Evaluation of encapsulated antagonistic yeasts using solution blow spraying for the control of fungi in common bean seeds (Phaseolus vulgaris L.)

ABSTRACT

The presence of fungi in common bean seeds is a recurring problem that compromises the physiological and sanitary quality of the plant material. In this context, antagonistic yeasts are presented as a sustainable alternative, but their application is limited due to their low stability during storage. Then, encapsulated bioformulations that preserve their viability and facilitate their use need to be developed. In this regard, the solution blow spraying technique is an aerohydrodynamic process that promotes the formation of microcapsules and preserves the viability of the encapsulated yeasts. Therefore, this study aimed to evaluate the efficacy of antagonistic yeasts encapsulated by solution blow spraying for controlling fungi in bean seeds. Five yeasts were preliminarily evaluated by in-vitro tests against fungi associated with bean seeds. After selecting the three yeasts to be tested on seeds, they were applied both in fresh and encapsulated form to evaluate their antifungal efficacy and verify that they did not affect seed germination or vigor. The results showed that Meyerozyma guilliermondii,Meyerozyma caribbica, and Yamadazyma mexicana had the greatest antagonistic potential against the evaluated fungi. The application of these yeasts, both fresh and encapsulated, reduced pathogen incidence during storage without affecting seed germination or vigor. These findings support the use of encapsulated antagonistic yeasts as a sustainable strategy for managing fungi in bean seeds.

Key words
Biocontrol; blotter test; germination; incidence

INTRODUCTION

Common beans (Phaseolus vulgaris L.) are one of the most important legume crops in Latin America due to their nutritional and economic value. In Brazil, domestic production for the 2024/2025 cycle was estimated at around 3 million tons (CONAB 2025). This food is an important source of protein, carbohydrates, and minerals, and provides various bioactive compounds associated with beneficial health effects.

Beans contain high levels of phytochemicals that confer antioxidant, antidiabetic, anticancer, cardioprotective, and anti-inflammatory properties, which have been widely documented in varieties consumed in regions such as China, India, and the Americas (Meenu et al. 2023). However, bean production and quality are affected by phytopathogenic fungi that attack both seeds during storage and plants in the field, mainly Fusarium, Aspergillus, and Penicillium, and to a lesser extent Colletotrichum, Cladosporium, and Alternaria (Costa and Scussel 2002, Lopes et al. 2018). These pathogens reduce seed viability and vigor, accelerate the physiological deterioration of seeds during storage, and can produce mycotoxins with implications for human health (Mills and Woods 1994, Martín et al. 2022).

Fungi can persist over winter in infected seeds or crop residues, serving as a primary source of inoculum (Matić et al. 2014, Naseri and Hemmati 2017). Traditionally, seeds are stored and treated with chemical fungicides to prevent infection, but their excessive use can lead to resistance and environmental problems (Pires et al. 2004, Castellanos et al. 2017). Therefore, sustainable strategies, including biological alternatives, need to be developed to ensure seed protection and crop health without compromising the environment. This approach also aligns with proposals to promote more sustainable agricultural systems through biological seed treatments (Sharma et al. 2015, Fess and Benedito 2018, Almeida et al. 2022).

In this context, biological control using antagonistic yeasts is emerging as an effective and environmentally safe strategy against fungal diseases. Yeasts have proven their effectiveness through various mechanisms of action, mainly by competing for nutrients and space, producing volatile organic compounds, and secreting enzymes that degrade the fungal cell wall (González-Gutiérrez et al. 2023). Various studies support the potential of using antagonistic yeasts to manage phytopathogens in seeds. For example, Pichia guilliermondii has inhibited the development of Fusarium fujikuroi in rice (Matić et al. 2014), and in wheat, Meyerozyma guilliermondii, Cyberlindnera saturnus, Cryptococcus carnescens, and Rhodotorula glutinis have been reported to be effective in reducing Fusarium spp. disease incidence (Podgórska-Kryszczuk et al. 2022).

However, the direct application of fresh yeast has limitations due to its low stability and difficulty in preservation, necessitating the development of encapsulated bioformulations to protect yeasts’ viability during storage (Aguirre-Güitrón et al. 2018). Traditionally, yeast microencapsulation has been carried out by spray drying (Aguirre-Güitrón et al. 2018, Orcen et al. 2024); nevertheless, emerging techniques such as solution blow spraying (SBSp) offer significant advantages.

SBSp is an aerohydrodynamic process that does not require high temperatures or electricity, unlike other encapsulation technologies such as spray drying or electrospraying, and preserves microbial viability. SBSp involves the simultaneous atomization of a polymer solution and a pressurized gas through a coaxial nozzle, in which the interaction between the gas and solution generates microdroplets to form microcapsules as the solvent evaporates (Covarrubias-Rivera et al. 2025). Its simplicity, low cost, and efficiency position it as a promising technique for yeast encapsulation with industrial and commercial potential.

Research has already been conducted on the use of yeast and encapsulated microbial agents in biocontrol. However, their specific application in seeds remains limited. Likewise, the use of emerging technologies such as SBSp for yeast encapsulation in this context has not been documented. Based on this, the hypothesis proposed that encapsulated yeast bioformulations can inhibit the growth of fungi associated with bean seeds. The objectives of the study were: to preliminarily evaluate antagonistic yeasts through in-vitro tests against fungi associated with bean seeds, to evaluate the effectiveness of fresh and encapsulated yeasts for controlling pathogens in bean seeds, and to determine whether the application of bioformulations compromises seedling germination or vigor, ensuring their safety for cultivation.

MATERIALS AND METHODS

Raw materials

The AG was purchased from Sigma Aldrich Co. (St. Louis, MO, United States of America), and the WPC was obtained from Davisco Foods International, Inc. (Le Sueur, MN, United States of America). It contains 80 g of protein, 9 g of lactose, 8 g of lipids, and 3 g of water and minerals per 100 g of product. Black beans (Phaseolus vulgaris L.) of the cultivar IAC VELOZ, category S2, harvest 2024/2024, obtained from a conventional producer (Cittolin Comércio de Cereais Ltda., Paraná, Brazil), were used.

Microorganisms

The yeasts M. guilliermondii LMA-Cp01 (GenBank: MZ254664), M. caribbica (GenBank JQ398674), and Yamadazyma mexicana LPa14 (GenBank: OM949962) were previously isolated from the epicarp of papaya (López-Cruz et al. 2020), mango (Bautista-Rosales et al. 2013), and avocado (González-Gutiérrez et al. 2023), respectively. The yeast isolate R. glutinis AH 14-3 (SisGen: A85CDA9) was isolated from rose (Carvalho et al. 2020). The yeast R. mucilaginosa [Tropical Culture Collection: 5470] was acquired from the Fundação André Tosello (Campinas, São Paulo, Brazil). The fungi Penicillium sp., Fusarium sp., and Aspergillus sp. correspond to the Fungal Collection of the Food Microbiology Laboratory, Center for Biological and Health Sciences, Universidade Estadual do Oeste do Paraná, Cascavel, Paraná, Brazil.

Preparation of yeast and fungal inoculants

The yeasts were reactivated in 50 mL of yeast extract-peptone-dextrose broth [YPD, containing yeast extract (10 g.L^-1), dextrose (20 g.L^-1), and casein peptone (20 g.L^-1)] and incubated in a rotary shaker (TE 1400, Tecnal, São Paulo, Brazil) at 28°C and 150 rpm for 36 h. The biomass was recovered by centrifugation at 2,100 × g (3,000 rpm, Fanem Centrifuge Model Baby, São Paulo, Brazil) at 25°C for 3 min. On the other hand, the fungi were grown on potato dextrose agar (PDA, Sigma Aldrich Co., St. Louis, MO, United States of America) and incubated at 28°C for seven days. Subsequently, the yeast cell and fungal spore suspensions were adjusted to 10⁸ cells/mL and 10⁵ spores/mL, respectively, using a hemocytometer (Covarrubias-Rivera et al. 2025).

In-vitro evaluation of yeasts against phytopathogenic fungi associated with seeds

The antagonistic potential of M. guilliermondii, M. caribbica, Y. mexicana, R. mucilaginosa, and R. glutinis was preliminarily evaluated by spreading 0.50 mL of cell suspensions previously adjusted to 10⁸ cells/mL on the surface of Petri dishes containing PDA agar. The plates were kept at 25°C for 1 h to allow the suspensions to dry. Subsequently, a 6-mm hole was made in the center of each plate. The inhibition assay was performed by inoculating 20 μL of a spore suspension (10⁵ spores/mL) of Penicillium spp., Fusarium spp., or Aspergillus spp. Plates inoculated only with the fungal suspensions under the same conditions were used as controls. The plates were incubated at 28°C for seven days, and the diameter of fungal growth was recorded to calculate the percentage of inhibition (Eq. 1) (González-Gutiérrez et al. 2024). Each treatment was evaluated with five replicates, and the experiment was repeated three times.

(1) $\mathrm{Inhibition}\mathrm{of}\mathrm{mycelial}\mathrm{growth}\left(\mathrm{\%}\right)=\left[\frac{C-T}{C}\right]\times 100$

Where C: the control; T: the mycelial growth diameter of the treatment

Preparation of the polymer solution and the yeast encapsulation process by solution blow spraying

The WPC solution was prepared at 16% (w/w) by dissolving the required amount in sterile distilled water at 25°C, with stirring for 2 h. Separately, a 2% (w/w) AG solution was prepared, gently stirred for 4 h, and then sterilized at 121°C for 15 min. The water evaporated during sterilization was replaced by adding sterile distilled water to adjust the mass. Once cooled, the AG solution was added to the WPC solution, with 5% (w/w) Tween 20 (Êxodo Científica, Sumaré, São Paulo, Brazil) as a surfactant. The polymer mixture was stirred until homogenized.

In a previous study, the composition of the powder bioformulation was optimized using a 3²-experimental design (Covarrubias-Rivera et al. 2025). Subsequently, the biomass of M. guilliermondii, M. caribbica, and Y. mexicana was independently resuspended in the polymer suspension at a ratio of 1:6 (w/w) fresh yeast:polymer solution. In each case, the cell concentration was adjusted to 10⁹ cells/mL by counting in a Neubauer chamber (González-Gutiérrez et al. 2024). A commercial airbrush (TimberTech model ABPST01) coupled to an air compressor (Motomil model CMI-7.6/24BR) was used for spraying. Environmental conditions were maintained at 20°C and 40–60% relative humidity. Six mL of the yeast suspension was placed in the airbrush reservoir and sprayed onto a collection plate. The operating conditions were: air pressure 0.3 MPa, nozzle diameter 0.5 mm, and distance between the needle and the collector 50 cm, selected to ensure jet stability and uniform microcapsule formation. Finally, the microcapsules obtained were collected and stored under refrigeration until use (Covarrubias-Rivera et al. 2025).

Evaluation of the health of seeds treated with yeast using the Blotter test

The antagonistic effect was preliminarily evaluated using fresh cell suspensions (10⁸ cells/mL) of M. guilliermondii, M. caribbica, Y. mexicana, and R. glutinis to assess their potential to inhibit the pathogens before proceeding to encapsulation. After this, the test was performed only with M. guilliermondii, M. caribbica, and Y. mexicana, which were selected for encapsulation. The bean seeds were treated with fresh and encapsulated cell suspensions (10⁸ cells/mL). For encapsulated yeasts, suspensions were prepared by hydrating 0.1 g of powder from each microencapsulated yeast formulation in 9.9 mL of distilled water, with continuous stirring for approximately 3 h to achieve the desired concentration for evaluation. For each treatment, a dose of 1.3 mL of the cell suspension was applied per 22 g of seeds (≈ 100 seeds). Afterwards, the seeds were placed in sterile plastic bags, manually homogenized for 1.5 min to ensure uniform distribution of the inoculum, and left to dry at room temperature for 90 min. Untreated seeds, seeds treated only with sterile distilled water, and seeds treated with empty capsules were used as controls.

Seed health was evaluated using the Blotter test, in accordance with the Seed Analysis Standards (Brasil 2009). For this purpose, 25 seeds were placed at equal distances on two sheets of sterile germinatest paper, moistened with a 0.1% (v/v) solution of 2,4-D (dimethylamine salt; 806 g.L^-1, acid equivalent: 670 g.L^-1) at 0.1% (v/v), applied in a proportion equivalent to 2.5 times the mass of the dry paper, using the commercial herbicide Nortox, and placed in gerbox boxes. The samples were incubated in a germination chamber (Tecnal model TE-391, Piracicaba, São Paulo, Brazil) at 25°C, with a 12 h photoperiod, for eight days.

At the end of incubation, the seeds were visually inspected and examined with a digital microscope (S04-600X) at 25× magnification to detect the presence of mycelium (Fig. 1) (Henning 2015) of Penicillium spp., Fusarium spp., or Aspergillus spp. With this information, the disease incidence (Eq. 2) and the relative frequency of the fungal genus visualized in the seeds (Eq. 3) were calculated. The evaluation was carried out at two time points: after the application of the treatments (day 0) and after 30 days of seed storage in craft paper bags under ambient conditions. Each treatment was evaluated using 400 seeds per replication, and the entire experiment was repeated independently three times. The yeast treatments were arranged in a 3 × 2 × 2 factorial design, in which the yeast species (M. guilliermondii, M. caribbica, and Y. mexicana), the form of yeast administration (fresh or encapsulated), and the storage period (0 or 30 days) served as experimental factors. In addition, untreated seeds, seeds treated with water, and seeds treated with empty capsules were included as control treatments.

Figure 1
Presumptive detection of Fusarium spp., Aspergillus spp., and Penicillium spp. in black bean seeds using the Blotter test.

(2) $\mathrm{Incidence}\mathrm{of}\mathrm{the}\mathrm{disease}\left(\mathrm{\%}\right)=\frac{Numberofinfectedseeds}{Totalnumberofseedsevaluated}\times 100$

(3) $\mathrm{Relative}\mathrm{frequency}\mathrm{of}\mathrm{fungus}\mathrm{gender}\left(\mathrm{\%}\right)=\left[\frac{N_{fungus}}{N_{infected}}\right]\times 100$

Where N_fungus: the number of infected seeds in which that genus was identified; N_infected: the total number of infected seeds (with any fungus).

Evaluation of antagonistic yeasts on bean seed germination and vigor

The treatments and application method were the same as those described in the mentioned methodology. The germination test was carried out in 5-L plastic trays (28 cm long, 18 cm wide, and 10 cm high), with evenly distributed 2,150 g of medium-grain sand, used for the first time, as substrate. The substrate was then moistened to 60% of its capacity before sowing, in accordance with the Seed Analysis Standards (Brasil 2009). The seeds were sown at a depth of 2 cm, distributed equidistantly. Irrigation was carried out daily using gravimetric control, maintaining the moisture level close to the initial value for nine days.

The tests were carried out under controlled environmental conditions, with a constant temperature of 22 ± 2°C and a photoperiod of 12 h. Seedling counts were performed according to the criteria of Marcos Filho (1999) and Schuab et al. (2006), in which the first emergence was considered when the cotyledons were above the sand level, and from that moment on, the number of seedlings that emerged daily was recorded without removing them. At the end of the experiment, the cumulative number of emerged seedlings per day was obtained, calculated by the difference from the previous day’s count. From these data, the emergence rate index (Eq. 4) was calculated according to Maguire (1962). The seedlings were then classified as normal, abnormal, or dead, according to the Seed Analysis Standards (Brasil 2009).

Based on this classification, the germination percentage (Eq. 5) was calculated, and the length of the root and aerial part (cm) was measured. To determine dry weight, the cotyledons were removed, placed in paper bags, and dried in an oven (TECNAL model TE-394/1) with forced air circulation at 60°C for 48 h. Finally, the dry material was weighed on an analytical balance (Shimadzu model BL320H), and the results were expressed in grams per seedling. The evaluation was carried out at two time points: immediately after treatment application (day 0) and after 30 days of storage. Each treatment was analyzed using 200 seeds per repetition, distributed in four replicates of 50 seeds, and the entire experiment was repeated in three independent trials.

(4) $\mathrm{Emergency}\mathrm{speed}\mathrm{index}=\frac{G_1}{N_1}+\frac{G_2}{N_2}+\cdots +\frac{G_n}{N_n}$

Where G: the number of emerged seedlings observed in the count; N: the number of days since sowing in each count.

(5) $\mathrm{Germination}\mathrm{rate}(\%)=\frac{Numberofnormalseedlingsemerged}{Numberofseedsplanted}$

Statistical analysis

Results are expressed as mean ± standard deviation. Data obtained from the in-vitro assays were analyzed using one-way analysis of variance (ANOVA). For seed evaluations, antagonistic yeast treatments were analyzed using a 3 × 2 × 2 factorial design, with yeast species, yeast application form (fresh or encapsulated), and seed storage period (0 or 30 days) as factors. Mean comparisons were performed using Fisher’s least significant difference (LSD) test (p < 0.05). Statistica v. 12, StatSoft, Inc., was used for statistical analysis.

RESULTS AND DISCUSSION

In-vitro antagonistic effect of yeasts on the mycelial growth of phytopathogenic fungi in bean seeds

A preliminary evaluation was conducted on the antagonistic effect of five yeast strains against phytopathogenic fungi of interest, commonly associated with the deterioration of bean seeds. In the in-vitro test, the yeasts exhibited varying degrees of mycelial inhibition (Figs. 2 and 3). Against Fusarium sp. (Fig. 3a), the highest inhibition percentage was observed in M. guilliermondii (77.67%), followed by M. caribbica (66.15%) and R. glutinis (62.53%), while Y. mexicana reached 42.98% and R. mucilaginosa showed the lowest inhibition (28.46%). In the case of Aspergillus sp. (Fig. 3b), the strains M. guilliermondii, M. caribbica, Y. mexicana, and R. glutinis recorded percentages above 94%, with no significant differences between them, in contrast to R. mucilaginosa, which showed only 56.09%. Likewise, for Penicillium sp. (Fig. 3c), these strains maintained high inhibition (> 86%, p > 0.05), whereas R. mucilaginosa reached only 30.29%.

Figure 2
Representative images of the in-vitro inhibition test with antagonistic yeasts (10⁸ cells/mL) against pathogenic fungi of interest in bean seeds.

Figure 3
In-vitro mycelial inhibition of (a) Fusarium sp., (b) Aspergillus sp., and (c) Penicillium sp. through the action of different yeast strains. Bars with different letters indicate significant differences between treatments according to Fisher’s least significant difference test (p < 0.05). Values represent the mean ± standard deviation.

These results showed that antagonistic efficacy in vitro depends on both the yeast and the fungus evaluated. M. guilliermondii, M. caribbica, R. glutinis, and Y. mexicana stood out against Aspergillus sp. and Penicillium sp., while against Fusarium sp., the response was more variable, with greater effectiveness using M. guilliermondii. This variability reflects differences in their spectrum of action depending on the phytopathogen and the mechanisms involved (Spadaro and Droby 2016). The biological control activity of yeasts may be associated with various mechanisms, including competition for nutrients and space, the production of hydrolytic enzymes, and the production of volatile compounds (Ayón-Macías et al. 2025, González-Gutiérrez et al. 2023). However, the specific mechanism of biological control through different food matrices, such as seeds, requires further clarification.

Commonly, yeast replication time is usually shorter than that of fungi, giving them an initial competitive advantage (Yan et al. 2021). In this context, the high inhibition observed against Aspergillus sp. and Penicillium sp. indicates broad-spectrum antifungal activity, which may be associated with the secretion of hydrolytic enzymes by yeasts, such as chitinases, proteases, and glucanases (Ayón-Macías et al. 2025). In contrast, the reduced inhibition against Fusarium sp. could be attributed to variations in cell wall structure, particularly in the content and proportions of chitin and glucans, which determine the susceptibility of the fungus to these enzymes (Nimrichter et al. 2016). On the other hand, the orange coloration observed in the boxes corresponding to R. mucilaginosa (Fig. 2) is due to the production of carotenoids (Li et al. 2022), which caused agar pigmentation. In addition, the yeast R. mucilaginosa showed lower efficacy against the tested fungi, which is consistent with the findings of Zara et al. (2021), who noted reduced biocontrol potential for this species. Consequently, R. mucilaginosa was not considered in subsequent tests.

Effectiveness of antagonistic yeasts in fresh and encapsulated form on disease incidence and fungal frequency in bean seeds

In the preliminary evaluation with fresh yeast suspensions on bean seeds, the biocontrol capacity of the strains studied was determined. In this assay, R. glutinis exhibited a disease incidence of 98.66%, demonstrating a lack of effectiveness. Therefore, it was excluded from further analyses. Consequently, the yeasts M. guilliermondii, M. caribbica, and Y. mexicana, in fresh and encapsulated form, were selected for subsequent evaluation (Tables 1 and 2). Disease incidence and the relative frequency of fungal genera were assessed at the beginning (0 day) and after 30 days of seed storage.

At the beginning of storage (Table 1), the controls showed a high disease incidence, with 86.04% in untreated seeds, 85.75% in seeds treated with distilled water, and 90.13% in seeds treated with empty capsules (p > 0.05), which showed the absence of antifungal effect of the empty capsules. Yeast treatments significantly reduced the disease incidence. When yeast was applied in its fresh form, all showed comparable efficacy (p > 0.05), with disease incidence values of 52.25% for Y. mexicana, 53.10% for M. guilliermondii, and 59.81% for M. caribbica. In contrast, the application of yeast in encapsulated form also reduced disease incidence, with no differences between strains, registering 68.44% for M. caribbica, 69.38% for M. guilliermondii, and 75.69% for Y. mexicana. This behavior is consistent with previous studies involving other antagonistic strains of M. guilliermondii, which have been effective against A. flavus in hazelnut seeds (Dikmetas et al. 2023) and F. fujikuroi in rice (Matić et al. 2014), mainly due to its rapid surface colonization and ability to compete for space and nutrients (Spadaro and Droby 2016). Likewise, the efficacy observed in treated seeds may be attributed to the ability of yeasts to form biofilms on the surface, as these act as a physical barrier that limits the development of pathogens (Lawal et al. 2025).

Table 1
Disease incidence and relative frequency of fungi in bean seeds treated with yeast and evaluated at the beginning and end of storage<tfn href="tfn01">*</tfn>.

Species	Form of application of the yeast	Seed storage period (days)	Disease incidence (%)	Frequency Fusarium spp. (%)	Frequency Aspergillus spp. (%)	Frequency Penicillium spp. (%)
M. guilliermondii	Fresh	0	53.10 ± 9.38deH	8.70 ± 3.34dF	15.68 ± 4.10cdDEF	84.95 ± 6.19aA
		30	74.14 ± 9.60abDE	47.18 ± 6.99bcCDE	8.46 ± 4.06deGH	73.76 ± 4.58abcdBC
	Encapsulated	0	69.38 ± 8.77abcE	15.12 ± 8.20dF	29.93 ± 5.14aA	72.10 ± 8.81bcdBC
		30	72.88 ± 9.34bDE	50.11 ± 10.63abBC	9.63 ± 5.15deFGH	79.79 ± 3.62abcAB
M. caribbica	Fresh	0	59.81 ± 10.78cdGH	8.82 ± 2.20dF	14.87 ± 1.03cdDEFG	85.57 ± 9.66abA
		30	72.29 ± 10.75abcDE	50.50 ± 2.92abBC	3.31 ± 4.25eH	70.75 ± 4.12cdeBC
	Encapsulated	0	68.44 ± 6.26abcEF	17.94 ± 2.6dF	27.60 ± 4.38abAB	73.45 ± 2.30abcdBC
		30	68.25 ± 6.71abEF	48.46 ± 9.47bcBCD	19.28 ± 3.07bcCDE	57.46 ± 9.25efE
Y. mexicana	Fresh	0	52.25 ± 9.88eH	48.05 ± 4.07abcBCD	14.65 ± 4.20cdeDEFG	69.03 ± 5.17defCD
		30	78.63 ± 4.96abBCD	60.28 ± 6.47aA	10.04 ± 1.95cdFGH	69.32 ± 2.06fCD
	Encapsulated	0	75.69 ± 7.44abcCDE	39.3 ± 2.41cDE	24.77 ± 8.41abABC	65.25 ± 9.24defCDE
		30	61.38 ± 10.54acFG	39.78 ± 9.30bcDE	13.02 ± 8.03deEFG	61.35 ± 7.44deDE
Controls
Untreated		0	86.04 ± 5.10AB	38.00 ± 3.56E	16.54 ± 3.10DEF	68.71 ± 6.06CD
		30	82.75 ± 4.40ABC	39.31 ± 10.49DE	19.39 ± 2.92CDE	73.70 ± 3.62BC
Control (H2O)		0	85.75 ± 7.36AB	45.2 ± 3.91CDE	21.00 ± 3.97BCD	65.24 ± 3.88CDE
		30	83.56 ± 4.45AB	57.00 ± 9.47AB	28.89 ± 6.92A	58.43 ± 1.53E
Capsules without yeast		0	90.13 ± 5.77A	42.66 ± 6.57CDE	31.60 ± 8.23A	65.39 ± 10.38CDE
		30	82.99 ± 3.54ABC	49.55 ± 7.30BC	14.60 ± 2.79DEFG	70.44 ± 6.68CD

• ^*
  The values represent the mean ± standard deviation across three independent replicates, each consisting of 16 boxes with 25 seeds (400 seeds per treatment).
• Lowercase letters correspond to comparisons among treatments included in the 3 × 2 × 2 factorial design, whereas uppercase letters correspond to comparisons between factorial treatments and control treatments using Fisher’s least significant difference test (p < 0.05).

After 30 days of storage, the disease incidence in the control seeds (untreated and water-treated) remained high (82.75 and 83.56%), showing no difference compared with the seeds at the start of storage (Table 1). Similarly, the application of capsules without yeast did not show a protective effect, confirming that the encapsulating material has no antifungal activity.

On the other hand, treatments with encapsulated yeasts maintained a reducing effect, although with variations between strains. Y. mexicana stood out, with the lowest disease incidence (61.38%), which represented a significant decrease compared to the controls. On the other hand, M. caribbica also showed a consistent reduction (68.25%), while M. guilliermondii showed intermediate values (72.88%). According to the analysis of variance (Table 2), the effectiveness of the treatments in reducing disease incidence was influenced by both the form of yeast application and the duration of seed storage. Furthermore, the interaction between these two factors (p < 0.05) indicated that the effectiveness of the yeasts (whether fresh or encapsulated) depended on storage time.

Table 2
Analysis of variance of the main effects and interactions of yeast species, yeast application method, and seed storage period on disease incidence and the frequency of the observed fungal genus<tfn href="tfn02">*</tfn>.

Source of variation	Disease incidence	Fusarium spp.	Aspergillus spp.	Penicillium spp.
Species (S)	NS	< 0.001	NS	< 0.001
Formulation (F)	0.002	NS	< 0.001	NS
Storage period (T)	< 0.001	< 0.001	< 0.001	0.0154
S × F	NS	NS	NS	0.031
S × T	NS	< 0.001	NS	NS
F × T	0.003	NS	0.0139	0.040
S × F × T	0.006	0.009	0.017	NS

• NS: not significant (p > 0.05);
• ^*
  values correspond to the p-values obtained from the factorial analysis of variance that evaluates the main effects of the yeast species (S), the form of application of the yeast (F), and the storage period of the treated seeds (T), as well as their interactions, on the germination percentage, the emergence rate index, the root length, the stem length, and the dry mass of the bean seedlings.

In particular, fresh yeast was more effective at the start of storage, whereas encapsulated formulations retained their antagonistic activity better after 30 days of seed storage, suggesting that encapsulation promoted cell viability, likely due to the protection provided by the polymer matrix. This difference in efficacy observed at the start of storage is attributed to encapsulated yeasts requiring a period of release and metabolic adaptation before exerting their antagonistic effect, which may delay their action relative to fresh yeasts. However, encapsulated formulations offer advantages as they facilitate handling, storage, and eventual commercialization compared to fresh yeast (López-Cruz et al. 2022, González-Gutiérrez et al. 2024). This behavior has been previously described in encapsulated systems with WPC and AG as wall materials, which provide cellular protection and allow for gradual release, thus constituting a key advantage for biocontrol applications (Covarrubias-Rivera et al. 2025).

Regarding the relative frequency of fungal genera in seeds at the beginning of storage, Penicillium spp. was the most prevalent through all treatments, ranging from 65.25 to 85.57%. On the other hand, Fusarium spp. and Aspergillus spp. showed greater variability. In particular, the frequency of Fusarium spp. was significantly lower in seeds treated with fresh M. guilliermondii (8.70%), whereas Aspergillus spp. frequency decreased with fresh Y. mexicana (14.65%). At the end of 30 days of storage, an increase in the detection of Fusarium spp. was observed in several treatments, especially in fresh Y. mexicana (60.28%). However, this genus was significantly reduced in seeds treated with fresh (47.18%) and encapsulated (50.11%) M. guilliermondii. The frequency of Aspergillus spp. decreased overall, with a more marked decrease in treatments with encapsulated M. caribbica (3.31%) and M. guilliermondii (9.63%). For its part, the fungus Penicillium spp. continued to be the predominant genus. This behavior reflects the specificity of interaction mechanisms, in which the effectiveness of antagonistic yeasts depends on the yeast strain and the phytopathogen involved (Zhang et al. 2020). A fungicide-treated control was not included in this study because the primary objective was to conduct an initial screening of the antifungal potential of encapsulated yeasts and to evaluate the encapsulation strategy itself.

However, it is important to note that the use of naturally contaminated seeds may introduce variability in the initial infection levels, which could influence the observed responses. Therefore, future studies should consider controlled inoculation approaches and fungicide-treated control to improve experimental consistency and reproducibility.

Effect of fresh and encapsulated yeasts on the germination and vigor of bean seeds

At the beginning of storage (Table 3), treatments with both fresh and encapsulated yeasts showed no significant differences in germination percentage compared to the control, with values ranging from 87.05 to 95.50%. The emergence rate index showed a similar trend, with the application of fresh M. guilliermondii (22.13) and M. caribbica (22.22) yeasts standing out. In terms of seedling growth, root and shoot length remained comparable between treatments, with slightly higher values observed when applying encapsulated M. caribbica (27.19 cm) for the shoot and fresh M. caribbica (15.62 cm) for root length. Dry mass remained stable in all treatments (0.156–0.183 g; p > 0.05).

Table 3
Percentage of germination, root and stem length, dry weight and emergence rate index in bean seeds treated with fresh yeasts and encapsulated during storage<tfn>*</tfn>.

Species	Form of application of the yeast	Seed storage period (days)	Germination percentage (%)	Emergency speed index	Root length (cm)	Length of the overhead part (cm)	Dry mass (g)
M. guilliermondii	Fresh	0	95.50 ± 3.00ab	22.13 ± 0.78abA	12.80 ± 0.38cdBC	26.15 ± 0.62abcA	0.183 ± 0.012abAB
		30	96.50 ± 3.00ab	19.67 ± 0.90cA	14.02 ± 0.40abABC	25.49 ± 1.36bcA	0.177 ± 0.030abAB
	Encapsulated	0	88.95 ± 8.41b	22.12 ± 2.24abA	11.94 ± 1.29dBC	27.57 ± 0.34aA	0.156 ± 0.007bB
		30	95.33 ± 4.16ab	19.36 ± 0.44cA	13.00 ± 2.21bcdABC	26.65 ± 1.24abcA	0.165 ± 0.010abAB
M. caribbica	Fresh	0	95.00 ± 5.03ab	22.22 ± 0.46aA	15.62 ± 1.57aA	27.19 ± 2.24aA	0.181 ± 0.014abAB
		30	95.25 ± 0.96ab	20.36 ± 0.26bcA	14.43 ± 1.18abcAB	25.70 ± 0.84cA	0.178 ± 0.014abAB
	Encapsulated	0	90.00 ± 6.73ab	19.67 ± 1.77cA	12.07 ± 0.89cdBC	27.43 ± 0.91abA	0.172 ± 0.019abAB
		30	93.33 ± 3.06ab	19.86 ± 0.62cA	12.55 ± 1.29cdBC	26.01 ± 1.37abcA	0.160 ± 0.013abAB
Y. mexicana	Fresh	0	95.25 ± 4.86ab	21.49 ± 1.47abA	13.21 ± 0.33bcdABC	26.02 ± 0.51abcA	0.174 ± 0.018abAB
		30	95.25 ± 3.77ab	19.90 ± 1.32cA	13.16 ± 2.19abcABC	27.67 ± 3.21abcA	0.184 ± 0.026abAB
	Encapsulated	0	87.05 ± 1.48ab	18.12 ± 0.28cA	11.48 ± 0.32dC	26.94 ± 0.61abA	0.160 ± 0.020abAB
		30	98.00 ± 2.31a	19.98 ± 1.34cA	13.66 ± 0.12bcABC	26.07 ± 0.99abcA	0.172 ± 0.015aAB
Controls
Untreated		0	91.75 ± 9.99A	19.59 ± 3.03bcA	12.87 ± 0.75bcBC	26.14 ± 0.77dA	0.167 ± 0.017aAB
		30	94.75 ± 2.99A	19.41 ± 1.12aA	12.48 ± 1.16bBC	25.77 ± 0.99abA	0.176 ± 0.020aAB
Control (H2O)		0	95.08 ± 1.25A	20.05 ± 0.39abcA	13.33 ± 0.96bBC	26.95 ± 0.94abcdA	0.171 ± 0.037a
		30	96.45 ± 2.53A	20.50 ± 0.86aA	13.28 ± 0.29abABC	25.12 ± 1.03bA	0.186 ± 0.022aA
Capsules without yeast		0	89.10 ± 6.93A	19.26 ± 1.47abcA	12.99 ± 0.57bcdABC	28.04 ± 1.60aA	0.163 ± 0.021aAB
		30	95.75 ± 4.35A	19.50 ± 1.10aA	13.36 ± 0.61abABC	25.01 ± 0.94bA	0.163 ± 0.008aAB

• ^*
  The values correspond to the mean ± standard deviation of three independent replicates, each consisting of four boxes with 50 seeds (200 seeds per treatment).
• Lowercase letters correspond to comparisons among treatments included in the 3 × 2 × 2 factorial design, whereas uppercase letters correspond to comparisons between factorial treatments and control treatments using Fisher’s least significant difference test (p < 0.05).

At the final seed storage stage (30 days; Table 4), the results confirmed the stability of the physiological parameters. The germination rate remained high (> 93%) for all evaluated treatments, with no differences compared to the control. The emergence rate index was uniform (19.36–20.50; p > 0.05) without compromising seedling emergence. In terms of development, the length of the aerial part remained in the range of 25.01–27.67 cm, being slightly higher when Y. mexicana was applied in its fresh form (27.67 cm), while root length did not show any relevant reductions. Dry mass remained constant between treatments (0.160–0.186 g; p > 0.05).

Table 4
Analysis of variance of the main effects and interactions of yeast species, yeast application method, and seed storage period on germination and seedling growth parameters of bean seeds<tfn href="tfn04">*</tfn>.

Source of variation	Germination percentage	Emergency speed index	Root length	Length of the overhead part	Dry mass
Species (S)	NS	NS	NS	NS	NS
Formulation (F)	NS	NS	< 0.001	NS	NS
Storage period (T)	NS	< 0.001	0.044	0.010	NS
S × F	NS	NS	NS	NS	NS
S × T	NS	NS	0.026	NS	NS
F × T	NS	0.036	NS	NS	NS
S × F × T	NS	NS	NS	NS	NS

• NS: not significant (p > 0.05);
• ^*
  values correspond to the p-values obtained from the factorial analysis of variance that evaluates the main effects of the yeast species (S), the form of application of the yeast (F), and the storage period of the treated seeds (T), as well as their interactions, on the germination percentage, the emergence rate index, the root length, the stem length and the dry mass of the bean seedlings.

According to analysis of variance (Table 4), the physiological parameters evaluated were not significantly affected by yeast species, the form of application (fresh or encapsulated), or their interactions, suggesting that the different antagonistic yeast treatments showed similar effects on seed physiological quality. Overall, the absence of negative effects on root and shoot growth suggests that antagonistic yeasts do not interfere with the physiological processes associated with seedling vigor. This behavior supports the compatibility between the bioformulations evaluated and seed physiology, an essential aspect for their implementation in sustainable agricultural systems (Dewedar and Ibrahim 2016). Furthermore, when evaluating the dry matter of the seedlings, there were no significant differences between treatments, indicating that the presence of yeasts did not affect the ability of the seeds to acquire and mobilize nutrients during the initial stages of seedling growth (Hesham and Mohamed 2011).

Future research could evaluate combined formulations that integrate yeasts with high antifungal activity together with strains or compounds with plant growth-promoting effects, such as phytohormone production, nutrient solubilization, or root development stimulation. This strategy would allow for the simultaneous optimization of phytosanitary protection and the physiological performance of seedlings. Overall, the results suggest yeast-seed interaction does not compromise plant physiology and opens the possibility of applying encapsulated yeasts as a safe application strategy in agricultural management programs.

CONCLUSION

The yeasts M. guilliermondii, M. caribbica, and Y. mexicana have significant antagonistic potential against fungi associated with bean seeds. The application of these yeasts, both fresh and encapsulated using SBSp, reduced the disease incidence of pathogens without affecting germination or seedling vigor, supporting their safety for cultivation. These findings support the use of encapsulated antagonistic yeasts as a sustainable strategy for pathogen control in bean seeds, with potential for implementation in sustainable agricultural programs.

ACKNOWLEDGMENTS

Not applicable.

• How to cite:
  Covarrubias-Rivera, L., Calderón-Santoyo, M., Pereira, R. A. S., Christ, D., Ragazzo-Sánchez, J. A. and Coelho, S. R. M. (2026).Evaluation of encapsulated antagonistic yeasts using solution blow spraying for the control of fungi in common bean seeds (Phaseolus vulgaris L.). Bragantia, 85, e20250265. https://doi.org/10.1590/1678-4499.20250265
• FUNDING
  Move La América
  Grant No. 88881.996902/2024-01
  Secretariat of Science, Humanities, Technology, and Innovation
  Grant No. 1153561
• DECLARATION OF USE OF ARTIFICIAL INTELLIGENCE TOOLS
  The authors declare that artificial intelligence (ChatGPT, OpenAI) was used exclusively for language editing and text revision. All scientific content, analyses, interpretations, and writing of the manuscript were performed by the authors.

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available on request from the corresponding author.

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Edited by

• Section Editor:
  Gabriel Constantino Blain https://orcid.org/0000-0001-8832-7734

Publication Dates

• Publication in this collection
  10 Aug 2026
• Date of issue
  2026

History

• Received
  03 Dec 2025
• Accepted
  27 May 2026