Alfalfa (Medicago sativa) means “father of all foods” belongs to Leguminosae family. It is a perennial herbaceous leguminous flowering plant known as Queen of Forage plant, lives upto 8 years. Due to its high protein content and fiber, they are cultivated worldwide and widely used as fodder for cows1. The crop is autorotated before reseeding due to its toxins having an allelopathic effect for other plants growth. The plant also has deep root system, thereby improving soil and water holding ability of plants. Alfalfa fixes high nitrogen than other plants due to its symbiotic association with microbes.
Medicago sativa, a therapeutic value plant has been reported for a number of phytopharmacological activities such as neuroprotective, hypocholesterolemic, antioxidant, antiulcer, antimicrobial, hypolipidemic, estrogenic and in the treatment of atherosclerosis, heart disease, stroke, cancer, diabetes2 and menopausal symptoms in women3-10.
The plant extract has been reported to possess bioactive compounds namely saponins, flavonoids, phytoestrogens, coumarins, alkaloids, amino acids, phytosterols, vitamins, digestive enzymes and terpenes11-13.
Based on preliminary functional diversity studies carried (unpublished results) on the isolation of microbes from the rhizosphere of alfalfa plant. Out of 32 isolates, this isolate was selected for its amylase, cellulase, protease and phosphate solubilization activities. Thus the study was aimed at isolating the functionally diverse organism from rhizosphere soil region of Alfalfa plant (Medicago sativa) and identifying the bioactive compound responsible for antibacterial action.
MATERIALS AND METHODS
Sample collection: Soil sample was collected from the rhizosphere region of Alfalfa plant fields during June 2016 from Sulur, Coimbatore, Tamilnadu, India. Studies were carried out from June, 2016 to March, 2017 (Fig. 1).
Isolation and identification of micro-organism: One gram of the collected soil samples were used for serial dilution to isolate microbes by spread plate method. To the nutrient agar plates, 0.1 mL of serially diluted samples (101 to 107) were plated, incubated at 37°C for 24-72 h. After incubation, bacterial isolates were checked for purity and preserved in glycerol stocks and as nutrient agar slants for further tests.
These colonies were observed for Gram’s nature and morphological characters such as size, shape, color, texture, opacity, elevation, margin and mobility.
They were further identified using biochemical methods as stated in Bergey’s manual for characterization which includes Indole, Methyl Red, VogesPrauskaeur, citrate, urease and TSI slants etc.
Antibacterial activity: The antimicrobial activities of crude extracts of all isolated bacteria were tested against bacterial pathogens by agar well diffusion method. Muller-Hinton agar (MHA) plates were prepared and the wells were made with sterile cork borer on the agar plates. The overnight grown nutrient broth cultures of all bacterial pathogens were uniformly swabbed on to the surface of MHA plates using sterile cotton swabs. Each 50 μL of cell free supernatants were aseptically incorporated into the well and the plates were incubated in an upright position at 37°C for 24 h. After incubation, the plates were observed for zone of inhibition.
Production and extraction of the bioactive compounds: For obtaining the large biomass, the active strain were inoculated into 1 L of nutrient agar medium and incubated in shaker at 30°C at 160 rpm for 36 h. After incubation, the media contents were centrifuged at 10,000 rpm for 10 min to obtain the cell free supernatant.
The cell free supernatant extracted with organic solvent-ethyl acetate and extraction carried out with 3 volume of solvent for 2 h by using rotary shaker supernatant fractions were flash evaporated at 45°C temperature to ensure complete removal of solvent and the extracts were evaporated to dryness. The resulting residues were dissolved in small amount of respective solvents and stored at -20°C until further purified.
Molecular identification and phylogenetic analysis of the bioactive compound
Genomic DNA isolation: DNA isolation from bacterial isolate performed according to the cold spring harbour lab protocol. Briefly, the isolates were grown in Nutrient Broth (Himedia, India) for 24 h days at 37°C, pelleted and washed in 1 mL Tris-EDTA (TE) buffer. The pellets were resuspended in 500 μL TE buffer containing 1 mg mL1 lysozyme. After incubation at room temperature for 2 h, 75 μL of 10% Sodium Dodecyl Sulfate (SDS) and 125 μL of 5 M NaCl were added to this mixture. The samples were centrifuged (10,000 rpm for 10 min at room temperature) and incubated in ice cold ethanol (-70°C) for 3 min, later in a 65°C water bath for 3 min and on ice for 10 min. RNase (200 μg mL1 of sample) added to the supernatant to remove RNA contamination and the mixture incubated at 37°C for 15 min.
Proteinase K (50 μg mL1 of sample) added to content and the mixture was incubated at 37°C for 30 min. An equal volume of phenol/chloroform/isoamyl alcohol (25:24:1) with upper aqueous phase recovered after centrifugation (10,000 rpm for 10 min at room temperature). To this an equal volume of chloroform/isoamyl alcohol (24:1) added and upper aqueous phase transferred to a new microfuge tubes after centrifugation (10,000 rpm for 5 min at room temperature). The DNA precipitated by mixing the aqueous phase with 50 μL of 3 M sodium acetate, 300 μL of ice-cold isopropyl alcohol and incubated at -20°C for 20 min. The DNA pelleted and washed twice with 70% ethanol. The pellet blot dried and resuspended in 40 μL of TE buffer and stored at -20°C.
Amplification of 16S rRNA gene: 16S rRNA genes were amplified from the extracted genomic DNA using the 8 F and 1541 R universal eubacterial primers designed to target the conserved regions in the genomic DNA of the isolates and amplify approximately 1.4 kb length gene. The forward primer 5`-AGAGTTTGATCCTGGCTCAG-3` and reverse primer 5`-AAGGAGGTGATCCAGCCGCA-3` were used for amplification.
The PCR mix contained 5 μL of 25×PCR buffer, 4 μL of 25 mM MgCl2, 5 μL of 5 μM 518 Forward Primer and 5 μL of 5 μM 800 Revers Primer, 5 μL of 1 mM dNTP’s, 0.5 μL of Taq DNA polymerase (Thermo Scientific, India) and 2 μL of genomic DNA.
The reaction volume adjusted and made up to a final volume of 50 μL with sterile double-distilled water and amplified in an automated thermal cycler (Vapo protect Pro S, Eppendorf). The PCR conditions were an initial denaturation stage at 95°C for 2 min, followed by 35 cycles of denaturation at 94°C for 45 sec, annealing at 55°C for 60 sec, extension at 72°C for 60 sec and a final extension step at 72°C for 10 min. Negative controls with no DNA template were included in all PCR experiments.
16S rRNA gene sequencing: The Polymerase Chain Reaction (PCR) products were purified with a Montage PCR Clean up kit (Millipore) as per the manufacturer’s instructions. The purified PCR products were then sequenced. Sequencing was performed by using Big Dye terminator cycle sequencing kit (Applied BioSystems, USA). Sequencing products were resolved on an Applied Biosystems model 3730XL automated DNA sequencing system (Xcelris Laboratories, India).
Phylogenetic analysis: The 16s rRNA sequence was blast using NCBI blast similarity search tool. The phylogeny analysis of query sequence with the closely related sequence of blast results performed followed by multiple sequence alignment. The program MUSCLE 3.7 used for multiple alignments of sequences. The resulting aligned sequences were cured using the program Gblocks 0.91b. This Gblocks eliminates poorly aligned positions and divergent regions (removes alignment noise). Finally, the program PhyML 3.0 aLRT used for phylogeny analysis and HKY85 as Substitution model.
FTIR analysis: The purified bacterial extract was subjected to FTIR spectroscopic analysis (Perkin Elmer Lambda), equipped with KBr beam splitter with DTGS (Deuterated triglycine sulfate) detector. The technique work on the fact that bonds and groups of bonds vibrate at characteristic frequencies. A molecule that is exposed to infrared rays absorbs infrared energy at frequencies, which are characteristic to that molecule.
GC-MS analysis: The Thermo MS DSQ II used for the analysis packed DB 35-MS capillary standard non-polar column and the components were separated using Helium as carrier gas at a flow of 1 mL min1. The injector temperature set at 260°C during the chromatographic run. The volume of sample injected 1 μL at an oven temperature of 70°C (6 min). Interpretations on mass spectrums of GC-MS were done using the database of National Institute Standard and Technology (NIST) having more than 62,000 patterns. The mass spectra of the unknown components will be compared with the spectrum of the known components stored in the NIST library. The name, molecular weight and structure of the components of the test materials will be ascertained.
RESULTS AND DISCUSSION
Antibacterial activity: The rhizosphere region of soil carries a heterogeneous group of microbial population which can play a vital role in soil function. The antibacterial activity of the crude extract of the isolate studied against 7 clinical pathogens (P. aeruginosa, Klebsiella sp, S. aureus, Proteus vulgaricus, S. pneumonia, E. coli and B. cereus ). The isolate showed significant zone of inhibition against Klebsiella sp. and S. aureus (18 and 17 mm) (Fig. 2).
Similar reports by researchers showed that the microbes isolated form rhizosphere region possessed significant anti-microbial activity. Ramakrishnan et al.14 reported the wide range of antibacterial activity of Streptomycetes sp. isolated from the rhizosphere soil of medicinal plants at Kolli hills of Tamil Nadu. Ryandini et al.15 isolated Streptomyces sp. from mangrove rhizosphere mud of rhizophora mucronata from east Segara Anakan mud and reported significant activity on multiple drug resistant bacteria.
Also Rajalakshmi and Mahesh16 reported antimicrobial activity of Aspergillus terrus isolated from rhizosphere region of medicinal plants in and around Kuttralam, Tirunelveli. Upon GC-MS analysis, ten compounds were identified and tetracontane was reported to be bioactive potential compound.
Molecular characterization of the isolates: The genomic DNA of the isolate isolated and subjected to 16S rRNA gene amplification for the species identification. PCR product of the length 1,400 bp purified and sequenced in Yaazhxenomics lab, Coimbatore. The 16S rRNA sequences of the isolate subjected to BLAST analysis using mega blast tool of GenBank (http://www.ncbi.nlm.n ih.gov/). Among different species comprising of closet neighbouring strains in NCBI-BLAST analysis used in the phylogenetic analysis. The phylogenetic trees were constructed based on the neighbour joining method and percentage differences in the genetic relationships between the neighbouring strains of the two samples were analyzed.
Results revealed that the 16S rRNA partial gene sequence of the isolate showed 97% similarity with B. horikoshii (Fig. 3, 4). The 16S rRNA gene sequence submitted to the Gene bank (NCBI, USA) and Genebank ID accession number MK226527 received.
FTIR analysis: Figure 5 depicted the FTIR analysis of the B. horikoshii extract showing strong peaks at 3417, 1643.35, 1097.5, 1658.78, 2926 and 715 cm1, respectively. Major group was found to be N-H stretching at 3417 cm1 (Table 1). The FTIR results elucidated an array of functional groups at a frequency ranges indicating the presence of functional groups corresponding to aromatic alkenes, aliphatic amines, compounds with aromatic rings, alkynes, amides, alcohols and phenols.
|Table 1:||FTIR analysis of B. horikoshii extract
||Antibacterial activity of AL5 against Klebsiella and S. aureus
||GCMS analysis of compounds obtained from B. horikoshii extract |
||Multiple alignment scores of Bacillus horikoshii
||Phylogenetic tree of B. horikoshii based on the 16S rRNA gene sequencing
The presence of such functional groups could be attributed to the bioactive nature of the partial fraction of B. horikoshii cell free supernatant.
GC-MS analysis: Figure 6 depicted the GC-MS analysis of B. horikoshii extract revealing presence of 31 peaks and 6 compounds were characterized and identified by comparison of the mass spectra of the constituents with the NIST library (Table 2, 3). The retention times (RT) are represented in minutes.
Stearyl alcohol, 11-octadecenal had highest intensity of 11.55% at retention time of 36.19 min. 11-Octadecenal has been reported to be present in essential oils from Launaea resedifolia L., possess antibacterial activity in range17 of 11-37 mm. Similarly the presence of 9-octadecenal observed in marine red alga Laurencia brandenii showed various biological activities18.
||FTIR spectrum of B. horikoshii extract
||GCMS spectrum analysis of B. horikoshii extract
|Table 3:||Activity of compounds identified in B. horikoshii extract
The next major constituent for B. horikoshii extract was found to be 4-Cyano-2H-1-benzothiopyran at retention time 10.4 min and intensity of 8.12%. 4-Cyano-2H-1-benzothiopyran, a bicyclic benzene reported to have antibacterial action, antimalarial and anti-tumor activities especially against colon cancer19,20. Fatty alcohol hexadecane with retention time of 15.49 min. The alkane hydrocarbon octadecane was found at the retention time of 19.59 min.
Similarly Bacillus strain isolated from the groundnut rhizosphere soil reported by Bharose and Gajera et al.21 had biocontrol activity against aflatoxin producing Aspergillus strain. GCMS analysis of Bacillus subtilis revealed presence of 55 compounds, out of which 2-hydroxy-4-phenyl-6-phenethyl pyrimidine (10%) identified as bioactive compound responsible for anti-fungal action.
Saranya Devi and Mohan22 studied the rhizosphere region of Casuarina equisetifolia and identified that Bacillus pumilus showed high inhibition against Fusarium oxysporum. The ethyl acetate extract of Bacillus pumilus on GC-MS analysis revealed presence of 19 compounds and Cis-3-chloro all alcohol and 9-octadecenamide as predominant bioactive compounds responsible for salt tolerance and antifungal activity.
Through this study we were able to isolate new antimicrobials against potent pathogens. Based on molecular identification the active isolate of rhizosphere region of Alfaalfa plant was identified as Bacillus horikoshii. The studies on the rhizosphere region isolate Bacillus horikoshii extract lead to identification of 11 volatile compounds and study on the antimicrobial activity showed that action to be due to presence of 11-octadecenal. Thus further in vitro and in vivo biological studies are required for anticancer medical applications in various fields.