Healthy plant parts, i.e., root, stem, and leaf, were collected from three individual plants of C. dichotoma growing in the Herbal Garden of Shri Mata Vaishno Devi University, Katra, Jammu and Kashmir (32.9418°N and 74.9541°E, elevation 754 m), India, in sterile polythene bags and brought immediately to the laboratory and processed to reduce the risk of contamination. The specimen was deposited at the Department of Botany, University of Jammu, with accession number 16613.

Plant samples were washed in sterile distilled water at every step of the surface sterilization process. To assess the effectiveness of the surface sterilization method, a sterility check was performed for every sample. For this surface sterilization, plant samples were placed on nutrient agar, incubated at 37°C for 48 h, and checked for feasible microbial growth. Plant samples were used for further experimentation only if no growth was observed (Hallmann et al., 2006).

The surface-sterilized plant segments were cut into approximately 6 mm diameter disks under aseptic conditions and placed on nutrient agar plates, and they were incubated for 48 h at 37°C in an inverted position. After 48 h, the plates were observed for bacterial growth surrounding the root, stem, and leaf sections. Endophytic bacteria emerging from the surface of plant segments were collected using an inoculation needle and further subcultured to obtain pure colonies. Single colonies acquired were streaked on fresh nutrient agar plates for further purification. These pure colonies were preserved in 50% glycerol stock and used for further experimental procedures.

All selected bacterial isolates were identified through morphological characteristics, such as colony color, margin, consistency, and texture, and microscopic characterization, such as gram staining, endospore staining, and motility. The biochemical and physiological tests like indole utilization, methyl red, and sugar utilization were performed according to standard procedures (Pielou, 1975; Smibert and Krieg, 1994).

The endophytic bacterial strains were screened for the presence of several enzyme activities, such as catalase, oxidase, amylase, cellulase, and lipase. The monitoring was done by streaking the selected endophytic bacteria on culture agar media with the substrate (Table 1 in Supplementary Material). For lipase, cellulase, and amylase activities, standard substrates like tributyrin, carboxymethyl cellulose, and starch were used, respectively. Enzyme activity was observed by flooding the plates with Congo red for cellulase and iodine reagent for amylase, and the presence of clear zones was confirmed for lipase. Catalase and oxidase activities of the bacterial isolates were determined by adding hydrogen peroxide (H₂O₂) and oxidase reagent (1% tetramethyl-p-phenylenediamine dihydrochloride), respectively, on the smear of freshly grown endophytic bacterial cultures in nutrient agar plates. The presence of oxygen bubbles and purple color, respectively, was considered as positive.

To examine the effect of sodium chloride on the growth of endophytic bacterial isolates, a nutrient agar medium was prepared by adding different concentrations of NaCl (0–12%). Endophytic bacterial strains were inoculated into the plates and incubated at 37°C for 6 days, and observations were recorded every 24 h.

All endophytic bacterial strains were grown in nutrient broth medium for 5 days at 37°C, and antimicrobial activity was determined by agar diffusion method against six pathogenic bacteria, namely, Staphylococcus aureus MTCC 737, Escherichia coli MTCC 1687, Bacillus subtilis MTCC 1789, Pseudomonas aeruginosa MTCC 1688, Klebsiella pneumonia MTCC 432, and Salmonella typhi MTCC 733. Each endophytic bacterial culture (10 ml) was centrifuged at 1,000 rpm for 10 min and the supernatant was used to determine antibacterial activity. Overnight grown cultures of the test organisms were spread by sterile cotton swabs onto the surface of the Muller Hinton agar plates. Wells (6 mm) were made and 100 μl of endophytic bacterial strain was added into it; an equal volume of sterile nutrient broth instead of bacterial endophyte was used as a negative control, and ampicillin was used as a positive control. All plates were wrapped with parafilm and incubated for 24 h at 37°C and observed for the zone of inhibition of pathogenic bacteria. Antibacterial activity was evaluated by measuring the diameter of the clear zone of inhibition (Mohamad et al., 2018).

Antibiotic susceptibility test of endophytic bacterial strains was performed by adopting the Kirby-Bauer disk diffusion method (Bauer, 1966). Overnight nutrient broth culture of endophytic bacteria was prepared, and all the endophytic bacterial isolates were inoculated into Muller Hinton agar plates using a sterile cotton swab. Standard antibiotic disks, such as ampicillin (10 μg/disk), streptomycin (30 μg/disk), neomycin (30 μg/disk), chloramphenicol (30 μg/disk), bacitracin (10 U/disk), rifampicin (5 μg/disk), erythromycin (15 μg/disk), kanamycin (30 μg/disk), amoxycillin (30 μg/disk), and tetracycline (30 μg/disk), were placed on Muller Hinton agar plates and incubated 37°C for 24 h. After incubation, the antibiotic susceptibility pattern was determined by measuring the inhibition zone. Based on the diameter of the zone of inhibition recorded to the nearest mm, the organisms were designated as sensitive, intermediate, and resistant following DIFCO Manual 10th edition (1984).

Bacterial isolates with high antibacterial activity were chosen for molecular identification using 16S rDNA sequence analysis. A loopful of freshly grown bacterial cells was dissolved in tubes with 500 μl of Tris-EDTA for DNA extraction. Then, 500 μl of phenol: chloroform: isoamyl alcohol (25:24:1) was added. This mixture was vortexed and centrifuged at 14,000 g for 5 min. The upper aqueous phase was transferred to a fresh tube, and 800 μl of chilled isopropanol was added. The samples were centrifuged again for 5 min at 14,000 g, and the supernatant was discarded. The pellets were dried at room temperature overnight before being eluted in 70 μl of TE buffer. A NanoDrop spectrophotometer was used to measure the absorbance at 260 nm to determine the concentration of genomic DNA. The primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′) were used to amplify the 16S rRNA gene. Total DNA (50–500 ng) was added to the PCR mix (30 μl), which contained 24 μl of DreamTaq PCR master mixture, 1 μl of 10 nm each primer, 2 μl of DNA, and 7.5 μl of 10X buffer in a total volume of 30 μl. This reaction was performed under the following conditions: one cycle of 94°C for 5 min, followed by 35 cycles of 94°C for 1 min, 55°C for 1 min, 72°C for 1 min, and a final extension of 10 min at 72°C. The amplified DNA products were separated and visualized under UV light using agarose gel electrophoresis. The amplicons were purified using the Genei Pure^TM quick PCR purification kit and quantified using a spectrophotometer at 260 nm. Biologia Research India sequenced the purified partial 16S rDNA amplicons. The sequences were assembled, edited, and aligned in MEGA11 before comparison with those in the GenBank database with the Basic Local Alignment Search Tool (https://blast.ncbi.nlm.nih.gov/Blast.cgi) to determine the sequence homology with closely related organisms. In this study, the microorganisms with the highest level of identity (100%) were chosen as the closest match, and isolated bacteria were classified to the species level based on the information of the closest microbes.

To analyze the various volatile bioactive constituents, an ethyl acetate extract of SMC212 was subjected to gas chromatography-mass spectrometry (GC-MS). The Indian Institute of Technology, Jammu, conducted the GC-MS analysis. SHIMADZU, QP2010 PLUS was used for GC-MS analysis. The injecting temperature was set to 250°C, the column temperature was set to 50°C, the pressure was set to 29.7 kPa, and the column flow rate was set to 0.72 ml/min. The sample's total running time was 40 min. The phytochemical compounds in ethyl acetate extract were identified based on retention time by matching MS with available standards using the Willey and NIST libraries. The constituents' names, molecular formulas, and molecular weights were determined.

Colonization rate was determined as the total number of plant segments colonized by bacteria divided by the total number of incubated samples. Isolation rate was calculated as the number of bacterial strains obtained from plant segments divided by the total number of incubated segments. The Shannon Weiner diversity index (H ^/) was calculated as:

where Pi is the relative abundance of species and i contributes to the total number of species (Di Bitetti, 2000). All of the experiments were carried out in triplicate, and the data reported are the averages of the results. The SPSS-22 statistical software (SPSS, Inc., Chicago, IL, USA) was used to calculate the means and standard deviation.

To get pure endophytes from the inner plant tissues of C. dichotoma, epiphytic microorganisms and other contamination must be removed or killed through the surface sterilization method. For this, plant samples (i.e., root, stem, and leaf) were treated separately by a different amalgam of chemical disinfectants. Method I (70% ethanol) was not found productive individually as a high percentage of contamination was noticed along with the growth of endophytes. While in Methods II, III, and IV, plant samples were treated with different mixtures and duration of sodium hypochlorite, ethanol, and mercuric chloride to attain a sufficient result. In Method III, mercuric chloride was successful in removing the contamination, yet the survival percentage of endophytic bacteria declined. Our result shows that only Method IV (0.1% Tween 20, 4% sodium hypochlorite for 10 min, and 70% ethanol for 3 min) was found effective for surface sterilization of C. dichotoma plant tissues, with no contamination and a high percentage of survival rate. Reports are indicative of surface sterilization methods used in dye-yielding plants for the isolation of endophytic bacteria with 70% ethanol for 6–8 min and 0.1% mercuric chloride for 5–10 min showing promising results (Khanam and Chandra, 2017). It was found that surface sterilization by 2% sodium hypochlorite and 0.1% Tween 20 for 3 min followed by ethanol 70% for 1 min was well-suited for the isolation of endophytic microorganisms from Acalypha indica (Ramalashmi et al., 2018). The results for optimization of surface sterilization are shown in Figure 1.

Segments of surface-sterilized root, stem, and leaf of C. dichotoma incubated on nutrient agar plates showed growth of morphologically distinct bacterial colonies surrounding the plant segments after 48–96 h. To avoid the collection of repetitive strains, a total of 33 endophytic bacteria were isolated in pure form from 170 segments (i.e., 62 root, 54 stem, and 54 leaf segments) of C. dichotoma of which 16, 9, and 8 isolates were obtained from root, stem, and leaf segments, respectively (Table 1; Figure 2 in Supplementary Material). Colonization frequency calculated in percentage was highest in leaf samples (37.03) but gradually decreased in stem (35.18) and root (32.25) samples, while the isolation rate was lower in leaf (0.14) as compared to stem (0.16) and root (0.25) samples. The Shannon Wiener diversity index showed that roots (0.85) of C. dichotoma harbor diverse types of endophytic bacteria as compared with stem (0.61) and leaf (0.54) which are in line with the available reports for Chinese medicinal plants Berberis poiretii, Eucommia ulmoides, and Rhus potanini showing a high colonization frequency (47–63%) and isolation rates (0.7–0.9) of endophytic fungi (Sun et al., 2008).

The bacterial endophytes of C. dichotoma were characterized based on morphological (Table 3 in Supplementary Material), microscopical, and biochemical features (Table 2); evaluation of enzymatic activity; and carbohydrate utilization (Table 3). Cultural characteristics of all the isolated endophytic bacteria showed that out of 33 isolates, 18 pigmented and 15 non-pigmented organisms were identified. Microscopic characteristics of all endophytic bacteria showed that out of 33 isolates, 18 were Gram-positive (5 cocci and 13 rods) and 15 were Gram-negative rods. Filamentous forms were not observed in either root, stem or leaf samples. Twelve isolates out of 33 were motile, and 8 isolates showed endospore formation, and they were suspected as Bacillus species. Enzyme profile of endophytic bacterial isolates showed that all endophytic bacteria produced oxidase, and 87% of them produced catalase. The isolates also showed lipase (69%), amylase (78%), cellulase (87%), nitrate reductase (39%), methyl red (30%), and indole (39%) production. The isolated endophytes were also screened for their ability to utilize carbohydrates, i.e., dextrose, fructose, sucrose, maltose, and lactose, in phenol red agar medium supplemented with 1% sugar. Dextrose was found to be the best-utilized carbon source by most of the bacterial isolates; bacterial isolates were moderate in fermenting fructose and sucrose, while very few isolates were fermenting maltose (6) and lactose (7).

Based on the micromorphological and physio-biochemical analysis, the isolated endophytic bacteria were tentatively identified as species of Bacillus, Pseudomonas, Paenibacillus, Acidomonas, Streptococcus, Ralstonia, Micrococcus, Staphylococcus, and Alcalignes. Similar endophytic bacteria have been reported from medicinal plants like Azadirachta indica, Gynura procumbens, Tephrosia apolline, Phyllanthus emblica, and Vitis vinifera (Baldan et al., 2014; Khan et al., 2014). However, several authors have reported the presence of endophytic bacteria inside plants belonging to the genera Pantoea, Bacillus, Microbacterium, Paenibacillus, and Sphingomonas (Rijavec et al., 2007).

The growth of isolated endophytic bacteria at different sodium chloride concentrations (Table 4) indicates that all the 33 isolates showed good growth at 0–6% NaCl concentration. Only 22 isolates showed sustained growth at 12% NaCl concentration. Thus, it can be inferred that an increase in NaCl concentration causes a proportional decrease in growth rate.

The antibiotic susceptibility pattern of the selected endophytic bacteria was determined against ten different antibiotics by the disk diffusion method, which shows that endophytic bacteria from root, stem, and leaf of C. dichotoma were mostly resistant to rifampicin, amoxicillin, and bacitracin, while they were susceptible to erythromycin and streptomycin. Out of 33 isolates, 10 and 7 bacteria showed an intermediate response to neomycin and ampicillin, respectively. Out of 33 isolates, 3 isolates were sensitive to rifampicin, 5 isolates were sensitive to amoxicillin and bacitracin, 8 isolates were sensitive to ampicillin, 14 isolates were sensitive to tetracycline and neomycin, 20 isolates were sensitive to chloramphenicol, 22 isolates were sensitive to kanamycin, 23 isolates were sensitive to erythromycin, and 25 isolates were sensitive to streptomycin. Antibiotic sensitivity patterns of isolated endophytic bacteria with zone size in mm are shown in Table 5.

Antibacterial properties of all isolated endophytic bacteria were assessed against clinical strains of both Gram-negative and Gram-positive bacteria. The endophytic bacteria which inhibited the growth of any of the test organisms were considered to have antibacterial activity, and the zone of inhibition length was measured in mm (Table 6). Out of 33 endophytes screened, the majority showed antibacterial activity against B. subtilis followed by K. pneumonia. Bacterial strains, i.e., SMC204, SMC212, SMC101, and SMC301, exhibited antibacterial activity among all the six test organisms. The best activity was expressed by SMC212 against S. aureus, B. subtilis, E. coli, S. typhi, K. pneumonia, and Pseudomonas aeruginosa with a zone of inhibition having a diameter of 35, 30, 20, 15, 15, and 13 mm, respectively. Although several reports showed the antimicrobial potential of endophytic fungi from medicinal plants (Kuo et al., 2021), the antimicrobial evaluation of endophytic bacteria is rare (Cardoso et al., 2020). Li et al. (2008) investigated endophytic Actinomycetes associated with medicinal plants in the rainforest of Yunnan, China, and recognized that endophytic Streptomyces exhibit antimicrobial activities against S. aureus, E. coli, and Candida albicans.

The 16S rRNA gene sequencing was used to characterize the endophytic bacterium SMC212, and a phylogenetic tree was constructed using MEGA 11 software. Using MEGA 11, the maximum likelihood tree of SMC 212, constructed based on 16s rDNA gene sequences analysis, was based on the Tamura 3-parameter model with the lowest BIC and highest AIC values. All spots with gaps and missing data were removed. Pairwise deletion was used to close gaps, and the estimated transition/transversion bias (R) was 2.2. The evolutionary history was inferred by using the maximum likelihood method and the bootstrap consensus tree inferred from 1,000 to 3,000 iterations. The evolutionary relationship is represented as a dendrogram (Figure 2), which clearly shows that SMC212 is related to Bacillus thuringiensis. To the best of our knowledge, this is the first article to report the isolation of an endophytic bacterium from C. dichotoma that has been identified and showed similarity to B. thuringiensis. The sequences obtained in this study have been deposited in GenBank under accession number OM320575. Numerous previous studies investigated the diversity of bacterial endophytes in medicinal plants (Hamayun et al., 2021). Pseudomonas sp., Paenibacillus sp., and Bacillus megaterium were previously identified as Korean ginseng root endophytes in the bacterial population isolated from Plectranthus tenuiflorus (Cho et al., 2007). Paenibacillus has been discovered as an endophyte in various woody plants such as coffee, pine, and poplar (Bent and Chanway, 2002).

The results pertaining to GC-MS analysis (Figure 3) of the ethyl acetate crude extract of B. thuringiensis OM320575 were analyzed using GC-MS which led to the identification of 38 different compounds. Table 7 shows the retention time (RT), molecular formula, and molecular weight. The NIST program was used to deconvolute the GC-MS data, and the obtained mass spectra were matched to entries in the compound library. The chemical structures of these compounds are also depicted in Figure 4 in the Supplementary Material. The compounds that occupied major percentage in the extract are dibutyl phthalate (32.53%) and eicosane (13.59%) at various time intervals; tetrapentacontane (6.88%) at various time intervals; heneicosane (6.58%) at various time intervals; hexadecane (5.57%); tetradecane (5.08%); silane, trichlorooctadecyl (2.6%); and 2,4-di-tert-butylphenol (2.27%). The bacterium B. thuringiensis OM320575 produced bioactive chemicals with antimicrobial, antioxidant, and anticancer properties, according to GC-MS analyses.