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Rajnish Prakash Singh and Prabhat Nath Jha, Molecular identification and characterization of rhizospheric bacteria for plant growth promoting ability, Int.J.Curr.Biotechnol., 2015, 3(7):12-18.

International Journal of Current Biotechnology ISSN: 2321 - 8371 Journal Homepage : http://ijcb.mainspringer.com

Molecular identification and characterization of rhizospheric bacteria for plant growth promoting ability Rajnish Prakash Singh* and Prabhat Nath Jha Department of Biological Sciences, Birla Institute of Technology and Science (BITS), Pilani 333031, Rajasthan, India.

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Article History: Received 13 July 2015 Received in revised form 19 July 2015 Accepted 26 July 2015 Available online 30 July 2015

Key words: QPGPR, Rhizosphere, ACC deaminase, PCR, Sorgastrum nutans, Desert region of Rajasthan, India.

A B S T R A C T The objective of this study was to isolate and characterize a rhizospheric bacterium from Sorgastrum nutans, growing around the desert region of Rajasthan (India). Plant growth promoting rhizobacteria (PGPR) are known to influence plant growth by various direct or indirect mechanisms. Isolated strain was tested for various PGP traits like 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity, phosphate solubilization, indole acetic acid production, production of siderophore, nitrogen fixation and ammonia production. Bio-control ability of isolate was screened by antagonistic activity against certain fungal/bacterial pathogens as well as Hydrogen cyanide HCN production. Isolated test organism was also biochemically characterized. Further identification of isolate was performed by PCR based 16S rRNA gene sequencing. Moreover evaluation of the isolate SNP-18 exhibiting multiple plant growth promoting (PGP) traits on soil/plant system is on-going to uncover their efficacy as effective PGPR.

Introduction Plant growth promoting rhizobacteria (PGPR) are a group of heterogeneous bacteria that can be found in the rhizosphere, at root surfaces and in association with roots, which can improve the extent or quality of plant growth directly and or indirectly. Term PGPR was first introduced in 1978 by Kloepper and colleagues. The direct promotion by PGPR occurs in terms of providing the plant with a plant growth promoting substances that is synthesized by the bacterium or facilitating the uptake of certain plant nutrients from the environment. The indirect promotion of plant growth occurs when PGPR prevent the deleterious effect of phytopathogenic microorganisms. In last few decades, a large group of bacteria including species of Alcaligens, Arthobacter, Azospirillum, Bacillus, Burkholderia, Pseudomonas, Klebsiella, Enterobacter, and Serratia have reported to enhance plant growth (Kloepper et al., 1989; Glick, 1995). However the exact mechanisms of PGPR mediated plant growth are not fully understood, but are thought to occurs through (i) the ability to produce plant growth regulators such as indoleacetic acid, gibberellic acid, cytokinins (Glick, 1995), (ii) asymbiotic N2 fixation (Boddey and Dobereiner, 1995), (iii) solubilization of mineral phosphates and other nutrients (Gaur, 1990) (iv) *Corresponding author. Email address: [email protected] Mobile: +91 9950206538

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antagonistic activity against phytopathogenic microorganisms by production of siderophores, antibiotics and HCN production (Scher and Baker, 1982). Most of the bacteria of genus Pseudomonas and Bacillus exploited as biocontrol agent. Some PGPR may promote plant growth indirectly by affecting symbiotic N2 fixation, nodulation or nodule occupancy (Fuhrmann and Wollum, 1989). In addition, plant growth promoting bacterial strains must be rhizospheric competent, for their survival and colonization in the rhizospheric soil (Cattelan et al., 1999). The current agricultural sector are suffering from various environmental challenges including climate/ weather conditions, abiotic stress conditions, characteristics of soil or deleterious activity of the indigenous microbial flora of the soil (Bent et al., 2001). According to FAO (Food and Agriculture Organization of United States) 20-40% global crop production severely affected every year due to activity of various phytopathogenic microorganism. Moreover, use of pesticides adversely affecting the ecological environment by increasing various kinds of pollution and drastic effect on soil fertility. Therefore use of microbial inoculants as bio-control agents could prevents plant pathogens through polyphasic mechanism like production of antibiotics or lytic enzymes competition with other soil micro-organisms for nutrients and induced systemic resistance. Hence use of beneficial rhizospheric bacteria could be used as an effective alternative to chemical

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fertilizers, pesticides and other agricultural supplements (Ashrafuzzaman et al., 2009). In addition, microorganism with ACC deaminase activity has an additional benefit on plants through reducing the stress inducible ethylene level in host plants (Karthikeyan et al., 2012; Jha et al., 2012). The effectiveness of ACC deaminase enzyme had been reported in various environmental stresses like flooding, heat stress, drought, metal contamination, organic pollutants, wounding, pathogen and insect infection (Glick et al., 2007; Morgan and Drew 1997). Sorgastrum nutans, commonly known as yellow Indian grass, belongs to family Poaceae. Yellow Indian grass is native to prairie habitats  and  tolerant  to  shade.  In  desert  of Rajasthan, it can be seen frequently growing in agricultural lands, therefore our aim was to isolate and characterize the rhizospheric bacteria associated with Sorgastrum nutans. Materials and Methods Isolation and screening for ACC deaminase activity For the isolation, rhizospheric soil of Sorgastrum nutans (1 g) was mixed with 50 ml of yeast extract mannitol broth (YEM) medium (g l -1: mannitol 10; K 2HPO 4 0.5; MgSO4.7H2O 0.2; NaCl 0.1; yeast extract 1; CaCO3 1; pH 7.0) (Vincent, 1970) and incubated at 30°C for 48 h. Dilution of this culture was finally placed on Dworkin and Foster (DF)-agar plate supplemented with 3mM ACC as unique nitrogen source. Finally luxuriantly growing bacterial isolate SNP-18 was selected and sub-cultured several times on DF-ACC agar plate to ensure its ability to use ACC as carbon and nitrogen sources. Strain was maintained in 15% glycerol at -80°C and was screened for their biochemical and PGP traits. Biochemical characterization Isolate was screened for various biochemical tests such as Gram staining, starch agar test, IMViC. Activities for some enzymes such as catalase, oxidase, lipase, amylase, protease, nitrate reductase were tested as per standard protocols (Prescott and Harley, 2002). Ability of the isolate to utilize various carbon sources was tested using carbohydrate utilization test kit (KB 009, Himedia, India). Sensitivity of isolate to various antibiotics namely gentamicin, ampicillin, erythromycin, kanamycin, tetracycline, streptomycin, and chloramphenicol was tested by antibiotic discs (HTM 002, Himedia India). Molecular identification and phylogenetic analysis Isolation of genomic DNA For isolation of genomic DNA (g-DNA), a singly colony was picked from the freshly grown culture of bacterial isolate and was grown in Luria-broth medium (Himedia, India) for overnight at 37°C with shaking at 150 rpm. Total genomic DNA of strain was isolated by Quiagen DNA isolation kit (Quiagen, USA). PCR amplification of 16S rRNA gene Identification of isolate SNP-18 was done by 16S rRNA gene sequencing. Genomic DNA of bacterial isolate was amplified with universal primer 27F1 (52 AGAGTTTGATCMTGGCTCAG-32 ) and 1494Rc (52 TACGGCTACCTTGTTACGAC-32 ) in a 25 µl reaction mixture containing 10 X buffer (with 2.5mM MgCl2) 2.5 µl, 20 pmole forward and reverse primer each 2.0 µl, dNTP mixture (2.5mM) 3.0 µl, 0.5 µl of Taq DNA polymerase (2.5 U), nuclease free water and 50 ng of DNA template. DNA samples were amplified on DNA thermalcycler (T 100, BioRad, USA). The PCR condition were as follows : initial denaturation for 3 min at 94°C, 30 13

cycles each consisting of denaturation for 1 min at 94°C, primer annealing for 1 min at 54 °C and extension at 72°C for 5 min and, a final elongation of 5 min at 72°C. An aliquot of 3-4 µl of PCR reaction product was electrophoresed on a 1% agarose gel containing ethidium bromide (0.5µg ml-1 in H2O) and the DNA bands were visualized under the UV light in a gel documentation system (BIORAD, India). Sequencing and phylogenetic analysis The resulted amplicon of size 1.5 Kb was purified using a DNA purification kit (Quiagen, USA) and sequencing was performed at Xcelris Genomics Labs Ltd. (Xcelris, India). The sequenced nucleotides were compared against GenBank database using the NCBI BLAST algorithm and deposited in the NCBI data base http:// www.ncbi.nlm.nih.gov/BLAST. Taxonomic affiliation of isolates was assigned using RDP database (http:// rdp.cme.msu.edu/seqmatch/seqmatch_intro.jsp) at 98% threshold of 16S rRNA gene sequence. A Phylogenetic analysis was done by using Software MEGA 6.0 (Tamura et al., 2013) and aligned using CLUSTAL-X. The pairwise evolutionary distance was constructed by NeighborJoining method with the bootstrap of 500 replicates to cluster the associated taxa. Plant growth promoting test ACC deaminase assay Bacterial isolate was grown in Tryptic soya broth (Himedia, India) to late log phase at 200 rpm for 24 h at 30 °C. The collected cells were harvested by centrifugation, washed with 0.1M Tris HCl (pH 7.6) and incubated overnight in 7.5 ml minimal medium containing 3 mM ACC as sole nitrogen source. The bacterial cells were returned to shaking water bath for induction of enzyme at 200 rpm for 24 h at 30 °C. Then cells were harvested by centrifugation, washed ubsequently with 0.1M Tris-HCl (pH 7.6) and resuspended in 0.1 M Tris-HCl (pH 8.5). Finally thirty µl toluene was added to cell suspension and homogenized for 30 s. At this point 100 µl of toluenized cells were set aside for protein assay and stored at 4 °C. The remaining toluenized cell suspension was used immediately for ACC deaminase assay. ACC deaminase activity was assayed by monitoring the amount of á-ketobutyric acid following the protocol of Penrose and Glick, (2003). The produced á-ketobutyrate was determined by comparing absorbance at 540 nm of test sample to standard curve of á-ketobutyrate (SigmaAldrich, USA) ranging between 0.1 to 1.0 nmol. Assay for indole-acetic acid (IAA) production The IAA production of bacterial isolates was estimated following the method of Gordon and Weber, (1951) with minor modification. Isolated strains were grown in Nutrient broth containing 100µg/ml Tryptophan for 72 h at 30 °C and kept on shaking at 180 rev/min. Fully grown cultures were centrifuged at 5000 rpm. The supernatant (2 ml) was mixed with two drops of orthophosphoric acid and 4ml of the Salkowski reagent (50 ml, 35% of perchloric acid, 1ml 0.5M FeCl3 solution). Development of pink colour indicates IAA production. Optical density was taken at 530nm with the help of UV-visible spectrophotometer (Jasco Corporation, Japan) and concentration of IAA in sample was determined from the standard curve of IAA. Gibberellic acid production test For gibberellic acid production bacterial isolate was grown in 100 ml NB medium at 30 °C for 72 h. Following the incubation period, cultures were centrifuged at 8,000g for 10 min and the pH of supernatant was adjusted to 2.5

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Figure – 1: Phylogenetic tree of Citrobacter freundii SNP-18 with other closely related Citrobacter sp

Table – 1: Biochemical characterization of isolate SNP-18

Characteristic (s) Gram test Indole MR VP Amylase Lipase Urease Catalase Nitrate reductase + positive, - negative Antibiotic resistance Chloramphenicol Tetracycline Kanamycin Gentamycin Vancomycin + sensitive, ++ resistant

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Activity + +

+ + ++ ++ ++

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Table – 2: Carbohydrate utilization pattern of strain SNP-18 Carbohydrates Lactose Xylose Maltose Fructose Dextrose Galactose Raffinose Trehalose Melibiose Sucrose L-Arabinose Mannose Inulin Sodium gluconate Glycerol Salicin Dulcitol Inositol Sorbitol Mannitol Adonitol Arabitol Erythritol α-Methyl-D-glucoside Rhamnose Cellobiose Melezitose α-Methyl-D-mannoside Xylitol ONPG Esculin hydrolysis D-Arabinose Citrate utilization Malonate utilization Sorbose

Test + + + + + + + + + + + + + + + + + + + + + + + +

Table – 3: Plant growth promoting traits of strain C. freundii SNP-18 Plant growth promoting traits

Activity

ACCD activity (nmol of α-KB mg-1 Pr.hr-1) 157.00 ± 2.7 IAA production (µg/ml) 0.214±0.023 Phosphate solubilization (µg/ml) 7.440± 0.850 Siderophore index Nitrogen fixation + HCN production +

+ indicates a positive reaction, - indicates a positive reaction Table – 4: Test of antagonistic activities of Citrobacter freundii SNP-18 against bacterial and fungal pathogens Bacteria

Activity

Zone of inhibition (mm)

Escherichia coli Staphylococcus aureus Bacillus Cereus Erwinia Carotovora Fungal species Fusarium oxysporum Fusarium moniliforme Fusarium graminearum Aspergillus flavus Penicillium citrium

---++

NA NA NA 12.2±0.34

++ ---++

13.1±0.21 NA NA 9.7±0.14 14.8±0.42

-- negative, + poor, ++ good; ±denote standard deviation; NA no activity

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using 1 N HCl and it was extracted with equal volume of ethyl acetate in a separating funnel. The extract was retreated with equal volume of ethyl acetate 2 to 3 times to get concentrated amount of gibberellic acid. To 1.5 ml of extract 0.2 ml of potassium ferrocyanide was added and centrifuged at 1,500g for 10 min. An equal volume of 30% HCl was added in the supernatant and incubated for 1 h at room temperature. The absorbance of the mixture was measured at 254 nm in a UV-Visible spectrophotometer (Jasco Corporation, Japan). The amount of gibberellic acid was calculated from the standard curve prepared in range of 10-100 µg ml-1 (Holbrook et al., 1961). Phosphate solubilization assay P-solubilization ability of isolate was tested using NBRIP (medium (Composition:g/l 10, glucose; 5, Ca3(PO4); 1, MgSO4.7H2O; 0.2, KCl; 1, NaCl; 5, NH4Cl; and 2% agar, pH 7.0) following the method of Mehta and Nautiyal (2001). Release of free phosphate was quantified according to the method of Marinetti (1962). For Quantification, standard curve was prepared using various concentration of K2HPO4. Qualitative estimation for P- solubilization was performed on NBRIP-agar plate. Bacterial culture was spotted on the medium and incubated at 30 °C for 48-72 h and observed for the presence of clear halo zone around the spot. Nitrogen fixation test Nitrogen fixation ability of strain was evaluating by growing on N- free JNFb medium following the standard protocol (Dobereiner et al., 1995). Composition of JNFbper liter was: malic acid, 5.0 g; K2HPO4, 0.6 g; KH2PO4, 1.8 g; MgSO4‡7H2O 0.2 g; NaCl, 0.1 g; CaCl2, 0.02 g; 0.5% bromothymol blue in 0.2 N KOH, 2 ml; vitamin solution, 1 ml; micronutrient solution, 2 ml; 1.64% Fe‡EDTA solution, 4 ml and KOH, 4.5 g. One-hundred milliliters of vitamin solution contained 10 mg of biotin and 20 mg of pyridoxolHCl. The micronutrient solution contained (per liter) 0.4 g; CuSO4, 0.12 g; ZnSO4‡7H2O, 1.4 g; H3BO3, 1.0 g; Na2MoO4.2H2O and 1.5 g; MnSO4.H2O, pH was adjusted to 5.8. Sub culturing was repeated several times to ensure diazotrophy in given isolate. Siderophore production: A 2µl overnight grown culture of isolate (106 CFU/ml) was spot inoculated on chrome azurole S (CAS) agar plates (Schwyn and Neiland, 1987) and incubated at 30 °C for 4-5 days. After growth, bacterial colony was observed for appearance of orange color around bacterial growth. Experiment was performed in triplicate. Test for ammonia production Bacterial isolate was tested for the production of ammonia in peptone water. Freshly grown cultures were inoculated in 10ml peptone water in each tube and incubated for 48– 72 h at 28±2 °C. Nessler’s reagent (0.5 ml) was added in each tube. Development of brown to yellow colour was a positive test for ammonia production (Cappuccino and Sherman, 1992). HCN production Isolates was screened for the production of hydrogen cyanide as per the method of Lorck (1948). Briefly, nutrient broth amended with 4.4 g glycinel-1 was prepared and isolated strain was streaked on modified agar plate. A Whatman filter paper no. 1 soaked in 2% sodium carbonate in 0.5% picric acid solution was placed in the top of the plate. Plates were sealed with parafilm and incubated at 28±2 °C for 4 days. Development of orange to red colour indicated HCN production.

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Antagonistic test Antagonistic activity was evaluated by using agar well diffusion method against plant pathogenic fungal species namely Aspergillus flavus, Fusarium oxysporum, Fusarium moniliforme, Fusarium graminearum and Penicillium citrium. Antagonistic activity against certain bacterial pathogens such as Bacillus cereus, Erwinia carotovora, Escherichia coli, and Staphylococcus aureus were also determined. Briefly, freshly grown culture of fungal and bacterial species was spread on tryptic soya broth and potato dextrose agar plates. Well size of 6 mm was made by metallic borer and filled with 108 CFU ml-1 of freshly grown culture of isolate. The plates were incubated for 7 days at 28 °C for fungal species and 24 h at 37 °C for bacteria. Antagonistic activity was determined by measuring zone of inhibition for which parameter used as

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