Potassium Effects on Improved Growth, Nodulation, and Nitrogen Fixation of Hairy Vetch1

J. Q. LYND, EDWARD A. HANLON, JR., AND GEORGE V. ODELL, JR.2

ABSTRACTSymbiotic N fixation by forage legumes presents practical

means for improved pasture productivity. However, soil fer-tility and plant nutrient deficiencies may greatly limit legumegrowth, nodulation, and nitrogenase activity levels.

Results from 3 years' field and greenhouse experiments wereevaluated to determine the influence of K and P levels withand without Ca on growth, nodulation, nitrogenase (CsHj. re-duction) and nodule cytosol components of 'Madison' hairyvetch (Vicia villosa Roth) grown in a siliceous thermic Psam-mentic Paleustalf (Eufaula). Significant increases in bothgrowth and seed yield resulted from application of 50 mg Pand 100 mg of K/kg soil. Nodulation and nitrogenase activity(C2H2 reduction) at anthesis (248 day age) were significantlyincreased with both P and PK treatments. Nitrogenase activitylevels correlated with nodule mass with highly significant in-creases resulting from the K treatment. Phosphorus contentof nodule cytosol was significantly increased with 0, 50, 100,150 mg P/kg soil in factorial combinations with 100 mg of Kand 400 mg of Ca/kg soil. Nitrogenase activity levels were in-creased with all K treatments and effects of K were highlysignificant influencing every parameter except cytosol Ca. Cy-tosol Na was reduced inversely to increased K content. Growthincreased in quadratic response to 0, 100, 200, and 300 mg ofK applied with and without 100 mg of P/kg soil. Highly sig-nificant linear increase in nodule weight, nitrogenase activity,and nodule cytosol K resulted with increased K levels withoutP interaction. Cytosol Ca, Mg, and P were significantly in-creased, and highly significant decreases in Na resulted withincreased K applications. The most reliable response surfaceby multiple regression with R2 = 0.738 was: nitrogenase = 48.36nodule weight + 0.1 K - 1.44 top weight - 0.04 Na + 0.01 Ca,C.V. = 27.4%. Nodule weight X nitrogenase correlations werehighly significant with negative correlations existing for cytosolNa and every other parameter.

Additional Index Words: Rhizobium leguminosarum Frank,pasture legumes, Vicia villosa, nodule cytosol.

Lynd, J. Q., E. A. Hanlon, Jr., and G. V. Odell, Jr. 1981. Po-tassium effects on improved growth, nodulation, and nitrogenfixation of hairy vetch. Soil Sci. Soc. Am. J. 45:302-306.

HAIRY VETCH (Vicia villosa Roth) is a desirablelegume for improved forage production when

grown both with annual cool season small grain com-binations and with perennial warm season bermuda-grass (Cynodon dactylon L. Pers.) pastures. Althoughthis legume is well adapted to a wide range in climateand soil conditions, productivity and symbiotic nitro-gen fixation are greatly influenced by soil fertility (3).

Earlier studies (6) were of particular value for sta-tistical evaluation of bermudagrass-vetch productionwith yearling steers grazing experimental pasturesestablished on contrasting land types with differentialsoil fertility and grazing regimes. These studies wereutilized for field evaluation of hairy vetch and severalclovers within bermudagrass pastures during 1946 to1961. Systematic observations for legume persistenceand productivity were continued during 1962 to 1975within the paddock areas under continuous intensivegrazing as a cow-calf ranch operation with no addi-tional soil fertility treatments. The favorable responseof hairy vetch to K fertilization in these studies hasbeen consistent resulting in greater productivity andreseeding persistence, particularly during years of ex-treme climatic stress with severe winter temperatureand droughty fall and winter months.

1 Journal article 3823 Oklahoma Agric. Exp. Stn., Stillwater,OK 74078. Received 21 July 1980. Approved 15 Oct. 1980.

2 Professor; Supervisor, Agronomic Service Laboratory (Agron-omy); and Professor (Biochemistry), respectively, OklahomaState Univ., Stillwater, OK 74078.

LYND ET AL.: POTASSIUM EFFECTS ON GROWTH, NODULATION, AND N FIXATION OF HAIRY VETCH 303

Quantitative determinations of nitrogen fixation bylegumes within grass-legume pasture swards have beendifficult and limited data are available for evaluationswith intensive grazing (3). Procedures for nitrogenasecytosol components and associated nodule enzymologyhave recently been developed that are well adapted toforage legumes (13, 16). However, suitable containertype culture techniques are requisite for quantitative,intact recovery of delicate nodule-root systems, with-out injury or loss of nodules, from legumes grown infield experiments (7).

The objective of these studies was to determine theinfluence of soil fertility levels on growth, seed yield,nodulation, nitrogenase activity (C2H2 reduction), andassociated nodule cytosol components of Madisonhairy vetch. This paper reports 3 years' results fromfield and greenhouse experiments evaluating factorialcombinations of potassium and phosphorus levels withand without Ca in a siliceous thermic PsammenticPaleustalf (Eufaula).

MATERIALS AND METHODSMadison hairy vetch plants were grown in the field during

1975 to 1978 within individual 15-cra diam by 19-cm deep plas-tic containers (3 kg of soil) and in the greenhouse in 11 by 14cm containers (1 kg of soil). Seed were inoculated at plantingwith 1 ml of liquid media of Rhizobium leguminosarum Frank,> 10" viable cells ml'1 cultured from ATCC 10314 (L. T. Leonardand L. W. Erdman, USDA) 3HOC1. The methods previouslyreported (7) were utilized for culture, growth, nodulation, andnitrogenase determinations. Plant day age of field plants asdays after seedling emergence was initiated 10-12 Sept. 1975;15-17 Sept. 1976; and 9-12 Sept. 1977. Detailed chemical andphysical analyses of the soil used were previously reported (5).The moderately fertile, siliceous thermic Psammentic Paleustalf(Eufaula), pH 6.1 had been cropped to small grains during theprevious 13 years. The 15-cm depth epipedon analysis was1.2% organic matter, 0.03% total N, 30.5 ppm available P, ex-changeable cations as meq/100 g soil; Ca 1.4, Mg 0.6, K 0.11,Na 0.06, with cation exchange capacity 2.88.3 Soil fertility treat-ments included monobasic calcium phosphate, Ca Ht(PO4)2, atlevels of 50 mg P/kg soil in the field studies and 50, 100, and150 mg P/kg soil in the greenhouse. Potassium treatments in-cluded potassium chloride, KC1, at 100 mg K/kg soil in thefield and 100, 200, and 300 mg K/kg soil in the greenhousestudies. Calcium as CaCO3 was applied as 400 mg Ca/kg soil inthe greenhouse experiments.

The field studies included O, P, and PK treatments (50 mgP and 100 mg K/kg soil) in a randomized block design. Three

"Laboratory procedures, Oklahoma State Univ., AgronomicServices Laboratory for Soil, Plant, & Water Analysis (revised1980).

replicates were harvested for each soil fertility treatment at 210,248 and 280 day age for each of the 3 years 1976, 1977, and1978. Determinations were made for top growth, fresh noduleweight, and nitrogenase activity levels (C2H2 reduction) for allplants at each sampling. Number of seed pods and seed weightper plant were determined at 280-day age.

Greenhouse studies included triplicate pot cultures for eachtreatment harvested at 48-day age with each experiment re-peated three times. The first series was a factorial with levels ofP; 0, 50, 100 and 150 mg P/kg soil combined with and withoutK at 100 mg/kg soil and with and without Ca at 400 mg/kgsoil. The second series was also a factorial with levels of K at0, 100, 200, and 300 mg K/kg soil combined with and withoutP at 100 mg/kg soil. All pots of the second series received Ca at400 mg/kg soil.

At harvest the root-nodule systems were separated, washedfree of soil, blotted with paper toweling to remove wash-waterand placed in serum cap bottles for nitrogenase activity deter-minations (C2H2 reduction) (15). Approximately 1 hour wasthe time lapse from field collection to the initiation of acety-lene incubations in the laboratory.

Acetylene reduction was determined using 0.1 atm C2H2 (lab.spec., purified grade, Linde Div., Union Carbide, Inc.). Ethy-lene production during incubation at 27°C was determined at30 min-intervals with a Perkin Elmer GC 3920 with 1.83 m by3.2 mm Poropak N 80/100 columns (Waters Assoc.). The ethyl-ene standard utilized for calibration and monitoring GC analyseswas the Scott Ev. Tech. 1,090 ppm ± 5% C2H4/Na (Supelco,Inc.).

Nodules were picked from the roots and weighed immediatelyfollowing the gas chromatography analyses. Nodule cytosol de-terminations by the methods of Vance et al. (16) were slightlymodified to separate the cell-free nodule extract. Aliquots ofthe fresh nodules were crushed within glass tubes g/ml (1:10ratio) in 0 to 5°C double distilled H2O. The filtered homo-genate was subjected to ultrasonic 7.3 pulse frequency in anice bath for 30 seconds using a PT 10 ST Willems Polytron(Brinkman Instruments, Inc.) and followed by refrigerated cen-trifugation at 12 X 103 g for 10 min. The clear, cell-free super-natant was aseptically transferred to sterile culture tubes andstored at 0 to 5°C. Following enzyme and cytosol componentanalyses, the residual nodule extracts were lyophilized for stor-age preservation using a Unitrap Model 10-100 (Virtis Co.).

The nodule cytosol components were determined using a Per-kin-Elmer 373 Atomic Absorption Flame Spectrophotometerwith K, Ca, and Mg in lanthanum chloride (0.1JV HC1) solu-tion and Na without the lanthanum addition. Nonconjugateand inorganic P were determined with the ascorbic acid oxida-tion method as phosphomolybdenum blue.

RESULTS AND DISCUSSIONPlant growth, determined as top dry weight includ-

ing seed pods, more than doubled from anthesis (248-day age) to maturity (280-day age) with signficant dif-ferences between soil fertility treatments (Table 1).A single exception was the nonsignificant response

Table 1—Effects of soil fertility treatment on growth, nodule weight, nitrogenase activity, and seed yields of Madison hairy vetch.

Dry top weight, g/plant

Fresh nodule weight,g/plant

Nitrogenase,fimolC2H4g"'hour1

Seed pod, no./plantSeed weight, g/plant

Plant age,dayst

210248280210248280210248280280

280

Nonfertilized

1976

S.OaJ12.2d23.56.99.2b8.0e

21.733.8c9.1

15.34.6

1977

7.424.873.43.4

10.3b8.3e

14.143.14.1

21.7

2.1a

1978

3.4a12.3d54.71.86.56.3e

28.031.4c15.311.0

1.4a

Avg

5.316.450.34.0x8.77.5z

21.3x36.19.5z

16.02.7

1976

6.4b13.630.16.8

lO.Oc6.9f

18.0a35.48.7e

28.76.5b

50 mg P/kg soil

1977

12.531.187.3

4.0a14.07.9f

19.5a53.7

7.8e32.06.7b

1978

5.0b21.651.32.9a7.9c7.6f

28.842.611.623.03.0

Avg

8.0x22.156.24.6x

10.67.5z

22.1x43.79.4z

27.95.4

50 mg P + 100 mg K/kg soil

1976

7.2c22.242.97.8

11. 4d6.4g

23.1b53.8

9.7f30.3

8.0

1977

13.638.097.1

4.616.96.8g

21.6b63.6d

7.3f40.0

9.2

1978

5.6c30.356.6

2.311. 2d8.5

26.0b66.8d16.934.76.7

Avg

8.8x30.265.5

4.9x13.27.2z

23.6x61.4ll.Sz35.0

8.0

f Days from seed emergence.t Means followed by the same letter are not significantly different at 0.05 level with Duncan's Multiple Range analyses. Letters a.. .g are used for plant day

age by year. Letters x.. .z pertains to 3-year averages.

304 SOIL SCI. SOC. AM. J., VOL. 45, 1981

to K at 210-day age with the initiation of acceleratedvegetative growth that occurred at that time. Theyear 1976-1977 was the most favorable for hairy vetchproductivity. Climatic conditions for the 1975-1976crop were least favorable, and variation in responseto soil fertility treatments of several plant parameterswas apparent between the 3 years of these field ex-periments.

Fresh nodule weight per plant was not significantlydifferent between soil fertility treatments except dur-ing the peak nitrogenase activity levels occurring atanthesis, 248-day age. The influence of K with in-creased development of cluster type nodules at thisstage of plant growth is illustrated in Fig. 1. Nitro-genase activity levels were correlated with nodule massat anthesis with highly significant increases apparentfrom the K treatment. Both nodule development andnitrogenase activity generally began acceleration onwell-drained soil sites at approximately 210- to 220-day age (7), with the most rapid plant growth occur-ring after that date. Nitrogenase activity levels de-clined rapidly following anthesis with nodules deterio-rating and sloughing from roots during seed pod de-velopment. The number of seed pods and the actualseed produced per plant did not significantly correlate,although both increased significantly with P and Kfertilization in these studies. Results from many previ-ous years' field observations substantiated that morefavorable reseeding of hairy vetch with greater standpersistence in bermudagrass pastures resulted with Kfertilization when available soil P was not limiting(6). Similar growth responses have been reported formany tropical and temperate pasture legumes grownon K-deficient soils (1, 14). Highly variable effectsobserved among different species were influenced bysoil pH and base cation levels (2, 9, 12).

Results from the initial greenhouse experiments inTable 2 summarize the effects of applied P levels infactorial combinations with K and Ca on growth, nodu-lation, nitrogenase, and nodule cytosol composition.Phosphorus content of nodule cytosol was significantly

increased by P levels with interactions apparent forK and Ca combinations. Effects of K were highly sig-nificant, influencing every parameter with the excep-tion of cytosol Ca content. Potassium was dominantwithin nodule cytosol having about 10 times higherconcentration than any of the other cations. The high-ly significant reduced Na content apparent with in-creased K levels was noted throughout all of thesestudies. Nitrogenase activity increased with K additionin all treatment combinations. Soil pH of culturesreceiving the Ca treatment was slightly alkaline, 7.3 to7.5, with all parameters significantly affected by Cawith exception of nitrogenase activity levels.

Interactions within and between treatment combina-tions as influenced by Ca are shown in Table 3. Sig-nificant correlation coefficients between parametersare summarized with and without Ca treatment andwith all P and K treatments pooled. Highly signifi-cant correlations common to differential Ca treatmentincluded top weight x nodule weight, nitrogenase XK, Na X K (negative), Ca X top weight, Mg X K(negative), and Mg X Na. The predictive equations,most reliable for the response surface of nitrogenaseactivity levels, were attained by stepwise regressionwith each independent parameter evaluated in variouscombination and sequence. These models were thentested by backward elimination with each computedas the last contributing variable within the model.

Table 2—Effects of applied P levels with K and Ca on growth,nodulation, nitrogenase activity, and base nutrient

composition of nodule cytosol, Madison hairy vetch.

Fig. 1—Highly significant increases in nodule development andnitrogenase activity levels of Madison hairy vetch resultedwith K fertilization when P and Ca were not limiting. Left,no K. Right, 200 mg K/kg soil. Both received 100 mg P and400 mg Ca/kg soil.

<t

Dry top weight,g/plant

Fresh nodules,mg/plant

Nitrogenase,fimol CSH4g-' hour1

K

Na

Ca

Mg

P

^ombina-ion treatmentt

0KCa

CaK0KCa

CaK0KCa

CaK

0KCa

CaK0KCa

CaK0KCa

CaK0KCa

CaK0KCa

CaK

Applied P levels, mg/kg soil• ———————————————————— Treatment

0 50 100 150 effect

1.69 1.69 1.73 1.782.05 1.77 2.12 1.85 K**1.83 1.86 1.75 1.91 Ca*2.55 2.44 2.18 2.38

648.6 583.5 509.4 551.9623.8 638.3 788.5 746.9 K**521.9 635.9 631.7 677.9 Ca*793.3 942.5 862.1 925.9

26.9 27.8 17.7 22.534.6 39.7 25.7 32.5 K***20.2 22.0 21.6 20.924.9 30.6 28.1 36.1

Cytosol composition, jig/g nodule3,475.0 3,712.0 3,681.0 3,361.05,368.0 5,274.0 5,335.0 5,154.0 K***3,049.0 2,373.0 2,701.0 2,563.0 Ca***5,210.0 5,471.0 5,371.0 5,208.0 K x Ca***

453.2 403.2 425.6 553.6135.1 100.2 103.2 098.2 K***753.3 755.1 578.7 615.0 Ca***133.4 120.3 128.1 105.3127.5 122.5 082.5 122.5140.0 117.5 122.5 095.0137.5 165.0 160.0 115.3 Ca***180.0 160.0 162.5 135.0143.8 149.0 140.8 175.3111.5 104.0 104.3 098.8 K***259.0 251.8 200.8 217.5 Ca***161.5 145.0 171.0 134.5 K x Ca*216.0 415.0 497.5 501.0 P***280.3 360.0 430.3 467.0 K***347.3 360.8 441.0 393.5 P x Ca***329.5 339.8 380.5 420.5 P x K***

*,**,*** Level of significant differences at P <0.05, 0.01, and 0.001, re-spectively.

t Applied levels were 100 mg K and 400 mg Ca/kg soil.

LYND ET AL.: POTASSIUM EFFECTS ON GROWTH, NODULATION, AND N FIXATION OF HAIRY VETCH 305

Table 3—Correlation coefficients between growth, nodulation, nitrogenase, and cytosol components of Madison hairy vetchwith and without Ca fertilization.

Applied Ca fertilization!

Topweight

Noduleweight

Cytosol analyses

NaseJ K Na Ca Mg

Top weightNodule weightNase

KNaCaMg

>, s

0.768***-0.497**

0.013-0.221

0.522**0.266

0.3650.102

-0.2870.573***0.244

0.2360.552**0.427*

0.485**-0.119

0.3460.158

0.440*0.642***0.1980.846***

-0.844***0.094

-0.584***0.3630.853***

0.497**0.2860.3570.026

-0.222

0.037

0.481**0.517**0.045

-0.579***0.828***0.099

*,**,*** Correlation significant levels = 0.05,0.01, and 0.001, respectively.t Ca applied 400 mg/kg soil as CaCOj with all pots receiving P and K fertilization.t Nase = nitrogenase activity levels as CjH, /unoUg fresh nodule hour"'.

The highly significant regression with parameter com-binations having the greatest R2 (0.576) was: nitro-genase activity (Nase g nodule"1 hour""1) = 51.0nodule weight + 0.08 K + 1.56 Ca - 73.4 top weight- 0.57 Na, C.V. - 42.8%. Nodule weight and cytosolK were dominate factors governing 40.2% of Nase vari-ability.

Others (4) have utilized top growth for estimatingnodule activity in selection for improved N fixationwith alfalfa. Large differences between legume specieswith contrasting growth characteristics are apparent(8). The negative relationship for top growth andnitrogenase levels of hairy vetch at anthesis may re-flect the profuse tillering and fibrous root system pre-viously reported (7). The physiological significanceof nodule cytosol has been of interest for investigationswith nodule components that may influence nitro-genase activity levels (10, 16). The base cation com-position of nodule cytosol in this study was found tobe influenced by the soil plant nutrient environmentas has been reported by others with analyses of foliage,stems, and seed (2, 9, 11, 12).

Effects of four K application levels with and withoutP, are summarized in Table 4. All cultures received400 mg Ca/kg soil as CaCO3, resulting in soil pH levelsof 7.4 to 7.6 for these studies. Growth as top weightincreased in quadratic response to increased levels ofapplied K yielding highly significant increases at allK levels with and without P additions. However, freshnodule weight increased linearly with increased Kapplications without significant P response. Nitro-genase activity levels paralleled nodule mass with high-ly significant linear response to K application levels.

The K content of nodule cytosol increased linearlywith over a four-fold highly significant response to the

Table 4—Effects of applied P with K levels on growth,nodulation, nitrogenase activity, and base nutrientcomposition of nodule cytosol, Madison hairy vetch.

P treat-ment

mg/kg soil 0

Applied K levels, mg/kg soil

100 200 300 effect

Dry top weight,g/plant

Fresh noduleweight,rag/plant

Nitrogenase,/imol C2H4g"' hour'1

0100

0100

0100

0.941.28

406.0350.7

14.1812.49

1.532.33

494.3612.0

30.5527.53

1.892.35

759.3778.9

42.1246.78

1.92 K***2.58 P***

864.7 K***820.7

59.26 K***60.39

Cytosol analysis, ^g/g noduleK

Na

Ca

Mg

P

0100

0100

0100

0100

0100

1,076.01,008.0

374.3451.5116.5123.5169.7172.8190.0298.0

3,244.03,932.0

243.3313.7144.2159.5166.3192.8210.0330.8

3,913.04,499.0

189.7175.0179.7178.2194.8238.0263.0339.8

4,528.04,530.0

166.4143.2208.9199.5218.2259.3286.8349.5

K***

K***

K***

K*P*K**p***

*,**,*** Level of significant differences = P <0.05, 0.01, and 0.001, re-spectively.

applied K levels. An inverse influence of increased Kto decreased Na content was highly significant, withCa, Mg, and P all significantly increased in the nodulecytosol with increased K application. Phosphorus ap-plication influenced significantly only P and Mg com-position without apparent interactions for treatmentlevels.

Interactions among parameters within and betweentreatment combinations as infleunced by applied K

Table 5—Correlation coefficients between growth, nodulation, nitrogenase, and cytosol components of Madison hairy vetchwith P and K fertilization.

P and K treatments t

Cytosol analyses

Nodule weight NaseJ Na Ca

****** Correlation significance levels = 0.05,0.01, and 0.001, respectively.t Phosphorus applied at 100 mg P/kg and K at 100,200, and 300 mg K/kg soil with results of all K levels pooled for these correlations.} Nase = nitrogenase activity levels as C2H, /imol/g fresh nodule hour'.

306 SOIL SCI. SOC. AM. J., VOL. 45, 1981

levels, with and without P, are summarized in Table5. Regardless of P treatment, highly significant corre-lation coefficients resulted for: nitrogenase activitylevels X nodule weight K x Na (negative), Mg X Na(negative) and significant nodule weight X Na (nega-tive). Negative correlations were apparent for everyparameter as a function of Na content. Multiple re-gression procedures were used to determine the mostreliable response surface equation: nitrogenase acti-vity levels (Nase) = 48.36 nodule weight + 0.01 K -1.44 top weight - 0.04 Na + 0.01 Ca, R2 = 0.738, C.V.= 27.4%. Previous research (11, 14) has shown thatincreased growth and nodulation with K fertilizationmay be attained if adequate soil P and Ca are avail-able for legumes. Other studies (9, 10) have also re-ported that high nitrogenase activity levels, adjunct tovigorous growth and nodule mass development, aredependent on sufficiency of available soil K. IncreasedK fertilizer applications apparently did ameliorate un-desirable Na effects for symbiotic nitrogen fixation inthese studies. Large differences in response to K bycontrasting tropical and temperate species indicateneed for careful evaluations (1, 2, 10).

Hairy vetch has desirable characteristics for produc-tive forage growth on acid, low-fertility soils; however,the actual nitrogen fixation at those sites may be great-ly limited (6). The marked response of Madison hairyvetch for nodule mass development and increased ni-trogenase activity levels with K fertilization is similarto responses reported for other legumes (9, 14).

The pot culture technique in these studies is wellsuited for nondestructive evaluations suitable for plantbreeding and genetic studies. Repotting the root sys-tem following nodule removal and defoliation pro-vides means to utilize outstanding individual plants infurther studies (7).

ACKNOWLEDGMENTWe are particularly thankful to Robert M. Ahring, Research

Agronomist, USDA-SEA-AR Southern Region for the contribu-tion of certified Madison hairy vetch seed. The assistance withthe computerized statistical analysis by Ronald W. McNew,Professor of Statistics, Oklahoma State Univ., is gratefully ac-knowledged.