Chitosan Nanoparticles Inactivate Alfalfa Mosaic Virus Replication and Boost Innate Immunity in Nicotiana glutinosa Plants

Abstract

Plant viral infection is one of the most severe issues in food security globally, resulting in considerable crop production losses. Chitosan is a well-known biocontrol agent against a variety of plant infections. However, research on combatting viral infections is still in its early stages. The current study investigated the antiviral activities (protective, curative, and inactivation) of the prepared chitosan/dextran nanoparticles (CDNPs, 100 µg mL^-1) on Nicotiana glutinosa plants. Scanning electron microscope (SEM) and dynamic light scattering analysis revealed that the synthesized CDNPs had a uniform, regular sphere shapes ranging from 20 to 160 nm in diameter, with an average diameter of 91.68 nm. The inactivation treatment was the most effective treatment, which resulted in a 100% reduction in the alfalfa mosaic virus (AMV, Acc# OK413670) accumulation level. On the other hand, the foliar application of CDNPs decreased disease severity and significantly reduced viral accumulation levels by 70.43% and 61.65% in protective and curative treatments, respectively, under greenhouse conditions. Additionally, the induction of systemic acquired resistance, increasing total carbohydrates and total phenolic contents, as well as triggering the transcriptional levels of peroxidase, pathogen-related protein-1, and phenylalanine ammonia-lyase were observed. In light of the results, we propose that the potential application of CDNPs could be an eco-friendly approach to enhance yield and a more effective therapeutic elicitor for disease management in plants upon induction of defense systems.

Keywords: alfalfa mosaic virus; antiviral activity; chitosan nanoparticles; gene expression.

Figure 1

Structural and compositional characterization of chitosan/dextran nanoparticles (CDNPs). (A) SEM image of the prepared CDNPs, (B) particle size distribution of the synthesized CDNPs using dynamic light scattering technique. (Bar = 1 µm at ×15,000), (C,D) FTIR spectra of pure chitosan and CDNPs, respectively.

Figure 2

(A) Calico symptoms on naturally AMV-infected potato (Solanum tuberosum L.) plants; (B) local lesions developed on Ch. amaranticolor leaves in response to AMV after mechanical inoculation at 5 dpi.

Figure 3

Phylogenetic tree based on the nucleotide sequence of the coat protein (CP) gene of the Egyptian alfalfa mosaic virus isolate OK413670, and other CP genes of AMV isolates retrieved from GenBank. The phylogeny was created using the UPGMA statistical approach and tested using the bootstrap method with 2000 replications.

Figure 4

Effect of chitosan/dextran nanoparticles (CDNPs) on the disease symptoms development on N. glutinosa leaves infected with AMV at 20 days post-inoculation. (A) Mock-inoculated control plants, (B) AMV-inoculated control plants, (C) plants treated with CDNPs (100 µg mL⁻¹) 24 h before inoculation of AMV (protective treatment), (D) plants treated with CDNPs (100 µg mL⁻¹) 24 h after inoculation of AMV (curative treatment), (E) plant treated with a mixture of CDNPs with the same amount of purified TMV and incubated for 1 h (inactivity treatment).

Figure 5

(A) A histogram showing the accumulation level of AMV in AMV-infected N. glutinosa plants at 22 dpi of different treatments. (B) Effect of chitosan/dextran nanoparticles (CDNPs) on total carbohydrates and total phenolics (C) of AMV-infected N. glutinosa plants at 22 dpi of different treatments. Data presented are means ± standard deviation (mean ± SD) of three biological replicates. Different letters indicate statistically significant differences among treatments according to Tukey’s honestly significant difference test (p < 0.05).

Figure 6

Effect of chitosan/dextran nanoparticles (CDNPs) on the relative expression level of peroxidase (POD) in AMV-infected N. glutinosa plants at 3, 6, 10, 15, and 20 dpi. (A) POD relative expression levels of different treatments at 3, 6, 10, 15, and 20 dpi. Data presented are means ± standard deviation (mean ± SD) of three biological replicates. Different letters indicate statistically significant differences among treatments according to Tukey’s honestly significant difference test (p_{time × treatment} < 0.05). (B–E) Simple linear regression (SLR) and quadratic polynomial regression analysis between POD relative expression and time post-inoculation of AMV-infected, protective-treated, curative-treated, and inactivating-treated plants, respectively. Open small circles present the row data (n = 3), solid lines present the SLR line, and polynomial regression models are presented as dashed-line. The 95% confidence intervals for the estimated regression are blue- and yellow-shaded. Regression equations, R², R²_adj, and p-value based on the F test (p < 0.05) were also obtained and presented within the graph.

Figure 7

Effect of chitosan/dextran nanoparticles (CDNPs) on the relative expression level of Pathogenesis-related protein 1 (PR-1) in AMV-infected N. glutinosa plants at 3, 6, 10, 15, and 20 dpi. (A) PR-1 relative expression levels of different treatments at 3, 6, 10, 15, and 20 dpi. Data presented are means ± standard deviation (mean ± SD) of three biological replicates. Different letters indicate statistically significant differences among treatments according to Tukey’s honestly significant difference test (p_{time × treatment} < 0.05). (B–E) Simple linear regression (SLR) and quadratic polynomial regression analysis between PR-1 relative expression and time post-inoculation of AMV-infected, protective-treated, curative-treated, and inactivating-treated plants, respectively. Open small circles present the row data (n = 3), solid lines present the SLR line, while polynomial regression models are presented as dashed-line. The 95% confidence intervals for the estimated regression are blue- and yellow-shaded. Regression equations, R², R²_adj, and p-value based on the F test (p < 0.05) were also obtained and presented within the graph.

Figure 8

Effect of chitosan/dextran nanoparticles (CDNPs) on the relative expression level of phenylalanine ammonia-lyase (PAL) in AMV-infected N. glutinosa plants at 3, 6, 10, 15, and 20 dpi. (A) PAL relative expression levels of different treatments at 3, 6, 10, 15, and 20 dpi. Data presented are means ± standard deviation (mean ± SD) of three biological replicates. Different letters indicate statistically significant differences among treatments according to Tukey’s honestly significant difference test (p_{time × treatment} < 0.05). (B–E) Simple linear regression (SLR) and quadratic polynomial regression analysis between PAL relative expression and time post-inoculation of AMV-infected, protective-treated, curative-treated, and inactivating-treated plants, respectively. Open small circles present the row data (n = 3), solid lines present the SLR line, while polynomial regression models are presented as dashed lines. The 95% confidence intervals for the estimated regression are blue- and yellow-shaded. Regression equations, R², R²_adj, and p-value based on the F test (p < 0.05) were also obtained and presented within the graph.