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Article

Effectiveness of Hexanoic Acid for the Management of Bacterial Spot on Tomato Caused by Xanthomonas perforans

by
Ketsira Pierre
1,
Naweena Thapa
2,
Qingchun Liu
1,
Mustafa Ojonuba Jibrin
1,
Jeffrey B. Jones
2 and
Shouan Zhang
1,*
1
Tropical Research and Education Center, Plant Pathology Department, University of Florida, Homestead, FL 33031, USA
2
Plant Pathology Department, University of Florida, Gainesville, FL 32611, USA
*
Author to whom correspondence should be addressed.
Agriculture 2025, 15(7), 695; https://doi.org/10.3390/agriculture15070695
Submission received: 3 March 2025 / Revised: 23 March 2025 / Accepted: 24 March 2025 / Published: 25 March 2025
(This article belongs to the Section Crop Protection, Diseases, Pests and Weeds)
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Abstract

:
Bacterial spot of tomato (BST), caused by Xanthomonas euvesicatoria pv. perforans (referred to as X. perforans thereafter), is widely distributed globally, including Florida, and reduces fruit quality and yield in tomato fields. Currently, copper-based bactericides are widely used for this disease control; however, the effectiveness of these treatments has diminished due to the emergence of copper-tolerant strains. Therefore, there is a need for novel chemical controls against BST. In this study, we investigated hexanoic acid (HA) as an alternative against copper-tolerant strains of X. perforans through laboratory, greenhouse, and field experiments. In vitro experiments demonstrated HA had a lower minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) compared to copper sulfate, with values of 512 and 1024 mg/L for HA versus 1024 and 2048 mg/L for copper sulfate. HA exhibited bactericidal activity within 1 h at 512 and 1024 mg/L. In greenhouse trials, HA applied at 512 and 1024 mg/L two days before inoculation significantly reduced disease severity compared to untreated controls and Kocide 3000 (copper hydroxide) + Penncozeb. However, field trials indicated that while HA reduced disease severity relative to the untreated control, it did not outperform the grower standard commercial bactericide ManKocide (copper hydroxide + mancozeb), nor did it improve total yield. Previous studies have shown the antimicrobial activity of HA against various other phytopathogens, but this study is the first to demonstrate the potential of hexanoic acid for controlling BST.

1. Introduction

In Florida, fresh market tomatoes are a valuable vegetable crop accounting for about USD 322.5 million annually [1]. Unfortunately, tomato production has seen a steady decline since the 2000s due to multiple factors, including the disease bacterial spot of tomato (BST) [2]. BST was first reported in South Africa by Ethel Doidge in 1921, who described the disease as a canker on tomato fruits [3]. Since then, BST has become one of the most harmful tomato diseases and is present worldwide [4,5]. In Florida, BST is almost exclusively caused by the bacterial phytopathogen X. euvesicatoria pv. perforans (referred to as X. perforans in this paper) [6,7,8,9]. Infected leaves are initially characterized by circular water-soaked lesions that turn dark brown to black and are often accompanied by a chlorotic halo [4,6,7,10] (Figure 1A). Eventually, lesions coalesce and result in yellowing of leaves (Figure 1B), which in turn leads to defoliation, significantly impacting yield and fruit quality [11] (Figure 1C,D). Therefore, effective management of this disease is critical to Florida’s tomato production.
Currently, tomato growers mainly rely on copper-based bactericides as chemical controls against X. perforans. As it stands, 99.8% of X. perforans strains isolated from tomato fields in Florida have developed copper tolerance [9]. Therefore, growers have to apply copper-based bactericides more frequently to manage this disease, but this does not guarantee improved efficacy and results in increased costs for BST management. The extensive use of copper-based bactericides has led to the establishment and spread of copper-resistant Xanthomonas strains [12,13] and the accumulation of copper metal in soils and groundwater, affecting the environment and plant health [14,15]. Additionally, X. perforans is a quickly evolving pathogen; therefore, breeding for disease resistance is difficult, and there are currently no commercially available resistant varieties [10]. To improve the efficacy of copper-based bactericides, growers usually tank mix with mancozeb, an ethylene bisdithiocarbamate (EBDC) fungicide, for enhanced control of copper bactericides [16,17,18,19]. However, it too has been experiencing diminished efficacy, and, more importantly, EBDC may be carcinogenic, thus facing further regulation on food crops in the future [20]. Therefore, there is a need for new effective chemical controls against X. perforans.
Recent studies have shown the potential of small molecule compounds as control agents against plant diseases caused by Xanthomonas spp. [18,21,22,23,24]. Small molecules are organic, non-peptide compounds of less than 1500 Daltons. They can be synthesized or derived from natural products [25]. In this study, we evaluated the efficacy of the small molecule, hexanoic acid (HA), for the control of copper-resistant strains of X. perforans. Although previous research has demonstrated the benefits of HA in mitigating disease caused by several plant pathogens in tomatoes, such as Pseudomonas syringae and Botrytis cinerea, when applied as a priming agent, it has not been explored for managing BST disease. Our study is the first to investigate using HA for managing BST in both greenhouse and field conditions [26,27,28,29]. HA is a natural short monocarboxylic acid present in strawberries, butter, and cheeses, which has been reported to promote plant defense response by inducing callose deposition and activating the salicylic acid and jasmonic acid pathways to induce resistance in the plant [26,27,28,29,30,31]. HA can also prime redox-related genes as well as pathogen-specific responses [31,32]. Based on these characteristics, we hypothesized that this small molecule could be an effective strategy for the control of BST. The aim of this study was to evaluate the in vitro effect of HA against X. perforans and its efficacy in managing BST in the greenhouse and field.

2. Materials and Methods

2.1. Chemicals

Hexanoic acid (HA) (≥99%, Sigma Aldrich, St. Louis, MO, USA) was used to test for its effects on the management of BST. Stock solutions of HA (1.0 × 104 mg/L) were prepared in sterile distilled water (SDW) and stored at 4 °C for further dilutions. For in vitro assays, copper stock solutions (2.0 × 101 mg/L) were prepared using copper (II) sulfate pentahydrate (CuSO4·5H2O) (Fisher Scientific, Hampton, NH, USA). For greenhouse experiments, Kocide 3000 (DuPont, Wilmington, DE, USA) containing 30% metallic Cu in the form of copper hydroxide [Cu(OH)2] was used as the copper treatment. Kocide 3000 at the label rate of 2.1 g/L and Penncozeb 75DF (United Phosphorus Inc., King of Prussia, PA, USA) at the rate of 1.2 g/L in SDW were prepared immediately before use. For field trials, ManKocide—with active ingredients consisting of 30% Cu present as Cu (OH)2 and 15% mancozeb (Valent BioSciences Corp., Long Grove, IL, USA)—was applied at 2.1 g/L as the copper bactericide treatment.

2.2. Preparation of X. perforans Bacterial Suspension

GEV 485, a copper-resistant strain of X. perforans isolated from infected tomato plants in Florida and obtained from the Jones Lab storage collection, was used to evaluate the antibacterial activity of HA in vitro and in greenhouse experiments. For field trials, QL, a copper-resistant strain of X. perforans isolated from tomato plants in Homestead, FL, was used. Bacterial strains stored at −80 °C in cryovials were streaked for individual colonies on nutrient agar (NA; Difco™ Sparks, MD, USA) and incubated for 48 h at 28 °C. Single colonies were streaked onto fresh NA plates to ensure bacterial purity. To activate the expression of copper resistance in GEV 485, the bacteria were streaked on NA amended with 20 ug/mL of copper by adding CuSO4·5H2O (Cu-NA plates) and incubated for 24 h at 28 °C. Loopfuls of bacterial cells were removed from the Cu-NA plate surface and suspended in a sterile 0.01 M MgSO4·7H2O solution. The bacterial concentration was adjusted to ~5.0 × 108 colony-forming units (CFUs)/mL based on an OD600 = 0.3.

2.3. In Vitro Assays

2.3.1. Determination of the MIC and MBC of Hexanoic Acid and Copper

The minimum inhibitory concentration (MIC) of HA and copper (CuSO4·5H2O) against GEV 485 was determined using a 96-well plate containing 100 μL of nutrient broth (NB) with individual wells containing copper or HA at various concentrations. Each well was inoculated with 100 μL of the GEV 485 suspension at 5.0 × 108 CFU/mL. The final concentrations in the wells were 16, 32, 64, 128, 256, 512, 1024, 2048, 4096, and 8192 mg/L for both HA and copper based on preliminary testing. Three wells, each with 100 μL of NB and 100 μL of the bacterial suspension, were used as the controls. Suspensions in all wells were thoroughly mixed by pipetting up and down. The plate was then covered with a lid and incubated under stationary conditions at 28 °C for 24 h. The MICs for HA and copper treatments were determined by observing the wells for bacterial growth, indicated by a cloudy appearance in the well, and the minimum concentration (mg/L) at which no bacterial growth was observed, indicated by a clear appearance in the well, was identified as the MIC. An aliquot of 20 μL of the suspension from the wells that gave a clear solution was pipetted on NA plates. The plates were incubated overnight at 28 °C to determine the minimum bactericidal concentration (MBC). The MBC is determined as the minimum concentration with no visible growth of the bacteria on the NA plates. The experiment was repeated two more times, with three replications for each treatment.

2.3.2. Antibacterial Activity of Hexanoic Acid Against the Copper-Resistant Strain of X. perforans GEV 485

To evaluate the antibacterial activity of HA over time, GEV 485 bacterial suspension was prepared as previously described. The final concentration of the suspension was adjusted to 105 CFU/mL. Then, 20 μL of this suspension was added to tubes containing 2 mL of HA at concentrations of 32, 64, 128, 256, 512, and 1024 mg/L based on the MIC and MBC assay. Additionally, three tubes containing 2 mL of sterile 0.01 M MgSO4·7H2O solution and 20 μL of the bacterial suspension served as the controls. All tubes were incubated at 28 °C on an orbital shaker at 150 rpm for 15 min, 1 h, 4 h, 8 h, and 24 h. At each time point, 50 μL of the suspension from each tube was plated on NA plates and incubated at 28 °C for 48 h. After incubation, the colonies from 50 μL of the suspension were counted, and total viable colonies in 1 mL of the bacterial suspension were calculated. Each treatment consisted of three replicates, and the experiment was repeated two times.

2.4. In Planta Assays

2.4.1. Effect of Hexanoic Acid on the Bacterial Spot of Tomato in the Greenhouse

For the greenhouse trial, treatments included HA at 70, 512, and 1024 mg/L (based on the in vitro results), Kocide 3000 at 2.1 g/L, Kocide 3000 (2.1 g/L) + Pencozeb at 1.2 g/L, and the untreated control (sterile tap water). Previous studies have shown that soil drenching with HA provides improved efficacy and prolonged disease control compared to foliar application [33,34]. Therefore, HA treatments were applied via soil drench, while all other treatments were applied to plants using standard plastic spray bottles with adjustable nozzles. Tomato (cv. Bonnie Best) plants were grown in seedling trays for two weeks and then transplanted to larger pots (15 cm), at which point plants were in the seedling stage. After 1–2 additional weeks of growth, the plants were then moved to a growth chamber and incubated at 28 °C, with a 12 h photoperiod and 80% relative humidity. Then, 50 mL of each rate of HA solution was applied to 3–4-week-old plants in the leaf development stage with a fourth pair of true leaves visible by drenching the soil 4 or 2 days prior to bacterial inoculation to prime the plants to induce their basal plant defense. Kocide 3000 and the mixture of Kocide 3000 + Penncozeb were sprayed on the tomato plants until runoff, and all plants were allowed to air dry for 4 h before bacterial inoculation. Three plants were drenched with SDW and served as the untreated control. The GEV-485 bacterial suspension (5.0 × 108 CFU/mL) was sprayed onto both sides of the leaves using a plastic spray bottle, and then plants were covered with plastic bags to ensure high humidity. Plastic bags were carefully removed two days post-inoculation, and the plants remained in the growth chamber for 24 h before they were moved to the greenhouse. In the greenhouse, the plants were exposed to the natural photoperiod and were irrigated daily, avoiding wetting the foliage. Disease severity and phytotoxicity (damage to plants caused by the chemical treatment) were evaluated every other day using the Horsfall–Barratt scale [35], starting from three days up to fifteen days post-inoculation. The area under the disease progress curve (AUDPC) was calculated based on disease severity rated during the experimental period. Three experiments were performed, and each treatment consisted of three replicates.

2.4.2. Effect of Hexanoic Acid on the Development of Tomato Bacterial Spot and Yield in the Field

Two field trials were conducted in calcareous soils to evaluate the efficacy of HA against BST in fall 2020 and spring 2021 at the University of Florida Tropical Research and Education Center in Homestead, Florida. The soil is a typical gravelly soil containing 33% soil and 67% pebbles (>2 mm) with 1.5% organic matter and a pH of 7.8. The geographic coordinates of the site ranged from 025°30′43″ to 025°30′51″ north latitude and 080°30′02″ to 080°30′03″ west longitude. Daily weather data (temperature, relative humidity, and rainfall) in Homestead was obtained from the Florida automated weather network (FAWN) database [36]. From fall 2020 to spring 2021, temperatures in Homestead ranged from 33.14 to 3.01 °C, average relative humidity was between 63 and 89%, and cumulative rainfall ranged from 0.23 to 13.65 inches [36]. In both field trials, the tomato cultivar Red Bounty was used because of its resistance to tomato chlorotic spot virus (TCSV), a predominant virus on tomatoes in South Florida [37]. A randomized block (RB) design was used for the field experiments with three replicates per treatment. The beds were 0.9 m wide and spaced 1.8 m apart. Four-week-old tomato seedlings were transplanted into raised beds covered with black polyethylene mulch at 0.6 m spacing in a single bed of 6.0 m in length for each plot. Each plot contained nine plants with 1.2 m between adjacent plots. The plots were maintained using commercial tomato production practices including regular irrigation, scheduled fertilizer injections, and other plant management practices throughout the field trials. Field treatments included HA at 500 mg/L (based on greenhouse data), ManKocide at 2.1 mg/L, or tap water for the untreated control, with all treatments applied once a week for eight weeks. About two weeks after transplanting, during which plants were in the leaf development stage, HA treatments were applied via soil drench (50 mL/plant) at the base of tomato plants. ManKocide and tap water were applied foliarly. All treatments were applied using Chapin manual pump backpack sprayers (4-gallon tank, 0.5 GPM flow rate, adjustable spray pattern, 20-inch wand, 48-inch hose), which were pressurized to 40–60 psi and calibrated for uniform application to plant surfaces. After the third foliar application, a bacterial suspension (108 CFU/mL) of X. perforans strain QL (copper-resistant strain isolated from tomato plants in Homestead, FL, USA) was applied to each plant until runoff using the before mentioned Chapin backpack sprayer. Once BST symptoms appeared (approx. 7–10 days after inoculation), bacterial spot disease severity was rated weekly in each plot as the percentage of symptomatic leaf tissue, and AUDPC was calculated based on disease severity rated during the experimental period. Tomato fruits were harvested twice from each trial and graded according to the USDA standard [38].

2.5. Statistical Analysis

Statistical analysis for in vitro data was performed using IBM SPSS Statistics software (version 22; Armonk, NY, USA) [39]. The data were analyzed by analysis of variance (ANOVA) for significant differences between the treatments, followed by pairwise comparison by the post hoc test using the Student–Newman–Keuls (SNK) method at p = 0.05. Data from the greenhouse experiments were analyzed for statistical significance using SAS (version 9.4, SAS Institute Inc., Cary, NC, USA) [40] in which a linear mixed model (PROC GLIMMIX) was used, with the fixed effects being the treatment rate combination, time, and their interaction with the experiment being treated as a random effect. An AR(1), an autoregressive order 1 correlation structure, was also included to count for repeated measures over the different time points. Tukey’s HSD post hoc test was used for pairwise comparison at p = 0.05. For field trial data, ANOVA was conducted, and comparisons were completed with Fisher’s Least Significant Difference (LSD) post hoc test in SAS (version 9.4, SAS Institute Inc., Cary, NC, USA) [40]. The significance level was set to 0.05.

3. Results

3.1. Determination of the MIC and MBC of Hexanoic Acid and Copper

A two-fold serial dilution of HA and copper was performed to achieve concentrations from 8192 to 16 mg/L. The MICs of hexanoic acid (HA) and copper (CuSO4) were 512 mg/L and 1024 mg/L, respectively (Table 1). HA at 16–256 mg/L and copper at 16–512 mg/L had no effects on bacterial growth. However, after plating on NA, copper at 2048 mg/L showed bactericidal effects, and the MBC of HA was determined as 1024 mg/L against the copper-resistant X. perforans strain GEV-485. These findings indicate that HA is more effective at lower concentrations than copper in vitro.

3.2. Antibacterial Activity of Hexanoic Acid Against the Copper-Resistant Strain of X. perforans GEV 485

Hexanoic acid (HA) at all concentrations except 32 mg/L showed antibacterial activity against the copper-resistant X. perforans strain GEV-485 at 24 h (p < 0.0001) (Figure 2). When GEV 485 was exposed to HA at higher concentrations, such as 1024 and 512 mg/L, bactericidal activity was observed within 1 h after exposure. At 256 mg/L, there was no bacterial growth after 4 h of exposure. HA at 128 mg/L exhibited bactericidal effects at 24 h; however, it was able to reduce the bacterial population by ~approx. 50% within 4 h. On the other hand, HA at lower concentrations of 32 and 64 mg/L did not inhibit bacterial growth at any time point, although HA at 64 mg/L did significantly reduce the bacterial population after 24 h of exposure (p < 0.0001). The control with the 0.01 M MgSO4·7H2O solution showed no effects on the bacterial population.

3.3. Effect of Hexanoic Acid on the Bacterial Spot of Tomato in the Greenhouse

In planta testing of hexanoic acid (HA) demonstrated its effectiveness in reducing bacterial spot disease, as indicated by the area under the disease progress curve (AUDPC) and disease severity (DS) (p = 0.0003 and 0.003, respectively) (Figure 3). HA at 512 and 1024 mg/L applied 2 days before inoculation (dbi) with X. perforans resulted in significantly lower DS compared to the untreated control and Kocide + Penncozeb. Copper treatments Kocide 3000 and Kocide + Penncozeb failed to significantly reduce the disease severity of bacterial spot. HA at 70, 512, and 1024 mg/L applied 2 dbi and HA at 512 mg/L applied 4 dbi, significantly reducing AUDPC compared to the untreated control (p ≤ 0.0013). None of the HA treatments showed a statistically significant difference in AUDPC compared to the copper treatments, though some HA treatments (such as HA applied at 2 dbi) reduced AUDPC by more than 50%. The highest levels of disease were observed in Kocide 3000, Kocide 3000+ Penncozeb, and the untreated controls. No phytotoxicity was detected in any treatments; the plants showed no necrosis or chlorosis associated with phytotoxicity following chemical treatments with HA and Kocide.

3.4. Effect of Hexanoic Acid on the Development of Tomato Bacterial Spot and Yield in the Field

In field trials conducted during 2020–2021, both HA at 0.5 g/L and ManKocide at 2.1 g/L led to significantly lower disease compared to the untreated control (Table 2). ManKocide applied at the label rate of 2.1 g/L provided the best overall control of the bacterial spot of tomato. In field trial 1, HA significantly reduced DS and suppressed disease progress compared to the untreated control but was less effective than ManKocide. HA had no effect on fruit yield, whereas ManKocide significantly increased fruit yield compared to both the untreated control and HA in field trial 1. In field trial 2, there was no significant difference between HA and ManKocide; however, both treatments significantly reduced DS compared to the untreated control. In both trials, HA and ManKocide successfully reduced disease progress, although ManKocide resulted in a lower AUDPC than HA. No significant effects on tomato fruit yield were observed from any treatments in field trial 2.

4. Discussion

Hexanoic acid (HA) was previously tested against several plant pathogens, including B. cinerea [26,32,33], P. syringae [41], Alternaria alternata [28,29], and X. citri [34]. These studies demonstrated HA’s ability to trigger defensive responses in plants, resulting in reduced disease. HA has the capability for broad-spectrum applications, as it has been shown to be effective against bacterial and fungal plant pathogens without causing phytotoxicity in crops. In our study, HA was able to reduce and inhibit X. perforans growth in a concentration-dependent manner. In vitro, HA at higher concentrations (512–1024 mg/L) exhibited bactericidal effects within 1 h of exposure, while at lower concentrations (64–256 mg/L), it maintained or reduced bacterial populations.
Our in vitro screenings revealed that HA was effective in eradicating X. perforans at half the concentration required for copper sulfate. This is noteworthy because while copper is widely used in agriculture, overreliance on this chemical has led to the development of copper resistance to pathogens and raised concerns about environmental impacts [7,14,42]. Although copper is a metal that can be reused indefinitely, it does not degrade in soils and can accumulate in areas with high copper usage, such as agricultural fields and groves [4,43,44,45]. This is particularly concerning in South Florida, where much of the state’s economically important agricultural activity occurs. HA, on the other hand, has the potential to provide a more sustainable alternative for managing bacterial spot in tomato production because it is readily biodegradable and has low bioaccumulation potential [46]. Additionally, a study has also reported that HA does not accumulate in the aerial part of the tomato plants but can still trigger the plant’s defense mechanisms, which then fight off pathogens, such as B. cinerea [27]. While caution is still necessary to prevent its potential adverse effects on the environment and non-target organisms until further work is performed to determine if HA is safe to use as a chemical control strategy, it remains an attractive alternative to conventional commercial active ingredients, like copper. This study did not evaluate the effects of HA on beneficial bacteria; however, its low toxicity, relatively low concentration for reducing disease, and biodegradability suggest it is likely safe for beneficial soil microbes, although further research is needed to confirm this.
Previous studies have also demonstrated the improved efficacy of HA when it was applied as a soil drench compared to foliar application [33,34]. In a study comparing the efficacy of HA applied via foliar spray versus soil drenching on nine-month-old ‘Pineapple’ sweet orange plants infected with X. citri, soil drenching consistently resulted in lower bacterial populations and better control of the disease [34]. Additionally, soil drenching with HA demonstrated prolonged disease control compared to foliar application [34]. This was because callose deposition was enhanced in drench-treated plants, acting as a physical barrier against invading pathogens, thus improving plant resistance [34,47]. Although this study did not evaluate the mode of action of HA against X. perforans in tomato, existing research suggests that HA consistently involves enhancing the jasmonic acid (JA) signaling and abscisic acid-mediated callose deposition, both of which are critical plant hormones responsible for responding to environmental stresses and stimuli [27,33,48]. In addition, HA induces callose accumulation in sweet orange, which is necessary for a cell wall-associated defense against pathogens, such as Xanthomonas citri [34]. Furthermore, HA triggers the rapid accumulation of reactive oxygen species (ROS), which are crucial for hypersensitive defense response, causing localized cell death in infected plant tissue to limit the spread of pathogens [31].
The results from our greenhouse studies further support soil drenching with HA, as it effectively reduced BST. Application of HA both at 2 and 4 days prior to inoculation reduced disease more than both Kocide 3000 and Kocide 3000 + Penncozeb, which are standard treatments for BST control. This is important given that copper hydroxide, the active ingredient in many commercial copper-based bactericides, has been reported to have reduced or no efficacy after its over 60 years of use against X. perforans [49]. Considering this, it is worth noting that other studies have reported the improved control of BST when small molecules such as NAC, IAN, carvacrol, piperidine, and pyrrolidine were applied in combination with copper-based bactericides Kocide and ManKocide [21,22,23,24]. Therefore, further studies are needed to assess the combined benefits, if any, of HA with copper-based bactericides.
Additionally, HA at 512 mg/L applied 4 dbi and significantly reduced AUDPC compared to the untreated control, although HA at 1024 mg/L did not. While we cannot provide a definitive answer, it is possible that treatment at higher concentrations may not always lead to better results. For instance, HA at 1024 mg/L 4 dbi might have been applied too early because the same treatment of 2 dbi significantly reduced disease progress compared to the control. Studies have demonstrated that chemical treatments can be effective at lower rates, but higher rates can result in diminished effectiveness or more disease [50,51]. Phytotoxicity is a common, though not universal, indicator of chemical treatments causing stress or damage to plants [52]. Therefore, it is crucial to determine the optimal concentration for sustainable disease management while minimizing potential harm to the plant.
Although numerous greenhouse studies have evaluated HA regarding its bactericidal activity [26,29,30,31,33,41], there is limited information on its performance in field conditions. Our field trials conducted during 2020–2021 showed that both HA and ManKocide significantly reduced BST compared to the untreated control. Although HA effectively lowered disease relative to the untreated control, it did not provide more effective control than ManKocide. While this result is not uncommon when testing new chemicals under field conditions, it is noteworthy that the strain of X. perforans (QL) used in the field differed from that used in in vitro and greenhouse studies (GEV 485). This, coupled with the differences in greenhouse and field environmental conditions, may have contributed to the varied efficacy of HA over copper-based bactericides in greenhouse vs. field conditions. Greenhouse trials evaluate chemicals in planta under controlled conditions to minimize the effects of confounding variables, such as environmental stress, which may have contributed to HA, showing better efficacy compared to Kocide. However, under field conditions, which more accurately reflect real-world scenarios, other factors such as weather, plant stress, soil composition, microbial diversity, and more can influence plant health and overall chemical performance, leading to HA’s reduced efficacy compared to Mankocide.
In field trial 1, ManKocide not only improved yield compared to HA and the untreated control but also demonstrated superior efficacy in disease management. However, in field trial 2, no significant differences in yield were detected among treatments, likely due to the higher disease pressure, which may have limited their effectiveness. This observation aligns with reports indicating that environmental conditions conducive to disease, such as elevated temperature and humidity, along with prolonged leaf wetness, can result in more severe disease and over 50% yield loss, even with current BST management practices [21]. The disease pressure in field trial 2, more than double that in trial 1, highlights the limitations of these chemicals under conditions favorable to disease development. It also emphasizes the importance of considering both disease control and yield when evaluating new treatment options.
While this study demonstrated the potential of HA for management BST, it also revealed limitations that must be considered in future work. Notably, field trials showed that while HA did reduce disease, it did not outperform ManKocide. ManKocide is an established formula with several ingredients. As HA demonstrated strong in vitro activities against X. perforans, future studies need to be conducted to investigate if HA applied as a soil drench in combination with foliar spray can improve disease control against BST. In addition, more research should investigate strategies to enhance the efficacy of chemical treatments under high disease pressure, considering factors such as environmental conditions, timing of treatments, weather forecasts, and chemical stability. Additionally, although HA is biodegradable, this study did not evaluate its impact on beneficial soil microbes, non-target organisms, or its broader ecological effects. Moving forward, future research should address these factors, especially if HA is to be considered a more environmentally friendly alternative to copper applications. Integrating HA into existing disease management programs could provide growers with an alternative option to the declining efficacy of copper-based bactericides. Although HA is considered affordable to produce, economic considerations, including the cost of production and applications, should be evaluated to determine its practicability for adoption in large-scale commercial tomato production. This comprehensive approach could better maintain or improve yield, thereby helping growers avoid financial losses due to plant disease.

5. Conclusions

This study evaluated hexanoic acid (HA) as a novel potential chemical against X. perforans, the pathogen causing bacterial spot on tomatoes. HA showed bactericidal effects in vitro and reduced disease in planta under both greenhouse and field conditions. While HA offers improvements over Kocide 3000 in the greenhouse, it does not yet match the effectiveness of established treatments, such as the copper bactericide ManKocide, in the field. Future research should focus on the improvement of its efficacy in the field through refining application techniques, exploring interactions with different pathogen strains, and investigating combinations with other treatments, providing more robust solutions for the management of BST and diseases caused by Xanthomonas spp. or closely related bacterial pathogens on other crops. With further research and refinement, we anticipate that HA could be an effective and sustainable ingredient for managing bacterial spot disease in tomato crops and beyond.

Author Contributions

Conceptualization, M.O.J., S.Z. and J.B.J.; methodology, N.T. and Q.L.; investigation, N.T. and Q.L.; validation, M.O.J., S.Z. and J.B.J.; formal analysis, K.P., N.T. and Q.L.; writing—original draft preparation, K.P. and N.T.; writing—review and editing, K.P., S.Z. and J.B.J.; visualization, K.P., N.T. and Q.L.; supervision, S.Z. and J.B.J. All authors have read and agreed to the published version of the manuscript.

Funding

This study was funded by the United States Department of Agriculture’s (USDA) Agricultural Marketing Service through the Florida Department of Agriculture and Consumer Services Specialty Crop Block Grant project (Award No. AM180100XXXXG046).

Institutional Review Board Statement

Not applicable.

Data Availability Statement

This manuscript includes all data produced or analyzed during this project.

Acknowledgments

The authors thank Yuanyuan Wang and the field crew at the UF/IFAS Tropical Research and Education Center for their assistance with the field trials. Special thanks go to Renato Carvalho for his assistance with the in vitro assays and James Colee for his support with the statistical analysis. We would also like to acknowledge the contributions of other Zhang and Jones lab members from 2020 to 2024 for their encouragement and support.

Conflicts of Interest

The authors declare no conflicts of interest.

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Agriculture 15 00695 g001
Figure 1. Disease symptoms of bacterial spot on tomatoes: (A) circular lesions surrounded by a chlorotic halo on the leaflet, (B) lesions merging and yellowing of the leaflet, (C) lesions on tomato fruit in fields, and (D) defoliation and yield loss due to bacterial spot of tomato. Photos by K. Pierre and N. Thapa.
Figure 1. Disease symptoms of bacterial spot on tomatoes: (A) circular lesions surrounded by a chlorotic halo on the leaflet, (B) lesions merging and yellowing of the leaflet, (C) lesions on tomato fruit in fields, and (D) defoliation and yield loss due to bacterial spot of tomato. Photos by K. Pierre and N. Thapa.
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Figure 2. In vitro activity of hexanoic acid against X. perforans at 15 min, 1 h, 4 h, 8 h, and 24 h. The experiment consisted of three replicates per treatment. The treatments included hexanoic acid at 32, 64, 128, 256, 512, and 1024 mg/L, and the 0.01 M MgSO4·7H2O solution served as the control. Error bars represent standard deviation. Based on ANOVA followed by the SNK post hoc test, different letters above the bars (a, b, c, d, and e) indicate statistical significance between treatments (p < 0.05), whereas the same letter above the bars represents no statistical difference. In vitro experiments were conducted twice.
Figure 2. In vitro activity of hexanoic acid against X. perforans at 15 min, 1 h, 4 h, 8 h, and 24 h. The experiment consisted of three replicates per treatment. The treatments included hexanoic acid at 32, 64, 128, 256, 512, and 1024 mg/L, and the 0.01 M MgSO4·7H2O solution served as the control. Error bars represent standard deviation. Based on ANOVA followed by the SNK post hoc test, different letters above the bars (a, b, c, d, and e) indicate statistical significance between treatments (p < 0.05), whereas the same letter above the bars represents no statistical difference. In vitro experiments were conducted twice.
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Figure 3. Effect of hexanoic acid and copper-based bactericides on the development of tomato bacterial spot in planta in greenhouse experiments. Hexanoic acid treatments included 70, 512, and 1024 mg/L either 2 days or 4 days before inoculation (dbi), and Kocide 3000 and Kocide 3000 + Pencozeb were included as copper controls. Sterile tap water was used for the untreated control. Error bars represent the standard deviation of three replicates. The area under the disease progress curve (AUDPC) was calculated using Horsfall–Barratt disease severity (DS) scale ratings collected every other day from the third day after inoculation until 15 days after inoculation. ANOVA followed by Tukey’s post hoc test was used to depict statistical significance (p < 0.05). Different letters above the bars indicate statistical significance between treatments, whereas the same letter above the bars represents no statistical difference. Lowercase letters (a, b) indicate significant differences in AUDPC, while capital letters (A, B) indicate statistical differences in final disease severity rating. The experiments were performed three times.
Figure 3. Effect of hexanoic acid and copper-based bactericides on the development of tomato bacterial spot in planta in greenhouse experiments. Hexanoic acid treatments included 70, 512, and 1024 mg/L either 2 days or 4 days before inoculation (dbi), and Kocide 3000 and Kocide 3000 + Pencozeb were included as copper controls. Sterile tap water was used for the untreated control. Error bars represent the standard deviation of three replicates. The area under the disease progress curve (AUDPC) was calculated using Horsfall–Barratt disease severity (DS) scale ratings collected every other day from the third day after inoculation until 15 days after inoculation. ANOVA followed by Tukey’s post hoc test was used to depict statistical significance (p < 0.05). Different letters above the bars indicate statistical significance between treatments, whereas the same letter above the bars represents no statistical difference. Lowercase letters (a, b) indicate significant differences in AUDPC, while capital letters (A, B) indicate statistical differences in final disease severity rating. The experiments were performed three times.
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Table 1. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of hexanoic acid (HA) and copper against the copper-resistant strain of X. perforans GEV 485.
Table 1. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of hexanoic acid (HA) and copper against the copper-resistant strain of X. perforans GEV 485.
Hexanoic Acid and Copper Concentrations (mg/L) a
Treatment1632641282565121024204840968192
Hexanoic Acid (HA)+++++MICMBC---
Copper (CuSO4)++++++MICMBC--
The MIC and MBC for HA were 512 and 1024 mg/L, respectively, and the MIC and MBC for copper were 1024 and 2048 mg/L, respectively. a A two-fold serial dilution was performed to achieve the concentrations of 8192—16 mg/L; ‘+’ indicates that the growth of bacteria was observed, while ‘-’ indicates that no bacteria growth was observed.
Table 2. Effect of hexanoic acid on the development of tomato bacterial spot and yield in the field.
Table 2. Effect of hexanoic acid on the development of tomato bacterial spot and yield in the field.
Trial 1 aTrial 2 a
TreatmentDS b (%)AUDPC cYield d (kg/plant)DS b (%)AUDPC cYield d (kg/plant)
Untreated control19.0 a212.6 a6.93 b49.4 a560.3 a2.85 a
Hexanoic acid 0.5 g/L13.1 b122.0 b7.07 b44.2 b441.0 b2.72 a
ManKocide 2.1 g/L10.8 c95.9 c8.83 a41.3 b390.1 c2.76 a
LSD0.051.9126.41.345.048.70.54
a Tomato cultivar used in both trials = red bounty. b DS = final rating for disease severity, the percentage of bacterial spot lesions on leaves of the entire plant. c The area under the disease progress curve (AUDPC) was calculated based on disease severity ratings throughout the experiment. d Total yield of harvested tomatoes from each field trial based on the USDA standard. Statistical analysis was performed in SAS (version 9.4) using ANOVA and an LSD post hoc test at p = 0.05. The different letters next to DS, AUDPC, and yield values indicate statistical significance between treatments, whereas the same letter represents no statistical difference.
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MDPI and ACS Style

Pierre, K.; Thapa, N.; Liu, Q.; Jibrin, M.O.; Jones, J.B.; Zhang, S. Effectiveness of Hexanoic Acid for the Management of Bacterial Spot on Tomato Caused by Xanthomonas perforans. Agriculture 2025, 15, 695. https://doi.org/10.3390/agriculture15070695

AMA Style

Pierre K, Thapa N, Liu Q, Jibrin MO, Jones JB, Zhang S. Effectiveness of Hexanoic Acid for the Management of Bacterial Spot on Tomato Caused by Xanthomonas perforans. Agriculture. 2025; 15(7):695. https://doi.org/10.3390/agriculture15070695

Chicago/Turabian Style

Pierre, Ketsira, Naweena Thapa, Qingchun Liu, Mustafa Ojonuba Jibrin, Jeffrey B. Jones, and Shouan Zhang. 2025. "Effectiveness of Hexanoic Acid for the Management of Bacterial Spot on Tomato Caused by Xanthomonas perforans" Agriculture 15, no. 7: 695. https://doi.org/10.3390/agriculture15070695

APA Style

Pierre, K., Thapa, N., Liu, Q., Jibrin, M. O., Jones, J. B., & Zhang, S. (2025). Effectiveness of Hexanoic Acid for the Management of Bacterial Spot on Tomato Caused by Xanthomonas perforans. Agriculture, 15(7), 695. https://doi.org/10.3390/agriculture15070695

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