BalayoganSivasankariandMarimuthuAnandharajdownloads.hindawi.com/journals/aag/2014/248591.pdf · In...

14
Research Article Isolation and Molecular Characterization of Potential Plant Growth Promoting Bacillus cereus GGBSTD1 and Pseudomonas spp. GGBSTD3 from Vermisources Balayogan Sivasankari and Marimuthu Anandharaj Department of Biology, Gandhigram Rural Institute-Deemed University, Gandhigram, Dindigul, Tamil Nadu 624 302, India Correspondence should be addressed to Balayogan Sivasankari; [email protected] Received 1 May 2014; Accepted 28 July 2014; Published 1 September 2014 Academic Editor: Tibor Janda Copyright © 2014 B. Sivasankari and M. Anandharaj. is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Vermicompost was prepared from leaf materials of Gliricidia sepium + Cassia auriculata + Leucaena leucocephala with cow dung (1 : 1 : 2) using Eudrilus eugeniae (Kinberg) and Eisenia fetida for 60 days. Nineteen bacterial strains which have the capability to fix nitrogen, solubilize inorganic phosphate, and produce phytohormones were isolated from vermicompost, vermisources, and earthworm (fore, mid, and hind) guts and tested for plant growth studies. Among the bacterial strains only five strains had both activities; among the five Bacillus spp. showed more nitrogen fixing activity and Pseudomonas spp. showed more phosphate solubilizing activity. Hence these bacterial strains were selected for further molecular analysis and identified Bacillus cereus GGBSTD1 and Pseudomonas spp. GGBSTD3. Plant growth studies use these two organisms separately and as consortium (Bacillus cereus + Pseudomonas spp.) in (1 : 1) ratio at different concentrations using Vigna unguiculata (L.) Walp. at different day intervals. e germination percent, shoot length, root length, leaf area, chlorophyll a content of the leaves, chlorophyll b content of the leaves, total chlorophyll content of the leaves, fresh weight of the whole plant, and dry weight of the whole plant were significantly enhanced by the consortium (Bacillus cereus + Pseudomonas spp.) of two organisms at 5 mL concentrations on the 15th day compared to others. 1. Introduction Nowadays, under the modern agricultural practices, chemi- cal fertilizers are used to boost the crop production. But the application of chemical fertilizers affects the total productiv- ity of the crops and in the long run the soil becomes sterile and unfit for cultivation practices. Hence, in order to enhance the fertility status of the soil, the natural way of feeding the soil with different types of organic inputs (composts, vermicomposts, biofertilizers, farmyard manure, etc.) has been developed so as to ensure sustained productivity [1]. However, a better understanding of nutrient cycling and the factors governing their decomposition in soil is imper- ative for implementing sustainable management practices. Nutrient cycling in soil involves chemical, biochemical, and physicochemical reactions, with the biochemical reactions being catalyzed by soil enzymes associated with viable cells of microbial origin and plant roots. erefore, any factor that affects soil microbial population will necessarily alter soil enzyme activity [2]. Sustainability of agricultural systems has become an important issue all over the world. Many of the issues of sustainability are related to soil quality and its change with time. It is well known that intensive cultivation has led to a rapid decline in organic matter and nutrient levels besides affecting soil physical properties. Conversely, management practices with organic materials influence agricultural sustainability by improving physical, chemical, and biological properties of soils [3]. Recently, there is increasing interest in the potential of vermicomposts, as plant growth media and as soil amend- ments. Vermicomposts are finely divided peat-like materials with high porosity, aeration, drainage, water-holding capac- ity, and microbial activity, which make them excellent soil amendments or conditioner. It is a sustainable source of Hindawi Publishing Corporation Advances in Agriculture Volume 2014, Article ID 248591, 13 pages http://dx.doi.org/10.1155/2014/248591

Transcript of BalayoganSivasankariandMarimuthuAnandharajdownloads.hindawi.com/journals/aag/2014/248591.pdf · In...

Page 1: BalayoganSivasankariandMarimuthuAnandharajdownloads.hindawi.com/journals/aag/2014/248591.pdf · In Vitro Nitrogen Fixing Activity. e bacterial isolates were screened for nitrogen

Research ArticleIsolation and Molecular Characterization of Potential PlantGrowth Promoting Bacillus cereus GGBSTD1 and Pseudomonasspp GGBSTD3 from Vermisources

Balayogan Sivasankari and Marimuthu Anandharaj

Department of Biology Gandhigram Rural Institute-Deemed University Gandhigram Dindigul Tamil Nadu 624 302 India

Correspondence should be addressed to Balayogan Sivasankari balayogansivasankarigmailcom

Received 1 May 2014 Accepted 28 July 2014 Published 1 September 2014

Academic Editor Tibor Janda

Copyright copy 2014 B Sivasankari and M Anandharaj This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Vermicompost was prepared from leaf materials of Gliricidia sepium + Cassia auriculata + Leucaena leucocephala with cow dung(1 1 2) using Eudrilus eugeniae (Kinberg) and Eisenia fetida for 60 days Nineteen bacterial strains which have the capabilityto fix nitrogen solubilize inorganic phosphate and produce phytohormones were isolated from vermicompost vermisourcesand earthworm (fore mid and hind) guts and tested for plant growth studies Among the bacterial strains only five strains hadboth activities among the five Bacillus spp showed more nitrogen fixing activity and Pseudomonas spp showed more phosphatesolubilizing activity Hence these bacterial strains were selected for further molecular analysis and identified Bacillus cereusGGBSTD1 and Pseudomonas spp GGBSTD3 Plant growth studies use these two organisms separately and as consortium (Bacilluscereus+Pseudomonas spp) in (1 1) ratio at different concentrations usingVigna unguiculata (L)Walp at different day intervalsThegermination percent shoot length root length leaf area chlorophyll a content of the leaves chlorophyll b content of the leaves totalchlorophyll content of the leaves fresh weight of the whole plant and dry weight of the whole plant were significantly enhanced bythe consortium (Bacillus cereus + Pseudomonas spp) of two organisms at 5mL concentrations on the 15th day compared to others

1 Introduction

Nowadays under the modern agricultural practices chemi-cal fertilizers are used to boost the crop production But theapplication of chemical fertilizers affects the total productiv-ity of the crops and in the long run the soil becomes sterileand unfit for cultivation practices Hence in order to enhancethe fertility status of the soil the natural way of feedingthe soil with different types of organic inputs (compostsvermicomposts biofertilizers farmyard manure etc) hasbeen developed so as to ensure sustained productivity [1]However a better understanding of nutrient cycling andthe factors governing their decomposition in soil is imper-ative for implementing sustainable management practicesNutrient cycling in soil involves chemical biochemical andphysicochemical reactions with the biochemical reactionsbeing catalyzed by soil enzymes associated with viable cells

of microbial origin and plant roots Therefore any factorthat affects soil microbial population will necessarily altersoil enzyme activity [2] Sustainability of agricultural systemshas become an important issue all over the world Manyof the issues of sustainability are related to soil qualityand its change with time It is well known that intensivecultivation has led to a rapid decline in organic matterand nutrient levels besides affecting soil physical propertiesConversely management practices with organic materialsinfluence agricultural sustainability by improving physicalchemical and biological properties of soils [3]

Recently there is increasing interest in the potential ofvermicomposts as plant growth media and as soil amend-ments Vermicomposts are finely divided peat-like materialswith high porosity aeration drainage water-holding capac-ity and microbial activity which make them excellent soilamendments or conditioner It is a sustainable source of

Hindawi Publishing CorporationAdvances in AgricultureVolume 2014 Article ID 248591 13 pageshttpdxdoiorg1011552014248591

2 Advances in Agriculture

macro- and micronutrients which enlivens the soil throughpartial substitution of the horticultural container mediaEnhancement in plant growth after substitution of soils orgreenhouse container media with composts is attributedto modifications in soil structure change in water avail-ability increased availability of macro- and micronutrientsstimulation of microbial activity and augmentation of theactivities of critical substances by microorganisms throughinteractions with earthworms [4] The vermicompost is richin available nutrients soil beneficial microbes and plantgrowth promoting substances

Research during the past few decades has led to theidentification of certain biological organisms and their prod-ucts that could potentially be used as fertilizer sources Thisstrategy of fertilizing the soil with biological sources has beenwidely accepted and recognized as a viable alternative to theapplication of chemical fertilizers Nitrogen (N) fixing andphosphate (P) solubilizing bacteria may be important forplant nutrition by increasingN andPuptake by the plants andplaying a significant role as plant growth promoting bacteriain the biofertilization of crops Increasing and extendingthe role of biofertilizers could reduce adverse environmentaleffects caused by chemical fertilizers Nitrogen is one ofthe basic requirements for the growth productivity andyield of plants On a worldwide basis it is estimated thatabout 175 million tons of nitrogen per year is added tosoil through biological nitrogen fixation Meanwhile super-phosphate fertilizer is expensive and is in short supply andhence biofertilizers can bridge the gap Soluble fertilizerphosphorus (P) is the worldrsquos second largest bulk agriculturalchemical under use and as such is absolutely essential for foodproduction However most soil phosphorus approximately95ndash99 percent is present in the form of insoluble phosphatesand hence cannot be utilized by plants [5] To increasethe availability of phosphorus to plants large amounts offertilizers are used on a regular basis But after applicationa large proportion of the fertilizer phosphorus is quicklytransferred to the insoluble form [6] Therefore a very littlepercentage of the applied phosphorus only is used makingcontinuous application necessary It has been reported thatmany soil fungi and bacteria can solubilize inorganic phos-phates [7] The Bacillus species offer several advantages overthe other genera because of their capacity to produce sporesin unfavorable environmental conditions This characteristicfacilitates the conversion of spore suspensions to powderformulations without killing bacteria [8] Vermicompost isrich in NPK plant growth promoters and other nutrientsand when applied the soil will be enriched with a varietyof nutrients that will become available for the indigenousmicroflora

The PGPR have been known to directly enhance plantgrowth by a variety of mechanisms namely fixation of atmo-spheric nitrogen that is transferred to the plant productionof siderophores that chelate iron and make it available tothe plant root solubilization of minerals such as phosphorusand synthesis of phytohormones [9] Plant growth promotingbacteria (PGPB) stimulate plant growth by nitrogen fixation[10] solubilization of nutrients [11] and production of growthhormones and 1-aminocyclopropane-1-carboxylate (ACC)

The present paper describes the potential of PGPR isolatesfrom vermisources and their plant growth stimulating activ-ity in pot trials under ambient conditions Further studies willclarify the potential use of these bacteria used as biofertilizers

The main objective of this study was to isolate iden-tify and characterize the potential plant growth promotingrhizobacteria from vermisources as well as evaluating theirplant growth potential under controlled environment andcharacterizing the isolated bacteria using various moleculartechniques These bacteria can be considered promisingcandidates for application in sustainable agriculturalmanage-ment

2 Materials and Methods

21 Vermicompost Preparation For the present study epigeicearthworms Eudrilus eugeniae (Kinberg) and Eisenia fetida(Savigny) were collected from the breeding stock of theDepartment of Biology GandhigramRural Institute-DeemedUniversity Gandhigram Tamil Nadu India and leaf mate-rials of Gliricidia sepium Jacq Leucaena leucocephala (Lam)De Wit and Cassia auriculata Linn were collected fromGandhigram campus The leaf materials were separatelysubjected to predigestion for 15 days by sprinkling wateron the heap and covering it with gunny bag and turning itperiodically in order to release out the initial heat producedduring decomposition of organic material The changes intemperature were observed every three days up to 15 daysThe vermibeds were prepared in plastic containers of 45 times35 times 15 cm size and the substrate was moistened to hold60ndash80 percent moisture and kept for 24 hours stabilization20 numbers of healthy clitellate E eugeniae and 30 numbersof E fetida were separately introduced in the vermibedsThe vermicomposting trials were carried out in the rearingroomwith the relative humidity and the temperature of 75ndash85percent and 26ndash28∘C respectively The substrate was turned(mixed) once in a week and maintained up to 60 days Theexperiment was carried out with three replicates for eachsubstrate with proper control [12]

22 Microbial Study The total microbial counts in termsof colony forming units (CFU) of bacteria in the vermi-composts vermicasts and earthworm (fore mid and hind)guts were determined every 15 days (0 15 30 45 and 60 d)using standard plate count method From the total colonyforming units only those bacterial colonies (19 bacterialcolonies)which showedpredominant growthwere restreakedonto appropriate agar medium to obtain pure cultures andsubjected to characterization and identification

23 Screening for Biofertilization Activities

231 In Vitro Nitrogen Fixing Activity The bacterial isolateswere screened for nitrogen fixing ability using nitrogen-freeliquidmedium [13] Nitrogen-freemediumwas prepared andsterilized The strains were inoculated separately on selectivenitrogen-free liquid medium All the test organisms wereincubated at 37∘C plusmn 2∘C for 7 days The nitrogen fixing

Advances in Agriculture 3

activities were observed on the basis of the formation ofturbidity in the flasks Among all the nineteen bacterialisolates tested only five bacterial isolates formed significantlyhigher rate of turbidity in the nitrogen-free liquid mediumand selected for further molecular and plant growth studies

232 In Vitro Phosphate Solubilization All bacterial iso-lates were screened for inorganic phosphate solubilizationusing Pikovskayarsquos agar medium the selective medium forphosphate solubilization test [14] Pikovskayarsquos agar mediumwas prepared and sterilized and the bacterial strains wereinoculated separately on the selective medium containing 05percent of (wv) tricalcium phosphate (Ca

3PO4) as complex

insoluble phosphate source All the test plates of the bacteriawere incubated at 37∘C plusmn 2∘C for 10 days After incubationfor up to 7 days at 30∘C formation of yellow halos andorclearing zones was evaluated The results were expressed assolubilization index (SI) and they were measured using thefollowing formula [15]

SI =Colony diameter + halozone diameter

Colony diameter (1)

Five bacterial strains which showed phosphate solubiliz-ing activity were identified and grouped as phosphate sol-ubilizing bacteria (PSB) The bacterial strain which showedhighest SI was selected for further molecular and plantgrowth studies

24 Production of Phytohormones (IAA) The bacterial cul-tures were inoculated in nutrient broth with tryptophan(5 120583gmL) and incubated at 28 plusmn 2∘C for 5 days Afterincubation cultures were centrifuged at 3000 rpm for 30minTwo milliliters of the supernatant was mixed with 2 drops oforthophosphoric acid and 4mL of Salkowskirsquos reagent (50mLof 35 perchloric acid + 1mL 05 FeCl

3) and incubated in the

dark for 25 minutes Development of pink colour indicatesindole-3-acetic acid (IAA) production The optical densitywas measured at 530 nm using Spectronic 200 (India) Thequantity of IAA production was estimated using standardIAA graph and expressed asmicrograms permilliliter [16 17]

25 Siderophore Production A qualitative assay ofsiderophore production was conducted in Chrome Azurol S(CAS) agar medium [18] CAS agar plates were prepared andspot-inoculated with test organism and incubated at 30∘C for3ndash5 days Change of blue color of the medium surroundingthe bacterial growth to fluorescent yellow indicated theproduction of siderophore Bacillus subtilis was chosen as apositive control [19 20]

26 16S rDNA Gene Amplification and Sequencing GenomicDNA from each isolate was extracted using the modifiedmethod of Smoker and Barnum [21] The extracted DNAwas dissolved in 20120583L TE buffer and used as the templatefor the PCR reactions PCR amplifications were performedin a total volume of 50 120583L by mixing 20 ng of the templateDNA with 25mM concentrations of each deoxynucleotide

triphosphate and 1 120583m of each universal primer of Bac8uf(51015840-AGAGTTTGATCCTGGCTCAG-31015840) and Univ1492r (51015840-CTACGGCTACCTTGTTACGA-31015840) described by Edwars etal [22] The thermocycling profile was carried out with aninitial denaturation at 95∘C (4min) followed by 30 cyclesof denaturation at 95∘C (1min) annealing at 56∘C (30 s)extension at 72∘C (1min) and a final extension at 72∘C(10min) in an Eppendorf Gradient thermocycler The PCRamplified rDNA were purified by using the Quick PCRpurification kit (Bangalore Genie India) 16S rRNA genesequence of the isolate was compared with 16S rRNA genesequences available by the BLAST search in the NCBIGenBank database (httpwwwncbinlmnihgov) The anal-ysis of alignment and homology of the partial nucleotidesequence of Bacillus sp was carried out by the basic localalignment search tool (BLAST) The PCR products werestored at 4∘C Aliquots of the PCR products were separatedby 2 agarose gel electrophoresis in TAE buffer (pH 80)A 100 bp DNA marker (Sigma Bangalore) was used as areference Gels were stainedwith ethidiumbromide (11 gmL)and visualized under UV light

27 Nucleotide Sequence Accession The sequence obtained inthis study was deposited in the GenBank (USA) nucleotidesequence database under the accessionnumberGQ413962 (Bcereus) and HM753262 (Pseudomonas spp)

28 Phylogenetic Analysis The 16S rRNA gene sequenceswere obtained in the present study and the taxonomicallyrelated Bacillus spp were retrieved from the NationalCenter from Biotechnology Information (NCBI) database(httpwwwncbinlmnihgov) All the sequences werealigned using multiple sequence alignment programCLUSTAL W developed by Higgins et al [23] The pairwiseevolutionary distances were computed using the methodof Kimura [24] The multiple distance matrix obtained wasthen used to construct phylogenetic trees using neighbourjoining method of Saitou and Nei [25] Phylogenetic treeswere constructed by using Mega 5 software (MolecularEvolutionary Genetic Analysis)

29 Prediction of RNA Secondary Structure The secondarystructure of 16S rDNA of Bacillus spp was predicted usingthe bioinformatics Genebee tool

210 Restriction Site Analysis in 16S rDNA The restrictionsites in DNA of Bacillus spp were analyzed using theNEB Cutter program version 20 tools available online athttptoolsnebcomNEBcutter2indexphp

211 Fermentation Process For the preparation of inocu-lum 50mL of nutrient broth medium (HiMedia MumbaiIndia) was inoculated with appropriate overnight bacte-rial culture and incubated at 37∘C for 24 h at 120 rpm(OD540= 045 10

7-108 CFUmLminus1) The cell-free super-natant was obtained by centrifuging the fermented broth at

4 Advances in Agriculture

10000 rpm for 10minThe supernatantwas analyzed for plantgrowth promotion at various concentrations

212 Evaluation of Plant Growth Promoting Activity underControlled Environment The nitrogen fixing bacteria (Bacil-lus spp) and the phosphate solubilizing bacteria (Pseu-domonas spp) were separately tested for plant growth pro-moting activity and also as consortium (1 1) at 5 differentconcentrations that is 1 2 3 4 and 5mL at three differentday intervals (5 10 and 15 days) with proper control throughExp1 Exp2 Exp3 and Exp0 respectively usingVigna unguic-ulata (L) Walp The seeds (30 seeds in each pot) were sownin plastic pots containing sterilized vermiculite and arrangedin a completely randomized factorial design The seedlingswere grown in a greenhouse at a temperature of 28ndash32∘Cand 85 relative humidity The pots were watered to 50water-holding capacity and were maintained at this moisturecontent by watering to weight every day and plant growthwasobserved for 15 days Parameters such as germination percentshoot length root length leaf area chlorophyll content of theleaves fresh weight of the whole plant and dry weight of thewhole plant were measured using standard procedure

213 Statistical Analysis The following statistical tools wereused for the analyses and interpretation of the data Theexperimental results are presented in the form of tablesand graphs using Microsoft Excel 2007 Data obtained fromthe different treatments were statistically analyzed using theone-way ANOVA and the mean values were compared bythe Duncanrsquos multiple range test for multiple comparisonsDifferences were considered significant at the 005 level usingOrigin software (Version 850) 2010 Origin Lab Corpora-tion

3 Results

31 Isolation and Characterization of Bacterial Isolates Atotal of nineteen bacterial strains were isolated from vermi-compost vermicast and earthworm (fore mid and hind)guts (Table 1) The morphological and biochemical char-acteristics of bacterial isolates were elucidated in Table 1All the isolates were tested for plant growth promotingactivity Among the 19 bacterial isolates only five bacterialstrains showed plant growth promoting activity Based oncolony morphology microscopic observations and culturalbiochemical and physiological properties the bacteriumwasgiven the name Bacillus spp Alcaligenes spp Erwinia sppSerratia spp or Pseudomonas sppThese five bacterial strainswere able to grow in the nitrogen-free medium and they werealso identified as phosphate solubilizing microorganisms onthe basis of their solubilization index (Table 2) Bacillus spphave the highest SI (3855) followed by Pseudomonas spp(3522) and Serratia spp (3045) All the isolates produceda significant amount of IAA from the medium in the rangeof 113 to 242120583gmL Bacillus spp showed higher IAAproduction (242120583gmL) and Serratia spp showed lower IAAproduction (113 120583gmL) All the strains (except Alcaligenes

spp) were positive for siderophore production showing ayellow zone on the CAS agar medium plate (Table 2)

32 Molecular Studies The partial 16S rRNA sequencescarried out in the present study for Bacillus spp andPseudomonas spp covered a stretch of approximately 1500nucleotides for each About half of the sequences found in theclone library showed only slight relationship to other knownsequences while the other half were highly similar (approx-imately 95 percent sequence identity) to other databaseentries for Bacillus spp and Pseudomonas spp Less than 05percent of all nucleotides was found to be unique within theconserved regions of the cloned sequence and could almostalways be related to reading errors in ambiguous regions ofthe sequencing gel On the basis of phylogenetic analysisof 16S rDNA partial sequences Bacillus spp GGBSTD1 isidentified as Bacillus cereus and Pseudomonas spp GGBSTD3is identified as Pseudomonas spp Phylogenetic analyses ofthe strains based on the neighbor joining method wererepresented in Figures 1 and 2 Among eubacteria the meanguanine and cytosine (G + C) content of genomic DNAvaries from approximately 25 to 75 That the bacterialgenomic G + C content is somehow related to phylogeny hasbeen suggested [26 27] The phylogenetic tree of eubacterial16S rRNA clearly indicates this relationship Gram-positivebacteria Bacillus subtilis have genomic G + C 42 Gram-negative bacteria with intermediate G + C content such asEscherichia coli (50) Serratia marcescens (58) Salmonellatyphimurium (51) and Pseudomonas fluorescens (60)belong to the common Gram-negative bacteria [28] Bacilluscereus have the G + C content 53 and A + T content 47similarly Pseudomonas spp having G + C content 53 andA + T content 47 Most functional RNA molecules havecharacteristic secondary structures that are highly conservedin evolution In this study RNA secondary structures ofBacillus cereus and the Pseudomonas spp were predictedBacillus cereus have free energy of minus2941 kkalmol andPseudomonas spp have free energy of minus2998 kkalmol

33 Evaluation of Biofertilization Abilities under ControlledConditions Germination percentage of Vigna unguiculataseeds sown in vermiculite supplemented with Bacillus cereus(Exp1) with Pseudomonas spp (Exp2) and with consortium(Exp3) (1 1 ratio) at five different concentrations (1 2 3 4and 5mL) and in the control (Exp0) is shown in Figure 3The highest germination percentage was observed in Exp3(consortium) at 5mL concentration compared to others Theincrease in germination percentage and in other growthparameters can be attributed to the combined effect of thenitrogen fixing and phosphate solubilizing microbes whichare able to fix atmospheric nitrogen and solubilize P andalso produce growth promoting substancesThese organismshave been isolated from vermisources and this observationindicates the reason for promotion and enhancement of cropgrowth and yield when vermicompost is applied It is a proofthat these symbioticmicrobes present in vermisources benefitthe soil fertility Observation on growth parameters such asshoot length root length and leaf area of V unguiculata

Advances in Agriculture 5

Table1

Predo

minantb

acteria

lstrains

isolated

from

verm

isourcesand

theirb

iochem

icalcharacteris

ticsT1-con

trolmdash

Gsepium

+L

leucocephala+

cowdu

ngin

(112)(worm

free)T

2mdashG

sepium

+L

leucocephala+cowdu

ngin

(112)+

EeugeniaeT3

mdashG

sepium

+L

leucocephala+cowdu

ngin

(112)+

EfetidaT4

-con

trol(2)mdashC

auric

ulata+L

leucocephala+cow

dung

in(112)(worm

free)T

5mdashC

auric

ulata+L

leucocephala+cowdu

ngin

(112)+

EeugeniaeT

6mdashC

auric

ulata+L

leucocephala+cowdu

ngin

(112)+

Efetida

Snu

mber

Samples

ource

Biochemicalcharacteris

ticsc

Nam

eofthe

strain

Gramrsquos

reactio

naCell

shape

Motility

bIndo

letest

Methyl

Red

test

Voges

Proskauer

test

Citrate

Utilization

test

Catalase

test

Urease

test

Gelatin

hydrolysis

test

Nitrate

redu

ctase

test

Starch

hydrolysis

test

Casein

hydrolysis

test

Glucose

utilizatio

ntest

1Ve

rmicom

post

(T2)

+Cocciminus

minus+

minusminus

+minus

++

minusminus

minusStaphylococcus

spp

2Ve

rmicom

post

(T3)

+Ro

d+

minusminus

minusminus

+minus

minus+

minusminus

+Co

rynebacterium

spp

3Ve

rmicom

post

(T5)

+Ro

dminus

++

++

+minus

++

+minus

+Ba

cillusspp

4Ve

rmicom

post

(T6)

+Cocciminus

++

minusminus

+minus

+minus

minusminus

+Micrococcusspp

5Ve

rmicast(T3

)+

Cocciminus

++

minusminus

minus+

++

+minus

minusKlebsiella

spp

6Ve

rmicast(T5

)+

Rod

minus+

minusminus

+minus

+minus

+minus

minus+

Streptococcusspp

7

Verm

icast(T6

)+

Cocciminus

+minus

++

minusminus

++

minusminus

+En

terobacter

spp

8Ea

rthw

orm

hind

gut(T2

)+

Cocciminus

minus+

+minus

+minus

minusminus

++

+Trico

ccocus

spp

9Ea

rthw

orm

hind

gut(T3

)+

Cocciminus

minus+

minus+

++

minus+

minusminus

minusYersiniaspp

10

Earthw

orm

hind

gut(T5

)+

Cocciminus

minusminus

minusminus

++

++

minusminus

minusMicrococcusspp

11

Verm

icom

post

(T1)

minusRo

dminus

++

minusminus

minusminus

++

minusminus

+Escherich

iaspp

12

Verm

icom

post

(T2)

minusRo

dminus

minus+

minusminus

minus+

++

minusminus

+Proteusspp

13

Verm

icom

post

(T4)

minusRo

dminus

minusminus

+minus

+minus

minus+

++

+Pa

racoccus

spp

14

Verm

icom

post

(T5)

minusRo

d+

+minus

minusminus

++

++

minusminus

minusPseudomonas

spp

15

Verm

icast(T2

)minus

Rod

+minus

+minus

++

+minus

+minus

minusminus

Citro

bacter

spp

16

Verm

icast(T3

)minus

Cocciminus

++

minusminus

+minus

+minus

minusminus

minusErwiniaspp

17Ve

rmicast(T5

)minus

Rod

++

++

++

minus+

minusminus

minusminus

Alcaligenesspp

18

Verm

icast(T6

)minus

Cocciminus

minus+

++

+minus

++

minusminus

minusSerratiaspp

19

Earthw

orm

mid

gut(T2

)minus

Rod

+minus

minusminus

+minus

minusminus

minusminus

minusminus

Pediococcusspp

a Gramrsquossta

ining(+)p

ositive(minus)n

egative

b Motility(+)

motile(minus)n

onmotile

c Biochem

icalcharacteris

tics(+)p

ositive(minus)n

egative

6 Advances in Agriculture

Bacillus cereus strain GGBSTD2 16S ribosomal RNA gene

Bacillus cereus strain SU-6 16S ribosomal RNA gene

Bacillus cereus strain JB0013 16S ribosomal RNA gene

Bacillus cereus isolate PN24 16S ribosomal RNA gene

Bacillus cereus strain JB0005 16S ribosomal RNA gene

Bacillus sp G2DM-85 16S ribosomal RNA gene

Bacillus cereus strain JBE0009 16S ribosomal RNA gene

Bacillus sp EB460 16S ribosomal RNA gene

Bacillus cereus strain 421-3R 16S ribosomal RNA gene

Bacillus sp EB447 16S ribosomal RNA gene

Bacillus sp EB470 16S ribosomal RNA gene

Bacillus sp EB421 16S ribosomal RNA gene

Bacillus sp EB431 16S ribosomal RNA gene

Bacillus sp EB462 16S ribosomal RNA gene

Bacillus sp EB429 16S ribosomal RNA gene

Bacillus cereus strain 421-4R 16S ribosomal RNA gene

Bacillus sp EB480 16S ribosomal RNA gene

Bacillus sp EB13 16S ribosomal RNA gene

Uncultured compost bacterium partial 16S rRNA gene

Bacillus thuringiensis gene for 16S rRNA

Figure 1 Neighbor joining phylogenetic tree of full 16S rRNA gene sequences showing the relationship among Bacillus cereus GGBSTD1and representatives of some related taxa The sequence data for several closely related Bacillus cultures were recovered from GenBank andincluded in the tree

Table 2 The phosphate solubilizing activity phytohormone (IAA) production and siderophores production of the bacterial isolates

Name of the organism Phosphate solubilization index (SI) IAA production (120583gmL)a Siderophores productionb

Bacillus spp 3855 242 +++Alcaligenes spp 2930 147 minus

Erwinia spp 2890 113 +Serratia spp 3045 168 ++Pseudomonas spp 3522 196 +++aIndole acetic acid production in culture media supplemented with tryptophan (5 120583gmL) after 5 daysbIn vitro siderophores production minus represents the absence of siderophores production and +++ represents gt10mm wide yellow-orange zone

is given in Tables 3 and 4 respectively The shoot length(1147 plusmn 006 cm) root length (1027 plusmn 006 cm) and leafarea (905 plusmn 026 cm2) of the plant get significantly (119875 lt0001) increased in Exp3 (consortium of Bacillus cereus +Pseudomonas spp [1 1]) at 5mL concentration on the 15thday compared to other experimentsThe chlorophyll a b andtotal chlorophyll content of the leaves on d 5 d 10 and d 15are given in Table 5 The chlorophyll a (010 plusmn 009mgg offresh leaf) chlorophyll b (1085 plusmn 016mgg of fresh leaf)and total chlorophyll (1095plusmn007mgg of fresh leaf) content

of leaves were significantly (119875 lt 0001) higher in Exp3(consortium of Bacillus cereus + Pseudomonas spp (1 1))at 5mL concentration on the 15th day compared to otherexperiments Observations on the fresh weight of the wholeplant are given in Table 6 The fresh weight of the wholeplant (092 plusmn 000 g) was significantly (119875 lt 0001) higherin Exp3 (consortium of Bacillus cereus + Pseudomonas spp(1 1)) at 5mL concentration on the 15th day compared toother experiments Observations on the dry weight of thewhole plant are given in Table 6 The dry weight of the whole

Advances in Agriculture 7

Pseudomonas fluorescens strain LMG 5825 16S ribosomal RNA gene

Pseudomonas fluorescens strain LMG 5829 16S ribosomal RNA gene

Pseudomonas fluorescens strain LMG 14676 16S ribosomal RNA gene

Pseudomonas sp W15Feb13 16S ribosomal RNA gene

Pseudomonas sp W15Feb28 16S ribosomal RNA gene

Pseudomonas sp p50 16S ribosomal RNA gene

Uncultured bacterium clone 661193 16S ribosomal RNA gene

Pseudomonas sp BSs20166 16S ribosomal RNA gene

Pseudomonas sp 28zhy partial 16S rRNA gene

Uncultured bacterium clone AYRV1-027 16S ribosomal RNA gene

Uncultured Pseudomonadaceae bacterium clone290cost002-P3L-2032 16S ribosomal RNA gene

Pseudomonas sp GGBSTD3 16S ribosomal RNA gene

Pseudomonas sp DK3 16S ribosomal RNA gene

Pseudomonas reactans strain AMP-13 16S ribosomal RNA gene

Figure 2 Neighbor joining phylogenetic tree of full 16S rRNA gene sequences showing the relationship among Pseudomonas spp GGBSTD3and representatives of some related taxa The sequence data for several closely related Pseudomonas cultures were recovered from GenBankand included in the tree

plant (009 plusmn 000 g) was significantly (119875 lt 0001) higherin Exp3 (consortium of Bacillus cereus + Pseudomonas spp(1 1)) at 5mL concentration on the 15th day compared toother experiments

4 Discussion

Inoculation of these newly identified nitrogen fixing andphosphate solubilizingmicrobes in the plant growthmediumenhanced various physiological activities culminating inhigher biomass production in V unguiculata Since this is anonhazardous way of fertilization of crop plants it is veryrelevant to a developing country like India This techniquecan save the farmers of India from the problem posed byhigh cost of fertilizers by offering a comparatively inexpensivealternative (biofertilizers) while additionally preventing thedegradation of the soil and thereby ensuring sustainable agri-culture Bess (1999) reported that the content in compostingneeds to be determined through the concentration of sixfunctional groups of microorganisms such as aerobic bacte-ria anaerobic bacteria fungi actinomycetes Pseudomonasand nitrogen fixing bacteria [29] Now there are ways toevaluate the concentrations of these organisms in the finishedcompost and that can serve as an interpretation guide to

determine the quality of the compost as an inoculant of soilmicroorganisms Grafff (1981) and Atlavinyte have reporteda higher P content in the cast than in the surrounding soil[30] Satchell (1983) has linked the phosphatase activity inthe gut of worms to the availability of bound P in soils Thebacterial strains which have the capability to fix nitrogenonly can grow in the nitrogen-free medium [31] Hencethe nitrogen fixing ability was tested in the present studybased on the growth of bacterial strain in nitrogen- freemedium Five bacteria strains showedmore nitrogen fixationamong all the five bacterial strains tested only one strain (ieBacillus spp) showed significantly more turbidity comparedto the other strains The ultimate source of the nitrogen usedby plants is N

2gas which constitutes 78 of the Earthrsquos

atmosphere Unfortunately higher plants cannot metabolizeN2directly into protein N

2gas must be converted to a plant

available form [32] isolated many species of nitrogen fixingBacillus (B megaterium B cereus B subtilis B licheniformisand B azotoformans) from the rhizospheric zone of ricefields in Central China The widely studied Bacillus genusrepresents one of the most diverse genera in the bacilli groupNumerous Bacillus and Paenibacillus strains express plantgrowth promoting (PGP) activities and a number of thesestrains have already been commercially developed as biolog-ical fungicides insecticides and nematicides or generic plant

8 Advances in Agriculture

Table 3 Shoot length and Root length ofVigna unguiculata grown on vermiculite supplemented with Bacillus cereus with Pseudomonas sppand with their consortium (1 1) at five different concentrations (1 2 3 4 and 5mL) on the 5th the 10th and the 15th day and in the control

Experimentsa Quantity of bacterial cultures added Shoot length (cm)b Root length (cm)b

5th day 10th day 15th day 5th day 10th day 15th day

Exp0

1mL 297 (ab) 397 (a) 497 (ab) 197 (de) 283 (ab) 397 (bcd)2mL 303 (bcd) 403 (ab) 500 (ab) 200 (abc) 287 (de) 400 (a)3mL 310 (abc) 407 (a) 503 (ab) 203 (de) 290 (abc) 403 (ab)4mL 310 (bcd) 410 (bcd) 507 (bcd) 207 (a) 297 (de) 410 (abc)5mL 313 (a) 413 (ab) 510 (ab) 210 (bcd) 300 (ab) 413 (de)

Exp1

1mL 340 (ab) 440 (abc) 540 (ab) 233 (abc) 327 (bcd) 433 (f)2mL 450 (ab) 540 (abc) 647 (ab) 333 (ab) 430 (a) 540 (abc)3mL 550 (de) 630 (ab) 750 (a) 440 (de) 550 (abc) 620 (a)4mL 653 (ab) 730 (a) 833 (ab) 550 (bcd) 653 (de) 750 (abc)5mL 740 (ab) 840 (abc) 923 (f) 640 (abc) 753 (ab) 827 (ab)

Exp2

1mL 277 (bcd) 357 (f) 440 (de) 160 (de) 217 (abc) 350 (f)2mL 343 (abc) 447 (ab) 553 (ab) 253 (abc) 340 (bcd) 427 (bcd)3mL 437 (de) 543 (abc) 633 (abc) 340 (ab) 443 (f) 533 (ab)4mL 547 (bcd) 647 (de) 733 (a) 420 (bcd) 550 (ab) 633 (ab)5mL 640 (f) 733 (bcd) 833 (bcd) 533 (ab) 630 (ab) 730 (bcd)

Exp3

1mL 530 (bcd) 647 (ab) 730 (ab) 433 (de) 550 (abc) 620 (ab)2mL 643 (ab) 740 (bcd) 827 (f) 547 (a) 630 (de) 733 (ab)3mL 737 (bcd) 840 (bcd) 927 (ab) 640 (ab) 740 (bcd) 840 (bcd)4mL 847 (abc) 930 (a) 1043 (abc) 740 (f) 833 (a) 937 (abc)5mL 957 (ab) 1030 (f) 1147 (bcd) 833 (abc) 940 (ab) 1027 (bcd)

aExp0 nutrient solution (control) Exp1 Bacillus cereus Exp2 Pseudomonas spp Exp3 consortium of Bacillus cereus + Pseudomonas spp (1 1)bMeanof two repeated experiments (30 plants)Different letterswithin parenthesis indicate significant difference between treatments for each growth parameterusing Duncanrsquos multiple range test (119875 = 005)

growth promoters The use of these strains in agriculturehas recently been reviewed [33]Those strains besides havingseveral PGP properties can also fix nitrogen and also dophosphate solubilization The symbiotic fixation of nitrogenthrough inoculation of legume crops with effective rhizobiais well known [34] Asymbiotic nitrogen fixing bacteriawhich live in the rhizosphere andor endophytically oftenincrease yields of crops Many bacterial species have nitrogenfixing properties including Bacillus spp Azotobacter sppAzospirillum spp Beijerinckia spp and Pseudomonas spp[35] Nitrogen fixing bacteria have been used in foliar appli-cations inmulberryThe solubilization index (SI) was formeddue to solubilization of insoluble phosphates by organic acidsecretion [36] El-Komy (2005) indicated that Pseudomonasfluorescence and Bacillus megaterium strains were the mostpowerful phosphate solubilizers on PVK plates as well as onPikovskayarsquos broth [37] The P concentration in Pikovskayarsquosbroth increased gradually achieving a peak on the sixth dayand declined slowly during the late days In this study pHvalues decreased gradually in PVK broth during the earlyincubation days and no revival was observed during laterdays for all the tested bacterial strains Several bacteria par-ticularly those belonging to the genus Bacillus spp convertinsoluble phosphate into soluble forms by secreting organicacids such as formic acid acetic acid propionic acid citricacid fumaric acid gluconic acid glyoxylic acid ketobutyric

acid malonic acid succinic acid and tartaric acid Theseacids lower the pH and bring about the dissolution of boundforms of phosphate Some of the hydroxyl acids may chelatewith calcium and iron resulting in effective solubilization andutilization of phosphates [38]

Beneduzi et al (2008) had isolated the plant growthpromoting strain SVPR30 and identified by 16S rRNA genesequence as Bacillus spp and they had tested it for plantgrowth through in vivo experiments [39] This strain wascharacterized as a high IAA producer able to solubilize phos-phate and also fix a considerably high amount of nitrogenThe inoculation of rice with Bacillus spp SVPR30 strainshowed a significant increase in the root and shoot partswhen compared with the controls within 15 and 30 daysafter sprouting Numerous Bacillus and Paenibacillus strainsexpressed plant growth promoting (PGP) activities and anumber of these strains have been commercially developedas generic plant growth promotersThe use of these strains inagriculture has recently been reviewed [33]

Various reports indicate that coinoculation of beneficialorganisms generally increased plant growth relative to singleinoculation with a sole beneficial organism [40] Most ofthe effects of the individual microorganisms coinoculationare additive although a synergistic effect has been reportedin some cases The average root to shoot ratio was higherin B subtilis treated yam minisetts in comparison to those

Advances in Agriculture 9

Table 4 Leaf area of Vigna unguiculata grown on vermiculite supplemented with Bacillus cereus with Pseudomonas spp and with theirconsortium (1 1) at five different concentrations (1 2 3 4 and 5mL) on the 5th the 10th and the 15th days and in the control

Experimentsa Quantity of bacterial cultures added Leaf area (cm2)b

5th day 10th day 15th day

Exp0

1mL 027 (ab) 094 (abc) 407 (b)2mL 035 (ab) 102 (ab) 419 (abc)3mL 040 (abc) 125 (ab) 471 (bcd)4mL 048 (ab) 138 (abc) 510 (ab)5mL 054 (abc) 161 (b) 538 (b)

Exp1

1mL 018 (bcd) 123 (b) 601 (abc)2mL 025 (ab) 138 (abc) 632 (ab)3mL 033 (abc) 150 (ab) 684 (b)4mL 038 (ab) 168 (ab) 686 (abc)5mL 044 (abc) 191 (bcd) 733 (ab)

Exp2

1mL 011 (b) 107 (ab) 534 (b)2mL 014 (ab) 119 (ab) 559 (ab)3mL 017 (abc) 127 (ab) 579 (ab)4mL 021 (ab) 153 (abc) 631 (abc)5mL 026 (ab) 166 (b) 667 (b)

Exp3

1mL 026 (bcd) 297 (ab) 753 (bcd)2mL 033 (b) 325 (ab) 787 (ab)3mL 040 (ab) 350 (bcd) 811 (bcd)4mL 044 (b) 384 (b) 861 (b)5mL 062 (ab) 400 (bcd) 905 (ab)

aExp0 nutrient solution (control) Exp1 Bacillus cereus Exp2 Pseudomonas spp Exp3 consortium of Bacillus cereus + Pseudomonas spp (1 1)bMeanof two repeated experiments (30 plants)Different letterswithin parenthesis indicate significant difference between treatments for each growth parameterusing Duncanrsquos multiple range test (119875 = 005)

0102030405060708090

100

Concentration of bacterial cultures (mL)1 2 3 4 5

Germination ()

Exp0

Exp1 Exp3

Exp2

Figure 3 Germination percentage ofVigna unguiculata seeds sownin vermiculite supplementedwithBacillus cereus with Pseudomonasspp and with their consortium at five different concentrations (1 23 4 and 5mL) and in the control Exp0 nutrient solution (control)Exp1 Bacillus cereus Exp2 Pseudomonas spp Exp3 consortium ofBacillus cereus + Pseudomonas spp (1 1)

not treated with the bacterial culture [41] In earlier reportsroot elongation was found to occur in Sesbania aculeata byinoculation with Azotobacter spp and Pseudomonas spp in

Vigna radiata by Pseudomonas putida [16] and in Pennisetumamericanum by Azospirillum brasilense [42] Esitken et al(2006) had found out that Bacillus OSU-142 and Pseu-domonas BA-8 alone or in combination had a great potentialto increase the growth yield and nutrition of sweet cherryplant [43] BacillusM3 BacillusOSU-142 andMicobacteriumFS01 separately or in combination were found to have greatpotential for use as plant growth promoting rhizobacteriato increase production in apples and in many other crops[44] These phosphate solubilizing microbes consist pre-dominantly of fungal bacterial and actinomycetes speciescollectively called phosphate solubilizing microorganismsmdashPSM [45] There are several microorganisms which canalso solubilize the cheaper sources of phosphorous such asrock phosphate Bacteria such as Bacillus are widely usedin plant production system and are important phosphorussolubilizing microorganisms resulting in improved growthyield of crops andmetabolic activities especially in synthesisof protein [34 46]

5 Conclusions

This study illustrates the isolation of plant growth promotingbacteria from vermisources screening under in vitro con-ditions for multiple PGPR traits and their evaluation undercontrolled conditions in a pot experiment The findings ofthis study indicated that the consortium of B cereus and

10 Advances in Agriculture

Table5Ch

loroph

yllcon

tent

ofleavesofVignaun

guicu

latagrow

non

verm

iculite

supp

lementedwith

Bacilluscereuswith

Pseudomonas

sppand

with

theircon

sortium

(11)atfi

vedifferent

concentrations

(1234and

5mL)

onthe5

thdaythe10thdayandthe15thdayandin

thec

ontro

l

Experim

entsa

Quantity

ofbacterial

Cultu

resa

dded

Chloroph

yllcon

tent

(mggof

fresh

leaf)b

5thday

10th

day

15th

day

Chloroph

ylla

Chloroph

yllb

Total

chloroph

yll

Chloroph

ylla

Chloroph

yllb

Total

chloroph

yll

Chloroph

ylla

Chloroph

yllb

Total

chloroph

yll

Exp0

1mL

021

(ab)

285

(abc)

307

(a)

020

(abc)

481

(a)

501

(ab)

015

(bcd)

612

(abc)

627

(bcd)

2mL

022

(abc)

293

(ab)

315

(abc)

021

(ab)

489

(ab)

510

(bcd)

012

(abc)

618

(ab)

630

(ab)

3mL

023

(abc)

301

(ab)

315

(abc)

022

(bcd)

497

(bcd)

518

(a)

014

(a)

633

(cde)

647

(bcd)

4mL

024

(ab)

308

(f)

332

(a)

022

(a)

504

(b)

527

(abc)

015

(bcd)

641

(cde)

656

(a)

5mL

025

(f)

316

(abc)

341

(bcd)

019

(ab)

511(ab)

530

(abc)

011(cde)

645

(ab)

650

(abc)

Exp1

1mL

035

(b)

400

(cde)

435

(abc)

011(b)

581

(cde)

593

(cde)

010

(abc)

704(abc)

715(f)

2mL

026

(abc)

462

(bcd)

488

(f)

011(abc)

647

(b)

658

(abc)

006

(ab)

779(bcd)

785(a)

3mL

021

(bcd)

526

(abc)

547

(ab)

008

(bcd)

718(ab)

726(bcd)

006

(f)

847

(f)

853

(ab)

4mL

008

(f)

587

(cde)

595

(b)

011(bcd)

778(abc)

789(abc)

004

(bcd)

933(b)

937(bcd)

5mL

026

(abc)

641

(bcd)

667

(abc)

025

(cde)

834

(cde)

859

(f)

007

(abc)

976(cde)

983(abc)

Exp2

1mL

030

(abc)

354

(ab)

384

(b)

012

(a)

545

(abc)

556

(ab)

007

(bcd)

681

(abc)

688

(bcd)

2mL

029

(bcd)

420

(f)

449

(abc)

011(bcd)

610

(ab)

621

(abc)

007

(abc)

744(f)

752(bcd)

3mL

022

(ab)

504

(abc)

527

(bcd)

015

(a)

677

(cde)

692

(bcd)

006

(bcd)

812

(ab)

817

(bcd)

4mL

010

(f)

566

(a)

576

(cde)

018

(abc)

744(f)

762(ab)

005

(ab)

885

(a)

890

(abc)

5mL

006

(bcd)

622

(abc)

628

(abc)

018

(b)

810

(abc)

828

(a)

004

(cde)

948(a)

952(f)

Exp3

1mL

027

(ab)

470

(bcd)

496

(cde)

009

(ab)

668

(b)

677

(cde)

010

(a)

814

(ab)

825

(a)

2mL

020

(ab)

548

(a)

568

(f)

003

(bcd)

746(bcd)

749(ab)

009

(ab)

887

(f)

897

(ab)

3mL

005

(bcd)

584

(abc)

589

(ab)

008

(bcd)

804

(abc)

812

(cde)

010

(ab)

953(abc)

963(bcd)

4mL

023

(f)

663

(ab)

686

(cde)

007

(ab)

870

(b)

877

(f)

010

(abc)

1019

(bcd)

1029(f)

5mL

022

(ab)

744(cde)

766(a)

009

(a)

932(abc)

940(ab)

010

(a)

1085(ab)

1095(abc)

a Exp0nu

trient

solutio

n(con

trol)

Exp1B

acillus

cereusE

xp2Pseudomonas

sppE

xp3consortiu

mof

Bacilluscereus+

Pseudomonas

spp(11)

b Meanof

tworepeated

experim

ents(30plants)

Differentletterswith

inparenthesis

indicatesig

nificantd

ifference

betweentre

atmentsfore

achgrow

thparameter

usingDun

canrsquos

multip

lerangetest(119875=005)

Advances in Agriculture 11

Table 6 Fresh weight and dry weight of whole plant of Vigna unguiculata grown on vermiculite supplemented with Bacillus cereus withPseudomonas spp and with their consortium (1 1) at five different concentrations (1 2 3 4 and 5mL) on the 5th the 10th and the 15 daysand in the control

Experimentsa Quantity of bacterial cultures added Fresh weight of the whole plant (g)b Dry weight of the whole plant (g)b

5th day 10th day 15th day 5th day 10th day 15th day

Exp0

1mL 058 (bcd) 061 (abc) 064 (ab) 006 (a) 006 (abc) 006 (ab)2mL 058 (ab) 061 (a) 064 (abc) 006 (bcd) 006 (ab) 006 (ab)3mL 058 (abc) 061 (ab) 064 (bcd) 006 (abc) 006 (bcd) 006 (b)4mL 041 (abc) 061 (ab) 064 (ab) 006 (abc) 006 (ab) 006 (abc)5mL 058 (abc) 061 (abc) 064 (bcd) 006 (a) 006 (bcd) 006 (def)

Exp1

1mL 062 (abc) 065 (abc) 068 (ab) 006 (bcd) 006 (ab) 007 (bcd)2mL 065 (bcd) 068 (bcd) 071 (def) 006 (abc) 007 (a) 007 (ab)3mL 068 (abc) 071 (def) 074 (bcd) 007 (abc) 007 (ab) 007 (abc)4mL 071 (ab) 074 (abc) 077 (abc) 007 (bcd) 007 (b) 008 (bcd)5mL 074 (abc) 077 (def) 080 (ab) 007 (def) 008 (a) 008 (b)

Exp2

1mL 056 (ab) 059 (bcd) 062 (ab) 006 (ab) 006 (ab) 006 (abc)2mL 059 (abc) 062 (abc) 065 (def) 006 (ab) 006 (abc) 006 (bcd)3mL 062 (bcd) 065 (a) 068 (bcd) 006 (abc) 006 (bcd) 007 (abc)4mL 065 (abc) 068 (ab) 071 (b) 006 (ab) 007 (abc) 007 (b)5mL 068 (abc) 071 (b) 074 (a) 007 (def) 007 (abc) 007 (bcd)

Exp3

1mL 074 (ab) 077 (ab) 080 (abc) 007 (a) 008 (bcd) 008 (ab)2mL 077 (bcd) 080 (a) 083 (bcd) 008 (a) 008 (bcd) 008 (abc)3mL 080 (bcd) 083 (bcd) 086 (abc) 008 (ab) 008 (b) 009 (b)4mL 083 (bcd) 086 (abc) 089 (ab) 008 (abc) 009 (ab) 009 (bcd)5mL 086 (ab) 089 (b) 092 (def) 009 (bcd) 009 (bcd 009 (b)

aExp0 nutrient solution (control) Exp1 Bacillus cereus Exp2 Pseudomonas spp Exp3 consortium of Bacillus cereus + Pseudomonas spp (1 1)bMeanof two repeated experiments (30 plants)Different letterswithin parenthesis indicate significant difference between treatments for each growth parameterusing Duncanrsquos multiple range test (119875 = 005)

Pseudomonas spp acts as a potential biofertilizer due to itsvarious plant growth promotion abilities such as solubiliza-tion of phosphate and production of IAA and siderophore itcan be developed as potential microbial inoculant for variouscrops

Conflict of Interests

The authors declared that there is no conflict of interestsregarding this paper

Acknowledgments

Theauthors are thankful to theDepartment of BiologyGand-higram Rural InstitutemdashDeemed University GandhigramThis research was funded by University Grants Commissionunder UGC Research Fellowship in Science for MeritoriousStudents (BSR) Grant no F4-12008

References

[1] U S Bhawalkar Vermiculture Ecotechnology EarthwormResearch Institute Pune India 2nd edition 1996

[2] S Kiss D M Bularda and D Radulescu ldquoBiological Signifi-cance of Enzymes Accumulated in SoilrdquoAdvances in Agronomyvol 27 pp 25ndash87 1975

[3] D L Karlen M J Mausbach J W Doran R G Cline R FHarris and G E Schuman ldquoSoil quality a concept definitionand framework for evaluationrdquo Soil Science Society of AmericaJournal vol 61 no 1 pp 4ndash10 1997

[4] R M Atiyeh C A Edwards S Subler and J Metzger ldquoEarth-worm processed organic wastes as components of horticulturalpotting media for growing manifold and vegetable seedlingsrdquoCompost Science and Utilization vol 8 no 3 pp 215ndash223 2000

[5] N Vassilev and M Vassileva ldquoBiotechnological solubilizationof rock phosphate on media containing agro-industrial wastesrdquoApplied Microbiology and Biotechnology vol 61 no 5-6 pp435ndash440 2003

[6] S A Omar ldquoThe role of rock-phosphate-solubilizing fungiand vesicular-arbusular-mycorrhiza (VAM) in growth of wheatplants fertilized with rock phosphaterdquo World Journal of Micro-biology and Biotechnology vol 14 no 2 pp 211ndash218 1998

[7] J A J Paul and T Daniel ldquoStandardization of sampling methodfor physical characterization of municipal solid wasterdquo AsianJournal of Water Environment and Pollution vol 5 no 1 pp95ndash98 2007

[8] R Lalloo D Maharajh J Gorgens and N Gardiner ldquoA down-stream process for production of a viable and stable Bacilluscereus aquaculture biological agentrdquo Applied Microbiology andBiotechnology vol 86 no 2 pp 499ndash508 2010

[9] B Sivasankari N Kavikumar and M Anandharaj ldquoIndole-3-acetic acid production and enhanced Plant growth promotionby Indigenous bacterial speciesrdquo Journal of Current Research inScience vol 1 no 5 pp 331ndash335 2013

12 Advances in Agriculture

[10] J Han L Sun X Dong et al ldquoCharacterization of a novel plantgrowth-promoting bacteria strain Delftia tsuruhatensis HR4both as a diazotroph and a potential biocontrol agent againstvarious plant pathogensrdquo Systematic and Applied Microbiologyvol 28 no 1 pp 66ndash76 2005

[11] H Rodrıguez and R Fraga ldquoPhosphate solubilizing bacte-ria and their role in plant growth promotionrdquo BiotechnologyAdvances vol 17 no 4-5 pp 319ndash339 1999

[12] T Daniel and N Karmegam ldquoBio-conversion of selected leaflitters using an African epigeic earthworm Eudrilus eugeniaerdquoEcology Environment and Conservation vol 5 no 3 pp 271ndash275 1999

[13] J Dobereiner I E Marriel and M Nery ldquoEcological distri-bution of Spirillum lipoferum Beijerinckrdquo Canadian Journal ofMicrobiology vol 22 no 10 pp 1464ndash1473 1976

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

[15] M Edi Premono A M Moawad and P L G Vlek ldquoEffect ofphosphate-solubilizing Pseudomonas putida on the growth ofmaize and its survival in the rhizosphererdquo Indonesian Journal ofCrop Science vol 11 pp 13ndash23 1996

[16] C L Patten and B R Glick ldquoBacterial biosynthesis of indole-3-acetic acidrdquo Canadian Journal of Microbiology vol 42 no 3pp 207ndash220 1996

[17] N Amaresan V Jayakumar K Kumar and N ThajuddinldquoIsolation and characterization of plant growth promotingendophytic bacteria and their effect on tomato (Lycopersiconesculentum) and chilli (Capsicum annuum) seedling growthrdquoAnnals of Microbiology vol 62 no 2 pp 805ndash810 2012

[18] B Schwyn and J B Neilands ldquoUniversal chemical assay forthe detection and determination of siderophoresrdquo AnalyticalBiochemistry vol 160 no 1 pp 47ndash56 1987

[19] P Rahi P Vyas S Sharma and A Gulati ldquoPlant growthpromoting potential of the fungus Discosia sp FIHB 571 fromtea rhizosphere tested on chickpea maize and peardquo IndianJournal of Microbiology vol 49 no 2 pp 128ndash133 2009

[20] K Kumar N Amaresan S Bhagat K Madhuri and R CSrivastava ldquoIsolation and characterization of rhizobacteriaassociated with coastal agricultural ecosystem of rhizospheresoils of cultivated vegetable cropsrdquo World Journal of Microbiol-ogy and Biotechnology vol 27 no 7 pp 1625ndash1632 2011

[21] J A Smoker and S R Barnum ldquoRapid small scale DNAisolation from filamentous cyanobacteriardquo FEMS MicrobiologyLetters vol 56 no 1 pp 119ndash122 1988

[22] U Edwars T Rogall H Blocker M Emde and E C BottgerldquoIsolation and direct complete nucleotide determination ofentire genes Characterization of a gene coding for 16S riboso-mal RNArdquoNucleic Acids Research vol 17 no 19 pp 7843ndash78531989

[23] D G Higgins A J Bleasby and R Fuchs ldquoCLUSTAL Vimproved software for multiple sequence alignmentrdquo ComputerApplications in the Biosciences vol 8 no 2 pp 189ndash191 1992

[24] M Kimura ldquoA simple method for estimating evolutionary ratesof base substitutions through comparative studies of nucleotidesequencesrdquo Journal ofMolecular Evolution vol 16 no 2 pp 111ndash120 1980

[25] N Saitou and M Nei ldquoThe neighbor-joining method a newmethod for reconstructing phylogenetic treesrdquo Molecular Biol-ogy and Evolution vol 4 no 4 pp 406ndash425 1987

[26] E Barbu K Y Lee and R Wahl ldquoContent of purine andpyrimidine base in desoxyribonucleic acid of bacteriardquo Annalesde lrsquoInstitut Pasteur vol 91 no 2 pp 212ndash224 1956

[27] N Sueoka ldquoVariation and heterogeneity of base compositionof deoxyribonucleic acids a compilation of old and new datardquoJournal of Molecular Biology vol 3 no 1 pp 31ndash40 1961

[28] H Hori and S Osawa ldquoEvolutionary change in 5S rRNAsecondary structure and a phylogenic tree of 352 5S rRNAspeciesrdquo BioSystems vol 19 no 3 pp 163ndash172 1986

[29] V Bess ldquoEvaluating microbiology of compost microbial con-tent of composting is helping the producers and growers tounderstand its role as a soil inoculant and plantrdquo BioCycleMagazine p 62 1999

[30] O Grafff ldquoPreliminary experiments of vermin composting ofdifferent waste materials using Eudrilus eugeniae Kinbergrdquo inWorkshop on the Role of Earthworms in the Stabilization ofOrganic Residues M Happelhof Ed pp 178ndash191 Beech LeafPress Kalamazoo Mich USA 1981

[31] J E Satchell Earthworm Ecology from Darwin to VermicultureChapman and Hall 1983

[32] G H Xie M Y Cai G C Tao and Y Steinberger ldquoCultivableheterotrophic N2-fixing bacterial diversity in rice fields in theYangtze River Plainrdquo Biology and Fertility of Soils vol 37 no 1pp 29ndash38 2003

[33] B BMcSpaddenGardener ldquoEcology ofBacillus and Paenibacil-lus spp in agricultural systemsrdquo Phytopathology vol 94 no 11pp 1252ndash1258 2004

[34] J Dobereiner ldquoBiological nitrogen fixation in the tropics socialand economic contributionsrdquo Soil Biology andBiochemistry vol29 no 5-6 pp 771ndash774 1997

[35] V M Reis F L Olivares and J Dobereiner ldquoImprovedmethodology for isolation of Acetobacter diazotrophicus andconfirmation of its endophytic habitatrdquoWorld Journal of Micro-biology and Biotechnology vol 10 no 4 pp 401ndash405 1994

[36] S Khalil N Ayub S Alam and F Latif ldquoOrganic acids pro-duction and phosphate solubilization by phosphate solubilizingmicroorganisms (PSM) under in vitro conditionsrdquo PakistanJournal of Biological Sciences vol 7 no 2 pp 187ndash196 2004

[37] H M A El-Komy ldquoCoimmobilization of Azospirillumlipoferum and Bacillus megaterium for successful phosphorusand nitrogen nutrition of wheat plantsrdquo Food Technology andBiotechnology vol 43 no 1 pp 19ndash27 2005

[38] N B Paul and W V B Sundara Rao ldquoPhosphate-dissolvingbacteria in the rhizosphere of some cultivated legumesrdquo Plantand Soil vol 35 no 1 pp 127ndash132 1971

[39] A Beneduzi D Peres L K Vargas M H Bodanese-Zanettiniand LM P Passaglia ldquoEvaluation of genetic diversity and plantgrowth promoting activities of nitrogen-fixing bacilli isolatedfrom rice fields in South Brazilrdquo Applied Soil Ecology vol 39no 3 pp 311ndash320 2008

[40] M P Raimam U Albino M F Cruz et al ldquoInteraction amongfree-living N-fixing bacteria isolated from Drosera villosa varvillosa and AM fungi (Glomus clarum) in rice (Oryza sativa)rdquoApplied Soil Ecology vol 35 no 1 pp 25ndash34 2007

[41] M R Swain S K Naskar and R C Ray ldquoIndole-3-acetic acidproduction and effect on sprouting of yam (Dioscorea rotundataL) minisetts by Bacillus subtilis isolated from culturable cow-dung microflorardquo Polish Journal of Microbiology vol 56 no 2pp 103ndash110 2007

[42] T M Tien M H Garkins and D H Hubbell ldquoPlant growthsubstances produced by Azospirillum brasilense and their effect

Advances in Agriculture 13

on the growth of pearl Millet Pennisetum americanum (L)rdquoApplied and EnvironmentalMicrobiology vol 37 no 5 pp 1016ndash1024 1979

[43] A Esitken L Pirlak M Turan and F Sahin ldquoEffects of floraland foliar application of plant growth promoting rhizobacteria(PGPR) on yield growth and nutrition of sweet cherryrdquo ScientiaHorticulturae vol 110 no 4 pp 324ndash327 2006

[44] H Karlidag A Esitken M Turan and F Sahin ldquoEffects of rootinoculation of plant growth promoting rhizobacteria (PGPR)on yield growth and nutrient element contents of leaves ofapplerdquo Scientia Horticulturae vol 114 no 1 pp 16ndash20 2007

[45] C S Nautiyal S Bhadauria P Kumar H Lal R Mondal andD Verma ldquoStress induced phosphate solubilization in bacteriaisolated fromalkaline soilsrdquoFEMSMicrobiology Letters vol 182no 2 pp 291ndash296 2000

[46] A K Halder A K Mishra P Bhattacharya and P KChakrabarthy ldquoSolubilization of inorganic phosphates byBradyrhizobiumrdquo Indian Journal of Experimental Biology vol29 pp 28ndash31 1991

Submit your manuscripts athttpwwwhindawicom

Nutrition and Metabolism

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Food ScienceInternational Journal of

Agronomy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Applied ampEnvironmentalSoil Science

Volume 2014

AgricultureAdvances in

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PsycheHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BiodiversityInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Plant GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biotechnology Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Forestry ResearchInternational Journal of

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Journal of BotanyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

EcologyInternational Journal of

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Veterinary Medicine International

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Cell BiologyInternational Journal of

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Evolutionary BiologyInternational Journal of

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Page 2: BalayoganSivasankariandMarimuthuAnandharajdownloads.hindawi.com/journals/aag/2014/248591.pdf · In Vitro Nitrogen Fixing Activity. e bacterial isolates were screened for nitrogen

2 Advances in Agriculture

macro- and micronutrients which enlivens the soil throughpartial substitution of the horticultural container mediaEnhancement in plant growth after substitution of soils orgreenhouse container media with composts is attributedto modifications in soil structure change in water avail-ability increased availability of macro- and micronutrientsstimulation of microbial activity and augmentation of theactivities of critical substances by microorganisms throughinteractions with earthworms [4] The vermicompost is richin available nutrients soil beneficial microbes and plantgrowth promoting substances

Research during the past few decades has led to theidentification of certain biological organisms and their prod-ucts that could potentially be used as fertilizer sources Thisstrategy of fertilizing the soil with biological sources has beenwidely accepted and recognized as a viable alternative to theapplication of chemical fertilizers Nitrogen (N) fixing andphosphate (P) solubilizing bacteria may be important forplant nutrition by increasingN andPuptake by the plants andplaying a significant role as plant growth promoting bacteriain the biofertilization of crops Increasing and extendingthe role of biofertilizers could reduce adverse environmentaleffects caused by chemical fertilizers Nitrogen is one ofthe basic requirements for the growth productivity andyield of plants On a worldwide basis it is estimated thatabout 175 million tons of nitrogen per year is added tosoil through biological nitrogen fixation Meanwhile super-phosphate fertilizer is expensive and is in short supply andhence biofertilizers can bridge the gap Soluble fertilizerphosphorus (P) is the worldrsquos second largest bulk agriculturalchemical under use and as such is absolutely essential for foodproduction However most soil phosphorus approximately95ndash99 percent is present in the form of insoluble phosphatesand hence cannot be utilized by plants [5] To increasethe availability of phosphorus to plants large amounts offertilizers are used on a regular basis But after applicationa large proportion of the fertilizer phosphorus is quicklytransferred to the insoluble form [6] Therefore a very littlepercentage of the applied phosphorus only is used makingcontinuous application necessary It has been reported thatmany soil fungi and bacteria can solubilize inorganic phos-phates [7] The Bacillus species offer several advantages overthe other genera because of their capacity to produce sporesin unfavorable environmental conditions This characteristicfacilitates the conversion of spore suspensions to powderformulations without killing bacteria [8] Vermicompost isrich in NPK plant growth promoters and other nutrientsand when applied the soil will be enriched with a varietyof nutrients that will become available for the indigenousmicroflora

The PGPR have been known to directly enhance plantgrowth by a variety of mechanisms namely fixation of atmo-spheric nitrogen that is transferred to the plant productionof siderophores that chelate iron and make it available tothe plant root solubilization of minerals such as phosphorusand synthesis of phytohormones [9] Plant growth promotingbacteria (PGPB) stimulate plant growth by nitrogen fixation[10] solubilization of nutrients [11] and production of growthhormones and 1-aminocyclopropane-1-carboxylate (ACC)

The present paper describes the potential of PGPR isolatesfrom vermisources and their plant growth stimulating activ-ity in pot trials under ambient conditions Further studies willclarify the potential use of these bacteria used as biofertilizers

The main objective of this study was to isolate iden-tify and characterize the potential plant growth promotingrhizobacteria from vermisources as well as evaluating theirplant growth potential under controlled environment andcharacterizing the isolated bacteria using various moleculartechniques These bacteria can be considered promisingcandidates for application in sustainable agriculturalmanage-ment

2 Materials and Methods

21 Vermicompost Preparation For the present study epigeicearthworms Eudrilus eugeniae (Kinberg) and Eisenia fetida(Savigny) were collected from the breeding stock of theDepartment of Biology GandhigramRural Institute-DeemedUniversity Gandhigram Tamil Nadu India and leaf mate-rials of Gliricidia sepium Jacq Leucaena leucocephala (Lam)De Wit and Cassia auriculata Linn were collected fromGandhigram campus The leaf materials were separatelysubjected to predigestion for 15 days by sprinkling wateron the heap and covering it with gunny bag and turning itperiodically in order to release out the initial heat producedduring decomposition of organic material The changes intemperature were observed every three days up to 15 daysThe vermibeds were prepared in plastic containers of 45 times35 times 15 cm size and the substrate was moistened to hold60ndash80 percent moisture and kept for 24 hours stabilization20 numbers of healthy clitellate E eugeniae and 30 numbersof E fetida were separately introduced in the vermibedsThe vermicomposting trials were carried out in the rearingroomwith the relative humidity and the temperature of 75ndash85percent and 26ndash28∘C respectively The substrate was turned(mixed) once in a week and maintained up to 60 days Theexperiment was carried out with three replicates for eachsubstrate with proper control [12]

22 Microbial Study The total microbial counts in termsof colony forming units (CFU) of bacteria in the vermi-composts vermicasts and earthworm (fore mid and hind)guts were determined every 15 days (0 15 30 45 and 60 d)using standard plate count method From the total colonyforming units only those bacterial colonies (19 bacterialcolonies)which showedpredominant growthwere restreakedonto appropriate agar medium to obtain pure cultures andsubjected to characterization and identification

23 Screening for Biofertilization Activities

231 In Vitro Nitrogen Fixing Activity The bacterial isolateswere screened for nitrogen fixing ability using nitrogen-freeliquidmedium [13] Nitrogen-freemediumwas prepared andsterilized The strains were inoculated separately on selectivenitrogen-free liquid medium All the test organisms wereincubated at 37∘C plusmn 2∘C for 7 days The nitrogen fixing

Advances in Agriculture 3

activities were observed on the basis of the formation ofturbidity in the flasks Among all the nineteen bacterialisolates tested only five bacterial isolates formed significantlyhigher rate of turbidity in the nitrogen-free liquid mediumand selected for further molecular and plant growth studies

232 In Vitro Phosphate Solubilization All bacterial iso-lates were screened for inorganic phosphate solubilizationusing Pikovskayarsquos agar medium the selective medium forphosphate solubilization test [14] Pikovskayarsquos agar mediumwas prepared and sterilized and the bacterial strains wereinoculated separately on the selective medium containing 05percent of (wv) tricalcium phosphate (Ca

3PO4) as complex

insoluble phosphate source All the test plates of the bacteriawere incubated at 37∘C plusmn 2∘C for 10 days After incubationfor up to 7 days at 30∘C formation of yellow halos andorclearing zones was evaluated The results were expressed assolubilization index (SI) and they were measured using thefollowing formula [15]

SI =Colony diameter + halozone diameter

Colony diameter (1)

Five bacterial strains which showed phosphate solubiliz-ing activity were identified and grouped as phosphate sol-ubilizing bacteria (PSB) The bacterial strain which showedhighest SI was selected for further molecular and plantgrowth studies

24 Production of Phytohormones (IAA) The bacterial cul-tures were inoculated in nutrient broth with tryptophan(5 120583gmL) and incubated at 28 plusmn 2∘C for 5 days Afterincubation cultures were centrifuged at 3000 rpm for 30minTwo milliliters of the supernatant was mixed with 2 drops oforthophosphoric acid and 4mL of Salkowskirsquos reagent (50mLof 35 perchloric acid + 1mL 05 FeCl

3) and incubated in the

dark for 25 minutes Development of pink colour indicatesindole-3-acetic acid (IAA) production The optical densitywas measured at 530 nm using Spectronic 200 (India) Thequantity of IAA production was estimated using standardIAA graph and expressed asmicrograms permilliliter [16 17]

25 Siderophore Production A qualitative assay ofsiderophore production was conducted in Chrome Azurol S(CAS) agar medium [18] CAS agar plates were prepared andspot-inoculated with test organism and incubated at 30∘C for3ndash5 days Change of blue color of the medium surroundingthe bacterial growth to fluorescent yellow indicated theproduction of siderophore Bacillus subtilis was chosen as apositive control [19 20]

26 16S rDNA Gene Amplification and Sequencing GenomicDNA from each isolate was extracted using the modifiedmethod of Smoker and Barnum [21] The extracted DNAwas dissolved in 20120583L TE buffer and used as the templatefor the PCR reactions PCR amplifications were performedin a total volume of 50 120583L by mixing 20 ng of the templateDNA with 25mM concentrations of each deoxynucleotide

triphosphate and 1 120583m of each universal primer of Bac8uf(51015840-AGAGTTTGATCCTGGCTCAG-31015840) and Univ1492r (51015840-CTACGGCTACCTTGTTACGA-31015840) described by Edwars etal [22] The thermocycling profile was carried out with aninitial denaturation at 95∘C (4min) followed by 30 cyclesof denaturation at 95∘C (1min) annealing at 56∘C (30 s)extension at 72∘C (1min) and a final extension at 72∘C(10min) in an Eppendorf Gradient thermocycler The PCRamplified rDNA were purified by using the Quick PCRpurification kit (Bangalore Genie India) 16S rRNA genesequence of the isolate was compared with 16S rRNA genesequences available by the BLAST search in the NCBIGenBank database (httpwwwncbinlmnihgov) The anal-ysis of alignment and homology of the partial nucleotidesequence of Bacillus sp was carried out by the basic localalignment search tool (BLAST) The PCR products werestored at 4∘C Aliquots of the PCR products were separatedby 2 agarose gel electrophoresis in TAE buffer (pH 80)A 100 bp DNA marker (Sigma Bangalore) was used as areference Gels were stainedwith ethidiumbromide (11 gmL)and visualized under UV light

27 Nucleotide Sequence Accession The sequence obtained inthis study was deposited in the GenBank (USA) nucleotidesequence database under the accessionnumberGQ413962 (Bcereus) and HM753262 (Pseudomonas spp)

28 Phylogenetic Analysis The 16S rRNA gene sequenceswere obtained in the present study and the taxonomicallyrelated Bacillus spp were retrieved from the NationalCenter from Biotechnology Information (NCBI) database(httpwwwncbinlmnihgov) All the sequences werealigned using multiple sequence alignment programCLUSTAL W developed by Higgins et al [23] The pairwiseevolutionary distances were computed using the methodof Kimura [24] The multiple distance matrix obtained wasthen used to construct phylogenetic trees using neighbourjoining method of Saitou and Nei [25] Phylogenetic treeswere constructed by using Mega 5 software (MolecularEvolutionary Genetic Analysis)

29 Prediction of RNA Secondary Structure The secondarystructure of 16S rDNA of Bacillus spp was predicted usingthe bioinformatics Genebee tool

210 Restriction Site Analysis in 16S rDNA The restrictionsites in DNA of Bacillus spp were analyzed using theNEB Cutter program version 20 tools available online athttptoolsnebcomNEBcutter2indexphp

211 Fermentation Process For the preparation of inocu-lum 50mL of nutrient broth medium (HiMedia MumbaiIndia) was inoculated with appropriate overnight bacte-rial culture and incubated at 37∘C for 24 h at 120 rpm(OD540= 045 10

7-108 CFUmLminus1) The cell-free super-natant was obtained by centrifuging the fermented broth at

4 Advances in Agriculture

10000 rpm for 10minThe supernatantwas analyzed for plantgrowth promotion at various concentrations

212 Evaluation of Plant Growth Promoting Activity underControlled Environment The nitrogen fixing bacteria (Bacil-lus spp) and the phosphate solubilizing bacteria (Pseu-domonas spp) were separately tested for plant growth pro-moting activity and also as consortium (1 1) at 5 differentconcentrations that is 1 2 3 4 and 5mL at three differentday intervals (5 10 and 15 days) with proper control throughExp1 Exp2 Exp3 and Exp0 respectively usingVigna unguic-ulata (L) Walp The seeds (30 seeds in each pot) were sownin plastic pots containing sterilized vermiculite and arrangedin a completely randomized factorial design The seedlingswere grown in a greenhouse at a temperature of 28ndash32∘Cand 85 relative humidity The pots were watered to 50water-holding capacity and were maintained at this moisturecontent by watering to weight every day and plant growthwasobserved for 15 days Parameters such as germination percentshoot length root length leaf area chlorophyll content of theleaves fresh weight of the whole plant and dry weight of thewhole plant were measured using standard procedure

213 Statistical Analysis The following statistical tools wereused for the analyses and interpretation of the data Theexperimental results are presented in the form of tablesand graphs using Microsoft Excel 2007 Data obtained fromthe different treatments were statistically analyzed using theone-way ANOVA and the mean values were compared bythe Duncanrsquos multiple range test for multiple comparisonsDifferences were considered significant at the 005 level usingOrigin software (Version 850) 2010 Origin Lab Corpora-tion

3 Results

31 Isolation and Characterization of Bacterial Isolates Atotal of nineteen bacterial strains were isolated from vermi-compost vermicast and earthworm (fore mid and hind)guts (Table 1) The morphological and biochemical char-acteristics of bacterial isolates were elucidated in Table 1All the isolates were tested for plant growth promotingactivity Among the 19 bacterial isolates only five bacterialstrains showed plant growth promoting activity Based oncolony morphology microscopic observations and culturalbiochemical and physiological properties the bacteriumwasgiven the name Bacillus spp Alcaligenes spp Erwinia sppSerratia spp or Pseudomonas sppThese five bacterial strainswere able to grow in the nitrogen-free medium and they werealso identified as phosphate solubilizing microorganisms onthe basis of their solubilization index (Table 2) Bacillus spphave the highest SI (3855) followed by Pseudomonas spp(3522) and Serratia spp (3045) All the isolates produceda significant amount of IAA from the medium in the rangeof 113 to 242120583gmL Bacillus spp showed higher IAAproduction (242120583gmL) and Serratia spp showed lower IAAproduction (113 120583gmL) All the strains (except Alcaligenes

spp) were positive for siderophore production showing ayellow zone on the CAS agar medium plate (Table 2)

32 Molecular Studies The partial 16S rRNA sequencescarried out in the present study for Bacillus spp andPseudomonas spp covered a stretch of approximately 1500nucleotides for each About half of the sequences found in theclone library showed only slight relationship to other knownsequences while the other half were highly similar (approx-imately 95 percent sequence identity) to other databaseentries for Bacillus spp and Pseudomonas spp Less than 05percent of all nucleotides was found to be unique within theconserved regions of the cloned sequence and could almostalways be related to reading errors in ambiguous regions ofthe sequencing gel On the basis of phylogenetic analysisof 16S rDNA partial sequences Bacillus spp GGBSTD1 isidentified as Bacillus cereus and Pseudomonas spp GGBSTD3is identified as Pseudomonas spp Phylogenetic analyses ofthe strains based on the neighbor joining method wererepresented in Figures 1 and 2 Among eubacteria the meanguanine and cytosine (G + C) content of genomic DNAvaries from approximately 25 to 75 That the bacterialgenomic G + C content is somehow related to phylogeny hasbeen suggested [26 27] The phylogenetic tree of eubacterial16S rRNA clearly indicates this relationship Gram-positivebacteria Bacillus subtilis have genomic G + C 42 Gram-negative bacteria with intermediate G + C content such asEscherichia coli (50) Serratia marcescens (58) Salmonellatyphimurium (51) and Pseudomonas fluorescens (60)belong to the common Gram-negative bacteria [28] Bacilluscereus have the G + C content 53 and A + T content 47similarly Pseudomonas spp having G + C content 53 andA + T content 47 Most functional RNA molecules havecharacteristic secondary structures that are highly conservedin evolution In this study RNA secondary structures ofBacillus cereus and the Pseudomonas spp were predictedBacillus cereus have free energy of minus2941 kkalmol andPseudomonas spp have free energy of minus2998 kkalmol

33 Evaluation of Biofertilization Abilities under ControlledConditions Germination percentage of Vigna unguiculataseeds sown in vermiculite supplemented with Bacillus cereus(Exp1) with Pseudomonas spp (Exp2) and with consortium(Exp3) (1 1 ratio) at five different concentrations (1 2 3 4and 5mL) and in the control (Exp0) is shown in Figure 3The highest germination percentage was observed in Exp3(consortium) at 5mL concentration compared to others Theincrease in germination percentage and in other growthparameters can be attributed to the combined effect of thenitrogen fixing and phosphate solubilizing microbes whichare able to fix atmospheric nitrogen and solubilize P andalso produce growth promoting substancesThese organismshave been isolated from vermisources and this observationindicates the reason for promotion and enhancement of cropgrowth and yield when vermicompost is applied It is a proofthat these symbioticmicrobes present in vermisources benefitthe soil fertility Observation on growth parameters such asshoot length root length and leaf area of V unguiculata

Advances in Agriculture 5

Table1

Predo

minantb

acteria

lstrains

isolated

from

verm

isourcesand

theirb

iochem

icalcharacteris

ticsT1-con

trolmdash

Gsepium

+L

leucocephala+

cowdu

ngin

(112)(worm

free)T

2mdashG

sepium

+L

leucocephala+cowdu

ngin

(112)+

EeugeniaeT3

mdashG

sepium

+L

leucocephala+cowdu

ngin

(112)+

EfetidaT4

-con

trol(2)mdashC

auric

ulata+L

leucocephala+cow

dung

in(112)(worm

free)T

5mdashC

auric

ulata+L

leucocephala+cowdu

ngin

(112)+

EeugeniaeT

6mdashC

auric

ulata+L

leucocephala+cowdu

ngin

(112)+

Efetida

Snu

mber

Samples

ource

Biochemicalcharacteris

ticsc

Nam

eofthe

strain

Gramrsquos

reactio

naCell

shape

Motility

bIndo

letest

Methyl

Red

test

Voges

Proskauer

test

Citrate

Utilization

test

Catalase

test

Urease

test

Gelatin

hydrolysis

test

Nitrate

redu

ctase

test

Starch

hydrolysis

test

Casein

hydrolysis

test

Glucose

utilizatio

ntest

1Ve

rmicom

post

(T2)

+Cocciminus

minus+

minusminus

+minus

++

minusminus

minusStaphylococcus

spp

2Ve

rmicom

post

(T3)

+Ro

d+

minusminus

minusminus

+minus

minus+

minusminus

+Co

rynebacterium

spp

3Ve

rmicom

post

(T5)

+Ro

dminus

++

++

+minus

++

+minus

+Ba

cillusspp

4Ve

rmicom

post

(T6)

+Cocciminus

++

minusminus

+minus

+minus

minusminus

+Micrococcusspp

5Ve

rmicast(T3

)+

Cocciminus

++

minusminus

minus+

++

+minus

minusKlebsiella

spp

6Ve

rmicast(T5

)+

Rod

minus+

minusminus

+minus

+minus

+minus

minus+

Streptococcusspp

7

Verm

icast(T6

)+

Cocciminus

+minus

++

minusminus

++

minusminus

+En

terobacter

spp

8Ea

rthw

orm

hind

gut(T2

)+

Cocciminus

minus+

+minus

+minus

minusminus

++

+Trico

ccocus

spp

9Ea

rthw

orm

hind

gut(T3

)+

Cocciminus

minus+

minus+

++

minus+

minusminus

minusYersiniaspp

10

Earthw

orm

hind

gut(T5

)+

Cocciminus

minusminus

minusminus

++

++

minusminus

minusMicrococcusspp

11

Verm

icom

post

(T1)

minusRo

dminus

++

minusminus

minusminus

++

minusminus

+Escherich

iaspp

12

Verm

icom

post

(T2)

minusRo

dminus

minus+

minusminus

minus+

++

minusminus

+Proteusspp

13

Verm

icom

post

(T4)

minusRo

dminus

minusminus

+minus

+minus

minus+

++

+Pa

racoccus

spp

14

Verm

icom

post

(T5)

minusRo

d+

+minus

minusminus

++

++

minusminus

minusPseudomonas

spp

15

Verm

icast(T2

)minus

Rod

+minus

+minus

++

+minus

+minus

minusminus

Citro

bacter

spp

16

Verm

icast(T3

)minus

Cocciminus

++

minusminus

+minus

+minus

minusminus

minusErwiniaspp

17Ve

rmicast(T5

)minus

Rod

++

++

++

minus+

minusminus

minusminus

Alcaligenesspp

18

Verm

icast(T6

)minus

Cocciminus

minus+

++

+minus

++

minusminus

minusSerratiaspp

19

Earthw

orm

mid

gut(T2

)minus

Rod

+minus

minusminus

+minus

minusminus

minusminus

minusminus

Pediococcusspp

a Gramrsquossta

ining(+)p

ositive(minus)n

egative

b Motility(+)

motile(minus)n

onmotile

c Biochem

icalcharacteris

tics(+)p

ositive(minus)n

egative

6 Advances in Agriculture

Bacillus cereus strain GGBSTD2 16S ribosomal RNA gene

Bacillus cereus strain SU-6 16S ribosomal RNA gene

Bacillus cereus strain JB0013 16S ribosomal RNA gene

Bacillus cereus isolate PN24 16S ribosomal RNA gene

Bacillus cereus strain JB0005 16S ribosomal RNA gene

Bacillus sp G2DM-85 16S ribosomal RNA gene

Bacillus cereus strain JBE0009 16S ribosomal RNA gene

Bacillus sp EB460 16S ribosomal RNA gene

Bacillus cereus strain 421-3R 16S ribosomal RNA gene

Bacillus sp EB447 16S ribosomal RNA gene

Bacillus sp EB470 16S ribosomal RNA gene

Bacillus sp EB421 16S ribosomal RNA gene

Bacillus sp EB431 16S ribosomal RNA gene

Bacillus sp EB462 16S ribosomal RNA gene

Bacillus sp EB429 16S ribosomal RNA gene

Bacillus cereus strain 421-4R 16S ribosomal RNA gene

Bacillus sp EB480 16S ribosomal RNA gene

Bacillus sp EB13 16S ribosomal RNA gene

Uncultured compost bacterium partial 16S rRNA gene

Bacillus thuringiensis gene for 16S rRNA

Figure 1 Neighbor joining phylogenetic tree of full 16S rRNA gene sequences showing the relationship among Bacillus cereus GGBSTD1and representatives of some related taxa The sequence data for several closely related Bacillus cultures were recovered from GenBank andincluded in the tree

Table 2 The phosphate solubilizing activity phytohormone (IAA) production and siderophores production of the bacterial isolates

Name of the organism Phosphate solubilization index (SI) IAA production (120583gmL)a Siderophores productionb

Bacillus spp 3855 242 +++Alcaligenes spp 2930 147 minus

Erwinia spp 2890 113 +Serratia spp 3045 168 ++Pseudomonas spp 3522 196 +++aIndole acetic acid production in culture media supplemented with tryptophan (5 120583gmL) after 5 daysbIn vitro siderophores production minus represents the absence of siderophores production and +++ represents gt10mm wide yellow-orange zone

is given in Tables 3 and 4 respectively The shoot length(1147 plusmn 006 cm) root length (1027 plusmn 006 cm) and leafarea (905 plusmn 026 cm2) of the plant get significantly (119875 lt0001) increased in Exp3 (consortium of Bacillus cereus +Pseudomonas spp [1 1]) at 5mL concentration on the 15thday compared to other experimentsThe chlorophyll a b andtotal chlorophyll content of the leaves on d 5 d 10 and d 15are given in Table 5 The chlorophyll a (010 plusmn 009mgg offresh leaf) chlorophyll b (1085 plusmn 016mgg of fresh leaf)and total chlorophyll (1095plusmn007mgg of fresh leaf) content

of leaves were significantly (119875 lt 0001) higher in Exp3(consortium of Bacillus cereus + Pseudomonas spp (1 1))at 5mL concentration on the 15th day compared to otherexperiments Observations on the fresh weight of the wholeplant are given in Table 6 The fresh weight of the wholeplant (092 plusmn 000 g) was significantly (119875 lt 0001) higherin Exp3 (consortium of Bacillus cereus + Pseudomonas spp(1 1)) at 5mL concentration on the 15th day compared toother experiments Observations on the dry weight of thewhole plant are given in Table 6 The dry weight of the whole

Advances in Agriculture 7

Pseudomonas fluorescens strain LMG 5825 16S ribosomal RNA gene

Pseudomonas fluorescens strain LMG 5829 16S ribosomal RNA gene

Pseudomonas fluorescens strain LMG 14676 16S ribosomal RNA gene

Pseudomonas sp W15Feb13 16S ribosomal RNA gene

Pseudomonas sp W15Feb28 16S ribosomal RNA gene

Pseudomonas sp p50 16S ribosomal RNA gene

Uncultured bacterium clone 661193 16S ribosomal RNA gene

Pseudomonas sp BSs20166 16S ribosomal RNA gene

Pseudomonas sp 28zhy partial 16S rRNA gene

Uncultured bacterium clone AYRV1-027 16S ribosomal RNA gene

Uncultured Pseudomonadaceae bacterium clone290cost002-P3L-2032 16S ribosomal RNA gene

Pseudomonas sp GGBSTD3 16S ribosomal RNA gene

Pseudomonas sp DK3 16S ribosomal RNA gene

Pseudomonas reactans strain AMP-13 16S ribosomal RNA gene

Figure 2 Neighbor joining phylogenetic tree of full 16S rRNA gene sequences showing the relationship among Pseudomonas spp GGBSTD3and representatives of some related taxa The sequence data for several closely related Pseudomonas cultures were recovered from GenBankand included in the tree

plant (009 plusmn 000 g) was significantly (119875 lt 0001) higherin Exp3 (consortium of Bacillus cereus + Pseudomonas spp(1 1)) at 5mL concentration on the 15th day compared toother experiments

4 Discussion

Inoculation of these newly identified nitrogen fixing andphosphate solubilizingmicrobes in the plant growthmediumenhanced various physiological activities culminating inhigher biomass production in V unguiculata Since this is anonhazardous way of fertilization of crop plants it is veryrelevant to a developing country like India This techniquecan save the farmers of India from the problem posed byhigh cost of fertilizers by offering a comparatively inexpensivealternative (biofertilizers) while additionally preventing thedegradation of the soil and thereby ensuring sustainable agri-culture Bess (1999) reported that the content in compostingneeds to be determined through the concentration of sixfunctional groups of microorganisms such as aerobic bacte-ria anaerobic bacteria fungi actinomycetes Pseudomonasand nitrogen fixing bacteria [29] Now there are ways toevaluate the concentrations of these organisms in the finishedcompost and that can serve as an interpretation guide to

determine the quality of the compost as an inoculant of soilmicroorganisms Grafff (1981) and Atlavinyte have reporteda higher P content in the cast than in the surrounding soil[30] Satchell (1983) has linked the phosphatase activity inthe gut of worms to the availability of bound P in soils Thebacterial strains which have the capability to fix nitrogenonly can grow in the nitrogen-free medium [31] Hencethe nitrogen fixing ability was tested in the present studybased on the growth of bacterial strain in nitrogen- freemedium Five bacteria strains showedmore nitrogen fixationamong all the five bacterial strains tested only one strain (ieBacillus spp) showed significantly more turbidity comparedto the other strains The ultimate source of the nitrogen usedby plants is N

2gas which constitutes 78 of the Earthrsquos

atmosphere Unfortunately higher plants cannot metabolizeN2directly into protein N

2gas must be converted to a plant

available form [32] isolated many species of nitrogen fixingBacillus (B megaterium B cereus B subtilis B licheniformisand B azotoformans) from the rhizospheric zone of ricefields in Central China The widely studied Bacillus genusrepresents one of the most diverse genera in the bacilli groupNumerous Bacillus and Paenibacillus strains express plantgrowth promoting (PGP) activities and a number of thesestrains have already been commercially developed as biolog-ical fungicides insecticides and nematicides or generic plant

8 Advances in Agriculture

Table 3 Shoot length and Root length ofVigna unguiculata grown on vermiculite supplemented with Bacillus cereus with Pseudomonas sppand with their consortium (1 1) at five different concentrations (1 2 3 4 and 5mL) on the 5th the 10th and the 15th day and in the control

Experimentsa Quantity of bacterial cultures added Shoot length (cm)b Root length (cm)b

5th day 10th day 15th day 5th day 10th day 15th day

Exp0

1mL 297 (ab) 397 (a) 497 (ab) 197 (de) 283 (ab) 397 (bcd)2mL 303 (bcd) 403 (ab) 500 (ab) 200 (abc) 287 (de) 400 (a)3mL 310 (abc) 407 (a) 503 (ab) 203 (de) 290 (abc) 403 (ab)4mL 310 (bcd) 410 (bcd) 507 (bcd) 207 (a) 297 (de) 410 (abc)5mL 313 (a) 413 (ab) 510 (ab) 210 (bcd) 300 (ab) 413 (de)

Exp1

1mL 340 (ab) 440 (abc) 540 (ab) 233 (abc) 327 (bcd) 433 (f)2mL 450 (ab) 540 (abc) 647 (ab) 333 (ab) 430 (a) 540 (abc)3mL 550 (de) 630 (ab) 750 (a) 440 (de) 550 (abc) 620 (a)4mL 653 (ab) 730 (a) 833 (ab) 550 (bcd) 653 (de) 750 (abc)5mL 740 (ab) 840 (abc) 923 (f) 640 (abc) 753 (ab) 827 (ab)

Exp2

1mL 277 (bcd) 357 (f) 440 (de) 160 (de) 217 (abc) 350 (f)2mL 343 (abc) 447 (ab) 553 (ab) 253 (abc) 340 (bcd) 427 (bcd)3mL 437 (de) 543 (abc) 633 (abc) 340 (ab) 443 (f) 533 (ab)4mL 547 (bcd) 647 (de) 733 (a) 420 (bcd) 550 (ab) 633 (ab)5mL 640 (f) 733 (bcd) 833 (bcd) 533 (ab) 630 (ab) 730 (bcd)

Exp3

1mL 530 (bcd) 647 (ab) 730 (ab) 433 (de) 550 (abc) 620 (ab)2mL 643 (ab) 740 (bcd) 827 (f) 547 (a) 630 (de) 733 (ab)3mL 737 (bcd) 840 (bcd) 927 (ab) 640 (ab) 740 (bcd) 840 (bcd)4mL 847 (abc) 930 (a) 1043 (abc) 740 (f) 833 (a) 937 (abc)5mL 957 (ab) 1030 (f) 1147 (bcd) 833 (abc) 940 (ab) 1027 (bcd)

aExp0 nutrient solution (control) Exp1 Bacillus cereus Exp2 Pseudomonas spp Exp3 consortium of Bacillus cereus + Pseudomonas spp (1 1)bMeanof two repeated experiments (30 plants)Different letterswithin parenthesis indicate significant difference between treatments for each growth parameterusing Duncanrsquos multiple range test (119875 = 005)

growth promoters The use of these strains in agriculturehas recently been reviewed [33]Those strains besides havingseveral PGP properties can also fix nitrogen and also dophosphate solubilization The symbiotic fixation of nitrogenthrough inoculation of legume crops with effective rhizobiais well known [34] Asymbiotic nitrogen fixing bacteriawhich live in the rhizosphere andor endophytically oftenincrease yields of crops Many bacterial species have nitrogenfixing properties including Bacillus spp Azotobacter sppAzospirillum spp Beijerinckia spp and Pseudomonas spp[35] Nitrogen fixing bacteria have been used in foliar appli-cations inmulberryThe solubilization index (SI) was formeddue to solubilization of insoluble phosphates by organic acidsecretion [36] El-Komy (2005) indicated that Pseudomonasfluorescence and Bacillus megaterium strains were the mostpowerful phosphate solubilizers on PVK plates as well as onPikovskayarsquos broth [37] The P concentration in Pikovskayarsquosbroth increased gradually achieving a peak on the sixth dayand declined slowly during the late days In this study pHvalues decreased gradually in PVK broth during the earlyincubation days and no revival was observed during laterdays for all the tested bacterial strains Several bacteria par-ticularly those belonging to the genus Bacillus spp convertinsoluble phosphate into soluble forms by secreting organicacids such as formic acid acetic acid propionic acid citricacid fumaric acid gluconic acid glyoxylic acid ketobutyric

acid malonic acid succinic acid and tartaric acid Theseacids lower the pH and bring about the dissolution of boundforms of phosphate Some of the hydroxyl acids may chelatewith calcium and iron resulting in effective solubilization andutilization of phosphates [38]

Beneduzi et al (2008) had isolated the plant growthpromoting strain SVPR30 and identified by 16S rRNA genesequence as Bacillus spp and they had tested it for plantgrowth through in vivo experiments [39] This strain wascharacterized as a high IAA producer able to solubilize phos-phate and also fix a considerably high amount of nitrogenThe inoculation of rice with Bacillus spp SVPR30 strainshowed a significant increase in the root and shoot partswhen compared with the controls within 15 and 30 daysafter sprouting Numerous Bacillus and Paenibacillus strainsexpressed plant growth promoting (PGP) activities and anumber of these strains have been commercially developedas generic plant growth promotersThe use of these strains inagriculture has recently been reviewed [33]

Various reports indicate that coinoculation of beneficialorganisms generally increased plant growth relative to singleinoculation with a sole beneficial organism [40] Most ofthe effects of the individual microorganisms coinoculationare additive although a synergistic effect has been reportedin some cases The average root to shoot ratio was higherin B subtilis treated yam minisetts in comparison to those

Advances in Agriculture 9

Table 4 Leaf area of Vigna unguiculata grown on vermiculite supplemented with Bacillus cereus with Pseudomonas spp and with theirconsortium (1 1) at five different concentrations (1 2 3 4 and 5mL) on the 5th the 10th and the 15th days and in the control

Experimentsa Quantity of bacterial cultures added Leaf area (cm2)b

5th day 10th day 15th day

Exp0

1mL 027 (ab) 094 (abc) 407 (b)2mL 035 (ab) 102 (ab) 419 (abc)3mL 040 (abc) 125 (ab) 471 (bcd)4mL 048 (ab) 138 (abc) 510 (ab)5mL 054 (abc) 161 (b) 538 (b)

Exp1

1mL 018 (bcd) 123 (b) 601 (abc)2mL 025 (ab) 138 (abc) 632 (ab)3mL 033 (abc) 150 (ab) 684 (b)4mL 038 (ab) 168 (ab) 686 (abc)5mL 044 (abc) 191 (bcd) 733 (ab)

Exp2

1mL 011 (b) 107 (ab) 534 (b)2mL 014 (ab) 119 (ab) 559 (ab)3mL 017 (abc) 127 (ab) 579 (ab)4mL 021 (ab) 153 (abc) 631 (abc)5mL 026 (ab) 166 (b) 667 (b)

Exp3

1mL 026 (bcd) 297 (ab) 753 (bcd)2mL 033 (b) 325 (ab) 787 (ab)3mL 040 (ab) 350 (bcd) 811 (bcd)4mL 044 (b) 384 (b) 861 (b)5mL 062 (ab) 400 (bcd) 905 (ab)

aExp0 nutrient solution (control) Exp1 Bacillus cereus Exp2 Pseudomonas spp Exp3 consortium of Bacillus cereus + Pseudomonas spp (1 1)bMeanof two repeated experiments (30 plants)Different letterswithin parenthesis indicate significant difference between treatments for each growth parameterusing Duncanrsquos multiple range test (119875 = 005)

0102030405060708090

100

Concentration of bacterial cultures (mL)1 2 3 4 5

Germination ()

Exp0

Exp1 Exp3

Exp2

Figure 3 Germination percentage ofVigna unguiculata seeds sownin vermiculite supplementedwithBacillus cereus with Pseudomonasspp and with their consortium at five different concentrations (1 23 4 and 5mL) and in the control Exp0 nutrient solution (control)Exp1 Bacillus cereus Exp2 Pseudomonas spp Exp3 consortium ofBacillus cereus + Pseudomonas spp (1 1)

not treated with the bacterial culture [41] In earlier reportsroot elongation was found to occur in Sesbania aculeata byinoculation with Azotobacter spp and Pseudomonas spp in

Vigna radiata by Pseudomonas putida [16] and in Pennisetumamericanum by Azospirillum brasilense [42] Esitken et al(2006) had found out that Bacillus OSU-142 and Pseu-domonas BA-8 alone or in combination had a great potentialto increase the growth yield and nutrition of sweet cherryplant [43] BacillusM3 BacillusOSU-142 andMicobacteriumFS01 separately or in combination were found to have greatpotential for use as plant growth promoting rhizobacteriato increase production in apples and in many other crops[44] These phosphate solubilizing microbes consist pre-dominantly of fungal bacterial and actinomycetes speciescollectively called phosphate solubilizing microorganismsmdashPSM [45] There are several microorganisms which canalso solubilize the cheaper sources of phosphorous such asrock phosphate Bacteria such as Bacillus are widely usedin plant production system and are important phosphorussolubilizing microorganisms resulting in improved growthyield of crops andmetabolic activities especially in synthesisof protein [34 46]

5 Conclusions

This study illustrates the isolation of plant growth promotingbacteria from vermisources screening under in vitro con-ditions for multiple PGPR traits and their evaluation undercontrolled conditions in a pot experiment The findings ofthis study indicated that the consortium of B cereus and

10 Advances in Agriculture

Table5Ch

loroph

yllcon

tent

ofleavesofVignaun

guicu

latagrow

non

verm

iculite

supp

lementedwith

Bacilluscereuswith

Pseudomonas

sppand

with

theircon

sortium

(11)atfi

vedifferent

concentrations

(1234and

5mL)

onthe5

thdaythe10thdayandthe15thdayandin

thec

ontro

l

Experim

entsa

Quantity

ofbacterial

Cultu

resa

dded

Chloroph

yllcon

tent

(mggof

fresh

leaf)b

5thday

10th

day

15th

day

Chloroph

ylla

Chloroph

yllb

Total

chloroph

yll

Chloroph

ylla

Chloroph

yllb

Total

chloroph

yll

Chloroph

ylla

Chloroph

yllb

Total

chloroph

yll

Exp0

1mL

021

(ab)

285

(abc)

307

(a)

020

(abc)

481

(a)

501

(ab)

015

(bcd)

612

(abc)

627

(bcd)

2mL

022

(abc)

293

(ab)

315

(abc)

021

(ab)

489

(ab)

510

(bcd)

012

(abc)

618

(ab)

630

(ab)

3mL

023

(abc)

301

(ab)

315

(abc)

022

(bcd)

497

(bcd)

518

(a)

014

(a)

633

(cde)

647

(bcd)

4mL

024

(ab)

308

(f)

332

(a)

022

(a)

504

(b)

527

(abc)

015

(bcd)

641

(cde)

656

(a)

5mL

025

(f)

316

(abc)

341

(bcd)

019

(ab)

511(ab)

530

(abc)

011(cde)

645

(ab)

650

(abc)

Exp1

1mL

035

(b)

400

(cde)

435

(abc)

011(b)

581

(cde)

593

(cde)

010

(abc)

704(abc)

715(f)

2mL

026

(abc)

462

(bcd)

488

(f)

011(abc)

647

(b)

658

(abc)

006

(ab)

779(bcd)

785(a)

3mL

021

(bcd)

526

(abc)

547

(ab)

008

(bcd)

718(ab)

726(bcd)

006

(f)

847

(f)

853

(ab)

4mL

008

(f)

587

(cde)

595

(b)

011(bcd)

778(abc)

789(abc)

004

(bcd)

933(b)

937(bcd)

5mL

026

(abc)

641

(bcd)

667

(abc)

025

(cde)

834

(cde)

859

(f)

007

(abc)

976(cde)

983(abc)

Exp2

1mL

030

(abc)

354

(ab)

384

(b)

012

(a)

545

(abc)

556

(ab)

007

(bcd)

681

(abc)

688

(bcd)

2mL

029

(bcd)

420

(f)

449

(abc)

011(bcd)

610

(ab)

621

(abc)

007

(abc)

744(f)

752(bcd)

3mL

022

(ab)

504

(abc)

527

(bcd)

015

(a)

677

(cde)

692

(bcd)

006

(bcd)

812

(ab)

817

(bcd)

4mL

010

(f)

566

(a)

576

(cde)

018

(abc)

744(f)

762(ab)

005

(ab)

885

(a)

890

(abc)

5mL

006

(bcd)

622

(abc)

628

(abc)

018

(b)

810

(abc)

828

(a)

004

(cde)

948(a)

952(f)

Exp3

1mL

027

(ab)

470

(bcd)

496

(cde)

009

(ab)

668

(b)

677

(cde)

010

(a)

814

(ab)

825

(a)

2mL

020

(ab)

548

(a)

568

(f)

003

(bcd)

746(bcd)

749(ab)

009

(ab)

887

(f)

897

(ab)

3mL

005

(bcd)

584

(abc)

589

(ab)

008

(bcd)

804

(abc)

812

(cde)

010

(ab)

953(abc)

963(bcd)

4mL

023

(f)

663

(ab)

686

(cde)

007

(ab)

870

(b)

877

(f)

010

(abc)

1019

(bcd)

1029(f)

5mL

022

(ab)

744(cde)

766(a)

009

(a)

932(abc)

940(ab)

010

(a)

1085(ab)

1095(abc)

a Exp0nu

trient

solutio

n(con

trol)

Exp1B

acillus

cereusE

xp2Pseudomonas

sppE

xp3consortiu

mof

Bacilluscereus+

Pseudomonas

spp(11)

b Meanof

tworepeated

experim

ents(30plants)

Differentletterswith

inparenthesis

indicatesig

nificantd

ifference

betweentre

atmentsfore

achgrow

thparameter

usingDun

canrsquos

multip

lerangetest(119875=005)

Advances in Agriculture 11

Table 6 Fresh weight and dry weight of whole plant of Vigna unguiculata grown on vermiculite supplemented with Bacillus cereus withPseudomonas spp and with their consortium (1 1) at five different concentrations (1 2 3 4 and 5mL) on the 5th the 10th and the 15 daysand in the control

Experimentsa Quantity of bacterial cultures added Fresh weight of the whole plant (g)b Dry weight of the whole plant (g)b

5th day 10th day 15th day 5th day 10th day 15th day

Exp0

1mL 058 (bcd) 061 (abc) 064 (ab) 006 (a) 006 (abc) 006 (ab)2mL 058 (ab) 061 (a) 064 (abc) 006 (bcd) 006 (ab) 006 (ab)3mL 058 (abc) 061 (ab) 064 (bcd) 006 (abc) 006 (bcd) 006 (b)4mL 041 (abc) 061 (ab) 064 (ab) 006 (abc) 006 (ab) 006 (abc)5mL 058 (abc) 061 (abc) 064 (bcd) 006 (a) 006 (bcd) 006 (def)

Exp1

1mL 062 (abc) 065 (abc) 068 (ab) 006 (bcd) 006 (ab) 007 (bcd)2mL 065 (bcd) 068 (bcd) 071 (def) 006 (abc) 007 (a) 007 (ab)3mL 068 (abc) 071 (def) 074 (bcd) 007 (abc) 007 (ab) 007 (abc)4mL 071 (ab) 074 (abc) 077 (abc) 007 (bcd) 007 (b) 008 (bcd)5mL 074 (abc) 077 (def) 080 (ab) 007 (def) 008 (a) 008 (b)

Exp2

1mL 056 (ab) 059 (bcd) 062 (ab) 006 (ab) 006 (ab) 006 (abc)2mL 059 (abc) 062 (abc) 065 (def) 006 (ab) 006 (abc) 006 (bcd)3mL 062 (bcd) 065 (a) 068 (bcd) 006 (abc) 006 (bcd) 007 (abc)4mL 065 (abc) 068 (ab) 071 (b) 006 (ab) 007 (abc) 007 (b)5mL 068 (abc) 071 (b) 074 (a) 007 (def) 007 (abc) 007 (bcd)

Exp3

1mL 074 (ab) 077 (ab) 080 (abc) 007 (a) 008 (bcd) 008 (ab)2mL 077 (bcd) 080 (a) 083 (bcd) 008 (a) 008 (bcd) 008 (abc)3mL 080 (bcd) 083 (bcd) 086 (abc) 008 (ab) 008 (b) 009 (b)4mL 083 (bcd) 086 (abc) 089 (ab) 008 (abc) 009 (ab) 009 (bcd)5mL 086 (ab) 089 (b) 092 (def) 009 (bcd) 009 (bcd 009 (b)

aExp0 nutrient solution (control) Exp1 Bacillus cereus Exp2 Pseudomonas spp Exp3 consortium of Bacillus cereus + Pseudomonas spp (1 1)bMeanof two repeated experiments (30 plants)Different letterswithin parenthesis indicate significant difference between treatments for each growth parameterusing Duncanrsquos multiple range test (119875 = 005)

Pseudomonas spp acts as a potential biofertilizer due to itsvarious plant growth promotion abilities such as solubiliza-tion of phosphate and production of IAA and siderophore itcan be developed as potential microbial inoculant for variouscrops

Conflict of Interests

The authors declared that there is no conflict of interestsregarding this paper

Acknowledgments

Theauthors are thankful to theDepartment of BiologyGand-higram Rural InstitutemdashDeemed University GandhigramThis research was funded by University Grants Commissionunder UGC Research Fellowship in Science for MeritoriousStudents (BSR) Grant no F4-12008

References

[1] U S Bhawalkar Vermiculture Ecotechnology EarthwormResearch Institute Pune India 2nd edition 1996

[2] S Kiss D M Bularda and D Radulescu ldquoBiological Signifi-cance of Enzymes Accumulated in SoilrdquoAdvances in Agronomyvol 27 pp 25ndash87 1975

[3] D L Karlen M J Mausbach J W Doran R G Cline R FHarris and G E Schuman ldquoSoil quality a concept definitionand framework for evaluationrdquo Soil Science Society of AmericaJournal vol 61 no 1 pp 4ndash10 1997

[4] R M Atiyeh C A Edwards S Subler and J Metzger ldquoEarth-worm processed organic wastes as components of horticulturalpotting media for growing manifold and vegetable seedlingsrdquoCompost Science and Utilization vol 8 no 3 pp 215ndash223 2000

[5] N Vassilev and M Vassileva ldquoBiotechnological solubilizationof rock phosphate on media containing agro-industrial wastesrdquoApplied Microbiology and Biotechnology vol 61 no 5-6 pp435ndash440 2003

[6] S A Omar ldquoThe role of rock-phosphate-solubilizing fungiand vesicular-arbusular-mycorrhiza (VAM) in growth of wheatplants fertilized with rock phosphaterdquo World Journal of Micro-biology and Biotechnology vol 14 no 2 pp 211ndash218 1998

[7] J A J Paul and T Daniel ldquoStandardization of sampling methodfor physical characterization of municipal solid wasterdquo AsianJournal of Water Environment and Pollution vol 5 no 1 pp95ndash98 2007

[8] R Lalloo D Maharajh J Gorgens and N Gardiner ldquoA down-stream process for production of a viable and stable Bacilluscereus aquaculture biological agentrdquo Applied Microbiology andBiotechnology vol 86 no 2 pp 499ndash508 2010

[9] B Sivasankari N Kavikumar and M Anandharaj ldquoIndole-3-acetic acid production and enhanced Plant growth promotionby Indigenous bacterial speciesrdquo Journal of Current Research inScience vol 1 no 5 pp 331ndash335 2013

12 Advances in Agriculture

[10] J Han L Sun X Dong et al ldquoCharacterization of a novel plantgrowth-promoting bacteria strain Delftia tsuruhatensis HR4both as a diazotroph and a potential biocontrol agent againstvarious plant pathogensrdquo Systematic and Applied Microbiologyvol 28 no 1 pp 66ndash76 2005

[11] H Rodrıguez and R Fraga ldquoPhosphate solubilizing bacte-ria and their role in plant growth promotionrdquo BiotechnologyAdvances vol 17 no 4-5 pp 319ndash339 1999

[12] T Daniel and N Karmegam ldquoBio-conversion of selected leaflitters using an African epigeic earthworm Eudrilus eugeniaerdquoEcology Environment and Conservation vol 5 no 3 pp 271ndash275 1999

[13] J Dobereiner I E Marriel and M Nery ldquoEcological distri-bution of Spirillum lipoferum Beijerinckrdquo Canadian Journal ofMicrobiology vol 22 no 10 pp 1464ndash1473 1976

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

[15] M Edi Premono A M Moawad and P L G Vlek ldquoEffect ofphosphate-solubilizing Pseudomonas putida on the growth ofmaize and its survival in the rhizosphererdquo Indonesian Journal ofCrop Science vol 11 pp 13ndash23 1996

[16] C L Patten and B R Glick ldquoBacterial biosynthesis of indole-3-acetic acidrdquo Canadian Journal of Microbiology vol 42 no 3pp 207ndash220 1996

[17] N Amaresan V Jayakumar K Kumar and N ThajuddinldquoIsolation and characterization of plant growth promotingendophytic bacteria and their effect on tomato (Lycopersiconesculentum) and chilli (Capsicum annuum) seedling growthrdquoAnnals of Microbiology vol 62 no 2 pp 805ndash810 2012

[18] B Schwyn and J B Neilands ldquoUniversal chemical assay forthe detection and determination of siderophoresrdquo AnalyticalBiochemistry vol 160 no 1 pp 47ndash56 1987

[19] P Rahi P Vyas S Sharma and A Gulati ldquoPlant growthpromoting potential of the fungus Discosia sp FIHB 571 fromtea rhizosphere tested on chickpea maize and peardquo IndianJournal of Microbiology vol 49 no 2 pp 128ndash133 2009

[20] K Kumar N Amaresan S Bhagat K Madhuri and R CSrivastava ldquoIsolation and characterization of rhizobacteriaassociated with coastal agricultural ecosystem of rhizospheresoils of cultivated vegetable cropsrdquo World Journal of Microbiol-ogy and Biotechnology vol 27 no 7 pp 1625ndash1632 2011

[21] J A Smoker and S R Barnum ldquoRapid small scale DNAisolation from filamentous cyanobacteriardquo FEMS MicrobiologyLetters vol 56 no 1 pp 119ndash122 1988

[22] U Edwars T Rogall H Blocker M Emde and E C BottgerldquoIsolation and direct complete nucleotide determination ofentire genes Characterization of a gene coding for 16S riboso-mal RNArdquoNucleic Acids Research vol 17 no 19 pp 7843ndash78531989

[23] D G Higgins A J Bleasby and R Fuchs ldquoCLUSTAL Vimproved software for multiple sequence alignmentrdquo ComputerApplications in the Biosciences vol 8 no 2 pp 189ndash191 1992

[24] M Kimura ldquoA simple method for estimating evolutionary ratesof base substitutions through comparative studies of nucleotidesequencesrdquo Journal ofMolecular Evolution vol 16 no 2 pp 111ndash120 1980

[25] N Saitou and M Nei ldquoThe neighbor-joining method a newmethod for reconstructing phylogenetic treesrdquo Molecular Biol-ogy and Evolution vol 4 no 4 pp 406ndash425 1987

[26] E Barbu K Y Lee and R Wahl ldquoContent of purine andpyrimidine base in desoxyribonucleic acid of bacteriardquo Annalesde lrsquoInstitut Pasteur vol 91 no 2 pp 212ndash224 1956

[27] N Sueoka ldquoVariation and heterogeneity of base compositionof deoxyribonucleic acids a compilation of old and new datardquoJournal of Molecular Biology vol 3 no 1 pp 31ndash40 1961

[28] H Hori and S Osawa ldquoEvolutionary change in 5S rRNAsecondary structure and a phylogenic tree of 352 5S rRNAspeciesrdquo BioSystems vol 19 no 3 pp 163ndash172 1986

[29] V Bess ldquoEvaluating microbiology of compost microbial con-tent of composting is helping the producers and growers tounderstand its role as a soil inoculant and plantrdquo BioCycleMagazine p 62 1999

[30] O Grafff ldquoPreliminary experiments of vermin composting ofdifferent waste materials using Eudrilus eugeniae Kinbergrdquo inWorkshop on the Role of Earthworms in the Stabilization ofOrganic Residues M Happelhof Ed pp 178ndash191 Beech LeafPress Kalamazoo Mich USA 1981

[31] J E Satchell Earthworm Ecology from Darwin to VermicultureChapman and Hall 1983

[32] G H Xie M Y Cai G C Tao and Y Steinberger ldquoCultivableheterotrophic N2-fixing bacterial diversity in rice fields in theYangtze River Plainrdquo Biology and Fertility of Soils vol 37 no 1pp 29ndash38 2003

[33] B BMcSpaddenGardener ldquoEcology ofBacillus and Paenibacil-lus spp in agricultural systemsrdquo Phytopathology vol 94 no 11pp 1252ndash1258 2004

[34] J Dobereiner ldquoBiological nitrogen fixation in the tropics socialand economic contributionsrdquo Soil Biology andBiochemistry vol29 no 5-6 pp 771ndash774 1997

[35] V M Reis F L Olivares and J Dobereiner ldquoImprovedmethodology for isolation of Acetobacter diazotrophicus andconfirmation of its endophytic habitatrdquoWorld Journal of Micro-biology and Biotechnology vol 10 no 4 pp 401ndash405 1994

[36] S Khalil N Ayub S Alam and F Latif ldquoOrganic acids pro-duction and phosphate solubilization by phosphate solubilizingmicroorganisms (PSM) under in vitro conditionsrdquo PakistanJournal of Biological Sciences vol 7 no 2 pp 187ndash196 2004

[37] H M A El-Komy ldquoCoimmobilization of Azospirillumlipoferum and Bacillus megaterium for successful phosphorusand nitrogen nutrition of wheat plantsrdquo Food Technology andBiotechnology vol 43 no 1 pp 19ndash27 2005

[38] N B Paul and W V B Sundara Rao ldquoPhosphate-dissolvingbacteria in the rhizosphere of some cultivated legumesrdquo Plantand Soil vol 35 no 1 pp 127ndash132 1971

[39] A Beneduzi D Peres L K Vargas M H Bodanese-Zanettiniand LM P Passaglia ldquoEvaluation of genetic diversity and plantgrowth promoting activities of nitrogen-fixing bacilli isolatedfrom rice fields in South Brazilrdquo Applied Soil Ecology vol 39no 3 pp 311ndash320 2008

[40] M P Raimam U Albino M F Cruz et al ldquoInteraction amongfree-living N-fixing bacteria isolated from Drosera villosa varvillosa and AM fungi (Glomus clarum) in rice (Oryza sativa)rdquoApplied Soil Ecology vol 35 no 1 pp 25ndash34 2007

[41] M R Swain S K Naskar and R C Ray ldquoIndole-3-acetic acidproduction and effect on sprouting of yam (Dioscorea rotundataL) minisetts by Bacillus subtilis isolated from culturable cow-dung microflorardquo Polish Journal of Microbiology vol 56 no 2pp 103ndash110 2007

[42] T M Tien M H Garkins and D H Hubbell ldquoPlant growthsubstances produced by Azospirillum brasilense and their effect

Advances in Agriculture 13

on the growth of pearl Millet Pennisetum americanum (L)rdquoApplied and EnvironmentalMicrobiology vol 37 no 5 pp 1016ndash1024 1979

[43] A Esitken L Pirlak M Turan and F Sahin ldquoEffects of floraland foliar application of plant growth promoting rhizobacteria(PGPR) on yield growth and nutrition of sweet cherryrdquo ScientiaHorticulturae vol 110 no 4 pp 324ndash327 2006

[44] H Karlidag A Esitken M Turan and F Sahin ldquoEffects of rootinoculation of plant growth promoting rhizobacteria (PGPR)on yield growth and nutrient element contents of leaves ofapplerdquo Scientia Horticulturae vol 114 no 1 pp 16ndash20 2007

[45] C S Nautiyal S Bhadauria P Kumar H Lal R Mondal andD Verma ldquoStress induced phosphate solubilization in bacteriaisolated fromalkaline soilsrdquoFEMSMicrobiology Letters vol 182no 2 pp 291ndash296 2000

[46] A K Halder A K Mishra P Bhattacharya and P KChakrabarthy ldquoSolubilization of inorganic phosphates byBradyrhizobiumrdquo Indian Journal of Experimental Biology vol29 pp 28ndash31 1991

Submit your manuscripts athttpwwwhindawicom

Nutrition and Metabolism

Journal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Food ScienceInternational Journal of

Agronomy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

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International Journal of

Microbiology

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Applied ampEnvironmentalSoil Science

Volume 2014

AgricultureAdvances in

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PsycheHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BiodiversityInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

GenomicsInternational Journal of

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Plant GenomicsInternational Journal of

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Biotechnology Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Forestry ResearchInternational Journal of

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Journal of BotanyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

EcologyInternational Journal of

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Veterinary Medicine International

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Cell BiologyInternational Journal of

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Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Page 3: BalayoganSivasankariandMarimuthuAnandharajdownloads.hindawi.com/journals/aag/2014/248591.pdf · In Vitro Nitrogen Fixing Activity. e bacterial isolates were screened for nitrogen

Advances in Agriculture 3

activities were observed on the basis of the formation ofturbidity in the flasks Among all the nineteen bacterialisolates tested only five bacterial isolates formed significantlyhigher rate of turbidity in the nitrogen-free liquid mediumand selected for further molecular and plant growth studies

232 In Vitro Phosphate Solubilization All bacterial iso-lates were screened for inorganic phosphate solubilizationusing Pikovskayarsquos agar medium the selective medium forphosphate solubilization test [14] Pikovskayarsquos agar mediumwas prepared and sterilized and the bacterial strains wereinoculated separately on the selective medium containing 05percent of (wv) tricalcium phosphate (Ca

3PO4) as complex

insoluble phosphate source All the test plates of the bacteriawere incubated at 37∘C plusmn 2∘C for 10 days After incubationfor up to 7 days at 30∘C formation of yellow halos andorclearing zones was evaluated The results were expressed assolubilization index (SI) and they were measured using thefollowing formula [15]

SI =Colony diameter + halozone diameter

Colony diameter (1)

Five bacterial strains which showed phosphate solubiliz-ing activity were identified and grouped as phosphate sol-ubilizing bacteria (PSB) The bacterial strain which showedhighest SI was selected for further molecular and plantgrowth studies

24 Production of Phytohormones (IAA) The bacterial cul-tures were inoculated in nutrient broth with tryptophan(5 120583gmL) and incubated at 28 plusmn 2∘C for 5 days Afterincubation cultures were centrifuged at 3000 rpm for 30minTwo milliliters of the supernatant was mixed with 2 drops oforthophosphoric acid and 4mL of Salkowskirsquos reagent (50mLof 35 perchloric acid + 1mL 05 FeCl

3) and incubated in the

dark for 25 minutes Development of pink colour indicatesindole-3-acetic acid (IAA) production The optical densitywas measured at 530 nm using Spectronic 200 (India) Thequantity of IAA production was estimated using standardIAA graph and expressed asmicrograms permilliliter [16 17]

25 Siderophore Production A qualitative assay ofsiderophore production was conducted in Chrome Azurol S(CAS) agar medium [18] CAS agar plates were prepared andspot-inoculated with test organism and incubated at 30∘C for3ndash5 days Change of blue color of the medium surroundingthe bacterial growth to fluorescent yellow indicated theproduction of siderophore Bacillus subtilis was chosen as apositive control [19 20]

26 16S rDNA Gene Amplification and Sequencing GenomicDNA from each isolate was extracted using the modifiedmethod of Smoker and Barnum [21] The extracted DNAwas dissolved in 20120583L TE buffer and used as the templatefor the PCR reactions PCR amplifications were performedin a total volume of 50 120583L by mixing 20 ng of the templateDNA with 25mM concentrations of each deoxynucleotide

triphosphate and 1 120583m of each universal primer of Bac8uf(51015840-AGAGTTTGATCCTGGCTCAG-31015840) and Univ1492r (51015840-CTACGGCTACCTTGTTACGA-31015840) described by Edwars etal [22] The thermocycling profile was carried out with aninitial denaturation at 95∘C (4min) followed by 30 cyclesof denaturation at 95∘C (1min) annealing at 56∘C (30 s)extension at 72∘C (1min) and a final extension at 72∘C(10min) in an Eppendorf Gradient thermocycler The PCRamplified rDNA were purified by using the Quick PCRpurification kit (Bangalore Genie India) 16S rRNA genesequence of the isolate was compared with 16S rRNA genesequences available by the BLAST search in the NCBIGenBank database (httpwwwncbinlmnihgov) The anal-ysis of alignment and homology of the partial nucleotidesequence of Bacillus sp was carried out by the basic localalignment search tool (BLAST) The PCR products werestored at 4∘C Aliquots of the PCR products were separatedby 2 agarose gel electrophoresis in TAE buffer (pH 80)A 100 bp DNA marker (Sigma Bangalore) was used as areference Gels were stainedwith ethidiumbromide (11 gmL)and visualized under UV light

27 Nucleotide Sequence Accession The sequence obtained inthis study was deposited in the GenBank (USA) nucleotidesequence database under the accessionnumberGQ413962 (Bcereus) and HM753262 (Pseudomonas spp)

28 Phylogenetic Analysis The 16S rRNA gene sequenceswere obtained in the present study and the taxonomicallyrelated Bacillus spp were retrieved from the NationalCenter from Biotechnology Information (NCBI) database(httpwwwncbinlmnihgov) All the sequences werealigned using multiple sequence alignment programCLUSTAL W developed by Higgins et al [23] The pairwiseevolutionary distances were computed using the methodof Kimura [24] The multiple distance matrix obtained wasthen used to construct phylogenetic trees using neighbourjoining method of Saitou and Nei [25] Phylogenetic treeswere constructed by using Mega 5 software (MolecularEvolutionary Genetic Analysis)

29 Prediction of RNA Secondary Structure The secondarystructure of 16S rDNA of Bacillus spp was predicted usingthe bioinformatics Genebee tool

210 Restriction Site Analysis in 16S rDNA The restrictionsites in DNA of Bacillus spp were analyzed using theNEB Cutter program version 20 tools available online athttptoolsnebcomNEBcutter2indexphp

211 Fermentation Process For the preparation of inocu-lum 50mL of nutrient broth medium (HiMedia MumbaiIndia) was inoculated with appropriate overnight bacte-rial culture and incubated at 37∘C for 24 h at 120 rpm(OD540= 045 10

7-108 CFUmLminus1) The cell-free super-natant was obtained by centrifuging the fermented broth at

4 Advances in Agriculture

10000 rpm for 10minThe supernatantwas analyzed for plantgrowth promotion at various concentrations

212 Evaluation of Plant Growth Promoting Activity underControlled Environment The nitrogen fixing bacteria (Bacil-lus spp) and the phosphate solubilizing bacteria (Pseu-domonas spp) were separately tested for plant growth pro-moting activity and also as consortium (1 1) at 5 differentconcentrations that is 1 2 3 4 and 5mL at three differentday intervals (5 10 and 15 days) with proper control throughExp1 Exp2 Exp3 and Exp0 respectively usingVigna unguic-ulata (L) Walp The seeds (30 seeds in each pot) were sownin plastic pots containing sterilized vermiculite and arrangedin a completely randomized factorial design The seedlingswere grown in a greenhouse at a temperature of 28ndash32∘Cand 85 relative humidity The pots were watered to 50water-holding capacity and were maintained at this moisturecontent by watering to weight every day and plant growthwasobserved for 15 days Parameters such as germination percentshoot length root length leaf area chlorophyll content of theleaves fresh weight of the whole plant and dry weight of thewhole plant were measured using standard procedure

213 Statistical Analysis The following statistical tools wereused for the analyses and interpretation of the data Theexperimental results are presented in the form of tablesand graphs using Microsoft Excel 2007 Data obtained fromthe different treatments were statistically analyzed using theone-way ANOVA and the mean values were compared bythe Duncanrsquos multiple range test for multiple comparisonsDifferences were considered significant at the 005 level usingOrigin software (Version 850) 2010 Origin Lab Corpora-tion

3 Results

31 Isolation and Characterization of Bacterial Isolates Atotal of nineteen bacterial strains were isolated from vermi-compost vermicast and earthworm (fore mid and hind)guts (Table 1) The morphological and biochemical char-acteristics of bacterial isolates were elucidated in Table 1All the isolates were tested for plant growth promotingactivity Among the 19 bacterial isolates only five bacterialstrains showed plant growth promoting activity Based oncolony morphology microscopic observations and culturalbiochemical and physiological properties the bacteriumwasgiven the name Bacillus spp Alcaligenes spp Erwinia sppSerratia spp or Pseudomonas sppThese five bacterial strainswere able to grow in the nitrogen-free medium and they werealso identified as phosphate solubilizing microorganisms onthe basis of their solubilization index (Table 2) Bacillus spphave the highest SI (3855) followed by Pseudomonas spp(3522) and Serratia spp (3045) All the isolates produceda significant amount of IAA from the medium in the rangeof 113 to 242120583gmL Bacillus spp showed higher IAAproduction (242120583gmL) and Serratia spp showed lower IAAproduction (113 120583gmL) All the strains (except Alcaligenes

spp) were positive for siderophore production showing ayellow zone on the CAS agar medium plate (Table 2)

32 Molecular Studies The partial 16S rRNA sequencescarried out in the present study for Bacillus spp andPseudomonas spp covered a stretch of approximately 1500nucleotides for each About half of the sequences found in theclone library showed only slight relationship to other knownsequences while the other half were highly similar (approx-imately 95 percent sequence identity) to other databaseentries for Bacillus spp and Pseudomonas spp Less than 05percent of all nucleotides was found to be unique within theconserved regions of the cloned sequence and could almostalways be related to reading errors in ambiguous regions ofthe sequencing gel On the basis of phylogenetic analysisof 16S rDNA partial sequences Bacillus spp GGBSTD1 isidentified as Bacillus cereus and Pseudomonas spp GGBSTD3is identified as Pseudomonas spp Phylogenetic analyses ofthe strains based on the neighbor joining method wererepresented in Figures 1 and 2 Among eubacteria the meanguanine and cytosine (G + C) content of genomic DNAvaries from approximately 25 to 75 That the bacterialgenomic G + C content is somehow related to phylogeny hasbeen suggested [26 27] The phylogenetic tree of eubacterial16S rRNA clearly indicates this relationship Gram-positivebacteria Bacillus subtilis have genomic G + C 42 Gram-negative bacteria with intermediate G + C content such asEscherichia coli (50) Serratia marcescens (58) Salmonellatyphimurium (51) and Pseudomonas fluorescens (60)belong to the common Gram-negative bacteria [28] Bacilluscereus have the G + C content 53 and A + T content 47similarly Pseudomonas spp having G + C content 53 andA + T content 47 Most functional RNA molecules havecharacteristic secondary structures that are highly conservedin evolution In this study RNA secondary structures ofBacillus cereus and the Pseudomonas spp were predictedBacillus cereus have free energy of minus2941 kkalmol andPseudomonas spp have free energy of minus2998 kkalmol

33 Evaluation of Biofertilization Abilities under ControlledConditions Germination percentage of Vigna unguiculataseeds sown in vermiculite supplemented with Bacillus cereus(Exp1) with Pseudomonas spp (Exp2) and with consortium(Exp3) (1 1 ratio) at five different concentrations (1 2 3 4and 5mL) and in the control (Exp0) is shown in Figure 3The highest germination percentage was observed in Exp3(consortium) at 5mL concentration compared to others Theincrease in germination percentage and in other growthparameters can be attributed to the combined effect of thenitrogen fixing and phosphate solubilizing microbes whichare able to fix atmospheric nitrogen and solubilize P andalso produce growth promoting substancesThese organismshave been isolated from vermisources and this observationindicates the reason for promotion and enhancement of cropgrowth and yield when vermicompost is applied It is a proofthat these symbioticmicrobes present in vermisources benefitthe soil fertility Observation on growth parameters such asshoot length root length and leaf area of V unguiculata

Advances in Agriculture 5

Table1

Predo

minantb

acteria

lstrains

isolated

from

verm

isourcesand

theirb

iochem

icalcharacteris

ticsT1-con

trolmdash

Gsepium

+L

leucocephala+

cowdu

ngin

(112)(worm

free)T

2mdashG

sepium

+L

leucocephala+cowdu

ngin

(112)+

EeugeniaeT3

mdashG

sepium

+L

leucocephala+cowdu

ngin

(112)+

EfetidaT4

-con

trol(2)mdashC

auric

ulata+L

leucocephala+cow

dung

in(112)(worm

free)T

5mdashC

auric

ulata+L

leucocephala+cowdu

ngin

(112)+

EeugeniaeT

6mdashC

auric

ulata+L

leucocephala+cowdu

ngin

(112)+

Efetida

Snu

mber

Samples

ource

Biochemicalcharacteris

ticsc

Nam

eofthe

strain

Gramrsquos

reactio

naCell

shape

Motility

bIndo

letest

Methyl

Red

test

Voges

Proskauer

test

Citrate

Utilization

test

Catalase

test

Urease

test

Gelatin

hydrolysis

test

Nitrate

redu

ctase

test

Starch

hydrolysis

test

Casein

hydrolysis

test

Glucose

utilizatio

ntest

1Ve

rmicom

post

(T2)

+Cocciminus

minus+

minusminus

+minus

++

minusminus

minusStaphylococcus

spp

2Ve

rmicom

post

(T3)

+Ro

d+

minusminus

minusminus

+minus

minus+

minusminus

+Co

rynebacterium

spp

3Ve

rmicom

post

(T5)

+Ro

dminus

++

++

+minus

++

+minus

+Ba

cillusspp

4Ve

rmicom

post

(T6)

+Cocciminus

++

minusminus

+minus

+minus

minusminus

+Micrococcusspp

5Ve

rmicast(T3

)+

Cocciminus

++

minusminus

minus+

++

+minus

minusKlebsiella

spp

6Ve

rmicast(T5

)+

Rod

minus+

minusminus

+minus

+minus

+minus

minus+

Streptococcusspp

7

Verm

icast(T6

)+

Cocciminus

+minus

++

minusminus

++

minusminus

+En

terobacter

spp

8Ea

rthw

orm

hind

gut(T2

)+

Cocciminus

minus+

+minus

+minus

minusminus

++

+Trico

ccocus

spp

9Ea

rthw

orm

hind

gut(T3

)+

Cocciminus

minus+

minus+

++

minus+

minusminus

minusYersiniaspp

10

Earthw

orm

hind

gut(T5

)+

Cocciminus

minusminus

minusminus

++

++

minusminus

minusMicrococcusspp

11

Verm

icom

post

(T1)

minusRo

dminus

++

minusminus

minusminus

++

minusminus

+Escherich

iaspp

12

Verm

icom

post

(T2)

minusRo

dminus

minus+

minusminus

minus+

++

minusminus

+Proteusspp

13

Verm

icom

post

(T4)

minusRo

dminus

minusminus

+minus

+minus

minus+

++

+Pa

racoccus

spp

14

Verm

icom

post

(T5)

minusRo

d+

+minus

minusminus

++

++

minusminus

minusPseudomonas

spp

15

Verm

icast(T2

)minus

Rod

+minus

+minus

++

+minus

+minus

minusminus

Citro

bacter

spp

16

Verm

icast(T3

)minus

Cocciminus

++

minusminus

+minus

+minus

minusminus

minusErwiniaspp

17Ve

rmicast(T5

)minus

Rod

++

++

++

minus+

minusminus

minusminus

Alcaligenesspp

18

Verm

icast(T6

)minus

Cocciminus

minus+

++

+minus

++

minusminus

minusSerratiaspp

19

Earthw

orm

mid

gut(T2

)minus

Rod

+minus

minusminus

+minus

minusminus

minusminus

minusminus

Pediococcusspp

a Gramrsquossta

ining(+)p

ositive(minus)n

egative

b Motility(+)

motile(minus)n

onmotile

c Biochem

icalcharacteris

tics(+)p

ositive(minus)n

egative

6 Advances in Agriculture

Bacillus cereus strain GGBSTD2 16S ribosomal RNA gene

Bacillus cereus strain SU-6 16S ribosomal RNA gene

Bacillus cereus strain JB0013 16S ribosomal RNA gene

Bacillus cereus isolate PN24 16S ribosomal RNA gene

Bacillus cereus strain JB0005 16S ribosomal RNA gene

Bacillus sp G2DM-85 16S ribosomal RNA gene

Bacillus cereus strain JBE0009 16S ribosomal RNA gene

Bacillus sp EB460 16S ribosomal RNA gene

Bacillus cereus strain 421-3R 16S ribosomal RNA gene

Bacillus sp EB447 16S ribosomal RNA gene

Bacillus sp EB470 16S ribosomal RNA gene

Bacillus sp EB421 16S ribosomal RNA gene

Bacillus sp EB431 16S ribosomal RNA gene

Bacillus sp EB462 16S ribosomal RNA gene

Bacillus sp EB429 16S ribosomal RNA gene

Bacillus cereus strain 421-4R 16S ribosomal RNA gene

Bacillus sp EB480 16S ribosomal RNA gene

Bacillus sp EB13 16S ribosomal RNA gene

Uncultured compost bacterium partial 16S rRNA gene

Bacillus thuringiensis gene for 16S rRNA

Figure 1 Neighbor joining phylogenetic tree of full 16S rRNA gene sequences showing the relationship among Bacillus cereus GGBSTD1and representatives of some related taxa The sequence data for several closely related Bacillus cultures were recovered from GenBank andincluded in the tree

Table 2 The phosphate solubilizing activity phytohormone (IAA) production and siderophores production of the bacterial isolates

Name of the organism Phosphate solubilization index (SI) IAA production (120583gmL)a Siderophores productionb

Bacillus spp 3855 242 +++Alcaligenes spp 2930 147 minus

Erwinia spp 2890 113 +Serratia spp 3045 168 ++Pseudomonas spp 3522 196 +++aIndole acetic acid production in culture media supplemented with tryptophan (5 120583gmL) after 5 daysbIn vitro siderophores production minus represents the absence of siderophores production and +++ represents gt10mm wide yellow-orange zone

is given in Tables 3 and 4 respectively The shoot length(1147 plusmn 006 cm) root length (1027 plusmn 006 cm) and leafarea (905 plusmn 026 cm2) of the plant get significantly (119875 lt0001) increased in Exp3 (consortium of Bacillus cereus +Pseudomonas spp [1 1]) at 5mL concentration on the 15thday compared to other experimentsThe chlorophyll a b andtotal chlorophyll content of the leaves on d 5 d 10 and d 15are given in Table 5 The chlorophyll a (010 plusmn 009mgg offresh leaf) chlorophyll b (1085 plusmn 016mgg of fresh leaf)and total chlorophyll (1095plusmn007mgg of fresh leaf) content

of leaves were significantly (119875 lt 0001) higher in Exp3(consortium of Bacillus cereus + Pseudomonas spp (1 1))at 5mL concentration on the 15th day compared to otherexperiments Observations on the fresh weight of the wholeplant are given in Table 6 The fresh weight of the wholeplant (092 plusmn 000 g) was significantly (119875 lt 0001) higherin Exp3 (consortium of Bacillus cereus + Pseudomonas spp(1 1)) at 5mL concentration on the 15th day compared toother experiments Observations on the dry weight of thewhole plant are given in Table 6 The dry weight of the whole

Advances in Agriculture 7

Pseudomonas fluorescens strain LMG 5825 16S ribosomal RNA gene

Pseudomonas fluorescens strain LMG 5829 16S ribosomal RNA gene

Pseudomonas fluorescens strain LMG 14676 16S ribosomal RNA gene

Pseudomonas sp W15Feb13 16S ribosomal RNA gene

Pseudomonas sp W15Feb28 16S ribosomal RNA gene

Pseudomonas sp p50 16S ribosomal RNA gene

Uncultured bacterium clone 661193 16S ribosomal RNA gene

Pseudomonas sp BSs20166 16S ribosomal RNA gene

Pseudomonas sp 28zhy partial 16S rRNA gene

Uncultured bacterium clone AYRV1-027 16S ribosomal RNA gene

Uncultured Pseudomonadaceae bacterium clone290cost002-P3L-2032 16S ribosomal RNA gene

Pseudomonas sp GGBSTD3 16S ribosomal RNA gene

Pseudomonas sp DK3 16S ribosomal RNA gene

Pseudomonas reactans strain AMP-13 16S ribosomal RNA gene

Figure 2 Neighbor joining phylogenetic tree of full 16S rRNA gene sequences showing the relationship among Pseudomonas spp GGBSTD3and representatives of some related taxa The sequence data for several closely related Pseudomonas cultures were recovered from GenBankand included in the tree

plant (009 plusmn 000 g) was significantly (119875 lt 0001) higherin Exp3 (consortium of Bacillus cereus + Pseudomonas spp(1 1)) at 5mL concentration on the 15th day compared toother experiments

4 Discussion

Inoculation of these newly identified nitrogen fixing andphosphate solubilizingmicrobes in the plant growthmediumenhanced various physiological activities culminating inhigher biomass production in V unguiculata Since this is anonhazardous way of fertilization of crop plants it is veryrelevant to a developing country like India This techniquecan save the farmers of India from the problem posed byhigh cost of fertilizers by offering a comparatively inexpensivealternative (biofertilizers) while additionally preventing thedegradation of the soil and thereby ensuring sustainable agri-culture Bess (1999) reported that the content in compostingneeds to be determined through the concentration of sixfunctional groups of microorganisms such as aerobic bacte-ria anaerobic bacteria fungi actinomycetes Pseudomonasand nitrogen fixing bacteria [29] Now there are ways toevaluate the concentrations of these organisms in the finishedcompost and that can serve as an interpretation guide to

determine the quality of the compost as an inoculant of soilmicroorganisms Grafff (1981) and Atlavinyte have reporteda higher P content in the cast than in the surrounding soil[30] Satchell (1983) has linked the phosphatase activity inthe gut of worms to the availability of bound P in soils Thebacterial strains which have the capability to fix nitrogenonly can grow in the nitrogen-free medium [31] Hencethe nitrogen fixing ability was tested in the present studybased on the growth of bacterial strain in nitrogen- freemedium Five bacteria strains showedmore nitrogen fixationamong all the five bacterial strains tested only one strain (ieBacillus spp) showed significantly more turbidity comparedto the other strains The ultimate source of the nitrogen usedby plants is N

2gas which constitutes 78 of the Earthrsquos

atmosphere Unfortunately higher plants cannot metabolizeN2directly into protein N

2gas must be converted to a plant

available form [32] isolated many species of nitrogen fixingBacillus (B megaterium B cereus B subtilis B licheniformisand B azotoformans) from the rhizospheric zone of ricefields in Central China The widely studied Bacillus genusrepresents one of the most diverse genera in the bacilli groupNumerous Bacillus and Paenibacillus strains express plantgrowth promoting (PGP) activities and a number of thesestrains have already been commercially developed as biolog-ical fungicides insecticides and nematicides or generic plant

8 Advances in Agriculture

Table 3 Shoot length and Root length ofVigna unguiculata grown on vermiculite supplemented with Bacillus cereus with Pseudomonas sppand with their consortium (1 1) at five different concentrations (1 2 3 4 and 5mL) on the 5th the 10th and the 15th day and in the control

Experimentsa Quantity of bacterial cultures added Shoot length (cm)b Root length (cm)b

5th day 10th day 15th day 5th day 10th day 15th day

Exp0

1mL 297 (ab) 397 (a) 497 (ab) 197 (de) 283 (ab) 397 (bcd)2mL 303 (bcd) 403 (ab) 500 (ab) 200 (abc) 287 (de) 400 (a)3mL 310 (abc) 407 (a) 503 (ab) 203 (de) 290 (abc) 403 (ab)4mL 310 (bcd) 410 (bcd) 507 (bcd) 207 (a) 297 (de) 410 (abc)5mL 313 (a) 413 (ab) 510 (ab) 210 (bcd) 300 (ab) 413 (de)

Exp1

1mL 340 (ab) 440 (abc) 540 (ab) 233 (abc) 327 (bcd) 433 (f)2mL 450 (ab) 540 (abc) 647 (ab) 333 (ab) 430 (a) 540 (abc)3mL 550 (de) 630 (ab) 750 (a) 440 (de) 550 (abc) 620 (a)4mL 653 (ab) 730 (a) 833 (ab) 550 (bcd) 653 (de) 750 (abc)5mL 740 (ab) 840 (abc) 923 (f) 640 (abc) 753 (ab) 827 (ab)

Exp2

1mL 277 (bcd) 357 (f) 440 (de) 160 (de) 217 (abc) 350 (f)2mL 343 (abc) 447 (ab) 553 (ab) 253 (abc) 340 (bcd) 427 (bcd)3mL 437 (de) 543 (abc) 633 (abc) 340 (ab) 443 (f) 533 (ab)4mL 547 (bcd) 647 (de) 733 (a) 420 (bcd) 550 (ab) 633 (ab)5mL 640 (f) 733 (bcd) 833 (bcd) 533 (ab) 630 (ab) 730 (bcd)

Exp3

1mL 530 (bcd) 647 (ab) 730 (ab) 433 (de) 550 (abc) 620 (ab)2mL 643 (ab) 740 (bcd) 827 (f) 547 (a) 630 (de) 733 (ab)3mL 737 (bcd) 840 (bcd) 927 (ab) 640 (ab) 740 (bcd) 840 (bcd)4mL 847 (abc) 930 (a) 1043 (abc) 740 (f) 833 (a) 937 (abc)5mL 957 (ab) 1030 (f) 1147 (bcd) 833 (abc) 940 (ab) 1027 (bcd)

aExp0 nutrient solution (control) Exp1 Bacillus cereus Exp2 Pseudomonas spp Exp3 consortium of Bacillus cereus + Pseudomonas spp (1 1)bMeanof two repeated experiments (30 plants)Different letterswithin parenthesis indicate significant difference between treatments for each growth parameterusing Duncanrsquos multiple range test (119875 = 005)

growth promoters The use of these strains in agriculturehas recently been reviewed [33]Those strains besides havingseveral PGP properties can also fix nitrogen and also dophosphate solubilization The symbiotic fixation of nitrogenthrough inoculation of legume crops with effective rhizobiais well known [34] Asymbiotic nitrogen fixing bacteriawhich live in the rhizosphere andor endophytically oftenincrease yields of crops Many bacterial species have nitrogenfixing properties including Bacillus spp Azotobacter sppAzospirillum spp Beijerinckia spp and Pseudomonas spp[35] Nitrogen fixing bacteria have been used in foliar appli-cations inmulberryThe solubilization index (SI) was formeddue to solubilization of insoluble phosphates by organic acidsecretion [36] El-Komy (2005) indicated that Pseudomonasfluorescence and Bacillus megaterium strains were the mostpowerful phosphate solubilizers on PVK plates as well as onPikovskayarsquos broth [37] The P concentration in Pikovskayarsquosbroth increased gradually achieving a peak on the sixth dayand declined slowly during the late days In this study pHvalues decreased gradually in PVK broth during the earlyincubation days and no revival was observed during laterdays for all the tested bacterial strains Several bacteria par-ticularly those belonging to the genus Bacillus spp convertinsoluble phosphate into soluble forms by secreting organicacids such as formic acid acetic acid propionic acid citricacid fumaric acid gluconic acid glyoxylic acid ketobutyric

acid malonic acid succinic acid and tartaric acid Theseacids lower the pH and bring about the dissolution of boundforms of phosphate Some of the hydroxyl acids may chelatewith calcium and iron resulting in effective solubilization andutilization of phosphates [38]

Beneduzi et al (2008) had isolated the plant growthpromoting strain SVPR30 and identified by 16S rRNA genesequence as Bacillus spp and they had tested it for plantgrowth through in vivo experiments [39] This strain wascharacterized as a high IAA producer able to solubilize phos-phate and also fix a considerably high amount of nitrogenThe inoculation of rice with Bacillus spp SVPR30 strainshowed a significant increase in the root and shoot partswhen compared with the controls within 15 and 30 daysafter sprouting Numerous Bacillus and Paenibacillus strainsexpressed plant growth promoting (PGP) activities and anumber of these strains have been commercially developedas generic plant growth promotersThe use of these strains inagriculture has recently been reviewed [33]

Various reports indicate that coinoculation of beneficialorganisms generally increased plant growth relative to singleinoculation with a sole beneficial organism [40] Most ofthe effects of the individual microorganisms coinoculationare additive although a synergistic effect has been reportedin some cases The average root to shoot ratio was higherin B subtilis treated yam minisetts in comparison to those

Advances in Agriculture 9

Table 4 Leaf area of Vigna unguiculata grown on vermiculite supplemented with Bacillus cereus with Pseudomonas spp and with theirconsortium (1 1) at five different concentrations (1 2 3 4 and 5mL) on the 5th the 10th and the 15th days and in the control

Experimentsa Quantity of bacterial cultures added Leaf area (cm2)b

5th day 10th day 15th day

Exp0

1mL 027 (ab) 094 (abc) 407 (b)2mL 035 (ab) 102 (ab) 419 (abc)3mL 040 (abc) 125 (ab) 471 (bcd)4mL 048 (ab) 138 (abc) 510 (ab)5mL 054 (abc) 161 (b) 538 (b)

Exp1

1mL 018 (bcd) 123 (b) 601 (abc)2mL 025 (ab) 138 (abc) 632 (ab)3mL 033 (abc) 150 (ab) 684 (b)4mL 038 (ab) 168 (ab) 686 (abc)5mL 044 (abc) 191 (bcd) 733 (ab)

Exp2

1mL 011 (b) 107 (ab) 534 (b)2mL 014 (ab) 119 (ab) 559 (ab)3mL 017 (abc) 127 (ab) 579 (ab)4mL 021 (ab) 153 (abc) 631 (abc)5mL 026 (ab) 166 (b) 667 (b)

Exp3

1mL 026 (bcd) 297 (ab) 753 (bcd)2mL 033 (b) 325 (ab) 787 (ab)3mL 040 (ab) 350 (bcd) 811 (bcd)4mL 044 (b) 384 (b) 861 (b)5mL 062 (ab) 400 (bcd) 905 (ab)

aExp0 nutrient solution (control) Exp1 Bacillus cereus Exp2 Pseudomonas spp Exp3 consortium of Bacillus cereus + Pseudomonas spp (1 1)bMeanof two repeated experiments (30 plants)Different letterswithin parenthesis indicate significant difference between treatments for each growth parameterusing Duncanrsquos multiple range test (119875 = 005)

0102030405060708090

100

Concentration of bacterial cultures (mL)1 2 3 4 5

Germination ()

Exp0

Exp1 Exp3

Exp2

Figure 3 Germination percentage ofVigna unguiculata seeds sownin vermiculite supplementedwithBacillus cereus with Pseudomonasspp and with their consortium at five different concentrations (1 23 4 and 5mL) and in the control Exp0 nutrient solution (control)Exp1 Bacillus cereus Exp2 Pseudomonas spp Exp3 consortium ofBacillus cereus + Pseudomonas spp (1 1)

not treated with the bacterial culture [41] In earlier reportsroot elongation was found to occur in Sesbania aculeata byinoculation with Azotobacter spp and Pseudomonas spp in

Vigna radiata by Pseudomonas putida [16] and in Pennisetumamericanum by Azospirillum brasilense [42] Esitken et al(2006) had found out that Bacillus OSU-142 and Pseu-domonas BA-8 alone or in combination had a great potentialto increase the growth yield and nutrition of sweet cherryplant [43] BacillusM3 BacillusOSU-142 andMicobacteriumFS01 separately or in combination were found to have greatpotential for use as plant growth promoting rhizobacteriato increase production in apples and in many other crops[44] These phosphate solubilizing microbes consist pre-dominantly of fungal bacterial and actinomycetes speciescollectively called phosphate solubilizing microorganismsmdashPSM [45] There are several microorganisms which canalso solubilize the cheaper sources of phosphorous such asrock phosphate Bacteria such as Bacillus are widely usedin plant production system and are important phosphorussolubilizing microorganisms resulting in improved growthyield of crops andmetabolic activities especially in synthesisof protein [34 46]

5 Conclusions

This study illustrates the isolation of plant growth promotingbacteria from vermisources screening under in vitro con-ditions for multiple PGPR traits and their evaluation undercontrolled conditions in a pot experiment The findings ofthis study indicated that the consortium of B cereus and

10 Advances in Agriculture

Table5Ch

loroph

yllcon

tent

ofleavesofVignaun

guicu

latagrow

non

verm

iculite

supp

lementedwith

Bacilluscereuswith

Pseudomonas

sppand

with

theircon

sortium

(11)atfi

vedifferent

concentrations

(1234and

5mL)

onthe5

thdaythe10thdayandthe15thdayandin

thec

ontro

l

Experim

entsa

Quantity

ofbacterial

Cultu

resa

dded

Chloroph

yllcon

tent

(mggof

fresh

leaf)b

5thday

10th

day

15th

day

Chloroph

ylla

Chloroph

yllb

Total

chloroph

yll

Chloroph

ylla

Chloroph

yllb

Total

chloroph

yll

Chloroph

ylla

Chloroph

yllb

Total

chloroph

yll

Exp0

1mL

021

(ab)

285

(abc)

307

(a)

020

(abc)

481

(a)

501

(ab)

015

(bcd)

612

(abc)

627

(bcd)

2mL

022

(abc)

293

(ab)

315

(abc)

021

(ab)

489

(ab)

510

(bcd)

012

(abc)

618

(ab)

630

(ab)

3mL

023

(abc)

301

(ab)

315

(abc)

022

(bcd)

497

(bcd)

518

(a)

014

(a)

633

(cde)

647

(bcd)

4mL

024

(ab)

308

(f)

332

(a)

022

(a)

504

(b)

527

(abc)

015

(bcd)

641

(cde)

656

(a)

5mL

025

(f)

316

(abc)

341

(bcd)

019

(ab)

511(ab)

530

(abc)

011(cde)

645

(ab)

650

(abc)

Exp1

1mL

035

(b)

400

(cde)

435

(abc)

011(b)

581

(cde)

593

(cde)

010

(abc)

704(abc)

715(f)

2mL

026

(abc)

462

(bcd)

488

(f)

011(abc)

647

(b)

658

(abc)

006

(ab)

779(bcd)

785(a)

3mL

021

(bcd)

526

(abc)

547

(ab)

008

(bcd)

718(ab)

726(bcd)

006

(f)

847

(f)

853

(ab)

4mL

008

(f)

587

(cde)

595

(b)

011(bcd)

778(abc)

789(abc)

004

(bcd)

933(b)

937(bcd)

5mL

026

(abc)

641

(bcd)

667

(abc)

025

(cde)

834

(cde)

859

(f)

007

(abc)

976(cde)

983(abc)

Exp2

1mL

030

(abc)

354

(ab)

384

(b)

012

(a)

545

(abc)

556

(ab)

007

(bcd)

681

(abc)

688

(bcd)

2mL

029

(bcd)

420

(f)

449

(abc)

011(bcd)

610

(ab)

621

(abc)

007

(abc)

744(f)

752(bcd)

3mL

022

(ab)

504

(abc)

527

(bcd)

015

(a)

677

(cde)

692

(bcd)

006

(bcd)

812

(ab)

817

(bcd)

4mL

010

(f)

566

(a)

576

(cde)

018

(abc)

744(f)

762(ab)

005

(ab)

885

(a)

890

(abc)

5mL

006

(bcd)

622

(abc)

628

(abc)

018

(b)

810

(abc)

828

(a)

004

(cde)

948(a)

952(f)

Exp3

1mL

027

(ab)

470

(bcd)

496

(cde)

009

(ab)

668

(b)

677

(cde)

010

(a)

814

(ab)

825

(a)

2mL

020

(ab)

548

(a)

568

(f)

003

(bcd)

746(bcd)

749(ab)

009

(ab)

887

(f)

897

(ab)

3mL

005

(bcd)

584

(abc)

589

(ab)

008

(bcd)

804

(abc)

812

(cde)

010

(ab)

953(abc)

963(bcd)

4mL

023

(f)

663

(ab)

686

(cde)

007

(ab)

870

(b)

877

(f)

010

(abc)

1019

(bcd)

1029(f)

5mL

022

(ab)

744(cde)

766(a)

009

(a)

932(abc)

940(ab)

010

(a)

1085(ab)

1095(abc)

a Exp0nu

trient

solutio

n(con

trol)

Exp1B

acillus

cereusE

xp2Pseudomonas

sppE

xp3consortiu

mof

Bacilluscereus+

Pseudomonas

spp(11)

b Meanof

tworepeated

experim

ents(30plants)

Differentletterswith

inparenthesis

indicatesig

nificantd

ifference

betweentre

atmentsfore

achgrow

thparameter

usingDun

canrsquos

multip

lerangetest(119875=005)

Advances in Agriculture 11

Table 6 Fresh weight and dry weight of whole plant of Vigna unguiculata grown on vermiculite supplemented with Bacillus cereus withPseudomonas spp and with their consortium (1 1) at five different concentrations (1 2 3 4 and 5mL) on the 5th the 10th and the 15 daysand in the control

Experimentsa Quantity of bacterial cultures added Fresh weight of the whole plant (g)b Dry weight of the whole plant (g)b

5th day 10th day 15th day 5th day 10th day 15th day

Exp0

1mL 058 (bcd) 061 (abc) 064 (ab) 006 (a) 006 (abc) 006 (ab)2mL 058 (ab) 061 (a) 064 (abc) 006 (bcd) 006 (ab) 006 (ab)3mL 058 (abc) 061 (ab) 064 (bcd) 006 (abc) 006 (bcd) 006 (b)4mL 041 (abc) 061 (ab) 064 (ab) 006 (abc) 006 (ab) 006 (abc)5mL 058 (abc) 061 (abc) 064 (bcd) 006 (a) 006 (bcd) 006 (def)

Exp1

1mL 062 (abc) 065 (abc) 068 (ab) 006 (bcd) 006 (ab) 007 (bcd)2mL 065 (bcd) 068 (bcd) 071 (def) 006 (abc) 007 (a) 007 (ab)3mL 068 (abc) 071 (def) 074 (bcd) 007 (abc) 007 (ab) 007 (abc)4mL 071 (ab) 074 (abc) 077 (abc) 007 (bcd) 007 (b) 008 (bcd)5mL 074 (abc) 077 (def) 080 (ab) 007 (def) 008 (a) 008 (b)

Exp2

1mL 056 (ab) 059 (bcd) 062 (ab) 006 (ab) 006 (ab) 006 (abc)2mL 059 (abc) 062 (abc) 065 (def) 006 (ab) 006 (abc) 006 (bcd)3mL 062 (bcd) 065 (a) 068 (bcd) 006 (abc) 006 (bcd) 007 (abc)4mL 065 (abc) 068 (ab) 071 (b) 006 (ab) 007 (abc) 007 (b)5mL 068 (abc) 071 (b) 074 (a) 007 (def) 007 (abc) 007 (bcd)

Exp3

1mL 074 (ab) 077 (ab) 080 (abc) 007 (a) 008 (bcd) 008 (ab)2mL 077 (bcd) 080 (a) 083 (bcd) 008 (a) 008 (bcd) 008 (abc)3mL 080 (bcd) 083 (bcd) 086 (abc) 008 (ab) 008 (b) 009 (b)4mL 083 (bcd) 086 (abc) 089 (ab) 008 (abc) 009 (ab) 009 (bcd)5mL 086 (ab) 089 (b) 092 (def) 009 (bcd) 009 (bcd 009 (b)

aExp0 nutrient solution (control) Exp1 Bacillus cereus Exp2 Pseudomonas spp Exp3 consortium of Bacillus cereus + Pseudomonas spp (1 1)bMeanof two repeated experiments (30 plants)Different letterswithin parenthesis indicate significant difference between treatments for each growth parameterusing Duncanrsquos multiple range test (119875 = 005)

Pseudomonas spp acts as a potential biofertilizer due to itsvarious plant growth promotion abilities such as solubiliza-tion of phosphate and production of IAA and siderophore itcan be developed as potential microbial inoculant for variouscrops

Conflict of Interests

The authors declared that there is no conflict of interestsregarding this paper

Acknowledgments

Theauthors are thankful to theDepartment of BiologyGand-higram Rural InstitutemdashDeemed University GandhigramThis research was funded by University Grants Commissionunder UGC Research Fellowship in Science for MeritoriousStudents (BSR) Grant no F4-12008

References

[1] U S Bhawalkar Vermiculture Ecotechnology EarthwormResearch Institute Pune India 2nd edition 1996

[2] S Kiss D M Bularda and D Radulescu ldquoBiological Signifi-cance of Enzymes Accumulated in SoilrdquoAdvances in Agronomyvol 27 pp 25ndash87 1975

[3] D L Karlen M J Mausbach J W Doran R G Cline R FHarris and G E Schuman ldquoSoil quality a concept definitionand framework for evaluationrdquo Soil Science Society of AmericaJournal vol 61 no 1 pp 4ndash10 1997

[4] R M Atiyeh C A Edwards S Subler and J Metzger ldquoEarth-worm processed organic wastes as components of horticulturalpotting media for growing manifold and vegetable seedlingsrdquoCompost Science and Utilization vol 8 no 3 pp 215ndash223 2000

[5] N Vassilev and M Vassileva ldquoBiotechnological solubilizationof rock phosphate on media containing agro-industrial wastesrdquoApplied Microbiology and Biotechnology vol 61 no 5-6 pp435ndash440 2003

[6] S A Omar ldquoThe role of rock-phosphate-solubilizing fungiand vesicular-arbusular-mycorrhiza (VAM) in growth of wheatplants fertilized with rock phosphaterdquo World Journal of Micro-biology and Biotechnology vol 14 no 2 pp 211ndash218 1998

[7] J A J Paul and T Daniel ldquoStandardization of sampling methodfor physical characterization of municipal solid wasterdquo AsianJournal of Water Environment and Pollution vol 5 no 1 pp95ndash98 2007

[8] R Lalloo D Maharajh J Gorgens and N Gardiner ldquoA down-stream process for production of a viable and stable Bacilluscereus aquaculture biological agentrdquo Applied Microbiology andBiotechnology vol 86 no 2 pp 499ndash508 2010

[9] B Sivasankari N Kavikumar and M Anandharaj ldquoIndole-3-acetic acid production and enhanced Plant growth promotionby Indigenous bacterial speciesrdquo Journal of Current Research inScience vol 1 no 5 pp 331ndash335 2013

12 Advances in Agriculture

[10] J Han L Sun X Dong et al ldquoCharacterization of a novel plantgrowth-promoting bacteria strain Delftia tsuruhatensis HR4both as a diazotroph and a potential biocontrol agent againstvarious plant pathogensrdquo Systematic and Applied Microbiologyvol 28 no 1 pp 66ndash76 2005

[11] H Rodrıguez and R Fraga ldquoPhosphate solubilizing bacte-ria and their role in plant growth promotionrdquo BiotechnologyAdvances vol 17 no 4-5 pp 319ndash339 1999

[12] T Daniel and N Karmegam ldquoBio-conversion of selected leaflitters using an African epigeic earthworm Eudrilus eugeniaerdquoEcology Environment and Conservation vol 5 no 3 pp 271ndash275 1999

[13] J Dobereiner I E Marriel and M Nery ldquoEcological distri-bution of Spirillum lipoferum Beijerinckrdquo Canadian Journal ofMicrobiology vol 22 no 10 pp 1464ndash1473 1976

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

[15] M Edi Premono A M Moawad and P L G Vlek ldquoEffect ofphosphate-solubilizing Pseudomonas putida on the growth ofmaize and its survival in the rhizosphererdquo Indonesian Journal ofCrop Science vol 11 pp 13ndash23 1996

[16] C L Patten and B R Glick ldquoBacterial biosynthesis of indole-3-acetic acidrdquo Canadian Journal of Microbiology vol 42 no 3pp 207ndash220 1996

[17] N Amaresan V Jayakumar K Kumar and N ThajuddinldquoIsolation and characterization of plant growth promotingendophytic bacteria and their effect on tomato (Lycopersiconesculentum) and chilli (Capsicum annuum) seedling growthrdquoAnnals of Microbiology vol 62 no 2 pp 805ndash810 2012

[18] B Schwyn and J B Neilands ldquoUniversal chemical assay forthe detection and determination of siderophoresrdquo AnalyticalBiochemistry vol 160 no 1 pp 47ndash56 1987

[19] P Rahi P Vyas S Sharma and A Gulati ldquoPlant growthpromoting potential of the fungus Discosia sp FIHB 571 fromtea rhizosphere tested on chickpea maize and peardquo IndianJournal of Microbiology vol 49 no 2 pp 128ndash133 2009

[20] K Kumar N Amaresan S Bhagat K Madhuri and R CSrivastava ldquoIsolation and characterization of rhizobacteriaassociated with coastal agricultural ecosystem of rhizospheresoils of cultivated vegetable cropsrdquo World Journal of Microbiol-ogy and Biotechnology vol 27 no 7 pp 1625ndash1632 2011

[21] J A Smoker and S R Barnum ldquoRapid small scale DNAisolation from filamentous cyanobacteriardquo FEMS MicrobiologyLetters vol 56 no 1 pp 119ndash122 1988

[22] U Edwars T Rogall H Blocker M Emde and E C BottgerldquoIsolation and direct complete nucleotide determination ofentire genes Characterization of a gene coding for 16S riboso-mal RNArdquoNucleic Acids Research vol 17 no 19 pp 7843ndash78531989

[23] D G Higgins A J Bleasby and R Fuchs ldquoCLUSTAL Vimproved software for multiple sequence alignmentrdquo ComputerApplications in the Biosciences vol 8 no 2 pp 189ndash191 1992

[24] M Kimura ldquoA simple method for estimating evolutionary ratesof base substitutions through comparative studies of nucleotidesequencesrdquo Journal ofMolecular Evolution vol 16 no 2 pp 111ndash120 1980

[25] N Saitou and M Nei ldquoThe neighbor-joining method a newmethod for reconstructing phylogenetic treesrdquo Molecular Biol-ogy and Evolution vol 4 no 4 pp 406ndash425 1987

[26] E Barbu K Y Lee and R Wahl ldquoContent of purine andpyrimidine base in desoxyribonucleic acid of bacteriardquo Annalesde lrsquoInstitut Pasteur vol 91 no 2 pp 212ndash224 1956

[27] N Sueoka ldquoVariation and heterogeneity of base compositionof deoxyribonucleic acids a compilation of old and new datardquoJournal of Molecular Biology vol 3 no 1 pp 31ndash40 1961

[28] H Hori and S Osawa ldquoEvolutionary change in 5S rRNAsecondary structure and a phylogenic tree of 352 5S rRNAspeciesrdquo BioSystems vol 19 no 3 pp 163ndash172 1986

[29] V Bess ldquoEvaluating microbiology of compost microbial con-tent of composting is helping the producers and growers tounderstand its role as a soil inoculant and plantrdquo BioCycleMagazine p 62 1999

[30] O Grafff ldquoPreliminary experiments of vermin composting ofdifferent waste materials using Eudrilus eugeniae Kinbergrdquo inWorkshop on the Role of Earthworms in the Stabilization ofOrganic Residues M Happelhof Ed pp 178ndash191 Beech LeafPress Kalamazoo Mich USA 1981

[31] J E Satchell Earthworm Ecology from Darwin to VermicultureChapman and Hall 1983

[32] G H Xie M Y Cai G C Tao and Y Steinberger ldquoCultivableheterotrophic N2-fixing bacterial diversity in rice fields in theYangtze River Plainrdquo Biology and Fertility of Soils vol 37 no 1pp 29ndash38 2003

[33] B BMcSpaddenGardener ldquoEcology ofBacillus and Paenibacil-lus spp in agricultural systemsrdquo Phytopathology vol 94 no 11pp 1252ndash1258 2004

[34] J Dobereiner ldquoBiological nitrogen fixation in the tropics socialand economic contributionsrdquo Soil Biology andBiochemistry vol29 no 5-6 pp 771ndash774 1997

[35] V M Reis F L Olivares and J Dobereiner ldquoImprovedmethodology for isolation of Acetobacter diazotrophicus andconfirmation of its endophytic habitatrdquoWorld Journal of Micro-biology and Biotechnology vol 10 no 4 pp 401ndash405 1994

[36] S Khalil N Ayub S Alam and F Latif ldquoOrganic acids pro-duction and phosphate solubilization by phosphate solubilizingmicroorganisms (PSM) under in vitro conditionsrdquo PakistanJournal of Biological Sciences vol 7 no 2 pp 187ndash196 2004

[37] H M A El-Komy ldquoCoimmobilization of Azospirillumlipoferum and Bacillus megaterium for successful phosphorusand nitrogen nutrition of wheat plantsrdquo Food Technology andBiotechnology vol 43 no 1 pp 19ndash27 2005

[38] N B Paul and W V B Sundara Rao ldquoPhosphate-dissolvingbacteria in the rhizosphere of some cultivated legumesrdquo Plantand Soil vol 35 no 1 pp 127ndash132 1971

[39] A Beneduzi D Peres L K Vargas M H Bodanese-Zanettiniand LM P Passaglia ldquoEvaluation of genetic diversity and plantgrowth promoting activities of nitrogen-fixing bacilli isolatedfrom rice fields in South Brazilrdquo Applied Soil Ecology vol 39no 3 pp 311ndash320 2008

[40] M P Raimam U Albino M F Cruz et al ldquoInteraction amongfree-living N-fixing bacteria isolated from Drosera villosa varvillosa and AM fungi (Glomus clarum) in rice (Oryza sativa)rdquoApplied Soil Ecology vol 35 no 1 pp 25ndash34 2007

[41] M R Swain S K Naskar and R C Ray ldquoIndole-3-acetic acidproduction and effect on sprouting of yam (Dioscorea rotundataL) minisetts by Bacillus subtilis isolated from culturable cow-dung microflorardquo Polish Journal of Microbiology vol 56 no 2pp 103ndash110 2007

[42] T M Tien M H Garkins and D H Hubbell ldquoPlant growthsubstances produced by Azospirillum brasilense and their effect

Advances in Agriculture 13

on the growth of pearl Millet Pennisetum americanum (L)rdquoApplied and EnvironmentalMicrobiology vol 37 no 5 pp 1016ndash1024 1979

[43] A Esitken L Pirlak M Turan and F Sahin ldquoEffects of floraland foliar application of plant growth promoting rhizobacteria(PGPR) on yield growth and nutrition of sweet cherryrdquo ScientiaHorticulturae vol 110 no 4 pp 324ndash327 2006

[44] H Karlidag A Esitken M Turan and F Sahin ldquoEffects of rootinoculation of plant growth promoting rhizobacteria (PGPR)on yield growth and nutrient element contents of leaves ofapplerdquo Scientia Horticulturae vol 114 no 1 pp 16ndash20 2007

[45] C S Nautiyal S Bhadauria P Kumar H Lal R Mondal andD Verma ldquoStress induced phosphate solubilization in bacteriaisolated fromalkaline soilsrdquoFEMSMicrobiology Letters vol 182no 2 pp 291ndash296 2000

[46] A K Halder A K Mishra P Bhattacharya and P KChakrabarthy ldquoSolubilization of inorganic phosphates byBradyrhizobiumrdquo Indian Journal of Experimental Biology vol29 pp 28ndash31 1991

Submit your manuscripts athttpwwwhindawicom

Nutrition and Metabolism

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Food ScienceInternational Journal of

Agronomy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Applied ampEnvironmentalSoil Science

Volume 2014

AgricultureAdvances in

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PsycheHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BiodiversityInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Plant GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biotechnology Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Forestry ResearchInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of BotanyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

EcologyInternational Journal of

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Veterinary Medicine International

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Cell BiologyInternational Journal of

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Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Page 4: BalayoganSivasankariandMarimuthuAnandharajdownloads.hindawi.com/journals/aag/2014/248591.pdf · In Vitro Nitrogen Fixing Activity. e bacterial isolates were screened for nitrogen

4 Advances in Agriculture

10000 rpm for 10minThe supernatantwas analyzed for plantgrowth promotion at various concentrations

212 Evaluation of Plant Growth Promoting Activity underControlled Environment The nitrogen fixing bacteria (Bacil-lus spp) and the phosphate solubilizing bacteria (Pseu-domonas spp) were separately tested for plant growth pro-moting activity and also as consortium (1 1) at 5 differentconcentrations that is 1 2 3 4 and 5mL at three differentday intervals (5 10 and 15 days) with proper control throughExp1 Exp2 Exp3 and Exp0 respectively usingVigna unguic-ulata (L) Walp The seeds (30 seeds in each pot) were sownin plastic pots containing sterilized vermiculite and arrangedin a completely randomized factorial design The seedlingswere grown in a greenhouse at a temperature of 28ndash32∘Cand 85 relative humidity The pots were watered to 50water-holding capacity and were maintained at this moisturecontent by watering to weight every day and plant growthwasobserved for 15 days Parameters such as germination percentshoot length root length leaf area chlorophyll content of theleaves fresh weight of the whole plant and dry weight of thewhole plant were measured using standard procedure

213 Statistical Analysis The following statistical tools wereused for the analyses and interpretation of the data Theexperimental results are presented in the form of tablesand graphs using Microsoft Excel 2007 Data obtained fromthe different treatments were statistically analyzed using theone-way ANOVA and the mean values were compared bythe Duncanrsquos multiple range test for multiple comparisonsDifferences were considered significant at the 005 level usingOrigin software (Version 850) 2010 Origin Lab Corpora-tion

3 Results

31 Isolation and Characterization of Bacterial Isolates Atotal of nineteen bacterial strains were isolated from vermi-compost vermicast and earthworm (fore mid and hind)guts (Table 1) The morphological and biochemical char-acteristics of bacterial isolates were elucidated in Table 1All the isolates were tested for plant growth promotingactivity Among the 19 bacterial isolates only five bacterialstrains showed plant growth promoting activity Based oncolony morphology microscopic observations and culturalbiochemical and physiological properties the bacteriumwasgiven the name Bacillus spp Alcaligenes spp Erwinia sppSerratia spp or Pseudomonas sppThese five bacterial strainswere able to grow in the nitrogen-free medium and they werealso identified as phosphate solubilizing microorganisms onthe basis of their solubilization index (Table 2) Bacillus spphave the highest SI (3855) followed by Pseudomonas spp(3522) and Serratia spp (3045) All the isolates produceda significant amount of IAA from the medium in the rangeof 113 to 242120583gmL Bacillus spp showed higher IAAproduction (242120583gmL) and Serratia spp showed lower IAAproduction (113 120583gmL) All the strains (except Alcaligenes

spp) were positive for siderophore production showing ayellow zone on the CAS agar medium plate (Table 2)

32 Molecular Studies The partial 16S rRNA sequencescarried out in the present study for Bacillus spp andPseudomonas spp covered a stretch of approximately 1500nucleotides for each About half of the sequences found in theclone library showed only slight relationship to other knownsequences while the other half were highly similar (approx-imately 95 percent sequence identity) to other databaseentries for Bacillus spp and Pseudomonas spp Less than 05percent of all nucleotides was found to be unique within theconserved regions of the cloned sequence and could almostalways be related to reading errors in ambiguous regions ofthe sequencing gel On the basis of phylogenetic analysisof 16S rDNA partial sequences Bacillus spp GGBSTD1 isidentified as Bacillus cereus and Pseudomonas spp GGBSTD3is identified as Pseudomonas spp Phylogenetic analyses ofthe strains based on the neighbor joining method wererepresented in Figures 1 and 2 Among eubacteria the meanguanine and cytosine (G + C) content of genomic DNAvaries from approximately 25 to 75 That the bacterialgenomic G + C content is somehow related to phylogeny hasbeen suggested [26 27] The phylogenetic tree of eubacterial16S rRNA clearly indicates this relationship Gram-positivebacteria Bacillus subtilis have genomic G + C 42 Gram-negative bacteria with intermediate G + C content such asEscherichia coli (50) Serratia marcescens (58) Salmonellatyphimurium (51) and Pseudomonas fluorescens (60)belong to the common Gram-negative bacteria [28] Bacilluscereus have the G + C content 53 and A + T content 47similarly Pseudomonas spp having G + C content 53 andA + T content 47 Most functional RNA molecules havecharacteristic secondary structures that are highly conservedin evolution In this study RNA secondary structures ofBacillus cereus and the Pseudomonas spp were predictedBacillus cereus have free energy of minus2941 kkalmol andPseudomonas spp have free energy of minus2998 kkalmol

33 Evaluation of Biofertilization Abilities under ControlledConditions Germination percentage of Vigna unguiculataseeds sown in vermiculite supplemented with Bacillus cereus(Exp1) with Pseudomonas spp (Exp2) and with consortium(Exp3) (1 1 ratio) at five different concentrations (1 2 3 4and 5mL) and in the control (Exp0) is shown in Figure 3The highest germination percentage was observed in Exp3(consortium) at 5mL concentration compared to others Theincrease in germination percentage and in other growthparameters can be attributed to the combined effect of thenitrogen fixing and phosphate solubilizing microbes whichare able to fix atmospheric nitrogen and solubilize P andalso produce growth promoting substancesThese organismshave been isolated from vermisources and this observationindicates the reason for promotion and enhancement of cropgrowth and yield when vermicompost is applied It is a proofthat these symbioticmicrobes present in vermisources benefitthe soil fertility Observation on growth parameters such asshoot length root length and leaf area of V unguiculata

Advances in Agriculture 5

Table1

Predo

minantb

acteria

lstrains

isolated

from

verm

isourcesand

theirb

iochem

icalcharacteris

ticsT1-con

trolmdash

Gsepium

+L

leucocephala+

cowdu

ngin

(112)(worm

free)T

2mdashG

sepium

+L

leucocephala+cowdu

ngin

(112)+

EeugeniaeT3

mdashG

sepium

+L

leucocephala+cowdu

ngin

(112)+

EfetidaT4

-con

trol(2)mdashC

auric

ulata+L

leucocephala+cow

dung

in(112)(worm

free)T

5mdashC

auric

ulata+L

leucocephala+cowdu

ngin

(112)+

EeugeniaeT

6mdashC

auric

ulata+L

leucocephala+cowdu

ngin

(112)+

Efetida

Snu

mber

Samples

ource

Biochemicalcharacteris

ticsc

Nam

eofthe

strain

Gramrsquos

reactio

naCell

shape

Motility

bIndo

letest

Methyl

Red

test

Voges

Proskauer

test

Citrate

Utilization

test

Catalase

test

Urease

test

Gelatin

hydrolysis

test

Nitrate

redu

ctase

test

Starch

hydrolysis

test

Casein

hydrolysis

test

Glucose

utilizatio

ntest

1Ve

rmicom

post

(T2)

+Cocciminus

minus+

minusminus

+minus

++

minusminus

minusStaphylococcus

spp

2Ve

rmicom

post

(T3)

+Ro

d+

minusminus

minusminus

+minus

minus+

minusminus

+Co

rynebacterium

spp

3Ve

rmicom

post

(T5)

+Ro

dminus

++

++

+minus

++

+minus

+Ba

cillusspp

4Ve

rmicom

post

(T6)

+Cocciminus

++

minusminus

+minus

+minus

minusminus

+Micrococcusspp

5Ve

rmicast(T3

)+

Cocciminus

++

minusminus

minus+

++

+minus

minusKlebsiella

spp

6Ve

rmicast(T5

)+

Rod

minus+

minusminus

+minus

+minus

+minus

minus+

Streptococcusspp

7

Verm

icast(T6

)+

Cocciminus

+minus

++

minusminus

++

minusminus

+En

terobacter

spp

8Ea

rthw

orm

hind

gut(T2

)+

Cocciminus

minus+

+minus

+minus

minusminus

++

+Trico

ccocus

spp

9Ea

rthw

orm

hind

gut(T3

)+

Cocciminus

minus+

minus+

++

minus+

minusminus

minusYersiniaspp

10

Earthw

orm

hind

gut(T5

)+

Cocciminus

minusminus

minusminus

++

++

minusminus

minusMicrococcusspp

11

Verm

icom

post

(T1)

minusRo

dminus

++

minusminus

minusminus

++

minusminus

+Escherich

iaspp

12

Verm

icom

post

(T2)

minusRo

dminus

minus+

minusminus

minus+

++

minusminus

+Proteusspp

13

Verm

icom

post

(T4)

minusRo

dminus

minusminus

+minus

+minus

minus+

++

+Pa

racoccus

spp

14

Verm

icom

post

(T5)

minusRo

d+

+minus

minusminus

++

++

minusminus

minusPseudomonas

spp

15

Verm

icast(T2

)minus

Rod

+minus

+minus

++

+minus

+minus

minusminus

Citro

bacter

spp

16

Verm

icast(T3

)minus

Cocciminus

++

minusminus

+minus

+minus

minusminus

minusErwiniaspp

17Ve

rmicast(T5

)minus

Rod

++

++

++

minus+

minusminus

minusminus

Alcaligenesspp

18

Verm

icast(T6

)minus

Cocciminus

minus+

++

+minus

++

minusminus

minusSerratiaspp

19

Earthw

orm

mid

gut(T2

)minus

Rod

+minus

minusminus

+minus

minusminus

minusminus

minusminus

Pediococcusspp

a Gramrsquossta

ining(+)p

ositive(minus)n

egative

b Motility(+)

motile(minus)n

onmotile

c Biochem

icalcharacteris

tics(+)p

ositive(minus)n

egative

6 Advances in Agriculture

Bacillus cereus strain GGBSTD2 16S ribosomal RNA gene

Bacillus cereus strain SU-6 16S ribosomal RNA gene

Bacillus cereus strain JB0013 16S ribosomal RNA gene

Bacillus cereus isolate PN24 16S ribosomal RNA gene

Bacillus cereus strain JB0005 16S ribosomal RNA gene

Bacillus sp G2DM-85 16S ribosomal RNA gene

Bacillus cereus strain JBE0009 16S ribosomal RNA gene

Bacillus sp EB460 16S ribosomal RNA gene

Bacillus cereus strain 421-3R 16S ribosomal RNA gene

Bacillus sp EB447 16S ribosomal RNA gene

Bacillus sp EB470 16S ribosomal RNA gene

Bacillus sp EB421 16S ribosomal RNA gene

Bacillus sp EB431 16S ribosomal RNA gene

Bacillus sp EB462 16S ribosomal RNA gene

Bacillus sp EB429 16S ribosomal RNA gene

Bacillus cereus strain 421-4R 16S ribosomal RNA gene

Bacillus sp EB480 16S ribosomal RNA gene

Bacillus sp EB13 16S ribosomal RNA gene

Uncultured compost bacterium partial 16S rRNA gene

Bacillus thuringiensis gene for 16S rRNA

Figure 1 Neighbor joining phylogenetic tree of full 16S rRNA gene sequences showing the relationship among Bacillus cereus GGBSTD1and representatives of some related taxa The sequence data for several closely related Bacillus cultures were recovered from GenBank andincluded in the tree

Table 2 The phosphate solubilizing activity phytohormone (IAA) production and siderophores production of the bacterial isolates

Name of the organism Phosphate solubilization index (SI) IAA production (120583gmL)a Siderophores productionb

Bacillus spp 3855 242 +++Alcaligenes spp 2930 147 minus

Erwinia spp 2890 113 +Serratia spp 3045 168 ++Pseudomonas spp 3522 196 +++aIndole acetic acid production in culture media supplemented with tryptophan (5 120583gmL) after 5 daysbIn vitro siderophores production minus represents the absence of siderophores production and +++ represents gt10mm wide yellow-orange zone

is given in Tables 3 and 4 respectively The shoot length(1147 plusmn 006 cm) root length (1027 plusmn 006 cm) and leafarea (905 plusmn 026 cm2) of the plant get significantly (119875 lt0001) increased in Exp3 (consortium of Bacillus cereus +Pseudomonas spp [1 1]) at 5mL concentration on the 15thday compared to other experimentsThe chlorophyll a b andtotal chlorophyll content of the leaves on d 5 d 10 and d 15are given in Table 5 The chlorophyll a (010 plusmn 009mgg offresh leaf) chlorophyll b (1085 plusmn 016mgg of fresh leaf)and total chlorophyll (1095plusmn007mgg of fresh leaf) content

of leaves were significantly (119875 lt 0001) higher in Exp3(consortium of Bacillus cereus + Pseudomonas spp (1 1))at 5mL concentration on the 15th day compared to otherexperiments Observations on the fresh weight of the wholeplant are given in Table 6 The fresh weight of the wholeplant (092 plusmn 000 g) was significantly (119875 lt 0001) higherin Exp3 (consortium of Bacillus cereus + Pseudomonas spp(1 1)) at 5mL concentration on the 15th day compared toother experiments Observations on the dry weight of thewhole plant are given in Table 6 The dry weight of the whole

Advances in Agriculture 7

Pseudomonas fluorescens strain LMG 5825 16S ribosomal RNA gene

Pseudomonas fluorescens strain LMG 5829 16S ribosomal RNA gene

Pseudomonas fluorescens strain LMG 14676 16S ribosomal RNA gene

Pseudomonas sp W15Feb13 16S ribosomal RNA gene

Pseudomonas sp W15Feb28 16S ribosomal RNA gene

Pseudomonas sp p50 16S ribosomal RNA gene

Uncultured bacterium clone 661193 16S ribosomal RNA gene

Pseudomonas sp BSs20166 16S ribosomal RNA gene

Pseudomonas sp 28zhy partial 16S rRNA gene

Uncultured bacterium clone AYRV1-027 16S ribosomal RNA gene

Uncultured Pseudomonadaceae bacterium clone290cost002-P3L-2032 16S ribosomal RNA gene

Pseudomonas sp GGBSTD3 16S ribosomal RNA gene

Pseudomonas sp DK3 16S ribosomal RNA gene

Pseudomonas reactans strain AMP-13 16S ribosomal RNA gene

Figure 2 Neighbor joining phylogenetic tree of full 16S rRNA gene sequences showing the relationship among Pseudomonas spp GGBSTD3and representatives of some related taxa The sequence data for several closely related Pseudomonas cultures were recovered from GenBankand included in the tree

plant (009 plusmn 000 g) was significantly (119875 lt 0001) higherin Exp3 (consortium of Bacillus cereus + Pseudomonas spp(1 1)) at 5mL concentration on the 15th day compared toother experiments

4 Discussion

Inoculation of these newly identified nitrogen fixing andphosphate solubilizingmicrobes in the plant growthmediumenhanced various physiological activities culminating inhigher biomass production in V unguiculata Since this is anonhazardous way of fertilization of crop plants it is veryrelevant to a developing country like India This techniquecan save the farmers of India from the problem posed byhigh cost of fertilizers by offering a comparatively inexpensivealternative (biofertilizers) while additionally preventing thedegradation of the soil and thereby ensuring sustainable agri-culture Bess (1999) reported that the content in compostingneeds to be determined through the concentration of sixfunctional groups of microorganisms such as aerobic bacte-ria anaerobic bacteria fungi actinomycetes Pseudomonasand nitrogen fixing bacteria [29] Now there are ways toevaluate the concentrations of these organisms in the finishedcompost and that can serve as an interpretation guide to

determine the quality of the compost as an inoculant of soilmicroorganisms Grafff (1981) and Atlavinyte have reporteda higher P content in the cast than in the surrounding soil[30] Satchell (1983) has linked the phosphatase activity inthe gut of worms to the availability of bound P in soils Thebacterial strains which have the capability to fix nitrogenonly can grow in the nitrogen-free medium [31] Hencethe nitrogen fixing ability was tested in the present studybased on the growth of bacterial strain in nitrogen- freemedium Five bacteria strains showedmore nitrogen fixationamong all the five bacterial strains tested only one strain (ieBacillus spp) showed significantly more turbidity comparedto the other strains The ultimate source of the nitrogen usedby plants is N

2gas which constitutes 78 of the Earthrsquos

atmosphere Unfortunately higher plants cannot metabolizeN2directly into protein N

2gas must be converted to a plant

available form [32] isolated many species of nitrogen fixingBacillus (B megaterium B cereus B subtilis B licheniformisand B azotoformans) from the rhizospheric zone of ricefields in Central China The widely studied Bacillus genusrepresents one of the most diverse genera in the bacilli groupNumerous Bacillus and Paenibacillus strains express plantgrowth promoting (PGP) activities and a number of thesestrains have already been commercially developed as biolog-ical fungicides insecticides and nematicides or generic plant

8 Advances in Agriculture

Table 3 Shoot length and Root length ofVigna unguiculata grown on vermiculite supplemented with Bacillus cereus with Pseudomonas sppand with their consortium (1 1) at five different concentrations (1 2 3 4 and 5mL) on the 5th the 10th and the 15th day and in the control

Experimentsa Quantity of bacterial cultures added Shoot length (cm)b Root length (cm)b

5th day 10th day 15th day 5th day 10th day 15th day

Exp0

1mL 297 (ab) 397 (a) 497 (ab) 197 (de) 283 (ab) 397 (bcd)2mL 303 (bcd) 403 (ab) 500 (ab) 200 (abc) 287 (de) 400 (a)3mL 310 (abc) 407 (a) 503 (ab) 203 (de) 290 (abc) 403 (ab)4mL 310 (bcd) 410 (bcd) 507 (bcd) 207 (a) 297 (de) 410 (abc)5mL 313 (a) 413 (ab) 510 (ab) 210 (bcd) 300 (ab) 413 (de)

Exp1

1mL 340 (ab) 440 (abc) 540 (ab) 233 (abc) 327 (bcd) 433 (f)2mL 450 (ab) 540 (abc) 647 (ab) 333 (ab) 430 (a) 540 (abc)3mL 550 (de) 630 (ab) 750 (a) 440 (de) 550 (abc) 620 (a)4mL 653 (ab) 730 (a) 833 (ab) 550 (bcd) 653 (de) 750 (abc)5mL 740 (ab) 840 (abc) 923 (f) 640 (abc) 753 (ab) 827 (ab)

Exp2

1mL 277 (bcd) 357 (f) 440 (de) 160 (de) 217 (abc) 350 (f)2mL 343 (abc) 447 (ab) 553 (ab) 253 (abc) 340 (bcd) 427 (bcd)3mL 437 (de) 543 (abc) 633 (abc) 340 (ab) 443 (f) 533 (ab)4mL 547 (bcd) 647 (de) 733 (a) 420 (bcd) 550 (ab) 633 (ab)5mL 640 (f) 733 (bcd) 833 (bcd) 533 (ab) 630 (ab) 730 (bcd)

Exp3

1mL 530 (bcd) 647 (ab) 730 (ab) 433 (de) 550 (abc) 620 (ab)2mL 643 (ab) 740 (bcd) 827 (f) 547 (a) 630 (de) 733 (ab)3mL 737 (bcd) 840 (bcd) 927 (ab) 640 (ab) 740 (bcd) 840 (bcd)4mL 847 (abc) 930 (a) 1043 (abc) 740 (f) 833 (a) 937 (abc)5mL 957 (ab) 1030 (f) 1147 (bcd) 833 (abc) 940 (ab) 1027 (bcd)

aExp0 nutrient solution (control) Exp1 Bacillus cereus Exp2 Pseudomonas spp Exp3 consortium of Bacillus cereus + Pseudomonas spp (1 1)bMeanof two repeated experiments (30 plants)Different letterswithin parenthesis indicate significant difference between treatments for each growth parameterusing Duncanrsquos multiple range test (119875 = 005)

growth promoters The use of these strains in agriculturehas recently been reviewed [33]Those strains besides havingseveral PGP properties can also fix nitrogen and also dophosphate solubilization The symbiotic fixation of nitrogenthrough inoculation of legume crops with effective rhizobiais well known [34] Asymbiotic nitrogen fixing bacteriawhich live in the rhizosphere andor endophytically oftenincrease yields of crops Many bacterial species have nitrogenfixing properties including Bacillus spp Azotobacter sppAzospirillum spp Beijerinckia spp and Pseudomonas spp[35] Nitrogen fixing bacteria have been used in foliar appli-cations inmulberryThe solubilization index (SI) was formeddue to solubilization of insoluble phosphates by organic acidsecretion [36] El-Komy (2005) indicated that Pseudomonasfluorescence and Bacillus megaterium strains were the mostpowerful phosphate solubilizers on PVK plates as well as onPikovskayarsquos broth [37] The P concentration in Pikovskayarsquosbroth increased gradually achieving a peak on the sixth dayand declined slowly during the late days In this study pHvalues decreased gradually in PVK broth during the earlyincubation days and no revival was observed during laterdays for all the tested bacterial strains Several bacteria par-ticularly those belonging to the genus Bacillus spp convertinsoluble phosphate into soluble forms by secreting organicacids such as formic acid acetic acid propionic acid citricacid fumaric acid gluconic acid glyoxylic acid ketobutyric

acid malonic acid succinic acid and tartaric acid Theseacids lower the pH and bring about the dissolution of boundforms of phosphate Some of the hydroxyl acids may chelatewith calcium and iron resulting in effective solubilization andutilization of phosphates [38]

Beneduzi et al (2008) had isolated the plant growthpromoting strain SVPR30 and identified by 16S rRNA genesequence as Bacillus spp and they had tested it for plantgrowth through in vivo experiments [39] This strain wascharacterized as a high IAA producer able to solubilize phos-phate and also fix a considerably high amount of nitrogenThe inoculation of rice with Bacillus spp SVPR30 strainshowed a significant increase in the root and shoot partswhen compared with the controls within 15 and 30 daysafter sprouting Numerous Bacillus and Paenibacillus strainsexpressed plant growth promoting (PGP) activities and anumber of these strains have been commercially developedas generic plant growth promotersThe use of these strains inagriculture has recently been reviewed [33]

Various reports indicate that coinoculation of beneficialorganisms generally increased plant growth relative to singleinoculation with a sole beneficial organism [40] Most ofthe effects of the individual microorganisms coinoculationare additive although a synergistic effect has been reportedin some cases The average root to shoot ratio was higherin B subtilis treated yam minisetts in comparison to those

Advances in Agriculture 9

Table 4 Leaf area of Vigna unguiculata grown on vermiculite supplemented with Bacillus cereus with Pseudomonas spp and with theirconsortium (1 1) at five different concentrations (1 2 3 4 and 5mL) on the 5th the 10th and the 15th days and in the control

Experimentsa Quantity of bacterial cultures added Leaf area (cm2)b

5th day 10th day 15th day

Exp0

1mL 027 (ab) 094 (abc) 407 (b)2mL 035 (ab) 102 (ab) 419 (abc)3mL 040 (abc) 125 (ab) 471 (bcd)4mL 048 (ab) 138 (abc) 510 (ab)5mL 054 (abc) 161 (b) 538 (b)

Exp1

1mL 018 (bcd) 123 (b) 601 (abc)2mL 025 (ab) 138 (abc) 632 (ab)3mL 033 (abc) 150 (ab) 684 (b)4mL 038 (ab) 168 (ab) 686 (abc)5mL 044 (abc) 191 (bcd) 733 (ab)

Exp2

1mL 011 (b) 107 (ab) 534 (b)2mL 014 (ab) 119 (ab) 559 (ab)3mL 017 (abc) 127 (ab) 579 (ab)4mL 021 (ab) 153 (abc) 631 (abc)5mL 026 (ab) 166 (b) 667 (b)

Exp3

1mL 026 (bcd) 297 (ab) 753 (bcd)2mL 033 (b) 325 (ab) 787 (ab)3mL 040 (ab) 350 (bcd) 811 (bcd)4mL 044 (b) 384 (b) 861 (b)5mL 062 (ab) 400 (bcd) 905 (ab)

aExp0 nutrient solution (control) Exp1 Bacillus cereus Exp2 Pseudomonas spp Exp3 consortium of Bacillus cereus + Pseudomonas spp (1 1)bMeanof two repeated experiments (30 plants)Different letterswithin parenthesis indicate significant difference between treatments for each growth parameterusing Duncanrsquos multiple range test (119875 = 005)

0102030405060708090

100

Concentration of bacterial cultures (mL)1 2 3 4 5

Germination ()

Exp0

Exp1 Exp3

Exp2

Figure 3 Germination percentage ofVigna unguiculata seeds sownin vermiculite supplementedwithBacillus cereus with Pseudomonasspp and with their consortium at five different concentrations (1 23 4 and 5mL) and in the control Exp0 nutrient solution (control)Exp1 Bacillus cereus Exp2 Pseudomonas spp Exp3 consortium ofBacillus cereus + Pseudomonas spp (1 1)

not treated with the bacterial culture [41] In earlier reportsroot elongation was found to occur in Sesbania aculeata byinoculation with Azotobacter spp and Pseudomonas spp in

Vigna radiata by Pseudomonas putida [16] and in Pennisetumamericanum by Azospirillum brasilense [42] Esitken et al(2006) had found out that Bacillus OSU-142 and Pseu-domonas BA-8 alone or in combination had a great potentialto increase the growth yield and nutrition of sweet cherryplant [43] BacillusM3 BacillusOSU-142 andMicobacteriumFS01 separately or in combination were found to have greatpotential for use as plant growth promoting rhizobacteriato increase production in apples and in many other crops[44] These phosphate solubilizing microbes consist pre-dominantly of fungal bacterial and actinomycetes speciescollectively called phosphate solubilizing microorganismsmdashPSM [45] There are several microorganisms which canalso solubilize the cheaper sources of phosphorous such asrock phosphate Bacteria such as Bacillus are widely usedin plant production system and are important phosphorussolubilizing microorganisms resulting in improved growthyield of crops andmetabolic activities especially in synthesisof protein [34 46]

5 Conclusions

This study illustrates the isolation of plant growth promotingbacteria from vermisources screening under in vitro con-ditions for multiple PGPR traits and their evaluation undercontrolled conditions in a pot experiment The findings ofthis study indicated that the consortium of B cereus and

10 Advances in Agriculture

Table5Ch

loroph

yllcon

tent

ofleavesofVignaun

guicu

latagrow

non

verm

iculite

supp

lementedwith

Bacilluscereuswith

Pseudomonas

sppand

with

theircon

sortium

(11)atfi

vedifferent

concentrations

(1234and

5mL)

onthe5

thdaythe10thdayandthe15thdayandin

thec

ontro

l

Experim

entsa

Quantity

ofbacterial

Cultu

resa

dded

Chloroph

yllcon

tent

(mggof

fresh

leaf)b

5thday

10th

day

15th

day

Chloroph

ylla

Chloroph

yllb

Total

chloroph

yll

Chloroph

ylla

Chloroph

yllb

Total

chloroph

yll

Chloroph

ylla

Chloroph

yllb

Total

chloroph

yll

Exp0

1mL

021

(ab)

285

(abc)

307

(a)

020

(abc)

481

(a)

501

(ab)

015

(bcd)

612

(abc)

627

(bcd)

2mL

022

(abc)

293

(ab)

315

(abc)

021

(ab)

489

(ab)

510

(bcd)

012

(abc)

618

(ab)

630

(ab)

3mL

023

(abc)

301

(ab)

315

(abc)

022

(bcd)

497

(bcd)

518

(a)

014

(a)

633

(cde)

647

(bcd)

4mL

024

(ab)

308

(f)

332

(a)

022

(a)

504

(b)

527

(abc)

015

(bcd)

641

(cde)

656

(a)

5mL

025

(f)

316

(abc)

341

(bcd)

019

(ab)

511(ab)

530

(abc)

011(cde)

645

(ab)

650

(abc)

Exp1

1mL

035

(b)

400

(cde)

435

(abc)

011(b)

581

(cde)

593

(cde)

010

(abc)

704(abc)

715(f)

2mL

026

(abc)

462

(bcd)

488

(f)

011(abc)

647

(b)

658

(abc)

006

(ab)

779(bcd)

785(a)

3mL

021

(bcd)

526

(abc)

547

(ab)

008

(bcd)

718(ab)

726(bcd)

006

(f)

847

(f)

853

(ab)

4mL

008

(f)

587

(cde)

595

(b)

011(bcd)

778(abc)

789(abc)

004

(bcd)

933(b)

937(bcd)

5mL

026

(abc)

641

(bcd)

667

(abc)

025

(cde)

834

(cde)

859

(f)

007

(abc)

976(cde)

983(abc)

Exp2

1mL

030

(abc)

354

(ab)

384

(b)

012

(a)

545

(abc)

556

(ab)

007

(bcd)

681

(abc)

688

(bcd)

2mL

029

(bcd)

420

(f)

449

(abc)

011(bcd)

610

(ab)

621

(abc)

007

(abc)

744(f)

752(bcd)

3mL

022

(ab)

504

(abc)

527

(bcd)

015

(a)

677

(cde)

692

(bcd)

006

(bcd)

812

(ab)

817

(bcd)

4mL

010

(f)

566

(a)

576

(cde)

018

(abc)

744(f)

762(ab)

005

(ab)

885

(a)

890

(abc)

5mL

006

(bcd)

622

(abc)

628

(abc)

018

(b)

810

(abc)

828

(a)

004

(cde)

948(a)

952(f)

Exp3

1mL

027

(ab)

470

(bcd)

496

(cde)

009

(ab)

668

(b)

677

(cde)

010

(a)

814

(ab)

825

(a)

2mL

020

(ab)

548

(a)

568

(f)

003

(bcd)

746(bcd)

749(ab)

009

(ab)

887

(f)

897

(ab)

3mL

005

(bcd)

584

(abc)

589

(ab)

008

(bcd)

804

(abc)

812

(cde)

010

(ab)

953(abc)

963(bcd)

4mL

023

(f)

663

(ab)

686

(cde)

007

(ab)

870

(b)

877

(f)

010

(abc)

1019

(bcd)

1029(f)

5mL

022

(ab)

744(cde)

766(a)

009

(a)

932(abc)

940(ab)

010

(a)

1085(ab)

1095(abc)

a Exp0nu

trient

solutio

n(con

trol)

Exp1B

acillus

cereusE

xp2Pseudomonas

sppE

xp3consortiu

mof

Bacilluscereus+

Pseudomonas

spp(11)

b Meanof

tworepeated

experim

ents(30plants)

Differentletterswith

inparenthesis

indicatesig

nificantd

ifference

betweentre

atmentsfore

achgrow

thparameter

usingDun

canrsquos

multip

lerangetest(119875=005)

Advances in Agriculture 11

Table 6 Fresh weight and dry weight of whole plant of Vigna unguiculata grown on vermiculite supplemented with Bacillus cereus withPseudomonas spp and with their consortium (1 1) at five different concentrations (1 2 3 4 and 5mL) on the 5th the 10th and the 15 daysand in the control

Experimentsa Quantity of bacterial cultures added Fresh weight of the whole plant (g)b Dry weight of the whole plant (g)b

5th day 10th day 15th day 5th day 10th day 15th day

Exp0

1mL 058 (bcd) 061 (abc) 064 (ab) 006 (a) 006 (abc) 006 (ab)2mL 058 (ab) 061 (a) 064 (abc) 006 (bcd) 006 (ab) 006 (ab)3mL 058 (abc) 061 (ab) 064 (bcd) 006 (abc) 006 (bcd) 006 (b)4mL 041 (abc) 061 (ab) 064 (ab) 006 (abc) 006 (ab) 006 (abc)5mL 058 (abc) 061 (abc) 064 (bcd) 006 (a) 006 (bcd) 006 (def)

Exp1

1mL 062 (abc) 065 (abc) 068 (ab) 006 (bcd) 006 (ab) 007 (bcd)2mL 065 (bcd) 068 (bcd) 071 (def) 006 (abc) 007 (a) 007 (ab)3mL 068 (abc) 071 (def) 074 (bcd) 007 (abc) 007 (ab) 007 (abc)4mL 071 (ab) 074 (abc) 077 (abc) 007 (bcd) 007 (b) 008 (bcd)5mL 074 (abc) 077 (def) 080 (ab) 007 (def) 008 (a) 008 (b)

Exp2

1mL 056 (ab) 059 (bcd) 062 (ab) 006 (ab) 006 (ab) 006 (abc)2mL 059 (abc) 062 (abc) 065 (def) 006 (ab) 006 (abc) 006 (bcd)3mL 062 (bcd) 065 (a) 068 (bcd) 006 (abc) 006 (bcd) 007 (abc)4mL 065 (abc) 068 (ab) 071 (b) 006 (ab) 007 (abc) 007 (b)5mL 068 (abc) 071 (b) 074 (a) 007 (def) 007 (abc) 007 (bcd)

Exp3

1mL 074 (ab) 077 (ab) 080 (abc) 007 (a) 008 (bcd) 008 (ab)2mL 077 (bcd) 080 (a) 083 (bcd) 008 (a) 008 (bcd) 008 (abc)3mL 080 (bcd) 083 (bcd) 086 (abc) 008 (ab) 008 (b) 009 (b)4mL 083 (bcd) 086 (abc) 089 (ab) 008 (abc) 009 (ab) 009 (bcd)5mL 086 (ab) 089 (b) 092 (def) 009 (bcd) 009 (bcd 009 (b)

aExp0 nutrient solution (control) Exp1 Bacillus cereus Exp2 Pseudomonas spp Exp3 consortium of Bacillus cereus + Pseudomonas spp (1 1)bMeanof two repeated experiments (30 plants)Different letterswithin parenthesis indicate significant difference between treatments for each growth parameterusing Duncanrsquos multiple range test (119875 = 005)

Pseudomonas spp acts as a potential biofertilizer due to itsvarious plant growth promotion abilities such as solubiliza-tion of phosphate and production of IAA and siderophore itcan be developed as potential microbial inoculant for variouscrops

Conflict of Interests

The authors declared that there is no conflict of interestsregarding this paper

Acknowledgments

Theauthors are thankful to theDepartment of BiologyGand-higram Rural InstitutemdashDeemed University GandhigramThis research was funded by University Grants Commissionunder UGC Research Fellowship in Science for MeritoriousStudents (BSR) Grant no F4-12008

References

[1] U S Bhawalkar Vermiculture Ecotechnology EarthwormResearch Institute Pune India 2nd edition 1996

[2] S Kiss D M Bularda and D Radulescu ldquoBiological Signifi-cance of Enzymes Accumulated in SoilrdquoAdvances in Agronomyvol 27 pp 25ndash87 1975

[3] D L Karlen M J Mausbach J W Doran R G Cline R FHarris and G E Schuman ldquoSoil quality a concept definitionand framework for evaluationrdquo Soil Science Society of AmericaJournal vol 61 no 1 pp 4ndash10 1997

[4] R M Atiyeh C A Edwards S Subler and J Metzger ldquoEarth-worm processed organic wastes as components of horticulturalpotting media for growing manifold and vegetable seedlingsrdquoCompost Science and Utilization vol 8 no 3 pp 215ndash223 2000

[5] N Vassilev and M Vassileva ldquoBiotechnological solubilizationof rock phosphate on media containing agro-industrial wastesrdquoApplied Microbiology and Biotechnology vol 61 no 5-6 pp435ndash440 2003

[6] S A Omar ldquoThe role of rock-phosphate-solubilizing fungiand vesicular-arbusular-mycorrhiza (VAM) in growth of wheatplants fertilized with rock phosphaterdquo World Journal of Micro-biology and Biotechnology vol 14 no 2 pp 211ndash218 1998

[7] J A J Paul and T Daniel ldquoStandardization of sampling methodfor physical characterization of municipal solid wasterdquo AsianJournal of Water Environment and Pollution vol 5 no 1 pp95ndash98 2007

[8] R Lalloo D Maharajh J Gorgens and N Gardiner ldquoA down-stream process for production of a viable and stable Bacilluscereus aquaculture biological agentrdquo Applied Microbiology andBiotechnology vol 86 no 2 pp 499ndash508 2010

[9] B Sivasankari N Kavikumar and M Anandharaj ldquoIndole-3-acetic acid production and enhanced Plant growth promotionby Indigenous bacterial speciesrdquo Journal of Current Research inScience vol 1 no 5 pp 331ndash335 2013

12 Advances in Agriculture

[10] J Han L Sun X Dong et al ldquoCharacterization of a novel plantgrowth-promoting bacteria strain Delftia tsuruhatensis HR4both as a diazotroph and a potential biocontrol agent againstvarious plant pathogensrdquo Systematic and Applied Microbiologyvol 28 no 1 pp 66ndash76 2005

[11] H Rodrıguez and R Fraga ldquoPhosphate solubilizing bacte-ria and their role in plant growth promotionrdquo BiotechnologyAdvances vol 17 no 4-5 pp 319ndash339 1999

[12] T Daniel and N Karmegam ldquoBio-conversion of selected leaflitters using an African epigeic earthworm Eudrilus eugeniaerdquoEcology Environment and Conservation vol 5 no 3 pp 271ndash275 1999

[13] J Dobereiner I E Marriel and M Nery ldquoEcological distri-bution of Spirillum lipoferum Beijerinckrdquo Canadian Journal ofMicrobiology vol 22 no 10 pp 1464ndash1473 1976

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

[15] M Edi Premono A M Moawad and P L G Vlek ldquoEffect ofphosphate-solubilizing Pseudomonas putida on the growth ofmaize and its survival in the rhizosphererdquo Indonesian Journal ofCrop Science vol 11 pp 13ndash23 1996

[16] C L Patten and B R Glick ldquoBacterial biosynthesis of indole-3-acetic acidrdquo Canadian Journal of Microbiology vol 42 no 3pp 207ndash220 1996

[17] N Amaresan V Jayakumar K Kumar and N ThajuddinldquoIsolation and characterization of plant growth promotingendophytic bacteria and their effect on tomato (Lycopersiconesculentum) and chilli (Capsicum annuum) seedling growthrdquoAnnals of Microbiology vol 62 no 2 pp 805ndash810 2012

[18] B Schwyn and J B Neilands ldquoUniversal chemical assay forthe detection and determination of siderophoresrdquo AnalyticalBiochemistry vol 160 no 1 pp 47ndash56 1987

[19] P Rahi P Vyas S Sharma and A Gulati ldquoPlant growthpromoting potential of the fungus Discosia sp FIHB 571 fromtea rhizosphere tested on chickpea maize and peardquo IndianJournal of Microbiology vol 49 no 2 pp 128ndash133 2009

[20] K Kumar N Amaresan S Bhagat K Madhuri and R CSrivastava ldquoIsolation and characterization of rhizobacteriaassociated with coastal agricultural ecosystem of rhizospheresoils of cultivated vegetable cropsrdquo World Journal of Microbiol-ogy and Biotechnology vol 27 no 7 pp 1625ndash1632 2011

[21] J A Smoker and S R Barnum ldquoRapid small scale DNAisolation from filamentous cyanobacteriardquo FEMS MicrobiologyLetters vol 56 no 1 pp 119ndash122 1988

[22] U Edwars T Rogall H Blocker M Emde and E C BottgerldquoIsolation and direct complete nucleotide determination ofentire genes Characterization of a gene coding for 16S riboso-mal RNArdquoNucleic Acids Research vol 17 no 19 pp 7843ndash78531989

[23] D G Higgins A J Bleasby and R Fuchs ldquoCLUSTAL Vimproved software for multiple sequence alignmentrdquo ComputerApplications in the Biosciences vol 8 no 2 pp 189ndash191 1992

[24] M Kimura ldquoA simple method for estimating evolutionary ratesof base substitutions through comparative studies of nucleotidesequencesrdquo Journal ofMolecular Evolution vol 16 no 2 pp 111ndash120 1980

[25] N Saitou and M Nei ldquoThe neighbor-joining method a newmethod for reconstructing phylogenetic treesrdquo Molecular Biol-ogy and Evolution vol 4 no 4 pp 406ndash425 1987

[26] E Barbu K Y Lee and R Wahl ldquoContent of purine andpyrimidine base in desoxyribonucleic acid of bacteriardquo Annalesde lrsquoInstitut Pasteur vol 91 no 2 pp 212ndash224 1956

[27] N Sueoka ldquoVariation and heterogeneity of base compositionof deoxyribonucleic acids a compilation of old and new datardquoJournal of Molecular Biology vol 3 no 1 pp 31ndash40 1961

[28] H Hori and S Osawa ldquoEvolutionary change in 5S rRNAsecondary structure and a phylogenic tree of 352 5S rRNAspeciesrdquo BioSystems vol 19 no 3 pp 163ndash172 1986

[29] V Bess ldquoEvaluating microbiology of compost microbial con-tent of composting is helping the producers and growers tounderstand its role as a soil inoculant and plantrdquo BioCycleMagazine p 62 1999

[30] O Grafff ldquoPreliminary experiments of vermin composting ofdifferent waste materials using Eudrilus eugeniae Kinbergrdquo inWorkshop on the Role of Earthworms in the Stabilization ofOrganic Residues M Happelhof Ed pp 178ndash191 Beech LeafPress Kalamazoo Mich USA 1981

[31] J E Satchell Earthworm Ecology from Darwin to VermicultureChapman and Hall 1983

[32] G H Xie M Y Cai G C Tao and Y Steinberger ldquoCultivableheterotrophic N2-fixing bacterial diversity in rice fields in theYangtze River Plainrdquo Biology and Fertility of Soils vol 37 no 1pp 29ndash38 2003

[33] B BMcSpaddenGardener ldquoEcology ofBacillus and Paenibacil-lus spp in agricultural systemsrdquo Phytopathology vol 94 no 11pp 1252ndash1258 2004

[34] J Dobereiner ldquoBiological nitrogen fixation in the tropics socialand economic contributionsrdquo Soil Biology andBiochemistry vol29 no 5-6 pp 771ndash774 1997

[35] V M Reis F L Olivares and J Dobereiner ldquoImprovedmethodology for isolation of Acetobacter diazotrophicus andconfirmation of its endophytic habitatrdquoWorld Journal of Micro-biology and Biotechnology vol 10 no 4 pp 401ndash405 1994

[36] S Khalil N Ayub S Alam and F Latif ldquoOrganic acids pro-duction and phosphate solubilization by phosphate solubilizingmicroorganisms (PSM) under in vitro conditionsrdquo PakistanJournal of Biological Sciences vol 7 no 2 pp 187ndash196 2004

[37] H M A El-Komy ldquoCoimmobilization of Azospirillumlipoferum and Bacillus megaterium for successful phosphorusand nitrogen nutrition of wheat plantsrdquo Food Technology andBiotechnology vol 43 no 1 pp 19ndash27 2005

[38] N B Paul and W V B Sundara Rao ldquoPhosphate-dissolvingbacteria in the rhizosphere of some cultivated legumesrdquo Plantand Soil vol 35 no 1 pp 127ndash132 1971

[39] A Beneduzi D Peres L K Vargas M H Bodanese-Zanettiniand LM P Passaglia ldquoEvaluation of genetic diversity and plantgrowth promoting activities of nitrogen-fixing bacilli isolatedfrom rice fields in South Brazilrdquo Applied Soil Ecology vol 39no 3 pp 311ndash320 2008

[40] M P Raimam U Albino M F Cruz et al ldquoInteraction amongfree-living N-fixing bacteria isolated from Drosera villosa varvillosa and AM fungi (Glomus clarum) in rice (Oryza sativa)rdquoApplied Soil Ecology vol 35 no 1 pp 25ndash34 2007

[41] M R Swain S K Naskar and R C Ray ldquoIndole-3-acetic acidproduction and effect on sprouting of yam (Dioscorea rotundataL) minisetts by Bacillus subtilis isolated from culturable cow-dung microflorardquo Polish Journal of Microbiology vol 56 no 2pp 103ndash110 2007

[42] T M Tien M H Garkins and D H Hubbell ldquoPlant growthsubstances produced by Azospirillum brasilense and their effect

Advances in Agriculture 13

on the growth of pearl Millet Pennisetum americanum (L)rdquoApplied and EnvironmentalMicrobiology vol 37 no 5 pp 1016ndash1024 1979

[43] A Esitken L Pirlak M Turan and F Sahin ldquoEffects of floraland foliar application of plant growth promoting rhizobacteria(PGPR) on yield growth and nutrition of sweet cherryrdquo ScientiaHorticulturae vol 110 no 4 pp 324ndash327 2006

[44] H Karlidag A Esitken M Turan and F Sahin ldquoEffects of rootinoculation of plant growth promoting rhizobacteria (PGPR)on yield growth and nutrient element contents of leaves ofapplerdquo Scientia Horticulturae vol 114 no 1 pp 16ndash20 2007

[45] C S Nautiyal S Bhadauria P Kumar H Lal R Mondal andD Verma ldquoStress induced phosphate solubilization in bacteriaisolated fromalkaline soilsrdquoFEMSMicrobiology Letters vol 182no 2 pp 291ndash296 2000

[46] A K Halder A K Mishra P Bhattacharya and P KChakrabarthy ldquoSolubilization of inorganic phosphates byBradyrhizobiumrdquo Indian Journal of Experimental Biology vol29 pp 28ndash31 1991

Submit your manuscripts athttpwwwhindawicom

Nutrition and Metabolism

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Food ScienceInternational Journal of

Agronomy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Applied ampEnvironmentalSoil Science

Volume 2014

AgricultureAdvances in

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PsycheHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BiodiversityInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Plant GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biotechnology Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Forestry ResearchInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of BotanyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

EcologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Veterinary Medicine International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Cell BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Page 5: BalayoganSivasankariandMarimuthuAnandharajdownloads.hindawi.com/journals/aag/2014/248591.pdf · In Vitro Nitrogen Fixing Activity. e bacterial isolates were screened for nitrogen

Advances in Agriculture 5

Table1

Predo

minantb

acteria

lstrains

isolated

from

verm

isourcesand

theirb

iochem

icalcharacteris

ticsT1-con

trolmdash

Gsepium

+L

leucocephala+

cowdu

ngin

(112)(worm

free)T

2mdashG

sepium

+L

leucocephala+cowdu

ngin

(112)+

EeugeniaeT3

mdashG

sepium

+L

leucocephala+cowdu

ngin

(112)+

EfetidaT4

-con

trol(2)mdashC

auric

ulata+L

leucocephala+cow

dung

in(112)(worm

free)T

5mdashC

auric

ulata+L

leucocephala+cowdu

ngin

(112)+

EeugeniaeT

6mdashC

auric

ulata+L

leucocephala+cowdu

ngin

(112)+

Efetida

Snu

mber

Samples

ource

Biochemicalcharacteris

ticsc

Nam

eofthe

strain

Gramrsquos

reactio

naCell

shape

Motility

bIndo

letest

Methyl

Red

test

Voges

Proskauer

test

Citrate

Utilization

test

Catalase

test

Urease

test

Gelatin

hydrolysis

test

Nitrate

redu

ctase

test

Starch

hydrolysis

test

Casein

hydrolysis

test

Glucose

utilizatio

ntest

1Ve

rmicom

post

(T2)

+Cocciminus

minus+

minusminus

+minus

++

minusminus

minusStaphylococcus

spp

2Ve

rmicom

post

(T3)

+Ro

d+

minusminus

minusminus

+minus

minus+

minusminus

+Co

rynebacterium

spp

3Ve

rmicom

post

(T5)

+Ro

dminus

++

++

+minus

++

+minus

+Ba

cillusspp

4Ve

rmicom

post

(T6)

+Cocciminus

++

minusminus

+minus

+minus

minusminus

+Micrococcusspp

5Ve

rmicast(T3

)+

Cocciminus

++

minusminus

minus+

++

+minus

minusKlebsiella

spp

6Ve

rmicast(T5

)+

Rod

minus+

minusminus

+minus

+minus

+minus

minus+

Streptococcusspp

7

Verm

icast(T6

)+

Cocciminus

+minus

++

minusminus

++

minusminus

+En

terobacter

spp

8Ea

rthw

orm

hind

gut(T2

)+

Cocciminus

minus+

+minus

+minus

minusminus

++

+Trico

ccocus

spp

9Ea

rthw

orm

hind

gut(T3

)+

Cocciminus

minus+

minus+

++

minus+

minusminus

minusYersiniaspp

10

Earthw

orm

hind

gut(T5

)+

Cocciminus

minusminus

minusminus

++

++

minusminus

minusMicrococcusspp

11

Verm

icom

post

(T1)

minusRo

dminus

++

minusminus

minusminus

++

minusminus

+Escherich

iaspp

12

Verm

icom

post

(T2)

minusRo

dminus

minus+

minusminus

minus+

++

minusminus

+Proteusspp

13

Verm

icom

post

(T4)

minusRo

dminus

minusminus

+minus

+minus

minus+

++

+Pa

racoccus

spp

14

Verm

icom

post

(T5)

minusRo

d+

+minus

minusminus

++

++

minusminus

minusPseudomonas

spp

15

Verm

icast(T2

)minus

Rod

+minus

+minus

++

+minus

+minus

minusminus

Citro

bacter

spp

16

Verm

icast(T3

)minus

Cocciminus

++

minusminus

+minus

+minus

minusminus

minusErwiniaspp

17Ve

rmicast(T5

)minus

Rod

++

++

++

minus+

minusminus

minusminus

Alcaligenesspp

18

Verm

icast(T6

)minus

Cocciminus

minus+

++

+minus

++

minusminus

minusSerratiaspp

19

Earthw

orm

mid

gut(T2

)minus

Rod

+minus

minusminus

+minus

minusminus

minusminus

minusminus

Pediococcusspp

a Gramrsquossta

ining(+)p

ositive(minus)n

egative

b Motility(+)

motile(minus)n

onmotile

c Biochem

icalcharacteris

tics(+)p

ositive(minus)n

egative

6 Advances in Agriculture

Bacillus cereus strain GGBSTD2 16S ribosomal RNA gene

Bacillus cereus strain SU-6 16S ribosomal RNA gene

Bacillus cereus strain JB0013 16S ribosomal RNA gene

Bacillus cereus isolate PN24 16S ribosomal RNA gene

Bacillus cereus strain JB0005 16S ribosomal RNA gene

Bacillus sp G2DM-85 16S ribosomal RNA gene

Bacillus cereus strain JBE0009 16S ribosomal RNA gene

Bacillus sp EB460 16S ribosomal RNA gene

Bacillus cereus strain 421-3R 16S ribosomal RNA gene

Bacillus sp EB447 16S ribosomal RNA gene

Bacillus sp EB470 16S ribosomal RNA gene

Bacillus sp EB421 16S ribosomal RNA gene

Bacillus sp EB431 16S ribosomal RNA gene

Bacillus sp EB462 16S ribosomal RNA gene

Bacillus sp EB429 16S ribosomal RNA gene

Bacillus cereus strain 421-4R 16S ribosomal RNA gene

Bacillus sp EB480 16S ribosomal RNA gene

Bacillus sp EB13 16S ribosomal RNA gene

Uncultured compost bacterium partial 16S rRNA gene

Bacillus thuringiensis gene for 16S rRNA

Figure 1 Neighbor joining phylogenetic tree of full 16S rRNA gene sequences showing the relationship among Bacillus cereus GGBSTD1and representatives of some related taxa The sequence data for several closely related Bacillus cultures were recovered from GenBank andincluded in the tree

Table 2 The phosphate solubilizing activity phytohormone (IAA) production and siderophores production of the bacterial isolates

Name of the organism Phosphate solubilization index (SI) IAA production (120583gmL)a Siderophores productionb

Bacillus spp 3855 242 +++Alcaligenes spp 2930 147 minus

Erwinia spp 2890 113 +Serratia spp 3045 168 ++Pseudomonas spp 3522 196 +++aIndole acetic acid production in culture media supplemented with tryptophan (5 120583gmL) after 5 daysbIn vitro siderophores production minus represents the absence of siderophores production and +++ represents gt10mm wide yellow-orange zone

is given in Tables 3 and 4 respectively The shoot length(1147 plusmn 006 cm) root length (1027 plusmn 006 cm) and leafarea (905 plusmn 026 cm2) of the plant get significantly (119875 lt0001) increased in Exp3 (consortium of Bacillus cereus +Pseudomonas spp [1 1]) at 5mL concentration on the 15thday compared to other experimentsThe chlorophyll a b andtotal chlorophyll content of the leaves on d 5 d 10 and d 15are given in Table 5 The chlorophyll a (010 plusmn 009mgg offresh leaf) chlorophyll b (1085 plusmn 016mgg of fresh leaf)and total chlorophyll (1095plusmn007mgg of fresh leaf) content

of leaves were significantly (119875 lt 0001) higher in Exp3(consortium of Bacillus cereus + Pseudomonas spp (1 1))at 5mL concentration on the 15th day compared to otherexperiments Observations on the fresh weight of the wholeplant are given in Table 6 The fresh weight of the wholeplant (092 plusmn 000 g) was significantly (119875 lt 0001) higherin Exp3 (consortium of Bacillus cereus + Pseudomonas spp(1 1)) at 5mL concentration on the 15th day compared toother experiments Observations on the dry weight of thewhole plant are given in Table 6 The dry weight of the whole

Advances in Agriculture 7

Pseudomonas fluorescens strain LMG 5825 16S ribosomal RNA gene

Pseudomonas fluorescens strain LMG 5829 16S ribosomal RNA gene

Pseudomonas fluorescens strain LMG 14676 16S ribosomal RNA gene

Pseudomonas sp W15Feb13 16S ribosomal RNA gene

Pseudomonas sp W15Feb28 16S ribosomal RNA gene

Pseudomonas sp p50 16S ribosomal RNA gene

Uncultured bacterium clone 661193 16S ribosomal RNA gene

Pseudomonas sp BSs20166 16S ribosomal RNA gene

Pseudomonas sp 28zhy partial 16S rRNA gene

Uncultured bacterium clone AYRV1-027 16S ribosomal RNA gene

Uncultured Pseudomonadaceae bacterium clone290cost002-P3L-2032 16S ribosomal RNA gene

Pseudomonas sp GGBSTD3 16S ribosomal RNA gene

Pseudomonas sp DK3 16S ribosomal RNA gene

Pseudomonas reactans strain AMP-13 16S ribosomal RNA gene

Figure 2 Neighbor joining phylogenetic tree of full 16S rRNA gene sequences showing the relationship among Pseudomonas spp GGBSTD3and representatives of some related taxa The sequence data for several closely related Pseudomonas cultures were recovered from GenBankand included in the tree

plant (009 plusmn 000 g) was significantly (119875 lt 0001) higherin Exp3 (consortium of Bacillus cereus + Pseudomonas spp(1 1)) at 5mL concentration on the 15th day compared toother experiments

4 Discussion

Inoculation of these newly identified nitrogen fixing andphosphate solubilizingmicrobes in the plant growthmediumenhanced various physiological activities culminating inhigher biomass production in V unguiculata Since this is anonhazardous way of fertilization of crop plants it is veryrelevant to a developing country like India This techniquecan save the farmers of India from the problem posed byhigh cost of fertilizers by offering a comparatively inexpensivealternative (biofertilizers) while additionally preventing thedegradation of the soil and thereby ensuring sustainable agri-culture Bess (1999) reported that the content in compostingneeds to be determined through the concentration of sixfunctional groups of microorganisms such as aerobic bacte-ria anaerobic bacteria fungi actinomycetes Pseudomonasand nitrogen fixing bacteria [29] Now there are ways toevaluate the concentrations of these organisms in the finishedcompost and that can serve as an interpretation guide to

determine the quality of the compost as an inoculant of soilmicroorganisms Grafff (1981) and Atlavinyte have reporteda higher P content in the cast than in the surrounding soil[30] Satchell (1983) has linked the phosphatase activity inthe gut of worms to the availability of bound P in soils Thebacterial strains which have the capability to fix nitrogenonly can grow in the nitrogen-free medium [31] Hencethe nitrogen fixing ability was tested in the present studybased on the growth of bacterial strain in nitrogen- freemedium Five bacteria strains showedmore nitrogen fixationamong all the five bacterial strains tested only one strain (ieBacillus spp) showed significantly more turbidity comparedto the other strains The ultimate source of the nitrogen usedby plants is N

2gas which constitutes 78 of the Earthrsquos

atmosphere Unfortunately higher plants cannot metabolizeN2directly into protein N

2gas must be converted to a plant

available form [32] isolated many species of nitrogen fixingBacillus (B megaterium B cereus B subtilis B licheniformisand B azotoformans) from the rhizospheric zone of ricefields in Central China The widely studied Bacillus genusrepresents one of the most diverse genera in the bacilli groupNumerous Bacillus and Paenibacillus strains express plantgrowth promoting (PGP) activities and a number of thesestrains have already been commercially developed as biolog-ical fungicides insecticides and nematicides or generic plant

8 Advances in Agriculture

Table 3 Shoot length and Root length ofVigna unguiculata grown on vermiculite supplemented with Bacillus cereus with Pseudomonas sppand with their consortium (1 1) at five different concentrations (1 2 3 4 and 5mL) on the 5th the 10th and the 15th day and in the control

Experimentsa Quantity of bacterial cultures added Shoot length (cm)b Root length (cm)b

5th day 10th day 15th day 5th day 10th day 15th day

Exp0

1mL 297 (ab) 397 (a) 497 (ab) 197 (de) 283 (ab) 397 (bcd)2mL 303 (bcd) 403 (ab) 500 (ab) 200 (abc) 287 (de) 400 (a)3mL 310 (abc) 407 (a) 503 (ab) 203 (de) 290 (abc) 403 (ab)4mL 310 (bcd) 410 (bcd) 507 (bcd) 207 (a) 297 (de) 410 (abc)5mL 313 (a) 413 (ab) 510 (ab) 210 (bcd) 300 (ab) 413 (de)

Exp1

1mL 340 (ab) 440 (abc) 540 (ab) 233 (abc) 327 (bcd) 433 (f)2mL 450 (ab) 540 (abc) 647 (ab) 333 (ab) 430 (a) 540 (abc)3mL 550 (de) 630 (ab) 750 (a) 440 (de) 550 (abc) 620 (a)4mL 653 (ab) 730 (a) 833 (ab) 550 (bcd) 653 (de) 750 (abc)5mL 740 (ab) 840 (abc) 923 (f) 640 (abc) 753 (ab) 827 (ab)

Exp2

1mL 277 (bcd) 357 (f) 440 (de) 160 (de) 217 (abc) 350 (f)2mL 343 (abc) 447 (ab) 553 (ab) 253 (abc) 340 (bcd) 427 (bcd)3mL 437 (de) 543 (abc) 633 (abc) 340 (ab) 443 (f) 533 (ab)4mL 547 (bcd) 647 (de) 733 (a) 420 (bcd) 550 (ab) 633 (ab)5mL 640 (f) 733 (bcd) 833 (bcd) 533 (ab) 630 (ab) 730 (bcd)

Exp3

1mL 530 (bcd) 647 (ab) 730 (ab) 433 (de) 550 (abc) 620 (ab)2mL 643 (ab) 740 (bcd) 827 (f) 547 (a) 630 (de) 733 (ab)3mL 737 (bcd) 840 (bcd) 927 (ab) 640 (ab) 740 (bcd) 840 (bcd)4mL 847 (abc) 930 (a) 1043 (abc) 740 (f) 833 (a) 937 (abc)5mL 957 (ab) 1030 (f) 1147 (bcd) 833 (abc) 940 (ab) 1027 (bcd)

aExp0 nutrient solution (control) Exp1 Bacillus cereus Exp2 Pseudomonas spp Exp3 consortium of Bacillus cereus + Pseudomonas spp (1 1)bMeanof two repeated experiments (30 plants)Different letterswithin parenthesis indicate significant difference between treatments for each growth parameterusing Duncanrsquos multiple range test (119875 = 005)

growth promoters The use of these strains in agriculturehas recently been reviewed [33]Those strains besides havingseveral PGP properties can also fix nitrogen and also dophosphate solubilization The symbiotic fixation of nitrogenthrough inoculation of legume crops with effective rhizobiais well known [34] Asymbiotic nitrogen fixing bacteriawhich live in the rhizosphere andor endophytically oftenincrease yields of crops Many bacterial species have nitrogenfixing properties including Bacillus spp Azotobacter sppAzospirillum spp Beijerinckia spp and Pseudomonas spp[35] Nitrogen fixing bacteria have been used in foliar appli-cations inmulberryThe solubilization index (SI) was formeddue to solubilization of insoluble phosphates by organic acidsecretion [36] El-Komy (2005) indicated that Pseudomonasfluorescence and Bacillus megaterium strains were the mostpowerful phosphate solubilizers on PVK plates as well as onPikovskayarsquos broth [37] The P concentration in Pikovskayarsquosbroth increased gradually achieving a peak on the sixth dayand declined slowly during the late days In this study pHvalues decreased gradually in PVK broth during the earlyincubation days and no revival was observed during laterdays for all the tested bacterial strains Several bacteria par-ticularly those belonging to the genus Bacillus spp convertinsoluble phosphate into soluble forms by secreting organicacids such as formic acid acetic acid propionic acid citricacid fumaric acid gluconic acid glyoxylic acid ketobutyric

acid malonic acid succinic acid and tartaric acid Theseacids lower the pH and bring about the dissolution of boundforms of phosphate Some of the hydroxyl acids may chelatewith calcium and iron resulting in effective solubilization andutilization of phosphates [38]

Beneduzi et al (2008) had isolated the plant growthpromoting strain SVPR30 and identified by 16S rRNA genesequence as Bacillus spp and they had tested it for plantgrowth through in vivo experiments [39] This strain wascharacterized as a high IAA producer able to solubilize phos-phate and also fix a considerably high amount of nitrogenThe inoculation of rice with Bacillus spp SVPR30 strainshowed a significant increase in the root and shoot partswhen compared with the controls within 15 and 30 daysafter sprouting Numerous Bacillus and Paenibacillus strainsexpressed plant growth promoting (PGP) activities and anumber of these strains have been commercially developedas generic plant growth promotersThe use of these strains inagriculture has recently been reviewed [33]

Various reports indicate that coinoculation of beneficialorganisms generally increased plant growth relative to singleinoculation with a sole beneficial organism [40] Most ofthe effects of the individual microorganisms coinoculationare additive although a synergistic effect has been reportedin some cases The average root to shoot ratio was higherin B subtilis treated yam minisetts in comparison to those

Advances in Agriculture 9

Table 4 Leaf area of Vigna unguiculata grown on vermiculite supplemented with Bacillus cereus with Pseudomonas spp and with theirconsortium (1 1) at five different concentrations (1 2 3 4 and 5mL) on the 5th the 10th and the 15th days and in the control

Experimentsa Quantity of bacterial cultures added Leaf area (cm2)b

5th day 10th day 15th day

Exp0

1mL 027 (ab) 094 (abc) 407 (b)2mL 035 (ab) 102 (ab) 419 (abc)3mL 040 (abc) 125 (ab) 471 (bcd)4mL 048 (ab) 138 (abc) 510 (ab)5mL 054 (abc) 161 (b) 538 (b)

Exp1

1mL 018 (bcd) 123 (b) 601 (abc)2mL 025 (ab) 138 (abc) 632 (ab)3mL 033 (abc) 150 (ab) 684 (b)4mL 038 (ab) 168 (ab) 686 (abc)5mL 044 (abc) 191 (bcd) 733 (ab)

Exp2

1mL 011 (b) 107 (ab) 534 (b)2mL 014 (ab) 119 (ab) 559 (ab)3mL 017 (abc) 127 (ab) 579 (ab)4mL 021 (ab) 153 (abc) 631 (abc)5mL 026 (ab) 166 (b) 667 (b)

Exp3

1mL 026 (bcd) 297 (ab) 753 (bcd)2mL 033 (b) 325 (ab) 787 (ab)3mL 040 (ab) 350 (bcd) 811 (bcd)4mL 044 (b) 384 (b) 861 (b)5mL 062 (ab) 400 (bcd) 905 (ab)

aExp0 nutrient solution (control) Exp1 Bacillus cereus Exp2 Pseudomonas spp Exp3 consortium of Bacillus cereus + Pseudomonas spp (1 1)bMeanof two repeated experiments (30 plants)Different letterswithin parenthesis indicate significant difference between treatments for each growth parameterusing Duncanrsquos multiple range test (119875 = 005)

0102030405060708090

100

Concentration of bacterial cultures (mL)1 2 3 4 5

Germination ()

Exp0

Exp1 Exp3

Exp2

Figure 3 Germination percentage ofVigna unguiculata seeds sownin vermiculite supplementedwithBacillus cereus with Pseudomonasspp and with their consortium at five different concentrations (1 23 4 and 5mL) and in the control Exp0 nutrient solution (control)Exp1 Bacillus cereus Exp2 Pseudomonas spp Exp3 consortium ofBacillus cereus + Pseudomonas spp (1 1)

not treated with the bacterial culture [41] In earlier reportsroot elongation was found to occur in Sesbania aculeata byinoculation with Azotobacter spp and Pseudomonas spp in

Vigna radiata by Pseudomonas putida [16] and in Pennisetumamericanum by Azospirillum brasilense [42] Esitken et al(2006) had found out that Bacillus OSU-142 and Pseu-domonas BA-8 alone or in combination had a great potentialto increase the growth yield and nutrition of sweet cherryplant [43] BacillusM3 BacillusOSU-142 andMicobacteriumFS01 separately or in combination were found to have greatpotential for use as plant growth promoting rhizobacteriato increase production in apples and in many other crops[44] These phosphate solubilizing microbes consist pre-dominantly of fungal bacterial and actinomycetes speciescollectively called phosphate solubilizing microorganismsmdashPSM [45] There are several microorganisms which canalso solubilize the cheaper sources of phosphorous such asrock phosphate Bacteria such as Bacillus are widely usedin plant production system and are important phosphorussolubilizing microorganisms resulting in improved growthyield of crops andmetabolic activities especially in synthesisof protein [34 46]

5 Conclusions

This study illustrates the isolation of plant growth promotingbacteria from vermisources screening under in vitro con-ditions for multiple PGPR traits and their evaluation undercontrolled conditions in a pot experiment The findings ofthis study indicated that the consortium of B cereus and

10 Advances in Agriculture

Table5Ch

loroph

yllcon

tent

ofleavesofVignaun

guicu

latagrow

non

verm

iculite

supp

lementedwith

Bacilluscereuswith

Pseudomonas

sppand

with

theircon

sortium

(11)atfi

vedifferent

concentrations

(1234and

5mL)

onthe5

thdaythe10thdayandthe15thdayandin

thec

ontro

l

Experim

entsa

Quantity

ofbacterial

Cultu

resa

dded

Chloroph

yllcon

tent

(mggof

fresh

leaf)b

5thday

10th

day

15th

day

Chloroph

ylla

Chloroph

yllb

Total

chloroph

yll

Chloroph

ylla

Chloroph

yllb

Total

chloroph

yll

Chloroph

ylla

Chloroph

yllb

Total

chloroph

yll

Exp0

1mL

021

(ab)

285

(abc)

307

(a)

020

(abc)

481

(a)

501

(ab)

015

(bcd)

612

(abc)

627

(bcd)

2mL

022

(abc)

293

(ab)

315

(abc)

021

(ab)

489

(ab)

510

(bcd)

012

(abc)

618

(ab)

630

(ab)

3mL

023

(abc)

301

(ab)

315

(abc)

022

(bcd)

497

(bcd)

518

(a)

014

(a)

633

(cde)

647

(bcd)

4mL

024

(ab)

308

(f)

332

(a)

022

(a)

504

(b)

527

(abc)

015

(bcd)

641

(cde)

656

(a)

5mL

025

(f)

316

(abc)

341

(bcd)

019

(ab)

511(ab)

530

(abc)

011(cde)

645

(ab)

650

(abc)

Exp1

1mL

035

(b)

400

(cde)

435

(abc)

011(b)

581

(cde)

593

(cde)

010

(abc)

704(abc)

715(f)

2mL

026

(abc)

462

(bcd)

488

(f)

011(abc)

647

(b)

658

(abc)

006

(ab)

779(bcd)

785(a)

3mL

021

(bcd)

526

(abc)

547

(ab)

008

(bcd)

718(ab)

726(bcd)

006

(f)

847

(f)

853

(ab)

4mL

008

(f)

587

(cde)

595

(b)

011(bcd)

778(abc)

789(abc)

004

(bcd)

933(b)

937(bcd)

5mL

026

(abc)

641

(bcd)

667

(abc)

025

(cde)

834

(cde)

859

(f)

007

(abc)

976(cde)

983(abc)

Exp2

1mL

030

(abc)

354

(ab)

384

(b)

012

(a)

545

(abc)

556

(ab)

007

(bcd)

681

(abc)

688

(bcd)

2mL

029

(bcd)

420

(f)

449

(abc)

011(bcd)

610

(ab)

621

(abc)

007

(abc)

744(f)

752(bcd)

3mL

022

(ab)

504

(abc)

527

(bcd)

015

(a)

677

(cde)

692

(bcd)

006

(bcd)

812

(ab)

817

(bcd)

4mL

010

(f)

566

(a)

576

(cde)

018

(abc)

744(f)

762(ab)

005

(ab)

885

(a)

890

(abc)

5mL

006

(bcd)

622

(abc)

628

(abc)

018

(b)

810

(abc)

828

(a)

004

(cde)

948(a)

952(f)

Exp3

1mL

027

(ab)

470

(bcd)

496

(cde)

009

(ab)

668

(b)

677

(cde)

010

(a)

814

(ab)

825

(a)

2mL

020

(ab)

548

(a)

568

(f)

003

(bcd)

746(bcd)

749(ab)

009

(ab)

887

(f)

897

(ab)

3mL

005

(bcd)

584

(abc)

589

(ab)

008

(bcd)

804

(abc)

812

(cde)

010

(ab)

953(abc)

963(bcd)

4mL

023

(f)

663

(ab)

686

(cde)

007

(ab)

870

(b)

877

(f)

010

(abc)

1019

(bcd)

1029(f)

5mL

022

(ab)

744(cde)

766(a)

009

(a)

932(abc)

940(ab)

010

(a)

1085(ab)

1095(abc)

a Exp0nu

trient

solutio

n(con

trol)

Exp1B

acillus

cereusE

xp2Pseudomonas

sppE

xp3consortiu

mof

Bacilluscereus+

Pseudomonas

spp(11)

b Meanof

tworepeated

experim

ents(30plants)

Differentletterswith

inparenthesis

indicatesig

nificantd

ifference

betweentre

atmentsfore

achgrow

thparameter

usingDun

canrsquos

multip

lerangetest(119875=005)

Advances in Agriculture 11

Table 6 Fresh weight and dry weight of whole plant of Vigna unguiculata grown on vermiculite supplemented with Bacillus cereus withPseudomonas spp and with their consortium (1 1) at five different concentrations (1 2 3 4 and 5mL) on the 5th the 10th and the 15 daysand in the control

Experimentsa Quantity of bacterial cultures added Fresh weight of the whole plant (g)b Dry weight of the whole plant (g)b

5th day 10th day 15th day 5th day 10th day 15th day

Exp0

1mL 058 (bcd) 061 (abc) 064 (ab) 006 (a) 006 (abc) 006 (ab)2mL 058 (ab) 061 (a) 064 (abc) 006 (bcd) 006 (ab) 006 (ab)3mL 058 (abc) 061 (ab) 064 (bcd) 006 (abc) 006 (bcd) 006 (b)4mL 041 (abc) 061 (ab) 064 (ab) 006 (abc) 006 (ab) 006 (abc)5mL 058 (abc) 061 (abc) 064 (bcd) 006 (a) 006 (bcd) 006 (def)

Exp1

1mL 062 (abc) 065 (abc) 068 (ab) 006 (bcd) 006 (ab) 007 (bcd)2mL 065 (bcd) 068 (bcd) 071 (def) 006 (abc) 007 (a) 007 (ab)3mL 068 (abc) 071 (def) 074 (bcd) 007 (abc) 007 (ab) 007 (abc)4mL 071 (ab) 074 (abc) 077 (abc) 007 (bcd) 007 (b) 008 (bcd)5mL 074 (abc) 077 (def) 080 (ab) 007 (def) 008 (a) 008 (b)

Exp2

1mL 056 (ab) 059 (bcd) 062 (ab) 006 (ab) 006 (ab) 006 (abc)2mL 059 (abc) 062 (abc) 065 (def) 006 (ab) 006 (abc) 006 (bcd)3mL 062 (bcd) 065 (a) 068 (bcd) 006 (abc) 006 (bcd) 007 (abc)4mL 065 (abc) 068 (ab) 071 (b) 006 (ab) 007 (abc) 007 (b)5mL 068 (abc) 071 (b) 074 (a) 007 (def) 007 (abc) 007 (bcd)

Exp3

1mL 074 (ab) 077 (ab) 080 (abc) 007 (a) 008 (bcd) 008 (ab)2mL 077 (bcd) 080 (a) 083 (bcd) 008 (a) 008 (bcd) 008 (abc)3mL 080 (bcd) 083 (bcd) 086 (abc) 008 (ab) 008 (b) 009 (b)4mL 083 (bcd) 086 (abc) 089 (ab) 008 (abc) 009 (ab) 009 (bcd)5mL 086 (ab) 089 (b) 092 (def) 009 (bcd) 009 (bcd 009 (b)

aExp0 nutrient solution (control) Exp1 Bacillus cereus Exp2 Pseudomonas spp Exp3 consortium of Bacillus cereus + Pseudomonas spp (1 1)bMeanof two repeated experiments (30 plants)Different letterswithin parenthesis indicate significant difference between treatments for each growth parameterusing Duncanrsquos multiple range test (119875 = 005)

Pseudomonas spp acts as a potential biofertilizer due to itsvarious plant growth promotion abilities such as solubiliza-tion of phosphate and production of IAA and siderophore itcan be developed as potential microbial inoculant for variouscrops

Conflict of Interests

The authors declared that there is no conflict of interestsregarding this paper

Acknowledgments

Theauthors are thankful to theDepartment of BiologyGand-higram Rural InstitutemdashDeemed University GandhigramThis research was funded by University Grants Commissionunder UGC Research Fellowship in Science for MeritoriousStudents (BSR) Grant no F4-12008

References

[1] U S Bhawalkar Vermiculture Ecotechnology EarthwormResearch Institute Pune India 2nd edition 1996

[2] S Kiss D M Bularda and D Radulescu ldquoBiological Signifi-cance of Enzymes Accumulated in SoilrdquoAdvances in Agronomyvol 27 pp 25ndash87 1975

[3] D L Karlen M J Mausbach J W Doran R G Cline R FHarris and G E Schuman ldquoSoil quality a concept definitionand framework for evaluationrdquo Soil Science Society of AmericaJournal vol 61 no 1 pp 4ndash10 1997

[4] R M Atiyeh C A Edwards S Subler and J Metzger ldquoEarth-worm processed organic wastes as components of horticulturalpotting media for growing manifold and vegetable seedlingsrdquoCompost Science and Utilization vol 8 no 3 pp 215ndash223 2000

[5] N Vassilev and M Vassileva ldquoBiotechnological solubilizationof rock phosphate on media containing agro-industrial wastesrdquoApplied Microbiology and Biotechnology vol 61 no 5-6 pp435ndash440 2003

[6] S A Omar ldquoThe role of rock-phosphate-solubilizing fungiand vesicular-arbusular-mycorrhiza (VAM) in growth of wheatplants fertilized with rock phosphaterdquo World Journal of Micro-biology and Biotechnology vol 14 no 2 pp 211ndash218 1998

[7] J A J Paul and T Daniel ldquoStandardization of sampling methodfor physical characterization of municipal solid wasterdquo AsianJournal of Water Environment and Pollution vol 5 no 1 pp95ndash98 2007

[8] R Lalloo D Maharajh J Gorgens and N Gardiner ldquoA down-stream process for production of a viable and stable Bacilluscereus aquaculture biological agentrdquo Applied Microbiology andBiotechnology vol 86 no 2 pp 499ndash508 2010

[9] B Sivasankari N Kavikumar and M Anandharaj ldquoIndole-3-acetic acid production and enhanced Plant growth promotionby Indigenous bacterial speciesrdquo Journal of Current Research inScience vol 1 no 5 pp 331ndash335 2013

12 Advances in Agriculture

[10] J Han L Sun X Dong et al ldquoCharacterization of a novel plantgrowth-promoting bacteria strain Delftia tsuruhatensis HR4both as a diazotroph and a potential biocontrol agent againstvarious plant pathogensrdquo Systematic and Applied Microbiologyvol 28 no 1 pp 66ndash76 2005

[11] H Rodrıguez and R Fraga ldquoPhosphate solubilizing bacte-ria and their role in plant growth promotionrdquo BiotechnologyAdvances vol 17 no 4-5 pp 319ndash339 1999

[12] T Daniel and N Karmegam ldquoBio-conversion of selected leaflitters using an African epigeic earthworm Eudrilus eugeniaerdquoEcology Environment and Conservation vol 5 no 3 pp 271ndash275 1999

[13] J Dobereiner I E Marriel and M Nery ldquoEcological distri-bution of Spirillum lipoferum Beijerinckrdquo Canadian Journal ofMicrobiology vol 22 no 10 pp 1464ndash1473 1976

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

[15] M Edi Premono A M Moawad and P L G Vlek ldquoEffect ofphosphate-solubilizing Pseudomonas putida on the growth ofmaize and its survival in the rhizosphererdquo Indonesian Journal ofCrop Science vol 11 pp 13ndash23 1996

[16] C L Patten and B R Glick ldquoBacterial biosynthesis of indole-3-acetic acidrdquo Canadian Journal of Microbiology vol 42 no 3pp 207ndash220 1996

[17] N Amaresan V Jayakumar K Kumar and N ThajuddinldquoIsolation and characterization of plant growth promotingendophytic bacteria and their effect on tomato (Lycopersiconesculentum) and chilli (Capsicum annuum) seedling growthrdquoAnnals of Microbiology vol 62 no 2 pp 805ndash810 2012

[18] B Schwyn and J B Neilands ldquoUniversal chemical assay forthe detection and determination of siderophoresrdquo AnalyticalBiochemistry vol 160 no 1 pp 47ndash56 1987

[19] P Rahi P Vyas S Sharma and A Gulati ldquoPlant growthpromoting potential of the fungus Discosia sp FIHB 571 fromtea rhizosphere tested on chickpea maize and peardquo IndianJournal of Microbiology vol 49 no 2 pp 128ndash133 2009

[20] K Kumar N Amaresan S Bhagat K Madhuri and R CSrivastava ldquoIsolation and characterization of rhizobacteriaassociated with coastal agricultural ecosystem of rhizospheresoils of cultivated vegetable cropsrdquo World Journal of Microbiol-ogy and Biotechnology vol 27 no 7 pp 1625ndash1632 2011

[21] J A Smoker and S R Barnum ldquoRapid small scale DNAisolation from filamentous cyanobacteriardquo FEMS MicrobiologyLetters vol 56 no 1 pp 119ndash122 1988

[22] U Edwars T Rogall H Blocker M Emde and E C BottgerldquoIsolation and direct complete nucleotide determination ofentire genes Characterization of a gene coding for 16S riboso-mal RNArdquoNucleic Acids Research vol 17 no 19 pp 7843ndash78531989

[23] D G Higgins A J Bleasby and R Fuchs ldquoCLUSTAL Vimproved software for multiple sequence alignmentrdquo ComputerApplications in the Biosciences vol 8 no 2 pp 189ndash191 1992

[24] M Kimura ldquoA simple method for estimating evolutionary ratesof base substitutions through comparative studies of nucleotidesequencesrdquo Journal ofMolecular Evolution vol 16 no 2 pp 111ndash120 1980

[25] N Saitou and M Nei ldquoThe neighbor-joining method a newmethod for reconstructing phylogenetic treesrdquo Molecular Biol-ogy and Evolution vol 4 no 4 pp 406ndash425 1987

[26] E Barbu K Y Lee and R Wahl ldquoContent of purine andpyrimidine base in desoxyribonucleic acid of bacteriardquo Annalesde lrsquoInstitut Pasteur vol 91 no 2 pp 212ndash224 1956

[27] N Sueoka ldquoVariation and heterogeneity of base compositionof deoxyribonucleic acids a compilation of old and new datardquoJournal of Molecular Biology vol 3 no 1 pp 31ndash40 1961

[28] H Hori and S Osawa ldquoEvolutionary change in 5S rRNAsecondary structure and a phylogenic tree of 352 5S rRNAspeciesrdquo BioSystems vol 19 no 3 pp 163ndash172 1986

[29] V Bess ldquoEvaluating microbiology of compost microbial con-tent of composting is helping the producers and growers tounderstand its role as a soil inoculant and plantrdquo BioCycleMagazine p 62 1999

[30] O Grafff ldquoPreliminary experiments of vermin composting ofdifferent waste materials using Eudrilus eugeniae Kinbergrdquo inWorkshop on the Role of Earthworms in the Stabilization ofOrganic Residues M Happelhof Ed pp 178ndash191 Beech LeafPress Kalamazoo Mich USA 1981

[31] J E Satchell Earthworm Ecology from Darwin to VermicultureChapman and Hall 1983

[32] G H Xie M Y Cai G C Tao and Y Steinberger ldquoCultivableheterotrophic N2-fixing bacterial diversity in rice fields in theYangtze River Plainrdquo Biology and Fertility of Soils vol 37 no 1pp 29ndash38 2003

[33] B BMcSpaddenGardener ldquoEcology ofBacillus and Paenibacil-lus spp in agricultural systemsrdquo Phytopathology vol 94 no 11pp 1252ndash1258 2004

[34] J Dobereiner ldquoBiological nitrogen fixation in the tropics socialand economic contributionsrdquo Soil Biology andBiochemistry vol29 no 5-6 pp 771ndash774 1997

[35] V M Reis F L Olivares and J Dobereiner ldquoImprovedmethodology for isolation of Acetobacter diazotrophicus andconfirmation of its endophytic habitatrdquoWorld Journal of Micro-biology and Biotechnology vol 10 no 4 pp 401ndash405 1994

[36] S Khalil N Ayub S Alam and F Latif ldquoOrganic acids pro-duction and phosphate solubilization by phosphate solubilizingmicroorganisms (PSM) under in vitro conditionsrdquo PakistanJournal of Biological Sciences vol 7 no 2 pp 187ndash196 2004

[37] H M A El-Komy ldquoCoimmobilization of Azospirillumlipoferum and Bacillus megaterium for successful phosphorusand nitrogen nutrition of wheat plantsrdquo Food Technology andBiotechnology vol 43 no 1 pp 19ndash27 2005

[38] N B Paul and W V B Sundara Rao ldquoPhosphate-dissolvingbacteria in the rhizosphere of some cultivated legumesrdquo Plantand Soil vol 35 no 1 pp 127ndash132 1971

[39] A Beneduzi D Peres L K Vargas M H Bodanese-Zanettiniand LM P Passaglia ldquoEvaluation of genetic diversity and plantgrowth promoting activities of nitrogen-fixing bacilli isolatedfrom rice fields in South Brazilrdquo Applied Soil Ecology vol 39no 3 pp 311ndash320 2008

[40] M P Raimam U Albino M F Cruz et al ldquoInteraction amongfree-living N-fixing bacteria isolated from Drosera villosa varvillosa and AM fungi (Glomus clarum) in rice (Oryza sativa)rdquoApplied Soil Ecology vol 35 no 1 pp 25ndash34 2007

[41] M R Swain S K Naskar and R C Ray ldquoIndole-3-acetic acidproduction and effect on sprouting of yam (Dioscorea rotundataL) minisetts by Bacillus subtilis isolated from culturable cow-dung microflorardquo Polish Journal of Microbiology vol 56 no 2pp 103ndash110 2007

[42] T M Tien M H Garkins and D H Hubbell ldquoPlant growthsubstances produced by Azospirillum brasilense and their effect

Advances in Agriculture 13

on the growth of pearl Millet Pennisetum americanum (L)rdquoApplied and EnvironmentalMicrobiology vol 37 no 5 pp 1016ndash1024 1979

[43] A Esitken L Pirlak M Turan and F Sahin ldquoEffects of floraland foliar application of plant growth promoting rhizobacteria(PGPR) on yield growth and nutrition of sweet cherryrdquo ScientiaHorticulturae vol 110 no 4 pp 324ndash327 2006

[44] H Karlidag A Esitken M Turan and F Sahin ldquoEffects of rootinoculation of plant growth promoting rhizobacteria (PGPR)on yield growth and nutrient element contents of leaves ofapplerdquo Scientia Horticulturae vol 114 no 1 pp 16ndash20 2007

[45] C S Nautiyal S Bhadauria P Kumar H Lal R Mondal andD Verma ldquoStress induced phosphate solubilization in bacteriaisolated fromalkaline soilsrdquoFEMSMicrobiology Letters vol 182no 2 pp 291ndash296 2000

[46] A K Halder A K Mishra P Bhattacharya and P KChakrabarthy ldquoSolubilization of inorganic phosphates byBradyrhizobiumrdquo Indian Journal of Experimental Biology vol29 pp 28ndash31 1991

Submit your manuscripts athttpwwwhindawicom

Nutrition and Metabolism

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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International Journal of

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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

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Applied ampEnvironmentalSoil Science

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AgricultureAdvances in

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PsycheHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BiodiversityInternational Journal of

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ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

GenomicsInternational Journal of

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Plant GenomicsInternational Journal of

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Biotechnology Research International

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Page 6: BalayoganSivasankariandMarimuthuAnandharajdownloads.hindawi.com/journals/aag/2014/248591.pdf · In Vitro Nitrogen Fixing Activity. e bacterial isolates were screened for nitrogen

6 Advances in Agriculture

Bacillus cereus strain GGBSTD2 16S ribosomal RNA gene

Bacillus cereus strain SU-6 16S ribosomal RNA gene

Bacillus cereus strain JB0013 16S ribosomal RNA gene

Bacillus cereus isolate PN24 16S ribosomal RNA gene

Bacillus cereus strain JB0005 16S ribosomal RNA gene

Bacillus sp G2DM-85 16S ribosomal RNA gene

Bacillus cereus strain JBE0009 16S ribosomal RNA gene

Bacillus sp EB460 16S ribosomal RNA gene

Bacillus cereus strain 421-3R 16S ribosomal RNA gene

Bacillus sp EB447 16S ribosomal RNA gene

Bacillus sp EB470 16S ribosomal RNA gene

Bacillus sp EB421 16S ribosomal RNA gene

Bacillus sp EB431 16S ribosomal RNA gene

Bacillus sp EB462 16S ribosomal RNA gene

Bacillus sp EB429 16S ribosomal RNA gene

Bacillus cereus strain 421-4R 16S ribosomal RNA gene

Bacillus sp EB480 16S ribosomal RNA gene

Bacillus sp EB13 16S ribosomal RNA gene

Uncultured compost bacterium partial 16S rRNA gene

Bacillus thuringiensis gene for 16S rRNA

Figure 1 Neighbor joining phylogenetic tree of full 16S rRNA gene sequences showing the relationship among Bacillus cereus GGBSTD1and representatives of some related taxa The sequence data for several closely related Bacillus cultures were recovered from GenBank andincluded in the tree

Table 2 The phosphate solubilizing activity phytohormone (IAA) production and siderophores production of the bacterial isolates

Name of the organism Phosphate solubilization index (SI) IAA production (120583gmL)a Siderophores productionb

Bacillus spp 3855 242 +++Alcaligenes spp 2930 147 minus

Erwinia spp 2890 113 +Serratia spp 3045 168 ++Pseudomonas spp 3522 196 +++aIndole acetic acid production in culture media supplemented with tryptophan (5 120583gmL) after 5 daysbIn vitro siderophores production minus represents the absence of siderophores production and +++ represents gt10mm wide yellow-orange zone

is given in Tables 3 and 4 respectively The shoot length(1147 plusmn 006 cm) root length (1027 plusmn 006 cm) and leafarea (905 plusmn 026 cm2) of the plant get significantly (119875 lt0001) increased in Exp3 (consortium of Bacillus cereus +Pseudomonas spp [1 1]) at 5mL concentration on the 15thday compared to other experimentsThe chlorophyll a b andtotal chlorophyll content of the leaves on d 5 d 10 and d 15are given in Table 5 The chlorophyll a (010 plusmn 009mgg offresh leaf) chlorophyll b (1085 plusmn 016mgg of fresh leaf)and total chlorophyll (1095plusmn007mgg of fresh leaf) content

of leaves were significantly (119875 lt 0001) higher in Exp3(consortium of Bacillus cereus + Pseudomonas spp (1 1))at 5mL concentration on the 15th day compared to otherexperiments Observations on the fresh weight of the wholeplant are given in Table 6 The fresh weight of the wholeplant (092 plusmn 000 g) was significantly (119875 lt 0001) higherin Exp3 (consortium of Bacillus cereus + Pseudomonas spp(1 1)) at 5mL concentration on the 15th day compared toother experiments Observations on the dry weight of thewhole plant are given in Table 6 The dry weight of the whole

Advances in Agriculture 7

Pseudomonas fluorescens strain LMG 5825 16S ribosomal RNA gene

Pseudomonas fluorescens strain LMG 5829 16S ribosomal RNA gene

Pseudomonas fluorescens strain LMG 14676 16S ribosomal RNA gene

Pseudomonas sp W15Feb13 16S ribosomal RNA gene

Pseudomonas sp W15Feb28 16S ribosomal RNA gene

Pseudomonas sp p50 16S ribosomal RNA gene

Uncultured bacterium clone 661193 16S ribosomal RNA gene

Pseudomonas sp BSs20166 16S ribosomal RNA gene

Pseudomonas sp 28zhy partial 16S rRNA gene

Uncultured bacterium clone AYRV1-027 16S ribosomal RNA gene

Uncultured Pseudomonadaceae bacterium clone290cost002-P3L-2032 16S ribosomal RNA gene

Pseudomonas sp GGBSTD3 16S ribosomal RNA gene

Pseudomonas sp DK3 16S ribosomal RNA gene

Pseudomonas reactans strain AMP-13 16S ribosomal RNA gene

Figure 2 Neighbor joining phylogenetic tree of full 16S rRNA gene sequences showing the relationship among Pseudomonas spp GGBSTD3and representatives of some related taxa The sequence data for several closely related Pseudomonas cultures were recovered from GenBankand included in the tree

plant (009 plusmn 000 g) was significantly (119875 lt 0001) higherin Exp3 (consortium of Bacillus cereus + Pseudomonas spp(1 1)) at 5mL concentration on the 15th day compared toother experiments

4 Discussion

Inoculation of these newly identified nitrogen fixing andphosphate solubilizingmicrobes in the plant growthmediumenhanced various physiological activities culminating inhigher biomass production in V unguiculata Since this is anonhazardous way of fertilization of crop plants it is veryrelevant to a developing country like India This techniquecan save the farmers of India from the problem posed byhigh cost of fertilizers by offering a comparatively inexpensivealternative (biofertilizers) while additionally preventing thedegradation of the soil and thereby ensuring sustainable agri-culture Bess (1999) reported that the content in compostingneeds to be determined through the concentration of sixfunctional groups of microorganisms such as aerobic bacte-ria anaerobic bacteria fungi actinomycetes Pseudomonasand nitrogen fixing bacteria [29] Now there are ways toevaluate the concentrations of these organisms in the finishedcompost and that can serve as an interpretation guide to

determine the quality of the compost as an inoculant of soilmicroorganisms Grafff (1981) and Atlavinyte have reporteda higher P content in the cast than in the surrounding soil[30] Satchell (1983) has linked the phosphatase activity inthe gut of worms to the availability of bound P in soils Thebacterial strains which have the capability to fix nitrogenonly can grow in the nitrogen-free medium [31] Hencethe nitrogen fixing ability was tested in the present studybased on the growth of bacterial strain in nitrogen- freemedium Five bacteria strains showedmore nitrogen fixationamong all the five bacterial strains tested only one strain (ieBacillus spp) showed significantly more turbidity comparedto the other strains The ultimate source of the nitrogen usedby plants is N

2gas which constitutes 78 of the Earthrsquos

atmosphere Unfortunately higher plants cannot metabolizeN2directly into protein N

2gas must be converted to a plant

available form [32] isolated many species of nitrogen fixingBacillus (B megaterium B cereus B subtilis B licheniformisand B azotoformans) from the rhizospheric zone of ricefields in Central China The widely studied Bacillus genusrepresents one of the most diverse genera in the bacilli groupNumerous Bacillus and Paenibacillus strains express plantgrowth promoting (PGP) activities and a number of thesestrains have already been commercially developed as biolog-ical fungicides insecticides and nematicides or generic plant

8 Advances in Agriculture

Table 3 Shoot length and Root length ofVigna unguiculata grown on vermiculite supplemented with Bacillus cereus with Pseudomonas sppand with their consortium (1 1) at five different concentrations (1 2 3 4 and 5mL) on the 5th the 10th and the 15th day and in the control

Experimentsa Quantity of bacterial cultures added Shoot length (cm)b Root length (cm)b

5th day 10th day 15th day 5th day 10th day 15th day

Exp0

1mL 297 (ab) 397 (a) 497 (ab) 197 (de) 283 (ab) 397 (bcd)2mL 303 (bcd) 403 (ab) 500 (ab) 200 (abc) 287 (de) 400 (a)3mL 310 (abc) 407 (a) 503 (ab) 203 (de) 290 (abc) 403 (ab)4mL 310 (bcd) 410 (bcd) 507 (bcd) 207 (a) 297 (de) 410 (abc)5mL 313 (a) 413 (ab) 510 (ab) 210 (bcd) 300 (ab) 413 (de)

Exp1

1mL 340 (ab) 440 (abc) 540 (ab) 233 (abc) 327 (bcd) 433 (f)2mL 450 (ab) 540 (abc) 647 (ab) 333 (ab) 430 (a) 540 (abc)3mL 550 (de) 630 (ab) 750 (a) 440 (de) 550 (abc) 620 (a)4mL 653 (ab) 730 (a) 833 (ab) 550 (bcd) 653 (de) 750 (abc)5mL 740 (ab) 840 (abc) 923 (f) 640 (abc) 753 (ab) 827 (ab)

Exp2

1mL 277 (bcd) 357 (f) 440 (de) 160 (de) 217 (abc) 350 (f)2mL 343 (abc) 447 (ab) 553 (ab) 253 (abc) 340 (bcd) 427 (bcd)3mL 437 (de) 543 (abc) 633 (abc) 340 (ab) 443 (f) 533 (ab)4mL 547 (bcd) 647 (de) 733 (a) 420 (bcd) 550 (ab) 633 (ab)5mL 640 (f) 733 (bcd) 833 (bcd) 533 (ab) 630 (ab) 730 (bcd)

Exp3

1mL 530 (bcd) 647 (ab) 730 (ab) 433 (de) 550 (abc) 620 (ab)2mL 643 (ab) 740 (bcd) 827 (f) 547 (a) 630 (de) 733 (ab)3mL 737 (bcd) 840 (bcd) 927 (ab) 640 (ab) 740 (bcd) 840 (bcd)4mL 847 (abc) 930 (a) 1043 (abc) 740 (f) 833 (a) 937 (abc)5mL 957 (ab) 1030 (f) 1147 (bcd) 833 (abc) 940 (ab) 1027 (bcd)

aExp0 nutrient solution (control) Exp1 Bacillus cereus Exp2 Pseudomonas spp Exp3 consortium of Bacillus cereus + Pseudomonas spp (1 1)bMeanof two repeated experiments (30 plants)Different letterswithin parenthesis indicate significant difference between treatments for each growth parameterusing Duncanrsquos multiple range test (119875 = 005)

growth promoters The use of these strains in agriculturehas recently been reviewed [33]Those strains besides havingseveral PGP properties can also fix nitrogen and also dophosphate solubilization The symbiotic fixation of nitrogenthrough inoculation of legume crops with effective rhizobiais well known [34] Asymbiotic nitrogen fixing bacteriawhich live in the rhizosphere andor endophytically oftenincrease yields of crops Many bacterial species have nitrogenfixing properties including Bacillus spp Azotobacter sppAzospirillum spp Beijerinckia spp and Pseudomonas spp[35] Nitrogen fixing bacteria have been used in foliar appli-cations inmulberryThe solubilization index (SI) was formeddue to solubilization of insoluble phosphates by organic acidsecretion [36] El-Komy (2005) indicated that Pseudomonasfluorescence and Bacillus megaterium strains were the mostpowerful phosphate solubilizers on PVK plates as well as onPikovskayarsquos broth [37] The P concentration in Pikovskayarsquosbroth increased gradually achieving a peak on the sixth dayand declined slowly during the late days In this study pHvalues decreased gradually in PVK broth during the earlyincubation days and no revival was observed during laterdays for all the tested bacterial strains Several bacteria par-ticularly those belonging to the genus Bacillus spp convertinsoluble phosphate into soluble forms by secreting organicacids such as formic acid acetic acid propionic acid citricacid fumaric acid gluconic acid glyoxylic acid ketobutyric

acid malonic acid succinic acid and tartaric acid Theseacids lower the pH and bring about the dissolution of boundforms of phosphate Some of the hydroxyl acids may chelatewith calcium and iron resulting in effective solubilization andutilization of phosphates [38]

Beneduzi et al (2008) had isolated the plant growthpromoting strain SVPR30 and identified by 16S rRNA genesequence as Bacillus spp and they had tested it for plantgrowth through in vivo experiments [39] This strain wascharacterized as a high IAA producer able to solubilize phos-phate and also fix a considerably high amount of nitrogenThe inoculation of rice with Bacillus spp SVPR30 strainshowed a significant increase in the root and shoot partswhen compared with the controls within 15 and 30 daysafter sprouting Numerous Bacillus and Paenibacillus strainsexpressed plant growth promoting (PGP) activities and anumber of these strains have been commercially developedas generic plant growth promotersThe use of these strains inagriculture has recently been reviewed [33]

Various reports indicate that coinoculation of beneficialorganisms generally increased plant growth relative to singleinoculation with a sole beneficial organism [40] Most ofthe effects of the individual microorganisms coinoculationare additive although a synergistic effect has been reportedin some cases The average root to shoot ratio was higherin B subtilis treated yam minisetts in comparison to those

Advances in Agriculture 9

Table 4 Leaf area of Vigna unguiculata grown on vermiculite supplemented with Bacillus cereus with Pseudomonas spp and with theirconsortium (1 1) at five different concentrations (1 2 3 4 and 5mL) on the 5th the 10th and the 15th days and in the control

Experimentsa Quantity of bacterial cultures added Leaf area (cm2)b

5th day 10th day 15th day

Exp0

1mL 027 (ab) 094 (abc) 407 (b)2mL 035 (ab) 102 (ab) 419 (abc)3mL 040 (abc) 125 (ab) 471 (bcd)4mL 048 (ab) 138 (abc) 510 (ab)5mL 054 (abc) 161 (b) 538 (b)

Exp1

1mL 018 (bcd) 123 (b) 601 (abc)2mL 025 (ab) 138 (abc) 632 (ab)3mL 033 (abc) 150 (ab) 684 (b)4mL 038 (ab) 168 (ab) 686 (abc)5mL 044 (abc) 191 (bcd) 733 (ab)

Exp2

1mL 011 (b) 107 (ab) 534 (b)2mL 014 (ab) 119 (ab) 559 (ab)3mL 017 (abc) 127 (ab) 579 (ab)4mL 021 (ab) 153 (abc) 631 (abc)5mL 026 (ab) 166 (b) 667 (b)

Exp3

1mL 026 (bcd) 297 (ab) 753 (bcd)2mL 033 (b) 325 (ab) 787 (ab)3mL 040 (ab) 350 (bcd) 811 (bcd)4mL 044 (b) 384 (b) 861 (b)5mL 062 (ab) 400 (bcd) 905 (ab)

aExp0 nutrient solution (control) Exp1 Bacillus cereus Exp2 Pseudomonas spp Exp3 consortium of Bacillus cereus + Pseudomonas spp (1 1)bMeanof two repeated experiments (30 plants)Different letterswithin parenthesis indicate significant difference between treatments for each growth parameterusing Duncanrsquos multiple range test (119875 = 005)

0102030405060708090

100

Concentration of bacterial cultures (mL)1 2 3 4 5

Germination ()

Exp0

Exp1 Exp3

Exp2

Figure 3 Germination percentage ofVigna unguiculata seeds sownin vermiculite supplementedwithBacillus cereus with Pseudomonasspp and with their consortium at five different concentrations (1 23 4 and 5mL) and in the control Exp0 nutrient solution (control)Exp1 Bacillus cereus Exp2 Pseudomonas spp Exp3 consortium ofBacillus cereus + Pseudomonas spp (1 1)

not treated with the bacterial culture [41] In earlier reportsroot elongation was found to occur in Sesbania aculeata byinoculation with Azotobacter spp and Pseudomonas spp in

Vigna radiata by Pseudomonas putida [16] and in Pennisetumamericanum by Azospirillum brasilense [42] Esitken et al(2006) had found out that Bacillus OSU-142 and Pseu-domonas BA-8 alone or in combination had a great potentialto increase the growth yield and nutrition of sweet cherryplant [43] BacillusM3 BacillusOSU-142 andMicobacteriumFS01 separately or in combination were found to have greatpotential for use as plant growth promoting rhizobacteriato increase production in apples and in many other crops[44] These phosphate solubilizing microbes consist pre-dominantly of fungal bacterial and actinomycetes speciescollectively called phosphate solubilizing microorganismsmdashPSM [45] There are several microorganisms which canalso solubilize the cheaper sources of phosphorous such asrock phosphate Bacteria such as Bacillus are widely usedin plant production system and are important phosphorussolubilizing microorganisms resulting in improved growthyield of crops andmetabolic activities especially in synthesisof protein [34 46]

5 Conclusions

This study illustrates the isolation of plant growth promotingbacteria from vermisources screening under in vitro con-ditions for multiple PGPR traits and their evaluation undercontrolled conditions in a pot experiment The findings ofthis study indicated that the consortium of B cereus and

10 Advances in Agriculture

Table5Ch

loroph

yllcon

tent

ofleavesofVignaun

guicu

latagrow

non

verm

iculite

supp

lementedwith

Bacilluscereuswith

Pseudomonas

sppand

with

theircon

sortium

(11)atfi

vedifferent

concentrations

(1234and

5mL)

onthe5

thdaythe10thdayandthe15thdayandin

thec

ontro

l

Experim

entsa

Quantity

ofbacterial

Cultu

resa

dded

Chloroph

yllcon

tent

(mggof

fresh

leaf)b

5thday

10th

day

15th

day

Chloroph

ylla

Chloroph

yllb

Total

chloroph

yll

Chloroph

ylla

Chloroph

yllb

Total

chloroph

yll

Chloroph

ylla

Chloroph

yllb

Total

chloroph

yll

Exp0

1mL

021

(ab)

285

(abc)

307

(a)

020

(abc)

481

(a)

501

(ab)

015

(bcd)

612

(abc)

627

(bcd)

2mL

022

(abc)

293

(ab)

315

(abc)

021

(ab)

489

(ab)

510

(bcd)

012

(abc)

618

(ab)

630

(ab)

3mL

023

(abc)

301

(ab)

315

(abc)

022

(bcd)

497

(bcd)

518

(a)

014

(a)

633

(cde)

647

(bcd)

4mL

024

(ab)

308

(f)

332

(a)

022

(a)

504

(b)

527

(abc)

015

(bcd)

641

(cde)

656

(a)

5mL

025

(f)

316

(abc)

341

(bcd)

019

(ab)

511(ab)

530

(abc)

011(cde)

645

(ab)

650

(abc)

Exp1

1mL

035

(b)

400

(cde)

435

(abc)

011(b)

581

(cde)

593

(cde)

010

(abc)

704(abc)

715(f)

2mL

026

(abc)

462

(bcd)

488

(f)

011(abc)

647

(b)

658

(abc)

006

(ab)

779(bcd)

785(a)

3mL

021

(bcd)

526

(abc)

547

(ab)

008

(bcd)

718(ab)

726(bcd)

006

(f)

847

(f)

853

(ab)

4mL

008

(f)

587

(cde)

595

(b)

011(bcd)

778(abc)

789(abc)

004

(bcd)

933(b)

937(bcd)

5mL

026

(abc)

641

(bcd)

667

(abc)

025

(cde)

834

(cde)

859

(f)

007

(abc)

976(cde)

983(abc)

Exp2

1mL

030

(abc)

354

(ab)

384

(b)

012

(a)

545

(abc)

556

(ab)

007

(bcd)

681

(abc)

688

(bcd)

2mL

029

(bcd)

420

(f)

449

(abc)

011(bcd)

610

(ab)

621

(abc)

007

(abc)

744(f)

752(bcd)

3mL

022

(ab)

504

(abc)

527

(bcd)

015

(a)

677

(cde)

692

(bcd)

006

(bcd)

812

(ab)

817

(bcd)

4mL

010

(f)

566

(a)

576

(cde)

018

(abc)

744(f)

762(ab)

005

(ab)

885

(a)

890

(abc)

5mL

006

(bcd)

622

(abc)

628

(abc)

018

(b)

810

(abc)

828

(a)

004

(cde)

948(a)

952(f)

Exp3

1mL

027

(ab)

470

(bcd)

496

(cde)

009

(ab)

668

(b)

677

(cde)

010

(a)

814

(ab)

825

(a)

2mL

020

(ab)

548

(a)

568

(f)

003

(bcd)

746(bcd)

749(ab)

009

(ab)

887

(f)

897

(ab)

3mL

005

(bcd)

584

(abc)

589

(ab)

008

(bcd)

804

(abc)

812

(cde)

010

(ab)

953(abc)

963(bcd)

4mL

023

(f)

663

(ab)

686

(cde)

007

(ab)

870

(b)

877

(f)

010

(abc)

1019

(bcd)

1029(f)

5mL

022

(ab)

744(cde)

766(a)

009

(a)

932(abc)

940(ab)

010

(a)

1085(ab)

1095(abc)

a Exp0nu

trient

solutio

n(con

trol)

Exp1B

acillus

cereusE

xp2Pseudomonas

sppE

xp3consortiu

mof

Bacilluscereus+

Pseudomonas

spp(11)

b Meanof

tworepeated

experim

ents(30plants)

Differentletterswith

inparenthesis

indicatesig

nificantd

ifference

betweentre

atmentsfore

achgrow

thparameter

usingDun

canrsquos

multip

lerangetest(119875=005)

Advances in Agriculture 11

Table 6 Fresh weight and dry weight of whole plant of Vigna unguiculata grown on vermiculite supplemented with Bacillus cereus withPseudomonas spp and with their consortium (1 1) at five different concentrations (1 2 3 4 and 5mL) on the 5th the 10th and the 15 daysand in the control

Experimentsa Quantity of bacterial cultures added Fresh weight of the whole plant (g)b Dry weight of the whole plant (g)b

5th day 10th day 15th day 5th day 10th day 15th day

Exp0

1mL 058 (bcd) 061 (abc) 064 (ab) 006 (a) 006 (abc) 006 (ab)2mL 058 (ab) 061 (a) 064 (abc) 006 (bcd) 006 (ab) 006 (ab)3mL 058 (abc) 061 (ab) 064 (bcd) 006 (abc) 006 (bcd) 006 (b)4mL 041 (abc) 061 (ab) 064 (ab) 006 (abc) 006 (ab) 006 (abc)5mL 058 (abc) 061 (abc) 064 (bcd) 006 (a) 006 (bcd) 006 (def)

Exp1

1mL 062 (abc) 065 (abc) 068 (ab) 006 (bcd) 006 (ab) 007 (bcd)2mL 065 (bcd) 068 (bcd) 071 (def) 006 (abc) 007 (a) 007 (ab)3mL 068 (abc) 071 (def) 074 (bcd) 007 (abc) 007 (ab) 007 (abc)4mL 071 (ab) 074 (abc) 077 (abc) 007 (bcd) 007 (b) 008 (bcd)5mL 074 (abc) 077 (def) 080 (ab) 007 (def) 008 (a) 008 (b)

Exp2

1mL 056 (ab) 059 (bcd) 062 (ab) 006 (ab) 006 (ab) 006 (abc)2mL 059 (abc) 062 (abc) 065 (def) 006 (ab) 006 (abc) 006 (bcd)3mL 062 (bcd) 065 (a) 068 (bcd) 006 (abc) 006 (bcd) 007 (abc)4mL 065 (abc) 068 (ab) 071 (b) 006 (ab) 007 (abc) 007 (b)5mL 068 (abc) 071 (b) 074 (a) 007 (def) 007 (abc) 007 (bcd)

Exp3

1mL 074 (ab) 077 (ab) 080 (abc) 007 (a) 008 (bcd) 008 (ab)2mL 077 (bcd) 080 (a) 083 (bcd) 008 (a) 008 (bcd) 008 (abc)3mL 080 (bcd) 083 (bcd) 086 (abc) 008 (ab) 008 (b) 009 (b)4mL 083 (bcd) 086 (abc) 089 (ab) 008 (abc) 009 (ab) 009 (bcd)5mL 086 (ab) 089 (b) 092 (def) 009 (bcd) 009 (bcd 009 (b)

aExp0 nutrient solution (control) Exp1 Bacillus cereus Exp2 Pseudomonas spp Exp3 consortium of Bacillus cereus + Pseudomonas spp (1 1)bMeanof two repeated experiments (30 plants)Different letterswithin parenthesis indicate significant difference between treatments for each growth parameterusing Duncanrsquos multiple range test (119875 = 005)

Pseudomonas spp acts as a potential biofertilizer due to itsvarious plant growth promotion abilities such as solubiliza-tion of phosphate and production of IAA and siderophore itcan be developed as potential microbial inoculant for variouscrops

Conflict of Interests

The authors declared that there is no conflict of interestsregarding this paper

Acknowledgments

Theauthors are thankful to theDepartment of BiologyGand-higram Rural InstitutemdashDeemed University GandhigramThis research was funded by University Grants Commissionunder UGC Research Fellowship in Science for MeritoriousStudents (BSR) Grant no F4-12008

References

[1] U S Bhawalkar Vermiculture Ecotechnology EarthwormResearch Institute Pune India 2nd edition 1996

[2] S Kiss D M Bularda and D Radulescu ldquoBiological Signifi-cance of Enzymes Accumulated in SoilrdquoAdvances in Agronomyvol 27 pp 25ndash87 1975

[3] D L Karlen M J Mausbach J W Doran R G Cline R FHarris and G E Schuman ldquoSoil quality a concept definitionand framework for evaluationrdquo Soil Science Society of AmericaJournal vol 61 no 1 pp 4ndash10 1997

[4] R M Atiyeh C A Edwards S Subler and J Metzger ldquoEarth-worm processed organic wastes as components of horticulturalpotting media for growing manifold and vegetable seedlingsrdquoCompost Science and Utilization vol 8 no 3 pp 215ndash223 2000

[5] N Vassilev and M Vassileva ldquoBiotechnological solubilizationof rock phosphate on media containing agro-industrial wastesrdquoApplied Microbiology and Biotechnology vol 61 no 5-6 pp435ndash440 2003

[6] S A Omar ldquoThe role of rock-phosphate-solubilizing fungiand vesicular-arbusular-mycorrhiza (VAM) in growth of wheatplants fertilized with rock phosphaterdquo World Journal of Micro-biology and Biotechnology vol 14 no 2 pp 211ndash218 1998

[7] J A J Paul and T Daniel ldquoStandardization of sampling methodfor physical characterization of municipal solid wasterdquo AsianJournal of Water Environment and Pollution vol 5 no 1 pp95ndash98 2007

[8] R Lalloo D Maharajh J Gorgens and N Gardiner ldquoA down-stream process for production of a viable and stable Bacilluscereus aquaculture biological agentrdquo Applied Microbiology andBiotechnology vol 86 no 2 pp 499ndash508 2010

[9] B Sivasankari N Kavikumar and M Anandharaj ldquoIndole-3-acetic acid production and enhanced Plant growth promotionby Indigenous bacterial speciesrdquo Journal of Current Research inScience vol 1 no 5 pp 331ndash335 2013

12 Advances in Agriculture

[10] J Han L Sun X Dong et al ldquoCharacterization of a novel plantgrowth-promoting bacteria strain Delftia tsuruhatensis HR4both as a diazotroph and a potential biocontrol agent againstvarious plant pathogensrdquo Systematic and Applied Microbiologyvol 28 no 1 pp 66ndash76 2005

[11] H Rodrıguez and R Fraga ldquoPhosphate solubilizing bacte-ria and their role in plant growth promotionrdquo BiotechnologyAdvances vol 17 no 4-5 pp 319ndash339 1999

[12] T Daniel and N Karmegam ldquoBio-conversion of selected leaflitters using an African epigeic earthworm Eudrilus eugeniaerdquoEcology Environment and Conservation vol 5 no 3 pp 271ndash275 1999

[13] J Dobereiner I E Marriel and M Nery ldquoEcological distri-bution of Spirillum lipoferum Beijerinckrdquo Canadian Journal ofMicrobiology vol 22 no 10 pp 1464ndash1473 1976

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

[15] M Edi Premono A M Moawad and P L G Vlek ldquoEffect ofphosphate-solubilizing Pseudomonas putida on the growth ofmaize and its survival in the rhizosphererdquo Indonesian Journal ofCrop Science vol 11 pp 13ndash23 1996

[16] C L Patten and B R Glick ldquoBacterial biosynthesis of indole-3-acetic acidrdquo Canadian Journal of Microbiology vol 42 no 3pp 207ndash220 1996

[17] N Amaresan V Jayakumar K Kumar and N ThajuddinldquoIsolation and characterization of plant growth promotingendophytic bacteria and their effect on tomato (Lycopersiconesculentum) and chilli (Capsicum annuum) seedling growthrdquoAnnals of Microbiology vol 62 no 2 pp 805ndash810 2012

[18] B Schwyn and J B Neilands ldquoUniversal chemical assay forthe detection and determination of siderophoresrdquo AnalyticalBiochemistry vol 160 no 1 pp 47ndash56 1987

[19] P Rahi P Vyas S Sharma and A Gulati ldquoPlant growthpromoting potential of the fungus Discosia sp FIHB 571 fromtea rhizosphere tested on chickpea maize and peardquo IndianJournal of Microbiology vol 49 no 2 pp 128ndash133 2009

[20] K Kumar N Amaresan S Bhagat K Madhuri and R CSrivastava ldquoIsolation and characterization of rhizobacteriaassociated with coastal agricultural ecosystem of rhizospheresoils of cultivated vegetable cropsrdquo World Journal of Microbiol-ogy and Biotechnology vol 27 no 7 pp 1625ndash1632 2011

[21] J A Smoker and S R Barnum ldquoRapid small scale DNAisolation from filamentous cyanobacteriardquo FEMS MicrobiologyLetters vol 56 no 1 pp 119ndash122 1988

[22] U Edwars T Rogall H Blocker M Emde and E C BottgerldquoIsolation and direct complete nucleotide determination ofentire genes Characterization of a gene coding for 16S riboso-mal RNArdquoNucleic Acids Research vol 17 no 19 pp 7843ndash78531989

[23] D G Higgins A J Bleasby and R Fuchs ldquoCLUSTAL Vimproved software for multiple sequence alignmentrdquo ComputerApplications in the Biosciences vol 8 no 2 pp 189ndash191 1992

[24] M Kimura ldquoA simple method for estimating evolutionary ratesof base substitutions through comparative studies of nucleotidesequencesrdquo Journal ofMolecular Evolution vol 16 no 2 pp 111ndash120 1980

[25] N Saitou and M Nei ldquoThe neighbor-joining method a newmethod for reconstructing phylogenetic treesrdquo Molecular Biol-ogy and Evolution vol 4 no 4 pp 406ndash425 1987

[26] E Barbu K Y Lee and R Wahl ldquoContent of purine andpyrimidine base in desoxyribonucleic acid of bacteriardquo Annalesde lrsquoInstitut Pasteur vol 91 no 2 pp 212ndash224 1956

[27] N Sueoka ldquoVariation and heterogeneity of base compositionof deoxyribonucleic acids a compilation of old and new datardquoJournal of Molecular Biology vol 3 no 1 pp 31ndash40 1961

[28] H Hori and S Osawa ldquoEvolutionary change in 5S rRNAsecondary structure and a phylogenic tree of 352 5S rRNAspeciesrdquo BioSystems vol 19 no 3 pp 163ndash172 1986

[29] V Bess ldquoEvaluating microbiology of compost microbial con-tent of composting is helping the producers and growers tounderstand its role as a soil inoculant and plantrdquo BioCycleMagazine p 62 1999

[30] O Grafff ldquoPreliminary experiments of vermin composting ofdifferent waste materials using Eudrilus eugeniae Kinbergrdquo inWorkshop on the Role of Earthworms in the Stabilization ofOrganic Residues M Happelhof Ed pp 178ndash191 Beech LeafPress Kalamazoo Mich USA 1981

[31] J E Satchell Earthworm Ecology from Darwin to VermicultureChapman and Hall 1983

[32] G H Xie M Y Cai G C Tao and Y Steinberger ldquoCultivableheterotrophic N2-fixing bacterial diversity in rice fields in theYangtze River Plainrdquo Biology and Fertility of Soils vol 37 no 1pp 29ndash38 2003

[33] B BMcSpaddenGardener ldquoEcology ofBacillus and Paenibacil-lus spp in agricultural systemsrdquo Phytopathology vol 94 no 11pp 1252ndash1258 2004

[34] J Dobereiner ldquoBiological nitrogen fixation in the tropics socialand economic contributionsrdquo Soil Biology andBiochemistry vol29 no 5-6 pp 771ndash774 1997

[35] V M Reis F L Olivares and J Dobereiner ldquoImprovedmethodology for isolation of Acetobacter diazotrophicus andconfirmation of its endophytic habitatrdquoWorld Journal of Micro-biology and Biotechnology vol 10 no 4 pp 401ndash405 1994

[36] S Khalil N Ayub S Alam and F Latif ldquoOrganic acids pro-duction and phosphate solubilization by phosphate solubilizingmicroorganisms (PSM) under in vitro conditionsrdquo PakistanJournal of Biological Sciences vol 7 no 2 pp 187ndash196 2004

[37] H M A El-Komy ldquoCoimmobilization of Azospirillumlipoferum and Bacillus megaterium for successful phosphorusand nitrogen nutrition of wheat plantsrdquo Food Technology andBiotechnology vol 43 no 1 pp 19ndash27 2005

[38] N B Paul and W V B Sundara Rao ldquoPhosphate-dissolvingbacteria in the rhizosphere of some cultivated legumesrdquo Plantand Soil vol 35 no 1 pp 127ndash132 1971

[39] A Beneduzi D Peres L K Vargas M H Bodanese-Zanettiniand LM P Passaglia ldquoEvaluation of genetic diversity and plantgrowth promoting activities of nitrogen-fixing bacilli isolatedfrom rice fields in South Brazilrdquo Applied Soil Ecology vol 39no 3 pp 311ndash320 2008

[40] M P Raimam U Albino M F Cruz et al ldquoInteraction amongfree-living N-fixing bacteria isolated from Drosera villosa varvillosa and AM fungi (Glomus clarum) in rice (Oryza sativa)rdquoApplied Soil Ecology vol 35 no 1 pp 25ndash34 2007

[41] M R Swain S K Naskar and R C Ray ldquoIndole-3-acetic acidproduction and effect on sprouting of yam (Dioscorea rotundataL) minisetts by Bacillus subtilis isolated from culturable cow-dung microflorardquo Polish Journal of Microbiology vol 56 no 2pp 103ndash110 2007

[42] T M Tien M H Garkins and D H Hubbell ldquoPlant growthsubstances produced by Azospirillum brasilense and their effect

Advances in Agriculture 13

on the growth of pearl Millet Pennisetum americanum (L)rdquoApplied and EnvironmentalMicrobiology vol 37 no 5 pp 1016ndash1024 1979

[43] A Esitken L Pirlak M Turan and F Sahin ldquoEffects of floraland foliar application of plant growth promoting rhizobacteria(PGPR) on yield growth and nutrition of sweet cherryrdquo ScientiaHorticulturae vol 110 no 4 pp 324ndash327 2006

[44] H Karlidag A Esitken M Turan and F Sahin ldquoEffects of rootinoculation of plant growth promoting rhizobacteria (PGPR)on yield growth and nutrient element contents of leaves ofapplerdquo Scientia Horticulturae vol 114 no 1 pp 16ndash20 2007

[45] C S Nautiyal S Bhadauria P Kumar H Lal R Mondal andD Verma ldquoStress induced phosphate solubilization in bacteriaisolated fromalkaline soilsrdquoFEMSMicrobiology Letters vol 182no 2 pp 291ndash296 2000

[46] A K Halder A K Mishra P Bhattacharya and P KChakrabarthy ldquoSolubilization of inorganic phosphates byBradyrhizobiumrdquo Indian Journal of Experimental Biology vol29 pp 28ndash31 1991

Submit your manuscripts athttpwwwhindawicom

Nutrition and Metabolism

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Food ScienceInternational Journal of

Agronomy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Applied ampEnvironmentalSoil Science

Volume 2014

AgricultureAdvances in

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PsycheHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BiodiversityInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Plant GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biotechnology Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Forestry ResearchInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of BotanyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

EcologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Veterinary Medicine International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Cell BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Page 7: BalayoganSivasankariandMarimuthuAnandharajdownloads.hindawi.com/journals/aag/2014/248591.pdf · In Vitro Nitrogen Fixing Activity. e bacterial isolates were screened for nitrogen

Advances in Agriculture 7

Pseudomonas fluorescens strain LMG 5825 16S ribosomal RNA gene

Pseudomonas fluorescens strain LMG 5829 16S ribosomal RNA gene

Pseudomonas fluorescens strain LMG 14676 16S ribosomal RNA gene

Pseudomonas sp W15Feb13 16S ribosomal RNA gene

Pseudomonas sp W15Feb28 16S ribosomal RNA gene

Pseudomonas sp p50 16S ribosomal RNA gene

Uncultured bacterium clone 661193 16S ribosomal RNA gene

Pseudomonas sp BSs20166 16S ribosomal RNA gene

Pseudomonas sp 28zhy partial 16S rRNA gene

Uncultured bacterium clone AYRV1-027 16S ribosomal RNA gene

Uncultured Pseudomonadaceae bacterium clone290cost002-P3L-2032 16S ribosomal RNA gene

Pseudomonas sp GGBSTD3 16S ribosomal RNA gene

Pseudomonas sp DK3 16S ribosomal RNA gene

Pseudomonas reactans strain AMP-13 16S ribosomal RNA gene

Figure 2 Neighbor joining phylogenetic tree of full 16S rRNA gene sequences showing the relationship among Pseudomonas spp GGBSTD3and representatives of some related taxa The sequence data for several closely related Pseudomonas cultures were recovered from GenBankand included in the tree

plant (009 plusmn 000 g) was significantly (119875 lt 0001) higherin Exp3 (consortium of Bacillus cereus + Pseudomonas spp(1 1)) at 5mL concentration on the 15th day compared toother experiments

4 Discussion

Inoculation of these newly identified nitrogen fixing andphosphate solubilizingmicrobes in the plant growthmediumenhanced various physiological activities culminating inhigher biomass production in V unguiculata Since this is anonhazardous way of fertilization of crop plants it is veryrelevant to a developing country like India This techniquecan save the farmers of India from the problem posed byhigh cost of fertilizers by offering a comparatively inexpensivealternative (biofertilizers) while additionally preventing thedegradation of the soil and thereby ensuring sustainable agri-culture Bess (1999) reported that the content in compostingneeds to be determined through the concentration of sixfunctional groups of microorganisms such as aerobic bacte-ria anaerobic bacteria fungi actinomycetes Pseudomonasand nitrogen fixing bacteria [29] Now there are ways toevaluate the concentrations of these organisms in the finishedcompost and that can serve as an interpretation guide to

determine the quality of the compost as an inoculant of soilmicroorganisms Grafff (1981) and Atlavinyte have reporteda higher P content in the cast than in the surrounding soil[30] Satchell (1983) has linked the phosphatase activity inthe gut of worms to the availability of bound P in soils Thebacterial strains which have the capability to fix nitrogenonly can grow in the nitrogen-free medium [31] Hencethe nitrogen fixing ability was tested in the present studybased on the growth of bacterial strain in nitrogen- freemedium Five bacteria strains showedmore nitrogen fixationamong all the five bacterial strains tested only one strain (ieBacillus spp) showed significantly more turbidity comparedto the other strains The ultimate source of the nitrogen usedby plants is N

2gas which constitutes 78 of the Earthrsquos

atmosphere Unfortunately higher plants cannot metabolizeN2directly into protein N

2gas must be converted to a plant

available form [32] isolated many species of nitrogen fixingBacillus (B megaterium B cereus B subtilis B licheniformisand B azotoformans) from the rhizospheric zone of ricefields in Central China The widely studied Bacillus genusrepresents one of the most diverse genera in the bacilli groupNumerous Bacillus and Paenibacillus strains express plantgrowth promoting (PGP) activities and a number of thesestrains have already been commercially developed as biolog-ical fungicides insecticides and nematicides or generic plant

8 Advances in Agriculture

Table 3 Shoot length and Root length ofVigna unguiculata grown on vermiculite supplemented with Bacillus cereus with Pseudomonas sppand with their consortium (1 1) at five different concentrations (1 2 3 4 and 5mL) on the 5th the 10th and the 15th day and in the control

Experimentsa Quantity of bacterial cultures added Shoot length (cm)b Root length (cm)b

5th day 10th day 15th day 5th day 10th day 15th day

Exp0

1mL 297 (ab) 397 (a) 497 (ab) 197 (de) 283 (ab) 397 (bcd)2mL 303 (bcd) 403 (ab) 500 (ab) 200 (abc) 287 (de) 400 (a)3mL 310 (abc) 407 (a) 503 (ab) 203 (de) 290 (abc) 403 (ab)4mL 310 (bcd) 410 (bcd) 507 (bcd) 207 (a) 297 (de) 410 (abc)5mL 313 (a) 413 (ab) 510 (ab) 210 (bcd) 300 (ab) 413 (de)

Exp1

1mL 340 (ab) 440 (abc) 540 (ab) 233 (abc) 327 (bcd) 433 (f)2mL 450 (ab) 540 (abc) 647 (ab) 333 (ab) 430 (a) 540 (abc)3mL 550 (de) 630 (ab) 750 (a) 440 (de) 550 (abc) 620 (a)4mL 653 (ab) 730 (a) 833 (ab) 550 (bcd) 653 (de) 750 (abc)5mL 740 (ab) 840 (abc) 923 (f) 640 (abc) 753 (ab) 827 (ab)

Exp2

1mL 277 (bcd) 357 (f) 440 (de) 160 (de) 217 (abc) 350 (f)2mL 343 (abc) 447 (ab) 553 (ab) 253 (abc) 340 (bcd) 427 (bcd)3mL 437 (de) 543 (abc) 633 (abc) 340 (ab) 443 (f) 533 (ab)4mL 547 (bcd) 647 (de) 733 (a) 420 (bcd) 550 (ab) 633 (ab)5mL 640 (f) 733 (bcd) 833 (bcd) 533 (ab) 630 (ab) 730 (bcd)

Exp3

1mL 530 (bcd) 647 (ab) 730 (ab) 433 (de) 550 (abc) 620 (ab)2mL 643 (ab) 740 (bcd) 827 (f) 547 (a) 630 (de) 733 (ab)3mL 737 (bcd) 840 (bcd) 927 (ab) 640 (ab) 740 (bcd) 840 (bcd)4mL 847 (abc) 930 (a) 1043 (abc) 740 (f) 833 (a) 937 (abc)5mL 957 (ab) 1030 (f) 1147 (bcd) 833 (abc) 940 (ab) 1027 (bcd)

aExp0 nutrient solution (control) Exp1 Bacillus cereus Exp2 Pseudomonas spp Exp3 consortium of Bacillus cereus + Pseudomonas spp (1 1)bMeanof two repeated experiments (30 plants)Different letterswithin parenthesis indicate significant difference between treatments for each growth parameterusing Duncanrsquos multiple range test (119875 = 005)

growth promoters The use of these strains in agriculturehas recently been reviewed [33]Those strains besides havingseveral PGP properties can also fix nitrogen and also dophosphate solubilization The symbiotic fixation of nitrogenthrough inoculation of legume crops with effective rhizobiais well known [34] Asymbiotic nitrogen fixing bacteriawhich live in the rhizosphere andor endophytically oftenincrease yields of crops Many bacterial species have nitrogenfixing properties including Bacillus spp Azotobacter sppAzospirillum spp Beijerinckia spp and Pseudomonas spp[35] Nitrogen fixing bacteria have been used in foliar appli-cations inmulberryThe solubilization index (SI) was formeddue to solubilization of insoluble phosphates by organic acidsecretion [36] El-Komy (2005) indicated that Pseudomonasfluorescence and Bacillus megaterium strains were the mostpowerful phosphate solubilizers on PVK plates as well as onPikovskayarsquos broth [37] The P concentration in Pikovskayarsquosbroth increased gradually achieving a peak on the sixth dayand declined slowly during the late days In this study pHvalues decreased gradually in PVK broth during the earlyincubation days and no revival was observed during laterdays for all the tested bacterial strains Several bacteria par-ticularly those belonging to the genus Bacillus spp convertinsoluble phosphate into soluble forms by secreting organicacids such as formic acid acetic acid propionic acid citricacid fumaric acid gluconic acid glyoxylic acid ketobutyric

acid malonic acid succinic acid and tartaric acid Theseacids lower the pH and bring about the dissolution of boundforms of phosphate Some of the hydroxyl acids may chelatewith calcium and iron resulting in effective solubilization andutilization of phosphates [38]

Beneduzi et al (2008) had isolated the plant growthpromoting strain SVPR30 and identified by 16S rRNA genesequence as Bacillus spp and they had tested it for plantgrowth through in vivo experiments [39] This strain wascharacterized as a high IAA producer able to solubilize phos-phate and also fix a considerably high amount of nitrogenThe inoculation of rice with Bacillus spp SVPR30 strainshowed a significant increase in the root and shoot partswhen compared with the controls within 15 and 30 daysafter sprouting Numerous Bacillus and Paenibacillus strainsexpressed plant growth promoting (PGP) activities and anumber of these strains have been commercially developedas generic plant growth promotersThe use of these strains inagriculture has recently been reviewed [33]

Various reports indicate that coinoculation of beneficialorganisms generally increased plant growth relative to singleinoculation with a sole beneficial organism [40] Most ofthe effects of the individual microorganisms coinoculationare additive although a synergistic effect has been reportedin some cases The average root to shoot ratio was higherin B subtilis treated yam minisetts in comparison to those

Advances in Agriculture 9

Table 4 Leaf area of Vigna unguiculata grown on vermiculite supplemented with Bacillus cereus with Pseudomonas spp and with theirconsortium (1 1) at five different concentrations (1 2 3 4 and 5mL) on the 5th the 10th and the 15th days and in the control

Experimentsa Quantity of bacterial cultures added Leaf area (cm2)b

5th day 10th day 15th day

Exp0

1mL 027 (ab) 094 (abc) 407 (b)2mL 035 (ab) 102 (ab) 419 (abc)3mL 040 (abc) 125 (ab) 471 (bcd)4mL 048 (ab) 138 (abc) 510 (ab)5mL 054 (abc) 161 (b) 538 (b)

Exp1

1mL 018 (bcd) 123 (b) 601 (abc)2mL 025 (ab) 138 (abc) 632 (ab)3mL 033 (abc) 150 (ab) 684 (b)4mL 038 (ab) 168 (ab) 686 (abc)5mL 044 (abc) 191 (bcd) 733 (ab)

Exp2

1mL 011 (b) 107 (ab) 534 (b)2mL 014 (ab) 119 (ab) 559 (ab)3mL 017 (abc) 127 (ab) 579 (ab)4mL 021 (ab) 153 (abc) 631 (abc)5mL 026 (ab) 166 (b) 667 (b)

Exp3

1mL 026 (bcd) 297 (ab) 753 (bcd)2mL 033 (b) 325 (ab) 787 (ab)3mL 040 (ab) 350 (bcd) 811 (bcd)4mL 044 (b) 384 (b) 861 (b)5mL 062 (ab) 400 (bcd) 905 (ab)

aExp0 nutrient solution (control) Exp1 Bacillus cereus Exp2 Pseudomonas spp Exp3 consortium of Bacillus cereus + Pseudomonas spp (1 1)bMeanof two repeated experiments (30 plants)Different letterswithin parenthesis indicate significant difference between treatments for each growth parameterusing Duncanrsquos multiple range test (119875 = 005)

0102030405060708090

100

Concentration of bacterial cultures (mL)1 2 3 4 5

Germination ()

Exp0

Exp1 Exp3

Exp2

Figure 3 Germination percentage ofVigna unguiculata seeds sownin vermiculite supplementedwithBacillus cereus with Pseudomonasspp and with their consortium at five different concentrations (1 23 4 and 5mL) and in the control Exp0 nutrient solution (control)Exp1 Bacillus cereus Exp2 Pseudomonas spp Exp3 consortium ofBacillus cereus + Pseudomonas spp (1 1)

not treated with the bacterial culture [41] In earlier reportsroot elongation was found to occur in Sesbania aculeata byinoculation with Azotobacter spp and Pseudomonas spp in

Vigna radiata by Pseudomonas putida [16] and in Pennisetumamericanum by Azospirillum brasilense [42] Esitken et al(2006) had found out that Bacillus OSU-142 and Pseu-domonas BA-8 alone or in combination had a great potentialto increase the growth yield and nutrition of sweet cherryplant [43] BacillusM3 BacillusOSU-142 andMicobacteriumFS01 separately or in combination were found to have greatpotential for use as plant growth promoting rhizobacteriato increase production in apples and in many other crops[44] These phosphate solubilizing microbes consist pre-dominantly of fungal bacterial and actinomycetes speciescollectively called phosphate solubilizing microorganismsmdashPSM [45] There are several microorganisms which canalso solubilize the cheaper sources of phosphorous such asrock phosphate Bacteria such as Bacillus are widely usedin plant production system and are important phosphorussolubilizing microorganisms resulting in improved growthyield of crops andmetabolic activities especially in synthesisof protein [34 46]

5 Conclusions

This study illustrates the isolation of plant growth promotingbacteria from vermisources screening under in vitro con-ditions for multiple PGPR traits and their evaluation undercontrolled conditions in a pot experiment The findings ofthis study indicated that the consortium of B cereus and

10 Advances in Agriculture

Table5Ch

loroph

yllcon

tent

ofleavesofVignaun

guicu

latagrow

non

verm

iculite

supp

lementedwith

Bacilluscereuswith

Pseudomonas

sppand

with

theircon

sortium

(11)atfi

vedifferent

concentrations

(1234and

5mL)

onthe5

thdaythe10thdayandthe15thdayandin

thec

ontro

l

Experim

entsa

Quantity

ofbacterial

Cultu

resa

dded

Chloroph

yllcon

tent

(mggof

fresh

leaf)b

5thday

10th

day

15th

day

Chloroph

ylla

Chloroph

yllb

Total

chloroph

yll

Chloroph

ylla

Chloroph

yllb

Total

chloroph

yll

Chloroph

ylla

Chloroph

yllb

Total

chloroph

yll

Exp0

1mL

021

(ab)

285

(abc)

307

(a)

020

(abc)

481

(a)

501

(ab)

015

(bcd)

612

(abc)

627

(bcd)

2mL

022

(abc)

293

(ab)

315

(abc)

021

(ab)

489

(ab)

510

(bcd)

012

(abc)

618

(ab)

630

(ab)

3mL

023

(abc)

301

(ab)

315

(abc)

022

(bcd)

497

(bcd)

518

(a)

014

(a)

633

(cde)

647

(bcd)

4mL

024

(ab)

308

(f)

332

(a)

022

(a)

504

(b)

527

(abc)

015

(bcd)

641

(cde)

656

(a)

5mL

025

(f)

316

(abc)

341

(bcd)

019

(ab)

511(ab)

530

(abc)

011(cde)

645

(ab)

650

(abc)

Exp1

1mL

035

(b)

400

(cde)

435

(abc)

011(b)

581

(cde)

593

(cde)

010

(abc)

704(abc)

715(f)

2mL

026

(abc)

462

(bcd)

488

(f)

011(abc)

647

(b)

658

(abc)

006

(ab)

779(bcd)

785(a)

3mL

021

(bcd)

526

(abc)

547

(ab)

008

(bcd)

718(ab)

726(bcd)

006

(f)

847

(f)

853

(ab)

4mL

008

(f)

587

(cde)

595

(b)

011(bcd)

778(abc)

789(abc)

004

(bcd)

933(b)

937(bcd)

5mL

026

(abc)

641

(bcd)

667

(abc)

025

(cde)

834

(cde)

859

(f)

007

(abc)

976(cde)

983(abc)

Exp2

1mL

030

(abc)

354

(ab)

384

(b)

012

(a)

545

(abc)

556

(ab)

007

(bcd)

681

(abc)

688

(bcd)

2mL

029

(bcd)

420

(f)

449

(abc)

011(bcd)

610

(ab)

621

(abc)

007

(abc)

744(f)

752(bcd)

3mL

022

(ab)

504

(abc)

527

(bcd)

015

(a)

677

(cde)

692

(bcd)

006

(bcd)

812

(ab)

817

(bcd)

4mL

010

(f)

566

(a)

576

(cde)

018

(abc)

744(f)

762(ab)

005

(ab)

885

(a)

890

(abc)

5mL

006

(bcd)

622

(abc)

628

(abc)

018

(b)

810

(abc)

828

(a)

004

(cde)

948(a)

952(f)

Exp3

1mL

027

(ab)

470

(bcd)

496

(cde)

009

(ab)

668

(b)

677

(cde)

010

(a)

814

(ab)

825

(a)

2mL

020

(ab)

548

(a)

568

(f)

003

(bcd)

746(bcd)

749(ab)

009

(ab)

887

(f)

897

(ab)

3mL

005

(bcd)

584

(abc)

589

(ab)

008

(bcd)

804

(abc)

812

(cde)

010

(ab)

953(abc)

963(bcd)

4mL

023

(f)

663

(ab)

686

(cde)

007

(ab)

870

(b)

877

(f)

010

(abc)

1019

(bcd)

1029(f)

5mL

022

(ab)

744(cde)

766(a)

009

(a)

932(abc)

940(ab)

010

(a)

1085(ab)

1095(abc)

a Exp0nu

trient

solutio

n(con

trol)

Exp1B

acillus

cereusE

xp2Pseudomonas

sppE

xp3consortiu

mof

Bacilluscereus+

Pseudomonas

spp(11)

b Meanof

tworepeated

experim

ents(30plants)

Differentletterswith

inparenthesis

indicatesig

nificantd

ifference

betweentre

atmentsfore

achgrow

thparameter

usingDun

canrsquos

multip

lerangetest(119875=005)

Advances in Agriculture 11

Table 6 Fresh weight and dry weight of whole plant of Vigna unguiculata grown on vermiculite supplemented with Bacillus cereus withPseudomonas spp and with their consortium (1 1) at five different concentrations (1 2 3 4 and 5mL) on the 5th the 10th and the 15 daysand in the control

Experimentsa Quantity of bacterial cultures added Fresh weight of the whole plant (g)b Dry weight of the whole plant (g)b

5th day 10th day 15th day 5th day 10th day 15th day

Exp0

1mL 058 (bcd) 061 (abc) 064 (ab) 006 (a) 006 (abc) 006 (ab)2mL 058 (ab) 061 (a) 064 (abc) 006 (bcd) 006 (ab) 006 (ab)3mL 058 (abc) 061 (ab) 064 (bcd) 006 (abc) 006 (bcd) 006 (b)4mL 041 (abc) 061 (ab) 064 (ab) 006 (abc) 006 (ab) 006 (abc)5mL 058 (abc) 061 (abc) 064 (bcd) 006 (a) 006 (bcd) 006 (def)

Exp1

1mL 062 (abc) 065 (abc) 068 (ab) 006 (bcd) 006 (ab) 007 (bcd)2mL 065 (bcd) 068 (bcd) 071 (def) 006 (abc) 007 (a) 007 (ab)3mL 068 (abc) 071 (def) 074 (bcd) 007 (abc) 007 (ab) 007 (abc)4mL 071 (ab) 074 (abc) 077 (abc) 007 (bcd) 007 (b) 008 (bcd)5mL 074 (abc) 077 (def) 080 (ab) 007 (def) 008 (a) 008 (b)

Exp2

1mL 056 (ab) 059 (bcd) 062 (ab) 006 (ab) 006 (ab) 006 (abc)2mL 059 (abc) 062 (abc) 065 (def) 006 (ab) 006 (abc) 006 (bcd)3mL 062 (bcd) 065 (a) 068 (bcd) 006 (abc) 006 (bcd) 007 (abc)4mL 065 (abc) 068 (ab) 071 (b) 006 (ab) 007 (abc) 007 (b)5mL 068 (abc) 071 (b) 074 (a) 007 (def) 007 (abc) 007 (bcd)

Exp3

1mL 074 (ab) 077 (ab) 080 (abc) 007 (a) 008 (bcd) 008 (ab)2mL 077 (bcd) 080 (a) 083 (bcd) 008 (a) 008 (bcd) 008 (abc)3mL 080 (bcd) 083 (bcd) 086 (abc) 008 (ab) 008 (b) 009 (b)4mL 083 (bcd) 086 (abc) 089 (ab) 008 (abc) 009 (ab) 009 (bcd)5mL 086 (ab) 089 (b) 092 (def) 009 (bcd) 009 (bcd 009 (b)

aExp0 nutrient solution (control) Exp1 Bacillus cereus Exp2 Pseudomonas spp Exp3 consortium of Bacillus cereus + Pseudomonas spp (1 1)bMeanof two repeated experiments (30 plants)Different letterswithin parenthesis indicate significant difference between treatments for each growth parameterusing Duncanrsquos multiple range test (119875 = 005)

Pseudomonas spp acts as a potential biofertilizer due to itsvarious plant growth promotion abilities such as solubiliza-tion of phosphate and production of IAA and siderophore itcan be developed as potential microbial inoculant for variouscrops

Conflict of Interests

The authors declared that there is no conflict of interestsregarding this paper

Acknowledgments

Theauthors are thankful to theDepartment of BiologyGand-higram Rural InstitutemdashDeemed University GandhigramThis research was funded by University Grants Commissionunder UGC Research Fellowship in Science for MeritoriousStudents (BSR) Grant no F4-12008

References

[1] U S Bhawalkar Vermiculture Ecotechnology EarthwormResearch Institute Pune India 2nd edition 1996

[2] S Kiss D M Bularda and D Radulescu ldquoBiological Signifi-cance of Enzymes Accumulated in SoilrdquoAdvances in Agronomyvol 27 pp 25ndash87 1975

[3] D L Karlen M J Mausbach J W Doran R G Cline R FHarris and G E Schuman ldquoSoil quality a concept definitionand framework for evaluationrdquo Soil Science Society of AmericaJournal vol 61 no 1 pp 4ndash10 1997

[4] R M Atiyeh C A Edwards S Subler and J Metzger ldquoEarth-worm processed organic wastes as components of horticulturalpotting media for growing manifold and vegetable seedlingsrdquoCompost Science and Utilization vol 8 no 3 pp 215ndash223 2000

[5] N Vassilev and M Vassileva ldquoBiotechnological solubilizationof rock phosphate on media containing agro-industrial wastesrdquoApplied Microbiology and Biotechnology vol 61 no 5-6 pp435ndash440 2003

[6] S A Omar ldquoThe role of rock-phosphate-solubilizing fungiand vesicular-arbusular-mycorrhiza (VAM) in growth of wheatplants fertilized with rock phosphaterdquo World Journal of Micro-biology and Biotechnology vol 14 no 2 pp 211ndash218 1998

[7] J A J Paul and T Daniel ldquoStandardization of sampling methodfor physical characterization of municipal solid wasterdquo AsianJournal of Water Environment and Pollution vol 5 no 1 pp95ndash98 2007

[8] R Lalloo D Maharajh J Gorgens and N Gardiner ldquoA down-stream process for production of a viable and stable Bacilluscereus aquaculture biological agentrdquo Applied Microbiology andBiotechnology vol 86 no 2 pp 499ndash508 2010

[9] B Sivasankari N Kavikumar and M Anandharaj ldquoIndole-3-acetic acid production and enhanced Plant growth promotionby Indigenous bacterial speciesrdquo Journal of Current Research inScience vol 1 no 5 pp 331ndash335 2013

12 Advances in Agriculture

[10] J Han L Sun X Dong et al ldquoCharacterization of a novel plantgrowth-promoting bacteria strain Delftia tsuruhatensis HR4both as a diazotroph and a potential biocontrol agent againstvarious plant pathogensrdquo Systematic and Applied Microbiologyvol 28 no 1 pp 66ndash76 2005

[11] H Rodrıguez and R Fraga ldquoPhosphate solubilizing bacte-ria and their role in plant growth promotionrdquo BiotechnologyAdvances vol 17 no 4-5 pp 319ndash339 1999

[12] T Daniel and N Karmegam ldquoBio-conversion of selected leaflitters using an African epigeic earthworm Eudrilus eugeniaerdquoEcology Environment and Conservation vol 5 no 3 pp 271ndash275 1999

[13] J Dobereiner I E Marriel and M Nery ldquoEcological distri-bution of Spirillum lipoferum Beijerinckrdquo Canadian Journal ofMicrobiology vol 22 no 10 pp 1464ndash1473 1976

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

[15] M Edi Premono A M Moawad and P L G Vlek ldquoEffect ofphosphate-solubilizing Pseudomonas putida on the growth ofmaize and its survival in the rhizosphererdquo Indonesian Journal ofCrop Science vol 11 pp 13ndash23 1996

[16] C L Patten and B R Glick ldquoBacterial biosynthesis of indole-3-acetic acidrdquo Canadian Journal of Microbiology vol 42 no 3pp 207ndash220 1996

[17] N Amaresan V Jayakumar K Kumar and N ThajuddinldquoIsolation and characterization of plant growth promotingendophytic bacteria and their effect on tomato (Lycopersiconesculentum) and chilli (Capsicum annuum) seedling growthrdquoAnnals of Microbiology vol 62 no 2 pp 805ndash810 2012

[18] B Schwyn and J B Neilands ldquoUniversal chemical assay forthe detection and determination of siderophoresrdquo AnalyticalBiochemistry vol 160 no 1 pp 47ndash56 1987

[19] P Rahi P Vyas S Sharma and A Gulati ldquoPlant growthpromoting potential of the fungus Discosia sp FIHB 571 fromtea rhizosphere tested on chickpea maize and peardquo IndianJournal of Microbiology vol 49 no 2 pp 128ndash133 2009

[20] K Kumar N Amaresan S Bhagat K Madhuri and R CSrivastava ldquoIsolation and characterization of rhizobacteriaassociated with coastal agricultural ecosystem of rhizospheresoils of cultivated vegetable cropsrdquo World Journal of Microbiol-ogy and Biotechnology vol 27 no 7 pp 1625ndash1632 2011

[21] J A Smoker and S R Barnum ldquoRapid small scale DNAisolation from filamentous cyanobacteriardquo FEMS MicrobiologyLetters vol 56 no 1 pp 119ndash122 1988

[22] U Edwars T Rogall H Blocker M Emde and E C BottgerldquoIsolation and direct complete nucleotide determination ofentire genes Characterization of a gene coding for 16S riboso-mal RNArdquoNucleic Acids Research vol 17 no 19 pp 7843ndash78531989

[23] D G Higgins A J Bleasby and R Fuchs ldquoCLUSTAL Vimproved software for multiple sequence alignmentrdquo ComputerApplications in the Biosciences vol 8 no 2 pp 189ndash191 1992

[24] M Kimura ldquoA simple method for estimating evolutionary ratesof base substitutions through comparative studies of nucleotidesequencesrdquo Journal ofMolecular Evolution vol 16 no 2 pp 111ndash120 1980

[25] N Saitou and M Nei ldquoThe neighbor-joining method a newmethod for reconstructing phylogenetic treesrdquo Molecular Biol-ogy and Evolution vol 4 no 4 pp 406ndash425 1987

[26] E Barbu K Y Lee and R Wahl ldquoContent of purine andpyrimidine base in desoxyribonucleic acid of bacteriardquo Annalesde lrsquoInstitut Pasteur vol 91 no 2 pp 212ndash224 1956

[27] N Sueoka ldquoVariation and heterogeneity of base compositionof deoxyribonucleic acids a compilation of old and new datardquoJournal of Molecular Biology vol 3 no 1 pp 31ndash40 1961

[28] H Hori and S Osawa ldquoEvolutionary change in 5S rRNAsecondary structure and a phylogenic tree of 352 5S rRNAspeciesrdquo BioSystems vol 19 no 3 pp 163ndash172 1986

[29] V Bess ldquoEvaluating microbiology of compost microbial con-tent of composting is helping the producers and growers tounderstand its role as a soil inoculant and plantrdquo BioCycleMagazine p 62 1999

[30] O Grafff ldquoPreliminary experiments of vermin composting ofdifferent waste materials using Eudrilus eugeniae Kinbergrdquo inWorkshop on the Role of Earthworms in the Stabilization ofOrganic Residues M Happelhof Ed pp 178ndash191 Beech LeafPress Kalamazoo Mich USA 1981

[31] J E Satchell Earthworm Ecology from Darwin to VermicultureChapman and Hall 1983

[32] G H Xie M Y Cai G C Tao and Y Steinberger ldquoCultivableheterotrophic N2-fixing bacterial diversity in rice fields in theYangtze River Plainrdquo Biology and Fertility of Soils vol 37 no 1pp 29ndash38 2003

[33] B BMcSpaddenGardener ldquoEcology ofBacillus and Paenibacil-lus spp in agricultural systemsrdquo Phytopathology vol 94 no 11pp 1252ndash1258 2004

[34] J Dobereiner ldquoBiological nitrogen fixation in the tropics socialand economic contributionsrdquo Soil Biology andBiochemistry vol29 no 5-6 pp 771ndash774 1997

[35] V M Reis F L Olivares and J Dobereiner ldquoImprovedmethodology for isolation of Acetobacter diazotrophicus andconfirmation of its endophytic habitatrdquoWorld Journal of Micro-biology and Biotechnology vol 10 no 4 pp 401ndash405 1994

[36] S Khalil N Ayub S Alam and F Latif ldquoOrganic acids pro-duction and phosphate solubilization by phosphate solubilizingmicroorganisms (PSM) under in vitro conditionsrdquo PakistanJournal of Biological Sciences vol 7 no 2 pp 187ndash196 2004

[37] H M A El-Komy ldquoCoimmobilization of Azospirillumlipoferum and Bacillus megaterium for successful phosphorusand nitrogen nutrition of wheat plantsrdquo Food Technology andBiotechnology vol 43 no 1 pp 19ndash27 2005

[38] N B Paul and W V B Sundara Rao ldquoPhosphate-dissolvingbacteria in the rhizosphere of some cultivated legumesrdquo Plantand Soil vol 35 no 1 pp 127ndash132 1971

[39] A Beneduzi D Peres L K Vargas M H Bodanese-Zanettiniand LM P Passaglia ldquoEvaluation of genetic diversity and plantgrowth promoting activities of nitrogen-fixing bacilli isolatedfrom rice fields in South Brazilrdquo Applied Soil Ecology vol 39no 3 pp 311ndash320 2008

[40] M P Raimam U Albino M F Cruz et al ldquoInteraction amongfree-living N-fixing bacteria isolated from Drosera villosa varvillosa and AM fungi (Glomus clarum) in rice (Oryza sativa)rdquoApplied Soil Ecology vol 35 no 1 pp 25ndash34 2007

[41] M R Swain S K Naskar and R C Ray ldquoIndole-3-acetic acidproduction and effect on sprouting of yam (Dioscorea rotundataL) minisetts by Bacillus subtilis isolated from culturable cow-dung microflorardquo Polish Journal of Microbiology vol 56 no 2pp 103ndash110 2007

[42] T M Tien M H Garkins and D H Hubbell ldquoPlant growthsubstances produced by Azospirillum brasilense and their effect

Advances in Agriculture 13

on the growth of pearl Millet Pennisetum americanum (L)rdquoApplied and EnvironmentalMicrobiology vol 37 no 5 pp 1016ndash1024 1979

[43] A Esitken L Pirlak M Turan and F Sahin ldquoEffects of floraland foliar application of plant growth promoting rhizobacteria(PGPR) on yield growth and nutrition of sweet cherryrdquo ScientiaHorticulturae vol 110 no 4 pp 324ndash327 2006

[44] H Karlidag A Esitken M Turan and F Sahin ldquoEffects of rootinoculation of plant growth promoting rhizobacteria (PGPR)on yield growth and nutrient element contents of leaves ofapplerdquo Scientia Horticulturae vol 114 no 1 pp 16ndash20 2007

[45] C S Nautiyal S Bhadauria P Kumar H Lal R Mondal andD Verma ldquoStress induced phosphate solubilization in bacteriaisolated fromalkaline soilsrdquoFEMSMicrobiology Letters vol 182no 2 pp 291ndash296 2000

[46] A K Halder A K Mishra P Bhattacharya and P KChakrabarthy ldquoSolubilization of inorganic phosphates byBradyrhizobiumrdquo Indian Journal of Experimental Biology vol29 pp 28ndash31 1991

Submit your manuscripts athttpwwwhindawicom

Nutrition and Metabolism

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Food ScienceInternational Journal of

Agronomy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Applied ampEnvironmentalSoil Science

Volume 2014

AgricultureAdvances in

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PsycheHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BiodiversityInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Plant GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biotechnology Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Forestry ResearchInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of BotanyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

EcologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Veterinary Medicine International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Cell BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Page 8: BalayoganSivasankariandMarimuthuAnandharajdownloads.hindawi.com/journals/aag/2014/248591.pdf · In Vitro Nitrogen Fixing Activity. e bacterial isolates were screened for nitrogen

8 Advances in Agriculture

Table 3 Shoot length and Root length ofVigna unguiculata grown on vermiculite supplemented with Bacillus cereus with Pseudomonas sppand with their consortium (1 1) at five different concentrations (1 2 3 4 and 5mL) on the 5th the 10th and the 15th day and in the control

Experimentsa Quantity of bacterial cultures added Shoot length (cm)b Root length (cm)b

5th day 10th day 15th day 5th day 10th day 15th day

Exp0

1mL 297 (ab) 397 (a) 497 (ab) 197 (de) 283 (ab) 397 (bcd)2mL 303 (bcd) 403 (ab) 500 (ab) 200 (abc) 287 (de) 400 (a)3mL 310 (abc) 407 (a) 503 (ab) 203 (de) 290 (abc) 403 (ab)4mL 310 (bcd) 410 (bcd) 507 (bcd) 207 (a) 297 (de) 410 (abc)5mL 313 (a) 413 (ab) 510 (ab) 210 (bcd) 300 (ab) 413 (de)

Exp1

1mL 340 (ab) 440 (abc) 540 (ab) 233 (abc) 327 (bcd) 433 (f)2mL 450 (ab) 540 (abc) 647 (ab) 333 (ab) 430 (a) 540 (abc)3mL 550 (de) 630 (ab) 750 (a) 440 (de) 550 (abc) 620 (a)4mL 653 (ab) 730 (a) 833 (ab) 550 (bcd) 653 (de) 750 (abc)5mL 740 (ab) 840 (abc) 923 (f) 640 (abc) 753 (ab) 827 (ab)

Exp2

1mL 277 (bcd) 357 (f) 440 (de) 160 (de) 217 (abc) 350 (f)2mL 343 (abc) 447 (ab) 553 (ab) 253 (abc) 340 (bcd) 427 (bcd)3mL 437 (de) 543 (abc) 633 (abc) 340 (ab) 443 (f) 533 (ab)4mL 547 (bcd) 647 (de) 733 (a) 420 (bcd) 550 (ab) 633 (ab)5mL 640 (f) 733 (bcd) 833 (bcd) 533 (ab) 630 (ab) 730 (bcd)

Exp3

1mL 530 (bcd) 647 (ab) 730 (ab) 433 (de) 550 (abc) 620 (ab)2mL 643 (ab) 740 (bcd) 827 (f) 547 (a) 630 (de) 733 (ab)3mL 737 (bcd) 840 (bcd) 927 (ab) 640 (ab) 740 (bcd) 840 (bcd)4mL 847 (abc) 930 (a) 1043 (abc) 740 (f) 833 (a) 937 (abc)5mL 957 (ab) 1030 (f) 1147 (bcd) 833 (abc) 940 (ab) 1027 (bcd)

aExp0 nutrient solution (control) Exp1 Bacillus cereus Exp2 Pseudomonas spp Exp3 consortium of Bacillus cereus + Pseudomonas spp (1 1)bMeanof two repeated experiments (30 plants)Different letterswithin parenthesis indicate significant difference between treatments for each growth parameterusing Duncanrsquos multiple range test (119875 = 005)

growth promoters The use of these strains in agriculturehas recently been reviewed [33]Those strains besides havingseveral PGP properties can also fix nitrogen and also dophosphate solubilization The symbiotic fixation of nitrogenthrough inoculation of legume crops with effective rhizobiais well known [34] Asymbiotic nitrogen fixing bacteriawhich live in the rhizosphere andor endophytically oftenincrease yields of crops Many bacterial species have nitrogenfixing properties including Bacillus spp Azotobacter sppAzospirillum spp Beijerinckia spp and Pseudomonas spp[35] Nitrogen fixing bacteria have been used in foliar appli-cations inmulberryThe solubilization index (SI) was formeddue to solubilization of insoluble phosphates by organic acidsecretion [36] El-Komy (2005) indicated that Pseudomonasfluorescence and Bacillus megaterium strains were the mostpowerful phosphate solubilizers on PVK plates as well as onPikovskayarsquos broth [37] The P concentration in Pikovskayarsquosbroth increased gradually achieving a peak on the sixth dayand declined slowly during the late days In this study pHvalues decreased gradually in PVK broth during the earlyincubation days and no revival was observed during laterdays for all the tested bacterial strains Several bacteria par-ticularly those belonging to the genus Bacillus spp convertinsoluble phosphate into soluble forms by secreting organicacids such as formic acid acetic acid propionic acid citricacid fumaric acid gluconic acid glyoxylic acid ketobutyric

acid malonic acid succinic acid and tartaric acid Theseacids lower the pH and bring about the dissolution of boundforms of phosphate Some of the hydroxyl acids may chelatewith calcium and iron resulting in effective solubilization andutilization of phosphates [38]

Beneduzi et al (2008) had isolated the plant growthpromoting strain SVPR30 and identified by 16S rRNA genesequence as Bacillus spp and they had tested it for plantgrowth through in vivo experiments [39] This strain wascharacterized as a high IAA producer able to solubilize phos-phate and also fix a considerably high amount of nitrogenThe inoculation of rice with Bacillus spp SVPR30 strainshowed a significant increase in the root and shoot partswhen compared with the controls within 15 and 30 daysafter sprouting Numerous Bacillus and Paenibacillus strainsexpressed plant growth promoting (PGP) activities and anumber of these strains have been commercially developedas generic plant growth promotersThe use of these strains inagriculture has recently been reviewed [33]

Various reports indicate that coinoculation of beneficialorganisms generally increased plant growth relative to singleinoculation with a sole beneficial organism [40] Most ofthe effects of the individual microorganisms coinoculationare additive although a synergistic effect has been reportedin some cases The average root to shoot ratio was higherin B subtilis treated yam minisetts in comparison to those

Advances in Agriculture 9

Table 4 Leaf area of Vigna unguiculata grown on vermiculite supplemented with Bacillus cereus with Pseudomonas spp and with theirconsortium (1 1) at five different concentrations (1 2 3 4 and 5mL) on the 5th the 10th and the 15th days and in the control

Experimentsa Quantity of bacterial cultures added Leaf area (cm2)b

5th day 10th day 15th day

Exp0

1mL 027 (ab) 094 (abc) 407 (b)2mL 035 (ab) 102 (ab) 419 (abc)3mL 040 (abc) 125 (ab) 471 (bcd)4mL 048 (ab) 138 (abc) 510 (ab)5mL 054 (abc) 161 (b) 538 (b)

Exp1

1mL 018 (bcd) 123 (b) 601 (abc)2mL 025 (ab) 138 (abc) 632 (ab)3mL 033 (abc) 150 (ab) 684 (b)4mL 038 (ab) 168 (ab) 686 (abc)5mL 044 (abc) 191 (bcd) 733 (ab)

Exp2

1mL 011 (b) 107 (ab) 534 (b)2mL 014 (ab) 119 (ab) 559 (ab)3mL 017 (abc) 127 (ab) 579 (ab)4mL 021 (ab) 153 (abc) 631 (abc)5mL 026 (ab) 166 (b) 667 (b)

Exp3

1mL 026 (bcd) 297 (ab) 753 (bcd)2mL 033 (b) 325 (ab) 787 (ab)3mL 040 (ab) 350 (bcd) 811 (bcd)4mL 044 (b) 384 (b) 861 (b)5mL 062 (ab) 400 (bcd) 905 (ab)

aExp0 nutrient solution (control) Exp1 Bacillus cereus Exp2 Pseudomonas spp Exp3 consortium of Bacillus cereus + Pseudomonas spp (1 1)bMeanof two repeated experiments (30 plants)Different letterswithin parenthesis indicate significant difference between treatments for each growth parameterusing Duncanrsquos multiple range test (119875 = 005)

0102030405060708090

100

Concentration of bacterial cultures (mL)1 2 3 4 5

Germination ()

Exp0

Exp1 Exp3

Exp2

Figure 3 Germination percentage ofVigna unguiculata seeds sownin vermiculite supplementedwithBacillus cereus with Pseudomonasspp and with their consortium at five different concentrations (1 23 4 and 5mL) and in the control Exp0 nutrient solution (control)Exp1 Bacillus cereus Exp2 Pseudomonas spp Exp3 consortium ofBacillus cereus + Pseudomonas spp (1 1)

not treated with the bacterial culture [41] In earlier reportsroot elongation was found to occur in Sesbania aculeata byinoculation with Azotobacter spp and Pseudomonas spp in

Vigna radiata by Pseudomonas putida [16] and in Pennisetumamericanum by Azospirillum brasilense [42] Esitken et al(2006) had found out that Bacillus OSU-142 and Pseu-domonas BA-8 alone or in combination had a great potentialto increase the growth yield and nutrition of sweet cherryplant [43] BacillusM3 BacillusOSU-142 andMicobacteriumFS01 separately or in combination were found to have greatpotential for use as plant growth promoting rhizobacteriato increase production in apples and in many other crops[44] These phosphate solubilizing microbes consist pre-dominantly of fungal bacterial and actinomycetes speciescollectively called phosphate solubilizing microorganismsmdashPSM [45] There are several microorganisms which canalso solubilize the cheaper sources of phosphorous such asrock phosphate Bacteria such as Bacillus are widely usedin plant production system and are important phosphorussolubilizing microorganisms resulting in improved growthyield of crops andmetabolic activities especially in synthesisof protein [34 46]

5 Conclusions

This study illustrates the isolation of plant growth promotingbacteria from vermisources screening under in vitro con-ditions for multiple PGPR traits and their evaluation undercontrolled conditions in a pot experiment The findings ofthis study indicated that the consortium of B cereus and

10 Advances in Agriculture

Table5Ch

loroph

yllcon

tent

ofleavesofVignaun

guicu

latagrow

non

verm

iculite

supp

lementedwith

Bacilluscereuswith

Pseudomonas

sppand

with

theircon

sortium

(11)atfi

vedifferent

concentrations

(1234and

5mL)

onthe5

thdaythe10thdayandthe15thdayandin

thec

ontro

l

Experim

entsa

Quantity

ofbacterial

Cultu

resa

dded

Chloroph

yllcon

tent

(mggof

fresh

leaf)b

5thday

10th

day

15th

day

Chloroph

ylla

Chloroph

yllb

Total

chloroph

yll

Chloroph

ylla

Chloroph

yllb

Total

chloroph

yll

Chloroph

ylla

Chloroph

yllb

Total

chloroph

yll

Exp0

1mL

021

(ab)

285

(abc)

307

(a)

020

(abc)

481

(a)

501

(ab)

015

(bcd)

612

(abc)

627

(bcd)

2mL

022

(abc)

293

(ab)

315

(abc)

021

(ab)

489

(ab)

510

(bcd)

012

(abc)

618

(ab)

630

(ab)

3mL

023

(abc)

301

(ab)

315

(abc)

022

(bcd)

497

(bcd)

518

(a)

014

(a)

633

(cde)

647

(bcd)

4mL

024

(ab)

308

(f)

332

(a)

022

(a)

504

(b)

527

(abc)

015

(bcd)

641

(cde)

656

(a)

5mL

025

(f)

316

(abc)

341

(bcd)

019

(ab)

511(ab)

530

(abc)

011(cde)

645

(ab)

650

(abc)

Exp1

1mL

035

(b)

400

(cde)

435

(abc)

011(b)

581

(cde)

593

(cde)

010

(abc)

704(abc)

715(f)

2mL

026

(abc)

462

(bcd)

488

(f)

011(abc)

647

(b)

658

(abc)

006

(ab)

779(bcd)

785(a)

3mL

021

(bcd)

526

(abc)

547

(ab)

008

(bcd)

718(ab)

726(bcd)

006

(f)

847

(f)

853

(ab)

4mL

008

(f)

587

(cde)

595

(b)

011(bcd)

778(abc)

789(abc)

004

(bcd)

933(b)

937(bcd)

5mL

026

(abc)

641

(bcd)

667

(abc)

025

(cde)

834

(cde)

859

(f)

007

(abc)

976(cde)

983(abc)

Exp2

1mL

030

(abc)

354

(ab)

384

(b)

012

(a)

545

(abc)

556

(ab)

007

(bcd)

681

(abc)

688

(bcd)

2mL

029

(bcd)

420

(f)

449

(abc)

011(bcd)

610

(ab)

621

(abc)

007

(abc)

744(f)

752(bcd)

3mL

022

(ab)

504

(abc)

527

(bcd)

015

(a)

677

(cde)

692

(bcd)

006

(bcd)

812

(ab)

817

(bcd)

4mL

010

(f)

566

(a)

576

(cde)

018

(abc)

744(f)

762(ab)

005

(ab)

885

(a)

890

(abc)

5mL

006

(bcd)

622

(abc)

628

(abc)

018

(b)

810

(abc)

828

(a)

004

(cde)

948(a)

952(f)

Exp3

1mL

027

(ab)

470

(bcd)

496

(cde)

009

(ab)

668

(b)

677

(cde)

010

(a)

814

(ab)

825

(a)

2mL

020

(ab)

548

(a)

568

(f)

003

(bcd)

746(bcd)

749(ab)

009

(ab)

887

(f)

897

(ab)

3mL

005

(bcd)

584

(abc)

589

(ab)

008

(bcd)

804

(abc)

812

(cde)

010

(ab)

953(abc)

963(bcd)

4mL

023

(f)

663

(ab)

686

(cde)

007

(ab)

870

(b)

877

(f)

010

(abc)

1019

(bcd)

1029(f)

5mL

022

(ab)

744(cde)

766(a)

009

(a)

932(abc)

940(ab)

010

(a)

1085(ab)

1095(abc)

a Exp0nu

trient

solutio

n(con

trol)

Exp1B

acillus

cereusE

xp2Pseudomonas

sppE

xp3consortiu

mof

Bacilluscereus+

Pseudomonas

spp(11)

b Meanof

tworepeated

experim

ents(30plants)

Differentletterswith

inparenthesis

indicatesig

nificantd

ifference

betweentre

atmentsfore

achgrow

thparameter

usingDun

canrsquos

multip

lerangetest(119875=005)

Advances in Agriculture 11

Table 6 Fresh weight and dry weight of whole plant of Vigna unguiculata grown on vermiculite supplemented with Bacillus cereus withPseudomonas spp and with their consortium (1 1) at five different concentrations (1 2 3 4 and 5mL) on the 5th the 10th and the 15 daysand in the control

Experimentsa Quantity of bacterial cultures added Fresh weight of the whole plant (g)b Dry weight of the whole plant (g)b

5th day 10th day 15th day 5th day 10th day 15th day

Exp0

1mL 058 (bcd) 061 (abc) 064 (ab) 006 (a) 006 (abc) 006 (ab)2mL 058 (ab) 061 (a) 064 (abc) 006 (bcd) 006 (ab) 006 (ab)3mL 058 (abc) 061 (ab) 064 (bcd) 006 (abc) 006 (bcd) 006 (b)4mL 041 (abc) 061 (ab) 064 (ab) 006 (abc) 006 (ab) 006 (abc)5mL 058 (abc) 061 (abc) 064 (bcd) 006 (a) 006 (bcd) 006 (def)

Exp1

1mL 062 (abc) 065 (abc) 068 (ab) 006 (bcd) 006 (ab) 007 (bcd)2mL 065 (bcd) 068 (bcd) 071 (def) 006 (abc) 007 (a) 007 (ab)3mL 068 (abc) 071 (def) 074 (bcd) 007 (abc) 007 (ab) 007 (abc)4mL 071 (ab) 074 (abc) 077 (abc) 007 (bcd) 007 (b) 008 (bcd)5mL 074 (abc) 077 (def) 080 (ab) 007 (def) 008 (a) 008 (b)

Exp2

1mL 056 (ab) 059 (bcd) 062 (ab) 006 (ab) 006 (ab) 006 (abc)2mL 059 (abc) 062 (abc) 065 (def) 006 (ab) 006 (abc) 006 (bcd)3mL 062 (bcd) 065 (a) 068 (bcd) 006 (abc) 006 (bcd) 007 (abc)4mL 065 (abc) 068 (ab) 071 (b) 006 (ab) 007 (abc) 007 (b)5mL 068 (abc) 071 (b) 074 (a) 007 (def) 007 (abc) 007 (bcd)

Exp3

1mL 074 (ab) 077 (ab) 080 (abc) 007 (a) 008 (bcd) 008 (ab)2mL 077 (bcd) 080 (a) 083 (bcd) 008 (a) 008 (bcd) 008 (abc)3mL 080 (bcd) 083 (bcd) 086 (abc) 008 (ab) 008 (b) 009 (b)4mL 083 (bcd) 086 (abc) 089 (ab) 008 (abc) 009 (ab) 009 (bcd)5mL 086 (ab) 089 (b) 092 (def) 009 (bcd) 009 (bcd 009 (b)

aExp0 nutrient solution (control) Exp1 Bacillus cereus Exp2 Pseudomonas spp Exp3 consortium of Bacillus cereus + Pseudomonas spp (1 1)bMeanof two repeated experiments (30 plants)Different letterswithin parenthesis indicate significant difference between treatments for each growth parameterusing Duncanrsquos multiple range test (119875 = 005)

Pseudomonas spp acts as a potential biofertilizer due to itsvarious plant growth promotion abilities such as solubiliza-tion of phosphate and production of IAA and siderophore itcan be developed as potential microbial inoculant for variouscrops

Conflict of Interests

The authors declared that there is no conflict of interestsregarding this paper

Acknowledgments

Theauthors are thankful to theDepartment of BiologyGand-higram Rural InstitutemdashDeemed University GandhigramThis research was funded by University Grants Commissionunder UGC Research Fellowship in Science for MeritoriousStudents (BSR) Grant no F4-12008

References

[1] U S Bhawalkar Vermiculture Ecotechnology EarthwormResearch Institute Pune India 2nd edition 1996

[2] S Kiss D M Bularda and D Radulescu ldquoBiological Signifi-cance of Enzymes Accumulated in SoilrdquoAdvances in Agronomyvol 27 pp 25ndash87 1975

[3] D L Karlen M J Mausbach J W Doran R G Cline R FHarris and G E Schuman ldquoSoil quality a concept definitionand framework for evaluationrdquo Soil Science Society of AmericaJournal vol 61 no 1 pp 4ndash10 1997

[4] R M Atiyeh C A Edwards S Subler and J Metzger ldquoEarth-worm processed organic wastes as components of horticulturalpotting media for growing manifold and vegetable seedlingsrdquoCompost Science and Utilization vol 8 no 3 pp 215ndash223 2000

[5] N Vassilev and M Vassileva ldquoBiotechnological solubilizationof rock phosphate on media containing agro-industrial wastesrdquoApplied Microbiology and Biotechnology vol 61 no 5-6 pp435ndash440 2003

[6] S A Omar ldquoThe role of rock-phosphate-solubilizing fungiand vesicular-arbusular-mycorrhiza (VAM) in growth of wheatplants fertilized with rock phosphaterdquo World Journal of Micro-biology and Biotechnology vol 14 no 2 pp 211ndash218 1998

[7] J A J Paul and T Daniel ldquoStandardization of sampling methodfor physical characterization of municipal solid wasterdquo AsianJournal of Water Environment and Pollution vol 5 no 1 pp95ndash98 2007

[8] R Lalloo D Maharajh J Gorgens and N Gardiner ldquoA down-stream process for production of a viable and stable Bacilluscereus aquaculture biological agentrdquo Applied Microbiology andBiotechnology vol 86 no 2 pp 499ndash508 2010

[9] B Sivasankari N Kavikumar and M Anandharaj ldquoIndole-3-acetic acid production and enhanced Plant growth promotionby Indigenous bacterial speciesrdquo Journal of Current Research inScience vol 1 no 5 pp 331ndash335 2013

12 Advances in Agriculture

[10] J Han L Sun X Dong et al ldquoCharacterization of a novel plantgrowth-promoting bacteria strain Delftia tsuruhatensis HR4both as a diazotroph and a potential biocontrol agent againstvarious plant pathogensrdquo Systematic and Applied Microbiologyvol 28 no 1 pp 66ndash76 2005

[11] H Rodrıguez and R Fraga ldquoPhosphate solubilizing bacte-ria and their role in plant growth promotionrdquo BiotechnologyAdvances vol 17 no 4-5 pp 319ndash339 1999

[12] T Daniel and N Karmegam ldquoBio-conversion of selected leaflitters using an African epigeic earthworm Eudrilus eugeniaerdquoEcology Environment and Conservation vol 5 no 3 pp 271ndash275 1999

[13] J Dobereiner I E Marriel and M Nery ldquoEcological distri-bution of Spirillum lipoferum Beijerinckrdquo Canadian Journal ofMicrobiology vol 22 no 10 pp 1464ndash1473 1976

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

[15] M Edi Premono A M Moawad and P L G Vlek ldquoEffect ofphosphate-solubilizing Pseudomonas putida on the growth ofmaize and its survival in the rhizosphererdquo Indonesian Journal ofCrop Science vol 11 pp 13ndash23 1996

[16] C L Patten and B R Glick ldquoBacterial biosynthesis of indole-3-acetic acidrdquo Canadian Journal of Microbiology vol 42 no 3pp 207ndash220 1996

[17] N Amaresan V Jayakumar K Kumar and N ThajuddinldquoIsolation and characterization of plant growth promotingendophytic bacteria and their effect on tomato (Lycopersiconesculentum) and chilli (Capsicum annuum) seedling growthrdquoAnnals of Microbiology vol 62 no 2 pp 805ndash810 2012

[18] B Schwyn and J B Neilands ldquoUniversal chemical assay forthe detection and determination of siderophoresrdquo AnalyticalBiochemistry vol 160 no 1 pp 47ndash56 1987

[19] P Rahi P Vyas S Sharma and A Gulati ldquoPlant growthpromoting potential of the fungus Discosia sp FIHB 571 fromtea rhizosphere tested on chickpea maize and peardquo IndianJournal of Microbiology vol 49 no 2 pp 128ndash133 2009

[20] K Kumar N Amaresan S Bhagat K Madhuri and R CSrivastava ldquoIsolation and characterization of rhizobacteriaassociated with coastal agricultural ecosystem of rhizospheresoils of cultivated vegetable cropsrdquo World Journal of Microbiol-ogy and Biotechnology vol 27 no 7 pp 1625ndash1632 2011

[21] J A Smoker and S R Barnum ldquoRapid small scale DNAisolation from filamentous cyanobacteriardquo FEMS MicrobiologyLetters vol 56 no 1 pp 119ndash122 1988

[22] U Edwars T Rogall H Blocker M Emde and E C BottgerldquoIsolation and direct complete nucleotide determination ofentire genes Characterization of a gene coding for 16S riboso-mal RNArdquoNucleic Acids Research vol 17 no 19 pp 7843ndash78531989

[23] D G Higgins A J Bleasby and R Fuchs ldquoCLUSTAL Vimproved software for multiple sequence alignmentrdquo ComputerApplications in the Biosciences vol 8 no 2 pp 189ndash191 1992

[24] M Kimura ldquoA simple method for estimating evolutionary ratesof base substitutions through comparative studies of nucleotidesequencesrdquo Journal ofMolecular Evolution vol 16 no 2 pp 111ndash120 1980

[25] N Saitou and M Nei ldquoThe neighbor-joining method a newmethod for reconstructing phylogenetic treesrdquo Molecular Biol-ogy and Evolution vol 4 no 4 pp 406ndash425 1987

[26] E Barbu K Y Lee and R Wahl ldquoContent of purine andpyrimidine base in desoxyribonucleic acid of bacteriardquo Annalesde lrsquoInstitut Pasteur vol 91 no 2 pp 212ndash224 1956

[27] N Sueoka ldquoVariation and heterogeneity of base compositionof deoxyribonucleic acids a compilation of old and new datardquoJournal of Molecular Biology vol 3 no 1 pp 31ndash40 1961

[28] H Hori and S Osawa ldquoEvolutionary change in 5S rRNAsecondary structure and a phylogenic tree of 352 5S rRNAspeciesrdquo BioSystems vol 19 no 3 pp 163ndash172 1986

[29] V Bess ldquoEvaluating microbiology of compost microbial con-tent of composting is helping the producers and growers tounderstand its role as a soil inoculant and plantrdquo BioCycleMagazine p 62 1999

[30] O Grafff ldquoPreliminary experiments of vermin composting ofdifferent waste materials using Eudrilus eugeniae Kinbergrdquo inWorkshop on the Role of Earthworms in the Stabilization ofOrganic Residues M Happelhof Ed pp 178ndash191 Beech LeafPress Kalamazoo Mich USA 1981

[31] J E Satchell Earthworm Ecology from Darwin to VermicultureChapman and Hall 1983

[32] G H Xie M Y Cai G C Tao and Y Steinberger ldquoCultivableheterotrophic N2-fixing bacterial diversity in rice fields in theYangtze River Plainrdquo Biology and Fertility of Soils vol 37 no 1pp 29ndash38 2003

[33] B BMcSpaddenGardener ldquoEcology ofBacillus and Paenibacil-lus spp in agricultural systemsrdquo Phytopathology vol 94 no 11pp 1252ndash1258 2004

[34] J Dobereiner ldquoBiological nitrogen fixation in the tropics socialand economic contributionsrdquo Soil Biology andBiochemistry vol29 no 5-6 pp 771ndash774 1997

[35] V M Reis F L Olivares and J Dobereiner ldquoImprovedmethodology for isolation of Acetobacter diazotrophicus andconfirmation of its endophytic habitatrdquoWorld Journal of Micro-biology and Biotechnology vol 10 no 4 pp 401ndash405 1994

[36] S Khalil N Ayub S Alam and F Latif ldquoOrganic acids pro-duction and phosphate solubilization by phosphate solubilizingmicroorganisms (PSM) under in vitro conditionsrdquo PakistanJournal of Biological Sciences vol 7 no 2 pp 187ndash196 2004

[37] H M A El-Komy ldquoCoimmobilization of Azospirillumlipoferum and Bacillus megaterium for successful phosphorusand nitrogen nutrition of wheat plantsrdquo Food Technology andBiotechnology vol 43 no 1 pp 19ndash27 2005

[38] N B Paul and W V B Sundara Rao ldquoPhosphate-dissolvingbacteria in the rhizosphere of some cultivated legumesrdquo Plantand Soil vol 35 no 1 pp 127ndash132 1971

[39] A Beneduzi D Peres L K Vargas M H Bodanese-Zanettiniand LM P Passaglia ldquoEvaluation of genetic diversity and plantgrowth promoting activities of nitrogen-fixing bacilli isolatedfrom rice fields in South Brazilrdquo Applied Soil Ecology vol 39no 3 pp 311ndash320 2008

[40] M P Raimam U Albino M F Cruz et al ldquoInteraction amongfree-living N-fixing bacteria isolated from Drosera villosa varvillosa and AM fungi (Glomus clarum) in rice (Oryza sativa)rdquoApplied Soil Ecology vol 35 no 1 pp 25ndash34 2007

[41] M R Swain S K Naskar and R C Ray ldquoIndole-3-acetic acidproduction and effect on sprouting of yam (Dioscorea rotundataL) minisetts by Bacillus subtilis isolated from culturable cow-dung microflorardquo Polish Journal of Microbiology vol 56 no 2pp 103ndash110 2007

[42] T M Tien M H Garkins and D H Hubbell ldquoPlant growthsubstances produced by Azospirillum brasilense and their effect

Advances in Agriculture 13

on the growth of pearl Millet Pennisetum americanum (L)rdquoApplied and EnvironmentalMicrobiology vol 37 no 5 pp 1016ndash1024 1979

[43] A Esitken L Pirlak M Turan and F Sahin ldquoEffects of floraland foliar application of plant growth promoting rhizobacteria(PGPR) on yield growth and nutrition of sweet cherryrdquo ScientiaHorticulturae vol 110 no 4 pp 324ndash327 2006

[44] H Karlidag A Esitken M Turan and F Sahin ldquoEffects of rootinoculation of plant growth promoting rhizobacteria (PGPR)on yield growth and nutrient element contents of leaves ofapplerdquo Scientia Horticulturae vol 114 no 1 pp 16ndash20 2007

[45] C S Nautiyal S Bhadauria P Kumar H Lal R Mondal andD Verma ldquoStress induced phosphate solubilization in bacteriaisolated fromalkaline soilsrdquoFEMSMicrobiology Letters vol 182no 2 pp 291ndash296 2000

[46] A K Halder A K Mishra P Bhattacharya and P KChakrabarthy ldquoSolubilization of inorganic phosphates byBradyrhizobiumrdquo Indian Journal of Experimental Biology vol29 pp 28ndash31 1991

Submit your manuscripts athttpwwwhindawicom

Nutrition and Metabolism

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Food ScienceInternational Journal of

Agronomy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Applied ampEnvironmentalSoil Science

Volume 2014

AgricultureAdvances in

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PsycheHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BiodiversityInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Plant GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biotechnology Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Forestry ResearchInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of BotanyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

EcologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Veterinary Medicine International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Cell BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Page 9: BalayoganSivasankariandMarimuthuAnandharajdownloads.hindawi.com/journals/aag/2014/248591.pdf · In Vitro Nitrogen Fixing Activity. e bacterial isolates were screened for nitrogen

Advances in Agriculture 9

Table 4 Leaf area of Vigna unguiculata grown on vermiculite supplemented with Bacillus cereus with Pseudomonas spp and with theirconsortium (1 1) at five different concentrations (1 2 3 4 and 5mL) on the 5th the 10th and the 15th days and in the control

Experimentsa Quantity of bacterial cultures added Leaf area (cm2)b

5th day 10th day 15th day

Exp0

1mL 027 (ab) 094 (abc) 407 (b)2mL 035 (ab) 102 (ab) 419 (abc)3mL 040 (abc) 125 (ab) 471 (bcd)4mL 048 (ab) 138 (abc) 510 (ab)5mL 054 (abc) 161 (b) 538 (b)

Exp1

1mL 018 (bcd) 123 (b) 601 (abc)2mL 025 (ab) 138 (abc) 632 (ab)3mL 033 (abc) 150 (ab) 684 (b)4mL 038 (ab) 168 (ab) 686 (abc)5mL 044 (abc) 191 (bcd) 733 (ab)

Exp2

1mL 011 (b) 107 (ab) 534 (b)2mL 014 (ab) 119 (ab) 559 (ab)3mL 017 (abc) 127 (ab) 579 (ab)4mL 021 (ab) 153 (abc) 631 (abc)5mL 026 (ab) 166 (b) 667 (b)

Exp3

1mL 026 (bcd) 297 (ab) 753 (bcd)2mL 033 (b) 325 (ab) 787 (ab)3mL 040 (ab) 350 (bcd) 811 (bcd)4mL 044 (b) 384 (b) 861 (b)5mL 062 (ab) 400 (bcd) 905 (ab)

aExp0 nutrient solution (control) Exp1 Bacillus cereus Exp2 Pseudomonas spp Exp3 consortium of Bacillus cereus + Pseudomonas spp (1 1)bMeanof two repeated experiments (30 plants)Different letterswithin parenthesis indicate significant difference between treatments for each growth parameterusing Duncanrsquos multiple range test (119875 = 005)

0102030405060708090

100

Concentration of bacterial cultures (mL)1 2 3 4 5

Germination ()

Exp0

Exp1 Exp3

Exp2

Figure 3 Germination percentage ofVigna unguiculata seeds sownin vermiculite supplementedwithBacillus cereus with Pseudomonasspp and with their consortium at five different concentrations (1 23 4 and 5mL) and in the control Exp0 nutrient solution (control)Exp1 Bacillus cereus Exp2 Pseudomonas spp Exp3 consortium ofBacillus cereus + Pseudomonas spp (1 1)

not treated with the bacterial culture [41] In earlier reportsroot elongation was found to occur in Sesbania aculeata byinoculation with Azotobacter spp and Pseudomonas spp in

Vigna radiata by Pseudomonas putida [16] and in Pennisetumamericanum by Azospirillum brasilense [42] Esitken et al(2006) had found out that Bacillus OSU-142 and Pseu-domonas BA-8 alone or in combination had a great potentialto increase the growth yield and nutrition of sweet cherryplant [43] BacillusM3 BacillusOSU-142 andMicobacteriumFS01 separately or in combination were found to have greatpotential for use as plant growth promoting rhizobacteriato increase production in apples and in many other crops[44] These phosphate solubilizing microbes consist pre-dominantly of fungal bacterial and actinomycetes speciescollectively called phosphate solubilizing microorganismsmdashPSM [45] There are several microorganisms which canalso solubilize the cheaper sources of phosphorous such asrock phosphate Bacteria such as Bacillus are widely usedin plant production system and are important phosphorussolubilizing microorganisms resulting in improved growthyield of crops andmetabolic activities especially in synthesisof protein [34 46]

5 Conclusions

This study illustrates the isolation of plant growth promotingbacteria from vermisources screening under in vitro con-ditions for multiple PGPR traits and their evaluation undercontrolled conditions in a pot experiment The findings ofthis study indicated that the consortium of B cereus and

10 Advances in Agriculture

Table5Ch

loroph

yllcon

tent

ofleavesofVignaun

guicu

latagrow

non

verm

iculite

supp

lementedwith

Bacilluscereuswith

Pseudomonas

sppand

with

theircon

sortium

(11)atfi

vedifferent

concentrations

(1234and

5mL)

onthe5

thdaythe10thdayandthe15thdayandin

thec

ontro

l

Experim

entsa

Quantity

ofbacterial

Cultu

resa

dded

Chloroph

yllcon

tent

(mggof

fresh

leaf)b

5thday

10th

day

15th

day

Chloroph

ylla

Chloroph

yllb

Total

chloroph

yll

Chloroph

ylla

Chloroph

yllb

Total

chloroph

yll

Chloroph

ylla

Chloroph

yllb

Total

chloroph

yll

Exp0

1mL

021

(ab)

285

(abc)

307

(a)

020

(abc)

481

(a)

501

(ab)

015

(bcd)

612

(abc)

627

(bcd)

2mL

022

(abc)

293

(ab)

315

(abc)

021

(ab)

489

(ab)

510

(bcd)

012

(abc)

618

(ab)

630

(ab)

3mL

023

(abc)

301

(ab)

315

(abc)

022

(bcd)

497

(bcd)

518

(a)

014

(a)

633

(cde)

647

(bcd)

4mL

024

(ab)

308

(f)

332

(a)

022

(a)

504

(b)

527

(abc)

015

(bcd)

641

(cde)

656

(a)

5mL

025

(f)

316

(abc)

341

(bcd)

019

(ab)

511(ab)

530

(abc)

011(cde)

645

(ab)

650

(abc)

Exp1

1mL

035

(b)

400

(cde)

435

(abc)

011(b)

581

(cde)

593

(cde)

010

(abc)

704(abc)

715(f)

2mL

026

(abc)

462

(bcd)

488

(f)

011(abc)

647

(b)

658

(abc)

006

(ab)

779(bcd)

785(a)

3mL

021

(bcd)

526

(abc)

547

(ab)

008

(bcd)

718(ab)

726(bcd)

006

(f)

847

(f)

853

(ab)

4mL

008

(f)

587

(cde)

595

(b)

011(bcd)

778(abc)

789(abc)

004

(bcd)

933(b)

937(bcd)

5mL

026

(abc)

641

(bcd)

667

(abc)

025

(cde)

834

(cde)

859

(f)

007

(abc)

976(cde)

983(abc)

Exp2

1mL

030

(abc)

354

(ab)

384

(b)

012

(a)

545

(abc)

556

(ab)

007

(bcd)

681

(abc)

688

(bcd)

2mL

029

(bcd)

420

(f)

449

(abc)

011(bcd)

610

(ab)

621

(abc)

007

(abc)

744(f)

752(bcd)

3mL

022

(ab)

504

(abc)

527

(bcd)

015

(a)

677

(cde)

692

(bcd)

006

(bcd)

812

(ab)

817

(bcd)

4mL

010

(f)

566

(a)

576

(cde)

018

(abc)

744(f)

762(ab)

005

(ab)

885

(a)

890

(abc)

5mL

006

(bcd)

622

(abc)

628

(abc)

018

(b)

810

(abc)

828

(a)

004

(cde)

948(a)

952(f)

Exp3

1mL

027

(ab)

470

(bcd)

496

(cde)

009

(ab)

668

(b)

677

(cde)

010

(a)

814

(ab)

825

(a)

2mL

020

(ab)

548

(a)

568

(f)

003

(bcd)

746(bcd)

749(ab)

009

(ab)

887

(f)

897

(ab)

3mL

005

(bcd)

584

(abc)

589

(ab)

008

(bcd)

804

(abc)

812

(cde)

010

(ab)

953(abc)

963(bcd)

4mL

023

(f)

663

(ab)

686

(cde)

007

(ab)

870

(b)

877

(f)

010

(abc)

1019

(bcd)

1029(f)

5mL

022

(ab)

744(cde)

766(a)

009

(a)

932(abc)

940(ab)

010

(a)

1085(ab)

1095(abc)

a Exp0nu

trient

solutio

n(con

trol)

Exp1B

acillus

cereusE

xp2Pseudomonas

sppE

xp3consortiu

mof

Bacilluscereus+

Pseudomonas

spp(11)

b Meanof

tworepeated

experim

ents(30plants)

Differentletterswith

inparenthesis

indicatesig

nificantd

ifference

betweentre

atmentsfore

achgrow

thparameter

usingDun

canrsquos

multip

lerangetest(119875=005)

Advances in Agriculture 11

Table 6 Fresh weight and dry weight of whole plant of Vigna unguiculata grown on vermiculite supplemented with Bacillus cereus withPseudomonas spp and with their consortium (1 1) at five different concentrations (1 2 3 4 and 5mL) on the 5th the 10th and the 15 daysand in the control

Experimentsa Quantity of bacterial cultures added Fresh weight of the whole plant (g)b Dry weight of the whole plant (g)b

5th day 10th day 15th day 5th day 10th day 15th day

Exp0

1mL 058 (bcd) 061 (abc) 064 (ab) 006 (a) 006 (abc) 006 (ab)2mL 058 (ab) 061 (a) 064 (abc) 006 (bcd) 006 (ab) 006 (ab)3mL 058 (abc) 061 (ab) 064 (bcd) 006 (abc) 006 (bcd) 006 (b)4mL 041 (abc) 061 (ab) 064 (ab) 006 (abc) 006 (ab) 006 (abc)5mL 058 (abc) 061 (abc) 064 (bcd) 006 (a) 006 (bcd) 006 (def)

Exp1

1mL 062 (abc) 065 (abc) 068 (ab) 006 (bcd) 006 (ab) 007 (bcd)2mL 065 (bcd) 068 (bcd) 071 (def) 006 (abc) 007 (a) 007 (ab)3mL 068 (abc) 071 (def) 074 (bcd) 007 (abc) 007 (ab) 007 (abc)4mL 071 (ab) 074 (abc) 077 (abc) 007 (bcd) 007 (b) 008 (bcd)5mL 074 (abc) 077 (def) 080 (ab) 007 (def) 008 (a) 008 (b)

Exp2

1mL 056 (ab) 059 (bcd) 062 (ab) 006 (ab) 006 (ab) 006 (abc)2mL 059 (abc) 062 (abc) 065 (def) 006 (ab) 006 (abc) 006 (bcd)3mL 062 (bcd) 065 (a) 068 (bcd) 006 (abc) 006 (bcd) 007 (abc)4mL 065 (abc) 068 (ab) 071 (b) 006 (ab) 007 (abc) 007 (b)5mL 068 (abc) 071 (b) 074 (a) 007 (def) 007 (abc) 007 (bcd)

Exp3

1mL 074 (ab) 077 (ab) 080 (abc) 007 (a) 008 (bcd) 008 (ab)2mL 077 (bcd) 080 (a) 083 (bcd) 008 (a) 008 (bcd) 008 (abc)3mL 080 (bcd) 083 (bcd) 086 (abc) 008 (ab) 008 (b) 009 (b)4mL 083 (bcd) 086 (abc) 089 (ab) 008 (abc) 009 (ab) 009 (bcd)5mL 086 (ab) 089 (b) 092 (def) 009 (bcd) 009 (bcd 009 (b)

aExp0 nutrient solution (control) Exp1 Bacillus cereus Exp2 Pseudomonas spp Exp3 consortium of Bacillus cereus + Pseudomonas spp (1 1)bMeanof two repeated experiments (30 plants)Different letterswithin parenthesis indicate significant difference between treatments for each growth parameterusing Duncanrsquos multiple range test (119875 = 005)

Pseudomonas spp acts as a potential biofertilizer due to itsvarious plant growth promotion abilities such as solubiliza-tion of phosphate and production of IAA and siderophore itcan be developed as potential microbial inoculant for variouscrops

Conflict of Interests

The authors declared that there is no conflict of interestsregarding this paper

Acknowledgments

Theauthors are thankful to theDepartment of BiologyGand-higram Rural InstitutemdashDeemed University GandhigramThis research was funded by University Grants Commissionunder UGC Research Fellowship in Science for MeritoriousStudents (BSR) Grant no F4-12008

References

[1] U S Bhawalkar Vermiculture Ecotechnology EarthwormResearch Institute Pune India 2nd edition 1996

[2] S Kiss D M Bularda and D Radulescu ldquoBiological Signifi-cance of Enzymes Accumulated in SoilrdquoAdvances in Agronomyvol 27 pp 25ndash87 1975

[3] D L Karlen M J Mausbach J W Doran R G Cline R FHarris and G E Schuman ldquoSoil quality a concept definitionand framework for evaluationrdquo Soil Science Society of AmericaJournal vol 61 no 1 pp 4ndash10 1997

[4] R M Atiyeh C A Edwards S Subler and J Metzger ldquoEarth-worm processed organic wastes as components of horticulturalpotting media for growing manifold and vegetable seedlingsrdquoCompost Science and Utilization vol 8 no 3 pp 215ndash223 2000

[5] N Vassilev and M Vassileva ldquoBiotechnological solubilizationof rock phosphate on media containing agro-industrial wastesrdquoApplied Microbiology and Biotechnology vol 61 no 5-6 pp435ndash440 2003

[6] S A Omar ldquoThe role of rock-phosphate-solubilizing fungiand vesicular-arbusular-mycorrhiza (VAM) in growth of wheatplants fertilized with rock phosphaterdquo World Journal of Micro-biology and Biotechnology vol 14 no 2 pp 211ndash218 1998

[7] J A J Paul and T Daniel ldquoStandardization of sampling methodfor physical characterization of municipal solid wasterdquo AsianJournal of Water Environment and Pollution vol 5 no 1 pp95ndash98 2007

[8] R Lalloo D Maharajh J Gorgens and N Gardiner ldquoA down-stream process for production of a viable and stable Bacilluscereus aquaculture biological agentrdquo Applied Microbiology andBiotechnology vol 86 no 2 pp 499ndash508 2010

[9] B Sivasankari N Kavikumar and M Anandharaj ldquoIndole-3-acetic acid production and enhanced Plant growth promotionby Indigenous bacterial speciesrdquo Journal of Current Research inScience vol 1 no 5 pp 331ndash335 2013

12 Advances in Agriculture

[10] J Han L Sun X Dong et al ldquoCharacterization of a novel plantgrowth-promoting bacteria strain Delftia tsuruhatensis HR4both as a diazotroph and a potential biocontrol agent againstvarious plant pathogensrdquo Systematic and Applied Microbiologyvol 28 no 1 pp 66ndash76 2005

[11] H Rodrıguez and R Fraga ldquoPhosphate solubilizing bacte-ria and their role in plant growth promotionrdquo BiotechnologyAdvances vol 17 no 4-5 pp 319ndash339 1999

[12] T Daniel and N Karmegam ldquoBio-conversion of selected leaflitters using an African epigeic earthworm Eudrilus eugeniaerdquoEcology Environment and Conservation vol 5 no 3 pp 271ndash275 1999

[13] J Dobereiner I E Marriel and M Nery ldquoEcological distri-bution of Spirillum lipoferum Beijerinckrdquo Canadian Journal ofMicrobiology vol 22 no 10 pp 1464ndash1473 1976

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

[15] M Edi Premono A M Moawad and P L G Vlek ldquoEffect ofphosphate-solubilizing Pseudomonas putida on the growth ofmaize and its survival in the rhizosphererdquo Indonesian Journal ofCrop Science vol 11 pp 13ndash23 1996

[16] C L Patten and B R Glick ldquoBacterial biosynthesis of indole-3-acetic acidrdquo Canadian Journal of Microbiology vol 42 no 3pp 207ndash220 1996

[17] N Amaresan V Jayakumar K Kumar and N ThajuddinldquoIsolation and characterization of plant growth promotingendophytic bacteria and their effect on tomato (Lycopersiconesculentum) and chilli (Capsicum annuum) seedling growthrdquoAnnals of Microbiology vol 62 no 2 pp 805ndash810 2012

[18] B Schwyn and J B Neilands ldquoUniversal chemical assay forthe detection and determination of siderophoresrdquo AnalyticalBiochemistry vol 160 no 1 pp 47ndash56 1987

[19] P Rahi P Vyas S Sharma and A Gulati ldquoPlant growthpromoting potential of the fungus Discosia sp FIHB 571 fromtea rhizosphere tested on chickpea maize and peardquo IndianJournal of Microbiology vol 49 no 2 pp 128ndash133 2009

[20] K Kumar N Amaresan S Bhagat K Madhuri and R CSrivastava ldquoIsolation and characterization of rhizobacteriaassociated with coastal agricultural ecosystem of rhizospheresoils of cultivated vegetable cropsrdquo World Journal of Microbiol-ogy and Biotechnology vol 27 no 7 pp 1625ndash1632 2011

[21] J A Smoker and S R Barnum ldquoRapid small scale DNAisolation from filamentous cyanobacteriardquo FEMS MicrobiologyLetters vol 56 no 1 pp 119ndash122 1988

[22] U Edwars T Rogall H Blocker M Emde and E C BottgerldquoIsolation and direct complete nucleotide determination ofentire genes Characterization of a gene coding for 16S riboso-mal RNArdquoNucleic Acids Research vol 17 no 19 pp 7843ndash78531989

[23] D G Higgins A J Bleasby and R Fuchs ldquoCLUSTAL Vimproved software for multiple sequence alignmentrdquo ComputerApplications in the Biosciences vol 8 no 2 pp 189ndash191 1992

[24] M Kimura ldquoA simple method for estimating evolutionary ratesof base substitutions through comparative studies of nucleotidesequencesrdquo Journal ofMolecular Evolution vol 16 no 2 pp 111ndash120 1980

[25] N Saitou and M Nei ldquoThe neighbor-joining method a newmethod for reconstructing phylogenetic treesrdquo Molecular Biol-ogy and Evolution vol 4 no 4 pp 406ndash425 1987

[26] E Barbu K Y Lee and R Wahl ldquoContent of purine andpyrimidine base in desoxyribonucleic acid of bacteriardquo Annalesde lrsquoInstitut Pasteur vol 91 no 2 pp 212ndash224 1956

[27] N Sueoka ldquoVariation and heterogeneity of base compositionof deoxyribonucleic acids a compilation of old and new datardquoJournal of Molecular Biology vol 3 no 1 pp 31ndash40 1961

[28] H Hori and S Osawa ldquoEvolutionary change in 5S rRNAsecondary structure and a phylogenic tree of 352 5S rRNAspeciesrdquo BioSystems vol 19 no 3 pp 163ndash172 1986

[29] V Bess ldquoEvaluating microbiology of compost microbial con-tent of composting is helping the producers and growers tounderstand its role as a soil inoculant and plantrdquo BioCycleMagazine p 62 1999

[30] O Grafff ldquoPreliminary experiments of vermin composting ofdifferent waste materials using Eudrilus eugeniae Kinbergrdquo inWorkshop on the Role of Earthworms in the Stabilization ofOrganic Residues M Happelhof Ed pp 178ndash191 Beech LeafPress Kalamazoo Mich USA 1981

[31] J E Satchell Earthworm Ecology from Darwin to VermicultureChapman and Hall 1983

[32] G H Xie M Y Cai G C Tao and Y Steinberger ldquoCultivableheterotrophic N2-fixing bacterial diversity in rice fields in theYangtze River Plainrdquo Biology and Fertility of Soils vol 37 no 1pp 29ndash38 2003

[33] B BMcSpaddenGardener ldquoEcology ofBacillus and Paenibacil-lus spp in agricultural systemsrdquo Phytopathology vol 94 no 11pp 1252ndash1258 2004

[34] J Dobereiner ldquoBiological nitrogen fixation in the tropics socialand economic contributionsrdquo Soil Biology andBiochemistry vol29 no 5-6 pp 771ndash774 1997

[35] V M Reis F L Olivares and J Dobereiner ldquoImprovedmethodology for isolation of Acetobacter diazotrophicus andconfirmation of its endophytic habitatrdquoWorld Journal of Micro-biology and Biotechnology vol 10 no 4 pp 401ndash405 1994

[36] S Khalil N Ayub S Alam and F Latif ldquoOrganic acids pro-duction and phosphate solubilization by phosphate solubilizingmicroorganisms (PSM) under in vitro conditionsrdquo PakistanJournal of Biological Sciences vol 7 no 2 pp 187ndash196 2004

[37] H M A El-Komy ldquoCoimmobilization of Azospirillumlipoferum and Bacillus megaterium for successful phosphorusand nitrogen nutrition of wheat plantsrdquo Food Technology andBiotechnology vol 43 no 1 pp 19ndash27 2005

[38] N B Paul and W V B Sundara Rao ldquoPhosphate-dissolvingbacteria in the rhizosphere of some cultivated legumesrdquo Plantand Soil vol 35 no 1 pp 127ndash132 1971

[39] A Beneduzi D Peres L K Vargas M H Bodanese-Zanettiniand LM P Passaglia ldquoEvaluation of genetic diversity and plantgrowth promoting activities of nitrogen-fixing bacilli isolatedfrom rice fields in South Brazilrdquo Applied Soil Ecology vol 39no 3 pp 311ndash320 2008

[40] M P Raimam U Albino M F Cruz et al ldquoInteraction amongfree-living N-fixing bacteria isolated from Drosera villosa varvillosa and AM fungi (Glomus clarum) in rice (Oryza sativa)rdquoApplied Soil Ecology vol 35 no 1 pp 25ndash34 2007

[41] M R Swain S K Naskar and R C Ray ldquoIndole-3-acetic acidproduction and effect on sprouting of yam (Dioscorea rotundataL) minisetts by Bacillus subtilis isolated from culturable cow-dung microflorardquo Polish Journal of Microbiology vol 56 no 2pp 103ndash110 2007

[42] T M Tien M H Garkins and D H Hubbell ldquoPlant growthsubstances produced by Azospirillum brasilense and their effect

Advances in Agriculture 13

on the growth of pearl Millet Pennisetum americanum (L)rdquoApplied and EnvironmentalMicrobiology vol 37 no 5 pp 1016ndash1024 1979

[43] A Esitken L Pirlak M Turan and F Sahin ldquoEffects of floraland foliar application of plant growth promoting rhizobacteria(PGPR) on yield growth and nutrition of sweet cherryrdquo ScientiaHorticulturae vol 110 no 4 pp 324ndash327 2006

[44] H Karlidag A Esitken M Turan and F Sahin ldquoEffects of rootinoculation of plant growth promoting rhizobacteria (PGPR)on yield growth and nutrient element contents of leaves ofapplerdquo Scientia Horticulturae vol 114 no 1 pp 16ndash20 2007

[45] C S Nautiyal S Bhadauria P Kumar H Lal R Mondal andD Verma ldquoStress induced phosphate solubilization in bacteriaisolated fromalkaline soilsrdquoFEMSMicrobiology Letters vol 182no 2 pp 291ndash296 2000

[46] A K Halder A K Mishra P Bhattacharya and P KChakrabarthy ldquoSolubilization of inorganic phosphates byBradyrhizobiumrdquo Indian Journal of Experimental Biology vol29 pp 28ndash31 1991

Submit your manuscripts athttpwwwhindawicom

Nutrition and Metabolism

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Food ScienceInternational Journal of

Agronomy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Applied ampEnvironmentalSoil Science

Volume 2014

AgricultureAdvances in

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PsycheHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BiodiversityInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Plant GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biotechnology Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Forestry ResearchInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of BotanyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

EcologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Veterinary Medicine International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Cell BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Page 10: BalayoganSivasankariandMarimuthuAnandharajdownloads.hindawi.com/journals/aag/2014/248591.pdf · In Vitro Nitrogen Fixing Activity. e bacterial isolates were screened for nitrogen

10 Advances in Agriculture

Table5Ch

loroph

yllcon

tent

ofleavesofVignaun

guicu

latagrow

non

verm

iculite

supp

lementedwith

Bacilluscereuswith

Pseudomonas

sppand

with

theircon

sortium

(11)atfi

vedifferent

concentrations

(1234and

5mL)

onthe5

thdaythe10thdayandthe15thdayandin

thec

ontro

l

Experim

entsa

Quantity

ofbacterial

Cultu

resa

dded

Chloroph

yllcon

tent

(mggof

fresh

leaf)b

5thday

10th

day

15th

day

Chloroph

ylla

Chloroph

yllb

Total

chloroph

yll

Chloroph

ylla

Chloroph

yllb

Total

chloroph

yll

Chloroph

ylla

Chloroph

yllb

Total

chloroph

yll

Exp0

1mL

021

(ab)

285

(abc)

307

(a)

020

(abc)

481

(a)

501

(ab)

015

(bcd)

612

(abc)

627

(bcd)

2mL

022

(abc)

293

(ab)

315

(abc)

021

(ab)

489

(ab)

510

(bcd)

012

(abc)

618

(ab)

630

(ab)

3mL

023

(abc)

301

(ab)

315

(abc)

022

(bcd)

497

(bcd)

518

(a)

014

(a)

633

(cde)

647

(bcd)

4mL

024

(ab)

308

(f)

332

(a)

022

(a)

504

(b)

527

(abc)

015

(bcd)

641

(cde)

656

(a)

5mL

025

(f)

316

(abc)

341

(bcd)

019

(ab)

511(ab)

530

(abc)

011(cde)

645

(ab)

650

(abc)

Exp1

1mL

035

(b)

400

(cde)

435

(abc)

011(b)

581

(cde)

593

(cde)

010

(abc)

704(abc)

715(f)

2mL

026

(abc)

462

(bcd)

488

(f)

011(abc)

647

(b)

658

(abc)

006

(ab)

779(bcd)

785(a)

3mL

021

(bcd)

526

(abc)

547

(ab)

008

(bcd)

718(ab)

726(bcd)

006

(f)

847

(f)

853

(ab)

4mL

008

(f)

587

(cde)

595

(b)

011(bcd)

778(abc)

789(abc)

004

(bcd)

933(b)

937(bcd)

5mL

026

(abc)

641

(bcd)

667

(abc)

025

(cde)

834

(cde)

859

(f)

007

(abc)

976(cde)

983(abc)

Exp2

1mL

030

(abc)

354

(ab)

384

(b)

012

(a)

545

(abc)

556

(ab)

007

(bcd)

681

(abc)

688

(bcd)

2mL

029

(bcd)

420

(f)

449

(abc)

011(bcd)

610

(ab)

621

(abc)

007

(abc)

744(f)

752(bcd)

3mL

022

(ab)

504

(abc)

527

(bcd)

015

(a)

677

(cde)

692

(bcd)

006

(bcd)

812

(ab)

817

(bcd)

4mL

010

(f)

566

(a)

576

(cde)

018

(abc)

744(f)

762(ab)

005

(ab)

885

(a)

890

(abc)

5mL

006

(bcd)

622

(abc)

628

(abc)

018

(b)

810

(abc)

828

(a)

004

(cde)

948(a)

952(f)

Exp3

1mL

027

(ab)

470

(bcd)

496

(cde)

009

(ab)

668

(b)

677

(cde)

010

(a)

814

(ab)

825

(a)

2mL

020

(ab)

548

(a)

568

(f)

003

(bcd)

746(bcd)

749(ab)

009

(ab)

887

(f)

897

(ab)

3mL

005

(bcd)

584

(abc)

589

(ab)

008

(bcd)

804

(abc)

812

(cde)

010

(ab)

953(abc)

963(bcd)

4mL

023

(f)

663

(ab)

686

(cde)

007

(ab)

870

(b)

877

(f)

010

(abc)

1019

(bcd)

1029(f)

5mL

022

(ab)

744(cde)

766(a)

009

(a)

932(abc)

940(ab)

010

(a)

1085(ab)

1095(abc)

a Exp0nu

trient

solutio

n(con

trol)

Exp1B

acillus

cereusE

xp2Pseudomonas

sppE

xp3consortiu

mof

Bacilluscereus+

Pseudomonas

spp(11)

b Meanof

tworepeated

experim

ents(30plants)

Differentletterswith

inparenthesis

indicatesig

nificantd

ifference

betweentre

atmentsfore

achgrow

thparameter

usingDun

canrsquos

multip

lerangetest(119875=005)

Advances in Agriculture 11

Table 6 Fresh weight and dry weight of whole plant of Vigna unguiculata grown on vermiculite supplemented with Bacillus cereus withPseudomonas spp and with their consortium (1 1) at five different concentrations (1 2 3 4 and 5mL) on the 5th the 10th and the 15 daysand in the control

Experimentsa Quantity of bacterial cultures added Fresh weight of the whole plant (g)b Dry weight of the whole plant (g)b

5th day 10th day 15th day 5th day 10th day 15th day

Exp0

1mL 058 (bcd) 061 (abc) 064 (ab) 006 (a) 006 (abc) 006 (ab)2mL 058 (ab) 061 (a) 064 (abc) 006 (bcd) 006 (ab) 006 (ab)3mL 058 (abc) 061 (ab) 064 (bcd) 006 (abc) 006 (bcd) 006 (b)4mL 041 (abc) 061 (ab) 064 (ab) 006 (abc) 006 (ab) 006 (abc)5mL 058 (abc) 061 (abc) 064 (bcd) 006 (a) 006 (bcd) 006 (def)

Exp1

1mL 062 (abc) 065 (abc) 068 (ab) 006 (bcd) 006 (ab) 007 (bcd)2mL 065 (bcd) 068 (bcd) 071 (def) 006 (abc) 007 (a) 007 (ab)3mL 068 (abc) 071 (def) 074 (bcd) 007 (abc) 007 (ab) 007 (abc)4mL 071 (ab) 074 (abc) 077 (abc) 007 (bcd) 007 (b) 008 (bcd)5mL 074 (abc) 077 (def) 080 (ab) 007 (def) 008 (a) 008 (b)

Exp2

1mL 056 (ab) 059 (bcd) 062 (ab) 006 (ab) 006 (ab) 006 (abc)2mL 059 (abc) 062 (abc) 065 (def) 006 (ab) 006 (abc) 006 (bcd)3mL 062 (bcd) 065 (a) 068 (bcd) 006 (abc) 006 (bcd) 007 (abc)4mL 065 (abc) 068 (ab) 071 (b) 006 (ab) 007 (abc) 007 (b)5mL 068 (abc) 071 (b) 074 (a) 007 (def) 007 (abc) 007 (bcd)

Exp3

1mL 074 (ab) 077 (ab) 080 (abc) 007 (a) 008 (bcd) 008 (ab)2mL 077 (bcd) 080 (a) 083 (bcd) 008 (a) 008 (bcd) 008 (abc)3mL 080 (bcd) 083 (bcd) 086 (abc) 008 (ab) 008 (b) 009 (b)4mL 083 (bcd) 086 (abc) 089 (ab) 008 (abc) 009 (ab) 009 (bcd)5mL 086 (ab) 089 (b) 092 (def) 009 (bcd) 009 (bcd 009 (b)

aExp0 nutrient solution (control) Exp1 Bacillus cereus Exp2 Pseudomonas spp Exp3 consortium of Bacillus cereus + Pseudomonas spp (1 1)bMeanof two repeated experiments (30 plants)Different letterswithin parenthesis indicate significant difference between treatments for each growth parameterusing Duncanrsquos multiple range test (119875 = 005)

Pseudomonas spp acts as a potential biofertilizer due to itsvarious plant growth promotion abilities such as solubiliza-tion of phosphate and production of IAA and siderophore itcan be developed as potential microbial inoculant for variouscrops

Conflict of Interests

The authors declared that there is no conflict of interestsregarding this paper

Acknowledgments

Theauthors are thankful to theDepartment of BiologyGand-higram Rural InstitutemdashDeemed University GandhigramThis research was funded by University Grants Commissionunder UGC Research Fellowship in Science for MeritoriousStudents (BSR) Grant no F4-12008

References

[1] U S Bhawalkar Vermiculture Ecotechnology EarthwormResearch Institute Pune India 2nd edition 1996

[2] S Kiss D M Bularda and D Radulescu ldquoBiological Signifi-cance of Enzymes Accumulated in SoilrdquoAdvances in Agronomyvol 27 pp 25ndash87 1975

[3] D L Karlen M J Mausbach J W Doran R G Cline R FHarris and G E Schuman ldquoSoil quality a concept definitionand framework for evaluationrdquo Soil Science Society of AmericaJournal vol 61 no 1 pp 4ndash10 1997

[4] R M Atiyeh C A Edwards S Subler and J Metzger ldquoEarth-worm processed organic wastes as components of horticulturalpotting media for growing manifold and vegetable seedlingsrdquoCompost Science and Utilization vol 8 no 3 pp 215ndash223 2000

[5] N Vassilev and M Vassileva ldquoBiotechnological solubilizationof rock phosphate on media containing agro-industrial wastesrdquoApplied Microbiology and Biotechnology vol 61 no 5-6 pp435ndash440 2003

[6] S A Omar ldquoThe role of rock-phosphate-solubilizing fungiand vesicular-arbusular-mycorrhiza (VAM) in growth of wheatplants fertilized with rock phosphaterdquo World Journal of Micro-biology and Biotechnology vol 14 no 2 pp 211ndash218 1998

[7] J A J Paul and T Daniel ldquoStandardization of sampling methodfor physical characterization of municipal solid wasterdquo AsianJournal of Water Environment and Pollution vol 5 no 1 pp95ndash98 2007

[8] R Lalloo D Maharajh J Gorgens and N Gardiner ldquoA down-stream process for production of a viable and stable Bacilluscereus aquaculture biological agentrdquo Applied Microbiology andBiotechnology vol 86 no 2 pp 499ndash508 2010

[9] B Sivasankari N Kavikumar and M Anandharaj ldquoIndole-3-acetic acid production and enhanced Plant growth promotionby Indigenous bacterial speciesrdquo Journal of Current Research inScience vol 1 no 5 pp 331ndash335 2013

12 Advances in Agriculture

[10] J Han L Sun X Dong et al ldquoCharacterization of a novel plantgrowth-promoting bacteria strain Delftia tsuruhatensis HR4both as a diazotroph and a potential biocontrol agent againstvarious plant pathogensrdquo Systematic and Applied Microbiologyvol 28 no 1 pp 66ndash76 2005

[11] H Rodrıguez and R Fraga ldquoPhosphate solubilizing bacte-ria and their role in plant growth promotionrdquo BiotechnologyAdvances vol 17 no 4-5 pp 319ndash339 1999

[12] T Daniel and N Karmegam ldquoBio-conversion of selected leaflitters using an African epigeic earthworm Eudrilus eugeniaerdquoEcology Environment and Conservation vol 5 no 3 pp 271ndash275 1999

[13] J Dobereiner I E Marriel and M Nery ldquoEcological distri-bution of Spirillum lipoferum Beijerinckrdquo Canadian Journal ofMicrobiology vol 22 no 10 pp 1464ndash1473 1976

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

[15] M Edi Premono A M Moawad and P L G Vlek ldquoEffect ofphosphate-solubilizing Pseudomonas putida on the growth ofmaize and its survival in the rhizosphererdquo Indonesian Journal ofCrop Science vol 11 pp 13ndash23 1996

[16] C L Patten and B R Glick ldquoBacterial biosynthesis of indole-3-acetic acidrdquo Canadian Journal of Microbiology vol 42 no 3pp 207ndash220 1996

[17] N Amaresan V Jayakumar K Kumar and N ThajuddinldquoIsolation and characterization of plant growth promotingendophytic bacteria and their effect on tomato (Lycopersiconesculentum) and chilli (Capsicum annuum) seedling growthrdquoAnnals of Microbiology vol 62 no 2 pp 805ndash810 2012

[18] B Schwyn and J B Neilands ldquoUniversal chemical assay forthe detection and determination of siderophoresrdquo AnalyticalBiochemistry vol 160 no 1 pp 47ndash56 1987

[19] P Rahi P Vyas S Sharma and A Gulati ldquoPlant growthpromoting potential of the fungus Discosia sp FIHB 571 fromtea rhizosphere tested on chickpea maize and peardquo IndianJournal of Microbiology vol 49 no 2 pp 128ndash133 2009

[20] K Kumar N Amaresan S Bhagat K Madhuri and R CSrivastava ldquoIsolation and characterization of rhizobacteriaassociated with coastal agricultural ecosystem of rhizospheresoils of cultivated vegetable cropsrdquo World Journal of Microbiol-ogy and Biotechnology vol 27 no 7 pp 1625ndash1632 2011

[21] J A Smoker and S R Barnum ldquoRapid small scale DNAisolation from filamentous cyanobacteriardquo FEMS MicrobiologyLetters vol 56 no 1 pp 119ndash122 1988

[22] U Edwars T Rogall H Blocker M Emde and E C BottgerldquoIsolation and direct complete nucleotide determination ofentire genes Characterization of a gene coding for 16S riboso-mal RNArdquoNucleic Acids Research vol 17 no 19 pp 7843ndash78531989

[23] D G Higgins A J Bleasby and R Fuchs ldquoCLUSTAL Vimproved software for multiple sequence alignmentrdquo ComputerApplications in the Biosciences vol 8 no 2 pp 189ndash191 1992

[24] M Kimura ldquoA simple method for estimating evolutionary ratesof base substitutions through comparative studies of nucleotidesequencesrdquo Journal ofMolecular Evolution vol 16 no 2 pp 111ndash120 1980

[25] N Saitou and M Nei ldquoThe neighbor-joining method a newmethod for reconstructing phylogenetic treesrdquo Molecular Biol-ogy and Evolution vol 4 no 4 pp 406ndash425 1987

[26] E Barbu K Y Lee and R Wahl ldquoContent of purine andpyrimidine base in desoxyribonucleic acid of bacteriardquo Annalesde lrsquoInstitut Pasteur vol 91 no 2 pp 212ndash224 1956

[27] N Sueoka ldquoVariation and heterogeneity of base compositionof deoxyribonucleic acids a compilation of old and new datardquoJournal of Molecular Biology vol 3 no 1 pp 31ndash40 1961

[28] H Hori and S Osawa ldquoEvolutionary change in 5S rRNAsecondary structure and a phylogenic tree of 352 5S rRNAspeciesrdquo BioSystems vol 19 no 3 pp 163ndash172 1986

[29] V Bess ldquoEvaluating microbiology of compost microbial con-tent of composting is helping the producers and growers tounderstand its role as a soil inoculant and plantrdquo BioCycleMagazine p 62 1999

[30] O Grafff ldquoPreliminary experiments of vermin composting ofdifferent waste materials using Eudrilus eugeniae Kinbergrdquo inWorkshop on the Role of Earthworms in the Stabilization ofOrganic Residues M Happelhof Ed pp 178ndash191 Beech LeafPress Kalamazoo Mich USA 1981

[31] J E Satchell Earthworm Ecology from Darwin to VermicultureChapman and Hall 1983

[32] G H Xie M Y Cai G C Tao and Y Steinberger ldquoCultivableheterotrophic N2-fixing bacterial diversity in rice fields in theYangtze River Plainrdquo Biology and Fertility of Soils vol 37 no 1pp 29ndash38 2003

[33] B BMcSpaddenGardener ldquoEcology ofBacillus and Paenibacil-lus spp in agricultural systemsrdquo Phytopathology vol 94 no 11pp 1252ndash1258 2004

[34] J Dobereiner ldquoBiological nitrogen fixation in the tropics socialand economic contributionsrdquo Soil Biology andBiochemistry vol29 no 5-6 pp 771ndash774 1997

[35] V M Reis F L Olivares and J Dobereiner ldquoImprovedmethodology for isolation of Acetobacter diazotrophicus andconfirmation of its endophytic habitatrdquoWorld Journal of Micro-biology and Biotechnology vol 10 no 4 pp 401ndash405 1994

[36] S Khalil N Ayub S Alam and F Latif ldquoOrganic acids pro-duction and phosphate solubilization by phosphate solubilizingmicroorganisms (PSM) under in vitro conditionsrdquo PakistanJournal of Biological Sciences vol 7 no 2 pp 187ndash196 2004

[37] H M A El-Komy ldquoCoimmobilization of Azospirillumlipoferum and Bacillus megaterium for successful phosphorusand nitrogen nutrition of wheat plantsrdquo Food Technology andBiotechnology vol 43 no 1 pp 19ndash27 2005

[38] N B Paul and W V B Sundara Rao ldquoPhosphate-dissolvingbacteria in the rhizosphere of some cultivated legumesrdquo Plantand Soil vol 35 no 1 pp 127ndash132 1971

[39] A Beneduzi D Peres L K Vargas M H Bodanese-Zanettiniand LM P Passaglia ldquoEvaluation of genetic diversity and plantgrowth promoting activities of nitrogen-fixing bacilli isolatedfrom rice fields in South Brazilrdquo Applied Soil Ecology vol 39no 3 pp 311ndash320 2008

[40] M P Raimam U Albino M F Cruz et al ldquoInteraction amongfree-living N-fixing bacteria isolated from Drosera villosa varvillosa and AM fungi (Glomus clarum) in rice (Oryza sativa)rdquoApplied Soil Ecology vol 35 no 1 pp 25ndash34 2007

[41] M R Swain S K Naskar and R C Ray ldquoIndole-3-acetic acidproduction and effect on sprouting of yam (Dioscorea rotundataL) minisetts by Bacillus subtilis isolated from culturable cow-dung microflorardquo Polish Journal of Microbiology vol 56 no 2pp 103ndash110 2007

[42] T M Tien M H Garkins and D H Hubbell ldquoPlant growthsubstances produced by Azospirillum brasilense and their effect

Advances in Agriculture 13

on the growth of pearl Millet Pennisetum americanum (L)rdquoApplied and EnvironmentalMicrobiology vol 37 no 5 pp 1016ndash1024 1979

[43] A Esitken L Pirlak M Turan and F Sahin ldquoEffects of floraland foliar application of plant growth promoting rhizobacteria(PGPR) on yield growth and nutrition of sweet cherryrdquo ScientiaHorticulturae vol 110 no 4 pp 324ndash327 2006

[44] H Karlidag A Esitken M Turan and F Sahin ldquoEffects of rootinoculation of plant growth promoting rhizobacteria (PGPR)on yield growth and nutrient element contents of leaves ofapplerdquo Scientia Horticulturae vol 114 no 1 pp 16ndash20 2007

[45] C S Nautiyal S Bhadauria P Kumar H Lal R Mondal andD Verma ldquoStress induced phosphate solubilization in bacteriaisolated fromalkaline soilsrdquoFEMSMicrobiology Letters vol 182no 2 pp 291ndash296 2000

[46] A K Halder A K Mishra P Bhattacharya and P KChakrabarthy ldquoSolubilization of inorganic phosphates byBradyrhizobiumrdquo Indian Journal of Experimental Biology vol29 pp 28ndash31 1991

Submit your manuscripts athttpwwwhindawicom

Nutrition and Metabolism

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Food ScienceInternational Journal of

Agronomy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Applied ampEnvironmentalSoil Science

Volume 2014

AgricultureAdvances in

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PsycheHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BiodiversityInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Plant GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biotechnology Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Forestry ResearchInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of BotanyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

EcologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Veterinary Medicine International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Cell BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Page 11: BalayoganSivasankariandMarimuthuAnandharajdownloads.hindawi.com/journals/aag/2014/248591.pdf · In Vitro Nitrogen Fixing Activity. e bacterial isolates were screened for nitrogen

Advances in Agriculture 11

Table 6 Fresh weight and dry weight of whole plant of Vigna unguiculata grown on vermiculite supplemented with Bacillus cereus withPseudomonas spp and with their consortium (1 1) at five different concentrations (1 2 3 4 and 5mL) on the 5th the 10th and the 15 daysand in the control

Experimentsa Quantity of bacterial cultures added Fresh weight of the whole plant (g)b Dry weight of the whole plant (g)b

5th day 10th day 15th day 5th day 10th day 15th day

Exp0

1mL 058 (bcd) 061 (abc) 064 (ab) 006 (a) 006 (abc) 006 (ab)2mL 058 (ab) 061 (a) 064 (abc) 006 (bcd) 006 (ab) 006 (ab)3mL 058 (abc) 061 (ab) 064 (bcd) 006 (abc) 006 (bcd) 006 (b)4mL 041 (abc) 061 (ab) 064 (ab) 006 (abc) 006 (ab) 006 (abc)5mL 058 (abc) 061 (abc) 064 (bcd) 006 (a) 006 (bcd) 006 (def)

Exp1

1mL 062 (abc) 065 (abc) 068 (ab) 006 (bcd) 006 (ab) 007 (bcd)2mL 065 (bcd) 068 (bcd) 071 (def) 006 (abc) 007 (a) 007 (ab)3mL 068 (abc) 071 (def) 074 (bcd) 007 (abc) 007 (ab) 007 (abc)4mL 071 (ab) 074 (abc) 077 (abc) 007 (bcd) 007 (b) 008 (bcd)5mL 074 (abc) 077 (def) 080 (ab) 007 (def) 008 (a) 008 (b)

Exp2

1mL 056 (ab) 059 (bcd) 062 (ab) 006 (ab) 006 (ab) 006 (abc)2mL 059 (abc) 062 (abc) 065 (def) 006 (ab) 006 (abc) 006 (bcd)3mL 062 (bcd) 065 (a) 068 (bcd) 006 (abc) 006 (bcd) 007 (abc)4mL 065 (abc) 068 (ab) 071 (b) 006 (ab) 007 (abc) 007 (b)5mL 068 (abc) 071 (b) 074 (a) 007 (def) 007 (abc) 007 (bcd)

Exp3

1mL 074 (ab) 077 (ab) 080 (abc) 007 (a) 008 (bcd) 008 (ab)2mL 077 (bcd) 080 (a) 083 (bcd) 008 (a) 008 (bcd) 008 (abc)3mL 080 (bcd) 083 (bcd) 086 (abc) 008 (ab) 008 (b) 009 (b)4mL 083 (bcd) 086 (abc) 089 (ab) 008 (abc) 009 (ab) 009 (bcd)5mL 086 (ab) 089 (b) 092 (def) 009 (bcd) 009 (bcd 009 (b)

aExp0 nutrient solution (control) Exp1 Bacillus cereus Exp2 Pseudomonas spp Exp3 consortium of Bacillus cereus + Pseudomonas spp (1 1)bMeanof two repeated experiments (30 plants)Different letterswithin parenthesis indicate significant difference between treatments for each growth parameterusing Duncanrsquos multiple range test (119875 = 005)

Pseudomonas spp acts as a potential biofertilizer due to itsvarious plant growth promotion abilities such as solubiliza-tion of phosphate and production of IAA and siderophore itcan be developed as potential microbial inoculant for variouscrops

Conflict of Interests

The authors declared that there is no conflict of interestsregarding this paper

Acknowledgments

Theauthors are thankful to theDepartment of BiologyGand-higram Rural InstitutemdashDeemed University GandhigramThis research was funded by University Grants Commissionunder UGC Research Fellowship in Science for MeritoriousStudents (BSR) Grant no F4-12008

References

[1] U S Bhawalkar Vermiculture Ecotechnology EarthwormResearch Institute Pune India 2nd edition 1996

[2] S Kiss D M Bularda and D Radulescu ldquoBiological Signifi-cance of Enzymes Accumulated in SoilrdquoAdvances in Agronomyvol 27 pp 25ndash87 1975

[3] D L Karlen M J Mausbach J W Doran R G Cline R FHarris and G E Schuman ldquoSoil quality a concept definitionand framework for evaluationrdquo Soil Science Society of AmericaJournal vol 61 no 1 pp 4ndash10 1997

[4] R M Atiyeh C A Edwards S Subler and J Metzger ldquoEarth-worm processed organic wastes as components of horticulturalpotting media for growing manifold and vegetable seedlingsrdquoCompost Science and Utilization vol 8 no 3 pp 215ndash223 2000

[5] N Vassilev and M Vassileva ldquoBiotechnological solubilizationof rock phosphate on media containing agro-industrial wastesrdquoApplied Microbiology and Biotechnology vol 61 no 5-6 pp435ndash440 2003

[6] S A Omar ldquoThe role of rock-phosphate-solubilizing fungiand vesicular-arbusular-mycorrhiza (VAM) in growth of wheatplants fertilized with rock phosphaterdquo World Journal of Micro-biology and Biotechnology vol 14 no 2 pp 211ndash218 1998

[7] J A J Paul and T Daniel ldquoStandardization of sampling methodfor physical characterization of municipal solid wasterdquo AsianJournal of Water Environment and Pollution vol 5 no 1 pp95ndash98 2007

[8] R Lalloo D Maharajh J Gorgens and N Gardiner ldquoA down-stream process for production of a viable and stable Bacilluscereus aquaculture biological agentrdquo Applied Microbiology andBiotechnology vol 86 no 2 pp 499ndash508 2010

[9] B Sivasankari N Kavikumar and M Anandharaj ldquoIndole-3-acetic acid production and enhanced Plant growth promotionby Indigenous bacterial speciesrdquo Journal of Current Research inScience vol 1 no 5 pp 331ndash335 2013

12 Advances in Agriculture

[10] J Han L Sun X Dong et al ldquoCharacterization of a novel plantgrowth-promoting bacteria strain Delftia tsuruhatensis HR4both as a diazotroph and a potential biocontrol agent againstvarious plant pathogensrdquo Systematic and Applied Microbiologyvol 28 no 1 pp 66ndash76 2005

[11] H Rodrıguez and R Fraga ldquoPhosphate solubilizing bacte-ria and their role in plant growth promotionrdquo BiotechnologyAdvances vol 17 no 4-5 pp 319ndash339 1999

[12] T Daniel and N Karmegam ldquoBio-conversion of selected leaflitters using an African epigeic earthworm Eudrilus eugeniaerdquoEcology Environment and Conservation vol 5 no 3 pp 271ndash275 1999

[13] J Dobereiner I E Marriel and M Nery ldquoEcological distri-bution of Spirillum lipoferum Beijerinckrdquo Canadian Journal ofMicrobiology vol 22 no 10 pp 1464ndash1473 1976

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

[15] M Edi Premono A M Moawad and P L G Vlek ldquoEffect ofphosphate-solubilizing Pseudomonas putida on the growth ofmaize and its survival in the rhizosphererdquo Indonesian Journal ofCrop Science vol 11 pp 13ndash23 1996

[16] C L Patten and B R Glick ldquoBacterial biosynthesis of indole-3-acetic acidrdquo Canadian Journal of Microbiology vol 42 no 3pp 207ndash220 1996

[17] N Amaresan V Jayakumar K Kumar and N ThajuddinldquoIsolation and characterization of plant growth promotingendophytic bacteria and their effect on tomato (Lycopersiconesculentum) and chilli (Capsicum annuum) seedling growthrdquoAnnals of Microbiology vol 62 no 2 pp 805ndash810 2012

[18] B Schwyn and J B Neilands ldquoUniversal chemical assay forthe detection and determination of siderophoresrdquo AnalyticalBiochemistry vol 160 no 1 pp 47ndash56 1987

[19] P Rahi P Vyas S Sharma and A Gulati ldquoPlant growthpromoting potential of the fungus Discosia sp FIHB 571 fromtea rhizosphere tested on chickpea maize and peardquo IndianJournal of Microbiology vol 49 no 2 pp 128ndash133 2009

[20] K Kumar N Amaresan S Bhagat K Madhuri and R CSrivastava ldquoIsolation and characterization of rhizobacteriaassociated with coastal agricultural ecosystem of rhizospheresoils of cultivated vegetable cropsrdquo World Journal of Microbiol-ogy and Biotechnology vol 27 no 7 pp 1625ndash1632 2011

[21] J A Smoker and S R Barnum ldquoRapid small scale DNAisolation from filamentous cyanobacteriardquo FEMS MicrobiologyLetters vol 56 no 1 pp 119ndash122 1988

[22] U Edwars T Rogall H Blocker M Emde and E C BottgerldquoIsolation and direct complete nucleotide determination ofentire genes Characterization of a gene coding for 16S riboso-mal RNArdquoNucleic Acids Research vol 17 no 19 pp 7843ndash78531989

[23] D G Higgins A J Bleasby and R Fuchs ldquoCLUSTAL Vimproved software for multiple sequence alignmentrdquo ComputerApplications in the Biosciences vol 8 no 2 pp 189ndash191 1992

[24] M Kimura ldquoA simple method for estimating evolutionary ratesof base substitutions through comparative studies of nucleotidesequencesrdquo Journal ofMolecular Evolution vol 16 no 2 pp 111ndash120 1980

[25] N Saitou and M Nei ldquoThe neighbor-joining method a newmethod for reconstructing phylogenetic treesrdquo Molecular Biol-ogy and Evolution vol 4 no 4 pp 406ndash425 1987

[26] E Barbu K Y Lee and R Wahl ldquoContent of purine andpyrimidine base in desoxyribonucleic acid of bacteriardquo Annalesde lrsquoInstitut Pasteur vol 91 no 2 pp 212ndash224 1956

[27] N Sueoka ldquoVariation and heterogeneity of base compositionof deoxyribonucleic acids a compilation of old and new datardquoJournal of Molecular Biology vol 3 no 1 pp 31ndash40 1961

[28] H Hori and S Osawa ldquoEvolutionary change in 5S rRNAsecondary structure and a phylogenic tree of 352 5S rRNAspeciesrdquo BioSystems vol 19 no 3 pp 163ndash172 1986

[29] V Bess ldquoEvaluating microbiology of compost microbial con-tent of composting is helping the producers and growers tounderstand its role as a soil inoculant and plantrdquo BioCycleMagazine p 62 1999

[30] O Grafff ldquoPreliminary experiments of vermin composting ofdifferent waste materials using Eudrilus eugeniae Kinbergrdquo inWorkshop on the Role of Earthworms in the Stabilization ofOrganic Residues M Happelhof Ed pp 178ndash191 Beech LeafPress Kalamazoo Mich USA 1981

[31] J E Satchell Earthworm Ecology from Darwin to VermicultureChapman and Hall 1983

[32] G H Xie M Y Cai G C Tao and Y Steinberger ldquoCultivableheterotrophic N2-fixing bacterial diversity in rice fields in theYangtze River Plainrdquo Biology and Fertility of Soils vol 37 no 1pp 29ndash38 2003

[33] B BMcSpaddenGardener ldquoEcology ofBacillus and Paenibacil-lus spp in agricultural systemsrdquo Phytopathology vol 94 no 11pp 1252ndash1258 2004

[34] J Dobereiner ldquoBiological nitrogen fixation in the tropics socialand economic contributionsrdquo Soil Biology andBiochemistry vol29 no 5-6 pp 771ndash774 1997

[35] V M Reis F L Olivares and J Dobereiner ldquoImprovedmethodology for isolation of Acetobacter diazotrophicus andconfirmation of its endophytic habitatrdquoWorld Journal of Micro-biology and Biotechnology vol 10 no 4 pp 401ndash405 1994

[36] S Khalil N Ayub S Alam and F Latif ldquoOrganic acids pro-duction and phosphate solubilization by phosphate solubilizingmicroorganisms (PSM) under in vitro conditionsrdquo PakistanJournal of Biological Sciences vol 7 no 2 pp 187ndash196 2004

[37] H M A El-Komy ldquoCoimmobilization of Azospirillumlipoferum and Bacillus megaterium for successful phosphorusand nitrogen nutrition of wheat plantsrdquo Food Technology andBiotechnology vol 43 no 1 pp 19ndash27 2005

[38] N B Paul and W V B Sundara Rao ldquoPhosphate-dissolvingbacteria in the rhizosphere of some cultivated legumesrdquo Plantand Soil vol 35 no 1 pp 127ndash132 1971

[39] A Beneduzi D Peres L K Vargas M H Bodanese-Zanettiniand LM P Passaglia ldquoEvaluation of genetic diversity and plantgrowth promoting activities of nitrogen-fixing bacilli isolatedfrom rice fields in South Brazilrdquo Applied Soil Ecology vol 39no 3 pp 311ndash320 2008

[40] M P Raimam U Albino M F Cruz et al ldquoInteraction amongfree-living N-fixing bacteria isolated from Drosera villosa varvillosa and AM fungi (Glomus clarum) in rice (Oryza sativa)rdquoApplied Soil Ecology vol 35 no 1 pp 25ndash34 2007

[41] M R Swain S K Naskar and R C Ray ldquoIndole-3-acetic acidproduction and effect on sprouting of yam (Dioscorea rotundataL) minisetts by Bacillus subtilis isolated from culturable cow-dung microflorardquo Polish Journal of Microbiology vol 56 no 2pp 103ndash110 2007

[42] T M Tien M H Garkins and D H Hubbell ldquoPlant growthsubstances produced by Azospirillum brasilense and their effect

Advances in Agriculture 13

on the growth of pearl Millet Pennisetum americanum (L)rdquoApplied and EnvironmentalMicrobiology vol 37 no 5 pp 1016ndash1024 1979

[43] A Esitken L Pirlak M Turan and F Sahin ldquoEffects of floraland foliar application of plant growth promoting rhizobacteria(PGPR) on yield growth and nutrition of sweet cherryrdquo ScientiaHorticulturae vol 110 no 4 pp 324ndash327 2006

[44] H Karlidag A Esitken M Turan and F Sahin ldquoEffects of rootinoculation of plant growth promoting rhizobacteria (PGPR)on yield growth and nutrient element contents of leaves ofapplerdquo Scientia Horticulturae vol 114 no 1 pp 16ndash20 2007

[45] C S Nautiyal S Bhadauria P Kumar H Lal R Mondal andD Verma ldquoStress induced phosphate solubilization in bacteriaisolated fromalkaline soilsrdquoFEMSMicrobiology Letters vol 182no 2 pp 291ndash296 2000

[46] A K Halder A K Mishra P Bhattacharya and P KChakrabarthy ldquoSolubilization of inorganic phosphates byBradyrhizobiumrdquo Indian Journal of Experimental Biology vol29 pp 28ndash31 1991

Submit your manuscripts athttpwwwhindawicom

Nutrition and Metabolism

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Food ScienceInternational Journal of

Agronomy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Applied ampEnvironmentalSoil Science

Volume 2014

AgricultureAdvances in

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PsycheHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BiodiversityInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Plant GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biotechnology Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Forestry ResearchInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of BotanyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

EcologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Veterinary Medicine International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Cell BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Page 12: BalayoganSivasankariandMarimuthuAnandharajdownloads.hindawi.com/journals/aag/2014/248591.pdf · In Vitro Nitrogen Fixing Activity. e bacterial isolates were screened for nitrogen

12 Advances in Agriculture

[10] J Han L Sun X Dong et al ldquoCharacterization of a novel plantgrowth-promoting bacteria strain Delftia tsuruhatensis HR4both as a diazotroph and a potential biocontrol agent againstvarious plant pathogensrdquo Systematic and Applied Microbiologyvol 28 no 1 pp 66ndash76 2005

[11] H Rodrıguez and R Fraga ldquoPhosphate solubilizing bacte-ria and their role in plant growth promotionrdquo BiotechnologyAdvances vol 17 no 4-5 pp 319ndash339 1999

[12] T Daniel and N Karmegam ldquoBio-conversion of selected leaflitters using an African epigeic earthworm Eudrilus eugeniaerdquoEcology Environment and Conservation vol 5 no 3 pp 271ndash275 1999

[13] J Dobereiner I E Marriel and M Nery ldquoEcological distri-bution of Spirillum lipoferum Beijerinckrdquo Canadian Journal ofMicrobiology vol 22 no 10 pp 1464ndash1473 1976

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

[15] M Edi Premono A M Moawad and P L G Vlek ldquoEffect ofphosphate-solubilizing Pseudomonas putida on the growth ofmaize and its survival in the rhizosphererdquo Indonesian Journal ofCrop Science vol 11 pp 13ndash23 1996

[16] C L Patten and B R Glick ldquoBacterial biosynthesis of indole-3-acetic acidrdquo Canadian Journal of Microbiology vol 42 no 3pp 207ndash220 1996

[17] N Amaresan V Jayakumar K Kumar and N ThajuddinldquoIsolation and characterization of plant growth promotingendophytic bacteria and their effect on tomato (Lycopersiconesculentum) and chilli (Capsicum annuum) seedling growthrdquoAnnals of Microbiology vol 62 no 2 pp 805ndash810 2012

[18] B Schwyn and J B Neilands ldquoUniversal chemical assay forthe detection and determination of siderophoresrdquo AnalyticalBiochemistry vol 160 no 1 pp 47ndash56 1987

[19] P Rahi P Vyas S Sharma and A Gulati ldquoPlant growthpromoting potential of the fungus Discosia sp FIHB 571 fromtea rhizosphere tested on chickpea maize and peardquo IndianJournal of Microbiology vol 49 no 2 pp 128ndash133 2009

[20] K Kumar N Amaresan S Bhagat K Madhuri and R CSrivastava ldquoIsolation and characterization of rhizobacteriaassociated with coastal agricultural ecosystem of rhizospheresoils of cultivated vegetable cropsrdquo World Journal of Microbiol-ogy and Biotechnology vol 27 no 7 pp 1625ndash1632 2011

[21] J A Smoker and S R Barnum ldquoRapid small scale DNAisolation from filamentous cyanobacteriardquo FEMS MicrobiologyLetters vol 56 no 1 pp 119ndash122 1988

[22] U Edwars T Rogall H Blocker M Emde and E C BottgerldquoIsolation and direct complete nucleotide determination ofentire genes Characterization of a gene coding for 16S riboso-mal RNArdquoNucleic Acids Research vol 17 no 19 pp 7843ndash78531989

[23] D G Higgins A J Bleasby and R Fuchs ldquoCLUSTAL Vimproved software for multiple sequence alignmentrdquo ComputerApplications in the Biosciences vol 8 no 2 pp 189ndash191 1992

[24] M Kimura ldquoA simple method for estimating evolutionary ratesof base substitutions through comparative studies of nucleotidesequencesrdquo Journal ofMolecular Evolution vol 16 no 2 pp 111ndash120 1980

[25] N Saitou and M Nei ldquoThe neighbor-joining method a newmethod for reconstructing phylogenetic treesrdquo Molecular Biol-ogy and Evolution vol 4 no 4 pp 406ndash425 1987

[26] E Barbu K Y Lee and R Wahl ldquoContent of purine andpyrimidine base in desoxyribonucleic acid of bacteriardquo Annalesde lrsquoInstitut Pasteur vol 91 no 2 pp 212ndash224 1956

[27] N Sueoka ldquoVariation and heterogeneity of base compositionof deoxyribonucleic acids a compilation of old and new datardquoJournal of Molecular Biology vol 3 no 1 pp 31ndash40 1961

[28] H Hori and S Osawa ldquoEvolutionary change in 5S rRNAsecondary structure and a phylogenic tree of 352 5S rRNAspeciesrdquo BioSystems vol 19 no 3 pp 163ndash172 1986

[29] V Bess ldquoEvaluating microbiology of compost microbial con-tent of composting is helping the producers and growers tounderstand its role as a soil inoculant and plantrdquo BioCycleMagazine p 62 1999

[30] O Grafff ldquoPreliminary experiments of vermin composting ofdifferent waste materials using Eudrilus eugeniae Kinbergrdquo inWorkshop on the Role of Earthworms in the Stabilization ofOrganic Residues M Happelhof Ed pp 178ndash191 Beech LeafPress Kalamazoo Mich USA 1981

[31] J E Satchell Earthworm Ecology from Darwin to VermicultureChapman and Hall 1983

[32] G H Xie M Y Cai G C Tao and Y Steinberger ldquoCultivableheterotrophic N2-fixing bacterial diversity in rice fields in theYangtze River Plainrdquo Biology and Fertility of Soils vol 37 no 1pp 29ndash38 2003

[33] B BMcSpaddenGardener ldquoEcology ofBacillus and Paenibacil-lus spp in agricultural systemsrdquo Phytopathology vol 94 no 11pp 1252ndash1258 2004

[34] J Dobereiner ldquoBiological nitrogen fixation in the tropics socialand economic contributionsrdquo Soil Biology andBiochemistry vol29 no 5-6 pp 771ndash774 1997

[35] V M Reis F L Olivares and J Dobereiner ldquoImprovedmethodology for isolation of Acetobacter diazotrophicus andconfirmation of its endophytic habitatrdquoWorld Journal of Micro-biology and Biotechnology vol 10 no 4 pp 401ndash405 1994

[36] S Khalil N Ayub S Alam and F Latif ldquoOrganic acids pro-duction and phosphate solubilization by phosphate solubilizingmicroorganisms (PSM) under in vitro conditionsrdquo PakistanJournal of Biological Sciences vol 7 no 2 pp 187ndash196 2004

[37] H M A El-Komy ldquoCoimmobilization of Azospirillumlipoferum and Bacillus megaterium for successful phosphorusand nitrogen nutrition of wheat plantsrdquo Food Technology andBiotechnology vol 43 no 1 pp 19ndash27 2005

[38] N B Paul and W V B Sundara Rao ldquoPhosphate-dissolvingbacteria in the rhizosphere of some cultivated legumesrdquo Plantand Soil vol 35 no 1 pp 127ndash132 1971

[39] A Beneduzi D Peres L K Vargas M H Bodanese-Zanettiniand LM P Passaglia ldquoEvaluation of genetic diversity and plantgrowth promoting activities of nitrogen-fixing bacilli isolatedfrom rice fields in South Brazilrdquo Applied Soil Ecology vol 39no 3 pp 311ndash320 2008

[40] M P Raimam U Albino M F Cruz et al ldquoInteraction amongfree-living N-fixing bacteria isolated from Drosera villosa varvillosa and AM fungi (Glomus clarum) in rice (Oryza sativa)rdquoApplied Soil Ecology vol 35 no 1 pp 25ndash34 2007

[41] M R Swain S K Naskar and R C Ray ldquoIndole-3-acetic acidproduction and effect on sprouting of yam (Dioscorea rotundataL) minisetts by Bacillus subtilis isolated from culturable cow-dung microflorardquo Polish Journal of Microbiology vol 56 no 2pp 103ndash110 2007

[42] T M Tien M H Garkins and D H Hubbell ldquoPlant growthsubstances produced by Azospirillum brasilense and their effect

Advances in Agriculture 13

on the growth of pearl Millet Pennisetum americanum (L)rdquoApplied and EnvironmentalMicrobiology vol 37 no 5 pp 1016ndash1024 1979

[43] A Esitken L Pirlak M Turan and F Sahin ldquoEffects of floraland foliar application of plant growth promoting rhizobacteria(PGPR) on yield growth and nutrition of sweet cherryrdquo ScientiaHorticulturae vol 110 no 4 pp 324ndash327 2006

[44] H Karlidag A Esitken M Turan and F Sahin ldquoEffects of rootinoculation of plant growth promoting rhizobacteria (PGPR)on yield growth and nutrient element contents of leaves ofapplerdquo Scientia Horticulturae vol 114 no 1 pp 16ndash20 2007

[45] C S Nautiyal S Bhadauria P Kumar H Lal R Mondal andD Verma ldquoStress induced phosphate solubilization in bacteriaisolated fromalkaline soilsrdquoFEMSMicrobiology Letters vol 182no 2 pp 291ndash296 2000

[46] A K Halder A K Mishra P Bhattacharya and P KChakrabarthy ldquoSolubilization of inorganic phosphates byBradyrhizobiumrdquo Indian Journal of Experimental Biology vol29 pp 28ndash31 1991

Submit your manuscripts athttpwwwhindawicom

Nutrition and Metabolism

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Food ScienceInternational Journal of

Agronomy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Applied ampEnvironmentalSoil Science

Volume 2014

AgricultureAdvances in

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PsycheHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BiodiversityInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Plant GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biotechnology Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Forestry ResearchInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of BotanyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

EcologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Veterinary Medicine International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Cell BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Page 13: BalayoganSivasankariandMarimuthuAnandharajdownloads.hindawi.com/journals/aag/2014/248591.pdf · In Vitro Nitrogen Fixing Activity. e bacterial isolates were screened for nitrogen

Advances in Agriculture 13

on the growth of pearl Millet Pennisetum americanum (L)rdquoApplied and EnvironmentalMicrobiology vol 37 no 5 pp 1016ndash1024 1979

[43] A Esitken L Pirlak M Turan and F Sahin ldquoEffects of floraland foliar application of plant growth promoting rhizobacteria(PGPR) on yield growth and nutrition of sweet cherryrdquo ScientiaHorticulturae vol 110 no 4 pp 324ndash327 2006

[44] H Karlidag A Esitken M Turan and F Sahin ldquoEffects of rootinoculation of plant growth promoting rhizobacteria (PGPR)on yield growth and nutrient element contents of leaves ofapplerdquo Scientia Horticulturae vol 114 no 1 pp 16ndash20 2007

[45] C S Nautiyal S Bhadauria P Kumar H Lal R Mondal andD Verma ldquoStress induced phosphate solubilization in bacteriaisolated fromalkaline soilsrdquoFEMSMicrobiology Letters vol 182no 2 pp 291ndash296 2000

[46] A K Halder A K Mishra P Bhattacharya and P KChakrabarthy ldquoSolubilization of inorganic phosphates byBradyrhizobiumrdquo Indian Journal of Experimental Biology vol29 pp 28ndash31 1991

Submit your manuscripts athttpwwwhindawicom

Nutrition and Metabolism

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Food ScienceInternational Journal of

Agronomy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Applied ampEnvironmentalSoil Science

Volume 2014

AgricultureAdvances in

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PsycheHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BiodiversityInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Plant GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biotechnology Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Forestry ResearchInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of BotanyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

EcologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Veterinary Medicine International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Cell BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Page 14: BalayoganSivasankariandMarimuthuAnandharajdownloads.hindawi.com/journals/aag/2014/248591.pdf · In Vitro Nitrogen Fixing Activity. e bacterial isolates were screened for nitrogen

Submit your manuscripts athttpwwwhindawicom

Nutrition and Metabolism

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Food ScienceInternational Journal of

Agronomy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Applied ampEnvironmentalSoil Science

Volume 2014

AgricultureAdvances in

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PsycheHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BiodiversityInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Plant GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biotechnology Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Forestry ResearchInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of BotanyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

EcologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Veterinary Medicine International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Cell BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


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AAG presentation

Gravity + AAG notes

Gravity + AAG notes

ALQ155 AAG Conversion Clinic

ALQ155 AAG Conversion Clinic

Aag khanaeybatoolpar

Aag khanaeybatoolpar

Aag ny 2012

Aag ny 2012

Jungle ki Aag

Jungle ki Aag

4th AAG Results

4th AAG Results

Aag.... Sholay Revisited

Aag.... Sholay Revisited

Company Profile (AAG)

Company Profile (AAG)

AAG Newsletter Vol#04

AAG Newsletter Vol#04

Crisis Cartography - AAG 2013

Crisis Cartography - AAG 2013

Regional AAG - 2008

Regional AAG - 2008

AAG Wealth Management

AAG Wealth Management

AAG Presention 58400 r2

AAG Presention 58400 r2

Aag He Aag by Anees Nagi

Aag He Aag by Anees Nagi

Acad Aag 2007

Acad Aag 2007

TITLE PAGE - ClinicalTrials.gov...AAg AAg AAg AAg AAg AAg AAg AAg AAg AAg AAg AAg AAg AAg AAg 2014N202021_00 CONFIDENTIAL RES117169 4 INVESTIGATOR PROTOCOL AGREEMENT PAGE I confirm

TITLE PAGE - ClinicalTrials.gov...AAg AAg AAg AAg AAg AAg AAg AAg AAg AAg AAg AAg AAg AAg AAg 2014N202021_00 CONFIDENTIAL RES117169 4 INVESTIGATOR PROTOCOL AGREEMENT PAGE I confirm

AAG Company Profile

AAG Company Profile

Aag 2012 gisedcop_md

Aag 2012 gisedcop_md

AAG 2014 (T3G)

AAG 2014 (T3G)