Production, Purification, and Application of a Biomolecule with Herbicidal Activity Produced by Fusarium fujikuroi in Submerged Cultivation

Abstract

This study investigated the production, purification, and evaluation of a microbial metabolite with herbicidal activity produced by Fusarium fujikuroi via submerged fermentation. The purified compound (PC) was obtained through organic solvent extraction and chromatographic purification, and assessed in bioassays using Raphanus sativus and Triticum aestivum as bioindicator plants. A concentration of 23 mg mL⁻¹ completely inhibited seed germination in 96-well plate assays, while the crude extract (EXT) and cell-free broth (CFB) allowed radicle protrusion but resulted in abnormal seedlings with chlorosis and reduced growth. Mathematical models estimated that concentrations of 16.0 mg mL⁻¹ for radish and 0.9 mg mL⁻¹ for wheat were sufficient to suppress germination with the PC. In substrate experiments, the PC at 6.4 and 64.0 mg mL⁻¹ did not inhibit germination but caused anomalies in radish and significantly reduced wheat seedling growth. In naturally infested soil, the PC maintained phytotoxicity symptoms for 21 days, and after 28 days, a concentration of 64.0 mg mL⁻¹ significantly reduced radish seedling growth. The results highlight the potential of the compound as a bioherbicide.

1. Introduction

Agriculture is critical for providing food and raw materials to the growing global population. Sustainable crop production requires protection against weeds, pests, and diseases. Biotic and abiotic factors can negatively impact crop development, compromising food security [1]. The main causes of reduced productivity are weeds, which grow in commercial cultivation fields, and compete for nutrients, water, sunlight, and space [2]. To illustrate the global impact of weeds, losses in India and the United States are estimated at US$11 billion and US$17.2 billion annually, respectively, mainly affecting economically significant crops such as soybean, maize, and beans [3,4,5,6].

Herbicides account for approximately 60% of all pesticides used globally and are the cornerstone of weed management in large-scale agricultural systems [7]. Despite their success in controlling weeds, there is growing concern over the environmental impacts of these chemical compounds [8]. Additionally, the number of documented cases of herbicide-resistant weeds increases annually, alarming policymakers, environmentalists, and farmers. To date, more than 500 distinct cases of herbicide resistance in weeds (considering specific species and mechanisms of action) have been reported worldwide. These cases involve resistance to 21 of the 31 known herbicide mechanisms of action and 168 distinct active ingredients [9]. Given the externalities associated with synthetic herbicides, it is essential to identify and test new natural compounds with the potential for controlling high-impact agricultural weeds.

Biomolecules produced by microorganisms, such as phytotoxins, have demonstrated significant herbicidal activity, positioning themselves as promising alternatives to traditional synthetic herbicides [10]. These microbial-derived molecules offer advantages such as excellent selectivity and lower environmental impact, contributing to more sustainable agricultural practices [11]. Moreover, the diversity of secondary metabolites produced by microorganisms broadens the spectrum of action against different weed species, reducing reliance on synthetic chemical compounds [12]. Developing bioherbicides based on these biomolecules can mitigate weed resistance and environmental contamination issues, promoting more ecological and efficient agriculture. Compared to chemical herbicides, phytotoxins exhibit unique mechanisms of action, making them highly attractive for discovering new bioherbicides [10,13,14]. Moreover, exploring microbial metabolites facilitates the identification of new biochemical pathways and molecular targets, driving innovation in sustainable crop management as Schmaltz et al. [15] emphasized.

Purifying biomolecules with herbicidal activity produced by microorganisms is a crucial step for characterizing the active compound and evaluating its biological activity. Previous studies conducted by the research group demonstrated the herbicidal activity of the fungus Fusarium fujikuroi [16,17,18] using concentrated cell-free culture broth and surfactant-containing formulations for the control of relevant weed species (Conyza sp.) and bioindicator plants such as Cucumis sativus and Sorghum bicolor. However, the need to isolate and identify the bioactive molecule responsible for the herbicidal activity of the culture broth motivated the development of this study. This work presents the production and purification process of a biomolecule synthesized by the phytopathogenic fungus F. fujikuroi in submerged cultivation, along with evaluation of its herbicidal activity using the bioindicator plants Raphanus sativus (radish) and Triticum aestivum (wheat). Furthermore, the study investigates the residual effect of the biomolecule in soil and its influence on the germination and development of R. sativus.

2. Materials and Methods

2.1. Storage and Maintenance of the Strain

The fungus Fusarium fujikuroi (MG 189928; Code: SO210) was isolated from plants exhibiting disease symptoms collected in areas of the Pampa biome (Santa Maria, RS, Brazil). The microorganism was isolated and stored in the Bioprocess Laboratory collection at the Chemical Engineering Department of UFSM [16]. The isolate was maintained at 4 °C in tubes containing colonized rice grains to preserve its viability. For each experiment, the microorganism was reactivated by transferring colonized rice grains to Petri dishes containing potato dextrose agar (PDA) medium. The inoculum for cultivation consisted of two fungal mycelium disks (7 mm in diameter) grown on these plates for 14 days in a BOD–type incubator at 25 °C with a 12-h photoperiod.

2.2. Submerged Cultivation

The fungal broth was produced via submerged cultivation. The cultivation medium composition (g L⁻¹) was protein hydrolysate (Transfertech, Erechim, Brazil) (15), sucrose (15), glucose (15), monobasic potassium phosphate (4), anhydrous calcium chloride (0.8), magnesium sulfate heptahydrate (0.6), ferrous sulfate (0.1), manganese sulfate monohydrate (0.016), zinc sulfate heptahydrate (0.014), and the antibiotic chloramphenicol (0.25). Nutrients were weighed on an analytical balance and dissolved in distilled water. The pH of the medium was adjusted to 5.0 using 2 N hydrochloric acid. Each 250 mL Erlenmeyer flask was filled with 100 mL of medium and sterilized in a vertical autoclave (Prismatec, Model CS 150, Itu, Brazil) for 20 min at 120 °C and 1 atm. After sterilization and cooling, each flask was inoculated with two fully developed fungal mycelium disks to initiate cultivation. Microorganism handling was performed in a Biological Safety Cabinet (Filterflux, Model SBIIA1-1266/4, Piracicaba, Brazil). Submerged cultivation was conducted in an orbital shaker (New Brunswick, Model Innova 44, Edison, CA, USA) at 28 °C, 150 rpm, and a 12-h photoperiod for 7 days. At the end of the cultivation period, the fungal broth was separated from the fungal biomass by centrifugation at 4000 rpm for 10 min (Eppendorf, Model 5430R, Hamburg, Germany). The supernatant was initially vacuum-filtered through qualitative filter paper, followed by filtration using nylon membranes (47 mm diameter) with 0.45 µm and 0.22 µm pore sizes (Merck Millipore, Massachusetts, MA, USA), obtaining a cell-free fermented broth (CFB). Part of the filtered broth was refrigerated for bioassays, while the remaining portion was used for organic extract production.

2.3. Extraction and Purification

The cell-free fermented broth (CFB) was extracted using ethyl acetate as the organic solvent at a 1:1 (v/v) ratio in two successive extractions, employing a separation funnel. The organic phase obtained from the extractions was dried using anhydrous magnesium sulfate, evaporated under low pressure and a temperature of 45 °C in a rotary evaporator (Solab, Model SL-126). The obtained extract (EXT) was quantified and divided into two portions: one for bioassays and another for compound separation and purification procedures.

The compounds were fractionated using column chromatography (CC) with a glass column (internal diameter 35 mm). Silica gel 60 Å (230–400 mesh, height = 14 cm) (Sigma Aldrich, São Paulo, Brazil) was used as the stationary phase. The elution was performed using a hexane (Hex) and isopropanol (iPrOH) solvent system. After packing the silica in the column, the extract, pre-adsorbed onto a portion of silica, was added to the top of the column. Initially, 30 mL of hexane was passed through, followed by 200 mL of the mobile phase composed of an 80:20 (v/v) Hex/iPrOH mixture. A total of 210 fractions (0.5 to 0.7 mL each) were collected and analyzed by thin-layer chromatography (TLC) using aluminum chromatoplates coated with silica gel 60 Å (F254, 0.5 mm) (Sigma Aldrich, São Paulo) and a mobile phase of Hex/iPrOH (80:20, v/v). Fractions 17 to 101 were pooled and evaporated, resulting in 0.186 g of the purified compound (PC).

2.4. Bioassays

2.4.1. Seed Preparation

Seeds of radish sparker ISLA^® (Raphanus sativus L.), free of pesticides and obtained from local commerce in Santa Maria-RS, and wheat seed (Triticum aestivum L.) variety BRS Belajoia, cultivated in a rural property in Santo Antônio das Missões-RS, were used for the bioassays. The seeds were stored at 4 °C to maintain viability. Before bioassays, seeds underwent aseptic treatment in the following order: immersion for 1 min in 70% ethanol, and then 1 min in 1% sodium hypochlorite solution, followed by two washes in sterile distilled water (1 min each).

2.4.2. Phytotoxic Potential of Crude Extract (EXT) and Purified Compound (PC) in 96-Well Microplates

This method was used due to the low volume of purified compound obtained, which limits the choices of other methods. For this assay, the EXT and PC, obtained from 2 L of fermented broth, were dissolved in 1% DMSO to achieve a concentration of 23.3 mg mL⁻¹. The cell-free filtered broth (CFB) was used directly without further dilution. DMSO (1%) and sterile distilled water were used as controls. The dose–response curve for the EXT and PC included concentrations of (mg mL⁻¹) 0.000233, 0.00233, 0.0233, 0.233, 2.33, and 23.30.

Experiments were conducted using 96-well microplates, each containing a single seed immersed in 100 µL of the respective sample. Twelve seeds were tested per treatment, with treatments performed in triplicate. The plates were sealed with microporous tape and incubated in a growth chamber (POL-EKO-APARATURA SP.J., Wodzisław, Poland) at 20 °C with a 12-h photoperiod for 7 days.

After incubation, germination percentages and normal seedling rates were evaluated. Seeds were considered germinated when a radicle longer than 1 mm was observed. Seedlings were classified as normal when all essential structures were fully developed, complete, proportional, and healthy as per RAS standards [19]. Data were analyzed for normality and homogeneity of variances, transformed when necessary, and subjected to ANOVA. Means were separated using Tukey’s test at a 5% significance level or fitted using regression analysis where applicable.

2.4.3. Phytotoxic Potential of the Purified Compound (PC) in the Pre-Emergence of R. sativus and T. aestivum

For this assay, the PC was obtained from 3 L of fermented broth (as described in Section 2.2). The sample was dissolved in 1% DMSO with six drops of Tween 80 to reach a concentration of 64.0 mg mL⁻¹ of PC. Controls included 1% DMSO, sterile distilled water, sterile distilled water with Tween 80, and a mixture of hexane and isopropanol. Concentrations of 6.4 and 64.0 mg mL⁻¹ were used for the PC in this experiment.

Transparent acrylic boxes (5 × 5 cm) were used for the assay. Each box contained 7 g of commercial peat-based substrate (Carolina Soil^®—Garden, Santa Cruz do Sul, Brazil), moistened with 7 mL of sterile distilled water. For each treatment, four boxes were used, each with four seeds. Radish and wheat were tested separately. For each treatment, 130 µL of the sample was applied over each seed, followed by substrate coverage. The boxes were sealed and incubated in a growth chamber (POL-EKO-APARATURA SP.J., Wodzisław, Poland) at 20 °C with a 12-h photoperiod for 6 days.

After incubation, normal emerged seedlings (%) and total length (root + shoot, in cm) were evaluated. Normal seedlings were classified based on the criteria mentioned in Section 2.4.2. Data were analyzed for normality and variance homogeneity and subjected to ANOVA. Means were separated using Tukey’s test at a 5% significance level.

2.4.4. Effect of the Purified Compound (PC) on Soil Infested with Weed Seeds

The soil used in this assay was collected from the surface layer (up to 10 cm depth) of an agricultural area in São Sepé-RS, historically infested with weed species such as barnyard grass (Echinochloa sp.), sedges (Cyperus sp.), and red rice (Oryza sativa), among others. After collection, the soil was manually sieved to remove plant debris. Seeds from the soil’s natural seed bank were used as bioindicators.

For this assay, the crude extract (EXT) and PC (obtained from 3 L of fermented broth) were dissolved in 1% DMSO to reach a 64.0 mg mL⁻¹ concentration. The cell-free broth (CFB) was used as obtained. Controls included 1% DMSO and sterile distilled water. Final concentrations of 6.4 mg mL⁻¹ and 64.0 mg mL⁻¹ were used for the EXT and PC.

Transparent acrylic boxes (5 × 5 cm) containing 25 g of soil and 5 mL of sterile distilled water were prepared. Each treatment was performed in quadruplicate (four boxes). A total of 520 µL of the sample was applied over the soil surface and distributed evenly in each corner of the box (130 µL per corner). The boxes were sealed and transferred to a growth chamber (POL-EKO-APARATURA SP.J., Wodzisław, Poland) maintained at 25 °C with a 12-h photoperiod for 21 days.

After 21 days, the total number of seedlings emerged per treatment was recorded. Data were analyzed for normality and variance homogeneity, followed by ANOVA, with means compared using Tukey’s test at a 5% significance level.

2.4.5. Residual Effect of the Purified Compound (PC) on Soil and Its Influence on the Emergence and Development of R. sativus Seedlings

This assay aimed to evaluate the residual effect of the PC 28 days after application (DAA). After removing seedlings from the previous experiment (Section 2.4.3), the soil in each acrylic box was homogenized. Seeds of radish, prepared as described in Section 2.4.1, were used.

The assays were conducted in transparent acrylic boxes (5 × 5 cm) containing 25 g of soil. To adjust moisture levels, 2 mL of sterile distilled water was added to each box. Four radish seeds were sown per box, 21 DAA, with each treatment replicated four times. Boxes were sealed and incubated for seven days in a growth chamber (POL-EKO-APARATURA SP.J., Wodzisław, Poland) maintained at 25 °C with a 12-h photoperiod.

The germination percentage and total seedling length were evaluated seven days after radish sowing (28 DAA). Data were analyzed for normality and variance homogeneity and subjected to ANOVA. Means were compared using Tukey’s test at a 5% significance level.

3. Results

3.1. Phytotoxic Potential of the Crude Extract (EXT) and Purified Compound (PC) in 96-Well Microplates

The reduced final volume and low concentration of the samples produced required miniaturized tests to be carried out in the initial screening. The treatment significantly affected seed germination and the production of normal seedlings in radish and wheat (Figure 1). Compared to the cell-free broth (CFB), extract (EXT), water, and DMSO, the purified compound (PC) at 23 mg mL⁻¹ caused complete inhibition of germination in both species. Wheat seeds exposed to CFB and EXT exhibited germination percentages similar to those of the controls, with seed coat rupture and radicle emergence. However, regarding the rate of normal seedlings, these treatments resulted in chlorotic seedlings, delayed foliage development, and inhibited growth. Consequently, the mean values for CFB, EXT, and PC were statistically similar for this variable.

Seed germination percentages are widely used as indicators of bioavailability and acute toxicity of various compounds [20]. Germination involves biochemical, physiological, and morphological transformations [21], and is divided into three phases. Phase I involves rapid water absorption and tissue expansion; Phase II corresponds to a hydration plateau characterized by increased cellular and metabolic activity culminating in radicle protrusion, the first visible sign of germination. Phase III marks seedling development. The data suggest that for both radish and wheat, the PC at 23 mg mL⁻¹ disrupted processes associated with Phase II. In contrast, the CFB and EXT affected Phase III as illustrated in Figure 2.

This behavior likely reflects the concentration of phytotoxic compounds, which tends to increase during purification, making the CFB the most diluted sample. The relationship between concentration, germination percentage, and seedling normality for radish is shown in Figure 3a–c and for wheat in Figure 4a,b. The results indicate that the highest EXT concentration of 23.0 mg mL⁻¹ was insufficient to inhibit seed germination in both species. Moreover, no biologically meaningful mathematical model could be adjusted to describe the behavior of wheat seeds exposed to the EXT.

In contrast, the adjusted models revealed that approximately 16.0 and 0.9 mg mL⁻¹ of the PC were sufficient to suppress seed germination in radish and wheat, respectively. Concentration effects were also evident in normal seedling establishment. The importance of using monocotyledonous and dicotyledonous bioindicator plants as models for assessing herbicidal activity is well-documented in early screenings [22] as sensitivity differences exist.

Additionally, the presence of multiple compounds in the EXT obtained via liquid–liquid extraction, as evidenced by preliminary thin-layer chromatography (TLC) analyses, leads to a low concentration of the herbicidal active ingredient in the sample. This explains the need for higher EXT concentrations to achieve the same control potential observed with the PC. This is even more pronounced for the CFB sample where the number of compounds is more significant, resulting in extremely low bioactive concentrations compared to purified samples [11].

Chromatographic techniques for purifying microbial metabolites are widely used and well-documented in the scientific literature [23,24,25,26,27]. Purification is essential not only for mode-of-action elucidation but also for detailed characterization of bioactive metabolites, enabling the development of optimized process engineering strategies to enhance the productivity and yield of target compounds.

3.2. Phytotoxic Potential of the Purified Compound (PC) on the Pre-Emergence of R. sativus and T. aestivum

It is common for compounds to exhibit herbicidal activity in plate-based screenings but fail to reproduce the same behavior in soil [28]. This discrepancy can partly be attributed to the physicochemical interactions between the molecules and the soil matrix, which may reduce their bioavailability. Consequently, there is a need to evaluate higher concentrations of compounds in soil-based assays compared to plate-based experiments where water serves as the sole delivery vehicle for bioherbicide.

When cultivated in acrylic boxes containing substrate, the percentage of normal seedlings and total seedling length were significantly influenced by the pre-emergence application of the PC samples at 6.4 and 64.0 mg mL⁻¹ (Figure 5). Although seed germination was not inhibited, both concentrations induced abnormalities in radish seedlings, such as chlorosis, cotyledon retention, and a drastic reduction in growth. While the PC application at 6.4 mg mL⁻¹ did not affect the normality of wheat seedlings compared to the controls, both concentrations resulted in a significant reduction in growth, with 64.0 mg mL⁻¹ exhibiting higher phytotoxicity.

Germination inhibition and changes in growth patterns are common outcomes in studies involving compounds with bioherbicidal potential. According to Hasan et al. [29], the phytotoxic effects on weed growth result in low levels of root cell division, nutrient uptake, and synthesis of growth hormones and pigments, as well as the development of reactive oxygen species (ROS) and other stress-related compounds, in addition to abnormal antioxidant activity. These symptoms are illustrated in Figure 6.

3.3. Effect of the Purified Compound (PC) on Soil Infested with Weed Seeds

The inconsistent efficacy of bioherbicides has posed challenges to their widespread application as it is influenced by factors such as bioactive compound content, the spectrum of weed control, formulation, and application method [29]. For this reason, it is essential to evaluate these compounds under conditions that resemble those encountered in crop fields. To this end, the samples were applied to soil collected from a soybean cultivation area and maintained in a growth chamber for 21 days.

At the end of this period, the number of emerged weed seedlings was counted, with a predominance of barnyard grass (Echinochloa spp.). However, no significant differences were observed between treatments (data not presented). Based on the symptoms observed in the seedlings (Figure 7), it can be inferred that the obtained results may be attributed to the origin of the seeds used in the assay. The soil seed bank serves as the primary source of persistent weed infestations, replenished in each cycle with new propagules, resulting in seeds with varying dormancy levels and germination fluxes [30].

3.4. Residual Effect of the Purified Compound (PC) in Soil and Its Influence on the Emergence and Development of R. sativus Seedlings

The persistence of phytotoxic symptoms, such as chlorosis and reduced weed growth, observed in the previous experiment, even 21 days after treatment application, indicated the potential persistence of the compounds in the soil. Consequently, the soil from each experimental unit was homogenized, and four radish seeds were sown as bioindicators.

Seven days after sowing (28 days after treatment application to the soil), the 64.0 mg mL⁻¹ concentration of the PC significantly reduced the length of R. sativus seedlings. The means for other treatments were similar to the controls (Figure 8). Figure 9 shows symptoms associated with exposure to a higher concentration of the PC.

Natural herbicides are often considered to have advantages due to their short half-life and low environmental persistence [31]. However, residual activity within the growing season is desirable as it extends the control period by inhibiting new emergence fluxes, reduces competition for essential growth resources, and minimizes the selection pressure for herbicide-resistant weeds [32].

Thus, the PC produced from the submerged cultivation of F. fujikuroi could be a bioherbicide. However, further studies are needed to identify and characterize its weed control spectrum and selectivity for commercially important crops.

4. Conclusions

The compounds produced via submerged fermentation of F. fujikuroi inhibit germination and affect the production of normal seedlings in the bioindicator species radish (R. sativus) and wheat (T. aestivum), with the observed effects being concentration-dependent for both the extract (EXT) and the purified compound (PC).

At 64 mg mL⁻¹ concentration, the PC persisted in the soil for 28 days post-application, resulting in phytotoxic symptoms in radish seedlings. This study demonstrated that the PC derived from submerged fermentation of F. fujikuroi has the potential for application as a bioherbicide applied in the pre-emergence of weeds. However, further studies are essential to validate its potential, including identifying and characterizing the compound, weed control spectrum, and selectivity for commercially relevant crops.