sequence from DG was compared to ‘Anna’ and other reference sequences in public databases. Consistent with Dr. Sherman’s 45 year old predictions, DG shares it’s most significant sequence similarity to ‘Anna’, and less with other low-chill varieties. The majority (~92%) of the total variants are in intergenic, upstream, downstream, or intronic regions suggesting recent divergence of ‘Anna’ and DG. The results are consistent with Dr. Sherman’s phenological data that suggest that DG possessed genetics more similar to Israeli genotypes than a chance seedling from Golden Delicious as legend describes. Most importantly, the study illustrates the power of genomic sequencing in selection of parents for low-chill apple crosses as well as debunking horticulutral methology.

Salinity is a major constraint on tomato crop production and is increasingly intensified by changing climate conditions. This study aimed to develop superior salt-tolerant tomato cultivars by evaluating genetic variation in salt tolerance, identifying associated single-nucleotide polymorphism (SNP) markers through genome-wide association studies (GWAS), and performing genomic prediction (GP). A total of 265 tomato accessions from the USDA germplasm collection were evaluated at the seedling stage under controlled greenhouse conditions with saline stress (200 mM NaCl). Nineteen accessions were identified as salt-tolerant, exhibiting leaf injury scores ≤3.0 (on a 1–5 scale) and chlorophyll reduction of

Lettuce (Lactuca sativa L.) is one of the most important leafy vegetable crops worldwide. Soil salinity adversely affects lettuce production leading to considerable yield losses. Identification of genetic loci controlling salt tolerance will facilitate molecular marker development and thereby assist breeders in developing lettuce cultivars with salt tolerance. Accordingly, we conducted a genome-wide association study (GWAS) to identify marker-trait association for salt tolerance at the seedling stage using 409 diverse lettuce accessions and 56,820 high-quality single nucleotide polymorphism (SNP) markers obtained through genotype-by-sequencing technology. Several statistical models, including GLM, MLM, FarmCPU, and BLINK were employed using the GAPIT version 3 software tool for GWAS. Based on three important seedling stage traits affected by salinity, i.e., shoot fresh weight (FW), shoot dry weight (DW) and chlorophyl index (SPAD), 13 significant salt tolerance related SNPs representing 10 QTLs were identified on lettuce chromosomes 1, 3, 4, 6, 7, 8 and 9. Notably, a major QTL on chromosome 4, encompassing four significant SNPs within a 116 bp region of the lettuce reference genome (v8), explained 49% of the phenotypic variation for FW. The identified salt tolerance-related QTLs provide a valuable resource for developing assays for marker-assisted selection to breed lettuce cultivars with improved salt tolerance.

Chile pepper (Capsicum annuum) is widely produced and consumed, but farmers face significant challenges associated with high temperature stress. Tolerance to high temperatures is a phenotype comprised of numerous component traits each of which contribute to the overall performance of the plant. Our aim was to identify the key mechanisms associated with heat stress response in the leaves and in the floral organs of chile pepper. One-month-old plants of heat-sensitive (AVPP1609-038) and -tolerant (AVPP1609-015) recombinant inbred line (RIL) of chile pepper were subjected to heat stress (38 and 28°C day and night temperatures) and control conditions (32 and 24°C day and night temperatures) in growth chambers with a 14-hour photoperiod. Leaf and floral bud samples were collected for RNA extraction at 11 and 18 days after treatment, respectively, with four biological replicates per tissue. Differentially expressed genes (DEGs) were identified by comparing tolerant and sensitive RILs across treatments and tissues. For the heat-tolerant AVPP1609-015 under heat stress, 1,118 DEGs were identified, with 649 specific to floral buds, 381 in leaves, and 88 shared between the two tissues. Biological processes such as RNA splicing and heat acclimation were predominantly upregulated in floral buds, while lipid catabolism was enhanced in leaves. Developmental processes were consistently suppressed in both tissues for the RILs under heat stress conditions. For the heat-tolerant AVPP1609-015 nuclease activity was strongly suppressed, likely to preserve nucleic acid integrity under heat stress. Hormonal regulation showed tissue specificity, with salicylic acid playing a pivotal role in leaves and ethylene in floral buds, potentially associated with flower abscission. Additionally, key transcription factors associated with heat tolerance were identified. While some mechanisms of heat tolerance were shared between tissues, distinct responses were observed as well, suggesting the need for different breeding approaches to enhance heat tolerance in vegetative and reproductive tissues of chile pepper. These findings provide valuable insights for developing heat-resilient chile pepper and a foundation for future research.

Herbicide tolerance in plants is an increasingly valuable trait due to the high labor and costs associated with weed control in agriculture. Herbicide application remains the most effective and widely used weed management strategy, making the development of tolerant plants essential. First discovered in the 1970s and commercially grown since 1984, herbicide resistant crops have become a key tool in agriculture, with increasing demand for new tolerant varieties. Chemical mutagenesis and CRISPR-mediated gene editing have been used to induce mutations and develop herbicide tolerant plants. Chemical mutagenesis involves treating plant tissue with mutagens such as ethyl methanesulfonate (EMS) to induce random mutations, followed by screening to identify tolerant mutants. This conventional approach has played a significant role in breeding programs and remains widely used for developing herbicide tolerant crops. EMS mutagenesis has successfully generated ALS-resistant varieties in several agronomic crops, including Clearfield® maize, rice, and wheat, which are resistant to imidazolinone (IMI) herbicides without being classified as genetically modified (GM). It is particularly effective for developing crops resistant to ALS- and ACCase-inhibiting herbicides, as these mutations typically require only minor changes in the target genes. CRISPR-mediated gene editing, using tools such as CRISPR-Cas9, base editing (CBE, ABE), and prime editing, enables precise modifications in plant genomes to confer herbicide tolerance. These advancements have revolutionized crop development through their efficiency, precision, and cost-effectiveness. By targeting herbicide receptor genes such as ALS, ACCase, and EPSPS, CRISPR-based systems have produced herbicide tolerant varieties in several agronomic crops. CRISPR is particularly valuable for engineering tolerance to non-selective herbicides, such as glyphosate, due to the complex genomic architecture of the EPSPS gene. Chemical mutagenesis facilitates the discovery of novel mutations and is particularly useful in understudied species lacking the genomic information required for CRISPR-based modification. In contrast, CRISPR-based genome editing provides a highly precise and efficient method for developing herbicide tolerant crops, especially when targeting complex genes. Integrating chemical mutagenesis with CRISPR-mediated gene editing expands the range of available herbicide tolerance traits and offers new opportunities for sustainable weed management. These advances in agronomic crops provide a strong foundation for extending herbicide tolerance studies to horticultural and specialty crops, where research has been more limited despite similar weed management issues.

Open to all attendees.

Developing integrated pest and pathogen management approach for controlled environment horticulture crops is of major impotance to safeguard growers interest, and community health and sustainability. The abilty to control temperature, light and to some extent humidity, creates optimal conditions for crop growth. However, the same suitable environment creates favorable opportunities for insect pest population growth, their associated viral diseases and fungal pathogens. Growers often rely on chemical insecticides to control CEH pests, leading to selective pressure and development of insecticide resistance in major CEH pests, such as aphids, thrips and whitelfies. This highlights the fact that insecticides are not reliable long-term pest control solution. Therefore, CEH specific effective pest and pathogen management approaches are urgently needed to move towards sustainable CEH crop production nationwide.

Speakers:

• Samantha Wilden, Cornell University
• Arash Kheroiding, Texas A&M AgriLife
• Krishna Bhattarai, Texas A&M AgriLife
• Erich Schoeller, University of Georgia
• Ruchika Kashyap, University of Georgia
• Andrew Ogden, University of Georgia
• Azlan Zahid, Texas A&M  AgriLife

Huanglongbing (HLB), associated with Candidatus Liberibacter asiaticus (CLas), continues to devastate citrus production worldwide. Individual protective covers (IPCs) offer a physical barrier against the Asian citrus psyllid, delaying CLas infection in young trees. However, once removed, trees become vulnerable to rapid infection. This study evaluates an integrated approach utilizing IPCs followed by systemic oxytetracycline (OTC) trunk injections to mitigate disease progression and sustain tree health. A field trial was established with ‘Valencia’ sweet orange (Citrus sinensis) grafted onto US-812 or US-942 rootstocks. Trees were either protected with IPCs for 18 months after planting (“late infected”) or had “open” IPCs (“early infected”). All trees received annual OTC injections in 2023 and 2024, beginning 10 months after IPC removal. CLas titers, OTC residues, tree growth, fruit yield and quality, leaf nutrient content, and root endorhizosphere microbial dynamics were assessed. Results indicate that IPCs successfully delayed infection, as late-infected trees maintained lower CLas titers for the first five months after IPC removal and exhibited denser canopies and larger scion and rootstock trunk diameters than early-infected trees. OTC injections significantly reduced CLas titers in leaves, with effects persisting for up to six months. However, no significant reductions in CLas titers were detected in fibrous roots, suggesting limited OTC translocation to the root system. Injected trees exhibited a greener canopy color, greater leaf size, higher chlorophyll content, higher yield, larger fruit size, and better juice quality compared to non-injected trees. No significant changes in leaf nutrient content were observed, and the root endorhizosphere microbial diversity remained unchanged across treatments, indicating minimal disruption to the microbial community due to the antibiotic. These findings provide evidence supporting the combination of IPCs and systemic delivery of OTC as an integrated strategy to manage HLB in new citrus plantings. IPCs delay the onset of infection, allowing trees to establish a healthy canopy and robust root system, while OTC injections aid in sustaining tree health post-infection. The integration of these practices provides a promising framework for establishing productive citrus orchards under HLB-endemic conditions.

Huanglongbing (HLB), associated with phloem-limited bacteria Candidatus Liberibacter asiaticus (CLas), is a major threat to citrus production worldwide, severely reducing tree health, yield, and fruit quality. In Florida, trunk injection of oxytetracycline (OTC) has emerged as a promising management strategy for HLB. Under this context, this study aimed to enhance the efficacy of OTC injection by evaluating different injection volumes, modes of injection (one or two-sided), and injection timing in HLB-affected ‘Valencia’ orange (Citrus sinensis) trees in southwest Florida. Two experiments were conducted with 8-year-old trees. The first experiment, implemented in two locations, assessed four combinations of different OTC volumes and modes of injection: 1) 50 mL (one-sided), 2) 100 mL (one-sided), 3) 100 mL (two-sided, 50 mL each side), and 4) 200 mL (two-sided, 100 mL each side), all delivering 0.825 g (a.i.) OTC (Rectify, AgroSource, Inc.) per tree. The second experiment compared early (May) and late (July) injections using 100 mL (two-sided, 50 mL each side), delivering 0.825 g (a.i.) OTC (Rectify) per tree. Injectors were placed on opposite sides of the trunk for the two-sided treatments. OTC solutions were prepared right before injection. In both experiments, treatments were compared against an untreated control. The experimental design was completely randomized, with 10 single-tree replications for the first experiment and 8 replications of 4 trees each for the second. Leaf samples were collected over time to analyze bacterial (CLas) titers and OTC residues, while canopy health, yield, and fruit quality were evaluated annually. For the second experiment, fruit development (size and peel color) was monitored monthly after OTC injection, and fruit yield and quality were assessed annually. Our results demonstrate that two-sided injection increases fruit yield more than one-sided injection and that the combination of two-sided injection and a larger volume of the injected solution may further enhance fruit yield compared to the untreated control. This improvement is attributed to better OTC distribution within the canopy, leading to greater bacterial suppression and tree recovery. Moreover, early (May) injection promotes fruit size by extending the fruit growth period, while late (July) injection enhances juice soluble solids content with a more compact fruit size. However, early injections result in greener fruit, which requires a longer maturation period to achieve the desired peel color and sugar content. These findings offer valuable insights for optimizing OTC trunk injection strategies to improve citrus health and productivity in HLB-affected citrus orchards.

The combined use of Individual Protective Covers (IPCs) and oxytetracycline (OTC) trunk injection offers a promising strategy for managing huanglongbing (HLB) in young citrus trees by reducing environmental stress and preventing Candidatus Liberibacter asiaticus (CLas) inoculation. This study evaluated whether young citrus trees that were grown without IPCs for the first two years after planting and severely affected by HLB can be rehabilitated by OTC injections, and whether IPCs provide additional benefits beyond OTC alone. ‘Valencia’ sweet orange trees on US-812 and US-942 rootstocks, were organized in a randomized complete block design with three treatments: 1) no injection no IPC, 2) OTC injection no IPC, and 3) OTC injection IPC. Each treatment-rootstock combination consisted of 14 single-tree replications. Trunk injections were performed annually for two years (2023-2024) using Chemjet tree injectors, with solutions delivered into the rootstock. Comprehensive data were collected on the yield, juice quality, bacterial titers, disease state, flushing patterns, and psyllid infestation. No significant differences were observed between the rootstocks except for the juice quality, which was better in combination with US-812. OTC-injected trees consistently exhibited higher yields and improved juice quality, with no additional benefits from IPCs. A significant decrease in CLas titers was noted starting three months post-injection. Injected trees under IPCs had fewer HLB symptoms in the second year, and injected trees exhibited a significant increase in canopy density compared with non-injected trees. Trees with IPCs flushed less frequently during summer and winter than trees without IPCs. The psyllid infestation rate was the same in injected and non-injected trees, indicating that OTC treatment does not affect host attractiveness. These findings highlight the potential of OTC trunk injection to rehabilitate young HLB-affected citrus trees and enhance tree productivity. The longer-term role of IPCs in young tree rehabilitation is still under investigation.

The systemic delivery of oxytetracycline (OTC) by trunk injection has emerged as a viable strategy to diminish the impacts of the bacterial disease Huanglongbing (HLB) in Florida. This study aimed to determine the optimal OTC dose and best time of delivery to enhance tree health and productivity in mature HLB-affected citrus trees. It also investigated whether OTC effects persist after one single injection compared to two consecutive annual injections. The study began in 2023 at a commercial citrus grove in Polk County, Florida, using 18-year-old ‘Valencia’ (Citrus sinensis) trees grafted onto Swingle (C. paradisi × Poncirus trifoliata) rootstock. Four OTC doses (0.55 g, 0.825 g, 1.1 g, and 1.65 g of active ingredient per tree) were evaluated alongside two application times, spring and late summer, with a non-injected control for comparison. The experiment was a randomized complete block design with eight replications containing four trees. A commercial OTC formulation (ReMedium TI, 95% oxytetracycline hydrochloride) registered in Florida was applied using commercial tree injectors (FlexInject). Doses were applied in 100 ml acidified water through a single injector, except for the highest dose, which was split between two injectors and applied in a volume of 75 ml on opposite sides of the trunk. All trees received their designated doses in year 1, except for the non-injected controls. Only half of the trees injected in year 1 received a second dose in year 2. Trees were harvested in March 2024 and February 2025. Fruits were collected for fruit and juice quality analysis and to determine OTC fruit residue levels. Regardless of the dose, all OTC-treated trees produced more, larger-sized fruits, more total soluble solids (TSS), and a higher TSS-to-acid ratio compared to non-injected controls. The best results were obtained with the highest dose applied by split injections. There were no major differences between spring and summer injections except for the TSS content, which was higher and the percent acid, which was lower when trees were injected in late summer. However, fruit OTC residues were above the allowed maximum after late summer injections, suggesting that injections should be completed by the end of spring. Trees injected for two consecutive years produced significantly more, better-quality fruits than trees injected only once in the first year. These results confirm the efficacy of OTC trunk injections to enhance productivity and fruit quality under HLB-endemic conditions and provide important guidelines regarding dose and timing of injections.

Citrus production in the State of Florida has been decimated by the disease Huanglongbing (HLB). This disease is associated with the phloem-limited, gram-negative bacteria Candidatus Liberibacter asiaticus (CLas) and is vectored by the Asian citrus psyllid (Diaphorina Citri). Since HLB was first identified in Florida in 2005 it spread rapidly and was considered endemic in the state by 2013, resulting in a nearly 90% decline in citrus fruit production. While breeding more tolerant or fully resistant cultivars is likely the best long-term solution, trunk injections of the antibiotic oxytetracycline were granted emergency use approval in October 2022 to combat the bacteria. Historically most of Florida’s citrus production has been sweet orange (Citrus sinensis (L.) Osbeck) cultivars including ‘Hamlin’ and ‘Valencia’ which were primarily destined for juice processing. Unfortunately, all known commercial sweet orange cultivars are susceptible to HLB infection leading to the rapid and drastic decrease in production across the state. Of the sweet orange cultivars that have been released by the University of Florida Institute of Food and Agricultural Sciences (UF-IFAS), Orie and Louise Lee (‘OLL’) lines have shown some degree of tolerance to HLB in cultivar field trials. In our experiment we evaluated the effects of rootstock selection and oxytetracycline (OTC) trunk injections on the fruit and juice quality of the ‘OLL-8’ sweet orange scion. Three of the evaluated rootstocks (‘US-942’, ‘US-897’, and ‘Swingle’) are industry standard diploids, while the other three rootstocks (‘UFR-2’, ‘UFR-4’, and ‘UFR-6’) are tetraploids released by the University of Florida. For each of these rootstocks there were trees that were injected with OTC and untreated control trees. Fruit were harvested at several time points and data were collected on juice quality attributes including titratable acidity in citric acid equivalents, total soluble solids, sugar/acid ratio, pounds-solids per box, and juice color, as well as fruit characteristics including mass, diameter, total yield, and premature fruit drop. The trees that were injected with OTC had significantly larger mean fruit diameter, higher mean ratio, and lower premature fruit drop than untreated controls. There was also evidence for statistically significant differences among rootstock selections in conjunction with the ‘OLL-8’ scion for each of the characteristics that were evaluated. These results indicate that rootstock selection and OTC-trunk injections can significantly improve fruit and juice quality of the ‘OLL-8’ sweet orange scion. However, more research is needed on the effects of OTC injections in different rootstock-scion combinations.

Citrus Huanglongbing (HLB), also known as citrus greening, is associated with Candidatus Liberibacter asiaticus (CLas), a phloem-limited bacterium transmitted by an insect vector, Asian Citrus Psyllid (ACP). Currently, there are no commercially viable treatment options available for HLB management. Trunk injection of Oxytetracycline (OTC) has been widely adopted as a treatment option in Florida. Currently, OTC is not approved for use in Texas citrus, and no OTC efficacy data is available. This study evaluated the efficacy of OTC in mitigating HLB and managing tree health in nine and 17-year old ‘Rio Red’ grapefruit (Citrus paradisi Macfadyen) trees on sour orange (Citrus aurantium L.) rootstock, on a randomized complete block design in two commercial orchards. RECTIFY™ (95% Oxytetracycline hydrochloride) was injected into tree trunks at two rates (0.55g and 1.1g per tree) using Chemjet® tree injectors during June-July 2023 and May 2024. Trees were evaluated for various attributes, including periodic (pre-treatment, three, six, and nine months after OTC injection) bacterial titer in leaf and root tissues and tree health monitoring (canopy color, canopy density, and HLB severity) for the same time intervals. Fruits collected at harvest maturity were evaluated for yield, fruit size, and juice quality (brix, titratable acidity, flavonoids, carotenoids). Additionally, pre-harvest fruit drop and trunk injection site damage were recorded. OTC residue was measured in whole fruit and juice using Ultra-High-Performance Liquid Chromatography coupled with tandem Mass Spectrometry (UHPLC-MS-TOF). Nine-year-old trees treated with 1.1g of OTC/tree in one of the sites showed a significant reduction of bacterial titers in leaf tissue. OTC treatment did not significantly improve yield. Canopy color showed noticeable improvement after the second OTC injection in the sites, whereas canopy density remained largely unaffected. After the second OTC injection, HLB severity and pre-harvest fruit drop were reduced, and improvement in fruit size was observed in 9-year-old trees. There is no significant improvement in juice quality. Damage caused by trunk injections healed rapidly in older trees than in younger trees. No OTC residue was detected in the juice and whole fruit at six months after the OTC injection. This is the first report on the efficacy of OTC for HLB management in Rio Red grapefruit in South Texas.

Huanglongbing (HLB, aka citrus greening) has devastated citrus production around the world, especially in Florida where the disease has been endemic since 2013. HLB is associated with phloem-limited bacteria, Candidatus Liberibacter asiaticus, vectored by the Asian citrus psyllid, Diaphorina citri. Elimination of the disease vector is difficult because of its abundance and the high costs of frequent insecticide applications. Foliar applied therapies to treat infected trees are largely ineffective because of the systemic distribution of the bacteria. Trunk injection of oxytetracycline (OTC) has emerged as a possible therapy to systemically deliver and target the pathogen and restore tree health. Since the registration of trunk injection of OTC for commercial use in Florida, our lab has conducted numerous field studies to assess the efficacy of this therapy on a large scale and study the influence of various factors such as tree age, rootstock, OTC dose, and time and method of application. Overall, all studies revealed significant improvements in fruit production along with increases in fruit and juice quality after one year of application. Yield improvements after two years of application were cumulative, reaching up to two-fold or more. The magnitude of improvements depended on various factors. Young trees generally responded better than older trees, and tree responses varied based on the rootstock. The efficacy of the therapy also depended on the dose of OTC administered to the trees, with higher doses generally generating larger effects. Moreover, delivering OTC on multiple sides of a tree instead of on only one side enhanced its distribution and therefore, efficacy. Late season injections improved the juice quality more but increased the OTC residue content in the fruits. The currently allowed residue level is 10 ppb, which was not exceeded when injections occurred in spring or early summer. OTC is phytotoxic and can have adverse effects on the tree. Efforts are underway to reduce or eliminate these negative effects and improve efficacy. Alternative therapies to be used instead of, or in combination with OTC, are also under evaluation. Taken together, the systemic delivery of OTC by trunk injection has emerged as a viable and economically feasible therapy to manage HLB in Florida and has been widely adopted. Nevertheless, it is regarded as a short-term solution until other solutions become available.

In Florida and worldwide, citrus production is threatened by Huanglongbing (HLB), associated with phloem-limited Candidatus Liberibacter asiaticus (CLas). Oxytetracycline delivery by trunk injection has been approved to treat commercial citrus groves affected by this endemic disease. However, the commercially available formulations require acidification of the active ingredient (oxytetracycline hydrochloride, OTC) to a pH of 1.8-2.0 to dissolve and stabilize the antibiotic, which may cause trunk damage, chloride toxicity, and limits compatibility with other compounds. A novel adjuvant has been developed, using EPA-approved ingredients to dissolve and stabilize OTC at a neutral pH while maintaining efficacy. This study compares the efficacy of neutralized and acidified solutions of OTC with and without the addition of ZnSO4 to optimize HLB management. The efficacy of Streptomycin (STM) as an alternative to or combined with OTC was also assessed. A field trial was conducted in a commercial citrus orchard in southwest Florida using seven-year-old ‘Valencia’ sweet orange trees grafted on X-639 rootstock. The experimental design was a randomized block with ten treatments and eight replications, each containing four trees. Treatments included combinations of OTC, STM, and ZnSO4 with or without the adjuvant, and appropriate controls. Trees were injected in June 2024 with 100 mL of treatment solution on one side of the trunk using commercial (FlexInject, TJ BioTech) injectors. Leaves were collected to assess CLas titers and bacterial suppression. Phytotoxic effects in the canopy and trunk damage at the injection site were evaluated. Treatments containing the adjuvant and/or OTC caused significant phytotoxicity and more trunk damage. Trees were harvested in March 2025. Fruit yield was higher for all treatments containing OTC compared to the controls . The highest yield was measured when trees were injected with a combination of OTC and STM. The findings of this study led to the redesign of the neutral pH adjuvant to minimize tree damage and enhance OTC efficacy. The optimized formulation will be tested using the same experimental design in upcoming studies.