The Water-Soluble Chitosan Derivative, N-Methylene Phosphonic Chitosan, Is an Effective Fungicide against the Phytopathogen Fusarium eumartii

Abstract

Chitosan has been considered an environmental-friendly polymer. However, its use in agriculture has not been extended yet due to its relatively low solubility in water. N-Methylene phosphonic chitosan (NMPC) is a water-soluble derivative prepared by adding a phosphonic group to chitosan. This study demonstrates that NMPC has a fungicidal effect on the phytopathogenic fungus Fusarium solani f. sp. eumartii (F. eumartii) judged by the inhibition of F. eumartti mycelial growth and spore germination. NMPC affected fungal membrane permeability, reactive oxygen species production, and cell death. Also, this chitosan-derivative exerted antifungal effects against two other phytopathogens, Botrytis cinerea, and Phytophthora infestans. NMPC did not affect tomato cell viability at the same doses applied to these phytopathogens to exert fungicide action. In addition to water solubility, the selective biological cytotoxicity of NMPC adds value in its application as an antimicrobial agent in agriculture.

Keywords: Fusarium solani f. sp. eumartii; N-methylene phosphonic chitosan derivative; Solanum lycopersicum; antifungal activity.

Fig. 1

Antimicrobial dose-dependent effect of N-methylene phosphonic chitosan (NMPC). (A) The values represent the percentages of total spores/sporangia present in each sample after the incubation with different concentrations of NMPC for 24 h. Each value is the mean ± SD of at least 3 independent experiments. (B) Representative images are shown. Scale bars = 22 μm (upper panels), 15 μm (middle panels), 30 μm (lower panels).

Fig. 2

N-Methylene phosphonic chitosan (NMPC) inhibits Fusarium solani f. sp. eumartii (F. eumartii) mycelial growth. F. eumartii was inoculated in a liquid potato dextrose broth medium with different concentrations of NMPC and incubated for 4 days. The quantification of the mycelial-dry weight is expressed as the percentage of control (100%). Each value is the mean ± SD of 3 independent experiments. Different letters point out statistically significant differences (Tukey’s test, P < 0.05).

Fig. 3

N-Methylene phosphonic chitosan (NMPC) mediates fungicidal action on Fusarium solani f. sp. eumartii (F. eumartii) spores. Spores were incubated with 1 or 5 μg/ml NMPC for 24 h and then plated on the Petri dishes containing fresh potato dextrose agar media to allow fungal growth. F. eumartti was grown for 3 days at 25°C. Values represent the percentage of colony-forming units compared to control (100%). Each value is the mean ± SD of at least 3 independent experiments. Different letters point out statistically significant differences (Tukey’s test, P < 0.05). Representative images are shown (inset).

Fig. 4

N-Methylene phosphonic chitosan (NMPC) affects Fusarium solani f. sp. eumartii (F. eumartii) cell viability. Fungal spores were incubated with 1 μg/ml or 5 μg/ml NMPC for 24 h and stained with propidium iodide. Dead spores are observed in red fluorescence. (A) Representative images are shown. The bright-field image for each treatment is shown below the respective fluorescent images. Scale bars = 22 μm. (B) Values represent the percentage of the stained spores present in each sample. Each value is the mean ± SD of at least 3 independent experiments. Different letters point out statistically significant differences (Tukey’s test, P < 0.05).

Fig. 5

N-Methylene phosphonic chitosan (NMPC) induces cell membrane permeabilization. Kinetic of cell membrane permeabilization by 5 μg/ml NMPC in fungal spores. Cell membrane permeabilization was visualized in Fusarium solani f. sp. eumartii (F. eumartii) spores by SYTOX Green probe. (A) Representative images are shown. Scale bars = 22 μm. (B) Values represent the percentage of green spores present in each sample. Each value is the mean ± SD of at least 3 independent experiments. Different letters point out statistically significant differences (Tukey’s test, P < 0.05).

Fig. 6

N-Methylene phosphonic chitosan (NMPC) induces H₂O₂ production in Fusarium solani f. sp. eumartii (F. eumartii) spores. Spores were treated with 2.5 or 5 μg/ml NMPC for 4 h before 3,3′-diaminobenzidine (DAB) staining and subjected to microscopic analysis. (A) Representative images are shown. Scale bars = 22 μm. (B) Values are expressed as a percentage of total spores present in each sample. Each value is the mean ± SD of at least 3 independent experiments. Different letters point out statistically significant differences (Tukey’s test, P < 0.05).

Fig. 7

N-Methylene phosphonic chitosan (NMPC) does not affect tomato cell viability. Suspension-cultured tomato cells were incubated with different concentrations of NMPC for 24 h and then stained with Evans blue. As negative and positive controls, water and 1% Triton X-100 respectively, were included. (A) Representative images of at least 3 independent experiments are shown. Scale bars = 20 μm. (B) Quantification of cell viability was estimated by recording Evans blue retention in tomato cells. Values are expressed as the percentage of water control (100%). Each value is the mean ± SD of at least 3 independent experiments. Different letters point out statistically significant differences (Tukey’s test, P < 0.05).