Nisin-based therapy: a realistic and eco-friendly biocontrol strategy to contrast Xylella fastidiosa subsp. pauca infections in planta

Abstract

The lack of sustainable strategies for combating Xylella fastidiosa (Xf) highlights the pressing need for novel practical antibacterial tools. In this study, Lactococcus lactis subsp. lactis strain ATCC 11454 (L. lactis), known for its production of nisin A, was in vitro tested against Xf subsp. pauca. Preliminary investigations showed that nisin A was involved in a strong antagonistic activity exhibited by L. lactis against Xf. Thus, the efficacy of nisin A was comprehensively assessed through a combination of in vitro and in planta experiments. In vitro investigations employing viable-quantitative PCR, spot assay, turbidity reduction assay, fluorescence microscopy, and transmission electron microscopy demonstrated nisin's robust bactericidal effect on Xf at a minimal lethal concentration of 0.6 mg/mL. Moreover, results from fluorescence and transmission electron microscopies indicated that nisin directly and rapidly interacts with the membranes of Xf cells, leading to the destruction of bacterial cells in few minutes. In in planta tests, nisin also demonstrated the ability to tackle Xf infections within Nicotiana benthamiana plants that remained asymptomatic 74 days post inoculation. Furthermore, RPLC-ESI-MS/MS analyses showed that nisin translocated to all parts of the plants and remains intact for up to 9 days. For the first time, this study underscores the nisin-based strategy as a realistic and eco-friendly approach to be further investigated against Xf infections in the field.

Keywords: RPLC-ESI-MS/MS; antimicrobial peptide; bacteria; biocontrol; electron microscopy; v-qPCR.

Figure 1

Schematic representation of Xylella fastidiosa and nisin inoculation sites.

Figure 2

Illustration outlining the nisin injection and the segmentation of the plant into five distinct sections.

Figure 3

BYCE plate showing the antagonistic activity of Lactococcus lactis subsp. lactis against Xylella fastidiosa subsp. pauca. (A,B) Xylella fastidiosa challenged with Lactococcus lactis subsp. lactis. (C,D) Xylella fastidiosa treated with PBS. Brackets indicate the inhibition zones. Arrows indicate the sites of treatments.

Figure 4

v-qPCR assay showing DNA amplification curves obtained from untreated Xf: (A) Xf (OD₆₀₀ = 0.32), (B) Xf (OD₆₀₀ = 0.16), (C) Xf (OD₆₀₀ = 0.08), (D) Xf (OD₆₀₀ = 0.04), (E) Xf (OD₆₀₀ = 0.02), and from nisin-treated Xf: (F) nisin 0.05 mg/mL, (G) nisin 0.1 mg/mL, (H) nisin 0.2 mg/mL, (I) nisin 0.4 mg/mL, (J) nisin 0.6 mg/mL, (K) nisin 0.8 mg/mL, (L) nisin 1 mg/mL, (M) nisin 1.5 mg/mL, (N) nisin 3 mg/mL, (O) nisin 6 mg/mL. (P) Sterile distilled water used as negative control.

Figure 5

BCYE agar plate showing the spot assay of nisin against Xf; (1) nisin 3 mg/mL-treated Xf; (2) nisin 1.5 mg/mL-treated Xf; (3) nisin 1 mg/mL-treated Xf; (4) nisin 0.8 mg/mL-treated Xf; (5) nisin 0.6 mg/mL-treated Xf; (6) Untreated Xf used as a positive control; (7) PBS used as a negative control.

Figure 6

Optical density values at 600 nm showing the reduction in Xylella fastidiosa cell growth at 1 h, 2 h and 3 h after the addition of nisin at 0.6 mg/mL. The histograms show the mean values of three experimental repetitions with standard deviation.

Figure 7

Fluorescent micrographs showing nisin-treated Xylella fastidiosa cells at 0.6 mg/mL. Green and red fluorescence represent viable and dead cells, respectively. Bar: 10 μm.

Figure 8

Transmission electron micrographs showing the impact of nisin on Xf cells. (A,B) Untreated Xf cells, used as control. (C–H) Nisin-treated Xf cells showing structural, cell wall, and cytoplasm alterations. Bar: 100 nm.

Figure 9

In planta assay showing the effect of nisin on Xylella fastidiosa subsp. pauca in Nicotiana benthamiana plants 74 dpi. (A) Healthy plants with no symptoms; (B) Xf-infected plants showing leaf scorch symptoms; (C) preventive nisin-treated Xf-infected plants with no symptoms; (D) curative nisin-treated Xf-infected plants with no symptoms.

Figure 10

TaqMan-based qPCR assay showing DNA amplification curves obtained from (A) genomic DNA of Xf used as positive control, (B) genomic DNA extracted from leaves of nisin-untreated Xf-infected Nicotiana benthamiana plants (positive controls), (C) genomic DNA extracted from leaves of Xf-infected Nicotiana benthamiana plants treated preventively and curatively with nisin and from healthy plants used as a negative control.

Figure 11

Chromatograms by RPLC-ESI-MS/MS in selected reaction monitoring (SRM) of nisin A in plant sap spiked at 30 ng/mL concentration (A) and in the stem of section 4 extracted after 24 h (B). Major product ions detected in CID-MS/MS spectrum of tetra-charged ion at m/z 839.3 (C). Values here reported are the means of three replicates.

Figure 12

Calibration curve of nisin A in plant sap; the x-axis indicates the concentration of nisin, and the y-axis indicates the area of the SRM chromatogram.