Cereal Nitrogen
Fixation
International Crops Research Institute for the Semi-Arid Tropics
The International Crops Research Institute for the Semi-Arid Tropics is a nonprofit scientific educational
institute receiving support from donors through the Consultative Group on International Agricultural
Research. Donors to I C R I S A T include governments and agencies of Australia, Belgium, Canada, Federal
Republic of Germany, Finland, France, India, Italy, Japan, Netherlands, Nigeria, Norway, People's
Republic of China, Sweden, Switzerland, United Kingdom, United States of America, and the following
international and private organizations: Arab Bank for Economic Development in Africa, Asian Development Bank, International Development Research Centre, International Fertilizer Development Center,
International Fund for Agricultural Development, The European Economic Community, The Ford
Foundation, The Leverhulme Trust, The Opec Fund for International Development, The Population
Council, The Rockefeller Foundation, The World Bank, and the United Nations Development Programme. Information and conclusions in this publication do not necessarily reflect the position of the
aforementioned governments, agencies, and international and private organizations.
Cover: Mucigel-secreting brace roots of sorghum grown at ICRISAT Center during the rainy season.
shows an electron micrograph of a nitrogen-fixing bacterium
A z o s p i r i l l u m brasilense (x 19 800).
Inset
Cereal Nitrogen Fixation
Proceedings o f the W o r k i n g G r o u p M e e t i n g
held a t I C R I S A T Center, I n d i a ,
9-12 O c t o b e r 1984
International Crops Research Institute for the Semi-Arid Tropics
Patancheru, Andhra Pradesh 502 324, India
1986
Correct citation: I C R I S A T (International Crops Research Institute for the Semi-Arid Tropics). 1986.
Cereal nitrogen fixation. Proceedings of the Working Group Meeting, 9-12 Oct 1984, I C R I S A T Center,
India. Patancheru, A.P. 502324, India: I C R I S A T .
Workshop
Coordinator
and
Scientific
Editor
S. P. W a n i
The opinions in this publication are those of the authors and not necessarily those of I C R I S A T . The
designations employed and the presentation of the material in this publication do not imply the expression
of any opinion whatsoever on the part of I C R I S A T concerning the legal status of any country, territory,
city, or area, or of its authorities, or concerning the delimitation of its frontiers or boundaries. Where trade
names are used this does not constitute endorsement of or discrimination against any product by the
Institute.
ISBN 92-9066-111-9
Contents
Foreword
Welcome
Address
Inaugural
Address
L.D.
Swindale
C.R.
Jackson
vii
1
J.S.
Kanwar
3
S.P.
Wani
7
N.S.
Subba Rao
Cereal Nitrogen Fixation: Problems
and
Potentialities
Cereal N i t r o g e n F i x a t i o n Research under the B N F
Coordinated
Project o f the I C A R
23
Associative Biological N i t r o g e n F i x a t i o n Research
at Haryana Agricultural University
B.S. K u n d u , K . R . D a d a r w a l ,
and P. T a u r o
Studies
on
the
Interactions
between
31
Azospirillum
and Pearl Millet
A.V.
Rao
and B. Venkateswarlu
37
Response of Pearl Millet to Inoculation w i t h
Azospirillwn
brasilense
at
Varying
Levels
of
Nitrogen
G.S. Jadhav, A . N . Giri,
and N.B.
Pawar
43
Rhizosphere Ecology and Nitrogen Fixation
of
Azospirillum
in
Pearl
Millet
D.
Purushothaman
and K.
Govindarajan
47
Response of S o r g h u m and Pearl Millet Genotypes
to
Azospirillum
and
Azotobacter
Inoculations
C.P. Ghonsikar, R.S. Raut,
and G.B.
Research on Cereal Nitrogen F i x a t i o n at I C R I S A T
S.P.
Rudraksha
Wani
53
55
Yield and Nitrogen Gains of S o r g h u m as Influenced
by
Azospirillum
brasilense
N.S. Subba Rao,
K.V.B.R.
Tilak, C.S. Singh,
and
Azotobacter
Inoculation:
Nitrogen
and Response of S o r g h u m C S H
P.V. N a i r
69
Economy
1
S.T. Shende, G.B. Rudraksha,
Rajani Apte, and R.S. Raut
75
Response of S o r g h u m Cultivars to Inoculation
with
D.
Azospirillum
Purushothaman
and G. Oblisami
N i t r o g e n T r a n s f o r m a t i o n s by A.
brasilense S t r a i n
12S
f r o m Sorghum Roots
Effect
of
brasilense
Azotobacter
Inoculations
chroococcum
and
and
Nitrogen
on
77
Bela S h u k l a a n d B.S. K u n d u
81
B.K. K o n d e and P.A. Shinde
85
Azospirillum
yields
of S o r g h u m , Maize, Pearl Millet, and Wheat
Root-Associated Nitrogen Fixation in Finger Millet
M . N . U p a d h y a y a , S.V. Hegde,
P.V. R a i , a n d S.P. W a n i
93
Heterotrophic Nitrogen Fixation as Influenced
V.
by Fertilizers in Rice-Soil Systems
Rajaramamohan Rao,
J.L.N. Rao, and T.K. Adhya
103
Effect of Certain Organic A m e n d m e n t s and Potassium on
the
Bacterization
of
Rice
with
Azotobacter
Chroococcum
N.N.
Prasad
107
K.R.
Krishna
111
Studies o n Vesicular A r b u s c u l a r M y c o r r h i z a i n Cereals
at I C R I S A T Center (abstract only)
T h e use o f E L I S A ( E n z y m e - L i n k e d I m m u n o s o r b e n t
Assay) f o r Q u a l i t y Assessment of Bacterial
Inoculants (abstract only)
P.T.C
Nambiar
113
Recommendations
115
M e e t i n g Organization and Participants
118
Foreword
A m o n g cereals, s o r g h u m a n d m i l l e t s t o g e t h e r p r o v i d e the highest c a l o r i e i n t a k e i n
A f r i c a n diets a n d r a n k t h i r d i n A s i a . These crops are g r o w n l a r g e l y o n r a i n f e d ,
m a r g i n a l soils b y r e s o u r c e - p o o r f a r m e r s w h o c a n n o t bear the cost o f n i t r o g e n o u s
fertilizers that can boost their yields.
A l t h o u g h a n i n e x h a u s t i b l e store o f n i t r o g e n exists i n the a t m o s p h e r e , m o s t plants
a r e n o t a b l e t o use i t d i r e c t l y . L e g u m e s h a v e b e e n k n o w n t o m a k e use o f t h i s n i t r o g e n
t h r o u g h association o f n i t r o g e n - f i x i n g bacteria, w h i c h f o r m nodules i n the roots o f
these p l a n t s a n d h a v e a s y m b i o t i c r e l a t i o n s h i p w i t h t h e m . I n r e c e n t y e a r s , r e s e a r c h h a s
indicated that certain bacteria l i v i n g near the roots of s o r g h u m a n d m i l l e t can also f i x
the a t m o s p h e r i c n i t r o g e n n o n s y m b i o t i c a l l y a n d p a r t i a l l y meet the n i t r o g e n requirem e n t o f these c r o p s .
C o n s e q u e n t l y , the c o m p l e x process o f n i t r o g e n f i x a t i o n i n s o r g h u m a n d m i l l e t has
a t t r a c t e d the a t t e n t i o n o f m a n y scientists i n I n d i a a n d a b r o a d . T h e studies are s t i l l i n
t h e i r p r e l i m i n a r y stages a n d t h e m e t h o d o l o g y i s s t i l l b e i n g p e r f e c t e d . W i t h a v i e w t o
share experiences, discuss m e t h o d s a n d t e c h n i q u e s , a n d a r r i v e a t a n agreed basis f o r
assessing r e s u l t s , a w o r k i n g g r o u p m e e t i n g w a s c o n v e n e d a t I C R I S A T C e n t e r d u r i n g
9-12 O c t o b e r 1984. A t o t a l o f 3 0 scientists t o o k p a r t i n the d e l i b e r a t i o n s , 1 9 f r o m
Indian institutions and 11 f r o m I C R I S A T .
Meetings such as this play an i m p o r t a n t role, developing interpersonal a n d profess i o n a l r e l a t i o n s h i p s a m o n g the scientists i n v o l v e d , w h i c h c a n lead t o m o r e m e a n i n g f u l
research a n d d e v e l o p m e n t strategies. I a m pleased t h a t I C R I S A T h o s t e d this m e e t i n g
o n c e r e a l n i t r o g e n fixation, a n d I b e l i e v e these p r o c e e d i n g s w i l l be a v a l u a b l e s o u r c e o f
i n f o r m a t i o n and inspiration to all concerned.
L . D . Swindale
Director General
vii
Welcome Address
C . R . Jackson
Ladies and Gentlemen:
W e are pleased t o have y o u here t o d a y . I w a n t t o w e l c o m e y o u o n b e h a l f o f
I C R I S A T , its m a n a g e m e n t , a n d o u r staff t o this i m p o r t a n t w o r k i n g g r o u p m e e t i n g o n
cereal n i t r o g e n f i x a t i o n .
M o s t of my concern as Director of International Cooperation in I C R I S A T is aimed
a t places outside I n d i a , p a r t i c u l a r l y i n A f r i c a . S o this m e e t i n g i s o f p a r t i c u l a r i m p o r tance t o m e , because I believe i t helps a i d the d e v e l o p m e n t o f t e c h n o l o g y t h a t w i l l
m e a n a great deal to I n d i a , to A f r i c a , a n d to a l l countries where the c u l t i v a t i o n of crops
is in some p a r t m a r g i n a l a n d of a subsistence n a t u r e .
A s y o u a l l k n o w , n i t r o g e n i s one o f the m o s t i m p o r t a n t factors l i m i t i n g cereal
p r o d u c t i o n i n t h e w o r l d a n d b i o l o g i c a l n i t r o g e n f i x a t i o n needs t o b e f u l l y e x p l o i t e d .
W e need t o u n d e r s t a n d i t a s w e l l a s possible, b u t n o t t o the e x t e n t t h a t w e u n d e r s t a n d
e v e r y t h i n g b e f o r e w e g o t o t h e f i e l d i n a t t e m p t i n g t o use t h i s p h e n o m e n o n .
T h e r e are m a n y parts o f the w o r l d t h a t are i n d i f f i c u l t y f r o m l a g g i n g f o o d supplies,
g r o w i n g p o p u l a t i o n s , etc. H e r e I w a n t t o e m p h a s i z e the p r o b l e m o f A f r i c a , p a r t i c u l a r l y W e s t A f r i c a , i n a t t e m p t i n g t o feed itself. T h e S a h e l i a n r e g i o n , a n d the r e g i o n a b i t
f u r t h e r S o u t h f r o m the Sahel are i n such great distress t h a t w e m u s t c o n t e m p l a t e the
m o v e m e n t o f p e o p l e o n a c o n t i n e n t a l scale t o p l a c e s w h e r e c o n d i t i o n s a r e b e t t e r f o r
growing food.
I a m g o i n g t o m a k e a n a p p e a l t o y o u t o give y o u r v e r y best t h o u g h t s t o this
p a r t i c u l a r subject o f n i t r o g e n f i x a t i o n i n cereal crops. W e m u s t m a k e early progress i n
this subject. It is n o t j u s t an academic subject. I w a n t to t r a n s m i t to y o u the u r g e n c y of
t h e p r o b l e m o f d e v e l o p i n g t h i s t e c h n o l o g y , s o t h a t i t c a n b e a p p l i e d o n a b r o a d scale
a n d w i t h d e p e n d a b i l i t y i n I n d i a , i n A f r i c a , a n d i n o t h e r parts o f the w o r l d . Y o u m u s t
g i v e y o u r b e s t t h o u g h t t o i t a n d see t h a t i t w o r k s i n t h e f i e l d . Y o u m u s t e x p e n d e v e r y
e f f o r t t o see t h a t s o m e t h i n g u s e f u l f o r h u m a n k i n d c o m e s o u t o f y o u r r e s e a r c h a n d o u t
o f the deliberations o f conferences l i k e this.
We cannot continue to rely solely on plant breeding to give us bigger a n d better
p l a n t s t h a t w i l l g r o w o n less a n d less w a t e r . T h e r e a r e o t h e r f a c t o r s i n t h e e q u a t i o n .
S o i l f e r t i l i t y i s o n e o f t h e m a n d a v e r y i m p o r t a n t o n e . W e c e r t a i n l y n e e d t o p u t t o use
e v e r y i n c r e m e n t o f k n o w l e d g e t h a t w e h a v e , s u c h a s t h e p r a c t i c a l use o f N 2 - f i x i n g
m i c r o o r g a n i s m s , t o g i v e u s a n a d d i t i o n a l i n c r e m e n t o f g r o w t h i n these v e r y h a r s h
e n v i r o n m e n t s where people are t r y i n g desperately to survive.
W i t h those few w o r d s o f encouragement, a n d w i t h the n o t i n g that y o u r p r o g r a m i s
g o i n g to cover some very i m p o r t a n t topics a n d perhaps reveal things never before
revealed to h u m a n k i n d , I w i s h y o u all l u c k in this conference. W e l c o m e . G i v e us y o u r
best efforts.
1
I n a u g u r a l Address
J. S. Kanwar
I t gives m e i m m e n s e pleasure t o e x t e n d m y w a r m w e l c o m e t o the p a r t i c i p a n t s o f this
m e e t i n g , w h o are a c t i v e l y engaged i n research o n N 2 f i x a t i o n b y cereals, specifically
s o r g h u m a n d millets. I feel this m e e t i n g is t i m e l y a n d i m p o r t a n t . S o r g h u m a n d m i l l e t
together p r o v i d e f o o d t o 700 m i l l i o n people o f the semi-arid tropics. T h e y are g r o w n
on 70 m i l l i o n ha, m o s t l y under rainfed conditions, often w i t h o u t nitrogenous fertilizers, a n d b y r e s o u r c e - p o o r f a r m e r s w h o c a n n o t a f f o r d m o n e t a r y i n p u t s . N e a r l y 9 5 % o f
m i l l e t a n d 6 0 % o f s o r g h u m g r a i n i s p r o d u c e d u n d e r e n v i r o n m e n t s w h e r e stress f r o m
l a c k o f m o i s t u r e , n u t r i e n t stress, a n d o t h e r p h y s i c a l a n d b i o t i c stresses s e r i o u s l y
constrain yields.
N i t r o g e n is a key element in increased c r o p p r o d u c t i o n , a n d a n y technique that w i l l
enhance nitrogen n u t r i t i o n o f the crop w i l l go a l o n g w a y i n increasing crop yields. T h e
Green R e v o l u t i o n in wheat a n d rice is n o t h i n g b u t the conversion of chemical energy
supplied t h r o u g h fertilizers i n t o b i o l o g i c a l products where h i g h - y i e l d i n g varieties act
as vehicles of this change. T h e greater the capacity of the variety to respond to heavier
doses o f n i t r o g e n , greater i s the y i e l d o b t a i n e d . T h u s , i t i s n o e x a g g e r a t i o n t h a t the
p r o b l e m o f f o o d deficit a n d m a l n u t r i t i o n i n the w o r l d i s really the p r o b l e m o f n i t r o g e n
starvation of the cereal crops.
Despite the fact t h a t a n i n e x h a u s t i b l e store o f N 2 exists i n the a t m o s p h e r e , m o s t
p l a n t s a r e n o t a b l e t o use i t d i r e c t l y . I n d u s t r i a l p l a n t s a r e c o n v e r t i n g a s m a l l f r a c t i o n o f
available N2 i n t o nitrogenous fertilizers, b u t it is recognized that despite man's
i n g e n u i t y a n d resources, the w o r l d is n o t able to meet m o r e t h a n a f r a c t i o n of the
n i t r o g e n r e q u i r e m e n t o f the crops. T h u s w e have t o l o o k t o other sources, o f w h i c h
biological nitrogen f i x a t i o n ( B N F ) is an i m p o r t a n t one. T h e dependance of fertilizer
n i t r o g e n p r o d u c t i o n o n fossil energy resources a n d the prospects o f d i m i n i s h e d
a v a i l a b i l i t y o f this costly i n p u t f o r fertilizer i n the years t o c o m e have a d d e d a n urgency
t o the study o f B N F .
W e recognize t h a t crops like s o r g h u m a n d millets are very p o o r c o m p e t i t o r s f o r the
a v a i l a b l e f e r t i l i z e r s , a n d m o r e s o u n d e r d r y - f a r m i n g c o n d i t i o n s , because o f the greater
risks i n v o l v e d a n d l o w e r payoff. Thus, for m o r e t h a n 60 m i l l i o n ha of s o r g h u m a n d
m i l l e t g r o w n i n the S A T , any b r e a k t h r o u g h i n B N F c o u l d b r i n g i n a r e v o l u t i o n i n
production.
T h e f i n d i n g b y D o b e r e i n e r o f n o n s y m b i o t i c N 2 f i x a t i o n b y associated bacteria i n
cereals, s t i m u l a t e d research i n t h i s subject a l l o v e r the w o r l d . W i t h the e s t a b l i s h m e n t
o f I C R I S A T , a special emphasis was l a i d o n N 2 - f i x a t i o n studies i n s o r g h u m a n d
m i l l e t s . C o n s i d e r a b l e progress has been m a d e i n o u r u n d e r s t a n d i n g o f this process, b u t
there are n u m e r o u s aspects t h a t need t h o r o u g h i n v e s t i g a t i o n before w e c a n t a k e this
technology t o farmers' fields. T h e standardization o f techniques t o measure N 2
f i x a t i o n i s t h e f i r s t p r o b l e m n e e d i n g a t t e n t i o n . I t i s r e c o g n i z e d t h a t unless r e l i a b l e ,
repeatable, a n d c o m p a r a b l e results are o b t a i n e d b y different w o r k e r s , n o v a l i d c o n c l u sions c a n be d r a w n .
3
A f t e r 1 0 years o f p a i n s t a k i n g research, I C R I S A T m i c r o b i o l o g i s t s are n o w reasonably confident that the methodologies they have developed can give reproducible
results f o r o t h e r scientists as w e l l . W h e t h e r it is a m e a s u r e m e n t of nitrogenase a c t i v i t y
w i t h a c e t y l e n e r e d u c t i o n m e t h o d o n seedlings g r o w n i n test t u b e s o r p o t s , o r
15
N
d i l u t i o n techniques, s i m i l a r results have been c l a i m e d . H o w e v e r , the q u e s t i o n still
r e m a i n s w h e t h e r the results o f the c o n t r o l l e d e n v i r o n m e n t s correlate w e l l w i t h the f i e l d
performance of different genotypes in different environments.
D r W a n i w i l l give y o u the details o f o u r B N F w o r k . I w i l l j u s t m a k e a m e n t i o n o f the
b r o a d objectives:
•
•
to standardize research techniques.
to study relative efficiency of N2 f i x a t i o n by different genotypes of s o r g h u m a n d
millets.
•
to study environmental factors affecting N2 fixation.
•
t o increase N 2 f i x a t i o n b y genetic m a n i p u l a t i o n o f the p l a n t , b y a g r o n o m i c m a n i p u l a t i o n of the c r o p , or by i n o c u l a t i n g w i t h effective bacterial cultures.
I n short, w e are interested i n i n c o r p o r a t i n g the desirable traits o f N 2 f i x a t i o n i n
h i g h - y i e l d i n g varieties a n d developing techniques a n d practices f o r e n h a n c i n g yields of
s o r g h u m a n d millets i n farmers' f i e l d s . O u r studies are n o t restricted t o N , a s w e are
also s t u d y i n g differences in activities of m y c o r r h i z a e associated w i t h different genotypes f o r release o f p h o s p h a t e s a n d its i n t e r a c t i o n s w i t h N 2 f i x a t i o n .
An External P r o g r a m Review panel that evaluated
ICRISAT's
w o r k earlier this
year, while appreciating our w o r k to date on B N F , observed that it is innovative but
s p e c u l a t i v e a n d needs t o b e i n t e n s i f i e d t o test a n d select t h e m o s t e f f e c t i v e p l a n t m i c r o o r g a n i s m associations. W e realize t h a t t o a c c o m p l i s h this task, w e need the
support of many competent microbiologists w o r k i n g under different environments,
using the same m e t h o d o l o g y .
I consider this meeting very t i m e l y as y o u c a n share y o u r experiences w i t h I C R I S A T
scientists a n d d e v e l o p a c o m m o n a p p r o a c h f o r f u r t h e r studies o n N 2 f i x a t i o n b y
s o r g h u m a n d millets. We are n o w entering a second phase in the study of N2 f i x a t i o n
b y cereals. D r W a n i a n d his colleagues have screened some g e r m p l a s m lines a n d rated
t h e m f o r their N 2 - f i x i n g efficiency. H o w e v e r , there is a large g e r m p l a s m resource that
still needs to be screened. S e c o n d l y , we are interested in t r a n s f e r r i n g these u s e f u l traits
t o a g r o n o m i c a l l y s u p e r i o r lines f o r a c h i e v i n g the yield a d v a n t a g e . T h e r e are some
indications t h a t this can be done, b u t we w o u l d like to have some critical studies m a d e
c o o p e r a t i v e l y s o a s t o b e sure a b o u t the f i n d i n g s . T h i s W o r k i n g G r o u p M e e t i n g s h o u l d
help us achieve the f o l l o w i n g objectives :
•
to share k n o w l e d g e a n d experience of n o n s y m b i o t i c N2 f i x a t i o n in s o r g h u m a n d
millet;
•
to accept standardized techniques f o r m a k i n g measurements of N2 fixation;
•
to develop a c o o r d i n a t e d a p p r o a c h f o r c o n d u c t i n g research f o r t r a n s f e r r i n g desirable traits to agronomically superior genotypes; and
•
t o s t u d y the effect o f e n v i r o n m e n t s o n n o n s y m b i o t i c N 2 f i x a t i o n i n cereals.
F o r a c h i e v i n g these objectives, i t i s essential t h a t this m e e t i n g identifies teams o f
scientists w o r k i n g i n d i f f e r e n t institutes t o t a k e u p c o o r d i n a t e d research, w h o agree t o
4
share experiences a n d materials. These should be interdisciplinary teams consisting of
geneticists, m i c r o b i o l o g i s t s , a n d a g r o n o m i s t s / soil scientists. I am c o n v i n c e d t h a t the
high hopes that have arisen—about i m p r o v i n g the nitrogen n u t r i t i o n of s o r g h u m and
millet a n d enhancing their p r o d u c t i o n — c a n be realized o n l y t h r o u g h interdisciplinary
teamwork.
I u n d e r s t a n d that all of y o u assembled here t o d a y are interested in such t e a m w o r k ,
b u t while studying N2 fixation y o u should not forget the interaction of the e n v i r o n m e n t a n d the effect o f o t h e r nutrients i n v o l v e d i n this process. T h e effect o f vesicular
arbuscular mycorrhiza in stimulating N2 fixation through enhanced availability of
phosphates has been observed. W h e t h e r this synergism c a n be enhanced t h r o u g h
m a n a g e m e n t of e n v i r o n m e n t s or genetic engineering is a q u e s t i o n t h a t needs investigat i o n . T h e r e is a l o t of basic w o r k t h a t needs to be d o n e . I find in this g r o u p s o m e
scientists w h o are i n v o l v e d i n such basic studies.
Q u i t e often the experiments are conducted under controlled environments w i t h o u t
a n y m o i s t u r e constraints. W h e t h e r the same results are possible u n d e r c o n d i t i o n s of
d r o u g h t stress a n d h i g h t e m p e r a t u r e needs i n v e s t i g a t i o n . T h e effect o f p h y s i c a l s o i l
e n v i r o n m e n t s a n d i n i t i a l s o i l - f e r t i l i t y level o n N 2 f i x a t i o n also needs t o b e s t u d i e d . I t i s
m y i m p r e s s i o n t ha t there are m o r e u n k n o w n factors t h a n the k n o w n ones i n f l u e n c i n g
n o n s y m b i o t i c N2 fixation by crops like s o r g h u m a n d millets a n d we need to investigate
further.
J u d g i n g f r o m the p r o g r a m t h a t m y colleagues have d r a w n u p f o r this m e e t i n g , I get
the feeling t h a t y o u w o u l d b e l o o k i n g a t a l l these p r o b l e m s . I realize t h a t m a n y o f y o u
are interested i n N 2 f i x a t i o n b y cereals o t h e r t h a n s o r g h u m a n d m i l l e t s . T h e r e are s o m e
papers r e l a t i n g to rice. Since the e n v i r o n m e n t s in w h i c h rice is g r o w n are v e r y different
f r o m those o f s o r g h u m a n d millets, the transferability o f the i n f o r m a t i o n f r o m rice t o
s o r g h u m a n d m i l l e t s a n d vice versa needs i n v e s t i g a t i o n . I t w o u l d b e desirable f o r y o u
t o extend y o u r discussions t o N 2 f i x a t i o n b y s o r g h u m a n d millets u n d e r c o n d i t i o n s o f
d r o u g h t stress a s w e l l . I u n d e r s t a n d t h a t t h e best e s t i m a t e s s h o w t h a t a b o u t 2 0 k g N
ha-1 could be added t h r o u g h bacterial association w i t h sorghum and millets. Even an
increase of 20 kg N ha-1 in all the s o r g h u m - a n d m i l l e t - p r o d u c i n g areas c a n m e a n a
t r e m e n d o u s increase i n the p r o d u c t i o n o f these cereals. W e w i l l b e d o i n g a great service
t o h u m a n i t y i f w e c a n d e m o n s t r a t e this effect o n f a r m e r s ' fields, u n d e r the e n v i r o n m e n t s w h e r e these c r o p s are g r o w n .
It w i l l be a real achievement if we succeed in t r a n s f e r r i n g this t r a i t to a g r o n o m i c a l l y
superior genotypes. I need h a r d l y emphasize that A f r i c a , m o r e t h a n I n d i a , is badly in
need o f this t e c h n o l o g y a n d w e are keenly interested i n the o u t c o m e o f y o u r researches.
I w i s h y o u a l l success i n y o u r d e l i b e r a t i o n s . I h a v e g r e a t p l e a s u r e i n i n a u g u r a t i n g t h i s
Working Group Meeting.
5
Cereal Nitrogen Fixation: Problems and Potentialities
S. P. Wani 1
Summary
Estimates
of
occurs
at
magnitudes
Long-term
estimating
nonsymbiotic
N
the
(ARA)
are
fixation
as
light,
is
of N2 fixed.
in
as
infield
of
bacteria
the
of
work
are
difficult
15
using
techniques
limitations
source
plant
examples.
involved
their performance,
the
plant
results
that
nitrogen
fixation
significance.
although
these
are
indicate
to
measure,
N
directly
are
discussed.
to
their
use
to
are
measure
necessary for
nitrogen fixation
Acetylene-reduction
in
quantification
assays
of nitrogen
studies.
moisture,
with
fixation
Techniques
there
about
through
influence
but
soil
of agronomic
employing
sensitive
illustrated
fixation
studies,
areas
production,
temperature,
Types
activity
be
crop
understanding
fixation
nitrogen
may
involved
well
nitrogen
that
very
Current
associative
balances for
amounts
and problems
and
Possibilities
breeding
and
of
and
of energy for
genotype,
are
methods
such
mode
of
age,
of improving
nitrogen fixation
and the
and
combined nitrogen
the
ability
of cereals
effect
of
on
nitrogen
to
support
discussed.
used
studies.
cereal
plant
to
isolate,
Problems
benefiting
count,
associated
crops from
with
and
test
selecting
inoculations
are
their
bacteria
discussed.
nitrogenase
for
field
Future
highlighted.
Introduction
o f cereal n i t r o g e n f i x a t i o n a n d t o i n d i c a t e the areas
needing further investigation.
N i t r o g e n i s the m o s t l i m i t i n g n u t r i e n t i n f o o d p r o d u c t i o n . T h e b i o l o g i c a l n i t r o g e n cycle ( F i g . 1 ) i s
responsible f o r a t u r n o v e r of 10 8 -10 9 t N a - 1 on e a r t h
i n w h i c h b i o l o g i c a l l y f i x e d N 2 i s one o f the i n p u t s .
M i c r o b i o l o g y of the Association
N o n s y m b i o t i c a n d associative N 2 f i x a t i o n i s c o n s i dered to occur at magnitudes that m a y be of agro-
Since W i n o g r a d s k y (1893) established t h a t C l o s t r i -
n o m i c significance ( D o b e r e i n e r 1978, K n o w l e s 1976,
dium pasteurianum c o u l d
M o o r e 1966, D a r t a n d W a n i 1982, W a n i e t a l . 1984).
Beijerinck
The apparent potential for biological nitrogen fixa-
the
list
(1901)
fix
described
atmospheric
the
first
N2 and
Azotobacter,
o f n i t r o g e n - f i x i n g bacteria has gone o n
t i o n ( B N F ) associated w i t h cereals exceeds its p r e -
i n c r e a s i n g ( B a l a n d r e a u 1983). M a n y d i f f e r e n t g e n -
sent u t i l i z a t i o n , b u t k n o w l e d g e i n t h i s f i e l d i s n o t
era a n d strains o f N 2 - f i x i n g bacteria c a n b e i s o l a t e d
e n o u g h t o e x p l o i t these associations f u l l y . T h e r e
f r o m the s o i l a n d the r o o t s . T h e d i f f i c u l t y i n s t u d y i n g
a p p e a r t o b e m a n y ways o f i n c r e a s i n g t h e c o n t r i b u -
the e c o l o g y o f N 2 - f i x i n g b a c t e r i a i s i n d e v i s i n g selec-
t i o n f r o m cereal n i t r o g e n f i x a t i o n . T h e a i m o f t h i s
t i v e m e d i a a n d n e w i s o l a t i o n procedures t o c o u n t the
r e v i e w i s t o evaluate the p r o b l e m s a n d p o t e n t i a l i t i e s
populations of particular organisms. Each labora-
1.
Microbiologist, Pearl Millet Improvement Program, I C R I S A T , Patancheru, A.P. 502 324, India.
I C R I S A T (International Crops Research Institute for the Semi-Arid Tropics). 1986. Cereal nitrogen fixation. Proceedings of the Working
Group Meeting, 9-12 Oct 1984, I C R I S A T Center, India. Patancheru, A.P. 502 324, India: I C R I S A T .
7
N2
Denitrification
Fixation
Protein
in
Ammonification
NO-3
NH3
organisms
Nitrification
NO-2
Assimilation
F i g u r e 1. T h e nitrogen cycle.
t o r y uses a p a r t i c u l a r set o f t e c h n i q u e s f o r g r o w i n g ,
s a m p l i n g , distance o f r o o t s f r o m t h e c r o w n , a n d
isolating, a n d c o u n t i n g N 2 - f i x i n g bacteria. Conse-
d i f f i c u l t y in recovering all the roots f r o m the soil.
q u e n t l y , e a c h l a b o r a t o r y has a t e n d e n c y t o c o n s i d e r
H o w e v e r , expressing t h e n u m b e r o f b a c t e r i a p e r u n i t
t h a t its o w n b a c t e r i u m has a d o m i n a n t r o l e i n N 2
mass o f a b o v e - g r o u n d p l a n t p a r t s p r o d u c e d m a y
fixation.
give
a
better
understanding
and
uniformity
in
expression.
Isolation and Enumeration
of N2-fixing Bacteria
Testing for N2-fixing Efficiency
T o o v e r c o m e t h e p r o b l e m o f selective c a r b o n s o u r c e
Sometimes reports of new organisms capable of fix-
in the m e d i u m as far as rhizosphere bacteria are
i n g a t m o s p h e r i c n i t r o g e n are l a t e r p r o v e d t o b e
c o n c e r n e d , use o f c a r b o n sources s i m i l a r t o t h o s e
u n t r u e because t h e c u l t u r e u n d e r test m a y n o t b e
present i n t h e r h i z o s p h e r e , i.e, r o o t e x u d a t e s , w o u l d
p u r e a n d even s l i g h t c o n t a m i n a t i o n b y a N 2 - f i x i n g
b e better. Use o f t h e ' s p e r m o s p h e r e m o d e l ' i s p r o m i s -
organism could
be sufficient to indicate fixation.
i n g f o r counting and isolating N 2 - f i x i n g bacteria
G r o w t h on a N-free m e d i u m is n o t a sufficient criter-
( T h o m a s - B a u z o n e t a l . 1982).
i o n f o r n i t r o g e n a s e a c t i v i t y ( H i l l a n d P o s t g a t e 1969)
A n o t h e r d i f f i c u l t y i n c o m p a r i n g t h e results o f v a r -
as some of the N-scavenging bacteria can g r o w on
ious groups is the w a y of expressing the n u m b e r of
traces o f N present i n t h e m e d i u m a n d s o m e N 2 -
b a c t e r i a . T h e s e are g e n e r a l l y expressed p e r g of
fixing bacteria cannot g r o w on a m e d i u m completely
rhizospheric soil. However, there is no clear d e f i n i -
free o f c o m b i n e d N ( W a t a n a b e a n d B a r r a q u i o 1979).
t i o n o f r h i z o s p h e r i c s o i l . E x p r e s s i n g t h e results p e r
The
15
N 2 i n c o r p o r a t i o n , even t h o u g h a d e f i n i t i v e test
u n i t mass o f r o o t does n o t solve t h e p r o b l e m e i t h e r ,
t o m e a s u r e N 2 f i x a t i o n i s t o o e x p e n s i v e a n d gener-
c o n s i d e r i n g t h e d i f f e r e n t types o f r o o t s f o u n d o n t h e
a l l y C 2 H 2 r e d u c t i o n a s a n i n d i r e c t assay t e c h n i q u e i s
s a m e p l a n t , s a m p l i n g m e t h o d , age o f t h e p l a n t a t
u s e d . T h i s t e c h n i q u e poses p r o b l e m s d u e t o t h e s h o r t
8
exposure p e r i o d t o C 2 H 2 r e d u c t i o n a n d also due t o
adsorption
o x i d a t i o n o f C 2 H 4 b y s o m e b a c t e r i a ( K n o w l e s 1981).
fluorescens.
M a n y N 2 - f i x i n g b a c t e r i a express n i t r o g e n a s e a c t i v -
and
ity o n l y w h e n they are i n sufficient numbers ( H a u k e -
tetrazolium-reducing bacteria in the roots of maize,
of
Rhizobium
Electron
millet
roots
trifolii
and
micrographs
and
optical
Pseudomonas
of P.
maximum
micrographs
of
P a c e w i c z o w a e t a l . 1970, B r o u z e s e t a l . 1971) a n d i n
w h e a t , a n d s o r g h u m suggest t h a t i n f e c t i o n o f t h e
enumeration experiments w i t h high dilutions this
c o r t e x a n d stale o f these r o o t s b y Azospirillum i s a t
c a n t a k e as l o n g as 3 weeks ( V i l l e m i n et a l . 1974). T h e
t h e p o i n t o f emergence o f l a t e r a l r o o t s ( P a t r i q u i n
established n i t r o g e n a s e a c t i v i t y c a n b e suppressed
a n d D o b e r e i n e r 1978; U m a l i - G a r c i a e t a l
by carbon or oxygen limitation so that the overall
1980; M a g a l h a s e e t a l . 1979). B a c t e r i a h a v e b e e n
period f o r expression of activity is very short. This
o b s e r v e d i n t o r n o r d i s r u p t e d r o o t cells ( S c h a n k e t
difficulty can be overcome by waiting long enough
a l . 1983) a n d also o b s e r v e d i n t e r c e l l u l a r l y b u t n o t
for
each
tube
containing
N2-fixing
bacteria
to
1978,
w i t h i n l i v i n g r o o t cells ( U m a l i - G a r c i a e t a l . 1980).
d e v e l o p a s u f f i c i e n t l y large p o p u l a t i o n a n d t h e n
a d d i n g fresh m e d i u m , a n d i n c u b a t i n g under C 2 H 2
( V i l l e m i n e t a l . 1974). A n o t h e r p o s s i b i l i t y i s t o i n c u bate t h e r e p l i c a t e tubes u n d e r 1 % C 2 H 2 a s s o o n a s
Estimates of Nonsymbiotic and
t h e y are i n o c u l a t e d ( B a l a n d r e a u 1983).
Associative N 2
Fixation
S o m e o f t h e m o s t - c o n v i n c i n g evidence t h a t n o n s y m -
Identification
of Bacteria
biotic nitrogen
fixation
may be important under
f i e l d c o n d i t i o n s has c o m e f r o m n i t r o g e n - b a l a n c e
A d e t a i l e d i d e n t i f i c a t i o n of b a c t e r i a i s o l a t e d is g e n -
studies.
erally overlooked by soil microbiologists. As i n d i -
Rothamsted, England estimated nonsymbiotic nit-
cated
r o g e n f i x a t i o n u p t o 18-20 k g N h a - 1 a - 1 i n p l o t s
by
Balandreau
(1983)
"many
soil
The
long-term
continuously
the
receiving no nitrogen fertilizer, a n d m o r e t h a n 39 kg
generic level based o n c o m m o n tests, e.g., o r g a n i s m s
N ha - 1 in p l o t s left to d e v e l o p n a t u r a l v e g e t a t i o n
f o r m i n g a pellicle in malate semisolid m e d i u m are
( J e n k i n s o n 1977, W i t t y e t a l . 1977) ( T a b l e 1).
identified
up
to
w h e a t since
1943
at
Sometimes
are
to
studies
m i c r o b i o l o g i s t s are n o t v e r y k e e n o n t a x o n o m y " .
organisms
cropped
N-balance
and
c a l l e d Azospirillum, w h e n there a r e o t h e r N 2 - f i x i n g
N i t r o g e n - b a l a n c e studies a r e a l s o a v a i l a b l e i n t h e
b a c t e r i a such a s some m e m b e r s o f e n t e r o b a c t e r i a -
tropics. In the old long-term, permanent-manurial
ceae a n d species o f Pseudomonas c a p a b l e o f f o r m -
e x p e r i m e n t a t C o i m b a t o r e , I n d i a , t h e r e was a net
i n g pellicle i n s e m i s o l i d m a l a t e m e d i u m . E i t h e r w a y ,
gain of N in b o t h c o n t r o l (no fertilizer) plots and
workers should not overlook detailed t a x o n o m y and
plots w i t h N- and P- fertilizer application ( K r i s h n a -
should avoid n a m i n g u n c o n f i r m e d cultures (Balan-
m o o r t h y a n d R a v i k u m a r 1973). S e v e r a l p o t e x p e r i -
d r e a u 1983).
ments w i t h s o r g h u m , pearl millet, finger millet
Occurrence of N2-fixing Bacteria
T a b l e 1 . S o m e estimates o f nitrogen f i x e d i n association
w i t h cereals a n d grasses based on N balance.
Large populations of heterotrophic bacteria capable
o f g r o w i n g o n N - f r e e m e d i a exist i n soils o f t h e
Nitrogen fixed
Crop
( k g N ha-1 a - 1 )
Reference
s e m i - a r i d t r o p i c s ( W a n i , i n press). I n g e n e r a l , m u l t i p l i c a t i o n o f s u c h b a c t e r i a a s w e l l a s selective p r o l i f e r a t i o n o f p a r t i c u l a r types occurs i n t h e r h i z o s p h e r e .
Nitrogen-fixing
bacteria
have
been observed
Maize
11.2
Wheat
18-23
D a r t a n d D a y 1975
Rice
30-60
F i r t h e t a l . 1973
to
K o y a m a a n d A p p 1979
a d h e r e v e r y closely t o t h e r o o t s a n d c o n s i d e r a b l e
n u m b e r s w e r e o b t a i n e d f r o m r o o t pieces s u r f a c e sterilized in 1% c h l o r a m i n e T f o r 1 h ( D a r t a n d W a n i
W a l c o t t e t a l 1977
Legume-free
grass s o d
1982). W h e t h e r t h e a s s o c i a t i o n b e t w e e n b a c t e r i a a n d
Grasses
plant roots is external or the bacteria invade the r o o t
Noncultivated
tissues i s n o t c l e a r l y k n o w n . U m a l i - G a r c i a e t a l .
(1980) f o u n d t h a t a d s o r p t i o n o f t h r e e s t r a i n s o f A .
brasilense t o m i l l e t r o o t h a i r s was b e t t e r t h a n t h e
S m i t h e t a l . 1954
(legume-free)
R y e grass
Finger millet
34
S m i t h e t a l . 1954
45
W h i t e e t a l . 1945
49
D a r t a n d D a y 1975
63
P a r k e r 1957
112-148
M o o r e 1963
9
( E l e u c i n e coracana), a n d N a p i e r b a j r a 21 ( P e n n i s e tum purpureum
x
P.
americanum) h a v e s h o w n s u b -
I n t h e t r o p i c s , h i g h rates o f d i n i t r o g e n f i x a t i o n
associated w i t h grasses have been r e p o r t e d . F o r
ha - 1
a-1
s t a n t i a l p o s i t i v e balances f o r N ( D a r t a n d W a n i
e x a m p l e , 90 kg N
1982; W a n i , i n press; U p a d h y a y a e t a l . 1986). A
F l u g g e ( D o b e r e i n e r e t a l . 1972), 2 k g N h a - 1 d - 1 w i t h
w i t h Paspalum notatum
p o s i t i v e N b a l a n c e o v e r a 4 - m o n t h p e r i o d f o r the s o i l
Zea mays L . ( v o n B u l o w a n d D o b e r e i n e r 1975), 3 - 6 3
p l a n t system i n p o t e x p e r i m e n t s w i t h f i n g e r m i l l e t
k g N h a - 1 season - 1 w i t h f l o o d e d rice ( Y o s h i d a a n d
was f o u n d , w h i c h e x t r a p o l a t e d t o a g a i n o f 1 1 2 - 1 4 8
A n c a j a s 1973), a n d 70 kg N ha - 1 a - 1 a l s o w i t h rice
k g N h a - 1 ( M o o r e 1963). S i m i l a r l y , p o s i t i v e N b a l -
( B a l a n d r e a u et a l . 1976). T h e highest rates (2 kg N
ance f o r f l o o d e d soils i n p o t s p l a n t e d t o rice h a v e
ha-1 d-1) of d i n i t r o g e n f i x a t i o n in the tropics were
been r e p o r t e d ( A p p e t a l . 1980). T h e s u b s t a n t i a l
o b t a i n e d b y u s i n g p r e i n c u b a t e d e x c i s e d - r o o t assays
c o n t r i b u t i o n o f B N F t o t h e N e c o n o m y o f t h e rice
( v o n B u l o w a n d D o b e r e i n e r 1975, D o b e r e i n e r 1978).
c r o p is w e l l d o c u m e n t e d .
T h e s h o r t f a l l s i n this assay m e t h o d a r e discussed
Azolla-Anabaena associa-
t i o n / b l u e - g r e e n algae, a n d p h o t o s y n t h e t i c b a c t e r i a
account f o r substantial contributions to total N
under A R A methods.
T h e r e are f e w r e p o r t s ( T a b l e 3 ) i n d i c a t i n g i n c o r -
i n p u t f o r t h e rice c r o p ( V e n k a t a r a m a n 1975, W a t a -
poration of
n a b e 1 9 8 1 , S i n g h 1981). A t t h e I n t e r n a t i o n a l R i c e
unequivocal
15
N 2 i n t o cereal p l a n t s t h a t p r o v i d e a n
p r o o f of biological nitrogen fixation
Research Institute ( I R R I ) , 23 rice crops were g r o w n
( R u s c h e l e t a l . 1975, D e - P o l l i e t a l . 1977, I t o e t a l .
o v e r 1 1 years w i t h o u t a d d i t i o n o f N w i t h n o a p p a r -
1980, G i l l e r et a l . 1984). T h e r e is a need to c o l l e c t
-1
m o r e d a t a i n the t r o p i c s t o e s t i m a t e t h e n i t r o g e n
c r o p - 1 were removed t h r o u g h g r a i n a n d straw ( W a t -
f i x a t i o n w i t h d i f f e r e n t cereals. T h i s c a n b e a c h i e v e d
ent d e c l i n e in s o i l N f e r t i l i t y . A b o u t 4 5 - 6 0 kg N ha
a n a b e a n d L e e 1977). U s i n g
15
N2 incorporation by
r h i z o s p h e r i c s o i l , i t has been d e m o n s t r a t e d t h a t r h i -
by c o n d u c t i n g l o n g t e r m N-balance trials in the field
a n d also by
15
N 2 i n c o r p o r a t i o n studies.
zospheric soil fixed f o u r - f o l d higher nitrogen t h a n
the nonrhizospheric soil. T h e
15
N2 incorporation in
t h e r h i z o s p h e r e also v a r i e d s i g n i f i c a n t l y d e p e n d i n g
M e t h o d o l o g y for Measurement of
o n t h e v a r i e t y ( C h a r y u l u e t a l . 1981).
N2
Fixation
G e n e r a l l y , l o w rates o f d i n i t r o g e n f i x a t i o n ( < 6 k g
N h a - 1 season - 1 ) have been r e p o r t e d f o r grasses, i n
T e c h n i q u e s used f o r m e a s u r e m e n t o f n i t r o g e n f i x a -
t e m p e r a t e c l i m a t e s ( T a b l e 2 ) ( N e l s o n e t a l . 1976,
t i o n associated w i t h f i e l d - a n d p o t - g r o w n p l a n t s c a n
T j e p k e m a a n d B u r r i s 1976, Pedersen e t a l . 1978).
b e b r o a d l y classified a s d i r e c t a n d i n d i r e c t .
H i g h e r f i x a t i o n rates o f u p t o 3 3 k g N h a - 1 i n 100
days f o r Cynodon dactylon ( L . ) pers. w e r e r e p o r t e d
f r o m T e x a s ( W e a v e r e t a l . 1980). F r o m t h e O r e g o n
Direct Techniques
w e t l a n d s Juncus ballicus p l a n t s w e r e r e p o r t e d to fix
u p t o 0.8 k g N h a - 1 d - 1 ( T j e p k e m a a n d E v a n s 1976).
T o t a l N by Kjeldahl analysis.
T o t a l - N measure-
T a b l e 2 . S o m e estimates o f nitrogen f i x e d , based o n acetylene-reduction activity ( A R A ) .
Plant
Period
kg N
Grasses i n C a l i f o r n i a
Season
<6
Oklahoma Oregon
ha-1
Reference
S t e y n a n d D e l w i c h e 1970
K a p u s t k a a n d R i c e 1978
Wisconsin and New Zealand
L i n e a n d L o u t i t 1973
Wheat and sorghum in Nebraska
N e l s o n e t a l . 1976
T j e p k e m a a n d B u r r i s 1976
P e d e r s e n e t a l . 1978
Sporobolis
heterolepsis
Cynodon
Juncus
dactylon
balticus
Paspalum
Zea
Oryza
notatum
mays
sativa
Pasture soils
10
Year
100 D a y s
Day
Season
Day
Season
9
33
0.8
90
T j e p k e m a a n d B u r r i s 1976
W e a v e r e t a l . 1980
T j e p k e m a a n d E v a n s 1976
D o b e r e i n e r e t a l . 1972
2
v o n B u l o w a n d D o b e r e i n e r 1975
3.6
Y o s h i d a a n d A n c a j a s 1973
Year
70
B a l a n d r e a u e t a l . 1976
Year
32
K o c h a n d O y a 1974
Table 3. Incorporation of
I 5
N 2 b y nonlegumes.
-1
% Ndfa
Incubation
N fixed (µg p l a n t )
time
Shoot
Root
D.decumbens
78 h
0.01
0.12
.
P.
30 h
0.001
0.02
-
Crop
S.
0.
S.
P.
notatum
officinarum
Soil
Shoot
Root
Soil
Reference
1
7
-
D e - P o l l i e t a l . 1977
0
1
-
D e - P o l l i e t a l . 1977
R u s c h e l e t a l . 1975
30 h
4.66
4.14
160
52
-
24 h
0.001
0.03
-
0
3
-
R u s c h e l et a l . 1978
72 h
0.15
0.27
-
124
46
-
R u s c h e l e t a l . 1981
I t o et a l . 1980
sativa
vulgare
americanum
7d
3.26
1.43
-
961
1005
-
7d
0.08
0.37
0.07
65
44
510
3d
0.003
0.35
0.01
6
95
16
E s k e w e t a l . 1981
3d
0.406
0.242
-
33
13
-
G i l l e r e t a l . 1984
3d
0.05
0.089
-
1.4
1.1
-
2.0
1.5
6.01
Y o s h i d a a n d Y o n e y a m a 1980
Grown further
5d
ments w i t h the K j e l d a h l m e t h o d w i t h small subsam-
bance o f r h i z o s p h e r i c i n t e g r i t y a n d u n r e p r e s e n t a t i v e
pies of a p a r t i c u l a r system enable N - a c c r e t i o n to be
n i t r o g e n u p t a k e b y the p l a n t . These p r o b l e m s have
However, as the K j e l d a h l
been o v e r c o m e r e c e n t l y w i t h a s i m p l e , i n e x p e n s i v e
analysis does n o t d i s t i n g u i s h N f r a c t i o n w i t h i n t h e
apparatus developed at I C R I S A T Center f o r expos-
t o t a l , i t i s essential t o c o n s t r u c t a n N - b a l a n c e sheet
i n g the p l a n t s t o
for estimating N input f r o m N2 fixation. Under field
c a n b e used t o establish the r a t i o o f C 2 H 2 r e d u c t i o n
c o n d i t i o n s such e x p e r i m e n t s are d i f f i c u l t t o c o n d u c t ,
to N2
a s t h e y need t o r u n f o r m o r e t h a n o n e season a n d
ments.
d e t e r m i n e d w i t h ease.
1 5
fixation
It
is
N 2 ( G i l l e r e t a l . 1984). T h i s m e t h o d
b u t i s n o t relevant t o f i e l d e x p e r i obviously
difficult
1 5
extrapolate
r e q u i r e a r i g o r o u s s a m p l i n g o f t h e s o i l i f t h e y are t o
amounts o f nitrogen f i x e d from
r e l i a b l y measure s o i l - N changes of 2 0 - 5 0 kg N ha - 1
studies o v e r a s h o r t p e r i o d t o a m o u n t s f i x e d o n a p e r
a-1
p l a n t o r p e r hectare basis.
( V a l l i s 1973). A l s o , e s t i m a t i o n o f N 2 loss b y
2
incorporation
T h e above l i m i t a t i o n can be overcome using the
denitrification is difficult under field conditions,
a l t h o u g h i t i s believed t o b e s m a l l u n d e r n o r m a l f i e l d
N
to
15
N isotope-dilution technique. Using this technique
s i t u a t i o n s w i t h l o w doses o f N f e r t i l i z e r a p p l i c a t i o n s
lines o f s o r g h u m a n d m i l l e t g r o w n i n p o t s c o n t a i n i n g
( G r e e n l a n d 1962). T h e l y s i m e t e r , a l t h o u g h a dis-
v e r m i c u l i t e were screened f o r t h e i r p o t e n t i a l t o f i x
t u r b e d system, enables m e a s u r e m e n t s o f N-accre-
N 2 ( G i l l e r e t a l . , 1986). I n s u c h e x p e r i m e n t s , e x t r a
t i o n a n d loss w i t h m o r e p r e c i s i o n . H o w e v e r , i t i s
care has t o b e t a k e n t o p r e v e n t t h e systems f r o m
d i f f i c u l t to regulate the water content of lysimeters,
getting contaminated w i t h
because the s o i l w i t h i n the l y s i m e t e r i s d e t a c h e d
such a s w a t e r o r the g r o w t h m e d i u m .
I 4
N f r o m o t h e r sources,
A n a l t e r n a t i v e m e t h o d t o measure N 2 f i x a t i o n
f r o m the w a t e r t a b l e .
w o u l d b e t o d e t e r m i n e differences i n n a t u r a l a b u n T h e use of N isotopes.
T h e use of i s o t o p e
l 3
N is
r e s t r i c t e d because o f its s h o r t h a l f - l i f e o f 1 1 m i n . T h e
stable i s o t o p e
15
N is preferred f o r measuring n i t r o -
dance o f
I 5
N a r i s i n g f r o m mass d i s c r i m i n a t i o n
effects r e s u l t i n g f r o m N 2 f i x a t i o n , N H 4 a s s i m i l a t i o n ,
and
I 5
N t r a n s p o r t . B u t a s t h e δ I 5 N has been r e p o r t e d
g e n f i x a t i o n . I t has been used w i t h sugarcane, t r o p i -
to vary considerably w i t h soil depth (Karamanos
c a l grasses, a n d r i c e , u s i n g c h a m b e r s t o enclose b o t h
a n d R e n n i e 1980), its use t o d e t e r m i n e N 2 f i x a t i o n
t h e p l a n t s a n d g r o w t h m e d i a ( R u s c h e l e t a l 1975,
may be limited.
D e - P o l l i e t a l . 1977, I t o e t a l . 1980). M a j o r d i f f i c u l ties w i t h s u c h e x p e r i m e n t s are t h e e n c l o s u r e o f
p l a n t s a n d changes i n e n v i r o n m e n t a l c o n d i t i o n s
Indirect Techniques
w i t h t h e necessary l o n g - t e r m i n c u b a t i o n s r e q u i r i n g
complex control equipment. Incubation chambers
A c e t y l e n e - r e d u c t i o n assays ( A R A ) .
have also been e v a c u a t e d t o r e m o v e t h e a i r b e f o r e
reduces n u m e r o u s c h e m i c a l analoges o f n i t r o g e n ,
Nitrogenase
i n t r o d u c t i o n o f gas m i x t u r e ( D e - P o l l i e t a l . 1977;
small molecules c o n t a i n i n g a t r i p l e b o n d ( T a b l e 4).
R u s c h e l e t a l . 1975, 1981). T h i s c o u l d l e a d t o d i s t u r -
A l l b i o l o g i c a l d i n i t r o g e n - f i x i n g systems tested t o
11
T a b l e 4. S o m e substrates reduced by nitrogenase.
Substrate
Products
Dinitrogen
N=N
NH3
Ammonia
Acetylene
HC=CH
H2C=CH2
Ethylene
Hydrogen cyanide
H-C=N
CH4+NH3
Methane,
M e t h y l isocyanide
CH3-N=C
CH3NH2+CH4
Methylamine,
H y d r o g e n azide
H-N-N=N
NH3+N2
A m m o n i a , nitrogen
Nitrous oxide
N=N-O
N2+H20
Nitrogen,
Hydrogen ion
H30+
H2+H20
Hydrogen,
ammonia
methane1
water
water
1. Ethylene, ethane, and propylene formed as minor products.
A l l reductions require A T P . For more detailed discussion and references, see Postgate (1972).
date have also reduced acetylene to ethylene. T h e use
o f f l a m e i o n i s a t i o n detector gas c h r o m a t o g r a p h y t o
measure the ethylene p r o d u c e d was first p r o p o s e d
b y H a r d y a n d K n i g h t (1967). T h e A R A i s a s i m p l e
b u t i n d i r e c t m e t h o d t o test nitrogenase a c t i v i t y ( B e r gersen 1970). T h e A R A i s a b o u t 10 3 times m o r e
sensitive t h a n I 5 N techniques a n d 10 6 t i m e s m o r e
sensitive t h a n the K j e l d a h l m e t h o d . E t h y l e n e c a n b e
separated c o m p l e t e l y f r o m C 2 H 2 , C H 4 , a n d a l l o t h e r
gases a n d C 2 H 2 a n d C 2 H 4 are easily a n d r a p i d l y
detected u s i n g gas c h r o m a t o g r a p h y . A s the A R A
does n o t measure transfer o f f i x e d N 2 f r o m the d i a z o t r o p h t o t h e associated c r o p p l a n t , i t c a n o n l y
i d e n t i f y w h e t h e r o r n o t nitrogenase a c t i v i t y i s p r e sent in a p a r t i c u l a r system. E x p e r i m e n t s w i t h 1 5 N are
s t i l l necessary t o d e m o n s t r a t e t h a t a g r i c u l t u r a l crops
d e r i v e s i g n i f i c a n t benefit f r o m N 2 f i x a t i o n . B e i n g a n
i n d i r e c t assay, t h e m a j o r d i f f i c u l t y w i t h the A R A i s
i n quantifying the amounts o f N 2 fixed over time.
The ratio of C 2 H 2 : N 2 reducing activity cannot be
assumed w i t h m u c h accuracy w i t h o u t a c t u a l tests
and is seldom experimentally determined (Knowles
1981). T h e o r e t i c a l l y , three moles o f C 2 H 2 are
reduced p e r m o l e o f N 2 reduced, h o w e v e r , C 2 H 2 : N 2
ratios v a r y i n g f r o m 1.5 to 6.9 f o r different systems
have been r e p o r t e d (Bergersen 1970, K n o w l e s 1981).
T h e s o l u b i l i t i e s o f C 2 H 2 a n d N 2 i n w a t e r are different
w h i c h makes i t d i f f i c u l t t o i n t e r p r e t the C 2 H 2 data.
P r o b l e m s w i t h the A R A can also b e e n c o u n t e r e d
w h e n l o w N 2 - f i x a t i o n rates are measured i n soils.
E t h y l e n e p r o d u c e d b y a n a e r o b i c bacteria c a n overestimate N 2 f i x a t i o n , a n d b a c t e r i a l o x i d a t i o n o f ethylene c a n reduce estimates o f f i x a t i o n (de B o n t 1976,
H a r v e y a n d U n s c o t t 1978, N o h r s t e d t 1975, W i t t y
1979). T h e p r o b l e m o f endogenous p r o d u c t i o n o f
ethylene i n t e r f e r i n g i n A R A c o u l d b e o v e r c o m e
u s i n g l 4 C 2 H 2 ( W i t t y 1979) o r the endogenous p r o d u c t i o n o f ethylene c a n b e measured b y suppressing
12
nitrogenase a c t i v i t y u s i n g C O , w h i c h stops n i t r o genase f u n c t i o n i n g w i t h o u t d a m a g i n g the p l a n t s
( N o h r s t e d t 1983).
T h e excised r o o t assay i n v o l v e s p r e i n c u b a t i o n f o r
8-18 h u n d e r reduced o x y g e n tension before e x p o sure t o C 2 H 2 ( D o b e r e i n e r a n d D a y 1975, N e y r a a n d
D o b e r e i n e r 1977). D u r i n g p r e i n c u b a t i o n o f r o o t s ,
considerable f e r m e n t a t i o n a n d p r o l i f e r a t i o n o f bacteria takes place r e s u l t i n g i n o v e r e s t i m a t i o n o f
nitrogenase a c t i v i t y ( O k o n e t a l . 1977, v a n B e r k u m
a n d B o h l o o l 1980, B a r b e r et a l . 1976). H o w e v e r ,
i m m e d i a t e r e d u c t i o n o f acetylene b y excised r o o t s
f r o m several grasses ( v a n B e r k u m a n d Sloger 1979)
a n d s o r g h u m a n d m i l l e t s ( D a r t a n d W a n i 1982) has
been r e p o r t e d . T h e difficulties i n c o m p l e t e recovery
o f the p l a n t r o o t s u n d e r f i e l d c o n d i t i o n s c o m p l i c a t e s
the i n t e r p r e t a t i o n a n d c o m p a r i s o n o f d a t a collected
b y different g r o u p s .
In-situ assays w i t h i n t a c t plants are c u m b e r s o m e
a n d t h e measurements are d i f f i c u l t t o i n t e r p r e t
( B a l a n d r e a u a n d D o m m e r g u e s 1973, Lee et a l . 1977,
T j e p k e m a a n d v a n B e r k u m 1977). S o i l - r o o t cores
r e m o v e d f r o m the field at harvest have been used f o r
m e a s u r i n g nitrogenase a c t i v i t y o f b o t h grasses a n d
g r a i n c r o p s ( D a y e t a l . 1975b; v a n B e r k u m a n d D a y
1980; W a n i e t a l . 1983; W a n i , i n press). T h e i n i t i a l
l a g p e r i o d varies f r o m 1 to 30 h w i t h s o i l cores,
d e p e n d i n g o n the t i m e r e q u i r e d f o r d i f f u s i o n o f C 2 H 2
t h r o u g h different s o i l types ( v a n B e r k u m a n d D a y
1980, W a n i et a l . 1984). H o w e v e r , large p l a n t - t o p l a n t v a r i a b i l i t y has been r e p o r t e d u s i n g t h i s techn i q u e ( D a r t a n d W a n i 1982, W a n i e t a l . 1983,
U p a d h y a y a 1984) a n d i t i s n o t clear w h e t h e r t h i s
reflects t h e n a t u r a l v a r i a t i o n . H o w e v e r , such v a r i a b i l i t y has been r e p o r t e d w i t h in-situ assays ( B a l a n d r e a u 1979) as w e l l as i n t a c t p l a n t assays in t h e
greenhouse ( W a n i e t a l . 1984). T h e factors responsible have been studied a n d as a result i m p r o v e m e n t s
in the soil-core assay technique have been m a d e
( W a n i et a l . 1983).
W i t h the i m p r o v e d soil-core ( p l a n t e d core) assay,
w h i c h involves g r o w i n g the p l a n t i n cores i n the f i e l d
f r o m 20 days after s o w i n g ( D A S ) t i l l assayed, signific a n t l y higher a c t i v i t y has been r e c o r d e d t h a n f o r the
plants g r o w n a n d sampled i n the n o r m a l ( d i s t u r b e d
core) w a y ( W a n i e t a l . 1983). I n some cases results o f
soil-core assays are e x t r a p o l a t e d to hectare basis on
the basis of core area ( N e l s o n et a l . 1976, W e a v e r et
a l . 1980) or p l a n t p o p u l a t i o n (Pedersen et a l . 1978).
H o w e v e r , such estimates c a n be correct o n l y w h e n
factors l i k e seasonal a n d d i u r n a l v a r i a t i o n s i n the
a c t i v i t y , s o i l m o i s t u r e , soil t e m p e r a t u r e , a n d f e r t i l i t y
status o f the soil are t a k e n i n t o a c c o u n t . I f i t i s
essential to e x t r a p o l a t e the soil-core assay results to
hectare basis, t h e n several cores at each assay t i m e
s h o u l d be t a k e n a n d the plants s h o u l d be assayed at
regular intervals t h r o u g h o u t the g r o w t h p e r i o d . T h e
a c t i v i t y at different c r o p - g r o w t h stages c a n be p l o t ted, a n d b y c o n s i d e r i n g the p e r i o d u n d e r each a c t i v i t y p o i n t , necessary corrections f o r d i u r n a l v a r i a t i o n
a n d C 2 H 2 : N 2 r e d u c t i o n r a t i o can b e made.
A n i n t a c t - p l a n t assay f o r p o t - g r o w n plants overcomes the p r o b l e m s faced w i t h soil-core assays, e.g.,
d e s t r u c t i o n o f the plants, m e c h a n i c a l d i s t u r b a n c e ,
tedious a n d t i m e - c o n s u m i n g operations, etc. ( W a n i
et a l . 1984). U s i n g this technique, genotypes c a n be
screened f o r t h e i r p o t e n t i a l t o s t i m u l a t e r h i z o s p h e r i c
nitrogenase a c t i v i t y a n d / o r v a r i o u s e n v i r o n m e n t a l
a n d b i o l o g i c a l factors affecting the a c t i v i t y can be
studied. S i m i l a r l y , f o r t u b e - g r o w n seedlings, i n t a c t p l a n t assays have been used f o r screening lines of
crops o r bacterial strains i n association w i t h the
plants f o r t h e i r nitrogenase a c t i v i t y ( W a n i , i n press).
These i n t a c t - p l a n t assays being n o n d e s t r u c t i v e are
p r o m i s i n g f o r screening plants w i t h h i g h a c t i v i t y .
Selected plants c a n t h e n be used in b r e e d i n g
programs.
Current
Understanding about
Factors
Affecting
Nonsymbiotic
a n d Associative N 2 F i x a t i o n
Energy Source
T h e basic u n s o l v e d p r o b l e m c o n c e r n i n g associative
n i t r o g e n f i x a t i o n i s the s u p p l y o f a n adequate energy
source. T h e types a n d n u m b e r s o f m i c r o o r g a n i s m s
present i n the rhizosphere are largely d e t e r m i n e d b y
energy sources a v a i l a b l e t h r o u g h r o o t exudates a n d
p l a n t debris ( R o v i r a 1965). R o o t exudates p l a y a n
i m p o r t a n t r o l e f o r rhizo s ph eric m i c r o f l o r a o f y o u n g
seedlings. As r o o t s age a n d die, cell debris becomes
the d o m i n a n t energy source. P l a n t - r o o t e x u d a t i o n is
affected by p l a n t species, c u l t i v a r , p l a n t age, l i g h t ,
t e m p e r a t u r e , p l a n t n u t r i t i o n , soil m o i s t u r e , m i c r o o r ganisms, a n d r o o t d a m a g e ( R o v i r a 1965, 1969).
These are generally the same factors t h a t affect associative n i t r o g e n f i x a t i o n ( D o b e r e i n e r a n d D a y 1975;
B a l a n d r e a u et a l . 1978; W a n i et a l . 1983, 1984). T h e
t o t a l loss o f c a r b o n f r o m r o o t s i s m u c h greater w h e n
c o m p a r e d t o the organic c a r b o n e x u d e d ( R o v i r a
1969, M a r t i n 1977, B a r b e r a n d M a r t i n 1976). U s i n g
g r o w t h a n d nitrogenase a c t i v i t y o f a z o s p i r i l l a a s
c r i t e r i a , q u a l i t a t i v e differences i n the r o o t exudates
o f s o r g h u m ( I C R I S A T 1983) a n d m i l l e t genotypes
( R a o a n d V e n k a t e s w a r l u 1986) have been s h o w n .
Photosynthesis
Several-fold higher nitrogenase a c t i v i t y has been
recorded w i t h intact s o r g h u m plants, a s c o m p a r e d t o
those whose tops were r e m o v e d p r i o r to assay ( W a n i
et a l . 1984). H o w e v e r , it is n o t clear w h e t h e r this
effect is d i r e c t l y related to p h o t o s y n t h a t e s u p p l y .
D i u r n a l v a r i a t i o n s i n nitrogenase a c t i v i t y associated w i t h grasses, s o r g h u m , m i l l e t , finger m i l l e t ,
Panicum
maximum, a n d Lolium perenne have been
r e p o r t e d ( D o b e r e i n e r a n d D a y 1975; B a l a n d r e a u
1975; W a n i et a l . 1983; W a n i , in press; U p a d h y a y a e t
a l . 1986). H o w e v e r , these studies do n o t s h o w a n y
clear r e l a t i o n s h i p between photosynthesis a n d r o o t associated N 2 f i x a t i o n because f l u c t u a t i o n s i n s o i l
t e m p e r a t u r e c o i n c i d e w i t h the cycle o f nitrogenase
a c t i v i t y d u r i n g the d a y - n i g h t cycle ( D a r t a n d W a n i
1982, v a n B e r k u m a n d B o h l o o l 1980). F u r t h e r
e x p e r i m e n t s c o n d u c t e d a t I C R I S A T Center w i t h
c o n t r o l l e d soil t e m p e r a t u r e d i d n o t s h o w d i u r n a l
v a r i a t i o n i n nitrogenase a c t i v i t y o f i n t a c t s o r g h u m
a n d m i l l e t plants ( W a n i , u n p u b l i s h e d results). S i g n i f i c a n t changes i n the rate o f root-associated C 2 H 2
r e d u c t i o n w i t h i n 1 5 m i n o f t r a n s f e r r i n g plants f r o m
s u n l i g h t i n t o the d a r k or vice versa have been
r e p o r t e d ( V a n B e r k u m a n d Sloger 1981), w h i c h s u g gests a s t r o n g l i n k between nitrogenase a c t i v i t y a n d
l i g h t incidence i n rice. H o w e v e r , the nitrogenase
a c t i v i t y o f the a b o v e - r e p o r t e d crops m a y b e i n d i rectly related to photosynthesis, as is evident f r o m
studies o f D o b e r e i n e r e t a l . (1973) w h o f o u n d t h a t
even t h o u g h n o d i u r n a l cycle o f nitrogenase was
observed in P. notatum, p r o l o n g e d i n c u b a t i o n s of
plants i n the d a r k reduced the a c t i v i t y .
13
Seasonal Variation
Combined Nitrogen and Phosphorus
Seasonal v a r i a t i o n s i n the nitrogenase a c t i v i t y o f
forage grasses, c o r n , s o r g h u m , m i l l e t , Setaria italica,
a n d Eleucine coracana have been r e p o r t e d u s i n g
excised-root or soil-core assays ( D o b e r e i n e r a n d
D a y 1975, v o n B u l o w a n d D o b e r e i n e r 1975, B a l a n d r e a u 1975, K a p u l n i k et a l . 1981, W a n i et a l . 1983,
U p a d h y a y a e t a l . 1986). W i t h c o r n m a x i m u m a c t i v i ties were r e c o r d e d at the 7 5 % s i l k i n g stage ( v o n
B u l o w a n d D o b e r e i n e r 1975), a n d w i t h s o r g h u m a n d
m i l l e t a t t h e late f l o w e r i n g / e a r l y g r a i n - f i l l i n g stage.
T h e a c t i v i t y was related t o ontogenetic d e v e l o p m e n t
o f the p l a n t ( W a n i e t a l . 1983). W h i l e s t u d y i n g seas o n a l p r o f i l e s o f nitrogenase a c t i v i t y , f e r t i l i z e r n i t r o gen s h o u l d be t a k e n i n t o c o n s i d e r a t i o n as it has been
s h o w n t h a t higher rates o f N a p p l i c a t i o n i n h i b i t the
nitrogenase a c t i v i t y associated w i t h cereals.
T h e presence of c o m b i n e d N affects the e n z y m e
nitrogenase. M a n i p u l a t i n g the times a n d m e t h o d s o f
N a p p l i c a t i o n a n d selection o f the p r o p e r f o r m o f
f e r t i l i z e r N , l i k e slow-release f o r m u l a t i o n s , m a y h e l p
t o harness m a x i m u m n i t r o g e n f i x a t i o n associated
w i t h these crops, w i t h o u t r e d u c i n g yields.
Phosphorus fertilizer application is required for
o p t i m u m growth and nitrogen-fixing activity by
a z o l l a a n d blue-green algae ( D e a n d M a n d a l 1956,
W a t a n a b e et a l . 1977). It is necessary to s t u d y the
effect of levels a n d f o r m s of P a n d K a n d o t h e r
elements o n nitrogenase a c t i v i t y o f cereals.
Temperature
A l i n e a r response to t e m p e r a t u r e has been observed
f r o m 10°C to 35°C for C 2 H 2 reduction activity of
Clostridium pasteurianum in c u l t u r e a n d f o r Azotobacter cell-free nitrogenase ( H a r d y et a l . 1968).
Increased C 2 H 2 r e d u c t i o n over t i m e b y grass cores
was a t t r i b u t e d t o w a r m i n g o f the s o i l ( N e l s o n e t a l .
1976). W i t h i n t a c t m i l l e t a n d s o r g h u m plants g r o w n
i n p o t s , s i g n i f i c a n t l y h i g h e r C 2 H 2 r e d u c t i o n activities
were r e c o r d e d a t 34° a n d 4 0 ° C t h a n w i t h the plants
i n c u b a t e d a t 2 9 ° C ( W a n i e t a l . 1984).
Soil Moisture
T h e rate o f nitrogenase a c t i v i t y b y the s o i l cores was
p o s i t i v e l y correlated w i t h s o i l m o i s t u r e a n d the rate
o f acetylene r e d u c t i o n increased e x p o n e n t i a l l y w i t h
linear increases i n s o i l m o i s t u r e ( D a y e t a l . 1975a).
S i m i l a r c o r r e l a t i o n s have been r e p o r t e d i n s o i l cores
o f grasslands (Vlassak e t a l . 1973) a n d s o r g h u m a n d
m i l l e t ( W a n i e t a l . 1983), a s also i n p o t - g r o w n
s o r g h u m a n d m i l l e t plants ( W a n i e t a l . 1984). I t i s
d i f f i c u l t t o p i n p o i n t h o w s o i l m o i s t u r e affects
nitrogenase a c t i v i t y as m a n y p l a n t processes t h a t
m a y influence t h i s a c t i v i t y are also affected b y s o i l m o i s t u r e levels. D a y e t a l . (1975a) h y p o t h e s i z e d t h a t
a s the level o f anaerobiosis i n s o i l c r u m b s a n d t h e
rhizosphere increases w i t h h i g h e r s o i l m o i s t u r e , t h e
p O 2 affects nitrogenase a c t i v i t y .
14
Plant Breeding
T h e m a j o r t h r u s t i n N 2 f i x a t i o n has been m i c r o b i o l o g i c a l i n o r i e n t a t i o n . E v e n t h o u g h the role o f p l a n t
genotype i n N 2 f i x a t i o n has been recognized, there i s
a p a u c i t y o f i n f o r m a t i o n o n the n a t u r e o f the genetic
i n v o l v e m e n t o f the host. T h e use o f n o n d e s t r u c t i v e
i n t a c t - p l a n t assays f o r m e a s u r i n g C 2 H 2 r e d u c t i o n
a c t i v i t y i n the greenhouse c o u p l e d w i t h 1 5 N isotope
d i l u t i o n technique a n d f u r t h e r testing f o r y i e l d
p o t e n t i a l u n d e r l o w - f e r t i l i t y field c o n d i t i o n s seems a
prospective p r o p o s a l f o r such studies.
Before b r e e d i n g m e t h o d s can p r o d u c e cereal lines
w i t h increased p o t e n t i a l f o r n i t r o g e n f i x a t i o n , i t i s
essential to u n d e r s t a n d the associations g o v e r n i n g
N 2 f i x a t i o n t r a i t s i n p a r t i c u l a r crops. T h e r e are several reports i n d i c a t i n g differences a m o n g s t genotypes o f s o r g h u m , m i l l e t , m i n o r m i l l e t s , rice a n d
forage grasses, a n d wheat, ( B o u t o n et a l . 1979;
D o b e r e i n e r 1966, 1970,1977; v o n B u l o w a n d D o b e reiner 1975; Pederson et a l . 1978; W a t a n a b e et a l .
1979; C h a r y u l u e t a l . 1981; D a r t a n d W a n i 1982;
U p a d h y a y a e t a l . 1986; W a n i , i n press). A t I C R I S A T Center, 1 8 o u t o f 248 m i l l e t lines tested s h o w e d
s i g n i f i c a n t l y h i g h nitrogenase a c t i v i t y ( > 4 6 0 n m o l
C 2 H 4 1 5 c m d i a m core - 1 h - 1 ) a n d t w o lines, G a m 7 3
a n d J 1407, were consistently active o v e r several
seasons. S i m i l a r l y , 1 5 o u t o f 334 lines o f f i e l d - g r o w n
s o r g h u m were consistently active i n three o r m o r e
seasons t h o u g h n o t o n each assay occasion. T h i s
m a y have been due t o u n f a v o r a b l e s o i l m o i s t u r e o r
o t h e r c o n d i t i o n s d u r i n g t h e season.
I n t h e E x - B o r n u p o p u l a t i o n o f m i l l e t , large p l a n t t o - p l a n t v a r i a b i l i t y f o r s t i m u l a t i n g nitrogenase
a c t i v i t y r a n g i n g f r o m 0 t o 1900 n m o l C 2 H 4 p l a n t - 1 h - 1
has been observed u s i n g i n t a c t p o t assay t e c h n i q u e .
W o r k o n s t a b i l i z i n g the character o f h i g h a n d l o w
nitrogenase a c t i v i t y i n this p o p u l a t i o n i s u n d e r w a y
t o s t u d y the i n h e r i t a n c e o f t h i s t r a i t ( I C R I S A T
1983). T h e r e is an urgent need to pursue b r e e d i n g
research f o r p r o d u c i n g lines w i t h increased p o t e n t i a l
for nitrogen fixation.
C r o p Responses t o I n o c u l a t i o n
T h e r e are several reports a b o u t f i e l d - a n d p o t - g r o w n
cereals i n o c u l a t e d w i t h N 2 - f i x i n g bacteria a n d these
have been reviewed ( B o d d e y a n d D o b e r e i n e r 1982).
M a n y re p o rt s s h o w statistically significant increases
i n cereal yields o r otherwise a n d also negative
responses. T h e m e c h a n i s m b y w h i c h the cereals
i n o c u l a t e d w i t h n i t r o g e n - f i x i n g bacteria derive the
benefit i s n o t clearly u n d e r s t o o d . H o w e v e r , k n o w l edge has a c c u m u l a t e d to i n d i c a t e the possible m e c h anisms i n v o l v e d . I t has been s h o w n t h a t N 2 - f i x i n g
a z o s p i r i l l a a n d Azotobacter benefit the i n o c u l a t e d
plants t h r o u g h b i o l o g i c a l n i t r o g e n f i x a t i o n ( C o h e n
et a l . 1980, D a r t a n d W a n i 1982, H e g a z i et a l . 1983,
N u r e t a l . 1980, O k o n 1982) a n d also b y e n h a n c i n g
r o o t - h a i r f o r m a t i o n a n d therefore, increased r o o t
u p t a k e c a p a c i t y caused b y the secretion o f g r o w t h
h o r m o n e s ( T i e n et a l . 1979, Vlassak a n d Reynders
1981). T h e extent t o w h i c h each o f the v a r i o u s p r o cesses c o n t r i b u t e s to y i e l d increase of i n o c u l a t e d
cereal plants remains to be assessed.
Areas of Future Research
These associative N 2 - f i x i n g systems need to be
u n d e r s t o o d i n d e t a i l , i n o r d e r t o f u l l y harness t h e i r
p o t e n t i a l benefits. W o r k in several areas needs to be
c o n t i n u e d w i t h v i g o r s o a s t o p u t the systems t o w o r k
a n d i m p r o v e t h e m f u r t h e r . M o r e precise estimates o f
the q u a n t i t y o f n i t r o g e n f i x e d are essential b y c o n d u c t i n g e x p e r i m e n t s using careful n i t r o g e n - b a l a n c e
studies a n d l 5 N -based techniques.
M e t h o d o l o g i e s f o r s t u d y i n g associative n i t r o g e n
f i x a t i o n w i t h i m p r o v e d methods o f measuring
nitrogenase a c t i v i t y (acetylene r e d u c t i o n ) , e.g.,
planted-core assays f o r field-grown p l a n t s , i n t a c t p l a n t assays f o r t u b e - g r o w n seedlings a n d p o t t e d
greenhouse plants, etc., a n d greater use of 1 5 N-based
techniques have been developed. H o w e v e r , this area
needs m o r e a t t e n t i o n a s i t i s i m p o r t a n t f r o m the
p o i n t o f v i e w o f better screening a n d selection
methods.
T h e r e is a need to l o o k at the b a c t e r i a l systems
i n v o l v e d i n associative symbiosis systems. B y
m a n i p u l a t i n g the c u l t u r e m e d i a a n d c u l t u r a l c o n d i tions like O2 concentration, p H , temperature, a n d
c o n c e n t r a t i o n s o f c a r b o n a n d o t h e r n u t r i e n t sources,
i t w i l l b e possible t o i d e n t i f y m a n y u n k n o w n o r g a nisms i n v o l v e d i n these associations. I n p a r t i c u l a r ,
w e need t o give t h o u g h t t o t h e r o l e o f n o n n i t r o g e n
fixers present in the rhizosphere, as r e p o r t s have
s h o w n synergism a m o n g s t n i t r o g e n f i x e r s a n d n o n n i t r o g e n fixers. These organisms m a y p l a y a n i m p o r tant role in manipulating oxygen concentration in
the rhizosphere, p H changes a n d m o r e o v e r , m a y
p r o v i d e energy substances b y m e t a b o l i z i n g the c o m p o u n d s w h i c h N 2 - f i x i n g bacteria c a n n o t use d i r e c t l y .
M o r e critical methods of isolation and identificat i o n are r e q u i r e d , as it is a p p a r e n t t h a t there are s t i l l
l i t t l e - k n o w n o r n e w f o r m s o f rhizosphere bacteria.
Greater s u p p o r t f o r t a x o n o m i c studies is essential.
T h e r o l e o f bacteria i n associative s y m b i o t i c systems
needs to be better u n d e r s t o o d at the basic level so as
t o seek i n f o r m a t i o n o n (1) l o c a t i o n o f the bacteria o n
r o o t s , (2) source o f energy f o r N 2 f i x a t i o n , (3) r o l e o f
p l a n t a f f i n i t y b e y o n d t h a t o f c a r b o h y d r a t e supplier,
(4) ecological factors g o v e r n i n g such associations,
(5) types o f bacteria i n v o l v e d , a n d (6) c r i t e r i a t o b e
used f o r selecting b a c t e r i a l strains f o r f i e l d - i n o c u l a t i o n studies.
A l t h o u g h several reports have i n d i c a t e d s i g n i f i cant p o s i t i v e responses t o c r o p i n o c u l a t i o n s i n f i e l d s ,
the d a t a are h i g h l y variable. T h e reasons f o r t h e
f a i l u r e to o b t a i n p o s i t i v e responses in some cases
m u s t be studied. Increased yields of field-grown
crops i n o c u l a t e d w i t h N 2 - f i x i n g bacteria a n d the
p o s s i b i l i t y o f i n t e r a c t i o n between host c u l t i v a r s a n d
bacterial strains indicate the need to select the m o s t suitable c o m b i n a t i o n s o f host c u l t i v a r s a n d b a c t e r i a l
strains. T h e r e is l i t t l e agreement on several p o i n t s .
F o r e x a m p l e : (1) Is there a host specificity f o r bacteria? (2) W h i c h bacteria s h o u l d be used as i n o c u l u m
a n d s h o u l d it be a single species or a m i x t u r e of
bacteria? (3) W h i c h m e t h o d o f i n o c u l a t i o n s h o u l d b e
used? (4) W h a t s h o u l d be the nature of a suitable
carrier? (5) W h a t c r i t e r i a s h o u l d be used f o r checki n g the q u a l i t y o f the i n o c u l a n t s produced?
I n f o r m a t i o n i s also essential t o p u t the system t o
w o r k . M o r e emphasis needs t o b e g i v e n t o studies
p e r t a i n i n g t o establishment a n d s u r v i v a l o f the
a d d e d i n o c u l u m i n the rhizosphere a n d also the
factors t h a t m i g h t affect the p e r f o r m a n c e o f the
a d d e d i n o c u l u m . W h a t i s the exact r o l e o f i n o c u l a t e d
bacteria i n increasing c r o p yields? A r e these solely
due t o N 2 - f i x a t i o n o r h o r m o n a l effects o r because o f
p r o t e c t i o n against p l a n t pathogens?
15
K n o w l e d g e a b o u t the a g r o n o m i c practices t h a t
c o u l d help increase N 2 f i x a t i o n u n d e r n o r m a l situations, as w e l l as w i t h i n o c u l a t i o n , w i l l go a l o n g w a y
i n i m p r o v i n g N 2 f i x a t i o n . T h e i n f o r m a t i o n o n the
r o l e o f o r g a n i c a m e n d m e n t s , synergistic levels o f
combined N , appropriate f o r m and method o f applic a t i o n o f c o m b i n e d N , effect o f o t h e r m a c r o - a n d
microelements o n N 2 f i x a t i o n , and interaction w i t h
o t h e r rhizospheric m i c r o o r g a n i s m s l i k e m y c o r r h i z a ,
w i l l help t o derive m a x i m u m possible benefits f r o m
associative N 2 f i x a t i o n .
Based on the available l i t e r a t u r e , it seems possible
t o i m p r o v e p l a n t genotypes i n a p r a c t i c a l w a y f o r
increased associative N 2 f i x a t i o n b y f o l l o w i n g r o u t i n e p l a n t b r e e d i n g m e t h o d s . N o t m a n y efforts have
been d i r e c t e d i n this l i n e , p r o b a b l y because o f l a c k o f
r o u t i n e assay m e t h o d s t o measure N 2 f i x a t i o n , w h i c h
c a n b e used f o r selection, a n d also l a c k o f i n f o r m a t i o n o n the i n h e r i t a n c e o f this p a r t i c u l a r t r a i t i n host
p l a n t . M o r e i n f o r m a t i o n i s r e q u i r e d o n basic
aspects, such a s m e c h a n i s m o f i n h e r i t a n c e o f the N 2
f i x a t i o n t r a i t i n host plants, c r i t e r i a t o b e used f o r
selecting lines w i t h h i g h N 2 f i x a t i o n p o t e n t i a l , a n d
b r e e d i n g m e t h o d s t o b e a d o p t e d . T h r o u g h concerted
efforts i n this d i r e c t i o n , i t w i l l b e possible t o select o r
breed host lines w i t h increased associative N 2 - f i x i n g
ability.
References
App,
A . , Bouldin,
D.R., D a r t , P . J . , a n d
Watanabe,
I . 1980. C o n s t r a i n t s t o b i o l o g i c a l n i t r o g e n f i x a t i o n i n soils
of the t r o p i c s . Pages 319-337 in P r i o r i t i e s f o r a l l e v i a t i n g
soil-related constraints to f o o d p r o d u c t i o n in the tropics.
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Discussion
H . L . S . Tandon:
W h y d o y o u tag the i n o c u l a t i o n w i t h the a v a i l a b i l i t y
of a seed d r i l l , w h i c h introduces a n o t h e r cost factor?
F a r m e r s n o t h a v i n g a d r i l l m a y s t i l l be interested in
the p r o v e n i n o c u l u m .
S.P.Wani:
T h e seed d r i l l is n o t a must, b u t if it is already in use
we are suggesting the use of the s l u r r y i n o c u l a t o r .
O t h e r w i s e h a n d a p p l i c a t i o n o f s l u r r y i s perfectly a l l
right.
H.L.S.Tandon:
W h e n d o y o u expect the B N F t e c h n o l o g y t o enter
o n - f a r m research p r o g r a m s , before it is available f o r
the S A T farmers?
S.P.Wani:
I n o c u l a t i o n t e c h n o l o g y s h o u l d be at the o n - f a r m test
stage in 3-4 years.
P.Tauro:
W h e n l i g h t has n o effect, w h y s h o u l d d e c a p i t a t i o n
have a n effect o n A R A ?
S.P.Wani:
As I m e n t i o n e d in the presentation, the m e c h a n i s m
of reduced a c t i v i t y soon after d e c a p i t a t i o n is n o t
u n d e r s t o o d yet. Because of i n j u r y to the p l a n t , the
roots m a y be secreting some c o m p o u n d s t h a t m i g h t
be d e t r i m e n t a l to the bacteria, resulting in reduced
activity.
G.S. Jadhav:
As stated in the presentation, the m o i s t u r e a n d N in
the s o i l s h o u l d be kept constant. H o w e v e r , they are
d y n a m i c w i t h t i m e . H o w can the response o f different strains i n the f i e l d b e c o m p a r e d , w h e n b o t h
m o i s t u r e a n d N are c h a n g i n g w i t h i n the season as
w e l l as between seasons?
S.P.Wani:
I referred to constant m o i s t u r e a n d N o n l y f o r the
experiments i n v o l v i n g g e r m p l a s m screening or seasonal measurement o f nitrogenase a c t i v i t y , a n d n o t
f o r the i n o c u l a t i o n experiments. I t w i l l b e impossible
to o b t a i n u n i f o r m m o i s t u r e a n d N d u r i n g the season
in the field. T h i s is suggested f o r c o n t r o l l e d greenhouse experiments alone.
G.S.Jadhav:
Has the water-suspension m e t h o d o f i n o c u l a t i o n
a p p l i c a t i o n been c o m p a r e d w i t h seed i n o c u l a t i o n ,
soil application, and F Y M - m i x e d f u r r o w
application?
S.P.Wani:
No.
S.V.Hegde:
D i d y o u e x a m i n e the r o o t s / r h i z o s p h e r e o f i n o c u lated a n d n o n i n o c u l a t e d pearl m i l l e t r e g a r d i n g the
establishment of i n o c u l a t e d Azospirillum or the
counts o f the b a c t e r i u m , t o p r o v e that the beneficial
effects are due to i n o c u l a t e d A z o s p i r i l l u m ?
S.P. W a n i :
N o , i t i s n o t possible t o study the establishment o f
the i n o c u l a t e d strains in the field unless we have
specific m a r k e r strains a s inoculants. N e i t h e r M P N
n o r o r d i n a r y p l a t i n g can give the desired results. A t
present we are s t a n d a r d i z i n g the E L I S A technique,
a n d if we can use this technique successfully we
i n t e n d t o study the establishment a n d s u r v i v a l o f the
i n o c u l a t e d s t r a i n i n the f i e l d .
B . K . Konde:
I t i s reported b y E g y p t i a n scientists t h a t l i q u i d i n o c u l a t i o n is superior to seed i n o c u l a t i o n in g r o u n d n u t s
where the seeds are treated w i t h fungicides. In o r d e r
t o a v o i d the direct contact o f fungicides w i t h r h i z o bia, l i q u i d i n o c u l a t i o n p r o v e d t o b e superior, b u t
m e t h o d s o f azospirilla i n o c u l a t i o n have n o t been
t r i e d a n d evaluated. T h e y , therefore, need to be
studied.
S.P.Wani:
I agree w i t h y o u r views t h a t w o r k o n i n o c u l a t i o n
methods of azospirilla needs to be done. A b o u t
s l u r r y i n o c u l a t i o n a t I C R I S A T Center, D r N a m b i a r
has observed t h a t f o r g r o u n d n u t s s l u r r y i n o c u l a t i o n
gives better results t h a n seed c o a t i n g , a n d this is
m a i n l y because the g r o u n d n u t seed cotyledons get
separated because o f w e t t i n g , resulting i n reduced
g e r m i n a t i o n . In a d d i t i o n , fungicides can be used at
sowing along w i t h rhizobial culture application.
21
Cereal Nitrogen F i x a t i o n Research under the B N F
Coordinated Project o f the I C A R
N . S . Subba Rao1
Summary
The
paper
Fixation
effects
obtained
lacunae
highlights
the
(BNF) project
of sorghum
on
the
existing
results
of the
and millets
isolation
in
our
by
of
field
Indian
experiments
Council
Azospirillum.
and establishment
knowledge
and
carried out
of Agricultural
The
review
of A z o s p i r i l l u m
the future
in
possibilities
under
Research
also
roots,
the
Biological
(ICAR)
on
encompasses
and it
of research
the
briefly
under
Nitrogen
inoculation
initial
results
indicates
the
the
project.
Introduction
T h e B N F c o o r d i n a t e d project has the f o l l o w i n g
mandate.
1 . I n t e n s i f i c a t i o n o f research o n b i o l o g i c a l n i t r o g e n
f i x a t i o n in a c o o r d i n a t e d manner.
2 . Survey a n d ecology o f N 2 - f i x i n g m i c r o o r g a nisms, e v a l u a t i o n o f benefits o f i n o c u l a t i o n a n d
i n t e r a c t i o n w i t h host g e r m p l a s m , pesticides, a n d
i n o r g a n i c fertilizers.
3. Genetic studies to enlarge host spectrum by m u t a t i o n , conjugation, transduction, and transformation.
4. S o m a t i c h y b r i d i z a t i o n , tissue-culture techniques,
a n d fusion o f protoplasts using host plants a n d
m i c r o b i a l cultures.
5. P h y s i o l o g i c a l studies to transfer available k n o w l edge at the f u n d a m e n t a l level on n i t r o g e n - f i x i n g
a n d related enzymes to the a p p l i e d side.
T h e project has 11 centers located a l l over I n d i a
( F i g . 1).
T h e present r e p o r t summarizes the fieldwork
done on cereal n i t r o g e n fixation under the project,
w i t h p a r t i c u l a r reference t o s o r g h u m a n d millets.
1.
INDIA
Centers
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
Delhi
Hisar
Varanasi
Kalyani
Jabalpur
Baroda
Parbhani
Cuttack
Bangalore ( I I S )
Bangalore
(UAS)
Coimbatore
Figure 1. Locations of the centers of I C A R ' s coordinated project on biological nitrogen fixation.
Project Director, Agro-Energy Center, Indian Agricultural Research Institute, New Delhi 110 012, India.
I C R I S A T (International Crops Research Institute for the Semi-Arid Tropics). 1986. Cereal nitrogen fixation. Proceedings of the Working
Group Meeting, 9-12 Oct 1984, I C R I S A T Center, India. Patancheru, A.P. 502 324, India: I C R I S A T .
23
Ecology of Diazotrophic Bacteria
I n r o o t c o l o n i z a t i o n studies w i t h k n o w n strains o f
Azospirillum i n association w i t h specially g r o w n
plants, the bacteria were f o u n d i n the r o o t hairs a n d
were f o u n d t o traverse u p w a r d s i n the stem, a s
observed u n d e r a l i g h t m i c r o s c o p e ( L a k s h m i et a l .
1977). In wheat, Azospirillum c o u l d be isolated f r o m
surface-sterilized r o o t s a n d stem segments ( K a v i m a n d a n et a l . 1978). Transverse sections of stem
showed the presence o f bacteria p r o m i n e n t l y i n p r o t o x y l e m a n d often i n m e t a x y l e m vessels ( L a k s h m i e t
a l . 1977). I n rice seedlings g r o w n u n d e r aseptic
nitrogen-free c o n d i t i o n s , Azospirillum i n o c u l a t i o n
increased r o o t biomass ( D e w a n a n d S u b b a R a o
1979).
Several centers u n d e r the B N F project i n i t i a t e d
w o r k o n the i s o l a t i o n a n d c h a r a c t e r i z a t i o n o f
n i t r o g e n - f i x i n g bacteria f r o m rice, wheat, barley,
s o r g h u m , a n d millets. Greater a t t e n t i o n was p a i d t o
the i s o l a t i o n a n d c h a r a c t e r i z a t i o n of Azospirillum
i n t o A. brasilense a n d A. lipoferum. Nitrogenase
activities of i n t a c t c u l t i v a t e d plants a n d weeds associated w i t h t h e m were measured. Azospirillum isolates were p u r i f i e d a n d acetylene-reduction assay
( A R A ) measurements i n pure cultures were made.
T h e a m o u n t o f n i t r o g e n f i x e d per g r a m o f c a r b o n
source was also q u a n t i f i e d by the K j e l d a h l m e t h o d .
4800
A-Control
B-Azospirillum
brasilense
C-40 k g N h a - 1 ( U r e a a t s o w i n g t i m e )
D-40 kg N ha-1 + A. brasilense
4400
1-Coimbatore
2-Dharwad
3-Hyderabad
4-Akola
5-Parbhani
6-Navasari
7-Indore
8-Udaipur
9-Pantnagar
10-All
I n d i a mean
4000
3600
3200
2800
2400
2000
1600
1200
800
400
0
A B C D
1
A B C D
2
AB C D
A B C D
AB C D
A BC D
AB C D
A B C D
3
4
5
6
7
8
AB C D
9
A B C D
10
Figure 2. Response of sorghum to inoculation with A. brasilense at different locations (average of 4 years' field
trials), 1979-82.
24
Response
of
Crops
F i g u r e 2 f o r s o r g h u m , F i g u r e 3 f o r pearl m i l l e t , a n d
F i g u r e 4 f o r finger m i l l e t .
F o r most crops, the y i e l d response to i n o c u l a t i o n
varied between locations. I n pearl m i l l e t also, the
yields were variable w i t h l o c a t i o n b u t i n o c u l a t i o n
p r o v e d to be beneficial even at a basal dose of 40 kg
N ha - 1 except at J o d h p u r , where fertilizer N d i d n o t
p r o v i d e a d d i t i o n a l benefits i n c o n j u n c t i o n w i t h bact e r i a l i n o c u l a t i o n . I n s o r g h u m also, the yields v a r i e d
w i t h locations b u t simple Azospirillum i n o c u l a t i o n s
increased yields i n six locations w i t h o r w i t h o u t
a d d i t i o n o f 4 0 k g N ha-1. A t f o u r locations ( A k o l a ,
Parbhani, Navsari, and lndore), addition of 40 kg N
ha" 1 decreased the benefits due to i n o c u l a t i o n .
A t t e m p t s are under w a y to understand the factors
responsible f o r the negative response to i n o c u l a t i o n
t o Azospirillum
Inoculation
H a v i n g isolated several strains of Azospirillum
( L a k s h m i k u m a r i et a l . 1976), systematic trials in the
p o t - c u l t u r e house were carried o u t to determine the
efficiency o f different strains i n increasing c r o p
yields ( S u b b a R a o e t a l . 1979), p a r t i c u l a r l y o f
s o r g h u m , p e a r l m i l l e t , finger m i l l e t (Eleucine coracana Gaertn.), a n d barley. Efficient strains of A.
brasilense capable of enhancing yields of these crops
were later tested under field c o n d i t i o n s under the A l l
I n d i a C o o r d i n a t e d C r o p I m p r o v e m e n t Projects o f
the I C A R f o r barley, s o r g h u m , a n d millets. T h e
results o b t a i n e d so far have been s u m m a r i z e d in
1-Durgapura
2-Jodhpur
3-Aurangabad
4-Gwalior
2800
2600
5-Kanpur
6-Hyderabad
7-Hisar
8-Jamnagar
A
Noninoculated
control
B
C
D
1/2 N a p p l i c a t i o n +
1/3 N
"
+
Full N
"
+
+
Azospirillum
inoculation
"
"
"
"
"
"
2400
2200
2000
1800
1600
1400
1200
1000
800
600
400
200
A B C D
A B C D
A B C D
A B C D
A B C D
A B C D
A B C D
1
2
3
4
5
6
7
A B C D
8
F i g u r e 3. Response of pearl m i l l e t to i n o c u l a t i o n w i t h A. brasilense at different locations (average of 3 years'
f i e l d trials), 1979-81.
25
Noninoculated
was a n s w e r e d b y c o n d u c t i n g a f i e l d e x p e r i m e n t a t
control
Azospirillum
brasilense
40 kg N ha -1 ( U r e a )
40 kg N ha
-1
+ A.
the P a r b h a n i center w i t h s o r g h u m a n d p e a r l m i l l e t
d u r i n g t h e 1983 r a i n y season. A s u m m a r y o f t h e d a t a
of the 1 9 8 2 / 8 3 e x p e r i m e n t s is presented in T a b l e s 1
brasilense
a n d 2 . T h e f o l l o w i n g c o n c l u s i o n s were d r a w n .
5
T a b l e 1. R o o t mass, shoot mass, r o o t : s h o o t r a t i o , root
v o l u m e , and yield of field-grown rainy-season sorghum
(Sorghum bicolor), as influenced by seed inoculation with
4
Azotobacter a n d Azospirillum at different stages of crop
g r o w t h in a field experiment, P a r b h a n i , rainy season 1983. 1
Non-
Parameter/
3
Days of
inoculated
Azoto-
sampling
control
bacter
Azospirillum
CD
at 5 %
R o o t mass
(g plant-1)
20
2
3.40
4.34 2
6.412
0.89
2
18.53 2
2.03
2.70
35
12.20
I6.41
50
16.05
20.77 2
21.77 2
65
17.82
20.47
22.86 2
3.77
80
15.20
15.90
15.26
NS3
32.66
37.60
S h o o t mass
(g p l a n t - 1 )
1
20
0
Bangalore
Dholi
Almora
50.93 2
7.64
2
156.26 2
15.84
35
111.80
139.33
50
165.13
184.86 2
I90.492
12.76
65
167.66
187.73
203.86 2
23.01
80
156.46
167.86 2
182.40 2
10.09
Dindori
Root:shoot ratio
Figure 4. Response of finger millet to inoculation
w i t h A.
brasilense.
i n the presence o f f e r t i l i z e r N a t c e r t a i n l o c a t i o n s .
Experiments under field conditions by coopera-
20
9.60
8.66
7.94
35
9.16
8.49
8.43
50
10.28
8.90
8.75
65
9.40
9.16
8.91
80
10.29
10.55
11.95
2.79
3.40
t o r s a t C o i m b a t o r e a n d H i s a r centers o f t h e p r o j e c t
have
also
shown
that
Azospirillum
inoculations
Root volume
( m L plant-1)
increased p e a r l m i l l e t y i e l d .
20
Effect
of Azotobacter
AzospiriUum
on
and
Root
and Shoot Measurements
35
9.05
50
13.85
4.76 2
0.89
2
13.68 2
1.62
17.09 2
17.76 2
2.20
12.21
65
14.50
16.36
19.26 2
80
13.18
14.06
13.93
NS
3237
3632
3817
NS
4581
5950 2
65152
802
Grain yield
2.11
(kg ha-1)
It is n o w recognized that n o n s y m b i o t i c n i t r o g e n fixing bacteria produce g r o w t h - p r o m o t i n g substan-
Fodder yield
-1
ces
(Tien
et
al.
1979)
in
addition
to
fixing
( k g ha )
a t m o s p h e r i c n i t r o g e n . O n e q u e s t i o n has o f t e n been
1 . M e a n o f f i v e p l a n t s , e a c h f r o m t h r e e r e p li c a t e d p l o t s .
p o s e d : w i l l seed i n o c u l a t i o n h e l p i n i n c r e a s i n g t h e
2 . S i g n i f i c a n t i ncrease o v e r c o n tr o l .
r o o t biomass under field conditions? T h i s question
3. N S = N o t s i gn i f i c a n t .
26
Table 2. S h o o t mass, root biomass, and yield of fieldgrown
pearl
enced
by
Azospirillum
millet
seed
(Pennisetum americanum), as influinoculation
with
Azotobacter
and
at different stages of crop g r o w t h in a field
experiment, P a r b h a n i , rainy season 1983. 1
Parameter/
Days of
sampling
control
ever, Azospirillum h a d a m o r e p r o n o u n c e d effect
on
root
biomass
and
shoot
dry
mass
than
Azotobacter.
2. T h e g r a i n y i e l d increase d u e to seed i n o c u l a t i o n
over the n o n i n o c u l a t e d c o n t r o l was 12.2% w i t h
Noninoculated
biomass a t d i f f e r e n t stages o f c r o p g r o w t h . H o w -
Azotobacter
Azospirillum
CD
at 5 %
Azotobacter
and
17.9%
with
Azospirillum,
but
the results were n o t s i g n i f i c a n t . H o w e v e r , b o t h
the o r g a n i s m s i n d i v i d u a l l y b r o u g h t a b o u t a sig-
S h o o t mass
n i f i c a n t increase i n f o d d e r y i e l d over the n o n i n o c -
(g plant-1)
ulated c o n t r o l .
20
22.27
31.65 2
32.382
35
72.46
94.412
88.00 2
9.01
50
89.38
103.432
97.78
10.48
65
67.58
86.83
78.98
NS3
80
59.66
79.752
67.662
9.44
6.62
Pearl Millet
1. Seed
inoculation
with
either
Azotobacter
or
Azospirillum increased s h o o t mass a n d r o o t b i o -
R o o t mass
mass s i g n i f i c a n t l y over the n o n i n o c u l a t e d c o n t r o l
(g plant-1)
a t all stages o f c r o p g r o w t h . I n p e a r l m i l l e t the
20
2.13
3.162
3.312
0.61
35
5.56
7.902
6.662
0.83
50
6.51
8.802
8.15
1.79
65
5.08
8.532
7.832
0.97
80
4.60
8.012
6.I82
0.92
response w i t h Azotobacter was better t h a n w i t h
Azospirillum
inoculation.
2. T h e g r a i n y i e l d increase due to seed i n o c u l a t i o n
over the n o n i n o c u l a t e d c o n t r o l was 41.8 w i t h
Azotobacter,
and
13.4% w i t h
Azospirillum i n o c -
u l a t i o n , but the results were not s i g n i f i c a n t .
Root:shoot ratio
20
10.45
10.01
9.78
35
13.00
11.82
13.21
50
13.72
11.75
11.99
65
13.30
10.77
10.08
80
12.96
9.95
10.94
3. Seed i n o c u l a t i o n w i t h Azotobacter or A z o s p i r i l lum s i g n i f i c a n t l y increased f o d d e r y i e l d o v e r the
noninoculated control.
These results i n d i c a t e t h a t bacterial i n o c u l a t i o n
results in increased r o o t biomass w i t h the possible
Root volume
consequent i m p r o v e d u p t a k e o f n u t r i e n t s f r o m the
( m L plant-1)
s o i l . T h e r e f o r e , the observed enhanced yields d u e to
20
1.96
2.612
2.732
0.58
i n o c u l a t i o n m a y be a t t r i b u t a b l e to f a c t o r s such as
35
4.38
6.582
6.032
0.83
growth-promoting
50
5.08
7.082
6.352
1.56
bacteria, i n a d d i t i o n t o t h e i r a b i l i t y t o f i x e l e m e n t a l
nitrogen.
65
5.38
8.382
7.062
0.83
80
5.23
9.752
6.802
1.03
substances
elaborated
by the
T h e a b i l i t y of A. brasilense to i m p r o v e r o o t b i o mass has also been s h o w n in p o t e x p e r i m e n t s w i t h
G r a i n yield
(kg
1036
1469
1175
NS
ha-1)
Fodder yield
finger
millet,
tum), a n d
4497
74722
50262
kodo
millet
(Paspalum
scrobicula-
I t a l i a n m i l l e t (Setaria italica).
732
(kg ha-1)
1. Mean of five plants, each from three replicated plots.
2. Significant increase over noninoculated c ontrol.
3. NS = Not significant.
Response of Pearl M i l l e t
t o Inoculation with V A M Fungi
and
Azospirillum
brasilense
I n these p o t e x p e r i m e n t s the vesicular a r b u s c u l a r
Sorghum
m y c o r r h i z a ( V A M ) f u n g i used f o r s o i l i n o c u l a t i o n
were
1.
Seed i n o c u l a t i o n w i t h Azotobacter or A z o s p i r i l lum s i g n i f i c a n t l y increases s h o o t mass a n d r o o t
Acaulospora
Glomus fasciculatum,
tions,
sp,
with
Gigaspora
A.
margarita,
brasilense
and
combina-
over the n o n i n o c u l a t e d c o n t r o l . T h e d r y -
27
matter
content
of
shoots,
root
biomass,
and
P - u p t a k e o f the c r o p were s t u d i e d ( T a b l e 3).
g r a i n a n d f o d d e r y i e l d increased s i g n i f i c a n t l y d u e t o
combined i n o c u l a t i o n (Table 4).
T a b l e 3. Response of pearl millet to inoculation w i t h A.
1
Table 4.
Influence o f A . brasilense a n d V A M ( G l o m u s
brasilense a n d V A M fungi i n potted plants , I A R I , N e w
fasciculatum) inoculation on root a n d shoot mass, r o o t
Delhi.
volume,
Treatment
Shoot
Root
Total
P
mass
mass
P
uptake
(g
plant-1)
(g
plant-1)
content
(mg
(%)
plant-1)
and
yield
of
sorghum
CSH
8R
in
a
field
experiment.
Treatments
Root
Grain
volume
(mL
yield
Fodder
yield
plant -1 )
(kg
ha-1)
(kg
ha -1 )
21.5
12.6
1990
4280
4.82
23.22
13.9
2150
4610
4.3
22.32
13.1
2100
4480
6.8 2
28.12
15.4
26602
56802
0.9
NS3
390
630
Root
mass1
Shoot
mass1
(g
plant -1 )
(g
plant -1 )
4.1
Noninoculated
Noninoculated
control (No V A M ,
no
A.
A.
brasilense)
brasilense
Acaulospora
sp
13.3
0.16
0.10
13.7
14.4
0.35 2
0.142
20.1
16.4
0.28 2
0.142
21.1
control
A.
brasilense
VAM
(G.
G.
margarita
16.4
0.25
0.142
fasciculatum)
21.2
A. brasilense +
G.
fasciculatum
Acaulospora
A.
G.
A.
G.
A.
sp
+
16.8
0.28
0.11
20.2
17.4
0.382
0.09
16.6
brasilense
margarita
(G.
CD
fasciculatum)
(P<0.05)
0.5
1. Sampled at 80 days of plant growth.
2. Significant increase over n oninocula ted control.
+
19.8 2
0.362
0,12
23.2
20.62
0.382
0.13
25.92
3. NS = Not significant.
brasilense
fasciculatum
+
Future Program
brasilense
L.S.D. (P<0.05)
NS3
0.14
1.
NS
0.14
0.012
NS
NS
NS
0.012
NS
0.012
NS
T h e areas o f research t h a t need a t t e n t i o n w i t h r e g a r d
Azospirillum
t o B N F aspects o f s o r g h u m a n d m i l l e t s are: (1) T h e
m o d e o f e n t r y o f the associated b a c t e r i a i n t o p l a n t
2 . V A M fungi
r o o t s has t o b e c l e a r l y u n d e r s t o o d . (2) T h e p r o c e dures f o r e n u m e r a t i o n o f b a c t e r i a i n the r h i z o s p h e r e ,
3. V A M x A z o -
3.12
0.10
NS
5.2
spirillum
o n the r o o t surface, a n d inside the r o o t s have t o b e
s t a n d a r d i z e d . (3) T h e best m e t h o d o f seed i n o c u l a t i o n has t o b e s t a n d a r d i z e d . (4) T h e r e l a t i v e benefits
1. Average of six replications.
2. Significant increase over corresponding control.
3. NS = N o t significant.
b y g r o w t h factors and N 2 f i x a t i o n i n plant g r o w t h
have to be q u a n t i t a t i v e l y assessed. (5) W h a t are t h e
g r o w t h f a c t o r s e l a b o r a t e d b y the b a c t e r i a i n t h e r o o t
system a n d i n t h e r h i z o s p h e r e , b o t h q u a l i t a t i v e l y
a n d q u a n t i t a t i v e l y ? (6) A r e h o s t - c u l t i v a r responses
Effect o f D u a l Inoculation with
A.brasilense
and
VAM
Fungi
to inoculants repeatable at different locations a n d ,
l i k e w i s e , c a n the i n t r i n s i c a b i l i t y o f c u l t i v a r s t o s h o w
h i g h A R A values i n i n t a c t - p l a n t systems b e r e p r o -
on Sorghum Growth and Yield
d u c e d u n d e r d i f f e r e n t c o n d i t i o n s ? A r e these c h a r a c -
I n a f i e l d e x p e r i m e n t a t P a r b h a n i , i t was o b s e r v e d
genetically to other cultivars?
teristics h e r i t a b l e ? A n d , i f so, c a n t h e y b e t r a n s f e r r e d
t h a t t h e m e a n r o o t a n d s h o o t mass o f s o r g h u m
In developing countries such as I n d i a , where
Azospirillum
sorghum a n d millets are g r o w n under l o w - i n p u t c o n -
a n d V A M i n o c u l a t i o n . T h e results a l s o s h o w e d t h a t
d i t i o n s , even m a r g i n a l increases i n c r o p y i e l d s t o t h e
plants
28
increased
significantly
due
to
equivalents o b t a i n a b l e by the a d d i t i o n of 20-30 kg N
ha -1 , are indeed w o r t h w h i l e . Hence, intensive f u t u r e
efforts o n B N F research aspects o f s o r g h u m a n d
m i l l e t s , a t b o t h the f u n d a m e n t a l a n d a p p l i e d levels,
would be rewarding. The fundamental w o r k in
f u t u r e requires emphasis on the genetics a n d m o l e c u l a r b i o l o g y o f d i a z o t r o p h i c bacteria associated w i t h
p l a n t r o o t s , o n experiments designed t o integrate
engineered n i t r o g e n - f i x i n g bacteria w i t h p l a n t p r o toplasts a n d tissue cultures, so as to i m p a r t higher
nitrogen-fixation potential to sorghum and millet
cultivars.
Discussion
O . P . Rupela:
Y o u r e p o r t t h a t a t P a r b h a n i , increase i n r o o t mass
was n o t i c e d due to i n o c u l a t i o n consistently f o r 3
years. W a s it a p o t or field trial? If it was a field t r i a l ,
h o w was the r o o t mass recovered a n d measured?
N . S . Subba R a o :
Yes, it was a field t r i a l . T h e r o o t system was carefully
d u g o u t a n d washed, a n d lateral r o o t s were recovered a n d measured b y displacement o f w a t e r i n a
m e a s u r i n g c y l i n d e r f o r v o l u m e , a n d later d r y mass
data were o b t a i n e d .
Acknowledgements
T h e a u t h o r wishes t o t h a n k w o r k e r s a t different
centers f o r t h e i r c o o p e r a t i o n a n d , m o r e p a r t i c u l a r l y ,
those i n v o l v e d i n f i e l d w o r k a t the P a r b h a n i
center—Drs R.S.Raut, G.B.Rudraksha, K . V . B.R.
T i l a k , a n d C . S . S i n g h , a n d the project c o o r d i n a t o r s
o f the s o r g h u m a n d m i l l e t p r o g r a m s o f the I C A R .
References
D e w a n , G . I . , and Subba R a o , N . S . 1979. Seed i n o c u l a t i o n
with
Azospirillum
brasilense a n d
Azotobacter chroococcum a n d the r o o t biomass of rice ( O r y z a sativa L . ) . P l a n t
a n d S o i l 53:295-302.
G.S. Jadhav:
T o realize the a g r o n o m i c significance o f N 2 f i x a t i o n
by n o n s y m b i o t i c bacteria it is very essential t o :
1. R e p o r t a l l s u p p l e m e n t a r y i n f o r m a t i o n , such as
i n i t i a l soil f e r t i l i t y , r a i n f a l l p a t t e r n , i r r i g a t i o n ,
soil type, a n d v a r i e t y o r c u l t i v a r used.
2 . I n d e t e r m i n i n g responses w e s h o u l d analyze y i e l d
data b o t h i n terms o f statistical significance a n d
e c o n o m i c benefits, in terms of N e c o n o m y a n d
y i e l d gain.
3 . A d a p t i v e trials o n farmers' f i e l d s s h o u l d b e c o n d u c t e d t o take N 2 - f i x a t i o n t e c h n o l o g y t o farmers
q u i c k l y , i t b e i n g l o w cost a n d e c o n o m i c a l l y a n d
ecologically profitable.
K a v i m a n d a n , S . K . , S u b b a R a o , N . S . , and M o h r i r ,
A . V . 1978. I s o l a t i o n of Spirillum lipoferum f r o m the
stems o f w h e a t a n d n i t r o g e n f i x a t i o n i n e n r i c h m e n t c u l tures. C u r r e n t Science 47:96-98.
O n the basis o f present k n o w l e d g e o f i n o c u l a t i o n
m e t h o d s of Azospirillum, w h i c h is the best a n d m o s t
e c o n o m i c a l m e t h o d o f i n o c u l a t i o n i n pearl m i l l e t ?
Lakshmi, V . , R a o , A . S . , Vijayalakshmi, K . , Lakshmikum a r i , M . , T i l a k , K . Y . B . R . , and Subba R a o , N . S . 1977.
E s t a b l i s h m e n t a n d s u r v i v a l of Spirillum lipoferum. P r o ceedings o f the I n d i a n A c a d e m y o f Sciences, S e c t i o n B
86:397-405.
N . S . Subba R a o :
Seed i n o c u l a t i o n as practiced f o r legume i n o c u l a t i o n
w i t h rhizobia.
L a k s h m i k u m a r i , M . , K a v i m a n d a n , S . K . , and Subba R a o ,
N . S . 1976. O c c u r r e n c e o f n i t r o g e n f i x i n g Spirillum i n
r o o t s o f rice, s o r g h u m , maize a n d o t h e r p l a n t s . I n d i a n
J o u r n a l o f E x p e r i m e n t a l B i o l o g y 14:638-639.
Subba R a o , N . S . , T i l a k , K . V . B . R . , Singh, C . S . , a n d
L a k s h m i k u m a r i , M . 1979. Response o f a few e c o n o m i c
species o f g r a m i n a c e o u s p l a n t s t o i n o c u l a t i o n w i t h A z o s p i rillum brasilense. C u r r e n t Science 48:133-134.
T i e n , T . M . , Gaskins, M . H . , and H u b b e l l , D . H . 1979.
P l a n t g r o w t h substances p r o d u c e d b y Azospirillum b r a s i lense a n d t h e i r effect o n the g r o w t h o f p e a r l m i l l e t (Pennisetum americanum L . ) . A p p l i e d
and
Environmental
M i c r o b i o l o g y 37:1016-1024.
J . V . D . K . Kumar Rao:
Y o u have r e p o r t e d significant responses t o A z o s p i rillum i n o c u l a t i o n at six o u t of n i n e centers u n d e r
the A l l I n d i a B N F project. W h a t i s t h e m e t h o d o f
i n o c u l a t i o n used i n these trials? W h a t i s the f o r m o f
i n o c u l a n t a n d the n a t u r e o f the carrier? W h a t i s the
viable-cell c o u n t i n the i n o c u l a n t a n d o n the seed
after i n o c u l a t i o n ?
N . S . Subba R a o :
Seed i n o c u l a t i o n was d o n e f r o m a finely p o w d e r e d
f a r m y a r d m a n u r e a n d s o i l i n o c u l a n t i n the r a t i o o f
1 : 1 , b y m a k i n g a n aqueous s l u r r y a n d also u s i n g
c a r b o x y m e t h y l cellulose as an adhesive (same as
29
r h i z o b i a l seed i n o c u l a t i o n ) . W e d i d n o t m a k e v i a b l e c e l l c o u n t s o n the seed, b u t w e m o n i t o r e d i n o c u l a n t
s u r v i v a l in this carrier. It h a d 10 9 cells even after 9
m o n t h s o f storage u n d e r r o o m c o n d i t i o n s .
S . V . Hegde:
Y o u m e n t i o n e d nitrogenase a c t i v i t y o f some c u c u r bits i n tissue c u l t u r e i n the absence o f a n y N 2 - f i x i n g
microorganisms.
1 . H o w was the c u c u r b i t
microorganisms?
plant
made
free
of
2 . W h a t was the level o f a c t i v i t y i n the a x e n i c
culture?
N . S . Subba R a o :
T h e c a l l i o f c u c u r b i t s e x h i b i t e d h i g h nitrogenase
a c t i v i t y w i t h o u t a n y bacteria i n the cells. T h e possib i l i t y o f n i f gene i n c o r p o r a t i o n i n t o the c h r o m o z o m e
of the p l a n t cell has been p o s t u l a t e d by P r o f . Sen a n d
his students at K a l y a n i U n i v e r s i t y , West Bengal.
P. Tauro:
R e g a r d i n g the K a l y a n i e x p e r i m e n t , w h a t was the
level o f acetylene r e d u c t i o n i n tissue culture?
N . S . Subba R a o :
E n d o g e n o u s ethylene p r o d u c t i o n is r u l e d o u t .
S.P. W a n i :
I s there a n y
response?
coordinated
trial
on inoculation
N . S . Subba R a o :
T h e r e are n o c o o r d i n a t e d t r i a l s . B u t w e are c o n d u c t i n g experiments at Parbhani and Coimbatore. We
have no facilities f o r large-scale testing.
P . A . Shinde:
D u r i n g the V I I F i v e Y e a r P l a n , p o s i t i o n s o f M i c r o biologists s h o u l d b e p r o p o s e d i n M i l l e t a n d
S o r g h u m projects o f the I C A R .
30
Associative Biological N i t r o g e n F i x a t i o n Research
at H a r y a n a A g r i c u l t u r a l University
B.S. K u n d u , K . R . D a d a r w a l , and P . T a u r o 1
Summary
Several
different
types
with
sorghum,
associated
as
determined
by
vary
widely
with
12S,
has
of bacteria
their
the
been found
brasilense.
It
pearl
to
also
dependent
nitrogen fixation.
used
the
in
cultivation
with
millet,
the
and
acetylene-reduction
isolates.
have
possesses
Its
of the
to
grown
activity
the
several
ARA
and
has
high
a
ARA
strong
is
above
been
The
and
the
as
C02,
levels
have
been found
quantity
the
isolates
identified
reduces
to
acetylene
Hisar.
hundred
hydrogenase,
insensitive
reduce
at
(ARA),
Among
of
conditions for
tested,
a
and
one
strain
optimal
sorghum
be
fixed,
A
RA
isolate,
of A z o s p i r i l l u m
carries
of inorganic
to
nitrogen
out
a
nitrogen
nitrategenerally
crops.
Introduction
O u r interest i n n i t r o g e n f i x a t i o n a t H a r y a n a A g r i c u l t u r a l U n i v e r s i t y ( H A U ) began i n the late 1970s
w h e n we isolated a s t r a i n of Pseudomonas azotogensis f r o m the r o o t s o f pearl m i l l e t t h a t c o u l d reduce
n i t r o g e n ( P r a b h a e t a l . 1980). Since t h e n this w o r k
has been strengthened a s p a r t o f a n I C A R c o o r d i nated research project o n B i o l o g i c a l N i t r o g e n F i x a t i o n ( B N F ) i n the D e p a r t m e n t o f M i c r o b i o l o g y . O u r
m a j o r interests are centered a r o u n d the i s o l a t i o n a n d
q u a n t i f i c a t i o n o f n i t r o g e n f i x a t i o n b y the v a r i o u s
bacteria f r o m the r o o t s o f s o r g h u m , p e a r l m i l l e t , a n d
wheat, three m a j o r crops g r o w n i n o r a r o u n d
Haryana.
Nitrogen Fixation in Pearl Millet
and Wheat
W e c a r r i e d o u t some i n i t i a l studies t o d e t e r m i n e the
t i m e o f d a y w h e n m a x i m u m n i t r o g e n f i x a t i o n occurs
a n d also the stage o f c r o p g r o w t h d u r i n g w h i c h
1.
ability
wheat
f i x a t i o n i s m a x i m u m . I n a l l studies r e p o r t e d h e r e i n ,
n i t r o g e n fixation was d e t e r m i n e d by m e a s u r i n g the
acetylene-reduction a c t i v i t y ( A R A ) .
In p e a r l m i l l e t , the in situ nitrogenase a c t i v i t y was
estimated at 50 days after s o w i n g ( f l o w e r i n g stage)
by the core assay m e t h o d . F o r this, s o i l cores of 15
c m d i a m e t e r a n d 2 2 c m d e p t h were r e m o v e d a l o n g
w i t h the p l a n t a n d transferred to 5 L a i r t i g h t plastic
buckets w i t h the p r o v i s i o n f o r i n j e c t i n g a n d s a m p l i n g acetylene. T h e cores were t h e n i n c u b a t e d i n
10% acetylene a t m o s p h e r e f o r 18 h at 3 0 - 3 2 ° C , after
w h i c h gas samples were w i t h d r a w n a n d analyzed f o r
the e x t e n t o f acetylene r e d u c t i o n . I n wheat, s i m i l a r
s o i l cores a r o u n d the plants were t a k e n a n d the
a d h e r i n g s o i l was r e m o v e d b y gentle s h a k i n g . T h e
t o p was c u t , a n d the r o o t s were t r a n s f e r r e d t o 500
m L assay j a r s a n d assayed a s before f o r A R A .
I n e x p e r i m e n t s t o d e t e r m i n e the d i u r n a l v a r i a t i o n
in nitrogen f i x a t i o n , we found that more than 25
µ moles o f acetylene was reduced per p l a n t s o i l core
in 24 h w h e n the p l a n t s were assayed a r o u n d n o o n ,
a n d f i x a t i o n was less either before o r after. I t was
f o u n d t h a t N 2 f i x a t i o n was m a x i m u m d u r i n g t h e
Associate Professor, National Fellow, and Professor of Eminence, Department of Microbiology, College of Basic Sciences and
Humanities, Haryana Agricultural University, Hisar, Haryana 125 004, India.
I C R I S A T (International Crops Research Institute for the Semi-Arid Tropics). 1986. Cereal nitrogen fixation. Proceedings of the W o r k i n g
Group Meeting, 9-12 Oct 1984, I C R I S A T Center, India. Patancheru, A . P . 502324, India: I C R I S A T .
31
p r e f l o w e r i n g stage a n d less thereafter. These
parameters were f o u n d t o b e s i m i l a r i n wheat. I t was
also f o u n d t h a t the extent o f A R A v a r i e d s i g n i f i c a n t l y w i t h the w h e a t v a r i e t y ; the h i g h - y i e l d i n g v a r ieties were generally ( b u t n o t always) f o u n d t o
d i s p l a y a h i g h e r level o f A R A .
Determination of M o s t Probable
N u m b e r ( M P N ) o f Nitrogen-fixing
B a c t e r i a Associated w i t h P e a r l M i l l e t
and Wheat
F o r the e s t i m a t i o n o f M P N , 1 0 varieties o f w h e a t
a n d 3 varieties of pearl m i l l e t were selected. P l a n t
samples were collected f r o m the n o n f e r t i l i z e d area o f
the u n i v e r s i t y f a r m d u r i n g the p r e f l o w e r i n g stage
a n d M P N estimated b o t h f r o m the r o o t washings
a n d f r o m the r o o t macerates. D e t a i l s o f the procedures f o l l o w e d are available f r o m the a u t h o r s . T h e
sterilized Dobereiner's semisolid m e d i u m was used
( D o b e r e i n e r e t a l . 1976) a n d the M P N was calcul a t e d f r o m the reference table o f H a r r i g a n a n d
M c C a n c e (1956), T h e r o o t s were washed g e n t l y f o r
r e m o v i n g the a d h e r i n g s o i l , cut i n t o 2-3 c m l o n g
pieces, a n d 5-g r o o t pieces were transferred to 45 mL
sterile saline a n d shaken a t r o o m t e m p e r a t u r e (283 0 ° C ) f o r l h . T h e washed r o o t pieces were t h e n
macerated i n sterilized m o r t a r a n d pestle a n d the
v o l u m e was made u p t o 5 0 m L . T e n - f o l d serial d i l u t i o n s f r o m r o o t w a s h i n g a n d r o o t macerate were
p r e p a r e d a n d used f o r M P N using Dobereiner's
semisolid m e d i u m ( D o b e r e i n e r et a l . 1976). F i v e
replicated tubes f r o m each d i l u t i o n were i n c u b a t e d
f o r 48 h at 3 0 ° C a n d t h e n 10% of the gas phase was
replaced w i t h acetylene.
I n wheat, the M P N i n r o o t washings v a r i e d f r o m
1.7 x 10 6 g - 1 of fresh r o o t s to 13 x 10 6 g - 1 , w h i l e in the
r o o t macerates, the n u m b e r v a r i e d between 0.6-5.5 x
10 6 g - 1 o f fresh r o o t weight. T h e h i g h - y i e l d i n g c u l t i vars W H 147, W H 157, H D 2009, a n d P 1030 h a d a
higher n u m b e r o f n i t r o g e n - f i x i n g bacteria associated w i t h t h e i r roots. I n contrast t o this, i n p e a r l
m i l l e t ( a rainy-season c r o p ) , the n u m b e r o f bacteria
f o u n d i n the r o o t washings were less t h a n i n the r o o t
macerates s u g g e s t i n g a p o s s i b l e r e l a t i o n s h i p
between the n u m b e r o f bacteria i n the r o o t zone a n d
the e n v i r o n m e n t a l t e m p e r a t u r e d u r i n g the g r o w t h
p e r i o d o f pearl m i l l e t . I t i s l i k e l y t h a t because o f the
h i g h t e m p e r a t u r e , a greater n u m b e r of bacteria are
f o u n d inside the r o o t s t h a n outside. I n w h e a t i t i s the
reverse, since the g r o w t h p e r i o d corresponds to a
32
t i m e w h e n the temperatures are moderate. I n b o t h
crops, however, there is a r e l a t i o n s h i p between the
n u m b e r o f bacteria f o u n d , the extent o f n i t r o g e n
f i x a t i o n , a n d the y i e l d p o t e n t i a l o f the v a r i e t y . These
results a l l o w e d us to c o n c l u d e t h a t a large n u m b e r of
bacteria associate w i t h the roots o f cereals g r o w n i n
this area.
F r o m the tubes t h a t showed A R A f r o m the above
e x p e r i m e n t , isolations were made o n N-free agar
m e d i u m , a n d 216 isolates f r o m p e a r l m i l l e t were
selected based o n c o l o n y m o r p h o l o g y , o u t o f w h i c h
183 were selected f o r f u r t h e r study. S i m i l a r l y , f r o m
wheat a n d s o r g h u m , a t o t a l of 146 cultures were
isolated f o r f u r t h e r study. T h e isolates i n c l u d e d b o t h
G r a m - p o s i t i v e a n d G r a m - n e g a t i v e bacteria b u t surp r i s i n g l y , except f o r one o r t w o cultures, a z o s p i r i l l a
were conspicuously absent. These cultures were
f o u n d t o v a r y w i d e l y i n t h e i r A R A , a n d the m e d i a
c o n d i t i o n s r e q u i r e d f o r t h e i r o p t i m a l A R A expression also v a r i e d ( B . S . K u n d u , u n p u b l i s h e d data).
T a b l e 1 shows the A R A o f some o f the b a c t e r i a l
isolates f r o m the roots o f the above three crops.
Isolate 12S f r o m s o r g h u m showed the highest A R A
under the assay c o n d i t i o n s ; b u t the value was m u c h
less t h a n the values r e p o r t e d f o r other bacteria isolated f r o m the roots o f cereals. T o check the p o t e n t i a l o f this o r g a n i s m w i t h the o t h e r cultures,
standard cultures were o b t a i n e d f r o m o t h e r l a b o r a tories a n d c u l t u r e d s i m i l a r l y a l o n g w i t h the t w o best
cultures f r o m each o f three crops. I t was f o u n d t h a t
the level o f A R A was highest w h e n c u l t u r e d i n semis o l i d m e d i u m a n d o n the 3 r d o r 4 t h d a y o f i n c u b a t i o n ( T a b l e 2). A m o n g a l l the cultures tested, isolate
12S h a d the highest a c t i v i t y a n d was considered to be
Table 1 . T h e A R A (nmoles C 2 H 4 tube - 1 d - 1 ) o f some isolates f r o m sorghum, pearl millet, a n d wheat.
Pearl millet
Isolate A R A
P M 1B
2B
3B
4B
5B
6B
7B
8B
9B
10B
MB
Sorghum
Wheat
31
84
906
996
756
276
252
ND
4
ND
4
1. ND = not detected.
Isolate
ARA
Isolate
ARA
W 1
12
168
12
24
19
29
17
190
20
12
14
S2
192
ND1
480
384
384
2
3
4
5
6
7
8
9
10
11
3
4
5
6
7
8
9
10
11
12
612
552
156
300
720
2160
T a b l e 2 . A R A (nmoles C 2 H 4 tube - 1 d - 1 ) level o f various
T a b l e 3.
nitrogen-fixing bacteria. 1
SP7.
G r o w t h rates of A. brasilense isolates 12S a n d
O . D . 600 n m a f t e r
A R A , days after i n o c u l a t i o n
6 d a y s at 3 0 °C
Bacteria
2
3
4
5
S.
itersonii
125
140
140
135
A.
brasilense,
1373
2420
2005
1595
s t r a i n SP 7
A.
6
7
95
861
602
brasilense,
s t r a i n 12S
1889
2595
2715
2990
1610
1365
I s o l a t e 8S
from sorghum
9
30
72
82
72
24
6
11
14
6
3
2
-
3
6
5
2
8
12
74
58
42
4
50
141
161
168
-
68
Isolate 4B
f r o m pearl millet
Treatment
12S
SP7
Control (-Malate)
0.02
0.09
+Malate
0.04
0.27
+Malate +Urea
0.05
0.38
+Malate + K N O 3
0.09
0.45
+Malate +NH4C1
0.09
0.48
Malate concentration: 5 gm L -1 ; N source: 5
µm.
Isolate 22B
f r o m pearl millet
Isolate 2 W
f r o m wheat
Isolate 8 W
f r o m wheat
1. Azotobacter and Rhizobia showed no A R A in this medium.
a s u p e r i o r c u l t u r e . It has been subsequently tested
f o r its A R A , c o m p a r e d w i t h A. brasilense SP7, a n d
f o u n d superior u n d e r a l l c o n d i t i o n s .
I t has been f o u n d t h a t A R A expression o f this
o r g a n i s m is highest w h e n it is c u l t u r e d in a semisolid
m e d i u m . F u r t h e r , f o r m a x i m a l expression the m e d i a
s h o u l d c o n t a i n m a l a t e as the c a r b o n source a n d
either v i t a m i n s o r yeast e x t r a c t . O p t i m u m p H
s h o u l d be 6-7 a n d the o p t i m u m t e m p e r a t u r e
3 0 ° ± 2 ° C u n d e r semisolid c o n d i t i o n s . Highest A R A
is detected w i t h i n 4-5 days.
T o estimate the n i t r o g e n - f i x i n g p o t e n t i a l o f isolate 12S, w e have t r i e d t o g r o w i t i n l i q u i d m e d i u m
f o r 24 h a n d t h e n transfer the cells to s e m i s o l i d
m e d i u m . By this it was possible to use a larger cell
mass a n d i n d u c e A R A earlier. U n d e r these c o n d i t i o n s , i t was possible t o r e c o r d higher A R A values,
f o r b o t h SP7 a n d 12S strains. W e believe t h a t this
s t r a i n has capacities higher t h a n 103 µ m o f A R A per
O D , w h i c h i s considerably higher t h a n a n y o t h e r
c u l t u r e tested so far.
U n l i k e SP7, the isolate 12S displays p o o r g r o w t h
under a l l c u l t u r a l c o n d i t i o n s ( T a b l e 3). U n t i l
recently, w e had n o m e d i u m i n w h i c h this c o u l d
g r o w u n i f o r m l y , b u t n o w w e have developed one
c o n t a i n i n g galactose, i n w h i c h this o r g a n i s m g r o w s
u n i f o r m l y as w e l l as possesses A R A a c t i v i t y
( B . S . K u n d u , u n p u b l i s h e d data). T h i s strain, l i k e
other strains of a z o s p i r i l l a , also possesses a s t r o n g
h y d r o g e n - o x i d i z i n g system that appears to be associated w i t h n i t r o g e n f i x a t i o n , since A R A a c t i v i t y i s
enhanced b y 3 1 % i n the presence o f 10% h y d r o g e n
( T a b l e 4). T h i s c u l t u r e has also been f o u n d to s h o w
a u t o t r o p h i c g r o w t h a n d the a b i l i t y t o reduce c a r b o n
d i o x i d e . T h e a b i l i t y t o f i x atmospheric n i t r o g e n ,
o x i d i z e h y d r o g e n , as w e l l as reduce c a r b o n d i o x i d e ,
makes this o r g a n i s m a n ideal choice f o r f u t u r e studies in associative symbiosis. T h e o n l y p r o b l e m is its
e x t r e m e l y slow rate o f g r o w t h .
A s s o c i a t i v e s y m b i o n t s perhaps have a role in soils
w i t h l o w n i t r o g e n levels o r under subsistence a g r i c u l t u r e . A l t h o u g h NO 3 -dependent N 2 r e d u c t i o n i s
k n o w n i n this o r g a n i s m , the n i t r o g e n m e t a b o l i s m o f
these o r g a n i s m s u n d e r c o n d i t i o n s of l o w s o i l N levels
becomes i m p o r t a n t . W e have e x a m i n e d its a b i l i t y t o
u t i l i z e different i n o r g a n i c f o r m s o f n i t r o g e n a n d its
relation to nitrogen fixation. When cultured in
T a b l e 4 . Enhancement o f A R A i n strain 12S a n d S P 7 o f
A. brasilense by hydrogen.
P h y s i o l o g i c a l Studies w i t h I s o l a t e 1 2 S
Isolate 12S was subjected to v a r i o u s tests, a n d it has
been f o u n d s i m i l a r to A. brasilense except in its
a n t i b i o t i c resistance p a t t e r n . W h i l e strain SP7 is
resistant to s t r e p t o m y c i n , s t r a i n 12S is resistant to
chloramphenicol.
ARA1
% increase
C2H2+H2
H 2 ase 2
in A R A
4220
5570
34
31
2564
2830
15
10
Strain
C2H2
12S
SP7
1. A R A : nm C 2 H 4 tube -1 d - 1
2. H 2 ase: µm H 2 consumed d -1 .
33
m e d i a c o n t a i n i n g 300 p p m n i t r a t e - N , this o r g a n i s m
carries o u t d e n i t r i f i c a t i o n . H o w e v e r , w h e n c u l t u r e d
u n d e r l o w e r levels of nitrat e N (60 p p m ) , it n o t o n l y
utilizes n i t r a t e b u t also fixes n i t r o g e n . A s c o m p a r e d
t o t h i s , A R A i s n o t detected a t c o n c e n t r a t i o n s o f
a m m o n i u m - N a s l o w a s 2 4 p p m . I n v i e w o f the fact
t h a t soils r a r e l y c o n t a i n such levels o f either n i t r a t e
o r a m m o n i c a l n i t r o g e n , w e can safely c o n c l u d e t h a t
u n d e r l o w - n i t r o g e n regimes, n i t r o g e n - f i x i n g a b i l i t y
of this o r g a n i s m is n o t affected.
F u t u r e Research Priorities
F u t u r e studies w i l l i n c l u d e e v a l u a t i o n o f strain 12S
under field c o n d i t i o n s i n s o r g h u m , pearl m i l l e t , a n d
wheat. A t present w e f i n d that its s u r v i v a l f o l l o w i n g
seed a p p l i c a t i o n is p o o r , a n d we have h a d d i f f i c u l t i e s
i n establishing its association w i t h any p a r t o f the
r o o t s . W e also have plans t o c o n t i n u e o u r b i o c h e m i cal studies w i t h this o r g a n i s m a n d t o understand this
o r g a n i s m genetically, w i t h a view to explore its usefulness i n the c u l t i v a t i o n o f cereals.
P o t - h o u s e a n d F i e l d Studies
Since isolate 12S was f o u n d to be very s i m i l a r to SP7
b u t h a d h i g h e r A R A , i t was decided t o test w h e t h e r i t
c o u l d associate w i t h the r o o t s o f some o f the crops
a n d f i x n i t r o g e n . I n pot-house experiments w i t h t w o
varieties of pearl m i l l e t , a significant response was
seen b o t h i n r o o t a n d shoot w e i g h t t o the seed a p p l i c a t i o n of these bacteria ( T a b l e 5). These observat i o n s led us to test the usefulness of this b a c t e r i u m
under field c o n d i t i o n s .
O u r studies have n o t s h o w n conclusively w h e t h e r
these bacteria excrete a n y g r o w t h - p r o m o t i n g substances. O u r estimates o f m a l a t e i n the r o o t s o f
s o r g h u m a n d pearl m i l l e t suggest a greater a c c u m u l a t i o n o f malate d u r i n g later stages o f g r o w t h w h e n
nitrogen fixation is m i n i m u m .
Acknowledgements
T h i s research was f i n a n c e d b y the I n d i a n C o u n c i l o f
A g r i c u l t u r a l Research ( I C A R ) , N e w D e l h i , a n d the
authors gratefully acknowledge the f i n a n c i a l
assistance.
References
Dobreiner, J . , M a r r e l I . E . , and N e r y , M . 1976. E c o l o g i c a l
d i s t r i b u t i o n of Spirillum lipoferum B e i j e r i n c k . C a n a d i a n
J o u r n a l o f M i c r o b i o l o g y 22:1464-1476.
T a b l e 5. Effect of seed inoculation in pearl millet cultivars.
Treatment
Control
Strain
Strain
Strain
CD
12S
4B
4B+22B
(P<0.05)
129
43
-
129
183
Parameter
Cultivars
N 2 - f i x e r s x 10 4 g - 1
HS 1
8
27
(Rhizosphere)
HS 4
54
246
N 2 - f i x e r s x 10 4 g - 1
HS 1
2
16
154
12
(Rhizosplane)
HS 4
134
134
162
222
HS 1
ND
Root
ARA
6.0
1.2
1.8
24.1
28.6
15.9
-
( C 2 H 4 g - 1 r o o t mass)
HS 4
D r y matter
HS 1(R)1
203
305
353
348
( m g / 3 plants)
H S 1(S) 2
580
726
883
773
HS 4(R)
435
643
570
630
72
H S 4(S)
683
900
883
953
240
12S: A. brasilense,
4B and 22B: Bacterial isolates f r o m pearl millet.
1. R = Root weight.
2. S = Shoot weight.
34
8.2
80.7
130
H a r r i g a n , W . F . , and M c C a n c e , M . E . 1956. P r o b a b i l i t y
tables f o r the e s t i m a t i o n o f b a c t e r i a l n u m b e r s b y the d i l u t i o n tube technique. In L a b o r a t o r y methods in m i c r o b i o l o g y . L o n d o n , U K : A c a d e m i c Press.
P r a b h a , S . , N a i r , S . K . , D a d a r w a l , K . R . , and T a u r o , P .
1980. Pseudomonas azotogensis: an associate s y m b i o n t of
Pennisetum americanum. P l a n t a n d S o i l 49:657-659.
Discussion
C . P . Ghonsikar:
W h i c h c a r b o h y d r a t e c o m p o u n d s were i d e n t i f i e d
besides m a l a t e a t a t i m e o f h i g h A R A a c t i v i t y ?
P. Tauro:
W e estimated malate e n z y m a t i c a l l y , a n d w e were
interested o n l y in malate since this c a r b o n source has
been f o u n d t o s u p p o r t o p t i m u m N 2 f i x a t i o n b y
Azospirillum.
N . S . Subba R a o :
O t h e r Kreb's cycle intermediates also need to be
studied.
P . Tauro:
O t h e r c a r b o n sources w i l l be tested. B u t m a l a t e c a n
be easily assayed e n z y m a t i c a l l y .
N . N . Prasad:
W h e n malate is less in r o o t extract, it increases w i t h
age w h i l e nitrogenase a c t i v i t y decreases w i t h age.
W h a t m a y b e the possible source o f c a r b o n f o r A z o s pirillum t o f i x a t m o s p h e r i c nitrogen? I n rice c r o p ,
w i t h o u t C4 p a t h w a y also, Azospirillum is effective
as r e p o r t e d by T N A U . So, I feel there is a need to
ascertain the n a t u r e o f c a r b o n c o m p o u n d s used b y
Azospirillum to f i x a t m o s p h e r i c n i t r o g e n .
P. Tauro:
We d i d n ' t study these aspects.
O . P . Rupela:
I n y o u r presentation y o u m e n t i o n e d that there was a
l o g 2 decrease i n p o p u l a t i o n o f i n o c u l a n t s t r a i n , i n
pots, w i t h i n a week. C o u l d y o u please give us some
figures? H o w d i d y o u estimate these p o p u l a t i o n s ?
P. Tauro
T h e r e was a decrease of l o g 2 ( f r o m 10 6 to 10 4 ) d u r i n g
the 1st week, w h i c h f u r t h e r decreased. A t the end o f
2 weeks, the s u r v i v a l was o n l y a b o u t 10 2 . T h e bacteria i n t r o d u c e d had a c h l o r a m p h e n i c o l m a r k e r .
35
Studies on the Interactions Between Azospirillum
and Pearl M i l l e t
A . V . R a o and B . Venkateswarlu1
Summary
Biologically
seed
active
and
stimulated
by
present
by
compounds—such
root
seed and
in
the
exudates
the
roots
A
marked
exhibited
amounts
of
were found
marked
increase
sandy
loam
and
with
in
consistent
A.
the
the
pearl
activity
compounds.
inadequate for
increase
in
Inoculation
grown
nitrogenase
carbon
sugars,
millet.
exudates,
exudates.
inoculation/ impregnation
lation,
root
of axenically
older plants.
as
of pearl
growth
acids,
and
was generally
related
significantly
enhanced
millet plants,
increase
and
in
Axenically
that
with
varied
However,
optimum
population
the
growth
and
organic
activity
to
the
the
the
carbon
exudation
was
activity
observed
in
in
the
upon
the
compounds
of various
compounds
more pronounced in
was
activity
root
addition
in
brasilense,
of organic
millet plants,
The
compounds present
organism
of A.
of roots
grown pearl
of the
acids—are present
quantity
effect
cultivars.
nitrogenase
was
and
nitrogenase
cell permeability
brasilense.
population
Azospirillum
amino
The
upon
was
in
related
the
zone,
as
root
there
of sucrose.
rhizoplane following
the
observed upon
inoculation
inocuto
the
exudates
was
a
Maximum
in
a
soil.
Introduction
Materials and Methods
Pearl m i l l e t , a n i m p o r t a n t c r o p o f the semi-arid
tropics ( S A T ) , has been reported to derive a g r o n o m ically significant a m o u n t s o f n i t r o g e n f r o m the
Azospirillum association ( S m i t h et a l . 1976). H o w ever, results of the i n o c u l a t i o n experiments have n o t
always been consistent (Barber et a l . 1979, N u r et a l .
1980, Vlassak a n d Reynders 1981, V e n k a t e s w a r l u
a n d R a o 1983). T h e causes f o r the p l a n t - g r o w t h
response f o l l o w i n g i n o c u l a t i o n are n o t clearly
k n o w n . T h e r e is also a lack of i n f o r m a t i o n on the
p h y s i o l o g i c a l interactions between the host p l a n t
a n d the m i c r o s y m b i o n t . I n the present study, a n
a t t e m p t has been made to investigate the interactions between the seed a n d r o o t exudates of pearl
m i l l e t w i t h Azospirillum a n d its i m p l i c a t i o n s in the
f o r m a t i o n o f the associative symbiosis.
T h e bacterial strain used in these experiments was
isolated f r o m the roots o f pearl m i l l e t a n d i d e n t i f i e d
as Azospirillum brasilense, f o l l o w i n g the m e t h o d
used by T a r r a n d et a l . (1978). T h e i n o c u l u m used in
these studies is a pure-cell suspension of 4 - d a y - o l d
A. brasilense ( O D = 0 . 4 g r o w n in n u t r i e n t b r o t h ) suspended in phosphate buffer.
Seed exudates were collected by s o a k i n g surfacesterilized seeds in d i s t i l l e d water f o r 24 h a n d t h o r o u g h l y rinsed w i t h d i s t i l l e d water. T h e exudates
were sterilized b y f i l t r a t i o n a n d stored a t 4 ° C . F o r
c o l l e c t i o n o f r o o t exudates, 12-day-old plants,
g r o w n a x e n i c a l l y in test tubes (25 x 200 m m ) c o n t a i n i n g acid-washed q u a r t z sand a n d watered w i t h
Hoagland's nutrient solution containing 50 p p m
a m m o n i u m n i t r a t e , were used. T h e exudates were
1.
Microbiologists, Central A r i d Zone Research Institute, Jodhpur, Rajasthan 342003, India.
I C R I S A T (International Crops Research Institute for the Semi-Arid Tropics). 1986, Cereal nitrogen fixation. Proceedings of the Working
Group Meeting, 9-12 Oct 1984, I C R I S A T Center, India. Patancheru, A.P. 502 324, India: I C R I S A T .
37
collected by rinsing the roots a n d the associated sand
i n d i s t i l l e d water, p o o l e d , p u r i f i e d , a n d concentrated
to represent 1 mL f o r every 10 seedlings.
T h e effect o f i n o c u l a t i o n w i t h A . brasilense o n the
p a t t e r n o f r o o t e x u d a t i o n was studied o n 3 - d a y - o l d
plants b y a d d i n g 1 m L pure-cell suspension t o each
tube. T h e c o n t r o l plants received the same a m o u n t
o f a u t o c l a v e d cells. R o o t exudates were p e r i o d i c a l l y
collected a n d analyzed f o r organic c a r b o n b y W a l k ley a n d B l a c k r a p i d t i t r a t i o n m e t h o d , t o t a l n i t r o g e n
b y m i c r o - K j e l d a h l m e t h o d , r e d u c i n g sugars b y N e l son's arseno-molybdate m e t h o d , a n d a m i n o n i t r o gen b y the n i n h y d r i n m e t h o d .
T o s t u d y the effect o f seed a n d r o o t exudates o n
the g r o w t h a n d nitrogenase a c t i v i t y of A. brasilense,
the exudates were i n c o r p o r a t e d i n the c u l t u r e m e d i a
i n different concentrations. F o r seed exudates,
Dobereiner's semisolid malate m e d i u m ( D o b e r e i n e r
a n d D a y 1976) w i t h 0.08% agar was used f o r g r o w t h ,
a n d w i t h 0.175% agar f o r nitrogenase a c t i v i t y . T h e
tubes after i n o c u l a t i o n were i n c u b a t e d a t 3 0 ° C f o r
7 2 h , a n d the g r o w t h was measured a s O D a t 520 n m .
F o r nitrogenase a c t i v i t y 2 days after i n c u b a t i o n , the
c o t t o n plugs were replaced b y Subaseals a n d 10% o f
the a i r was replaced w i t h C 2 H 2 . E t h y l e n e p r o d u c e d
2 4 h after i n c u b a t i o n was estimated b y a n A I M I L N u c o n gas c h r o m a t o g r a p h , e m p l o y i n g a P o r o p a k - T
c o l u m n (2 m * 0.003 m) w i t h N 2 as c a r r i e r gas. F o r
r o o t exudates, a malate l i q u i d m e d i u m , c o n t a i n i n g
o n l y 5 0 % o f the m a l i c a c i d r e c o m m e n d e d b u t supplem e n t e d w i t h 5 0 p p m a m m o n i u m sulfate, was used,
w h i l e f o r nitrogenase a c t i v i t y a semisolid malate
m e d i u m c o n t a i n i n g 5 0 % o f the r e c o m m e n d e d level
o f m a l i c a c i d was u t i l i z e d .
T o study the p e r m e a b i l i t y o f pearl m i l l e t r o o t s ,
one set of tubes was i n o c u l a t e d w i t h 1 mL cell suspension of A. brasilense on the 3 r d day, w h i l e the
c o n t r o l received the same a m o u n t o f a u t o c l a v e d
cells. A t 1 5 days after s o w i n g ( D A S ) , samples o f
fresh r o o t s were w r a p p e d in a cheese c l o t h a n d k e p t
i n d i s t i l l e d water. T h e contents were shaken f o r 5
m i n a n d the c o n d u c t i v i t y o f the b a t h i n g s o l u t i o n was
measured at 1 h intervals. F u r t h e r the r o o t m a t e r i a l
f r o m c o n t r o l plants was i m p r e g n a t e d w i t h p u r e cell
suspension of A. brasilense u n d e r v a c u u m , a n d the
p e r m e a b i l i t y was tested as described above.
T o measure nitrogenase a c t i v i t y o f p e a r l m i l l e t
plants, 1 m L o f i n o c u l u m a n d 4 m L o f 1 % sucrose i n
n u t r i e n t s o l u t i o n were a d d e d t o one set o f 3 - d a y - o l d
seedlings in a sterilized sand a n d v e r m i c u l i t e g r o w t h
m e d i u m , w h i l e the second set received the i n o c u l u m
a n d 4 m L o f n u t r i e n t s o l u t i o n . Nitrogenase a c t i v i t y
( C 2 H 2 r e d u c t i o n ) o f 2 1 - d a y - o l d plants was measured
38
after i n c u b a t i o n f o r 2 4 h w i t h 10% C 2 H 2 . A p p r o priate c o n t r o l s were m a i n t a i n e d to get absolute
activity.
To estimate Azospirillum p o p u l a t i o n s , the 3-dayo l d plants were i n o c u l a t e d w i t h 1 m L o f cell suspens i o n ( 1 0 8 cells m L - 1 ) . T h e p o p u l a t i o n s o f
Azospirillum i n b o t h the rhizosphere a n d n o n r h i z o s phere (tubes c o n t a i n i n g the g r o w t h m e d i u m b u t
w i t h o u t plants) were d e t e r m i n e d at 3-day intervals
by d i l u t i o n plate technique, using a malate-agar
m e d i u m supplemented w i t h 500 p p m a m m o n i u m
sulfate. To assess the establishment of this bacteria
f o l l o w i n g i n o c u l a t i o n , the n u m b e r s of Azospirillum
i n different r o o t zones, i.e., rhizosphere, r h i z o p l a n e ,
washed a n d crushed r o o t s , a n d surface-sterilized
a n d crushed r o o t s were estimated b y the M P N
m e t h o d f r o m 3 0 - d a y - o l d plants g r o w i n g i n a sandy
loam soil.
Results
P e a r l m i l l e t seed exudates c o n t a i n e d v a r i a b l e
a m o u n t s o f sugars a n d a m i n o acids, a n d the differences a m o n g c u l t i v a r s were n o t significant ( T a b l e 1).
R e d u c i n g sugars were m u c h higher t h a n a m i n o acids
i n a l l the c u l t i v a r s .
Seed exudates s t i m u l a t e d the g r o w t h of A. brasilense ( T a b l e 2). T h e increase in g r o w t h was n o t
d i r e c t l y p r o p o r t i o n a l t o the c o n c e n t r a t i o n o f the
exudates. T h e g r o w t h s t i m u l a t i o n appeared t o b e
due to the C a n d N c o m p o u n d s present in the e x u dates. T h e nitrogenase a c t i v i t y also increased w i t h
the a d d i t i o n o f exudates u p t o 9 % ( T a b l e 2). T h e r e
was a profuse g r o w t h in the semisolid m e d i u m , pos-
T a b l e 1 . Biochemical composition o f seed exudates o f
three pearl millet cultivars.
Compound
(mg/2.5 m L
Pearl millet cultivars
o f seed
exudates)
BJ
104
M
78
V.No. 2
Organic
carbon
3.10 ± 0.85 1
3.60 ± 1.12
3.50 ± 1.09
0.62 ± 0.028
0.66 ± 0.024
0.52 ± 0.037
0.32 ± 0.018
0.26 ± 0.017
0.31
0.53 ± 0.031
0.47 ± 0.024
0.54 ± 0.027
Reducing
sugars
Amino
nitrogen
± 0.021
Total
nitrogen
1. Mean ± Standard deviation.
T a b l e 2. Effect of seed exudates f r o m pearl millet cultivars on g r o w t h a n d nitrogenase activity of A. brasilense.
Concentration
Pearl millet cultivar
of seed
e x u d a t e s (%)
Parameter
Control
3
Growth (OD)
6
BJ 104
M 78
V No.2
Mean
0.22
-
0.28
0.28
0.33
0.296
0.29
0.35
0.37
0.337
9
-
0.30
0.35
0.41
0.353
12
-
0.31
0.36
0.41
0.360
Mean
-
0.295
0.355
0.380
0.349
Nitrogenase
activity
3
139
142
140
(nmol C2H4
6
-
160
186
198
181.3
tube-1 h-1)
9
-
14!
212
170
174.3
12
-
110
245
168
174.5
Mean
-
137.5
196.0
169.0
167.6
CD
(P<
0.05)
76
N 2 ase
Growth
0.016
Cultivar
Concentration
Interaction
C o n t r o l v s Rest
140.4
12.0
0.018
13.8
NS1
23.9
0.023
17.6
1. NS = Not significant.
sibly due t o the presence o f considerable n i t r o g e n o u s
c o m p o u n d s i n the exudates. S i m i l a r effects were
observed on the g r o w t h of A. brasilense w i t h i n d i v i d u a l a m i n o acids ( R a o a n d V e n k a t e s w a r l u 1982).
A t concentrations exceeding 9%, the a c t i v i t y was
reduced b u t g r o w t h was unaffected, i n d i c a t i n g the
repression o f the a c t i v i t y b y the a m i n o n i t r o g e n . T h i s
is evident f r o m the fact t h a t the a c t i v i t y was n o t
repressed even at higher concentrations w i t h c u l t i v a r
M 78, whose exudates c o n t a i n e d r e l a t i v e l y less of
total and amino nitrogen.
T h e data i n d i c a t e d t h a t pearl m i l l e t seed exudates
have a favorable effect on the g r o w t h of A. brasilense a n d m i g h t help i n the m u l t i p l i c a t i o n o f the
i n o c u l a t e d bacteria on the seed. Hence, s o a k i n g
seeds before i n o c u l a t i o n , as r e c o m m e n d e d f o r r h i z o b i a l i n o c u l a t i o n , m a y n o t be necessary f o r p e a r l
millet.
The data on exudation of various organic c o m p o u n d s b y p e a r l m i l l e t roots i n d i c a t e d t h a t m a r k e d
q u a n t i t a t i v e differences exist a m o n g c u l t i v a r s i n the
e x u d a t i o n p a t t e r n ( F i g . 1). T h e exudates o f c u l t i v a r
B J 104 c o n t a i n e d the highest a m o u n t s o f o r g a n i c
c a r b o n a n d r e d u c i n g sugars, f o l l o w e d b y c u l t i v a r
M B H 110. Q u a l i t a t i v e analysis o f the exudates b y
t h i n - l a y e r c h r o m a t o g r a p h y s h o w e d the presence o f
a m i n o acids l i k e g l u t a m i c a c i d , t r y p t o p h a n e , cysteine, a n d asparagine, a n d sugars l i k e glucose, f r u c -
BJ 1 0 4
MBH 1 1 0
MP 15
MBH 1 1 8
600
60
400
40
200
20
0
Reducing
0
Organic carbon
80
6
60
4
40
2
0
0
Total
Figure
sugars
1.
nitrogen
Amino
nitrogen
Chemical composition of root exudates
f r o m f o u r pearl millet varieties.
39
tose, a n d sucrose. O r g a n i c acids l i k e c i t r a t e , m a l a t e ,
a n d succinate were also detected, b u t i n v e r y s m a l l
quantities.
T h e g r o w t h a n d nitrogenase a c t i v i t y of A. brasilense were s t i m u l a t e d b y the i n c o r p o r a t i o n o f r o o t
exudates in a m e d i u m c o n t a i n i n g a s u b o p t i m a l level
o f c a r b o n source. T h e g r o w t h enhancement v a r i e d
w i t h the v a r i e t y a n d was generally related t o the
a m o u n t o f c a r b o n c o m p o u n d s present i n the e x u dates ( T a b l e 3 ) .
The e x u d a t i o n of organic compounds by the roots
o f a x e n i c a l l y g r o w n pearl m i l l e t was greatly
increased in the presence of A. brasilense ( T a b l e 4).
Inoculated plants exuded m a r k e d l y higher amounts
o f o r g a n i c c a r b o n , r e d u c i n g sugars, a n d t o t a l a n d
a m i n o n i t r o g e n . Increased r o o t e x u d a t i o n i n the
presence o f N 2 - f i x i n g bacteria has also been f o u n d i n
s o r g h u m ( L e e a n d G a s k i n s 1982) a n d i n w h e a t ( B e c k
a n d G i l m o u r 1983). Changes i n the p e r m e a b i l i t y a n d
a l t e r a t i o n i n r o o t m e t a b o l i s m m i g h t b e some o f the
reasons f o r the enhanced r o o t e x u d a t i o n u p o n i n o c u l a t i o n ( R o v i r a 1965), T h e enhancement i n the r o o t
e x u d a t i o n o f v a r i o u s c o m p o u n d s i n t h e present
s t u d y was essentially due to the increased p e r m e a b i l i t y o f the r o o t cells, w h i c h increased b y 8.5% o v e r the
c o n t r o l , w h i l e w i t h the direct i m p r e g n a t i o n w i t h
p u r e cells of A. brasilense, the increase was a b o u t
4 0 % , i n d i c a t i n g some p h y s i o l o g i c a l i n t e r a c t i o n
between r o o t s a n d the b a c t e r i u m at the cellular level
a n d / o r i n j u r y t o the r o o t s caused d u e t o v a c u u m
impregnation.
The
population
o f Azospirillum
increased
m a r k e d l y a n d r e m a i n e d m u c h higher i n the r h i z o s phere a s c o m p a r e d t o the n o n r h i z o s p h e r e , i n d i c a t i n g
t h a t t h e o r g a n i c c o m p o u n d s i n the r o o t exudates
aided the m u l t i p l i c a t i o n of Azospirillum. T h e
nitrogenase a c t i v i t y o f p e a r l m i l l e t cultivars was gene r a l l y related t o the organic c a r b o n a n d r e d u c i n g
T a b l e 3.
T a b l e 4 . Changes
in
r o o t e x u d a t i o n pattern i n pearl
millet, as influenced by inoculation w i t h A. brasilense.
Compound
A g e o f the plant ( D A S )
(µg/10
seedlings)
12
15
Mean
412
466
492
456.7
36.64
I n o c u l a t e d 481
548
590
539.7
49.40
Treatment
8
SD
Organic
carbon
Control
Total
nitrogen1
Control
59
67
75
67.0
9.91
Inoculated
68
79
88
78.3
9.99
Control
41
52
66
53
12.07
Inoculated
58
56
75
63
10.08
Reducing
sugars1
Amino
nitrogen1
Control
3.9
4.8
5.6
4.77
0.97
Inoculated
4.6
5.9
6.8
5.77
1.12
1. Significant at 5% level using Kruskal Wallis test.
sugars released in the r o o t exudates. H o w e v e r , these
results are f r o m t w o d i f f e r e n t sets o f e x p e r i m e n t s .
T h e close r e l a t i o n s h i p observed between the o r g a n i c
c a r b o n i n the exudates a n d the g r o w t h o f A z o s p i r i l lum i n v i t r o a n d nitrogenase a c t i v i t y o f the i n t a c t
p l a n t s emphasized the role o f r o o t exudates i n the
genetic v a r i a t i o n i n nitrogenase a c t i v i t y . T h e c a r b o n
c o m p o u n d s present i n the exudates were, h o w e v e r ,
f o u n d t o b e inadequate f o r o p t i m u m a c t i v i t y a s there
was a spurt in the a c t i v i t y in a l l the 4 c u l t i v a r s w h e n
an e x t r a n e o u s c a r b o n source (sucrose) was s u p p l i e d
i n the r o o t zone ( T a b l e 5). S i m i l a r results have been
r e p o r t e d w i t h wheat ( L e t h b r i d g e a n d D a v i d s o n
1983).
Studies u n d e r field c o n d i t i o n s in a sandy l o a m soil
have also i n d i c a t e d that Azospirillum establishes in
the roots o f pearl m i l l e t u p o n i n o c u l a t i o n , a n d m a x i m u m M P N c o u n t s were f o u n d i n the rhizosphere
G r o w t h of A. brasilense as influenced by r o o t
exudates o f pearl millet cultivars.
Concentration
T a b l e 5. Nitrogenase activity of pearl millet cultivars, as
influenced by sucrose.
G r o w t h ( O D ) with cultivar
of root
BJ
MP
MBH
MBH
N 2 ase a c t i v i t y
e x u d a t e s (%)
104
15
110
118
(nmoles C 2 H 4 h-1 tube-1)
0 (Control)
0.202
-
-
-
5
0.218
0.209
0.215
0.210
10
0.236
0.221
0.231
0.225
15
0.259
0.235
0.250
0.242
20
0.281
0.262
0.273
0.269
SE±
40
0.033
Without
With
Cultivar
sucrose
sucrose
B J 104
21
49
M P 15
13
33
MBH
110
14
45
MBH
118
13
46
T a b l e 6. L o c a l i z a t i o n of Azospirillum in different root
zones of pearl millet, as influenced by inoculation.
M P N of Azospirillum (x 10 4 )
Plant-'
R o o t zone
g-1 d r y s o i l
Rhizosphere
Control
Inoculated
3.46 ± 0 . 4 7 1 5.50 ± 0.93
9.17 ± 1.41 11.00 ± 2.10
Rhizoplane
Control
Inoculated
-
Washed and
crushed roots
Control
Inoculated
-
Surface-sterilized
crushed roots
Control
Inoculated
-
-
-
g-1 dry root
42.20 ± 4.57
72.60 ± 8.78
0.39 ± 0.07
4.25 ± 0.96
3.03 ± 0.58
28.30 ± 3.89
1.02 ± 0.13
6.00 ± 1.13
7.90 ± 1.03
40.10 ± 5.79
0.33 ± 0.05
0.45 ± 0.09
2.46 ± 0.39
3.01 ± 0.47
1. ± standard deviation.
( T a b l e 6). T h e p o p u l a t i o n was m u c h higher i n
washed roots t h a n i n surface-sterilized roots, i n d i c a t i n g a greater c o n c e n t r a t i o n of the organisms on
the r o o t surface. S i m i l a r observations were recorded
in maize ( O k o n et a l . 1977). I n o c u l a t i o n s i g n i f i c a n t l y
increased the n u m b e r s in a l l the r o o t zones, but the
highest response was n o t e d in the r h i z o p l a n e . T h e r e
was a n i n e - f o l d increase in the p o p u l a t i o n in the
r h i z o p l a n e o n u n i t r o o t d r y weight basis. T h i s again
indicates t h a t the p o p u l a t i o n b u i l d u p largely takes
place on the r o o t surface.
F r o m these results, it m a y be c o n c l u d e d t h a t the
r o o t exudates o f pearl m i l l e t p l a y a n i m p o r t a n t r o l e
in the establishment of A. brasilense in the r o o t zone,
resulting in the successful f o r m a t i o n of the associative symbiosis.
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d e s c r i p t i o n s of a new genus Azospirillum gen. n o v . a n d t w o
species, Azospirillum lipoferum ( B e i j e r i n c k ) c o m b . n o v .
a n d Azospirillum brasilense sp. n o v . C a n a d i a n J o u r n a l of
M i c r o b i o l o g y 24:967-980.
Venkateswarlu, B . , and R a o , A . V . 1983. Response o f p e a r l
m i l l e t to i n o c u l a t i o n w i t h different strains of Azospirillum
brasilense. P l a n t a n d S o i l 74:379-386.
Vlassak, K . , and Reynders, L . 1981. A g r o n o m i c aspects o f
b i o l o g i c a l d i n i t r o g e n f i x a t i o n b y Azospirillum spp. i n t e m perate r e g i o n . Pages 93-100 in A s s o c i a t i v e N 2 - f i x a t i o n :
proceedings o f the I n t e r n a t i o n a l W o r k s h o p o n A s s o c i a t i v e
N 2 - f i x a t i o n , 2-6 J u l 1979, P i r a c i c a b a , B r a z i l ( V o s e , P . B . ,
a n d R u s c h e l , A . P . , eds.). V o l . 1 . B o c a R a t o n , F l o r i d a ,
U S A : C R C Press.
41
Discussion
N . S . Subba R a o :
W h a t w i l l be the s t o r y i n r o o t exudates o f a n y o t h e r
plant?
A.V.Rao:
W e have n o t studied the r o o t exudates o f o t h e r
plants. H o w e v e r , there are differences i n e x u d a t i o n
a m o n g plants because o f the v a r i a t i o n i n p h o t o s y n thetic p a t h w a y s a m o n g these plants.
P.V. Rai:
W h i l e the c o n c e n t r a t i o n o f seed exudate i s l i n e a r t o
Azospirillum p o p u l a t i o n increase, w h y does it
i n h i b i t the nitrogenase a c t i v i t y a t 6 % c o n c e n t r a t i o n
o f exudate?
A.V.Rao:
P r o b a b l y the n i t r o g e n c o n t e n t of seed exudate at
p r o p o r t i o n s higher t h a n 6 % i s i n h i b i t o r y t o
nitrogenase a c t i v i t y . Because of the N c o n c e n t r a t i o n , the p o p u l a t i o n o f Azospirillum i s m o r e b u t the
nitrogenase is i n h i b i t e d .
P.V.Rai:
W h a t is the difference between seed exudate a n d
r o o t exudate?
A.V.Rao:
W a s h e d seeds were soaked i n w a t e r a n d the c h e m i cals t h a t leaked f r o m seeds were called seed e x u dates.
R o o t exudates were collected after
germination.
P . V . Rai:
W h e n the Azospirillum p o p u l a t i o n is so w e l l m a i n t a i n e d b y the r o o t exudate, w h y does i t enter i n t o the
root?
A.V.Rao:
I t i s the character o f Azospirillum t o enter a n d c o l onize the endo-rhizosphere ( H i s t o s p h e r e ) . U n l i k e
Azotobacter, it is an associative s y m b i o t i c bacter i u m . T h i s o r g a n i s m i s observed u n d e r the e p i d e r m a l
layers a n d w i t h i n t h e c o r t e x .
B.S.Kundu:
N 2 - f i x a t i o n efficiency i s n o t c o r r e l a t e d t o d r y w e i g h t
b u t g r o w t h - p r o m o t i n g substance. S o w e s h o u l d l o o k
f o r organisms secreting m o r e o f g r o w t h - p r o m o t i n g
substance, a n d n o t f o r N 2 f i x a t i o n .
42
A.V.Rao:
I n case o f Azospirillum, g r o w t h regulators i n a d d i t i o n t o N 2 f i x a t i o n are responsible f o r enhanced d r y
m a t t e r a n d g r a i n y i e l d . E v e n i n legumes, there i s n o
c o r r e l a t i o n between the nitrogenase a c t i v i t y at a n y
stage a n d the y i e l d increase u p o n i n o c u l a t i o n .
B . K . Konde:
A t w h a t stage o f p l a n t g r o w t h d o y o u estimate A z o spirillum p o p u l a t i o n to d e t e r m i n e its l o c a l i z a t i o n ?
A.V.Rao:
A t 3 0 days o f p l a n t g r o w t h .
Response of Pearl M i l l e t to Inoculation w i t h Azospirillum
brasilense at V a r y i n g Levels of N i t r o g e n
G.S. Jadhav, A . N . G i r i , and N . B . Pawar1
Summary
Investigations carried out during 1979 to 1983 on black soils at Aurangabad and mixed red and
black soils at Vaijapur revealed that Azospirillum inoculation of seed alone increased the yield of
pearl millet by 14-20% over the control. A synergistic effect of Azospirillum inoculation with
nitrogen on increased yield was also observed, though it was not significant. On both soil types,
the highest yield increase was due to A. brasilense inoculation of seed at sowing + 40 kg N ha-1
applied in two splits. The effect of rainfall on response is discussed. The future strategy of research
for realizing the agronomic significance of Azospirillum inoculation in pearl millet-based c r o p ping systems is presented.
Introduction
I n experiments c o n d u c t e d under the A l l I n d i a C o o r d i n a t e d M i l l e t s I m p r o v e m e n t Project ( A I C M I P )
w i t h Azospirillum brasilense, the yields of pearl
m i l l e t increased s i g n i f i c a n t l y due to i n o c u l a t i o n at
D u r g a p u r a , were n o n s i g n i f i c a n t a t H i s a r , a n d d i d
n o t increase a t K a n p u r a n d J a m n a g a r ( A I C M I P
1982). T h u s , the results of field experiments have
been e x t r e m e l y variable. T h e i n f o r m a t i o n o n pearl
m i l l e t response to Azospirillum i n o c u l a t i o n is
meager, especially w i t h a n d w i t h o u t N a p p l i c a t i o n i n
M a h a r a s h t r a state. I n this paper, research o n p e a r l
m i l l e t response to i n o c u l a t i o n w i t h A. brasilense
c o n d u c t e d a t the A I C M I P Center, A u r a n g a b a d , a n d
at V a i j a p u r in M a h a r a s h t r a state is presented.
m i x e d red a n d b l a c k soils a t V a i j a p u r , t o study the
effect of A. brasilense a n d fertilizer n i t r o g e n on g r a i n
y i e l d o f pearl m i l l e t h y b r i d B J 104 a n d its e c o n o m i c s .
T h e i n i t i a l s o i l - f e r t i l i t y status o f e x p e r i m e n t a l
soils is given in T a b l e 1.
A l l the experiments reported here were c o n d u c t e d
d u r i n g the r a i n y season. E i g h t treatments ( T a b l e 2)
were replicated f o u r times in a r a n d o m i z e d - b l o c k
T a b l e 1 . I n i t i a l fertility status o f e x p e r i m e n t a l soils i n
Aurangabad and Vaijapur, Maharashtra.
Aurangabad
Parameter
1979
O r g a n i c c a r b o n (%)
pH
Materials and Methods
1.
1981
1983
0.18
0.38
0.49
0.27
8.2
8.31
8.3
8.2
32.4
35.0
Available phosphorus
( k g ha- 1 )
F i e l d experiments were l a i d o u t d u r i n g 1979-81 o n
b l a c k soils a t A u r a n g a b a d a n d d u r i n g 1982-83 o n
1980
Vaijapur
11.6
14.4
Available potassium
(kg
ha-1)
448
448
347
350
Agronomist, Seed Production Specialist, and Plant Pathologist, Bajra Research Station, Marathwada Agricultural University, Paithan
Road, Aurangabad, Maharashtra, India.
I C R I S A T (International Crops Research Institute for the Semi-Arid Tropics). 1986. Cereal nitrogen fixation. Proceedings of the W o r k i n g
Group Meeting, 9-12 Oct 1984, I C R I S A T Center, India. Patancheru, A . P . 502 324, India: I C R I S A T .
43
T a b l e 2. Effect of
Azospirillum
brasilense a n d nitrogen on grain yield ( k g h a - 1 ) of pearl millet at A u r a n g a b a d and V a i j a p u r ,
Maharashtra.
Vaijapur
Aurangabad
Increase
Increase
over
1979
Treatments
1. C o n t r o l
1
2.
A.
3.
13.3 k g N ha-1
brasilense
4 . 13.3 k g N
ha-1
+ A.
brasilense
5. 20 k g N h a - '
6. 20 k g N
ha-1
+ A.,
brasilense
7. 4 0 k g N h a - '
8. 4 0 k g N h a - ' + A.,
brasilense
SE
CV
(%)
1980
1981
over
Pooled
control
average
(%)
1982
726
1431
1861
1834
1702
—
Pooled
control
1983
average
(%)
630
678
—
1789
2128
1882
1933
14
904
732
818
20.0
2053
1899
1998
1983
17
867
940
903
33.0
2151
2083
1825
2020
19
859
897
878
30.0
2173
2081
2254
2084
22
770
980
875
30.0
2464
2185
2102
2184
28
844
1147
996
47.0
2339
2250
2254
2284
34
807
1170
955
41.0
2421
2513
2085
2340
38
659
1250
955
41.0
±77
±193
±163
±166
6.4
15.8
16.30
—
±33
—
8.2
±88
18.3
1. Noninoculated and no nitrogen.
design. T h e c r o p was s o w n at a 45 cm x 10 cm
spacing. N i t r o g e n was applied as urea in t w o e q u a l
splits. H a l f the dose of N as per the treatment a n d 30
k g P 2 0 5 ha - 1 was a p p l i e d a t s o w i n g . T h e r e m a i n i n g
h a l f N was a p p l i e d at t i l l e r i n g . P e a r l m i l l e t seeds
were i n o c u l a t e d w i t h carrier-based i n o c u l a n t of A.
brasilense p r i o r to s o w i n g (as per the procedure of
S u b b a R a o 1981). C u l t u r a l operations were carried
o u t as a n d w h e n r e q u i r e d .
14% over the c o n t r o l . T h e i n t e r a c t i o n o f A z o s p i r i l lum w i t h n i t r o g e n was n o t statistically significant.
Seasonal r a i n f a l l at A u r a n g a b a d d u r i n g the three
years ranged between 605 a n d 690 m m . H o w e v e r ,
the response to Azospirillum i n o c u l a t i o n a l o n g w i t h
N a p p l i c a t i o n was significant o n l y d u r i n g 1979. T h i s
m a y be due to the l o w organic c a r b o n a n d general
l o w f e r t i l i t y of the field selected in 1979 ( T a b l e 1), as
A. brasilense is active in l o w - f e r t i l i t y soil ( D o b e reiner a n d D a y 1976).
Results
Vaijapur
Aurangabad
In 1979, i n o c u l a t i o n w i t h Azospirillum w i t h o u t N
a p p l i c a t i o n enhanced g r a i n y i e l d significantly over
the n o n i n o c u l a t e d c o n t r o l w i t h n o n i t r o g e n . T h e
a p p l i c a t i o n of 40 kg N ha - 1 + Azospirillum p r o d u c e d
the highest g r a i n y i e l d , w h i c h was o n par w i t h 4 0 k g
N ha -1 a n d 20 kg N ha -1 + Azospirillum i n o c u l a t i o n .
I n o c u l a t i o n w i t h A. brasilense p r o d u c e d s i g n i f i c a n t l y higher g r a i n y i e l d over the respective n o n inoculated controls only in combination w i t h 20 kg
N ha - 1 .
W i t h few exceptions, s i m i l a r b u t n o n s i g n i f i c a n t
increases in y i e l d were observed due to various treatments d u r i n g 1980 a n d 1981 ( T a b l e 2). P o o l e d data
analysis f o r three years revealed t h a t the A z o s p i r i l lum i n o c u l a t i o n alone increased the g r a i n y i e l d by
44
T h e treatment differences were significant d u r i n g
b o t h the years ( T a b l e 2). In 1982 Azospirillum i n o c u l a t i o n of seed at the t i m e of s o w i n g recorded the
highest g r a i n y i e l d , b u t the results were rather
inconsistent.
D u r i n g 1983 the g r a i n yields due to 20 a n d 40 kg N
ha -1
w i t h Azospirillum, as w e l l as 40 kg N ha -1
w i t h o u t Azospirillum, were c o m p a r a b l e a n d signific a n t l y higher t h a n a l l other treatments. T h o u g h
there i s inconsistency i n the results, d a t a f o r t w o
years indicated t h a t Azospirillum i n o c u l a t i o n of
seed at the t i m e of s o w i n g p r o d u c e d 2 0 % increase in
g r a i n y i e l d over the c o n t r o l ; the response to A z o s p i rillum i n o c u l a t i o n w i t h the a p p l i c a t i o n of 20 a n d 40
kg N ha - 1 was positive. T h e percent y i e l d increase
due to Azospirillum i n o c u l a t i o n w i t h or w i t h o u t N
a p p l i c a t i o n was m o r e m a r k e d i n the l o w - f e r t i l i t y ,
m i x e d red a n d black soil a t V a i j a p u r t h a n o n the
relatively fertile black soil at A u r a n g a b a d .
bacteria associations f o r increased yields c a n be
developed.
Acknowledgement
Economics
Inoculation
of Azospirillum
D a t a f r o m A u r a n g a b a d o n Azospirillum i n o c u l a t i o n reveal that, under p r e v a i l i n g prices o f different
i n p u t s , a p p l i c a t i o n o f 4 0 k g N ha - 1 i n t w o equal splits
+ Azospirillum i n o c u l a t i o n of seed is e c o n o m i c a l ;
the highest gross returns per ha (Rs 957) a n d net
p r o f i t (Rs 752) were o b t a i n e d f r o m this treatment.
T h u s i n o c u l a t i o n w i t h A. brasilense s i g n i f i c a n t l y
increased pearl m i l l e t y i e l d i n some years b u t n o t i n
others. W i t h few exceptions, the synergistic effect o f
Azospirillum a n d n i t r o g e n was observed at 20 a n d
40 kg N ha - 1 . T h e i n i t i a l s o i l - f e r t i l i t y status, r a i n f a l l ,
soil moisture, and possibly the n i t r o g e n - t r a n s f o r m a t i o n processes in the soil are responsible f o r v a r y i n g
degrees of responses observed in this study. T h e
increases i n c r o p yields w h e n inoculated w i t h A z o s pirillum species a n d further increases w h e n this is
supplemented w i t h 20 a n d 40 kg N ha - 1 finds s u p p o r t
f r o m the studies by S m i t h et a l . (1978), S u b b a R a o
(1981), a n d D a r t a n d W a n i (1982). T h e synergistic
effect of Azospirillum a n d fertilizer N m a y be a t t r i b uted t o a d d i t i o n a l N 2 f i x a t i o n and possible p r o d u c t i o n of plant harmones by A. brasilense at s m a l l
doses of n i t r o g e n a p p l i c a t i o n ( T i e n et a l . 1979).
Problems and Future Research
Needs
M o s t o f the farmers i n the S A T practice different
f o r m s o f i n t e r c r o p p i n g w i t h pearl m i l l e t . Pigeonpea
a n d g r o u n d n u t are established intercrops w i t h p e a r l
m i l l e t . T h e p o s s i b i l i t y of using Azospirillum a n d
Rhizobium i n o c u l a n t s , a l o n g w i t h a basal starter
dose of fertilizer n i t r o g e n , needs to be studied in
m i l l e t - l e g u m e i n t e r c r o p p i n g systems. T h e m a i n
a g r o n o m i c p r o b l e m w i t h the use of Azospirillum or
a n y other i n o c u l a n t i s the c o m p e t i t i o n i t w i l l face
f r o m physical, chemical, a n d b i o l o g i c a l factors i n
field situations. T h u s , there is a need to c o n d u c t a
large n u m b e r o f f a r m trials t o study the effects o f
e n v i r o n m e n t a l factors o n the efficiency o f n i t r o g e n
fixation by Azospirillum, so t h a t a viable a g r o t e c h n o l o g y f o r better m a n i p u l a t i o n o f these n o v e l p l a n t -
T h e authors w i s h t o t h a n k I C A R , N e w D e l h i , f o r
f i n a n c i a l s u p p o r t a n d the H e a d , D i v i s i o n o f M i c r o b i o l o g y , I A R I , N e w D e l h i , for s u p p l y i n g A z o s p i r i l lum culture. T h e y also acknowledge the technical
assistance received f r o m S . D . D h a w a n d k a r , B . G . H i wale, a n d D . T . G a r a d .
References
A I C M I P ( A l l I n d i a Coordinated Millets I m p r o v e m e n t
Project). 1982. Progress r e p o r t o f the A l l I n d i a C o o r d i n a t e d M i l l e t s I m p r o v e m e n t Project ( I C A R ) . P u n e ,
M a h a r a s h t r a , I n d i a : College o f A g r i c u l t u r e .
D a r t , P J . , and W a n i , S.P. 1982. N o n - s y m b i o t i c n i t r o g e n
f i x a t i o n a n d s o i l f e r t i l i t y . Pages 3-27 in N o n - s y m b i o t i c
n i t r o g e n f i x a t i o n a n d o r g a n i c m a t t e r i n the t r o p i c s . S y m p o sia paper 1 . T r a n s a c t i o n s o f the 12th I n t e r n a t i o n a l C o n gress o f S o i l Science, 8-16 Feb 1982, N e w D e l h i , I n d i a . N e w
D e l h i , I n d i a : I n d i a n A g r i c u l t u r a l Research I n s t i t u t e ,
Dobereiner, J . , and D a y , J . M . 1976. A s s o c i a t i v e s y m b i oses i n t r o p i c a l grasses: c h a r a c t e r i s a t i o n o f m i c r o o r g a nisms a n d d i n i t r o g e n - f i x i n g sites. Pages 518-538 in
Proceedings o f the F i r s t I n t e r n a t i o n a l S y m p o s i u m o n N i t r o g e n F i x a t i o n , 3-7 J u n 1974, P u l l m a n , W a s h i n g t o n , U S A
( N e w t o n , W . E . , a n d N y a m n , C . J . , eds.). V o l . 2 . P u l l m a n ,
W a s h i n g t o n , U S A : W a s h i n g t o n State U n i v e r s i t y Press.
S m i t h , R . L . , Schank, S.C., B o u t o n , J . H . , a n d Quesenberry, K . H . 1978. Y i e l d increases o f t r o p i c a l grasses after
i n o c u l a t i o n w i t h Spirillum lipoferum. Pages 380-385 in
E n v i r o n m e n t a l r o l e o f n i t r o g e n - f i x i n g blue-green algae a n d
a s y m b i o t i c bacteria ( G r a n h a l l , U . , ed.). E c o l o g i c a l B u l l e t ins n o . 26. S t o c k h o l m , Sweden: S w e d i s h N a t u r a l Research
Council.
Subba R a o , N . S .
1981. Response o f c r o p s t o Azospirillum
i n o c u l a t i o n i n I n d i a . Pages 137-144 i n A s s o c i a t i v e N 2 f i x a t i o n : proceedings o f the I n t e r n a t i o n a l W o r k s h o p o n
A s s o c i a t i v e N 2 - f i x a t i o n , 2-6 J u l 1979, P i r a c i c a b a , B r a z i l
( V o s e , P . B . , a n d R u s c h e l , A . P . , eds.). V o l . 1 . B o c a R a t o n ,
F l o r i d a , U S A : C R C Press.
T i e n , T . M . , Gaskins, M . H . , a n d H u b b e l , D . H . 1979. P l a n t
g r o w t h substances p r o d u c e d by Azospirillum brasilense
a n d t h e i r effect o n the g r o w t h o f p e a r l m i l l e t ( P e n n i s e t u m
americanum L . ) . A p p l i e d a n d E n v i r o n m e n t a l M i c r o b i o l o g y 37:1016-1024.
45
Rhizosphere Ecology and Nitrogen F i x a t i o n
of Azospirillum in P e a r l M i l l e t
D . Purushothaman and K . Govindarajan1
Summary
This paper describes the qualitative and quantitative occurrence of
nitrogen-fixing
bacteria,
particularly A z o s p i r i l l u m , in the root environments of pearl millet. In the rhizosphere, organisms
like A z o t o b a c t e r , Beijerinckia, ard D e r x i a were present in low density; however, the population
of A z o s p i r i l l u m was considerably high in the rhizoplane and rhizosphere. A z o s p i r i l l u m colonized
to the extent of 1-8% of the total heterotrophic bacteria. An analysis of 400 isolates of A z o s p i r i l l u m revealed that both A. l i p o f e r u m (79%) and A. brasilense (21%) were present in the root
environments.
The nitrogen-fixing ability of these isolates ranged from 3 to 24.8 mg N g-1 malic acid, which
corresponded to 50-450 n moles of ARA. The data on the population dynamics of A z o s p i r i l l u m in
the roots of pearl millet revealed that certain drug-resistant mutants were more competitive than
the native strain of A z o s p i r i l l u m in colonizing the rhizoplane and rhizosphere. In general, the
density of A z o s p i r i l l u m was maximum at the tillering and flowering stages of the crop. The root
exudates
of pearl millet
contained fairly high
concentrations of soluble sugars and amino
nitrogen.
Introduction
D i v e r g e n t views have been expressed on the use of
Azospirillum as an i n o c u l a n t in a g r i c u l t u r e ( B r o w n
1982). H o w e v e r , it is generally believed t h a t A z o s p i rillum, l i v i n g i n r h i z o c o e n o t i c a s s o c i a t i o n w i t h
several graminaceous crops, has a considerable
p o t e n t i a l f o r i m p r o v i n g yields i n extensive low-cost
f a r m i n g systems i n the tropics. I n this paper, the
rhizosphere ecology of Azospirillum spp a n d the
n i t r o g e n - f i x i n g p o t e n t i a l of its isolates are described
w i t h relevance t o pearl m i l l e t .
Materials and Methods
T h e drug-resistant m u t a n t s , str r a n d C A M r were
developed i n o u r l a b o r a t o r y b y exposure t o the
1.
c h e m i c a l m u t a g e n , e t h y l methane sulfonate ( E M S ) .
T h e y were resistant to 500 µ g ml - 1 of s t r e p t o m y c i n
and chloramphenicol. The nonnitrogen-fixing
m u t a n t , S3, was developed f r o m the parent c u l t u r e ,
C D ( A T C C : 29729, a p i n k isolate) a t the M i c r o b i o l ogy a n d C e l l Science L a b o r a t o r y , U n i v e r s i t y o f
Florida, USA.
A large n u m b e r of A z o s p i r i l l u m cultures were
isolated f r o m the r o o t tissues o f pearl m i l l e t g r o w n a t
the M i l l e t Breeding S t a t i o n o f the T N A U f o l l o w i n g
the e n r i c h m e n t technique detailed by D o b e r e i n e r
a n d D a y (1976).
Y o u n g a n d fresh r o o t bits, surface-sterilized w i t h
8 0 % e t h a n o l a n d washed w i t h sterile d i s t i l l e d water,
were p l u n g e d i n t o malate semisolid m e d i u m ( B a l d a n i a n d D o b e r e i n e r 1980) a n d supplemented w i t h
5 0 m g L - 1 o f yeast extract ( N F b ) . A l o o p f u l o f the
characteristic subsurface pellicle was streaked on
Associate Professor and Assistant Professor, Center of Advanced Studies in Agricultural Microbiology, T a m i l Nadu Agricultural
University, Coimbatore, T a m i l Nadu 641 003, India.
I C R I S A T (International Crops Research Institute for the Semi-Arid Tropics). 1986. Cereal nitrogen fixation. Proceedings of the Working
Group Meeting, 9-12 Oct 1984, I C R I S A T Center, India. Patancheru, A . P . 502 324, India: I C R I S A T .
47
N F b agar plates. T h e single colonies were p u r i f i e d
f u r t h e r b y streaking o n p o t a t o agar ( B M S ) a n d the
t y p i c a l p i n k , w r i n k l e d colonies were e x a m i n e d
m i c r o s c o p i c a l l y a n d transferred to trypticase soyagar slants. T h e species of Azospirillum isolates
were i d e n t i f i e d as per T a r r a n d et a l . (1978).
E n u m e r a t i o n o f the t o t a l h e t e r o t r o p h i c bacteria i n
the r o o t e n v i r o n m e n t s o f pearl m i l l e t was d o n e f o l l o w i n g the technique o f P r a m e r a n d S c h m i d t (1966).
E n u m e r a t i o n of Azotobacter was done by using
W a k s m a n m e d i u m no.77, o f Beijerinckia b y using
Jensen's m e d i u m , a n d of Derxia sp by using C o m pelo a n d Dobereiner's m e d i u m . T h e t o t a l d i a z o t r o p h s were enumerated b y using the m e d i u m o f
W a t a n a b e a n d B a r r a q u i o (1979). T h e most p r o b a b l e
n u m b e r ( M P N ) technique was used f o r d e t e r m i n i n g
the p o p u l a t i o n s of Azospirillum ( H e g a z i et al. 1979).
E a r t h e n pots (30 cm dia) c o n t a i n i n g red s o i l : c o m post 1:1 ( v / v ) a t 14 kg pot - 1 were used f o r p o t c u l t u r e
studies ( o r g a n i c c a r b o n 0.580%, t o t a l n i t r o g e n
1.12%, pH 7.0 a n d E . C . 0.46 m m h o s cm - 1 ). T h e soil
received a basal dose of 20 kg ha - 1 of N, 30 kg ha - 1 of
P2O5, and 30 kg ha-1 of K 2 O .
T h e seeds o f the pearl m i l l e t c u l t i v a r , X 3 , were
surface sterilized w i t h 0 . 1 % s o d i u m h y p o c h l o r i t e
a n d treated w i t h the peat-based i n o c u l u m o f Az os pirillum ( c o n t a i n i n g ca. 10 x 10 8 cells g - 1 ), using j a g g e r y
s o l u t i o n as an adhesive. A i r - d r i e d seeds were s o w n in
the pots. Each p o t also received a soil i n o c u l u m of
2 g of Azospirillum at the t i m e of s o w i n g . S t a n d a r d
a g r o n o m i c a n d c u l t i v a t i o n practices were f o l l o w e d .
A t desired intervals, the plants were u p r o o t e d a n d
rhizosphere a n d r h i z o p l a n e samples were collected
( P r a m e r a n d S c h m i d t 1966).
T h e drug-resistant m u t a n t s used in the study were
enumerated on O k o n ' s m e d i u m c o n t a i n i n g 500 µ g
mL - 1 of the respective a n t i b i o t i c ( O k o n et a l . 1977).
As S3 was a n a t u r a l l y p i n k p i g m e n t e d m u t a n t , it was
easy t o c o u n t t h e m o n trypticase soy agar m e d i u m .
F o r the e s t i m a t i o n o f N 2 f i x a t i o n , the isolates were
g r o w n i n the N F b semisolid m e d i u m (supplemented
w i t h 100 m g o f g l u t a m i c a c i d L - 1 ) f o r 7 days a t 30° C .
T o t a l n i t r o g e n was d e t e r m i n e d b y the m i c r o K j e l d a h l m e t h o d ( H u m p h r i e s 1956).
Acetylene r e d u c t i o n a c t i v i t y ( A R A ) o f 3-day-old
cultures, g r o w n i n 3 0 m l o f N free-semisolid malate
m e d i u m , was estimated b y i n c u b a t i n g the cultures
u n d e r 1 % acetylene f o r 6 h . I n the case o f the r o o t associated A R A , freshly collected r o o t s free f r o m
soil particles were t a k e n in the flasks a n d the assay
c o n d u c t e d s i m i l a r l y ( P u r u s h o t h a m a n et a l . 1979).
R o o t exudates were collected by the m e t h o d desc r i b e d b y D a z z o a n d H r a b a k ( 1 9 8 1 ) . A f t e r 7 , 14, a n d
48
21 days of seed g e r m i n a t i o n , the s o l u t i o n was aseptically collected, clarified by c e n t r i f u g a t i o n , a n d the
v o l u m e was suitably reduced. T o t a l sugars a n d
a m i n o n i t r o g e n i n the r o o t exudate were d e t e r m i n e d
f o l l o w i n g the methods o f Nelson (1944) a n d M o o r e
a n d Stein (1948).
Results
A n u m b e r of organisms l i k e Azotobacter, Beijerinckia, Derxia, a n d Azospirillum were observed in
the rhizosphere o f pearl m i l l e t . E x c e p t i n g A z o s p i r i l lum, the p o p u l a t i o n s of Azotobacter, Beijerinckia,
a n d Derxia were very p o o r in the rhizosphere a n d in
the r h i z o p l a n e ( T a b l e 1). Azospirillum was f o u n d to
occupy the roots in the rhizosphere a n d the r h i z o plane to the extent of 8% ( T a b l e 2). T h e r h i z o p l a n e
had higher c o l o n i z a t i o n by Azospirillum t h a n the
rhizosphere. Perhaps the a b u n d a n t m u c i g e l on the
r o o t surface i s helpful i n the a t t a c h m e n t o f m i c r o o r ganisms on to the rhizoplane. One of the reasons f o r
the intense m i c r o b i a l a c t i v i t y in the r o o t zone is the
e x u d a t i o n o f energy-rich and g r o w t h - p r o m o t i n g
organic c o m p o u n d s ( N e w m a n 1978, B r o w n 1982). I t
has been estimated t h a t 12-18% of the p h o t o s y n t h e t i c a l l y fixed c a r b o n is released as organic c o m p o u n d s
i n the r o o t e x u d a t i o n (Beck a n d G i l m o u r 1983). I n
o u r study, the a m o u n t o f t o t a l soluble sugars i n the
r o o t exudates ranged f r o m 0.6 to 2.25% a n d the
a m i n o n i t r o g e n varied f r o m 0.21 to 0.43%. It is
evident f r o m the study t h a t the rhizosphere a n d
r h i z o p l a n e are p o t e n t i a l sites f o r the c o l o n i z a t i o n of
Azospirillum. T h e p o p u l a t i o n s o f d i a z o t r o p h s i n
general a n d Azospirillum in p a r t i c u l a r , were h i g h at
the t i l l e r i n g a n d f l o w e r i n g stages o f the c r o p .
T h e species d i s t r i b u t i o n of Azospirillum in the
Table 1. Distribution
o f nitrogen-fixing organisms i n
pearl millet at flowering.
N o . o f bacteria g-1 d r y wt.
Organisms
Azotobacter
Azospirillum
Beijerinckia
sp
sp
sp
Rhizosphere
Rhizoplane
(soil)
(roots)
7.27 x 103
2.8 x 103
5
12.8 x 10.5
103
26.4 x 103
6.30
14.2
x 10
x
x 102
1.3 x 102
Others (unidentified)
15.25 x 105
6.8 x 105
Total heterotrophs
28.6
x 107
4 2 . 0 x 106
Derxia
sp
2.6
T a b l e 2.
Percentage distribution of Azospirillum a n d other diazotrophs to total bacteria in the roots of pearl millet.
G r o w t h phase o f c r o p
Root
environment
Seedling
Tillering
Flowering
Harvest
6.14
0.69
Mean
Rhizosphere
Azospirillum
2.39
1.6
Other diazotrophs
22.7
24.9
39.8
24.6
2.70
28.0
Rhizoplane
Azospirillum
0.8
4.2
8.62
Other diazotrophs
3.8
12.6
18.12
roots of pearl m i l l e t revealed t h a t A. lipoferum c o n stituted 7 9 % a n d A. brasilense 2 1 % . B o t h n i r + a n d
n i r strains were present a n d the n i r + isolates predominated (Fig.1).
A b o u t 8 0 % o f the isolates had very p o o r N 2 - f i x i n g
capacity (3-10 mg N g - 1 m a l i c acid)); w h i l e f o r 0.8%
isolates N 2 f i x e d was i n the range o f 21-25 m g N g - 1 o f
m a l i c acid ( T a b l e 3). T h e A R A , w h i c h also varied
considerably f r o m 5 0 t o 450 nmoles C 2 H 4 isolate - 1
h - 1 , was l o w i n 7 9 % o f the isolates a n d h i g h i n o n l y
9% isolates.
T h e drug-resistant m u t a n t s in general e x h i b i t e d a
m u c h higher c o l o n i z i n g a b i l i t y b o t h i n the r h i z o sphere a n d i n the r h i z o p l a n e ( T a b l e 4). T h e native
p o p u l a t i o n was p o o r i n c o m p e t i t i v e c o l o n i z a t i o n i n
the rhizosphere; however, on the r h i z o p l a n e t h e i r
n u m b e r was f a i r l y h i g h . It is of interest to observe
that the n o n n i t r o g e n - f i x i n g m u t a n t , S3, also possessed a m u c h better c o l o n i z i n g a b i l i t y t h a n the
Azospirillum
A.
0.22
3.46
11.33
10.8
native isolates. T h e root-associated A R A o f p e a r l
m i l l e t inoculated w i t h C A M r i s also f o u n d t o b e
significantly higher ( T a b l e 5).
Recently, increased c o l o n i z a t i o n of Rhizobium
phaseoli in the rhizosphere of bean has been
achieved b y the use o f fungicide-resistant strains a n d
T a b l e 3 . N i t r o g e n f i x a t i o n a n d A R A b y 4 0 0 isolates o f
Azospirillum spp f r o m the roots of pearl millet.
N2 fixation
Distri-
range ( m g N
bution
g-1 malic acid)
(%)
1
1-5
II
6-10
III
IV
Group
V
Isolates
lipoferum
ARA
Distri-
(n mol C 2 H 4 butrion
tube-1 h-1)
<%)
56.8
10-50
46.5
21.6
51-100
33.0
11-15
12.5
101-200
12.5
16-20
8.3
201-400
6.8
21-25
0.8
401-500
2.2
(400)
A.
(79%)
brasilense
(21%)
nir+
nir-
nir+
nir-
(58%)
(21%)
(10%)
(11%)
nir + :
Converts
NO3
to
NO2
and beyond NO2
n i r - : C o n v e r t s NO3 to NO2 and n o t beyond
Figure 1. Distribution of Azospirillum spp in the rhizosphere of pearl millet.
49
also b y a p p l y i n g fungicides t o the s o i l ( M e n d e z C a s t r o a n d A l e x a n d e r 1983).
T h e s t r i k i n g increase i n the root-associated A R A
o f p e a r l m i l l e t , i n o c u l a t e d w i t h the m u t a n t strains, i s
n o d o u b t due t o the increased c o l o n i z a t i o n o f the
r o o t e n v i r o n m e n t w i t h efficient n i t r o g e n - f i x i n g
strains. T h i s opens up newer hopes f o r e x p l o i t i n g the
potentials o f Azospirillum i n n i t r o g e n f i x a t i o n .
T h o u g h w e c o u l d n o t trace the n u t r i e n t release i n the
r o o t exudates over a l o n g p e r i o d , the data collected
over 21 days clearly illustrate the a v a i l a b i l i t y of an
a b u n d a n t energy source i n the v i c i n i t y o f roots.
Recent studies o n the surface c o l o n i z a t i o n o f r o o t b y
m i c r o o r g a n i s m s have indicated t h a t o n l y 4-10% o f
the r o o t area is c o l o n i z e d by m i c r o o r g a n i s m s ( R u s sel 1977).
Table 4. Percent colonization of rhizosphere and rhizoplane of pearl millet by drug-resistant mutants and native
Azospirillum.
Locus and
stage o f c r o p
Rhizosphere
seedling
tillering
flowering
harvest
Mean
Native
isolate
Drug-resistant mutants
Strr
C A M r N 2 ase-S3 M e a n
0.20
3.60
3.60
0.31
14.24
14.92
14.92
12.19
13.26
29.09
27.12
14.30
3.7
5.5
43.1
25.0
1.92
14.06
20.94
19.32
Rhizoplane
seedling
tillering
flowering
harvest
2.7
14.8
17.8
6.9
24.2
61.4
44.7
25.0
3.7
58.9
40.2
24.6
4.3
20.5
10.0
3.7
Mean
10.55
28.82
31.85
9.62
7.85
13.27
22.18
12.95
17.05
19.85
23.72
15.05
Table 5. T h e A R A of pearl millet root as influenced by
inoculation w i t h different Azospirillum strains.
Strain
CAM
ARA
( n moles C 2 H 4
g-1 root h-1)
r
Strr
S3
Noninocuiated control
1. Significantly different from the control (P<0.05).
50
168.76 1
185.80 1
22.60
23.35
References
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51
Response of S o r g h u m a n d P e a r l M i l l e t Genotypes
to Azospirillum a n d Azotobacter Inoculations
C . P . Ghonsikar, R . S . R a u t , and G.B. Rudraksha1
Summary
Sorghum and pearl millet genotypes were tested in afield trial for their response to A z o s p i r i l l u m
and A z o t o b a c t e r inoculations. A z o s p i r i l l u m inoculation increased grain yield of sorghum by 18%
over the control as compared to 12% yield increase with A z o t o b a c t c r . Fodder yields in sorghum
and pearl millet reflected a significant influence of A z o t o b a c t e r and A z o s p i r i l l u m inoculations.
Sorghum hybrid CSH 9 and pearl millet genotypes MP 21 and Bangaon showed relatively better
response to A z o t o b a c t e r and A z o s p i r i l l u m inoculation than other genotypes. The results imply
that nitrogen-fixing organisms like A z o t o b a c t e r and A z o s p i r i l l u m showed an ability to enhance
biomass production in sorghum and pearl millet. However, it is necessary to locate specificity of
interaction between an isolate and host-plant species.
Introduction
N i t r o g e n s u p p l y t o s o r g h u m a n d pearl m i l l e t i s gene r a l l y a l i m i t i n g factor, p a r t i c u l a r l y w h e n they are
g r o w n o n l o w - f e r t i l i t y soils, a n d the e x p l o i t a t i o n o f
rhizospheric n i t r o g e n f i x a t i o n m a y p r o v e beneficial
f o r c r o p i m p r o v e m e n t . F i e l d e x p e r i m e n t s have
p r o v i d e d l i m i t e d i n f o r m a t i o n a n d there is a great
v a r i a b i l i t y i n the responses t o i n o c u l a t i o n s . I n o c u l a t i o n resulted i n increased yields, a l t h o u g h such
responses were v a r i a b l e d e p e n d i n g on the N status of
the soil a n d v a r i e t y o f the c r o p p l a n t e d . T h i s paper
reports the effect of Azotobacter a n d Azospirillum
i n o c u l a t i o n o n g r o w t h ( r o o t a n d s h o o t mass) a n d
y i e l d o f s o r g h u m a n d pearl m i l l e t u n d e r f i e l d
conditions.
i n o c u l a t e d either w i t h Azotobacter chroococcum or
Azospirillum brasilense, a n d n o n i n o c u l a t e d plants
served as a check t r e a t m e n t . T h e c r o p received 40 kg
N ha - 1 a n d 60 kg P 2 O 5 ha - 1 at the t i m e of s o w i n g . In
a n o t h e r f i e l d t r i a l , pearl m i l l e t c u l t i v a r s B a n g a o n , B J
104, B K 560, a n d M P 2 1 were p l a n t e d w i t h s i m i l a r
treatments. T h e c r o p received 25 kg N ha - 1 a n d 50 kg
P 2 O 5 ha - 1 . These t w o trials were c o n d u c t e d d u r i n g
the r a i n y season ( J u n - O c t ) in 6.0 m x 4.5 m plots
o r g a n i z e d i n a f a c t o r i a l R B D w i t h three replicates.
R o o t a n d s h o o t masses were measured p e r i o d i c a l l y
t o assess biomass p r o d u c t i o n . R o o t v o l u m e was
d e t e r m i n e d b y displacement o f w a t e r v o l u m e i n a
m e a s u r i n g c y l i n d e r . F i n a l l y , g r a i n a n d f o d d e r yields
were recorded at harvest.
Results
Materials and Methods
Sorghum cultivars C S H 1, C S H 5, C S H 6, C S H 9,
a n d S P V 297 were s o w n o n a t y p i c V e r t i s o l f i e l d w i t h
p H 8.2, O . C . 0.62%, C . E . C . 4 2 m e q 100 g - 1 s o i l a n d
h i g h m o i s t u r e - r e t e n t i o n capacity. T h e seeds were
1.
Sorghum
I n o c u l a t i o n o f s o r g h u m plants w i t h Azospirillum
increased r o o t mass by 2 9 % , s h o o t mass by 3 0 % , a n d
r o o t v o l u m e b y 3 9 % over the respective n o n i n o c u lated c o n t r o l s . T h e Azotobacter i n o c u l a t i o n i n -
Associate Dean, PG School, and Microbiologists, Department of Microbiology, Marathwada Agricultural University, Parbhani,
Maharashtra 431 402, India.
I C R I S A T (International Crops Research Institute for the Semi-Arid Tropics). 1986. Cereal nitrogen fixation. Proceedings of the W o r k i n g
Group Meeting, 9-12 Oct 1984, I C R I S A T Center, India. Patancheru, A . P . 502324, India: I C R I S A T .
53
Table
1.
spiriiium
on
Effect of inoculation with Azotobacter a n d Azothe
growth
of sorghum
during
the rainy
Table
2.
Effect o f inoculation w i t h Azotobacter a n d A z o -
spirillum o n the g r o w t h o f pearl m i l l e t , P a r b h a n i , rainy
season.
season.
Days after sowing
35
50
65
Days after sowing
Treatment
20
80
No inoculation
3.4
12.2
16.0
17.8
15.2
4.3
16.4
20.7
20.5
15.9
Treatment
M e a n r o o t mass ( g p l a n t - 1 )
Azotobacter
chroccoccum
brasilense
CD
(P<0.05)
18.5
21.7
22.9
15.3
0.89
2.0
2.7
3.7
NS1
No inoculation
32.6
111.8
165.1
167.6 156.0
chroccoccum
37.6
139.3
184.8
187.3
167.8
brasilense
CD
(P<0.05)
CD
(P<0.05)
156.2
190.5
203.8
182.5
7.6
15.8
12.7
23.0
10.1
2.1
5.5
6.5
5.1
4.6
3.2
7.9
8.8
8.5
8.01
3.3
0.6
6.6
0.83
8.1
1.72
7.8
0.99
6.18
0.91
— M e a n s h o o t mass ( g p l a n t - 1 ) —
No inoculation
22.3
72.7
89.4
67.6
59.6
31.6
94.4
103.4
86.8
79.7
32.4
8.6
88.0
9.0
97.8
10.4
78.9
NS1
67.6
9.4
chroccoccum
Azospirillwn
50.9
80
hrasilense
Azotobacter
Azospirillum
65
chroccoccum
M e a n s h o o t mass ( g p l a n t - 1 ) - - - - - - - - - - - - - Azotobacter
50
M e a n r o o t mass ( g p l a n t - 1 )
Azospirillum
6.4
35
No inoculation
Azotobacter
Azospirillum
20
brasilense
CD
(P<0.05)
1, NS = Not significant.
1. NS = Not significant.
creased the r o o t mass by 18%, s h o o t mass by 15%,
a n d r o o t v o l u m e b y 14% over the c o n t r o l ( T a b l e 1).
T h e observed enhancement i n r o o t a n d s h o o t mass
o f s o r g h u m , p a r t i c u l a r l y i n e a r l y g r o w t h stages a s a
result of Azotobacter a n d Azospirillum i n o c u l a t i o n ,
m a y b e ascribed t o g r o w t h - p r o m o t i n g substances
( L a k s h m i k u m a r i et a l . 1976, Shende et a l . 1977). T h e
12.2% increase i n g r a i n y i e l d o f s o r g h u m b y A z o t o bacter a n d 18.0% increase w i t h Azospirillum i n o c u l a t i o n was, however, statistically n o n s i g n i f i c a n t . T h e
m e a n increases i n f o d d e r y i e l d o f s o r g h u m genotypes
d u e to Azotobacter ( 3 2 % ) a n d Azospirillum ( 4 3 % )
i n o c u l a t i o n s were s i g n i f i c a n t l y greater t h a n n o n i n o c u l a t e d s o r g h u m p l o t s . A p p a r e n t l y Azotobacter
a n d Azospirillum i n o c u l a t i o n s influence biomass
p r o d u c t i o n greatly, b u t i t i s n o t s i g n i f i c a n t l y
reflected i n the g r a i n y i e l d .
addition to their k n o w n nitrogen-fixing ability. A z o tobacter i n o c u l a t i o n in general p r o d u c e d 0.23 t h a - 1
a d d i t i o n a l g r a i n over its n o n i n o c u l a t e d check, w h i l e
Azospirillum gave 0.14 t ha -1 a d d i t i o n a l g r a i n . T h i s
a d v a n t a g e h o w e v e r , was n o t s t a t i s t i c a l l y s i g n i f i c a n t .
Azotobacter i n o c u l a t i o n increased f o d d e r y i e l d of
pearl m i l l e t s i g n i f i c a n t l y a n d its effect was better
t h a n t h a t o f Azospirillum.
T h i s r e p o r t has s h o w n p o s i t i v e effects o f A z o t o bacter a n d Azospirillum i n o c u l a t i o n s to s o r g h u m
a n d p e a rl m i l l e t . I t m a y b e necessary t o i d e n t i f y
specificity between an isolate a n d h o s t - p l a n t species
t o realize the p o t e n t i a l o f r h i z o s p h e r i c n i t r o g e n f i x ers f o r increasing c r o p yields.
Pearl Millet
B o t h the bacteria s t i m u l a t e d r o o t g r o w t h s i g n i f i c a n t l y a n d the effect o f Azotobacter o n r o o t mass
was m o r e t h a n t h a t o f Azospirillum ( T a b l e 2 ) . T h e
i n o c u l a t e d p l a n t s also reflected h i g h e r r o o t v o l u m e
t h a n noninoculated plants. T h e inoculated plants
p r o d u c e d n e a r l y 1 4 % h i g h e r s h o o t mass t h a n n o n i n o c u l a t e d checks. T h e benefits f r o m such m i c r o o r ganisms m a y b e v i e w e d a s effects o f p l a n t g r o w t h
substances ( M i s h u s t i n a n d S h i l i n i k o v a 1969), i n
54
References
L a k s h m i k u m a r i , M . , K a v i m a n d a n , S . K . , and S u b b a R a o ,
N . S . 1976. O c c u r r e n c e o f n i t r o g e n f i x i n g Spirillum i n
roots of rice, s o r g h u m , maize a n d other plants. I n d i a n
J o u r n a l o f E x p e r i m e n t a l B i o l o g y 14:638-639.
Mishustin, E . N . , and S h i l n i k o v a , V . K . 1969. T h e b i o l o g i cal fixation of atmospheric nitrogen by free-living bacteria.
Pages 82-109 i n S o i l b i o l o g y : reviews o f research. P a r i s ,
France: U N E S C O .
Shende, S . T . , A p t e , R . G . , a n d Singh, T . 1977. I n f l u e n c e o f
Azotobacter o n g e r m i n a t i o n o f rice a n d c o t t o n seeds. C u r r e n t Science 46:675.
Research o n Cereal N i t r o g e n F i x a t i o n a t I C R I S A T
S.P.
Wani1
Summary
The acetylene-reduction assay for nitrogenase activity has shown that the roots of sorghum and
pearl millet stimulate N2 fixation, A soil-core assay used for measuring nitrogenase activity of
field-grown plants showed large plant-to-plant
variability.
An improved core assay (planted core
assay) developed at ICRISAT Center showed higher activity than that recorded by regular core
assay. In greenhouse assay methods plants with shoots sustained higher activity than the ones
whose tops were cut before the assay, Nitrogenase activity varied throughout the day, as well as
over the season. Activity was maximum at the late flowering or early grain-filling stage and it was
related to the ontogenetic development of the plant. Activity was favored in moist, warm (ca
35° C) soil and decreased with high levels of fertilizer N.
Genotypic variation in sorghum and pearl millet lines for stimulating rhizospheric nitrogenase
activity was observed.
Out of 284 pearl millet lines tested, 18 lines stimulated high nitrogenase
activity (>460 nmol C2H4 h-1 15 cm diam core-1) in the rhizosphere. Two lines, Gam 73 and J
1407, were consistently active over several seasons. Similarly, 28 of 334 sorghum lines tested had
high nitrogenase activity (>460 nmol C2H4 h-1 15 cm diam core-1).
At ICRISAT Center, pot-culture experiments with sorghum and pearl millet grown in a
low-fertility Alfisol,
or in unsterilized washed vermiculite,
showed substantial positive balances
for N. Long-term N balance studies in the field with sorghum and pearl millet cultivars are
continuing.
Using l5N2 it has been shown that 20-days-old sorghum seedlings fix N2 in the rhizosphere, and
part of it is taken up by the plant within 3 days after exposure. The 15N isotope dilution technique
has been evaluated for studying genotypic variation in sorghum and pearl millet cultivars for N2
fixation.
Many different kinds of bacteria closely associated with the roots of sorghum and pearl millet
plants show nitrogenase activity.
Responses to inoculation in terms of increased dry-matter
production and N uptake have been observed in pot experiments with sorghum and pearl millet
grown in Alfisols and vermiculite. In five out of nine field trials with pearl millet cultivars,
inoculations
with N2-fixing bacteria significantly increased the grain and plant dry-matter yields
in all the cultivars.
Introduction
T h e s e m i - a r i d t r o p i c s ( S A T ) p r o d u c e 6 0 % o f the
w o r l d ' s s o r g h u m , a n d 9 5 % o f the w o r l d ' s m i l l e t s ,
f r o m a t o t a l c r o p p e d area o f a b o u t 7 0 m i l l i o n h a .
B o t h cereals r e s p o n d t o i n p u t s o f N , yet a l m o s t a l l
the p r o d u c t i o n i s w i t h o u t the use o f fertilizer. T h e
s o i l f e r t i l i t y w i t h respect t o N depends o n the rate o f
b i o l o g i c a l t u r n o v e r o f n i t r o g e n a n d the a m o u n t s o f
nitrogen added to soil t h r o u g h fertilizer, manure,
1. Microbiologist, Pearl Millet Improvement Program, I C R I S A T , Patancheru, A . P . 502 324, India.
I C R I S A T (International Crops Research Institute for the Semi-Arid Tropics). 1986. Cereal nitrogen fixation, Proceedings of the W o r k i n g
Group Meeting, 9-12 Oct 1984, I C R I S A T Center, India. Patancheru, A . P . 502324, India: I C R I S A T .
55
and biological nitrogen fixation.
T h e o v e r a l l objectives o f the I C R I S A T research
p r o g r a m t h a t concentrates o n b i o l o g i c a l N 2 f i x a t i o n
associated w i t h s o r g h u m a n d m i l l e t are
1 . T o q u a n t i f y the a m o u n t s o f N 2 - f i x a t i o n a n d t o
gauge its i m p o r t a n c e i n r e l a t i o n t o o t h e r N i n p u t s
t h a t sustain cereal c r o p p r o d u c t i o n i n the S A T .
2 . T o seek ways o f e n h a n c i n g this a c t i v i t y b y
genetic, a g r o n o m i c , o r m i c r o b i o l o g i c a l m e t h o d s .
Development of Acetylene Reduction
Assay ( A R A ) f o r M e a s u r i n g
Nitrogenase Activity
C e n t r a l t o o u r research i s the d e v e l o p m e n t o f suitable assay techniques to measure nitrogenase
activity.
A soil-core assay m e t h o d has been s t a n d a r d i z e d
f o r measuring acetylene-reduction activity ( A R A )
associated w i t h f i e l d - g r o w n plants. S o i l cores c o n t a i n i n g p l a n t r o o t s are t a k e n i n m e t a l c y l i n d e r s (15
c m diameter, 2 2 c m length) w i t h a s l i t t l e soil d i s t u r bance as possible, a n d i n c u b a t e d in sealed 6 L plastic
vessels u n d e r an atmosphere of ca 15% acetylene in
a i r ( F i g . 1). Gas samples are t a k e n after 1 a n d 6 h a n d
later analyzed f o r ethylene p r o d u c t i o n b y gas c h r o m a t o g r a p h y . T h e a c t i v i t y o f such cores i s u s u a l l y
linear w i t h l i t t l e o r n o l a g p e r i o d p r i o r t o the onset o f
nitrogenase a c t i v i t y . W i t h t h i s assay t e c h n i q u e , w e
e n c o u n t e r e d large p l a n t - t o - p l a n t v a r i a b i l i t y i n
nitrogenase a c t i v i t y . W e have s t u d i e d the factors
responsible f o r such p l a n t - t o - p l a n t v a r i a b i l i t y a n d
m o d i f i e d o u r techniques a c c o r d i n g l y .
M e c h a n i c a l disturbance. T h e cores s a m p l e d a n d
t r a n s p o r t e d w i t h p r e c a u t i o n s h a d t h r e e - f o l d greater
a c t i v i t y t h a n the cores sampled a n d t r a n s p o r t e d
normally.
T i m e l a g between severing t h e p l a n t t o p s a n d injecti n g C 2 H 2 . T h e highest a c t i v i t y was r e c o r d e d w h e n
the t i m e l a g was least, i.e., a t 0.5 h . A n increase i n the
t i m e t a k e n t o inject t h e C 2 H 2 s i g n i f i c a n t l y reduced
the a c t i v i t y r e c o r d e d , w i t h a s i g n i f i c a n t negative
correlation (r = -0.421, P < 0 . 0 1 ) .
Temperature. M o s t a c t i v i t y was r e c o r d e d w h e n
the s o i l cores were i n c u b a t e d a t 3 5 ° C ( W a n i e t a l .
1983).
Core size.
The activity varied f r o m field to field
a n d the cores t a k e n o v e r t h e c r o w n o f t h e p l a n t
56
c o n t a i n e d between 1 3 a n d 5 0 % o f the t o t a l a c t i v i t y o f
the p l a n t d e p e n d i n g o n its age ( R a o a n d D a r t 1981).
T i m e o f sampling. T h i s s i g n i f i c a n t l y affected
nitrogenase a c t i v i t y o f f i e l d - g r o w n p e a r l m i l l e t
h y b r i d N H B 3 . N i t r o g e n a s e a c t i v i t y increased f r o m
0915 to 1815 h d u r i n g the p h o t o p e r i o d a n d t h e n
declined d u r i n g the n i g h t u n t i l 0715 h the n e x t m o r n i n g . T h e a c t i v i t y changed l i t t l e between 0915 a n d
1500 h. H o w e v e r , s i g n i f i c a n t l y higher nitrogenase
a c t i v i t y was r e c o r d e d f o r the plants sampled between
1715 a n d 2115 h t h a n at 1115 h.
Soil moisture. T h e effect o f s o i l m o i s t u r e o n
nitrogenase a c t i v i t y was s t u d i e d i n t w o ways: (1) b y
u s i n g the line-source i r r i g a t i o n system a n d (2) b y
a d d i n g w a t e r t o cores j u s t before assay. B o t h
m e t h o d s gave significant c o r r e l a t i o n s between s o i l
m o i s t u r e a n d nitrogenase a c t i v i t y .
O b v i o u s l y , i t i s necessary t o c o n t r o l a s m a n y o f the
a b o v e variables a s possible i f a n assay o f f i e l d - g r o w n
plants is to w o r k .
T h e a c t i v i t y r e c o r d e d w i t h the i m p r o v e d soil-core
assay t e c h n i q u e was s i g n i f i c a n t l y h i g h e r t h a n t h a t
r e c o r d e d w i t h the r e g u l a r soil-core assay t e c h n i q u e .
Nitrogenase activity of field-grown pearl millet
h y b r i d N H B 3 estimated b y b o t h the assay m e t h o d s
i n d i c a t e d t h a t activities o f m o s t o f the plants estim a t e d by the r e g u l a r core assay ranged f r o m 0 to 20
n m o l C 2 H 4 p l a n t - 1 h - 1 c o m p a r e d w i t h 100-250 n m o l
C 2 H 4 p l a n t - 1 h - 1 , f o r the i m p r o v e d p l a n t e d - c o r e
assay. M e a n a c t i v i t y recorded f o r p l a n t e d cores was
167 n m o l C 2 H 4 p l a n t - 1 h - 1 , s i g n i f i c a n t l y h i g h e r t h a n
the r e g u l a r core a c t i v i t y o f 1 8 n m o l C 2 H 4 p l a n t - 1 h - 1 .
A s plants aged, a c t i v i t y o f the r e g u l a r cores d e c l i n e d
m o r e t h a n t h a t o f p l a n t e d cores ( W a n i e t a l . 1983).
S i m i l a r results were o b t a i n e d w i t h s o r g h u m lines a s
s h o w n i n T a b l e 1 ( I C R I S A T 1983).
Because soil-core assays are t i m e c o n s u m i n g a n d
s o m e w h a t v a r i a b l e , w e have d e v e l o p e d a l t e r n a t i v e
assay systems — one where seedlings are g r o w n in
test tubes, a n d a n o t h e r where p l a n t s are g r o w n i n
pots.
T h e test t u b e c u l t u r e t e c h n i q u e has been develo p e d m a i n l y t o test the effect o f host g e n o t y p e a n d
b a c t e r i a l c u l t u r e o n nitrogenase a c t i v i t y . P l a n t s are
g r o w n i n 2 5 m m x 200 m m tubes, w i t h a s m a l l t u b e
a t t a c h e d t o t h e side near the b o t t o m . T h e tubes are
f i l l e d w i t h 20-25 m L o f g r o w t h m e d i u m , s o i l , sand,
s a n d : F Y M , v e r m i c u l i t e , o r nitrogen-free agar,
covered a n d p a i n t e d t o keep l i g h t f r o m the r o o t
m e d i u m . P l a n t s are g r o w n inside the t u b e , w h i c h i s
p l u g g e d w i t h c o t t o n w o o l u n t i l t h e assay, t h e n w i t h a
Figure 1. Steps in the soil-core assay technique for
measuring nitrogenase activity of field-grown
plants: (a) cutting plant at ground level and scraping
of algal growth on soil surface; (b) driving metal core
around plant roots; (c) lifting out soil-root core from
the ground; (d) putting the soil core in plastic container; and (e) sealing container with P V C tape and
injecting acetylene gas for incubation.
57
T a b l e 1 . Nitrogenase activity (nmoles C 2 H 4 p l a n t - 1 h - 1 ) o f
sorghum cultivars as estimated by regular-core a n d p l a n t ed-core assay methods a t I C R I S A T Center. 1
Cultivar
Regular-core
Planted-core
assay
assay
I S 1057
24
IS 2207
41
2101
253
IS 9180
33
295
I S 2638
33
316
I S 2391
38
682
I S 3951
37
448
I S 3949
30
267
CSV 5
61
335
Soil
20
119
35 b
535 c
Mean
1.
Average of 4 replicated cores. L o g transformation (of nmoles
C 2 H 4 + 1) used to analyze data. Figures appended with letters
vary significantly ( P < 0 . 0 5 ) f r o m each other.
r u b b e r S u b a seal; the side t u b e is also closed w i t h a
S u b a seal. A c e t y l e n e gas, e q u i v a l e n t t o 15% o f the
free v o l u m e in the tube, is injected t h r o u g h the b o t t o m S u b a seal.
Eleven lines o f pearl m i l l e t were s o w n a n d one
c o n t r o l tube w i t h o u t seed i n o c u l a t e d w i t h a r h i z o spheric extract of field-grown m i l l e t to p r o v i d e a
m i x e d i n o c u l u m . Differences between cultivars i n
s t i m u l a t i n g nitrogenase a c t i v i t y were a p p a r e n t by 20
days after s o w i n g ( D A S ) a n d r a n k i n g s were s i m i l a r
i n different g r o w t h media. T h e nitrogenase a c t i v i t y
associated w i t h pearl m i l l e t seedlings g r o w n i n verm i c u l i t e test tubes varies w i t h the c u l t u r e o f o r g a nisms used. A t 1 4 D A S a m a x i m u m a c t i v i t y o f 2 1
nmoles C 2 H 4 p l a n t - 1 d - 1 was o b t a i n e d w i t h a c u l t u r e
o f Derxia sp. T h e v a r i a b i l i t y a m o n g plants i n this
system was m u c h less t h a n f o r f i e l d - g r o w n p l a n t s . I t
m a y b e possible t o screen pearl m i l l e t lines m o r e
r e l i a b l y f o r differences i n their a b i l i t y t o s t i m u l a t e
nitrogenase a c t i v i t y in such a system.
S i m i l a r l y , the a c t i v i t y o f s o r g h u m C S H 6 seedlings i n o c u l a t e d w i t h Azospirillum
lipoferum.
A,
brasilense, Azotobacter chroococcum, a n d
Derxia
spp a t 8 D A S was e x a m i n e d . T h e highest a c t i v i t y o f
364 n m o l C 2 H 4 p l a n t - 1 d - 1 was o b t a i n e d w i t h plants
inoculated
with
Azotobacter chroococcum.
The
a c t i v i t y in the n o n i n o c u l a t e d c o n t r o l plants was 1.2
n m o l C 2 H 4 p l a n t - 1 d - 1 ( I C R I S A T 1980).
T h e a b o v e t e c h n i q u e was f u r t h e r i m p r o v e d b y
a l l o w i n g the shoots t o g r o w outside t h e test t u b e . A t
assay t i m e , S u b a seal a n d silicone r u b b e r were used
58
t o seal the gas phase a r o u n d the r o o t m e d i u m , w i t h
the s h o o t r e m a i n i n g free i n the a i r . T h i s m e t h o d
a g a i n p e r m i t t e d differences to be detected between
pearl m i l l e t lines b y 2 0 D A S . L i k e w i s e , m a x i m u m
f i e l d a c t i v i t y o f nine pearl m i l l e t lines recorded w i t h
r e g u l a r soil-core assays over three to six seasons a n d
the a c t i v i t y recorded w i t h seedlings g r o w n i n tubes
f i l l e d w i t h sand:farmyard m a n u r e (97:3 w / w ) a n d
v e r m i c u l i t e were p o s i t i v e l y correlated ( r = 0 . 6 5 ,
r = 0 . 6 7 , P < 0 . 0 5 ) ( I C R I S A T 1984).
We have also developed a nitrogenase assay
m e t h o d f o r i n t a c t plants i n pots, where o n l y the r o o t
system i n the p o t i s exposed t o acetylene. Q u i c k e r
a n d better d i f f u s i o n o f C 2 H 2 a n d several times
greater nitrogenase a c t i v i t y was observed w h e n acetylene was injected at the b o t t o m of the p o t t h a n at
the t o p . I n t a c t - p l a n t assays w i t h s o r g h u m a n d m i l l e t
showed linear rates of C 2 H 4 p r o d u c t i o n up to 16 h
w i t h a s m a l l l a g i n the b e g i n n i n g ( F i g . 2). T h e t i m e course assays i n d i c a t e d the f e a s i b i l i t y of m e a s u r i n g
C 2 H 2 r e d u c t i o n b y intact s o r g h u m a n d m i l l e t plants
g r o w n in pots. A f t e r 16 h C 2 H 4 p r o d u c t i o n increased
s l i g h t l y , suggesting that the i n c u b a t i o n p e r i o d f o r
e s t i m a t i n g A R A b y i n t a c t - p l a n t assays s h o u l d b e
less t h a n 16 h. In o u r assays we use 6-h i n c u b a t i o n
f o r e s t i m a t i n g A R A . T h e i n i t i a l lag p e r i o d f o r C 2 H 4
p r o d u c t i o n was possibly because o f the t i m e
70
60
50
40
30
20
10
0
5
20
10
15
Incubation time (h)
25
30
F i g u r e 2. Relationship between ethylene production
a n d i n c u b a t i o n t i m e ( h ) d u r i n g t h e assay o f i n t a c t
s o r g h u m plants. Bars represent ± S E .
r e q u i r e d f o r C 2 H 2 d i f f u s i o n t h r o u g h o u t the r o o t
m e d i u m i n the p o t .
H i g h e r A R A was associated w i t h the i n t a c t plants
t h a n w i t h decapitated plants. M e a n nitrogenase
a c t i v i t y of 4 9 - d a y - o l d plants across the 15 s o r g h u m
c u l t i v a r s w i t h i n t a c t shoots was 625 nmoles C 2 H 4
p l a n t - 1 h - 1 s i g n i f i c a n t l y higher ( P < 0 . 0 5 ) t h a n the
a c t i v i t y w i t h decapitated plants (247 nmoles C 2 H 4
p l a n t - 1 h - 1 ) . T h e r e was n o significant i n t e r a c t i o n
between the assay methods a n d the s o r g h u m c u l t i vars. There was a 26-times r e d u c t i o n in the a c t i v i t y
o f these c u l t i v a r s due t o d e c a p i t a t i o n o f t h e i r shoots
w i t h 7 6 - d a y - o l d plants as c o m p a r e d w i t h 2.5 times
r e d u c t i o n w i t h 4 9 - d a y - o l d plants. T h i s h i g h e r a c t i v i t y m i g h t be due to a c o n t i n u o u s s u p p l y of p h o t o s y n thate to the roots f r o m the intact shoot, or the
p h y s i o l o g i c a l processes of the roots m i g h t have been
d i s t u r b e d because o f d e c a p i t a t i o n . F u r t h e r studies
w i l l be required ( W a n i et a l . 1984).
W i t h plants g r o w n i n sand, higher nitrogenase
a c t i v i t y was observed w i t h increasing F Y M concent r a t i o n s . T h e increased a c t i v i t y m a y be due to better
p l a n t g r o w t h , w i t h increased F Y M c o n c e n t r a t i o n s
p r o v i d i n g m o r e root-surface area a n d m o r e r o o t
exudates (derived f r o m an increased r o o t mass) f o r
c o l o n i z a t i o n b y N 2 - f i x i n g bacteria. F u r t h e r e x p e r i m e n t a t i o n i s required t o understand the role o f F Y M
i n s t i m u l a t i n g nitrogenase a c t i v i t y associated w i t h
plants. Increased a c t i v i t y was recorded w i t h the
temperature increasing f r o m 2 7 ° C t o 3 3 ° C . S o i l
m o i s t u r e had a large effect on nitrogenase a c t i v i t y of
s o r g h u m a n d m i l l e t i n the i n t a c t - p l a n t assay.
T h e i n t a c t - p l a n t assay was tested f o r e s t i m a t i n g
nitrogenase a c t i v i t y associated w i t h 15 s o r g h u m c u l tivars, a n d w e c o u l d differentiate c u l t i v a r s w i t h h i g h
a n d l o w associated a c t i v i t y . T h e v a r i a b i l i t y between
plants g r o w n a n d assayed in pots was m u c h less t h a n
between f i e l d - g r o w n plants ( W a n i et a l . 1984). T h i s
suggested the p r o m i s e of using this technique f o r
screening s o r g h u m a n d m i l l e t c u l t i v a r s f o r t h e i r
potential to fix atmospheric nitrogen biologically,
after s t u d y i n g the r e l a t i o n s h i p between i n t a c t - p l a n t
assay a n d field assay methods. It p e r m i t s a study of
the a c t i v i t y a t different g r o w t h stages o f the p l a n t ,
c o l l e c t i o n of selfed seed f r o m the plants, a n d crossi n g between plants t h a t c o u l d be used f o r p r o d u c i n g
test h y b r i d s . M a x i m u m expressions o f the a c t i v i t y
c o u l d b e o b t a i n e d b y g r o w i n g plants i n 3 % F Y M
m i x e d w i t h sand o r A l f i s o l s o i l , b y w a t e r i n g the
plants t o 60-70% w a t e r - h o l d i n g capacity ( W H C ) , b y
m a i n t a i n i n g the t e m p e r a t u r e o f the p l a n t g r o w t h
m e d i u m a t a r o u n d 33° C , a n d b y n o t d e c a p i t a t i n g the
shoot.
H o s t Genetic Differences
in Promoting N2-fixation
D i f f e r e n t genotypes o f b o t h s o r g h u m a n d p e a r l
m i l l e t s h o w differences i n s t i m u l a t i n g nitrogenase
a c t i v i t y . A t I C R I S A T Center, 135 o u t o f the 284
m i l l e t lines tested s t i m u l a t e d nitrogenase a c t i v i t y
t h a t was m o r e t h a n t w i c e t h a t o f soil w i t h o u t p l a n t
roots. Eighteen lines s t i m u l a t e d h i g h nitrogenase
a c t i v i t y ( > 4 6 0 n m o l C 2 H 4 1 5 c m d i a m core - 1 h - 1 ) .
T w o lines, G a m 73 a n d J 1407, were consistently
active over several seasons ( I C R I S A T 1978, 1980,
1981; D a r t a n d W a n i 1982). Large p l a n t - t o - p l a n t
v a r i a b i l i t y i n nitrogenase a c t i v i t y was observed i n
the E x - B o r n u p o p u l a t i o n , r a n g i n g f r o m 0 to 1900
n m o l C 2 H 4 p l a n t - 1 h - 1 . W o r k o n s t a b i l i z i n g the character o f h i g h a n d l o w nitrogenase a c t i v i t y i n this
p o p u l a t i o n i s under w a y t o study the inheritance o f
this t r a i t ( I C R I S A T 1984).
I n s o r g h u m , 2 8 o u t o f 334 f i e l d - g r o w n lines tested
had h i g h nitrogenase a c t i v i t y ( > 4 6 0 n m o l C 2 H 4 1 5
c m d i a m core - 1 h - 1 ) associated w i t h t h e i r roots. T h e
active lines came f r o m I n d i a (12 o u t of 104 tested),
West A f r i c a ( 6 o f 36), East a n d C e n t r a l A f r i c a ( 5 o f
63), S o u t h A f r i c a ( 6 o f 29), U S A ( 2 o f 39), T h a i l a n d
(1 of 2), a n d J a p a n (1 of 3). H o w e v e r , 167 lines
s t i m u l a t e d nitrogenase a c t i v i t y of at least 100 n m o l
C 2 H 4 core - 1 h - 1 , w h i c h was m o r e t h a n t w i c e the m e a n
a c t i v i t y o f soil cores w i t h o u t p l a n t roots (range 0-40
n m o l C 2 H 4 core - 1 h - 1 ) . Fifteen lines have been c o n sistently active in three or m o r e seasons, t h o u g h they
are n o t consistently active on each assay occasion
d u r i n g the season ( I C R I S A T 1978, D a r t a n d W a n i
1982).
Several lines of m i n o r millets i n c l u d i n g Eleusine
coracana, Panicum sp, P. miliaceurn, a n d Setaria
italica have been screened f o r nitrogenase a c t i v i t y .
T h e p r o p o r t i o n o f active lines i n m i n o r m i l l e t s was
m o r e t h a n t h a t w i t h pearl m i l l e t lines ( I C R I S A T
1979, 1980). Several t r o p i c a l grasses b e l o n g i n g to the
genera
Brachiaria,
Cenchrus,
Chloris,
Cymbopogon,
Dicanthium,
Euchlaena,
Panicum, Pennisetum, Setaria, a n d Sorghum have been g r o w n
w i t h o u t a d d i t i o n o f a n y n i t r o g e n fertilizer over 7
years i n a n o b s e r v a t i o n p l o t . T h i r t y - f o u r o u t o f 4 8
entries were very active in s t i m u l a t i n g N 2 - f i x a t i o n , as
observed f r o m the data o b t a i n e d f r o m soil-core
assays w i t h these grasses. Some of the entries, e.g.,
Pennisetum purpureum a n d a cross between P. p u r pureum a n d P. americanum ( N a p i e r bajra), h a d
r e m a r k a b l y h i g h nitrogenase a c t i v i t y , reaching as
m u c h as 300 g N ha -1 d -1 ( I C R I S A T 1978).
59
Measurement of N2-fixation
Use o f
15
N techniques
15
N 2 Incorporation. I n the 1 5 N 2 i n c o r p o r a t i o n techn i q u e a p l a n t enclosed in a c o n t a i n e r is exposed to
15
N 2 , a n d after i n c u b a t i o n , p l a n t tissues are e x a m i n e d f o r a b o v e - n o r m a l c o n c e n t r a t i o n s o f the heavy
isotope. S o r g h u m C S H 5 seedlings g r o w n i n tubes i n
s a n d : F Y M ( 9 7 : 3 w / w ) m i x t u r e using a n i n t a c t - t u b e
assay system were exposed t o I 5 N 2 , b y e x c h a n g i n g
the gas i n the r o o t m e d i u m i n the test tubes b y w a t e r
displacement ( I C R I S A T 1983). T h e o x y g e n c o n t e n t
o f the r o o t zone was m o n i t o r e d a n d m a i n t a i n e d a t
2 0 % . A f t e r e x p o s i n g 2 0 - d a y - o l d seedlings t o labelled
15
N 2 in this f a s h i o n f o r 3 days, I 5 N was detected in the
g r o w t h m e d i u m (0.005 1 5 N a t o m % excess). Seven
days after the l a b e l l e d gas was r e m o v e d , the 1 5 N
a t o m % excess in the plants h a d increased considerab l y w i t h 0.029 1 5 N a t o m % excess i n the r o o t s a n d
0.019 I 5 N a t o m % excess in shoots ( I C R I S A T 1983).
V a r i a t i o n i n I 5 N due t o differences i n b o t h n a t u r a l
a b u n d a n c e a n d a n a l y t i c a l errors can a c c o u n t f o r a t
the m o s t ± 0.010 a t o m % I 5 N excess (Bergersen
1970).
A n a p p a r a t u s has been developed t o i n t r o d u c e gas
m i x t u r e i n t o the r o o t - g r o w t h c h a m b e r b y p u r g i n g
w i t h C O 2 f o l l o w e d b y a b s o r p t i o n o f the C O 2 w i t h
soda l i m e ( G i l l e r et a l . 1984) as suggested by W i t t y
a n d D a y (1978), thus a v o i d i n g f l o o d i n g the r o o t
zone w i t h w a t e r t o transfer the gas used i n the prev i o u s e x p e r i m e n t . U s i n g this device 2 4 - d a y - o l d
s o r g h u m seedlings were exposed t o I 5 N 2 . T h e results
c l e a r l y d e m o n s t r a t e d t h a t (1) detectable a m o u n t s o f
I5
N2 were f i x e d i n the rhizosphere o f s o r g h u m seed-
T a b l e 2.
I5
lings a n d that (2) f i x e d 1 5 N was i n c o r p o r a t e d i n t o the
p l a n t r o o t s a n d shoots w i t h i n 3 days o f i n i t i a l e x p o sure to the gas ( T a b l e 2). A f t e r a f u r t h e r week of
g r o w t h the i n c o r p o r a t i o n o f I 5 N i n the r o o t s a n d i n
the shoots h a d a l m o s t d o u b l e d . T h e a c t u a l a m o u n t
of I 5 N 2 fixed d u r i n g the exposure p e r i o d a n d the
a m o u n t w h i c h was i n c o r p o r a t e d i n t o the p l a n t r o o t s
a n d shoots can be estimated by c o n v e r t i n g the
results i n t o µ g N fixed, using the f o l l o w i n g f o r m u l a :
N fixed (µ g) =
15
N a t o m % excess in p l a n t
T o t a l plant N (µ g) x
15 N
a t o m % excess in gas phase
l5
N dilution. T h e isotope d i l u t i o n p r i n c i p l e has
been used in p o t e x p e r i m e n t s f o r d i f f e r e n t i a t i n g lines
of sorghum and millet for their potential to fix
a t m o s p h e r i c n i t r o g e n . S i x lines each o f s o r g h u m a n d
m i l l e t were g r o w n i n pots i n a washed v e r m i c u l i t e :sand m i x t u r e (1:1 w / w ) a n d watered w i t h 1 0 p p m N
s o l u t i o n w i t h 10 I 5 N a t o m % excess d a i l y as r e q u i r e d .
T h e percentage o f N 2 f i x e d b y a n i n d i v i d u a l line was
c a l c u l a t e d b y c o m p a r i n g i t t o the line s h o w i n g the
highest I 5 N a t o m % c o n t e n t a s c o n t r o l ( I C R I S A T
1984, G i l l e r et a l . 1986). S o r g h u m l i n e IS 801 d e r i v e d
2 7 % , a n d m i l l e t line D 180 17%, of t o t a l p l a n t N f r o m
B N F , i n c o m p a r i s o n w i t h l o w nitrogenase s t i m u l a t i n g s o r g h u m l i n e I S 3003 a n d p e a r l m i l l e t I C H 107.
H o w e v e r , i n these e x p e r i m e n t s considerable d i l u t i o n
o f a d d e d I 5 N was observed i n a l l the lines i n c o m p a r i son w i t h the I 5 N c o n t e n t o f the s o l u t i o n a d d e d ,
suggesting t h a t a source o f 1 4 N o t h e r t h a n f i x a t i o n
m a y b e present i n the system (seed o r v e r m i c u l i t e ) . I t
was n o t e d t h a t I 4 N released f r o m v e r m i c u l i t e d i l u t e d
N incorporated into 21-day-old sorghum C S H 5 plants after exposure of r o o t systems to
N 2 1 for 72 h.
Fixed N2
P l a n t age
D r y mass
Total N
(mg plant-1)
( m g plant-1)
at harvest
(days)
I5
Atom%
I5
N
excess
incorporated
(µg
plant-1)
24
7.2 3
10 ± 1.2
Shoot
264 ±
7.0 ± 0.28
0.056 ± 0.006
Root
2 4 6 ± 17.4
4.2 ± 0.26
0.056 ± 0.003
Total
5 1 0 ± 19.1
11.2 ± 0.33
Shoot
4 0 0 ± 13.4
5.9 ± 0.54
0.102 ± 0.022
16 ± 4 . 7
Root
673 ± 56.5
5.8 ± 0.52
0.073 ± 0.016
11 ± 3 . 2
Total
1073 ± 62.7
11.7 ± 0.96
6 ±0.5
16 ± 1.5
33
I5
1.
Mean enrichment 40.3 atom %
2.
3.
Calculated as total N x atom % I5 N excess in the plant divided by atom % I 5 N excess in gas.
Values are means of five and four replicates for the 24- and 33-day harvests, respectively, ± S.E.
60
N excess.
27 ± 7.9
15
N added. Therefore, the c o m p a r i s o n was m a d e t o
the line w i t h the highest I 5 N content. I n such e x p e r i ments e x t r a care has to be t a k e n to prevent the
systems g e t t i n g c o n t a m i n a t e d w i t h 1 4 N f r o m o t h e r
sources such a s w a t e r o r the g r o w t h m e d i u m . A n
a l t e r n a t i v e a p p r o a c h is to use u n i f o r m l y I 5 N labelled
s o i l i n pots f o r g r o w i n g lines t o screen f o r t h e i r
potential to fix atmospheric nitrogen ( I C R I S A T
1983).
N i t r o g e n - b a l a n c e Studies
A n o t h e r aspect o f o u r w o r k deals w i t h measurement
o f N 2 f i x a t i o n associated w i t h s o r g h u m , m i l l e t , a n d
related species, a n d transferred to the c r o p . We are
a p p r o a c h i n g this b y experiments using 1 5 N e n r i c h m e n t , b y nitrogen-balance experiments i n p o t c u l ture, a n d b y the m u c h m o r e d i f f i c u l t , l o n g - t e r m field
experiments.
Pot Experiments
W e have concentrated s o far o n establishing n i t r o gen balances associated w i t h s o r g h u m , m i l l e t , a n d
N a p i e r bajra (Pennisetum purpureum x P. americanum) g r o w n i n p o t c u l t u r e i n v e r m i c u l i t e media w i t h
a n d w i t h o u t added N fertilizer, a n d i n soil l o w i n
n i t r o g e n . These e x p e r i m e n t s have established t h a t
there is a s m a l l b u t measurable N 2 f i x a t i o n by bacteria associated w i t h the roots of these plants, w h i c h is
t a k e n u p b y the p l a n t d u r i n g the g r o w i n g season, a n d
some o f w h i c h also remains i n the r o o t i n g m e d i u m .
I n one e x p e r i m e n t s o r g h u m was g r o w n t o m a t u r i t y w i t h o u t f e r t i l i z e r N i n washed v e r m i c u l i t e . T h e
plants were r a t o o n e d t w i c e a n d g r a i n harvests t a k e n ,
w i t h t o t a l d r y - m a t t e r p r o d u c t i o n of 195 g p o t - 1 of 10
plants o r i g i n a l l y s o w n . I n a n o t h e r e x p e r i m e n t w i t h
s o r g h u m g r o w n i n v e r m i c u l i t e f o r 4 9 days, the N
balance s h o w e d a considerable increase in N in
p l a n t e d b u t n o t i n u n p l a n t e d pots ( D a r t a n d W a n i
1982, I C R I S A T 1980). T h e p o s i t i v e N balance
across a l l i n o c u l a t i o n treatments was 269 m g p o t - 1 o f
five p l a n t s w h e n no fertilizer N was a p p l i e d , a n d 124
mg p o t - 1 w h e n 53 mg N p o t - 1 was a p p l i e d . A substantial p r o p o r t i o n of this N was g a i n e d by the r o o t
m e d i u m ( 3 3 % o f the t o t a l N balance f o r z e r o - N
t r e a t m e n t ) , a l t h o u g h this m a y result f r o m f i n e r o o t s
missed f r o m the r o o t sample.
In another pot experiment w i t h sorghum C S H 5
g r o w n i n a n unsterilized A l f i s o l w i t h three different
levels of a d d e d n i t r o g e n , a considerable balance f o r
N due to i n o c u l a t i o n over the c o n t r o l was observed
( T a b l e 3). A m a x i m u m m e a n balance across the
n i t r o g e n levels f o r N over u n p l a n t e d t r e a t m e n t of
331 m g p o t - 1 was observed i n case o f i n o c u l a t i o n
w i t h A. lipoferum ( I C M 1001). A d d i t i o n of 20 kg N
ha - 1 equivalen t resulted in h i g h e r balance f o r N
across the i n o c u l a t i o n treatments c o m p a r e d to no N
a d d i t i o n a n d a d d i t i o n of 40 kg N ha - 1 equivalent
( I C R I S A T 1984).
P e a r l m i l l e t g r o w n i n v e r m i c u l i t e i n p o t s also
a t t a i n e d a p o s i t i v e N balance of 109 mg p o t - 1 c o n t a i n i n g five plants w i t h o u t a d d e d N fertilizer. T h e r e
was a p o s i t i v e N balance o f 9 6 m g p o r - 1 w i t h n i t r o g e n
a d d e d equivalent to 20 kg N ha - 1 ( D a r t a n d W a n i
1982). S i m i l a r l y , in a p o t t r i a l w i t h m i l l e t BJ 104
g r o w n in an unsterilized A l f i s o l there was a signific a n t l y higher positive n i t r o g e n balance ( o v e r
u n p l a n t e d treatments) due t o i n o c u l a t i o n w i t h N 2 fixing bacteria over the n o n i n o c u l a t e d c o n t r o l . A
T a b l e 3. N i t r o g e n balance w i t h sorghum C S H 5 inoculated w i t h nitrogen-fixing bacteria. 1
Total dry
Culture
Nitrogen in
Net
total dry
nitrogen
G r a i n mass
matter
matter
balance
(g p o t 1 )
(g pot-1)
(mg pot-1)
(mg pot-1)
Azospirillum
lipoferum
13.9
79.9
307
331
Azotobacter
chroococcum
12.1
75.4
259
226
14.9
79.9
270
150
11.6
66.9
223
113
Napier bajra root extract ( N B R E )
Control
(noninoculated)
SE±
c v %
1.
0.64
16.8
2.52
11.6
8.7
11.8
56.5
95
Plants were grown in the greenhouse in pots containing nonsterilized Alfisol. A l o n g with inoculation with bacteria, three nitrogen levels
(0, 20, and 40 kg N ha -1 ) were used and each treatment was replicated four times.
61
positive n i t r o g e n balance o f 322 m g p o t - 1 over a n
u n p l a n t e d c o n t r o l was r e c o r d e d in a t r e a t m e n t i n o c ulated w i t h Azospirillum lipoferum across the n i t r o gen levels. A m a x i m u m N balance of 240 mg p o t - 1
was observed across the i n o c u l a t i o n t r e a t m e n t s t h a t
received 2 0 k g N h a - 1 equivalent. A d d i t i o n o f 4 0 k g
N ha - 1 reduced the balance s i g n i f i c a n t l y ( I C R I S A T
1984). These f i n d i n g s i n d i c a t e d that a d d i t i o n o f n i t rogen a t l o w e r rates enhanced N 2 f i x a t i o n t h a t
resulted i n h i g h e r positive balance, a n d higher rates
of N application inhibited N2 fixation.
C u t t i n g s o f the N a p i e r bajra h y b r i d N B 2 1 were
g r o w n in vermiculite and an A l f i s o l . The cuttings
g r e w w i t h o u t added N f e r t i l i z e r t o a b o u t 150 c m i n
7 2 days before being r a t o o n e d . A t f i n a l harvest a t
194 days, the e x t r a N a c c u m u l a t e d in the s o i l
a m o u n t e d t o 216 m g N p o t - 1 f o r a single p l a n t w i t h o u t a d d e d N w i t h t o t a l p o s i t i v e balance o f 539 m g N ,
a n d 368 mg N p l a n t - 1 w i t h 20 kg ha - 1 added N w i t h
the t o t a l positive balance o f 657 m g N . F o r the
vermiculite rooting medium, N accumulation w i t h
zero N t r e a t m e n t was 167 mg N in the m e d i u m a n d
361 m g N p o s i t i v e o v e r a l l balance ( D a r t a n d W a n i
1982).
Field Experiments
A l o n g - t e r m field e x p e r i m e n t was started in 1978 in
c o l l a b o r a t i o n w i t h the S o i l C h e m i s t r y a n d F e r t i l i t y
s u b p r o g r a m to measure the N balance in s o r g h u m
p r o d u c t i o n in an A l f i s o l under rainfed conditions.
T h e same eight c u l t i v a r s w i t h either h i g h nitrogenase
a c t i v i t y o r h i g h N u p t a k e u n d e r l o w f e r t i l i t y , are
g r o w n each year on the same plots. F e r t i l i z e r N is
added a t the rate o f 0 , 20, a n d 4 0 k g ha - 1 . M e a n
i n i t i a l N c o n t e n t o f the t o p 0-15 c m o f the s o i l
w i t h o u t fertilizer N was 0.040% N , 0.056% i n the
15-30 c m zone, a n d 0.053% a t 30-90 c m . A l l a b o v e g r o u n d p l a n t m a t e r i a l is r e m o v e d at harvest. T h e r e
were significant differences between the c u l t i v a r s i n
g r a i n y i e l d a n d N u p t a k e f r o m the second season
o n w a r d s . D u r i n g the 6 t h year o f this e x p e r i m e n t
( r a i n y season 1983), c u l t i v a r s also v a r i e d s i g n i f i c a n t l y across the N levels in t o t a l d r y - m a t t e r p r o d u c t i o n . D u r i n g the 7 t h year o f the e x p e r i m e n t ( r a i n y
season 1984), a u n i f o r m c r o p of pearl m i l l e t was
g r o w n . The total dry-matter yield of pearl millet on
plots where c u l t i v a r s C S H 5 a n d I S 2333 were g r o w n
p r e v i o u s l y was at p a r across the n i t r o g e n levels.
T h e c u m u l a t i v e n i t r o g e n u p t a k e t h r o u g h aboveg r o u n d p l a n t parts f r o m 1978 to 1983 (except 1981)
62
i n d i c a t e d t h a t highest n i t r o g e n u p t a k e a m o n g s t the
s o r g h u m c u l t i v a r s across t h e a p p l i e d n i t r o g e n levels
was i n the case o f C S H 5 (230 k g ha - 1 ) a n d t h e l o w e st
was i n the case o f I S 2333 (180 k g ha - 1 ) ( I C R I S A T
1985). These results suggest t h a t s o r g h u m c u l t i v a r s
do vary for their N 2 - f i x i n g ability. However, we w i l l
have a clearer p i c t u r e of this w h e n s o i l - N changes
over the first years of the e x p e r i m e n t are measured.
Samples are c u r r e n t l y b e i n g processed. L e a c h i n g
losses a n d i n p u t s of N t h r o u g h r a i n f a l l are l i k e l y to
be s m a l l .
I n another long-term N-balance trial a t I C R I S A T
Center, several t r o p i c a l grasses are g r o w n f o r 7 years
w i t h o u t a d d i n g a n y fertilizer N . T h e c r o p receives 4 0
k g P 2 O 5 h a - 1 a - 1 a n d i s i r r i g a t e d d u r i n g the d r y
season. T h e m a x i m u m d r y - m a t t e r p r o d u c t i o n has
been o b t a i n e d w i t h the N a p i e r bajra h y b r i d N B 2 1
where an equivalent of 370 t ha - 1 d r y m a t t e r , c o n t a i n i n g an equivalent of 2752 kg N have been harvested in 7 years ( T a b l e 4). A p r e l i m i n a r y s o i l
s a m p l i n g up to a d e p t h of 90 cm after 5.5 years
showed n o difference i n N content o f the s o i l f r o m
the p l o t s where N B 2 1 was g r o w n a n d where the l o w
d r y - m a t t e r - p r o d u c i n g e n t r y was g r o w n . These
results suggest that entries l i k e NB 21 are d e r i v i n g
some o f t h e i r r e q u i r e m e n t o f N t h r o u g h B N F i n
a d d i t i o n t o the s o i l - N p o o l . T h o r o u g h soil s a m p l i n g
i n these plots w i l l a l l o w c o m p u t a t i o n o f the exact
a m o u n t s of N fixed by these entries over the years.
C o n t r i b u t i o n to N B a l a n c e
by Blue-green Algae
Crusts of blue-green algae develop on the soil surface o f m a n y c r o p p e d f i e l d s d u r i n g the r a i n y season
a n d after i r r i g a t i o n ( D a r t a n d W a n i 1982, I C R I S A T
1983). T h e p r e d o m i n a n t heterocystous algae, seen
f o r m i n g mats o n fields a t I C R I S A T Center, were
t w o Anabaena species a n d Nostocmuscorum. O t h e r
Nostoc species as w e l l as algae b e l o n g i n g to the
genera
Calothrix,
Aphanothece, Microsystis, Lyngbya, a n d Oscillatoria were also observed ( I C R I S A T
1983). O n A l f i s o l s , the g r o w t h a n d N 2 - f i x i n g a c t i v i t y
o f those algae u n d e r s o r g h u m a n d m i l l e t was genera l l y l o w , b u t u n d e r t r o p i c a l grasses such as Pennisetum purpureum, the mats m a y be very active,
d e p e n d i n g o n the wetness o f the s o i l surface, a n d the
extent o f the p l a n t canopy. A c t i v i t y decreases
r a p i d l y as the s o i l surface dries o u t , v i r t u a l l y ceasing
3 days after w e t t i n g of the s o i l surface if the r a d i a t i o n
levels are h i g h . Nitrogenase a c t i v i t y of these a l g a l
T a b l e 4. D r y - m a t t e r p r o d u c t i o n and nitrogen u p t a k e by forage grass spp in l o n g - t e r m nitrogen-balance t r i a l . 1
Grass
Pennisetum
P.
P.
x
P.
purpureum
NB
JVM-2
squamulatum
Cenchrus
Panicum
Setaria
Chloris
Panicum
Pennisetum
21
Production
Nitrogen
Uptake
cumulative
per d a y
cumulative
per day
(years)
(t ha-1)
( k g ha-1)
( k g ha-1)
( k g ha-1)
6.94
370.7
146.3
2752
1.09
6.94
211.0
83.3
1760
0.69
6.94
165.5
65.3
1189
0.47
ciliaris
6.94
164.3
64.9
1235
0.49
maximum
6.00
152.0
69.0
1080
0.49
6.94
119.0
47.0
836
0.33
6.94
98.9
39.0
617
0.24
6.31
96.3
41.8
706.6
0.31
antidotale
6.5
43.0
18.1
318
0.13
rupellii
6.5
41.7
17.6
312
0.13
anceps
Pennisetum
1.
americanum
purpureum
D r y matter
Age
mezinium
gayana
Estimated f r o m 6 m 2 net harvest area, harvested 19 times, crop is irrigated during the dry season and 40 kg P 2 O 5 ha -1 is added every year.
mats w h e n e x t r a p o l a t e d to a surface-area basis
ranged f r o m 24 to 119 mg N f i x e d m - 2 d - 1 , c o m p a r e d
w i t h o n l y 0.5 to 1.6 mg N m - 2 d - 1 f o r surface soil
w i t h o u t visible algal g r o w t h . T h e algal m a t covered
u p t o 2 9 % o f the soil surface ( D a r t a n d W a n i 1982),
a t w h i c h level this extrapolates t o a n u p p e r estimate
of f i x a t i o n of 28 kg N ha - 1 a - 1 , assuming 80 days at
the a b o v e level o f a c t i v i t y f o r a n i r r i g a t e d p e r e n n i a l
grass c r o p .
observed between the a m o u n t o f exudate a n d r o o t
a n d / o r shoot g r o w t h o f seedlings g r o w n i n axenic
l i q u i d c u l t u r e , a n d the r a n k i n g o f the cultivars i n o c u lated w i t h A. lipoferum for nitrogenase a c t i v i t y d i d
n o t correlate w i t h the a m o u n t s o f s o l u b l e e x u d a t e
measured. S i m i l a r l y , this r a n k i n g also differed f r o m
that f o r g r o w t h and a c t i v i t y of A. lipoferum in synthetic m e d i a ( I C R I S A T 1984).
Combined Nitrogen
F a c t o r s A f f e c t i n g Associative
N 2 Fixation
N i t r o g e n f i x a t i o n associated w i t h s o r g h u m a n d
m i l l e t is affected by p l a n t g e n o t y p e , r o o t exudates,
seasonal a n d d i u r n a l v a r i a t i o n , soil type, s o i l m o i s ture, t e m p e r a t u r e , levels o f c o m b i n e d n i t r o g e n , a n d
o r g a n i c c a r b o n i n the s o i l . S o m e o f these factors
have been discussed u n d e r v a r i o u s assay techniques
a n d o n l y those n o t discussed earlier are described
below.
Root Exudates
Q u a l i t a t i v e differences i n the soluble exudates o f
seedlings o f d i f f e r e n t s o r g h u m genotypes were d e m o n s t r a t e d b y v a r i a t i o n i n g r o w t h a n d nitrogenase
a c t i v i t y o f a given bacterial c u l t u r e in semisolid synthetic m e d i a c o n t a i n i n g r o o t exudates as the sole
c a r b o n source. O n l y the a z o s p i r i l l a cultures g r e w
w e l l ; the o t h e r organisms tested grew p o o r l y , w i t h
l i t t l e nitrogenase a c t i v i t y . N o c o r r e l a t i o n was
T h e presence of c o m b i n e d N affects the enzyme
nitrogenase. W i t h s o r g h u m plants g r o w n i n tubes
filled w i t h washed sand, nitrogenase a c t i v i t y was
d r a s t i c a l l y reduced w h e n the plants were fed w i t h
above 15 p p m N in s o l u t i o n ( I C R I S A T 1984).
Nitrogenase a c t i v i t y o f s o r g h u m plants i n the f i e l d
was s t i m u l a t e d due to a d d i t i o n of 20 kg N ha - 1 over
the n o - N a d d i t i o n t r e a t m e n t , b u t 40 kg N ha - 1
reduced the a c t i v i t y . W i t h m i l l e t BJ 104 plants
g r o w n i n pots f i l l e d w i t h A l f i s o l , mean nitrogenase
a c t i v i t y t h r o u g h o u t the g r o w t h p e r i o d was signific a n t l y h i g h e r w h e n the plants were watered w i t h 10
a n d 2 5 p p m N i n s o l u t i o n d a i l y , t h a n w i t h the plants
w h i c h received no n i t r o g e n a n d also at 100 p p m N
s o l u t i o n d a i l y . A p p l i c a t i o n o f a basal dose o f 20, 40,
a n d 80 kg N ha - 1 equivalent d i d n o t affect the a c t i v i t y
significantly.
Nitrogen-fixing Bacteria
T h e m a r k e d differences i n nitrogenase a c t i v i t y o f
s o r g h u m a n d m i l l e t between fields m a y be p a r t l y due
63
t o differences i n t h e i r m i c r o b i a l p o p u l a t i o n s , suggesting t h a t i t m a y b e possible t o o b t a i n responses t o
i n o c u l a t i o n w i t h bacteria. W e c o m p a r e d different
m e t h o d s o f e s t i m a t i n g bacterial p o p u l a t i o n s a n d isol a t i n g N 2 - f i x i n g bacteria f r o m r o o t o r soil samples.
T h e N-free m e d i a t h a t c o n t a i n e d sucrose a n d m a l a t e
as the c a r b o n source were used to c o m p a r e three
m e t h o d s o f i s o l a t i n g a n d q u a n t i f y i n g the bacteria.
C o u n t s o f p r e s u m p t i v e n i t r o g e n fixers (4.5 x 10 8 i n
sucrose a n d 2.5 x 10 8 i n malate m e d i a g - 1 o f r o o t )
were h i g h e r u n d e r the d i l u t i o n plate c o u n t m e t h o d
t h a n counts estimated b y most p r o b a b l e n u m b e r
( M P N ) m e t h o d i n semisolid media ( 5 x 10 5 i n sucrose
a n d 5 x 10 4 g - 1 r o o t in malate m e d i u m ) , a n d higher
t h a n axenic p l a n t tubes (5 x 10 5 g - 1 r o o t ) . Recovery
o f nitrogenase-positive bacterial isolates was 4 3 %
higher w i t h sucrose a n d 3 0 % higher w i t h malate b y
the M P N m e t h o d . Recovery o f nitrogenase-positive
bacterial isolates f r o m M P N w i t h axenic plants was
3 7 % using malate m e d i u m ( T a b l e 5 ; I C R I S A T
1984).
I n the p l a n t e n r i c h m e n t c u l t u r e technique f o r isol a t i n g N 2 fixers, sterile seedlings o f s o r g h u m o r m i l l e t
are g r o w n o n a c a r b o n a n d N-free m e d i u m i n test
tubes, a n d i n o c u l a t e d w i t h a d i l u t i o n series o f the s o i l
o r c u l t u r e u n d e r test. M o s t p r o b a b l e n u m b e r estimates o f N 2 - f i x i n g bacteria are m a d e f r o m the A R A
T a b l e 5 . E s t i m a t i o n a n d isolation o f n i t r o g e n - f i x i n g bacteria f r o m r o o t samples by different methods. 1
Number of
Total
Method
counts
isolates
Total Active
Dilution and plating
Sucrose m e d i u m
4.5 x 108
10
0
Malate medium
2.5 x 108
10
0
M P N i n semisolid m e d i u m
Sucrose
5 x 105
7
3
Malate
5 x 104
10
3
4
0
5
0
8
3
6
1
M P N w i t h plants
5 x 105
Isolations on
Sucrose m e d i u m
Malate medium
1.
Roots of active Ex-Bornu plants were macerated and serial
dilutions were used for plating or inoculating semisolid media
in bottles containing 6 mL medium or Ex-Bornu plants grown
on Fahraeu's medium. The plants were grown i n 6 m L Fahraeu's medium in 25 x 200 mm plant tubes.
64
o f these tubes, a n d f r o m direct p l a t i n g o f the highest
positive d i l u t i o n s . A second or t h i r d selection f r o m
this p o p u l a t i o n can be made by a g a i n g o i n g t h r o u g h
the process o f d i l u t i o n a n d r e i n o c u l a t i o n o f sterile
seedlings. W i t h this technique, w i t h the increasing
e n r i c h m e n t d u r i n g each generation, the n u m b e r o f
c o l o n y types decreased a n d the p r o p o r t i o n o f
nitrogenase-positive bacteria increased.
L a r g e p o p u l a t i o n s o f bacteria capable o f g r o w i n g
i n a i r o n N-free m e d i a exist i n s o i l . Use o f the
acetylene-reduction assay indicated nitrogenase
a c t i v i t y f o r a b o u t 6 0 % o f these p r e s u m p t i v e N 2 f i x ers. T h e n u m b e r o f c o l o n y types a n d the p o p u l a t i o n
sizes depended on the selection of m e d i a a n d v a r i e d
w i t h the c a r b o n source. A b o u t f o u r times a s m a n y
bacteria g r e w on a sucrose-based m e d i u m t h a n on a
malate-based m e d i u m . A d d i n g a s m a l l a m o u n t o f
yeast extract (100 m g L - 1 ) d o u b l e d t h e i r n u m b e r o n
b o t h the media. T h e n u m b e r i n the t o p 4 0 c m o f s o i l
was a b o u t 10 times greater t h a n t h a t in the 40-to-60
cm zone. T h e r e were over a m i l l i o n E n t e r o b a c t e r i a ceae g - 1 s o i l , several species of w h i c h are k n o w n to
f i x n i t r o g e n anaerobically.
S t i m u l a t i o n o f p r e s u m p t i v e , aerobic N 2 fixers
occurs i n the rhizosphere o f several plants. T h e
n u m b e r o f easily recognizable types o f bacteria f r o m
the r o o t surface t h a t grew on N-free media was 10
times greater t h a n the p o p u l a t i o n g r o w i n g i n the s o i l
a w a y f r o m the roots. A selection f o r p a r t i c u l a r types
o f bacteria also occurred i n the r o o t zone, r e s u l t i n g
i n less t h a n h a l f the types f o u n d i n the b u l k s o i l .
S o m e bacteria were f o u n d very closely b o u n d t o the
r o o t a n d perhaps even i n the r o o t tissues. A f t e r
s h a k i n g the r o o t w i t h glass beads to r e m o v e surfaceattached bacteria a n d t h e n t h o r o u g h l y s t e r i l i z i n g the
r o o t surface w i t h 1% c h l o r o m i n e T f o r 1 h, we recovered m o r e t h a n 400 000 bacteria g - 1 o f fresh r o o t
f r o m the r o o t macerate ( D a r t a n d W a n i 1982).
A survey of 200 sites in the t r a d i t i o n a l pearl m i l l e t g r o w i n g areas i n n o r t h w e s t e r n I n d i a i n d i c a t e d t h a t
t o t a l p o p u l a t i o n o f organisms capable o f g r o w i n g o n
a N-free sucrose m e d i u m supplemented w i t h 50 mg
L - 1 yeast extrac t ( Y E ) , ranged f r o m 10 7 to 10 8 g - 1 s o i l
( D a r t a n d W a n i 1982). Nitrogenase a c t i v i t y was
detected i n 4 2 % o f the 3760 isolates made f r o m the
h i g h e s t - d i l u t i o n plates. E v e r y soil c o n t a i n e d o r g a nisms w h i c h p r o d u c e d pellicles a n d reduced C 2 H 2 o n
a malate m e d i u m , w i t h a M P N o f N 2 fixers v a r y i n g
f r o m 10 3 t o 10 5 g - 1 s o i l . S o m e o f these soils d i d n o t
c o n t a i n Azospirillum, a n d Enterobacteriaceae a n d
Pseudomonas were c o m m o n l y isolated f r o m the
malate e n r i c h m e n t cultures. T h e isolates f r o m the
sucrose-based m e d i u m c o u l d be classified i n t o at
least seven different genera of N 2 - f i x i n g bacteria,
i n c l u d i n g types w h i c h are s t i l l to be i d e n t i f i e d . Enterobacter cloacae was the m o s t - c o m m o n isolate.
S o m e Pseudomonas types were also nitrogenase
positive. T h e r e were at least 10 6 a c t i m o n y c e t e - l i k e
organisms g - 1 soil i n every sample a n d o f the 229
isolates, 70 had nitrogenase a c t i v i t y on sucrose + YE
m e d i u m b u t o n subsequent p u r i f i c a t i o n they lost the
a c t i v i t y . T h e isolates o b t a i n e d f r o m this s t u d y c o u l d
be classified on c o l o n y m o r p h o l o g y i n t o at least 22
different g r o u p s . H o w e v e r , they represent o n l y the
m o s t - n u m e r o u s organisms able t o g r o w o n t w o
m e d i a ( D a r t a n d W a n i 1982, I C R I S A T 1982).
S o m e o f these cultures lose a c t i v i t y d u r i n g p u r i f i c a t i o n a n d s u b c u l t u r i n g . In l a b o r a t o r y studies a synergistic effect on nitrogenase a c t i v i t y was f o u n d
w h e n t w o l o w - f i x i n g p u r e cultures were m i x e d
together. C u l t u r e s of Erwinia herbicola a n d Enterobacter cloacae each g r e w on N-free m e d i a , b u t were
inactive w h e n assayed f o r C 2 H 2 r e d u c t i o n a c t i v i t y .
W h e n b o t h cultures were g r o w n together, a h i g h
a c t i v i t y was measured b y C 2 H 2 r e d u c t i o n (101
nmoles C 2 H 4 b i j o u b o t t l e - 1 h - 1 ) ( I C R I S A T 1982).
S i m i l a r l y , Azotobacter chroococcum in p u r e c u l t u r e
alone h a d nitrogenase a c t i v i t y o f 5 0 nmoles C 2 H 4
b i j o u - 1 h - 1 , b u t w h e n it was g r o w n w i t h Erwinia
herbicola the a c t i v i t y increased to 121 nmoles C 2 H 4
b i j o u - 1 h - 1 ( I C R I S A T 1982).
season. I n o c u l a t i o n o f s o r g h u m c u l t i v a r s w i t h
n i t r o g e n - f i x i n g bacteria resulted i n 2-10% increase
i n g r a i n yields across the c u l t i v a r s o v e r the n o n i n o c ulated c o n t r o l . H o w e v e r the increases were n o t stat i s t i c a l l y significant. A s i m i l a r t r e n d was observed
f o r t o t a l d r y - m a t t e r p r o d u c t i o n ( I C R I S A T 1985).
D u r i n g 1982-1984, nine f i e l d trials were c o n d u c t e d a t I C R I S A T Center a n d d i f f e r e n t l o c a t i o n s
i n I n d i a t o study t h e response o f m i l l e t c u l t i v a r s t o
i n o c u l a t i o n w i t h n i t r o g e n - f i x i n g bacteria. I n a l l the
t r i a l s i n o c u l a t i o n increased the g r a i n a n d t o t a l d r y m a t t e r yields o f m i l l e t . H o w e v e r significant y i e l d
increases due t o i n o c u l a t i o n were observed i n five
trials ( T a b l e 6). T h e s u m m a r y table indicates t h a t u p
t o 3 0 % increase i n m e a n y i e l d over the c o n t r o l was
observed i n the test c u l t i v a r s due t o i n o c u l a t i o n w i t h
N 2 - f i x i n g bacteria.
I n a l l the experiments, p l a n t d r y - m a t t e r yields also
f o l l o w e d trends s i m i l a r t o t h a t o f g r a i n y i e l d . I n o c u lations increased p l a n t N u p t a k e i n a l l the e x p e r i ments except where A. brasilense ( S P 7) was used. In
a few cases g r a i n N c o n t e n t was increased d u e to
i n o c u l a t i o n s . I n a l l the t r i a l s , trends f o r host c u l t i v a r
and bacterial strain interactions for grain and plant
d r y - m a t t e r yields were observed; however, s i g n i f i cant i n t e r a c t i o n f o r g r a i n a n d p l a n t d r y - m a t t e r
yields was observed i n one t r i a l each o n l y .
Responses t o I n o c u l a t i o n
Acknowledgements
I n p o t - c u l t u r e studies, the response o f s o r g h u m a n d
m i l l e t t o i n o c u l a t i o n w i t h N 2 - f i x i n g bacteria varied
w i t h the g r o w t h m e d i u m a n d a m o u n t o f N fertilizer
added. T h e responses o f s o r g h u m g r o w n i n pots i n
nonsterile A l f i s o l , t o i n o c u l a t i o n w i t h Azospirillum
lipoferum a n d N B R E , a m i x e d c u l t u r e , were a g r a i n
y i e l d increase o f 2 2 % a n d t o t a l d r y m a t t e r increase o f
2 9 % . T h e y i e l d increase o c c u r r e d even w h e n the
e q u i v a l e n t o f 4 0 k g N h a - 1 was a d d e d ( I C R I S A T
1982). N i t r o g e n - b a l a n c e studies i n d i c a t e d a p o s i t i v e
N balance over the c o n t r o l due to i n o c u l a t i o n s
( T a b l e 3). I n a f i e l d t r i a l o n a n A l f i s o l w i t h the three
sorghum hybrids C S H 1, C S H 5, and C S H 9 inoculated w i t h l i q u i d peat c u l t u r e suspension o f N 2 - f i x i n g
bacteria, we observed increased d r y - m a t t e r p r o d u c t i o n ( P < 0 . 1 ) o f a l l three h y b r i d s i n o c u l a t e d w i t h A .
lipoferum ( I C M 1001) a n d a N B R E c u l t u r e ( I C R I S A T 1983).
A n o t h e r field t r i a l w i t h three s o r g h u m c u l t i v a r s
C S H 5 , C S H 9 , a n d S P V 351 a n d 1 0 i n o c u l a t i o n
treatments was c o n d u c t e d d u r i n g the 1984 r a i n y
D r P.J. D a r t i n i t i a t e d the Cereal M i c r o b i o l o g y p r o gram at I C R I S A T and Drs R . V . Subba Rao, D . B .
Godse, a n d J . A . K i p e - N o l t i n i t i a t e d some o f the
studies discussed in this paper. T h e results f o r N
balances f r o m e x p e r i m e n t s are p a r t o f a c o l l a b o r a t i o n w i t h D r s K . L . Saharawat, T . J . R e g o , a n d F . R
B i d i n g e r . E x c e l l e n t technical assistance was received
f r o m S. Chandrapalaiah, U . K . A v a l a k k i , M . N .
Upadhyaya, and K. Prakash Hebbar.
References
Bergersen,
between
F . J . 1970.
nitrogen
The
fixation
quantitative
relationship
a n d the a c e t y l e n e - r e d u c t i o n
assay. A u s t r a l i a n J o u r n a l o f B i o l o g i c a l Sciences 23:10151025.
D a r t , P . J . , and W a n i , S.P. 1982. N o n - s y m b i o t i c n i t r o g e n
f i x a t i o n a n d s o i l f e r t i l i t y . Pages 3-27 in N o n - s y m b i o t i c
nitrogen fixation and organic matter in the tropics. S y m p o -
65
T a b l e 6. A s u m m a r y of p e a r l millet i n o c u l a t i o n experiments conducted at different locations d u r i n g 1 9 8 2 - 8 4 .
Location and
season
Soil type
I C R I S A T Center
Alfisol
Cultivars
2 . I P 2787
4.
Percentage
treatment
increase1
A.lipoferum
(1)
A.brasilense
I C M S 7819
S u m m e r 1982
Inoculation
6.05
(SP
7)
-6.5
Remarks
For plant dry matter
significant interaction
( P = 0 . 0 5 ) between cultivar
NBRE
and culture was noted.
I C R I S A T Center Alfisol
3 . I P 2787
6.
I C M S 7703
R a i n y 1982
A.lipoferum
A.lipoferum
W C C 75
3. B J 104
MBH
For grain Nb content,
significant
6.0
A.brasilense
I C R I S A T Center Alfisol
14.5
21.02
NBRE
A.chroococcum
S u m m e r 1983
(1)
( I C M 1001)
5.
110
M E B H 23/81
(ICM
(SP
A.lipoferum
2001)
7)
-11.8
(1)
3. B J 104
MBH
R a i n y 1983
5.
(ICM 1001)
A.chroococcum
(ICM
2001)
1001)
(ICM
4 . B J 104
7.
5.5
-
A.lipoferum
( I C M 1001)
A.brasilense
(1)
W C C 75
A.brasilense
(2)
I C M S 7703
A b l + Ab2
B K 560
R a i n y 1984
6.0
2001)
NBRE
I C R I S A T Center Alfisol
6.0
-
(1)
(ICM
A.chroococcum
M E B H 23/81
A.chroococcum
10.7 2
0.7
(ICM
2001)
R a i n y 1984
Bhavanisagar
M i x e d Red
and black
Alfisol
R a i n y 1984
Parbhani4
Vertisol
R a i n y 1984
4 . B J 104
5.
R a i n y 1984
Vertisol
( I C M 1001)
B K 560
W C C 75
A.chroococcum
I C M S 7703
NBRE
4 . B J 104
5.
(2)
7.6
yield between cultivars
a n d inoculants was
11.6 2
A.lipoferum
W C C 75
A.chroococcum
I C M S 7703
NBRE
7.
6.1
(ICM
6.1
(SL
A.lipoferum
16.5 2
33)
(ICM
18.7 2
2001)
( I C M 1001)
6,1
.
24.52
M P 21
A.brasilense
(1)
19.3 2
W C C 75
A.brasilense
(2)
29.52
4 . B K 560
Ab1
5.
+ Ab2
10.5
A.chroococcum
30.62
NBRE
27.8 2
A.lipoferum
( I C M 1001)
RHR 1
A.brasilense
W C C 75
A.chroococcum
I C M S 7703
NBRE
1.
Increase in mean grain yield across the cultivars.
2.
Significantly (P = 0.05) higher over the control.
(SL
(ICM
3.
In cooperation wi t h G.S. Jadhav, Marathwada Agricultural University, Parbhani.
In cooperation with N.S. Subba Rao and K.V.B.R. Tilak, I A R I , New Delhi.
5.
In cooperation w ith S.D. Ugale, Mahatma Phule Agricultural University, Rahuri.
9.4
7.1
33)
4.
66
2001)
( I C M 1001)
A.brasilense
4 . B J 104
25.62
17.4
B K 560
I C M S 7703
Rahuri5
A.lipoferum
A.brasilense
2001)
Significant
interaction for grain
4.8
NBRE
Vaijapur3
cultures was observed.
12.4
A.lipoferum
AMpoferum
110
interaction between
cultivars and
5.0
A.lipoferum
NBRE
I C R I S A T Center Alfisol
14.0
13.9
6.7
noted.
sia papers 1 . T r a n s a c t i o n s o f the 1 2 t h I n t e r n a t i o n a l C o n gress o f S o i l Science, 8-16 F e b 1982, N e w D e l h i , I n d i a . N e w
D e l h i , I n d i a : I n d i a n A g r i c u l t u r a l Research I n s t i t u t e .
Giller, K . E . , D a y , J . M . , D a r t , P . J . , and W a n i , S.P.
1984.
A m e t h o d f o r m e a s u r i n g t h e transfer o f f i x e d n i t r o g e n f r o m
free-living bacteria t o higher plants using
15
N 2 . Journal of
M i c r o b i o l o g i c a l M e t h o d s 2(6):307-316.
Giller, K . E . , W a n i , S.P., and D a y , J . M . 1986. Use o f
i s o t o p e d i l u t i o n t o measure n i t r o g e n f i x a t i o n associated
w i t h t h e r o o t s o f s o r g h u m a n d m i l l e t genotypes. P l a n t a n d
S o i l 90:255-263.
I C R I S A T ( I n t e r n a t i o n a l Crops Research Institute for the
S e m i - A r i d Tropics). 1978. A n n u a l r e p o r t 1977-78. P a t a n -
W a n t , S . P . , U p a d h y a y a , M . N . , a n d D a r t , P . J . 1984. A n
i n t a c t p l a n t assay f o r e s t i m a t i n g n i t r o g e n a s e a c t i v i t y ( C 2 H 2
reduction) of sorghum a n d millet plants g r o w n in pots.
P l a n t a n d S o i l 82:15-29.
W i t t y , J . F . , a n d D a y , J . M . 1978. Use o f 1 5 N 2 i n e v a l u a t i n g
a s y m b i o t i c N 2 f i x a t i o n . Pages 135-150 i n I s o t o p e s i n b i o logical d i n i t r o g e n f i x a t i o n : proceedings o f a n A d v i s o r y
G r o u p M e e t i n g organized b y the J o i n t F A O / I A E A D i v i s i o n o f A t o m i c E n e r g y i n F o o d a n d A g r i c u l t u r e , 21-25
N o v 1977, V i e n n a , A u s t r i a . V i e n n a , A u s t r i a : I n t e r n a t i o n a l
A t o m i c Energy Agency.
Discussion
c h e r u , A . P . 502 324, I n d i a : I C R I S A T .
I C R I S A T ( I n t e r n a t i o n a l Crops Research Institute for the
S e m i - A r i d Tropics). 1979. A n n u a l r e p o r t 1978. P a t a n c h e r u , A . P . 502 324, I n d i a : I C R I S A T .
P.Tauro:
C o n s i d e r i n g t h a t m i l l e t s are g r o w n w i t h m i n i m u m
a g r o n o m i c practices, w h a t i n o c u l a t i o n t e c h n i q u e
w o u l d y o u recommend?
I C R I S A T ( I n t e r n a t i o n a l Crops Research Institute for the
S e m i - A r i d Tropics). 1980. A n n u a l r e p o r t 1979. P a t a n cheru, A . P . 502324, India: I C R I S A T .
I C R I S A T ( I n t e r n a t i o n a l Crops Research Institute for the
S e m i - A r i d Tropics). 1981. A n n u a l r e p o r t 1980. P a t a n c h e r u , A . P . 502 324, I n d i a : I C R I S A T .
I C R I S A T ( I n t e r n a t i o n a l Crops Research Institute for the
S e m i - A r i d Tropics). 1982. A n n u a l r e p o r t 1981. P a t a n c h e r u , A . P . 502 324, I n d i a : I C R I S A T .
I C R I S A T ( I n t e r n a t i o n a l Crops Research Institute for the
S e m i - A r i d Tropics). 1983. A n n u a l r e p o r t 1982. P a t a n cheru, A . P . 502324, India: I C R I S A T .
I C R I S A T ( I n t e r n a t i o n a l Crops Research Institute for the
S e m i - A r i d Tropics). 1984. A n n u a l r e p o r t 1983. P a t a n cheru, A . P . 502324, India: I C R I S A T .
I C R I S A T ( I n t e r n a t i o n a l Crops Research Institute for the
S e m i - A r i d Tropics). 1985. A n n u a l r e p o r t 1984. P a t a n c h e r u , A . P . 502 324, I n d i a : I C R I S A T .
S.P.Wani:
Seed i n o c u l a t i o n i s t h e best, b u t i f w e f i n d t h a t s l u r r y
i n o c u l a t i o n is s u p e r i o r w e w i l l h a v e t o o p t f o r i t . A t
present
we
are
comparing
various
methods
of
inoculation.
P.Tauro:
I s a n y t h i n g k n o w n a b o u t the N-scavenging a b i l i t y o f
d i f f e r e n t grasses?
S.P.Wani:
N o . But w i t h t h o r o u g h soil sampling we should be in
a p o s i t i o n t o f i n d o u t t h e status o f N s o u r c e i n t h e
soil.
P.Tauro:
C o u l d N r e c o v e r y u p t o 2000 k g / 5 y r b e m o r e d u e t o
scavenging t h a n due t o B N F ?
R a o , R . V . S . , and D a r t , P . J . 1981. N i t r o g e n f i x a t i o n assoc i a t e d w i t h s o r g h u m a n d m i l l e t . Pages 169-177 i n A s s o c i a t i v e N 2 - f i x a t i o n : proceedings o f the I n t e r n a t i o n a l
W o r k s h o p o n A s s o c i a t i v e N 2 - f i x a t i o n , 2-6 J u l y 1979, P i r a c i c a b a , B r a z i l ( V o s e , P . B . , a n d R u s c h e l , A . P . , eds.). V o l . 1 .
B o c a R a t o n , F l o r i d a , U S A : C R C Press.
S.P.Wani:
N o . A s the i n i t i a l s o i l s a m p l i n g a n d s a m p l i n g a f t e r 5
years h a v e n o t s h o w n N differences t o t h i s e x t e n t , w e
c a n i n f e r t h a t s o i l N was n o t t h e source. R e m o v a l o f
2000 k g N f r o m the s o i l w i l l be easily d e t e c t e d b y
W a n i , S . P . ( I n press.) N i t r o g e n f i x a t i o n p o t e n t i a l i t i e s o f
K j e l d a h l analysis.
s o r g h u m a n d m i l l e t . In N e w trends in biological nitrogen
f i x a t i o n ( S u b b a R a o , N . S . , ed.). N e w D e l h i , I n d i a : O x f o r d
and 1BH Publishing Co.
W a n i , S.P., D a r t , P . J . , a n d U p a d h y a y a , M . N . 1983. F a c t o r s a f f e c t i n g n i t r o g e n a s e a c t i v i t y ( C 2 H 2 r e d u c t i o n ) associated w i t h s o r g h u m and m i l l et estimated using the soil
c o r e assay. C a n a d i a n J o u r n a l o f M i c r o b i o l o g y 29:10631069.
P.Tauro:
Have
you
noticed
any
negative
response
to
inoculation?
S.P.Wani:
Yes,
in
the
case
of inoculation
with
Azospirillum
brasilense.
67
B . K . Konde:
W h a t a d d e d advantages d i d y o u observe f r o m s l u r r y
i n o c u l u m o v e r seed i n o c u l a t i o n ?
S.P.Wani:
I d o n ' t t h i n k so, as generally basal dressings w i t h
P 2 O 5 are d o n e i n a l l the t r i a l s .
S.P.Wani:
Joseph Thomas:
I a m impressed b y the p a i n s t a k i n g e x p e r i m e n t s w i t h
i m p r o v e d f i e l d techniques. T h e q u a n t i t y o f n i t r o g e n
recovered b y N a p i e r grass w o r k s o u t t o 465 k g ha - 1 .
D o y o u have a n y 1 5 N d a t a t o substantiate this?
B y u s i n g s l u r r y i n o c u l a t i o n (peat c u l t u r e suspended
in water), more bacterial i n o c u l u m can be added in
the f i e l d a s c o m p a r e d t o seed i n o c u l a t i o n . A n o t h e r
advantage i s t h a t i n d r o u g h t - p r o n e areas, this a d d i t i o n a l m o i s t u r e helps i n the establishment o f the
p l a n t s in the early stage. S u c h an effect has been
observed at V a i j a p u r w h e r e there were no rains at a l l
after s o w i n g , a n d o n the e n t i r e s t a t i o n o n l y the i n o c u l a t i o n t r i a l w h e r e l i q u i d suspension was a p p l i e d has
c o m e u p w e l l , whereas a l l o t h e r trials s o w n i n the
n o r m a l w a y resulted i n p a t c h y p l a n t establishment.
B.K.Konde:
W h e n d o y o u i n o c u l a t e the plants?
S.P.Wani:
G e n e r a l l y a t the t i m e o f s o w i n g i f the t r i a l i s h a n d
s o w n , o t h e r w i s e s o o n after emergence, i.e., 5-6 days
after s o w i n g , i f the t r i a l i s t r a c t o r s o w n .
G . Oblisami:
W h a t i s the cost o f l i q u i d i n o c u l a t i o n o f A z o s p i r i l
lum t o s o r g h u m w h e n c o m p a r e d t o seed
inoculation?
S.P.Wani:
W e have n o t w o r k e d o u t the exact costs o f s l u r r y
inoculation, but if sowing is done by a bullockd r a w n i m p l e m e n t , the cost w i l l b e a b o u t R s . 30-40. A
s i m p l e , inexpensive device has been d e v e l o p e d by
o u r F a r m P o w e r a n d E q u i p m e n t scientists f o r t h i s
p u r p o s e i n g r o u n d n u t , a n d i t c a n b e used f o r o t h e r
c r o p s also.
O . P . Rupela:
I n c o n t i n u a t i o n o f D r . T a u r o ' s questions, was t h e
negative effect s t a t i s t i c a l l y significant?
S.P.Wani:
T h e negative effects w i t h A. brasilense ( S P 7) i n o c u l a t i o n were consistent i n t w o t r i a l s w h e r e 4 c u l t i v a r s
were t r i e d , a n d i n one t r i a l t h e r e d u c t i o n was s t a t i s t i c a l l y significant.
G.S. M u r t h y :
W h e n t h e r e i s n o increase i n t h e g r a i n y i e l d b u t there
i s a n increase i n t o t a l biomass y i e l d , perhaps o t h e r
n u t r i e n t s such a s p h o s p h o r u s a n d z i n c are b e c o m i n g
l i m i t i n g factors.
68
S.P.Wani:
No.
S . V . Hegde:
D i d y o u have a n u n p l a n t e d s o i l system f o r N balance study? W h a t was the N g a i n due to p l a n t i n g
per se?
S.P.Wani:
Yes, w e d o m a i n t a i n a n u n p l a n t e d c o n t r o l i n a l l
N - b a l a n c e t r i a l s . T h e N g a i n due to p l a n t i n g a l o n e
was 113 mg N p o t - 1 s o w n w i t h s o r g h u m , a n d 27 mg
N p o t - 1 s o w n w i t h m i l l e t . These gains are across the 3
N levels used.
S.V.Hegde:
I s i t a p p r o p r i a t e s c i e n t i f i c a l l y t o use N a p i e r bajra
r o o t e x t r a c t ( N B R E ) , w h i c h i s a m i x t u r e o f several
m i c r o o r g a n i s m s a n d w h i c h c o u l d also change every
t i m e the c u l t u r e i s o b t a i n e d f r o m bajra r o o t ? Instead
c a n w e n o t isolate the o r g a n i s m o r o r g a n i s m s
responsible i n p u r e c u l t u r e a n d use them?
S.P.Wani:
I agree w i t h y o u t h a t N B R E i s a c o m p l e x c u l t u r e a n d
there w i l l b e p r o b l e m s i n p r o d u c i n g a n d m a i n t a i n i n g
the q u a l i t y of such a c o m p l e x i n o c u l u m . I d o n ' t
t h i n k N B R E c a n b e r e c o m m e n d e d f o r general use.
We started u s i n g N B R E as a test m i x t u r e a n d as it
gave p o s i t i v e responses w e c o n t i n u e d w i t h i t . I t c a n n o t b e used unless k n o w n bacteria f r o m N B R E
m i x e d t o g e t h e r p e r f o r m e q u a l l y w e l l . W e are s t u d y i n g the c o m p o s i t i o n o f N B R E i n terms o f m i c r o b i a l
c o m p o n e n t s a n d there are at least 14-15 d i f f e r e n t
bacteria i n v o l v e d w h i c h s h o u l d b e e q u a l l y g o o d i n
performance. This w i l l take m o r e time and I am not
sure w h e t h e r i n the near f u t u r e i t w i l l be possible o r
n o t . C o m i n g t o t h e q u e s t i o n o f m a i n t a i n i n g the same
c o m p o s i t i o n , w e a l w a y s use the s t a r t i n g m a t e r i a l f o r
preparing N B R E f r o m our long-term N-balance
t r i a l a n d t i l l n o w w e have observed p o s i t i v e
responses w i t h N B R E p r e p a r e d a t d i f f e r e n t t i m e s .
Y i e l d a n d N i t r o g e n Gains of S o r g h u m as Influenced
by Azospirillum brasilense
N . S . Subba R a o , K . V . B . R . T i l a k , C.S. Singh, and P . V . N a i r 1
Summary
Initial
pot
experiments
on
seed
inoculation
brasilense
demonstrated
increased
better
than
CSH
In field
years,
the
mean
nitrogen)
also
afield
in
soil
and
with
P
inoculation
spora
rilium
in
that
treatments
low
inoculation
Growth
to
experiment,
pronounced at
seed
increase
was equivalent
noticed
In
6.
margarita,
grain
with
A.
with
the
brasilense.
The
to
on
a
nine
centers
in
14.6
to
21.7
mycorrhizal
fasciculatum),
in combination
were
significant
with
the
kg
Azospirilium
CSH
in
5
responding
India for four
noninoculated
ha-1.
N
N
increased
successive
control
(no
This trend was
kg
uptake,
A.
which
N uptake
was
by plants
more
due
to
-1
N ha .
phosphorus-deficient
vesicular-arbuscular
responses
over
increased
the
nonsterile,
with
cultivar
application.
general,
varied from
the
of 15-20
of N
resulted
In
bicolor)
with
inoculation
levels
of sorghum
sorghum
and G l o m u s
due
at
the application
nitrogen.
brasilense
of
by
conducted
increasing
of applied
uptake
yield
obtainable
(Sorghum
production,
experiments
inoculation
levels
of sorghum
dry-matter
soil
(VAM) fungi
were
(Acaulospora,
with seed inoculation
brasilense
+
improved
G.
with
margarita
by
GigaAzospiand A.
brasilense + G. f a s c i c u l a t u m .
Introduction
D o b e r e i n e r (1975) a n d S u b b a R a o e t a l . ( 1 9 7 9 a ) .
T h e s t r a i n s w e r e i d e n t i f i e d as A. brasilense a c c o r d -
T h e present s t u d y was c o n d u c t e d t o (1) e v a l u a t e t h e
i n g t o T a r r a n d e t a l . (1978). T h e n i t r o g e n - f i x i n g
response
a b i l i t y o f d i f f e r e n t strains
of
sorghum
to
Azospirilium
brasilense
inoculation, alone and i n c o m b i n a t i o n w i t h graded
was e s t i m a t e d
by the
m i c r o - K j e l d a h l m e t h o d ( J a c k s o n 1967).
levels o f f e r t i l i z e r n i t r o g e n i n p o t s a n d i n f i e l d c o n d i -
Seeds o f s o r g h u m h y b r i d s C S H 5 a n d C S H 6 w e r e
t i o n s a n d (2) t o f i n d o u t the c o m b i n e d effects o f seed
i n o c u l a t e d w i t h a carrier-based ( s o i l + F Y M 1:1)
i n o c u l a t i o n w i t h A.
c u l t u r e of A. brasilense c o n t a i n i n g e f f i c i e n t s t r a i n s ,
brasilense a n d s o i l i n o c u l a t i o n
w i t h V A M fungi o n the g r o w t h a n d P uptake o f
f o l l o w i n g the m e t h o d o u t l i n e d b y Subba R a o e t a l
sorghum.
( 1 9 7 9 a , b ) . N o n i n o c u l a t e d seeds w e r e c o a t e d w i t h
s t e r i l i z e d ( h e a t - k i l l e d ) carrier-based c u l t u r e .
T w o p o t culture experiments were conducted
Materials and Methods
u s i n g 1 2 k g n o n s t e r i l i z e d a l l u v i a l s o i l o f p H 7.3.
Azospirilium was i s o l a t e d f r o m t h e r o o t s o f 1 8 c u l t i -
s o r g h u m cultivars ( C S H 5 a n d C S H 6) w i t h three
v a t e d s o r g h u m varieties g r o w n i n breeders' p l o t s b y
s t r a i n s of A. brasilense (S 3 S 1 2 , a n d S 1 8 ) s i n g l y a n d in
f o l l o w i n g the methods described b y v o n B u l o w a n d
c o m b i n a t i o n w a s s t u d i e d . A b a s a l dose o f 2 0 k g N
I n the f i r s t experiment, a n interaction o f t w o
1. Project Director, Agro-Energy Center, Senior Microbiologist, Scientist, and P h D Student, Division of Microbiology, Indian Agricultural Research Institute, New D e l h i 110 012, India.
I C R I S A T (International Crops Research Institute for the Semi-Arid Tropics). 1986. Cereal nitrogen fixation. Proceedings of the W o r k i n g
Group Meeting, 9-12 Oct 1984, I C R I S A T Center, India. Patancheru, A . P . 502 324, India: I C R I S A T .
69
ha - 1 was a p p l i e d to a l l the t r e a t m e n t s . P h o s p h o r u s
( P 2 O 5 ) a n d p o t a s s i u m ( K 2 O ) was a p p l i e d a t the rate
o f 4 0 k g h a - 1 . I n the second e x p e r i m e n t , t h e effect o f
i n o c u l a t i o n o n C S H 6 was studied w i t h five levels o f
f e r t i l i z e r N (0, 20, 40, 60, a n d 80 kg ha - 1 ) a p p l i e d at
seeding. I n the t h i r d e x p e r i m e n t , the c o m b i n e d
effects of seed i n o c u l a t i o n w i t h A. brasilense a n d s o i l
i n o c u l a t i o n w i t h different V A M f u n g i ( A c a u l o s p o r a
sp, Gigaspora margarita, a n d Glomus fasciculatum)
were s t u d i e d o n s o r g h u m c u l t i v a r C S H 5 . N i t r o g e n
was a p p l i e d a t the rate o f 2 0 k g ha - 1 , a n d p o t a s h
( K 2 O ) a t 6 0 k g ha - 1 , a t t h e t i m e o f seeding. I n a l l p o t
experiments, d r y matter produced by 75-day-old
p l a n t s ( p r e f l o w e r i n g stage) was d e t e r m i n e d . I n a l l
e x p e r i m e n t s , N was a p p l i e d as urea, P 2 O 5 as single
superphosphate ( S S P ) , a n d K 2 O as p o t a s s i u m
chloride.
F i e l d e x p e r i m e n t s were c o n d u c t e d a t d i f f e r e n t
locations in I n d i a on a simple randomized-block
design. T h e effect o f seed i n o c u l a t i o n o f s o r g h u m
c u l t i v a r C S H 5 w i t h a carrier-based ( s o i l : F Y M - 1 : 1 )
c u l t u r e of A. brasilense was tested at a l l l o c a t i o n s .
T h e i n o c u l a t i o n effects were s t u d i e d i n the presence
a n d absence o f g r a d e d levels o f N a t the t i m e o f
sowing. Phosphorus (P2O5) and potassium ( K 2 O )
were a p p l i e d as single superphosphate a n d potass i u m c h l o r i d e a t the rate o f 4 0 k g h a - 1 each a t t h e
t i m e o f s o w i n g . G r a i n a n d s t r a w yields a t the t i m e o f
c r o p m a t u r i t y (120 days after s o w i n g ) were determ i n e d . A t the D e l h i center also, a f i e l d e x p e r i m e n t
was l a i d o u t o n a s a n d y - l o a m s o i l o f p H 7.3. T h e p l o t
size was 10 m 2 (4 x 2 . 5 m ) a n d the t r e a t m e n t s were
r e p l i c a t e d f o u r times.
E n u m e r a t i o n o f t h e Azospirillum was d o n e 2 0
days after s o w i n g i n t h e r h i z o s p h e r e - s o i l samples
a n d r o o t s separately, f o l l o w i n g the m o s t - p r o b a b l e
n u m b e r ( M P N ) m e t h o d a s per the tables r e p r o d u c e d
b y A l e x a n d e r (1965).
T h e nitrogen content in root, shoot, and grain
samples was d e t e r m i n e d b y the m o d i f i e d K j e l d a h l
m e t h o d ( J a c k s o n 1967) a n d expressed as a percentage o n o v e n - d r y basis.
T h e m a x i m u m m e a n y i e l d increase was r e c o r d e d
by the c u l t u r e S 1 2 , closely f o l l o w e d by S 3 +S 1 2 , even
t h o u g h there was n o s i g n i f i c a n t difference between
these t w o treatments. S i m i l a r l y , n o s i g n i f i c a n t d i f ference c o u l d be n o t e d a m o n g t h e t r e a t m e n t s S 3 , S 18 ,
S 3 +S 1 8 , S 1 2 +S 1 8 , a n d S 3 +S 1 2 +S 1 8 , i n d i c a t i n g t h a t there
was n o a d d e d effect o n the g r o w t h o f s o r g h u m p l a n t s
by using a m i x e d i n o c u l u m containing more than
one isolate of Azospirillum. T h e response of c u l t i v a r
C S H 5 to Azospirillum i n o c u l a t i o n was better t h a n
t h a t o f C S H 6 . T h i s t y p e o f g e n o t y p i c differences
have a l r e a d y been r e p o r t e d w i t h lines o f m a i z e ( v o n
Bulow
and
Dobereiner
1975)
and
Digitaria
decumbens a n d Panicum maximum ( S m i t h et a l .
1978).
T h e response o f s o r g h u m t o N was l i n e a r u p t o 8 0
kg N ha - 1 u n d e r n o - i n o c u l a t i o n t r e a t m e n t s w h i l e
w i t h i n o c u l a t i o n , y i e l d increases were n o t i c e d o n l y
up to the 60 kg N ha - 1 level ( T a b l e 1). No differences
c o u l d b e n o t i c e d between the i n o c u l a t e d a n d n o n i n o c u l a t e d t r e a t m e n t s u n d e r the highest level of N
(80 k g h a - 1 ) , suggesting the p o s s i b i l i t y o f a n i n t e r f e r ence w i t h f u n c t i o n i n g o f nitrogenase i n the presence
o f c o m b i n e d N ( D a y e t a l . 1975, W e i e r 1980). T h e
d a t a i n T a b l e 1 i n d i c a t e t h a t the increased d r y m a t t e r y i e l d o b t a i n e d b y the a p p l i c a t i o n o f 2 0 k g N
ha - 1 c o u l d also be achieved by inexpensive
inoculation.
Inoculations w i t h azospirilla brought about an
8 - f o l d increase i n its r h i z o s p h e r i c p o p u l a t i o n ( T a b l e
2). S i m i l a r l y p l a n t r o o t s o f i n o c u l a t e d p l a n t s c o n t a i n
Table
1 . D r y m a t t e r ( g p o t - 1 ) o f s o r g h u m a s affected b y
inoculation with
fertilizer
Azospirillum
strains in c o m b i n a t i o n w i t h
nitrogen. 1
Nitrogen ( k g ha-1)
Treatment
0
20
40
60
80
Mean
Without
inoculation
135
147
158
166
173
156
146
152
172
178
170
164 2
165.0
172.0
171.5
With
inoculation
Results
Mean
levels
of nitrogen
A t o t a l of 18 strains (S 1 -S 1 8 ) were isolated f r o m t h e
roots of different sorghum cultivars using semisolid
m a l a t e m e d i u m . T h e efficiency o f N 2 f i x a t i o n a m o n g
t h e isolates r a n g e d f r o m 8 to 17 mg N g - 1 c a l c i u m
malate. M a x i m u m N 2 f i x a t i o n rate o f 16.8 m g N g - 1
c a l c i u m m a l a t e was estimated i n three c u l t u r e s S 3 ,
S12, and S18 g r o w n for 72 h.
70
CD
140.5 149.5
(P<0.05)
CV(%)
12.60
5.68
7.3
1.
Average of four r e p l i c a t i o n .
2.
Significantly (P < 0 . 0 5 ) different f r o m corresponding noninoculated controls.
Table
2.
Numbers
of Azospirillum
associated
with
the
rhizosphere a n d roots of s o r g h u m as affected by inoculation
w i t h Azospirillum strain S 1 2 .
M P N g - 1 soil
Treatment
Rhizosphere
N0 101
2.8 x 103
2
2.2 x 104
N0 11
Root
N0 10 (nonsterilized)
2.1 x 104
N0 10 (sterilized)
1.8 x 10 2
N 0 1 1 (nonsterilized)
3.5 x 105
N0 11 (sterilized)
3.5 x 102
1.
2.
N 0 1 0 : Noninoculated control w i t h o ut fertilizer nitrogen.
N 0 1 1 : Without fertilizer N but inoculated with Azospirillum.
17-fold h i g h e r bacterial p o p u l a t i o n t h a n the r o o t s o f
n o n i n o c u l a t e d plants. S t e r i l i z a t i o n o f the r o o t s w i t h
c h l o r a m i n e - T f o r 3 0 m i n s h a r p l y decreased the
n u m b e r o f Azospirilla, i n d i c a t i n g t h a t m a n y o f the
bacteria were associated w i t h the r o o t tissue. T h a t
t h i s decrease was o f the o r d e r o f a 1000-fold i n
treated p l a n t s as against a 100-fold in n o n t r e a t e d
ones s h o w e d t h a t the r o o t s o f i n o c u l a t e d p l a n t s h a r b o r e d a s u b s t a n t i a l n u m b e r o f b a c t e r i a l cells o n the
o u t e r r o o t tissue.
T h e evidence f r o m this s t u d y i n d i c a t e d t h a t A z o spirillum m a y b e f a v o r e d i n the r h i z o p l a n e o f a n
Table
3.
i n o c u l a t e d s o r g h u m c r o p . I t i s a d m i t t e d t h a t the
c r i t e r i a f o r i d e n t i f y i n g t h e o r g a n i s m was n o t perfect
i n the absence o f a c e t y l e n e - r e d u c t i o n assay ( A R A )
o r fluorescence a n t i b o d y ( F A ) techniques. B u t t h e
o v e r a l l analysis o f d a t a p o i n t s t o the p o s s i b i l i t y o f
increasing the p o p u l a t i o n s o f n i t r o g e n - f i x i n g A z o spirillum in the rhizosphere as w e l l as in the r o o t s of
a s o r g h u m c r o p b y p o p u l a t i o n o f selected strains.
I n m u l t i l o c a t i o n a l trials u n d e r different a g r o c l i m a t i c c o n d i t i o n s o f I n d i a , the increase i n y i e l d d u e t o
i n o c u l a t i o n was significant at 4 o u t of 9 centers
d u r i n g the years 1979-1982 ( T a b l e 3). T h e m e a n
increase i n g r a i n y i e l d due t o seed i n o c u l a t i o n o v e r
the c o n t r o l , averaged o v e r a l l t r i a l s o v e r 4 years,
a m o u n t e d t o 18.7%, a l m o s t e q u i v a l e n t t o the a p p l i c a t i o n of 15-20 kg N ha - 1 as urea. I n o c u l a t i o n in the
presence of 40 kg N ha - 1 s i g n i f i c a n t l y increased g r a i n
y i e l d over the c o r r e s p o n d i n g c o n t r o l a t t w o o u t o f
n i n e centers ( C o i m b a t o r e a n d P a n t n a g a r ) .
In a field t r i a l at D e l h i , 60 kg N ha - 1 + i n o c u l a t i o n
was f o u n d s u p e r i o r t o a l l o t h e r t r e a t m e n t s ( T a b l e 4 ) .
I n o c u l a t i o n c o u l d n o t b r i n g significant benefits i f n o
N was a p p l i e d . S t o v e r y i e l d i n s o r g h u m o b t a i n e d
w i t h 20 kg N ha - 1 a p p l i c a t i o n c o u l d also be achieved
by Azospirillum i n o c u l a t i o n . It was also o b s e r v e d
t h a t i n o c u l a t i o n a l o n g w i t h g r a d e d levels o f N
resulted i n 7.6-13.5% increase i n stover y i e l d s . T h e
effect was m o r e significant (15.6%) i n c o m b i n a t i o n
w i t h 20 kg N ha - 1 .
Several w o r k e r s r e p o r t e d N gains by f r e e - l i v i n g
G r a i n y i e l d ( k g h a - 1 ) o f s o r g h u m C S H 5 a s a f f e c t e d b y i n o c u l a t i o n w i t h Azospirillum brasilense a n d f e r t i l i z e r N a t
nine locations in India.
4 0 k g N ha-1 +
Noninoculated
Location
control
Azospirillum
Azospirillum
brasilense
4 0 k g N ha-1
CD
brasilense
(P<0.05)
3250
Udaipur
2640
31901
4480
4580
Coimbatore
31901
34101
4170
47801
475
Dharwad
1650
20701
3020
3160
405
Hyderabad
3410
4010
4340
4390
875
690
770
2070
1990
250
880
1150
2510
2180
285
1670
1830
1960
1860
350
Akola
Parbhani
Navsari
Indore
1470
1660
2450
2180
450
Pantnagar
1950
2400
2430
2810
375
Mean
1920
2280
3050
3110
-
CV(%)
9.5
1. Significant increase over corresponding noninoculated control.
71
T a b l e 4.
G r a i n a n d stover yields ( k g h a - 1 ) of s o r g h u m , as affected by i n o c u l a t i o n with Azospirillum in c o m b i n a t i o n w i t h
fertilizer n i t r o g e n , I A R I , N e w D e l h i .
Nitrogen ( k g ha-1)
Treatment
Parameter
Grain yield
0
60
Mean
Without inoculation
3260
3920
4460
5119
4190
W i t h inoculation
3400
47901
53801
58201
48501
3330
4360
4920
5469
Mean
Stover yield
40
20
CD
(P<0.05)
CV
(%)
1.62
325
14.5
Without inoculation
7120
9110
10240
11460
9480
With inoculation
7990
105301
11 370 t
12340
105501
7550
9810
10810
11890
Mean
CD
(P<0.05)
CV
(%)
481
961
11.5
1. Significantly different f r o m corresponding noninoculated treatments.
bacteria r a n g i n g f r o m 34-165 kg N ha - 1 a - 1 ( B a l t e n sperger e t a l . 1978). T h e basic questions w i t h r e g a r d
t o the c o n t r i b u t i o n o f Azospirillum t o the increased
N status of i n o c u l a t e d plants s t i l l r e m a i n to be satisf a c t o r i l y answered. T h e d a t a d o i n d i c a t e t h a t N
u p t a k e was m o r e i n i n o c u l a t e d s o r g h u m t h a n i n t h e
c o r r e s p o n d i n g n o n i n o c u l a t e d ones, a n d this effect
was m o r e p r o n o u n c e d i n c o m b i n a t i o n w i t h 2 0 k g N
ha -1 ( T a b l e 5).
A m a x i m u m g a i n of 21.7 kg N ha - 1 by the p l a n t
was o b t a i n e d by i n o c u l a t i o n w i t h 20 kg N ha - 1 over
the c o r r e s p o n d i n g n o n i n o c u l a t e d t r e a t m e n t . I n o c u -
T a b l e 5.
l a t i o n also resulted in a g a i n of a b o u t 15 kg N ha - 1
b o t h u n d e r the the zero N a n d 60 kg N levels.
T h o u g h the above figures represent o n l y a p p r o x i m a t e values, these rates o f N 2 f i x a t i o n c o u l d v e r y
w e l l be c o r r e l a t e d w i t h the N savings n o t e d f r o m
increased g r a i n yields due t o i n o c u l a t i o n .
S o i l i n o c u l a t i o n w i t h m y c o r r h i z a l f u n g i increased
the d r y m a t t e r o f r o o t s , shoots, p h o s p h o r u s c o n c e n t r a t i o n in plants, and phosphorus uptake by
s o r g h u m . Seed i n o c u l a t i o n w i t h A. brasilense in
c o n j u n c t i o n w i t h Gigaspora margarita or Glomus
fasciculatum
produced significantly more dry-
T o t a l n i t r o g e n u p t a k e in s o r g h u m under different c o m b i n a t i o n s of
Azospirillum
brasilense a n d fertilizer n i t r o g e n ,
I A R I , New Delhi.
Increase i n
Nitrogen uptake (kg ha )
Treatment
kg N
ha
-1
N u p t a k e by
-1
A. brasilense
Root
Stover
Grain
inoculation
Total
( k g ha-1)
0
N1
3.0
31.9
41.6
76.5
-
0
I2
3.7
40.2
47.2
91.1
14.6
20
N
3.8
48.5
53.3
105.6
-
20
I
4.1
57.5
65.7
127.3
21.7
40
N
4.0
55.9
61.8
121.7
-
40
I
3.8
63.1
73.8
141.7
20.0
60
N
4.5
69.00
73.7
147.1
-
60
I
5.2
70.75
86.3
162.3
15.2
1, N = Noninoculated.
2. I = Inoculated.
72
T a b l e 6. Response of sorghum C S H 5 to inoculation w i t h
Treatment
Azospirillum
brasilense a n d V A M f u n g i .
S h o o t mass
R o o t mass
Total
phosphorus
content
(g plant-1)
(g plant-1)1
(%)
Phosphorus
uptake
( m g plant-1)
14.5
0.18
0.10
14.5
18.5
17.2
17.5
17.8
17.8
0.32 2
0.12
0.15
0.15 2
0.16*
0.15 2
0.15 2
0.17 2
21.9
Noninoculated control (no V A M and
no A. brasilense)
A.
brasilense
Acaulospora
sp
Gigaspora
margarita
Glomus
fasciculatum
Acaulospora + A.
brasilense
G. margarita + A. brasilense
G. fasciculatum + A. brasilense
20.52
20.8 2
0.22
0.25
0.22
0.25
0.34 2
0.37 2
Azospirilium
brasilense
V A M fungi
A.brasilense x V A M f u n g i i n t e r a c t i o n
NS3
NS
2.12
CD
0.12
NS
0.11
25.8
25.5
26.7
26.5
28.52
32.52
(P<0.05)
NS
0.04
0.03
NS
NS
5.3
1. Average of six replicated pots; each pot had four plants.
2. Significant increase over corresponding control.
3. NS = Not significant.
m a t t e r c o n t e n t o f r o o t s , shoots, P c o n t e n t o f shoots,
a n d P u p t a k e o f s o r g h u m t h a n the n o n m y c o r r h i z a l
treatments ( T a b l e 6).
Azospirillum i n o c u l a t i o n has been s h o w n to
increase the r o o t biomass ( D e w a n a n d S u b b a R a o
1979) a n d therefore, t h e significant increase i n d r y m a t t e r p r o d u c t i o n a n d r o o t biomass b y a c o m b i n a t i o n of seed i n o c u l a t i o n w i t h A. brasilense a n d s o i l
i n o c u l a t i o n w i t h Gigaspora margarita a n d / o r G l o mus fasciculatum c o u l d be a t t r i b u t e d to an increased
P t r a n s p o r t by the m y c o r r h i z a l fungus a i d e d by an
increased r o o t biomass ( T a b l e 6).
References
A l e x a n d e r , M . 1965. M o s t - p r o b a b l e - n u m b e r m e t h o d f o r
m i c r o b i a l p o p u l a t i o n s . Pages 1467-1472 i n M e t h o d s o f s o i l
analysis, ( B l a c k , C . A . , E v a n s , D . D . , W h i t e , J . L . , E n s m i n ger, L . E . , a n d C l a r k , F . E . , eds.). [ I n P t . ] M a d i s o n , W i s c o n sin, U S A : A m e r i c a n Society o f A g r o n o m y .
Baltensperger, A . A . , Schank, S . C . , S m i t h , R . L . , L i t t e l l ,
R . C , B o u t o n , J . H . , a n d D u d e c k , A . E . 1978. Effect o f
inoculation
w i t h Azospirillum a n d
Azotobacter on t u r f t y p e b e r m u d a genotypes. C r o p Science 18:1043-1045.
D a y , J . M . , H a r r i s , D . , D a r t , P J . , and
van B e r k u m ,
P . 1975. T h e B r o a d b a l k e x p e r i m e n t . A n i n v e s t i g a t i o n o f
n i t r o g e n gains f r o m n o n - s y m b i o t i c f i x a t i o n . Pages 71-84 i n
Nitrogen f i x a t i o n by free-living micro-organisms (Stewart,
W.D.P.,ed.). Cambridge, U K : Cambridge University
Press.
D e w a n , G . I . , a n d Subba R a o , N . S . 1979. Seed i n o c u l a t i o n
with
Azospirillum
brasilense a n d
Azotobacter chroococcum a n d the r o o t b i o m a s s of rice (Oryza sativa L . ) . P l a n t
a n d S o i l 53:295-302.
Jackson, M X . 1967. S o i l c h e m i c a l analysis. N e w D e l h i ,
I n d i a : Prentice H a l l .
S m i t h , R . L . , Schank, S . C . , B o u t o n , J . H . , and Quesenberry, K . H . 1978. Y i e l d increases o f t r o p i c a l grasses after
i n o c u l a t i o n w i t h Spirillum lipoferum. Pages 380-385 in
E n v i r o n m e n t a l r o l e o f n i t r o g e n - f i x i n g blue-green algae a n d
a s y m b i o t i c b a c t e r i a ( G r a n h a l l , U . , ed.). E c o l o g i c a l B u l l e t i n s n o . 2 6 . S t o c k h o l m , S w e d e n : S w e d i s h N a t u r a l Science
Research C o u n c i l .
Subba R a o , N . S . , Tilak, K . V . B . R . , Lakshmikumari, M . ,
and Singh, C . S .
1979a. Azospirillum a n e w b a c t e r i a l f e r t i l izer. I n d i a n F a r m i n g 30:3-5.
S u b b a R a o , N . S , T i l a k , K . V . B . R . , Singh, C . S . , a n d L a k s h m i k u m a r i , M . 1979b. Response o f a f e w e c o n o m i c species
o f g r a m i n a c e o u s p l a n t s t o i n o c u l a t i o n w i t h Azospirillum
brasilense. C u r r e n t Science 48:133-134.
73
T a r r a n d , J.J., Krieg, N . R . , and Dobereiner, J. 1978. A
t a x o n o m i c study of the Spirillum lipoferum g r o u p , w i t h
descriptions of a new genus, Azospirillum gen. n o v . a n d
t w o species, Azospirillum lipoferum ( B e i j e r i n c k ) c o m b .
n o v . a n d Azospirillum brasilense sp. n o v . C a n a d i a n J o u r n a l o f M i c r o b i o l o g y 24:967-980.
von Bulow, J . F . W . , a n d Dobereiner, J . 1975. P o t e n t i a l f o r
n i t r o g e n f i x a t i o n i n maize genotypes i n B r a z i l . Proceedings
o f the N a t i o n a l A c a d e m y o f Sciences o f the U n i t e d States
o f A m e r i c a 72:2389-2393.
Weier, K . L . 1980. N i t r o g e n f i x a t i o n associated w i t h
grasses. T r o p i c a l Grasslands 14:194-201.
Discussion
G . Oblisami:
W h a t i s the l o a d o f V A M - f u n g a l i n o c u l a n t t o the s o i l
i n o c u l a t i o n f o r sorghum?
K . V . B . R . Tilak:
T h e i n o c u l u m c a r r i e d 100-125 c h l a m y d o s p o r e s per
50 g of i n o c u l u m .
Joseph T h o m a s :
T h e d a t a o f D r T i l a k s h o w i n g consistent increase i n
r o o t mass on Azospirillum i n o c u l a t i o n are s i g n i f i cant. T h i s is s i m i l a r to a m y c o r r h y z a l effect l e a d i n g
t o enhanced u p t a k e o f n u t r i e n t s . T h a t this i s really s o
c a n b e d e m o n s t r a t e d b y a p p r o p r i a t e w o r k a t centers
like I A R I and I C R I S A T .
K . V . B . R . Tilak:
V A M i s k n o w n t o increase o t h e r n u t r i e n t s l i k e Z n ,
Fe, C u , etc., a p a r t f r o m P h o s p h o r u s . S o i t needs t o
be e x a m i n e d .
S.P. W a n i :
I n y o u r d a t a y o u refer t o N balance, w h i c h i s n o t
correct. It is increased N u p t a k e a n d n o t the N g a i n
or N balance.
K . V . B . R . Tilak:
Yes, this m a y be considered as N u p t a k e .
P.V. Rai:
W h a t was the i n o c u l a t i o n m e t h o d f o l l o w e d i n the
dual
inoculum
of Azospirillum
brasilense a n d
VAM?
74
K . V . B . R . Tilak:
Seed i n o c u l a t i o n o f A . brasilense a n d V A M was
m i x e d w i t h soil p r i o r to sowing.
B . K . Konde:
D i d y o u i n o c u l a t e Azospirillum a n d V A M a t the
same time?
K . V . B . R . Tilak:
Yes, a t the t i m e o f s o w i n g .
H . L . S . Tandon:
W h y is there so m u c h emphasis on s t u d y i n g N b a l ance or N g a i n by t o t a l N analysis of the s o i l w h e n
the N changes are e x t r e m e l y s m a l l as c o m p a r e d to
t o t a l N present in s o i l , a n d it w o u l d be very d i f f i c u l t
t o separate real gains f r o m a n a l y t i c a l errors o r
variability?
K . V . B . R . Tilak:
W e have n o t g i v e n m u c h emphasis o n soil N . B u t soil
N content is to be t a k e n i n t o c o n s i d e r a t i o n w h i l e
c a l c u l a t i n g the benefits of t o t a l N status d u e to i n o c u l a t i o n w i t h the N 2 f i x e r .
Azotobacter Inoculation: N i t r o g e n E c o n o m y
a n d Response o f S o r g h u m C S H 1
S . T . Shende1, G . B . R u d r a k s h a 2 , R a j a n i A p t e 1 , a n d R . S . R a u t 2
Summary
Field
trials
conducted
revealed that
substantially
decreased
e.g.,
and
appeared
60,
to
with
consistently.
gradually
30,
during
inoculation
with
The
increased
and 90 kg N
reveal
a
the
saving
1979,
1980,
Azotobacter
inoculation
doses
ha-1,
of 30
effect
N
1981
rainy
with
seasons
(M4),
was
of N-fertilizer.
inoculation
kg
and
chroococcum
with
sorghum
CSH
1
increased grain and fodder yields
more
at
lower
N-fertilizer
levels
at
all
levels
of nitrogen
application,
But
and
A z o t o b a c t e r showed a complementary effect and
ha-1.
Introduction
Inconsistent results w i t h Azotobacter chroococcum,
p a r t i c u l a r l y i n a g r o n o m i c a l e x p e r i m e n t s , have been
a t t r i b u t e d t o the l o w p o p u l a t i o n o f the o r g a n i s m
b o t h i n s o i l a n d c r o p rhizosphere ( M i s h u s t i n a n d
S h i l n i k o v a 1969, B r o w n 1974). H o w e v e r , there arc
re p o rt s t h a t i n o c u l a t i o n w i t h f r e e - l i v i n g n i t r o g e n f i x i n g bacteria enhanced c r o p yields, i n c l u d i n g t h a t
o f cereals ( M e s h r a m a n d Shende 1982, Shende a n d
A p t e 1982, S u b b a R a o e t a l . 1982, W a n i e t a l . 1985).
T h i s paper re p o rt s the results o f f i e l d t r i a l s c o n ducted w i t h sorghum under agroclimatic conditions
f o r three consecutive r a i n y seasons a t P a r b h a n i i n
Maharashtra.
Materials and Methods
F i e l d t r i a l s were c o n d u c t e d a t P a r b h a n i d u r i n g t h e
1979, 1980, a n d 1981 r a i n y seasons. T h e e x p e r i m e n t s
were l a i d o u t i n r a n d o m i z e d - b l o c k designs i n c l u d i n g
f o u r levels o f n i t r o g e n , i.e., 0 , 30, 6 0 , a n d 9 0 k g N
ha - 1 . I n a l l these e x p e r i m e n t s , s o r g h u m h y b r i d v a r i e t y C S H 1 was used. A u n i f o r m basal dose o f 4 0 k g
P 2 O 5 a n d 40 kg K 2 O ha - 1 was a p p l i e d . N i t r o g e n was
g i v e n a s urea i n t w o s p l i t a p p l i c a t i o n s .
T h e s t r a i n o f Azotobacter chroococcum ( M 4 )
used was isolated f r o m the rhizosphere o f m a i z e
p l a n t . T h i s s t r a i n i s c h r o m o g e n i c a n d was f o u n d t o
f i x 1.76 mg N g - 1 sucrose a n d synthesize i n d o l e acetic
acid ( I A A ) and G L S compounds.
Results
D u r i n g 1979, the f o d d e r y i e l d o f 8830 k g ha - 1 ,
o b t a i n e d by a c o m b i n e d t r e a t m e n t of 60 kg N ha - 1
a n d i n o c u l a t i o n w i t h Azotobacter, was s i g n i f i c a n t l y
h i g h e r t h a n the 8020 k g h a - 1 o b t a i n e d w i t h a p p l i c a t i o n of 60 kg N ha - 1 alone, a n d was at p a r w i t h the
f o d d e r y i e l d o b t a i n e d w i t h the a p p l i c a t i o n o f 9 0 k g N
ha -1 ( T a b l e 1).
I n o c u l a t i o n w i t h Azotobacter a l o n e increased
g r a i n y i e l d f r o m 3180 t o 3680 k g h a - 1 (15.7%), w h i c h
was statistically a t p a r w i t h the 3890 k g h a - 1 o b t a i n e d
w i t h t h e a p p l i c a t i o n o f 3 0 k g N ha - 1 . T h u s , there i s a n
i n d i c a t i o n o f n i t r o g e n saving u p t o 3 0 k g N ha - 1 . A t
h i g h e r levels o f a p p l i e d N , the y i e l d increases d u e t o
Azotobacter were n o t statistically s i g n i f i c a n t . T h e
c o m b i n e d t r e a t m e n t appeared t o b e advantageous
even at the 90 kg N ha - 1 a p p l i c a t i o n .
A s i m i l a r t r e n d was seen d u r i n g 1980. A p p l i c a t i o n
o f Azotobacter s i g n i f i c a n t l y raised t h e g r a i n y i e l d
1. Senior Microbiologist and Scientist, Division of Microbiology, Indian Agricultural Research Institute, New Delhi 110 012, India,
2. Microbiologists, Department of Microbiology, Marathwada Agricultural University, Parbhani, Maharashtra 431 402, India.
I C R I S A T (International Crops Research Institute for the Semi-Arid Tropics). 1986. Cereal nitrogen fixation. Proceedings of the W o r k i n g
Group Meeting, 9-12 Oct 1984, I C R I S A T Center, India. Patancheru, A . P . 502 324, India: I C R I S A T .
75
Table
1.
Effect o f Azotobacter ( M 4 ) inoculation a n d lev-
els o f nitrogen o n grain a n d fodder yields o f sorghum C S H
1 , P a r b h a n i , rainy seasons 1 9 7 9 - 8 1 .
Increase
Grain
Fodder
in grain
yield
yield
yield
(kg ha-1) ( k g ha-1)
Treatment
(%)
R a i n y season 1979
Control
3180
5980
-
Azotobacter
3680
6540
15.7
3890
6970
-
4220
7600
8.6
3 0 k g N. ha-1
30 k g N ha-1 +
Azotobacter
6 0 k g N ha-1
60 k g N ha-1 +
Azotobacter
90 k g N ha-1
9 0 k g N ha-1 +
Azotobacter
SE
4100
8020
-
4280
8830
4.53
4670
8760
-
4960
9070
6.4
±1 3 7
±2 0 1
R a i n y season 1980
Control
1960
-
5510
Azotobacter
2620
7200
30 k g N ha-1
2830
8400
-
3170
9080
12.1
3 0 k g N ha-1 +
Azotobacter
60 k g N ha-1
6 0 k g N ha-1 +
Azotobacter
9 0 k g N ha-1
9 0 k g N ha-1 +
Azotobacter
SE
33.6
3510
9600
-
3620
10460
3.0
4170
10710
-
4320
12680
3.6
±1 7 0
±4 0 6
1660
4890
Azotobacter
2050
5740
1980
5740
30 k g N ha-1
3 0 k g N ha
-1
+
Azotobacter
6 0 k g N ha-1 +
SE
-
-
7370
7370
47.7
Azotobacter
3390
8660
3460
8490
61.3
-
Azotobacter
3810
9600
3.5
±1 5 0
±4 0 0
-
f r o m 1960 to 2620 kg ha - 1 (33.6% increase), w h e n
fertilizer N was n o t added. I n c o m b i n a t i o n s w i t h N ,
Azotobacter increased the y i e l d b u t differences were
a g a i n n o t s i g n i f i c a n t . B u t i t c o u l d b e seen v e r y
clearly t h a t 30 kg N ha - 1 c o u l d be saved at each level
o f n i t r o g e n a p p l i c a t i o n i f seed i n o c u l a t i o n w i t h A z o tobacter was p r a c t i c e d .
D u r i n g 1981, g r a i n as w e l l as f o d d e r yields
increased m a r k e d l y w i t h Azotobacter i n o c u l a t i o n .
F o d d e r y i e l d increased w i t h i n o c u l a t i o n a t a l l levels
76
References
273.
23.2
2930
9 0 k g N ha-1
9 0 k g N ha-1 +
The authors wish to thank Dr N . S . Subba Rao,
H e a d , D i v i s i o n o f M i c r o b i o l o g y , I A R I , and
Marathwada Agricultural University for providing
the facilities f o r these e x p e r i m e n t s .
M e s h r a m , S . U . , and Shende, S . T . 1982. Response o f
m a i z e to Azotobacter chroococcum. P l a n t a n d S o i l 69:265-
2100
6 0 k g N ha-1
Acknowledgements
B r o w n , M . E . 1974. Seed b a c t e r i z a t i o n . A n n u a l R e v i e w o f
P h y t o p a t h o l o g y 12:181-197.
R a i n y season 1981
Control
o f n i t r o g e n a p p l i c a t i o n , b u t increases were h i g h w i t h
30-60 kg N ha -1 . G r a i n y i e l d increased s i g n i f i c a n t l y
w i t h i n o c u l a t i o n , g i v i n g 390 kg ha - 1 or 23.2% m o r e
t h a n the c o n t r o l . I n o c u l a t i o n alone was c o m p a r a b l e
to a p p l i c a t i o n of 30 or 60 kg N ha - 1 . As in the
p r e v i o u s 2 years, a c o m b i n e d t r e a t m e n t of i n o c u l a t i o n together w i t h a p p l i c a t i o n of 30 a n d 60 N kg ha - 1
was the best t r e a t m e n t b o t h f o r f o d d e r a n d g r a i n
yields.
T h u s , the c o m b i n e d a p p l i c a t i o n o f Azotobacter
w i t h 60 kg N ha - 1 in the f o r m of urea was the best
t r e a t m e n t , to harness the effect of Azotobacter in
o r d e r t o increase g r a i n a n d f o d d e r yields a n d a t the
same t i m e t o save a b o u t 3 0 k g h a - 1 o f f e r t i l i z e r N .
Mishustin, E . N . , and Shilnikova, V . K . 1969. T h e b i o l o g i cal f i x a t i o n o f a t m o s p h e r i c n i t r o g e n b y f r e e - l i v i n g bacteria.
Pages 82-109 in S o i l b i o l o g y : reviews of research. Paris,
France: U N E S C O .
S h e n d e , S . T . , a n d A p t e , R . G . 1982. Azotobacter
i n o c u l a t i o n — a remunerative i n p u t for agricultural crops.
Pages 532-543 in Proceedings of the N a t i o n a l S y m p o s i u m
o n B i o l o g i c a l N i t r o g e n F i x a t i o n , 25-27 Feb 1982, N e w
D e l h i , I n d i a . B o m b a y , I n d i a : B h a b h a A t o m i c Research
Centre.
Subba R a o , N . S . , T i l a k , K . V . B . R . , Singh, C.S., and G a u t a m , R . C . 1982. C r o p y i e l d a n d n i t r o g e n c o n t e n t o f p e a r l
m i l l e t (Pennisetum americanum) in response to A z o p i r i l ium brasilense. Pages 507-516 in Proceedings of the
N a t i o n a l S y m p o s i u m o n B i o l o g i c a l N i t r o g e n F i x a t i o n , 252 7 F e b 1982, N e w D e l h i , I n d i a . B o m b a y , I n d i a : B h a b h a
A t o m i c Research C e n t r e .
W a n i , S.P., Chandrapalaiah, S., and D a r t , P . J . 1985.
Response o f p e a r l m i l l e t c u l t i v a r s t o i n o c u l a t i o n w i t h
n i t r o g e n - f i x i n g bacteria. E x p e r i m e n t a l A g r i c u l t u r e 21:175182.
Response of S o r g h u m Cultivars to I n o c u l a t i o n
with
Azospirillum
D . Purushothaman and G . Oblisami1
Summary
This study presents evidence of wide variations among sorghum genotypes in supporting nitrogen
fixation.
Quantitatively and qualitatively,
the isolates of A z o s p i r i l l u m differ in their interaction
with
cultivars.
Based on
the root-associated acetylene-reduction activity (ARA),
19 sorghum
cultivars were classified into high ARA and low ARA genotypes. Inoculation with an efficient
strain (CSH 24) of A z o s p i r i l l u m enhanced the ARA of only certain cultivars. Some cultivars
when inoculated exhibited negative response in ARA.
In root-bit preincubation test also,
the
cultivars CSH 5 and Co.24 recorded high ARA while K.tall and USH 1 showed negligible
increase.
In afield study,
A z o s p i r i l l u m inoculation significantly increased the grain yield (13.1%)
of cultivar Co. 24.
Introduction
O f the m a n y cereals g r o w n i n I n d i a , s o r g h u m has
been consistently f o u n d to respond to Azospirillum
i n o c u l a t i o n ( A 1 C R P B N F 1980). T h e response has
been m u c h greater a t l o w levels o f n i t r o g e n t h a n a t
higher levels. Perhaps one of the m o s t significant
a n d e n c o u r a g i n g f i n d i n g s i n r e l a t i o n t o associative
N 2 f i x a t i o n i s t h a t there appear t o b e v a r i e t a l d i f f e r ences i n responses t o i n o c u l a t i o n . I n this paper, the
responses o f different genotypes o f s o r g h u m
[Sorghum bicolor L. ( M o e n c h ) ] t o i n o c u l a t i o n w i t h
Azospirillum are
presented.
Materials and Methods
T h e isolates o f Azospirillum were c u l t u r e d f o l l o w i n g
the root-tissue e n r i c h m e n t technique. T h e isolates
were p u r i f i e d b y s t r e a k i n g over p o t a t o - m a l i c acid
1.
m e d i u m . T h e e n u m e r a t i o n of Azospirillum was
d o n e f o l l o w i n g the m o s t - p r o b a b l e n u m b e r ( M P N )
technique using N-free m a l i c acid m e d i u m ( H e g a z i
et a l . 1979).
M u d pots of 36 cm diameter were filled w i t h 5 kg p o t - 1
redloam soil:sand:farmyard manure ( F Y M )
( 5 : 5 : l v / v ) m i x t u r e ( p H 7.2; t o t a l n i t r o g e n 0.085%,
organic c a r b o n 0.48%). Before raising the c r o p , 20
kg N ha - 1 as urea, 40 kg P 2 0 5 ha - 1 as superphosphate,
a n d 40 kg K 2 0 ha - 1 as potassium c h l o r i d e were
applied.
S o i l samples f r o m the rhizosphere a n d r h i z o p l a n e
were collected f o l l o w i n g standard procedures ( P r a mer a n d S c h m i d t 1966, L y n c h 1982).
Azospirillum isolates g r o w n i n 3 0 m L m a l i c a c i d
semisolid m e d i u m f o r 7 2 h i n special 100 m L
restricted-neck Erlenmeyer flasks were assayed f o r
A R A by i n c u b a t i n g under 1% acetylene f o r 6 h at
28°C
I n the case o f root-associated A R A , freshly c o l lected r o o t samples were placed i n 100 m L E r l e n -
Associate Professor and Head, Center of Advanced Studies in Agricultural Microbiology, Tamil Nadu Agricultural University,
Coimbatore, T a m i l Nadu 641 003, India.
ICR1SAT (International Crops Research Institute for the Semi-Arid Tropics). 1986. Cereal nitrogen fixation. Proceedings of the W o r k i n g
Group Meeting, 9-12 Oct 1984, I C R I S A T Center, India. Patancheru, A.P. 502 324, India: I C R I S A T .
77
meyer f l a s k ( r o o t s t o occupy 20-25% o f the v o l u m e )
a n d s i m i l a r l y incubated under 1% acetylene f o r 6 h
( P u r u s h o t h a m a n et a l . 1979).
T h e p r o m i s i n g isolate C S H 2 4 was g r o w n u n d e r
static c o n d i t i o n s i n m a l i c acid b r o t h m e d i u m , supplemented w i t h 100 mg g l u t a m i c a c i d L - 1 . A f t e r 5 days
o f g r o w t h the b r o t h was m i x e d w i t h sterilized peat
s o i l neutralized w i t h c a l c i u m carbonate. T h e peatbased i n o c u l u m c o n t a i n e d 12 x 10 8 cells g - 1 on d r y
mass basis. Surface-sterilized seeds of the s o r g h u m
genotypes were treated w i t h the i n o c u l u m , u s i n g
jaggery s o l u t i o n as the sticker. E a c h seed after treatm e n t carried 32 x 10 6 cells.
T h e field experiment was conducted at the M i l l e t
Breeding Station, T a m i l N a d u A g r i c u l t u r a l University ( T N A U ) , C o i m b a t o r e , o n a sandy l o a m s o i l w i t h
a p H o f 6.9, 0.026% t o t a l n i t r o g e n , a n d 0.64%
organic c a r b o n . T h e p l o t size of 6 m x 5 m was
replicated thrice in a r a n d o m i z e d - b l o c k design. T e n
packets (2.5 kg) of the i n o c u l u m ha - 1 were used f o r
the f i e l d study. A t the t i m e o f t h i n n i n g the c r o p
(15-20 days after sowing), the b i o f e r t i l i z e r was sidedressed. T h e c r o p received t w o life-saving irrigations.
Results
T e n o u t o f 1 9 s o r g h u m genotypes differed signific a n t l y i n t o t a l Azospirillum counts i n the r h i z o sphere at the f l o w e r i n g stage. C u l t i v a r s C o . 2 4 a n d
C o . 19 registered a higher c o u n t of Azospirillum in
the r h i z o p l a n e ( T a b l e 1). These differences m a y arise
f r o m the q u a l i t a t i v e a n d q u a n t i t a t i v e changes i n the
r o o t exudates u p o n w h i c h the e n e r g y - d e m a n d i n g
d i a z o t r o p h s are dependant ( H u b b e l l 1977). T h e
mean A R A o f isolates f r o m genotypes ranged f r o m
1 6 t o 360 nmoles o f C 2 H 4 h - 1 , s h o w i n g over 2 0 - f o l d
v a r i a t i o n . I n general, isolates f r o m C S H 5 , C o . 19,
C o . 24, U S H 1, a n d C o . 18 recorded s u b s t a n t i a l l y
Table 1 . Most-probable number ( M P N ) o f
Azospirillum
sp in the roots of some s o r g h u m cultivars.
Rhizosphere
Cultivar
(x
104)g-1
Rhizoplane
(x 104) g-1
Co.19
22.6
Co.20
56.2
7.2
Co.24
84.6
24.6
4.8
15.8
CSH 4
CD(P<0.05)
78
11.84
42.6
6.16
T a b l e 2. T h e ARA of Azospirillum isolates f r o m different
sorghum
cultivars.
No.of
isolates
Cultivar
tested
Mean A R A
(nmole
C2H4
h-1 flask-1)
CSH 5
10
360 ± 26
Co.19
20
312 ± 18
Co.24
25
280 ± 21
USH 1
15
220 ± 26
Co.18
16
211 ± 23
Co.20
12
190 ± 16
Co.21
15
105 ± 12
CSH 6
10
99 ± 12
Co.22
15
87 ± 16
CSV 3
20
65 ± 11
CSV 4
15
65 ± 18
CSH 4
10
18 ± 4
K.tall
10
16 ± 8
higher A R A t h a n the others ( T a b l e 2). T h o u g h the
isolates were m a i n t a i n e d on N - r i c h m e d i u m f o r several generations, the v a r i a t i o n i n A R A c o u l d o n l y b e
traceable to the host genotypes. A l t h o u g h in vitro
d e t e r m i n a t i o n s o f A R A d o n o t reflect the field perf o r m a n c e of these isolates, these d a t a suggest the
v a r i a t i o n a m o n g the isolates. T h e genotypes differed
s i g n i f i c a n t l y i n respect o f the root-associated A R A
d e t e r m i n e d u n d e r n o n i n o c u l a t e d c o n d i t i o n s at the
f l o w e r i n g stage ( T a b l e 3). Genotypes that s u p p o r t e d
h i g h A R A were C o . 18, C o . 24, C o . 20, C S H 5 , C o .
19, a n d U S H 1 . T h e reason f o r l o w A R A observed
w i t h some varieties m i g h t be the presence of i n e f f i cient strains. It s h o u l d be p o i n t e d o u t that c u l t i v a r s
Co.24, C S H 5, C o . 2 0 , C o . 18, a n d C o . 19 possessed
h i g h A R A a n d also a reasonably large n u m b e r o f
Azospirilla.
A l l the 1 0 c u l t i v a r s tested responded t o A z o s p i r i l lum i n o c u l a t i o n . H o w e v e r , i n o c u l a t i o n d i d n o t
increase the A R A s i g n i f i c a n t l y , except i n genotypes
Co.24, C S H 5 , C o . 18, a n d C o . 2 0 . C u l t i v a r s C o . 2 1 ,
C S H 4 , a n d U S H 1 registered negative responses i n
A R A w i t h i n o c u l a t i o n ( T a b l e 4 ) . I n general, p r e i n c u b a t i o n of r o o t s w i t h energy source f o r 6 h g r e a t l y
increased A R A . T h e m a x i m u m A R A was observed
w i t h Co.24 a n d C S H 5 w h i l e K . t a l l a n d U S H 1 had
l o w A R A . W i t h o u t p r e i n c u b a t i o n the c u l t i v a r s
recorded p o o r A R A ( T a b l e 5). A s the tissues were
n o t surface-sterilized b u t were u n i f o r m l y washed t o
r e m o v e the a d h e r i n g s o i l particles, Azospirillum
f o u n d entrapped i n the m u c i g e l w o u l d n o t have been
lost; also, those d w e l l i n g w i t h i n the c o r t i c a l cells
w o u l d r e s p o n d t o the energy-source s u p p l y a n d the
cell n u m b e r w o u l d have considerably increased. I t
T a b l e 5 . Effect o f preincubation o f washed s o r g h u m r o o t
bits w i t h malic acid o n A R A .
A R A (nmole C 2 H 4 g-1 dry root h-1)
Cultivar
T a b l e 3.
Root-associated nitrogenase activity of s o r g h u m
cultivars at the f l o w e r i n g stage.
Nitrogenase activity
(nmole C 2 H 4 g
Preincubation
No preincubation
Co.24
288 ±
19
143 ±
8
CSH 5
320 ±
16
76 ±
7
K tall
60 ±
8
USH 1
75 ±
10
43 ± 12
68 ±
8
1. ± Standard deviation of the mean.
-1
dry root h-1)
Cultivar
C o . 18
327
C o . 24
318
C o . 20
268
T a b l e 6 . G r a i n yield ( t h a 1 ) o f sorghum C o . 2 4 a s i n f l u -
CSH 5
241
enced by Azospirillum inoculation a n d fertilizer N.
C o . 19
216
USH 1
212
Nitrogen
C o . 21
120
applied
Non-
CSH 6
112
kg ha-1
inoculated
USV 3
105
0
1.7
1.9
1.8
+ 12.7
92
10
2.0
2.1
2.04
+
C o . 22
68
20
2.0
2.3
2.15
+ 13.7
296
68
CSV 3
K.tall
48
CSH 4
42
USV 1
38
CSV 5
36
CSV 4
35
269
34
A S 3280
30
CD
T a b l e 4 . T h e A R A o f s o r g h u m cultivars a s influenced b y
Azospirillum
inoculation.
R o o t associated A R A
(nmole C 2 H 4 g-1 root h-1)
Cultivar
C h a n g e (%)
Noninoculated
Inoculated
C o . 18
217
268
+ 23.7
C o . 19
126
176
+ 39.0
C o . 20
168
186
+ 10.7
C o . 21
146
123
- 5.8
C o . 22
62
84
+ 34.8
C o . 24
308
426
+ 38.0
K.tall
65
68
+
4.9
CSH 4
44
38
-
13.6
CSH 5
198
268
+ 35.2
USH 1
142
112
- 21.1
%
Inoculated
Mean
increase
7.0
30
2.3
2.5
2.44
+ 11.5
40
2.4
2.7
2.55
+ 15.2
50
2.4
2.8
2.6
+ 16.3
2.1
2.4
-
+ 13.1
Mean
12.72
(P<0.05)
G r a i n yield(t ha-1)
c o u l d be argued t h a t the cultivars C o . 2 4 a n d C S H 5
p e r m i t t e d a higher rate o f m u l t i p l i c a t i o n o f the d i a z o t r o p h d u r i n g the i n c u b a t i o n p e r i o d t h a n the o t h e r
genotypes. A l t e r n a t i v e l y , as i n d i c a t e d earlier, the
presence o f m o r e efficient strains w i t h C o . 2 4 a n d
C S H 5 w o u l d have reflected i n the increased A R A .
T h o u g h p r e i n c u b a t i o n leads t o o v e r e s t i m a t i o n o f
n i t r o g e n f i x a t i o n , it does give us an idea of the
b e h a v i o r o f varieties.
In a field e x p e r i m e n t , the g r a i n y i e l d increased
s i g n i f i c a n t l y due t o Azospirillum i n o c u l a t i o n . O n a n
average, the increase i n g r a i n y i e l d was 1 3 . 1 % . G r a i n
y i e l d w i t h 20 or 30 kg N ha - 1 + Azospirillum i n o c u l a t i o n was 2.4 t ha - 1 , w h i c h was equivalent to t h a t
o b t a i n e d w i t h 50 kg N ha - 1 w i t h o u t i n o c u l a t i o n
( T a b l e 6).
I n c o n c l u s i o n , i t m a y b e said t h a t s o r g h u m genotypes d i f f e r m a r k e d l y i n h a r b o r i n g d i a z o t r o p h s b o t h
in quantity and in quality. In a combination of
p o t e n t i a l genotypes w i t h a p p r o p r i a t e , efficient
strains of Azospirillum, one can d e f i n i t e l y get g o o d
responses.
79
References
A I C R P B N F ( A l l I n d i a C o o r d i n a t e d Research Project o n
B i o l o g i c a l N i t r o g e n F i x a t i o n ) . 1980. P r o j e c t coordinators' report. New Delhi, India: Indian Council of
A g r i c u l t u r a l Research.
H e g a z i , N . A . , A m e r , H . , and M o n i b , M . 1979. E n u m e r a t i o n of N 2 - f i x i n g spirilla. Soil Biology and Biochemistry
11:437-438.
H u b b e l l , D . H . 1977. P l a n t r o o t a n d b i o l o g i c a l n i t r o g e n
f i x a t i o n . Proceedings, S o i l a n d C r o p Science S o c i e t y o f
F l o r i d a 36:37-40.
L y n c h , J . M . 1982. I n t e r a c t i o n s between b a c t e r i a a n d
plants in the r o o t e n v i r o n m e n t . Pages 1-23 in B a c t e r i a a n d
plants ( R h o d e s - R o b e r t , M . E . , a n d S k i n n e r , F . A . , eds.).
L o n d o n , U K : A c a d e m i c Press.
P r a m e r , D , and Schmidt, E . L . 1966. E x p e r i m e n t a l s o i l
m i c r o b i o l o g y . M i n n e a p o l i s , M i n n e s o t a , U S A : Burgess
Publishing Co.
P u r u s h o t h a m a n , D . , Gunasekaran, S . a n d O b l i s a m i ,
G . 1979. N i t r o g e n f i x a t i o n b y Azospirillum i n some t r o p i cal p l a n t s . Proceedings o f the I n d i a n N a t i o n a l Science
A c a d e m y , P a r t B 46:713-717.
80
Nitrogen Transformations by A. brasilense Strain 12S
f r o m S o r g h u m Roots
Bela S h u k l a a n d B . S . K u n d u 1
Summary
One of the isolates of A.
brasilense (12S) from sorghum roots showed enhanced nitrogenase
activity and cell growth with NO-3 (<60 µg NO-3 mL-1) and NO-2 ( < 3 0 µg NO-2 mL-1) under
microaerophilic
conditions.
Both
denitrification
and ammonification
were
observed simultane+
-1
ously in this organism. At low levels of ammonium (<20 µg NH 4 mL ) and urea (60 µg mL-1),
growth and nitrogenase activity were improved.
These compounds were also transformed to
gaseous forms
of nitrogen.
Introduction
Azospirillum spp are capable of several n i t r o g e n
t r a n s f o r m a t i o n s : (1) n i t r a t e can be u t i l i z e d by assimi l a t o r y n i t r a t e a n d n i t r i t e reductases; (2) d i s s i m i l a t o r y n i t r a t e r e d u c t i o n is carried o u t u n d e r anaerobic
c o n d i t i o n s , w i t h NO - 3 as an electron acceptor, a n d is
reduced t o n i t r i t e , n i t r o u s o x i d e , n i t r o g e n , o r a m m o n i u m ; a n d (3) nitrate-dependent n i t r o g e n f i x a t i o n
occurs under severe o x y g e n l i m i t a t i o n s . I n the l i g h t
of these observations, the present i n v e s t i g a t i o n was
u n d e r t a k e n t o study the n i t r o g e n t r a n s f o r m a t i o n s b y
A. brasilense (12S), effect of n i t r o g e n sources on cell
g r o w t h , t o t a l n i t r o g e n under m i c r o a e r o p h i l i c c o n d i t i o n s , a n d levels w h i c h are c r i t i c a l f o r expression o f
nitrogenase a c t i v i t y .
Materials and Methods
T h e s o r g h u m r o o t isolate A. brasilense (12S) was a
l o c a l isolate m a i n t a i n e d b y regular transfers o n
m o d i f i e d (0.2 g yeast e x t r a c t replaced v i t a m i n s ) m a l ate m e d i u m agar slants ( D o b e r e i n e r a n d D a y 1976).
1.
T h e c u l t u r e was m u l t i p l i e d i n b r o t h m e d i u m i n c u bated at 30° ± 2 ° C a n d 0.1 mL c u l t u r e h a v i n g 10 8
cells mL - 1 was used as i n o c u l u m f o r the studies.
T h e n i t r o g e n t r a n s f o r m a t i o n s studies were c o n ducted u s i n g v a r i a b l e concentrations o f NO - 3 , NO - 2 ,
N H + 4 , a n d NH + 2 (urea) in malate semisolid m e d i u m .
T h e tubes c o n t a i n i n g 5 m L m e d i u m were i n o c u l a t e d
w i t h 0.1 m L i n o c u l u m a n d i n c u b a t e d a t 3 0 ° ± 2 ° C .
T h e presence o f N O - 3 , N O - 2 , N H - 4 urea, N 2 O , t o t a l
n i t r o g e n , a n d cell p r o t e i n were d e t e r m i n e d at 0, 3,
a n d 6 days after i n c u b a t i o n .
F o r the e s t i m a t i o n o f various f o r m s o f n i t r o g e n ,
the c u l t u r e m e d i u m after A R A a n d N 2 O assay was
centrifuged at 10 000 r p m , a n d the supernatent was
used f o r v a r i o u s estimations. N i t r a t e c o n c e n t r a t i o n s
were measured by usin g salicylic acid ( C a t a l d o et a l .
1975) a n d N O 2 b y d e v e l o p i n g c o l o r w i t h s u l f a n i l i c
a c i d a n d a - N - n a p h t h y l ethylene d i a m i n e h y d r o c h l o ride ( N i c h o l a s a n d N a s o n 1957). T h e a m m o n i u m
estimations were m a d e by Nesselerization ( J a c k s o n
1967) a n d urea b y the procedure o f D o u g l a s a n d
B r e m n e r (1970). T o t a l n i t r o g e n was d e t e r m i n e d b y
the c o n v e n t i o n a l m i c r o - K j e l d h a l m e t h o d ( J a c k s o n
1967). T o t a l cell p r o t e i n was measured b y f u r t h e r
MSc Student and Associate Professor, Department of Microbiology, College of Basic Sciences and Humanities, Haryana Agricultural
University, Hisar, Haryana 125 004, India.
I C R I S A T (International Crops Research Institute for the Semi-Arid Tropics). 1986. Cereal nitrogen fixation. Proceedings of the W o r k i n g
Group Meeting, 9-12 Oct 1984, I C R I S A T Center, India. Patancheru, A.P. 502 324, India: I C R I S A T .
81
digesting the cell mass i n I N N a O H a n d later determ i n i n g the a m o u n t o f s o lu b il i zed p r o t e i n b y the
procedure o f L o w r y e t a l . (1951).
T h e nitrogenase a c t i v i t y was measured by reduct i o n of acetylene to ethylene using a d u a l - c o l u m n
N u c o n 5500 gas c h r o m a t o g r a p h . T h e tubes were
i n c u b a t e d under 10% C 2 H 2 f o r 24 h a n d the samples
were analyzed f o r C 2 H 4 p r o d u c e d .
N i t r o u s o x i d e was analyzed f r o m the tubes t h a t
were subjected t o A R A assay using gas T C D c h r o m a t o g r a p h ( f i t t e d w i t h p o r a p a k N c o l u m n ) , using
H 2 as carrier gas (30 mL min - 1 ), at a o v e n temperat u r e of 100° ± 5 ° C . N i t r o u s o x i d e f o r reference was
prepared b y reacting zinc w i t h d i l u t e H N O 3 ( 1 : 1 ) i n
an a i r t i g h t container. T h e N 2 O peaks were detected
b y c o m p a r i n g w i t h r e p o r t e d results a n d r e t e n t i o n
t i m e was recorded.
T a b l e 1 . Effect o f n i t r a t e , nitrite, a m m o n i u m , a n d urea o n
nitrogenase activity ( n moles C 2 H 4 d - 1 m L - 1 ) .
Levels
(µg
Nitrate
(NOT)
Nitrite
(NOT)
Ammonium
(NH+4)
Results
A. brasilense isolate 12S showed h i g h e r A R A t h a n
A. brasilense (Sp7a) a n d differed p h y s i o l o g i c a l l y
f r o m i t . T h e added n i t r a t e n i t r o g e n was d e n i t r i f i e d t o
n i t r i t e a t a l l levels (60-1200 µ g N 0 3 m L - 1 ) a n d
f u r t h e r r e d u c t i o n was n o t i c e d o n l y a t l o w e r levels o n
longer i n c u b a t i o n ( T a b l e 1). S i m i l a r observations
have been r e p o r t e d earlier using a z o s p i r i l l a strains
(Scott e t a l . 1979, N e y r a a n d V a n B e r k u m 1977,
N e l s o n a n d K n o w l e s 1978). Nitrogenase a c t i v i t y was
s l i g h t l y increased b y n i t r a t e a d d i t i o n s ( < 6 0 µ g N 0 3
m L - 1 ) , T h e i n h i b i t i o n o f nitrogenase a t higher levels
o f N O 3 m a y b e due t o N O 2 a c c u m u l a t i o n t h a t subseq u e n t l y b l o c k s N O 3 - d e p e n d e n t A R A . A slight g a i n
i n t o t a l cell p r o t e i n was n o t i c e d o n l y a t l o w e r levels
o f N O 3 ( < 6 0 µ g m L - 1 , T a b l e 2). N i t r a t e d i d n o t
increase the t o t a l cell p r o t e i n a p p r e c i a b l y , w h i c h
indicates the n o n u t i l i z a t i o n o f this c o m p o u n d u n d e r
m i c r o a e r o p h i l i c c o n d i t i o n s f o r cell g r o w t h , but i s
used as an e l e c t r o n acceptor. T o t a l n i t r o g e n s h o w e d
a m a r g i n a l g a i n at l o w levels a n d a s m a l l loss at
higher levels. A s m a l l increase in cell mass a n d t o t a l
n i t r o g e n a t the end o f the e x p e r i m e n t m a y b e due t o
n i t r o g e n f i x a t i o n . A t higher c o n c e n t r a t i o n s o f
n i t r a t e , the loss i n t o t a l n i t r o g e n was due t o f u r t h e r
d e g r a d a t i o n of NO 3 to N 2 O a n d N 2 .
It was f o u n d t h a t NO 2 was reduced to N 2 O at 60
µ g N O 2 m L - 1 a n d A R A was i m p r o v e d , b u t a t h i g h e r
levels the nitrogenase was repressed. A t l o w e r levels,
slight increase i n t o t a l cell p r o t e i n was also n o t i c e d .
T h e increase in cell mass was less after 3 days t h a n at
the earlier stage. T o t a l n i t r o g e n showed a m a r g i n a l
82
mL-1)
Days of incubation
3
6
0
698
282
60
710
380
300
ND1
ND
600
ND
ND
1200
ND
ND
0
792
334
30
870
351
60
910
380
90
ND
ND
120
ND
ND
150
ND
ND
430
0
647
10
699
499
20
676
460
40
ND
395
100
ND
ND
Amide
0
726
423
(NH+2)
30
759
480
60
752
468
120
ND
256
300
ND
ND
1. ND = Not detected.
g a i n up to 6 days of i n c u b a t i o n o n l y at l o w levels; at
higher c o n c e n t r a t i o n s a slight loss was observed.
Precise q u a n t i t a t i v e d e t e r m i n a t i o n s of N 2 O c o u l d
n o t be m a d e because of n o n a v a i l a b i l i t y of a s t a n d a r d
n i t r o u s o x i d e . A m m o n i u m was also f o u n d a t the end
of the e x p e r i m e n t , suggesting c o n v e r s i o n of NO - 3 to
N H + 4 . I n v i e w o f the fact that n i t o g e n f i x a t i o n does
n o t o c c u r u n d e r these c o n d i t i o n s , the presence o f
a m m o n i u m i s m o s t l i k e l y due t o a m m o n i f i c a t i o n .
S i m i l a r observations have also been r e p o r t e d by
Scott e t a l . (1979). N i t r o g e n - f i x i n g bacteria w i t h the
a b i l i t y t o assimilate N O - 3 w i l l n o t use this c o m p o u n d
f o r n i t r o g e n f i x a t i o n . H o w e v e r u n d e r o x y g e n levels
t o o l o w t o a l l o w r e s p i r a t i o n , nitrate-dependent acetylene r e d u c t i o n c a n occur. I n the e x p e r i m e n t s
where n i t r i t e was used as a substrate, s i m i l a r results
were f o u n d a n d n i t r i t e was used as e l e c t r o n acceptor.
I t therefore appears t h a t b o t h n i t r a t e a n d n i t r i t e
r e s p i r a t i o n i n this o r g a n i s m are c o u p l e d t o A R A .
T h e added a m m o n i c a l n i t r o g e n was u t i l i z e d by A.
brasilense 12S r a p i d l y , a n d o n the 3 r d d a y o f i n c u b a t i o n n o residual a m m o n i u m was f o u n d i n the
Table 2. Effect of nitrate, nitrite, a m m o n i u m , and amideN o n cell g r o w t h , p r o t e i n , a n d t o t a l n i t r o g e n .
T o t a l cell
protein
(µg mL
Levels
(µg
mL-1)
0
-1
Total nitrogen
(µg mL-1)
)
Days
3
6
0
Days
3
6
282
310
108
115
100
325
340
113
120
120
36
50
96
170
312
46
60
93
160
312
53
69
91
154
32
39
46
55
48
57
60
55
59
66
Nitrate
0
100
(NO-3)
60
300
600
100
98
98
1200
110
0
30
100
120
110
110
130
100
318
357
350
355
423
370
116
147
104
120
147
95
0
10
20
40
100
100
90
90
100
110
286
378
437
450
512
352
396
468
494
520
0
30
60
100
110
90
90
100
270
407
475
568
619
310
470
535
600
660
Nitrite
(NO-2)
60
90
120
150
Ammonium
(NH+4)
Amide
(NH+2)
120
300
66
75
296
55
62
65
50
54
54
53
68
80
36
48
67
74
48
111
115
83
110
42
58
73
103
44
68
77
50
78
89
104
191
98
182
193
66
76
81
m e d i u m a t l o w e r c o n c e n t r a t i o n s . A n appreciable
increase i n A R A was n o t i c e d u p t o 2 0 µ g N H + 4 m L - 1
o v e r the c o n t r o l . T h i s m a y b e because o f m o r e cell
mass available f o r acetylene r e d u c t i o n . T h e a m m o n i u m a d d e d at higher levels showed a loss in t o t a l
nitrogen, indicating oxidation of a m m o n i u m to
gaseous n i t r o g e n w h i c h was c o n f i r m e d by gas c h r o m a t o g r a p h y . At the 40 µ g NH + 4 m L - 1 level, n i t r o g e nase was depressed on the 6 t h d a y a n d n o a c t i v i t y
was detected o n the 3 r d day. M a r k e d increase i n
t o t a l cell p r o t e i n was n o t i c e d a t a l l levels o f a m m o n i u m c o m p a r e d t o the c o n t r o l . H o w e v e r , the effect
was less at h i g h e r levels. A s m a l l g a i n in t o t a l n i t r o gen was f o u n d at less t h a n 20 µ g N H J mL - 1 w i t h a
loss at 100 µ g NH + 4 m L - 1 .
U r e a was c o n v e r t e d t o a m m o n i u m a n d n o residu a l urea was detected on the 6 t h d a y even at a
c o n c e n t r a t i o n o f 300 µ g m L - 1 . T h e presence o f active
urease was n o t i c e d as the a d d e d urea was c o n v e r t e d
i n t o a m m o n i u m even o n the 3 r d d a y o f i n c u b a t i o n .
A m m o n i u m formed further showed transformations s i m i l a r t o those reported above. A R A was
enhanced over the c o n t r o l w i t h added urea u p t o 6 0
µ g on the 3 r d day, a n d was detected at 120 µ g on the
6 t h day. A t a l l concentrations, a n appreciable
increase i n cell p r o t e i n was n o t i c e d . T h e t o t a l n i t r o gen content increased m a r g i n a l l y at l o w levels, b u t at
higher c o n c e n t r a t i o n s a small loss was f o u n d .
A l l the n i t r o g e n o u s c o m p o u n d s tested s h o w e d a n
increase i n A R A a t l o w e r levels, w h i c h i s a n a d d i t i o n a l benefit of using this o r g a n i s m in the presence
o f added n i t r o g e n . T h e i n h i b i t o r y levels are a b o v e
the r e c o m m e n d e d field doses, therefore, a loss in
a d d e d n i t r o g e n w i l l n o t occur. Rather, a n increase i n
p o p u l a t i o n s w i l l help i n m o r e n i t r o g e n f i x a t i o n . Use
of A. hrasilense 12S can therefore e n r i c h n i t r o g e n
c o n t e n t by d i n i t r o g e n f i x a t i o n , even w h e n the levels
o f c o m b i n e d n i t r o g e n are l o w a n d the o x y g e n level i s
o p t i m a l i n the soil. A l t e r n a t i v e l y , this m a y c o n t r i b ute t o losses o f n i t r o g e n b y d e n i t r i f i c a t i o n w h e n the
concentration of combined nitrogen is high and o x y gen is l i m i t i n g .
References
C a t a l d o , D . A . , H a r o o n , M L , Schrader, L . W . , and Youngs,
V . L . 1975. R a p i d c o l o r i m e t r i c d e t e r m i n a t i o n o f n i t r a t e i n
p l a n t tissue b y n i t r a t i o n o f salicylic a c i d . C o m m u n i c a t i o n s
i n S o i l Science a n d P l a n t A n a l y s i s 6:71-80.
Dobereiner, J . , and D a y , J . M . 1976. A s s o c i a t i v e s y m b i oses i n t r o p i c a l grasses: c h a r a c t e r i z a t i o n o f m i c r o o r g a n i s m s a n d d i n i t r o g e n - f i x i n g sites. Pages 518-538 in
Proceedings o f the F i r s t I n t e r n a t i o n a l S y m p o s i u m o n N i t r o g e n F i x a t i o n , 3-7 J u n 1974, P u l l m a n , W a s h i n g t o n , U S A
( N e w t o n , W . E . , a n d N y m a n , C . J . , eds.). V o l . 2 . P u l l m a n ,
W a s h i n g t o n , U S A : W a s h i n g t o n State U n i v e r s i t y Press.
Douglas, L . A . , and Bremner, J . M . 1970. E x t r a c t i o n a n d
c o l o r i m e t r i c d e t e r m i n a t i o n o f urea i n soils. Proceedings,
S o i l Science Society o f A m e r i c a 34:859-862.
Jackson, M . L . 1967. S o i l c h e m i c a l analysis. N e w D e l h i ,
India: Prentice H a l l .
L o w r y , P . H . , Rosenbrough, N . J . , F a r r , A . L . , and R a n d a l l ,
R J.
1951. P r o t e i n m e a s u r e m e n t w i t h the f o l i n p h e n o l rea-
gent. J o u r n a l o f B i o l o g i c a l C h e m i s t r y 193:265-275.
Nelson, L . M . , a n d Knowles, R . 1978. Effect o f o x y g e n a n d
nitrate on nitrogen fixation and denitrification by Azospirillum brasilense g r o w n in c o n t i n u o u s c u l t u r e . C a n a d i a n
J o u r n a l o f M i c r o b i o l o g y 24:1395-1403.
N e y r a , C . A . , and van B e r k u m , P . 1977. N i t r a t e r e d u c t i o n
and
nitrogenase a c t i v i t y in Spirillum lipoferum. C a n a d i a n
J o u r n a l o f M i c r o b i o l o g y 23:306-310.
83
Nicholas, D . J . D . , a n d N a s o n , A . 1957. D e t e r m i n a t i o n o f
n i t r a t e a n d n i t r i t e . M e t h o d s i n E n z y m o l o g y 3:981-984.
Scott, D . B . , Scott, C . A . , a n d Dobereiner, J . 1979.
N i t r o g e n a s e a c t i v i t y a n d n i t r a t e r e s p i r a t i o n i n Azospirillum spp. A r c h i v e s o f M i c r o b i o l o g y 121:141-145.
84
Effect of Azotobacter chroococcum a n d Azospirillum
brasilense Inoculations and N i t r o g e n on Yields
o f S o r g h u m , M a i z e , P e a r l M i l l e t , and W h e a t
B . K . Konde and P . A . Shinde1
Summary
Field
experiments
In
general
were
conducted
inoculation
intermediate
levels
of
with
pearl
millet,
and
along
with
one-third
or
yields,
thus
significantly
A.
saving
superior
chroococcum,
a
third
to
and
in
N,
wheat
over
level
recommended
noninoculated
others
both
1979-82
rainy
chroococcum
significantly
two-third
of
the
the
Azotobacter
fertilizer
maize,
during
or
increased
fertilizer
control.
cultures
N.
In
were
Azospirillum
grain
the fully fertilized
of recommended
and postrainy
and
noninoculated
N,
generally
Inoculation
with
some
cases,
A.
at par
with
each
seasons
on
brasilense,
stover
resulted
either
along
yields
treatments.
of
brasilense
Vertisols.
of
Either
in
with
sorghum,
culture,
increased
crop
cultures
was
the
was
superior
to
other.
Introduction
Materials and Methods
Several w o r k e r s have r e p o r t e d that f i e l d i n o c u l a t i o n
w i t h Azospirillum u n d e r diverse s o i l a n d e n v i r o n m e n t a l c o n d i t i o n s resulted i n 1 0 - 8 1 % increase i n
cereal c r o p yields. Y i e l d s o b t a i n e d w i t h i n o c u l a t i o n ,
a l o n g w i t h i n t e r m e d i a t e levels o f f e r t i l i z e r N , were
r e p o r t e d t o b e higher t h a n i n f u l l y f e r t i l i z e d n o n i n o c ulated p l o t s (Desale 1980, K a p u l n i k et a l . 1982,
S u b b a R a o et a l . 1982, P a t i l 1983, Desale a n d K o n d e
1984, Z a m b r e et a l . 1984). These results suggest t h a t
it is possible to save a p a r t of v a l u a b l e f e r t i l i z e r N by
Azospirillum
inoculation.
T h e present paper reports the results o f f i e l d
experiments conducted at M a h a t m a Phule A g r i c u l t u r a l U n i v e r s i t y ( M P A U ) , R a h u r i , I n d i a , o n the
effects of Azotobacter chroococcum a n d A z o s p i r i l lum brasilense i n o c u l a t i o n w i t h g r a d e d levels of fert i l i z e r N o n g r a i n a n d stover yields o f s o r g h u m ,
maize, pearl m i l l e t , a n d wheat.
T h e carrier-based i n o c u l a n t of Azospirillum b r a s i lense ( A b ) was o b t a i n e d f r o m the D e p a r t m e n t o f
M i c r o b i o l o g y , I n d i a n A g r i c u l t u r a l Research I n s t i tute ( I A R I ) , N e w D e l h i . T h e lignite-based i n o c u lants of Azospirillum brasilense ( A M P 5) i s o l a t e d
f r o m the rhizosphere o f pearl m i l l e t a n d A . brasilense ( C 2 ) (10 9 cells g - 1 ) isolated f r o m the r o o t s o f
Cynodon dactylon were prepared in this d e p a r t m e n t . T h e carrier-based i n o c u l a n t of Azotobacter
chroococcum (10 8 cells g - 1 ) was o b t a i n e d f r o m t h e
A g r i c u l t u r a l B a c t e r i o l o g i s t , College o f A g r i c u l t u r e ,
Pune, M a h a r a s h t r a , I n d i a .
T h e seeds were treated w i t h the i n o c u l a n t s o f
respective organisms at the rate of 250 g (10 kg
seed) -1 as per the m e t h o d described by S u b b a R a o
(1981).
T h e e x p e r i m e n t s were c o n d u c t e d o n V e r t i s o l s ( p H
7.0-7.5, t o t a l N 0.03-0.05%, o r g a n i c C 0.60%) d u r i n g
1. Associate Professor and Head, Department of Agricultural Microbiology and Plant Pathology, Mahatma Phule Agricultural University,
Rahuri, Maharashtra 413 722, India.
I C R I S A T (International Crops Research Institute for the Semi-Arid Tropics). 1986. Cereal nitrogen fixation. Proceedings of the W o r k i n g
Group Meeting, 9-12 Oct 1984, I C R I S A T Center. India. Patancheru, A . P . 502 324, India: I C R I S A T .
85
the 1979-82 r a i n y a n d p o s t r a i n y seasons, u s i n g fact o r i a l r a n d o m i z e d - b l o c k designs ( R B D ) w i t h three
t o f o u r r e p l i c a t i o n s . T h e p l o t size v a r i e d f r o m 9.2 m 2
t o 20.26 m 2 , d e p e n d i n g u p o n t h e c r o p . F o r s o r g h u m
a n d maize, 6 2 k g h a - 1 each o f P 2 O 5 a n d K 2 O were
a p p l i e d a t s o w i n g t o a l l p l o t s . T h e n i t r o g e n levels 0 ,
33, 66, a n d 100 k g N h a - 1 were a p p l i e d i n t w o s p l i t
doses, i.e., t w o - t h i r d a t s o w i n g a n d the r e m a i n i n g
o n e - t h i r d one m o n t h after s o w i n g .
I n the p e a r l m i l l e t e x p e r i m e n t , a basal dose o f 3 0
k g P 2 O 5 h a - 1 was a p p l i e d , w h i l e the n i t r o g e n a t 20,
4 0 , 60, 80, a n d 100% of r e c o m m e n d e d dose ( i . e . , 12,
2 4 , 36, 48, a n d 6 0 k g N ha - 1 ) was a p p l i e d i n t w o e q u a l
doses, one a t s o w i n g a n d the o t h e r o n e m o n t h later.
I n the w h e a t e x p e r i m e n t , P 2 O 5 (60 k g h a - 1 ) a n d
K 2 O (50 kg ha - 1 ) were a p p l i e d at s o w i n g , w h i l e N was
a p p l i e d i n t w o s p l i t doses, i.e., h a l f a t s o w i n g a n d the
rest 2 5 days after s o w i n g ( D A S ) .
In a l l cases, N was a p p l i e d as urea, P 2 O 5 as single
superphosphate, a n d K 2 O a s m u r i a t e o f p o t a s h . A l l
crops received a l i g h t i r r i g a t i o n s o o n after s o w i n g .
Weeding, interculturing, and plant-protection opera t i o n s were c a r r i e d o u t a s a n d w h e n r e q u i r e d . T h e
stover a n d g r a i n yields were recorded f r o m each p l o t
at harvest.
Results
Sorghum
Increases i n g r a i n yields a l o n g w i t h b a c t e r i z a t i o n
o b t a i n e d at the 66 a n d 100 kg N ha - 1 levels were n o t
statistically significant. M a x i m u m percentage
increase in g r a i n y i e l d was observed at the 33 kg N
ha - 1 level a l o n g w i t h b a c t e r i z a t i o n t h a n at h i g h e r N
levels ( T a b l e 1). T h i s s h o w e d that higher level of N
fertilizer a p p l i c a t i o n was d e t r i m e n t a l t o n i t r o g e n fixing bacteria. H o w e v e r , higher g r a i n y i e l d was
o b t a i n e d at 66 kg N ha - 1 a l o n g w i t h b a c t e r i z a t i o n
t h a n w i t h 100 kg N ha - 1 alone. T h i s i n d i c a t e d t h a t 66
kg N ha - 1 c o u l d be c o m p a t i b l e w i t h either c u l t u r e s ,
a n d there appeared to be a p o s s i b i l i t y of s a v i n g 33 kg
N ha - 1 . A c r o s s the n i t r o g e n levels, i n o c u l a t i o n s w i t h
b o t h the cultures s i g n i f i c a n t l y increased g r a i n y i e l d
o v e r the c o n t r o l a n d n o significant difference c o u l d
T a b l e 1. G r a i n a n d stover yields of sorghum as influenced by inoculation w i t h nitrogen-fixing bacteria a n d fertilizer N.
N level ( k g h a - 1 )
0
Culture
66
33
100
Mean
4.52
-1
- G r a i n yield (t ha )
Control
A
chroococcum
(Ac)
4.00
4.19
4.67
5.20
5.04
5.65
5.94
6.01
(26.00)1
A.
brasilense
(Ab)
5.14
(28.50)
(34.84)
5.76
(37.47)
6.09
(30.41)
(15.58)
6.17
(18.65)
NS2
CD
4.72
Mean
CD
(27.19)
5.20
5.57
5.66
(25.22)
5.79
(28.09)
0.52
5.80
0.52
(P<0.05)
Stover yield (t ha-1)
Control
A.
chroococcum
(AC)
5.50
6.00
8.00
8.01
6.67
8.59
8.81
9.02
(21.12)
A.
brasilense
(Ac)
7.51
(36.54)
CD
(P<0.05)
1. % increase over control.
2. NS = Not significant.
86
9.76
(62.66)
(P<0.05)
Mean
CD
(43.16)
(10.12)
(12.60)
8.12
8.27
(20.20)
11.01
12.01
10.07
(36.62)
(49.93)
(46.36)
1.43
6.56
6.88
9.27
0.72
0.72
9.68
be n o t i c e d between the t w o bacteria. T h e results f o r
the i n t e r a c t i o n between cultures a n d N levels were
also n o t s t a t i s t i c a l l y s i g n i f i c a n t . I n general, trends
s i m i l a r t o g r a i n yields were also observed i n the case
of stover yields. T h e Azospirillum i n o c u l a t i o n was
f o u n d to be s i g n i f i c a n t l y s u p e r i o r to Azotobacter,
since i t p r o d u c e d t w o tonnes m o r e stover y i e l d t h a n
Azotobacter.
T h e results f o r i n t e r a c t i o n between
cultures a n d N levels were s i g n i f i c a n t .
t r o l , a n d n o significant difference c o u l d b e n o t i c e d
between t h e m . T h e results o f i n t e r a c t i o n f o r c u l t u r e s
a n d N levels were s t a t i s t i c a l l y s i g n i f i c a n t . T h e results
f o r stover y i e l d also s h o w e d a s i m i l a r t r e n d to those
f o r g r a i n yields.
Pearl Millet
B a c t e r i z a t i o n u n d e r i n t e r m e d i a t e levels ( 3 6 , 4 8 , a n d
60 kg ha - 1 ) of N fertilizer a p p l i c a t i o n s i g n i f i c a n t l y
increased g r a i n y i e l d ( T a b l e 3). H i g h e s t percentage
increase in g r a i n y i e l d was o b t a i n e d at the 48 kg N
level, a l o n g w i t h bacterization, w i t h either o f t h e
Azospirillum strains. T h e yields o b t a i n e d at 48 kg N
a l o n g w i t h b a c t e r i z a t i o n were h i g h e r t h a n w i t h 6 0 k g
N ha - 1 alone. T h u s b o t h strains h a d better c o m p a t i b i l i t y w i t h the 48 kg N level a n d s h o w e d the p o s s i b i l i t y of saving 12 kg N ha - 1 . Differences b e t w e e n t h e
t w o strains as also f o r the i n t e r a c t i o n b e t w e e n t h e
strains a n d N levels were s t a t i s t i c a l l y i n s i g n i f i c a n t .
T h e results f o r stover yields s h o w e d a s i m i l a r t r e n d
t o those o b t a i n e d f o r g r a i n yields.
Maize
A significant increase in g r a i n y i e l d c o u l d be
o b t a i n e d w i t h 66 a n d 100 kg N ha - 1 a l o n g w i t h
i n o c u l a t i o n , but n o t w i t h 33 kg N ha - 1 a p p l i c a t i o n
a l o n g w i t h i n o c u l a t i o n ( T a b l e 2). Differences
between 66 kg a n d 100 kg N a l o n g w i t h b a c t e r i z a t i o n
were n o t s i g n i f i c a n t . T h i s s h o w e d t h a t b o t h the c u l tures were c o m p a t i b l e at the 66 kg N level a n d i n d i cated the p o s s i b i l i t y of saving 33 kg N ha - 1 .
Azotobacter
a n d Azospirillum p r o d u c e d 4 9 % a n d
5 5 % h i g h e r g r a i n yields over the n o n i n o c u l a t e d c o n -
T a b l e 2 . G r a i n a n d stover yields o f maize a s influenced b y inoculation w i t h nitrogen-fixing bacteria a n d fertilizer N .
N level ( k g h a - 1 )
Culture
0
33
66
100
Mean
3.88
Grain yield (t ha-1)
Control
A.
chroococcum
(Ac)
3.43
3.95
3.98
4.15
3.60
5.60
6.98
7.07
(4.95)1
A. brasilense ( A b )
3.90
(13.70)
(41.86)
5.81
(47.18)
7.20
(80.90)
(70.36)
7.27
(75.18)
3.64
Mean
5.12
6.05
5.81
(49.74)
6.40
(55.67)
1.33
2.65
CD (P<0.05)
CD
(75.38)
6.16
1.33
(P<0.05)
Stover yields (t ha-1)
Control
A.
chroococcum
(Ac)
3.90
5.12
6.08
7.22
5.58
4.71
6.22
7.47
7.94
6.58
(22.86)
(9.97)
(17.92)
(5.36)
A.
hrasilense
CD
(Ab)
6.53
7.94
8.55
6.95
(7.38)
(27.53)
(30.60)
(18.42)
(24.55)
(P<0.05)
0.52
1.04
(P<0.05)
Mean
CD
(21.48)
4.80
4.47
5.%
7.16
7.90
NS2
1 . % increase over control.
2. NS = Not significant.
87
T a b l e 3 . G r a i n a n d stover yields o f pearl millet a s influenced b y inoculation w i t h n i t r o g e n - f i x i n g bacteria a n d fertilizer N .
N level ( k g ha-1)
12
Culture
24
36
48
60
Mean
2.10
G r a i n yield (t ha-1)
Control
A.
A.
brasilense ( A M P
brasilense
CD
5)
(Ab)
1.70
1.82
2.19
2.30
2.48
1.80
1.94
2.32
2.65
2.60
2.26
(5.88)1
(6.59)
(5.94)
(4.84)
(7.62)
1.78
1.92
2.30
(4.77)
(5.49)
(5.02)
1.76
1.89
2.27
Stover y i e l d (t
Control
brasilense ( A M P
brasilense
2.63
2.27
(6.05)
(8.09)
0.16
2.56
2.57
-
2.79
0.26
CD(P<0.05)
A.
2.72
(18.26)
NS2
(P<0.05)
Mean
A.
(15.22)
5)
(Ab)
ha-1)
—
2.53
2.55
2.60
3.07
3.22
2.60
2.64
2.73
3.36
3.42
2.95
(2.77)
(3.53)
(5.00)
(9.44)
(6.21)
(5.74)
2.59
2.63
2.75
(2.37)
(3.14)
(5.76)
CD
3.50
(14.01)
3.50
2.99
(6.69)
(7.17)
NS
NS
2.57
Mean
CD
2.61
2.69
3.31
3.38
0.16
(P<0.05)
1. % increase over control.
2. NS = Not significant.
Wheat
( T a b l e 4). T h e i n t e r a c t i o n effects were f o u n d t o b e
statistically significant. T h e i n t e r a c t i o n effect o f the
120 kg N ha - 1 , level w i t h Azospirillum (4.19 t ha - 1 )
a n d Azotobacter (4.01 t ha - 1 ) was at p a r a n d s u p e r i o r
to a l l other remaining treatment combinations. The
highest g r a i n y i e l d was o b t a i n e d at 120 kg N ha - 1 , b u t
T h e Azospirillum i n o c u l a t i o n recorded a m e a n g r a i n
y i e l d of 3.04 t ha - 1 over different levels of N f e r t i l i z e r
a p p l i c a t i o n , f o l l o w e d by Azotobacter, 2.90 t ha - 1 , as
c o m p a r e d t o 2.50 t h a - 1 i n the n o n i n o c u l a t e d c o n t r o l
T a b l e 4 . G r a i n yield o f wheat a s influenced b y i n o c u l a t i o n w i t h n i t r o g e n - f i x i n g bacteria a n d fertilizer N .
N level ( k g h a - ' )
12
Culture
24
36
48
60
Mean
Grain yield (t ha-1)
Control
A.
A.
chroococcum
brasilense
(Ac)
(Ab)
1.58
2.07
2.53
2.91
3.41
2.54
1.66
2.40
2.97
3.47
4.01
2.90
(5.06)'
(15.94)
(17.39)
(19.24)
(17.59)
1.76
2.49
(11.39)
CD
(P<0.05)
1. % increase over control.
88
(23.32)
3.66
(25.77)
4.19
(22.87)
0.18
(P<0.05)
Mean
CD
(20.28)
3.12
1.67
2.32
2.87
3.35
0.093
(16.00)
3.04
(21.60)
0.093
3.87
highest increase over n o n i n o c u l a t e d c o n t r o l due t o
Azotobacter was 19.24%
a n d due to Azospirillum
25.77%, i n c o m b i n a t i o n w i t h 9 0 k g N ha - 1 . T h i s i s
o b v i o u s , since increase in fertilizer doses at a c e r t a i n
level decreases a crop's response. I n o c u l a t i o n w i t h
Azotobacter a n d Azospirillum over N levels p r o d u c e d 1 6 a n d 2 1 % h i g h e r g r a i n yields o n a n average
over the n o n i n o c u l a t e d c o n t r o l .
Acknowledgements
T h e a u t h o r s express t h e i r sincere t h a n k s t o D r N . S .
Subba R a o , H e a d , D i v i s i o n o f M i c r o b i o l o g y , I A R I ,
New Delhi, and Dr P.L. Patil, Agricultural Bacteriologist, College o f A g r i c u l t u r e , P u n e , f o r s u p p l y o f
Azospirillum a n d Azotobacter i n o c u l a n t s . T h e y also
express t h e i r t h a n k s t o M s M . A . Z a m b r e , M r P . H .
P a t i l , a n d late M r A . G . Desale, p o s t g r a d u a t e s t u dents on whose research this paper has been based,
f o r t h e i r help i n the present i n v e s t i g a t i o n .
Conclusions
T h e results i n d i c a t e t h a t a b o u t o n e - t h i r d o f the
r e c o m m e n d e d level of fertilizer N c o u l d be saved if
seed b a c t e r i z a t i o n w i t h either cultures is d o n e before
s o w i n g . T h i s i s o f p a r t i c u l a r significance t o those
farmers w h o are n o t i n a p o s i t i o n t o invest i n f e r t i l i z ers. I n a d d i t i o n t o the k n o w n benefits f r o m associat i v e n i t r o g e n f i x a t i o n ( W a n i 1986), the increase i n
c r o p yields have also been a t t r i b u t e d to the p r o d u c t i o n o f g r o w t h - p r o m o t i n g substances b y either o f
these organisms ( T i e n et a l . 1979, Vlassak a n d
Reynders 1981) a n d also due to the p o s i t i v e effects of
Azospirillum r o o t d e v e l o p m e n t a n d r o o t u p t a k e o f
m i n e r a l s a n d w a t e r ( K a p u l n i k e t a l . 1982). I n the
present i n v e s t i g a t i o n Azospirillum brasilense was
f o u n d to be either s u p e r i o r to A. chroococcum or at
p a r w i t h i t . T h e i n t e r a c t i o n effects o n g r a i n yields
between N fertilizer levels a n d seed b a c t e r i z a t i o n
w i t h either cultures were observed to be i n s i g n i f i c a n t
i n s o r g h u m a n d pearl m i l l e t , b u t significant i n maize
a n d wheat.
P r i o r i t i e s f o r F u t u r e Research
Based o n results o f the present e x p e r i m e n t s the f o l l o w i n g lines o f w o r k are suggested:
1. Response of c r o p genotypes to Azospirillum
s h o u l d be tested u n d e r f i e l d c o n d i t i o n s . T h i s
i n f o r m a t i o n w o u l d b e useful f o r b r e e d i n g genotypes h a v i n g enhanced n i t r o g e n - f i x i n g a b i l i t y .
2 . I s o l a t i o n a n d screening o f d i a z o t r o p h i c a z o s p i r i l l a strains h a v i n g h i g h nitrogenase a c t i v i t y f r o m
a w i d e range of hosts i n c l u d i n g cereals, vegetables, grasses, sugarcane, p o t a t o , ginger, t u r m e r i c ,
etc., s h o u l d be u n d e r t a k e n .
3. C o m p a t i b i l i t y of Azospirillum w i t h seed-dressi n g fungicides, insecticides, a n d f e r t i l i z e r n i t r o g e n s h o u l d be s t u d i e d .
References
Desale, A . G . 1980. Effect o f seed b a c t e r i z a t i o n w i t h A z o tobacter a n d Azospirillum cultures u n d e r v a r i o u s levels of
nitrogen on growth, and yield of sorghum and maize.
M . S c . thesis, M a h a t m a P h u l e A g r i c u l t u r a l U n i v e r s i t y ,
Rahuri, Maharashtra, India.
Desale, A . G . , and K o n d e , B . K . 1984. Response o f
s o r g h u m t o seed b a c t e r i z a t i o n w i t h n i t r o g e n levels. J o u r n a l
o f M a h a r a s h t r a A g r i c u l t u r a l U n i v e r s i t i e s 9:169-170.
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Y . 1982. T h e effect o f Azospirillum i n o c u l a t i o n o n g r o w t h
a n d y i e l d o f c o r n . Israel J o u r n a l o f B o t a n y 31:247-255.
P a t i l , P . H . 1983. Response o f some pear l m i l l e t g e n o t y p e s
t o Azospirillum i n o c u l a t i o n a n d effect o f i n o c u l a t i o n u n d e r
g r a d e d levels o f n i t r o g e n o n g r o w t h a n d y i e l d o f p e a r l m i l l e t
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University, Rahuri, Maharashtra, India.
Subba R a o , N . S . 1981. Response o f crops t o Azospirillum
i n o c u l a t i o n i n I n d i a . Pages 137-144 i n A s s o c i a t i v e N 2 f i x a t i o n : proceedings o f the I n t e r n a t i o n a l W o r k s h o p o n
A s s o c i a t i v e N 2 - f i x a t i o n , 2-6 J u l 1979, P i r a c i c a b a , B r a z i l
( V o s e , P . B . , a n d R u s c h e l , A . P . , eds.). V o l . 1 . B o c a R a t o n ,
F l o r i d a , U S A : C R C Press.
Subba R a o , N . S . , T i l a k , K . V . B . R . , Singh, C.S., and G a u t a m , R . C . 1982. C r o p y i e l d a n d n i t r o g e n c o n t e n t o f p e a r l
m i l l e t ( P e n n i s e t u m americanum) in response to A z o s p i r i l lum brasilense. Pages 507-516 in Proceedings of the
National Symposium on Biological Nitrogen F i x a t i o n , 252 7 F e b 1982, N e w D e l h i , I n d i a . B o m b a y , I n d i a : B h a b h a
A t o m i c Research C e n t r e .
T i e n , T . M . , Gaskins, M . H . , and H u b b e l l , D . H . 1979.
P l a n t g r o w t h substances p r o d u c e d by Azospirillum brasilense a n d t h e i r effect o n the g r o w t h o f p e a r l m i l l e t (Pennisetum americanum
L.).
Applied and Environmental
M i c r o b i o l o g y 37:1016-1024.
89
Vlassak, K., and Reynders, L. 1981. Azospirillum r h i z o coenoses in a g r i c u l t u r a l p r a c t i c e . Page 4 9 4 in C u r r e n t perspectives i n n i t r o g e n f i x a t i o n : proceedings o f t h e
I n t e r n a t i o n a l S y m p o s i u m o n N i t r o g e n F i x a t i o n , 1-5 D e c
1980, C a n b e r r a , A u s t r a l i a ( G i b s o n , A . H . , a n d N e w t o n ,
W . E . , eds.). C a n b e r r a , A u s t r a l i a : A u s t r a l i a n A c a d e m y o f
Science.
N . S . Subba R a o :
A t the h i g h e r level o f f e r t i l i z e r n i t r o g e n a p p l i c a t i o n ,
the a m o u n t of f e r t i l i z e r N left b e h i n d u n u t i l i z e d is
n o t k n o w n . S i m p l e a r i t h m e t i c c a l c u l a t i o n o f savings
is c o n v e n i e n t to present, b u t d e d u c t i o n s f o r r e s i d u a l
n i t r o g e n m a y have t o b e d o n e .
W a n i , S . P . 1986. C e r e a l n i t r o g e n f i x a t i o n : p r o b l e m s a n d
p o t e n t i a l i t i e s . Pages 7-21 in C e r e a l n i t r o g e n f i x a t i o n . P r o ceedings o f the W o r k i n g G r o u p M e e t i n g , 9-12 O c t 1984,
I C R I S A T C e n t e r , I n d i a . P a t a n c h e r u , A . P . 502 324, I n d i a :
I n t e r n a t i o n a l C r o p s Research I n s t i t u t e f o r the S e m i - A r i d
Tropics.
B . K . Konde:
W e l l , w e have n o t estimated the q u a n t u m o f f e r t i l izer n i t r o g e n left b e h i n d ; h o w e v e r , this suggestion
w i l l be taken into consideration while planning
further experiments.
Z a m b r e , M . A . , K o n d e , B . K . , a n d Sonar, K . R . 1984.
Effect of Azotobacter chroococcum a n d Azospirillum brasilense i n o c u l a t i o n u n d e r g r a d e d levels o f n i t r o g e n o n
g r o w t h a n d y i e l d o f w h e a t . P l a n t a n d S o i l 79:61-67.
S.P. W a n i :
H i g h e r N percentage in p l a n t tissues a n d increased
biomass w i l l result i n very h i g h N u p t a k e . T h e d a t a
need to be rechecked f o r u n u s u a l l y h i g h values.
Discussion
B . K . Konde:
Yes, 1 agree w i t h y o u r c o m m e n t s b u t can't d e n y the
facts t h a t w e observed. H o w e v e r , i n f u t u r e e x p e r i ments, we shall take due care to ensure the p r o p e r N
e s t i m a t i o n o f p l a n t tissues.
G.S.Murty:
A t w h a t level o f N - a p p l i c a t i o n was the saving o f
o n e - t h i r d t o t w o - t h i r d o f the r e c o m m e n d e d n i t r o g e n
dose observed, 2 0 k g ha - 1 , 4 0 k g h a - 1 , o r m o r e ? T h e
r e c o m m e n d e d dose f o r s o r g h u m a n d maize is 100 kg
N ha - 1 .
B . K . Konde:
S a v i n g of o n e - t h i r d N (33 kg N ha - 1 ) has been
observed i n s o r g h u m a n d maize a n d a saving o f 2 0 %
N (12 kg N ha - 1 ) in p e a r l m i l l e t a n d o n e - t h i r d N (30
kg N ha - 1 ) in wheat.
P.V. Rai:
W e need t o l o o k f o r the q u a l i t y o f p r o t e i n i n the
grains f r o m the i n o c u l a t e d t r e a t m e n t , a l t h o u g h i t
a l w a y s gives h i g h e r p r o t e i n .
B . K . Konde:
T h i s m a y b e i n c l u d e d i n o u r research projects. Yes,
we have also a l r e a d y estimated the p r o t e i n c o n t e n t
o f g r a i n s i n a l l c r o p s , b u t the data presented are
c o n f i n e d t o f o d d e r a n d g r a i n yields a n d N c o n t e n t i n
p l a n t tissues, o w i n g t o l i m i t a t i o n o f t i m e .
G . S . Jadhav:
S m a l l p l o t size m a y b e the reason f o r v e r y h i g h y i e l d .
B . K . Konde:
T h e net p l o t size r a n g e d f r o m 9 t o 2 0 m 2 .
90
V . R . Rao:
I n y o u r studies o n s o r g h u m , the percentage N i n
p l a n t tissues due t o i n o c u l a t i o n increased f r o m 0 . 3 %
i n the c o n t r o l t o 0.8%. C o u l d y o u t h r o w some l i g h t
o n this?
B . K . Konde:
T h i s increase i s a t t r i b u t e d t o b i o l o g i c a l l y f i x e d N ,
besides the fertilizer n i t r o g e n s u p p l i e d t h r o u g h the
soil i n the f o r m o f urea. These data, a s suggested b y
o t h e r m e m b e r s , w i l l b e checked.
S . T . Shende:
W h y was the i n o c u l a t i o n effect a t par w i t h h i g h
n i t r o g e n a p p l i c a t i o n ? W a s i t due t o l o w efficiency o f
N a p p l i c a t i o n d u r i n g that season?
B . K . Konde:
I t i s a t t r i b u t e d t o the reason t h a t h i g h e r level o f N
f e r t i l i z e r a p p l i c a t i o n m i g h t have depressed the
nitrogenase a c t i v i t y o f the o r g a n i s m s , a l t h o u g h
increased levels of N f e r t i l i z e r a l o n e also increased
yields a n d t h e i r parameters.
G . Oblisami:
I n y o u r d a t a o n i n o c u l a t i o n o f maize w i t h A . chroococcum a n d Azospirillum brasilense, the zero level of
f e r t i l i z e r n i t r o g e n gave h i g h e r g r a i n yields t h a n 100%
fertilizer n i t r o g e n alone. H o w d o y o u e x p l a i n this
phenomenon?
B . K . Konde:
I t h i n k the reasons m i g h t be (1) h i g h o r g a n i c m a t t e r
( o r g a n i c C 0.6%) c o n t e n t of the s o i l , (2) h i g h rates of
fertilizer n i t r o g e n depressing nitrogenase a c t i v i t y ,
a n d (3) m o r e g r o w t h - p r o m o t i n g substances being
p r o d u c e d i n the absence o f h i g h e r levels o f fertilizer
N.
91
Root-Associated N i t r o g e n F i x a t i o n i n Finger M i l l e t
M . N . Upadhyaya1, S.V. Hegde2, P.V. R a i 2 , and S.P. W a n i 3
Summary
This
paper
presents
nitrogen fixation
finger
activity
cultivars
(2)
in
plant
root-associated
millet
yields
and
study
with
N
ha
to four
over
pure
no
uptake
from
were
Indaf 7,
over
and
the
three
diurnal
soil,
with
levels
All
of
cultivars
wash,
and
the
applied
SRI
had
nitrogen
highest
and
but
showed
over
activity
root
cultures,
a
in
net
gain
earlyand
or
dryby
counts
during
macerate.
The
Inoculation
increased
the field.
higher
affected
diazotroph
Enterobacter.
not
(4)
was further
unsterilized
diazotroph
greenhouse,
group
nitrogenase
of 53
of nitrogenase
cultivars,
system
and
assay
levels
between
soil-root
of root-associated
nitrogenase
low
medium-maturity
Pseudomonas
NBRE
in
(1)
between
millet
root
of
control
in
aspects
Soil-root
variability
activity
changes.
some
indicated
relationship
of finger
on
Gaertn).
high
higher
species
out
groups
(3)
(5)
activity
root-adhering
in
coracana
apparent
and
inoculation
-1
lines,
moisture,
cultivar
N
(6)
carried
maturity
variability,
stage
nitrogen fixers
finger
kg
(Eleucine
Nitrogenase
soil
reproductive
millet
and
production.
of investigations
belonging
plant-to-plant
groups,
age,
the
high
hybrids
late-maturity
matter
results
in finger
millet
activity,
the
of
above-ground
Nitrogen-balance
equivalent
to
63-9.4
inoculation.
Introduction
F i n g e r m i l l e t (Eleusine coracana G a e r t n ) is an
i m p o r t a n t f o o d c r o p i n the s e m i - a r i d t r o p i c s ( S A T ) ,
p a r t i c u l a r l y i n I n d i a a n d Eastern A f r i c a . T h e c r o p
ranks f o u r t h i n area ( 5 m i l l i o n ha) a n d p r o d u c t i o n
(4.5 m i l l i o n t ) a m o n g m i l l e t s i n the w o r l d . I n d i a
accounts f o r 4 5 % o f the w o r l d p r o d u c t i o n .
Contributions of nonsymbiotic nitrogen fixation
associated w i t h finger m i l l e t a n d o t h e r m i l l e t s ,
t h o u g h felt to be s u b s t a n t i a l , have been w i d e r a n g i n g
(20-148 kg N ha - 1 ) d u e to different m e t h o d o l o g i e s
used b y different w o r k e r s ( M o o r e 1963, J e n k i n s o n
1977, D a r t a n d W a n i 1982). Studies were c o n d u c t e d
a t the U n i v e r s i t y o f A g r i c u l t u r a l Sciences, B a n g a lore, o n (1) root-associated nitrogenase a c t i v i t y o f 5 3
finger m i l l e t c u l t i v a r s , (2) occurrence o f d i a z o t r o p h s ,
(3) i n o c u l a t i o n response, (4) factors affecting n i t r o gen fixation, a n d (5) n i t r o g e n balance.
Screening G e r m p l a s m
F i f t y - t h r e e c u l t i v a r s representing b o t h p u r e - l i n e
selections a n d h y b r i d s i n f o u r m a t u r i t y g r o u p s w e r e
screened f o r t h e i r a b i l i t y t o s t i m u l a t e nitrogenase
a c t i v i t y ( C 2 H 2 r e d u c t i o n ) i n the rhizosphere a t t h e
f l o w e r i n g a n d p o s t f l o w e r i n g g r o w t h stages ( T a b l e
1). U n p l a n t e d soil a n d finger m i l l e t c u l t i v a r s I n d a f 5
were i n c l u d e d a s checks i n each g r o u p . T h e p l a n t s
were g r o w n i n p o l y t h e n e bags f i l l e d w i t h 3.3 k g s o i l .
T h e s o i l surface i n each b a g was covered w i t h a 5 - c m
t h i c k g r a v e l , a n d the bags were k e p t i m m e r s e d in a
sand bed t o a v o i d algal g r o w t h a t the s o i l surface a n d
1. P h D Student, Australian National University, Canberra, Australia.
2.
Senior Microbiologist, and Professor and Head, Department of Agricultural Microbiology, University of Agricultural Sciences, Gandhi
3.
Microbiologist, Pearl Millet improvement Program, I C R I S A T , Patancheru, A . P . 502 324, India.
Krishi Vignana Kendra ( G K V K ) , Bangalore, Karnataka 560 065, India.
I C R I S A T (International Crops Research Institute f o r the Semi-Arid Tropics). 1986. Cereal nitrogen fixation. Proceedings of the W o r k i n g
G r o u p Meeting, 9-12 Oct 1984, I C R I S A T Center, India. Patancheru, A . P . 502 324, India: I C R I S A T .
93
a t the interface o f s o i l a n d bag. A basal dose o f 5 0 k g
each o f N , P 2 O 5 , a n d K 2 O h a - 1 e q u i v a l e n t was a p p l i e d t h r o u g h a m m o n i u m sulfate, superphosphate,
a n d p o t a s s i u m c h l o r i d e respectively. P l a n t s were
watered a s r e q u i r e d w i t h t a p w a t e r a n d one d a y
before assay the m o i s t u r e c o n t e n t of t h e bags was
adjusted t o 70-80% w a t e r - h o l d i n g capacity ( W H C ) .
F o r nitrogenase assay, t h e m e t h o d f o l l o w e d b y W a n i
et a l . (1983) was used.
T h e general level o f nitrogenase a c t i v i t y associated w i t h 5 3 finger m i l l e t c u l t i v a r s was l o w , a n d the
g r o u p means r a n g e d f r o m 2 t o 267 n m o l e s C 2 H 4 h - 1
b a g - 1 or 1 to 59 n m o l e s h - 1 g - 1 r o o t mass ( T a b l e 1).
R a n k i n g o f c u l t i v a r s based o n r o o t a c t i v i t y d i f f e r e d
w h e n expressed a s g - 1 r o o t t h a n o n b a g - 1 basis. T h e
m e a n nitrogenase a c t i v i t y o f 0.2-13 n m o l e s h - 1 b a g - 1
i n the u n p l a n t e d s o i l suggested s t i m u l a t i o n o f
nitrogenase a c t i v i t y b y the p l a n t . Increased n i t r o g e nase a c t i v i t y i n A s s a y I I o v e r A s s a y I i n m a t u r i t y
groups I and II indicated higher activity d u r i n g postf l o w e r i n g g r o w t h stages. T h e m a x i m u m nitrogenase
a c t i v i t y o f i n d i v i d u a l plants i n this s t u d y was 488
n m o l e s C 2 H 4 h - 1 bag - 1 . T h i s i s m u c h l o w e r t h a n the
a c t i v i t y o f 1064 nmoles C 2 H 4 h - 1 core - 1 r e c o r d e d f o r
f i n g e r m i l l e t c u l t i v a r P R 202 a t I C R I S A T Center
( I C R I S A T 1978) a n d 374 nmoles C 2 H 4 h - 1 g - 1 d r y
mass by D o m m e r g u e s et a l . (1973).
W i t h i n a cultivar, plant-to-plant variation in
nitrogenase a c t i v i t y f r o m 1 0 t o 488 nmoles C 2 H 4 h - 1
bag - 1 i n c u l t i v a r T N A U 169 a n d 5-359 nmoles C 2 H 4
h - 1 b a g - 1 i n c u l t i v a r I n d a f 5 was observed. S u c h
v a r i a t i o n s have also been r e c o r d e d i n s o r g h u m a n d
pearl m i l l e t ( W a n i e t a l . 1983).
O f t h e 5 3 c u l t i v a r s tested, the a c t i v i t y was m o r e
T a b l e 1 . Nitrogenase activity (nmoles C 2 H 4 h - 1 ) 1 o f soil-root system o f f i n g e r millet cultivars d u r i n g t w o assays. 1
Maturity
Assay 1
group
Assay II
Range/Cultivar
(Days to 5 0 %
DAS3
Cultivar
flowering)
bag-1
g-1
2
root
Range/Cultivar2
DAS
bag-1
g-1 root
10-138
4-46
I
V L 1 1 4 , V L 1 1 5 , V L 116
12-70
2-30
(56-62)
V L 1 1 8 , V L 110,
R E C 13-1
R E C 13-1
R E C 45-1
R E C 45-1
P E S 83-2,PES 400
R E C 45-1
V L 115
P E S 83-2
P E S 83-2
55
62
(68)
(38)
R A U 3,R A U 7,RAU 5,RAU 2,
R E C 4 5 - 1 , R E C 13-1
II
VL
56-267
8-59
(63-70)
P R 1 7 7 , P R 1103,
T N A U 160 T N A U 160
V L 117
P R 1103
P R 1 0 9 1 , P E S 176,
T N A U 169 T N A U 169
T N A U 169 P R 1091
117, I n d a f 9 , H R 3 7 4 ,
70
2-30
1-11
92
(13)
T N A U 1 6 9 , T N A U 167,
(131)
T N A U 193, T N A U 2 5 6 ,
T N A U 160, T N A U 2 9 4
1 5 4 , H R 2823
ND4
ND
94
21-166
3-24
HI
HR 919,HR
(71-78)
R O H 2, H R 1541,
P R 230
P R 230
P u r n a , P R 230, PR 202,
B 7-7-43
B 7-7-43
B 7 - 7 - 4 3 , P E S 110, J N R 9 8 1 - 1
J N R 852,JNR
(85)
1008
IV
I n d a f 1, I n d a f 3, VR 550,
19-12
4-32
(79-89)
I n d a f 7, I n d a f 8,
ND
ND
99
Indaf 8
Indaf 8
I n d a f 1 0 , I n d a f 1 1 , V R 530
I E 1012
I n d a f 10
K M 13, I E 1012, U 6 , U 10
( 5 1 ) A 104
1. Mean of 4 replications. Indaf 5 and implanted soil were used as checks. Nitrogenase activity of unplanted control ranged f r o m 0.2 to 13 nm
C 2 H 4 bag - 1 .
2. Mean nitrogenase activity.
3. P A S = Days after sowing.
4. ND = N o t determined.
94
t h a n 300 n m o l e C 2 H 4 h - 1 b a g - 1 i n 1 2 c u l t i v a r s ,
between 200 to 300 in 8 c u l t i v a r s , between 100 to 200
n m o l in 20 c u l t i v a r s , a n d less t h a n 100 in 13 c u l t i v a r s .
S u c h v a r i a b i l i t y f o r nitrogenase a c t i v i t y a m o n g s t
finger m i l l e t c u l t i v a r s has also been observed earlier
( I C R I S A T 1978). T h e t o p five c u l t i v a r s , based o n
m a x i m u m a c t i v i t y , were T N A U 169, T N A U 256,
P u r n a , I E 1012, a n d I n d a f 9 , i n t h a t o r d e r . Least
a c t i v i t y was f o u n d i n R A U 7 . Generally, h y b r i d
selections s h o w e d h i g h e r nitrogenase a c t i v i t y t h a n
p u r e - l i n e selections ( T a b l e 2).
The m e d i u m - m a t u r i t y cultivars (groups I I and
I I I ) had h i g h e r nitrogenase a c t i v i t y t h a n either e a r l y (group I) or late-maturity cultivars (group I V ) .
M i s h r a e t a l . (1980) h a d also f o u n d t h a t m e d i u m m a t u r i t y c u l t i v a r s were m o r e p r o d u c t i v e t h a n e a r l y
o r late types, based o n t h e i r screening o f 400 f i n g e r
m i l l e t cultivars.
i n c l u d e d . S i x r a n d o m l y selected p l a n t s were assayed
f o r nitrogenase at 22, 32, 44, 59, 68, 8 1 , 105, a n d 129
days after s o w i n g ( D A S ) , f o l l o w i n g the m e t h o d
m e n t i o n e d earlier.
Nitrogenase a c t i v i t y increased f r o m 2 2 D A S ( 9
n m o l C 2 H 4 h - 1 bag - 1 ) t o peak a t 6 8 D A S (106 n m o l
C 2 H 4 h - 1 bag - 1 ) a n d t h e n decreased t o 3 6 n m o l e s
C 2 H 4 h - 1 bag - 1 a t 105 D A S ( F i g . 1).
F o r e s t i m a t i n g the counts o f d i a z o t r o p h s , t w o
r a n d o m l y selected bags each, i.e., p l a n t e d a n d
u n p l a n t e d , were selected each t i m e after assay.
Increase i n nitrogenase a c t i v i t y o f the s o i l - r o o t syst e m closely f o l l o w e d increase o f d i a z o t r o p h c o u n t s
u p t o 6 8 days, p a r t i c u l a r l y the d i a z o t r o p h c o u n t o f
nonsterilized and sterilized r o o t samples. U n l i k e the
nitrogenase a c t i v i t y t h a t decreased 6 8 D A S , t h e d i a z o t r o p h c o u n t r e m a i n e d largely u n c h a n g e d . Assay
a t 6 8 D A S gave b o t h m a x i m u m nitrogenase a c t i v i t y
(129 n m o l C 2 H 4 h - 1 bag - 1 ) a s w e l l a s m a x i m u m d i a z o t r o p h c o u n t o f sterilized ( 4 x 10 9 g - 1 r o o t ) a n d
n o n s t e r i l i z e d (2 x 10 11 g - 1 r o o t ) macerate.
I n studies w i t h c u l t i v a r I n d a f 5 , the d i a z o t r o p h
c o u n t s were i n general h i g h e r d u r i n g the r e p r o d u c tive stage t h a n d u r i n g the vegetative stage. R o o t a d h e r i n g s o i l gave the m a x i m u m d i a z o t r o p h c o u n t
(8.5 x 10 9 ) g - 1 d r y r o o t , f o l l o w e d by n o n s t e r i l i z e d
r o o t macerate (3.4 x 10 9 ), a n d r o o t w a s h (3.1 x 10 9 ).
Surface-sterilization o f r o o t reduced the c o u n t f r o m
3.4 x 10 9 to 1.6 x 10 8 (g r o o t mass) -1 f o r 5 m i n
s t e r i l i z a t i o n , a n d 1.3 x 10 6 (g r o o t mass) -1 f o r 60 m i n
s t e r i l i z a t i o n . T h e n u m b e r o f c o l o n y types o f d i a z o t r o p h s d i d n o t increase f r o m the vegetative t o r e p r o -
Seasonal Variation in Diazotroph Counts
and Nitrogenase Activity
Plants o f c u l t i v a r I n d a f 5 were s i m i l a r l y g r o w n i n
p o l y t h e n e bags, except that 2.75 kg soil was used in
this e x p e r i m e n t . T h e plants were i n o c u l a t e d w i t h 50
m L r h i z o s p h e r e - s o i l e x t r a c t . T h i s e x t r a c t was prepared by m i x i n g 1 kg rhizospheric soil collected
f r o m the field w i t h 4 L N-free sucrose m e d i u m ,
i n c u b a t e d f o r 42 h at r o o m t e m p e r a t u r e , filtered
t h r o u g h a cheese c l o t h , a n d f i n a l l y m a d e up to 10 L
using water. A n u n p l a n t e d t r e a t m e n t was also
T a b l e 2. Screening of pure-line a n d h y b r i d selections of different origin for the root-associated nitrogenase a c t i vi t y .
N o . of cultivars having
No. of
Highest n i t r o -
nitrogenase activity
culti-
(nmol C 2 H 4 plant-1 h-1)
vars
active
(nmol C2H4
cultivar
plant-1 h-1)
Pedigree
Karnataka
Hybrid
15
5
4
2
4
Indaf 9
361
Tamil Nadu
Hybrid
6
3
0
3
0
T N A U 169
488
A n d h r a Pradesh
Pure line
7
0
1
3
3
P R 1091
250
M a d h y a Pradesh
Pure line
5
1
1
2
1
J N R 852
354
Uttar Pradesh
Pure line
11
1
2
6
2
P E S 83-2
324
2
200-300
100-200
Bihar
Pure line
4
0
0
2
Orissa
Pure line
2
1
0
1
Exotic collection
Pure line
3
1
0
1
Total
53
12
(23)*
8
(15)
<100
genase a c t i v i t y
Origin
tested
>300
Most
1
20
13
(37)
(25)
RAU 5
183
B 7-7-43
314
I E 1012
403
1. Figures in parentheses are percentages.
95
e n t l y o n N-free m e d i u m were f i n a l l y o b t a i n e d i n
pure cultures.
W h e n the 4 0 d i a z o t r o p h isolates f r o m c u l t i v a r
I n d a f 5 were screened f o r A R A , a l l b u t 3 isolates
d u c t i v e g r o w t h stage. A m a x i m u m o f 1 5 c o l o n y
types were observed i n n o n r h i z o s p h e r i c s o i l , a n d
surface-sterilized r o o t macerate s h o w e d 5 to 6 c o l o n y types. A t o t a l o f 4 0 isolates t h a t g r e w consist-
120
Soil-root a c t i v i t y
Soil a c t i v i t y
S h o o t mass
R o o t mass
100
80
50
60
40
40
30
20
20
10
0
0
22
32
44
59
Age
68
81
105
129
(days)
F i g u r e 1. Seasonal pattern of nitrogenase activity a n d shoot a n d r o o t mass of finger millet cultivar I n d a f 5.
96
p r o d u c e d less t h a n 2 n m o l e s C 2 H 4 h - 1 t u b e - 1 . O f
these three isolates, N o . 38 a n d 45 were i d e n t i f i e d as
Enterobacter
cloacae a n d Isolate N o . 23 was
Azospirillum-like.
T h e rest of the isolates were species of Pseudomonas or Enterobacter cloacae. I s o lates f r o m f i n g e r m i l l e t were c o m p a r e d w i t h three
reference cultures, i.e., N B R E ( n a p i e r bajra r o o t
e x t r a c t ) , A. lipoferum ( 4 A B L ) , a n d Azospirillum
lipoferum ( I C M 1001) f o r in-vitro a c t i v i t y . T h e c u l tures N B R E a n d 4 A B L s h o w e d f o u r - f o l d m o r e
a c t i v i t y t h a n the isolates f r o m finger m i l l e t . H o w ever, E. cloacae s h o w e d h i g h e r a c t i v i t y t h a n A.
lipoferum. H i g h e s t a c t i v i t y (115 nmoles C 2 H 4 h - 1
t u b e - 1 ) was r e c o r d e d i n the N B R E c u l t u r e .
Effect of Soil Moisture
Effect o f m o i s t u r e o n nitrogenase a c t i v i t y o f finger
m i l l e t I n d a f 5 was studied by m a n i p u l a t i n g the m o i s t u r e regime i n the s o i l a n d b y u s i n g a n i n t a c t - p l a n t
assay m e t h o d ( W a n i et a l . 1984). T h e plants were
g r o w n in s o i l in plastic bags, a n d a basal dose of 25
kg N a n d 50 kg each of P 2 O 5 a n d K 2 O ha - 1 t h r o u g h
urea, superphosphate, a n d p o t a s s i u m c h l o r i d e was
a p p l i e d . T i l l the m o i s t u r e - l e v e l treatments were
i m p o s e d , a l l the plants were w a t e r e d u n i f o r m l y .
N i t r o g e n a s e a c t i v i t y of the s o i l - r o o t system as
affected b y six m o i s t u r e levels ( 2 0 % W H C t o 9 0 %
W H C ) increased w i t h increasing s o i l m o i s t u r e .
T h e r e was n o significant change i n nitrogenase a c t i v i t y between m o i s t u r e levels v a r y i n g f r o m 19% t o 4 7 %
W H C . L i n e a r regression o f average nitrogenase
a c t i v i t y against s o i l - m o i s t u r e c o n t e n t i n d i c a t e d a
significant c o r r e l a t i o n ( r = 0 . 7 8 8 ) .
Diurnal Variation
T e n p l a n t s o f c u l t i v a r I n d a f 5 were g r o w n i n 6 - L
plastic p o t s c o n t a i n i n g 6 kg a l l u v i a l s o i l . A basal
dose of 25 kg N a n d 50 kg each of P 2 O 5 a n d K 2 O was
a p p l i e d . T h e c o n d i t i o n s f o r g r o w i n g p l a n t s were
s i m i l a r t o those described b y W a n i e t a l . (1984).
N i t r o g e n a s e a c t i v i t y was estimated u s i n g i n t a c t p l a n t assay t e c h n i q u e ( W a n i et a l . 1984). A c e t y l e n e
gas was injected at 1200 a n d gas samples were c o l lected at 2 h i n t e r v a l s up to 2400. S o i l t e m p e r a t u r e in
the p o t a t each assay t i m e was r e c o r d e d . T h e d i u r n a l
p a t t e r n o f nitrogenase a c t i v i t y i s s h o w n i n F i g u r e 2 .
M a x i m u m a c t i v i t y o f 104 n m o l C 2 H 4 h - 1 p l a n t - 1 was
r e c o r d e d between 10 am a n d 12 n o o n .
105
Nitrogenase
Soil
activity
temperature
90
80
32
70
28
60
24
50
20
40
16
30
20
10
Day
Night
Day
0
Time o f day ( h )
F i g u r e 2 . D i u r n a l p a t t e r n o f nitrogenase a c t i v i t y o f a n
i n t a c t - p l a n t system o f finger m i l l e t c u l t i v a r I n d a f 5 .
Response to Inoculation with NitrogenFixing Bacteria
Response o f f i n g e r m i l l e t c u l t i v a r I n d a f 7 t o seed
i n o c u l a t i o n w i t h d i a z o t r o p h cultures ( o b t a i n e d f r o m
I C R I S A T C e n t e r ) was studied at 25 a n d 50 kg ha - 1
levels o f a p p l i e d n i t r o g e n , u n d e r greenhouse a n d
f i e l d situations. I n t h e greenhouse, p l a n t s were
raised i n 2 5 c m x 1 5 c m i r o n cores f i l l e d w i t h 3/75 k g
a l l u v i a l s o i l . T h e basal dose consisted o f a p a r t o f the
N a l o n g w i t h 5 0 k g each o f P 2 O 5 a n d K 2 O . T h e
r e m a i n i n g N was a p p l i e d as a t o p dressing. In each
case, a single p l a n t was g r o w n . T h e seeds were
soaked f o r 18 h in b r o t h c u l t u r e of bacteria as per t h e
t r e a t m e n t a n d d r i e d . A t s o w i n g , each c o r e was i n o c ulated w i t h 8 mL b r o t h culture. The noninoculated
b r o t h served a s the c o n t r o l . Surface s o i l i n the cores
was c o v e r e d w i t h g r a v e l a n d the m o i s t u r e c o n t e n t i n
the cores was m a i n t a i n e d a t a r o u n d 7 0 % W H C .
T h e g r a i n a n d s t r a w yields increased s i g n i f i c a n t l y
97
over the c o n t r o l due t o i n o c u l a t i o n w i t h N B R E
(37%) a n d S R I (35%) ( F i g . 3). I n o c u l a t i o n w i t h A .
l i p o f e r u m ( 4 A B L ) increased l e a f n u m b e r b u t n o t
p l a n t h e i g h t over the c o n t r o l . N i t r o g e n alone o r
interaction of N x inoculation had no significant
effect o n y i e l d parameters. N i t r o g e n c o n t e n t o f g r a i n
increased 5 0 % f r o m i n o c u l a t i o n w i t h N B R E a n d
4 7 % w i t h S R I ; total uptake o f nitrogen b y the plant
increased 7 2 % w i t h N B R E a n d 6 6 % w i t h S R I . I n o c u l a t i o n h a d n o effect o n n i t r o g e n c o n t e n t o f straw.
I n t e r a c t i o n of N x i n o c u l a t i o n ( N B R E ) increased N
c o n t e n t o f g r a i n b y 1 % , w h i l e N alone h a d n o effect.
I n the f i e l d e x p e r i m e n t , a l l the cultures used i n the
p o t t r i a l except the S R I isolate were used. T h e experi m e n t was c o n d u c t e d f o l l o w i n g r a n d o m i z e d - b l o c k
design a n d 2 m x 2 m p l o t s f o r each t r e a t m e n t ,
replicated f o u r times. T h e N , P , a n d K treatments
were as in the case of the p o t e x p e r i m e n t . Seeds were
1. Noninoculated
control
2 . Azospirillum
3. 4 ABL
lipoferum
soaked in p e a t - i n o c u l a n t suspension f o r 18 h, a i r
d r i e d a n d s o w n i n r o w s 22.5 c m apart. E a c h p l o t was
i n o c u l a t e d a t s o w i n g w i t h p e a t - i n o c u l a n t suspension
( 1 g peat i n o c u l a n t suspended i n 500 m L w a t e r )
u n i f o r m l y . I n o c u l a t i o n o f finger m i l l e t w i t h a l l the
cultures o f N 2 - f i x i n g bacteria increased g r a i n o r
straw yields by 8-10% over the c o n t r o l plants, b u t the
increase was n o t significant ( F i g . 4). A p p l i c a t i o n o f
N also gave n o n s i g n i f i c a n t y i e l d increases over the
c o n t r o l plants. H i g h e s t a b o v e - g r o u n d y i e l d (5552 k g
ha - 1 ) was o b t a i n e d w i t h A . L i p o f e r u m ( I C M 1001)
i n o c u l a t i o n + 50 kg N ha - 1 , f o l l o w e d by N B R E c u l t u r e + 25 kg N ha - 1 (5168 kg ha - 1 ). N i t r o g e n c o n t e n t
o f f i n g e r m i l l e t s t r a w increased b y 0.3% due t o i n o c u l a t i o n w i t h N B R E a n d N u p t a k e increased b y 1 2 k g
ha - 1 ( F i g . 5). A p p l i c a t i o n of 50 kg N ha - 1 increased
g r a i n n i t r o g e n u p t a k e b y 2 5 k g ha - 1 , b u t h a d n o
effect o n straw n i t r o g e n c o n t e n t .
4. NBRE
5. Sharda
ragi
isolate
(SRI)
15
100
Grain
Straw
12
80
9
60
6
40
3
20
0
0
1
2
3
4
5
1
2
3
4
5
Figure 3. Response of finger millet cultivar I n d a f 7 to inoculation w i t h diazotrophs in a pot trial.
98
Grain
Straw
a t 2 5 k g N ha-1
a t 2 5 k g N ha-1
-1
a t 50 k g N ha
Mean
Grain
-1
a t 5 0 k g N ha
Straw
inoculation
Mean
70
inoculation
5500
60
5000
50
4500
40
4000
30
3500
20
3000
10
2500
a
Control
0
C o n t r o l A.
lipoferum
4ABL
NBRE
Figure 4. Response of field-grown finger millet cul-
Figure
millet
5.
A.
lipoferum
Nitrogen
cultivar
4ABL
uptake
Indaf 7
as
NBRE
by field-grown finger
influenced
by
nitrogen
levels a n d i n o c u l a t i o n s w i t h d i a z o t r o p h s .
t i v a r I n d a f 7 w i t h n i t r o g e n levels t o i n o c u l a t i o n w i t h
diazotrophs.
Nitrogen Balance
N i t r o g e n balances (postharvest N - p r e s o w i n g N in
soil) due to the g r o w t h of finger m i l l e t I n d a f 5 as
affected b y p l a n t i n g , i n o c u l a t i o n , a n d three levels o f
n i t r o g e n (0, 25, a n d 5 0 k g ha - 1 ) were studied i n p o t
c u l t u r e i n the greenhouse ( F i g . 6). M a x i m u m posit i v e n i t r o g e n balance (200 m g p o r - 1 ) was f o u n d i n
i n o c u l a t e d finger m i l l e t at the 0 level a p p l i e d - N ,
w h i c h also s h o w e d the least loss of s o i l n i t r o g e n (5
m g p o t - 1 ) . M a x i m u m loss o f s o i l n i t r o g e n was f o u n d
i n the u n p l a n t e d p o t w i t h 5 0 k g h a - 1 a p p l i e d N . F i n a l
N-balances i n i n o c u l a t e d a n d p l a n t e d systems were
200, 168, a n d 198 m g p o t - 1 . I n the u n p l a n t e d system,
there were negative balances o f 1 1 , 58, a n d 9 3 m g
pot - 1 respectively at the 0, 25, a n d 50 kg a p p l i e d - N
levels. E v e n t h o u g h i n o c u l a t e d pots s h o w e d a h i g h e r
balance (188 m g p o r 1 ) t h a n the n o n i n o c u l a t e d p o t
(161 m g p o t - 1 ) , the difference was n o t s i g n i f i c a n t .
P l a n t i n g I n d a f 5 resulted i n a n N g a i n o f 175 m g
pot - - 1 , whereas in the u n p l a n t e d system there was a
loss of 49 mg N p o t - 1 . T h e system gains of N in p o t s
due t o i n o c u l a t i o n e x t r a p o l a t e d t o a net g a i n o f 6.3,
7.6, a n d 9.4 kg N ha - 1 , respectively, at the 0, 25, a n d
50 kg ha - 1 a p p l i e d - N levels.
References
D a r t , P . J . , and W a n i , S.P. 1982. N o n - s y m b i o t i c n i t r o g e n
f i x a t i o n a n d s o i l f e r t i l i t y . Pages 3-27 in N o n - s y m b i o t i c
n i t r o g e n f i x a t i o n a n d o r g a n i c m a t t e r i n the t r o p i c s . S y m p o sia papers 1 . T r a n s a c t i o n s o f t h e 12th I n t e r n a t i o n a l C o n gress o f S o i l Science, 8-16 Feb 1982, N e w D e l h i , I n d i a . N e w
D e l h i , I n d i a : I n d i a n A g r i c u l t u r a l Research I n s t i t u t e .
D a y , J . M . , Neves, M . C . P . , and
Dobereiner, J . 1975.
N i t r o g e n a s e a c t i v i t y o n t h e r o o t s o f t r o p i c a l forage grasses.
S o i l B i o l o g y a n d B i o c h e m i s t r y 7:107-112.
99
Inoculated
No N
Noninoculated
25
kg
N
ha-1
Sown
50
kg
N
ha-1
Not
sown
200
(+)ve
160
120
80
40
0
40
(-)ve
80
120
Figure 6. Nitrogen balance in p o t - g r o w n finger millet cultivar I n d a f S as affected by nitrogen a n d inoculation
(95-day-old plants).
G . , and
H a r d y , R . W . F . , B u r n s , R . C . , a n d H o l s t e n , R . D . 1973.
W e i n h a r d , P . 1973. N o n - s y m b i o t i c n i t r o g e n f i x a t i o n i n
A p p l i c a t i o n s o f t h e a c e t y l e n e - e t h y l e n e assay f o r m e a s u r e -
Dommergues,
Y.,
Balandreau,
J., Rinaudo,
t h e r h i z o s p h e r e o f r i c e , m a i z e a n d d i f f e r e n t t r o p i c a l grasses.
ment of nitrogen fixation. Soil Biology and Biochemistry
S o i l B i o l o g y a n d B i o c h e m i s t r y 5:83-89.
5:47-81.
100
I C R I S A T ( I n t e r n a t i o n a l Crops Research Institute for the
S e m i - A r i d Tropics). 1978. A n n u a l r e p o r t 1977-78. P a t a n c h e r u , A . P 502 324, I n d i a : I C R I S A T .
Jenkinson, D . S . 1977. T h e n i t r o g e n e c o n o m y o f B r o a d b a l k e x p e r i m e n t s . I . N i t r o g e n balance i n the e x p e r i m e n t s .
R e p o r t f o r 1976, R o t h a m s t e d E x p e r i m e n t a l S t a t i o n 2:103109.
M i s h r a , H . P . , P a t n a i k , M . C . , and N a y a k , B . K . 1980.
V a r i a t i o n i n q u a n t i t a t i v e characters a n d t h e i r a s s o c i a t i o n
w i t h qualitative traits i n f i n g e r millet. I n d i a n J o u r n a l o f
A g r i c u l t u r a l Sciences 50:298-301.
M o o r e , A . W . 1963. N i t r o g e n f i x a t i o n i n l a t o s o l i c s o i l
u n d e r grass. P l a n t a n d S o i l 19:127-138.
Rennie, R . J . 1981. A single m e d i u m f o r the i s o l a t i o n o f
acetylene r e d u c i n g ( d i n i t r o g e n f i x i n g ) bacteria f r o m soils.
C a n a d i a n J o u r n a l o f M i c r o b i o l o g y 27:8-14.
W a n i , S.P., D a r t , P . J . , and U p a d h y a y a , M . N . 1983. F a c t o r s a f f e c t i n g nitrogenase a c t i v i t y ( C 2 H 2 r e d u c t i o n ) associated w i t h s o r g h u m a n d m i l l e t e s t i m a t e d u s i n g the s o i l
core assay. C a n a d i a n J o u r n a l o f M i c r o b i o l o g y 29:10631069.
W a n i , S.P., U p a d h y a y a , M . N . , and D a r t , P . J . 1984. A n
i n t a c t p l a n t assay f o r e s t i m a t i n g nitrogenase a c t i v i t y ( C 2 H 2
reduction) of sorghum and millet plants g r o w n in pots.
P l a n t a n d S o i l 82:15-29.
101
Heterotrophic N i t r o g e n F i x a t i o n as Influenced
by Fertilizers in Rice-Soil Systems
V . Rajaramamohan R a o , J . L . N . R a o , and T . K . Adhya1
Summary
The
rice
We
rhizosphere-soil
investigated
conditions,
the
effects
employing gas
level
form,
and
with
the
rhizosphere.
activity,
the
the
rice
extent
plant.
ence
on
nitrogenase,
from
this
laboratory,
the
nitrogen
nitrogenase
of
economy
of fertilizer
The
the
while
varying
under
nitrogen
an
the
the
sources
phosphate
urgent
of
with
phosphate
rock
is
on
by
several
soil
nitrogenase
under
laboratory
and
field
assay.
Studies
indicate
that
the
the
nitrogenase
associated
enhanced
nitrogenase
acetylene-reduction
application
stimulation
there
affected
of mineral fertilizers
chromatographic
method
Among
is
was
need to
impact
application
urea
method
less
F e r t i l i z e r i s one o f the key i n p u t s i n c r o p p r o d u c t i o n ,
b u t the i n f o r m a t i o n o n its influence o n n i t r o g e n
fixation in the rice rhizosphere soil is rather l i m i t e d .
It has been w e l l established that nitrogenase is
depressed by f e r t i l i z e r n i t r o g e n . H o w e v e r , u n d e r
f i e l d c o n d i t i o n s , the effects o f n i t r o g e n f e r t i l i z e r
either s i n g l y o r i n c o m b i n a t i o n w i t h o t h e r n u t r i e n t s
could be complicated owing to plant absorption and
o t h e r f i e l d factors. I n f o r m a t i o n o n the effect o f
p h o s p h o r u s a p p l i c a t i o n o n s o i l nitrogenase i n
l o w l a n d - r i c e soils is also n o t available. We have
e x a m i n e d the p o t e n t i a l n i t r o g e n - f i x i n g a c t i v i t y i n
rhizosphere s o i l as influenced by different sources of
f e r t i l i z e r n i t r o g e n , p h o s p h o r u s , a n d c e r t a i n management practices under field and l a b o r a t o r y
conditions.
Materials and Methods
A f i e l d e x p e r i m e n t was c o n d u c t e d to evaluate the
influence o f f o r m s a n d m e t h o d s o f a p p l i c a t i o n o f
of
and
significantly
application
and
superphosphate
effective.
evaluate
of improved
Introduction
influenced
briquettes
tested,
environmental
Based
the
exerted
on
this
the overall contribution of
rice
cultural factors.
growth
phase
of
stimulatory
influ-
and earlier
research
N2
fixation
to
technology.
urea n i t r o g e n o n the nitrogenase a c t i v i t y o f t h e
rhizospheric s o i l . N i t r o g e n was a p p l i e d as urea p r i l l s
a n d urea briquettes at a rate of 40 kg N ha - 1 , in
a d d i t i o n t o 2 0 k g h a - 1 each o f P 2 O 5 a n d K 2 O , t o a l l
treatments. Urea briquettes were a p p l i e d b e h i n d t h e
p l o w o r t o s h a l l o w w a t e r between the r o w s a m o n t h
after s o w i n g . T h e treatments w i t h p r i l l e d urea
i n c l u d e d broadcast a n d i n c o r p o r a t i o n , urea a p p l i e d
b e h i n d the p l o w a n d between seed r o w s . W a t e r was
a l l o w e d to a c c u m u l a t e in the field a f o r t n i g h t after
g e r m i n a t i o n , a n d the level reached a b o u t 5 0 c m b y
6 0 days after s o w i n g ( D A S ) . T h e water level v a r i e d
between 4 0 c m a n d 5 0 c m u p t o 120 D A S a n d
g r a d u a l l y declined thereafter.
I n a l l treatments, rhizospheric s o i l ( 2 g fresh w t )
was collected p e r i o d i c a l l y f r o m three p l a n t s f r o m
each p l o t a n d transferred t o 125 x 1 6 m m B - D V a c u tainer ( N e w Jersey) tubes f o r acetylene ( C 2 H 2 )
r e d u c t i o n analysis. T h e i n c u b a t i o n a n d analysis
were c o n d u c t e d as per the procedures described by
N a y a k e t a l . (1980), M a h a p a t r a a n d R a o (1981), a n d
R a o et al. (1983). H i g h - p u r i t y n i t r o g e n served as a
c a r r i e r gas at a flow rate of 30 mL min - 1 .
T h e effect o f different levels a n d sources o f p h o s -
1. Microbiologist, U G C Teacher Fellow, and Microbiologist, Central Rice Research Institute, Cuttack, Orissa 753 006, India.
I C R I S A T (International Crops Research Institute for the Semi-Arid Tropics). 1986, Cereal nitrogen fixation. Proceedings of the W o r k i n g
Group Meeting, 9-12 Oct 1984, I C R I S A T Center, India. Patancheru, A . P . 502324, India: I C R I S A T .
103
p h o r u s o n s o i l nitrogenase a c t i v i t y was e v a l u a t e d i n
a l a b o r a t o r y i n c u b a t i o n study. A l l u v i a l a n d P deficient a l k a l i n e soils were a i r d r i e d , screened
t h r o u g h < 2 m m sieve, a n d 5 g placed i n B - D v a c u t a i n e r tubes. O n e set o f soils was f l o o d e d (1.5 c m
s t a n d i n g w a t e r ) a n d t h e o t h e r was k e p t a t 5 0 % w a t e r h o l d i n g c a p a c i t y ( W H C ) A l l treatments were r e p l i cated three times. P h o s p h o r u s was a p p l i e d as
K 2 H P O 4 to p r o v i d e 0, 10, 20, 40, 80, a n d 100 p p m P
levels. I n a n o t h e r e x p e r i m e n t r o c k phosphate, d i c a l c i u m phosphate, a n d superphosphate were a p p l i e d
t o y i e l d f i n a l concentrations o f 0 , 20, 40, a n d 6 0 p p m
P . N i t r o g e n a s e a c t i v i t y was d e t e r m i n e d a t d i f f e r e n t
i n t e r v a l s . T h e r e m a i n i n g a v a i l a b l e P was e s t i m a t e d
as Olsen's P.
T h e p o p u l a t i o n s o f different g r o u p s o f n i t r o g e n f i x i n g m i c r o o r g a n i s m s i n the soils u n d e r different
treatments were c o u n t e d b y c o n v e n t i o n a l seriald i l u t i o n techniques o n N-free m e d i a . Azospirillum
sp was c o u n t e d f o l l o w i n g the m e t h o d suggested by
O k o n e t a l . (1977), a n d anaerobic n i t r o g e n fixers a n d
Azotobacter b y the p r o c e d u r e o f R a o e t a l (1973).
Results presented are the means o f f i v e replicates f o r
Azospirillum a n d anaerobic N 2 f i x e r s , a n d three
replicates f o r Azotobacter.
Results
N i t r o g e n a s e was, i n general, s t i m u l a t e d b y the a p p l i c a t i o n o f 4 0 k g N ha - 1 , p a r t i c u l a r l y d u r i n g the i n i t i a l
5 4 D A S ( T a b l e 1). Nitrogenase a c t i v i t y was s i g n i f i c a n t l y ( P < 0 . 0 5 ) reduced w h e n the urea b r i q u e t t e s
were a p p l i e d t o s t a n d i n g s h a l l o w w a t e r , whereas i t
was h i g h w h e n the briquettes were a p p l i e d either
b e h i n d the p l o w o r between the r o w s . Perhaps the
increased N c o n c e n t r a t i o n in the r h i z o s p h e r e s o l u t i o n c o u l d have reduced the n i t r o g e n a v a i l a b i l i t y ,
r e s u l t i n g i n a r e d u c t i o n i n a c t i v i t y w h e n t h e urea was
a p p l i e d t o s t a n d i n g s h a l l o w w a t e r . I n fact, a p p l i c a t i o n o f urea b r i q u e t t e s t o the r o o t zone prevents
n i t r o g e n loss a n d results i n the slower release o f
n i t r o g e n . T h u s , the s l o w e r s u p p l y o f l o w levels o f
n i t r o g e n m i g h t have f a v o r e d nitrogenase a c t i v i t y .
A d d e d - N i s k n o w n t o s t i m u l a t e s o i l nitrogenase
( K n o w l e s a n d D e n i k e 1974, T r o l l e d e n i e r 1977, C h a r y u l u et a l . 1981).
U r e a briquettes resulted in a s i g n i f i c a n t ( P < 0 . 0 5 )
increase i n g r a i n y i e l d , c o m p a r e d w i t h the c o n t r o l , i n
a l l m e t h o d s o f a p p l i c a t i o n . T h e nitrogenase was,
h o w e v e r , affected b y t h e m e t h o d o f N a p p l i c a t i o n .
T h u s , n o a p p a r e n t c o r r e l a t i o n existed between t h e
104
T a b l e 1 . Nitrogenase activity ( n m o l C 2 H 4 g - 1 soil d - 1 ) i n
the rhizosphere as influenced by f o r m a n d m e t h o d of n i t r o gen a p p l i c a t i o n .
Days after sowing
Treatment
(40 k g N ha-1)
Without
30
47
54
76
88
102
580
420
390
250
150
210
640
510
450
320
230
170
760
450
430
310
480
170
740
430
400
140
150
190
570
130
200
180
170
190
620
620
540
360
310
370
±38
±25
±33
±27
±27
±20
N
fertilizer
Urea prills
Applied
between
seed r o w s
Broadcast and
incorporated
Applied
behind
the p l o u g h
Urea briquettes
Applied in
shallow water
Applied
behind
the p l o u g h
SE
nitrogenase a n d the c r o p y i e l d u n d e r these
situations.
O u r results i n d i c a t e t h a t p h o s p h o r u s a d d i t i o n
s t i m u l a t e d nitrogenase a c t i v i t y i n the t w o soils
( T a b l e 2), a n d the s t i m u l a t i o n was m o r e p r o n o u n c e d
under nonflooded conditions. The stimulation by
p h o s p h o r u s was m o r e a p p a r e n t i n the a l l u v i a l s o i l .
T h u s the effect o f p h o s p h o r u s o n nitrogenase
depended o n the w a t e r regime a n d s o i l t y p e . These
differences c o u l d b e a t t r i b u t e d t o the a l t e r a t i o n s i n
the a v a i l a b l e P u n d e r t w o w a t e r regimes in the soils.
I n fact, the a v a i l a b l e P disappeared faster i n the
P-deficient s o i l t h a n i n a l l u v i a l soil.
A d d i t i o n o f superphosphate a n d d i c a l c i u m p h o s phate stimulated nitrogenase activity almost
t h r o u g h o u t i n c u b a t i o n , w h i l e the r o c k p h o s p h a t e
h a d a n i n n o c u o u s effect ( T a b l e 3). S u p e r p h o s p h a t e
was s u p e r i o r over o t h e r P-sources i n e n h a n c i n g s o i l
nitrogenase a n d c r o p yields o n this a l l u v i a l s o i l .
A d d i t i o n of phosphorus in alluvial soil had little
effect o n the p o p u l a t i o n s o f Azospirillum, a n a e r o b i c
N 2 - f i x e r s , a n d Azotobacter ( T a b l e 4 ) . I n c o n t r a s t , i n
a P-deficient s o i l , the a d d i t i o n o f phosphate s t i m u lated the p o p u l a t i o n o f Azospirillum a n d A z o t o b a c ter. T h e s t i m u l a t i o n was m o r e a p p a r e n t at the 10
p p m P level. T h e P-deficient s o i l , however, h a d
h i g h e r n u m b e r s o f Azotobacter t h a n the a l l u v i a l
s o i l . Changes i n the m i c r o b i a l p o p u l a t i o n s a n d levels
of a v a i l a b l e P m i g h t be responsible f o r changes in
nitrogenase a c t i v i t y i n the soils.
O u r results suggest t h a t rhizosphere s o i l n i t r o g e n ase i s influenced b y the m o d e o f a p p l i c a t i o n o f f e r t i l izer n i t r o g e n . T h e level a n d source o f p h o s p h o r u s
a p p l i e d t o rice soils u n d e r n o n f l o o d e d a n d sub-
merged c o n d i t i o n s also e x h i b i t e d p r o f o u n d i n f l u ence o n nitrogenase a c t i v i t y a n d the p o p u l a t i o n o f
n i t r o g e n fixers. T h i s s t u d y opens up a f u r t h e r need to
manipulate fertilizer-management practices to
e x p l o i t the benefit o f b i o l o g i c a l n i t r o g e n f i x a t i o n f o r
rice u n d e r these s i t u a t i o n s .
Acknowledgement
T a b l e 2. I n f l u e n c e of phosphate on soil nitrogenase activity ( n m o l C 2 H 4 g - 1 soil d - 1 ) i n t w o soils under f l o o d e d a n d
T h e a u t h o r s w i s h t o t h a n k D r H . K . Pande, D i r e c t o r ,
C R R I , f o r p r o v i d i n g the facilities t o c o n d u c t this
research.
n o n f l o o d e d conditions.
Days of incubation
11
P-level
F1
ppm
16
NF2
F
30
25
NF
F
NF
F
NF
A l l u v i a l soil
0
30
80
30
100
40
80
10
40
100
50
90
40
90
T a b l e 3 . Effect o f P-sources o n soil nitrogenase ( n m o l
ND3
C 2 H 4 g - 1 soil d - 1 ) in a submerged alluvial soil.
20
70
590
40
200
50
70
40
80
310
40
160
40
30
80
90
290
50
170
80
20
P-source
100
70
430
70
60
130
20
Control
L S D (P<0.05)
18
97
10
34
10
11
(P<0.01)
25
136
15
48
15
15
Rock
P-deficient alkaline soil
80
120
100
40
60
60
70
30
10
80
490
100
250
120
120
90
130
20
80
130
140
70
110
100
120
90
40
150
150
120
140
100
70
100
50
80
250
170
150
180
90
110
70
50
100
170
200
200
60
140
10
100
20
LSD (P<0.05)
48
105
30
37
33
21
17
24
(P<0.01)
67
147
43
52
46
30
24
34
8
14
19
22
25
29
0
40
150
90
40
40
30
ppm
phosphate
0
Days of incubation
P-level
Dicalcium
phosphate
Super
phosphate
20
20
150
110
50
50
40
40
80
90
40
30
10
30
60
70
130
60
50
30
30
40
20
100
157
130
50
50
40
90
160
130
40
40
30
60
100
160
150
80
50
50
40
20
100
110
150
50
40
40
120
180
210
50
40
30
60
150
160
140
40
40
40
19
14
LSD(P<0.05)
34
31
29
21
(P<0.01)
47
42
40
29
1. F = Flooded.
2. NF = Nonflooded.
N S 1 19
1. NS = Not significant.
3. ND = N o t determined .
T a b l e 4. P o p u l a t i o n of N 2 - f i x i n g microorganisms as influenced by the phosphate level in flooded soils ( 1 2 days
incubation).
Nitrogen-fixing population g-1 dry soil
Soil
P-level
Azospirillum
(ppm)
(x
F1
Alluvial
0
10
80
P-deficient
alkaline
16
2.8
16
0
0.5
10
13.0
80
2.8
Anaerobic N
10 6 )
NF2
F
Azotobacter
(x105)
fixers
NF
(x104)
F
NF
0.14
2.4
1.4
5
0.60
2.8
1.7
2
1.8
0.12
2.8
2.4
3
6.5
0.45
1.4
0.24
47
9
0.17
0.24
122
21
1.7
0.17
98
14
13.5
0.18
7.5
1. F = Flooded.
2. NF = Nonflooded.
105
References
C h a r y u l u , P . B . B . N . , N a y a k , D . N . , and R a o , V . R . 1981.
N i t r o g e n i n c o r p o r a t i o n b y rhizosphere s o i l : influence o f
rice variety, organic matter and combined nitrogen. Plant
a n d S o i l 59:399-405.
Knowles, R . , and D e n i k e , D . 1974. Effects o f a m m o n i u m
n i t r i t e a n d n i t r a t e - n i t r o g e n o n anaerobic nitrogenase a c t i v i t y i n s o i l . S o i l B i o l o g y a n d B i o c h e m i s t r y 6:353-358.
f i x a t i o n . T h e deleterious effect of higher levels of N
o n nitrogenase i s alleviated b y the a p p l i c a t i o n o f
higher levels of P to the system.
D . Purushothaman:
C o u l d y o u t e l l u s the response o f rice genotypes t o
i n o c u l a t i o n w i t h Azospirillum'!
V.R.Rao:
M a h a p a t r a , R . N . , and R a o , V . R . 1981. I n f l u e n c e o f h e x a c h l o r o c y c l o h e x a n e o n the nitrogenase a c t i v i t y o f rice
rhizosphere s o i l . P l a n t a n d S o i l 59:473-477.
W e have l i m i t e d i n f o r m a t i o n o n the genotype
response to Azospirillum i n o c u l a t i o n w i t h regard to
yield.
N a y a k , D . N . , Pasalu I . C . , and R a o , V . R . 1980. I n f l u e n c e
o f n a t u r a l a n d synthetic pesticides o n n i t r o g e n f i x a t i o n
( C 2 H 2 r e d u c t i o n ) i n the rice rhizosphere. C u r r e n t Science
49:118-119.
U.S. K u n d u :
W h a t i s the percentage o f azospirilla i n the rice
rhizosphere?
O k o n , Y . , Albrecht, S . L . , and B u m s , R . H . 1977. M e t h o d s
f o r g r o w i n g Spirillum lipoferum a n d f o r c o u n t i n g i t i n p u r e
c u l t u r e a n d i n association w i t h plants. A p p l i e d a n d E n v i r o n m e n t a l M i c r o b i o l o g y 33:85-88.
R a o , J . L . N . , Pasalu, I . C . , and R a o , V . R . 1983. N i t r o g e n
f i x a t i o n ( C 2 H 2 r e d u c t i o n ) i n the rice rhizosphere s o i l a s
i n f l u e n c e d b y pesticides a n d m e t h o d s o f t h e i r a p p l i c a t i o n .
J o u r n a l o f A g r i c u l t u r a l Science ( U K ) 100:637-642.
R a o , V . R . , Kalininskaya, T . A . , and M i l l e r , Y . M . 1973.
T h e a c t i v i t y o f n o n - s y m b i o t i c n i t r o g e n f i x a t i o n i n soils o f
r i c e fields s t u d i e d w i t h I 5 N . M i k r o b i o l o g i y a 42:729-734.
Trolledenier, G . 1977. I n f l u e n c e o f some e n v i r o n m e n t a l
factors o n n i t r o g e n f i x a t i o n i n the rhizosphere o f rice. P l a n t
a n d S o i l 47:203-217.
Discussion
N . S . Subba R a o :
H o w d o y o u estimate Rhizosphere i n the rice p l a n t
i n wet land?
V.R.Rao:
T h e rhizosphere c o l l e c t i o n a n d s a m p l i n g f r o m wetl a n d soils o f t e n pose several p r o b l e m s . We consider
the soil f r a c t i o n attached to the rice r o o t s as the
rhizosphere.
G.S.Murthy:
D i d y o u s t u d y the i n d i v i d u a l effects o f N a n d P o n
nitrogenase a c t i v i t y , or t h e i r c o m b i n e d effect?
V.R.Rao:
W e have i n f o r m a t i o n o n the independent a n d c o m b i n e d effects o f N a n d P o n r i c e rhizosphere N 2
106
V.R.Rao:
Azospirillum i s one o f the N 2 f i x e r s associated w i t h
rice. C o n v i n c i n g q u a n t i t a t i v e data o n the percentage
o f a z o s p i r i l l a i n rice rhizosphere are n o t available.
C . P . Ghonsikar:
W h a t i s the r e l i a b i l i t y o f A R A m e t h o d o l o g y i n w e t l a n d c o n d i t i o n s o f rice cultures? A n y difficulties i n
assay techniques? If there are, w h a t special techniques are adopted?
V.R.Rao:
A R A m e t h o d i n w e t l a n d c o n d i t i o n s c o u l d a t best b e
q u a l i t a t i v e a n d is often e m p l o y e d f o r screening a n d
e v a l u a t i n g the N 2 - f i x i n g p o t e n t i a l under such c o n d i tions. T h e r e are several difficulties associated w i t h
this assay system l i k e gas d i f f u s i o n , i n i t i a l lag, s o l u b i l i t y , etc. T h i s technique s t i l l needs p e r f e c t i o n a n d
i m p r o v e m e n t s are i n progress.
P. Tauro:
W h a t i s the n u m b e r o f azospirilla i n the rice soil?
V.R.Rao:
10 6 g - 1 soil.
Effect of Certain Organic Amendments
and Potassium on the Bacterization
of Rice w i t h Azotobacter chroococcum
N . N . Prasad1
Summary
effect
of
Sesbania,
bacterization
The
of
Oryza
ments,
gave
Sesbania
31.1%
of neem
Azotobacter
increase
cake
application
+
with
increase
in
increase
in
over
application
markedly
application
K
K
glyricidia
sativa
the
in
levels.
The
sunhemp
with
Azotobacter
gave
a 31. 7%
nonamended
combination
augmented
Azotobacter
and
L.
the
indicated
(green
increase
control.
with
increase
Azotobacter population
The
results
in
studied.
also
showed
inoculation
population
in
and paddy
was
straw
Among
on
the
in grain yield and Glyricidia +
Azotobacter
Azotobacter
an
manures),
chroococcum
grain
the
in
and
on
the
straw
rhizosphere
the
Azotobacter
beneficial effects
rice yield.
Neem
rhizosphere.
yields
of rice
the
treat-
cake
Potassium
of rice
with
an
increased
with
an
levels.
Introduction
N i t r o g e n fixation is an energy-consuming process
a n d the a v a i l a b i l i t y o f c a r b o h y d r a t e i n soil assumes
m u c h significance f o r the f i x a t i o n o f a t m o s p h e r i c
nitrogen. Under field conditions, application of
organic amendments, therefore, becomes essential
f o r the e x p l o i t a t i o n o f the n i t r o g e n - f i x i n g p o t e n t i a l
o f Azotobacter. S o m e o f the data o b t a i n e d o n the
b a c t e r i z a t i o n of rice (Oryza sativa L . ) w i t h A.
chroococcum are s u m m a r i z e d below.
Results
I n studies o n the effect o f green manures a n d p a d d y
straw o n the b a c t e r i z a t i o n o f rice w i t h Azotobacter
at A n n a m a l a i U n i v e r s i t y , Sesbania + Azotobacter
gave the m a x i m u m increase i n g r a i n y i e l d (31.7%)
over the c o n t r o l ( T a b l e 1), w h i l e g l y r i c i d i a + A z o t o bacter gave a 31.1 % increase a n d s u n h e m p + A z o t o -
bacter gave a 2 3 . 1 % increase in g r a i n yields ( P r a k a s h
a n d Prasad 1980).
O r g a n i c amendments a l o n g w i t h Azotobacter
have also increased the straw y i e l d a n d 1000 g r a i n
mass. Interestingly, the a d d i t i o n o f o r g a n i c a m e n d m e n t c o m b i n a t i o n s w i t h Azotobacter i n o c u l a t i o n
have m a r k e d l y augmented shoot n i t r o g e n c o n t e n t ,
w h e n c o m p a r e d to either Azotobacter i n o c u l a t i o n
or organic amendments alone ( P r a k a s h 1977).
W o r k done a t A n n a m a l a i U n i v e r s i t y revealed t h a t
w i t h Azotobacter i n o c u l a t i o n the o p t i m u m level o f
fertilizer N c o u l d be reduced f r o m 120 to 90 kg ha - 1
f o r the rice c r o p ( P r a k a s h 1977). T h e o r g a n i c - m a t t e r
a p p l i c a t i o n was also f o u n d to be beneficial to the
Azotobacter i n o c u l a t i o n . To investigate this aspect
further, Azotobacter was i n o c u l a t e d at three levels
o f g l y r i c i d i a a p p l i c a t i o n a t t w o levels o f n i t r o g e n
( N a g a r a j a n 1978). T h e results revealed the p o s s i b i l i t y of reducing the rate of fertilizer N to 60 kg N ha - 1
f r o m 9 0 k g N ha - 1 , i f supplemented w i t h g l y r i c i d a
a p p l i c a t i o n at 7.5 t ha - 1 a n d i n o c u l a t e d w i t h Azoto-
1. Professor and Head, Department of Microbiology, Annamalai University, Annamalai Nagar, Tamil Nadu 608 002, India.
I C R I S A T (International Crops Research Institute for the Semi-Arid Tropics). 1986. Cereal nitrogen fixation. Proceedings of the W o r k i n g
Group Meeting, 9-12 Oct 1984, I C R I S A T Center, India. Patancheru, A . P . 502 324, India: I C R I S A T .
107
T a b l e 1 . Effect o f organic amendments ( 6 t h a - 1 ) a n d A z o -
Table
tobacter inoculation on grain yield of rice.
on grain yield of rice.
3.
Treatment
Increase
Increase
over
Grain
over
respective
yield
control
control
(%)
(%)
ha-1)
(kg
Treatment
Control
3136
-
-
Azotobacter
3526
12.5
12.5
Sesbania
3835
22.3
-
Sesbania
4124
31.7
9.4
3526
12.5
-
4109
31.1
18.6
+
Azotobacter
Glyricidia
Glyricidia
+
Azotobacter
+
Azotobacter
Sunhemp
Sunhemp
3467
10.6
-
3860
23.1
12.5
Paddy straw
3089
-1.4
-
Paddy
3610
15.1
16.8
CD
s t r a w + Azotobacter
Effect of neem cake and Azotobacter inoculation
Grain
Change
N e e m cake
Nitrogen
Azoto-
yield
over
(t ha-1)
( k g ha-1)
bacter1
(kg ha-1)
control2
0
90
4650
+9.4
6
90
4764
+12.1
8.5
90
4816
+13.3
12.5
90
4848
+14.1
25.0
90
4895
+15.2
0
90
NI
3884
-8.6
0
120
NI
4250
-
1.1 = Inoculated, NI = Not inoculated.
2. Control = 120 kg N ha-1 only.
0.48
(P<0.05)
bacter ( T a b l e 2). At the 90 kg N ha - 1 level, a d d i t i o n
of 7.5 t ha - 1 g l y r i c i d i a gave a 32.1% increase in g r a i n
y i e l d , w h i l e w i t h 60 kg N ha - 1 + 7.51 ha - 1 g l y r i c i d i a ,
g r a i n y i e l d increased by 27.8%.
T h e effect o f neem cake o n b a c t e r i z a t i o n o f rice
(var.
white ponni) w i t h Azotobacter d u r i n g the
samba (northeast m o n s o o n ) season ( O c t 1979-Feb
1980) was studied ( S a h u l H a m e e d 1980). N e e m cake
was applied at 0, 6, 8.5, 12.5, a n d 25 t ha -1 w i t h
Azotobacter i n o c u l a t i o n . N i t r o g e n a p p l i c a t i o n at
120 kg N ha - 1 , w i t h o u t Azotobacter i n o c u l a t i o n or
neem cake, served as the c o n t r o l .
T h e results have s h o w n t h a t neem cake a p p l i c a t i o n i n c o m b i n a t i o n w i t h Azotobacter i n o c u l a t i o n
on rice can give g r a i n y i e l d increases of 12.1-15.2%
a n d straw y i e l d increases of 15.9-18.7% ( T a b l e 3).
T h e neem cake a p p l i c a t i o n m a r k e d l y a u g m e n t e d
the Azotobacter p o p u l a t i o n in the rhizosphere at 75
D A S . T h e increase i n p o p u l a t i o n o f this o r g a n i s m
was up to 41.8 x 10 3 at 25 t ha -1 neem cake, as
c o m p a r e d to 22.3 x 10 3 w i t h no neem cake a p p l i c a t i o n ( T a b l e 4).
We studied the effect of graded levels of p o t a s s i u m
a p p l i c a t i o n o n the a z o t o b a c t e r i z a t i o n o f rice (var.
white ponni). Potassium a p p l i c a t i o n increased g r a i n
a n d s t r a w yields of rice as also the 1000-grain mass.
T h e a p p l i c a t i o n of K w i t h Azotobacter i n o c u l a t i o n
augmented the Azotobacter p o p u l a t i o n in the r h i z o sphere of rice. T h e p o p u l a t i o n s increased to 20.46 x
10 3 at 200 kg K 2 O ha -1 as c o m p a r e d to 12.1 x 10 3
w i t h o u t K a p p l i c a t i o n ( T a b l e 5).
Table
4.
Effect of neem cake a n d Azotobacter inoculation
on rhizosphere p o p u l a t i o n of Azotobacter (x 10 3 g - 1 ovenT a b l e 2.
Effect
of
glyricidia
and
N
w i t h Azotobacter
dry soil).
chroococcum inoculation on grain a n d straw yields of rice.
Treatments
Treatment
N e e m cake
Glyricidi a
Nitrogen
G r a i n yield
Straw yield
(t h a - 1 )
(kg ha-1)
(kg ha-1)
( k g ha-1)
0
90
5029
14275
0
60
4611
13618
2.5
90
5325
15702
2.5
60
4942
13672
5.0
90
6019
16197
5.0
60
5710
15043
7.5
90
6642
17460
7.5
60
6425
16583
108
(t ha-1)
Nitrogen
( k g ha-1)
Days after
Azato
bacter1
transplantation
15
35
55
75
0
90
I
13.8
17.7
19.6
22.3
6
90
I
16.3
19.5
23.2
26.1
29.3
8.5
90
I
14.0 21.9
26.5
12.5
90
I
20.1
23.9
28.5
31.9
25.0
90
I
2 6 . 4 29.9
34.8
41.8
0
90
NI
12.6
15.1
17.2
18.5
0
120
NI
11.2
13.2
15.7
17.8
1. 1 = Inoculated, NI = N o t inoculated.
T a b l e 5. Effect of potassium application w i t h Azotobacter chroococcum inoculation on the rhizosphere population of Azotobacter (x 10 3 g - 1 oven-dry soil).
Potassium
( k g K2O h a - 1 )
0
50
100
150
200
D a y s after t r a n s p l a n t a t i o n
15
30
45
8.48
10.68
12.82
13.14
15.25
10.28
12.84
14.28
16.14
19.28
12.12
14.86
16.24
18.45
20.46
References
N a g a r a j a n , R . 1978. Studies o n c e r t a i n aspects o f b a c t e r i z a t i o n of rice (Oryza sativa L . ) w i t h Azotobacter c h r o o c o c cum B e i j . M . S c . t h e s i s , A n n a m a l a i U n i v e r s i t y ,
Annamalainagar, T a m i l Nadu, India.
Prakash, R . 1977. Studies o n the effect o f b a c t e r i z a t i o n o f
r i c e ( O r y z a sativa L . ) a n d c e r t a i n factors i n f l u e n c i n g the
g r o w t h of Azotobacter in vitro a n d in s o i l . M . S c . thesis,
Annamalai University, Annamalainagar, T a m i l Nadu,
India.
P r a k a s h , R , and Prasad, N . N . 1980. Effect o f c e r t a i n
o r g a n i c a m e n d m e n t s o n the efficiency o f i n o c u l a t i o n w i t h
Azotobacter. Pages 90-94 in Aspects of b i o l o g i c a l n i t r o g e n
f i x a t i o n : proceedings o f the T h i r d S o u t h e r n R e g i o n a l C o n ference o n M i c r o b i a l I n o c u l a n t s i n C r o p P r o d u c t i o n , 1-2
A p r 1977, D h a r w a d , K a r n a t a k a , I n d i a . B a n g a l o r e , K a r n a t a k a : U n i v e r s i t y o f A g r i c u l t u r a l Sciences.
Sahul H a m e e d , M . 1980. Studies o n the effect o f c e r t a i n
o r g a n i c m a t t e r a m e n d m e n t s a n d c r o p v a r i e t y o n the bacteri z a t i o n of rice (Oryza sativa L . ) w i t h Azotobacter c h r o o c occum B e i j . M . S c . thesis, A n n a m a l a i U n i v e r s i t y ,
Annamalainagar, T a m i l Nadu, India.
109
Studies on Vesicular Arbuscular M y c o r r h i z a
in Cereals at I C R I S A T Center
K . R . Krishna1
Abstract
The
vesicular
grown
in
variation
tion
in
Fungal
technique
1.
it
could
differed
pearl
based
be
millet,
is
genotypes
colonization,
widespread
tested
which
(P<0.05)
further
both
sorghum
and pearl
millet
three field
locations
showed a
large
ranged between
25
male-sterile
lines,
between
that
on
across
mycorrhizal
colonization
and 56%.
Root
restorer
lines,
is
plant-genotype
colonizaand
their
dependent
heritable.
in
their
indicating
on phosphorus
improved performance
symbiosis
millet
significantly
indicating
isolates
and
mycorrhizal
Pearl
mycorrhizal
varied
crosses,
that
sorghum
with
mean
percentage
derived
and
arbuscular
SAT regions.
on
ability
a
need
estimation
sorghum,
to
to
in
stimulate
screen
bleeding
is
and
sap,
growth
select
which
and phosphorus
fungi
could
for
be
uptake
improved
used
on
response.
to
both
A
select fungi
described.
Microbiologist, Pearl Millet Improvement Program, I C R I S A T , Patancheru, A.P. 502 324, India.
I C R I S A T (International Crops Research Institute for the Semi-Arid Tropics), 1986. Cereal nitrogen fixation. Proceedings of the Working
Group Meeting, 9-12 Oct 1984, I C R I S A T Center, India. Patancheru, A.P. 502324, India: I C R I S A T .
111
T h e use o f E L I S A (Enzyme-Linked Immunosorbent
Assay) for Q u a l i t y Assessment of Bacterial Inoculants
P . T . C . Nambiar1
Abstract
major
reasons
inoculants.
One
of
the
The
conventional plant-infection
carrier-based
inoculants.
control
of
Rhizobium
The
test
ELISA
necessary
a
single
modified for
1.
plant
results
to
has
can
test
ELISA
the
the
for
At
the
ICRISAT
to
be
obtained
quality
quality
of
is
of
25-30
assessment
2
we
often
have
days for
days.
inoculum
the
of
same
other
informers'
is
The plant-infection
within
eight
technology
Center,
grown for
needed for
BNF
technique
inoculants.
be
plate
failure
fields
used
standardized
technique
is
packets
number
bacterial
hundred
by
the
of peat
and
the
poor
enumerate
ELISA
time
the plant-infection
One
is
to
for
quality
rhizobia
the
quality
consuming and laborious.
technique
assessment,
sixty-eight
siratro plants
plant-infection
packets.
of
in
technique,
This
technique
while
while
can
are
only
be
inoculants.
Microbiologist, Groundnut Improvement Program, I C R I S A T , Patancheru, A.P. 502 324, India.
I C R I S A T (International Crops Research Institute for the Semi-Arid Tropics). 1986. Cereal nitrogen fixation. Proceedings of the Working
Group Meeting, 9-12 Oct 1984, I C R I S A T Center, India. Patancheru, A.P. 502 324, India: I C R I S A T .
113
Recommendations
T h e need to have f a i r l y u n i f o r m procedures for measuring the a m o u n t of n i t r o g e n
f i x e d b y s o r g h u m a n d m i l l e t s u n d e r i n situ c o n d i t i o n s w a s r e c o g n i z e d a s a p r e r e q u i s i t e
f o r e m b a r k i n g o n m o r e i n t e n s i v e r e s e a r c h o n t h e B N F t e c h n o l o g y o f these c r o p s . T h e
acetylene-reduction m e t h o d ( A R A ) , e m p l o y i n g whole plants either b y axenic p l a n t
cultures i n o c u l a t e d w i t h N 2 - f i x i n g bacteria o r b y n o n d i s t u r b e d soil cores o r intact
p l a n t assay, w a s c o n s i d e r e d t o b e a p p r o p r i a t e f o r q u a l i t a t i v e l y e s t i m a t i n g t h e n i t r o g e nase a c t i v i t y a n d , h e n c e , t h e N 2 - f i x i n g a b i l i t y o f these p l a n t s . T h e p l a n t e d s o i l c o r e
m e t h o d , s t a n d a r d i z e d b y I C R I S A T s c i e n t i s t s , w a s e n d o r s e d t o m e e t t h e assay r e q u i r e ments for this purpose.
S i n c e t h e t i m e o f i n c u b a t i o n w i t h a c e t y l e n e gas c a n b e a v a r i a b l e f a c t o r i n s u c h
assays, i t i s r e c o m m e n d e d t h a t t h e t i m e c o u r s e assay b e t e r m i n a t e d w h e n t h e l i n e a r i t y
o f t h e r e a c t i o n b e g i n s t o cease. W h e n e v e r q u a n t i t a t i v e m e a s u r e m e n t s a r e n e e d e d , t h e
15
N d i l u t i o n t e c h n i q u e has to be resorted t o , c h o o s i n g a suitable n o n f i x i n g c o n t r o l
plant.
I n s o f a r a s p o t c u l t u r e o r field e x p e r i m e n t s are c o n c e r n e d , the t o t a l b i o m a s s o f the
plant ( r o o t , shoot, a n d grain), i n d i v i d u a l l y or collectively, m a y be t a k e n as the
y a r d s t i c k t o measure the o v e r a l l benefits f r o m i n o c u l a t i o n w i t h bacterial cultures.
S u c h a m e a s u r e m e n t w o u l d take i n t o a c c o u n t the benefits d e r i v e d b y N 2 f i x a t i o n a s
w e l l a s b y o t h e r factors, such a s g r o w t h - p r o m o t i n g substances o r e n h a n c e d n u t r i e n t
uptake c o n c o m m i t a n t w i t h i m p r o v e d r o o t g r o w t h . T h e design a n d conduct o f the f i e l d
e x p e r i m e n t s s h o u l d be left to the a g r o n o m i s t s , b u t m i c r o b i o l o g i s t s have to supervise
i n o c u l a t i o n procedures a n d w a t c h the progress o f experiments t o record o u t b r e a k o f
pests a n d diseases, w h i c h m a y l i m i t o r m a s k b e n e f i t s f r o m B N F . I n these e x p e r i m e n t s ,
fertilizer nitrogen should be kept w i t h i n 30-40 kg N ha-1.
There c a n b e b o t h l o n g - t e r m a n d s h o r t - t e r m approaches t o future research o n B N F
technology for sorghum and millets.
T h e l o n g - t e r m approaches m a y include the f o l l o w i n g :
1. To search extensively f o r cultivars that e x h i b i t consistently h i g h nitrogenase
a c t i v i t y a n d p l a n t b i o m a s s o u t p u t u n d e r d i v e r s e stress c o n d i t i o n s i n t h e s e m i a r i d tropics, w i t h a view to eventually transfer the beneficial traits to cultivated
varieties t h r o u g h intensive plant-breeding methods. T h i s line o f w o r k m a y also
include designing o f breeding procedures t o f i n d o u t i f the h i g h p r o d u c t i o n
a t t r i b u t e o f plants are inheritable.
2 . T o e x p l o r e v a r i e t a l i n t e r a c t i o n w i t h s p e c i f i c b a c t e r i a l species o r s t r a i n s .
3 . T o exchange b a c t e r i a l g e r m p l a s m a n d m a i n t a i n t h e m a t different centers.
T h e s h o r t - t e r m strategies m a y i n c l u d e the f o l l o w i n g :
1 . T o test t h e e f f i c i e n c y o f s u p e r i o r b a c t e r i a l c u l t u r e s i n e n h a n c i n g t h e y i e l d
potential of s o r g h u m a n d millets under field conditions in the semi-arid tropics.
115
2 . T o test v a r i o u s m e t h o d s o f i n o c u l a t i o n u n d e r f i e l d c o n d i t i o n s t o e v o l v e a
suitable m e t h o d .
3. To qualitatively a n d quantitatively define the extent of g r o w t h - p r o m o t i n g substances p r o d u c e d b y e f f i c i e n t s t r a i n s o f b a c t e r i a i n p u r e c u l t u r e s a n d i n t h e
rhizosphere of crops.
4 . T o intensify w o r k o n identification o f hitherto undefined N 2 - f i x i n g bacteria i n
sorghum and millets.
5 . T o q u a n t i t a t i v e l y estimate the p r e p o n d e r a n c e o f several types o f N 2 - f i x i n g
bacteria in the r o o t region, by standardizing the e n u m e r a t i o n procedures.
116
117
Meeting Organization
Organizing Committee
F.R. Bidinger, K . K . L e e , S.P. W a n i
Session Chairpersons
Rapporteurs
P.V. Rai
P.T.C. Nambiar
P. Tauro
R.P. Thakur
Joseph Thomas
J . V . D . K . Kumar Rao
K . K . Lee
S.B. Chavan
G. Oblisami
O.P. Rupela
N . N . Prasad
S. Chandrapal
C. Johansen
J . V . D . K . Kumar Rao
N.S. Subba Rao
S. Pande and P. Soman
Participants
C.P. Ghonsikar
Associate Dean, PG School
Department of Microbiology
Marathwada Agricultural University
Parbhani
Maharashtra 431 402
India
B.K. Konde
Associate Professor
Department of Microbiology and Plant Pathology
Mahatma Phule Agricultural University
Rahuri
Maharashtra 413 722
India
S.V. Hegde
Senior Microbiologist
Department of Agricultural Microbiology
University of Agricultural Sciences
Gandhi Krishi Vignana Kendra ( G K V K )
Bangalore
Karnataka 560 065
India
B.S. Kundu
Associate Professor
Department of Microbiology
College of Basic Sciences and Humanities
Haryana Agricultural University
Hisar
Haryana 125 004
India
G .S.Jadhav
Agronomist
Bajra Research Station
Marathwada Agricultural University
Paithan Road
Aurangabad
Maharashtra
India
G.S. Murthy
Chief Agronomist
Coromandel Fertilizers L t d
126, Sarojini Devi Road
P.B.No.1589
Secunderabad
Andhra Pradesh 500 003
India
118
K . C . Mohan Kumar
Department of Microbiology
Andhra Pradesh Agricultural University
Rajendranagar
Hyderabad
Andhra Pradesh 500 030
India
G. Oblisami
Head
Centre of Advanced Studies
in Agricultural Microbiology
T a m i l Nadu Agricultural University
Coimbatore
T a m i l Nadu 641 003
India
H . Polasa
Professor and Head
Department of Microbiology
Osmania University
Hyderabad
Andhra Pradesh 500 007
India
N . N . Prasad
Professor and Head
Department of Microbiology
Annamalai University
Annamalai Nagar
T a m i l Nadu 608 002
India
D . Purushothaman
Associate Professor
Center of Advanced Studies in
Agricultural Microbiology
Tamil Nadu Agricultural University
Coimbatore
Tamil Nadu 641 003
India
P.V. Rai
Professor and Head
Department of Agricultural Microbiology
University of Agricultural Sciences
Gandhi Krishi Vignana Kendra ( G K V K )
Bangalore
Karnataka 560 065
India
A . V . Rao
Microbiologist
Central A r i d Zone Research Institute
Jodhpur
Rajasthan 342 003
India
V. Rajaramamohan Rao
Microbiologist
Central Rice Research Institute
Cuttack
Orissa 753 006
India
S.T. Shende
Senior Microbiologist
Division of Microbiology
IARI
New Delhi 110 012
India
P.A. Shinde
Head
Department of Agricultural Microbiology
and Plant Pathology
Mahatma Phule Agricultural University
Rahuri
Maharashtra 413 722
India
N.S. Subba Rao
Project Director
Agro-Energy Center
IARI
New Delhi 110 012
India
H . L . S . Tandon
Director
Fertilizer Development and
Consultation Organization
New Delhi 110 048
India
P. Tauro
Professor of Eminence
Department of Microbiology
College of Basic Sciences and Humanities
Haryana Agricultural University
Hisar .
Haryana 125 004
India
Joseph Thomas
Biology and Agriculture Division
Bhabha Atomic Research Centre
Trombay
Bombay
Maharashtra 400 085
India
119
K.V.B.R. Tilak
Senior Microbiologist
Division of Microbiology
IARI
New Delhi 110 012
India
I C R I S A T Participants
J.S. Kanwar, Director of Research
C.R. Jackson, Director of International Cooperation
K . K . Lee, Principal Cereals Microbiologist
S.P. Wani, Microbiologist (Millet)
K . R . Krishna, Microbiologist (Millet)
S.B. Chavan, Plant Breeder (Millet)
C. Johansen, Principal Agronomist (Pulses)
O.P. Rupela, Agronomist (Microbiology, Pulses)
J.V.D.K. Kumar Rao, Agronomist (Microbiology, Pulses)
J . H . Williams, Principal Plant Physiologist (Groundnut)
P.T.C. Nambiar, Microbiologist (Groundnut)
Acknowledgement
T h e organizers express t h e i r sincere t h a n k s t o D r H . L . S . T a n d o n , F e r t i l i z e r D e v e l o p m e n t a n d C o n s u l t a t i o n
O r g a n i z a t i o n , N e w D e l h i , I n d i a , f o r useful advice, a n d t o G . S a t y a k u m a r i , N i r m a l a K u m a r , K . V . H a n u m a n t h a R a o , the I n f o r m a t i o n Services staff, a n d the S o r g h u m a n d M i l l e t s I n f o r m a t i o n Center staff ( a l l I C R I S A T )
f o r t h e i r h e l p i n the e d i t i n g a n d t y p i n g o f these proceedings.
120
RA-00097
ICRISAT
International Crops Research Institute for the Semi-Arid Tropics
Patancheru, Andhra Pradesh 502 324, India
ISBN 92-9066-111-9
Printed at ICRISAT Center
ICR 85-0043