Plant and Soil 78, 381-391 (1984). Ms.

5514
9 1984 Martinus Ni]hoff/Dr W. Junk Publishers, The Hague. Printed in the Netherlands.

Aluminium toxicity and nodulation of Trifofium repens

M. WOOD, J.E. COOPER* and A.J. HOLDING*
Department o f Agricultural and Food Bacteriology, The Queen's University o f Belfast, and
Agricultural and Food Bacteriology Research Division, Department o f Agriculture for Northern
Ireland*, Newforge Lane, Belfast BT9 5PX, Northern Ireland

Received 30 May 1983. Revised November 1983

Key words Aluminium Calcium Nodulation pH Phosphate Polymeric hydrolysis

Rhizobium Rhizosphere Root elongation Root hairs Trifoliumrepens White clover

Summary Effects of aluminium on the Trifolium repens war Huia-Rhizobium trifolii strain
HP3 symbiosis were studied using an axenic solution-culture system. With 10 ~zM phosphate,
5 0 # M aluminium reduced or inhibited root elongation at pH <5.0, root hair formation at
pH < 5 . 0 - 5 . 5 , and Rhizobium multiplication in the rhizosphere and nodule formation at
pH < 6.0. In the absence of aluminium, root elongation and root hair formation were reduced
at pH < 4.3, and Rhizobium multiplication and nodule formation were inhibited at pH < 5.0.
Root hair formation was more sensitive to aluminium at pH < 5 than was root elongation. No
effect of aluminium on Rhizobium multiplication and nodule formation at pH < 5 was detected
because both were sensitive to pH alone.
At pH 5.5 most of the aluminum changed immediately to a form which was susceptib!e to
low-speed centrifugation, but which was detected by the aluminon method of analysis, and after
24 h a precipitate formed. The concentration of phosphate was reduced also, to approximately
1 #M. Toxicity was overcome by either increasing the phosphate concentration from 10 to
50/~M, or by increasing the pH to 6.0 and the calcium concentration to 1000/~M.

Introduction
Early studies implicated aluminium in acid soil infertility 1' 2 and this
has been confirmed for certain soils in Australia 3, the southeastern
United States 4 and southern Brazil s. Studies on the chemistry of acid
mineral soils show them to be aluminium-saturated systems 6, with most
of the acidity being due to hydrolysis of the various forms of
aluminium present 7.
The developmental stages of legume-Rhizobium symbioses are
particularly sensitive to low pH and high aluminium concentrations.
Aluminium depresses nodulation of Stylosanthes but does not affect
nitrogen fixation in plants previously nodulated in the absence of
aluminium 8, 9 Aluminium and low pH are more important than
calcium deficiency and high manganese concentrations (the other
principal soil acidity factors) in limiting growth of slow-growing
rhizobia in laboratory media 1~ and nodulation of Trifolium repens by
Rhizobium trifolii n. Aluminium also inhibits the growth of R. trifolii
in the rhizosphere of T. repens and depresses nodule formation at

381
382 WOOD, COOPER AND HOLDING

pH 5.5, at which aluminium precipitates out of solution n . The form of

aluminium under these conditions depends m a i n l y upon the con-
centration of hydroxide and phosphate ions z~. This paper reports the
results of a more detailed study of the effects of aluminium, calcium
and phosphate on the T. r e p e n s - R . t r i f o l i i symbiosis in the pH range
4--6.

Materials and methods

Four experiments were carried out under axenic conditions using the solution-culture
method of Wood, Cooper and Holding 13, in a Fisons 600G3/TTL growth cabinet at 15~ with a
20 h day and 4 h night. Surface-sterilised seed of T. repens was used together with R. trifolii
strain HP311 . Measurements of root elongation, root hair formation, Rhizobium numbers in the
rhizosphere and nodule formation were as described previously 1~, 13.
The first experiment examined the response of the symbiosis to calcium and aluminium over
a wider range of pH than that previously reported H. A complete factorial design of 5 pH • 2
calcium X 2 aluminium was used with the following treatment levels: pH 4.0, 4.5, 5.0, 5.5,6.0;
50 and 1000/~M calcium (as CaC12 ' 2H20); 0 and 50 ~tM aluminium (as A1K (SO4)2 9 12H20).
Two replicate growth assemblies were included per treatment.
The second experiment examined the response of the symbiosis to 5 0 ~ M aluminium at
pH 5.5 with 1000 ~M calcium and 10, 50 and 100 ~tM phosphate (as Nail 2PO4-2H20). Three
replicate growth assemblies were included per treatment.
The third experiment examined the response of the symbiosis to aluminium concentrations
< 50~M, and to calcium, at pH 4.5. A complete factorial design of 2 calcium • 6 aluminium
was used with the following treatment levels at pH 4.5 : 50 and 1000 ~M calcium; 0, 10, 20, 30,
40, 50/zM aluminium. Two replicate growth assemblies were included per treatment.
In the final experiment the concentrations of aluminium and phosphate in the rooting
solution at pH 4.3 and 5.5 were determined by chemical analysis. Growth assemblies were
prepared with 1000~zM calcium and the following treatments: pH 4.3, 5 0 ~ M aluminium;
pH 5.5, no aluminium; pH 5,5, 50 # M aluminium. The solutions were shaken, then sampled at
4, 24 and 48 h after adjusting the pH. The samples were centrifuged at 2000 g for 20 min and
the supernatant analysed for aluminium and phosphate. In addition, a pH5.5, 5 0 g M
aluminium treatment was sampled, after shaking, at 0, 45 and 120 rain and either centrifuged
and analysed for aluminium, or analysed immediately without centrifugation. This treatment
was then left undisturbed for a further 21 h, the upper part of the solution sampled without
shaking or centrffugation, and analysed for aluminium.
Attempts were made initially to measure the concentration of aluminium using a Perkin
Elmer 306 atomic absorption spectrophotometer, coupled to a heated graphite atomiser, but
this method gave inconsistent results. This is in agreement with an earlier report ~4 of the unsuit-
ability of this method for routine aluminium analysis. The colorimetric aluminon method of
Hsu ~s with the additional use of ascorbic acid to eliminate interference from iron 16 was found
to be satisfactory. The method was used as described b y Jayman and Sivasubramanian ~6, but
standard solutions were prepared from A1K(S04) 2 9 12H20. Phosphate was measured by the
method of Watanabe and Olsen 17 . In both analyses absorbance was measured using a Pye
Unicam SP-6-500 spectrophotometer.

Results

R o o t elongation
Analysis of variance of the root elongation data from the first experi-
ment showed that all of the main factors had significant effects on root
ALUMINIUM TOXICITY AND NODULATION OF WHITE CLOVER 383

Table 1. Effect of pH and calcium and aluminium concentrations on root elongation and root
hair formation by T. repens vax Huia, and nodule formation by R. trifolii strain HP3 (2 obser-
vations per treatment; SE root elongation means 1.9)
pH
4.0 4.5 5.0 5.5 6.0
Aluminium Calcium
(#M) (#M) RE RH N RE RH N RE RH N RE RH N RE RH N
0 50 15 0 24 + 0 22 + 0 21 + 83 22 + 50
1000 19 -- 0 28 + 0 26 + 50 .22 + 83 22 + 100
50 50 2 -- 0 6 0 18 -- 0 28 + 17 26 - 17
1000 19 0 18 0 24 -- 0 26 + 0 19 - 100
RE, root elongation (mm); RH, presence (+) or absence (--) of root hairs after 10 days; N,
percentage plants nodulated after 10 days.

elongation; these effects were included in the significant ( P < 0.05)

interaction between pH, calcium and aluminium. The results (Table 1)
show that in the absence of aluminium root elongation was reduced at
pH4.0, particularly with 50/~M calcium, but was unaffected at
pH > 4.5. 50/~M aluminium was severely toxic to the roots at pH 4.0
and 4.5 with 50 ~M calcium. The stunted roots with laterals appearing
as peg-like projections are characteristic aluminium toxicity symp-
toms 11,18. An increase in the calcium concentration to 1000#M com-
pensated for the adverse effects of 50/aM aluminium at pH < 5 . 5 0 #M
aluminium had no effect on root elongation at pH 5.0-6.0, in agree-
ment with the earlier report for pH 5.511 . This response by root
elongation to 50/aM aluminium at pH 5.5 was unaffected by 10
100#M phosphate (Table 2).
Analysis of variance of the root elongation data from the third
experiment showed that there was a significant (P < 0.01) interaction
between calcium and aluminium for root elongation at pH 4.5. Table 3
shows that in the presence of aluminium root elongation was greater
with 1000/aM than with 50/aM calcium. This effect of calcium was
particularly pronounced with 30, 40 and 50/~M aluminium. With
50 tam calcium there was a progressive decrease in root elongation as
the concentration of aluminium was increased from 10 to 50 ~tM. With
Table 2. Effect of phosphate concentration at pH 5.5 with 5 0 ~ M aluminium on root elong-
ation and the presence (+) or absence (--) of root hairs after 10 days by T. repens vat Huh, and
nodule formation (percentage plants nodulated 10 days after inoculation) by R. trifolii strain
HP3 (3 observations per mean; SE mot elongation means 1.2)
Phosphate (#M)
10 50 100
Root elongation (ram) 23 20 20
Root hair formation + + +
Percentage plants nodulated 0 44 67
384 WOOD, COOPER AND HOLDING

Table 3. Effect of calcium and aluminium concentrations at pH 4 s on root elongation and root
hair formation by T. repens vat Huia (2 observations per treatment; SE root elongation means
1.4)
Aluminium 0zM)
0 10 20 30 40 50

Calcium (I~M) RE RH RE RH RE RH RE RH RE RH RE RH
50 23 + 22- 19- 15 -- 6 -- 3 --
1000 19 + 27 + 25 -- 24- 16- 17 --

RE root elongation (mm)

RH presence (+) or absence (--) of root hairs after 10 days

1000gM calcium the increase from 10 to 30taM aluminium had no

effect, and 40 and 50 gM aluminium reduced root elongation.

Root hair formation

The results (Table 1) show that root hair formation was unaffected
at pH 6.0 and 5.5 in the presence and absence of aluminium, and at
pH 5.0 and 4.5 in the absence of aluminium. There were no root hairs
at 4.0 with both levels of calcium and aluminium, nor at pH 4.5 and 5.0
with 50taM aluminium. Root hairs were formed at pH 5.5 with 50/~M
aluminium and 10-100 taM phosphate (Table 2).
At pH 4.5 there was an interaction between calcium and aluminium
(Table 3). In the absence of aluminium root hairs were formed with
both levels of calcium, in agreement with the results from the first
experiment (Table 1). There were no root hairs with 20-50taM
aluminium at both levels of calcium nor with 10 gM aluminium and
50 gM calcium, However the inhibitory effect of 10/aM aluminium was
overcome by increasing the calcium concentration to 1000 p.M.

Rhizosphere population of Rhizobium

Analysis of variance of the Rhizobium data from the first experiment
showed that all of the main factors had significant effects on the
Rhizobium population, and were included in the significant (P < 0.001)
interaction between pH, calcium, aluminium and time (Fig 1). At
pH 4.0 and 4.5, with both levels of calcium and aluminium, the popu-
lation was stable or decreased. A similar response was reported for this
symbiosis at pH 4.3 and 4.7 al. There was reduced multiplication of
Rhizobium in the absence of aluminium at pH 5.0 with 1000taM
calcium, but no growth with 50/~M calcium. At pH 5.0 with 50 taM/
aluminium the aluminium precipitated out of solution, and the
Rhizobium population decreased with both levels of calcium.
The response of the Rhizobium population to pH 5.5 was the same
 ALUMINIUM TOXICITY AND NODULATION OF WHITE CLOVER 385

pH 4.0 50yM calcium pH/.,.0= lO00/uM calcium

2 0 If'--------
i~ I 9
[~,I 7 " - ~._~__.____
I or ~= ,~
01
0
I
1 2
I I
3 4
I
5
I I0 ,
~I 2 3 ~ 5

pH 4.5 50jan calcium pH 4.5 IO00)JM calcium

~of><.><~: _ _ . ~ _ , _ _ , f
F ~ , _9 _ , __.
@_-------. 9 9
1.0F ~ / o ~ o / o
3[ I I I I I I I I I I I
0 1 2 3 Z, 5 0 1 2 3 l. 5

3"0FpH 5.0 50juM calcium r pH 5.0 IO00)JM calcium 9 9

i- t o~ ~

s
'or I " "
8 o~ ~ ; , ~ ~ o I ; 3 ~'J' i. 5
T
,~ 5.0 " pH 5 5 50}aM calcium "pH 5.5 IO00~uM calcium

J
"2
L3 O~ 0 O~ j 9
d 4.0 ./
(-
3.0
?, 0j O
2.0

1.0
~.I.~. "~'I\
l l l
, \,/,
1 2 3 ~ 5 1 2 3 ~ 5

5.0
pH 6.0 50)aMcalcium o / ~ "PH 6.0 IO00/uM calcium e ~-'~~
t..0

3.0
~.~'~ ,...-I._"
. S . ~. .~~176
,~"

2.0 ~*.,-.._.__, ~, ~ I I--I 50/uM

1.0

I I I~,I, _n I I I 1 I
1 2 3 z, 5 0 1 2 3 ~ 5
Time (days) Time (days)

Fig. 1. Effect of pH and calcium and aluminium concentration on the numbers of R. trifolii
strain HP3 in the zhizospheze of T. repens vat Huia (2 Observations per mean; SE means 0.29).
For A1 concentrations See Legend in the figure.
386 WOOD, COOPER AND HOLDING

5.0 Phosphate
C
.9 I--I IO)JM .-~
~5 &.O 0--, 50pM t~---~I"

3.0
ul

2. 2.o i
0
d
1.0 9 9_ _ / I ~
0
-J 0 I I I I 1
0 I 2 3 4 5
Time (days}
Fig. 2. Effect of phosphate concentration on the numbers ofR. trifolii strain HP3 in the thizo-
sphere of T. repens var Huia at pH 5.5 with 5 0 u M aluminium (3 Observations per mean; SE
means 0.24).

as that reported previously11; there was exponential growth in the

absence of aluminium and a decrease in the population with 50/~M
aluminium and both levels of calcium. The same response was observed
at pH 6.0 with both levels of calcium in the absence of aluminium, but
the toxic effect of 50 pM aluminium was overcome by increasing the
calcium concentration from 50 to 1000 pM.
Phosphate had a significant ( P < 0.001) effect on the Rhizobium
population and the results (Fig 2) show that the inhibitory effect of
50/~M aluminium at pH 5.5 with 10 pM phosphate was overcome by
increasing the phosphate concentration to 50 or 100/~M (compare the
growth with that under non-stressed conditions in Fig 1).
There was no effect of calcium or aluminium on number of
Rhizobium in the rhizosphere at pH 4.5; the Rhizobium population
decreased with both levels of calcium and all levels of aluminium, in
agreement with the result from the first experiment (Fig 1).

Nodule formation
The results for nodule formation (Table 1) show inhibition at pH 4.0
and 4.5 with both levels of calcium and aluminium. The response at
pH 4.5 was confirmed with O-50pM aluminium in the third experi-
ment. Nodulation is also inhibited at pH 4.3 and 4.7 under the same
conditions la, No nodules were formed at pH5.0 with 50/aM
aluminium and both levels of calcium, nor were there any in the
absence of aluminium at pH 5.0 with 50gM calcium. However 50% of
the plants were nodulated when the calcium concentration was
increased to t 000/sM.
The nodulation response at pH 5.5 was the same as that previously
reportedH; 83 percent of the plants were nodulated in the absence of
aluminium, but only 0 - 1 7 percent with 50taM aluminium. A similar
ALUMINIUM TOXICITY AND NODULATION OF WHITE CLOVER 387

response was observed at pH 6.0 with 50 taM calcium, but the adverse
effect of 50tsM aluminium was overcome by raising the calcium con-
centration t o 1000/aM.
An increase in'the phosphate concentration from 10 to 50 gM caused
an increase in t h e percentage plants nodulated at pH 5.5 with 50/~M
aluminium from 0 to 44 percent, and with 100/~M phosphate 67
percent of the plants were nodulated (Table 2).

Alumlnium and phosphate analyses

The results (T~ble 4a) show that at pH 4.3 all of the aluminium and
phosphate was detected by the analyses (no precipitate was observed).
There was no difference between the phosphate values at pH 4.3 in the
presence of aluminium and at pH 5.5 without aluminium. With 50 uM
aluminium at pH5.5 a precipitate was observed after 24h. Although
there was no visible precipitate after 4h, the concentrations of
aluminium and phosphate in the supernatant decreased to 5.5 and
1.3/aM respectively. Similar levels were found after 24 and 48 h.
Aluminium analyses after shorter times (Table 4b) showed that the
concentration of aluminium in the supernatant of a centrifuged sample
was reduced to 4~0 ~M after 20 min. When the solution was not centri-
fuged, all of the aluminium was detected after 2 h. However, when the
solution was left undisturbed for a further 21 h the precipitate which
formed settled out of the upper part of the solution and the residual
concentration of aluminium was 8 ~3/.

Table 4. Aluminium and phosphate analyses of rooting solution (a) with different levels of pH
and aluminium concentration, with eentrifugation prior to analysis; (b) at pH 5.5 with 50 u M
aluminium, with and without eentdfugation prior to analysis (2 observations per mean)
(a)
pH Aluminium ~ M ) Analysis Time (h)
4 24 48
5.5 0 Aluminium (#M) 0 0 0
Phosphate 0tM) 10.5 11.2 9.4
5.5 50 Aluminium 0zM) 5 .5 4.0 2.0
Phosphate (#M) 1.3 2.7 0.8
4.3 50 Aluminium ~ M ) 51.0 46.0 53.0
Phosphate (pM) 10.5 11.0 8.7
O)
Time (rain)
0 20 45 65 120 145
Centrffugation -- + -- + _ +
Aluminium ~ M ) 50.5 4.0 47.5 4.0 47.5 5.0
388 WOOD, COOPER AND HOLDING

Table 5. Critical pH values in the absence and presence of aluminium, with 10 # M phosphate,
for four stages in the development of the symbiosis between T. repens and R. trifolii
Stage Critical pH value
No aluminium 50 uM aluminium
Root elongation 4.3 5.0
Root hair formation 4.3 5.0-5.5
Rhizobium multiplication 5.0 6.0
Nodule formation 5.0 6.0

Discussion

Critical pH values
These results, together with those reported previously xa, enable
critical pH values to be defined, both in the absence and presence of
aluminium, for some of the stages leading to nodulation of T. repens by
R. trifolii (Table 5). In some instances the critical pH value is that value
at which an increase in the calcium concentration from 50 to 1000 taM
overcomes the otherwise inhibitory effect of that pH. These critical
values show that at pH 4.3-5.0 root elongation and root hair formation
are limited by aluminium rather than by pH, whereas Rhizobium multi-
plication and nodule formation are limited by pH alone. With 50 ~M
aluminium the pH must be raised to 5.0 for optimum root growth and
root hair formation, whereas pH 6.0 is required with 10 taM phosphate
for optimum growth of the rhizosphere population of Rhizobium and
nodule formation.

Implications for acid soils

These results suggest that in a soil of pH < 5.0 with a high organic
matter content (therefore a low level of exchangeable aluminium 19)
nodulation of white clover could be limited by lack of multiplication
and nodulating ability of the Rhizobium, rather than by growth and
development of the roots of the host plant. If the pH of this soil is
raised to pH > 5.0 then nodulation should occur. However, in a soil of
pH < 5.0 with a low organic matter content (therefore a high level of
exchangeable aluminium 19) development of the symbiosis could be
limited by both poor root growth and root hair formation, and lack of
multiplication and nodulating ability of the Rhizobium. For nodulation
to occur in such a soil, either the pH must be raised to 5.5 by the
addition of lime (which also supplies calcium) and the phosphate con-
centration increased to 50 taM, or with a lower concentration of phos-
phate the pH must be raised to 6.0. This is supported by an observation
that the addition of phosphate to an acid brown earth limed to pH 5.5
ALUMINIUM TOXICITY AND NODULATION OF WHITE CLOVER 389

improved the nodulation response by white clover (Holding and

Rangeley, unpublished). The phosphate concentration in the soil
solution surrounding plant roots is in the range 0.1-1.0 #M 2~ however,
so aluminium toxicity will probably be eliminated more effectively by
raising the pH to around 6.0.

Critical aluminiurn concentrations and toxicity mechanisms

The critical aluminium concentration for root elongation at pH 4.5
was 30/~M, whereas the value for root hair formation was 10#M. These
results suggest that root hair formation is more sensitive to aluminium
at pH < 5 than is root elongation, but both parameters are equally
sensitive to low pH. The mechanism of aluminium toxicity towards
plants at pH < 5 is not clearly understood, but several mechanisms have
been proposed. Aluminium toxicity has often been associated with
reduced uptake of several nutrients, especially calcium and phos-
phate 2x. Aluminium can also inhibit cell division by interfering with
mitosis is and may reduce sugar phosphorylation in root metabolism 22.
The main beneficial effect of calcium addition on root elongation at
pH < 5 was in the removal or partial removal of the inhibitory effect of
aluminium, but calcium had little effect on root hair formation at
pH < 5 with aluminium present. Some of the effect of calcium could be
in decreasing the activity of aluminium ions in solution rather than in
affecting the physiology of the plant 23.
It proved impossible to detect an effect of aluminium on Rhizobium
multiplication and nodule formation at pH < 5 because both were
inhibited by pH alone. This contrasts with the response of slow-growing
strains of Rhizobium (e.g. those belonging to the 'cowpea miscellany')
which are generally more tolerant of low pH than fast-growing R. trifolii
strains 24, and which show reduced multiplication with 50#M
aluminium at pH 4.52s, and reduced nodulation at pH 4.5 with 125 pM
aluminium 8. In all treatments where Rhizobium multiplication was
reduced or inhibited, there was a reduction in nodule formation. In an
axenic solution-culture system multiplication of Rhizobium in the
rhizosphere may not be necessary for nodule formation, but in a com-
petitive environment multiplication may be of more importance in
ensuring nodule formation by a particular strain.
The mechanism of the toxic effect of 50/sM aluminium on
Rhizobium multiplication and nodule formation at pH 5.5 is unclear.
It is generally agreed that at p H > 7 aluminium is present as
AI(0H)a(H20)~ and at p H < 4 ad Al(H20)6 a+26 As the ratio of
hydroxyl to aluminium ions is increased in the range pH 4 - 7 , dissolved
species containing aluminium and hydroxyl ions are formed. When the
390 WOOD, COOPER AND HOLDING

supply Of hydroxyl ions is great enough a precipitate of aluminium

hydroxide is formed. However, there is little agreement about the form
of these intermediate dissolved species. There is good evidence in
support of a polymeric hydrolysis scheme 27 which proposes the
existence of positively.charged polynuclear complexes in which the
ratio of hydroxyl to aluminium ions need not be integral. Before the
aluminium is completely neutralised by hydroxyl ions the residual
positive charge on the polymers must be balanced by counter-anions
such as phosphate, and the concentration of these anions can affect the
stability of the polymers formed 12.
The chemical analyses show that as soon as the pH of the rooting
solution was raised from 4.3 to 5.5 most of the aluminium was changed
to a form which was susceptible to low-speed centrifugation, but which
was also detected by the aluminon method of analysis(Table 4). This
included an acid-hydrolysis step, and the aluminon reagent was
buffered at pH 4.1, so the aluminium which remained after centri-
fugation (approximately 5/aM) was not necessarily in a monomeric
form ie the same form as at pH 4.3. The concentration of phosphate
was reduced, together with that of aluminium, to approximately 1 laM.
In view of the recent report that Rhizobium can multiply in the
absence of an external source of phosphate 28, it seems unlikely that the
death of Rhizobium at pH 5.5 with 50/aM aluminium was due to an
indirect effect of reducing the concentration of phosphate in solution.
The removal of this toxic effect by increasing the phosphate concen-
tration from 10 to 50/aM was probably due to an acceleration of the
neutralisation of aluminium by hydroxyl and phosphate ions. The
effect of aluminium on R. trifolii at pH 5 - 6 is being investigated
further.

Acknowledgements The authors are grateful to Dr D J Kilpatfick for assistance with experi-
mental design and statistical analysis of data, and to the Ministry of Agriculture, Fisheries and
Food for providing the senior author with a Postgraduate Studentship.

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