Mycosynthesis and Application of Fe2O3 Nanoparticles to Diminish Fruit Rot Diseases by Maintaining their Composition and Pertinent Organoleptic Properties

Abstract

Fruit rot diseases pose significant threats to the agricultural sector by impacting the production of fruits. The economy of Pakistan heavily relies on agriculture and the fruit rot diseases are particularly concerning, especially for high-value fruits like peaches, apples, and strawberries. These fruits hold substantial commercial value and contribute in export and local livelihoods. Addressing these challenges requires the adaptation of innovative technologies for the sustainable production of these plants. In the last decade, nanotechnology has become one of the most utilized technologies in the food and agricultural industry. This study was planned to optimize the mycosynthesis and application of iron oxide nanoparticles (Fe₂O₃ NPs) to control fruit rot diseases of peaches, apples and strawberries. For better understanding, this study has been described in three parts. In the first section, surveys were conducted to collect apple fruit with typical brown rot symptoms. Diseased fruit parts were cultured in Petri plates containing potato dextrose agar media. After 3-5 days, mycelial mass could be observed in Petri plates and based on its morphological, microscopic and molecular analyses, it was identified as Fusarium oxysporum. For the control of this disease, a known beneficial fungus (Trichoderma harzianum) was used to synthesize Fe2O3 NPs. For this purpose, FeCl3 was mixed in the potato dextrose broth extract of T. harzianum and calcinated to synthesize Fe2O3 NPs. Before the application of these mycosynthesized Fe2O3 NPs to control brown rot disease of apple, sophisticated techniques were used for their characterization. FTIR spectroscopy showed the presence of different reducing and stabilizing compounds on the surface of Fe2O3 NPs. The average size (17.78 nm) of these mycosynthesized Fe2O3 NPs was revealed by X-ray diffraction (XRD) analysis. The purity of Fe2O3 NPs was determined by Energy‐dispersive X‐ray (EDX) which showed strong presence of iron, while scanning electron microscopy (SEM) exhibited a high degree of polydispersity. These characterized NPs were used for foliar application, and it resulted in significant reduction of brown rot symptoms of apple. Moreover, NPs-treated fruit maintained better biochemical composition and greater organoleptic properties than untreated fruit. The superior quality of NPstreated apple fruit was evident by the higher presence of soluble solids, sugars and ascorbic acid. These results depicted the great potential of these NPs in controlling brown rot of apple. In the second part of this study, aflatoxin contamination was assessed in infected peach (Prunus persica L.) fruit and this infection and aflatoxin production was controlled by the application of mycosynthesized Fe2O3 NPs. For this purpose, diseased peach fruit were collected, and the disease-causing pathogen was isolated on SDA. By observing the morphology of isolated pathogen on Pteri plates and under microscope, and by analyzing its 16S sequence, the diseasecausing pathogen was identified as Aspergillus flavus. To see the aflatoxin-producing ability of this isolated strain of A. flavus, in vitro, it was cultured in Petri plates, on SDA. After one week, fully grown fungus was observed under UV light at 365 nm wavelength. These Petri plates were then exposed to the vapors of ammonium hydroxide (31%), which changed the colony color to light grey, and confirmed the presence of aflatoxin. To control peach-fruit disease, Fe2O3 NPs were synthesized in the filtrate of a T. harzianum (as described in the previous section) and characterized. FTIR spectrum revealed the attachment of secondary amines, alcohol and alkyne with Fe2O3 NPs. XRD analysis confirmed their nano-size, and the SEM analysis revealed their spherical shape. The EDX spectrum displayed strong signals of iron (74.38%), indicating the successful formation of Fe2O3 NPs. These NPs inhibited mycelial growth in Petri plates, in vitro, and the highest growth inhibition (65.4%) was exhibited by 1 mg/ml concentration of NPs. The concentration performed best in controlling fruit rot of peach, in vivo. In infected NPs-treated peach fruit, higher amounts of soluble solids, sucrose, total sugar and ascorbic acid were observed than infected and untreated control fruit. Similarly, treated fruit displayed higher titratable acidity and firmness than untreated peach fruit. In these infected fruits, the production of aflatoxin was observed by using three reliable methods including thin layer chromatography (TLC), enzyme-linked immunosorbent assay (ELISA) and high-performance liquid chromatography (HPLC). All these techniques verified the variable production of aflatoxins in NPs-treated and untreated fruits. Treatment of peach fruit with Fe2O3 NPs not only decreased the disease infestation of peach fruit but also resulted in less production of aflatoxins. In infected and untreated control fruit, the maximum production of aflatoxins was detected. In the third and last section of this study, Fe2O3 NPs were applied to increase the shelflife of strawberries at room temperature. As described in the first section, Fe2O3 NPs were mycosynthesized in the cell-free extract of T. harzianum. UV–vis spectroscopy at 420 nm indicated the synthesis of NPs and FTIR spectroscopy showed the presence of amines, aromatics, and alkenes on the surface of Fe2O3 NPs. XRD analysis determined their nano-size, while EDX analysis confirmed the significant presence (68%) of iron. SEM microscopy verified the spherical flowery-crystalline morphology of Fe2O3 NPs. Healthy strawberries were treated with variable doses (0.1, 0.25, 0.50, 0.75, 1.0, and 1.5 mg/ml) of Fe2O3 NPs and their effects on the postharvest quality attributes of strawberries were monitored for six days. Though all concentrations of superparamagnetic Fe2O3 NPs contributed to enhance their shelf life, 1 mg/ml concentration of Fe2O3 NPs performed best to inhibit the weight loss and preserve their titratable acidity. Application of Fe2O3 NPs also helped strawberries to sustain good quality by maintaining higher ascorbic acid contents and superior firmness. Conclusive findings of this study signify the environment-friendly use of Fe₂O₃ NPs for the management of fruit rot diseases of peaches, apples and strawberries. T. harzianum is well known for its biocontrol activities. Using the metabolites of this fungus to synthesize NPs is a next-level utilization of this human-gut friendly fungus. Through the mycosynthesis process, these nanoparticles offer an eco-friendly alternative to conventional practices of disease control and management. The findings of this thesis encourage the use of nanotechnology in controlling fruit diseases and provide valuable insights for future research and practical applications. Fe₂O₃ NPs are produced in the powder form, which make their application very convenient in the field. As the world is continuously seeking innovative solutions to enhance its agricultural productivity and sustainability, Fe₂O₃ NPs could play a pivotal role in protecting valuable fruit crops and ensuring food security.