Influence of Organic Nitrogen on Soil Nitrogen, Nodulation, Nitrogen Fixation,and Yield of Soybeans1

D. F. BEZDICEK, R. F. MULFORD, AND B. H. MAGEE2

ABSTRACTNitrogen was applied on a previous rye (Secale Cereale L.)

cover crop for later determination of the effect of organic Non soybean (Glycine max L.) N2-fixation rate, plant dry mat-ter, N uptake, and yield. Nitrogen was applied at 0, 56, 112,and 224 kg N/ha as NH4NO3 or urea either on rye in mid-March or at soybean planting time in a 4 x 2 x 2 factorialdesign. In both years of study, N2-fixation rate as estimatedfrom acetylene reduction was reduced throughout the seasonby increased fertilization rate. For 1 year, interactions showedthat N2-fixation rate and nodule mass were stimulated late inthe season from increased N fertilizer applied to the rye butnot at soybean planting. Interactions between NH4NO3 andurea and time of application showed that NH4NO3 as com-pared with urea applied on rye significantly increased soybeandry matter, N uptake, and yield. Urea applied at plantingincreased these parameters in comparison with NH4NO3. Theobserved increases in dry matter, N uptake and yield fromrye-applied NH4NO3 were not accompanied by a correspond-ing increase in N2-fixation rate or nodule mass. This wouldsuggest that the N2 fixing system was not responsible for theincrease of dry matter, N uptake, and yield. Soil organic N wasnot measurably influenced by the treatments imposed.

Additional Index Words: Glycine max., N2 fixation efficiency,nodule mass, acetylene reduction method.

Y IELD RESPONSES from inorganic N applications to soy-beans have been contradictory. Lathwell and Evans

(12) reported that soybean yield was closely related toaccumulated plant N and that N applied at bloom stageincreased yield. Other studies of yield increases from N fer-tilizer have been reported by Pettiet (16), Bhangoo andAlbritton (5), Cartter and Hartwig (7), and Ohlrogge(14). However, numerous studies, both published and un-published, have shown negative response of N fertilizationon soybean yield. Factors such as availability of soil N, rateand time of application, placement, type of carrier, andeffectiveness of native or applied rhizobia influence theresponsiveness of soybeans to N fertilizer.

Better agreement is found on the inhibitory effects ofinorganic N on legume N2 fixation. Soybeans have theoption of obtaining N from mineralized soil N, fertilizer N,or from the symbiotic N2-fixing system. Early work byFred, Baldwin, and McCoy (8) showed that nodulation andsubsequently N2 fixation can be inhibited by added inor-ganic N. Later experiments (13) with labeled 15N showed

1 Contribution no. 4795 and Scientific Article no. A1880 ofthe Maryland Agr. Exp. Sta., Dep. of Agronomy. Presented be-fore Div. S-3, Soil Science Society of America, Miami, Florida,1 Nov. 1972. Received 27 April 1973. Approved 31 Oct. 1973.2 Formerly Assistant Professor, Dep. of Agronomy, Univ. ofMaryland, now Associate Professor, Dep. of Agronomy, Wash-ington State Univ., Pullman; Faculty Research Assistant; andAgricultural Aide, Dep. of Agronomy, Univ. of Maryland, re-spectively.

that as inorganic N was increased, total plant N assimilatedby symbiotic N2 fixation decreased. Similar results wereobtained with labeled 15N in nutrient culture (1, 2), Morerecently, N2 fixation inhibition by inorganic N has beendemonstrated with the C2H2-C2H4 method (10).

Several studies (2, 9, 15) have suggested that smallamounts of combined N have increased N2 fixation or pro-duced yield increases presumably by stimulating plantgrowth prior to active nodulation and fixation. Hardy andHolsten reported that soybean meal used as a form of Nfertilizer did not markedly affect N2 fixation but producedyield increases of greater than 20% (R. W. F. Hardy andR. D. Holsten. 1971. Nitrogen fertilization of soybeans:Effects of N fixation and yield. Agron. Abstracts p. 82.).

Other investigations (2, 12, 13, 16) have suggested thatcombined N is necessary for maximum growth response.Although many of these studies show that combined N nor-mally reduces the amount of N2 fixed symbiotically, Har-per and Cooper (11) have shown that nodulation was notaffected when combined N was placed away from the nod-ule zone but was reduced when uniform incorporation wasattempted. These and other studies suggest that the N2-fix-ation system may be stimulated from combined N whenapplied either at critical time periods or away from thenodule zone.

The application of these observations would favor theuse of N in some organic form where N would be releasedat a rate slow enough to supply plant needs at critical timeperiods but not at a rate that would inhibit N2 fixation.Present investigations were initiated to determine if organicN residues applied from a previously-fertilized rye cropwould influence N2-fixation rate and other parameters.

MATERIALS AND METHODSThe experimental site was located on Evesboro loamy sand

at the Poplar Hill Research Farm near Quantico, Maryland.Rye (Secale cereale L.) was planted as a cover crop in the fallof 1969 and 1970. Yearly spring fertilizer application was ap-plied in a 2 x 2 x 4 factorial design as follows with four repli-cations: type of fertilizers, NH4NO3 and urea; time of applica-tion, on rye in March and at soybean planting; and fertilizerrate, 0, 56, 112, and 224 kg N/ha broadcast. Individual four-row plots were 3.1 m by 6.7 m. 'Kent' soybeans Glycine max L.Merr) were planted in 1970 and 1971 on 24 May after the ryeresidues were shredded and turned under. Total dry residue ofrye was approximately 4,600 and 2,400 kg/ha for rye-appliedN and no-rye N, respectively.

Soil samples were taken periodically from each plot and ana-lyzed for pH and extracted with 0.05JV HC1-0.025A' H2SO4 foranalysis of Ca, Mg, P, and K by the methods used in the Mary-land Soil Testing Laboratory (3). Soil NH4

+ and NO3~ wereextracted with IN KC1 and determined by steam distillation asdescribed by Bremner (6). Soil organic N was determinedafter 0.01M CaCl2 extraction and autoclaving according to themethod of Stanford and DeMar (17).

Nitrogen fixation rate of soybean nodules was determined bythe acetylene reduction (C2H2-C2H4) method described byHardy et al. (10) with the following modifications. One-foot

268

BEZDICEK ET AL.: EFFECT OF ORGANIC N ON SOIL N, AND NODULATION, FIXATION, AND YIELD OF SOYBEANS 269

(0.305-m) sections of row containing soybean plants were re-moved randomly from the center two rows of each plot fol-lowed by separating the roots from the tops. The nodulatedroots were placed in either 500- or 1,000-ml jars depending onthe volume of roots. The containers were attached to a 12-jarmanifold system whereby air was removed by a vacuum pumpand replaced with a mixture containing 75% Ar and 25% O2.The evacuation process was repeated four times except that thelast addition of the Ar-O2 mixture was added to less than atmos-pheric pressure to accommodate the addition of acetylene gas.One-hundred milliliters of purified acetylene gas were addedwith a syringe to the jars to bring the final pressure to atmos-pheric or slightly above. Final partial pressures for C2H2, O2,and Ar were 0.10, 0.22, and 0.68 atm, respectively. After incu-bation for 40 min, approximately 5 ml were removed with plas-tic syringes or blood vacuum tubes. Blood vacuum tubes werefound to be superior because gas samples could be stored with-out significant loss of sample. Ethylene production was deter-mined by injection of 0.5 ml into an Aerograph Model 200 gaschromatograph equipped with an H2-flame ionization detector.Acetylene and ethylene were separated on a 0.3 cm by 45 cmcopper column containing Porapak type N, 80-100 mesh. Injec-tor and detector temperatures were 75 and 100C, respectively.Purified N2 carrier gas at 26 ml/min provided retention timesfor C2H4 and C2H2 of 0.5 and 1.0 min, respectively. Standardgases were obtained from the Matheson Company. Impuritiescontained in C2H2 were removed with a concentrated H2SO4scrubber. Total moles C2H2 -> C2H4 were calculated fromknown standards on an hour-hectare basis after computation ofnecessary volume and land-area corrections.

Nodule mass and number were determined on roots obtainedfrom the 0.305-m row sample. Total soybean top growth (drymatter) was determined on the tops of the same sample. Planttops were analyzed for total Kjeldahl N; whereas samples weredigested with HC1O3-HNO3 and analyzed for P, Ca, Mg, and Kby methods used in the Maryland Soil Testing Laboratory (4).Soybean yield was determined from two 4.9-m rows.

RESULTS AND DISCUSSION

In 1970, N2(C2H2)-fixation rate and nodule mass weredetermined on 18 and 31 August. Interactions between vari-ables imposed were generally not significant for either ofthe dates studied. Main effects observed for fertilizationrate, time, and carrier on 31 August are shown in Table 1.Both N2(C2H2)-fixation rate and nodule mass appeared todecrease with increased fertilization rate. Nitrogen signifi-cantly decreased nodule mass. These observations are con-

Table 1—Nitrogen fixation rate and nodule mass on 31 Augustand soybean yield as influenced by fertilization rate, time

of fertilizer application, and type of fertilizer carrierat Poplar Hill, 1970

Fertilizertreatment

Fertilization rate (R)kgN/ha

056

112224

Time of application (T)N on rye

N at plantingFertilizer carrier (C)

NH4NOSUrea

N3 (CgH^fbcatlon rateMoles CjH2 — CjHj/hour per ha

2.82. 82.41.5

2.62. 1

2.22.5

Nodulemass

355"297229140

280229

252257

Soybeanseedyield

•kg/ha ————

1,8821,9491,8351.767

1,9151,801

1,8681,848

** Significantly different at the \% level within each factor studied. Means without sub-scripts are nonsignificant.

sistent with many other studies reported in the literature.Nitrogen fixation rate and nodule mass appeared to de-crease but not significantly from N applied at plantingbut not on rye. Soybean yield was not significantly influ-enced by any of the treatments imposed. Data obtained for18 and 31 August are similar. Therefore, the data forAugust 18 are not reported. Soil data were not determinedin 1970. Below-average rainfall during the summer of 1970may have accounted for the lack of response to treatments.

The 1971 studies included soil N measured at two datesin addition to nodule and plant N data measure at fivedates. The influence of fertilizer treatments on soil N andsoybean seed yield is shown in Table 2. Both soil NH4

+ andNO3" at 36 days significantly increased with increased fer-tilization rate. Nitrogen applied at planting significantlyincreased NH4

+ and NO3~ when compared with rye-appliedN. Soil NH4

+ and NO3~ were significantly higher from Napplied as urea when compared with the NH4NO3 treat-ment. Soil values at 107 days after planting were not in-fluenced by fertilizer treatments except that soil NO3~ wassignificantly increased by increase in N application rate.Organic N appeared to increase from the earlier samplingdate. A slight increase in organic N was noted for NH4NO3

when compared with urea.

Table 2—Soil NH4 + , NO3~, and organic N at two dates and soybean yield as influenced by fertilization rate, time of fertilizationapplication, and type of fertilizer carrier at Poplar Hill, 1971

SollN36 days after planting

Fertilizertreatment

Fertilization rate <B)kg/ ha

056

112224

Time of application (T)N on rye

N at planting

Fertilizer carrier (C)NH.NO,

Urea

NHj

l.Ot"1.33.3

12.3

0. 8t"8.0

2.3t"6.7

NO,-

5. 8f8.0

13.122.8

6.5J"18.0

11. Of13.5

OrganicN

70817784

7878

8076

107 days after planting

NHf

1.91.71.71.9

1.81.8

1.71.8

NOT

1.8*2.01.92.2

1.92.0

2.01.9

OrganicN

121140163151

155133

158f130

Soybeanseed yield

kg/ha

2.386J2,2782,3252,218

2.392J"2,204

2,278t2,325

Significantly different at the 5% level within each factor studied.** Significantly different at the 1% level within each factor studied.t Significantly different at the 10% level within each factor studied.j Significant Interactions of 5% or greater observed between another factor In the same column.

270 SOIL SCI. SOC. AMER. PROC., VOL. 38, 1974

56 112FERTILIZER N-kg/ho

224 ON RYE AT PLANTINGTIME OF APPLICATION

56 112FERTILIZER N-kg/ha

224

56 112FERTILIZER N-kg/ha

224 224 ON RYE AT PLANTINGTIME OF APPLICATION

56 112FERTILIZER N-kg/ha

Fig. 1—Significant interactions observed for soil NH4 + , soil NOs~, and soybean N2(C2Ha)-fixation rate at 36 days after planting.Symbols indicate the following: R X C, N fertilization rate and fertilizer carrier; R X T, N fertilization rate and time of appli-cation; and T X C, time of application and fertilizer carrier.

Significant interactions observed at 36 days for soil NH4 +

and soil NO3~ indicated in Table 2 and for N2 (C2H2)-fixation rate are shown in Fig. la-/. The rate X carrier(R X C) interaction in Fig. \a shows that the increase insoil NH4

+ observed with increased fertilization rate waslargely from urea rather than from NH4NO3. Furthermore,the rate X time (R x T) interactions in Fig. \b and leshow that both soil NH4

+ and NO3~ were highest for Napplied at planting, but were considerably lower when ap-plied to the rye. Thus, the increases in soil NH4

+ and NO3~with fertilizer rate observed in Table 2 were due largely tourea rather than NH4NO3 applied at planting. As shown inFig. le and If, soil NH4

+ and NO3~ levels were higher atplanting from urea than NH4NO3. The significant R X Tinteraction for N2(C2H2)-fixation rate (Fig. Id) showsthat fixation rate was decreased to a smaller extent whenN was applied to the rye rather than at planting. Similarinteractions not shown were obtained for nodule mass.

The influence of fertilization treatments on soybean nod-ule mass, N2(C2H2)-fixation rate, and N2 fixation efficiencyis shown in Table 3. Nodule mass was significantly de-creased by increased fertilization rate and by the proximityof N application to planting at all dates reported. No signif-icant differences in nodule mass were found between Nsources. The significant interactions between fertilizationrate and time of application at 36 and 107 days showedthat the greatest reduction in nodule mass was from Napplied at planting.

Nitrogen fixation rate, which followed a similar patternto nodule mass, generally increased through the season to90 days when a decrease was noted. Significant decreases inN2 fixation rate were noted with increased fertilization rateand from the proximity of N application to planting. Nitro-gen sources had little influence on N2 fixation rate exceptat 107 days where fixation was significantly greater forurea in comparison to NH4NO3. The significant interaction

Table 3—Soybean nodule mass, N»(C2H2)—fixation rate, and Nj fixation efficiency as influenced by fertilization rate, time of fer-tilizer application, and type of fertilizer carrier at Poplar Hill, 1971

Fertilizertreatment

Fertilization rate (R)kg/ha

056

112224

Time of application (T)N on rye

N at planting

Fertilizer carrier (C)NH,NOj

Urea

Nodule masskg/ha

36

176t"815016

81f"46

5770

49 72

293" t233 t160 t56 J

238" |133 t

183 t188 t

90

499"505366222

456"339

419376

107

599t"414433343

563t"312

400474

Nj(CjHj) - fixation ratemoles CjHj — CjHj/hour per ha

36

1.6t"1.10.80.2

1.2T"0.7

0.81.1

Days after plantli49 72

2.7f2.21.50.6

2. 2f"1.3

1.81.7

1.7"1.01.20.8

1.4T*1.0

1.2f1.2

90

5.6f6.04.13.5

5.2f4.4

5.34.3

107

3.9T3.23.42.8

4.22.5

2.83.8

Fixation efficiencypmoles C&t — CaH^/g nodule hour

36

15131514

1513

1414

49

1199

12

911

912

72

1ttt

tt

»t

90

12121214

1212

13t12

107

7101010

711

8*11

Significantly different at the 5% level within each factor studied.** Significantly different at the 1% level within each factor studied.t Significant Interactions of 5% or greater observed between another factor In the same column.t Data not taken.

BEZDICEK ET AL.: EFFECT OF ORGANIC N ON SOIL N, AND NODULATION, FIXATION, AND YIELD OF SOYBEANS 271

shown at 72 days between time of fertilizer application andN source showed that NH4NO3 decreased N2 fixation ratemore than did urea on rye; whereas, at planting, urea de-creased fixation relative to NH4NO3.

The most frequent and significant interaction observedfor N2 fixation rate was between N fertilization rate andthe time of N application. These are shown in Fig. 2 for 49,90, and 107 days after planting. The same R X T interac-tion for 36 days was previously presented in Fig. Id. Fixa-tion rate was reduced with increased fertilizer N at plantingfor all sampling dates. However, fixation rate was reducedto a smaller degree at 49 days when N was applied to therye. A slight increase in N2 fixation rate was observed at 90days from 56 kg N/ha applied to the previous rye crop.However at 107 days after planting, N2 fixation rate wasstimulated from rye N at rates up to 224 kg/ha; whereas,fixation rate was drastically decreased by N applied at soy-bean planting. These results suggest that N combined organ-ically in the rye released N at a rate sufficient to stimulatethe N2 fixation system late in the season. It is interesting tonote that rye-applied N at 56 kg/ha slightly increased thefixation rate at all sampling dates.

Nitrogen fixation efficiency was not significantly influ-enced by any of the fertilizer treatments except at 107 dayswhere a higher value was found for urea in comparison toNH4NO3 (Table 3). Fixation efficiency was generallyhigher earlier in the season but decreased at the last sam-pling date. Stimulation of N2 fixation by rye-applied N(Fig. 2) was not apparently a result of increased N2 fixa-tion efficiency since similar fertilizer rate X time interac-tions were not obtained for fixation efficiency. However,since similar fertilizer rate X time interactions obtained forN2 fixation were observed for nodule mass at 36 and 107days, the stimulation of N2 fixation by rye-applied N waslikely from increased nodule mass. The significant increasein soybean seed yield (Table 2) from rye-applied N mayhave been due to the increased N2 fixation rate late in theseason. However, influence of other nutrients or favorableenvironmental conditions produced from the rye-applied Ncannot be dismissed.

Percent plant N, N uptake, and dry matter accumulationof soybeans as influenced by fertilizer treatments are shownin Table 4. Plant N was significantly increased by addedfertilizer N and by the proximity of N application to plant-

6.0

5.0

oeg

i 4.0

3.0

2.0

1.0

FERTILIZER N-k9/ha0 56 112 224

ON AT ON AT ON ATRYE PLANTING RYE PLANTING RYE PLANTING

49 90 107AGE (DAYS)

Fig. 2—Relation between N2_(C2H2) -fixation rate and time offertilizer N application as influenced by N application rateafter 49, 90, and 107 days after planting.

ing. No significant differences in plant N were observedbetween N sources. Few significant differences in N uptakeand dry matter were noted with increased fertilizer N ex-cept N uptake at 72 days was significantly increased. Sig-nificant differences between N sources were obtained onlyat 107 days where N uptake and dry matter were signifi-cantly higher from urea application. In addition to theseobservations, N2 fixation rate at 107 days was also signifi-cantly higher from urea application relative to NH4NO3(Table 3). It is interesting to note the apparently greaternodule mass for urea at 107 days. Whether the greater drymatter production from the urea application placed a Ndemand on the plant or whether N was limiting is not im-mediately known. However, it does appear that N was notlimiting plant growth since the plant N composition at 107days was approximately 3.6% for all treatments (Table 4).It is difficult to determine why the stimulation of dry mat-ter, N uptake, and N2 fixation efficiency for the urea ap-plication were not evident until late in the season. A late-rainy season in 1972 did promote dry matter productionand N2 fixation later than usually observed. Higher, butnot significant values for nodule mass, N2 fixation rate, Nuptake, and dry matter observed for NH4NO3 at 90 days

Table 4—Percent plant N, N uptake, and dry matter accumulation of soybeans as influenced by fertilization rate, time of fertilizerapplication, and type of fertilizer carrier at Poplar Hill, 1971

Fertilizertreatment

Fertilization rate.(H)kg/ha

056

112224

Plant N - %

36

3.43.64.03.9

49

3.5"3.73.64.0

72 90 107

3.1* 3.4 3.63.1 3.4 3.53. 2 3. 2 3. 53.4 3.3 3.6

N uptake -

36 49

18t 5620 6220 5921 66

Days al72

108*119120160

kg/haEter pla

90

199206194185

Dry matter

107

263271245275

36

513534491526

49

1,5861,6631,6331,651

72

3,5693,7843,7543,978

- kg/ha

90

5,8196,1635,9485,603

107

7,2407,7157,1127,629

Time of application (T)N on rye

N at planting

Fertilizer carrier (C)NHjNQ,

Urea

3.63.8

3.63.8

3.6«3.9

3.63.8

3.1*3.3

3.63.6

3. 2 3.33.2 3.3

3.63.6

1920

19t20

59t62

58T63

161135

129124

206t185

205t186

271255

243*283

509 l,646t 3,848526 1,616 3,698

521 l,603t 3,745509 1.659 3,797

6,163f 7,7155,603 7,155

6,189t 6,892"5.573 7.965

Significantly different at the 5% level within eaeh factor studied.* Significantly different at the 1% level within each factor studied.

Significant Interactions of 5% or greater observed between another factor In the same column.

272 SOIL SCI. SOC. AMER. PROC., VOL. 38, 1974

Table 5—Soybean plant N uptake, dry matter accumulation, and seed yield as influenced by two methods of fertilizer applicationand two fertilizer sources at Poplar Hill, 1971

Fertilizerapplication

N on ryeN at planting

49NH4N03

6253

N uptakeBays after planting

Urea NH,NO3

56 23071 180

Dry miittcrDays after planting

90Urea

183190

NH4NOj

1,7581,448

49Urea

kg/ha ———1,5301,789

90NHjNOj

6,9265,344

Urea

5,3445,780

YieldNH4NO3

2,5472,224

Urea

2,4722,400

Data represent significant Interactions (>. 05) between fertilizer application time and sources. Main effects are shown In Tables 2 and 4.

suggest that these parameters reached a peak earlier in theseason.

Although changes in soybean dry matter and total Nuptake were seldom significantly influenced by either timeof fertilizer application or N source, significant interactionsdid occur at 49 and 90 days (Table 4). These and a similarinteraction for soybean yield are shown in Table 5. Ammo-nium nitrate as compared with urea applied to the rye in-creased these parameters in all cases; whereas, decreaseswere observed when NH4NO3 was applied at planting. Theyield increases from rye-applied NH4NO3 are related tosimilar increases in dry matter and N uptake. This wouldsuggest that the rye-applied N increased yield as a result ofthe increased dry matter production. Similar and significanttime X carrier interactions (not shown) were observed fortotal soybean P and K uptake at 49 and 90 days. Thus, theincreased dry matter production observed from rye-appliedNH4NO3 may have been influenced by nutrients otherthan N such as P or K.

The time X carrier interactions observed for dry matter,N uptake, and yield were not accompanied by similar cor-responding interactions for N2 fixation rate. A significanttime X carrier interaction (Table 3) for N2 fixation ratewas observed at 72 days. However, the fixation rate wasgreater for urea applied to rye than for NH4NO3 applied atplanting which is the reverse of the above interactions. Thiswould suggest that the observed increases in dry matter, Nuptake, and yield shown in Table 5 from rye-appliedNH4NO3 were not directly due to stimulation of the N2fixing system but rather from the soil system. Apparently,the soil environment was favorably influenced by the appli-cation of NH4NO3 but not from urea applied to the rye.The conducive factors may be related to nutrients otherthan N since uptake of both P and K were increased fromthe rye-applied NH4NO3.

SUMMARY AND CONCLUSIONS

Application of N to rye preceding a soybean crop wasshown to significantly reduce the inhibition of N2(C2H2)-fixation rate when compared to N fertilizer applied at soy-bean planting. Significant interactions showed that N ap-plied at rates to 224 kg N/ha on rye increased N2 fixationrate late in 1971 when compared to similar rates applied atsoybean planting. Slight stimulation of N2 fixation was ob-served throughout the season from 56 kg N/ha applied tothe rye. Significant differences in dry matter, N uptake,and yield were observed between fertilizer carrier and timeof application. These interactions showed that dry matter,N uptake and soybean yield were significantly increased by

NH4NO3 relative to urea applied to the rye. The growthand yield increases observed could not be accounted for bystimulation of N2 fixation system. Total uptake of P and Kwas also increased from NH4NO3 application to rye whichsuggests that the rye-applied N responses on a subsequentsoybean crop may not be due to N alone. These results sug-gest that low rates of organically-applied N can stimulatethe symbiotic N2 fixing system throughout the growing sea-son. Higher rates were shown to stimulate fixation later inthe season. Application of inorganic N at soybean plantingsignificantly reduced N2 fixation throughout the growingseason. Soybean seed yields were significantly higher fromrye-applied N relative to inorganic N applied at soybeanplanting.

ARDAKANI ET AL.I AMMONIUM AND NITRITE OXIDATION IN A SOIL FIELD PLOT 273