Isolation and characterization of bacterial endophytes from the roots of Cassia tora L

Abstract

In the present investigation, five endophytic bacterial strains isolated from the roots of Cassia tora L. were identified as Bacillus subtilis, Agrobacterium tumefaciens, Bacillus sp., Pseudomonas putida, and Pseudomonas sp. based on biochemical characteristics as well as 16S rRNA gene sequencing. Isolates were screened for plant growth promoting traits, antibacterial activity, antifungal activity, antibiotic sensitivity, and salinity tolerance. The majority of the endophytic strains produced phytohormone indole acetic acid (IAA), ammonia, and also solubilized phosphate. Siderophore and HCN production were observed in A. tumefaciens, P. putida, and Pseudomonas sp. The antibiotic sensitivity profile indicated that the isolates were resistant to chloramphenicol, while highly susceptible to neomycin and streptomycin. Bacterial endophytes gave a definite stamp on their antibacterial activity against Escherichia coli and Klebsiella pneumoniae and antifungal activity against Pythium ultimum, Aureobasidium pullulans, and Alternaria alternata. Pseudomonas sp. showed maximum salt tolerance (6 % NaCl) contrary to A. tumefaciens that was least tolerant.

Introduction

Plants constitute a diverse niche for microorganisms as the host for pathogens, symbionts, epiphytes, and endophytes. Endophytes are microorganisms that colonize the host tissues and establish a relationship where both partners get a benefit from their interactions (Reiter and Sessitsch 2006). Some bacteria and fungi enter the plants as endophytes and do not harm them by establishing symbiotic, mutualistic, commensalistic, and trophobiotic relationships (Nair and Padmavathy 2014). The populations of endophytes are generally less than rhizospheric bacteria because roots favor growth of various microbial communities. Bacterial endophytes play an important role in plants' metabolic activities. They trigger various biochemical pathways in the host plant by producing physiologically important chemical compounds. They produce a diverse range of secondary metabolites with vital medicinal values, and can be used in medicine, agriculture, and industry (Strobel and Daisy 2003; Ryan et al. 2008; Christina et al. 2013). Moreover, they actively participate in plant protection against environmental stresses and make them more adaptable (Nagarajkumar et al. 2004; Rosenblueth and Martinez-Romero 2006). Bacterial endophytes interact with plants through the production of phytohormones, siderophores, ammonia, HCN, phosphate solubilization (Compant et al. 2005; Rajkumar et al. 2006), and through lytic enzymes production (Nagarajkumar et al. 2004). They have a superior role in plant growth promotion compared to rhizobacteria (Conrath et al. 2006; Ryan et al. 2008), and colonize the ecological niche similar to that of phytopathogens, which makes them suitable as biocontrol agents (Berg et al. 2005). Furthermore, siderophores excreted by them find extensive applications in agriculture. Bacterial endophytes have been exploited using biotechnological approaches for the production of antibiotics, immunosuppressants, anticancer compounds, and other potent secondary metabolites (Strobel and Daisy 2003) as the case of Bacillus subtilis, Bacillus sp., Bacillus cereus, and Lysinibacillus sp. isolated from Miquelia dentata, which produce Camptothecine (CPT) in vitro, a potent anticancer drug (Shweta et al. 2013). Among the several endophytic actinomycetes isolated from Trewia nudilora, Streptomyces sp. 5B and Streptomyces sp. M27m3 produce ansamycins (Arachevaleta et al. 1989). Beside this, they are broadly involved in the bioremediation of pollutants. The efficiency of biodegradation depends upon the activity of bacterial genes required for enzymatic breakdown of contaminants (Germaine et al. 2006; Kaimi et al. 2007; Yousaf et al. 2011).

Medicinal plants provide valuable therapeutic agents in traditional medicines used at the global level for human health. Cassia tora L. (family Caesalpiniaceae) is the small, annual, medicinal herb, distributed throughout tropical and subtropical zones of the world as a weed. Its medicinal properties have been described in the ancient Ayurvedic literature. This plant has great therapeutic potential because of the wide range of medicinally vital bioactive compounds, including anthraquinones, chrysophanol, emodin, chalcone, rhein, euphol, basseol, etc. These compounds are antiprolifrative, hypolipidemic immunostimulatory, anticancers, antimutagenic and hepatoprotective properties and thus C. tora is used as the traditional to cure for various diseases such as dermatitis, diabetes, cough, cold and fever, etc. (Jain and Patil 2010; Meena et al. 2010).

In this study, we tried to explore the endophytic bacterial communities in the roots of C. tora L. and their role in plant growth promoting activities along with antibiotic sensitivity, antibacterial and antifungal activities as well as the capability of inducing salt tolerance.

Materials and methods

Collection of plant material

Cassia tora L. plant samples were collected from the Banaras Hindu University campus, India (20° 18^’ N and 80° 36^’ E, elevation 80.71 m), in triplicate. Plants were uprooted and roots thoroughly washed in running tap water to remove the adhering soil, then dipped in surface disinfectant (3 % H₂O₂, 3 min) and vigorously shaken. This was followed by washing them with sterile distilled water, and finally they were dipped in 70 % ethanol (3-5 min). Further, roots were treated with 1 % hypochlorite (2 min) and rinsed with sterile Milli-Q water. The freshly sterilized roots were sliced into small pieces and transferred to nutrient agar plates and incubated (25 °C) for 3-4 days.

Characterization of bacterial isolates

Endophytic bacterial isolates were characterized on the basis of colony morphology and biochemical and molecular analysis. The morphological and biochemical characteristics of the isolates were examined according to the Bergey’s Manual of Determinative Bacteriology (Holt et al. 1994).

16S rRNA gene amplification and sequencing

Genomic DNA was isolated using the GeneiPure^TM bacterial DNA purification kit (GeNei^TM, Bangaluru, India) following the manufacturer’s protocol. Universal eubacterial primers F-D1 5′-CCGAATTCGTCGACAACAGAGTTTGATCCTGGCTCAG-3′ and R-D1 5′-CCCGGGATCCAAGCTTAAGGAGGTGATCCAGCC-3′ (Kumar et al. 2006) were used to amplify the 1500 bp region of the 16S rRNA gene using a thermal cycler (BioRad, USA). Amplification products were resolved on agarose-gel (1.5 %), and visualized using a gel documentation system (Alfa Imager, Alfa Innotech Corporation, USA). The amplicons were purified using a GeneiPure^TM quick PCR purification kit (GeNei^TM, Bangaluru, India) and quantified at 260 nm using calf thymus DNA as a control. The purified partial 16S rDNA amplicon was sequenced in an Applied Biosystems 3130 Genetic Analyzer (Applied Biosystems®, USA).

Analysis of 16S rDNA sequences

The partial sequences were compared with sequences available from DNA databases, and sequences showing >99 % similarity were retrieved by the Nucleotide Basic Local Alignment Search Tool (BLAST N) program available at the National Center for Biotechnology Information (NCBI) BLAST server (www.ncbi.nlm.nih.gov/BLAST). The sequences were aligned using the multiple sequences alignment tool, Clustal X 2.1 version. The phylogenetic and molecular evolutionary analysis was conducted using MEGA 5.1 (Tamura et al. 2011).

PGP traits analysis

Production of IAA

Bacteria were cultured (25 ± 2 °C) for 48 h in the nutrient media supplemented with 100 and 200 μg/ml of L-Tryptophan and then centrifuged (8000 rpm, 10 min). Supernatant (2 ml) was mixed with two drops of orthophosphoric acid and 4 ml of the Salkowski reagent (50 ml, 35 % of perchloric acid, 1 ml 0.5 M, FeCl₃ solution) and production of IAA was confirmed by the development of pink color (Brick et al. 1991).

Phosphate solubilization

The bacterial isolates were inoculated at three to four places on the Pikovskaya media containing tricalcium phosphate in a plate and incubated (28 ± 2 °C) for 2-3 days (Pikovskaya 1948). The development of a clear halo zone around bacterial isolates indicated positive phosphate solubilization activity.

Ammonia production

Freshly grown test bacterial isolates were inoculated in 10 ml peptone water in the tube and incubated (28 ± 2 °C) for 48 h. Nessler^,s reagent (0.5 ml) was added in each tube and the development of a brown to yellow color indicated ammonia production (Cappuccino and Sherman 1992).

Siderophore production

The cultured bacterial isolates were spotted on a Chrome azurol S agar plate (Schwyn and Neilands 1987). The development of a yellow orange halo zone around the bacterial spot indicated siderophore production.

HCN production

Bacterial isolates were streaked on petri plates of solidified King’s B medium (1954) and a single disc of filter paper was placed in the lid of each petri plate. The plates were then sealed with parafilm® and incubated (25 ± 2 °C) for 72 h. The color change in the filter paper from deep yellow to dark brown was visually assessed for production of HCN (Bakker and Schippers 1987).

Antibiotic sensitivity test

An antibiotic sensitivity test was performed using antibiotic impregnated discs (6 mm dia.) containing streptomycin, tetracycline, chloramphenicol, amoxicillin, and neomycin using the Kirby Bauer disc-diffusion assay (Bauer et al. 1996) at the antibiotic dose of 30 μg/disc. The diameter of the inhibition zone was recorded and organisms were categorized as resistant, intermediate, and sensitive following the DIFCO Manual 10th edition (1984).

Antibacterial activity

All bacterial endophytes were screened for their antibacterial properties following the cross-streak assay method (Williston et al. 1947) against Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus. Nutrient agar was inoculated with bacterial endophytes as a single streak at the centre of the petri plate and incubated for seven days (30 °C). The overnight grown cultures of the test organisms were streaked at a right angle to the endophyte and observed for growth/inhibition after 24–48 h of incubation (30 °C). The size of the inhibition zone was measured to the nearest mm.

Antifungal activity

Endophytic bacteria were tested against three mould strains for their fungistatic activity using a 24 h culture of separate strains grown in nutrient broth at 28 °C. Fungal test cultures were prepared on PDA medium plates. They were inoculated with three separate 15 drops of each bacterial culture spotted in rows on the agar. The plates were incubated at room temperature for seven days and the fungal growth inhibition scored (Owen and Hundley 2004).

Salt tolerance

To assay the salt tolerance of endophytic bacterial isolates, 20 μl aliquots of the 24 h old test culture were inoculated with 1 % protease peptone and a sequential series of 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, and 10 % NaCl. Cultures were incubated at 28 °C, and their growth measured after 24-48 h by an absorbance at 600 nm.

Results

Physio-biochemical characterization

A total of 16 endophytic bacterial isolates were isolated from the internal tissues of Cassia tora L. roots. No bacterial growth occurred at the rhizoplane of the sterilized roots. Six out of 16 isolates were gram-positive. All the bacterial isolates were rod shaped and positive for catalase, oxidase, and starch hydrolysis activity, except three, which were later identified as strain CT1 (Bacillus subtilis). All the isolated strains were able to ferment glucose whereas three isolates (CT3) fermented lactose, mannitol, and D-mannose. Sucrose was fermented by three isolates (CT3, CT4 and CT5) and maltose by seven (CT2 and CT3), while nine isolates (CT2, CT3 and CT5) reduced nitrate and six produced H₂S (CT2 and CT3) (Table 1).

Table. 1 Biochemical characterization of selected bacterial isolates of Cassia tora L

Phylogenetic analysis

The nucleotide sequences derived from 16S r RNA have been submitted to GenBank. On the basis of sequence analysis, bacterial isolates were assigned to five species, namely, Bacillus subtilis CT1 (KJ011524), Agrobacterium tumefaciens CT2 (KJ011525), Bacillus sp. CT3 (KJ011526), Pseudomonas putida CT4 (KJ011527), and Pseudomonas sp. CT5 (KJ011528) (Table 2). A phylogenetic tree was constructed by the neighbor-joining method using a distance matrix from an alignment tool (Fig. 1).

Table 2 Closest relative of the isolated strains as revealed by 16S rRNA gene sequencing

Fig. 1

Phylogenetic tree constructed from 16S rRNA gene sequences of the entire bacterial strains (CT1-CT5) using th neighbor-joining approach. Each number on a branch indicates the bootstrap confidence values corresponding to the scale bar of branch lengths. GenBank accession numbers of nucleotide sequences are shown along with the name of the bacterial species. Phylogenetic analyses were conducted in MEGA 5.1

PGP traits analysis

All the bacterial isolates produced IAA, with a maximum for bacterial strain CT2 and a minimum for CT5. In case of siderophore and HCN production, three strains, CT2, CT4, and CT5, were positive for the test. All the bacterial strains solubilized tricalcium phosphate on Pikoskaya nutrient agar petri plates, and were positive for ammonia production (Table 3).

Table 3 Characterization of selected bacterial isolates for plant growth promoting rhizobacteria (PGPR traits)

Antibiotic sensitivity test

The antibiotic sensitivity pattern of the endophytic bacterial isolates was determined against five different antibiotics by the disc diffusion method. The results showed that bacterial endophytes from root tissues of Cassia tora L. were mostly resistant to chloramphenicol (Table 4), while being sensitive to neomycin followed by streptomycin and amoxycillin. Strain CT5 showed high resistance to three of five antibiotics whereas strains CT2 and CT1 were the most sensitive ones.

Table 4 Antibiotic sensitivity of endophytic isolates

Antibacterial activity

Antibacterial properties of all the bacterial endophytes were assessed against Escherichia coli, Staphylococcus aureus, and Klebsiella pneumoniae. All endophytes were active against E. coli and K. pneumoniae as determined by the inhibition zones (Table 5), with strains CT1 and CT3 being the most active. None of the isolates were active against S. aureus.

Table 5 Antibacterial activity of endophytic isolates

Antifungal activity

To assess the fungistatic activity, five fungal species, namely, Exophiala mesophila, Alternaria alternata, Byssochlamys fulva, Aureobasidium pullulans, and Pythium ultimum were used. Fungistatic activity was determined as the zones of growth inhibition area on the agar plate containing applied bacteria . All the isolates proved to be antifungal, while strain CT1 was active against all the tested moulds; the other isolates were selective by activity against one or more species (Table 6).

Table 6 Fungistatic behaviour of endophytic isolates

Salt stress

Salinity tolerance was observed among the bacterial isolates as evident from their growth at high NaCl concentrations. All the isolates were tolerant with CT2 being the most efficient and CT5 as the least tolerant, respectively. The bacterial strains CT4 and CT5 showed enhanced growth up to 5 % NaCl, while a decrease was observed in 6 % NaCl. On the other hand, growth was almost negligible in 6-10 % of NaCl for all the test strains (Table 1).

Discussion

The spatial distribution of endophytic genera normally depends on seasonal variation, rainfall, soil parameters, location of plants, plant age, and the genotypes (Lamit et al. 2014). In this study, we tested only one genotype of Cassia tora L. from three locations, which did not represent the real description of culturable endophytic diversity. On the basis of morphological and biochemical characteristics, endophytic bacteria from C. tora L. roots could be grouped into three phyla belonging to three different genera, namely, Bacillus (Firmicutes), Agrobacterium (Proteobacteria), and Pseudomonas (Gamma proteobacteria). The 16S rRNA gene sequences of the isolates assigned the endophytic bacterial population into three clads and five species, namely, Bacillus subtilis CT1, Agrobacterium tumefaciens CT2, Bacillus sp. CT3, Pseudomonas putida CT4, and Pseudomonas sp. CT5. These species have already been reported as endophytes of many plants (Goryluk et al. 2009; Khan and Doty 2009; Ma et al. 2009; Liu et al. 2010; Rashid et al. 2012).

Endophytes like Bacillus subtilis, Bacillus sp., and Pseudomonas putida are known for their ability to produce indole acetic acid (IAA) (Khan and Doty 2009; He et al. 2010). Indeed, in our findings, all the strains including Pseudomonas sp. CT5 and Agrobacterium tumefaciens CT2 have also synthesized IAA in considerable amounts in the presence of tryptophan, and this can be helpful for the plants in terms of growth (Rajkumar et al. 2006). A. tumefaciens CT2, P. putida CT4, and Pseudomonas sp. CT5 strains produce siderophores, and may benefit the iron-deficient plants by chelating Fe⁺³ from complex matter and making it available to plants for growth promotion (Khan and Doty 2009). He et al. (2010) reported that phosphate solubilizing bacteria (PSB) are frequent in the rhizosphere, and they could be used as microbial inoculants in agriculture and forestry. All the endophytic strains isolated presently behaved as PSB by solubilizing phosphate and, thus, representing an aid to utilization of the phosphate reserves, fixed soil phosphorus, and the phosphates applied to enhance the crop yields (Khan et al. 2006). Phosphate solubilization has already been reported in Bacillus sp. and Pseudomonas putida (Rajkumar et al. 2006). All the isolated strains produced ammonia (NH₃) as the secondary metabolite, and can be taken as the important trait of plant growth promoting rhizobacteria (PGPR) able to influence plant growth indirectly (Wani et al. 2007). On exposure to complex nitrogen sources, endophytic bacteria accumulate, synthesize, and supply nitrogen to the host plant and promote root and shoot elongation and, thus, increases plant biomass (Marques et al. 2010). Bacterial strains CT2 (A. tumefaciens), CT4 (P. putida), and CT5 (Pseudomonas sp.) also produced hydrogen cyanide (HCN), the desired quality of PGPR. By synthesizing HCN, some rhizobacteria inhibit plant disease development and strengthen the host’s disease resistance mechanism (Schippers et al. 1990; Whipps 2001).

Many endophytic bacteria exhibit antibiotic characteristics that inhibit the growth of an antagonistic bacterium. Amongst all the bacterial isolates, Bacillus subtilis CT1, Agrobacterium tumefaciens CT2, and Bacillus sp. CT3 were susceptible while Pseudomonas sp. CT5 was resistant to all the antibiotics used except tetracycline (Table 4). The bacterial isolate CT5 of Cassia tora L. was resistant to chloramphenicol and amoxycillin in contrast to the Pseudomonas sp. from Andrographis paniculata, which was resistant to only amoxycillin (Arunachalam and Gayathri 2010). Such contrasting results indicate that the behavior of bacterial endophytes may vary from plant to plant and from species to species depending on the environmental conditions (Nair and Padmavathy 2014). The antibiotic properties of endophytic bacteria increase the host plant resistance to pathogens and promote their growth (Bhore et al. 2010).

Natural products from the endophytic microbes inhibit or kill a wide range of harmful disease-causing bacteria, fungi, and protozoans that affect plants, humans, and animals (Demain 1981). Pseudomonas produced a variety of antibiotics such as 2, 4-diacetylphloroglucinol (DAPG), pyrrolnitrin, and HCN (Whipps 2001), whereas Bacillus sp. produced many kinds of antibiotics including bacitracin. The present investigation revealed that all the isolated strains had antibacterial properties (Table 5), which can be helpful in controlling the growth of pathogenic species and improve colonization resistance (Sullivan et al. 2001).

All the test organisms chosen for antifungal activity were virulent pathogens and caused severe infection in living organisms. It is reported that Exophiala mesophila is pathogenic to human beings; Aureobasidium pullulans and Byssochlamys fulva are responsible for fruit rot and Alternaria alternata produced mycotoxins (Fassiatova 1983; Porteous et al. 2003). Pythium ultimum causes the serious damping-off disease in plants. Bacillus and Pseudomonas protect plants against the damping-off disease and increase the seedling emergence rate after inoculation of soybean seeds (Leon et al. 2009). Our finding showed that all the bacterial isolates were antifungal against the abovementioned pathogens (Table 6). They, thus, may be helpful to the host to resist the virulent fungi. Antagonistic interactions between bacteria and fungi are of particular relevance for soil functioning and also the ecosystem-level processes (Jousset et al. 2014).

Endophytic bacteria are proficient in triggering the physiological changes in plants to promote growth and development against environmental stresses (Conrath et al. 2006). Previous reports suggested that even at the high salt concentration or at high ionic strength, endophytes successively proliferate inside the host (Bal et al. 2013). Salt tolerance has been observed in all the endophytic isolates of Cassia tora L. (Table 1) as they could grow in media containing 2 % NaCl. An increase in the NaCl concentration to 3 % was growth inhibitory for all the strains with the strain CT2 (A. tumefaciens) being most sensitive. Further increment in the salt concentrations up to 5 % was lethal to strain CT3 (Bacillus sp.) with some populations of strain CT4 (P. putida) and CT5 (Pseudomonas sp.) surviving in up to 6 % NaCl in contrast to salt tolerance in Pseudomonas sp. (4 %) and Bacillus sp. (2 %) as reported by Rashid et al. (2012). The trend indicates that these endophytes may reside and multiply in plants growing at high salt limits or high ionic strength and may possibly provide salt tolerance to the host.

Conclusion

Bacterial endophytes from the roots of Cassia tora L. are novel and diverse. They display PGP traits of variable degrees to fulfill the requirements of the host through synthesis of phytohormone indole acetic acid (IAA), ammonia, siderophores, HCN, and by solubilization of phosphate. These endophytes can be used as a bioresource for the production of the above bioactive compounds. All the strains were resistant to selected antibiotics, and possess antimicrobial potential, thus, they can also be used as the potent antimicrobial agents against pathogenic or opportunistic microorganisms. In addition, their high salt tolerance may help plants become more adapted to salt stress.