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D E D I C A T I O N
For a love pure and true:
To my dear wife, Joyce, and the c h i l d r e n  w ho  had 
to bear my long a b s e n c e  from home.
TO GOD BE THE GLORY.
i i
STUDIES ON THE D E V E L O P M E N T  OF RHIZOBIUM I N O C U L U M  U S I N G  M O S S  AS 
C AR RI ER FOR BAMBAR A GROUN D N U T ,  VIGHA SUBTERRAHEA (L) V ER DC .
A Th esi s P r e s e n t e d  by 
J O S E P H  ADD O A M P O F O  
In Parti al  F u l f i l m e n t  
of the R e q u i r e m e n t s  for the D e g r e e  
M A S T E R  OF P H I L O S O P H Y  
of the U n i v e r s i t y  of G h ana
OCTOBER, 1991
From: The D e p a r t m e n t  of B o t a n y
U n i v e r s i t y  of Ghana  
L e g o n .
i i i
I h e r e b y  declare that the w o r k  p r e s e n t e d  in this thesis:
"S TU DI ES ON THE D E V E L O P M E N T  OF R H I Z O B I U M  
I N O C U L U M  U S I N G  MO SS AS C A R R I E R  FOR 
B A M B A R A  GROUND NU T. V I G N A  S U B T E R R A N E A  
(L) VER D C . "
was done e n t i r e l y  by me in the D e p a r t m e n t  of Botan y, U n i v e r s i t y  
of Ghana Legon from A u g u s t  1990 to A u g u s t  1991.
This wo rk has nev e r  be en p r e s e n t e d  e i t h e r  in p a r t  or 
c om ple tel y, for any d e g r e e  of this U n i v e r s i t y  or e l s e w h e r e .
J.A. AM POF O,  B •S c •(H o n s )D i p •Ed 
U n i v e r s i t y  of Ghana 
L e g o n .
Date : f. [ l. ~  t
P RO F E S S O R  G.C. CLERK 
S UP ER V I S O R
Date
nov. 2 7  [ 9 ?\
A B S T R A C T
i v
R e s ea rc h was c a r r i e d  out to d e v e l o p  an e f f e c t i v e  i n o c u l u m  
carrier of the n o d u l e  b a c t e r i u m ,  Rhizobium sp., for the 
i n o cu la ti on of seeds of b a m b a r a  g r o u n d n u t  [Vigna subterranea (L.) 
Verde, comb. nov. = Voandzeia subterranea (L.) T h o uars ] f r o m  a 
local m a te ri al. Five out of 16 s t r a i n s  of Rhizobium sp. 
isolated from soils of legu me farms at eig h t  l o c a l i t i e s  w i t h i n  
a radius of 180 km from L e g o n  were us ed in e x p e r i m e n t s  to test 
the e f f i c i e n c y  of the c a r r i e r  that was d e v e l o p e d .  P l a n t s  
infected by these five s t r a i n s  g r e w  well, had h i g h  d r y  w e i g h t s  
and high n i t r o g e n  c o n t e n t  of the sh oot s and f ormed m a n y  la rge  
n o d u l e s .
A good Rhizobium c a r r i e r  was d e v e l o p e d  from the moss, 
Brachymenium sp. H a r v e s t e d  t ha ll i w e r e  s u n - d r i e d  for 10 days and 
de com p o s e d  for 15 days. W h e n  stored  at r o o m  t e m p e r a t u r e ,  the 
Rhizobium-p o p u l a t i o n  of i n o c u l a t e d  mo ss c o m p o s t  of 50 per cent 
m o i s t u r e  co nte nt rose to a m a x i m u m  of 4.0 0 x 1 0 ^  cel l s  per
gram of comp ost after 10 we eks. The p o p u l a t i o n  r e m a i n e d  
s t a t i o n a r y  t h e r e a f t e r  till the end of the 14th w e e k  w i t h o u t  
showing any signs of a u t o l y s i s .  Ste r i l e  c o m p o s t  i n o c u l a t e d  and 
stored at room t e m p e r a t u r e  for two w e e k s  w h e n  the Rhizobium
ii
p o p u l a t i o n  rose to a p p r o x i m a t e l y  1 x 10 ^ p e r  g r a m  of c o m p o s t  
was used to inocu la te the seeds in the v a r i o u s  e x p e r i m e n t s .
Ex-Ada and E x - T a m a l e  v a r i e t i e s  of b a m b a r a  g r o u n d n u t
ino cul ated with the five st r a i n s  of Rhizobium sp. all n o d u l a t e d .  
Plants of Ex-Ada v a r i e t y  d e v e l o p e d  best at low light i n t e n s i t y
(1100 - 2200 lux at 9.00 am, 40 30 - 6200 lux at n o o n  and 1600 -
1900 lux at 3.00 pm). M e d i u m  light i n t e n s i t y  (1400 - 2 8 0 0  lux 
at 9.00 am, 6400 - 9100 lux at n o o n  and 2900 - 3500 lux at
3.00) was less favo u r a b l e  and hi gh light i n t e n s i t y  (5300 - 6200 
lux at 9.00 am, 8800- 10000 lux at noon and 4600 - 6400 lux at
3.00 pm) was least fa v o u r a b l e .  A f ter 30 days, the r e s p e c t i v e
Vmean dry we i g h t s  of the pl ants at the low, m e d i u m  and h i g h  
light i n t e n s i t i e s  were 1,62 - 2.91g, 1.33 - 1.8 2 g  and 0,91 -
l,41g; and the r e s p e c t i v e  m e a n  n u m b e r  of n o d u l e s  per p l a n t  was 
54 - 78, 42 - 51 and 16 - 21.
Plants of E x - T a m a l e  v a r i e t y  w a t e r e d  on ce  in two days 
showed the best growth. Tho s e  w a t e r e d  o n c e  in four da ys  s h o w e d  
m o d e r a t e  growth, and g r o w t h  of p lants  w a t e r e d  once in six days 
was g r e a t l y  reduced. Aft e r  30 days, the r e s p e c t i v e  m e a n  dry 
w ei g h t s  of plants w a t e r e d  onc e in two, four and six days w e r e
I.00 - 1.68g, 0.64 - 0 . 9 4 g  and 0.48 - 0.76g, and the r e s p e c t i v e  
mean n u m b e r  of no d u l e s  per plant was 43 - 56, 16 - 25 and 8 -
II. In both tests, c o n d i t i o n s  of l i ght and m o i s t u r e  w h i c h  
e n c o u r a g e d  h ig her n o d u l a t i o n  also i n d u c e d  the f o r m a t i o n  of 
larger nodules.
It was con c l u d e d  that c o m p o s t  of Brachyseniua sp. is a good 
Rhizobium ca rrier  and can be used for r o u t i n e  i n o c u l a t i o n  of 
b a m b a r a  g r o u n d n u t  seeds. To d e r i v e  the m a x i m u m  b e n e f i t  fr om 
the i n ocula ti on, it is d e s i r a b l e  to i d e n t i f y  the be st s t r a i n  of 
Rhizobium sp, for each v a r i e t y  of b a m b a r a  g r o u n d n u t .
C O N T E N T S
Page
I. I N T R O D U C T I O N  A N D  L I T E R A T U R E  R E V I E W  .......................... *
II. M A T E R I A L S  AND G E N E R A L  M E T H O D S  ................................  14
i. M a t e r i a l s ....................................................
a. Soils ..........           14
b. Bam b a r a  g r o u n d n u t  seeds ....................  .... 15
c. Rhi zob ium s t r a i n ..................................... 15
d. D e c o m p o s e d  Moss m a t e r i a l  . . . ............    16
ii. Gen e r a l  M e t h o d s   ............... . . ... .    17
a. R a i s i n g  of b a m b a r a  g r o u n d n u t  p l a n t s ........ . 17
b. S e l e c t i o n  of v i a b l e  seeds for p l a n t i n g  .... 17
c. C o n d i t i o n s  for g r o w i n g  the p l a n t s ........... .. 17
d. Cu l t u r e  m e d i a .................................... 17
1. Ye a s t  e x t r a c t - m a n n i t o l  agar (YAM) ......  18
2. Congo Red yea st e x t r a c t - m a n n i t o l  agar.. 18
3. Yea s t  e x t r a c t - m a n n i t o  1 b r o t h  (YEM) .... 18
4. S e e d l i n g  A g a r ..................................  18
5. Sachs' s o l u t i o n ...............................  19
e. Me t h o d s  of s t e r i l i z a t i o n  .... ................... 19
f. C h e m i c a l s  .... ••••       20
g. C h a r a c t e r i z a t i o n  of soil s a m p l e s .......... . 20
1. D e t e r m i n a t i o n  of m i n e r a l  f r a c t i o n  ....... 20
2. E s t i m a t i o n  of o x i d i z a b l e  o r g a n i c  m a t t e r
in soil: W a\k 1 e y and B l a c k !s Rapid 
T i t r a t i o n  M e t h o d  .............................  22
3. E s t i m a t i o n  of total N i t r o g e n  in soil .. 23
h. D e t e r m i n a t i o n  of N i t r o g e n  c o n t e n t  of pl a n t s .  24
i. Iso l a t i o n  of Rhizobium s t r a i n s ................. .. 24
j. P r e p a r a t i o n  of d e c o m p o s e d  moss m a t e r i a l  .... 25
k.. P r e p a r a t i o n  of the Rhizobium i n o c u l u m ....  2 6
vi
1, V i a b l e  Total Cell C o u n t s  of i n o c u l a t e d  m o s s . .  26
m. P l a n t i n g  Sheds of d i f f e r e n t  light i n t e n s i t i e s  26
n. A s s e s s m e n t  of e xtent  of g r o w t h  of i n o c u l a t e d
p l a n t s .................    27
o. D e t e r m i n a t i o n  of plant dry w e i g h t ...... 27
p. M e a s u r e m e n t  of pH  .......... •••• .... ••• ••• 27
q. S t a t i s t i c a l  a n a l y s i s  ....   28
r. E x p e r i m e n t a l  p r e c a u t i o n s  ...• ..... ....... 28
III. E X P E R I M E N T A L  D E T A I L S
A. D e t e r m i n a t i o n  of p r o p e r t i e s  of the d i f f e r e n t  soil 
samples. ...•     . .  .  *  ............ ••••• 29
B. Is o l a t i o n  of st rai ns  of Rhizobium sp. from the 
dif f e r e n t  soil samples ...• ...•   31
C. C o m p a r a t i v e  i n f e c t i v e  a b i l i t y  of five s t r a i n s  of
Rhizobium sp. i s o la ted from b a m b a r a  g r o u n d n u t
p l a n t s ..................       32
D. P r e p a r a t i o n  of the <v*oss c o m p o s t  as a c a r r i e r  and
study of shelf life of i n o c u l u m  c a r r i e r ................33
E. N o d u l a t i o n  of pl ants i n o c u l a t e d  w i t h  d i f f e r e n t
strains of Rhizobiim sp, in mo ss  c a r r i e r  un d e r  
d if f e r e n t  w a te r stress c o n d i t i o n s  ....................  34
F. N o d u l a t i o n  of p la nts i n o c u l a t e d  w i t h  d i f f e r e n t
strains of Rhizobium sp. in mo ss  c a r r i e r  u n d e r
d i f f e r e n t  light i n t e n s i t i e s  ...........................  36
IV. R E S U L T S
A. D e t e r m i n a t i o n  of p r o p e r t i e s  of the d i f f e r e n t
soil samples .... .............. ....   38
B. I solatio n of st rai ns  of Rhizobium sp. from the 
d i ffer en t soil samples  ...............................45
C. C o m p a r a t i v e  i n f e c t i v e  a b i l i t y  of five st r a i n s  of
Rhizobium sp. isolate d from b a m b a r a  g r o u n d n u t
p l a n t s .............. ci
vi i
Page
D. P r e p a r a t i o n  of the moss c o m p o s t  as a c a r r i e r
and study of shelf life of i n o c u l u m  c a r r i e r  ••• 55
E. N o d u l a t i o n  of plants  i n o c u l a t e d  w i t h  d i f f e r e n t
strains of Rhizobium sp. in moss c a r r i e r  u n d e r
d i f f e r e n t  wa t e r  stress c o n d i t i o n s  ...............  62
1. Mean leaf n u m b e r  per p l a n t  •••• .... .... 75
2. Mean size of m i d d l e  l e a f l e t  ... .... .... 87
3. Me an dry w e i g h t  of p l a n t ................ .... 98
4. Mean n u m b e r  of n o d u l e s  per p l a n t ...........  110
5. Size of n o d u l e s ...................................  121
F. N o d u l a t i o n  of plants i n o c u l a t e d  w i t h  d i f f e r e n t
strains of Rhizobium sp. in m o s s  c a r r i e r  u n d e r
dif f e r e n t  light i n t e n s i t i e s  ....................  128
1. Mean n u m b e r  of leaves per p l a n t ..  143
2. Mean len gth and w i d t h  of m i d - l e a f l e t s  ... 154
3. Mean dry w e i g h t  of p l a n t s ....................  165
4. Mean n u m b e r  of n o d u l e s  per p l a n t ...........  176
5. Size of n o d u l e s   ...............................  187
V. G E N E R A L  D I S C U S S I O N  ...........................................  194
VI. S U M M A R Y ........................................................... 208
VII. A C K N O W L E D G E M E N T ................................................ 215
V I I I . L I T E R A T U R E  C I TE D .............................................. 217
IX. A P P E N D I X .........................................................  227
A. Days for w a t e r i n g  b a m b a r a  g r o u n d n u t ,  E x - T a m a l e  
variety, p l a n t .................. ..................... 227
B. The Pre sc ott T r i a n g l e  s h o w i n g  the r e l a t i o n s h i p  
between c o n t e n t s  of c l a y ; silt and sand in 
d e t e r m i n i n g  the d i f f e r e n t  ki nds of soil ........ 228
Page
ix
C. D i s t r i b u t i o n  of n o d u l e  size in two v a r i e t i e s
of ba m b a r a  g r o u n d n u t  plants r a i s e d  in soils
from legume plots from d i f f e r e n t  l o c a l i t i e s  ....
D. D i s t r i b u t i o n  of n o d u l e  size of b a m b a r a  g r o u n d n u t
Ex-Ada v a r iety , ra ise d on fS e e d l i n g  A g a r 1 .,. ...
E. G ro wth of five s t r a i n s  of Rhizobiua sp. in 
i n o c u l a t e d  moss c a r r i e r  stored at room
t e m p e r a t u r e  for 14 we e k s  .....................................
F. D i s t r i b u t i o n  of n o d u l e  size of b a m b a r a  g r o u n d n u t  
plants grown u n de r d i f f e r e n t  w a t e r i n g  r e g i m e s  ........
G. R e c o r d i n g s  of light i n t e n s i t i e s  d u r i n g  g r o w t h
of bam b a r a  g r o u n d n u t  plants, E x - A d a  v a r i e t y ,  
under Sheds 1, 2, and 3 from A p r i l  10, 1991 
to May 14, 1991 ...........................................
H. D i s t r i b u t i o n  of n o d u l e  size of b a m b a r a  g r o u n d n u t  
plants grown u n de r d i f f e r e n t  light i n t e n s i t i e s  for 
30 days  ................      # .....................
Page
229
230
231
233
236
237
I N T R O D U C T I O N  AND L I T E R A T U R E  R E V I E W
The i n c r e a s i n g  d e m a n d  for food and a nima l feed has in 
the past been met l a r g e l y  t h r o u g h  e x p a n d i n g  the area of 
land under c u l t i v a t i o n .  But as the a v a i l a b i l i t y  of
u n c u l t i v a t e d  a ra bl e lands b e c o m e s  lim ite d,  e m p h a s i s  mu st 
now be shifted to r a i s i n g  the p r o d u c t i v i t y  of land a l r e a d y  
under c u l t i v a t i o n .  I m p r o v e d  c u l t i v a t i o n  m e t h o d s ,
m a i n t e n a n c e  of soil fe rt il ity, use of h i g h  y i e l d i n g  
strains and i n t r o d u c t i o n  of n e w  cro ps are some of the 
r e c o m m e n d e d  ways of i m p r o v i n g  p r o d u c t i v i t y .
These r e c o m m e n d a t i o n s  are p a r t i c u l a r l y  d i r e c t e d  at 
d e v e l o p i n g  c o u n t r i e s  w h e r e  food p r o d u c t i o n  lags far b e h i n d  
food demand. In these areas, p r o t e i n  d e f i c i e n c y  is still 
a seri ous problem. A n i m a l  p r o t e i n  is s e l d o m  a f f o r d a b l e  by 
the po orer se ction of the s o c i e t i e s ,  so l e g u m e s  u s u a l l y  
pr o v i d e  the ch ief and s o m e t i m e s  the on ly s o u r c e  of 
protein. All le gumes are rich in p r o t e i n  ( 20 -4 0 per 
cent), iron and the B- v i t a m i n s ,  w h i c h  m a k e  th em  e x c e l l e n t  
food even wh en eat e n  in small a m o u n t s  ( A y k r o y d  and 
Do ughty, 1964). Some have be en fo und  to c o n t a i n  c a r o t e n e  
(Vi tam in A) and some, for example, gr o u n d n u  t (Arachis hypogea 
L.), have the ex tra v a l u e  of b e i n g  ri ch in oil.
Ta nno ns and U l l a h  (1969) i n d i c a t e d  that the n u t r i t i v e  
v al ue de riv ed  from a d i e t a r y  p r o t e i n  dep e n d s  not o n l y  on 
its q u a n t i t y  in the diet but also on its q u a lit y,  
d e t e r m i n e d  by its amino acid c o m p o s i t i o n .  A c c o r d i n g  to 
Ta nnons  and U l lah (op.cit.) ot her fa c t o r s  w h i c h  a f f e c t  the 
n u t r i t i v e  val ue of e d i b l e  l e g u m i n o u s  seeds i n c l u d e  the 
c o n c e n t r a t i o n  of c o n s t i t u e n t  h a e m a g g l u t i n i s  and t r y p s i n  
inhibitors.
Be as it may, d e v e l o p i n g  c o u n t r i e s  are not just 
p r o d u c i n g  e no ugh of the legumes, d e s p i t e  the e n o r m o u s  
vari eti es, for example, b a m b a r a  g r o u n d n u t  (Vigna subterranea 
L. Verde = Voandzeia subterranea (L) T h ouars) , c o w p e a  (Vigna
1
unguiculata L.), groun d n u t ,  p i g e o n  pea (Cajanus cajan
M i l ls p.) locust bean ( C e r a t o n i a  s i l i q u a  L*), w i n g e d  bean 
(P s o p h o c a r p u s  t e t r a g o n o l o b u s  (L.) DC), etc., a v a i l a b l e  to 
them. Results of seven c o m m u n i t y  su r v e y s  that e x a m i n e d
25,000 c h i l d r e n  in A f r i c a  r e v e a l e d  a range of 1.7 - 7.8
per cent of sever e P r o t e i n - e n e r g y  m a l n u t r i t i o n  (PEM) and 
5 #4 - 44,9 per cent of m o d e r a t e  forms. R o u g h  e s t i m a t e s  by 
the World H ea lt h O r g a n i z a t i o n  (WHO) b a s e d  on m e d i u m  v a l u e s  
of the total n u m b e r  of c h i l d r e n  a f f e c t e d  by the d i s e a s e  in 
A frica  as rep or t e d  by Food and A g r i c u l t u r e  O r g a n i z a t i o n  
(F.A.O.) ( 1988 ), are 2.7 m i l l i o n  of severe, and 16.3
m i l l i o n  of m o d e r a t e  forms of PEM. PEM c o v e r s  a wi de 
s p e c t r u m  of p a t h o l o g i c a l  c o n d i t i o n s ,  r a n g i n g  b e t w e e n  the 
ex tr em e forms, 'Kwashiorkor' (a m a l n u t r i t i o n  d i s ea se, 
p r i m a r i l y  of c hild re n, c aused  by severe p r o t e i n  
def i c i e n c y ,  u s u a l l y  o c c u r r i n g  w h e n  the chi ld is w e a n e d  
from the breast; the sy mpt o m s  are r e t a r d e d  growth, c h a n g e s  
in skin and hair p i g m e n t a t i o n ,  d i s t e n s i o n  of the a b d o m e n  
(pot belly), edema, di arr h o e a ,  a n a e m i a  and d e r m a t o s e s )  on 
the one hand, and ’n u t r i t i o n a l  m a r a s m u s '  (a c o n d i t i o n  of 
ex t r e m e  m a l n u t r i t i o n  and e m a c i a t i o n ,  o c c u r r i n g  c h i e f l y  in 
young children, c h a r a c t e r i z e d  by p r o g r e s s i v e  w a s t i n g  of 
s u b c u t a n e o u s  ti ssue and m u s c l e )  on the other. A large 
p r o p o r t i o n  of PEM ca use s an i n t e r m e d i a t e  cond ition 
b e t w e e n  the two, w h i c h  is r e f e r r e d  to as 'q a r a s m i c  
k w a s h i o r k o r ’. E v i d e n c e  from d e v e l o p i n g  c o u n t r i e s
i nd ic ates that most c h i l d r e n  w i t h  PEM are c o n s u m i n g  diets  
d e f i c i e n t  in bo th  e n e r g y  and p r o t e i n s  (F.A.O., 1988).
Le gu me s when used as a cover crop p r o t e c t  the soil,
i n c rease soil f e r t i l i t y  by m i n e r a l i z a t i o n  of their 
abs c i s s e d  leaves and add c o m b i n e d  n i t r o g e n  t h r o u g h  
sy mb io tic n i t r o g e n - f i x a t i o n .  All these r esul t in an 
i m p r o v e m e n t  of crops. Skerraan (1977), for e x a mple,  
repo rted that at the Sisal R e s e a r c h  Sta t i o n  at T a n g a  in 
Tanza nia , a cover crop of t r o pical  k u d z u  or puero  (Pueraria 
phaseoloides (Roxb.) Benth.), g r o w i n g  as an i n t e r - r o w  crop 
with sisal i ncrea se d the yield  of sisal by 48 per cent.
2 -
3It has earlier been d e m o n s t r a t e d  that leg) ues, as co v e r  crop, 
also e n c o u r a g e d  earlier u n f u r l i n g  of sisal leaves and add e d  635 
kg of n i t rogen  per h e c t a r e  to the soil, e q u i v a l e n t  to the 
a p p l i c a t i o n  of 1.4 tons of urea per h e c t a r e  ( R i j k e b u s c h ,  1967).
The pra ctic e of a l l e y - c r o p p i n g  has b e c o m e  w i d e s p r e a d  in 
the Tropics in recent years. A l t e r n a t i n g  rows of c r o p s  and 
legume trees are raised as a means of i n c r e a s i n g  crop  yiel d. 
The legume trees en rich the soil and at the same time p r o t e c t  
the soil. In ce rtain cases the speci es of le gumes p l a n t e d  are 
also fodder for goats and sheep.
The L e g u m i n o s a e  have long been k n o w n  as p l a n t s  w h i c h  in 
nat ure  are as so ciated  w i t h  s y m b i o t i c  b a c t e r i a .  A l t h o u g h  this 
a s s o c i a t i o n  is now, by the n ew  e c o l o g i c a l  t e r m i n o l o g y ,  m o r e  
c o r r e c t l y  a mutu al ism, the t r a d i t i o n a l  name, s y m b i o s i s ,  has 
pe r s i s t e d  till today, and is w i d e l y  used. The b a c t e r i a  o c cu r 
in nodules on the roots in this m u t u a l  r e l a t i o n s h i p .  The 
Le g u m i n o s a e  contai ns three large s u b d i v i s i o n s  or fa m i l i e s .  
These are the M i m o s a c e a e  and the C a e s a l p i n i a c e a e , w h i c h  are 
m o s t l y  tr opi c a l  in d i s t r i b u t i o n ,  and the P a p i l i o n a c e a e  w h i c h  
cont ains both tropical and t e m p e r a t e  species . Not all leg u m e s  
nodulate. The ability  to do so be i n g  least p r o n o u n c e d  in the 
C a e s a l p i n i a c e a e  in w h ic h about 75 p e r c e n t  of the s p e c i e s  lack 
nodu les  (Allen and Baldwin, 1954). In con tr ast, n o d u l a t i o n  is 
almost un i v e r s a l  among the P a p i l i o n a c e a e  species, w h i l e  the 
M i m o s a c e a e  occ upy an i n t e r m e d i a t e  p os ition. N o d u l e  s h ape and 
size vary c o n s i d e r a b l y  from speci es to species. A n n u a l s  
us u a l l y  have large no du les grouped  about the t a p r o o t  or the 
first order lateral roots. The n o d u l e s  of p e r e n n i a l s  tend to 
be smaller and more w i d e l y  d i s t r i b u t e d  over the root system. 
New nodules  are formed t h r o u g h o u t  the g r o w i n g  s e a s o n  and old 
nodu les  are sloughed off. The b a c t e r i a  of legume n o d u l e s  
bel ong  to the genus Rhizobium.
Two broad groups of a n g i o s p e r m s  po s s e s s  root n o d u l e s , o n e  
c o m p r i s i n g  the legumes and the other, c e r t a i n  n o n - l e g u m e s .  The 
b a c t e r i a l  compon e n t s  are d i f f e r e n t  in the two a s s o c i a t i o n s .
4Rout n o d u l e s  are found in seven n o n - l e g u m e  a n g i o s p e r m  f a mili es , 
na mel y, Betu la ceae, C a s u a r i n a c e a e , C o r i a r i a c e a e , E l e a g n a c e a e ,  
M y r i c a c e a e ,  R h a m n a c e a e  and R o sac ea e. The ir a s s o c i a t e  o r g a n i s m  
is an ac t i n o m y c e t e .  Norri s (1956) a d v a n c e d  the h y p o t h e s i s  that 
t;he p r o m i s c u o u s  "c owpea type" Rhizobium u s u a l l y  a s s o c i a t e d  w i t h  
the n o d u l a t i o n  of t r o p i c a l  legumes is the a n c e s t r a l  and t y p i c a l  
organism, and s u g g e s t e d  that it is here that the n o r m  of the 
s ym bi osis is to be found.
The c u l t i v a t i o n  of leg um i n o u s  crops is a ba s i c  f e a t u r e  of 
most of the p e r m a n e n t  a g r i c u l t u r a l  sy stem s of the wo rld. F r o m  
the point of v i e w  of the a g r o n o m i s t ,  the l egume - Rhizobium 
symbios is  is u n d o u b t e d l y  the mo st i m p o r t a n t  b i o l o g i c a l  
m e c h a n i s m  for add ing n i t r o g e n  to the s o i l - p l a n t  s y s t e m  
(Skerman, 1977). It is not s u r p r i s i n g  that Rhizobium t e c h n o l o g y  
has, th erefo re , b e c o m e  a s p e c i a l i z e d  field of study  in r e l a t i o n  
to the L e g u m i n o s a e ,
The str ains of Rhizobium species ha ve be en  found to d i f f e r  
in several  p r o p e r t i e s  by wh i c h  they are c o m m o n l y  c h a r a c t e r i z e d .  
They d iffer  m a i n l y  in their c u l t u r a l  and s e r o l o g i c a l  
pr o per ti es, b a c t e r i o p h a g e  s u s c e p t i b i l i t y ,  a b i l i t y  to form 
nodu les  on p a r t i c u l a r  ho st species, s y m b i o t i c  n i t r o g e n - f i x i n g  
e f f e c t i v e n e s s ,  and in other c h a r a c t e r i s t i c s  such as the n u m b e r  
of nodules  formed on the host plant (Vincent, 1974). So wli  and 
Mytton (1986) have also shown that the Rhizobium s pe c i e s  are
host spec if ic and each speci es c o n s i s t s  of sev e r a l  str ai ns.
Some of these strains of the same sp ec ies also v a r y  in their 
ab i l i t y  to induce n odule  fo r m a t i o n  and to fix n i t r o g e n .  Th ey 
d e m o n s t r a t e d  this in e x p e r i m e n t s  u s i n g  st rains of Rhizobium
leguminosarum from d i f f e r e n t  a g r i c u l t u r a l  l o c a t i o n s  and Vicia faba 
v a r i e t y  minor, as host. They, thus, c o n f i r m e d  e a r l i e r  f i n d i n g s  
of M y t t o n  and L i v e s e y  (1983) who used c lo ve r v a r i e t i e s  and 
d i f f e r e n t  isolates of Rhizobium species. Burton, A l l e n  and
Berger (1952) p r o vided  an i n t e r e s t i n g  as pec t of this p h e n o m e n o n  
many years earlier. Th ey found that isol at es of Rhizobium 
phaseoli from di f f e r e n t  nod u l e s  on the same plant v a r i e d  wi d e l y .
5Out of 85 strains iso la ted, only 12 (14.1 p e r c e n t )  wer e
ef fe ct ive.
The d e v e l o p m e n t  of a legume - Rhizobium s y m b i o s i s  thus 
inv olv es ma ny spe cific i n t e r a c t i o n s  b e t w e e n  the Rhizobium 
species and the legume host. E f f e c t i v e  ( n i t r o g e n - f i x i n g )  
no dules  are formed w h e n  the host v a r i e t y  and Rhizobium s t r a i n  
are g e n e t i c a l l y  c o m p a t i b l e ,  and the a p p r o p r i a t e  i n t e r a c t i o n s  
occur d u r i n g  each stage of n o d u l e  d e v e l o p m e n t .  C o n v e r s e l y ,  
i n e f f e c t i v e  ( n on -f ixing)  n o d u l e s  are the r e s u l t  of i n t e r a c t i o n s  
b e t w e e n  g e n e t i c a l l y  i n c o m p a t i b l e  p a r t n e r s .  Not s u r p r i s i n g l y ,  
the s t r u c t u r e  of i n e f f e c t i v e  n o d u l e s  is f r e q u e n t l y  ab n o r m a l .  
The n a t u r e  of the s t r u c t u r a l  a b n o r m a l i t i e s  and the s tag es  at 
wh ich they appear, v a r y  from one i n e f f e c t i v e  legume  - Rhizobium 
c o m b i n a t i o n  to anot he r ( M a c K e n z i e  and Jordan, 1974; Mosse, 
1964; Pa nkhurs t,  1974).
It fol lows that the a s s o c i a t i o n  can onl y d e v e l o p  if the 
right s trai n of the a p p r o p r i a t e  Rhizobium species is p r e s e n t .  
But the p r e s e n c e  of the co rrec t st rain in e v er y l o c a l i t y  c a n n o t  
be g u a r a n t e e d .  The an swer to the p r o b l e m  is to i n t r o d u c e  the 
bacter i u m .
The i n t r o d u c t i o n  of an e f f e c t i v e  s train of Rhizobium can 
mean the d i f f e r e n c e  b e t w e e n  a s u c c e s s f u l  d e v e l o p m e n t  and 
c o m p l e t e  fai lur e of the crop. E a r l y  a t t e m p t s  to g r o w  le g u m e s  
in new areas invo lved the s p r e a d i n g  of soil from the o r i g i n a l  
ha b i t a t  c o n t a i n i n g  the b a c t e r i u m  s tr ai n over the n e w  land to be 
planted. Later, cul t u r e  of the Rhizobium species in v a r i o u s  
liquid or on s em isolid n u t r i e n t  m e d i a  was ap p l i e d  e x t e r n a l l y  to 
the seeds prior to sowing. This p r o c e d u r e  was r e q u i r e d  for not 
only areas being c u l t i v a t e d  for the first time w i t h  legumes, 
but also for old farm lands wh i c h  h a v e  not been p l a n t e d  w i t h  
legumes for some time. For, Rhizobium sp ec ie s are f a c u l t a t i v e  
s ym bi onts able to live as n or ma l c o m p o n e n t s  of the soil 
m i c r o f l o r a  in the t e m p o r a r y  ab s e n c e  of their hosts, but their 
c o n t i n u e d  ex i s t e n c e  as f r e e - l i v i n g  h e t e r o t r o p h s  dep e n d s  on the 
p r e se nce of the host root (Skerman, 1977).
6As k n o w l e d g e  of the Rhizobium s train  r e q u i r e m e n t s  of each 
p a r t i c u l a r  legume in cre a s e d ,  it b e c a m e  n e c e s s a r y  to e s t a b l i s h  
type or r eferen ce  c u l t u r e s  of each in the l a b o r a t o r y ,  and from 
these st arter  c u l tures are p r e p a r e d  for use in the field as
co m m e r c i a l  ino cul ants.  The aim of i n o c u l a t i n g  the seed is to 
coat it with s u f f i c i e n t l y  high n u m b e r  of cells of the b a c t e r i u m  
to p r o v i d e  an early and e f f e c t i v e  n o d u l a t i o n  of the s e e d l i n g .  
V i n c e n t  (1970) e s t i m a t e d  that 300 cells per seed we re a d e q u a t e  
to i n i tiate in fe ction, but in c u r r e n t  p r a c t i c e  m u c h  h i g h e r  
numbers  are used, and a range of m a t e r i a l s  has be en a d o p t e d  as 
c a r riers of the b a c t e r i u m  cells.
F i n e l y  ground peat is now be i n g  g e n e r a l l y  u s e d  in m a n y  
cou n t r i e s  as a ca rr ie r in the p r e p a r a t i o n  of le gum e i n o c u l a n t s .  
Subba Rao ( 1977 ) d e s c r i b e d  ho w this is done in A u s t r a l i a  and
other c o u n t r i e s .  In Au st ra l i a ,  peat is h a r v e s t e d ,  d r ie d in the 
field and ground to pass th ro ugh a 200 m e s h  sieve. This is
then n e u t r a l i z e d  wi th c a l c i u m  c a r b o n a t e  (CaCO^) and p a c k e d  in 
low d e n s i t y  p o l y t h e n e  bags after w h i c h  they are s t e r i l i z e d  by 
gamma rays at a dose of 5 0  - as r a d i a t i o n  is
c o n s i d e r e d  to be s u p e r i o r  to a u t o c l a v i n g .  The Rhizobium s t r a i n  
is grown in Yeast  M a n n i t o l  Bro th (YEM) to a t t a i n  a hi gh 
p o p u l a t i o n  level. The r e c o m m e n d e d  m i n i m u m  v i a b l e  co u n t  in
A u s t r a l i a  is 500x10^ v i a b l e  r h i z o b i a  per ml, a l t h o u g h  in
p r a c t i c e  the nu mbe rs  u s u a l l y  reach the ra nge of 1,000 - 4, 0 0 0  x 
10^ v i a b l e  cells per ml. The br o t h  is added to the s t e r i l i z e d  
peat at a p p r o p r i a t e  peat volume: b r o t h  v o l u m e  ratio that wi ll
raise the m o i s t u r e  con t e n t  of the peat to 60 p e r ce nt, u s i n g  a 
syringe. The p u n c t u r e  mad e by the s y r i n g e  n e e d l e  is se aled
with a d h e s i v e  tape, and the c o n tents  of the bag are m i x e d  by
ro l l i n g  the bag in the hand. The bag w i t h  its c o n t e n t s  is 
in c u b a t e d  at 2 6°C for two weeks and then stored at 4°C. At the 
end of the incubation period, the peat would contain 10^ - 10® Rhizobium
cells per gram of peat. N o n - s t e r i l e  peat can also be 
i n o c u l a t e d  and used. M e c h a n i c a l  m i x e r s  are used to mi x the 
peat with the broth, and the final d e s i r e d  m o i s t u r e  c o n t e n t  is 
45 to 50 per cent on a wet - w ei gh t basis. The peat - Rhizobium 
m i x t u r e  is then sieved th rou gh  a c oarse  sieve to r emove  lumps,
7m a t u r e d  for four days at 26°C in trays cov e r e d  wit h p o l y t h e n e  
sheets and then p a c k a g e d  in p o l y t h e n e  bags*
The p r e p a r a t i o n  di f f e r s  in some d e t a i l s  in ot her 
co untri es . In the U n i t e d  States of A m e r i c a  (U.S.A.),  for
example, the bro t h  is spr a y e d  onto po w d e r e d ,  n e u t r a l i z e d  and
f l a s h - d r i e d  peat (pa rtial  s t e r i l i z a t i o n )  w h i l e  it is be i n g  
a g i ta ted in a r ib bo n or p a d d l e - t y p e  b a t c h - m i x e r .  A f t e r  mi xing , 
the i n o c u l a n t  is spread in thin layers on the floor for 48 to 
72 hours at 22-24°C. The produc t is then m i l l e d  to b r e a k  up
a g g r e g a t e s  and b ec om e f in el y p u l v e r i z e d  and then p a c k e d  into 
p o l y t h e n e  bags. Inst ead of a s s e s s i n g  the bacter-ium p o p u l a t i o n  
by the Total V i a b l e  Count method, the load of b a c t e r i u m  cells 
may be d e t e r m i n e d  in a ver y d i f f e r e n t  way. This a l t e r n a t i v e  
m e t h o d  is termed the " g r o w - o u t "  test. I n o c u l a t e d  seeds are 
pl anted  in st erile sand or v e r m i c u l i t e  s u p p l i e d  w i t h  n u t r i e n t s  
other than n i t r o g e n  and the plants are h a r v e s t e d  af ter  5 - 6  
weeks. The roots are e x a m i n e d  for n o d u l e s  and the e f f i c a c y  of 
the i n o c u l a n t  assessed. If 90 p e r c e n t  or m o r e  of the p la nt s 
have one or mo re  n o d u l e s  on or n e a r  the p r i m a r y  root, the 
sample is c o n s i d e r e d  s a t i s f a c t o r y ;  w i t h  67 to 90 per cent 
plants w i t h  one or m o r e  nodules,  the sa mple is c o n s i d e r e d  fair. 
Samples g iv in g less than 67 per cent n o d u l a t e d  p l a n t s  are
u n s a t i s f a c t o r y  (Burton, M a r t i n e z  and Curley, 1972).
In India, p o w de red peat or lig n i t e  (a soft m a t e r i a l  quite 
si milar to coal) n e u t r a l i z e d  with CaCO^ is p a s s e d  t h r o u g h  a 200 
mes h sieve and then a u t o c l a v e d  at 151b p r e s s u r e  (103.5 
k i l o p a s c a l )  for four hours. Upon cooling, a shake c u l t u r e  is 
added to the peat in such a p r o p o r t i o n  so as to p r o d u c e  a 
pr odu ct wi th  40 per cent m o i s t u r e  con ten t.  Aft er c u r i n g  for a 
few hours at room t e m p e r a t u r e  the p r o d u c t  is p a c k e d  in 
p o l y t h e n e  bags. The p r e p a r a t i o n  u s u a l l y  c o n t a i n s  a b a c t e r i u m  
p o p u l a t i o n  of 10 x 107 in it ially , 10 x 1 08 after four we eks , and 
1 0 x 1 010 after 12 weeks (Subba Rao, 1977).
8The m i n i m u m  s t a n d a r d s  for v i a b l e  r h i z o b i a  v a r y  in other 
c ou nt r i e s .  Burton, M a r t i n e z  and C u r l e y  (1972) r e p o r t e d  3 x 
108for C z e c h o s l o v a k i a ,  4 - 25 x 109 for Holland, 1 x 108 for New
Ze a l a n d  and 5 - 10 x 107 for Russia.
In the i n o c u l a t i n g  pro ce ss, the pea t m i x t u r e  is m a d e  into
a slurry wit h water, but p r e f e r a b l y  w i t h  a d i l u t e  s u s p e n s i o n  of 
a d h esive (1 to 2 per cent Me l l o f o s ,  15 per cent gum a r a b i c )  for 
greater a d h e r e n c e  (Sk erman, 1977). The seeds are m i x e d
t h o r o u g h l y  in the peat - a d h e s i v e  slurry. F i n e l y  g r o u n d  
c a l c i u m  c a r b o n a t e  (CaCO^) is then a p p l i e d  to the i n o c u l a t e d  
seed while it is still wet and the seed is ro lled in the p o w d e r  
so as to o b t a i n  u n i f o r m  c o a t i n g  of lime over it. The p e l l e t e d  
seed may be sown i m m e d i a t e l y  but, if a b s o l u t e l y  n e c e s s a r y ,  may 
be stor ed up to two to three wee k s  at t e m p e r a t u r e s  not 
e x c e e d i n g  1 8°C (Subba R a o , 1977),
It is im p o r t a n t  that for the i n o c u l a n t  to work, it mu st  
firmly a d h e r e  to the seed. As a n o r m a l  p r o c e d u r e ,  t h e r e f o r e ,  
ad h e s i v e s  are used d u r i n g  the a p p l i c a t i o n  of the i n o c u l a n t .  
The a d h e s i v e s  in cl ud e gum ar abic (40% w t / v o l )  ( K u n e l i u s  and 
Umesh, 1975), c a r b o x y m e t h y 1 c e l l u l o s e  (4% w t / v o l )  (P h i l p o t t s ,  
1982) and wall paper glue (10% wt /vo l ) ,  and ot hers such as corn 
syrup, honey, p o w d e r e d  m i l k  and e v a p o r a t e d  m i l k  (E l e g b a  and 
Renn i e , 19 8 4 ).
Qu a l i t y  of legume i n o c u l a n t  is ba s e d  on m a n y  factors: 
a b i l i t y  of the Rhizobium species to n o d u l a t e  and fix n i t r o g e n ;
p r e s e n c e  of a d e q u a t e  n u m b e r s  of v i a b l e  e f f e c t i v e  Rhizobium cell
in the i n o c u l a n t  to induce  inf ec tion;  l o n g e v i t y  of the cells  in 
the i n o c u l a n t  under st or ag e c o n d i t i o n s ;  the p u r i t y  of the 
inoculum; and the ease of app li c a t i o n .
The l i t e r a t u r e  p r o v i d e s  a long list of le gu mes p r e s e n t l y  
i n o c u l a t e d  be for e sowing as a ro utine p ract ic e. Th ey  in c l u d e  
the fo l l o w i n g  species: Adesmia DC., Aeschynomene L., Alysicarpus
Neck., Anthyllis Riv., Arachis Lin., Cajanus DC., Canavalia DC., 
Cassia Tourn. ex L., Centrosema Benth., Cicer (T ourn.) L., Desmodium
9D e s v . , Glycine L . , Hedysarum (Tourn.) L . , Indigofera L . , Lablab 
Ad an s., Lens (Tourn.) L., Leucaena Benth., Lotononis E c k l . e t  
Zeyh., Lupinus(Tourn. ) L., Medicago Tourn. ex L., Melilotus T o u r n
ex Hall, Onobrychis L., Ornithopus L., Phaselous (T ou rn .) L.,
Pisum( T o u r n . ) L., Poeraria DC, Sesbania Scop., Stylosanthes SW. ,
Tephrosia Pers., Teramnus Sw., Trifolium (To urn .) L, Vicia T our n. ex 
L., Vigna Savi, and Zornia J.F. Gmel. (Skerman , 19 77).
I n o c u l a t i o n  is so i n e x p e n s i v e  that once the c o r r e c t  type
of c a r r i e r  has be en d e v e l o p e d ,  it co uld be a d o p t e d  by local
farmers. Peat - bas e d  c u l t u r e s  are m o s t  p o p u l a r  b e c a u s e  of the 
p r o t e c t i o n  the peat o ff er s the Rhizobium specieS'j" r e s u l t i n g  in 
high post - i n o c u l a t i o n  survi val. H o w e v e r ,  h i g h  q u a l i t y  peat 
is not e a s i l y  av a i l a b l e  in some parts of the world, e s p e c i a l l y  
in the Tropics. T h i s  has led to the s earch  for s u i t a b l e  
a l t e r n a t i v e  Rhizobium-c arr ier m a t e r i a l s  in p lace s w h e r e  peat is 
n a  av aila bl e.
In 1968 and 1969 , solid ba s e d  i n o c u l a n t s  w i t h  wet land 
or ga ni c soil as a peat s u b s t i t u t e  w e r e  tried in Z i m b a b w e  
(Ryder, 1984). The m a t e r i a l  was not s a t i s f a c t o r y  and was 
a ba nd one d. In o c u l a n t  u s i n g  f inel y g r o u n d  r o t t e d  m a i z e  cobs 
(known as c o b - e a r t h ) , in hi gh  d e n s i t y  p o l y t h e n e  (HDPE) bags was 
also d e v e l o p e d  on an e x p e r i m e n t a l  ba sis in Z i m b a b w e  (Corby, 
1976). The c o b - e a r t h  p r o v e d  a s u i t a b l e  base. H o w e v e r ,  the 
n a t u r a l  f o r m a t i o n  of c o b - e a r t h  takes as long as four years,  and 
a t t em pt s to speed its d e c o m p o s i t i o n  p r o v e d  futile. W o r k  on 
this m a t e r i a l  was also d i s c o n t i n u e d  (Ryder, 1984).
Ba g a s s e  and its fine dust c o m p o n e n t  b a g a s i l l o  h a v e  be en  
tested in Zimbabwe. B a g a s s e  i n h i b i t e d  m u l t i p l i c a t i o n  of 
Rhizobium sp ecies and the c u l t u r e s  did not s u r v i v e  on it. 
However, b a g a s i l l o  was found to be v e r y  s u i t a b l e  for the g r o w t h  
of: R. japonicum for soybean. I n o c u l a n t s  for so yb eans, g r o u n d n u t s  
and other c o m m e r c i a l l y  i m p o r t a n t  m e m b e r s  of the c o w p e a  group, 
f i n e - s t e m  stylo (Stylosanthes &  r€cfeci ( i n e e n s i s )  j and lu c e r n e  
(Medicago satina Linn.) are now  p r o d u c e d  on the b a g a s i l l o  ca r r i e r  
at G r a s s l a n d s  R e s e a r c h  Station, Zim ba bwe.
10
Fi lte r mud from sugar mi l l s  is c u r r e n t l y  b e i n g  us ed as a 
ca rr ie r for ino c u l a n t  p r o d u c t i o n  at the N a i r o b i  Rhizobium 
M I R C E N  ( M i c r o b i o l o g i c a l  R e s o u r c e s  Cen t r e )  (Anyango, 1984). It 
is a b y - p r o d u c t  o b t a i n e d  d u r i n g  the f i l t r a t i o n  and 
c l a r i f i c a t i o n  p r o c e s s e s  of cane juice. F i l t e r  m ud c o n s i s t s  of 
fine fibres, mud solids, c h e m i c a l  s u b s t a n c e s  and has a very  
high water  h o l d i n g  c a p a c i t y  (Philp otts, 1976). The a d o p t i o n  of 
filter mud foll ow ed e a r l i e r  r e s e a r c h  s t u d i e s  at the U n i v e r s i t y  
of Na i r o b i  on local ly  a v a i l a b l e  m a t e r i a l s ;  i n c l u d i n g  b a g a s s e ,  
bag a s s e  and c h a r c o a l  dust, c of fee husks, coir dust, d i a t o m i t e ,  
filter mud and sa wdus t in w h i c h  f ilter mud p r o v e d  to be the 
most p r o m i s i n g  (Anyango, 1984). R e p o r t s  on its'-suitability as 
a Rhizobium - car r i e r  ha ve also come from ot h e r  sou r c e s  (Talik  
and Subba R a o , 1974; U r i y o  and C h o w d h u r  y, 1979).
This w o r k  was done to d e v e l o p  a local m a t e r i a l  w h i c h  will 
f a c i l i t a t e  wi de a d o p t i o n  of the i n o c u l a t i o n  m e t h o d  also in Wes t 
Africa. Secondly, it may re sul t in the d e v e l o p m e n t  of a m e t h o d  
of i m p r o v i n g  the p r o d u c t i v i t y  of b a m b a r a  g r o u n d n u t .
The m o s t  im p o r t a n t  l e g u m i n o u s  crops in terms of p r o d u c t i o n  
and c o n s u m p t i o n  in A f r i c a  are in the order, g r o u n d n u t ,  c o w p e a  
and b a m b a r a  g r o u n d n u t  (S el lschop, 1962). The same ord e r  of 
i m p o r t a n c e  of these three crops has be en r e p o r t e d  in Gh a n a  
(Guerts, 1962). A c c o r d i n g  to S t a n t o n  (1968), b a m b a r a
g r o u n d n u t s  are pop u l a r  in the N o r t h e r n  dri e r  areas of We st 
A fr ic a whe r e  the soil is too poor for the c u l t i v a t i o n  of 
gro u n d n u t s ,  and Br a m m e r  (1962) r e p o r t e d  that b a m b a r a  g r o u n d n u t  
is am ong the dr ought r e s i s t a n t  crops s u i t a b l e  for the s a v a n n a  
o c h r o s o l s .  The i m p o r t a n c e  of b a m b a r a  g r o u n d n u t  in t r o p i c a l  
ag r i c u l t u r e  e s p e c i a l l y  those of arid s a v a n n a s  is thus o b v ious.
Ba m b a r a  g r o u n d n u t  has va r i o u s  local names in d i f f e r e n t  
parts of A f r i c a  and Europe. The F r e n c h  call it " v o a n d z o u "  and 
in Ken y a  and T a n z a n i a  flnj u g a  ma we" (Sw ah ili) (F.A.O., 1988) In
N o r t h e r n  Ghana, it is c o m m o n l y  k n o w n  as "semie".
11
It is n ative to Wes t A f r i c a  and is c u l t i v a t e d  t h r o u g h o u t  
Africa; from Senegal to Kenya, from the S ah ara to South Afr ica , 
and Mal a g a s y .  The g r e a t e s t  v a r i a t i o n  of types is t h o u g h t  to
occur in Bur k i n a  Faso, Togo, the m i d d l e  belt of N i g eri a,
T a n z a n i a  and Zambia, It is also found in C e n t r a l  and So uth 
America, in parts of As ia  ( P h i l i p p i n e s  and I n d o n e s i a )  and 
N o r t h e r n  A u s t r i a l i a  (F.A.O., 1988),
The va r i o u s  na mes of b a m b a r a  g r o u n d n u t  in d i f f e r e n t  parts 
of A fr ica have been used as a c r i t e r i o n  to trace the o r i g i n  of 
the plant (Stanton, 1968). A c c o r d i n g  to R u s s e l  (1960),
Voandzeia whi c h  is the former gen e r i c  na me  came fr om  the 
d e f o r m a t i o n  of a M a l a g a s y  term " v o a n j o "  w h i c h  m e a n s  the seed 
that sa tisf ie s, R&s s e 1^36^)the r e f o r e , b e l i e v e d  the crop
or i g i n a t e d  from Mal a g a s y .  C o b b l e y  and S te ele (1976), howev e r ,  
o b s er ve d that Bam b a r a  is a d i s t r i c t  in the u p p e r  N i g e r  near 
T i m b u k t u  and, th erefore, the crop is p r o b a b l y  of W e s t  A f r i c a n  
origin. They further  a rgu ed  that b a m b a r a  g r o u n d n u t  is w i l d  in
i s o la te d l o ca tions in the sav a n n a  zone in We st  A f r i c a  and may
have been d o m e s t i c a t e d  around the head w a t e r  of the Niger
river,
Bam b a r a  gro u n d n u t  is an an nual herb w i t h  e i t h e r  op en  or
bunched forms, w i t h  h i g h l y  b r a n c h i n g  short stem that roo ts at 
the nodes. It forms s u b t e r r a n e a n  pods just b e n e a t h  the s u r f a c e  
of the ground. The leaves are c o m p o u n d  w i t h  th ree l e a f l e t s  
which are u s u a l l y  held u p r i g h t  on a long p e t io le. The l e a f l e t s  
are e l l i p t i c  to obovate, smooth, d a r k - g r e e n  above, p a l e r  b e l o w  
with entire margin. The m i d d l e  leaf l e t  is u s u a l l y  l arger than 
the two lateral ones. The flowers hav e y e l l o w  or r e d d i s h  pi nk  
petals and fruits are round or ovoid pods c o n t a i n i n g  one or two 
seeds. The near s p h e r i c a l  seeds, about 1,5cm di a m e t e r ,  are 
smooth and ve ry hard wh en  dried. The seeds are of d i f f e r e n t  
colours, cream, brown, m o t t l e d  or b l a c k - e y e d ,  d e p e n d i n g  on the 
variety.
12
The seeds are a v a l u a b l e  food. A l t h o u g h  they h a v e  less 
oil (6 to 12 per cent) and p r o t e i n  (14 to 24 per cent) than 
g r o u n d n u t s ,  they have more c a r b o h y d r a t e ,  and m a k e  a we ll 
b a l a n c e d  food, with a c a l o r i f i c  val u e  eq ual  to that of a h i g h -  
q u a l i t y  cereal grain. The pro t e i n  is r e l a t i v e l y  hi gh  in l ys ine 
and try pt ophan , so m a kes an e x c e l l e n t  m i x  w i t h  c e r e a l s .  
M o d e r a t e  amount of B vit a m i n s ,  and small amou nt of m i n e r a l s  and 
v i t a m i n  A are pre s e n t  (F.A.O., 1988). In Ghana, the seeds are
soaked for 24 ho urs b e f o r e  cooking, then b o i l e d  u n t i l  soft.
They are then made into a type of p o r r i d g e  w i t h  some of the
beans f l o at ing in it ("abo boe") • The p o r r i d g e  is oft e n
s w eete ne d wi th sugar.
In Cameroon, they are ground, r a w  or boi le d, and added to 
soups and stews. In T a n z a n i a  the b o i l e d  seeds are c r u s h e d  and 
m ix ed with  g r o u n d n u t  paste, whi l e  in M a l a g a s y ,  they are oft e n  
added to mea t stew wi th  rice, or eat e n  w i t h  g r e e n  l e afy
ve get a b l e .  The b a m b a r a  g r o u n d n u t  flour has a c h e s t n u t  f l a v o u r  
when b oi le d and in S o u t h e r n  Africa, the flour is m i x e d  w i t h  oil 
or b utte r and eaten w i t h  meat, or it is mad e into b a ll s or 
cakes. It is s o me times b oi le d wi th m a i z e  or m i l l e t  fl our to 
form a stiff do ugh  which, when  salted and m a d e  into balls, wi ll  
keep for sev era l days (F .A .0.,1988). The crop serves A f r i c a  
very well.
A f r i c a n  farmers u s u a l l y  i n t e r c r o p  b a m b a r a  g r o u n d n u t  w i t h  
other crops. The crop is raised from seeds. T h e y  are gr own
d uri ng the rainy season, wi th so wing c a r r i e d  out w i t h  the ear l y
rains. In the drier re gio ns of the Trop ic s, r a i n f a l l  is of ten
the l i m i t i n g  factor in crop pr o d u c t i o n ,  not on ly  b e c a u s e  of its 
s c a rc ity but also bec a u s e  of the v e r y  h i g h  e v a p o r a t i v e  d e m a n d  
(M acCartney , No rthwood, Dagg and Dawson, 1971). The g r o w t h  and 
yield of ba m b a r a  g r o u n d n u t s  have be en  found to be v e r y
se nsitive  to m o i s t u r e  stress (Billaz and Ochs, 1961; H o l for d,  
1971 ) .
13
Ma ny  factors affect n o d u l e  fo rmat io n, such as soil 
moistur e,  pH and the a v a i l a b i l i t y  o f ( t o x i c i t y  of m i n e r a l s  
(Jardin, 1982), but the a v a i l a b i l i t y  of p h o s p h o r u s ,  
c a r b o h y d r a t e  ser ving as a sour ce of e l e c t r o n s  for n i t r o g e n a s e  
activity, and an a d e q u a t e  plant w a t e r  status can be s i n g l e d  out 
as the most li mit i n g  to n o d u l e  f u n c t i o n  in le gumes (P hillips , 
1980; Sprent, 1972). Wh i l e  d r o u g h t  has b e e n  shown to a f f e c t  
n o d u l a t i o n  d i r e c t l y  by d e s i c c a t i n g  d e l i c a t e  n o d u l e  ti s s u e s  
(Sprent, 1986), low soil w a t e r  p o t e n t i a l  i n t e r a c t s  w i t h  
p h o s p h o r u s  a v a i l a b i l i t y  (Bonetti, M o n t a n h e i r o  and Saito, 1984) 
to pro d u c e  indir e c t  e ffect on n o d u l e  a c t i v i t y  m e d i a t e d  by the 
host plant.
Subba Rao (1977) r e p o r t e d  that root t e m p e r a t u r e  a f f e c t e d  
n o d u l a t i o n  and i n f e c t i o n  p r o c e s s e s  in c l o v e r s  (Trifolium sp) 
grown on agar slopes in e n v i r o n m e n t  c o n t r o l l e d  g r o w t h  c a b i n e t s .  
T e m p e r a t u r e s  b e l o w  10°C and ab ove  34°C r e t a r d e d  root ha ir
i n f e c t i o n  by Rhizobium. O p t i m u m  t e m p e r a t u r e  for b a c t e r i a l
tissue f o r m a t i o n  in the n o d u l e s  was 20 - 30°C, but n i t r o g e n
fix at io n could take place from 12 to 32°C. A m o n g  the t r o p i c a l  
legumes, ef fec ts  of day t e m p e r a t u r e  on root n o d u l a t i o n  ha ve 
been st udi ed in so yb ea n (Glycine max L.) and B en gal g r a m  (Cicer
arietinum (Tourn.) L.) in pot trails u s i n g  s e l e c t e d  Rhizobium 
strains. One of the b a c t e r i a l  st ra ins was mo st e f f e c t i v e  at
33°C on soybean, whi le others show ed no d i f f e r e n c e  in 
e f f e c t i v e n e s s  at 21 to 33°C. In B e n g a l  gram, non e of the 
b a c t e r i a l  strains p r o d u c e d  nod u l e s  at t e m p e r a t u r e s  abo v e  32°C. 
N i t r o g e n a s e  a c t i v i t y  was best in the t e m p e r a t u r e  range of 24 to 
3 3°C .
In the i n v e s t i g a t i o n  d e s c r i b e d  in this thesis, an
i nocul an t ca rri er  was p r e p a r e d  w i t h  the moss, Brachymenium s p . , 
and its e f f i c a c y  was tested in a n u m b e r  of e x p e r i m e n t s  u s i n g  a 
n umb er of strains of Rhizobium sp. from d i f f e r e n t  legume 
g ro w i n g  lo caliti es  and two b a m b a r a  g r o u n d n u t  v a r i e t i e s ,  E x - A d a  
and Ex-Ta mal e.
14
I I . M A T E R I A L S  AND G E N E R A L  M E T H O D S
i . M A T E R I A L S
(a ) S o i l s :
Soils were c o l l e c t e d  for two d i f f e r e n t  types of 
e x p e r i m e n t  s ;
(1) Rhizobium sp ecies  used s u b s e q u e n t l y  for this 
i n v e s t i g a t i o n  were i s o l a t e d  from soils c o l l e c t e d  in 
Se p t e m b e r  and October, 1990 , from e-ight d i f f e r e n t  
farms w h ic h were un der c u l t i v a t i o n  w i t h  c o w p e a  and 
s o y b e a n s :
1. a c ow pe a farm at As h i a m a n ,  2 5 . 4 k m  east of
Legon;
2. a c owpea  farm at the U n i v e r s i t y  of Ghana
A g r i c u l t u r a l  R e s e a r c h  Station, at Kpong,
80km n o r t h - e a s t  of Legon;
3. a c o w p e a  e x p e r i m e n t a l  plot in the T e a c h i n g
G ard en of the B o t a n y  D e p a r t m e n t ,  U n i v e r s i t y  
of Ghana, Legon;
4. a c o w p e a  plot at the U n i v e r s i t y  of Gha n a
A g r i c u l t u r a l  R e s e a r c h  S t a t i o n  at Nu ng ua,
12km n o r t h - e a s t  of Legon;
5. a s o y a b e a n  plot at the U n i v e r s i t y  of Gh a n a
A g r i c u l t u r a l  R e s e a r c h  Sta t i o n  at Nungua,
12km n o r t h - e a s t  Legon;
6 . a co wpea plot of the Pl ant P r o t e c t i o n  Unit,
M i n i s t r y  of A g r i c u l t u r e ,  at Pok ua se, 19km
n o r t h - w e s t  of Legon;
7. a c ow pe a farm at Shia shie, 4km sou t h  of
Legon; and
8 . a c owpe a plot (plot 46) at the W e i j a
I r r i g a t i o n  Pro j e c t  Site, 24km s o u t h - w e s t  of 
Legon.
A s u f f i c i e n t l y  large q u a n t i t y  of the soil 
collected  at each site to a de pth  of 1 0 cm, was 
con vey ed to the l a b o r a t o r y  in a large p o l y t h e n e  bag 
(100 x 75cm) and sieved wi th  a 2mm - m e s h  just b e f o r e  
use.
(2) Loa m soil used for other e x p e r i m e n t s  was 
s u p pl ied by the Gr ounds and G a r d e n  D i v i s i o n  of the 
D e p a r t m e n t  of Botany, U n i v e r s i t y  of Ghana, Legon.
(b ) B a m b a r a  g r o u n d n u t  seeds
Seeds of two c o m m o n l y  c u l t i v a t e d  v a r i e t i e s  in
the country, E x - A d a  and E x - T a m a l e ,  we re  p u r c h a s e d  
from the M a k o l a  M a r k e t  in A c c r a  and st ore d in small 
p o l y t h e n e  bags (26 x 39cm) in the r e f r i g e r a t o r  (4°C) 
until needed. The E x- Ada v a r i e t y  is m o s t l y
c u l t i v a t e d  in the s o u t h e r n  part of Ghana, in the
Vo lta  Region. The seeds are cr e a m  in c olo ur  and 0.8
1.2cm in diameter . The E x - T a m a l e  v a r i e t y  is 
c u l t i v a t e d  in the n o r t h e r n  part of Gh a n a  and the
seeds are also cream  c o l o u r e d  but are b l a c k - e y e d .  
They are s l i g h t l y  larger, 1.0 - 1. 6cm in d i a m e t e r .
P r e l i m i n a r y  g e r m i n a t i o n  tests showed that the 
samples p u r c h a s e d  were h i g h l y  vi ab le. A p e r c e n t a g e  
g e r m i n a t i o n  of 90 - 96 per cent was o b t a i n e d  in the
tests.
(c ) R h i z o b i u m  st rains
Strains of Rhizobium sp eci es use d we re i s o l a t e d  
from the soil samples c o l l e c t e d  from the ei ght  
d i ffer en t legume plots. These st rains hav e been 
a c c o r d i n g l y  named in this thesis after their so urces 
of origin, viz., Ashiam an, Kpong, Legon, N u n g u a  1, 
Nu ngu a 2, Pokuase, S h i ashie  and Wei j a  strains.
- 1 5 -
16
The two b a m b a r a  g r o u n d n u t  v a r i e t i e s  were p l a n t e d  
in the soil samples from the ei ght sites, and the 
strains of Rhizobium sp. i s o l a t e d  from e f f e c t i v e  
no du le s w h i c h  d e v e l o p e d  on the roots as d e s c r i b e d  
later und er G e n e r a l  M e t h o d s .  The c o n t e n t s  of the 
no du le s were s t r e a k e d  on Pet ri p lates  of Co n g o  Red 
Yea s t  M a n n i t o l  Agar m e d i u m  and i n c u b a t e d  at 30°C for 
5 days. The c o l o n i e s  of Rhizobium w h i c h  d e v e l o p e d  
were s u b - c u l t u r e d  to o b t a i n  pure c u lt ures, again, on 
Yea s t  M a n n i t o l  Agar. The c u l t u r e s  w e r e  then
t r a n s f e r r e d  to Ye ast  M a n n i t o l  Agar slants in 
M c C a r t n e y  tubes and i n c u b a t e d  at 3 0°C for 5 days 
after w h i c h  st e r i l e  liquid p a r a f f i n  was p o u r e d  onto 
the slants. The pure c u l t u r e s  were k e p t  at room 
t e m p e r a t u r e .
(d) D e c o m p o s e d  Moss m a t e r i a l
Both d e c o m p o s e d  and fr esh mo ss  m a t e r i a l  of 
Brachymenium sp. was c o l l e c t e d  from E f f i d u a s e  and New 
Zongo, all in K o f o r i d u a ,  8 5 . 0 k m  n o r t h - w e s t  from 
Legon. Brachymenium sp. was found to be p a r t i c u l a r l y  
abundant on old, w e a t h e r e d  c ement blocks.
The m a t e r i a l  was t h o r o u g h l y  s u n - d r i e d  at the 
D e p a r t m e n t  of B ot any and kept in s ea le d bl a c k  
p o l y t h e n e  bags (90 x 60cm) and ke pt in the l a b o r a t o r y  
until needed.
i i . G E N E R A L  M E T H O D S
(a ) R a i s i n g  of B a m b a r a  g r o u n d n u t  p la nt s
The two v a r i e t i e s  of b a m b a r a  g r o u n d n u t s ,  E x - A d a  
and Ex-T ama le, were grown in the v a r i o u s  e x p e r i m e n t s  
in b l ac k p o l y t h e n e  bags, 20c m h i g h  and 1 2 , 5 c m  in 
di ameter.  The p o l y t h e n e  bags w e r e  f il le d w i t h
i d en tical w e i g h t s  of the a p p r o p r i a t e  p l a n t i n g  
m a t e r i a l  up to a depth of 18cm. D r a i n a g e  holes,
0 .5 c m  in d i a m e t e r  were m a d e  at the b o t t o m  of the 
p o l y t h e n e  b a g s .
(b ) S e l e c t i o n  of v i a b l e  seeds for p l a n t i n g
U n d a m a g e d  b a m b a r a  g r o u n d n u t  seeds of c l o s e l y  
si mil ar sizes were s u r f a c e - s t e r i l i z e d  by i m m e r s i n g  
for fitfe min u t e s  in 0.1 per cent m e r c u r i c  c h l o r i d e  
(HgCl) s o l ution and then w a s h e d  in six c h a n g e s  of 
st eri le d i s t i l l e d  water. The seeds wer e ne xt  w a s h e d  
in 70 per cent Et han ol  for three m i n utes,  and r in se d 
twice wi th st erile d i s t i l l e d  water.
The st erile  seeds we re  then p la ced on w a t e r  agar 
(1.0% agar) in large Petri plat es (14cm d i a m e t e r )  and 
i nc ub ated at room t e m p e r a t u r e  for three days. The 
v i g o r o u s l y  g e r m i n a t i n g  seeds we re  s e l e c t e d  for 
p 1 a n t i n g .
(c ) C o n d i t i o n s  for g r o w i n g  the p lants
The b a m b a r a  g r o u n d n u t  pl ants were gro w n  un der 
va rio us c o n d i t i o n s  and these wo u l d  be d e s c r i b e d  at 
a p p r o p r i a t e  places in Cha p t e r  III - E x p e r i m e n t a l  
De t a i 1 s .
(d ) C u l t u r e  m e d i a
D i f f e r e n t  me d i a  were used for d i f f e r e n t  p u r p o s e s  
at var ious stages of the work. The c o m p o s i t i o n  of 
media used were as follows:
- 17 -
Yeast ext ract ~ m a n n i t o l  agar (YMA)
Yeast e x t r a c t - m a n n i t o 1 agar (YMA) as d e s c r i b e d  by 
Fred, Baldwin, and McCoy, (1932) was used for the rou t i n e
c u l t i v a t i o n  of the Rhizobium sp.
Mann itol  ...............................  1 Og
I^HPC^ ......................................  0*5g
M g S O ^ l ^ O  .................................  0 .2 g
NaCl   O.lg
Yeast ext rac t (eg. Difco, O x o i d )   0.4g
Agar .........................................  15g
D is ti lled water ............................  1L
The m e d i u m  was a u t o c l a v e d  at 1.1kg cm ^ at 1 2 1°C for 15 
minutes .
Congo Red Ye a s t  e x t r a c t - m a n n i t o l  agar
This c o n t a i n e d  the same i n g r e d i e n t s  as Y e a s t  e x t r a c t -  
m a n n i t o l  agar and s u p p l e m e n t e d  with 10ml of 1/400 aqu e o u s  
solu ti on of Congo red s t e r i l i z e d  s e p a r a t e l y  and added 
asept ically to give a final Congo red c o n c e n t r a t i o n  of 
25gml~^ just be fore use (Hahn, 1966).
Yeast e x t r a c t - m a n n i t o l  bro t h  ( Y E M )
This con t a i n e d  the same i n g r e d i e n t s  as Y e a s t  e x t r a c t -  
m a n n i t o l  agar but Agar was e x c l u d e d  and 4. 0g  C a l c i u m
c ar b o n a t e  ( C a C 0^ ) added.
S e e dl in g Agar
A n i t r o g e n - f r e e  plant n u t r i e n t  s o l ution was used to 
make d e e p - l a y e r e d  s e e dl in g agar in la r g e - s i z e d  test tube 
(19.0 x 3,5cm), a c c o r d i n g  to the m et hod of F a h r a e u s  
(1957). This m e d i u m  contained:
19
CaCl^I^O .............................  °*lg
MgS04.7H20 ................................  0 . 12 g
k h 2p o4 ...............................  ° ’lg
Na2HP04 . 12H20 .................................  0.1 5 g
Ferric c i t r a t e . . . . ..... ................ . 0 .005g
Agar ................................ 1 5 S
Di s t i l l e d  wa t e r  ................... .......
The m e d i u m  was s t e r i l i z e d  at 1.1kg cm-2 s t eam 
pres su re at 121°C for 15 m i n u t e s .
5. Sachs * s o l u t i o n
Normal c o m p l e t e  S a c h s ’ s o l u t i o n  (Sachs, 1860) 
used in w a t e r i n g  the b a m b a r a  g r o u n d n u t  pl ant s
c on t a i n e d  :
C a l c i u m  sulp hate ..........    0 . 2 5 g
C a l c i u m  p h o s p h a t e  .............    0. 2 5 g
M a g n e s i u m  s u l p h a t e  ........ . . .............  0 . 25g
Sodium c h l o r i d e  ............................. 0.0 8g
P o t a s s i u m  n i t r a t e   ......................  0.7 0g
Ferric c h l o r i d e   ......    Tr ace
Dis t i l l e d  water ............................  1L
(e ) Met h o d s  of s t e r i l i z a t i o n
The seeds of the two v a r i e t i e s  of b a m b a r a  g r o u n d n u t  
and their no dules were s u r f a c e - s t e r i l i z e d  as d e s c r i b e d  
ear lie r at (b).
Nu tr ient  media, d i s t i l l e d  water, M c C a r t n e y  tubes, 
m e a s u r i n g  cylinder, E r l e n m e y e r  flasks, glass rods and 
pi pe ttes  were s t e r i l i z e d  by a u t o c l a v i n g  at 1 .1kg c m -2 
steam pre ss ure at 121°C for 15 m i n u t e s .  C otton wo ol  plugs 
of E r l e n m e y e r  flasks c o n t a i n i n g  m e d i a  or d i s t i l l e d  w a ter 
were t e m p o r a r i l y  co v e r e d  with g re as e paper to p r e v e n t  the 
p e n e t r a t i o n  of any c o n d e n s e d  wa t e r  d u r i n g  a u t o c l a v i n g .  
Pipettes, m e a s u r i n g  c y l i n d e r s  and glass rods w e r e  also 
wr a p p e d  in grease paper be for e a u t o c l a v i n g .
Petri dishes (7, 9 and 14cm d i a m e t e r )  wer e s t e r i l i z e d  
by h e a t i n g  at 160°C for 6 hours in an e l e c t r i c  oven.
Tips of forceps, i n o c u l a t i n g  n e e d l e s  and loops were 
flamed to red- he at and a i r - c o o l e d  b e f o r e  use.
The i n o c u l a t i n g  room was s t e r i l i z e d  by s p r a y i n g  
h e a v i l y  wi th 5 per cent aq ueous d ett ol  s o l u t i o n  and 
a llo wed to stand for 10 m i n u t e s  b e f o r e  use.
The p l a n t i n g  soil was a u t o c l a v e d  in s t r o n g  p o l y t h e n e  
sacks at 1.1kg cm-^ steam p r e s s u r e  at 1 2 1°C for 30 
minu te s. A u t o c l a v i n g  was r e p e a t e d  three times b e f o r e  the 
soil was u s e d .
The d e c o m p o s e d  moss m a t e r i a l  used as Rhizobium c a r r i e r  
was a u t o c l a v e d  at 1.1kg cm-^ at 1 2 1°C for 4 hours.
C h e m i c a l s
All c he micals  used in the p r e p a r a t i o n  of m e d i a  were 
ei the r of the ' A n a l a r ' or the 1B D H 1 ( B r itish  Dr ug H ouse) 
g r a d e .
C h a r a c t e r i z a t i o n  of soil samples
1 . D e t e r m i n a t i o n  of m i n e r a l  f r a c t i o n ;H y d r o m e t e r  m e t h o d
The m e t h o d  d e s c r i b e d  by Day (1965) was used. A 
h y d r o m e t e r  was c a l i b r a t e d  by p l a c i n g  100ml C a l g o n  
so lut i o n  (C al c i u m  h e x a m e t a p h o s p h a t e , 41g; S o d i u m
car bonate, 9 g ; d i s t i l l e d  water, 1L) into a 1L 
m e a s u r i n g  c y l inder and the v o l u m e  made up to one 
litre with d i s t i l l e d  wa ter of a t e m p e r a t u r e  of 20°C. 
The solu tion was t h o r o u g h l y  m i x e d  and the h y d r o m e t e r  
was then put into it. The scale r e a d i n g  (RL) was 
r e c o r d e d .
21
Two samples of 40g w e i g h t  of a i r - d r i e d  sieved (with a 
2.0mm mesh) soil, were c a r e f u l l y  m e a s u r e d  out. One lot was 
dried o v e r n i g h t  at 105°C in an oven and the o v e n - d r y  w e i g h t  
recorded. The second lot was m i x e d  wi th  100ml of C a l g o n  
sol ut io n and stirred in an e l e c t r i c  m o t o r  m i x e r  for 5 - 1 0  
min ute s. A few drops of amyl alc o h o l  we re added  to p r e v e n t  
frothing. The m i x t u r e  was then t r a n s f e r r e d  into a 1L m e a s u r i n g  
c y l in der and the v o l u m e  ma de  up to one litre w i t h  d i s t i l l e d  
water at a t e m p e r a t u r e  of 20°C, The m i x t u r e  was s t i r r e d  w i t h  
the plugger and the h y d r o m e t e r  put g e n t l y  into the m i x t u r e .
The h y d r o m e t e r  rea d i n g  (R) was r e c o r d e d  a f te r 30 se c o n d s  
■ 1 again after one minute. W i t h o u t  r e m i x i n g  the s u s p e n s i o n ,
further h y d r o m e t e r  r e a di ngs were made after 3, 10, 30, 90, 270
and 720 m i n ut es. After the last r e a d i n g  the s u s p e n s i o n  was 
st irred and poured into a 450 u m - m e s h  sieve and w a s h e d  un der  
the r u n n i n g  tap. The resid u a l  soil p a r t i c l e s  were s c o o p e d  out, 
dried in an oven o v e r n i g h t  at 105°C and w e i g h e d  to o b t a i n  
we igh t of total sand. The e x p e r i m e n t  was c a r r i e d  out at 20°C.
The c o n c e n t r a t i o n  of s u s p e n s i o n  in g / l i t r e  (C) was
c a l c u l a t e d  usi n g  the equation:
C - R - RL
And the s u mmatio n p e r c e n t a g e  (P) from the equa tion:
P = 100 (c/co)
wh ere Co is the o v e n - d r y  w eig ht  of soil in g / l i t r e  of
s us pen sio n.
22
The c o r r e s p o n d i n g  p a r t i c l e  sizes w e r e  c a l c u l a t e d  fro m the 
e q u a t i o n :
X (microns) * °/(t)^
wh ere 0 is the s e d i m e n t a t i o n  p a r a m e t e r  from Ta ble  of v a l u e s  of 
0 ( D a y 3 1956) us i n g  c o r r e s p o n d i n g  v a l u e s  of R. The l e t t e r  ft ’ 
is the s e d i m e n t a t i o n  time in m i n u t e s .
A graph of P ag ai nst X was p l o t t e d  on a s emi -1 o gar i thm i c
axis. The s u m m a t i o n  p e r c e n t a g e  (P) at d i f f e r e n t  v a l u e s  of X 
were read. V al ues for P2 , P6> P ^  and wer e p r o v i d e d  by X
values of 2 , 6 , 20 and 60 r e s p e c t i v e l y .
P e r c e n t a g e  clay = P2
P e r c e n t a g e  fine silt = - P2
P e r c e n t a g e  m e d i u m  silt = P2Q ~ P5
P e r c e n t a g e  c oarse  silt = P^q __ P ^
P e r c e n t a g e  total sand = 1 00-(% clay +
% total silt)
2. E s t i m a t i o n  of o x i d i z a b l e  o r g a n i c  m a t t e r  in soils 
I
Wajcley and B l a c k ' s  R a p i d  T i t r a t i o n  m e t h o d
The m e t h o d  d e s c r i b e d  by H e s s e  (1972) was e m p l o y e d .  
Half a gram of sieved a i r - d r y  soil (using 0 . 5 m m  mesh)  was 
put in a 500ml E r l e n m e y e r  flask and 10ml of IN P o t a s s i u m  
dichroraate s o l u t i o n  (49.04g l ^C^Oy in 100ml of d i s t i l l e d  
w ate r) added, foll owed by 2 0 ml of c o n c e n t r a t e d  s u l p h u r i c  
acid (98 per cent). The co nt en t of the flask was a g i t a t e d  
for one m in ut e and al lowed  to stand for 30 m i n u t e s  on 
asb estos  sheet. Two h u n d r e d  m i l l i l i t r e s  of d i s t i l l e d  
w a t e r ; 10ml of p h o s p h o r i c  acid, and 1 .0ml of d i p h e n y l a m i n e  
i n d i c a t o r  so lut i o n  (0.5g d i p h e n y 1 a m i n e ; 100ml c o n c e n t r a t e d
S u l p h u r i c  acid and 20ml d i s t i l l e d  w ate r)  we re  then added 
in that order. The c o n tents of the flasks we re  t i t r a t e d  
a gai nst IN Ferrou s a m m o n i u m  s u l p h a t e  (8 7 . 4 4 g  Fer r o u s  
a m m o n i u m  sulphate; 500ml, d i s t i l l e d  water; 20ml 
c o n c e n t r a t e d  su l p h u r i c  acid mad e up to one litre of 
sol uti on) until the co lour cha n g e d  from o ra nge to gre en 
and fin all y to r e d d i s h - b r o w n .  A b l an k d e t e r m i n a t i o n  made 
wi thout  soil, served to s t a n d a r d i s e  the Fer r o u s  a m m o n i u m
23 -
s u l p h a t e  solution. P e r c e n t a g e  o x i d i z a b l e  o r g a n i c  c a r b o n  
was cal cu l a t e d  from the formula:
% Organic carbon = (blank titre - actual titre) x 0.3 x N
weight of soil
where N is the n o r m a l i t y  of Fe r r o u s  a m m o n i u m  s u l p h a t e  
solution.
P e r c e n t a g e  o x i d i z a b l e  o r g a n i c  m a t t e r  was o b t i a n e d  by 
m u l t i p l y i n g  the v a lue of p e r c e n t a g e  o x i d i z a b l e  c a r b o n  by 
the factor 1.724.
3. E s t i m a t i o n  of Total N i t r o g e n  in soil
The m ethod d e s c r i b e d  by H e s s e  (1972) was e m p l o y e d .  
One gram of a i r - d a y  soil was p l a c e d  in 300ml K j e l d a h l  
flask and m o i s t e n e d  w i t h  a few dr ops  of d i s t i l l e d  water. 
A sp atul a f u l  of c a t a l y s t  (l^SO^, 10g; C u S O ^ S ^ O ,  l.Og;
Selenium, l.Og) was added, f o l l o w e d  by 10ml of
c o n c e n t r a t e d  s u l p h u r i c  acid and d i g e s t e d  over an e l e c t r i c  
h ea te r for 2 \  hours or unt i l  the d i g e s t  c l a r i f i e d .  The
digest was allowed to cool and the v o l u m e  m a d e  up to 1 0 0 ml
with d i s t i l l e d  w a ter in a v o l u m e t r i c  flask. A m o u n t s  of 
5ml each of the dil u t e d  digest were p i p e t t e d  into M a r k h a m  
d i s t i l l a t i o n  ap p a r a t u s  and 2ml of 50% S o d i u m  h y d r o x i d e  
sol ut io n added. The m i x t u r e  was d i s t i l l e d  and the 
d i s t i l l a t e  col l e c t e d  in 4ml of 2% Boric acid - i n d i c a t o r  
(2 0 g Boric acid c r y stal s d i s s o l v e d  in 900ml hot wa t e r ) ,  
cooled and a mi xed  i n d i c a t o r  s o l u t i o n  ( p r e p a r e d  by
d i s s o l v i n g  O.lg b r o m o c r e s o l  green and 0.0 7 g  m e t h y l  red in 
100ml 95 per cent Et han o l )  added un t i l  the colour  of the 
Boric a c i d - i n d i c a t o r  chan g e d  from green to pink. The 
d i s t i l l a t e  was then t i t rate d a g a i n s t  0. 0 1 N  S u l p h u r i c  acid. 
Values pr e s e n t e d  in this thesis are means of three 
re p l i c a t e  titres.
24
The whole pr ocess  was r e p e a t e d  u s i n g  0 . 2 g  ca ne  sugar 
in place of soil to cor r e c t  for any n i t r o g e n  c o m p o u n d  
pr ese nt in the reagents . T o tal n i t r o g e n  in soil s ample  
was d e t e r m i n e d  from the eq ua tion:
% Nitrogen = Meq. of acid x Meq. of N x Vol. of extract x 100 
weight of sample x volume of diluted digest
(h ) D e t e r m i n a t i o n  of N i t r o g e n  c o n t e n t  of plant s
The same p r o c e d u r e  d e s c r i b e d  ab o v e  for the e s t i m a t i o n  
of total n i t r o g e n  in soil was used, to d e t e r m i n e  the 
n i t r o g e n  co nt ent of dried and p o w d e r e d  pl a n t  m a t e r i a l .
(i ) I s o l a t i o n  of R h i z o b i u m  stra ins
The root systems of the two b a m b a r a  g r o u n d n u t  
v a r i e t i e s  were w a s h e d  c a r e f u l l y  under g e n t l e  r u n n i n g  
water. Well formed, and firm n o d u l e s  on the tap root of 
each plant were c a r e f u l l y  d e t a c h e d  for the i s o l a t i o n  of 
the bacterium. The no d u l e s  were put in st e r i l e  d i s t i l l e d  
water c o n t i a n i n g  a little clean a c i d - w a s h e d  sand in 
M c C a r t n e y  tubes and the tubes v i g o r o u s l y  s haken to r emo ve  
gross surface c o n t a m i n a t i o n .
The nodul es  were next s u r f a c e - s t e r i l i z e d  for five 
m i n u t e s  in 0.1% HgCl so lut i o n  and r e p e a t e d l y  ri nse d in six 
changes of sterile d i s t i l l e d  water. They we re f u r t h e r  put 
in 70 per cent Et hano l for three m i n u t e s  and f i n a l l y  
rinsed with sterile d i s t i l l e d  water#
whe r e ;
Meq. of acid N o r m a l i t y  of acid x Tit r e  
v o l u m e
N o r m a l i t y  of acid
Meq- of N
Vol. of ex tract
0 . 0 1
0.014
50ml
25
The no dul es  were then c r u s h e d  w i t h  a s t e r i l e  glass 
rod in a few drops of st e r i l e  d i s t i l l e d  w a te r in s t e r i l e  
M c C a r t n e y  tubes. Five serial d i l u t i o n s  of 1/10, 1/100,
1 /1 0 0 0 , 1 /1 0 0 0 0 , and 1 / 1 0 0 0 0 0  of the s u s p e n s i o n  were
p r e p a r e d  and the 1 / 1 00 00 0 d i l u t i o n  s t r e a k e d  on Petri
plates of Congo red Y MA m e d i u m  and i n c u b a t e d  at 30°C, for 
five d a y s .
At the end of the i n c u b a t i o n  period, i s o l a t e d
col on ie s of the Rhizobium sp. w e r e  s u b - c u l t u r e d  on YMA 
slants in M c C a r t n e y  tubes. The M c C a r t n e y  tubes w i t h  the 
pure c u l tures  were filled w i t h  s t e r i l e  l iquid  p a r a f f i n  to 
c o m p l e t e l y  s u b me rg e the slant and st ored in the 
r e f r i g e r a t o r  (4°C) .
(j ) P r e p a r a t i o n  of d e c o m p o s e d  mo ss m a t e r i a l
The fresh Brachymenium sp. m a t e r i a l  c o l l e c t e d  from 
K o f o r i d u a  was s u n - d r i e d  for 30days. It was then w a s h e d  in
a bowl of tap wa ter  to remove the a d h e r i n g  soil p a r t i c l e s
and fragm ents of fo reign plant ma tter.  The bu lk  was next 
cured by sto r i n g  in sealed b l a c k  p o l y t h e n e  bag at room 
te m p e r a t u r e  for 15 days.
After curing, the moss m a t e r i a l  was s u n - d r i e d  for 10 
days and ground to pass thr o u g h  a 200 um mesh.
The ground moss m a t e r i a l  was n e u t r a l i z e d  w i t h  C a l c i u m  
c a r b o n a t e  (CaCO^) p ow de r and a l i q u o t e s  of lOg w e r e  put 
into p o l y p r o p y l e n e  bags, 1 2 . 5 c m  hi gh  and 15cm di a m e t e r ^  
and the mo uth of the ba^gs tied w i t h  twine.
These were a u t o c l a v e d  at l.£kg c m - 2 at 121°C for four 
h ° u r s / the period also used for peat and lig n i t e  (Subba 
Rao, 1977 ). The bags wi th their c o n t e n t s  were s to red in 
the r e f r i g e r a t o r  (4°C) until needed.
26
(k) P r e p a r a t i o n  of the R h i z o b i u m  i n ocul um .
Shake c u l t u r e s  of Rhizobium sp. we re grown in Y e a s t  
e x t r a c t - m a n n i t o l  broth in 250 ml E r l e n m e y e r  f lask s for 10 
days. A half, X , and 1/g d i l u t i o n s  of the s u s p e n s i o n  of 
each strain were p r ep ar ed, and we re used, t o g e t h e r  w i t h  
the u n d i l u t e d  s u s p e n s i o n  as s e p a r a t e  t r e a t m e n t s ,  to 
i n ocul at e the cured moss m a t e r i a l .
(l ) V i a b l e  To tal  Cell C o u n t  of I n o c u l a t e d  Moss.
A p p r o p r i a t e  d i l u t i o n s  of the s u s p e n s i o n  of the 
i no cu l a t e d  moss m a t e r i a l  we re p r e p a r e d  and 1ml a l i q u o t e s
were of each d i l u t i o n  used to i n o c u l a t e  i n d i v i d u a l  Petri 
plates of Congo red YMA med ium. The p l a t e s  w e r e  i n c u b a t e d  
at 30^C for 48 hours and the n u m b e r  of c o l o n i e s  that 
d e v e l o p e d  on a plate was count ed. The n u m b e r  of ce lls in 
the o r i g i n a l  s u s p e n s i o n  was o b t a i n e d  by m u l t i p l y i n g  the 
mean number of c o l on ie s per pl ate by the d i l u t i o n  factor.
(m ) P l a n t i n g  sheds of d i f f e r e n t  light i n t e n s i t i e s .
Three sheds were c o n s t r u c t e d  w i t h  p l y w o o d  b a t t e n s  in 
such a way as to give shades of d i f f e r e n t  light 
inten s i t i e s .  Each was 8m long, 1 wide and lm h i g h  w i t h  
battens on the four sides and the top. The sheds stood
para ll el to each other on a c o n c r e t e  p l a t f o r m  w i t h  the two 
ends facing east and west. The b a t t e n s  of shed No.l were 
2.5cm apart. The bat t e n s  of shed N o . 2 were 5cm apart. 
Shed No. 3 had no ba tt ens and p la nt s p l a c e d  in this were
fully exposed to sunlight. All the sheds we re  c o v e r e d  on 
the top and the east and west sides w i t h  p o l y t h e n e  sheets 
to keep off rain. The two r e m a i n i n g  sides w e r e  left
u n c o v e r e d  to allow free c i r c u l a t i o n  of air. The n o r t h e r n  
sides of the two sides of the two sheds w i t h  b a t t e n s  were 
loosely  nail ed so that they could be e as ily r e m o v e d  wh en 
w a t e r i n g  or m e a s u r i n g  the light in te nsity.
The light i n t e n s i t y  und e r  the three sheds was 
m e a s u r e d  every other day w i t h  an ” Eel P o r t a b l e  
P h o t o e l e c t r i c  p h o t o m e t e r  (Evans E l e c t r o s e l e n i u m  Ltd. 
Hal ste ad, E n gla nd ). Thr ee m e a s u r e m e n t s  were m a d e  in each 
shed, 2m apart al ong the m e d i a n  e a s t - w e s t  t ransec t,  and 
the mean c a l culated .
(n ) A s s e s s m e n t  of e x t e n t  of g r o w t h  of 
e x p e r i m e n t a l  pl ants.
The f o l l o w i n g  re co rd s w e r e  m a d e  of the b a m b a r a  
g r o u n d n u t  pl ants for the a s s e s s m e n t  of the e f f e c t s  of the 
trea tment s :
1. Number of leaves.
2. L e n g t h  and w i dth of the m i d d l e  leaflet.
3. N umb er  and d i a m e t e r s  of the n o d u l e s  .
4. Dry w e i g h t  of the plant.
5. N i t r o g e n  co n t e n t  of plant top.
(o ) D e t e r m i n a t i o n  of pl ant  dry w e i g h t
U p r o o t e d  plants were t h o r o u g h l y  w a s h e d  u n d e r  the tap 
to remove all soil part ic l e s .  Th ey w e r e  then put
i n d i v i d u a l l y  in me tal  pans and d r ie d in an e l e c t r i c  oven 
at 8 0° C for 48 hours and then w e i g h e d  w i t h  P r e c i s a  300C 
W e i g h i n g  m a c h i n e  (PAG. D E R L I K O N  AG., Zurich, S w i t z e r l a n d ) .
(p ) M e a s u r e m e n t  of pH.
H y d r o g e n  ion c o n c e n t r a t i o n  of soil s u s p e n s i o n ,  
po w der ed  moss s u s p e n s i o n  and the m e d i a  we re  m e a s u r e d  w i t h  
a Pye U n i c a m  Model 290 pH met e r  (EDT I n s t r u m e n t s  Ltd, 
Dover England ) .
- 27 -
28
(q ) S t a t i s t i c a l  a n aly se s.
Results were s t a t i s t i c a l l y  a n a l y s e d  whe r e
a p p r o p r i a t e .
(r ) E x p e r i m e n t a l  p r e c a u t i o n s .
1 . G l a s s w a r e  were kept  s c r u p u l o u s l y  clean.
G l a s s w a r e  w h i c h  had a l r e a d y  b e e n  c l e a n e d  w i t h  w a t e r  
and d e t e r g e n t s  we re  r inse d s e v e r a l  times u n d e r  the 
tap and three times w i t h  d i s t i l l e d  w a t e r  and all o w e d
to drain dry b e f o r e  use or s t e r i l i z a t i o n .
2. The room used for r a i s i n g  the .plants in the
’S e e d l i n g  Agar' tubes was s p r a y e d  t h o r o u g h l y  w i t h  5
fc» f- C-(\ C
per cent aq ue ous s o l u t i o n ^ a n d  a l l o w e d  to st and for a
day b e f o r e  b e in g used.
3. Rhizobium su s p e n s i o n  for p l a t i n g  was al ways
shaken w i t h  W h i r l i M i x e r  TM (F ISONS S c i e n t i f i c  
E qu ip men t, England) for one m i n u t e  and sa mple 
w i t h d r a w n  i m m e d i a t e l y  t h e r e a f t e r  for pla ting.
4. Soil for g r o w i n g  the pl ant s was a u t o c l a v e d  just
before u s e .
5. To pre v e n t  cross c o n t a m i n a t i o n  w i t h  d i f f e r e n t  
strains or d i f f e r e n t  d e n s i t i e s  of s u s p e n s i o n s  of the 
same strain, hands were w a s h e d  in w a r m  w a t e r  and then 
with 70 per cent Et ha nol and r ins ed  s e v e r a l  times 
with ste rile d i s t i l l e d  w a t e r  w h e n  the i n o c u l a t e d  
seeds were being planted.
29
III. E X P E R I M E N T A L  D E T A I L S
A . D E T E R M I N A T I O N  OF P R O P E R T I E S  OF 
THE D I F F E R E N T  SOIL SAM P L E S
The first e x e r c i s e  w h i c h  was c a r r i e d  out was to 
d e t e r m i n e  some of the p r o p e r t i e s  of the ei ght s a m p l e s  of 
soil c o l l e c t e d  from legume farms at A s h i a m a n ,  Kpong,
Legon, Nungua, Pok uase, S h i a s h i e  and Weija.
N o d u l a t i o n  is a f f e c t e d  by a v a r i e t y  of e d a p h i c  
factors, some of wh i c h  act t h r o u g h  their i n f l u e n c e  on the 
n u t r i t i o n  of the host. For example, c a l c i u m  and
p h o s p h o r u s  play an imp o r t a n t  part in the le gume - Rhizobium 
re latio ns hip. P h o s p h o r u s  d e f i c i e n t  p la nt s do not n o d u l a t e  
p r o p e r l y  and in a d d i t i o n  an a d e q u a t e  s u p p l y  of soil
ph osph o r u s  helps to m a i n t a i n  the p o p u l a t i o n  of n o d u l e
b a c t e r i a  in the soil at a h i g h  level. C a l c i u m  is
im portant  for the n u t r i t i o n  of bot h leg ume s and b a c t e r i a
(An der son and M o y e , 1952). Plants w h i c h  are g r o s s l y
n i t r o g e n  d e f i c i e n t  n o d u l a t e  s p a r i n g l y  or not at all w h i l e  
a high level of a v a i l a b i l i t y  of soil n i t r o g e n  d e p r e s s e s  
n o d u l a t i o n  (Vincent, 1965) and re du ce s fix at io n. 
F ur th e r m o r e ,  an i n c r e a s e  in soil pH i n c r e a s e s  r h i z o b i a l  
nu mb er s and res ults in good n o d u l a t i o n  (Bond, 1951). Any 
large di f f e r e n c e s  in the p r o p e r t i e s  of the eig h t  soil 
sa mples may cause s i g n i f i c a n t  d i f f e r e n c e s  in n o d u l a t i o n .
The n i t r o g e n  content, or g a n i c  m a t t e r  content, pH of the 
soil, soil min e r a l  fraction, and Rhizobium p o p u l a t i o n
counts, of each sample were a ss es sed.
Soil N i t r o g e n  Content  Test
This was ca rr ied out with each of the ei ght  soil 
samples. Five grams of each soil sample were d i g e s t e d  in 
each of three K j e ldah l flasks, as d e s c r i b e d  under 
'Material and Methods'. Each of the three r e p l i c a t e s  was 
d i s t i l l a t e d  s e p a r a t e l y  and tit ra ted. The m e a n  of the
three titres was then d e t e r m i n e d .  The m e a n  titre was used 
to ca l c u l a t e  the P e r c e n t a g e  N i t r o g e n  of the soil sample.
Or ga n i c  M a t t e r  C o n t e n t
The or ganic  m a t t e r  co nt e n t  of 0.5g soil was 
d e t e r m i n e d  by first e s t a b l i s h i n g  the p e r c e n t a g e  C a r b o n  
co ntent whi c h  was then m u l t i p l i e d  by 1 .724 to gi ve  the 
P e r c e n t a g e  Or ga ni c M a t t e r  of the sample. Thr e e  tests were 
made for each soil sample, a c c o r d i n g  to the m e t h o d  
d e s c r i b e d  un der ’M a t e r i a l s  and M e t ho ds' to p r o v i d e  three 
titres from the t i t r a t i o n  of the d i g e s t e d  soil. The mean 
titre value was then used to c a l c u l a t e  the p e r c e n t a g e  
ca rbon content, w h ich was used for the d e t e r m i n a t i o n  of a 
single value of P e r c e n t a g e  Or ganic M a t t e r  C o n te nt .
pH of Soil
The pH of soil s o l ut ion p r e p a r e d  w i t h  sam p l e s  of soil 
from each l o c a l i t y  was d e t e r m i n e d .  Three  d e t e r m i n a t i o n s  
were made for each soil sample and the m e a n  pH c a l c u l a t e d .  
The soil s o l ut io n was p r e p a r e d  by s h a k i n g  40g soil in 100 
ml d i s t i l l e d  wa t e r  wi th  an e l e c t r i c  m o t o r  m i x e r  for one 
hour, and the s u p e r n a t a n t  used for the pH m e a s u r e m e n t  
after the large pa r t i c l e s  had settled.
Soil M i n e r a l  F r a c t i o n
The m e t h o d  us ed has been fully  d e s c r i b e d  u n d e r  
'Materials and M eth od s'. The o r i g i n a l  s o l u t i o n  for the 
d e t e r m i n a t i o n  of the m i n e r a l  f r a c t i o n  c o n s i s t e d  of 40g 
soil and 100 ml C al go n solution.
Three d e t e r m i n a t i o n s  were ca r r i e d  out w i t h  each of 
the eight soil samples, whi c h  p r o v i d e d  three v a l u e s  for 
each min e r a l  fraction. The Table of Res u l t s  (Table 1) 
co nt ai ns the means of each set of three d e t e r m i n a t i o n s .
- 30 -
I S O L A T I O N  OF ST R A I N S  OF R H I Z O B I U M  S P » -FROM 
THE D I F F E R E N T  SOIL SA MP LES
The two v a r i e t i e s  of b a m b a r a  g r o u n d n u t  plants, E x - A d a  
and Ex- T a m a l e  were pl a n t e d  in each of the ei g h t  soil 
samples. Bl ack p o l y t h e n e  bags, 20 cm h i g h  and 1 2 . 5 c m  
diameter, wi th d r a i n a g e  holes at the b o t t o m  we re f i l l e d  up 
to 18cm high wi th  the d i f f e r e n t  soil samples.
Five seeds were sowed in each bag, and the s e e d l i n g s  
were thinned to one after they had emerged.  T h e r e  wer e 
five re pl icates for each soil s a m p l e  and pl a n t  v a r i e t y .  
The bags were labelled a p p r o p r i a t e l y  and a r r a n g e d  
randomly, on the eas t e r n  v e r a n d a h  of p o s t - g r a d u a t e  
la boratory. The plants, th erefor e,  r e c e i v e d  full s u n l i g h t  
up to 11.00 am each day and w e r e  p r o t e c t e d  from rains. 
They were wat e r e d  d a i l y  w i t h  20ml tap w a t e r  per bag. Once 
a week, each bag receiv ed, in additio n,  10ml S a c h s 1 
sol ution  to augment the n u t r i e n t  c o n t e n t  in the soil. The 
plants were sown on S e p t e m b e r  17, 1990 and h a r v e s t e d  on
October 21, 1990.
After the plants had be en  h a r v e s t e d  and the roots 
th orou g h l y  washed, the nod u l e s  we re d e t a c h e d  to be cou n t e d  
and measur ed, and the largest ones of each lot s e l e c t e d
for isol ati on of the Rhizobium sp. f o l l o w i n g  the m e t h o d
d es cr ibed under ’M a t e r i a l s  and M e t h o d s 1.
To provide a d d i t i o n a l  det a i l s  on the p e r f o r m a n c e  of 
the plants in the va ri ous soil samples, the dry w e i g h t s  of
the plants and their r e l at iv e p e r c e n t a g e  n i t r o g e n  con t e n t
were determined.
- 31 -
C * C O M P A R A T I V E  I N E F F E C T I V E  A B I L I T Y  OF F I V E  S T R A I N S  OF
R H I Z O B I U M  SP, I S O L A T E D  F R O M  B A M B A R A  G R O U N D N U T  P L A N T S
The soil p r o p e r t i e s  st u d i e d  in E x p e r i m e n t  4 d i f f e r e d  
among the eight soil samples. The e xt en t of n o d u l a t i o n  of 
the plants g r o w i n g  in the d i f f e r e n t  soils was, ther ef o r e ,  
d e t e r m i n e d  by other fac tors in a d d i t i o n  to the a b i l i t y  of 
the strains to in fec t b a m b a r a  g r o u n d n u t .  The n o d u l e  -
forming abi l i t i e s  of the str a i n s  i s o l a t e d  fr om  n o d u l e s  
formed in the d i f f e r e n t  soil s a m p l e s  were, theref o r e ,  
co mp ar ed in this e x p e r i m e n t  u n d e r  the same g r o w i n g  
condi t i o n .
Broth c u l t u r e s  were raise d w i t h  strain s of Rhizobium 
sp. iso lated from the n o d u l e s  and us ed  to i n o c u l a t e  tubes 
of 'Seedling Agar'.
The Large b o i l i n g  tubes w e r e  fi lle d to a d e p t h  of
1 2. 0c m with the ’S e e d l i n g  A g a r ’ . A pie ce of n o n - a b s o r b e n t  
co tton wool was i n s e r t e d  into each tube to just abo v e  the 
surface of the m e d i u m  and the tube was then p l u g g e d  w i t h  a 
n o n - a b s o r b e n t  c otton wool and a u t o c l a v e d .
The m e d i u m  was al lo wed to set after a u t o c l a v i n g  wi th  
the tube in an u p r i g h t  p o s i t i o n  and a l l o w e d  to stand for
24 hours. Each tube was i n o c u l a t e d  w i t h  1.0ml of 1 0-day
old cul ture br oth  of the a p p r o p r i a t e  strain. The tip of 
the pipette  c o n t a i n i n g  the i n o c u l u m  was p u s h e d  t h r o u g h  the 
n o n - a b s o r b e n t  co tton wool i n s e r t e d  into the b o i l i n g  tube 
and the i n o cu lu m placed d i r e c t l y  on the surf a c e  of the 
’Seed ling Agar' medium.
A 2-day old s u r f a c e - s t e r i l i z e d  and g e r m i n a t i n g  
ba m b a r a  seed of the Ex -Ad a v a r i e t y  was a s e p t i c a l l y  placed 
up ri gh t on the wad of co tton wool in side the tube, p u s h i n g  
the radicle thr ough the c ot to n wool so that it to u c h e d  the
- 32 -
33
1 S e e dl ing A g a r 1 medium . The c o t t o n  wool plug was then put 
back in place, and the 12-cm  c o l u m n  of the 'Se e d l i n g  A g a r ’ 
co ver ed with bl a c k  p o l y t h e n e  sheet. For the c o n t r o l  1ml 
st eril i z e d  d i s t i l l e d  water was us ed  i n s t e a d  of b r o t h  
culture. Three r e p l i c a t e s  of each strain, and the c o n t r o l  
were set up. The tubes were kept in the l a b o r a t o r y  ne ar 
the wi nd ows to r e c e i v e  a d e q u a t e  light, from N o v e m b e r  11, 
1990 to D e c e m b e r  18 , 1990. On N o v e m b e r  20, 1990 the
co tton wool plugs were r e m o v e d  to a l l o w  the pl an ts to 
extend out of the tubes.
The plants were e x a m i n e d  at the end of the 
e xp eri men t, and
(a) plant with nod u l e s  were id e n t i f i e d ,
(b) no dules on n o d u l a t i n g  plants  we re counted, and
(c) tops of n o d u l a t i n g  plants w e r e  dried at 80°C for 48
hours and weig he d.
D. P R E P A R A T I O N  OF THE M O S S  C O M P O S T  AS C A R R I E R  A ND  
S TU D Y  OF SHELF L I F E  ON I N O C U L U M  C A R R I E R
The i d e n t i f i c a t i o n  of p r o m i s i n g  Rhizobium st ra in s
among those isolat ed  was f o l l o w e d  by the p r e p a r a t i o n  of
Brachymenium sp. co mpost as Rhizobium i n o c u l u m  c a r r i e r
a c c o r d i n g  to the p r o c e d u r e  o u t l i n e  in 'M a t e r i a l  and 
M ethod  s
The sterile co mp os t in tAe p o l y t h e n e  bags was 
in ocul a t e d  with u n d i l u t e d ,  \ and 1 /Q d i l u t i o n s  of the
br oth culture, us ing  a v ol ume b r o t h  cu l t u r e  that a d j u s t e d  
the m o i s t u r e  content of the co m p o s t  to 50 per cent. By a 
pre vi ou s d e t e r m i n a t i o n ,  it was e s t a b l i s h e d  that 2 0 ml of 
the broth ino culum br o u g h t  lOg of the c o m p o s t  to 50 per 
cent m o i s t u r e  content. Subba Rao (1977) us ed a
p r e p a r a t i o n  with 45-60 per cent m o i s t u r e  content.
34
After the i n o c u l u m  had been added and the m o u t h  of the
p o l y t h e n e  bag tied again with twine, the bag was k n e a d e d  wi th
fingers to mix the c o n t e n t s  t h o r o u g h l y .  Th ey  were
a p p r o p r i a t e l y  labelled and left in the i n o c u l a t i n g  ro om  at 28°C 
for two weeks to mature, a v i a b l e  cell co unt was made, and then 
used in the va ri ous s u b s e q u e n t  n o d u l a t i o n  tests. T h o s e  w h i c h  
were not used i m m e d i a t e l y  and those u s e d  in s t u d i e s  on the
shelf life of i n o c u l a t e d  moss c a r r i e r  we re s t o r e d  in the
r e f r i g e r a t o r  (4°C) until needed.
Subba Rao (1977) r e p o r t e d  that he o b t a i n e d  the h i g h e s t
o
Rhizobium cell count of 10 x 10 per gr am after four we eks
storage. It was c o n s i d e r e d  n e c e s s a r y  to find out w h e t h e r  the
period of i n c u b a t i o n  of two wee k s  a d o p t e d  here  co uld  be
exten ded  if de s i r e d  w i t h o u t  a u t o l y s i s  and d e c l i n e  of the cell
popul a t i o n  set ting in.
Inoculate d bags c o n t a i n i n g  moss w i t h  i n o c u l a  of the
di f f e r e n t  c o n c e n t r a t i o n s  we re kept in the l a b o r a t o r y  at ro om  
t e m peratur e (28°C). Samples were w i t h d r a w n  at 7-d a y  i n t e r v a l s  
over 14 weeks for the d e t e r m i n a t i o n  of the cell p o p u l a t i o n ,  
using Congo red YMA for the total v i a b l e  cell co unt tests.
E. N O D U L A T I O N  OF P L A N T S  I N O C U L A T E D  W I T H  D I F F E R E N T  
ST R A I N S  OF R H I Z O B I U M  SP. IN M O S S  C A R R I E R  U N D E R  
D I F F E R E N T  W A T E R  S TRESS  C O N D I T I O N S
Since m o i s t u r e  co nten t is k n o w n  to a f f e c t  the
de v e l o p m e n t  of legum in ous pl ants (B e n t h l e n f a l v a y , Brown, 
Mihara and Stafford, 1987; S e li skar, 1987), it was
selected as one of the factors that could be u s e d  in 
trials to test the e f f i c i e n c y  of Brachymenium sp. c o m p o s t  as 
Rhizobium sp. i n o c u l u m  carrier. Besides, the v i a b i l i t y  of 
rhizo b i a  is affe ct ed by m o i s t u r e  c o n t e n t  of the soil and 
rhizobia are s e nsi ti ve to e x c e s s i v e  d r y i n g  wh en e x p o s e d  to 
open air. However, small nu mb er s su rvive for a long time
35
in air -d ried soil, p r o b a b l y  in the film of h y g r o s c o p i c  
mo i s t u r e  wh ich su rr o u n d  the soil p a r t i c l e s .  E xcess  wa t e r  
may limit a e r a t i o n  and he n c e  s u r v i v a l  of the b a c t e r i a .  
Sk erman  (1977) r e p o r t e d  that m a x i m u m  g r o w t h  and n o d u l a t i o n  
of legumes g e n e r a l l y  occur in soil w i t h  a w a t e r  c o n t e n t  
b e t w e e n  75 and 85 per cent of its wat e r  - h o l d i n g  
capa cit y. This test was c a r r i e d  out u s i n g  E x - T a m a l e  
v a r i e t y  wh ich  is c u l t i v a t e d  m o s t l y  in N o r t h e r n  G h a n a  w h e r e  
low soil m o i s t u r e  levels oc cur  for a g r e a t e r  part of the 
year.
The barabara g r o u n d n u t  pl ant s we re  r aised in soil of 
three di f f e r e n t  w a ter regimes S u r f a c e  - s t e r i l i z e d  b a m b a r a  
gr o u n d n u t  seeds p r e - g e r m i n a t e d  on 1% wa t e r  agar were 
co ate d with the moss i n o c u l u m  car ri er , by s t i r r i n g  30 
seeds in a m i x t u r e  of 3g of moss c a r r i e r  ino cu lum, 1.2ml
of 40 per cent gum arabic and l.Og CaCO^. The i n o c u l a t e d
seeds were spread in large Petri di shes for one h o u r  to 
a ll ow the i n o c u l u m  to dry p r o p e r l y  b e f o r e  they w e r e  sown. 
G e r m i n a t e d  seeds for the co nt rol we re not i n o c u l a t e d .  The 
seeds were sown in equal v o l u m e s  of the a u t o c l a v e d  soil at 
5 seeds per pot w h i c h  were thi n n e d  to one per pot after 
g e r m i n a t i n g .
The plan te d seeds were d i v i d e d  into three batches:  
Batch N o . 1 was w a t e r e d  each ot her day; B a tc h N o . 2 was
w a t e r e d  once in four days; and Batch  N o . 3 was w a t e r e d  once 
in six days. The time table for w a t e r i n g  is sho w n  in 
A p p e n d i x  A. The pots were a r r a n g e d  r a n d o m l y  on a v e r a n d a  
of the l a b o r a t o r y  facing east and r e c e i v e d  full s u n l i g h t  
each m o r n i n g  till 11.00 am. There we re  five r e p l i c a t e s
for each treatment.
36
The plants were h a r v e s t e d  30 days after g e r m i n a t i o n  
(grown from M a rch 13, 1991 to A p ri l 17, 1991) and (a) size 
of the m iddle  leaf let s, (b) n u m b e r  of le aves on each 
plant, (c) dry w e i g h t  of the plants,  and (d) n u m b e r  and 
di a m e t e r s  of the nodu les, of ea ch t r e a t m e n t  and c o n t r o l  
we re recorded,
F. N O D U L A T I O N  OF P L A N T S  I N O C U L A T E D  W I T H  D I F F E R E N T  
S TR A I N S  OF R H I Z O B I U M  SP. IN M O S S  C A R R I E R  U N D E R  
D I F F E R E N T  L I G H T  I N T E N S I T I E S
An o p t i m u m  light i n t e n s i t y  is n e c e s s a r y  for m a x i m u m  
n o d u l a t i o n  and n i t r o g e n  fixati on . This m ay  be r e l a t e d  to 
the o p t i m u m  c a r b o h y d r a t e  levels in the pl ant s. For it has 
been shown that, u n der s ha ded c o n d i t i o n s  s p r a y i n g  the 
plants with sugar s o l u t i o n  i n c r e a s e s  n i t r o g e n  f i x a t i o n
(Skerman, 1977). The b a m b a r a  g r o u n d n u t  p l a n t s  were, in
this exp er iment,  gro wn u n der  three light i n t e n s i t i e s  to 
find out w h i c h  of the three w o u l d  be c o n s i d e r e d  o p t i m u m  
for this crop. The E x - A d a  v a r i e t y  was us ed for this
e x p e r i m e n t  as it is the v a r i e t y  gro w n  w i d e l y  u n d e r  m i x e d  
f arm ing in s o u thern Ghana.
Inocu l a t e d  g e r m i n a t e d  seeds, p r e p a r e d  as in
E x p e r i m e n t  E, we re  pl ant ed  in the b l a c k  p o l y t h e n e  bags and 
put in the sheds of the three light i n t e n s i t i e s .  The 
co ntr ol bags were pla n t e d  w i t h  u n i n o c u l a t e d  g e r m i n a t e d  
seeds. Th ere  were five r e p l i c a t e s  of ea ch  t r e a t m e n t .  The 
plants were wat e r e d  da ily  in the late a f t e r n o o n  w i t h  tap 
water .
Light i n t e n s i t i e s  in the three sheds wer e m e a s u r e d
every other day with the ' E e l 1 P o r t a b l e  P h o t o e l e c t r i c  
Ph otometer , at 9.00 am, 12.00 no on and 3.00 pm.
The plants were h a r v e s t e d  af ter 30 days ( gr ow n from
April 10, 1991 to May 15, 1991). In this e x p e r i m e n t  also
(a) size of the m i d d l e  leaf let s, (b) n u m b e r  of l e a v e s  on 
each plant, (c) dry w e i g h t  of the plants, and (d) n u m b e r  
and d iam et ers of the nodu les, of each  t r e a t m e n t  and
control, were record ed.
- 37 -
38
VI, R E S U L T S  * 
D E T E R M I N A T I O H  OF P R O P E R T I E S  OF TH E D I F F E R E N T  S O I L  SAMP LES.
The results of this e x p e r i m e n t  p r e s e n t e d  in T a b l e  la 
show that the pH's of the soils we re  simi lar. The pH of 
the eight soil samples ra nged from pH 6.48 (Legon) to pH
6.84 (Weija). Also wi th the e x c e p t i o n  of Kpong, L e g o n  and 
W ei ja soils, the soil samples were sandy loam, as shown in 
Table lb, and d e t e r m i n e d  by the P r e s c o t t  T r i a n g l e  
(A ppendix A) from the 'Particle size d i s t r i b u t i o n '  da ta in 
Table la. In contrast, the P e r c e n t a g e  N i t r o g e n  and Mean 
Pe rcen t a g e  Or ganic matter v a r i e d  c o n s i d e r a b l y .  The
P e r c e n t a g e  N i t r o g e n  of the A s h i a m a n  soil was only 0.025 
per cent, while  it was as hi gh  as 0. 1 3 2  per cent in the 
Pok uas e soil. The Kpo ng and Le g o n  soils w h i c h  came next 
to the latter had a p p r o x i m a t e l y  on ly h a l f  the level of 
P e r c e n t a g e  N i t r o g e n  in the P o k u a s e  soil.
Li kewise, while the h i g h e s t  Me an  P e r c e n t a g e  O r g a n i c  
Matter rec orded was 4.513 per cent in the P o k u a s e  soil, 
the least wh ich o c c u r r e d  in the A s h i a m a n  soil was as low 
as 0.468 per cent.
There was a clear r e l a t i o n s h i p  b e t w e e n  P e r c e n t a g e  
N i t ro ge n and Mean P e r c e n t a g e  O r g a n i c  Ma tt er. P e r c e n t a g e  
N i t r o g e n  inc re ased wi th  i n c r e a s i n g  Me an  P e r c e n t a g e  O r g a n i c  
Ma tter. Thus, the A s h i a m a n  soil had the lowest P e r c e n t a g e  
N i t ro ge n and Mean P e r c e n t a g e  Org a n i c  Matter, and P o k u a s e  
soil had the hi gh est P e r c e n t a g e  N i t r o g e n  and Me an  
P er ce n t a g e  Organic Matter.
39
Total viable  count st ud i e s  u s i n g  Co n g o  red YMA 
produced the Rhizobium sp* p o p u l a t i o n s  i n d i c a t e d  in Ta ble
2. There were high p o p u l a t i o n s ,  b e t w e e n  111 x 10^ and 108 
x 10 cells per gram of soil in soils from Kpong, Legon, 
Nungua(l), Po ku as e and Weija. The r e m a i n i n g  so ils  had 
very low p o p u l a t i o n s  not e x c e e d i n g  20 x 10^ per gr am of 
soil. P h o t o g r a p h s  of Congo red Y MA i n o c u l a t e d  with
s u s p en si ons of soils from the eight l o c a l i t i e s  and 
incubated  at 30^C for 5 days are sho wn in Pl at es la and 
lb.
TABLE la
Some p r o p e r t i e s  of soils from l egume plots from d i f f e r e n t  
l o c a l i t i e s .
- 40 -
Source 
of soil
| P a r t i c l e  size 
| d i s t r i b u t i o n  
| (% by w t )
%
Me an 
Org a n i c  
Mat ter
Mean
pH
| Sand Silt Clay Ni trogen (%)
Ashiaman
1
82.33
1
3.42 14.25 0.025 0.468 6.60
Kpong
1
| 51.10 
1
9.65 39.25 0.067 2.009 6 . 80
Legon
1
| 74.83
I
3.42 21.75 0.069 1 . 733 6.48
Nungua ( 1 )
1
| 75.24 
1
5.51 19.25 0.049 1.472 6.67
N u n g u a (2 )
1
| 80.68
1
3 .82 15.50 0.039 0.977 6 .82
Pokua s e
1
82.30
1
0.95 16.75 0.132 4.513 6 .76
Shiashie
1
| 81.30 
1
4.45 14.25 0.028 0.578 6.66
Weija
1
| 69.73 6.02 24.25 0.055 2.064 6.84
41
Kinds of soils o b t a i n e d  from l eg ume plots from d i f f e r e n t  
local iti es as d e t e r m i n e d  w i t h  the P r e s c o t t  Tr i a n g l e .
(Based on data of P a r t i c l e  size d i s t r i b u t i o n  in T a b l e  la)
TABLE lb
Source of Soil Type of Soil
Ash i aman S an dy loam
Kpong Sa ndy clay
Legon San dy clay loam
Nu ngu a (1) Sandy loam
N un gua ( 2 ) Sandy loam
Pokuas e Sandy loam
Shiashie Sandy loam
Wei j a Sandy cl ay  loam.
42
TABLE 2
Rhizobium p o p u l a t i o n  in soils from the d i f f e r e n t  
local iti es .
Source 
o f 
Soil
Mean No. of Rhizobium
p o p u l a t i o n  ( X 10^) 
g ~ * soil
Ash i araan 1 5
Kpong 180
Legon 138
Nungua (1) 112
Nungua (2) 20
Pokuase 111
Shiashie 19
Weija 165
- 43 -
l a _ t o p : Inocula of N u n g u a  (2) soil (lef t) and
Pokuase soil (Right)#
BOTTOM: Inocula of A s h i a m a n  soil (Left) and
Shiashie soil (Right).
lb. TOP: Ino cula of W e i j a  soil (Left) and L e g o n  soil
( R i g h t ).
BOTTOM: Inocula of W e i j a  soil (Left) and L e g o n  soil
( R i g h t ).
PLATE 1. Photographs of Petri p l a t e s  of C o n g o  red YMA 
inoculated with sus pe nsions  of soils from ei g h t  d i f f e r e n t  
localities showing colo nies of Rhizobium sp. a f ter i n c u b a t i o n  at
3 0°C for 5 days. (X 1/3).
k k
B , I S O L A T I O N  OR S T R A I N S  OF RHIZOBIUM sp.
F R O M  THE D I F F E R E N T  SOIL SA MP LES
The p r e c e e d i n g  tests sh owed that Rhizobium s pe ci es 
were present in all the eight  soils. This e x p e r i m e n t
showed that they were cap a b l e  of i n f e c t i n g  b a m b a r a
g r o u n d n u t  plants and c a u s i n g  n o d u l a t i o n  w h e n  the plants 
were raised in the soils. But they did this to v a r y i n g
degrees. Most of the o b s e r v a t i o n s  and res u l t s  of the
e xp er i m e n t  are r e c o r d e d  in Table 3 and Figs. 1 and 2.
On the basis of plant g r o w t h  and the e x t e n t  of
n o d u l a t i o n  r e c or ded in Ta ble 3, the pl ant s co u l d  be 
s ep ar ate d into three groups as follows:
G ro u p  1 . Plants w h ic h g r e w  l u x u r i a n t l y  w i t h  deep-
green foliage, had h i g h e s t  Mean Shoot dry
weight, (between 1.80g and 2.36g) and four of
them had mean root dry w e i g h t  (be tw een 0 . 31 g and
0•3 9 g ). They also formed the h i g h e s t  n u m b e r  of
nodules. The barabara g r o u n d n u t  v a r i e t i e s  and
a s s o c i a t e d  Rhizobium sp eci es were:
i Ex-Ada variety and Legon Rhizobium sp. s train
ii E x-Ad a v a r i e t y  and Wei j a  Rhizobium sp.
strain
iii Ex - T a m a l e  v a r i e t y  and Kp o n g  Rhizobium sp.
strain
iv E x - T a m a l e  v a r i e t y  and N u n g u a ( l )  Rhizobium
s p . strain
v E x - T a m a l e  v a r i e t y  and P o k u a s e  Rhizobium sp.
strain
Their no dul es  were the la rg est with
r e s p e c t i v e  mean d i a m e t e r s  of 1.81, 1.41, 2.47
1.50 and 1.80mm. The c 1 a s s-d i ame t e r s of these 
no dul es are p r e s e n t e d  in the h i s t o g r a m s  in Fig.
2 .
- 45 -
Gr o u p  2
Group 3
Plants with m o d e r a t e  g r o w t h  and n o d u l a t i o n :
i Ex-Ada v a r i e t y  and K p o n g  Rhizobium sp. s t r a i n  
ii Ex-Ad a v a r i e t y  and N u n g u a ( l )  Rhizobium sp. s train 
iii Ex -Ad a v a r i e t y  and P o k u a s e  Rhizobium sp. s t r a i n  
iv E x - T a m a l e  v a r i e t y  and L e g o n  Rhizobium sp. strain 
v E x - T a m a l e  v a r i e t y  and W e i j a  Rhizobium sp. strain.
Plants wi th st unted  g r o w t h  and y e l l o w i s h - g r e e n  
foliage and formed the s m a l l e s t  n u m b e r  of m e a n  n u m b e r  
of no dules per plant, 3 - 1 0  n o d ules,  c o m p a r e d  to 30 
- 44 mean no dules per plant of the a s s o c i a t i o n s  of
Group 1.
i Ex-Ada v a r i e t y  and A s h i a m a n  Rhizobium sp. s train  
ii Ex-A da v a r i e t y  and N u n g u a ( 2 )  Rhizobium sp. strain 
iii Ex -Ada v a r i e t y  and S h i a s h i e  Rhizobium sp. st rain 
iv E x - T a m a l e  v a r i e t y  and A s h i a m a n  Rhizobium sp.
strain
v E x - T a m a l e  v a r i e t y  and N u n g u a ( 2 )  Rhizobium sp.
Strain
vi E x - T a m a l e  v a r i e t y  and Sh ias h i e  Rhizobium sp.
strain.
It was ob vious  that i n f e c t i o n  by s t r a i n s  of 
Rhizobium sp. of soils from A sh iaman,  N u n g u a ( 2 )  and 
Shiashie plots was poor and we re not used a n y m o r e  in 
the subs eq u e n t  i n v e s t i g a t i o n s .  It can also be seen 
from Table 3 and Fig. 1 that the lowest P e r c e n t a g e  
Ni t rog en  con te nt of the shoots was r e c o r d e d  in the 
plants, both Ex -Ada and E x — Ta male v a r i e t i e s ,  gro wn in 
the A s h i a m a n  and S h i ashie soils
- 4 6 -
Fig. 1 i l l u s t r a t e s  the r e l a t i o n s h i p  b e t w e e n  e x t e n t  of 
n o d u l a t i o n  of the plants  of the v a r i o u s  va r i s t r a in
a s s o c i at ions,  their dry w e i g h t s  and per cent n i t r o g e n  of their 
shoots.
i •
There was a h i g h l y  s i g n i f i c a n t  p o s i t i v e  c o r r e l a t i o n  
be t V e e n  the me an dry w e i g h t  of shoot and the p e r c e n t a g e  
n i t ro gen of the shoot (r = 0.8616; ty ^  = 12.5130; P<O . 0 0 1 ) .
There was no c o r r e l a t i o n  be t w e e n  the m e a n  dry w e i g h t  of root
and the p e r c e n t a g e  n i t r o g e n  of the shoot (r = 0 . 1 947).
C o n s i d e r i n g  all the o b s e r v a t i o n s ,  it was d e c i d e d  that the
Rhizobium sp. strains of the a s s o c i a t i o n s  of Group 1 w o u l d  be
used in all the s u b s e q u e n t  e x p e r i m e n t s .  These  are:
(a) Kpong Rhizobium sp. strain in n o d u l e s  of E x - T a m a l e
var ie ty
(b) Legon Rhizobium sp. strain in nod u l e s  of E x - A d a
var ie ty
(c) Nungua( l)  Rhizobium sp, strain in n o d u l e s  of E x - T a m a l e  
va ri et y
(d) Pokuase Rhizobium sp, strain  in no d u l e s  of E x - T a m a l e
var i e ty
(e) Weija Rhizobium sp, strai n in no d u l e s  of E x - A d a
variety.
- 4 7 -
- 48 -
TAB L E  3
G row th and n o d u l a t i o n  of two v a r i e t i e s  of B a m b a r a  
g ro u n d n u t  raised in soils from legu me plots from d i f f e r e n t  
l oc al i t i e s  for 30 days under n o r m a l  d a y / n i g h t  regime.
Source
of
Soil
Plant
variety
Mean
Dry weight (g)(+ 5 
of plant
Shoot root
5.E) %
Nitrogen
of
shoot
Mean No. 
of nodules 
per plant 
(to the nearest 
whole no.)
Ashiaman Ex-Ada 0.44 + 0.01 0.06 + 0.01 0.37 5
Ex-Tamale 0.61 +, 0.15 0.08 + 0.01 0.29 2
Kpong Ex-Ada 0.39 + 0.06 0.07 + 0.02 0.40 11
Ex-Tamale 2.36 _+ 0.37 0.38 + 0.03 0.46 38
Legon Ex-Ada 2.03 + 0.35 0.39 + 0.05 0.49 41
Ex-Tamale 0.51 _+ 0.03 0.14 + 0.02 0.41 14
Nungua Ex-Ada 0.72 + 0.12 0.11 + 0.02 0.37 11
(1) Ex-Tamale 1.81 _+ 0.19 0.33 + 0.05 0.48 32
Nungua Ex-Ada 0.35 + 0.06 0.07 + 0.01 0.39 10
(2) Ex-Tamale 0.35 _+ 0.13 0.09 + 0.01 0.41 8
Pokuase Ex-Ada 0.67 + 0.12 0.07 + 0.01 0.37 12
Ex-Tamale 2.08 +_ 0.37 0.31 + 0.06 0.53 30
Shiashie Ex-Ada 0.34 + 0.17 0.07 + 0.01 0.33 3
Ex-Tamale 0.61 +_ 0.15 0.11 + 0.02 0.38 3
Weija Ex-Ada 1.80 + 0.59 0.10 + 0.00 0.49 44
Ex-Tamale 0.85 i  0.05 0.38 + 0.04 0.40 17
49 -
Fig. 1 The graph of nodulating ability of two varieties of Bambara 
groundnut plants raised in soil samples for 30 days.
A - Ashtnman soil; K - Kpong soil; L - Legon soil;
N l - Nun^ur. (-Piot 1) Soil N2 - Nungua (Plot 2) soil P - Pokuase soil;
S - Shiashie soil; w - Weija soil
- 50
60
30
60
2 O
60
20
60
20
60
20
1----1
E i  -  A DA  V A R I E T Y  
I N L E G O N  S O I L
Ex -  ADA V A R I E T Y  IN 
W E I J A  S O I L
Ex -  T A M A L E  V A R I E T Y  
IN K P O N G  S O I L
Ex “  T A M A L E  V A R I E T Y  
IN N U N G U A  ( I  J S O I L
Ex  -  T A M A L E  V A R I E T Y  
IN  P O K U A 5 E  S O I L
I 2 3 4 5 6
C L A S S  D I A M E T E R  
I: 0-56 — 1.05  mm 2: 1.06 — 1. 5 5mm
3: I - 56 —  2 .0  5 mm 4.' 2 . 0 6 - 2 . 5 S m m
5: 2 .56  — 3 -05 mm 6. 3 06*“ 3 - 5 S » »
Fig. 2
Distribution of class-diameters of nodules of
plants of the Ex-Ada and Ex-Tamale v a r i e t i e s , j  groundnut
Kpong, Legon, Nungua(l), Pokuase and Weija plots £ 0 ^ 3 0 ^  
under normal day/night regime. P J° dayS
51
C. C O M P A R A T I V E  I N F E C T I V E  A B I L I T Y  OF TH E F I V E  S T R A T H S  OF 
R H I Z O B I U M  gp. I S O L A T E D  F R O M  B A M B A R A  G R O U N D N U T  P L A N T S
fS e e d l i n g  Agar' proved to be a good m e d i u m  for this 
e x p e r i m e n t  in w h i c h  the i n f e c t i v i t y  of the five s t r a i n s  of 
Rhizobium sp, was studied. E x - A d a  v a r i e t y  pl ants
i n o c u l a t e d  wi th the Gro u p  1 st r a i n s  of Rhizobium sp, of 
E x p e r i m e n t  B all g r e w  well as can be o b s e r v e d  in Plate 2,
The me an dry w e i g h t  of the shoots of the d i f f e r e n t  
t re atm ent s, mean n u m b e r  of n o d u l e s  w h i c h  d e v e l o p e d  on the 
pl ants and the mea n d i a m e t e r  of the n o d u l e s  are t a b u l a t e d  
in Ta ble  4, Fig. 3 shows the n o d u l e  c l a s s - d i a m e t e r  
d i s t r i b u t i o n  for the v a r i o u s  t r e a t m e n t s .
The v al ues for me an  shoot  dry w e i g h t s  w e r e  quite 
close; the least was 0, 41g  for pl ants i n o c u l a t e d  wi th 
P o k u a s e - E x - T a m a l e  strain, and the h i g h e s t  was 0 , 4 9 g  for 
plants i n o c u l a t e d  with the L e g o n - E x - A d a  strain. The r e  was 
no s i g n i f i c a n t  d i f f e r e n c e  b e t w e e n  the m e a n  dry w e i g h t s  of 
the i n o c u l a t e d  plants. Th ey  were, h o w eve r,  all
s i g n i f i c a n t l y  d i f f e r e n t  from the v a l u e  for the c o n tro l.  
Va lues for the m e a n  n u m b e r  of n o d u l e s  per pl a n t  for the 
five t r e a t m e n t s  also fell w i t h i n  the n a r r o w  ran g e  of 
28-34.
There was, howe ve r, c o n s i d e r a b l e  v a r i a t i o n  in me an 
d i a m e t e r s  of the nod ules.  The h i s t o g r a m s  in Fig. 3 also 
i l l u s t r a t e  this very  well. The g r e a t e s t  m e a n  d i a m e t e r  of
1 .46mm  of plants i n o c u l a t e d  w i t h  the 1K p o n g - E x - T a m a l e 1 
strain is far gr ea ter (39 per cent in c r e a s e )  than the 
s m a ll es t mean d i a m e t e r  of 1 . 0 5 m m  of pl ants i n o c u l a t e d  with 
the L e g o n - E x - A d a  strain.
The con trol u n i n o c u l a t e d  p la nt s we re sma l l e r  in size 
and they did not nodulate.
- 52 -
TAB L E  4
Dry weight and extent of n o d u l a t i o n  of i n o c u l a t e d  Barabara 
g r ou ndnut plants, Ex-Ada  variety, raise d on ’S e e d l i n g  Agar' for
20 days at 28°C.
Strain of Rhizobium
sp from nodules of |
1
1
1
I
Mean Dry 
w eight  
of shoot 
(g>
Mean No. 
of Nod u l e s  
per plant 
(to the 
n ea r e s t  
w ho l e  no . )
Me an
D i a m e t e r  
of 
N o d u l e s  
( m m )
Ex-Ada in Legon soil
1
0. 4 9a 28 1.10
1
Ex-Ada in Weija soil | 0 . 4 5a 34 1 .23
Ex-T ama le in Kpong soil 0 . 44a 30 1 .05
Ex-Tamale in N u ng ua (l) soil| 0 . 4 7a 33 1.46
Ex-Tamale in Pokuase soil |
I
0 . 4 la 30 1.42
U n inoc ul ated plants | 
(control)
1
0 . 2 5b 0 -
By the ca lc ul a t e d  C o n f i d e n c e  Limits at 95% (Kershaw, 1973) 
means bearin g the same letter are not s i g n i f i c a n t l y  d i f f e r e n t .
From
- 53 -
Left: 1. I n o c u l a t i o n  with  P o k u a s e  s tra in  of Rhizobium sp
2. I n o c u l a t i o n  with L e gon strain  of Rhizobium sp.
3. I n o c u l a t i o n  wit h N u n g u a ( l )  st rai n of Rhizobium
s p .
4. I n o c u l a t i o n  wi th Kp o n g  s tr ain of Rhizobium sp.
5. I n o c u l a t i o n  wi th W e i j a  s tr ain of Rhizobium sp.
6 . U n i n o c u l a t e d  (Control).
PLATE 2. P h o t o g r a p h  of B a m b a r a  g r o u n d u n t  plant, Ex- 
Ada v a r i e t y  grown in fS e e d l i n g  Agar* i n o c u l a t e d  w i t h  
d i f f e r e n t  strains of Rhizobium sp., un der n ormal d a y / n i g h t  
regime at room t e m p e r a t u r e  for 12 days. (X 1/5).
54 -
e o
4 0
frO
4 0
eo
4 0
D
o
LJ
a
u. o
o
to
4 0
8 0
4 O
R H I  2 O P I U M  S T R A I N  F R OM
n o d u l e s  O F
E  x — ADA  IN  L E G O N  
S O I L
Ex -  ADA IN 
W E I J A  SO I L
E x ~  T AMA L E  IN 
K PO f i G  S p | L
Ex - T AMA L E  IN
NU NGU A ( I  ) SO m
I x - T A M A L E  IN 
P O K U A S E  S O I L
I 2 3 4 S
C L A S S  D I A M E T E R
|; 0 2 4  — O . 65 n • 2: O 66 -  | . 05aa i
3 10  6 — I 4 5 m b  4 1.46 — I- •9m r
* ' ™ d l y  2 0  d a , " o i d
P R E P A R A T I O N  OF THE M O S S  C O M P O S T  AS C A R R I E R  AND 
S TU D Y  OF SHELF L I F E  OF I N O C U L U M  C A R R I E R
Cell p o p u l a t i o n s  of the five st r a i n s  of Rhizobium
species s e l ec ted for the rest of this r e s e a r c h  e v i d e n t l y  
m u l t i p l i e d  at d i f f e r e n t  rates d u r i n g  10 days of i n c u b a t i o n  
in Y e a s t - e x t r a c t  m a n n i t o l  (YEM) Broth. The p o p u l a t i o n s  
ranged from 619 x 105 cells m l -1 (Wei ja str ain ) to 973 x 
105 cells ml”1 (Nungua (1) strain), as shown  in Ta b l e  5.
The five st rains fell into two groups. L e g o n  and 
Nung ua( l) strains sh owed c o n s i d e r a b l y  g r e a t e r  p o p u l a t i o n s  
than Kpong, Po k u a s e  and W e i j a  strains.
U nd il uted, and and 1/8 d i l u t i o n s  of the v a r i o u s
br oth c u l tu res we re us ed  in i n o c u l a t i n g ,  i n d i v i d u a 1 l y -
s amp les of the g ro un d moss compo s t .  The i n o c u l a t e d  
co mpost  was i n c u b a t e d  for two w e e k s  at ro om t e m p e r a t u r e .
V i a b l e  cell counts of the Rhizobium sp. of the v a r i o u s  
strains c u l tu red in the moss c a r r i e r  for two w e e k s  are 
pr e s e n t e d  in Table 6 .
Each c o n t a i n e d  lOg of mo ss  comp ost. To this was 
added 20ml of b r o t h  c u l t u r e  w h i c h  b r o u g h t  the m o i s t u r e  
con ten t to 50 per cent. By m i x i n g  the c o n t e n t s  of the 
bags very thor oughly, l.Og of c o m p o s t  could be a s s u m e d  to 
co nt ai n 2.0ml of the c u l t u r e  s u s p e n s i o n  added. It was 
possible, the refore, to d e t e r m i n e  the a p p r o x i m a t e  in it ial 
cell p o p u l a t i o n  at the b e g i n n i n g  of i n c u b a t i o n .  T h es e 
c a l c u l a t e d  va lues have been r e c o r d e d  in Tab l e  6 to show 
the extent of gr owt h of the r h i z o b i a  in each tr ea t m e n t .  
There were three r e m a r k a b l e  fi nd ings. First, g r o w t h  of 
the p o p u l a t i o n  of all strains was tre m e n d o u s .  S e cond ly , 
inocula of the four d i f f e r e n t  c o n c e n t r a t i o n s  p r o d u c e d  
almost similar p o p u l a t i o n  levels at the end of 14 days,
and thirdly, p o p u l a t i o n  counts, of strain s w i t h  in itia l
- 55 -
56
smaller cell nu mbers  (Ex-A da in W e i j a  soil, E x - T a m a l e  in K p o n g  
soil and Ex- T a m a l e  in Po k u a s e  soil) and those of s t r a i n s  w i t h  
initial greater cell n u m b e r s  (Ex -Ad a in L e g o n  soil and Ex- 
Tamale in Nungua(l)  soil) we re  quite close.
Shelf life studies made wit h moss c o m p o s t  i n o c u l a t e d  wi th 
u n d i l u t e d  broth cu lt u r e  and h a l f - d i l u t e d  bro t h  c u l t u r e  only, 
showed gradual i n c rea se  in p o p u l a t i o n  c oun ts  wi th all and h a l f ­
diluted broth cu ltu re  the st rains of Rhizobiua sp. in the moss 
carrier w i t h i n  14 weeks. There was no sign of a u t o l y s i s  in the 
14 weeks, but by the 10th w e e k  all the st rains had r e a c h e d  the 
phase of S t a t i o n a r y  Growth.
Plate 3 shows a b u n d a n t  g rowt h of the cells of the v a r i o u s  
strains when s u s p e n s i o n s  of the v a r i o u s  mo ss c a r r i e r s  were 
pre pa re d after six w e e k s 1 sto ra ge and p l a t e d  on Con g o  red Y M A . 
The w e e k l y  p o p u l a t i o n  counts r e c o r d e d  are p r e s e n t e d  g r a p h i c a l l y  
in Fig. 4, while the v alues a pp ea r in A p p e n d i c e s .
C o n t a m i n a t i o n  of the moss c a r r i e r  d u r i n g  s t o r a g e  was 
ne gligible . The ve ry few c o n t a m i n a n t s  could be i d e n t i f i e d  
bec aus e they beco me s t r o n g l y  c o l o u r e d  w i t h  the dye. No att e m p t  
was made to ide nti fy  them, nor to q u a n t i f y  their n u m be rs .
5 7
Po pul a t i o n  of strains of Rhizobium sp, gr own  in b r o t h  of 
YEM m ed ium at 30°C for 10 days.
TABLE 5
Strains of Rhizobium s p . 
f r om nodules of
Mean N o • of 
p o p u l a t i o n .
Rhizobium
(X105) ml-1
Ex-Ada in Legon soil 852
Ex-Ada in Weija soil 619
Ex-T a m a 1e in Kpong soil 675
Ex-Tamale in N u n g u a (1 ) soil 973
Ex-T a m a 1e in Pokuase soil 676
- 58
T A B L E  6
M u l t i p l i c a t i o n  of strains Rhizobium sp. in mos s c a r r i e r  
inc ub ated  at 3 0°C for 2 weeks.
Strains of 
Rhizobium s p
from no dule s of
I n o culum
c o n c e n t r a t i o n
A p p r o x i m a t  e 
in it ia l 
P o p u l a t  ion 
(X109 ) 
g-1 ca r r i e r
Mean N o . of 
Rhizobium
p o p u l a t  ion 
.present ( XI 0 ^  
g”1 c a r r i e r
Ex-Ada in Legon 
soil
U n d i l u t e d  
\ D i l utio n 
X D i l u t i o n  
Dilut i o n
0.0852
0.0426
0.0213
0.0107
242
192
207
215
Ex-Ad a in We i j a Und ilu ted 0. 0619 240
soil \ D i l uti on 0 .031 0 189
X D i l u t i o n 0.0155 199
^g D i l u t i o n 0 .0 07 7 210
Ex-Tamale  in Kpong Und ilu ted 0.0675 246
soil \ D i l ut ion 0 .0 33 8 194
X D i l u t i o n 0.0169 213
^g D i l uti on 0.0084 243
Ex Tamale in Und ilu t ed 0.0973 221
N u n g u a (1) soil \ Dilu tion 0 .0 48 7 196
X D i l ution 0.0243 228
ly/g D i l ut io n 0 .0122 206
Ex-Ta ma le in Und ilu ted 0.0676 216
Pokuase soil \ D i l ut ion 0 .0 33 8 190
X Dil ut io n 0.0169 224
Dilution 0.0085 195
- 59 -
3a TOP :
BOTTOM
Inocula of K p o n g - E x - T a m a l e  s t r a i n  (Left)  and
Pokuase-Ex-Taraale s tra in  (Right).
Inocula of K p o n g - E x - T a m a l e  s t r a i n  (Lef t) and
P o k u a s e - E x - T a m a l e  s trai n (Rig ht) u s i n g  the \  
dilutions as inocula, (X /^) •
3b TOP : Inocula of W e i j a - E x - A d a  s t r a i n  (Left), 
Le gon - E x - A d a  strain (Middl e)  and 
N u n g u a (1)- E x - T a m a l e  s t r a i n  (Rig ht),
BOTTOM: Inocula of W e i j a - E x - A d a  s t r a i n  (Left),
L e g o n - E x - A d a  strain (Mi ddl e)  and
N u n g u a (1)- E x - T a m a l e  s t r a i n  (Right), u s i n g  the 
dilutions as inocula. (X^).
PLATE 3. Photographs of Petri p l a t e s  of C o n g o  red YMA 
inoculated with suspension s from the m o s s  c a r r i e r  a f t e r  6 weeks 
on the shelf. Inc ubation at 30°C for 5 days.
60
M
EA
N
 
NO
 
OF
 
C
O
LO
N
IE
S 
PE
R 
ML
 
(I
X 
lO
9 
)
4 ft • IO 12 |4
TIME ( Wt« ks )
* ® ’ U n d 11 u t « d c o n c e n t r a t i o n
- o - o ~  1/2 D i l u t i o n
1* i K •  ^ Population t oi live strains oi Rhi?obium sp. in inoculated 
moss carriers stored at room temperature for 14 weeks*
62
E. N O D U L A T I O N  OF P L A N T S  I N O C U L A T E D  W I T H  D I F F E R E N T  
ST RAINS  OF R H I Z O B I U M  SP. IN M O S S  C A R R I E R  U N D E R  
D I F F E R E N T  W A T E R  S TRESS C O N D I T I O N S
U n i n o c u l a t e d  con trol plants g r e w  p o o r l y  and did not 
form any nod ules.  They formed few leaves, only 5 - 6 in 
number, which  were y e 1 1 o w i s h - g r e e n  in colour, and the 
plants were g e n e r a l l y  stunted. Those r e c e i v i n g  w a t e r  each 
other day p r o du ce d the largest le afl e t s  of w h i c h  the 
middle leaflet m e a s u r e d  on the a v e r a g e  3,36 cm long and 
1.57 cm wide. Those wa t e r e d  once in four days and once in 
six days pro duce d smaller leaflets.
All the i n o c u l a t e d  plants of the v a r i o u s  w a t e r i n g  
treatments, on the other hand, n o d u l a t e d .  They had 
greater me an dry weight, they p r o d u c e d  mo re leaves w h i c h  
were d e e p - g r e e n  in colour (adaxial surfa ce) and la rger 
leaflets. The resul ts o b t a i n e d  are shown in T a b l e s  7-11.
The amount of water r e c e i v e d  by the pl ants g r e a t l y  
affe cte d them in ma ny ways. It a f f e c t e d  the dry w e i g h t s  
of the plants, the n umb er  of leaves (Plates 4 and 5) and 
the number of no dul es  formed. The v alu es  r e c o r d e d  for 
these were h i g h e s t  for plants w a t e r e d  at 2 -d ay in t e r v a l s  
and lowest for plants w a t e r e d  at 6 -day in tervals . Plants 
wa te re d at 2-day i n te rv als had m e a n  dry w e i g h t s  fr om l.OOg 
to 1 . 6 8 g , p r o du ce d 10 - 15 leaves and formed 43 - 56 
nod ule s per plant. The c o r r e s p o n d i n g  v alu es  for p la nt s 
wa te re d at 4-d ay int er va ls were 0.64 - 0.94g, 8 - 1 2  
leaves and 16 - 25 nodules. And for plants w a t e r e d  at 6 - 
day intervals, 0.48 - 0.96g, 7 - 1 0  leaves and 8 - 1 1  
l e a v e s .
- 63 -
T A B L E  7a
Leaf d e v e l o p m e n t  of Ba m b a r a  g r o u n d n u t ,  v a r i e t y  E x - T a m a l e ,  
in oc ul ated with K p o n g - E x - T a m a l e  s train  grown u n d e r  n or mal
da y / n i g h t  regime at d i f f e r e n t water stresses •
C o n c e n t r a t i o n  of 
Initial Broth 
Cu lture Ino culum  
of moss Carrier
Mean No. of leaves 
per plant (to the 
n e a r e s t  whole no.) 
at f o l l o w i n g  water 
s tr e s s e s *
Mean length and 
of m i d - l e a f l e t  ( 
f o l l o w i n g  w a te r 
s t r e s s e s *
wi d t h  
c m ) at
2 4 6 2 4 6
undilut ed 15 12 9 6.13x2.29 6 .00x2.10 6.32x2.08
\ Dilu ti on 11 11 8 6.13x2.44 6.32x2.49 6.35x2.03
\ Dil ution 11 11 8 6.36x2.37 6.09x2.18 5.63x2.05
^/g Dil ution 1 10 8 8 |5.45x2.06 6.61x2.01 6.04x1.89
Un i n o c u 1 a ted 
(CONTROL)
6 6 5 3.36x1.57 2.29x1.31 1.57x0.91
2 - watered once in 2 days,
4 “ watered once in 4 days
6 = watered once in 6 days (see Appendix A)
TABLE 7b
Dry weight and e xtent of n o d u l a t i o n  of B a m b a r a  g r o u n d n u t ,  
v a r i e t y  Ex-Ta mal e, pl ant s i n o c u l a t e d  w i t h  K p o n g - E x - T a m a l e  
strain grown und er n o r m a l  d a y / n i g h t  r e g i m e  at d i f f e r e n t  w a te r 
stresses .
C o n c e n t r a t i o n  of 
Initial Broth 
Cu lture  Inoc ulum 
of moss Carrier
Mean Dry wt. per 
pl ant  (g) at 
foil owing water 
s t r e s s e s *
Mea n 
plant 
who le 
wat e r
No. of N o d u l e s  per 
(to the n e a r e s t  
no.) at f o l l o w i n g  
s t r e s s e s *
2 4 6 2 4 6
Und ilu t ed 1.44 
+ 0.39
0.94 
+ 0.25
0.70 
+ 0.22
55 19 9
\ D i l u t i o n 1 .26 
+ 0.33
0.78 
+ 0.23
0.74 
+ 0.22
49 18 9
\ D i l ut ion 1 . 00 
+ 0.27
0.69 
+ 0.21
0.72 
+ 0.24
49 18 10
/g D i l utio n 1.10 
+ 0.29
0.90 
+ 0.28
0.62 
+ 0.17
43 16 10
U n i n o c u l a  ted 
(CONTROL)
0.54 
+ 0.16
0.32 
+ 0.14
0.11 
+ 0.03
0 0 0
2 = watered once in 2 days
4 - watered once in 4 days
6 = watered once in 6 days (see Appendix A)
TABLE 8a
Leaf development of Bambara groundnut, variety Ex-Tamale,
inoculated with Legon-Ex-Ada strain grown under normal
day/night regime at different water stresses.
C o n c e n t r a t i o n  of 
Initial Broth 
Cul tur e I n o cul um  
of moss Carrier
Mean No. of leaves 
per plant (to the 
n e a r e s t  whole no.) 
at f o l l o w i n g  wa ter 
st re sses*
Mean le ngt h and 
of m i d - l e a f l e t  ( 
f o l l o w i n g  wat e r  
s t r e s s e s *
wi dt h 
c m ) at
2 A 6 2 A 6
Und ilu t ed 12 10 8 8.18x2.76 5.A5x2.31 5.55x2.05
\ Dilut ion 10 9 8 8.50x2.7A 7.06x2.31 5.98x2.26
\ Dilutio n 11 10 9 6.52x2.30 5.61x2.19 5.90x1.89
V g  Dilu tion 1 1 1 9 8 6.75x2.33 6.25x2.11 5.A2x2.08
U n i n o c u l a t e d
(CONTROL)
6 6 5 3.36x1.57 2.29x1.31 1.57x0.91
2 = watered once in 2 days.
A - watered once in A days
6 = watered once in 6 days (see Appendix A)
TABLE 8b
Dry Weight  and ex tent of n o d u l a t i o n  of B a m b a r a  g r o u n d n u t ,  
v a r i e t y  Ex-T amale, plan ts i n o c u l a t e d  w i t h  L e g o n - E x - A d a  strain 
grown under norma l d a y / n i g h t  r eg im e at d i f f e r e n t  wa t e r  
stresses.
C o n c e n t r a t i o n  of 
Initial Broth 
Culture I n o cu lum 
of moss Carrier
Mean Dry w t . per 
pl ant  (g) at 
f o l l o w i n g  water 
str e s s e s *
Mean 
plant 
who 1 e 
water
No. of N o d u l e s  per 
(to the n e a r e s t  
no . ) at f o 1 l ow in g 
s t r e s s e s *
2 4 6 2 4 6
U n dilu te d 1 . 59 0.88 0.64 44 20 11
±0.44 ±0.28 ± 0 . 1 8
\ Dil ution 1.10 0.90 0.59 52 18 9
+ 0.28 ±0.29 ± 0.21
\ Dilut io n 1 .49 0.84 0.69 51 16 8
± 0. 44 ±0.29
HCM•O+1
^/g D i l ution 1 .36 0.69 0.68 50 20 1 1
±0.41
<
r•
O+1 ± 0.20
Un i n o c u l a t e d 0.54 0.32 0 . 1 1 0 0 0
(CONTROL) ±0. 1 6 ± 0 . 1 4 ±0.03
2 = watered once in 2 days
4 - watered once in 4 days
6 - watered once in 6 days (see Appendix A)
- 67 -
T A B L E  9a
Leaf d e v e l o p m e n t  of Barabara gr ou nd n u t ,  v a r i e t y  E x - T a m a l e ,  
i no cu lated with N u n g u a  (1) E x - T a m a l e  stra in  grown u n d e r  n ormal 
d a y/ night regime at d i f f e r e n t  water stresses.
C o n c e n t r a t i o n  of 
Initial Broth 
Culture Ino culu m 
of moss Carrier
Mean No. of leaves 
per plant (to the 
ne a r e s t  whole no.) 
at fo l l o w i n g  water 
s tress es *
| Mean  length and 
| of m i d - l e a f l e t  ( 
| f o l l o w i n g  water 
| st r e s s e s *
wi d th 
c m ) at
2 4 6 2 4 6
U ndilu te d 12 10 8 |6.98x2.79 5.61x2.14 5.27x2.05
\ Dil uti on 14 10 8 |5.82x2.18 5.06x2.02 5.57x2.39
X Dil uti on 10 9 7 |6 .66x2.33 5,20x2.19 5.60x1.93
*/g Dilution 1 12 9 8 |6.02x2.27 6.06x2 u 16 6.43x2.07
Un i n o c u l a  ted 
(CONTROL)
6 6 5 |3.36x1.57 2.29x1.31 1.57x0.91
2 watered once in 2 days.
4 watered once in 4 days
6 = watered once in 6 days (see Appendix A)
TABLE 9b
Dry weight and ex tent of n o d u l a t i o n  of B a m b a r a  gr ou n d n u t ,  
v ar i e t y  Ex-Ta male, plants i n o c u l a t e d  w i t h  N u n g u a (1)- E x - T a m a l e  
strain grown under no rma l d a y / n i g h t  r egime  at d i f f e r e n t  wa ter 
st resses .
C o n c e n t r a t i o n  of 
Initial Broth 
Culture Inocu l u m  
of moss Carrier
. Me an Dry w t • per 
plant (g) at 
f o l l o w i n g  water 
st r e s s e s *
Me an 
plant 
who 1 e 
wa ter
No. of N o d u l e s  per 
(to the n e a r e s t  
n o .) at f o 1 lowing 
s t r e s s e s *
2 4 6 2 4 6
u n dilu te d 1 .27 
±0. 30
0.85
±0.24
0.72
± 0.21
47 19 10
\ Dil ution 1.17
i 0 . 2 8
0.69
+.0.21
0.67
± 0.21
44 18 11
\ Dilution 1 .22 
±0 . 3 8
0.67
± 0.22
0.58 
±0 .20
50 18 10
*/g Dil ut io n 1.25 
+ 0.38
0.64
± 0.21
0.70 
+ 0.19
49 18 9
Un i n o c u l a  ted 
(CONTROL)
0 . 54 
+ 0.16
0.32
±0.14
0.11 
+ 0.03
0 0 0
2 « watered once in 2 days
4 = watered once in 4 days
6 = watered once in 6 days (see Appendix A)
69
TABLE 10a
Leaf development of Bambara groundnut, variety Ex-Tamale,
inoculated with Pokuase-Ex-Tamale strain grown under normal
day/night regime at different water stresses.
C o n c e n t r a t i o n  of 
Initial Broth 
Culture Ino culu m 
of moss Carrier
Mean No. of leaves 
per plant (to the 
n e a r e s t  whole no.) 
at f o llowi ng  water 
s tress es *
M e a n  le ngth and 
of m i d - l e a f l e t  ( 
f o l l o w i n g  wa ter 
st r e s s e s *
w i d t h  
cm) at
2 4 6 2 4 6
u n dilu te d 12 11 9 6.55x2.51 6.47x2.26 4.88x1.94
\ Dil uti on 13 10 9 6.81x2.28 5.90x1.98 5.41x1.98
\ Dil ut io n 13 9 10 6.62x2.78 6.34x2.05 6.26x1.88
/g Dil uti on 1 1° 10 9 7.19x2.59 6.18x2.55 5.30x2.09
Un inoculated 
(CONTROL)
6 6 5 3.36x1.57 2.29x1.31 1.57x0.91
2 watered once in 2 days.
4 watered once in 4 days
6 = watered once in 6 days (see Appendix A)
70
Dry weight and extent of n o d u l a t i o n  of Bambara gr ou nd n u t ,  
va rie ty Ex-Tamale, plants i n o c u l a t e d  with P o k u a s e - E x - T a m a l e  
strain grown under n or ma l d a y / n i g h t  r eg im e at d i f f e r e n t  water 
stresses .
TABLE 10b
Co n c e n t r a t i o n  of 
Initial Broth 
Culture Inoc ulum 
of moss Ca rrier
Mean Dry wt. per 
plant (g) at 
f o 1 lowing water 
s tr e s s e s *
Mean 
plant 
who le 
w at er
No. of N o d u l e s  per 
(to the n e a r e s t  
no . ) at f o l l o w i n g  
s tr e s s e s *
2 4 6 2 4 6
Undiluted 1.43 0.88 0.76 44 19 11
±0.43 + 0.23 ±0 . 3 3
\ Dilu tion 1 .39 0.70 0.57 51 19 9
±0.41 ± 0 .19 ±0.16
X  Dilution 1.29 0.64 0.61 56 20 9
±0.36 + 0.17 ± 0 . 2 3
1 /g Dil uti on 1 .32 0.74 0.48 45 19 9
±0.31 + 0 .22 + 0.11
Uninoculated 0. 54 0.32 0.11 0 0 0
(CONTROL) ±0.16
r—i•
o+1 ± 0. 0 3
2 = watered once in 2 days
4 = watered once in 4 days
6 = watered once in 6 days (see Appendix A)
71
TABLE 11a
Leaf development of Bambara groundnut, variety Ex-Tamale,
inoculated with Weija-Ex-Ada strain grown under normal
day/night regime at different water stresses.
Cone en tration of 
Initial Broth 
Culture Ino culu m 
of moss Carrier
Mean No. of leaves 
per plant (to the 
n e a r e s t  whole no.) 
at f o l l o w i n g  water 
s tress es *
Me an  le ngt h and 
of m i d - l e a f l e t  ( 
f o l l o w i n g  wa ter 
s t r e s s e s *
w i d t h  
c m ) at
2 4 6 2 4 6
Undi lut ed 13 11 9 6.13x2.48 4.76x2„03 5.63x2.21
\ Dil ution 12 10 9 6.52x2.55 5.31x2.16 6.43x2.05
\ Dil ution 13 8 9 6.03x2.37 5.97x2.20 6.02x1.90
V g  Dilu ti on 12 11 9 |7.02x2.17 5.80x2.13 5.99x2.13
Un in oculate d 
(CONTROL )
6 6 5 3.36x1.57 2.29x1.31 1.57x0.91
2 = watered once in 2 days.
4 = watered once in 4 days
6 watered once in 6 days (see Appendix A)
72
Dry we ight and extent of n o d u l a t i o n  of Bam b a r a  grou nd n u t ,  
v a r i e t y  Ex-Tamale, plants i n o c u l a t e d  wi th W e i j a - E x - A d a  strain 
grown under n or mal d ay/nig ht  regime at d i f f e r e n t  w a ter
s t r e s s e s .
TABLE lib
C o n c e n t r a t i o n  of 
Initial Broth 
Culture Inocul um 
of moss Carrier
Mean Dry wt. per 
plant (g) at 
f o 1 lowing water 
str e s s e s *
Me an  
plant 
who 1 e 
wat e r
No, of N o d u l e s  per 
(to the n e a r e s t  
n o ,) at f o l l o w i n g  
st r e s s e s *
2 4 6 2 4 6
und ilu ted 1 .68 0.71 0,73 51 17 9
±0 .43 ± 0.2 2 + 0.22
\ Dilutio n 1.65 0. 76 0.51 53 19 11
±0 . 4 5 ±0.23 ±0.16
\ Dilution 1 . 24 0.77 0.68 53 18 10
± 0 . 3  3 ± 0.20 + 0.20
^/g Dil ution | 1.48 0.65 0.69 1 5 5 25 10
±0 .38 ± 0 . 1 9 ± 0.2 0
Unino cu lated 0.54 0 .32 0.11 0 0 0
(CONTROL) ±0 .16 + 0.14 + 0.03
2 = wa ter ed once in 2 days
4 = watered once in 4 days
6 = watered once in 6 days (see A p p e n d i x  A)
73 -
PLATE 4. Photog r a p h  showin g barabara g r o u n d n u t  plants w a t e r e d  
at di f f e r e n t  intervals after i n o c u l a t i o n  w i t h  
Rhizobium s p . ( x 1 / g )
Left: W a t e r e d  at 2-day in te rvals.
Middle: Wat e r e d  at 4-day intervals .
Right: Watered at 6-day intervals.
- 7 4 -
PLATE 5. P h o t o g r a p h  sh ow in g 30 day old b a m b a r a  g r o u n d n u t
plants w a t e r e d  at d i f f e r e n t  i nte rv als after 
i n o c u l a t i o n  w i t h  Rhizobium sp.
Note the d i f f e r e n c e s  in d e v e l o p m e n t  of both shoot and 
root systems, (x /g) )
Left: W a t e r e d  at 2- day in te rvals.
Middle: W a t e r e d  at 4-day inte rva ls.
Right: Watered at 6-day intervals.
75
Me an leaf n umbe r per plant
A n a l y s e s  of V a r i a n c e  (Two -w ay A n ov a with r e p l i c a t i o n )
showed that there was si g n i f i c a n t  e f f e c t  of the w a t e r  stress 
and i n o c u l a t i o n  on the me an leaf n u m b e r  for all the five
st rai ns of Rhizobium sp. used at bo th 5 per cent and 1/ "'per cent
levels of sig n i f i c a n c e .  There was, how ev er, no s i g n i f i c a n t
effect of the i n t e r a c t i o n  of the two t r e a t m e n t s  on the leaf
number d e v e l o p m e n t  and so the two t r e a t m e n t s  w e r e  a ct ing
i n d e p e n d e n t  of each ot her  (Tables 12a, 13a, 14a, 15a, and 16a).
The Dunca n ' s  New M u l t i p l e  Range Test of Tables 12b, 13b,
14b, 15b and 16b showed that for each s tr ai n of Rhizobium sp.
the four initial i n o c u l u m  de n s i t i e s  fell in one group and they 
were not s i g n i f i c a n t l y  d i f f e r e n t  from each other at b o t h  5 per 
cent and 1 per cent levels of s i g n i f i c a n c e ,  but w e r e  t o g et her 
d iffer en t from the con trol.
The effect of w a ter stress can be s u m m a r i s e d  a£- follows:
(a) The ef fects  of the three wat e r  re gi me s were
s i g n i f i c a n t l y  d i f f e r e n t  from each other at both 5 per cent 
and 1 per cent levels of s i g n i f i c a n c e  in plants
in ocul a t e d  wit h N u ngua( l)  - E x - T a m a l e  strain.
(b) In plants i n o c u l a t e d  w i t h  K p o n g - E x - T a m a l e  strain, the
eff ect s of the three wat e r  reg i m e s  were s i g n i f i c a n t l y  
d i f f e r e n t  from each other at 5 per c e n t  level of 
s ig ni ficanc e,  but at 1 per cent level of s i g n i f i c a n c e  
e ffe cts of w a t e r i n g  at 4 and 6 -d ay  i nt ervals  we re not
s i g n i f i c a n t l y  d i f f e r e n t  from each other.
76 -
(c) In plants i n o c u l a t e d  w i t h  strains of P o k u a s e - E x -
Tamale and W e i j a - E x - A d a , ef fe c t s  of d i f f e r e n t  w a t e r i n g
times were not s i g n i f i c a n t l y  d i f f e r e n t  at 5 per cent level
ce>ik
of signifi ca nce, but at 1 per level plan ts w a t e r e d  at 2 -
A
day inte rvals p r o d u c e d  a s i g n i f i c a n t l y  d i f f e r e n t  e ffec t
from the other two treatm ents.
(d) In plants i n o c u l a t e d  wi th  s t r a i n  of L e g o n - E x - A d a ,  the
e ffe cts were not s i g n i f i c a n t l y  d i f f e r e n t  at b o t h  5 per 
cent and 1 per cent levels of s i g n i f i c a n c e .
77
(Plants i n o c u l a t e d  with K p o n g - E x - T a m a l e  strain).
TABLE 12a
Analysis of variance (Two-way Anova with replication) for
data of Table 7a, Mean leaf number per plant.
Source 
o f
V a r i a t i o n
| Sura
1 of 
I squares
I
Degree 
o f 
freedom
Mean
squares
F
va lue
Water stress
1
| 122.75
1
2 61.375 8.249 * ★
Inoculat ion
1
| 292.75 
1
4 73.188 9.837 •* *
Water stress 
& I n o c u l a t i o n
1
1
| 41.25
I
8 5.156 0.693 NS
Error
1
| 44 6,4 0
I
60 7.440 -
T O T A L
1
| 903.15 
1
74 - -
** S i g n if ic ant at 1% level of s i g n i f i c a n c e
NS Non-Significant
- 78 -
TABLE 12b
(Data of Table 12a)
Du nc an 's New M u l t i p l e  Range Test for means of leaf n um be r 
of Ba m b a r a  g r ou nd nut plants su b j e c t e d  to
a) Water stress
5%
1%
b ) inocula ti on
Co nt ro l
5%
1%
Note: Any two means not u n d e r s c o r e d  by the same line
are s i g n i f i c a n t l y  d i f f e r e n t .  Any two means
u n d e r s c o r e d  by the same line are not s i g n i f i c a n t l y  
d i f f e r e n t .
2 : 2-day w a t e r i n g  interval 6 : 6-day w a t e r i n g  i n t er val
4: 4-day  w a t e r i n g  interval
A: Undiluted concentration C: \ dilution
B: h dilution D : 1/8 dilution
79
TABLE 13a
Analysis of variance (Two-way Anova with replication) for
data of Table 8a, Mean leaf number per plant.
(Plant i n o c u l a t e d  wi th L e g o n - E x - A d a  strain).
Source 
o f
V a r i a t i o n
| Sura
1 of 
| squares
Degree 
o f
f reedom
Mean
squares
F
v al u e
Water stress
1
| 77.31
I
2 38.655 5.315 **
I n o c ul at ion
1
| 165.87 
I
4 41.468 5.701 **
Water stress 
& I n o c u l a t i o n
1
1
| 11.09 
1
8 1.386 0.191 NS
Error
1
| 43 6.4 0
I
60 7.273 -
T O T A L
1
| 690.67 
1
74 - -
* ■* S i g n i f i c a n t at 1% level of s i g n i f i c a n c e
NS N o n - S i g n i f i c a n t
TABLE 13b
(Data of Table 13a)
Duncan's  New M u l t i p l e  Range Test for means of leaf n u m b e r  
of Ba m b a r a  gro u n d n u t  plants s ubj ec ted to
a) Water stress
- 80 -
b ) ino culat  ion
Control
5%
1%
Note: Any two means not u n d e r s c o r e d  by the same line
are s i g n i f i c a n t l y  diffe r e n t .  Any two means
u n d e r s c o r e d  by the same line are not s i g n i f i c a n t l y  
different.
2 : 2 -day w a t e r i n g  int erval 6 : 6-day w a t e r i n g  i n t erva l
4: 4-day w a t e r i n g  interval
A: Undiluted concentration C: \ dilution
B: ^ dilution D: \/8 dilution
81
TABLE 14a
Analysis of variance (Two-way Anova with replication) for
data of Table 9a. Mean leaf number per plant.
(Plant in ocul a t e d  wit h N u n g u a (1)- E x - T a m a l e  strain).
Source 
o f
V a r i a t i o n
| Sum
1 of 
| squares
Degree 
o f
f reedom
Mean
squares
F
va lue
Water stress
1
| 148.88
I
2 74.440 21.309  **
I n o cu la ti on
1
| 219.87
1
4 54.968 15.735 **
Water stress 
& In o c u l a t i o n
1
1
| 29.65
1
8 3 . 706 1.061 NS
Error
1
| 209.60 
1
60 3.493 -
T O T A L
1
| 608.00 
1
74 - -
** S i g n i f i c a n t at 1 % level of s i g n i f i c a n c e
NS Non-Significant
82
TABLE 14b
a) Water stress
(Data of Table 14a )
Duncan's New Multiple Range Test for means of leaf number
of Bambara groundnut plants subjected to
5%
1 %
b) inoculation
Control
5%
1%
Note: Any two means not underscored by the same line
are significantly different. Any two means
underscored by the same line are not significantly 
different.
2: 2-day watering interval 
4: 4-day watering interval
A: Undiluted concentration
B: \ dilution
6: 6-day watering interval
C: \ dilution
D: 1/8 dilution
83
(Plant inocul at ed w i t h  P o k u a s e - E x - T a m a l e  strain).
TABLE 15a
Analysis of variance (Two-way Anova with replication) for
data of Table 10a. Mean leaf number per plant.
Source 
o f
Va r i a t i o n
Sum 
o f 
squares
Degree 
o f 
f r eed om
Mean
squares
F
va lue
Water stress 116.27 2 58.135 7.234 * *
In oc ul ation 254.94 4 63 . 735 8.468 * *
Water stress 
& I n o cul at ion 41 .86 8 5 .233 0.695 NS
Error 4 51. 60 60 7.527 -
T O T A L 864.67 74 - -
** Significant at 1% level of significance
NS Non-Significant
TABLE 15b
(Data of Table 15a)
Duncan's New Multiple Range Test for means of leaf number 
of Bambara groundnut plants subjected to
a) Water stress
- 84 -
b ) inoculat ion
Control
5%
1%
Note: Any two means not underscored by the same line
are significantly different. Any two means
underscored by the same line are not significantly 
different.
2: 2 day watering interval 6: 6-day watering interval
4: 4-day watering interval
A: Undiluted concentration C: \ dilution
B: 5s dilution D: 1/8 dilution
8 5
TABLE 16a
Analysis of variance (Two-way Anova with replication) for
data of Table 11a. Mean leaf number per plant#
(Plant inoculated with Weija-Ex-Ada strain).
Source 
o f
Variation
| Sum
1 0 f
j squares
Degree 
o f 
freed om
Mean 
square s
F
value
Water stress
1
| 122.75 
]
2 61.375 9.433 **
Inoculation
1
| 253.79 
t
4 63.448 9.751 **
Water stress 
& Inocula t ion
I
1
| 45.65 
[
8 5.706 0.877 NS
Error
I
| 390.40 
1
60 6. 507 -
T O T A L
1
| 812.59
— J____
74 - -
** Significant at 1% level of s igni f ic anc e
NS Non-Significant
86
a) Water stress
TABLE 16b
(Data of Table 16a)
D u n c a n ’s New Multiple Range Test for means of leaf number
of Bambara groundnut plants subjected to
2 4 6
________________________________________________  5%
1%
b) inoculat ion
A B C D Control
5%
1%
Note: Any two means not underscored by the same line
are significantly different. Any two means
underscored by the same line are not significantly 
different.
2: 2-day watering interval 6: 6-day watering interval
4: 4-day watering interval
A: Undiluted concentration C: \ dilution
B: h dilution D : 1/8 dilution
87
Mean size of middle leaflets.
It was remarkable that water stress had a great effect on the size of 
the leaflets of the uninoculated plants (Tables 7a, 8a, 9a, 10a, 11a), and 
the mean length of middle leaflets of plants watered at 2 day intervals was 
more than double that of plants watered at 6-day intervals. Even though
the differences in the inoculated plants were not that accentuated,
Analyses of Variance (Two-way Anova with replication) in Tables 17a, 18a, 
19a, 20a, 21a showed there was significant effect of the water stress and
inoculation on the leaflet sizes at both 5 per cent and 1 per cent levels 
of significance. There was a significant effect of their interaction at 
both 5 per cent and 1 per cent levels of significance for Legon-Ex-Ada, 
Nungua(1)-Ex-Tamale and Weija-Ex-Ada strains, a significant effect at 5 per 
cent level of significance only for Pokuase-Ex-Taraale strain, and a non­
significant effect for Kpong-Ex-Tamale strain.
By the Duncan's New Multiple Range Test shown in Tables 17b, 18b,
19b, 20b and 21b, there was no effect of the initial inoculum density at 
both 5 per cent and 1 per cent levels of significance for Kpong-Ex- 
Tamale, Nungua(l)-Ex-Tamale, Pokuase-Ex-Taraale and Weija-Ex-Ada strains. 
For the Legon-Ex-Ada strain, the initial inoculum densities separated into 
two groups, undiluted and \  dilution in one, and \  and 1/8 dilution in the 
other.
In the case of relationship between water stress and leaflet size,
the Duncan's New Multiple Range Test also in Tables 17b, 18b, 19b, 20b and
21b, showed that the effects of the three watering regimes were 
significantly different at both 5 per cent and 1 per cent levels of 
significance with plants inoculated with Legon-Ex-Ada, Pokuase-Ex-Tamale, 
and Weija-Ex-Ada strains; the effect of 2-day watering was significantly
different from the effects of 4-day and 6-day watering at both 5 per cent 
and 1 per cent levels of significance for plants inoculated with 
Nungua(1)-Ex—Tamale strain; and the effects of the three watering regimes 
were not significantly different at both 5 per cent and 1 per cent levels 
of significance for plants inoculated with Kpong—Ex—Tamale strain.
88
(Plant in ocu l a t e d  with K p o n g - E x - T a m a l e  strain).
TABLE 17a
Analysis of variance (Two-way Anova with replication) for
data of Table 7a, Mean size of middle leaflets
Source 
o f
Variation
| Sura
1 0 f
| squares
Degree 
o f 
freedom
Mean
squares
F
va 1 ue
Water stress
1
100.30
1
2 50.150 9.166 **
Inocu1 at i on
1
| 1666.30
|
4 416.575 76.141 **
Water stress 
& Inoculation
1
1
| 81.01 
1
8 10.126 1.851 NS
Error
1
| 492.40
i _________
90 5.471 -
T O T A L
1
| 2339.75
_! _ _
104 - -
* * Significant at 1% level of significance
NS Non-Significant
- 89 -
TABLE 17b
(Data of Ta ble 17a)
D u n can's New M u l t i p l e  Range Test for me ans  of lea f l e t  size 
of Bam b a r a  g r o u n d n u t  plants su b j e c t e d  to
a) Water stress
b ) ino cul at ion
Control
5%
1%
Note: Any two means not u n d e r s c o r e d  by the same line
are s i g n i f i c a n t l y  d i f f e r e n t .  Any two means
u n d e r s c o r e d  by the same line are not s i g n i f i c a n t l y  
di fferent.
2 : 2-day w a t e r i n g  inte rval 6 : 6 -day w a t e r i n g  i n t e r v a l
4: 4-day w a t e r i n g  interval
A: Undiluted concentration C: \ dilution
B: \ dilution D; x/8 dilution
90
(Plant i n o c u l a t e d  w i t h  L e g o n - E x - A d a  strain).
TABLE 18a
Analysis of variance (Two-way Anova with replication) for
data of Table 8a. Mean size of middle leaflets
Source 
o f
V a r i a t i o n
Sum 
o f 
squares
D eg ree 
o f 
freedom
Me an 
squares
F
value
Water stress 890.05 2 44 5. 02 5 55.855 **
I n o c u l a t i o n 2407.18 4 601 . 795 7 5.532 **
Water stress 
& Inoc ulat ion 386.63 8 48.329 0.066 **
Error 717.07 90 7.967 -
T O T A L 44 00.93 104 - -
Significant at 1% level of significance
i
91
a) Water stress
TABLE 18b
(Data of Table 18a)
Duncan's New Multiple Range Test for means of leaflet size
of Bambara groundnut plants subjected to
2 4 6
5%
1%
b) in o c u l a t i o n
A B C D Control
5%
1%
Note: Any two means not u n d e r s c o r e d  by the same line
are s i g n i f i c a n t l y  d i f f e r e n t .  Any two means
u n d e r s c o r e d  by the same line are not s i g n i f i c a n t l y  
different.
2 : 2 -day w a t e r i n g  interval 6 : 6 -day w a t e r i n g  i n t er val
4: 4-day w a t e r i n g  interval
A: Undiluted concentration C: \ dilution
B: \ dilution d ; 1/8 dilution
92
TABLE 19a
Analysis of variance (Two-way Anova with replication) for
data of Table 9a. Mean size of middle leaflets
(Plant in oc ul a t e d  with N u n g u a (1)- E x - T a m a l e  strain).
Source 
o f
V a r i a t i o n
Sum 
o f 
sq uar es
Degree 
o f 
freedom
I
Me an 
sq uares
F
v a lue
Water stress 222 . 1 1 2 111.055 3 5. 206 **
I n o c u l a t i o n 1635.53 4 40 8.883 12 9.621 **
Water stress 
& Inocul at  ion 23 5 . 04 8 29 .38 9.314 **
Error 283.90 90 3.154 -
T O T A L 2376.58 104
.
-
Significant at 1% level of significance
TABLE 19b
(Data of Table 19a)
Dunc an 's New M u l t i p l e  Range Test for means of le aflet size 
of Ba mb ara g rou nd nut plants s ub je cted to
a) Water stress
- 93 -
b ) in o c u l a t i o n
5%
1%
D Control
5%
1%
Note: Any two means not u n d e r s c o r e d  by the same line
are s i g n i f i c a n t l y  di ff e r e n t .  Any two means
u n d e r s c o r e d  by the same line are not s i g n i f i c a n t l y  
different.
2 : 2-day w a t e r i n g  interval
4: 4-day w a t e r i n g  interval
A: U ndilute d c o n c e n t r a t i o n
B: \ diluti on
6 : 6 -day w a t e r i n g  i n t erval
C: \ dilution
D: 1/8 dilution
93
a) Water stress
TABLE 19b
(Data of Table 19a)
Duncan's New Multiple Range Test for means of leaflet size
of Barabara groundnut plants subjected to
2 4 6
_________________________  5%
1%
b) i n o c ul at ion
A B C D Control
5%
1%
Note: Any two means not u n d e r s c o r e d  by the same line 
are s i g n i f i c a n t l y  di fferent. Any two means
u n d e r s c o r e d  by the same line are not s i g n i f i c a n t l y  
different.
2-day w a t e r i n g  interval 
4-day w a t e r i n g  interval 
Undil ut ed c o n c e n t r a t i o n
6 : 6 -day w a t e r i n g  int erval
C: \ dilution
D: 1/8 dilution
94
A n a ly si s of v a r i a n c e  (Two-wa y A n o v a  with r e p l i c a t i o n )  for 
data of Table 10a. Mean size of m i d d l e  leaflets
i
(Plant inoc u l a t e d  w i t h  P o k u a s e - E x - T a m a l e  strain).
TABLE 20a
Source 
o f
V a r i a t i o n
| Sum
1 of 
1 squares
1
Degree 
o f 
freedom
Mean
squares
F
va lue
Water stress
1
| 628.65 
1
2 3 14. 325 71 .483 * *
I n o c u 1 at i on
1
| 1966.79 
I
4 4 91 . 6 9 8 111 .820 * *
Water stress 
& I n o c u l a t i o n
1
1
| 93.00 
1
8 11.625 2.644 *
Error
1
| 395.75 
1
90 4.397 -
T O T A L
1
| 3084.19
1
104 - -
* * S i g n i f i c a n t at 1 % l e v e 1 of s i g n i f i c a n c e
Significant at 5% level of significance
- 95
TAB L E  20b
(Data of Table 20a)
D u n ca n's New M u l t i p l e  Range Test for means of le a f l e t  size 
of Barabara g r ou ndnut plants sub j e c t e d  to
a) Water stress
5%
1%
b) i n o c u l a t i o n
Control
5%
1%
Note: Any two means not u n d e r s c o r e d  by the same line
are s i g n i f i c a n t l y  di fferen t.  Any two means
u n d e r s c o r e d  by the same line are not s i g n i f i c a n t l y  
different.
2 : 2 -day w a t e r i n g  interval 6 : 6 -day w a t e r i n g  int er v a l
4: 4- day w a t e r i n g  interval
A: Undiluted concentration C: \ dilution
B: \ dilution D: 1/8 dilution
- 96
TABLE 21a
A n a l y s i s  of v a r i a n c e  (Tw o-way An o v a  wit h r e p l i c a t i o n )  for 
data of Table 11a, Me an  size of m i d d l e  leaflets
(Plant i n o c u l a t e d  wi th W e i j a - E x - A d a  strain).
Source 
o f
V a r i a t i o n
| Sum 
| o f 
| square s 
1
Degree 
o f 
freedom
Mean
squares
F
va lue
Wa ter stress
1
| 259.82 
1
2 12 9.9 10 62 .537 **
I n o c u 1 at ion
1
] 1716.07 
|
4 429.0 18 2 06 . 5 2 3  **
Water stress 
& I n o c u l a t i o n
t
1
| 83.66 
1
8 10.458 5.034 **
Error
1
| 186.96 
1
90 2.077 -
T O T A L
1
| 2246.51 
1 .......
104 - -
** Significant at 1% level of significance
97
a) Water stress
TABLE 21b
(Data of Table 21a)
Duncan's New Multiple Range Test for means of leaflet size
of Bambara groundnut plants subjected to
2 4 6
5%
1%
b) in o c u l a t i o n
A B C D C o n t r o 1
5%
1%
Note: Any two me ans  not u n d e r s c o r e d  by the same line
are s i g n i f i c a n t l y  d i f f e r e n t .  Any two means
u n d e r s c o r e d  by the same line are not s i g n i f i c a n t l y  
di fferent,
2 : 2-day w a t e r i n g  int erval 6 : 6 -day w a t e r i n g  i n t e r v a l
4: 4-day w a t e r i n g  interval
A: Undiluted concentration C: ^ d i l u t i o n
B: \ dilution D: 1/8 dilution
98
Mean dry weight  of pl ants.
There was m a r k e d  ef fec t of i n o c u l a t i o n  on the m e a n  dry
weight of the b a m b a r a  g r o u n d n u t  plants as there was above 30
per cent increas e of all the i n o c u l a t e d  pl ants over the
uninocu lat ed (contr ol) plant.
Ana lys es of V a r i a n c e  (Two-way  A n ov a with r e p l i c a t i o n )  test 
of Tables 22a, 23a, 24a, 25a and 26a sh owed s i g n i f i c a n t  e ff ect
of the tr eatment s on dry we ight at 5 per cent and 1 per cent 
levels of s i g n i f i c a n c e  for all the i n o c u l a t e d  plants. There
was, however, no s i g n i f i c a n t  effect of the i n t e r a c t i o n  of the 
two treatments on the dry weight.
There were no s i g n i f i c a n t  d i f f e r e n c e s  b e t w e e n  the ef fe ct s 
of the di f f e r e n t  ini t i a l  i n o c u l u m  d e n s i t i e s  at both 5 per cent 
and 1 per cent levels of s i g n i f ic an ce, u s i n g  the D u n c a n ' s  New
Multiple Range Test p r e s e n t e d  in Tables 22b, 23b, 24b, 25b and
26b .
In case of the w a ter stress treatmen ts , the three w a t e r i n g  
regimes had s i g n i f i c a n t l y  dif f e r e n t  ef f e c t s  at 1 per cent 
level of s i g n i f i c a n c e  for plants i n o c u l a t e d  with the K p o n g - E x -  
Tamale strain, but at 5 per cent level of s i g n i f i c a n c e  there 
was no si g n i f i c a n t  d i f f e r e n c e  b e t w e e n  the ef fects of the 2 -day
and 4-day w a t e r i n g  in terval s.  With pl ants i n o c u l a t e d  w i t h  the
remaining four strains, there was no s i g n i f i c a n t  d i f f e r e n c e  
between the eff ects of the 4-day and 6- d a y  w a t e r i n g  t r e a t m e n t s  
at both 5 per cent and 1 per cent levels of s i g n i f i c a n c e .
99
TABLE 22a
Analysis of variance (Two-way Anova with replication) for
data of Table 7a. Mean dry weight.
(Plant inocu l a t e d  w i t h  K p o n g - E x - T a m a l e  strain).
Source | 
of | 
V ar ia tio n | 
1.
Sum 
o f 
squares
Degree
of
freedom
Mean
squares
F
v al u e
1
Water stress | 
1
13.10 2 6.550 8 .7 47 **
1
Inoculation | 
1
17.59 4 4.398 5.873 **
Water stress |
& Inoculation | 
I
1.92 8 0.240 0.321 NS
1
Error |
I
44.93 60 0.749 -
1
T O T A L  | 
1
77.54 74 - -
** Significant at 1% level of significance
NS Non-Significant
TA BLE  22b
(Data of Table 22a)
Duncan's New M u l t i p l e  Range Test for means of dry w e i g h t  
of Barabara g r ou ndnut plants su b j e c t e d  to
a) Water stress
- 100 -
b) i n o c u l a t i o n
Control
5%
1%
Note: Any two means not u n d e r s c o r e d  by the same line
are s i g n i f i c a n t l y  diffe re nt. Any two means
u n d e r s c o r e d  by the same line are not s i g n i f i c a n t l y  
di f f e r e n t .
2 : 2 -day w a t e r i n g  interval 6 : 6 -day w a t e r i n g  i n t erval
4: 4-day w a t e r i n g  interval
A: Undiluted concentration C: \ dilution
B: H dilution D; 1/8 dilution
101 -
TABLE 23a
An a 1ys i s of v a r i a n c e  (Two-way A no v a  with r e p l i c a t i o n )  for
data of Table 8b ♦ Mean dry we i gh t .
(Plant in oculat ed  w i t h  Legon -E x- Ad a s t r a i n ).
Source | Sura De gree
o f 1 of o f Mean F
V a r i a t i o n 1 squares 
1
fr eed om sq uares va lue
Water stress
1
| 2 A . 7 7 
!
2 12.385 18.952 **
I n o c ulati on
1
| 19.60 
1
4 4. 900 7.498 **
Water stress
1
1
& In o c u l a t i o n | 3.36 
1
8 0.420 0.643  NS
Error
1
| 39.21 
1
60 0.654 -
T O T A L
1
| 86.94 
1
74 - -
** Significant at 1% level of significance
NS Non-Significant
-  1 0 2  -  
TA BLE  23b 
(Data of Ta ble 23a)
D u n c a n ’s New M u l t i p l e Range Test for means of dry w e i g h t
0 f Ba mba ra gr o u n d n u t  plants sub j e c t e d  to
a ) Water stress
2 4 6
5%
1%
b) i n o culati on
A B C D Control
5%
1%
Note: Any two means not u n d e r s c o r e d  by the same line
are s i g n i f i c a n t l y  d i f f e r e n t .  Any two means
u n d e r s c o r e d  by the same line are not s i g n i f i c a n t l y  
d i f f e r e n t .
2 : 2-day w a t e r i n g  interval 6 : 6 -day w a t e r i n g  in ter v a l
4: 4-day w a t e r i n g  interval
A: Undiluted concentration C: \ dilution
B: H dilution D: !/8 dilution
103
(Plant in oculate d wi th N u n g u a (1 )- E x - T a m a l e  strain).
TABLE 24a
Analysis of variance (Two-way Anova with replication) for
data of Table 9b, Mean dry weight.
Source 
o f
Va ria t ion
Sum
1 of 
| squares
De gre e 
o f 
fr e e d o m
Mean
squares
F
va lue
Water stress
1
| 16.41
1
2 8.205 8.838 **
Inoculat ion
1
| 14.96 
1
4 3.740 4.028 **
Water stress 
& Inoculat io n
1
1
| 1.11 
1
8 0.139 0.1 5 0  NS
Error
1
| 55.71
I
60 0.928 -
T O T A L
1
| 88.19
1
74 - -
** Si g n i f i c a n t at 1 % level of s i gni f ic anc e
NS Non-Significant
104
TABLE 24b
(Data of Table 24a)
Duncan's New Multiple Range Test for means of dry weight
of Bambara groundnut plants subjected to
a) Water stress
2 4 6
5%
1%
b) in oc ul ation
A B C D Control
5%
1%
Note: Any two means not u n d e r s c o r e d  by the same line
are s i g n i f i c a n t l y  d i f f e r e n t .  Any two means
u n d e r s c o r e d  by the same line are not s i g n i f i c a n t l y  
different.
2 : 2 -day w a t e r i n g  interval 6 : 6 -day w a t e r i n g  i n t er va l
4: 4-day w a t e r i n g  interval
A: Undiluted concentration C: \ dilution
B: i dilution d ; 1/8 dilution
- 105
T AB LE 25a
An alysis of v a r i a n c e  (Two-way An ova w i t h  r e p l i c a t i o n )  for 
data of Table 10b. Mean dry weight.
(Plant inoc ul ated w i t h  Pokuase-Ex-Taraale strain).
Source | 
of | 
V ariat io n |
Sum 
o f 
squares
Degree 
o f 
fr eed om
Mean
squares
F
v al ue
1
Water stress 23. 75 2 11.875 27.164 * ★
1
Inoculat ion | 19.03 4 4.758 10.883 ★ *
Water stress [
& Ino cul ation  | 
1
2.42 8 0. 303 0 . 692 NS
Error |
I
26.23 60 0.437 -
1
T O T A L  | 
1
71.43 74 - -
** Significant at 1% level of significance
NS Non-Significant
T AB LE 25b
(Data of Table 25a)
D u n c a n ’s New M u l t i p l e  Range Test for means  of dry w e i g h t  
of Bambara g roun dn ut plants s u b j e c t e d  to
a ) Water s tress
- 107 -
b ) inoculat ion
Co nt ro l
5%
1%
Note: Any two means not u n d e r s c o r e d  by the same line
are s i g n i f i c a n t l y  d i f f e r e n t .  Any two means
u n d e r s c o r e d  by the same line are not s i g n i f i c a n t l y  
d i f f e r e n t .
2 : 2-day w a t e r i n g  interval 
4: 4-day w a t e r i n g  interval
A: Undiluted concentration
B : \ dilution
6 : 6 -day w a t e r i n g  i n t e r v a l
C: % dilution
D : 1/8 dilution
108 -
TABLE 26a
An aly sis of v a r i a n c e  (Two-way A n o v a  w i t h  r e p l i c a t i o n )  for 
data of Table lib. Mean dry weight.
(Plant inoculated wit h W e i j a - E x - A d a  strain).
Source 
o f
V ar ia tio n
| Sum
1 of 
| squares
Degree 
of 
f reed om
Mean
squares
F
v al u e
Water stress
1
| 36.47 
l
2 18.235 52.100 * *
Inoculation
1
| 20.25 
I
4 5.063 1 4.4 64 ■k *
Water stress 
& In oculation
I
1
| 5.25 
1
8 0.656 1 . 875 NS
Error
1
| 21.00 
1
60 0.350 -
T O T A L
1
| 82.97 
1
74 - -
** Significant at 1% level of significance
NS Non-Significant
TA BLE  26b
(Data of Table  26a)
Duncan's New M u l t i p l e  Range Test for me ans  of dry w eight  
of Bambara g r ou nd nut plants s u b j e c t e d  to
a) Water stress
- 109 -
2 4 6
_________________________  5%
1%
b) in oc ula tio n
A B C D Co n t r o l
5%
1%
Note: Any two means not u n d e r s c o r e d  by the same line 
are s i g n i f i c a n t l y  d i f f e r e n t .  Any two means
u n d e r s c o r e d  by the same line are not s i g n i f i c a n t l y  
d i f f e r e n t .
2 : 2 -day w a t e r i n g  interval 
4: 4-day w a t e r i n g  interval
6 -day w a t e r i n g  i n t e r v a l
A: Undiluted concentration
B: \ dilution
C: \ d i l u t i o n
D: 1/8 d i l u t i o n
Mean nu mber of nod u l e s  per plant
Finally the result of A n a l y s e s  of V a r i a n c e  Tests ( T w o- wa y 
Anova with repli c a t i o n )  pr e s e n t e d  in T a b l e s  27a, 28a, 29a, 30a
and 31a showed that there was s i g n i f i c a n t  ef fect at b o t h  5 per 
cent and 1 per cent levels of s i g n i f i c a n c e  of the two 
treatments, that is water stress and inocu l a t i o n ,  as wel l as 
their i n t e raction  on the mean n u m b e r  of n o d u l e s  fo rmed by the 
barabara g round nu t plants.
Tables 27b, 28b, 29b, 30b and 31b show the re s u l t s  of
Duncan's New M u l t i p l e  Range Test. I n o c u l a t i o n  had a clear 
effect, and the ef fect of all of the i n o c u l a t i o n s  w e r e  not 
s i g n i f icantly  d i f f e r e n t  from each oth e r  but d i f f e r e n t  from 
effects of the controls  at both 5 per cent and 1 per cent 
levels of si gni fic ance.
The effects of the three w a t e r i n g  re g i m e s  were 
significan tl y d i f f e r e n t  from each ot her at both 5 per cent and 
1 per cent levels for plants i n o c u l a t e d  wi th N u n g u a ( 1) -Ex- 
Tamale and P o k u a s e - E x - T a m a l e  strains. The ef fects of the three 
levels of w a t ering  were also d i f f e r e n t  from each other at 1 
per cent level of s i g n i f i c a n c e  for pl ant s i n o c u l a t e d  w i t h  
K p o n g - E x - T a m a l e  and L e g o n - E x - A d a  strains, but at 5 per cent 
level of s i gnif ic ance there was no s i g n i f i c a n t  d i f f e r e n c e s  
between the effects of 4-d ay and 6-d ay  w a t e r i n g  regimes. 
Plants inoculate d with W e i j a - E x - A d a  st rain s how ed  n o n ­
significant dif f e r e n c e s  be tween the e f f e c t  of 4 -day and 6 -day 
wat eri ng regimes at both 5 per cent and 1 per cent levels of 
s i gn i f i c anc e .
- 110 -
Ill
(Plants i n o c u l a t e d  wit h K p o n g - E x - T a m a l e  strain).
TABLE 27a
Analysis of variance (Two-way Anova with replication) for
data of Table 7b. Mean number of nodules.
Source | 
o f | 
v ar ia tio n
Sura 
o f 
squares
D eg re e 
o f 
fre e d o m
Mean - 
squares
F
va l u e
1
Water stress |
I
13904.35 2 6952.175 120.725 * *
1
I n o c ul at ion j
1
7918.59 4 1979 . 648 3 4.3 77 * *
Water stress &
In oc ula tio n |
1
3664.85 8 458 . 1 0 6 7.955 ★ *
Error |
1
3455.20 60 57.587 -
TOTAL | 
______  1
28 94 2. 99 74 - -
* * Significant at 1% level of significance.
- 112 -
TA B L E  27b
(Datfi( of Table 27a)
D u n c a n ’s New M u l t i p l e  Range Test for mea n s  of n o d u l e  
number of Barabara g r o u n d n u t  plants s u b j e c t e d  to
a ) water stress
2 4 6
5%
1%
b) inocula t ion
A B C D C o n t r o 1
5%
1%
N °te: Any two means not u n d e r s c o r e d  by the same line
are s i g n i f i c a n t l y  d i f f e r e n t .  Any two means
und e r s c o r e d  by the same line are not s i g n i f i c a n t l y  
different,
2 : 2-day w a t erin g interval 6 : 6 -day w a t e r i n g  i n t e r v a l
4: 4-day w a t e r i n g  interval
A: Undiluted concentration
B: \ dilution
C: \ dilution
D: ^/g dilution.
113
TABLE 28a
Analysis of variance (Two-way Anova with replication) for
data of Table 8b. Mean number of nodules.
(Plants i n o c u l a t e d  wi th  L e g o n - E x - A d a  strain).
Source | 
o f | 
v ar ia tio n |
Sum 
o f
squares
Degree 
o f
fr e e d o m
Me an 
squares
F
v a lue
1
Water stress | 13911.15 2 6955.575 120.785  * *
1
Inoculat ion | 8015.66 4 2 0 0 3. 91 5 3 4 . 7 9 8  * *
Water stress & | 
Inoculat i on | 3716.18 8 464.523 8.067 * *
Error |
1
3200.80 60 53.347 -
TOTAL |
1
28843.79 74 - -
* * Significant at 1% level of significance.
TABLE 28b
a) water stress
(Data of Table 28a)
Duncan's New Multiple Range Test for means of nodule
number of Bambara groundnut plants subjected to
2 4 6
5%
1%
b) in oculation
A B C D Con t r o l
5%
1%
Note: Any two means not u n d e r s c o r e d  by the same 1 :
s i g n i f i c a n t l y  diffe ren t. Any two me ans  u n d e r s c o r e d  
by the same line are not s i g n i f i c a n t l y  d i f f e r e n t .
2 : 2-day w a t e r i n g  interval 6 : 6 -day w a t e r i n g  i n t e r v a l
4: 4-day w a t e r i n g  interval
A: Und il uted c o n c e n t r a t i o n  C: \ d i l ution
B: * dilution D . 1/ d i l u t i o n .
115
Analysis of v a r i a n c e  (Two-wa y A n ova w i t h  r e p l i c a t i o n )  for
data of Table 9b. Mean number of nodules .
(Plants in oc ul a t e d  with N u n g u a (1 )- E x - T a m a l e  strain).
TABLE 29a
Source 
o f
va riation
Sum 
o f 
squares
Degree 
o f 
f r e ed ora
Mean
squares
F
va lue
Water stress 1 2346 . 1 6 2 617 3. 080 106. 9 8 6  * *
Inoculation 7672.53 4 1918.133 33 .243 * *
Water stress & 
Inocula t ion 3179.39 8 397.424 6.888 * *
Error 3462.00 60 57.700 -
TOTAL 266 60.00 74 - -
* * Significant at 1% level of significance.
TABLE 29b
(Datfl of Table 29a)
D u n c a n ’s New Multiple Range Test for means of nodule
number of Bambara groundnut plants subjected to
) water stress
2 4 6
5%
1%
b) in oc ulation
A B C D Co nt ro l
5%
1%
Note: Any two means not u n d e r s c o r e d  by the same line 
are s i g n i f i c a n t l y  di ff er e n t .  Any two means
un d e r s c o r e d  by the same line are not s i g n i f i c a n t l y  
di f f e r e n t .
2 : 2-day w a t e r i n g  inter val 6 : 6 -day w a t e r i n g  i n t e r v a l
4: 4-day w a t er in g interval
A: Un d i l u t e d  c o n c e n t r a t i o n  C: \ d i l u t i o n
B : \ dilution D: /g dilution.
117 -
T A B L E  30a
Ana lys is of v a r i a n c e  (Tw o-w ay A n o v a  w i t h  r e p l i c a t i o n )  for 
data of Table 10b, Mean n umb er  of nodule s.
(Plants inocu l a t e d  with Pokuase-Ex-Taraale strain).
Source 
o f
vari ati on
1
Sura 
o f
squares
Degree
of
fr ee do m
Mean 
square s
F
va l u e
Water stress
1
| 1383 0.3 2 2 6915.160 10 7 . 9 5 9  * *
Inoculation | 8154.99 4 2038.748 31 .829 * *
Water stress & 
Inoculation
1
| 3771.81 
1
8 47 1. 47 6 7.361 * *
Error
1
| 3843.21 
1
60 64.054 -
TOTAL
1
| 296 00.33
1
74 - -
* * Significant at 1% level of significance.
118 -
TAB L E  30b
(Datfl of Table 30a)
Duncan's New M u l t i p l e  Range Test for me ans of n o d u l e  
number of Barabara g r o u n d n u t  plants s u b j e c t e d  to
a) water stress
2 4 6
5%
1%
b) inoculat ion
A B C D Co ntrol
5%
1%
Note: Any two means not u n d e r s c o r e d  by the same line
are s i g n i f i c a n t l y  di ffer e n t .  Any two me ans
un de rs cored by the same line are not s i g n i f i c a n t l y  
different.
2 . 2-day w a t er ing interval 6 : 6 -day w a t e r i n g  i n t erval
4: 4-day w a t er ing interval
A: Undil uted c o n c e n t r a t i o n  C: \ d i l u t i o n
\ dilution D: /g dilution.
119
Analysis of v a r i a n c e  (Two-way A n o v a  w i t h  r e p l i c a t i o n )  for 
data of Table lib. Mean number of nodule s.
(PLants i n o c u l a t e d  with W e i j a - E x - A d a  strain).
TABLE 31a
Source 
o f
variation
Sum
of
squares
Degree
of
fr ee do m
Mean
squares
F
v al u e
Water stress 16000.88 2 80 00. 4 4 0 7 4.250  * *
Inoculation 9208 . 13 4 2302.033 21.365 * *
Water stress & 
Inoculation 4095.39 8 511 .924 4.751 * *
Error 6465 . 00 60 107.750 -
TOTAL 35 76 9,40 74 - -
* * Significant at 1% level of significance.
120 -
TAB L E  31b
(Datfc of Tab le 31a)
Duncan's New M u l t i p l e  Range Test for means of n o d u l e  
number of Bambara g r o u n d n u t  plants s u b j e c t e d  to
a ) water s tress
b ) inoculat ion
Control
5%
1%
5%
Note: Any two means not u n d e r s c o r e d  by the same line are
sig n i f i c a n t l y  different . Any two me ans u n d e r s c o r e d  
by the same line are not s i g n i f i c a n t l y  di ffe r e n t .
2 : 2-day w a t e r i n g  interval 6 : 6 -day w a t e r i n g  i n t e r v a l
4 : 4-day wat erin g interval
A: U n di luted c o n c e n t r a t i o n  C: \ d i l u t i o n
B: \ dilution D : V o  dilution.
121
Size of nodules
M e asur em ents of the d i am et ers of the no d u l e s  s ho we d that 
in all the treatments n odu le  size was a f f e c t e d  by the a m o u n t  of 
water the plants received. The data in Table 32 and the 
histograms in Figs. 5 - 9  show that plants w a t e r e d  at 2-day 
intervals formed the largest no dule s and those w a t e r e d  at 6 — day 
intervals had the small es t nodules.
- 122 -
TA BLE 32
Mean diam eters of nod ules formed by Bam b a r a  g r o u n d n u t  
plants grown under d i f f e r e n t  w a t e r i n g  re gimes for 30 days.
Initial Mean di am eters (c m ) of
Rhizobium dens i ty n o d u l e s  of plants w a t e r e d
o f once in
Inoculum Ino cu lu m 2 days 4 days 6 days
Kpo ng -Ex- Ta male Und iluted 2 .62 1.48 0.69
strain \ d i l ut io n 2.68 0.79 0.79
\ dil ut io n 2.38 1.84 0.77
V g  dilutio n 2.56 0.88 0.83
Legon -Ex -Ada U n d i l u t e d 2.11 1 .90 0.87
strain \ dil utio n 2.30 0.99 0.85
\ dilu tion 2.26 1 .04 0.64
*/g dil ut ion 2.42 1.05 0.58
Nu ngu a(l) -E x- Tamal e Und iluted 2.79 2.13 0.59
strain \ dilut ion 2.38 0.96 0.71
\ dilutio n 2.48 0.73 0. 74
V g  dilut ion 2.31 0.92 0.67
Poku ase -Ex-Tam ale Und iluted 3 .01 0.99 0.85
s train \ dilu tion 2.64 0.87 0.77
\ dil utio n 2.24 1 .02 0.69
V g  dil uti on 2.23 0.97 0.62
Wei j a-Ex-Ad a Und ilu ted 2.75 0.88 0.82
strain \ dilu tio n 2.39 1 .06 0.81
\ dilution 2.89 1.12 0.61
*/g dil ution 2.14 0.85 0. 74
- 123 -
50
O
IOO
I O O
1 __ Do f  Int « r v a l A — Day lnt« f val 6 —  D a y  I n t «  r vo I
r D EL n
> 
u 
z
“  501-
O  
w 
a
u_
J =La
IOO
50- -
o
LOO
h n
50-
-f=* T“-r a .
I 2 *  A 5 6 7
T " T  '>
I 2 3 4 5 6 7
i:
2:
3-
4:
I 2 3 4 5 6 7 
C L A S S  D I A M E T E R
O -  1*00 m ft 5*. 4.01 — S .O O n n
I 0 l - 2 . 0 0 » m  6 '
2 .01— 3-00  nn  7.
J . O I  - 4 . 0 0 m
5 0 1  — 6-0 0 «m  
6.01 - 7 - O O m
Fi« 5 Distribution oi class-diametecs of nodules of Bambara 
groundnut plants, Ex-Tamale variety, inoculated with Kponc- 
•x-Iamale strain of Rhizobium sp. and grown under different 
water stress regimes for 30 days. Seeds inoculated with 
moss carrier with different initial Broth culture.
1/8
 
D
ilu
ti
on
 
1/4
 
D
ilu
ti
on
 
1/2
 
D
ilu
ti
on
 
U
n
d
il
u
te
d
«Fig-.-
- 124
100
50
IOO
5 0
IOO
5 0 P
O
IOO
5 0
2 — Day I n t e r v a l Do y I n t i r v o I 6 — D a y  ( n t i r v o l
r Q l
in
! k ▼ i r i t i t t, 3 3 4 5 6 7 I J  3 4  5 6 7 I 2 3 4 * 6 7
CLASS  D I AMETER  
0  — 1 0  Own 5 4 . 0 1 - 5  0 0 . *
2: 1.01 — 2 . 0 0 * 1  6 5.01 *— ft 0 0 « »
j ;  2 .01 — 3-0 0 » »  7.' 6 . 0 1— 7 . 0 0 « «
4;  3 .0  1 —4 . OO am
6 Distribution of class-diameters o'! nodules of Bambara
groundnut plants, Ex-Tamale variety, inoculated with
Legon-hx-Ada strain ol Rhizobium sp. and grown under
different water stress regimes for 30 days. Seeds
inocylated with moss carrier with different initial Broth culture.
P
E
R
C
E
N
T
A
G
E
 
F
R
E
Q
U
E
N
C
Y
- 125 '
I OO
50
IOO
50
I OO
5 0
IOO
50
if — Do y I n t e r v a l 4 — Do y I n t e r v a l
D
r 1 , □a.
m t K . = L
CL
I 2 3 4 S 6 7 r— r 3 4 5 6 7I 2
C L A S S  D IAM E T E R  
I: O — I .OOmm 5: 4-01 — 5 . 0 0  mm
2 :  I - O I  — 2 . 0 0  mm 6. 5 01  — 6 . 0 0  i rm
\
3:  2 .01 — 3 0 0  m m 7. 6 . 0 1 —  7 . 0 0 m m
4:  3 . 0 1 - 4 . OOmm
Fig 7 Distribution of class-diameters of nodules of
groundnut plants, Ex-Tamale variety, inoculatedwith 
Kungua ( 1) -Ex-Tamale strain of Rhizobxun. sp and grow,^ 
■under different! water stress regimes for 3C d y. .; • 
inoculated with moss carrier with different mit .
culture.
P
E
R
C
E
N
T
A
G
E
 
F
R
E
Q
U
E
N
C
Y
- 126 -
l OO-
5 0
O '
I OO-
50 '
o
100
5 0 -  -
I O
2 — D q j  l n t * r v o
r a ,
4 — Day  I nU r v o l
n
a _ Dor 1 ^ 11 < vo 1
Q
r T 1
I u
r "i  r » i 
1 2  3 4 5 6 7
C L A S S  D IAM E T E R
t: 0 -- 1. OO aim 5 ‘- 4.01 —
2 : 1.01 — 2 .0 0  mm 6. 5 . 0  1 —
3: 2.01 — 3-OOmm 7.* 6 0  1 —
i— <— m r i *
I 2 3 4 5 6 7
5 0  0  mm
6 . O O  m m
7 . O O  m m
A: 3 0 l - 4 . 0 0 m m
« Distribution of c ] ass-d i M e t e r s  o] nodule, o, Kan,b,r;1 
P*kuaseUExPT ant? ’ tX- ramale vari« y ,  inoculated with
inoculated with carrier with d i ] 1 e re.n ”i i!n i a ] S h
1/0
 
D
il
ut
io
n 
1/4
 
D
il
ut
io
n
P
E
R
C
E
N
T
A
G
E
 
F
R
E
Q
U
E
N
C
Y
- 127 -
2 — Da y  l nt «  r t d  I
I OO
5 0  -
O
I OO
50
\
o
I OO
5 0
r n
4 *- D a y  I n t e r v a l 6 —  D a y  l n t « r  v o  I
i
= □ =
I OO
5 0 -
2Y tT  i i f
I 2 3 4 5 6 7
Fig. 9
I 2  3  4  5  6  7
C L A S S  D I A M E T E R  
t :  o  - I . O O M  5 : 4 . q i  —  5 . 0 0 1 * «
2 .’  1 . 0 1  —  2 . 0 0  mm 6 - 3 . 0 1  —  6  • O O  M m
3 . 2 .0 1 -  J - O O m m  7 : 6  0 1 —  7  0  O  m m
4 : 3  0 1 -  4 . 0 0 mm
Distribution of class-diameters of nodules of Bambara 
groundnut plants, Ex-Tamale variety,- inoculated with 
Weija-Ex-Ada strain of Rhizobium sp, and grown under 
different water stress regumes for 30 days. Seeds 
inoculated wtih moss carrier with different initial 
Broth culture
I/S
 
D
IL
U
TI
O
N
 
1/4
 
D
IL
U
T
IO
N
 
1/
2 
D
IL
U
T
IO
N
 
U
N
D
IL
U
T
E
D
N O D U L A T I O N  OF P L A N T S  I N O C U L A T E D  W I T H  D I F F E R E N T  
STRAINS OF R H I Z O B I U M  S P « IN M O S S  C A R R I E R  U N D E R  
D I F F E R E N T  L I G H T  I N T E N S I T I E S
Using daylig ht , a f l u c t u a t i o n  in light i n t e n s i t y  
should be e x p ect ed  each day w i t h  the h i g h e s t  i n t e n s i t y  
occur ri ng ar ound mid- da y. It wi ll  be errone o u s ,
therefore, to pre s e n t  a single  v a l u e  of light i n t e n s i t y  
for each of the three t r e atm en ts. The graphs of light 
in te nsities  at 9.00 am, 12.00 n o o n  and 3.00 pm in Fig. 10 
show that for Shed 1, p r o v i d i n g  the lowest light
intensity, i n t e n s i t y  at 9.00 am was b e t w e e n  1100 and 2200 
lux, at 12.00 noon, it was b e t w e e n  4030 and 6200 lux, and 
it was be t w e e n  1600 and 1900 lux at 3.00 pm. The
c o r r e s p o n d i n g  v alu es  for Shed 2 we re 1400 to 2800, 6400 to 
9100 and 2900 to 3500 lux, r e s p e c t i v e l y .  And for Shed 3, 
5300 to 6200, 8800 to 10000, and 46 00 to 6400 lux. The
three treat ments will be r e f e r r e d  to in the text as low 
light intensity; m e d i u m  light i n t e n s i t y  and h i g h  light 
intensity, for c o n d i t i o n s  und e r  Shed 1, 2, and 3
r e s p e c t i v e l y  (Plate 6 ).
I no c u l a t i o n  of the b a m b a r a  g r o u n d n u t  p lan ts  w i t h  the 
moss carrier was very e f f e c t i v e  un der  all the three 
di ffe re nt light i n t e n s i t i e s  u n d e r  w h i c h  the p l a n t s  w e r e
grown. The in ocul a t e d  plants g r e w  we ll  and those u n der 
the two lower light i n tens it ies, Sheds 1 and 2, h ad green 
foliage. The plants raised u n d e r  the h i g h e s t  light 
intensity, Shed 3, showed some y e l l o w i n g  of the leaves. 
The u n i n o c u l a t e d  (control) plants, also, had y e l l o w i s h -  
green foliage (Plate 7).
- 128 -
129
Light i n t e n s i t y  a f f ected both i n o c u l a t e d  and 
u n i n o c u l a t e d  plants. In the con t r o l  set of plants, the 
mean numbers of leaves per plant, the size of the m i d d l e  
leaflets, and the me an dry w e i g h t s  of the p lant s d e c r e a s e d  
with increase in light in tensity. The n u m b e r  of le aves in 
the in oc ulated plants was a p p a r e n t l y  not a f f e c t e d  by light 
in ten si ty w i t h i n  the range used in this i n v e s t i g a t i o n .  
The size of the m i d d l e  leaflets, the dry w e i g h t s  of the 
plants and the nu mber of nodules, however, d e c r e a s e d  w i t h  
i nc re asi ng light intensi ty. The v alues r e c o r d e d  are 
prese nte d in Tables  33 - 37,
LI
G
H
T
 
IN
T
E
N
S
IT
Y
 
(L
u
x
)
10,000  
6(3 00
2,000 
0
10)000
6 ,000
2,00 0 
O
10,000 
6,00 0
2,000 
O
IO  14 I I  22 26 30  4 • 12
|* A P R I L ----------- 4< MAY   M
“  5 h • d I — L o w  119 h t I n t e n s i t y
- o - o  -  Shed  11 — Med ium 11 ^ ht I n t e n s i t y  
t  9 Shed  111 High l i gh t  I n t e n s i t y
Fig. 10 Recording of light intensities under sheds 1, 2 and 3 at 9.00
a.m., 12.00 noon and 3.00 p.m. during period of growth of Bambara 
groundnut plants, Ex-Ada variety, inoculated with different 
strains af Rhizobium sp.
- 130 -
9 . 0 0  o ^
12 . 0 0  noon
3 -OO p
131
PLATE 6 : Ph oto gr aph showing the sheds of three light
in tensities with the r e m o v a b l e  sides ' j f  the two sheds 
with battens taken off to show the grow i n g  b a m b a r a  
groundnut plants. (xl/48)
Left: High light int e n s i t y  (Shed 3)
Middle: M ed iu m light i n t e n s i t y  (Shed 2)
Right Low light intensity (Shed 1)
132
PLATE 7: P h o t o g r a p h  sh owin g barabara g r o u n d n u t  plants
raised under dif f e r e n t  light i n t e n s i t i e s  after 
in o cul at ion with Rhizobium sp. ^
Left: Low light i n t e n s i t y  (Shed 1)
Middle: M e d i u m  light i n t e n s i t y  (Shed 2)
R i g h t : High light intensity (Shed 3)
- 133
T AB L E  33a
Leaf d e v e l o p m e n t  of b a m b a r a  gr ou ndnut, v a r i e t y  Ex-Ada, 
inoculated with K p o n g - E x - T a m a l e  s t r a i n  grown und e r  n o r m a l  
day/night regime at d i f f e r e n t  light inten s i t i e s .
Co n c e nt ration of 
Initial Broth 
Culture Ino cul um 
of moss Carrier
Mean No. of leaves 
per plant (to the 
nearest  who l e  no.) 
at f o l l o w i n g  light 
in tens ities*
Me an  length and 
o f m i d - l e a f l e t  ( 
f o l l o w i n g  light 
i n t e n s i t i e s .*
wid th 
c m ) at
L M H L M H
Und ilu ted 25 25 26 6.09x2.70 5.47x2.27 4.90x1.74
\ Dilution 28 27 26 6.14x2.31 5.70x2.31 5.07x1.79
\ Dilution 28 27 27 6.89x2.84 5.59x2.33 5.26x1.96
*/g Dilution 27 26 28 5.60x2.34 5.79x2.49 5.16x1.64
Uninocula ted 
(CONTROL)
21 19 11 3.00x1.40 2.20x1.19 2.00x0.98
L: Low light in t e n s i t y
M: Medium light int en si ty
H: High light intensity
134
Dry weight and extent of n o d u l a t i o n  of B a m b a r a  g r o u n d n u t ,  
variety Ex-Ada, plants i n o c u l a t e d  w i t h  K p o n g - E x - T a m a 1e s tr ai n 
grown under normal da y / n i g h t  r e g i m e  at d i f f e r e n t  light 
intensities.
TABLE 33b
Co n c e n tra ti on of 
Initial Broth 
Culture Ino cul um 
of moss Carrier
Mean Dry wt. per 
plant (g) at 
f o l l o w i n g  light 
i n t e n s i t i e s *
L M H L M H
Und ilu t ed
1
| 1.91
1+0.26
1
1.49 
± 0 . 3  5
1.21 
± 0.2 6
63 47 16
\ Dilution
1
| 1.77
1+0.30
1
1 . 50 
±0.3 3
1.18 
±0.3 7
65 48 18
\ Dilution
1
| 1 .62
1+0.30
1
1.35
±0.29
1.17 
± 0.2 2
66 48 20
V g  Dilution
I
| 1.64
1+0.34
1
1.40 
± 0 . 3  8
1.15
± 0.21
59 43 21
Uninoculated
(CONTROL)
1
| 0.67 
1+0.14
1
0.53
±0 .13
0.29
±0.06
0 0 0
M e a n  No. of N o d u l e s  per 
pl ant  (to the n e a r e s t  
wh o l e  no.) at f o l l o w i n g
L: Low light inte nsity
M: Me dium light int en si ty
H: High light intensity
- 135 -
T AB L E  34a
Leaf de v e l o p m e n t  of Bam b a r a  gr ou nd n u t ,  v a r i e t y  Ex-Ada, 
ino culated with L e g o n - E x - A d a  st rai n grown u n der n or ma l
da y/night regime a t dif f e r e n t light i n t e n s i t i e s .
Co n c e n t r a t i o n  of 
Initial Broth 
Culture Ino cul um 
of moss Carrier
Mean No, of leaves 
per plant (to the 
ne a r e s t  wh ole  no,) 
at fo l l o w i n g  light 
i n t e n s i t i e s *
| M e a n  le ngth and 
| of m i d - l e a f l e t  ( 
| f o l l o w i n g  light 
| i n t e n s i t i e s .*
wi d th 
cm) at
L M H L M H
Und iluted 26 23 26 |6.21x2.36 5,90x2.16 4.80x1.64
\ Dil ution 25 22 26 |5.83x2.50 5.67x2.21 4.93x1.53
\ Dilution 25 24 26 |6.10x2.36 5.46x2.00 4.77x1.56
1/g Dilution | 27 25 26 |5.70x2.28 5.66x2.14 4.84x1.56
U n inoc ul at ed
(CONTROL)
21 19 11 |3.00x1.4 2.20x1.19 2.00x0.98
L: Low light in t e n s i t y
M: Medium light i nten si ty
H: High light intensity
136
Dry weight and extent of n o d u l a t i o n  of B a m b a r a  g r o u n d n u t ,  
var iet y Ex-Ada, plants i n o c u l a t e d  w i t h  L e g o n - E x - A d a  s tr ai n 
grown under norm al d a y / n i g h t  regi me at d i f f e r e n t  light 
i n t e n s i t i e s •
TABLE 34b
C o n c e n t r a t i o n  of 
Initial Broth 
Culture Inoc ulum 
of moss Carrier
Mean Dry wt. per 
plant (g ) at 
f o l l o w i n g  light 
i n t e n s i t i e s *
Me an 
plant 
who le 
light
No. of N o d u l e s  per 
(to the n e a r e s  t 
no . ) at f o l l o w i n g  
intens i t i e s .
L M H L M H
Und iluted 1 .73 
+ 0.28
1.46 
+ 0.32
1.17 
±0.2 9
65 43 18
\ Dilution 1 .82 
±0. 35
1.43 
+ 0.21
1.41 
± 0 . 3  8
60 42 21
\ Dilution 1 .77 
+ 0.30
1.46 
+ 0.24
1.06
± 0.21
58 49 17
V g  Dil uti on 1 .69 
+ 0.31
1 .50 
+ 0.31
1 .10 
± 0.21
67 43 20
Un i n o c u 1 ated 
(CONTROL)
0.67
±0.14
0. 53 
± 0 . 1 3
0.29
±0 . 0 6
0 0 0
Low light i nt ensity 
Medium light in t e n s i t y  
High light int en sity
- 137 -
T A BL E 35a
Leaf d e v e l o p m e n t  of Bamba ra  grou nd n u t ,  v a r i e t y  Ex-Ada, 
inocu lat ed with N u n g u a (1)- E x - T a m a l e  s trai n grown  u n d e r  n o r m a l
day/ nig ht regime at di f f e r e n t light intens i t i e s .
C o n c e n t r a t i o n  of 
Initial Broth 
Culture Inoc ulum 
of moss Carrier
Mean No. of leaves 
per plant (to the 
ne ar es t whole no.) 
at f o l l o w i n g  light 
intens ities*
| Mean length  and 
| of m i d - l e a f l e t  ( 
| f o l l o w i n g  light 
| i n t e n s i t i e s . *
wi dt h 
cm ) a t
L M H L M H
Und ilu ted 26 25 27 |7.83x2.79 5.41x2.36 4.57x1.60
\ Dilution 26 26 28 |6.21x2.49 4.91x1.94 5.24x1.76
\ Dilu tion 28 27 28 |6.31x2.40 5.46x2.13 4.96x1.59
/g Dilution 1 27 25 26 |6.51x2.66 5.41x2.16 5.08x2.00
Un inoculated 
(CONTROL)
21 19 11 |3.00x1.40 2.20x1.19 2.00x0.98
L: Low light in t e n s i t y
M: Medium light i ntens it y
H: High light intensity
TABLE 35b
Dry weight and extent of n o d u l a t i o n  of B a m b a r a  groun d n u t ,  
va rie ty Ex-Ada, pl ants i n o c u l a t e d  wi th  N u n g u a (1 )- E x - T a m a  1e 
strain grown under no rmal d a y / n i g h t  re gime at d i f f e r e n t  light 
intensities.
- 138 -
C o n c e n t r a t i o n  of 
Initial Broth 
Culture Ino cu lum 
of moss Carrier
Me an Dry wt. per 
plant (g) at 
fo l l o w i n g  light 
i n t e n s i t i e s *
Mean
p la nt
w ho le
light
No. of N o d u l e s  per 
(to the n e a r e s t  
n o .) at f o l l o w i n g  
i n t e n s i t i e s .
L M H L M H
Und iluted 1.93 1 .46 1 . 24 68 45 17
+ 0.47 + 0.28 + 0 .22
\ Dilution 1 . 75 1.43 1.17 67 43 19
+ 0,38 1
 + o ± 0  . 24
\ Dilution 1 .84 1 .82 1.12 66 44 17
+ 0.28 + 0.35 + 0 .24
V g  Dilution 2.91 1.58 0.91 64 43 19
_^ 0 . 6 1 + 0.26 + 0.19
Un i n o c u 1 ated 0.67 0.53 0.29 0 0 0
(CONTROL) + 0,14 + 0.13 + 0 .06
L: Low light intens it y
M: Medi um light i nt ensity
H: High light intensity
- 139 -
T AB LE 36a
Leaf d e v e l o p m e n t  of bam b a r a  groun d n u t ,  v a r i e t y  Ex-Ada, 
inocul ate d with P o k u a s e - E x - T a m a l e  strain grown un d e r  n o r m a l
day/night regime at d i f f e r e n t light i n t e n s i t i e s .
C o n c e n t r a t i o n  of 
Initial Broth 
Culture Ino culum 
of moss Carrier
Mean No, of leaves 
per plant (to the 
nea r e s t  whole no.) 
at f o ll owing light 
i n t e n s i t i e s *
Me an  length and 
of m i d - l e a f l e t  ( 
f o l l o w i n g  light 
i n t e n s i t i e s . *
w i d t h  
c m ) at
L M H L M H
Und iluted 25 29 25 6.87x2.57 5.77x2.36 5.01x1.77
\ Dilu tion 28 26 27 6.47x2.43 5.51x2.17 5.63x1.79
\ Dilution 27 28 27 5.67x2.27 5.86x2.30 5.71x2.16
1 /q Dilution 1 27 27 27 |6.01x2.49 5.37x2.01 5.11x1.77
U n inoc ul at ed
(CONTROL)
21 19 11 3.00x1.40 2.20x1.19 2.00x0.98
L: Low light i ntensi ty
M: Medium light i nte ns ity
H: High light intensity
140
Dry weight and ex tent of n o d u l a t i o n  of Ba m b a r a  grou nd nut, 
variety Ex-Ada, plants i n o c u l a t e d  wi th  P o k u a s e - E x - T a m a 1e strain 
grown under normal d a y / n i g h t  r egime  at d i f f e r e n t  light 
intensities.
TABLE 36b
Co n c e n t r a t i o n  of 
Initial Broth 
Culture Inoculum 
of moss Carrier
Mean Dry wt. per 
plant (g ) at 
f o l l o w i n g  light 
i n t e n s i t i e s *
Me an 
plant 
who 1 e 
light
No. of N o d u l e s  per 
(to the n e a r e s t  
n o .) at f o l l o w i n g  
i n t e n s i t i e s .
L M H L M H
Un diluted 2 . 70 
± 0  . 2 8
1.48 
±0.3 0
1.18
± 0. 2 3
70 49 18
\ Dilution 1 .62 
+ 0.33
1 .38 
±0 .34
1.19 
± 0 . 2  4
78 50 18
V Dilution 1 .72 
± 0 . 2  9
1.33
±0.29
1 .05 
± 0.2 2
66 51 19
V g  Dilution 1 . 79 
± 0 . 3  3
1 . 52 
±0.3 5
1.02 
± 0.2 6
64 45 20
Un inoculated 
(CONTROL)
0.67 
+ 0.14
0.53
±0 . 1 3
0.29 
± 0.0 6
0 0 0
L: Low light in te nsity
M : Medium light i n te nsity
H: High light intensity
- 141 -
T A BLE 37a
Leaf d e v e l o p m e n t  of bam b a r a  ground n u t ,  v a r i e t y  Ex-Ada, 
in oculate d with W e i j a - E x - T a m a  1e s trai n grown und e r  n orma l
day/ nig ht regime at di f f e r e n t light in ten s i t i e s .
C o n c e n t r a t i o n  of 
Initial Broth 
Culture Ino cu lu m 
of moss Carrier
Mean No. of leaves 
per plant (to the 
n e a r e s t  whole no.) 
at f o l l o w i n g  light 
i n t e n s i t i e s *
Mean length and 
of m i d - l e a f l e t  ( 
f o l l o w i n g  light 
intens i t i e s .*
wid t h  
c m ) at
L M H L M H
Und ilu ted 26 26 26 7.49x3.00 5.81x2.56 5.00x1.64
\ Dilution 28 26 27 6.33x2.73 6.03x2.19 5.06x1.75
\ Dilution 26 28 26 6.30x2.50 6.07x2.66 5.24x1.89
1 Dilution 27 26 26 [8.56x2.93 5.63x1.90 5.53x1.94
Uninoculate d
(CONTROL)
21 19 11 3.00x1.40 2.20x1.19 2.00x0.98
L: Low light i n tensity
M: Me diu m light in t e n s i t y
H: High light intensity
142
Dry we ight and extent of n o d u l a t i o n  of Ba m b a r a  g r o u n d n u t ,  
var iet y Ex-Ada, plants i n o c u l a t e d  with Wei ja-Ex-Taraa1 e s train  
grown under no rma l d a y / n i g h t  regime at d i f f e r e n t  light 
intensities.
T A B L E  37b
C o n c e n t r a t i o n  of 
Initial Broth 
Culture Inocu lu m 
o f mo ss Carrier
Mean Dry wt. per 
plant (g) at 
f o 1 lowing 1 ight 
i n t e n s i t i e s *
Mean 
plant 
who 1 e 
light
N o . of N o d u l e s  per 
(to the n e a r e s t  
no . ) at f o l l o w i n g  
i n t e n s i t i e s .
L M H L M H
Und ilu t e d 1 .94 
± 0 . 4  8
1.48 
+ 0.35
0.98
± 0 . 2 7
67 44 18
\ Dilution 1.81 
± 0 . 4  3
1 .46 
±0.3 3
1.06 
± 0 . 3  0
60 46 20
h Dilution 1 .73 
± 0  .26
1.48
±0.29
1 .23 
± 0.2 6
62 44 19
l /q Dil ution 1.81 
+ 0.25
1 .52 
+ 0.38
1.09 
+ 0.20
54 44 19
Un in oc u l a t e d
(CONTROL)
0.67
± 0 . 1 4
0.53
± 0 . 1 3
0.29 
± 0.0 6
0 0 0
L: Low light in t e n s i t y
M: Me dium light i nten si ty
H: High light intensity
14 3
The data were ana ly se d s t a t i s t i c a l l y  and the r e s u l t s  can 
be s u m m a r i s e d  as follows:
Mean n u m b e r  of leaves per plant
The p e rt in ent data appear in Tables 33a, 34a, 35a, 36a and
37a. The Analyses  of V a r i a n c e  (Two-way Ano v a  wi th  r e p l i c a t i o n )  
in Tables 38a, 39a, 40a, 41a and 42a i n d i c a t e  that there was no 
sig nific an t effect of light i n t e n s i t y  on me an leaf n u m b e r  
produced by the i n o c u l a t e d  plants. There was s i g n i f i c a n t  
effect of i n o c u l a t i o n  on mean leaf n u m b e r  at both 5 per cent 
and ) per cent levels of si g n i f i c a n c e .  The e ff ect of the 
in t e ractio n of light i n t e nsitie s and i n o c u l a t i o n  on m e a n  leaf 
number was si g n i f i c a n t  at only the f )  ) per cent level of 
significance.
R esu lts of the Dun can's New M u l t i p l e  Range Test for means 
of leaf nu mbe r in Tables 38b, 39b, 40b, 41b and 42b sho wed that 
the mean numbers of leaves of i n o c u l a t e d  plants wi th  d i f f e r e n t  
initial inoculu m de n s i t i e s  were not s i g n i f i c a n t l y  d i f f e r e n t  
from each other, but all were d i f f e r e n t  at both 5 per cent and 
\ per cent levels of s i g n i f i c a n c e  from the mean n u m b e r  of 
leaves of the u n i n o c u l a t e d  plants.
IAA
TABLE 38a
Analysis of variance (Two-way Anova with replication) for
data of Table 33a. Mean leaf number.
(Plant inoculated with Kpong-Ex-Tama1e strain)
Sourc e 
o f
v ar ia tion
| Sum
1 0 f
| squares
Degree 
o f
f r ee d om
Mean
squares
F
val u e
Light intens ity
1
| 65.31
1
2 32.65 5 2 . 705 NS
In oculati on
1
| 1163.39 
1
A 2 9 0 . 8A8 2 A .090 * *
Light in t e n s i t y  & 
In oculatio n
1
1
| 2A5.09 
I
8 30.636 2 . 538 *
Error
1
| 72A.A0 
1
60 12.073 -
TOTAL
1
| 2198.19 
1
7 A - -
** S i g n i f i c a n t  at 1% level of si gnific an ce.
NS No n - s i gn i f i c an t .
* Significant at 5% level of significance.
145
(Data of Table 38a)
D u n c a n ’s New M u l t i p l e  Range Test for means of leaf n um be r 
of Ba mb ara g r oundnu t plants subje cted to
i n o culatio n
TABLE 38b
A B C D  Control
5%
1%
Note: Any two means not u n d e r s c o r e d  by the same line are 
s i g n i f i c a n t l y  different* Any two means u n d e r s c o r e d  
by the same line are not s i g n i f i c a n t l y  di ff er ent.
A: U n d i l u t e d  c o n c e n t r a t i o n C : \ dilut i o n
B: \ d i l ut io n D : V g  dilution.
146
TABLE 39a
Analysis of variance (Two-way Anova with replication) for
data of Table 34a. Mean leaf number.
(Plants inoculated with Legon-Ex-Ada strain)
Source 
of | 
va r i a t i o n  |
Sura 
o f
squares
Degree 
o f 
freedom
Mean
squares
F
value
1
Light in t e n s i t y  | 79 .04 2 39 .52 2.662 NS
1
Ino cu la tion
1
849.28 4 212.32 14.301 **
Light i nt en sity & 
oc u l a t i o n 291.36 8 36.42 2.453 *
Error
l
890.80 60 14.847 -
1
TOTAL | 
.. .. 1
2110.48 74 - -
** Sig n i f i c a n t  at 1% level of si g n i f i c a n c e .
* Significant at 5% level of significance.
NS Non-significant,
147
(Data of Table 39a)
D u n c a n ’s New M u l t i p l e  Range Test for means of leaf n um ber 
of Ba mbara gro un dn ut plants s ubje ct ed to
in o c u l a t i o n
TABLE 39b
A B C D  Control
5%
1%
Note : Any two means not u n d e r s c o r e d  by the same line are 
s i g n i f i c a n t l y  different. Any two means u n d e r s c o r e d  
by the same line are not s i g n i f i c a n t l y  di fferen t.
A: U n d i l u t e d  c o n c e n t r a t i o n C : \ d i l u t i o n
di lu tion D : V g  d i l u t i o n .
148
TABLE 40a
Analysis of variance (Two-way Anova with replication) for
data of Table 35a. Mean leaf number.
(Plant inoculated with N u n g u a (1)-Ex-Tamale strain)
Source 
o f
variation
| Sum 
1 of
1 squares
Degree 
o f 
f re edo m
Mean
squares
F
value
Light intens ity
1
| 27.23
I
2 13.615 0.917 NS
Inocul ati on
1
| 1158.72
I
4 289.680 19.512 * ★
L -»h t in tens i ty & 
In oculation
1
1
| 286.64
1
8 35.830 2.413 *
Error
1
| 643.60
i
60 10.727 -
TOTAL
1
| 2116.19 
1
74 - -
** S i g n ifica nt  at 1% level of si gnifica nc e.
* Significant at 5% level of significance.
NS Non - s i gn i f i c an t .
149
TABLE 39b
(Data of Table 40a)
Dun ca n' s New M u l t i p l e  Range Test for me ans  of leaf n u m b e r  
of Bam bara g r oundn ut  plants subjecte d to
i n o c ul at ion
A B C D  Co nt rol
5%
1%
Note: Any two means not u n d e r s c o r e d  by the same line are 
s i g n i f i c a n t l y  different. Any two means u n d e r s c o r e d  
by the same line are not s i g n i f i c a n t l y  d i f f e r e n t .
A: U n d i l u t e d  c o n c e n t r a t i o n C : \ d i l u t i o n
B: \ dilution D : V g  d ilut i o n .
150
TABLE 41a
Analysis of variance (Two-way Anova with replication) for
data of Table 36a. Mean leaf number.
(Plant inocu l a t e d  with P o k u a s e - E x - T a m a l e  strain)
Source | 
of | 
variation |
Sura 
o f
squares
Degree 
o f 
freedom
Mean
squares
F
value
1
Light intensity | 90.72 2 45.360 2.966 NS
Inoculation | 1246.99 4 311 .748 20.385 ★ ■*
1
Light intensity & | 
Inoculation | 262.21 8 32.776 2 .143 *
Error |
1
917.60 60 15.293 -
TOTAL |
. 1
2523.52 74 - -
** Si g n i f i c a n t  at 1% level of si g n i f i c a n c e ,
* Significant at 5% level of significance.
NS Non- significant .
151
(Data of Table 41a)
Dun can 's New M u l t i p l e  Range Test for means of leaf n u m b e r  
of Bam bar a g ro un dnut plants s u bject ed  to
i n o c ul at ion
TABLE 41b
A B C D  Control
5%
1%
Note: Any two means not u n d e r s c o r e d  by the same line are 
s i g n i f i c a n t l y  different. Any two means u n d e r s c o r e d  
by the same line are not s i g n i f i c a n t l y  d i f f e r e n t .
A: U n d i l u t e d  c o n c e n t r a t i o n C : \ d i l u t i o n
B: \ dilution D : ■^/q dilution.
152
TABLE 42a
Analysis of variance (Two-way Anova with replication) for
data of Table 37a. Mean leaf number.
(Plant inoculated with Weija-Ex-Taraale strain)
Source 
of | 
variation
Sura 
o f
squa res
Degree 
o f 
freedom
Mean 
square s
F
va lue
1
Light int ensity
1
52.16 2 26.080 2.192 NS
1
In oculatio n j
1
1085.25 4 271.313 22.799 * -k
1
Light int en sity &| 
I n o c u 1 a t i on
1
266.91 8 33.364 2 . 804 ★
1
Error
..1
714 .00 60 11 .900 -
1
TOTAL
1
2118.32 74 - -
** Sig n i f i c a n t  at 1% level of si gn ificanc e.
* Significant at 5% level of significance.
NS Non- significant .
153
(Data of Table 42a)
D u n c a n ’s New M u l t i p l e  Range Test for means of leaf n um ber 
of Ba mba ra g roundnu t plants s u bj ec ted to
in o c u l a t i o n
TABLE 42b
A B C D Contr ol
5%
1%
Note: Any two means not u n d e r s c o r e d  by the same line are 
s i g n i f i c a n t l y  different. Any two means u n d e r s c o r e d  
by the same line are not s i g n i f i c a n t l y  diffe r e n t .
A: U n d i l u t e d  c o n c e n t r a t i o n C : \ d i l u t i o n
B : dilution D: 1/Q dilution.
154
Mean length and wi dth of m i d - l e a f l e t s
Ana lys es of V a r i a n c e  (Two-way Ano v a  wi th r e p l i c a t i o n )  as 
pres ent ed in Tables 43a, 44a, 45a, 46a and 47a for the me a n s  of 
leaflet sizes showed there was s i g n i f i c a n t  effe ct of all the 
treatments applied and their i n t e r a c t i o n  on m e a n  l e a f l e t  size 
for all the five strains at both 5 per cent and 1 per cent 
levels of s ign if icance.
Duncan's  New M u l t i p l e  Range Test of Tables 43b, 44b, 45b,
46b and 47b show that sizes of l e a fle ts  of all i n o c u l a t e d  
plants were s i g n i f i c a n t l y  larger than those of u n i n o c u l a t e d  
plants at both 5 per cent and 1 per cent levels of 
significance.
Also plants under low, m e d i u m  and hi gh light i n t e n s i t i e s  
produced leaves w h i c h  diff er ed s i g n i f i c a n t l y  in size from each 
other at both 5 per cent and 1 per cent levels of
s i g n i f i c a n c e .
- 155 -
TABLE 43a
Anal ysi s of v a r i a n c e  (Two- way A n o v a  wi th r e p l i c a t i o n )  for 
data of Table 33b. Mean size of raid- 1 e a f 1e t .
(Plant inocu lated w i t h  Kpong- E x - T a m a 1e strain)
Source 
o f
variation
Sum
1 of
squares
D eg re e 
o f 
fr e e d o m
Mean
squares
F
value
Light intensity
1
| 623.38 
1
2 311.690 11 2. 40 6 ★ *
Inoculation
1
| 1663.46
1
4 415.865 149.975 ★
Light intens i ty & 
Inoculation
1
1
| 180.15
1
8 22.519 8.121 * ★
Error
1
| 249.56
L
90 2.773 -
TOTAL
1
| 2716.55 
1
104 - -
Si g nif ic ant at 1% level of s i g n i f i c a n c e .
- 156 -
TABLE 43b
(Data of Table 43a) 
Duncan's New M u l t i p l e  Range Test for means 
of Bambara gro u n d n u t  plants s ubje ct ed to
a) d if fe rent light i n t e nsitie s
of le a f l e t  size
1 2 3
5%
1%
b) ino cu lation
A B C D Control
5%
1%
Note: Any two means not u n d e r s c o r e d  by the same line are 
s i g n i f i c a n t l y  different. Any two means u n d e r s c o r e d  
by the same line are not s i g n i f i c a n t l y  differ e n t .
1 : Under Shed 1 3: Und e r  Shed 3
2 : Unde r Shed 2
A : U n d i l u t e d  c o n c e n t r a t i o n C: \ d i l u t i o n
B : \ dilu tion D: ^/g dil ut ion.
- 157 -
TABLE 44a
Anal ys is of v a r i a n c e  (Two-way Ano v a  wit h r e p l i c a t i o n )  for 
data of Table 34b, Me an size of m i d - 1e a f 1 e t .
(Plant in oc ulated with Kp ong - Ex-Taraale s t r a i n )
Source 
o f
variation
| Sura 
1 of
| squares
Degree 
o f 
fr ee do m
Mean
squares
F
va lue
Light intensit y
1
| 597.04
1
2 298.52 11 2.1 13 * *
Inoc ula tion
1
| 1168.55 
1
4 292.138 109.716 * *
L i <> h t i ntensi ty  & 
Inoculation
1
1
| 88.14
I
8 11.018 4. 138 * *
Error
1
| 239.64 
1
90 2.663 -
TOTAL
1
| 2093.37
1
104 - -
Significant at 1% level of significance.
158
TABLE 44b
(Data of Table 44a)
D u n c a n ' s  N e w M u l t i p l e  R a n g e  T e s t  for m e a n s  of l e a f l e t  s iz e  
of B a m b a r a  g r o u n d n u t  p l a n t s  s u b j e c t e d  to
a) d i f f e r e n t  l i g h t  i n t e n s i t i e s
1 2 3
5%
1%
b ) inoculation
A B C D Control
5%
1%
Note : Any two m e a n s  not u n d e r s c o r e d  by the s a m e  line are 
s i g n i f i c a n t l y  d i f f e r e n t .  A n y  two m e a n s  u n d e r s c o r e d  
by the same line are not s i g n i f i c a n t l y  d i f f e r e n t .
1 : Unde r Shed 1
2 : Under Shed 2
A: Undiluted concentration
B: \ dilution
3: U n der Shed 3
C: \  dilution
D: V o  dilution
TABLE 45a
An al ys is of v a r i a n c e  (Two-way A n ov a w i t h  r e p l i c a t i o n )  for 
data of Table 35b. Mean size of m i d - l e a f l e t .
(Plant in oc ulated  w i t h  N u n g u a ( 1 ) - E x - T a m a l e  strain)
- 159 -
Source 
o f
variation
Sura 
o f
squares
Degree 
o f 
freedom
Mean
squares
F
value
Light intensity 1025.10 2 512.550 108.430 **
inocula t ion 1626.62 4 406.655 86.028 **
Light intensity & 
Inoculation 310.83 8 38.854 8.220 **
Error 425.43 90 4.727 -
TOTAL 3387.98 104 - -
* * Significant at 1% level of
i
s i gn i f i c anc e .
TA BLE  45b
(Data of Table 45a)
D u n c a n ’s New M u l t i p l e  Range Test for m e ans of l e a f l e t  size 
of Bambara g r ou nd nut plan ts sub j e c t e d  to
- 160 -
a) differen t light i n t e n s i t i e s
1 2 3
5%
1%
b ) inocul ation
A B C D Control
5%
1%
Note: Any two means not u n d e r s c o r e d  by the same line are 
s i g n i f i c a n t l y  di ff erent.  Any two mea ns u n d e r s c o r e d  
by the same line are not s i g n i f i c a n t l y  d i f f e r e n t .
1 : Unde r Shed 1 3: U n d e r  Shed 3
2: Under Shed 2
A: Un d i l u t e d  c o n c e n t r a t i o n C: \ d i l u t i o n
B : \ dilution
D : */g d iluti on .
- 161 -
TABLE 46a
A n a lysis of v a r i a n c e  (Two-way Anovaj w i t h  r e p l i c a t i o n )  for 
data of Table 36b. Mean size of m i d - l e a f l e t .
(Plant in oc ulated w i t h  Pok uase- E x - T a m a l e strain)
Source 
o f
variation
| Sum 
1 of
1 squares
Degree
of
fre e d o m
Mean
squares
F
value
Light intensity
1
| 353.88 
1
2 176.940 4 5 . 5 9 0 **
Inoculation
1
| , 16 04. 80 
1
4 401 . 2 0 0 103.372 * *
Light int en sity & 
Inoculation
1
1
| 165.47
1
8 20.684 5.329 **
Error
1
| 349.30 
1
90 3.881 -
TOTAL
1
| 2473.45
J _  -
104 - -
Significant at 1% level of significance.
TABLE 46b
(Data of Table 46a)
Duncan's New M u l t i p l e  Range Test for mea ns of leafle t size 
of Ba mba ra  g ro undnut plants s u b j e c t e d  to
a) d if fe rent light in t e n s i t i e s
- 162 -
5%
1%
b) inocul ation
Co nt ro l
5%
1%
Note: Any two means not u n d e r s c o r e d  by the same line are
s i g n i f i c a n t l y  di ff erent. Any two me ans u n d e r s c o r e d  
by the same line are not s i g n i f i c a n t l y  d i f f e r e n t .
1: Under Shed 1 3: U n d e  r Shed 3
2: Under Shed 2
A: U n dilute d c o n c e n t r a t i o n C: \ d i l u t i o n
B : \ dilution D: ^/o dilution
- 163 -
TA BLE  47a
Analysis of v a r i a n c e  (Two-way Ano v a  wi th r e p l i c a t i o n )  for 
data of Table 37b. Mean size of m i d - l e a f l e t .
(Plant in oculate d w i t h  We i j a- E x - T a m a l e strain)
Source 
o f
variation
| Sura 
1 of
| squares
Degree 
o f 
fr e e d o m
Mean
squares
F
va lu e
Light intensity
1
| 1386.66
I
2 693.330 178 . 642 ★ *
Inoculat ion
1
| 2296.21 
1
4 574.053 147.909 * *
Light intensity & 
Inoculation
1
1
| 655.92
1
8 81.990 21.125 ■* *
Error
1
| 583.60 
1
90 6.484 -
TOTAL
1
| 49 22.39 
1 ...................
104 - -
Significant at 1% level of significance.
164
TABLE 47b
(Data of Table 47a)
Duncan's New M u l t i p l e  Range Test for means of l e a f l e t  size 
of Bambara grou ndn ut plants su b j e c t e d  to
a) d iffe re nt light i n t ens it ies
1 2 3
5%
1%
b) inoculation
A B C D Control
5%
1%
Note : Any two means not u n d e r s c o r e d by the same line are
si g n i f i c a n t l y  di ff erent. Any two me ans  u n d e r s c o r e d  
by the same line are not s i g n i f i c a n t l y  d i f f e r e n t .
1 : Under Shed 1 
2: Und e r Shed 2 
A: Un d i l u t e d  c o n c e n t r a t i o n
B : \ d ilut ion
3: Und e r Shed 3
C : \ d i l u t i o n
D : V g  dilution.
165
Mean dry w e i g h t  of pl ants
Res ult s of A n a l y s e s  of V a r i a n c e  test (Two-way  A n o v a  with 
repli cat ion) of Tabl es 48a, 49a, 50a, 51a and 52a sh ow that
there was s i g n i f i c a n t  ef fect for the two t r e a tm en ts, light 
int ensity and i n oc ulation , on the mea n dry w e i g h t s  at bo th 5 
per cent and 1 per cent levels of s i g n i f i c a n c e .  The r e  was, 
however, no s i g n i f i c a n t  ef fe ct of their i n t e r a c t i o n .
Tables 48b, 49b, 50b, 51b and 52b are the D u n c a n ' s  New
Multiple Range Test carried  out for the m e a n  dry w e i g h t s .  The 
values for plants of the var i o u s  ini t i a l  i n o c u l u m  d e n s i t i e s  of
all the five strains were d i f f e r e n t  at b o t h  5 per cent and 1
per cent levels of s i g n i f i c a n c e  from that of the con t r o l
plants.
The effects of light in t e n s i t y  were:
a. The effects of the three light i n t e n s i t i e s  on plants 
inoculated with L e g o n - E x - A d a ,  P o k u a s e - E x - T a m a l e ,  and 
W e i j a - E x - A d a  strains we re  s i g n i f i c a n t l y  d i f f e r e n t  
from each other at 5 per cent level of s i g n i f i c a n c e ,  
but at the 1 per cent level of s i g n i f i c a n c e  eff e c t s  
of m e d i u m  and high light i n t e n s i t i e s  w e r e  not 
s i g n i f i c a n t l y  different .
b. Effects of the three light i n t e n s i t i e s  were
s i g n i f i c a n t l y  d i f f e r e n t  from each other at both  5 per 
cent and 1 per cent levels of s i g n i f i c a n c e  for
plants in ocu la ted w i t h  K p o n g - E x - T a m a l e  and
N u n u g u a ( 1 )- E x - T a m a l e  strains.
16 6 -
TABLE 48a
Analysis of v a r i a n c e  (Two-way Ano v a  with r e p l i c a t i o n )  for 
data of Table 33a, Mean dry weight of plants.
(Plant i n o c u l a t e d  w i t h  K p o n g - E x - T a m a l e  s t r a i n )
Source j 
of |
variation |
Sum 
o f
squares
Degree 
o f 
freedom
Mean
squares
F
value
1
Light intensit y |
1
14.23 2 7.115 7.974 **
I
Inoculat ion |
1
44 . 54 4 11.315 12.479 **
1
Light intensity & |
Inocula t ion |
1
0.69 8 0.086 0.097 NS
1
Error |
1
53 . 54 60 0.892 -
1
TOTAL |
!
113 .00 74 - -
** Significant at 1 % level of sign if ic a n c e .
NS Non-significant
167
(Data of Table 48a)
D u n c a n ’s New M u l t i p l e  Range Test for me ans of of dry 
weights of Bambara g r o u n d n u t  plants s u b j e c t e d  to
a) d i fferen t light i n t en sities
TABLE 48b
1 2 3
5%
1%
b) i n o c ul at ion
A B C D Control
5%
1%
Note : Any two mean s not u n d e r s c o r e d  by the same 1 ine are
s i g n i f i c a n t l y  different . Any two mea ns u n d e r s c o r e d  
by the same line are not s i g n i f i c a n t l y  dif fe r e n t .
1: Under Shed 1 
2 : Under Shed 2
3: U n der Shed 3
A: Un d i l u t e d  c o n c e n t r a t i o n
B: \ dilution
C: \ d i l u t i o n
D ' V q  dilut i o n .
168
TABLE 49a
Analysis of variance (Two-way Anova with replication) for
data of Table 34a. Mean dry weight of plants.
(Plant inoculated with Legon-Ex-Ada strain)
Source | 
of | 
variation |
Sura 
o f
squares
Degree 
o f
fr ee do m
Mean
squares
F
val u e
1
Light i ntensit y | 14.05 2 7.025 8.982 * *
1
Inocula t i on |
1
43 . 36 4 10.840 13.859 * *
1
Light int en sity & | 
Inoculat ion | 3.92 8 0.490 0.627 NS
Error |
1
46.93 60 0.782 -
1
TOTAL |
1
108.26 74 - -
** Significant at 1% level of significance.
NS Non-significant
TABLE 49b
(Data of Table 49a)
Dun ca n' s New M u l t i p l e  Range Test for mea ns of of dry 
weights of Bambara g r o u n d n u t  plants s u b j e c t e d  to
a) di f f e r e n t  light i n t e nsitie s
- 169 ~
b ) inoculat  ion
D Control
5%
1%
Note : Any two means not u n d e r s c o r e d  by the same line are 
s i g n i f i c a n t l y  different. Any two means  u n d e r s c o r e d  
by the same line are not s i g n i f i c a n t l y  d i f f e r e n t .
1: Under Shed 1 Un d e r  Shed 3
2 : Under Shed 2
A: U n d i l u t e d  c o n c e n t r a t i o n C: \ d i l u t i o n
B : \ d ilut ion D: V o  dilution
- 170 -
T AB LE 50a
Analysis  of v a r i a n c e  (Two-way A n o v a  w i t h  r e p l i c a t i o n )  for 
data of Table 35a. Me an dry w eight of plants.
(Plant in oculate d with Nungua( l) - E x - T a m a l e strain)
Source 
o f
var i at ion
| Sura 
1 of
| squares
Degree 
o f 
fr ee do m
Mean
squares
F
va lue
Light int en sity
1
| 23.44
1
2 11 .720 7.850 * *
Inocula t ion
1
| 45.24 
1
4 11.310 7.575 * *
Light i ntensi ty  & 
Inocula t ion
1
1
| 2.11 
1
8 0.264 0.177 NS
Error
1
| 89.58
l
60 1.493 -
TOTAL
1
| 160.37
i
74 - -
** Significant at 1% level of significance.
NS Non-significant
171
(Data of Table 50a)
Duncan's New M u l t i p l e  Range Test for means of of dry 
weights of Bambara gro u n d n u t  plants su b j e c t e d  to
a) d iff er ent light int en sities
TABLE 50b
1 2 3
5%
1%
b) i n o c ulation
A B C D Control
5%
1%
Note: Any two means not u n d e r s c o r e d  by the same line are 
s i g n i f i c a n t l y  different. Any two means u n d e r s c o r e d  
by the same line are not s i g n i f i c a n t l y  d i f f e r e n t .
1: Und er Shed 1 3: Und e r Shed 3
2: Under Shed 2
A: U n d i l u t e d  c o n c e n t r a t i o n C: \ d i l u t i o n
B: \ dilution D: V g  dilution.
- 172 -
TABLE 51a
An aly sis of v a r i a n c e  (Two-way A n o v a  w i t h  r e p l i c a t i o n )  for 
data of Table 36a. Me an dry w e i g h t  of plants.
(Plant inoculat ed wi th  K pong - Ex - T a m a l e s t r a i n )
Source 
o f
variation
| Sum 
1 of
1 squares
D egree
of
fr e e d o m
Mean 
square s
F
v al ue
Light int ensity
1
| 18.98
1
2 9.490 10.477 * *
Inoculat ion
1
| 42.50
1
4 10.625 11.730 * *
Light intensity & 
Inocula t i on
1
1
| 2.28 
1
8 0.285 0.315 NS
Error
1
| 54.35
J
60 0.906 -
TOTAL
1
| 118.11 
1
74 - -
** Significant at 1% level of significance.
NS Non-significant
TABLE 51b
(Data of Table 51a)
Duncan's New M u l t i p l e  Range Test for mea n s  of of dry 
weights of Barabara g r o u n d n u t  plants s u b j e c t e d  to
a) dif fe rent light intens it ies
- 173 -
b ) inoculat ion
Control
5%
1%
Note: Any two means not u n d e r s c o r e d  by the same line are
s i g n i f i c a n t l y  di ff erent. Any two means u n d e r s c o r e d  
by the same line are not s i g n i f i c a n t l y  d i f f e r e n t .
1: Under Shed 1 U n de r Shed 3
2 : Under Shed 2
A: U ndilut ed  c o n c e n t r a t i o n C: \ d i l u t i o n
B: \ dilution D: /g dilution
174
TABLE 52a
Analysis of variance (Two-way Anova with replication) for
data of Table 37a. Mean dry weight of plants.
(Plant inoculated with Weija-Ex-Ada strain)
Source 
o f
variation
Sura 
o f
squares
Degree 
o f 
f reed om
Mean
squares
F
v a lue
Light intens ity 22 .04 2 11 .020 10.196 * ★
Ino culation 45.14 4 11.285 10.441 ★ *
Light inte nsity & 
Inoculation 2.09 8 0.261 0. 242 NS
Error 64 . 85 60 1.081 -
TOTAL 134.12 74 - -
* * Signi ficant at 1 % level of s i gn i f ic anc e .
NS Non-significant
175
(Data of Table 52a)
Duncan's New M u l t i p l e  Range Test for means of of dry 
weights of Bambara g r o u n d n u t  plants s u b j e c t e d  to
a) d i fferen t light int e n s i t i e s
TABLE 52b
5%
1 %
b) inoculatio n
Co ntr ol
5%
1%
Note: Any two means not u n d e r s c o r e d  by the same line are
s i g n i f i c a n t l y  di ff erent. Any two mea ns u n d e r s c o r e d  
by the same line are not s i g n i f i c a n t l y  d i f f e r e n t .
1: Under Shed 1 3: U n d e r  Shed 3
2: Under Shed 2
A: U n dilut ed  c o n c e n t r a t i o n C: \ d i l u t i o n
B: \ dilution D : /g dilution
176
Mean n u m b e r  of n o d u l e s  per plant
No d u l a t i o n  did not take place in the u n i n o c u l a t e d  plants 
exposed to all three light intens ities.
The mean nu mber of nodules of i n o c u l a t e d  p lants ranged  
between 54 and 78 , 42 and 51, and 16 and 21 for p l a n t s  r ai sed
under low, m e d i u m  and high light in te ns i t i e s ,  r e s p e c t i v e l y .  
The mean number of no d u l e s  formed by the i n o c u l a t e d  plant s 
appear in Tables 33b, 34b, 35b, 36b and 37b.
The Two-way Anova with r e p l i c a t i o n  m e t h o d  was a g ai n used 
to find the s i g n i f i c a n t  effect of light intens it y, and 
inoculation, and the in t e r a c t i o n  of the two on n o d u l a t i o n .
Tables 53a, 54a, 55a, 56a and 57a i n d i c a t e  that there was
significant effect of all light i n t e n s i t i e s  and i n o c u l a t i o n  and 
their int era ct ions for plants i n o c u l a t e d  with the five strains, 
at both 5 per cent and 1 per cent levels of s i g n i f i c a n c e .
The Duncan's New M u l t i p l e  Range Test for means of n od ule
number presented in Tables 53b, 54b, 55b, 56b and 57b showed
significant effect of i n o c u l a t i o n  at bo th  5 per cent and 1 
per cent levels of s i g n i f icanc e.
Mean numbers of no dules  formed und e r  the three light 
intensities were all s i g n i f i c a n t l y  d i f f e r e n t  from each oth er at 
both 5 per cent and 1 per cent levels of s i g n i f i c a n c e .
- 177 -
TABLE 53a
An a lys is  of v a r i a n c e  (Tw o-way  A n o v a  w i t h  r e p l i c a t i o n )  for 
data of Table 33b, Mean number of n o d u l e s  per plant.
(Plant in oc ulated w i t h  Kp ong- Ex - T a m a l e s t r a i n )
Source 
o f
var iat ion
| Sura 
| o f
1 squares
Degree 
o f 
fre e d o m
Mean
squares
F
v a l u e
Light inte nsi ty
1
| 16032.2 
1
2 80 16 .1 0 8 7.132  **
Inoc ula tion
1
| 22038.9 
1
4 5509.72 59.888 **
Light in tensity  & 
Inoculat ion
I
1
| 4212.3 
1
8 526.54 5.723 **
Error
1
| 5520.0 
1
60 92.00 -
TOTAL
1
| 4 7 803.4 
1 _
74 - -
Significant at 1% level of significance.
- 178 -
TABLE 53b
(Data of Table 43a)
Dun can's  New M u l t i p l e  Range Test for me ans  of n o d u l e  
number of Bam bara g r o u n d n u t  plants s u b j e c t e d  to
a) dif f e r e n t  light int ensiti es
1 2 3
5%
1%
M  in o c u l a t i o n
A B C D Control
5%
1%
Note : Any two means not u n d e r s c o r e d  by the same line are 
s i g n i f i c a n t l y  different. Any two me ans u n d e r s c o r e d  
by the same line are not s i g n i f i c a n t l y  d i f f e r e n t .
1: Under Shed 1 3 ; Un d e r  Shed 3
2: Under Shed 2
A: U n d i l u t e d  c o n c e n t r a t i o n  C: \ d i l u t i o n
B: ^ d i l u t i o n  ^ . 1 / A .,, .
/g  d i l u t j i o n
179
An al ysis  of v a r i a n c e  (Two-way An o v a  w i t h  r e p l i c a t i o n )  for 
data of Table 34b, Mean nu mber of n o d u l e s  per plant.
(Plant in oculate d with L e g o n - E x - A d a  strain)
TABLE 54a
Source 
o f
v a riat io n
| Sura
1 0 f
squares
Degree 
o f 
freedom
Mean 
square s
F
va lue
Light intens ity
1
| 15097.0 
I
2 7548.52 85.571 •k it
Ino cu la tion
1
| 21065.8
1
4 5266.45 59.701 *  *
Light i n te nsity & 
Ino cu la tion
1
1
| 4173.1
1
8 521.64 5.913 *  St
Error
1
| 5292.8
J _____________
60 88.21 -
TOTAL
1
| 45628 . 7
- 1__________
74 - -
Significant at 1% level of significance.
180
(Data of Table 54a)
Duncan's New M u l t i p l e  Range Test for me ans of n od ul e 
number of Barabara g r o u n d n u t  plants s u b j e c t e d  to
a) d iffer en t light intensit ies
TABLE 54b
1 2 3
5%
1%
b) inoculat io n
A B C D Control
5%
1%
Note : Any two means not und er s cored by the same line are
s i g n i f i c a n t l y  different. Any two means u n d e r s c o r e d  
by the same line are not s i g n i f i c a n t l y  di ff er e n t .
1: Under Shed 1 3: Under Shed 3
2: Und e r Shed 2
A: U n d i l u t e d  c o n c e n t r a t i o n
B : \ dilution
C: \ d i l u t i o n
D : *Vg dilution
- 181 -
TABLE 55a
Anal ysis of v a r i a n c e  (Two-way A n o v a  w i t h  r e p l i c a t i o n )  for 
data of Table 35b. Mean number of n o d u l e s  per plant.
(Plant in oc ulated wi th N u n g u a (1) - E x - T a m a l e s t r a i n )
Source 
o f
var ia tion
| Sum
1 0 f
1 squares
Degree 
of 
f re edom
Mean
squares
F
va l u e
Light intensi ty
1
| 18466.6 
1
2 9233.3 1 18. 32 **
In oc ul ation
1
| 21895.7 
1
4 5473.9 70.15 **
Light in t e n s i t y  & 
Inoculat ion
1
1
| 4677.8 
1
8 584.7 7.493 **
Error
1
| 4682.0 60 78.0 -
TOTAL
1
| 49722.1 
1
74 - -
Significant at 1% level of significance*
- 182
TABLE 55b
(Data of Table 55a)
Duncan's New M u l t i p l e  Range Test for means of n odule 
number of Bambara g r o u n d n u t  plants su b j e c t e d  to
a) dif fe re nt light int ens ities
1 2 3
5%
1%
b) in oculat ion
A B C D Co ntrol
5%
1%
Note : Any two means not und er s cored by the same line are
s i g n i f i c a n t l y  different. Any two means u n d e r s c o r e d  
by the same line are not s i g n i f i c a n t l y  di ff e r e n t .
1: Under Shed 1 3 ; U n de r Shed 3
2: Unde r Shed 2
A: Und i l u t e d  c o n c e n t r a t i o n  C: \ d i l u t i o n
B: ^ d i l u t i o n  n . 1/ j ., ..
/g d i l u t i o n
- 183 -
TABLE 56a
An al ys is of v a r i a n c e  (Two-way Ano v a  wit h r e p l i c a t i o n )  for 
data of Table 36b. Mean number of nodu l e s  per plant.
(Plant inoculated with Pokuase- E x -T amale strain)
Source 
o f
variation
| Sum 
1 of
1 squares
Degree 
o f
freedom
Mean
squares
F
v al u e
Light intensity
1
| 20670.6 
1
2 10335.3 10 2.57 **
I n o c u 1 a t i on
1
| 2 5 2 7 4.1
I
4 6318.5 62.71 **
Light inten sit y & 
Inocula t ion
1
1
| 5576.3
1
8 697 .0 6. 918 **
Error
1
| 6045.6
J
60 100. 7 -
TOTAL
1
| 57566.7 74 - -
Significant at 1% level of significance.
184
TABLE 56b
(Data of Table 56a)
Duncan's New M u l tipl e Range Test for neans of n o d u l e  
number of Bambara gr o u n d n u t  plants su b j e c t e d  to
a) d iffer en t light intensit ies
1 2 3
5%
1%
b) inoculat ion
A B C D Control
5%
1%
Note : Any two means not u n d e r s c o r e d by the same line are
si g n i f i c a n t l y  different. Any two mea ns u n d e r s c o r e d  
by the same line are not s i g n i f i c a n t l y  diff er e n t ,
1: Under Shed 1 3 : Under Shed 3
2: Under Shed 2
A: Undiluted  con c e n t r a t i o n  C: \ d i l u t i o n
B: H dilution D ; 1/ dilution.
185
Analysis  of v a r i a n c e  (Two-way A n ova w i t h  r e p l i c a t i o n )  for 
data of Table 37b, Mean number of n o d u l e s  per plant.
(Plant in oc ulated with W e i j a - E x - A d a  strain)
TABLE 57a
Source 
o f
var iat ion
| Sum
1 of 
| squares
Degree 
o f 
freedom
Mean
squares
F
va lue
Light intensit y
1
| 13871.2
I
2 6935.6 63.05 **
Inoculat ion
1
| 20682.9 
1
4 5170.7 4 7.006 **
Light i nt en sity & 
I n o c u 1 at ion
1
1
| 3812.2
1
8 476 .5 4 .3 3 2  **
Error
1
| 6600.8
1 ......
60 110.0 -
TOTAL
1
| 44967.2
.1 _
74 - -
Significant at 1% level of significance.
TABLE 57b
(Data of Table 57a)
Du nca n's New M u l t i p l e  Range Test for me ans of n o d u l e  
number of Bam bara gro u n d n u t  plants s u b j e c t e d  to
a) d if ferent  light i n t e nsitie s
1 2 3
5%
1%
b) i n o c ul at ion
A B C D Co ntrol
5%
Note: Any two means not u n d e r s c o r e d  by the same line are
s i g n i f i c a n t l y  dif fe rent.  Any two means u n d e r s c o r e d  
by the same line are not s i g n i f i c a n t l y  different.
Is Under Shed 1 3: Under Shed 3
2: Under Shed 2
A: Un d i l u t e d  c o n c e n t r a t i o n  C: \ d i l u t i o n
B: ^ d i l u t i o n  n « 1 / j
D; /8 dilution.
Size of nodules
Light i nt en sity also affected the sizes of the nodul es , as 
can be seen from the mean diame te rs of nod u l e s  of the vari o u s  
t re at men ts in Table 58, and the h i s t o g r a m s  of class - d i a m e t e r s  
of the nodules in Figs. 11 - 15,
Nod ules formed by plants raised under L ow  light i n t e n s i t y  
had c l a s s - d i a m e t e r s  ra n g i n g  from 2.51 to 4.21cm. Tho s e  formed 
by plants raised under m e d i u m  and high light i n t e n s i t i e s  ra nge d 
from 1.79 to 2.63cm, and 0.87 to 1.95cm, r e s p e c tive ly .
- 188 -
TABLE 58
Mean d iameter s of nodule s formed by B a m b a r a  g r o u n d n u t
plants grown under d i f f e r e n t  light i n t e n s i t i e s  for 30 days .
Initial Me an  di a m e t e r s (cm ) of
Rhizobium den sit y nodules of plants grown
o f under
In o c u 1um Ino culum Shed 1 Shed 2 Shed 3
K p o n g - E x - T a m a l e U n d i l u t e d 3,20 2.06 1 . 84
strain \ d ilution 2.51 2.31 0.89
\ dil ution 2.84 2.27 1.82
V o  dilutio n 2.63 1.96 1.89
Le gon - E x - A d  a Und iluted 3.32 2.63 1.62
strain \ d ilut ion 2.96 2.36 1.63
\ dil ut io n 3.12 2.13 1 .74
V g  dil utio n 3.56 2.32 1.12
Nungua(l)-Ex-Taraale Und iluted 3.22 2.25 1 .78
strain \ dil ut io n 2.74 1 .35 0.99
\ dil ut io n 2.67 2.35 1 .10
*/g dilu ti on 3.67 1 .79 1 .00
P o k u a s e - E x - T a m a l e Und iluted 2.91 1.97 0.87
strain \ dilut io n 2.79 2.33 1 . 89
\ dilution 2.59 1 .88 1.89
/o dilution 2.67 2.42 1.67
We i j a - E x - A d a Und iluted 3.60 1.67 1 .65
strain \ dilut io n 2.74 2.04 1 .25
\ dil ution 4.12 2.38 1 .36
V q dil uti on 2.71 2.36 1.95
PE
R
C
EN
T
A
G
E 
F
R
E
Q
U
E
N
C
Y
- 189 -
I o o
50
I O O
5 0 '
• O
I OO
5 0
I O O
50 -
8 H € D S H E D  2 S H E D  3
i □ a
□ O H
□ E 1— *— i n
m - r= *
I 2 3 4 5 6 7
I I i f
I 2 3 4 5 6 7 
CLASS  D I A M E T E R
U
I 2  3  4  5 6  7
i: o  - lOOmm 5: 4.01 — 5-OOmm
2: I .OI -  2.00  AM 6" 5 . 0 | ~ 6-00 mm
3: 2.01 — 3 -00 mm 7. 6.01 — 7 .00  mm
4: 301 — 4 . 0 0nm
Fig. 11 Distribution class-diameters of nodules of Bambara
groundnut plants, Ex-Ada variety, inoculated with Kpong- 
Ex-Tamale strain of Rhizobium sp. and grown under different 
light intensities for 30 days. Seeds inoculated with moss 
carrier with different initial Broth culture.
Fig.
- 190 -
I O — I .O O IK IK
2: 1.0 I -  2.00mm
3 2.01  — 3 ,  OO m m
A:  3-01 — 4. OOmra
5: 4.01 — 5 . 0 0 * 1
6'  5.01 —  6 OO mm
I !  6.01 —  7. OO  * m
2 Distribution of class-diameters of nodules of Bambara groundnut
plants, Ex-Ada variety, inoculated with Legon-Ex-Ada strain of
Shizobiun, sp and grown under different light intensities for
3° days Seeds inoculated with moss carrier with different 
initial Broth culture.
(/
•
D
il
u
ti
o
n
 
1/
4 
D
il
u
ti
o
n
 
1/
2 
D
il
ut
io
n 
U
nd
il
ut
ed
PE
R
C
EN
T
A
G
E
 
F
R
E
Q
U
E
N
C
Y
IOO
30-
IOO
5 0
IOO
5Q -
I OO
SHED  I
- 191 -
SHED 7
□ J L fTfTTL
5 H E 0 3
z D d n . a
r i h - ^
r
Q £ L T r - n
p : u n n  \
2 3 4 3 6 7  I 2 3 4 5 6 7
CLASS  D IAM ETER
i: O -  I OO mm 5: 4.01 -  5 OOn»M
2 : 1.01 - 2  O O m m 6‘. 5.01 — 6 . 0 0 mm
3: 2.01 — 3-OO i r 7'. 6 01 —  7 . 0 0 mm
4: 3 .0 1— 4 . 0 0  mm
Fig. 13 Distribution of class-diameters of nodules ol' Bambara
groundnut plants, Ex-Ada variety, inoculated with Nungua(l) 
Ex-Tamale strain of Rhizobium sp and grown under different 
light intensities for 30 days. Seeds inoculated with moss 
carrier with different initial Broth culture.
PE
RC
EN
TA
G
E 
FR
EQ
U
EN
C
Y
- 192 -
i: O - I . O O a a  s: 4 .01  -  S . O O m
a: 1.01— a.ooa* «: 5.01 - t o o m
3: S . O I _ J . O O « i »  7: 6.01 — 7. OO  ■■
4: 3 01 -4.00mm
Fig. 14 Distribution of class-diameters of nodules of Bambara ground­
nut plants, Ex-Ada variety, inoculated with Pokuase-Ex- 
Tamale strain of Rhizobium sp and growth under different 
light intensities for 30 days. Seeds inoculated with moss 
carrier with different initial Broth culture.
1/8
 
D
ilu
ti
on
 
1/4
 
D
ilu
ti
on
 
1/
2 
Di
lu
ti
on
 
U
nd
il
u
te
d
PE
RC
EN
TA
G
E 
FR
EQ
U
EN
C
Y
193 -
IOO
50
IOO
J O
IOO
50
IOO
S H K D  I
r^lThn
5 Ht  D 2
d H
5 H F D 1
e Q I L a .
-«-rl LtfcL l a r t
T V - n  ,
1 1  1 4 5 6 7 4 5 6 7 2 3 4 5 6 7
C L A S S  O I A M E  T ^ R
I! O -  1 . 0 0 * 1
a :  t . o i _ a . o oM «  
j : a.ot  — J . O O am  
4: 3-01 —4 . 0 0  Da
s:  4 . 0 1 - J . O O . .
* '  S O|f — 6 OOmm  
7: 6-01 — 7. O O a n
Fig.15 Distribution of class-diameters of nodules of Bambara
groundnut plants, Ex-Ada variety inoculated with Weija—Ex- 
Ada strain of Rhizobium sp and grown under different 
light intensities for 30 days. Seeds inoculated with 
carrier with different initial Broth culture.
moss
1/
8.
 D
il
ut
io
n 
1/4
 
D
il
u
ti
on
 
1/
2 
D
il
ut
io
n 
U
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d
il
u
te
d
V, G E N E R A L  D I S C U S S I O N
Barabara g r oundn ut  is an i mp or tant legume crop in T r o p i c a l  
Africa and comes only third after c o w p e a  and grou n d n u t .  It is 
a sa vann a plant and there can be no limit to a v a i l a b l e  land for 
its c u ltivat io n. It is dr ought r e s i s t a n t  and y i e l d s  a
re a s o n a b l y  good crop even when grown on poor soils. In spite 
of these attrib utes, very little r e s e a r c h  has been c a r r i e d  out 
on the crop to support the industry, n e i t h e r  has the a c r e a g e  
under c u l t i v a t i o n  i nc reased  over the years. Indeed, an FAO 
report (FAO, 1988) poi nte d out that barabara g r o u n d n u t  
p r o d u c t i o n  has d e c l i n e d  in recent years and it is be i n g
replaced by an e x p a n d i n g  gro u n d n u t  p r o du ct ion. May be if the
di f f e r e n t  die t a r y  p r e p a r a t i o n s  of the pro du ct in the i n d i v i d u a l
African co u n t r i e s  di ff us e throu g h o u t  the p r o d u c i n g  count r i e s ,  
its usage in each cou n t r y  will b r o a d e n  and e n c o u r a g e  i n c r e a s e d  
produc tio n.
The range of us age is striking. For example, in Ghana,
the beans are soaked, boiled and made into a type of p o r r i d g e  
which is sweetened  with sugar. In Cameroun, they are used raw 
or, after cooking, ground and added to soups and stews. In 
Tanzania, the boiled  seeds are crushed, gr o u n d n u t  paste added, 
and the mix t u r e  eaten wit h other diets. In Mal a g a s y ,  they are 
often added to stew with rice or eaten with a gr e e n  leafy 
ve ge tab le,  and in Sou th ern Africa the seed flour is m i x e d  wit h 
oil or butter and eaten with meat, or ma de into cakes.
C u l t i v a t i o n  of barabara g r ou ndnut on ve ry large scale will 
o b v i o u s l y  face cer tain problems. The plant is a t t a c k e d  by few 
diseases, the most serious ones are Fusarium wilt and leaf spot 
by Cercospora canescens. However, the plant is a t t acke d by ma ny 
pests. A ttack by root knot ne m a t o d e s  (Meloidogyne sp.) is 
wi despread . Leaf hop pers for example, Empoasca facialis and Hilda
- 194 -
- 195 -
potruclis are im portant in Natal and Ta nz anian, r e s p e c t i v e l y .  
In Ghana, the plant is attacl<2d by larvae of the b u t t e r f l y ,  
Dracrisia maculosa, and in U ga nda by larvae of Lamprosema indicata
(FAO, 1988).
Cl i mat ic  and eda phi c factors can also af fect p r o d u c t i v i t y , 
the major factors be ing  nu trients, water and light. A s ea rch 
through the relevant li te ra t u r e  r e c orded  no stu die s on the 
n u t r i t i o n a l  r e q u i r e m e n t s  of barabara gro un dnut.
N i t r o g e n  was present in all the soils from the eight 
localities. The hig he st recorded me an  p e r c e n t a g e  was 0.13 2 per 
cent (soil from Pokuase) and the lowest mean p e r c e n t a g e  of 
0 . 025 per cent (Ash iam an soil) (see Table la). Me an total 
n i t r o g e n  co ntent in soils r e c o m m e n d e d  for good pla nt g r o w t h  
(Bremner and Edwards, 1965 ) ranges from 0.05 per cent to 0.25 
per cent. The soil samples wh i c h  were used w i t h  n i t r o g e n  
content wi thin this range were those from Kpong, Legon, 
Nungua(l), Pokuase and Weija. The plants raised in these soils 
c o i n c i d e n t a l l y  grew better, with mean dry we ig ht s r a n g i n g  from
0.46g to 2.74g, than those raised in the n i t r o g e n - p o o r  soils of 
Ashiaman, Nun gu a(2) and Shiashie  with only 0.50 and 0.69g; 0.42 
and 0,44g; and 0.41 and 0.72g mean dry we ig hts (Ex-Ada and Ex- 
Tamale v a r i et ie s), r e s p e c t i v e l y  (see Table 3).
It was found in this i n v e s t i g a t i o n  that se ver e w a te r 
stress n a t u r a l l y  gr e a t l y  affected gr owt h and n o d u l a t i o n  of the 
ba mba ra g r o u n d n u t  plant. What is imp or ta nt in the p r e s e n t  
o b s e r v a t i o n  is the r e l a t i o n s h i p  b e t w e e n  the d e g r e e  of the 
effect and the interval of wate rin g. Plant dry w e i g h t s  and 
number of nodules formed are two features w h ich sho wed a 
un if or m re latio ns hip. The data in Tables 7 to 11 i n d i c a t e d  
that the mean dry weight of plants wat e r e d  once in two days was 
a p p r o x i m a t e l y  two times that of plants w a t e r e d  once in six 
days. The ratio of mean nu mbe r of no du le s of b a m b a r a  g r o u n d n u t
196
plants wa tered once in two, four and six days was a p p r o x i m a t e l y  
1 : 2 : 4 - 5 .
Water affects plant grow th by a f f e c t i n g  inte r n a 1 
p h y s i o l o g i c a l  p roces se s and c o n d it ions. G ro wt h of p la nt s is 
con troll ed  by rates of cell divis i o n  and e n l a r g e m e n t  and by the 
supply of or ganic and i n or ga nic c o m p o u n d s  r e q uired  for the 
synthesis of new p r o t o p l a s m  and cell walls. Cg.11 e n l a r g e m e n t  
is p a r t i c u l a r l y  de p e n d e n t  on at least a m i n i m u m  d e g r e e  of cell 
tugor, and stem and leaf e l o n g a t i o n  are q u i c k l y  c h e c k e d  or 
stopped by water def ic its (Miller, 1965). This is well 
ill us tr ated by the sizes of the m i d d l e  leaflets of barabara 
groundnut plants rec orded in Tables 7 to 11.
De cr e a s e  in wa ter co nte nt  i n v a r i a b l y  reduce s the rate of 
p h o t o s y n t h e s i s  (Brix, 1962) and this leads to d e c r e a s e  in dry 
weight. Con s e q u e n t l y ,  dry weig h t s  of the barabara g r o u n d n u t
plants d e cr eased w i t h  an inc reas e in the interva l of w a t e r i n g .
\
Values of mean dry we i g h t s  for plants w a t e r e d  once in two, four 
and six days, re sp ective ly , were 0.54 - 1.68g, 0.32 - 0.9 4 g  and
0.11 - 0.76g (values for both i n o c u l a t e d  and u n i n o c u l a t e d
plants) (see Table 7 - 11).
Mo i s t u r e  def icits  have been found to prevent the syn t h e s i s  
of protei ns  by many obs e r v e r s  (eg, Bar n e t t  and Naylor, 1966; 
Petrie and Wood, 1938; Yarooh, 1958). Studies on the a b i l i t y  
of A r i z o n a  Co mmo n and Coastal Be r m u d a  grass (Cynodon dactylon (L) 
Pers) to sy nt hesize amino acids and pro teins d u r i n g  w a te r 
stress by Barnett and Naylor (1966) showed that amino acids 
were c o n t i n u a l l y  sy n t h e s i z e d  d u r i n g  the wa ter  stress 
treatments, but protein syn th esis was inhibite d and pro t e i n  
levels de creased. Water stress induced a 10- to 1 0 0 -fold 
ac c u m u l a t i o n  of free asp aragine.
197
Gates and Bonner (1960) showed that the am ount of both DNA 
and RNA per leaf dec r e a s e d  under wa t e r  deficits,  a l t h o u g h  they 
were similar in the water - d e f i c i e n t  and co n t r o l  t r e a t m e n t s  of 
tomatoes on a dry w e i g h t  basis. The d e c r e a s e  in n u c l e i c  acids 
per leaf is i n t i m a t e l y  relat ed to the slower g r o w t h  rate per 
leaf. Gates and Bonner (1960) a t t e m p t e d  to v e r i f y  w h e t h e r  this 
de cre ase was due to d e c r e a s e d  sy n t h e s i s  in r e s p o n s e  to w a t e r  
deficits. Their results showed that the rate of
i n c o r p o r a t i o n  into nu c l e i c  acids und e r  w a t e r  d e f i c i t s  is 
probably due to a c c e l e r a t e d  d e s t r u c t i o n  ra the r than d e c r e a s e d  
synthesis. Since pr o t e i n  s yn th esis is re la ted to RNA, the 
effects of a m o i s t u r e  def i c i t  on n u c l e i c  acids will r ed uce the 
rate of pro t e i n  s y nth es is ind ir ectly. B a m b a r a  g r o u n d n u t  plants 
watered once in 4 days and 6 days w h i c h  gr ew less v i g o r o u s l y  
might have s u f fe red i m p a i r m e n t  of p r o t e i n  synt hes is.
Water shortage also impairs au xin p r o d u c t i o n  so that cell 
enl argem en t and growt h is s u p p r e s s e d  (Treshow, 1970). L i m i t e d  
cell e n l a r g e m e n t  leads to re duce d leaf areas, s h o r t e n e d  
internodes, and stunt ed and r o s ette d plants.
There was m ar ked e ff ec t of light i n t e n s i t y  on the g row th  
and d e v e l o p m e n t  of the b a m b a r a  g r o u n d n u t  plants (see T ables 33 
- 37). D i f f e r e n t  levels of light i n t e n s i t y  w i t h i n  the range
used in this i n v e s t i g a t i o n  s u p p o r t e d  d e v e l o p m e n t  of the plants 
to d i ff erent degrees.
It is well kn own  that in the u n d e r g r o w t h  of fo rests where 
the light in tensity is low, the shrubs and gra s s e s  d e v e l o p  
broad leaves wh ich  trap m a x i m u m  light in the dim light. In 
this wo rk  the largest leaflets were p r o d u c e d  by p lants  e x p o s e d
198 -
to the lowest light intensi ty. The mean leaflet d i m e n s i o n s  of 
5.60 x 2.27 - 8.56 x 3.00 cm were re du ce d to 4.91 x 1.90 - 6.07 
x 2.66 cm under the m e d i u m  light i n t e n s i t y  and still fur t h e r  to 
4.57 x 1.53 - 5.71 x 2.16 cm under the h i g h e s t  light i n t e n s i t y  
(see Tables 33 - 37).
However, the lowest light i n t e n s i t y  p r o v i d e d  in this w o r k  
seems a p p a r e n t l y  to be the best for the g rowt h of b a m b a r a  
groundnut plants. F o r ; the mean dry w e i g h t s  of the plants, of 
lowest light in t e n s i t y  thr ough the m e d i u m  to the h i g h e s t  light 
in tensity were 1.62 - 2.91g, 1 .33 - 1 . 82g and 0.91 - 1.41g,
r es pective ly. The c o r r e s p o n d i n g  me an n um be r of n o d u l e s  per 
plant were also 54 - 78, 42 - 51 and 16 - 21, r e s p e c t i v e l y .
Light i n t e n s i t y  d i r e c t l y  af fect p h o t o s y n t h e s i s .  M a n y  
studies (eg. Hall and R a o , 1986) have shown that p h o t o s y n t h e s i s
becomes less e f f i c i e n t  under high light in tensity. J u d g i n g  
from the p r o d u c t i v i t y  of the b a m b a r a  g r o u n d n u t  plants, light 
intensities bet w e e n  8800 and 10000 lux have p rov ed  to be 
un fav o u r a b l e  to the plants. Too hi gh  a light i n t e n s i t y  
depresses p h o t o s y n t h e t i c  rate th ro ugh a rapid p h o t o - o x i d a t i o n  
of c h l o r o p h y l 1.
The low light i n t e n s i t y  1100 to 6000 lux w h i c h  has 
supported the best g rowth  in the g r o w t h  of b a m b a r a  g r o u n d n u t  
plants, may be quite close to the light i n t e n s i t y  the crop 
en counters under mixed fa rmi ng  c o n d i t i o n s  in Ghana. The 
findings also point out that in N o r t h e r n  parts of Ghana whe r e  
it is grown as a m o n o c r o p  fully e x p o s e d  to sunl ight, the 
p r o d u c t i v i t y  of the plants falls short of their p o t e n t i a l .  
Further work must be ca rr ie d out to d e t e r m i n e  the lowest light 
inte nsi ty capable of s u p p o r t i n g  good p r o d u c t i v i t y .  This 
k nowle dg e is important in d e c i d i n g  sp a c i n g  in mix e d  farms and 
ac ceptabl e levels of shading of b a m b a r a  g r o u n d n u t  plants.
199
Even though this e x p e r i m e n t  was not c a r r i e d  out long 
enough to include f r u iting it is r e a s o n a b l e  to a ss ume that the 
larger the plant the gre ate r will be the n umber of fl owers and, 
therefore, the higher the yield. Plants at the low light 
inten sit y wh ich p r o d u c e d  the largest plants (see Plate 7) are 
most likely to give the best yield*
Le gu me s stand apart from other crops b e c a u s e  of their ne ed  
for m utual ba c t e r i a l  part ner s, the Rhizobium sp. in a v e r y  
special r e l a t i onshi p.  The sy m b i o t i c  a s s o c i a t i o n  i n v o l v i n g  
legumes and Rhizobium sp. leads to the f i x a t i o n  of a t m o s p h e r i c  
nitr ogen in a high en ergy - c o n s u m i n g  pro c e s s  c a t a l y s e d  by the 
enzyme n i t r o g e n a s e  and wh i c h  uses ATP of r e s p i r a t i o n .  The mo de  
of i nfec ti on of clover by the n o d u l e  b a c t e r i a  has been c l o s e l y  
studied by N ut man (1958), A later a c c o u n t  of the a c t u a l  cell 
entry was later p r e s e n t e d  by Jordan, Gri n y e r  and C o u l t e r  
(1963 ) .
The n o d u l e - r o o t  c o m p l e x  f o r m a t i o n  inv olves three phases:
(a) nodu le d e v e l o p m e n t ,  (b) b a c t e r o i d  form at ion, and (c) 
l eg ha e m o g l o b i n  synthesis.
Legume roots exude v i t a m i n  B w h i c h  s t i m u l a t e s  g r o w t h  of 
Rhizobium sp. in their r h i z o s p h e r e .  The root p r o d u c e s  in 
addition t r y p t o p h a n  wh i c h  the b a c t e r i u m  c o n vert s into Indole 
Acetic Acid (IAA), The IAA causes c h a r a c t e r i s t i c  c u r l i n g  and 
sometimes b r a n c h i n g  of the root ha irs . P o l y s a c c h a r i d e s  of 
Rhizobium slime at the same time in du ce s the p r o d u c t i o n  of the 
wall a t t a c k i n g  enzyme, p o l y g a l a c t u r o n a s e  (PG) by the legume 
root. IAA and PG combine to soften the apex of the root hair 
causing i n v a g i n a t i o n  of the wall at the apex to start the 
formation of an infection thread - a c e l l u l o s e  tube fi lle d w i t h  
muc i 1 a g e .
200
The Rhizobium cells multiply rapidly at this stage to 
produce small spherical flagellated cells referred to as 
swarraer cells, which swim into the mucilage of the infection 
thread. The infection thread begins to grow towards the base 
of the root hair led by the root hair nucleus* When the 
infection thread establishes contact with the wall between the 
epidermal cell of the root hair and the adjacent cortical wall, 
the same process of wall invagination takes place admitting the 
infection thread into the cortical cell.
The legume cortex, remarkably contains tetraploid cells 
among the diploid cells which are the destination of the 
infection threads. When the infection thread eventually enters 
a tetraploid cell, it branches profusely, and numerous vesicles 
appear on its external surface* The swarmer cells migrate into 
these vesicles, which are in turn blebbed off into the cell. 
The cell also encloses the detached vesicles in its cytoplasm 
with its own synthesized membrane. After this the tetraploid 
cells are stimulated to divide repeatedly to form the primodiura 
nodule. The nodule grows considerably and pushes out of the 
root assuming the characteristic nodule shape.
The swarraer cells are then modified to make them 
functional nitrogen fixers. They lose their flagella and grow 
into swollen mishapen and even branching forms - the 
bacteroids. The bacteroids contain the nitrogenase and fix 
nitrogen.
Paradoxically, the oxygen which will be required for 
respiration to generate ATP for nitrogen fixation inactivates 
nitrogenase. Nitrogenase contains two proteins; Protein 1 
which consists of Molybdenum, non-heme iron and labile sulphur.
201
It is called Mo 1 y b d o f e r r e d o x i n , and is not p a r t i c u l a r l y  
s en si tive to oxygen; and Protei n 2 w h i c h  c o n s i s t s  of no 
M o l y bd en um, n o n -h eme iron and less labile sulphur. It is 
called A z o f e r r e d o x i n  and is very s e n s i t i v e  to oxygen.
The tetra p l o i d  cells of the n o d u l e  pl ay  a cr u c i a l  role of 
p r o t e c t i n g  n i t r o g e n a s e  from o xygen injury. They c o n t a i n  a 
h a e m o g l o b i n  - 1 e g h a e m o g 1obin - whi c h  has st ron g a f f i n i t y  to
oxygen and binds the o xyge n in the nodule, r e l e a s i n g  it 
grad ua ll y to the b a c t e r o i d  for r e s p i r a t i o n  and ATP g e n e r a t i o n .  
The nodule thus p r o vi de s the proper  e n v i r o n m e n t  to pro t e c t  
n i t r o g e n a s e  from oxygen.
A s s o c i a t i o n  of a b a c t e r i u m  wi th  a legume h a p p e n s  b e c a u s e  
part of the genetic i n f o r m a t i o n  for s y n t h e s i z i n g  the n e c e s s a r y  
h a e m o g l o b i n  is pre s e n t  in the b a c t e r i u m ,  and part is coded by 
genes in the plant. This e x p lains the s p e c i f i c i t y  of the 
r e lati on ship be t w e e n  the Rhizobium species. P r e s e n t l y ,  the 
recognised Legu me group - Rhizobium sp. a s s o c i a t i o n s  are as 
shown in Table 59.
- 202 -
TA BLE  59 CR OSS  - I N O C U L A T I O N G R O U P S *  OF R H I Z O B I U M
Rhizobium s p . C r o s s - i n o c u l a t i o n  
g r o u p i n g s .
Legume types
R. Leguminosarum 
R. phaseoli 
R. trifolii 
R. meliloti 
R. lupini 
R. japonicum 
Rhizobium sp.
Pea group 
Bean group 
Cl ove r group 
Al f a l f a  group 
Lu pin i group 
So ybe an group 
Co wpe a group
Pisum, Vicia,, Lens
Phaseolus
Trifolium
Helilotus, Hedicago, Trigonella 
Lupini, Orinthopus 
Glycine
Vigna, Arachis
* The p r i n c i p l e  of c r o s s - i n o c u 1 ati on g r o u p i n g  is bas e d  on 
the ab i l i t y  of an isolate of Rhizobium sp. to form n o d u l e s  
in a li mited g en er a of sp ecies of legumes r e l a t e d  to one 
an other (Subba Rao, 1977).
A single i nfecti ve  sp ecies of Rhizobium is g e n e r a l l y  able 
to infect se veral  d i f f e r e n t  plant species. This s p e c i f i c i t y  
appears to reside in a single b a c t e r i a l  gene or in a n u m b e r  of 
closely linked genes, b e c a u s e  the a b i l i t y  to i nf ec t a 
particular plant group can be t r a n s f e r r e d  from one s p e c i e s  of 
Rhizobium to ano ther by the p r o c e s s  of DNA - m e d i a t e d  
tra nsformation.
The barabara g r o u n d n u t  has been r e p o r t e d  to be n o d u l a t e d  in 
various ge ogr a p h i c a l  areas (Allen and Allen, 1981) but 
co m p a r a t i v e l y  little a t t e n t i o n  has be en gi ven  to its Rhizobium 
affin iti es or its n i t r o g e n  - f ix ing ca p a c i t y .  Stu d i e s  by 
Soraasegaran, Abaidoo and K u m a g a  (1990) show ed that s tra in  TAL 
169 w h ich was derived from n o d u l e s  of Vigna unguiculata was 
c o n s i s t e n t l y  effective on all g e n o t y p e s  of b a m b a r a  g r o u n d n u t .
203
The genus Rhizobium c ompri se s of two main groups, the fast 
and slow growers, d i s t i n g u i s h e d  by their r e l a t i v e  speed of 
growth and change of pH induced in YMA. The 'fast grow er s 
have a Mean G e n e r a t i o n  Time un d e r  the mo st  f a v o u r a b l e  
co ndition s of about 2 - A hours and pr o d u c e  d e t e c t a b l e  c o l o n i e s  
on YMA in 2 - 3 days, and large, gum my c o l o n i e s  up to 5mm
diameter, or more, by 5 days at 2 5°C. the Me an G e n e r a t i o n  Time 
of the slow growers is more likely to be 6 - 8  hou r and 
colonies seldom exceed 1 - 2mm d i a m e t e r  after 10 days (Subba
Rao, 1977 ). This work showed that all the five st r a i n s  used 
produced d e t e c t a b l e  c o l onies in 2 - 3 days s u g g e s t i n g  they were 
all fast g r o w e r s .
Be cause  of the s p e c i f i c i t y  of the a s s o c i a t i o n ,  the 
d if fer enc es in the n o d u l a t i o n  a b i l i t y  of b a m b a r a  g r o u n d n u t  
plants in the d i f f e r e n t  soil samples shown in Table 3, r a n g i n g  
from a mean of three no dules per plant in S h i a s h i e  soil to as 
high as 4 4 plant in W e ij a soil; can be a t t r i b u t e d  to
differ enc es in level of p o p u l a t i o n s  of the a p p r o p r i a t e  str ain 
of Rhizobium sp. Na tura l l y ,  there co uld be some other
co nt ribu ti ng  factors such as soil n u t r i e n t  levels, pH, etc.
The v a r i a b i l i t y  of Rhizobium sp. p o p u l a t i o n  has made the 
in oculation of legumes with their a s s o c i a t e  strains a r o u t i n e  
agric ult ural pra ctice in many  c o u n t r i e s  as has be en  m e n t i o n e d  
in the I n t r o d u c t i o n  and L i t e r a t u r e  Review. A t t e n t i o n  is 
usually given to (a) the n u m b e r  of v i a b l e  r h i z o b i a  supplied, 
(b) p urit y of the culture, and (c) a b i l i t y  of the r h i z o b i a  to 
no dul ate ef f e c t i v e l y  the p a r t i c u l a r  l eg um e plant. Maj o r  
c o n s i d e r a t i o n  is given to the last, b e c a u s e  the only 
d i s t i n c t i o n  between rhiz ob ia and c e r t a i n  re lated  b a c t e r i a  is 
the ab i l i t y  to produce nodu les (Burton, M a r t i n e z  and Curley, 
19 70.) .
204
The pr od uc t i o n  of r hi zo bial c u l tu res on a large scale 
pre par ed in sta in less steel ferraenter ves s e l s  wit h a d e q u a t e
fa cilit ie s for a e r at ion and a g i t a t i o n  of the c u l t u r e  me dium, A 
medium of yeast extract m a n n i t o l  broth is c o m m o n l y  used and the 
orga nis ms are grown for 96 hours. The f e r m e n t e r  is s t e r i l i z e d  
with p r e s s u r i z e d  steam and the air used for a e r a t i o n  of the 
culture m e d i u m  is s t e r i l i z e d  by filt ers b e f o r e  it is i n t r o d u c e d  
into the cul ture medium.
In India, in the ab sence of t r a d i t i o n a l  f e r m e n t e r s ,  the 
rhizobia are raised in shake c u l t u r e s  u s i n g  1L or 2L glass
bottles or flasks (Sahni, 1976),
U n a v a i l a b i l i t y  of a steel f e r m e n t e r  should, t h e r e f o r e ,  not 
be a c o n s t r a i n t  in the i n o c u l a t i o n  p ra ctice.  What m a t t e r s  is 
an eff e c t i v e  ca rrier by w h i c h  the i n o c u l u m  will be a p p l i e d  to 
the seed with the aid of a good a d hesive.  E s s e n t i a l l y ,  a 
suitable m a t e r i a l  for use as a c a r r i e r  must be able to r etain  
the v i a b i l i t y  of the r h i z o b i a  over long periods . It m u s t  also 
decompose in a r e l a t i v e l y  short time, have a high w a t e r  h o l d i n g  
capacity and should not c o n t a i n  s u b s t a n c e s  that are i n h i b i t o r y  
to the r h i z o b i a .
Peat pos s e s s e s  these imp o r t a n t  c h a r a c t e r i s t i c s  and has 
been the t r a di tional m a t e r i a l  used as a carrier. E f f i c i e n t
su bstitute s have been ado p t e d  in m a n y  areas w h i c h  lack peat.
These inc lude bagasse, b a g a s s i l o ,  c offe e husk, coir dust, 
filter mud (a by-pr o d u c t  from sugar cane fa c t o r i e s ) ,  li g n i t e  
and sawdust. By the pr ese nt  i n v e s t i g a t i o n ,  d e c o m p o s e d  moss 
(Brachymenium sp.) has been added to the list of s u b s t i t u t e s .
The moss compost d i s c o v e r e d  in this i n v e s t i g a t i o n  took 
only 15 days to decompose. it a p p a r e n t l y  c o n t a i n e d  no
inhib i t o r y  comp ounds as the r h i zobia did not only s u r v i v e  in it
205
for 14 weeks at n ormal a t m o s p h e r i c  t e m p e r a t u r e  but also 
m u l t i p l i e d  in it (see Fig. 4). A S t a t i o n a r y  Pha se of g r o w t h  
was att ai ned by the tenth week, but there was no sign of 
au tolysis by the end of the st orage p e r i o d  of 14 weeks.
The rhi zo bial p o p u l a t i o n s  in i n o c u l a t e d  moss c o m p o s t  at 
room te m p e r a t u r e  rea ched a level of 1 x 1 0 ^  cells per g r a m  of 
compost in 2 weeks (see Tab le 6). This c o m p a r e s  v e r y  we ll  wi th  
po pu la tions achi eved wi th other ca r r i e r s .  P o p u l a t i o n s  of 1 x 
10 cells per gram after 12 days has be en r e p o r t e d  u s i n g
o
ba gas il lo (Ryder, 1984), of 1 x 10 cells per gr am for filter 
mud in 14 days (Anyango, 1984), and 1 x 10^ cells per g r a m  for 
lignite (Subba Rao, 1977).
The ex t e n s i v e  results in Tables 12 - 58 of e x p e r i m e n t s  in 
which the in oc u l a t e d  b a m b a r a  g r o u n d n u t  pl ants e it her r e c e i v e d  
diff ere nt amounts of w a te r or we re e x p o s e d  to d i f f e r e n t  light 
in tensitie s showed that ino c u l a  in the moss c a r r i e r  w e r e  ve ry  
effective. For example, whe r e a s  the n u m b e r  of n o d u l e s  fo rme d 
by Ex -A da var i e t y  plants in Leg o n  soil was 41 and the m e a n  
nodule dia me te r was 1 .81mm  (see Table 3), plant s i n o c u l a t e d  
with strain of Rhizobium sp. isol at ed from these n o d u l e s  and 
i:. .orporated into the moss car r i e r  formed a v e r a g e l y  67 n o d u l e s  
per plant (see Table 34b) wi th  a m e a n  d i a m e t e r  of 3 . 5 6 m m  (see 
Table 58).
Simil arl y, the mean n u m b e r  of n o d u l e s  per plant fo rmed by 
Ex -Ta ma le va ri ety of b a m b a r a  g r o u n d n u t  g r o w i n g  in K p o n g  soil 
was AA (see Table 3) and the me an  n o d u l e  d i a m e t e r  was 2.A7mm. 
Using moss carrier c o n t a i n i n g  K p o n g - E x - T a m a l e  s tr ain of 
Rhizobium sp. to inoc ula te E x - T a m a l e  v a r i e t y  of b a m b a r a  plants, 
the mean number of nodules per plant was 55 (see Table 7B) and 
the mean nodule diameter  was 2 .6 8mm (see Table 32).
206
In o c u l a t i o n  of new plots for r a i s i n g  b a m b a r a  g r o u n d n u t  has 
been tried by Dadson, Brooks and W u t o h  (1987) and by Stanton, 
Doughty, O r r a c c a - T e t t e h  and S te ele (1966) w i t h  s u c ce ss * 
Inocu lat ion of the seeds with moss car r i e r  should be r e g a r d e d  
as a much more c o n v e n i e n t  prac tice.
It is w o r t h y  of no te that the two b a m b a r a  g r o u n d n u t  
varieties, Ex -Ad a and E x - T a m a l e ,  s howed s i g n i f i c a n t l y  d i f f e r e n t  
responses when they we re  pla n t e d  in the same s am ple of soil 
(see Table 3). This s t r e s s e s  the idea of legume - Rhizobium sp. 
genetical c o m p a t i b i l i t y .  It is, t h e r e f o r e ,  i m p o r t a n t  to take 
into account the i n t e r a c t i o n  of host v a r i e t y  and s t r a i n  of 
Rhizobium sp. in any future at tem p t s  at c o m m e r c i a l  p r o d u c t i o n  of 
moss car rier for b a m b a r a  grou ndn ut.
Since Brachymenium sp. has turned out to be a good c a r r i e r ; 
a survey of its h a b i t a t s  must be c a r r i e d  out. The bes t among  
them can c o n s e q u e n t l y  be c o n s e r v e d  and p r o p e r l y  m a n a g e d  as 
sources for moss m at erial.
Finally, it has been cle a r l y  shown that there are m a n y  
different strains of Rhizobium sp. a s s o c i a t e d  w i t h  b a m b a r a  
gr ou ndn ut in Ghana. The most e f f i c i e n t  among those i s o l a t e d  
during the course of this wo rk  were tested und e r  d i f f e r e n t  
watering regimes and light intens i t i e s .  They may not n e c e s s a r y  
be the best of all the a v a i l a b l e  st ra in s pre s e n t  in G h a n a i a n  
soils, as the c o l l e c t i o n  of soils for the e x p e r i m e n t  was 
limited to an area w i t h i n  a radius of only 180 km from Legon. 
This leaves u n sa mpled the vast areas of the N o r t h e r n  part of 
the c o u n t r y  where b a m b a r a  g r o u n d n u t  is i n t e n s i v e l y  grown* 
Isolation  from all bam b a r a  gr o w i n g  re gi ons in Ghana  s h o u l d  be 
an e s s e n t i a l  future exer cis e. It is only w h e n  the best str a i n s  
have been ide nt i f i e d  for each plant v a r i e t y  that the g r e a t e s t  
benefit can be derived from ba mbar a g r o u n d n u t  seed i n o c u l a t i o n .
207
It may be c o n c l u d e d  that c o m p o s t  of Brachymenium sp. is a 
good Rhizobium ca r r i e r  and can be used for ro ut ine i n o c u l a t i o n  
of barabara g r o u n d n u t  seeds b e f o r e  p l a n t i n g  to e n h a n c e  
nodula tio n. C o m m e r c i a l  p r e p a r a t i o n  of the i n o c u l u m  c a r r i e r  can 
be emb ar ked upon after:
(a) the best strain of Rhizobium sp. for each barabara g r o u n d n u t  
va r i e t y  has been id e n t i f i e d ,  and
(b) the best hab it a t s  of Brachymeniw sp. have been i d e n t i f i e d ,  
and plans for c o n s e r v a t i o n  and c o n t r o l l e d  u t i l i z a t i o n  of 
the species have bee n w o r k e d  out.
208
Strains of Rhizobium sp. we re o b t a i n e d  from n o d u l e s  of 
ba mb ar a g r o u n d n u t  plants w h i c h  w e r e  gr own in soils 
co l l e c t e d  from legume plots at Ash i a m a n ,  Kpong, Legon, 
Nungua, Pokuase, S h i a s h i e  and Weija.
The pH of soils ranged from pH 6,48 to 6.88.
Soils from Leg o n  and Wei j a  we re  s a n d y - c l a y  lo a m y  soils; 
those from A shiam an , Nungua, P o k u a s e  and S h i a s h i e  w e r e  
sandy loam soils; and soil from Kpong, sandy c l a y  soil.
The p e r c e n t a g e  n i t r o g e n  c o n t e n t  of the soils was, in 
as c e n d i n g  order: A s hiaman , 0.025; Shiashie,  0.028; N u n g u a
(plot 2), 0.039; N u n g u a  (plot 1), 0.049; Weija, 0.055;
Kpong, 0.067; Legon, 0.069; and Poku as e, 0.132 per cent.
The mean p e r c e n t a g e  or ga ni c m a t t e r  of the soils was, in 
a s cend in g order: A s hiam an , 0.468; Sh ia shie, 0.578; N u n g u a
(plot 2). 0.977; N u n g u a  (plot 1), 1.472; Legon, 1.733;
Kpong, 2.009; Weija, 2.064; Pok uase, 4. 5 1 3  per cent.
Total viable cell count st u d i e s  u s i n g  C o ng o Red YMA 
indicated the p r e s e n c e  of Rhizobium sp in all the eight 
soil samples. Soils from Kpong, Legon, N u n g u a ( p l o t  1), 
Pokuase and Weija had high p o p u l a t i o n s  of 180 x 104, 138
X 104> 112 X  104> i n  x 104 and 165 x 1 04 cells per g r a m  of
soil, re spectiv ely. Soils from A s h i a m a n ,  N u n g u a ( p l o t  2) 
and Shiashie had ve ry low p o p u l a t i o n s ,  not e x c e e d i n g  20 x 
104 cells per gram of soil.
VI SUMMARY
P l a n t s  of the two v a r i e t i e s  of b a m b a r a  g r o u n d n u t ,  E x - A d a  
and E x - T a m a l e ,  g r o w i n g  in the e i g h t  soil s a m p l e s  c o u l d  be 
s e p a r a t e d  into t h r e e  c a t e g o r i e s  on the b a s i s  of e x t e n t  of 
g r o w t h  and n o d u l a t i o n .
G r oup 1 : L u x u r i a n t l y  g r o w i n g  p lants w i t h  d e e p - g r e e n
foliage and most a b u n d a n t  n o d ules . The pla nt
v a r i e t y  and Rhizobium sp, strain  a s s o c i a t i o n
wer e :
i. E x - A d a  v a r i e t y  and Leg o n  strain
ii. E x-Ada  v a r i e t y  and W e i j a  strain  
iii. E x - T a m a l e  v a r i e t y  and K p o n g  st rain 
iv. E x - T a m a l e  v a r i e t y  and N u n g u a ( p l o t  1) s tr ai n 
v. E x - T a m a l e  v a r i e t y  and P o k u a s e  strain.
Group 2 : Plants w h i c h  g r e w  m o d e r a t e l y  well and n o d u l a t i o n
was i n t e r m e d i a t e .  The plant v a r i e t y  - Rhizobium
sp. s tr ain a s s o c i a t i o n  were:
i. E x- Ada v a r i e t y  and K p o n g  s train
ii. E x - A d a  v a r i e t y  and N u n g u a ( l )  s tr ai n
iii. E x - A d a  v a r i e t y  and P o k u a s e  strain
iv. E x - T a m a l e  v a r i e t y  and L e g o n  strain
v. E x - T a m a l e  v a r i e t y  and W e i j a  strain.
G ro u p  3 : Stunted plants with y e l l o w i s h - g r e e n  f o l i a g e  and
the lowest mean n u m b e r  of n o d u l e s  per plant. 
The plant v a r i e t y  - Rhizobium sp, s train
a s s o c i a t i o n  were:
i, E x - A d a  v a r i e t y  and A s h i a m a n  s trai n
ii, E x - A d a  v a r i e t y  and N u n g u a ( p l o t  2) s t r a i n
iii. E x - A d a  v a r i e t y  and S h i a s h i e  strain
iv. E x - T a m a l e  v a r i e t y  and A s h i a m a n  s train
v. E x - T a m a l e  v a r i e t y  and N u n g u a ( 2 )  s tr ai n
vi. E x - T a m a l e  v a r i e t y  and S h i a s h i e  strain.
The five strains of Rhizobium sp. of Gro up 1 i n f e c t e d  Ex-
Ada var i e t y  plants grown in ?S e e d l i n g  A g a r 1, and ca used 
nodulati on . The pl ants formed  b e t w e e n  28 and 34 n o d u l e s  
per plant, and had b e t w e e n  0.41 and 0.4 9 g  me an shoot dry
weights .
U n d i l u t e d  and \ and V g  d i l u t i o n s  of the b r o t h  c u l t u r e s
of the five s e l ec ted st rai ns of Rhizobium sp. w e r e  used in 
inoculating, i n d i v i d u a l l y ,  sa mples of the g r o u n d  moss 
compost at a ratio of 20ml b r o t h  to lOg c o m p o s t  w h i c h  
brought the m o i s t u r e  c o n t e n t  to 50 per cent.
All the five str ains m u l t i p l i e d  r a p i d l y  in the moss
carrier and cell co unt  of 1 x 1 0 ^  cells per g r a m  of 
compost was r e c orded  after i n c u b a t i o n  at 30°C for two 
weeks. Secondly the in it ial i n o c u l a  of the four d i f f e r e n t  
c o n c e n t r a t i o n s  p r o d u c e d  almost s i m i l a r  p o p u l a t i o n  levels 
at the end of 14 d a y s .
211
11. Shelf life studies on the moss c a r r i e r  i n o c u l a t e d  w i t h  the 
u n d i l u t e d  and ^ - d i l u t e d  br o t h  c u l t u r e s  only, s how ed  
gr adual  increase in p o p u l a t i o n  counts w i t h  all the str a i n s  
of Rhizobium sp. in the moss c a r r i e r  over 10 weeks. The 
ma x i m u m  p o p u l a t i o n  level was t h e r e a f t e r  m a i n t a i n e d  until  
the 14th week w i t h o u t  a u t o l y s i s  s e t t i n g  in.
12. Moss car riers c o n t a i n i n g  the five st rains of Rhizobium sp. 
and gum arabic as a d h e s i v e  were used to i n o c u l a t e  Ex- 
Tamale va ri ety of b a m b a r a  g r o u n d n u t  p lants w h i c h  we re then 
wa tered  eith er once in two days or once in four days or 
once in six d a y s .
13. The plants were raised in st e r i l e  loamy soil and c o n t r o l  
plants were not i n o c ula te d. O b s e r v a t i o n s  m a d e  can be 
su mmari se d as follows:
(a) Co ntr ol  plants did not n o d u l a t e  and w e r e  g e n e r a l l y  
st unted with y e l l o w i s h - g r e e n  leaves,
(b) All the i n o c u l a t e d  plants n o d u l a t e d .
(c) In oc u l a t e d  plants w a t e r e d  once in two days had m e a n  
dry we ights b e t w e e n  1.00 and 1.68g; p r o d u c e d  10 to 15 
leaves per plant and formed 43 to 56 n o d u l e s  per 
plant, after 30 days. The m i d d l e  l e a fl ets m e a s u r e d
a verag el y 5.45 - 8.50 x 2.06 2.79cm.
(d) In oc ulated  plants wa t e r e d  once in four days had m e a n  
dry weights b e t w e e n  0.64 and 0.94g; p r o d u c e d  8 to 12 
leaves per plant; and formed 16 to 25 n o d u l e s  per
plant, after 30 days. The m i d d l e  lea fl et s m e a s u r e d
a v erag el y 4.76 - 7.06 x 1.98 - 2.55cm.
212
(e) Inoculated pl ants w a t e r e d  once in six days had mea n
dry weights b e t w e e n  0.48 and 0.96g; p r o d u c e d  7 to 10 
leaves per plant; and f orm ed  8 to 11 n o d u l e s  per
plant, after 30 days. The m i d d l e  l e a f l e t s  m e a s u r e d  
a v e r a g e l y  4*88 — 6.43 x 1.88 — 2.39cm.
(f) U n i n o c u l a t e d  c o n t r o l  p la nt s also r e s p o n d e d  to 
w a t e r i n g  tr eatmen t.
i. Those w a t e r e d  once in two days had m e a n  dry
w eight  of 0.5 4 g ; and p r o d u c e d  6 leaves per
plant, after 30 days. The m i d d l e  l e a fl ets
m e a s u r e d  a v e r a g e l y  3.36 x 1.57cm.
ii. Those w a t e r e d  once in four days had m e a n  dry
we igh t of 0.3 2 g  and p r o d u c e d  6 leaves per plant, 
after 30 days. The m i d d l e  lea f l e t  m e a s u r e d  
a v e r a g e l y  2.29 x 1.31cm.
iii. Those w a t e r e d  once in six days had m e a n  dry
w eight  of O.llg; and p r o d u c e d  5 leaves per
plant, after 30 days. The m i d d l e  l e a f l e t s
m e a s u r e d  a v e r a g e l y  1.57 x 0.91cm.
14. Moss car riers  c o n t a i n i n g  the five st ra ins of Rhizobium sp. 
and gum arabic as a d h e s i v e  we re used to i n o c u l a t e  E x - A d a  
v a r i e t y  of barabara g r o u n d n u t  pl ants w h i c h  w e r e  then 
ex posed  to light of d i f f e r e n t  i n t e n s i t i e s ,  namely, low, 
m e d i u m  and high light i n tens it ies.
15. The low light i n t e n s i t y  tr e a t m e n t  was 1 1 0 0 - 2 2 0 0  lux at 
9.00am, 4030 6200 lux at noon and 1 6 0 0 - 1 9 0 0  lux at
3.00pm; the m e d i u m  light i n t e n s i t y  tr e a t m e n t  was 1 4 0 0 - 2 8 0 0  
lux at 9.0 0 a m , 64 0 0 - 9 1 0 0  lux at noon and 2 9 0 0 - 3 5 0 0  lux at 
3.00pm; and the high light i n t e n s i t y  tr e a t m e n t  was 5300 — 
6200 lux at 9.00am, 8 8 0 0 - 1 0 0 0 0  lux at noon and 4 6 0 0 - 6 4 0 0  
lux at 3 . 0 0 p m .
1 6 .
17.
A p p r o x i m a t e l y  the same n umbe r of l ea ves w e r e  formed by the 
ino culat ed  plants under the three light i n t e n s i t i e s .  
Other features of the plants, h o w e v e r ,  d i f f e r e d  w i t h  light 
intensity. To summarise :
(a) In oculat ed  plants un d e r  low light i n t e n s i t y  had m e a n  
dry wei g h t s  b e t w e e n  1.62 and 2.91g; and f or med 54 to
78 nod ul es per plant, af ter  30 days. The m i d d l e
leaflets m e a s u r e d  a v e r a g e l y  5 . 60 - 8 . 56 x 2 . 27 -
3,00cm.
(b) Inoc u l a t e d  plan ts un der m e d i u m  light i n t e n s i t y  had 
mean dry w e i g h t s  b e t w e e n  1.33 and 1.8 2 g ; and formed 
42 to 51 n o d u l e s  per plant, after 30 days. The
middle leafl et s m e a s u r e d  a v e r a g e l y  4.91 - 6.07 x
1 . 9 0 - 2 . 66cm.
(c) In oculate d plants under hi gh light i n t e n s i t y  had m e a n  
dry weights b e t w e e n  0.91 and 1 •5 4 g ; and fo rmed 16 to
21 nodules per plant, after 30 days. The m i d d l e
leaflets m e a s u r e d  a v e r a g e l y  4.57 - 5.71 x 1.53
2 . 16cm.
The u n i n o c u l a t e d  co nt rol plants w h i c h  did not n o d u l a t e ,  
however, showed an effect of d i f f e r e n t  light i n t e n s i t i e s  
and n um ber of leaves formed. Bri efly,  in the u n i n o c u l a t e d  
plants :
(a) those under low light i n t e n s i t y  had m e a n  dry w e i g h t
of 0.6 7 g ; and p r o duced 21 leaves per plant, after 30
days. The m i d d l e  lea fle ts m e a s u r e d  a v e r a g e l y  3.00 x
1.40 c m .
-  2 1 3  -
2 1 4
(b) those under m e d i u m  light i n t e n s i t y  had m e a n  dry
weight of 0•5 3 g ; and p r o d u c e d  19 leaves per plant, 
after 30 days. The m i d d l e  l e a flets  m e a s u r e d
a ve r a g e l y  2.20 x 1.19 cm.
(c) those under high light i n t e n s i t y  had m e a n  dry w e i g h t  
of 0.29g; and p r o d u c e d  11 lea ves per plant, a f ter 30 
days. The m i d d l e  l e a flets  m e a s u r e d  a v e r a g e l y  2. 00 x 
0.98 c m .
18. With both light i n t e n s i t y  and soil m o i s t u r e  c o n t e n t  tests, 
the size of no dules was d i r e c t l y  r e l a t e d  to the n u m b e r  of 
no du le s formed. The larger the n u m b e r  of n o d u l e s  per 
plant the larger the n odule size.
A C K N O W L E D G E M E N T
I desire to express my d e e p e s t  a p p r e c i a t i o n  and g r a t i t u d e  
to my supervisor, Prof, G.C. Clerk, a l u m i n a r y  and r e n o w n e d  
Botanist, who su g g e s t e d  this p r o b l e m  and g ui de d me w i t h  ke en  
interest d ur ing the c o u r s e  of this i n v e s t i g a t i o n ,  and for the 
valuable c r i t i c i s m  and s u g g e s t i o n s  d u r i n g  the p r e p a r a t i o n  of 
this thesis.
I am grate ful to Mr, M, De igo  and Mr, F.O.K. Seku for 
t' ir technical ass is tance.
My sincere thanks go to Prof, Y. A h e n k o r a h ,  Dr- Dua- 
Yentumi and Dr, O w u s u - B e n o a h , all of the Soil S c i e n c e  
Department for a l l o w i n g  me to use f a c i l i t i e s  at the D e p a r t m e n t  
for ana ly sing my soil smaples, and to Mr, Julius N a r t e n o r ,  a 
technician of the D e p a r t m e n t  and Mr, A n i p a  of V . R . B . P .  for 
their as si st a n c e  d u r i n g  the analyses , I thank Dr- F, Kuraaga 
and Mr. M, Elegba, both of the Crop S c i e n c e  D e p a r t m e n t ,  for 
their as si stance  and advice. I also thank Mr, D i c k s o n  A b u g a r i  
of the Food and N u t r i t i o n  D e p a r t m e n t  for g r i n d i n g  the moss 
compost into pow der ed form.
I am also grat ef ul to Mr- Anim, the D e p a r t m e n t a l  driver, 
for his as sistan ce  wi th field c o l l e c t i o n  of soil fr om  the 
various loc alities, and to Daniel and Dia n a  C l er k for a s s i s t i n g  
in the co llection, I thank Ben Owusu  Addo, my b r o ther,  in 
a s sist in g me to collect the moss m at erial,
I ac k n o w l e d g e  with sincere thanks the ma j o r  f i n a n c i a l  
support prov id ed by the G o v e r n m e n t  of Ghana  for this work. My 
thanks also go to Messrs O d o i - L a r b i  and O d o i - D a r k o  for their 
support, and to the m a n a g e m e n t  and staff of Kay M i c r o - D a t a  
Services Ltd., Accra, most e s p e c i a l l y  Mr. G o d w i n  Addo and Miss 
Joyce Afo a k w a  for typing and pri nt in g of the m a n u s c r i p t .
-  2 1 5  -
216
Special thanks go to the head of B o t a n y  D e p a r t m e n t ,  
Dr.G.T. Odaratten, and all lec tu res and staff of the D e p a r t m e n t  
for their as sist a n c e  in d i v e r s e  forms.
Finally, I am g r e a t l y  i n d e b t e d  to my wife, Joyce, for her 
prayers, support, and u n d e r s t a n d i n g  d u r i n g  the c o u r s e  of this 
work, and to my parent s,  Mr. S. Addo A m p o f o  and Mrs. M. 
Obiribea Ampofo, for their i n spi ra tion.
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- 226 -
- 227 “
A P P L N O I X  A
PAYS  FOR WATER ING  -  B AMB A R A  G R O U N D N U T  V A R I E T Y  Ex  — T A M A L E
i h T L R v a l D A Y S
M A R C H A P R I L
13 14 15 16 17 I B  19 2 0  21 22 23 24  2 5  26 2 7  20  2 9  3 0  31 1 2 3 4  5 6  7 e  9 I O  I I  12 13 14 15 16 17
, - O i V  ^  
6 - C a Y  [•' |
M  f f l  f f l  f f l  f f l  
□  □  □  E
□  □ □ □ □ □ □ □  
m  m  u  m  
□  □
1
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- 1
228
API’KNDIX H
4
The Prescott Triangle showing the relationship between contents 
of clay, silt and sand in determining the different kinds of 
soil.
- 229 -
A P P E K D I X  C
Distribution of nodule size in two varieties of Bambara 
groundnut plants raised in soils from legume plots from 
different localities for 30 days under normal day/night regime.
(Data for Fig. 2) .
Source Plant
Percentage frequency of 
nodule in class-diameter (mm)
of
Soil
variety
0.56.1.05 1.06-1.55 1.56-2.05 2.06-2. 55 2.56-3.05 3.06-3.5
Kpong Ex-Tamale 8.7 0 10.9 28.3 32.6 19.6
Legon Ex-Ada 0 13.0 71.7 15.2 0 0
Nungua
(i)
Ex-Tamale 17.4 39.1 30.4 10.9 2.2 0
Pokuase Ex-Tamale 0 39.1 34.8 17.4 8.7 0
Wei ja Ex-Ada 2.2 73.9 19.6 4.4 0 0
230
A P P E N D I X  D
D i s t r i b u t i o n  of n o d u l e  size of B a m b a r a  g r o u n d n u t ,  E x - A d a  v a r i e t y ,  
raised on 'Seedling Agar' for 20 days at 2 8°C.
(Data for Fig. 3)
Percentage frequency of
Strain of Rhizobium | nodule in class- diameter (ram)
sp. from nodules of
0.26-0.65 0.66-1.05 1.06-1.45 1.46-1.85 1.86-2.25
Ex-Ada in Legon soil 7.1 46.4 32.1 14.3 0
Ex-Ada in Weija soil 7.1 17.9 39.3 35.7 0
Ex-Tamale in Kpong 
soil 28.6 17.9 32.1 21.4 0
Ex-Tamale in Nungua(l) 
soil
0 10.7 21.4 60.7 7.1
Ex-Tamale in Pokuase 
soil
0 21.4 21.4 46.4 10.7
A P P E N D I X  Et
Growth of five strai ns of Rhizobium sp. in i n o c u l a t e d  moss 
carrier stored at room t e m p e r a t u r e  for 14 weeks*
(Data for Fig, 4)
Strains of |Population of the various strains of
- 231 -
Rhizobium s p . Inoc ulum | Rhizobium sp. after the following
from nodules of concentration number of weeks: (xlO^)
4 5 6 7 8 9
Ex-Ad a in Legon soil Undiluted 252 269 300 380 380 381
\ Dilution 200 208 273 342 348 365
Ex-Ada in Weija soil Undiluted 246 292 311 337 341 368
\ Dilution 220 232 312 356 366 379
Ex-Tamale in Kpong Undiluted 246 250 279 280 280 316
soil
\ Dilution 220 241 247 261 270 270
Ex-Tamale in Nungua(l) Undiluted 224 224 272 301 377 381
soil
\ Dilution 206 225 280 317 325 365
Ex-Tamale in Pokuase Undiluted 226 255 289 331 355 356
soil
\ Dilution 210 229 270 297 340 349
— 232
A P P E N D I X E?
Growth of five st rains  of Rhizobium s p . in i n o c u l a t e d  mo
carrier stored at room t e m p e r a t u r e for 14 w e e k s .
(Data for Fig. 4 ) (C o n t 'd ).
Strains of Population of the various strains o
Rhizobium sp. I n o c u l u m Rhizobium sp. after the following
from nodules of concentration number of weeks: (xl0°)
10 11 12 13 14
Ex-Ada in Legon soil Undiluted 387 392 421 429 429
\ Dilution 389 413 417 417 432
Ex-Ada in Weija soil Undiluted 383 416 417 433 435
\ Dilution 385 392 420 430 442
Ex-Tamale in Kpong Undiluted 318 332 364 365 382
soil
\ Dilution 271 287 293 335 336
E Tamale in Nungua(l) Undiluted 400 405 423 425 427
soil
\ Dilution 373 384 395 408 422
Ex-Tamale in Pokuase Undiluted | 394 396 408 415 425
soil j
*5 Dilution | 380 403 416 418 418
A P P E N D I X  F,
Di s t r i b u t i o n  of n o d u l e  size of B a m b a r a  g r o u n d n u t  p l a n t s  
grown under 2-day w a t e r i n g  int e r v a l s  for 30 days,
(Data for Figs 5 - 9)
- 233 -
Rhizobium
Inoculum
Initial 
density of 
Inoculum
|Percentage frequency of 
|nodule in class-diaraeters 
1 (cm)
»-*
 
o i
o o
1.01-
2.00
2.01-
3.00
3.01-
4.00
4.01-
5.00
5.01-
6.00
f».01-
7.00
Kpong-Ex-Tamale Undiluted 1 7-1 23.2 57.1 12.5 0 0 0
strain \ Dilution | 6.8 30.5 40.7 15.3 5.1 1.7 0
\ Dilution | 3.5 45.6 38.6 12.3 0 0 0
/g Dilution 1 1-7
1
31.0 60.3 1.7 5.2 0 0
Legon-Ex-Ada Undiluted | 32.2 37.3 6.8 16.9 3.4 1.7 1,7
strain \ Dilution | 10.2 45.8 23.7 18.6 1.7 0 0
\ Dilution | 20.3 20.3 44.1 15.3 0 0 0
V g  Dilution | 14.0 
1
31.6 28.1 21.1 5.3 0 0
Nungua(1)-Ex-Tamale Undiluted | 3.8 35.8 18.9 32.1 9.4 0 0
strain \ Dilution | 12.3 13.6 33.3 19.3 3.5 0 0
\ Dilution | 25.9 19.0 25.9 19.0 8.6 1.7 0
V g  Dilution | 39.0 
1
18.6 18.6 3.4 15.3 5.1 0
Pokuase-Ex-Tamale Undiluted 1 5-4 10.7 60.7 19.6 3.6 0 0
strain \ Dilution | 3.4 15.5 70.7 6.9 3.4 0 0
\ Dilution | 5.0 48.3 35.0 10.0 1.7 0 0
V g  Dilution | 14.5 22.6 54.8 8.1 0 0 0
Weija-Ex-Ada Undiluted | 10.2 35.6 15.3 3.22 6.8 0 0
strain \ Dilution | 15.0 23.3 38.3 20.0 3.3 0 0
\ Dilution | 3.6 20.0 50.9 10.9 12.7 1.8 0
/g Dilution | 5.5
J____
52.7 32.7 9.1 0 0 0
-  2 3 4  -
APPENDIX F0.
Di st ri b u t i o n  of n odu le  size of B a m b a r a  g r o u n d n u t  p l a n t s  grown 
under 4-day w a t er in g i n t e r v a l s  for 30 days.
(Data for Figs 5 - 9)
Rhizobium
Inoculum
Initial 
density of 
Inoculum
|Percentage frequency of 
|nodule in class-diameters 
I (cm)
»— 
o
 i
o o
1.01-
2.00
2.01-
3.00
3.01-
4.00
4.01-
5.00
5.01-
6.00
6,01-
7,00
Kpong-Ex-Tamale Undiluted | 6.5 48.4 32.3 12.9 0 0 0
strain \ Dilution | 68.2 31.8 0 0 0 0 0
\ Dilution | 10.3 61.5 28.2 0 0 0 0
V g  Dilution | 81.8 
1
18.2 0 0 0 0 0
Legon-Ex-Ada Undiluted 1 7-4 74.1 14.8 0 3.7 0 0
strain \ Dilution | 70.6 29.4 0 0 0 0 0
\ Dilution | 66.7 33.3 0 0 0 0 0
^/g Dilution | 70.6 
1
23.5 5.9 0 0 0 0
NunguaC1)-Ex-Tamale Undiluted | 15.4 38.5 38.5 7.7 0 0 0
strain \ Dilution | 77.8 16.7 5.6 0 0 0 0
\ Dilution | 91.7 8.3 0 0 0 0 0
V g  Dilution | 80.0
I
10.0 10.0 0 0 0 0
Pokuase-Ex-Taraale Undiluted | 84.0 8.0 8.0 0 0 0 0
strain \ Dilution | 83.3 16.7 0 0 0 0 0
\ Dilution | 53.6 42.9 3.6 0 0 0 0
^/g Dilution | 67.6 
1
32.4 0 0 0 0 0
Weija-Ex-Ada Undiluted | 72.4 27.6 0 0 0 0 0
strain \ Dilution | 61.0 36.6 2.4 0 0 0 0
\ Dilution | 47.7 50.8 1.5 0 0 0 0
^/g Dilution | 75.0 
1
25.0 0 0 0 0 0
- 235 -
APPEHDIX F „
D i s t r i b u t i o n  of n o d u l e  size of B a m b a r a  g r o u n d n u t  pl ants gr o w n  
under 6-day w a t e r i n g  i n t e r v a l s  for 30 days.
(Data for Figs 5 - 9)
Rhizobium
Inoculum
Initial 
density of 
Inoculum
[Percentage frequency of 
|nodule in class-diaraeters 
1 (cm)
►-*
 
o i
jg
1.01-
2.00
2.01-
3.00
3.01-
4.00
4.01-
5.00
5.01-
6.00
6.01-
7,00
Kpong-Ex-Tamale Undiluted | 96.3 3.7 0 0 0 0 0
strain \ Dilution | 100 0 0 0 0 0 1)
\ Dilution | 91.7 8.3 0 0 0 0 0
■^/g Dilution | 91.7
1
8.3 0 0 0 0 0
Legon-Ex-Ada Undiluted | 81.8 8.2 0 0 0 0 0
strain \ Dilution | 75.0 25.0 0 0 0 0 0
\ Dilution | 100 0 0 0 0 0 0
^/g Dilution | 100 
I
0 0 0 0 0 0
Nungua(1)-Ex-Tamale Undiluted | 100 0 0 0 0 0 0
st- ■ in \ Dilution | 90.7 9.3 0 0 0 0 0
\ Dilution | 93.3 6.7 0 0 0 0 0
^ /q Dilution | 93.8
I
6.2 0 0 0 0 0
Pokuase-Ex-Tamale Undiluted | 47.4 42.1 10.5 0 0 0 0
strain \ Dilution | 53.8 38.5 7.7 0 0 0 0
\ Dilution | 100 0 0 0 0 0 0
■^/g Dilution 1 100 
1
0 0 0 0 0 0
Weija-Ex-Ada Undiluted • 03 22.2 0 0 0 0 0
strain \ Dilution | 80.0 20.0 0 0 0 0 0
\ Dilution | 100 0 0 0 0 0 0
■^/g Dilution 00 U1 • "•4 14.3 0 0 0 0 0
- 236 -
APPENDIX G .
Recordings of light intensities during growth of Bambara groundnut plants, Ex- 
Ada variety, under Sheds 1, 2 and 3 on the following dates: (X10) Lux,
(Data for Fig, 10)
Date
9. 00 am 12 ,00 noon -3.00 pm
SHED SHED S H E D ........
1 2 3 1 2 3 1 - 2- 3
10/4/91 160 230 600 410 640 893 183 290 593
12/4/91 129 190 573 573 690 880 190 290 473
14/4/91 213 260 553 556 846 966 183 303 550
16/4/91 129 180 590 410 686 1000 176 290 620
18/4/91 220 280 600 620 853 1000 190 303 620
20/4/91 129 190 590 573 846 966 176 300 610
22/4/91 110 140 530 543 846 966 180 303 460
24/4/91 213 240 600 573 900 1000 180 350 500
26/4/91 160 223 620 403 810 1000 170 300 630
28/4/91 190 210 590 543 846 920 176 310 630
30/4/91 129 190 580 593 900 966 190 350 570
2/5/91 175 230 590 573 830 1000 180 310 600
4/5/91 203 273 600 593 910 1000 190 350 610
6/5/91 200 270 530 580 846 920 180 310 550
8/5/91 110 240 580 550 890 966 190 290 593
10/5/91 160 250 610 600 900 1000 160 .300 640
12/5/91 168 260 600 410 846 1000 190 340 610
14/5/91 160 280 550 550 853 893 180 310 550
- 237 -
APPENDIX H p
D i s t r i b u t i o n  of n odul e size of Barabara g r o u n d n u t  p l a n t s  gro w n  
under Low light in t e n s i t y  for 30 days*
(Data for Figs 11 - 15)
Rhizobium
Inoculum
Initial 
density of 
Inoculum
|Percentage frequency of 
|nodule in class-diameters 
1 (cm)
*-*
 
o
 i
o o
1.01-
2.00
2.01-
3.00
3,01-
4,00
4.01-
5,00
5.01-
6.00
6.01-
7.00
Kpong-Ex-Taraale Undiluted | 2.9 17.1 34.3 28,6 15.7 0 1 .4
strain \ Dilution | 41.4 15.7 14.3 5,7 20.0 2.9 0
\ Dilution | 20.0 20.0 22.9 28,6 2,9 5,7 0
V q Dilution | 30.0
I
41.4 20.0 7,1 0 1.4 0
Legon-Ex-Ada Undiluted | 2.9 31.4 11.4 24,3 25.7 4.3 0
s . ain \ Dilution 1 5-7 32.9 21,4 27,1 8,6 1.4 2.9
\ Dilution | 24,3 21.4 8,6 8,6 11.4 12.9 12.9
V g  Dilution | 2.9 
1
8,6 37,1 28,6 14.3 4.3 4.3
Nungua(1)-Ex-Taraale Undiluted | 22.9 40,0 22,9 5,7 2.9 2.9 2.9
strain \ Dilution | 68.6 5,7 17,1 2.9 5.7 0 0
\ Dilution | 22.9 32,9 24,3 11.4 7.1 1,4 0
V g  Dilution 1 0 7,1 37,1 27.1 20.0 5,7 2,9
Pokuase-Ex-Tamale Undiluted | 27.1 38,6 14,3 14.3 5.7 0 0
strain \ Dilution | 22.9 35,7 20,0 10.0 8.6 1.4 0
\ Dilution 1 *-3 28,6 54,3 10.0 2.9 0 0
/g Dilution 1 1-4
1
40,0 31,4 15.7 8,6 0 0
Weija-Ex-Ada Undiluted 1 4,3 18,6 21,4 24.3 18,6 11.4 1,4
strain \ Dilution | 11,4 15,7 54,3 12,9 2,9 2,9 0
\ Dilution | 2,9 5,7 11,4 47,1 18,6 10,0 4,3
/g Dilution | 22,9 
1
38,6 15,7 14,3 2,9 5,7 0
- 238 -
APPENDIX B q .
Di s t r i b u t i o n  of n o d u l e  size of B a m b a r a  g r o u n d n u t  p l a n t s  gro wn 
under Me dia n light i n t e n s i t y  for 30 days.
(Data for Figs 11 - 15)
Rhizobium Initial 1 Percentage frequency of
density of |nodule in class-diameters
Inoculum Inoculum I (cm)
1 °- 1.01- 2.01- 3.01- 4.01- 5.01- 6.01-
| 1.00 2.00 3.00 4.00 5.00 6.00 7.00
Kpong-Ex-Tamale Undiluted 1 7*1 27.1 18.6 31.4 11.4 4.3 0
strain \ Dilution | 41.4 45.7 7.1 0 4.3 1.4 0
\ Dilution | 27.1 35.7 17.1 7.1 8.6 2.9 0
^/g Dilution 12.9 54.3 22.9 5.7 0 0 0
Legon-Ex-Ada Undiluted | 18.6 21.4 25.6 14.3 11.4 0 0
strain \ Dilution | 24.3 24.3 31.4 8.6 11.4 0 0
\ Dilution 1 5-7 12.9 41.4 22.9 12.9 1.4 0
V g  Dilution 20.0 25.7 45.7 5.7 2.9 0 0
Nungua(1)-Ex-Taraale Undiluted | 20.0 14.3 22.9 18.6 21.4 2.9 0
strain \ Dilution | 60.0 31.4 8.6 0 0 0 0
\ Dilution | 34.3 22.9 14.3 24.3 4.3 0 0
V g  Dilution 51.4 8.6 28.6 11.4 0 0 0
Pokuase-Ex-Tamale Undiluted | 82.9 14.3 2.9 0 0 0 0
strain \ Dilution | 2.9 28.6 54.3 8.6 5.7 0 0
\ Dilution | 11.4 61.4 20.0 5.7 0 0 0
*/g Dilution 2.9 40.0 37.1 17.1 2.9 0 0
Weija-Ex-Ada Undiluted | 45.7 37.1 5.7 11.4 0 0 0
strain \ Dilution 1 5.7 28.6 54.3 5.7 5.7 0 0
\ Dilution | 37.1 34.3 14.3 5.7 2.9 2.9 2,.9
V g  Dilution 14.3 34.3 37.1 14.3 0 0 0
- zjy -
APPENDIX H^»
D i s t r i b u t i o n  of n o d u l e  size of B a m b a r a  g r o u n d n u t  p la nt s grown 
under High light i n t e n s i t y  for 30 days.
(Data for Figs 11 - 15)
Rhizobium
Inoculum
Initial 
density of 
Inoculum
[Percentage frequency of 
|nodule in class-diameters 
I (cm)
1 °“
| 1.00
1.01-
2.00
2.01-
3.00
3.01-
4.00
4.01-
5.00
5.01-
6.00
6. 
7.
01-
00
Kpong-Ex-Tamale Undiluted | 44.3 24.3 17.1 14.3 0 0 0
strain \ Dilution | 80.0 20.0 0 0 0 0 0
\ Dilution | 17.1 35.7 32.9 11.4 2.9 0 0
*/g Dilution | 22.9 45.7 28.6 2.9 0 0 0
Legon-Ex-Ada Undiluted | 37.1 62.9 0 0 0 0 0
strain \ Dilution | 22.9 68.6 8.6 0 0 0 0
\ Dilution 1 5-7 34.3 37.1 20.0 0 2.9 0
V o  Dilution | 65.7 
1
34.3 0 0 0 0 0
Nungua(1)-Ex-Taraale Undiluted | 42.9 14.3 22.9 14.3 5.7 0 0
strain \ Dilution | 97.1 2.9 0 0 0 0 0
\ Dilution | 81.4 10.0 2.9 5.7 0 0 0
^ /o Dilution | 85.7 
1
11.4 2.9 0 0 0 0
Pokuase-Ex-Tamale Undiluted | 88.6 8.6 2.8 0 0 0 0
strain \ Dilution | 22.9 37.1 40.0 0 0 0 0
\ Dilution | 27.1 32.9 40.00 0 0 0 0
/g Dilution | 2.9 
1
31.4 48.6 17.1 0 0 0
Weija-Ex-Ada Undiluted | 30.0 50.0 20.0 0 0 0 0
strain \ Dilution | 45.7 52.9 1.4 0 0 0 0
\ Dilution | 8.6 50.0 18.6 12.9 7.1 2.9 0
/a Dilution I 45.7 14.3 28.6 11.4 0 0 0