Symbiotic N 15 N Tracer Technique Pueraria phaseoloides...

Research ArticleSymbiotic N2-Fixation Estimated by the 15N Tracer Techniqueand Growth of Pueraria phaseoloides (Roxb) Benth Inoculatedwith Bradyrhizobium Strain in Field Conditions

Papa Saliou Sarr1 Judith Wase Okon2 Didier Aime Boyogueno Begoude3 Shigeru Araki1

Zacheacutee Ambang2 Makoto Shibata4 and Shinya Funakawa4

1Center for African Area Studies Graduate School of Asian and African Area Studies Kyoto University46 Shimoadachi-cho Yoshida Sakyo-ku Kyoto 606-8501 Japan2Department of Plant Biology Faculty of Science University of Yaounde I PO Box 812 Yaounde Cameroon3Regional Biocontrol and Applied Microbiology Laboratory Institute of Agricultural Research for Development (IRAD)PO Box 2067 Yaounde Cameroon4Graduate School of Agriculture Soil Science Laboratory Kyoto University Kitashirakawa Oiwake-cho Sakyo-kuKyoto 606-8502 Japan

Correspondence should be addressed to Papa Saliou Sarr pesseus77yahoofr

Received 4 December 2015 Accepted 4 January 2016

Academic Editor Juan Manuel Ruiz-Lozano

Copyright copy 2016 Papa Saliou Sarr et alThis is an open access article distributed under theCreativeCommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

This field experiment was established in Eastern Cameroon to examine the effect of selected rhizobial inoculation on N2-fixation

and growth of Pueraria phaseoloides Treatments consisted of noninoculated and Bradyrhizobium yuanmingense S3-4-inoculatedPueraria with three replications each Ipomoea batatas as a non-N

2-fixing reference was interspersed in each Pueraria plot All

the twelve plots received 2 gNm2 of 15N ammonium sulfate 10 atom excess At harvest dry matter yields and the nitrogenderived from atmospheric N

2-fixation (Ndfa) of inoculated Pueraria were significantly (119875 lt 005) higher (81 and 1083

resp) than those of noninoculated Pueraria The inoculation enhanced nodule dry weight 244-fold Consequently the harvestedN significantly (119875 lt 005) increased by 83 in inoculated Pueraria resulting from the increase in N

2-fixation and soil N uptake A

loss of 55 to 60 of the N fertilizer was reported and 36 to 40 of it was immobilized in soil Here we demonstrated that both N2-

fixing potential of P phaseoloides and soil N uptake are improved through field inoculations using efficient bradyrhizobial speciesIn practice the inoculation contributes to maximize N input in soils by the cover croprsquos biomass and represent a good strategy toimprove soil fertility for subsequent cultivation

1 Introduction

The genus Pueraria originates from South Asia SoutheastAsia China Japan and parts of Oceania [1] and is nowwidespread throughout the humid tropics Perennial legumecover crops such as Pueraria phaseoloides are commonlygrown between the rows trees or used in improved fallowsin order to reduce soil erosion improve soil fertility andincrease the growth and yield of associated plants [2ndash4]Leguminous crops are a source of nitrogen and contributeto an increase in the nitrogen uptake of the nonleguminousassociated crop [5] A major advantage of growing a legume

cover crop is its ability to fix N2symbiotically and to

accumulate nitrogen in the plantation agroecosystem Pphaseoloides fixes nitrogen in symbiosis with Bradyrhizobiumstrains and is classified as ldquopromiscuous ineffectiverdquo [6]Nitrogen fixation is a process by which nitrogen in theatmosphere is converted into ammonium [7] Both the plantand the rhizobia benefit from such a symbiotic relationshipNitrogen is the most commonly deficient nutrient in manysoils around the world and it is the most commonly suppliedplant nutrient However the supply of nitrogen throughfertilizers has severe environmental concerns Atmosphericnitrogen fixed by the symbiotic association between soil

Hindawi Publishing CorporationScientificaVolume 2016 Article ID 7026859 10 pageshttpdxdoiorg10115520167026859

2 Scientifica

bacteria (rhizobia) and crop legumes is likely to be in theorder of 20 to 22 million tons of nitrogen each year [8]

In Eastern Cameroon short-term Pueraria fallows oftwo years are commonly practiced to restore soil fertilityfor subsequent cultivation of cassava which is a staple foodin the area Yield increments of cassava are related to soilquality improvement which includes soil erosion limitationincrease of soil organic carbon and nitrogen following a gooddecomposition of Pueraria litters The phylogenetic diversityof nitrogen-fixing bacteria in the root nodules of Puerariain Eastern Cameroon was previously investigated [9] Usingmolecular biology techniques these authors identified threegroups of Bradyrhizobium species corresponding to B spB yuanmingense and B elkanii that nodulate Puerariaphaseoloides By assessing the nitrogen fixation potential ofthese three groups using the Acetylene Reduction Activity(ARA) method it was found that strains belonging to Byuanmingense showed significant higher efficiencyThe selec-tion of appropriate rhizobial microsymbionts is becominga complex procedure due to the fact that several legumesspecies can be nodulated by single rhizobia [10] Yet the sym-biotic association between the legumes and their microbialsymbionts plays a significant role in agriculture worldwide byreducing ca 100 million metric tons of atmospheric nitrogensaving US$ 8 billionyear on fertilizer N [11 12]The commonapproach to improving the symbiotic fixation of nitrogenand legume productivity using superior or very effectiveexotic rhizobial strains as inoculants often fails to achievethe desired responses [13 14] The failure has been attributedto the poor competitiveness of the introduced rhizobia [15]the nonspecificity of the bacteria and the occurrence andresistance to stress of ineffective indigenous strains in soils[16] To improve the N

2-fixation and growth of Pueraria the

use of rhizobia selected from indigenous community withcapacity for nodulation and strong nitrogen-fixing activity isbelieved to be an important agronomic approach

Pueraria phaseoloides is used in the tropics as a forage andcover crop but rhizobial inoculations of this legume in fieldconditions have not been widely tested The importance ofthis work is to improve the nitrogen fixation potential andgrowth of Pueraria through field inoculations with effectivestrains of Bradyrhizobium enabling enhancement of theinput of nitrogen in the soil via biological process to shortenthe fallow period of Pueraria and put the land in faster use aswell as reduce environmental hazards that the use of chemicalfertilizers to increase yields often poses In this paper wereport on the effects of inoculating Puerariawith the selectedefficient B yuanmingense S3-4 strain [9] on its N

2-fixation

dry biomass production total N uptake nitrogen fertilizeruse efficiency and soil fertility through a field experimentSymbiotic nitrogen fixation was estimated using the 15Ntracer technique and Ipomoea batatas (L) Lam was used as anonfixing reference plant

2 Materials and Methods

21 Study Site The study was carried out in Andom village(5∘151015840N 13∘301015840E) located in Eastern Cameroon which is inthe forest-savanna transition zone [17 18] The elevation of

the plateau is 650ndash700m asl and its basement consists ofmetamorphic rocks The annual rainfall range of this areais 1350ndash1550mm and the distribution of rainfall is bimodalaccording to fieldmeasurement carried out from 2008 to 2013[19] Rains begin in mid-March a short dry season followsfrom mid-July to the end of August and then the main rainyseason extends from September to the middle of NovemberThe mean annual temperature in the region is 25∘C TheDecemberndashFebruary period is the driest part of the yearwhenthe relative humidity may be higher than 60 [20] The soilpH in the cultivated land varies from 482 to 517 with averagelevels of organic C total N and available P of 181 0113and 457 ppm respectively and a CN ratio of 1591 [21]Soils are classified as Typic Kandiudox according to USDAclassification system [22] The dominant forest vegetation isAlbizia zygia which dominates the tree layer The dominantspecies in the savanna are the perennial grasses Pennisetumsetaceum Imperata cylindrica and Chromolaena odorata [1718] The experiment was set on a cleared natural fallow landpreviously dominated by Chromolaena odorata There havebeen no planted legumes including Pueraria and no historyof rhizobial inoculation was observed in the site

22 Experimental Design Treatments consisted of inoculatedPueraria and noninoculated Pueraria each replicated threetimes in a complete randomized block design Ipomoeabatatas plots were interspersed in each Pueraria plot as non-N2-fixing references giving a total of 12 plots (4m2 plotminus1)

with a planting density of 9 plants per 15m times 15m Ibatataswas used as a reference crop as it shares with Puerariasimilar patterns of N uptake from the soil and will obtainsimilar 15N enrichment in the soil-derived N [23] The useof a reference crop is important in estimating the amountof nitrogen derived from the atmosphere (Ndfa) of legumesEach Pueraria-Ipomoea block was separated to others by a2m interval (Figure 1) Pueraria seeds were sown on 9August2014 at 50 cm spacing Uniform Ipomoea seedlings of 10 cmlength were obtained from farmersrsquo fields and transplanted intheir corresponding plots at 50 cm spacing two weeks afterPueraria seeds were sown

23 Rhizobial Inoculation and Application of 15N LabelledAmmonium Sulfate Prior to this field experiment rhizobialstrains were isolated from Pueraria nodules collected fromthe experimental site and other neighboring fields andsubjected to DNA-based molecular analysis [9] The resultsshowed that the native strains for Pueraria in the experimen-tal field clustered into 3 groups corresponding to Bradyrhizo-bium yuanmingense B elkanii and B sp By assessing theirnitrogen fixation potential using the Acetylene ReductionAssay (ARA) it appeared the B yuanmingense strains andS3-4 in particular showed a significant highest potentialTherefore Bradyrhizobium yuanmingense S3-4 identified asan efficient Pueraria symbiont [9] was used in this fieldexperiment From glycerol stock it was streaked on YMAagar plates [24] for one week After colony developmentone pure colony was grown in 20mL YMB for anotherweek to produce the inoculum The obtained solution of2094 times 109 cellsmLminus1 was diluted 1000 times with sterilized

Scientifica 3

IPIP

NIP1

NIP2

IpB2IpB1

IpB3

IpB5 IpB6

IP1

IpB4 NIP3

2m

2m

2m

Figure 1 Experimental design IP = inoculated Pueraria NIP = noninoculated Pueraria and IpB = Ipomoea batatas IP and NIP treatmentswere each replicated 3 times giving a total of 6 Pueraria plots Ipomoea plots (six) were interspersed in each Pueraria plot as non-N

2-fixing

references Plotrsquos size was 4m2 In each plot plant spacing was 50 cm corresponding to 9 hills per 15m times 15m for both Pueraria and IpomoeaFor Pueraria seeds were sown but thinned to 3 plants per hill after the seedlings were well established while for Ipomoea uniform seedlingsof 10 cm length were transplanted two weeks after the sowing of Pueraria seeds Pueraria inoculation was performed using the effective strainB yuanmingense S3-4 (2094 times 108 cells per hill) three weeks after seedrsquos sowing All plots received 2 gNm2 of 15N labelled ammonium sulfate10 atom excess applied at 1 and a half months after Pueraria seeds were sown Plots were regularly weeded throughout the rain-fed proneexperiment that lasted 4 months

water to give a concentration of 2094 times 106 cellsmLminus1 Theinoculation was carried 3 weeks after the sowing of Puerariaseeds (29 August 2014) when seedlings were well establishedAbove each Pueraria hill (in the three inoculated Puerariaplots) 100mL of the diluted inoculum corresponding to2094 times 108 cells hillminus1 was applied carefully One and a halfmonths after the starting of the experience when all cropswere well established (22 September 2014) nitrogen fertilizeras 15N labelled ammonium sulfate enriched with 10 atomexcess was applied at the rate of 2 gNm2 When estimatingthe nitrogen fixation of legumes using labelled fertilizer Nlower rates of N fertilizer are preferably applied to avoidlimiting the nitrogen fixation potential Some authors [25 26]

also used the same rate of 2 gNm2 during their studies on theN2-fixation of cowpea and Pueraria in field conditions Prior

to the fertilizer application Pueraria seeds were thinned tothree seedlings per hill As 2 gNm2 corresponds to 9524 gof ammonium sulfate per square meter the 12 plots inour experimental site required 475 g of ammonium sulfateConsidering the loss that may occur during the applicationof the fertilizer solution we considered making a solutionfor 13 plots (495 g of ammonium sulfate) This amount wasdissolved in 78 liters of water after which a 6-liter solutionwascarefully spread in each plot using a sprayer Due to the highcost of labelled fertilizers we limited the experimental unit toa 4m2 area per plot After the fertilizer solution was applied

4 Scientifica

a thin layer of soil was spread above the plots to mimicthe atmospheric volatilization of the fertilizer A portion ofthe solution was stored in a small bottle for further analysisof the exact atom percent excess of the fertilizer solutionThroughout the experimental period plots were carefullyand regularly weeded to avoid removing the 15N labelledammonium sulfate outside the plots

24 Data Collection P phaseoloides litter begins to accu-mulate on the soil surface about 6 months after establish-ment followed by subsequent mineralization [27] To avoidentering the period of Pueraria leaves drop-off which wouldalter the results this experiment lasted fourmonths (AugustndashDecember 2014) At term the 9 central plants of each plot(to avoid the border effect) were harvested (Figure 1) Thefresh shoots were separated from the fresh roots in eachplot and their biomass was weighed Pueraria root noduleswere separated from the roots in each plot and their freshweight was recorded Fresh subsamples of known weightswere collected from the shoot biomass and root biomassof each Pueraria and Ipomoea plot and together with thefresh nodules per Pueraria plot they were oven-dried at70∘C for 48 hours in laboratory Conversion calculationswere made to determine the dry matter yields of inoculatedPueraria noninoculated Pueraria and Ipomoea Compositesoil samples were collected from each plot at the 0ndash15 cmhorizon air dried and used for nutrient analysis

The dried plant samples were ground at lt05mm andused for analysis Total N was determined on the finelyground plant materials using 50mg samples The standardwas 30mg L-glutamic acid Samples were packed in tin cap-sules and analyzed on a Carlo Erba NA1500NC elementalanalyzer [28] Total N () of soil samples was determinedfollowing the same procedure except that the amount of500mg was used Total carbon was determined using a dry-combustion method with a CN analyzer (VarioMax CHNElementar Analysensysteme GmbH Hanau Germany) The15Nenrichment in soil and plantmaterialswas determined onan isotope ratiomass spectrometer (Delta FinniganMAT 251Bremen Germany) coupled on-line to an elemental analyzer[28] The 15N fertilizer solution used in this experiment wasalso analyzed to record the exact 15N atom excess

25 Calculations The 15N atom excess in plant materialswas obtained by using the 15N atom provided duringthe analysis and the atmospheric natural 15N abundance(03662) by applying the equation

15N atom excess = 15N atom

minus natural 15N abundance(1)

In addition the total nitrogen (TN gm2) fertilizer nitrogenrecovery (gm2) fertilizer nitrogen use efficiency (NUE)the percentage of N crops that derived from the fertilizer(Ndff) nitrogen derived from atmosphere (Ndfa) andnitrogen derived from soil (Ndfs) were determined

Most calculations in this paper were according to theInternational Atomic Energy Agency The fertilizer nitrogen

recovery which corresponds to the amount of nitrogen inplants that derived from the applied nitrogen (Ndff) wascalculated as

Ndff (gm2) = (TN times Ndff100) (2)

The Ndff was calculated using the following equation

Ndff = (15N atom excess of plant sample15N atom excess of fertilizer

)

times 100

(3)

The TN and NUE were derived from the following calcula-tions

TN (gm2) = (DM times N100) (4)

DM (gm2) corresponds to the dry matter yield

NUE () = (Ndff (gm2)

Applied N (gm2)) times 100 (5)

The proportion of Pueraria nitrogen derived from the atmo-sphere (Ndfa) was calculated using the 15N isotope dilutionequation [29]

Ndfa = (1 minus NdfffixNdff ref) times 100 (6)

In (6) Ndfffix is the Ndff of the N2-fixing plant and Ndff ref

the Ndff of the non-N2-fixing reference As for Pueraria

the sources of nitrogen are the soil the fertilizer and theatmosphere Ndfs was calculated as

Ndfs = 100 minusNdff minusNdfa (7)

For the non-N2-fixing Ipomoea reference crop

Ndfs = 100 minusNdff (8)

The TN (gm2) and Ndff (gm2) in roots shoots and nodulesof Pueraria were determined separately The sum of TN inshoots roots and nodules gave the total nitrogen contentin the plant while the sum of Ndff in the three componentsyielded the totalNdff in the plant For Ipomoea theTN (gm2)in roots and shoots were summed to give the plant totalnitrogen and similarly the total nitrogen derived from thefertilizer was the sum of those in shoots and rootsThese totalvalues were used in the final calculations To calculate theNdfa in a Pueraria plot (Ndfa of plant or Ndfa of residualsoil) we considered the Ndff (Ndfa of plant or Ndfa ofresidual soil) of the Ipomoea grown in interspersed plots

The available amount of nitrogen in the soil [total soil N(gm2)] in terms of equivalents units of ammonium sulfatewas estimated in each plot By using a labelled fertilizersource it is possible to determine the plant available amountof nutrient in the soil and this amount is expressed rela-tively to the amount available in the fertilizer source Some

Scientifica 5

Table 1 Growth characteristics of plants (inoculated and noninoculated Pueraria Ipomoea) after 4-month cultivation

Treatments Fresh weight (gm2) Dry weight (gm2)Shoot Root Nodule Total Shoot Root Nodule Total

I Pueraria 3033b 120 2667b 318b 640b 036 0690b 6829bNI Pueraria 1440a 070 1148a 152a 351a 021 0283a 3748aIpomoea 1797a 443 mdash 224ab 299a 123 mdash 4220abSign Trait (119875

005) lowast ns lowast lowast lowast ns lowast lowast

I Pueraria = inoculated Pueraria NI Pueraria = noninoculated Pueraria Sign Trait = significant trait at 5 probability level Nod mass = nodule masslowastsignificant at 119875 lt 005 and ns = nonsignificant One-way ANOVA was performed using CropStat ver 72In a column values with different letters (a b) are statistically different at the indicated significant trait

authors [30ndash32] assumed that when a plant is confrontedwith two or more sources of a nutrient element the nutrientuptake from each of these sources is proportional to theamounts available in each source Implicit in this assumptionis the fact that there is a complete mixture of the isotopes orisotopic equilibria In this situation the following equationwas proposed

(Plant Ndff

Applied fertilizer N) = (

Plant NdfsTotal soil N

) (9)

Therefore

Total soil N (gm2)

= (Applied fertilizer N times plant Ndfs

plant Ndff)

(10)

N immobilization that corresponds to the portion of fertilizerN remaining in the soil at harvest is calculated as

N immobilization () = ( Soil NdffApplied N

) times 100 (11)

Following that the amount of fertilizer N lost during the trialwas calculated as follows

N loss (gm2) = applied N

minus (soil Ndff + plant Ndff) (12)

Finally N loss values can be transformed into percentagesAll data were subjected to analysis of variance (ANOVA)

using the CropStat 72 software (International Rice ResearchInstitute Manila Philippines) at a probability level of 5Mean separation was performed using Fisherrsquos least signifi-cant difference (LSD) test whenever a significant result (119875 lt005) was obtained

3 Results and Discussion

31 Growth Response of Plants to Bradyrhizobial Inoculationand Fertilizer Application Fresh and dry matter yields ofPueraria and Ipomoea are reported in Table 1 It shows apositive effect (119875 lt 005) of inoculation with Bradyrhizobiumyuanmingense S3-4 on P phaseoloides growth 4 months afterseed sowing Total fresh and dry weights were significantly

higher in inoculated Pueraria than in noninoculated Puer-aria This difference originated from the difference in shootproduction given that no significant difference in rootrsquos yieldwas recorded between the inoculated and the noninoculatedPueraria The inoculation contributed to increase the totalfresh and dry weights of Pueraria 202- and 181-fold respec-tively The 181-fold increase of the dry weight of P phase-oloides receiving the inoculation was 81 higher comparedto that of Puerariawithout inoculationThe nodule fresh anddry weights were also significantly (119875 lt 005) improved 232-and 244-fold by the inoculation respectively The increasein nodule dry weight from 0283 gm2 in the noninoculatedPueraria to 0690 gm2 in the inoculated Pueraria after 4-month growth (Table 1) could derive from the increase innodule number or bigger nodule size In this study the nodulenumber was not counted at the field during the harvest due tohigh numbers but rather their total fresh and dry weights perplot were recorded However it is well known that maturedbigger nodules with stronger hemoglobin activity are betterdrivers for increased N

2-fixation in legume crops [33] rather

than a high number of small immature nodules It wasfound that theN

2-fixation in groundnutwas stimulated along

with growth of nodules by application of another externalsource as millet straw [34] Similarly Pueraria plants with agreatermass of effective nodules tend to develop greater plantbiomass

In addition although the main goal of this work was tocompare the effect of the inoculation on Pueraria growthcharacteristics and N

2-fixation we observed that the dry

matter production of the reference crop (I batatas) was notsignificantly different from that of Pueraria

Table 2 shows the nitrogen status in plants at harvestTotalNuptake in the reference crop (Ipomoea) was not signif-icantly different from that of the noninoculated Pueraria butwas significantly lower than that of the inoculated PuerariaIn addition the harvested total N harvested from inoculatedPueraria was significantly higher than that harvested fromnoninoculated Pueraria at 5 probability level The higher Ncontent in inoculated Pueraria could be expected since it isstrongly related to the higher dry matter production in thistreatment An overall increase of 0825 gm2 was obtained ininoculated plots within four months To understand whetherthis increase in total N was a consequence of the obtainedimprovement of the N

2-fixation itself the total N in inoc-

ulated and noninoculated Pueraria plants was fractionatedinto nitrogen derived from soil (Ndfs) nitrogen derived

6 Scientifica

Table 2 N status in plants (inoculated and noninoculated Pueraria Ipomoea) at harvest

Treatments Total N Ndff Ndfs Ndfa NUEgm2

I Pueraria 1818b 0616a 5677a 3709 561bNI Pueraria 0993a 0768a 6692a 2624 368aIpomoea 0784a 1027b 8973b mdash 388aSign Trait (119875

005) lowastlowast lowastlowast lowastlowast ns lowast

I Pueraria = inoculated Pueraria NI Pueraria = noninoculated Pueraria Sign Trait = significant trait at 5 probability level and ns = nonsignificantlowastsignificant at 119875 lt 005 lowastlowastsignificant at 119875 lt 001 Ndff = nitrogen derived from fertilizer Ndfs = nitrogen derived from soil Ndfa = nitrogen derived fromatmosphere and NUE = fertilizer nitrogen use efficiency One-way ANOVA was performed using CropStat ver 72In a column values with different letters (a b) are statistically different at the indicated significant trait

from fertilizer (Ndff) and nitrogen derived from atmosphere(Ndfa) and that in Ipomoea was fractionated into Ndfs andNdff and results are shown in Figure 2We observed that as anon-N

2-fixing reference crop Ipomoea extractedmost of its N

from soil The contribution of the N2-fixation in the plantrsquos N

pool was significantly higher (119875 lt 005) in inoculated thanin noninoculated Pueraria The nitrogen derived from N

2-

fixation accounted for 2625 in noninoculated Pueraria and3708 in inoculated Pueraria (Table 2) The results of plantN fractionation indicated that the surplus of 0825 gNm2in the inoculated Pueraria did not derive only from theimprovement of N

2-nitrogen fixation While +0388 gNm2

(47) in inoculated Pueraria was added by the N2-fixation

the others 0398 gNm2 (483) and 0039 gNm2(461)derived from the soil and applied fertilizer N respectivelyThis clearly indicates that the inoculation of Pueraria withB yuanmingense S3-4 not only enhances the N

2-fixation

but also may make a positive contribution in facilitation ofa better uptake of N from the soil and from the appliedfertilizer For instance Table 2 shows better fertilizer N useefficiency of the inoculated Pueraria than the noninoculatedPueraria It is important to note that as the harvested plantswere located in the middle of each plot there was no riskof nutrient uptake from the external soil environment whichwould result in erratic data due to the dilution of the isotopeFurthermore the interspersed design where each Puerariaplot was next to a plot of the reference Ipomoea reduced thefertility gradient that may exist in the experimental site

The dual increase of N fromN2-fixation and soil N uptake

in inoculated Pueraria as shown in Figure 2 is not surprisingand agrees with Butler and Ladd [35] These authors alsofound that the amount of N

2fixed in medic (Medicago

littoralis) and pea (Pisum sativum) increased together withincreasing uptake of soil N in different soils when amountsof available N did not exceed 35 120583gN gminus1 soil for medic and74120583gN gminus1 soil for pea The amount of soil N in the Puerariaplots of this study (Table 4) was higher (21mg gminus1 on average)than those values reported by Butler and Ladd [35] HoweverVesterager et al [27] reported that apparently P phaseoloidesis tolerant towards the elevated inorganic N levels in the soilwhich is an important quality for a cover crop in order toaccumulate N in plantation systems Moreover by studyingthe effect of litter application on the N

2-fixation of Pueraria

these authors found a higher amount of total N in the plants

2

18

16

14

12

1

08

06

04

02

0IP NIP

PlantsIpomoea

Har

veste

d pl

ant N

(gm

2)

NdffNdfsNdfa

ab

01122

10624

0644

00737

0664

0256

0706

00776

Figure 2 Sources and amount of plantN (gm2) harvested per treat-ment during the four-month experiment period Ndfs = nitrogenderived from soilNdff=nitrogenderived from fertilizer andNdfa =nitrogen derived from atmosphere IP = inoculated Pueraria NIP =noninoculated Pueraria In a column values correspond to theamount of N (gm2) per N source (soil fertilizer and atmosphere)One-way ANOVA using CropStat ver 72 was applied to the sameN source for IP NIP and Ipomoea and no significant difference wasobserved except for Ndfa between IP and NIP with different letters

supplied with litter since the total uptake of soil N increasedconsiderably by litter application Similarly to their findingthe effect of the fertilizer N in our experiment was slightlyimproved by the bradyrhizobial inoculation as indicated bythe NUE and described above

Table 3 shows theN status in shoots roots and nodules ofinoculated Pueraria noninoculated Pueraria and IpomoeaIt indicates that the inoculation did not influence the Nconcentration in P phaseoloides Therefore the 83 increasein total N of inoculated Pueraria compared to noninoculatedPueraria as shown in Table 2 was a result of the higher drymatter yield (Table 1) which resulted from a combination of(i) higher N

2-fixation (ii) soil N uptake and (iii) a better use

efficiency of the fertilizer NIn the present study the contribution of the symbiotic

N2-fixation to the total N in P phaseoloides (26ndash37) was

Scientifica 7

Table 3 Effect of bradyrhizobial inoculation on plant N status after 4-month growth

Treatments N 15N atom excess Total N (gm2)Shoot Root Nodule Shoot Root Nodule Shoot Root Nodule

I Pueraria 271 118b 48 0615a 0773 0474 1743b 0042 0033NI Pueraria 273 120b 43 0742a 0893 0485 0953a 0027 0013Ipomoea 256 069a 0995b 0880 0783a 0084Sign Trait (119875

005) ns lowast lowast lowast ns lowast ns ns lowast ns ns

I Pueraria = inoculated Pueraria NI Pueraria = noninoculated Pueraria Sign Trait = significant trait at 5 probability level and ns = nonsignificantlowastsignificant at 119875 lt 005 lowastlowastlowastsignificant at 119875 lt 0001 One-way ANOVA was performed using CropStat ver 72In a column values with different letters (a b) are statistically different at the indicated significant trait

Table 4 Soil N status in inoculated and noninoculated Pueraria and Ipomoea plots at harvest

Treatments OrgC N CN Total N Ndfs Ndfa Ndff N imb N loss gm2 gm2 gm2 gm2 gm2

I Pueraria 308 0212 1452 1833 9177a 1690 415 068 408 074 370 5712 114NI Pueraria 300 0209 1433 1788 9480b 1695 114 021 407 073 365 6005 12Ipomoea 298 0214 1395 1955 9584b 1874 mdash mdash 416 081 405 5573 112Sign Trait (119875

005) ns ns ns ns lowast ns ns ns ns ns ns ns ns

I Pueraria = inoculated Pueraria NI Pueraria = noninoculated Pueraria Sign Trait = significant trait at 5 probability level and ns = nonsignificantlowastsignificant at 119875 lt 005 Composite soil samples were collected from each plot at harvest and subjected to chemical analysis for organic carbon (OrgC) N() and 15N atom excess using the methods described in the text Following that (3) (6) (7) or (8) in the text was used to calculate soil Ndff () Ndfa() and Ndfs () Soil total N is the average N in treatments at the end of the trial and has three origins (Ndfs = soil nitrogen derived from soil Ndfa = soilnitrogen derived from atmosphere and Ndff = soil nitrogen derived from fertilizer) Total N is calculated based on (10) in the text N imb = N immobilization(it corresponds to the percentage of the fertilizer N that remains in the soil at the end of the experiment out of the initially applied 2 gm2 cf (11)) One-wayANOVA was performed using CropStat ver 72 N loss is the fertilizer nitrogen lost in the system during the experiment via denitrification or leaching orvolatilizationIn a column values with different letters (a b) are statistically different at the indicated significant trait

lower than that found by some authors in pot [27] and infield [23 36] conditions which reached 70 P phaseoloideswithout inoculation was estimated by 15N isotope dilutionto fix between 60 and 80 of the N it accumulated ina 2-year-old palm plantation in Malaysia amounting to151 kgNhaminus1 yearminus1 [26] Galiana et al [37] inoculatedAcaciamangium trees with two effective Bradyrhizobium sp strainsand estimated their nitrogen fixation by the 15N naturalabundance method and reported that 55ndash62 of N derivedfrom atmosphere in the inoculated trees while the noninoc-ulated control trees fixed 41 of their nitrogen The lowerN2-fixation level in our experiment could partly be related

to the lower plant density (9 plants15m times 15m) we usedfor the purpose of the trial The most adequate spacing forPueraria for a fast soil cover was estimated to be 25 cm [38]which would result in a density of 25 plants125 times 125mThe shortage of rains during the first weeks of the experimentat the end of August 2014 (data not shown) that led towatering of the plants could be another reason Furthermoregiven that our experiment was set on a cleared natural fallowland adequate N amount from the soil and the subsequentmineralization of residual litters may also have lowered thenitrogen fixation potential High inorganic N concentrationsare known to inhibit N

2-fixation [39] However variation

exists in the sensitivity of symbiotic N2-fixation to combined

N between and within legume species [40 41] For instancewith C caeruleum no response to inoculation with severalrhizobial strains was found in four experiments in Malaysia

even though the inoculant strains formed over 70 of thenodules in some cases [23] In further experiments inocu-lation of C caeruleum and Pueraria with different strains ofBradyrhizobium also failed to improve their growth [42] Insome cases the failure of cover crop establishment has beenlinked to the lack of suitable inoculants

The 15N atom excess in Pueraria (inoculated or non-inoculated) shootswas found to be lower than that in Ipomoea(Table 3) This is to be expected since the N obtained byPueraria from the soil and fixation from the atmospherehas reduced the 14N15N ratio in the tissues [41] of thelegume crop contrary to the nonnodulating reference cropAlthough it was not significant the 15Nenrichment in shootsroots and nodules of Pueraria slightly decreased by thebradyrhizobial inoculation justifying the slight reductionof the 14N15N ratio and increase in N

2-fixation in these

three plant components In P phaseoloides the enrichmentwas higher in roots than in shoots and nodules A similarresult was obtained in a study of the symbiotic N

2-fixation

of P phaseoloides as influenced by litter mineralization usingthe grass Axonopus compressus as a non-N

2-fixing reference

crop [27] This has been attributed to a relatively higherdilution of labelled 15N in shoots of legumes than in rootsby symbiotically fixed N [35 43] Our results agree withOghoghorie and Pate [44] who found that symbiotically fixedN accumulated in shoots and nodules whereas the rootsderived most of its N from mineral sources The pattern ofhigher enrichment in shoots than in roots in I batatasmight

8 Scientifica

be a result of variations in the enrichment in available Nwith time Such variations may be affected by soil type plantspecies and length of the growth period [27] The total N inshoots roots and nodules reported in Table 3 indicates thatrather than the roots and the nodules the shoots explain betterthe difference in total dry matter yield and total N harvestbetween the inoculated and the noninoculated Pueraria Forthis reasonmany studies using the 15N enrichment techniquefocus on analyzing only the aboveground biomass

32 Uptake of Fertilizer and Soil N The recoveries of labelledfertilizer N (Ndff) in inoculated Pueraria noninoculatedPueraria and Ipomoeawere 616 778 and 1027 respec-tively (Table 2)The percentage of fertilizerN recovered in thenonfixing Ipomoea crop was significantly higher than that inthe legume cropThis is particularly important since non-N

2-

fixing references require more nitrogen from other sourcessuch as applied fertilizer N compared to legumes that fixatmospheric nitrogen as another source of N However therecovery of labelled fertilizer N was very low compared tothe results of other studies It reached 63 for P phaseoloidesand 71 in the A compressus reference [27] However theNdff in this study was similar to that obtained in cowpeagrown in field conditions [25] The low recovery of labelledfertilizer N in the present study which correlated with thelow NUE () could be related to the high loss (55ndash60) oflabelled fertilizer N from the top 15 cm of soils (Table 4)Nitrogen can become unavailable to the crop by leaching oras gaseous losses due to denitrification andor volatilizationThe concept of fertilizer use efficiency implies not only themaximum uptake of the applied nutrient by the crop but alsothe availability of the applied nutrient under variable climaticand edaphic conditions Although this study was carried outto determine the nitrogen fixation of Pueraria following fieldinoculations it is important to study the efficient use offertilizers in order to obtain the highest possible yield with aminimum fertilizer application Moreover 36 to 40 of thelabelled fertilizer N was immobilized in the soil (Table 4)Immobilization refers to the incorporation of nitrate andexchangeable ammonium into organic forms that are notavailable to plants This occurs as microorganisms competewith plants for available nitrogen which results in a decreasein the nitrogen availability for the crop The low recovery ofthe labelled fertilizer N justified the high proportions ()of Ndfs in plant materials It was significantly higher in thereference crop (8973) than in the inoculated (5677) andthe noninoculated (6692) Pueraria as shown in Table 2Although no significant difference was observed in theNdfs between the inoculated and noninoculated Puerariabiomass there was an overall increase in the amount of Nuptake (gm2) from soil This is in contradiction with otherstudies which demonstrated that plants tend to extract lessernutrients from the soil stock when other sources are availableIn this regard Rees et al [45] reported a reduced uptakeof soil N following application of legume residues with ahigh N content A reduction of the recovery of 15N labelledfertilizer by application of legume residues with a low CNratio was also reported [46] Although the analysis was not

significant at 5 probability level the residual soil N mainlyderived from the original soil solution small N portions wererecovered from the applied ammonium sulfate fertilizer butalso from theN

2fixed and returned to the soil within the four-

month growth

4 Conclusion

The inoculation with an effective bradyrhizobial strain stim-ulated the growth of field grown P phaseoloides and increasedthe total N amount via an increase of the amounts ofN derived from symbiotic N

2-fixation and from soil The

positive effect of the inoculation on the nodulation pheno-type (increase of nodule dry weight) and N

2-fixation of P

phaseoloides shown in this study indicates that the introducedB yuanmingense S3-4 was competitive with indigenousstrains in field conditions However to find out whetherthis improvement was mainly due to the inoculation theidentification of infection with the used strain in the noduleswould provide more accurate information This study isimportant in that it is the first report describing the nitrogenfixation potential and growth of inoculated P phaseoloidesin field conditions with an effective Bradyrhizobium strainin Eastern Cameroon Furthermore it showed the positivecontribution of field inoculations in maximizing N input insoils during fallows for subsequent cultivationThe subject ofthe study has some relevance to soil fertility management inCameroon which is one of the limiting factors of agriculturalproduction

Conflict of Interests

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

Acknowledgments

This work was supported by the Science and Technol-ogy Research Partnership for Sustainable Development(SATREPS) program of the Japan Science and Technol-ogy Agency (JST) and the Japan International CooperationAgency (JICA) which funded the Cameroon Forest-SavannaSustainability (FOSAS) project To SATREPS the authorsexpress their sincere gratitudeThe authors also would like tothank Mr Dieudonne Ndong the Field Research Technicianof the FOSAS project for his valuable support during thefield work They also acknowledge Mr Shinichi Watanabefrom the Soil Science Laboratory of the Graduate School ofAgriculture Kyoto University for his support during the 15Nisotopic analyses

References

[1] B Heider E Fischer T Berndl and R Schultze-Kraft ldquoAnalysisof genetic variation among accessions of Pueraria montana(Lour) Merr var lobata and Pueraria phaseoloides (Roxb)Benth based on RAPD markersrdquo Genetic Resources and CropEvolution vol 54 no 3 pp 529ndash542 2007

Scientifica 9

[2] W J Broughton ldquoEffect of various covers on soil fertility underHevea brasiliensis muell arg and on growth of the treerdquo Agro-Ecosystems vol 3 pp 147ndash170 1976

[3] B G Cook B C Pengelly S D Brown et al ldquoTropical foragesan interactive selection tool Lablab purpureusrdquo CSIRODPIampF(Qld) CIAT and ILRI Brisbane Australia 2005 httpwwwtropicalforagesinfokeyForagesMediaHtmlLablabpurpureushtm

[4] M Unkovich D Herridge M People et al Measuring Plant-Associated Nitrogen Fixation in Agricultural Systems vol 136 ofACIAR Monograph 2008

[5] K Fujita S Ogata K Matsumoto T Masuda G K Ofosu-Budu and K Kuwata ldquoNitrogen transfer and dry matterproduction in soybean and sorghum mixed cropping system atdifferent population densitiesrdquo Soil Science and Plant Nutritionvol 36 no 2 pp 233ndash241 1990

[6] R Sylvester-Bradley D Mosquera N M Asakawa and GSanchez ldquoPromiscuity and responses to rhizobial inoculationof tropical kudzu (Pueraria phaseoloides)rdquo Field Crops Researchvol 27 no 3 pp 267ndash279 1991

[7] J Postgate Nitrogen Fixation Cambridge University PressCambridge UK 3rd edition 1998

[8] D F Herridge M B Peoples and R M Boddey ldquoGlobal inputsof biological nitrogen fixation in agricultural systemsrdquo Plantand Soil vol 311 no 1-2 pp 1ndash18 2008

[9] P S Sarr S Araki D A Begoude et al ldquoPhylogeny and nitrogenfixation potential of Bradyrhizobium species isolated from thelegume cover crop Pueraria phaseoloides (Roxb) Benth inEastern Cameroonrdquo Soil Science and Plant Nutrition 2015

[10] J G Howieson ldquoThe host-rhizobia relationshiprdquo in GeneticResources of Mediterranean Pasture and Forage Legumes SJ Bennett and P S Cocks Eds Current Plant Science andBiotechnology in Agriculture pp 96ndash106 Springer RotterdamThe Netherlands 1999

[11] Anonymous ldquoFrist step to reduce plant need for nitrogen fer-tilizer uncoveredrdquo 2013 httpwwwsciencedailycomreleases201309130927183314htm

[12] J G Howieson R J Yates K J Foster D Real and R B BesierldquoProspects for the future use of legumesrdquo in Nitrogen-FixingLeguminous Symbioses M J Dilworth E K James J I Sprentand W E Newton Eds vol 7 of Nitrogen Fixation OriginsApplications and Research Progress pp 363ndash394 SpringerDordrecht The Netherlands 2008

[13] J Brockwell P J Bottomley and J E Thies ldquoManipulation ofrhizobia microflora for improving legume productivity and soilfertility a critical assessmentrdquo Plant and Soil vol 174 no 1-2pp 143ndash180 1995

[14] K M Vlassak and J Vanderleyden ldquoFactors influencing noduleoccupancy by inoculant rhizobiardquo Critical Reviews in PlantSciences vol 16 no 2 pp 163ndash229 1997

[15] J G Streeter ldquoFailure of inoculant rhizobia to overcomethe dominance of indigenous strains for nodule formationrdquoCanadian Journal of Microbiology vol 40 no 7 pp 513ndash5221994

[16] M V B Figueiredo J J Vilar H A Burity and F P De FrancaldquoAlleviation of water stress effects in cowpea by Bradyrhizobiumspp inoculationrdquo Plant and Soil vol 207 no 1 pp 67ndash75 1998

[17] B Guillet G Achoundong J Y Happi et al ldquoAgreementbetween floristic and soil organic carbon isotope (13C12C 14C)indicators of forest invasion of savannas during the last centuryin Cameroonrdquo Journal of Tropical Ecology vol 17 no 6 pp 809ndash832 2001

[18] B Mertens and E F Lambin ldquoLand-cover-change trajectoriesin Southern Cameroonrdquo Annals of the Association of AmericanGeographers vol 90 no 3 pp 467ndash494 2000

[19] M Shibata S Sugihara and S Funakawa ldquoSoil nitrogendynamics in tropical seasonal forests of Cameroon a compari-son of two forests under different soil conditionsrdquo in ProgressReport 2013 Forest-Savanna Sustainability Project CameroonA Shioya and K Hayashi Eds pp 369ndash377 FOSAS ProjectIRAD Yaounde Cameroon 2014

[20] T A Johannes M B Vabi and D KMalaa ldquoAdoption of maizeand cassava production technologies in the Forest-Savannahzone of Cameroon implications for poverty reductionrdquo WorldApplied Sciences Journal vol 11 no 2 pp 196ndash209 2010

[21] P S Sarr S Araki and ENjukwe ldquoInteractions between cassavavarieties and soil characteristics in crop production in EasternCameroonrdquo African Study Monograph vol 34 no 4 pp 187ndash202 2013

[22] Soil Survey Staff Keys to Soil Taxonomy United States Depart-ment of Agriculture Natural Resources Conservation ServiceWashington DC USA 10th edition 2006

[23] A IkramMN Sudin and E S Jensen ldquoEstimatingN2-fixation

by Pueraria phaseoloides in Rubber interrows using the 15Nisotopic dilution techniquerdquo Journal ofNatural Rubber Researchvol 10 no 3 pp 199ndash208 1995

[24] J M Vincent A Manual for the Practical Study of the Root-Nodule Bacteria Blackwell Scientific Oxford UK 1970

[25] P S Sarr M Khouma M Sene A Guisse A N Badiane andT Yamakawa ldquoEffect of pearl millet-cowpea cropping systemson nitrogen recovery nitrogen use efficiency and biologicalfixation using the 15N tracer techniquerdquo Soil Science and PlantNutrition vol 54 no 1 pp 142ndash147 2008

[26] A R Zaharah H A H Sharifuddin M N Razley and AK Mohd Saidi ldquoMeasurement of nitrogen fixed by Puerariaphaseoiloides by N-15 dilution techniquerdquo Pertanika vol 9 no1 pp 45ndash49 1986

[27] J M Vesterager S Oslashsterby E S Jensen and J K SchjoerringldquoSymbiotic N2-fixation by the cover crop Pueraria phaseoloidesas influenced by litter mineralizationrdquo Plant and Soil vol 177no 1 pp 1ndash10 1995

[28] E S Jensen ldquoEvaluation of automated analysis of 15N and totalN in plant material and soilrdquo Plant and Soil vol 133 no 1 pp83ndash92 1991

[29] M Fried and V Middelboe ldquoMeasurement of amount ofnitrogen fixed by a legume croprdquo Plant and Soil vol 47 no 3pp 713ndash715 1977

[30] M Fried and L A Dean ldquoA concept concerning the measure-ment of available soil nutrientsrdquo Soil Science vol 73 no 4 pp263ndash272 1952

[31] H Broeshart ldquoQuantitativemeasurement of fertilizer uptake bycropsrdquo Netherland Journal of Agricultural Science vol 22 pp245ndash254 1974

[32] P B Vose Introduction to Nuclear Techniques in Agronomy andPlant Biology Pergamon Press New York NY USA 1980

[33] P S Sarr S Fujimoto and T Yamakawa ldquoNodulation nitrogenfixation and growth of rhizobia-inoculated cowpea (Vignaunguiculata LWalp) in relationwith external nitrogen and lightintensityrdquo International Journal of Plant Biology and Researchvol 3 no 1 article 1025 2015

[34] F-P Rebafka B J Ndunguru and H Marschner ldquoCrop residueapplication increases nitrogen fixation and dry matter produc-tion in groundnut (Arachis hypogaea L) grown on an acid sandy

10 Scientifica

soil inNigerWest Africardquo Plant and Soil vol 150 no 2 pp 213ndash222 1993

[35] J H A Butler and J N Ladd ldquoSymbiotically-fixed and soil-derived nitrogen in legumes grown in pots in soils with differentamounts of available nitraterdquo Soil Biology and Biochemistry vol17 no 1 pp 47ndash55 1985

[36] G Cadisch R Sylvester-Bradley and J Nosberger ldquo 15N-basedestimation of nitrogen fixation by eight tropical forage-legumesat two levels of PK supplyrdquo Field Crops Research vol 22 no 3pp 181ndash194 1989

[37] A Galiana P Balle A NrsquoGuessan Kanga andAM DomenachldquoNitrogen fixation estimated by the 15N natural abundancemethod in Acacia mangium Willd Inoculated with Bradyrhi-zobium sp and grown in silvicultural conditionsrdquo Soil Biologyand Biochemistry vol 34 no 2 pp 251ndash262 2002

[38] A Perin J G M Guerra M G Teixeira and E Zonta ldquoSoilcover and nutrient accumulation of two perennial legumes asfunctions of spacing and planting densitiesrdquo Revista Brasileirade Ciencia do Solo vol 28 no 1 pp 207ndash213 2004 (Portuguese)

[39] J Streeter and P P Wong ldquoInhibition of legume noduleformation and N

2fixation by nitraterdquo Critical Reviews in Plant

Sciences vol 7 no 1 pp 1ndash23 1988[40] B J Carroll and A Mathews ldquoNitrate inhibition of nodulation

in legumesrdquo inMolecular Biology of Symbiotic Nitrogen FixationP M Gresshoff Ed pp 159ndash180 CRC Press Boca Raton FlaUSA 1990

[41] G Hardarson F Zapata and S K A Danso ldquoEffect of plantgenotype and nitrogen fertilizer on symbiotic nitrogen fixationby soybean cultivarsrdquo Plant and Soil vol 82 no 3 pp 397ndash4051984

[42] F A Wahab R A Date R J Roughley A Ikram and SChong Kewi ldquoResponse of perennial leguminous cover cropsin Malaysia to inoculation with Bradyrhizobiumrdquo TropicalGrasslands vol 23 no 1 pp 1ndash7 1989

[43] E F Henzell A E Martin P J Ross and K P HaydockldquoIsotopic studies on the uptake of nitrogen by pasture plantsIV Uptake of nitrogen from labelled plant material by Rhodesgrass and Siratrordquo Australian Journal of Agricultural Researchvol 19 no 1 pp 65ndash77 1968

[44] C G O Oghoghorie and J S Pate ldquoThe nitrate stress syndromeof the nodulated field pea (Pisum arvense L) Techniques formeasurement and evaluation in physiological termsrdquo Plant andSoil vol 35 no 1 pp 185ndash202 1971

[45] R M Rees L Yan and M Ferguson ldquoThe release and plantuptake of nitrogen from some plant and animal manuresrdquoBiology and Fertility of Soils vol 15 no 4 pp 285ndash293 1993

[46] S J Corak M S Smith and C T MacKown ldquoFate of 15Nlabelled legume and ammoniumnitrogen sources in a soil-plantsystemrdquoCommunications in Soil Science and Plant Analysis vol23 no 5-6 pp 631ndash642 1992

Submit your manuscripts athttpwwwhindawicom

Nutrition and Metabolism

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Food ScienceInternational Journal of

Agronomy


International Journal of



Microbiology

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Applied ampEnvironmentalSoil Science

Volume 2014

AgricultureAdvances in


PsycheHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BiodiversityInternational Journal of


ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

GenomicsInternational Journal of


Plant GenomicsInternational Journal of


Biotechnology Research International


Forestry ResearchInternational Journal of


Journal of BotanyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

EcologyInternational Journal of


Veterinary Medicine International


Cell BiologyInternational Journal of


Evolutionary BiologyInternational Journal of


2 Scientifica

bacteria (rhizobia) and crop legumes is likely to be in theorder of 20 to 22 million tons of nitrogen each year [8]

In Eastern Cameroon short-term Pueraria fallows oftwo years are commonly practiced to restore soil fertilityfor subsequent cultivation of cassava which is a staple foodin the area Yield increments of cassava are related to soilquality improvement which includes soil erosion limitationincrease of soil organic carbon and nitrogen following a gooddecomposition of Pueraria litters The phylogenetic diversityof nitrogen-fixing bacteria in the root nodules of Puerariain Eastern Cameroon was previously investigated [9] Usingmolecular biology techniques these authors identified threegroups of Bradyrhizobium species corresponding to B spB yuanmingense and B elkanii that nodulate Puerariaphaseoloides By assessing the nitrogen fixation potential ofthese three groups using the Acetylene Reduction Activity(ARA) method it was found that strains belonging to Byuanmingense showed significant higher efficiencyThe selec-tion of appropriate rhizobial microsymbionts is becominga complex procedure due to the fact that several legumesspecies can be nodulated by single rhizobia [10] Yet the sym-biotic association between the legumes and their microbialsymbionts plays a significant role in agriculture worldwide byreducing ca 100 million metric tons of atmospheric nitrogensaving US$ 8 billionyear on fertilizer N [11 12]The commonapproach to improving the symbiotic fixation of nitrogenand legume productivity using superior or very effectiveexotic rhizobial strains as inoculants often fails to achievethe desired responses [13 14] The failure has been attributedto the poor competitiveness of the introduced rhizobia [15]the nonspecificity of the bacteria and the occurrence andresistance to stress of ineffective indigenous strains in soils[16] To improve the N

2-fixation and growth of Pueraria the

use of rhizobia selected from indigenous community withcapacity for nodulation and strong nitrogen-fixing activity isbelieved to be an important agronomic approach

Pueraria phaseoloides is used in the tropics as a forage andcover crop but rhizobial inoculations of this legume in fieldconditions have not been widely tested The importance ofthis work is to improve the nitrogen fixation potential andgrowth of Pueraria through field inoculations with effectivestrains of Bradyrhizobium enabling enhancement of theinput of nitrogen in the soil via biological process to shortenthe fallow period of Pueraria and put the land in faster use aswell as reduce environmental hazards that the use of chemicalfertilizers to increase yields often poses In this paper wereport on the effects of inoculating Puerariawith the selectedefficient B yuanmingense S3-4 strain [9] on its N

2-fixation

dry biomass production total N uptake nitrogen fertilizeruse efficiency and soil fertility through a field experimentSymbiotic nitrogen fixation was estimated using the 15Ntracer technique and Ipomoea batatas (L) Lam was used as anonfixing reference plant

2 Materials and Methods

21 Study Site The study was carried out in Andom village(5∘151015840N 13∘301015840E) located in Eastern Cameroon which is inthe forest-savanna transition zone [17 18] The elevation of

the plateau is 650ndash700m asl and its basement consists ofmetamorphic rocks The annual rainfall range of this areais 1350ndash1550mm and the distribution of rainfall is bimodalaccording to fieldmeasurement carried out from 2008 to 2013[19] Rains begin in mid-March a short dry season followsfrom mid-July to the end of August and then the main rainyseason extends from September to the middle of NovemberThe mean annual temperature in the region is 25∘C TheDecemberndashFebruary period is the driest part of the yearwhenthe relative humidity may be higher than 60 [20] The soilpH in the cultivated land varies from 482 to 517 with averagelevels of organic C total N and available P of 181 0113and 457 ppm respectively and a CN ratio of 1591 [21]Soils are classified as Typic Kandiudox according to USDAclassification system [22] The dominant forest vegetation isAlbizia zygia which dominates the tree layer The dominantspecies in the savanna are the perennial grasses Pennisetumsetaceum Imperata cylindrica and Chromolaena odorata [1718] The experiment was set on a cleared natural fallow landpreviously dominated by Chromolaena odorata There havebeen no planted legumes including Pueraria and no historyof rhizobial inoculation was observed in the site

22 Experimental Design Treatments consisted of inoculatedPueraria and noninoculated Pueraria each replicated threetimes in a complete randomized block design Ipomoeabatatas plots were interspersed in each Pueraria plot as non-N2-fixing references giving a total of 12 plots (4m2 plotminus1)

with a planting density of 9 plants per 15m times 15m Ibatataswas used as a reference crop as it shares with Puerariasimilar patterns of N uptake from the soil and will obtainsimilar 15N enrichment in the soil-derived N [23] The useof a reference crop is important in estimating the amountof nitrogen derived from the atmosphere (Ndfa) of legumesEach Pueraria-Ipomoea block was separated to others by a2m interval (Figure 1) Pueraria seeds were sown on 9August2014 at 50 cm spacing Uniform Ipomoea seedlings of 10 cmlength were obtained from farmersrsquo fields and transplanted intheir corresponding plots at 50 cm spacing two weeks afterPueraria seeds were sown

23 Rhizobial Inoculation and Application of 15N LabelledAmmonium Sulfate Prior to this field experiment rhizobialstrains were isolated from Pueraria nodules collected fromthe experimental site and other neighboring fields andsubjected to DNA-based molecular analysis [9] The resultsshowed that the native strains for Pueraria in the experimen-tal field clustered into 3 groups corresponding to Bradyrhizo-bium yuanmingense B elkanii and B sp By assessing theirnitrogen fixation potential using the Acetylene ReductionAssay (ARA) it appeared the B yuanmingense strains andS3-4 in particular showed a significant highest potentialTherefore Bradyrhizobium yuanmingense S3-4 identified asan efficient Pueraria symbiont [9] was used in this fieldexperiment From glycerol stock it was streaked on YMAagar plates [24] for one week After colony developmentone pure colony was grown in 20mL YMB for anotherweek to produce the inoculum The obtained solution of2094 times 109 cellsmLminus1 was diluted 1000 times with sterilized

Scientifica 3

IPIP

NIP1

NIP2

IpB2IpB1

IpB3

IpB5 IpB6

IP1

IpB4 NIP3

2m

2m

2m


2-fixing





4 Scientifica













)

times 100

(3)
















Scientifica 5







(Plant Ndff



) (9)

Therefore

Total soil N (gm2)


plant Ndff)

(10)



) times 100 (11)



















6 Scientifica










2-





2-fixation








2

18

16

14

12

1

08

06

04

02

0IP NIP

PlantsIpomoea

Har

veste

d pl

ant N

(gm

2)

NdffNdfsNdfa

ab

01122

10624

0644

00737

0664

0256

0706

00776







Scientifica 7


















2-fixation in these


2-fixation


2-fixing reference


8 Scientifica



2-




month growth

4 Conclusion




2-fixation of P




Acknowledgments


References


Scientifica 9



































10 Scientifica


















Journal of




Agronomy





Microbiology




Volume 2014
























Scientifica 3

IPIP

NIP1

NIP2

IpB2IpB1

IpB3

IpB5 IpB6

IP1

IpB4 NIP3

2m

2m

2m


2-fixing





4 Scientifica













)

times 100

(3)
















Scientifica 5







(Plant Ndff



) (9)

Therefore

Total soil N (gm2)


plant Ndff)

(10)



) times 100 (11)



















6 Scientifica










2-





2-fixation








2

18

16

14

12

1

08

06

04

02

0IP NIP

PlantsIpomoea

Har

veste

d pl

ant N

(gm

2)

NdffNdfsNdfa

ab

01122

10624

0644

00737

0664

0256

0706

00776







Scientifica 7


















2-fixation in these


2-fixation


2-fixing reference


8 Scientifica



2-




month growth

4 Conclusion




2-fixation of P




Acknowledgments


References


Scientifica 9



































10 Scientifica


















Journal of




Agronomy





Microbiology




Volume 2014
























4 Scientifica













)

times 100

(3)
















Scientifica 5







(Plant Ndff



) (9)

Therefore

Total soil N (gm2)


plant Ndff)

(10)



) times 100 (11)



















6 Scientifica










2-





2-fixation








2

18

16

14

12

1

08

06

04

02

0IP NIP

PlantsIpomoea

Har

veste

d pl

ant N

(gm

2)

NdffNdfsNdfa

ab

01122

10624

0644

00737

0664

0256

0706

00776







Scientifica 7


















2-fixation in these


2-fixation


2-fixing reference


8 Scientifica



2-




month growth

4 Conclusion




2-fixation of P




Acknowledgments


References


Scientifica 9



































10 Scientifica


















Journal of




Agronomy





Microbiology




Volume 2014
























Scientifica 5







(Plant Ndff



) (9)

Therefore

Total soil N (gm2)


plant Ndff)

(10)



) times 100 (11)



















6 Scientifica










2-





2-fixation








2

18

16

14

12

1

08

06

04

02

0IP NIP

PlantsIpomoea

Har

veste

d pl

ant N

(gm

2)

NdffNdfsNdfa

ab

01122

10624

0644

00737

0664

0256

0706

00776







Scientifica 7


















2-fixation in these


2-fixation


2-fixing reference


8 Scientifica



2-




month growth

4 Conclusion




2-fixation of P




Acknowledgments


References


Scientifica 9



































10 Scientifica


















Journal of




Agronomy





Microbiology




Volume 2014
























6 Scientifica










2-





2-fixation








2

18

16

14

12

1

08

06

04

02

0IP NIP

PlantsIpomoea

Har

veste

d pl

ant N

(gm

2)

NdffNdfsNdfa

ab

01122

10624

0644

00737

0664

0256

0706

00776







Scientifica 7


















2-fixation in these


2-fixation


2-fixing reference


8 Scientifica



2-




month growth

4 Conclusion




2-fixation of P




Acknowledgments


References


Scientifica 9



































10 Scientifica


















Journal of




Agronomy





Microbiology




Volume 2014
























Scientifica 7


















2-fixation in these


2-fixation


2-fixing reference


8 Scientifica



2-




month growth

4 Conclusion




2-fixation of P




Acknowledgments


References


Scientifica 9



































10 Scientifica


















Journal of




Agronomy





Microbiology




Volume 2014
























8 Scientifica



2-




month growth

4 Conclusion




2-fixation of P




Acknowledgments


References


Scientifica 9



































10 Scientifica


















Journal of




Agronomy





Microbiology




Volume 2014
























Scientifica 9



































10 Scientifica


















Journal of




Agronomy





Microbiology




Volume 2014
























10 Scientifica


















Journal of




Agronomy





Microbiology




Volume 2014


























Journal of




Agronomy





Microbiology




Volume 2014
























Symbiotic N 15 N Tracer Technique Pueraria phaseoloides...

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