Impact of Phosphate Solubilizing Bacteria and Phosphorus Application on Forage Yield and Quality of Berseem in West BengalD.C.Roy1,M.Ray2,N.K.Tudu3andC.K.Kundu2

1Department of ILFC, WBUAFS, 37 K.B. Sarani, Kolkata-700037, India. 2Department of Agronomy, Bidhan Chandra Krishi Viswavidyalaya, Kalyani-741235, Nadia, West Bengal, India.3Nadia Krishi Vigyan Kendra, Bidhan Chandra Krishi Viswavidyalaya, Mohanpur, Nadia-741252, West Bengal, India.

Corresponding author: dcroy09@gmail.com

Paper No. 323 Received: 2 September 2014 Accepted: 6 May 2015 Published: 29 June 2015

Abstract

Phosphorusavailabilityisincreasedthroughtheuseofphosphatesolubilizingbacteriaforenhancingtheforageyieldandimprovingthequalityofberseemfodder.AfieldexperimentwasconductedinthefodderfarmofWestBengalUniversityofAnimalandFisherySciences,Belgachiaduringrabi,season2013-14. The cropwas sownwith inoculated (I1) andun-inoculated (I0) seeds of berseem (Trifolium alexandrinumL.)of varietywardenandwasgivenphosphorusfertilizeratthelevels40(P1),60(P2),80(P3)and100(P4)kgP2O5ha-1intheformofSSPinRCBDwiththreereplicationsinplotsofnetsize6mX2m.PSBinoculationsignificantlyincreasesgreenforageanddrymatteryieldbyincreasingplantheight,no.ofbranches,leaf-stemratio,etc.AllthequalitytraitsexceptashcontentanddrymatterpercentageweresignificantlyaffectedbyPSBinoculation.Highergreenforageyield(320.4qtha-1),drymatteryield(39.2qtha-1),crudeproteinyield(7.89qtha-1),crudeproteinpercentage(19.28%),ethylextract(3.32%)andnitrogenfreeextract(41.75%)wererecordedfromtheplotsinoculatedwithPSB.Applicationofphosphorusfertilizerssignificantlyinfluencedthegreenforageanddrymatteryieldofberseem,leaf-stemratio,etc.thoughplantheightandno.ofbrancheswerenot-significant.Allthequalityparameters i.e.CP,CF,EE,NFE,exceptashpercentageweresignificantlyinfluencedbyphosphorusapplication.Best resultswereobtainedwith80kgP2O5ha-1 in all the cases.Regarding interactioneffect,higheryieldsofbetterqualitygreen forageofberseemwereobtained in I1P3 i.e. inoculationwithPSBwithphosphorusapplicationof80kgP2O5ha-1.

Highlights

• Phosphatesolubilizingbacteriaenhancestheforageyieldandimprovethequality• Phosphorusapplicationattherate80kgP2O5ha-1gavebetterresult

Keywords:PSB,phosphorus,berseem,crudeprotein,crudefibre

Foddercropsplayavitalroleinagriculturebecause,thesupplyofnutritiousfoddersinsufficientamountis a basic requirement for livestock to fulfill theincreasingdemandofmilk, butter and other dairybyproductsforutilizationbyhumanbeings.Dueto

ever increasinghumanpopulationpressure, arablelandmainly used for food and fodder productionis limited only to 4.60% of the total cultivableland. At present, the country faces a net deficitof 35% green fodder, 10% dry crop residues and

AGRONOMY

International Journal of Agriculture, Environment and BiotechnologyCitation: IJAEB: 8(2): 315-321 June 2015DOI Number: 10.5958/2230-732X.2015.00039.X©2015 New Delhi Publishers. All rights reserved

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Table 1. Effect of PSB and different levels of phosphorus on plant growth and forage yield of berseem

Treatment Plant height (cm) No. of branches per plant Leaf stem ratio Green forage yield (qt. ha-1)

Dry matter yield (qt. ha-1)

InoculationI0 51.60 8.78 0.88 302.5 36.8I1 52.30 9.52 1.09 320.4 39.2S.Em (±) 0.15 0.13 0.03 2.38 0.22C.D. 0.64 0.56 0.15 10.26 0.95Phosphorus [P] (Kg ha-1)P1 51.93 9.11 0.87 297.1 37.0P2 51.94 9.14 0.99 311.6 37.5P3 51.98 9.19 1.08 321.7 39.4P4 51.95 9.17 1.01 315.4 38.1S.Em (±) 0.25 0.15 0.06 5.12 0.40C.D. NS NS 0.14 12.54 0.98I X PI0P1 51.58 8.72 0.74 288.5 36.6I0P2 51.59 8.77 0.88 302.1 36.4I0P3 51.63 8.82 0.98 312.6 38.1I0P4 51.60 8.81 0.92 306.8 36.1I1P1 52.28 9.48 1.00 305.7 37.8I1P2 52.29 9.50 1.10 321.2 38.2I1P3 52.33 9.56 1.17 330.8 40.7I1P4 52.30 9.54 1.11 323.9 40.1S.Em (±) 0.33 0.36 0.11 7.71 0.44C.D. NS NS 0.26 18.87 1.08

I0 - Seeds un-inoculated with PSB, I1 - Seeds inoculated with PSB, P1 – 40 kg P2O5 ha-1, P2-60 kg P2O5 ha-1, P3 – 80 kg P2O5 ha-1, and P4 - 100 kg P2O5 ha-1,

33% feeds (Planning Commission of India 2012;HandbookofAgriculture2009).Berseem(TrifoliumalexandrinumL.) isoneofthemost importantrabifodder crop. It is considered as themost potentialcrop from productivity as well as maintenance ofsoil fertility. It contains about 20-24% protein atgreenstages(BarikandTiwari1998).Thisnutritious,succulentandpalatablefodderisavailableforfairlylongperiodduringwinter,springandearlysummer(ChaterjeeandDas1989).

In India, though the soil and climatic conditionsare favorable for berseem production, but its per

hectare yield of fodder is very low. There aremany constraints for low yield of berseem, butimproper fertilizer application is considered to bea major limiting factor. The correct and judiciousfertilizer application can enhance yield up to50% (Zia et al. 1991 ) and can also improve thequality of fodder (Keshwa and Singh, 1992). Ina plant, phosphorus is a common component ofmany organic compounds. Phosphorus deficiencyhowever significantly reduce the plant growth(Marschner 1997). Phosphorus plays an importantroleinphotosynthesisand

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Table 2. Effect of PSB and different levels of phosphorus on various quality parameters of berseem

Treatment Dry matter (%)

Crude Protein yield (qt. ha-1)

Crude Protein (%)

Crude Fibre (%) Ash (%) Ethyl Extract

(%)Nitrogen Free Extract (%)

InoculationI0 12.18 6.57 18.59 22.92 14.35 3.29 40.85I1 12.30 7.89 19.28 21.03 14.62 3.32 41.75S.Em (±) 0.09 0.13 0.14 0.15 0.11 0.005 0.18C.D. NS 0.57 0.60 0.65 NS 0.020 0.77Phosphorus [P] (Kg ha-1)P1 12.17 7.02 18.66 22.83 14.45 3.20 40.87P2 12.19 7.12 18.74 21.99 14.46 3.28 41.54P3 12.32 7.60 19.33 21.03 14.53 3.41 41.72P4 12.28 7.19 19.02 22.06 14.51 3.35 41.08S.Em (±) 0.05 0.20 0.21 0.37 0.13 0.011 0.24C.D. 0.13 0.48 0.52 0.91 NS 0.028 0.58I X P

I0P1 12.11 6.35 18.28 23.80 14.30 3.17 40.45I0P2 12.13 6.46 18.41 22.92 14.33 3.27 41.07I0P3 12.25 6.89 19.01 21.97 14.39 3.39 41.24I0P4 12.23 6.58 18.66 22.99 14.38 3.33 40.64I1P1 12.24 7.69 19.03 21.86 14.60 3.22 41.29I1P2 12.24 7.79 19.07 21.06 14.59 3.28 42.00I1P3 12.39 8.25 19.65 20.08 14.66 3.41 42.20I1P4 12.33 7.83 19.37 21.12 14.63 3.37 41.51S.Em (±) 0.13 0.54 0.49 0.63 0.16 0.13 0.38C.D. NS 1.32 1.21 1.55 NS NS 0.93

I0 - Seeds un-inoculated with PSB, I1 - Seeds inoculated with PSB, P1 – 40 kg P2O5 ha-1, P2-60 kg P2O5 ha-1, P3 – 80 kg P2O5 ha-1, and P4 - 100 kg P2O5 ha-1,

the synthesis of nucleic acids, lipids, proteinsand other important compounds (Guinn 1984).Thephosphorus is immobile in thesoilandduetoits immobility the unused phosphorus applied asfertilizer that remains in thesoilbecomesavailablefor the next crops (Read et al. 1973).According toHamidandSarwar(1977),thecropusesonly15-33%oftheappliedPandtherestresults inthebuildupof residual P. At present, domestic production ofP fertilizers is insufficient to fulfill the country’sdemand,hencelargeamountofPfertilizersneededtobeimportedattheexpenditureofmassiveforeignexchange.Moreover,worldresourcesofeconomical

P (Rock phosphate) may be washed-out by 2050(Vanceet .al.,2003).Thereisanincreasingtrendtoreplace the chemical fertilizers by amicrobial self-propagating source of essential plant nutrients.The practice of microbial inoculation is becomingpopular throughout theworldduetobeingsimpletouseandhavenosideeffects.

The role of microorganisms in solubilizing theinorganic phosphate was known as early as 1903(Kuecey et al. 1989). Phosphorus unavailability isone of the important factors limiting yield of thecrops.Phosphorusavailability is increasedthrough

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the use of phosphate solubilizing bacteria (PSB),and itmay enhance the forage yield and improvethe quality of berseem. The microorganism,involved in solubilizing phosphorus are searchingsolublephosphorus andpromotes the efficiencyofbiologicalnitrogenfixationwhichresultsingreateraccessibility to other trace elements by producingplantgrowthpromoting substancesandultimatelyplantgrowthisenhanced(Gyaneshwaretal.,2002).Phosphate solubilizing bacteria (PSB) producesorganicacidslikelactate,oxalate,succinate,tartarate,acetate, gluconate, glycolate, citrate, ketogluconateetc.(Gyaneshwaret al. 1998;Puenteet al. 2004).Themicroorganismcapableofsolubilizngphosphatecanproduce and release organic acids and protons intheirsurroundingsthatdecreasethepHinthatareaandthussolubilizecalcium-phosphoruscomplexes.Due to these organic acids, PO4

-2 is exchanged byacidanionorischelatedandthusbecomesdissolvedmineralphosphate(BajpalandRao1971).

The reports on interactive effects of chemicalphosphorus applied, and phosphate solubilizingbacteria (PSB) on berseem is lacking in our zone.Therefore,theexperimentwasdesignedtoevaluatethe impact of phosphate solubilizing bacteria(PSB) inoculationanddifferent levelsofphosphatefertilizerongrowth,forageyieldandforagequalityofberseeminWestBengalcondition.

Materials and Methods

A field experiment to study the impact of PSBinoculationandphosphorusapplicationofberseemwas conducted in the fodder farm ofWest BengalUniversityofAnimalandFisherySciences,Belgachiafodder farmduring rabi, season 2013-14. The cropsownwithinoculated(I1)andun-inoculated(I0)seedsof berseem (var. warden) was given phosphorusfertilizeratthelevels40(P1),60(P2),80(P3)and100(P4)kgP2O5ha-1intheformofsinglesuperphosphate(SSP).The experimentwas laidout in randomizedcompleteblockdesign(RCBD)withthreereplicationsmeasuringanetplotsizeof6mX2m.theseedsofberseemvar.wardenwereinoculatedwithPSBpeatmixerandbroadcastedinawellpreparedseedbed

inmiddleofNovember.Abasaldoseof20kgNha-1 and30kgKha-1weregivenduringlandpreparation.All the other cultural practices were kept normaland uniform for all the treatments. The crop washarvested 70days a sowingatpod formation.Thegrowthparameterslikeplantheight,leafstemratioand number of branches per plant were recordedby randomly selecting ten plants from each plot.The plant height was measured with the help ofmeasuring tape from ground level to highest leaftip.Fordrymatterpercentage,thesamplewasdriedin shade and dried to electric oven at 70°C up toa period till constant weight was achieved. Thedrymatterpercentagewasusedastoolformeasuringthe total dry matter yield.A fraction of dry masswas taken and grinded and then itwas preservedin polythene bags for quality analysis. Qualityparameters like dry matter (DM), crude protein(CP), crude fibre (CF), ether extract (EE) total ash(TA)andnitrogenfreeextract(NFE)ofthesampleswere determined according to Association of theOfficial Analytical Chemist (AOAC) (Anonymous1990). The dry matter was determined by dryingthe samples at 80°C till constant weight. Crudeproteinwasestimatedbymicro ‘Kjeldhal’method.Thepercentofnitrogen indicatedtheestimationofCP. The ether extract in a samplewas determinedbyextractingwithdiethyletherat60°Cin‘Soxhlet’sapparatus’. For crude fibre, sample was reflexedfirstwith1.25%H2SO4andsubsequentlywith1.25%NaOH for 30 min each to dissolve the acid andalkali solublecomponentpresent in it.TheresiduecontainingCFwasdried toa constantweight, andthedriedresiduewasignitedinmufflefurnace,lossofweightonignitionwascalculatedtoexpressitasCF.Forash,samplewasignitedinmufflefurnaceat550°Ctoburnalltheorganicmatterandleftoverwasweighed as ash. The Nitrogen Free Extract (NFE)was calculated by subtracting the sum of CP, EE,CFandash from the sampleweightondrymatterbasis.Theobservationsongrowth,yieldandqualityparameters were calculated using SPSS software,IBMInc.2009andleastsignificantwascomputedatp≤0.05asdescribedinGomezandGomez(1984).

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Results and Discussion

Growth parameters and yields

It isclearfromthetable1thatPSBinoculationhassignificant effect on growth parameters like plantheight,numberofbranchesperplantandleafstemratio. PSB inoculated plots recorded significantlyhigherplantheight (52.30cm);no.ofbranchesperplant(9.52)andleafstemratio(1.09).Similareffectof PSB on cluster bean was reported by Ayub et al. (2013). Chintapalli et al. (2012) recorded thesignificant effect of Rhizobium + PSB on plantheight of berseem. El-Gizawy andMehasan (2004)also reported significant increase in plant heightand number of branches per plant of chickpea byPSB inoculation.Ramana et al. (2010) reported thatthe inoculation with PSB increased leaf area offrenchbean significantly.However,non-significantdifferencewas found for phosphorus on the plantheightandnumberofbranchesperplantofberseem(Table1).Thepossibleargumentmaybeduetolesscompetition of the plants for nutrients particularlyphosphorus.Theresultsareincloseagreementswiththat of Saeed et al. (2011).However, application ofphosphorus resulted significant effect on leaf stemratio of berseem (Table 1) and better results (1.08)obtained with phosphorus application of 80 kg ha-1P2O5.Ayubet al.(2012)alsoreportedasignificanteffect of phosphorus on leaf stem ratio of clusterbean.

Significantly higher green forage yield (320.4 qt ha-1)anddrymatteryield(39.2qtha-1)wererecordedfromtheplots,inoculatedwithPSB(Table1)andthatwasmainlyduetogreatervaluesofplantheight,no.of branches per plant and leaf stem ratio. SimilareffectofPSBongreenforageanddrymatteryieldofclusterbeanhadbeenobservedbyAyubet al.(2013).Chintapalli et al. (2012) recorded maximum greenforage yield of berseemwhen the crop inoculatedwith Rhizobium and PSB along with FYM. Effectof different levels of phosphorus application ongreenforageyield,aswellasdrymatteryield,wasalso found significant. Higher green forage yield(321.7 qt ha-1) and dry matter yield (39.4 qt ha-1)

wereobtainedwithphosphorusapplicationof80kg ha-1.IncreaseinPlevelsfrom40kgha-1to80kgha-1 graduallyincreasedthegreenforageanddrymatteryieldof berseem. IncreaseofP level beyond80kgha-1adverselyaffectsthegreenforageanddrymatteryield and the same were statistically at par withthose achieved with the application of 80 kg P2O5 ha-1. Maximum green forage and dry matterproductionat80kgP2O5ha-1mightbetheresultsofhigherplantheight,no. of branchesperplant, leafstemratioandofcourse,thebetterrootdevelopmentwhich provides a better habitat for the activity ofbiological nitrogen fixing bacteria. The significanteffectondrymatteryieldofclusterbeanatvariouslevels of P had also been reported byAyub et al.(2012) and Chauhan and Bajpal (1979). Interactioneffect of PSB andphosphorus on plant height andno.ofbranchesperplantwerefoundnotsignificant.Significantlyhighergreenforageyield(330.8qt.ha-1),drymatteryield(40.7qt.ha-1)andleafstemratio(1.17)wereobtainedfromtheplotsinoculatedwithPSB and having phosphorus application of 80 kgha-1mightbeduetobettercropgrowth,higherplantheight and more no. of branches per plant. Ayubet al. (2013) also reported significant effect of PSBinoculation andP application on green forage anddrymatteryieldinclusterbean.

Quality parameters

All the quality traits except ash content and drymatterpercentageweresignificantlyaffectedbyPSBinoculation.PSBinoculationexhibitedhighervaluesforcrudeproteinyield(7.89qt.ha-1),crudeproteinpercentage (19.28%), ethyl extract (3.32), nitrogenfree extract (41.75) and lowest crude fibre (21.03),than uninoculation (Table 2). Ayub et al. (2013)recordedhigherCPandEEfromthePSBinoculationcropsofclusterbean.RugheimandAbdelganj(2012)also reported the significantly higher values ofCPandNFEinPSBinoculationinfavabean.

Application of phosphorus was significant for allqualityparametersexcept theashcontent. IncreaseinP levels from40kgha-1 to 80 kgha-1 graduallyincreased thedrymatterpercentage, crudeprotein

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yield, crude protein percentage, ethyl extract andnitrogen free extract of berseem and increase of Plevel beyond 80 kg ha-1 adversely affected thosequality traits. However, the effect of P applicationfrom40kgha-1to80kgha-1oncrudefibrepercentageof berseem was just opposite. Higher dry matterpercentage (12.32), crude protein yield (7.60 qt. ha-1),crudeproteinpercentage(19.33%),ethylextract(3.41%) and nitrogen free extract (41.72%) wereobtainedwithPapplicationof80kgha-1 (Table2).However,significantlylowestcrudefibrepercentage(21.03%) was recorded with 80 kg P2O5 ha-1 andhighest (22.83%) with 40 kg P2O5 ha-1. Turk et al. (2007)alsoreportedadecreaseincrudefibrecontentwithincreasethelevelsofphosphorusinvetchcrops.Ayub et al. (2013) reported the significant effect ofP application on drymatter percentage and crudefibrecontentofclusterbean.Grewalet al.(2004)andYadav et al. (2011) also recorded significant effectof P on crude protein percentage of cluster bean.Betterquality green forageproducedwith 80kgPha-1mightbetheresultsofbetterrootdevelopmentwhich provides a better habitat for the activityof biological nitrogen fixing bacteria. The higherrootmass exploits the soil fromsurroundingmoreeffectively and improves the nutrients availabilityforplants.

Interaction effect of PSB and phosphorus on drymatter percentage, ash content and ethyl extractwasfoundnotsignificant.Significantlyhighercrudeproteinyield(8.25qt.ha-1),crudeproteinpercentage(19.65%), nitrogen free extract (42.20%) and lowestcrudefibre (20.08%)were obtained from the plots,inoculated with PSB and having phosphorusapplicationof80kgha-1(Table2).

Conclusion

From the results of the present study, it can beconcluded that phosphorus solubilizing bacterialinoculation alongwith phosphorus application areessential forobtaininghigheryieldofgoodqualityfodder of berseem. For obtaining higher yield ofgood quality fodder of berseem, it seems better toinoculate its seed with phosphorus solubilizing

bacteria (PSB)andapplyphosphorusat the rateof80kgha-1underWestBengalcondition.

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