MINISTEmDE LA RECEiERCHE NATIONAL ACADW OF ...
MINISTEmDE LA RECEiERCHE
NATIONAL ACADW OF SC!IENCES
SCIBNTIFIQUE ET TEZHNIQUE
BOSTID
""-WV
USA
INSTITUT SENEGALAIS DE
s.-m----*-
RECRERCBES AGRICO~S
-aD-w-
DEP- DES REw1m
SUR LES PBODUCTIONS VEGET~ALES
-.N--
IMPROVING THE YIELD AND
BIOLOGXCAL NITROGEN FIXATION OF
BAMBARA GROUNDNUT
Ref. CRG GRANT No. BNF-SN-2-84-21
AMI & PROGRESS REPORT
Principal Investigator
Mamadou GUEYB
Soi1 microbiologist
Octobre 1985
Centre National de Rechekhes Agronomiques de
1.0GENEZ&LBACKGlXXJND
2-OBJECTIVES
3 - MATElUiLS AN-D METHODS
3.1 - Field screening bambara groundnnt cultivars
3.1,1 - Screening cultivars - native Rhizobium symbiosis
3.1.2 - Rhizobium isolation from bambara groundnut nodules
3.2 - Assessment of nitrogen fixation
3.2.1 - Experhnent in sterile soi1
3.2.2 - Exp@riment in non sterile soi1
4 - RESULTS
4.1 - F'.eld experimwt
4.1.1 - Relative Effectiveness
4.1.2 - Rhizobitm strains isolation
4.1.3 - Disease incidence
4.2 - Pot experimant
5 - ONGOINGREWARCH
5.1 - ScreenFng bambara groundnut cultivars
5.2 - Assessment of nitrogen fixation by bambara groundnut
6 - COLL&BORATIoN AM(XG INSTITUTES
7 - S-Y
Appendice 1
Appendlc? II
1 - GENI?AL BACKGROUI~
In the developping countries, specially at the semi arid tropics in
Africa, the food availability and quality is a social and political problem. It is
very important for those countries to develop the research on the nutritional
crops which are the resources for the future. On this point of view, Voandzeia
subterranea, also known as Bambara groundnut in Mali, Gajanga (1) in Senegal is
a materiel of choice.,
In 1983, Senegal Institute of Agricultural Research (SSRA) had submitted
a proposa1 to National Academy of Scient s (NAS) for studying the Biological Nitro-s
gen Fixation by bambara groundnut (Voandzeia subterranea) in the framework of
West Lfrica MIRCEN. I:n December 1983, Dr Michael DOW had a long discussion with
the principal investigator and his collaborators. The discussion was about the
objectives and methodology of the grant.
In May, 1984, the NAS approved to ISRA a two-year grant to carry out
a research project entitled "lmproving the YLeld and Biological Nitrogen Fixation
of Bambara groundnut.
2 - 0EJEX!T1vEs
The main objective of the project is to improve the yield of bambara
groundnut through the nitrogen fixation. Because it is an underexploited legume,
it is necessary to constitute first an important germplasm of bambara groundnut
and a collection of effective Rhizobium strains. This is why the objectives of
the first phase of this project are :
i- Obtain promising bambara groundnut germplasm in local markets and
from international collections.
2- Determine responsiveness and Select pure strains based on phenotype
and yield.
3- Screen rhizobial strains, including some isolated locally and some
obtained from international collections,
4- Observe possible diseases on bambara groundnut.
(1) Gajanga /-gadjangas-/ : Name of bambara groundnut in rxinciaml na+~-n=.~
3
3 - MATERIAIS LND METHODS
-..
3.1 - Field screening bambara groundnut cultivars :
3.1,1 - srecning cultivars - Rhizobium spiosis :
Fiold experiments were carried out in differont experimental stations
in Senegal. Different bambara groundnut cultivars were hand sown in 7.5 x 1.5m
plots arranged randomly. rlhe cultivars were sown in two treatments : without
(-NI and with (i-N) nitrogen fertilization (ca, 50kg urea - N&a). Al.1 plots
received phosphorus and potassium fertilizat~on (ca, 60kg P205/ha and 12Okg KCl/ha
respectively). Six weeks aft ,r planting, the Relative Effectiveness (R.E.) of
each host plant - indigenous Rhizobium strains combination was evaluated by the
following formula :
X of ten samples in the -iN bloc
R.E :=
x 100
X of ten saplcs in the + N bloc
In this formula X points out shoot, root and nodule dry weights, speci-
fit N2- asc activity expressed as acetylene reduction activity (ARA) or nitro-
gen content (R%I.
3.1.2 - Rhizobium isolation from bambara groundnut nodules :
Nodules were selected from each plot indicated above. Nodule surface
should thcn be stcrilized by first rinsing in 95% ethanol and than soaking in
0.1% H(fl~< and rinsing in stcrile water to rernove residual sterilant. Aseptical-
ly, each nodule was cutted in half and a portion of the bacterial tissu was remo-
ved with a sterile needle, and streaked for isolation on yC!:st mannitol agar
plates. Plates were incubated at 2Vi. until colonies were evident.
3.2 - Assessment of nitrogen fixation :
Pot experiment was carried out for assessing the nitrogen fixation by
two bambara groundnut oultivars using 15N methods. Surface sterilized seeds were
germinated in sterile sand for 3 days. Gorminated seed.s were then transplanted
into a pot containing 7kg of autoclaved soi.1 for the f,irst part, and into a pot
containing 7kg of non sterile soi1 for the second part.
4
3.2.1 - Experiment in sterilc soi.1 :
There are fivc treatmants in sterilo soi1 :
- 3ambara groundnut non inoculated witn Rhizobium (R) or mycorrhize
04) 0 Ammonium sulfate CNN, 5% atom excess) Will be supplied at 20 kg/ha :
O20 treatment.
- Rambara groundnut non inoculated with R or H. Ammonium sulfate
(15N, 1% atom excess) Will be supplied at 100 kg/ha : OIW treatment.
- Bambaua groundnut inoc:lated with I&izobium only. Ammonium sulfate
(15N, 5% atom excess) Will be supplied at 20 kg/ha : R2* treatment,,
- Bambara groundnut inoculated with mycorhize only. Ammonium sulfate
(l5N, 5% atom excess) Will be supplied at 20 kg/ha : M2* tieatment.
- Bambara groundnut inoculated with Rhizobium and mycorrhize, Ammonium
sulfate (15N, 5% atom excess) wilrh be supplied at 20 kg/ha : R&IZ0
treatment.
3.2-2 - Experiment in non sterile soi.1 :
There are the same treatments as above and four another treatments which
are :
- Non nodulating groundnut (Aracbid hypogaea) cultivar non inoculated
with i?hizobir.un or mycorhize. Lmmonium sul.fate (l"N, 5% atom excecs)
Will be supplied at 20 kg/ha : A2* traatment.
- Ncm Roduloting Asachis hypogaea cultivar non inoculated with Rhizo-
bium or mycorrhize, Ammonium sulfate (15N, 1% atom excess) will be
supplied at 100 kg/ha : AIOO trearmrnt.
- Non nodulating soybean cultivar non inoculated with Rhlzobium or
mycorrhize, Ammonium sulfate (15N, 5% atom excess) will be supplied
at 20 kg/ha : S20 treatment,
- Non nodulating soybean cultivar non inoculated with Rhizobium or
mycorrhize. Ammonium sulfate (l5N, 1% atom excess) Will be supplieàl
at 100 kg/ha : S loo treatment.
5
For inoculating the seedlings with Xhizobium, each seedling was inocula-
ted with Iml of lihizobium culture strain TAL 1000 containing 10' Cells per ml.
For inoculating the seedlings with mycorrhize each seedling was inoculated with
IcXnl of a suspension of endomycorrhize, strain Glomus mosseae. The Glomus mosseae
inoculum was prepared by blending ca 1CQg (fresh weight) of infected roots of non
nodulated cowpca, hyphae and spores in I liter of water.
4.1 - Field experiment :
The first field screening experiment carried out from January to Eiarch,
1985 included only eight cultivars obtained from S .uth 'of Cameroun (cv. S.C.)
Mali (cv. 83-126 ; 83-127 ; 83-129 ; 83-133 ; 83-131) and Senegal (cv. 78-1, '79-I).
4.1.1 - Relative effectiveness :
Six wceks after planting, plants wcrc pulled up. Shoot and nodule dry
weights, i;:2 nase activity expressed as acctylene reduction activity (ARA) and ni-
trogen content are presented in Tables 1 and 2.
Rclitive effectiveness of the eight bambara groundnut cultivars is shown
in Fig. 1. Cultivars 78-I and 83-130 grew better than the other cultivars whica had
a similar growth (Fig. IA). A11 cultivars nodulated moderately with indigenous
rhizobial population in the -N and +H blocs (fig. IB). Nodulation was observed on
both principal and lateral roots. Nodules had the typica:t beige colour but a low
percentage of them (ca 2%) had purple colour, Eaglesham et al. (1982) and Gueye
em-
(1983) made similar observations on Signa unguiculata. Although Fig. IB shows that
nodule dry weight of: cv. 83-126 and South Cameroun (S.C.) was higher, the NL-ase
activity expressed as specific ARA was highcr for cv. 83-129, 83-131 and S.C.
Fig. lC,. Howevcr there no difference in relative effectiveness based on the shoot
nitrogen content (Fig, ID) and root nitrogen content (Fig. IE) of the eight culki-
vars, Cultivars 83-131 and S.C. had also thc higher seed yield (Fig, ID).
Table 1.
Shoot and nodule dry weights, specific AR.&, nitrogen content and disease incidence of e$ght
cultivars of barnbara groundnut cultivated in field in the -N bloc.
Shoot and root dry weights
Kodule dry
Specific :]LRF Nitrogen content (R%)
Disease inci-
(g/lO plan%3)
weight
(Umole C,H4/
dence*
mg/lOplants)
h/mgI '-
Shoot
Fttxe-
(%)
-... .
.
.-_
_
-. .
cu1tivars
Shoot
Root
Whole plant
-.
76-l
73.70 a
1.82 a
75.52
114 a
340.45 a
2.67 a
1.37 a
10*15 a
7n
tz3-i
45.37 b
1.75 a
47.12
97 a
315.79 a
2.82 a
1.32 a
II.32 a
83-126
90.25 c
2.37 a
92 -62
214 b
127.11 b
2.75 a
1.05 a
4.15 a
83-127
70.12 a
1.75 a
71.87
309 c
73.43 b
2.90 a
1.70 a
12.80 a
83-129
70.12 a
2.32 a
72.44
92 a
1500.87 c
2.78 a
1.18 a
13.15 a
83-130
101.85 c
2.20 a
104.05
200b
246.99 d
2.73 a
0.93 a
10.80 a
83-131
77.55 a
2.45 a
80.00
230 0
169.55 b
2.76 a
1.66 a
7.10 a
S.C.
59.32 ab
1.62 a
60.95
155 2
420.55 a
2.67 a
1.37 a
6.22 a
Values followed by the some letter in each column do not differ significantly (P = O,Ol),
UXJNCAN, 1985).
9 Simple Variante Test
Table 2,
Shoot and nodule dry weic, ts, specific AFW, nitrogen content and disease incidence of eight
cultivars of bambara groundnut cultivated in field in the +N bloc,
Shoot and root dry weights
Nodule dry
Specific ARA
Nitrogen Content (N%)
(g/lO plants)
weight
(ii. mole C2H4/
Biscase inci-
(mg/lO.plants)
h/mg)
Shoot
Root
dence *
Cultivars
Shoot
Root
Wholc plants
(%)
-II
--
78-l
38.60 a
1.42 a
40.02
104 a
755.66 a
2.75 a
1.55 a
7.88 a
79-l
36.12 i:
1.70 a
38.52
286 b
247.37 b
2.96 a
1.46 a
5.80 a
83-126
57.95 b
2.05 a
60.02
160 a
332.85 c
2.81 a
1.11 a
5.40 a
83-127
43.40 a
1.95 a
45.20
290 b
182.44 d
2,88 a
1.78 a
9.30 a
83-129
47.62 ab
2.07 a
49.67
427 c
612.61 a
3.02 iî.
1.42 a
5.90 a
83-130
48.02 ab
2.00 a
49.45
270 b
371.58c
2.7~1 a
0.94 a
2,# a
83-131
46.87 ab
1.97 a
39.00
1975 d
80.12 e
2.82 a
1.52 a
6.40 a
S.C.,
38.10 a
1.3s a
39.45
136 a
294.48 bC
2.88 a
1 .4ô a
9.22 a
Vaiues followed by the same letter in each column do not differ significantly (P = 0101)
+ Simple Variante Test
6
4.1.2 - RhizoBium strains isolati& :
- -
One Rhizobium strain was isolated from each plot. 16 strains were ob-
tained and teszd by plant infection test using ~~acxotyloma africacum. These
strains were stored at - 80°C in giycerol in West Africa MIRCEN Rhizobium co.llec-
tion. The more effective and more competitive strains ,will then be used in ficld
triais. Later, duplicates Will be send to one of our collaborators for furthor
characterisations and other tests.
4.1.3 0~ Disease incidence :
.-
During the experim?nt, a disease was observed on collar plants. Prelimi-
nary microscopic observations showed that this dis?ase is due to a fungus which
should be .E'usarium monilifonne, However, therc is no difference in disease inci-
dence of the cight cultivars in each treatment (Tables 1 and 2) e
4.2 -- Pot axperimenr& :
Wa did not get results on assessment of nitrogen fixation by bambara
15
groundnut using
N methods. Plants died alter 30 days of growth because of disea-
se described as above.
5 - Oti GOIIG RESEGRCH
5.1 - Screening bambara groundnut cuitivars :
Ziftur many investigations arround Senegal and requests of some research
institutes like University of Maryland Eastcrn Shore (t3!4ESII we got 25 cultivms
of bambara groundnut including the eight cultivars described above.Thcse cultivars
are from Cameroun. (11, Mali (5), Senegal (41, and Togo 1115). During the raining
saison in Senegal (from July to October 19R5), we ciurled out a field screening
of these 25 cultivars using the Relative Effcctuveness method, Results Will be
available in January 19816.
With UMES collaboration, we Will get 75 cultivars of Bambara groundnut
at October, 1986. From February to May, 1986 we Will carry out a third field
screening of the a11 cultivars constitutiag our bambara groundnut gcrmplnsm. We
Will Select the three best cultivars for further mm-mrimm+~~
7
5 . 2 - Assessment (of nîtrogen fixation by bambara groundnut :
Pot experiment Will be carrîed out again from November to December, 1985,
Nîtrogen content of shoots and roots Will be determîned at ORSTOM soi1 microbîo-
logy lajboratory in Dakar. This laboratory îs well equiped in mass spectrometer.
Results Will be available in April, 1986.
Our grant is conducted in the framework of the West Afrîca P3IRCEX. The
first collaboration is done in this regîonal infrastructure for getting culti.vars
of bambara groundnut from our collaborators in SJest Afrîcao
~he second collaboration is with OR3iZQN laboratory in Dakar. This labo-
"
ratory is well equiped for a11 types of microbiologîcal manipulations,
We havc- also a thrîd collaboration with the Clnîversity Of biaryland Eastern
Shore (UMJW 0 ~!e havc to carry out together bambara graundnut inoculation trials.
Results of thc 1384 triais are available. 1985 ones Will be avaîlable at December,
1986.
7 - SUMMiiY
The grant was awarded to Bambey National Agricultural Research Center
of the Senegal Instîtute Agricultural Research to study the improvement of yield
and Biological Nîtrogczn Fixation of Bambara groundnut (Voandzeia subterranea) I) The
objectives of the first phase of thîs grant werc :
l/ *" Obtaîn promîsîng bambara groundnut gormplasme in local markets and
from international collections,
2/ - Detcrmî~?e responsîveness and sclcct pure! strains based on phenoltype
and yield.
31 - Scr^cn rhizobial strains, includiq some isolated locally and some
obtained from internat'o la1 collections,
4/ - Observe possible diseases on bambarn groundnut.
Requcsts for getting bambara groundnut germplasm were sent to Drs, Ravoh
Itrarîvo (Madagascar Island) Ethnar and Mergaai (Togo) , i)akora (Ghana) and Hidayat
(Indonesîa). j?&q-ua"Jlv WQ ham-3 4 m n-.-L.^-- --~~ '
--
8
Mali, 1 from Cameroun and 4 from Sencgal, The problun for constituting a bambara
groLnd..iut germplasm i s that there is a very little amount seed of diffcront cul-
tivars in old collections as in Togo, Ghana, and Madagascar, bocal multiplication
of these cultivars is oftcn necessary. This is the case of Dr. Mtrjeai's collection
which will send to us 75 cultivars at the end of 1985 raining season (with the
help of the Univaxsity of Maryland Eastern Ghorc 1. Ca. Ravoh Itrarivo (Madagascar}
also Will send to us many cultivars upon an. irriz,ort licence 1 supplied.
Nevcrthelcss we carried out during 1935 dry season onefield cxperimcnt
based on scrcaning bambara groundnut - Nhizobium symbiosis. The results of this
experiment
are presented at 1985 E%?+S mleeting. During this experiment
including eight cultivars a disease was obscrved on some plants which well grew
during on month. Thus the leaves withered as a vater stress, then plants died.
Preliminary observations under a dissecting microscope showed that is probably
a fungal diseaso causdd bly Fusarium sp. Studies on diseases ob bambara groundnut
are reeded.
A similar Gcx,pcrimcnt including 25 cultivars is now carrying out during
the raining season.
A seccnd expcriment about the assessment of N2 fixation by two cultivars
of bambara groundnut u!Sing 151$ methods was startcd in .April, 1985. Howcver this
experinent was not finLshcd because of plant discase mentioned above.
Plan for the next pcriod :
II-
On goirq and future research activitics invo:Lve continuation of coï-lec-
tion and field screening of harnbara groundnut cultivars, sutdy the effectircness
and competitivcnass of a11
Hhizobium collected, the assessment of 1J2 fixation
using 15N mcthods, Thon, after, a new proposa1 Will be s&mitted to E\\sES for the
second phase of thc grant.
APPEEJDICE 1
Equi~ent received (up to October, 1985)
-
Incubator
Autostill
pH meter
Shaker
Centrifuge
Freezer
Refrigerator
Glass ware
10
APPENDICE II
1 - TRXNING AND CONSULTAï4TS'VISIT
Programme teaching is not include in the grant. But a consultant visit
will be held during the second year of the project carrying out. Consultant Will
Dr. P. SOMASEGARAD from NIFTAL project in Bawaii or Dr, W. KEYSER from Beltsville
university.
2 - ME~Z’INGS ATTENDED
We have at,tended at BOSTID - NAS meeting only. During the first meeting
at Chiang Mai in Thailand,
we presented our project. During the second o:ne
in Bawaii,
We.
presented the first results of fileld screening of bambara
groundnut cultivars.
3 - PUBLICATIONS
Three publications Will be submitted to MIRCEN Journal of Applicd Micro-
'biology and Biotechnoiogy.
- Assessment of N2 fixation by bambara groundnut using 15N method SO
- Field scrcening of bambara groundnut cultivars
- Bambara groundnut inoculation trials (in collaboration with the Univer-
sity of MnryLand Eastern Shore) m
CAPTION FOR FIGUrnS
Fig. 1 : Re:a...ive Effectiveness (R.E.1 of eight bambara groundnut cultivars
1n :
LE, based on plant dry weight
1B :
R.E. based on nodule dry wciyht
1c :
R-E, based on specific ARA
1D :
R.E. based on shoot id%
1E :
R.E. based out root NB
1E :
R,E. based on seed yield
f
7
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NATIONAL ACADW OF SC!IENCES
SCIBNTIFIQUE ET TEZHNIQUE
BOSTID
""-WV
USA
INSTITUT SENEGALAIS DE
s.-m----*-
RECRERCBES AGRICO~S
-aD-w-
DEP- DES REw1m
SUR LES PBODUCTIONS VEGET~ALES
-.N--
IMPROVING THE YIELD AND
BIOLOGXCAL NITROGEN FIXATION OF
BAMBARA GROUNDNUT
Ref. CRG GRANT No. BNF-SN-2-84-21
AMI & PROGRESS REPORT
Principal Investigator
Mamadou GUEYB
Soi1 microbiologist
Octobre 1985
Centre National de Rechekhes Agronomiques de
1.0GENEZ&LBACKGlXXJND
2-OBJECTIVES
3 - MATElUiLS AN-D METHODS
3.1 - Field screening bambara groundnnt cultivars
3.1,1 - Screening cultivars - native Rhizobium symbiosis
3.1.2 - Rhizobium isolation from bambara groundnut nodules
3.2 - Assessment of nitrogen fixation
3.2.1 - Experhnent in sterile soi1
3.2.2 - Exp@riment in non sterile soi1
4 - RESULTS
4.1 - F'.eld experimwt
4.1.1 - Relative Effectiveness
4.1.2 - Rhizobitm strains isolation
4.1.3 - Disease incidence
4.2 - Pot experimant
5 - ONGOINGREWARCH
5.1 - ScreenFng bambara groundnut cultivars
5.2 - Assessment of nitrogen fixation by bambara groundnut
6 - COLL&BORATIoN AM(XG INSTITUTES
7 - S-Y
Appendice 1
Appendlc? II
1 - GENI?AL BACKGROUI~
In the developping countries, specially at the semi arid tropics in
Africa, the food availability and quality is a social and political problem. It is
very important for those countries to develop the research on the nutritional
crops which are the resources for the future. On this point of view, Voandzeia
subterranea, also known as Bambara groundnut in Mali, Gajanga (1) in Senegal is
a materiel of choice.,
In 1983, Senegal Institute of Agricultural Research (SSRA) had submitted
a proposa1 to National Academy of Scient s (NAS) for studying the Biological Nitro-s
gen Fixation by bambara groundnut (Voandzeia subterranea) in the framework of
West Lfrica MIRCEN. I:n December 1983, Dr Michael DOW had a long discussion with
the principal investigator and his collaborators. The discussion was about the
objectives and methodology of the grant.
In May, 1984, the NAS approved to ISRA a two-year grant to carry out
a research project entitled "lmproving the YLeld and Biological Nitrogen Fixation
of Bambara groundnut.
2 - 0EJEX!T1vEs
The main objective of the project is to improve the yield of bambara
groundnut through the nitrogen fixation. Because it is an underexploited legume,
it is necessary to constitute first an important germplasm of bambara groundnut
and a collection of effective Rhizobium strains. This is why the objectives of
the first phase of this project are :
i- Obtain promising bambara groundnut germplasm in local markets and
from international collections.
2- Determine responsiveness and Select pure strains based on phenotype
and yield.
3- Screen rhizobial strains, including some isolated locally and some
obtained from international collections,
4- Observe possible diseases on bambara groundnut.
(1) Gajanga /-gadjangas-/ : Name of bambara groundnut in rxinciaml na+~-n=.~
3
3 - MATERIAIS LND METHODS
-..
3.1 - Field screening bambara groundnut cultivars :
3.1,1 - srecning cultivars - Rhizobium spiosis :
Fiold experiments were carried out in differont experimental stations
in Senegal. Different bambara groundnut cultivars were hand sown in 7.5 x 1.5m
plots arranged randomly. rlhe cultivars were sown in two treatments : without
(-NI and with (i-N) nitrogen fertilization (ca, 50kg urea - N&a). Al.1 plots
received phosphorus and potassium fertilizat~on (ca, 60kg P205/ha and 12Okg KCl/ha
respectively). Six weeks aft ,r planting, the Relative Effectiveness (R.E.) of
each host plant - indigenous Rhizobium strains combination was evaluated by the
following formula :
X of ten samples in the -iN bloc
R.E :=
x 100
X of ten saplcs in the + N bloc
In this formula X points out shoot, root and nodule dry weights, speci-
fit N2- asc activity expressed as acetylene reduction activity (ARA) or nitro-
gen content (R%I.
3.1.2 - Rhizobium isolation from bambara groundnut nodules :
Nodules were selected from each plot indicated above. Nodule surface
should thcn be stcrilized by first rinsing in 95% ethanol and than soaking in
0.1% H(fl~< and rinsing in stcrile water to rernove residual sterilant. Aseptical-
ly, each nodule was cutted in half and a portion of the bacterial tissu was remo-
ved with a sterile needle, and streaked for isolation on yC!:st mannitol agar
plates. Plates were incubated at 2Vi. until colonies were evident.
3.2 - Assessment of nitrogen fixation :
Pot experiment was carried out for assessing the nitrogen fixation by
two bambara groundnut oultivars using 15N methods. Surface sterilized seeds were
germinated in sterile sand for 3 days. Gorminated seed.s were then transplanted
into a pot containing 7kg of autoclaved soi.1 for the f,irst part, and into a pot
containing 7kg of non sterile soi1 for the second part.
4
3.2.1 - Experiment in sterilc soi.1 :
There are fivc treatmants in sterilo soi1 :
- 3ambara groundnut non inoculated witn Rhizobium (R) or mycorrhize
04) 0 Ammonium sulfate CNN, 5% atom excess) Will be supplied at 20 kg/ha :
O20 treatment.
- Rambara groundnut non inoculated with R or H. Ammonium sulfate
(15N, 1% atom excess) Will be supplied at 100 kg/ha : OIW treatment.
- Bambaua groundnut inoc:lated with I&izobium only. Ammonium sulfate
(15N, 5% atom excess) Will be supplied at 20 kg/ha : R2* treatment,,
- Bambara groundnut inoculated with mycorhize only. Ammonium sulfate
(l5N, 5% atom excess) Will be supplied at 20 kg/ha : M2* tieatment.
- Bambara groundnut inoculated with Rhizobium and mycorrhize, Ammonium
sulfate (15N, 5% atom excess) wilrh be supplied at 20 kg/ha : R&IZ0
treatment.
3.2-2 - Experiment in non sterile soi.1 :
There are the same treatments as above and four another treatments which
are :
- Non nodulating groundnut (Aracbid hypogaea) cultivar non inoculated
with i?hizobir.un or mycorhize. Lmmonium sul.fate (l"N, 5% atom excecs)
Will be supplied at 20 kg/ha : A2* traatment.
- Ncm Roduloting Asachis hypogaea cultivar non inoculated with Rhizo-
bium or mycorrhize, Ammonium sulfate (15N, 1% atom excess) will be
supplied at 100 kg/ha : AIOO trearmrnt.
- Non nodulating soybean cultivar non inoculated with Rhlzobium or
mycorrhize, Ammonium sulfate (15N, 5% atom excess) will be supplied
at 20 kg/ha : S20 treatment,
- Non nodulating soybean cultivar non inoculated with Rhizobium or
mycorrhize. Ammonium sulfate (l5N, 1% atom excess) Will be supplieàl
at 100 kg/ha : S loo treatment.
5
For inoculating the seedlings with Xhizobium, each seedling was inocula-
ted with Iml of lihizobium culture strain TAL 1000 containing 10' Cells per ml.
For inoculating the seedlings with mycorrhize each seedling was inoculated with
IcXnl of a suspension of endomycorrhize, strain Glomus mosseae. The Glomus mosseae
inoculum was prepared by blending ca 1CQg (fresh weight) of infected roots of non
nodulated cowpca, hyphae and spores in I liter of water.
4.1 - Field experiment :
The first field screening experiment carried out from January to Eiarch,
1985 included only eight cultivars obtained from S .uth 'of Cameroun (cv. S.C.)
Mali (cv. 83-126 ; 83-127 ; 83-129 ; 83-133 ; 83-131) and Senegal (cv. 78-1, '79-I).
4.1.1 - Relative effectiveness :
Six wceks after planting, plants wcrc pulled up. Shoot and nodule dry
weights, i;:2 nase activity expressed as acctylene reduction activity (ARA) and ni-
trogen content are presented in Tables 1 and 2.
Rclitive effectiveness of the eight bambara groundnut cultivars is shown
in Fig. 1. Cultivars 78-I and 83-130 grew better than the other cultivars whica had
a similar growth (Fig. IA). A11 cultivars nodulated moderately with indigenous
rhizobial population in the -N and +H blocs (fig. IB). Nodulation was observed on
both principal and lateral roots. Nodules had the typica:t beige colour but a low
percentage of them (ca 2%) had purple colour, Eaglesham et al. (1982) and Gueye
em-
(1983) made similar observations on Signa unguiculata. Although Fig. IB shows that
nodule dry weight of: cv. 83-126 and South Cameroun (S.C.) was higher, the NL-ase
activity expressed as specific ARA was highcr for cv. 83-129, 83-131 and S.C.
Fig. lC,. Howevcr there no difference in relative effectiveness based on the shoot
nitrogen content (Fig, ID) and root nitrogen content (Fig. IE) of the eight culki-
vars, Cultivars 83-131 and S.C. had also thc higher seed yield (Fig, ID).
Table 1.
Shoot and nodule dry weights, specific AR.&, nitrogen content and disease incidence of e$ght
cultivars of barnbara groundnut cultivated in field in the -N bloc.
Shoot and root dry weights
Kodule dry
Specific :]LRF Nitrogen content (R%)
Disease inci-
(g/lO plan%3)
weight
(Umole C,H4/
dence*
mg/lOplants)
h/mgI '-
Shoot
Fttxe-
(%)
-... .
.
.-_
_
-. .
cu1tivars
Shoot
Root
Whole plant
-.
76-l
73.70 a
1.82 a
75.52
114 a
340.45 a
2.67 a
1.37 a
10*15 a
7n
tz3-i
45.37 b
1.75 a
47.12
97 a
315.79 a
2.82 a
1.32 a
II.32 a
83-126
90.25 c
2.37 a
92 -62
214 b
127.11 b
2.75 a
1.05 a
4.15 a
83-127
70.12 a
1.75 a
71.87
309 c
73.43 b
2.90 a
1.70 a
12.80 a
83-129
70.12 a
2.32 a
72.44
92 a
1500.87 c
2.78 a
1.18 a
13.15 a
83-130
101.85 c
2.20 a
104.05
200b
246.99 d
2.73 a
0.93 a
10.80 a
83-131
77.55 a
2.45 a
80.00
230 0
169.55 b
2.76 a
1.66 a
7.10 a
S.C.
59.32 ab
1.62 a
60.95
155 2
420.55 a
2.67 a
1.37 a
6.22 a
Values followed by the some letter in each column do not differ significantly (P = O,Ol),
UXJNCAN, 1985).
9 Simple Variante Test
Table 2,
Shoot and nodule dry weic, ts, specific AFW, nitrogen content and disease incidence of eight
cultivars of bambara groundnut cultivated in field in the +N bloc,
Shoot and root dry weights
Nodule dry
Specific ARA
Nitrogen Content (N%)
(g/lO plants)
weight
(ii. mole C2H4/
Biscase inci-
(mg/lO.plants)
h/mg)
Shoot
Root
dence *
Cultivars
Shoot
Root
Wholc plants
(%)
-II
--
78-l
38.60 a
1.42 a
40.02
104 a
755.66 a
2.75 a
1.55 a
7.88 a
79-l
36.12 i:
1.70 a
38.52
286 b
247.37 b
2.96 a
1.46 a
5.80 a
83-126
57.95 b
2.05 a
60.02
160 a
332.85 c
2.81 a
1.11 a
5.40 a
83-127
43.40 a
1.95 a
45.20
290 b
182.44 d
2,88 a
1.78 a
9.30 a
83-129
47.62 ab
2.07 a
49.67
427 c
612.61 a
3.02 iî.
1.42 a
5.90 a
83-130
48.02 ab
2.00 a
49.45
270 b
371.58c
2.7~1 a
0.94 a
2,# a
83-131
46.87 ab
1.97 a
39.00
1975 d
80.12 e
2.82 a
1.52 a
6.40 a
S.C.,
38.10 a
1.3s a
39.45
136 a
294.48 bC
2.88 a
1 .4ô a
9.22 a
Vaiues followed by the same letter in each column do not differ significantly (P = 0101)
+ Simple Variante Test
6
4.1.2 - RhizoBium strains isolati& :
- -
One Rhizobium strain was isolated from each plot. 16 strains were ob-
tained and teszd by plant infection test using ~~acxotyloma africacum. These
strains were stored at - 80°C in giycerol in West Africa MIRCEN Rhizobium co.llec-
tion. The more effective and more competitive strains ,will then be used in ficld
triais. Later, duplicates Will be send to one of our collaborators for furthor
characterisations and other tests.
4.1.3 0~ Disease incidence :
.-
During the experim?nt, a disease was observed on collar plants. Prelimi-
nary microscopic observations showed that this dis?ase is due to a fungus which
should be .E'usarium monilifonne, However, therc is no difference in disease inci-
dence of the cight cultivars in each treatment (Tables 1 and 2) e
4.2 -- Pot axperimenr& :
Wa did not get results on assessment of nitrogen fixation by bambara
15
groundnut using
N methods. Plants died alter 30 days of growth because of disea-
se described as above.
5 - Oti GOIIG RESEGRCH
5.1 - Screening bambara groundnut cuitivars :
Ziftur many investigations arround Senegal and requests of some research
institutes like University of Maryland Eastcrn Shore (t3!4ESII we got 25 cultivms
of bambara groundnut including the eight cultivars described above.Thcse cultivars
are from Cameroun. (11, Mali (5), Senegal (41, and Togo 1115). During the raining
saison in Senegal (from July to October 19R5), we ciurled out a field screening
of these 25 cultivars using the Relative Effcctuveness method, Results Will be
available in January 19816.
With UMES collaboration, we Will get 75 cultivars of Bambara groundnut
at October, 1986. From February to May, 1986 we Will carry out a third field
screening of the a11 cultivars constitutiag our bambara groundnut gcrmplnsm. We
Will Select the three best cultivars for further mm-mrimm+~~
7
5 . 2 - Assessment (of nîtrogen fixation by bambara groundnut :
Pot experiment Will be carrîed out again from November to December, 1985,
Nîtrogen content of shoots and roots Will be determîned at ORSTOM soi1 microbîo-
logy lajboratory in Dakar. This laboratory îs well equiped in mass spectrometer.
Results Will be available in April, 1986.
Our grant is conducted in the framework of the West Afrîca P3IRCEX. The
first collaboration is done in this regîonal infrastructure for getting culti.vars
of bambara groundnut from our collaborators in SJest Afrîcao
~he second collaboration is with OR3iZQN laboratory in Dakar. This labo-
"
ratory is well equiped for a11 types of microbiologîcal manipulations,
We havc- also a thrîd collaboration with the Clnîversity Of biaryland Eastern
Shore (UMJW 0 ~!e havc to carry out together bambara graundnut inoculation trials.
Results of thc 1384 triais are available. 1985 ones Will be avaîlable at December,
1986.
7 - SUMMiiY
The grant was awarded to Bambey National Agricultural Research Center
of the Senegal Instîtute Agricultural Research to study the improvement of yield
and Biological Nîtrogczn Fixation of Bambara groundnut (Voandzeia subterranea) I) The
objectives of the first phase of thîs grant werc :
l/ *" Obtaîn promîsîng bambara groundnut gormplasme in local markets and
from international collections,
2/ - Detcrmî~?e responsîveness and sclcct pure! strains based on phenoltype
and yield.
31 - Scr^cn rhizobial strains, includiq some isolated locally and some
obtained from internat'o la1 collections,
4/ - Observe possible diseases on bambarn groundnut.
Requcsts for getting bambara groundnut germplasm were sent to Drs, Ravoh
Itrarîvo (Madagascar Island) Ethnar and Mergaai (Togo) , i)akora (Ghana) and Hidayat
(Indonesîa). j?&q-ua"Jlv WQ ham-3 4 m n-.-L.^-- --~~ '
--
8
Mali, 1 from Cameroun and 4 from Sencgal, The problun for constituting a bambara
groLnd..iut germplasm i s that there is a very little amount seed of diffcront cul-
tivars in old collections as in Togo, Ghana, and Madagascar, bocal multiplication
of these cultivars is oftcn necessary. This is the case of Dr. Mtrjeai's collection
which will send to us 75 cultivars at the end of 1985 raining season (with the
help of the Univaxsity of Maryland Eastern Ghorc 1. Ca. Ravoh Itrarivo (Madagascar}
also Will send to us many cultivars upon an. irriz,ort licence 1 supplied.
Nevcrthelcss we carried out during 1935 dry season onefield cxperimcnt
based on scrcaning bambara groundnut - Nhizobium symbiosis. The results of this
experiment
are presented at 1985 E%?+S mleeting. During this experiment
including eight cultivars a disease was obscrved on some plants which well grew
during on month. Thus the leaves withered as a vater stress, then plants died.
Preliminary observations under a dissecting microscope showed that is probably
a fungal diseaso causdd bly Fusarium sp. Studies on diseases ob bambara groundnut
are reeded.
A similar Gcx,pcrimcnt including 25 cultivars is now carrying out during
the raining season.
A seccnd expcriment about the assessment of N2 fixation by two cultivars
of bambara groundnut u!Sing 151$ methods was startcd in .April, 1985. Howcver this
experinent was not finLshcd because of plant discase mentioned above.
Plan for the next pcriod :
II-
On goirq and future research activitics invo:Lve continuation of coï-lec-
tion and field screening of harnbara groundnut cultivars, sutdy the effectircness
and competitivcnass of a11
Hhizobium collected, the assessment of 1J2 fixation
using 15N mcthods, Thon, after, a new proposa1 Will be s&mitted to E\\sES for the
second phase of thc grant.
APPEEJDICE 1
Equi~ent received (up to October, 1985)
-
Incubator
Autostill
pH meter
Shaker
Centrifuge
Freezer
Refrigerator
Glass ware
10
APPENDICE II
1 - TRXNING AND CONSULTAï4TS'VISIT
Programme teaching is not include in the grant. But a consultant visit
will be held during the second year of the project carrying out. Consultant Will
Dr. P. SOMASEGARAD from NIFTAL project in Bawaii or Dr, W. KEYSER from Beltsville
university.
2 - ME~Z’INGS ATTENDED
We have at,tended at BOSTID - NAS meeting only. During the first meeting
at Chiang Mai in Thailand,
we presented our project. During the second o:ne
in Bawaii,
We.
presented the first results of fileld screening of bambara
groundnut cultivars.
3 - PUBLICATIONS
Three publications Will be submitted to MIRCEN Journal of Applicd Micro-
'biology and Biotechnoiogy.
- Assessment of N2 fixation by bambara groundnut using 15N method SO
- Field scrcening of bambara groundnut cultivars
- Bambara groundnut inoculation trials (in collaboration with the Univer-
sity of MnryLand Eastern Shore) m
CAPTION FOR FIGUrnS
Fig. 1 : Re:a...ive Effectiveness (R.E.1 of eight bambara groundnut cultivars
1n :
LE, based on plant dry weight
1B :
R.E. based on nodule dry wciyht
1c :
R-E, based on specific ARA
1D :
R.E. based on shoot id%
1E :
R.E. based out root NB
1E :
R,E. based on seed yield
f
7
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