research article evaluation of phytase producing bacteria ......solubilization index (s.i.) was...

Research ArticleEvaluation of Phytase Producing Bacteria forTheir Plant Growth Promoting Activities

Prashant Singh1 Vinod Kumar12 and Sanjeev Agrawal1

1 Department of Biochemistry College of Basic Sciences and Humanities G B Pant University of Agriculture and TechnologyPantnagar 263145 India

2 Akal School of Biotechnology Eternal University Baru Sahib Sirmaur 173101 India

Correspondence should be addressed to Vinod Kumar sangwanvinodyahoocom

Received 31 July 2013 Revised 5 November 2013 Accepted 6 November 2013 Published 23 January 2014

Academic Editor Michael A Cotta

Copyright copy 2014 Prashant Singh et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Bacterial inoculants are known to possess plant growth promoting abilities and have potential as liquid biofertilizer applicationFour phytase producing bacterial isolates (phytase activity in the range of 0076ndash0174UmL) identified as Advenella species (PB-05 PB-06 and PB-10) and Cellulosimicrobium sp PB-09 were analyzed for their plant growth promoting activities like siderophoreproduction IAA production HCN production ammonia production phosphate solubilization and antifungal activity All isolateswere positive for the above characteristics except for HCN productionThe solubilization index for phosphorus on Pikovskaya agarplates was in the range of 2ndash4 Significant amount of IAA (719 to 3503 120583gmL) production and solubilized phosphate (18953 to74684 120583gmL) was noticed by these isolates at different time intervals Besides that a greenhouse study was also conducted withIndian mustard to evaluate the potential of these isolates to promote plant growth Effect of seed bacterization on various plantgrowth parameters and P uptake by plant were used as indicators The plant growth promoting ability of bacterial isolates in potexperiments was correlated to IAA production phosphate solubilization and other in vitro tests On the basis of present findingsisolate PB-06 was most promising in plant growth promotion with multiple growth promoting characteristics

1 Introduction

It is well known that a considerable number of bacterialspecies mostly those associated with the plant rhizosphereare able to exert a beneficial effect upon plant growth There-fore their use as biofertilizers or control agents for agricultureimprovement has been a focus of numerous researchers fora number of years Bacterial inoculants have been used toincrease plant yields in several countries and commercialproducts are currently available For example in India sev-eral biofertilizers are commercially produced and employedwith different crops mostly using strains of AzotobacterRhizobium Azospirillum and Burkholderia Several possi-ble mechanisms have been proposed including suppres-sion of diseases caused by plant pathogens [1] competitionwith pathogenic microorganisms by colonizing roots [2]production of plant-growth-regulating substances such asindole-3-acetic acid (IAA) [3] and lowering ethylene lev-els in root cells Plant-stimulatory effects exerted by plant

growth promoting bacteria (PGPB) might also be due toan enhanced availability of limited plant nutrients suchas nitrogen phosphorus B-vitamins and amino acids inthe rhizosphere caused by phosphate-solubilizing and dia-zotrophic bacteria [4 5] Improved phosphorus nutritionis achievable by ldquomobilizationrdquo of phosphorus as insolubleinorganic polyphosphates and phytate which accounts for20ndash50 of the total soil organic phosphorus [6] A number ofPGPB are efficient in phytostimulation and biofertilizationand as biocontrol agents but in most cases difficulties inobtaining successful formulations and insufficient knowledgeof the basic molecular principles of their action has hinderedtheir commercial use [7]

P is an important plant macronutrient making up forabout 02 of a plantrsquos dry weight It is a component of keymolecules such as nucleic acids phospholipids andATP andconsequently plants cannot grow without a reliable supplyof this nutrient P is also involved in controlling key enzymereactions and in the regulation of metabolic pathways After

Hindawi Publishing CorporationInternational Journal of MicrobiologyVolume 2014 Article ID 426483 7 pageshttpdxdoiorg1011552014426483

2 International Journal of Microbiology

N P is the second most frequently limiting macronutrientfor plant growth In the rhizosphere the conversion of theinsoluble forms of inorganic P to a form accessible by plantsis achieved by phosphate-solubilizing bacteria (PSB) whichrelease phosphates meanly by organic acids releasing How-ever organic P forms particularly phytates are predominantin most soils (10ndash50 of total P) and must be mineralizedby phytases (myo-inositol hexakisphosphate phosphohydro-lases) to be available P for plants [8] In this context bacteriawith both activities production of organic acids to solubiliseinorganic P and production of phytase to mineralize phytatehave been isolated from rhizosphere [9 10] and proposed aspotential plant growth promoting rhizobacteria (PGPR) tobe used in soil with high content of organic P Studies havealso revealed that phytase-producing rhizobacteria (PPB) notonly harbor the ability to mineralize phytate but also harborother PGPR activities such as the production of indole aceticacid siderophore volatiles and ammonia [11 12] Indianmustard (Brassica juncea) is an important oil seed crop whichproduces an edible mustard oil used in vegetables picklesand so forth at a large scaleThe present study was conductedwith the following objectives of characterization of phosphatesolubilizing bacterial isolates for plant growth promotingpotential in vitro and a greenhouse study for the ability ofselected isolates to promote plant growth ofmustard (Brassicajuncea) Recently a similar study was conducted with mus-tard usingAchromobacter spBacillus sp andTetrathiobactorsp [13]

2 Material and Method

21 Chemicals Reagents and Media Microbiological medialike Pikovskaya agar Luria Broth (LB) media and nutrientagar media were procured from HiMedia Pvt Ltd IndiaOther chemicals of interests used were of analytical gradePreviously isolated phytase producing bacterial strains (PB-05 PB-06 PB-09 and PB-10) in our lab was taken for thefollowing study [14]

22 Production and Determination of Phytase Activity Theisolates were tested for their ability to produce phytase on thephytase screeningmedium (PSM) described byKerovuo et al[15]The streaked plates were incubated overnight at 37∘Candthe translucent region of the plate gave a visual indicationof extracellular phytase production Phytase activity in liquidPSM media was determined by following the method ofEngelen et al [16] as used in Kumar et al [13]

23 Analysis of Phosphate SolubilizationEfficiency Phosphatesolubilization by bacterial isolates was done by the method ofPikovskaya [17] Plates weremade in duplicate for each bacte-rial isolate using Pikovskaya agar medium Bacterial culturewas point inoculated at the centre of Pikovskaya agar plateand incubated in incubator at 28∘C for 7 d The plates werethen examined for halo zone around bacterial culture andsolubilization index (SI) was calculated as SI = (colonydiameter + halo zone diameter)colony diameter [18]

Phosphate solubilizing efficiency of bacterial cultureswas quantified in specified NBRIP (national botanicalresearch institute phosphate glucose 10 gL Ca

3(PO4)24 gL

MgCl2sdot6H2O 5 gL MgSO

4sdot7H2O 025 gL KCl 02 gL and

NH4(SO4)201 gL) media using the method of Nautiyal [4]

24 Screening for Other Plant Growth Promoting Activities

241 Indole Acetic Acid Siderophore Hydrogen CyanideOrganic Acid and Ammonia Production Indole acetic acid(IAA) production by bacterial isolates was determined in LBbroth supplemented with L-Tryptophan (500 120583gmL) at 2448 and 72 h as described by Patten and Glick [19] For thisbacterial cells were removed by centrifugation at 10000 rpmfor 5min at 4∘C One mL of the supernatant was mixedwith 4mL of Salkowskirsquos reagent in the ratio of 1 4 andincubated at room temperature for 20min Development ofa pink colour indicated indoles The absorbance of super-natant mixture (supernatant + Salkowskirsquos reagent) for indoleproduction was measured at 530 nm and quantity of indoleswas determined by comparison with a standard curve usingan IAA standard graph Siderophore production was deter-mined by using blue indicator dye and chrome azurol S agar[20] Bacterial isolates exhibiting orange halo zone on chromeazurol S agar after 5 d of incubation at 28∘C were consideredpositive for the production of siderophores For hydrogencyanide (HCN) production the methodology described byBakker and Schippers [21] was used Isolates were grownon plates of tryptic soy agar (10) amended with glycine(44 g Lminus1) and FeCl

3sdotH2O (03mM) A change from yellow

to orange red brown or reddish brown was recorded asan indication of weak moderate or strongly cyanogenicpotential respectively Organic acid production potential ofdifferent isolates was analyzed using thin layer chromatog-raphy (TLC) on Silica-G (Merck chemicals) gel plates usingdifferent solvent systems Finally ammonia production testwas performed by growing selected isolated in peptone waterfor 72 h at 30∘C Change in colour after addition of 1mLNesslerrsquos reagent (K

2HgI4 14) in each tube was observed

The presence of faint yellow colour indicates small amountof ammonia and deep yellow to brownish colour indicatesmaximum ammonia production

242 Antifungal Activity Antagonistic nature of bacterialisolate against A brassicaceae was determined by employ-ing dual culture technique Briefly bacterial isolates werestreaked at the centre of 90mm petriplate containing PDAand incubated for 36 h at 25 plusmn 2∘C A plug of fungus wasthen placed on the edge of petriplate at both sides of streakedculture Plates were incubated at 25 plusmn 2∘C for 7 dThe radii ofthe fungal colony towards and away from the bacterial colonywere measured The percentage of growth inhibition wascalculated using the formula inhibition = (119877minus119903)119877 where 119903is the radius of the fungal colony opposite the bacterial colonyand 119877 is the maximum radius of the fungal colony away fromthe bacterial colony [22]

International Journal of Microbiology 3

Table 1 Plant growth promoting characters of phytase producing bacterial isolates

Isolates Characterization (staining) Production assay Phosphorus solubilization

Gramrsquos Endospore Siderophore Ammonia HCN Catalase Ca3(PO4)2 Phytate Solubilization index(Ca3(PO4)2)

Control NA NA minus minus minus minus minus minus 0PB-05 minus + + ++ minus + + + 2PB-06 minus minus + ++ minus + +++ + 4PB-09 minus minus ++ + minus + + + 2PB-10 minus minus + ++ minus + ++ + 25

25 Greenhouse Experiment

251 Seeds and Soil Seeds of Brassica juncea were surfacesterilized with 1 sodium hypochlorite for 30 sec and rinsedwith double distilled water thrice before use in greenhouseexperiment Soil used in the experiment was collected fromthe experimental farmof the university the soil was dried andsieved to 2mm before mixing it with cow dung manure (3 1)and autoclaved for 1 h prior to pot experiment The chemicalcharacteristics of soil mixture were also analyzed usedsoil mixture contained 2352 kg ha1 potassium 2007 kg haminus1nitrogen 2822 kg haminus1 phosphorus 15 organic acid con-tent 0171 milisimon electrical conductivity and 674 pH andthe texture of soil was silt clay loam

252 Seed Bacterization and Pot Experiment Bacterial cellswere inoculated in 25mL LB in 100mL conical flask andincubated at 37∘C 120 rpm Colony forming units (cfu) werecounted and bacterial inoculums containing approximately1 times 108 cfumL were used for seed bacterization CMC(100mg) was added to flask containing culture inoculum asadhesive material Ten gram of seeds was soaked in bacterialsuspension for 12 h on a rotary shaker at 150 rpm Thebacterial suspension was drained off and the seeds were driedovernight aseptically in laminar air flow Seeds soaked indistilled water amended with CMC served as control Theplants were grown in greenhouse under a daynight cycle of168 h 2520∘C and 60 relative humidity Pot soil (soildecomposed cow dungmanure at 3 1 vv) was filled into pots(20 cm diameter) Ten bacterized seeds of Brassicawere sownin each pot in three replicates

253 Phosphorus Content Estimation 30 d old seedlingswere collected by uprooting the plants carefully withoutdamaging the root system and analyzed for phosphoruscontent Seedlings were air dried grounded and digestedin 15mL HClO

4and 5mL HNO

3and phosphorus content

was measured using spectrophotometric vanado-molybdatemethod [23] Rhizosphere soil samples were collected fromeach treatment and analyzed for variation in pH and availablephosphorous content by following Olsenrsquos method [24]

26 Characterization of Bacterial Isolates Bacterial isolateswere taken from the culture during log phase for gram stain-ing and were examined for endospore formation during sta-tionary phase Genetic characterization based on 16S rRNA

gene sequence was also done Briefly genomic DNA fromselected isolates was extracted as described byNeumann et al[25] and PCR amplification of 16S rRNA gene was carriedout by using primers RDNA-1A (51015840-AGA GTT TGA TCCTGG CTC AG-31015840) and RDNA-1B (51015840-AAG GAG GTG ATCCAG CCG CA-31015840) The PCR was done as follows a hot-startof 94∘C for 3min followed by 35 cycles of 94∘C for 1min 54∘Cfor 1min 72∘C for 15min and a final extension for 10min at72∘C Amplified PCR products were purified with QIAquickGel Extraction kit (Qiagen Germany) and sequenced in anautomated DNA sequencer (Applied Biosystems 3730) atDNA Sequencing Facility University of Delhi (South Cam-pus) New Delhi India The sequences obtained were com-pared with sequences in the NCBI GenBank database usingblastn program (httpblastncbinlmnihgovBlastcgi) andthen deposited in NCBI

3 Result and Discussion

31 Characterization of Bacterial Isolates All studied bac-terial cultures were gram negative Out of four bacterialisolates PB-05 were found to be endospore forming andshowed green colour under microscope while the remain-ing bacterial isolates that is PB-06 PB-09 and PB-10were nonendospore forming (Table 1) Based on 16S rDNAgene sequences three isolates (PB-05 PB-06 PB-10) wereidentified as Advenella species (GenBank accession numberJN6308081 JN6308091 and JQ7274331 for PB-05 PB-06and PB-10 resp) while the fourth isolate was identified asCellulosimicrobium sp PB-09 (GenBank accession numberJN6308061) In another study byKumar et al [13] the isolatesused for similar study were identified as Achromobactor spPB-01 Bacillus sp PB-13 and Tetrathiobactor sp PB-03

32 Phytase Production and Phosphate Solubilization byBacterial Isolates In the soil 20ndash80 of phosphate is inorganic form [8] and plant may poorlynot possess an innateability to acquire phosphorus directly from soil phytate[26] Hence these isolated PSBrsquos were analyzed for theirphytase activity which degrades the soil phytate to lowerphosphate esters which are available to plants All of the fourbacterial cultures showed translucent region around colonieson phytase screening medium described by Kerovuo et al[15] In liquid PSM media all bacterial isolates were foundto be positive for phytase production Highest phytase activ-ity was shown by PB-06 (0174UmL) followed by PB-10


Table 2 Effect of seed bacterization on different plant growth parameters after 30 days of germination under greenhouse study

Bacterial isolates Fresh weight Dry weight Root length Shoot length P content Antifungal activity Phytase activity(g) (g) (cm) (cm) (mgg) inhibition (UmL)

Control 0182 plusmn 0003 0039 plusmn 0002 6740 plusmn 0050 3540 plusmn 0029 1430 plusmn 204 000 0000PB-05 1380 plusmn 0029 0455 plusmn 0001 13340 plusmn 0136 5720 plusmn 0091 2270 plusmn 300 000 0076PB-06 1030 plusmn 0018 0160 plusmn 0005 13380 plusmn 0115 6380 plusmn 0152 2205 plusmn 959 1875 0174PB-09 0629 plusmn 0002 0082 plusmn 0003 10620 plusmn 0096 4200 plusmn 0118 3020 plusmn 407 000 0129PB-10 1016 plusmn 0027 0132 plusmn 0007 14300 plusmn 0114 4460 plusmn 0067 1780 plusmn 601 2500 0161

(0161 UmL) PB-09 (0129UmL) and PB-05 (0076UmL)(Table 2) Isolate PB-06 predominantly produced higherlevel of phytase Nutrient source and concentration greatlyinfluence the bacterial growth and enzyme production Highphytate content in the mediummight induce the synthesis ofphytase enzyme where 5mM concentration of sodium phy-tate induced the phytase production Phytase has beenisolated and characterized earlier from several gram-positiveand gram negative soil bacteria for example Bacillus subtilis[15] Acetobactor sp [13] B laevolacticus [27] Klebsiella ter-rigena [28] Pseudomonas sp [29] and Enterobacter sp [30]Idriss et al [31] reported that the extracellular phytase fromBamyloliquefaciens FZB45 promotes growth ofmaize seedlingsunder in vitro conditions In another study Kumar et al [13]also reported phytase producing bacterial isolates promotingplant growth of Indian mustard

The bacterial isolates solubilized tricalcium phosphate inPikovskaya media Advenella sp PB-06 showed highest solu-bilization index of 4 while Cellulosimicrobium sp PB-09 andAdvenella sp PB-10 showed the least solubilization index of 2Advenella sp PB-05 showed the intermediate solubilizationindex of 25 (Table 1) Since the direct measurement of Psolubilization in broth assay is likely to give more reliableresults than regular plate assay isolates were also testedfor their ability to solubilize tricalcium phosphate (TCP) inNBRIP medium at 24 h 48 h and 72 h and were found tosolubilize tricalcium phosphate with an increase from 24 hto 72 h in case of PB-09 while for other isolates it increasesupto 48 h then it remains constant Maximum phosphatesolubilization shown by PB-09 at 72 h was 74684120583gmLLeast phosphate solubilization shown by PB-05 at 24 h was18953 120583gmL (Figure 1) A direct correlation of P-solubilizingactivity in solid and liquid media was found in all testedisolates They showed better P solubilizing activity in liquidmedium compared to solidmediumOn plates the isolates P-solubilizing zone around the bacterial colony varied from 08to 20 cm in radius while in liquid medium it varied from18953 to 74684 120583gmLminus1 using TCP as a source of insolublePThe final pH of the medium in which this strain was grownincreased from 7 to 874 after incubation Similar studies havebeen conducted by Alikhani et al [32] where different iso-lates of rhizobia from Iranian soils were tested for their abilityto dissolve inorganic and organic phosphates In contrastthey observed the drop in pH of the culture filtrate withthe release of soluble orthophosphate which indicated theimportance of organic acid production in the mobilizationprocess In a similar study Kumar et al [13] have also studied

0

5

10

15

20

25

30

35

40

Control PB-05 PB-06 PB-09 PB-10Bacterial isolates

IAA

cont

ent (120583

gm

L)

24h48h

72h

Figure 1 Effect of time on phosphate solubilization by bacterialisolates

three phytase producing isolates for their P solubilizationabilities on solid medium as well as liquid NBRIP media Inthat case solubilization index and P solubilized were in therange of 129ndash20 and 193ndash642 120583gmLminus1 respectively

33 Plant Growth Promoting Activities of Bacterial IsolatesPGPB can exert a direct effect on plant growth other thanthemechanism of phosphate solubilization like production ofphytohormones biological nitrogen fixation enhancing theavailability of other trace elements and increased iron nutri-tion through iron-chelating siderophores and volatile com-pounds that affects the plant signaling pathways Addition-ally by antibiosis competition for space and nutrients andinduction of systemic resistance in plant against a broadspectrum of root and foliar pathogens might also contributeto enhanced plant growth and metabolism [33] Character-ization of different traits of rhizobacteria was the commonprocedure employed while screening and selecting PGPBrsquos[13 34] In our study too these phytase producing bacterialisolates were characterized for different traits like IAA pro-duction siderophoreHCN antibiosis and their plant growthpromoting ability

All bacterial cultures were IAA producing as indicated byobserved pink colour in flasks of all cultures (except PB-09at 24 h and control) At 48 h and 72 h Cellulosimicrobium sp


0

100

200

300

400

500

600

700

800


24h48h

72h

Phos

phor

us co

nten

t (120583

gm

L)

Figure 2 Effect of time on IAA production by bacterial isolates

PB-09 also produced IAA as indicated by pink colour of cul-ture IAA level in media increased from 24 h to 72 h in case ofCellulosimicrobium sp PB-09 while inAdvenella species thatis PB-05 PB-06 and PB-10 it decreases from 24 h to 72 hMaximum IAAproductionwas shown byAdvenella sp PB-10(3503 120583gmL) followed by Advenella sp PB-06 (260 120583gmL)at 24 h while minimumwas shown by Cellulosimicrobium spPB-09 (719 120583gmL) at 48 h followed by Advenella sp PB-06(962120583gmL) at 72 h (Figure 2)These isolates released greaterquantities of IAA in the presence of a physiological precursortryptophan in culture medium Production of IAA variesgreatly among different species and is also influenced byculture conditions growth stage and availability of substrate[35] Leinhos and Vacek [36] reported IAA production byPseudomonas and Acinetobacter isolated from wheat and ryerhizosphere ranging from 001 to 398mgmLminus1 Bacterialisolates obtained from the rhizosphere of various plants havebeen shown to produce IAA in pure culture Most of theisolates in this study produced higher IAA in the presence ofthe precursor L-Trp

All the selected isolates were found positive forsiderophores and ammonia production but negative forHCNproduction (Table 1) In our studies none of the isolates hasshown HCN production (shows antifungal activity) whichmight be due to variation in growth parameters liketemperature nutrient availability and growth pattern Allisolates showed NH

3production at a high extent due to N

2

availability which increased to plant roots and plant couldsynthesize nitrate for uptake When bacterial isolates onPDA plate were challenge inoculated with pathogenic fungilike Alternaria brassicacae isolates PB-06 and PB-10 affordedsome degree of inhibition against fungal growth andshowed antagonism against A brassicaceae under in vitroexperiments Bacterial isolates PB-06 and PB-10 showed1875 and 25 fungal (A brassicacae) growth inhibition

respectively Bacterial isolates PB-05 and PB-09 showed noantifungal activity in dual culture technique (Table 2) Theprotection offered by the isolates may be due to inducedsystemic resistance But in other isolates no fungal growthinhibition observed provided resistance over the isolates

34 Greenhouse Studies Phosphorus solubilization is oneof the important mechanisms through which PGPB isolatespromote plant growth but this is not the only way ofplant growth promotionThere are several other mechanismslike direct stimulation production of gibberellins cytokininACC deaminase and volatile compounds that are alsoreported previously [37] which were not characterized inthe present study Brassica seeds bacterized with rhizo-spheric isolates showed significant increased shoot lengthroot length fresh weight and dry weight over controlAlso all bacterial isolates inoculated plants showed anincreased inorganic P content in seedlings Advenella spPB-06 inoculated plant showed highest increase in rootlength (638 cm) and Advenella sp PB-10 inoculated plantsshowed highest increase in shoot length (1430 cm) (Table 2)It was assumed that one or many of these traits may beinvolved in the plant growth promoting activity by isolatesPB-05 PB-06 PB-09 and PB-10 Our result showed thesuccessful screening of the bacterial isolates for in vitrosolubilization of inorganic phosphate IAA production andtheir effects on fresh weight dry weight root length andshoot length of Brassica seedlings Seedlings containingCellulosimicrobium sp PB-09 showed highest P contentof 302mgg Maximum available P in soil enhanced byAdvenella sp PB-06 was 877mgKg soil Seedlings raisedfrom seeds bacterized with phytase positive PGPB isolatesshowed higher accumulation of phosphorus content thanuninoculated control Among them isolate PB-09 treatmentshowed significant increase in accumulation of phosphorousin comparison with uninoculated Isolate PB-09 showedsignificant increase in phosphorus accumulation in seedlingswhile isolate PB-05 was found significantly increasing dryweight of seedlings in comparison to control (Table 2) Sev-eral rhizobacteria can solubilize inorganic phosphate fromsoil and such bacteria improve solubilization of unavailablesoil phosphate resulting in a high efficiency of phosphorususe When rhizosphere soil samples of 30 d old seedlingswith all PSRB isolates were analyzed not much variationsin pH was observedThere were no significant changes in pHof soil samples collected from different seedlings

In conclusion All studied phytase-producing bacteriafrom Himalayan soils showed ability to harbor diverse plantgrowth promoting activities including production of phy-tase ammonia siderophores and indole acetic acid releasingof P from insoluble inorganic phosphates and inhibitionof phytopathogen R solani Also the inoculation of Brassicajuncea seeds with Advenella species (PB-05 PB-06 and PB-10) showed improved P content and growth of Brassicajuncea The present study clearly revealed that all isolatestested in this study had the ability to solubilize inorganicphosphate producing phytase ammonia and IAA and


increased availability of P IAA and ammonia leading toincreased plant growth These isolates may be further char-acterized using molecular approaches and changes in expres-sion of related genes for in-depth understanding of detailedmechanism of plant growth promotion The isolates mayalso be used in development of a suitable liquid biofertilizeremploying a consortium of such kind of plant beneficialmicrobes

Conflict of Interests

On behalf of all contributing authors it is declared that thereis no conflict of interests regarding this paper

Acknowledgments

The authors gratefully acknowledge department of scienceand technology for providing infrastructural facility in theform of DST-FIST Grant to Department of BiochemistryVinod Kumar is grateful to Indian Council of AgricultureResearch (ICAR) for providing financial assistance in theformof ICAR-SRF Support provided byDr Bhupendra SinghKharayat Department of Plant Pathology is duly acknowl-edged

References

[1] K P Smith J Handelsman and RM Goodman ldquoGenetic basisin plants for interactions with disease-suppressive bacteriardquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 96 no 9 pp 4786ndash4790 1999

[2] L CDekkers C C Phoelich L VanDer Fits andB J J Lugten-berg ldquoA site-specific recombinase is required for competitiveroot colonization by Pseudomonas fluorescens WCS365rdquo Pro-ceedings of the National Academy of Sciences of the United Statesof America vol 95 no 12 pp 7051ndash7056 1998

[3] O Steenhoudt and J Vanderleyden ldquoAzospirillum a free-living nitrogen-fixing bacterium closely associated with grassesgenetic biochemical and ecological aspectsrdquo FEMS Microbiol-ogy Reviews vol 24 no 4 pp 487ndash506 2000

[4] C S Nautiyal ldquoAn efficient microbiological growth mediumfor screening phosphate solubilizing microorganismsrdquo FEMSMicrobiology Letters vol 170 no 1 pp 265ndash270 1999

[5] H Rozycki H Dahm E Strzelczyk and C Y Li ldquoDiazotrophicbacteria in root-free soil and in the root zone of pine (Pinussylvestris L) and oak (Quercus robur L)rdquo Applied Soil Ecologyvol 12 no 3 pp 239ndash250 1999

[6] A E Richardson P A Hadobas and J E Hayes ldquoExtracellularsecretion of Aspergillus phytase from Arabidopsis roots enablesplants to obtain phosphorus from phytaterdquo Plant Journal vol25 no 6 pp 641ndash649 2001

[7] G V Bloemberg and B J J Lugtenberg ldquoMolecular basisof plant growth promotion and biocontrol by rhizobacteriardquoCurrentOpinion in Plant Biology vol 4 no 4 pp 343ndash350 2001

[8] A E Richardson P A Hadobas J E Hayes C P Orsquohara andR J Simpson ldquoUtilization of phosphorus by pasture plants sup-plied withmyo-inositol hexaphosphate is enhanced by the pres-ence of soil micro-organismsrdquo Plant and Soil vol 229 no 1 pp47ndash56 2001

[9] M A Jorquera M T Hernandez Z Rengel P Marschner andM De La Luz Mora ldquoIsolation of culturable phosphobacteriawith both phytate-mineralization and phosphate-solubilizationactivity from the rhizosphere of plants grown in a volcanic soilrdquoBiology and Fertility of Soils vol 44 no 8 pp 1025ndash1034 2008

[10] M A Jorquera D E Crowley P Marschner et al ldquoIdentifica-tion of 120573-propeller phytase-encoding genes in culturable Paeni-bacillus and Bacillus spp from the rhizosphere of pasture plantson volcanic soilsrdquo FEMSMicrobiology Ecology vol 75 no 1 pp163ndash172 2011

[11] O A Martınez M A Jorquera D E Crowley and M L de laMora ldquoInfluence of nitrogen fertilisation on pasture culturablerhizobacteria occurrence and the role of environmental factorson their potential PGPR activitiesrdquo Biology and Fertility of Soilsvol 47 no 8 pp 875ndash885 2011

[12] B Saharan and V Nehra ldquoPlant growth promoting rhizobac-teria a critical reviewrdquo Life Science and Medicine Research vol2011 pp 1ndash17 2011

[13] V Kumar P Singh M A Jorquera et al ldquoIsolation of phytase-producing bacteria from Himalayan soils and their effect ongrowth and phosphorus uptake of Indian mustard (Brassicajuncea)rdquo World Journal of Microbiology and Biotechnology vol29 no 8 pp 1361ndash1369 2013

[14] P Kumar Production and characterization of bacterial phytaseand its assessment as feed additive [PhD thesis] Departmentof Biochemistry G B Pant University of Agriculture andTechnology Uttrakhand India 2010

[15] J Kerovuo M Lauraeus P Nurminen N Kalkkinen and JApajalahti ldquoIsolation characterizationmolecular gene cloningand sequencing of a novel phytase from Bacillus subtilisrdquoApplied and Environmental Microbiology vol 64 no 6 pp2079ndash2085 1998

[16] A J Engelen F C van der Heeft P H Randsdorp and E LSmit ldquoSimple and rapid determination of phytase activityrdquo Jour-nal of AOAC International vol 77 no 3 pp 760ndash764 1994

[17] R I Pikovskaya ldquoMobilization of phosphorus in soil in connec-tion with vital activity of somemicrobial speciesrdquoMicrobiologyvol 17 pp 362ndash370 1948

[18] M Edi-Premono Moawad and P L G Vleck ldquoEffect of phos-phate solubilizing Pseudomonas putida on the growth of maizeand its survival in the rhizosphererdquo Indonesian Journal of CropScience vol 11 pp 13ndash23 1996

[19] C L Patten and B R Glick ldquoRole of Pseudomonas putidaindoleacetic acid in development of the host plant root systemrdquoApplied and Environmental Microbiology vol 68 no 8 pp3795ndash3801 2002

[20] B Schwyn and J B Neilands ldquoUniversal chemical assay for thedetection and determination of siderophoresrdquo Analytical Bio-chemistry vol 160 no 1 pp 47ndash56 1987

[21] A W Bakker and B Schippers ldquoMicrobial cyanide productionin the rhizosphere in relation to potato yield reduction andPseudomonas SPP-mediated plant growth-stimulationrdquo SoilBiology and Biochemistry vol 19 no 4 pp 451ndash457 1987

[22] E E Idris D J Iglesias M Talon and R Borriss ldquoTryptophan-dependent production of Indole-3-Acetic Acid (IAA) affectslevel of plant growth promotion by Bacillus amyloliquefaciensFZB42rdquoMolecular Plant-Microbe Interactions vol 20 no 6 pp619ndash626 2007

[23] A J Engelen F C Van Der Heeft P H G Randsdorp W A CSomers J Schaefer and B J C van der Vat ldquoDetermination ofphytase activity in feed by a colorimetric enzymatic method


collaborative interlaboratory studyrdquo Journal of AOAC Interna-tional vol 84 no 3 pp 629ndash633 2001

[24] A G Murugesan and C Rajakumari ldquoEnvironmental scienceand biotechnology theory and techniquesrdquo Microbiology vol144 pp 1565ndash1573 2006

[25] B Neumann A Pospiech and H U Schairer ldquoRapid isolationof genomic DNA from gram-negative bacteriardquo Trends inGenetics vol 8 no 10 pp 332ndash333 1992

[26] R Greiner and M L Alminger ldquoStereospecificity of myo-inositol hexakisphosphate dephosphorylation by phytate-degrading enzymes of cerealsrdquo Journal of FoodBiochemistry vol25 no 3 pp 229ndash248 2001

[27] H K Gulati B S Chadha and H S Saini ldquoProduction andcharacterization of thermostable alkaline phytase from Bacilluslaevolacticus isolated from rhizosphere soilrdquo Journal of Indus-trial Microbiology and Biotechnology vol 34 no 1 pp 91ndash982007

[28] R Greiner E Haller U Konietzny and K Jany ldquoPurificationand characterization of a phytase from Klebsiella terrigenardquoArchives of Biochemistry and Biophysics vol 341 no 2 pp 201ndash206 1997

[29] A E Richardson and P A Hadobas ldquoSoil isolates of Pseu-domonas spp that utilize inositol phosphatesrdquo Canadian Jour-nal of Microbiology vol 43 no 6 pp 509ndash516 1997

[30] S J Yoon Y J Choi H K Min et al ldquoIsolation and identifi-cation of phytase-producing bacterium Enterobacter sp 4 andenzymatic properties of phytase enzymerdquo Enzyme and Micro-bial Technology vol 18 no 6 pp 449ndash454 1996

[31] E E Idriss O Makarewicz A Farouk et al ldquoExtracellular phy-tase activity of Bacillus amyloliquefaciens FZB45 contributes toits plant-growth-promoting effectrdquoMicrobiology vol 148 no 7pp 2097ndash2109 2002

[32] H A Alikhani N Saleh-Rastin and H Antoun ldquoPhosphatesolubilization activity of rhizobia native to Iranian soilsrdquo Plantand Soil vol 287 no 1-2 pp 35ndash41 2006

[33] P Gyaneshwar G N Kumar L J Parekh and P S Poole ldquoRoleof soilmicroorganisms in improvingPnutrition of plantsrdquoPlantand Soil vol 245 no 1 pp 83ndash93 2002

[34] A J Cattelan P G Hartel and J J Fuhrmann ldquoScreening forplant growth-promoting rhizobacteria to promote early soy-bean growthrdquo Soil Science Society of America Journal vol 63 no6 pp 1670ndash1680 1999

[35] A Muller H Hillebrand and E W Weiler ldquoIndole-3-aceticacid is synthesized from L-tryptophan in roots of Arabidopsisthalianardquo Planta vol 206 no 3 pp 362ndash369 1998

[36] V Leinhos and O Vacek ldquoBiosynthesis of auxins by phosphate-solubilizing rhizobacteria from wheat (Triticum aestivum) andrye (Secale cereale)rdquoMicrobiological Research vol 149 no 1 pp31ndash35 1994

[37] A R Podile and G K Kishor ldquoPlant growth promoting rhi-zobacteriardquo in Plant Associated Bacteria S S GnanamanickamEd pp 195ndash230 Springer Dordrecht The Netherlands 2006

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


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International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


N P is the second most frequently limiting macronutrientfor plant growth In the rhizosphere the conversion of theinsoluble forms of inorganic P to a form accessible by plantsis achieved by phosphate-solubilizing bacteria (PSB) whichrelease phosphates meanly by organic acids releasing How-ever organic P forms particularly phytates are predominantin most soils (10ndash50 of total P) and must be mineralizedby phytases (myo-inositol hexakisphosphate phosphohydro-lases) to be available P for plants [8] In this context bacteriawith both activities production of organic acids to solubiliseinorganic P and production of phytase to mineralize phytatehave been isolated from rhizosphere [9 10] and proposed aspotential plant growth promoting rhizobacteria (PGPR) tobe used in soil with high content of organic P Studies havealso revealed that phytase-producing rhizobacteria (PPB) notonly harbor the ability to mineralize phytate but also harborother PGPR activities such as the production of indole aceticacid siderophore volatiles and ammonia [11 12] Indianmustard (Brassica juncea) is an important oil seed crop whichproduces an edible mustard oil used in vegetables picklesand so forth at a large scaleThe present study was conductedwith the following objectives of characterization of phosphatesolubilizing bacterial isolates for plant growth promotingpotential in vitro and a greenhouse study for the ability ofselected isolates to promote plant growth ofmustard (Brassicajuncea) Recently a similar study was conducted with mus-tard usingAchromobacter spBacillus sp andTetrathiobactorsp [13]

2 Material and Method

21 Chemicals Reagents and Media Microbiological medialike Pikovskaya agar Luria Broth (LB) media and nutrientagar media were procured from HiMedia Pvt Ltd IndiaOther chemicals of interests used were of analytical gradePreviously isolated phytase producing bacterial strains (PB-05 PB-06 PB-09 and PB-10) in our lab was taken for thefollowing study [14]

22 Production and Determination of Phytase Activity Theisolates were tested for their ability to produce phytase on thephytase screeningmedium (PSM) described byKerovuo et al[15]The streaked plates were incubated overnight at 37∘Candthe translucent region of the plate gave a visual indicationof extracellular phytase production Phytase activity in liquidPSM media was determined by following the method ofEngelen et al [16] as used in Kumar et al [13]

23 Analysis of Phosphate SolubilizationEfficiency Phosphatesolubilization by bacterial isolates was done by the method ofPikovskaya [17] Plates weremade in duplicate for each bacte-rial isolate using Pikovskaya agar medium Bacterial culturewas point inoculated at the centre of Pikovskaya agar plateand incubated in incubator at 28∘C for 7 d The plates werethen examined for halo zone around bacterial culture andsolubilization index (SI) was calculated as SI = (colonydiameter + halo zone diameter)colony diameter [18]

Phosphate solubilizing efficiency of bacterial cultureswas quantified in specified NBRIP (national botanicalresearch institute phosphate glucose 10 gL Ca

3(PO4)24 gL

MgCl2sdot6H2O 5 gL MgSO

4sdot7H2O 025 gL KCl 02 gL and

NH4(SO4)201 gL) media using the method of Nautiyal [4]

24 Screening for Other Plant Growth Promoting Activities

241 Indole Acetic Acid Siderophore Hydrogen CyanideOrganic Acid and Ammonia Production Indole acetic acid(IAA) production by bacterial isolates was determined in LBbroth supplemented with L-Tryptophan (500 120583gmL) at 2448 and 72 h as described by Patten and Glick [19] For thisbacterial cells were removed by centrifugation at 10000 rpmfor 5min at 4∘C One mL of the supernatant was mixedwith 4mL of Salkowskirsquos reagent in the ratio of 1 4 andincubated at room temperature for 20min Development ofa pink colour indicated indoles The absorbance of super-natant mixture (supernatant + Salkowskirsquos reagent) for indoleproduction was measured at 530 nm and quantity of indoleswas determined by comparison with a standard curve usingan IAA standard graph Siderophore production was deter-mined by using blue indicator dye and chrome azurol S agar[20] Bacterial isolates exhibiting orange halo zone on chromeazurol S agar after 5 d of incubation at 28∘C were consideredpositive for the production of siderophores For hydrogencyanide (HCN) production the methodology described byBakker and Schippers [21] was used Isolates were grownon plates of tryptic soy agar (10) amended with glycine(44 g Lminus1) and FeCl

3sdotH2O (03mM) A change from yellow

to orange red brown or reddish brown was recorded asan indication of weak moderate or strongly cyanogenicpotential respectively Organic acid production potential ofdifferent isolates was analyzed using thin layer chromatog-raphy (TLC) on Silica-G (Merck chemicals) gel plates usingdifferent solvent systems Finally ammonia production testwas performed by growing selected isolated in peptone waterfor 72 h at 30∘C Change in colour after addition of 1mLNesslerrsquos reagent (K

2HgI4 14) in each tube was observed

The presence of faint yellow colour indicates small amountof ammonia and deep yellow to brownish colour indicatesmaximum ammonia production

242 Antifungal Activity Antagonistic nature of bacterialisolate against A brassicaceae was determined by employ-ing dual culture technique Briefly bacterial isolates werestreaked at the centre of 90mm petriplate containing PDAand incubated for 36 h at 25 plusmn 2∘C A plug of fungus wasthen placed on the edge of petriplate at both sides of streakedculture Plates were incubated at 25 plusmn 2∘C for 7 dThe radii ofthe fungal colony towards and away from the bacterial colonywere measured The percentage of growth inhibition wascalculated using the formula inhibition = (119877minus119903)119877 where 119903is the radius of the fungal colony opposite the bacterial colonyand 119877 is the maximum radius of the fungal colony away fromthe bacterial colony [22]










4and 5mL HNO














0

5

10

15

20

25

30

35

40


IAA

cont

ent (120583

gm

L)

24h48h

72h






0

100

200

300

400

500

600

700

800


24h48h

72h

Phos

phor

us co

nten

t (120583

gm

L)





2









Acknowledgments


References















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology










4and 5mL HNO














0

5

10

15

20

25

30

35

40


IAA

cont

ent (120583

gm

L)

24h48h

72h






0

100

200

300

400

500

600

700

800


24h48h

72h

Phos

phor

us co

nten

t (120583

gm

L)





2









Acknowledgments


References















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology







0

5

10

15

20

25

30

35

40


IAA

cont

ent (120583

gm

L)

24h48h

72h






0

100

200

300

400

500

600

700

800


24h48h

72h

Phos

phor

us co

nten

t (120583

gm

L)





2









Acknowledgments


References















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

100

200

300

400

500

600

700

800


24h48h

72h

Phos

phor

us co

nten

t (120583

gm

L)





2









Acknowledgments


References















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





Acknowledgments


References















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

research article evaluation of phytase producing bacteria ......solubilization index (s.i.) was...

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