Particle-size dependent bactericidal activity of magnesium oxide against Xanthomonas perforans and bacterial spot of tomato

Bacterial spot, caused by Xanthomonas spp., is a highly destructive disease of tomatoes worldwide. Copper (Cu) bactericides are often ineffective due to the presence of Cu-tolerant strains. Magnesium oxide (MgO) is an effective alternative to Cu bactericides against Xanthomonas spp. However, the effects of particle size on bactericidal activity and fruit elemental levels are unknown. In this study, nano (20 nm) and micron (0.3 and 0.6 µm) size MgO particles were compared for efficacy. Nano MgO had significantly greater in vitro bactericidal activity against Cu-tolerant X. perforans than micron MgO at 25–50 µg/ml. In field experiments nano and micron MgO applied at 200 and 1,000 µg/ml were evaluated for disease control. Nano MgO at 200 µg/ml was the only treatment that consistently reduced disease severity compared to the untreated control. Inductively Coupled Plasma Optical Emission Spectroscopy revealed that nano MgO applications did not significantly alter Mg, Cu, Ca, K, Mn, P and S accumulation compared to fruits from the untreated plots. We demonstrated that although both nano MgO and micron MgO had bactericidal activity against Cu-tolerant strains in vitro, only nano MgO was effective in bacterial spot disease management under field conditions.

Tomato (Solanum lycopersicum) is an economically important crop in the United States and worldwide. Just in 2017, the total tomatoes production amounted to 12.5 million metric tons in the United States. The value of this crop totaled $1.67 billion dollars 1 . Bacterial spot is one of the most damaging diseases that can cause major yield reductions in the tomato market around the world, especially in where the high humidity and temperatures create a favorable environment [2][3][4] . Bacterial spot disease of tomato is caused by four distinct Xanthomonas species 5 . In Florida, which is the largest fresh market tomato producer in the United States 1 , X. perforans is the dominant causal agent of bacterial spot of tomato. Although the disease has been around since its discovery in South Africa in 1914 6 , effective disease management strategies for bacterial spot are currently limited. Given that Florida's tomato production industry has a long history with bacterial spot disease, the pathogen has developed resistance toward bactericides including streptomycin 7,8 and copper (Cu) 2,9 . Cu-tolerant Xanthomonas strains were isolated in the 1960s, as grower's noticed the diminishing efficacy of Cu bactericides 9 . Subsequently it was found that addition of ethylene-bis-dithiocarbamates (EBDC) to Cu bactericides provided better disease control and improved Cu solubility 9,10 . Since Cu-tolerant Xanthomonas strains are sensitive to Cu-EBDC, this option remains the standard treatment for tomato producers in Florida and elsewhere. However, when environmental conditions are optimal for disease development, even Cu-EBDC is ineffective against bacterial spot disease of tomato 11,12 . Efforts to identify alternatives to Cu-EBDC have been extensive over the last two decades. For instance, bacteriophages have been extensively studied and are commercially available for managing bacterial spot disease [13][14][15][16] . However, bacteriophages are highly sensitive to environmental factors, which can decrease their Results Bactericidal activity of different particle size of MgO compared with Cu bactericide. The bactericidal activity was confirmed by the viability assay ( Fig. 1A-F). Bacterial mortality was nearly 100% (red fluorescence) after treatment with 100 µg/ml MgO (20 nm, 0.3 and 0.6 µm) (Fig. 1D-F) for 4 h similar to the heat treated positive control (Fig. 1B). In comparison, the Cu bactericide (Kocide 3000) (Fig. 1C) had 80.7% alive cells (green fluorescence), which was similar to the untreated control (80% alive cells) (Fig. 1A). effect of Mgo particle size on in vitro growth of X. perforans. Both nano and micron (20 nm, 0.3, and 0.6 µm) MgO had significant antimicrobial activity at 100 µg/ml against the Cu-tolerant strain, X. perforans GEV485 after 4 h ( Fig. 2A,B). The minimum bactericidal concentration (MBC) for nano MgO against Cu-tolerant X. perforans strain was 25-50 µg/ml, whereas the MBC for micron MgO (0.3 and 0.6 µm) was 100 µg/ ml ( Fig. 2A,B).
Comparison of field efficacy of MgO with Cu and Cu-EBDC for management of tomato bacterial spot. In the first field experiment, conducted during spring 2016 in Quincy, FL, plants that received either concentration of nano MgO (20 nm) (1,000 µg/ml or 200 µg/ml) had significantly less disease compared to the untreated control, but were not different from the other treatments (Table 1). Of the two larger size MgO particle sizes tested, only 0.3 µm MgO at 1,000 µg/ml significantly reduced disease severity compared to the untreated control. Both Cu bactericide and Cu-EBDC were not significantly different from the control. Treatment applications did not cause any phytotoxicity on the tomato plants (data not shown).
In the second trial during fall 2016 at Quincy, FL, (Table 1), both concentrations (1,000 and 200 µg/ml) of the nano-MgO (20 nm) significantly reduced disease severity compared to the untreated control, whereas the grower's standard Cu-EBDC did not significantly reduce disease in the field trials compared to the untreated control ( Table 1). Neither of the micron MgO (0.3 µm and 0.6 µm) treatments showed significant disease reduction compared to the untreated control in this trial (Table 1). No phytotoxicity was observed for any of the treatments in this experiment (data not shown). There were no significant impacts on total yield due to MgO treatments in both field trials ( Table 2). Accumulation of metals in harvested tomato fruits treated with MgO. In the spring 2016 trial (Table 3), there were no significant differences for any of the elements (Al, B, Ca, Cu, Fe, K, Mg, Mn, Mo, Na, P, S, and Zn) (Table S1) when comparing nano MgO (20 nm) treated fruit with the untreated controls. Similarly, fruit collected from micron MgO (0.3 µm and 0.6 µm) treated plots, at both concentrations of 0.3 µm MgO and 1,000 µg/ml of 0.6 µm MgO had no significant differences in elemental concentration compared to the untreated control. As for Cu bactericide (Kocide 3000) and the grower standard (Cu-EBDC), both treatments showed significantly higher Cu concentrations relative to the untreated control. The Cu bactericide treated fruits contain Cu that is twice as high (4.5 mg/kg higher) as the untreated fruit. For the fruit collected in fall 2016 trial (Tables 3), 1,000 µg/ml of 0.3 µm MgO treatment showed significantly higher Al content in the peel, with approximately 4 mg/kg more Al in dry weight compared to the untreated control (Table 3). However, Al did not significantly accumulate in either whole fruit or flesh for the 1,000 µg/ml of 0.3 µm MgO. Fruit receiving 1,000 µg/ml 0.3 µm MgO also contained significantly higher levels of Ca in the whole fruit (+8 mg/kg more in fresh weight) and peel (+0.043 mg/kg more in fresh weight) compared to the untreated control. The Cu-EBDC treatment showed significantly higher Ca content (+12.26 mg/kg more in fresh weight); values were at nearly two-fold of that accumulated in the untreated fruit (Table 3). Unlike the spring trial, Cu content of the fruit was not significantly impacted by the Kocide 3000 or Cu-EBDC treatment.

Discussion
In this study, we compared nano and micron size MgO for in vitro bactericidal activity to copper-tolerant X. perforans bacterial cells. We demonstrated in the viability assay that both nano (20 nm) and micron (0.3 µm and 0.6 µm) MgO at concentrations as low as 100 µg/ml had high bactericidal activity (100% percent reduction) after 4 h. In comparison, Cu bactericide (Kocide 3000) was similar (19.3% mortality) to the untreated control (20% mortality). These results indicate that both nano (20 nm) and micron (0.3 µm and 0.6 µm) MgO at 100 µg/ml were more effective against Cu-tolerant X. perforans compared to Cu bactericide. However, in the in vitro assay, the MBC of nano-MgO (20 nm) against Cu-tolerant X. perforans was 25-50 µg/ml, whereas the MBC of micron  93% to 97%, when the MgO nanoparticle size decreased from 69 nm to 26 nm. However, that report focused on the MgO nanoparticles less than 100 nm, whereas in this study, we focused on evaluating antibacterial activity of nano (20 nm) and micron MgO (0.3 µm and 0.6 µm) against Cu-tolerant X. perforans.
Since both nano and micron MgO showed bactericidal activity against Cu-tolerant X. perforans in vitro, we compared the effectiveness of these materials with Cu bactericide and grower standard Cu-EBDC in the field. According to the field experiments, only nano MgO (20 nm) as low at 200 µg/ml provided significant disease reduction consistently compared with the untreated control (P < 0.05) in both 2016 Spring and Fall field trials in Quincy, FL. In 2016 Fall field trial, nano MgO (20 nm) even provided greater disease control than the grower's standard Cu-EBDC (P < 0.05). Although both nano (20 nm) and micron (0.3 µm and 0.6 µm) MgO had antibacterial activity in vitro, the field trial experiments showed that only nano MgO (20 nm) could significantly reduce disease severity in the field.
In the past decade, nano MgO particles have been shown to have antimicrobial activity against several mammalian pathogens 51,52 . Additionally, MgO nanoparticles (~50 ± 10 nm) at concentrations as low as 100 μg/ml resulted in high inhibition rates of fungal spore germination of several fungal plant pathogens including Alternaria alternata, Fusarium oxysporum, Rhizopus stolonifer, and Mucor plumbeus 53 . Recently, a limited number of studies have explored utilizing Mg nanomaterials to manage bacterial pathogens 23,54 . Based on these studies, antibacterial mechanisms were proposed for MgO nanoparticle against bacterial cell at the nano-bio interface. Liao et al. used transmission electron microscopy (TEM) to show that MgO nanoparticles could cause membrane These findings demonstrate the potential of utilizing MgO nanoparticles to manage plant pathogens in agriculture systems. Furthermore, MgO is a more sustainable treatment option since, unlike Cu, it is not on the list of the EPA's Toxic Release Inventory (TRI) Program or in the Integrated Risk Information System 36 . Last but not least, by using MgO nanoparticles as alternatives to Cu bactericide would reduce the selective pressure on the developing Cu-tolerant X. perforans in the field.
The fate of engineered nanomaterials is a concern along with approaches involving material release into the environment. Due to the use of MgO in medical field such as cancer research, studies investigating the toxicity of MgO nanoparticles toward mammals have been conducted 55 The study showed that MgO nanoparticles (8 nm) did not cause significant membrane damage on Caco-2 cells in the cytotoxicity study. However, in order to fit the National Nanotechnology Initiative (NNI), which supports responsible development of nanotechnology, studies on the fate and effects of nanoparticle MgO in the environment are limited [37][38][39][40] ; such work should be done to ensure the sustainability of such approaches as part of nano-enabled agriculture.
The metallic elemental composition derived from nano and micron MgO treatments in fruit is of relevant concern to the research community and general public. As shown in this study, there were no significant differences for any of the elements (Al, B, Ca, Cu, Fe, K, Mg, Mn, Mo, Na, P, S, and Zn) (  www.nature.com/scientificreports www.nature.com/scientificreports/ treated fruit with the untreated controls. This finding is consistent with our previous study in which the nano MgO treatments did not impact accumulation of elemental concentration in the fruit compared to the untreated control 23 . In 2016 Spring trial (Table 3), similar to nano MgO (20 nm), micron MgO (0.3 µm and 0.6 µm) did not alter the elemental composition compared to fruits in the untreated control. However, fruits collected from Cu bactericide (Kocide 3000) and the grower standard (Cu-EBDC) treated plots showed significantly higher Cu concentrations (+0.2 mg/kg in fresh weight) relative to the untreated control. The study by Liao et al. also revealed that Cu-EBDC treated fruits had significantly greater Cu accumulation based on fresh weight more than the untreated control. Though consumption of tomato treated with Cu bactericide might lead to more Cu exposure, the concentration is still within the daily dietary limit 57,58 . In addition, unlike the spring trial, the Cu concentration of fruit was not significantly impacted by the Kocide 3000 or Cu-EBDC treatment in the fall trial. Former studies suggest that different environmental conditions such as crop variety, heavy metal exposure time, and location may affect Cu accumulation or elemental composition in crops [59][60][61] . Although applying Cu bactericides may increase Cu content in the fruit slightly without immediate risk to the consumer, field runoff containing Cu still pose ecological risk to non-target aquatic organisms [62][63][64] . In addition, Cu accumulation in the soil will potential cause phytotoxicity to tomato plants 65,66 . Thus it is still critical to find effective alternatives against bacterial spot to avoid intense Cu application in agriculture system.
In conclusion, although both micron (0.3 µm and 0.6 µm) and nano (20 nm) MgO have bactericidal activity against Cu-tolerant X. perforans in vitro, whereas micron MgO did not significantly reduce disease severity as effectively as nano MgO in the field. Importantly, the efficacy of MgO against bacterial spot disease of tomato is size dependent. Nano MgO bactericide still has great potential to become an alternative to Cu bactericides against bacterial spot disease of tomato as long as regulatory clearance can be obtained.

Materials and Methods
Bacterial strains and storage. X. perforans strain GEV485 (Cu-tolerant), isolated from tomato in Florida, was used in this study. Bacterial cells from pure cultures of these strains were suspended in sterile 30% glycerol solution and stored at −80 °C. Prior to use, bacteria were grown on nutrient agar (NA) medium (BBL, Becton Dickinson and Co., Cockeysville, MD) at 28 °C and were transferred every 24 to 48 h. Bacterial cells were collected from cultures grown on NA for 24 h, suspended in 0.01 M MgSO 4 , and the suspensions were adjusted to A 600 = 0.3 at λ = 600 nm (~5 × 10 8 CFU/ml). In vitro experiment evaluating minimum inhibitory concentration. X. perforans strain GEV485 (Cu-tolerant) was cultured from −80 °C storage and was suspended in sterile tap water and suspensions were diluted to 10 5 CFU/ml. Twenty micoliters of the bacterial suspension were transferred to 2 mL of MgO (20 nm, 0.3 and 0.6 µm) at different concentrations (100, 50, 25, and 12.5 µg/ml) in glass tubes. Sterile tap water served as the control. The tubes were incubated on a shaker (200 rpm) at 28 °C. Fifty microliters were sampled from each tube and plated on nutrient agar. Bacterial colonies were counted on each plate and converted to colony forming units (CFU)/ml.  www.nature.com/scientificreports www.nature.com/scientificreports/ Viability assay evaluating bactericidal activity. X. perforans strain GEV485 was used for the viability assay. Bacterial cells were incubated in nutrient broth (BBL, Becton Dickinson and Co., Cockeysville, MD) at 28 °C on a shaker at 300 rpm for 16 h to log phase. Bacterial cells were pelleted by centrifugation (16,872 × g for 10 min) and resuspended in 0.01 M MgSO 4 , and the suspensions were adjusted to A 600 = 0.3 at λ = 600 nm (~5 × 10 8 CFU/ml). Then 4.5 mL of the bacterial suspension were transferred to 500 μl of the following treatments in sterile glass tubes: 3 particle sizes of MgO (i.e., 20 nm, 0.3, or 0.6 µm), Cu bactericide (Kocide ® 3000 (DuPont, Wilmington, DE)) at 1,000 µg/ml. Sterilized tap water served as the control. The tubes were incubated on a shaker (300 rpm) at 28 °C for 4 h. After washing with 1 mL 0.85% NaCl twice, 1 ml samples from each tube were stained using the LIVE/DEAD BacLight Bacterial Viability kit (L7007, Molecular Probes, Invitrogen). The stain was a mixture of 1.5 ml Component A (SYTO 9 dye, 1.67 mM/Propidium iodide, 1.67 mM) with 1.5 ml Component B (SYTO 9 dye, 1.67 mM/Propidium iodide, 18.3 mM). Following addition of the stain, the sample was incubated in darkness for 15 min at room temperature. Micrographs were taken on a Nikon Eclipse Ti inverted microscope (Nikon, Melville, NY) at ×40 fluorescent optics using NIS-Elements imaging software (Ver To ensure adequate disease development in the field plots, a suspension of Cu-tolerant X. perforans strain GEV485 was adjusted to 5 × 10 8 CFU/ml in deionized water and was applied to the foliage in the field by spraying the 1 st , 8 th , and 15 th plant in each plot. One liter of each treatment was applied to each plot weekly with CO 2 pressurized spray boom with five nozzles until one week before fruit harvest. Plants were assessed for bacterial spot disease severity and phytotoxicity using the Horsfall-Barratt disease severity scale 70 every week after inoculation until harvest. The area under disease progress curve (AUDPC) was then calculated 71 . There were four replications per treatment and the experiment was conducted three times. Twelve out of fifteen plants, excluding the two towards the two ends of plots, were harvested for assessing the yield. Mature green or early breaker stage fruit were harvested and graded by USDA standards 72 . At least two harvests were made for each field experiment, which is common for fresh market tomato production in Florida. elemental analysis of the fruits. At harvest, five medium-sized mature-green stage fruits, with diameters between 5.72 and 6.43 cm according to the USDA standards [72][73][74] were collected from each of the treatments. The fruit were harvested from outside of the canopy from the first and the last plants of each of four plots from the 2016 Spring and Fall Quincy trial at 7 days after final application. The harvested fruit were hand-washed and sent to Department of Analytical Chemistry, The Connecticut Agricultural Experiment Station, New Haven, CT, USA. Four to eight grams of fresh tomato fruit with peel and flesh, and four to eight grams each of peel only and flesh only samples were dried in an electric oven at 70 °C for 48 h. Dried samples were pre-digested overnight with 2 ml of concentrated nitric acid and 2 ml of H 2 O 2 . After the pre-digestion step, these samples were digested at 115 °C for 45 min and then cooled to room temperature. The samples were filtered through cotton plugs and the volume was adjusted to 50 ml. The samples were stored at room temperature until analysis. Al, B, Ca, Cu, Fe, K, Mg, Mn, Mo, Na, P, S, and Zn concentrations were determined by Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES) using the Atom Scan 16 (Thermo-Jarrell Ash, Franklin, MA, USA). Analysis was performed following the methods described in previous studies 75-77 . Statistical analysis. The data collected from the in vitro assays and field experiments were evaluated for statistical significance using ANOVA followed by pair-wise comparisons using either the Least Significant Difference (LSD) for field studies, and the Student Newman-Keuls (SNK) method for in vitro and elemental accumulation experiments in IBM ® SPSS ® Statistics Version 22. A p-value of 0.05 was used to evaluate significance.