Antimicrobial activity of Epsilon-Poly-L-lysine against phytopathogenic bacteria

Abstract

Antimicrobial peptides (AMPs) are components of immune defense in many organisms, including plants. They combat pathogens due to their antiviral, antifungal and antibacterial properties, and are considered potential therapeutic agents. An example of AMP is Epsilon-Poly-L-lysine (EPL), a polypeptide formed by ~ 25 lysine residues with known antimicrobial activity against several human microbial pathogens. EPL presents some advantages such as good water solubility, thermal stability, biodegradability, and low toxicity, being a candidate for the control of phytopathogens. Our aim was to evaluate the antimicrobial activity of EPL against four phytobacterial species spanning different classes within the Gram-negative phylum Proteobacteria: Agrobacterium tumefaciens (syn. Rhizobium radiobacter), Ralstonia solanacearum, Xanthomonas citri subsp. citri (X. citri), and Xanthomonas euvesicatoria. The minimum inhibitory concentration (MIC) of the peptide ranged from 80 μg/ml for X. citri to 600 μg/ml for R. solanacearum and X. euvesicatoria. Two hours of MIC exposure led to pathogen death due to cell lysis and was enough for pathogen clearance. The protective and curative effects of EPL were demonstrated on tomato plants inoculated with X. euvesicatoria. Plants showed less disease severity when sprayed with EPL solution, making it a promising natural product for the control of plant diseases caused by diverse Proteobacteria.

Figure 1

Inhibition of bacterial growth by spotting assay. Each bacterium was grown on LB agar plates and different concentrations of the peptide (70–1,000 μg/ml) were tested in different spots to determine the minimum inhibitory concentration of EPL able to inhibit bacterial growth. Water was used as negative control. The plates were placed at 28 °C for two days and evaluated. Three biological replicates were done.

Figure 2

Growth inhibition curves in the presence of EPL treatment at MIC. Aliquots were taken in 30-min intervals, during 2 h, serially diluted and plated. Plates were kept for two days at 28 °C when CFU were counted. The numbers of CFU/ml are the averages for three replicates plating from each sample, and the error bars represent the standard deviations. Percentage of cell mortality was calculated as the ratio of cell counts in the treated group with EPL to those in the control group.

Figure 3

Cell viability measured by fluorescence emitted by bacterial cells treated with EPL at MIC for one hour. SYTO 9 was added to the bacterial suspensions and incubated for 15 min in the dark. Samples were excited at 470 nm and emission spectrum (490–700 nm) was recorded. A lower emitted fluorescence near 530 nm indicates a lower number of viable cells. Controls included water-only treatment (non EPL-treated) and EPL with fluorophore (without bacterial cells). Values shown are the average of three biological replicates.

Figure 4

Overlap of fluorescence images of bacterial cells stained with SYTO 9 and propidium iodide for 15 min in the dark. Bacterial cells were treated with EPL at MIC for one hour or water as control. The red fluorescence is emitted because of an interaction between propidium iodide and DNA when the cytoplasmic membrane is damaged, whereas bacteria with intact cell membranes are impermeable to propidium iodide and display green fluorescence. Images shown are representative of three biological replicates. Scale bar = 10 μm.

Figure 5

The effect of EPL on the bacterial cell surface. Scanning electron microscopy of bacterial cells treated with EPL at MIC for 1 h. No-EPL (water) treatment was used as control. Scanning electron microscope was operated at 10 kV. Scale bar = 1 μm.

Figure 6

Enhanced protection against bacterial spot in tomatoes sprayed with EPL solution. Twelve Santa Cruz-Kada tomato plants were analyzed for each condition after 30 days of X. euvesicatoria inoculation. The figure shows representative leaves of three tomato plants treated with EPL (onefold MIC or twofold MIC) 2 days before inoculation (a), and tomato plants treated with EPL (onefold MIC or twofold MIC) 2 days post-inoculation (b).