Présentation d'#Aeschynomene Afraspera$ qui est une légumineuse tropicale qui se caractérise par de nombreuses nodulations sur la tige, fixateur d'azote. Grâce à ce potentiel de fixateur d'azote, #Aeschynomene Afraspera$ pourrait être utilisé en tant qu'engrais vert pour la rizicultur

Alazard, Didier

Duhoux, E.

Horizon / Pleins textes

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Bio1 Fertil Soils (1987) 4:61-66 
Biology and Fertility 
, Ofsoils 
Nitrogen-fixing stem nodules on Aeschynomene afraspera 
:D. Alazardl and E. Duhoux' 
i ORSTOM, Laboratoire de Microbiologie, BP 1386, Dakar, Sénégal 
'Département de Biologie Végétale, Université de Dakar, Dakar, Sénégal 
Summary. Aeschynomene afraspera is a wild annual 
legume growing in periodically waterlogged soils in 
western Africa. This legume is characterized by a pro- 
fuse stem nodulation. Nodules are formed on the 
stem at the emergence of lateral root primordia, 
called nodulation sites. These sites are irregularly dis- 
tributed on vertical rows all along the stem and 
branches. Stem nodules are hemispherically shaped. 
Their outside is dark green and they contain a red-pig- 
mented central zone. Stem nodules exhibit a high ni- 
trogen-fixing potential. Acetylene reduction assays re- 
sult in stem nodule activity of 309 pmol C2H4g-' dry 
nodule h- l. Field-grown stem nodulated Aeschyno- 
inene accumulated more N (51 g N m-' in 10 weeks) 
than the root nodulated one. Because of this nitrogen- 
fixing potential and its ability to grow in waterlogged 
conditions, A. afraspera could probably be intro- 
duced into tropical rice cropping systems. 
Key words: Stem nodulation - Aeschynomene afra- 
spera - Legume - Nitrogen fixation - Acetylene 
reduction assay (ARA) 
i 
The phenomenon of stem nodulation has been report- 
ed in three genera of legumes: Aeschynomene, Neptu- 
nia and Sesbania. These plants are hydrophytes, 
growing wildly in flooded areas, marshes and 
riversides. 
Sesbania rostrata (Dreyfus and Dommergues 
1981) and Neptunia oleracea (Schaede 1940) are the 
only stem nodulated plants within their genera, 
whereas this characteristic is relatively widespread 
within the g&uSÃeschyño 
Offprint requests to: D. Alazard 
Aerial stem nodules were first described on 
Aeschynonzene aspera L. (Hagerup 1928). Several' 
studies on this topic have since been published (Von 
Suessenguth and Beyerle 1936; Arora 1954; Barrios 
and Gonzales 1971; Yatazawa and Yoshida 1979; 
Eaglesham and Szalay 1983; Alazard 1985). Today 15 
species of Aeschynomene developing stem nodules are 
known. Aeschynonzene spp. fall into three cross-in- 
oculation groups according to their effective nodula- 
tion response patterns with strains of Rhizobium iso- 
lated from stem and root nodules (Alazard 1985). 
The present paper refers to earlier investigations 
on stem nodulation of A .  afraspera. This plant is 
characterized by a stem nodulation which is more ex- 
tensive than that of other stem nodulated Aeschyno- 
mene species. A. afraspera J. Leonard is an annual 
plant, 1 .O - 1.5 m tall, which grows well in soils which 
are subject to seasonal flooding. This plant is wide- 
spread in tropical Africa and is described as a closely 
related species of A. aspera L., native of tropical 
India and Malaysia (Berhaut 1971). 
. 
Aeschynoinene afraspera shows a high sensitivity 
to climatic variations, particularly to temperature and 
photoperiod. At the end of the rainy season in Sene: 
gal (13 h photoperiod; day temperature 32"C), 3 
months after seed germination, plants are 2 m tall and 
at the flowering stage. However, during the cold and 
dry season, (11 h photoperiod; day temperature 
23 OC), flowering takes place when plants are 60 cm in 
height and 6 weeks old. 
The structure of stem nodules of A .  afraspera is 
described here. The nitrogen-fixing potential of this 
plant is evaluated in order to determine its use in trop- 
ical agriculture. 
Fonds Documentaire ORSTOM 
Cote: 6~~15129 Ex: 5 
62 D. Alazard and E. Duhoux: Stem nodulation of Aeschynomene afraspera 
Materials and methods 
Rhizobiuin strain. A Rhizobium strain, designated as ORS 322, was 
isolated from stem nodules of A.  afruspera using standard tech- 
niques (Vincent 1970). Its ability to induce stem and root nodules 
on A.  afraspera was confirmed. The stock culture was maintained 
at 4 "C on agar slants containing yeast extract and mannitol (YM). 
Cultures were grown at 30°C in YM broth. 
Plant culture and inoculation. Seeds of Aeschynomene afraspera 
were surface sterilized by immersion in concentrated H$O, for 
30 min followed by 10 washes in sterile water. This treatment also 
scarified seeds and improved germination. Seeds were germinated 
in Petri dishes with 1% water agar. 
Seedlings were transplanted in plastic pots containing sterile soil 
2 days later. The soil used was of a sandy type, pH 7.0 (vernacular 
name: Dior soil) from Senegal with C ,  N and P contents of 0.40'70, 
0.025% and 0.037% respectively. Plants were grown in water- 
logged conditions unless otherwise stated in a greenhouse under 
natural light supplemented with 250 W high-intensity discharge 
lamps, with a photoperiod of 13 h, dayhight temperatures of 
35 "C/30°C and a mean humidity of 90%. To induce root nodula- 
tion, a drop of a 3-day-old YM culture (10' cells m1-l) of Rhizobi- 
um strain ORS 322 was added to the soil in each pot. Stems were in- 
oculated by applying a tenfold dilution of the same culture onto the 
stem with a small spraying bottle or a sterile brush. 
, 
Histology of stem nodules and nodulation sites. Stem nodule sam- 
ples and uninoculated sites were fixed overnight in 3% glutaralde- 
hyde in 0.2M cacodylate buffer, pH 7.0, at 4°C. After washing in 
cacodylate buffer they were dehydrated in an ethanol series and em- 
bedded in Paraplast (+) (Brunswich Co.) or Epon. Serial thin-sec- 
tions were stained with Regaud's iron-hematoxylin stain or 0.1% 
toluidine blue. 
Acetylene reduction activity (ARA). Plants were grown in 15-cm- 
diameter plastic pots containing 2 kg sandy soil supplemented with 
a nutrient solution (Hewitt 1966) containing 30 ppm N. This en- 
ables a normal growth until stem and root inoculation, 5 weeks af- 
ter sowing. Four weeks after inoculation, nodule-bearing stem por- 
tions and root systems were assayed separately for nitrogen-fixing 
activity by the acetylene reduction method (Hardy et al. 1968). 
Stem and root nodules were carefully excised at the stem and root 
surface, oven dried over night at 8OoC, cooled in a dessicator and 
weighed. 
Nitrogen accumulation. Plants were grown in plastic cylinders 
made of commercially available polyvinylchloride drainage tubes 
(inside diameter, 30 cm; length, 50 cm) vertically driven into the 
field. These cylinders were bottom closed and contained 35 kg 
sandy soil supplemented with 1.6 g K2HP04. Three seedlings of A. 
afraspera were transplanted into each cylinder. The soil was mois- 
tened for 3 days after transplantation and then kept waterlogged. 
Fig. 1. Nitrogen-fixing nodules (N) and uninoculated nodulation 
sites (S) on the stem of Aeschynomene afraspera. Bar represents 
1 cm 
Fig. 2. Unrolled stem of A.  afraspera showing nodulation sites dis- 
tributed along dense vertical rows. Bar represents 1 cm 
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D. Alazard and E. Duhoux: Stem nodulation of Aeschynonzene afras] 
- . _  
Fig. 3. Transverse section of an uninoculated nodulation site con- 
stituted by a root primordium breaking through the stem epider- 
mis. The root primordium forms a circular cavity around it. C cir- 
cular cavity; E epidermis; EC flattened epidermal cells: SC stem 
cortex. Bar represents 0.1 mm 
All the plants were root inoculated 1 week later. Half of the plants 
$vas stem inoculated 3 and 5 weeks after sowing. The other half was 
not stem inoculated (control). At harvest, 5 weeks after the last in- 
oculation, all plant material was oven dried for 48 h at 80°C and 
weighed. The dry material was ground and mixed, and the total ni- 
trogen content was determined by Kjeldahl digestion (Bremner 
1960) using a Büchi automated Kjeldahl system. 
Results and discussion 
Stem inoculation of Aeschynornene afraspera 
Artificial stem inoculation of Aeschynorneiie afrape- 
ra by a single application of rhizobia onto the stem 
produced a regular nodulation (Fig. 1). In controlled 
conditions, nodules appeared only on those parts of 
the stem where inoculum had been externally applied. 
Infection did not develop from inoculated parts of the 
plant to uninoculated parts through the stem vascular 
bundles. Repeated stem inoculation (4 times over 2 
months) of a growing plant until 1.5 m in height re- 
sulted in a mean weight of nodules of about 12 g fresh 
weight (data not shown). Waterlogging was not a pre- 
7era 63 
Fig. 4. Stem of young A. afraspera plant cultivated and immersed 
in water for 10 days. Most root primordia have evolved into adven- 
titious rootlets. Bar represents 1 cm 
requisite for stem nodulation (Eaglesham and Szalay 
1983). 
Aesclzyiiorizeize afraspera growing in natural habi- 
tats exhibits a considerable number of aerial stem 
nodules distributed at random along the principal 
stem and lateral branches. Dust, rain and wind seem 
to play a significant role in natural stem inoculation. 
A few nodules occur also on the roots of the plants. 
Because of the poor root nodulation, the presence of 
stem nodules can be viewed as an alternative adapta- 
tion to wet environmental conditions (Hagerup 1928). 
The nodulation sites 
The stem of A .  afraspera bore round, small swellings, 
no larger than 1 mm in diameter, distributed along 
dense vertical rows (Fig. 2). These swellings were 
called the nodulation sites. On the same row, nodula- 
tion sites were gradually distributed, more frequently 
at the base of the stem than at the top. At half-height, 
a 1.20-m-long plant bore about 10 sites cm-’. 
64 D. Alazard and E. Duhoux: Stem nodulation of Aeschynomene afraspera 
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Fig. 5. Cross section of a 15-day-old stem nodule showing two dark 
areas of infected tissue on both sides of remaining vascular bundles 
of the root primordium. Note the hemispherical shape of the nod- 
ule with a broad attachment to the stem. Toluidine blue 0.1%; 
Paraplast (+) block. Bar represents 1 mm 
Fig. 6. Part of the infected zone of a IO-day-old nodule. Infected 
cells contain numerous rhizobia (R), prominent nucleus (N) and 
vacuoles (V). Regaud's iron-haematoxylin stain; Epon block; Bar 
represents 10 pm 
Nodulation sites of A. afraspera always included 
an adventitious root primordium. Sites located below 
the two upper internodes were generally mature for 
rhizobia1 infection. Thin sections showed that the 
root primordium broke down through the epidermal 
dome (Fig. 3). The apex of the root primordium was 
overlayed with a thin skin of flattened epidermal cells 
(Fig. 3). The large base of the epidermal dome was 
continuous with the stem cortex. In immature sites, at 
the top of the plant, the root primordium was still em- 
bedded in the cortical tissues of the stem and nodule 
development was never observed. 
When immersed in water the cuttings of stem or 
branches developed abundant adventitious roots 
(Fig. 4). 
Description of stem nodules 
Stem nodules were visible 4 - 5 days after inoculation. 
They were fully developed 3 weeks later, forming 
prominent swellings under the stem epidermis. Nod- 
ules were very numerous and were sometimes contigu- 
ous (Fig. 1). The overall morphology of A. afraspera 
nodules, was that of the determinate type (Goodchild 
1977). Mature nodules were hemispherically shaped 
with a broad attachment to the stem and appeared 
flattened against the stem. They were 3 - 5 mm in 
transverse diameter and 2 - 3 mm high. Nodules were 
dark green in external appearance and contained a 
red-pigmented central tissue, suggesting the presence 
of leghemoglobin. Cross-sections of young nodules 
showed two distinct infected areas on both sides of 
vascular connections of the root primordium (Fig. 5), 
indicating that each of these infected areas results 
probably from a separate infection origin within the 
infection site. The central tissue of the nodule never 
contained any uninfected cells. Mature infected cells 
presented a prominent nucleus and vacuoles (Fig. 6). 
Nitrogen-fixing potential of A. afraspera 
Acetylene reduction activity (ARA) values of stem 
and root nodules of A. afraspera are shown in Table 
1. Activities in both cases were linear over a 1.5-h as- 
say period and depended on the age of the nodules. 
Nodules were assayed 4 weeks after inoculation when 
nodules had higher specific activities. Aeschynomene 
afraspera exhibited a high nitrogen-fixing capacity. 
Specific ARA of stem nodules was 309 pmol C2H4 
g-' nodule dry wt. h-I, an efficiency similar to that 
of other stem nodules, Sesbania rostrata (Dreyfus and 
Dommergues 1981) and Aeschynomene scabra (Ea- 
glesham and Szalay 1983), which have ARAS of 259 
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D. Alazard and E. Duhoux: Stem nodulation of Aeschynonzene afrapera 65 
Table 1. Nodulation and acetylene reduction activity (ARA) of 9-week-old plants of A. afrapera inoculated with Rhizobium ORS 322. The 
nodule-bearing stem portion was that between the second and the fourth node above cotyledons 
Part of plant Nodules per plant ARA Specific ARA 
(pmol C2H4 plant-' h-') (pmol C2H4 g-' nodule dry wt. h-l)  
Dry wt. (mg) Number 
Stem portion 605 2 57 145 f 12 l87k 13 
Root system , 43&i1 1 9 f 4  5 f 2 . 5  
309 * 27 
116%23 
All data are means of five replicates f standard deviation 
Table 2. Effect of stem inoculation upon nitrogen fixation by A. afrapera grown in field conditions 
Treatment Plant dry weight 
(g plant-') 
Nitrogen content of plants 
(o70 per dry wt.) (mg N plant-') 
Stem and root inoculated 
Root inoculated only 
46&3.5 
8.0k1.3 
2.97* 0.15 
1.97k 0.10 
1366k 125 
158f 18 
All data are means of five replicates k standard deviation 
and 327 pmol C2H4 g-' nodule dry wt. h-' respec- 
tively. The specific ARA of stem nodules is higher 
than that of root nodules of field-grown grain le- 
gumes such as Vigna, Vicia or Lupinus, which range 
from 180 to 250 pmol C2H4 g-' nodule dry wt. h-' 
(Graham and Chatel 1983). Aeschynomene root nod- 
ulation was poor in waterlogged conditions as shown 
in Table 1. Root nodules were small and their specific 
ARA significantly lower than that of the stem nod- 
ules. The development of root nodules and their ni- 
trogen-fixing activity was probably affected by water- 
logging. 
An estimation of nitrogen fixation by stem nod- 
ules of A. afraspera using the difference method (Wil- 
liams et al. 1977) is presented in Table 2. When har- 
vested, stem nodulated plants were about 1.2 m in 
height while plants without stem nodules were only 
0.5 m tall. A. afraspera bearing stem nodules had a 
relatively high nitrogen content of about 3% (dry 
wt.). The total N content of stem inoculated plants 
was 1366 mg N whereas that of plants without stem 
nodules was only 158mg N. By difference, the net 
fixation by stem nodules was calculated as 1208 mg N 
plant-', representing about 90% of the total N con- 
tent of the stem inoculated plant. It can be noted that 
the nitrogen fixation value may be an overestimate be- 
cause the difference method assumes that the root sys- 
tems are comparable in the two treatments. Stem in- 
oculation could provide sufficient nitrogen to support 
additional root growth, resulting in a higher soil ni- 
trogen uptake. In that case, the difference method 
would overestimate nitrogen fixation. 
Nevertheless, the maximum estimate of nitrogen 
fixation by stem nodulated plants based on the cylin- 
der experiment (3 plants 0.07 m-2) was 51.3 g N m-2, 
a higher value than that obtained with Sesbanìa ro- 
strata, 30 g N m-2, for 2 months (Rinaudo and Mou- 
diongui 1985), indicating that Aesclzynoinene afraspe- 
ra is one of the most active nitrogen-fixing legumes. 
These data suggest that A. afraspera might have 
an important agronomic value as a green manure 
crop, particularly in the rice production system of 
tropical regions. A. afraspera is a rapidly growing 
plant which could grow in paddy fields of tropical Af- 
rich at the beginning of the rice season while rice seed- 
lings are still in the nursery beds. Furthermore, this 
plant is able to fix nitrogen under waterlogged or 
flooded soil conditions, which are usually unfavour- 
able for legumes. Studies in this direction are in pro- 
gress. 
Acknowledgments. The authors thank Dr. Y .  R. Dommergues for 
his critical review of the manuscript. This work was supported by a 
grant from the EEC under contract No. TSD-081F. 
References 
Alazard D (1985) Stem and root nodulation in Aeschynomene spp. 
Appl Environ Microbio1 50:732- 734 
Arora N (1954) Morphological development of the root and stem 
nodules of Aeschynomene indica L. Phytomorphology 
Barrios S, Gonzalez V (1971) Rhizobia1 symbiosis on Venezuelan 
Berhaut J (1971) Flore illustrée du Sénégal. Dicotylédones. Tome 
Bremner JM (1960) Determination of nitrogen in soil by the 
Dreyfus BL, Dommergues YR (1981) Nitrogen-fixing nodules 
4:211-216 
savannas. Plant and Soil 34:707 - 719 
V. Légumineuses papillonacées. Clairafrique, Dakar 
Kjeldahl method. J Agri Sci 55:ll-  33 
66 
6 
D. Alazard and E. Duhoux: Stem nodulation of Aeschynomene afraspera 
induced by Rhizobium on the stems of the tropical legume 
Sesbania rostrata. FEMS Microbio1 Lett 10:313 - 317 
Eaglesham ARJ, Szalay AA (1983) Aerial stem nodules on Aeschy- 
nomene spp. Plant Sci Lett 29:265 - 272 
Graham PH, Chatel DL (1983) Agronomy. In: Broughton WJ (ed) 
Nitrogen fixation, vol 3. Oxford University Press, Oxford, pp 
Goodchild DJ (1977) The ultrastructure of root nodules in relation 
to nitrogen fixation. Int Rev Cytol [Suppl] 6:125 - 139 
Hagerup O (1928) En hygrofil baelplante (Aeschynomene aspera 
L.) med bakterieknolde paa staengelen. Dan Bot Ark 15:1-9 
Hardy RWF, Hosten RD, Jackson EK, Burns RC (1968) The 
acetylene-ethylene assay for N-fixation: laboratory and field 
evaluation. Plant Physiol 43:1185 - 1207 
Hewitt EJ (1966) Sand and water culture methods used in the study 
of plant nutrition. Tec Comm N 22. Commonwealth Agricul- 
tural Bureaux, Farnham Royal, England 
Lison M (1960) Histochimie et cytologie animale. Principes et mé- 
thodes. Gauthier-Villars, Paris 
56 - 98 
Rinaudo G,  Moudiongui A (1985) Fixation de l'azote par Sesbania 
rostrata: son utilisation comme engrais vert. Bull Res Agr 
Gembloux (in press) 
Schaede R (1940) Die Knöllchen der adventiven Wasserwurzeln von 
Neptunia oleraced und ihre Bakteriensymbiose. Planta 31:l- 21 
Von Suessenguth K, Beyerle R (1936) Über Bakterienknöllchen am 
Spross von Aeschynomene paniculata. Willd Hedwigia 75: 
Vincent JM (1970) A manual for the practical study of root nodule 
bacteria. IBP Handbook N 15, Blackwell, Oxford 
Williams WA, Jones MB, Delwiche CC (1977) Clover N-fixation 
measurement by total N-difference and NA-values in lysimeterk 
Agron J 69:1023 - 1024 
Yatazawa M, Yoshida S (1979) Stem nodules in Aeschynomene 
indica and their capacity of nitrogen fixation. Plant Physiol 45: 
234 - 237 
293 - 295 
Received June 27, 1986 


Nitrogen-fixing stem nodules on Aeschynomene afraspera

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