THE SULFUR REQUIREMENT OF THE RED CLOVER PLANT.*

BY W. E. TOTTINGHAM.

(From the Laboratory of Agricultural Chemistry, University of Wisconsin, Madison.)

PLATE 1.

(Received for publication, September 23, 1918.)

Several years ago, Hart and Peterson’ called attention to the apparent deficiency of sulfur in certain types of soil, as related to the demands made upon this element by some species of agri- cultural plants. More recently, Hart and the write? have reported increased production of dry matter by plants of various species which had received calcium and sodium sulfates in soil cultures conducted in a greenhouse. This effect was particularly striking in the case of common red clover (Trifolium pratense), which gave a greater response to calcium sulfate than to sodium sulfate. Other investigators, notably Shedd,’ have obtained similar beneficial effects from sulfates with soil cultures. P&z4 devoted special attention to the effects of calcium sulfate upon the growth of red clover. He found that the development of red clover bacteria, as well as of the young host plant, was stim- ulated by this source of sulfur.

The present paper deals with the response of the common red clover plant to different forms and planes of sulfur supply, under conditions better controlled than the ordinary soil culture. As nutrient medium, use has been made of the well known nutrient

* Published with the permission of the ‘Director of the Wisconsin Agri- cultural Experiment Station.

1 Hart, E. B., and Peterson, W. H., J. Am. Chem. Sot., 1911, xxxiii, 549:

? Hart, E. B., and Tottingham, W. E., J. Agfic. Research, 1916, v, 233. 3 Shedd, 0. M., Kentuclcy Agric. Exp. Station, Bull. i74, 1914, 595. 1 Pitz, W., J. Agric. Research, 1916, v, 771.

429

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430 Sulfur Requirement of Red Clover

solution of Knop,5 with a total salt concentration of 0.2 per cent, by volume. It is quite possible that this solution may be im- proved for the growth of clover by readjusting its salt content, as Shive and also the writer have found with wheat;6 but, until such readjustment should be tested, it seemed t,o be as suitable for the present purpose as any other nutrient solution.

The methods followed with the water cultures were those pre- viously, employed by the writer,6 except that the seeds were germinated in pure sand, instead of over wat,er, until the seedlings were large enough for fixing in the culture vessels. About 10 days were required for this stage of development. In order t,o avoid the high hydrogen ion content which would result from the use of KH2P0, as the only source of phosphorus, this salt and KzHP04 wcrc added to the solution in cquimolecular pro- portions, each in one-half the usual molecular concentration of the KH,POb. Where CaS04 and CaC03 were employed they were added in the solid form when the solution was transferred to the culture vessels. Each culture vessel (1 pint jar of the Mason pattern) had a capacity of about 425 cc. and supported nine seedlings at the beginning of each culture series. The nutrient solutions were renewed after intervals of 3 days.

The plan of treatment of the solutions involved both the par- tial and complete replacement of MgSOd with. Mg(NO&. To the sulfur-free solution produced in the latter case, sulfur was also restored in the form of tither Na&O* or CaSO+ each being made molecularly equivalent to the MgS04 of the unmodified Knop solution. Still other portions of the sulfur-free solution were modified by adding either NaN03 or CaC03, in twice mo- lecular and molecular proportions respectively, as compared with the usual amount of MgSO.+. These last modified solutions served as controls for determining the effects of ‘the sodium and extra calcium added elsewhere in the form of sulfates.

The method followed with the sand cultures was that of irri- gation with a renewed nutrient solution, as previously employed by McCall.7 Glazed, stoneware jars, about 16.0 cm. in diameter

j Icnop, W., Landw. Versuchssta., 1865, vii, 93. ‘: Tottingham, W. E., Phgsiol. Researches, 1913-17, i, 133. Shivc, .J.

\\'., ibid., 327. i McCall, A. G., Soil Science, 1916, ii, 207.

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W. E. Tottingham

and 11.0 cm. deep (2 quart jars), which conveniently held 3.5 kilos of fine sand, were employed as culture vessels. The inner surface of these jars was coated with paraffin, to prevent con- tamination of the nutrient solution by solut,es from the stone- ware. The sand employed consisted of spheroidal particles, most of which passed through a sieve with 70 meshes to the inch but were retained by one of 80 meshes. It was the product designated “banding sand” by the Ottawa Silica Company of Illinois. Although containing only traces of impurities, it was washed thoroughly with water before use. For the purposes of maintaining alkalinity and supplying iron, 3.0 gm. of CaC03 and 1.0 gm. of Fe&, were mixed with the portion of sand for each culture. Phosphorus was added to the nutrient solution in the form of KZHPOJ. The application of nutrient solution was gradually increased from 25 to 100 cc. per culture at each renewal, and the period between renewals was gradually reduced from 4 to 2 days. As measured by Hilgard’s method,* the water- holding capacity of the sand here employed was 23.4 per cent, by weight. By daily weighings, in connection with the use of a suction pump, the plane of total water content of all of the cul- tures was made uniform and was increased gradually from 20 to 22 per cent, by weight, of the dry sand. It was possible to meas- ure approximately the loss of water through the plants, as the seedlings and irrigating funnel were sealed about with a mixture of Vaseline and paraffin, but the data obtained showed no signi- ficant variations.

As none of the plants produced flowers in any of the culture series, it was difficult to select a stage of growth approximating maturity. Each series was harvested, however, when pro- nounced death of the basal leaves seemed to indicate a decided decline of growth with all of the cultures. After determining the average length of the three or four longest roots, and washing the latter parts, the plants were severed at the crown of the roots. The tops and roots were then dried at about lOO”C., for weighing. Except in the case of the sand cultures, the finely ground tops of the duplicate cultures were mixed for determining the average nitrogen content. In some cases, determinations of nitrogen in

8 Hilgard, E. IV., Soils, New York, 1906, 208-209.

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432 Sulfur Requirement of Red Clover

t’he form of nitrates were also made. Total nitrogen was deter- mined by the Gunning method9 and nitrate nitrogen was deter- mined by precipitation with nitron.lO It seemed possible that the data derived from these analyses would indicate disturbances of protein synthesis, which might follow deficiencies in the sulfur supply of these clover plants.

Series 1 was conducted with water cultures. It covered a much longer period of growth than the succeeding series. After 37 days of growth the cultures were transferred to jars of a capacity of about 850 cc. (1 quart jars of the Mason pattern). The widely varying number of,plant,s which survived in the different cultures tends to make the results of questionable value. However, the latter are unchanged in general trend if considered on the basis of unit plant per culture; and they are confirmed, in some re- spects, by the succeeding .and more uniform series. The results appear in Table I.

Series 2, also conducted with water cultures, was disturbed by attacks from thrips. This may account partly for its compara- tively early apparent maturing. Its more restricted growth period may, in turn, account for its less marked differences in yields, as compared with the other series. The results of this series appear in Table II.

Series 3, conducted with sand cultures, reached apparent ma- turity comparatively quickly. The seedlings were 10 days older than usual when transferred to the cultures, having been nour- ished for this period by irrigation with the sulfur-free modifica- tion of Knop’s solution. Only six plants were started in each culture. The entire series was conducted upon a rotating table, after the manner of Shive’l, to render uniform the effects of aerial environment with all of the cultures. The results appear in Table III.

A comparative survey of the results of these three series of cultures shows that 0.9 of the usual amount of MgS04 in Knop’s solution could be displaced, under the conditions of the water

9 Official and provisional methods of analysis, Assn. O&id Agric. Chemists, U. S. Dept. Agric., Bureau of Chem., Bull. 107 (revised), 1907.

10 Treadwell, F. P., Analytical chemistry, New York, 4th edition, 1915, ii, 451-453.

11 Shive, Physiol. Researches, 1913-17, i, 344345.

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TABL

E I.

Yiel

d an

d Ni

troge

n Co

nten

t of

Re

d Cl

over

(T

rijol

ium

pr

aten

se)

Grow

n 1%

Da

ys

(Mar.

IS

-July

13

, 19

16)

in

Knop

’s So

lutio

n an

d iV

fodi

$cat

ions

Th

ereo

f.

1 2 3 4 5 6 7 8 9 10

11

12

13

14

15

16

17

18

I De

scrip

tion

of

nutri

ent

med

ium

.

Unm

odifie

d Kn

op

solu

tion.

3.9

MgSO

a of

Kn

op

solu

tion

repla

ced

by

Mg(N

O&.

Tota

l Mg

SOa

of

Knop

so

lutio

n re

place

d by

Mg(N

Odz.

Sam

e as

5 a

nd

6 +

Na2S

Od

= Mg

SOd

of

Knop

so

lutio

n.

Sam

e as

5 a

nd

6 +

Na.N

Oz

= M

gS04

of

Kn

op

solu

tion.

Sa

me

as

5 an

d 6

+ Ca

S04

* Mg

SOd

of

Knop

so

lutio

n.

Sam

e as

5

and

6 +

CaC0

3 -̂

MgS

Ol

of

Knop

so

lutio

n.

Sam

e as

5

and

6 +

CaSO

d ==

3

X Mg

SOa

of

Knop

so

lutio

n.

Sam

e as

5

and

6 +

CaC0

3 =

3 X

MgSO

d of

Kn

op

solu

tion.

No.

of

plant

s m

a-

ture

d.

Yield

of

dr

y Yi

eld

of

dry

tops

. ro

ots.

gm.

perc

ent

gm.

per c

ent

3.25

2 -

0.92

4 -

3.58

5 10

0 1.

008

100

3.41

4 --

0.94

2 -

3.38

4 99

0.

730

87

2.22

0 -

1.24

8 -

1.95

2 61

1.

304

132

3.38

8 -

1.04

4 -

3.07

5 95

1.

113

112

2.34

0 -

1.11

6 -

2.02

2 64

1.

038

111

4.39

2 -

0.87

6 -

4.38

6 12

8 1.

194

107

2.07

2 -

1.20

8 -

2.00

4 60

1.

266

128

4.35

2 -

1.20

0 -

3.70

8 11

8 0.

904

109

2.45

4 -

1.43

4 -

2.49

6 72

1.

520

153

--

-

1 &x&

ml

leng

tt of

ro

ots.

Mua

l

mm

.

119

103

137

175

250

225

.131

11

9 16

2 22

5 14

4 13

7 17

5 28

7 10

0 17

5 22

5 20

0

Reln-

tiv

e.

P ce

nt

- 100

141

214

113

- 175

127

208

124

- 192

1 I lbt

al nit

roge

n of

to

ps.

er c

at’

3.47

- 3.63

4.46

3.65

- 3.76

3.84

4.69

3.63

3.47

0.12

3 10

3

0.09

3 78

0.11

8 99

0.08

2 69

-

-

0.16

9 14

2

0.09

6 81

0.14

6 12

3 -

-

0.08

6 72

* Av

erag

e va

lue

of

dupl

icate

cu

lture

s.

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TABL

E II.

Yiel

d an

d Ni

troge

n Co

nten

t of

Re

d Cl

over

Gr

own

64 Da

ys

(Apr

. IS

-Jun

e 16

, 19

16)

in

Knop

’s So

lutio

n an

d M

odiJi

catio

ns

Ther

eof.

1 2 Un

mod

ified

Knop

so

lutio

n.

3 0.

9 of

MgS

O,

of K

nop

solu

tion

repl

aced

by

4

Mg(

NO&.

5 To

tal

MgS

04

of K

nop

solu

tion

repl

aced

by

6

Mg(

NOdz

.

7 8 9 10

11

12

13

14

Sam

e as

5 a

nd 6

+

NatS

Od

= M

gS04

of

Kno

p 9

1.54

4 so

lutio

n.

8 1.

6311

Sam

e as

5 a

nd 6

+

NaN0

3 =

MgS

04

of K

nop

8 1.

1581

so

lutio

n.

9 0.

950

Sam

e as

5 a

nd 6

+

CaSO

n =

MgS

04

of K

nop

9 1.

271

solu

tion.

. 9

1.39

1

Sam

e as

5 a

nd 6

+

CaC0

3 *

MgS

04

of K

nop

solu

tion.

Desc

riptio

n of

nutrie

nt m

edium

.

* Av

erag

e va

lue

of d

uplic

ate

cultu

res.

t

Conv

erte

d to

eq

uiva

lent

fo

r nin

e pl

ants

,

-7

L,“,’

Yield

of

dry

tops.

ma-

tu

red.

ht

uai.

-__

gm.

P

9 1.

354

9 1.

367

Rela-

tiv

e.

erca

t

- 100

9 1.

388

8 1.

2891

t

9 0.

955

9 0.

861

- 0.

429

- 12

5 98

0.

471

95

106

67

9 0.

888

9 1.

046

-

- 117

- 77

98

- 71 -

Yield

of

dry

Maxim

al len

gtl

roots

. of

roots

.

- 1 To

tal

nitrog

en

of top

s.

Actua

l. p;

;- Ac

tua

1. --

--

gm.

per

cent

mm

. P

0.51

2 -

137

0.43

1 10

0 14

4

I- Re

la-

tive.

ercen

t rET

cm

&,

100

3.03

82

- 3.40

0.47

6 -

125

0.47

0 10

0 13

5 92

4.

03

0.53

5 -

150

- 0.

572

117

150

106

3.20

0.50

9 -

160

- -

0.53

3 10

8 13

7 10

6 3.

91

0.51

7 -

0.55

9 11

4 10

0 12

8

131

137

- .81 95

- 3.24

0.49

9 -

0.52

1 10

8 - 3.90

____

gm

.* pe

r ce

nt*

- -

0.04

1 10

0

- -

0.04

6 11

2

- -

0.03

7 90

- _

0.05

1 12

4

- -

0.04

1 10

0

- -

0.04

3 10

5

- -

0.03

8 93

_.

- -

,,> ,,

> -

- _

,j I

- .~

-

,,

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TABL

E III

.

Yiel

d an

d Ni

troge

n Co

nten

t of

Re

d Cl

over

Gr

own

54

Days

(J

an.

Y-M

ar.

2,

1918

) in

Sa

nd

Irriga

ted

with

Kn

op’s

Solu

tion

and

Mod

i,fica

tions

Th

ereo

f, wi

th

CaC0

3 Ad

ded.

CultA

m

No. 1 2 3 4 5 6 7 8

Desc

riptio

n of

nu

trien

t m

ediu

m

Unm

odifie

d Kn

op

solu

tion.

0.99

of

Mg

SOn

of

Knop

so

lutio

n re

place

d by

Mg

NMz.

Tota

l M

gS04

of

Kn

op

solu

tion

repla

ced

by

Mg(N

Odz.

Sam

e as

5 a

nd

6 +

CaS0

4 *

MgS

04

pf

Knop

so

lutio

n.

No

of

plant

s m

a-

ture

d. G

0 6 6 6 6 6 6

? htunl.

Re

la-

tive.

Yield

of

dr

y to

ps.

&?m

. ?r

ce?t

t

3.66

3.

66

100

2.88

-

3.18

83

1.50

2.

22

51

5.04

4.

92

136

Yield

of

dr

y ro

ots.

Mua

l

c7m

.t 1.

80

1.96

2.08

1.

94

0.48

1.

12

2.76

3.

08

%Jt

.-

er c

ent

100

107 43

- 155

-

1\

A

* -

Iaxim

al

leng

tt of

ro

ots.

.ctua

1

mm

.

169

138

163

125

150

200

169

169

_-

Rela-

tiv

e.

Actu

al.

Rela-

tiv

e.

ET cen

t 100 94

- 114

- 110

1 I

.-

lbtal

nitro

gen

of

tops

.

e-ce

nt

4.24

4.

25

4.11

3.

45

3.43

4.

31

4.04

4.

00

gm.*

pm

cent

* m

, 0.

156

100

ti z

0.11

4 73

2’

F5

- -

0.07

4 47

B

0.20

1 12

9

* Av

erag

e va

lue

of

dupl

icate

cu

lture

s. t

Air-d

ried;

co

ntai

ning

sa

nd.

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Sulfur Requirement of Red Clover

cultures, without serious1.y disturbing the growth of the red clover plant. In the sand cultures, displacement of 0.99 of the usual amount of MgS04 led to a .considerably decreased produc- tion of dry matter of the stems and leaves. Displacement of the total MgSO, led to decreases of 33 to 49 per cent in the pro- duction of dry tops in the various culture series. Addition of sulfur to the modified sulfur-free Knop’s solution, in the forms of Na&SOb and CaSO*, resulted in increased growth of the tops of the clover plant. In the majority of these cases the growth was decidedly superior to that with the unmodified Knop’s solution. The results of the control cultures with NaN03 and CaC03 show that this effect is not attributable to the cations of these added sulfates, when supplied in certain other salts.

As regards the roots, the results are extremely variable. With the water cultures of Series 1 the effect of the various sulfates was to reduce the length of roots, as compared with the sulfur-free modification of Knop’s solution, but this was not true of the other series of cultures.

The amount of nitrogen absorbed by these cultures fluctuated more or less parallel to the yields of dry tops, both of these values decreasing with the decrease of sulfur supply. It would seem logical to conclude that a deficiency of sulfur here operated to decrease the synthesis of protein and restrict the elaboration of plant tissue, with a concomitant decrease in the assimilation of nitrogen from .the culture medium. That the percentage of assimilation of the absorbed nitrogen was not greatly different in the various cultures of each series here dealt with, is shown by the following percentages of the total nitrogen in the form of nitrate, as determined by analysis: Series 1, Cultures 1 and 2, 7.2 per cent; Cultures 3 and 4, 11.0 per cent. Series 3, Cultures 1 and 2, 7.5 per cent; Cultures 5 and 6, 6.2 per cent; Cultures 7 and 8, 7.5 per cent.

On the whole, the results of these cultures show a superiority of Na&SOh and CaSO* over MgSO+ as sources of sulfur for the red clover plant in water and sand cultures. This might be expected to follow the known toxic properties of magnesium salts. In harmony with the results previously reported from soil cul- tures2 CaS04 has proved an especially efficient source of sulfur.

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W. E. Tottingham 437

In view of the fact that all of the nutrient media which here supplied sulfur were liberally supplied with calcium, it appears that CaSOc had peculiar efficiency as a molecular complex.

SUMMARY.

The data presented here show that, for these experimental conditions, between 0.1 and 0.01 of the usual amount of MgSO, of Knop’s solution was as efficient as the usual amount for the growth of red clover, when the balance of the MgS04 was re- placed by Mg(NO&. The yield of dry tops of this plant was only one-half to two-thirds as great with MgSOa, wholly replaced by Mg(NO& as with the unmodified Knop’s solution. Addition of Na&304 and CaS04 to the sulfur-free modification cf Knop’s solution, in amounts equivalent to the sulfur of the unmodified solution, produced yields of dry tops superior to those of the latter solution. In this respect, CaS04 was very efficient. From the nature of the various nutrient media employed, it appears that the sulfur of this salt functioned in the molecular combina- tion in which it was supplied. The more or less parallel fluctua- tions of plane of sulfur supply, weight of nitrogen assimilated, and yield of dry tops, of these clover plants, indicate that a deficiency of sulfur supply restricted growth by limiting the synthesis of protein.

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438 Sulfur Requirement of Red Clover

EXPLANATION OF PLATE 1.

FIG. 1. Series 1. Water cultures of Trifolium pratelzse after 99 days of growth. Plants 1 and 2, unmodified Knop’s solution. Nos. 5 and 6, total MgS04 of Knop’s solution replaced by Mg(NOz)z. Plants 7 and 8, same as Nos. 5 and 6, plus NasSOa = MgSOa of Knop’s solution. Plants 11 and 12, same as Nos. 5 and 6, plus CaS04 = MgSOd of Knop’s solution. Plants 13 and 14, same as Nos. 5 and 6, plus CaC03 * MgSOd of Knop’s solution.

FIG. 2. Series 3. Sand cultures of Trifolium pratense after 53 days of growth. Plants 1 and 2, unmodified Knop’s solution. Nos. 3 and 4, 0.99 of MgS04 of Knop’s solution replaced by Mg(NO&. Plants 5 and 6, total MgS04 of Knop’s solution replaced by Mg(NOz)z. Plants 7 and 8, same as Nos. 5 and 6, plus CaS04 = MgS04 of Knop’s solution.

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THE JOURNAL OF BIOLOGICAL CHEMISTRY. VOL. XXXVI. PLATE 1.

FIG. 1.

I 5 7 0 ”

FIG. 2.

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W. E. TottinghamRED CLOVER PLANT

THE SULFUR REQUIREMENT OF THE

1918, 36:429-438.J. Biol. Chem. 

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