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Advances in agronomy volume 29


ADVANCES IN

AGRONOMY
VOLUME 29


CONTRIBUTORS TO THIS VOLUME

ESHELBRESLER

K. R. CHRISTIAN
C. S. COOPER

R. C. DALAL
J. DOBEREINER
D. R. GRIFFITH
GURDEVS. KHUSH
T. MAEDA
CARLOSA. NEYRA
S. D. PARSONS

C. B. RICHEY

W. R. SCOWCROFT

H. TAKENAKA
B. P. WARKENTIN


ADVANCES IN

AGRONOMY
Prepared under the Auspices of the
AMERICAN SOCIETY OF AGRONOMY

VOLUME 29

Edited by N. C . BRADY
International Rice Research Institute
Manila, Philippines
ADVISORY BOARD

w.L. COLVILLE, CHAIRMAN
G. W. KUNZE D. G. BAKER D. E. WEIBEL
G. R. DUTT H. J. GORZ
M. STELLY, EX OFFICIO,
ASA Headquarters
1977

ACADEMIC PRESS 9 New York San Francisco London
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CONTENTS

............................
PREFACE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
CONTRIBUTORS TO VOLUME 29

iX

xi

NITROGEN FIXATION I N GRASSES

Carlos A. Neyra and J . Dobereiner
I . Introduction

....................................
.................
111. Bacteriology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
IV. Factors Affecting Nitrogen Fixation in Grasses . . . . . . . . . . . . . .
V . General Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Note Added in Proof . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I1. Nitrogen Fixation in C-3 and C-4 Grasses

1
3
12
26
30
33
38

SOMATIC CELL GENETICS AND PLANT IMPROVEMENT

W . R. Scowcroft
I.
I1.
111.
IV.
V.
VI .
VII .

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Plant Cell Tissue Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Anther Culture and Haploids . . . . . . . . . . . . . . . . . . . . . . . . .
Mutant Isolation and Selection . . . . . . . . . . . . . . . . . . . . . . . .
Plant Cell Protoplasts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Genetic Transformation in F'lants .......................
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

39
40
44
48
55
61
73
74

SOIL ORGANIC PHOSPHORUS
R. C. Dalal
I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I1. Organic Phosphorus Content of Soil .....................
111. Nature of Soil Organic Phosphorus ......................
IV Organic Phosphorus in Soil Solution .....................
V . Organic Phosphorus Turnover in Soil ....................
VI . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

.

83
84
86
90
96
112
113


vi

CONTENTS

GROWTH OF THE LEGUME SEEDLING

C. S. Cooper

I.
I1.
I11.
IV .
V.
VI .
VII .
VIII .

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Physiological Predetermination . . . . . . . . . . . . . . . . . . . . . . . .
Germination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Stages of Seedling Development . . . . . . . . . . . . . . . . . . . . . . .
Improvement of Legume Seedling Vigor . . . . . . . . . . . . . . . . . .
Seedbed Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Seeding Forage Legumes . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Seeding Management Practices . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

119
120
121
123
130
130
131
137
137

YIELDS AND CULTURAL ENERGY REQUIREMENTS FOR CORN
AND SOYBEANS WITH VARIOUS TILLAGE-PLANTING SYSTEMS

C . B . Richey. D . R . Griffith. and S . D . Parsons

I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I1. Tillage-Planting Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I11. Influence of Tillage-Planting System on Yields . . . . . . . . . . . . .
IV . Yield Factors Influenced by Tillage-Planting System . . . . . . . . . .
V . Energy Requirements for Various Tillage-Planting Systems . . . . .
Vl . Projecting Energy Savings with Reduced Tillage . . . . . . . . . . . . .
VII . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

141
143
147
157
169
178
180
180

EFFECTS OF THE ENVIRONMENT ON THE GROWTH OF ALFALFA

K . R . Christian
I . Introduction

....................................

.............
111. Shoot Growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
IV. Root Growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I1. Genetic Variation in Response t o Environment

V . Environmental Factors and Vegetative Growth . . . . . . . . . . . . . .
VI . Phases in Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Vll . Plant Associations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
VI11 . Genetic Adaptation t o Environment .....................
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

183
185
186
189
191
209
214
217
219


CONTENTS

vii

PHYSICAL PROPERTIES OF ALLOPHANE SOILS

T . Maeda. H . Takenaka. and B . P. Warkentin
I.
I1.
I11.
IV .
V.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Index Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Structure of Allophane Soils . . . . . . . . . . . . . . . . . . . . . . . . . .
Physical Characteristics of Allophane Soils . . . . . . . . . . . . . . . .
Soil Engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

229
232
241
246
253
261

DISEASE A N D INSECT RESISTANCE IN RICE

Gurdev S. Khush
I.
I1.
111.
IV .
V.
VI .

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Disease Resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Insect Resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Developing Varieties with Multiple Resistance . . . . . . . . . . . . . .
Stability of Resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

265
266
301
322
329
333
333

T R IC K LE-D R IP IR R IG A T I0N: PR INC IPL ES A N D
APPLICATION TO SOIL-WATER MANAGEMENT

Eshel Bresler

I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I1. Potential Advantages of Trickle Irrigation . . . . . . . . . . . . . . . . .
111. Problems in Practical Use . . . . . . . . . . . . . . . . . . . . . . . . . . . .
IV . Modeling of Water and Salt Flows . . . . . . . . . . . . . . . . . . . . . .
V . Soil-Water Regime during Trickle Infiltration . . . . . . . . . . . . . . .
VI . Solute Distribution during Infiltration . . . . . . . . . . . . . . . . . . .
VII . Application of Infiltration Models to the Design of
Trickle Irrigation Systems . . . . . . . . . . . . . . . . . . . . . . . . . . .
VIII . Water Management in Marginal Soils . . . . . . . . . . . . . . . . . . . . .
List of Symbols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

376
387
389
391

SUBJECTINDEX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

395

344
345
351
353
367
372


This Page Intentionally Left Blank


CONTR I BUT0 RS
Numbers in parentheses indicate the pages on which the authors’ contributions begin.

ESHEL BRESLER (343), Division of Soil Physics, lnstitute of Soils and Water,
Agricultural Research Organization, Volcani Center, Bet Dagan, Israel
K. R. CHRISTIAN (183), Division of Plant Industry, Commonwealth Scientific and Industrial Organization, Canberra, Australia
C. S. COOPER (1 19), Agricultural Research Service, United States Department
of Agriculture, Bozeman, Montana
R. C. DALAL (83), Department of Agronomy and Soil Science, University of
New England, Armidale, N. S. W., Australia
J. DOBEREINER ( l ) , EMBRAPA, Campo Grande, Rio de Janeiro, Brazil
D. R. GRIFFITH (14 I), Purdue Agricultural Experiment Station, Lafayette,
Indiana
GURDEV S . KHUSH (265), International Rice Research Institute, Los BaCos,
Philippines
T . MAEDA (229), Department of Agricultural Engineering, Hokkaido University, Sapporo, Japan
CARLOS A. NEYRA (l), EMBRAPA, Campo Grande, Ria de Janeiro, Brazil
S. D. PARSONS (141), Purdue Agricultural Experiment Station, Lafayette,
Indiana
C. B. RICHEY (141), Purdue Agn’cultural Experiment Station, Lafayette,
Indiana
W. R. SCOWCROFT (39), Division of Plant Zndusty, Commonwealth Scientific
and Industrial Organization, Canberra, Australia
H. TAKENAKA (229), Department of Agricultural Engineering, University of
Tokyo, Tokyo, Japan
B. P. WARKENTIN (229), Department of Renewable Resources, McGill University, Montreal, Canada


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PREFACE
The internationality of science has never been more evident than it is today.
And the need for international exchange of scientific findings is as pressing in
soil and crop science as in any other field. Meeting the world's food needs while
simultaneously maintaining an environment suitable for humankind as well as
other animal species is among the primary objectives of soil and crop scientists
throughout the world. These objectives can be attained only if there is rapid
and effective interchange of scientific information from one country to another.
This volume follows the pattern that has characterized recent issues of
Advances in Agronomy. The subjects addressed are of international concern.
Likewise, the authors are from diverse backgrounds and nationalities.
While all of the papers in this volume provide information of concern to those
who produce food, four have rather direct implications for food production.
The review of research on nitrogen fixation on grasses, on the pests of rice, on
trickle-drip irrigation, and on somatic cell techniques for plant improvement
each identify unique means of increasing world food supplies. The authors have
appropriately emphasized past accomplishments, current research constraints,
and potential for future progress.
Two papers deal with soil characteristics. One provides an upto-date review of
soil organic phosphorus research. The other is concerned with allophane, an
important soil mineral, the knowledge of which has been quite limited. In a
third soils-oriented paper, energy-saving minimum tillage techniques are discussed. This is a research area that will receive increasing attention in the future
because of rising energy costs.
Research on forage crops is the focus of two papers, one concerned with the
growth of legume seedlings and the second with the influence of the environment on alfalfa growth. Both reviews will be helpful for animal-oriented food
production systems.
Soil and crop scientists throughout the world are indebted to the 14 authors
who have prepared these important papers. Their contributions will undoubtedly
stimulate future research efforts in the subject areas covered in this volume.
N. C. BRADY

xi


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NITROGEN FIXATION I N GRASSES
Carlos A. Neyra and J. Dobereiner
EMBRAPA, Campo Grande, Rio de Janeiro, Brazil

I. Introduction ..................................................
11. Nitrogen Fixation in C-3 and C-4 Grasses . . . . . . . . . . . . . . . .. . . . . . . . . .
A. C-4 Grass Systems . . . . . . . . . . . . . . . . . . ~. . . . . ~. . . . . . . . . . . . .
B. C-3 Grass Systems . , . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . ... .. . . . . . . . . . .. . . ...
C. Miscellaneous Systems . . . . . .
. ..... . .
111. Bacteriology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
A. Beijerinckia . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . , . . . . . . . . . . ..
. . .. . . . .
B. Azotobacter paspali .
C. Spirillum lipoferum . . .. . . . . , . . . . . . . . . . . . . . . . . . . . . . . , . .
IV. Factors Affecting Nitrogen Fixation in Grasses . . . . . . . . . . . . . . . . . . . . . .
A. Seasonal and Diurnal Fluctuations . . . . . . . . . . . . . . . . . . . . . . . . . . . .
B. PlantGenotype ..............................................
. .. .. . . . . .. .. .. . . . . . . .. .. . . . . . . . . .. . . . .
C. Temperature . .
D. Oxygen ....................................................
. . .. . . . . . . . . . . . . . . .. . . . . . . .
E. Combined Nitrogen . . . . . . . .
V. General Discussion . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References ....................................................
Note Added in Proof . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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9
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38

I. Introduction

Population growth and changing dietary habits have led to an increased
demand for protein for human consumption. Combined nitrogen (N) and biological Nzfutation represent the major inputs of N for crop and protein yield.
While some plants, notably grain crops, have relied mostly on combined N
sources, some other plants, notably legumes, have the capability of being at least
partially self-sufficient through symbiotic Nz fixation. As to the nonbiological
inputs, increased use of fertilizer N is probably the most important single factor
that has enabled cereal grain production to increase significantly in recent years
(Hardy, 1976). It is also predicted that increasing cereal grain production at the
world level will require the use of increasing amounts of fertilizer N (Hardy,
1976). However, in the less developed countries the availability and the high
prices of fertilizer N are limiting factors for its use on a large scale. In addition,
in tropical regions considerable amounts of N, mostly in the form of NO3 are
lost from the soil by leaching (da Eira et al., 1968).
1


TABLE I

N, Fixation and Incorporation in Digitaria decurnbens and Paspalurn notaturn Grown in Pots for 2 Weeksa
N, fixed

Plant species and amendment
Digifaria decurnbens cv. transvala without sucrose*

D. decumbens cv. slenderstem with 0.5% sucrose

Paspalurn notaturn cv. batatais without sucroseb

P. notaturn cv. batatis with 0.5% sucrose

Atoms %
excess l 5 N

Part of
plant

Total N in
plants (mglpot)

Roots
Rhizomes
Stems
Leaves
Total

1.78
2.74
4.32
7.34
16.18

0.151
0.146
0.021
0.007

Roots
Rhizomes
Stems
Leaves
Total

1.40
4.06
1.80
4.74
12.00

0.582
0.709
0.073
0.010

Roots
Rhizomes
Leaves
Total

2.89
2.62
6.03

0.563
0.703
0.070

11.54

Roots
Rhizomes
Leaves
Total

2.71
3.25
3.85
9.81

-

-

-

1.021
1.392
0.053
-

(fig/pot)

2.69
4.00
0.91
0.5 1
8.1 1
8.15
28.79
1.31
0.47
38.72

16.34
15.28
2.97
34.59
27.56
44.39
2.06
74.01

( d g roots
+ rhizomes)
-

-

10.73
-

-

33.64
-

24.39
-

43.33

'Summarized from De-Polli et al. (1977). See Soil Biol. Biochern. 9, 119-1 23. Used by permission.
'Values from D. decumbens are from single pots but those from P. nofafumare from duplicate pots. All pots were incubated in the same jar
for 72 hours (15-hOur light and 9-hour dark periods) in a gas mixture containing an average 42.8% N, (enrichment I5N, 85.5%), 2.6% O , ,
3.2% CO,, and 51.4% A. The cv. for the mass spectrometer analyses was 0.40-2.62%.


NITROGEN FIXATION 1N GRASSES

3

Although improved technologies of fertilizer N production and increased
efficiency of fertilizer use by plants could make more N available for the plants,
nevertheless alternative technologies should be found to lessen the dependence
of plants on fertilizer N. To develop N self-sufficiency in forage grasses and grain
crops may constitute a major breakthrough in the years ahead. Efforts along
these lines may include the incorporation of nifgenes into cells that normally do
not fur N2 (Brill, 1974) or the development of already present plant-bacteria
associations. Dixon and Postgate (1972) demonstrated the possibility of transferring nifgenes to bacteria; Giles and Whitehead (1976) have demonstrated that
Nz -fixing bacteria can be incorporated directly into protoplasts of a mycorrhizal
fungus. This could be of tremendous importance in the infection process of
plant roots by N2 -fixing bacteria.
Recent findings (Rinaudo et al., 1971; Dobereiner et al., 1972a; Dobereiner
and Day, 1976; von Bulow and Dobereiner, 1975) have revealed already existing
associations of tropical grasses with N2 -fixing bacteria which under favorable
conditions may be contributing significantly to the nitrogen economy of these
plants. Although it is premature to predict the actual contribution of Nz
fixation to plant nitrogen, it is of major importance to mention that incorporation of ''N2 into plant tissues has recently been demonstrated (Table I).
Many of the tropical grasses able to support significant nitrogenase activity
possess the photosynthetic C-4 pathway (Day et al., 1975b). The amount of
light required to saturate photosynthesis and the maximum photosynthetic rate
attainable are much greater in C-4 than in C-3 plants (Chollet and Ogren, 1975).
At high light intensities and low temperatures the rate of photosynthesis is
essentially the same in C-3 and C 4 species, but at higher temperatures C 4 plants
show higher photosynthetic rates. Furthermore, losses of carbon due to photorespiration are minimal in C-4 plants (Chollet and Ogren, 1975). This evidence
suggests that tropical grasses may be very efficient in harvesting light energy for
nitrogen fixation.
Maximization of N2 futation in tropical grass-bacteria associations and the
elaboration of agronomic practices to enhance or promote N2 fixation in grasses
will depend on the identification of the various limiting factors controlling this
process under field conditions.
In this review we intend to give an interpretative account of recent developments in this rapidly expanding field and to discuss in more detail some of our
work which is not yet available in the literature.

II. Nitrogen Fixation in C-3and C-4 Grasses

Nitrogen-futing bacteria are widely distributed in soils, and it has been suggested for a long time that major contributions to the system could be expected


4

CARLOS A . NEYRA AND J. DOBEREINER

(Beijerinck, 1925; Schroder, 1932; Krasil'nikov, 1968; Dobereiner, 1966; AbdEl-Malek, 1971; and many others). Various anaerobic, facultative, and aerobic
bacteria are capable of fixing nitrogen in soil, in the rhizosphere, and in roots.
Interest in aerobic organisms has been generally greater because aerobic metabolism is more efficient, and because agricultural soils, with some exceptions (e.g.,
paddy rice), are well aerated. In most systems the availabitity of energy and
carbon substrates represents the major limiting factors to biological N2 fixation.
Nitrogen fixation of importance in soils has only been demonstrated after the
addition of carbon substrates (Mishustin, 1970; Brouzes et al., 1971; Abd-ElMalek, 1971) or when growing plants release part of their photosynthates
(Dobereiner and Alvahydo, 1959; Dobereiner, 1961; Dobereiner et ul., 1972a;
Day et ul, 1975b). In addition, plant root exudates can play an important role
in the establishment and maintenance of the rhizosphere population (Rovira,
1965b).
A good example of solar energy utilization for N2 fixation is the legume
symbiosis, where the energy requirement for nitrogen fixation is equivalent to
the requirement for nitrate reduction (Minchin and Pate, 1973; Gibson, 1976).
However, photosynthate availability is still considered a major limiting factor for
N2 fixation in soybeans (Quebedeaux et ul., 1975).
On the other hand some tropical grasses can grow and produce constant yields
without addition of nitrogen fertilizer to the soils, and it was suspected for many
years that substantial Nz fixation occurred in these systems (Parker, 1957;
Moore, 1966; Dobereiner, 1966). Because of their photosynthetic characteristics
(see Section I), most of these plants are in a favorable position with regard to
photosynthate availability for growth and N2 fixation. In the last 5 years the
evidence for Nz fixation in grasses has accumulated rapidly. The results obtained
by several authors for field-grown tropical plants are summarized in Table 11.
Although the measurement of uptake of "N-enriched N z represents the most
satisfactory method for evaluation of N2 fixation, the introduction of the
acetylene reduction method has represented a major breakthrough in the evaluation of N, fixation both in the laboratory and under field conditions. The work
of Schollhorn and Burris (1967) and Dillworth (1966) suggested that the rate of
acetylene reduction may be used as an index of the rate of Nz fixation. The
reduction of acetylene to ethylene (C, H2-C2 H4) and the measurement of
ethylene by gas chromatography has been extensively used for the assessment of
N2 fixation in grass-bacteria associations. The reader is referred to the literature
for further details on the use of "N as a tracer and for the acetylene reduction
method (Burris and Wilson, 1957; Stewart et uL, 1967; Hardy et al., 1968, 1973;
Burris, 1972, 1974; Dart et ul., 1972).
Several procedures for the assessment of N2 fixation by acetylene reduction
have been adopted. A general procedure for assaying excised roots was described


NITROGEN FIXATION IN GRASSES

5

for Paspalurn notaturn (Dobereiner et al., 1972a) and Dig'taria decurnbens
(Abrantes et al., 1975), and the same procedure with slight modifications has
been used for several other forage grasses and grain crops (von Biilow and
Dobereiner, 1975; Day et al., 1975b; van Berkum and Neyra, 1976; Sloger and
Owens, 1976). Steel cylinders of small diameter are very useful for taking cores
of small grasses from the field (Day et al., 1975a; Abrantes et al., 1975). A
variety of devices have also been described for in situ measurements of N2
fixation under field conditions (Balandreau et al., 1974; Balandreau, 1975;
Watanabe and Kuk-Ki-Lee, 1975).
A. C-4 GRASS SYSTEMS

I . Paspalurn notatum
The first tropical C-4 grass-bacterial association to be studied in detail was
that of Paspalurn notaturn-Azotobacter paspali. Five ecotypes in this grass
(tetraploid types) show a very specific association with Azotobacter paspali
(Dobereiner, 1966; Dobereiner and Campelo, 1971). Of the 33 ecotypes or
cultivars studied, only five (tetraploid types) stimulated A. paspali growth in the
rhzosphere. Establishment of the bacteria on the roots takes several months and
inoculation does not accelerate this rhizosphere association (Dobereiner and
Campelo, 1971). Field plants, transplanted with adhering soil into vermiculite
and watered with nitrogen-free nutrient solution, fixed 80 mg N per pot in 2
months, the amount necessary for normal growth (Dobereiner and Day, 1975).
CzH2 reduction assays with intact soil plant cores correlated well with excised
roots extracted from the soil and assayed after overnight preincubation in low
p 0 2 (Dobereiner et al., 1972a). Paspalurn notatum grown in sand, from seeds,
did not show A. paspafi establishment, except when glucose was added (Kass et
al., 1971). It is possible that besides A. paspali other microorganisms (e.g.,
mycorrhizal fungi) may be involved in the establishment of the association.
Mosse (1972) observed very intensive mycorrhizal infection of this grass. Inoculation of irradiated Brazilian soils with Endogone spores resulted in large
increases in forage yield in Paspalurn notaturn (Mosse, 1972).
Localization of A. paspali has been suggested to be in the mucagel layer
outside the root (Dobereiner et al., 1972a). The correlation of root piece
nitrogenase activity and enrichment culture activity in A . paspali sucrose
medium was highly significant (r = .08l) (Dobereiner and Day, 1974) when the
same root pieces were used for inoculation of the enrichment medium. Estimates
of nitrogen fixation in intact soil plant cores (10 cm #) by the CzH2 reduction
method were calculated to be 340 g N/ha per day. "N2 assays in smaller vessels
extrapolated to 110 g N/ha per day (calculated from data by De-Polli, 1976).


6

CARLOS A. NEYRA AND J. DOBEREINER

2. Sugar Cane
In many parts of the world this crop has been grown in monoculture for more
than 100 years without addition of nitrogen fertilizer and a survey in SBo Paulo
(Brazil) revealed that only half of the fields with this crop responded to nitrogen
fertilizer even if PK was also supplied (Verdade, 1967). Selective stimulation of
the nitrogen-fixing Beijerinckia under sugar cane vegetation and positive rhizosphere effects have been shown (Dobereiner, 196 1). Assays with the acetylene
reduction method indicate that in this crop only a minor part of the N2 is fixed
in or on the roots and most of it in the rhizosphere or in the soil (Dobereiner et
al., 1972b; Ruschel, 1976). Rain water can carry leaf exudates into the soil
which enhance Beijerinckia growth (Dobereiner and Alvahydo, 1959). Maximal
soil nitrogenase activities were found in rhizosphere soil and between the rows,
where the canopy closes (Dobereiner et al., 1972b). Sugar cane seedlings exposed to 's N2 indicated fixation, incorporation, and translocation of nitrogen
to the leaves (Ruschel et al., 1976). Spirillum lipofem8mdoes not appear to be
stimulated in the sugar cane rhizosphere (Dobereiner, 1976a) and this supports
the prevalence of Beijerinckia spp. as the major N2 fixer in this plant.

3. Digitaria decumbens
This grass contains several cultivars of agronomic importance, e.g., Pangola,
Transvala, and Slenderstem. These three grasses were grown in our experimental
fields from November 1973 to May 1975 (two summers, one winter) and
showed mean nitrogen yields of 1S O , 1.48, and 1.40 kg/ha per day, respectively.
The nitrogen gain of the soil (0-20 cm depth) calculated from Kjeldahl analyses
before and after this period was 405, 216, and 468 g/ha per day, respectively
(Schank et al., 1975). Intact soil plant core assays in the summer 1975 showed
nitrogenase activities equivalent to 880, 480, and 970 g N/ha per day, respectively (Day and Dobereiner, unpublished data). Similar values (1460 ? 85 g
N/ha per day for Transvala and 1326 g N/ha per day for Slenderstem) have been
estimated from the data of De-Polli (1976). In lsN2 experiments significant
incorporation and translocation was shown in both species (Table I).
The N2 -fixing bacteria most commonly associated with Digitaria decumbens is
Spirrillum lipofemm. In several experiments, significant correlations of root
piece nitrogenase activity with S. lipofemm enrichment culture activity were
found, suggesting that S. lipofemm is the major organism responsible for
nitrogenase activity on roots (Dobereiner and Day, 1976). The most active root
pieces showed strongly reducing sites within the cortex, where cells packed with
tetrazolium-reducing bacteria were found. Inactive root pieces did not show such
sites (Dobereiner and Day, 1976).


TABLE I1
Potential of N, Fixation in Field-Grown Tropical Forage Grasses Associated with N, -Fixing Bacteria

N, -ase activity
C, H, /h/g
Plant species
Andropogen gayanus (C, )
Aizdropogen spp. (C,)
Brachiaria mutica (C,)
B. rugulosa (C,)
B. brachylopha (C,)
Bulbostylis aphylanthoides
Cynoden dactilon (C,)
Cynoden dactilon (C, )
Cyperus rotundus (C, )
Cypents sp. (?)
Cyperus obtusiflorus (?)
Digitaria decumbens (C,)
Hyparrhenia rufa (C,)
Hyparrhenia rufa (C,)
Hyparrhenia dissoluta (?)
Melinis minutiflora (C,)
Panicum maximum (C, j
Panicum maximum (C,)
Paspalum notatum (C,)
Paspalum comersenii (?)
Pennisentm purpureum (C, 1
Pennisetum purpureum ( C , )

Country

Roots

Soil

References

Nigeria
Ivory Coast
Brazil
Brazil
Ivory Coast
Ivory Coast
Brazil
Nigeria
Brazil
Nigeria
Ivory Coast
Brazil
Brazil
Nigeria
Ivory Coast
Brazil
Brazil
Nigeria
Brazil
Nigeria
Brazil
Nigeria

15-270
50-380
150-750
5-150
100-140
74
11-269
10- 50

-

Day and Dart (personal communication)
Balandreau e t al. (1973)
Dobereiner and Day (1975)
Dobereiner and Day (1975)
Balandreau et al. (1973)
Balandreau et al. (1973)
nobereiner and Day (1975)
Day and Dart (personal communication)
Dobereiner et al. (1975)
Day and Dart (personal communication)
Balandreauet al. (1973)
Dobereiner and Day (1975)
Dobereiner and Day (1975)
Day and Dart (personal communication)
Balandreau e t al. (1 973)
Dobereiner and Day (1975)
Dobereiner and Day (1975)
Day and Dark (personal communication)
Dobereiner and Day (1975)
Day and Dart (personal communication)
Dobereiner and Day (1975)
Day and Dart (personal communication)

-

0
-

0-0.07
-

10- 30

-

2
30-620
21-404
20- 30
30-140
10- 15
13- 41
20-299
75
2-283
25-30
5-954
60

-

nMinimal and maximal values obtained with excised preincubated roots.

-

0-0.35
0-0.15
-

04.19
04.15
-

04.33
-

04.09
-


8

CARLOS A. NEYRA AND J. DOBEREINER

4. Other Forage Grasses
Excised root assays have shown that several other tropical C-4 forage grasses
are able to fur N2 (Table 11). In a 3-year experiment in Nigeria, soil under fallow
of Panicum maximum contained 0.18% N (15 cm depth) while fallows under
legumes (Leucena glauca and Cajanus cajan) contained only 0.13%. This difference corresponds to 250 kg N/ha per year (Greenland, 1975). This illustrates
the tremendous importance of such a fallow crop for the nitrogen balance of
tropical soils even if only part of this amount was due to bic Jgical N2 fixation
and the remaining to prevention from leaching or denitrification. Only limited
results from core assays are available. Balandreau and Villemin (1973) estimated
N2 fnation (C2 H,) rates of 10-1 5 kg N/ha per year (in situ assays) in Ivory Coast
savannas where Panicum maximum and Andropogon sp. were predominant.
These authors found N,-fixing aerobes to be predominant in the rhizosphere but
did not identify them or relate them specifically to root nitrogenase activity. A
survey of S. lipoferum occurrence in various parts of Brazil revealed a high
incidence of this organism where Panicum maximum replaced virgin forest
(Diibereiner et al., 1976). In another experiment (six sites, 10 samples each)
there was a significant difference in S. lipoferum incidence between forage
grasses. Panicum maximum and Brachiaria mutica were the most favorable and
Hypawhenia rufa the least (Dobereiner, 1976b).
The mode of infection of Panicum maximum by Spirillum lipoferum has been
investigated by electron microscopy in axenic seedlings (Garcia et al., 1976). The
bacteria were observed on the root surface within 24 hours and in the middle
lamellae of the root cells within a week. No intracellular infection was observed
even after 1 month. These authors have suggested that S. lipoferum enters the
roots with the aid of pectolytic enzymes.
5. Grain Crops

Maize and sorghum represent two of the major grain crops in the world. High
nitrogenase activities (up to 9000 nmoles CzH4/g roots per hour) were found on
excised, preincubated maize and sorghum roots in a lowland soil in Rio de
Janeiro State (von Bulow and Dobereiner, 1975). Other estimates by this
method range between 100 and 2000 nmoles CzH4/g roots per hour (von Bulow
and Dobereiner, 1975; Abrantes et nl., 1976; Barber et al., 1976; Okon et al.,
1977a; Sloger and Owens, 1976). However, very low or no activities were
reported from soil plant core and in situ assays (Balandreau and Dommergues,
1973; Barber et a l , 1976; Burris, 1976; Tjepkema and van Berkum, personal
communication; for discussion on this discrepancy see Section V).
In Rio de Janeiro (von Bulow and Dobereiner, 1975), Brasilia and Londrina
(Peres, Nery, and Dobereiner, unpublished data) Spirillum lipoferum was found


NITROGEN FIXATION IN GRASSES

9

to be abundant in all Nz-fixing maize and sorghum roots examined. Sloger and
Owens (1976) also report isolation of this organism from maize roots grown in
Beltsville, Maryland, while it was not found in Wisconsin or Oregon (Burris et al.,
1976; Barber et al., 1976). Field-grown maize plants in Wisconsin inoculated
with strains of S. lipofemm isolated from Digitaria roots in Brazil, showed
establishment of the bacteria inside the roots (Burris, 1976; Dobereiner et al.,
1976). Inoculated plants showed higher nitrogenase activity than uninoculated
ones, while nitrogen-fertilized plants had no activity (Burris et al., 1976; Barber
et al., 1976). The total number of bacteria in surface-sterilized maize roots was
similar t o the number of S. lipofemm in the inoculated maize roots (Okon et al.,
1976a).
Significant correlations (p = 0.01) between maize root piece activities and
enrichment culture activities were only obtained when the roots were previously
surface-sterilized (von Bulow and Db'bereiner, 1975). Detailed studies on the
localization of Spirillum lipofemm in maize and sorghum roots are not yet
available. Maize plants grown in sterilized sand and soil collected in Wisconsin,
showed nitrogenase activities when inoculated with S. lipofemm. The organism
was reisolated from surface-sterilized roots (Burris et al., 1976). Effects on plant
growth and nitrogen incorporation however were not significant in these experiments. In Oregon attempts to isolate Nz-furing bacteria from maize plants
yielded Enterobacter cloacae (Raju et al., 1972).

B. C-3 GRASS SYSTEMS
1. Rice

There is little doubt as to the substantial contribution of biological Nz
fixation to the N economy of this most important grain crop. For instance, a
total of 23 rice crops, in an 11-year experiment at the International Rice
Research Institute in the Philippines, were obtained from a nonfertilized field
with no apparent decline in the nitrogen fertility of the soil. About 45 to 60 kg
N/ha per crop were removed through straw and grain (Watanabe and Kuk-KiLee, 1975). This represents a substantial amount of N which had to be replaced
in order to maintain the fertility level of the soil. Blue-green algae and photosynthetic bacteria account for a large part of the Nz fixation in paddy rice
(Watanabe and Kuk-Ki-Lee, 1975; Elnawamy, 1976). This subject has been
reviewed elsewhere (Stewart, 1976; Venkataraman, 1975).
Bacterial Nz f i a t i o n in intact rice cultures grown in test tubes has been shown
by Rinaudo et al. (1971) by Kjeldahl analyses and acetylene reduction. For the
latter method, plants were removed and assayed after 24 hours of preincubation
under anaerobic conditions. The results obtained by the two methods were in


10

CARLOS A. NEYRA AND J . DOBEREINER

good agreement. Excised root assays of field-grown rice roots (Yoshida, 1971a;
Yoshida and Ancajas, 1973) confirmed “rhizosphere N2 fixation.” Results from
intact soil plant systems in the field gave about 50 to 200 g N/ha per day at the
flowering stage, by the nonalgal component. The algae were separated by
removing the flooding water and assayed separately for N2 fixation (Watanabe,
1976). Balandreau (1975) reported that 25 to 30 kg N/ha can be fixed for the
growing season by the nonalgal component.
Bacterial counts indicate that Beijerinckia sp. and Enterobacter cloacae are the
most common N2-furing bacteria in the rhizosphere of rice (Yoshida, 1971b;
Balandreau, 1975). However, the methods used by these authors would not
reveal Spirillum lipoferum. When various types of roots were compared, mature
roots with many laterals were the most active ones (Hamad-Fares et aZ., 1976).
Such roots, surface-sterilized for 1 hour with 1% chloramin T yielded almost
pure cultures of an organism with properties resembling S. lipoferum (Diem et
al., 1976). However, most of the nitrogen fixation in the rice system has been
attributed to rhizosphere soil rather than roots themselves (Yoshida and Ancajas,
1973). Higher numbers of aerobic than of anaerobic N2-fixing bacteria in the
rhizosphere of rice were also found by Dommergues et al. (1973) and Watanabe
and Kuk-Ki-Lee (1975). Aerobic or microaerophilic N2-fixing bacteria were also
found to be prevalent in roots of a salt marsh grass (Patriquin, 1976). Methaneoxidizing bacteria which are able to fix Nz were also found in rice paddies. The
large amount of CH4 which can accumulate in these soils, should not be
overlooked as a potential carbon source for Nz fixation (De Bont et aZ., 1976).
However, O2 diffusion seems a limiting factor for this system. Inhibition of CH4
oxidation by C2H2 and consequent interference in C2 H2 reduction complicate
estimates of Nz fixation where these organisms are present (De Bont and
Mulder, 1976). Very high numbers (up to 3.6 X lo7) of N2-fixingCH4-oxidizing
organisms were found in the rice rhizosphere (De Bont el al., 1976).

2. Wheal
A nitrogen balance study in the famous Broadbalk continuous wheat experiment carried out from 1843 to 1967 in England, showed an average annual gain
of 34 kg N/ha, of which 24 kg N/ha were removed with straw and grain (Jenkinson, 1973). However, values extrapolated from C2H2 reduction assays on cores
were much lower (2 to 3 kg N/ha per year) (Day et al., 1975a). It was also
shown that nitrogenase activity of soil cores containing wheat was significantly
higher than in bare soil (Day et al., 1975a).
Wheat cores assayed in Oregon have been calculated to fm 2 g N/ha per day
(Barger et al., 1976). Much higher nitrogenase activities have been observed in
wheat cores assayed in Rio de Janeiro (Table 111). Similar results were obtained
with cores from several wheat cultivars grown in pots in Parana (Brazil) (New
and Abrantes, personal communication). Excised root assays underestimated, by


11

NITROGEN FIXATION IN GRASSES

TABLE 111
Nitrogenase Activity in 10 Intact Wheat Cores (cv. Sonora) Collected at Random in the
Field, at Flowering Stage

Mean 2 most active cores
Mean 5 intermediate cores
Mean 3 least active cores
Mean all cores

nmol
C, H, /hour/core

g N, /day
10 cm@core

2641
1137
180

597 x
276 X
44 x 10-6

-

-

g N, /daylhaa

506
238
38
229

aEstimate by the theoretical C, H, :N, 3: 1 ratio from 24-hour rates based on the @ of
10-cm area of the cores corrected for 15-cm distance between rows.

about one-half, the core activities but showed significant correlations with the
core assays (r = 0.86 in Rio de Janeiro and r = 0.87 in Parana).
In the Broadbalk experiment a large part of Nz fixation was attributed to
blue-green algae but root nitrogenase activity was attributed t o anaerobic or
facultative bacteria (Day et al., 1975a). Barber el al. (1976) isolated N,,-fixing
strains of Enterobacter cloacae, Bacillus macerans, and B. polymyxa from wheat
roots in Oregon. On the other hand, enrichment cultures in semisolid N-free
malate medium inoculated with surface-sterilized wheat roots obtained from
different locations in Brazil (Rio de Janeiro, Parana, and Brasilia) yielded almost
100% positive samples of SpiriZlum lipofemm. Samples from R o Grande d o Sul
(extreme south of Brazil) showed that only 20% of the root samples were
positive for this organism. Attempts to correlate root piece nitrogenase activity
with enrichment culture activity in wheat have been unsuccessful.
Larson and Neal (1976) described a highly specific association of a facultative
Bacillus sp. with a disomic chromosome substitution line of wheat. The Bacillus
was isolated from a soil where wheat had been growing for 30 years without
nitrogen fertilizer. The rhlzosphere of this wheat line contained also more
nitrate-reducing bacteria and a lower total number of microorganisms. In
monoxenic culture, the bacterium closely associated itself with the root surface.
Abundant numbers of bacterial cells were found on the root surface as well as in
the intercellular spaces between the cortical root cells. Rovira (1965a) reported
establishment of an N,-fixing Bacillus sp. in wheat. Recent fine structure studies
by Foster and Rovira (1976) showed active penetration of wheat cortex cell
walls by bacteria, including Bacillus sp., at the flowering stage.

C. MISCELLANEOUS SYSTEMS

In addition to the N, -Axing systems previously described, which all bear some
relation to agricultural crops, a number of water plants and weeds have been


12

CARLOS A. NEYRA AND J. DOBEREINER

shown to exhibit substantial nitrogenase activity. An understanding of these
systems may help to clarify others of more immediate agricultural importance.
The tropical marine angiosperms K5alassia testudinum, Syringodium Jiliforme,
and Diplanthera wrightii and the temperate Zostera manna fixed an amount of
nitrogen sufficient for growth (Patriquin and Knowles, 1972). Thalassia testudinum Nz furation reached 100 to 500 kg N/ha per year. Conversion factors of
CzH2 reduction estimates as compared with estimates by lsNz incorporation
were close to the theoretical value 3 (2.6 to 4.6). A good correlation of numbers
of anaerobic Nz -furing bacteria and nitrogenase activity in glucose-amended
sediments was obtained, but aerobic N2 fixers were 50 to 300 times more
abundant in the rhizosphere than in the sediment and the authors concluded
that organisms other than Azotobacter and Clostridium are the predominant
nitrogen furers in these systems. Spartina altemiflora a C-4 grass from Canadian
salt marshes (Patriquin, 1976) was shown to have an association similar to that
described for Digifaria (Dobereiner and Day, 1976). Tetrazolium-reducing bacteria, similar to S. lipofemm, were found to be concentrated in the outer and
inner cortex layer of the roots. Nz-fixing aerobic bacteria resembling S. lipoferum were also isolated from Potamogeton Jilifonnis roots grown in Scottish
lakes (Silvester-Bradley, personal communication). Kgh nitrogenase activity has
alwo been observed in excised roots of mangroves (Rhizophora mangle and two
other species; Silver et al., 1976) and in intact soil plant cores of Juncus balticus
(Barber et al., 1976) and several inulin-containing plants (Dahlia pinnata and
others; Jain and Vlassak, 1975; Vlassak and Jain, 1976).
I I I . Bacteriology

Nitrogen-fixing bacteria which have been found in association with grasses are
all capable of fixing nitrogen in soil or culture medium without the plant.
Therefore, they are generally included in the group of “free-living Nz-fixing
bacteria” (Mulder and Brotonegoro, 1974). An excellent up-to-date review on
the entire group has been given by these authors and we will therefore restrict
this chapter to bacteria for which specific associations with tropical grasses have
been shown. Several species of Nz-fixing bacteria have been isolated from the
rhizosphere of temperate plants, e.g., the facultative Enterobacter cloacae, other
Enterobacter spp., and members of the Klebsiella aerobacter group (Raju et al.,
1972; Evans et al., 1972; Barber et al., 1976; Balandreau, 1975) but have not
been shown to be involved in nitrogenase activity on roots or in the rhizosphere.
A microaerophilic N2-fixing bacterium has been isolated from Digitmia sanguinalis with characteristics very similar to s. lipoferum (Barber and Evans,
1976).
The Azotobacter spp. (except A. paspali) are found mainly in the outer
rhizosphere of plants and can be very abundant under warm arid conditions


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