Balandreau, J.

Dommergues, Yvon

Rinaudo, Gérard

Weinhard, P.

Horizon / Pleins textes

Soil Bid. Biocbem. Vol. 5, pp. 83-89. Pergamon Press 1973. Printed in Great Britain 
NON-SYMBIOTIC NITROGEN FIXATION IN THE RHIZO- 
SPHERES OF RICE, MAIZE AND DIFFERENT TROPICAL 
GRASSES 
Y. DOMMERGUES, J. BALANDREAU, G.RINAUDO and PIERRETTE WEINHARD 
Centre de Pédologie du CNRS, 54, VandoeuvrelBs-Nancy, France 
Office de la Recherche Scientifique et Technique Outre-Mer, Dakar, Sénégal 
(Accepted 1 March 1972) 
Summary-Nitrogenase activity estimated in the rhizospheres of rice, maize and different 
tropical grasses grown under controlled laboratory conditions was shown to depend upon 
plant species. High nitrogenase activity (2000-6000 nmoles CzH4 h-' g-' dry root) occurred 
in rice rhizosphere, this activity being only 10 times lower than that of symbi0ticsystems;inthe 
rhizosphere of many other grasses grown in a similar way nitrogenase activity was as low as 10 
nmoles C2H4 h-l  g-' dry root. The influence of soil type on nitrogenase activity was im- 
pressive; but the exact nature of the factors implicated could not be established. A rather weak 
flush of nitrogenase activity in the rhizosphere occurred in the early stage of the plant growth; it 
was probably due to the exudation of compounds from the seedandlasted 2 or 4 daysaccording 
to the size of the seed. When the plant entered into its intense photosynthetic phase, the nitro- 
genase activity gradually increased. When the shoots were severed, nitrogenase activity in the 
rhizosphere ceased. Nitrogenase activity in the rhizosphere responded greatly to light in- 
tensity. Extrapolation of these laboratory findings to the field is discussed. 
INTRODUCTION 
THE ECOLOGICAL significance of non-symbiotic nitrogen fixation in the rhizosphere is one 
of the most controversial problems in the field of soil biology. Some authors, who have 
studied the role of Azotobacter in the rhizosphere, think that the contribution of this 
bacterium to nitrogen enrichment is negligible (Mishustin and Shilnikova, 1969). On the 
other hand, it has been suggested that, at least under tropical climates and in the rhizosphere 
of some plants, non-symbiotic nitrogen fixation must not be overlooked (Döbereiner, 1968). 
For 2 yr, we have been studying this problem using acetylene reduction assay in the case 
of laboratory grown plants (Hauke-Pacewiczowa et al., 1970; Rinaudo et al., 1971; Balan- 
dreau et al., 1971 ; Weinhard et al., 1971). This paper presents results from a number of 
recent experiments in which some major factors affecting nitrogenase activity have been 
examined in the rhizosphere of various plants. 
MATEXIAL,S AND METHODS 
Soils 
Most of the soils used were tropical and were sampled to a depth of approximately 10 cm 
(Table 1). 
83 
84 Y. DOMMERGUES, J. BALANDREAU, G. FUNAUDO AND PIERRETTE WEINHARD 
TABLE 1. DESCRIPTION OF EXPERIMENTAL SOILS 
Soil no. Soil type Origin 
C N Clay 
PH (%> (%> C:N (%> 
1 
2 
3 
4 
5 
6 
7 
Oxic fluvent 
Oxic fluvent 
Oxic fluvent 
Oxisol 
Ustipsamment 
Ustipsament 
Typic eutrochrept 
Dabou, Ivory Coast 4.1 4.70 0.29 16.4 63 
Abengourou, Ivory Coast 4’2 3.17 0.23 13.8 29 
Yamoussokro, Ivory Coast 5.8 1.78 0.15 11.8 27 
Ambatobe, Tananarive, 4.2 0.95 0.074 12.8 21 
Madagascar 
Lamto, Ivory Coast 6.0 0.83 0.07 11.8 5 
Bambey, Senegal 4.5 0.17 0.014 12.0 2 
Pixérécourt, France 7.8 3.25 0.29 11.2 23 
NB-After waterlogging, the p H s  of soils 1, 2 and 3 increased up to 6.3, 6.6 and 6.9. 
Plants 
Plants used were rice (var. IR8), Eleusine coracana, maize (var. INRA 260); Paspalum 
virgatum, Pennisetum purpuretim, Panicum maximum from O. R. S .T. O. M. experimental 
station at Adiopodoumé (Ivory Coast); Cyperus zollingeri, Cyperus obtus@orus from 
Lamto I.B.P. station (Ivory Coast). Rice, maize and Eleusine coracana plants were obtained 
from seeds, other plants from root-stocks. The plants were cultivated in cabinets under the 
following conditions : light intensity, 30,000 lux, 16 h illumination per day ; temperature, 
28 &- 2°C; relative air humidity, 70-90 per cent. The rice soils were waterlogged and, in the 
case of Paspahm virgatum, soils were kept at water holding capacity. The other plants were 
grown in plastic containers placed in jars that were one-half to two-thirds fìlled with water. 
For each treatment, there were 5-6 replicates. 
Acetylene reduction assays 
The technique was similar to that of Hardy et al. (1968) and Stewart et al. (1967) 
except that test materials were exposed to a gas mixture containing 90 per cent Ar and 18 
per cent C2H2, previous experiments having shown that omission of oxygen did not impede 
CzHz reduction (Rinaudo et al., 1971). This technique was used with two systems: (1) 
‘soil + plant’ systems (rhizosphere soils) extracted in one piece from the growth vessels; 
(2) ‘control soil’ systems (non-rhizosphere soils), where no plants had been grown. These 
materials were exposed at 30 f 1°C to gas mixture, in the dark, for about 4, 7, 20, 24 and 
30 h. Maximum CzHz reduction rates, expressed as nmoles C,H, h-l g-l dry weight of 
soil or root, were calculated from time-course curves of C2H, reduction. The rhizosphere 
effect was measured as the difference between the activities of ‘soil + plant’ and of soil 
alone. The latter was usually negligible. Preliminary experiments showed little difference 
between tests in which the ‘soil + plant’ was assayed in the growth containers (Dr. J. 
Döbereiner, personal communication), or extracted and assayed in one piece as described 
above. 
RESULTS 
Because of the great variability of nitrogenase activity, it is difficult to classify the rhizo- 
sphere effects according to plant species. However preliminary results (Table 2) Shoa that 
these effects in decreasing order are rice > Eleusine coracana, Paspalum virgatum, Cyperus 
zollingerì and Cyperais obtusfloriis > Panicum purpureum and Panicum maximum. 
NITROGEN FIXATION IN RHIZOSPHERES 85 
TABLE 2. NITROGENASE ACTIVITY IN THE RHIZOSPHERES OF RICE AND DIFFERENT TROPICAL 
GRASSES 
Age of plant Soil Nitrogenase activity 
Plant species (months) no. (nmoles C2H4 h-l 8-l dry root)* 
Rice e 1  1 1800 -+ 274 
N 1  2 1190 f 140 
N 1  3 1430 f 205 
2: 0.3 1 6153 f l516t 
Eleusine coracana e 1  5 374 f 165 
N 1  6 1.3 & 0.5 
Paspalum virgatum 4 4 528 f 48 
8 4 345 f 163 
Pennisetuìn purpureuin 2 4 4 f 0.7 
Panicum inaxiìnuìn 2 5 10 & 1.1 
Cypei-lrs zollingeri 3 5 162 f 47 
6 5 110 & 25 
Cyperus obtusiflorus 6 5 352 & 66 
* f Standard deviation. 
t Maximum value from Fig. 3. 
When nitrogenase activity was expressed on a root weight basis, time curves, for seed- 
lings growing at 30,000 lux, showed two stages: (1) stage of increase of activity which lasted 
5-10 days, (2) stage of stabilization (Fig. 1) or decrease (Fig. 3). At 3000 lux, a flush of 
nitrogenase activity occurred during the early stage (Fig. 2). When nitrogenase activity was 
expressed on a soil weight basis, time curves, for seedlings growing at 30,000 lux, showed 
increasing activity throughout the experiment except for a short interval (Fig. 1). 
T 
Age of plant (days) 
I I  
u 4 5  14 22 27 
(a) (b) 
FIG. l(a). Time course of nitrogenase activity (per g dry root) in the rhizospheres of rice 
(0-0) or maize (0-0) seedlings grown at 30,000 lux in soil 7. (b) Time course of 
nitrogenase activity (per g dry soil) in the same rhizospheres of rice (@---O) and maize 
(m-m) seedlings grown in the same conditions. Limits show standard error of means. 
86 Y. DOMMERGUES, J. BALANDREAU, G. RINAUDO AND PIERRETI'E WEINHARD 
10000 
8000 
6000 
4000 
2000 
O 
- 
e 
v 
N 
I 
O 
I \  
1 I I  1 '  I/ \
/! ----*------ Age of plant (daz )  
2 4 8 o 6, 
FIO. 2. Time course of nitrogenase activity (per g dry root) in the rhizosphere of young maize 
seedlings grown at 30,000 lux (0-0) and at 3000 lux (e --- O )  in soil 7. The flush 
which occurs at the 8th day is probably induced by root exudates supplied by seed reserves. 
Limits show standard erkor of means. 
The influence of soil type on nitrogenase activity could be considerable; for instance in 
the case of Elezrsine coracana, this activity was 100 times greater in soil 5 than in soil 6 
(Table 2). 
Rice seedlings were grown under normal light (30,000 lux) during a 10-day period, after 
which half of them were shaded, thus reducing the light to about 3000 lux. Time-curves of 
nitrogenase activity expressed on a root weight basis showed that within 24 h, shading had 
reduced nitrogenase activity to one-sixth of the unshaded value (Fig. 3). 
When shoots of rice grown in soil 1 were severed, nitrogenase activity in the rhizosphere 
decreased sharply (Fig. 4). Within 5 days from cutting, nitrogenase activity expressed on a 
soil weight basis was negligible. Identical results had been obtained previously with rice 
grown in the same conditions in soils 2 and 3 (Rinaudo, 1970). 
DISCUSSION AND CONCLUSION 
Evidence of nitrogenase activity in the rhizosphere * 
Nitrogenase activity could be demonstrated in the rhizosphere of most of the plants we 
studied. This activity was rather low in some cases, but in the case of rice it reached values 
as high as 2000 or 6000 nmoles C2H4 h-l g-' root (dry weight). Compared with results 
NITROGEN FIXATION IN RHIZOSPHERES 81 
6000 
5000 
4000 
3000 
2000 
1000 
O 
> , , ~ ~ y ~ - + ~  -;I- , ‘ y l g e  of planf(day2 
4 6 i 0  I I  13 47 20 24 27 32 
FIG. 3. Time course of nitrogenase activity (per g dry root) in the rhizosphere of rice seedlings 
grown in soil 1 at 30,000 lux (0-0). The line (O - - - O )  represents the time course of 
nitrogenase activity in the rhizosphere after the seedlings had been shaded (3000 lux). Limits 
show standard error of means. 
obtained by Yoshida and Ancajas (1970) and Döbereiner et al. (1971), the latter figures seem 
abnormally high. In our experiments the soils may have been contaminated by nitrogen- 
fixing algae; but this objection cannot stand, because the C2H2 assays were carried out in 
the dark and C2H2 reduction-did not decrease with time. Moreover non-rhizosphere soil 
controls always showed very low nitrogenase activity. 
It is interesting to compare nitrogenase activities of efficient non-symbiotic nitrogen- 
fixing systems (such as rice rhizospheres) with those of well-known symbiotic nitrogen- 
fixing systems (i.e. legume or non-legume nodules); in the former nitrogenase activity can 
reach 1000-6000 nmoles C2H, h-l  g-l dry root, in the latter, 10,000-80,000 nmoles CzH4 
h-l g-l dry nodule (Hardy et al., 1968; Wheeler, 1969; Schwinghamer et al., 1970; DÖ- 
bereiner et al., 1971). Expressed on a root weight basis, nitrogenase activity in efficient non- 
symbiotic systems was thus about one-tenth of that of symbiotic systems (Weinhard et al., 
1971). 
-Factors afectigg nitrogenase activity in the rhizosphere 
Since the microflora on and around the roots is dependent very largely for its supply of 
organic nutrients on exudation .from the roots (Rovira; 1965), it was predictable that 
88 Y. DOMMERGUES, J. BALANDREAU, G. RINAUDO AND PIERRETTE WEINHARD 
30 
O 
FIG. 4. Time course of nitrogenase activity (per g dry soil) in the rhizosphere of intact rice 
seedlings (0-0) and in the rhizosphere of decapitated rice seedlings (8  - - - 0 )  grown in 
soil 1. Stems were severed when seedlings were 14 or 21 days old. 
factors controlling exudation would also control nitrogenase activity. Our experiments 
have confirmed this assumption. 
Some plants species, especially rice, induced a very favorable environment; on the con- 
trary, others, such as Pennisetum purpureum or Panicum maximum, were much less favour- 
able for nitrogenase activity in the rhizosphere. 
During the first stages of growth, time-curves of nitrogenase activity in the rhizosphere of 
seedlings appeared to depend on light intensity. In the case of well-lit seedlings (30,000 lux) 
nitrogenase activity expressed on a root weight basis, increased progressively with age. In 
the case of shaded seedlings (3000 lux), this activity occurred as a flush followed by a sharp 
decrease. As this flush was not light-dependent, presumably it was induced by root exudates 
supplied by seed reserves (Balandreau et al., 1971). When plants were older (more than 
10-20 days in our experiments), nitrogenase activity, estimated on a root weight basis, 
decreased. Thus in the older parts of rhizospheres, nitrogenase activity would probably be 
less than in younger regions. 
Light intensity, by affecting the photosynthetic process, is known to influence the quali- 
tative and quantitative nature of root exudates (Rovira, 1965; Barber, 1969). This fact 
explains that shading caused a dramatic decrease of nitrogenase activity within 24 h; it 
also explains how shoot removal similarly reduced this activity within a few days. 
The influence of soil type on nitrogenase activity in the rhizosphere is considerable, but 
we do not yet know the exact nature of the factors implicated. Are they biological, physical 
or purely chemical? Oxygen deficiency is a most important factor (cf. Hauke-Pacewiczowa 
et al., 1970), but other factors such as phosphorus and organic matter content, must not be 
overlooked. 
Validity of the acetylene reduction assay 
The use of the acetylene reduction assay for estimation of nitrogen-fixing activity implies 
that the quantity of acetylene reduced is directly related to the amount of nitrogen that could 
have been reduced. One might expect that, since 6 electrons are required for the reduction 
. 
NITROGEN FIXATION IN RHIZOSPHERES 89 
c 
of a nitrogen molecule and 2 for an acetylene molecule, the ratio ‘CzH2 : Nz7 would be 3 : 1. 
But it has been shown that, in number of living systems, this ratio could be different from 
this theoretical value (Bergersen, 1970; Dr. M. J. Dilworth, personal communication). That 
is why Bergersen (1970) emphasized the necessity of establishing a calibration for this 
relationship for every system studied. This calibration in the case of rice rhizosphere has 
given values close to 3 (Rinaudo and Dommergues, 1971). We have assumed that this value 
would also be characteristic of rhizospheres of other plants, but thisassumption will have to 
be corroborated. 
Extrapolation of laboratory measures to tlie field 
Results obtained in controlled environment cabinets cannot be extrapolated to the field 
without taking into account (1) in situ climatic and edaphic conditions (2) the quantity of 
really active roots. Our knowledge of the ecology of nitrogen-fixing organisms is still not 
sufficient to allow us to make these corrections. The best way to solve the problem would be 
to use in situ methods to estimate nitrogenase activity. Results of preliminary laboratory 
experiments suggest that maximum biological nitrogen-inputs are to be found in eco- 
systems with the following features : waterlogged soil, sunny climate, and root systems that 
form large amounts of exudates. Rice fields possess these features. 
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Non-symbiotic nitrogen fixation in the rhizospheres of rice, maize and different tropical grasses

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