/ Nitrogen fixation in bambara groundnut, j ...
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Nitrogen fixation in bambara groundnut,
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Voandzeia subterranea (L.) Thouars
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Mamadou Gueye” and Lucien M. Bordeleau*
Received 19 February 1988, accepted 21 March 1988
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Introduction
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Bambara groundnut (Voundzeia subteerruneu (L.) Thouars) is a desirable legume
for Africa semi-arid trop54 agriculture. Like cowpeas, the bambara groundnut
nodulates freely in tropical soils. Although it produces a nutritious food, the bambara
groundnut remains one of the crops most neglected by science (Nationa! Academy of
Sciences, 1979).
The most important feature of legumes is their ability to fix atmospheric nitrogen
and considerable efforts have been expended towards increasing the eticiency of its 1
symbiotic association with Rhizobium. Now, it is accepted that seed inoculation with
:
superior Rhirobium strains is necessary with most legumes. However, cowpea seed
mocuiation is usually unsutmssful because of competition between introduced and W
indigenous Rhizobium strains in the soil. Therefore, an important step towards 1
improved nitrogen fixation is to isolate and identify competitive indigenous Rhizobia i
with a view to using ellte strains as inoculants (Mulongoy et’af., 1980).
/
This paper presents data ‘on screening field bambara groundnut varieties, mtrogen
fixing ability of indigenous Rhizobium strains and evaluation of interactions between
host plant and Rhizobium strain.
Materiafs and methods
Field variera1 screening.
Trials were done at two sites in Senegai during the 1985
rainy season. The sites differed in both rainfall (Bambey, 377 mm annum-‘;.Nioro,
532 mm annum-‘) and in soi1 type (Table 1). Twenty four cultivars of bambara
groundnut (listed’in Fig. 1) were hand sown in a randomized block design replicated
four times. The cultivars were sown in two treatments: without (-N) and with (+N)
nitrogen fertihzation (50 kg urea/ha). Al1 plots received 60 kg PzO.Jha and 120 kg
KWha. Six weeks after planting, the relative effectiveness (RE) of each host planti
indigenous Rhizobium strain combination was evaluated as follows (Mulongoy et al.,
1980):
’ MIRCEN-CNRA, B.P. 53, Bambey, Senegal.
’ Canada Agriculture, Research Statron, 2560 Hochelaga blvd, Sainte-Foy, Canada, GlV 2.U. 1
ôOxford Ilniversity Press 1988 }
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Table P Lharacteristics
of soi1 at Bambey and Nioro experimental stations
mm
Composition (%)
____..
.
. . -
-_- .l.._l_-.- .---
Soi1
Total C
Total N
Availablc P
Clay
L.oam
S a n d
PH (HZ~)
(OIsen’)
---.-
Bambey
2910 290
1 2 1
4
2
96
7.x
_ Nioro
4160
'
310
137
b
3
90
5.8
‘Olsen ef al. (1954).
‘weight’ of ten samples in the -N block
RE =
x 100
‘weight’ of ten samples in the -t-N block
1
In this formula, ‘weight’ is either of shoot, root or nodule dry weight or shoot nitrogen
1
content.
I
Nitrogen fixing ability of indigenous Rhizobium strains.
The samples were taken in
each treatrnent from five well-developed plants and still having immature pods and 45
well-developed, pink nodules were sampled from the tap roots. Rhizobium strains
were isolated according to standard procedures (Vincent, 1970). Isolated rhizobia
were cultured on agar slants made from yeast extrac
altos medium. Nitrogen fixing
ability of the isolates were compared
q
in terms of s Ôot dry- weight of bambara
groundnut cultivar 85001 used as test plant and cultivated in Leonard jar assemblies
(Leonard, 1943). Seeds were sterilized using 0.01% (WV) HgC12 for 3 min and
germinate,d in sterile sand for 3 d; each seedling was then transplanted into a jar
assembly. One ml suspension of rhizobial isolate broth (approx. 109 cells) was
added aselptically to the seedlings for ail treatments except for an uninoculated control
and plants were grown in a greenhouse. Nitrogen-free IIe-itt solution (Hewitt, 1946)
diluted with water (1:3 vlv) was added once a week during the growing period. After
35,d growth, plant tops were clipped off, dried and weighed, for dry matter production.
Greenhouse evaluation of host plant-Rhizobium straiti interactions. Both the
indigenous and Niftal Rhizobium strains (Iisted in Table 3) were selected on the basis
of their effectiveness. The Niftal strains - TAL 22, TAL; 169 and TAL 569 - were
supplied by the NifIA!. project. Hawaii, USA and; served as standards for
comparison against the indigenous ones. Twelve bambara groundnut cultivars
also Iistedl in Table 3 were used as host plants. Seed :sterilization of each genotype,
germination, transplantation, inoculation and watering wére conducted as described
above an’d seeds were planted in Leonard jars. Treatments were arranged in a
randomized block design replicated five times. After 50 d$ )ol growth, the plants
were harvcsted. Plant tops were dried and weighed. Nodulation was scored taking
into account the number (N), the interna1 colour (C) and the size (S) of nodules.
The noduiation index (Nod. 1.) was calculated as follows:
Nod. 1. =NxCxS
Nodule number was rated on a scale from 0 (no nodule) to 3 (many nodules). Nodule
interna1 colour was from 0 (white) to 1 (red) and nodule size was from 1 (small
nodule) to 2 (big nodule).
Field varietal screening
At Bambey station the cultivais 83-127, Sud Cameroun, Sarakawa. 3, Sarakawa 10
and Lassa 1 had a relative effectiveness (RE), based on shoot dry weight, below 40%
0
(Fig. 1) while at Nioro station, all cultivars had thezame RE of over 40% (Fig. 2).
At Bambey,station, only cultivar Sarakawa 10 had ai RE, based on root dry weight,
,
below 50Y$at Nioro station, all cultivars had the same RE of over 50%. Relative
Effectiveness, based on nodule dry weight, was much lower at Bambey than at Nioro:
it was in the range of $0 lto 120% at Bambey for cultivars 83-131, Ketao 3, Alheride 1,
Lassa 1 and Lassa 4 and in the range of 360 to 480% at Nioro for cultivars 83-129,
83-131, Sud Cameroun, Sarakawa 2, Ketao 9, V2 and 85002. Relative Effectiveness,
based on shoot nitrog;en content, was over 50% for all cultivars except for
Awandjelo 1 at Bambey station.
These results indicated that Bambey and Nioro rhizobial population established a
moderately effective symbiosis with the cultivars of bambara groundnut used. Among
these cultivars, 12 grew better at both the locations without nitrogen fertilizer
(Table 2) indicating their ability to set an effective symbiosis in the two different field
conditions.
Table 2 Shoot dry weight (g/lO plants) of the perfonned cultivars of bambara groundnut in field
at Bambey and Nioro experimental stations
Bambara Cultivars
Bambey
Nioro --_-.- _..._.. -_. - ..__. ._
79-1
55.5b
b
l
83-126
62.2b
Zb
,
83-129
55.gb
39h
/
83-131
56Sb
30.2b
l
Sarakawa 1
52.8;
31.1b
Awancljelo 1
40.Sab
Ketao 2
63:5
31.7b
Ketao 3
66 2ab
32.9b
Alheride 1
68:Tb
43.9”
Lassa 3
78.5’
38 6ab
v2
61.5b
41:3Pb
85001
56.5b
42.0”
--
-
-~-
Values foltowed by the same letter in each column do not differ significantiy at the 0.05 level by
DUNCAN’s multiple range test (1955).
Effectiveness of indigenous Rhizobium strains isolated from bambara groundnut
nodules
Bambara groundnut cultivars sampled were moderately nodulated on both the tap
and lateral roots. Many nodules appeared to be effective as indicated by interior
colour. Thirty one isolates were SO obtained: (Ii to Ist, Fig. 3). The isolates were slow-
growing and formed small (diam. < 1 mm), dry colonies on yeast mannitol agar. Four
isolates (12, ii3, 122 and I& were lodged in West Africa MIRCEN (MAO) Rhizobium
culture collection because of their high effectiveness with cultivar 85001 which was
from Senegal and had good RE values at both Bambey and Nioro experimental
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Nnokmv3ans
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0
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1
1
2
VM’i’RWVS
OBMNVMV
VM’0WKi
VMM
VMvnvtlvs
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VMkW’tiVS
VMVNAIVS
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statior . These isolates were named MAO 113, MAO 118, MAO 121 and MAO 126,
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respef,tively, and are indicated by an asterisk in Fig. 3.
Evaludon of host cultivar-Rhizobium strains interactions
Rhizobium strains used were compared for their effectiveness to nodulate the most
promising cultivars of bambara groundnut selected in field varietal screening. Nodules
were found in the top 5 cm of root system on ah plants except controls which were ah
nodule free. Nodulation index and the effectiveness varied greatly with the strains
(Table 3). Strains MAO 113 and MAO 118 exhibited effective nodulation on six and
three cultivars respectively while strains TAL 22, TAL 169 and TAL 569 wera
effective on five, four and three cultivars respectively. Ah Rhizobium strains were
effective on cultivar 83-131 exceptstrain TAL 169 which exhibited partial nodulation.
Al1 Rhizobium strains were partially effective on cultivar Ketao 3. Similarly,
Rhizobium strains MAO 113 and MAO 118 were ineffective on cultivar V2 whila
strains TAL 22, TAL 169 and TAL 569 were partially effective on this cultivar. Strains
,I
:‘.1: ’ I-....
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 ineffective on cultivars :Ketao 2, Alheride 1 and Lassa 3,
Strain TAL 22 presented the widest effectiveness spectrum: it was ftrlly’effective on
42% and partially effective on 58% of ah cultivars studied, followed by strain MAO
113. These rësults indicated that plant genotype had an important effect on nodulation
of bambara gioundnut.
Although Corbin et al., (1977) had proposed a scbring system to classify nodulation
and Elordeleau et al., (1981) had reported a method for scoring nodulation, we
adopted another method. The reliability of this mefhod was indicated by the fact that
nodulation index and shoot dry weight (Table 3) showed the same trend of
LG
.‘,
:
effectiveness in host plant-Rhizobiurn strain combinations.
Discwision
f
Many studies have reported an improvement of biological nitrogen fixation resulting
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from legume cultivar selection (Heichel, 1982; Minchin et al., 1978; Zary et al., 1978)
However, selection for increased nitrogen fixation (annot be achieved by selection of
the legume alone; both host genotype and rhizobia! strain influence nitrogen fixation
(Arrendell, et al., 1985; Hohenberg ei al., 1982; Islkib et al., 1980; Zary et al., 1978).
Most of the studies reported on plant genotype Rhizobium strain interactions
indica.ted differences between cultivars of the sami: species in ability to fix nitrogen
using introduced rhizobial strains. Because it is difficult to introduce and establish new
Rhizobium strains into fields containing indigenous strains (Brockwell, 1980), it is
important to isolate competitive and adapted strains, which cari nodulate effectively
most cultivars even strains are not widely distributed in the soi]. Instead of introducing
new Rhizobium strains, the isolated adapted Rhizobium strains may be re-introduced
into the field so that they Will compete against indigenous strains which are les$
numerous. Our results show that a11 Rhizobr’um strains including introduced
(NifI’AL) and indigenous strains nodulated ah cultivars. When the hosts and rhizobial
strains were arranged in an increasing order of effectiveness, one indigenous
Rhizobium MAO 113 exhibited the highest effective association with the cultiva&
M A 0 1 1 8 1
*
L
r
.
/
MAO 121 1
*
l
l
MAO 126
t*
.
l
Fig. 3 Shoot dry weight of’ Voundzeiusubtemuuu
cultivated in Léonard jar assembly and inoculated with 31
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rhizobial isolates. Treatm’ents with ??differ significantly with the control at the 0.05 level by DUNCAN’s i
multiple range test (1955).
i
Table 3 Shoot dry weight (SDW) @/plant) and nodulation index (Nod. 1.) of 12 bambara groundnut cultivars inoculated by cowpea
strains from West ,MIRCEN Africa (MAO) and NifTAL oroiect
i(
,- “. .
M;a;oTT5
_ .- - MA~ _ ..---
TGrïtrol--~
TAI,---
.- - - - TAI, 1 6 9
------ ----rAr -
Bambara
.-
---- -1--
Cultivars
SDW’
Nod. 1.’
SDW
Nod. 1.
SDW
Nod. 1.
SDW
Nod. 1.
SDW
Nod. 1.
SDW
Nod. 1.
-.
--1;41mr-..e. ---.
.~
79-l
0.9R
nn
211”
.-r-.------- ~.pï--E------~----- ‘~--
1,ga --- e
83-126
1.1’ nn
1.5”‘” E
1.6”” c
1.4&
e
1.8’ E
1P
e
83-129
1.0’ nn
1.6’*
E
l.PX
e
1.4t”
e
l.lbe
1
1.9” E
83-131
1.0” nn
1.6Rh
E
1.P
F
1.8”” E
1.5”h
c
1.8” E
Sarakawa 1
1.2” nn
1.6”h
E
1.6””
c
1.8’h
E
l.Sah
e
1 Yh
e
Awandjelo 1
0.9”
nn
1.1’ 1
1.2’”
1
l.Sahc
e
1.9”
E
1.P
e
Ketao 2
0.9” nn
1.6”” E
1 P
.‘
f
l.+
e
1.5”b
e
l.lh
1
Ketao 3
1.2” nn
1.F
e
l.Vhc’
c
1.4hc
e
l.Ssh
e
1.6’h
e
Alheride 1
1.2”
n
n
f.3”1”
e.
<
1.6”‘” e
1.8””
E
l 4& e
1.1”
i
Lassa 3
1 .O”
nn
1.7”
E
l.lh
1
1.9”” E
111”
e
1.1”
1
v2
1.0” nn
1.5ah 1
l.lh
1
1.2c
e
0.Y
e
1.P
85001
0.9a nn
1.4”hc
e
2.0” E
1.5”’
e
1.9’
E
1.8’
E
b
_
_
_-.--.
_.__
--.---
‘. ~&~s~~l&dby the same lctter in each column do not dlffer at thi 0.05 levcl by Duncan’s multiple range test (1955). a: highly effective; ab: partiaily
effective; b: incffective.
f
z nn: non-nodulated; E: highly effective nodlation (-
Z= 4); e: partialiy effective nodulation (2 4 Nod. 1. < 4); 1: ineffective nodulation (Nod. 1. < 2).
_~ .._..__... -.
_-. .-_-
_ -_
-
Therefore, strain MAO 113 is expected to give better nitrogen fixation under field
conditions.
The strategy to improve nitrogen fixation by legumes in general is to first improve
genotype and then select for best Rhizobium strains to increase nitrogen fixation.
Selection of host plants with good general effectiveness in nitrogen fixing ability is an
other avenue (Wynne (et al. 1980). The strategy most likely is to select in each field
condition the most effective nitrogen fixing host plant-Rhizobium strain combination.
In our study, the higher relative effectiveness obtained with cultivars at Nioro
experimental station might be due to better soi1 conditions (higher organic matter)
that are favourable to this plant (Tardieux, 1975). However, the determination of the
relative effectiveness of host plant$ndigenous Rhizobium strains combinations
permitted us to Select the best combinations in each area. Greenhouse evaluation of
these combinations is the initial phase of the process of increasing nitrogen fixation
through Rhizobium strains selection. The second phase was to investigate field
response of bambara g.roundnut to inoculation with the selected Rhizobium strains.
Effect of inoculation with strains MAO 113, MAO 118, TAL 22 and TAL 569 on the
grain yield of 79-1 and 83-131 was obtained in two Senegalese soi1 (Gueyeb%j@.
However, longterm efforts Will be required to breed for improving nitrogen fixation in
bambara groundnut.
Acknowledgements
This research was supported by grant no. BNF-SN-2-84-21 from the U.S. BOSTID-
National Academy of Sciences. The authors thank Alioune GNING and Omar
TOURE for valuable technical assistance.
References
ARRENDELL, S., WYNNE, J. C., ELKAN, G. H. and ISLEIB, T:G. 1985 Variation for nitrogen
fixation progenies of a Virginia x Spanish peanut cross.
25, 865-869.
BORDELEAU, L. M., GIRO~X, M., OUELLET, R. and ANTOUN, H. 1981 Effet du soufre et de
l’azote sur la fixation symbiotique d’azote chez les plantules de luzerne (Medicugo saliva L.).
Canadian Journal of Plant Science 61, 639-W.
BROCKWELL,
J. 1980 Experiments with trop and pasture legumes - Principles and practice. In
Methodes for evaluating biological nitrogen fîxation. Edited by Bergersen, F. J. pp. 417-490.
Chichester, New York, Brisbane, Toronto: John Wiley and Sons.
CALDWELL, B. E. 1966 Inheritance of a strain-specific ine-ffective nodulation in soybean. &op
Science 6, 427-428.
C~RBIN, E. J., BROCKWELL,
J. and GAULT, R. R. 1977 Nodulation studies on chickpea (Citer
arietinum). Australian Journal of Experimental
Agriculfural
Nusbandry 17, 124-134.
DUNCAN, D. B. 1955 Multiple range and multiple test. Biometrics 11, i-42.
GUEYE, M. 1987 Effect of inoculation with different Rhizobium strains on grain yield of
bambara groundnut (Voandzeia subterruneu (L.) Thouars) in two senegalese soils. Paper
presented at the symposium on the contribution of biological nitrogen fixation to plant
production. 3-7 August., 1987. Bogor, Indonesia.
HEICHEL, G. H. 1982 Breeding alfalfa for improved nitrogen fixation: a physiological
perspective. Iowa State Journal of Research 56, 255-280.
HE~, E. J. 1966 Sand and water culture methods used in the study of plant nutrition.
Technical communication No. 22 (2nd ed.) 547 pp. Commonwealth agricultural bureaux.
London.
HOHENDERC;,
J. S.. MUNNS, D. N. and TUCKER, C. L. 1982 RIiirobium-host
specihcttjes m
coccineus L. and Phuseolus vulgarlr L. Trop Science 22, 445-459.
Is~.faa, T. ci., WYNNE, J. C., ELKAN, G. H. and SCHNEEWEIS,
T. J. 1980 Quantitative genetics
aspects of nitrogen fixation in peanut (Arachis hypogueu L.). Peunur Science 7, 101-105.
LEONARD, L. 7’. 1943 A simple assembly for use in the testing of culture of rhizobia. Journalof
Racteriology 45, 523-527.
MINCHIN, F. R., SUMMERFNU, R. J. and EAGI.FSHAM, A. R. J. 1978 Plant genotype Rhirobium
strains interactions in cowpea Vignu unguiculuta (L.) Walp.). Tropical Agriculfure (Trinidad)
55, 107-115.
MuI.~N(;~Y, K., AYANABA, A. and PU.VER, E. 1980 Exploiting the diversity in the cowpea-
rhizohia symbiosis for increased cowpea production. In Proceedings of the Global Impact of
Applied Microhiology held at Lagos; Part 2. Rhizosphere biology. pp. 121-125. Academic
Press. London.
NATIONAI. ACA»EMY OF SCIENCES, 1979 Pulses. In Tropical Legumes: resources for the future.
pp. 47-53. National Academy of Sciences, Washington D.C.
OLSEN, S. R., C~LE, L. V., WATANABE, F. S. and DEAN, L. A. 1954 Estimation of available
phosphate in soils by extraction with sodium bicarbonate. Circulur of U.S. Depurtment of
Agricuffure
No. 939.
SEETIN, M. W. and BARNFS, D. K. 1977 Variation among alfalfa genotypes for acetylene
-i
‘.._
.’
reduction. Crop Science 17, 783-787.
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.
TARDIEU, M. 1957 les cultures d’appoint dans la zone du CRA Bambey. BuIletin Agronomique.
Annules du Centre de Recherches Agronomiques de Bumbey au Senegul 17, 5-54.
VINCENT, J. M. 1970 A manual for the practical study of the root-nodule bacteria. I.B.P.
Handbook No. 15 Blackwell Scientific Publications. Oxford and Edinburgh. 164 pp.
WYNNE, J. C., ELKAN, G. I-I., MEISNER, C. M., SCHNEEWEIS, T. J. and LIGON, J. M. 1980
Greenhouse evaluation of strains of Rhizobium for peanut. Agronomy Journal 72,645-649.
ZARY, K. W., MILLER, Jr., J. C., WEAVER, R. W. and BARNES, L. W. 1978 Intraspecific
vanability for nitrogen fixation in southernpea (V@u ungu+data
(L.) Walp). Journal of
Americun Sociaty for ffortirrtlfure Science 103, 8(6-KOK.
Summary
.:. ,. : ,‘,
TO improve biological nitrogen fixation in bambara groundut, (Voandzeiu subterrunea (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 cuhivars were screened during this study. Both indigenous and introduced Niftal
Rhizobium strains were used during a second study on host cultivars X Rhizobium strains
interactions. Nodulation index and shoot dry weight were used to assess the efficiency. The
widest effectiveness spectrum was ohscrved with thr indigenous strain MAO 113 and the
introduced strain TAL 22.
Résumé
Fixarion d’azote dans la noix de Batnbara (Voandzeia subterrilna CL.)- Thouars)
Dans le but d’améliorer la fixation biologique d’azote dans la noix de Bambara (Voundzeiu
subterranea (L.) Thouars), l’efficacité relative de 24 cultivars a été étudiée sur le terrain dans
deux stations exp&imentales
choisies pour leurs conditions écologiques différentes. Trente-et-
une souches de Rhizobium ont été isolées et 12 cultivnrs ont été testées au cours de cette étude.
Tant les souches indigènes de Rhizobium que celles Niftal introduites ont été utilisCes au cours
d’une deuxième 6tude sur les interractions
entre les souches de Rhizobium et les cultivars-
hôtes. L’indice de nodulation et le poids sec de la pousse ont été utilisées pour l’établissement
de I’efficience. Le spectre d’efficience le plus large a été observé avec la souche indigène MAO
113 et la souche introduite TAI. 22.
Resumen
Afin de meiorar la hiaci6n biohkica de nitrr5eeno en el cacahuete de Bambara (Voundzeia
\\
sub~erranea*(L.)
Thouars), se reakr un estu&o de su efectividad relativa en campo en dos
estaciones experimentales seleccionadas por sus distintas condiciones ecol6gicas. Durante este
estudio se aislaron treintaiuna cepas de Rhizobium y se probaron doce cultivares del huésped.
En un segundo ensayo se estudiaron las interacciones huesped xcepa de Rhizobium para 10 cual
se utilizaron cepas de Rhizobium Niftal tanto indigenas como introducidas. La eficiencia se
valor determinando el indice de nodulacion y el peso seco de la parte aerea. El espectro mas
amplio de etkacia se albserv6 con la cepa indfgena MAO 113 y la introducida TAL 22.
Nitrogen fixation in bambara groundnut,
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Voandzeia subterranea (L.) Thouars
‘1
c%blcbdo~~~~
!i
Mamadou Gueye” and Lucien M. Bordeleau*
Received 19 February 1988, accepted 21 March 1988
,),.
. . . . ; i‘;::..
::::
Introduction
‘,Y’>’ .. ,’ .
Bambara groundnut (Voundzeia subteerruneu (L.) Thouars) is a desirable legume
for Africa semi-arid trop54 agriculture. Like cowpeas, the bambara groundnut
nodulates freely in tropical soils. Although it produces a nutritious food, the bambara
groundnut remains one of the crops most neglected by science (Nationa! Academy of
Sciences, 1979).
The most important feature of legumes is their ability to fix atmospheric nitrogen
and considerable efforts have been expended towards increasing the eticiency of its 1
symbiotic association with Rhizobium. Now, it is accepted that seed inoculation with
:
superior Rhirobium strains is necessary with most legumes. However, cowpea seed
mocuiation is usually unsutmssful because of competition between introduced and W
indigenous Rhizobium strains in the soil. Therefore, an important step towards 1
improved nitrogen fixation is to isolate and identify competitive indigenous Rhizobia i
with a view to using ellte strains as inoculants (Mulongoy et’af., 1980).
/
This paper presents data ‘on screening field bambara groundnut varieties, mtrogen
fixing ability of indigenous Rhizobium strains and evaluation of interactions between
host plant and Rhizobium strain.
Materiafs and methods
Field variera1 screening.
Trials were done at two sites in Senegai during the 1985
rainy season. The sites differed in both rainfall (Bambey, 377 mm annum-‘;.Nioro,
532 mm annum-‘) and in soi1 type (Table 1). Twenty four cultivars of bambara
groundnut (listed’in Fig. 1) were hand sown in a randomized block design replicated
four times. The cultivars were sown in two treatments: without (-N) and with (+N)
nitrogen fertihzation (50 kg urea/ha). Al1 plots received 60 kg PzO.Jha and 120 kg
KWha. Six weeks after planting, the relative effectiveness (RE) of each host planti
indigenous Rhizobium strain combination was evaluated as follows (Mulongoy et al.,
1980):
’ MIRCEN-CNRA, B.P. 53, Bambey, Senegal.
’ Canada Agriculture, Research Statron, 2560 Hochelaga blvd, Sainte-Foy, Canada, GlV 2.U. 1
ôOxford Ilniversity Press 1988 }
!
.’
,”
.:.
.
. . &
.I
..&..
. .
<
Table P Lharacteristics
of soi1 at Bambey and Nioro experimental stations
mm
Composition (%)
____..
.
. . -
-_- .l.._l_-.- .---
Soi1
Total C
Total N
Availablc P
Clay
L.oam
S a n d
PH (HZ~)
(OIsen’)
---.-
Bambey
2910 290
1 2 1
4
2
96
7.x
_ Nioro
4160
'
310
137
b
3
90
5.8
‘Olsen ef al. (1954).
‘weight’ of ten samples in the -N block
RE =
x 100
‘weight’ of ten samples in the -t-N block
1
In this formula, ‘weight’ is either of shoot, root or nodule dry weight or shoot nitrogen
1
content.
I
Nitrogen fixing ability of indigenous Rhizobium strains.
The samples were taken in
each treatrnent from five well-developed plants and still having immature pods and 45
well-developed, pink nodules were sampled from the tap roots. Rhizobium strains
were isolated according to standard procedures (Vincent, 1970). Isolated rhizobia
were cultured on agar slants made from yeast extrac
altos medium. Nitrogen fixing
ability of the isolates were compared
q
in terms of s Ôot dry- weight of bambara
groundnut cultivar 85001 used as test plant and cultivated in Leonard jar assemblies
(Leonard, 1943). Seeds were sterilized using 0.01% (WV) HgC12 for 3 min and
germinate,d in sterile sand for 3 d; each seedling was then transplanted into a jar
assembly. One ml suspension of rhizobial isolate broth (approx. 109 cells) was
added aselptically to the seedlings for ail treatments except for an uninoculated control
and plants were grown in a greenhouse. Nitrogen-free IIe-itt solution (Hewitt, 1946)
diluted with water (1:3 vlv) was added once a week during the growing period. After
35,d growth, plant tops were clipped off, dried and weighed, for dry matter production.
Greenhouse evaluation of host plant-Rhizobium straiti interactions. Both the
indigenous and Niftal Rhizobium strains (Iisted in Table 3) were selected on the basis
of their effectiveness. The Niftal strains - TAL 22, TAL; 169 and TAL 569 - were
supplied by the NifIA!. project. Hawaii, USA and; served as standards for
comparison against the indigenous ones. Twelve bambara groundnut cultivars
also Iistedl in Table 3 were used as host plants. Seed :sterilization of each genotype,
germination, transplantation, inoculation and watering wére conducted as described
above an’d seeds were planted in Leonard jars. Treatments were arranged in a
randomized block design replicated five times. After 50 d$ )ol growth, the plants
were harvcsted. Plant tops were dried and weighed. Nodulation was scored taking
into account the number (N), the interna1 colour (C) and the size (S) of nodules.
The noduiation index (Nod. 1.) was calculated as follows:
Nod. 1. =NxCxS
Nodule number was rated on a scale from 0 (no nodule) to 3 (many nodules). Nodule
interna1 colour was from 0 (white) to 1 (red) and nodule size was from 1 (small
nodule) to 2 (big nodule).
Field varietal screening
At Bambey station the cultivais 83-127, Sud Cameroun, Sarakawa. 3, Sarakawa 10
and Lassa 1 had a relative effectiveness (RE), based on shoot dry weight, below 40%
0
(Fig. 1) while at Nioro station, all cultivars had thezame RE of over 40% (Fig. 2).
At Bambey,station, only cultivar Sarakawa 10 had ai RE, based on root dry weight,
,
below 50Y$at Nioro station, all cultivars had the same RE of over 50%. Relative
Effectiveness, based on nodule dry weight, was much lower at Bambey than at Nioro:
it was in the range of $0 lto 120% at Bambey for cultivars 83-131, Ketao 3, Alheride 1,
Lassa 1 and Lassa 4 and in the range of 360 to 480% at Nioro for cultivars 83-129,
83-131, Sud Cameroun, Sarakawa 2, Ketao 9, V2 and 85002. Relative Effectiveness,
based on shoot nitrog;en content, was over 50% for all cultivars except for
Awandjelo 1 at Bambey station.
These results indicated that Bambey and Nioro rhizobial population established a
moderately effective symbiosis with the cultivars of bambara groundnut used. Among
these cultivars, 12 grew better at both the locations without nitrogen fertilizer
(Table 2) indicating their ability to set an effective symbiosis in the two different field
conditions.
Table 2 Shoot dry weight (g/lO plants) of the perfonned cultivars of bambara groundnut in field
at Bambey and Nioro experimental stations
Bambara Cultivars
Bambey
Nioro --_-.- _..._.. -_. - ..__. ._
79-1
55.5b
b
l
83-126
62.2b
Zb
,
83-129
55.gb
39h
/
83-131
56Sb
30.2b
l
Sarakawa 1
52.8;
31.1b
Awancljelo 1
40.Sab
Ketao 2
63:5
31.7b
Ketao 3
66 2ab
32.9b
Alheride 1
68:Tb
43.9”
Lassa 3
78.5’
38 6ab
v2
61.5b
41:3Pb
85001
56.5b
42.0”
--
-
-~-
Values foltowed by the same letter in each column do not differ significantiy at the 0.05 level by
DUNCAN’s multiple range test (1955).
Effectiveness of indigenous Rhizobium strains isolated from bambara groundnut
nodules
Bambara groundnut cultivars sampled were moderately nodulated on both the tap
and lateral roots. Many nodules appeared to be effective as indicated by interior
colour. Thirty one isolates were SO obtained: (Ii to Ist, Fig. 3). The isolates were slow-
growing and formed small (diam. < 1 mm), dry colonies on yeast mannitol agar. Four
isolates (12, ii3, 122 and I& were lodged in West Africa MIRCEN (MAO) Rhizobium
culture collection because of their high effectiveness with cultivar 85001 which was
from Senegal and had good RE values at both Bambey and Nioro experimental
:
::;
1.
<.. :
.
. . ,f
i
.. .
.
.
.
:_ ,”
.
..Y. ;
.<.<. .
.:
:
.,
r’,
.,.; .b
:.:
;.
:.. ‘1.
;:
:‘-
a 7
..-
Y
-j
.
,..
.1
4 ,
v-4
.>...
.y”.,,>”
,
,.
*
,_
Nnokmv3ans
iN~Otl3k’WLl
L
2
0
01
1
1
2
VM’i’RWVS
OBMNVMV
VM’0WKi
VMM
VMvnvtlvs
L
VMkW’tiVS
VMVNAIVS
ovH3.uHl
MIMHlV
2 c
6
LtFism
Em
Pv5sv-l
OVl’dw ovm
OVlV?4
92L%a L2l-m
621-B OPE8
IEl-ca
looS8
2ms8
L-8L
L-ô.!
ClflS
w
:,.
:
,
statior . These isolates were named MAO 113, MAO 118, MAO 121 and MAO 126,
d
respef,tively, and are indicated by an asterisk in Fig. 3.
Evaludon of host cultivar-Rhizobium strains interactions
Rhizobium strains used were compared for their effectiveness to nodulate the most
promising cultivars of bambara groundnut selected in field varietal screening. Nodules
were found in the top 5 cm of root system on ah plants except controls which were ah
nodule free. Nodulation index and the effectiveness varied greatly with the strains
(Table 3). Strains MAO 113 and MAO 118 exhibited effective nodulation on six and
three cultivars respectively while strains TAL 22, TAL 169 and TAL 569 wera
effective on five, four and three cultivars respectively. Ah Rhizobium strains were
effective on cultivar 83-131 exceptstrain TAL 169 which exhibited partial nodulation.
Al1 Rhizobium strains were partially effective on cultivar Ketao 3. Similarly,
Rhizobium strains MAO 113 and MAO 118 were ineffective on cultivar V2 whila
strains TAL 22, TAL 169 and TAL 569 were partially effective on this cultivar. Strains
,I
:‘.1: ’ I-....
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 ineffective on cultivars :Ketao 2, Alheride 1 and Lassa 3,
Strain TAL 22 presented the widest effectiveness spectrum: it was ftrlly’effective on
42% and partially effective on 58% of ah cultivars studied, followed by strain MAO
113. These rësults indicated that plant genotype had an important effect on nodulation
of bambara gioundnut.
Although Corbin et al., (1977) had proposed a scbring system to classify nodulation
and Elordeleau et al., (1981) had reported a method for scoring nodulation, we
adopted another method. The reliability of this mefhod was indicated by the fact that
nodulation index and shoot dry weight (Table 3) showed the same trend of
LG
.‘,
:
effectiveness in host plant-Rhizobiurn strain combinations.
Discwision
f
Many studies have reported an improvement of biological nitrogen fixation resulting
.:_.
_’
from legume cultivar selection (Heichel, 1982; Minchin et al., 1978; Zary et al., 1978)
However, selection for increased nitrogen fixation (annot be achieved by selection of
the legume alone; both host genotype and rhizobia! strain influence nitrogen fixation
(Arrendell, et al., 1985; Hohenberg ei al., 1982; Islkib et al., 1980; Zary et al., 1978).
Most of the studies reported on plant genotype Rhizobium strain interactions
indica.ted differences between cultivars of the sami: species in ability to fix nitrogen
using introduced rhizobial strains. Because it is difficult to introduce and establish new
Rhizobium strains into fields containing indigenous strains (Brockwell, 1980), it is
important to isolate competitive and adapted strains, which cari nodulate effectively
most cultivars even strains are not widely distributed in the soi]. Instead of introducing
new Rhizobium strains, the isolated adapted Rhizobium strains may be re-introduced
into the field so that they Will compete against indigenous strains which are les$
numerous. Our results show that a11 Rhizobr’um strains including introduced
(NifI’AL) and indigenous strains nodulated ah cultivars. When the hosts and rhizobial
strains were arranged in an increasing order of effectiveness, one indigenous
Rhizobium MAO 113 exhibited the highest effective association with the cultiva&
M A 0 1 1 8 1
*
L
r
.
/
MAO 121 1
*
l
l
MAO 126
t*
.
l
Fig. 3 Shoot dry weight of’ Voundzeiusubtemuuu
cultivated in Léonard jar assembly and inoculated with 31
j’
rhizobial isolates. Treatm’ents with ??differ significantly with the control at the 0.05 level by DUNCAN’s i
multiple range test (1955).
i
Table 3 Shoot dry weight (SDW) @/plant) and nodulation index (Nod. 1.) of 12 bambara groundnut cultivars inoculated by cowpea
strains from West ,MIRCEN Africa (MAO) and NifTAL oroiect
i(
,- “. .
M;a;oTT5
_ .- - MA~ _ ..---
TGrïtrol--~
TAI,---
.- - - - TAI, 1 6 9
------ ----rAr -
Bambara
.-
---- -1--
Cultivars
SDW’
Nod. 1.’
SDW
Nod. 1.
SDW
Nod. 1.
SDW
Nod. 1.
SDW
Nod. 1.
SDW
Nod. 1.
-.
--1;41mr-..e. ---.
.~
79-l
0.9R
nn
211”
.-r-.------- ~.pï--E------~----- ‘~--
1,ga --- e
83-126
1.1’ nn
1.5”‘” E
1.6”” c
1.4&
e
1.8’ E
1P
e
83-129
1.0’ nn
1.6’*
E
l.PX
e
1.4t”
e
l.lbe
1
1.9” E
83-131
1.0” nn
1.6Rh
E
1.P
F
1.8”” E
1.5”h
c
1.8” E
Sarakawa 1
1.2” nn
1.6”h
E
1.6””
c
1.8’h
E
l.Sah
e
1 Yh
e
Awandjelo 1
0.9”
nn
1.1’ 1
1.2’”
1
l.Sahc
e
1.9”
E
1.P
e
Ketao 2
0.9” nn
1.6”” E
1 P
.‘
f
l.+
e
1.5”b
e
l.lh
1
Ketao 3
1.2” nn
1.F
e
l.Vhc’
c
1.4hc
e
l.Ssh
e
1.6’h
e
Alheride 1
1.2”
n
n
f.3”1”
e.
<
1.6”‘” e
1.8””
E
l 4& e
1.1”
i
Lassa 3
1 .O”
nn
1.7”
E
l.lh
1
1.9”” E
111”
e
1.1”
1
v2
1.0” nn
1.5ah 1
l.lh
1
1.2c
e
0.Y
e
1.P
85001
0.9a nn
1.4”hc
e
2.0” E
1.5”’
e
1.9’
E
1.8’
E
b
_
_
_-.--.
_.__
--.---
‘. ~&~s~~l&dby the same lctter in each column do not dlffer at thi 0.05 levcl by Duncan’s multiple range test (1955). a: highly effective; ab: partiaily
effective; b: incffective.
f
z nn: non-nodulated; E: highly effective nodlation (-
Z= 4); e: partialiy effective nodulation (2 4 Nod. 1. < 4); 1: ineffective nodulation (Nod. 1. < 2).
_~ .._..__... -.
_-. .-_-
_ -_
-
Therefore, strain MAO 113 is expected to give better nitrogen fixation under field
conditions.
The strategy to improve nitrogen fixation by legumes in general is to first improve
genotype and then select for best Rhizobium strains to increase nitrogen fixation.
Selection of host plants with good general effectiveness in nitrogen fixing ability is an
other avenue (Wynne (et al. 1980). The strategy most likely is to select in each field
condition the most effective nitrogen fixing host plant-Rhizobium strain combination.
In our study, the higher relative effectiveness obtained with cultivars at Nioro
experimental station might be due to better soi1 conditions (higher organic matter)
that are favourable to this plant (Tardieux, 1975). However, the determination of the
relative effectiveness of host plant$ndigenous Rhizobium strains combinations
permitted us to Select the best combinations in each area. Greenhouse evaluation of
these combinations is the initial phase of the process of increasing nitrogen fixation
through Rhizobium strains selection. The second phase was to investigate field
response of bambara g.roundnut to inoculation with the selected Rhizobium strains.
Effect of inoculation with strains MAO 113, MAO 118, TAL 22 and TAL 569 on the
grain yield of 79-1 and 83-131 was obtained in two Senegalese soi1 (Gueyeb%j@.
However, longterm efforts Will be required to breed for improving nitrogen fixation in
bambara groundnut.
Acknowledgements
This research was supported by grant no. BNF-SN-2-84-21 from the U.S. BOSTID-
National Academy of Sciences. The authors thank Alioune GNING and Omar
TOURE for valuable technical assistance.
References
ARRENDELL, S., WYNNE, J. C., ELKAN, G. H. and ISLEIB, T:G. 1985 Variation for nitrogen
fixation progenies of a Virginia x Spanish peanut cross.
25, 865-869.
BORDELEAU, L. M., GIRO~X, M., OUELLET, R. and ANTOUN, H. 1981 Effet du soufre et de
l’azote sur la fixation symbiotique d’azote chez les plantules de luzerne (Medicugo saliva L.).
Canadian Journal of Plant Science 61, 639-W.
BROCKWELL,
J. 1980 Experiments with trop and pasture legumes - Principles and practice. In
Methodes for evaluating biological nitrogen fîxation. Edited by Bergersen, F. J. pp. 417-490.
Chichester, New York, Brisbane, Toronto: John Wiley and Sons.
CALDWELL, B. E. 1966 Inheritance of a strain-specific ine-ffective nodulation in soybean. &op
Science 6, 427-428.
C~RBIN, E. J., BROCKWELL,
J. and GAULT, R. R. 1977 Nodulation studies on chickpea (Citer
arietinum). Australian Journal of Experimental
Agriculfural
Nusbandry 17, 124-134.
DUNCAN, D. B. 1955 Multiple range and multiple test. Biometrics 11, i-42.
GUEYE, M. 1987 Effect of inoculation with different Rhizobium strains on grain yield of
bambara groundnut (Voandzeia subterruneu (L.) Thouars) in two senegalese soils. Paper
presented at the symposium on the contribution of biological nitrogen fixation to plant
production. 3-7 August., 1987. Bogor, Indonesia.
HEICHEL, G. H. 1982 Breeding alfalfa for improved nitrogen fixation: a physiological
perspective. Iowa State Journal of Research 56, 255-280.
HE~, E. J. 1966 Sand and water culture methods used in the study of plant nutrition.
Technical communication No. 22 (2nd ed.) 547 pp. Commonwealth agricultural bureaux.
London.
HOHENDERC;,
J. S.. MUNNS, D. N. and TUCKER, C. L. 1982 RIiirobium-host
specihcttjes m
coccineus L. and Phuseolus vulgarlr L. Trop Science 22, 445-459.
Is~.faa, T. ci., WYNNE, J. C., ELKAN, G. H. and SCHNEEWEIS,
T. J. 1980 Quantitative genetics
aspects of nitrogen fixation in peanut (Arachis hypogueu L.). Peunur Science 7, 101-105.
LEONARD, L. 7’. 1943 A simple assembly for use in the testing of culture of rhizobia. Journalof
Racteriology 45, 523-527.
MINCHIN, F. R., SUMMERFNU, R. J. and EAGI.FSHAM, A. R. J. 1978 Plant genotype Rhirobium
strains interactions in cowpea Vignu unguiculuta (L.) Walp.). Tropical Agriculfure (Trinidad)
55, 107-115.
MuI.~N(;~Y, K., AYANABA, A. and PU.VER, E. 1980 Exploiting the diversity in the cowpea-
rhizohia symbiosis for increased cowpea production. In Proceedings of the Global Impact of
Applied Microhiology held at Lagos; Part 2. Rhizosphere biology. pp. 121-125. Academic
Press. London.
NATIONAI. ACA»EMY OF SCIENCES, 1979 Pulses. In Tropical Legumes: resources for the future.
pp. 47-53. National Academy of Sciences, Washington D.C.
OLSEN, S. R., C~LE, L. V., WATANABE, F. S. and DEAN, L. A. 1954 Estimation of available
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reduction. Crop Science 17, 783-787.
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Summary
.:. ,. : ,‘,
TO improve biological nitrogen fixation in bambara groundut, (Voandzeiu subterrunea (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 cuhivars were screened during this study. Both indigenous and introduced Niftal
Rhizobium strains were used during a second study on host cultivars X Rhizobium strains
interactions. Nodulation index and shoot dry weight were used to assess the efficiency. The
widest effectiveness spectrum was ohscrved with thr indigenous strain MAO 113 and the
introduced strain TAL 22.
Résumé
Fixarion d’azote dans la noix de Batnbara (Voandzeia subterrilna CL.)- Thouars)
Dans le but d’améliorer la fixation biologique d’azote dans la noix de Bambara (Voundzeiu
subterranea (L.) Thouars), l’efficacité relative de 24 cultivars a été étudiée sur le terrain dans
deux stations exp&imentales
choisies pour leurs conditions écologiques différentes. Trente-et-
une souches de Rhizobium ont été isolées et 12 cultivnrs ont été testées au cours de cette étude.
Tant les souches indigènes de Rhizobium que celles Niftal introduites ont été utilisCes au cours
d’une deuxième 6tude sur les interractions
entre les souches de Rhizobium et les cultivars-
hôtes. L’indice de nodulation et le poids sec de la pousse ont été utilisées pour l’établissement
de I’efficience. Le spectre d’efficience le plus large a été observé avec la souche indigène MAO
113 et la souche introduite TAI. 22.
Resumen
Afin de meiorar la hiaci6n biohkica de nitrr5eeno en el cacahuete de Bambara (Voundzeia
\\
sub~erranea*(L.)
Thouars), se reakr un estu&o de su efectividad relativa en campo en dos
estaciones experimentales seleccionadas por sus distintas condiciones ecol6gicas. Durante este
estudio se aislaron treintaiuna cepas de Rhizobium y se probaron doce cultivares del huésped.
En un segundo ensayo se estudiaron las interacciones huesped xcepa de Rhizobium para 10 cual
se utilizaron cepas de Rhizobium Niftal tanto indigenas como introducidas. La eficiencia se
valor determinando el indice de nodulacion y el peso seco de la parte aerea. El espectro mas
amplio de etkacia se albserv6 con la cepa indfgena MAO 113 y la introducida TAL 22.