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
.
,
.
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
.
,
.