Assessment of the Biocontrol Efficacy of Silver Nanoparticles Synthesized by Trichoderma asperellum Against Infected Hordeum vulgare L. Germination

Abstract

This study investigated the biosynthesis, statistical optimization, characterization, and biocontrol activity of silver nanoparticles (AgNPs) produced by newly isolated Trichoderma sp. The Trichoderma asperellum strain TA-3N was identified based on the ITS gene sequence, together with its phenotypic characteristics (GenBank accession number: OM321439). The color change from light yellow to brown after the incubation period indicates AgNPs biosynthesis. The UV spectrum revealed a single peak with the maximum absorption at 453 nm, indicating that T. asperellum produces AgNPs effectively. A Rotatable Central Composite Design (RCCD) was used to optimize the biosynthesis of AgNPs using the aqueous mycelial-free filtrate of T. asperellum. The optimal conditions for maximum AgNPs biosynthesis (156.02 µg/mL) were predicted theoretically using the desirability function tool and verified experimentally. The highest biosynthetic produced AgNPs by T. asperellum reached 160.3 µg/mL using AgNO₃ concentration of 2 mM/mL, initial pH level of 6, incubation time of 60 h, and biomass weight of 6 g/100 mL water. SEM and TEM imaging revealed uniform spherical shape particles that varied in size between 8.17 and 17.74 nm. The synthesized AgNPs have a Zeta potential value of -9.51 mV. FTIR analysis provided insights into the surface composition of AgNPs, identifying various functional groups such as N-H, -OH, C-H, C=O, and the amide I bond in proteins. Cytotoxicity and genotoxicity assays demonstrated that AgNPs in combination with T. asperellum can mitigate the toxic effects of Fusarium oxysporum on barley. This intervention markedly enhanced cell division rates and decreased chromosomal irregularities. The results indicate that AgNPs synthesized by T. asperellum show the potential as an eco-friendly and efficient method for controlling plant diseases. Further studies are necessary to investigate their possible use in the agricultural sector.

Keywords: Trichoderma asperellum; barley; biocontrol activity; biosynthesis; characterization; cyto-genotoxicity assay; silver nanoparticles; statistical optimization.

Figure 1

Morphological and structural identification of Trichoderma sp. strain TA-3N: (A) characteristic growth of Trichoderma sp. strain TA-3N on PDA medium after 7 days of incubation at 25 °C; (B) microscopic features displaying septate and branched mycelium along with conidia observed by light microscopy at 100×; and (C,D) scanning electron microscopy (SEM) images.

Figure 2

Phylogenetic tree of Trichoderma sp. strain TA-3N: This phylogenetic tree was constructed using a sequence from ITS regions of Trichoderma sp. strain TA-3N and closely related species. The tree was constructed utilizing 1000 bootstrap replicates, with accession numbers for the sequences indicated in parentheses.

Figure 3

The color change demonstrates the biosynthesis of AgNPs using T. asperellum. Bottle (A) represents the control (aqueous mycelial-free filtrate without AgNO₃); Bottle (B) shows the test flask (aqueous mycelial-free filtrate with AgNO₃) after 72 h of incubation. (C) displays the UV-Vis spectral scan (ranging from 300 to 1000 nm) of the biosynthesized AgNPs.

Figure 4

Three-dimensional plots showing the mutual effects of AgNO₃ conc. (X₁), initial pH level (X₂), incubation time (X₃), and biomass weight (X₄) on AgNPs biosynthesis by T. asperellum. (A–C) show the effect of AgNO₃ concentration on AgNPs biosynthesis when interacting with initial pH level, incubation period and biomass weight; respectively. (A,D,E) show the effect of initial pH level on AgNPs biosynthesis when interacting with AgNO₃ concentration, incubation time, and biomass weight; respectively. (B,D,F) show the effect of incubation time on AgNPs biosynthesis when interacting with the AgNO₃ concentration, initial pH level and biomass weight; respectively. (C,E,F) show the effect of biomass weight on AgNPs biosynthesis when interacting with the AgNO₃ concentration, initial pH level and incubation time; respectively.

Figure 5

(A) Normal probability plot of internally studentized residuals. (B) A plot of internally studentized residuals versus predicted values. (C) Plot of predicted versus actual. (D) Box–Cox plot of model transformation of silver nanoparticle biosynthesis by T. asperellum as affected by AgNO₃ conc. (X₁), initial pH level (X₂), incubation time (X₃), and biomass weight (X₄).

Figure 6

The optimization plot displayed the desirability function and the optimum predicted values of AgNP biosynthesis by T. asperellum.

Figure 7

Biosynthesized AgNPs by T. asperellum: (A) scanning electron microscopy (SEM), (B,C) transmission electron microscopy (TEM) micrographs, and (D) energy-dispersive X-ray spectroscopy (EDX) analysis demonstrating the elemental composition of native silver.

Figure 8

AgNPs synthesized by T. asperellum exhibit (A) Zeta potential and (B) Fourier transform infrared (FTIR) spectrum of various functional groups responsible for stabilizing or capping AgNPs.

Figure 9

Seedling germination traits of H. vulgare under different concentrations of AgNPs, F. oxysporum treatment, silver nitrate, and a combination of these treatments. Abbreviations: T₁, Dist water (control); T₂, spore suspension of F. oxysporum; T₃, silver nitrate solution 1 mM; T₄, (10 mg/L of AgNPs); T₅, (20 mg/L of AgNPs); T₆, (30 mg/L of AgNPs); T₇, 10 mg/L of AgNPs and spore suspension of F. oxysporum; T₈, 20 mg/L of AgNPs and spore suspension of F. oxysporum; T₉, (30 mg/L of AgNPs and spore suspension of F. oxysporum).

Figure 10

Various chromosome aberrations observed in H. vulgare under different treatments for 24 h: (A) diploid cell metaphase (2n =14) after (T₂), (B) stickiness metaphase after (T₁), (C) ring at metaphase after (T₂), (D) disturbed metaphase after (T₅), (E) large non-congression at metaphase after (T8), (F) small non-congression at metaphase after (T₉), (G) bridge at anaphase after (T₃), (H) laggard anaphase after T₉, (I) late separation anaphase after T₉, (J) oblique anaphase after T₂, (K) diagonal anaphase after T₄, (L) disturbed anaphase after T₄, (M) oblique telophase and abnormal cell size after T₅, (N) late separation telophase after T₂, (O) diagonal telophase after T₁, and (P) bridge telophase after T₂ with magnification 1000× except M at 400×.