Combined application of bio-organic phosphate and phosphorus solubilizing bacteria (Bacillus strain MWT 14) improve the performance of bread wheat with low fertilizer input under an arid climate

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h tt p : / / w w w . b j m i c r o b i o l . c o m . b r /

Environmental

Microbiology

Combined

application

of

bio-organic

phosphate

and

phosphorus

solubilizing

bacteria

(Bacillus

strain

MWT

14)

improve

the

performance

of

bread

wheat

with

low

fertilizer

input

under

an

arid

climate

Muhammad

Tahir

a

_,

_Umaira

_Khalid

c

_,

_Muhammad

_Ijaz

b,∗

_,

_Ghulam

_Mustafa

_Shah

a

_,

Muhammad

Asif

Naeem

a

_,

_Muhammad

_Shahid

d

_,

_Khalid

_Mahmood

e

_,

_Naveed

_Ahmad

a

_,

Fazal

Kareem

c

a_COMSATS_Institute_of_Information_Technology,_Department_of_{Environmental}_Sciences,_Vehari,_Pakistan b_Bahauddin_Zakariya_University,_Bahadur_Sub-Campus,_College_of_Agriculture,_Layyah,_Pakistan c_Bahauddin_Zakariya_University,_Department_of_Agronomy,_Multan,_Pakistan

d_Government_College_University,_Department_of_{Bioinformatics}_and_{Biotechnology,}_Faisalabad,_Pakistan e_Aarhus_University,_Faculty_of_Science_and_Technology,_Department_of_{Agro-ecology,}_Aarhus,_Denmark

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received19April2017 Accepted12November2017 Availableonline24April2018 AssociateEditor:JerriZilli

a

b

s

t

r

a

c

t

Thisstudywasaimedtoinvestigatetheeffectofbio-organicphosphateeitheraloneorin combinationwithphosphorussolubilizingbacteriastrain(BacillusMWT-14)onthegrowth andproductivityoftwowheatcultivars(Galaxy-2013andPunjab-2011)alongwith recom-mended(150–100NPkgha−1)andhalfdose(75–50NPkgha−1)offertilizers.Thecombined applicationofbio-organicphosphateandthephosphoroussolubilizingbacteriastrainat eitherfertilizerlevelsigniﬁcantlyimprovedthegrowth,yieldparametersand productiv-ityofbothwheatcultivarscomparedtonon-inoculatedcontroltreatments.Thecultivar Punjab-2011 producedthehigherchlorophyllcontents,cropgrowthrate,andthestraw yieldathalfdoseofNPfertilizer; whileGalaxy-2013,with thecombinedapplicationof bio-organicphosphateandphosphoroussolubilizingbacteriaunderrecommendedNP fer-tilizer dose. Combined over both NP fertilizer levels, the combineduse of bio-organic phosphate andphosphorous solubilizing bacteria enhanced the grain yieldof cultivar Galaxy-2013by54.3%andthatofcultivarPunjab-2011by83.3%.Thecombined applica-tionofbio-organicphosphate andphosphoroussolubilizingbacteria alsoincreasedthe

∗ _{Corresponding}_author.

E-mail:muhammad.ijaz@bzu.edu.pk(M.Ijaz).

https://doi.org/10.1016/j.bjm.2017.11.005

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Keywords: Phosphorous Phosphoroussolubilizing bacteria Bioavailability Rhizosphere Lowfertilizerinput

populationofphosphoroussolubilizingbacteria,thesoilorganicmatterandphosphorous contentsinthesoil.Inconclusion,thecombinedapplicationofbio-organicphosphateand phosphoroussolubilizingbacteriaoffersaneco-friendlyoptiontoharvestthebetterwheat yieldwithlowfertilizerinputunderaridclimate.

licenses/by-nc-nd/4.0/).

Introduction

Breadwheat(TriticumaestivumL.)isconsumedasstapleby thebillionsacrosstheglobeowningtoitsgreatercaloriesand proteincontentsascomparedtothe othercereals.1,2

How-ever,theaverageyieldofbreadwheatperunitareaisverylow thanitsactualpotentialinmanydevelopingcountriesofthe worldincludingthePakistan.Manyfactorssuchasimproper sowingtime,poorseedquality,poorirrigation/nutrient man-agementandthelowsoilorganicmatter(SOM)contents,1–8

areresponsibleforthislowproductivityofbreadwheat. Amongthesefactors,theSOMisverycrucialasitaffects theavailabilityofplantnutrients.3_The_SOM_contents_of_about

3% are considered essential for the suitable soil physical condition,bettercationexchangecapacityandthenutrient availabilitytoobtainthehighercropyields.3,9 _However,_the

SOMcontentsofthePakistanisoilsare>0.5%owingtohigh temperature.3,10 _which_decreases_the_availability_of_nitrogen

and phosphorous dueto reductioninthe cation exchange capacity.11–13

Phosphorous is crucial element required for the plant growth and development.14–16 _The _unique _source_of

phos-phorous fertilizerisrock phosphatewhich isacomplex of phosphateandcalcium.17_However,_the_production_of

elemen-tal phosphorous, phosphoric acid, and other phosphorous basedfertilizerfrom therockphosphate isitselfanenergy consumingprocess.17–19

Inthisaspect,theconversionofrockphosphatethrough biochemical means into solubleand readily available state whichmaydirectlybefedtoplantsisausefultechnique.17

Thebestavailable,ecosystemfriendly,andcheapertechnique toconverttherockphosphateintoplantavailablephosphateis theuseofcompost(agriculturewaste)andbacteria.20–23_This

biochemicalprocesshaslowenergyinputdemandanddoes notrequirethechemicalcatalyst.Theendproductobtained in this process is rich in available phosphorous and SOM contents,17_with_optimum_carbon_(C)_and_N_ratio_(C:N).

Therhizosphericsoilisdominatedbythediversebacterial communities.24–28_These_bacterial_communities_are_involved

indevelopingthemutualinteractionwithplantrootsandthus improvethegrowthanddevelopmentofplantsthrough vari-ousmechanisms.19,29–31_These_mechanisms_include_biological

nitrogen ﬁxation (BNF), production of growth hormones, improvementintheavailabilityofthenutrientsparticularly phosphorous,andthebio-controlofplantpathogens.19,31,32

ThebacterialstrainsbelongingtogeneraAzospirillum, Azo-tobacter,Bacillus,Enterobacter,Pseudomonas andPantoea have beenisolatedfromtherhizosphereofvariouscropsandare reportedtoimprovetheplantgrowth.28,31,33–38_Among_these,

Bacillusiswellknowngenuswhosemultiplestrainspossess thepotentialtopromotethegrowthofwheat.28,31,36,39

As the bio-organic phosphate (BOP) is rich in soluble phosphorous, its addition to soil may improve the plant growth moreefﬁciently as compared withthe use of syn-thetic phosphorousfertilizersourcessuchasdiammonium phosphateandsinglesuperphosphate.Furthermore,theuse ofphosphatesolubilizingbacteria(PSB)alongwithsynthetic phosphorousfertilizermayimprovetheproductivityofcrops byincreasingthesolubilityofthephosphorousinthesoil.As theBOPisrichintheorganicmatter,itmayfurtherimprove theavailabilityofothernutrientstotheplantsandmaythus provide thesuitable conditionsforthesurvival, multiplica-tion and functioningofthebeneﬁcial rhizosphere bacteria. Thesebacteriainturnincreasethesoilfertilitythroughthe process of bio-mineralization.40 _The _PSB _solubilize_the _soil

phosphorousthroughreleasingtheorganicacids(e.g.acetic acid,formicacid,lacticacid,glycolicacidandsuccinicacids, etc.)andthecarboxylandhydroxylgroupsoftheorganicacids produced by PSB,chelate the phosphate bounded cations, therebyconvertingphosphorousintosolubleforms.41

AdditionofPSBstimulatestheprocessofphosphorous sol-ubilizationbothinsoilaswellasinBOP,asthephosphorous solubilizationismajoractivityofPSB.However,tothebestof ourknowledge,verylimitedinformationisavailableaboutthe effectofcombinedapplicationofBOPandPSBonsoilfertility aswellasonthegrowthandproductivityofcontrastingwheat cultivarsgrownatdifferentfertilizerlevels.Forthisstudy,we hypothesizedthattheuseofPSBalongwithBOPandNP fer-tilizermayenhancethesolublephosphoruswithinthesoil. Thespeciﬁcobjectiveofthisstudywastocheckthecombined effectofBOPandPSBonsoilfertilityandproductivityofbread wheatunderdifferentlevelsofNPfertilizer.

Materials

and

methods

Preparationofbioorganicphosphate

Bioorganicphosphate(enrichedwithavailablephosphorous and organic mattercontents)waspreparedusingcompost, biogasresidueandrockphosphatebyadoptingtheprocedure asdescribedbyNarayanan17_with_some_{modiﬁcations.}_For_this

purpose,rockphosphate(freefromsilicawith26%total phos-phorous)waspulverizedandreﬁnedtogetauniformsizeof 75microns.Thecompostwaspreparedaftercompostingof cropandfruitresiduesfor15days.Thebiogas residuewas obtainedfromalocalbiogasplantwhichusespoultrywaste andanimalwastei.e.cowdung.Thereafter,boththebiogas residuesandcompostwereair-driedintrayandgroundedin

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aballmill(CAMstarterTypeQS5-15A-500V 15A,China)to passthrougha1.0mmsieve.Thegroundmaterialwasmixed withoreofrockphosphateintheratioof1:2.Theblendwas thensuspendedinawaterstirredtankwithawater-solidratio of7:3.Thereactionwascarriedoutinagitatedstirredtank (bioreactor,CEMMkII,ArmﬁeldLtd,UK)usingtheconditions proposedbyNarayanan.17_Bacterial_culture_of_phosphate

sol-ubilizingbacterial(PSB)strainBacillusMWT-14,42_was_added_to

thereactorkeepingtheinoculumstrengthof107_cfu/mL_and

tankvolumeof3–5%.

Thewholeprocesswascarriedoutinthreesteps.Atthe ﬁrststep,slurry(mixtureofbiogasresidue,compostandrock phosphate)was addedslowly and continuouslytothe bio-reactor(CEMMkII,ArmﬁeldLtd,UK)toallowthefermentation bykeepingtankonagitation for7–10daysattemperature, pressure andpH conditionsas mentionedbyNarayanan.17

Afterthis step,PSB strain Bacillus MWT-14 was inoculated to the bioreactor and kept on continuous agitation for an extra5daystoallowthediffusionofatmosphericoxygen/air intothereactor.After5days,bacterialculture(107_cfu/mL)_of

nitrogenﬁxingbacteria Azospirillum(unpublished data)was inoculatedtothereactor;and thereactorwaskepton agi-tationforanextra5days.Afterthecomplete fermentation process,theproductwas ﬁlteredtoseparatethe solid por-tion.Thissolid portionwasdried,groundandanalyzedon spectrophotometertomeasuretheconcentrationofsoluble phosphoroususingtheproceduredescribedpreviously.43

Concentrationoforganicmatterinthesolidmaterialwas determined by using the procedure proposed by Allison44

withsomemodiﬁcations;2.0gweightofthesamplewasused insteadof1.0gin500mLconicalﬂaskand5.0mLofK2Cr2O7

wasaddedtoavoidtheearlycolorchange.Thisﬁnalproduct wasnamedasBOP(16.5%P2O5and25%organicmatterwith

19:1C:Nratio).

Preparationofinoculumandﬁeldexperimentation

BacterialstrainBacillussp.MWT-14(accessionno.KT933232) with known phosphate solubilization and growth promot-ing potential,42 _was _inoculated _to _nutrient _broth ₍₂₅₀_mL)

and was kept on shaking (150rpm) for 24h at a temper-ature of 30±2◦C. Inoculum strength was determined on spectrophotometerbymeasuringthe opticaldensityofthe grownbacterialcultureandwasmaintainedat107_cfu_mL–1_.

Further,we re-determined the strength ofinoculum using serialdilutionplatecountmethod.31_The_reason_for_selecting

theBacillus sp.MWT-14 wasthat this strainshowed maxi-mumphosphoroussolubilizationactivityandimprovedthe wheatgrowthinourpreviousstudy.42_The_BOP_@₁₂₅_kg_ha−1

andPSBstrainwereappliedassoletreatmentaswellasin combinedformalongwithrecommended(150–100NPkgha−1, respectively)andhalfoftherecommended(75–50NPkgha−1, respectively)dosesintwobreadwheatcultivars(Galaxy-2013 andPunjab-2011).Urea(46%N)anddiammoniumphosphate (46%P,18%N)wereusedasourceofnitrogenand phospho-rous,respectively.Potassiumwasappliedatauniformrate of100K2Okgha−1 inall experimentalplotsusing sulphate

ofpotassium(K2SO4)assource.ThefulldoseofBOP,andK

wassoilappliedatthetimeofsowing.However,nitrogenwas

appliedinthreeequalsplitsatsowing,tilleringand dough stage.

Theexperimentwasconductedduringthe year2015–16 attheexperimentalfarmofCollegeofAgriculture,Bahauddin ZakariyaUniversity,BahadurSub-Campus,Layyah(latitudeof 30.9;longitudeof70.9andaltitudeof143mabovesealevel). Theexperimentwaslaidoutinrandomizedcompleteblock design(RCBD)infactorialarrangementwiththreereplications maintainingagrossplotsizeof4m×1.8m.

Forbacterialinoculation,thebacterialcultureofthestrain

Bacillussp.MWT-14wasmixedwithsterilizedﬁlter-mudand the seeds of boththe bread wheat cultivarswere pelleted withthismixturecontainingsterilizedﬁlter-mudandinoculla (107_cfu/g_of_carrier)._Inoculated_seeds_were_sown_at_a_rate_of

120kgha−1usingamanualhanddrill.

All theotheragronomic practiceswere keptuniformfor alltheexperimentaltreatments.Physico-chemical character-isticsofexperimentalsoilweredeterminedbeforeconducting andafterharvestingtheexperiment.Siltloamtexturedsoil, with EC 2.8dsm−1, pH 7.9, organic matter 0.33%,available phosphorouscontents 9.0mgkg−1 ofdry soil,andavailable Ncontents0.083%,wasusedinthisstudy.Themagnesium contentsofthePakistanisoilsare1000–8500ppm.

Recording

of

data

Populationofphosphatesolubilizingbacteria

Thesoilsamplesofeachexperimentalunitwerecollectedin sterilizedplasticbagsfromtherhizosphereofwheatat dif-ferentgrowthstagesi.e.tillering[at30–40daysaftersowing (DAS)], booting (60–70 DAS)and grain ﬁlling stage(100–120 DAS), and werestored at4◦C forfurtherprocessing. Popu-lationofPSBwasmeasuredusingplatecountserialdilution method.45_For_this_purpose,_0.1_g_of_the_collected_rhizosphere

soil(ofeachexperimentalunit)wassuspendedintesttube containing9.0mLsterilizedsalinesolution(0.8%,w/v).Serial dilutionswerepreparedupto10−5 and100␮Lofeach dilu-tion was spread on the plates containing Pikovskaya agar medium,46_supplemented_with_tri-calcium_phosphate_(TCP)_as

insolublephosphoroussource.Theplateswereincubatedat 30±2◦Cfor48–72h(h).Thenumbersofcolonyformingunits (cfu)showingtransparenthalozoneformation(indicates sol-ubilizationofinsolublephosphorous)were countedandlog valueswereobtainedtogetthebacterialpopulation(cfu/gdry soil).

Cropallometry

Data onplantgrowth parametersviz.leafarea index(LAI), cropgrowthrate(CGR),netassimilationrate(NAR)and the chlorophyllcontentswererecordedfortnightlystartingfrom 40DASofthewheatcrop.Ahomogenousarea(0.5m2₎_was_cut

randomlytorecordthegrowthparameters.TorecordtheLAI, totalareaoftheseparatedleaveswasmeasuredbyusingaleaf areameter(DTAreaMeter,ModelMK2,DeltaTDevices, Cam-bridge,UK).TheLAIwascalculatedbydividingthetotalareaof theleavestothetotalgroundareawhichhadbeenharvested forthesampling.DataonCGR,wasrecordedafterharvesting

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thecropsamplesfromanareaof0.5m2_at₁₅_days_interval_and

wereweighedfreshlyandthenoven-driedat70±5◦Cfor48h. Thereafter,CGRandNARwerecalculatedbyfollowingthe pro-tocolasdescribedbyHunt.47_The_chlorophyll_contents_were

measuredusingthechlorophyll meter(SPAD502,Spectrum Technologies,Inc,Aurora,IL)ateach15daysinterval.

Morphologicalandyieldparameters

Atmaturity,spikebearingtillers(fertileorreproductivetillers) fromanareaof1m2_area_of_each_plot_were_counted.

There-after,twentyplantsfromeach plotwererandomlyselected tomeasuretheplantheight,averagenumberofspikeletsper spikeand number of grainsper spike. Thebiological yield oftheeachtreatmentwasmeasuredbyharvestingthearea of3.0m2 _from _the _each _plot. _The _harvested _samples_were

sun-dried for a week, and were weighed. To measure the grainyield,thedriedbundleswere threshedandthegrains were separated from the straw. Theseparated grains were weighedand the grainweightwasrecorded.From seedlot ofeachplot,threerandomsamplesof1000grainswere col-lected,weighedand averaged togetthe1000-grainweight. Thestrawyieldwascalculatedbysubtractingthegrainyield fromthe biologicalyield.Theharvestindexwascalculated astheratioofgrainyieldtothebiologicalyieldexpressedin percentage.

Statisticalanalysis

Thecollecteddatawasstatisticallyanalyzedusingthe soft-ware “Statistix (8.1version, Tallahassee, Florida)” using the ﬁsheranalysisofvariancetechnique.Thedifferenceamong the treatments were compared using the least signiﬁcant differencetestat5%probabilitylevel.48 _The_co-relation

co-efﬁcientswere drawntoobservetherelationshipofgrowth parametersofbreadwheatcultivarswiththe grainyieldof wheatusingthesoftware“Statistix(8.1version,Tallahassee, Florida)

Results

Cropallometry

Sole or combined application of BOP or PSB strain Bacillus

MWT-14withrecommendeddoseofNP(150–100NPkgha−1), and halfoftherecommendeddoseofNP(75–50NPkgha−1) fertilizersigniﬁcantly(p≤0.05)increasedtheLAI,CGR,NAR and thechlorophyll contentsofbothbreadwheatcultivars thantheirrespectivenon-inoculatedcontrol(Figs.1–4).

In Galaxy-2013, the highest LAI was recorded with BOP+PSBtreatmentalongwiththerecommendedNP fertil-izer(i.e.NPat150–100kgha−1);inPunjab-2011,theLAIwas themaximumwithBOP+PSBalongwiththehalfofthe recom-mendeddoseofNPfertilizer(75–50kgha−1)at90DAS(Fig.1). InGalaxy-2013,thehighestCGRwasrecordedwithBOP+PSB along with recommended NP fertilizer (150–100kgha−1);in Punjab-2011,theCGRwasthemaximumwithBOP+PSBalong withrecommendeddoseofNPfertilizer(150–100kgha−1),and halfoftherecommendeddoseofNPfertilizer(75–50kgha−1) at90DAS(Fig.2).Likewise,inGalaxy-2013,thehighestNAR was recorded withBOP+PSB along withthe recommended dose of NP fertilizer (150–100kgha−1); in Punjab-2011, the NARwasalmostthesimilarinBOP+PSBalongwith recom-mended(NPat150–100kgha−1)andhalfoftherecommended dose of NP fertilizer (75–50kgha−1) (Fig. 3). Similarly, in Galaxy-2013,thehighestchlorophyllcontentswererecorded with BOP+PSB along with the recommended dose of the NPfertilizer(150–100kgha−1);inPunjab-2011,thechlorophyll contentswerealmostsimilarwithBOP+PSBalongwith rec-ommended (150–100kgha−1)andhalf ofthe recommended doseoftheNPfertilizer(75–50kgha−1)(Fig.4).AtagivenNP level,Galaxy-2013showedrelativelygreaterLAI,CGR,NARand chlorophyllcontentsthanPunjab-2011(Figs.1–4).

PopulationofPSB

Population in terms of colony forming units (cfu) of PSB recorded at three different growth stages i.e. tillering,

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 F1 F2 F1 F2 F1 F2 F1 F2 F1 F2 F1 F2

60 DAS 75 DAS 90 DAS 60 DAS 75 DAS 90 DAS

Galaxy-2013 Punjab-2011 Le a f a re a i nd ex Treatments

Non-inoculated PSB BOP PSB+BOP

Fig.1–Leafareaindexofbreadwheatcultivarsasinﬂuencedbybio-organicphosphate,PSBanddifferentlevelsofNP chemicalfertilizers;F1=halfoftherecommendedNPfertilizerlevel(N–Pat75–50kgha−1);F2=recommendedNPfertilizer level(N–Pat150–100kgha−1);PSB=phosphatesolubilizingbacteria;BOP=bio-organicphosphate.

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0 10 20 30 40 50 60 70 F1 F2 F1 F2 F1 F2 F1 F2 F1 F2 F1 F2

60 DAS 75 DAS 90 DAS 60 DAS 75 DAS 90 DAS

Galaxy-2013 Punjab-2011 Cr o p g ro wt h r a te (g m -2 da y -1) Treatments

Fig.2–Cropgrowthrateofbreadwheatcultivarsasinﬂuencedbybio-organicphosphate,PSBanddifferentlevelsofNP chemicalfertilizers;F1=halfoftherecommendedNPfertilizerlevel(N–Pat75–50kgha−1);F2=recommendedNPfertilizer level(N–Pat150–100kgha−1);PSB=phosphatesolubilizingbacteria;BOP=bio-organicphosphate.

0 1 2 3 4 5 6 7 8 9 10 N on -i n ocu la te d PS B BO P PS B+ BO P N on -i n ocu la te d PS B BO P PS B+ BO P

Galaxy-2013 Punjab-2011

Net a ss im il ati o n r a te (g m -2 da y -1) Treatments F1 F2

Fig.3–Netassimilationrateofbreadwheatcultivarsasinﬂuencedbybio-organicphosphate,PSBanddifferentlevelsofNP chemicalfertilizers;F1=halfoftherecommendedNPfertilizerlevel(N–Pat75–50kgha−1);F2=recommendedNPfertilizer level(N–Pat150–100kgha−1);PSB=phosphatesolubilizingbacteria;BOP=bio-organicphosphate.

-5 0 5 10 15 20 25 30 35 40 45 50 F1 F2 F1 F2 F1 F2 F1 F2 F1 F2 F1 F2

40-55 DAS 55-70 DAS 70-85 DAS 40-55 DAS 55-70 DAS 70-85 DAS

T o ta l ch lo ro ph y ll content ind ex Treatments

Fig.4–Totalchlorophyllcontentsofbreadwheatcultivarsasinﬂuencedbybio-organicphosphate,PSBanddifferentlevels ofNPchemicalfertilizers;F1=halfoftherecommendedNPfertilizerlevel(N–Pat75–50kgha−1);F2=recommendedNP fertilizerlevel(N–Pat150–100kgha−1);PSB=phosphatesolubilizingbacteria;BOP=bio-organicphosphate.

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0.0 2.0 4.0 6.0 8.0 10.0 12.0 F1 F2 F1 F2 F1 F2 F1 F2 F1 F2 F1 F2

Tillering Booting Grain filling Tillering Booting Grain filling

B a c te ra il p o p u la tio n ( c fu /g o f s o il) Treatments

Fig.5–Populationdynamicsofphosphatesolubilizingbacteriainrhizosphereofbreadwheatcultivarsasinﬂuencedby bio-organicphosphate,PSBanddifferentlevelsofNPchemicalfertilizers;F1=halfoftherecommendedNPfertilizerlevel (N–Pat75–50kgha−1);F2=recommendedNPfertilizerlevel(N–Pat150–100kgha−1);PSB=phosphatesolubilizingbacteria; BOP=bio-organicphosphate.

booting and grain filling stageshowed anincreasing trend fromtilleringtobooting,anddecreasedatgrainfillingstage forall theexperimentaltreatments(Fig.5). InGalaxy-2013, thehighestpopulationofPSB(averagedoverthreesampling stages)was recordedwithBOP+PSBalong withthe recom-mendeddoseofNPfertilizer(150–100kgha−1);inPunjab-2011, thepopulationofPSB(averagedoverthreesamplingstages) wasalmostsimilarwithBOP+PSBalongwithrecommended (NPat150–100kgha−1)andhalfoftherecommendeddoseof NPfertilizer(75–50kgha−1).Overall,thepopulationofPSBwas thehighestatbootingstage(8.67cfupergramsoil)followedby grainfilling(6.0cfupergramsoil)andtilleringstage(5.36cfu pergramsoil)(Fig.5).

Morphologicalandyieldparameters

Thisstudyindicatedthatthree-wayinteractionofbreadwheat cultivarswithfertilizerlevelsandthePSBwassigniﬁcantfor allthe morphological/yieldparameters,grain yield,harvest index,SOM,soilphosphorousandsoilN(Table1).Atagiven NPlevel,soleorcombinedapplicationofthePSB orBOP to boththewheatcultivarsshowedanimprovementinthe mor-phological/yieldparameters,grainyield,harvestindex,SOM, andthesoilphosphorouswithrespecttotheirnon-inoculated control(Table1).

The highest plant height, number of productive tillers, spikeletsperspike,grainsperspike,1000-grainweight, bio-logicalyield,strawyieldandtheharvestindexwasrecorded withtheBOP+PSB,inGalaxy-2013andPunjab-2011,withthe recommended (NP at 150–100kgha−1) and half ofthe rec-ommendeddoseofNPfertilizer(75–50kgha−1)respectively. Howeverthistreatmentcombinationwasstatisticallysimilar withthesole applicationofBOP alongwiththehalf ofthe recommendeddoseofNPfertilizer(75–50kgha−1)in Punjab-2011forplantheight;combinedapplicationofBOP+PSBalong withtherecommendeddoseofNPfertilizer(150–100kgha−1) inPunjab-2011forgrainperspike,1000-grainweight, biolog-icalyield,grainyield,strawyieldandharvestindex;andsole

applicationofPSBalongwithhalfoftherecommendeddose ofNPfertilizer(75–50kgha−1)inPunjab-2011fortheharvest index(Table1).

Soilproperties

Thisexperimentrevealedsigniﬁcant(p<0.05)increaseinSOM andsoilphosphorouscontentsduetosoleorcombined appli-cation of BOP and PSB over non-inoculated control plots

(Table 1). The highest SOM was recorded with the

appli-cation of BOP+PSB along with the recommended (NP at 150–100kgha−1)and half of the recommended dose of NP fertilizer (75–50kgha−1) in both of bread wheat cultivars. In both bread wheat cultivars, the phosphorous contents were the maximum with recommended dose of NP fer-tilizer (150–100kgha−1) and that was statistically similar with the half of the recommended dose of NP fertilizer (75–50kgha−1)inGalaxy-2013.Althoughthedifferenceswere non-signiﬁcant,thehighestsoilNcontentswerenotedwith combinedapplicationofBOPandPSBathighNPfertilizer lev-elsinbothbreadwheatcultivars(Table1).

ThecorrelationpredictedthattheLAI,CGRandthe chloro-phyll contentswere stronglypositively correlated with the grainyieldofwheatatbothfertilizerlevelsinbothofthebread wheatcultivars(Table2).

Discussion

In the present study, soil properties were substantially improved with the application of BOP along with the PSB whichwasvisiblethroughimprovementinsoilOMandsoil phosphorous contents.Thisimprovementin soil phospho-rouswasduetopositiveinﬂuenceofPSBwhich ultimately enhancedthesolubilityandreleaseofphosphorousfromNP fertilizer inthe rhizosphere ofthe bothbread wheat culti-vars.Severalpreviousstudieshavereportedimprovementin SOMcontentsandnutrientbalanceduetosupplementation

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Table1–Plantheight,numberofproductivetillers,spikeletsperspike,grainsperspike,1000-grainyield,biological yield,grainyield,strawyield,harvestindexoftwowheatcultivars,andthesoilorganicmatter,phosphorousand nitrogenatwheatharvestasaffectedbytheapplicationofbio-organicphosphateandphosphoroussolubilizingbacteria.

Treatments Galaxy-2013 Punjab-2011 Galaxy-2013 Punjab-2011 Galaxy-2013 Punjab-2011

N–P(kgha−1₎ _N–P_(kg_ha−1₎ _N–P_(kg_ha−1₎ _N–P_(kg_ha−1₎ _N–P_(kg_ha−1₎ _N–P_(kg_ha−1₎

75–50 150–100 75–50 150–100 75–50 150–100 75–50 150–100 75–50 150–100 75–50 150–100

Plantheight(cm) Numberofproductivetillers(m−2₎ _Spikelets_per_spike

Control 93.3e 94.6de 87.8a–c 85.3bc 257e 311c–e 98b 89b 15.3g 16.5ef 14.8d 15.2c

PSB 97.9c–e 104.1bc 83.3c 90.1a–c 336b–d 303de 123b 116b 16.1f 18.1b 15.9c 15.9c

BOP 102.6b–d104.6bc 92.5ab 87.1a–c 362bc 373b 122b 117b 16.8de 17.2cd 15.7c 15.5c

PSB+BOP 107.7b 123.1a 94.5a 87.3a-c 390b 474a 174a 109b 17.7bc 20.2a 18.0a 17.5b

LSD(p≤0.05) 9.3 7.9 38.2 44.0 0.59 0.50

Grainsperspike 1000-grainweight(g) Biologicalyield(Mgha−1)

Control 30.6g 33.0ef 29.0b 29.6b 44.7e 47.9c–e 38.7c 40.6bc 5.5d 6.4cd 5.1cd 5.4c

PSB 32.2f 36.2b 31.4ab 31.8ab 46.9de 52.7b 41.2b 42.2ab 6.8c 7.6bc 5.9bc 6.4b

BOP 33.6de 34.4cd 31.0ab 31.4ab 49.1b–e 50.1b–d 44.1ab 42.9ab 6.9c 7.6bc 6.0b 6.2b

PSB+BOP 35.4bc 40.4a 32.0a 32.0a 51.6bc 58.9a 46.5a 46.7a 7.9b 8.9a 7.6a 7.7a

LSD(p≤0.05) 0.59 0.50 4.57 4.00 0.43 0.41

Grainyield(Mgha−1) Strawyield(Mgha−1) Harvestindex(%)

Control 2.5bc 3.0b 2.0b 2.2ab 3.0cd 3.4c 3.1bc 3.2bc 45.5e 46.9d 39.2d 40.7c

PSB 3.3b 3.8ab 2.5ab 2.8ab 3.5bc 3.8b 3.4ab 3.6ab 48.5c 50.0ab 42.4a 43.8.ab

BOP 3.4b 3.8ab 2.5ab 2.7ab 3.5bc 3.8b 3.5ab 3.5ab 49.3b 50.0ab 41.7b 43.5b

PSB+BOP 4.0ab 4.5a 3.8a 3.9a 3.9b 4.4a 3.8a 3.8a 50ab 50.6a 50.0a 50.6a

LSD(p≤0.05) 0.43 0.43 0.42 0.44 0.50 0.42

Soilorganicmatter(%) Phosphorous(mgkg−1) Nitrogen(%)

Control 0.33d 0.33d 0.33c 0.32c 9.1cd 9.0cd 9.2c 9.1d 0.083 0.083 0.081 0.082

PSB 0.38c 0.39bc 0.39bc 0.38bc 9.3c 9.5b 9.5c 9.3c 0.085 0.086 0.085 0.084

BOP 0.40bc 0.41b 0.41b 0.39bc 9.5b 9.5b 9.5c 9.2c 0.085 0.085 0.086 0.084

PSB+BOP 0.45a 0.48a 0.46a 0.47a 9.8ab 10.3a 9.9b 10.3a 0.095 0.099 0.098 0.099

LSD(p≤0.05) 0.03 0.02 0.2 0.3 NS NS

Figuresofmaineffectsandinteractionssharingthesamecaseletterdonotdiffersigniﬁcantlyatp≤0.05;BOP,bio-organicphosphorous;PSB, phosphoroussolubilizingbacteria;N,nitrogen;P,phosphorous.

ofsoilwithbio-organiccompounds,23,49–51_and_PSB._However,

mostofthestudiesarerestrictedtosoleapplicationofPSB orbio-organicsforsoilreconditioningandcropproductivity. Inthe presentstudy,the combinedapplicationofBOP and PSB atagivenfertilizer level wasa betterchoicethan the soleapplicationofBOPandPSBinboththebreadwheat cul-tivars. Thiswas dueto thefact that BOP and PSB showed synergisticbehavior,improved theperformanceand multi-pliedthe effectofeachother. Indeed,the PSB improvethe efﬁciencyoftheBOPbyimprovingthePavailability,andBOP providestheoptimumconditionsforthemultiplicationofPSB (increasedpopulationofPSB5.61–9.83cfu/gdry soil;Fig. 5).

Moreover, the bio-organics(e.g. compost) actsasthe main sourceofeasilyavailablenutrients,substanceswithgrowth promoting potential and improves the population ofplant growth promoting rhizobacterialike N ﬁxers,phosphorous solubilizerand cellulosedecomposers,23,52 _as_was_observed

inthisstudy.ThepopulationofPSBwassigniﬁcantlyhigher withthecombinedapplicationofBOPandPSBirrespectiveof theNPfertilizerdose.Indeed,theapplicationofBOPenriched with organic carbon providedthe sourceofenergy to bac-teria and increasedthemultiplication rateofrhizobacteria due towhich the population ofPSB was enhancedin this study.

Table2–Correlationco-ofﬁciantsofleafareaindex,chlorophyllcontentsandcropgrowthratewiththegrainyieldoftwo breadwheatcultivarsasaffectedbyapplicationofPGPRsatrecommendedandhalfdoseoffertilizer.

Treatments Galaxy-2013 Punjab-2011

N–P(kgha−1) N–P(kgha−1)

75–50 150–100 75–50 150–100

Leafareaindex 0.99 0.82 0.99 0.82

Chlorophyllcontents 0.86 0.97 0.88 0.95

Cropgrowthrate 0.93 0.90 0.82 0.81

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Thisstudy indicated that the chlorophyll contents, LAI, CGRandNARofboththebreadwheatcultivarsunderanarid climateweresigniﬁcantlyimprovedwiththeapplicationof BOP andPSB;the combinedapplicationbeing most beneﬁ-cial.Indeed,theapplicationofBOPtothesoilimprovesthe availabilityanduptakeofthenutrientslikeN,phosphorous, KandZntothegrowingplantswhichultimatelystimulates theplantgrowthwhichwasvisiblethroughimprovementin CGR,LAI and NAR inthis study.Moreover, the inoculation ofbacterialstrainBacillusMWT-14(withknownphosphorous solubilizationandIAAproducingactivity;Hussainetal.42₎_also

multipliedthe effect ofBOP byincreasingthe PSB popula-tion(Fig.5)andphosphoroussolubilitywhichcontributedto improvedperformance ofboth breadwheat cultivars. Sev-eralpaststudieshavereportedtheimprovedactivityandan increaseinthe population ofPGPRsinthe soilswithgood SOMandthepivotalroleofPGPRsinimprovingthe phospho-roussolubilization,18-20,49-56_which_ultimately _resulted_in_an

increaseinthecholrophyllcontents,57,58_the_LAI,_CGR_and_NAR

inthisstudy.

AtagivenNPlevel,Galaxy-2013showedrelativelygreater LAI,CGR, NARand chlorophyll contentsthan Punjab-2011. ThisbetterperformanceofGlaxy-2013than Punjab-2011in termsofgrowthparametersmightbeduetoinheritedgenetic potentialofthiswheatcultivartoproducemorebiomassata givenNPlevels.

Inour study, the variable responseof bread wheat cul-tivars to the BOP and PSB treatments were observed. The cultivar Galaxy-2013 performed better with the combined applicationofBOPandPSBatrecommendedNPfertilizerlevel (150–100kgha−1);theperformanceofPunjab-2011wasalmost similaratrecommendedandhalfoftherecommendeddoseof NPfertilizer.ThisindicatesthatthecultivarPunjab-2011can begrownatlowfertilizerinputwiththeuseofBOPandPSB withoutanycompromiseonthegrainyield.

ImprovementinLAI,CGRandNARﬁnallyresultedin bet-terplantheightofbothbreadwheatcultivars.Highestplant heightandthemoreLAIandCGRduetothecombined appli-cationofBOPandPSBultimatelyenhancedthetotalbiomass andstrawyieldofbothbreadwheatcultivars.Bettergrowth attributesduetocombinedapplicationofBOPandPSBalso resultedinthe morenumber ofproductivetillersand bet-terallocation ofphotosynthatesfrom sourcetosinkwhich enhancedthegrainsperspikeand1000-grainweight.More numberofproductivetillersandimprovedgrainsperspike and1000-grainweightthusproducedthehighestgrainyield. Improvementingrowthandyieldattributesduetodueto inoc-ulationwithPGPR arewelldocumentedinseveralprevious studies.28,30,31,37,38,59,60

Conclusion

Thesoilfertility,growthandproductivityofbreadwheat cul-tivarswassigniﬁcantlyincreasedwiththeapplicationofBOP orPSB;theeffectsbeingmorepronouncedwiththecombined applicationoftheboth.Indeed,theapplicationofBOPandPSB enhancedthesoilSOMandsoilphosphorouswhichworked assoilconditionerthusimprovingthegrowthofbothbread wheatcultivarswhich wasvisiblethroughimprovementin

LAI,CGRandNAR.Improvedgrowthofthebothbreadwheat cultivarsduetocombined applicationofBOP and PSB ulti-mately enhanced the morphological and yield parameters (number ofproductivetillers,1000-grainweight,grains per spike)whichultimately enhancedthegrainyieldatagiven fertilizer level. Beinga stresstolerant cultivar,Punjab-2011 performedefﬁcientlyduetothecombinedapplicationofBOP andPSBatthehalfoftherecommendedlevelofNPfertilizer. Incontrast,Galaxy-2013showedpositiveresponseintermsof plantgrowthandgrainyieldatarecommendeddoseofNP fer-tilizerlevel.ItisrecommendedthattheBOPshouldbeapplied incombinationwithPSBtomaximizethebeneﬁts.Thus,the combineduseofBOPandPSBatlowfertilizerinputmightbe quiteusefultoincreasethesoilfertility,thecropgrowthand theproductivityofbreadwheat.Thiswillleadtoward elimi-nationoftheenvironmentalpollutionthroughminimizingthe manufacturinganduseofthesyntheticfertilizers.

Conﬂicts

of

interest

Theauthorsdeclarenoconﬂictsofinterest.

Acknowledgements

ThetechnicalassistanceofMr.AliAhmad(LabAssistant)in performinglabworkatBZU,Multanisacknowledged.Special thankstoMr.Wazeer,Zeshan,AsgharandHusnainfortheir kindhelpinconductingﬁeldexperimentsanddatacollection. IamgratefultomanagementteamofHexonGroupof Com-panies,Multanforprovidingthematerialtoprovidethelab facilitiesforformulatingthebio-organics.

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