Entrapment of antimicrobial compounds in a metal matrix for crop protection

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.

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).

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.

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×.

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.

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.

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.

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.

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.