Research ArticleDiversity Biocontrol and Plant Growth Promoting Abilities ofXylem Residing Bacteria from Solanaceous Crops

Gauri A Achari12 and Raman Ramesh1

1 ICAR Research Complex for Goa Old Goa Goa 403 402 India2Department of Microbiology Goa University Taleigao Plateau Goa 403206 India

Correspondence should be addressed to Gauri A Achari achari gauri 24yahoocoin

Received 17 January 2014 Revised 6 April 2014 Accepted 26 April 2014 Published 19 May 2014

Academic Editor Daniele Daffonchio

Copyright copy 2014 G A Achari and R RameshThis is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Eggplant (Solanum melongena L) is one of the solanaceous crops of economic and cultural importance and is widely cultivated inthe state of Goa India Eggplant cultivation is severely affected by bacterial wilt caused by Ralstonia solanacearum that colonizes thexylem tissue In this study 167 bacteria were isolated from the xylem of healthy eggplant chilli and Solanum torvum Sw by vacuuminfiltration and maceration Amplified rDNA restriction analysis (ARDRA) grouped these xylem residing bacteria (XRB) into 38haplotypes Twenty-eight strains inhibited growth of R solanacearum and produced volatile and diffusible antagonistic compoundsand plant growth promoting substances in vitro Antagonistic strains XB86 XB169 XB177 andXB200 recorded a biocontrol efficacygreater than 85 against BW and exhibited 12ndash22 increase in shoot length in eggplant in the greenhouse screening 16S rRNAbased identification revealed the presence of 23 different bacterial genera XRB with high biocontrol and plant growth promotingactivities were identified as strains of Staphylococcus sp Bacillus sp Streptomyces sp Enterobacter sp and Agrobacterium sp Thisstudy is the first report on identity of bacteria from the xylem of solanaceous crops having traits useful in cultivation of eggplant

1 Introduction

Ralstonia solanacearum is a vascular wilt pathogen thatbelongs to the 120573 subdivision of the Proteobacteria [1] and isone of the most destructive plant pathogens causing bacterialwilt (BW) in many crop plants It has broad host range andinfects around 54 plant families and 450 plant species [2]Thispathogen also has a wide geographical distribution rangingfrom tropical subtropical andwarm temperate regions of theworld [3] Cultivation of eggplant in the coastal state of GoaIndia is severely affected by BW leading to 30ndash100 crop loss[4]The bacterium infects the plant through root cracks at thesite of root emergence Subsequently the intercellular spacesof the root cortex and vascular parenchyma are colonizedCell wall degrading exoenzymes disrupt the cell walls andfacilitate its entry in the vascular system [5] Inside the xylemvessels the bacterial populations rapidly reach very highlevels of 1010 cellscm of stem [6] High cell density andproduction of high molecular weight exopolysaccharides by

R solanacearum lead to clogging of xylem vessels wiltingand eventually death of plant

Xylem of healthy plants has been reported to be colonizedby endophytic xylem residing bacteria (XRB) at low popula-tion levels and has been isolated from xylem of various cropsnamely citrus [7] sugar beets [8] maize [9] alfalfa [10]grape [11 12] and Bermuda grass [13] Several endophyticbacteria have been reported to originate from the rhizospheresoil initially entering the host plant during germination andradicle development through wounds or by colonizing thecracks formed in lateral root junctions when the endodermisand casparian strips are disrupted thus gaining an easy accessto the stele [14 15] After their initial entry depending onthe endophytic colonization ability bacteria may remainlocalized in the roots [16] or colonize intercellular spacesand vascular system [11] and move to the stems [17] Fewendophytes have been reported to be able to migrate toaerial plant parts through the vascular system passively withthe transpirational flow or through additional assistance by

Hindawi Publishing CorporationInternational Journal of MicrobiologyVolume 2014 Article ID 296521 14 pageshttpdxdoiorg1011552014296521

2 International Journal of Microbiology

production of cell wall degrading enzymes [18 19] Thesesystemically migrated endophytes have been isolated fromleaves [20] inflorescence [21] fruits [22] and seeds [23]

Among the several methods of plant disease manage-ment biocontrol plays an important role particularly in thecontrol of soil borne diseases Biocontrol agents may beused as an alternative pathogen management strategy orcan be combined with other management practices Biolog-ical control not only helps in suppressing the disease andincreasing crop yield but also has importance in reducing theenvironmental pollution due to use of chemical pesticides[24] Several studies have shown that endophytic bacteriacan be used as biocontrol agents against plant pathogensThe capability of colonizing internal host tissues and abilityto produce volatile and diffusible substances which inhibitpathogen induction of systemic resistance in the plant anddirectly or indirectly promoting plant growth have madeendophytes a valuable tool in agriculture to improve crop per-formance [18] Endophytic biocontrol agents isolated frompotato [25] tomato chilli [26] and eggplant [27] have beenused for management of BW However the wilt preventionability of xylem residing bacteria of solanaceous crops thatshare an ecological nichewith theBWpathogenhas remainedunexploredThis studywas undertaken to identify and screenbacteria isolated from the xylem of eggplant chilli and Storvum for their biocontrol activities against R solanacearumand growth promotion abilities in eggplant

2 Materials and Methods

21 Isolation of Xylem Residing Bacteria from Eggplant Chilliand S torvum

211 Collection of Xylem Sap Apparently healthy plants werecollected from the major vegetable growing locales in NorthGoa and South Goa districts of the coastal state of Goa IndiaEggplant samples were from two different varieties namelyBW susceptible and BW resistant variety Chilli sampleswere from the locally grown cultivar which is moderatelysusceptible to BW Wild eggplant S torvum is known tobe naturally resistant to BW and was sampled from a fieldin ICAR Research Complex for Goa India Stem pieces of13ndash15 cm length were surface sterilized by dipping in 01mercuric chloride for 1min and rinsed several times in sterilewater Wash water used for rinsing each surface sterilizedstem piece was plated onto Tryptic soy agar (TSA) (HiMedia LaboratoriesMumbai) to confirm surface sterilizationof each stem piece Xylem sap was extracted by vacuuminfiltration as described earlier [7 28] Briefly after thesurface sterilization of stem one cm piece from each end wasdiscarded Epidermis and cortex fromeach endwere removedand the vascular cylinder was fitted to sterile glass tubingattached in a rubber cork To the other end of the stem piecea sterile plastic tubing was attached that could hold at least500120583L of 1 X phosphate buffered saline (PBS) (NaCl 8 gLKCl 02 gL Na

2HPO4sdot2H2O 144 gL and KH

2PO4024 gL

pH 74) The cork with plant sample attached was thenfitted onto a Buchner flask For extraction of PBS through

the xylem vessels a suction pressure 8mbar was applied usinga diaphragm pump MPC101Z (Ilmvac GmbH Germany) Atotal of four successive infiltrations using 500120583L of PBS wereperformed for each sampleThe sapwas collected directly in asterile test tube placed inside the Buchner flask Alternativelymacerationtrituration was performed for isolation of theXRB from young eggplant and chilli samples which had thinand soft stems The epidermis and cortex from the surfacesterilized stem piece were removed aseptically to expose thevascular bundles The decorticated pieces were macerated ina sterile mortar and pestle using 2mL of sterile 1X PBS

212 Isolation One hundred 120583L of the vacuum in-filteredsap ormaceratewas plated ontoTSAormedium523 [29]Theplates were incubated at 28plusmn 2∘C for 5 days Different coloniesfrom isolation plates were selected based on differences intheir shape color and texture and purified onto medium523 Pure cultures of the xylem residing bacteria (XRB) thusobtained were maintained at minus80∘C as glycerol stocks forlong term and 4∘C for temporary storage

22 Amplified rDNA Restriction Analysis (ARDRA) ARDRAwas performed to determine the genetic diversity of the XRBin the collection Genomic DNA from the XRB was extractedas described byWilson [30] Quality and quantity of theDNAwere measured using Nanodrop 1000 (Thermo ScientificUSA) 16S rRNA gene was amplified using universal primers27F (51015840-AGAGTTTGATCCTGGCTCAG-31015840) and 1492R (51015840-GGTTACCTTGTTACGACTT-31015840) Twenty 120583L reaction mixcontained 1X PCR buffer 075 units of Taq DNA polymerase(SigmaAldrichUSA) 200120583MdNTPs (SigmaAldrichUSA)05120583M each primer (Chromous Biotech Bangalore India)and 50 ng120583L of genomic DNA Amplifications were carriedout on Eppendorf Mastercycler Pro Thermal cycler (Eppen-dorf Germany) Amplification cycle included a denaturationstep of 94∘C for 5min followed by 32 cycles of denaturationat 94∘C for 30s annealing at 55∘C for 40s extension at 72∘Cfor 1min and a final extension at 72∘C for 10min Theamplification of the 1500 bp PCR product was determined byelectrophoresis on 08 agarose gel Fifteenmicroliters of thePCR product was digested with one unit of MspI (ThermoScientific USA) for 4 h at 37∘C Restriction fragments wereseparated on a 2 agarose gel in 1X Tris-acetate EDTA buffercontaining 05 120583gmL ethidium bromide at 60 volts for 2 hGel was documented using Alpha Imager (Alpha InnotechInc USA) ARDRA restriction fingerprints were comparedvisually and scored manually as 1 for presence and 0 forabsence of fragment and the binary data was entered inthe NT Edit software version 11 b (Applied Biostatistics IncUSA) The similarity matrix derived using the binary data ofARDRA restriction fragmentwas subjected to cluster analysisusing unweighted pair group method for arithmetic average(UPGMA) using Dice coefficient in the NTSYSpc 202i soft-ware (Applied Biostatistics Inc USA) Subsequent to analysisseveral clusters were obtained Each cluster consisted ofXRB having an identical restriction fragment profile Strainshaving very unique restriction profile remained separableand independent clusters Clusters obtained were denoted

International Journal of Microbiology 3

as haplotypes Haplotypes were delineated at 80 similarityvalues of Dice coefficient and numbered asM80-1 toM80-38Representative strains from each haplotype were identified by16S rRNA gene sequencing

23 Antagonism Towards R solanacearum

231 In Vitro Inhibition Bioassay One hundred and sixty-seven XRB were screened for inhibition of the BW pathogenR solanacearum strain Rs-09-100 was isolated from BWinfected eggplant cultivated in Goa India and was usedfor screening in vitro and in planta Rs-09-100 belongs tophylotype I race 1 and biovar 3 of theR solanacearum speciescomplex The strain is pathogenic to eggplant cv Agassaimand causes 100 wilt within 15 days after inoculation undergreenhouse conditions (data not shown) Bioassay was per-formed by the agar well method as described by Rameshand Phadke [27] Briefly single colony of R solanacearumand XRB was grown in 5mL CPG broth (Casein hydrolysate10 gL Peptone 100 gL and Glucose 50 gL) and Kingrsquos Bbroth (Peptone 200 gL K

2HPO4 15 gL MgSO

4sdot 7H2O

15 gL and Glycerol 100mLL) respectively at 28 plusmn 2∘C for48 h with constant shaking at 140 rpm One hundred andfifty microliters of R solanacearum was seeded every 100mLmolten cooled Kingrsquos B agar mixed well and poured intoplates After the plates solidified three wells were made ineach plate by removing a circular agar piece with the help ofcork borer (8mm diameter) Twenty-five 120583L of culture brothof XRB containing 80 Log CFUmL was added into each ofthe three wells All the plates were incubated at 28 plusmn 2∘C for48 h Plates were observed for inhibition of R solanacearumZones of inhibition were measured as radius in mm from theedge of the agar well Strains that were found antagonisticto R solanacearum were screened for in vitro productionof antagonistic compounds and plant growth promotingsubstances and identified by 16S rRNA gene sequencing

24 Production of Volatile and Diffusible AntagonisticSubstances by XRB

241 Hydrogen Cyanide (HCN) Production Antagonisticstrains were tested for HCN production ability in presence ofglycine as described by Saraf et al [31] a slight modificationbeing the use of broth for the HCN test Immediately afterinoculation of strains in Kingrsquos B broth containing 44 gL ofglycine sterile filter paper strips dipped in picric acid solutionwere introduced taking care that the strips did not touch themedium and walls of the tube The tubes were sealed withparafilm and incubated at 28 plusmn 2∘C for 4 days with constantshaking at 140 rpmThe color change of the filter paper stripsfrom yellow to brick red during incubation indicated theproduction of HCN

242 Ammonia Production To detect ammonia productionantagonistic XRB were grown in peptone water (peptone200 gL NaCl 50 gL) with constant shaking at 140 rpm for48 h at 28 plusmn 2∘C Ammonia production was determined usingNesslerrsquos reagent as described by Marques et al [32]

243 Acetoin Production Acetoin production by antagonis-tic isolates was tested in Voges Proskauer broth (peptone70 gL K

2HPO450 gL dextrose 50 gL pH 70) After

incubation for 30 h at 28 plusmn 2∘C at 140 rpm onemL each of 5120572 napthol and 40KOHwere added to the culture andmixedwell Appearance of red coloration indicated production ofacetoin [33]

244 Siderophore Production Antagonistic XRB were testedfor siderophore production on a medium containing chromeazurol S (CAS) [34] Isolates producing orange haloes on theblue green colored medium after incubation at 28 plusmn 2∘C for48 h were positive for siderophore production

25 Production of Growth Promoting Substances bythe Antagonistic XRB

251 Indole Acetic Acid (IAA) Production Antagonisticstrains were tested for their ability to produce phytohormoneIAA in presence of tryptophan as described by Gordon andPaleg [35] Briefly strains were grown in nutrient brothamended with 100mgL of tryptophan for 30 h at 28 plusmn 2∘Cat 140 rpmThe supernatants were obtained by centrifugationat 6200 g for 10min One mL of supernatant was mixed withone mL of Salkowskyrsquos reagent (50mL 35 perchloric acid1mL 05M FeCl

3) Themixture was allowed to stand at room

temperature for five minutes and the absorbance was readat 530 nm A standard curve was prepared using analyticalgrade IAA and the concentrations of IAA in the culturesupernatants of XRB were estimated based on the curve

252 1-Aminocyclopropane-1-carboxylate (ACC) DeaminaseActivity Antagonistic strains were tested for their abilityto produce enzyme ACC deaminase as per the methoddescribed by Godinho et al [36] Strains were streaked onDworkin and Fosterrsquos DF salts agar containing 30mM ACCand incubated at 28 plusmn 2∘C for 7 days Ability of the strainsto grow on the medium containing ACC as a sole nitrogensource was indicative of ACC deaminase production

253 Phosphate Solubilization Antagonistic strains weretested for phosphate solubilization by a method describedby Godinho et al [36] All strains were spot inoculated onPikovskayarsquos agar plates (Hi Media Laboratories Mumbai)Plates were incubated at 28 plusmn 2∘C for 48 h Transparent zonesaround the growth of XRB on the opaquewhitemediumwereindicative of solubilisation phosphate

26 Greenhouse Experiments

261 Biocontrol Efficacy (BCE) of Antagonistic XRB Twenty-eight strains of XRBwere selected based on in vitro inhibitionof R solanacearum in the agar well bioassay Strains wereevaluated for controlling BW in seedlings of wilt susceptibleeggplant cv Agassaim under greenhouse conditions Thirty-day-old seedlings raised in nonsterile soil in greenhousewere transplanted in pots filled with standard nonsterilepot mixture (soil sand farmyard manure at 2 1 1 ratio)

4 International Journal of Microbiology

Ten mL suspension of antagonistic XRB (80 Log CFUmL)in sterile 1 X PBS was applied per seedling by soil drenchingEach treatment consisted of two replicates with two potsper replication and five seedlings per pot Twenty days aftertreatment with the antagonistic XRB the seedlings were chal-lenged by inoculating 10mL suspension of R solanacearumstrain Rs-09-100 (70 Log CFUmL) by soil drenching Plantsnot treated with XRB but challenged with R solanacearumserved as control Plants were maintained with suitablewatering and percentage of plants infected by wilt was noteduntil 25 days after challenging with R solanacearum Abilityof the XRB to prevent wilt in eggplant was expressed asbiocontrol efficacy (BCE) and was determined using theformula BCE = ([percent disease in control] minus [percentdisease in treatment]percent disease in control) times 100 [37]Strains with BCE greater than 25 were evaluated for theireffect on growth in eggplant under greenhouse conditions

262 Growth Promotion Ability of XRB Sixteen strainsof XRB exhibiting biocontrol efficacies greater than 25were studied for their effect on growth in eggplant Abilityto increase shoot length in wilt susceptible eggplant cvAgassaim was used as a measure to evaluate their growthpromotion efficacy under greenhouse conditions Thirty-day-old seedlings raised in nonsterile soil in greenhousewere transplanted in pots filled with standard nonsterile potmixture (soil sand farmyardmanure at 2 1 1 ratio) TenmLsuspension of XRB (80 Log CFUmL) in sterile 1 X PBSwas applied per seedling by soil drenching Each treatmentconsisted of two replicates with two pots per replication andfive seedlings per pot Plants were maintained with suitablewatering and plant height was measured from the soil level tothe shoot tip 40 days postinoculation Ability of antagonisticXRB to increase shoot length in eggplant was expressed asgrowth promotion efficacy (GPE) using the formula ([shootlength increase in treatment] minus [shoot length increase incontrol]shoot length increase in control) times 100 [38]

27 16S rRNA Gene Sequencing and Sequence Analysis Rep-resentative strains from each of the 38 ARDRA haplotypesand XRB exhibiting antagonism to R solanacearum wereselected for identification A total of 55 strains were cho-sen for identification Fragments of the 16S rRNA geneof size 1500 bp were amplified as described above in theARDRA section Amplicons were purified using GeneJetPCRpurification kit (Thermo Scientific USA) and sequencedusing 27F and 1492R primers (Xcelris Labs Pvt LtdIndia) Partial 16S rRNA gene sequences (about 1200 nt)obtained were matched against the sequences available inthe nucleotide database from National Center for Biotech-nology Information (httpwwwncbinlmnihgovBLAST)using the BLASTn (Basic Local Alignment Search Tool)program

3 Results

31 Isolation of Bacteria from the Xylem of Eggplant Chilli andS torvum In this study bacteria could be constantly isolated

from the xylem of eggplant chilli and S torvum by vacuuminfiltration andmaceration techniques Bacterial counts fromeach isolation ranged from 10 to 102 CFUmL of xylem sap ormacerate Colonies appeared between 24 and 120 h of aerobicincubation at 28plusmn2∘CAmongst 167 isolates obtained 99wereGram-negative rods (5928) and 68 were Gram-positivebacteria (4072) comprising of 42 rods (6176) 25 cocci(3676) and one filamentous actinomycete (Table 1)

32 ARDRA Analysis ARDRA generated three to six restric-tion fragments of the 16S rRNA gene amplified from theXRB Analysis of ARDRA profiles by UPGMA using Dicersquoscoefficient dividedXRBwith identical restriction profiles intoseveral groups At 80 similarity values of Dice coefficient167 strains of XRB were grouped into 38 haplotypes Hostbased analysis of ARDRA revealed that 89 strains isolatedfromBW susceptible eggplant were grouped in 31 haplotypes36 strains from BW resistant eggplant into 17 haplotypes 33strains from chilli into 19 haplotypes and 9 strains from Storvum into 7 haplotypes respectively (Table 1) A detailedrepresentation of the ARDRA based analysis of 167 strainsis presented in Table 2 Based on the ARDRA analysis ofthe collection of XRB 153 strains were distributed over 24different haplotypes and 14 XRB strains had unique profileswhich formed 14 independent haplotypes with one strain ineach Antagonistic strains (119899 = 28) were distributed over 14different ARDRA haplotypes wherein 6 antagonists formedindependent haplotypes Nonantagonistic strains were dis-tributed over 24 haplotypes Haplotypes M80-9 and M80-15were shared amongst BW susceptible and resistant eggplantchilli and S torvum Eleven haplotypes were unique to BWsusceptible eggplant Haplotypes M80-1 M8-12 and M80-38 were unique to BW resistant eggplant whereas haplotypesM80-13 M80-19 and M80-26 comprised of strains isolatedfrom chilli Haplotype M80-36 had a strain isolated from Storvum Other haplotypes were a combination of strains iso-lated from different plant species These results indicate thatbacterial communities from the xylem of mainly eggplantand chilli cultivated in different locations in Goa comprise ofdiverse bacteria However it is observed that each ARDRAgroup consists of bacteria from different plant species andplants collected from different locales In addition there arecertain XRB unshared between each of the plant species thatform unique haplotypes Moreover strains with biocontrolability (BCE gt 25) were restricted to only 8 haplotypesnamely M80-6 M80-7 M80-10 M80-15 M80-20 M80-29M80-31 and M80-36 (Table 2) Haplotypes M80-6 M80-7M80-29 M80-31 M80-36 and M80-38 comprised of XRBwith GPE gt 10 Interestingly the strains with BCE gt 25and GPE gt 10 within these haplotypes were from BWresistant eggplant (Table 1)

33 Antagonism towards R solanacearum

331 In Vitro Bioassay and Production of Volatile and Dif-fusible Antagonistic Compounds Plate based bioassay wasused for rapid screening of antagonism of XRB towards R

International Journal of Microbiology 5

Table1Overviewof

diversity

andfunctio

nalityof

XRBob

tained

from

eggplantchilliand

Storvum

Planth

ost

Num

bero

fsamples

Totalisolates

obtained

Gram-negative

Gram-positive

Haplotypesa

obtained

Antagon

istic

strainsb

Antagon

isticstrains

with

BCEgt25

Biocon

trolstrains

with

GPEgt10

Metho

dof

isolatio

n

BWsusceptib

leeggplant

2489

5336

3116

52

VIo

rMBW

resistant

eggplant

1236

2016

176

65

VIo

rMCh

illi(Ca

psicu

mannu

um)

733

2013

193

21

Mon

ly

Solanu

mtorvum

29

63

73

31

VIo

nly

Total

45167

9968

2816

9a H

aplotypesw

ered

etermined

usingARD

RA

b Antagon

ismto

Rsolana

cearum

teste

din

vitro

asdescrib

edin

Section2

BCE(biocontrolefficacy)a

ndGPE

(growth

prom

otioneffi

cacy)w

ered

etermined

underg

reenho

usec

onditio

nsin

eggplant

asdescrib

edin

Section2

VIVa

cuum

infiltration

Mm

aceration

6 International Journal of Microbiology

Table 2 Haplotypes of XRB based on ARDRA withMspI at 80 similarity plant host biocontrol and growth promotion activities

Haplotypenumbera

Number of strainsin each haplotype Plant host Strains selected for

identificationNumber of biocontrolstrains with BCE gt 25

Number of strainswith GPE gt 10

M80-1 1 RE XB159 0 0M80-2 1 SE XB34 0 0M80-3 1 SE XB66 0 0M80-4 2 SE C XB40 0 0M80-5lowast 1 SE XB140 0 0

M80-6lowast 10 SE RE C XB177 XB157 XB93XB169 XB153 XB170 4 3

M80-7lowast 6 SE RE C XB1 XB86 2 2M80-8 4 SE RE XB22 XB190 0 0M80-9lowast 8 SE RE ST C XB99 XB100 0 0M80-10lowast 10 SE RE C XB196 XB103 1 0M80-11 9 SE RE C XB137 0 0M80-12 1 RE XB158 0 0M80-13 1 C XB87 0 0M80-14 5 SE C XB88 0 0M80-15lowast 5 SE RE ST C XB70 1 0M80-16 3 SE XB47 0 0M80-17 2 SE XB35 0 0M80-18 1 SE XB41 0 0M80-19 2 C XB94 0 0M80-20lowast 13 SE RE C XB8 XB20 XB27 2 0M80-21 5 SE ST C XB25 0 0M80-22 1 SE XB53 0 0M80-23 5 SE RE C XB98 0 0M80-24 11 SE RE ST XB161 0 0M80-25 4 SE RE XB188 0 0M80-26 1 C XB168 0 0M80-27lowast 2 SE C XB126 XB7 0 0M80-28lowast 5 SE RE XB122 0 0M80-29lowast 9 SE RE C XB165 1 1M80-30 15 SE RE C XB167 XB109 0 0

M80-31lowast 11 SE ST C XB123 XB203 XB62XB114 XB202 3 1

M80-32 5 SE ST C XB92 0 0M80-33 1 SE XB37 0 0M80-34 1 SE XB36 0 0M80-35 2 SE XB64 0 0M80-36lowast 1 ST XB200 1 1M80-37lowast 1 SE XB134 0 0M80-38lowast 1 RE XB197 1 1aHaplotype based on ARDRA analysis usingMspI at 80 similarity values of Dice coefficientlowastHaplotype comprises of bacteria showing antagonistic property in the bioassaysSE bacterial wilt susceptible eggplant RE bacterial wilt resistant eggplant ST Solanum torvum C chilli plantBCE biocontrol efficacy GPE growth promotion efficacy determined as described in Section 2

International Journal of Microbiology 7

solanacearum strain Rs-09-100 Results of the in vitro screen-ing against R solanacearum revealed that 28 amongst 167XRB exhibited antagonism towards the pathogen (Table 3)Amongst the antagonists 16 were strains from BW suscepti-ble eggplant 6 from BW resistant eggplant and 3 each fromchilli and S torvum (Table 1) Amongst the 28 antagonists 7strains namely XB62 XB99 XB100 XB114 XB122 XB196XB197 and XB202 formed larger inhibition zones againstR solanacearum ranging from 40mm to 817mm (Table 3)The majority of the antagonistic strains (119899 = 16) producedinhibition zones ranging from 20mm to 383mm XB27XB134 XB165 and XB169 formed smaller inhibition zonesranging from 15mm to 20mm However 139 strains ofXRB did not inhibit R solanacearum in the bioassay testTwenty-eight antagonistic XRBwere screened for productionof volatile inhibitory compounds namely acetoin HCNammonia and diffusible siderophore molecules in vitro(Table 3) Acetoin production was observed in 3214 of theisolates Bacterial isolates XB7 and XB122 were found toproduce both HCN as well as siderophores XB62 XB93 andXB170 produced HCN whereas XB114 XB140 and XB203produced siderophores only XB93 XB99 XB123 XB134 andXB140 produced ammonia

34 Production of Plant Growth Promoting Substances byAntagonistic XRB Results of the screening of antagonisticXRB for in vitro production of several plant growth pro-moting compounds is presented in Table 3 Majority of theantagonistic strains produced the phytohormone IAA withconcentrations ranging from 1591 120583gmL to 64591120583gmLXB202 was found to be the best ACC deaminase producingstrain based on its luxuriant growth on DF salts mediumsupplemented with 30mM ACC as sole nitrogen sourceOther ACC deaminase producing strains include XB1 XB62XB86 and XB140 Scarce growth of XB165 and XB200 wasobserved onDF saltsmedium 6428of the strains producedphosphate solubilizing organic acids as indicated by clearhaloes on Pikovskayarsquos agar plate

35 Greenhouse Experiments

351 Suppression of Bacterial Wilt by Antagonistic XRBAbility to suppress BW was assessed as the difference in thepercentage of wilt in XRB treated plants with respect to wilt inuntreated control andwas expressed as the biocontrol efficacy(BCE) of the antagonists BCE of 16 strains with valuesranging from 286 to 100 is presented in Figure 1 Plantstreated with strains XB86 XB169 and XB177 were free fromBWand hence recorded 100 biocontrol efficacy Treatmentswith XB170 XB197 XB200 XB202 and XB203 recorded 30percent or less wilt incidence (70 to 85 BCE) XB1 XB27XB70 XB93 and XB123 treatments recorded BCE between429 and 571 BCE of 286 was recorded in XB20 andXB165 treatments However 12 antagonistic XRB recordedBCEof 25 andwere least effective inwilt protection Furtherit is observed that all the antagonistic strains originating fromBW resistant eggplant and S torvum exhibited BCE greaterthan 25 Five antagonistic strains from BW susceptible

minus2000

000

2000

4000

6000

8000

10000

12000

XB1

XB20

XB27

XB70

XB86

XB93

XB123

XB165

XB169

XB170

XB177

XB196

XB197

XB200

XB202

XB203

Con

trol

Isolates

Biocontrol efficacy (BCE)Growth promotion efficacy (GPE)

BCE

and

GPE

()

Figure 1 Biocontrol and plant growth promotion efficacies of selectXRB in eggplant BCE biocontrol efficacy determined 25 days afterchallenging with R solanacearum GPE growth promotion efficacydetermined 40 days after treatmentwithXRB Percent BCE andGPEcalculated using formulae BCE = ([percent disease in control] minus[percent disease in treatment]percent disease in control) times 100 andGPE= ([shoot length increase in treatment]minus [shoot length increasein control]shoot length increase in control) times 100 respectivelyUninoculated control had BCE and GPE values of 000 XB86XB169 and XB177 had BCE of 10000 in all the replicationsBars indicate mean values of BCE and GPE error bars indicatestandard deviation

eggplant and three fromchilli were effective in preventingwiltin eggplant (Table 1)

352 Growth Promotion by Antagonistic XRB Increase inshoot length of eggplant (40 days after treatment) observedin XRB treated plants in relation to untreated control wasexpressed as growth promotion efficacy (GPE) and is shownin Figure 1 Amongst the 16 strains which were effective inpreventing wilt six strains exhibited the highest increase inshoot length as indicated by their GPE values in the rangeof 139ndash223 Seven XRB recorded a GPE value rangingfrom 14 to 129 However strains XB27 XB196 and XB203stunted shoot growth in eggplant in comparison to untreatedcontrol When the source of XRB is considered 5555strains that exhibited GPE greater than 10 were isolatedfrom BW resistant eggplant Whereas only two antagonistsfrom BW susceptible plant and one each from chilli and Storvum were able to promote growth in eggplant (Table 1)Strains with GPE gt 10 belonged to six different ARDRAhaplotypes (Table 2)

36 Identification of XRB by 16S rRNA Gene Sequencing16S rRNA gene sequences of XRB were used to identify thediverse xylem inhabitants and antagonistic strains Identityof 55 XRB based on 16S rRNA gene sequencing and theirGenBank accessions are presented in Table 4 Overall 23

8 International Journal of Microbiology

Table3

Invitro

inhibitio

nof

Rsolana

cearum

andprod

uctio

nof

antagonisticc

ompo

unds

andplantg

rowth

prom

otingsubstances

byXR

B

Strain

Haplotype

Inhibitio

nof

Rsolana

cearum

Volatile

anddiffu

sibleinhibitory

compo

unds

Plantgrowth

prom

otingsubstances

Radius

inmm

HCN

Ammon

iaAc

etoin

Sideroph

ore

Phosph

ates

olub

ilisatio

nAC

Cdeam

inasea

IAA

b120583gmL

XB1

M80-7

283plusmn029

minusminus

minusminus

minus++

+10500plusmn707

XB7

M80-27

333plusmn058

+minus

minus+

+minus

4773plusmn321

XB8

M80-20

333plusmn029

minusminus

minusminus

+minus

1909plusmn129

XB20

M80-20

327plusmn040

minusminus

minusminus

+minus

2545plusmn171

XB27

M80-20

183plusmn029

minusminus

minusminus

+minus

1591plusmn107

XB62

M80-31

817plusmn076

+minus

minusminus

+++

++7318plusmn493

XB70

M80-15

383plusmn058

minusminus

minusminus

minusminus

17182plusmn1157

XB86

M80-7

233plusmn076

minusminus

minusminus

minus++

+6682plusmn450

XB93

M80-6

317plusmn029

+minus

+minus

+minus

5727plusmn386

XB99

M80-9

683plusmn058

minus+

+minus

+minus

32136plusmn2164

XB100

M80-9

450plusmn050

minus+

+minus

+minus

23864plusmn1607

XB114

M80-31

650plusmn050

minusminus

minus+

+minus

8909plusmn600

XB122

M80-28

433plusmn058

+minus

minus+

+minus

6682plusmn450

XB123

M80-31

300plusmn087

minus+

+minus

+minus

41682plusmn2807

XB126

M80-27

283plusmn029

minusminus

+minus

+minus

6045plusmn407

XB134

M80-37

157plusmn012

minus+

+minus

+minus

64591plusmn4350

XB140

M80-5

367plusmn076

minus+

minus+

+++

++5091plusmn343

XB153

M80-6

317plusmn029

minusminus

+minus

+minus

15591plusmn1050

XB157

M80-6

250plusmn100

minusminus

+minus

+minus

18455plusmn1243

XB165

M80-29

157plusmn012

minusminus

minusminus

minus+

19091plusmn1286

XB169

M80-6

153plusmn006

minusminus

minusminus

minusminus

1591plusmn107

XB170

M80-6

351plusmn002

+minus

minusminus

+minus

2864plusmn193

XB177

M80-6

199plusmn049

minusminus

minusminus

minusminus

7636plusmn514

XB196

M80-10

393plusmn012

minusminus

minusminus

minusminus

3500plusmn236

XB197

M80-38

433plusmn029

minusminus

+minus

minusminus

4136plusmn279

XB200

M80-36

187plusmn023

minusminus

minusminus

minus++

16864plusmn1136

XB202

M80-31

467plusmn021

minusminus

minusminus

+++

+8273plusmn557

XB203

M80-31

367plusmn029

minusminus

minusminus

minusminus

7000plusmn471

Inhibitio

nzonesa

remeanof

threereplications

andshow

ingsta

ndarddeviation

Inhibitio

nzone

was

measuredas

radius

from

theou

tere

dgeof

wellAlltheexperim

entswerecond

uctedat28plusmn2∘C

+indicates

presence

oftraitminusindicatesabsence

oftraitinplateb

ased

assaysaLevelsofAC

Cdeam

inasea

ctivitiesbasedon

grow

thon

plateb

ased

assayd

enoted

as+forlessg

rowth+++

form

oderateg

rowth+++

forlux

uriant

grow

thand

++++

forh

ighlyluxu

riant

grow

thbIAAwas

estim

ated

incultu

refiltra

teandexpressedas120583gmLusinganalyticalgradeIAAas

stand

ardvalues

indicatemeanandsta

ndarddeviation

International Journal of Microbiology 9

2545

2545

364

364

1273 2909

ActinobacteriaFirmicutesAlpha proteobacteria

Beta proteobacteriaGamma proteobacteriaBacteroidetes

(a)

Num

ber o

f XRB

Actin

obac

teria

Firm

icut

es

Alp

ha p

rote

obac

teria

Beta

pro

teob

acte

ria

Gam

ma p

rote

obac

teria

Bact

eroi

dete

s

Number of antagonistic XRB in each phylumsubdivision

Phylasubdivision

Total number of XRB belonging to each phylumsubdivision

25

20

15

10

5

0

2

14

9

16

4

7

1

2

1

2

11

14

(b)

Figure 2 (a) Distribution of XRB (phylumsubdivision level) identified by 16S rRNA gene sequencing Values indicate percentages of strainsbelonging to each phylasubdivision amongst the 55 identified strains (b) Distribution of antagonistic and nonantagonistic strains of XRB ineach phylumsubdivision

different genera of bacteria were identified Major gen-era identified are Bacillus sp (11 strains) Enterobacter sp(6 strains) Microbacterium sp (5 strains) Staphylococcussp (5 strains) Pseudomonas sp (5 strains) and Agrobac-terium sp (3 strains) Additional genera identified includ-ing Micrococcus sp Sphingomonas sp Flavobacterium spChryseobacterium sp Burkholderia sp and Xenophilus spare listed in Table 4 Based on the identification phylumProteobacteria consisting of Gram-negative bacteria of sub-divisions Alpha Proteobacteria (1273) Beta Proteobacteria(364) and Gamma Proteobacteria (2545) were predom-inant (4181 strains identified) followed by phyla Firmi-cutes (2909) Actinobacteria (2545) and Bacteroidetes(364) (Figure 2(a))

Eleven antagonistic strains identified belonged toGammasubdivision of Proteobacteria consisting of five strainseachof Enterobacter sp and fluorescent and nonfluorescent Pseu-domonas sp and one strain of Pantoea eucrina (XB126)(Figure 2(b)) Nine antagonists identified were of phylaFirmicutes consisting of Staphylococcus sp (5 strains) andBacillus sp (4 strains) Agrobacterium strains XB1 XB86 andXB165 Sphingomonas sp (XB197) of the Alpha Proteobacte-ria Streptomyces sp (XB200) and Janibacter melonis (XB70)of phyla Actinobacteria were found to be antagonistic Addi-tional antagonistic XRB include Burkholderia sp (XB140) of120573 Proteobacteria and Flavobacterium sp (XB203) of phylaBacteroidetes (Figure 2(b))

37 Statistical Analysis The statistical analysis of percent-age wilts and shoot length of eggplant was performedusing Web Agri Statistical Package (WASP) version 20(httpwwwicargoaresinwasp20indexphp)

4 Discussion

Eggplant and chilli not only are of economic and culturalimportance but also are common ingredients in the cuisinethroughout India In the coastal state of GoaR solanacearumhas been reported to be a destructive pathogen in cultivationof eggplant and chilli [4] Isolation of biocontrol agentsagainst the BW pathogen has been commonly restricted toendophytic tissue and plant rhizosphere [26 27] Studieson xylem colonizing endophytes were undertaken becausewe speculated existence of interactions between the XRBand vascular wilt pathogen R solanacearum during xylemcolonization Our study reveals the diversity biocontrolpotential and identity of endophytic xylem colonizers fromsolanaceous crops cultivated in Goa India A total of 167bacteria were isolated from the xylem of eggplant chilliand S torvum with Gram-negative bacteria (5928) pre-dominating in the collection Congruent to our observationGardner et al [7] and Bell et al [11] have earlier reportedisolation of more number of Gram-negative rod shapedbacteria from xylem of citrus and grapevine using vacuuminfiltration Scholander pressure bomb was found to beuseful in extraction of diverse bacterial genera from xylemtissues in contrast to trituration methods that yielded highernumber of Gram-positive rod shaped bacteria [12] ThoughScholander pressure bomb was not used in this study acombination of vacuum infiltration [7 11] and triturationof decorticated stems [10 12] was employed with an aimto isolate diverse XRB from xylem tissues This is the firststudy reporting the use of vacuum infiltration and triturationmethods for isolating xylem residing bacteria from eggplantchilli and S torvum

Traditionally bacteria have been characterized andgrouped based on colony morphology and biochemical

10 International Journal of Microbiology

Table 4 List of XRB identified by partial 16S rRNAgene sequencing their plant host accession numbers closestNCBImatch and similarity

Strain Plant host Accession number Closest NCBI match similarityXB1lowast SE KF447383 Agrobacterium tumefaciens 99XB7lowast SE KF447384 Pseudomonas aeruginosa 99XB8lowast SE KF447385 Staphylococcus haemolyticus 99XB20lowast SE KF447386 Staphylococcus haemolyticus 100XB22 SE KF447387 Brevibacterium casei 99XB25 C KF447388 Enterobacter sp 95XB27lowast SE KF447389 Staphylococcus haemolyticus 99XB34 SE KF447390 Curtobacterium sp 99XB35 SE KF447391 Microbacterium sp 99XB36 SE KF447392 Xenophilus sp 99XB37 SE KF447393 Chryseobacterium sp 99XB40 SE KF447394 Micrococcus luteus 89XB41 SE KF447395 Bacillus sp 99XB47 SE KF447396 Bacillus sp 99XB53 SE KF447397 Micrococcus sp 99XB62lowast SE KF447398 Pseudomonas sp 99XB64 SE KF447399 Bacillus thuringiensis 100XB66 SE KF447400 Pectobacterium carotovorum 99XB70lowast SE KF447401 Janibacter melonis 99XB86lowast C KF447402 Agrobacterium tumefaciens 99XB87 C KF447403 Bacillus barbaricus 99XB88 C KF447404 Bacillus sp 99XB92 C KF447405 Brevundimonas vesicularis 99XB93lowast C KF447406 Bacillus safensis 100XB94 C KF447407 Bacillus sp 100XB98 C KF447408 Microbacterium sp 100XB99lowast SE KF447409 Enterobacter sp 99XB100lowast SE KF447410 Enterobacter cloacae 98XB103 SE KF447411 Brachybacterium phenoliresistens 99XB109 SE KF447412 Rhodococcus corynebacterioides 99XB114lowast SE KF447413 Pseudomonas stutzeri 99XB122lowast SE KF913446 Pseudomonas aeruginosa 100XB123lowast SE KF447414 Enterobacter sp 99XB126lowast C KF447415 Pantoea eucrina 99XB134lowast SE KF447416 Enterobacter sp 99XB137 SE KF447417 Klebsiella sp 99XB140lowast SE KF447418 Burkholderia sp 99XB153lowast SE KF447419 Bacillus amyloliquefaciens 99XB157lowast SE KF447420 Bacillus subtilis 100XB158 RE KF447421 Bosea sp 99XB159 RE KF447422 Microbacterium xylanilyticum 99XB161 RE KF447423 Microbacterium aurum 99XB165lowast RE KF447424 Agrobacterium sp 99XB167 RE KF447425 Microbacterium aurum 99XB168 RE KF447426 Bacillus aryabhattai 100XB169lowast RE KF447427 Staphylococcus gallinarum 99XB170lowast RE KF447428 Staphylococcus sp 99XB177lowast RE KF447429 Bacillus cereus 99XB188 RE KF447430 Sphingomonas sp 99XB190 RE KF447431 Brevibacterium casei 99

International Journal of Microbiology 11

Table 4 Continued

Strain Plant host Accession number Closest NCBI match similarityXB196lowast RE KF447432 Enterobacter kobei 99XB197lowast RE KF447433 Sphingomonas sp 98XB200lowast ST KF913447 Streptomyces sp 100XB202lowast ST KF447434 Pseudomonas sp 99XB203lowast ST KF447435 Flavobacterium sp 99lowastStrain is antagonistic to R solanacearum based on bioassaySE bacterial wilt susceptible eggplant RE bacterial wilt resistant eggplant C chilli ST Solanum torvum

tests However whole genome fingerprinting or PCR-RFLPbased methods are rapid tools for determining geneticdiversity of bacteria in a given collection ARDRA which isa type of PCR based RFLP method has been used widely inestimating genetic diversity endophytic bacterial populationsand clustering genetically similar strains [39ndash41] In additionto earlier reports our study demonstrates the usefulnessof ARDRA as a tool to cluster genetically identical strainsof endophytic XRB isolated from eggplant chilli andS torvum In our study 9161 strains (119899 = 153) weregrouped in 24 haplotypes by using ARDRA The majority ofthese haplotypes represent a combination of XRB isolatedfrom different solanaceous plants from diverse localesThese results indicate that xylem of eggplant chilli andS torvum largely bears similar population of XRB whichcan efficiently cross colonize eggplant chilli or S torvumNevertheless 839 strains had unique ARDRA fingerprintand formed separable haplotypes This observation leads toa conclusion that a minor population of xylem inhabitantsare restricted to a specific plant species and cannot easilycross colonize xylem of other solanaceous plants Plantsare known to selectively support endophytic colonizationby specific bacteria [14] However factors that determinethe selection of xylem colonists or the ability of XRBto colonize eggplant chilli and S torvum in this studyremain unknown Interestingly the structure of endophyticcommunity in Nicotiana attenuata a member of Solanaceaefamily is shown to be influenced by soil composition andethylene homeostasis [40] Earlier evidence has shown thatcolonization by endophytic bacteria is also governed by plantgenotype as well as root exudates [42]

Only 1677 XRB out of 167 were antagonistic to Rsolanacearum based on in vitro assays Antagonistic XRBproduced volatile and diffusible inhibitory compoundsnamely HCN ammonia and acetoin and siderophoresThese substances have been long known to be involvedin disease suppression and indirect growth promotion inplants [43ndash46] These mechanisms possibly played a rolein the evident biocontrol effect against BW exhibited bythe XRB in the greenhouse screening Endophytic bacteriahave been known to have plant growth promoting traitsnamely production of IAA ACC deaminase and phosphatesolubilization [25 33 36] These traits were detected in themajority of antagonistic XRB tested in this study and mayhave resulted in the observed increase in shoot length ofeggplant in our greenhouse experiments In contrast strains

XB20 XB196 and XB203 suppressed growth in eggplantunder greenhouse conditions however no visible symptomsof disease were observed Vascular plugging and productionof certainmetabolites toxic to plant cells but not cell viabilitymay have resulted in stunted shoot in eggplant [11 47]

Evaluation of efficacy of antagonistic organisms to sup-press the plant diseases under greenhouse conditions isone of the key steps for selecting a potential biocontrolagent for disease management [48] Endophytic strains fromBW susceptible varieties of eggplant have been shown toprevent wilt and promote growth in eggplant earlier [27]Our greenhouse screening shows that 3846 of antagonisticXRB with biocontrol efficacies greater than 40 were isolatesfrom BW resistant varieties of eggplantThis raises a questionwhether the bacteria from resistant varieties are involved inBW resistance and whether BW resistant varieties are ableto selectively influence xylem colonization by antagonisticbacteria Presence of higher number of endophytes withantagonistic abilities was reported in BW resistant varieties oftomato as compared to susceptible varieties and their role inresistance to BWwas proposed [49] Similar observations oncorrelation of resistance of potato to soft rot and endophyticbacteria have been reported [50]Thus BW resistant varietiescan be considered a better host for isolating potential biocon-trol strains for management of bacterial wilt

Identification of 55 XRB strains by 16S rRNA genesequencing revealed the presence of 23 diverse genera ofbacteria belonging to 4 phyla of Eubacteria Strains belongingto phyla Firmicutes Actinobacteria and 120574 subdivision ofProteobacteria were the major xylem colonists identified inthis study Several genera of bacteria belonging to these phylahave also been reported to be present in endophytic tissuesand xylem of a variety of other agricultural and horticulturalplant species [14 51] In addition the majority of the antag-onists identified belonged to Enterobacter sp Pseudomonassp Bacillus sp and Staphylococcus sp Congruent to ourresults several researchers have reported bacteria isolatedfrom solanaceous crops and belonging to similar genera tobe antagonistic to R solanacearum [26 51ndash53] HoweverFlavobacterium spand Janibacter melonis identified in thisstudy have never been previously reported to be inhibitoryto R solanacearum Large population of the xylem inhabitingbacterial flora accounting for 8323 exhibited no antago-nism towards R solanacearum Nonantagonistic XRB wereidentified predominantly as Microbacterium sp Endophyticpersistence and nematicidal activities of Microbacterium sp

12 International Journal of Microbiology

have been reported [54 55] Therefore the collection of XRBisolated in this study can be screened for inhibitory activitiesagainst other important agricultural pests

Though few strains namely XB86 XB169 and XB177exhibited plant beneficial properties in this study their use-fulness in plant disease control remains to be seen XB86has been identified as Agrobacterium tumefaciens the crowngall disease pathogen and its deployment as biocontrolagent is uncertain XB169 (Staphylococcus gallinarum) andXB177 (Bacillus cereus) are reported as opportunistic animalpathogens and thus unsuitable for field applications Strep-tomyces sp has earlier been reported as antagonistic to Rsolanacearum and tested for management of wilt in potatoand tomato [56 57] XB200 (Streptomyces sp) is one of theXRB high BCE and GPE it could be explored further forbiocontrol of bacterial wilt after additional characterizationand field evaluation

5 Conclusion

This study is the first report on the identity of novel anddiverse XRB colonizing the xylem of eggplant chilli and Storvum XRB particularly from BW resistant varieties werefound to protect eggplant from bacterial wilt and enhancedgrowth in eggplant in the greenhouse screening Thereforethe repertoire of XRB reported in this study may be usefulfor cultivation of eggplant in BW affected areas

Conflict of Interests

The authors hereby declare that they have no conflict ofinterests regarding the publication of this paper

Acknowledgments

The authors greatly acknowledge financial support fromIndian Council of Agricultural Research (ICAR) New DelhiIndia through ldquoOutreach project on Phytopthora Fusariumand Ralstonia diseases of horticultural and field cropsrdquo-(PhytoFuRa) and the Director ICAR Research Complex forGoa India for the facilities

References

[1] E Yabuuchi Y KosakoHOyaizu et al ldquoProposal ofBurkholde-ria gen nov and transfer of seven species of the genusPseudomonas homology group II to the new genus with thetype species Burkholderia cepacia (Palleroni and Holmes 1981)comb novrdquo Microbiology and Immunology vol 36 no 12 pp1251ndash1275 1992

[2] E Wicker L Grassart R Coranson-Beaudu et al ldquoRalstoniasolanacearum strains from Martinique (French West Indies)exhibiting a new pathogenic potentialrdquo Applied and Environ-mental Microbiology vol 73 no 21 pp 6790ndash6801 2007

[3] Y Liu A Kanda K Yano et al ldquoMolecular typing of Japanesestrains of Ralstonia solanacearum in relation to the ability toinduce a hypersensitive reaction in tobaccordquo Journal of GeneralPlant Pathology vol 75 no 5 pp 369ndash380 2009

[4] R Ramesh ldquoField evaluation of biological control agents for themanagement of Ralstonia solanacearum in Brinjalrdquo Journal ofMycology and Plant Pathology vol 36 no 2 pp 327ndash328 2006

[5] J Vasse P Frey and A Trigalet ldquoMicroscopic studies ofintercellular infection and protoxylem invasion of tomatoroots by Pseudomonas solanacearumrdquoMolecular Plant-MicrobeInteractions vol 8 no 2 pp 241ndash251 1995

[6] S Genin and C Boucher ldquoRalstonia solanacearum secrets of amajor pathogen unveiled by analysis of its genomerdquo MolecularPlant Pathology vol 3 no 3 pp 111ndash118 2002

[7] J M Gardner A W Feldman and R M Zablotowicz ldquoIdentityand behavior of xylem-residing bacteria in rough lemon roots ofFlorida citrus treesrdquo Applied and Environmental Microbiologyvol 43 no 6 pp 1335ndash1342 1982

[8] M J Jacobs W M Bugbee and D A Gabriellson ldquoEnu-meration location and characterization of endophytic bacteriawithin sugar beet rootsrdquoCanadian Journal of Botany vol 63 no7 pp 1262ndash1265 1985

[9] D G Patriquin and J Dobereiner ldquoLight microscopy observa-tions of tetrazolium-reducing bacteria in the endorhizosphereof maize and other grasses in Brazilrdquo Canadian Journal ofMicrobiology vol 24 no 6 pp 734ndash742 1978

[10] S Gagne C Richard H Rousseau and H Antoun ldquoXylem-residing bacteria in alfalfa rootsrdquo Canadian Journal of Micro-biology vol 33 no 11 pp 996ndash1000 1987

[11] C R Bell G A Dickie W L G Harvey and J W Y FChan ldquoEndophytic bacteria in grapevinerdquo Canadian Journal ofMicrobiology vol 41 no 1 pp 46ndash53 1995

[12] J Hallmann J W Kloepper and R Rodrıguez-Kabana ldquoAppli-cation of Scholander pressure bomb to studies on endophyticbacteria of plantsrdquo Canadian Journal of Microbiology vol 43no 5 pp 411ndash416 1997

[13] J S Lampel G L Canter M B Dimock et al ldquoIntegrativecloning expression and stability of the cryIA(c) gene fromBacillus thuringiensis subsp kurstaki in a recombinant strain ofClavibacter xyli subsp cynodontisrdquo Applied and EnvironmentalMicrobiology vol 60 no 2 pp 501ndash508 1994

[14] M Rosenblueth and E Martınez-Romero ldquoBacterial endo-phytes and their interactions with hostsrdquo Molecular Plant-Microbe Interactions vol 19 no 8 pp 827ndash837 2006

[15] P R Hardoim L S van Overbeek and J D van ElsasldquoProperties of bacterial endophytes and their proposed role inplant growthrdquo Trends in Microbiology vol 16 no 10 pp 463ndash471 2008

[16] R Aravind A Kumar S J Eapen and K V RamanaldquoEndophytic bacterial flora in root and stem tissues of blackpepper (Piper nigrum L) genotype isolation identificationand evaluation against Phytophthora capsicirdquo Letters in AppliedMicrobiology vol 48 no 1 pp 58ndash64 2009

[17] J Kuklinsky-Sobral W L Araujo R Mendes I O Geraldi AA Pizzirani-Kleiner and J L Azevedo ldquoIsolation and charac-terization of soybean-associated bacteria and their potential forplant growth promotionrdquo Environmental Microbiology vol 6no 12 pp 1244ndash1251 2004

[18] S Compant B Duffy J Nowak C Clement and E A BarkaldquoUse of plant growth-promoting bacteria for biocontrol ofplant diseases principles mechanisms of action and futureprospectsrdquoApplied and Environmental Microbiology vol 71 no9 pp 4951ndash4959 2005

[19] E T Thorne B M Young G M Young et al ldquoThe structureof xylem vessels in grapevine (Vitaceae) and a possible passive

International Journal of Microbiology 13

mechanism for the systemic spread of bacterial diseaserdquo Amer-ican Journal of Botany vol 93 no 4 pp 497ndash504 2006

[20] W T Seo W J Lim E J Kim H D Yun Y H Lee and KM Cho ldquoEndophytic bacterial diversity in the young radishand their antimicrobial activity against pathogensrdquo Journal ofApplied Biological Chemistry vol 53 no 4 pp 493ndash503 2010

[21] S Compant H Kaplan A Sessitsch J Nowak E Ait Barkaand C Clement ldquoEndophytic colonization ofVitis vinifera L byBurkholderia phytofirmans strain PsJN from the rhizosphere toinflorescence tissuesrdquo FEMS Microbiology Ecology vol 63 no1 pp 84ndash93 2005

[22] P S B Miguel J C Delvaux M N V de Oliveira et al ldquoDiver-sity of endophytic bacteria in the fruits of Coffea canephorardquoAfrican Journal of Microbiology Research vol 7 no 7 pp 586ndash594 2013

[23] A Lopez-Lopez M A Rogel E Ormeno-Orrillo J Martınez-Romero and E Martınez-Romero ldquoPhaseolus vulgaris seed-borne endophytic community with novel bacterial species suchas Rhizobium endophyticum sp novrdquo Systematic and AppliedMicrobiology vol 33 no 6 pp 322ndash327 2010

[24] M Lwin and S L Ranamukhaarachchi ldquoDevelopment of bio-logical control of Ralstonia solanacearum through antagonisticmicrobial populationsrdquo International Journal of Agriculture andBiology vol 8 no 5 pp 657ndash660 2006

[25] F Rasche E Marco-Noales H Velvis et al ldquoStructural char-acteristics and plant-beneficial effects of bacteria colonizing theshoots of field grown conventional and geneticallymodifiedT4-lysozyme producing potatoesrdquo Plant and Soil vol 289 no 1-2pp 123ndash140 2006

[26] N Amaresan V Jayakumar K Kumar and N ThajuddinldquoEndophytic bacteria from tomato and chilli their diversity andantagonistic potential against Ralstonia solanacearumrdquoArchivesof Phytopathology and Plant Protection vol 45 no 3 pp 344ndash355 2012

[27] R Ramesh and G S Phadke ldquoRhizosphere and endophyticbacteria for the suppression of eggplant wilt caused byRalstoniasolanacearumrdquo Crop Protection vol 37 pp 35ndash41 2012

[28] W J French R G Christis and D L Stassi ldquoRecovery ofrickettsialike bacteria by vacuum infiltration of peach tissuesaffected with phony diseaserdquo Phytopathology vol 67 pp 945ndash948 1977

[29] P R Viss E M Brooks and J A Driver ldquoA simplified methodfor the control of bacterial contamination in woody plant tissueculturerdquo In Vitro Cellular andDevelopmental Biology vol 27 no1 article 42 1991

[30] K Wilson ldquoPreparation of genomic DNA from bacteriardquo inCurrent Protocols in Molecular Biology F M Ausubel R BrentR E Kingston et al Eds pp 241ndash245 John Wiley amp SonsNew York NY USA 1997

[31] M Saraf AThakkar U PandyaM Joshi and J Parikh ldquoPoten-tial of plant growth promoting microorganisms as biofertilizersand biopesticides and itrsquos exploitation in sustainable agricul-turerdquo Journal of Microbiology and Biotechnology Research vol3 no 5 pp 54ndash62 2013

[32] A P G C Marques C Pires H Moreira A O S S Rangeland P M L Castro ldquoAssessment of the plant growth promotionabilities of six bacterial isolates using Zea mays as indicatorplantrdquo Soil Biology and Biochemistry vol 42 no 8 pp 1229ndash1235 2010

[33] X Hao P Xie L Johnstone S J Miller C Rensingand G Wei ldquoGenome sequence and mutational analysis of

plant-growth-promoting bacteriumAgrobacterium tumefaciensCCNWGS0286 isolated from a zinc-lead mine tailingrdquo Appliedand Environmental Microbiology vol 78 no 15 pp 5384ndash53942012

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

[35] S A Gordon and L G Paleg ldquoQuantitative measurement ofindole acetic acidrdquo Plant Physiology vol 10 pp 37ndash48 1957

[36] A Godinho R Ramesh and S Bhosle ldquoBacteria from sanddunes of Goa promoting growth in eggplantrdquoWorld Journal ofAgricultural Sciences vol 6 no 5 pp 555ndash564 2010

[37] J-H Guo H-Y Qi Y-H Guo et al ldquoBiocontrol of tomato wiltby plant growth-promoting rhizobacteriardquo Biological Controlvol 29 no 1 pp 66ndash72 2004

[38] N Aliye C Fininsa and Y Hiskias ldquoEvaluation of rhizo-sphere bacterial antagonists for their potential to bioprotectpotato (Solanum tuberosum) against bacterial wilt (Ralstoniasolanacearum)rdquo Biological Control vol 47 no 3 pp 282ndash2882008

[39] L LagaceM PitreM Jacqeus andDRoy ldquoIdentification of thebacterial community ofmaple sap by using amplified ribosomalDNA (rDNA) restriction analysis and rDNA sequencingrdquoApplied and Environmental Microbiology vol 70 no 4 pp2052ndash2060 2004

[40] H H Long D G Sonntag D D Schmidt and I T BaldwinldquoThe structure of the culturable root bacterial endophyte com-munity of Nicotiana attenuata is organized by soil compositionand host plant ethylene production and perceptionrdquo NewPhytologist vol 185 no 2 pp 554ndash567 2010

[41] E Lopez-Fuentes V M Ruız-Valdiviezo E Martınez-RomeroF A Gutierrez-Miceli L Dendooven and R Rincon-RosalesldquoBacterial community in the roots and rhizosphere of Hyper-icum silenoides Juss 1804rdquo African Journal of MicrobiologyResearch vol 6 no 11 pp 2704ndash2711 2012

[42] M Fang R J Kremer P P Motavalli and G Davis ldquoBacterialdiversity in rhizospheres of nontransgenic and transgenic cornrdquoApplied and EnvironmentalMicrobiology vol 71 no 7 pp 4132ndash4136 2005

[43] C R Howell and R D Stiphanovic ldquoProduction of ammonia byEnterobacter cloaceae and its possible role in biological controlof Pythium pre-emergence damping-off by the bacteriumrdquoEcology and Epidemiology vol 78 no 8 pp 1075ndash1078 1988

[44] A Ramette M Frapolli G Defago and Y Moenne-LoccozldquoPhylogeny of HCN synthase-encoding hcnBC genes in bio-control fluorescent pseudomonads and its relationship withhost plant species and HCN synthesis abilityrdquoMolecular Plant-Microbe Interactions vol 16 no 6 pp 525ndash535 2003

[45] I Loaces L Ferrando and A F Scavino ldquoDynamics diversityand function of endophytic siderophore-producing bacteria inricerdquoMicrobial Ecology vol 61 no 3 pp 606ndash618 2011

[46] Y C Kim J Leveau B B M Gardener E A Pierson L SPierson and C-M Ryu ldquoThe multifactorial basis for planthealth promotion by plant-associated bacteriardquo Applied andEnvironmental Microbiology vol 77 no 5 pp 1548ndash1555 2011

[47] J M Gardner J L Chandler and A W Feldman ldquoGrowthresponses and vascular plugging of citrus inoculated withrhizobacteria and xylem-resident bacteriardquo Plant and Soil vol86 no 3 pp 333ndash345 1985

[48] C Pliego C Ramos A de Vicente and F M Cazorla ldquoScreen-ing for candidate bacterial biocontrol agents against soilborne

14 International Journal of Microbiology

fungal plant pathogensrdquoPlant and Soil vol 340 no 1-2 pp 505ndash520 2011

[49] H Feng Y Li and Q Liu ldquoEndophytic bacterial communi-ties in tomato plants with differential resistance to Ralstoniasolanacearumrdquo African Journal of Microbiology Research vol 7no 15 pp 1311ndash1318 2013

[50] A V Sturz B R Christie B G Matheson W J Arsenaultand N A Buchanan ldquoEndophytic bacterial communities inthe periderm of potato tubers and their potential to improveresistance to soil-borne plant pathogensrdquo Plant Pathology vol48 no 3 pp 360ndash369 1999

[51] A V Sturz B R Christie and J Nowak ldquoBacterial endophytespotential role in developing sustainable systems of crop produc-tionrdquo Critical Reviews in Plant Sciences vol 19 no 1 pp 1ndash302000

[52] R Ramesh A A Joshi and M P Ghanekar ldquoPseudomonadsmajor antagonistic endophytic bacteria to suppress bacterialwilt pathogenRalstonia solanacearum in the eggplant (Solanummelongena L)rdquo World Journal of Microbiology and Biotechnol-ogy vol 25 no 1 pp 47ndash55 2009

[53] A A Nawangsih I Damayanti S Wiyono and J G KartikaldquoSelection and characterization of endophytic bacteria as bio-control agents of tomato Bacterial Wilt DiseaserdquoHayati Journalof Biosciences vol 18 no 2 pp 66ndash70 2011

[54] R Vargas-Ayala R Rodrıguez-Kabana G Morgan-Jones J AMcInroy and J W Kloepper ldquoShifts in soil microflora inducedby velvetbean (Mucuna deeringiana) in cropping systems tocontrol root-knot nematodesrdquo Biological Control vol 17 no 1pp 11ndash22 2000

[55] D K Zinniel P Lambrecht N B Harris et al ldquoIsolationand characterization of endophytic colonizing bacteria fromagronomic crops and prairie plantsrdquoApplied and EnvironmentalMicrobiology vol 68 no 5 pp 2198ndash2208 2002

[56] F Lemessa and W Zeller ldquoScreening rhizobacteria for biolog-ical control of Ralstonia solanacearum in Ethiopiardquo BiologicalControl vol 42 no 3 pp 336ndash344 2007

[57] M S El-AlbyadM A El-Sayed A R El-Shanshoury andNHEl-Batanouny ldquoEffect of culture conditions on the antimicrobialactivities of UV-mutants of Streptomyces corchorusii and Sspiroverticillatus against bean and banana wilt pathogensrdquoMicrobiological Research vol 151 no 2 pp 201ndash211 1996

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International Journal of

Microbiology

2 International Journal of Microbiology

production of cell wall degrading enzymes [18 19] Thesesystemically migrated endophytes have been isolated fromleaves [20] inflorescence [21] fruits [22] and seeds [23]

Among the several methods of plant disease manage-ment biocontrol plays an important role particularly in thecontrol of soil borne diseases Biocontrol agents may beused as an alternative pathogen management strategy orcan be combined with other management practices Biolog-ical control not only helps in suppressing the disease andincreasing crop yield but also has importance in reducing theenvironmental pollution due to use of chemical pesticides[24] Several studies have shown that endophytic bacteriacan be used as biocontrol agents against plant pathogensThe capability of colonizing internal host tissues and abilityto produce volatile and diffusible substances which inhibitpathogen induction of systemic resistance in the plant anddirectly or indirectly promoting plant growth have madeendophytes a valuable tool in agriculture to improve crop per-formance [18] Endophytic biocontrol agents isolated frompotato [25] tomato chilli [26] and eggplant [27] have beenused for management of BW However the wilt preventionability of xylem residing bacteria of solanaceous crops thatshare an ecological nichewith theBWpathogenhas remainedunexploredThis studywas undertaken to identify and screenbacteria isolated from the xylem of eggplant chilli and Storvum for their biocontrol activities against R solanacearumand growth promotion abilities in eggplant

2 Materials and Methods

21 Isolation of Xylem Residing Bacteria from Eggplant Chilliand S torvum

211 Collection of Xylem Sap Apparently healthy plants werecollected from the major vegetable growing locales in NorthGoa and South Goa districts of the coastal state of Goa IndiaEggplant samples were from two different varieties namelyBW susceptible and BW resistant variety Chilli sampleswere from the locally grown cultivar which is moderatelysusceptible to BW Wild eggplant S torvum is known tobe naturally resistant to BW and was sampled from a fieldin ICAR Research Complex for Goa India Stem pieces of13ndash15 cm length were surface sterilized by dipping in 01mercuric chloride for 1min and rinsed several times in sterilewater Wash water used for rinsing each surface sterilizedstem piece was plated onto Tryptic soy agar (TSA) (HiMedia LaboratoriesMumbai) to confirm surface sterilizationof each stem piece Xylem sap was extracted by vacuuminfiltration as described earlier [7 28] Briefly after thesurface sterilization of stem one cm piece from each end wasdiscarded Epidermis and cortex fromeach endwere removedand the vascular cylinder was fitted to sterile glass tubingattached in a rubber cork To the other end of the stem piecea sterile plastic tubing was attached that could hold at least500120583L of 1 X phosphate buffered saline (PBS) (NaCl 8 gLKCl 02 gL Na

2HPO4sdot2H2O 144 gL and KH

2PO4024 gL

pH 74) The cork with plant sample attached was thenfitted onto a Buchner flask For extraction of PBS through

the xylem vessels a suction pressure 8mbar was applied usinga diaphragm pump MPC101Z (Ilmvac GmbH Germany) Atotal of four successive infiltrations using 500120583L of PBS wereperformed for each sampleThe sapwas collected directly in asterile test tube placed inside the Buchner flask Alternativelymacerationtrituration was performed for isolation of theXRB from young eggplant and chilli samples which had thinand soft stems The epidermis and cortex from the surfacesterilized stem piece were removed aseptically to expose thevascular bundles The decorticated pieces were macerated ina sterile mortar and pestle using 2mL of sterile 1X PBS

212 Isolation One hundred 120583L of the vacuum in-filteredsap ormaceratewas plated ontoTSAormedium523 [29]Theplates were incubated at 28plusmn 2∘C for 5 days Different coloniesfrom isolation plates were selected based on differences intheir shape color and texture and purified onto medium523 Pure cultures of the xylem residing bacteria (XRB) thusobtained were maintained at minus80∘C as glycerol stocks forlong term and 4∘C for temporary storage

22 Amplified rDNA Restriction Analysis (ARDRA) ARDRAwas performed to determine the genetic diversity of the XRBin the collection Genomic DNA from the XRB was extractedas described byWilson [30] Quality and quantity of theDNAwere measured using Nanodrop 1000 (Thermo ScientificUSA) 16S rRNA gene was amplified using universal primers27F (51015840-AGAGTTTGATCCTGGCTCAG-31015840) and 1492R (51015840-GGTTACCTTGTTACGACTT-31015840) Twenty 120583L reaction mixcontained 1X PCR buffer 075 units of Taq DNA polymerase(SigmaAldrichUSA) 200120583MdNTPs (SigmaAldrichUSA)05120583M each primer (Chromous Biotech Bangalore India)and 50 ng120583L of genomic DNA Amplifications were carriedout on Eppendorf Mastercycler Pro Thermal cycler (Eppen-dorf Germany) Amplification cycle included a denaturationstep of 94∘C for 5min followed by 32 cycles of denaturationat 94∘C for 30s annealing at 55∘C for 40s extension at 72∘Cfor 1min and a final extension at 72∘C for 10min Theamplification of the 1500 bp PCR product was determined byelectrophoresis on 08 agarose gel Fifteenmicroliters of thePCR product was digested with one unit of MspI (ThermoScientific USA) for 4 h at 37∘C Restriction fragments wereseparated on a 2 agarose gel in 1X Tris-acetate EDTA buffercontaining 05 120583gmL ethidium bromide at 60 volts for 2 hGel was documented using Alpha Imager (Alpha InnotechInc USA) ARDRA restriction fingerprints were comparedvisually and scored manually as 1 for presence and 0 forabsence of fragment and the binary data was entered inthe NT Edit software version 11 b (Applied Biostatistics IncUSA) The similarity matrix derived using the binary data ofARDRA restriction fragmentwas subjected to cluster analysisusing unweighted pair group method for arithmetic average(UPGMA) using Dice coefficient in the NTSYSpc 202i soft-ware (Applied Biostatistics Inc USA) Subsequent to analysisseveral clusters were obtained Each cluster consisted ofXRB having an identical restriction fragment profile Strainshaving very unique restriction profile remained separableand independent clusters Clusters obtained were denoted

International Journal of Microbiology 3

as haplotypes Haplotypes were delineated at 80 similarityvalues of Dice coefficient and numbered asM80-1 toM80-38Representative strains from each haplotype were identified by16S rRNA gene sequencing

23 Antagonism Towards R solanacearum

231 In Vitro Inhibition Bioassay One hundred and sixty-seven XRB were screened for inhibition of the BW pathogenR solanacearum strain Rs-09-100 was isolated from BWinfected eggplant cultivated in Goa India and was usedfor screening in vitro and in planta Rs-09-100 belongs tophylotype I race 1 and biovar 3 of theR solanacearum speciescomplex The strain is pathogenic to eggplant cv Agassaimand causes 100 wilt within 15 days after inoculation undergreenhouse conditions (data not shown) Bioassay was per-formed by the agar well method as described by Rameshand Phadke [27] Briefly single colony of R solanacearumand XRB was grown in 5mL CPG broth (Casein hydrolysate10 gL Peptone 100 gL and Glucose 50 gL) and Kingrsquos Bbroth (Peptone 200 gL K

2HPO4 15 gL MgSO

4sdot 7H2O

15 gL and Glycerol 100mLL) respectively at 28 plusmn 2∘C for48 h with constant shaking at 140 rpm One hundred andfifty microliters of R solanacearum was seeded every 100mLmolten cooled Kingrsquos B agar mixed well and poured intoplates After the plates solidified three wells were made ineach plate by removing a circular agar piece with the help ofcork borer (8mm diameter) Twenty-five 120583L of culture brothof XRB containing 80 Log CFUmL was added into each ofthe three wells All the plates were incubated at 28 plusmn 2∘C for48 h Plates were observed for inhibition of R solanacearumZones of inhibition were measured as radius in mm from theedge of the agar well Strains that were found antagonisticto R solanacearum were screened for in vitro productionof antagonistic compounds and plant growth promotingsubstances and identified by 16S rRNA gene sequencing

24 Production of Volatile and Diffusible AntagonisticSubstances by XRB

241 Hydrogen Cyanide (HCN) Production Antagonisticstrains were tested for HCN production ability in presence ofglycine as described by Saraf et al [31] a slight modificationbeing the use of broth for the HCN test Immediately afterinoculation of strains in Kingrsquos B broth containing 44 gL ofglycine sterile filter paper strips dipped in picric acid solutionwere introduced taking care that the strips did not touch themedium and walls of the tube The tubes were sealed withparafilm and incubated at 28 plusmn 2∘C for 4 days with constantshaking at 140 rpmThe color change of the filter paper stripsfrom yellow to brick red during incubation indicated theproduction of HCN

242 Ammonia Production To detect ammonia productionantagonistic XRB were grown in peptone water (peptone200 gL NaCl 50 gL) with constant shaking at 140 rpm for48 h at 28 plusmn 2∘C Ammonia production was determined usingNesslerrsquos reagent as described by Marques et al [32]

243 Acetoin Production Acetoin production by antagonis-tic isolates was tested in Voges Proskauer broth (peptone70 gL K

2HPO450 gL dextrose 50 gL pH 70) After

incubation for 30 h at 28 plusmn 2∘C at 140 rpm onemL each of 5120572 napthol and 40KOHwere added to the culture andmixedwell Appearance of red coloration indicated production ofacetoin [33]

244 Siderophore Production Antagonistic XRB were testedfor siderophore production on a medium containing chromeazurol S (CAS) [34] Isolates producing orange haloes on theblue green colored medium after incubation at 28 plusmn 2∘C for48 h were positive for siderophore production

25 Production of Growth Promoting Substances bythe Antagonistic XRB

251 Indole Acetic Acid (IAA) Production Antagonisticstrains were tested for their ability to produce phytohormoneIAA in presence of tryptophan as described by Gordon andPaleg [35] Briefly strains were grown in nutrient brothamended with 100mgL of tryptophan for 30 h at 28 plusmn 2∘Cat 140 rpmThe supernatants were obtained by centrifugationat 6200 g for 10min One mL of supernatant was mixed withone mL of Salkowskyrsquos reagent (50mL 35 perchloric acid1mL 05M FeCl

3) Themixture was allowed to stand at room

temperature for five minutes and the absorbance was readat 530 nm A standard curve was prepared using analyticalgrade IAA and the concentrations of IAA in the culturesupernatants of XRB were estimated based on the curve

252 1-Aminocyclopropane-1-carboxylate (ACC) DeaminaseActivity Antagonistic strains were tested for their abilityto produce enzyme ACC deaminase as per the methoddescribed by Godinho et al [36] Strains were streaked onDworkin and Fosterrsquos DF salts agar containing 30mM ACCand incubated at 28 plusmn 2∘C for 7 days Ability of the strainsto grow on the medium containing ACC as a sole nitrogensource was indicative of ACC deaminase production

253 Phosphate Solubilization Antagonistic strains weretested for phosphate solubilization by a method describedby Godinho et al [36] All strains were spot inoculated onPikovskayarsquos agar plates (Hi Media Laboratories Mumbai)Plates were incubated at 28 plusmn 2∘C for 48 h Transparent zonesaround the growth of XRB on the opaquewhitemediumwereindicative of solubilisation phosphate

26 Greenhouse Experiments

261 Biocontrol Efficacy (BCE) of Antagonistic XRB Twenty-eight strains of XRBwere selected based on in vitro inhibitionof R solanacearum in the agar well bioassay Strains wereevaluated for controlling BW in seedlings of wilt susceptibleeggplant cv Agassaim under greenhouse conditions Thirty-day-old seedlings raised in nonsterile soil in greenhousewere transplanted in pots filled with standard nonsterilepot mixture (soil sand farmyard manure at 2 1 1 ratio)

4 International Journal of Microbiology

Ten mL suspension of antagonistic XRB (80 Log CFUmL)in sterile 1 X PBS was applied per seedling by soil drenchingEach treatment consisted of two replicates with two potsper replication and five seedlings per pot Twenty days aftertreatment with the antagonistic XRB the seedlings were chal-lenged by inoculating 10mL suspension of R solanacearumstrain Rs-09-100 (70 Log CFUmL) by soil drenching Plantsnot treated with XRB but challenged with R solanacearumserved as control Plants were maintained with suitablewatering and percentage of plants infected by wilt was noteduntil 25 days after challenging with R solanacearum Abilityof the XRB to prevent wilt in eggplant was expressed asbiocontrol efficacy (BCE) and was determined using theformula BCE = ([percent disease in control] minus [percentdisease in treatment]percent disease in control) times 100 [37]Strains with BCE greater than 25 were evaluated for theireffect on growth in eggplant under greenhouse conditions

262 Growth Promotion Ability of XRB Sixteen strainsof XRB exhibiting biocontrol efficacies greater than 25were studied for their effect on growth in eggplant Abilityto increase shoot length in wilt susceptible eggplant cvAgassaim was used as a measure to evaluate their growthpromotion efficacy under greenhouse conditions Thirty-day-old seedlings raised in nonsterile soil in greenhousewere transplanted in pots filled with standard nonsterile potmixture (soil sand farmyardmanure at 2 1 1 ratio) TenmLsuspension of XRB (80 Log CFUmL) in sterile 1 X PBSwas applied per seedling by soil drenching Each treatmentconsisted of two replicates with two pots per replication andfive seedlings per pot Plants were maintained with suitablewatering and plant height was measured from the soil level tothe shoot tip 40 days postinoculation Ability of antagonisticXRB to increase shoot length in eggplant was expressed asgrowth promotion efficacy (GPE) using the formula ([shootlength increase in treatment] minus [shoot length increase incontrol]shoot length increase in control) times 100 [38]

27 16S rRNA Gene Sequencing and Sequence Analysis Rep-resentative strains from each of the 38 ARDRA haplotypesand XRB exhibiting antagonism to R solanacearum wereselected for identification A total of 55 strains were cho-sen for identification Fragments of the 16S rRNA geneof size 1500 bp were amplified as described above in theARDRA section Amplicons were purified using GeneJetPCRpurification kit (Thermo Scientific USA) and sequencedusing 27F and 1492R primers (Xcelris Labs Pvt LtdIndia) Partial 16S rRNA gene sequences (about 1200 nt)obtained were matched against the sequences available inthe nucleotide database from National Center for Biotech-nology Information (httpwwwncbinlmnihgovBLAST)using the BLASTn (Basic Local Alignment Search Tool)program

3 Results

31 Isolation of Bacteria from the Xylem of Eggplant Chilli andS torvum In this study bacteria could be constantly isolated

from the xylem of eggplant chilli and S torvum by vacuuminfiltration andmaceration techniques Bacterial counts fromeach isolation ranged from 10 to 102 CFUmL of xylem sap ormacerate Colonies appeared between 24 and 120 h of aerobicincubation at 28plusmn2∘CAmongst 167 isolates obtained 99wereGram-negative rods (5928) and 68 were Gram-positivebacteria (4072) comprising of 42 rods (6176) 25 cocci(3676) and one filamentous actinomycete (Table 1)

32 ARDRA Analysis ARDRA generated three to six restric-tion fragments of the 16S rRNA gene amplified from theXRB Analysis of ARDRA profiles by UPGMA using Dicersquoscoefficient dividedXRBwith identical restriction profiles intoseveral groups At 80 similarity values of Dice coefficient167 strains of XRB were grouped into 38 haplotypes Hostbased analysis of ARDRA revealed that 89 strains isolatedfromBW susceptible eggplant were grouped in 31 haplotypes36 strains from BW resistant eggplant into 17 haplotypes 33strains from chilli into 19 haplotypes and 9 strains from Storvum into 7 haplotypes respectively (Table 1) A detailedrepresentation of the ARDRA based analysis of 167 strainsis presented in Table 2 Based on the ARDRA analysis ofthe collection of XRB 153 strains were distributed over 24different haplotypes and 14 XRB strains had unique profileswhich formed 14 independent haplotypes with one strain ineach Antagonistic strains (119899 = 28) were distributed over 14different ARDRA haplotypes wherein 6 antagonists formedindependent haplotypes Nonantagonistic strains were dis-tributed over 24 haplotypes Haplotypes M80-9 and M80-15were shared amongst BW susceptible and resistant eggplantchilli and S torvum Eleven haplotypes were unique to BWsusceptible eggplant Haplotypes M80-1 M8-12 and M80-38 were unique to BW resistant eggplant whereas haplotypesM80-13 M80-19 and M80-26 comprised of strains isolatedfrom chilli Haplotype M80-36 had a strain isolated from Storvum Other haplotypes were a combination of strains iso-lated from different plant species These results indicate thatbacterial communities from the xylem of mainly eggplantand chilli cultivated in different locations in Goa comprise ofdiverse bacteria However it is observed that each ARDRAgroup consists of bacteria from different plant species andplants collected from different locales In addition there arecertain XRB unshared between each of the plant species thatform unique haplotypes Moreover strains with biocontrolability (BCE gt 25) were restricted to only 8 haplotypesnamely M80-6 M80-7 M80-10 M80-15 M80-20 M80-29M80-31 and M80-36 (Table 2) Haplotypes M80-6 M80-7M80-29 M80-31 M80-36 and M80-38 comprised of XRBwith GPE gt 10 Interestingly the strains with BCE gt 25and GPE gt 10 within these haplotypes were from BWresistant eggplant (Table 1)

33 Antagonism towards R solanacearum

331 In Vitro Bioassay and Production of Volatile and Dif-fusible Antagonistic Compounds Plate based bioassay wasused for rapid screening of antagonism of XRB towards R

International Journal of Microbiology 5

Table1Overviewof

diversity

andfunctio

nalityof

XRBob

tained

from

eggplantchilliand

Storvum

Planth

ost

Num

bero

fsamples

Totalisolates

obtained

Gram-negative

Gram-positive

Haplotypesa

obtained

Antagon

istic

strainsb

Antagon

isticstrains

with

BCEgt25

Biocon

trolstrains

with

GPEgt10

Metho

dof

isolatio

n

BWsusceptib

leeggplant

2489

5336

3116

52

VIo

rMBW

resistant

eggplant

1236

2016

176

65

VIo

rMCh

illi(Ca

psicu

mannu

um)

733

2013

193

21

Mon

ly

Solanu

mtorvum

29

63

73

31

VIo

nly

Total

45167

9968

2816

9a H

aplotypesw

ered

etermined

usingARD

RA

b Antagon

ismto

Rsolana

cearum

teste

din

vitro

asdescrib

edin

Section2

BCE(biocontrolefficacy)a

ndGPE

(growth

prom

otioneffi

cacy)w

ered

etermined

underg

reenho

usec

onditio

nsin

eggplant

asdescrib

edin

Section2

VIVa

cuum

infiltration

Mm

aceration

6 International Journal of Microbiology

Table 2 Haplotypes of XRB based on ARDRA withMspI at 80 similarity plant host biocontrol and growth promotion activities

Haplotypenumbera

Number of strainsin each haplotype Plant host Strains selected for

identificationNumber of biocontrolstrains with BCE gt 25

Number of strainswith GPE gt 10

M80-1 1 RE XB159 0 0M80-2 1 SE XB34 0 0M80-3 1 SE XB66 0 0M80-4 2 SE C XB40 0 0M80-5lowast 1 SE XB140 0 0

M80-6lowast 10 SE RE C XB177 XB157 XB93XB169 XB153 XB170 4 3

M80-7lowast 6 SE RE C XB1 XB86 2 2M80-8 4 SE RE XB22 XB190 0 0M80-9lowast 8 SE RE ST C XB99 XB100 0 0M80-10lowast 10 SE RE C XB196 XB103 1 0M80-11 9 SE RE C XB137 0 0M80-12 1 RE XB158 0 0M80-13 1 C XB87 0 0M80-14 5 SE C XB88 0 0M80-15lowast 5 SE RE ST C XB70 1 0M80-16 3 SE XB47 0 0M80-17 2 SE XB35 0 0M80-18 1 SE XB41 0 0M80-19 2 C XB94 0 0M80-20lowast 13 SE RE C XB8 XB20 XB27 2 0M80-21 5 SE ST C XB25 0 0M80-22 1 SE XB53 0 0M80-23 5 SE RE C XB98 0 0M80-24 11 SE RE ST XB161 0 0M80-25 4 SE RE XB188 0 0M80-26 1 C XB168 0 0M80-27lowast 2 SE C XB126 XB7 0 0M80-28lowast 5 SE RE XB122 0 0M80-29lowast 9 SE RE C XB165 1 1M80-30 15 SE RE C XB167 XB109 0 0

M80-31lowast 11 SE ST C XB123 XB203 XB62XB114 XB202 3 1

M80-32 5 SE ST C XB92 0 0M80-33 1 SE XB37 0 0M80-34 1 SE XB36 0 0M80-35 2 SE XB64 0 0M80-36lowast 1 ST XB200 1 1M80-37lowast 1 SE XB134 0 0M80-38lowast 1 RE XB197 1 1aHaplotype based on ARDRA analysis usingMspI at 80 similarity values of Dice coefficientlowastHaplotype comprises of bacteria showing antagonistic property in the bioassaysSE bacterial wilt susceptible eggplant RE bacterial wilt resistant eggplant ST Solanum torvum C chilli plantBCE biocontrol efficacy GPE growth promotion efficacy determined as described in Section 2

International Journal of Microbiology 7

solanacearum strain Rs-09-100 Results of the in vitro screen-ing against R solanacearum revealed that 28 amongst 167XRB exhibited antagonism towards the pathogen (Table 3)Amongst the antagonists 16 were strains from BW suscepti-ble eggplant 6 from BW resistant eggplant and 3 each fromchilli and S torvum (Table 1) Amongst the 28 antagonists 7strains namely XB62 XB99 XB100 XB114 XB122 XB196XB197 and XB202 formed larger inhibition zones againstR solanacearum ranging from 40mm to 817mm (Table 3)The majority of the antagonistic strains (119899 = 16) producedinhibition zones ranging from 20mm to 383mm XB27XB134 XB165 and XB169 formed smaller inhibition zonesranging from 15mm to 20mm However 139 strains ofXRB did not inhibit R solanacearum in the bioassay testTwenty-eight antagonistic XRBwere screened for productionof volatile inhibitory compounds namely acetoin HCNammonia and diffusible siderophore molecules in vitro(Table 3) Acetoin production was observed in 3214 of theisolates Bacterial isolates XB7 and XB122 were found toproduce both HCN as well as siderophores XB62 XB93 andXB170 produced HCN whereas XB114 XB140 and XB203produced siderophores only XB93 XB99 XB123 XB134 andXB140 produced ammonia

34 Production of Plant Growth Promoting Substances byAntagonistic XRB Results of the screening of antagonisticXRB for in vitro production of several plant growth pro-moting compounds is presented in Table 3 Majority of theantagonistic strains produced the phytohormone IAA withconcentrations ranging from 1591 120583gmL to 64591120583gmLXB202 was found to be the best ACC deaminase producingstrain based on its luxuriant growth on DF salts mediumsupplemented with 30mM ACC as sole nitrogen sourceOther ACC deaminase producing strains include XB1 XB62XB86 and XB140 Scarce growth of XB165 and XB200 wasobserved onDF saltsmedium 6428of the strains producedphosphate solubilizing organic acids as indicated by clearhaloes on Pikovskayarsquos agar plate

35 Greenhouse Experiments

351 Suppression of Bacterial Wilt by Antagonistic XRBAbility to suppress BW was assessed as the difference in thepercentage of wilt in XRB treated plants with respect to wilt inuntreated control andwas expressed as the biocontrol efficacy(BCE) of the antagonists BCE of 16 strains with valuesranging from 286 to 100 is presented in Figure 1 Plantstreated with strains XB86 XB169 and XB177 were free fromBWand hence recorded 100 biocontrol efficacy Treatmentswith XB170 XB197 XB200 XB202 and XB203 recorded 30percent or less wilt incidence (70 to 85 BCE) XB1 XB27XB70 XB93 and XB123 treatments recorded BCE between429 and 571 BCE of 286 was recorded in XB20 andXB165 treatments However 12 antagonistic XRB recordedBCEof 25 andwere least effective inwilt protection Furtherit is observed that all the antagonistic strains originating fromBW resistant eggplant and S torvum exhibited BCE greaterthan 25 Five antagonistic strains from BW susceptible

minus2000

000

2000

4000

6000

8000

10000

12000

XB1

XB20

XB27

XB70

XB86

XB93

XB123

XB165

XB169

XB170

XB177

XB196

XB197

XB200

XB202

XB203

Con

trol

Isolates

Biocontrol efficacy (BCE)Growth promotion efficacy (GPE)

BCE

and

GPE

()

Figure 1 Biocontrol and plant growth promotion efficacies of selectXRB in eggplant BCE biocontrol efficacy determined 25 days afterchallenging with R solanacearum GPE growth promotion efficacydetermined 40 days after treatmentwithXRB Percent BCE andGPEcalculated using formulae BCE = ([percent disease in control] minus[percent disease in treatment]percent disease in control) times 100 andGPE= ([shoot length increase in treatment]minus [shoot length increasein control]shoot length increase in control) times 100 respectivelyUninoculated control had BCE and GPE values of 000 XB86XB169 and XB177 had BCE of 10000 in all the replicationsBars indicate mean values of BCE and GPE error bars indicatestandard deviation

eggplant and three fromchilli were effective in preventingwiltin eggplant (Table 1)

352 Growth Promotion by Antagonistic XRB Increase inshoot length of eggplant (40 days after treatment) observedin XRB treated plants in relation to untreated control wasexpressed as growth promotion efficacy (GPE) and is shownin Figure 1 Amongst the 16 strains which were effective inpreventing wilt six strains exhibited the highest increase inshoot length as indicated by their GPE values in the rangeof 139ndash223 Seven XRB recorded a GPE value rangingfrom 14 to 129 However strains XB27 XB196 and XB203stunted shoot growth in eggplant in comparison to untreatedcontrol When the source of XRB is considered 5555strains that exhibited GPE greater than 10 were isolatedfrom BW resistant eggplant Whereas only two antagonistsfrom BW susceptible plant and one each from chilli and Storvum were able to promote growth in eggplant (Table 1)Strains with GPE gt 10 belonged to six different ARDRAhaplotypes (Table 2)

36 Identification of XRB by 16S rRNA Gene Sequencing16S rRNA gene sequences of XRB were used to identify thediverse xylem inhabitants and antagonistic strains Identityof 55 XRB based on 16S rRNA gene sequencing and theirGenBank accessions are presented in Table 4 Overall 23

8 International Journal of Microbiology

Table3

Invitro

inhibitio

nof

Rsolana

cearum

andprod

uctio

nof

antagonisticc

ompo

unds

andplantg

rowth

prom

otingsubstances

byXR

B

Strain

Haplotype

Inhibitio

nof

Rsolana

cearum

Volatile

anddiffu

sibleinhibitory

compo

unds

Plantgrowth

prom

otingsubstances

Radius

inmm

HCN

Ammon

iaAc

etoin

Sideroph

ore

Phosph

ates

olub

ilisatio

nAC

Cdeam

inasea

IAA

b120583gmL

XB1

M80-7

283plusmn029

minusminus

minusminus

minus++

+10500plusmn707

XB7

M80-27

333plusmn058

+minus

minus+

+minus

4773plusmn321

XB8

M80-20

333plusmn029

minusminus

minusminus

+minus

1909plusmn129

XB20

M80-20

327plusmn040

minusminus

minusminus

+minus

2545plusmn171

XB27

M80-20

183plusmn029

minusminus

minusminus

+minus

1591plusmn107

XB62

M80-31

817plusmn076

+minus

minusminus

+++

++7318plusmn493

XB70

M80-15

383plusmn058

minusminus

minusminus

minusminus

17182plusmn1157

XB86

M80-7

233plusmn076

minusminus

minusminus

minus++

+6682plusmn450

XB93

M80-6

317plusmn029

+minus

+minus

+minus

5727plusmn386

XB99

M80-9

683plusmn058

minus+

+minus

+minus

32136plusmn2164

XB100

M80-9

450plusmn050

minus+

+minus

+minus

23864plusmn1607

XB114

M80-31

650plusmn050

minusminus

minus+

+minus

8909plusmn600

XB122

M80-28

433plusmn058

+minus

minus+

+minus

6682plusmn450

XB123

M80-31

300plusmn087

minus+

+minus

+minus

41682plusmn2807

XB126

M80-27

283plusmn029

minusminus

+minus

+minus

6045plusmn407

XB134

M80-37

157plusmn012

minus+

+minus

+minus

64591plusmn4350

XB140

M80-5

367plusmn076

minus+

minus+

+++

++5091plusmn343

XB153

M80-6

317plusmn029

minusminus

+minus

+minus

15591plusmn1050

XB157

M80-6

250plusmn100

minusminus

+minus

+minus

18455plusmn1243

XB165

M80-29

157plusmn012

minusminus

minusminus

minus+

19091plusmn1286

XB169

M80-6

153plusmn006

minusminus

minusminus

minusminus

1591plusmn107

XB170

M80-6

351plusmn002

+minus

minusminus

+minus

2864plusmn193

XB177

M80-6

199plusmn049

minusminus

minusminus

minusminus

7636plusmn514

XB196

M80-10

393plusmn012

minusminus

minusminus

minusminus

3500plusmn236

XB197

M80-38

433plusmn029

minusminus

+minus

minusminus

4136plusmn279

XB200

M80-36

187plusmn023

minusminus

minusminus

minus++

16864plusmn1136

XB202

M80-31

467plusmn021

minusminus

minusminus

+++

+8273plusmn557

XB203

M80-31

367plusmn029

minusminus

minusminus

minusminus

7000plusmn471

Inhibitio

nzonesa

remeanof

threereplications

andshow

ingsta

ndarddeviation

Inhibitio

nzone

was

measuredas

radius

from

theou

tere

dgeof

wellAlltheexperim

entswerecond

uctedat28plusmn2∘C

+indicates

presence

oftraitminusindicatesabsence

oftraitinplateb

ased

assaysaLevelsofAC

Cdeam

inasea

ctivitiesbasedon

grow

thon

plateb

ased

assayd

enoted

as+forlessg

rowth+++

form

oderateg

rowth+++

forlux

uriant

grow

thand

++++

forh

ighlyluxu

riant

grow

thbIAAwas

estim

ated

incultu

refiltra

teandexpressedas120583gmLusinganalyticalgradeIAAas

stand

ardvalues

indicatemeanandsta

ndarddeviation

International Journal of Microbiology 9

2545

2545

364

364

1273 2909

ActinobacteriaFirmicutesAlpha proteobacteria

Beta proteobacteriaGamma proteobacteriaBacteroidetes

(a)

Num

ber o

f XRB

Actin

obac

teria

Firm

icut

es

Alp

ha p

rote

obac

teria

Beta

pro

teob

acte

ria

Gam

ma p

rote

obac

teria

Bact

eroi

dete

s

Number of antagonistic XRB in each phylumsubdivision

Phylasubdivision

Total number of XRB belonging to each phylumsubdivision

25

20

15

10

5

0

2

14

9

16

4

7

1

2

1

2

11

14

(b)

Figure 2 (a) Distribution of XRB (phylumsubdivision level) identified by 16S rRNA gene sequencing Values indicate percentages of strainsbelonging to each phylasubdivision amongst the 55 identified strains (b) Distribution of antagonistic and nonantagonistic strains of XRB ineach phylumsubdivision

different genera of bacteria were identified Major gen-era identified are Bacillus sp (11 strains) Enterobacter sp(6 strains) Microbacterium sp (5 strains) Staphylococcussp (5 strains) Pseudomonas sp (5 strains) and Agrobac-terium sp (3 strains) Additional genera identified includ-ing Micrococcus sp Sphingomonas sp Flavobacterium spChryseobacterium sp Burkholderia sp and Xenophilus spare listed in Table 4 Based on the identification phylumProteobacteria consisting of Gram-negative bacteria of sub-divisions Alpha Proteobacteria (1273) Beta Proteobacteria(364) and Gamma Proteobacteria (2545) were predom-inant (4181 strains identified) followed by phyla Firmi-cutes (2909) Actinobacteria (2545) and Bacteroidetes(364) (Figure 2(a))

Eleven antagonistic strains identified belonged toGammasubdivision of Proteobacteria consisting of five strainseachof Enterobacter sp and fluorescent and nonfluorescent Pseu-domonas sp and one strain of Pantoea eucrina (XB126)(Figure 2(b)) Nine antagonists identified were of phylaFirmicutes consisting of Staphylococcus sp (5 strains) andBacillus sp (4 strains) Agrobacterium strains XB1 XB86 andXB165 Sphingomonas sp (XB197) of the Alpha Proteobacte-ria Streptomyces sp (XB200) and Janibacter melonis (XB70)of phyla Actinobacteria were found to be antagonistic Addi-tional antagonistic XRB include Burkholderia sp (XB140) of120573 Proteobacteria and Flavobacterium sp (XB203) of phylaBacteroidetes (Figure 2(b))

37 Statistical Analysis The statistical analysis of percent-age wilts and shoot length of eggplant was performedusing Web Agri Statistical Package (WASP) version 20(httpwwwicargoaresinwasp20indexphp)

4 Discussion

Eggplant and chilli not only are of economic and culturalimportance but also are common ingredients in the cuisinethroughout India In the coastal state of GoaR solanacearumhas been reported to be a destructive pathogen in cultivationof eggplant and chilli [4] Isolation of biocontrol agentsagainst the BW pathogen has been commonly restricted toendophytic tissue and plant rhizosphere [26 27] Studieson xylem colonizing endophytes were undertaken becausewe speculated existence of interactions between the XRBand vascular wilt pathogen R solanacearum during xylemcolonization Our study reveals the diversity biocontrolpotential and identity of endophytic xylem colonizers fromsolanaceous crops cultivated in Goa India A total of 167bacteria were isolated from the xylem of eggplant chilliand S torvum with Gram-negative bacteria (5928) pre-dominating in the collection Congruent to our observationGardner et al [7] and Bell et al [11] have earlier reportedisolation of more number of Gram-negative rod shapedbacteria from xylem of citrus and grapevine using vacuuminfiltration Scholander pressure bomb was found to beuseful in extraction of diverse bacterial genera from xylemtissues in contrast to trituration methods that yielded highernumber of Gram-positive rod shaped bacteria [12] ThoughScholander pressure bomb was not used in this study acombination of vacuum infiltration [7 11] and triturationof decorticated stems [10 12] was employed with an aimto isolate diverse XRB from xylem tissues This is the firststudy reporting the use of vacuum infiltration and triturationmethods for isolating xylem residing bacteria from eggplantchilli and S torvum

Traditionally bacteria have been characterized andgrouped based on colony morphology and biochemical

10 International Journal of Microbiology

Table 4 List of XRB identified by partial 16S rRNAgene sequencing their plant host accession numbers closestNCBImatch and similarity

Strain Plant host Accession number Closest NCBI match similarityXB1lowast SE KF447383 Agrobacterium tumefaciens 99XB7lowast SE KF447384 Pseudomonas aeruginosa 99XB8lowast SE KF447385 Staphylococcus haemolyticus 99XB20lowast SE KF447386 Staphylococcus haemolyticus 100XB22 SE KF447387 Brevibacterium casei 99XB25 C KF447388 Enterobacter sp 95XB27lowast SE KF447389 Staphylococcus haemolyticus 99XB34 SE KF447390 Curtobacterium sp 99XB35 SE KF447391 Microbacterium sp 99XB36 SE KF447392 Xenophilus sp 99XB37 SE KF447393 Chryseobacterium sp 99XB40 SE KF447394 Micrococcus luteus 89XB41 SE KF447395 Bacillus sp 99XB47 SE KF447396 Bacillus sp 99XB53 SE KF447397 Micrococcus sp 99XB62lowast SE KF447398 Pseudomonas sp 99XB64 SE KF447399 Bacillus thuringiensis 100XB66 SE KF447400 Pectobacterium carotovorum 99XB70lowast SE KF447401 Janibacter melonis 99XB86lowast C KF447402 Agrobacterium tumefaciens 99XB87 C KF447403 Bacillus barbaricus 99XB88 C KF447404 Bacillus sp 99XB92 C KF447405 Brevundimonas vesicularis 99XB93lowast C KF447406 Bacillus safensis 100XB94 C KF447407 Bacillus sp 100XB98 C KF447408 Microbacterium sp 100XB99lowast SE KF447409 Enterobacter sp 99XB100lowast SE KF447410 Enterobacter cloacae 98XB103 SE KF447411 Brachybacterium phenoliresistens 99XB109 SE KF447412 Rhodococcus corynebacterioides 99XB114lowast SE KF447413 Pseudomonas stutzeri 99XB122lowast SE KF913446 Pseudomonas aeruginosa 100XB123lowast SE KF447414 Enterobacter sp 99XB126lowast C KF447415 Pantoea eucrina 99XB134lowast SE KF447416 Enterobacter sp 99XB137 SE KF447417 Klebsiella sp 99XB140lowast SE KF447418 Burkholderia sp 99XB153lowast SE KF447419 Bacillus amyloliquefaciens 99XB157lowast SE KF447420 Bacillus subtilis 100XB158 RE KF447421 Bosea sp 99XB159 RE KF447422 Microbacterium xylanilyticum 99XB161 RE KF447423 Microbacterium aurum 99XB165lowast RE KF447424 Agrobacterium sp 99XB167 RE KF447425 Microbacterium aurum 99XB168 RE KF447426 Bacillus aryabhattai 100XB169lowast RE KF447427 Staphylococcus gallinarum 99XB170lowast RE KF447428 Staphylococcus sp 99XB177lowast RE KF447429 Bacillus cereus 99XB188 RE KF447430 Sphingomonas sp 99XB190 RE KF447431 Brevibacterium casei 99

International Journal of Microbiology 11

Table 4 Continued

Strain Plant host Accession number Closest NCBI match similarityXB196lowast RE KF447432 Enterobacter kobei 99XB197lowast RE KF447433 Sphingomonas sp 98XB200lowast ST KF913447 Streptomyces sp 100XB202lowast ST KF447434 Pseudomonas sp 99XB203lowast ST KF447435 Flavobacterium sp 99lowastStrain is antagonistic to R solanacearum based on bioassaySE bacterial wilt susceptible eggplant RE bacterial wilt resistant eggplant C chilli ST Solanum torvum

tests However whole genome fingerprinting or PCR-RFLPbased methods are rapid tools for determining geneticdiversity of bacteria in a given collection ARDRA which isa type of PCR based RFLP method has been used widely inestimating genetic diversity endophytic bacterial populationsand clustering genetically similar strains [39ndash41] In additionto earlier reports our study demonstrates the usefulnessof ARDRA as a tool to cluster genetically identical strainsof endophytic XRB isolated from eggplant chilli andS torvum In our study 9161 strains (119899 = 153) weregrouped in 24 haplotypes by using ARDRA The majority ofthese haplotypes represent a combination of XRB isolatedfrom different solanaceous plants from diverse localesThese results indicate that xylem of eggplant chilli andS torvum largely bears similar population of XRB whichcan efficiently cross colonize eggplant chilli or S torvumNevertheless 839 strains had unique ARDRA fingerprintand formed separable haplotypes This observation leads toa conclusion that a minor population of xylem inhabitantsare restricted to a specific plant species and cannot easilycross colonize xylem of other solanaceous plants Plantsare known to selectively support endophytic colonizationby specific bacteria [14] However factors that determinethe selection of xylem colonists or the ability of XRBto colonize eggplant chilli and S torvum in this studyremain unknown Interestingly the structure of endophyticcommunity in Nicotiana attenuata a member of Solanaceaefamily is shown to be influenced by soil composition andethylene homeostasis [40] Earlier evidence has shown thatcolonization by endophytic bacteria is also governed by plantgenotype as well as root exudates [42]

Only 1677 XRB out of 167 were antagonistic to Rsolanacearum based on in vitro assays Antagonistic XRBproduced volatile and diffusible inhibitory compoundsnamely HCN ammonia and acetoin and siderophoresThese substances have been long known to be involvedin disease suppression and indirect growth promotion inplants [43ndash46] These mechanisms possibly played a rolein the evident biocontrol effect against BW exhibited bythe XRB in the greenhouse screening Endophytic bacteriahave been known to have plant growth promoting traitsnamely production of IAA ACC deaminase and phosphatesolubilization [25 33 36] These traits were detected in themajority of antagonistic XRB tested in this study and mayhave resulted in the observed increase in shoot length ofeggplant in our greenhouse experiments In contrast strains

XB20 XB196 and XB203 suppressed growth in eggplantunder greenhouse conditions however no visible symptomsof disease were observed Vascular plugging and productionof certainmetabolites toxic to plant cells but not cell viabilitymay have resulted in stunted shoot in eggplant [11 47]

Evaluation of efficacy of antagonistic organisms to sup-press the plant diseases under greenhouse conditions isone of the key steps for selecting a potential biocontrolagent for disease management [48] Endophytic strains fromBW susceptible varieties of eggplant have been shown toprevent wilt and promote growth in eggplant earlier [27]Our greenhouse screening shows that 3846 of antagonisticXRB with biocontrol efficacies greater than 40 were isolatesfrom BW resistant varieties of eggplantThis raises a questionwhether the bacteria from resistant varieties are involved inBW resistance and whether BW resistant varieties are ableto selectively influence xylem colonization by antagonisticbacteria Presence of higher number of endophytes withantagonistic abilities was reported in BW resistant varieties oftomato as compared to susceptible varieties and their role inresistance to BWwas proposed [49] Similar observations oncorrelation of resistance of potato to soft rot and endophyticbacteria have been reported [50]Thus BW resistant varietiescan be considered a better host for isolating potential biocon-trol strains for management of bacterial wilt

Identification of 55 XRB strains by 16S rRNA genesequencing revealed the presence of 23 diverse genera ofbacteria belonging to 4 phyla of Eubacteria Strains belongingto phyla Firmicutes Actinobacteria and 120574 subdivision ofProteobacteria were the major xylem colonists identified inthis study Several genera of bacteria belonging to these phylahave also been reported to be present in endophytic tissuesand xylem of a variety of other agricultural and horticulturalplant species [14 51] In addition the majority of the antag-onists identified belonged to Enterobacter sp Pseudomonassp Bacillus sp and Staphylococcus sp Congruent to ourresults several researchers have reported bacteria isolatedfrom solanaceous crops and belonging to similar genera tobe antagonistic to R solanacearum [26 51ndash53] HoweverFlavobacterium spand Janibacter melonis identified in thisstudy have never been previously reported to be inhibitoryto R solanacearum Large population of the xylem inhabitingbacterial flora accounting for 8323 exhibited no antago-nism towards R solanacearum Nonantagonistic XRB wereidentified predominantly as Microbacterium sp Endophyticpersistence and nematicidal activities of Microbacterium sp

12 International Journal of Microbiology

have been reported [54 55] Therefore the collection of XRBisolated in this study can be screened for inhibitory activitiesagainst other important agricultural pests

Though few strains namely XB86 XB169 and XB177exhibited plant beneficial properties in this study their use-fulness in plant disease control remains to be seen XB86has been identified as Agrobacterium tumefaciens the crowngall disease pathogen and its deployment as biocontrolagent is uncertain XB169 (Staphylococcus gallinarum) andXB177 (Bacillus cereus) are reported as opportunistic animalpathogens and thus unsuitable for field applications Strep-tomyces sp has earlier been reported as antagonistic to Rsolanacearum and tested for management of wilt in potatoand tomato [56 57] XB200 (Streptomyces sp) is one of theXRB high BCE and GPE it could be explored further forbiocontrol of bacterial wilt after additional characterizationand field evaluation

5 Conclusion

This study is the first report on the identity of novel anddiverse XRB colonizing the xylem of eggplant chilli and Storvum XRB particularly from BW resistant varieties werefound to protect eggplant from bacterial wilt and enhancedgrowth in eggplant in the greenhouse screening Thereforethe repertoire of XRB reported in this study may be usefulfor cultivation of eggplant in BW affected areas

Conflict of Interests

The authors hereby declare that they have no conflict ofinterests regarding the publication of this paper

Acknowledgments

The authors greatly acknowledge financial support fromIndian Council of Agricultural Research (ICAR) New DelhiIndia through ldquoOutreach project on Phytopthora Fusariumand Ralstonia diseases of horticultural and field cropsrdquo-(PhytoFuRa) and the Director ICAR Research Complex forGoa India for the facilities

References

[1] E Yabuuchi Y KosakoHOyaizu et al ldquoProposal ofBurkholde-ria gen nov and transfer of seven species of the genusPseudomonas homology group II to the new genus with thetype species Burkholderia cepacia (Palleroni and Holmes 1981)comb novrdquo Microbiology and Immunology vol 36 no 12 pp1251ndash1275 1992

[2] E Wicker L Grassart R Coranson-Beaudu et al ldquoRalstoniasolanacearum strains from Martinique (French West Indies)exhibiting a new pathogenic potentialrdquo Applied and Environ-mental Microbiology vol 73 no 21 pp 6790ndash6801 2007

[3] Y Liu A Kanda K Yano et al ldquoMolecular typing of Japanesestrains of Ralstonia solanacearum in relation to the ability toinduce a hypersensitive reaction in tobaccordquo Journal of GeneralPlant Pathology vol 75 no 5 pp 369ndash380 2009

[4] R Ramesh ldquoField evaluation of biological control agents for themanagement of Ralstonia solanacearum in Brinjalrdquo Journal ofMycology and Plant Pathology vol 36 no 2 pp 327ndash328 2006

[5] J Vasse P Frey and A Trigalet ldquoMicroscopic studies ofintercellular infection and protoxylem invasion of tomatoroots by Pseudomonas solanacearumrdquoMolecular Plant-MicrobeInteractions vol 8 no 2 pp 241ndash251 1995

[6] S Genin and C Boucher ldquoRalstonia solanacearum secrets of amajor pathogen unveiled by analysis of its genomerdquo MolecularPlant Pathology vol 3 no 3 pp 111ndash118 2002

[7] J M Gardner A W Feldman and R M Zablotowicz ldquoIdentityand behavior of xylem-residing bacteria in rough lemon roots ofFlorida citrus treesrdquo Applied and Environmental Microbiologyvol 43 no 6 pp 1335ndash1342 1982

[8] M J Jacobs W M Bugbee and D A Gabriellson ldquoEnu-meration location and characterization of endophytic bacteriawithin sugar beet rootsrdquoCanadian Journal of Botany vol 63 no7 pp 1262ndash1265 1985

[9] D G Patriquin and J Dobereiner ldquoLight microscopy observa-tions of tetrazolium-reducing bacteria in the endorhizosphereof maize and other grasses in Brazilrdquo Canadian Journal ofMicrobiology vol 24 no 6 pp 734ndash742 1978

[10] S Gagne C Richard H Rousseau and H Antoun ldquoXylem-residing bacteria in alfalfa rootsrdquo Canadian Journal of Micro-biology vol 33 no 11 pp 996ndash1000 1987

[11] C R Bell G A Dickie W L G Harvey and J W Y FChan ldquoEndophytic bacteria in grapevinerdquo Canadian Journal ofMicrobiology vol 41 no 1 pp 46ndash53 1995

[12] J Hallmann J W Kloepper and R Rodrıguez-Kabana ldquoAppli-cation of Scholander pressure bomb to studies on endophyticbacteria of plantsrdquo Canadian Journal of Microbiology vol 43no 5 pp 411ndash416 1997

[13] J S Lampel G L Canter M B Dimock et al ldquoIntegrativecloning expression and stability of the cryIA(c) gene fromBacillus thuringiensis subsp kurstaki in a recombinant strain ofClavibacter xyli subsp cynodontisrdquo Applied and EnvironmentalMicrobiology vol 60 no 2 pp 501ndash508 1994

[14] M Rosenblueth and E Martınez-Romero ldquoBacterial endo-phytes and their interactions with hostsrdquo Molecular Plant-Microbe Interactions vol 19 no 8 pp 827ndash837 2006

[15] P R Hardoim L S van Overbeek and J D van ElsasldquoProperties of bacterial endophytes and their proposed role inplant growthrdquo Trends in Microbiology vol 16 no 10 pp 463ndash471 2008

[16] R Aravind A Kumar S J Eapen and K V RamanaldquoEndophytic bacterial flora in root and stem tissues of blackpepper (Piper nigrum L) genotype isolation identificationand evaluation against Phytophthora capsicirdquo Letters in AppliedMicrobiology vol 48 no 1 pp 58ndash64 2009

[17] J Kuklinsky-Sobral W L Araujo R Mendes I O Geraldi AA Pizzirani-Kleiner and J L Azevedo ldquoIsolation and charac-terization of soybean-associated bacteria and their potential forplant growth promotionrdquo Environmental Microbiology vol 6no 12 pp 1244ndash1251 2004

[18] S Compant B Duffy J Nowak C Clement and E A BarkaldquoUse of plant growth-promoting bacteria for biocontrol ofplant diseases principles mechanisms of action and futureprospectsrdquoApplied and Environmental Microbiology vol 71 no9 pp 4951ndash4959 2005

[19] E T Thorne B M Young G M Young et al ldquoThe structureof xylem vessels in grapevine (Vitaceae) and a possible passive

International Journal of Microbiology 13

mechanism for the systemic spread of bacterial diseaserdquo Amer-ican Journal of Botany vol 93 no 4 pp 497ndash504 2006

[20] W T Seo W J Lim E J Kim H D Yun Y H Lee and KM Cho ldquoEndophytic bacterial diversity in the young radishand their antimicrobial activity against pathogensrdquo Journal ofApplied Biological Chemistry vol 53 no 4 pp 493ndash503 2010

[21] S Compant H Kaplan A Sessitsch J Nowak E Ait Barkaand C Clement ldquoEndophytic colonization ofVitis vinifera L byBurkholderia phytofirmans strain PsJN from the rhizosphere toinflorescence tissuesrdquo FEMS Microbiology Ecology vol 63 no1 pp 84ndash93 2005

[22] P S B Miguel J C Delvaux M N V de Oliveira et al ldquoDiver-sity of endophytic bacteria in the fruits of Coffea canephorardquoAfrican Journal of Microbiology Research vol 7 no 7 pp 586ndash594 2013

[23] A Lopez-Lopez M A Rogel E Ormeno-Orrillo J Martınez-Romero and E Martınez-Romero ldquoPhaseolus vulgaris seed-borne endophytic community with novel bacterial species suchas Rhizobium endophyticum sp novrdquo Systematic and AppliedMicrobiology vol 33 no 6 pp 322ndash327 2010

[24] M Lwin and S L Ranamukhaarachchi ldquoDevelopment of bio-logical control of Ralstonia solanacearum through antagonisticmicrobial populationsrdquo International Journal of Agriculture andBiology vol 8 no 5 pp 657ndash660 2006

[25] F Rasche E Marco-Noales H Velvis et al ldquoStructural char-acteristics and plant-beneficial effects of bacteria colonizing theshoots of field grown conventional and geneticallymodifiedT4-lysozyme producing potatoesrdquo Plant and Soil vol 289 no 1-2pp 123ndash140 2006

[26] N Amaresan V Jayakumar K Kumar and N ThajuddinldquoEndophytic bacteria from tomato and chilli their diversity andantagonistic potential against Ralstonia solanacearumrdquoArchivesof Phytopathology and Plant Protection vol 45 no 3 pp 344ndash355 2012

[27] R Ramesh and G S Phadke ldquoRhizosphere and endophyticbacteria for the suppression of eggplant wilt caused byRalstoniasolanacearumrdquo Crop Protection vol 37 pp 35ndash41 2012

[28] W J French R G Christis and D L Stassi ldquoRecovery ofrickettsialike bacteria by vacuum infiltration of peach tissuesaffected with phony diseaserdquo Phytopathology vol 67 pp 945ndash948 1977

[29] P R Viss E M Brooks and J A Driver ldquoA simplified methodfor the control of bacterial contamination in woody plant tissueculturerdquo In Vitro Cellular andDevelopmental Biology vol 27 no1 article 42 1991

[30] K Wilson ldquoPreparation of genomic DNA from bacteriardquo inCurrent Protocols in Molecular Biology F M Ausubel R BrentR E Kingston et al Eds pp 241ndash245 John Wiley amp SonsNew York NY USA 1997

[31] M Saraf AThakkar U PandyaM Joshi and J Parikh ldquoPoten-tial of plant growth promoting microorganisms as biofertilizersand biopesticides and itrsquos exploitation in sustainable agricul-turerdquo Journal of Microbiology and Biotechnology Research vol3 no 5 pp 54ndash62 2013

[32] A P G C Marques C Pires H Moreira A O S S Rangeland P M L Castro ldquoAssessment of the plant growth promotionabilities of six bacterial isolates using Zea mays as indicatorplantrdquo Soil Biology and Biochemistry vol 42 no 8 pp 1229ndash1235 2010

[33] X Hao P Xie L Johnstone S J Miller C Rensingand G Wei ldquoGenome sequence and mutational analysis of

plant-growth-promoting bacteriumAgrobacterium tumefaciensCCNWGS0286 isolated from a zinc-lead mine tailingrdquo Appliedand Environmental Microbiology vol 78 no 15 pp 5384ndash53942012

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

[35] S A Gordon and L G Paleg ldquoQuantitative measurement ofindole acetic acidrdquo Plant Physiology vol 10 pp 37ndash48 1957

[36] A Godinho R Ramesh and S Bhosle ldquoBacteria from sanddunes of Goa promoting growth in eggplantrdquoWorld Journal ofAgricultural Sciences vol 6 no 5 pp 555ndash564 2010

[37] J-H Guo H-Y Qi Y-H Guo et al ldquoBiocontrol of tomato wiltby plant growth-promoting rhizobacteriardquo Biological Controlvol 29 no 1 pp 66ndash72 2004

[38] N Aliye C Fininsa and Y Hiskias ldquoEvaluation of rhizo-sphere bacterial antagonists for their potential to bioprotectpotato (Solanum tuberosum) against bacterial wilt (Ralstoniasolanacearum)rdquo Biological Control vol 47 no 3 pp 282ndash2882008

[39] L LagaceM PitreM Jacqeus andDRoy ldquoIdentification of thebacterial community ofmaple sap by using amplified ribosomalDNA (rDNA) restriction analysis and rDNA sequencingrdquoApplied and Environmental Microbiology vol 70 no 4 pp2052ndash2060 2004

[40] H H Long D G Sonntag D D Schmidt and I T BaldwinldquoThe structure of the culturable root bacterial endophyte com-munity of Nicotiana attenuata is organized by soil compositionand host plant ethylene production and perceptionrdquo NewPhytologist vol 185 no 2 pp 554ndash567 2010

[41] E Lopez-Fuentes V M Ruız-Valdiviezo E Martınez-RomeroF A Gutierrez-Miceli L Dendooven and R Rincon-RosalesldquoBacterial community in the roots and rhizosphere of Hyper-icum silenoides Juss 1804rdquo African Journal of MicrobiologyResearch vol 6 no 11 pp 2704ndash2711 2012

[42] M Fang R J Kremer P P Motavalli and G Davis ldquoBacterialdiversity in rhizospheres of nontransgenic and transgenic cornrdquoApplied and EnvironmentalMicrobiology vol 71 no 7 pp 4132ndash4136 2005

[43] C R Howell and R D Stiphanovic ldquoProduction of ammonia byEnterobacter cloaceae and its possible role in biological controlof Pythium pre-emergence damping-off by the bacteriumrdquoEcology and Epidemiology vol 78 no 8 pp 1075ndash1078 1988

[44] A Ramette M Frapolli G Defago and Y Moenne-LoccozldquoPhylogeny of HCN synthase-encoding hcnBC genes in bio-control fluorescent pseudomonads and its relationship withhost plant species and HCN synthesis abilityrdquoMolecular Plant-Microbe Interactions vol 16 no 6 pp 525ndash535 2003

[45] I Loaces L Ferrando and A F Scavino ldquoDynamics diversityand function of endophytic siderophore-producing bacteria inricerdquoMicrobial Ecology vol 61 no 3 pp 606ndash618 2011

[46] Y C Kim J Leveau B B M Gardener E A Pierson L SPierson and C-M Ryu ldquoThe multifactorial basis for planthealth promotion by plant-associated bacteriardquo Applied andEnvironmental Microbiology vol 77 no 5 pp 1548ndash1555 2011

[47] J M Gardner J L Chandler and A W Feldman ldquoGrowthresponses and vascular plugging of citrus inoculated withrhizobacteria and xylem-resident bacteriardquo Plant and Soil vol86 no 3 pp 333ndash345 1985

[48] C Pliego C Ramos A de Vicente and F M Cazorla ldquoScreen-ing for candidate bacterial biocontrol agents against soilborne

14 International Journal of Microbiology

fungal plant pathogensrdquoPlant and Soil vol 340 no 1-2 pp 505ndash520 2011

[49] H Feng Y Li and Q Liu ldquoEndophytic bacterial communi-ties in tomato plants with differential resistance to Ralstoniasolanacearumrdquo African Journal of Microbiology Research vol 7no 15 pp 1311ndash1318 2013

[50] A V Sturz B R Christie B G Matheson W J Arsenaultand N A Buchanan ldquoEndophytic bacterial communities inthe periderm of potato tubers and their potential to improveresistance to soil-borne plant pathogensrdquo Plant Pathology vol48 no 3 pp 360ndash369 1999

[51] A V Sturz B R Christie and J Nowak ldquoBacterial endophytespotential role in developing sustainable systems of crop produc-tionrdquo Critical Reviews in Plant Sciences vol 19 no 1 pp 1ndash302000

[52] R Ramesh A A Joshi and M P Ghanekar ldquoPseudomonadsmajor antagonistic endophytic bacteria to suppress bacterialwilt pathogenRalstonia solanacearum in the eggplant (Solanummelongena L)rdquo World Journal of Microbiology and Biotechnol-ogy vol 25 no 1 pp 47ndash55 2009

[53] A A Nawangsih I Damayanti S Wiyono and J G KartikaldquoSelection and characterization of endophytic bacteria as bio-control agents of tomato Bacterial Wilt DiseaserdquoHayati Journalof Biosciences vol 18 no 2 pp 66ndash70 2011

[54] R Vargas-Ayala R Rodrıguez-Kabana G Morgan-Jones J AMcInroy and J W Kloepper ldquoShifts in soil microflora inducedby velvetbean (Mucuna deeringiana) in cropping systems tocontrol root-knot nematodesrdquo Biological Control vol 17 no 1pp 11ndash22 2000

[55] D K Zinniel P Lambrecht N B Harris et al ldquoIsolationand characterization of endophytic colonizing bacteria fromagronomic crops and prairie plantsrdquoApplied and EnvironmentalMicrobiology vol 68 no 5 pp 2198ndash2208 2002

[56] F Lemessa and W Zeller ldquoScreening rhizobacteria for biolog-ical control of Ralstonia solanacearum in Ethiopiardquo BiologicalControl vol 42 no 3 pp 336ndash344 2007

[57] M S El-AlbyadM A El-Sayed A R El-Shanshoury andNHEl-Batanouny ldquoEffect of culture conditions on the antimicrobialactivities of UV-mutants of Streptomyces corchorusii and Sspiroverticillatus against bean and banana wilt pathogensrdquoMicrobiological Research vol 151 no 2 pp 201ndash211 1996

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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International Journal of

Volume 2014

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International Journal of

Microbiology

International Journal of Microbiology 3

as haplotypes Haplotypes were delineated at 80 similarityvalues of Dice coefficient and numbered asM80-1 toM80-38Representative strains from each haplotype were identified by16S rRNA gene sequencing

23 Antagonism Towards R solanacearum

231 In Vitro Inhibition Bioassay One hundred and sixty-seven XRB were screened for inhibition of the BW pathogenR solanacearum strain Rs-09-100 was isolated from BWinfected eggplant cultivated in Goa India and was usedfor screening in vitro and in planta Rs-09-100 belongs tophylotype I race 1 and biovar 3 of theR solanacearum speciescomplex The strain is pathogenic to eggplant cv Agassaimand causes 100 wilt within 15 days after inoculation undergreenhouse conditions (data not shown) Bioassay was per-formed by the agar well method as described by Rameshand Phadke [27] Briefly single colony of R solanacearumand XRB was grown in 5mL CPG broth (Casein hydrolysate10 gL Peptone 100 gL and Glucose 50 gL) and Kingrsquos Bbroth (Peptone 200 gL K

2HPO4 15 gL MgSO

4sdot 7H2O

15 gL and Glycerol 100mLL) respectively at 28 plusmn 2∘C for48 h with constant shaking at 140 rpm One hundred andfifty microliters of R solanacearum was seeded every 100mLmolten cooled Kingrsquos B agar mixed well and poured intoplates After the plates solidified three wells were made ineach plate by removing a circular agar piece with the help ofcork borer (8mm diameter) Twenty-five 120583L of culture brothof XRB containing 80 Log CFUmL was added into each ofthe three wells All the plates were incubated at 28 plusmn 2∘C for48 h Plates were observed for inhibition of R solanacearumZones of inhibition were measured as radius in mm from theedge of the agar well Strains that were found antagonisticto R solanacearum were screened for in vitro productionof antagonistic compounds and plant growth promotingsubstances and identified by 16S rRNA gene sequencing

24 Production of Volatile and Diffusible AntagonisticSubstances by XRB

241 Hydrogen Cyanide (HCN) Production Antagonisticstrains were tested for HCN production ability in presence ofglycine as described by Saraf et al [31] a slight modificationbeing the use of broth for the HCN test Immediately afterinoculation of strains in Kingrsquos B broth containing 44 gL ofglycine sterile filter paper strips dipped in picric acid solutionwere introduced taking care that the strips did not touch themedium and walls of the tube The tubes were sealed withparafilm and incubated at 28 plusmn 2∘C for 4 days with constantshaking at 140 rpmThe color change of the filter paper stripsfrom yellow to brick red during incubation indicated theproduction of HCN

242 Ammonia Production To detect ammonia productionantagonistic XRB were grown in peptone water (peptone200 gL NaCl 50 gL) with constant shaking at 140 rpm for48 h at 28 plusmn 2∘C Ammonia production was determined usingNesslerrsquos reagent as described by Marques et al [32]

243 Acetoin Production Acetoin production by antagonis-tic isolates was tested in Voges Proskauer broth (peptone70 gL K

2HPO450 gL dextrose 50 gL pH 70) After

incubation for 30 h at 28 plusmn 2∘C at 140 rpm onemL each of 5120572 napthol and 40KOHwere added to the culture andmixedwell Appearance of red coloration indicated production ofacetoin [33]

244 Siderophore Production Antagonistic XRB were testedfor siderophore production on a medium containing chromeazurol S (CAS) [34] Isolates producing orange haloes on theblue green colored medium after incubation at 28 plusmn 2∘C for48 h were positive for siderophore production

25 Production of Growth Promoting Substances bythe Antagonistic XRB

251 Indole Acetic Acid (IAA) Production Antagonisticstrains were tested for their ability to produce phytohormoneIAA in presence of tryptophan as described by Gordon andPaleg [35] Briefly strains were grown in nutrient brothamended with 100mgL of tryptophan for 30 h at 28 plusmn 2∘Cat 140 rpmThe supernatants were obtained by centrifugationat 6200 g for 10min One mL of supernatant was mixed withone mL of Salkowskyrsquos reagent (50mL 35 perchloric acid1mL 05M FeCl

3) Themixture was allowed to stand at room

temperature for five minutes and the absorbance was readat 530 nm A standard curve was prepared using analyticalgrade IAA and the concentrations of IAA in the culturesupernatants of XRB were estimated based on the curve

252 1-Aminocyclopropane-1-carboxylate (ACC) DeaminaseActivity Antagonistic strains were tested for their abilityto produce enzyme ACC deaminase as per the methoddescribed by Godinho et al [36] Strains were streaked onDworkin and Fosterrsquos DF salts agar containing 30mM ACCand incubated at 28 plusmn 2∘C for 7 days Ability of the strainsto grow on the medium containing ACC as a sole nitrogensource was indicative of ACC deaminase production

253 Phosphate Solubilization Antagonistic strains weretested for phosphate solubilization by a method describedby Godinho et al [36] All strains were spot inoculated onPikovskayarsquos agar plates (Hi Media Laboratories Mumbai)Plates were incubated at 28 plusmn 2∘C for 48 h Transparent zonesaround the growth of XRB on the opaquewhitemediumwereindicative of solubilisation phosphate

26 Greenhouse Experiments

261 Biocontrol Efficacy (BCE) of Antagonistic XRB Twenty-eight strains of XRBwere selected based on in vitro inhibitionof R solanacearum in the agar well bioassay Strains wereevaluated for controlling BW in seedlings of wilt susceptibleeggplant cv Agassaim under greenhouse conditions Thirty-day-old seedlings raised in nonsterile soil in greenhousewere transplanted in pots filled with standard nonsterilepot mixture (soil sand farmyard manure at 2 1 1 ratio)

4 International Journal of Microbiology

Ten mL suspension of antagonistic XRB (80 Log CFUmL)in sterile 1 X PBS was applied per seedling by soil drenchingEach treatment consisted of two replicates with two potsper replication and five seedlings per pot Twenty days aftertreatment with the antagonistic XRB the seedlings were chal-lenged by inoculating 10mL suspension of R solanacearumstrain Rs-09-100 (70 Log CFUmL) by soil drenching Plantsnot treated with XRB but challenged with R solanacearumserved as control Plants were maintained with suitablewatering and percentage of plants infected by wilt was noteduntil 25 days after challenging with R solanacearum Abilityof the XRB to prevent wilt in eggplant was expressed asbiocontrol efficacy (BCE) and was determined using theformula BCE = ([percent disease in control] minus [percentdisease in treatment]percent disease in control) times 100 [37]Strains with BCE greater than 25 were evaluated for theireffect on growth in eggplant under greenhouse conditions

262 Growth Promotion Ability of XRB Sixteen strainsof XRB exhibiting biocontrol efficacies greater than 25were studied for their effect on growth in eggplant Abilityto increase shoot length in wilt susceptible eggplant cvAgassaim was used as a measure to evaluate their growthpromotion efficacy under greenhouse conditions Thirty-day-old seedlings raised in nonsterile soil in greenhousewere transplanted in pots filled with standard nonsterile potmixture (soil sand farmyardmanure at 2 1 1 ratio) TenmLsuspension of XRB (80 Log CFUmL) in sterile 1 X PBSwas applied per seedling by soil drenching Each treatmentconsisted of two replicates with two pots per replication andfive seedlings per pot Plants were maintained with suitablewatering and plant height was measured from the soil level tothe shoot tip 40 days postinoculation Ability of antagonisticXRB to increase shoot length in eggplant was expressed asgrowth promotion efficacy (GPE) using the formula ([shootlength increase in treatment] minus [shoot length increase incontrol]shoot length increase in control) times 100 [38]

27 16S rRNA Gene Sequencing and Sequence Analysis Rep-resentative strains from each of the 38 ARDRA haplotypesand XRB exhibiting antagonism to R solanacearum wereselected for identification A total of 55 strains were cho-sen for identification Fragments of the 16S rRNA geneof size 1500 bp were amplified as described above in theARDRA section Amplicons were purified using GeneJetPCRpurification kit (Thermo Scientific USA) and sequencedusing 27F and 1492R primers (Xcelris Labs Pvt LtdIndia) Partial 16S rRNA gene sequences (about 1200 nt)obtained were matched against the sequences available inthe nucleotide database from National Center for Biotech-nology Information (httpwwwncbinlmnihgovBLAST)using the BLASTn (Basic Local Alignment Search Tool)program

3 Results

31 Isolation of Bacteria from the Xylem of Eggplant Chilli andS torvum In this study bacteria could be constantly isolated

from the xylem of eggplant chilli and S torvum by vacuuminfiltration andmaceration techniques Bacterial counts fromeach isolation ranged from 10 to 102 CFUmL of xylem sap ormacerate Colonies appeared between 24 and 120 h of aerobicincubation at 28plusmn2∘CAmongst 167 isolates obtained 99wereGram-negative rods (5928) and 68 were Gram-positivebacteria (4072) comprising of 42 rods (6176) 25 cocci(3676) and one filamentous actinomycete (Table 1)

32 ARDRA Analysis ARDRA generated three to six restric-tion fragments of the 16S rRNA gene amplified from theXRB Analysis of ARDRA profiles by UPGMA using Dicersquoscoefficient dividedXRBwith identical restriction profiles intoseveral groups At 80 similarity values of Dice coefficient167 strains of XRB were grouped into 38 haplotypes Hostbased analysis of ARDRA revealed that 89 strains isolatedfromBW susceptible eggplant were grouped in 31 haplotypes36 strains from BW resistant eggplant into 17 haplotypes 33strains from chilli into 19 haplotypes and 9 strains from Storvum into 7 haplotypes respectively (Table 1) A detailedrepresentation of the ARDRA based analysis of 167 strainsis presented in Table 2 Based on the ARDRA analysis ofthe collection of XRB 153 strains were distributed over 24different haplotypes and 14 XRB strains had unique profileswhich formed 14 independent haplotypes with one strain ineach Antagonistic strains (119899 = 28) were distributed over 14different ARDRA haplotypes wherein 6 antagonists formedindependent haplotypes Nonantagonistic strains were dis-tributed over 24 haplotypes Haplotypes M80-9 and M80-15were shared amongst BW susceptible and resistant eggplantchilli and S torvum Eleven haplotypes were unique to BWsusceptible eggplant Haplotypes M80-1 M8-12 and M80-38 were unique to BW resistant eggplant whereas haplotypesM80-13 M80-19 and M80-26 comprised of strains isolatedfrom chilli Haplotype M80-36 had a strain isolated from Storvum Other haplotypes were a combination of strains iso-lated from different plant species These results indicate thatbacterial communities from the xylem of mainly eggplantand chilli cultivated in different locations in Goa comprise ofdiverse bacteria However it is observed that each ARDRAgroup consists of bacteria from different plant species andplants collected from different locales In addition there arecertain XRB unshared between each of the plant species thatform unique haplotypes Moreover strains with biocontrolability (BCE gt 25) were restricted to only 8 haplotypesnamely M80-6 M80-7 M80-10 M80-15 M80-20 M80-29M80-31 and M80-36 (Table 2) Haplotypes M80-6 M80-7M80-29 M80-31 M80-36 and M80-38 comprised of XRBwith GPE gt 10 Interestingly the strains with BCE gt 25and GPE gt 10 within these haplotypes were from BWresistant eggplant (Table 1)

33 Antagonism towards R solanacearum

331 In Vitro Bioassay and Production of Volatile and Dif-fusible Antagonistic Compounds Plate based bioassay wasused for rapid screening of antagonism of XRB towards R

International Journal of Microbiology 5

Table1Overviewof

diversity

andfunctio

nalityof

XRBob

tained

from

eggplantchilliand

Storvum

Planth

ost

Num

bero

fsamples

Totalisolates

obtained

Gram-negative

Gram-positive

Haplotypesa

obtained

Antagon

istic

strainsb

Antagon

isticstrains

with

BCEgt25

Biocon

trolstrains

with

GPEgt10

Metho

dof

isolatio

n

BWsusceptib

leeggplant

2489

5336

3116

52

VIo

rMBW

resistant

eggplant

1236

2016

176

65

VIo

rMCh

illi(Ca

psicu

mannu

um)

733

2013

193

21

Mon

ly

Solanu

mtorvum

29

63

73

31

VIo

nly

Total

45167

9968

2816

9a H

aplotypesw

ered

etermined

usingARD

RA

b Antagon

ismto

Rsolana

cearum

teste

din

vitro

asdescrib

edin

Section2

BCE(biocontrolefficacy)a

ndGPE

(growth

prom

otioneffi

cacy)w

ered

etermined

underg

reenho

usec

onditio

nsin

eggplant

asdescrib

edin

Section2

VIVa

cuum

infiltration

Mm

aceration

6 International Journal of Microbiology

Table 2 Haplotypes of XRB based on ARDRA withMspI at 80 similarity plant host biocontrol and growth promotion activities

Haplotypenumbera

Number of strainsin each haplotype Plant host Strains selected for

identificationNumber of biocontrolstrains with BCE gt 25

Number of strainswith GPE gt 10

M80-1 1 RE XB159 0 0M80-2 1 SE XB34 0 0M80-3 1 SE XB66 0 0M80-4 2 SE C XB40 0 0M80-5lowast 1 SE XB140 0 0

M80-6lowast 10 SE RE C XB177 XB157 XB93XB169 XB153 XB170 4 3

M80-7lowast 6 SE RE C XB1 XB86 2 2M80-8 4 SE RE XB22 XB190 0 0M80-9lowast 8 SE RE ST C XB99 XB100 0 0M80-10lowast 10 SE RE C XB196 XB103 1 0M80-11 9 SE RE C XB137 0 0M80-12 1 RE XB158 0 0M80-13 1 C XB87 0 0M80-14 5 SE C XB88 0 0M80-15lowast 5 SE RE ST C XB70 1 0M80-16 3 SE XB47 0 0M80-17 2 SE XB35 0 0M80-18 1 SE XB41 0 0M80-19 2 C XB94 0 0M80-20lowast 13 SE RE C XB8 XB20 XB27 2 0M80-21 5 SE ST C XB25 0 0M80-22 1 SE XB53 0 0M80-23 5 SE RE C XB98 0 0M80-24 11 SE RE ST XB161 0 0M80-25 4 SE RE XB188 0 0M80-26 1 C XB168 0 0M80-27lowast 2 SE C XB126 XB7 0 0M80-28lowast 5 SE RE XB122 0 0M80-29lowast 9 SE RE C XB165 1 1M80-30 15 SE RE C XB167 XB109 0 0

M80-31lowast 11 SE ST C XB123 XB203 XB62XB114 XB202 3 1

M80-32 5 SE ST C XB92 0 0M80-33 1 SE XB37 0 0M80-34 1 SE XB36 0 0M80-35 2 SE XB64 0 0M80-36lowast 1 ST XB200 1 1M80-37lowast 1 SE XB134 0 0M80-38lowast 1 RE XB197 1 1aHaplotype based on ARDRA analysis usingMspI at 80 similarity values of Dice coefficientlowastHaplotype comprises of bacteria showing antagonistic property in the bioassaysSE bacterial wilt susceptible eggplant RE bacterial wilt resistant eggplant ST Solanum torvum C chilli plantBCE biocontrol efficacy GPE growth promotion efficacy determined as described in Section 2

International Journal of Microbiology 7

solanacearum strain Rs-09-100 Results of the in vitro screen-ing against R solanacearum revealed that 28 amongst 167XRB exhibited antagonism towards the pathogen (Table 3)Amongst the antagonists 16 were strains from BW suscepti-ble eggplant 6 from BW resistant eggplant and 3 each fromchilli and S torvum (Table 1) Amongst the 28 antagonists 7strains namely XB62 XB99 XB100 XB114 XB122 XB196XB197 and XB202 formed larger inhibition zones againstR solanacearum ranging from 40mm to 817mm (Table 3)The majority of the antagonistic strains (119899 = 16) producedinhibition zones ranging from 20mm to 383mm XB27XB134 XB165 and XB169 formed smaller inhibition zonesranging from 15mm to 20mm However 139 strains ofXRB did not inhibit R solanacearum in the bioassay testTwenty-eight antagonistic XRBwere screened for productionof volatile inhibitory compounds namely acetoin HCNammonia and diffusible siderophore molecules in vitro(Table 3) Acetoin production was observed in 3214 of theisolates Bacterial isolates XB7 and XB122 were found toproduce both HCN as well as siderophores XB62 XB93 andXB170 produced HCN whereas XB114 XB140 and XB203produced siderophores only XB93 XB99 XB123 XB134 andXB140 produced ammonia

34 Production of Plant Growth Promoting Substances byAntagonistic XRB Results of the screening of antagonisticXRB for in vitro production of several plant growth pro-moting compounds is presented in Table 3 Majority of theantagonistic strains produced the phytohormone IAA withconcentrations ranging from 1591 120583gmL to 64591120583gmLXB202 was found to be the best ACC deaminase producingstrain based on its luxuriant growth on DF salts mediumsupplemented with 30mM ACC as sole nitrogen sourceOther ACC deaminase producing strains include XB1 XB62XB86 and XB140 Scarce growth of XB165 and XB200 wasobserved onDF saltsmedium 6428of the strains producedphosphate solubilizing organic acids as indicated by clearhaloes on Pikovskayarsquos agar plate

35 Greenhouse Experiments

351 Suppression of Bacterial Wilt by Antagonistic XRBAbility to suppress BW was assessed as the difference in thepercentage of wilt in XRB treated plants with respect to wilt inuntreated control andwas expressed as the biocontrol efficacy(BCE) of the antagonists BCE of 16 strains with valuesranging from 286 to 100 is presented in Figure 1 Plantstreated with strains XB86 XB169 and XB177 were free fromBWand hence recorded 100 biocontrol efficacy Treatmentswith XB170 XB197 XB200 XB202 and XB203 recorded 30percent or less wilt incidence (70 to 85 BCE) XB1 XB27XB70 XB93 and XB123 treatments recorded BCE between429 and 571 BCE of 286 was recorded in XB20 andXB165 treatments However 12 antagonistic XRB recordedBCEof 25 andwere least effective inwilt protection Furtherit is observed that all the antagonistic strains originating fromBW resistant eggplant and S torvum exhibited BCE greaterthan 25 Five antagonistic strains from BW susceptible

minus2000

000

2000

4000

6000

8000

10000

12000

XB1

XB20

XB27

XB70

XB86

XB93

XB123

XB165

XB169

XB170

XB177

XB196

XB197

XB200

XB202

XB203

Con

trol

Isolates

Biocontrol efficacy (BCE)Growth promotion efficacy (GPE)

BCE

and

GPE

()

Figure 1 Biocontrol and plant growth promotion efficacies of selectXRB in eggplant BCE biocontrol efficacy determined 25 days afterchallenging with R solanacearum GPE growth promotion efficacydetermined 40 days after treatmentwithXRB Percent BCE andGPEcalculated using formulae BCE = ([percent disease in control] minus[percent disease in treatment]percent disease in control) times 100 andGPE= ([shoot length increase in treatment]minus [shoot length increasein control]shoot length increase in control) times 100 respectivelyUninoculated control had BCE and GPE values of 000 XB86XB169 and XB177 had BCE of 10000 in all the replicationsBars indicate mean values of BCE and GPE error bars indicatestandard deviation

eggplant and three fromchilli were effective in preventingwiltin eggplant (Table 1)

352 Growth Promotion by Antagonistic XRB Increase inshoot length of eggplant (40 days after treatment) observedin XRB treated plants in relation to untreated control wasexpressed as growth promotion efficacy (GPE) and is shownin Figure 1 Amongst the 16 strains which were effective inpreventing wilt six strains exhibited the highest increase inshoot length as indicated by their GPE values in the rangeof 139ndash223 Seven XRB recorded a GPE value rangingfrom 14 to 129 However strains XB27 XB196 and XB203stunted shoot growth in eggplant in comparison to untreatedcontrol When the source of XRB is considered 5555strains that exhibited GPE greater than 10 were isolatedfrom BW resistant eggplant Whereas only two antagonistsfrom BW susceptible plant and one each from chilli and Storvum were able to promote growth in eggplant (Table 1)Strains with GPE gt 10 belonged to six different ARDRAhaplotypes (Table 2)

36 Identification of XRB by 16S rRNA Gene Sequencing16S rRNA gene sequences of XRB were used to identify thediverse xylem inhabitants and antagonistic strains Identityof 55 XRB based on 16S rRNA gene sequencing and theirGenBank accessions are presented in Table 4 Overall 23

8 International Journal of Microbiology

Table3

Invitro

inhibitio

nof

Rsolana

cearum

andprod

uctio

nof

antagonisticc

ompo

unds

andplantg

rowth

prom

otingsubstances

byXR

B

Strain

Haplotype

Inhibitio

nof

Rsolana

cearum

Volatile

anddiffu

sibleinhibitory

compo

unds

Plantgrowth

prom

otingsubstances

Radius

inmm

HCN

Ammon

iaAc

etoin

Sideroph

ore

Phosph

ates

olub

ilisatio

nAC

Cdeam

inasea

IAA

b120583gmL

XB1

M80-7

283plusmn029

minusminus

minusminus

minus++

+10500plusmn707

XB7

M80-27

333plusmn058

+minus

minus+

+minus

4773plusmn321

XB8

M80-20

333plusmn029

minusminus

minusminus

+minus

1909plusmn129

XB20

M80-20

327plusmn040

minusminus

minusminus

+minus

2545plusmn171

XB27

M80-20

183plusmn029

minusminus

minusminus

+minus

1591plusmn107

XB62

M80-31

817plusmn076

+minus

minusminus

+++

++7318plusmn493

XB70

M80-15

383plusmn058

minusminus

minusminus

minusminus

17182plusmn1157

XB86

M80-7

233plusmn076

minusminus

minusminus

minus++

+6682plusmn450

XB93

M80-6

317plusmn029

+minus

+minus

+minus

5727plusmn386

XB99

M80-9

683plusmn058

minus+

+minus

+minus

32136plusmn2164

XB100

M80-9

450plusmn050

minus+

+minus

+minus

23864plusmn1607

XB114

M80-31

650plusmn050

minusminus

minus+

+minus

8909plusmn600

XB122

M80-28

433plusmn058

+minus

minus+

+minus

6682plusmn450

XB123

M80-31

300plusmn087

minus+

+minus

+minus

41682plusmn2807

XB126

M80-27

283plusmn029

minusminus

+minus

+minus

6045plusmn407

XB134

M80-37

157plusmn012

minus+

+minus

+minus

64591plusmn4350

XB140

M80-5

367plusmn076

minus+

minus+

+++

++5091plusmn343

XB153

M80-6

317plusmn029

minusminus

+minus

+minus

15591plusmn1050

XB157

M80-6

250plusmn100

minusminus

+minus

+minus

18455plusmn1243

XB165

M80-29

157plusmn012

minusminus

minusminus

minus+

19091plusmn1286

XB169

M80-6

153plusmn006

minusminus

minusminus

minusminus

1591plusmn107

XB170

M80-6

351plusmn002

+minus

minusminus

+minus

2864plusmn193

XB177

M80-6

199plusmn049

minusminus

minusminus

minusminus

7636plusmn514

XB196

M80-10

393plusmn012

minusminus

minusminus

minusminus

3500plusmn236

XB197

M80-38

433plusmn029

minusminus

+minus

minusminus

4136plusmn279

XB200

M80-36

187plusmn023

minusminus

minusminus

minus++

16864plusmn1136

XB202

M80-31

467plusmn021

minusminus

minusminus

+++

+8273plusmn557

XB203

M80-31

367plusmn029

minusminus

minusminus

minusminus

7000plusmn471

Inhibitio

nzonesa

remeanof

threereplications

andshow

ingsta

ndarddeviation

Inhibitio

nzone

was

measuredas

radius

from

theou

tere

dgeof

wellAlltheexperim

entswerecond

uctedat28plusmn2∘C

+indicates

presence

oftraitminusindicatesabsence

oftraitinplateb

ased

assaysaLevelsofAC

Cdeam

inasea

ctivitiesbasedon

grow

thon

plateb

ased

assayd

enoted

as+forlessg

rowth+++

form

oderateg

rowth+++

forlux

uriant

grow

thand

++++

forh

ighlyluxu

riant

grow

thbIAAwas

estim

ated

incultu

refiltra

teandexpressedas120583gmLusinganalyticalgradeIAAas

stand

ardvalues

indicatemeanandsta

ndarddeviation

International Journal of Microbiology 9

2545

2545

364

364

1273 2909

ActinobacteriaFirmicutesAlpha proteobacteria

Beta proteobacteriaGamma proteobacteriaBacteroidetes

(a)

Num

ber o

f XRB

Actin

obac

teria

Firm

icut

es

Alp

ha p

rote

obac

teria

Beta

pro

teob

acte

ria

Gam

ma p

rote

obac

teria

Bact

eroi

dete

s

Number of antagonistic XRB in each phylumsubdivision

Phylasubdivision

Total number of XRB belonging to each phylumsubdivision

25

20

15

10

5

0

2

14

9

16

4

7

1

2

1

2

11

14

(b)

Figure 2 (a) Distribution of XRB (phylumsubdivision level) identified by 16S rRNA gene sequencing Values indicate percentages of strainsbelonging to each phylasubdivision amongst the 55 identified strains (b) Distribution of antagonistic and nonantagonistic strains of XRB ineach phylumsubdivision

different genera of bacteria were identified Major gen-era identified are Bacillus sp (11 strains) Enterobacter sp(6 strains) Microbacterium sp (5 strains) Staphylococcussp (5 strains) Pseudomonas sp (5 strains) and Agrobac-terium sp (3 strains) Additional genera identified includ-ing Micrococcus sp Sphingomonas sp Flavobacterium spChryseobacterium sp Burkholderia sp and Xenophilus spare listed in Table 4 Based on the identification phylumProteobacteria consisting of Gram-negative bacteria of sub-divisions Alpha Proteobacteria (1273) Beta Proteobacteria(364) and Gamma Proteobacteria (2545) were predom-inant (4181 strains identified) followed by phyla Firmi-cutes (2909) Actinobacteria (2545) and Bacteroidetes(364) (Figure 2(a))

Eleven antagonistic strains identified belonged toGammasubdivision of Proteobacteria consisting of five strainseachof Enterobacter sp and fluorescent and nonfluorescent Pseu-domonas sp and one strain of Pantoea eucrina (XB126)(Figure 2(b)) Nine antagonists identified were of phylaFirmicutes consisting of Staphylococcus sp (5 strains) andBacillus sp (4 strains) Agrobacterium strains XB1 XB86 andXB165 Sphingomonas sp (XB197) of the Alpha Proteobacte-ria Streptomyces sp (XB200) and Janibacter melonis (XB70)of phyla Actinobacteria were found to be antagonistic Addi-tional antagonistic XRB include Burkholderia sp (XB140) of120573 Proteobacteria and Flavobacterium sp (XB203) of phylaBacteroidetes (Figure 2(b))

37 Statistical Analysis The statistical analysis of percent-age wilts and shoot length of eggplant was performedusing Web Agri Statistical Package (WASP) version 20(httpwwwicargoaresinwasp20indexphp)

4 Discussion

Eggplant and chilli not only are of economic and culturalimportance but also are common ingredients in the cuisinethroughout India In the coastal state of GoaR solanacearumhas been reported to be a destructive pathogen in cultivationof eggplant and chilli [4] Isolation of biocontrol agentsagainst the BW pathogen has been commonly restricted toendophytic tissue and plant rhizosphere [26 27] Studieson xylem colonizing endophytes were undertaken becausewe speculated existence of interactions between the XRBand vascular wilt pathogen R solanacearum during xylemcolonization Our study reveals the diversity biocontrolpotential and identity of endophytic xylem colonizers fromsolanaceous crops cultivated in Goa India A total of 167bacteria were isolated from the xylem of eggplant chilliand S torvum with Gram-negative bacteria (5928) pre-dominating in the collection Congruent to our observationGardner et al [7] and Bell et al [11] have earlier reportedisolation of more number of Gram-negative rod shapedbacteria from xylem of citrus and grapevine using vacuuminfiltration Scholander pressure bomb was found to beuseful in extraction of diverse bacterial genera from xylemtissues in contrast to trituration methods that yielded highernumber of Gram-positive rod shaped bacteria [12] ThoughScholander pressure bomb was not used in this study acombination of vacuum infiltration [7 11] and triturationof decorticated stems [10 12] was employed with an aimto isolate diverse XRB from xylem tissues This is the firststudy reporting the use of vacuum infiltration and triturationmethods for isolating xylem residing bacteria from eggplantchilli and S torvum

Traditionally bacteria have been characterized andgrouped based on colony morphology and biochemical

10 International Journal of Microbiology

Table 4 List of XRB identified by partial 16S rRNAgene sequencing their plant host accession numbers closestNCBImatch and similarity

Strain Plant host Accession number Closest NCBI match similarityXB1lowast SE KF447383 Agrobacterium tumefaciens 99XB7lowast SE KF447384 Pseudomonas aeruginosa 99XB8lowast SE KF447385 Staphylococcus haemolyticus 99XB20lowast SE KF447386 Staphylococcus haemolyticus 100XB22 SE KF447387 Brevibacterium casei 99XB25 C KF447388 Enterobacter sp 95XB27lowast SE KF447389 Staphylococcus haemolyticus 99XB34 SE KF447390 Curtobacterium sp 99XB35 SE KF447391 Microbacterium sp 99XB36 SE KF447392 Xenophilus sp 99XB37 SE KF447393 Chryseobacterium sp 99XB40 SE KF447394 Micrococcus luteus 89XB41 SE KF447395 Bacillus sp 99XB47 SE KF447396 Bacillus sp 99XB53 SE KF447397 Micrococcus sp 99XB62lowast SE KF447398 Pseudomonas sp 99XB64 SE KF447399 Bacillus thuringiensis 100XB66 SE KF447400 Pectobacterium carotovorum 99XB70lowast SE KF447401 Janibacter melonis 99XB86lowast C KF447402 Agrobacterium tumefaciens 99XB87 C KF447403 Bacillus barbaricus 99XB88 C KF447404 Bacillus sp 99XB92 C KF447405 Brevundimonas vesicularis 99XB93lowast C KF447406 Bacillus safensis 100XB94 C KF447407 Bacillus sp 100XB98 C KF447408 Microbacterium sp 100XB99lowast SE KF447409 Enterobacter sp 99XB100lowast SE KF447410 Enterobacter cloacae 98XB103 SE KF447411 Brachybacterium phenoliresistens 99XB109 SE KF447412 Rhodococcus corynebacterioides 99XB114lowast SE KF447413 Pseudomonas stutzeri 99XB122lowast SE KF913446 Pseudomonas aeruginosa 100XB123lowast SE KF447414 Enterobacter sp 99XB126lowast C KF447415 Pantoea eucrina 99XB134lowast SE KF447416 Enterobacter sp 99XB137 SE KF447417 Klebsiella sp 99XB140lowast SE KF447418 Burkholderia sp 99XB153lowast SE KF447419 Bacillus amyloliquefaciens 99XB157lowast SE KF447420 Bacillus subtilis 100XB158 RE KF447421 Bosea sp 99XB159 RE KF447422 Microbacterium xylanilyticum 99XB161 RE KF447423 Microbacterium aurum 99XB165lowast RE KF447424 Agrobacterium sp 99XB167 RE KF447425 Microbacterium aurum 99XB168 RE KF447426 Bacillus aryabhattai 100XB169lowast RE KF447427 Staphylococcus gallinarum 99XB170lowast RE KF447428 Staphylococcus sp 99XB177lowast RE KF447429 Bacillus cereus 99XB188 RE KF447430 Sphingomonas sp 99XB190 RE KF447431 Brevibacterium casei 99

International Journal of Microbiology 11

Table 4 Continued

Strain Plant host Accession number Closest NCBI match similarityXB196lowast RE KF447432 Enterobacter kobei 99XB197lowast RE KF447433 Sphingomonas sp 98XB200lowast ST KF913447 Streptomyces sp 100XB202lowast ST KF447434 Pseudomonas sp 99XB203lowast ST KF447435 Flavobacterium sp 99lowastStrain is antagonistic to R solanacearum based on bioassaySE bacterial wilt susceptible eggplant RE bacterial wilt resistant eggplant C chilli ST Solanum torvum

tests However whole genome fingerprinting or PCR-RFLPbased methods are rapid tools for determining geneticdiversity of bacteria in a given collection ARDRA which isa type of PCR based RFLP method has been used widely inestimating genetic diversity endophytic bacterial populationsand clustering genetically similar strains [39ndash41] In additionto earlier reports our study demonstrates the usefulnessof ARDRA as a tool to cluster genetically identical strainsof endophytic XRB isolated from eggplant chilli andS torvum In our study 9161 strains (119899 = 153) weregrouped in 24 haplotypes by using ARDRA The majority ofthese haplotypes represent a combination of XRB isolatedfrom different solanaceous plants from diverse localesThese results indicate that xylem of eggplant chilli andS torvum largely bears similar population of XRB whichcan efficiently cross colonize eggplant chilli or S torvumNevertheless 839 strains had unique ARDRA fingerprintand formed separable haplotypes This observation leads toa conclusion that a minor population of xylem inhabitantsare restricted to a specific plant species and cannot easilycross colonize xylem of other solanaceous plants Plantsare known to selectively support endophytic colonizationby specific bacteria [14] However factors that determinethe selection of xylem colonists or the ability of XRBto colonize eggplant chilli and S torvum in this studyremain unknown Interestingly the structure of endophyticcommunity in Nicotiana attenuata a member of Solanaceaefamily is shown to be influenced by soil composition andethylene homeostasis [40] Earlier evidence has shown thatcolonization by endophytic bacteria is also governed by plantgenotype as well as root exudates [42]

Only 1677 XRB out of 167 were antagonistic to Rsolanacearum based on in vitro assays Antagonistic XRBproduced volatile and diffusible inhibitory compoundsnamely HCN ammonia and acetoin and siderophoresThese substances have been long known to be involvedin disease suppression and indirect growth promotion inplants [43ndash46] These mechanisms possibly played a rolein the evident biocontrol effect against BW exhibited bythe XRB in the greenhouse screening Endophytic bacteriahave been known to have plant growth promoting traitsnamely production of IAA ACC deaminase and phosphatesolubilization [25 33 36] These traits were detected in themajority of antagonistic XRB tested in this study and mayhave resulted in the observed increase in shoot length ofeggplant in our greenhouse experiments In contrast strains

XB20 XB196 and XB203 suppressed growth in eggplantunder greenhouse conditions however no visible symptomsof disease were observed Vascular plugging and productionof certainmetabolites toxic to plant cells but not cell viabilitymay have resulted in stunted shoot in eggplant [11 47]

Evaluation of efficacy of antagonistic organisms to sup-press the plant diseases under greenhouse conditions isone of the key steps for selecting a potential biocontrolagent for disease management [48] Endophytic strains fromBW susceptible varieties of eggplant have been shown toprevent wilt and promote growth in eggplant earlier [27]Our greenhouse screening shows that 3846 of antagonisticXRB with biocontrol efficacies greater than 40 were isolatesfrom BW resistant varieties of eggplantThis raises a questionwhether the bacteria from resistant varieties are involved inBW resistance and whether BW resistant varieties are ableto selectively influence xylem colonization by antagonisticbacteria Presence of higher number of endophytes withantagonistic abilities was reported in BW resistant varieties oftomato as compared to susceptible varieties and their role inresistance to BWwas proposed [49] Similar observations oncorrelation of resistance of potato to soft rot and endophyticbacteria have been reported [50]Thus BW resistant varietiescan be considered a better host for isolating potential biocon-trol strains for management of bacterial wilt

Identification of 55 XRB strains by 16S rRNA genesequencing revealed the presence of 23 diverse genera ofbacteria belonging to 4 phyla of Eubacteria Strains belongingto phyla Firmicutes Actinobacteria and 120574 subdivision ofProteobacteria were the major xylem colonists identified inthis study Several genera of bacteria belonging to these phylahave also been reported to be present in endophytic tissuesand xylem of a variety of other agricultural and horticulturalplant species [14 51] In addition the majority of the antag-onists identified belonged to Enterobacter sp Pseudomonassp Bacillus sp and Staphylococcus sp Congruent to ourresults several researchers have reported bacteria isolatedfrom solanaceous crops and belonging to similar genera tobe antagonistic to R solanacearum [26 51ndash53] HoweverFlavobacterium spand Janibacter melonis identified in thisstudy have never been previously reported to be inhibitoryto R solanacearum Large population of the xylem inhabitingbacterial flora accounting for 8323 exhibited no antago-nism towards R solanacearum Nonantagonistic XRB wereidentified predominantly as Microbacterium sp Endophyticpersistence and nematicidal activities of Microbacterium sp

12 International Journal of Microbiology

have been reported [54 55] Therefore the collection of XRBisolated in this study can be screened for inhibitory activitiesagainst other important agricultural pests

Though few strains namely XB86 XB169 and XB177exhibited plant beneficial properties in this study their use-fulness in plant disease control remains to be seen XB86has been identified as Agrobacterium tumefaciens the crowngall disease pathogen and its deployment as biocontrolagent is uncertain XB169 (Staphylococcus gallinarum) andXB177 (Bacillus cereus) are reported as opportunistic animalpathogens and thus unsuitable for field applications Strep-tomyces sp has earlier been reported as antagonistic to Rsolanacearum and tested for management of wilt in potatoand tomato [56 57] XB200 (Streptomyces sp) is one of theXRB high BCE and GPE it could be explored further forbiocontrol of bacterial wilt after additional characterizationand field evaluation

5 Conclusion

This study is the first report on the identity of novel anddiverse XRB colonizing the xylem of eggplant chilli and Storvum XRB particularly from BW resistant varieties werefound to protect eggplant from bacterial wilt and enhancedgrowth in eggplant in the greenhouse screening Thereforethe repertoire of XRB reported in this study may be usefulfor cultivation of eggplant in BW affected areas

Conflict of Interests

The authors hereby declare that they have no conflict ofinterests regarding the publication of this paper

Acknowledgments

The authors greatly acknowledge financial support fromIndian Council of Agricultural Research (ICAR) New DelhiIndia through ldquoOutreach project on Phytopthora Fusariumand Ralstonia diseases of horticultural and field cropsrdquo-(PhytoFuRa) and the Director ICAR Research Complex forGoa India for the facilities

References

[1] E Yabuuchi Y KosakoHOyaizu et al ldquoProposal ofBurkholde-ria gen nov and transfer of seven species of the genusPseudomonas homology group II to the new genus with thetype species Burkholderia cepacia (Palleroni and Holmes 1981)comb novrdquo Microbiology and Immunology vol 36 no 12 pp1251ndash1275 1992

[2] E Wicker L Grassart R Coranson-Beaudu et al ldquoRalstoniasolanacearum strains from Martinique (French West Indies)exhibiting a new pathogenic potentialrdquo Applied and Environ-mental Microbiology vol 73 no 21 pp 6790ndash6801 2007

[3] Y Liu A Kanda K Yano et al ldquoMolecular typing of Japanesestrains of Ralstonia solanacearum in relation to the ability toinduce a hypersensitive reaction in tobaccordquo Journal of GeneralPlant Pathology vol 75 no 5 pp 369ndash380 2009

[4] R Ramesh ldquoField evaluation of biological control agents for themanagement of Ralstonia solanacearum in Brinjalrdquo Journal ofMycology and Plant Pathology vol 36 no 2 pp 327ndash328 2006

[5] J Vasse P Frey and A Trigalet ldquoMicroscopic studies ofintercellular infection and protoxylem invasion of tomatoroots by Pseudomonas solanacearumrdquoMolecular Plant-MicrobeInteractions vol 8 no 2 pp 241ndash251 1995

[6] S Genin and C Boucher ldquoRalstonia solanacearum secrets of amajor pathogen unveiled by analysis of its genomerdquo MolecularPlant Pathology vol 3 no 3 pp 111ndash118 2002

[7] J M Gardner A W Feldman and R M Zablotowicz ldquoIdentityand behavior of xylem-residing bacteria in rough lemon roots ofFlorida citrus treesrdquo Applied and Environmental Microbiologyvol 43 no 6 pp 1335ndash1342 1982

[8] M J Jacobs W M Bugbee and D A Gabriellson ldquoEnu-meration location and characterization of endophytic bacteriawithin sugar beet rootsrdquoCanadian Journal of Botany vol 63 no7 pp 1262ndash1265 1985

[9] D G Patriquin and J Dobereiner ldquoLight microscopy observa-tions of tetrazolium-reducing bacteria in the endorhizosphereof maize and other grasses in Brazilrdquo Canadian Journal ofMicrobiology vol 24 no 6 pp 734ndash742 1978

[10] S Gagne C Richard H Rousseau and H Antoun ldquoXylem-residing bacteria in alfalfa rootsrdquo Canadian Journal of Micro-biology vol 33 no 11 pp 996ndash1000 1987

[11] C R Bell G A Dickie W L G Harvey and J W Y FChan ldquoEndophytic bacteria in grapevinerdquo Canadian Journal ofMicrobiology vol 41 no 1 pp 46ndash53 1995

[12] J Hallmann J W Kloepper and R Rodrıguez-Kabana ldquoAppli-cation of Scholander pressure bomb to studies on endophyticbacteria of plantsrdquo Canadian Journal of Microbiology vol 43no 5 pp 411ndash416 1997

[13] J S Lampel G L Canter M B Dimock et al ldquoIntegrativecloning expression and stability of the cryIA(c) gene fromBacillus thuringiensis subsp kurstaki in a recombinant strain ofClavibacter xyli subsp cynodontisrdquo Applied and EnvironmentalMicrobiology vol 60 no 2 pp 501ndash508 1994

[14] M Rosenblueth and E Martınez-Romero ldquoBacterial endo-phytes and their interactions with hostsrdquo Molecular Plant-Microbe Interactions vol 19 no 8 pp 827ndash837 2006

[15] P R Hardoim L S van Overbeek and J D van ElsasldquoProperties of bacterial endophytes and their proposed role inplant growthrdquo Trends in Microbiology vol 16 no 10 pp 463ndash471 2008

[16] R Aravind A Kumar S J Eapen and K V RamanaldquoEndophytic bacterial flora in root and stem tissues of blackpepper (Piper nigrum L) genotype isolation identificationand evaluation against Phytophthora capsicirdquo Letters in AppliedMicrobiology vol 48 no 1 pp 58ndash64 2009

[17] J Kuklinsky-Sobral W L Araujo R Mendes I O Geraldi AA Pizzirani-Kleiner and J L Azevedo ldquoIsolation and charac-terization of soybean-associated bacteria and their potential forplant growth promotionrdquo Environmental Microbiology vol 6no 12 pp 1244ndash1251 2004

[18] S Compant B Duffy J Nowak C Clement and E A BarkaldquoUse of plant growth-promoting bacteria for biocontrol ofplant diseases principles mechanisms of action and futureprospectsrdquoApplied and Environmental Microbiology vol 71 no9 pp 4951ndash4959 2005

[19] E T Thorne B M Young G M Young et al ldquoThe structureof xylem vessels in grapevine (Vitaceae) and a possible passive

International Journal of Microbiology 13

mechanism for the systemic spread of bacterial diseaserdquo Amer-ican Journal of Botany vol 93 no 4 pp 497ndash504 2006

[20] W T Seo W J Lim E J Kim H D Yun Y H Lee and KM Cho ldquoEndophytic bacterial diversity in the young radishand their antimicrobial activity against pathogensrdquo Journal ofApplied Biological Chemistry vol 53 no 4 pp 493ndash503 2010

[21] S Compant H Kaplan A Sessitsch J Nowak E Ait Barkaand C Clement ldquoEndophytic colonization ofVitis vinifera L byBurkholderia phytofirmans strain PsJN from the rhizosphere toinflorescence tissuesrdquo FEMS Microbiology Ecology vol 63 no1 pp 84ndash93 2005

[22] P S B Miguel J C Delvaux M N V de Oliveira et al ldquoDiver-sity of endophytic bacteria in the fruits of Coffea canephorardquoAfrican Journal of Microbiology Research vol 7 no 7 pp 586ndash594 2013

[23] A Lopez-Lopez M A Rogel E Ormeno-Orrillo J Martınez-Romero and E Martınez-Romero ldquoPhaseolus vulgaris seed-borne endophytic community with novel bacterial species suchas Rhizobium endophyticum sp novrdquo Systematic and AppliedMicrobiology vol 33 no 6 pp 322ndash327 2010

[24] M Lwin and S L Ranamukhaarachchi ldquoDevelopment of bio-logical control of Ralstonia solanacearum through antagonisticmicrobial populationsrdquo International Journal of Agriculture andBiology vol 8 no 5 pp 657ndash660 2006

[25] F Rasche E Marco-Noales H Velvis et al ldquoStructural char-acteristics and plant-beneficial effects of bacteria colonizing theshoots of field grown conventional and geneticallymodifiedT4-lysozyme producing potatoesrdquo Plant and Soil vol 289 no 1-2pp 123ndash140 2006

[26] N Amaresan V Jayakumar K Kumar and N ThajuddinldquoEndophytic bacteria from tomato and chilli their diversity andantagonistic potential against Ralstonia solanacearumrdquoArchivesof Phytopathology and Plant Protection vol 45 no 3 pp 344ndash355 2012

[27] R Ramesh and G S Phadke ldquoRhizosphere and endophyticbacteria for the suppression of eggplant wilt caused byRalstoniasolanacearumrdquo Crop Protection vol 37 pp 35ndash41 2012

[28] W J French R G Christis and D L Stassi ldquoRecovery ofrickettsialike bacteria by vacuum infiltration of peach tissuesaffected with phony diseaserdquo Phytopathology vol 67 pp 945ndash948 1977

[29] P R Viss E M Brooks and J A Driver ldquoA simplified methodfor the control of bacterial contamination in woody plant tissueculturerdquo In Vitro Cellular andDevelopmental Biology vol 27 no1 article 42 1991

[30] K Wilson ldquoPreparation of genomic DNA from bacteriardquo inCurrent Protocols in Molecular Biology F M Ausubel R BrentR E Kingston et al Eds pp 241ndash245 John Wiley amp SonsNew York NY USA 1997

[31] M Saraf AThakkar U PandyaM Joshi and J Parikh ldquoPoten-tial of plant growth promoting microorganisms as biofertilizersand biopesticides and itrsquos exploitation in sustainable agricul-turerdquo Journal of Microbiology and Biotechnology Research vol3 no 5 pp 54ndash62 2013

[32] A P G C Marques C Pires H Moreira A O S S Rangeland P M L Castro ldquoAssessment of the plant growth promotionabilities of six bacterial isolates using Zea mays as indicatorplantrdquo Soil Biology and Biochemistry vol 42 no 8 pp 1229ndash1235 2010

[33] X Hao P Xie L Johnstone S J Miller C Rensingand G Wei ldquoGenome sequence and mutational analysis of

plant-growth-promoting bacteriumAgrobacterium tumefaciensCCNWGS0286 isolated from a zinc-lead mine tailingrdquo Appliedand Environmental Microbiology vol 78 no 15 pp 5384ndash53942012

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

[35] S A Gordon and L G Paleg ldquoQuantitative measurement ofindole acetic acidrdquo Plant Physiology vol 10 pp 37ndash48 1957

[36] A Godinho R Ramesh and S Bhosle ldquoBacteria from sanddunes of Goa promoting growth in eggplantrdquoWorld Journal ofAgricultural Sciences vol 6 no 5 pp 555ndash564 2010

[37] J-H Guo H-Y Qi Y-H Guo et al ldquoBiocontrol of tomato wiltby plant growth-promoting rhizobacteriardquo Biological Controlvol 29 no 1 pp 66ndash72 2004

[38] N Aliye C Fininsa and Y Hiskias ldquoEvaluation of rhizo-sphere bacterial antagonists for their potential to bioprotectpotato (Solanum tuberosum) against bacterial wilt (Ralstoniasolanacearum)rdquo Biological Control vol 47 no 3 pp 282ndash2882008

[39] L LagaceM PitreM Jacqeus andDRoy ldquoIdentification of thebacterial community ofmaple sap by using amplified ribosomalDNA (rDNA) restriction analysis and rDNA sequencingrdquoApplied and Environmental Microbiology vol 70 no 4 pp2052ndash2060 2004

[40] H H Long D G Sonntag D D Schmidt and I T BaldwinldquoThe structure of the culturable root bacterial endophyte com-munity of Nicotiana attenuata is organized by soil compositionand host plant ethylene production and perceptionrdquo NewPhytologist vol 185 no 2 pp 554ndash567 2010

[41] E Lopez-Fuentes V M Ruız-Valdiviezo E Martınez-RomeroF A Gutierrez-Miceli L Dendooven and R Rincon-RosalesldquoBacterial community in the roots and rhizosphere of Hyper-icum silenoides Juss 1804rdquo African Journal of MicrobiologyResearch vol 6 no 11 pp 2704ndash2711 2012

[42] M Fang R J Kremer P P Motavalli and G Davis ldquoBacterialdiversity in rhizospheres of nontransgenic and transgenic cornrdquoApplied and EnvironmentalMicrobiology vol 71 no 7 pp 4132ndash4136 2005

[43] C R Howell and R D Stiphanovic ldquoProduction of ammonia byEnterobacter cloaceae and its possible role in biological controlof Pythium pre-emergence damping-off by the bacteriumrdquoEcology and Epidemiology vol 78 no 8 pp 1075ndash1078 1988

[44] A Ramette M Frapolli G Defago and Y Moenne-LoccozldquoPhylogeny of HCN synthase-encoding hcnBC genes in bio-control fluorescent pseudomonads and its relationship withhost plant species and HCN synthesis abilityrdquoMolecular Plant-Microbe Interactions vol 16 no 6 pp 525ndash535 2003

[45] I Loaces L Ferrando and A F Scavino ldquoDynamics diversityand function of endophytic siderophore-producing bacteria inricerdquoMicrobial Ecology vol 61 no 3 pp 606ndash618 2011

[46] Y C Kim J Leveau B B M Gardener E A Pierson L SPierson and C-M Ryu ldquoThe multifactorial basis for planthealth promotion by plant-associated bacteriardquo Applied andEnvironmental Microbiology vol 77 no 5 pp 1548ndash1555 2011

[47] J M Gardner J L Chandler and A W Feldman ldquoGrowthresponses and vascular plugging of citrus inoculated withrhizobacteria and xylem-resident bacteriardquo Plant and Soil vol86 no 3 pp 333ndash345 1985

[48] C Pliego C Ramos A de Vicente and F M Cazorla ldquoScreen-ing for candidate bacterial biocontrol agents against soilborne

14 International Journal of Microbiology

fungal plant pathogensrdquoPlant and Soil vol 340 no 1-2 pp 505ndash520 2011

[49] H Feng Y Li and Q Liu ldquoEndophytic bacterial communi-ties in tomato plants with differential resistance to Ralstoniasolanacearumrdquo African Journal of Microbiology Research vol 7no 15 pp 1311ndash1318 2013

[50] A V Sturz B R Christie B G Matheson W J Arsenaultand N A Buchanan ldquoEndophytic bacterial communities inthe periderm of potato tubers and their potential to improveresistance to soil-borne plant pathogensrdquo Plant Pathology vol48 no 3 pp 360ndash369 1999

[51] A V Sturz B R Christie and J Nowak ldquoBacterial endophytespotential role in developing sustainable systems of crop produc-tionrdquo Critical Reviews in Plant Sciences vol 19 no 1 pp 1ndash302000

[52] R Ramesh A A Joshi and M P Ghanekar ldquoPseudomonadsmajor antagonistic endophytic bacteria to suppress bacterialwilt pathogenRalstonia solanacearum in the eggplant (Solanummelongena L)rdquo World Journal of Microbiology and Biotechnol-ogy vol 25 no 1 pp 47ndash55 2009

[53] A A Nawangsih I Damayanti S Wiyono and J G KartikaldquoSelection and characterization of endophytic bacteria as bio-control agents of tomato Bacterial Wilt DiseaserdquoHayati Journalof Biosciences vol 18 no 2 pp 66ndash70 2011

[54] R Vargas-Ayala R Rodrıguez-Kabana G Morgan-Jones J AMcInroy and J W Kloepper ldquoShifts in soil microflora inducedby velvetbean (Mucuna deeringiana) in cropping systems tocontrol root-knot nematodesrdquo Biological Control vol 17 no 1pp 11ndash22 2000

[55] D K Zinniel P Lambrecht N B Harris et al ldquoIsolationand characterization of endophytic colonizing bacteria fromagronomic crops and prairie plantsrdquoApplied and EnvironmentalMicrobiology vol 68 no 5 pp 2198ndash2208 2002

[56] F Lemessa and W Zeller ldquoScreening rhizobacteria for biolog-ical control of Ralstonia solanacearum in Ethiopiardquo BiologicalControl vol 42 no 3 pp 336ndash344 2007

[57] M S El-AlbyadM A El-Sayed A R El-Shanshoury andNHEl-Batanouny ldquoEffect of culture conditions on the antimicrobialactivities of UV-mutants of Streptomyces corchorusii and Sspiroverticillatus against bean and banana wilt pathogensrdquoMicrobiological Research vol 151 no 2 pp 201ndash211 1996

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Zoology

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International Journal of

Microbiology

4 International Journal of Microbiology

Ten mL suspension of antagonistic XRB (80 Log CFUmL)in sterile 1 X PBS was applied per seedling by soil drenchingEach treatment consisted of two replicates with two potsper replication and five seedlings per pot Twenty days aftertreatment with the antagonistic XRB the seedlings were chal-lenged by inoculating 10mL suspension of R solanacearumstrain Rs-09-100 (70 Log CFUmL) by soil drenching Plantsnot treated with XRB but challenged with R solanacearumserved as control Plants were maintained with suitablewatering and percentage of plants infected by wilt was noteduntil 25 days after challenging with R solanacearum Abilityof the XRB to prevent wilt in eggplant was expressed asbiocontrol efficacy (BCE) and was determined using theformula BCE = ([percent disease in control] minus [percentdisease in treatment]percent disease in control) times 100 [37]Strains with BCE greater than 25 were evaluated for theireffect on growth in eggplant under greenhouse conditions

262 Growth Promotion Ability of XRB Sixteen strainsof XRB exhibiting biocontrol efficacies greater than 25were studied for their effect on growth in eggplant Abilityto increase shoot length in wilt susceptible eggplant cvAgassaim was used as a measure to evaluate their growthpromotion efficacy under greenhouse conditions Thirty-day-old seedlings raised in nonsterile soil in greenhousewere transplanted in pots filled with standard nonsterile potmixture (soil sand farmyardmanure at 2 1 1 ratio) TenmLsuspension of XRB (80 Log CFUmL) in sterile 1 X PBSwas applied per seedling by soil drenching Each treatmentconsisted of two replicates with two pots per replication andfive seedlings per pot Plants were maintained with suitablewatering and plant height was measured from the soil level tothe shoot tip 40 days postinoculation Ability of antagonisticXRB to increase shoot length in eggplant was expressed asgrowth promotion efficacy (GPE) using the formula ([shootlength increase in treatment] minus [shoot length increase incontrol]shoot length increase in control) times 100 [38]

27 16S rRNA Gene Sequencing and Sequence Analysis Rep-resentative strains from each of the 38 ARDRA haplotypesand XRB exhibiting antagonism to R solanacearum wereselected for identification A total of 55 strains were cho-sen for identification Fragments of the 16S rRNA geneof size 1500 bp were amplified as described above in theARDRA section Amplicons were purified using GeneJetPCRpurification kit (Thermo Scientific USA) and sequencedusing 27F and 1492R primers (Xcelris Labs Pvt LtdIndia) Partial 16S rRNA gene sequences (about 1200 nt)obtained were matched against the sequences available inthe nucleotide database from National Center for Biotech-nology Information (httpwwwncbinlmnihgovBLAST)using the BLASTn (Basic Local Alignment Search Tool)program

3 Results

31 Isolation of Bacteria from the Xylem of Eggplant Chilli andS torvum In this study bacteria could be constantly isolated

from the xylem of eggplant chilli and S torvum by vacuuminfiltration andmaceration techniques Bacterial counts fromeach isolation ranged from 10 to 102 CFUmL of xylem sap ormacerate Colonies appeared between 24 and 120 h of aerobicincubation at 28plusmn2∘CAmongst 167 isolates obtained 99wereGram-negative rods (5928) and 68 were Gram-positivebacteria (4072) comprising of 42 rods (6176) 25 cocci(3676) and one filamentous actinomycete (Table 1)

32 ARDRA Analysis ARDRA generated three to six restric-tion fragments of the 16S rRNA gene amplified from theXRB Analysis of ARDRA profiles by UPGMA using Dicersquoscoefficient dividedXRBwith identical restriction profiles intoseveral groups At 80 similarity values of Dice coefficient167 strains of XRB were grouped into 38 haplotypes Hostbased analysis of ARDRA revealed that 89 strains isolatedfromBW susceptible eggplant were grouped in 31 haplotypes36 strains from BW resistant eggplant into 17 haplotypes 33strains from chilli into 19 haplotypes and 9 strains from Storvum into 7 haplotypes respectively (Table 1) A detailedrepresentation of the ARDRA based analysis of 167 strainsis presented in Table 2 Based on the ARDRA analysis ofthe collection of XRB 153 strains were distributed over 24different haplotypes and 14 XRB strains had unique profileswhich formed 14 independent haplotypes with one strain ineach Antagonistic strains (119899 = 28) were distributed over 14different ARDRA haplotypes wherein 6 antagonists formedindependent haplotypes Nonantagonistic strains were dis-tributed over 24 haplotypes Haplotypes M80-9 and M80-15were shared amongst BW susceptible and resistant eggplantchilli and S torvum Eleven haplotypes were unique to BWsusceptible eggplant Haplotypes M80-1 M8-12 and M80-38 were unique to BW resistant eggplant whereas haplotypesM80-13 M80-19 and M80-26 comprised of strains isolatedfrom chilli Haplotype M80-36 had a strain isolated from Storvum Other haplotypes were a combination of strains iso-lated from different plant species These results indicate thatbacterial communities from the xylem of mainly eggplantand chilli cultivated in different locations in Goa comprise ofdiverse bacteria However it is observed that each ARDRAgroup consists of bacteria from different plant species andplants collected from different locales In addition there arecertain XRB unshared between each of the plant species thatform unique haplotypes Moreover strains with biocontrolability (BCE gt 25) were restricted to only 8 haplotypesnamely M80-6 M80-7 M80-10 M80-15 M80-20 M80-29M80-31 and M80-36 (Table 2) Haplotypes M80-6 M80-7M80-29 M80-31 M80-36 and M80-38 comprised of XRBwith GPE gt 10 Interestingly the strains with BCE gt 25and GPE gt 10 within these haplotypes were from BWresistant eggplant (Table 1)

33 Antagonism towards R solanacearum

331 In Vitro Bioassay and Production of Volatile and Dif-fusible Antagonistic Compounds Plate based bioassay wasused for rapid screening of antagonism of XRB towards R

International Journal of Microbiology 5

Table1Overviewof

diversity

andfunctio

nalityof

XRBob

tained

from

eggplantchilliand

Storvum

Planth

ost

Num

bero

fsamples

Totalisolates

obtained

Gram-negative

Gram-positive

Haplotypesa

obtained

Antagon

istic

strainsb

Antagon

isticstrains

with

BCEgt25

Biocon

trolstrains

with

GPEgt10

Metho

dof

isolatio

n

BWsusceptib

leeggplant

2489

5336

3116

52

VIo

rMBW

resistant

eggplant

1236

2016

176

65

VIo

rMCh

illi(Ca

psicu

mannu

um)

733

2013

193

21

Mon

ly

Solanu

mtorvum

29

63

73

31

VIo

nly

Total

45167

9968

2816

9a H

aplotypesw

ered

etermined

usingARD

RA

b Antagon

ismto

Rsolana

cearum

teste

din

vitro

asdescrib

edin

Section2

BCE(biocontrolefficacy)a

ndGPE

(growth

prom

otioneffi

cacy)w

ered

etermined

underg

reenho

usec

onditio

nsin

eggplant

asdescrib

edin

Section2

VIVa

cuum

infiltration

Mm

aceration

6 International Journal of Microbiology

Table 2 Haplotypes of XRB based on ARDRA withMspI at 80 similarity plant host biocontrol and growth promotion activities

Haplotypenumbera

Number of strainsin each haplotype Plant host Strains selected for

identificationNumber of biocontrolstrains with BCE gt 25

Number of strainswith GPE gt 10

M80-1 1 RE XB159 0 0M80-2 1 SE XB34 0 0M80-3 1 SE XB66 0 0M80-4 2 SE C XB40 0 0M80-5lowast 1 SE XB140 0 0

M80-6lowast 10 SE RE C XB177 XB157 XB93XB169 XB153 XB170 4 3

M80-7lowast 6 SE RE C XB1 XB86 2 2M80-8 4 SE RE XB22 XB190 0 0M80-9lowast 8 SE RE ST C XB99 XB100 0 0M80-10lowast 10 SE RE C XB196 XB103 1 0M80-11 9 SE RE C XB137 0 0M80-12 1 RE XB158 0 0M80-13 1 C XB87 0 0M80-14 5 SE C XB88 0 0M80-15lowast 5 SE RE ST C XB70 1 0M80-16 3 SE XB47 0 0M80-17 2 SE XB35 0 0M80-18 1 SE XB41 0 0M80-19 2 C XB94 0 0M80-20lowast 13 SE RE C XB8 XB20 XB27 2 0M80-21 5 SE ST C XB25 0 0M80-22 1 SE XB53 0 0M80-23 5 SE RE C XB98 0 0M80-24 11 SE RE ST XB161 0 0M80-25 4 SE RE XB188 0 0M80-26 1 C XB168 0 0M80-27lowast 2 SE C XB126 XB7 0 0M80-28lowast 5 SE RE XB122 0 0M80-29lowast 9 SE RE C XB165 1 1M80-30 15 SE RE C XB167 XB109 0 0

M80-31lowast 11 SE ST C XB123 XB203 XB62XB114 XB202 3 1

M80-32 5 SE ST C XB92 0 0M80-33 1 SE XB37 0 0M80-34 1 SE XB36 0 0M80-35 2 SE XB64 0 0M80-36lowast 1 ST XB200 1 1M80-37lowast 1 SE XB134 0 0M80-38lowast 1 RE XB197 1 1aHaplotype based on ARDRA analysis usingMspI at 80 similarity values of Dice coefficientlowastHaplotype comprises of bacteria showing antagonistic property in the bioassaysSE bacterial wilt susceptible eggplant RE bacterial wilt resistant eggplant ST Solanum torvum C chilli plantBCE biocontrol efficacy GPE growth promotion efficacy determined as described in Section 2

International Journal of Microbiology 7

solanacearum strain Rs-09-100 Results of the in vitro screen-ing against R solanacearum revealed that 28 amongst 167XRB exhibited antagonism towards the pathogen (Table 3)Amongst the antagonists 16 were strains from BW suscepti-ble eggplant 6 from BW resistant eggplant and 3 each fromchilli and S torvum (Table 1) Amongst the 28 antagonists 7strains namely XB62 XB99 XB100 XB114 XB122 XB196XB197 and XB202 formed larger inhibition zones againstR solanacearum ranging from 40mm to 817mm (Table 3)The majority of the antagonistic strains (119899 = 16) producedinhibition zones ranging from 20mm to 383mm XB27XB134 XB165 and XB169 formed smaller inhibition zonesranging from 15mm to 20mm However 139 strains ofXRB did not inhibit R solanacearum in the bioassay testTwenty-eight antagonistic XRBwere screened for productionof volatile inhibitory compounds namely acetoin HCNammonia and diffusible siderophore molecules in vitro(Table 3) Acetoin production was observed in 3214 of theisolates Bacterial isolates XB7 and XB122 were found toproduce both HCN as well as siderophores XB62 XB93 andXB170 produced HCN whereas XB114 XB140 and XB203produced siderophores only XB93 XB99 XB123 XB134 andXB140 produced ammonia

34 Production of Plant Growth Promoting Substances byAntagonistic XRB Results of the screening of antagonisticXRB for in vitro production of several plant growth pro-moting compounds is presented in Table 3 Majority of theantagonistic strains produced the phytohormone IAA withconcentrations ranging from 1591 120583gmL to 64591120583gmLXB202 was found to be the best ACC deaminase producingstrain based on its luxuriant growth on DF salts mediumsupplemented with 30mM ACC as sole nitrogen sourceOther ACC deaminase producing strains include XB1 XB62XB86 and XB140 Scarce growth of XB165 and XB200 wasobserved onDF saltsmedium 6428of the strains producedphosphate solubilizing organic acids as indicated by clearhaloes on Pikovskayarsquos agar plate

35 Greenhouse Experiments

351 Suppression of Bacterial Wilt by Antagonistic XRBAbility to suppress BW was assessed as the difference in thepercentage of wilt in XRB treated plants with respect to wilt inuntreated control andwas expressed as the biocontrol efficacy(BCE) of the antagonists BCE of 16 strains with valuesranging from 286 to 100 is presented in Figure 1 Plantstreated with strains XB86 XB169 and XB177 were free fromBWand hence recorded 100 biocontrol efficacy Treatmentswith XB170 XB197 XB200 XB202 and XB203 recorded 30percent or less wilt incidence (70 to 85 BCE) XB1 XB27XB70 XB93 and XB123 treatments recorded BCE between429 and 571 BCE of 286 was recorded in XB20 andXB165 treatments However 12 antagonistic XRB recordedBCEof 25 andwere least effective inwilt protection Furtherit is observed that all the antagonistic strains originating fromBW resistant eggplant and S torvum exhibited BCE greaterthan 25 Five antagonistic strains from BW susceptible

minus2000

000

2000

4000

6000

8000

10000

12000

XB1

XB20

XB27

XB70

XB86

XB93

XB123

XB165

XB169

XB170

XB177

XB196

XB197

XB200

XB202

XB203

Con

trol

Isolates

Biocontrol efficacy (BCE)Growth promotion efficacy (GPE)

BCE

and

GPE

()

Figure 1 Biocontrol and plant growth promotion efficacies of selectXRB in eggplant BCE biocontrol efficacy determined 25 days afterchallenging with R solanacearum GPE growth promotion efficacydetermined 40 days after treatmentwithXRB Percent BCE andGPEcalculated using formulae BCE = ([percent disease in control] minus[percent disease in treatment]percent disease in control) times 100 andGPE= ([shoot length increase in treatment]minus [shoot length increasein control]shoot length increase in control) times 100 respectivelyUninoculated control had BCE and GPE values of 000 XB86XB169 and XB177 had BCE of 10000 in all the replicationsBars indicate mean values of BCE and GPE error bars indicatestandard deviation

eggplant and three fromchilli were effective in preventingwiltin eggplant (Table 1)

352 Growth Promotion by Antagonistic XRB Increase inshoot length of eggplant (40 days after treatment) observedin XRB treated plants in relation to untreated control wasexpressed as growth promotion efficacy (GPE) and is shownin Figure 1 Amongst the 16 strains which were effective inpreventing wilt six strains exhibited the highest increase inshoot length as indicated by their GPE values in the rangeof 139ndash223 Seven XRB recorded a GPE value rangingfrom 14 to 129 However strains XB27 XB196 and XB203stunted shoot growth in eggplant in comparison to untreatedcontrol When the source of XRB is considered 5555strains that exhibited GPE greater than 10 were isolatedfrom BW resistant eggplant Whereas only two antagonistsfrom BW susceptible plant and one each from chilli and Storvum were able to promote growth in eggplant (Table 1)Strains with GPE gt 10 belonged to six different ARDRAhaplotypes (Table 2)

36 Identification of XRB by 16S rRNA Gene Sequencing16S rRNA gene sequences of XRB were used to identify thediverse xylem inhabitants and antagonistic strains Identityof 55 XRB based on 16S rRNA gene sequencing and theirGenBank accessions are presented in Table 4 Overall 23

8 International Journal of Microbiology

Table3

Invitro

inhibitio

nof

Rsolana

cearum

andprod

uctio

nof

antagonisticc

ompo

unds

andplantg

rowth

prom

otingsubstances

byXR

B

Strain

Haplotype

Inhibitio

nof

Rsolana

cearum

Volatile

anddiffu

sibleinhibitory

compo

unds

Plantgrowth

prom

otingsubstances

Radius

inmm

HCN

Ammon

iaAc

etoin

Sideroph

ore

Phosph

ates

olub

ilisatio

nAC

Cdeam

inasea

IAA

b120583gmL

XB1

M80-7

283plusmn029

minusminus

minusminus

minus++

+10500plusmn707

XB7

M80-27

333plusmn058

+minus

minus+

+minus

4773plusmn321

XB8

M80-20

333plusmn029

minusminus

minusminus

+minus

1909plusmn129

XB20

M80-20

327plusmn040

minusminus

minusminus

+minus

2545plusmn171

XB27

M80-20

183plusmn029

minusminus

minusminus

+minus

1591plusmn107

XB62

M80-31

817plusmn076

+minus

minusminus

+++

++7318plusmn493

XB70

M80-15

383plusmn058

minusminus

minusminus

minusminus

17182plusmn1157

XB86

M80-7

233plusmn076

minusminus

minusminus

minus++

+6682plusmn450

XB93

M80-6

317plusmn029

+minus

+minus

+minus

5727plusmn386

XB99

M80-9

683plusmn058

minus+

+minus

+minus

32136plusmn2164

XB100

M80-9

450plusmn050

minus+

+minus

+minus

23864plusmn1607

XB114

M80-31

650plusmn050

minusminus

minus+

+minus

8909plusmn600

XB122

M80-28

433plusmn058

+minus

minus+

+minus

6682plusmn450

XB123

M80-31

300plusmn087

minus+

+minus

+minus

41682plusmn2807

XB126

M80-27

283plusmn029

minusminus

+minus

+minus

6045plusmn407

XB134

M80-37

157plusmn012

minus+

+minus

+minus

64591plusmn4350

XB140

M80-5

367plusmn076

minus+

minus+

+++

++5091plusmn343

XB153

M80-6

317plusmn029

minusminus

+minus

+minus

15591plusmn1050

XB157

M80-6

250plusmn100

minusminus

+minus

+minus

18455plusmn1243

XB165

M80-29

157plusmn012

minusminus

minusminus

minus+

19091plusmn1286

XB169

M80-6

153plusmn006

minusminus

minusminus

minusminus

1591plusmn107

XB170

M80-6

351plusmn002

+minus

minusminus

+minus

2864plusmn193

XB177

M80-6

199plusmn049

minusminus

minusminus

minusminus

7636plusmn514

XB196

M80-10

393plusmn012

minusminus

minusminus

minusminus

3500plusmn236

XB197

M80-38

433plusmn029

minusminus

+minus

minusminus

4136plusmn279

XB200

M80-36

187plusmn023

minusminus

minusminus

minus++

16864plusmn1136

XB202

M80-31

467plusmn021

minusminus

minusminus

+++

+8273plusmn557

XB203

M80-31

367plusmn029

minusminus

minusminus

minusminus

7000plusmn471

Inhibitio

nzonesa

remeanof

threereplications

andshow

ingsta

ndarddeviation

Inhibitio

nzone

was

measuredas

radius

from

theou

tere

dgeof

wellAlltheexperim

entswerecond

uctedat28plusmn2∘C

+indicates

presence

oftraitminusindicatesabsence

oftraitinplateb

ased

assaysaLevelsofAC

Cdeam

inasea

ctivitiesbasedon

grow

thon

plateb

ased

assayd

enoted

as+forlessg

rowth+++

form

oderateg

rowth+++

forlux

uriant

grow

thand

++++

forh

ighlyluxu

riant

grow

thbIAAwas

estim

ated

incultu

refiltra

teandexpressedas120583gmLusinganalyticalgradeIAAas

stand

ardvalues

indicatemeanandsta

ndarddeviation

International Journal of Microbiology 9

2545

2545

364

364

1273 2909

ActinobacteriaFirmicutesAlpha proteobacteria

Beta proteobacteriaGamma proteobacteriaBacteroidetes

(a)

Num

ber o

f XRB

Actin

obac

teria

Firm

icut

es

Alp

ha p

rote

obac

teria

Beta

pro

teob

acte

ria

Gam

ma p

rote

obac

teria

Bact

eroi

dete

s

Number of antagonistic XRB in each phylumsubdivision

Phylasubdivision

Total number of XRB belonging to each phylumsubdivision

25

20

15

10

5

0

2

14

9

16

4

7

1

2

1

2

11

14

(b)

Figure 2 (a) Distribution of XRB (phylumsubdivision level) identified by 16S rRNA gene sequencing Values indicate percentages of strainsbelonging to each phylasubdivision amongst the 55 identified strains (b) Distribution of antagonistic and nonantagonistic strains of XRB ineach phylumsubdivision

different genera of bacteria were identified Major gen-era identified are Bacillus sp (11 strains) Enterobacter sp(6 strains) Microbacterium sp (5 strains) Staphylococcussp (5 strains) Pseudomonas sp (5 strains) and Agrobac-terium sp (3 strains) Additional genera identified includ-ing Micrococcus sp Sphingomonas sp Flavobacterium spChryseobacterium sp Burkholderia sp and Xenophilus spare listed in Table 4 Based on the identification phylumProteobacteria consisting of Gram-negative bacteria of sub-divisions Alpha Proteobacteria (1273) Beta Proteobacteria(364) and Gamma Proteobacteria (2545) were predom-inant (4181 strains identified) followed by phyla Firmi-cutes (2909) Actinobacteria (2545) and Bacteroidetes(364) (Figure 2(a))

Eleven antagonistic strains identified belonged toGammasubdivision of Proteobacteria consisting of five strainseachof Enterobacter sp and fluorescent and nonfluorescent Pseu-domonas sp and one strain of Pantoea eucrina (XB126)(Figure 2(b)) Nine antagonists identified were of phylaFirmicutes consisting of Staphylococcus sp (5 strains) andBacillus sp (4 strains) Agrobacterium strains XB1 XB86 andXB165 Sphingomonas sp (XB197) of the Alpha Proteobacte-ria Streptomyces sp (XB200) and Janibacter melonis (XB70)of phyla Actinobacteria were found to be antagonistic Addi-tional antagonistic XRB include Burkholderia sp (XB140) of120573 Proteobacteria and Flavobacterium sp (XB203) of phylaBacteroidetes (Figure 2(b))

37 Statistical Analysis The statistical analysis of percent-age wilts and shoot length of eggplant was performedusing Web Agri Statistical Package (WASP) version 20(httpwwwicargoaresinwasp20indexphp)

4 Discussion

Eggplant and chilli not only are of economic and culturalimportance but also are common ingredients in the cuisinethroughout India In the coastal state of GoaR solanacearumhas been reported to be a destructive pathogen in cultivationof eggplant and chilli [4] Isolation of biocontrol agentsagainst the BW pathogen has been commonly restricted toendophytic tissue and plant rhizosphere [26 27] Studieson xylem colonizing endophytes were undertaken becausewe speculated existence of interactions between the XRBand vascular wilt pathogen R solanacearum during xylemcolonization Our study reveals the diversity biocontrolpotential and identity of endophytic xylem colonizers fromsolanaceous crops cultivated in Goa India A total of 167bacteria were isolated from the xylem of eggplant chilliand S torvum with Gram-negative bacteria (5928) pre-dominating in the collection Congruent to our observationGardner et al [7] and Bell et al [11] have earlier reportedisolation of more number of Gram-negative rod shapedbacteria from xylem of citrus and grapevine using vacuuminfiltration Scholander pressure bomb was found to beuseful in extraction of diverse bacterial genera from xylemtissues in contrast to trituration methods that yielded highernumber of Gram-positive rod shaped bacteria [12] ThoughScholander pressure bomb was not used in this study acombination of vacuum infiltration [7 11] and triturationof decorticated stems [10 12] was employed with an aimto isolate diverse XRB from xylem tissues This is the firststudy reporting the use of vacuum infiltration and triturationmethods for isolating xylem residing bacteria from eggplantchilli and S torvum

Traditionally bacteria have been characterized andgrouped based on colony morphology and biochemical

10 International Journal of Microbiology

Table 4 List of XRB identified by partial 16S rRNAgene sequencing their plant host accession numbers closestNCBImatch and similarity

Strain Plant host Accession number Closest NCBI match similarityXB1lowast SE KF447383 Agrobacterium tumefaciens 99XB7lowast SE KF447384 Pseudomonas aeruginosa 99XB8lowast SE KF447385 Staphylococcus haemolyticus 99XB20lowast SE KF447386 Staphylococcus haemolyticus 100XB22 SE KF447387 Brevibacterium casei 99XB25 C KF447388 Enterobacter sp 95XB27lowast SE KF447389 Staphylococcus haemolyticus 99XB34 SE KF447390 Curtobacterium sp 99XB35 SE KF447391 Microbacterium sp 99XB36 SE KF447392 Xenophilus sp 99XB37 SE KF447393 Chryseobacterium sp 99XB40 SE KF447394 Micrococcus luteus 89XB41 SE KF447395 Bacillus sp 99XB47 SE KF447396 Bacillus sp 99XB53 SE KF447397 Micrococcus sp 99XB62lowast SE KF447398 Pseudomonas sp 99XB64 SE KF447399 Bacillus thuringiensis 100XB66 SE KF447400 Pectobacterium carotovorum 99XB70lowast SE KF447401 Janibacter melonis 99XB86lowast C KF447402 Agrobacterium tumefaciens 99XB87 C KF447403 Bacillus barbaricus 99XB88 C KF447404 Bacillus sp 99XB92 C KF447405 Brevundimonas vesicularis 99XB93lowast C KF447406 Bacillus safensis 100XB94 C KF447407 Bacillus sp 100XB98 C KF447408 Microbacterium sp 100XB99lowast SE KF447409 Enterobacter sp 99XB100lowast SE KF447410 Enterobacter cloacae 98XB103 SE KF447411 Brachybacterium phenoliresistens 99XB109 SE KF447412 Rhodococcus corynebacterioides 99XB114lowast SE KF447413 Pseudomonas stutzeri 99XB122lowast SE KF913446 Pseudomonas aeruginosa 100XB123lowast SE KF447414 Enterobacter sp 99XB126lowast C KF447415 Pantoea eucrina 99XB134lowast SE KF447416 Enterobacter sp 99XB137 SE KF447417 Klebsiella sp 99XB140lowast SE KF447418 Burkholderia sp 99XB153lowast SE KF447419 Bacillus amyloliquefaciens 99XB157lowast SE KF447420 Bacillus subtilis 100XB158 RE KF447421 Bosea sp 99XB159 RE KF447422 Microbacterium xylanilyticum 99XB161 RE KF447423 Microbacterium aurum 99XB165lowast RE KF447424 Agrobacterium sp 99XB167 RE KF447425 Microbacterium aurum 99XB168 RE KF447426 Bacillus aryabhattai 100XB169lowast RE KF447427 Staphylococcus gallinarum 99XB170lowast RE KF447428 Staphylococcus sp 99XB177lowast RE KF447429 Bacillus cereus 99XB188 RE KF447430 Sphingomonas sp 99XB190 RE KF447431 Brevibacterium casei 99

International Journal of Microbiology 11

Table 4 Continued

Strain Plant host Accession number Closest NCBI match similarityXB196lowast RE KF447432 Enterobacter kobei 99XB197lowast RE KF447433 Sphingomonas sp 98XB200lowast ST KF913447 Streptomyces sp 100XB202lowast ST KF447434 Pseudomonas sp 99XB203lowast ST KF447435 Flavobacterium sp 99lowastStrain is antagonistic to R solanacearum based on bioassaySE bacterial wilt susceptible eggplant RE bacterial wilt resistant eggplant C chilli ST Solanum torvum

tests However whole genome fingerprinting or PCR-RFLPbased methods are rapid tools for determining geneticdiversity of bacteria in a given collection ARDRA which isa type of PCR based RFLP method has been used widely inestimating genetic diversity endophytic bacterial populationsand clustering genetically similar strains [39ndash41] In additionto earlier reports our study demonstrates the usefulnessof ARDRA as a tool to cluster genetically identical strainsof endophytic XRB isolated from eggplant chilli andS torvum In our study 9161 strains (119899 = 153) weregrouped in 24 haplotypes by using ARDRA The majority ofthese haplotypes represent a combination of XRB isolatedfrom different solanaceous plants from diverse localesThese results indicate that xylem of eggplant chilli andS torvum largely bears similar population of XRB whichcan efficiently cross colonize eggplant chilli or S torvumNevertheless 839 strains had unique ARDRA fingerprintand formed separable haplotypes This observation leads toa conclusion that a minor population of xylem inhabitantsare restricted to a specific plant species and cannot easilycross colonize xylem of other solanaceous plants Plantsare known to selectively support endophytic colonizationby specific bacteria [14] However factors that determinethe selection of xylem colonists or the ability of XRBto colonize eggplant chilli and S torvum in this studyremain unknown Interestingly the structure of endophyticcommunity in Nicotiana attenuata a member of Solanaceaefamily is shown to be influenced by soil composition andethylene homeostasis [40] Earlier evidence has shown thatcolonization by endophytic bacteria is also governed by plantgenotype as well as root exudates [42]

Only 1677 XRB out of 167 were antagonistic to Rsolanacearum based on in vitro assays Antagonistic XRBproduced volatile and diffusible inhibitory compoundsnamely HCN ammonia and acetoin and siderophoresThese substances have been long known to be involvedin disease suppression and indirect growth promotion inplants [43ndash46] These mechanisms possibly played a rolein the evident biocontrol effect against BW exhibited bythe XRB in the greenhouse screening Endophytic bacteriahave been known to have plant growth promoting traitsnamely production of IAA ACC deaminase and phosphatesolubilization [25 33 36] These traits were detected in themajority of antagonistic XRB tested in this study and mayhave resulted in the observed increase in shoot length ofeggplant in our greenhouse experiments In contrast strains

XB20 XB196 and XB203 suppressed growth in eggplantunder greenhouse conditions however no visible symptomsof disease were observed Vascular plugging and productionof certainmetabolites toxic to plant cells but not cell viabilitymay have resulted in stunted shoot in eggplant [11 47]

Evaluation of efficacy of antagonistic organisms to sup-press the plant diseases under greenhouse conditions isone of the key steps for selecting a potential biocontrolagent for disease management [48] Endophytic strains fromBW susceptible varieties of eggplant have been shown toprevent wilt and promote growth in eggplant earlier [27]Our greenhouse screening shows that 3846 of antagonisticXRB with biocontrol efficacies greater than 40 were isolatesfrom BW resistant varieties of eggplantThis raises a questionwhether the bacteria from resistant varieties are involved inBW resistance and whether BW resistant varieties are ableto selectively influence xylem colonization by antagonisticbacteria Presence of higher number of endophytes withantagonistic abilities was reported in BW resistant varieties oftomato as compared to susceptible varieties and their role inresistance to BWwas proposed [49] Similar observations oncorrelation of resistance of potato to soft rot and endophyticbacteria have been reported [50]Thus BW resistant varietiescan be considered a better host for isolating potential biocon-trol strains for management of bacterial wilt

Identification of 55 XRB strains by 16S rRNA genesequencing revealed the presence of 23 diverse genera ofbacteria belonging to 4 phyla of Eubacteria Strains belongingto phyla Firmicutes Actinobacteria and 120574 subdivision ofProteobacteria were the major xylem colonists identified inthis study Several genera of bacteria belonging to these phylahave also been reported to be present in endophytic tissuesand xylem of a variety of other agricultural and horticulturalplant species [14 51] In addition the majority of the antag-onists identified belonged to Enterobacter sp Pseudomonassp Bacillus sp and Staphylococcus sp Congruent to ourresults several researchers have reported bacteria isolatedfrom solanaceous crops and belonging to similar genera tobe antagonistic to R solanacearum [26 51ndash53] HoweverFlavobacterium spand Janibacter melonis identified in thisstudy have never been previously reported to be inhibitoryto R solanacearum Large population of the xylem inhabitingbacterial flora accounting for 8323 exhibited no antago-nism towards R solanacearum Nonantagonistic XRB wereidentified predominantly as Microbacterium sp Endophyticpersistence and nematicidal activities of Microbacterium sp

12 International Journal of Microbiology

have been reported [54 55] Therefore the collection of XRBisolated in this study can be screened for inhibitory activitiesagainst other important agricultural pests

Though few strains namely XB86 XB169 and XB177exhibited plant beneficial properties in this study their use-fulness in plant disease control remains to be seen XB86has been identified as Agrobacterium tumefaciens the crowngall disease pathogen and its deployment as biocontrolagent is uncertain XB169 (Staphylococcus gallinarum) andXB177 (Bacillus cereus) are reported as opportunistic animalpathogens and thus unsuitable for field applications Strep-tomyces sp has earlier been reported as antagonistic to Rsolanacearum and tested for management of wilt in potatoand tomato [56 57] XB200 (Streptomyces sp) is one of theXRB high BCE and GPE it could be explored further forbiocontrol of bacterial wilt after additional characterizationand field evaluation

5 Conclusion

This study is the first report on the identity of novel anddiverse XRB colonizing the xylem of eggplant chilli and Storvum XRB particularly from BW resistant varieties werefound to protect eggplant from bacterial wilt and enhancedgrowth in eggplant in the greenhouse screening Thereforethe repertoire of XRB reported in this study may be usefulfor cultivation of eggplant in BW affected areas

Conflict of Interests

The authors hereby declare that they have no conflict ofinterests regarding the publication of this paper

Acknowledgments

The authors greatly acknowledge financial support fromIndian Council of Agricultural Research (ICAR) New DelhiIndia through ldquoOutreach project on Phytopthora Fusariumand Ralstonia diseases of horticultural and field cropsrdquo-(PhytoFuRa) and the Director ICAR Research Complex forGoa India for the facilities

References

[1] E Yabuuchi Y KosakoHOyaizu et al ldquoProposal ofBurkholde-ria gen nov and transfer of seven species of the genusPseudomonas homology group II to the new genus with thetype species Burkholderia cepacia (Palleroni and Holmes 1981)comb novrdquo Microbiology and Immunology vol 36 no 12 pp1251ndash1275 1992

[2] E Wicker L Grassart R Coranson-Beaudu et al ldquoRalstoniasolanacearum strains from Martinique (French West Indies)exhibiting a new pathogenic potentialrdquo Applied and Environ-mental Microbiology vol 73 no 21 pp 6790ndash6801 2007

[3] Y Liu A Kanda K Yano et al ldquoMolecular typing of Japanesestrains of Ralstonia solanacearum in relation to the ability toinduce a hypersensitive reaction in tobaccordquo Journal of GeneralPlant Pathology vol 75 no 5 pp 369ndash380 2009

[4] R Ramesh ldquoField evaluation of biological control agents for themanagement of Ralstonia solanacearum in Brinjalrdquo Journal ofMycology and Plant Pathology vol 36 no 2 pp 327ndash328 2006

[5] J Vasse P Frey and A Trigalet ldquoMicroscopic studies ofintercellular infection and protoxylem invasion of tomatoroots by Pseudomonas solanacearumrdquoMolecular Plant-MicrobeInteractions vol 8 no 2 pp 241ndash251 1995

[6] S Genin and C Boucher ldquoRalstonia solanacearum secrets of amajor pathogen unveiled by analysis of its genomerdquo MolecularPlant Pathology vol 3 no 3 pp 111ndash118 2002

[7] J M Gardner A W Feldman and R M Zablotowicz ldquoIdentityand behavior of xylem-residing bacteria in rough lemon roots ofFlorida citrus treesrdquo Applied and Environmental Microbiologyvol 43 no 6 pp 1335ndash1342 1982

[8] M J Jacobs W M Bugbee and D A Gabriellson ldquoEnu-meration location and characterization of endophytic bacteriawithin sugar beet rootsrdquoCanadian Journal of Botany vol 63 no7 pp 1262ndash1265 1985

[9] D G Patriquin and J Dobereiner ldquoLight microscopy observa-tions of tetrazolium-reducing bacteria in the endorhizosphereof maize and other grasses in Brazilrdquo Canadian Journal ofMicrobiology vol 24 no 6 pp 734ndash742 1978

[10] S Gagne C Richard H Rousseau and H Antoun ldquoXylem-residing bacteria in alfalfa rootsrdquo Canadian Journal of Micro-biology vol 33 no 11 pp 996ndash1000 1987

[11] C R Bell G A Dickie W L G Harvey and J W Y FChan ldquoEndophytic bacteria in grapevinerdquo Canadian Journal ofMicrobiology vol 41 no 1 pp 46ndash53 1995

[12] J Hallmann J W Kloepper and R Rodrıguez-Kabana ldquoAppli-cation of Scholander pressure bomb to studies on endophyticbacteria of plantsrdquo Canadian Journal of Microbiology vol 43no 5 pp 411ndash416 1997

[13] J S Lampel G L Canter M B Dimock et al ldquoIntegrativecloning expression and stability of the cryIA(c) gene fromBacillus thuringiensis subsp kurstaki in a recombinant strain ofClavibacter xyli subsp cynodontisrdquo Applied and EnvironmentalMicrobiology vol 60 no 2 pp 501ndash508 1994

[14] M Rosenblueth and E Martınez-Romero ldquoBacterial endo-phytes and their interactions with hostsrdquo Molecular Plant-Microbe Interactions vol 19 no 8 pp 827ndash837 2006

[15] P R Hardoim L S van Overbeek and J D van ElsasldquoProperties of bacterial endophytes and their proposed role inplant growthrdquo Trends in Microbiology vol 16 no 10 pp 463ndash471 2008

[16] R Aravind A Kumar S J Eapen and K V RamanaldquoEndophytic bacterial flora in root and stem tissues of blackpepper (Piper nigrum L) genotype isolation identificationand evaluation against Phytophthora capsicirdquo Letters in AppliedMicrobiology vol 48 no 1 pp 58ndash64 2009

[17] J Kuklinsky-Sobral W L Araujo R Mendes I O Geraldi AA Pizzirani-Kleiner and J L Azevedo ldquoIsolation and charac-terization of soybean-associated bacteria and their potential forplant growth promotionrdquo Environmental Microbiology vol 6no 12 pp 1244ndash1251 2004

[18] S Compant B Duffy J Nowak C Clement and E A BarkaldquoUse of plant growth-promoting bacteria for biocontrol ofplant diseases principles mechanisms of action and futureprospectsrdquoApplied and Environmental Microbiology vol 71 no9 pp 4951ndash4959 2005

[19] E T Thorne B M Young G M Young et al ldquoThe structureof xylem vessels in grapevine (Vitaceae) and a possible passive

International Journal of Microbiology 13

mechanism for the systemic spread of bacterial diseaserdquo Amer-ican Journal of Botany vol 93 no 4 pp 497ndash504 2006

[20] W T Seo W J Lim E J Kim H D Yun Y H Lee and KM Cho ldquoEndophytic bacterial diversity in the young radishand their antimicrobial activity against pathogensrdquo Journal ofApplied Biological Chemistry vol 53 no 4 pp 493ndash503 2010

[21] S Compant H Kaplan A Sessitsch J Nowak E Ait Barkaand C Clement ldquoEndophytic colonization ofVitis vinifera L byBurkholderia phytofirmans strain PsJN from the rhizosphere toinflorescence tissuesrdquo FEMS Microbiology Ecology vol 63 no1 pp 84ndash93 2005

[22] P S B Miguel J C Delvaux M N V de Oliveira et al ldquoDiver-sity of endophytic bacteria in the fruits of Coffea canephorardquoAfrican Journal of Microbiology Research vol 7 no 7 pp 586ndash594 2013

[23] A Lopez-Lopez M A Rogel E Ormeno-Orrillo J Martınez-Romero and E Martınez-Romero ldquoPhaseolus vulgaris seed-borne endophytic community with novel bacterial species suchas Rhizobium endophyticum sp novrdquo Systematic and AppliedMicrobiology vol 33 no 6 pp 322ndash327 2010

[24] M Lwin and S L Ranamukhaarachchi ldquoDevelopment of bio-logical control of Ralstonia solanacearum through antagonisticmicrobial populationsrdquo International Journal of Agriculture andBiology vol 8 no 5 pp 657ndash660 2006

[25] F Rasche E Marco-Noales H Velvis et al ldquoStructural char-acteristics and plant-beneficial effects of bacteria colonizing theshoots of field grown conventional and geneticallymodifiedT4-lysozyme producing potatoesrdquo Plant and Soil vol 289 no 1-2pp 123ndash140 2006

[26] N Amaresan V Jayakumar K Kumar and N ThajuddinldquoEndophytic bacteria from tomato and chilli their diversity andantagonistic potential against Ralstonia solanacearumrdquoArchivesof Phytopathology and Plant Protection vol 45 no 3 pp 344ndash355 2012

[27] R Ramesh and G S Phadke ldquoRhizosphere and endophyticbacteria for the suppression of eggplant wilt caused byRalstoniasolanacearumrdquo Crop Protection vol 37 pp 35ndash41 2012

[28] W J French R G Christis and D L Stassi ldquoRecovery ofrickettsialike bacteria by vacuum infiltration of peach tissuesaffected with phony diseaserdquo Phytopathology vol 67 pp 945ndash948 1977

[29] P R Viss E M Brooks and J A Driver ldquoA simplified methodfor the control of bacterial contamination in woody plant tissueculturerdquo In Vitro Cellular andDevelopmental Biology vol 27 no1 article 42 1991

[30] K Wilson ldquoPreparation of genomic DNA from bacteriardquo inCurrent Protocols in Molecular Biology F M Ausubel R BrentR E Kingston et al Eds pp 241ndash245 John Wiley amp SonsNew York NY USA 1997

[31] M Saraf AThakkar U PandyaM Joshi and J Parikh ldquoPoten-tial of plant growth promoting microorganisms as biofertilizersand biopesticides and itrsquos exploitation in sustainable agricul-turerdquo Journal of Microbiology and Biotechnology Research vol3 no 5 pp 54ndash62 2013

[32] A P G C Marques C Pires H Moreira A O S S Rangeland P M L Castro ldquoAssessment of the plant growth promotionabilities of six bacterial isolates using Zea mays as indicatorplantrdquo Soil Biology and Biochemistry vol 42 no 8 pp 1229ndash1235 2010

[33] X Hao P Xie L Johnstone S J Miller C Rensingand G Wei ldquoGenome sequence and mutational analysis of

plant-growth-promoting bacteriumAgrobacterium tumefaciensCCNWGS0286 isolated from a zinc-lead mine tailingrdquo Appliedand Environmental Microbiology vol 78 no 15 pp 5384ndash53942012

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

[35] S A Gordon and L G Paleg ldquoQuantitative measurement ofindole acetic acidrdquo Plant Physiology vol 10 pp 37ndash48 1957

[36] A Godinho R Ramesh and S Bhosle ldquoBacteria from sanddunes of Goa promoting growth in eggplantrdquoWorld Journal ofAgricultural Sciences vol 6 no 5 pp 555ndash564 2010

[37] J-H Guo H-Y Qi Y-H Guo et al ldquoBiocontrol of tomato wiltby plant growth-promoting rhizobacteriardquo Biological Controlvol 29 no 1 pp 66ndash72 2004

[38] N Aliye C Fininsa and Y Hiskias ldquoEvaluation of rhizo-sphere bacterial antagonists for their potential to bioprotectpotato (Solanum tuberosum) against bacterial wilt (Ralstoniasolanacearum)rdquo Biological Control vol 47 no 3 pp 282ndash2882008

[39] L LagaceM PitreM Jacqeus andDRoy ldquoIdentification of thebacterial community ofmaple sap by using amplified ribosomalDNA (rDNA) restriction analysis and rDNA sequencingrdquoApplied and Environmental Microbiology vol 70 no 4 pp2052ndash2060 2004

[40] H H Long D G Sonntag D D Schmidt and I T BaldwinldquoThe structure of the culturable root bacterial endophyte com-munity of Nicotiana attenuata is organized by soil compositionand host plant ethylene production and perceptionrdquo NewPhytologist vol 185 no 2 pp 554ndash567 2010

[41] E Lopez-Fuentes V M Ruız-Valdiviezo E Martınez-RomeroF A Gutierrez-Miceli L Dendooven and R Rincon-RosalesldquoBacterial community in the roots and rhizosphere of Hyper-icum silenoides Juss 1804rdquo African Journal of MicrobiologyResearch vol 6 no 11 pp 2704ndash2711 2012

[42] M Fang R J Kremer P P Motavalli and G Davis ldquoBacterialdiversity in rhizospheres of nontransgenic and transgenic cornrdquoApplied and EnvironmentalMicrobiology vol 71 no 7 pp 4132ndash4136 2005

[43] C R Howell and R D Stiphanovic ldquoProduction of ammonia byEnterobacter cloaceae and its possible role in biological controlof Pythium pre-emergence damping-off by the bacteriumrdquoEcology and Epidemiology vol 78 no 8 pp 1075ndash1078 1988

[44] A Ramette M Frapolli G Defago and Y Moenne-LoccozldquoPhylogeny of HCN synthase-encoding hcnBC genes in bio-control fluorescent pseudomonads and its relationship withhost plant species and HCN synthesis abilityrdquoMolecular Plant-Microbe Interactions vol 16 no 6 pp 525ndash535 2003

[45] I Loaces L Ferrando and A F Scavino ldquoDynamics diversityand function of endophytic siderophore-producing bacteria inricerdquoMicrobial Ecology vol 61 no 3 pp 606ndash618 2011

[46] Y C Kim J Leveau B B M Gardener E A Pierson L SPierson and C-M Ryu ldquoThe multifactorial basis for planthealth promotion by plant-associated bacteriardquo Applied andEnvironmental Microbiology vol 77 no 5 pp 1548ndash1555 2011

[47] J M Gardner J L Chandler and A W Feldman ldquoGrowthresponses and vascular plugging of citrus inoculated withrhizobacteria and xylem-resident bacteriardquo Plant and Soil vol86 no 3 pp 333ndash345 1985

[48] C Pliego C Ramos A de Vicente and F M Cazorla ldquoScreen-ing for candidate bacterial biocontrol agents against soilborne

14 International Journal of Microbiology

fungal plant pathogensrdquoPlant and Soil vol 340 no 1-2 pp 505ndash520 2011

[49] H Feng Y Li and Q Liu ldquoEndophytic bacterial communi-ties in tomato plants with differential resistance to Ralstoniasolanacearumrdquo African Journal of Microbiology Research vol 7no 15 pp 1311ndash1318 2013

[50] A V Sturz B R Christie B G Matheson W J Arsenaultand N A Buchanan ldquoEndophytic bacterial communities inthe periderm of potato tubers and their potential to improveresistance to soil-borne plant pathogensrdquo Plant Pathology vol48 no 3 pp 360ndash369 1999

[51] A V Sturz B R Christie and J Nowak ldquoBacterial endophytespotential role in developing sustainable systems of crop produc-tionrdquo Critical Reviews in Plant Sciences vol 19 no 1 pp 1ndash302000

[52] R Ramesh A A Joshi and M P Ghanekar ldquoPseudomonadsmajor antagonistic endophytic bacteria to suppress bacterialwilt pathogenRalstonia solanacearum in the eggplant (Solanummelongena L)rdquo World Journal of Microbiology and Biotechnol-ogy vol 25 no 1 pp 47ndash55 2009

[53] A A Nawangsih I Damayanti S Wiyono and J G KartikaldquoSelection and characterization of endophytic bacteria as bio-control agents of tomato Bacterial Wilt DiseaserdquoHayati Journalof Biosciences vol 18 no 2 pp 66ndash70 2011

[54] R Vargas-Ayala R Rodrıguez-Kabana G Morgan-Jones J AMcInroy and J W Kloepper ldquoShifts in soil microflora inducedby velvetbean (Mucuna deeringiana) in cropping systems tocontrol root-knot nematodesrdquo Biological Control vol 17 no 1pp 11ndash22 2000

[55] D K Zinniel P Lambrecht N B Harris et al ldquoIsolationand characterization of endophytic colonizing bacteria fromagronomic crops and prairie plantsrdquoApplied and EnvironmentalMicrobiology vol 68 no 5 pp 2198ndash2208 2002

[56] F Lemessa and W Zeller ldquoScreening rhizobacteria for biolog-ical control of Ralstonia solanacearum in Ethiopiardquo BiologicalControl vol 42 no 3 pp 336ndash344 2007

[57] M S El-AlbyadM A El-Sayed A R El-Shanshoury andNHEl-Batanouny ldquoEffect of culture conditions on the antimicrobialactivities of UV-mutants of Streptomyces corchorusii and Sspiroverticillatus against bean and banana wilt pathogensrdquoMicrobiological Research vol 151 no 2 pp 201ndash211 1996

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

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BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

International Journal of Microbiology 5

Table1Overviewof

diversity

andfunctio

nalityof

XRBob

tained

from

eggplantchilliand

Storvum

Planth

ost

Num

bero

fsamples

Totalisolates

obtained

Gram-negative

Gram-positive

Haplotypesa

obtained

Antagon

istic

strainsb

Antagon

isticstrains

with

BCEgt25

Biocon

trolstrains

with

GPEgt10

Metho

dof

isolatio

n

BWsusceptib

leeggplant

2489

5336

3116

52

VIo

rMBW

resistant

eggplant

1236

2016

176

65

VIo

rMCh

illi(Ca

psicu

mannu

um)

733

2013

193

21

Mon

ly

Solanu

mtorvum

29

63

73

31

VIo

nly

Total

45167

9968

2816

9a H

aplotypesw

ered

etermined

usingARD

RA

b Antagon

ismto

Rsolana

cearum

teste

din

vitro

asdescrib

edin

Section2

BCE(biocontrolefficacy)a

ndGPE

(growth

prom

otioneffi

cacy)w

ered

etermined

underg

reenho

usec

onditio

nsin

eggplant

asdescrib

edin

Section2

VIVa

cuum

infiltration

Mm

aceration

6 International Journal of Microbiology

Table 2 Haplotypes of XRB based on ARDRA withMspI at 80 similarity plant host biocontrol and growth promotion activities

Haplotypenumbera

Number of strainsin each haplotype Plant host Strains selected for

identificationNumber of biocontrolstrains with BCE gt 25

Number of strainswith GPE gt 10

M80-1 1 RE XB159 0 0M80-2 1 SE XB34 0 0M80-3 1 SE XB66 0 0M80-4 2 SE C XB40 0 0M80-5lowast 1 SE XB140 0 0

M80-6lowast 10 SE RE C XB177 XB157 XB93XB169 XB153 XB170 4 3

M80-7lowast 6 SE RE C XB1 XB86 2 2M80-8 4 SE RE XB22 XB190 0 0M80-9lowast 8 SE RE ST C XB99 XB100 0 0M80-10lowast 10 SE RE C XB196 XB103 1 0M80-11 9 SE RE C XB137 0 0M80-12 1 RE XB158 0 0M80-13 1 C XB87 0 0M80-14 5 SE C XB88 0 0M80-15lowast 5 SE RE ST C XB70 1 0M80-16 3 SE XB47 0 0M80-17 2 SE XB35 0 0M80-18 1 SE XB41 0 0M80-19 2 C XB94 0 0M80-20lowast 13 SE RE C XB8 XB20 XB27 2 0M80-21 5 SE ST C XB25 0 0M80-22 1 SE XB53 0 0M80-23 5 SE RE C XB98 0 0M80-24 11 SE RE ST XB161 0 0M80-25 4 SE RE XB188 0 0M80-26 1 C XB168 0 0M80-27lowast 2 SE C XB126 XB7 0 0M80-28lowast 5 SE RE XB122 0 0M80-29lowast 9 SE RE C XB165 1 1M80-30 15 SE RE C XB167 XB109 0 0

M80-31lowast 11 SE ST C XB123 XB203 XB62XB114 XB202 3 1

M80-32 5 SE ST C XB92 0 0M80-33 1 SE XB37 0 0M80-34 1 SE XB36 0 0M80-35 2 SE XB64 0 0M80-36lowast 1 ST XB200 1 1M80-37lowast 1 SE XB134 0 0M80-38lowast 1 RE XB197 1 1aHaplotype based on ARDRA analysis usingMspI at 80 similarity values of Dice coefficientlowastHaplotype comprises of bacteria showing antagonistic property in the bioassaysSE bacterial wilt susceptible eggplant RE bacterial wilt resistant eggplant ST Solanum torvum C chilli plantBCE biocontrol efficacy GPE growth promotion efficacy determined as described in Section 2

International Journal of Microbiology 7

solanacearum strain Rs-09-100 Results of the in vitro screen-ing against R solanacearum revealed that 28 amongst 167XRB exhibited antagonism towards the pathogen (Table 3)Amongst the antagonists 16 were strains from BW suscepti-ble eggplant 6 from BW resistant eggplant and 3 each fromchilli and S torvum (Table 1) Amongst the 28 antagonists 7strains namely XB62 XB99 XB100 XB114 XB122 XB196XB197 and XB202 formed larger inhibition zones againstR solanacearum ranging from 40mm to 817mm (Table 3)The majority of the antagonistic strains (119899 = 16) producedinhibition zones ranging from 20mm to 383mm XB27XB134 XB165 and XB169 formed smaller inhibition zonesranging from 15mm to 20mm However 139 strains ofXRB did not inhibit R solanacearum in the bioassay testTwenty-eight antagonistic XRBwere screened for productionof volatile inhibitory compounds namely acetoin HCNammonia and diffusible siderophore molecules in vitro(Table 3) Acetoin production was observed in 3214 of theisolates Bacterial isolates XB7 and XB122 were found toproduce both HCN as well as siderophores XB62 XB93 andXB170 produced HCN whereas XB114 XB140 and XB203produced siderophores only XB93 XB99 XB123 XB134 andXB140 produced ammonia

34 Production of Plant Growth Promoting Substances byAntagonistic XRB Results of the screening of antagonisticXRB for in vitro production of several plant growth pro-moting compounds is presented in Table 3 Majority of theantagonistic strains produced the phytohormone IAA withconcentrations ranging from 1591 120583gmL to 64591120583gmLXB202 was found to be the best ACC deaminase producingstrain based on its luxuriant growth on DF salts mediumsupplemented with 30mM ACC as sole nitrogen sourceOther ACC deaminase producing strains include XB1 XB62XB86 and XB140 Scarce growth of XB165 and XB200 wasobserved onDF saltsmedium 6428of the strains producedphosphate solubilizing organic acids as indicated by clearhaloes on Pikovskayarsquos agar plate

35 Greenhouse Experiments

351 Suppression of Bacterial Wilt by Antagonistic XRBAbility to suppress BW was assessed as the difference in thepercentage of wilt in XRB treated plants with respect to wilt inuntreated control andwas expressed as the biocontrol efficacy(BCE) of the antagonists BCE of 16 strains with valuesranging from 286 to 100 is presented in Figure 1 Plantstreated with strains XB86 XB169 and XB177 were free fromBWand hence recorded 100 biocontrol efficacy Treatmentswith XB170 XB197 XB200 XB202 and XB203 recorded 30percent or less wilt incidence (70 to 85 BCE) XB1 XB27XB70 XB93 and XB123 treatments recorded BCE between429 and 571 BCE of 286 was recorded in XB20 andXB165 treatments However 12 antagonistic XRB recordedBCEof 25 andwere least effective inwilt protection Furtherit is observed that all the antagonistic strains originating fromBW resistant eggplant and S torvum exhibited BCE greaterthan 25 Five antagonistic strains from BW susceptible

minus2000

000

2000

4000

6000

8000

10000

12000

XB1

XB20

XB27

XB70

XB86

XB93

XB123

XB165

XB169

XB170

XB177

XB196

XB197

XB200

XB202

XB203

Con

trol

Isolates

Biocontrol efficacy (BCE)Growth promotion efficacy (GPE)

BCE

and

GPE

()

Figure 1 Biocontrol and plant growth promotion efficacies of selectXRB in eggplant BCE biocontrol efficacy determined 25 days afterchallenging with R solanacearum GPE growth promotion efficacydetermined 40 days after treatmentwithXRB Percent BCE andGPEcalculated using formulae BCE = ([percent disease in control] minus[percent disease in treatment]percent disease in control) times 100 andGPE= ([shoot length increase in treatment]minus [shoot length increasein control]shoot length increase in control) times 100 respectivelyUninoculated control had BCE and GPE values of 000 XB86XB169 and XB177 had BCE of 10000 in all the replicationsBars indicate mean values of BCE and GPE error bars indicatestandard deviation

eggplant and three fromchilli were effective in preventingwiltin eggplant (Table 1)

352 Growth Promotion by Antagonistic XRB Increase inshoot length of eggplant (40 days after treatment) observedin XRB treated plants in relation to untreated control wasexpressed as growth promotion efficacy (GPE) and is shownin Figure 1 Amongst the 16 strains which were effective inpreventing wilt six strains exhibited the highest increase inshoot length as indicated by their GPE values in the rangeof 139ndash223 Seven XRB recorded a GPE value rangingfrom 14 to 129 However strains XB27 XB196 and XB203stunted shoot growth in eggplant in comparison to untreatedcontrol When the source of XRB is considered 5555strains that exhibited GPE greater than 10 were isolatedfrom BW resistant eggplant Whereas only two antagonistsfrom BW susceptible plant and one each from chilli and Storvum were able to promote growth in eggplant (Table 1)Strains with GPE gt 10 belonged to six different ARDRAhaplotypes (Table 2)

36 Identification of XRB by 16S rRNA Gene Sequencing16S rRNA gene sequences of XRB were used to identify thediverse xylem inhabitants and antagonistic strains Identityof 55 XRB based on 16S rRNA gene sequencing and theirGenBank accessions are presented in Table 4 Overall 23

8 International Journal of Microbiology

Table3

Invitro

inhibitio

nof

Rsolana

cearum

andprod

uctio

nof

antagonisticc

ompo

unds

andplantg

rowth

prom

otingsubstances

byXR

B

Strain

Haplotype

Inhibitio

nof

Rsolana

cearum

Volatile

anddiffu

sibleinhibitory

compo

unds

Plantgrowth

prom

otingsubstances

Radius

inmm

HCN

Ammon

iaAc

etoin

Sideroph

ore

Phosph

ates

olub

ilisatio

nAC

Cdeam

inasea

IAA

b120583gmL

XB1

M80-7

283plusmn029

minusminus

minusminus

minus++

+10500plusmn707

XB7

M80-27

333plusmn058

+minus

minus+

+minus

4773plusmn321

XB8

M80-20

333plusmn029

minusminus

minusminus

+minus

1909plusmn129

XB20

M80-20

327plusmn040

minusminus

minusminus

+minus

2545plusmn171

XB27

M80-20

183plusmn029

minusminus

minusminus

+minus

1591plusmn107

XB62

M80-31

817plusmn076

+minus

minusminus

+++

++7318plusmn493

XB70

M80-15

383plusmn058

minusminus

minusminus

minusminus

17182plusmn1157

XB86

M80-7

233plusmn076

minusminus

minusminus

minus++

+6682plusmn450

XB93

M80-6

317plusmn029

+minus

+minus

+minus

5727plusmn386

XB99

M80-9

683plusmn058

minus+

+minus

+minus

32136plusmn2164

XB100

M80-9

450plusmn050

minus+

+minus

+minus

23864plusmn1607

XB114

M80-31

650plusmn050

minusminus

minus+

+minus

8909plusmn600

XB122

M80-28

433plusmn058

+minus

minus+

+minus

6682plusmn450

XB123

M80-31

300plusmn087

minus+

+minus

+minus

41682plusmn2807

XB126

M80-27

283plusmn029

minusminus

+minus

+minus

6045plusmn407

XB134

M80-37

157plusmn012

minus+

+minus

+minus

64591plusmn4350

XB140

M80-5

367plusmn076

minus+

minus+

+++

++5091plusmn343

XB153

M80-6

317plusmn029

minusminus

+minus

+minus

15591plusmn1050

XB157

M80-6

250plusmn100

minusminus

+minus

+minus

18455plusmn1243

XB165

M80-29

157plusmn012

minusminus

minusminus

minus+

19091plusmn1286

XB169

M80-6

153plusmn006

minusminus

minusminus

minusminus

1591plusmn107

XB170

M80-6

351plusmn002

+minus

minusminus

+minus

2864plusmn193

XB177

M80-6

199plusmn049

minusminus

minusminus

minusminus

7636plusmn514

XB196

M80-10

393plusmn012

minusminus

minusminus

minusminus

3500plusmn236

XB197

M80-38

433plusmn029

minusminus

+minus

minusminus

4136plusmn279

XB200

M80-36

187plusmn023

minusminus

minusminus

minus++

16864plusmn1136

XB202

M80-31

467plusmn021

minusminus

minusminus

+++

+8273plusmn557

XB203

M80-31

367plusmn029

minusminus

minusminus

minusminus

7000plusmn471

Inhibitio

nzonesa

remeanof

threereplications

andshow

ingsta

ndarddeviation

Inhibitio

nzone

was

measuredas

radius

from

theou

tere

dgeof

wellAlltheexperim

entswerecond

uctedat28plusmn2∘C

+indicates

presence

oftraitminusindicatesabsence

oftraitinplateb

ased

assaysaLevelsofAC

Cdeam

inasea

ctivitiesbasedon

grow

thon

plateb

ased

assayd

enoted

as+forlessg

rowth+++

form

oderateg

rowth+++

forlux

uriant

grow

thand

++++

forh

ighlyluxu

riant

grow

thbIAAwas

estim

ated

incultu

refiltra

teandexpressedas120583gmLusinganalyticalgradeIAAas

stand

ardvalues

indicatemeanandsta

ndarddeviation

International Journal of Microbiology 9

2545

2545

364

364

1273 2909

ActinobacteriaFirmicutesAlpha proteobacteria

Beta proteobacteriaGamma proteobacteriaBacteroidetes

(a)

Num

ber o

f XRB

Actin

obac

teria

Firm

icut

es

Alp

ha p

rote

obac

teria

Beta

pro

teob

acte

ria

Gam

ma p

rote

obac

teria

Bact

eroi

dete

s

Number of antagonistic XRB in each phylumsubdivision

Phylasubdivision

Total number of XRB belonging to each phylumsubdivision

25

20

15

10

5

0

2

14

9

16

4

7

1

2

1

2

11

14

(b)

Figure 2 (a) Distribution of XRB (phylumsubdivision level) identified by 16S rRNA gene sequencing Values indicate percentages of strainsbelonging to each phylasubdivision amongst the 55 identified strains (b) Distribution of antagonistic and nonantagonistic strains of XRB ineach phylumsubdivision

different genera of bacteria were identified Major gen-era identified are Bacillus sp (11 strains) Enterobacter sp(6 strains) Microbacterium sp (5 strains) Staphylococcussp (5 strains) Pseudomonas sp (5 strains) and Agrobac-terium sp (3 strains) Additional genera identified includ-ing Micrococcus sp Sphingomonas sp Flavobacterium spChryseobacterium sp Burkholderia sp and Xenophilus spare listed in Table 4 Based on the identification phylumProteobacteria consisting of Gram-negative bacteria of sub-divisions Alpha Proteobacteria (1273) Beta Proteobacteria(364) and Gamma Proteobacteria (2545) were predom-inant (4181 strains identified) followed by phyla Firmi-cutes (2909) Actinobacteria (2545) and Bacteroidetes(364) (Figure 2(a))

Eleven antagonistic strains identified belonged toGammasubdivision of Proteobacteria consisting of five strainseachof Enterobacter sp and fluorescent and nonfluorescent Pseu-domonas sp and one strain of Pantoea eucrina (XB126)(Figure 2(b)) Nine antagonists identified were of phylaFirmicutes consisting of Staphylococcus sp (5 strains) andBacillus sp (4 strains) Agrobacterium strains XB1 XB86 andXB165 Sphingomonas sp (XB197) of the Alpha Proteobacte-ria Streptomyces sp (XB200) and Janibacter melonis (XB70)of phyla Actinobacteria were found to be antagonistic Addi-tional antagonistic XRB include Burkholderia sp (XB140) of120573 Proteobacteria and Flavobacterium sp (XB203) of phylaBacteroidetes (Figure 2(b))

37 Statistical Analysis The statistical analysis of percent-age wilts and shoot length of eggplant was performedusing Web Agri Statistical Package (WASP) version 20(httpwwwicargoaresinwasp20indexphp)

4 Discussion

Eggplant and chilli not only are of economic and culturalimportance but also are common ingredients in the cuisinethroughout India In the coastal state of GoaR solanacearumhas been reported to be a destructive pathogen in cultivationof eggplant and chilli [4] Isolation of biocontrol agentsagainst the BW pathogen has been commonly restricted toendophytic tissue and plant rhizosphere [26 27] Studieson xylem colonizing endophytes were undertaken becausewe speculated existence of interactions between the XRBand vascular wilt pathogen R solanacearum during xylemcolonization Our study reveals the diversity biocontrolpotential and identity of endophytic xylem colonizers fromsolanaceous crops cultivated in Goa India A total of 167bacteria were isolated from the xylem of eggplant chilliand S torvum with Gram-negative bacteria (5928) pre-dominating in the collection Congruent to our observationGardner et al [7] and Bell et al [11] have earlier reportedisolation of more number of Gram-negative rod shapedbacteria from xylem of citrus and grapevine using vacuuminfiltration Scholander pressure bomb was found to beuseful in extraction of diverse bacterial genera from xylemtissues in contrast to trituration methods that yielded highernumber of Gram-positive rod shaped bacteria [12] ThoughScholander pressure bomb was not used in this study acombination of vacuum infiltration [7 11] and triturationof decorticated stems [10 12] was employed with an aimto isolate diverse XRB from xylem tissues This is the firststudy reporting the use of vacuum infiltration and triturationmethods for isolating xylem residing bacteria from eggplantchilli and S torvum

Traditionally bacteria have been characterized andgrouped based on colony morphology and biochemical

10 International Journal of Microbiology

Table 4 List of XRB identified by partial 16S rRNAgene sequencing their plant host accession numbers closestNCBImatch and similarity

Strain Plant host Accession number Closest NCBI match similarityXB1lowast SE KF447383 Agrobacterium tumefaciens 99XB7lowast SE KF447384 Pseudomonas aeruginosa 99XB8lowast SE KF447385 Staphylococcus haemolyticus 99XB20lowast SE KF447386 Staphylococcus haemolyticus 100XB22 SE KF447387 Brevibacterium casei 99XB25 C KF447388 Enterobacter sp 95XB27lowast SE KF447389 Staphylococcus haemolyticus 99XB34 SE KF447390 Curtobacterium sp 99XB35 SE KF447391 Microbacterium sp 99XB36 SE KF447392 Xenophilus sp 99XB37 SE KF447393 Chryseobacterium sp 99XB40 SE KF447394 Micrococcus luteus 89XB41 SE KF447395 Bacillus sp 99XB47 SE KF447396 Bacillus sp 99XB53 SE KF447397 Micrococcus sp 99XB62lowast SE KF447398 Pseudomonas sp 99XB64 SE KF447399 Bacillus thuringiensis 100XB66 SE KF447400 Pectobacterium carotovorum 99XB70lowast SE KF447401 Janibacter melonis 99XB86lowast C KF447402 Agrobacterium tumefaciens 99XB87 C KF447403 Bacillus barbaricus 99XB88 C KF447404 Bacillus sp 99XB92 C KF447405 Brevundimonas vesicularis 99XB93lowast C KF447406 Bacillus safensis 100XB94 C KF447407 Bacillus sp 100XB98 C KF447408 Microbacterium sp 100XB99lowast SE KF447409 Enterobacter sp 99XB100lowast SE KF447410 Enterobacter cloacae 98XB103 SE KF447411 Brachybacterium phenoliresistens 99XB109 SE KF447412 Rhodococcus corynebacterioides 99XB114lowast SE KF447413 Pseudomonas stutzeri 99XB122lowast SE KF913446 Pseudomonas aeruginosa 100XB123lowast SE KF447414 Enterobacter sp 99XB126lowast C KF447415 Pantoea eucrina 99XB134lowast SE KF447416 Enterobacter sp 99XB137 SE KF447417 Klebsiella sp 99XB140lowast SE KF447418 Burkholderia sp 99XB153lowast SE KF447419 Bacillus amyloliquefaciens 99XB157lowast SE KF447420 Bacillus subtilis 100XB158 RE KF447421 Bosea sp 99XB159 RE KF447422 Microbacterium xylanilyticum 99XB161 RE KF447423 Microbacterium aurum 99XB165lowast RE KF447424 Agrobacterium sp 99XB167 RE KF447425 Microbacterium aurum 99XB168 RE KF447426 Bacillus aryabhattai 100XB169lowast RE KF447427 Staphylococcus gallinarum 99XB170lowast RE KF447428 Staphylococcus sp 99XB177lowast RE KF447429 Bacillus cereus 99XB188 RE KF447430 Sphingomonas sp 99XB190 RE KF447431 Brevibacterium casei 99

International Journal of Microbiology 11

Table 4 Continued

Strain Plant host Accession number Closest NCBI match similarityXB196lowast RE KF447432 Enterobacter kobei 99XB197lowast RE KF447433 Sphingomonas sp 98XB200lowast ST KF913447 Streptomyces sp 100XB202lowast ST KF447434 Pseudomonas sp 99XB203lowast ST KF447435 Flavobacterium sp 99lowastStrain is antagonistic to R solanacearum based on bioassaySE bacterial wilt susceptible eggplant RE bacterial wilt resistant eggplant C chilli ST Solanum torvum

tests However whole genome fingerprinting or PCR-RFLPbased methods are rapid tools for determining geneticdiversity of bacteria in a given collection ARDRA which isa type of PCR based RFLP method has been used widely inestimating genetic diversity endophytic bacterial populationsand clustering genetically similar strains [39ndash41] In additionto earlier reports our study demonstrates the usefulnessof ARDRA as a tool to cluster genetically identical strainsof endophytic XRB isolated from eggplant chilli andS torvum In our study 9161 strains (119899 = 153) weregrouped in 24 haplotypes by using ARDRA The majority ofthese haplotypes represent a combination of XRB isolatedfrom different solanaceous plants from diverse localesThese results indicate that xylem of eggplant chilli andS torvum largely bears similar population of XRB whichcan efficiently cross colonize eggplant chilli or S torvumNevertheless 839 strains had unique ARDRA fingerprintand formed separable haplotypes This observation leads toa conclusion that a minor population of xylem inhabitantsare restricted to a specific plant species and cannot easilycross colonize xylem of other solanaceous plants Plantsare known to selectively support endophytic colonizationby specific bacteria [14] However factors that determinethe selection of xylem colonists or the ability of XRBto colonize eggplant chilli and S torvum in this studyremain unknown Interestingly the structure of endophyticcommunity in Nicotiana attenuata a member of Solanaceaefamily is shown to be influenced by soil composition andethylene homeostasis [40] Earlier evidence has shown thatcolonization by endophytic bacteria is also governed by plantgenotype as well as root exudates [42]

Only 1677 XRB out of 167 were antagonistic to Rsolanacearum based on in vitro assays Antagonistic XRBproduced volatile and diffusible inhibitory compoundsnamely HCN ammonia and acetoin and siderophoresThese substances have been long known to be involvedin disease suppression and indirect growth promotion inplants [43ndash46] These mechanisms possibly played a rolein the evident biocontrol effect against BW exhibited bythe XRB in the greenhouse screening Endophytic bacteriahave been known to have plant growth promoting traitsnamely production of IAA ACC deaminase and phosphatesolubilization [25 33 36] These traits were detected in themajority of antagonistic XRB tested in this study and mayhave resulted in the observed increase in shoot length ofeggplant in our greenhouse experiments In contrast strains

XB20 XB196 and XB203 suppressed growth in eggplantunder greenhouse conditions however no visible symptomsof disease were observed Vascular plugging and productionof certainmetabolites toxic to plant cells but not cell viabilitymay have resulted in stunted shoot in eggplant [11 47]

Evaluation of efficacy of antagonistic organisms to sup-press the plant diseases under greenhouse conditions isone of the key steps for selecting a potential biocontrolagent for disease management [48] Endophytic strains fromBW susceptible varieties of eggplant have been shown toprevent wilt and promote growth in eggplant earlier [27]Our greenhouse screening shows that 3846 of antagonisticXRB with biocontrol efficacies greater than 40 were isolatesfrom BW resistant varieties of eggplantThis raises a questionwhether the bacteria from resistant varieties are involved inBW resistance and whether BW resistant varieties are ableto selectively influence xylem colonization by antagonisticbacteria Presence of higher number of endophytes withantagonistic abilities was reported in BW resistant varieties oftomato as compared to susceptible varieties and their role inresistance to BWwas proposed [49] Similar observations oncorrelation of resistance of potato to soft rot and endophyticbacteria have been reported [50]Thus BW resistant varietiescan be considered a better host for isolating potential biocon-trol strains for management of bacterial wilt

Identification of 55 XRB strains by 16S rRNA genesequencing revealed the presence of 23 diverse genera ofbacteria belonging to 4 phyla of Eubacteria Strains belongingto phyla Firmicutes Actinobacteria and 120574 subdivision ofProteobacteria were the major xylem colonists identified inthis study Several genera of bacteria belonging to these phylahave also been reported to be present in endophytic tissuesand xylem of a variety of other agricultural and horticulturalplant species [14 51] In addition the majority of the antag-onists identified belonged to Enterobacter sp Pseudomonassp Bacillus sp and Staphylococcus sp Congruent to ourresults several researchers have reported bacteria isolatedfrom solanaceous crops and belonging to similar genera tobe antagonistic to R solanacearum [26 51ndash53] HoweverFlavobacterium spand Janibacter melonis identified in thisstudy have never been previously reported to be inhibitoryto R solanacearum Large population of the xylem inhabitingbacterial flora accounting for 8323 exhibited no antago-nism towards R solanacearum Nonantagonistic XRB wereidentified predominantly as Microbacterium sp Endophyticpersistence and nematicidal activities of Microbacterium sp

12 International Journal of Microbiology

have been reported [54 55] Therefore the collection of XRBisolated in this study can be screened for inhibitory activitiesagainst other important agricultural pests

Though few strains namely XB86 XB169 and XB177exhibited plant beneficial properties in this study their use-fulness in plant disease control remains to be seen XB86has been identified as Agrobacterium tumefaciens the crowngall disease pathogen and its deployment as biocontrolagent is uncertain XB169 (Staphylococcus gallinarum) andXB177 (Bacillus cereus) are reported as opportunistic animalpathogens and thus unsuitable for field applications Strep-tomyces sp has earlier been reported as antagonistic to Rsolanacearum and tested for management of wilt in potatoand tomato [56 57] XB200 (Streptomyces sp) is one of theXRB high BCE and GPE it could be explored further forbiocontrol of bacterial wilt after additional characterizationand field evaluation

5 Conclusion

This study is the first report on the identity of novel anddiverse XRB colonizing the xylem of eggplant chilli and Storvum XRB particularly from BW resistant varieties werefound to protect eggplant from bacterial wilt and enhancedgrowth in eggplant in the greenhouse screening Thereforethe repertoire of XRB reported in this study may be usefulfor cultivation of eggplant in BW affected areas

Conflict of Interests

The authors hereby declare that they have no conflict ofinterests regarding the publication of this paper

Acknowledgments

The authors greatly acknowledge financial support fromIndian Council of Agricultural Research (ICAR) New DelhiIndia through ldquoOutreach project on Phytopthora Fusariumand Ralstonia diseases of horticultural and field cropsrdquo-(PhytoFuRa) and the Director ICAR Research Complex forGoa India for the facilities

References

[1] E Yabuuchi Y KosakoHOyaizu et al ldquoProposal ofBurkholde-ria gen nov and transfer of seven species of the genusPseudomonas homology group II to the new genus with thetype species Burkholderia cepacia (Palleroni and Holmes 1981)comb novrdquo Microbiology and Immunology vol 36 no 12 pp1251ndash1275 1992

[2] E Wicker L Grassart R Coranson-Beaudu et al ldquoRalstoniasolanacearum strains from Martinique (French West Indies)exhibiting a new pathogenic potentialrdquo Applied and Environ-mental Microbiology vol 73 no 21 pp 6790ndash6801 2007

[3] Y Liu A Kanda K Yano et al ldquoMolecular typing of Japanesestrains of Ralstonia solanacearum in relation to the ability toinduce a hypersensitive reaction in tobaccordquo Journal of GeneralPlant Pathology vol 75 no 5 pp 369ndash380 2009

[4] R Ramesh ldquoField evaluation of biological control agents for themanagement of Ralstonia solanacearum in Brinjalrdquo Journal ofMycology and Plant Pathology vol 36 no 2 pp 327ndash328 2006

[5] J Vasse P Frey and A Trigalet ldquoMicroscopic studies ofintercellular infection and protoxylem invasion of tomatoroots by Pseudomonas solanacearumrdquoMolecular Plant-MicrobeInteractions vol 8 no 2 pp 241ndash251 1995

[6] S Genin and C Boucher ldquoRalstonia solanacearum secrets of amajor pathogen unveiled by analysis of its genomerdquo MolecularPlant Pathology vol 3 no 3 pp 111ndash118 2002

[7] J M Gardner A W Feldman and R M Zablotowicz ldquoIdentityand behavior of xylem-residing bacteria in rough lemon roots ofFlorida citrus treesrdquo Applied and Environmental Microbiologyvol 43 no 6 pp 1335ndash1342 1982

[8] M J Jacobs W M Bugbee and D A Gabriellson ldquoEnu-meration location and characterization of endophytic bacteriawithin sugar beet rootsrdquoCanadian Journal of Botany vol 63 no7 pp 1262ndash1265 1985

[9] D G Patriquin and J Dobereiner ldquoLight microscopy observa-tions of tetrazolium-reducing bacteria in the endorhizosphereof maize and other grasses in Brazilrdquo Canadian Journal ofMicrobiology vol 24 no 6 pp 734ndash742 1978

[10] S Gagne C Richard H Rousseau and H Antoun ldquoXylem-residing bacteria in alfalfa rootsrdquo Canadian Journal of Micro-biology vol 33 no 11 pp 996ndash1000 1987

[11] C R Bell G A Dickie W L G Harvey and J W Y FChan ldquoEndophytic bacteria in grapevinerdquo Canadian Journal ofMicrobiology vol 41 no 1 pp 46ndash53 1995

[12] J Hallmann J W Kloepper and R Rodrıguez-Kabana ldquoAppli-cation of Scholander pressure bomb to studies on endophyticbacteria of plantsrdquo Canadian Journal of Microbiology vol 43no 5 pp 411ndash416 1997

[13] J S Lampel G L Canter M B Dimock et al ldquoIntegrativecloning expression and stability of the cryIA(c) gene fromBacillus thuringiensis subsp kurstaki in a recombinant strain ofClavibacter xyli subsp cynodontisrdquo Applied and EnvironmentalMicrobiology vol 60 no 2 pp 501ndash508 1994

[14] M Rosenblueth and E Martınez-Romero ldquoBacterial endo-phytes and their interactions with hostsrdquo Molecular Plant-Microbe Interactions vol 19 no 8 pp 827ndash837 2006

[15] P R Hardoim L S van Overbeek and J D van ElsasldquoProperties of bacterial endophytes and their proposed role inplant growthrdquo Trends in Microbiology vol 16 no 10 pp 463ndash471 2008

[16] R Aravind A Kumar S J Eapen and K V RamanaldquoEndophytic bacterial flora in root and stem tissues of blackpepper (Piper nigrum L) genotype isolation identificationand evaluation against Phytophthora capsicirdquo Letters in AppliedMicrobiology vol 48 no 1 pp 58ndash64 2009

[17] J Kuklinsky-Sobral W L Araujo R Mendes I O Geraldi AA Pizzirani-Kleiner and J L Azevedo ldquoIsolation and charac-terization of soybean-associated bacteria and their potential forplant growth promotionrdquo Environmental Microbiology vol 6no 12 pp 1244ndash1251 2004

[18] S Compant B Duffy J Nowak C Clement and E A BarkaldquoUse of plant growth-promoting bacteria for biocontrol ofplant diseases principles mechanisms of action and futureprospectsrdquoApplied and Environmental Microbiology vol 71 no9 pp 4951ndash4959 2005

[19] E T Thorne B M Young G M Young et al ldquoThe structureof xylem vessels in grapevine (Vitaceae) and a possible passive

International Journal of Microbiology 13

mechanism for the systemic spread of bacterial diseaserdquo Amer-ican Journal of Botany vol 93 no 4 pp 497ndash504 2006

[20] W T Seo W J Lim E J Kim H D Yun Y H Lee and KM Cho ldquoEndophytic bacterial diversity in the young radishand their antimicrobial activity against pathogensrdquo Journal ofApplied Biological Chemistry vol 53 no 4 pp 493ndash503 2010

[21] S Compant H Kaplan A Sessitsch J Nowak E Ait Barkaand C Clement ldquoEndophytic colonization ofVitis vinifera L byBurkholderia phytofirmans strain PsJN from the rhizosphere toinflorescence tissuesrdquo FEMS Microbiology Ecology vol 63 no1 pp 84ndash93 2005

[22] P S B Miguel J C Delvaux M N V de Oliveira et al ldquoDiver-sity of endophytic bacteria in the fruits of Coffea canephorardquoAfrican Journal of Microbiology Research vol 7 no 7 pp 586ndash594 2013

[23] A Lopez-Lopez M A Rogel E Ormeno-Orrillo J Martınez-Romero and E Martınez-Romero ldquoPhaseolus vulgaris seed-borne endophytic community with novel bacterial species suchas Rhizobium endophyticum sp novrdquo Systematic and AppliedMicrobiology vol 33 no 6 pp 322ndash327 2010

[24] M Lwin and S L Ranamukhaarachchi ldquoDevelopment of bio-logical control of Ralstonia solanacearum through antagonisticmicrobial populationsrdquo International Journal of Agriculture andBiology vol 8 no 5 pp 657ndash660 2006

[25] F Rasche E Marco-Noales H Velvis et al ldquoStructural char-acteristics and plant-beneficial effects of bacteria colonizing theshoots of field grown conventional and geneticallymodifiedT4-lysozyme producing potatoesrdquo Plant and Soil vol 289 no 1-2pp 123ndash140 2006

[26] N Amaresan V Jayakumar K Kumar and N ThajuddinldquoEndophytic bacteria from tomato and chilli their diversity andantagonistic potential against Ralstonia solanacearumrdquoArchivesof Phytopathology and Plant Protection vol 45 no 3 pp 344ndash355 2012

[27] R Ramesh and G S Phadke ldquoRhizosphere and endophyticbacteria for the suppression of eggplant wilt caused byRalstoniasolanacearumrdquo Crop Protection vol 37 pp 35ndash41 2012

[28] W J French R G Christis and D L Stassi ldquoRecovery ofrickettsialike bacteria by vacuum infiltration of peach tissuesaffected with phony diseaserdquo Phytopathology vol 67 pp 945ndash948 1977

[29] P R Viss E M Brooks and J A Driver ldquoA simplified methodfor the control of bacterial contamination in woody plant tissueculturerdquo In Vitro Cellular andDevelopmental Biology vol 27 no1 article 42 1991

[30] K Wilson ldquoPreparation of genomic DNA from bacteriardquo inCurrent Protocols in Molecular Biology F M Ausubel R BrentR E Kingston et al Eds pp 241ndash245 John Wiley amp SonsNew York NY USA 1997

[31] M Saraf AThakkar U PandyaM Joshi and J Parikh ldquoPoten-tial of plant growth promoting microorganisms as biofertilizersand biopesticides and itrsquos exploitation in sustainable agricul-turerdquo Journal of Microbiology and Biotechnology Research vol3 no 5 pp 54ndash62 2013

[32] A P G C Marques C Pires H Moreira A O S S Rangeland P M L Castro ldquoAssessment of the plant growth promotionabilities of six bacterial isolates using Zea mays as indicatorplantrdquo Soil Biology and Biochemistry vol 42 no 8 pp 1229ndash1235 2010

[33] X Hao P Xie L Johnstone S J Miller C Rensingand G Wei ldquoGenome sequence and mutational analysis of

plant-growth-promoting bacteriumAgrobacterium tumefaciensCCNWGS0286 isolated from a zinc-lead mine tailingrdquo Appliedand Environmental Microbiology vol 78 no 15 pp 5384ndash53942012

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

[35] S A Gordon and L G Paleg ldquoQuantitative measurement ofindole acetic acidrdquo Plant Physiology vol 10 pp 37ndash48 1957

[36] A Godinho R Ramesh and S Bhosle ldquoBacteria from sanddunes of Goa promoting growth in eggplantrdquoWorld Journal ofAgricultural Sciences vol 6 no 5 pp 555ndash564 2010

[37] J-H Guo H-Y Qi Y-H Guo et al ldquoBiocontrol of tomato wiltby plant growth-promoting rhizobacteriardquo Biological Controlvol 29 no 1 pp 66ndash72 2004

[38] N Aliye C Fininsa and Y Hiskias ldquoEvaluation of rhizo-sphere bacterial antagonists for their potential to bioprotectpotato (Solanum tuberosum) against bacterial wilt (Ralstoniasolanacearum)rdquo Biological Control vol 47 no 3 pp 282ndash2882008

[39] L LagaceM PitreM Jacqeus andDRoy ldquoIdentification of thebacterial community ofmaple sap by using amplified ribosomalDNA (rDNA) restriction analysis and rDNA sequencingrdquoApplied and Environmental Microbiology vol 70 no 4 pp2052ndash2060 2004

[40] H H Long D G Sonntag D D Schmidt and I T BaldwinldquoThe structure of the culturable root bacterial endophyte com-munity of Nicotiana attenuata is organized by soil compositionand host plant ethylene production and perceptionrdquo NewPhytologist vol 185 no 2 pp 554ndash567 2010

[41] E Lopez-Fuentes V M Ruız-Valdiviezo E Martınez-RomeroF A Gutierrez-Miceli L Dendooven and R Rincon-RosalesldquoBacterial community in the roots and rhizosphere of Hyper-icum silenoides Juss 1804rdquo African Journal of MicrobiologyResearch vol 6 no 11 pp 2704ndash2711 2012

[42] M Fang R J Kremer P P Motavalli and G Davis ldquoBacterialdiversity in rhizospheres of nontransgenic and transgenic cornrdquoApplied and EnvironmentalMicrobiology vol 71 no 7 pp 4132ndash4136 2005

[43] C R Howell and R D Stiphanovic ldquoProduction of ammonia byEnterobacter cloaceae and its possible role in biological controlof Pythium pre-emergence damping-off by the bacteriumrdquoEcology and Epidemiology vol 78 no 8 pp 1075ndash1078 1988

[44] A Ramette M Frapolli G Defago and Y Moenne-LoccozldquoPhylogeny of HCN synthase-encoding hcnBC genes in bio-control fluorescent pseudomonads and its relationship withhost plant species and HCN synthesis abilityrdquoMolecular Plant-Microbe Interactions vol 16 no 6 pp 525ndash535 2003

[45] I Loaces L Ferrando and A F Scavino ldquoDynamics diversityand function of endophytic siderophore-producing bacteria inricerdquoMicrobial Ecology vol 61 no 3 pp 606ndash618 2011

[46] Y C Kim J Leveau B B M Gardener E A Pierson L SPierson and C-M Ryu ldquoThe multifactorial basis for planthealth promotion by plant-associated bacteriardquo Applied andEnvironmental Microbiology vol 77 no 5 pp 1548ndash1555 2011

[47] J M Gardner J L Chandler and A W Feldman ldquoGrowthresponses and vascular plugging of citrus inoculated withrhizobacteria and xylem-resident bacteriardquo Plant and Soil vol86 no 3 pp 333ndash345 1985

[48] C Pliego C Ramos A de Vicente and F M Cazorla ldquoScreen-ing for candidate bacterial biocontrol agents against soilborne

14 International Journal of Microbiology

fungal plant pathogensrdquoPlant and Soil vol 340 no 1-2 pp 505ndash520 2011

[49] H Feng Y Li and Q Liu ldquoEndophytic bacterial communi-ties in tomato plants with differential resistance to Ralstoniasolanacearumrdquo African Journal of Microbiology Research vol 7no 15 pp 1311ndash1318 2013

[50] A V Sturz B R Christie B G Matheson W J Arsenaultand N A Buchanan ldquoEndophytic bacterial communities inthe periderm of potato tubers and their potential to improveresistance to soil-borne plant pathogensrdquo Plant Pathology vol48 no 3 pp 360ndash369 1999

[51] A V Sturz B R Christie and J Nowak ldquoBacterial endophytespotential role in developing sustainable systems of crop produc-tionrdquo Critical Reviews in Plant Sciences vol 19 no 1 pp 1ndash302000

[52] R Ramesh A A Joshi and M P Ghanekar ldquoPseudomonadsmajor antagonistic endophytic bacteria to suppress bacterialwilt pathogenRalstonia solanacearum in the eggplant (Solanummelongena L)rdquo World Journal of Microbiology and Biotechnol-ogy vol 25 no 1 pp 47ndash55 2009

[53] A A Nawangsih I Damayanti S Wiyono and J G KartikaldquoSelection and characterization of endophytic bacteria as bio-control agents of tomato Bacterial Wilt DiseaserdquoHayati Journalof Biosciences vol 18 no 2 pp 66ndash70 2011

[54] R Vargas-Ayala R Rodrıguez-Kabana G Morgan-Jones J AMcInroy and J W Kloepper ldquoShifts in soil microflora inducedby velvetbean (Mucuna deeringiana) in cropping systems tocontrol root-knot nematodesrdquo Biological Control vol 17 no 1pp 11ndash22 2000

[55] D K Zinniel P Lambrecht N B Harris et al ldquoIsolationand characterization of endophytic colonizing bacteria fromagronomic crops and prairie plantsrdquoApplied and EnvironmentalMicrobiology vol 68 no 5 pp 2198ndash2208 2002

[56] F Lemessa and W Zeller ldquoScreening rhizobacteria for biolog-ical control of Ralstonia solanacearum in Ethiopiardquo BiologicalControl vol 42 no 3 pp 336ndash344 2007

[57] M S El-AlbyadM A El-Sayed A R El-Shanshoury andNHEl-Batanouny ldquoEffect of culture conditions on the antimicrobialactivities of UV-mutants of Streptomyces corchorusii and Sspiroverticillatus against bean and banana wilt pathogensrdquoMicrobiological Research vol 151 no 2 pp 201ndash211 1996

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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International Journal of

Volume 2014

Zoology

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

6 International Journal of Microbiology

Table 2 Haplotypes of XRB based on ARDRA withMspI at 80 similarity plant host biocontrol and growth promotion activities

Haplotypenumbera

Number of strainsin each haplotype Plant host Strains selected for

identificationNumber of biocontrolstrains with BCE gt 25

Number of strainswith GPE gt 10

M80-1 1 RE XB159 0 0M80-2 1 SE XB34 0 0M80-3 1 SE XB66 0 0M80-4 2 SE C XB40 0 0M80-5lowast 1 SE XB140 0 0

M80-6lowast 10 SE RE C XB177 XB157 XB93XB169 XB153 XB170 4 3

M80-7lowast 6 SE RE C XB1 XB86 2 2M80-8 4 SE RE XB22 XB190 0 0M80-9lowast 8 SE RE ST C XB99 XB100 0 0M80-10lowast 10 SE RE C XB196 XB103 1 0M80-11 9 SE RE C XB137 0 0M80-12 1 RE XB158 0 0M80-13 1 C XB87 0 0M80-14 5 SE C XB88 0 0M80-15lowast 5 SE RE ST C XB70 1 0M80-16 3 SE XB47 0 0M80-17 2 SE XB35 0 0M80-18 1 SE XB41 0 0M80-19 2 C XB94 0 0M80-20lowast 13 SE RE C XB8 XB20 XB27 2 0M80-21 5 SE ST C XB25 0 0M80-22 1 SE XB53 0 0M80-23 5 SE RE C XB98 0 0M80-24 11 SE RE ST XB161 0 0M80-25 4 SE RE XB188 0 0M80-26 1 C XB168 0 0M80-27lowast 2 SE C XB126 XB7 0 0M80-28lowast 5 SE RE XB122 0 0M80-29lowast 9 SE RE C XB165 1 1M80-30 15 SE RE C XB167 XB109 0 0

M80-31lowast 11 SE ST C XB123 XB203 XB62XB114 XB202 3 1

M80-32 5 SE ST C XB92 0 0M80-33 1 SE XB37 0 0M80-34 1 SE XB36 0 0M80-35 2 SE XB64 0 0M80-36lowast 1 ST XB200 1 1M80-37lowast 1 SE XB134 0 0M80-38lowast 1 RE XB197 1 1aHaplotype based on ARDRA analysis usingMspI at 80 similarity values of Dice coefficientlowastHaplotype comprises of bacteria showing antagonistic property in the bioassaysSE bacterial wilt susceptible eggplant RE bacterial wilt resistant eggplant ST Solanum torvum C chilli plantBCE biocontrol efficacy GPE growth promotion efficacy determined as described in Section 2

International Journal of Microbiology 7

solanacearum strain Rs-09-100 Results of the in vitro screen-ing against R solanacearum revealed that 28 amongst 167XRB exhibited antagonism towards the pathogen (Table 3)Amongst the antagonists 16 were strains from BW suscepti-ble eggplant 6 from BW resistant eggplant and 3 each fromchilli and S torvum (Table 1) Amongst the 28 antagonists 7strains namely XB62 XB99 XB100 XB114 XB122 XB196XB197 and XB202 formed larger inhibition zones againstR solanacearum ranging from 40mm to 817mm (Table 3)The majority of the antagonistic strains (119899 = 16) producedinhibition zones ranging from 20mm to 383mm XB27XB134 XB165 and XB169 formed smaller inhibition zonesranging from 15mm to 20mm However 139 strains ofXRB did not inhibit R solanacearum in the bioassay testTwenty-eight antagonistic XRBwere screened for productionof volatile inhibitory compounds namely acetoin HCNammonia and diffusible siderophore molecules in vitro(Table 3) Acetoin production was observed in 3214 of theisolates Bacterial isolates XB7 and XB122 were found toproduce both HCN as well as siderophores XB62 XB93 andXB170 produced HCN whereas XB114 XB140 and XB203produced siderophores only XB93 XB99 XB123 XB134 andXB140 produced ammonia

34 Production of Plant Growth Promoting Substances byAntagonistic XRB Results of the screening of antagonisticXRB for in vitro production of several plant growth pro-moting compounds is presented in Table 3 Majority of theantagonistic strains produced the phytohormone IAA withconcentrations ranging from 1591 120583gmL to 64591120583gmLXB202 was found to be the best ACC deaminase producingstrain based on its luxuriant growth on DF salts mediumsupplemented with 30mM ACC as sole nitrogen sourceOther ACC deaminase producing strains include XB1 XB62XB86 and XB140 Scarce growth of XB165 and XB200 wasobserved onDF saltsmedium 6428of the strains producedphosphate solubilizing organic acids as indicated by clearhaloes on Pikovskayarsquos agar plate

35 Greenhouse Experiments

351 Suppression of Bacterial Wilt by Antagonistic XRBAbility to suppress BW was assessed as the difference in thepercentage of wilt in XRB treated plants with respect to wilt inuntreated control andwas expressed as the biocontrol efficacy(BCE) of the antagonists BCE of 16 strains with valuesranging from 286 to 100 is presented in Figure 1 Plantstreated with strains XB86 XB169 and XB177 were free fromBWand hence recorded 100 biocontrol efficacy Treatmentswith XB170 XB197 XB200 XB202 and XB203 recorded 30percent or less wilt incidence (70 to 85 BCE) XB1 XB27XB70 XB93 and XB123 treatments recorded BCE between429 and 571 BCE of 286 was recorded in XB20 andXB165 treatments However 12 antagonistic XRB recordedBCEof 25 andwere least effective inwilt protection Furtherit is observed that all the antagonistic strains originating fromBW resistant eggplant and S torvum exhibited BCE greaterthan 25 Five antagonistic strains from BW susceptible

minus2000

000

2000

4000

6000

8000

10000

12000

XB1

XB20

XB27

XB70

XB86

XB93

XB123

XB165

XB169

XB170

XB177

XB196

XB197

XB200

XB202

XB203

Con

trol

Isolates

Biocontrol efficacy (BCE)Growth promotion efficacy (GPE)

BCE

and

GPE

()

Figure 1 Biocontrol and plant growth promotion efficacies of selectXRB in eggplant BCE biocontrol efficacy determined 25 days afterchallenging with R solanacearum GPE growth promotion efficacydetermined 40 days after treatmentwithXRB Percent BCE andGPEcalculated using formulae BCE = ([percent disease in control] minus[percent disease in treatment]percent disease in control) times 100 andGPE= ([shoot length increase in treatment]minus [shoot length increasein control]shoot length increase in control) times 100 respectivelyUninoculated control had BCE and GPE values of 000 XB86XB169 and XB177 had BCE of 10000 in all the replicationsBars indicate mean values of BCE and GPE error bars indicatestandard deviation

eggplant and three fromchilli were effective in preventingwiltin eggplant (Table 1)

352 Growth Promotion by Antagonistic XRB Increase inshoot length of eggplant (40 days after treatment) observedin XRB treated plants in relation to untreated control wasexpressed as growth promotion efficacy (GPE) and is shownin Figure 1 Amongst the 16 strains which were effective inpreventing wilt six strains exhibited the highest increase inshoot length as indicated by their GPE values in the rangeof 139ndash223 Seven XRB recorded a GPE value rangingfrom 14 to 129 However strains XB27 XB196 and XB203stunted shoot growth in eggplant in comparison to untreatedcontrol When the source of XRB is considered 5555strains that exhibited GPE greater than 10 were isolatedfrom BW resistant eggplant Whereas only two antagonistsfrom BW susceptible plant and one each from chilli and Storvum were able to promote growth in eggplant (Table 1)Strains with GPE gt 10 belonged to six different ARDRAhaplotypes (Table 2)

36 Identification of XRB by 16S rRNA Gene Sequencing16S rRNA gene sequences of XRB were used to identify thediverse xylem inhabitants and antagonistic strains Identityof 55 XRB based on 16S rRNA gene sequencing and theirGenBank accessions are presented in Table 4 Overall 23

8 International Journal of Microbiology

Table3

Invitro

inhibitio

nof

Rsolana

cearum

andprod

uctio

nof

antagonisticc

ompo

unds

andplantg

rowth

prom

otingsubstances

byXR

B

Strain

Haplotype

Inhibitio

nof

Rsolana

cearum

Volatile

anddiffu

sibleinhibitory

compo

unds

Plantgrowth

prom

otingsubstances

Radius

inmm

HCN

Ammon

iaAc

etoin

Sideroph

ore

Phosph

ates

olub

ilisatio

nAC

Cdeam

inasea

IAA

b120583gmL

XB1

M80-7

283plusmn029

minusminus

minusminus

minus++

+10500plusmn707

XB7

M80-27

333plusmn058

+minus

minus+

+minus

4773plusmn321

XB8

M80-20

333plusmn029

minusminus

minusminus

+minus

1909plusmn129

XB20

M80-20

327plusmn040

minusminus

minusminus

+minus

2545plusmn171

XB27

M80-20

183plusmn029

minusminus

minusminus

+minus

1591plusmn107

XB62

M80-31

817plusmn076

+minus

minusminus

+++

++7318plusmn493

XB70

M80-15

383plusmn058

minusminus

minusminus

minusminus

17182plusmn1157

XB86

M80-7

233plusmn076

minusminus

minusminus

minus++

+6682plusmn450

XB93

M80-6

317plusmn029

+minus

+minus

+minus

5727plusmn386

XB99

M80-9

683plusmn058

minus+

+minus

+minus

32136plusmn2164

XB100

M80-9

450plusmn050

minus+

+minus

+minus

23864plusmn1607

XB114

M80-31

650plusmn050

minusminus

minus+

+minus

8909plusmn600

XB122

M80-28

433plusmn058

+minus

minus+

+minus

6682plusmn450

XB123

M80-31

300plusmn087

minus+

+minus

+minus

41682plusmn2807

XB126

M80-27

283plusmn029

minusminus

+minus

+minus

6045plusmn407

XB134

M80-37

157plusmn012

minus+

+minus

+minus

64591plusmn4350

XB140

M80-5

367plusmn076

minus+

minus+

+++

++5091plusmn343

XB153

M80-6

317plusmn029

minusminus

+minus

+minus

15591plusmn1050

XB157

M80-6

250plusmn100

minusminus

+minus

+minus

18455plusmn1243

XB165

M80-29

157plusmn012

minusminus

minusminus

minus+

19091plusmn1286

XB169

M80-6

153plusmn006

minusminus

minusminus

minusminus

1591plusmn107

XB170

M80-6

351plusmn002

+minus

minusminus

+minus

2864plusmn193

XB177

M80-6

199plusmn049

minusminus

minusminus

minusminus

7636plusmn514

XB196

M80-10

393plusmn012

minusminus

minusminus

minusminus

3500plusmn236

XB197

M80-38

433plusmn029

minusminus

+minus

minusminus

4136plusmn279

XB200

M80-36

187plusmn023

minusminus

minusminus

minus++

16864plusmn1136

XB202

M80-31

467plusmn021

minusminus

minusminus

+++

+8273plusmn557

XB203

M80-31

367plusmn029

minusminus

minusminus

minusminus

7000plusmn471

Inhibitio

nzonesa

remeanof

threereplications

andshow

ingsta

ndarddeviation

Inhibitio

nzone

was

measuredas

radius

from

theou

tere

dgeof

wellAlltheexperim

entswerecond

uctedat28plusmn2∘C

+indicates

presence

oftraitminusindicatesabsence

oftraitinplateb

ased

assaysaLevelsofAC

Cdeam

inasea

ctivitiesbasedon

grow

thon

plateb

ased

assayd

enoted

as+forlessg

rowth+++

form

oderateg

rowth+++

forlux

uriant

grow

thand

++++

forh

ighlyluxu

riant

grow

thbIAAwas

estim

ated

incultu

refiltra

teandexpressedas120583gmLusinganalyticalgradeIAAas

stand

ardvalues

indicatemeanandsta

ndarddeviation

International Journal of Microbiology 9

2545

2545

364

364

1273 2909

ActinobacteriaFirmicutesAlpha proteobacteria

Beta proteobacteriaGamma proteobacteriaBacteroidetes

(a)

Num

ber o

f XRB

Actin

obac

teria

Firm

icut

es

Alp

ha p

rote

obac

teria

Beta

pro

teob

acte

ria

Gam

ma p

rote

obac

teria

Bact

eroi

dete

s

Number of antagonistic XRB in each phylumsubdivision

Phylasubdivision

Total number of XRB belonging to each phylumsubdivision

25

20

15

10

5

0

2

14

9

16

4

7

1

2

1

2

11

14

(b)

Figure 2 (a) Distribution of XRB (phylumsubdivision level) identified by 16S rRNA gene sequencing Values indicate percentages of strainsbelonging to each phylasubdivision amongst the 55 identified strains (b) Distribution of antagonistic and nonantagonistic strains of XRB ineach phylumsubdivision

different genera of bacteria were identified Major gen-era identified are Bacillus sp (11 strains) Enterobacter sp(6 strains) Microbacterium sp (5 strains) Staphylococcussp (5 strains) Pseudomonas sp (5 strains) and Agrobac-terium sp (3 strains) Additional genera identified includ-ing Micrococcus sp Sphingomonas sp Flavobacterium spChryseobacterium sp Burkholderia sp and Xenophilus spare listed in Table 4 Based on the identification phylumProteobacteria consisting of Gram-negative bacteria of sub-divisions Alpha Proteobacteria (1273) Beta Proteobacteria(364) and Gamma Proteobacteria (2545) were predom-inant (4181 strains identified) followed by phyla Firmi-cutes (2909) Actinobacteria (2545) and Bacteroidetes(364) (Figure 2(a))

Eleven antagonistic strains identified belonged toGammasubdivision of Proteobacteria consisting of five strainseachof Enterobacter sp and fluorescent and nonfluorescent Pseu-domonas sp and one strain of Pantoea eucrina (XB126)(Figure 2(b)) Nine antagonists identified were of phylaFirmicutes consisting of Staphylococcus sp (5 strains) andBacillus sp (4 strains) Agrobacterium strains XB1 XB86 andXB165 Sphingomonas sp (XB197) of the Alpha Proteobacte-ria Streptomyces sp (XB200) and Janibacter melonis (XB70)of phyla Actinobacteria were found to be antagonistic Addi-tional antagonistic XRB include Burkholderia sp (XB140) of120573 Proteobacteria and Flavobacterium sp (XB203) of phylaBacteroidetes (Figure 2(b))

37 Statistical Analysis The statistical analysis of percent-age wilts and shoot length of eggplant was performedusing Web Agri Statistical Package (WASP) version 20(httpwwwicargoaresinwasp20indexphp)

4 Discussion

Eggplant and chilli not only are of economic and culturalimportance but also are common ingredients in the cuisinethroughout India In the coastal state of GoaR solanacearumhas been reported to be a destructive pathogen in cultivationof eggplant and chilli [4] Isolation of biocontrol agentsagainst the BW pathogen has been commonly restricted toendophytic tissue and plant rhizosphere [26 27] Studieson xylem colonizing endophytes were undertaken becausewe speculated existence of interactions between the XRBand vascular wilt pathogen R solanacearum during xylemcolonization Our study reveals the diversity biocontrolpotential and identity of endophytic xylem colonizers fromsolanaceous crops cultivated in Goa India A total of 167bacteria were isolated from the xylem of eggplant chilliand S torvum with Gram-negative bacteria (5928) pre-dominating in the collection Congruent to our observationGardner et al [7] and Bell et al [11] have earlier reportedisolation of more number of Gram-negative rod shapedbacteria from xylem of citrus and grapevine using vacuuminfiltration Scholander pressure bomb was found to beuseful in extraction of diverse bacterial genera from xylemtissues in contrast to trituration methods that yielded highernumber of Gram-positive rod shaped bacteria [12] ThoughScholander pressure bomb was not used in this study acombination of vacuum infiltration [7 11] and triturationof decorticated stems [10 12] was employed with an aimto isolate diverse XRB from xylem tissues This is the firststudy reporting the use of vacuum infiltration and triturationmethods for isolating xylem residing bacteria from eggplantchilli and S torvum

Traditionally bacteria have been characterized andgrouped based on colony morphology and biochemical

10 International Journal of Microbiology

Table 4 List of XRB identified by partial 16S rRNAgene sequencing their plant host accession numbers closestNCBImatch and similarity

Strain Plant host Accession number Closest NCBI match similarityXB1lowast SE KF447383 Agrobacterium tumefaciens 99XB7lowast SE KF447384 Pseudomonas aeruginosa 99XB8lowast SE KF447385 Staphylococcus haemolyticus 99XB20lowast SE KF447386 Staphylococcus haemolyticus 100XB22 SE KF447387 Brevibacterium casei 99XB25 C KF447388 Enterobacter sp 95XB27lowast SE KF447389 Staphylococcus haemolyticus 99XB34 SE KF447390 Curtobacterium sp 99XB35 SE KF447391 Microbacterium sp 99XB36 SE KF447392 Xenophilus sp 99XB37 SE KF447393 Chryseobacterium sp 99XB40 SE KF447394 Micrococcus luteus 89XB41 SE KF447395 Bacillus sp 99XB47 SE KF447396 Bacillus sp 99XB53 SE KF447397 Micrococcus sp 99XB62lowast SE KF447398 Pseudomonas sp 99XB64 SE KF447399 Bacillus thuringiensis 100XB66 SE KF447400 Pectobacterium carotovorum 99XB70lowast SE KF447401 Janibacter melonis 99XB86lowast C KF447402 Agrobacterium tumefaciens 99XB87 C KF447403 Bacillus barbaricus 99XB88 C KF447404 Bacillus sp 99XB92 C KF447405 Brevundimonas vesicularis 99XB93lowast C KF447406 Bacillus safensis 100XB94 C KF447407 Bacillus sp 100XB98 C KF447408 Microbacterium sp 100XB99lowast SE KF447409 Enterobacter sp 99XB100lowast SE KF447410 Enterobacter cloacae 98XB103 SE KF447411 Brachybacterium phenoliresistens 99XB109 SE KF447412 Rhodococcus corynebacterioides 99XB114lowast SE KF447413 Pseudomonas stutzeri 99XB122lowast SE KF913446 Pseudomonas aeruginosa 100XB123lowast SE KF447414 Enterobacter sp 99XB126lowast C KF447415 Pantoea eucrina 99XB134lowast SE KF447416 Enterobacter sp 99XB137 SE KF447417 Klebsiella sp 99XB140lowast SE KF447418 Burkholderia sp 99XB153lowast SE KF447419 Bacillus amyloliquefaciens 99XB157lowast SE KF447420 Bacillus subtilis 100XB158 RE KF447421 Bosea sp 99XB159 RE KF447422 Microbacterium xylanilyticum 99XB161 RE KF447423 Microbacterium aurum 99XB165lowast RE KF447424 Agrobacterium sp 99XB167 RE KF447425 Microbacterium aurum 99XB168 RE KF447426 Bacillus aryabhattai 100XB169lowast RE KF447427 Staphylococcus gallinarum 99XB170lowast RE KF447428 Staphylococcus sp 99XB177lowast RE KF447429 Bacillus cereus 99XB188 RE KF447430 Sphingomonas sp 99XB190 RE KF447431 Brevibacterium casei 99

International Journal of Microbiology 11

Table 4 Continued

Strain Plant host Accession number Closest NCBI match similarityXB196lowast RE KF447432 Enterobacter kobei 99XB197lowast RE KF447433 Sphingomonas sp 98XB200lowast ST KF913447 Streptomyces sp 100XB202lowast ST KF447434 Pseudomonas sp 99XB203lowast ST KF447435 Flavobacterium sp 99lowastStrain is antagonistic to R solanacearum based on bioassaySE bacterial wilt susceptible eggplant RE bacterial wilt resistant eggplant C chilli ST Solanum torvum

tests However whole genome fingerprinting or PCR-RFLPbased methods are rapid tools for determining geneticdiversity of bacteria in a given collection ARDRA which isa type of PCR based RFLP method has been used widely inestimating genetic diversity endophytic bacterial populationsand clustering genetically similar strains [39ndash41] In additionto earlier reports our study demonstrates the usefulnessof ARDRA as a tool to cluster genetically identical strainsof endophytic XRB isolated from eggplant chilli andS torvum In our study 9161 strains (119899 = 153) weregrouped in 24 haplotypes by using ARDRA The majority ofthese haplotypes represent a combination of XRB isolatedfrom different solanaceous plants from diverse localesThese results indicate that xylem of eggplant chilli andS torvum largely bears similar population of XRB whichcan efficiently cross colonize eggplant chilli or S torvumNevertheless 839 strains had unique ARDRA fingerprintand formed separable haplotypes This observation leads toa conclusion that a minor population of xylem inhabitantsare restricted to a specific plant species and cannot easilycross colonize xylem of other solanaceous plants Plantsare known to selectively support endophytic colonizationby specific bacteria [14] However factors that determinethe selection of xylem colonists or the ability of XRBto colonize eggplant chilli and S torvum in this studyremain unknown Interestingly the structure of endophyticcommunity in Nicotiana attenuata a member of Solanaceaefamily is shown to be influenced by soil composition andethylene homeostasis [40] Earlier evidence has shown thatcolonization by endophytic bacteria is also governed by plantgenotype as well as root exudates [42]

Only 1677 XRB out of 167 were antagonistic to Rsolanacearum based on in vitro assays Antagonistic XRBproduced volatile and diffusible inhibitory compoundsnamely HCN ammonia and acetoin and siderophoresThese substances have been long known to be involvedin disease suppression and indirect growth promotion inplants [43ndash46] These mechanisms possibly played a rolein the evident biocontrol effect against BW exhibited bythe XRB in the greenhouse screening Endophytic bacteriahave been known to have plant growth promoting traitsnamely production of IAA ACC deaminase and phosphatesolubilization [25 33 36] These traits were detected in themajority of antagonistic XRB tested in this study and mayhave resulted in the observed increase in shoot length ofeggplant in our greenhouse experiments In contrast strains

XB20 XB196 and XB203 suppressed growth in eggplantunder greenhouse conditions however no visible symptomsof disease were observed Vascular plugging and productionof certainmetabolites toxic to plant cells but not cell viabilitymay have resulted in stunted shoot in eggplant [11 47]

Evaluation of efficacy of antagonistic organisms to sup-press the plant diseases under greenhouse conditions isone of the key steps for selecting a potential biocontrolagent for disease management [48] Endophytic strains fromBW susceptible varieties of eggplant have been shown toprevent wilt and promote growth in eggplant earlier [27]Our greenhouse screening shows that 3846 of antagonisticXRB with biocontrol efficacies greater than 40 were isolatesfrom BW resistant varieties of eggplantThis raises a questionwhether the bacteria from resistant varieties are involved inBW resistance and whether BW resistant varieties are ableto selectively influence xylem colonization by antagonisticbacteria Presence of higher number of endophytes withantagonistic abilities was reported in BW resistant varieties oftomato as compared to susceptible varieties and their role inresistance to BWwas proposed [49] Similar observations oncorrelation of resistance of potato to soft rot and endophyticbacteria have been reported [50]Thus BW resistant varietiescan be considered a better host for isolating potential biocon-trol strains for management of bacterial wilt

Identification of 55 XRB strains by 16S rRNA genesequencing revealed the presence of 23 diverse genera ofbacteria belonging to 4 phyla of Eubacteria Strains belongingto phyla Firmicutes Actinobacteria and 120574 subdivision ofProteobacteria were the major xylem colonists identified inthis study Several genera of bacteria belonging to these phylahave also been reported to be present in endophytic tissuesand xylem of a variety of other agricultural and horticulturalplant species [14 51] In addition the majority of the antag-onists identified belonged to Enterobacter sp Pseudomonassp Bacillus sp and Staphylococcus sp Congruent to ourresults several researchers have reported bacteria isolatedfrom solanaceous crops and belonging to similar genera tobe antagonistic to R solanacearum [26 51ndash53] HoweverFlavobacterium spand Janibacter melonis identified in thisstudy have never been previously reported to be inhibitoryto R solanacearum Large population of the xylem inhabitingbacterial flora accounting for 8323 exhibited no antago-nism towards R solanacearum Nonantagonistic XRB wereidentified predominantly as Microbacterium sp Endophyticpersistence and nematicidal activities of Microbacterium sp

12 International Journal of Microbiology

have been reported [54 55] Therefore the collection of XRBisolated in this study can be screened for inhibitory activitiesagainst other important agricultural pests

Though few strains namely XB86 XB169 and XB177exhibited plant beneficial properties in this study their use-fulness in plant disease control remains to be seen XB86has been identified as Agrobacterium tumefaciens the crowngall disease pathogen and its deployment as biocontrolagent is uncertain XB169 (Staphylococcus gallinarum) andXB177 (Bacillus cereus) are reported as opportunistic animalpathogens and thus unsuitable for field applications Strep-tomyces sp has earlier been reported as antagonistic to Rsolanacearum and tested for management of wilt in potatoand tomato [56 57] XB200 (Streptomyces sp) is one of theXRB high BCE and GPE it could be explored further forbiocontrol of bacterial wilt after additional characterizationand field evaluation

5 Conclusion

This study is the first report on the identity of novel anddiverse XRB colonizing the xylem of eggplant chilli and Storvum XRB particularly from BW resistant varieties werefound to protect eggplant from bacterial wilt and enhancedgrowth in eggplant in the greenhouse screening Thereforethe repertoire of XRB reported in this study may be usefulfor cultivation of eggplant in BW affected areas

Conflict of Interests

The authors hereby declare that they have no conflict ofinterests regarding the publication of this paper

Acknowledgments

The authors greatly acknowledge financial support fromIndian Council of Agricultural Research (ICAR) New DelhiIndia through ldquoOutreach project on Phytopthora Fusariumand Ralstonia diseases of horticultural and field cropsrdquo-(PhytoFuRa) and the Director ICAR Research Complex forGoa India for the facilities

References

[1] E Yabuuchi Y KosakoHOyaizu et al ldquoProposal ofBurkholde-ria gen nov and transfer of seven species of the genusPseudomonas homology group II to the new genus with thetype species Burkholderia cepacia (Palleroni and Holmes 1981)comb novrdquo Microbiology and Immunology vol 36 no 12 pp1251ndash1275 1992

[2] E Wicker L Grassart R Coranson-Beaudu et al ldquoRalstoniasolanacearum strains from Martinique (French West Indies)exhibiting a new pathogenic potentialrdquo Applied and Environ-mental Microbiology vol 73 no 21 pp 6790ndash6801 2007

[3] Y Liu A Kanda K Yano et al ldquoMolecular typing of Japanesestrains of Ralstonia solanacearum in relation to the ability toinduce a hypersensitive reaction in tobaccordquo Journal of GeneralPlant Pathology vol 75 no 5 pp 369ndash380 2009

[4] R Ramesh ldquoField evaluation of biological control agents for themanagement of Ralstonia solanacearum in Brinjalrdquo Journal ofMycology and Plant Pathology vol 36 no 2 pp 327ndash328 2006

[5] J Vasse P Frey and A Trigalet ldquoMicroscopic studies ofintercellular infection and protoxylem invasion of tomatoroots by Pseudomonas solanacearumrdquoMolecular Plant-MicrobeInteractions vol 8 no 2 pp 241ndash251 1995

[6] S Genin and C Boucher ldquoRalstonia solanacearum secrets of amajor pathogen unveiled by analysis of its genomerdquo MolecularPlant Pathology vol 3 no 3 pp 111ndash118 2002

[7] J M Gardner A W Feldman and R M Zablotowicz ldquoIdentityand behavior of xylem-residing bacteria in rough lemon roots ofFlorida citrus treesrdquo Applied and Environmental Microbiologyvol 43 no 6 pp 1335ndash1342 1982

[8] M J Jacobs W M Bugbee and D A Gabriellson ldquoEnu-meration location and characterization of endophytic bacteriawithin sugar beet rootsrdquoCanadian Journal of Botany vol 63 no7 pp 1262ndash1265 1985

[9] D G Patriquin and J Dobereiner ldquoLight microscopy observa-tions of tetrazolium-reducing bacteria in the endorhizosphereof maize and other grasses in Brazilrdquo Canadian Journal ofMicrobiology vol 24 no 6 pp 734ndash742 1978

[10] S Gagne C Richard H Rousseau and H Antoun ldquoXylem-residing bacteria in alfalfa rootsrdquo Canadian Journal of Micro-biology vol 33 no 11 pp 996ndash1000 1987

[11] C R Bell G A Dickie W L G Harvey and J W Y FChan ldquoEndophytic bacteria in grapevinerdquo Canadian Journal ofMicrobiology vol 41 no 1 pp 46ndash53 1995

[12] J Hallmann J W Kloepper and R Rodrıguez-Kabana ldquoAppli-cation of Scholander pressure bomb to studies on endophyticbacteria of plantsrdquo Canadian Journal of Microbiology vol 43no 5 pp 411ndash416 1997

[13] J S Lampel G L Canter M B Dimock et al ldquoIntegrativecloning expression and stability of the cryIA(c) gene fromBacillus thuringiensis subsp kurstaki in a recombinant strain ofClavibacter xyli subsp cynodontisrdquo Applied and EnvironmentalMicrobiology vol 60 no 2 pp 501ndash508 1994

[14] M Rosenblueth and E Martınez-Romero ldquoBacterial endo-phytes and their interactions with hostsrdquo Molecular Plant-Microbe Interactions vol 19 no 8 pp 827ndash837 2006

[15] P R Hardoim L S van Overbeek and J D van ElsasldquoProperties of bacterial endophytes and their proposed role inplant growthrdquo Trends in Microbiology vol 16 no 10 pp 463ndash471 2008

[16] R Aravind A Kumar S J Eapen and K V RamanaldquoEndophytic bacterial flora in root and stem tissues of blackpepper (Piper nigrum L) genotype isolation identificationand evaluation against Phytophthora capsicirdquo Letters in AppliedMicrobiology vol 48 no 1 pp 58ndash64 2009

[17] J Kuklinsky-Sobral W L Araujo R Mendes I O Geraldi AA Pizzirani-Kleiner and J L Azevedo ldquoIsolation and charac-terization of soybean-associated bacteria and their potential forplant growth promotionrdquo Environmental Microbiology vol 6no 12 pp 1244ndash1251 2004

[18] S Compant B Duffy J Nowak C Clement and E A BarkaldquoUse of plant growth-promoting bacteria for biocontrol ofplant diseases principles mechanisms of action and futureprospectsrdquoApplied and Environmental Microbiology vol 71 no9 pp 4951ndash4959 2005

[19] E T Thorne B M Young G M Young et al ldquoThe structureof xylem vessels in grapevine (Vitaceae) and a possible passive

International Journal of Microbiology 13

mechanism for the systemic spread of bacterial diseaserdquo Amer-ican Journal of Botany vol 93 no 4 pp 497ndash504 2006

[20] W T Seo W J Lim E J Kim H D Yun Y H Lee and KM Cho ldquoEndophytic bacterial diversity in the young radishand their antimicrobial activity against pathogensrdquo Journal ofApplied Biological Chemistry vol 53 no 4 pp 493ndash503 2010

[21] S Compant H Kaplan A Sessitsch J Nowak E Ait Barkaand C Clement ldquoEndophytic colonization ofVitis vinifera L byBurkholderia phytofirmans strain PsJN from the rhizosphere toinflorescence tissuesrdquo FEMS Microbiology Ecology vol 63 no1 pp 84ndash93 2005

[22] P S B Miguel J C Delvaux M N V de Oliveira et al ldquoDiver-sity of endophytic bacteria in the fruits of Coffea canephorardquoAfrican Journal of Microbiology Research vol 7 no 7 pp 586ndash594 2013

[23] A Lopez-Lopez M A Rogel E Ormeno-Orrillo J Martınez-Romero and E Martınez-Romero ldquoPhaseolus vulgaris seed-borne endophytic community with novel bacterial species suchas Rhizobium endophyticum sp novrdquo Systematic and AppliedMicrobiology vol 33 no 6 pp 322ndash327 2010

[24] M Lwin and S L Ranamukhaarachchi ldquoDevelopment of bio-logical control of Ralstonia solanacearum through antagonisticmicrobial populationsrdquo International Journal of Agriculture andBiology vol 8 no 5 pp 657ndash660 2006

[25] F Rasche E Marco-Noales H Velvis et al ldquoStructural char-acteristics and plant-beneficial effects of bacteria colonizing theshoots of field grown conventional and geneticallymodifiedT4-lysozyme producing potatoesrdquo Plant and Soil vol 289 no 1-2pp 123ndash140 2006

[26] N Amaresan V Jayakumar K Kumar and N ThajuddinldquoEndophytic bacteria from tomato and chilli their diversity andantagonistic potential against Ralstonia solanacearumrdquoArchivesof Phytopathology and Plant Protection vol 45 no 3 pp 344ndash355 2012

[27] R Ramesh and G S Phadke ldquoRhizosphere and endophyticbacteria for the suppression of eggplant wilt caused byRalstoniasolanacearumrdquo Crop Protection vol 37 pp 35ndash41 2012

[28] W J French R G Christis and D L Stassi ldquoRecovery ofrickettsialike bacteria by vacuum infiltration of peach tissuesaffected with phony diseaserdquo Phytopathology vol 67 pp 945ndash948 1977

[29] P R Viss E M Brooks and J A Driver ldquoA simplified methodfor the control of bacterial contamination in woody plant tissueculturerdquo In Vitro Cellular andDevelopmental Biology vol 27 no1 article 42 1991

[30] K Wilson ldquoPreparation of genomic DNA from bacteriardquo inCurrent Protocols in Molecular Biology F M Ausubel R BrentR E Kingston et al Eds pp 241ndash245 John Wiley amp SonsNew York NY USA 1997

[31] M Saraf AThakkar U PandyaM Joshi and J Parikh ldquoPoten-tial of plant growth promoting microorganisms as biofertilizersand biopesticides and itrsquos exploitation in sustainable agricul-turerdquo Journal of Microbiology and Biotechnology Research vol3 no 5 pp 54ndash62 2013

[32] A P G C Marques C Pires H Moreira A O S S Rangeland P M L Castro ldquoAssessment of the plant growth promotionabilities of six bacterial isolates using Zea mays as indicatorplantrdquo Soil Biology and Biochemistry vol 42 no 8 pp 1229ndash1235 2010

[33] X Hao P Xie L Johnstone S J Miller C Rensingand G Wei ldquoGenome sequence and mutational analysis of

plant-growth-promoting bacteriumAgrobacterium tumefaciensCCNWGS0286 isolated from a zinc-lead mine tailingrdquo Appliedand Environmental Microbiology vol 78 no 15 pp 5384ndash53942012

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

[35] S A Gordon and L G Paleg ldquoQuantitative measurement ofindole acetic acidrdquo Plant Physiology vol 10 pp 37ndash48 1957

[36] A Godinho R Ramesh and S Bhosle ldquoBacteria from sanddunes of Goa promoting growth in eggplantrdquoWorld Journal ofAgricultural Sciences vol 6 no 5 pp 555ndash564 2010

[37] J-H Guo H-Y Qi Y-H Guo et al ldquoBiocontrol of tomato wiltby plant growth-promoting rhizobacteriardquo Biological Controlvol 29 no 1 pp 66ndash72 2004

[38] N Aliye C Fininsa and Y Hiskias ldquoEvaluation of rhizo-sphere bacterial antagonists for their potential to bioprotectpotato (Solanum tuberosum) against bacterial wilt (Ralstoniasolanacearum)rdquo Biological Control vol 47 no 3 pp 282ndash2882008

[39] L LagaceM PitreM Jacqeus andDRoy ldquoIdentification of thebacterial community ofmaple sap by using amplified ribosomalDNA (rDNA) restriction analysis and rDNA sequencingrdquoApplied and Environmental Microbiology vol 70 no 4 pp2052ndash2060 2004

[40] H H Long D G Sonntag D D Schmidt and I T BaldwinldquoThe structure of the culturable root bacterial endophyte com-munity of Nicotiana attenuata is organized by soil compositionand host plant ethylene production and perceptionrdquo NewPhytologist vol 185 no 2 pp 554ndash567 2010

[41] E Lopez-Fuentes V M Ruız-Valdiviezo E Martınez-RomeroF A Gutierrez-Miceli L Dendooven and R Rincon-RosalesldquoBacterial community in the roots and rhizosphere of Hyper-icum silenoides Juss 1804rdquo African Journal of MicrobiologyResearch vol 6 no 11 pp 2704ndash2711 2012

[42] M Fang R J Kremer P P Motavalli and G Davis ldquoBacterialdiversity in rhizospheres of nontransgenic and transgenic cornrdquoApplied and EnvironmentalMicrobiology vol 71 no 7 pp 4132ndash4136 2005

[43] C R Howell and R D Stiphanovic ldquoProduction of ammonia byEnterobacter cloaceae and its possible role in biological controlof Pythium pre-emergence damping-off by the bacteriumrdquoEcology and Epidemiology vol 78 no 8 pp 1075ndash1078 1988

[44] A Ramette M Frapolli G Defago and Y Moenne-LoccozldquoPhylogeny of HCN synthase-encoding hcnBC genes in bio-control fluorescent pseudomonads and its relationship withhost plant species and HCN synthesis abilityrdquoMolecular Plant-Microbe Interactions vol 16 no 6 pp 525ndash535 2003

[45] I Loaces L Ferrando and A F Scavino ldquoDynamics diversityand function of endophytic siderophore-producing bacteria inricerdquoMicrobial Ecology vol 61 no 3 pp 606ndash618 2011

[46] Y C Kim J Leveau B B M Gardener E A Pierson L SPierson and C-M Ryu ldquoThe multifactorial basis for planthealth promotion by plant-associated bacteriardquo Applied andEnvironmental Microbiology vol 77 no 5 pp 1548ndash1555 2011

[47] J M Gardner J L Chandler and A W Feldman ldquoGrowthresponses and vascular plugging of citrus inoculated withrhizobacteria and xylem-resident bacteriardquo Plant and Soil vol86 no 3 pp 333ndash345 1985

[48] C Pliego C Ramos A de Vicente and F M Cazorla ldquoScreen-ing for candidate bacterial biocontrol agents against soilborne

14 International Journal of Microbiology

fungal plant pathogensrdquoPlant and Soil vol 340 no 1-2 pp 505ndash520 2011

[49] H Feng Y Li and Q Liu ldquoEndophytic bacterial communi-ties in tomato plants with differential resistance to Ralstoniasolanacearumrdquo African Journal of Microbiology Research vol 7no 15 pp 1311ndash1318 2013

[50] A V Sturz B R Christie B G Matheson W J Arsenaultand N A Buchanan ldquoEndophytic bacterial communities inthe periderm of potato tubers and their potential to improveresistance to soil-borne plant pathogensrdquo Plant Pathology vol48 no 3 pp 360ndash369 1999

[51] A V Sturz B R Christie and J Nowak ldquoBacterial endophytespotential role in developing sustainable systems of crop produc-tionrdquo Critical Reviews in Plant Sciences vol 19 no 1 pp 1ndash302000

[52] R Ramesh A A Joshi and M P Ghanekar ldquoPseudomonadsmajor antagonistic endophytic bacteria to suppress bacterialwilt pathogenRalstonia solanacearum in the eggplant (Solanummelongena L)rdquo World Journal of Microbiology and Biotechnol-ogy vol 25 no 1 pp 47ndash55 2009

[53] A A Nawangsih I Damayanti S Wiyono and J G KartikaldquoSelection and characterization of endophytic bacteria as bio-control agents of tomato Bacterial Wilt DiseaserdquoHayati Journalof Biosciences vol 18 no 2 pp 66ndash70 2011

[54] R Vargas-Ayala R Rodrıguez-Kabana G Morgan-Jones J AMcInroy and J W Kloepper ldquoShifts in soil microflora inducedby velvetbean (Mucuna deeringiana) in cropping systems tocontrol root-knot nematodesrdquo Biological Control vol 17 no 1pp 11ndash22 2000

[55] D K Zinniel P Lambrecht N B Harris et al ldquoIsolationand characterization of endophytic colonizing bacteria fromagronomic crops and prairie plantsrdquoApplied and EnvironmentalMicrobiology vol 68 no 5 pp 2198ndash2208 2002

[56] F Lemessa and W Zeller ldquoScreening rhizobacteria for biolog-ical control of Ralstonia solanacearum in Ethiopiardquo BiologicalControl vol 42 no 3 pp 336ndash344 2007

[57] M S El-AlbyadM A El-Sayed A R El-Shanshoury andNHEl-Batanouny ldquoEffect of culture conditions on the antimicrobialactivities of UV-mutants of Streptomyces corchorusii and Sspiroverticillatus against bean and banana wilt pathogensrdquoMicrobiological Research vol 151 no 2 pp 201ndash211 1996

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

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BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

International Journal of Microbiology 7

solanacearum strain Rs-09-100 Results of the in vitro screen-ing against R solanacearum revealed that 28 amongst 167XRB exhibited antagonism towards the pathogen (Table 3)Amongst the antagonists 16 were strains from BW suscepti-ble eggplant 6 from BW resistant eggplant and 3 each fromchilli and S torvum (Table 1) Amongst the 28 antagonists 7strains namely XB62 XB99 XB100 XB114 XB122 XB196XB197 and XB202 formed larger inhibition zones againstR solanacearum ranging from 40mm to 817mm (Table 3)The majority of the antagonistic strains (119899 = 16) producedinhibition zones ranging from 20mm to 383mm XB27XB134 XB165 and XB169 formed smaller inhibition zonesranging from 15mm to 20mm However 139 strains ofXRB did not inhibit R solanacearum in the bioassay testTwenty-eight antagonistic XRBwere screened for productionof volatile inhibitory compounds namely acetoin HCNammonia and diffusible siderophore molecules in vitro(Table 3) Acetoin production was observed in 3214 of theisolates Bacterial isolates XB7 and XB122 were found toproduce both HCN as well as siderophores XB62 XB93 andXB170 produced HCN whereas XB114 XB140 and XB203produced siderophores only XB93 XB99 XB123 XB134 andXB140 produced ammonia

34 Production of Plant Growth Promoting Substances byAntagonistic XRB Results of the screening of antagonisticXRB for in vitro production of several plant growth pro-moting compounds is presented in Table 3 Majority of theantagonistic strains produced the phytohormone IAA withconcentrations ranging from 1591 120583gmL to 64591120583gmLXB202 was found to be the best ACC deaminase producingstrain based on its luxuriant growth on DF salts mediumsupplemented with 30mM ACC as sole nitrogen sourceOther ACC deaminase producing strains include XB1 XB62XB86 and XB140 Scarce growth of XB165 and XB200 wasobserved onDF saltsmedium 6428of the strains producedphosphate solubilizing organic acids as indicated by clearhaloes on Pikovskayarsquos agar plate

35 Greenhouse Experiments

351 Suppression of Bacterial Wilt by Antagonistic XRBAbility to suppress BW was assessed as the difference in thepercentage of wilt in XRB treated plants with respect to wilt inuntreated control andwas expressed as the biocontrol efficacy(BCE) of the antagonists BCE of 16 strains with valuesranging from 286 to 100 is presented in Figure 1 Plantstreated with strains XB86 XB169 and XB177 were free fromBWand hence recorded 100 biocontrol efficacy Treatmentswith XB170 XB197 XB200 XB202 and XB203 recorded 30percent or less wilt incidence (70 to 85 BCE) XB1 XB27XB70 XB93 and XB123 treatments recorded BCE between429 and 571 BCE of 286 was recorded in XB20 andXB165 treatments However 12 antagonistic XRB recordedBCEof 25 andwere least effective inwilt protection Furtherit is observed that all the antagonistic strains originating fromBW resistant eggplant and S torvum exhibited BCE greaterthan 25 Five antagonistic strains from BW susceptible

minus2000

000

2000

4000

6000

8000

10000

12000

XB1

XB20

XB27

XB70

XB86

XB93

XB123

XB165

XB169

XB170

XB177

XB196

XB197

XB200

XB202

XB203

Con

trol

Isolates

Biocontrol efficacy (BCE)Growth promotion efficacy (GPE)

BCE

and

GPE

()

Figure 1 Biocontrol and plant growth promotion efficacies of selectXRB in eggplant BCE biocontrol efficacy determined 25 days afterchallenging with R solanacearum GPE growth promotion efficacydetermined 40 days after treatmentwithXRB Percent BCE andGPEcalculated using formulae BCE = ([percent disease in control] minus[percent disease in treatment]percent disease in control) times 100 andGPE= ([shoot length increase in treatment]minus [shoot length increasein control]shoot length increase in control) times 100 respectivelyUninoculated control had BCE and GPE values of 000 XB86XB169 and XB177 had BCE of 10000 in all the replicationsBars indicate mean values of BCE and GPE error bars indicatestandard deviation

eggplant and three fromchilli were effective in preventingwiltin eggplant (Table 1)

352 Growth Promotion by Antagonistic XRB Increase inshoot length of eggplant (40 days after treatment) observedin XRB treated plants in relation to untreated control wasexpressed as growth promotion efficacy (GPE) and is shownin Figure 1 Amongst the 16 strains which were effective inpreventing wilt six strains exhibited the highest increase inshoot length as indicated by their GPE values in the rangeof 139ndash223 Seven XRB recorded a GPE value rangingfrom 14 to 129 However strains XB27 XB196 and XB203stunted shoot growth in eggplant in comparison to untreatedcontrol When the source of XRB is considered 5555strains that exhibited GPE greater than 10 were isolatedfrom BW resistant eggplant Whereas only two antagonistsfrom BW susceptible plant and one each from chilli and Storvum were able to promote growth in eggplant (Table 1)Strains with GPE gt 10 belonged to six different ARDRAhaplotypes (Table 2)

36 Identification of XRB by 16S rRNA Gene Sequencing16S rRNA gene sequences of XRB were used to identify thediverse xylem inhabitants and antagonistic strains Identityof 55 XRB based on 16S rRNA gene sequencing and theirGenBank accessions are presented in Table 4 Overall 23

8 International Journal of Microbiology

Table3

Invitro

inhibitio

nof

Rsolana

cearum

andprod

uctio

nof

antagonisticc

ompo

unds

andplantg

rowth

prom

otingsubstances

byXR

B

Strain

Haplotype

Inhibitio

nof

Rsolana

cearum

Volatile

anddiffu

sibleinhibitory

compo

unds

Plantgrowth

prom

otingsubstances

Radius

inmm

HCN

Ammon

iaAc

etoin

Sideroph

ore

Phosph

ates

olub

ilisatio

nAC

Cdeam

inasea

IAA

b120583gmL

XB1

M80-7

283plusmn029

minusminus

minusminus

minus++

+10500plusmn707

XB7

M80-27

333plusmn058

+minus

minus+

+minus

4773plusmn321

XB8

M80-20

333plusmn029

minusminus

minusminus

+minus

1909plusmn129

XB20

M80-20

327plusmn040

minusminus

minusminus

+minus

2545plusmn171

XB27

M80-20

183plusmn029

minusminus

minusminus

+minus

1591plusmn107

XB62

M80-31

817plusmn076

+minus

minusminus

+++

++7318plusmn493

XB70

M80-15

383plusmn058

minusminus

minusminus

minusminus

17182plusmn1157

XB86

M80-7

233plusmn076

minusminus

minusminus

minus++

+6682plusmn450

XB93

M80-6

317plusmn029

+minus

+minus

+minus

5727plusmn386

XB99

M80-9

683plusmn058

minus+

+minus

+minus

32136plusmn2164

XB100

M80-9

450plusmn050

minus+

+minus

+minus

23864plusmn1607

XB114

M80-31

650plusmn050

minusminus

minus+

+minus

8909plusmn600

XB122

M80-28

433plusmn058

+minus

minus+

+minus

6682plusmn450

XB123

M80-31

300plusmn087

minus+

+minus

+minus

41682plusmn2807

XB126

M80-27

283plusmn029

minusminus

+minus

+minus

6045plusmn407

XB134

M80-37

157plusmn012

minus+

+minus

+minus

64591plusmn4350

XB140

M80-5

367plusmn076

minus+

minus+

+++

++5091plusmn343

XB153

M80-6

317plusmn029

minusminus

+minus

+minus

15591plusmn1050

XB157

M80-6

250plusmn100

minusminus

+minus

+minus

18455plusmn1243

XB165

M80-29

157plusmn012

minusminus

minusminus

minus+

19091plusmn1286

XB169

M80-6

153plusmn006

minusminus

minusminus

minusminus

1591plusmn107

XB170

M80-6

351plusmn002

+minus

minusminus

+minus

2864plusmn193

XB177

M80-6

199plusmn049

minusminus

minusminus

minusminus

7636plusmn514

XB196

M80-10

393plusmn012

minusminus

minusminus

minusminus

3500plusmn236

XB197

M80-38

433plusmn029

minusminus

+minus

minusminus

4136plusmn279

XB200

M80-36

187plusmn023

minusminus

minusminus

minus++

16864plusmn1136

XB202

M80-31

467plusmn021

minusminus

minusminus

+++

+8273plusmn557

XB203

M80-31

367plusmn029

minusminus

minusminus

minusminus

7000plusmn471

Inhibitio

nzonesa

remeanof

threereplications

andshow

ingsta

ndarddeviation

Inhibitio

nzone

was

measuredas

radius

from

theou

tere

dgeof

wellAlltheexperim

entswerecond

uctedat28plusmn2∘C

+indicates

presence

oftraitminusindicatesabsence

oftraitinplateb

ased

assaysaLevelsofAC

Cdeam

inasea

ctivitiesbasedon

grow

thon

plateb

ased

assayd

enoted

as+forlessg

rowth+++

form

oderateg

rowth+++

forlux

uriant

grow

thand

++++

forh

ighlyluxu

riant

grow

thbIAAwas

estim

ated

incultu

refiltra

teandexpressedas120583gmLusinganalyticalgradeIAAas

stand

ardvalues

indicatemeanandsta

ndarddeviation

International Journal of Microbiology 9

2545

2545

364

364

1273 2909

ActinobacteriaFirmicutesAlpha proteobacteria

Beta proteobacteriaGamma proteobacteriaBacteroidetes

(a)

Num

ber o

f XRB

Actin

obac

teria

Firm

icut

es

Alp

ha p

rote

obac

teria

Beta

pro

teob

acte

ria

Gam

ma p

rote

obac

teria

Bact

eroi

dete

s

Number of antagonistic XRB in each phylumsubdivision

Phylasubdivision

Total number of XRB belonging to each phylumsubdivision

25

20

15

10

5

0

2

14

9

16

4

7

1

2

1

2

11

14

(b)

Figure 2 (a) Distribution of XRB (phylumsubdivision level) identified by 16S rRNA gene sequencing Values indicate percentages of strainsbelonging to each phylasubdivision amongst the 55 identified strains (b) Distribution of antagonistic and nonantagonistic strains of XRB ineach phylumsubdivision

different genera of bacteria were identified Major gen-era identified are Bacillus sp (11 strains) Enterobacter sp(6 strains) Microbacterium sp (5 strains) Staphylococcussp (5 strains) Pseudomonas sp (5 strains) and Agrobac-terium sp (3 strains) Additional genera identified includ-ing Micrococcus sp Sphingomonas sp Flavobacterium spChryseobacterium sp Burkholderia sp and Xenophilus spare listed in Table 4 Based on the identification phylumProteobacteria consisting of Gram-negative bacteria of sub-divisions Alpha Proteobacteria (1273) Beta Proteobacteria(364) and Gamma Proteobacteria (2545) were predom-inant (4181 strains identified) followed by phyla Firmi-cutes (2909) Actinobacteria (2545) and Bacteroidetes(364) (Figure 2(a))

Eleven antagonistic strains identified belonged toGammasubdivision of Proteobacteria consisting of five strainseachof Enterobacter sp and fluorescent and nonfluorescent Pseu-domonas sp and one strain of Pantoea eucrina (XB126)(Figure 2(b)) Nine antagonists identified were of phylaFirmicutes consisting of Staphylococcus sp (5 strains) andBacillus sp (4 strains) Agrobacterium strains XB1 XB86 andXB165 Sphingomonas sp (XB197) of the Alpha Proteobacte-ria Streptomyces sp (XB200) and Janibacter melonis (XB70)of phyla Actinobacteria were found to be antagonistic Addi-tional antagonistic XRB include Burkholderia sp (XB140) of120573 Proteobacteria and Flavobacterium sp (XB203) of phylaBacteroidetes (Figure 2(b))

37 Statistical Analysis The statistical analysis of percent-age wilts and shoot length of eggplant was performedusing Web Agri Statistical Package (WASP) version 20(httpwwwicargoaresinwasp20indexphp)

4 Discussion

Eggplant and chilli not only are of economic and culturalimportance but also are common ingredients in the cuisinethroughout India In the coastal state of GoaR solanacearumhas been reported to be a destructive pathogen in cultivationof eggplant and chilli [4] Isolation of biocontrol agentsagainst the BW pathogen has been commonly restricted toendophytic tissue and plant rhizosphere [26 27] Studieson xylem colonizing endophytes were undertaken becausewe speculated existence of interactions between the XRBand vascular wilt pathogen R solanacearum during xylemcolonization Our study reveals the diversity biocontrolpotential and identity of endophytic xylem colonizers fromsolanaceous crops cultivated in Goa India A total of 167bacteria were isolated from the xylem of eggplant chilliand S torvum with Gram-negative bacteria (5928) pre-dominating in the collection Congruent to our observationGardner et al [7] and Bell et al [11] have earlier reportedisolation of more number of Gram-negative rod shapedbacteria from xylem of citrus and grapevine using vacuuminfiltration Scholander pressure bomb was found to beuseful in extraction of diverse bacterial genera from xylemtissues in contrast to trituration methods that yielded highernumber of Gram-positive rod shaped bacteria [12] ThoughScholander pressure bomb was not used in this study acombination of vacuum infiltration [7 11] and triturationof decorticated stems [10 12] was employed with an aimto isolate diverse XRB from xylem tissues This is the firststudy reporting the use of vacuum infiltration and triturationmethods for isolating xylem residing bacteria from eggplantchilli and S torvum

Traditionally bacteria have been characterized andgrouped based on colony morphology and biochemical

10 International Journal of Microbiology

Table 4 List of XRB identified by partial 16S rRNAgene sequencing their plant host accession numbers closestNCBImatch and similarity

Strain Plant host Accession number Closest NCBI match similarityXB1lowast SE KF447383 Agrobacterium tumefaciens 99XB7lowast SE KF447384 Pseudomonas aeruginosa 99XB8lowast SE KF447385 Staphylococcus haemolyticus 99XB20lowast SE KF447386 Staphylococcus haemolyticus 100XB22 SE KF447387 Brevibacterium casei 99XB25 C KF447388 Enterobacter sp 95XB27lowast SE KF447389 Staphylococcus haemolyticus 99XB34 SE KF447390 Curtobacterium sp 99XB35 SE KF447391 Microbacterium sp 99XB36 SE KF447392 Xenophilus sp 99XB37 SE KF447393 Chryseobacterium sp 99XB40 SE KF447394 Micrococcus luteus 89XB41 SE KF447395 Bacillus sp 99XB47 SE KF447396 Bacillus sp 99XB53 SE KF447397 Micrococcus sp 99XB62lowast SE KF447398 Pseudomonas sp 99XB64 SE KF447399 Bacillus thuringiensis 100XB66 SE KF447400 Pectobacterium carotovorum 99XB70lowast SE KF447401 Janibacter melonis 99XB86lowast C KF447402 Agrobacterium tumefaciens 99XB87 C KF447403 Bacillus barbaricus 99XB88 C KF447404 Bacillus sp 99XB92 C KF447405 Brevundimonas vesicularis 99XB93lowast C KF447406 Bacillus safensis 100XB94 C KF447407 Bacillus sp 100XB98 C KF447408 Microbacterium sp 100XB99lowast SE KF447409 Enterobacter sp 99XB100lowast SE KF447410 Enterobacter cloacae 98XB103 SE KF447411 Brachybacterium phenoliresistens 99XB109 SE KF447412 Rhodococcus corynebacterioides 99XB114lowast SE KF447413 Pseudomonas stutzeri 99XB122lowast SE KF913446 Pseudomonas aeruginosa 100XB123lowast SE KF447414 Enterobacter sp 99XB126lowast C KF447415 Pantoea eucrina 99XB134lowast SE KF447416 Enterobacter sp 99XB137 SE KF447417 Klebsiella sp 99XB140lowast SE KF447418 Burkholderia sp 99XB153lowast SE KF447419 Bacillus amyloliquefaciens 99XB157lowast SE KF447420 Bacillus subtilis 100XB158 RE KF447421 Bosea sp 99XB159 RE KF447422 Microbacterium xylanilyticum 99XB161 RE KF447423 Microbacterium aurum 99XB165lowast RE KF447424 Agrobacterium sp 99XB167 RE KF447425 Microbacterium aurum 99XB168 RE KF447426 Bacillus aryabhattai 100XB169lowast RE KF447427 Staphylococcus gallinarum 99XB170lowast RE KF447428 Staphylococcus sp 99XB177lowast RE KF447429 Bacillus cereus 99XB188 RE KF447430 Sphingomonas sp 99XB190 RE KF447431 Brevibacterium casei 99

International Journal of Microbiology 11

Table 4 Continued

Strain Plant host Accession number Closest NCBI match similarityXB196lowast RE KF447432 Enterobacter kobei 99XB197lowast RE KF447433 Sphingomonas sp 98XB200lowast ST KF913447 Streptomyces sp 100XB202lowast ST KF447434 Pseudomonas sp 99XB203lowast ST KF447435 Flavobacterium sp 99lowastStrain is antagonistic to R solanacearum based on bioassaySE bacterial wilt susceptible eggplant RE bacterial wilt resistant eggplant C chilli ST Solanum torvum

tests However whole genome fingerprinting or PCR-RFLPbased methods are rapid tools for determining geneticdiversity of bacteria in a given collection ARDRA which isa type of PCR based RFLP method has been used widely inestimating genetic diversity endophytic bacterial populationsand clustering genetically similar strains [39ndash41] In additionto earlier reports our study demonstrates the usefulnessof ARDRA as a tool to cluster genetically identical strainsof endophytic XRB isolated from eggplant chilli andS torvum In our study 9161 strains (119899 = 153) weregrouped in 24 haplotypes by using ARDRA The majority ofthese haplotypes represent a combination of XRB isolatedfrom different solanaceous plants from diverse localesThese results indicate that xylem of eggplant chilli andS torvum largely bears similar population of XRB whichcan efficiently cross colonize eggplant chilli or S torvumNevertheless 839 strains had unique ARDRA fingerprintand formed separable haplotypes This observation leads toa conclusion that a minor population of xylem inhabitantsare restricted to a specific plant species and cannot easilycross colonize xylem of other solanaceous plants Plantsare known to selectively support endophytic colonizationby specific bacteria [14] However factors that determinethe selection of xylem colonists or the ability of XRBto colonize eggplant chilli and S torvum in this studyremain unknown Interestingly the structure of endophyticcommunity in Nicotiana attenuata a member of Solanaceaefamily is shown to be influenced by soil composition andethylene homeostasis [40] Earlier evidence has shown thatcolonization by endophytic bacteria is also governed by plantgenotype as well as root exudates [42]

Only 1677 XRB out of 167 were antagonistic to Rsolanacearum based on in vitro assays Antagonistic XRBproduced volatile and diffusible inhibitory compoundsnamely HCN ammonia and acetoin and siderophoresThese substances have been long known to be involvedin disease suppression and indirect growth promotion inplants [43ndash46] These mechanisms possibly played a rolein the evident biocontrol effect against BW exhibited bythe XRB in the greenhouse screening Endophytic bacteriahave been known to have plant growth promoting traitsnamely production of IAA ACC deaminase and phosphatesolubilization [25 33 36] These traits were detected in themajority of antagonistic XRB tested in this study and mayhave resulted in the observed increase in shoot length ofeggplant in our greenhouse experiments In contrast strains

XB20 XB196 and XB203 suppressed growth in eggplantunder greenhouse conditions however no visible symptomsof disease were observed Vascular plugging and productionof certainmetabolites toxic to plant cells but not cell viabilitymay have resulted in stunted shoot in eggplant [11 47]

Evaluation of efficacy of antagonistic organisms to sup-press the plant diseases under greenhouse conditions isone of the key steps for selecting a potential biocontrolagent for disease management [48] Endophytic strains fromBW susceptible varieties of eggplant have been shown toprevent wilt and promote growth in eggplant earlier [27]Our greenhouse screening shows that 3846 of antagonisticXRB with biocontrol efficacies greater than 40 were isolatesfrom BW resistant varieties of eggplantThis raises a questionwhether the bacteria from resistant varieties are involved inBW resistance and whether BW resistant varieties are ableto selectively influence xylem colonization by antagonisticbacteria Presence of higher number of endophytes withantagonistic abilities was reported in BW resistant varieties oftomato as compared to susceptible varieties and their role inresistance to BWwas proposed [49] Similar observations oncorrelation of resistance of potato to soft rot and endophyticbacteria have been reported [50]Thus BW resistant varietiescan be considered a better host for isolating potential biocon-trol strains for management of bacterial wilt

Identification of 55 XRB strains by 16S rRNA genesequencing revealed the presence of 23 diverse genera ofbacteria belonging to 4 phyla of Eubacteria Strains belongingto phyla Firmicutes Actinobacteria and 120574 subdivision ofProteobacteria were the major xylem colonists identified inthis study Several genera of bacteria belonging to these phylahave also been reported to be present in endophytic tissuesand xylem of a variety of other agricultural and horticulturalplant species [14 51] In addition the majority of the antag-onists identified belonged to Enterobacter sp Pseudomonassp Bacillus sp and Staphylococcus sp Congruent to ourresults several researchers have reported bacteria isolatedfrom solanaceous crops and belonging to similar genera tobe antagonistic to R solanacearum [26 51ndash53] HoweverFlavobacterium spand Janibacter melonis identified in thisstudy have never been previously reported to be inhibitoryto R solanacearum Large population of the xylem inhabitingbacterial flora accounting for 8323 exhibited no antago-nism towards R solanacearum Nonantagonistic XRB wereidentified predominantly as Microbacterium sp Endophyticpersistence and nematicidal activities of Microbacterium sp

12 International Journal of Microbiology

have been reported [54 55] Therefore the collection of XRBisolated in this study can be screened for inhibitory activitiesagainst other important agricultural pests

Though few strains namely XB86 XB169 and XB177exhibited plant beneficial properties in this study their use-fulness in plant disease control remains to be seen XB86has been identified as Agrobacterium tumefaciens the crowngall disease pathogen and its deployment as biocontrolagent is uncertain XB169 (Staphylococcus gallinarum) andXB177 (Bacillus cereus) are reported as opportunistic animalpathogens and thus unsuitable for field applications Strep-tomyces sp has earlier been reported as antagonistic to Rsolanacearum and tested for management of wilt in potatoand tomato [56 57] XB200 (Streptomyces sp) is one of theXRB high BCE and GPE it could be explored further forbiocontrol of bacterial wilt after additional characterizationand field evaluation

5 Conclusion

This study is the first report on the identity of novel anddiverse XRB colonizing the xylem of eggplant chilli and Storvum XRB particularly from BW resistant varieties werefound to protect eggplant from bacterial wilt and enhancedgrowth in eggplant in the greenhouse screening Thereforethe repertoire of XRB reported in this study may be usefulfor cultivation of eggplant in BW affected areas

Conflict of Interests

The authors hereby declare that they have no conflict ofinterests regarding the publication of this paper

Acknowledgments

The authors greatly acknowledge financial support fromIndian Council of Agricultural Research (ICAR) New DelhiIndia through ldquoOutreach project on Phytopthora Fusariumand Ralstonia diseases of horticultural and field cropsrdquo-(PhytoFuRa) and the Director ICAR Research Complex forGoa India for the facilities

References

[1] E Yabuuchi Y KosakoHOyaizu et al ldquoProposal ofBurkholde-ria gen nov and transfer of seven species of the genusPseudomonas homology group II to the new genus with thetype species Burkholderia cepacia (Palleroni and Holmes 1981)comb novrdquo Microbiology and Immunology vol 36 no 12 pp1251ndash1275 1992

[2] E Wicker L Grassart R Coranson-Beaudu et al ldquoRalstoniasolanacearum strains from Martinique (French West Indies)exhibiting a new pathogenic potentialrdquo Applied and Environ-mental Microbiology vol 73 no 21 pp 6790ndash6801 2007

[3] Y Liu A Kanda K Yano et al ldquoMolecular typing of Japanesestrains of Ralstonia solanacearum in relation to the ability toinduce a hypersensitive reaction in tobaccordquo Journal of GeneralPlant Pathology vol 75 no 5 pp 369ndash380 2009

[4] R Ramesh ldquoField evaluation of biological control agents for themanagement of Ralstonia solanacearum in Brinjalrdquo Journal ofMycology and Plant Pathology vol 36 no 2 pp 327ndash328 2006

[5] J Vasse P Frey and A Trigalet ldquoMicroscopic studies ofintercellular infection and protoxylem invasion of tomatoroots by Pseudomonas solanacearumrdquoMolecular Plant-MicrobeInteractions vol 8 no 2 pp 241ndash251 1995

[6] S Genin and C Boucher ldquoRalstonia solanacearum secrets of amajor pathogen unveiled by analysis of its genomerdquo MolecularPlant Pathology vol 3 no 3 pp 111ndash118 2002

[7] J M Gardner A W Feldman and R M Zablotowicz ldquoIdentityand behavior of xylem-residing bacteria in rough lemon roots ofFlorida citrus treesrdquo Applied and Environmental Microbiologyvol 43 no 6 pp 1335ndash1342 1982

[8] M J Jacobs W M Bugbee and D A Gabriellson ldquoEnu-meration location and characterization of endophytic bacteriawithin sugar beet rootsrdquoCanadian Journal of Botany vol 63 no7 pp 1262ndash1265 1985

[9] D G Patriquin and J Dobereiner ldquoLight microscopy observa-tions of tetrazolium-reducing bacteria in the endorhizosphereof maize and other grasses in Brazilrdquo Canadian Journal ofMicrobiology vol 24 no 6 pp 734ndash742 1978

[10] S Gagne C Richard H Rousseau and H Antoun ldquoXylem-residing bacteria in alfalfa rootsrdquo Canadian Journal of Micro-biology vol 33 no 11 pp 996ndash1000 1987

[11] C R Bell G A Dickie W L G Harvey and J W Y FChan ldquoEndophytic bacteria in grapevinerdquo Canadian Journal ofMicrobiology vol 41 no 1 pp 46ndash53 1995

[12] J Hallmann J W Kloepper and R Rodrıguez-Kabana ldquoAppli-cation of Scholander pressure bomb to studies on endophyticbacteria of plantsrdquo Canadian Journal of Microbiology vol 43no 5 pp 411ndash416 1997

[13] J S Lampel G L Canter M B Dimock et al ldquoIntegrativecloning expression and stability of the cryIA(c) gene fromBacillus thuringiensis subsp kurstaki in a recombinant strain ofClavibacter xyli subsp cynodontisrdquo Applied and EnvironmentalMicrobiology vol 60 no 2 pp 501ndash508 1994

[14] M Rosenblueth and E Martınez-Romero ldquoBacterial endo-phytes and their interactions with hostsrdquo Molecular Plant-Microbe Interactions vol 19 no 8 pp 827ndash837 2006

[15] P R Hardoim L S van Overbeek and J D van ElsasldquoProperties of bacterial endophytes and their proposed role inplant growthrdquo Trends in Microbiology vol 16 no 10 pp 463ndash471 2008

[16] R Aravind A Kumar S J Eapen and K V RamanaldquoEndophytic bacterial flora in root and stem tissues of blackpepper (Piper nigrum L) genotype isolation identificationand evaluation against Phytophthora capsicirdquo Letters in AppliedMicrobiology vol 48 no 1 pp 58ndash64 2009

[17] J Kuklinsky-Sobral W L Araujo R Mendes I O Geraldi AA Pizzirani-Kleiner and J L Azevedo ldquoIsolation and charac-terization of soybean-associated bacteria and their potential forplant growth promotionrdquo Environmental Microbiology vol 6no 12 pp 1244ndash1251 2004

[18] S Compant B Duffy J Nowak C Clement and E A BarkaldquoUse of plant growth-promoting bacteria for biocontrol ofplant diseases principles mechanisms of action and futureprospectsrdquoApplied and Environmental Microbiology vol 71 no9 pp 4951ndash4959 2005

[19] E T Thorne B M Young G M Young et al ldquoThe structureof xylem vessels in grapevine (Vitaceae) and a possible passive

International Journal of Microbiology 13

mechanism for the systemic spread of bacterial diseaserdquo Amer-ican Journal of Botany vol 93 no 4 pp 497ndash504 2006

[20] W T Seo W J Lim E J Kim H D Yun Y H Lee and KM Cho ldquoEndophytic bacterial diversity in the young radishand their antimicrobial activity against pathogensrdquo Journal ofApplied Biological Chemistry vol 53 no 4 pp 493ndash503 2010

[21] S Compant H Kaplan A Sessitsch J Nowak E Ait Barkaand C Clement ldquoEndophytic colonization ofVitis vinifera L byBurkholderia phytofirmans strain PsJN from the rhizosphere toinflorescence tissuesrdquo FEMS Microbiology Ecology vol 63 no1 pp 84ndash93 2005

[22] P S B Miguel J C Delvaux M N V de Oliveira et al ldquoDiver-sity of endophytic bacteria in the fruits of Coffea canephorardquoAfrican Journal of Microbiology Research vol 7 no 7 pp 586ndash594 2013

[23] A Lopez-Lopez M A Rogel E Ormeno-Orrillo J Martınez-Romero and E Martınez-Romero ldquoPhaseolus vulgaris seed-borne endophytic community with novel bacterial species suchas Rhizobium endophyticum sp novrdquo Systematic and AppliedMicrobiology vol 33 no 6 pp 322ndash327 2010

[24] M Lwin and S L Ranamukhaarachchi ldquoDevelopment of bio-logical control of Ralstonia solanacearum through antagonisticmicrobial populationsrdquo International Journal of Agriculture andBiology vol 8 no 5 pp 657ndash660 2006

[25] F Rasche E Marco-Noales H Velvis et al ldquoStructural char-acteristics and plant-beneficial effects of bacteria colonizing theshoots of field grown conventional and geneticallymodifiedT4-lysozyme producing potatoesrdquo Plant and Soil vol 289 no 1-2pp 123ndash140 2006

[26] N Amaresan V Jayakumar K Kumar and N ThajuddinldquoEndophytic bacteria from tomato and chilli their diversity andantagonistic potential against Ralstonia solanacearumrdquoArchivesof Phytopathology and Plant Protection vol 45 no 3 pp 344ndash355 2012

[27] R Ramesh and G S Phadke ldquoRhizosphere and endophyticbacteria for the suppression of eggplant wilt caused byRalstoniasolanacearumrdquo Crop Protection vol 37 pp 35ndash41 2012

[28] W J French R G Christis and D L Stassi ldquoRecovery ofrickettsialike bacteria by vacuum infiltration of peach tissuesaffected with phony diseaserdquo Phytopathology vol 67 pp 945ndash948 1977

[29] P R Viss E M Brooks and J A Driver ldquoA simplified methodfor the control of bacterial contamination in woody plant tissueculturerdquo In Vitro Cellular andDevelopmental Biology vol 27 no1 article 42 1991

[30] K Wilson ldquoPreparation of genomic DNA from bacteriardquo inCurrent Protocols in Molecular Biology F M Ausubel R BrentR E Kingston et al Eds pp 241ndash245 John Wiley amp SonsNew York NY USA 1997

[31] M Saraf AThakkar U PandyaM Joshi and J Parikh ldquoPoten-tial of plant growth promoting microorganisms as biofertilizersand biopesticides and itrsquos exploitation in sustainable agricul-turerdquo Journal of Microbiology and Biotechnology Research vol3 no 5 pp 54ndash62 2013

[32] A P G C Marques C Pires H Moreira A O S S Rangeland P M L Castro ldquoAssessment of the plant growth promotionabilities of six bacterial isolates using Zea mays as indicatorplantrdquo Soil Biology and Biochemistry vol 42 no 8 pp 1229ndash1235 2010

[33] X Hao P Xie L Johnstone S J Miller C Rensingand G Wei ldquoGenome sequence and mutational analysis of

plant-growth-promoting bacteriumAgrobacterium tumefaciensCCNWGS0286 isolated from a zinc-lead mine tailingrdquo Appliedand Environmental Microbiology vol 78 no 15 pp 5384ndash53942012

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

[35] S A Gordon and L G Paleg ldquoQuantitative measurement ofindole acetic acidrdquo Plant Physiology vol 10 pp 37ndash48 1957

[36] A Godinho R Ramesh and S Bhosle ldquoBacteria from sanddunes of Goa promoting growth in eggplantrdquoWorld Journal ofAgricultural Sciences vol 6 no 5 pp 555ndash564 2010

[37] J-H Guo H-Y Qi Y-H Guo et al ldquoBiocontrol of tomato wiltby plant growth-promoting rhizobacteriardquo Biological Controlvol 29 no 1 pp 66ndash72 2004

[38] N Aliye C Fininsa and Y Hiskias ldquoEvaluation of rhizo-sphere bacterial antagonists for their potential to bioprotectpotato (Solanum tuberosum) against bacterial wilt (Ralstoniasolanacearum)rdquo Biological Control vol 47 no 3 pp 282ndash2882008

[39] L LagaceM PitreM Jacqeus andDRoy ldquoIdentification of thebacterial community ofmaple sap by using amplified ribosomalDNA (rDNA) restriction analysis and rDNA sequencingrdquoApplied and Environmental Microbiology vol 70 no 4 pp2052ndash2060 2004

[40] H H Long D G Sonntag D D Schmidt and I T BaldwinldquoThe structure of the culturable root bacterial endophyte com-munity of Nicotiana attenuata is organized by soil compositionand host plant ethylene production and perceptionrdquo NewPhytologist vol 185 no 2 pp 554ndash567 2010

[41] E Lopez-Fuentes V M Ruız-Valdiviezo E Martınez-RomeroF A Gutierrez-Miceli L Dendooven and R Rincon-RosalesldquoBacterial community in the roots and rhizosphere of Hyper-icum silenoides Juss 1804rdquo African Journal of MicrobiologyResearch vol 6 no 11 pp 2704ndash2711 2012

[42] M Fang R J Kremer P P Motavalli and G Davis ldquoBacterialdiversity in rhizospheres of nontransgenic and transgenic cornrdquoApplied and EnvironmentalMicrobiology vol 71 no 7 pp 4132ndash4136 2005

[43] C R Howell and R D Stiphanovic ldquoProduction of ammonia byEnterobacter cloaceae and its possible role in biological controlof Pythium pre-emergence damping-off by the bacteriumrdquoEcology and Epidemiology vol 78 no 8 pp 1075ndash1078 1988

[44] A Ramette M Frapolli G Defago and Y Moenne-LoccozldquoPhylogeny of HCN synthase-encoding hcnBC genes in bio-control fluorescent pseudomonads and its relationship withhost plant species and HCN synthesis abilityrdquoMolecular Plant-Microbe Interactions vol 16 no 6 pp 525ndash535 2003

[45] I Loaces L Ferrando and A F Scavino ldquoDynamics diversityand function of endophytic siderophore-producing bacteria inricerdquoMicrobial Ecology vol 61 no 3 pp 606ndash618 2011

[46] Y C Kim J Leveau B B M Gardener E A Pierson L SPierson and C-M Ryu ldquoThe multifactorial basis for planthealth promotion by plant-associated bacteriardquo Applied andEnvironmental Microbiology vol 77 no 5 pp 1548ndash1555 2011

[47] J M Gardner J L Chandler and A W Feldman ldquoGrowthresponses and vascular plugging of citrus inoculated withrhizobacteria and xylem-resident bacteriardquo Plant and Soil vol86 no 3 pp 333ndash345 1985

[48] C Pliego C Ramos A de Vicente and F M Cazorla ldquoScreen-ing for candidate bacterial biocontrol agents against soilborne

14 International Journal of Microbiology

fungal plant pathogensrdquoPlant and Soil vol 340 no 1-2 pp 505ndash520 2011

[49] H Feng Y Li and Q Liu ldquoEndophytic bacterial communi-ties in tomato plants with differential resistance to Ralstoniasolanacearumrdquo African Journal of Microbiology Research vol 7no 15 pp 1311ndash1318 2013

[50] A V Sturz B R Christie B G Matheson W J Arsenaultand N A Buchanan ldquoEndophytic bacterial communities inthe periderm of potato tubers and their potential to improveresistance to soil-borne plant pathogensrdquo Plant Pathology vol48 no 3 pp 360ndash369 1999

[51] A V Sturz B R Christie and J Nowak ldquoBacterial endophytespotential role in developing sustainable systems of crop produc-tionrdquo Critical Reviews in Plant Sciences vol 19 no 1 pp 1ndash302000

[52] R Ramesh A A Joshi and M P Ghanekar ldquoPseudomonadsmajor antagonistic endophytic bacteria to suppress bacterialwilt pathogenRalstonia solanacearum in the eggplant (Solanummelongena L)rdquo World Journal of Microbiology and Biotechnol-ogy vol 25 no 1 pp 47ndash55 2009

[53] A A Nawangsih I Damayanti S Wiyono and J G KartikaldquoSelection and characterization of endophytic bacteria as bio-control agents of tomato Bacterial Wilt DiseaserdquoHayati Journalof Biosciences vol 18 no 2 pp 66ndash70 2011

[54] R Vargas-Ayala R Rodrıguez-Kabana G Morgan-Jones J AMcInroy and J W Kloepper ldquoShifts in soil microflora inducedby velvetbean (Mucuna deeringiana) in cropping systems tocontrol root-knot nematodesrdquo Biological Control vol 17 no 1pp 11ndash22 2000

[55] D K Zinniel P Lambrecht N B Harris et al ldquoIsolationand characterization of endophytic colonizing bacteria fromagronomic crops and prairie plantsrdquoApplied and EnvironmentalMicrobiology vol 68 no 5 pp 2198ndash2208 2002

[56] F Lemessa and W Zeller ldquoScreening rhizobacteria for biolog-ical control of Ralstonia solanacearum in Ethiopiardquo BiologicalControl vol 42 no 3 pp 336ndash344 2007

[57] M S El-AlbyadM A El-Sayed A R El-Shanshoury andNHEl-Batanouny ldquoEffect of culture conditions on the antimicrobialactivities of UV-mutants of Streptomyces corchorusii and Sspiroverticillatus against bean and banana wilt pathogensrdquoMicrobiological Research vol 151 no 2 pp 201ndash211 1996

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

8 International Journal of Microbiology

Table3

Invitro

inhibitio

nof

Rsolana

cearum

andprod

uctio

nof

antagonisticc

ompo

unds

andplantg

rowth

prom

otingsubstances

byXR

B

Strain

Haplotype

Inhibitio

nof

Rsolana

cearum

Volatile

anddiffu

sibleinhibitory

compo

unds

Plantgrowth

prom

otingsubstances

Radius

inmm

HCN

Ammon

iaAc

etoin

Sideroph

ore

Phosph

ates

olub

ilisatio

nAC

Cdeam

inasea

IAA

b120583gmL

XB1

M80-7

283plusmn029

minusminus

minusminus

minus++

+10500plusmn707

XB7

M80-27

333plusmn058

+minus

minus+

+minus

4773plusmn321

XB8

M80-20

333plusmn029

minusminus

minusminus

+minus

1909plusmn129

XB20

M80-20

327plusmn040

minusminus

minusminus

+minus

2545plusmn171

XB27

M80-20

183plusmn029

minusminus

minusminus

+minus

1591plusmn107

XB62

M80-31

817plusmn076

+minus

minusminus

+++

++7318plusmn493

XB70

M80-15

383plusmn058

minusminus

minusminus

minusminus

17182plusmn1157

XB86

M80-7

233plusmn076

minusminus

minusminus

minus++

+6682plusmn450

XB93

M80-6

317plusmn029

+minus

+minus

+minus

5727plusmn386

XB99

M80-9

683plusmn058

minus+

+minus

+minus

32136plusmn2164

XB100

M80-9

450plusmn050

minus+

+minus

+minus

23864plusmn1607

XB114

M80-31

650plusmn050

minusminus

minus+

+minus

8909plusmn600

XB122

M80-28

433plusmn058

+minus

minus+

+minus

6682plusmn450

XB123

M80-31

300plusmn087

minus+

+minus

+minus

41682plusmn2807

XB126

M80-27

283plusmn029

minusminus

+minus

+minus

6045plusmn407

XB134

M80-37

157plusmn012

minus+

+minus

+minus

64591plusmn4350

XB140

M80-5

367plusmn076

minus+

minus+

+++

++5091plusmn343

XB153

M80-6

317plusmn029

minusminus

+minus

+minus

15591plusmn1050

XB157

M80-6

250plusmn100

minusminus

+minus

+minus

18455plusmn1243

XB165

M80-29

157plusmn012

minusminus

minusminus

minus+

19091plusmn1286

XB169

M80-6

153plusmn006

minusminus

minusminus

minusminus

1591plusmn107

XB170

M80-6

351plusmn002

+minus

minusminus

+minus

2864plusmn193

XB177

M80-6

199plusmn049

minusminus

minusminus

minusminus

7636plusmn514

XB196

M80-10

393plusmn012

minusminus

minusminus

minusminus

3500plusmn236

XB197

M80-38

433plusmn029

minusminus

+minus

minusminus

4136plusmn279

XB200

M80-36

187plusmn023

minusminus

minusminus

minus++

16864plusmn1136

XB202

M80-31

467plusmn021

minusminus

minusminus

+++

+8273plusmn557

XB203

M80-31

367plusmn029

minusminus

minusminus

minusminus

7000plusmn471

Inhibitio

nzonesa

remeanof

threereplications

andshow

ingsta

ndarddeviation

Inhibitio

nzone

was

measuredas

radius

from

theou

tere

dgeof

wellAlltheexperim

entswerecond

uctedat28plusmn2∘C

+indicates

presence

oftraitminusindicatesabsence

oftraitinplateb

ased

assaysaLevelsofAC

Cdeam

inasea

ctivitiesbasedon

grow

thon

plateb

ased

assayd

enoted

as+forlessg

rowth+++

form

oderateg

rowth+++

forlux

uriant

grow

thand

++++

forh

ighlyluxu

riant

grow

thbIAAwas

estim

ated

incultu

refiltra

teandexpressedas120583gmLusinganalyticalgradeIAAas

stand

ardvalues

indicatemeanandsta

ndarddeviation

International Journal of Microbiology 9

2545

2545

364

364

1273 2909

ActinobacteriaFirmicutesAlpha proteobacteria

Beta proteobacteriaGamma proteobacteriaBacteroidetes

(a)

Num

ber o

f XRB

Actin

obac

teria

Firm

icut

es

Alp

ha p

rote

obac

teria

Beta

pro

teob

acte

ria

Gam

ma p

rote

obac

teria

Bact

eroi

dete

s

Number of antagonistic XRB in each phylumsubdivision

Phylasubdivision

Total number of XRB belonging to each phylumsubdivision

25

20

15

10

5

0

2

14

9

16

4

7

1

2

1

2

11

14

(b)

Figure 2 (a) Distribution of XRB (phylumsubdivision level) identified by 16S rRNA gene sequencing Values indicate percentages of strainsbelonging to each phylasubdivision amongst the 55 identified strains (b) Distribution of antagonistic and nonantagonistic strains of XRB ineach phylumsubdivision

different genera of bacteria were identified Major gen-era identified are Bacillus sp (11 strains) Enterobacter sp(6 strains) Microbacterium sp (5 strains) Staphylococcussp (5 strains) Pseudomonas sp (5 strains) and Agrobac-terium sp (3 strains) Additional genera identified includ-ing Micrococcus sp Sphingomonas sp Flavobacterium spChryseobacterium sp Burkholderia sp and Xenophilus spare listed in Table 4 Based on the identification phylumProteobacteria consisting of Gram-negative bacteria of sub-divisions Alpha Proteobacteria (1273) Beta Proteobacteria(364) and Gamma Proteobacteria (2545) were predom-inant (4181 strains identified) followed by phyla Firmi-cutes (2909) Actinobacteria (2545) and Bacteroidetes(364) (Figure 2(a))

Eleven antagonistic strains identified belonged toGammasubdivision of Proteobacteria consisting of five strainseachof Enterobacter sp and fluorescent and nonfluorescent Pseu-domonas sp and one strain of Pantoea eucrina (XB126)(Figure 2(b)) Nine antagonists identified were of phylaFirmicutes consisting of Staphylococcus sp (5 strains) andBacillus sp (4 strains) Agrobacterium strains XB1 XB86 andXB165 Sphingomonas sp (XB197) of the Alpha Proteobacte-ria Streptomyces sp (XB200) and Janibacter melonis (XB70)of phyla Actinobacteria were found to be antagonistic Addi-tional antagonistic XRB include Burkholderia sp (XB140) of120573 Proteobacteria and Flavobacterium sp (XB203) of phylaBacteroidetes (Figure 2(b))

37 Statistical Analysis The statistical analysis of percent-age wilts and shoot length of eggplant was performedusing Web Agri Statistical Package (WASP) version 20(httpwwwicargoaresinwasp20indexphp)

4 Discussion

Eggplant and chilli not only are of economic and culturalimportance but also are common ingredients in the cuisinethroughout India In the coastal state of GoaR solanacearumhas been reported to be a destructive pathogen in cultivationof eggplant and chilli [4] Isolation of biocontrol agentsagainst the BW pathogen has been commonly restricted toendophytic tissue and plant rhizosphere [26 27] Studieson xylem colonizing endophytes were undertaken becausewe speculated existence of interactions between the XRBand vascular wilt pathogen R solanacearum during xylemcolonization Our study reveals the diversity biocontrolpotential and identity of endophytic xylem colonizers fromsolanaceous crops cultivated in Goa India A total of 167bacteria were isolated from the xylem of eggplant chilliand S torvum with Gram-negative bacteria (5928) pre-dominating in the collection Congruent to our observationGardner et al [7] and Bell et al [11] have earlier reportedisolation of more number of Gram-negative rod shapedbacteria from xylem of citrus and grapevine using vacuuminfiltration Scholander pressure bomb was found to beuseful in extraction of diverse bacterial genera from xylemtissues in contrast to trituration methods that yielded highernumber of Gram-positive rod shaped bacteria [12] ThoughScholander pressure bomb was not used in this study acombination of vacuum infiltration [7 11] and triturationof decorticated stems [10 12] was employed with an aimto isolate diverse XRB from xylem tissues This is the firststudy reporting the use of vacuum infiltration and triturationmethods for isolating xylem residing bacteria from eggplantchilli and S torvum

Traditionally bacteria have been characterized andgrouped based on colony morphology and biochemical

10 International Journal of Microbiology

Table 4 List of XRB identified by partial 16S rRNAgene sequencing their plant host accession numbers closestNCBImatch and similarity

Strain Plant host Accession number Closest NCBI match similarityXB1lowast SE KF447383 Agrobacterium tumefaciens 99XB7lowast SE KF447384 Pseudomonas aeruginosa 99XB8lowast SE KF447385 Staphylococcus haemolyticus 99XB20lowast SE KF447386 Staphylococcus haemolyticus 100XB22 SE KF447387 Brevibacterium casei 99XB25 C KF447388 Enterobacter sp 95XB27lowast SE KF447389 Staphylococcus haemolyticus 99XB34 SE KF447390 Curtobacterium sp 99XB35 SE KF447391 Microbacterium sp 99XB36 SE KF447392 Xenophilus sp 99XB37 SE KF447393 Chryseobacterium sp 99XB40 SE KF447394 Micrococcus luteus 89XB41 SE KF447395 Bacillus sp 99XB47 SE KF447396 Bacillus sp 99XB53 SE KF447397 Micrococcus sp 99XB62lowast SE KF447398 Pseudomonas sp 99XB64 SE KF447399 Bacillus thuringiensis 100XB66 SE KF447400 Pectobacterium carotovorum 99XB70lowast SE KF447401 Janibacter melonis 99XB86lowast C KF447402 Agrobacterium tumefaciens 99XB87 C KF447403 Bacillus barbaricus 99XB88 C KF447404 Bacillus sp 99XB92 C KF447405 Brevundimonas vesicularis 99XB93lowast C KF447406 Bacillus safensis 100XB94 C KF447407 Bacillus sp 100XB98 C KF447408 Microbacterium sp 100XB99lowast SE KF447409 Enterobacter sp 99XB100lowast SE KF447410 Enterobacter cloacae 98XB103 SE KF447411 Brachybacterium phenoliresistens 99XB109 SE KF447412 Rhodococcus corynebacterioides 99XB114lowast SE KF447413 Pseudomonas stutzeri 99XB122lowast SE KF913446 Pseudomonas aeruginosa 100XB123lowast SE KF447414 Enterobacter sp 99XB126lowast C KF447415 Pantoea eucrina 99XB134lowast SE KF447416 Enterobacter sp 99XB137 SE KF447417 Klebsiella sp 99XB140lowast SE KF447418 Burkholderia sp 99XB153lowast SE KF447419 Bacillus amyloliquefaciens 99XB157lowast SE KF447420 Bacillus subtilis 100XB158 RE KF447421 Bosea sp 99XB159 RE KF447422 Microbacterium xylanilyticum 99XB161 RE KF447423 Microbacterium aurum 99XB165lowast RE KF447424 Agrobacterium sp 99XB167 RE KF447425 Microbacterium aurum 99XB168 RE KF447426 Bacillus aryabhattai 100XB169lowast RE KF447427 Staphylococcus gallinarum 99XB170lowast RE KF447428 Staphylococcus sp 99XB177lowast RE KF447429 Bacillus cereus 99XB188 RE KF447430 Sphingomonas sp 99XB190 RE KF447431 Brevibacterium casei 99

International Journal of Microbiology 11

Table 4 Continued

Strain Plant host Accession number Closest NCBI match similarityXB196lowast RE KF447432 Enterobacter kobei 99XB197lowast RE KF447433 Sphingomonas sp 98XB200lowast ST KF913447 Streptomyces sp 100XB202lowast ST KF447434 Pseudomonas sp 99XB203lowast ST KF447435 Flavobacterium sp 99lowastStrain is antagonistic to R solanacearum based on bioassaySE bacterial wilt susceptible eggplant RE bacterial wilt resistant eggplant C chilli ST Solanum torvum

tests However whole genome fingerprinting or PCR-RFLPbased methods are rapid tools for determining geneticdiversity of bacteria in a given collection ARDRA which isa type of PCR based RFLP method has been used widely inestimating genetic diversity endophytic bacterial populationsand clustering genetically similar strains [39ndash41] In additionto earlier reports our study demonstrates the usefulnessof ARDRA as a tool to cluster genetically identical strainsof endophytic XRB isolated from eggplant chilli andS torvum In our study 9161 strains (119899 = 153) weregrouped in 24 haplotypes by using ARDRA The majority ofthese haplotypes represent a combination of XRB isolatedfrom different solanaceous plants from diverse localesThese results indicate that xylem of eggplant chilli andS torvum largely bears similar population of XRB whichcan efficiently cross colonize eggplant chilli or S torvumNevertheless 839 strains had unique ARDRA fingerprintand formed separable haplotypes This observation leads toa conclusion that a minor population of xylem inhabitantsare restricted to a specific plant species and cannot easilycross colonize xylem of other solanaceous plants Plantsare known to selectively support endophytic colonizationby specific bacteria [14] However factors that determinethe selection of xylem colonists or the ability of XRBto colonize eggplant chilli and S torvum in this studyremain unknown Interestingly the structure of endophyticcommunity in Nicotiana attenuata a member of Solanaceaefamily is shown to be influenced by soil composition andethylene homeostasis [40] Earlier evidence has shown thatcolonization by endophytic bacteria is also governed by plantgenotype as well as root exudates [42]

Only 1677 XRB out of 167 were antagonistic to Rsolanacearum based on in vitro assays Antagonistic XRBproduced volatile and diffusible inhibitory compoundsnamely HCN ammonia and acetoin and siderophoresThese substances have been long known to be involvedin disease suppression and indirect growth promotion inplants [43ndash46] These mechanisms possibly played a rolein the evident biocontrol effect against BW exhibited bythe XRB in the greenhouse screening Endophytic bacteriahave been known to have plant growth promoting traitsnamely production of IAA ACC deaminase and phosphatesolubilization [25 33 36] These traits were detected in themajority of antagonistic XRB tested in this study and mayhave resulted in the observed increase in shoot length ofeggplant in our greenhouse experiments In contrast strains

XB20 XB196 and XB203 suppressed growth in eggplantunder greenhouse conditions however no visible symptomsof disease were observed Vascular plugging and productionof certainmetabolites toxic to plant cells but not cell viabilitymay have resulted in stunted shoot in eggplant [11 47]

Evaluation of efficacy of antagonistic organisms to sup-press the plant diseases under greenhouse conditions isone of the key steps for selecting a potential biocontrolagent for disease management [48] Endophytic strains fromBW susceptible varieties of eggplant have been shown toprevent wilt and promote growth in eggplant earlier [27]Our greenhouse screening shows that 3846 of antagonisticXRB with biocontrol efficacies greater than 40 were isolatesfrom BW resistant varieties of eggplantThis raises a questionwhether the bacteria from resistant varieties are involved inBW resistance and whether BW resistant varieties are ableto selectively influence xylem colonization by antagonisticbacteria Presence of higher number of endophytes withantagonistic abilities was reported in BW resistant varieties oftomato as compared to susceptible varieties and their role inresistance to BWwas proposed [49] Similar observations oncorrelation of resistance of potato to soft rot and endophyticbacteria have been reported [50]Thus BW resistant varietiescan be considered a better host for isolating potential biocon-trol strains for management of bacterial wilt

Identification of 55 XRB strains by 16S rRNA genesequencing revealed the presence of 23 diverse genera ofbacteria belonging to 4 phyla of Eubacteria Strains belongingto phyla Firmicutes Actinobacteria and 120574 subdivision ofProteobacteria were the major xylem colonists identified inthis study Several genera of bacteria belonging to these phylahave also been reported to be present in endophytic tissuesand xylem of a variety of other agricultural and horticulturalplant species [14 51] In addition the majority of the antag-onists identified belonged to Enterobacter sp Pseudomonassp Bacillus sp and Staphylococcus sp Congruent to ourresults several researchers have reported bacteria isolatedfrom solanaceous crops and belonging to similar genera tobe antagonistic to R solanacearum [26 51ndash53] HoweverFlavobacterium spand Janibacter melonis identified in thisstudy have never been previously reported to be inhibitoryto R solanacearum Large population of the xylem inhabitingbacterial flora accounting for 8323 exhibited no antago-nism towards R solanacearum Nonantagonistic XRB wereidentified predominantly as Microbacterium sp Endophyticpersistence and nematicidal activities of Microbacterium sp

12 International Journal of Microbiology

have been reported [54 55] Therefore the collection of XRBisolated in this study can be screened for inhibitory activitiesagainst other important agricultural pests

Though few strains namely XB86 XB169 and XB177exhibited plant beneficial properties in this study their use-fulness in plant disease control remains to be seen XB86has been identified as Agrobacterium tumefaciens the crowngall disease pathogen and its deployment as biocontrolagent is uncertain XB169 (Staphylococcus gallinarum) andXB177 (Bacillus cereus) are reported as opportunistic animalpathogens and thus unsuitable for field applications Strep-tomyces sp has earlier been reported as antagonistic to Rsolanacearum and tested for management of wilt in potatoand tomato [56 57] XB200 (Streptomyces sp) is one of theXRB high BCE and GPE it could be explored further forbiocontrol of bacterial wilt after additional characterizationand field evaluation

5 Conclusion

This study is the first report on the identity of novel anddiverse XRB colonizing the xylem of eggplant chilli and Storvum XRB particularly from BW resistant varieties werefound to protect eggplant from bacterial wilt and enhancedgrowth in eggplant in the greenhouse screening Thereforethe repertoire of XRB reported in this study may be usefulfor cultivation of eggplant in BW affected areas

Conflict of Interests

The authors hereby declare that they have no conflict ofinterests regarding the publication of this paper

Acknowledgments

The authors greatly acknowledge financial support fromIndian Council of Agricultural Research (ICAR) New DelhiIndia through ldquoOutreach project on Phytopthora Fusariumand Ralstonia diseases of horticultural and field cropsrdquo-(PhytoFuRa) and the Director ICAR Research Complex forGoa India for the facilities

References

[1] E Yabuuchi Y KosakoHOyaizu et al ldquoProposal ofBurkholde-ria gen nov and transfer of seven species of the genusPseudomonas homology group II to the new genus with thetype species Burkholderia cepacia (Palleroni and Holmes 1981)comb novrdquo Microbiology and Immunology vol 36 no 12 pp1251ndash1275 1992

[2] E Wicker L Grassart R Coranson-Beaudu et al ldquoRalstoniasolanacearum strains from Martinique (French West Indies)exhibiting a new pathogenic potentialrdquo Applied and Environ-mental Microbiology vol 73 no 21 pp 6790ndash6801 2007

[3] Y Liu A Kanda K Yano et al ldquoMolecular typing of Japanesestrains of Ralstonia solanacearum in relation to the ability toinduce a hypersensitive reaction in tobaccordquo Journal of GeneralPlant Pathology vol 75 no 5 pp 369ndash380 2009

[4] R Ramesh ldquoField evaluation of biological control agents for themanagement of Ralstonia solanacearum in Brinjalrdquo Journal ofMycology and Plant Pathology vol 36 no 2 pp 327ndash328 2006

[5] J Vasse P Frey and A Trigalet ldquoMicroscopic studies ofintercellular infection and protoxylem invasion of tomatoroots by Pseudomonas solanacearumrdquoMolecular Plant-MicrobeInteractions vol 8 no 2 pp 241ndash251 1995

[6] S Genin and C Boucher ldquoRalstonia solanacearum secrets of amajor pathogen unveiled by analysis of its genomerdquo MolecularPlant Pathology vol 3 no 3 pp 111ndash118 2002

[7] J M Gardner A W Feldman and R M Zablotowicz ldquoIdentityand behavior of xylem-residing bacteria in rough lemon roots ofFlorida citrus treesrdquo Applied and Environmental Microbiologyvol 43 no 6 pp 1335ndash1342 1982

[8] M J Jacobs W M Bugbee and D A Gabriellson ldquoEnu-meration location and characterization of endophytic bacteriawithin sugar beet rootsrdquoCanadian Journal of Botany vol 63 no7 pp 1262ndash1265 1985

[9] D G Patriquin and J Dobereiner ldquoLight microscopy observa-tions of tetrazolium-reducing bacteria in the endorhizosphereof maize and other grasses in Brazilrdquo Canadian Journal ofMicrobiology vol 24 no 6 pp 734ndash742 1978

[10] S Gagne C Richard H Rousseau and H Antoun ldquoXylem-residing bacteria in alfalfa rootsrdquo Canadian Journal of Micro-biology vol 33 no 11 pp 996ndash1000 1987

[11] C R Bell G A Dickie W L G Harvey and J W Y FChan ldquoEndophytic bacteria in grapevinerdquo Canadian Journal ofMicrobiology vol 41 no 1 pp 46ndash53 1995

[12] J Hallmann J W Kloepper and R Rodrıguez-Kabana ldquoAppli-cation of Scholander pressure bomb to studies on endophyticbacteria of plantsrdquo Canadian Journal of Microbiology vol 43no 5 pp 411ndash416 1997

[13] J S Lampel G L Canter M B Dimock et al ldquoIntegrativecloning expression and stability of the cryIA(c) gene fromBacillus thuringiensis subsp kurstaki in a recombinant strain ofClavibacter xyli subsp cynodontisrdquo Applied and EnvironmentalMicrobiology vol 60 no 2 pp 501ndash508 1994

[14] M Rosenblueth and E Martınez-Romero ldquoBacterial endo-phytes and their interactions with hostsrdquo Molecular Plant-Microbe Interactions vol 19 no 8 pp 827ndash837 2006

[15] P R Hardoim L S van Overbeek and J D van ElsasldquoProperties of bacterial endophytes and their proposed role inplant growthrdquo Trends in Microbiology vol 16 no 10 pp 463ndash471 2008

[16] R Aravind A Kumar S J Eapen and K V RamanaldquoEndophytic bacterial flora in root and stem tissues of blackpepper (Piper nigrum L) genotype isolation identificationand evaluation against Phytophthora capsicirdquo Letters in AppliedMicrobiology vol 48 no 1 pp 58ndash64 2009

[17] J Kuklinsky-Sobral W L Araujo R Mendes I O Geraldi AA Pizzirani-Kleiner and J L Azevedo ldquoIsolation and charac-terization of soybean-associated bacteria and their potential forplant growth promotionrdquo Environmental Microbiology vol 6no 12 pp 1244ndash1251 2004

[18] S Compant B Duffy J Nowak C Clement and E A BarkaldquoUse of plant growth-promoting bacteria for biocontrol ofplant diseases principles mechanisms of action and futureprospectsrdquoApplied and Environmental Microbiology vol 71 no9 pp 4951ndash4959 2005

[19] E T Thorne B M Young G M Young et al ldquoThe structureof xylem vessels in grapevine (Vitaceae) and a possible passive

International Journal of Microbiology 13

mechanism for the systemic spread of bacterial diseaserdquo Amer-ican Journal of Botany vol 93 no 4 pp 497ndash504 2006

[20] W T Seo W J Lim E J Kim H D Yun Y H Lee and KM Cho ldquoEndophytic bacterial diversity in the young radishand their antimicrobial activity against pathogensrdquo Journal ofApplied Biological Chemistry vol 53 no 4 pp 493ndash503 2010

[21] S Compant H Kaplan A Sessitsch J Nowak E Ait Barkaand C Clement ldquoEndophytic colonization ofVitis vinifera L byBurkholderia phytofirmans strain PsJN from the rhizosphere toinflorescence tissuesrdquo FEMS Microbiology Ecology vol 63 no1 pp 84ndash93 2005

[22] P S B Miguel J C Delvaux M N V de Oliveira et al ldquoDiver-sity of endophytic bacteria in the fruits of Coffea canephorardquoAfrican Journal of Microbiology Research vol 7 no 7 pp 586ndash594 2013

[23] A Lopez-Lopez M A Rogel E Ormeno-Orrillo J Martınez-Romero and E Martınez-Romero ldquoPhaseolus vulgaris seed-borne endophytic community with novel bacterial species suchas Rhizobium endophyticum sp novrdquo Systematic and AppliedMicrobiology vol 33 no 6 pp 322ndash327 2010

[24] M Lwin and S L Ranamukhaarachchi ldquoDevelopment of bio-logical control of Ralstonia solanacearum through antagonisticmicrobial populationsrdquo International Journal of Agriculture andBiology vol 8 no 5 pp 657ndash660 2006

[25] F Rasche E Marco-Noales H Velvis et al ldquoStructural char-acteristics and plant-beneficial effects of bacteria colonizing theshoots of field grown conventional and geneticallymodifiedT4-lysozyme producing potatoesrdquo Plant and Soil vol 289 no 1-2pp 123ndash140 2006

[26] N Amaresan V Jayakumar K Kumar and N ThajuddinldquoEndophytic bacteria from tomato and chilli their diversity andantagonistic potential against Ralstonia solanacearumrdquoArchivesof Phytopathology and Plant Protection vol 45 no 3 pp 344ndash355 2012

[27] R Ramesh and G S Phadke ldquoRhizosphere and endophyticbacteria for the suppression of eggplant wilt caused byRalstoniasolanacearumrdquo Crop Protection vol 37 pp 35ndash41 2012

[28] W J French R G Christis and D L Stassi ldquoRecovery ofrickettsialike bacteria by vacuum infiltration of peach tissuesaffected with phony diseaserdquo Phytopathology vol 67 pp 945ndash948 1977

[29] P R Viss E M Brooks and J A Driver ldquoA simplified methodfor the control of bacterial contamination in woody plant tissueculturerdquo In Vitro Cellular andDevelopmental Biology vol 27 no1 article 42 1991

[30] K Wilson ldquoPreparation of genomic DNA from bacteriardquo inCurrent Protocols in Molecular Biology F M Ausubel R BrentR E Kingston et al Eds pp 241ndash245 John Wiley amp SonsNew York NY USA 1997

[31] M Saraf AThakkar U PandyaM Joshi and J Parikh ldquoPoten-tial of plant growth promoting microorganisms as biofertilizersand biopesticides and itrsquos exploitation in sustainable agricul-turerdquo Journal of Microbiology and Biotechnology Research vol3 no 5 pp 54ndash62 2013

[32] A P G C Marques C Pires H Moreira A O S S Rangeland P M L Castro ldquoAssessment of the plant growth promotionabilities of six bacterial isolates using Zea mays as indicatorplantrdquo Soil Biology and Biochemistry vol 42 no 8 pp 1229ndash1235 2010

[33] X Hao P Xie L Johnstone S J Miller C Rensingand G Wei ldquoGenome sequence and mutational analysis of

plant-growth-promoting bacteriumAgrobacterium tumefaciensCCNWGS0286 isolated from a zinc-lead mine tailingrdquo Appliedand Environmental Microbiology vol 78 no 15 pp 5384ndash53942012

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

[35] S A Gordon and L G Paleg ldquoQuantitative measurement ofindole acetic acidrdquo Plant Physiology vol 10 pp 37ndash48 1957

[36] A Godinho R Ramesh and S Bhosle ldquoBacteria from sanddunes of Goa promoting growth in eggplantrdquoWorld Journal ofAgricultural Sciences vol 6 no 5 pp 555ndash564 2010

[37] J-H Guo H-Y Qi Y-H Guo et al ldquoBiocontrol of tomato wiltby plant growth-promoting rhizobacteriardquo Biological Controlvol 29 no 1 pp 66ndash72 2004

[38] N Aliye C Fininsa and Y Hiskias ldquoEvaluation of rhizo-sphere bacterial antagonists for their potential to bioprotectpotato (Solanum tuberosum) against bacterial wilt (Ralstoniasolanacearum)rdquo Biological Control vol 47 no 3 pp 282ndash2882008

[39] L LagaceM PitreM Jacqeus andDRoy ldquoIdentification of thebacterial community ofmaple sap by using amplified ribosomalDNA (rDNA) restriction analysis and rDNA sequencingrdquoApplied and Environmental Microbiology vol 70 no 4 pp2052ndash2060 2004

[40] H H Long D G Sonntag D D Schmidt and I T BaldwinldquoThe structure of the culturable root bacterial endophyte com-munity of Nicotiana attenuata is organized by soil compositionand host plant ethylene production and perceptionrdquo NewPhytologist vol 185 no 2 pp 554ndash567 2010

[41] E Lopez-Fuentes V M Ruız-Valdiviezo E Martınez-RomeroF A Gutierrez-Miceli L Dendooven and R Rincon-RosalesldquoBacterial community in the roots and rhizosphere of Hyper-icum silenoides Juss 1804rdquo African Journal of MicrobiologyResearch vol 6 no 11 pp 2704ndash2711 2012

[42] M Fang R J Kremer P P Motavalli and G Davis ldquoBacterialdiversity in rhizospheres of nontransgenic and transgenic cornrdquoApplied and EnvironmentalMicrobiology vol 71 no 7 pp 4132ndash4136 2005

[43] C R Howell and R D Stiphanovic ldquoProduction of ammonia byEnterobacter cloaceae and its possible role in biological controlof Pythium pre-emergence damping-off by the bacteriumrdquoEcology and Epidemiology vol 78 no 8 pp 1075ndash1078 1988

[44] A Ramette M Frapolli G Defago and Y Moenne-LoccozldquoPhylogeny of HCN synthase-encoding hcnBC genes in bio-control fluorescent pseudomonads and its relationship withhost plant species and HCN synthesis abilityrdquoMolecular Plant-Microbe Interactions vol 16 no 6 pp 525ndash535 2003

[45] I Loaces L Ferrando and A F Scavino ldquoDynamics diversityand function of endophytic siderophore-producing bacteria inricerdquoMicrobial Ecology vol 61 no 3 pp 606ndash618 2011

[46] Y C Kim J Leveau B B M Gardener E A Pierson L SPierson and C-M Ryu ldquoThe multifactorial basis for planthealth promotion by plant-associated bacteriardquo Applied andEnvironmental Microbiology vol 77 no 5 pp 1548ndash1555 2011

[47] J M Gardner J L Chandler and A W Feldman ldquoGrowthresponses and vascular plugging of citrus inoculated withrhizobacteria and xylem-resident bacteriardquo Plant and Soil vol86 no 3 pp 333ndash345 1985

[48] C Pliego C Ramos A de Vicente and F M Cazorla ldquoScreen-ing for candidate bacterial biocontrol agents against soilborne

14 International Journal of Microbiology

fungal plant pathogensrdquoPlant and Soil vol 340 no 1-2 pp 505ndash520 2011

[49] H Feng Y Li and Q Liu ldquoEndophytic bacterial communi-ties in tomato plants with differential resistance to Ralstoniasolanacearumrdquo African Journal of Microbiology Research vol 7no 15 pp 1311ndash1318 2013

[50] A V Sturz B R Christie B G Matheson W J Arsenaultand N A Buchanan ldquoEndophytic bacterial communities inthe periderm of potato tubers and their potential to improveresistance to soil-borne plant pathogensrdquo Plant Pathology vol48 no 3 pp 360ndash369 1999

[51] A V Sturz B R Christie and J Nowak ldquoBacterial endophytespotential role in developing sustainable systems of crop produc-tionrdquo Critical Reviews in Plant Sciences vol 19 no 1 pp 1ndash302000

[52] R Ramesh A A Joshi and M P Ghanekar ldquoPseudomonadsmajor antagonistic endophytic bacteria to suppress bacterialwilt pathogenRalstonia solanacearum in the eggplant (Solanummelongena L)rdquo World Journal of Microbiology and Biotechnol-ogy vol 25 no 1 pp 47ndash55 2009

[53] A A Nawangsih I Damayanti S Wiyono and J G KartikaldquoSelection and characterization of endophytic bacteria as bio-control agents of tomato Bacterial Wilt DiseaserdquoHayati Journalof Biosciences vol 18 no 2 pp 66ndash70 2011

[54] R Vargas-Ayala R Rodrıguez-Kabana G Morgan-Jones J AMcInroy and J W Kloepper ldquoShifts in soil microflora inducedby velvetbean (Mucuna deeringiana) in cropping systems tocontrol root-knot nematodesrdquo Biological Control vol 17 no 1pp 11ndash22 2000

[55] D K Zinniel P Lambrecht N B Harris et al ldquoIsolationand characterization of endophytic colonizing bacteria fromagronomic crops and prairie plantsrdquoApplied and EnvironmentalMicrobiology vol 68 no 5 pp 2198ndash2208 2002

[56] F Lemessa and W Zeller ldquoScreening rhizobacteria for biolog-ical control of Ralstonia solanacearum in Ethiopiardquo BiologicalControl vol 42 no 3 pp 336ndash344 2007

[57] M S El-AlbyadM A El-Sayed A R El-Shanshoury andNHEl-Batanouny ldquoEffect of culture conditions on the antimicrobialactivities of UV-mutants of Streptomyces corchorusii and Sspiroverticillatus against bean and banana wilt pathogensrdquoMicrobiological Research vol 151 no 2 pp 201ndash211 1996

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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International Journal of

Volume 2014

Zoology

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GenomicsInternational Journal of

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Signal TransductionJournal of

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ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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International Journal of

Microbiology

International Journal of Microbiology 9

2545

2545

364

364

1273 2909

ActinobacteriaFirmicutesAlpha proteobacteria

Beta proteobacteriaGamma proteobacteriaBacteroidetes

(a)

Num

ber o

f XRB

Actin

obac

teria

Firm

icut

es

Alp

ha p

rote

obac

teria

Beta

pro

teob

acte

ria

Gam

ma p

rote

obac

teria

Bact

eroi

dete

s

Number of antagonistic XRB in each phylumsubdivision

Phylasubdivision

Total number of XRB belonging to each phylumsubdivision

25

20

15

10

5

0

2

14

9

16

4

7

1

2

1

2

11

14

(b)

Figure 2 (a) Distribution of XRB (phylumsubdivision level) identified by 16S rRNA gene sequencing Values indicate percentages of strainsbelonging to each phylasubdivision amongst the 55 identified strains (b) Distribution of antagonistic and nonantagonistic strains of XRB ineach phylumsubdivision

different genera of bacteria were identified Major gen-era identified are Bacillus sp (11 strains) Enterobacter sp(6 strains) Microbacterium sp (5 strains) Staphylococcussp (5 strains) Pseudomonas sp (5 strains) and Agrobac-terium sp (3 strains) Additional genera identified includ-ing Micrococcus sp Sphingomonas sp Flavobacterium spChryseobacterium sp Burkholderia sp and Xenophilus spare listed in Table 4 Based on the identification phylumProteobacteria consisting of Gram-negative bacteria of sub-divisions Alpha Proteobacteria (1273) Beta Proteobacteria(364) and Gamma Proteobacteria (2545) were predom-inant (4181 strains identified) followed by phyla Firmi-cutes (2909) Actinobacteria (2545) and Bacteroidetes(364) (Figure 2(a))

Eleven antagonistic strains identified belonged toGammasubdivision of Proteobacteria consisting of five strainseachof Enterobacter sp and fluorescent and nonfluorescent Pseu-domonas sp and one strain of Pantoea eucrina (XB126)(Figure 2(b)) Nine antagonists identified were of phylaFirmicutes consisting of Staphylococcus sp (5 strains) andBacillus sp (4 strains) Agrobacterium strains XB1 XB86 andXB165 Sphingomonas sp (XB197) of the Alpha Proteobacte-ria Streptomyces sp (XB200) and Janibacter melonis (XB70)of phyla Actinobacteria were found to be antagonistic Addi-tional antagonistic XRB include Burkholderia sp (XB140) of120573 Proteobacteria and Flavobacterium sp (XB203) of phylaBacteroidetes (Figure 2(b))

37 Statistical Analysis The statistical analysis of percent-age wilts and shoot length of eggplant was performedusing Web Agri Statistical Package (WASP) version 20(httpwwwicargoaresinwasp20indexphp)

4 Discussion

Eggplant and chilli not only are of economic and culturalimportance but also are common ingredients in the cuisinethroughout India In the coastal state of GoaR solanacearumhas been reported to be a destructive pathogen in cultivationof eggplant and chilli [4] Isolation of biocontrol agentsagainst the BW pathogen has been commonly restricted toendophytic tissue and plant rhizosphere [26 27] Studieson xylem colonizing endophytes were undertaken becausewe speculated existence of interactions between the XRBand vascular wilt pathogen R solanacearum during xylemcolonization Our study reveals the diversity biocontrolpotential and identity of endophytic xylem colonizers fromsolanaceous crops cultivated in Goa India A total of 167bacteria were isolated from the xylem of eggplant chilliand S torvum with Gram-negative bacteria (5928) pre-dominating in the collection Congruent to our observationGardner et al [7] and Bell et al [11] have earlier reportedisolation of more number of Gram-negative rod shapedbacteria from xylem of citrus and grapevine using vacuuminfiltration Scholander pressure bomb was found to beuseful in extraction of diverse bacterial genera from xylemtissues in contrast to trituration methods that yielded highernumber of Gram-positive rod shaped bacteria [12] ThoughScholander pressure bomb was not used in this study acombination of vacuum infiltration [7 11] and triturationof decorticated stems [10 12] was employed with an aimto isolate diverse XRB from xylem tissues This is the firststudy reporting the use of vacuum infiltration and triturationmethods for isolating xylem residing bacteria from eggplantchilli and S torvum

Traditionally bacteria have been characterized andgrouped based on colony morphology and biochemical

10 International Journal of Microbiology

Table 4 List of XRB identified by partial 16S rRNAgene sequencing their plant host accession numbers closestNCBImatch and similarity

Strain Plant host Accession number Closest NCBI match similarityXB1lowast SE KF447383 Agrobacterium tumefaciens 99XB7lowast SE KF447384 Pseudomonas aeruginosa 99XB8lowast SE KF447385 Staphylococcus haemolyticus 99XB20lowast SE KF447386 Staphylococcus haemolyticus 100XB22 SE KF447387 Brevibacterium casei 99XB25 C KF447388 Enterobacter sp 95XB27lowast SE KF447389 Staphylococcus haemolyticus 99XB34 SE KF447390 Curtobacterium sp 99XB35 SE KF447391 Microbacterium sp 99XB36 SE KF447392 Xenophilus sp 99XB37 SE KF447393 Chryseobacterium sp 99XB40 SE KF447394 Micrococcus luteus 89XB41 SE KF447395 Bacillus sp 99XB47 SE KF447396 Bacillus sp 99XB53 SE KF447397 Micrococcus sp 99XB62lowast SE KF447398 Pseudomonas sp 99XB64 SE KF447399 Bacillus thuringiensis 100XB66 SE KF447400 Pectobacterium carotovorum 99XB70lowast SE KF447401 Janibacter melonis 99XB86lowast C KF447402 Agrobacterium tumefaciens 99XB87 C KF447403 Bacillus barbaricus 99XB88 C KF447404 Bacillus sp 99XB92 C KF447405 Brevundimonas vesicularis 99XB93lowast C KF447406 Bacillus safensis 100XB94 C KF447407 Bacillus sp 100XB98 C KF447408 Microbacterium sp 100XB99lowast SE KF447409 Enterobacter sp 99XB100lowast SE KF447410 Enterobacter cloacae 98XB103 SE KF447411 Brachybacterium phenoliresistens 99XB109 SE KF447412 Rhodococcus corynebacterioides 99XB114lowast SE KF447413 Pseudomonas stutzeri 99XB122lowast SE KF913446 Pseudomonas aeruginosa 100XB123lowast SE KF447414 Enterobacter sp 99XB126lowast C KF447415 Pantoea eucrina 99XB134lowast SE KF447416 Enterobacter sp 99XB137 SE KF447417 Klebsiella sp 99XB140lowast SE KF447418 Burkholderia sp 99XB153lowast SE KF447419 Bacillus amyloliquefaciens 99XB157lowast SE KF447420 Bacillus subtilis 100XB158 RE KF447421 Bosea sp 99XB159 RE KF447422 Microbacterium xylanilyticum 99XB161 RE KF447423 Microbacterium aurum 99XB165lowast RE KF447424 Agrobacterium sp 99XB167 RE KF447425 Microbacterium aurum 99XB168 RE KF447426 Bacillus aryabhattai 100XB169lowast RE KF447427 Staphylococcus gallinarum 99XB170lowast RE KF447428 Staphylococcus sp 99XB177lowast RE KF447429 Bacillus cereus 99XB188 RE KF447430 Sphingomonas sp 99XB190 RE KF447431 Brevibacterium casei 99

International Journal of Microbiology 11

Table 4 Continued

Strain Plant host Accession number Closest NCBI match similarityXB196lowast RE KF447432 Enterobacter kobei 99XB197lowast RE KF447433 Sphingomonas sp 98XB200lowast ST KF913447 Streptomyces sp 100XB202lowast ST KF447434 Pseudomonas sp 99XB203lowast ST KF447435 Flavobacterium sp 99lowastStrain is antagonistic to R solanacearum based on bioassaySE bacterial wilt susceptible eggplant RE bacterial wilt resistant eggplant C chilli ST Solanum torvum

tests However whole genome fingerprinting or PCR-RFLPbased methods are rapid tools for determining geneticdiversity of bacteria in a given collection ARDRA which isa type of PCR based RFLP method has been used widely inestimating genetic diversity endophytic bacterial populationsand clustering genetically similar strains [39ndash41] In additionto earlier reports our study demonstrates the usefulnessof ARDRA as a tool to cluster genetically identical strainsof endophytic XRB isolated from eggplant chilli andS torvum In our study 9161 strains (119899 = 153) weregrouped in 24 haplotypes by using ARDRA The majority ofthese haplotypes represent a combination of XRB isolatedfrom different solanaceous plants from diverse localesThese results indicate that xylem of eggplant chilli andS torvum largely bears similar population of XRB whichcan efficiently cross colonize eggplant chilli or S torvumNevertheless 839 strains had unique ARDRA fingerprintand formed separable haplotypes This observation leads toa conclusion that a minor population of xylem inhabitantsare restricted to a specific plant species and cannot easilycross colonize xylem of other solanaceous plants Plantsare known to selectively support endophytic colonizationby specific bacteria [14] However factors that determinethe selection of xylem colonists or the ability of XRBto colonize eggplant chilli and S torvum in this studyremain unknown Interestingly the structure of endophyticcommunity in Nicotiana attenuata a member of Solanaceaefamily is shown to be influenced by soil composition andethylene homeostasis [40] Earlier evidence has shown thatcolonization by endophytic bacteria is also governed by plantgenotype as well as root exudates [42]

Only 1677 XRB out of 167 were antagonistic to Rsolanacearum based on in vitro assays Antagonistic XRBproduced volatile and diffusible inhibitory compoundsnamely HCN ammonia and acetoin and siderophoresThese substances have been long known to be involvedin disease suppression and indirect growth promotion inplants [43ndash46] These mechanisms possibly played a rolein the evident biocontrol effect against BW exhibited bythe XRB in the greenhouse screening Endophytic bacteriahave been known to have plant growth promoting traitsnamely production of IAA ACC deaminase and phosphatesolubilization [25 33 36] These traits were detected in themajority of antagonistic XRB tested in this study and mayhave resulted in the observed increase in shoot length ofeggplant in our greenhouse experiments In contrast strains

XB20 XB196 and XB203 suppressed growth in eggplantunder greenhouse conditions however no visible symptomsof disease were observed Vascular plugging and productionof certainmetabolites toxic to plant cells but not cell viabilitymay have resulted in stunted shoot in eggplant [11 47]

Evaluation of efficacy of antagonistic organisms to sup-press the plant diseases under greenhouse conditions isone of the key steps for selecting a potential biocontrolagent for disease management [48] Endophytic strains fromBW susceptible varieties of eggplant have been shown toprevent wilt and promote growth in eggplant earlier [27]Our greenhouse screening shows that 3846 of antagonisticXRB with biocontrol efficacies greater than 40 were isolatesfrom BW resistant varieties of eggplantThis raises a questionwhether the bacteria from resistant varieties are involved inBW resistance and whether BW resistant varieties are ableto selectively influence xylem colonization by antagonisticbacteria Presence of higher number of endophytes withantagonistic abilities was reported in BW resistant varieties oftomato as compared to susceptible varieties and their role inresistance to BWwas proposed [49] Similar observations oncorrelation of resistance of potato to soft rot and endophyticbacteria have been reported [50]Thus BW resistant varietiescan be considered a better host for isolating potential biocon-trol strains for management of bacterial wilt

Identification of 55 XRB strains by 16S rRNA genesequencing revealed the presence of 23 diverse genera ofbacteria belonging to 4 phyla of Eubacteria Strains belongingto phyla Firmicutes Actinobacteria and 120574 subdivision ofProteobacteria were the major xylem colonists identified inthis study Several genera of bacteria belonging to these phylahave also been reported to be present in endophytic tissuesand xylem of a variety of other agricultural and horticulturalplant species [14 51] In addition the majority of the antag-onists identified belonged to Enterobacter sp Pseudomonassp Bacillus sp and Staphylococcus sp Congruent to ourresults several researchers have reported bacteria isolatedfrom solanaceous crops and belonging to similar genera tobe antagonistic to R solanacearum [26 51ndash53] HoweverFlavobacterium spand Janibacter melonis identified in thisstudy have never been previously reported to be inhibitoryto R solanacearum Large population of the xylem inhabitingbacterial flora accounting for 8323 exhibited no antago-nism towards R solanacearum Nonantagonistic XRB wereidentified predominantly as Microbacterium sp Endophyticpersistence and nematicidal activities of Microbacterium sp

12 International Journal of Microbiology

have been reported [54 55] Therefore the collection of XRBisolated in this study can be screened for inhibitory activitiesagainst other important agricultural pests

Though few strains namely XB86 XB169 and XB177exhibited plant beneficial properties in this study their use-fulness in plant disease control remains to be seen XB86has been identified as Agrobacterium tumefaciens the crowngall disease pathogen and its deployment as biocontrolagent is uncertain XB169 (Staphylococcus gallinarum) andXB177 (Bacillus cereus) are reported as opportunistic animalpathogens and thus unsuitable for field applications Strep-tomyces sp has earlier been reported as antagonistic to Rsolanacearum and tested for management of wilt in potatoand tomato [56 57] XB200 (Streptomyces sp) is one of theXRB high BCE and GPE it could be explored further forbiocontrol of bacterial wilt after additional characterizationand field evaluation

5 Conclusion

This study is the first report on the identity of novel anddiverse XRB colonizing the xylem of eggplant chilli and Storvum XRB particularly from BW resistant varieties werefound to protect eggplant from bacterial wilt and enhancedgrowth in eggplant in the greenhouse screening Thereforethe repertoire of XRB reported in this study may be usefulfor cultivation of eggplant in BW affected areas

Conflict of Interests

The authors hereby declare that they have no conflict ofinterests regarding the publication of this paper

Acknowledgments

The authors greatly acknowledge financial support fromIndian Council of Agricultural Research (ICAR) New DelhiIndia through ldquoOutreach project on Phytopthora Fusariumand Ralstonia diseases of horticultural and field cropsrdquo-(PhytoFuRa) and the Director ICAR Research Complex forGoa India for the facilities

References

[1] E Yabuuchi Y KosakoHOyaizu et al ldquoProposal ofBurkholde-ria gen nov and transfer of seven species of the genusPseudomonas homology group II to the new genus with thetype species Burkholderia cepacia (Palleroni and Holmes 1981)comb novrdquo Microbiology and Immunology vol 36 no 12 pp1251ndash1275 1992

[2] E Wicker L Grassart R Coranson-Beaudu et al ldquoRalstoniasolanacearum strains from Martinique (French West Indies)exhibiting a new pathogenic potentialrdquo Applied and Environ-mental Microbiology vol 73 no 21 pp 6790ndash6801 2007

[3] Y Liu A Kanda K Yano et al ldquoMolecular typing of Japanesestrains of Ralstonia solanacearum in relation to the ability toinduce a hypersensitive reaction in tobaccordquo Journal of GeneralPlant Pathology vol 75 no 5 pp 369ndash380 2009

[4] R Ramesh ldquoField evaluation of biological control agents for themanagement of Ralstonia solanacearum in Brinjalrdquo Journal ofMycology and Plant Pathology vol 36 no 2 pp 327ndash328 2006

[5] J Vasse P Frey and A Trigalet ldquoMicroscopic studies ofintercellular infection and protoxylem invasion of tomatoroots by Pseudomonas solanacearumrdquoMolecular Plant-MicrobeInteractions vol 8 no 2 pp 241ndash251 1995

[6] S Genin and C Boucher ldquoRalstonia solanacearum secrets of amajor pathogen unveiled by analysis of its genomerdquo MolecularPlant Pathology vol 3 no 3 pp 111ndash118 2002

[7] J M Gardner A W Feldman and R M Zablotowicz ldquoIdentityand behavior of xylem-residing bacteria in rough lemon roots ofFlorida citrus treesrdquo Applied and Environmental Microbiologyvol 43 no 6 pp 1335ndash1342 1982

[8] M J Jacobs W M Bugbee and D A Gabriellson ldquoEnu-meration location and characterization of endophytic bacteriawithin sugar beet rootsrdquoCanadian Journal of Botany vol 63 no7 pp 1262ndash1265 1985

[9] D G Patriquin and J Dobereiner ldquoLight microscopy observa-tions of tetrazolium-reducing bacteria in the endorhizosphereof maize and other grasses in Brazilrdquo Canadian Journal ofMicrobiology vol 24 no 6 pp 734ndash742 1978

[10] S Gagne C Richard H Rousseau and H Antoun ldquoXylem-residing bacteria in alfalfa rootsrdquo Canadian Journal of Micro-biology vol 33 no 11 pp 996ndash1000 1987

[11] C R Bell G A Dickie W L G Harvey and J W Y FChan ldquoEndophytic bacteria in grapevinerdquo Canadian Journal ofMicrobiology vol 41 no 1 pp 46ndash53 1995

[12] J Hallmann J W Kloepper and R Rodrıguez-Kabana ldquoAppli-cation of Scholander pressure bomb to studies on endophyticbacteria of plantsrdquo Canadian Journal of Microbiology vol 43no 5 pp 411ndash416 1997

[13] J S Lampel G L Canter M B Dimock et al ldquoIntegrativecloning expression and stability of the cryIA(c) gene fromBacillus thuringiensis subsp kurstaki in a recombinant strain ofClavibacter xyli subsp cynodontisrdquo Applied and EnvironmentalMicrobiology vol 60 no 2 pp 501ndash508 1994

[14] M Rosenblueth and E Martınez-Romero ldquoBacterial endo-phytes and their interactions with hostsrdquo Molecular Plant-Microbe Interactions vol 19 no 8 pp 827ndash837 2006

[15] P R Hardoim L S van Overbeek and J D van ElsasldquoProperties of bacterial endophytes and their proposed role inplant growthrdquo Trends in Microbiology vol 16 no 10 pp 463ndash471 2008

[16] R Aravind A Kumar S J Eapen and K V RamanaldquoEndophytic bacterial flora in root and stem tissues of blackpepper (Piper nigrum L) genotype isolation identificationand evaluation against Phytophthora capsicirdquo Letters in AppliedMicrobiology vol 48 no 1 pp 58ndash64 2009

[17] J Kuklinsky-Sobral W L Araujo R Mendes I O Geraldi AA Pizzirani-Kleiner and J L Azevedo ldquoIsolation and charac-terization of soybean-associated bacteria and their potential forplant growth promotionrdquo Environmental Microbiology vol 6no 12 pp 1244ndash1251 2004

[18] S Compant B Duffy J Nowak C Clement and E A BarkaldquoUse of plant growth-promoting bacteria for biocontrol ofplant diseases principles mechanisms of action and futureprospectsrdquoApplied and Environmental Microbiology vol 71 no9 pp 4951ndash4959 2005

[19] E T Thorne B M Young G M Young et al ldquoThe structureof xylem vessels in grapevine (Vitaceae) and a possible passive

International Journal of Microbiology 13

mechanism for the systemic spread of bacterial diseaserdquo Amer-ican Journal of Botany vol 93 no 4 pp 497ndash504 2006

[20] W T Seo W J Lim E J Kim H D Yun Y H Lee and KM Cho ldquoEndophytic bacterial diversity in the young radishand their antimicrobial activity against pathogensrdquo Journal ofApplied Biological Chemistry vol 53 no 4 pp 493ndash503 2010

[21] S Compant H Kaplan A Sessitsch J Nowak E Ait Barkaand C Clement ldquoEndophytic colonization ofVitis vinifera L byBurkholderia phytofirmans strain PsJN from the rhizosphere toinflorescence tissuesrdquo FEMS Microbiology Ecology vol 63 no1 pp 84ndash93 2005

[22] P S B Miguel J C Delvaux M N V de Oliveira et al ldquoDiver-sity of endophytic bacteria in the fruits of Coffea canephorardquoAfrican Journal of Microbiology Research vol 7 no 7 pp 586ndash594 2013

[23] A Lopez-Lopez M A Rogel E Ormeno-Orrillo J Martınez-Romero and E Martınez-Romero ldquoPhaseolus vulgaris seed-borne endophytic community with novel bacterial species suchas Rhizobium endophyticum sp novrdquo Systematic and AppliedMicrobiology vol 33 no 6 pp 322ndash327 2010

[24] M Lwin and S L Ranamukhaarachchi ldquoDevelopment of bio-logical control of Ralstonia solanacearum through antagonisticmicrobial populationsrdquo International Journal of Agriculture andBiology vol 8 no 5 pp 657ndash660 2006

[25] F Rasche E Marco-Noales H Velvis et al ldquoStructural char-acteristics and plant-beneficial effects of bacteria colonizing theshoots of field grown conventional and geneticallymodifiedT4-lysozyme producing potatoesrdquo Plant and Soil vol 289 no 1-2pp 123ndash140 2006

[26] N Amaresan V Jayakumar K Kumar and N ThajuddinldquoEndophytic bacteria from tomato and chilli their diversity andantagonistic potential against Ralstonia solanacearumrdquoArchivesof Phytopathology and Plant Protection vol 45 no 3 pp 344ndash355 2012

[27] R Ramesh and G S Phadke ldquoRhizosphere and endophyticbacteria for the suppression of eggplant wilt caused byRalstoniasolanacearumrdquo Crop Protection vol 37 pp 35ndash41 2012

[28] W J French R G Christis and D L Stassi ldquoRecovery ofrickettsialike bacteria by vacuum infiltration of peach tissuesaffected with phony diseaserdquo Phytopathology vol 67 pp 945ndash948 1977

[29] P R Viss E M Brooks and J A Driver ldquoA simplified methodfor the control of bacterial contamination in woody plant tissueculturerdquo In Vitro Cellular andDevelopmental Biology vol 27 no1 article 42 1991

[30] K Wilson ldquoPreparation of genomic DNA from bacteriardquo inCurrent Protocols in Molecular Biology F M Ausubel R BrentR E Kingston et al Eds pp 241ndash245 John Wiley amp SonsNew York NY USA 1997

[31] M Saraf AThakkar U PandyaM Joshi and J Parikh ldquoPoten-tial of plant growth promoting microorganisms as biofertilizersand biopesticides and itrsquos exploitation in sustainable agricul-turerdquo Journal of Microbiology and Biotechnology Research vol3 no 5 pp 54ndash62 2013

[32] A P G C Marques C Pires H Moreira A O S S Rangeland P M L Castro ldquoAssessment of the plant growth promotionabilities of six bacterial isolates using Zea mays as indicatorplantrdquo Soil Biology and Biochemistry vol 42 no 8 pp 1229ndash1235 2010

[33] X Hao P Xie L Johnstone S J Miller C Rensingand G Wei ldquoGenome sequence and mutational analysis of

plant-growth-promoting bacteriumAgrobacterium tumefaciensCCNWGS0286 isolated from a zinc-lead mine tailingrdquo Appliedand Environmental Microbiology vol 78 no 15 pp 5384ndash53942012

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

[35] S A Gordon and L G Paleg ldquoQuantitative measurement ofindole acetic acidrdquo Plant Physiology vol 10 pp 37ndash48 1957

[36] A Godinho R Ramesh and S Bhosle ldquoBacteria from sanddunes of Goa promoting growth in eggplantrdquoWorld Journal ofAgricultural Sciences vol 6 no 5 pp 555ndash564 2010

[37] J-H Guo H-Y Qi Y-H Guo et al ldquoBiocontrol of tomato wiltby plant growth-promoting rhizobacteriardquo Biological Controlvol 29 no 1 pp 66ndash72 2004

[38] N Aliye C Fininsa and Y Hiskias ldquoEvaluation of rhizo-sphere bacterial antagonists for their potential to bioprotectpotato (Solanum tuberosum) against bacterial wilt (Ralstoniasolanacearum)rdquo Biological Control vol 47 no 3 pp 282ndash2882008

[39] L LagaceM PitreM Jacqeus andDRoy ldquoIdentification of thebacterial community ofmaple sap by using amplified ribosomalDNA (rDNA) restriction analysis and rDNA sequencingrdquoApplied and Environmental Microbiology vol 70 no 4 pp2052ndash2060 2004

[40] H H Long D G Sonntag D D Schmidt and I T BaldwinldquoThe structure of the culturable root bacterial endophyte com-munity of Nicotiana attenuata is organized by soil compositionand host plant ethylene production and perceptionrdquo NewPhytologist vol 185 no 2 pp 554ndash567 2010

[41] E Lopez-Fuentes V M Ruız-Valdiviezo E Martınez-RomeroF A Gutierrez-Miceli L Dendooven and R Rincon-RosalesldquoBacterial community in the roots and rhizosphere of Hyper-icum silenoides Juss 1804rdquo African Journal of MicrobiologyResearch vol 6 no 11 pp 2704ndash2711 2012

[42] M Fang R J Kremer P P Motavalli and G Davis ldquoBacterialdiversity in rhizospheres of nontransgenic and transgenic cornrdquoApplied and EnvironmentalMicrobiology vol 71 no 7 pp 4132ndash4136 2005

[43] C R Howell and R D Stiphanovic ldquoProduction of ammonia byEnterobacter cloaceae and its possible role in biological controlof Pythium pre-emergence damping-off by the bacteriumrdquoEcology and Epidemiology vol 78 no 8 pp 1075ndash1078 1988

[44] A Ramette M Frapolli G Defago and Y Moenne-LoccozldquoPhylogeny of HCN synthase-encoding hcnBC genes in bio-control fluorescent pseudomonads and its relationship withhost plant species and HCN synthesis abilityrdquoMolecular Plant-Microbe Interactions vol 16 no 6 pp 525ndash535 2003

[45] I Loaces L Ferrando and A F Scavino ldquoDynamics diversityand function of endophytic siderophore-producing bacteria inricerdquoMicrobial Ecology vol 61 no 3 pp 606ndash618 2011

[46] Y C Kim J Leveau B B M Gardener E A Pierson L SPierson and C-M Ryu ldquoThe multifactorial basis for planthealth promotion by plant-associated bacteriardquo Applied andEnvironmental Microbiology vol 77 no 5 pp 1548ndash1555 2011

[47] J M Gardner J L Chandler and A W Feldman ldquoGrowthresponses and vascular plugging of citrus inoculated withrhizobacteria and xylem-resident bacteriardquo Plant and Soil vol86 no 3 pp 333ndash345 1985

[48] C Pliego C Ramos A de Vicente and F M Cazorla ldquoScreen-ing for candidate bacterial biocontrol agents against soilborne

14 International Journal of Microbiology

fungal plant pathogensrdquoPlant and Soil vol 340 no 1-2 pp 505ndash520 2011

[49] H Feng Y Li and Q Liu ldquoEndophytic bacterial communi-ties in tomato plants with differential resistance to Ralstoniasolanacearumrdquo African Journal of Microbiology Research vol 7no 15 pp 1311ndash1318 2013

[50] A V Sturz B R Christie B G Matheson W J Arsenaultand N A Buchanan ldquoEndophytic bacterial communities inthe periderm of potato tubers and their potential to improveresistance to soil-borne plant pathogensrdquo Plant Pathology vol48 no 3 pp 360ndash369 1999

[51] A V Sturz B R Christie and J Nowak ldquoBacterial endophytespotential role in developing sustainable systems of crop produc-tionrdquo Critical Reviews in Plant Sciences vol 19 no 1 pp 1ndash302000

[52] R Ramesh A A Joshi and M P Ghanekar ldquoPseudomonadsmajor antagonistic endophytic bacteria to suppress bacterialwilt pathogenRalstonia solanacearum in the eggplant (Solanummelongena L)rdquo World Journal of Microbiology and Biotechnol-ogy vol 25 no 1 pp 47ndash55 2009

[53] A A Nawangsih I Damayanti S Wiyono and J G KartikaldquoSelection and characterization of endophytic bacteria as bio-control agents of tomato Bacterial Wilt DiseaserdquoHayati Journalof Biosciences vol 18 no 2 pp 66ndash70 2011

[54] R Vargas-Ayala R Rodrıguez-Kabana G Morgan-Jones J AMcInroy and J W Kloepper ldquoShifts in soil microflora inducedby velvetbean (Mucuna deeringiana) in cropping systems tocontrol root-knot nematodesrdquo Biological Control vol 17 no 1pp 11ndash22 2000

[55] D K Zinniel P Lambrecht N B Harris et al ldquoIsolationand characterization of endophytic colonizing bacteria fromagronomic crops and prairie plantsrdquoApplied and EnvironmentalMicrobiology vol 68 no 5 pp 2198ndash2208 2002

[56] F Lemessa and W Zeller ldquoScreening rhizobacteria for biolog-ical control of Ralstonia solanacearum in Ethiopiardquo BiologicalControl vol 42 no 3 pp 336ndash344 2007

[57] M S El-AlbyadM A El-Sayed A R El-Shanshoury andNHEl-Batanouny ldquoEffect of culture conditions on the antimicrobialactivities of UV-mutants of Streptomyces corchorusii and Sspiroverticillatus against bean and banana wilt pathogensrdquoMicrobiological Research vol 151 no 2 pp 201ndash211 1996

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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International Journal of

Microbiology

10 International Journal of Microbiology

Table 4 List of XRB identified by partial 16S rRNAgene sequencing their plant host accession numbers closestNCBImatch and similarity

Strain Plant host Accession number Closest NCBI match similarityXB1lowast SE KF447383 Agrobacterium tumefaciens 99XB7lowast SE KF447384 Pseudomonas aeruginosa 99XB8lowast SE KF447385 Staphylococcus haemolyticus 99XB20lowast SE KF447386 Staphylococcus haemolyticus 100XB22 SE KF447387 Brevibacterium casei 99XB25 C KF447388 Enterobacter sp 95XB27lowast SE KF447389 Staphylococcus haemolyticus 99XB34 SE KF447390 Curtobacterium sp 99XB35 SE KF447391 Microbacterium sp 99XB36 SE KF447392 Xenophilus sp 99XB37 SE KF447393 Chryseobacterium sp 99XB40 SE KF447394 Micrococcus luteus 89XB41 SE KF447395 Bacillus sp 99XB47 SE KF447396 Bacillus sp 99XB53 SE KF447397 Micrococcus sp 99XB62lowast SE KF447398 Pseudomonas sp 99XB64 SE KF447399 Bacillus thuringiensis 100XB66 SE KF447400 Pectobacterium carotovorum 99XB70lowast SE KF447401 Janibacter melonis 99XB86lowast C KF447402 Agrobacterium tumefaciens 99XB87 C KF447403 Bacillus barbaricus 99XB88 C KF447404 Bacillus sp 99XB92 C KF447405 Brevundimonas vesicularis 99XB93lowast C KF447406 Bacillus safensis 100XB94 C KF447407 Bacillus sp 100XB98 C KF447408 Microbacterium sp 100XB99lowast SE KF447409 Enterobacter sp 99XB100lowast SE KF447410 Enterobacter cloacae 98XB103 SE KF447411 Brachybacterium phenoliresistens 99XB109 SE KF447412 Rhodococcus corynebacterioides 99XB114lowast SE KF447413 Pseudomonas stutzeri 99XB122lowast SE KF913446 Pseudomonas aeruginosa 100XB123lowast SE KF447414 Enterobacter sp 99XB126lowast C KF447415 Pantoea eucrina 99XB134lowast SE KF447416 Enterobacter sp 99XB137 SE KF447417 Klebsiella sp 99XB140lowast SE KF447418 Burkholderia sp 99XB153lowast SE KF447419 Bacillus amyloliquefaciens 99XB157lowast SE KF447420 Bacillus subtilis 100XB158 RE KF447421 Bosea sp 99XB159 RE KF447422 Microbacterium xylanilyticum 99XB161 RE KF447423 Microbacterium aurum 99XB165lowast RE KF447424 Agrobacterium sp 99XB167 RE KF447425 Microbacterium aurum 99XB168 RE KF447426 Bacillus aryabhattai 100XB169lowast RE KF447427 Staphylococcus gallinarum 99XB170lowast RE KF447428 Staphylococcus sp 99XB177lowast RE KF447429 Bacillus cereus 99XB188 RE KF447430 Sphingomonas sp 99XB190 RE KF447431 Brevibacterium casei 99

International Journal of Microbiology 11

Table 4 Continued

Strain Plant host Accession number Closest NCBI match similarityXB196lowast RE KF447432 Enterobacter kobei 99XB197lowast RE KF447433 Sphingomonas sp 98XB200lowast ST KF913447 Streptomyces sp 100XB202lowast ST KF447434 Pseudomonas sp 99XB203lowast ST KF447435 Flavobacterium sp 99lowastStrain is antagonistic to R solanacearum based on bioassaySE bacterial wilt susceptible eggplant RE bacterial wilt resistant eggplant C chilli ST Solanum torvum

tests However whole genome fingerprinting or PCR-RFLPbased methods are rapid tools for determining geneticdiversity of bacteria in a given collection ARDRA which isa type of PCR based RFLP method has been used widely inestimating genetic diversity endophytic bacterial populationsand clustering genetically similar strains [39ndash41] In additionto earlier reports our study demonstrates the usefulnessof ARDRA as a tool to cluster genetically identical strainsof endophytic XRB isolated from eggplant chilli andS torvum In our study 9161 strains (119899 = 153) weregrouped in 24 haplotypes by using ARDRA The majority ofthese haplotypes represent a combination of XRB isolatedfrom different solanaceous plants from diverse localesThese results indicate that xylem of eggplant chilli andS torvum largely bears similar population of XRB whichcan efficiently cross colonize eggplant chilli or S torvumNevertheless 839 strains had unique ARDRA fingerprintand formed separable haplotypes This observation leads toa conclusion that a minor population of xylem inhabitantsare restricted to a specific plant species and cannot easilycross colonize xylem of other solanaceous plants Plantsare known to selectively support endophytic colonizationby specific bacteria [14] However factors that determinethe selection of xylem colonists or the ability of XRBto colonize eggplant chilli and S torvum in this studyremain unknown Interestingly the structure of endophyticcommunity in Nicotiana attenuata a member of Solanaceaefamily is shown to be influenced by soil composition andethylene homeostasis [40] Earlier evidence has shown thatcolonization by endophytic bacteria is also governed by plantgenotype as well as root exudates [42]

Only 1677 XRB out of 167 were antagonistic to Rsolanacearum based on in vitro assays Antagonistic XRBproduced volatile and diffusible inhibitory compoundsnamely HCN ammonia and acetoin and siderophoresThese substances have been long known to be involvedin disease suppression and indirect growth promotion inplants [43ndash46] These mechanisms possibly played a rolein the evident biocontrol effect against BW exhibited bythe XRB in the greenhouse screening Endophytic bacteriahave been known to have plant growth promoting traitsnamely production of IAA ACC deaminase and phosphatesolubilization [25 33 36] These traits were detected in themajority of antagonistic XRB tested in this study and mayhave resulted in the observed increase in shoot length ofeggplant in our greenhouse experiments In contrast strains

XB20 XB196 and XB203 suppressed growth in eggplantunder greenhouse conditions however no visible symptomsof disease were observed Vascular plugging and productionof certainmetabolites toxic to plant cells but not cell viabilitymay have resulted in stunted shoot in eggplant [11 47]

Evaluation of efficacy of antagonistic organisms to sup-press the plant diseases under greenhouse conditions isone of the key steps for selecting a potential biocontrolagent for disease management [48] Endophytic strains fromBW susceptible varieties of eggplant have been shown toprevent wilt and promote growth in eggplant earlier [27]Our greenhouse screening shows that 3846 of antagonisticXRB with biocontrol efficacies greater than 40 were isolatesfrom BW resistant varieties of eggplantThis raises a questionwhether the bacteria from resistant varieties are involved inBW resistance and whether BW resistant varieties are ableto selectively influence xylem colonization by antagonisticbacteria Presence of higher number of endophytes withantagonistic abilities was reported in BW resistant varieties oftomato as compared to susceptible varieties and their role inresistance to BWwas proposed [49] Similar observations oncorrelation of resistance of potato to soft rot and endophyticbacteria have been reported [50]Thus BW resistant varietiescan be considered a better host for isolating potential biocon-trol strains for management of bacterial wilt

Identification of 55 XRB strains by 16S rRNA genesequencing revealed the presence of 23 diverse genera ofbacteria belonging to 4 phyla of Eubacteria Strains belongingto phyla Firmicutes Actinobacteria and 120574 subdivision ofProteobacteria were the major xylem colonists identified inthis study Several genera of bacteria belonging to these phylahave also been reported to be present in endophytic tissuesand xylem of a variety of other agricultural and horticulturalplant species [14 51] In addition the majority of the antag-onists identified belonged to Enterobacter sp Pseudomonassp Bacillus sp and Staphylococcus sp Congruent to ourresults several researchers have reported bacteria isolatedfrom solanaceous crops and belonging to similar genera tobe antagonistic to R solanacearum [26 51ndash53] HoweverFlavobacterium spand Janibacter melonis identified in thisstudy have never been previously reported to be inhibitoryto R solanacearum Large population of the xylem inhabitingbacterial flora accounting for 8323 exhibited no antago-nism towards R solanacearum Nonantagonistic XRB wereidentified predominantly as Microbacterium sp Endophyticpersistence and nematicidal activities of Microbacterium sp

12 International Journal of Microbiology

have been reported [54 55] Therefore the collection of XRBisolated in this study can be screened for inhibitory activitiesagainst other important agricultural pests

Though few strains namely XB86 XB169 and XB177exhibited plant beneficial properties in this study their use-fulness in plant disease control remains to be seen XB86has been identified as Agrobacterium tumefaciens the crowngall disease pathogen and its deployment as biocontrolagent is uncertain XB169 (Staphylococcus gallinarum) andXB177 (Bacillus cereus) are reported as opportunistic animalpathogens and thus unsuitable for field applications Strep-tomyces sp has earlier been reported as antagonistic to Rsolanacearum and tested for management of wilt in potatoand tomato [56 57] XB200 (Streptomyces sp) is one of theXRB high BCE and GPE it could be explored further forbiocontrol of bacterial wilt after additional characterizationand field evaluation

5 Conclusion

This study is the first report on the identity of novel anddiverse XRB colonizing the xylem of eggplant chilli and Storvum XRB particularly from BW resistant varieties werefound to protect eggplant from bacterial wilt and enhancedgrowth in eggplant in the greenhouse screening Thereforethe repertoire of XRB reported in this study may be usefulfor cultivation of eggplant in BW affected areas

Conflict of Interests

The authors hereby declare that they have no conflict ofinterests regarding the publication of this paper

Acknowledgments

The authors greatly acknowledge financial support fromIndian Council of Agricultural Research (ICAR) New DelhiIndia through ldquoOutreach project on Phytopthora Fusariumand Ralstonia diseases of horticultural and field cropsrdquo-(PhytoFuRa) and the Director ICAR Research Complex forGoa India for the facilities

References

[1] E Yabuuchi Y KosakoHOyaizu et al ldquoProposal ofBurkholde-ria gen nov and transfer of seven species of the genusPseudomonas homology group II to the new genus with thetype species Burkholderia cepacia (Palleroni and Holmes 1981)comb novrdquo Microbiology and Immunology vol 36 no 12 pp1251ndash1275 1992

[2] E Wicker L Grassart R Coranson-Beaudu et al ldquoRalstoniasolanacearum strains from Martinique (French West Indies)exhibiting a new pathogenic potentialrdquo Applied and Environ-mental Microbiology vol 73 no 21 pp 6790ndash6801 2007

[3] Y Liu A Kanda K Yano et al ldquoMolecular typing of Japanesestrains of Ralstonia solanacearum in relation to the ability toinduce a hypersensitive reaction in tobaccordquo Journal of GeneralPlant Pathology vol 75 no 5 pp 369ndash380 2009

[4] R Ramesh ldquoField evaluation of biological control agents for themanagement of Ralstonia solanacearum in Brinjalrdquo Journal ofMycology and Plant Pathology vol 36 no 2 pp 327ndash328 2006

[5] J Vasse P Frey and A Trigalet ldquoMicroscopic studies ofintercellular infection and protoxylem invasion of tomatoroots by Pseudomonas solanacearumrdquoMolecular Plant-MicrobeInteractions vol 8 no 2 pp 241ndash251 1995

[6] S Genin and C Boucher ldquoRalstonia solanacearum secrets of amajor pathogen unveiled by analysis of its genomerdquo MolecularPlant Pathology vol 3 no 3 pp 111ndash118 2002

[7] J M Gardner A W Feldman and R M Zablotowicz ldquoIdentityand behavior of xylem-residing bacteria in rough lemon roots ofFlorida citrus treesrdquo Applied and Environmental Microbiologyvol 43 no 6 pp 1335ndash1342 1982

[8] M J Jacobs W M Bugbee and D A Gabriellson ldquoEnu-meration location and characterization of endophytic bacteriawithin sugar beet rootsrdquoCanadian Journal of Botany vol 63 no7 pp 1262ndash1265 1985

[9] D G Patriquin and J Dobereiner ldquoLight microscopy observa-tions of tetrazolium-reducing bacteria in the endorhizosphereof maize and other grasses in Brazilrdquo Canadian Journal ofMicrobiology vol 24 no 6 pp 734ndash742 1978

[10] S Gagne C Richard H Rousseau and H Antoun ldquoXylem-residing bacteria in alfalfa rootsrdquo Canadian Journal of Micro-biology vol 33 no 11 pp 996ndash1000 1987

[11] C R Bell G A Dickie W L G Harvey and J W Y FChan ldquoEndophytic bacteria in grapevinerdquo Canadian Journal ofMicrobiology vol 41 no 1 pp 46ndash53 1995

[12] J Hallmann J W Kloepper and R Rodrıguez-Kabana ldquoAppli-cation of Scholander pressure bomb to studies on endophyticbacteria of plantsrdquo Canadian Journal of Microbiology vol 43no 5 pp 411ndash416 1997

[13] J S Lampel G L Canter M B Dimock et al ldquoIntegrativecloning expression and stability of the cryIA(c) gene fromBacillus thuringiensis subsp kurstaki in a recombinant strain ofClavibacter xyli subsp cynodontisrdquo Applied and EnvironmentalMicrobiology vol 60 no 2 pp 501ndash508 1994

[14] M Rosenblueth and E Martınez-Romero ldquoBacterial endo-phytes and their interactions with hostsrdquo Molecular Plant-Microbe Interactions vol 19 no 8 pp 827ndash837 2006

[15] P R Hardoim L S van Overbeek and J D van ElsasldquoProperties of bacterial endophytes and their proposed role inplant growthrdquo Trends in Microbiology vol 16 no 10 pp 463ndash471 2008

[16] R Aravind A Kumar S J Eapen and K V RamanaldquoEndophytic bacterial flora in root and stem tissues of blackpepper (Piper nigrum L) genotype isolation identificationand evaluation against Phytophthora capsicirdquo Letters in AppliedMicrobiology vol 48 no 1 pp 58ndash64 2009

[17] J Kuklinsky-Sobral W L Araujo R Mendes I O Geraldi AA Pizzirani-Kleiner and J L Azevedo ldquoIsolation and charac-terization of soybean-associated bacteria and their potential forplant growth promotionrdquo Environmental Microbiology vol 6no 12 pp 1244ndash1251 2004

[18] S Compant B Duffy J Nowak C Clement and E A BarkaldquoUse of plant growth-promoting bacteria for biocontrol ofplant diseases principles mechanisms of action and futureprospectsrdquoApplied and Environmental Microbiology vol 71 no9 pp 4951ndash4959 2005

[19] E T Thorne B M Young G M Young et al ldquoThe structureof xylem vessels in grapevine (Vitaceae) and a possible passive

International Journal of Microbiology 13

mechanism for the systemic spread of bacterial diseaserdquo Amer-ican Journal of Botany vol 93 no 4 pp 497ndash504 2006

[20] W T Seo W J Lim E J Kim H D Yun Y H Lee and KM Cho ldquoEndophytic bacterial diversity in the young radishand their antimicrobial activity against pathogensrdquo Journal ofApplied Biological Chemistry vol 53 no 4 pp 493ndash503 2010

[21] S Compant H Kaplan A Sessitsch J Nowak E Ait Barkaand C Clement ldquoEndophytic colonization ofVitis vinifera L byBurkholderia phytofirmans strain PsJN from the rhizosphere toinflorescence tissuesrdquo FEMS Microbiology Ecology vol 63 no1 pp 84ndash93 2005

[22] P S B Miguel J C Delvaux M N V de Oliveira et al ldquoDiver-sity of endophytic bacteria in the fruits of Coffea canephorardquoAfrican Journal of Microbiology Research vol 7 no 7 pp 586ndash594 2013

[23] A Lopez-Lopez M A Rogel E Ormeno-Orrillo J Martınez-Romero and E Martınez-Romero ldquoPhaseolus vulgaris seed-borne endophytic community with novel bacterial species suchas Rhizobium endophyticum sp novrdquo Systematic and AppliedMicrobiology vol 33 no 6 pp 322ndash327 2010

[24] M Lwin and S L Ranamukhaarachchi ldquoDevelopment of bio-logical control of Ralstonia solanacearum through antagonisticmicrobial populationsrdquo International Journal of Agriculture andBiology vol 8 no 5 pp 657ndash660 2006

[25] F Rasche E Marco-Noales H Velvis et al ldquoStructural char-acteristics and plant-beneficial effects of bacteria colonizing theshoots of field grown conventional and geneticallymodifiedT4-lysozyme producing potatoesrdquo Plant and Soil vol 289 no 1-2pp 123ndash140 2006

[26] N Amaresan V Jayakumar K Kumar and N ThajuddinldquoEndophytic bacteria from tomato and chilli their diversity andantagonistic potential against Ralstonia solanacearumrdquoArchivesof Phytopathology and Plant Protection vol 45 no 3 pp 344ndash355 2012

[27] R Ramesh and G S Phadke ldquoRhizosphere and endophyticbacteria for the suppression of eggplant wilt caused byRalstoniasolanacearumrdquo Crop Protection vol 37 pp 35ndash41 2012

[28] W J French R G Christis and D L Stassi ldquoRecovery ofrickettsialike bacteria by vacuum infiltration of peach tissuesaffected with phony diseaserdquo Phytopathology vol 67 pp 945ndash948 1977

[29] P R Viss E M Brooks and J A Driver ldquoA simplified methodfor the control of bacterial contamination in woody plant tissueculturerdquo In Vitro Cellular andDevelopmental Biology vol 27 no1 article 42 1991

[30] K Wilson ldquoPreparation of genomic DNA from bacteriardquo inCurrent Protocols in Molecular Biology F M Ausubel R BrentR E Kingston et al Eds pp 241ndash245 John Wiley amp SonsNew York NY USA 1997

[31] M Saraf AThakkar U PandyaM Joshi and J Parikh ldquoPoten-tial of plant growth promoting microorganisms as biofertilizersand biopesticides and itrsquos exploitation in sustainable agricul-turerdquo Journal of Microbiology and Biotechnology Research vol3 no 5 pp 54ndash62 2013

[32] A P G C Marques C Pires H Moreira A O S S Rangeland P M L Castro ldquoAssessment of the plant growth promotionabilities of six bacterial isolates using Zea mays as indicatorplantrdquo Soil Biology and Biochemistry vol 42 no 8 pp 1229ndash1235 2010

[33] X Hao P Xie L Johnstone S J Miller C Rensingand G Wei ldquoGenome sequence and mutational analysis of

plant-growth-promoting bacteriumAgrobacterium tumefaciensCCNWGS0286 isolated from a zinc-lead mine tailingrdquo Appliedand Environmental Microbiology vol 78 no 15 pp 5384ndash53942012

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

[35] S A Gordon and L G Paleg ldquoQuantitative measurement ofindole acetic acidrdquo Plant Physiology vol 10 pp 37ndash48 1957

[36] A Godinho R Ramesh and S Bhosle ldquoBacteria from sanddunes of Goa promoting growth in eggplantrdquoWorld Journal ofAgricultural Sciences vol 6 no 5 pp 555ndash564 2010

[37] J-H Guo H-Y Qi Y-H Guo et al ldquoBiocontrol of tomato wiltby plant growth-promoting rhizobacteriardquo Biological Controlvol 29 no 1 pp 66ndash72 2004

[38] N Aliye C Fininsa and Y Hiskias ldquoEvaluation of rhizo-sphere bacterial antagonists for their potential to bioprotectpotato (Solanum tuberosum) against bacterial wilt (Ralstoniasolanacearum)rdquo Biological Control vol 47 no 3 pp 282ndash2882008

[39] L LagaceM PitreM Jacqeus andDRoy ldquoIdentification of thebacterial community ofmaple sap by using amplified ribosomalDNA (rDNA) restriction analysis and rDNA sequencingrdquoApplied and Environmental Microbiology vol 70 no 4 pp2052ndash2060 2004

[40] H H Long D G Sonntag D D Schmidt and I T BaldwinldquoThe structure of the culturable root bacterial endophyte com-munity of Nicotiana attenuata is organized by soil compositionand host plant ethylene production and perceptionrdquo NewPhytologist vol 185 no 2 pp 554ndash567 2010

[41] E Lopez-Fuentes V M Ruız-Valdiviezo E Martınez-RomeroF A Gutierrez-Miceli L Dendooven and R Rincon-RosalesldquoBacterial community in the roots and rhizosphere of Hyper-icum silenoides Juss 1804rdquo African Journal of MicrobiologyResearch vol 6 no 11 pp 2704ndash2711 2012

[42] M Fang R J Kremer P P Motavalli and G Davis ldquoBacterialdiversity in rhizospheres of nontransgenic and transgenic cornrdquoApplied and EnvironmentalMicrobiology vol 71 no 7 pp 4132ndash4136 2005

[43] C R Howell and R D Stiphanovic ldquoProduction of ammonia byEnterobacter cloaceae and its possible role in biological controlof Pythium pre-emergence damping-off by the bacteriumrdquoEcology and Epidemiology vol 78 no 8 pp 1075ndash1078 1988

[44] A Ramette M Frapolli G Defago and Y Moenne-LoccozldquoPhylogeny of HCN synthase-encoding hcnBC genes in bio-control fluorescent pseudomonads and its relationship withhost plant species and HCN synthesis abilityrdquoMolecular Plant-Microbe Interactions vol 16 no 6 pp 525ndash535 2003

[45] I Loaces L Ferrando and A F Scavino ldquoDynamics diversityand function of endophytic siderophore-producing bacteria inricerdquoMicrobial Ecology vol 61 no 3 pp 606ndash618 2011

[46] Y C Kim J Leveau B B M Gardener E A Pierson L SPierson and C-M Ryu ldquoThe multifactorial basis for planthealth promotion by plant-associated bacteriardquo Applied andEnvironmental Microbiology vol 77 no 5 pp 1548ndash1555 2011

[47] J M Gardner J L Chandler and A W Feldman ldquoGrowthresponses and vascular plugging of citrus inoculated withrhizobacteria and xylem-resident bacteriardquo Plant and Soil vol86 no 3 pp 333ndash345 1985

[48] C Pliego C Ramos A de Vicente and F M Cazorla ldquoScreen-ing for candidate bacterial biocontrol agents against soilborne

14 International Journal of Microbiology

fungal plant pathogensrdquoPlant and Soil vol 340 no 1-2 pp 505ndash520 2011

[49] H Feng Y Li and Q Liu ldquoEndophytic bacterial communi-ties in tomato plants with differential resistance to Ralstoniasolanacearumrdquo African Journal of Microbiology Research vol 7no 15 pp 1311ndash1318 2013

[50] A V Sturz B R Christie B G Matheson W J Arsenaultand N A Buchanan ldquoEndophytic bacterial communities inthe periderm of potato tubers and their potential to improveresistance to soil-borne plant pathogensrdquo Plant Pathology vol48 no 3 pp 360ndash369 1999

[51] A V Sturz B R Christie and J Nowak ldquoBacterial endophytespotential role in developing sustainable systems of crop produc-tionrdquo Critical Reviews in Plant Sciences vol 19 no 1 pp 1ndash302000

[52] R Ramesh A A Joshi and M P Ghanekar ldquoPseudomonadsmajor antagonistic endophytic bacteria to suppress bacterialwilt pathogenRalstonia solanacearum in the eggplant (Solanummelongena L)rdquo World Journal of Microbiology and Biotechnol-ogy vol 25 no 1 pp 47ndash55 2009

[53] A A Nawangsih I Damayanti S Wiyono and J G KartikaldquoSelection and characterization of endophytic bacteria as bio-control agents of tomato Bacterial Wilt DiseaserdquoHayati Journalof Biosciences vol 18 no 2 pp 66ndash70 2011

[54] R Vargas-Ayala R Rodrıguez-Kabana G Morgan-Jones J AMcInroy and J W Kloepper ldquoShifts in soil microflora inducedby velvetbean (Mucuna deeringiana) in cropping systems tocontrol root-knot nematodesrdquo Biological Control vol 17 no 1pp 11ndash22 2000

[55] D K Zinniel P Lambrecht N B Harris et al ldquoIsolationand characterization of endophytic colonizing bacteria fromagronomic crops and prairie plantsrdquoApplied and EnvironmentalMicrobiology vol 68 no 5 pp 2198ndash2208 2002

[56] F Lemessa and W Zeller ldquoScreening rhizobacteria for biolog-ical control of Ralstonia solanacearum in Ethiopiardquo BiologicalControl vol 42 no 3 pp 336ndash344 2007

[57] M S El-AlbyadM A El-Sayed A R El-Shanshoury andNHEl-Batanouny ldquoEffect of culture conditions on the antimicrobialactivities of UV-mutants of Streptomyces corchorusii and Sspiroverticillatus against bean and banana wilt pathogensrdquoMicrobiological Research vol 151 no 2 pp 201ndash211 1996

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

International Journal of Microbiology 11

Table 4 Continued

Strain Plant host Accession number Closest NCBI match similarityXB196lowast RE KF447432 Enterobacter kobei 99XB197lowast RE KF447433 Sphingomonas sp 98XB200lowast ST KF913447 Streptomyces sp 100XB202lowast ST KF447434 Pseudomonas sp 99XB203lowast ST KF447435 Flavobacterium sp 99lowastStrain is antagonistic to R solanacearum based on bioassaySE bacterial wilt susceptible eggplant RE bacterial wilt resistant eggplant C chilli ST Solanum torvum

tests However whole genome fingerprinting or PCR-RFLPbased methods are rapid tools for determining geneticdiversity of bacteria in a given collection ARDRA which isa type of PCR based RFLP method has been used widely inestimating genetic diversity endophytic bacterial populationsand clustering genetically similar strains [39ndash41] In additionto earlier reports our study demonstrates the usefulnessof ARDRA as a tool to cluster genetically identical strainsof endophytic XRB isolated from eggplant chilli andS torvum In our study 9161 strains (119899 = 153) weregrouped in 24 haplotypes by using ARDRA The majority ofthese haplotypes represent a combination of XRB isolatedfrom different solanaceous plants from diverse localesThese results indicate that xylem of eggplant chilli andS torvum largely bears similar population of XRB whichcan efficiently cross colonize eggplant chilli or S torvumNevertheless 839 strains had unique ARDRA fingerprintand formed separable haplotypes This observation leads toa conclusion that a minor population of xylem inhabitantsare restricted to a specific plant species and cannot easilycross colonize xylem of other solanaceous plants Plantsare known to selectively support endophytic colonizationby specific bacteria [14] However factors that determinethe selection of xylem colonists or the ability of XRBto colonize eggplant chilli and S torvum in this studyremain unknown Interestingly the structure of endophyticcommunity in Nicotiana attenuata a member of Solanaceaefamily is shown to be influenced by soil composition andethylene homeostasis [40] Earlier evidence has shown thatcolonization by endophytic bacteria is also governed by plantgenotype as well as root exudates [42]

Only 1677 XRB out of 167 were antagonistic to Rsolanacearum based on in vitro assays Antagonistic XRBproduced volatile and diffusible inhibitory compoundsnamely HCN ammonia and acetoin and siderophoresThese substances have been long known to be involvedin disease suppression and indirect growth promotion inplants [43ndash46] These mechanisms possibly played a rolein the evident biocontrol effect against BW exhibited bythe XRB in the greenhouse screening Endophytic bacteriahave been known to have plant growth promoting traitsnamely production of IAA ACC deaminase and phosphatesolubilization [25 33 36] These traits were detected in themajority of antagonistic XRB tested in this study and mayhave resulted in the observed increase in shoot length ofeggplant in our greenhouse experiments In contrast strains

XB20 XB196 and XB203 suppressed growth in eggplantunder greenhouse conditions however no visible symptomsof disease were observed Vascular plugging and productionof certainmetabolites toxic to plant cells but not cell viabilitymay have resulted in stunted shoot in eggplant [11 47]

Evaluation of efficacy of antagonistic organisms to sup-press the plant diseases under greenhouse conditions isone of the key steps for selecting a potential biocontrolagent for disease management [48] Endophytic strains fromBW susceptible varieties of eggplant have been shown toprevent wilt and promote growth in eggplant earlier [27]Our greenhouse screening shows that 3846 of antagonisticXRB with biocontrol efficacies greater than 40 were isolatesfrom BW resistant varieties of eggplantThis raises a questionwhether the bacteria from resistant varieties are involved inBW resistance and whether BW resistant varieties are ableto selectively influence xylem colonization by antagonisticbacteria Presence of higher number of endophytes withantagonistic abilities was reported in BW resistant varieties oftomato as compared to susceptible varieties and their role inresistance to BWwas proposed [49] Similar observations oncorrelation of resistance of potato to soft rot and endophyticbacteria have been reported [50]Thus BW resistant varietiescan be considered a better host for isolating potential biocon-trol strains for management of bacterial wilt

Identification of 55 XRB strains by 16S rRNA genesequencing revealed the presence of 23 diverse genera ofbacteria belonging to 4 phyla of Eubacteria Strains belongingto phyla Firmicutes Actinobacteria and 120574 subdivision ofProteobacteria were the major xylem colonists identified inthis study Several genera of bacteria belonging to these phylahave also been reported to be present in endophytic tissuesand xylem of a variety of other agricultural and horticulturalplant species [14 51] In addition the majority of the antag-onists identified belonged to Enterobacter sp Pseudomonassp Bacillus sp and Staphylococcus sp Congruent to ourresults several researchers have reported bacteria isolatedfrom solanaceous crops and belonging to similar genera tobe antagonistic to R solanacearum [26 51ndash53] HoweverFlavobacterium spand Janibacter melonis identified in thisstudy have never been previously reported to be inhibitoryto R solanacearum Large population of the xylem inhabitingbacterial flora accounting for 8323 exhibited no antago-nism towards R solanacearum Nonantagonistic XRB wereidentified predominantly as Microbacterium sp Endophyticpersistence and nematicidal activities of Microbacterium sp

12 International Journal of Microbiology

have been reported [54 55] Therefore the collection of XRBisolated in this study can be screened for inhibitory activitiesagainst other important agricultural pests

Though few strains namely XB86 XB169 and XB177exhibited plant beneficial properties in this study their use-fulness in plant disease control remains to be seen XB86has been identified as Agrobacterium tumefaciens the crowngall disease pathogen and its deployment as biocontrolagent is uncertain XB169 (Staphylococcus gallinarum) andXB177 (Bacillus cereus) are reported as opportunistic animalpathogens and thus unsuitable for field applications Strep-tomyces sp has earlier been reported as antagonistic to Rsolanacearum and tested for management of wilt in potatoand tomato [56 57] XB200 (Streptomyces sp) is one of theXRB high BCE and GPE it could be explored further forbiocontrol of bacterial wilt after additional characterizationand field evaluation

5 Conclusion

This study is the first report on the identity of novel anddiverse XRB colonizing the xylem of eggplant chilli and Storvum XRB particularly from BW resistant varieties werefound to protect eggplant from bacterial wilt and enhancedgrowth in eggplant in the greenhouse screening Thereforethe repertoire of XRB reported in this study may be usefulfor cultivation of eggplant in BW affected areas

Conflict of Interests

The authors hereby declare that they have no conflict ofinterests regarding the publication of this paper

Acknowledgments

The authors greatly acknowledge financial support fromIndian Council of Agricultural Research (ICAR) New DelhiIndia through ldquoOutreach project on Phytopthora Fusariumand Ralstonia diseases of horticultural and field cropsrdquo-(PhytoFuRa) and the Director ICAR Research Complex forGoa India for the facilities

References

[1] E Yabuuchi Y KosakoHOyaizu et al ldquoProposal ofBurkholde-ria gen nov and transfer of seven species of the genusPseudomonas homology group II to the new genus with thetype species Burkholderia cepacia (Palleroni and Holmes 1981)comb novrdquo Microbiology and Immunology vol 36 no 12 pp1251ndash1275 1992

[2] E Wicker L Grassart R Coranson-Beaudu et al ldquoRalstoniasolanacearum strains from Martinique (French West Indies)exhibiting a new pathogenic potentialrdquo Applied and Environ-mental Microbiology vol 73 no 21 pp 6790ndash6801 2007

[3] Y Liu A Kanda K Yano et al ldquoMolecular typing of Japanesestrains of Ralstonia solanacearum in relation to the ability toinduce a hypersensitive reaction in tobaccordquo Journal of GeneralPlant Pathology vol 75 no 5 pp 369ndash380 2009

[4] R Ramesh ldquoField evaluation of biological control agents for themanagement of Ralstonia solanacearum in Brinjalrdquo Journal ofMycology and Plant Pathology vol 36 no 2 pp 327ndash328 2006

[5] J Vasse P Frey and A Trigalet ldquoMicroscopic studies ofintercellular infection and protoxylem invasion of tomatoroots by Pseudomonas solanacearumrdquoMolecular Plant-MicrobeInteractions vol 8 no 2 pp 241ndash251 1995

[6] S Genin and C Boucher ldquoRalstonia solanacearum secrets of amajor pathogen unveiled by analysis of its genomerdquo MolecularPlant Pathology vol 3 no 3 pp 111ndash118 2002

[7] J M Gardner A W Feldman and R M Zablotowicz ldquoIdentityand behavior of xylem-residing bacteria in rough lemon roots ofFlorida citrus treesrdquo Applied and Environmental Microbiologyvol 43 no 6 pp 1335ndash1342 1982

[8] M J Jacobs W M Bugbee and D A Gabriellson ldquoEnu-meration location and characterization of endophytic bacteriawithin sugar beet rootsrdquoCanadian Journal of Botany vol 63 no7 pp 1262ndash1265 1985

[9] D G Patriquin and J Dobereiner ldquoLight microscopy observa-tions of tetrazolium-reducing bacteria in the endorhizosphereof maize and other grasses in Brazilrdquo Canadian Journal ofMicrobiology vol 24 no 6 pp 734ndash742 1978

[10] S Gagne C Richard H Rousseau and H Antoun ldquoXylem-residing bacteria in alfalfa rootsrdquo Canadian Journal of Micro-biology vol 33 no 11 pp 996ndash1000 1987

[11] C R Bell G A Dickie W L G Harvey and J W Y FChan ldquoEndophytic bacteria in grapevinerdquo Canadian Journal ofMicrobiology vol 41 no 1 pp 46ndash53 1995

[12] J Hallmann J W Kloepper and R Rodrıguez-Kabana ldquoAppli-cation of Scholander pressure bomb to studies on endophyticbacteria of plantsrdquo Canadian Journal of Microbiology vol 43no 5 pp 411ndash416 1997

[13] J S Lampel G L Canter M B Dimock et al ldquoIntegrativecloning expression and stability of the cryIA(c) gene fromBacillus thuringiensis subsp kurstaki in a recombinant strain ofClavibacter xyli subsp cynodontisrdquo Applied and EnvironmentalMicrobiology vol 60 no 2 pp 501ndash508 1994

[14] M Rosenblueth and E Martınez-Romero ldquoBacterial endo-phytes and their interactions with hostsrdquo Molecular Plant-Microbe Interactions vol 19 no 8 pp 827ndash837 2006

[15] P R Hardoim L S van Overbeek and J D van ElsasldquoProperties of bacterial endophytes and their proposed role inplant growthrdquo Trends in Microbiology vol 16 no 10 pp 463ndash471 2008

[16] R Aravind A Kumar S J Eapen and K V RamanaldquoEndophytic bacterial flora in root and stem tissues of blackpepper (Piper nigrum L) genotype isolation identificationand evaluation against Phytophthora capsicirdquo Letters in AppliedMicrobiology vol 48 no 1 pp 58ndash64 2009

[17] J Kuklinsky-Sobral W L Araujo R Mendes I O Geraldi AA Pizzirani-Kleiner and J L Azevedo ldquoIsolation and charac-terization of soybean-associated bacteria and their potential forplant growth promotionrdquo Environmental Microbiology vol 6no 12 pp 1244ndash1251 2004

[18] S Compant B Duffy J Nowak C Clement and E A BarkaldquoUse of plant growth-promoting bacteria for biocontrol ofplant diseases principles mechanisms of action and futureprospectsrdquoApplied and Environmental Microbiology vol 71 no9 pp 4951ndash4959 2005

[19] E T Thorne B M Young G M Young et al ldquoThe structureof xylem vessels in grapevine (Vitaceae) and a possible passive

International Journal of Microbiology 13

mechanism for the systemic spread of bacterial diseaserdquo Amer-ican Journal of Botany vol 93 no 4 pp 497ndash504 2006

[20] W T Seo W J Lim E J Kim H D Yun Y H Lee and KM Cho ldquoEndophytic bacterial diversity in the young radishand their antimicrobial activity against pathogensrdquo Journal ofApplied Biological Chemistry vol 53 no 4 pp 493ndash503 2010

[21] S Compant H Kaplan A Sessitsch J Nowak E Ait Barkaand C Clement ldquoEndophytic colonization ofVitis vinifera L byBurkholderia phytofirmans strain PsJN from the rhizosphere toinflorescence tissuesrdquo FEMS Microbiology Ecology vol 63 no1 pp 84ndash93 2005

[22] P S B Miguel J C Delvaux M N V de Oliveira et al ldquoDiver-sity of endophytic bacteria in the fruits of Coffea canephorardquoAfrican Journal of Microbiology Research vol 7 no 7 pp 586ndash594 2013

[23] A Lopez-Lopez M A Rogel E Ormeno-Orrillo J Martınez-Romero and E Martınez-Romero ldquoPhaseolus vulgaris seed-borne endophytic community with novel bacterial species suchas Rhizobium endophyticum sp novrdquo Systematic and AppliedMicrobiology vol 33 no 6 pp 322ndash327 2010

[24] M Lwin and S L Ranamukhaarachchi ldquoDevelopment of bio-logical control of Ralstonia solanacearum through antagonisticmicrobial populationsrdquo International Journal of Agriculture andBiology vol 8 no 5 pp 657ndash660 2006

[25] F Rasche E Marco-Noales H Velvis et al ldquoStructural char-acteristics and plant-beneficial effects of bacteria colonizing theshoots of field grown conventional and geneticallymodifiedT4-lysozyme producing potatoesrdquo Plant and Soil vol 289 no 1-2pp 123ndash140 2006

[26] N Amaresan V Jayakumar K Kumar and N ThajuddinldquoEndophytic bacteria from tomato and chilli their diversity andantagonistic potential against Ralstonia solanacearumrdquoArchivesof Phytopathology and Plant Protection vol 45 no 3 pp 344ndash355 2012

[27] R Ramesh and G S Phadke ldquoRhizosphere and endophyticbacteria for the suppression of eggplant wilt caused byRalstoniasolanacearumrdquo Crop Protection vol 37 pp 35ndash41 2012

[28] W J French R G Christis and D L Stassi ldquoRecovery ofrickettsialike bacteria by vacuum infiltration of peach tissuesaffected with phony diseaserdquo Phytopathology vol 67 pp 945ndash948 1977

[29] P R Viss E M Brooks and J A Driver ldquoA simplified methodfor the control of bacterial contamination in woody plant tissueculturerdquo In Vitro Cellular andDevelopmental Biology vol 27 no1 article 42 1991

[30] K Wilson ldquoPreparation of genomic DNA from bacteriardquo inCurrent Protocols in Molecular Biology F M Ausubel R BrentR E Kingston et al Eds pp 241ndash245 John Wiley amp SonsNew York NY USA 1997

[31] M Saraf AThakkar U PandyaM Joshi and J Parikh ldquoPoten-tial of plant growth promoting microorganisms as biofertilizersand biopesticides and itrsquos exploitation in sustainable agricul-turerdquo Journal of Microbiology and Biotechnology Research vol3 no 5 pp 54ndash62 2013

[32] A P G C Marques C Pires H Moreira A O S S Rangeland P M L Castro ldquoAssessment of the plant growth promotionabilities of six bacterial isolates using Zea mays as indicatorplantrdquo Soil Biology and Biochemistry vol 42 no 8 pp 1229ndash1235 2010

[33] X Hao P Xie L Johnstone S J Miller C Rensingand G Wei ldquoGenome sequence and mutational analysis of

plant-growth-promoting bacteriumAgrobacterium tumefaciensCCNWGS0286 isolated from a zinc-lead mine tailingrdquo Appliedand Environmental Microbiology vol 78 no 15 pp 5384ndash53942012

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

[35] S A Gordon and L G Paleg ldquoQuantitative measurement ofindole acetic acidrdquo Plant Physiology vol 10 pp 37ndash48 1957

[36] A Godinho R Ramesh and S Bhosle ldquoBacteria from sanddunes of Goa promoting growth in eggplantrdquoWorld Journal ofAgricultural Sciences vol 6 no 5 pp 555ndash564 2010

[37] J-H Guo H-Y Qi Y-H Guo et al ldquoBiocontrol of tomato wiltby plant growth-promoting rhizobacteriardquo Biological Controlvol 29 no 1 pp 66ndash72 2004

[38] N Aliye C Fininsa and Y Hiskias ldquoEvaluation of rhizo-sphere bacterial antagonists for their potential to bioprotectpotato (Solanum tuberosum) against bacterial wilt (Ralstoniasolanacearum)rdquo Biological Control vol 47 no 3 pp 282ndash2882008

[39] L LagaceM PitreM Jacqeus andDRoy ldquoIdentification of thebacterial community ofmaple sap by using amplified ribosomalDNA (rDNA) restriction analysis and rDNA sequencingrdquoApplied and Environmental Microbiology vol 70 no 4 pp2052ndash2060 2004

[40] H H Long D G Sonntag D D Schmidt and I T BaldwinldquoThe structure of the culturable root bacterial endophyte com-munity of Nicotiana attenuata is organized by soil compositionand host plant ethylene production and perceptionrdquo NewPhytologist vol 185 no 2 pp 554ndash567 2010

[41] E Lopez-Fuentes V M Ruız-Valdiviezo E Martınez-RomeroF A Gutierrez-Miceli L Dendooven and R Rincon-RosalesldquoBacterial community in the roots and rhizosphere of Hyper-icum silenoides Juss 1804rdquo African Journal of MicrobiologyResearch vol 6 no 11 pp 2704ndash2711 2012

[42] M Fang R J Kremer P P Motavalli and G Davis ldquoBacterialdiversity in rhizospheres of nontransgenic and transgenic cornrdquoApplied and EnvironmentalMicrobiology vol 71 no 7 pp 4132ndash4136 2005

[43] C R Howell and R D Stiphanovic ldquoProduction of ammonia byEnterobacter cloaceae and its possible role in biological controlof Pythium pre-emergence damping-off by the bacteriumrdquoEcology and Epidemiology vol 78 no 8 pp 1075ndash1078 1988

[44] A Ramette M Frapolli G Defago and Y Moenne-LoccozldquoPhylogeny of HCN synthase-encoding hcnBC genes in bio-control fluorescent pseudomonads and its relationship withhost plant species and HCN synthesis abilityrdquoMolecular Plant-Microbe Interactions vol 16 no 6 pp 525ndash535 2003

[45] I Loaces L Ferrando and A F Scavino ldquoDynamics diversityand function of endophytic siderophore-producing bacteria inricerdquoMicrobial Ecology vol 61 no 3 pp 606ndash618 2011

[46] Y C Kim J Leveau B B M Gardener E A Pierson L SPierson and C-M Ryu ldquoThe multifactorial basis for planthealth promotion by plant-associated bacteriardquo Applied andEnvironmental Microbiology vol 77 no 5 pp 1548ndash1555 2011

[47] J M Gardner J L Chandler and A W Feldman ldquoGrowthresponses and vascular plugging of citrus inoculated withrhizobacteria and xylem-resident bacteriardquo Plant and Soil vol86 no 3 pp 333ndash345 1985

[48] C Pliego C Ramos A de Vicente and F M Cazorla ldquoScreen-ing for candidate bacterial biocontrol agents against soilborne

14 International Journal of Microbiology

fungal plant pathogensrdquoPlant and Soil vol 340 no 1-2 pp 505ndash520 2011

[49] H Feng Y Li and Q Liu ldquoEndophytic bacterial communi-ties in tomato plants with differential resistance to Ralstoniasolanacearumrdquo African Journal of Microbiology Research vol 7no 15 pp 1311ndash1318 2013

[50] A V Sturz B R Christie B G Matheson W J Arsenaultand N A Buchanan ldquoEndophytic bacterial communities inthe periderm of potato tubers and their potential to improveresistance to soil-borne plant pathogensrdquo Plant Pathology vol48 no 3 pp 360ndash369 1999

[51] A V Sturz B R Christie and J Nowak ldquoBacterial endophytespotential role in developing sustainable systems of crop produc-tionrdquo Critical Reviews in Plant Sciences vol 19 no 1 pp 1ndash302000

[52] R Ramesh A A Joshi and M P Ghanekar ldquoPseudomonadsmajor antagonistic endophytic bacteria to suppress bacterialwilt pathogenRalstonia solanacearum in the eggplant (Solanummelongena L)rdquo World Journal of Microbiology and Biotechnol-ogy vol 25 no 1 pp 47ndash55 2009

[53] A A Nawangsih I Damayanti S Wiyono and J G KartikaldquoSelection and characterization of endophytic bacteria as bio-control agents of tomato Bacterial Wilt DiseaserdquoHayati Journalof Biosciences vol 18 no 2 pp 66ndash70 2011

[54] R Vargas-Ayala R Rodrıguez-Kabana G Morgan-Jones J AMcInroy and J W Kloepper ldquoShifts in soil microflora inducedby velvetbean (Mucuna deeringiana) in cropping systems tocontrol root-knot nematodesrdquo Biological Control vol 17 no 1pp 11ndash22 2000

[55] D K Zinniel P Lambrecht N B Harris et al ldquoIsolationand characterization of endophytic colonizing bacteria fromagronomic crops and prairie plantsrdquoApplied and EnvironmentalMicrobiology vol 68 no 5 pp 2198ndash2208 2002

[56] F Lemessa and W Zeller ldquoScreening rhizobacteria for biolog-ical control of Ralstonia solanacearum in Ethiopiardquo BiologicalControl vol 42 no 3 pp 336ndash344 2007

[57] M S El-AlbyadM A El-Sayed A R El-Shanshoury andNHEl-Batanouny ldquoEffect of culture conditions on the antimicrobialactivities of UV-mutants of Streptomyces corchorusii and Sspiroverticillatus against bean and banana wilt pathogensrdquoMicrobiological Research vol 151 no 2 pp 201ndash211 1996

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

12 International Journal of Microbiology

have been reported [54 55] Therefore the collection of XRBisolated in this study can be screened for inhibitory activitiesagainst other important agricultural pests

Though few strains namely XB86 XB169 and XB177exhibited plant beneficial properties in this study their use-fulness in plant disease control remains to be seen XB86has been identified as Agrobacterium tumefaciens the crowngall disease pathogen and its deployment as biocontrolagent is uncertain XB169 (Staphylococcus gallinarum) andXB177 (Bacillus cereus) are reported as opportunistic animalpathogens and thus unsuitable for field applications Strep-tomyces sp has earlier been reported as antagonistic to Rsolanacearum and tested for management of wilt in potatoand tomato [56 57] XB200 (Streptomyces sp) is one of theXRB high BCE and GPE it could be explored further forbiocontrol of bacterial wilt after additional characterizationand field evaluation

5 Conclusion

This study is the first report on the identity of novel anddiverse XRB colonizing the xylem of eggplant chilli and Storvum XRB particularly from BW resistant varieties werefound to protect eggplant from bacterial wilt and enhancedgrowth in eggplant in the greenhouse screening Thereforethe repertoire of XRB reported in this study may be usefulfor cultivation of eggplant in BW affected areas

Conflict of Interests

The authors hereby declare that they have no conflict ofinterests regarding the publication of this paper

Acknowledgments

The authors greatly acknowledge financial support fromIndian Council of Agricultural Research (ICAR) New DelhiIndia through ldquoOutreach project on Phytopthora Fusariumand Ralstonia diseases of horticultural and field cropsrdquo-(PhytoFuRa) and the Director ICAR Research Complex forGoa India for the facilities

References

[1] E Yabuuchi Y KosakoHOyaizu et al ldquoProposal ofBurkholde-ria gen nov and transfer of seven species of the genusPseudomonas homology group II to the new genus with thetype species Burkholderia cepacia (Palleroni and Holmes 1981)comb novrdquo Microbiology and Immunology vol 36 no 12 pp1251ndash1275 1992

[2] E Wicker L Grassart R Coranson-Beaudu et al ldquoRalstoniasolanacearum strains from Martinique (French West Indies)exhibiting a new pathogenic potentialrdquo Applied and Environ-mental Microbiology vol 73 no 21 pp 6790ndash6801 2007

[3] Y Liu A Kanda K Yano et al ldquoMolecular typing of Japanesestrains of Ralstonia solanacearum in relation to the ability toinduce a hypersensitive reaction in tobaccordquo Journal of GeneralPlant Pathology vol 75 no 5 pp 369ndash380 2009

[4] R Ramesh ldquoField evaluation of biological control agents for themanagement of Ralstonia solanacearum in Brinjalrdquo Journal ofMycology and Plant Pathology vol 36 no 2 pp 327ndash328 2006

[5] J Vasse P Frey and A Trigalet ldquoMicroscopic studies ofintercellular infection and protoxylem invasion of tomatoroots by Pseudomonas solanacearumrdquoMolecular Plant-MicrobeInteractions vol 8 no 2 pp 241ndash251 1995

[6] S Genin and C Boucher ldquoRalstonia solanacearum secrets of amajor pathogen unveiled by analysis of its genomerdquo MolecularPlant Pathology vol 3 no 3 pp 111ndash118 2002

[7] J M Gardner A W Feldman and R M Zablotowicz ldquoIdentityand behavior of xylem-residing bacteria in rough lemon roots ofFlorida citrus treesrdquo Applied and Environmental Microbiologyvol 43 no 6 pp 1335ndash1342 1982

[8] M J Jacobs W M Bugbee and D A Gabriellson ldquoEnu-meration location and characterization of endophytic bacteriawithin sugar beet rootsrdquoCanadian Journal of Botany vol 63 no7 pp 1262ndash1265 1985

[9] D G Patriquin and J Dobereiner ldquoLight microscopy observa-tions of tetrazolium-reducing bacteria in the endorhizosphereof maize and other grasses in Brazilrdquo Canadian Journal ofMicrobiology vol 24 no 6 pp 734ndash742 1978

[10] S Gagne C Richard H Rousseau and H Antoun ldquoXylem-residing bacteria in alfalfa rootsrdquo Canadian Journal of Micro-biology vol 33 no 11 pp 996ndash1000 1987

[11] C R Bell G A Dickie W L G Harvey and J W Y FChan ldquoEndophytic bacteria in grapevinerdquo Canadian Journal ofMicrobiology vol 41 no 1 pp 46ndash53 1995

[12] J Hallmann J W Kloepper and R Rodrıguez-Kabana ldquoAppli-cation of Scholander pressure bomb to studies on endophyticbacteria of plantsrdquo Canadian Journal of Microbiology vol 43no 5 pp 411ndash416 1997

[13] J S Lampel G L Canter M B Dimock et al ldquoIntegrativecloning expression and stability of the cryIA(c) gene fromBacillus thuringiensis subsp kurstaki in a recombinant strain ofClavibacter xyli subsp cynodontisrdquo Applied and EnvironmentalMicrobiology vol 60 no 2 pp 501ndash508 1994

[14] M Rosenblueth and E Martınez-Romero ldquoBacterial endo-phytes and their interactions with hostsrdquo Molecular Plant-Microbe Interactions vol 19 no 8 pp 827ndash837 2006

[15] P R Hardoim L S van Overbeek and J D van ElsasldquoProperties of bacterial endophytes and their proposed role inplant growthrdquo Trends in Microbiology vol 16 no 10 pp 463ndash471 2008

[16] R Aravind A Kumar S J Eapen and K V RamanaldquoEndophytic bacterial flora in root and stem tissues of blackpepper (Piper nigrum L) genotype isolation identificationand evaluation against Phytophthora capsicirdquo Letters in AppliedMicrobiology vol 48 no 1 pp 58ndash64 2009

[17] J Kuklinsky-Sobral W L Araujo R Mendes I O Geraldi AA Pizzirani-Kleiner and J L Azevedo ldquoIsolation and charac-terization of soybean-associated bacteria and their potential forplant growth promotionrdquo Environmental Microbiology vol 6no 12 pp 1244ndash1251 2004

[18] S Compant B Duffy J Nowak C Clement and E A BarkaldquoUse of plant growth-promoting bacteria for biocontrol ofplant diseases principles mechanisms of action and futureprospectsrdquoApplied and Environmental Microbiology vol 71 no9 pp 4951ndash4959 2005

[19] E T Thorne B M Young G M Young et al ldquoThe structureof xylem vessels in grapevine (Vitaceae) and a possible passive

International Journal of Microbiology 13

mechanism for the systemic spread of bacterial diseaserdquo Amer-ican Journal of Botany vol 93 no 4 pp 497ndash504 2006

[20] W T Seo W J Lim E J Kim H D Yun Y H Lee and KM Cho ldquoEndophytic bacterial diversity in the young radishand their antimicrobial activity against pathogensrdquo Journal ofApplied Biological Chemistry vol 53 no 4 pp 493ndash503 2010

[21] S Compant H Kaplan A Sessitsch J Nowak E Ait Barkaand C Clement ldquoEndophytic colonization ofVitis vinifera L byBurkholderia phytofirmans strain PsJN from the rhizosphere toinflorescence tissuesrdquo FEMS Microbiology Ecology vol 63 no1 pp 84ndash93 2005

[22] P S B Miguel J C Delvaux M N V de Oliveira et al ldquoDiver-sity of endophytic bacteria in the fruits of Coffea canephorardquoAfrican Journal of Microbiology Research vol 7 no 7 pp 586ndash594 2013

[23] A Lopez-Lopez M A Rogel E Ormeno-Orrillo J Martınez-Romero and E Martınez-Romero ldquoPhaseolus vulgaris seed-borne endophytic community with novel bacterial species suchas Rhizobium endophyticum sp novrdquo Systematic and AppliedMicrobiology vol 33 no 6 pp 322ndash327 2010

[24] M Lwin and S L Ranamukhaarachchi ldquoDevelopment of bio-logical control of Ralstonia solanacearum through antagonisticmicrobial populationsrdquo International Journal of Agriculture andBiology vol 8 no 5 pp 657ndash660 2006

[25] F Rasche E Marco-Noales H Velvis et al ldquoStructural char-acteristics and plant-beneficial effects of bacteria colonizing theshoots of field grown conventional and geneticallymodifiedT4-lysozyme producing potatoesrdquo Plant and Soil vol 289 no 1-2pp 123ndash140 2006

[26] N Amaresan V Jayakumar K Kumar and N ThajuddinldquoEndophytic bacteria from tomato and chilli their diversity andantagonistic potential against Ralstonia solanacearumrdquoArchivesof Phytopathology and Plant Protection vol 45 no 3 pp 344ndash355 2012

[27] R Ramesh and G S Phadke ldquoRhizosphere and endophyticbacteria for the suppression of eggplant wilt caused byRalstoniasolanacearumrdquo Crop Protection vol 37 pp 35ndash41 2012

[28] W J French R G Christis and D L Stassi ldquoRecovery ofrickettsialike bacteria by vacuum infiltration of peach tissuesaffected with phony diseaserdquo Phytopathology vol 67 pp 945ndash948 1977

[29] P R Viss E M Brooks and J A Driver ldquoA simplified methodfor the control of bacterial contamination in woody plant tissueculturerdquo In Vitro Cellular andDevelopmental Biology vol 27 no1 article 42 1991

[30] K Wilson ldquoPreparation of genomic DNA from bacteriardquo inCurrent Protocols in Molecular Biology F M Ausubel R BrentR E Kingston et al Eds pp 241ndash245 John Wiley amp SonsNew York NY USA 1997

[31] M Saraf AThakkar U PandyaM Joshi and J Parikh ldquoPoten-tial of plant growth promoting microorganisms as biofertilizersand biopesticides and itrsquos exploitation in sustainable agricul-turerdquo Journal of Microbiology and Biotechnology Research vol3 no 5 pp 54ndash62 2013

[32] A P G C Marques C Pires H Moreira A O S S Rangeland P M L Castro ldquoAssessment of the plant growth promotionabilities of six bacterial isolates using Zea mays as indicatorplantrdquo Soil Biology and Biochemistry vol 42 no 8 pp 1229ndash1235 2010

[33] X Hao P Xie L Johnstone S J Miller C Rensingand G Wei ldquoGenome sequence and mutational analysis of

plant-growth-promoting bacteriumAgrobacterium tumefaciensCCNWGS0286 isolated from a zinc-lead mine tailingrdquo Appliedand Environmental Microbiology vol 78 no 15 pp 5384ndash53942012

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

[35] S A Gordon and L G Paleg ldquoQuantitative measurement ofindole acetic acidrdquo Plant Physiology vol 10 pp 37ndash48 1957

[36] A Godinho R Ramesh and S Bhosle ldquoBacteria from sanddunes of Goa promoting growth in eggplantrdquoWorld Journal ofAgricultural Sciences vol 6 no 5 pp 555ndash564 2010

[37] J-H Guo H-Y Qi Y-H Guo et al ldquoBiocontrol of tomato wiltby plant growth-promoting rhizobacteriardquo Biological Controlvol 29 no 1 pp 66ndash72 2004

[38] N Aliye C Fininsa and Y Hiskias ldquoEvaluation of rhizo-sphere bacterial antagonists for their potential to bioprotectpotato (Solanum tuberosum) against bacterial wilt (Ralstoniasolanacearum)rdquo Biological Control vol 47 no 3 pp 282ndash2882008

[39] L LagaceM PitreM Jacqeus andDRoy ldquoIdentification of thebacterial community ofmaple sap by using amplified ribosomalDNA (rDNA) restriction analysis and rDNA sequencingrdquoApplied and Environmental Microbiology vol 70 no 4 pp2052ndash2060 2004

[40] H H Long D G Sonntag D D Schmidt and I T BaldwinldquoThe structure of the culturable root bacterial endophyte com-munity of Nicotiana attenuata is organized by soil compositionand host plant ethylene production and perceptionrdquo NewPhytologist vol 185 no 2 pp 554ndash567 2010

[41] E Lopez-Fuentes V M Ruız-Valdiviezo E Martınez-RomeroF A Gutierrez-Miceli L Dendooven and R Rincon-RosalesldquoBacterial community in the roots and rhizosphere of Hyper-icum silenoides Juss 1804rdquo African Journal of MicrobiologyResearch vol 6 no 11 pp 2704ndash2711 2012

[42] M Fang R J Kremer P P Motavalli and G Davis ldquoBacterialdiversity in rhizospheres of nontransgenic and transgenic cornrdquoApplied and EnvironmentalMicrobiology vol 71 no 7 pp 4132ndash4136 2005

[43] C R Howell and R D Stiphanovic ldquoProduction of ammonia byEnterobacter cloaceae and its possible role in biological controlof Pythium pre-emergence damping-off by the bacteriumrdquoEcology and Epidemiology vol 78 no 8 pp 1075ndash1078 1988

[44] A Ramette M Frapolli G Defago and Y Moenne-LoccozldquoPhylogeny of HCN synthase-encoding hcnBC genes in bio-control fluorescent pseudomonads and its relationship withhost plant species and HCN synthesis abilityrdquoMolecular Plant-Microbe Interactions vol 16 no 6 pp 525ndash535 2003

[45] I Loaces L Ferrando and A F Scavino ldquoDynamics diversityand function of endophytic siderophore-producing bacteria inricerdquoMicrobial Ecology vol 61 no 3 pp 606ndash618 2011

[46] Y C Kim J Leveau B B M Gardener E A Pierson L SPierson and C-M Ryu ldquoThe multifactorial basis for planthealth promotion by plant-associated bacteriardquo Applied andEnvironmental Microbiology vol 77 no 5 pp 1548ndash1555 2011

[47] J M Gardner J L Chandler and A W Feldman ldquoGrowthresponses and vascular plugging of citrus inoculated withrhizobacteria and xylem-resident bacteriardquo Plant and Soil vol86 no 3 pp 333ndash345 1985

[48] C Pliego C Ramos A de Vicente and F M Cazorla ldquoScreen-ing for candidate bacterial biocontrol agents against soilborne

14 International Journal of Microbiology

fungal plant pathogensrdquoPlant and Soil vol 340 no 1-2 pp 505ndash520 2011

[49] H Feng Y Li and Q Liu ldquoEndophytic bacterial communi-ties in tomato plants with differential resistance to Ralstoniasolanacearumrdquo African Journal of Microbiology Research vol 7no 15 pp 1311ndash1318 2013

[50] A V Sturz B R Christie B G Matheson W J Arsenaultand N A Buchanan ldquoEndophytic bacterial communities inthe periderm of potato tubers and their potential to improveresistance to soil-borne plant pathogensrdquo Plant Pathology vol48 no 3 pp 360ndash369 1999

[51] A V Sturz B R Christie and J Nowak ldquoBacterial endophytespotential role in developing sustainable systems of crop produc-tionrdquo Critical Reviews in Plant Sciences vol 19 no 1 pp 1ndash302000

[52] R Ramesh A A Joshi and M P Ghanekar ldquoPseudomonadsmajor antagonistic endophytic bacteria to suppress bacterialwilt pathogenRalstonia solanacearum in the eggplant (Solanummelongena L)rdquo World Journal of Microbiology and Biotechnol-ogy vol 25 no 1 pp 47ndash55 2009

[53] A A Nawangsih I Damayanti S Wiyono and J G KartikaldquoSelection and characterization of endophytic bacteria as bio-control agents of tomato Bacterial Wilt DiseaserdquoHayati Journalof Biosciences vol 18 no 2 pp 66ndash70 2011

[54] R Vargas-Ayala R Rodrıguez-Kabana G Morgan-Jones J AMcInroy and J W Kloepper ldquoShifts in soil microflora inducedby velvetbean (Mucuna deeringiana) in cropping systems tocontrol root-knot nematodesrdquo Biological Control vol 17 no 1pp 11ndash22 2000

[55] D K Zinniel P Lambrecht N B Harris et al ldquoIsolationand characterization of endophytic colonizing bacteria fromagronomic crops and prairie plantsrdquoApplied and EnvironmentalMicrobiology vol 68 no 5 pp 2198ndash2208 2002

[56] F Lemessa and W Zeller ldquoScreening rhizobacteria for biolog-ical control of Ralstonia solanacearum in Ethiopiardquo BiologicalControl vol 42 no 3 pp 336ndash344 2007

[57] M S El-AlbyadM A El-Sayed A R El-Shanshoury andNHEl-Batanouny ldquoEffect of culture conditions on the antimicrobialactivities of UV-mutants of Streptomyces corchorusii and Sspiroverticillatus against bean and banana wilt pathogensrdquoMicrobiological Research vol 151 no 2 pp 201ndash211 1996

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

International Journal of Microbiology 13

mechanism for the systemic spread of bacterial diseaserdquo Amer-ican Journal of Botany vol 93 no 4 pp 497ndash504 2006

[20] W T Seo W J Lim E J Kim H D Yun Y H Lee and KM Cho ldquoEndophytic bacterial diversity in the young radishand their antimicrobial activity against pathogensrdquo Journal ofApplied Biological Chemistry vol 53 no 4 pp 493ndash503 2010

[21] S Compant H Kaplan A Sessitsch J Nowak E Ait Barkaand C Clement ldquoEndophytic colonization ofVitis vinifera L byBurkholderia phytofirmans strain PsJN from the rhizosphere toinflorescence tissuesrdquo FEMS Microbiology Ecology vol 63 no1 pp 84ndash93 2005

[22] P S B Miguel J C Delvaux M N V de Oliveira et al ldquoDiver-sity of endophytic bacteria in the fruits of Coffea canephorardquoAfrican Journal of Microbiology Research vol 7 no 7 pp 586ndash594 2013

[23] A Lopez-Lopez M A Rogel E Ormeno-Orrillo J Martınez-Romero and E Martınez-Romero ldquoPhaseolus vulgaris seed-borne endophytic community with novel bacterial species suchas Rhizobium endophyticum sp novrdquo Systematic and AppliedMicrobiology vol 33 no 6 pp 322ndash327 2010

[24] M Lwin and S L Ranamukhaarachchi ldquoDevelopment of bio-logical control of Ralstonia solanacearum through antagonisticmicrobial populationsrdquo International Journal of Agriculture andBiology vol 8 no 5 pp 657ndash660 2006

[25] F Rasche E Marco-Noales H Velvis et al ldquoStructural char-acteristics and plant-beneficial effects of bacteria colonizing theshoots of field grown conventional and geneticallymodifiedT4-lysozyme producing potatoesrdquo Plant and Soil vol 289 no 1-2pp 123ndash140 2006

[26] N Amaresan V Jayakumar K Kumar and N ThajuddinldquoEndophytic bacteria from tomato and chilli their diversity andantagonistic potential against Ralstonia solanacearumrdquoArchivesof Phytopathology and Plant Protection vol 45 no 3 pp 344ndash355 2012

[27] R Ramesh and G S Phadke ldquoRhizosphere and endophyticbacteria for the suppression of eggplant wilt caused byRalstoniasolanacearumrdquo Crop Protection vol 37 pp 35ndash41 2012

[28] W J French R G Christis and D L Stassi ldquoRecovery ofrickettsialike bacteria by vacuum infiltration of peach tissuesaffected with phony diseaserdquo Phytopathology vol 67 pp 945ndash948 1977

[29] P R Viss E M Brooks and J A Driver ldquoA simplified methodfor the control of bacterial contamination in woody plant tissueculturerdquo In Vitro Cellular andDevelopmental Biology vol 27 no1 article 42 1991

[30] K Wilson ldquoPreparation of genomic DNA from bacteriardquo inCurrent Protocols in Molecular Biology F M Ausubel R BrentR E Kingston et al Eds pp 241ndash245 John Wiley amp SonsNew York NY USA 1997

[31] M Saraf AThakkar U PandyaM Joshi and J Parikh ldquoPoten-tial of plant growth promoting microorganisms as biofertilizersand biopesticides and itrsquos exploitation in sustainable agricul-turerdquo Journal of Microbiology and Biotechnology Research vol3 no 5 pp 54ndash62 2013

[32] A P G C Marques C Pires H Moreira A O S S Rangeland P M L Castro ldquoAssessment of the plant growth promotionabilities of six bacterial isolates using Zea mays as indicatorplantrdquo Soil Biology and Biochemistry vol 42 no 8 pp 1229ndash1235 2010

[33] X Hao P Xie L Johnstone S J Miller C Rensingand G Wei ldquoGenome sequence and mutational analysis of

plant-growth-promoting bacteriumAgrobacterium tumefaciensCCNWGS0286 isolated from a zinc-lead mine tailingrdquo Appliedand Environmental Microbiology vol 78 no 15 pp 5384ndash53942012

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

[35] S A Gordon and L G Paleg ldquoQuantitative measurement ofindole acetic acidrdquo Plant Physiology vol 10 pp 37ndash48 1957

[36] A Godinho R Ramesh and S Bhosle ldquoBacteria from sanddunes of Goa promoting growth in eggplantrdquoWorld Journal ofAgricultural Sciences vol 6 no 5 pp 555ndash564 2010

[37] J-H Guo H-Y Qi Y-H Guo et al ldquoBiocontrol of tomato wiltby plant growth-promoting rhizobacteriardquo Biological Controlvol 29 no 1 pp 66ndash72 2004

[38] N Aliye C Fininsa and Y Hiskias ldquoEvaluation of rhizo-sphere bacterial antagonists for their potential to bioprotectpotato (Solanum tuberosum) against bacterial wilt (Ralstoniasolanacearum)rdquo Biological Control vol 47 no 3 pp 282ndash2882008

[39] L LagaceM PitreM Jacqeus andDRoy ldquoIdentification of thebacterial community ofmaple sap by using amplified ribosomalDNA (rDNA) restriction analysis and rDNA sequencingrdquoApplied and Environmental Microbiology vol 70 no 4 pp2052ndash2060 2004

[40] H H Long D G Sonntag D D Schmidt and I T BaldwinldquoThe structure of the culturable root bacterial endophyte com-munity of Nicotiana attenuata is organized by soil compositionand host plant ethylene production and perceptionrdquo NewPhytologist vol 185 no 2 pp 554ndash567 2010

[41] E Lopez-Fuentes V M Ruız-Valdiviezo E Martınez-RomeroF A Gutierrez-Miceli L Dendooven and R Rincon-RosalesldquoBacterial community in the roots and rhizosphere of Hyper-icum silenoides Juss 1804rdquo African Journal of MicrobiologyResearch vol 6 no 11 pp 2704ndash2711 2012

[42] M Fang R J Kremer P P Motavalli and G Davis ldquoBacterialdiversity in rhizospheres of nontransgenic and transgenic cornrdquoApplied and EnvironmentalMicrobiology vol 71 no 7 pp 4132ndash4136 2005

[43] C R Howell and R D Stiphanovic ldquoProduction of ammonia byEnterobacter cloaceae and its possible role in biological controlof Pythium pre-emergence damping-off by the bacteriumrdquoEcology and Epidemiology vol 78 no 8 pp 1075ndash1078 1988

[44] A Ramette M Frapolli G Defago and Y Moenne-LoccozldquoPhylogeny of HCN synthase-encoding hcnBC genes in bio-control fluorescent pseudomonads and its relationship withhost plant species and HCN synthesis abilityrdquoMolecular Plant-Microbe Interactions vol 16 no 6 pp 525ndash535 2003

[45] I Loaces L Ferrando and A F Scavino ldquoDynamics diversityand function of endophytic siderophore-producing bacteria inricerdquoMicrobial Ecology vol 61 no 3 pp 606ndash618 2011

[46] Y C Kim J Leveau B B M Gardener E A Pierson L SPierson and C-M Ryu ldquoThe multifactorial basis for planthealth promotion by plant-associated bacteriardquo Applied andEnvironmental Microbiology vol 77 no 5 pp 1548ndash1555 2011

[47] J M Gardner J L Chandler and A W Feldman ldquoGrowthresponses and vascular plugging of citrus inoculated withrhizobacteria and xylem-resident bacteriardquo Plant and Soil vol86 no 3 pp 333ndash345 1985

[48] C Pliego C Ramos A de Vicente and F M Cazorla ldquoScreen-ing for candidate bacterial biocontrol agents against soilborne

14 International Journal of Microbiology

fungal plant pathogensrdquoPlant and Soil vol 340 no 1-2 pp 505ndash520 2011

[49] H Feng Y Li and Q Liu ldquoEndophytic bacterial communi-ties in tomato plants with differential resistance to Ralstoniasolanacearumrdquo African Journal of Microbiology Research vol 7no 15 pp 1311ndash1318 2013

[50] A V Sturz B R Christie B G Matheson W J Arsenaultand N A Buchanan ldquoEndophytic bacterial communities inthe periderm of potato tubers and their potential to improveresistance to soil-borne plant pathogensrdquo Plant Pathology vol48 no 3 pp 360ndash369 1999

[51] A V Sturz B R Christie and J Nowak ldquoBacterial endophytespotential role in developing sustainable systems of crop produc-tionrdquo Critical Reviews in Plant Sciences vol 19 no 1 pp 1ndash302000

[52] R Ramesh A A Joshi and M P Ghanekar ldquoPseudomonadsmajor antagonistic endophytic bacteria to suppress bacterialwilt pathogenRalstonia solanacearum in the eggplant (Solanummelongena L)rdquo World Journal of Microbiology and Biotechnol-ogy vol 25 no 1 pp 47ndash55 2009

[53] A A Nawangsih I Damayanti S Wiyono and J G KartikaldquoSelection and characterization of endophytic bacteria as bio-control agents of tomato Bacterial Wilt DiseaserdquoHayati Journalof Biosciences vol 18 no 2 pp 66ndash70 2011

[54] R Vargas-Ayala R Rodrıguez-Kabana G Morgan-Jones J AMcInroy and J W Kloepper ldquoShifts in soil microflora inducedby velvetbean (Mucuna deeringiana) in cropping systems tocontrol root-knot nematodesrdquo Biological Control vol 17 no 1pp 11ndash22 2000

[55] D K Zinniel P Lambrecht N B Harris et al ldquoIsolationand characterization of endophytic colonizing bacteria fromagronomic crops and prairie plantsrdquoApplied and EnvironmentalMicrobiology vol 68 no 5 pp 2198ndash2208 2002

[56] F Lemessa and W Zeller ldquoScreening rhizobacteria for biolog-ical control of Ralstonia solanacearum in Ethiopiardquo BiologicalControl vol 42 no 3 pp 336ndash344 2007

[57] M S El-AlbyadM A El-Sayed A R El-Shanshoury andNHEl-Batanouny ldquoEffect of culture conditions on the antimicrobialactivities of UV-mutants of Streptomyces corchorusii and Sspiroverticillatus against bean and banana wilt pathogensrdquoMicrobiological Research vol 151 no 2 pp 201ndash211 1996

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

14 International Journal of Microbiology

fungal plant pathogensrdquoPlant and Soil vol 340 no 1-2 pp 505ndash520 2011

[49] H Feng Y Li and Q Liu ldquoEndophytic bacterial communi-ties in tomato plants with differential resistance to Ralstoniasolanacearumrdquo African Journal of Microbiology Research vol 7no 15 pp 1311ndash1318 2013

[50] A V Sturz B R Christie B G Matheson W J Arsenaultand N A Buchanan ldquoEndophytic bacterial communities inthe periderm of potato tubers and their potential to improveresistance to soil-borne plant pathogensrdquo Plant Pathology vol48 no 3 pp 360ndash369 1999

[51] A V Sturz B R Christie and J Nowak ldquoBacterial endophytespotential role in developing sustainable systems of crop produc-tionrdquo Critical Reviews in Plant Sciences vol 19 no 1 pp 1ndash302000

[52] R Ramesh A A Joshi and M P Ghanekar ldquoPseudomonadsmajor antagonistic endophytic bacteria to suppress bacterialwilt pathogenRalstonia solanacearum in the eggplant (Solanummelongena L)rdquo World Journal of Microbiology and Biotechnol-ogy vol 25 no 1 pp 47ndash55 2009

[53] A A Nawangsih I Damayanti S Wiyono and J G KartikaldquoSelection and characterization of endophytic bacteria as bio-control agents of tomato Bacterial Wilt DiseaserdquoHayati Journalof Biosciences vol 18 no 2 pp 66ndash70 2011

[54] R Vargas-Ayala R Rodrıguez-Kabana G Morgan-Jones J AMcInroy and J W Kloepper ldquoShifts in soil microflora inducedby velvetbean (Mucuna deeringiana) in cropping systems tocontrol root-knot nematodesrdquo Biological Control vol 17 no 1pp 11ndash22 2000

[55] D K Zinniel P Lambrecht N B Harris et al ldquoIsolationand characterization of endophytic colonizing bacteria fromagronomic crops and prairie plantsrdquoApplied and EnvironmentalMicrobiology vol 68 no 5 pp 2198ndash2208 2002

[56] F Lemessa and W Zeller ldquoScreening rhizobacteria for biolog-ical control of Ralstonia solanacearum in Ethiopiardquo BiologicalControl vol 42 no 3 pp 336ndash344 2007

[57] M S El-AlbyadM A El-Sayed A R El-Shanshoury andNHEl-Batanouny ldquoEffect of culture conditions on the antimicrobialactivities of UV-mutants of Streptomyces corchorusii and Sspiroverticillatus against bean and banana wilt pathogensrdquoMicrobiological Research vol 151 no 2 pp 201ndash211 1996

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology