Anthracnose disease attributed to various Colletotrichum species, manifests as a fungal infection affecting postharvest fruits. Characterized by dark, sunken lesions often encircled by a red or brown halo, anthracnose compromises fruit quality by inducing mushiness or sponginess in the flesh, thereby altering texture and flavour and promoting significant decay to fruits. Infected fruits experience a shortened shelf life due to heightened susceptibility to rot and spoilage, translating into financial setbacks for producers and retailers while diminishing marketability and economic value (Alegbeleye et al., 2022). Moreover, anthracnose can persist and exacerbate throughout storage and transportation, resulting in substantial postharvest losses.
Colletotrichum gloeosporioides, a common fungal pathogen that significant contributor to anthracnose, a destructive postharvest disease in fruits. Upon infiltrating fruits, C. gloeosporioides breaches the fruit epidermis via wounds, natural openings, or directly through the intact surface. Once established, the fungus proliferates, giving rise to characteristic dark, sunken lesions on the fruit's exterior. This colonization initiates the breakdown of cell walls and membranes, culminating in tissue decay and softening (Oliviera Silva et al., 2022). Furthermore, C. gloeosporioides secretes enzymes and toxins that facilitate invasion and nutrient acquisition from the host, exacerbating the harm inflicted. As the infection advances, the fruit becomes increasingly susceptible to secondary infections and rapid deterioration, ultimately leading to substantial economic losses for growers and distributors (Peralta-Ruiz et al., 2023). Hence, stringent postharvest handling and storage practices are imperative to mitigate these losses.
However, due to the lack of cultivars resistant to anthracnose, in practice, application of biological and synthetic fungicides with different active ingredients such as imazalil, fludioxonil, and thiabendazole have been used for controlling anthracnose in fruit postharvest (Bhatta, 2021; Ezzouggari et al., 2024). Prolonged exposure of these fungicides at high concentration can cause skin and eye irritation, as well as gastrointestinal symptoms such as nausea and vomiting (Tao et al., 2020). While biological based fungicides consist of complex components that their intense flavors affect the fruit quality and high melting point of the natural extracts usually requires specific carrier to dissolve them and hence may limit their uses as antimicrobials in various products, since the practical application of natural antimicrobials often requires sufficient concentration. Considering the increase of consumer awareness on human health and environmental issues, there is an urgent need to develop efficient, sustainable, and eco-friendly agents to mitigate the postharvest anthracnose of mango.
Several postharvest management strategies, including the utilization of films and edible coatings, have been developed to combat anthracnose in fruit. However, the adoption of various packing materials is constrained by safety concerns, inadequate water barrier properties, and the potential presence of hazardous chemicals that could compromise food safety (Amirullah et al., 2022). However, edible coating materials like lipids, polysaccharides, and proteins have drawbacks in their water vapour barrier, unpleasant flavors, sensitivity to environmental conditions, and potential contamination with other substances that limit their applications (Iniguez-Moreno et al., 2021; Silva et al., 2011). These have a substantial impact on their performances by making the fruit more vulnerable to disease attack and changing the fruit's overall quality. Thus, a quest for novel, environmentally friendly treatments with good water barrier properties is essential to solve the above challenges.
This study proposes hydrophobic deep eutectic oil-in-water nanoemulsion (HyDEN) as a promising edible coating material due to its notable hydrophobic, antioxidant, and antibacterial properties, effectively combating postharvest anthracnose. Hydrophobic deep eutectic solvents (HDES), as elucidated by van Osch et al. (2019) and Martins et al. (2018), are derived from solid fatty acids and terpenes components, rendering them insoluble in water. However, the challenge of their low solubility in water is addressed by transforming them into an emulsion-based system, such as a nanoemulsion, as highlighted by Zeng et al. (2021). Upon nano-emulsification, optimized HDES-in-water nanoemulsion, as demonstrated by Syed et al. (2022), exhibits enhanced synergistic antibacterial activity against E. coli and S. aureus, alongside heightened efficacy against C. acnes. Furthermore, Zeng et al. (2021) discovered that nano-emulsified HDES displays intensified antibacterial activity, influenced by oil phase properties, particle size, and droplet size. Additionally, as observed by Gidado et al. (2023), nanoemulsified HDES-in-water demonstrates potent antifungal activity against C. gloeosporioides, attributed to its robust mode of action, binding affinity, and rapid onset compared to both HDES and commercial fungicide.
Understanding the mechanism of action underlying the antifungal activity of HyDEN is crucial for elucidating their efficacy and potential application in postharvest disease management. Specifically, targeting on analysing physical changes in the membrane structure of C. gloeosporioides such as mycelial growth inhibition, spore germination, cellular leakage of soluble sugar, and electrical conductivity. The mechanism of cellular leakage involves the disruption of membrane permeability barriers that normally control the movement of substances across the membrane. When the membrane is damaged, these barriers are compromised, allowing molecules to diffuse freely in and out of the cell. Soluble sugars and ions present within the cell can then leak out into the surrounding environment, leading to an increase in electrical conductivity (Cooper, 2000). When an antifungal agent is introduced to fungal cells such as C. gloeosporioides, it can target specific components of the cell membrane or cellular machinery, leading to membrane disruption and spore germination inhibition. Antifungal agents may disrupt the membrane by interfering with its structure, altering lipid composition, or inhibiting key enzymes involved in membrane synthesis or maintenance. As a result of membrane disruption, several cellular components may leak out of the fungal cells. Soluble sugars and ions are among the substances that can leak out of the cell when the membrane integrity is compromised. This leakage occurs because the damaged membrane can no longer regulate the movement of molecules in and out of the cell effectively (Huang et al., 2019).
The objective of this study is to investigate the impact of HyDEN on mechanisms associated with cell membrane degradation and disruption of C. gloeosporioides. Specifically, the study aims to examine the effects of HyDEN on mycelial growth inhibition, cell membrane permeability, spore germination, and cellular leakage of soluble sugars and electrical conductivity, which are indicative of cell membrane integrity and fungal cell viability. By elucidating these mechanisms, the study seeks to provide insights into the antifungal properties of HyDEN and its potential application for controlling anthracnose disease in postharvest fruit.