Biosynthesis of Trichoderma harzianum mediated silver nanoparticles (AgNPs), their characterization and in vitro antimicrobial potential against Fusarium oxysporum f. sp. lycopersici and Sclerotium rolfsii

Introduction

Synthesis of nanoparticles represents a cornerstone of nanotechnological advancement. Silver nanoparticles (AgNPs) have been thoroughly investigated in recent decades, and their application in agriculture offers an alternative protective measure against diseases and pests, which could lessen our reliance on environmentally harmful fungicides and pesticides. Species belonging to genus Trichoderma are commonly acknowledged for their role in biological control of plant pathogens. These filamentous fungi exhibit mycoparasitic behavior through mechanisms such as hyphal coiling and secretion of cell wall-degrading enzymes (Zin and Badaluddin, 2020) [16] . Additionally, Trichoderma species possess NADHdependent enzymes, incorporating nitrate reductase that hold significant importance in nanoparticle synthesis and stabilization (Muhammad et al. 2021) [11] . Prior investigations have illustrated that AgNPs produced through Trichoderma spp. exhibit strong inhibitory effects against a range of phytopathogenic fungi and bacteria (Elgorban et al. 2016; Chandrasekhar et al. 2016) [5,2] . Biosynthesis AgNPs was carried out by using the extracellular culture filtrate of fungal biocontrol agent Trichoderma harzianum and their characterization was done by using UV-Visible spectroscopy and Transmission Electron Microscopy (TEM) available at Eco-friendly Disease Management and Beneficial Microbes Research Laboratory, MPKV, Rahuri (M.S) India.

Materials and Methods

Chemical

Silver nitrate (AgNO₃) used in the present investigation was procured from Hi-Media Laboratories Pvt. Ltd. The molecular weight of AgNO₃ is 169.87 g mol⁻¹.

2.2 Synthesis of silver nanoparticles from culture filtrate of fungal culture 90 mL of aqueous solution of 5mM Silver nitrate (AgNO3) (equivalent to 170 ppm) was mixed with 10 mL of T. harzianum, culture filtrate for the synthesis of silver nanoparticles in a 250 mL conical flask. The conical flask was covered with the aluminum foil and the whole mixture was incubated at room temperature for 24 hrs. The color change of silver nitrate from colorless to brown color was considered as indicator of formation of silver nanoparticles through reduction of silver ions from Ag + to Ag 0 . The silver nanoparticles solution was centrifuged at 10000 rpm for 20 minutes to separate the AgNPs. The resultant pellet was kept in hot air oven overnight at 60 °C to make a fine powder of nanoparticles. The stepwise protocol employed for the biosynthesis of silver nanoparticles using Trichoderma harzianum culture filtrate is illustrated in Plate 1.

Plate 1: Photographs showing biosynthesis of silver nanoparticles from Trichoderma harzianum culture filtrate.

2.3

Characterization of Biosynthesized Silver Nanoparticles Silver nanoparticles synthesized by T. harzianum culture filtrate was analyzed using visual observation in color change and techniques such as UV-visible spectroscopy and Transmission Electron Microscopy (TEM).

Visual observation in color change

Visual observation in color change and color intensity during the incubation period of 24 hrs. was recorded.

UV-visible spectroscopy

UV-Visible spectroscopy analysis was done by using Evolution 201 UV-Visible Spectrophotometer available at Department of Soil science, NRC on Grape, Pune. The reduction of silver ions were monitored after 24 hrs. by recording the UV-Vis spectrum of the reaction medium after diluting a small aliquot of the sample into deionized water. The spectra of the surface plasmon resonance of AgNPs in the reaction mixture was recorded using UV-Vis spectrophotometer at wavelengths between 200 to 800 nm.

Transmission electron microscopy (TEM)

Transmission electron microscopy (TEM) was utilized for the analysis of shape and size and used to image the synthesized AgNPs. TEM analysis of the sample was carried out at Eco-friendly Disease Management and Beneficial Microbes Research Laboratory, MPKV, Rahuri.

Culture Collection

The pure cultures of test fungal pathogens included Fusarium oxysporum f. sp. lycopersici, the causal organism of Fusarium wilt of tomato, and Sclerotium rolfsii responsible for stem rot disease of groundnut were obtained from Department of Plant Pathology and Agricultural Microbiology, Mahatma Phule Krishi Vidyapeeth (MPKV), Rahuri, Maharashtra and the fungal pathogens were maintained on Potato Dextrose Agar (PDA) medium.

2.5

In vitro antimicrobial potential of Trichoderma harzianum mediated biosynthesized silver nanoparticles (AgNPs) against test fungal plant pathogens Silver nanoparticles synthesized by T. harzianum culture was evaluated under in vitro conditions against the test fungal plant pathogens by poisoned food technique, using PDA as basal medium to observe the inhibitory effect of these AgNPs on the mycelial growth of test fungal plant pathogens. Double strength PDA medium (Potatoes-400 g, Dextrose-40 g, Agar 40 g, Distilled Water-1000 mL) was prepared by doubling the amount of constituents except distilled water and the medium was sterilized at 15-20 psi pressure for 20 minutes. Simultaneously, different concentrations (@ 10 ppm, 25 ppm, 50 ppm, 75 ppm and 100 ppm) of silver nanoparticles were prepared in sterilized distilled water so as to get desired concentration of AgNPs after mixing the AgNPs solutions in the double strength media in the 250 ml flask. The PDA added with the AgNPs was shaken well to mix thoroughly and then poured into the Petri plates and allowed to solidify. A small culture bit of 5 mm size of seven days old culture of the test pathogen was cut with a sterile cork borer and picked up with the help of sterilized inoculation needle and then inoculated in the centre of each Petri plate under aseptic conditions in the laminar air flow and incubated at 27 ± 1 °C for a period of 7 days. The Petri plates poured with double strength PDA medium without amending AgNPs was served as untreated control. Observations on radial mycelial growth/colony diameter (mm) of the test pathogens was recorded after 7 days. Per cent inhibition of mycelial growth of the test pathogens over untreated control was calculated.

Results and Discussion

Characterization of Biosynthesized AgNPs from Trichoderma harzianum Culture Filtrate Visual Observation (Color Change)

The reaction mixture comprising Trichoderma harzianum culture filtrate and silver nitrate solution exhibited a gradual but pronounced color change from colorless/light yellow to dark brown during the incubation period of 24 hrs. The highest dark color intensity (+++) was recorded for T. harzianum-mediated synthesis confirming efficient bioreduction. The color change observed in Trichodermamediated synthesis is a well-recognized qualitative indicator of AgNPs formation These results validate previous results by Singh and Raja, 2011 [14] ; Elamawi et al. 2018 [3] ; El-Wakil, 2020 [6] ; El-Ashmony et al. 2022 [4] where brown coloration was considered a primary indicator of AgNP formation using Trichoderma spp. culture filtrates.

UV-Visible Spectroscopy

UV-Visible spectrophotometric analysis of Trichoderma harzianum mediated silver nanoparticles revealed a distinct, sharp and well-defined absorption peak at 428.87 nm, with a maximum absorbance value of 2.143 confirming the formation of nanosized silver particles as graphically showed in graph Figure 1. Peak was narrow and symmetrical, indicating homogeneous particle size distribution and minimal aggregation. The observed SPR peak lies within the characteristic wavelength range reported for biologically synthesized AgNPs and indicates uniform particle size distribution. Comparable absorption peaks were onserved earlier for Trichoderma-mediated AgNP synthesis, ranging from 385 to 435 nm, validating the present findings by Elamawi et al. 2018 [3] ; Kaman and Dutta, 2019 [9] ; El-Wakil, 2020 [6] ; El-Ashmony et al. 2022 [4] ; Narware et al. 2023 [12] . [3] ; Kaman and Dutta, 2019 [9] ; El-Wakil, 2020 [6] ; El-Ashmony et al. 2022 [4] , confirming the reliability of the biosynthesis protocol employed in the present investigation.

Plate 2: TEM Micrograph showing the relatively spherical shape silver nanoparticles with a size range of 13.28 to 15.12 nm, synthesized by using Trichoderma harzianum culture filtrate. The present findings clearly demonstrate the strong antifungal potential of Trichoderma harzianum mediated AgNPs against F. oxysporum f. sp. lycopersici. The progressive increase in growth inhibition with increasing AgNPs concentration indicates a direct relationship between nanoparticle dose and antifungal efficacy. At higher concentrations, AgNPs likely interact more effectively with fungal cell walls and membranes, leading to structural damage, disruption of membrane permeability, and interference with metabolic processes, ultimately suppressing hyphal extension. The pronounced inhibition observed at 75 and 100 ppm suggests that higher nanoparticle density enhances contact with fungal hyphae, resulting in increased cellular stress and growth arrest. The superior efficacy of Trichoderma-derived AgNPs may also be attributed to the presence of fungal metabolites acting as capping agents, which could synergistically enhance nanoparticle toxicity. The results of the present investigation are in close agreement with earlier reports. Al-Abboud (2018) [1] documented complete growth inhibition of F. oxysporum at 80 ppm utilizing AgNPs biosynthesized from T. harzianum. Similarly, Elamawi et al. (2018) [3] reported high inhibition percentages of AgNPs prepered by Trichoderma longibrachiatum against various fungal pathogens, involving F. oxysporum. El-Wakil (2020) [6] demonstrated significant antifungal activity of AgNPs synthesized from various Trichoderma species against Fusarium spp., with T. hamatum showing the highest inhibitory response. Recent findings by Thepbandit et al. (2024) [15] further support the present results, reporting 83.66 % inhibition of Fusarium solani by T. harzianum-derived AgNPs. Additionally, the antifungal effectiveness observed in this study is inline with previous investigations conducted by Jo et al. (2009) [8] , Kim et al. (2012) [10] , Jolanta et al. (2013) [7] and Ouda (2014) [13] , who collectively demonstrated the broad-spectrum antifungal action of AgNPs against plant pathogenic fungi. Overall, the results confirm that AgNPs derived from Trichoderma harzianum demonstrate strong antifungal action against F. oxysporum f. sp. lycopersici, with higher concentrations showing significantly enhanced inhibitory effects.

in vitro

Plate 3: Photograph showing in vitro evaluation of antifungal potential of Trichoderma harzianum mediated biosynthesized silver nanoparticles (AgNPs) against Fusarium oxysporum f. sp. lycopersici. Previous research provides corroborative evidence for the present observations. Kaman and Dutta (2019) [9] documented substantial growth inhibition of S. rolfsii when silver nanoparticles derived from Trichoderma asperellum were applied, with inhibition exceeding 70 per cent at 100 ppm concentration. Their findings demonstrated that sclerotial fungi are particularly sensitive to nanosilver, a trend that was clearly reflected in the present study. Investigations conducted by El-Ashmony et al. (2022) [4] further emphasized the antifungal potential of Trichodermabased AgNPs, where nanoparticles synthesized from T. harzianum caused substantial suppression of Sclerotinia sclerotiorum and S. rolfsii. The observed reduction in linear mycelial growth across varying concentrations indicated that the antifungal action of AgNPs is not limited to a single pathogen but extends to multiple soil-borne fungi with similar infection strategies. The heightened antifungal response against S. rolfsii might be because of dual action of AgNPs and antifungal metabolites produced by Trichoderma species. These metabolites, including hydrolytic enzymes and secondary antifungal compounds, may become associated with the nanoparticle surface, thereby enhancing penetration and disrupting fungal cellular integrity. Consequently, the present findings reaffirm the suitability of Trichoderma harzianum-mediated silver nanoparticles as an effective biological alternative for controlling Sclerotium rolfsii.

In vitro Evaluation of Antifungal Potential of

Conclusion

Successful biosynthesis of AgNPs was attained utilizing

Trichoderma harzianum culture filtrate. Trichoderma-mediated biosynthesized AgNPs demonstrated significant antifungal efficacy against Fusarium oxysporum f. sp. lycopersici and Sclerotium rolfsii. The significant in vitro inhibition of test pathogens underscores the potential of this biosynthesized AgNPs as a sustainable, eco-friendly component of integrated disease management strategies, offering an alternative to reduce reliance on synthetic chemical pesticides.

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