Diurnal Changes in Nitrogen Fixation in the Root Nodules of Casuarina

Diurnal Changes in Nitrogen Fixation in the Root Nodules of CasuarinaAuthor(s): G. Bond and Anne H. MackintoshSource: Proceedings of the Royal Society of London. Series B, Biological Sciences, Vol. 192, No.1106 (Dec. 31, 1975), pp. 1-12Published by: The Royal SocietyStable URL: http://www.jstor.org/stable/76978 .

Accessed: 07/05/2014 03:32

Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at .http://www.jstor.org/page/info/about/policies/terms.jsp

.JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range ofcontent in a trusted digital archive. We use information technology and tools to increase productivity and facilitate new formsof scholarship. For more information about JSTOR, please contact [email protected].

.

The Royal Society is collaborating with JSTOR to digitize, preserve and extend access to Proceedings of theRoyal Society of London. Series B, Biological Sciences.

http://www.jstor.org

This content downloaded from 169.229.32.138 on Wed, 7 May 2014 03:32:06 AMAll use subject to JSTOR Terms and Conditions

http://www.jstor.org/action/showPublisher?publisherCode=rsl

http://www.jstor.org/stable/76978?origin=JSTOR-pdf

http://www.jstor.org/page/info/about/policies/terms.jsp


Proc. B. Soc. Lond. B. 192, 1-12 (1975) Printed in Great Britain

Diurnal changes in nitrogen fixation in the root nodules of Casuarina

BY G. BOND, F.R.S. AND ANNE H. MACKINTOSH

Botany Department, University of Glasgow

(Received 7 July 1975)

Measurements were made of the rate of nitrogen fixation and of the carbohydrate content of nodules detached at different times of day from plants of Casuarina cunninghamiana growing in water culture under semi- natural conditions in a greenhouse lit by daylight and subject to considerable diurnal variation in temperature also. The 15N method was used for the measurement of fixation. A distinction was made between 'fixation potential' - the rate shown by nodules incubated under 15N at a standard favourable temperature, and 'actual fixation', namely the rate shown by nodules incubated at the temperature prevailing in the greenhouse at the time. It was assumed that the values obtained for actual fixation, though known to be considerably below those prevailing in the nodules at the times of detachment, would be proportional to the latter.

Actual fixation was low in the early morning, but soon rose and remained at a relatively high level for several hours centred about mid- afternoon, and then fell. A separate study showed that the rate of fixation increased steadily over the temperature range 10-36 ?C, and indicated that temperature fluctuations in the greenhouse were partly responsible for the diurnal changes in the rate of fixation. An additional factor, presumably light intensity acting through its effect on the supply of

carbohydrates to the nodules, also appeared to be involved, but although evidence was obtained that during periods of low light or darkness the fixation potential was related to the level of nodular carbohydrates, over the part of the day when actual fixation was high the nodules showed a low carbohydrate content. The possible explanation of this situation is considered.

1. INTRODUCTION

The examination of nodulated plants growing under natural or semi-natural

conditions for diurnal variations in the rate of nitrogen fixation, and the further

analysis of any variations detected, cannot fail to add to knowledge of the physio-

logical functioning of such plants. Though it is not the method used in the present

study, the introduction of the acetylene assay as an indirect means of measuring fixation has been followed by several examinations of nodulated plants for such

diurnal rhythms, but for brevity's sake only such of these studies as related to

non-leguminous nodulated plants will be noted here. In first-year plants of Alnus glutinosa and Myrica gale growing in a greenhouse

which on the days of his experiments was kept at near constant temperature,

Vol. I92. B. (3I December I975) [ I ]



G. Bond and Anne H. Mackintosh

Wheeler (i969, 1971) found that maximum rates of acetylene reduction were shown over the midday period, and under the circumstances this was attributed to the effect of light intensity. No relation could, however, be detected between the indicated rates of fixation and the gross level of carbohydrates in the nodules at different times of day, but evidence was provided that a substantial proportion of the nodular carbohydrate was unavailable for use in fixation, and that the controlling factor was the rate of ingress of new photosynthates into the nodules. It was shown by 14C experiments that in the plants studied, translocation from the leaves to the nodules can be a rapid process and is likely to be considerable around midday; the incoming carbohydrates appear to be used quickly in fixation and associated processes, so that the gross level of nodular carbohydrates is not significantly affected.

Subsequently attempts have been made to detect light-induced diurnal changes in non-leguminous nodulated plants under field conditions. Akkermans (I97I) found a higher mean rate of acetylene reduction in daytime than at night in nodules excised from a tree of Alnus glutinosa and incubated at a standard temperature during assay, while Fessenden, Knowles & Brouzes (I973), in similar tests on nodules from bushes of MIyrica asplenifolia, found no evidence of any immediate

light-dependence on the part of the nodules; the authors of both papers stress the magnitude of the sampling error with field material and the difficulty of drawing any firm conclusion. Soil temperature records given by Akkermans (I97I) for Alnus and Hippophae sites indicate that diurnal variations in fixation induced

by the temperature factor are very likely to occur in the field. The present study, to which Bond (I971) made a brief interim reference, grew

out of tests made in 1966 to establish whether there was any particular time of day when nodules of greenhouse-grown plants of Casuarina, which were being used in conjunction with 15N in studies of the effect of various factors on fixation, should be detached in order to obtain the most rapid fixation. The results (see later) prompted further experiments which were carried out as plants became available from 1967 to 1972. Despite some disadvantages attached to the 15N method, in particular that in using it attention has, for reasons of economy, to be confined to detached nodules, the use of the method was continued in the present study so that all the data would be comparable.

In addition to the different methodology and greenhouse management (see below), the present study differs from those of Wheeler (i969, 1971) in that it relates to another genus - one whose species are adapted to tropical rather than temperate conditions, while in addition the plants at the time of use were older and much larger. Although it is a large and important genus there has hitherto been little experimental study of the nodules of Casuarina.

2



Diurnal changes in nitrogen fixation in Casuarina

2. MATERIALS AND METHODS

(a) Plant culture

Except in the initial experiment the nodules studied were taken from plants of Casuarina cunninghamiana Miq. growing in a greenhouse lit by daylight and

subject to a considerable diurnal fluctuation in temperature also. According to Coyne (1973) this is the largest-growing of the Australian species of Casuarina, attaining a height of up to 35 m and a trunk diameter of up to 1.3 m. It is common in many parts of eastern and northeastern Australia and occurs typically near streams and rivers where it has the useful effect of stabilizing their banks. Seedlings raised from seed obtained from Vilmorin-Andrieux, Paris, were set up in May in water culture in Crone's solution made up to a nitrogen-free formula, but for the first month the seedlings were supplied with 10 mg/l of ammonium-nitrate nitro-

gen. Then they were changed to nitrogen-free solution and inoculated by applying to their roots an aqueous suspension of crushed nodules taken from older plants of the same species. Nodules appeared after 3-4 weeks and after a further interval the plants began to grow strongly.

The experiments were made with nodules taken from plants in August of their second year of growth, when their attributes were approximately as follows:

height 1 m, dry mass per plant 16 g, nodule dry mass per plant 1 g, total nitrogen content per plant 350 mg. At the time of use the plants were growing three per two-litre jar.

(b) Measurement of rate of fixation As indicated already, these measurements were made on nodules in the detached

condition. Those used on each occasion of fixation measurement were usually drawn from 3-5 plants, undersized plants being avoided. Only a proportion of the nodules present on each plant was taken and the nodules were combined to give a bulk sample of 4.5 g fresh mass. These particular plants and their remaining nodules were then discarded, unless the nodules were required for carbohydrate estimations. After thorough mixing (facilitated by prior removal of the longer nodule-roots) the bulk sample was divided equally between three 28 ml bottles which were then evacuated and refilled to 1 atm (105 Pa) with a gas mixture containing 10 % nitrogen, 20 % oxygen and 70 % argon. The time elapsing between the beginning of nodule picking and the final gassing was 15-20 min. Where what will be termed the 'fixation potential' was being measured, the nitrogen in the gas mixture contained 31 atoms % excess '1N, and the nodules were incubated under the gas mixture for 3 h at 24 ?C. Where it was sought to assess the 'actual fixation', nitrogen with 60 atoms % excess 15N was used and the nodule samples (protected from direct sunlight) were placed on the greenhouse bench for 2 h at the prevailing temperature(s). At the end of the period the nodules were immersed in cone. sulphuric acid, preparatory to their subjection to the salicylic acid- thiosulphate modification of the Kjeldahl process (Horwitz I955). The distillates

I-2

3




from that process were analysed by mass spectrometer for 15N content by Dr C. W. Crane, Queen Elizabeth Hospital, Birmingham.

The results of the measurements of fixation rates will be expressed as total fixation (14N and 15N) per unit of nodule nitrogen; there was no discernible diurnal variation in the magnitude of the latter. The maximum enrichment found in the nodule samples was 0.496 and the minimum (except in the darkening experiment -

see later) 0.018 atom %. Several samples of nodules, analysed without exposure to excess 15N, showed the normal content of 15N, i.e. 0.370 atom %.

In some experiments the triplicate samples of nodules exposed to 15N were

separately analysed for 15N content, and least significant differences (P = 0.05) calculated by analysis of variance. In other experiments the distillates from the

Kjeldahl estimations on the triplicate samples were combined prior to 15N assay, but it is reasonable to assume that least significant differences in these experiments will be proportionately similar to those calculated as above, where they were of the order of 30 % of the mean of all the observations.

(c) Estimation of nodular carbohydrates

Nodule samples for this purpose were quick-frozen in tubes immersed in ethanol-

dry ice mixture and stored at - 20 ?C until analysed. They were then freeze-dried

and triplicate samples analysed for sugars and polysaccharides by the method of

McCready, Guggolz, Silviera & Owens (I950), involving extraction with ethanol

and dilute perchloric acid respectively, followed by the colorimetric assay of the

extracts after treatment with anthrone reagent. The two fractions will subse-

quently be designated 'soluble' and 'reserve' respectively, and amounts present will be expressed in terms of glucose and on the basis of nodule dry matter.

Statistical treatment of these results was carried out as for the fixation data.

(d) General

Light intensity was measured with an 'EEL' light meter, which is sensitive to

wavelengths in the range 250-730 nm. Quoted times of day are in British Summer

Time, i.e. G.M.T. plus 1 h. The aim was that experiments on diurnal variation

should be done on sunny days, when there would be marked changes in greenhouse

temperature and light intensity, but the need for advance preparations necessitated

reliance on weather forecasts. Regrettably fixation measurements during night- time were impracticable, mainly because the assistance of several people was

needed for the rapid collection of nodules. Further details of methods for particular experiments are given later.

3. DATA OBTAINED

(a) Diurnal variation in fixation potential

In 1966 Dr C. Rodriguez-Barrueco, Centro de Edafologia y Biologia Aplicada, Salamanca, who was then working in the authors' laboratory, determined the

4




fixation potential of nodules of Casuarina torulosa at four different times during a sunny day on which by early afternoon the temperature in the greenhouse reached 27 ?C and the light intensity 75000 lx, sunrise being at 06h03 and sunset at 20h34. As shown in figure 1, between 07h00 and llhOO fixation potential fell to a just significant extent, but then began to rise slowly and by 19h00 had

regained the early morning level.

4-

0

0

0 -

2- 0 1.s.d.

I ! I I ! I

08th0() 1 2hOO 16h00 20h00 24h00

time of nodule detachment

FIGURE 1. Diurnal variations in fixation potential of Casuarina nodules. O, C. torulosa; O, C. cunninghamiana. The least significant difference (l.s.d.) shown refers to the first species. Approximate times of sunrise and sunset in both experiments were 06h00 and 20h30 respectively.

A similar experiment, except that nodules of C. cunninghamiana were used, was later carried out by the present authors. On the day of the experiment the sky was cloudy until midday but then cleared somewhat, giving a light intensity of 21500 lx and a temperature of 30 ?C in the greenhouse. As will be seen in figure 1, considerably larger changes in fixation potential were shown in this experiment. Between 07h00 and 1 hOO potential fell by half, and although, as in the above experiment, this was eventually followed by a return to the high level of the early morning, there is now an indication of a temporary rise in potential in the early afternoon. Though statistical treatment was not possible in this experiment, for reasons given, the levels of significance in other experiments suggest that this temporary rise is significant. It also seems likely that fixation potential remains high overnight.

(b) Diurnal variation in actual fixation As noted already, these experiments differed from those just described chiefly

in that the nodule samples were held in the greenhouse while under 15N, at the temperature(s) prevailing there.

The results of a first experiment are shown in figure 2. The day was unexpectedly

5



6 G. Bond and Anne H. Mackintosh

overcast, so that light intensity and temperature attained only moderate heights, as indicated. None the less significant changes were found in fixation rates, a

doubling being shown during the morning, repeated in reverse in the evening. Two further experiments confirmed that actual fixation is considerably higher

around the middle of the day than it is earlier or later. In one of these, at 15h00, when light intensity was 80000 lx and temperature 33 ?C, the fixation rate was close to 4 ,pg N per mg nodule N per hour, but had fallen to 1.9 [Lg at 23h00 (3 h after sunset). At 08h00 on the following morning the rate was 1.5 [g.

10000-

4, x , ,A . 30

5000- , o- . o\ =

i 3- * I

1-

. I

0I /

06h04, sunset 20h30. b0O

with records of light intensity (O) and temperature (x) within greenhouse. Sunrise

004, sunset 2h3.\ -

Carbohydrate estimations were made on nodule samples collected at the same times and from the same plants as those whose actual fixation rates were reported in the preceding subsection. The results are combined in relative form in figure 3; in preparing this a value of 100 was assigned to the level of soluble carbohydrate found at 23h r in each of the three experiments, and mean relative values calculated for other times of day. Data for reserve carbohydrates were expressed as

percentages of the corresponding values for the soluble fraction, and these too

were averaged for a given time of day. The results show that the levels of the




soluble and possibly of the reserve fraction also were significantly higher in the

early morning and late evening than during the middle of the day. The mean absolute value for the soluble carbohydrate fraction in Casuarina

nodules at 23h00 was 34 mg per g nodule dry matter, and for the reserve fraction 7.5 mg. At the same time in the evening Wheeler (I969, I97I) found the total

sugars in the nodules of Alnus glutinosa to be 58 mg per g nodule dry matter and the reserve fraction to be 150 mg. Thus the Casuarina nodule has a much lower level of the latter fraction; in corroboration it can be said that starch is much less in evidence in sections of those nodules than in the case of Alnus.

[ soluble carbohydrates I s.d{

'

reserve .s

100- lo -

o 80-

.

X 60-

-0 40-

C 20- a> 20-

07h00 llhOO 15h00 19h00 23h00 time of nodule detachment

FIGURE 3. Changes in carbohydrate content of Casuarina nodules during the day, presented in relative form (see text).

(d) Effect of temperature on rate of fixation This aspect was examined in order to assess the extent to which changes in

greenhouse temperature could explain the changes in actual fixation rate reported in ?3(b). Nodules taken from plants at 14h00 were incubated under 31 atoms % 15N for 3 h at five temperatures within the range likely to be experienced within the greenhouse, with three samples at each temperature. Initially a sufficient stock of nodules for the whole experiment was collected from 13 plants and well mixed. The results are shown in figure 4. A steady rise in fixation rate occurred with increasing temperature, with no suggestion of a falling-off even at 36 ?C.

Suitable calculation from figure 4 yielded the following Q10 values: 10-20 ?C, 2.8; 15-25 ?C, 1.8; 20-30 ?C, 1.6; 25-35 ?C, 1.4.

(e) Effect of darkening of plants on fixation

Although it is obvious that the continuance of fixation in the nodules is depen- dent in the long-run on a supply of photosynthates and thus on an adequate exposure of the plant to light, in the present connection it was necessary to explore

7




he time relation of the dependence. To this end the effect of artificial darkening of plants on the fixation potential of their nodules was studied, with accompanying carbohydrate analyses.

On the starting day of the experiment (day 0), the fixation potential of nodules detached at 12h00 from plants which had been under normal conditions was determined in the usual way, and at the same time similar plants were placed in an area

1.5-

s;

z l.o- a)

0-c

0 b0

4 0.5' a)

10 20 30

temperature/?C

l.s.d.

40

FIGURE 4. Effect of temperature of incubation on fixation in detached Casuarina nodules.

?

I 2

a) f4

Si

o

) 1

a)

z fx

* fixation

I

0 I I

[ soluble * r reserve - carbohydrates-- .carbohydrates

I 1 2 3

days from start

i 4 5

1 . ..

. .,::

.....

30 -c>

a)

-20

a),

-1

Ga

-20 9 bo

0.)

S

FIGURE 5. Effects of darkening on fixation potential and carbohydrate content of Casuarina nodules. The data for day 0 and day 4 relate to the nodules of plants kept under normal greenhouse conditions.

8

;:.

.

-

: :

;;;

r ?. ??

?? .?

. .

;.*.

I




within the greenhouse from which light was completely excluded by suitable screens. On each of the three following days and again on the fifth day, nodule

samples were taken from two of the darkened plants at 12h00 for the determination of the fixation potential and carbohydrate content, the two plants being then discarded. All the plants were of perfectly normal appearance when withdrawn from darkness. On the fourth day samples of nodules from plants which had remained under normal conditions were assayed.

The results are shown in figure 5. The data for the nodules from normal plants assayed on days 0 and 4 are in good agreement and show that fixation activity and carbohydrate content were not drifting for seasonal reasons. The nodules from darkened plants examined on day 1 - after 24 h darkness - showed a potential which was little more than half that recorded on day 0. On day 2 the potential was similar in value to that for day 0, but by day 3 it was of much lower value and

by day 5 was almost insignificant, marked reductions in soluble carbohydrate also

being shown on those two days, and on the fifth day in the reserve fraction also.

Although it is not impossible that fixation potential in the nodules of darkened

plants should first fall and then recover, the authors prefer to suspect that the low result on day 1 was due to an increased sampling error consequent on the collection of nodules from only two plants on each occasion in this experiment.

4. DIscussioN

Previous work (Bond I96I) had shown that after detachment the nodules of Casuarina retain the faculty for fixation relatively strongly, and that they fix

nitrogen at a steady rate for some hours, but in connection with the present study it was desired to establish how this rate compared with that shown prior to detachment. Suitable harvests and Kjeldahl analysis of one-year-old plants of C. cunninghaminana growing in water culture in the greenhouse showed that during May the mean rate of fixation was 13 ,ig N per mg nodule N per hour. It is estimated that by August - the month in which the present experiments were carried out - the rate of fixation in the above terms will have fallen to some value such as 8 ,tg, since under the local conditions the main flush of summer growth is over. The mean rate of actual fixation shown in the present experiments by nodules detached at various times of day is about 2 ,ig, i.e. 25 % of the estimated activity of nodules on the plant. The conditions during exposure to 15N may have been partly responsible for this discrepancy, particularly in that the level of nitrogen provided in the gas mixture (10 %) may have been somewhat sub-optimal, as is certainly true for alder nodules (Bond 1959). Oxygen depletion during incubation is another factor which might limit fixation, but calculations based on the rate of respiration of the nodules and the effect of oxygen tension on fixation (Bond 1961) suggest that this did not occur to any serious extent in the present study. Apart from any effect of the conditions of incubation it appears that detachment itself causes a fairly immediate drop in fixation. In the present study the reasonable

9




assumption is made that the rate of fixation shown by detached nodules, although it is for various reasons below that prevailing immediately prior to detachment, will usually be proportional to the latter.

The data reported in ?3 (b) for actual fixation rates in nodules detached from plants growing under normal greenhouse conditions show a clear diurnal variation, with activity rising during the morning, remaining at a relatively high level for several hours, and then falling to the low level shown in the early morning, with the likelihood that this low level persisted overnight. It can be concluded from the separate examination of the effect of temperature on fixation - which showed that fixation at 30 ?0 was over three times as rapid as at 10 ?0 - that this factor played

o 3-

o / \

b 1- ^ - . 1 , 51 . t .I . i

08h00 12h00 16h00 20h00 24h00 time of nodule detachment

FIGUTRE 6. The upper graph (0) is reproduced from figure 2 and shows the observed diurnal variations in actual fixation by Casuarina nodules. The lower graph (0) shows the degree of variation that can be explained solely on the basis of changes in greenhouse temperature during the day.

a part in the production of these diurnal changes. However, when as in figure 6 the observed values of actual fixation are compared with those expected solely on the basis of temperature fluctuations, calculated by using the Q10 values reported in ? 3 (d), it becomes obvious that some other factor is supplementing temperature.

Here attention naturally turns to light intensity, which through its possible effect on the flow of photosynthates into the nodules might boost fixation in the

morning, with a corresponding fall in the evening. Hence it is necessary to examine the data for evidence of any relation between the rate of fixation and the level of nodular carbohydrate. The data in figure 1 show that the fixation potential rises in the evening as actual fixation falls, and it is difficult to avoid the conclusion that this build-up in potential is due to the continued arrival of photosynthates which

cannot, owing to the prevailing low temperatures, be much used in fixation. In

agreement with this is the rise in nodular carbohydrate revealed by actual analysis (figure 3). Further, soon after sunrise the potential starts to fall as actual fixation -

again partly as a result of temperature changes - increases, and it seems obvious that this fall is due to the utilization of the accumulated carbohydrates in fixation and associated processes, as is again confirmed by the carbohydrate data. Thus,

10




taking into account also the results of the darkening experiment, it can be said that during periods of low light intensity or darkness there is evidence of a direct relation between nodular capacity for fixation and the carbohydrate level.

However, during daytime there is a puzzling situation in that high rates of fixation (actual) are associated with low levels of nodular carbohydrates. As already noted, Wheeler (1969, 1971) was confronted by a similar situation in alder, but provided evidence that in daytime, especially over the midday period, there was a rapid influx of photosynthates into the nodules, and in view of the analytical data he suggested that these photosynthates were utilized as soon as they arrived in the nodule. A difficulty arises in attempting to extend this explanation to the Casuarina data, for although it was reasonable for Wheeler - whose observations were made on nodulated root systems separated from the tops - to assume that there was still sufficient photosynthate in transit through the upper part of the root system to explain the high rate of fixation shown around midday, in a detached nodule the amount in transit must be much smaller and would soon be used up; that it could account for the excess of fixation - over and above that explainable by temperature - during the afternoon, and the temporary rise in fixation potential shown in figure 1, is debatable. No alternative explanation has, however, occurred to the authors.

The results of the darkening experiment (figure 5) - if the doubtful value for fixation recorded on day 1 is set aside -indicate that a serious reduction in fixation potential only occurred after 48-72 h darkness. The degree of persistence of fixation in the nodules of these darkened plants of Casuarina stands in marked contrast to that in alder, where Wheeler (I97I) found fixation to be extinguished after only 24 h darkness. His plants were some 4 months old and 20 cm tall, compared with 16 months and 1 m respectively for the Casuarina studied here. The present authors have found that in 4-month-old plants of Hippophae rhamnoides L. the fixation potential had fallen to 1/5 of its original rate after 24 h darkness, and in plants of Myrica cerifera L. of similar age to 1/10 after 48 h. This rather close light-dependence of fixation is probably typical of young non-legume nodulated plants. It seems that in the older, larger, plants of Casuarina used here, there are sufficient carbohydrate reserves in the plants to maintain normal levels in active, growing parts such as the nodules for at least 2 days of light deprival.

The data on the effect of temperature on fixation in Casuarina nodules indicate that the optimum is at least 36 ?C. Benecke (I970) reported an optimum of 20 ?C in Alnus viridis nodules, a similar value being recorded by Akkermans (197I) for field-grown nodules of A nus glutinosa and Hippophai rhamnoides. Wheeler (i97I) found an optimum of 25 ?C in greenhouse-grown nodules of A. glutinosa, a value also found by the present authors for corresponding nodules of H. rhamnoides. According to Coyne (1973) temperatures of 30-35 ?C are experienced in summer in regions of Australia where C. cunninghamiana occurs, so that a greater tolerance towards temperature is to be expected in the nodules than in those of the non- legumes of temperate climates mentioned above.

11



12 G. Bond and Anne H. Mackintosh

This work was facilitated by the award of a research scholarship by the Science Research Council to A.H.M. and subsequently of a grant by the British Com- mittee for the International Biological Programme. Advice given by Dr C. T. Wheeler on the estimation of carbohydrates is also acknowledged.

REFERENCES

Akkermans, A. D. L. 1971 Nitrogen fixation and nodulation of Alnus and Hippophai under natural conditions. Doctoral thesis, University of Leiden, The Netherlands.

Benecke, U. 1970 Nitrogen fixation by Alnus viridis (Chaix) DC. Pl. Soil 33, 30-48. Bond, G. I959 In Utilization of nitrogen and its compounds by plants (ed . K. . Porter).

Thirteenth Symp. Soc. exp. Biol. Cambridge University Press. Bond, G. I96I The oxygen relation of nitrogen fixation in root nodules. Z. allg. Mikrobiol.

1, 93-99. Bond, G. 1971 In Biological nitrogen fixation in natural and agricultural habitats (ed. T. A. Lie

& E. G. Mulder). PI. Soil (special volume). Coyne, P. D. I973 Some aspects of the autecology of casuarinas, with particular reference to

nitrogen fixation. Ph.D. thesis, Australian National University, Canberra. Fessenden, R. J., Knowles, R. & Brouzes, R. 1973 Acetylene-ethylene assay studies on

excised root nodules of Myrica asplenifolia L. Proc. Soil Sci. Soc. Am. 37, 893-898. H-orwitz, W. (ed.) 1955 Official methods of analysis of the Association of Official Agricultural

Chemists, 8th ed. Washington, D.C.: Ass. Off. Agr. Chem. McCready, R. M., Guggolz, J., Silviera, V. & Owens, H. S. I950 The determination of starch

and amylose in vegetables. Analyt. Chem. 22, 1156-1158. Wheeler, C. T. 1969 The diurnal fluctuations in nitrogen fixation in the nodules of Alnus

glutinosa and Myrica gale. New Phytol. 68, 675-682. VVheeler, C. T. 1971 The causation of the diurnal changes in nitrogen fixation in the nodules

of Alnus glutinosa. New Phytol. 70, 487-495.



Documents

Diurnal Changes in Nitrogen Fixation in the Root Nodules of Casuarina