Electrospun Biocarriers with Immobilized Yeasts for Eco-Friendly Biocontrol of Fusarium graminearum

Abstract

This study reports, for the first time, the successful application of chitosan oligosaccharide (COS) and 2-hydroxyethyl cellulose (HEC) coatings on electrospun poly(3-hydroxybutyrate) (PHB) materials for the immobilization of non-conventional yeast strains with fungal biocontrol potential. The coatings enhanced the surface wettability of PHB fibers, facilitating efficient yeast adhesion and viability maintenance. Among the tested strains, Pichia acaciae YD6 was newly isolated and characterized, while Pichia fermentans YP6 and Zygosaccharomyces bailii YE1 had previously been identified as endophytic colonizers. All three strains demonstrated high adaptability, efficient immobilization, and antagonistic activity, confirming their potential for biocontrol applications. COS-coated PHB fibers promoted greater colony expansion than those coated with HEC. Antifungal assays of the yeast-containing biocarriers showed significant inhibition of F. graminearum growth. These findings underscore the potential of PHB-based fibrous materials as sustainable, bioactive carriers for yeast immobilization, with desirable biological properties. This approach offers a promising and eco-friendly strategy for pest control and bioactive agent delivery in agricultural applications.

1. Introduction

Plant diseases, especially those caused by fungal pathogens, pose a serious threat to global food security by affecting key crops such as wheat, maize, and rice [1,2,3,4]. In particular, cultivated wheat is highly susceptible to fungal infections like Fusarium graminearum due to its limited genetic diversity [5,6]. The widespread use of synthetic pesticides in agriculture, intended to control these diseases, has led to long-term harmful consequences for both human health and the environment [7,8,9]. Technologies that harness naturally occurring organisms, such as biocontrol agents, are emerging as viable alternatives to chemical pesticides. These beneficial microorganisms help manage plant diseases by inhibiting pathogen growth [10], promoting plant health [11], or enhancing the host’s defenses [12]. Among these biocontrol agents, yeasts are gaining particular attention for their potential to combat a wide range of plant pathogens. They can antagonize various phytopathogens, offering a promising alternative to reduce dependence on harmful chemical treatments [13]. Yeast-based biocontrol agents, particularly species from the genera Saccharomyces, Zygosaccharomyces, and Pichia, have demonstrated significant antifungal activity against Fusarium solani [14,15,16]. Similarly, fungal antagonists such as Trichoderma spp. have shown efficacy in reducing disease severity [17,18,19]. These findings underscore the potential of yeast-based agents as integral components of sustainable, integrated pest management strategies, aimed at reducing reliance on chemical pesticides and enhancing crop protection in agriculture.

Electrospinning is a versatile technique used to create nanofibers from various polymers, capable of encapsulating living cells, including beneficial yeasts. Yeasts, known for their metabolic versatility and ability to produce bioactive compounds, can be effectively protected and stabilized within the fibrous matrix, enabling their controlled release and prolonged activity [20,21]. There are scarce studies revealing the successful immobilization of yeast in polyvinyl alcohol nanofibers via electrospinning, followed by cross-linking. The immobilized yeasts remained viable and active [22]. Furthermore, efficient immobilization of Saccharomyces cerevisiae on poly(3-hydroxybutyrate) (PHB)/konjac-glucomannan nanofiber membranes produced by solution blow-spinning was achieved. These membranes exhibited ~88% immobilization efficiency, maintained yeast viability, and supported fermentation performance across multiple batches [23]. In this regard, biopolymers derived from organic sources, such as PHB, are increasingly considered promising for agricultural applications. PHB, known for its biodegradability, offers an environmentally friendly alternative to traditional plastics, which is particularly beneficial in agricultural settings where materials often remain in the environment [24,25]. When combined with other polymers, PHB can be used alongside Bacillus subtilis in electrospun materials, providing a promising approach for developing effective biocontrol formulations, as demonstrated in our previous studies [26,27]. Moreover, PHB exhibits favorable mechanical properties, such as tensile strength, tensile modulus, and melting temperature, comparable to those of some synthetic polymers, including polypropylene and polyethylene [28].

Building on the potential of electrospinning for plant disease management, this study presents, for the first time, the development of fibrous PHB-based materials incorporated with diverse yeasts using a combination of electrospinning and dip-coating methods. The novel materials were characterized by SEM to observe yeast morphology and fiber structure. Additionally, water contact angle measurements were conducted to assess the physical properties of the developed mats. The viability of the incorporated yeasts was confirmed through microbiological tests, demonstrating their potential as stable and effective biocontrol agents. This work also advances biological control strategies by utilizing COS- and HEC-immobilized yeasts against F. graminearum, a major threat to cereal crops. By integrating these yeast-based materials into sustainable disease management, this study contributes to the development of innovative approaches within integrated pest management, aiming to reduce reliance on chemical pesticides and mitigate the impact of F. graminearum on global food production.

2. Materials and Methods

2.1. Materials

Poly(3-hydroxybutyrate) (PHB, average Mw 330,000 g/mol, Biomer, Schwalbach, Germany), 2-hydroxyethyl cellulose (HEC, average Mw ~250,000 g/mol, Sigma-Aldrich, Darmstadt, Germany), and chitosan oligosaccharide (COS, average Mw 3000–5000 g/mol, Kitto Life Co., Ltd., Pyeongtaek-si, Gyeonggi-do, Republic of Korea) were used. Merck (Darmstadt, Germany) supplied N,N-dimethylformamide (DMF) and chloroform (CHCl₃). Deionized (DI) water was used in all experiments. All reagents were used as received without further purification.

Yeast Malt Agar (YMA) medium (containing 5 g/L yeast extract, 10 g/L malt extract, and 20 g/L agar) and Sabouraud Dextrose Agar were obtained from Himedia (Mumbai, India). Yeast Peptone Dextrose (YPD) agar (containing 10 g/L yeast extract, 20 g/L glucose, 20 g/L peptone, and 20 g/L agar) was procured from Merck KGaA (Darmstadt, Germany). Fusarium graminearum NBIMCC 2214 was obtained from the Bulgarian National Bank for Industrial Microorganisms and Cell Cultures (NBIMCC).

2.2. Preparation of Fibrous Materials

A 14% (w/v) PHB solution was electrospun to create fibrous PHB materials. Using a reflux condenser, PHB was dissolved in a chloroform/N,N-dimethylformamide (4:1 v/v) solvent system at a temperature of 60 °C. Subsequently, the solution was transferred into a 20 mL plastic syringe equipped with a 20-gauge metal needle and linked to a high-voltage power supply set to 25 kV. The distance from the needle tip to the grounded rotating collector (45 mm in diameter) was 25 cm. The collector rotation speed was set to 2000 rpm. To maintain a controlled flow rate of 3 mL/h, a syringe pump (NE-300, New Era Pump Systems Inc., Farmingdale, NY, USA) was used. Electrospinning was carried out for 6 h at 25 °C and 51% relative humidity. The resulting fibrous materials were then placed in a vacuum desiccator with heating (Vacuo-Temp, J.P. Selecta, Barcelona, Spain) at 30 °C to remove residual solvents.

Dip-coating of the electrospun PHB mats was carried out using COS and HEC aqueous solutions (0.5 wt%), as well as suspensions containing immobilized yeasts (10% v/v with respect to the polysaccharides). PHB discs (12 mm in diameter) were placed in these formulations for 30 min. Following treatment, the samples were withdrawn, gently blotted to remove surface moisture, and left to dry at room temperature until reaching a constant weight. In this way, two types of fibrous carriers were prepared: PHB mats coated with either COS or HEC, denoted as PHB/COS and PHB/HEC, respectively; and PHB mats coated with COS or HEC along with different yeast strains, denoted as PHB/COS/Y and PHB/HEC/Y, respectively.

2.3. Fibrous Materials Characterization

Scanning electron microscopy (SEM, Jeol JSM-5510, Tokyo, Japan) was used to observe the morphology of the obtained fibers and yeasts. Prior to SEM analysis, the samples were coated with gold using a Jeol JFC-1200 fine coater (JEOL Co., Ltd., Tokyo, Japan) under vacuum conditions. SEM images were captured at various magnifications to analyze the surface morphology and fiber structure. To quantify the mean fiber diameter and standard deviation, ImageJ software (v. 1.53e, Wayne Rasband, National Institutes of Health, Bethesda, MD, USA) was used. From multiple SEM micrographs, at least 30 fibers were measured to ensure a comprehensive evaluation of the fibrous materials.

The hydrophobic/hydrophilic behavior of the materials was evaluated using an Easy Drop DSA20E drop shape analysis system (Krüss GmbH, Hamburg, Germany). Static contact angle measurements were performed using the sessile drop method. A 7 µL droplet of DI water was carefully deposited onto the sample surface via a computer-controlled dosing system. The contact angles were determined through image analysis, with ten measurements taken at different locations on each sample. The mean contact angle value represents the average of these measurements. All experiments were conducted at a controlled temperature of 20 °C.

2.4. Yeast and Fungal Pathogen Isolation, Identification, and Cultivation

Among the isolated strains, P. acaciae YD6—newly identified in this study—along with P. fermentans YP6 and Z. bailii YE1, were obtained from various plants after surface sterilization, following established procedures [14,15]. The plant material was macerated in 100 mL of phosphate buffer under aseptic conditions with continuous shaking. The suspension was filtered through sterile filter paper, and 0.2 mL aliquots were inoculated onto yeast malt agar (5 g/L yeast extract, 10 g/L malt extract, 20 g/L agar) (Himedia, Mumbai, India) supplemented with chloramphenicol to enumerate endophytic yeasts. Plates were incubated at 25 °C in the dark for 5–7 days.

For molecular identification, genomic DNA was extracted using the HiPurA fungal DNA purification kit (Himedia, India) following the manufacturer’s instructions. Yeast species were identified through 18S rRNA sequencing and BLAST analysis (v. 2.16.0). rRNA sequences were amplified via PCR using 2 µL each of 10 µM ITS-5 and NL-4 primers and 0.5 µL Taq polymerase (5 U/µL, Canvax, Spain). PCR conditions were carried out according to our previously established procedure [14]. Sequencing was performed by Macrogen (Seoul, South Korea), and nucleotide sequences were analyzed using BLAST (v. 2.16.0) on the NCBI platform.

Fusarium graminearum (NBIMCC 2214) was cultivated at temperature of 25 °C on yeast extract peptone dextrose (YPD) agar (10 g/L yeast extract, 20 g/L glucose, 20 g/L peptone, 20 g/L agar) (Merck KGaA, Darmstadt, Germany) for 7 days, following established methods [29].

2.5. Sporulation and Radial Growth of Immobilized Yeasts

Yeasts reproduce vegetatively (by budding or simple division) and sexually (through spore formation). Budding is the primary method of reproduction. In sexual reproduction, two cells fuse, and their nuclei divide multiple times within the cell. A protective envelope then forms around four to eight ascospores, allowing yeasts to survive unfavorable conditions. To induce sporulation in the yeast strains YD6, YE1, and YP6, they were cultivated in 100 mL of pre-sporulation sterile medium containing glucose (20 g), (NH₄)₂SO₄ (2 g), KH₂PO₄ (2 g), yeast extract (5 g), and distilled water up to 1000 mL. Cultivation was carried out for three days at 30 °C. Once the cultures reached a concentration of 1 × 10⁶ CFU/mL, they were centrifuged at 5000 g and preserved for immobilization in COS and HEC solutions. To assess yeast viability from the fibrous PHB/COS/Y or PHB/HEC/Y discs (12 mm diameter), samples were cultured on Sabouraud Dextrose Agar (Himedia, Mumbai, India) and incubated at 30 °C. Growth was evaluated on the third day post-inoculation. Radial growth experiments were conducted in triplicate (n = 3), and statistical significance was assessed using one-way ANOVA (p < 0.05). Statistical analysis of yeast colony diameters compared COS- and HEC-coated biocarriers for each yeast strain. Data are presented as mean ± standard deviation from three biological replicates.

2.6. Antifungal Activity Assessed by Double-Layer Agar Assay

The antifungal activity of the isolated yeast strains YD6, YP6, and YE1, immobilized on PHB carriers coated with either COS or HEC, was evaluated against Fusarium graminearum using the agar diffusion method. Yeast-immobilized fibrous PHB disks (12 mm diameter) were placed onto YPD agar plates previously inoculated with F. graminearum strain 2214. The plates were incubated at 28 °C for 7 days. Antifungal activity was assessed by measuring the diameter of mycelial growth in comparison to control plates without yeast. Experiments were performed in triplicate, and results are expressed as mean ± standard deviation (SD). The methodology was based on a previously described protocol [30].

2.7. Statistical Analysis

One-way analysis of variance (ANOVA) was conducted to assess differences in colony diameters of yeast strains immobilized on COS- and HEC-coated biocarriers. Statistical analysis was performed using R software version 4.3.2 (R Core Team, 2023) [31]. Tukey’s Honest Significant Difference (HSD) test was applied for post hoc comparisons. Statistical significance was set at p < 0.05. Results are presented as mean ± standard deviation (SD) based on three independent biological replicates. Different lowercase letters indicate statistically significant differences between treatments within the same yeast strain.

3. Results and Discussion

Recently, we demonstrated that PHB coated with chitosan and cellulose derivative-based fibrous biohybrid materials effectively preserves the viability of the encapsulated biocontrol agent Bacillus subtilis during storage and supports its normal growth upon exposure to moisture [26,27]. To the best of our knowledge, reports on the incorporation of yeasts into PHB-based materials remain scarce. Therefore, in the present study, electrospun PHB-based biocarriers with immobilized yeasts were developed for eco-friendly fungal biocontrol, using a combination of electrospinning and dip-coating techniques. Among the yeast strains, P. acaciae YD6 was newly isolated and characterized in this study, while P. fermentans YP6 and Z. bailii YE1 were obtained from various plant sources. For dip-coating, water-soluble chitosan oligosaccharides (COS) and hydroxyethyl cellulose (HEC) were selected due to their favorable properties, making them ideal candidates for protective and delivery coatings in biohybrid systems designed for microbial applications. For clarity, PHB mats coated with COS and different yeast strains will be referred to as PHB/COS/Y, while those coated with HEC and yeast strains will be designated as PHB/HEC/Y.

3.1. Morphological Analysis by SEM

The SEM micrographs provide detailed insights into the morphology and structural characteristics of the fabricated PHB-based fibrous materials. For the uncoated PHB fibers (Figure 1a), the SEM image reveals uniform, cylindrical fibers without defects and pores along their length. The mean fiber diameter was measured to be 378 nm ± 81 nm. These observations indicate that the electrospinning process under the selected conditions successfully produced consistent and high-quality fibers. On the other hand, the PHB fibers coated with COS (Figure 1b) show a distinct change in morphology. The mean fiber diameter increased to 455 nm ± 119 nm. This enlargement in diameter indicates that the dip-coating process successfully applied a layer of COS onto the surface of the PHB fibers, making them thicker than the uncoated fibers. Similarly, the PHB fibers coated with HEC (Figure 1c) demonstrate an increase in fiber diameter, with a mean value of 490 nm ± 130 nm. This further increase in diameter compared to both the uncoated and COS-coated fibers suggests that the HEC coating resulted in a more significant thickening effect. Additionally, the SEM images reveal the formation of thin films between the fibers, which arise due to the higher viscosity of HEC solution used for dip-coating. These films play a crucial role in improving inter-fiber bonding and enhancing the mechanical integrity of the fibrous mat. Furthermore, the presence of these films could contribute to the improved mechanical properties of the coated fibers, such as increased tensile strength and flexibility, making the material more suitable for various applications. Overall, these SEM results clearly demonstrate the influence of the coating process on the morphology and dimensions of the PHB fibers. The effective deposition of COS and HEC coatings is evident from the increased fiber diameters and the formation of films between fibers, which align with the anticipated outcomes of the fabrication process. These findings underscore the efficacy of the dip-coating technique in altering the surface properties of electrospun PHB fibers while preserving their structural integrity and enhancing their mechanical properties.

P. acaciae YD6, P. fermentans YP6, and Z. bailii YE1 are non-conventional yeast strains with promising potential for biotechnological applications, particularly in biocontrol and sustainable agriculture. These yeasts exhibit distinct physiological and morphological traits enabling them to thrive under diverse environmental conditions, making them strong candidates for immobilization on functional materials such as polyhydroxybutyrate (PHB) nanofiber carriers. P. acaciae YD6 is well known for its robust fermentation capacity and high adaptability, while P. fermentans YP6 demonstrates enhanced competitiveness in nutrient-rich environments. In contrast, Z. bailii YE1 shows advantages in high-density immobilization and controlled release systems, highlighting its suitability for applications requiring prolonged functionality.

SEM analysis of the three yeast species revealed distinct morphological characteristics, supported by precise measurements compared to standard dimensions, highlighting their potential for various biotechnological applications. The cells of P. acaciae YD6 exhibited an ellipsoidal to ovoidal shape with a mean length of 3.27 ± 0.67 μm and width of 2.22 ± 0.43 μm (Figure 1d). These measurements fall at the lower end of the size range reported for P. acaciae under standard growth conditions. Early taxonomic descriptions indicate cell dimensions of approximately 1.5–6.0 μm in length and 2.5–11.0 μm in width, varying with the growth medium and phase [32]. The observed discrepancies are likely attributable to differences in cultivation conditions, such as nutrient availability, growth stage, and temperature, as yeast cell size is known to be influenced by both environmental factors and cell-cycle dynamics [33]. The ellipsoidal morphology observed is consistent with typical features of the Pichia genus, which commonly exhibits ovoid to elongate cells with multilateral budding [34]. Cells of P. fermentans YP6 appeared larger, with mean length 3.62 ± 0.55 μm and width 2.69 ± 0.41 μm (Figure 1e). The measured sizes are consistent with the data in literature describing P. fermentans cells as ovoid to ellipsoidal, typically ranging from approximately 3 to 6 μm in length under various conditions [35]. In contrast, Z. bailii YE1 (Figure 1f) exhibited the smallest cell dimensions, with a mean length of 3.12 ± 0.43 μm and width of 2.17 ± 0.41 μm, indicating a compact morphology. Reported cell size ranges for Z. bailii under standard culture conditions vary from approximately 3.5–6.5 μm in length and 4.5–11.5 μm in width, with cells typically described as spheroidal to ellipsoidal depending on the specific growth environment [36]. The SEM measurements obtained in this study fall near or slightly below the lower end of these ranges, likely due to cultivation in minimal or stress-mimicking media, where nutrient limitation is known to reduce average cell size. A more compact morphology may facilitate efficient encapsulation within biodegradable matrices such as PHB fibers, enhancing carrier packing density, stability, and sustained biological activity. In all cases, the measured cell dimensions were cross-referenced with established yeast morphology data to highlight how non-conventional yeasts often display smaller or more variable sizes compared to conventional strains. The SEM-derived measurements confirmed the distinct physical properties of each yeast strain and further demonstrated their suitability for targeted applications in sustainable agriculture, biocontrol, and biotechnology.

COS and HEC coatings have been reported to enhance cell adhesion and protect immobilized microorganisms. Chitosan-coated electrospun PHB fibers form a thin, uniform film that increases surface hydrophilicity and introduces additional binding sites for microbial cells, thereby improving immobilization efficiency and cell retention under flow or mechanical stress [27,37]. Similarly, cellulose-based coatings create a hydrated, protective environment that mitigates desiccation and shear damage, consistent with previous studies on enzyme and microbial immobilization using biopolymer composites [38]. These trends align closely with our observations for P. acaciae YD6, P. fermentans YP6, and Z. bailii YE1 on PHB fibers, supporting the conclusion that the applied immobilization strategy effectively preserves cell integrity and metabolic activity across diverse yeast genera.

SEM images in Figure 2 illustrate the successful immobilization of all three yeast strains on PHB fibers coated with COS/Y or HEC/Y. The uniform distribution of ellipsoidal cells across the coated fibers indicates strong adherence, likely facilitated by electrostatic and hydrogen-bonding interactions between yeast cell wall components (e.g., mannoproteins, glucans), and the polymer coatings. Both COS and HEC act as film-forming agents, generating a continuous matrix on the PHB surface that provides structural support and a protective interface for yeast cell anchoring. This matrix not only enhances cell attachment but also shields immobilized microorganisms from physical stress and desiccation. SEM observations confirm that the yeast cells are firmly attached to the coated fibers and exhibit early signs of budding, indicative of active metabolism and ongoing cell-cycle progression. The coatings further reinforce the structural integrity of the yeast–fiber interface, minimizing cell detachment and maintaining high local cell density—an essential attribute for effective biotechnological and biocontrol applications. The early budding observed is consistent with previous reports on immobilized cells, supporting the functional viability of the immobilization strategy [22,23].

Species-specific traits directly influence immobilization outcomes. The larger cell size and surface area of P. fermentans facilitate more extensive contact with the carrier surface, increasing adhesion strength; however, each cell also occupies more space, which favors applications in nutrient-rich fermentations where high local cell density supports rapid nutrient uptake. P. acaciae, with its moderate cell size and robust fermentation capacity, demonstrates balanced immobilization performance across diverse conditions. In contrast, the compact morphology of Z. bailii is particularly advantageous for high-density immobilization; its smaller cells pack more uniformly within fiber meshes, maximizing cell loading and enabling sustained release in controlled-delivery systems. Similar benefits of smaller yeast cells have been reported for immobilization on porous carriers, where compact morphology enhances packing density and structural stability [38].

Overall, the SEM analysis confirmed the successful immobilization of all three yeast strains on PHB fibers coated with either COS/Y or HEC/Y. The uniform distribution of yeast cells across the coated surfaces, along with the presence of visible budding, indicated effective attachment and sustained metabolic activity on the functionalized carriers. These results align with the observed species-specific morphological traits, where differences in cell size and shape influenced packing density, adhesion, and spatial organization on the fibers. The choice of coating material further affected immobilization outcomes: COS facilitated more uniform cell coverage due to its high affinity for yeast cell wall components, while HEC enhanced structural integrity and provided a stable matrix for prolonged retention. These findings confirm the distinct physical characteristics of P. fermentans, P. acaciae, and Z. bailii, and reinforce their potential for targeted applications in sustainable agriculture, biocontrol, and biotechnology.

3.2. Surface Wettability Analysis of Electrospun PHB Mats

One key factor influencing biofilm adhesion or detachment is the degree of surface wettability. The hydrophilic–hydrophobic balance and surface characteristics of the host material significantly affect the adherence of bacteria, fungi, and yeasts. Since the prepared fibrous materials are intended for contact with microorganisms, it is essential to evaluate their surface wettability by measuring the water contact angle (WCA) of the electrospun PHB mats. It is well established that PHB fibers produced by electrospinning exhibit hydrophobic properties [39]. Digital images of water droplets on the surfaces of uncoated PHB mats and those coated with COS and HEC, along with their respective WCA values, are presented in Figure 3. As shown in Figure 3a, the electrospun uncoated PHB mat has a WCA of 97.3° ± 3.9°, confirming its hydrophobic nature. In contrast, the COS- and HEC-coated PHB mats (Figure 3b,c) exhibited a WCA of 0°, indicating immediate absorption of the water droplets upon contact. This observation confirms that coating PHB mats with COS or HEC imparts superhydrophilic properties to the surface. These surface modifications significantly reduced the contact angle, thereby enhancing the surface characteristics for microbial adhesion.

3.3. Molecular Identification and Taxonomic Classification of Yeast Strains

The yeast strains YP6 and YE1 were previously isolated and characterized in earlier studies conducted by our research group [14,15]. In contrast, strain YD6 was newly isolated and identified in the present study. Its identification was performed through molecular profiling based on 18S rRNA gene sequencing. Total genomic DNA was extracted from fresh cultures, and PCR amplification was carried out using universal primers targeting conserved eukaryotic regions. The resulting PCR products (~1100 bp) were purified and sequenced bidirectionally. The obtained sequences were analyzed using the BLAST algorithm against the NCBI GenBank database. Species-level identification was based on sequence similarity and phylogenetic clustering. The analysis confirmed that strain YD6 belongs to Pichia acaciae, supported by a high sequence identity (>99%) with reference sequences. This identification was further validated using the expanded Saccharomycetales dataset in the NCBI database, which includes many recently described species within the Saccharomycota phylum (

Table 1. Identification of yeast strains and their GenBank accession details.

Strain Name	Identification	% Similarity	GenBank Accession Number
YD6 1	P. acaiae 1	99	MW756319
YP6 2	P. fermentans 2	100	MZ798453.1
YE1 3	Z. bailii 3	100	OL904963

¹ This study. ² Ref. [14]. ³ Ref. [15].

).

3.4. Comparative Radial Growth of Immobilized Yeast Strains

The yeast strains P. acaciae YD6, P. fermentans YP6, and Z. bailii YE1 each exhibit distinct physiological traits and environmental adaptability. Their growth behavior after immobilization on PHB-coated with COS or HEC carriers provides insights into their metabolic flexibility and potential applications in biological control and industrial biotechnology. Growth tests on yeast malt agar supplemented with fibrous PHB/COS/Y and PHB/HEC/Y materials showed visible radial expansion of colonies from inoculation disks (Figure 4). Colonies formed on COS-coated matrices (Figure 4 bottom) were generally larger and more morphologically complex than those on HEC-coated materials (Figure 4 top). For instance, P. acaciae YD6 displayed greater radial growth and more irregular, proliferative edges on PHB/COS, in contrast to the smoother and more restricted colonies on PHB/HEC (Figure 4b). Similar trends were observed for P. fermentans YP6 and Z. bailii YE1, with COS coatings consistently supporting broader colony development (Figure 4c,d).

Obviously, all three strains were successfully immobilized in both fibrous biocarriers and displayed measurable growth, with COS generally supporting larger colony diameters. Notably, Z. bailii YE1 exhibited the most vigorous growth, particularly on the PHB/COS carrier, suggesting enhanced metabolic activity or superior compatibility with this matrix—potentially due to improved nutrient diffusion, greater matrix porosity, or intrinsic bioactivity. Quantitative analysis (

Table 2. Growth response of P. acaciae YD6, P. fermentans YP6, and Z. bailii YE1 after inoculation.

Biocarrier	Strain	Colony Diameter, mm
PHB/COS/YD6	P. acaciae YD6	18.50 ± 1.95 b
PHB/HEC/YD6	P. acaciae YD6	15.23 ± 1.08 c
PHB/COS/YP6	P. fermentans YP6	19.05 ± 1.88 b
PHB/HEC/YP6	P. fermentans YP6	16.45 ± 2.14 b,c
PHB/COS/YE1	Z. bailii YE1	18.03 ± 0.92 a
PHB/HEC/YE1	Z. bailii YE1	15.30± 1.99 a

Notes: a, b, c indicate statistically significant groupings.

) confirmed these trends. PHB/COS carriers promoted significantly larger colony diameters for YD6 (p = 0.025) and YE1 (p = 0.019) compared to their HEC-coated counterparts. Although the difference for YP6 was not statistically significant (p = 0.176), the same directional trend was observed. These results demonstrate that COS coatings generally enhance yeast proliferation on fibrous PHB scaffolds. The enhanced growth observed on COS-coated carriers may stem from several factors, including improved matrix porosity, increased surface hydrophilicity, and the bioactivity of COS. COS is known to mimic components of fungal cell walls, such as chitin and β-glucans, and may stimulate yeast enzymatic activity or signaling pathways that promote growth [40,41]. Additionally, previous studies suggest that yeasts capable of metabolizing chitin-derived substrates can exhibit chitinase or β-1,3-glucanase activity, contributing to fungal biocontrol through enzymatic degradation of pathogenic cell walls [42,43,44]. The pronounced growth of Z. bailii YE1 on PHB/COS may thus reflect an intrinsic compatibility with COS-based matrices or a greater enzymatic capacity to utilize complex oligosaccharides.

In contrast, growth on PHB/HEC fibers—though generally lower, remains notable. HEC is a cellulose derivative, and its use as a coating implies potential cellulolytic or β-glucosidase activity by the immobilized strains. Such enzymatic capabilities are relevant for biomass conversion, biofilm formation, and colonization of plant surfaces, all of which are desirable traits in sustainable agricultural and industrial systems [45,46].

These findings underscore the functional versatility of the tested yeast strains and highlight COS as a superior coating material for supporting growth in immobilized systems. To our knowledge, this is one of the first studies to evaluate yeast immobilization by direct fibrous coating with COS and HEC, revealing strain-specific growth patterns and potential mechanisms that warrant further investigation. Future work will focus on characterizing the enzymatic profiles of these strains, their persistence under environmental stress, and their competitive interactions with plant pathogens.

3.5. Antifungal Activity of Immobilized Yeasts Against Fusarium Graminearum

To evaluate the antifungal potential of the immobilized yeast formulations, agar diffusion assays were conducted using P. acaciae YD6, P. fermentans YP6, and Z. bailii YE1 strains immobilized on PHB carriers coated with either COS or HEC. Untreated F. graminearum cultures served as the negative control. A key mechanism of yeast-mediated biocontrol is competition for nutrients and space, whereby yeasts colonize ecological niches (e.g., wound sites) and deprive pathogens of essential resources [47,48,49].

As shown in Figure 5a, untreated F. graminearum exhibited unrestricted growth and characteristic reddish pigmentation, primarily due to aurofusarin—a polyketide pigment co-regulated with trichothecene mycotoxins [50,51]. In contrast, all immobilized yeast formulations significantly inhibited fungal growth and reduced pigment intensity. Such pigment suppression is indicative of altered secondary metabolism, often correlated with decreased mycotoxin biosynthesis under competitive or stress-inducing conditions [52,53,54]. Reduction in red pigmentation was observed across all yeast-carrier combinations, with inhibition zones ranging from 16 to 26 mm (Figure 5b–d). Z. bailii YE1 on PHB/COS and PHB/HEC exhibited the most pronounced effects, characterized by restricted colony development and near-complete loss of pigmentation. These observations are consistent with the activity of zygocin, a proteinaceous killer toxin produced by Z. bailii known to inhibit a broad spectrum of fungi [55,56]. Similarly, P. fermentans YP6 produced strong inhibition zones, aligning with reports of killer toxin production by related Pichia species such as P. membranifaciens and P. kudriavzevii [57,58]. Though P. acaciae YD6 displayed comparatively weaker inhibition, its consistent activity supports its role in biocontrol, potentially through mechanisms such as volatile organic compound (VOC) production or enzyme secretion [59,60].

The observed antifungal effects reflect the multifactorial nature of yeast antagonism. Documented mechanisms include competition for nutrients and micronutrients (e.g., iron via siderophores), production of hydrolytic enzymes (chitinases, glucanases), secretion of VOCs, and the release of killer toxins. VOCs and enzyme activities have been shown to inhibit F. graminearum growth and reduce mycotoxin production. Additionally, nutrient competition may restrict biosynthetic precursors (e.g., acetyl-CoA) or alter the microenvironment (pH, osmotic pressure), suppressing secondary metabolism [43,52,53].

Control experiments using COS and HEC solutions alone (Figure S1, Supplementary Material) confirmed that the coatings themselves did not inhibit fungal growth, suggesting that the antifungal activity stems from the immobilized yeast. While COS has mild antimicrobial effects at higher concentrations, its role here is primarily as a bioadhesive matrix enhancing yeast adhesion, viability, and possibly metabolic output [61,62,63]. COS and HEC coatings improve matrix compatibility, protect against desiccation and mechanical stress, and preserve metabolic activity—factors critical for sustained biocontrol function. SEM analysis confirmed that immobilized yeast cells retained metabolic activity, as evidenced by budding structures and robust surface attachment (Figure 2). These morphological observations are consistent with prior findings where PHB-based biocarriers maintained yeast viability and supported active metabolite secretion [23]. COS and HEC coatings also contributed to carrier biocompatibility, supporting sustained release and high local cell densities—essential traits for bioactive agricultural coatings or seed treatments [62].

Although we did not directly identify specific antifungal metabolites or toxin genes in this study, the strong inhibition zones, loss of fungal pigmentation, and morphological evidence of metabolic activity strongly suggest secretion of killer toxins or related bioactive compounds. Known examples include zygocin (Z. bailii) [55], PMKT (P. membranifaciens) [57], mycocins (W. anomalus) [64], and the K1 and K2 toxins from S. cerevisiae [65], which target cell wall components and membrane integrity in susceptible fungi.

These results demonstrate that immobilization on PHB/COS and PHB/HEC carriers preserves and potentially enhances the antagonistic activity of non-conventional yeast strains against F. graminearum. Notably, COS-based formulations, especially those incorporating Z. bailii YE1, yielded the most effective outcomes and represent a promising platform for sustainable crop protection. Potential applications include seed coatings, postharvest biocontrol, and soil amendments to suppress Fusarium-related diseases. Future work should include metabolomic profiling and gene expression analysis to identify the antifungal compounds involved, along with greenhouse and field-scale studies to validate efficacy under realistic conditions. Key focus areas include persistence in soil, interaction with native microbiota, colonization efficiency, and compatibility with crop physiology and agricultural practices.

4. Conclusions

This study demonstrates, for the first time, the successful development of electrospun PHB-based fibrous carriers coated with COS and HEC as effective carriers for the immobilization of non-conventional yeast strains with biocontrol potential against Fusarium graminearum. The applied coatings significantly enhanced the surface wettability of PHB fibers, enabling efficient yeast adhesion and viability retention. Among the two coatings, COS proved greater colony expansion than HEC. The yeast strains—P. acaciae YD6 (newly isolated), P. fermentans YP6, and Z. bailii YE1, showed high adaptability, successful immobilization, and strong antagonistic activity. An antifungal assay confirmed that immobilized yeasts retained potent killer toxin activity and effectively inhibited fungal growth and pigmentation. These findings validate the potential of PHB/COS/Y and PHB/HEC/Y fibrous biocarriers as sustainable platforms for delivering bioactive agents in agricultural settings. This pioneering approach introduces a novel biotechnological platform that integrates biodegradable polymers with functional coatings to develop targeted, environmentally friendly biocontrol strategies. The findings demonstrate significant potential for applications in sustainable agriculture, postharvest protection, and microbial formulations, while laying the groundwork for future research focused on scalability and field deployment.

Although this study focused on F. graminearum, the broader application of COS-based yeast biocarriers against a diverse range of fungal pathogens merits further exploration. Future research will prioritize in vivo and in planta evaluations, such as detached leaf assays and greenhouse trials, to validate the efficacy and environmental robustness of these biocarriers under realistic conditions. Additionally, studies will investigate large-scale production methods, formulation stability, and delivery mechanisms. Field trials will be conducted to assess performance within the complex interactions of plants, pathogens, and environmental factors, thereby facilitating the translation of this technology into practical, sustainable crop protection solutions.