Research Article Phylogenetic Relationship of Phosphate...

Research ArticlePhylogenetic Relationship of Phosphate Solubilizing Bacteriaaccording to 16S rRNA Genes

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 30 June 2014 Revised 2 September 2014 Accepted 10 September 2014

Academic Editor Qaisar Mahmood

Copyright copy 2015 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

Phosphate solubilizing bacteria (PSB) can convert insoluble form of phosphorous to an available form Applications of PSB asinoculants increase the phosphorus uptake by plant in the field In this study isolation and precise identification of PSBwere carriedout in Malaysian (Serdang) oil palm field (University Putra Malaysia) Identification and phylogenetic analysis of 8 better isolateswere carried out by 16S rRNA gene sequencing in which as a result five isolates belong to the Beta subdivision of Proteobacteriaone isolate was related to the Gama subdivision of Proteobacteria and two isolates were related to the Firmicutes Bacterial isolatesof 6upmr 2upmr 19upmnr 10upmr and 24upmr were identified as Alcaligenes faecalis Also bacterial isolates of 20upmnr and17upmnr were identified as Bacillus cereus and Vagococcus carniphilus respectively and bacterial isolates of 31upmr were identifiedas Serratia plymuthica Molecular identification and characterization of oil palm strains as the specific phosphate solubilizer canreduce the time and cost of producing effective inoculate (biofertilizer) in an oil palm field

1 Introduction

Phosphorus is the least available essential nutrients whereits concentrations are less than many other nutrients in soilSince available phosphorus seldom exceeds 10 120583M in soilthe lack of that is common in most soil [1 2] Availableinorganic P in soil solution is 2120583M and it is several orders ofsize lower than that in plant tissues (5ndash20mM) In the otherhand aluminum iron and calcium interact strongly withP and make it unavailable to plants Also twenty to eightypercent of phosphate in soil is in organic form [3] it should bemineralized into inorganic form before it becomes availablefor plant up-taking Several bacterial and fungal species werereported as phosphate solubilizer in crops and some of themwere used as biofertilizers in agricultural fields [1]

With the aid of molecular technologies the study ofmicrobial ecology and understanding the place of microor-ganisms in society were made easy [4ndash7]These days interestfor molecular identification of bacteria based on the betterunderstanding of their biological role in keeping a sustainablebiosphere has been increased [8ndash11] Sequence analysis of

16S rRNA gene can find phylogenetic relationships betweenbacteria [11ndash15]

Studies have shown that diversity of microorganism insoil is large [16]Therefore the aim for identifying communi-ties ofmicroorganismswith key roles in specific soil chemicalprocesses such as phosphate solubilization is important insoil microbiology [17ndash21] The objective of this study wasto isolate identify and characterize phosphate solubilizingbacteria from rhizosphere and nonrhizosphere of oil palm inUniversity Putra Malaysiarsquos soil (Serdang)

2 Materials and Methods

21 Soil Sampling Soil samples were collected from oilpalm plantation at University Putra Malaysia Serdang (+2∘591015840 966610158401015840 +101∘ 431015840 23541610158401015840) The samples were takenrandomly from mature oil palm plantations (eight years)at a depth of 15ndash20 cm Sampling was done in a mannerwhich ensure that no cross contamination can occur betweenrhizosphere and nonrhizosphere samples Soil samples were

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 201379 5 pageshttpdxdoiorg1011552015201379

2 BioMed Research International

Table 1 Soil analysis

Sample 120583ggpH C N P K Ca Mg Cu Fe Mn Zn

UPMNR 54a 16b 01b 398b 38b 402a 81b 089a 089a 3b 2b

UPMR 50b 58a 043a 453a 129a 337b 102a 052b 052a 10a 6a

In each column followed by different letters are significantly different (119875 lt 005) according to ANOVA test performed with SPSS 101 SoftwareUPMNR UPM nonrhizosphere and UPMR UPM rhizosphere

then analyzed to determine the chemical elements includingNitrogen [22] Phosphorus (P) [23] Potassium (K) Calcium(Ca) Magnesium (Mg) [24] Zinc (Zn) Iron (Fe) Manganese(Mn) and Copper (Cu) [25] (Table 1)

22 Isolation of Phosphate Solubilizing Bacteria Bacterialisolates were isolated from soil samples by using root-freesoil as nonrhizosphere and also by using soil which issurrounding the root of oil palm as Rhizosphere Ten gramsof soil was diluted in 95mL of sterile water to form a serialdilution up to 10minus6 Then 01mL of the final three dilutionswas individually plated on Pikovskaya (PVK) media [26]Bacteria which represent clearing halozone on the plates wereselected and purified on PVK media [27] The eight bacterialisolates were selected for further study based on the highperformance of phosphate solubilization [28]

23 Identification of Phosphate Solubilizing Bacteria Using16S rRNA Gene All eight isolates were grown in LuriaBroth Agar (LBA) media Bacteria were grown at 28∘C for18 hours in a shaking incubator (Amersham PharmaciaBiotech) (200 rpm) [29] Genomic DNA was extracted frombacterial isolates using a commercial kit (Qiagen Miniprep27104 Matrix Technologies Cooperation USA) accordingto the manufacturerrsquos instructions Then DNA was storedat minus20∘C The concentration and purity of the extractedDNA were determined spectrophotometrically [29] Twouniversal oligonucleotide forward and reverse primers weresynthesized based on the standard 16S rRNA gene sequence[30] The primers used to amplify the 16S rDNA sampleswere forward primer -51015840-CCGAATTCGTCGACAACA-GAGTTTGATCCTGGCTCAG-31015840 and reverse primer -51015840-CCCGGATCCAAGCTTACGGCTACCTTGTTACGACTT-31015840

24 PCR Amplification PCR amplification was performed ina total volume of 50120583L mixture It contained 5120583L of 10x PCRbuffer 05 120583L of 10x dNTP 1 120583L of forward and 1 120583L of reverseprimer 13 120583L of template DNA (10 ng) 1 120583L of Taq DNApolymerase and sterile distilled water adjusted to 50 120583L Thetubes were subjected to 35 cycles in a thermal cycler with thefollowing program initial denaturation at 95∘C for 3 minuteswhich was followed with 35-cycle consisting of denaturationat 94∘C for 30 seconds annealing at 60∘C for 30 seconds andelongation at 72∘C for 2 minutesThe reaction was completedwith an extension step at 72∘C for 5 minutes All sampleswere removed from the thermal cycler and stored at minus20∘CThe PCR product was determined by electrophoresis with

loading 8120583L of PCR product on to 1 agarose gel The gelwas stained with ethidium bromide and photographed usinggel-documentation system (Hoefer PS 500XT) [29]The PCRproducts of 16S rRNA gene of each sample were purifiedby the PCR purification kit (Vivantis GF-PC-100 Malaysia)following the manufacturerrsquos instructions The 16S rRNAgene products of eight bacteria isolates were sequenced Theclean PCR product was subjected to cycle sequencings inboth directions using the universal primers The sequencingwas done by ABAPRISM Dye Terminator Cycle Sequenc-ing method (NHK Bioscience Solutions Sdn Bhd) Thenucleotide sequenceswere edited using the softwareChromasand compared with published sequences in the NationalCenter for Biotechnology Information Genbank using theBLAST software Phylogenetical analyses of 16S rRNA genesequences were aligned using the software CLUSTAL W 18The phylogenetical analysis was conducted using the Neigh-bor Joining Method Then the output trees were performedwith the software Molecular Evolutionary Genetics Analysisversion 40 (MEGA 4) [29]

3 Results and Discussion

31 16S rRNA Gene Analysis Two universal oligonucleotideswere used to determine and identify the 16S rRNA gene for allisolates The primer amplified the gene successfully from allof the phosphate solubilizing bacterial isolates although therewere no obvious variations in the size of rRNA gene productsbetween the eight bacterial isolates The size of the 16S rRNAgene product of all isolated bacteria in this study was about14 Kb to the relative DNA size marker (Figure 1) The 16SrRNA gene sequence of phosphates solubilizing bacteria thatwas isolated from oil palm soil was compared with Genbankand received the accession numbers (Table 2) The 16S rRNAgene sequences allowed separation between isolates at thespecies level

It is also important to consider that for the identifi-cation of isolates from oil palm soil it is not necessaryto sequence the whole 1500 bp length and thus partialsequencing can provide necessary information even thoughthe whole sequencing that includes the entire 1500 bp regionmight be useful to distinguish between particular strains

Comparison of the partial 16S rDNA sequence of eightisolates with Genbank database showed that they belong totwo taxonomic lineages Five isolates belonged to the Betasubdivision of Proteobacteria one isolate was Gama subdi-vision of Proteobacteria and two isolates were FirmicutesBacterial isolates of 6upmr 2upmr 19upmnr 10upmr and24upmr were identified as Alcaligenes faecalis Also bacterial

BioMed Research International 3

Table 2 Identification and relationship of PSB isolates based on 16S rDNA marker data

Name Kingdom Phylum Class Order Family Genus SpeciesAccessionnumber(NCBI)

17upmnr Bacteria Firmicutes Bacilli Lactobacillales Enterococcaceae Vagococcus V carniphilus KJ78345220upmnr Bacteria Firmicutes Bacilli Bacillales Bacillaceae Bacillus B cereus KJ72960219upmnr Bacteria Proteobacteria Betaproteobacteria Burkholderiales Alcaligenaceae Alcaligenes A faecalis KJ7485936upmr Bacteria Proteobacteria Betaproteobacteria Burkholderiales Alcaligenaceae Alcaligenes A faecalis KJ7296082upmr Bacteria Proteobacteria Betaproteobacteria Burkholderiales Alcaligenaceae Alcaligenes A faecalis KJ74858610upmr Bacteria Proteobacteria Betaproteobacteria Burkholderiales Alcaligenaceae Alcaligenes A faecalis KJ74858524upmr Bacteria Proteobacteria Betaproteobacteria Burkholderiales Alcaligenaceae Alcaligenes A faecalis KJ74858731upmr Bacteria Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Serratia S plymuthica KJ729609

isolates of 20upmnr and 17upmnr were identified as Bacilluscereus and Vagococcus carniphilus respectively and bacterialisolates of 31upmr was identified as Serratia plymuthica(Table 2) Before it had been reported that PseudomonasBacillus and Rhizobium strains are the most powerful andabundant strains of bacterial phosphate solubilizers [31] Alsothe genera of Aspergillus Penicillium Klebsiella Burkholde-ria and Staphylococcus are better phosphate solubilizersin Colombia oil palm plantation [13] However BacillusRhodococcus Arthrobacter Serratia Chryseobacterium Delf-tia Gordonia and Phyllobacterium genus were better inTaiwan [32] Furthermore the phylogenetic diversity ofphosphate solubilizing bacteria (PSB) distributed in soil ofChinawas characterized andmembers of Proteobacteriaweredominant Most of the isolates were associated with thegenera of Burkholderia Pseudomonas Acinetobacter Enter-obacter Pantoea Serratia Klebsiella Leclercia Raoultella andCedecea [33] Also researcher reported that most phosphatesolubilizing bacteria (PSB) in a croppasture rotation inUruguay were related to the genera of Burkholderia Acine-tobacter and genus Pseudomonas [34]

The 16S rRNA gene analysis revealed that all of isolatesbelong to the genera Alcaligenes Serratia Bacillus andVagococcus The most often observed species of the betaProteobacter genus was Alcaligenes Sequences from eightisolates were completely or higher than 98 similar to other16S rRNA sequences from database The isolates 19upmnr6upmr 2upmr 24upmr and 31upmr had 99 and 17upmnrisolate had 98 similarity with other isolates in gene bankdatabase (NCBI) (Table 2)

The phylogenetic analysis based on the partial 16S rRNAgene sequencing could classify the three main taxonomiclineages (Figure 2)The sequences obtained fromAlcaligenesSerratia Bacillus and Vagococcus genera formed separatedbranches from one another There are three phylogenybranches that belong to Alcaligenes strains (6upmr 10upmr2upmr 19upmnr and 24upmr) Serratia (31upmr) bacil-lus (20upmnr) and Vagococcus (17upmnr) (Figure 2) Allisolates form three distinct clusters based on near full-length 16S rRNA gene sequence analysis Cluster C1 belongsto Alcaligenes strains that were isolated from rhizosphereand nonrhizosphere environment at Serdang (UPM) soilComparison of sequences revealed a greater genetic diversity

14

1 2 3 4 5 6 7 8

Figure 1 PCR products of 16S rDNA (Line 1 = 20upmnr Line 2 =19upmnr Line 3 = 6upmr Line 4 = 10upmr Line 5 = 31upmr Line 6= 2upmr Line 7 = 24upmr and Line 8 = 17upmnr)

in Alcaligenes strains Isolate 31upmr from UPM rhizosphereformed separate cluster (C2) while the other showed closerelationship with each other (cluster C3) These resultssuggested that theAlcaligenes genus grouped at cluster C1 wasdiverse and needed another molecular marker to distinguishbetween them

It suggested that a physiological stress or effects of envi-ronment led to selection of less diverse communities in bacte-rial populations and general suppression of high solubilizingactivity The results showed the differences in the isolatersquossequences in two areas of sampling while most isolatedbacteria from nonrhizosphere and rhizosphere belonged toAlcaligenes species Rhizosphere bacteria were more diversebecause of the population compared with nonrhizosphere

4 Conclusion

In conclusion conservation region in the 16S rRNA genesequence could identify all isolates of phosphate solubiliz-ing bacteria isolated from oil palm soil successfully Thissequence can serve as a good molecular chronometer foridentification of phosphate solubilizing bacteria with noprevious knowledge The degree of gene conservation isconsidered to be a significant part of cell identificationThis study also shows that partial sequencing can providestatistically valid measurements for evolutionary distances ofphosphate solubilizing isolates Assigning a numerical valueto the rate of change in phosphate solubilizing isolates canmake taxonomic groups for all isolates Furthermore it shows


Alcaligenes faecalis strain (6upmr)Alcaligenes faecalis strain (10upmr)Alcaligenes faecalis strain (2upmr)Alcaligenes faecalis strain (19upmnr)Alcaligenes faecalis strain (24upmr)Serratia plymuthica strain (31upmr)Bacillus cereus strain (20upmnr)Vagococcus carniphilus strain (17upmnr)

C1

C2

C3

000002004006008010012

Figure 2 The dendrograms of cluster analysis showing the identification and relationship of UPMrsquos PSB isolates based on 16S rRNA gene

that the rate of change in phosphate solubilizing isolates canbe different at University Putra field Finally no gene hasshown as broad applicability over all the taxonomic groupsas the 16S rRNA gene Thus if the objective would be toidentify an unknown isolates the 16S rRNA gene sequenceis an excellent and extensively used choice

Conflict of Interests

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

References

[1] K G Raghothama ldquoPhosphate acquisitionrdquo Annual Review ofPlant Biology vol 50 pp 665ndash693 1999

[2] H Yadav R K Gothwal V K Nigam S Sinha-Roy andP Ghosh ldquoOptimization of culture conditions for phosphatesolubilization by a thermo-tolerant phosphate-solubilizing bac-terium Brevibacillus sp BISR-HY65 isolated from phosphateminesrdquo Biocatalysis and Agricultural Biotechnology vol 2 no3 pp 217ndash225 2013

[3] A Jungk B Seeling and J Gerke ldquoMobilization of differentphosphate fractions in the rhizosphererdquo Plant and Soil vol 155-156 no 1 pp 91ndash94 1993

[4] F Ali Shah Q Mahmood M M Shah A Pervez and S AAsad ldquoMicrobial ecology of anaerobic digesters the key playersof anaerobiosisrdquo The Scientific World Journal vol 2014 ArticleID 183752 21 pages 2014

[5] P Lidder and A Sonnino ldquoChapter 1mdashbiotechnologies for themanagement of genetic resources for food and agriculturerdquo inAdvances in Genetics T F Stephen F Goodwin and C D JayEds pp 1ndash167 Academic Press 2012

[6] V V Shenoy and G M Kalagudi ldquoEnhancing plant phos-phorus use efficiency for sustainable croppingrdquo BiotechnologyAdvances vol 23 no 7-8 pp 501ndash513 2005

[7] J F BQuerido J Agirre G AMarti DMAGuerin andM SSilva ldquoMolecular techniques for dicistrovirus detection withoutRNA extraction or purificationrdquoBioMedResearch Internationalvol 2013 Article ID 218593 3 pages 2013

[8] R G Burns J L DeForest J Marxsen et al ldquoSoil enzymesin a changing environment current knowledge and futuredirectionsrdquo Soil Biology amp Biochemistry vol 58 pp 216ndash2342013

[9] S G Dastager W-J Li I Saadoun and M Miransari ldquoMicro-bial diversity-sustaining earth and industryrdquo Applied and Envi-ronmental Soil Science vol 2011 Article ID 459195 2 pages 2011

[10] R Farina A Beneduzi A Ambrosini et al ldquoDiversity of plantgrowth-promoting rhizobacteria communities associated with

the stages of canola growthrdquo Applied Soil Ecology vol 55 pp44ndash52 2012

[11] A Krimitzas I Pyrri V N Kouvelis E Kapsanaki-Gotsiand M A Typas ldquoA phylogenetic analysis of greek isolatesof Aspergillus species based on morphology and nuclear andmitochondrial gene sequencesrdquo BioMed Research Internationalvol 2013 Article ID 260395 18 pages 2013

[12] K-YKimM-HKoH Liu et al ldquoPhylogenetic relationships ofPseudorasbora Pseudopungtungia and Pungtungia (TeleosteiCypriniformes Gobioninae) inferred from multiple nucleargene sequencesrdquo BioMed Research International vol 2013Article ID 347242 6 pages 2013

[13] E Acevedo T Galindo-Castaneda F Prada M Navia and HM Romero ldquoPhosphate-solubilizing microorganisms associ-ated with the rhizosphere of oil palm (Elaeis guineensis Jacq)in Colombiardquo Applied Soil Ecology vol 80 pp 26ndash33 2014

[14] S A Ahmad M Y Shukor N A Shamaan W P M Cormackand M A Syed ldquoMolybdate reduction to molybdenum blueby an antarctic bacteriumrdquo BioMed Research International vol2013 Article ID 871941 10 pages 2013

[15] T Jagielski J Van Ingen N Rastogi J Dziadek P K Mazurand J Bielecki ldquoCurrent methods in the molecular typing ofMycobacterium tuberculosis and other Mycobacteriardquo BioMedResearch International vol 2014 Article ID 645802 21 pages2014

[16] A Felske A Wolterink R van Lis and A D L AkkermansldquoPhylogeny of the main bacterial 16S rRNA sequences indrentse A grassland soils (The Netherlands)rdquo Applied andEnvironmental Microbiology vol 64 no 3 pp 871ndash879 1998

[17] M A Kertesz E Fellows and A Schmalenberger ldquoRhizobacte-ria andplant sulfur supplyrdquo inAdvances inAppliedMicrobiologyS S Allen I Laskin and M G Geoffrey Eds pp 235ndash268Academic Press 2007

[18] A Beneduzi D Peres P B da Costa M H Bodanese Zanettiniand L M P Passaglia ldquoGenetic and phenotypic diversity ofplant-growth-promoting bacilli isolated from wheat fields insouthern Brazilrdquo Research in Microbiology vol 159 no 4 pp244ndash250 2008

[19] J Ghyselinck S L S Velivelli K Heylen et al ldquoBioprospectingin potato fields in the Central Andean Highlands screening ofrhizobacteria for plant growth-promoting propertiesrdquo System-atic and Applied Microbiology vol 36 no 2 pp 116ndash127 2013

[20] G Tsiamis D Karpouzas A Cherif and K MavrommatisldquoMicrobial diversity for biotechnologyrdquo BioMed Research Inter-national vol 2014 Article ID 845972 3 pages 2014

[21] C Spampinato and D Leonardi ldquoMolecular fingerprints toidentify Candida speciesrdquo BioMed Research International vol2013 Article ID 923742 10 pages 2013


[22] J M Bremner ldquoDetermination of nitrogen in soil by theKjeldahl methodrdquo Journal of Agricultural Sciences vol 55 pp11ndash13 1960

[23] R H Bray and L T Kurtz ldquoDetermination of total organic andavailable forms of phosphorus in soilsrdquo Soil Science vol 59 no1 pp 339ndash445 1945

[24] A C Chang A L Page and B-J Koo ldquoBiogeochemistry ofphosphorus iron and trace elements in soils as influencedby soil-plant-microbial interactionsrdquo in Developments in SoilScience P M H J M B A Violante and L Gianfreda Edspp 43ndash57 Elsevier 2002

[25] W L Lindsay andW A Norvell ldquoDevelopment of a DTPA soiltest for zinc iron manganese and copperrdquo Soil Science Societyof America Journal vol 42 no 3 pp 421ndash428 1978

[26] C A Oliveira V M C Alves I E Marriel et al ldquoPhosphatesolubilizingmicroorganisms isolated from rhizosphere ofmaizecultivated in an oxisol of the Brazilian Cerrado Biomerdquo SoilBiology and Biochemistry vol 41 no 9 pp 1782ndash1787 2009

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

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

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

[30] 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

[31] B C Behera S K Singdevsachan R R Mishra S K Duttaand H N Thatoi ldquoDiversity mechanism and biotechnology ofphosphate solubilisingmicroorganism inmangrovemdasha reviewrdquoBiocatalysis and Agricultural Biotechnology vol 3 no 2 pp 97ndash110 2014

[32] Y P Chen P D Rekha A B Arun F T Shen W-A Lai and CC Young ldquoPhosphate solubilizing bacteria from subtropical soiland their tricalcium phosphate solubilizing abilitiesrdquo AppliedSoil Ecology vol 34 no 1 pp 33ndash41 2006

[33] P-X Yang L Ma M-H Chen et al ldquoPhosphate solubilizingability and phylogenetic diversity of bacteria from p-rich soilsaround dianchi lake drainage area of Chinardquo Pedosphere vol22 no 5 pp 707ndash716 2012

[34] G Azziz N Bajsa T Haghjou et al ldquoAbundance diversityand prospecting of culturable phosphate solubilizing bacteriaon soils under crop-pasture rotations in a no-tillage regime inUruguayrdquo Applied Soil Ecology vol 61 pp 320ndash326 2012

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


Table 1 Soil analysis

Sample 120583ggpH C N P K Ca Mg Cu Fe Mn Zn

UPMNR 54a 16b 01b 398b 38b 402a 81b 089a 089a 3b 2b

UPMR 50b 58a 043a 453a 129a 337b 102a 052b 052a 10a 6a

In each column followed by different letters are significantly different (119875 lt 005) according to ANOVA test performed with SPSS 101 SoftwareUPMNR UPM nonrhizosphere and UPMR UPM rhizosphere

then analyzed to determine the chemical elements includingNitrogen [22] Phosphorus (P) [23] Potassium (K) Calcium(Ca) Magnesium (Mg) [24] Zinc (Zn) Iron (Fe) Manganese(Mn) and Copper (Cu) [25] (Table 1)

22 Isolation of Phosphate Solubilizing Bacteria Bacterialisolates were isolated from soil samples by using root-freesoil as nonrhizosphere and also by using soil which issurrounding the root of oil palm as Rhizosphere Ten gramsof soil was diluted in 95mL of sterile water to form a serialdilution up to 10minus6 Then 01mL of the final three dilutionswas individually plated on Pikovskaya (PVK) media [26]Bacteria which represent clearing halozone on the plates wereselected and purified on PVK media [27] The eight bacterialisolates were selected for further study based on the highperformance of phosphate solubilization [28]

23 Identification of Phosphate Solubilizing Bacteria Using16S rRNA Gene All eight isolates were grown in LuriaBroth Agar (LBA) media Bacteria were grown at 28∘C for18 hours in a shaking incubator (Amersham PharmaciaBiotech) (200 rpm) [29] Genomic DNA was extracted frombacterial isolates using a commercial kit (Qiagen Miniprep27104 Matrix Technologies Cooperation USA) accordingto the manufacturerrsquos instructions Then DNA was storedat minus20∘C The concentration and purity of the extractedDNA were determined spectrophotometrically [29] Twouniversal oligonucleotide forward and reverse primers weresynthesized based on the standard 16S rRNA gene sequence[30] The primers used to amplify the 16S rDNA sampleswere forward primer -51015840-CCGAATTCGTCGACAACA-GAGTTTGATCCTGGCTCAG-31015840 and reverse primer -51015840-CCCGGATCCAAGCTTACGGCTACCTTGTTACGACTT-31015840

24 PCR Amplification PCR amplification was performed ina total volume of 50120583L mixture It contained 5120583L of 10x PCRbuffer 05 120583L of 10x dNTP 1 120583L of forward and 1 120583L of reverseprimer 13 120583L of template DNA (10 ng) 1 120583L of Taq DNApolymerase and sterile distilled water adjusted to 50 120583L Thetubes were subjected to 35 cycles in a thermal cycler with thefollowing program initial denaturation at 95∘C for 3 minuteswhich was followed with 35-cycle consisting of denaturationat 94∘C for 30 seconds annealing at 60∘C for 30 seconds andelongation at 72∘C for 2 minutesThe reaction was completedwith an extension step at 72∘C for 5 minutes All sampleswere removed from the thermal cycler and stored at minus20∘CThe PCR product was determined by electrophoresis with

loading 8120583L of PCR product on to 1 agarose gel The gelwas stained with ethidium bromide and photographed usinggel-documentation system (Hoefer PS 500XT) [29]The PCRproducts of 16S rRNA gene of each sample were purifiedby the PCR purification kit (Vivantis GF-PC-100 Malaysia)following the manufacturerrsquos instructions The 16S rRNAgene products of eight bacteria isolates were sequenced Theclean PCR product was subjected to cycle sequencings inboth directions using the universal primers The sequencingwas done by ABAPRISM Dye Terminator Cycle Sequenc-ing method (NHK Bioscience Solutions Sdn Bhd) Thenucleotide sequenceswere edited using the softwareChromasand compared with published sequences in the NationalCenter for Biotechnology Information Genbank using theBLAST software Phylogenetical analyses of 16S rRNA genesequences were aligned using the software CLUSTAL W 18The phylogenetical analysis was conducted using the Neigh-bor Joining Method Then the output trees were performedwith the software Molecular Evolutionary Genetics Analysisversion 40 (MEGA 4) [29]

3 Results and Discussion

31 16S rRNA Gene Analysis Two universal oligonucleotideswere used to determine and identify the 16S rRNA gene for allisolates The primer amplified the gene successfully from allof the phosphate solubilizing bacterial isolates although therewere no obvious variations in the size of rRNA gene productsbetween the eight bacterial isolates The size of the 16S rRNAgene product of all isolated bacteria in this study was about14 Kb to the relative DNA size marker (Figure 1) The 16SrRNA gene sequence of phosphates solubilizing bacteria thatwas isolated from oil palm soil was compared with Genbankand received the accession numbers (Table 2) The 16S rRNAgene sequences allowed separation between isolates at thespecies level

It is also important to consider that for the identifi-cation of isolates from oil palm soil it is not necessaryto sequence the whole 1500 bp length and thus partialsequencing can provide necessary information even thoughthe whole sequencing that includes the entire 1500 bp regionmight be useful to distinguish between particular strains

Comparison of the partial 16S rDNA sequence of eightisolates with Genbank database showed that they belong totwo taxonomic lineages Five isolates belonged to the Betasubdivision of Proteobacteria one isolate was Gama subdi-vision of Proteobacteria and two isolates were FirmicutesBacterial isolates of 6upmr 2upmr 19upmnr 10upmr and24upmr were identified as Alcaligenes faecalis Also bacterial








14

1 2 3 4 5 6 7 8




4 Conclusion




C1

C2

C3

000002004006008010012





References












































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








14

1 2 3 4 5 6 7 8




4 Conclusion




C1

C2

C3

000002004006008010012





References












































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



C1

C2

C3

000002004006008010012





References












































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Research Article Phylogenetic Relationship of Phosphate...

Documents

Transcript of Research Article Phylogenetic Relationship of Phosphate...