MG/1 D REPUBLIQUE DU SENEGAL. 1 NSTITUT...
MG/1 D
REPUBLIQUE DU SENEGAL.
1
NSTITUT SENEGALAIS DE
R;ECHERCHES AGRICOLES
FrINISTERE DE LA RECHERCHE
SCIENTIFIQUE E.T TECHNIQUE
DEPARTEMENT DE RECHERCHES SUR LES
PRODUCTIONS VEGETALES
PROGRAMME MIRCEN
NITROGEN FIXATION IN
LEGUMES
Part 1.
RHIZOEXUM INOCULANTS
Part 2.
ROLE Of MIRCEN
by
Mamadou CUEYE
I n t h e framewark o f
(1’
gional Training Course on Microbiological and
Chc ical Assessment of Ir’ater
Quality in Relation
ta Water Supply, Sanitation and Irrigation.
BANJUL, Thc Gambia
Centre National de Recherches AgronomiW$*E
nr,tfJb
24 November 12, 1983
dEi Bambsy
P
fitif ZOBIUI1 1 NOZUL,‘;NiS
?? ???
?? ?
- 1 -
INTRODUCTION
1 - Nitroqen cyclti (Fis* 1)
Leguminous crops play
an important rolc 13s a supplier of
Pcotoin for
third world
poopls. In many CBS~S, soils
lack available nitroqon which is a majcr
limiting fector for plant groivth and yicld.
H owever , plants cari not Fix thr frac air nitrogen
urithout tho help
of effective F4hizobia in tho
root systoms nodulas ('l). Nitrogen issutid from
Plant reskdues or
animal uiastes (2) is minoralized (3) bofore
its assimilation
by plants under NH; or
NO3 form (4). In naturel
conditions, plant growth
dapuns
on microbial actiui tias
in the soil.. Even through thure is a
heawy dressing cf
fertilizar
from industriel fixatiun (51, soi1 microorganisms contributu ta in-
creasu
amount of nitrogon
assimilatcd by plants.
Soi1 NH; and NO; content intcrwane in four process :
i )
Ammonia and nitrate production : ammonia
production (3) is a stage of
ths
mineralization. Different hetcrotrophic microorqanisms contribute at its
prodtition.
H oulever,
specific autotrophic bactoria produco
the nitrati; (6).
NH; t 3/2 Or2 ---$NO;+ 2Ht + H20 (Nitrosomonas)
N02- + 1/2 0:2 ,----+ NOJ
(Ni trobacter)
ii)
T h e reoroanitation ( 7 )
: Use of tho minera1 nitroqen NH: by
hetero-
trophic bacteria
for their proteinic synthesis.
iii) The colloidal solution of tho clêy-humus complex play an important ~$1.
in tha ammonia absorption (8)
and the chomical fixation (9),
iv) The nitragon wsstes by volatilization (IO), lixiviation in soi1 (11)
and chemicol or biological deni trification
(12).
2- Biolooical ni troqen fixation
plants of leguminous and actinorhizal
crops fix the free air nitro-
wn modiated by Rhizobia. This process is
callod Biclogical Nitroqon Fixation
(BNF) : housed in plant nodulus,
Rhizobia obtaint food and sncrgy
from thoir
host ;
Rhizobia in turn,
fix atmosphsric nitrogon
urhich is thon metabolizdd
by tho plant host.
It is ~011 astablished that tho noduli! is a structurti iesued from th.,
proliforation of cortical root ~611s infectad
by Rhizobiô. Insids
the nodule, tt ._
bacteroibare
cnclosud within mcmbranz cnvtilopes
and are
tho site of nitrogon
fixation.
Nitrogen
fixation is
an anaerobic process
and tho laghaomoqlobin rc;g;‘-
latc3s the Dxygen. According to following diagram
(Fig. 21, nitrogenaso is thl?
enzyme urhich mediatos thc reduction
of N2 to NH3.
This enzyme is made up of tutic
components : ono urith both iron (FI:) and molybdenum (Mo) and tho second urith i:L'
(Fe) only. The first stable product in BNF is ammonia. Thd ammonium ion NH: pro-
cod is assimilated through
GS-GOGAT (GlutDmind Synthatas~-Glutamate synthetasej
pathway for
aminoacids and proteins synthesis.
- ? -
Thcrefart!,
BNF pk:rmitç pl?nt gruurth without hawing recwrsz to L!X-
pansiwe
çhemical f,?rtilizors.
1 t is e
truu nitroqon
uconomy. This oxplainç why
many countrii:s, i?sp,2cially
th:: duveloping countrii;s~,
malfi: an effort f‘cr pronc-
tiny w-td improving 6iW
by culturinq
iihizc;bia in laburstarios and prrïducing
s-_I_
RhlzGbium inoculnnts.
--"-
BIOL.OGICAL
FIXATION
( 1 )
\\
\\ .
0
(5 1 INDUSTRIAL
.
\\
NH3
FIXATION
\\
( 1 2 1 DEMINERALIZATION
t
( 1 0 ) ‘VOLATILIZATION
.-
(111 LIXIVIATION
( 3 1
/
NITRIFICATION ( 6 1
(8 1 ADSORPTION
!
CURVED
ARROWS
(- ) B I O L O G I C A L
PROCESS
STRAIGHT ARROWS
1-o) C H E M I C A L
PROCESS
E L E C T R O N S DONOR
PLANTS
ELECTRONS
PROTEINS
3 A”
t
AMINOACIDS
x3A’
2
GLUTAMATE
GOGAT
î OXOGLUTARATE
ENERGY SOURCE
TIC, 2
PHOTOSYNTHESIS
A = ELECTRONS
ACCEPTOR
- 3 -
? -
Rhizobiurn strain selection
7‘he f i r s t step
i n legume inoculant manufacture is to obtain effec-
tiwe nitroqen t'ixing strains of Ahizobia for the legrimes to be inoculated.
The attributes needed
in an inoculant strain are :
(i) the ability to form nitro+nfixing nodules on tke host for which it is
recommended,
(ii.) the hiqh compotitiveness in nodules formation and
(iii) the survival
and the multiplication of Rhizobia in soi1 (DATE, 19'76).
-
1.1 - Nitroqen fixinq nodules formation
The effectiweness of a Rhizobium strain is its ability tc fix nitro-
gen within the nodules. Jhe effoctiveness is influenced by intrinsic factors
(variatiomwlthin
a crclss inoculation group) and extrinsic factoEs (environ-
mental conditions :
pH, varying soi1 types, soi1 nitrogon level, interactions
with indigenous soi1 microorganism@),
1 . 2 -
Competitiveness of Hhizobia in nodule formation
An important characteristic in strain
Ahizobium selcction is the
ability of a strain
to successfully compete for nodule formation with the nativ<:
rhirobial
population &ich is generally less effeotiva.
1 . 3 -
Survival of rhizobium
The most effective and most competitiue gizobium must multiply and
survive
within soil.
2 -
Cultivation and massive production of Rhizobia
2.1 - Cultivation of Rhizobia
Rhizobia grows in a routine medfum whose composition is given in thi-:
appendix. Rhizobia cari be cultured in shake culture or
in glass bottles, or in
any of a
luide variety
of fermentes. Monosaccharides
and disacchari,des are
thr
most
common source
of carbon
for Rhizobiurn isolates although some slow grouiing
s t r a i n s przfer
pentoscc (arabinoskz)
instcad of mannitol or sucrose (DATE, 1974).
2 . 2 - Massive production
of rhirobium
'TO obtain a ghizobiurn culturt? of
a high concentration, the ferment&
i s used. Prier
to the cultivstion of Rhizobium in a ferment&, it is usually
recommended to start,
first, a preliminary c u l t u r e
i n a small
flask:. Later,
th:;
s t a r t e r
culture is thon transferrcd ascptically to the
production fermenter at
tho rata of 1 $
by volumt?. Culture is aeretcd
by sterile air.
An oxygen parti21
prossurs
of O.ÎS atmosphuro is optimum and &izobia grow test in the range of
25 to 32°C.
Thcrc BTO diffrzrcnt t y p e s o f
f e r m e n t e r : from large fermenters
with
compliclttid d!ivisEs
(EICI'RTÛN,
1 9 6 7 ) t o
simple f'erment@s u s c d i n AuetIalian
(DATL,
1974),
(Fig. 5). Accorcling
to Tustraiian c?xperidnce,
autoclavable fcrmcntation
I N O C U L A T I O N P O I N T
FILTER
--- AIR OUT LE1
- A I R INLE:T
--
-c
--
-
-
-
-v
r1--
-
--
F E R M E N T E R -, ---
-
-
-
-
--
-
Fb OUT LET FOR BROTH
- 4 -
unit i.3 hf!t;tds tharr SC:~.'
opr!r~ting lomplicatcd fermenters requirincj stuarn
steriliz:3tion.
3 - 1 noculen t c:-,rrier s
Thc qualitics of o r;ood carrier inoculant naterial ari? : (1) highly
absorptivc and casy to procoss ; ( 2 ) N o n - t o x i c to Rhi.zobia ; ( 3 ) Easy to sttiri-
-
-
lizo ;
(4) Availabla in adoquatc amounts ; (5) InaxPc?nsiwe and (6) good adhtz-
sion to soeds (BURTON, 1981).
Many mEttiriâ1s can bc IJSC?~ as terriers : poat, charcoal, clay, ligntu,
compost, straw,
pulverizcd minural:, such as vermiculitc, and polyacrylamido
(DoMMERGuES et al., 1973).
-
-
peat has boen tho most commonly used baso
“or
commercial inoculants.
It is a9ret?d that as an inoculant bool-:,
stcrilu ptiat i s s u p e r i o r t c
n o n stGrili,
Peat and that heat tri;at:?d or .lutoclave!j psat is battur
than air
driod Pcat.
4
- Ouality control
Thc aim of Eizobium inoculant quality contrnl is ta avoid problcms
during production and storag;;
lnd 31.~0 to ensurc
the high quelity cultures.
Howvswcr, it is
nuccssary tc standardizc
thU i n o c u l a n t . F o r a particular local
conditions, th, number of vi,~bl~~ c~.lls in Rhizobium inoculant standsrd must b:?
suporior tO just adequati
numbr:r of Rhi-robium for nooulating thc legumo in
sterila conditions. Within thi: limits of inoculum tc::hnoloQy (i.e. tho many
factors
Ihich affect inoculant's quality), trop agroi-Iomic c a r a c t a r i s t i e s
dc-
tarmins
inoculant standard. plany rcsnarchcrs
recomme8-bdad an inoculum wvt)i.ch
urould providc approximatively
1 0 , 0 0 0 to 1 0 0 , 0 0 0 viabl.e Rhizobia p e r
sced f o r
3 good inoculation (table 1).
'Table ? : Number o f
viable Ahizobium cells required per seed.
(BORDELEAU, L.
and PREVOST, D. 1'7181).
Crop
Secd size
Plumber of viable
(Numbcr secds/kq)
cells per seed
-t
Alfalfa
i
Clover
?03
Bird’s-foot
Sainfoin
T ntisrinediate
104
5 0 , 0 0 0 - 2 0 0 , 0 0 0
Beans
Largo
105
Peas
< 3 0 , 0 0 0
Soybeans
-s-
4
- Aqrononic importance
Inoculation succoss~s of failures in tho field have been shown
(HAM et al., 1976
-
-
; BALASUNDARAM and SUBBA RAO, 1977; SUBBA RAO, 1976).
Failures tci obtain the desirod
rssponse may be
due to (i) thc
prasence than introduced offectiuc'flhizobia strains, (ii) the presenco of
antagonistç of Rhizobia rvhich minimizc the numbor of nhizobia in the rhi-
zophere and (iii) soi1 canditions which limit symblosis : acidity, alkalinity,
high nitrogcn leva1
in soi.1 or othar
factors.
The use of ;Echizobial cultures
in legume ostablishment has been
uidely rwcognized,
especially in arcas urhere
indiganous ffhizobia bave beon
found to bu inndequate.
CONCLUSION
1
n many countries, especially in the developing countries,
grain
legums i s p r o d u c e d mainly b y small farmers
iuho u s e m u l t i p l e c r o p p i n g systzms
and minimal
technical imputs. Tho osscntial rolc
of thc Microbiolugical Ru-
sources Centcrs
(MIRCEN) is to pramotc
the uso of inoculant tuchnclogy in
these developing countrics (sea part 2).
PART 2 : ROLE Of MIRCEN
ROLE OF MIRCEti IN
DEVELOPING COLINTRIES
I
M=I> -z: -= ..= -= -= -= -= .--*: -= -
/
Developing countries are today facing with
mnny difficultios as
insufficiency of food, unvironmontal pollution, enorgy crisis. In somo of
l
these countries, hunger abats social and political unrest.
1
In the prosant fact,
science of microbiology offers a wido fiold
I
of applications of micrcbiological technologies in thu developing countries
1
because it takes actions in agricultural productivitty,
food and onergy pro-
)
duction, hcalth, ztc...
i
I n fuod p r o d u c t i o n ,
single ce11 protcin
(SZP) bas a t t r n c t e d
c o n -
/
sidorable interost as a
group of
fuods rich
in protein,
producsd by non-
agricultural
maans and hopefully lcss oxpensivo than convantional protein-
rich meterials
such as soybean moal.
Use of SCP is yet limited. It is uscd
for
animale nutrition. Tho micrcarganisms
ussd for
SCP production include
b a c t a r i a , yeast
a n d algso.
'dood is the more
important combustible usod in develcping countrics.
Frankia is an actinomycot which cari nodulato Casuarina oquisetifoli-, a nitro-
gen fixing tree.
In
the other
hand, one of the important
biological rasearch is the
.
production, of cooking gas
from agricultural wastes.
Soi.1
and mater pollution require microbiological
technologies :
effluent wjter
coming f’rom facturias must
degreted through microbial
actions
to avcid watar
pollution.
T h r o u g h microbial a c t i v i t i e s , ure cari obtain
a n t i b o d i e s , I n d i s p e n s a -
ble enzymes like proteass, amylase,
collulase and vitarnins which are a11 impor-
tant to our
daily living.
Metabolits produced by soi1 microorganisms
improve plant groth.
Crctp yield cari be improved
by domtisticating biological nitrogen
fixation in
ordcr to reduce
the use of expcnsiva nitrogon fertilizurs.
Science
of microbiology is
alsa applicd for
protecting crops against
diseoses
modiated by Eaccillus thurinqiansis, Pseudomonas septica, C1ostridi.m
Erfrinqens
and cthor sntomtipathogen microcrgonisms.
Thesc exomPlos illustratr:
the role
of microorganisms
in thu develüP-
ment sf third world countrias
and tho importance
of culture
collocticn of
micruorganisms.
This ia why UNEP/UNESCO/ICRO pane1 on microbiology
has launch.2
the Microbiolugical Rcsources
Centet (MIRCEN) prcgrams which are
designed tc :
a) - Provide the infrastructure for a uicrld
nstwork which would incor-
porato rogional and intarrogianal co-operating laboratories
geared to
thc
management, distribution 2nd utilization of thc micxnbisl
gene Pool i
b)
- Reinforcc ,?ffurts rolating to
thc const:rvation
o f micrucrganisms,
with
emphasis on Rhizobium gcne pools,
-
-
in developing countries, with an
agrarian
base ;
c> - Foster
thc development of now inoxpensive technologies rative to
the rcgion ;
r.1) - Promote th.z applications of microbiology in ordor to strengthen
rural economiès ;
0)
- serve as focal centors for the training
of manpower
and the dif-
fusion of microbiological
knowlcdgc.
In Si?ncgûl,
the ISRA (1) agriculture1 ond forestry resoarch ccnters
at
Bambey and Dakar-Hann, raspoctively,
have been dosignated as thlr West Afri-
ca MIRCEN. That MIRCEN will focus on establishing a regional
mic.robial cul-
ture collection to serve
tha agricultural
production.
Important species
of microorganisms
of economic significsncs, which
are
usod in ths preparntion of biofcrtilizor
inoculum production (Par
example,
Rhizobium, F rankia,
Myc o-rrhiza) , and
bioonergy goneration (for example,
biogosj
Uri11
be centralized for prossrvation
and use primarily
in Senegal. At a later
stage, co-opnrating laboraties in neighbouring countries of Mali, IJpper Voltn,
Gambia, Cuinea, Niger, Chad and Togo Uri11 benefit from
the culture
collection
at the MIRCEN.
Tha MIRCEN will also undertake collaborative research projects,
recommend ragional f
ollowships, or ganizo short- term training and eventually
attract
international contracts for resesrch
work in
tho region.
(?) ISRA : Institut SerIégalais de Recherches Agronomiques.
Scnogclese Instituto of Agronomie Researches.
REFERENCE
BALASUNDAHAM,
V.R. nnd SUBBA RAU, N.S., 3977. A roview
of development of
rhizobial inoculants for soybeans in India. Fertilizer NE. 22,
42-46.
BORDELEAU, L. and PREVOST, D., 1971. Quality control program
by fodcral govarn-
ment agencios and mathod of tcsting leguma inoculant and pre-inOcuLatoc!,
sced products
in Canado. In Prac. 8th North
American J.H.
Stophens!
-
PP*
566-579.
BURTON, 3.C. - 1967. fjlhizobium culture
and use. In Micrubial
technology (Eds.
J.
Peppler) Rainhold Publishing Corporation, N~UI York
pp. l-33.
BURTON, J.C.,
1981. RJzobium inoculant5 for
developing countries.
Trop. Aqriz.
(Trinidad).
SB, 291-295.
BURTON, J.C.,
1982. Modem concepts in lugume inoculation. In Biological Nitri-
gen Fixation Technology for Tropical Agriculture.(Eds
P.H. Grahem
an4 S.C. Harris) pp*
105-114. CIAT, Cali.
DATE, R.A. - 1970. Microbiological problems
in the inoculation
and nodulatian
in legumes. plant and soil. 32, 703-725.
DATE, R.A. 1974. Legume inoculant producticn. Proc. INSA. 40 (B), 667-686.
DATE, R.A. 1976. Principels
of Rhizobium strain
selection. In
Symbiotic nitrL-
gen fixation in plants.
(Eds P.S. Nutman). Cambridge Univ. Press,
Cambridge, England pp. 137-150.
DATE, R.A. and ROUGHLEY, R.J., 1977. Preperation
of legume ssed inoculants.
In A treatise on :jinitrogen fixation. Sec. IV, Agronomy and Ecolcgy.
vds R.W.F. Hardy an3 A.H. Gibson) pp. 243-276. Wiloy, N~UI York.
DOMMERGUES, Y
.R., DIEM,
H.G. an3 DIVIES, C., 1979. Polyacrylamida
entrapped
Rhizobium as an inoculant for legumes. Appl. Environ. Microbicl. 37:
779-7Bl.
HAM, C.E., LAWN, R.J. and BRUN, W.A., 1976. Influence of inoculation, nitrvgtin
fcrtilizer
and photosynthctic sources -
sink manipulations of fiell!
grourn
soyboans, In Symbictic Nitroqen
Fixation in Plants.. (Eds P.S.
Nutman) pp. 239-253. Combridqo
University Press.
SUBEA RAO, N.S., 1976. Ficld rPGp’onee
of lcgumca in India to inocultiti&l end'
furtilizor
ePpli:otion. In Symbictic hlitrogon Fixation in plante.
(Eds.
P.S. Nutman). pp. 255-268 Cambridgcl Univcreity pross.
Appënilix
: Routine modium for growth of Rhiz(jbium
Yeast-Extract Mannitol brsth.
0.5 g
Mg 304, 7H20
0.2 g
NaCl
0.1 g
Mannitol
10
%
Y cast-ex tract
1 9
Distillod wator
1000 ml
Agar
15 9
pH = 6.8
.
,
.