Herbicidal Activity of Baccharis trimera Extract on Oryza sativa L. and Cyperus ferax

Abstract

This study evaluated the application of the aqueous extract from Baccharis trimera (Less) DC. in the control of weed species Oryza sativa L. and Cyperus ferax Rich. during the germination and early development stages. Extracts were obtained through pressurized liquid extraction using kinetic assays. Shorter extraction times (1 to 10 min) showed extracts with higher inhibitory effects on seed germination, shoot and root lengths, and fresh and dry mass of the plants. The survival of treated plants was also affected, especially during the early stages of development, reaching up to 40% of mortality. HPLC analysis identified phenolic compounds such as ferulic acid, rutin, quercitrin, and quercetin, with higher concentrations found in the extracts obtained at shorter times of extraction. The reduction in these compounds over longer extraction times was correlated with decreased inhibitory activity. The results indicate that the aqueous extract of B. trimera (Less) DC. holds potential for ecological weed management, standing out as a viable alternative to reduce weed resistance to synthetic herbicides.

1. Introduction

Weeds are any vegetation that grows in undesirable places. With the growth of agriculture, weeds have emerged as significant competitors, evolving in a persistent ecological struggle for space, light, water, and nutrients [1]. In the cultivation of rice, species such as Oryza sativa L. (weed biotypes) and Cyperus ferax Rich. represent a major challenge to crop productivity. These weed species are highly competitive due to their rapid growth potential and lifestyle similar to that of the agricultural species [2]. Weeds are a significant source of biological risks that impair both rice yield and quality [3,4,5]. Chemical weed control is often more efficient and less labor-intensive compared to mechanical or cultural methods, making it a preferred choice in modern agriculture [6].

However, the excessive use of herbicides has led to serious environmental impacts and human health risks, in addition to contributing to the development of resistance in weed species [7]. Sustainable alternatives to weed control are essential to reduce reliance on chemicals [8]. In this context, the application of inhibitory compounds emerges as a promising alternative because plants release chemical substances that interfere with the growth and development of other species, potentially affecting germination, root and seedling growth, cell division, protein synthesis, and respiration [9].

Baccharis trimera (Less) DC., a plant native to South America and belonging to the Asteraceae family, is known for its secondary metabolites with potential inhibitory effects. Studies indicate that compounds found in B. trimera (Less) DC. can inhibit the growth of various plants, including both cultivated and invasive species [10]. There was a negative inhibitory effect of the crude aqueous extract of B. trimera (Less) DC. plants on the germination, vigor, and growth of vegetable loofah seedlings [11].

Research indicates that phenolic acids can suppress the initial growth of invasive species such as Ambrosia artemisiifolia [12]. Phenolic compounds are secondary metabolites widely found in plants. They have antioxidant properties and act in defense against pathogens and environmental stresses. Chemical compounds can affect seed germination, growth, and development of neighboring plants by interrupting several physiological mechanisms, such as photosynthesis, respiration, and water and hormonal balance [13].

Studies involving the inhibition of weeds are of great interest to agriculture because many of the chemical substances have potential for use in controlling crop weeds and can inhibit germination and plant growth [14]. Currently, natural products of plant origin with inhibitory activity have been studied as alternatives to synthetic herbicides in weed management [15].

The bioactivity of natural plant products may be related to the extraction method used since this influences the composition, availability, and purity of the compounds, as well as the efficacy of the material obtained [16]. The extraction process releases the components of the plant matrix structure to obtain extracts with a high concentration of compounds, present in small quantities in the natural solid matrix of plants [17]. The adoption of extraction methods using green technologies has been expanding, considering the benefits in economic, environmental, and yield aspects [18]. The pressurized liquid extraction (PLE) method is a green technology because it can use GRAS (Generally Recognized As Safe) solvents, such as water, in addition to being an alternative for the extraction of natural products [19]. The main advantage of PLE over conventional extraction methods is that pressurized solvents remain in the liquid phase when taken to temperatures above their boiling points under atmospheric pressure conditions [20].

Thus, this study aimed to evaluate the inhibitory effect of the aqueous extract of B. trimera (Less) DC. obtained by PLE on the germination and early development of Oryza sativa L. and Cyperus ferax Rich., contributing to expanding knowledge on the potential use of B. trimera (Less) DC. extract in the ecological control of weeds.

2. Materials and Methods

2.1. Collection and Processing of Plant Material

The branches of B. trimera (Less) DC. were collected in October 2024, in the morning, from a rural area in the municipality of Manoel Viana, state of Rio Grande do Sul, Brazil, located at the geographical coordinates −29.570922° latitude and −55.493954° longitude. After collection, the plant material was transported to the Phytotechnics Laboratory of the Instituto Federal Farroupilha (IFFar—Brazil). A 2.5% sodium hypochlorite solution was used to clean and sanitize at a ratio of 10 mL per liter of water, and the branches were submerged for 10 min. The material was dried in an oven at 45 °C for 72 h. After complete drying, the branches were ground in an industrial blender, resulting in smaller-sized particles, and stored under refrigeration until the preparation of extracts.

2.2. Extraction by PLE

Aqueous extract was obtained by PLE using an apparatus available at the Biomass and Biofuel Laboratory (L2B) of the Federal University of Santa Maria (UFSM), Brazil, following the procedure reported by Confortin [16]. For each extraction assay in a useful vessel volume of 81 mL, 10 g of the previously processed plant material of B. trimera (Less) DC. was used with water as the solvent. The operational conditions were set: water flow rate of 10 mL/min, temperature of 60 °C, and pressure of 10 MPa.

The extracts were collected at regular intervals: from the first to the tenth minute, samples were collected every minute; from the tenth minute onward, collections were made every 5 min (15, 20, 25, and 30 min), totaling 30 min of extraction. All experimental assays (for each collection time) were conducted in triplicate, maintaining constant the temperature, pressure, and flow rate throughout the process. After preparation, the aqueous extracts were stored in amber bottles, labeled with the corresponding collection time, stored at 4 °C, and applied to the weeds within 72 h. Although no chemical degradation analyses were performed during storage of extracts, they were stored under refrigeration and in the absence of light, aimed at preserving the bioactivity of the extract.

2.3. Application of Extracts in the Pre- and Post-Germination of Weeds

For evaluating the effects during the pre-germination phase, seeds of O. sativa L. and C. ferax Rich. were used, following the procedures established by the Seed Analysis Rules (RASs) [21]. The assays were conducted in Gerbox-type boxes. The bottom was filled with two sheets of Germitest paper, previously moistened with the corresponding plant extract for each extraction time. The applied volume was equivalent to 2.5 times the weight of the Germitest paper. For each treatment, 25 seeds (O. sativa L. or C. ferax Rich.) were placed into each box, with three replications per treatment. Distilled water was used as the negative control. The boxes were placed in a B.O.D. chamber with a constant temperature of 25 °C and a 12 h photoperiod for 21 days. Germination counts were carried out on the seventh, fourteenth, and twenty-first days after setting up the tests. The Germination Speed Index (GSI) was calculated through Equation (1).

GSI = ∑ (n_i/t_i)

(1)

where “n_i” is the number of seeds that germinated at time ‘i’, and “t_i” is the time after installation of the test. In the post-germination phase, applications of the aqueous extract of B. trimera (Less.) DC. occurred at 15 and 30 days after sowing (DAS). According to the BBCH (Biologische Bundesanstalt, Bundessortenamt und Chemische Industrie) scale, 15 and 30 DAS represent stages 12–13 (two to three fully expanded leaves) and 15–21 (five to six leaves, beginning of tillering), respectively. The objective was to evaluate the effect of the extract on the development of species at different growth stages. For each treatment, 15 plants were placed in plastic trays filled with pine bark substrate, MecPlant brand, manufactured by MecPrec (Telêmaco Borba, Paraná, Brazil). All trays were identified with the respective treatments. The tests were conducted in triplicate for both species and growth stages.

The extracts were applied via foliar spraying using a volume of 2.5 mL per tray (equivalent to 100 L/ha) with the help of a manual sprayer. The volume was calculated based on the equivalence of terrestrial application per hectare, adjusted proportionally to the area of the tray. After application, the trays were kept in a greenhouse for 21 days (Figure 1). At the end of this period, the plants were collected, and the length of the shoot and root parts was evaluated using a ruler. Fresh and dry weights of the plants were also determined on an analytical balance (model AS5500C, Marte Scales and Precision Equipment Ltda., São Paulo, Brazil). For the fresh weight, the material was weighed immediately after collection. Then, it was placed in brown paper envelopes and taken to an oven, where it remained for 72 h at 65 °C to determine the dry weight. Plant mortality was quantified by direct counting of live individuals in each experimental unit, performed 21 days after application of the aqueous extract of B. trimera (Less) DC. Each unit started with 15 sown plants. The survival percentage was calculated as the ratio between the number of live plants after the experimental period and the initial number of plants, multiplied by 100, according to Equation (2).

Live plants (%) = (Number of live plants/15) × 100

(2)

2.4. Chromatographic Analysis

The chromatographic analysis of the aqueous extract of B. trimera (Less) DC. was performed using High-Performance Liquid Chromatography (HPLC) to identify the chemical compounds. For this evaluation, three distinct extraction times (1, 15, and 30 min) were selected to observe variations in the chemical profile throughout the extraction process.

The analyses were carried out using a Prominence LC-20AT HPLC system (Shimadzu, Kyoto, Japan), equipped with a UV-VIS SPD-20A detector and a CTO-20A column oven. The system was operated with LabSolutions software, version 5.86. Compound separation was performed using a C-18 column (250 mm × 4.6 mm; 5 µm particle diameter), maintained at a constant temperature of 40 °C. The mobile phase consisted of two solvents: water acidified with 0.1% phosphoric acid (Solvent A) and methanol (Solvent B). Detection of analytes was carried out at wavelengths of 280 nm and 360 nm. This method was adapted based on the work of Zadra [22].

2.5. Statistical Analysis

The experiment was conducted in a completely randomized design, with a three-factor factorial arrangement. Statistical analysis of the data was performed using Sisvar software, version 5.8. The results were subjected to analysis of variance (ANOVA), and treatment means were compared using the Scott–Knott test at a 5% significance level (p-value ≤ 0.05).

3. Results

3.1. Germination

The inhibitory effects of the aqueous extract of B. trimera (Less) DC. on the seed germination of O. sativa L. and C. ferax Rich. are presented in

Table 1. Germination of O. sativa L. and C. ferax Rich. seeds after application of B. trimera (Less) DC. extract.

Extraction Time (min)	Germination of O. sativa L. (%)	GSI (O. sativa L.)	Germination of C. ferax Rich. (%)	GSI (C. ferax Rich.)
1	24.67 ± 1.88 a	1.20 a	19.33 ± 1.70 a	1.00 a
2	24.33 ± 0.94 a	1.33 a	14.66 ± 1.89 a	0.86 a
3	21.33 ± 1.24 a	1.14 a	17.67 ± 1.89 a	0.94 a
4	26.34 ± 0.94 a	1.42 a	20.66 ± 3.30 a	1.02 a
5	27.00 ± 2.82 a	1.36 a	19.00 ± 1.41 a	1.03 a
6	23.00 ± 1.41 a	1.34 a	19.00 ± 2.83 a	1.02 a
7	26.33 ± 2.49 a	1.36 a	21.34 ± 3.09 a	1.04 a
8	27.00 ± 1.41 a	1.51 a	20.67 ± 0.47 a	1.17 a
9	27.00 ± 2.16 a	1.39 a	23.00 ± 1.41 b	1.25 a
10	27.67 ± 3.39 a	1.47 a	24.00 ± 2.94 b	1.30 a
15	35.33 ± 4.64 b	1.87 b	30.00 ± 4.55 c	1.54 a
20	38.33 ± 7.31 b	1.97 b	34.66 ± 4.99 d	1.80 b
25	40.00 ± 5.65 b	2.09 b	39.00 ± 4.55 d	2.06 b
30	41.66 ± 6.18 b	2.19 b	42.33 ± 4.11 d	2.19 b
Control	58.67 ± 0.02 c	3.15 c	57.00 ± 0.17 e	3.30 c
Coefficient of variation (%)	13.84	26.15	13.67	28.47

Different letters within the same column indicate a significant difference at a 95% confidence level; GSI: Germination Speed Index.

. It was observed that all treatments with the plant extract inhibited the germination of both species when compared to the control (distilled water), highlighting the inhibitory potential of the extract during the early developmental stages of these plants.

For the germination of O. sativa L. seeds, the most pronounced inhibitory effects were observed after applying extracts obtained between 1 and 10 min of extraction. The application of extracts collected at 1–10 min resulted in the lowest germination percentage. Extracts obtained between 15 and 30 min showed effects on germination rates ranging from 35.33% to 41.66%, still significantly lower than the control, which had a germination rate of 58.67%.

In the case of C. ferax Rich. seeds, the aqueous extract of B. trimera (Less) DC. also demonstrated an inhibitory effect. The lowest germination rates (between 14.67% and 24.00%) were recorded for samples of weeds after applying extracts collected between 1 and 10 min. For samples applied with aqueous extracts obtained in extraction times between 15 and 30 min, germination ranged from 30.00% to 42.33%, remaining lower than the control, which showed a germination rate of 57.00%.

For O. sativa L., the GSI values remained below 1.5 at extraction times between 1 and 10 min, with a gradual increase after 15 min, reaching 2.19 at 30 min. However, all treatments presented lower GSI than the control (3.15), evidencing the inhibitory action of the extract, regardless of the extraction time. A similar behavior was observed for C. ferax Rich., whose GSI values ranged from 0.86 to 1.30 at the initial times, and increased with longer extraction times, reaching 2.19 at 30 min, also below the control value (3.30).

Overall, extracts obtained during the initial extraction times (1 to 10 min) caused the lowest germination percentages for both weed species evaluated. Although extracts from later extraction times presented higher germination rates than the early ones, most of them remained below the control, reinforcing the inhibitory potential of the aqueous extract of B. trimera (Less) DC.

3.2. Shoot Length

Figure 2 presents the results of the effects of the aqueous extract on the shoot length of O. sativa L. and C. ferax Rich. plants, evaluated at 15 and 30 DAS. It can be observed that all extraction times of the aqueous extract of B. trimera (Less) DC. resulted in reduced shoot length in O. sativa L. plants compared to the control. Extracts obtained between 1 and 10 min resulted in shoot lengths smaller than 15 cm, with no statistical differences among them. However, these treatments differed significantly from those obtained between 15 and 30 min, which were statistically different from the control.

For plants evaluated at 30 DAS, treatments with aqueous extracts resulted in shorter shoot lengths than the control. Statistically significant differences were observed among the treatments, with extracts obtained after 15 min showing mean values closer to the control, although still significantly lower. In both evaluated stages (15 and 30 DAS), O. sativa L. plants showed a clear statistical separation between early (1 to 10 min) and late (15 to 30 min) extraction times.

For C. ferax Rich. plants at 15 DAS, the application of extracts obtained up to 15 min caused lower shoot length averages in weeds compared to the control. For extraction times of 20, 25, and 30 min, the means did not differ statistically from the control, indicating a reduction in the inhibitory effect during these periods. For the 30-day-old plants, all treatments resulted in shorter shoot lengths than the control, with statistically significant differences among treatments. Extraction times of 8, 10, 20, 25, and 30 min showed no statistical differences among themselves, according to the Scott–Knott test at a 5% significance level.

3.3. Root Length

Figure 3 represents the average root length of O. sativa L. at 15 and 30 DAS, indicating that all extract treatments resulted in shorter root lengths compared to the control. The lowest averages of lengths were observed in samples applied with extracts obtained between 1 and 10 min, highlighting a more pronounced inhibitory efficacy of compounds obtained in the initial extraction periods. The data confirm statistically significant differences between all treatments and the control, for both 15- and 30-day-old plants. For C. ferax Rich. plants at 15 DAS, most treatments significantly reduced root length compared to the control, except for the extract obtained at 30 min, which did not show a statistical difference. The shortest root lengths were recorded for the application of extracts obtained at 1, 2, and 3 min, with values below 1 cm, indicating a strong inhibitory effect of these early-stage extracts.

In the case of 30-day-old C. ferax Rich. plants (Figure 3), all treatments differed statistically from the control, reinforcing the phytotoxic potential of B. trimera (Less) DC. aqueous extract, even at more advanced stages of development. Samples applied with extracts obtained at 10, 20, 25, and 30 min showed no significant differences, suggesting a potential plateau in inhibitory activity during the later extraction stages. However, extracts obtained between 1 and 4 min maintained the lowest average effects on root lengths, suggesting that the highest concentration and activity of inhibitory compounds are found in the earliest minutes of the extraction process.

3.4. Fresh and Dry Biomass

The average fresh biomass of O. sativa L. and C. ferax Rich. plants, measured at 15 and 30 DAS, is shown in Figure 4. For 15-day-old O. sativa L. plants, all treatments with the aqueous extract of B. trimera (Less) DC. resulted in lower fresh biomass averages than the control, with variations according to extraction time. Weeds applied with extracts obtained between 1 and 10 min presented statistically significant mass reductions, while those from 15 to 30 min did not differ significantly from the control. In 30-day-old plants (Figure 4), only treatments at 1, 2, 3, 4, and 6 min showed statistically significant differences from the control, with the lowest mean value recorded at the 1 min treatment, indicating stronger phytotoxic action of the extract at the early stages of extraction.

For C. ferax Rich. plants at 15 DAS, treatments with extracts obtained from 1 to 15 min were statistically different than the control, especially for the 1 to 8 min extracts, which produced the lowest fresh biomass averages. In contrast, extracts obtained from 20 min onward did not differ from the control. In 30-day-old plants (Figure 4), all extract treatments were statistically different from the control, with the lowest averages recorded at 1 and 2 min.

Regarding dry biomass (Figure 5), data from 15-day-old O. sativa L. plants show that treatments with extracts obtained at 1, 2, 3, 5, 6, 8, 9, and 10 min significantly reduced dry biomass compared to the control. The remaining extraction times (4, 7, 15, 20, 25, and 30 min) did not cause statistical differences.

For 30-day-old O. sativa L. plants, extracts obtained at 1, 2, 3, 4, 6, and 8 min caused significant reductions in dry biomass, while the others were not statistically different from the control. For C. ferax Rich. plants at both 15 and 30 DAS, all treatments presented lower dry biomass averages than the control, with the lowest values observed for extracts obtained between 1 and 10 min. These results reinforce the inhibitory potential of B. trimera (Less) DC. aqueous extract, especially at the initial extraction times, effectively reducing biomass accumulation in the evaluated weed species.

3.5. Live Plants

The percentage of live plants of O. sativa L. and C. ferax Rich. at 15 and 30 DAS is shown in Figure 6.

For O. sativa L. plants at 15 days after germination, the lowest survival rates were observed in treatments with extracts obtained at 2 and 9 min of extraction. In 30-day-old plants, the inhibitory effect was less pronounced, with the 2 min extract resulting in the lowest number of surviving plants. Regarding C. ferax Rich. at 15 DAS, the extracts obtained at 1 and 4 min led to the lowest survival rates. For the 30-day-old plants, the lowest percentage of living plants was also recorded in the treatment with the 1 min extract. Overall, the lowest survival percentages were found in the younger plants (15 DAS) and in treatments using extracts from the shortest extraction times.

3.6. Bioactive Compounds in the Aqueous Extract of Baccharis trimera (Less) DC

The chromatographic analysis of the aqueous extract of B. trimera (Less) DC. enabled the identification of four main compounds (

Table 2. Total percentage of each compound identified in the aqueous extract of B. trimera (Less) DC.

Compound	Composition in the Extract (%)
	Extract from 1 min	Extract from 15 min	Extract from 30 min
Ferulic acid	8.69	7.99	1.27
Rutin	9.58	8.78	1.66
Quercitrin	8.51	8.16	1.76
Quercitin	2.75	9.10	1.36

): ferulic acid (peak A), rutin (peak B), quercitrin (peak C), and quercetin (peak D). These analyses were conducted on extracts obtained at 1, 15, and 30 min of extraction. The results indicate a decreasing trend in the concentration of these compounds over time. It was observed that the phenolic content was higher in the extracts obtained in the initial minutes of extraction, especially at 1 min.

4. Discussion

The results obtained in this study demonstrate the inhibitory activity of the aqueous extract of B. trimera (Less) DC. on the weeds O. sativa L. and C. ferax Rich. during the germination phase and post-emergent development. The bioactivities of extracts varied according to the extraction time, the phenological stage of the plants, and the morphophysiological parameters analyzed.

During the germination phase, it was observed that extracts obtained at the early extraction times (1 to 10 min) were more efficient in inhibiting seed germination of both species, with significantly lower percentages than the control. Increases in B. trimera (Less) DC. extract concentrations resulted in the inhibition of root growth and germination speed of Lactuca sativa seeds [10]. The presence of phenolic compounds such as ferulic acid, rutin, quercitrin, and quercetin may have contributed to this inhibition, as these bioactive compounds are known to interfere with essential physiological processes for germination, such as protein synthesis and enzymatic activity. Phenolic compounds can cause oxidative stress, lipid peroxidation, and changes in cell walls, which can lead to cell death in several species [23].

Applying the aqueous extract of B. trimera (Less) DC. significantly affected the germination rate (calculated through GSI) of O. sativa L. and C. ferax Rich. seeds, indicating its potential inhibitory effect on these weed species. These results suggest that B. trimera (Less) DC. extract negatively interferes with the initial development of seeds of both species, most likely due to the presence of phenolic compounds and other secondary metabolites with inhibitory activity, which reinforces its potential as a bioherbicidal agent in sustainable weed management strategies. Extracts from Ageratum conyzoides and Borreria alata inhibited soybean seed germination and growth, with varying potency based on the concentration of allelochemicals [2]. Extracts of Capsicum baccatum showed an inhibitory effect on the pre-emergence of Cucumis sativus and Hovenia dulcis [17]. Leaf extracts of Eucalyptus camaldulensis and Coleus barbatus showed inhibitory effects on germination and GSI of the weed E. plana [19]. Aqueous methanolic extracts of whole plants of Ludiwigia decurrens showed inhibitory activity against O. sativa and rice weeds Echinochloa crus-galli and Monochoria vaginalis [24].

In the post-germination stage, the application of the extracts promoted significant changes in the morphological development of the plants, reducing shoot and root lengths, and fresh and dry biomass. Gusman [25] highlighted that the presence of secondary metabolites in B. trimera (Less) DC. can modulate the growth of other species under competitive conditions. Shadab [26] demonstrated that extracts from Lepidium didymium reduced the germination and growth of Lens culinaris and Melilotus alba. Similarly, B. trimera (Less) DC. extracts obtained between 1 and 10 min showed the most intense effects on the development of the evaluated weed species. The effects were more evident at 15 DAS, suggesting that early developmental stages are more susceptible to inhibitory action.

The effects of extracts on root lengths were also evident, negatively impacting the root growth of O. sativa L. and C. ferax Rich. It is consistent with the findings reported by Konig [27], in which the inhibition of root and shoot development of Eragrostis plana treated with B. trimera (Less) DC. extract was reached. Similar effects were reported by Pinheiro [28], who used the essential oil of Hesperozygis ringens, which affected germination and seedling growth in soybeans.

Inhibitory effects of Heliotropium indicum extract were reported on the shoot and root growth of cucurbit crops, indicating that these compounds act differently depending on the species [29]. The negative effects on root growth may have relevant practical implications for weed management and agricultural productivity. Regarding biomass, both fresh and dry mass of treated plants were significantly reduced, especially in treatments with extracts from 1 to 10 min [30]. Similar results using Setaria faberi extracts were reached, which negatively affected early seedling development in lettuce, impacting both length and dry/fresh weights [31].

Weed control is an essential practice in agriculture, especially in crops of great economic importance such as rice, soybeans, and corn, for example. In rice crops, weeds such as red rice and reed are highly aggressive and difficult to control, requiring efficient management strategies. These invasive species compete directly with rice plants for light, water, and nutrients, considerably reducing the yield of this crop [32].

Herbicides are tools for phytosanitary security in agricultural areas, but their excessive use can cause problems in agricultural production systems and a negative impact on human health and the environment [33]. Effective weed control is essential to ensure the productivity, profitability, and sustainability of agricultural systems. Phytochemical compounds and their biological efficacy can be used as bioherbicides in weed management strategies [34,35].

Weed survival was also affected. Extracts obtained during the initial minutes of extraction reduced the number of live plants, especially in C. ferax Rich. plants at 15 DAS, evidencing the phytotoxic potential of the extract in younger plants. Inhibitory compounds can increase cell membrane permeability, cause metabolic disturbances, and consequently lead to cell death in crops such as mustard and rapeseed [36,37]. On the other hand, the low mortality rate observed in 30-day-old plants highlights a possible physiological or adaptive resistance to the compound. Although the extract interferes with development, it is not sufficiently lethal in more advanced stages.

This behavior is similar to what occurs with many synthetic herbicides, whose effectiveness has been compromised by the emergence of resistant populations due to repeated and selective use. Such resistance reinforces the complexity of controlling weeds such as O. sativa L. and C. ferax Rich., highlighting the need for integrated management strategies. Phytochemical compounds represent a promising solution for managing ecological challenges and herbicide resistance [38].

Herbicidal activities against weed species have been reported for extracts of Canavalia ensiformis, Cirsium setosum, Cynara cardunculus, Juglans nigra, Lantana camara, and Ocimum basilicum, causing reduced growth and development, inhibiting germination, inducing oxidative stress, and decreasing the production of roots, leaves, and cotyledons [39]. These findings reinforce the viability of inhibitory compounds as sustainable tools in weed management.

Unlike many synthetic herbicides, which typically act through a single specific target site, allelochemicals, such as phenolics, often exert their effects through multiple modes of action. Phenolics can disrupt cellular processes such as RNA and protein synthesis, mitochondrial function, and cell division, which are crucial for plant growth [40]. This broader and nonspecific activity may help slow or reduce the evolution of resistance in weed populations.

Although statistical significance was observed for several variables, it is also important to assess the biological relevance of these effects. Modest reductions, such as a 10% reduction in biomass, may not substantially affect weed competitiveness under field conditions. However, reductions in germination or initial growth greater than 30%, as observed with short extraction times, suggest a more significant potential to suppress weed establishment during critical stages of crop development. Therefore, interpretation of results should consider not only statistical significance but also the practical implications of the observed effects for weed management.

Chromatographic analysis by HPLC allowed the biological effects observed to be related to the presence of phenolic compounds in the extract. As the extraction time increased, the concentrations declined, suggesting possible dilution due to prolonged exposure to the solvent under constant pressure and temperature. This reduction in bioactive compound concentration over time may reduce the bioactivities of the extracts, as compounds such as ferulic acid, rutin, and quercetin are well known for their inhibitory and antioxidant activities.

Therefore, the variation in concentration in the chemical profile as a function of extraction time may be directly related to the inhibitory efficiency observed in the biological assays. The levels of ferulic acid, rutin, quercitrin, and quercetin were higher in extracts obtained at 1 min, decreasing progressively with longer extraction times. This suggests that the inhibitory efficiency of the extracts is directly associated with the concentration of these compounds, underscoring the importance of extraction time in obtaining more effective extracts. The chemical components identified in this study are consistent with those reported in the literature. De Almeida [41] detected rutin, quercetin, and luteolin in aqueous extracts of B. trimera (Less) DC. Rabelo [42] also reported the presence of flavonoids and ferulic acid derivatives, and Da Silva [43] identified quercitrin, rutin, and kaempferol in aqueous extracts of the same species.

Even using only water as a solvent, bioactive compounds were extracted. Although organic solvents such as methanol and ethanol are widely used, aqueous extraction is a safe and effective environmental alternative. Using PLE, which combines high pressure and moderate temperature, may have contributed to the efficient release of these compounds. According to Barik [44], this technique stands out for its high efficiency, shorter extraction time, and reduced environmental impact, making it a promising alternative for obtaining plant-based bioactive extracts.

Therefore, the results indicate that the aqueous extract of B. trimera (Less) DC., especially when obtained through short protection times (up to 10 min), has high potential as an inhibitory agent for the management of specific plant species, such as O. sativa L. and C. ferax Rich. The combined prevention of germination, growth reduction and biomass suppression reinforces its potential use as a natural bioherbicide. Although the extract has shown promising effects under controlled conditions, its practical application in agricultural systems still faces important challenges. The results obtained in the laboratory cannot fully reflect environmental variability, such as interactions with soil microbiota, photodegradation or persistence of the compounds under field conditions.

The environmental persistence of plant extracts rich in phenolic compounds, such as that of B. trimera (Less) DC., has only been minimally explored and is of great relevance for their practical application as bioherbicides. Future studies should explore the extract performance in field environments, the retention time of these extracts in soil, investigate their chemical stability over time, and develop efficient formulations that improve their efficacy and shelf life. Furthermore, it is essential to evaluate the effects of B. trimera (Less) DC. extracts on crop species and non-target organisms to ensure environmental safety and predict their use as a sustainable management strategy for specific plants.

5. Conclusions

The results presented in this study demonstrate that the aqueous extract of B. trimera (Less) DC. has significant inhibitory potential on the weed species O. sativa L. and C. ferax Rich. in the pre-germination and post-germination phases. Extracts obtained at the initial extraction times (1 to 10 min) were the most efficient in inhibiting germination. For germinated seeds, the extracts reduced shoot and root lengths and decreased the fresh and dry biomass of the evaluated weeds. These effects are associated with the higher concentrations of phenolic compounds, such as ferulic acid, rutin, quercitrin, and quercetin, which were found in higher concentrations in the extracts obtained at shorter extraction times.

The reduction in the number of surviving plants, especially in the early stages of development, reinforces the potential of the extract as a natural phytotoxic agent, most likely representing a promising alternative to synthetic herbicides in weed management. The results indicate that the efficiency of the extract is related not only to the target species and the developmental stage but also to the extraction time of the plant material. Thus, this work contributes to advancing the knowledge about the inhibitory potential of native species like B. trimera (Less) DC. and emphasizes the importance of seeking sustainable alternatives in weed control.

The results correspond to laboratory conditions, while additional studies are needed to validate their practical application. Future research should evaluate the efficacy of the extract under field conditions, its persistence in the environment, and its safety for non-target organisms. In addition, it is essential to study formulation strategies that increase the stability, bioactivity, and feasibility of using the extract as a base for a bioherbicide formulation.