Research ArticleSpecific Genomic Fingerprints of Phosphate SolubilizingPseudomonas Strains Generated by Box Elements

Mohammad Bagher Javadi Nobandegani Halimi Mohd Saud and Wong Mui Yun

Institute Tropical Agriculture Universiti Putra Malaysia 43400 Serdang Selangor Malaysia

Correspondence should be addressed to Mohammad Bagher Javadi Nobandegani mbjavadi2002yahoocom

Received 10 July 2014 Revised 13 September 2014 Accepted 27 September 2014 Published 15 December 2014

Academic Editor Heide Schatten

Copyright copy 2014 Mohammad Bagher Javadi Nobandegani et al This is an open access article distributed under the CreativeCommons Attribution License which permits unrestricted use distribution and reproduction in any medium provided theoriginal work is properly cited

Primers corresponding to conserved bacterial repetitive of BOX elements were used to show that BOX-DNA sequences are widelydistributed in phosphate solubilizing Pseudomonas strains Phosphate solubilizing Pseudomonas was isolated from oil palm fields(tropical soil) inMalaysia BOX elements were used to generate genomic fingerprints of a variety of Pseudomonas isolates to identifystrains that were not distinguishable by other classification methods BOX-PCR that derived genomic fingerprints was generatedfrom whole purified genomic DNA by liquid culture of phosphate solubilizing Pseudomonas BOX-PCR generated the phosphatesolubilizing Pseudomonas specific fingerprints to identify the relationship between these strains This suggests that distribution ofBOX elementsrsquo sequences in phosphate solubilizing Pseudomonas strains is the mirror image of their genomic structureThereforethis method appears to be a rapid simple and reproducible method to identify and classify phosphate solubilizing Pseudomonasstrains and it may be useful tool for fast identification of potential biofertilizer strains

1 Introduction

Biofertilizer industry is facingwith the important challenge toidentify potential strains of each species rapidly and preciselyIn this regard the Pseudomonas species have shown betterperformance comparing to others [1] Pseudomonas strainswithin the species cannot be reliably distinguished by theircellular metabolisms or other phenotypic characteristics [2ndash5] Therefore strains classification is mostly based on oneor more host plants [6] Based on the phosphate solubi-lizing ability which is expressed in wide distribution inPseudomonas species this classification cannot be conclusiveand is open to alternative interpretations [7ndash10] Severalattempts such as fatty acids profiling [11 12] genomic andplasmid DNA analysis [10 13ndash19] and protein analysis [512 20] have been used to classify strains and overcome thisproblem even though these techniques are time-consumingexpensive or sometimes sensitive to use in routine labworks Thus it could be useful to find the fast rapid and

precise identification method to detect the most reliableand promising strains within the lot of strains which weredetected as phosphate solubilizing Pseudomonas on the basisof genomic fingerprinting approach

Families of repetitive DNA sequences which were dis-persed throughout the whole genome of various bacterialspecies were studied recently [21 22] One hundred fifty-fourbase-pair sequences which were determined as BOX-element[23] repetitive DNA sequences have been studied in moredetail These repetitive DNA sequences play an importantrole with the potential to construct stem-loop structure inthe organization of bacterial genome [21 24 25] Bacterialgenomic organization is thought to be shaped by selectiontherefore the distribution of BOX-elementsrsquo sequences canbe indicative of the structure and evolution of the bacterialgenome [21 24 25] On the basis of this theory and the clonalnature and population dynamics of bacteria [9 13 18 26ndash28] it can be theorized and assumed that each evolutionaryline or strains have a unique distribution or arrangement of

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 496562 7 pageshttpdxdoiorg1011552014496562

2 BioMed Research International

BOX repetitive sequences throughout the genomes and thatenables us to generate specific genomic fingerprints of eachisolate (strain)

In this paper the ability of the PCR technique withthe BOX-element corresponding primers to generate specificDNA fingerprints of phosphate solubilizing Pseudomonasspecies is demonstrated Also this technique can be a poten-tial tool for identification of the phylogenic relationshipsbetween the best phosphates solubilizing Pseudomonas forbiofertilizer industry application

2 Materials and Methods

21 Bacterial Isolates All phosphate solubilizing Pseudomo-nas isolates used in this study were isolated from the rhi-zosphere and nonrhizosphere of different locations (UPM-Semenyih-Dengkil oil palm fields) in Malaysia by using themodification method described by Nautiyal 1999 [29] Theisolates have been systematically identified by 16S rRNAmethod as Pseudomonas sp and are listed in Table 1 Allisolates were stored at minus80∘C in glycerol stock and streakedon nutrients agar for further applications [30]

22 Bacterial DNA Preparation Genomic DNA was extract-ed from isolated bacteria using a commercial kite (QiagenMiniprep 27104 Matrix Technologies Cooperation USA)according to the manufacturerrsquos instructions Bacteria cellswere grown overnight at 28∘C in LB broth with shakingOne milliliter of bacterial fresh culture was transferred to15mL microcentrifuge tube and centrifuged at 10000timesg for4 minutes at 4∘C The supernatant was discarded and thebacterial pellet resuspended in 100120583L 1x bactozymVortexingthe mixture was resulted in a homogenous suspension andthen the mixture was incubated at 50∘C for 30 minutes Fourhundred of DNAZOl solutions (TalronBiotech USA) wereadded to the lysate bacterial suspension and then it wasmixedfor 30 seconds and then incubated at room temperature for5 minutes DNA was precipitated by adding 03mL of 100ethanol and mixed by inversion for 15 seconds and thenstored at room temperature for 5 minutes Then the sampleswere transferred into a column that was assembled in a cleancollection tube provided by the company the samples werecentrifuged at 10000timesg for 1minuteThe columnwaswashedwith 750120583L of washing buffer (provided by Qiagen kit) andcentrifuged at 10000timesg two times for 1 minute each Columnwas placed into the clean microcentrifuge tube and 50 120583L TEbuffer was added directly onto column membrane and themixture stood for 2 minutes Again the tube was centrifugedat 10000timesg for 1min to eluteDNADNAwas stored atminus20∘C

23 PCR Amplification and Separation of DNA Bands Theprimer sequences corresponding to BOX elements (41)(51015840-CTACGGCAAGGCGACGCTGACG-31015840) and 16S rDNA[31] (f51015840-CCGAATTCGTCGACAACAGAGTTTGATCC-TGGCTCAG-31015840 and r51015840-CCCGGATCCAAGCTTACG-GCTACCTTGTTACGACTT-31015840) were synthesized atBioSynTech Sdn Bhd (HICOM Glenmarie Industrial Park40150 Selangor DE Malaysia) PCR condition for 16S rDNA

that was used had been described byWeisburg et al 1991 [31]by some modifications PCR amplification for 16S rDNA wasperformed by thermal cycler with the following programinitial denaturation was at 95∘C for 3 minutes followed by35 cycles consisting of denaturation at 94∘C for 30 secondsannealing at 60∘C for 30 seconds and elongation at 72∘Cfor 2 minutes and then the reaction was finished with anextension step at 72∘C for 5 minutes The BOX-PCR protocoland amplification were described by Versalovic et al 1993[22] After the reactions 8 to 10 120583L of the REP-PCR productswas separated on 1 agarose gels strained with ethidiumbromide and photographed by using gel-documentationsystem (Hoefer PS 500XT) [32]

24 Cluster Analysis (CA) For cluster analysis (CA) of dataa matrix was used to generate a genetic distance For thatEuclidean and Jaccard coefficient similarity matrix were usedand then dendrogram of relationship was generated throughunweighted pair-group method average (UPGMA) using thesoftware package NTSYS-pc program [33]

3 Results

Two universal oligonucleotides were used to determine andidentify the 16S rRNA gene for all isolatesThe primers ampli-fied the gene successfully from all phosphate solubilizingbacteria isolates It was seen that there were not obviousvariations in the size of rRNA gene products between thesix isolated bacteria and the size of the 16S rRNA geneproduct of all isolated bacteria investigated in this studywas approximately 14 Kb to the relative DNA size marker(Figure 1(a))

Comparing the partial 16S rDNA sequence from thesix bacterial isolates with sequences from the data base(NCBI) showed that they belong to the gamma subdivision ofProteobacteria phylum 18DNR 41DNR 22DNR 31SR and8SR bacterial isolates were classified in Pseudomonas genusas a Pseudomonas sp however 5DNR was identified as aPseudomonas fluorescens Sequences from these isolates were98 or more similar to other 16S rDNA sequences from database (Table 1)

The phylogenetic analysis based on the partial 16S rDNAsequencing was able to discriminate the two main taxo-nomic lineages using DNA neighbor phylogenetic tree pro-gram (Figure 2(a)) Within the main lineage the sequencesobtained from the bacterial strains associated with 31SR wereformed in the branch separated from the sequences of otherbacteria that were isolated from soil This feature was clearwithin the branch enclosing the sequences belonging to otherisolates There were six phylogeny branches that belonged toPseudomonas strains and they showed more than 99094similarity with each other (Figure 2(a))

Neighbor-joining analysis revealed the presence of twowell resolved lineages according to 16S rDNA sequenceanalysis designated clusters (A) and (B) (Figure 2(a)) Cluster(A) included 18DNR 41DNR 22DNR 8SR and 5DNRbacterial isolatesThe similarity between themwasmore than9996 Cluster (B) included the one species of Pseudomonas

BioMed Research International 3

Table1Ph

osph

ates

olub

ilizing

Pseudomonas

isolates

Num

ber

Isolate

Nam

eAc

cessionnu

mber(NCB

I)Ph

ylum

Class

Order

Family

Genus

118DNR

Pseudomonas

sp

KJ748597

Proteobacteria

Gam

maproteob

acteria

Pseudo

mon

adales

Pseudo

mon

adaceae

Pseudomonas

25D

NR

Pseudomonas

fluorescens

KJ748598

Proteobacteria

Gam

maproteob

acteria

Pseudo

mon

adales

Pseudo

mon

adaceae

Pseudomonas

341DNR

Pseudomonas

sp

KJ783454

Proteobacteria

Gam

maproteob

acteria

Pseudo

mon

adales

Pseudo

mon

adaceae

Pseudomonas

422DNR

Pseudomonas

sp

KJ729599

Proteobacteria

Gam

maproteob

acteria

Pseudo

mon

adales

Pseudo

mon

adaceae

Pseudomonas

531SR

Pseudomonas

sp

KJ748596

Proteobacteria

Gam

maproteob

acteria

Pseudo

mon

adales

Pseudo

mon

adaceae

Pseudomonas

68SR

Pseudomonas

sp

KJ783451

Proteobacteria

Gam

maproteob

acteria

Pseudo

mon

adales

Pseudo

mon

adaceae

Pseudomonas

4 BioMed Research International

(a)

10kb

15 kbp

250bp

(b)

Figure 1 PCR products of 16S rDNA (a) and BOX element (b) (Line 1 = 18DNR Line 2 = 5DNR Line 3 = 41DNR Line 4 = 22DNR Line 5 =31SR and Line 6 = 8SR)

006 000

(A)

(B)

22DNR (Pseudomonas sp)18DNR (Pseudomonas sp)

8SR (Pseudomonas sp)41DNR (Pseudomonas sp)31SR (Pseudomonas sp)

5DNR (Pseudomonasfluorescens)

(a)

(A2)

(A1)(A)

(B)

040 055 070 085 100

Coefficient

31SR5DNR41DNR22DNR8SR18DNR

(b)

Figure 2 Dendrogram of phosphate solubilizing Pseudomonas bacteria based on the 16S rDNA (a) and BOX-PCR marker (b)

sp (31SR) Gene sequences of 16S rDNA from phosphate sol-ubilizing Pseudomonas bacteria were grouped within gammaproteobacteria

31 BOX-PCR Analysis PCR fingerprints with the BOXelement primer (BOX-PCR) revealed species-specific bandpatterns for the various isolates of phosphate solubilizingPseudomonas DNA fingerprints obtained from BOX-PCRof extracted genomic DNA yielded comparable patternsBOX-PCR as a precise molecular marker allowed betterdiscrimination than 16S rDNA sequencing between strainswithin Pseudomonas species (isolates) BOX-PCR of theseisolates revealed six different fingerprint profiles among sixisolates of phosphate solubilizing bacteria isolated from oilpalm soil (Figure 1(b))0

PCR with BOX-PCR primer and chromosomal DNAfrom the strains yielded multiple distinct DNA productsof sizes ranging from approximately 300 to 5000 bp TheBOX-PCR patterns of Pseudomonas species designates werefound to be highly related to one another (gt40) 18DNRand 22DNR and 8SR Pseudomonas bacterial isolates whichwere identified as a Pseudomonas spwere found to be highlyrelated to one another (gt70) but very distinct from 41DNR(Pseudomonas sp) 5DNR (Pseudomonas fluorescens) and31SR (Pseudomonas sp) (Figure 2(b))

The BOX-PCR marker similarities using Jaccardrsquos coef-ficient were calculated using the data analysis The matrixof coefficient was then used to cluster the similar accessionbased on BOX-PCR data and then to construct the dendro-gram of relationship through the UPGMA The dendrogramshowing the genetic relationship of the isolates is presentedin Figure 2(b)

The cluster analysis of Pseudomonas species based on theBOX-PCR identified two major groups ((A) (B)) at geneticdistance = 060 (Figure 2) Cluster (A) contained threeisolates with the calculation of different locations (SemenyihDengkil) isolates 31SR 5DNR and 41DNR within this clus-ter In cluster (A) there was two subclusters which included(A1) (31SR 5DNR) and (A2) (41DNR) Cluster (B) wasformed by three isolates In this cluster isolates 22DNR 8SRand 18DNR were very similar to each other based on the16S rDNA sequencing however there was less than 70similarity based on the BOX-PCR It could reveal that theywere different strains Clusters (A) and (B) together formed amain cluster at genetic distance 04

4 Discussion

In this study we have demonstrated that BOX elements asrepetitive sequences were present in the genome of phosphate

BioMed Research International 5

solubilizing Pseudomonas isolates confirming and extendingthe conclusion of Versalovic et al 1991 [34] and Akkermanset al 1995 [35] and these sequences are virtually ubiquitousWehave also demonstrated that the BOX-PCRprotocolswereparticularly suitable for the rapid molecular characterizationof phosphate solubilizing Pseudomonas bacteria especiallyat the strains level The BOX-PCR protocol clearly had thepotential to differentiate between isolates including thosethat were not easily distinguished by other phenotypic andphylogenetic techniques such as 16S rDNA

The data presented here suggested that BOX-PCR couldbe a useful tool for identification of phosphate solubilizingPseudomonaspurposes in industrial biofertilizers technologySimilar outcomes have been made about the utility of BOX-PCR in human pathology [22 36 37]

Several circumstances must be considered if BOX-PCRis to be useful for the proper identification of unknownphosphate solubilizing Pseudomonas isolates Firstly thecharacteristic of BOX-like sequences on gel and thereforethe genomic fingerprint patterns generated by BOX-PCRmust be established over time and distance Comparison ofthe genomic fingerprint profiles of phosphate solubilizingPseudomonas isolates within a tropical areas separated bytime or distance supports the idea that the profiles remainstable This similarity of fingerprint profiles of isolates bytime has been noted by others too [37] Secondly the BOX-PCR technique must be able to distinguish among relatedbacterial strains with sufficient declaration and it also shouldbe reproducible

41 Supporting the Reproducibility of the BOX-PCR Proto-col Large numbers of phosphate solubilizing Pseudomonasisolates have determined that there was homogeneity offingerprint profiles within each isolate It was systematicallyshown that BOX in general could determine that the differ-ences between phosphate solubilizing Pseudomonas isolatesand substantial polymorphism were detected Therefore thedistribution of BOX sequences was an accurate reflection ofgenomic structure

Other PCR-based genomic fingerprinting techniqueshave been described and demonstrated to use for differ-entiating bacteria and diagnostic purposes [38ndash42] Thereare some fundamental differences between BOX-PCR andRAPD-DNA analyses The major difference lies in the lengthof the primers and the consequent PCR conditions RAPDanalysis relies on the use of primers with arbitrary sequences[43 44] whereas BOX-PCR involves the use of primersof 22 bp with high homology to repetitive sequences [34]The BOX primer permits the use of more inflexible PCRconditions which in turn reduce experimental variation andPCR artefacts

As noted by others the BOX-PCR technique is veryuseful for bacterial strain identification however the utilityof that for bacterial taxonomy may be limited to closelyrelated strains [36 45 46] Protein profile analysis or fattyacid profile analysis [12] serologic testing [47] and rRNAgene restriction patterns [10] support the distinctiveness ofBOX-PCR analysis

The BOX-PCR fingerprint profiles between the two iso-lates contain many bands of equal mobility and rely onthe concept that selection for a specialized niche affectsgenome organization [25] and that corresponds to a uniquedistribution of repetitive sequences in the bacterial genome

The BOX-PCR of phosphate solubilizing Pseudomonasisolates technique may be limited to phylogenetic analysisand it effectively differentiates between two evolutionary linesclassified within the same taxon

5 Conclusion

In conclusion we have found that BOX sequences werewidespread in phosphate solubilizing Pseudomonas isolatesand could be used to generate genomic fingerprints withinPseudomonas species Unique fingerprint profiles generatedby BOX-PCR could be exploited for identification purposesand for discerning evolutionary lines of phosphate solubiliz-ing Pseudomonas in oil palm fields Revealing the populationdiversity of phosphate solubilizing Pseudomonas isolatesin turn had implications for the implementation of themfor biofertilizers industry programs disease managementstrategies and ecological and epidemiological studies

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

References

[1] S Nikolaki and G Tsiamis ldquoMicrobial diversity in the era ofomic technologiesrdquo BioMed Research International vol 2013Article ID 958719 15 pages 2013

[2] DW Dye ldquoThe inadequacy of the usual determinative tests forthe identification of Xanthomonas spprdquoNew Zealand Journal ofScience vol 5 pp 393ndash416 1962

[3] N J Palleroni Bergeyrsquos Manual of Systematic Bacteriology vol1 TheWilliams ampWilkins Co Baltimore Md USA 1894

[4] M Van den Mooter and J Swings ldquoNumerical analysis of 295phenotypic features of 266 Xanthomonas strains and relatedstrains and an improved taxonomy of the genusrdquo InternationalJournal of Systematic Bacteriology vol 40 no 4 pp 348ndash3691990

[5] E van Zyl and P L Steyn ldquoDifferentiation of phytopathogenicPseudomonas and Xanthomonas species and pathovars bynumerical taxonomy and protein gel electrophoregramsrdquo Sys-tematic and Applied Microbiology vol 13 no 1 pp 60ndash71 1990

[6] J M Young J F Bradbury R E Davis et al ldquoNomenclaturalrevisions of plant pathogenic bacteria and list of names 1980ndash1988rdquo Plant Pathology vol 70 pp 213ndash221 1991

[7] DWGabriel and R de Fuyter ldquoRFLP analysis and gene taggingfor bacterial identification and taxonomyrdquo in Molecular PlantPathology A Practical Approach M J M S J Gurr and D JBowles Eds vol 1 pp 51ndash66 IRL Press New York NY USA1992

[8] D W Gabriel M T Kingsley J E Hunter and T GottwaldldquoReinstatement of Xanthomonas citri (ex Hasse) and X phaseoli(ex Smith) to species and reclassification of all X campestris

6 BioMed Research International

pv citri strainsrdquo International Journal of Systematic Bacteriologyvol 39 no 1 pp 14ndash22 1989

[9] G R Lazo R Roffey and D W Gabriel ldquoPathovars ofXanthomonas campestris are distinguishable by restrictionfragment-length polymorphismrdquo International Journal of Sys-tematic Bacteriology vol 37 pp 214ndash221 1987

[10] Y Berthier V Verdier J-L Guesdon et al ldquoCharacterization ofXanthomonas campestris pathovars by rRNA gene restrictionpatternsrdquo Applied and Environmental Microbiology vol 59 no3 pp 851ndash859 1993

[11] D E Stead ldquoGrouping of plant-pathogenic and some otherPseudomonas spp by using cellular fatty acid profilesrdquo Interna-tional Journal of Systematic Bacteriology vol 42 no 2 pp 281ndash295 1992

[12] L Vauterin P Yang B Hoste B Pot J Swings and KKersters ldquoTaxonomy of xanthomonads from cereals and grassesbased on SDS-PAGE of proteins fatty acid analysis and DNAhybridizationrdquo Journal of General Microbiology vol 138 no 7pp 1467ndash1477 1992

[13] T P Denny M N Gilmour and R K Selander ldquoGeneticdiversity and relationships of two pathovars of Pseudomonassyringaerdquo Journal of General Microbiology vol 134 no 7 pp1949ndash1960 1988

[14] J S Hartung and E L Civerolo ldquoGenomic fingerprints ofXanthomonas campestris pv citri strains from Asia SouthAmerica and Floridardquo Phytopathology vol 77 pp 282ndash2851987

[15] D C Hildebrand N J Palleroni and M N Schroth ldquoDeoxyri-bonucleic acid relatedness of 24 xanthomonad strains rep-resenting 23 Xanthomonas campestris pathovars and Xan-thomonas fragariaerdquoTheJournal of Applied Bacteriology vol 68no 3 pp 263ndash269 1990

[16] G J King ldquoPlasmid analysis and variation in Pseudomonassyringaerdquo Journal of Applied Bacteriology vol 67 no 5 pp 489ndash496 1989

[17] G R Lazo and D W Gabriel ldquoConservation of plasmidDNA sequences and pathovar identification of strains of Xan-thomonas campestrisrdquo Phytopathology vol 77 pp 448ndash4531987

[18] J E Leach F F White M L Rhoads and H Leung ldquoA repet-itive DNA sequence differentiates Xanthomonas campestnis pvoryzae from other pathovars of X campestrisrdquoMolecular Plant-Microbe Interactions Journal vol 3 pp 238ndash246 1990

[19] P C Pecknold and R G Grogan ldquoDeoxyribonucleic acidhomology groups among phytopathogenic Pseudomonasspeciesrdquo International Journal of Systematic Bacteriology vol23 no 2 pp 111ndash121 1973

[20] L Vauterin R Vantomme B Pot B Hoste J Swings and KKersters ldquoTaxonomic analysis of Xanthomonas campestris pvbegoniae and X campestris pv pelargonii by means of phy-topathological phenotypic protein electrophoretic and DNAhybridization methodsrdquo Systematic and Applied Microbiologyvol 13 no 2 pp 166ndash176 1990

[21] J R Lupski andGMWeinstock ldquoShort interspersed repetitiveDNA sequences in prokaryotic genomesrdquo Journal of Bacteriol-ogy vol 174 no 14 pp 4525ndash4529 1992

[22] J Versalovic V Kapur E OMason Jr et al ldquoPenicillin-resistantStreptococcus pneumoniae strains recovered in Houston identi-fication and molecular characterization of multiple clonesrdquoTheJournal of Infectious Diseases vol 167 no 4 pp 850ndash856 1993

[23] B Martin O Humbert M Camara et al ldquoA highly conservedrepeated DNA element located in the chromosome of Strepto-coccus pneumoniaerdquo Nucleic Acids Research vol 20 no 13 pp3479ndash3483 1992

[24] S Krawiec ldquoConcept of a bacterial speciesrdquo InternationalJournal of Systematic Bacteriology vol 35 no 2 pp 217ndash2201985

[25] S Krawiec and M Riley ldquoOrganization of the bacterial chro-mosomerdquo Microbiological Reviews vol 54 no 4 pp 502ndash5391990

[26] M Achtman and G Pluschke ldquoClonal analysis of descent andvirulence among selected Escherichia colirdquo Annual Review ofMicrobiology vol 40 pp 185ndash210 1986

[27] DW Gabriel J E HunterM T Kingsley JWMiller andG RLazo ldquoClonal population structure of Xanthomonas campestrisand genetic diversity among citrus canker strainsrdquo MolecularPlant-Microbe Interactions Journal vol 1 pp 59ndash65 1988

[28] R K Selander and JMMusser Population Genetics of BacterialPathogenesis Academic Press San Diego Calif USA 1990

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

[30] M B Javadinobandegani Distribution and Biochemical andGenotypic Comparison of Phosphate Solubilizing Bacteria in OilPalm Soils Department of Agriculture Technology UniversityPutra Malaysia Selangor Malaysia 2008

[31] W GWeisburg S M Barns D A Pelletier and D J Lane ldquo16Sribosomal DNA amplification for phylogenetic studyrdquo Journalof Bacteriology vol 173 no 2 pp 697ndash703 1991

[32] J Sambrook Molecular Cloning A Laboratory Manual ColdSpringHarbor Laboratory Press Cold SpringHarbor NY USA2001

[33] F J Rolf NTSYS-PC Numerical Taxonomy And MultivariateAnalysis SystemVersion 210 Applied Biostatistics 2000

[34] J Versalovic T Koeuth and J R Lupski ldquoDistribution ofrepetitive DNA sequences in eubacteria and application tofingerprinting of bacterial genomesrdquo Nucleic Acids Researchvol 19 no 24 pp 6823ndash6831 1991

[35] AD L Akkermans J D van Elsas and F J de BruijnMolecularMicrobial Ecology Manual Kluwer Academic Dordrecht TheNetherlands 1995

[36] C R Woods Jr J Versalovic T Koeuth and J R LupskildquoAnalysis of relationships among isolates of Citrobacter diversusby using DNA fingerprints generated by repetitive sequence-based primers in the polymerase chain reactionrdquo Journal ofClinical Microbiology vol 30 no 11 pp 2921ndash2929 1992

[37] C R Woods J Versalovic T Koeuth and J R Lupski ldquoWhole-cell repetitive element sequence-based polymerase chain reac-tion allows rapid assessment of clonal relationships of bacterialisolatesrdquo Journal of Clinical Microbiology vol 31 no 7 pp 1927ndash1931 1993

[38] M R Cancilla I B Powell A J Hillier and B E DavidsonldquoRapid genomic fingerprinting of Lactococcus lactis strainsby arbitrarily primed polymerase chain reaction with p32 andfluorescent labelsrdquo Applied and Environmental Microbiologyvol 58 no 5 pp 1772ndash1775 1992

[39] A Fekete J A Bantle S M Halling and R W Stich ldquoAmplifi-cation fragment length polymorphism in Brucella strains by useof polymerase chain reaction with arbitrary primersrdquo Journal ofBacteriology vol 174 no 23 pp 7778ndash7783 1992

BioMed Research International 7

[40] S Araujo I S Henriques S M Leandro A Alves A Pereiraand A Correia ldquoGulls identified as major source of fecalpollution in coastal waters a microbial source tracking studyrdquoScience of the Total Environment vol 470-471 pp 84ndash91 2014

[41] H Guo Z Mao H Jiang et al ldquoCommunity analysis ofplant growth promoting rhizobacteria for apple treesrdquo CropProtection vol 62 pp 1ndash9 2014

[42] L Zhu H Xu Y Zhang G Fu P Q Wu and Y Li ldquoBOX-PCRand PCR-DGGE analysis for bacterial diversity of a naturallyfermented functional food (Enzyme)rdquo Food Bioscience vol 5pp 115ndash122 2014

[43] J Welsh and M McClelland ldquoFingerprinting genomes usingPCR with arbitrary primersrdquoNucleic Acids Research vol 18 no24 pp 7213ndash7218 1990

[44] J G KWilliams A R Kubelik K J Livak J A Rafalski and SV Tingey ldquoDNApolymorphisms amplified by arbitrary primersare useful as geneticmarkersrdquoNucleic Acids Research vol 18 no22 pp 6531ndash6535 1990

[45] F J Louws D W Fulbright C T Stephens and F J DeBruijn ldquoSpecific genomic fingerprints of phytopathogenic Xan-thomonas and Pseudomonas pathovars and strains generatedwith repetitive sequences and PCRrdquoApplied and EnvironmentalMicrobiology vol 60 no 7 pp 2286ndash2295 1994

[46] A K Judd M Schneider M J Sadowsky and F J de BruijnldquoUse of repetitive sequences and the polymerase chain reac-tion technique to classify genetically related Bradyrhizobiumjaponicum serocluster 123 strainsrdquo Applied and EnvironmentalMicrobiology vol 59 no 6 pp 1702ndash1708 1993

[47] A A Benedict A M Alvarez and J Berestecky ldquoPathovarspecific monoclonal antibodies for Xanthomonas campestrispv oryzae and for Xanthomonas campestris pv oryzicolardquoPhytopathology vol 79 pp 322ndash328 1989

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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International Journal of

Microbiology

2 BioMed Research International

BOX repetitive sequences throughout the genomes and thatenables us to generate specific genomic fingerprints of eachisolate (strain)

In this paper the ability of the PCR technique withthe BOX-element corresponding primers to generate specificDNA fingerprints of phosphate solubilizing Pseudomonasspecies is demonstrated Also this technique can be a poten-tial tool for identification of the phylogenic relationshipsbetween the best phosphates solubilizing Pseudomonas forbiofertilizer industry application

2 Materials and Methods

21 Bacterial Isolates All phosphate solubilizing Pseudomo-nas isolates used in this study were isolated from the rhi-zosphere and nonrhizosphere of different locations (UPM-Semenyih-Dengkil oil palm fields) in Malaysia by using themodification method described by Nautiyal 1999 [29] Theisolates have been systematically identified by 16S rRNAmethod as Pseudomonas sp and are listed in Table 1 Allisolates were stored at minus80∘C in glycerol stock and streakedon nutrients agar for further applications [30]

22 Bacterial DNA Preparation Genomic DNA was extract-ed from isolated bacteria using a commercial kite (QiagenMiniprep 27104 Matrix Technologies Cooperation USA)according to the manufacturerrsquos instructions Bacteria cellswere grown overnight at 28∘C in LB broth with shakingOne milliliter of bacterial fresh culture was transferred to15mL microcentrifuge tube and centrifuged at 10000timesg for4 minutes at 4∘C The supernatant was discarded and thebacterial pellet resuspended in 100120583L 1x bactozymVortexingthe mixture was resulted in a homogenous suspension andthen the mixture was incubated at 50∘C for 30 minutes Fourhundred of DNAZOl solutions (TalronBiotech USA) wereadded to the lysate bacterial suspension and then it wasmixedfor 30 seconds and then incubated at room temperature for5 minutes DNA was precipitated by adding 03mL of 100ethanol and mixed by inversion for 15 seconds and thenstored at room temperature for 5 minutes Then the sampleswere transferred into a column that was assembled in a cleancollection tube provided by the company the samples werecentrifuged at 10000timesg for 1minuteThe columnwaswashedwith 750120583L of washing buffer (provided by Qiagen kit) andcentrifuged at 10000timesg two times for 1 minute each Columnwas placed into the clean microcentrifuge tube and 50 120583L TEbuffer was added directly onto column membrane and themixture stood for 2 minutes Again the tube was centrifugedat 10000timesg for 1min to eluteDNADNAwas stored atminus20∘C

23 PCR Amplification and Separation of DNA Bands Theprimer sequences corresponding to BOX elements (41)(51015840-CTACGGCAAGGCGACGCTGACG-31015840) and 16S rDNA[31] (f51015840-CCGAATTCGTCGACAACAGAGTTTGATCC-TGGCTCAG-31015840 and r51015840-CCCGGATCCAAGCTTACG-GCTACCTTGTTACGACTT-31015840) were synthesized atBioSynTech Sdn Bhd (HICOM Glenmarie Industrial Park40150 Selangor DE Malaysia) PCR condition for 16S rDNA

that was used had been described byWeisburg et al 1991 [31]by some modifications PCR amplification for 16S rDNA wasperformed by thermal cycler with the following programinitial denaturation was at 95∘C for 3 minutes followed by35 cycles consisting of denaturation at 94∘C for 30 secondsannealing at 60∘C for 30 seconds and elongation at 72∘Cfor 2 minutes and then the reaction was finished with anextension step at 72∘C for 5 minutes The BOX-PCR protocoland amplification were described by Versalovic et al 1993[22] After the reactions 8 to 10 120583L of the REP-PCR productswas separated on 1 agarose gels strained with ethidiumbromide and photographed by using gel-documentationsystem (Hoefer PS 500XT) [32]

24 Cluster Analysis (CA) For cluster analysis (CA) of dataa matrix was used to generate a genetic distance For thatEuclidean and Jaccard coefficient similarity matrix were usedand then dendrogram of relationship was generated throughunweighted pair-group method average (UPGMA) using thesoftware package NTSYS-pc program [33]

3 Results

Two universal oligonucleotides were used to determine andidentify the 16S rRNA gene for all isolatesThe primers ampli-fied the gene successfully from all phosphate solubilizingbacteria isolates It was seen that there were not obviousvariations in the size of rRNA gene products between thesix isolated bacteria and the size of the 16S rRNA geneproduct of all isolated bacteria investigated in this studywas approximately 14 Kb to the relative DNA size marker(Figure 1(a))

Comparing the partial 16S rDNA sequence from thesix bacterial isolates with sequences from the data base(NCBI) showed that they belong to the gamma subdivision ofProteobacteria phylum 18DNR 41DNR 22DNR 31SR and8SR bacterial isolates were classified in Pseudomonas genusas a Pseudomonas sp however 5DNR was identified as aPseudomonas fluorescens Sequences from these isolates were98 or more similar to other 16S rDNA sequences from database (Table 1)

The phylogenetic analysis based on the partial 16S rDNAsequencing was able to discriminate the two main taxo-nomic lineages using DNA neighbor phylogenetic tree pro-gram (Figure 2(a)) Within the main lineage the sequencesobtained from the bacterial strains associated with 31SR wereformed in the branch separated from the sequences of otherbacteria that were isolated from soil This feature was clearwithin the branch enclosing the sequences belonging to otherisolates There were six phylogeny branches that belonged toPseudomonas strains and they showed more than 99094similarity with each other (Figure 2(a))

Neighbor-joining analysis revealed the presence of twowell resolved lineages according to 16S rDNA sequenceanalysis designated clusters (A) and (B) (Figure 2(a)) Cluster(A) included 18DNR 41DNR 22DNR 8SR and 5DNRbacterial isolatesThe similarity between themwasmore than9996 Cluster (B) included the one species of Pseudomonas

BioMed Research International 3

Table1Ph

osph

ates

olub

ilizing

Pseudomonas

isolates

Num

ber

Isolate

Nam

eAc

cessionnu

mber(NCB

I)Ph

ylum

Class

Order

Family

Genus

118DNR

Pseudomonas

sp

KJ748597

Proteobacteria

Gam

maproteob

acteria

Pseudo

mon

adales

Pseudo

mon

adaceae

Pseudomonas

25D

NR

Pseudomonas

fluorescens

KJ748598

Proteobacteria

Gam

maproteob

acteria

Pseudo

mon

adales

Pseudo

mon

adaceae

Pseudomonas

341DNR

Pseudomonas

sp

KJ783454

Proteobacteria

Gam

maproteob

acteria

Pseudo

mon

adales

Pseudo

mon

adaceae

Pseudomonas

422DNR

Pseudomonas

sp

KJ729599

Proteobacteria

Gam

maproteob

acteria

Pseudo

mon

adales

Pseudo

mon

adaceae

Pseudomonas

531SR

Pseudomonas

sp

KJ748596

Proteobacteria

Gam

maproteob

acteria

Pseudo

mon

adales

Pseudo

mon

adaceae

Pseudomonas

68SR

Pseudomonas

sp

KJ783451

Proteobacteria

Gam

maproteob

acteria

Pseudo

mon

adales

Pseudo

mon

adaceae

Pseudomonas

4 BioMed Research International

(a)

10kb

15 kbp

250bp

(b)

Figure 1 PCR products of 16S rDNA (a) and BOX element (b) (Line 1 = 18DNR Line 2 = 5DNR Line 3 = 41DNR Line 4 = 22DNR Line 5 =31SR and Line 6 = 8SR)

006 000

(A)

(B)

22DNR (Pseudomonas sp)18DNR (Pseudomonas sp)

8SR (Pseudomonas sp)41DNR (Pseudomonas sp)31SR (Pseudomonas sp)

5DNR (Pseudomonasfluorescens)

(a)

(A2)

(A1)(A)

(B)

040 055 070 085 100

Coefficient

31SR5DNR41DNR22DNR8SR18DNR

(b)

Figure 2 Dendrogram of phosphate solubilizing Pseudomonas bacteria based on the 16S rDNA (a) and BOX-PCR marker (b)

sp (31SR) Gene sequences of 16S rDNA from phosphate sol-ubilizing Pseudomonas bacteria were grouped within gammaproteobacteria

31 BOX-PCR Analysis PCR fingerprints with the BOXelement primer (BOX-PCR) revealed species-specific bandpatterns for the various isolates of phosphate solubilizingPseudomonas DNA fingerprints obtained from BOX-PCRof extracted genomic DNA yielded comparable patternsBOX-PCR as a precise molecular marker allowed betterdiscrimination than 16S rDNA sequencing between strainswithin Pseudomonas species (isolates) BOX-PCR of theseisolates revealed six different fingerprint profiles among sixisolates of phosphate solubilizing bacteria isolated from oilpalm soil (Figure 1(b))0

PCR with BOX-PCR primer and chromosomal DNAfrom the strains yielded multiple distinct DNA productsof sizes ranging from approximately 300 to 5000 bp TheBOX-PCR patterns of Pseudomonas species designates werefound to be highly related to one another (gt40) 18DNRand 22DNR and 8SR Pseudomonas bacterial isolates whichwere identified as a Pseudomonas spwere found to be highlyrelated to one another (gt70) but very distinct from 41DNR(Pseudomonas sp) 5DNR (Pseudomonas fluorescens) and31SR (Pseudomonas sp) (Figure 2(b))

The BOX-PCR marker similarities using Jaccardrsquos coef-ficient were calculated using the data analysis The matrixof coefficient was then used to cluster the similar accessionbased on BOX-PCR data and then to construct the dendro-gram of relationship through the UPGMA The dendrogramshowing the genetic relationship of the isolates is presentedin Figure 2(b)

The cluster analysis of Pseudomonas species based on theBOX-PCR identified two major groups ((A) (B)) at geneticdistance = 060 (Figure 2) Cluster (A) contained threeisolates with the calculation of different locations (SemenyihDengkil) isolates 31SR 5DNR and 41DNR within this clus-ter In cluster (A) there was two subclusters which included(A1) (31SR 5DNR) and (A2) (41DNR) Cluster (B) wasformed by three isolates In this cluster isolates 22DNR 8SRand 18DNR were very similar to each other based on the16S rDNA sequencing however there was less than 70similarity based on the BOX-PCR It could reveal that theywere different strains Clusters (A) and (B) together formed amain cluster at genetic distance 04

4 Discussion

In this study we have demonstrated that BOX elements asrepetitive sequences were present in the genome of phosphate

BioMed Research International 5

solubilizing Pseudomonas isolates confirming and extendingthe conclusion of Versalovic et al 1991 [34] and Akkermanset al 1995 [35] and these sequences are virtually ubiquitousWehave also demonstrated that the BOX-PCRprotocolswereparticularly suitable for the rapid molecular characterizationof phosphate solubilizing Pseudomonas bacteria especiallyat the strains level The BOX-PCR protocol clearly had thepotential to differentiate between isolates including thosethat were not easily distinguished by other phenotypic andphylogenetic techniques such as 16S rDNA

The data presented here suggested that BOX-PCR couldbe a useful tool for identification of phosphate solubilizingPseudomonaspurposes in industrial biofertilizers technologySimilar outcomes have been made about the utility of BOX-PCR in human pathology [22 36 37]

Several circumstances must be considered if BOX-PCRis to be useful for the proper identification of unknownphosphate solubilizing Pseudomonas isolates Firstly thecharacteristic of BOX-like sequences on gel and thereforethe genomic fingerprint patterns generated by BOX-PCRmust be established over time and distance Comparison ofthe genomic fingerprint profiles of phosphate solubilizingPseudomonas isolates within a tropical areas separated bytime or distance supports the idea that the profiles remainstable This similarity of fingerprint profiles of isolates bytime has been noted by others too [37] Secondly the BOX-PCR technique must be able to distinguish among relatedbacterial strains with sufficient declaration and it also shouldbe reproducible

41 Supporting the Reproducibility of the BOX-PCR Proto-col Large numbers of phosphate solubilizing Pseudomonasisolates have determined that there was homogeneity offingerprint profiles within each isolate It was systematicallyshown that BOX in general could determine that the differ-ences between phosphate solubilizing Pseudomonas isolatesand substantial polymorphism were detected Therefore thedistribution of BOX sequences was an accurate reflection ofgenomic structure

Other PCR-based genomic fingerprinting techniqueshave been described and demonstrated to use for differ-entiating bacteria and diagnostic purposes [38ndash42] Thereare some fundamental differences between BOX-PCR andRAPD-DNA analyses The major difference lies in the lengthof the primers and the consequent PCR conditions RAPDanalysis relies on the use of primers with arbitrary sequences[43 44] whereas BOX-PCR involves the use of primersof 22 bp with high homology to repetitive sequences [34]The BOX primer permits the use of more inflexible PCRconditions which in turn reduce experimental variation andPCR artefacts

As noted by others the BOX-PCR technique is veryuseful for bacterial strain identification however the utilityof that for bacterial taxonomy may be limited to closelyrelated strains [36 45 46] Protein profile analysis or fattyacid profile analysis [12] serologic testing [47] and rRNAgene restriction patterns [10] support the distinctiveness ofBOX-PCR analysis

The BOX-PCR fingerprint profiles between the two iso-lates contain many bands of equal mobility and rely onthe concept that selection for a specialized niche affectsgenome organization [25] and that corresponds to a uniquedistribution of repetitive sequences in the bacterial genome

The BOX-PCR of phosphate solubilizing Pseudomonasisolates technique may be limited to phylogenetic analysisand it effectively differentiates between two evolutionary linesclassified within the same taxon

5 Conclusion

In conclusion we have found that BOX sequences werewidespread in phosphate solubilizing Pseudomonas isolatesand could be used to generate genomic fingerprints withinPseudomonas species Unique fingerprint profiles generatedby BOX-PCR could be exploited for identification purposesand for discerning evolutionary lines of phosphate solubiliz-ing Pseudomonas in oil palm fields Revealing the populationdiversity of phosphate solubilizing Pseudomonas isolatesin turn had implications for the implementation of themfor biofertilizers industry programs disease managementstrategies and ecological and epidemiological studies

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

References

[1] S Nikolaki and G Tsiamis ldquoMicrobial diversity in the era ofomic technologiesrdquo BioMed Research International vol 2013Article ID 958719 15 pages 2013

[2] DW Dye ldquoThe inadequacy of the usual determinative tests forthe identification of Xanthomonas spprdquoNew Zealand Journal ofScience vol 5 pp 393ndash416 1962

[3] N J Palleroni Bergeyrsquos Manual of Systematic Bacteriology vol1 TheWilliams ampWilkins Co Baltimore Md USA 1894

[4] M Van den Mooter and J Swings ldquoNumerical analysis of 295phenotypic features of 266 Xanthomonas strains and relatedstrains and an improved taxonomy of the genusrdquo InternationalJournal of Systematic Bacteriology vol 40 no 4 pp 348ndash3691990

[5] E van Zyl and P L Steyn ldquoDifferentiation of phytopathogenicPseudomonas and Xanthomonas species and pathovars bynumerical taxonomy and protein gel electrophoregramsrdquo Sys-tematic and Applied Microbiology vol 13 no 1 pp 60ndash71 1990

[6] J M Young J F Bradbury R E Davis et al ldquoNomenclaturalrevisions of plant pathogenic bacteria and list of names 1980ndash1988rdquo Plant Pathology vol 70 pp 213ndash221 1991

[7] DWGabriel and R de Fuyter ldquoRFLP analysis and gene taggingfor bacterial identification and taxonomyrdquo in Molecular PlantPathology A Practical Approach M J M S J Gurr and D JBowles Eds vol 1 pp 51ndash66 IRL Press New York NY USA1992

[8] D W Gabriel M T Kingsley J E Hunter and T GottwaldldquoReinstatement of Xanthomonas citri (ex Hasse) and X phaseoli(ex Smith) to species and reclassification of all X campestris

6 BioMed Research International

pv citri strainsrdquo International Journal of Systematic Bacteriologyvol 39 no 1 pp 14ndash22 1989

[9] G R Lazo R Roffey and D W Gabriel ldquoPathovars ofXanthomonas campestris are distinguishable by restrictionfragment-length polymorphismrdquo International Journal of Sys-tematic Bacteriology vol 37 pp 214ndash221 1987

[10] Y Berthier V Verdier J-L Guesdon et al ldquoCharacterization ofXanthomonas campestris pathovars by rRNA gene restrictionpatternsrdquo Applied and Environmental Microbiology vol 59 no3 pp 851ndash859 1993

[11] D E Stead ldquoGrouping of plant-pathogenic and some otherPseudomonas spp by using cellular fatty acid profilesrdquo Interna-tional Journal of Systematic Bacteriology vol 42 no 2 pp 281ndash295 1992

[12] L Vauterin P Yang B Hoste B Pot J Swings and KKersters ldquoTaxonomy of xanthomonads from cereals and grassesbased on SDS-PAGE of proteins fatty acid analysis and DNAhybridizationrdquo Journal of General Microbiology vol 138 no 7pp 1467ndash1477 1992

[13] T P Denny M N Gilmour and R K Selander ldquoGeneticdiversity and relationships of two pathovars of Pseudomonassyringaerdquo Journal of General Microbiology vol 134 no 7 pp1949ndash1960 1988

[14] J S Hartung and E L Civerolo ldquoGenomic fingerprints ofXanthomonas campestris pv citri strains from Asia SouthAmerica and Floridardquo Phytopathology vol 77 pp 282ndash2851987

[15] D C Hildebrand N J Palleroni and M N Schroth ldquoDeoxyri-bonucleic acid relatedness of 24 xanthomonad strains rep-resenting 23 Xanthomonas campestris pathovars and Xan-thomonas fragariaerdquoTheJournal of Applied Bacteriology vol 68no 3 pp 263ndash269 1990

[16] G J King ldquoPlasmid analysis and variation in Pseudomonassyringaerdquo Journal of Applied Bacteriology vol 67 no 5 pp 489ndash496 1989

[17] G R Lazo and D W Gabriel ldquoConservation of plasmidDNA sequences and pathovar identification of strains of Xan-thomonas campestrisrdquo Phytopathology vol 77 pp 448ndash4531987

[18] J E Leach F F White M L Rhoads and H Leung ldquoA repet-itive DNA sequence differentiates Xanthomonas campestnis pvoryzae from other pathovars of X campestrisrdquoMolecular Plant-Microbe Interactions Journal vol 3 pp 238ndash246 1990

[19] P C Pecknold and R G Grogan ldquoDeoxyribonucleic acidhomology groups among phytopathogenic Pseudomonasspeciesrdquo International Journal of Systematic Bacteriology vol23 no 2 pp 111ndash121 1973

[20] L Vauterin R Vantomme B Pot B Hoste J Swings and KKersters ldquoTaxonomic analysis of Xanthomonas campestris pvbegoniae and X campestris pv pelargonii by means of phy-topathological phenotypic protein electrophoretic and DNAhybridization methodsrdquo Systematic and Applied Microbiologyvol 13 no 2 pp 166ndash176 1990

[21] J R Lupski andGMWeinstock ldquoShort interspersed repetitiveDNA sequences in prokaryotic genomesrdquo Journal of Bacteriol-ogy vol 174 no 14 pp 4525ndash4529 1992

[22] J Versalovic V Kapur E OMason Jr et al ldquoPenicillin-resistantStreptococcus pneumoniae strains recovered in Houston identi-fication and molecular characterization of multiple clonesrdquoTheJournal of Infectious Diseases vol 167 no 4 pp 850ndash856 1993

[23] B Martin O Humbert M Camara et al ldquoA highly conservedrepeated DNA element located in the chromosome of Strepto-coccus pneumoniaerdquo Nucleic Acids Research vol 20 no 13 pp3479ndash3483 1992

[24] S Krawiec ldquoConcept of a bacterial speciesrdquo InternationalJournal of Systematic Bacteriology vol 35 no 2 pp 217ndash2201985

[25] S Krawiec and M Riley ldquoOrganization of the bacterial chro-mosomerdquo Microbiological Reviews vol 54 no 4 pp 502ndash5391990

[26] M Achtman and G Pluschke ldquoClonal analysis of descent andvirulence among selected Escherichia colirdquo Annual Review ofMicrobiology vol 40 pp 185ndash210 1986

[27] DW Gabriel J E HunterM T Kingsley JWMiller andG RLazo ldquoClonal population structure of Xanthomonas campestrisand genetic diversity among citrus canker strainsrdquo MolecularPlant-Microbe Interactions Journal vol 1 pp 59ndash65 1988

[28] R K Selander and JMMusser Population Genetics of BacterialPathogenesis Academic Press San Diego Calif USA 1990

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

[30] M B Javadinobandegani Distribution and Biochemical andGenotypic Comparison of Phosphate Solubilizing Bacteria in OilPalm Soils Department of Agriculture Technology UniversityPutra Malaysia Selangor Malaysia 2008

[31] W GWeisburg S M Barns D A Pelletier and D J Lane ldquo16Sribosomal DNA amplification for phylogenetic studyrdquo Journalof Bacteriology vol 173 no 2 pp 697ndash703 1991

[32] J Sambrook Molecular Cloning A Laboratory Manual ColdSpringHarbor Laboratory Press Cold SpringHarbor NY USA2001

[33] F J Rolf NTSYS-PC Numerical Taxonomy And MultivariateAnalysis SystemVersion 210 Applied Biostatistics 2000

[34] J Versalovic T Koeuth and J R Lupski ldquoDistribution ofrepetitive DNA sequences in eubacteria and application tofingerprinting of bacterial genomesrdquo Nucleic Acids Researchvol 19 no 24 pp 6823ndash6831 1991

[35] AD L Akkermans J D van Elsas and F J de BruijnMolecularMicrobial Ecology Manual Kluwer Academic Dordrecht TheNetherlands 1995

[36] C R Woods Jr J Versalovic T Koeuth and J R LupskildquoAnalysis of relationships among isolates of Citrobacter diversusby using DNA fingerprints generated by repetitive sequence-based primers in the polymerase chain reactionrdquo Journal ofClinical Microbiology vol 30 no 11 pp 2921ndash2929 1992

[37] C R Woods J Versalovic T Koeuth and J R Lupski ldquoWhole-cell repetitive element sequence-based polymerase chain reac-tion allows rapid assessment of clonal relationships of bacterialisolatesrdquo Journal of Clinical Microbiology vol 31 no 7 pp 1927ndash1931 1993

[38] M R Cancilla I B Powell A J Hillier and B E DavidsonldquoRapid genomic fingerprinting of Lactococcus lactis strainsby arbitrarily primed polymerase chain reaction with p32 andfluorescent labelsrdquo Applied and Environmental Microbiologyvol 58 no 5 pp 1772ndash1775 1992

[39] A Fekete J A Bantle S M Halling and R W Stich ldquoAmplifi-cation fragment length polymorphism in Brucella strains by useof polymerase chain reaction with arbitrary primersrdquo Journal ofBacteriology vol 174 no 23 pp 7778ndash7783 1992

BioMed Research International 7

[40] S Araujo I S Henriques S M Leandro A Alves A Pereiraand A Correia ldquoGulls identified as major source of fecalpollution in coastal waters a microbial source tracking studyrdquoScience of the Total Environment vol 470-471 pp 84ndash91 2014

[41] H Guo Z Mao H Jiang et al ldquoCommunity analysis ofplant growth promoting rhizobacteria for apple treesrdquo CropProtection vol 62 pp 1ndash9 2014

[42] L Zhu H Xu Y Zhang G Fu P Q Wu and Y Li ldquoBOX-PCRand PCR-DGGE analysis for bacterial diversity of a naturallyfermented functional food (Enzyme)rdquo Food Bioscience vol 5pp 115ndash122 2014

[43] J Welsh and M McClelland ldquoFingerprinting genomes usingPCR with arbitrary primersrdquoNucleic Acids Research vol 18 no24 pp 7213ndash7218 1990

[44] J G KWilliams A R Kubelik K J Livak J A Rafalski and SV Tingey ldquoDNApolymorphisms amplified by arbitrary primersare useful as geneticmarkersrdquoNucleic Acids Research vol 18 no22 pp 6531ndash6535 1990

[45] F J Louws D W Fulbright C T Stephens and F J DeBruijn ldquoSpecific genomic fingerprints of phytopathogenic Xan-thomonas and Pseudomonas pathovars and strains generatedwith repetitive sequences and PCRrdquoApplied and EnvironmentalMicrobiology vol 60 no 7 pp 2286ndash2295 1994

[46] A K Judd M Schneider M J Sadowsky and F J de BruijnldquoUse of repetitive sequences and the polymerase chain reac-tion technique to classify genetically related Bradyrhizobiumjaponicum serocluster 123 strainsrdquo Applied and EnvironmentalMicrobiology vol 59 no 6 pp 1702ndash1708 1993

[47] A A Benedict A M Alvarez and J Berestecky ldquoPathovarspecific monoclonal antibodies for Xanthomonas campestrispv oryzae and for Xanthomonas campestris pv oryzicolardquoPhytopathology vol 79 pp 322ndash328 1989

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

BioMed Research International 3

Table1Ph

osph

ates

olub

ilizing

Pseudomonas

isolates

Num

ber

Isolate

Nam

eAc

cessionnu

mber(NCB

I)Ph

ylum

Class

Order

Family

Genus

118DNR

Pseudomonas

sp

KJ748597

Proteobacteria

Gam

maproteob

acteria

Pseudo

mon

adales

Pseudo

mon

adaceae

Pseudomonas

25D

NR

Pseudomonas

fluorescens

KJ748598

Proteobacteria

Gam

maproteob

acteria

Pseudo

mon

adales

Pseudo

mon

adaceae

Pseudomonas

341DNR

Pseudomonas

sp

KJ783454

Proteobacteria

Gam

maproteob

acteria

Pseudo

mon

adales

Pseudo

mon

adaceae

Pseudomonas

422DNR

Pseudomonas

sp

KJ729599

Proteobacteria

Gam

maproteob

acteria

Pseudo

mon

adales

Pseudo

mon

adaceae

Pseudomonas

531SR

Pseudomonas

sp

KJ748596

Proteobacteria

Gam

maproteob

acteria

Pseudo

mon

adales

Pseudo

mon

adaceae

Pseudomonas

68SR

Pseudomonas

sp

KJ783451

Proteobacteria

Gam

maproteob

acteria

Pseudo

mon

adales

Pseudo

mon

adaceae

Pseudomonas

4 BioMed Research International

(a)

10kb

15 kbp

250bp

(b)

Figure 1 PCR products of 16S rDNA (a) and BOX element (b) (Line 1 = 18DNR Line 2 = 5DNR Line 3 = 41DNR Line 4 = 22DNR Line 5 =31SR and Line 6 = 8SR)

006 000

(A)

(B)

22DNR (Pseudomonas sp)18DNR (Pseudomonas sp)

8SR (Pseudomonas sp)41DNR (Pseudomonas sp)31SR (Pseudomonas sp)

5DNR (Pseudomonasfluorescens)

(a)

(A2)

(A1)(A)

(B)

040 055 070 085 100

Coefficient

31SR5DNR41DNR22DNR8SR18DNR

(b)

Figure 2 Dendrogram of phosphate solubilizing Pseudomonas bacteria based on the 16S rDNA (a) and BOX-PCR marker (b)

sp (31SR) Gene sequences of 16S rDNA from phosphate sol-ubilizing Pseudomonas bacteria were grouped within gammaproteobacteria

31 BOX-PCR Analysis PCR fingerprints with the BOXelement primer (BOX-PCR) revealed species-specific bandpatterns for the various isolates of phosphate solubilizingPseudomonas DNA fingerprints obtained from BOX-PCRof extracted genomic DNA yielded comparable patternsBOX-PCR as a precise molecular marker allowed betterdiscrimination than 16S rDNA sequencing between strainswithin Pseudomonas species (isolates) BOX-PCR of theseisolates revealed six different fingerprint profiles among sixisolates of phosphate solubilizing bacteria isolated from oilpalm soil (Figure 1(b))0

PCR with BOX-PCR primer and chromosomal DNAfrom the strains yielded multiple distinct DNA productsof sizes ranging from approximately 300 to 5000 bp TheBOX-PCR patterns of Pseudomonas species designates werefound to be highly related to one another (gt40) 18DNRand 22DNR and 8SR Pseudomonas bacterial isolates whichwere identified as a Pseudomonas spwere found to be highlyrelated to one another (gt70) but very distinct from 41DNR(Pseudomonas sp) 5DNR (Pseudomonas fluorescens) and31SR (Pseudomonas sp) (Figure 2(b))

The BOX-PCR marker similarities using Jaccardrsquos coef-ficient were calculated using the data analysis The matrixof coefficient was then used to cluster the similar accessionbased on BOX-PCR data and then to construct the dendro-gram of relationship through the UPGMA The dendrogramshowing the genetic relationship of the isolates is presentedin Figure 2(b)

The cluster analysis of Pseudomonas species based on theBOX-PCR identified two major groups ((A) (B)) at geneticdistance = 060 (Figure 2) Cluster (A) contained threeisolates with the calculation of different locations (SemenyihDengkil) isolates 31SR 5DNR and 41DNR within this clus-ter In cluster (A) there was two subclusters which included(A1) (31SR 5DNR) and (A2) (41DNR) Cluster (B) wasformed by three isolates In this cluster isolates 22DNR 8SRand 18DNR were very similar to each other based on the16S rDNA sequencing however there was less than 70similarity based on the BOX-PCR It could reveal that theywere different strains Clusters (A) and (B) together formed amain cluster at genetic distance 04

4 Discussion

In this study we have demonstrated that BOX elements asrepetitive sequences were present in the genome of phosphate

BioMed Research International 5

solubilizing Pseudomonas isolates confirming and extendingthe conclusion of Versalovic et al 1991 [34] and Akkermanset al 1995 [35] and these sequences are virtually ubiquitousWehave also demonstrated that the BOX-PCRprotocolswereparticularly suitable for the rapid molecular characterizationof phosphate solubilizing Pseudomonas bacteria especiallyat the strains level The BOX-PCR protocol clearly had thepotential to differentiate between isolates including thosethat were not easily distinguished by other phenotypic andphylogenetic techniques such as 16S rDNA

The data presented here suggested that BOX-PCR couldbe a useful tool for identification of phosphate solubilizingPseudomonaspurposes in industrial biofertilizers technologySimilar outcomes have been made about the utility of BOX-PCR in human pathology [22 36 37]

Several circumstances must be considered if BOX-PCRis to be useful for the proper identification of unknownphosphate solubilizing Pseudomonas isolates Firstly thecharacteristic of BOX-like sequences on gel and thereforethe genomic fingerprint patterns generated by BOX-PCRmust be established over time and distance Comparison ofthe genomic fingerprint profiles of phosphate solubilizingPseudomonas isolates within a tropical areas separated bytime or distance supports the idea that the profiles remainstable This similarity of fingerprint profiles of isolates bytime has been noted by others too [37] Secondly the BOX-PCR technique must be able to distinguish among relatedbacterial strains with sufficient declaration and it also shouldbe reproducible

41 Supporting the Reproducibility of the BOX-PCR Proto-col Large numbers of phosphate solubilizing Pseudomonasisolates have determined that there was homogeneity offingerprint profiles within each isolate It was systematicallyshown that BOX in general could determine that the differ-ences between phosphate solubilizing Pseudomonas isolatesand substantial polymorphism were detected Therefore thedistribution of BOX sequences was an accurate reflection ofgenomic structure

Other PCR-based genomic fingerprinting techniqueshave been described and demonstrated to use for differ-entiating bacteria and diagnostic purposes [38ndash42] Thereare some fundamental differences between BOX-PCR andRAPD-DNA analyses The major difference lies in the lengthof the primers and the consequent PCR conditions RAPDanalysis relies on the use of primers with arbitrary sequences[43 44] whereas BOX-PCR involves the use of primersof 22 bp with high homology to repetitive sequences [34]The BOX primer permits the use of more inflexible PCRconditions which in turn reduce experimental variation andPCR artefacts

As noted by others the BOX-PCR technique is veryuseful for bacterial strain identification however the utilityof that for bacterial taxonomy may be limited to closelyrelated strains [36 45 46] Protein profile analysis or fattyacid profile analysis [12] serologic testing [47] and rRNAgene restriction patterns [10] support the distinctiveness ofBOX-PCR analysis

The BOX-PCR fingerprint profiles between the two iso-lates contain many bands of equal mobility and rely onthe concept that selection for a specialized niche affectsgenome organization [25] and that corresponds to a uniquedistribution of repetitive sequences in the bacterial genome

The BOX-PCR of phosphate solubilizing Pseudomonasisolates technique may be limited to phylogenetic analysisand it effectively differentiates between two evolutionary linesclassified within the same taxon

5 Conclusion

In conclusion we have found that BOX sequences werewidespread in phosphate solubilizing Pseudomonas isolatesand could be used to generate genomic fingerprints withinPseudomonas species Unique fingerprint profiles generatedby BOX-PCR could be exploited for identification purposesand for discerning evolutionary lines of phosphate solubiliz-ing Pseudomonas in oil palm fields Revealing the populationdiversity of phosphate solubilizing Pseudomonas isolatesin turn had implications for the implementation of themfor biofertilizers industry programs disease managementstrategies and ecological and epidemiological studies

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

References

[1] S Nikolaki and G Tsiamis ldquoMicrobial diversity in the era ofomic technologiesrdquo BioMed Research International vol 2013Article ID 958719 15 pages 2013

[2] DW Dye ldquoThe inadequacy of the usual determinative tests forthe identification of Xanthomonas spprdquoNew Zealand Journal ofScience vol 5 pp 393ndash416 1962

[3] N J Palleroni Bergeyrsquos Manual of Systematic Bacteriology vol1 TheWilliams ampWilkins Co Baltimore Md USA 1894

[4] M Van den Mooter and J Swings ldquoNumerical analysis of 295phenotypic features of 266 Xanthomonas strains and relatedstrains and an improved taxonomy of the genusrdquo InternationalJournal of Systematic Bacteriology vol 40 no 4 pp 348ndash3691990

[5] E van Zyl and P L Steyn ldquoDifferentiation of phytopathogenicPseudomonas and Xanthomonas species and pathovars bynumerical taxonomy and protein gel electrophoregramsrdquo Sys-tematic and Applied Microbiology vol 13 no 1 pp 60ndash71 1990

[6] J M Young J F Bradbury R E Davis et al ldquoNomenclaturalrevisions of plant pathogenic bacteria and list of names 1980ndash1988rdquo Plant Pathology vol 70 pp 213ndash221 1991

[7] DWGabriel and R de Fuyter ldquoRFLP analysis and gene taggingfor bacterial identification and taxonomyrdquo in Molecular PlantPathology A Practical Approach M J M S J Gurr and D JBowles Eds vol 1 pp 51ndash66 IRL Press New York NY USA1992

[8] D W Gabriel M T Kingsley J E Hunter and T GottwaldldquoReinstatement of Xanthomonas citri (ex Hasse) and X phaseoli(ex Smith) to species and reclassification of all X campestris

6 BioMed Research International

pv citri strainsrdquo International Journal of Systematic Bacteriologyvol 39 no 1 pp 14ndash22 1989

[9] G R Lazo R Roffey and D W Gabriel ldquoPathovars ofXanthomonas campestris are distinguishable by restrictionfragment-length polymorphismrdquo International Journal of Sys-tematic Bacteriology vol 37 pp 214ndash221 1987

[10] Y Berthier V Verdier J-L Guesdon et al ldquoCharacterization ofXanthomonas campestris pathovars by rRNA gene restrictionpatternsrdquo Applied and Environmental Microbiology vol 59 no3 pp 851ndash859 1993

[11] D E Stead ldquoGrouping of plant-pathogenic and some otherPseudomonas spp by using cellular fatty acid profilesrdquo Interna-tional Journal of Systematic Bacteriology vol 42 no 2 pp 281ndash295 1992

[12] L Vauterin P Yang B Hoste B Pot J Swings and KKersters ldquoTaxonomy of xanthomonads from cereals and grassesbased on SDS-PAGE of proteins fatty acid analysis and DNAhybridizationrdquo Journal of General Microbiology vol 138 no 7pp 1467ndash1477 1992

[13] T P Denny M N Gilmour and R K Selander ldquoGeneticdiversity and relationships of two pathovars of Pseudomonassyringaerdquo Journal of General Microbiology vol 134 no 7 pp1949ndash1960 1988

[14] J S Hartung and E L Civerolo ldquoGenomic fingerprints ofXanthomonas campestris pv citri strains from Asia SouthAmerica and Floridardquo Phytopathology vol 77 pp 282ndash2851987

[15] D C Hildebrand N J Palleroni and M N Schroth ldquoDeoxyri-bonucleic acid relatedness of 24 xanthomonad strains rep-resenting 23 Xanthomonas campestris pathovars and Xan-thomonas fragariaerdquoTheJournal of Applied Bacteriology vol 68no 3 pp 263ndash269 1990

[16] G J King ldquoPlasmid analysis and variation in Pseudomonassyringaerdquo Journal of Applied Bacteriology vol 67 no 5 pp 489ndash496 1989

[17] G R Lazo and D W Gabriel ldquoConservation of plasmidDNA sequences and pathovar identification of strains of Xan-thomonas campestrisrdquo Phytopathology vol 77 pp 448ndash4531987

[18] J E Leach F F White M L Rhoads and H Leung ldquoA repet-itive DNA sequence differentiates Xanthomonas campestnis pvoryzae from other pathovars of X campestrisrdquoMolecular Plant-Microbe Interactions Journal vol 3 pp 238ndash246 1990

[19] P C Pecknold and R G Grogan ldquoDeoxyribonucleic acidhomology groups among phytopathogenic Pseudomonasspeciesrdquo International Journal of Systematic Bacteriology vol23 no 2 pp 111ndash121 1973

[20] L Vauterin R Vantomme B Pot B Hoste J Swings and KKersters ldquoTaxonomic analysis of Xanthomonas campestris pvbegoniae and X campestris pv pelargonii by means of phy-topathological phenotypic protein electrophoretic and DNAhybridization methodsrdquo Systematic and Applied Microbiologyvol 13 no 2 pp 166ndash176 1990

[21] J R Lupski andGMWeinstock ldquoShort interspersed repetitiveDNA sequences in prokaryotic genomesrdquo Journal of Bacteriol-ogy vol 174 no 14 pp 4525ndash4529 1992

[22] J Versalovic V Kapur E OMason Jr et al ldquoPenicillin-resistantStreptococcus pneumoniae strains recovered in Houston identi-fication and molecular characterization of multiple clonesrdquoTheJournal of Infectious Diseases vol 167 no 4 pp 850ndash856 1993

[23] B Martin O Humbert M Camara et al ldquoA highly conservedrepeated DNA element located in the chromosome of Strepto-coccus pneumoniaerdquo Nucleic Acids Research vol 20 no 13 pp3479ndash3483 1992

[24] S Krawiec ldquoConcept of a bacterial speciesrdquo InternationalJournal of Systematic Bacteriology vol 35 no 2 pp 217ndash2201985

[25] S Krawiec and M Riley ldquoOrganization of the bacterial chro-mosomerdquo Microbiological Reviews vol 54 no 4 pp 502ndash5391990

[26] M Achtman and G Pluschke ldquoClonal analysis of descent andvirulence among selected Escherichia colirdquo Annual Review ofMicrobiology vol 40 pp 185ndash210 1986

[27] DW Gabriel J E HunterM T Kingsley JWMiller andG RLazo ldquoClonal population structure of Xanthomonas campestrisand genetic diversity among citrus canker strainsrdquo MolecularPlant-Microbe Interactions Journal vol 1 pp 59ndash65 1988

[28] R K Selander and JMMusser Population Genetics of BacterialPathogenesis Academic Press San Diego Calif USA 1990

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

[30] M B Javadinobandegani Distribution and Biochemical andGenotypic Comparison of Phosphate Solubilizing Bacteria in OilPalm Soils Department of Agriculture Technology UniversityPutra Malaysia Selangor Malaysia 2008

[31] W GWeisburg S M Barns D A Pelletier and D J Lane ldquo16Sribosomal DNA amplification for phylogenetic studyrdquo Journalof Bacteriology vol 173 no 2 pp 697ndash703 1991

[32] J Sambrook Molecular Cloning A Laboratory Manual ColdSpringHarbor Laboratory Press Cold SpringHarbor NY USA2001

[33] F J Rolf NTSYS-PC Numerical Taxonomy And MultivariateAnalysis SystemVersion 210 Applied Biostatistics 2000

[34] J Versalovic T Koeuth and J R Lupski ldquoDistribution ofrepetitive DNA sequences in eubacteria and application tofingerprinting of bacterial genomesrdquo Nucleic Acids Researchvol 19 no 24 pp 6823ndash6831 1991

[35] AD L Akkermans J D van Elsas and F J de BruijnMolecularMicrobial Ecology Manual Kluwer Academic Dordrecht TheNetherlands 1995

[36] C R Woods Jr J Versalovic T Koeuth and J R LupskildquoAnalysis of relationships among isolates of Citrobacter diversusby using DNA fingerprints generated by repetitive sequence-based primers in the polymerase chain reactionrdquo Journal ofClinical Microbiology vol 30 no 11 pp 2921ndash2929 1992

[37] C R Woods J Versalovic T Koeuth and J R Lupski ldquoWhole-cell repetitive element sequence-based polymerase chain reac-tion allows rapid assessment of clonal relationships of bacterialisolatesrdquo Journal of Clinical Microbiology vol 31 no 7 pp 1927ndash1931 1993

[38] M R Cancilla I B Powell A J Hillier and B E DavidsonldquoRapid genomic fingerprinting of Lactococcus lactis strainsby arbitrarily primed polymerase chain reaction with p32 andfluorescent labelsrdquo Applied and Environmental Microbiologyvol 58 no 5 pp 1772ndash1775 1992

[39] A Fekete J A Bantle S M Halling and R W Stich ldquoAmplifi-cation fragment length polymorphism in Brucella strains by useof polymerase chain reaction with arbitrary primersrdquo Journal ofBacteriology vol 174 no 23 pp 7778ndash7783 1992

BioMed Research International 7

[40] S Araujo I S Henriques S M Leandro A Alves A Pereiraand A Correia ldquoGulls identified as major source of fecalpollution in coastal waters a microbial source tracking studyrdquoScience of the Total Environment vol 470-471 pp 84ndash91 2014

[41] H Guo Z Mao H Jiang et al ldquoCommunity analysis ofplant growth promoting rhizobacteria for apple treesrdquo CropProtection vol 62 pp 1ndash9 2014

[42] L Zhu H Xu Y Zhang G Fu P Q Wu and Y Li ldquoBOX-PCRand PCR-DGGE analysis for bacterial diversity of a naturallyfermented functional food (Enzyme)rdquo Food Bioscience vol 5pp 115ndash122 2014

[43] J Welsh and M McClelland ldquoFingerprinting genomes usingPCR with arbitrary primersrdquoNucleic Acids Research vol 18 no24 pp 7213ndash7218 1990

[44] J G KWilliams A R Kubelik K J Livak J A Rafalski and SV Tingey ldquoDNApolymorphisms amplified by arbitrary primersare useful as geneticmarkersrdquoNucleic Acids Research vol 18 no22 pp 6531ndash6535 1990

[45] F J Louws D W Fulbright C T Stephens and F J DeBruijn ldquoSpecific genomic fingerprints of phytopathogenic Xan-thomonas and Pseudomonas pathovars and strains generatedwith repetitive sequences and PCRrdquoApplied and EnvironmentalMicrobiology vol 60 no 7 pp 2286ndash2295 1994

[46] A K Judd M Schneider M J Sadowsky and F J de BruijnldquoUse of repetitive sequences and the polymerase chain reac-tion technique to classify genetically related Bradyrhizobiumjaponicum serocluster 123 strainsrdquo Applied and EnvironmentalMicrobiology vol 59 no 6 pp 1702ndash1708 1993

[47] A A Benedict A M Alvarez and J Berestecky ldquoPathovarspecific monoclonal antibodies for Xanthomonas campestrispv oryzae and for Xanthomonas campestris pv oryzicolardquoPhytopathology vol 79 pp 322ndash328 1989

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

4 BioMed Research International

(a)

10kb

15 kbp

250bp

(b)

Figure 1 PCR products of 16S rDNA (a) and BOX element (b) (Line 1 = 18DNR Line 2 = 5DNR Line 3 = 41DNR Line 4 = 22DNR Line 5 =31SR and Line 6 = 8SR)

006 000

(A)

(B)

22DNR (Pseudomonas sp)18DNR (Pseudomonas sp)

8SR (Pseudomonas sp)41DNR (Pseudomonas sp)31SR (Pseudomonas sp)

5DNR (Pseudomonasfluorescens)

(a)

(A2)

(A1)(A)

(B)

040 055 070 085 100

Coefficient

31SR5DNR41DNR22DNR8SR18DNR

(b)

Figure 2 Dendrogram of phosphate solubilizing Pseudomonas bacteria based on the 16S rDNA (a) and BOX-PCR marker (b)

sp (31SR) Gene sequences of 16S rDNA from phosphate sol-ubilizing Pseudomonas bacteria were grouped within gammaproteobacteria

31 BOX-PCR Analysis PCR fingerprints with the BOXelement primer (BOX-PCR) revealed species-specific bandpatterns for the various isolates of phosphate solubilizingPseudomonas DNA fingerprints obtained from BOX-PCRof extracted genomic DNA yielded comparable patternsBOX-PCR as a precise molecular marker allowed betterdiscrimination than 16S rDNA sequencing between strainswithin Pseudomonas species (isolates) BOX-PCR of theseisolates revealed six different fingerprint profiles among sixisolates of phosphate solubilizing bacteria isolated from oilpalm soil (Figure 1(b))0

PCR with BOX-PCR primer and chromosomal DNAfrom the strains yielded multiple distinct DNA productsof sizes ranging from approximately 300 to 5000 bp TheBOX-PCR patterns of Pseudomonas species designates werefound to be highly related to one another (gt40) 18DNRand 22DNR and 8SR Pseudomonas bacterial isolates whichwere identified as a Pseudomonas spwere found to be highlyrelated to one another (gt70) but very distinct from 41DNR(Pseudomonas sp) 5DNR (Pseudomonas fluorescens) and31SR (Pseudomonas sp) (Figure 2(b))

The BOX-PCR marker similarities using Jaccardrsquos coef-ficient were calculated using the data analysis The matrixof coefficient was then used to cluster the similar accessionbased on BOX-PCR data and then to construct the dendro-gram of relationship through the UPGMA The dendrogramshowing the genetic relationship of the isolates is presentedin Figure 2(b)

The cluster analysis of Pseudomonas species based on theBOX-PCR identified two major groups ((A) (B)) at geneticdistance = 060 (Figure 2) Cluster (A) contained threeisolates with the calculation of different locations (SemenyihDengkil) isolates 31SR 5DNR and 41DNR within this clus-ter In cluster (A) there was two subclusters which included(A1) (31SR 5DNR) and (A2) (41DNR) Cluster (B) wasformed by three isolates In this cluster isolates 22DNR 8SRand 18DNR were very similar to each other based on the16S rDNA sequencing however there was less than 70similarity based on the BOX-PCR It could reveal that theywere different strains Clusters (A) and (B) together formed amain cluster at genetic distance 04

4 Discussion

In this study we have demonstrated that BOX elements asrepetitive sequences were present in the genome of phosphate

BioMed Research International 5

solubilizing Pseudomonas isolates confirming and extendingthe conclusion of Versalovic et al 1991 [34] and Akkermanset al 1995 [35] and these sequences are virtually ubiquitousWehave also demonstrated that the BOX-PCRprotocolswereparticularly suitable for the rapid molecular characterizationof phosphate solubilizing Pseudomonas bacteria especiallyat the strains level The BOX-PCR protocol clearly had thepotential to differentiate between isolates including thosethat were not easily distinguished by other phenotypic andphylogenetic techniques such as 16S rDNA

The data presented here suggested that BOX-PCR couldbe a useful tool for identification of phosphate solubilizingPseudomonaspurposes in industrial biofertilizers technologySimilar outcomes have been made about the utility of BOX-PCR in human pathology [22 36 37]

Several circumstances must be considered if BOX-PCRis to be useful for the proper identification of unknownphosphate solubilizing Pseudomonas isolates Firstly thecharacteristic of BOX-like sequences on gel and thereforethe genomic fingerprint patterns generated by BOX-PCRmust be established over time and distance Comparison ofthe genomic fingerprint profiles of phosphate solubilizingPseudomonas isolates within a tropical areas separated bytime or distance supports the idea that the profiles remainstable This similarity of fingerprint profiles of isolates bytime has been noted by others too [37] Secondly the BOX-PCR technique must be able to distinguish among relatedbacterial strains with sufficient declaration and it also shouldbe reproducible

41 Supporting the Reproducibility of the BOX-PCR Proto-col Large numbers of phosphate solubilizing Pseudomonasisolates have determined that there was homogeneity offingerprint profiles within each isolate It was systematicallyshown that BOX in general could determine that the differ-ences between phosphate solubilizing Pseudomonas isolatesand substantial polymorphism were detected Therefore thedistribution of BOX sequences was an accurate reflection ofgenomic structure

Other PCR-based genomic fingerprinting techniqueshave been described and demonstrated to use for differ-entiating bacteria and diagnostic purposes [38ndash42] Thereare some fundamental differences between BOX-PCR andRAPD-DNA analyses The major difference lies in the lengthof the primers and the consequent PCR conditions RAPDanalysis relies on the use of primers with arbitrary sequences[43 44] whereas BOX-PCR involves the use of primersof 22 bp with high homology to repetitive sequences [34]The BOX primer permits the use of more inflexible PCRconditions which in turn reduce experimental variation andPCR artefacts

As noted by others the BOX-PCR technique is veryuseful for bacterial strain identification however the utilityof that for bacterial taxonomy may be limited to closelyrelated strains [36 45 46] Protein profile analysis or fattyacid profile analysis [12] serologic testing [47] and rRNAgene restriction patterns [10] support the distinctiveness ofBOX-PCR analysis

The BOX-PCR fingerprint profiles between the two iso-lates contain many bands of equal mobility and rely onthe concept that selection for a specialized niche affectsgenome organization [25] and that corresponds to a uniquedistribution of repetitive sequences in the bacterial genome

The BOX-PCR of phosphate solubilizing Pseudomonasisolates technique may be limited to phylogenetic analysisand it effectively differentiates between two evolutionary linesclassified within the same taxon

5 Conclusion

In conclusion we have found that BOX sequences werewidespread in phosphate solubilizing Pseudomonas isolatesand could be used to generate genomic fingerprints withinPseudomonas species Unique fingerprint profiles generatedby BOX-PCR could be exploited for identification purposesand for discerning evolutionary lines of phosphate solubiliz-ing Pseudomonas in oil palm fields Revealing the populationdiversity of phosphate solubilizing Pseudomonas isolatesin turn had implications for the implementation of themfor biofertilizers industry programs disease managementstrategies and ecological and epidemiological studies

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

References

[1] S Nikolaki and G Tsiamis ldquoMicrobial diversity in the era ofomic technologiesrdquo BioMed Research International vol 2013Article ID 958719 15 pages 2013

[2] DW Dye ldquoThe inadequacy of the usual determinative tests forthe identification of Xanthomonas spprdquoNew Zealand Journal ofScience vol 5 pp 393ndash416 1962

[3] N J Palleroni Bergeyrsquos Manual of Systematic Bacteriology vol1 TheWilliams ampWilkins Co Baltimore Md USA 1894

[4] M Van den Mooter and J Swings ldquoNumerical analysis of 295phenotypic features of 266 Xanthomonas strains and relatedstrains and an improved taxonomy of the genusrdquo InternationalJournal of Systematic Bacteriology vol 40 no 4 pp 348ndash3691990

[5] E van Zyl and P L Steyn ldquoDifferentiation of phytopathogenicPseudomonas and Xanthomonas species and pathovars bynumerical taxonomy and protein gel electrophoregramsrdquo Sys-tematic and Applied Microbiology vol 13 no 1 pp 60ndash71 1990

[6] J M Young J F Bradbury R E Davis et al ldquoNomenclaturalrevisions of plant pathogenic bacteria and list of names 1980ndash1988rdquo Plant Pathology vol 70 pp 213ndash221 1991

[7] DWGabriel and R de Fuyter ldquoRFLP analysis and gene taggingfor bacterial identification and taxonomyrdquo in Molecular PlantPathology A Practical Approach M J M S J Gurr and D JBowles Eds vol 1 pp 51ndash66 IRL Press New York NY USA1992

[8] D W Gabriel M T Kingsley J E Hunter and T GottwaldldquoReinstatement of Xanthomonas citri (ex Hasse) and X phaseoli(ex Smith) to species and reclassification of all X campestris

6 BioMed Research International

pv citri strainsrdquo International Journal of Systematic Bacteriologyvol 39 no 1 pp 14ndash22 1989

[9] G R Lazo R Roffey and D W Gabriel ldquoPathovars ofXanthomonas campestris are distinguishable by restrictionfragment-length polymorphismrdquo International Journal of Sys-tematic Bacteriology vol 37 pp 214ndash221 1987

[10] Y Berthier V Verdier J-L Guesdon et al ldquoCharacterization ofXanthomonas campestris pathovars by rRNA gene restrictionpatternsrdquo Applied and Environmental Microbiology vol 59 no3 pp 851ndash859 1993

[11] D E Stead ldquoGrouping of plant-pathogenic and some otherPseudomonas spp by using cellular fatty acid profilesrdquo Interna-tional Journal of Systematic Bacteriology vol 42 no 2 pp 281ndash295 1992

[12] L Vauterin P Yang B Hoste B Pot J Swings and KKersters ldquoTaxonomy of xanthomonads from cereals and grassesbased on SDS-PAGE of proteins fatty acid analysis and DNAhybridizationrdquo Journal of General Microbiology vol 138 no 7pp 1467ndash1477 1992

[13] T P Denny M N Gilmour and R K Selander ldquoGeneticdiversity and relationships of two pathovars of Pseudomonassyringaerdquo Journal of General Microbiology vol 134 no 7 pp1949ndash1960 1988

[14] J S Hartung and E L Civerolo ldquoGenomic fingerprints ofXanthomonas campestris pv citri strains from Asia SouthAmerica and Floridardquo Phytopathology vol 77 pp 282ndash2851987

[15] D C Hildebrand N J Palleroni and M N Schroth ldquoDeoxyri-bonucleic acid relatedness of 24 xanthomonad strains rep-resenting 23 Xanthomonas campestris pathovars and Xan-thomonas fragariaerdquoTheJournal of Applied Bacteriology vol 68no 3 pp 263ndash269 1990

[16] G J King ldquoPlasmid analysis and variation in Pseudomonassyringaerdquo Journal of Applied Bacteriology vol 67 no 5 pp 489ndash496 1989

[17] G R Lazo and D W Gabriel ldquoConservation of plasmidDNA sequences and pathovar identification of strains of Xan-thomonas campestrisrdquo Phytopathology vol 77 pp 448ndash4531987

[18] J E Leach F F White M L Rhoads and H Leung ldquoA repet-itive DNA sequence differentiates Xanthomonas campestnis pvoryzae from other pathovars of X campestrisrdquoMolecular Plant-Microbe Interactions Journal vol 3 pp 238ndash246 1990

[19] P C Pecknold and R G Grogan ldquoDeoxyribonucleic acidhomology groups among phytopathogenic Pseudomonasspeciesrdquo International Journal of Systematic Bacteriology vol23 no 2 pp 111ndash121 1973

[20] L Vauterin R Vantomme B Pot B Hoste J Swings and KKersters ldquoTaxonomic analysis of Xanthomonas campestris pvbegoniae and X campestris pv pelargonii by means of phy-topathological phenotypic protein electrophoretic and DNAhybridization methodsrdquo Systematic and Applied Microbiologyvol 13 no 2 pp 166ndash176 1990

[21] J R Lupski andGMWeinstock ldquoShort interspersed repetitiveDNA sequences in prokaryotic genomesrdquo Journal of Bacteriol-ogy vol 174 no 14 pp 4525ndash4529 1992

[22] J Versalovic V Kapur E OMason Jr et al ldquoPenicillin-resistantStreptococcus pneumoniae strains recovered in Houston identi-fication and molecular characterization of multiple clonesrdquoTheJournal of Infectious Diseases vol 167 no 4 pp 850ndash856 1993

[23] B Martin O Humbert M Camara et al ldquoA highly conservedrepeated DNA element located in the chromosome of Strepto-coccus pneumoniaerdquo Nucleic Acids Research vol 20 no 13 pp3479ndash3483 1992

[24] S Krawiec ldquoConcept of a bacterial speciesrdquo InternationalJournal of Systematic Bacteriology vol 35 no 2 pp 217ndash2201985

[25] S Krawiec and M Riley ldquoOrganization of the bacterial chro-mosomerdquo Microbiological Reviews vol 54 no 4 pp 502ndash5391990

[26] M Achtman and G Pluschke ldquoClonal analysis of descent andvirulence among selected Escherichia colirdquo Annual Review ofMicrobiology vol 40 pp 185ndash210 1986

[27] DW Gabriel J E HunterM T Kingsley JWMiller andG RLazo ldquoClonal population structure of Xanthomonas campestrisand genetic diversity among citrus canker strainsrdquo MolecularPlant-Microbe Interactions Journal vol 1 pp 59ndash65 1988

[28] R K Selander and JMMusser Population Genetics of BacterialPathogenesis Academic Press San Diego Calif USA 1990

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

[30] M B Javadinobandegani Distribution and Biochemical andGenotypic Comparison of Phosphate Solubilizing Bacteria in OilPalm Soils Department of Agriculture Technology UniversityPutra Malaysia Selangor Malaysia 2008

[31] W GWeisburg S M Barns D A Pelletier and D J Lane ldquo16Sribosomal DNA amplification for phylogenetic studyrdquo Journalof Bacteriology vol 173 no 2 pp 697ndash703 1991

[32] J Sambrook Molecular Cloning A Laboratory Manual ColdSpringHarbor Laboratory Press Cold SpringHarbor NY USA2001

[33] F J Rolf NTSYS-PC Numerical Taxonomy And MultivariateAnalysis SystemVersion 210 Applied Biostatistics 2000

[34] J Versalovic T Koeuth and J R Lupski ldquoDistribution ofrepetitive DNA sequences in eubacteria and application tofingerprinting of bacterial genomesrdquo Nucleic Acids Researchvol 19 no 24 pp 6823ndash6831 1991

[35] AD L Akkermans J D van Elsas and F J de BruijnMolecularMicrobial Ecology Manual Kluwer Academic Dordrecht TheNetherlands 1995

[36] C R Woods Jr J Versalovic T Koeuth and J R LupskildquoAnalysis of relationships among isolates of Citrobacter diversusby using DNA fingerprints generated by repetitive sequence-based primers in the polymerase chain reactionrdquo Journal ofClinical Microbiology vol 30 no 11 pp 2921ndash2929 1992

[37] C R Woods J Versalovic T Koeuth and J R Lupski ldquoWhole-cell repetitive element sequence-based polymerase chain reac-tion allows rapid assessment of clonal relationships of bacterialisolatesrdquo Journal of Clinical Microbiology vol 31 no 7 pp 1927ndash1931 1993

[38] M R Cancilla I B Powell A J Hillier and B E DavidsonldquoRapid genomic fingerprinting of Lactococcus lactis strainsby arbitrarily primed polymerase chain reaction with p32 andfluorescent labelsrdquo Applied and Environmental Microbiologyvol 58 no 5 pp 1772ndash1775 1992

[39] A Fekete J A Bantle S M Halling and R W Stich ldquoAmplifi-cation fragment length polymorphism in Brucella strains by useof polymerase chain reaction with arbitrary primersrdquo Journal ofBacteriology vol 174 no 23 pp 7778ndash7783 1992

BioMed Research International 7

[40] S Araujo I S Henriques S M Leandro A Alves A Pereiraand A Correia ldquoGulls identified as major source of fecalpollution in coastal waters a microbial source tracking studyrdquoScience of the Total Environment vol 470-471 pp 84ndash91 2014

[41] H Guo Z Mao H Jiang et al ldquoCommunity analysis ofplant growth promoting rhizobacteria for apple treesrdquo CropProtection vol 62 pp 1ndash9 2014

[42] L Zhu H Xu Y Zhang G Fu P Q Wu and Y Li ldquoBOX-PCRand PCR-DGGE analysis for bacterial diversity of a naturallyfermented functional food (Enzyme)rdquo Food Bioscience vol 5pp 115ndash122 2014

[43] J Welsh and M McClelland ldquoFingerprinting genomes usingPCR with arbitrary primersrdquoNucleic Acids Research vol 18 no24 pp 7213ndash7218 1990

[44] J G KWilliams A R Kubelik K J Livak J A Rafalski and SV Tingey ldquoDNApolymorphisms amplified by arbitrary primersare useful as geneticmarkersrdquoNucleic Acids Research vol 18 no22 pp 6531ndash6535 1990

[45] F J Louws D W Fulbright C T Stephens and F J DeBruijn ldquoSpecific genomic fingerprints of phytopathogenic Xan-thomonas and Pseudomonas pathovars and strains generatedwith repetitive sequences and PCRrdquoApplied and EnvironmentalMicrobiology vol 60 no 7 pp 2286ndash2295 1994

[46] A K Judd M Schneider M J Sadowsky and F J de BruijnldquoUse of repetitive sequences and the polymerase chain reac-tion technique to classify genetically related Bradyrhizobiumjaponicum serocluster 123 strainsrdquo Applied and EnvironmentalMicrobiology vol 59 no 6 pp 1702ndash1708 1993

[47] A A Benedict A M Alvarez and J Berestecky ldquoPathovarspecific monoclonal antibodies for Xanthomonas campestrispv oryzae and for Xanthomonas campestris pv oryzicolardquoPhytopathology vol 79 pp 322ndash328 1989

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

BioMed Research International 5

solubilizing Pseudomonas isolates confirming and extendingthe conclusion of Versalovic et al 1991 [34] and Akkermanset al 1995 [35] and these sequences are virtually ubiquitousWehave also demonstrated that the BOX-PCRprotocolswereparticularly suitable for the rapid molecular characterizationof phosphate solubilizing Pseudomonas bacteria especiallyat the strains level The BOX-PCR protocol clearly had thepotential to differentiate between isolates including thosethat were not easily distinguished by other phenotypic andphylogenetic techniques such as 16S rDNA

The data presented here suggested that BOX-PCR couldbe a useful tool for identification of phosphate solubilizingPseudomonaspurposes in industrial biofertilizers technologySimilar outcomes have been made about the utility of BOX-PCR in human pathology [22 36 37]

Several circumstances must be considered if BOX-PCRis to be useful for the proper identification of unknownphosphate solubilizing Pseudomonas isolates Firstly thecharacteristic of BOX-like sequences on gel and thereforethe genomic fingerprint patterns generated by BOX-PCRmust be established over time and distance Comparison ofthe genomic fingerprint profiles of phosphate solubilizingPseudomonas isolates within a tropical areas separated bytime or distance supports the idea that the profiles remainstable This similarity of fingerprint profiles of isolates bytime has been noted by others too [37] Secondly the BOX-PCR technique must be able to distinguish among relatedbacterial strains with sufficient declaration and it also shouldbe reproducible

41 Supporting the Reproducibility of the BOX-PCR Proto-col Large numbers of phosphate solubilizing Pseudomonasisolates have determined that there was homogeneity offingerprint profiles within each isolate It was systematicallyshown that BOX in general could determine that the differ-ences between phosphate solubilizing Pseudomonas isolatesand substantial polymorphism were detected Therefore thedistribution of BOX sequences was an accurate reflection ofgenomic structure

Other PCR-based genomic fingerprinting techniqueshave been described and demonstrated to use for differ-entiating bacteria and diagnostic purposes [38ndash42] Thereare some fundamental differences between BOX-PCR andRAPD-DNA analyses The major difference lies in the lengthof the primers and the consequent PCR conditions RAPDanalysis relies on the use of primers with arbitrary sequences[43 44] whereas BOX-PCR involves the use of primersof 22 bp with high homology to repetitive sequences [34]The BOX primer permits the use of more inflexible PCRconditions which in turn reduce experimental variation andPCR artefacts

As noted by others the BOX-PCR technique is veryuseful for bacterial strain identification however the utilityof that for bacterial taxonomy may be limited to closelyrelated strains [36 45 46] Protein profile analysis or fattyacid profile analysis [12] serologic testing [47] and rRNAgene restriction patterns [10] support the distinctiveness ofBOX-PCR analysis

The BOX-PCR fingerprint profiles between the two iso-lates contain many bands of equal mobility and rely onthe concept that selection for a specialized niche affectsgenome organization [25] and that corresponds to a uniquedistribution of repetitive sequences in the bacterial genome

The BOX-PCR of phosphate solubilizing Pseudomonasisolates technique may be limited to phylogenetic analysisand it effectively differentiates between two evolutionary linesclassified within the same taxon

5 Conclusion

In conclusion we have found that BOX sequences werewidespread in phosphate solubilizing Pseudomonas isolatesand could be used to generate genomic fingerprints withinPseudomonas species Unique fingerprint profiles generatedby BOX-PCR could be exploited for identification purposesand for discerning evolutionary lines of phosphate solubiliz-ing Pseudomonas in oil palm fields Revealing the populationdiversity of phosphate solubilizing Pseudomonas isolatesin turn had implications for the implementation of themfor biofertilizers industry programs disease managementstrategies and ecological and epidemiological studies

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

References

[1] S Nikolaki and G Tsiamis ldquoMicrobial diversity in the era ofomic technologiesrdquo BioMed Research International vol 2013Article ID 958719 15 pages 2013

[2] DW Dye ldquoThe inadequacy of the usual determinative tests forthe identification of Xanthomonas spprdquoNew Zealand Journal ofScience vol 5 pp 393ndash416 1962

[3] N J Palleroni Bergeyrsquos Manual of Systematic Bacteriology vol1 TheWilliams ampWilkins Co Baltimore Md USA 1894

[4] M Van den Mooter and J Swings ldquoNumerical analysis of 295phenotypic features of 266 Xanthomonas strains and relatedstrains and an improved taxonomy of the genusrdquo InternationalJournal of Systematic Bacteriology vol 40 no 4 pp 348ndash3691990

[5] E van Zyl and P L Steyn ldquoDifferentiation of phytopathogenicPseudomonas and Xanthomonas species and pathovars bynumerical taxonomy and protein gel electrophoregramsrdquo Sys-tematic and Applied Microbiology vol 13 no 1 pp 60ndash71 1990

[6] J M Young J F Bradbury R E Davis et al ldquoNomenclaturalrevisions of plant pathogenic bacteria and list of names 1980ndash1988rdquo Plant Pathology vol 70 pp 213ndash221 1991

[7] DWGabriel and R de Fuyter ldquoRFLP analysis and gene taggingfor bacterial identification and taxonomyrdquo in Molecular PlantPathology A Practical Approach M J M S J Gurr and D JBowles Eds vol 1 pp 51ndash66 IRL Press New York NY USA1992

[8] D W Gabriel M T Kingsley J E Hunter and T GottwaldldquoReinstatement of Xanthomonas citri (ex Hasse) and X phaseoli(ex Smith) to species and reclassification of all X campestris

6 BioMed Research International

pv citri strainsrdquo International Journal of Systematic Bacteriologyvol 39 no 1 pp 14ndash22 1989

[9] G R Lazo R Roffey and D W Gabriel ldquoPathovars ofXanthomonas campestris are distinguishable by restrictionfragment-length polymorphismrdquo International Journal of Sys-tematic Bacteriology vol 37 pp 214ndash221 1987

[10] Y Berthier V Verdier J-L Guesdon et al ldquoCharacterization ofXanthomonas campestris pathovars by rRNA gene restrictionpatternsrdquo Applied and Environmental Microbiology vol 59 no3 pp 851ndash859 1993

[11] D E Stead ldquoGrouping of plant-pathogenic and some otherPseudomonas spp by using cellular fatty acid profilesrdquo Interna-tional Journal of Systematic Bacteriology vol 42 no 2 pp 281ndash295 1992

[12] L Vauterin P Yang B Hoste B Pot J Swings and KKersters ldquoTaxonomy of xanthomonads from cereals and grassesbased on SDS-PAGE of proteins fatty acid analysis and DNAhybridizationrdquo Journal of General Microbiology vol 138 no 7pp 1467ndash1477 1992

[13] T P Denny M N Gilmour and R K Selander ldquoGeneticdiversity and relationships of two pathovars of Pseudomonassyringaerdquo Journal of General Microbiology vol 134 no 7 pp1949ndash1960 1988

[14] J S Hartung and E L Civerolo ldquoGenomic fingerprints ofXanthomonas campestris pv citri strains from Asia SouthAmerica and Floridardquo Phytopathology vol 77 pp 282ndash2851987

[15] D C Hildebrand N J Palleroni and M N Schroth ldquoDeoxyri-bonucleic acid relatedness of 24 xanthomonad strains rep-resenting 23 Xanthomonas campestris pathovars and Xan-thomonas fragariaerdquoTheJournal of Applied Bacteriology vol 68no 3 pp 263ndash269 1990

[16] G J King ldquoPlasmid analysis and variation in Pseudomonassyringaerdquo Journal of Applied Bacteriology vol 67 no 5 pp 489ndash496 1989

[17] G R Lazo and D W Gabriel ldquoConservation of plasmidDNA sequences and pathovar identification of strains of Xan-thomonas campestrisrdquo Phytopathology vol 77 pp 448ndash4531987

[18] J E Leach F F White M L Rhoads and H Leung ldquoA repet-itive DNA sequence differentiates Xanthomonas campestnis pvoryzae from other pathovars of X campestrisrdquoMolecular Plant-Microbe Interactions Journal vol 3 pp 238ndash246 1990

[19] P C Pecknold and R G Grogan ldquoDeoxyribonucleic acidhomology groups among phytopathogenic Pseudomonasspeciesrdquo International Journal of Systematic Bacteriology vol23 no 2 pp 111ndash121 1973

[20] L Vauterin R Vantomme B Pot B Hoste J Swings and KKersters ldquoTaxonomic analysis of Xanthomonas campestris pvbegoniae and X campestris pv pelargonii by means of phy-topathological phenotypic protein electrophoretic and DNAhybridization methodsrdquo Systematic and Applied Microbiologyvol 13 no 2 pp 166ndash176 1990

[21] J R Lupski andGMWeinstock ldquoShort interspersed repetitiveDNA sequences in prokaryotic genomesrdquo Journal of Bacteriol-ogy vol 174 no 14 pp 4525ndash4529 1992

[22] J Versalovic V Kapur E OMason Jr et al ldquoPenicillin-resistantStreptococcus pneumoniae strains recovered in Houston identi-fication and molecular characterization of multiple clonesrdquoTheJournal of Infectious Diseases vol 167 no 4 pp 850ndash856 1993

[23] B Martin O Humbert M Camara et al ldquoA highly conservedrepeated DNA element located in the chromosome of Strepto-coccus pneumoniaerdquo Nucleic Acids Research vol 20 no 13 pp3479ndash3483 1992

[24] S Krawiec ldquoConcept of a bacterial speciesrdquo InternationalJournal of Systematic Bacteriology vol 35 no 2 pp 217ndash2201985

[25] S Krawiec and M Riley ldquoOrganization of the bacterial chro-mosomerdquo Microbiological Reviews vol 54 no 4 pp 502ndash5391990

[26] M Achtman and G Pluschke ldquoClonal analysis of descent andvirulence among selected Escherichia colirdquo Annual Review ofMicrobiology vol 40 pp 185ndash210 1986

[27] DW Gabriel J E HunterM T Kingsley JWMiller andG RLazo ldquoClonal population structure of Xanthomonas campestrisand genetic diversity among citrus canker strainsrdquo MolecularPlant-Microbe Interactions Journal vol 1 pp 59ndash65 1988

[28] R K Selander and JMMusser Population Genetics of BacterialPathogenesis Academic Press San Diego Calif USA 1990

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

[30] M B Javadinobandegani Distribution and Biochemical andGenotypic Comparison of Phosphate Solubilizing Bacteria in OilPalm Soils Department of Agriculture Technology UniversityPutra Malaysia Selangor Malaysia 2008

[31] W GWeisburg S M Barns D A Pelletier and D J Lane ldquo16Sribosomal DNA amplification for phylogenetic studyrdquo Journalof Bacteriology vol 173 no 2 pp 697ndash703 1991

[32] J Sambrook Molecular Cloning A Laboratory Manual ColdSpringHarbor Laboratory Press Cold SpringHarbor NY USA2001

[33] F J Rolf NTSYS-PC Numerical Taxonomy And MultivariateAnalysis SystemVersion 210 Applied Biostatistics 2000

[34] J Versalovic T Koeuth and J R Lupski ldquoDistribution ofrepetitive DNA sequences in eubacteria and application tofingerprinting of bacterial genomesrdquo Nucleic Acids Researchvol 19 no 24 pp 6823ndash6831 1991

[35] AD L Akkermans J D van Elsas and F J de BruijnMolecularMicrobial Ecology Manual Kluwer Academic Dordrecht TheNetherlands 1995

[36] C R Woods Jr J Versalovic T Koeuth and J R LupskildquoAnalysis of relationships among isolates of Citrobacter diversusby using DNA fingerprints generated by repetitive sequence-based primers in the polymerase chain reactionrdquo Journal ofClinical Microbiology vol 30 no 11 pp 2921ndash2929 1992

[37] C R Woods J Versalovic T Koeuth and J R Lupski ldquoWhole-cell repetitive element sequence-based polymerase chain reac-tion allows rapid assessment of clonal relationships of bacterialisolatesrdquo Journal of Clinical Microbiology vol 31 no 7 pp 1927ndash1931 1993

[38] M R Cancilla I B Powell A J Hillier and B E DavidsonldquoRapid genomic fingerprinting of Lactococcus lactis strainsby arbitrarily primed polymerase chain reaction with p32 andfluorescent labelsrdquo Applied and Environmental Microbiologyvol 58 no 5 pp 1772ndash1775 1992

[39] A Fekete J A Bantle S M Halling and R W Stich ldquoAmplifi-cation fragment length polymorphism in Brucella strains by useof polymerase chain reaction with arbitrary primersrdquo Journal ofBacteriology vol 174 no 23 pp 7778ndash7783 1992

BioMed Research International 7

[40] S Araujo I S Henriques S M Leandro A Alves A Pereiraand A Correia ldquoGulls identified as major source of fecalpollution in coastal waters a microbial source tracking studyrdquoScience of the Total Environment vol 470-471 pp 84ndash91 2014

[41] H Guo Z Mao H Jiang et al ldquoCommunity analysis ofplant growth promoting rhizobacteria for apple treesrdquo CropProtection vol 62 pp 1ndash9 2014

[42] L Zhu H Xu Y Zhang G Fu P Q Wu and Y Li ldquoBOX-PCRand PCR-DGGE analysis for bacterial diversity of a naturallyfermented functional food (Enzyme)rdquo Food Bioscience vol 5pp 115ndash122 2014

[43] J Welsh and M McClelland ldquoFingerprinting genomes usingPCR with arbitrary primersrdquoNucleic Acids Research vol 18 no24 pp 7213ndash7218 1990

[44] J G KWilliams A R Kubelik K J Livak J A Rafalski and SV Tingey ldquoDNApolymorphisms amplified by arbitrary primersare useful as geneticmarkersrdquoNucleic Acids Research vol 18 no22 pp 6531ndash6535 1990

[45] F J Louws D W Fulbright C T Stephens and F J DeBruijn ldquoSpecific genomic fingerprints of phytopathogenic Xan-thomonas and Pseudomonas pathovars and strains generatedwith repetitive sequences and PCRrdquoApplied and EnvironmentalMicrobiology vol 60 no 7 pp 2286ndash2295 1994

[46] A K Judd M Schneider M J Sadowsky and F J de BruijnldquoUse of repetitive sequences and the polymerase chain reac-tion technique to classify genetically related Bradyrhizobiumjaponicum serocluster 123 strainsrdquo Applied and EnvironmentalMicrobiology vol 59 no 6 pp 1702ndash1708 1993

[47] A A Benedict A M Alvarez and J Berestecky ldquoPathovarspecific monoclonal antibodies for Xanthomonas campestrispv oryzae and for Xanthomonas campestris pv oryzicolardquoPhytopathology vol 79 pp 322ndash328 1989

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

6 BioMed Research International

pv citri strainsrdquo International Journal of Systematic Bacteriologyvol 39 no 1 pp 14ndash22 1989

[9] G R Lazo R Roffey and D W Gabriel ldquoPathovars ofXanthomonas campestris are distinguishable by restrictionfragment-length polymorphismrdquo International Journal of Sys-tematic Bacteriology vol 37 pp 214ndash221 1987

[10] Y Berthier V Verdier J-L Guesdon et al ldquoCharacterization ofXanthomonas campestris pathovars by rRNA gene restrictionpatternsrdquo Applied and Environmental Microbiology vol 59 no3 pp 851ndash859 1993

[11] D E Stead ldquoGrouping of plant-pathogenic and some otherPseudomonas spp by using cellular fatty acid profilesrdquo Interna-tional Journal of Systematic Bacteriology vol 42 no 2 pp 281ndash295 1992

[12] L Vauterin P Yang B Hoste B Pot J Swings and KKersters ldquoTaxonomy of xanthomonads from cereals and grassesbased on SDS-PAGE of proteins fatty acid analysis and DNAhybridizationrdquo Journal of General Microbiology vol 138 no 7pp 1467ndash1477 1992

[13] T P Denny M N Gilmour and R K Selander ldquoGeneticdiversity and relationships of two pathovars of Pseudomonassyringaerdquo Journal of General Microbiology vol 134 no 7 pp1949ndash1960 1988

[14] J S Hartung and E L Civerolo ldquoGenomic fingerprints ofXanthomonas campestris pv citri strains from Asia SouthAmerica and Floridardquo Phytopathology vol 77 pp 282ndash2851987

[15] D C Hildebrand N J Palleroni and M N Schroth ldquoDeoxyri-bonucleic acid relatedness of 24 xanthomonad strains rep-resenting 23 Xanthomonas campestris pathovars and Xan-thomonas fragariaerdquoTheJournal of Applied Bacteriology vol 68no 3 pp 263ndash269 1990

[16] G J King ldquoPlasmid analysis and variation in Pseudomonassyringaerdquo Journal of Applied Bacteriology vol 67 no 5 pp 489ndash496 1989

[17] G R Lazo and D W Gabriel ldquoConservation of plasmidDNA sequences and pathovar identification of strains of Xan-thomonas campestrisrdquo Phytopathology vol 77 pp 448ndash4531987

[18] J E Leach F F White M L Rhoads and H Leung ldquoA repet-itive DNA sequence differentiates Xanthomonas campestnis pvoryzae from other pathovars of X campestrisrdquoMolecular Plant-Microbe Interactions Journal vol 3 pp 238ndash246 1990

[19] P C Pecknold and R G Grogan ldquoDeoxyribonucleic acidhomology groups among phytopathogenic Pseudomonasspeciesrdquo International Journal of Systematic Bacteriology vol23 no 2 pp 111ndash121 1973

[20] L Vauterin R Vantomme B Pot B Hoste J Swings and KKersters ldquoTaxonomic analysis of Xanthomonas campestris pvbegoniae and X campestris pv pelargonii by means of phy-topathological phenotypic protein electrophoretic and DNAhybridization methodsrdquo Systematic and Applied Microbiologyvol 13 no 2 pp 166ndash176 1990

[21] J R Lupski andGMWeinstock ldquoShort interspersed repetitiveDNA sequences in prokaryotic genomesrdquo Journal of Bacteriol-ogy vol 174 no 14 pp 4525ndash4529 1992

[22] J Versalovic V Kapur E OMason Jr et al ldquoPenicillin-resistantStreptococcus pneumoniae strains recovered in Houston identi-fication and molecular characterization of multiple clonesrdquoTheJournal of Infectious Diseases vol 167 no 4 pp 850ndash856 1993

[23] B Martin O Humbert M Camara et al ldquoA highly conservedrepeated DNA element located in the chromosome of Strepto-coccus pneumoniaerdquo Nucleic Acids Research vol 20 no 13 pp3479ndash3483 1992

[24] S Krawiec ldquoConcept of a bacterial speciesrdquo InternationalJournal of Systematic Bacteriology vol 35 no 2 pp 217ndash2201985

[25] S Krawiec and M Riley ldquoOrganization of the bacterial chro-mosomerdquo Microbiological Reviews vol 54 no 4 pp 502ndash5391990

[26] M Achtman and G Pluschke ldquoClonal analysis of descent andvirulence among selected Escherichia colirdquo Annual Review ofMicrobiology vol 40 pp 185ndash210 1986

[27] DW Gabriel J E HunterM T Kingsley JWMiller andG RLazo ldquoClonal population structure of Xanthomonas campestrisand genetic diversity among citrus canker strainsrdquo MolecularPlant-Microbe Interactions Journal vol 1 pp 59ndash65 1988

[28] R K Selander and JMMusser Population Genetics of BacterialPathogenesis Academic Press San Diego Calif USA 1990

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

[30] M B Javadinobandegani Distribution and Biochemical andGenotypic Comparison of Phosphate Solubilizing Bacteria in OilPalm Soils Department of Agriculture Technology UniversityPutra Malaysia Selangor Malaysia 2008

[31] W GWeisburg S M Barns D A Pelletier and D J Lane ldquo16Sribosomal DNA amplification for phylogenetic studyrdquo Journalof Bacteriology vol 173 no 2 pp 697ndash703 1991

[32] J Sambrook Molecular Cloning A Laboratory Manual ColdSpringHarbor Laboratory Press Cold SpringHarbor NY USA2001

[33] F J Rolf NTSYS-PC Numerical Taxonomy And MultivariateAnalysis SystemVersion 210 Applied Biostatistics 2000

[34] J Versalovic T Koeuth and J R Lupski ldquoDistribution ofrepetitive DNA sequences in eubacteria and application tofingerprinting of bacterial genomesrdquo Nucleic Acids Researchvol 19 no 24 pp 6823ndash6831 1991

[35] AD L Akkermans J D van Elsas and F J de BruijnMolecularMicrobial Ecology Manual Kluwer Academic Dordrecht TheNetherlands 1995

[36] C R Woods Jr J Versalovic T Koeuth and J R LupskildquoAnalysis of relationships among isolates of Citrobacter diversusby using DNA fingerprints generated by repetitive sequence-based primers in the polymerase chain reactionrdquo Journal ofClinical Microbiology vol 30 no 11 pp 2921ndash2929 1992

[37] C R Woods J Versalovic T Koeuth and J R Lupski ldquoWhole-cell repetitive element sequence-based polymerase chain reac-tion allows rapid assessment of clonal relationships of bacterialisolatesrdquo Journal of Clinical Microbiology vol 31 no 7 pp 1927ndash1931 1993

[38] M R Cancilla I B Powell A J Hillier and B E DavidsonldquoRapid genomic fingerprinting of Lactococcus lactis strainsby arbitrarily primed polymerase chain reaction with p32 andfluorescent labelsrdquo Applied and Environmental Microbiologyvol 58 no 5 pp 1772ndash1775 1992

[39] A Fekete J A Bantle S M Halling and R W Stich ldquoAmplifi-cation fragment length polymorphism in Brucella strains by useof polymerase chain reaction with arbitrary primersrdquo Journal ofBacteriology vol 174 no 23 pp 7778ndash7783 1992

BioMed Research International 7

[40] S Araujo I S Henriques S M Leandro A Alves A Pereiraand A Correia ldquoGulls identified as major source of fecalpollution in coastal waters a microbial source tracking studyrdquoScience of the Total Environment vol 470-471 pp 84ndash91 2014

[41] H Guo Z Mao H Jiang et al ldquoCommunity analysis ofplant growth promoting rhizobacteria for apple treesrdquo CropProtection vol 62 pp 1ndash9 2014

[42] L Zhu H Xu Y Zhang G Fu P Q Wu and Y Li ldquoBOX-PCRand PCR-DGGE analysis for bacterial diversity of a naturallyfermented functional food (Enzyme)rdquo Food Bioscience vol 5pp 115ndash122 2014

[43] J Welsh and M McClelland ldquoFingerprinting genomes usingPCR with arbitrary primersrdquoNucleic Acids Research vol 18 no24 pp 7213ndash7218 1990

[44] J G KWilliams A R Kubelik K J Livak J A Rafalski and SV Tingey ldquoDNApolymorphisms amplified by arbitrary primersare useful as geneticmarkersrdquoNucleic Acids Research vol 18 no22 pp 6531ndash6535 1990

[45] F J Louws D W Fulbright C T Stephens and F J DeBruijn ldquoSpecific genomic fingerprints of phytopathogenic Xan-thomonas and Pseudomonas pathovars and strains generatedwith repetitive sequences and PCRrdquoApplied and EnvironmentalMicrobiology vol 60 no 7 pp 2286ndash2295 1994

[46] A K Judd M Schneider M J Sadowsky and F J de BruijnldquoUse of repetitive sequences and the polymerase chain reac-tion technique to classify genetically related Bradyrhizobiumjaponicum serocluster 123 strainsrdquo Applied and EnvironmentalMicrobiology vol 59 no 6 pp 1702ndash1708 1993

[47] A A Benedict A M Alvarez and J Berestecky ldquoPathovarspecific monoclonal antibodies for Xanthomonas campestrispv oryzae and for Xanthomonas campestris pv oryzicolardquoPhytopathology vol 79 pp 322ndash328 1989

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

BioMed Research International 7

[40] S Araujo I S Henriques S M Leandro A Alves A Pereiraand A Correia ldquoGulls identified as major source of fecalpollution in coastal waters a microbial source tracking studyrdquoScience of the Total Environment vol 470-471 pp 84ndash91 2014

[41] H Guo Z Mao H Jiang et al ldquoCommunity analysis ofplant growth promoting rhizobacteria for apple treesrdquo CropProtection vol 62 pp 1ndash9 2014

[42] L Zhu H Xu Y Zhang G Fu P Q Wu and Y Li ldquoBOX-PCRand PCR-DGGE analysis for bacterial diversity of a naturallyfermented functional food (Enzyme)rdquo Food Bioscience vol 5pp 115ndash122 2014

[43] J Welsh and M McClelland ldquoFingerprinting genomes usingPCR with arbitrary primersrdquoNucleic Acids Research vol 18 no24 pp 7213ndash7218 1990

[44] J G KWilliams A R Kubelik K J Livak J A Rafalski and SV Tingey ldquoDNApolymorphisms amplified by arbitrary primersare useful as geneticmarkersrdquoNucleic Acids Research vol 18 no22 pp 6531ndash6535 1990

[45] F J Louws D W Fulbright C T Stephens and F J DeBruijn ldquoSpecific genomic fingerprints of phytopathogenic Xan-thomonas and Pseudomonas pathovars and strains generatedwith repetitive sequences and PCRrdquoApplied and EnvironmentalMicrobiology vol 60 no 7 pp 2286ndash2295 1994

[46] A K Judd M Schneider M J Sadowsky and F J de BruijnldquoUse of repetitive sequences and the polymerase chain reac-tion technique to classify genetically related Bradyrhizobiumjaponicum serocluster 123 strainsrdquo Applied and EnvironmentalMicrobiology vol 59 no 6 pp 1702ndash1708 1993

[47] A A Benedict A M Alvarez and J Berestecky ldquoPathovarspecific monoclonal antibodies for Xanthomonas campestrispv oryzae and for Xanthomonas campestris pv oryzicolardquoPhytopathology vol 79 pp 322ndash328 1989

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