Development of Natural Fungicidal Agricultural Defensives Using Microbial Glycolipid and Vegetable Oil Blends

Abstract

The use of pesticides causes significant environmental problems, which drives the search for natural and non-toxic alternatives. In this study, a glycolipid biosurfactant (BS), produced by the yeast Starmerella bombicola ATCC 22214, was utilized as an active ingredient in natural agricultural defensive blends. The mixtures were tested for their fungicidal potential against phytopathogenic fungi isolated from fruits such as papaya, orange, and banana, demonstrating strong inhibition of fungal growth. The genera Penicillium, Colletotrichum, and Aspergillus were the pathogens present in the deterioration of the fruits used in the experiment. The biosurfactant was produced in a fermenter, yielding 10 g/L and reducing the surface tension to 31.56 mN/m, with a critical micelle concentration (CMC) of 366 mg/L. Blends of BS with oleic acid (T1) and lemongrass oil (T2) were found to be effective in controlling fungi. Additionally, the phytotoxicity of these formulations was assessed using Cucumis anguria (gherkin) seeds, where the blend of BS with castor oil (T4) showed the best performance, promoting seed germination. These results indicate the potential of such mixtures as natural alternatives for fungal control in plants and for application in sustainable agricultural systems.

1. Introduction

Agriculture plays a fundamental role in the development and sustainability of human society. According to the Food and Agriculture Organization of the United Nations (FAO) [1], agriculture is responsible for providing approximately 80% of the food consumed by the global population. A notable surge has been observed in the global agrochemical market to address the escalating need for food to sustain the increased human populace. This escalating demand has spurred the development and widespread adoption of synthetic agrochemicals for pest and weed management across various crops [2]. To address this demand while promoting sustainable development, international agreements and regulatory frameworks were established to encourage the responsible use of natural resources and mitigate the impacts of climate change. These efforts have influenced the focus on intensifying and sustaining agricultural production for the coming decades [3,4].

Agrochemicals are chemical substances that can perform the biological control of weeds or unwanted weeds (herbicides) [5] or even protect agricultural products against fungi (fungicides) [6] and pests such as rodents and insects (pesticides) [7]. According to the Food and Agriculture Organization of the United Nations (FAO), between 1990 and 2020, the global consumption of agrochemicals increased from 1.7 million tons to 2.7 million tons [1]. Currently, the control of phytopathogens relies mainly on high doses of chemical pesticides, the extensive use of which has impacted various ecosystems [4]. Pesticides are not biodegradable, or they are degraded into non-toxic compounds with great difficulty. Many of these products are dumped into water bodies, causing fish poisoning through bioaccumulation in their tissues or contaminating foods such as vegetables [5,8,9]. Therefore, searching for bioproducts or green products to obtain agro-defensive products is necessary to reduce the presence of toxic agrochemicals in agribusiness [8].

Natural agricultural defensives are pesticides of biological origin that can be obtained from plant, fungal, or bacterial extracts. Because of their biological origin, they have reduced toxicity and high biodegradability [10,11,12]. Essential oils and plant extracts are the most used botanical products as biopesticides. Typically, lipids and fatty acids act as solvents, which, when combined with emulsifiers, help stabilize the active ingredients [13].

Agrochemical formulations require surfactants, which are essential for their preparation, maintenance of physical stability, and improvement of biological performance. Chemical surfactants are widely used in agriculture as aids in crop production [14,15]. In this context, the importance of sustainability in using renewable resources and improving products brings to light the possibility of replacing chemical surfactants with their green counterparts [16].

From this perspective, biosurfactants have emerged as a promising green alternative because of their versatility and antimicrobial properties. Biosurfactants, such as rhamnolipids and lipopeptides, have demonstrated effectiveness in plant protection, especially against Gram-positive bacteria and phytopathogenic fungi, significantly contributing to disease control in agriculture [17]. These compounds operate through various mechanisms, including altering the permeability of pathogen cell membranes, leading to their inactivation. Furthermore, biosurfactants, being biodegradable and non-toxic, represent a sustainable alternative to synthetic agrochemicals, which pose environmental risks [4].

Green surfactants are surface-active substances with amphipathic characteristics, i.e., having both a hydrophilic and a hydrophobic portion, and antimicrobial properties that can be extracted from plants and/or can be obtained from the metabolism of microorganisms (so-called biosurfactants) or by chemical synthesis using natural extracts (biobased surfactants) [18,19]. Microbial biosurfactants, because of their advantageous characteristics for agro-industrial activities, have garnered attention for potential applications as biopesticides, biofertilizers, biostimulants, biodispersants, and soil bioremediators, among other versatile uses [4]. However, research on biosurfactants in the agricultural field, when compared with that in the environmental, health, cosmetic, and food areas, is still little explored [10,12,20,21,22,23].

Advances in the research and development of biopesticides significantly mitigate environmental damage caused by synthetic pesticide residues and promote sustainable agriculture. Despite the numerous advantages of biopesticides, biofertilizers, biostimulants, and biodispersants, their main challenge lies in commercialization. The barriers preventing the widespread adoption of natural agricultural pesticides include the high cost of refined commercial products, difficulty in meeting global market demand, variability in standard preparation methods and guidelines, the need to determine active ingredient dosages, the sensitivity of biopesticides to various environmental factors, and their ephemeral nature and slow action, among other challenges [2,4,24,25].

Recent studies have also demonstrated the potential of vegetable oils for use in natural agricultural defensive formulations. Essential oils, such as lemongrass and tea tree oil, have recognized fungicidal properties, and when combined with other materials, they can enhance efficacy in controlling phytopathogens [13]. However, the combination of biosurfactants with various vegetable oils for the development of agricultural defensives remains a relatively underexplored area, especially regarding their application in real-world agricultural settings.

Based on this background, this research aimed to investigate the application of the glycolipid produced by the yeast Starmerella bombicola ATCC^® 22214™ in the development of botanical pesticides, aiming to achieve a natural agricultural defensive formulation that could offer fungicidal protection to plants.

2. Materials and Methods

2.1. Microorganism, Maintenance, and Inoculum

The yeast Starmerella bombicola ATCC^® 22214™ obtained from the American Type Culture Collection (ATCC) was the strain used to produce the glycolipid biosurfactant. Yeast maintenance was performed on Yeast Mold Agar (YMA) medium. To obtain the inoculum, cells were activated in a medium containing 100 g/L glucose, 10 g/L yeast extract, and 0.1 g/L urea at 30 °C and 200 rpm for 24 h. The inoculum was standardized at 0.5 g/L of cells by reading at 600 nm and correlated with a previously obtained biomass concentration curve [26].

2.2. Biosurfactant Production

The glycolipid production medium, prepared as described by Hipólito et al. [26], had the following composition: 150 g/L glucose, 219.5 g/L oleic acid, and 2.5 g/L yeast extract. Biosurfactant production was carried out in a 1 L fermenter (Tec-Bio-Plus, Tecnal, Piracicaba, SP, Brazil) provided with temperature (28 °C) and pH (7.0 ± 0.2) control. The inoculum was standardized as described before, and the runs were carried out at an agitation of 200 rpm and aeration of 1 vvm. At the end of cultivation (168 h), the cell broth was centrifuged (10,000× g, 15 min at 20 °C), and the supernatant was subjected to extraction.

2.3. Surface Tension Determination

The surface tension of the biosurfactant was measured with an automatic tensiometer (KSV Sigma 700, Helsinki, Finland) using the Du Noüy ring method. The magnitude was measured by immersing the platinum ring in the cell-free broth, recording the force required to pull it through the air–liquid interface.

2.4. Biosurfactant Extraction

The biosurfactant was extracted and partially purified by liquid–liquid extraction with an organic solvent. Cells were removed by centrifugation (10,000× g, 15 min at 20 °C) to obtain the cell-free broth. The cell-free broth was extracted twice with an equal volume of ethyl acetate and shaken vigorously in a separatory funnel. The lower aqueous phase and the upper ethyl acetate phase were collected separately, and the former was extracted twice more with ethyl acetate. Ethyl acetate extracts were combined, and the solvent was evaporated under vacuum using a rotary evaporator (Tecnal, Piracicaba, SP, Brazil) to obtain crude biosurfactant and residual oil. The residual oil was removed by washing it three times with n-hexane, while the crude biosurfactant was decanted after washing with n-hexane [27]. The biosurfactant produced by the yeast was subjected to evaporation drying and stored for the formulation development stage.

2.5. Critical Micelle Concentration (CMC) Determination

The critical micelle concentration (CMC) was determined by measuring the surface tension of the dilutions of the isolated biosurfactant in distilled water up to a constant value. Stabilization was allowed to occur until the standard deviation of 15 successive measurements was less than 0.4 mN/m. The CMC was obtained by plotting surface tension against surfactant concentration and expressed as mg/L of biosurfactant.

2.6. Thin Layer Chromatography (TLC)

TLC was used as the method to confirm the production of glycolipids by the yeast Starmerella bombicola ATCC^® 22214™. Thin-layer chromatography (TLC) was conducted following the protocol outlined by Smyth et al. [28], employing the isolated biosurfactant. The TLC plates were developed using a mobile phase consisting of water/methanol/chloroform (0.2:1.5:6.5 v/v/v). Detection was achieved using p-Anisaldehyde as the spray reagent.

2.7. Blends of Natural Agricultural Defensives

The oils used in the blends of agricultural defensives were purchased in stores specializing in natural products from local and national markets in the city of Recife, PE, Brazil. The combination of biosurfactants with natural and essential oils was thoughtfully chosen based on scientific evidence of their antimicrobial properties, effectiveness as natural insecticides, and beneficial effects on plant growth, development, and protection [29]. The emulsifiable concentration type blend was prepared from the glycolipid biosurfactant dissolved in distilled water at 0.1% (v/v). Before incorporating lemongrass (Cymbopogon flexuosus) and tea tree (Melaleuca alternifolia) essential oils, sunflower vegetable oil was added as a neutral carrier. Commercial neem (Azadirachta indica) was used for comparative purposes as it is used as a fungicide on plants. Castor oil (Ricinus communis L.) was also tested, and distilled water was used as a control. The compositions of the tested blends are listed in

Table 1. Composition of the tested blends of natural agricultural defensives.

Treatment	Composition
T1	Biosurfactant + oleic acid + distilled water
T2	Biosurfactant + lemongrass oil + distilled water
T3	Biosurfactant + tea tree oil + distilled water
T4	Biosurfactant + castor oil + distilled water
T5	Biosurfactant + commercial neem
T6	Commercial neem
T7	Distilled water (control)
T8	Biosurfactant + distilled water

. All liquid blends were prepared without heating, with continuous stirring for more than 5 min using a Vortex magnetic stirrer until the formation of a single phase. The pH of the blends was adjusted to 6.8, the same value found in the commercial formulation. To maintain a reduced number of treatments, the biosurfactant was tested only at the 0.1% (v/v) concentration in all blends. The vegetable oil was used at concentrations of 10% (v/v) and essential oils at 1% (v/v). The final volume was completed with water up to 500 mL in all blends.

2.8. Assessment of the Antifungal Potential of the Formulated Agricultural Defensives

For this test, phytopathogenic fungi were isolated, using the indirect isolation technique, from papaya (Carica papaya L.), orange (Citrus sinensis (L.) Osbeck), and banana (Musa spp.) fruits obtained from a local fruit and vegetable store. Tissue fragments were removed from the border region between the injured area and the healthy area of the fruit, as this is the area where the pathogen is most active. Necrotic areas in the center of the lesions usually contain a high population of saprophytes and were avoided. These fungi were used to detect the presence of antifungal activity in the formulated pesticides. In this study, identification was not performed at the molecular level, but only phenotypically through identification keys and macro and microscopic visualization of fungal structures [30].

The isolated fungi were cultivated and maintained in Potato Dextrose Agar (PDA) at 28 °C. For the antifungal analysis, 1 mL of each agricultural defensive formulation was added to the PDA medium, contained in different Erlenmeyer flasks. The solutions were mixed with agar using a magnetic stirrer and poured into Petri dishes for solidification. With the plates prepared in the previous step, 7 mm discs of fungal mycelia (~10⁶ spores per disc) were transferred to the middle of the plates. These were incubated at 28 ± 2 °C for 5 days. Plates containing only PDA medium, and fungi were used as controls.

2.9. Determination of the Phytotoxicity of Agricultural Defensives Blends Using the Plate Germination Test

Cucumis anguria (gherkin) seeds were used for the germination and root growth method described by Yerushalmi et al. [31]. The gherkin was used because it is a species that grows quickly under controlled conditions, is economically important for family farming, and is also parasitized by various microorganisms, including fungi. All the agricultural biodefensives formulated according to Table 1 were tested. The treatments were placed separately in Petri dishes with 10 seeds, and the plates were incubated for 120 h at 28 °C. The number of germinated seeds was then counted, and the root length was measured from the hypocotyl transition point to the tip of the root. The germination index (GI, %), which is one of the most used ways to characterize the phytotoxicity of a compound, was calculated according to the equation below (1):

GI (%) = [(% seed germination) × (% root growth)]/100

(1)

where

% seed germination = [(% treatment germination)/(% control germination)] × 100

% root growth = [(average growth in treatment)/(average growth in control)] × 100.

The samples were analyzed in triplicate.

2.10. Determination of the Phytotoxicity of Agricultural Defensive Blends in the Seedbed

The same method described in the previous section was carried out. Cultivation trays with up to 15 cells were filled with Biokashi balanced organic substrate (Biomix, São Paulo, SP, Brazil) completely free of pests and diseases, with balanced pH (5.5–5.8), ready for use in seedlings in general organic cultivations. According to the manufacturer, this substrate, composed of coconut powder or fiber, ground, and pine bark, is rich in macro- and micronutrients. The treatments were placed separately in seedbeds with 3 seeds per cell, that is, cavities in the growing trays used for seeding and germination. The seeds were incubated for 7 to 14 days with artificial light in a closed environment with a 12 h on/12 h off light cycle. After this period, the number of germinated seeds was counted, and the length of the roots was measured from the hypocotyl transition point to the tip of the root. GI was determined in the same way as described in Section 2.9.

2.11. Determination of the Dispersion Capacity (Spread) of Blends on the Surface

For this test, 40 µL of the natural agricultural defensive blends (Table 1) were applied to a single point on 50 × 50 mm² paraffin sheets. Measurements were taken using a caliper. Triplicate samples were measured, and the droplet diameters were compared with that of the blank (sample T7).

2.12. Post-Harvest Shelf-Life Test

The post-harvest shelf-life test is usually carried out to evaluate the ability of a natural agrochemical formulation to extend the post-harvest shelf life of sensitive plant products, such as leafy crops. Samples from uninjured parts of Lactuca sativa var. capitata (lettuce) were selected and kept in Petri dishes, where they received the agricultural defensives described in Table 1. The treatments were sprayed on the adaxial surfaces of the leaves using a manual spray system. Then, the plates with the leaves covered by the treatments were stored at 28 °C. The evaluation was carried out visually every 24 h for 3 days, and during this interval, the presence of injuries on the leaf surfaces was analyzed in comparison with the blank (T7).

2.13. Statistical Analysis

All experiments were performed with at least three replications. The data were subjected to the one-way analysis of variance (ANOVA) significance test, and the difference between treatments was compared using the Tukey test (p ≤ 0.05). All data were presented as means and standard deviations using Origin 8.0 and Microsoft Excel 6.0 software.

3. Results and Discussion

3.1. Biosurfactant Production, CMC, and TLC

The biosurfactant produced in a bioreactor yielded 10 g/L and showed a surface tension of 31.56 mN/m. Previous studies on the synthesis of glycolipids indicate that the yield of sophorolipids varies depending on both the strain and the carbon sources. For instance, a sophorolipid from Starmerella bombicola ATCC 22214 was produced from corn straw with a yield of 27.45 g/L [32], while a maximum sophrolipid production of 40.82 g/L was recently reported for S. bombicola BCC5426 [33] using soybean meal as a carbon source at pH 4.5 in a 5 L fermenter. The extracted biosurfactant had a CMC of 366 mg/L (Figure 1), a value compatible with those described in the literature for glycolipid biosurfactants [34].

As revealed by the TLC chromatogram (Figure 2), the chemical nature of the biosurfactant was predicted as sophorolipid in comparison with the Rf values mentioned in the literature [35]. Spots with an Rf value between 0.11 and 0.6 were also observed in the test sample, indicating the presence of glycolipids, probably of the sophorolipid type.

Sophorolipids constitute one of the most studied classes of glycolipid-type biosurfactants [36,37]. Their structures are formed by a disaccharide called sophorose (2-O-β-D-glucopyranosyl-D-glucopyranose) linked to a long-chain hydroxy fatty acid (C16 to C18) by a glycosidic bond between the sugar anomeric C atom and the hydroxyl group of the fatty acid. Sophorolipids are generally a mixture of at least six to nine types of hydrophobic moieties with lactone form, which is preferable in many applications [38]. The surface tension of these biomolecules is around 33 mN/m.

Among the yeasts that produce them, S. bombicola is one of the most productive that can meet possible industrial demand [37]. Bioprocesses described in the literature for sophorolipid production include submerged batch and fed-batch fermentations conducted in shake flasks and bioreactors [39,40].

The sophorolipids market was projected to reach USD 5.52 billion by 2022, which indicates that it is the largest global market for microbial biosurfactants [41]. The applications of sophorolipids include their use mainly as cleaning and cosmetic and bioremediation agents, although research in the agricultural field is still very limited [4,42]. Industrial-relevant strains that synthesize sophorolipids, rhamnolipids lipopeptides, and trehalolipids can effectively produce high quantities of biosurfactants, improving sustainability in sustainable agriculture [43].

3.2. Antifungal Potential of Natural Agricultural Defensives

Figure 3 illustrates the results of the defense potential of blends against phytopathogenic fungi isolated directly from the diseased fruit species, namely, papaya (Carica papaya L.), orange (Citrus sinensis (L.) Osbeck), and banana (Musa spp.), acquired from a local fruit and vegetable store in Brazil. It was possible to isolate many fungi from different genera. According to the morphological characteristics, the genera Penicillium, Colletotrichum, and Aspergillus were the pathogens present in the deterioration of the fruits used in the experiment.

After 5 days, treatment with the blend T1 (biosurfactant + oleic acid) showed significant inhibition of fungal growth, which can be explained by the ability of oleic acid to disrupt fungal membrane integrity. Similarly, the blend T2 (biosurfactant + lemongrass oil) demonstrated strong antifungal action, possibly due to the presence of citral, a compound known to inhibit ergosterol synthesis in fungi [44]. However, the blend T4, containing castor oil, was less effective, suggesting that the combination of this oil with the biosurfactant may not have generated the necessary synergy to inhibit fungal growth. This result may be related to the chemical composition of castor oil, which may not have the same fungicidal properties found in essential oils such as lemongrass. In this respect, it is noteworthy that lemongrass at concentrations of up to 1% (v/v) was already shown to have a fungicidal activity higher than 80% [44] and that, according to Abubacker and Devi [45], oleic acid, present in olive oil and palm oil, also has excellent characteristics as a fungicide and bactericide.

In the current scenario, plant-derived biopesticides are gaining prominence because of their ability to specifically target the desired pest, their environmental compatibility, natural degradation capability, and safety for both humans and other forms of life [46].

The fungi isolated from the surfaces of fruits (papaya, orange, and banana) presented colonies of different colors, predominantly white, black, and greenish. The colonies exhibited a cottony and aerial texture, indicating the development of hyphae on the surface of the medium. In some samples, the edges of the colonies were well-defined, while others showed more diffuse and irregular growth. It is important to emphasize that the characterization of the fungi was performed qualitatively to evaluate the presence of antifungal activity in the mixtures tested. Detailed identification of fungal species was not the focus of this study and is suggested for future work with a greater emphasis on taxonomic classification. Thus, the visual comparison suggests the presence of three distinct types of fungi grown only with distilled water (T7), one of which grew more abundantly. The formulation containing tea tree oil as the oily ingredient (T3) also resulted in the growth of these three fungi, although to a lesser extent. Samples containing the biosurfactant, distilled water, and oleic acid (T1) or lemongrass oil (T2) did not show fungal growth. This promising result suggests a high level of applicability of both blends as biofungicides. On the other hand, the formulation containing castor oil as an oily ingredient (T4) allowed the abundant growth of one type of fungus and, to a lesser extent, another in the central area of the plaque.

In the sample treated with biosurfactant combined with commercial neem (T5), two different types of fungi were detected, whose growth was abundant and weak, respectively. Compared with that obtained using only commercial neem (T6), this result demonstrated a better inhibition efficiency against fungi. Still, as the concentration of neem in the T5 formulation was lower than in the T6 formulation, the former lost its inhibition efficiency, proving to be unviable. The sample treated with the T6 formulation still showed the growth of a fungus in its initial phase, suggesting that the T1 and T2 blends would possibly have an even greater commercial potential. Finally, the sample treated with the biosurfactant isolated in distilled water (T8) was heavily contaminated by one type of fungus, but not by the other two fungi detected applying the different blends, thus suggesting that inhibition can be improved by increasing the biosurfactant concentration.

The fungal growth observed in the T5 blend (biosurfactant + neem oil) can be attributed to a possible negative interaction between the active compounds of neem oil and the biosurfactant. This interaction may have reduced the fungicidal efficacy of neem oil, which can be explained by some potential mechanisms such as (i) interference in the solubility or availability of neem oil by the presence of the biosurfactant. Since this oil contains active compounds such as azadirachtin, which is responsible for its insecticidal and antifungal properties, the use of surfactants, including biosurfactants, may have modified the dispersion of azadirachtin, reducing its bioavailability to interact with fungi and, consequently, its efficacy. (ii) Commercial neem oil already contains compounds in its formulation that can act as emulsifiers. The introduction of an additional biosurfactant may have caused an imbalance in the ideal emulsification ratio of the active compounds, reducing their fungicidal effect. (iii) The combination of biosurfactants with certain vegetable oils may create chemical incompatibilities, leading to the degradation or inactivation of the bioactive components of neem oil.

Patent documents suggest the utilization of vegetable oils, such as soybean oil, as an adjunct to substances exhibiting fungicidal, bactericidal, insecticidal, and herbicidal properties in both prophylactic and therapeutic interventions against plant pathologies induced by fungi, bacteria, and insects in agricultural settings. These efforts aim to provide an environmentally sustainable alternative with a favorable toxicological profile [47,48]. Biosurfactants produced from microbes like bacteria and fungi have antimicrobial activity against plant phytopathogens and are promising biocontrol agents for sustainable agriculture [49]. Sophorolipids derived from Wickerhamiella domercqiae were evaluated against fungal and oomycete pathogens, including Fusarium oxysporum and Pythium ultimum. Results demonstrated inhibition of mycelial growth and spore germination of these pathogens [50].

3.3. Phytotoxicity of Formulated Agricultural Defensives

The germination test was used to evaluate the possible phytotoxicity of the agricultural biodefensives, i.e., their capability of interfering with Cucumis anguria (gherkin) seed germination. The experiments lasted 120 h (5 days), which corresponds to the average germination time of gherkin seeds. As shown in Figure 4, no C. anguria seed germination took place after most of the treatments containing the biosurfactant. Of all treatments, the formulation containing the biosurfactant and castor oil (T4) showed the best performance, allowing for the highest seed germination. This result is consistent with the observation that castor oil added to clayey or sandy soil without nutrients had low toxicity to five plant species [51]. On the contrary, it can be visually seen that no germination occurred in the sample plates containing the blends T1, T2, T3, T5, and T8 (with biosurfactant) during the analysis period. This is confirmed in

Table 2. Length of Cucumis anguria (gherkin) roots (10 seeds) in Petri dishes after treatment with the tested agricultural biodefensive blends. T1 = biosurfactant + oleic acid + distilled water, T2 = biosurfactant + lemongrass oil + distilled water, T3 = biosurfactant + tea tree oil + distilled water, T4 = biosurfactant + castor oil + distilled water, T5 = biosurfactant + commercial neem, T6 = commercial neem, T7 = distilled water, and T8 = biosurfactant + distilled water. Values in a column followed by different letters are significantly different according to the least significant difference test at p < 0.05.

Treatment	Root Length (mm)
T1	5.0 ± 3.2 d
T2	0.0 ± 0.0 d
T3	0.0 ± 0.0 d
T4	21.4 ± 14.0 c
T5	5.0 ± 1.8 d
T6	34.8 ± 26.4 b
T7	48.3 ± 15.2 a
T8	2.4 ± 2.48 d

by statistically coincident results of these treatments according to the Tukey test.

The length of the roots in the plate containing the T4 formulation (biosurfactant + castor oil) was the closest to those observed using the commercial formulation (T6) and the control (T7).

The impressive performance of the T4 formulation (biosurfactant + castor oil) in the germination tests can be attributed to several key factors. Castor oil serves as an excellent source of nutrients, primarily due to the presence of ricinoleic acid. This compound is known to enhance water retention in the soil, which facilitates better nutrient absorption by seeds. As a result, castor oil creates a more conducive environment for germination. In addition to the benefits provided by castor oil, the biosurfactant plays a crucial role in mitigating potential toxicity. It helps to distribute castor oil uniformly throughout the substrate, thereby preventing localized phytotoxic effects that could hinder seed development. Furthermore, the biosurfactant may enhance the overall structure of the substrate. By increasing its porosity and aeration, it contributes to improved root development and promotes successful seed germination.

These effects can be seen more clearly in Figure 5, which shows the germination index (GI, %) obtained after each treatment compared to distilled water taken as a control (T7).

It can be seen that none of the blends ensured higher GI than distilled water. However, this does not necessarily mean that they are toxic to the seeds. Most samples did not allow more than 50% germination, and the sample treated with the T4 formulation showed growth close to half that of the control (Figure 5). Given these results, it was also possible to observe that it is essential to carry out toxicity tests with different concentrations to determine the efficacy, selectivity, and safety of a biodefensive formulation, providing accurate information for developing phytosanitary products for specific applications.

3.4. Germination and Growth in Seedbed

This test aimed to identify whether agricultural defensives would have any type of inhibition of C. anguria (gherkin) germination in seedbeds simulating natural growth, as well as any delay or improvement in plant growth due to the assimilation of nutrients present in the substrate. As illustrated in Figure 6, all samples in this test were able to develop.

Some seeds developed more than others, and even the samples that did not develop in the phytotoxicity test showed excellent results. Given the calculations developed (

Table 3. Length of Cucumis anguria (gherkin) roots in three seedbeds after treatment with the tested agricultural biodefensive blends. T1 = biosurfactant + oleic acid + distilled water, T2 = biosurfactant + lemongrass oil + distilled water, T3 = biosurfactant + tea tree oil + distilled water, T4 = biosurfactant + castor oil + distilled water, T5 = biosurfactant + commercial neem, T6 = commercial neem, T7 = distilled water, and T8 = biosurfactant + distilled water. Values in a column followed by different letters are significantly different according to the least significant difference test at p < 0.05.

Treatment	Root Length (mm)
T1	60.98 ± 1.78 b,c
T2	50.29 ± 0.50 d
T3	74.42 ± 3.79 a
T4	59.27 ± 2.83 c
T5	63.96 ± 1.91 b
T6	58.13 ± 0.51 c
T7	40.21 ± 2.14 e
T8	57.64 ± 0.84 c

), it is possible to identify which results were statistically different according to the Tukey test.

The samples containing the blends T1 and T5 showed a statistically coincident length of their roots, and the same occurred with blends T1, T4, T6, and T8.

The results of GI obtained in seedbeds (Figure 7) clearly indicate that all treatments behaved better than distilled water taken as a control (GI = 80%), highlighting a high potential for nutrient aggregation and contribution to seed germination.

Suppose these results are correlated with those of the phytotoxicity tests (Section 3.3). In that case, treatment with blends T2 (biosurfactant + lemongrass oil + distilled water) and T3 (biosurfactant + tea tree oil + distilled water) made it difficult for seeds to germinate on Petri dishes where no additional nutrients were present, i.e., under conditions not favorable for the plant. On the other hand, when applied to soil containing nutrients (tests in seedbeds), they allowed very promising results, with the T3 formulation performing even better than the T8 one (biosurfactant + distilled water). These results suggest that products applied under natural conditions are unlikely to be toxic to plants and will function as additives to improve the assimilation of soil nutrients. However, to confirm these deductions, biochemical tests are necessary, such as the characterization of total proteins and the determination of the activities of peroxidase and polyphenol oxidase, among other enzymes.

The gherkin (Cucumis anguria L.) is a cucurbitaceous plant native to East Africa. It produces light green fruits covered with soft spines, measuring 5 to 7 cm in length and 3 to 4 cm in diameter. Particularly cherished in the culinary traditions of the north and northeast regions of Brazil, it is highly sensitive to low temperatures and frost, preferably cultivated during the warmer seasons, with a growth cycle of approximately 70 days. Despite being a resilient vegetable, relatively resistant to pests and diseases compared with other leafy greens, the gherkin is susceptible to productivity and quality losses due to parasitism by various microorganisms, including fungi, oomycetes, viruses, bacteria, and nematodes [52]. Biosurfactants in sustainable agriculture can improve nutrient status, increase wettability, and achieve more even dissemination of complex nutrients, potentially enhancing crop productivity [53]. Biosurfactants can also stimulate plant immunity, presenting an alternative strategy to mitigate diseases induced by phytopathogens [54].

3.5. Dispersion Capacity (Spreading) of Blends on the Surface

This assay of dispersion capacity assessment indicates which of the formulated agricultural pesticides would have the best surface area coverage of plants when applied as a fungicide. Figure 8 shows the radius measurement procedure to check the dispersion of a known volume dispersed on the paraffin sheet.

After applying the Tukey test, it was found that the blends T6 and T7 (control) led to statistically coincident dispersions with each other as well as the blends T2 and T8 between them. Figure 9 was generated from these determinations, which graphically represents the percentage values of the comparison of each treatment in relation to the control (formulation T7) obtained from the dispersion test.

All blends had a better spreading capacity than the control (T7) or the commercial product (T6) (Figure 9) because of the surface-active action of the biosurfactant present in their composition, with the best results observed for blends T2, T3, T5, and T8. As the T5 formulation is a commercial product, which already has emulsifiers in its composition that are not stated on the label, the biosurfactant addition was likely to improve its surfactant power, although with minimal difference compared to the diluted biosurfactant alone (T8).

Agrochemical formulations necessitate surfactant adjuvants for both physical stability and biological efficacy. Incorporation of adjuvants into the spray solution induces a notable decrease in droplet contact angle, thereby enhancing the spreading and wetting area [4]. A greater spreading capacity of an agricultural pesticide would allow for reducing the amount of product to be applied. This result may be achieved by the addition of a biosurfactant thanks to its capability to reduce the surface tension of the liquid, thereby promoting a larger coverage area. Such an effect of the biosurfactant is directly related to the efficiency of the treatment [55].

3.6. Post-Harvest Shelf-Life

The post-harvest shelf-life test aimed to evaluate the possibility of the blends protecting or harming sensitive products in some way, thus lengthening or shortening their shelf-life. Figure 10 shows the results of this test carried out on leaves of Lactuca sativa var. capitata (lettuce), which were selected because of the sensitivity and rapid absorption of water on their surface that lead this vegetable to rapid deterioration in places of sale.

In the images, it is possible to observe that only distilled water (T7—control) did not induce any type of injury, followed by formulation T6, which caused less damage than the others. Since they were the only blends without the biosurfactant, it is possible to infer that the presence of the biosurfactant in the concentration tested accelerated the process of vegetable deterioration, even in the presence of oils. Moreover, only castor oil (T4) reduced the negative impact caused by the biosurfactant, whereas neem oil (T5) intensified it. It is important to address that there is no direct evidence from studies that biosurfactants cause mechanical damage to the leaf cuticle. Instead, biosurfactants may increase water diffusion through the cuticle and attract moisture, which may indirectly affect the properties of the cuticle but does not necessarily cause mechanical damage [56].

It would be important to investigate which formulation components may contribute to accelerating plant degradation. This may include analysis of the active ingredients, adjuvants, and other components present in the formulation [57]. Therefore, T2, T3, and T4 would be the blends that, even containing the biosurfactant, could have their composition adjusted to carry out new, more in-depth tests. Given the results obtained here, discussions about the possible interaction between these components and a vegetable, as well as their individual and combined effects on food deterioration, can provide valuable insights for future improvements in the formulation. Additional tests with different fruits and vegetables can also provide more expressive and significant results, with indications for the use of these blends in specific products.

4. Conclusions

The glycolipid derived from Starmerella bombicola ATCC 22214 was obtained through fermentation using glucose and oleic acid as carbon sources. This glycolipid was then utilized as an active ingredient and/or adjuvant in agro-defensive blends, in combination with other green pesticides. These blends were evaluated for various parameters, including seed germination, dispersion of active ingredients on leaf surfaces, post-harvest preservation of plant products, efficacy against phytopathogens, and potential phytotoxicity. The resulting blends demonstrated a range of application possibilities, suggesting that incorporating the glycolipid as an agro-defensive agent alongside other natural active ingredients can enhance the effects already documented in the literature for similar compounds, either by improving their efficacy or mitigating their adverse effects. Notably, nearly all proposed blends exhibited potential for specific applications.

For example, blend T1 (biosurfactant + oleic acid) and blend T2 (biosurfactant + lemongrass oil) showed significant promise as fungicides, effectively inhibiting the growth of fungi present on the tested fruits. Blend T3 (biosurfactant + tea tree oil) may serve as a beneficial soil additive that supports plant growth. Additionally, blend T4 (biosurfactant + castor oil) emerged as a promising soil additive, demonstrating success in germination tests while reducing toxicity. Conversely, blend T5 (biosurfactant + neem oil) did not yield promising results under the conditions tested in this study.

Overall, it can be concluded that combining biosurfactants with other agents enhances the interaction between plants and soil nutrients, leading to improved soil properties. This synergy can result in more effective disease control and ultimately enhance plant health and yield, thereby maximizing crop production.

Future studies will focus on utilizing this biosurfactant under various conditions and against identified phytopathogenic fungi. Additionally, exploring biosurfactants from alternative sources and testing formulations at different concentrations for various fruits and vegetables will be crucial for obtaining more insightful results and advancing this biotechnological field.