Nitrogen fixation in bar bara groundnut, Voandzeia...
Nitrogen fixation in bar bara groundnut,
Voandzeia subterranea L.) Thouars
Mamadou Gueye’ and Lucien 4. Bordeleau*
Received 19 February 1988, accepted 21 M~I
1988
Introduction
Bambara groundnut (Vocrndzeia suhterrarze~ L.) Thouars) is a desirable legume
for Africa semi-arid tropical agriculture. L : cowpeas, the bambara groundnut
nodulates freely in tropical soils. Although it l Iduces a nutritious food, the bambara
groundnut remains one of the crops most neg
ted by science (National Academy of
Sciences, 1979).
The most important feature of legumes is 1 ir ability to fix atmospheric nitrogen
and considerable efforts have been expended Iwards increasing the efficiency of its
symbiotic association with Rhizobium. Now,
s accepted that seed inoculation with
superior Rhizobium strains is necessary with
ost legumes. However, cowpea seed
inoculation is usually unsuccessful because 0 aompetition between introduced and
indigenous Rhizobium strains in the soi]. r zrefore. an important step towards
improved nitrogen fixation is to isolate and id
tify competitive indigenous Rhizobia
with a view to using elite strains as inoculant
Mulongoy et al., 1980).
This paper presents data on screening field
imbara groundnut varieties, nitrogen
fixing ability of indigenous Rhizobium strains
Id evaluation of interactions between
host plant and Rhizobium strain.
Materials and methods
Fieki varietal screenitzg.
Tr,ials were done a
wo sites in Senegal during the 1985
rainy season. The sites differed in both rainf:
(Bambey, 377 mm annum-‘; Nioro.
S32 mm annum-‘) and in soi1 type (Table
, Twenty four cultivai-s of bambara
groundnut (listed in Fig. 1) were hand sown i i randomized block design replicated
four times. The cultivars were sown in two tn
ments: without (-N) and with (+N)
nitmgen fertilization (50 kg urea/ha). Ail pic
received 60 kg PzOs/ha and 120 kg
KCllha. Six weeks after planting, the relative
ifectiveness (RE) of each host plant/
indigenous Rhizohiztm strain combination wa!
valuatcd as follows (Mulongoy er a/. ,
1980):
’ MIRCEN-CNRA. B.P. 53, Barnbcy. Senegal.
’ Canada Agriculture. Research Station, 2560 Hoche&
Ivd, Sainte-Foy. Canada. GlV 253
0 Oxford University Press 1988
366 M . Guey & 1,. M. Bord&~~
Table 1 Characferistics of soi1 at Banbey ad Nioro cxperimental st.ations
wm
Total N
A,‘a’t;rtde 1’
(C Is,31”)
--~
290
121
310
137
‘weight’ oF ;en samples in the -N block
RE =
- -
- - - x 1 0 0
‘weight’ OC ;cn samples in the +N Hock
In this formula, ‘weight’ is either of shoot, root or nodule dry wcight or shoot nitrogen
content.
Nitrogcn ,fi.xing ubility of indigenou? IRhizobium struins.
The samples wcre taken in
each trcatment from five well-developcd plants and still having, immature pods and 4.5
well-devcloped, pink nodules werr: <;ampled Crom the tap roc,&. Rhizobium strains
were isolated according to standa -ci procedures (Vincent, lc~70). Isolated rhizobia
were cultured on agar slants made f:om yeast extract/mannitol rnedium. Nitrogen fixing
ability of the isolates were compared in terms of shoot dry weight of bambara
groundnut cultivar X5001 used as tl:s\\t plant and cultivated in IL.conard jar assemblies
(Leonard, 1943). Seeds were stelilized using 0.01% (w/v) HgCl, for 3 min and
germinatcd in sterile sand for 3 d; each Seedling was then Itransplanted into a jar
assembly. One ml suspension of rhizobial isolate broth (approx. 10” cells) was
added aseptically to the seedlings 1’~ r a11 treatments except for an uninoculated control
and plants were grown in a grcenhousc. Nitrogen-free Hewitt solution (Hewitt, 1966)
diluted with water ( 1:3 v/v) wus ad-led once a wcek during the growing period. After
35 d growth. plant tops were clipped off, dried and weighed for dry matter production.
Gwenhousc evuluutiora o f host pl’unt-Rhizohium strain interrrctiorrs.Both the
indigenous and Niftal Rhizohium s:r.:lins (listed in Table 3) werc selected on the basis
of their effectiveness. The Niftal ittains - TAI, 22, TAI., 169 and TAL 569 - were
supplied by the NiffAL project, Hawaii, USA and served as standards for
comparison against the indigencu; ones. Twelve bambara groundnut cultivars
also listed in Table 3 were used a:; ;lost plants. Seed sterilization of each genotype,
germination, transplantation, inocMion and watering were conducted as described
above and seeds were planted ir Leonard jars. Treatmcnts were arranged in a
randomized block design replicated five times. After SO days of growth, the plants
were harvested. Plant tops were ( rjed and weighed. Nodulation was scored taking
into account the number (N), the interna] colour (C) and the size (S) of nodules.
The nodulation index (Nod. 1.) W;IS calculated as follows:
Nol. 1. = N x C x S
Nodule number was rated on a sca.e from 0 (no nodule) to 3 (many nodules). Nodule
interna1 colour was from 0 (whit,:) to 1 (red) and nodule size was from 1 (small
nodule) to 2 (big nodule).
Nitrogen fixation in groundtlut 367
Kesults
Field varietal scrcening
At Bambey station the cultivars 83-127, Su’ Cameroun, Sarakawa 3, Sarakawa 10
and Lassa 1 had a relative effectiveness (RE) )ased on shoot dry weight, below 40%
(Fig. 1 j while at Nioro st,ation, a11 cultivars 1 d the same RE of over 40% (Fig. 2).
At Bambey station, only cultivar Sarakawa 1 had an RE, based on root dry weight,
below SO%. At Nioro station, a11 cultivars h I the same RE of over W%. Relative
Effectiveness, based on nodule dry weight , w
much lower at Bambey than at Nioro:
it was in the range of 80 to 120% at Bambey fl
cultivars 83-131, Ketao 3, Alheride 1,
Lassa 1 and Lassa 4 and in the range of 360 ) 480% at Nioro for cultivars 83-129.
X3-131. Sud Cameroun, Sarakawa 2. Ketao ! V2 and 85002. Relative Effectiveness,
based on shoot nitrogen content, was o r 50% for a11 cultivars except for
Awandjelo 1 at Bambey station.
These results indicated that Bambey and r )ro rhizobial population established a
moderately effective symbiosis with the cultiv
s of bambara groundnut used. Among
these cultivars, 12 grew better at both th
locations without nitrogen fertilizer
(Table 2) indicating their ability to set an effe ive symbiosis in the two different field
conditions.
Table 2 Shoot dry,weight (g/LO plants) of the perfo
led cultivars of bambara groundnut in field
at Bamhey and Nmro experimental stations
Bambara Cultivars
E nbey
Nioro
79-1
5 sh
30.4h
83- 126
c 2 ”
33.6”
x3-129
5 Sh
39sYh
83-131
5 sh
30.2h
Sarakawa 1
5 3”
31.1h
Awandjelo 1
6 ?”
40.Yh
Ketao 2
6 ih
31.7h
Ketao 3
6 )ah
32.9h
Alheride 1
6 ;A,
4
43.9”
Lassa 3
7 Y
38.fYh
v2
6 5’
41.3””
8500 1
5 jh
42.0”
Values followed by the same letter in each column
1 not differ significantly at the 0.05 level by
Duncan’s.multiple
range test (19%).
Effec’tiveness of indigenous Rhizohium str&
s isolated from bamhara groundnut
nodules
Bambara groundnut cultivars sampied were ,oderately nodulated on both the tap
and lateral roots. Many nodules appeared I be effective as indicated by interior
colour. Thirty one isolates were SO obtained: (
to 13*, Fig. 3). The isolates were slow-
growing and formed small (diam. < 1 mm), d colonies on yeast mannitol agar. Four
isolates (12, 113, IZ2 and lJo) were lodged in WI t Africa MIRCEN (MAO) Rhizobium
culture collection because of their high effet veness with cultivar 85001 which was
from Senegal and had good RE values at
)th Bambey and Nioro experimental
140 ta)
140
04
130 i
130
i20
110
100
90I
8 0
70
60
50
40
30
20
10
-
0
520~ J
IJU=
4 8 0
120-
s ,401
110-
lOO-
go-
80.
Cultivars
Cultivars
Fig. 1 Distribution of the Relative Effectiveness (RE) of 21 cultivars of Voandxiu s~~hf~~rtneu at Bamhey cxperimental station. A: RE hased on
shoot dry weight; B: RE hascd on root dry weight; C: RE hased on nodule dry weight; D: RE based on shoot nitrogen content.
140 tb)
130 i
120
110
100
5 90
90
B 80
80
2 70
70
<L> 60
60
g 50
50
40
40
g 30
30
20
20
10
10
0
-i
140 td'
î
130
120
110
-410090706050
40
30
20
1 0
0
Fig. 2 Distribution of the Relative Effectiveness (RE) of 24 cultivars of Voandzeiu suhlerrunea at Nioro experimental
station. A: RE based on shoot
dry weight: B: RE based on root dry weight; C: RE based on nodule dry weight: D: RE based on shoot nitrogen content.
370 M. Gueye & L. M. Bordelecu
stations. These isolates were named MAO ‘113, MAO 118. M.AO 121 amI MAO 126.
respectively, and are indicated by an asterisk in Fig. 3.
Evaluation of host cultivar-Rhizoh lum strains inteructiorzs
Rhizohium strains used were comaared for their effectiveness to nodulate the most
promising cultivars of bambara groundnut selected in held varietal screening. Nodules
were found in the top 5 cm of root svstem on all plants exccpt controls which were ail
nodule free. Nodulation index ani the effectiveness varicd greatly with the strains
(Table 3). Strains MAO 113 and hfA0 118 exhibited effcctivl: nodulation on six and
three cultivars respectively while strains TAL 22, TAL 169 and TAL 569 were
effective on five, four and three culltivars respectively. Al1 Rhizohium strains were
effective on cultivar 83-131 except sbrain TAL 169 which cxhibitecl partial nodulation.
,411 Rhizobium strains were parti,ally effective on cultivar Ketao 3. Similarly,
Rhizobium strains MAO 113 and h4AO 118 were ineffective on cultivar V2 while
strains TAL 22, TAL 169 and TAI, 5169 were partially effective on this cultivar. Strains
MAO 113 and TAL 169 were ineffective on cultivars Awandjelo 1 and 83-129
respectively; strain MAO 118 was ineffective on cultivars Awandjelo 1. Lassa 3 and
V2; strain TAL 569 was ineffectilre on cultivars Ketao 2. Alheride 1 and Lassa 3.
Strain TAL 22 presented the widejt effectiveness spectrum: il was fully effective on
42% and partially effective on Sgt?, faf a11 cultivars studied, followed by strain MAO
113. These results indicated that phnt genotype had an important effect on nodulation
of bambara groundnut .
Although Corbin et al., (1977) had proposed a scoring system to classify nodulation
and Bordeleau et al., (1981) had reported a method for s’coring nodulation, we
adopted another method. The reliab,ility of this method was indicated by the fact that
nodulation index and shoot dry weight (Table 3) showed the same trend of
effectiveness in host plant-Rhizobiuan strain combinations.
Discussion
Many studies have reported an iml ovement of biological nitrogen fixation resulting
from legume cultivar selection (Hei tel, 1982; Minchin et ~11.. 1978; Zary et ul., 1978).
However, selection for increased n .ogen fixation cannot be achieved by sclection of
the legume alone; both host genot: e and rhizobial strain influence nitrogen fixation
(Arrendell, et al., 1985; Hohenberl ?t ul., 1982; Isleib et rd., 1980; Zary et LII., 1978).
Most of the studies reported or plant genotype Rhizohi,um strain interactions
indicated differences between cuit
f the same species in ability to fix nitrogen
using introduced rhizobial strains. E
ause ii is difficult IO introduce and establish new
Rhizobium strains into fields cent
ing indigenous strains (Brockwell. 198(l), it is
important to isolate competitive ar
adapted strains, which cari nodulate effectively
most cultivars even strains are not v
ly distributed in the mil. Instead of introducing
new Rhizobium strains, the isolatec
apted Rhizobium strains may be re-introduced
into the field SO that they Will c c
te against indigenous strains which are less
numerous. Our results show th
a11 Rhizobium strains including introduced
(Niff AL) and indigenous strains nc
ted all cultivars. When the hosts and rhizobial
strains were arranged in an inc asing order of effectiveness, one indigenous
Rhizobium MAO 113 exhibited th
hest effective association with thc cultivars.
Nitrogen fixation in groundnut 371
Control
11
l2 M A O 1 1 3
13
14
15
l6
17
18
19
110
Ill
I 12
1,3 MAO 118
114
115
116
117
118
119
120
121
122
MAO 121
*
123
I 24
125
126
127
126
129
130
MAO 126
*
131
Fig. 3 Shoot dry weight of Voandzeia
subterrunea cultivated i Leonard jar assembly and inoculated with 31
rhizobial isolates. Treatments with * differ significantly wi h the control at the 0.05 level by Duncan’s
multiple range test (1955).
!
Table 3 Shoot dry weight (SDW) (g/plant) and nodulation index (Nod. 1.) of 12 bambara groundnut cultivars inoculated by cowpea
%
Rhizobium strains from West MIRCEN Africa (MAO) and NiffAL uroiect
P
Control
MAO 113
MAO 118
TAL 22
T A L 164,
T A L 569
Rambara
Cultivars
SDW’ Nod. 1.’
SDW Nod. 1.
SDW Nod. 1.
SDW Nod. 1 .
SDW
‘Vod.
1.
SDW
Nod. 1 .
79-1
(1 9"
nn
E
-_--.--.-_ -. 7 0;’
E
1.8;’
E
-.~-~-._I- 1.8”
c
g:- ; 7:
:.:"
nTr
L
e
!.P’
f
1 5-F'
F
lPh
P
X3-129
1 . 0 ”
nn
E
e
1 a”<
c
1.1”
1
1.9”
E
83-131
1 .O”
nn
E
E
l.Vh
E
1 .s;*h
c
1.8” E
Sarakawa 1
1.2”
nn
E
e
1 ,8””
E
I.5”h
e
1 5””
Awandjelo 1
0.9”
nn
1
1
1.s‘+c
e
1.9”
E
,,5:11’
;
Ketao 2
o.9i’
nn
E
c
l.S”h’
e
151,‘~
e
1.1“
1
Ketao 3
, ,3”
nn
e
e
1.4’” e
1 .y” e
l.fP‘
e
Alheride 1
1.2”
nn
e
1 .P
E
,,$‘h’
e
1.1“
1
Lassa 3
1.0”
ml
L
1
1.9’+
E
1.1’”
c
1.1“
1
\\‘2
I .o;’
nn
1
1
l.-’
r
0.9’
e
1.p
e
XSOOI
0.9”
nn
c
E
1.5””
e
1.Y
E
1.r;”
E
’ Values followed bv thc same letter in each column do net differ 3t the 0.05 level hy Duncan‘s multiple range test (1955). a: highly effective: ah: par’ially
effective; b: ineffective.
’ nn: non-nodulated; E: highly effective nodlation ( 3 4j; e: partially effective nodulation (2 .
c Nod. 1. i 1). 1. incffcctivc raiulaîion (Nod. 1. < 2).
t
.
*
.’
Nitrogen jixation in groundturt 373
Thcrefore, strain MAO Il3 is expected
gen fixation under field
conditions.
The strategy to improve nitrogen fixation b
urnes in general is to first improve
gcnotype and then Select for best Rhizohiu
ains to increase nitrogen fixation.
Selection of host plants with good general effe
eness in nitrogen fixing ability is an
other avenue (Wynne et al. 1980). The most
ly strategy is to Select in each Iield
condition the most effective nitrogen fixing ho
ant-Rhizohiutn strain combination.
In our study, the higher relative effecti
ohtained with cultivars at Nioro
experimental station might he due to bett
conditions (higher organic matter)
that are favourable to this plant (Tardieux,
However, the dctermination of the
relative effectiveness of hlost plant/indige
Rhizobium strains combinations
permitted us to selcct the best combinations
ch area. Greenhouse evaluation of
these combinations is the initial phase of the
cess of increasing nitrogen fixation
through Rhizobium strains selection. The s
d phase was to investigate field
response of bambara groundnut to inoculatior
h the selected Rhizohium strains.
Effcct of inoculation with st:rains MAO Il
118, TAL 22 and TAL 569 on the
grain yield of 79- 1 and 83-131 was obta
wo Senegalese soi1 (Gueye 1987).
However. long term efforts Will be require
ed for improving nitrogen fixation
in bambara groundnut.
Acknowledgements
This research was supported by grant no. BN
-1-84-31 from the U.S. BOSTID-
National Academy of Sciences. The authors t
k Alioune Gning and Omar Toure
for valuable technical assistance.
References
AKKENIXIJ., S., WYNNE, J. C.. EI.KAN. G. fi. and
IR. T. Ci. 1985 Variation for nitrogen
fixation progenies of a Virginia x Spanish peanu
C‘rop Science 25, 865-869.
BOKDEI.EAI.I, L. M., GIKO~X, M.. OUFXIXT, R.
TOUN, H. 1981 Effet du soufre et de
l’azote sur la fixation symbiotique d’azote chez
tules de luzerne (Medicago sarivu L.).
(‘~~~~ion Journul of Plunt Science 61, (739
BROCK~EI.I.. J. 1980 Experiments with trop and pa
lcgumes - Princ@es and practice. In
Methw1e.y for evalurrting hiological nitrogen ,fixutio
zd by Bergersen. F. J. pp. 4174YO.
Chichester, New York, Brisbane. Toronto: John
C.AI.DWEI.I.. B. E. 1966 Inheritance of a strain-speci
ective nodulation in soybean. Crop
Science 6, 427-428.
COKHIN, E. J., BROCKWEIJ,. J. and C~AU~X, R
studies on chickpea (Cicer
uietimrm).
Arrstralïan Journd of Experitne
bandry 17, 126-134.
DUIXXK, D. B. 1955 Multiple range and mul
GUEYE, M. 1987 Effect of inoculation with
m strains on grain yield of
bambara groundnut (Voandzeia srtbterranea (L.)
uars) in two senegalese soils. Paper
presented at the symposium on the contribution
biological nitrogen fixation to plant
production. 3-7 August, 198’7. Bogor, Indonesia
HEICHEI., G. H. 1982 Breeding alfalfa for imp
trogen fixation: a physiological
perspective. Iowa State Journal of Research 56, 2
HEWITT. E. J. 1966 Sand and water culture meth
in the study of plant nutrition.
Technical communication No. 22 (2nd ed.) 547
monwealth agricultural bureaux.
London.
r
l
c
l
!
(
374 M. Gueye & L. M. Bordeleau
HOHENBERG, .J. S., MUNNS, D. N. 2 nj TUCKER. C. L. 1982 Rhiïohilrm-host specificities in
Phuseolus coccineus L. and Phaseclcts vrtlgaris L. Crop Science 22, 445-459.
I,XEIB. T. G., WYNNE, J. C., EI.KAN, CI. H. and SCHNFXWEIS, T. J. 1980 Ouantitative
genetics
aspects of nitrogen fixation in pear u,t (Arachis hypogaea L.). PUIZII~ Science 7, 101-105.
L.EON.I\\RI), L. T. 1943 A simple assem 31,~ for use in the testing of culture of rhizobia. Journal oj
Bacteriology 45, 523-527.
NINCHIN, F. R., SUMMERFIELV, R. J. ;m,d EAGLESHAM, A. R. J. 1978 Plant genotype Rhizobium
strains interactions in cowpea Vigru! r:mguiculuta (L.) Walp.). Tropical Agriculture (Trinidad)
55, 107-l 1.5.
MULONGOY, K.. AYANABA, A. and FUILVEK, E. 1980 Exploiting the diversity in the cowpea-
rhizobia symbiosis for increased CO ,vpea production. In Proceedings of the Global Impact of
Applied Microbiology held at Lagos; Part 2. Rhizosphere hiology. pp. 121- 125. ,4cademic
Press. London.
NATIONAL ACAVEMY OF SCIENCES, 1979 Pulses. In Tropical Legumes: resources for the future.
pp. 47-53. National Academy of Sci[ences, Washington D.C.
OLSEN, S. R.. C~LE, L. V., W;\\TANPBI:, F. S. and DEAN, L. A. 1054 Estimation of available
phosphate in soils by extraction with sodium bicarbonate. Circwlur of U.S. Department qf
Agriculture No. 939.
Annales du Centre de
VINCENT. J. M. 1970 A
study of the root-nodule bacteria. J.B.P.
Handbook No. 15
IEAVER, R. W. and BARNES, 1,. W. 1978 Intraspecific
Walp). .iournul qf
Summary
TO improve biological nitrogen fixation in bambara groundut (Voandzeiu subterraneu (L.)
Thouars), the relative effectiveness of 24 cultivars was studied in the field at two experimental
stations selected for their different ecological conditions. Thirty one Rhizobium strains were
isolated and 12 cultivars were screene3 ,during this study. Both indigl,:nous and introduced Niftal
Rhizobium strains were used durit-q a~ second study on host cultivars x Rhizobium strains
interactions. Nodulation index and shoot dry weight were used to assess the efficiency. The
widesî effectiveness spectrum was ( b’served with thc indigenous strain MAO Il3 and the
introduced strain TAL 22.
Résumé
l?xution d’nzote dans lu noix de Jufmhuru (Voandzeia suhterrana (L.j Thouars)
Dans le but d’améliorer la fixation G)logique d’azote dans In noix dc Bambara (Voundzek
.xubterraneu (L.) Thouars), l’efficacitl! :.elative de 23 cultivars a Cté etudiée sur le terrain dans
deux stations expérimentales choisit:! pour leurs conditions kologiques diffkentcs. Trente-et-
une souches tle Rhizobium ont Cté isolrSes et 12 cultivars ont t%é test& au cours de cette Etude.
Tant les souches indigènes de RhizoEibm que celles Niftal introduitçs ont kté utilisées au cours
d’une deuxième étude sur les interractions
entre les souches de Rhizohiurn ct les cultivars-
hOtes. L’indice de nodulation et le p>i,ds sec de la pousse ont ét& utilisés pour l’tirahlissrment
de l’cffcience. Le spectre d’efficienct. 1: plus large a étC observ6 avec la souche indigène MAO
113 et la souche introduite TAL 22.
1 Nitrogen jixation in groundnut 375
Resumen
Afin de mejorar la fijacion biologica de nitroge
n el cacahuete de Bambara (Voandzeiu
suhterrurm (L.) Thouars), se realizo un estudio
u efectividad relativa en campo en dos
estaciones experimentales seleccionadas por sus d
as condiciones ecologicas. Durante este
estudio se aislaron treintaiuna cepas de Rhi
n doce cultivares del huésped.
En un segundo ensayo se estudiaron las inte
epa de Rhizobiztm para 10 cual
se utilizaron cepas de Rhizohium Niftal ta
introducidas. La eficiencia se
valoro determinando el indice de nodulacio
a parte aerea. El espectro mas
amplio de eficacia se observîr con la cepa i
y la introducida TAL 22.
Voandzeia subterranea L.) Thouars
Mamadou Gueye’ and Lucien 4. Bordeleau*
Received 19 February 1988, accepted 21 M~I
1988
Introduction
Bambara groundnut (Vocrndzeia suhterrarze~ L.) Thouars) is a desirable legume
for Africa semi-arid tropical agriculture. L : cowpeas, the bambara groundnut
nodulates freely in tropical soils. Although it l Iduces a nutritious food, the bambara
groundnut remains one of the crops most neg
ted by science (National Academy of
Sciences, 1979).
The most important feature of legumes is 1 ir ability to fix atmospheric nitrogen
and considerable efforts have been expended Iwards increasing the efficiency of its
symbiotic association with Rhizobium. Now,
s accepted that seed inoculation with
superior Rhizobium strains is necessary with
ost legumes. However, cowpea seed
inoculation is usually unsuccessful because 0 aompetition between introduced and
indigenous Rhizobium strains in the soi]. r zrefore. an important step towards
improved nitrogen fixation is to isolate and id
tify competitive indigenous Rhizobia
with a view to using elite strains as inoculant
Mulongoy et al., 1980).
This paper presents data on screening field
imbara groundnut varieties, nitrogen
fixing ability of indigenous Rhizobium strains
Id evaluation of interactions between
host plant and Rhizobium strain.
Materials and methods
Fieki varietal screenitzg.
Tr,ials were done a
wo sites in Senegal during the 1985
rainy season. The sites differed in both rainf:
(Bambey, 377 mm annum-‘; Nioro.
S32 mm annum-‘) and in soi1 type (Table
, Twenty four cultivai-s of bambara
groundnut (listed in Fig. 1) were hand sown i i randomized block design replicated
four times. The cultivars were sown in two tn
ments: without (-N) and with (+N)
nitmgen fertilization (50 kg urea/ha). Ail pic
received 60 kg PzOs/ha and 120 kg
KCllha. Six weeks after planting, the relative
ifectiveness (RE) of each host plant/
indigenous Rhizohiztm strain combination wa!
valuatcd as follows (Mulongoy er a/. ,
1980):
’ MIRCEN-CNRA. B.P. 53, Barnbcy. Senegal.
’ Canada Agriculture. Research Station, 2560 Hoche&
Ivd, Sainte-Foy. Canada. GlV 253
0 Oxford University Press 1988
366 M . Guey & 1,. M. Bord&~~
Table 1 Characferistics of soi1 at Banbey ad Nioro cxperimental st.ations
wm
Total N
A,‘a’t;rtde 1’
(C Is,31”)
--~
290
121
310
137
‘weight’ oF ;en samples in the -N block
RE =
- -
- - - x 1 0 0
‘weight’ OC ;cn samples in the +N Hock
In this formula, ‘weight’ is either of shoot, root or nodule dry wcight or shoot nitrogen
content.
Nitrogcn ,fi.xing ubility of indigenou? IRhizobium struins.
The samples wcre taken in
each trcatment from five well-developcd plants and still having, immature pods and 4.5
well-devcloped, pink nodules werr: <;ampled Crom the tap roc,&. Rhizobium strains
were isolated according to standa -ci procedures (Vincent, lc~70). Isolated rhizobia
were cultured on agar slants made f:om yeast extract/mannitol rnedium. Nitrogen fixing
ability of the isolates were compared in terms of shoot dry weight of bambara
groundnut cultivar X5001 used as tl:s\\t plant and cultivated in IL.conard jar assemblies
(Leonard, 1943). Seeds were stelilized using 0.01% (w/v) HgCl, for 3 min and
germinatcd in sterile sand for 3 d; each Seedling was then Itransplanted into a jar
assembly. One ml suspension of rhizobial isolate broth (approx. 10” cells) was
added aseptically to the seedlings 1’~ r a11 treatments except for an uninoculated control
and plants were grown in a grcenhousc. Nitrogen-free Hewitt solution (Hewitt, 1966)
diluted with water ( 1:3 v/v) wus ad-led once a wcek during the growing period. After
35 d growth. plant tops were clipped off, dried and weighed for dry matter production.
Gwenhousc evuluutiora o f host pl’unt-Rhizohium strain interrrctiorrs.Both the
indigenous and Niftal Rhizohium s:r.:lins (listed in Table 3) werc selected on the basis
of their effectiveness. The Niftal ittains - TAI, 22, TAI., 169 and TAL 569 - were
supplied by the NiffAL project, Hawaii, USA and served as standards for
comparison against the indigencu; ones. Twelve bambara groundnut cultivars
also listed in Table 3 were used a:; ;lost plants. Seed sterilization of each genotype,
germination, transplantation, inocMion and watering were conducted as described
above and seeds were planted ir Leonard jars. Treatmcnts were arranged in a
randomized block design replicated five times. After SO days of growth, the plants
were harvested. Plant tops were ( rjed and weighed. Nodulation was scored taking
into account the number (N), the interna] colour (C) and the size (S) of nodules.
The nodulation index (Nod. 1.) W;IS calculated as follows:
Nol. 1. = N x C x S
Nodule number was rated on a sca.e from 0 (no nodule) to 3 (many nodules). Nodule
interna1 colour was from 0 (whit,:) to 1 (red) and nodule size was from 1 (small
nodule) to 2 (big nodule).
Nitrogen fixation in groundtlut 367
Kesults
Field varietal scrcening
At Bambey station the cultivars 83-127, Su’ Cameroun, Sarakawa 3, Sarakawa 10
and Lassa 1 had a relative effectiveness (RE) )ased on shoot dry weight, below 40%
(Fig. 1 j while at Nioro st,ation, a11 cultivars 1 d the same RE of over 40% (Fig. 2).
At Bambey station, only cultivar Sarakawa 1 had an RE, based on root dry weight,
below SO%. At Nioro station, a11 cultivars h I the same RE of over W%. Relative
Effectiveness, based on nodule dry weight , w
much lower at Bambey than at Nioro:
it was in the range of 80 to 120% at Bambey fl
cultivars 83-131, Ketao 3, Alheride 1,
Lassa 1 and Lassa 4 and in the range of 360 ) 480% at Nioro for cultivars 83-129.
X3-131. Sud Cameroun, Sarakawa 2. Ketao ! V2 and 85002. Relative Effectiveness,
based on shoot nitrogen content, was o r 50% for a11 cultivars except for
Awandjelo 1 at Bambey station.
These results indicated that Bambey and r )ro rhizobial population established a
moderately effective symbiosis with the cultiv
s of bambara groundnut used. Among
these cultivars, 12 grew better at both th
locations without nitrogen fertilizer
(Table 2) indicating their ability to set an effe ive symbiosis in the two different field
conditions.
Table 2 Shoot dry,weight (g/LO plants) of the perfo
led cultivars of bambara groundnut in field
at Bamhey and Nmro experimental stations
Bambara Cultivars
E nbey
Nioro
79-1
5 sh
30.4h
83- 126
c 2 ”
33.6”
x3-129
5 Sh
39sYh
83-131
5 sh
30.2h
Sarakawa 1
5 3”
31.1h
Awandjelo 1
6 ?”
40.Yh
Ketao 2
6 ih
31.7h
Ketao 3
6 )ah
32.9h
Alheride 1
6 ;A,
4
43.9”
Lassa 3
7 Y
38.fYh
v2
6 5’
41.3””
8500 1
5 jh
42.0”
Values followed by the same letter in each column
1 not differ significantly at the 0.05 level by
Duncan’s.multiple
range test (19%).
Effec’tiveness of indigenous Rhizohium str&
s isolated from bamhara groundnut
nodules
Bambara groundnut cultivars sampied were ,oderately nodulated on both the tap
and lateral roots. Many nodules appeared I be effective as indicated by interior
colour. Thirty one isolates were SO obtained: (
to 13*, Fig. 3). The isolates were slow-
growing and formed small (diam. < 1 mm), d colonies on yeast mannitol agar. Four
isolates (12, 113, IZ2 and lJo) were lodged in WI t Africa MIRCEN (MAO) Rhizobium
culture collection because of their high effet veness with cultivar 85001 which was
from Senegal and had good RE values at
)th Bambey and Nioro experimental
140 ta)
140
04
130 i
130
i20
110
100
90I
8 0
70
60
50
40
30
20
10
-
0
520~ J
IJU=
4 8 0
120-
s ,401
110-
lOO-
go-
80.
Cultivars
Cultivars
Fig. 1 Distribution of the Relative Effectiveness (RE) of 21 cultivars of Voandxiu s~~hf~~rtneu at Bamhey cxperimental station. A: RE hased on
shoot dry weight; B: RE hascd on root dry weight; C: RE hased on nodule dry weight; D: RE based on shoot nitrogen content.
140 tb)
130 i
120
110
100
5 90
90
B 80
80
2 70
70
<L> 60
60
g 50
50
40
40
g 30
30
20
20
10
10
0
-i
140 td'
î
130
120
110
-410090706050
40
30
20
1 0
0
Fig. 2 Distribution of the Relative Effectiveness (RE) of 24 cultivars of Voandzeiu suhlerrunea at Nioro experimental
station. A: RE based on shoot
dry weight: B: RE based on root dry weight; C: RE based on nodule dry weight: D: RE based on shoot nitrogen content.
370 M. Gueye & L. M. Bordelecu
stations. These isolates were named MAO ‘113, MAO 118. M.AO 121 amI MAO 126.
respectively, and are indicated by an asterisk in Fig. 3.
Evaluation of host cultivar-Rhizoh lum strains inteructiorzs
Rhizohium strains used were comaared for their effectiveness to nodulate the most
promising cultivars of bambara groundnut selected in held varietal screening. Nodules
were found in the top 5 cm of root svstem on all plants exccpt controls which were ail
nodule free. Nodulation index ani the effectiveness varicd greatly with the strains
(Table 3). Strains MAO 113 and hfA0 118 exhibited effcctivl: nodulation on six and
three cultivars respectively while strains TAL 22, TAL 169 and TAL 569 were
effective on five, four and three culltivars respectively. Al1 Rhizohium strains were
effective on cultivar 83-131 except sbrain TAL 169 which cxhibitecl partial nodulation.
,411 Rhizobium strains were parti,ally effective on cultivar Ketao 3. Similarly,
Rhizobium strains MAO 113 and h4AO 118 were ineffective on cultivar V2 while
strains TAL 22, TAL 169 and TAI, 5169 were partially effective on this cultivar. Strains
MAO 113 and TAL 169 were ineffective on cultivars Awandjelo 1 and 83-129
respectively; strain MAO 118 was ineffective on cultivars Awandjelo 1. Lassa 3 and
V2; strain TAL 569 was ineffectilre on cultivars Ketao 2. Alheride 1 and Lassa 3.
Strain TAL 22 presented the widejt effectiveness spectrum: il was fully effective on
42% and partially effective on Sgt?, faf a11 cultivars studied, followed by strain MAO
113. These results indicated that phnt genotype had an important effect on nodulation
of bambara groundnut .
Although Corbin et al., (1977) had proposed a scoring system to classify nodulation
and Bordeleau et al., (1981) had reported a method for s’coring nodulation, we
adopted another method. The reliab,ility of this method was indicated by the fact that
nodulation index and shoot dry weight (Table 3) showed the same trend of
effectiveness in host plant-Rhizobiuan strain combinations.
Discussion
Many studies have reported an iml ovement of biological nitrogen fixation resulting
from legume cultivar selection (Hei tel, 1982; Minchin et ~11.. 1978; Zary et ul., 1978).
However, selection for increased n .ogen fixation cannot be achieved by sclection of
the legume alone; both host genot: e and rhizobial strain influence nitrogen fixation
(Arrendell, et al., 1985; Hohenberl ?t ul., 1982; Isleib et rd., 1980; Zary et LII., 1978).
Most of the studies reported or plant genotype Rhizohi,um strain interactions
indicated differences between cuit
f the same species in ability to fix nitrogen
using introduced rhizobial strains. E
ause ii is difficult IO introduce and establish new
Rhizobium strains into fields cent
ing indigenous strains (Brockwell. 198(l), it is
important to isolate competitive ar
adapted strains, which cari nodulate effectively
most cultivars even strains are not v
ly distributed in the mil. Instead of introducing
new Rhizobium strains, the isolatec
apted Rhizobium strains may be re-introduced
into the field SO that they Will c c
te against indigenous strains which are less
numerous. Our results show th
a11 Rhizobium strains including introduced
(Niff AL) and indigenous strains nc
ted all cultivars. When the hosts and rhizobial
strains were arranged in an inc asing order of effectiveness, one indigenous
Rhizobium MAO 113 exhibited th
hest effective association with thc cultivars.
Nitrogen fixation in groundnut 371
Control
11
l2 M A O 1 1 3
13
14
15
l6
17
18
19
110
Ill
I 12
1,3 MAO 118
114
115
116
117
118
119
120
121
122
MAO 121
*
123
I 24
125
126
127
126
129
130
MAO 126
*
131
Fig. 3 Shoot dry weight of Voandzeia
subterrunea cultivated i Leonard jar assembly and inoculated with 31
rhizobial isolates. Treatments with * differ significantly wi h the control at the 0.05 level by Duncan’s
multiple range test (1955).
!
Table 3 Shoot dry weight (SDW) (g/plant) and nodulation index (Nod. 1.) of 12 bambara groundnut cultivars inoculated by cowpea
%
Rhizobium strains from West MIRCEN Africa (MAO) and NiffAL uroiect
P
Control
MAO 113
MAO 118
TAL 22
T A L 164,
T A L 569
Rambara
Cultivars
SDW’ Nod. 1.’
SDW Nod. 1.
SDW Nod. 1.
SDW Nod. 1 .
SDW
‘Vod.
1.
SDW
Nod. 1 .
79-1
(1 9"
nn
E
-_--.--.-_ -. 7 0;’
E
1.8;’
E
-.~-~-._I- 1.8”
c
g:- ; 7:
:.:"
nTr
L
e
!.P’
f
1 5-F'
F
lPh
P
X3-129
1 . 0 ”
nn
E
e
1 a”<
c
1.1”
1
1.9”
E
83-131
1 .O”
nn
E
E
l.Vh
E
1 .s;*h
c
1.8” E
Sarakawa 1
1.2”
nn
E
e
1 ,8””
E
I.5”h
e
1 5””
Awandjelo 1
0.9”
nn
1
1
1.s‘+c
e
1.9”
E
,,5:11’
;
Ketao 2
o.9i’
nn
E
c
l.S”h’
e
151,‘~
e
1.1“
1
Ketao 3
, ,3”
nn
e
e
1.4’” e
1 .y” e
l.fP‘
e
Alheride 1
1.2”
nn
e
1 .P
E
,,$‘h’
e
1.1“
1
Lassa 3
1.0”
ml
L
1
1.9’+
E
1.1’”
c
1.1“
1
\\‘2
I .o;’
nn
1
1
l.-’
r
0.9’
e
1.p
e
XSOOI
0.9”
nn
c
E
1.5””
e
1.Y
E
1.r;”
E
’ Values followed bv thc same letter in each column do net differ 3t the 0.05 level hy Duncan‘s multiple range test (1955). a: highly effective: ah: par’ially
effective; b: ineffective.
’ nn: non-nodulated; E: highly effective nodlation ( 3 4j; e: partially effective nodulation (2 .
c Nod. 1. i 1). 1. incffcctivc raiulaîion (Nod. 1. < 2).
t
.
*
.’
Nitrogen jixation in groundturt 373
Thcrefore, strain MAO Il3 is expected
gen fixation under field
conditions.
The strategy to improve nitrogen fixation b
urnes in general is to first improve
gcnotype and then Select for best Rhizohiu
ains to increase nitrogen fixation.
Selection of host plants with good general effe
eness in nitrogen fixing ability is an
other avenue (Wynne et al. 1980). The most
ly strategy is to Select in each Iield
condition the most effective nitrogen fixing ho
ant-Rhizohiutn strain combination.
In our study, the higher relative effecti
ohtained with cultivars at Nioro
experimental station might he due to bett
conditions (higher organic matter)
that are favourable to this plant (Tardieux,
However, the dctermination of the
relative effectiveness of hlost plant/indige
Rhizobium strains combinations
permitted us to selcct the best combinations
ch area. Greenhouse evaluation of
these combinations is the initial phase of the
cess of increasing nitrogen fixation
through Rhizobium strains selection. The s
d phase was to investigate field
response of bambara groundnut to inoculatior
h the selected Rhizohium strains.
Effcct of inoculation with st:rains MAO Il
118, TAL 22 and TAL 569 on the
grain yield of 79- 1 and 83-131 was obta
wo Senegalese soi1 (Gueye 1987).
However. long term efforts Will be require
ed for improving nitrogen fixation
in bambara groundnut.
Acknowledgements
This research was supported by grant no. BN
-1-84-31 from the U.S. BOSTID-
National Academy of Sciences. The authors t
k Alioune Gning and Omar Toure
for valuable technical assistance.
References
AKKENIXIJ., S., WYNNE, J. C.. EI.KAN. G. fi. and
IR. T. Ci. 1985 Variation for nitrogen
fixation progenies of a Virginia x Spanish peanu
C‘rop Science 25, 865-869.
BOKDEI.EAI.I, L. M., GIKO~X, M.. OUFXIXT, R.
TOUN, H. 1981 Effet du soufre et de
l’azote sur la fixation symbiotique d’azote chez
tules de luzerne (Medicago sarivu L.).
(‘~~~~ion Journul of Plunt Science 61, (739
BROCK~EI.I.. J. 1980 Experiments with trop and pa
lcgumes - Princ@es and practice. In
Methw1e.y for evalurrting hiological nitrogen ,fixutio
zd by Bergersen. F. J. pp. 4174YO.
Chichester, New York, Brisbane. Toronto: John
C.AI.DWEI.I.. B. E. 1966 Inheritance of a strain-speci
ective nodulation in soybean. Crop
Science 6, 427-428.
COKHIN, E. J., BROCKWEIJ,. J. and C~AU~X, R
studies on chickpea (Cicer
uietimrm).
Arrstralïan Journd of Experitne
bandry 17, 126-134.
DUIXXK, D. B. 1955 Multiple range and mul
GUEYE, M. 1987 Effect of inoculation with
m strains on grain yield of
bambara groundnut (Voandzeia srtbterranea (L.)
uars) in two senegalese soils. Paper
presented at the symposium on the contribution
biological nitrogen fixation to plant
production. 3-7 August, 198’7. Bogor, Indonesia
HEICHEI., G. H. 1982 Breeding alfalfa for imp
trogen fixation: a physiological
perspective. Iowa State Journal of Research 56, 2
HEWITT. E. J. 1966 Sand and water culture meth
in the study of plant nutrition.
Technical communication No. 22 (2nd ed.) 547
monwealth agricultural bureaux.
London.
r
l
c
l
!
(
374 M. Gueye & L. M. Bordeleau
HOHENBERG, .J. S., MUNNS, D. N. 2 nj TUCKER. C. L. 1982 Rhiïohilrm-host specificities in
Phuseolus coccineus L. and Phaseclcts vrtlgaris L. Crop Science 22, 445-459.
I,XEIB. T. G., WYNNE, J. C., EI.KAN, CI. H. and SCHNFXWEIS, T. J. 1980 Ouantitative
genetics
aspects of nitrogen fixation in pear u,t (Arachis hypogaea L.). PUIZII~ Science 7, 101-105.
L.EON.I\\RI), L. T. 1943 A simple assem 31,~ for use in the testing of culture of rhizobia. Journal oj
Bacteriology 45, 523-527.
NINCHIN, F. R., SUMMERFIELV, R. J. ;m,d EAGLESHAM, A. R. J. 1978 Plant genotype Rhizobium
strains interactions in cowpea Vigru! r:mguiculuta (L.) Walp.). Tropical Agriculture (Trinidad)
55, 107-l 1.5.
MULONGOY, K.. AYANABA, A. and FUILVEK, E. 1980 Exploiting the diversity in the cowpea-
rhizobia symbiosis for increased CO ,vpea production. In Proceedings of the Global Impact of
Applied Microbiology held at Lagos; Part 2. Rhizosphere hiology. pp. 121- 125. ,4cademic
Press. London.
NATIONAL ACAVEMY OF SCIENCES, 1979 Pulses. In Tropical Legumes: resources for the future.
pp. 47-53. National Academy of Sci[ences, Washington D.C.
OLSEN, S. R.. C~LE, L. V., W;\\TANPBI:, F. S. and DEAN, L. A. 1054 Estimation of available
phosphate in soils by extraction with sodium bicarbonate. Circwlur of U.S. Department qf
Agriculture No. 939.
Annales du Centre de
VINCENT. J. M. 1970 A
study of the root-nodule bacteria. J.B.P.
Handbook No. 15
IEAVER, R. W. and BARNES, 1,. W. 1978 Intraspecific
Walp). .iournul qf
Summary
TO improve biological nitrogen fixation in bambara groundut (Voandzeiu subterraneu (L.)
Thouars), the relative effectiveness of 24 cultivars was studied in the field at two experimental
stations selected for their different ecological conditions. Thirty one Rhizobium strains were
isolated and 12 cultivars were screene3 ,during this study. Both indigl,:nous and introduced Niftal
Rhizobium strains were used durit-q a~ second study on host cultivars x Rhizobium strains
interactions. Nodulation index and shoot dry weight were used to assess the efficiency. The
widesî effectiveness spectrum was ( b’served with thc indigenous strain MAO Il3 and the
introduced strain TAL 22.
Résumé
l?xution d’nzote dans lu noix de Jufmhuru (Voandzeia suhterrana (L.j Thouars)
Dans le but d’améliorer la fixation G)logique d’azote dans In noix dc Bambara (Voundzek
.xubterraneu (L.) Thouars), l’efficacitl! :.elative de 23 cultivars a Cté etudiée sur le terrain dans
deux stations expérimentales choisit:! pour leurs conditions kologiques diffkentcs. Trente-et-
une souches tle Rhizobium ont Cté isolrSes et 12 cultivars ont t%é test& au cours de cette Etude.
Tant les souches indigènes de RhizoEibm que celles Niftal introduitçs ont kté utilisées au cours
d’une deuxième étude sur les interractions
entre les souches de Rhizohiurn ct les cultivars-
hOtes. L’indice de nodulation et le p>i,ds sec de la pousse ont ét& utilisés pour l’tirahlissrment
de l’cffcience. Le spectre d’efficienct. 1: plus large a étC observ6 avec la souche indigène MAO
113 et la souche introduite TAL 22.
1 Nitrogen jixation in groundnut 375
Resumen
Afin de mejorar la fijacion biologica de nitroge
n el cacahuete de Bambara (Voandzeiu
suhterrurm (L.) Thouars), se realizo un estudio
u efectividad relativa en campo en dos
estaciones experimentales seleccionadas por sus d
as condiciones ecologicas. Durante este
estudio se aislaron treintaiuna cepas de Rhi
n doce cultivares del huésped.
En un segundo ensayo se estudiaron las inte
epa de Rhizobiztm para 10 cual
se utilizaron cepas de Rhizohium Niftal ta
introducidas. La eficiencia se
valoro determinando el indice de nodulacio
a parte aerea. El espectro mas
amplio de eficacia se observîr con la cepa i
y la introducida TAL 22.