Rhizosphere soil samples were collected in an agricultural field with healthy, flowering Mimosa pudica L. in Leran Wetan Village, Palang District, Tuban Regency. Composite sampling technique was used to collect samples in five different points around the root zone at a depth of 10-20 cm (Sriwulan et al., 2022). Soil adhering to the roots was collected by gently brushing it off with a sterile brush and then mixed thoroughly (composited) to form one representative sample (Koyama et al., 2021). This composite sample was placed in a sterile plastic bag, labeled, and transported to the laboratory in a cool box for subsequent analysis.

Isolation and characterization of rhizospheric bacteria from the collected soil sample were carried out and began with a serial dilution. An aliquot of the composite soil sample was weighed 25 g and added to 225 mL of sterile 0.85% physiological NaCl solution in an Erlenmeyer flask (Jie et al., 2025). This suspension was shaken using an orbital shaker for 15 minutes at 120 rpm to obtain a 10⁻¹ dilution. A serial dilution was then performed up to a 10⁻⁶ dilution.

Bacterial inoculation was carried out by taking 0.1 ml of suspension at 10⁻⁵ and 10⁻⁶ dilutions. Inoculation was carried out using the pour plate method on NA media. Incubation was carried out at room temperature (28 ± 2°C) for 2x24 hours. The growing bacterial colonies were observed, and colonies with different morphological characteristics were purified using the plate streak method on NA media. The isolates were then characterized based on colony morphology, gram staining, and biochemical characterization using the catalase test.

The propagation of Fusarium spp. isolates was carried out by inoculating pure cultures using the agar block transfer method(Ekwomadu & Mwanza, 2023). A 5 mm diameter agar block containing Fusarium spp. mycelium was taken aseptically using a sterile cork borer. The block was then placed in the center of fresh PDA media and incubated at room temperature (28 ± 2°C) for 5-7 days.

The antifungal activity test of Mimosa pudica rhizosphere bacterial isolates against Fusarium spp. was carried out using the dual culture method on PDA media (Boulahouat et al., 2023). A 5mm agar block containing 7-day-old Fusarium spp. mycelium was placed on one side of a petri dish containing PDA media. On the opposite side, each purified isolate of Mimosa pudica rhizosphere bacteria was streaked, except for the control (no test bacterial isolate was streaked). Each bacterial isolate was repeated 3 times. The petri dish was then incubated at room temperature (28 ± 2°C) for 7 days.

The inhibition of fungal growth was observed. The radial growth diameter of Fusarium spp. towards the bacterial isolate was measured and compared to the growth diameter in the control plate (S. Wang et al., 2024). The percentage of inhibition (IP) was calculated using the following formula:

IP (%) = [(dc - dt) / dc] x 100%

Where:

IP = Inhibition Percentage

dc = Radial growth diameter of the fungus in the control plate (mm)

dt = Radial growth diameter of the fungus towards the bacterial isolate in the treatment plate (mm)

The inhibition percentage (IP) data obtained for each bacterial isolate were analyzed quantitative descriptive to identify which isolate exhibited the strongest antagonistic activity. Data was processed using SPSS 25.0 program in analysis of Variance (ANOVA) one-way test with confidence limit of 95% or p=0.05. If there is a noticeable difference effect between IP and isolate typed bacteria, further post-hoc Tukey's HSD test used to provided the highest mean inhibition percentage.

The bacterial isolates from the rhizosphere of Mimosa pudica L. were characterized based on colony morphology, microscopic characteristics, and biochemical tests. The characterization revealed six distinct isolates, and the characteristics were presented inTable 1. As shown in Table 1, the six isolates exhibited diverse characteristics. These observed characteristics were used as a basis for the preliminary genus-level identification of the isolates.

The characteristics of isolates PM1, PM3, and PM5 are indicative of the genus Bacillus. Bacteria from this genus initially often form punctiform colonies with entire edges (after short incubation periods). However, with longer incubation (typically 48 to 72 hours), these colonies can develop into larger, circular or irregular forms, sometimes appearing wrinkled due to endospore formation (Turenne et al., 2015). The punctiform colony with an entire edge observed for PM1 in this study is likely because characterization was performed after 24 hours of incubation. This morphological change was already observed in isolate PM3 and PM5, which can be attributed to differing growth rates among bacterial strains, even within the same genus. Bacillus isolates on Nutrient Agar (NA) generally appear translucent,  white, cream, or brown. This group consists of Gram-positive, rod-shaped cells and tests positive for catalase (Bergey, 1994). Soil is a common habitat for this bacterial genus, which aligns with the origin of the sample in this research, rhizosphere soil of Mimosa pudica L.

The characterization results for isolate PM2, as shown in Table 1, characteristics pointing towards the genus Cellulomonas. This genus is characterized by short rod-shaped cells or cocci (in some species), which may align to form V-shaped structures. They are Gram-positive, catalase-positive, often motile with one or more flagella, do not form spores, are non-acid-fast, are facultative anaerobes, possess cellulolytic activity, and are commonly found in soil and other organic matter (Bergey, 1994)(Wu et al., 2023).

The isolate PM4 exhibits characteristics indicative of bacteria belonging to the genus Arthrobacter. This genus is characterized by rod-shaped cells that undergo a rod-to-coccus cycle upon aging, a Gram-positive reaction, motility in some species, an inability to form endospores, an aerobic metabolism, a positive catalase test, and an optimal growth temperature of 25–30 °C. Arthrobacter species are widely distributed in nature, particularly in soil habitats ((Busse et al., 2015); (Khairani et al., 2019); (Roy & Kumar, 2020)).

Meanwhile, the characteristics of the PM6 isolate indicate a character that points to the genus Micrococcus. This genus is mixed with a group of bacteria that are gram-positive, have round cells that are paired, form tetrads, or grouped irregularly (Wang et al., 2021). The results of the gram staining obtained by the PM6 isolate appear purple, indicating that it is a gram-positive bacteria and has coccus-shaped cells arranged in irregular groups. Micrococcus colonies on NA media appear yellow or red, where the PM6 isolate shows a yellow isolate color. Bacteria from the Micrococcus genus also show positive catalase results with one of their habitats in soil (Bergey, 1994).

The data in Table 2 shows that the six bacterial isolates obtained from the rhizosphere of Mimosapudica in this study were able to inhibit Fusarium spp., with an inhibition percentage ranging from 61.99% to 81.83%. These results indicate that all isolates obtained in this study have antifungal activity against Fusarium spp.

The inhibition percentage data shown in Table 2 were analyzed using one-way ANOVA statistical analysis, which obtained a significant value of 0.01 < alpha 0.05. This indicates that differences in bacterial isolates affect their antifungal activity as indicated by their inhibition percentage. Meanwhile, further test results using the Tukey HSD test showed that Isolate PM2 provided the highest average inhibition percentage (81.83%), but was not significantly different from isolates PM6, PM1, and PM3.

These results indicate that the six isolates from the rhizosphere of Mimosapudica plants have the potential to be developed as biocontrol agents, particularly against Fusarium spp. Rhizosphere bacteria capable of inhibiting the growth of pathogenic fungal isolates generally possess the ability to produce antibiotic compounds, siderophores, lytic enzymes, or a combination of these mechanisms (Sriwulan et al., 2022).

Several bacterial species from the genus Bacillus are known to produce antibiotic compounds that can damage the cell membranes of pathogenic fungi, inhibit cell wall formation, and disrupt the metabolic processes of these pathogenic fungi (Puspitasari, 2023). Consistent with this finding, isolates PM1, PM3, and PM5 in this study, belonging to the genus Bacillus, were able to inhibit the growth of Fusarium spp. with inhibition rates of 70.96%, 70.14%, and 61.99%, respectively

Meanwhile, isolate PM5 produced the lowest inhibition percentage, at 61.99%. However, this figure still demonstrates the isolate's promising potential for development as a biocontrol agent. Several species of the genus Bacillus are known to produce the enzymes chitinase and glucanase, which can damage fungal cell walls (Dawoud et al., 2020). FFurthermore, Bacillus amyloliquefaciens is also known to stimulate host plants to increase their protective abilities against pathogen attacks ((Luo et al., 2022); (Ngalimat et al., 2021); (Zalila-Kolsi et al., 2023)).

The PM2 isolate, which demonstrated the highest antifungal activity in this in vitro test, is indicated as belonging to the genus Celullomonas. Bacteria from this genus exhibit antifungal activity through several mechanisms, including the ability to produce lytic enzymes that can damage fungal structures, such as chitinase and glucanase. These enzymes break down chitin and glucan, the main components and essential components of fungal cell walls. Furthermore, the metabolic activity of Celullomonas bacteria also alters microenvironmental conditions, particularly pH and oxygen levels. This will affect fungal survival. The ability of these bacteria to produce cellulase enzymes also allows them to release nutrients that can stimulate the growth of other antagonists (Ontañon et al., 2021)(Wu et al., 2023).

Meanwhile, the antifungal mechanism demonstrated by the PM4 isolate may occur through its ability to produce siderophore compounds. This will make it difficult for Fusarium spp. to obtain iron ions and will disrupt metabolic processes. Furthermore, bacterial groups from the Arthrobacter genus are also known to have the ability to colonize rapidly, thereby outcompeting Fusarium spp. in the process of nutrient competition (Roy & Kumar, 2020). Several Arthrobacter species have been reported to be able to produce antibiotic compounds, volatile compounds, and lytic enzymes such as chitinase and glucanase that can inhibit fungal growth. This group of fungi can also induce systemic plant resistance . The same mechanism was also shown by the group of bacteria from the genus Micrococcus which in the study was represented by the isolate PM6 (Wang et al., 2021).