In-vitro Screening of B. cepacia; C. freundii and S. marcescens for Antagonistic Efficacy

1Department of Molecular & Cellular Engineering, Sam Higginbottom University of Agriculture, Technology and Sciences, Allahabad 211007, U.P. India, 2 Directorates of Research, Sam Higginbottom University of Agriculture, Technology and Sciences, Allahabad 211007, U.P. India, 3Centre for Transgenic Studies, Sam Higginbottom University of Agriculture, Technology and Sciences, Allahabad 211007, U.P. India.

Quality and quantity of food are going to be important challenges to overcome in coming future.Exceeding population growth rate requires production of more agricultural products and to inevitably move towards increased production per unit area.Since fertilizer management is considered as one of the main factors of sustainable agriculture, gradual replacement of chemical fertilizers with biological fertilizers is quite inevitable since biofertilizers are environment friendly and costeffective.Elucidating non-chemical control methods to reduce postharvest decay is becoming increasingly important (Conway et al. 2004).Naturally occurring antagonists on host surfaces are a promising component of biological crop protection (De Costa and Erabadupitiya 2005).The rhizosphere, the narrow zone of soil that surrounds and influences the plant roots, is home to a large number of microorganisms and is considered to be one that can have profound effects on the growth, nutrition and health of plants in agro-ecosystems (Berendsen et al. 2012).Bacteria able to colonize plant root systems and promote plant growth are referred to as plant growth promoting rhizobacteria (PGPR) (Kloepper and Schroth 1978).PGPR can affect plant growth by a wide range of mechanisms such as solubilization of inorganic phosphate, production of phytohormones, siderophore and organic acids, lowering of plant ethylene levels, nitrogen fixation and biocontrol of plant diseases (Rani et al. 2012).Along with the promotion of plant growth, PGPR also help in sustainable agricultural development, thereby protecting the environment and thus help in maintaining the ecological balance.Therefore, the use of such beneficial bacteria as biofertilizers and biocontrol agents has attracted increased interest worldwide in attempts to achieve sustainability in agriculture (Datta et al. 2011).
The Biocontrol bacterial species generally employ an array of mechanisms such as antibiosis, competition, production of hydrocyanic acid (HCN), siderophore and antifungal compounds to antagonize pathogens (Singh et al. 2010).Various rhizobacteria, including for example Burkholderia cepacia, Staphylococcus epidermidis, and strains of the Bacillus subtilis group, stimulate plant growth by the emission of volatile organic compounds such as hydrogen cyanide and ammonia (Bitas et al. 2013).Voisard et al. (1989) reported that HCN production by the rhizobacteria plays a significant role in the biological control of pathogens by acting as an inducer of plant resistance.It was also reported that HCN indirectly influences plant growth promotion (Kumar et al. 2012).Afsharmanesh et al. (2010) suggested that fungal growth is mainly inhibited by HCN production and siderophore production.This in turn can indirectly enhance the plant growth by keeping plant healthy and disease free (Glick and Pasternak 2003).Additionally, secretion of siderophore and production of chitinases, glucanases, cellulases, lipases and other lytic enzymes by PGPR can affect plant growth as these are also found to suppress the growth of fungal pathogens (Kloepper et al. 1980).Siderophores can be defined as small peptidic molecules containing side chains and functional groups that can provide a high-affinity set of ligands to coordinate ferric ions (Crosa and Walsh 2002).Siderophores are secreted to solubilize iron from their surrounding environments, forming a complex ferric-siderophore that can move by diffusion and be returned to the cell surface (Andrews et al. 2003).The potent siderophore, pyoverdin, for example, can inhibit the growth of bacteria and fungi that present less potent siderophores in iron-depleted media in vitro (Kloepper et al. 1980a).A pseudobactin siderophore produced by P. putida B10 strain was also able to suppress Fusarium oxysporum in soil deficient in iron; this suppression was lost when the soil was replenished with iron, a condition that represses the production of iron chelators by microorganisms (Kloepper et al. 1980b).Burkholderia cepacia has shown antagonistic activity against a broad range of plant pathogens (Cartwright and Benson 1995).Several studies suggested that pyrrolnitrin production by B. cepacia as well as other Pseudomonas spp, is closely associated with biocontrol of plant diseases (Hwang et al. 2002).Serratia species, usually found in diverse natural environments, have been found to reduce disease severity of various foliar and soilborne diseases.Among Serratia species, S. marcescens was reported as an important bacterium that has the ability to induce systemic resistance to various pathogens and to exhibit antifungal activity against several soil-borne fungi like Rizoctonia solani and Phytophthora infestans that causes stem canker, black scurf in potato and late blight or potato blight disease respectively.
The disease protection measures of medicinal plants are still restricted to the application of various chemical fungicides which strictly do not fit with the basic theory of usefulness of herbal drugs.Also, the residual effects of different chemicals eventually contaminate the purity of such plant drugs and are also of serious concern from environmental point of view (Sharma et al. 2004).Many researchers have reported that effective colonization by PGPR contributed to the successful suppression of plant pathogens and thus these can be considered as potential use of biocontrol agents as replacements for agrochemicals (Guo et al. 2007).In recent years, certain plant associated rhizobacteria belonging to various genera like Pseudomonas, Burkholderia and Bacillus have drawn attention because of their potential to suppress the soil borne pathogens causing diseases in different crops (Cazorla et al. 2007).Due to excessive use of various agrochemicals including various pesticides of different chemical families in crop production, these beneficial microorganisms and their physiological activities important to soil fertility are adversely affected (Srinivas et al. 2008).Bacteria strains isolated from soils that have been contaminated with various biochemicals, including pesticides, are increasingly being used for bioremediation (reclaiming the soil by inoculating it with organisms able to degrade certain compounds which are detrimental to other life forms).In other words, the bacteria are using specific pesticides to meet their energy needs, i.e., using them as food (Hernández et al. 2008).To overcome the deleterious effects of pesticides on plants and of soil fumigants on soil fertility, the treatment of seeds with PGPR could be used as a bio-inoculant which displays a wide range of tolerance to pesticides and exhibit PGP activities under pesticide-stress (Ahemad and Khan 2010).

METHODOLOGY Isolation of antagonistic rhizobacterial strains
When Indian mustard plant was found, a 6"X 2.5"X 5" soil sample was collected, the soil was serially diluted and appropriate dilutions (10 -3 , 10 -5 , 10 -7 ) were spread plated on nutrient agar medium.The plates were incubated at 38°C and developed colonies were identified on the basis of cultural, morphological characteristics as described in the Bergey's Manual of Determinative Bacteriology (Holt et al. 1994).Selective colonies were taken and streaked on selective media for individual strains and kept in incubator at 38°C for 24 h to obtain colonies.

Identification of specific bacterial species
The bacterial colonies on incubated plates were identified on the basis of cultural, morphological and biochemical characteristics as described in the Bergey's Manual of Determinative Bacteriology (Holt et al. 1994).

Cultural and Morphological Characteristics
The isolates were identified on the basis of different colony characteristics like diameter, consistency, colour, texture, elevation, margin etc.The organism was subjected to Gram's staining (Holt et al. 1994) for observing the morphological characteristics such as the shape and arrangement (clusters or chains) of cells, and gram-reaction.

Screening of Rhizobacteria for plant growth promoting traits Siderophore production
For siderophore production, 1µl of overnight raised rhizobacterial culture in LB broth were spotted on Chrome Azurol S (CAS) agar plates and incubated at 37°C for 48 h.Plates were observed for the appearance of orange halo around the bacterial colony (Ali et al. 2013).

HCN Production
For qualitative estimation of HCN, isolate was streaked on nutrient agar plate supplemented with 4% glycine.A whatman filter paper soaked in a solution of 2% Na 2 CO 3 and 0.5% picric acid was placed between base and lid of petriplate and incubated at 37°C in inverted position for 48 h and observed for color change from yellow to orange brown (Ali et al. 2013).

Chitinase test
For the Chitinase test spot inoculation of isolated strain was made on chitin agar plate amended with 2% phenol red and incubated for 48 h at 37°C.Presence of clear zone around the culture indicates the chitinase activity (Holt et al. 1994).

Protease production
The bacterial species were spot inoculated on nutrient agar containing casein and gelatin.The zone of hydrolysis was observed for the protease production (Pandey et al. 2013).

Lipase test
Lipolytic activity was carried out on trimethoprim plate and observation of halogen was considered as positive for lipase production.

Antibiotic sensitivity test
Isolate bacterial strains were tested for its resistance against standard antibiotics namely gentamicin (30 µg), ampicillin (10 µg), erythomycin (10 µg), kanamycin (5 µg), tetracyclin (10 µg), vancomycin (25 µg), and chloramphenicol (10 µg) by the antibiotic sensitivity assay.Briefly, the bacterial cultures were swabbed onto NA media plates.The standard antibiotic disc (6 mm) was placed over the media surface and the plates were incubated at 37 °C for 24 h.The experiment was done in triplicate.The results were interpreted on the basis of the diameter of inhibition zone using the zone size

Antagonistic test
Biocontrol ability of the isolate was evaluated by fungal mycelial inhibition by well diffusion method against the bacterial (E.coli and S.aureus) and fungal (R.solani and P.infestans) pathogens.After the solidification of potato dextrose agar (PDA) 100µl of fungal spore suspension in 0.85% sterile saline was spread on the solidified plate.Well diameter of 6mm was made by metallic borer and filled with overnight grown culture of isolate (1 × 108cfu) and kept for incubation at 28 °C for seven days.

Bacterial DNA Isolation
DNA was isolated using Phenol Chloroform Method.From the overnight grown liquid culture 1.5 ml of culture was taken in micro centrifuge tube and centrifuged at 8000 rpm for 5 mins and pellet the bacterial cells.Pellets were suspended in 900µl of the TE buffer.1/10 volume of 10% SDS solution (900µl solution then add 100µl of solution into it) was then added and tubes were kept at 50-60ºC under water bath for approximately 2 hours.Mix 600µl of Phenol:Chloroform:Isoamyl alcohol(25:24:1) to it and shake gently by repeatedly inverting the tube.White precipitate appeared after mixing, this mixture was added to the tube and solution became creamy orange (white due to protein precipitation and orange due to phenol).Kept for 5 min and then centrifuged at 10000 rpm for 10 min.Upon centrifugation 3 layers were appeared.Upper transparent aqueous layer was collected in fresh new eppendorf tube and discard the lower layer.Kept the eppendorf tube in refrigerator to cool it for 10-15 min.Double volume of chilled propanol was added to the aqueous DNA solution drop by drop from its wall.After adding DNA precipitate out from the solution and which was kept again on 0ºC for 15 min.After precipitation, tubes were centrifuged at 10000rpm for 10 min and supernatant was discarded.DNA pellet was dried in air and dissolved in 50µl TE buffer and then run on gel with 1kb ladder (Ali et al. 2013).

16S rRNA amplification
For molecular characterization, bacterial genomic DNA was quantified at 260/280nm.The 16SrRNA gene was amplified by PCR using forward 5'-CCGAATTCGTCGACAACAG AGTTTGATCCTGGCTCAG-3' and reverse p r i m e r 5 ' -C C C G G G AT C C A A G C T TA C G GCTACCTTGTTACGACTT-3 primers under standard conditions (initial denaturation 94°C for 3 min, 35 cycles of denaturation at 94°C for 30 sec, annealing at 60°C for 30s, extension at 72°C for 60s, and final extension at 72°C for 7 min).The PCR product was found the size of 1424, 1487 and 1306 bp for Burkholderiacepacia, Citrobacter feurendii and Serratia marcescens respectively and was purified and sequenced (Applied Biosystems, New Delhi).The sequence of 16S rRNA genes of all three isolates were obtained and compared with the existing database of 16S rRNA gene and submitted to Gene Bank of NCBI.

Isolation of rhizobacterial strains from soil samples
In the present study three rhizobacterial strains (i.e.Burkholderia cepacia, Citrobacter freundii, Serratia marcescens) were isolated by spread plate technique on selective media from 10 -3 dilution of rhizospheric soil of mustard plant.Mustard plant rhizospheric soil was also used as sample for isolation of PGPR by (Lugtenberg and Kamilova 2009).Hiltner (1904) discovered that the  1).Previous study reported that PGPR isolated from Indian mustard rhizospheric soil, possessed ACC deaminase enzyme and promoted root proliferation significantly (Belimov et al. 2001).Dubey and Maheshwari (2010) isolated genera of Azatobacter, Bacillus, Burkholderia from soil which supply nitrogen to legume plants.

Screening of Rhizobacteria for plant growth promoting traits
Screening of bacterial isolates for their ability to promote plant growth.Rhizobacteria exhibit several plant growth promoting characteristics like phosphate solubilization, Nitrogen fixation, production of siderophore, IAA, Ninhydrin and ACC deaminase (Fig. 1

Indole acetic acid production test
Indole acetic acid test was conducted (Fig. 1a) that showed 4.22 µg/ml concentration of Burkholderia cepacia, while lesser concentration values of 2.42 and 1.86 µg/ml (Fig. 1a

Gibberellic acid production test
All three isolated bacterial strains were tested for gibberellic acid production where Burkholderia cepacia showed concentration i.e. 0.68µg/ml, while Serratia marcescens and Citrobacter freundii showed 0.24 and 0.32 µg/ ml (Fig. 1b) concentration respectively.From the graph (Fig. 1b) it can be observed that Burkholderia cepacia showed more concentration compare to other two isolates.Our results support the concept that the growth promotion in plants induced by Azospirillum infection may occur by a combination  showing that 20-40 pg/ml were produced (Bottini et al. 1989).The same gibberellins were found in similar amounts in cultures of A. brasilense (Janzen et al. 1992).The GAs pools in roots and nodules were of similar size, indicating that Rhizobium does not make a major contribution to GAs content in infected tissues (Atzorn et al. 1988).

ACC determination by colorimetric Ninhydrin assay
Ethylene glycol is used as solvent stabilizing ninhydrin reagent and for colour development.Ascorbic acid addition into ninhydrin reagent prevents oxidation of hydrindantin.Values for absorbance of ACC solutions were found in the rage 0.015 to 0.3 mmol/1 at 570 nm after the reaction was highly correlated with ACC concentrations (R 2 = 0.999) and resulted in linear calibration curves by standard ninhydrin assay.Conc. of 0.05, 0. PGPR ACC deaminase activity, but provides a screening method with a relatively short turnaround time.Determination of ACC concentrations in rhizobacterial isolate after certain incubation period in the DF-ACC medium, by both assays were statistically identical at the 0.05 confidence level.
Ali et al. (2014) conducted study on Pseudomonas isolates and found maximum 3.22 mg/mg protein EPS production (from Rdgp10 strain) while another strain (BriP15) produced 2.18 mg/mg protein EPS.Hartel and Alexander (1986) observed a significant correlation between the amount of EPS produced by cowpea Bradyrhizobium strains and desiccation tolerance.Glick et al. (1998) established that PGPB that have ACC deaminase activity help plants to withstand stress (biotic or abiotic) by reducing the level of the stress hormone ethylene through the activity of enzyme ACC deaminase, which hydrolyzes ACC into á-ketobutyrate and ammonia instead of ethylene.

Isolation and sequence characterization of Rhizobacterial 16S rRNA gene
Rhizobacterial genomic DNA was extracted and run on agarose gel with 1kb ladder.All three bacterial cultures showed genomic DNA size greater than 10kb size (Fig. 7).Spectrophotometric analysis of samples was done to quantify rhizobacterial genomic DNA concentration and found 1.53 µg/ml for Burkholderia cepacia; 1.38 µg/ml for Serratia marcescens; 1.35 µg/ml for Citrobacter freundii .DNA from the above mentioned 03 Rhizobia strains were PCR amplified and found the size of 1424, 1487 and 1306 bp for Burkholderia cepacia; Citrobacter freundii; Serratia marcescens respectively (Fig. 7).All three amplified products were then sequenced for 16S rRNA at Applied Biosystems using the oligonucleotide primers i.e. 16S forward primer: 5'-CCGAATTCGTCGACAA CAGAGTTTGATCCTGGCTCAG-3' and 16S reverse primer: 5'-CCCGGGATCCAAGCTTACG GCTACCTTGTTACGACTT-3.After sequencing of all three bacterial isolates, sequences were submitted to DNA Database Bank of Japan (DDBJ), a collaborator of NCBI and obtained accession number i.e.LC169488 , LC169489 and LC169490 for Burkholderia cepacia; Serratia marcescens; Citrobacter freundii respectively for all three isolates.Analysis of these three sequences by comparison with the sequences of GenBank database indicated that 26 strains were Rhizobium leguminasarum with > 99% identities with the type strain (i.e.1398/1402 bp).PGPR that exhibit ACC deaminase enzymatic activity support plants to survive under stress (biotic or abiotic) by lowering stress ethylene concentration by hydrolyzing ACC (immediate precursor of ethylene) into a-ketobutyrate and ammonia (Arshad et al. 2007).Variations in ACC deaminase enzymatic activity potential of the strains were noted in drought stress.In a previous research secretion of enzyme ACC deaminase by Pseudomonas strain SorgP4 was confirmed by acdS gene amplification using the same primer as reported earlier obtained a partial acdS gene from Pseudomonas isolate SorgP4 (Farajzadeh et al. 2010).acdS gene that codes for enzyme ACC deaminase has been earlier isolated from some rhizobacteria from rhizosphere of plant with or without abiotic stresses (Jha et al. 2009).Rhizobacteria capable of producing ACC deaminase are also known to posses potential to elevate growth of plants especially under stressed conditions such as flooding, heavy metals, high salinity and drought, thus acdS gene is a useful candidate gene for developing bio-inoculants to manage abiotic stress in plants.(Ali et al. 2013) Bacterial 16S rDNA sequences are generally considered as highly conserved (Woese 1987).Evolutionary or taxonomic relationships among bacteria and rhizobacteria are usually estimated through 16S rDNA sequence comparisons.According to the 16S rRNA sequences of our three rhizobacteria strains which exhibit ACC deaminase enzymatic activity, they are identified to be Burkholderia cepacia; Serratia marcescens; Citrobacter freundii.
Similar sequences generally code for similar structure; similar structure are considered to be associated in similar function.Therefore BLAST2 was performed to evaluate the association of ribosomal genes and growth promotion (Table 2) among these three species.In contrast to our notion of similar sequence to similar function, the sequences were found relatively divergent.Therefore, to search for more similar homologs of individual sequences, BLASTN was performed with an output value of 10.

CONCLUSION
Three rhizobacterial strains were isolated from soil sample of mustard plant rhizosphere.These isolates were subjected morphological and biochemical characterization followed by screened for plant growth promoting efficiency using several biochemical tests like phosphate solubilization, siderophore production, ammonia production, nitrogen fixation along with confirmatory tests like ninhydrin production, gibberellic acid production and indole acetic acid production tests.Our isolates were also screened for their ability to perform biocontrol activity using some antagonistic tests like HCN, chitinase, cellulase and lipase production tests.Genomic DNA of all three cultures were isolated and amplified through PCR with suitable primers.PCR products were then sequenced for 16s RNA gene and results were successfully submitted to DDBJ/NCBI under allotted accession numbers and bioinformatics based phylogenetic analysis was done to estimated 16s RNA sequence similarities of our isolates with those in the database and their evolutionary relationship.Based on molecular characterization selected three rhizobacterial strains were identified as Burkholderia cepacia, Serratia marcescens and Citrobacter freundii and these isolates were found capable for plant growth promoting activity and possessed significant antagonistic efficacy.Antibiotic susceptibility profile of B. cepacia, C. freundii and S. marcescens against various antibiotics namely gentamicin, ampicillin, erythomycin, kanamycin, tetracyclin, vancomycin and chloramphenicol was recorded.The ultimate achievement of these inoculates will be to be use as bio fertilizers that are cost effective and do not hinder soil fertility unlike chemical fertilizers.
Further research and understanding of mechanism of PGPR mediated phyto-stimulation would pave the way to find out more competent rhizobacterial strains which may work under diverse agroecological conditions.
) were recorded for Citrobacter freundii, Serratia marcescens respectively.Results of Ali et al., 2013 showed 41-47 µg/ml concentration of IAA with species of Pseudomonas.In another research V. paradoxus 5C-2 was seen positive for indoles production in vitro (Jiang et al. 2012).IAA production varies with bacterial isolates and concentration of tryptophan, although intrinsic IAA production ability of rhizospheric microorganism is regulated by the availability of precursors and uptake of rhizospheric IAA by plant(Arshad and Frankenberger 1993).(Belimov et al. 2009) reported that V. paradoxus 5C-2 strain elevated xylem abscisic acid concentrations in pea plant under drought conditions.Sharma et al. (2013) concluded in his study that all rhizobacterial isolates produced IAA, Bacillus produced (11.2-22.8µg/ml) and Pseudomonas isolates (4.4-21.6 µg/ml).

Table 1 .
Cultural, morphological and biochemical characteristics of B. cepacia, C. freundii and S. marcescens isolate

Table 2 .
Homology based study for selected isolates