Neem Essential Oil as an Antifungal Agent against Phyllosticta citricarpa

Abstract

The fungus Phyllosticta citricarpa is a quarantine phytopathogen responsible for causing citrus black spot (CBS) disease. To export fruits to CBS-free countries, they must undergo a sanitation process to ensure disease control. In this study, neem essential oil (NEO) was tested against P. citricarpa for the first time as an alternative sanitizer. In vitro experiments were conducted to determine the inhibition concentration of NEO for P. citricarpa, and the mode of action of the essential oil was evaluated. In vivo assays were performed to simulate the sanitization process used in packinghouses. NEO was characterized by GC-MS/MS. The results revealed that NEO at 100 μL·mL^-1 exhibited a similar inhibitory effect as copper oxychloride, suppressing 89.68 ± 1.14% of fungal mycelium growth. Fluorescence microscopy experiments demonstrated that NEO functions by disrupting the cytoplasmic membrane of fungal hyphae, leading to their death within 30 minutes of contact with NEO. GC-MS/MS characterization revealed a high presence of phenolic compounds, which serve as the primary antifungal agents responsible for the action against fungal hyphae. In vivo assays showed that NEO at 100 μL·mL^-1 also reduced microorganisms (CFU mL^-1) by 93.00 ± 3.88% compared to the negative control. Overall, the results demonstrate that NEO can effectively serve as an alternative sanitizer against P. citricarpa in citrus packinghouses. Our findings allow future studies to explore the use of NEO for sanitizing other fruits and combating different phytopathogens to broaden its potential application in fruit sanitation for export.

Figure 1

Comparative fungal growth of P. citricarpa, by area measured in the ImageJ software, after contact with different concentrations of NEO (40, 60, 80, and 100 μL·mL⁻¹). PC was 90 g·L⁻¹ of copper oxychloride, and NC was deionized water with 1% of DMSO. The photos of fungal growth of P. citricarpa on YPD agar plates are showed below of the graph, where A is NC; B is PC; C is NEO at 40 μL·mL⁻¹ of concentration; D is NEO at 60 μL·mL⁻¹ of concentration; E is NEO at 80 μL·mL⁻¹ of concentration; F is NEO at 100 μL·mL⁻¹ of concentration. Bars represent the average values of fungal growth area (cm²); whiskers indicate the SD of the mean of three independent experiments used to verify experimental errors.

Figure 2

Percentage of P. citricarpa hyphae with the permeabilized cytoplasmic membrane. Hyphae with intact membranes are colored green, while the permeabilized membranes are colored red. The concentration of NEO was at 100 μL·mL⁻¹, and the control was a sterilized saline solution (NaCl 0.87%) with 1% of DMSO. The pictures showed were at the overlay of Tx Red/eGFP and phase contrast images, with a magnification of ×100. The experiment was performed thrice and at least 50 hyphae were evaluated per experiment (n > 150). Horizontal bars are the average percentage; whiskers are the average SD for verification of experimental errors.

Figure 3

NEO spectral by GC-MS/MS.

Figure 4

Molecular structure of compounds associated with the antifungal activity by NEO.

Figure 5

Colony forming units rescued from the surface of citrus fruits after the sanitization assays at the final time of 40 days. Bars represent the averages of rescued cells; whiskers indicate the SD of the means. Three independent experiments were performed. Data showing the same letters are not significantly different from each other based on the nonparametrical statistical analysis of Kruskal–Wallis (Dunn), with three degrees of freedom. Negative control (NC) were fruits washed using sterile deionized water with 1% of DMSO; positive control (PC) were fruits sanitized with copper oxychloride at 90 g·L⁻¹ of concentration; NEO was applied at 100 μL·mL⁻¹ of concentration with 1% of DMSO as a vehicle. The H = 56.5859; p < 0.05 for NC to PC/NEO; the Z critical value was equal to 2.394 for all treatments; the Z-score value was 0.8957 for PC when compared with NEO, and the Z-score value was greater than 6.00 for NC when compared with PC or NEO.