
==== Front
Microb Biotechnol
Microb Biotechnol
10.1111/(ISSN)1751-7915
MBT2
Microbial Biotechnology
1751-7915
John Wiley and Sons Inc. Hoboken

10.1111/1751-7915.70005
MBT270005
MICROBIO-2024-002-RA.R3
Research Article
Research Article
Entrapment of antimicrobial compounds in a metal matrix for crop protection
Copper composites as crop protection agent
Brill et al.
Brill Aya 1 2
Menagen Barak 3
Malach Einav 1
Zelinger Einat 4
Avnir David 3
Burdman Saul https://orcid.org/0000-0001-9544-7371
2 saul.burdman@mail.huji.ac.il

Hayouka Zvi https://orcid.org/0000-0003-3582-4029
1 zvi.hayouka@mail.huji.ac.il

1 Institute of Biochemistry, Food Science and Nutrition, The Robert H. Smith Faculty of Agriculture, Food and Environment The Hebrew University of Jerusalem Rehovot Israel
2 Department of Plant Pathology and Microbiology, Institute of Environmental Sciences, The Robert H. Smith Faculty of Agriculture, Food and Environment The Hebrew University of Jerusalem Rehovot Israel
3 Institute of Chemistry The Hebrew University of Jerusalem Jerusalem Israel
4 Interdepartmental Core Facility, Robert H. Smith Faculty of Agriculture, Food and Environment The Hebrew University of Jerusalem Rehovot Israel
* Correspondence
Zvi Hayouka, Institute of Biochemistry, Food Science and Nutrition, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot, Israel.
Email: zvi.hayouka@mail.huji.ac.il
Saul Burdman, Department of Plant Pathology and Microbiology, Institute of Environmental Sciences, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot, Israel.
Email: saul.burdman@mail.huji.ac.il

13 9 2024
9 2024
17 9 10.1111/mbt2.v17.9 e7000502 1 2024
08 8 2024
© 2024 The Author(s). Microbial Biotechnology published by John Wiley & Sons Ltd.
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the terms of the http://creativecommons.org/licenses/by-nc-nd/4.0/ License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made.

Abstract

Agricultural yields are often limited by damage caused by pathogenic microorganisms, including plant‐pathogenic bacteria. The chemical control options to cope with bacterial diseases in agriculture are limited, predominantly relying on copper‐based products. These compounds, however, possess limited efficacy. Therefore, there is an urgent need to develop novel technologies to manage bacterial plant diseases and reduce food loss. In this study, a new antimicrobial agent was developed using a doping method that entraps small bioactive organic molecules inside copper as the metal matrix. The food preservative agent lauroyl arginate ethyl ester (ethyl lauroyl arginate; LAE) was chosen as the doped organic compound. The new composites were termed LAE@[Cu]. Bactericidal assays against Acidovorax citrulli, a severe plant pathogen, revealed that LAE and copper in the composites possess a synergistic interaction as compared with each component individually. LAE@[Cu] composites were further characterised in terms of chemical properties and in planta assays demonstrated their potential for further development as crop protection agents.

In this study, a new antimicrobial agent was developed using a doping method that entraps small bioactive organic molecules inside copper as the metal matrix. Bactericidal assays against Acidovorax citrulli, a severe plant pathogen, revealed that the composites possess a synergistic antimicrobial activity in vitro and in planta, demonstrating their great potential for further study as new crop protection agents.

Israel Ministry of Agriculture and Rural DevelopmentIsrael Ministry of Science and TechnologyICA in Israel Foundation source-schema-version-number2.0
cover-dateSeptember 2024
details-of-publishers-convertorConverter:WILEY_ML3GV2_TO_JATSPMC version:6.4.8 mode:remove_FC converted:13.09.2024
Brill, A. , Menagen, B. , Malach, E. , Zelinger, E. , Avnir, D. , Burdman, S. et al. (2024) Entrapment of antimicrobial compounds in a metal matrix for crop protection. Microbial Biotechnology, 17 , e70005. Available from: 10.1111/1751-7915.70005
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pmcINTRODUCTION

Crop yields are highly reduced by diseases caused by microorganisms (Savary et al., 2019; Sundin et al., 2016). Plant‐pathogenic bacteria are important causal agents of diseases in plants with almost all major crops being severely affected by one or more bacterial diseases (De La Fuente & Burdman, 2011; Ficke et al., 2018; Mahlein & Oerke, 2012). While a large diversity of fungicides is available for management of plant diseases caused by fungi, chemical control of bacterial plant diseases in agriculture is highly limited (Gitaitis & Walcott, 2007). To date, chemical control of these diseases predominantly relies on copper (Cu)‐based products (Katsoulas et al., 2020; Lamichhane et al., 2018; Malandrakis et al., 2019; Sundin et al., 2016; Yu et al., 2023). These compounds are easily washed from the plant surface and possess limited efficacy. Therefore, frequent sprays with high Cu concentrations are required, often leading to phytotoxicity and emergence of Cu‐resistant bacterial strains (Behlau et al., 2011; Worthington et al., 2012). The need of frequent sprays also increases production costs and environmental contamination, without necessarily providing adequate protection. Accordingly, there is an urgent need to develop novel technologies to manage bacterial plant diseases and reduce food loss.

In this study, a new antimicrobial compound was tested, which is based on the entrapment of organic molecules in a metal matrix (generally termed organic molecule@metal) (Avnir, 2014; Bauer Stern et al., 2019; Ben‐Knaz Wakshlak et al., 2016). The entrapment of organic molecules with antimicrobial activity in a matrix of bactericidal metals like Cu or silver may lead to the generation of novel composites with antimicrobial activities that are substantially higher than those exerted by the individual components (Ben‐Knaz et al., 2010, 2013). This study presents the synthesis and characterisation of novel composites that contain lauroyl arginate ethyl ester (ethyl lauroyl arginate; LAE) doped in a matrix of Cu, thus termed LAE@[Cu]. LAE is an ethyl ester arginine derivative, in which the amino terminus is acylated with lauric acid (Singare & Mhatre, 2012). LAE was selected for several reasons: (1) it possesses strong antimicrobial activity against a wide range of bacteria (Becerril et al., 2013; Kim et al., 2017; Li et al., 2024; Rodriguez et al., 2004) as well as different fungi (Becerril et al., 2023; Chen et al., 2022; Xu et al., 2018); (2) it is readily available and relatively cheap; (3) it is widely used as a food preservative (Known as E‐243) (Becerril et al., 2013; Hou et al., 2023) and (4) no reports are available in the scientific literature on potential applications of LAE as a crop protection agent. This study presents the chemical characterisation of the LAE@[Cu] composites and demonstrates their strong bactericidal activity towards A. citrulli. Moreover, the ability of these composites to reduce symptoms caused by this pathogen in planta is demonstrated. Overall, all data demonstrate the potential of LAE@[Cu] composites, as crop protection agents.

EXPERIMENTAL PROCEDURES

Bacterial strain and growth conditions

The bacterial strains that were used in this study are: Acidovorax citrulli strain M6 (Burdman et al., 2005), Xanthomonas perforans strain 97‐2 (Timilsina et al., 2019), Clavibacter michiganensis strain NCPPB 382 (Gartemann et al., 2003) and Streptomyces scabies strain Av (Lerat et al., 2009). Unless otherwise stated, cells were grown in Nutrient Broth (NB; Difco) medium (13 g/L) at 30°C and stored at −80°C in NB containing 25% glycerol. For bactericidal assays (see below), bacteria were incubated in PBS (8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4 and 0.24 g KH2PO4; pH 7.3).

Minimal inhibitory concentration (MIC) assays

MIC values of LAE, benzoic acid and lactic acid were determined by a broth microdilution method as described (Hayouka et al., 2013). Briefly, bacteria were grown for 24 h in NB at 30°C with shaking (180 rpm) and then diluted into fresh NB to an optical density at 595 nm (OD595) of 0.1. Samples of this culture were mixed with equal volumes of NB containing different concentrations of the tested compounds in each well of a sterile 96‐well plate (Corning 3650). Bacterial growth was determined in a Tecan Infinite Pro Plate reader at OD595. The MIC values were the lowest concentrations of the different compounds that caused inhibition of bacterial growth (over 60% bacterial growth inhibition). MIC values were determined as the average obtained from three independent experiments (each comprising three replicates per compounds concentration). The highest tested concentration was 200 μg/mL.

Preparation of composites

For synthesis of LAE@[Cu], 200 mg CuSO4 (98%; Arcos organic) (1.25 × 10−3 mol) were dissolved in 1 mL double distilled water (DDW). Zinc (99.8%; Sigma‐Aldrich) was added at an equal molar concentration as Cu (81 mg). After 30 s, LAE (98% ethyl‐Nᶛ‐lauroyl‐L‐arginate; Alfa chemistry) was added at different concentrations (1.03, 2.06 or 4.12 mM) and the solution was stirred at room temperature for 24 h. The resulting composites were then filtrated with a 0.22‐μm membrane and dried under vacuum overnight. Control composites (@[Cu]) were synthesised in the same manner, without the addition of LAE.

Thermal gravimetric analysis (TGA)

Analysis was conducted with a TGA/SDTA 851e, from 50°C to 800°C, at heating rate of 10°C per minute under N2.

Elemental analysis

This analysis was conducted with a Thermo Flash EA 1112 instrument. Samples (2–3 mg) were wrapped in a tinfoil capsule and used without any pre‐treatment. The samples were placed in a MAS 200 Autosampler drum, where they were purged with helium. Each sample, in turn, was dropped into the combustion reactor: a vertical quartz tube, maintained at 1020°C and packed with two separate layers of catalysts, which facilitated the next stages of the process: oxidation, reduction, and chromatographic separation.

Energy dispersive spectroscopy (EDS)

Analysis was carried out using a low‐vacuum Jeol IT‐100 SEM instrument. Samples were placed on a stub using carbon tape, and the stub was placed in the SEM holder. The images of uncoated samples were taken with an accelerating voltage of 20 kV at low vacuum (LV) mode 30 Pa and a backscattered electron image. EDS was done using a Jeol incorporated system (Borkow et al., 2022).

X‐ray diffraction (XRD)

Analysis of LAEx1.0@[Cu] composites by XRD was performed using a D8 Advance diffractometer apparatus (Bruker AXS) with secondary Graphite monochromator, 2° Sollers slits and a 0.2‐mm receiving slit. The powder samples were placed on low background quartz sample holders. The patterns from 5° to 85° 2θ were recorded at room temperature using CuKα radiation (λ = 0.15418 nm) under the following measurement conditions: tube voltage of 40 kV, tube current of 40 mA, step scan mode with a step size of 0.02° 2θ and a counting time of 1 s per step. The Scherrer equation was used to obtain the crystalline size from the experimental XRD data. The instrumental broadening was determined using LaB6 powder (NIST SRM 660).

Bactericidal assays

Bactericidal activity of LAE@[Cu] composites at various concentrations was tested in PBS containing ~107 colony forming units (CFU)/mL A. citrulli cells. The suspensions were incubated at 30°C with shaking for 30 min. Then, viable bacteria were determined following plating of serial dilutions on NA plates. Controls included @[Cu] (the composite‐like compound without LAE) and the commercial bactericide Kocide® 2000 (Gadot Agro; active compound: copper hydroxide), at the same Cu concentration as in the composites. An additional control was LAE at 1.25 ppm, which is the estimated concentration of LAE in 25 ppm of the LAEx1.0@[Cu] composite as observed by TGA analysis.

Assessment of bacterial cells by SEM

Bacterial cells exposed to different treatments were incubated with 4% glutaraldehyde solution on small glasses covered with poly‐lysine. Dehydration was done in increasing ethanol concentrations (25%, 50%, 75%, 95%, 100% ethanol in deionized water), 5 min (twice) for each concentration. Drying was performed with a critical point dryer (CPD BAL‐TEC CPD‐030) and gold coating was performed with Gold Sputter Coater (Polaron SEM Coating Unit). Then the samples were coated with iridium. The analysis was carried out on a Sirion (FEI) high‐resolution (HR) Jeol 7800 SEM instrument.

Release profile of LAE from LAEx1.0@[Cu]

For generation of a standard curve with different concentrations of LAE, 23 g NH4SCN were mixed with 11 g Co(NO3)2 in 26 mL of DDW to generate Co(SCN)4 −2. Fifty millilitre‐solutions of LAE at different concentrations (0.5, 1, 2, 3, 4, 5 and 10 ppm) were prepared. Then, 10 mL Co(SCN)4 −2 and 5 mL 1,2 dichloroethane were added to the LAE solutions. All compounds were mixed and shaken for 5 min in a separatory funnel. Then the two phases were separated. The LAE‐Co(SCN)4 −2 complex was soluble at the organic phase (1,2 dichloroethane). One millilitre from this phase was taken for OD measurement at 623 nm. For determination of LAE release profile, 400 ppm LAEx1.0@[Cu] (20 mg composite in 50 mL DDW) were incubated at 30°C with shaking and then analysed as described above.

Release profile of Cu+2 ions from LAEx1.0@[Cu] and @[Cu]

Solutions containing 400 ppm LAEx1.0@[Cu] or @[Cu] (20 mg in 50 mL DDW) were incubated at 30°C with shaking. From each flask, 1.5 mL samples were taken at different times, mixed with 1.5 mL DDW. Then, samples were diluted 3 times, and the concentration of Cu+2 was measured on a dual‐view High‐Resolution ICP‐OES spectrometer PlasmaQuant, Analytik Jena (Antonoglou et al., 2017; Sureshkumar et al., 2010).

Determination of the type of the interaction between LAE and Cu in the composites

Bactericidal assays were used to assess the type of the interaction between LAE and copper in the composites in terms of killing ability. Results from the aforementioned bactericidal assays were used to calculate the Combination Index (CI), using two methods: The highest single agent approach (Lehár et al., 2007), in which the CI is determined as: CI=maxEA, EBEAB

The response additivity approach (Slinker, 1998), in which the CI is calculated as follows: CI=EA+EBEAB

In the above equations, EA is the effect observed by the @[Cu], EB is the effect observed by LAE, and EAB is the effect observed for the composites. CI values smaller, equal or greater then 1, reflect synergism, additive effect or antagonism, respectively (Foucquier & Guedj, 2015).

In planta inoculation experiments

Melon (Cucumis melo) plants (cv. AN‐305; Origene Seeds) were grown in a growth chamber at 28°C (day: night, 14:10 h) in 10‐cm diameter/12‐cm height pots (three or four seedlings per pot) containing a commercial soil for 3 weeks. Then the three youngest fully developed leaves of each plant were sprayed with 200 ppm of LAEx1.0@[Cu] composites. Additional treatments were 200 ppm @[Cu], 10 ppm LAE (corresponding to the expected entrapped concentration of LAE in 200 ppm LAEx1.0@[Cu]) and Kocide® 2000, which was applied at the recommended concentration for management of the disease (2 mg/mL; 2000 ppm). As controls, the foliage was sprayed with DDW. After 4 h, the leaves were spray‐inoculated with a suspension of A. citrulli at ~106 CFU/mL until run‐off or DDW to assess cytotoxic effects. During the first 24 h after inoculation the plants were covered with plastic bags to increase humidity and thus infection. Disease severity was determined 5 days after inoculation using the following disease severity scale: 0, no symptoms; 1, few minor lesions; 2, few small necrotic spots; 3, increased necrotic spots in less than 25% of the leaf area; 4, increased necrotic spots in 75% to 100% of the leaf area; and 5, increased necrotic spots in more than 50% of the leaf area or dead leaf (Figure 7A).

Seed inoculation assays

Melon seeds (cv. Rachel; Hazera Genetics) were inoculated with A. citrulli by incubating them for 18 h in a ~ 105 CFU/mL bacterial suspension. To promote penetration of bacteria into the seeds, vacuum was applied three times during the first two hours of incubation as described (Dutta et al., 2016), using a solid PTFE vacuum pump (KNF Laboport). Control seeds were treated with DDW at similar conditions. Seeds were then treated with 400 ppm LAEx1.0@[Cu] or [@Cu], 20 ppm LAE or DDW, under vacuum (applied as indicated above) and stirring for 24 h. Seeds were then transferred to Petri dishes coated with cotton wool and seed paper (10 seeds per plate). The Petri dishes were wrapped with parafilm to increase humidity and they were kept in a greenhouse at 28°C. The amount of water in the plates was checked and adjusted daily. After 12 days, germination percentages and disease severity were determined. For disease, the following scale was used: 0, no foliar symptoms; 1, necrotic lesions on 0%–25% of cotyledons; 2, necrotic lesions on 25%–50% of cotyledons; 3, necrotic lesions on 50%–75% of cotyledons; and 4, necrotic lesions on 75%–100% of cotyledons. Germination percentages were determined for each treatment. To assess cytotoxic effects, at the first vacuum stage, 8 seeds (per treatment) were exposed to DDW. Then, seeds were treated with 400 ppm LAEx1.0@[Cu] or [@Cu], 20 ppm LAE or DDW, under vacuum, and were treated as described above.

RESULTS AND DISCUSSION

LAE inhibits growth of A. citrulli

The ability of a variety of antimicrobial agents to inhibit the growth of A. citrulli strain M6 was tested. These experiments revealed that among the tested compounds, only LAE had a strong bacteriostatic activity towards A. citrulli, with an average MIC value of 16 μg/mL (Figure S1). Therefore, LAE was selected as the antimicrobial agent to be entrapped in Cu‐based composites.

Synthesis of LAE@[Cu] composites and their chemical characterization

Synthesis of LAE@[Cu] composites was achieved as shown in Equation 1. LAE was dissolved in an aqueous solution containing Cu+2 and zinc (Zn) as reducing agent. Zinc was chosen as the reducing agent due to its ability to promote fast reduction at room temperature as recently described (Bauer Stern et al., 2019). LAE was added at different concentrations. The solution was stirred at 25°C for 24 h, filtrated, washed and dried under vacuum at room temperature overnight to generate the composites. As controls, composites were generated in the absence of LAE, which were named @[Cu] (Table 1). (1) CuSO4aq+Zns+LAEaq➔LAE@Cus+ZnSO4aq

Equation 1. Chemical reaction for synthesis of LAE@[Cu].

TABLE 1 Synthesis of LAE@[Cu] composites prepared with different concentrations of LAE.

Composite	CuSO4 (mg)	LAE (mg)	Zn (mg)	DDW (mL)	LAE concentration (mM) a	% of LAE entrapment b	
LAEx1.0@[Cu]	200	5.2	81	3	4.12	5.0	
LAEx0.5@[Cu]	200	2.6	81	3	2.06	4.5	
LAEx0.25@[Cu]	200	1.3	81	3	1.03	1.0	
@[Cu] c	200	0	81	3	0	0	
Abbreviation: DDW, double distilled water.

a LAE concentration (mM) in the reaction solution.

b Approximate percentages of LAE entrapment [% (w/w) relative to the LAE@[Cu] composite] in the composites as determined by different methods.

c For synthesis of @[Cu] (control), no LAE was added.

Chemical characterisation of the synthesised composites was accomplished by different methods, including thermal gravimetric analysis (TGA), elemental analysis, energy dispersive spectroscopy (EDS) and X‐ray diffraction (XRD). TGA allowed determining the percentage of LAE that was entrapped into the metal matrix. The percentages [% (w/w)] were ~5%, ~4.5% and ~1% in LAEx1.0@[Cu], LAEx0.5@[Cu] and LAEx0.25@[Cu], respectively (Table 1; Figure 1A). Elemental analysis provides the percentage of organic elements in a metal mixture. Elemental analyses of the tested composites inferred that LAEx1.0@[Cu], LAEx0.5@[Cu] and LAEx0.25@[Cu] contained 4.4%, 1.1% and 0.7% LAE, respectively (Figure 1B). Thus, supporting the results from the TGA analysis, the weight reduction of LAE × 1.0@[Cu] obtained was ~5%, similar to elemental assay results, which revealed that the sum of organic elements (N, C, H, and S) from LAE × 1.0@[Cu] was 4.4%. EDS analysis showed that the composites contained traces of Zn, sulphur (S) and oxygen (O) (Figure S2). XRD revealed that the composites contained ~20% of cuprite in both LAEx1.0@[Cu] and @[Cu] (Figure S3). The copper composite is named @[Cu] with square bracket to emphasize the presence of cuprite.

FIGURE 1 Entrapment of LAE in a copper matrix. (A) Entrapment illustration: copper ions were reduced by metallic zinc in the presence of LAE to form aggregated copper nanocrystals with the LAE molecules entrapped in the copper matrix. (B) Structure of ethyl lauroyl arginate (LAE).

The composite morphology was characterised by scanning electron microscopy (SEM). Representative images of LAEx1.0@[Cu] and @[Cu] are shown in Figure 2. In both cases, the aggregate sizes were within the microscopic scale, varying from a few to tens of micrometres. SEM images also revealed that most LAEx1.0@[Cu] aggregates had a spherical shape, which was not characteristic of aggregates in the @[Cu] samples (Figure 2). In addition, Fourier‐transform infrared spectroscopy (FTIR) was performed; the LAE signal was not observed in LAEx1.0@[Cu] spectrum because it is buried inside the composites (data not shown).

FIGURE 2 Composition characterisation of the composites. (A) Determination of the percentage of organic material of four composites with different entrapment amounts of LAE (including @Cu, without LAE, as control; see Table 1) by thermal gravimetric analysis (TGA). Analyses were conducted with a TGA/SDTA 851e analyser. Results are from one representative experiment out of three with similar results. (B) Determination of the concentrations of nitrogen, carbon, hydrogen and sulphur in the composites by elemental analyses using a Thermo Flash EA 1112 instrument. Results are averages and standard deviations of two replicates per sample.

Bactericidal activity of LAE@[Cu] composites

Bactericidal activity of the different composites towards A. citrulli was assessed as a model bacterium. A. citrulli is the causal agent of bacterial fruit blotch (BFB) of cucurbits (Burdman & Walcott, 2012). This disease represents a serious threat to cucurbit production worldwide, and especially, to watermelon and melon (Zivanovic & Walcott, 2017). To date, there are no reliable sources of disease resistance to this pathogen, and chemical control of this disease has only limited efficiency (Bahar & Burdman, 2010; Burdman & Walcott, 2012). The bactericidal activity of LAE@[Cu] composites at a concentration of 25 ppm was tested in phosphate‐buffered saline (PBS) containing ~107 CFU/mL (Figure 3A). LAEx1.0@[Cu] and LAEx0.5@[Cu] showed the highest levels of bactericidal activity. After 30 min of incubation, these treatments killed almost all bacterial cells, with a ~7 log reduction of viable cells. The LAEx0.25@[Cu] composite also showed a strong bactericidal effect, but lower than the two other composites (~5 log reduction). The activity of @[Cu] was very similar to that of Kocide® 2000, a Cu‐based commercial bactericide (~4 log reduction). Bactericidal activity of LAE alone was tested, at 1.25 ppm (a concentration similar to that of LAE that was entrapped in the LAEx1.0@[Cu] composite). Under tested conditions, 1.25 ppm of LAE showed a mild bactericidal activity (~2 log reduction) (Figure 3A).

FIGURE 3 Morphology of the composites as observed by scanning electron microscopy (SEM). (A) LAEX1.0@[Cu], three different magnifications, from left to right: 5000×, 30,000× and 100,000×. (B) @[Cu], three different magnifications, from left to right: 5000×, 20,000× and 100,000×.

The effect of the composites concentrations on bactericidal activity was explored. Bacteria were treated with two concentrations of LAEx1.0@[Cu], 12.5 and 25 ppm (Figure 3B). The bactericidal activity of 25 ppm LAEx1.0@[Cu] was substantially stronger than the activity of the same composite at 12.5 ppm (~7 and ~5 log reduction, respectively). These experiments also supported that the two components of the LAE@[Cu] composites interact synergistically in terms of bactericidal activity compared to the activity of each component individually (Figure 3A,B). A similar assay was performed towards three pathogenic bacteria: X. perforans (Gram‐negative); the causal agent of bacterial spot disease of tomato and pepper, C. michiganensis (Gram‐positive); the causal agent of bacterial canker and wilt disease of tomato and S. scabies (Gram‐positive); the causal agent of scab disease of potato. Figure S4 shows that LAEx1.0@[Cu] composites possess strong bactericidal activity against X. perforans and C. michiganensis. In contrast, the composites possessed mild efficacy towards S. scabies with ~1 log reduction in bacteria load.

To further validate the synergistic interaction between Cu and LAE in the composites, the bactericidal activity of LAEx1.0@[Cu] was compared with a mixture containing the different components at similar concentrations than in the composites. Bactericidal assays were done as described above, but in these assays, treatments included bacteria that were exposed to LAE at the doped concentration combined with 25 ppm @[Cu] or with CuSO4 (equivalent to 25 ppm Cu+2) (Figure 3C). In contrast to the ~7 log reduction of viable cells observed for 25 ppm LAEx1.0@[Cu], the combination of CuSO4 + LAE and @[Cu] + LAE mixtures led to log reductions of only ~3 and ~1, respectively. These results validated the synergistic interaction between Cu+2 ions and LAE in the composites and imply that the composite structure and composition have an impact on its antimicrobial activity. The synergistic nature of the interaction between Cu and LAE in the composites was further demonstrated by determination of Combination Index (CI) values using the Highest Single Agent and the Response Additivity methods (Foucquier & Guedj, 2015; Lehár et al., 2007; Slinker, 1998; Topman‐Rakover et al., 2020) (Table 2).

TABLE 2 LAEx1.0@[Cu] is characterized by the occurrence of a synergistic effect between LAE and Cu.

LAEx1.0@[Cu] concentration (ppm) a	CI value b	
The highest single agent	The response additivity	
25	0.58	0.79	
12.5	0.41	0.65	
a The calculations are based on bactericidal assays described in Figure 3B.

b CI, combination index. CI values smaller than 1 reflect a synergistic interaction (see explanation in Section 2).

Assessment of release of LAE and copper ions from the composites

To gain insight into the mode of action of the composites, the release of LAE and Cu+2 ions from LAEx1.0@[Cu] was evaluated. LAE molecules were detected using a colorimetric technique. The method is based on the use of Co(SCN)4 −2 that reacts with LAE to form a LAE‐Co(SCN)4 −2 complex, which can be measured by a spectrophotometer (Pezo et al., 2012). To characterise the release profile, a standard curve with different concentrations of LAE was generated (Figure 4A). Samples were collected for LAE measurements at different times.

FIGURE 4 Bactericidal assays of composites towards A. citrulli M6. (A) Bactericidal effects of three composites carrying different doping amounts of LAE. Treatment concentrations were 25 ppm except for LAE that was tested at 1.25 ppm (corresponding to the concentration of this compound in LAEx1.0@[Cu]). (B) Bactericidal effects of LAEx1.0@[Cu] at different concentrations (12.5 and 25 ppm). (C) Bactericidal effect of LAEx1.0@[Cu] as compared with the individual composite compounds individually or combined. Concentrations of LAE and CuSO4 in the different treatments corresponded to the expected concentrations of LAE and Cu in 25 ppm LAEx1.0@[Cu]. In these experiments, bacteria were exposed to the treatments for 30 min. All results represent averages and standard errors of three different experiments.

The maximum amount of LAE that was released from LAEx1.0@[Cu] composites under tested conditions was 10% of the composite's doped concentration (~2 ppm LAE from 400 ppm LAEx1.0@[Cu]), with most LAE being released during the first hours of incubation (Figure 4B).

The release of Cu+2 ions was assessed by Inductively Coupled Plasma‐Optical Emission Spectrometry (ICP‐OES) (Kilbride et al., 2006) under similar conditions. The maximum amount of Cu+2 that was released from LAEx1.0@[Cu] was ~0.6%, equivalent to ~2.5 ppm, and this was similar to the measured release of Cu+2 from the composites without LAE (@[Cu]; ~0.9%, equivalent to ~3.6 ppm) (Figure 5).

FIGURE 5 Release profile of LAE from LAEx1.0@[Cu] composites. (A) A standard calibration curve with different concentrations of LAE. (B) LAE release profile from 400 ppm LAEx1.0@[Cu] (20 mg composite in 50 mL DDW) along the incubation time. Data are averages and standard errors of three different experiments.

The above results indicate that, under the tested conditions, the release of both Cu+2 and LAE from LAEx1.0@[Cu] is relatively low. While these findings were quite surprising considering the high bactericidal activity of the composites, they suggest that it is not likely that the antimicrobial activity of the composites is only due to Cu+2 or LAE release. The hypothesis was that the composites may act as bioactive beads, with direct contact between them and bacterial cells to induce the antimicrobial activity.

Assessing damage of composites to the bacterial membrane

To assess the effect of LAEx1.0@[Cu] composites on the structure and shape of bacterial membranes, A. citrulli cells were exposed to 25 ppm LAEx1.0@[Cu] for 0.5 h, then cells were observed using SEM. Representative images are shown in Figure 6, and additional images are shown in Figure S5. Vesicle‐like structures were observed on the surface of some of the bacteria exposed to LAEx1.0@[Cu] after 0.5 h of incubation. The images showed that LAEx1.0@[Cu] may cause small pores, from which bacterial content might leak. Treatment with @[Cu] caused a mild effect on the bacterial surface, with most of the cells surviving the treatment after the 0.5 h incubation period (~0.5 log reduction). Bacterial cells that were exposed to 1.25 ppm LAE showed mixed phenotypes: while some of the cells looked injured, others did not seem to be affected. In contrast, the cells were substantially damaged after exposure to 25 ppm LAE.

FIGURE 6 Release profile of Cu+2 ions from LAEX1.0@[Cu] and @[Cu] as determined by the ICP‐OES method. Samples were taken at different time intervals (1.5, 3, 6, 9 and 24 h). Data are average and standard errors of two experiments with two replicates per time point.

LAE is known for its ability to induce the outer membrane disruption (Becerril et al., 2013; Ma et al., 2020; Rodriguez et al., 2004). As a surfactant, it is capable of disintegrating cell membranes at very low concentrations, damaging the membrane potential and cell permeability and consequently leading to bacterial death (Asker et al., 2009; Becerril et al., 2013; Yang et al., 2019). Cu+2 ions can damage the bacterial cell membrane, promote DNA damage and disrupt vital bacterial enzyme function, all leading to bacterial cell death (Cioffi et al., 2005; Dupont et al., 2011; El‐Ghamry et al., 2023; Flemming & Trevors, 1989; Ren et al., 2009). The hypothesis was that the bactericidal effect of the LAE@[Cu] composites is caused by a synergistic effect between LAE and copper. It is likely that LAE damages the bacterial outer membrane to promote faster and enhanced entry of copper ions into the bacterial cells.

Assessment of the composites' potential to control disease

Based on the potent antimicrobial effect of LAEx1.0@[Cu] composites on A. citrulli, the melon‐pathosystem was selected as a model to assess the potential of the composites as crop protection agents. Plants were sprayed with 200 ppm LAEx1.0@[Cu] or [@Cu], 10 ppm LAE (corresponding to the expected concentration of LAE in 200 ppm LAEx1.0@[Cu]) or 2 mg/mL of the Cu‐based commercial bactericide Kocide® 2000. As mentioned above, Cu‐based compounds are the most common bactericides for management of bacterial plant diseases in agriculture, including BFB caused by A. citrulli (Lamichhane et al., 2018; Singare & Mhatre, 2012; Sundin et al., 2016). As controls, the foliage was sprayed with DDW. After 4 h, the leaves were spray‐inoculated with a suspension of A. citrulli M6 at ~106 CFU/mL. Disease severity was determined 5 days after inoculation, using a 0‐to‐5 scale (Figure 7A; details in Section 2). The cytotoxicity of all treatments was examined by spraying melon leaves with the different treatments without bacterial inoculation. No cytotoxicity symptoms could be detected under tested conditions (representative leaves are shown in Figure S6A).

FIGURE 7 Morphology of A. citrulli cells exposed to LAEx1.0@[Cu]. Images were taken by SEM after incubation of ~107 CFU/mL of A. citrulli M6 for 0.5 h in PBS at 30°C. Bars represent 100 nm.

The results of three independent experiments are summarised in Figure 7B,C. The LAEx1.0@[Cu] composites significantly (p < 0.05) reduced disease severity as compared with non‐treated controls. Remarkably, the level of disease severity reduction of the composites (43%) did not differ statistically from that exerted by the Kocide® 2000 treatment (51%). Treatments with Cu (@[Cu]) and LAE alone also significantly (p < 0.05) reduced disease severity as compared with controls, but at lower levels than the LAEx1.0@[Cu] and Kocide® 2000 treatments (Figure 7C). These results confirm that the strong bactericidal activity of LAEx1.0@[Cu] and the synergistic interaction between Cu and LAE in the composites also occur in planta.

Assessment of the ability of the composites to reduce infection of contaminated seeds

Many plant‐pathogenic bacterial species are seedborne, namely, they have the ability to be transmitted by seeds (Dutta et al., 2014). The antimicrobial activity of LAEx1.0@[Cu] composites on melon seeds infected with A. citrulli was assessed. Importantly, A. citrulli is a seedborne pathogen that spreads rapidly under warm and wet conditions (Hopkins et al., 2009). As similar as for many other bacterial diseases, infected seeds are considered as the most important source of primary BFB inoculum and the main factor that led to global spread of BFB disease (Burdman & Walcott, 2012; Latin et al., 1995). Moreover, in the case of BFB, under optimal conditions for disease development, yields might be severely compromised even when only one or a few single seedling is infected at planting (Bahar et al., 2009; Bahar & Burdman, 2010).

Melon seeds were inoculated with a suspension of A. citrulli at ~105 CFU/mL by vacuum infiltration to mimic internal seed infection, as described by Dutta et al. (2016). Then, infected seeds were treated with the composites and the doping compounds for 24 h under vacuum conditions. Treatments included 400 ppm LAEx1.0@[Cu] or [@Cu], 20 ppm LAE (corresponding to the expected concentration of LAE in 200 ppm LAEx1.0@[Cu]) and DDW as negative control. The seeds were transferred to Petri dishes coated with wet cotton wool and seed paper, which were kept in a greenhouse at 28°C. After 12 days, germination percentage and disease severity were evaluated as described in Section 2. The results of two independent experiments are summarised in Figure 8. LAEx1.0@[Cu] significantly (p < 0.0001) reduced disease severity by 94% as compared with non‐treated controls (seeds treated with DDW in a similar manner as the inoculation treatment). The @[Cu] and LAE treatments reduced disease severity by only 14% and 3%, respectively. Seed germination was not damaged by composite treatment as germination percentages were 70, 62, 77 and 62 for LAEx1.0@[Cu], @[Cu], LAE and for the non‐treated seeds (DDW), respectively. In addition, no visible phytotoxic effects were detected in seedlings emerging from non‐infected seeds that were treated with LAEx1.0@[Cu], @[Cu] or LAE (Figure S6B). Overall, these results indicate that seed treatment with LAE@[Cu] composites reduces the incidence of BFB (Figure 9).

FIGURE 8 Effect of LAEx1.0@[Cu] on disease severity of melon leaves inoculated with A. citrulli. (A) Representative leaves of the different disease severity scores. (B) Average disease severity scores of the different treatments as measured 5 days after inoculation. Results represent averages and standard errors from 3 independent experiments, in which each treatment contained 20–40 leaves (replicates). The data were statistically analysed by one‐way analysis of variance (ANOVA) and Tukey's honest significant difference (HSD) test. Different letters indicate statistically significant differences (p < 0.05). (C) Effects of the different treatments on disease severity as expressed in percentage of disease inhibition.

FIGURE 9 Effect of LAEx1.0@Cu on melon seeds inoculated with A. citrulli. (A) Representative seedlings of the disease severity scale. (B) Values are averages (black medial line) of two different experiments with replicates (n) from 34 to 46 seeds per each treatment. The data were statistically analysed by Kruskal–Wallis test, Dunn's multiple comparison. *** indicate statistically significant differences (p < 0.0001) between LAEx1.0@[Cu] to DDW control. LAEx1.0@[Cu] statistically significant different from all other treatments (p ≤ 0.0003). Each dot represents a seed, bars represent mean ± SD, n = 34–46.

CONCLUSIONS

This study reports the synthesis and chemical characterisation of a novel type of composites, LAE@[Cu]. The composites possess highly bactericidal ability and demonstrate their potential as crop protection agents. This study also provides a proof‐of‐concept demonstrating the notion that composites that involve organic molecules entrapped in a metal matrix can be exploited as antimicrobials for applications in agriculture and in other fields. In future studies, it will be important to assess the bacterial ability to develop resistance against the LAE@[Cu], and particularly, in comparison with the level of resistance developed against the individual composite components.

AUTHOR CONTRIBUTIONS

Aya Brill: Formal analysis; investigation; writing – original draft. Barak Menagen: Methodology. Einav Malach: Methodology. Einat Zelinger: Formal analysis; methodology. David Avnir: Conceptualization; formal analysis; methodology. Saul Burdman: Conceptualization; supervision; Writing – review and editing. Zvi Hayouka: Conceptualization; Supervision; writing – review and editing.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflict of interests.

Supporting information

Data S1.

ACKNOWLEDGEMENTS

The authors acknowledge funding from the Israel Ministry of Agriculture and Rural Development and the Israel Ministry of Science and Technology. In addition, this research was also partially supported by grant from the ICA in Israel Foundation.

DATA AVAILABILITY STATEMENT

The data that supports the findings of this study are available in the supplementary material of this article.
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