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Review

Nano-Biofungicides and Bio-Nanofungicides: State of the Art of Innovative Tools for Controlling Resistant Phytopathogens

by
José Sebastian Dávila Costa
1,* and
Cintia Mariana Romero
1,2,*
1
Planta Piloto de Procesos Industriales Microbiológicos—(PROIMI-CONICET), Av. Belgrano y Pasaje Caseros, Tucumán T4001 MVB, Argentina
2
Facultad de Bioquímica, Química y Farmacia, Universidad Nacional de Tucumán (UNT), Ayacucho 471, Tucumán T4001 MVB, Argentina
*
Authors to whom correspondence should be addressed.
Biophysica 2025, 5(2), 15; https://doi.org/10.3390/biophysica5020015
Submission received: 26 February 2025 / Revised: 28 March 2025 / Accepted: 16 April 2025 / Published: 22 April 2025
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Abstract

:
Fungal diseases represent a significant threat to global agriculture, leading to substantial crop losses and endangering food security worldwide. Conventional chemical fungicides, while effective, are increasingly criticized for their detrimental environmental impacts, including soil degradation, water contamination, and the disruption of non-target organisms. Additionally, the overuse of these fungicides has accelerated the emergence of resistant fungal strains, further challenging disease management strategies. In response to these issues, bio-nanofungicides and nano-biofungicides have emerged as a cutting-edge solution, combining biocompatibility, environmental safety, and enhanced efficacy. These advanced formulations integrate bio-based agents, such as microbial metabolites or plant extracts, with nanotechnology to improve their stability, controlled release, and targeted delivery. Chitosan, silica, and silver nanoparticles were extensively studied for their ability to encapsulate bioactive compounds or because of their outstanding antifungal activity, while minimizing environmental residues. Recent studies demonstrated the potential of nano-based fungicides to address critical gaps in sustainable agriculture, with promising applications in integrated pest management systems. Here, we summarize the last advances in the development of bio-nanofungicides and nano-biofungicides and analyze the main differences between them. In addition, challenges such as large-scale production, regulatory approval, and comprehensive risk assessments are discussed.

Graphical Abstract

1. Introduction

The growing global population and the phenomenon of climate change are two of the principal concerns of the contemporary world. Every year, the necessity for innovative and eco-friendly technologies to enhance the efficiency of industrial activities increases. The development of materials through nanotechnology has gained attention because of their exceptional physicochemical properties. Indeed, nanomaterials have emerged as innovative tools for improving various industrial processes and products.
For several years, advances in nanotechnology were related to the study of non-biological physicochemical processes. For instance, the synthesis of nanoparticles was one of the most developed topics, as evidenced by the high number of scientific publications [1,2,3]. Firstly, nanoparticles were synthesized through non-biological methods. In other words, organic and inorganic precursors, reducing agents, and stabilizers, often under extreme temperature and pH conditions, were combined in order to produce nanoparticles [4]. Unfortunately, even to this day, stability and toxicity are two of the biggest unsolved problems of chemically synthesized nanoparticles [5].
The necessity to search for eco-friendly alternatives for the chemical synthesis of nanoparticles has led to the exploration of the potential of microorganisms. The first scientific report related to the biological synthesis of nanoparticles was published in 2002. In that year, Kowshik et al. reported the extracellular synthesis of silver nanoparticles using a metal-tolerant yeast strain. This study highlighted the relevance and advantages of the biological synthesis of nanoparticles, for instance, the simplicity of particle separation and the environmentally friendly nature of the process [6].
Nanotechnology, particularly through the synthesis of nanoparticles, has had a positive impact on science and different industries. Currently, solar energy systems, electronic devices, and skin-protecting sunscreens are based on nanotechnology. Despite these advances, the use of nanoparticles in agriculture, whether chemically or biologically synthesized, still has not reached its full potential. A major challenge in modern agriculture is the resistance that fungal phytopathogens have developed in recent years.
In the present review, we analyzed and discussed two interesting nanotechnological alternatives to face fungal disease and resistance in crops. The difference between nano-biofungicides and bio-nanofungicides is discussed in this work.

2. Global Impact of Fungal Diseases in Agriculture

Fungal plant pathogens are the primary cause of plant diseases, leading to substantial crop losses globally. Fungicide resistance is a problem of global concern, exacerbated by the emergence of fungicide-resistant strains that compromise the control of diseases. Despite current treatments, fungal diseases cost pre-harvest crops an estimated 10–23% of crop losses per year. Post-harvest losses add 10–20% more. These pathogens impact a variety of crops such as rice, wheat, maize, and soybean [7]. Resistant fungal phytopathogens provoke serious economic losses to crops every year, estimated at USD 60 billion [8].
The severity of fungal diseases may vary each year depending on the environmental conditions, the success of disease control measures, and the development of fungicide-resistant strains. Brazil and the United States are among the major soybean producers globally. In Brazil, for instance, Asian Soybean Rust disease caused by Phakopsora pachyrhizi is the most damaging disease for soybean, with yield losses reaching up to 90% (if not managed properly). The extensive occurrence of this pathogen requires the application of a large amount of fungicide, increasing production costs and THE environmental impact [9]. In the United States, Frogeye Leaf Spot caused by Cercospora sojina led to significant yield reductions each year. For instance, between 2013 and 2017, in Midwestern states, the estimated losses increased from 460,000 to 7.6 million bushels, indicating a growing threat to soybean growers [10].

3. Fungicide Resistance in Phytopathogenic Fungi

The widespread use of synthetic chemical fungicides has driven the evolution of resistant fungal phytopathogens. The resistance to fungicides is an adaptive ability of pathogens to survive and proliferate in the presence of fungicides that were previously effective in controlling them. This phenomenon is a major challenge in agriculture, threatening crop yields and food security [11]. Fungicide resistance mechanisms are well documented in the existing literature [12,13,14]; therefore, this review will not address them further.

3.1. Current Fungicides and Strategies to Combat Fungal Disease and Resistance

Nowadays, farmers use different strategies to manage fungal phytopathogen resistance. The rotation of crops, the use of fungicides with different mechanisms of action, and the optimization of dose to maintain effectiveness and reduce selection pressure are probably the most common strategies. Awarded for the continuous and strong development of fungicide resistance, the agrochemical companies invest in their R&D sector’s to discover the following: 1. fungicides with new mechanisms of action to target resistant pathogens; 2. the effective combination of active ingredients with different mechanisms of action to prevent the easy development of resistance; 3. cocktails of fungicides containing several active ingredients to combat different pathogens simultaneously.
The most common molecules used by the agrochemical companies for the formulation of fungicides are listed in Table 1 alongside their mechanisms of action. As mentioned above, the combination of active ingredients with different mechanisms of action is a widely adopted strategy to control resistance. For instance, formulations containing 400 g/L of Mefentrifluconazole + Pyraclostrobin or 450 g/L of Bixafen + Prothioconazole + Trifloxystrobin are currently available in the market.
The negative environmental impacts of chemical fungicides are well known. They can have residual effects on ecosystems, non-target organisms, and biodiversity in general. Interesting articles addressing this topic have already been published [15,16].

4. The Era of Nanotechnology in Agriculture

In recent years, the scientific community has shown a significantly increased interest in applying nanotechnology to agriculture. Consequently, this growing focus has fueled meaningful discussions and innovation aimed at achieving safer and more sustainable agricultural practices. Although nanotechnology presents a significant opportunity to enhance both productivity and sustainability in agriculture, challenges such as potential toxicity, bioaccumulation, and regulatory frameworks continue to generate diverse opinions among researchers [17,18]. The scientific community generally recognizes nanotechnology’s beneficial contributions to agriculture, such as addressing biotic and abiotic stresses in various crops [19], improving yields through the application of nano-pesticides and fertilizers [20], and reducing postharvest losses to preserve food security. Nevertheless, the adoption of nanomaterials in agriculture still introduces risks, highlighting the necessity for clear regulatory frameworks. Nanotoxicity arises from factors like nanoparticle size, aggregation, chemical composition, crystallinity, surface characteristics, and potential impurities from manufacturing processes. These properties may trigger oxidative stress, genotoxicity, or carcinogenic effects on plants, soil organisms, animals, and humans. To mitigate these risks, rigorous safety assessment and standardized validation protocols are essential. While there is no single dedicated regulatory framework specifically governing nanomaterials globally, existing legislation from various regulatory bodies provides some guidance.
In the United States, regulatory oversight for nanotechnology primarily resides with the Food and Drug Administration (FDA) and the Environmental Protection Agency (EPA). Under the Toxic Substances Control Act (TSCA), both agencies collaborate in establishing frameworks for evaluating nanomaterials, obligating the industry to report nanomaterial-related information every five years since 1986. Additionally, voluntary initiatives like the EPA’s Nanoscale Materials Stewardship Program (NMSP), established in 2008, have helped track and assess nanomaterial use, providing valuable safety data [21] and clarified the use of nanotechnology in organic agriculture by classifying engineered nanomaterials as synthetic substances. Approval requires the submission of a formal petition, evaluation by the National Organic Standards Board (NOSB), and a period for public commentary before inclusion on the USDA’s National List. Instead of establishing separate definitions, the USDA adheres to existing standards from the FDA and EPA. This approach ensures a balance between the potential benefits of nanotechnology, rigorous safety assessment, and public transparency [22,23]. In the European Union (EU), several entities, including the European Commission (EC), European Chemicals Agency (ECHA), and European Food Safety Authority (EFSA), have integrated guidelines relevant to nanomaterials within broader regulations. Key legislative frameworks include the Registration, Evaluation, Authorization, and Restriction of Chemicals (REACH), alongside other regulations such as (EC) No 1107/2009, (EC) 258/97, (EC) 1332/2008, and directives like 2002/46/EC and 2009/125/EC. These collectively address various aspects of nanomaterial use and safety assessment [21,24].
Effective regulatory preparedness is critical for safely leveraging nanotechnology’s potential in agriculture. Proactive, integrative, and adaptive regulatory approaches are required to adequately address emerging nanomaterials and ensure responsible innovation [25].

Nanoparticles as Fungicides

Metallic nanoparticles were widely studied for their antimicrobial properties. Indeed, thousands of articles reporting on the chemical or biological synthesis of nanoparticles were published. The number of articles is drastically reduced when the term “nanofungicide” is included in the search filters. According to the National Library of Medicine (Pubmed), only 16 articles related to nanofungicides were published from 2019 to 2024. However, using a stronger database like scite.ai, the number of publications increased to 64. Many of these works were aimed at synthesizing and characterizing metallic nanoparticles in order to evaluate their antifungal activity. Different kinds of chemically synthesized nanoparticles were reported to combat phytopathogenic fungi strains. For example, the Ni0.5Al0.5Fe2O4 nanoparticles with a size between 60 and 80 nm were effective against Fusarium oxysporum at a low concentration (0.5 mg/mL) [26]. Similarly, zinc and copper oxide nanoparticles were able to control the late blight disease of potatoes caused by Phytophthora infestans under greenhouse conditions [27]. The chemical synthesis of nanoparticles is often considered a highly contaminant method. In recent years, biological methods gained attention because of their synthesis efficiency, environmental compatibility, and good performance of their nanoparticles against phytopathogens. Recently, Bharose et al. [28] reported the bacteria-mediated green synthesis of silver nanoparticles and their antifungal potential against Aspergillus flavus. A small number of articles reported the application of biological nanoparticles in the field under non-controlled conditions. Silver nanoparticles extracellularly synthesized by the strain Amycolatopsis tucumanensis were capable of controlling one of the most important diseases in sugarcane, the Red Stripe, caused by Acidovorax avenae subsp. avenae [29].

5. Current Nano-Based Alternatives to Manage Fungal Disease and Resistance in Agriculture

As mentioned in Section 2, diseases caused by fungal-resistant strains provoke a negative impact on several crops and, subsequently, on the global economy. Agrochemical companies are continuously developing strategies to control fungal resistance as well as new fungicide formulations. However, the environmental impact of these formulations is extremely high (see Section 3.1 and Section 8). In recent years, several scientific studies were conducted in order to develop efficient fungicide formulations with the lowest possible environmental impact.

5.1. Comparison Between Traditional Fungicides and Nano-Based Alternatives in Real-World Agricultural Settings

The agricultural sector has witnessed an increasing focus on the development of novel fungicides, particularly nano-based alternatives, as a response to the shortcomings associated with traditional chemical fungicides. This shift is largely driven by the detrimental effects of prolonged fungicide use, such as the emergence of resistant fungal strains, environmental toxicity, and adverse impacts on human health. The comparison between traditional fungicides and nano-based alternatives reveals critical insights into their effectiveness and application in real-world agricultural settings. Nano-fungicides demonstrate promising advantages in terms of efficacy, reduced environmental impact, and sustainability. With ongoing research advancements, it is crucial for agricultural policies and practices to adapt, embracing these innovative solutions to safeguard food security and ecological integrity; a comparison is shown in Table 2.

5.2. Nano-Biofungicides and Bio-Nanofungicides

The terms nano-biofungicide and bio-nanofungicide are often used as synonyms. However, it is quite far from reality. Both refer to fungicides that combine nanotechnology with biological agents but are grouped into different categories. See Table 3 for a comprehensive comparison.
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Figure 1. Nano-biofungicide composed of conventional synthetic fungicides such as pyraclostrobin, difenoconazole, etc., nanoencapsulated with synthetic or natural polymers, lipids, or inorganic materials.
Figure 1. Nano-biofungicide composed of conventional synthetic fungicides such as pyraclostrobin, difenoconazole, etc., nanoencapsulated with synthetic or natural polymers, lipids, or inorganic materials.
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Figure 2. Bio-nanofungicide composed of a metallic core and a biological portion known as bio-corona (hard and soft).
Figure 2. Bio-nanofungicide composed of a metallic core and a biological portion known as bio-corona (hard and soft).
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5.2.1. Nano-Biofungicides

Nano-biofungicides are created by nanoencapsulating conventional synthetic fungicides such as pyraclostrobin and epoxiconazole, essentially resulting in a nanoencapsulated active ingredient with fungicidal activity. (Figure 1). Synthetic and natural polymers, lipid-based systems, and inorganic nanomaterials were reported as suitable materials for the synthesis of nano-biofungicide [30,31]. In the last decade, several studies were focused on designing successful formulations of nano-biofungicides. For instance, Bence et al. [32] reported the co-encapsulation of citral and the synthetic fungicide cyproconazole using solid lipid and chitosan nanoparticles. This formulation showed a high efficiency with a minimum inhibitory concentration of 1.56 μg mL−1. Moreover, Yu et al. [33] formulated a multi-stimuli-responsive nanocapsule delivery system loaded with pyraclostrobin. Interestingly, this system exhibited pH/laccase-responsive targeting against Botrytis disease, enabling the intelligent release of pyraclostrobin [33]. The encapsulation of synthetic fungicides to create nano-biofungicides represents a significant advance for modern agriculture.
One of the main advantages of nano-biofungicides is the improvement in the stability of the active ingredients. Traditional fungicides are often degraded when exposed to light and moisture. Nanoencapsulation provides protection to the active compounds, allowing for a controlled release, which improves their efficacy and reduces the number of applications [34,35]. In addition, by reducing the particle size of the active ingredients, nano-biofungicides increase their surface/volume ratio, which improves the absorption by plant tissues. This increased efficacy means that lower doses can achieve the same or better results compared to conventional formulations while reducing the environmental impact [19,36].

5.2.2. Bio-Nanofungicides

Bio-nanofungicides are metallic nanoparticles synthesized biogenically using microorganisms, plant extracts, or enzymes. [37,38]. A bio-nanofungicide is composed of a metallic core, usually reduced silver or copper, and a biological portion (derived from the microorganism, plant, or enzyme used in the synthesis). The biological portion, known as the bio-corona, is the main component of the bio-nanofungicide and forms two layers around the metallic core. (Figure 2). The main advantage of bio-nanofungicides compared with nano-biofungicides and traditional fungicides is precisely the presence of the bio-corona, which improves the effectiveness, stability, and delivery mechanisms, and reduces the negative environmental impact [39,40].
The bio-corona of bio-nanofungicides is a complex and dynamic structure composed of proteins, lipids, carbohydrates, and/or nucleic acids. The bio-corona molecules can be classified into a “hard” corona, which consists of tightly bound molecules, and a “soft” corona, which includes loosely associated molecules that can exchange dynamically (Figure 2) [41]. The precise composition of the bio-corona may vary depending on the biological agent used for the synthesis of the bio-nanofungicide. For instance, the fungus Macrophomina phaseolina synthesized silver nanoparticles with a bio-corona composed of 46 different proteins. Further analysis revealed that 60% of these proteins are hydrolytic enzymes and 21% are oxidoreductases [42]. Using a Streptomyces bacterium, Paterlini et al. [43] also synthetized silver nanoparticles and identified bio-corona proteins. In this work, molecular modeling and docking studies were performed to predict the interaction of bio-corona proteins with the surface of the nanoparticle. Thus, a structural model where proteins like aminopeptidase, tellurium resistant, and superoxide dismutase appeared linked to the surface of the nanoparticle was proposed [43]. These studies represented a significant advance in the understanding of proteins’ bio-corona composition. The identification and quantification of more complex molecules such as lipoproteins, glycolipids, and terpens, among others, were not yet addressed. Thus, metabolomics and proteomics studies conducted in parallel might be an excellent approach to elucidate the complete structure of the bio-coronas. The structure and composition of bio-coronas are crucial to understanding the interaction of bio-nanofungicides with phytopathogens and the environment.

6. Mechanisms of Action of Nano-Biofungicides and Bio-Nanofungicides

The mechanisms of action of nano-biofungicides and bio-nanofungicides are crucial to understand their efficiency in the management of fungal phytopathogens. As described in Section 5, both types of formulations are based on nanotechnology and biological agents, although in different manners, leading to distinct mechanisms of action.
In nano-biofungicides, the mechanism of action against phytopathogens will depend on the synthetic active ingredient that was nano-encapsulated. For instance, pyraclostrobin inhibits mitochondrial respiration, and epoxiconazole blocks the production of ergosterol, which is necessary for fungal cell membranes. Instead, in bio-nanofungicides, the active ingredient is the result of a synergism between the metallic core and the bio-corona.

6.1. Surface Interaction and Adhesion

In a foliar application, the leaf surface is the first barrier that fungicides have to face. Leaf surface is typically covered with a hydrophobic waxy cuticle that serves as a barrier to water and other substances. The ability of nano-biofungicides and bio-nanofungicides to pass through this barrier depends on several factors (Figure 3). Due to their small size, in general, both are able to navigate the complex microstructure of the leaf surface more effectively than conventional formulations. This can lead to better adhesion and retention on the leaf, increasing the probability of successful pathogen control [44]. In terms of interaction and adhesion with the leaf surface, the bio-corona of bio-nanofungicides represents an advantage. As described above, bio-coronas are complex structures composed of different types of biomolecules that give them an amphipathic characteristic. This diversity of molecules makes the interaction between the bio-nanofungicide and different types of leaves more efficient, allowing its application in a wide range of crops.

6.2. Penetration and Uptake

Once adhered to the leaf surface, nano-bio- and bio-nanofungicides must penetrate the cuticle to reach the underlying tissues where pathogens reside. The size of nano-biofungicide and bio-nanofungicide particles is fundamental to facilitating this process. In general, small particles can exploit the natural openings in the leaf surface, such as stomata and trichomes [45]. The polar pathway of penetration involves trichomes, hydathodes, necrosis spots, and stomata. In turn, in the nonpolar pathway, the leaf cuticle and its pores are crucial. In comparison with stomata and hydathodes, the cuticle covers a bigger surface in the leaves, making it the major structure for nano-fungicides delivery to plants through foliar application. The penetration of nano-biofungicides through the polar or nonpolar pathway depends on the chemical nature of the material used for the nanoencapsulation of the active ingredient. Bio-nanofungicides can use both pathways because of the amphipathic nature of their bio-coronas [42,43]. This versatility represents a significant advantage over nano-biofungicides and other fungicide formulations, which often require adjuvants to enhance penetration.
Hydathodes are secreting pores directly connected to the vasculature system. Although the information about the potential entry of nano-formulations in plant leaves through hydathodes is limited, two interesting studies demonstrated that the charge of the nano-molecules is crucial. CeO2 nanoparticles with a size of 8 nm were accumulated inside the hydathodes of lettuce leaves [46]. On the contrary, the negatively charged nanoparticulated polymer poly(ε-caprolactone) penetrated Brassica juncea leaves through hydathode apertures [47]. The role of stomata in the uptake of nano-formulations has been studied more. In this case, the size and charge of the nano-molecule is also important. Though the accumulation of different nano-molecules in stomata was observed, this does not guarantee entry into the plant; it could be a “dead-end” if translocation to other tissues does not occur. Bio-nanofungicide molecules have more chances of overcoming this barrier thanks to their charge and size distribution. The nonpolar uptake pathway involves close contact with the waxy layer on the cuticle, where bio-nanofungicides have the advantage again [48]. After crossing the cuticle, nano-formulations have to cross several barriers through the mesophyll before reaching the vasculature system (see the comprehensive review by Avellan et al., 2021) [49].

6.3. Release and Efficacy

After penetration, nano-biofungicides start the controlled release of active ingredients into the leaf tissue. Indeed, this is a key benefit of nano-biofungicides based on organic, matrices which provide sustained action against fungal pathogens [50]. Several studies were conducted in order to understand the release behavior of these molecules [51]. For instance, chitosan–carrageenan nanoparticles loaded with the fungicide mancozeb released 47% of the fungicide over six days, while solid lipid nanoparticles achieved a slightly higher release of 51% [52]. Recently, Li et al. [53] introduced an interesting molecule, a dual-function nano-pesticide. This intelligent delivery system, containing a fungicide and plant immune response molecules, represents a significant advance in the release and efficacy of fungicides in agriculture. Despite the significant advances, the controlled release of active ingredients from nano-biofungicides and their efficacy require further study, especially for their application in the field. The controlled release of a dose of fungicide can be set for a certain type of phytopathogen in a period of time. However, under non-experimental conditions, several phytopathogens often coexist, which may require different rates of fungicide release. A released dose of fungicide can be effective for a group of phytopathogens but promote resistance in others.
In bio-nanofungicides, antimicrobial activity is principally mediated by the metallic portion of the molecule, which interacts with and inhibits pathogens within the leaf tissue. In this case, the release of the metallic portion is not controlled but may be highly influenced by the presence of the bio-coronas. In recent years, several studies have been focused on understanding not only the structural role of bio-coronas but also their contribution to the antimicrobial activity [54,55,56]. The main conclusion is that the molecules that form the bio-coronas enhance the antimicrobial activity of bio-nanofungicides. However, the mechanisms and specific molecules involved in this antimicrobial synergism remain unclear. This is not unreasonable, if we consider that we are talking about an extremely complex structure in terms of its composition and that, being dynamic, it can alter its composition depending on the environment in which it is located.
Understanding this synergism between the metallic portion and bio-coronas may contribute to optimizing the use of bio-nanofungicides and fungal disease management in crops.

7. Improving Antifungal Strategies by Integrating Nanoparticles with Chemical Fungicides

An interesting alternative is the combination of nanoparticles with chemical fungicides, a strategy that significantly enhances antifungal efficacy and reduces the environmental impact associated with traditional agrochemicals. Recent studies have demonstrated synergistic effects when combining copper oxide nanoparticles (CuONPs) with conventional fungicides such as tebuconazole and iprodione. For instance, the combined use of CuONPs and tebuconazole reduced the minimum inhibitory concentration (MIC) required to control Botrytis cinerea and Fusarium oxysporum by up to eight times compared to CuONPs alone. Similarly, combining CuONPs with iprodione notably decreased the concentrations needed for the effective control of B. cinérea [57].
Studies involving silver nanoparticles (AgNPs), synthesized by both green and chemical methods, have also shown significant synergistic effects when combined with fungicides like carbendazim, mancozeb, and thiram. Green-synthesized AgNPs exhibited better performance than chemically synthesized ones, demonstrating a lower MIC when combined with fungicides. This indicates that the smaller size and biogenic nature of the nanoparticles likely enhance fungicide penetration and efficacy [58].
Biogenic copper or silver nanoparticles, due to their active surface and enhanced stability from antioxidant biological-derived compounds, offer multiple advantages, including reduced toxicity and improved environmental compatibility compared to chemically synthesized nanoparticles. Combining them with traditional fungicides could mitigate pathogen resistance through simultaneous action from different antifungal mechanisms [58,59].
Despite these significant advances, further studies under real field conditions are essential to validate the results and comprehensively evaluate the stability, environmental safety, and economic feasibility of these nanoparticle–fungicide combinations on a larger scale.

8. Environmental Impact—Soil Interaction

The incorporation of nano-molecules in agriculture has raised environmental concerns. Despite their high efficiency, bio-nanofungicides and nano-biofungicides represent a new technology whose long-term environmental impacts remain uncertain.
Nanotechnology can enhance nutrient delivery and soil fertility through the stimulation of soil enzyme activities. For instance, carbon nanoparticles (CNPs) applied to soil improve plant growth in a dose-dependent manner. At optimal concentrations (200 mg/kg), these nanoparticles significantly increased plant height, biomass yield, nutrient uptake, and nutrient use efficiency in corn. This improvement was attributed to the increased availability of nitrogen and phosphorus in the soil and the enhanced activity of soil enzymes such as urease, dehydrogenase, and phosphatase [60]. Additionally, nanoparticles can enhance plant growth and soil health through their interactions with rhizospheric bacteria, emphasizing the importance of applying appropriate doses. For example, the application of sulfidised silver nanoparticles (Ag2S-NPs) at controlled concentrations (5.9 mg Ag kg⁻1) showed better plant growth, demonstrated minimal toxicity toward microbial communities, suggesting safe and effective use in agriculture [61,62].
Compared to conventional fungicides, nano-formulations contain significantly lower concentrations of active ingredients, thus reducing their environmental impact. The main concern regarding nano-formulations relates to their metallic components, particularly silver and copper, which will be the focus of this discussion.
Bio-nanofungicides, described in Section 5.2.2, possess a metallic core surrounded by two biomolecule layers forming a bio-corona. These bio-coronas significantly influence their interactions with the environment [40]. A primary interaction occurs between bio-nanofungicides and soil particles. Humic substances, constituting 40–80% of total soil organic matter, can be divided into humic acids (HAs) and fulvic acids (FAs) [63]. HAs, which are less soluble and more hydrophobic than FAs, contain higher carbon and nitrogen levels and numerous reactive functional groups, such as carboxyl and phenolic groups. Soil organic matter also includes proteins, polysaccharides, lipids, and other organic compounds with functional groups like sulfhydryl and aromatic groups, enhancing their capacity to bind different surfaces. Indeed, studies have demonstrated natural interactions between bio-corona molecules in bio-nanofungicides and soil humic and fulvic acids [64]. During these interactions, soft-corona molecules are often spontaneously replaced by humic or fulvic acids, significantly increasing the size of bio-nanofungicide particles, reducing bioavailability, and consequently decreasing metallic toxicity. Thus, metal-based bio-nanofungicides, at proper concentrations, can positively influence soil properties beneficial to plant growth, including cation exchange capacity, water retention, total organic carbon, and nitrogen and phosphorus availability [64].
Soil microorganisms are indispensable for ecological cycling. Several bacterial phyla, including Actinobacteria, Proteobacteria, Acidobacteria, Verrucomicrobia, Firmicutes, Bacteroidetes, Gemmatimonadetes, Nitrospira, Chloroflexi, and Planctomycetes, positively correlate with agricultural soil quality and crop yield [65]. In a relevant study, agricultural soil treated with 100 mg/kg of silver bio-nanofungicide showed increased relative abundances of Alphaproteobacteria, Betaproteobacteria, and Actinobacteria, while only Bacteroidetes decreased. These findings suggest bio-nanofungicides are less toxic, or even beneficial, compared to conventional fungicides at the tested dose [66]. Similar results were observed by Lin et al. [67], where Fe-based bio-nanofungicide increased the abundance of Saccharibacteria, Proteobacteria, and Actinobacteria, likely linked to iron oxidation and reduction processes.
As discussed, bio-nanofungicides represent a promising new technology for agriculture. Due to their nano-biological nature, their environmental impact depends on chemical–biological characteristics, interactions with the environment, and applied doses. Although multiple studies confirm their non-toxic potential, evidence highlights the importance of case-by-case evaluations.

9. Conclusions

Bio-nanofungicides and nano-biofungicides represent a promising advancement in sustainable agriculture, providing effective alternatives to conventional chemical fungicides with a reduced environmental impact. By leveraging nanotechnology, these innovative formulations enhance the stability, bioavailability, and targeted delivery of bioactive compounds, thereby improving antifungal efficacy and minimizing chemical residues. Despite their significant potential, several critical challenges must be overcome to enable widespread adoption. These include addressing issues related to toxicity, regulatory approval, and field application.
Current studies highlight limitations in toxicity assessments, often lacking comprehensive environmental evaluations that could elucidate long-term ecological impacts. Furthermore, bio-based nanoparticles require optimized methods to achieve consistent efficacy and stability at industrial production levels. The scarcity of extensive field application studies underscores the need for broader evaluations across diverse agricultural conditions, ensuring practical effectiveness and reliability.
Future research should therefore emphasize developing standardized protocols for toxicity testing, including long-term ecological assessments. Research into the interactions between bio-nanofungicides and complex environmental matrices will further clarify their behavior and environmental fate, supporting informed and sustainable agricultural practices. Addressing these research priorities will significantly enhance the practical deployment and acceptance of nano-based fungicide technologies in sustainable agriculture.

Author Contributions

J.S.D.C.: Conceptualization, Investigation, Writing—Original Draft, Funding Acquisition. C.M.R.: Conceptualization, Writing—Review and Editing, Funding Acquisition. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by PICT 2019-00742 and PICT 2021-GRF-TII-00124.

Data Availability Statement

The data presented in this study are available on request from the corresponding author.

Conflicts of Interest

The authors declare no conflict of interest.

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Biophysica 05 00015 g003
Figure 3. Surface interaction, penetration, and uptake of bio-nanofungicides and nano-biofungicides.
Figure 3. Surface interaction, penetration, and uptake of bio-nanofungicides and nano-biofungicides.
Biophysica 05 00015 g003
Table 1. Chemical molecules often used in fungicide formulations.
Table 1. Chemical molecules often used in fungicide formulations.
Fungicides
Chemical ClassActive IngredientMode of Action
Strobilurins (QoI)Azoxystrobin, Pyraclostrobin, TrifloxystrobinInhibits mitochondrial respiration in fungi.
Triazoles (DMI)Tebuconazole, Propiconazole, Difenoconazole, Protioconazole, MefentrifluconazoleInhibits ergosterol biosynthesis, disrupting fungal cell membranes.
Carboxamides (SDHI)Boscalid, Fluxapyroxad, Penthiopyrad, BixafemInhibits fungal respiration by targeting succinate dehydrogenase.
InorganicCopper Sulfate, SulfurMulti-site activity, interfering with several fungal processes.
DithiocarbamatesMancozeb, ThiramMulti-site contact activity.
PhosphonatesFosetyl-Al, Phosphorus AcidInduces plant defenses and inhibits fungal growth.
PhenylamidesMefenoxam, MetalaxylInhibits RNA synthesis in fungi.
Table 2. Comparison between traditional fungicides vs. nano based.
Table 2. Comparison between traditional fungicides vs. nano based.
FeatureTraditional FungicidesNano-Based Alternatives
Active Ingredient DeliveryBroad application and systemic or contact action.Targeted delivery to plant surfaces or fungal pathogens.
Application DosageTypically, higher doses are required for effective control.Potential for significantly lower doses due to enhanced delivery and efficacy.
Application FrequencyOften requires repeated applications throughout the growing season.Potential for reduced application frequency due to enhanced persistence and controlled release.
Mechanism of ActionPrimarily targets broad metabolic pathways in fungi.Can involve targeted disruption of specific fungal processes, enhanced adhesion, and direct antimicrobial activity of nanoparticles. Multi-target effect.
EfficacyGenerally effective but prone to resistance development with overuse.Show promise for improved efficacy, including against fungicide-resistant strains.
Environmental FateCan persist in soil and water, leading to contamination.Potential for reduced environmental persistence due to lower application rates and targeted delivery. Environmental fate of nanoparticles is still under investigation.
Resistance DevelopmentOveruse is a significant driver of fungicide resistance.Potential to mitigate resistance development through novel mechanisms and targeted action.
CostGenerally, lower initial cost per unit.Generally, higher initial production cost. Potential for lower overall cost due to reduced application frequency and dosage.
RegulationWell-established regulatory frameworks in most regions.Regulatory frameworks are still evolving for nano-based pesticides in many regions.
Commercial AvailabilityWidely available and commonly used in agriculture.Currently, limited commercial availability, with ongoing research and development.
Public PerceptionGenerally accepted due to a long history of use, though concerns about environmental impact exist.Public perception is evolving, with potential concerns about the safety and long-term effects of nanotechnology in agriculture.
Long-Term EffectsWell-documented long-term environmental and ecological effects.Long-term environmental and ecological effects of many nanomaterials are still under investigation.
Table 3. Comparison between nano-biofungicides and bio-nanofungicides.
Table 3. Comparison between nano-biofungicides and bio-nanofungicides.
FeatureNano-Biofungicides (Figure 1)Bio-Nano Fungicides (Figure 2)
Primary Active IngredientConventional synthetic fungicides (e.g., pyraclostrobin and epoxiconazole)Metals in their elemental form (Ag0 and Cu0) and biomolecules (lipids, proteins, carbohydrates, etc.)
Nanotechnology RoleNanoencapsulation of synthetic fungicides for improved delivery, stability, and controlled release.Nanotechnology merges elemental metals and biomolecules to form stable, efficient, and versatile biological nanoparticles.
Core CompositionNanoencapsulated synthetic chemical fungicide.Metallic nanoparticle core (e.g., silver and copper) surrounded by a biological “bio-corona”.
Bio-CoronaTypically, not present. The nano aspect is the enclosure of the chemical fungicide.Present, complex structure of proteins, lipids, carbohydrates, etc. Classified as “hard” and “soft” corona layers.
Mechanism of ActionEnhanced delivery of chemical fungicide directly to the pathogen.Enhanced delivery of biological control agents and direct effect of metallic nanoparticles. Additionally, influenced by the bio-corona.
Environmental ImpactPotential for reduced chemical usage but still involves synthetic chemicals.Generally considered more environmentally friendly due to the use of biological agents.
Key AdvantagesIncreased stability, controlled release, improved absorption, reduced dosage.Enhanced effectiveness due to bio-corona, improved stability, targeted delivery, and reduced environmental impact.
ExamplesNanocapsules containing pyraclostrobin, lipid nanoparticles with cyproconazole.Silver or copper nanoparticles synthesized using plant extracts or microorganisms, with associated bio-corona.
Research FocusFocused on optimizing the efficiency and controlled release of chemical-based fungicides.Focused on understanding and optimizing the structure and function of the bio-corona.
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Dávila Costa, J.S.; Romero, C.M. Nano-Biofungicides and Bio-Nanofungicides: State of the Art of Innovative Tools for Controlling Resistant Phytopathogens. Biophysica 2025, 5, 15. https://doi.org/10.3390/biophysica5020015

AMA Style

Dávila Costa JS, Romero CM. Nano-Biofungicides and Bio-Nanofungicides: State of the Art of Innovative Tools for Controlling Resistant Phytopathogens. Biophysica. 2025; 5(2):15. https://doi.org/10.3390/biophysica5020015

Chicago/Turabian Style

Dávila Costa, José Sebastian, and Cintia Mariana Romero. 2025. "Nano-Biofungicides and Bio-Nanofungicides: State of the Art of Innovative Tools for Controlling Resistant Phytopathogens" Biophysica 5, no. 2: 15. https://doi.org/10.3390/biophysica5020015

APA Style

Dávila Costa, J. S., & Romero, C. M. (2025). Nano-Biofungicides and Bio-Nanofungicides: State of the Art of Innovative Tools for Controlling Resistant Phytopathogens. Biophysica, 5(2), 15. https://doi.org/10.3390/biophysica5020015

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