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  ORIGINAL PAPER Plant growth promoting potential of endophytic bacteria isolatedfrom  Piper nigrum B. Jasim  ã C. John Jimtha  ã Mathew Jyothis  ã E. K. Radhakrishnan Received: 27 August 2012/Accepted: 27 February 2013   Springer Science+Business Media Dordrecht 2013 Abstract  Piper nigrum  is an interesting plant to study theendophytic microbial factors affecting plant growth becauseof its unique features. Endophytic bacterial isolation fromthe plant resulted in the isolation of twelve bacterial isolateswhich were screened for various plant growth promotingproperties like phosphate solubilization, ACC deaminaseproduction, siderophore production etc. Interestingly, sevenisolates were found to have IAA biosynthetic potential.Bacterial isolates with multiple plant growth promotingproperties were studied for their growth promoting effect on Vigna radiata  seedlings. This resulted in the identificationof   Klebsiella  sp. (PnB 10) and  Enterobacter   sp. (PnB 11) asthe isolates with excellent growth promoting properties.The results confirm promising applications of the endo-phytic bacterial isolates obtained in the study and also theirpossible growth promoting effect in  P. nigrum . Keywords  Endophytic bacteria    Plant growthregulation    HPLC    16S rDNA sequencing Introduction Endophytic bacteria are ubiquitous with a rich biodiversityand unexplored biosynthetic potential (Strobel and Daisy2003; Ryan et al. 2008). By occupying the localized point of entry or by spreading within the plant, they produce anarray of bioactive metabolites and hydrolytic enzymes tosurvive in the unique chemical environment of the hostplant (Strobel 2003). In addition, their metabolic activitiescan contribute to the health, growth and development of plants. This can be direct growth promotion effects throughthe production of phytohormones, N2 fixation, synthesis of ACC (1-aminocyclopropane-1-carboxylate) deaminase,phosphate solubilization and also through the production of antimicrobial metabolites or siderophores to inhibit path-ogenic microorganisms (Xie et al. 1996; Bashan et al.1991; Glick et al. 1998; Leong 1986). Endophyticmicrobialproductscanhavesignificanteffectonplants.Microbialproductionofauxinsisknowntotriggerincrease in cell elongation, cell division and differentiationin various plants (Cleland 1990). Microbial population alsoperform phosphate solubilisation by the secretion of organicacids which convert the insoluble phosphates into solublemonobasic and dibasic ions and thereby making it availableto plants (Taurian et al. 2010; Goldstein 1986). ACC deaminase production by plant associated bacteria promotesplant growth by regulating the synthesis of ethylene andthereby reducing its harmful effects (Glick et al. 1998;Madhaiyan et al. 2006; Penrose et al. 2001; Mayak et al. 2004; Grincko and Glick  2001). Siderophore producing endophytic bacteria can restrict the growth of plant patho-gens because of their strong affinity towards Fe(III) (Berget al. 2005; Li et al. 2008; Yang et al. 2009). As the plant growth promoting properties of endophyticbacteria can vary, it is important to study such propertiesfrom microbial populations associated with economicallyimportant and physiologically unique plants. In the currentstudy, endophytic bacterial isolates from  Piper nigrum were investigated for plant growth promoting potential.Twelve bacterial strains belonging to various genera wereisolated, identified and studied for the presence of variousplant growth promoting compounds. Since studies on such B. Jasim    C. John Jimtha    M. Jyothis   E. K. Radhakrishnan ( & )School of Biosciences, Mahatma Gandhi University,Priyadharshini Hills PO, Kottayam Dist 686560, Kerala, Indiae-mail:  1 3 Plant Growth RegulDOI 10.1007/s10725-013-9802-y  aspects from  P. nigrum  is very limited, the study is sig-nificant and novel in its approach. Materials and methods Isolation and characterization of endophytic bacteriaPepper plants ( Piper nigrum ) were collected from Nava- jyothisree Karunakara Guru Research Centre for Ayurvedaand Siddha, Uzhavoor, Kottayam. The stem pieces of  P. nigrum  were cleaned with water and were cut into 1 to2 cm long pieces. This was followed by treatment withTween 80 for 10 min and wash with distilled water. Thesamples were further treated with sodium hypochlorite(with 1 % availabe chlorine) for 10 min and subsequentlywith 70 % ethanol for 1 min. This was followed by washwith sterilized distilled water and the final wash wasspread plated onto nutrient agar plate (peptone 5 g, beef extract 2 g, yeast extract 3 g, NaCl 5 g, agar 18 g anddH 2 O 1L, pH 7.0) and S agar plate (Dextrose 20 g, caseinhydrolysate 4 g, K  2 HPO 4  0.5 g, MgSO 4  7H 2 O 0.2 g,CaCl 2  0.1 g, ferric citrate 10 mg, CoSO 4  7H 2 O,CuSO 4 5H 2 O 0.1 mg, H 3 BO 3  1.5 mg, MnSO 4  H 2 O 0.8 mg,(NH 4 ) 6 Mo 7 O 24  4H 2 O 0.2 mg, ZnSO 4 .7H 2 O 0.6 mg, agar6 g and dH 2 O 1L, pH 7.0) as control. The surface sterilizedmaterials were further macerated in sterile phosphatebuffer saline (NaCl 8 g, KCl 0.2 g, Na 2 HPO 4  1.44 g,KH 2 PO 4  0.24, dh 2 O 1L, pH 7.4) and was serially diluted toa 10 - 3 dilution. From this, 100  l L was plated ontoNutrient agar and S agar plates. The experimental platesand control were incubated at room temperature for 5 daysand observed periodically for bacterial growth. Morpho-logically different colonies were selected and used forfurther studies.Identification of the isolates by 16S rDNA sequencingGenomic DNA isolation of the bacterial strains obtainedwas conducted as per the method described by Ausubelet al. (1995). The isolates were cultured overnight in Luria–Bertani broth and the cells were collected by centrifuga-tion. The cells were then resuspended in 567  l L of TEbuffer followed by lysis using 30  l L of 10 % SDS and3  l L of 20 mg/mL protienase K. The mixture was thenincubated for 1 h at 37   C. After which the lysate wasmixed thoroughly with 100  l L of 5 M sodium chloride and700  l L chloroform: isoamyl alcohol (24:1) and this wascentrifuged at 7,500 9 g  for 10 min. The aqueous layer wasthen transferred to a fresh tube and equal volume of iso-propanol was added. This was then inverted several timesand centrifuged again at 7,500 9 g  for 10 min. The pelletwas finally washed in 70 % ethanol (v/v) and air dried atroom temperature. The dried DNA pellet was resuspendedin 100  l L TE buffer and visualized on a 0.8 % agarose gel(w/v). The isolated genomic DNA was used as template forPCR amplification of 16S rDNA using primers 16SF (5 0 -AgA gTT TgA TCM Tgg CTC-3 0 ) and 16SR (5 0 -AAg gAggTg WTC CAR CC-3 0 ) as per the previous reports of Chunand Goodfellow (1995). PCR was performed at a finalvolume of 50  l L reaction containing 50 ng of genomicDNA, 20 pmol of each primer, 1.25 units of   Taq  DNApolymerase (Bangalore Genei), 200  l M of each dNTPsand 1X PCR buffer. PCR was carried out for 35 cycles in aMycycler TM (Bio-Rad, USA) with the initial denaturationfor 3 min at 94   C, cyclic denaturation for 30 s at 94   C,annealing for 30 s at 58   C and extension for 2 min at72   C with a final extension for 7 min at 72   C. The PCRproduct formation was confirmed by agarose gel electro-phoresis and the product was further purified for its use asthe template for sequencing PCR using Big Dye Termi-nator Sequence Reaction Ready Mix (Applied Biosystem).The sequence data thus obtained was further subjected toBLAST analysis (Zhang et al. 2000). The phylogeneticanalysis of the 16SrDNA sequence was conducted byMEGA 5 using neighbor-joining method with 1,000 boot-strap replicates (Tamura et al. 2011).Screening of the isolates for plant growth promoting(PGP) properties Phosphate solubilization The bacterial isolates were screened for phosphate solu-bilising property using the procedure described by Surangeet al. (1997). In this, the isolates were inoculated on to thePikovskaya medium (glucose 10 g, Ca 3 (PO 4 ) 2  5 g,(NH 4 ) 2 SO 4  0.5 g, NaCl 0.2 g, MgSO 4  7H 2 O 0.1 g, KCl0.2 g, FeSO 4  7H 2 O 0.002 g, yeast extract 0.5 g,MnSO 4  2H 2 O 0.002 g, agar 20, d.H 2 O 1L) containing2.4 mg/mL bromophenol blue. The media inoculated withthe isolates were incubated for 48 h and observed for theformation yellow zone around the colony.  ACC deaminase production The ACC deaminase production of the isolates wasscreened using the methods described by Dworkin andFoster (1958). For this, the isolates were inoculated on toDF salt minimal medium (KH 2 P0 4  4 g, Na 2 HPO 4  6 g,MgSO 4 .7H 2 O 0.2 g, FeSO 4 .7H 2 O 0.1 g, H 3 BO 3  10  l g,MnSO 4  10  l g, ZnSO 4  70  l g, CuSO 4  50  l g, MoO 3  10  l g,Glucose 2 g, gluconic acid 2 g, citric acid 2 g, agar 12 g,d.H 2 O 1L) amended with 2 g/L ammonium sulphate. Thepresence of bacterial growth in the media after incubationwas considered as positive result. Plant Growth Regul  1 3  Siderophore production The isolates were checked for the production of sidero-phores on Blue agar CAS medium containing chrome az-urol S (CAS) and hexadecyltrimethylammonium bromide(HDTMA) as indicators (Schwyn and Neilands 1987). Themedium was prepared by adding 100 mL of MM9 saltsolution (KH 2 PO 4  15 g, NaCl 25 g, NH 4 Cl 50 g, dH 2 O500 mL) to 750 mL of dH 2 O. This was followed byaddition of 32.24 g piperazine-N, N 0 -bis 2- ethanesulfonicacid (PIPES) after bringing the pH up to 6. This was fol-lowed by addition of 1.5 % agar and the medium wasautoclaved. After this, medium was cooled to 50   C fol-lowed by the addition of 30 mL filter sterilized 10 % ca-saminoacid solution, 10 mL of 20 % glucose solution and100 mL of dye solution (prepared by mixing 50 mL of 0.06 g Chrome azurol S with 9 mL of 0.0027 g of FeCl 3 -6H 2 O dissolved in 10 mL of 10 mM HCl). This was thenmixed with 0.073 g of HDTMA in 40 mL of dH 2 O toobtain a blue color by adding the mixture along the glasswall with enough agitation to mix the solution. The blueagar medium was aseptically poured on to sterile plates andallowed to solidify. All the bacterial isolates obtained wereinoculated into the CAS medium and incubated at 28   Cfor 24 h. Development of yellowish orange halo around thecolonies was taken as the indication for the production of siderophore. Screening of isolates for IAA production For screening the bacterial isolates for IAA production, theisolates were inoculated into 20 mL of nutrient brothsupplemented with 0.2 % (w/v) of   L -tryptophan and incu-bated for 10 days at 28   C. Then the culture supernatantwas collected by centrifugation at 3,000 rpm for 20 minand the supernatant was used for detection of indole 3acetic acid, as per the methods described previously byRahman et al. (2010). For preliminary screening, one mLof the supernatant was mixed with 2 mL of Salkowskireagent and were incubated in dark and observed for theintensity of red color formed. Uninoculated growth med-ium was used as negative control. The IAA positive iso-lates were further inoculated into 200 mL of nutrient brothsupplemented with 0.2 % (w/v) of   L -tryptophan and wasfurther incubated for 10 days at 28   C. The supernatantfrom the incubated culture was collected by centrifugationat 1,000  9  g for 20 min. The collected supernatant wasfurther acidified to pH 2.5–3.0 with 1 N HCl and this wasfollowed by extraction with ethyl acetate and vacuum dryin a rotary evaporator at 40   C. The dried product thusobtained was further dissolved in 1 mL of methanol(MeOH) and stored at  - 20   C. Presence of IAA in theconcentrated sample was analyzed by reverse-phase HPLCon a Supelcosil LC-18 column with a flow rate of 1 mL min - 1 as described by Jensen et al. (1995). For theelution, a mixture of H 2 O and MeOH (60:40), both con-taining 0.5 % acetic acid was used. Elution was monitoredat 260 nm by shimadzu UV–Vis Detector model SPD 10A. Effect of the endophytic extracts on growth promotionof Vigna radiata seedlings Both the culture filtrate and methanolic extract of theselected bacterial strains were used for the growth pro-motion studies on  Vigna radiata  seedlings. The selectionwas based on their multiple plant growth promotingproperties like phosphate solubilization, ACC deaminase,siderophore and IAA production. Seeds of   V. radiata  weresurface sterilized using NaOCl (with 0.5 % (v/v) availablechlorine) for 10 min and were subsequently washed with Table 1  Identification of the endophytic bacterial isolates by 16SrDNA similarity searchNameof theisolateSubmitted 16S rDNAsequence to NCBIunder the accessionnumberClosest NCBImatch withaccession numberPercentageof identityPnB 1 JQ886792  Bacillus firmus (HQ397586)99PnB 2 JQ886793  Paenibacillusdendritiformis (GU117660)99PnB 3 JQ886794  Pseudomonashibiscicola (JN082269)99PnB 4 JQ886795  Bordetella avium (EU082156)99PnB 5 JQ886796  Bacillus flexus (FJ226761)99PnB 6 JQ886797  Stenotrophomonasmaltophilia (AY682648)100PnB 7 JQ900142  Bacilluslicheniformis (JN189125)100PnB 8 JQ907393  Klebsiella pneumoniae (HQ259959)100PnB 9 JQ907394  Enterobacter cloacae (HQ220157)99PnB10JQ907395  Enterobacter   sp.(FJ646661)99PnB11JQ907396  Klebsiella oxytoca (GU253335)99PnB12JQ907398  Pantoeaagglomerans (FJ592998)100Plant Growth Regul  1 3  Fig. 1  The phylogeneticanalysis of partial 16S rDNAsequence of the isolatesobtained in the study along withsequences from NCBI usingMEGA 5 with neighbor joiningmethodPlant Growth Regul  1 3

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