Lactic acid bacteria (LAB) have been used from ancient times to fight against food spoilage organisms in bread, vegetables and dairy products. The probiotic organism used in fermented and probiotic products can be considered bio-controllers due to their activity against pathogens and their “generally recognized as safe - GRAS” status. In recent years, there has been an increased emphasis on identifying novel LAB strains with antimicrobial activity for food bio-protection. LAB are inhabiting from a variety of natural sources, including decomposing plant matter, vegetables, fruits, dairy products, fermented foods and beverages, silages, juices, sewage and the gastrointestinal tracts and mucosal surfaces of humans and animals. Despite their wide distribution, identifying LAB remains difficult. 16S rDNA sequencing methods allow precise strain-level identification, whereas traditional phenotypic approaches often lack reliability. Consequently, molecular taxonomy and whole-genome sequencing have become the most effective tools for distinguishing LAB at the species level (Siedler et al., 2019).
       
Lactic acid bacterial culture have been used as biological control agents on plant diseases in chilli (Colletotrichum capsici), tomato (Fusarium oxysporum) and cucumber (Pythium ultimum) (El-Mabrok et al., 2012). Tomato is the second most important vegetable crop next to potato grown around the world. Adaptability to a wide range of climatic and soil conditions enables its worldwide cultivation. In our country the maximum production of tomato occurs in Uttar Pradesh followed by Karnataka, Punjab, West Bengal and Assam (Singh et al., 2023). Tomato is affected by 200 known diseases of diverse causes and etiologies. Among them Fusarium oxysporum is the major seed-borne fungal pathogens which adversely affect the tomato production both under greenhouse and field conditions.
       
The present study aimed to isolate bacterial species from rhizosphere region of plants belonging to the solanaceous family, biochemical and molecular characterization were carried out. Further, beneficial bacterial species were selected for their antagonistic activity against phytopathogenic  bacteria and fungi. The antibiotic sensitivity of bacterial strains was evaluated against the major classes of antibiotics.

Collection of soil samples
 
The present study was conducted from June 2024 to July 2025. About 100 g of soil samples from the rhizosphere region of solanaceous crops (chilli, brinjal and tomato) were collected in polythene bags from different geographical regions of Belagavi district, Karnataka and were labelled, carried to the Biotechnology laboratory, Davangere University, Davangere, Karnataka, India and stored at 4^oC for further use (Fig 1).


 
Isolation of lactic acid bacteria from soil samples
 
About 1 g of rhizosphere soil was suspended in 9 mL sterile saline and diluted serially from 10^-1 to 10^-7. 0.1 mL aliquot of higher dilutions (10^-5 and 10^-7) were spread onto de Man, Rogosa and Sharpe (MRS) plates and incubated at 37^oC for 24-48 h (De Man et al., 1960; Chadli et al., 2024). After incubation, bacterial colony was selected based on their morphology and colony character according to Bergey’s manual of determinative bacteriology (Nwamaioha and Ibrahim 2018; Habib et al., 2020). The pure colonies were maintained by sub culturing twice on MRS agar and stored at 4^oC.
 
Lactic acid estimation
 
Inoculum preparation
 
Isolated bacterial strains were screened for lactic acid production using MRS broth. In the current study, inoculum was prepared through transference of 2% stock culture to growth medium (MRS). Incubation temperature was 35± 1^oC for 24-48 h at 150 rpm (Mulaw et al., 2019).
 
Determination of lactic acid in cultural liquid
 
Cultural liquid was separated from cells by centrifugation. The supernatant was diluted 20-fold with deionized water. Lactic acid in the sample was determined by spectro-photometric method for determination of lactic acid. The concentration of lactic acid was calculated using calibration curve.
 
Genotypic Identification
 
For DNA extraction, the bacterial isolates were grown in 50 mL of MRS broth under aerobic conditions for 48 h. Further, DNA was isolated and purified using Bacterial DNA purification kit (Genei, India). Isolated DNA was amplified with 16S rRNA specific primers and sequenced at Chromos Biotech Pvt. Ltd. Bangalore. 16S rRNA gene sequencing was aligned with the reference sequence published on the NCBI database using the BLAST algorithm. 
 
Biochemical tests
 
The identification of the selected LAB isolates was carried out by various biochemical tests by streaking the cultures on Nutrient Agar medium (Hi Media, Bangalore) and then incubating for 48 h at aerobic condition at 37^oC.
 
Catalase test
 
The test is performed by tube or slide method by mixing the colony of bacteria with few drops of 3% H₂O₂ on slide or to the test tube and looking for bubble formation within 10 seconds (Winn et al., 2006).
 
Oxidase test
 
This test is performed by impregnation of 1% tetra-methyl-p-phenylenediamine dihydrochloride acting as artificial electron donor into a filter paper and dried. The bacterial colonies are smeared on paper strip and check for colour change within 10 sec (Winn et al., 2006).
 
Indole test
 
Production of indoles by isolated bacteria was assayed as described by Patten and Glick (1996). Bacterial isolates were inoculated in Luria broth (LB) (1/10 strength) with L-tryptophan (500 μg/ml), incubated at 28±2^oC for 48 h followed by centrifugation at 8,000 rpm for 10 min. One mL of supernatant was mixed with 4 mL of Salkowski’s reagent in 1:4 ratio and incubated at room temperature for 20 min, pink colour indicated presence of indoles (Kochar et al., 2013).
 
Phosphate solubilisation test
 
The ability of isolated bacteria to solubilize phosphate was determined on Pikovskaya’s medium. Petri plates containing Pikovskaya’s medium were inoculated with isolated bacteria incubated at 28±2^oC, analyzed for clear zone around bacterial colony up to 10 days. The bacterium, which showed zone of clearance on repeated subculture onto Pikovskaya’s medium was considered positive for phosphate solubilization.
 
Lactose fermentation
 
The isolated carbohydrates are tested for lactose fermentation. Overnight-grown lactic acid bacterial culture was used to prepare new inoculation tubes. A single colony from each culture were streaked onto MRS agar supplemented with phenol red (0.05 g/L). The plates were incubated at 30^oC for 24 h.  Acid production by isolates were confirmed by colour change of  medium from red to yellow (Esmail et al., 2014).
 
H₂S production
 
The hydrogen sulphide (H₂S) production of isolates were determined using Triple Sugar Iron (TSI) agar (HiMedia, India). TSI slants of selected isolates were prepared as per the manufacturer’s guidelines. Bacterial colonies from each isolate were inoculated by stabbing the butt and streaking slant surface. The tubes were then incubated at 37^oC for 24 h. Appearance of black pigmentation in butt region resulting from ferrous sulphide formation as a positive indication of H₂S production (Madushanka et al., 2025).
 
Lysine decarboxylase
 
Lysine decarboxylase activity of selected isolates were assessed using modified decarboxylase medium. Basal medium, adjusted to pH 5.3 and supplemented with 0.5% (w/v) L-lysine monohydrochloride, while medium without lysine served as control. Actively growing cultures were first adapted in basal medium without amino acids for 5 days at 30^oC. Subsequently, 0.2 mL of adapted culture inoculated into lysine-supplemented and control media. Anaerobic conditions were maintained using sterile liquid paraffin and tubes were incubated at 30^oC for 3 days (Mete et al., 2017).
 
Citrate utilization
 
Citrate utilization of selected isolates were determined following Sirisha et al. (2021) method. Isolates were inoculated into Simmons citrate agar and incubated at 37^oC for 24 h. After the incubation, blue colour of appearance indicated the positive test for citrate utilization and was recorded.
 
Isolation of Phyto-pathogenic fungi
 
Phyto-pathogenic fungi were isolated from infected plant samples, including chilli, tomato and brinjal growing region around Belagavi region of Karnataka during field survey. Fungal pathogens were confirmed by observing the colony morphology under a stereomicroscope and conidial morphology under compound microscope.
       
The plant samples were washed with distilled water and followed by surface-sterilization using 1% sodium hypochlorite for 2 min and placed aseptically on potato dextrose agar (PDA).  The colonies of different shape and colours were sub-cultured on PDA incubated at 28^oC for 5 to 7 days and pure culture of each colony was maintained and stored at 4^oC.
 
Antibacterial activity
 
The antibacterial potential of LAB strains were evaluated against pathogenic bacterial species, i.e., X. campetris, E. coli and Pseudomonas. 100 mL of cell free supernatant from 24h old cultures of lactic acid bacteria were subjected to the antibacterial study using agar well method (Matevosyan et al., 2019; Pooja et al., 2024) and results were represented as zone of inhibition.

Antifungal activity
 
The antifungal activity was evaluated by spreading the fungi on PDA plates. All the LAB isolates were screened for the production of antifungal compounds. The phytopathogenic fungi F. oxysporum, A. niger, Penicillium spp., Botrytis spp., Verticella spp. and A. alternata were used as target organisms.  The LAB isolates were screened for antifungal activity on MRS agar plates. The formation of clear zone indicates the presence of antifungal compounds (Dahham et al., 2010).
 
Antibiotic susceptibility test
 
The disc diffusion method was used to assess the susceptibility of isolated LAB to six antibiotics, viz., ampicillin, kanamycin, tetracycline, vancomycin, rifampicin and penicillin. The bacterial strains were characterized as either resistant or sensitive to a specific antibiotic according to the specifications of the Clinical and Standard Laboratory Institute, 2009; (Gundappa et al., 2024).

A total eleven different bacterial strains were isolated from chilli, brinjal and tomato growing soil samples (Table 1) by using MRS media. Four Acinetobacter species were isolated from chilli, brinjal growing soil. Three Bacillus species were isolated, Microbacterium was isolated from chilli, Enterobacterium was isolated from the tomato L. lactis and L. fermentum isolated from tomato and brinjal growing soil.


       
The morphological characteristics of the selected isolate were noted majority of the isolates showed white and rough colonies on MRS Agar. Acinetobacter spp. is gram-negative and cocci in shape. The Bacillus spp., L. lactis and L. fermentum are gram-positive. Microbacterium and Enterobacterium spp. are spherical and are gram-negative in nature.  Similarly, Maany et al. (2019) isolated a total of 41 LAB from the soil of healthy and diseased tomato and Phaseolus plants using MRS agar.
       
The production of lactic acid in the isolated bacterial strains were determined by spectrophotometric method. Concentration of lactic acid ranged from 51.148 mg/L to 5311.11 mg/L. The lowest concentration was reported in S1 Acinetobacter schindleri and highest concentration was reported in S11 L. lactis (Fig 2).


       
A similar work was carried out by Tolieng et al. (2017) observed that lactic acid producing bacteria isolated from soil and tree barks in Thailand and showed Lactococcus produced the L-lactic acid (72.32-77.47 g/L) with 100% optical purity.  Enterococcus hirae produced (31.56-34.86 g/L), Bacillus coagulans produced 48.48 and 93.51 g/L of L-lactic acid with high optical purity (99.56%). Sporoacto bacillus produced D-lactic acid (87.64 g/L).
       
The potential probiotic isolates were identified through 16S rRNA gene sequencing. All eleven isolates produced an amplicon of approximately 1500 bp. 16S rRNA analysis revealed that identified strains S8, S1, S6 and S9 showed homology to (94.95%) Acinetobacter spp. JNG7 (99.37%) and its different strains. Similarly, S4, S7 and S5 showed homology to the (84.07%) Bacillus cereus strain F3-50, (99.58%) Bacillus velezensis strain 8-4 and (97.48%) Bacillus subtilis strain IP18. S3 and S2 showed homology to (99.29%) Enterobacter spp. strain LJ68, (98.15%) Microbacterium spp. HBUM178923. Strains S10 and S11 showed homology to (99%) Limosilactobacillus fermentum and (99%) Lactobacillus lactis. Similarly, 16S rRNA sequencing identified LAB isolates from winter salad pickle, a total 8 species showed Pediococcus pentosaceus (51.1%) and Lactobacillus plantarum (20%), W. cibaria (11.1%), L. fermentum (6.7%), E. faecium (4.4%), E. faecalis (2.2%), Leuconostoc citreum (2.2%) and Leuconostoc mesenteroides subsp. mesenteroides (2.2%) homology (Saeedi et al., 2015).
       
The biochemical tests results revealed that B. cereus, B. subtilis, B. velezensis showed positive and L. fermentum, L. lactis showed negative to catalase test. For oxidase test, B. subtilis showed negative, the remaining isolates showed positive. For indole, L. fermentum, L. lactis showed positive and B. cereus, B. subtilis, B. velezensis showed negative. For phosphate solubilization, B. cereus, B. subtilis showed positive and B. velezensis, L. fermentum, L. lactis showed negative. Similarly, all isolates showed positive in lactose fermentation test and negative to the H₂S production. For lysine decarboxylase test B. velezensis showed negative and the remaining isolates showed positive results. B. cereus, B. subtilis showed positive and B. velezensis, L. fermentum, L. lactis showed negative results (Table 2). Madushanka et al. (2025) observed that none of the isolates showed hydrogen sulphide production and gas production from glucose fermentation. Mete et al. (2017) determined Lysine carboxylase activity of isolates and observed seven isolates showed negative results.


       
Phyto-pathogenic fungi such as Fusarium oxysporum (F2), Alternaria alternata (G2), Rhizopus oryzae (TPS1), Rhizopus arrhizus (TPS2) and Fusarium spp. (YECA) were isolated from infected plants. Pure cultures were examined for colony and microscopic characteristics. Morphological characterization confirmed that the infected plant tissues were associated with selected isolates.
       
Genotypic identification was performed using 18S rDNA sequencing, sequencing data were compared with the reference sequence published on the NCBI database using the BLAST algorithm. The analysed strain G2 showed 88.35% similarity to A. alternata, F2 strain showed 94.82% similarity to F. oxysporum. TPFS1 and TPFS2 strains showed 99.55% and 99.11% similar to R. oryzae and R. arrhizus. The YECA strain showed 99.91% similarity to Fusarium spp. Thilagam et al. (2018) reported A. alternate. F. oxysporum, F. solani, A.  flavus, Colletotricum spp. in infected plant parts such as roots, stems, leaves, flowers and fruits from Tamil Nadu region.
       
Selected bacterial supernatants were examined for antibacterial activity against X. campetris, E. coli and Pseudomonas spp. B. cereus supernatant showed no zone against X. campetris and E. coli completely inhibited against Pseudomonas. B. subtilis and B. velezensis exhibit weak inhibitions, producing small zones (1-7 mm) against test organisms. L. fermentum supernatant completely inhibited against Xanthomonas and also higher concentrations of Pseudomonas, but it showed strong antibacterial potential (15-31 mm) against E. coli. L. lactis spp. supernatant also showed strong antibacterial activity against all tested organisms by producing large (18-33 mm) zone of inhibition, but at higher concentrations showed complete inhibition against Pseudomonas spp. (Table 3).


       
Four species of lactic acid bacteria were subjected to antimicrobial activity, among four L. lactis subsp. lactis showed the strongest inhibition 14 mm zone against E. coli. Weakest activity was observed with neutralized supernatants of L. saki and L. plantarum (8.33 mm) against Shigella dysenteriae and S. aureus (Chakoosari et al., 2015).
       
Antifungal assay showed variations in zone of inhibition of bacterial supernatant against Penicillium spp. F. oxysporum, Botrytis spp., Verticillium and A. niger. B. cereus supernatant moderate inhibition against Penicillium (4-7 mm), F. oxysporum (4-13 mm) and Botrytis spp. (4-9 mm), but no activity against Verticillium and A. niger. Whereas, B. subtilis supernatant showed activity against Verticillium (8-12 mm) and A. niger (3-5 mm), but no activity against remaining test organisms. B. velezensis supernatant showed zone of inhibition against Penicillium (3-6 mm) and Botrytis spp. (8-9 mm) and no zone of inhibition against F. oxysporum, Verticillium and A. niger.  L. fermentum exhibits antifungal activity by showing zones 6-15 mm to Penicillium, 5-18 mm to F. oxysporum, 6-21 mm to Verticillium, 11-19 mm to A. niger and no activity to Botrytis spp. L. lactis showed strong antifungal activity with zone of inhibition 10-26 mm against   F. oxysporum, 8-20 mm against Penicillium, 9-18 mm against A. niger and also showed complete inhibition against Verticillium and no activity against Botrytis spp. (Table 4). Magnusson et al. (2003) reported four LAB isolates showed significant antifungal activity against five Candida species, with inhibition zones ranging from 10.0 to 17.2 mm. The strongest effect (22.0 mm) was observed for isolate HH (L. curvatus) against C. glabrata ATCC2001. L. curvatus (HH) also inhibited C. parapsilosis (15.6 mm) and C. tropicalis (14.7 mm), while P. pentosaceus (HM) showed strong inhibition against C. krusei (17.2 mm), C. glabrata (16.0 mm) and C. albicans (13.3 mm). L. plantarum (HS) effectively suppressed C. albicans (15.3 mm) and C. krusei (13.1 mm).


               
Antibiotic assay revealed that L. lactis exhibited highest sensitivity with large inhibition zones (19-44 mm) against all tested antibiotics. B. cereus also showed strong activity to ciprofloxacin (30 mm), levofloxacin (29 mm), cefixime (29 mm) and gentamicin (27 mm). L. fermentum showed good susceptibility to multiple antibiotics, with clear zones of inhibition for ampicillin (25 mm), cefixime (15 mm), ciprofloxacin (19 mm), gentamicin (15 mm) and levofloxacin (16 mm) and no inhibition to tetracycline. B. subtilis and B. velezensis showed moderate susceptibility; both showed no inhibition to ampicillin and cefixime and showed 16 mm zones to gentamicin and tetracycline for ciprofloxacin 24 mm, 18 mm and 20 mm, 21 mm zone of inhibition for levofloxacin, respectively (Fig 3). A study by Onuoha et al. (2016) evaluated antibiotic sensitivity of E. coli, Klebsiella spp., S. aureus and Shigella spp. against eight antibiotics, including ceftazidime, ampicillin, amoxicillin- clavulanic acid, ciprofloxacin, cefuroxime, meropenem, ofloxacin and sulfamethoxazole. Their findings showed that meropenem was most effective antibiotic against all tested organisms, followed by the fluoroquinolones (ciprofloxacin and ofloxacin), which also exhibited strong inhibitory activity. Cefuroxime inhibited all isolates except Shigella spp., while the remaining antibiotics displayed sensitivity only against two of the tested organisms.

In the present study, an attempt was made to isolate lactic acid bacteria collected from soil samples of different geographical regions. A total 11 bacterial isolates, including L. fermentum, L. lactis, Acinetobacter, Bacillus, Microbacterium and Enterobacter species, were identified through morphological, biochemical and 16S rRNA gene sequencing methods. The study identified L. fermentum and L. lactis as strong lactic acid-producing strains with significant antibacterial and antifungal activity against major phytopathogens. These findings suggest that LAB isolates hold promising potential as eco-friendly biocontrol agents for sustainable crop protection.

The present study was supported by the Department of Studies in Biotechnology, Davangere University, Davangere, for conducting research activities.

Disclaimers
 
The views and conclusions expressed in this article are solely of the authors and do not necessarily represent the views of their affiliated institutions.
 
Informed consent
 
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The authors declare that there are no conflicts of interest regarding the publication of this article.