THE INTERACTION OF CALCIUM AND HYDROGEN IONS IN THE
NODULATION O F SUBTERRANEAN CLOVER
By J. F. LONERAGAN*
and E. J. DOWLING*
manuscript received Jalzuclry 9, 19581
Summary
A study was made of the effects of calcium and hydrogen ions on the nodulation
of subterranean clover (Trifolium subterraneum L.) in water culture. A compound
interaction was found.
At pH 4.0 or less no nodules were formed a t any calcium concentration. At
a 0.01mx calcium concentration no nodules were formed a t any pH used. Above
these critical values, almost maximum nodulation could be obtained by an increase
in either calcium concentration or pH, so that each factor was to a large degree
replaceable by the other.
Calcium and hydrogen ions in the range of concentrations which produced
these marked interactions on nodulation had no measurable effect on plant growth
(nitrate was supplied in solution). The effects of calcium and hydrogen ions on the
growth of the Rhizobium strain used werealso markedly different from their effects on
nodulation. Hydrogen ions had a dominating effect on Rhizobium growth, while
calcium ions had no effect a t any pH and were required a t most in trace amounts.
Hydrogen ions depressed calcium uptake by the plants. I t is suggested that,
in the range of concentrations where they were replaceable, the effects of calcium
and hydrogen ions on nodulation were through their influence on the level of caloium
in the plants. I t is concluded that the calcium requirement for nodulation of subterranean clover is higher than for growth of the host plant or for growth of Rhizobium.
Soil acidity and soil calcium have been recognized for many years as factors
influencing the nodulation and growth of legumes. Albrecht (1933) studied the
effect of different amounts of soil calcium on the nodulation of soybean over a pH
range of 4.0-6.5. The calcium supply was adjusted by mixing different amounts of
calcium-hydrogen clays of the required pH with quartz sand. At and below pH
5.0 nodules did not form a t any level of calciun~. At a p H of 5.5 there was a marked
effect of calcium supply on nodulation and this effect increased as the soils became
less acid.
On the other hand the effect of calcium supply on the growth of lettuce and
tomato plants in nutrient solutions was shown by Arnon and Johnson (1942) to be
much less a t pH 6 than a t more acid reactions (pH 4 and 5 ) . This effect was attributed
to a depression of calcium uptake by hydrogen ions (Arnon, Fratzke, and Johnson
1942). Schmehl, Peech, and Bradfield (1952) also observed a depressing action of
hydrogen ions on calcium uptake by lucerne. Thus the interaction of calcium and
hydrogen ions on plant growth and calcium uptake by plants is in direct contrast
to that for nodulation.
*Division of Plant Industry, C.S.I.R.O., Canberra.
CALCIUM AND HYDROGEN IONS IN CLOVER NODULATION
465
With the recent use of trace amounts of lime (as a coating on the seed) to
establish subterranean clover on acid soils on the Southern Tableland of New South
Wales (Loneragan et cd. 1955) it seemed desirable to obtain a better understanding of
the interaction of calcium and hydrogen ions on the nodulation and growth of this
plant. This paper describes the results of investigations on the interaction of these
factors on the nodulation and growth of subterranean clover grown in water culture.
Subterranean clover (Trifoliurn subterraneum L. var. Bacchus Marsh) seeds
were germinated on cheesecloth over water. After 9 days, six plants were transferred
to each of a number of 3-litre beakers containing nutrient solution. The basal nutrient
concentrations were: potassium phosphate l m ~ potassium
,
nitrate 5 m ~ ,magnesium sulphate lmiv, boric acid 1 5 p ~ ,copper sulphate 0 . 1 p ~ ,molybdic acid
0 . 0 3 p ~manganese
,
chloride 3 p ~zinc
, sulphate 0 . 3 p ~iron
,
1 8 p as
~ the chelate of the
dipotassium salt of ethylenediaminetetra-acetic acid. Treatments were imposed by
varying the level of calcium sulphate and by titrating the solutions to the required
pH daily with 0 . 1 sulphuric
~
acid solution. The solutions were aerated continuously
and the beakers u-ere covered to protect the roots from light.
Each beaker 11-as inoculated with Rhizobium after the plants had been growing
in solution for 1 day. Rhixobium t7,ifolii strain NA30 was used in all experiments.
The bacteria were grown on agar slopes consisting of 10 ml of the Rhizobium nutrient
solution described below, with additions of calcium sulphate (to give a concentration
of l m ~ and
) agar. After 4-7 days' growth a t 25°C the bacteria on the surface of
the slope were suspended in 10 ml of sterile, calcium-free, inorganic nutrient solution.
Plate counts of the number of bacteria in suspension gave consistent values of about
lo9 per ml. One ml of this suspension was diluted to 100 ml with calcium-free,
inorganic nutrient solution, and 1 ml of this dilution was further diluted to 1 litre.
One ml of this final dilution, containing about lo4 bacteria, was added to each beaker.
The plants were harvested soon after macroscopic nodules had formed, 12
days after transferring to treatments. Nodules were counted a t harvest. Six experiments are reported. Three experiments were conducted to examine the effect of
varying calcium sulphate and pH on the number of nodules formed. In experiments 1 and 2 there were two replicates. Experiment 3 had five replicates. A
fourth experiment was carried out in triplicate to compare the effects of magnesium
sulphate and calcium chloride with those of calcium sulphate on nodulation at pH 4.5.
Experiments 5 and 6 were designed to study the interaction of calcium and
hydrogen ions on the growth of Rhizobium. Sterile nutrient solutions identical in
inorganic composition to the above were used: to each litre of solution 1g of Difco
yeast extract and 5 g of mannitol were added. The yeast extract contained 0.016
m-moles of calcium per gram. The experiments were conducted in 100-ml Erlenmeyer flasks containing 10 ml of nutrient solution. The same procedure was
used for inoculation of the flasks as was used for the inoculation of the beakers
except that a sterile, calcium-free medium containing yeast extract and mannitol
was used for all dilutions. Experiment 5 was conducted in duplicate and each
flask received about lo6 bacteria
of the bacteria on the slope). Experiment 6
466
J. E. LONERAGAN AND E. J. DOWLING
of the bacteria
was carried out in triplicate and each flask received lo4 bacteria
on the slope). The maximum possible carry-over of calcium in the inoculum would
contribute a concentration of calcium in the flasks no greater than 0 . l P ~in experiment 5 and 0 . 0 0 1 p ~in experiment 6. After inoculation the flasks were placed on
an oscillating shaker in a constant temperature room a t 23°C for 4 days (experiment 5)
or 3 days (experiment 6). Growth was measured by determining culture density in a
nephelometer. Estimates of the number of bacteria both by plant infection dilution
technique and by direct counts indicated that growth became just detectable in the
nephelometer a t lo6 cells per ml, and that further increases in cell numbers were
directly proportional to cell density.
I
pH:
I
4.0
CALCIUM LEVEL:
Fig. 1.-The
I
J
I
6.0
5.0
10
0-0
EXPERIMENT 3
EXPERIMENT 2
EXPERIMENT 1
mM
I
I
I
I
5,O
4.0
I
I
I
6.0
4.0
5.0
.-----. .
7
mM
#..
1 mM
0-
0.1 mM
-0
I
6.0
I
-.
0.01 mM
0-.
-.
effect of p H on the nodulation of plants a t different levels of calcium.
111. RESULTS
(a) Nodulation
The effects of calcium and hydrogen ion concentrations on the percentage of
plants nodulated and on the number of nodules per plant for experiments 1, 2, and
3 are shown in Figure 1.
At a pH of 4.0 no plants were nodulated a t any concentration of calcium
sulphate. At a pH of 4.5 no nodules were formed a t low calcium sulphate levels
but there were marked effects of increasing calcium sulphate concentration resulting
in the nodulation of all plants at a lOmM calcium sulphate concentration. Also a t
a 0 . O l m ~concentration no plants were nodulated a t any pH, but a t a O . l m ~concentration there were marked effects of increasing pH, from 0-50 per cent. nodu-
467
CALCIUM AND HYDROGEN IONS IN CLOVER NODULATION
lation at pH 4.5 to 93-100 per cent. nodulated plants a t pH 6.0. Again a t a l m ~
calcium sulphate concentration there was a consistent positive effect of increasing
pH on the percentage of plants nodulated.
The data for the number of nodules per plant follow the same pattern as those
for the percentage of nodulated plants. Either increasing the calcium sulphate
concentration from 0 - l m x or l m to~ l O m ~a t pH 4.5, or increasing the pH from
4.5 to 6.0 a t 0 . l m ~or l m calcium
~
sulphate, produced a significant increase in the
number of nodules. All experiments showed evidence of a negative interaction
between the calcium sulphate treatment and the pH. This interaction was not
TABLE1
THE EFFECT OF CALCIUM SULPHATE, CALCIUM CHLORIDE, AND MAGNESIUM SULPHATE CONCENTRATIONS AT
pH 4.5
ON NODULATION AND GROWTH OF SUBTERRANEAN CLOVER PLANTS
1
Treatment
Kodules
I
Calcium
Sulphate
(mN)
Calcium
Chloride
(mM)
1
10
1
1
0
0
9
0
I1
,
Magnesium
Sulphate
(mM)
*Increase significant at P
Dry Weight (mglplant)
1
1
(No. per
plant)
83
100
1
1
1
10
=
I
1
33
13
9
0.05.
statistically significant for any single experiment but was found to be significant
when the data for all three experiments were combined. The effect of increasing the
calcium sulphate concentration from l m to~ 1 0 m was
~ significantly less a t pH 6.0
than a t pH 4.5 (P= 0.01).
Effects of calcium sulphate, calcium chloride, and magnesium sulphate in
varying concentrations are shown for experiment 4 in Table 1. Treatment details
are given in the table, together with data for the percentage of plants nodulated,
the mean number of nodules per plant, the dry weight of roots, and the dry weight
of shoots.
From Table 1 it is clear that calcium stimulates nodulation when present either
as the chloride or as the sulphate, and that high levels of sulphate as the magnesium
salt do not have any effect. The effects of calcium sulphate on nodulation reported
in the first experiments must be attributed to a specific effect of the calcium ion.
(6) Plant Growth
Dry weight data for shoots and roots of experiment 3 are given in Table 2.
Plants of all pH treatments a t 0 . 0 1 m ~calcium concentration were severely calciumdeficient and growth was markedly retarded.
468
J. F. LONERAGAN AND E. J. DOWLING
A pH of 3.5 also significantly reduced root and shoot dry weight a t O.lmw
calcium, and root dry weight a t l O m ~
calcium. A pH of 4.0 reduced root dry weight
at O . l m ~calcium, but did not significantly affect root eight a t the higher calcium
levels or shoot weight at any calcium level.
From pH 4.5 to 6.0, and from a calcium concentration of O . l m ~to lomix,
there were no significant effects of either calcium or hydrogen ions on the dry weight
production of either roots or shoots. I t is remarkable that growth was so unresponsive
to these concentrations of calcium and hydrogen ions which had such marked effects
on nodulation.
INTERACTION O F CALCIU4I AND H Y D R O G E N I O X S O N T H E GROWTH O F SUBTERRANE-LN CLOVER
Data
Shoot dry weightv (mglplant)
Root dry weight$ (mglplant)
!
I
I
1
Calcium
sulphate
0.01
0.1
1
0.01
!
I
'
-7
7.60
I
10.05
2.13
1
-
248
2.55
2.78
I
*hlinimum significant difference: a t P
=
0.01, 2.42;
?Not determined.
$Minimum significant difference: a t P
=
0.01, 0.98; a t P
(c) Rhizobium
8.33
12.1
i'd::
1
2.50
3.60
2.83
3.23
p
2.10
I
1
1005
11.46
L 3
p
10
~ ;i 1
-----
at P
=
=
1
-
-
p
1
8.28
11.1
11.1
12.2
2.55
3.20
3.20
3.23
-
3.55
3.15
-
I
0.05, 1.81.
0.05, 0.73.
Growth
The results of experiment 5, conducted to examine the effects of calcium and
hydrogen ions on the growth of Rhizobium, are presented in Figure 2. I t is clear
from this figure that increasing the calcium level from O v l m ~to 1 0 m had
~ no effect
on the growth of Rhizobium at any pH. On the other hand, pH had a large effect
on growth a t all calcium levels. There was no evidence of growth 4 days after
inoculation a t pH 4.0 and 4.5: at pH 5.0, 5.5, and 6.0, very dense suspensions of
bacteria were produced. Observations showed that prior to harvest, growth a t pH
6.0 was greater than a t pH 5.5, which in turn was greater than a t pH 5.0, so that
from pH 4.5 to pH 6.0 growth rate steadily increased with decreasing acidity. The
reason for identical yields a t harvest was probably that some other factor had
become limiting. By inoculating test plants with cultures grown a t pH 4.0 it was
shown that the bacteria were still viable after 4 days.
At no stage were any effects of calcium on growth observed. Experiment 6
showed that a t pH 6.0 solutions to which no calcium, other than that present in the
CALCIUM AND HYDROGEN IONS IN CLOVER NODULATION
469
yeast extract (0.016m~),was added produced very vigorous growth: in 3 days
cultures to which no calcium was added grew from lo4 cells per ml to 1.0 x 1010 cells
per ml, while O.lm;\l-calcium cultures produced 1.1 x 1010 cells per ml.
CALCIUM LEVEL
. - - -..
.x.
I
d
.............
0-
Fig. 2.-The
-
0.1d
effect of pH on the growth of Rhizobium a t different levels
of calcium.
IV. DISCUSSION
The data presented in this paper demonstrate a compound interaction (both
positive and negative) between increasing calcium and increasing pH for nodulation.
The data show that there are critical levels of calcium and pH below which both
factors are essential for the nodulation of subterranean clover plants. Above these
critical levels, almost maximum nodulatjon may be obtained by an increase in either
factor, so that each is to a large degree replaceable by the other. These data are in
good agreement with the findings of Arnon and Johnson (1942) for the effects of
these factors on the growth of tomato and lettuce plants. I t seems then that the
interaction of calcium and hydrogen ions is similar for the nodulation of legumes and
for the growth of plants. The observations of Albrecht (1933) on nodulation of
soybean, showing an increasing effect of soil calcium with decreasing acidity, are in
agreement with the results obtained in the present experiments over part of the range
of calcium and hydrogen ion levels.
I n the experiments presented in this paper the plants were supplied with
sufficient nitrate to allow full growth of the host plant independent of symbiosis.
470
J. F. LONERAGAN AND E. J. D O T ~ I N O
At the concentration used, this ion might have caused some depression of nodulation, which, however, would not affect the conclusions drawn.
The effect of calcium ions in counteracting the depressing effect of hydrogen
ions on the growth of plants mas found only a t extreme acidity in these experiments
with clover. The dry weight of shoots a t p H 3.5 and 4.0 was less a t O.lmiu than
a t 10mx calcium concentration, but the difference is only significant a t p H 3.5
(P= 0.05). Over the ranges of calcium concentrations from O . l m ~to lorn21 and of
p H from 4.5 to 6.0, which produced such a marked negative interaction on nodulation, the gro~vthof plants was remarkably uniform. Clearly the striking effects
of these treatments on nodulation cannot be attributed to secondary effects through
plant growth. I n addition the Rhizobium strain used showed no detectable growth
a t p H 4.5 but shoued large progressive responses to f~lrtlierdecreases in acidity.
This striking effect of p H on the growth rate of Rhizobium could explain the stimulating
effect of increasing p H on the nodulation of plants a t a O.lm3~or l m x calcium concentration. However, it does not directly explain the stimulating effect of 1 O m ~
calcium on nodulation a t p H 4.5 since there mas no detectable gromth of Rhizobium
a t this p H and this calcium level. The increase in ionic strength of the nutrient
solution might increase the p H a t the root surface of those plants a t a 1 0 m calcium
~
concentration compared with those a t lower concentrations. However, the p H
would not rise above that of the ambient solution, so that any effect of calcium on
nodulation a t p H 4.5 cannot be interpreted in terms of a shift in the p H of the
rhizosphere and a consequent increase in growth rate of the bacteria there. Moreover,
magnesium ions did not stimulate nodulation. The processes involved in the stimulation of nodulation by a lOmr\.r calcium concentration a t p H 4.5 must therefore
be those of infection or early nodule development.
An explanation for the interchangeability of calcium and hydrogen ions in
this process may be found in the work of Albrecht and Davis (1929),who showed that
calcium-starved soybean seedlings transplanted into a well-inoculated acid soil
developed very few nodules compared with transplanted seedlings which had
previously received calcium. This suggested that the level of calcium in the plant
lvas important in the nodulation of legumes. Such an effect of plant calcium on
nodulation is consistent both with the knomn effects of hydrogen ions in suppressing
calcium uptake by plants and with the effects of calcium and hydrogen ions on
nodulation reported here. Calcium uptake was depressed by hydrogen ions in
these experiments; the shoots of those plants grolln a t a l m n ~calcium concentration
contained significantly less calcium a t p H 4.5 (0.48 per cent. dry wt.) than a t p H
6.0 (0.69 per cent.) ( P = 0.05).
It is suggested then that the negative interaction for nodulation between increasing calcium and decreasing hydrogen ions reported in this paper may be
explained by the effect of these ions on the level of calcium in the plants. This
conclusion implies that nodulation has a higher requirement for calcium than does
gromth of either Rhizobium or host plant. This extra calcium could function a t any
stage of infection or early nodule development. It is of interest in this connexion
that Nutman (1956) has suggested an intimate association between the Rhizobium
CALCIUM AND HYDROGEN IONS I N CLOVER NODULATION
47 1
and the primary cell wall during infection, and that calcium is known to modify the
properties of the plant cell wall (Burstrom 1952; Tagawa and Bonner 1957).
The failure of calcium-deficient plants to form nodules is consistent with this
conclusion. However, the reduction in the dry weight of both roots and shoots a t
very low calcium levels was marked, and the absence of nodules on these plants
might therefore have resulted from a secondary effect on plant growth. Similarly,
extreme acidity (pH 3.5) reduced plant growth and nodulation. Hoxvever, a t pH
4.0 no nodules formed at the highest level of calcium in spite of the fact that there
was no significant reduction in the yield of these plants. At pH 4.0 there was no
apparent growth of Rhizobium although cells remained viable for at least 4 days in
pure culture. That acidity may prevent nodulation even in the presence of living
bacteria was shown by Cabezas de Herrerra (1956)who maintained lucerne Rhizobium
in culture solution at pH 4.0 and found that the bacteria were unable to infect the host
unIess returned to culture solution a t pH 6-6.
The finding that the Rhizobium strain used in these experiments grew vigorously
in the complete absence of calcium other than that present as impurity in the
medium is of considerable interest. Norris (1956) has postulated that Rhizobium
associated with Trifolizm has a high requirement of calcium for growth. This
conclusion was based on the results of experiments by JIcCalla (1937) and hiorris
(1966), which showed that high levels of calcium were needed in colloidal clay
cultures for the maximum growth of lucerne Rhizobium, in contrast with the soybean Rhizobiunx, which grew with no calcium other than that present as impurity.
However, the amount of calcium needed by Iucerne Rhizobium in these colIoida1
clay cultures was more than a thousandfold greater than the concentration of
calcium present as an impurity in the control cultures of the present experiments.
Our result therefore throm doubt on whether the Rhizobium associated with
Trifolium has in fact a high calcium requirement. I t suggests further that the
difference between the calcium nutrition of the lucerne Rhizobium and that of the
soybean Rhizobium may not be one of calcium requirement for metabolism but
rather a difference in the reaction of these organisms to calcium in the presence of a
clay colloid.
I t seems reasonable to anticipate that the conditions of extreme acidity and of
lorn7 calcium supply which severely retarded the grovth of subterranean clover plants
in these experiments might be encountered in soils. I n such soils subterranean clover
would not grow well even if the plants were nodulated (e.g. by use of a coat of lime
on the seed). A further problem under conditions of extreme acidity would be the
nodulation of the legume in years after the establishment of the pasture, for our
results show that at extreme acidity nodulation is inhibited even a t very high levels
of calcium.
On the other hand the data presented in this paper show that there is a wide
range of conditions in respect to calciun~and hydrogen ion concentrations within
which nodulation of subterranean clover is inhibited to a varying degree but throughout which growth is not affected. Soils whose calcium and hydrogen ion concentrations fall within these limits can be expected to support maximum growth of
subterranean clover without the addition of lime, although the use of lime may
472
J. F. LONERAGAN AND E. J. DOWLING
be obligatory to achieve nodulation. The use of trace amounts of lime in the form of
pellets around the seed (Loneragan et ul. 1955) should be sufficient to achieve nodulation and maximuni production on these soils. This conclusion is in keeping with
the results of Anderson and Moge (1952) who pointed out that on the more acid soils
of the Southern Tableland of New South Wales, where responses to lime occurred in
the early years of sown pasture, there TTas a continuous and substantial improvement
in nodulation and yield of subterranean clover over a period of five seasons where
no lime was used. The lime was apparently needed on these soils only for nodulation
in the year of establishment.
Mr. G. A. BlcIntyre, Division of illathematical Statistics, C.S.I.R.O., has
been most helpful with advice and statistical treatment of data. We also wish to
thank Mr. J. Brockwell and Mr. F. J. Bergersen of the Division of Plant Industry,
C.S.I.R.O., for help and advice v-ith bacteriological techniques, Mr. D. J. David for
the calcium analyses, and Dr. A. J. Anderson for his advice and criticism a t all
stages of the investigations. Mr. K. C. illarshall of the New South Wales Department of Agriculture kindly supplied the culture of Rhizobium trifolii strain NA30.
VI. REVERENCES
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TV. E., and JOHNSON,
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T . M. (1937).-Behaviour of legume bacteria (Rhizobium) in relation t o exchangeable
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JV. R., PEECH,M., and BRADFIELD,
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