research article application of potential phosphate...

Hindawi Publishing CorporationThe Scientific World JournalVolume 2013 Article ID 272409 10 pageshttpdxdoiorg1011552013272409

Research ArticleApplication of Potential Phosphate-Solubilizing Bacteriaand Organic Acids on Phosphate Solubilization from PhosphateRock in Aerobic Rice

Qurban Ali Panhwar1 Shamshuddin Jusop1 Umme Aminun Naher23

Radziah Othman12 and Mohd Ismail Razi2

1 Department of Land Management Faculty of Agriculture Universiti Putra Malaysia 43400 Serdang Selangor Malaysia2 Institute of Tropical Agriculture Universiti Putra Malaysia 43400 Serdang Selangor Malaysia3 Bangladesh Rice Research Institute Gazipur Bangladesh

Correspondence should be addressed to Radziah Othman radziahupmedumy

Received 1 July 2013 Accepted 22 August 2013

Academic Editors P Andrade and D Zhou

Copyright copy 2013 Qurban Ali Panhwar et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

A study was conducted at Universiti Putra Malaysia to determine the effect of phosphate-solubilizing bacteria (PSB) and organicacids (oxalic amp malic) on phosphate (P) solubilization from phosphate rock (PR) and growth of aerobic rice Four rates of eachorganic acid (0 10 20 and 30mM) and PSB strain (Bacillus sp) were applied to aerobic rice Total bacterial populations amountof P solubilization P uptake soil pH and root morphology were determined The results of the study showed significantly high Psolubilization in PSB with organic acid treatments Among the two organic acids oxalic acid was found more effective comparedto malic acid Application of oxalic acid at 20mM along with PSB16 significantly increased soluble soil P (2839mg kgminus1) plant Puptake (078 P potminus1) and plant biomass (3326mg) Addition of organic acids with PSB and PR had no influence on soil pH duringthe planting period A higher bacterial population was found in rhizosphere (878 log

10

cfu gminus1) compared to the nonrhizosphereand endosphere regions The application of organic acids along with PSB enhanced soluble P in the soil solution improved rootgrowth and increased plant biomass of aerobic rice seedlings without affecting soil pH

1 Introduction

Phosphate-solubilizing bacteria play a vital role in P sol-ubilization by producing organic acids Additionally soilscontain low molecular weight organic acids with one ormore carboxylic groups and some of these acids like citrateoxalate acetate malate isocitrate and tartrate Plant rootexudates and microorganisms produce organic acids anddegrade complex organic molecules [1 2] Organic acidsperform many functions in the soil such as root nutrientacquisition mineral weathering microbial chemotaxis andmetal detoxification They play an important role in themobilization of soil P and enhance P bioavailability [3 4]with decreasing P adsorption and dissolution of insoluble Pcompounds such as Ca Fe and Al phosphates [3] Organic

acid exudation from roots is considered an important mech-anism for plants to adapt in P-deficient environments [5]The mechanism involves mobilization of unavailable P inthe soil by organic acids [6] The role of organic acids inP solubilization is highly soil dependent On the contraryone of the P solubilization mechanisms of microbes is theproduction of organic acids [7] A number of organic acidssuch as lactic citric 2-ketogluconic malic oxalic malonictartaric and succinic have been identified that have chelatingproperties [8] Evidence showed that addition of organic acidsto soils increased plant P uptake [9]

The PR is an alternative and natural source of P Thedissolution of P from PR is required for the availabilityof P Many factors affect the dissolution of PR in soilsuch as chemical composition particle size of the PR soil

2 The Scientific World Journal

0

5

10

15

20

25

30

0 10 20 30Rate of oxalic acid (mM)

Solu

ble P

(mg k

gminus1)

Noninoculated (+PR) Inoculated (+PR)Noninoculated (minusPR) Inoculated (minusPR)

(a)

0

5

10

15

20

25

30

0 10 20 30Rate of malic acid (mM)

Solu

ble P

(mg k

gminus1)

Inoculated (+PR)Inoculated (minusPR)

Noninoculated (+PR)Noninoculated (minusPR)

(b)

Figure 1 Effect of (a) oxalic acid (b) malic acid on P solubilization Bars indicate standard error 119899 = 5

characteristics pHH2

PO4

minus andCa2

+ [10] Dissolution of PRin acid soils can be explained as follows Khasawneh and Doll[11]

Ca10

(PO4

)6

F2

+ 12H+ 997904rArr 10Ca2

+

+ 6H2

PO4

minus

+ 2Fminus (1)

The equation specifies that the rate of dissolution isdetermined by the concentration of protons (H+) and theconcentration of reaction products of Ca

2

+ and H2

PO4

Inacidic soils improvement of P nutrition to plants by directapplication of PR as P fertilizer has been considered [12]Phosphate rocks are affluent in calcium phosphate complexesand are soluble in acidic soil environments Either microbialreleased organic acids or any acidic condition may favor Psolubilization from PR Hence the present study was under-taken to determine the effect of different rates of organic acidswith phosphate-solubilizing bacteria on P solubilization andtheir effect on growth of aerobic rice

2 Materials and Methods

The experiment was conducted under in vitro conditionPSB16 (Bacillus sp) isolated form aerobic rice rhizosphere[13] which was tested to produce indoleacetic acid P solubi-lization organic acids and siderophore production in in vitrocondition The isolated strain was identified using 16S rRNAgene sequencing with accession number JX103827 Four rateseach of oxalic and malic acids (0 10 20 and 30mM) andPR (Christmas Island Rock Phosphate) at 60 kg P

2

O5

haminus1were applied The aerobic rice (var M9) was grown in thegrowth chamber for 40 days Total bacterial populationsP solubilization soil pH root morphology and agronomicparameters were recorded after 40 days of growth

21 Seed Surface Sterilization Inoculation and Growth ofRice Seedlings The surface sterilized seven days old seedlingswere transplanted into pots containing sterilized soil (500 g)with 4 uniform seedlings per pot Plants were grown for 40

days in a growth chamber with 12 h lightdark cycle at 29 plusmn1∘C temperature Approximately 5times 109mLminus1 of live washedbacterial cells of Bacillus sp (PSB16) were used as inoculumin each bacterial treatment

22 Determination of Bacterial Population Plant Biomassand Plant Tissue P The total bacterial population was deter-mined from rhizosphere nonrhizosphere and endosphereof aerobic rice plants After harvest soil available P wasdetermined using Bray 2 [14] and total plant tissue P wasanalyzed by the wet digestion method [15]

23 Determination of Root Development The root length(cm) total surface (cm2) and root volume (cm3) were quanti-fied using a scanner (Expression 1680 Epson) equipped witha 2 cm depth plexiglass tank (20times 30 cm) filled with UP H

2

O[16]

24 Data Analysis The experiment was conducted with thethree factors in four replicates in a completely randomizeddesign Data obtained were statistically analyzed using theSAS software program (Version 92) and treatment meanswere compared using Tukeyrsquos test (119875 lt 005)

3 Results

31 Effect of Organic Acids and PSB on P Solubilization andPlant P Uptake Higher values of solubilized P were foundin PSB inoculated treatments with organic acids applicationSignificantly high amount of solubilized P (3151) wasfound in PSB inoculated treatments with 20mM oxalic acid(Figure 1(a))

Between the two acids P solubilization was found higherin oxalic compared to malic acid at various rates In malicacid treatments higher P solubilization (2157mg kgminus1) wasobserved with 10mM of malic acid in PSB inoculated treat-ments (Figure 1(b))The amount of P solubilization and plant

The Scientific World Journal 3

0

1

2

3

4

5

6

7

8

9


NonrhizosphereRhizosphereEndosphere

Without PRPo

pula

tion

(log10

cfu g

minus1)

(a)

0

1

2

3

4

5

6

7

8

9


With PR

Popu

latio

n (log10

cfu g

minus1)


(b)

0

1

2

3

4

5

6

7

8

9


Without PR

Popu

latio

n (log10

cfu g

minus1)


(c)

0

1

2

3

4

5

6

7

8

9


With PR

Popu

latio

n ( log10

cfu g

minus1)


(d)

Figure 2 Effect of organic acids on PSB16 population (a) oxalic acid without PR (b) oxalic acid with PR (c) malic acid without PR (d)malic acid with PR Bars indicate standard error 119899 = 5

P uptake differed with the bacterial inoculation and rate oforganic acid application

The application of organic acids also affected plant Puptake Significantly higher plant P uptake was observed inPSB inoculated treatments addedwith organic acids (Table 1)Among both the highest values of plant P uptake was foundin PSB with oxalic acid at 20mM (078 P potminus1) followed by30mM (075 P potminus1) concentrations respectively

32 Effect of Organic Acids on Bacterial Populations Theaddition of organic acids influenced the bacterial populationSignificantly (119875 lt 005) higher populations were found inthe rhizosphere (878 log

10

cfu gminus1) while lower populationswere found in nonrhizosphere soils (540 log

10

cfu gminus1) Thehighest rhizosphere population (865 log

10

cfu gminus1) was foundin the 30mM oxalic acid treatment (Figures 2(a) and 2(b))In the case of malic acid a significantly higher population


0

5

10

15

20

25

30

35


Plan

t bio

mas

s (m

g)

Inoculated (+PR)Inoculated (minusPR)Noninoculated (minusPR) Noninoculated (+PR)

(a)

0

5

10

15

20

25

30

35


Plan

t bio

mas

s (m

g)

Inoculated (+PR)Inoculated (minusPR)Noninoculated (+PR)Noninoculated (minusPR)

(b)

Figure 3 Effect of organic acids (a) oxalic acid (b) malic acid on plant biomass Bars indicate standard error 119899 = 5

Table 1 Effect of organic acids on plant P uptake

Dose oforganicacid (mM)

Plant P uptake (mg P potminus1)Oxalic acid Malic acid

PSB noninoculated Treatments PSB inoculated Treatments PSB noninoculated Treatments PSB inoculated TreatmentsRate of PR (kg haminus1)

0 60 0 60 0 60 0 600 021e 029b 025f 036c 021d 028b 023e 033c10 024d 030b 029e 048b 022d 029b 025d 039a20 027c 037a 032d 078a 024cd 034a 027d 037ab30 028bc 039a 037c 075a 026c 036a 032c 036bMeans within the same column followed by the same letters are not significantly different at P le 005

was found in the rhizosphere at 30mMacid without PR treat-ment (878 log

10

cfu gminus1) whereas in nonrhizosphere soil thehighest population was recorded at 10mM acid without PR(654 log

10

cfu gminus1) However the endosphere population wasnot influenced by the addition of organic acids (Figures 2(c)and 2(d)) Solubilization of P by PSB in the rhizosphereis a continuous process The addition of organic acids hada positive influence on the PSB16 population A higherpopulation was found with the addition of organic acidscombined with PR The changes in PSB population occurredmostly in the nonrhizosphere region and it becomes lowerwith the addition of oxalic acid treatments

33 Effect of Organic Acids and PSB on Plant Height and PlantBiomass There were significant differences found betweenthe organic acids and their various rates Significantly (119875 lt005) high values of plant height were observed in the PSBinoculated treatments Among both acids the highest plantheight (23 cm) was observed in oxalic acid (20mM) with PRand PSB16 compared to malic acid (Table 2)

Inoculation of PSB with PR showed higher plant biomassthan noninoculated treatments (Figure 3) Application oforganic acids with PSB16 and PR significantly increasedthe plant biomass and comparatively oxalic acid producedhigher plant biomass than malic acid The highest plantbiomass (3326mg) was recorded in PSB inoculated plantsat 20mM oxalic acid concentration with PR (Figure 3(a))Application of malic acid increased biomass at 10mM andshowed no further increase at higher levels (Figure 3(b))

34 Effect of Organic Acids and PSB on Soil pH The soil pHduring the planting period was not much affected (Figures4 and 5) Slightly lower pH values were observed in PSBinoculated compared to noninoculated treatments Amongboth acids a higher decrease in pH values was found with theaddition ofmalic acid with PSB inoculationThe instability oforganic acid or soil buffering system might have an effect inregulating the soil pH However slight decreases were foundwhich could be due to the influence of organic acids to changepH in the rhizospheric regions


60

62

64

66

68

70

72

10 20 30 40Days

pH

OA0OA10

OA20OA30

(a) Noninoculated without PR

60

62

64

66

68

70

72

10 20 30 40Days

pH

OA0OA10

OA20OA30

(b) PSB inoculated without PR

60

62

64

66

68

70

72

10 20 30 40Days

pH

OA0OA10

OA20OA30

(c) Noninoculated with PR

60

62

64

66

68

70

72

10 20 30 40Days

pH

OA0OA10

OA20OA30

(d) PSB inoculated with PR

Figure 4 Effect of oxalic acid (OA) on soil pH Bars indicate standard error 119899 = 5

Table 2 Effect of organic acids on plant height of aerobic rice seedling


Plant height (cm)Oxalic acid Malic acid


0 60 0 60 0 60 0 600 1283c 17ab 16c 20b 1283d 17a 16c 20a10 15b 19a 21b 22ab 1467c 18a 19a 20a20 17ab 19a 213b 23a 16b 17a 17b 1933a30 17ab 1833a 21b 21b 1633ab 17a 1767b 1930aMeans within the same column followed by the same letters are not significantly different at 119875 le 005

35 Effect of Organic Acids and PSB on Root DevelopmentRoot development in aerobic rice was influenced by the appli-cation of organic acids PSB and PRThe highest root lengthsurface area and root volume were found in treatmentswith organic acids PR and PSB16 inoculations (Figures 6

and 7) Among both acids oxalic acid produced higher rootgrowth Significantly (119875 lt 005) higher root length (764 cm)root surface area (236 cm2) and root volume (011 cm3) werefound in oxalic acid at 20mM with PR and PSB16 inoculatedtreatments (Figures 6 and 7) External application of organic


60

62

64

66

68

70

72

10 20 30 40Days

pH

MA0MA10

MA20MA30


60

62

64

66

68

70

72

10 20 30 40Days

pH

MA0MA10

MA20MA30


60

62

64

66

68

70

72

10 20 30 40Days

pH

MA0MA10

MA20MA30


60

62

64

66

68

70

72

10 20 30 40Days

pH

MA0MA10

MA20MA30


Figure 5 Effect of malic acid (MA) on soil pH Bars indicate standard error 119899 = 5

acids along with PSB enhanced soluble P in the solution andthis had a positive impact on root growth

4 Discussion

The PSB and organic acid solubilized higher values of P inaerobic rice However high amounts of solubilized P wereobserved in PSB inoculated with oxalic acid applicationThese results are in agreement with the findings of Asea et al[17] who noted that an application of oxalic acid was effectivefor P solubilization Similar results were observed by Wei etal [18] who observed that oxalic acids are more prominentin solubilizing P compared to other organic acids Strongbinding abilities of oxalic and citric acids have been provenas the most competent agents to solubilize soil P [1] Theapplication of organic acids also affected plant P uptake

The bacterial population was influenced with the addi-tion of organic acids The population was varied in both

organic acids at different rates in plant rhizosphere and non-rizosphere population Thus it might be due direct contactof the acids in soil and could be able to perform prominentlyfor the P solubilization Moreover solubilization of P by PSBin the rhizosphere is a continuous process PSB solubilizeinsoluble P by several mechanisms such as acidificationchelation and exchange reactions [19] It was reported thatPSB solubilized PR as well as di-calcium phosphate and about20ndash50 times less organic acids secreted by PSB were requiredfor P solubilization Furthermore PSB strains (Citrobacterkoseri and Bacillus coagulans) have been proven to solubilizePRwithmany organic acids [20]The release of P is extremelysoil dependent with higher concentrations of organic acidsrequired to mobilize major quantities of P into the soilsolution [21]

The changes in PSB population occurred mostly inthe nonrhizosphere region and it becomes lower with theaddition of oxalic acid treatments This could happen due to


0

2

4

6

8

10


Root

leng

th (c

m)


(a)

0

05

1

15

2

25

3

35

4

45


Root

surfa

ce (c

m2)


(b)

0

002

004

006

008

01

012


Root

vol

ume (

cm3)


(c)Figure 6 Effect of oxalic acid on (a) root length (b) root surface and (c) Root volume Bars indicate standard error 119899 = 5

the transfer of bacteria from the nonrhizosphere to the rhi-zosphere zone as the rhizospheric zone is a source of organiccarbon which is needed for microbial activity Cannon etal [22] pointed out that there was a significant increase insoil and plant tissue P where oxalic acid was applied and isreadily degraded by microorganisms The increase in solubleP in the solution and increase in plant biomass proved thatapplication of organic acids along with PSB16 had a positiveeffect on plant and microbial growth

The organic acids with PSB16 and PR increased theplant biomass Besides P solubilization activity PSB liberatesphytohormone (IAA) that might have an influence on rootgrowth The extensive root system increased nutrient uptakefrom the surroundings which increased plant biomass [23]The organic acids serve as a source of carbon for the

microorganisms and subsequently affect the rhizospheremicrobial population as well as plant growth [24]

The application of organic acid PSB16 and PR slightlyaffected the soil pH values This could be due to the soilbuffering system and it did not affect much the change of soilpH The slight reductions were observed in the rhizospherethat could be due to the influence of organic acid applicationsThese results are consistent with the findings of Zeng et al[25] who reported that the organic acid have significantpositive correlation pH of the rhizosphere of rice plantsfurthermore when PR is added to soil (alfisols) mostlyorganic acids brought about a drop in pH for P released [26]

The plant root development in aerobic rice was affectedby the application of organic acids PSB and PR Externalapplication of organic acids along with PSB enhanced soluble


0

2

4

6

8

10


Root

leng

th (c

m)


(a)

0

05

1

15

2

25

3


Root

surfa

ce (c

m2)


(b)

0

001

002

003

004

005

006

007

008



Root

Vol

ume (

cm2)


(c)

Figure 7 Effect of malic acid on (a) root length (b) root surface and (c) root volume Bars indicate standard error 119899 = 5

P in the solution and this had a positive impact on rootgrowth These results are in agreement with the findings ofSrivastava et al [27] who reported that addition of organicacid with PR brought release of P and showed positiveeffect on plant growth Moreover Hoffland et al [28] foundorganic acids in root exudates which were highly efficientin increasing P release from PR The root development andplant biomass were correlated with the higher availabilityof P moreover PSB application may also have some otherbeneficial effects like phytohormones production

5 Conclusion

The application of organic acids and PSB16 showed differ-ences in P solubilization from PR Among both acids oxalicacid showed better results when compared to malic acidThe PSB16 population and soil pH were not affected by theapplication of organic acids Application of oxalic acid at20mM along with PSB16 significantly increased soluble Prelease from PR In conclusion addition of organic acidswith PSB increased the solubility of PR and had a significant


effect on the growth of aerobic rice However at the higherconcentration of oxalic acid application may present a healthrisk especially for children

Acknowledgments

The authors are grateful to Universiti Putra Malaysia andLongterm Research Grant Scheme (LRGS) fund for FoodSecurity providing the financial support for this project

References

[1] D L Jones ldquoOrganic acids in the rhizospheremdasha criticalreviewrdquo Plant and Soil vol 205 no 1 pp 25ndash44 1998

[2] R S Yadav and J C Tarafdar ldquoPhytase and phosphataseproducing fungi in arid and semi-arid soils and their efficiencyin hydrolyzing different organic P compoundsrdquo Soil Biology andBiochemistry vol 35 no 6 pp 745ndash751 2003

[3] N S Bolan R Naidu S Mahimairaja and S BaskaranldquoInfluence of low-molecular-weight organic acids on the solu-bilization of phosphatesrdquo Biology and Fertility of Soils vol 18no 4 pp 311ndash319 1994

[4] L Strom A G Owen D L Godbold and D L Jones ldquoOrganicacid mediated P mobilization in the rhizosphere and uptake bymaize rootsrdquo Soil Biology and Biochemistry vol 34 no 5 pp703ndash710 2002

[5] C R Chen L M Condron and Z H Xu ldquoImpacts of grasslandafforestationwith coniferous trees on soil phosphorus dynamicsand associatedmicrobial processes a reviewrdquoForest Ecology andManagement vol 255 no 3-4 pp 396ndash409 2008

[6] Y Wang Y He H Zhang J Schroder C Li and D ZhouldquoPhosphate mobilization by citric tartaric and oxalic acids ina clay loam ultisolrdquo Soil Science Society of America Journal vol72 no 5 pp 1263ndash1268 2008

[7] B B Jana ldquoDistribution pattern and role of phosphate sol-ubilizing bacteria in the enhancement of fertilizer value ofrock phosphate in aquaculture ponds State-of-the-artrdquo in FirstInternational Meeting on Microbial Phosphate SolubilizationE Velazquez and C Rodrıguez-Barrueco Eds pp 229ndash238Springer Salamanca Spain 2002

[8] R M N Kucey H H Janzen and M E Leggett ldquoMicrobiallymediated increases in plant-available phosphorusrdquo Advances inAgronomy vol 42 pp 199ndash228 1989

[9] N V Hue ldquoEffects of organic acidsanions on P sorptionand phytoavailability in soils with different mineralogiesrdquo SoilScience vol 152 no 6 pp 463ndash471 1991

[10] M M Hanafi J K Syers and N S Bolan ldquoLeaching effect onthe dissolution of two phosphate rocks in acid soilsrdquo Soil ScienceSociety of America Journal vol 56 no 4 pp 1325ndash1330 1992

[11] F E Khasawneh and E C Doll ldquoThe use of phosphate rock fordirect application to soilsrdquo Advances in Agronomy vol 30 pp159ndash206 1979

[12] S S S Rajan J H Watkinson and A G Sinclair ldquoPhosphaterocks for direct application to soilsrdquoAdvances in Agronomy vol57 pp 77ndash159 1996

[13] Q A Panhwar R Othman Z A Rahman S Meon andM R Ismail ldquoIsolation and characterization of phosphate-solubilizing bacteria from aerobic ricerdquo African Journal ofBiotechnology vol 11 no 11 pp 2711ndash2719 2012

[14] R H Bray and L T Kurtz ldquoDetermination of total organic andavailable forms of phosphorus in soilsrdquo Soil Science vol 59 pp39ndash45 1945

[15] J LHavlin andPN Soltanpour ldquoAnitric acid plant tissue digestmethod for use with inductively coupled plasma spectrometryrdquoCommunications in Soil Science and Plant Analysis vol 1 pp969ndash980 1980

[16] H El Zemrany S Czarnes P D Hallett S Alamercery R Ballyand L J Monrozier ldquoEarly changes in root characteristics ofmaize (Zea mays) following seed inoculation with the PGPRAzospirillum lipoferum CRT1rdquo Plant and Soil vol 291 no 1-2pp 109ndash118 2007

[17] P E A Asea R M N Kucey and J W B Stewart ldquoInorganicphosphate solubilization by two Penicillium species in solutionculture and soilrdquo Soil Biology and Biochemistry vol 20 no 4pp 459ndash464 1988

[18] L L Wei C R Chen and Z H Xu ldquoThe effect of low-molecular-weight organic acids and inorganic phosphorusconcentration on the determination of soil phosphorus by themolybdenum blue reactionrdquo Biology and Fertility of Soils vol45 no 7 pp 775ndash779 2009

[19] A Zaidi M S Khan M Ahemad M Oves and P A WanildquoRecent advances in plant growth promotion by phosphate-solubilizing microbesrdquo inMicrobial Strategies for Crop Improve-ment M S Khan A Zaidi and J Musarrat Eds pp 15ndash24Springer Berlin Germany 2009

[20] P Gyaneshwar G N Kumar and L J Parekh ldquoEffect of buffer-ing on the phosphate-solubilizing ability of microorganismsrdquoWorld Journal of Microbiology and Biotechnology vol 14 no 5pp 669ndash673 1998

[21] D L Jones and P R Darrah ldquoRe-sorption of organic com-pounds by roots of Zea mays L and its consequences in therhizosphere III Characteristics of sugar influx and effluxrdquoPlantand Soil vol 178 no 1 pp 153ndash160 1996

[22] J P Cannon E B Allen M F Allen L M Dudley and J JJurinak ldquoThe effects of oxalates produced by Salsola tragus onthe phosphorus nutrition of Stipa pulchrardquo Oecologia vol 102no 3 pp 265ndash272 1995

[23] U A Naher R Othman Z H Shamsuddin H M Saud M RIsmail and K A Rahim ldquoEffect of root exuded specific sugarson biological nitrogen fixation and growth promotion in rice(Oryza sativa)rdquoAustralian Journal of Crop Science vol 5 no 10pp 1210ndash1217 2011

[24] U A Naher O Radziah M S Halimi Z H Shamsuddin andI M Razi ldquoEffect of inoculation on root exudates carbon sugarand amino acids production of different rice varietiesrdquo ResearchJournal of Microbiology vol 3 no 9 pp 580ndash587 2008

[25] F Zeng S Chen Y Miao F Wu and G Zhang ldquoChanges oforganic acid exudation and rhizosphere pH in rice plants underchromium stressrdquo Environmental Pollution vol 155 no 2 pp284ndash289 2008

[26] J E Cunningham and C Kuiack ldquoProduction of citric andoxalic acids and solubilization of calcium phosphate by Peni-cillium bilaiirdquo Applied and Environmental Microbiology vol 58no 5 pp 1451ndash1458 1992

[27] S Srivastava M T Kausalya G Archana O P Rupela andG Naresh-Kumar ldquoEfficacy of organic acid secreting bacteriain solubilization of rock phosphate in acidic alfisolsrdquo in FirstInternationalMeeting onMicrobial Phosphate Solubilization 16ndash19 July 2002 E Velazquez and C Rodrıguez-Barrueco Eds pp117ndash124 Springer Salamanca Spain 2003


[28] E Hoffland G R Findenegg and J A Nelemans ldquoSolubiliza-tion of rock phosphate by rapemdashII local root exudation oforganic acids as a response to P-starvationrdquo Plant and Soil vol113 no 2 pp 161ndash165 1989

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Volume 2014

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Enzyme Research



Microbiology


0

5

10

15

20

25

30


Solu

ble P

(mg k

gminus1)

Noninoculated (+PR) Inoculated (+PR)Noninoculated (minusPR) Inoculated (minusPR)

(a)

0

5

10

15

20

25

30


Solu

ble P

(mg k

gminus1)



(b)

Figure 1 Effect of (a) oxalic acid (b) malic acid on P solubilization Bars indicate standard error 119899 = 5

characteristics pHH2

PO4

minus andCa2

+ [10] Dissolution of PRin acid soils can be explained as follows Khasawneh and Doll[11]

Ca10

(PO4

)6

F2

+ 12H+ 997904rArr 10Ca2

+

+ 6H2

PO4

minus

+ 2Fminus (1)

The equation specifies that the rate of dissolution isdetermined by the concentration of protons (H+) and theconcentration of reaction products of Ca

2

+ and H2

PO4

Inacidic soils improvement of P nutrition to plants by directapplication of PR as P fertilizer has been considered [12]Phosphate rocks are affluent in calcium phosphate complexesand are soluble in acidic soil environments Either microbialreleased organic acids or any acidic condition may favor Psolubilization from PR Hence the present study was under-taken to determine the effect of different rates of organic acidswith phosphate-solubilizing bacteria on P solubilization andtheir effect on growth of aerobic rice

2 Materials and Methods

The experiment was conducted under in vitro conditionPSB16 (Bacillus sp) isolated form aerobic rice rhizosphere[13] which was tested to produce indoleacetic acid P solubi-lization organic acids and siderophore production in in vitrocondition The isolated strain was identified using 16S rRNAgene sequencing with accession number JX103827 Four rateseach of oxalic and malic acids (0 10 20 and 30mM) andPR (Christmas Island Rock Phosphate) at 60 kg P

2

O5

haminus1were applied The aerobic rice (var M9) was grown in thegrowth chamber for 40 days Total bacterial populationsP solubilization soil pH root morphology and agronomicparameters were recorded after 40 days of growth

21 Seed Surface Sterilization Inoculation and Growth ofRice Seedlings The surface sterilized seven days old seedlingswere transplanted into pots containing sterilized soil (500 g)with 4 uniform seedlings per pot Plants were grown for 40

days in a growth chamber with 12 h lightdark cycle at 29 plusmn1∘C temperature Approximately 5times 109mLminus1 of live washedbacterial cells of Bacillus sp (PSB16) were used as inoculumin each bacterial treatment

22 Determination of Bacterial Population Plant Biomassand Plant Tissue P The total bacterial population was deter-mined from rhizosphere nonrhizosphere and endosphereof aerobic rice plants After harvest soil available P wasdetermined using Bray 2 [14] and total plant tissue P wasanalyzed by the wet digestion method [15]

23 Determination of Root Development The root length(cm) total surface (cm2) and root volume (cm3) were quanti-fied using a scanner (Expression 1680 Epson) equipped witha 2 cm depth plexiglass tank (20times 30 cm) filled with UP H

2

O[16]

24 Data Analysis The experiment was conducted with thethree factors in four replicates in a completely randomizeddesign Data obtained were statistically analyzed using theSAS software program (Version 92) and treatment meanswere compared using Tukeyrsquos test (119875 lt 005)

3 Results

31 Effect of Organic Acids and PSB on P Solubilization andPlant P Uptake Higher values of solubilized P were foundin PSB inoculated treatments with organic acids applicationSignificantly high amount of solubilized P (3151) wasfound in PSB inoculated treatments with 20mM oxalic acid(Figure 1(a))

Between the two acids P solubilization was found higherin oxalic compared to malic acid at various rates In malicacid treatments higher P solubilization (2157mg kgminus1) wasobserved with 10mM of malic acid in PSB inoculated treat-ments (Figure 1(b))The amount of P solubilization and plant


0

1

2

3

4

5

6

7

8

9



Without PRPo

pula

tion

(log10

cfu g

minus1)

(a)

0

1

2

3

4

5

6

7

8

9


With PR

Popu

latio

n (log10

cfu g

minus1)


(b)

0

1

2

3

4

5

6

7

8

9


Without PR

Popu

latio

n (log10

cfu g

minus1)


(c)

0

1

2

3

4

5

6

7

8

9


With PR

Popu

latio

n ( log10

cfu g

minus1)


(d)





10


10


10



0

5

10

15

20

25

30

35


Plan

t bio

mas

s (m

g)


(a)

0

5

10

15

20

25

30

35


Plan

t bio

mas

s (m

g)


(b)








10


10






60

62

64

66

68

70

72

10 20 30 40Days

pH

OA0OA10

OA20OA30


60

62

64

66

68

70

72

10 20 30 40Days

pH

OA0OA10

OA20OA30


60

62

64

66

68

70

72

10 20 30 40Days

pH

OA0OA10

OA20OA30


60

62

64

66

68

70

72

10 20 30 40Days

pH

OA0OA10

OA20OA30











60

62

64

66

68

70

72

10 20 30 40Days

pH

MA0MA10

MA20MA30


60

62

64

66

68

70

72

10 20 30 40Days

pH

MA0MA10

MA20MA30


60

62

64

66

68

70

72

10 20 30 40Days

pH

MA0MA10

MA20MA30


60

62

64

66

68

70

72

10 20 30 40Days

pH

MA0MA10

MA20MA30




4 Discussion






0

2

4

6

8

10


Root

leng

th (c

m)


(a)

0

05

1

15

2

25

3

35

4

45


Root

surfa

ce (c

m2)


(b)

0

002

004

006

008

01

012


Root

vol

ume (

cm3)









0

2

4

6

8

10


Root

leng

th (c

m)


(a)

0

05

1

15

2

25

3


Root

surfa

ce (c

m2)


(b)

0

001

002

003

004

005

006

007

008



Root

Vol

ume (

cm2)


(c)



5 Conclusion




Acknowledgments


References





































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

1

2

3

4

5

6

7

8

9



Without PRPo

pula

tion

(log10

cfu g

minus1)

(a)

0

1

2

3

4

5

6

7

8

9


With PR

Popu

latio

n (log10

cfu g

minus1)


(b)

0

1

2

3

4

5

6

7

8

9


Without PR

Popu

latio

n (log10

cfu g

minus1)


(c)

0

1

2

3

4

5

6

7

8

9


With PR

Popu

latio

n ( log10

cfu g

minus1)


(d)





10


10


10



0

5

10

15

20

25

30

35


Plan

t bio

mas

s (m

g)


(a)

0

5

10

15

20

25

30

35


Plan

t bio

mas

s (m

g)


(b)








10


10






60

62

64

66

68

70

72

10 20 30 40Days

pH

OA0OA10

OA20OA30


60

62

64

66

68

70

72

10 20 30 40Days

pH

OA0OA10

OA20OA30


60

62

64

66

68

70

72

10 20 30 40Days

pH

OA0OA10

OA20OA30


60

62

64

66

68

70

72

10 20 30 40Days

pH

OA0OA10

OA20OA30











60

62

64

66

68

70

72

10 20 30 40Days

pH

MA0MA10

MA20MA30


60

62

64

66

68

70

72

10 20 30 40Days

pH

MA0MA10

MA20MA30


60

62

64

66

68

70

72

10 20 30 40Days

pH

MA0MA10

MA20MA30


60

62

64

66

68

70

72

10 20 30 40Days

pH

MA0MA10

MA20MA30




4 Discussion






0

2

4

6

8

10


Root

leng

th (c

m)


(a)

0

05

1

15

2

25

3

35

4

45


Root

surfa

ce (c

m2)


(b)

0

002

004

006

008

01

012


Root

vol

ume (

cm3)









0

2

4

6

8

10


Root

leng

th (c

m)


(a)

0

05

1

15

2

25

3


Root

surfa

ce (c

m2)


(b)

0

001

002

003

004

005

006

007

008



Root

Vol

ume (

cm2)


(c)



5 Conclusion




Acknowledgments


References





































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

5

10

15

20

25

30

35


Plan

t bio

mas

s (m

g)


(a)

0

5

10

15

20

25

30

35


Plan

t bio

mas

s (m

g)


(b)








10


10






60

62

64

66

68

70

72

10 20 30 40Days

pH

OA0OA10

OA20OA30


60

62

64

66

68

70

72

10 20 30 40Days

pH

OA0OA10

OA20OA30


60

62

64

66

68

70

72

10 20 30 40Days

pH

OA0OA10

OA20OA30


60

62

64

66

68

70

72

10 20 30 40Days

pH

OA0OA10

OA20OA30











60

62

64

66

68

70

72

10 20 30 40Days

pH

MA0MA10

MA20MA30


60

62

64

66

68

70

72

10 20 30 40Days

pH

MA0MA10

MA20MA30


60

62

64

66

68

70

72

10 20 30 40Days

pH

MA0MA10

MA20MA30


60

62

64

66

68

70

72

10 20 30 40Days

pH

MA0MA10

MA20MA30




4 Discussion






0

2

4

6

8

10


Root

leng

th (c

m)


(a)

0

05

1

15

2

25

3

35

4

45


Root

surfa

ce (c

m2)


(b)

0

002

004

006

008

01

012


Root

vol

ume (

cm3)









0

2

4

6

8

10


Root

leng

th (c

m)


(a)

0

05

1

15

2

25

3


Root

surfa

ce (c

m2)


(b)

0

001

002

003

004

005

006

007

008



Root

Vol

ume (

cm2)


(c)



5 Conclusion




Acknowledgments


References





































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


60

62

64

66

68

70

72

10 20 30 40Days

pH

OA0OA10

OA20OA30


60

62

64

66

68

70

72

10 20 30 40Days

pH

OA0OA10

OA20OA30


60

62

64

66

68

70

72

10 20 30 40Days

pH

OA0OA10

OA20OA30


60

62

64

66

68

70

72

10 20 30 40Days

pH

OA0OA10

OA20OA30











60

62

64

66

68

70

72

10 20 30 40Days

pH

MA0MA10

MA20MA30


60

62

64

66

68

70

72

10 20 30 40Days

pH

MA0MA10

MA20MA30


60

62

64

66

68

70

72

10 20 30 40Days

pH

MA0MA10

MA20MA30


60

62

64

66

68

70

72

10 20 30 40Days

pH

MA0MA10

MA20MA30




4 Discussion






0

2

4

6

8

10


Root

leng

th (c

m)


(a)

0

05

1

15

2

25

3

35

4

45


Root

surfa

ce (c

m2)


(b)

0

002

004

006

008

01

012


Root

vol

ume (

cm3)









0

2

4

6

8

10


Root

leng

th (c

m)


(a)

0

05

1

15

2

25

3


Root

surfa

ce (c

m2)


(b)

0

001

002

003

004

005

006

007

008



Root

Vol

ume (

cm2)


(c)



5 Conclusion




Acknowledgments


References





































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


60

62

64

66

68

70

72

10 20 30 40Days

pH

MA0MA10

MA20MA30


60

62

64

66

68

70

72

10 20 30 40Days

pH

MA0MA10

MA20MA30


60

62

64

66

68

70

72

10 20 30 40Days

pH

MA0MA10

MA20MA30


60

62

64

66

68

70

72

10 20 30 40Days

pH

MA0MA10

MA20MA30




4 Discussion






0

2

4

6

8

10


Root

leng

th (c

m)


(a)

0

05

1

15

2

25

3

35

4

45


Root

surfa

ce (c

m2)


(b)

0

002

004

006

008

01

012


Root

vol

ume (

cm3)









0

2

4

6

8

10


Root

leng

th (c

m)


(a)

0

05

1

15

2

25

3


Root

surfa

ce (c

m2)


(b)

0

001

002

003

004

005

006

007

008



Root

Vol

ume (

cm2)


(c)



5 Conclusion




Acknowledgments


References





































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

2

4

6

8

10


Root

leng

th (c

m)


(a)

0

05

1

15

2

25

3

35

4

45


Root

surfa

ce (c

m2)


(b)

0

002

004

006

008

01

012


Root

vol

ume (

cm3)









0

2

4

6

8

10


Root

leng

th (c

m)


(a)

0

05

1

15

2

25

3


Root

surfa

ce (c

m2)


(b)

0

001

002

003

004

005

006

007

008



Root

Vol

ume (

cm2)


(c)



5 Conclusion




Acknowledgments


References





































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

2

4

6

8

10


Root

leng

th (c

m)


(a)

0

05

1

15

2

25

3


Root

surfa

ce (c

m2)


(b)

0

001

002

003

004

005

006

007

008



Root

Vol

ume (

cm2)


(c)



5 Conclusion




Acknowledgments


References





































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



Acknowledgments


References





































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology










Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

research article application of potential phosphate...

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