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Advances in agronomy volume 31


ADVANCES IN

AGRONOMY
VOLUME 31


CONTRIBUTORS TO THIS VOLUME
J.-C. FOURNIER

UMESHC. GUFTA

M. G. HALE

L. D. MOORE
K . NEMETH
P. H. NYE
GINETTE
SIMON-SYLVESTRE
R. R. SMITH
N . L. TAYLOR

G. L. TERMAN


ADVANCES IN

AGRONOMY
Prepared irr cooperation with the

AMERICAN
SOCIETY
OF AGRONOMY

VOLUME 31
Edited by

N. C . BRADY

International Rice Research Institute
Manila, Philippines

ADVISORY BOARD

H. J . GORZ,CHAIRMAN
K . M. K I N G R. B. GROSSMANT. M . STARLING
J . B. POWELL J . W . BIGGAR
M. STELLY,
EX

OFFICIO,

ASA Headquarters
1979

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CONTENTS
CONTRIBUTORS
TO VOLUME
31 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
PREFACE
.....................................................

ix
xi

EFFECTS OF PESTICIDES ON THE SOIL MICROFLORA

Ginette Simon-Sylvestre and J . C . Fournier
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I1 . Methodology Applied in the Study of the Effects of Pesticides on
the Soil Microflora . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1

3

111. Effects of Pesticides on the Microorganisms and on the Total

Activity of the Soil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
IV . Effects of Pesticides on the Biological Cycles of the Soil . . . . . .
.............
V . Action on Pathogenic Microorganisms . . . . .
VI . Effect of Pesticides on the Microflora Respo
Degradation . . . . . . . . . .
............................
VII . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

26
35
47
63
80
81

FACTORS AFFECTING ROOT EXUDATION II: 1970-1978

M . G . Hale and L . D . Moore
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I1 . Plant Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
111. Effects of Environmental Factors . . . . . . . . . . . . . . . . . . . . . . . . . .
IV . Foliar Application of Chemicals . . . . . . . . . . . . . . . . . . . . . . . . . . .
V . Biotic Factors Affecting Root Exudation ....................
VI . Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

93
95
103
107
111
120
120

RED CLOVER BREEDING AND GENETICS

N . L . Taylor and R . R . Smith
I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I1 . Taxonomy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
111. Reproduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
IV . Heritability and Gene Action . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
V

125
127
129
131


vi

CONTENTS

V.
VI .
VII .
VIII .

Sources of Genetic Variation . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Alteration of Populations through Selection and Hybridization . .
Use of Selected Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Maintenance of Genetic Stability during Seed Multiplication . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

133
138
144
148
149

THE AVAILABILITY OF NUTRIENTS IN THE SOIL AS DETERMINED BY
ELECTRO-ULTRAFILTRATION (EUF)

K . Nemeth
I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

155

IV . The EUF Values Required for Optimal Plant Nutrition and Their
Calculation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
V . Conclusions for Practical Soil Analysis . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

179
185
186

11. Problems of Conventional Soil Testing Practice . . . . . . . . . . . . . . 156
111. Electro-ultrafiltration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
158

VOLATILIZATION LOSSES OF NITROGEN AS AMMONIA FROM SURFACE-APPLIED
FERTILIZERS. ORGANIC AMENDMENTS. AND CROP RESIDUES

G . L . Terman

I . Introduction ...........................................

189

I1 * Measurement of NH3 Volatilization Losses . . . . . . . . . . . . . . . . . . 191
111. NH, Losses from NHq-N and Urea Fertilizers, Including Moisture

and Temperature Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
NH, Losses from Urea . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
NH3 Losses from Anhydrous NH, and NHIOH . . . . . . . . . . . . . . .
NH, Losses from Organic Amendments and Crop Residues . . . .
NH3 Losses from Flooded Soils . . . . . . . . . . . . . . . . . . . . . . . . . . .
NH, Losses in Forest Fertilization . . . . . . . . . . . . . . . . . . . . . . . . .
IX . NH, Sorption by Soils and Vegetation . . . . . . . . . . . . . . . . . . . . . .
X . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

IV .
V.
VI .
VII .
VIII .

193
206
210
212
214
216
218
219
220

DIFFUSION OF IONS AND UNCHARGED SOLUTES IN SOILS AND SOIL CLAYS

P . H . Nye

I . The Diffusion Process and Its Range .......................
11. The Mechanism of Ion Movement . . . . . . . . . . . . . . . . . . . . . . . . .

225
227


CONTENTS

vii

111. Diffusion of Adsorbed Ions in Soil Clays and Clay-Type Minerals
Diffusion of Ions and Molecules in Soil ....................

IV .
V.
v1.
VII .
VIII .

229
246
Prediction of Diffusion Coefficients in Soil . . . . . . . . . . . . . . . . . . 253
Volatile Solutes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
261
Methods of Measurement of lon Diffusion Coefficients in Soil . . 263
Diffusion in Practice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
266
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
269

BORON NUTRITION OF CROPS

Umesh C . Gupta
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I1 . Boron-Containing Fertilizers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
111. Methods of Determining Boron in Plants and Soils . . . . . . . . . . . .
IV . Role of Boron in Plants. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
V . Factors Affecting Boron Requirement and Uptake in Plants . . . . .
VI . Deficient. Sufficient. and Toxic Levels of Boron in Plants . . . . .
VII . Deficiency and Toxicity Symptoms of Boron in Plants . . . . . . . .
VIII . Summary and Future Research Needs . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

273
275
276
280
283
292
293
302
303

SUBJECT
INDEX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

309


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CONTRIBUTORS
Number\ in parentheses indicate the pages on which the authors’ contributions begin

J.- C . FOURNIER ( I ) , National Institute of Agricultural Research, Department
of Soil Science, Laboratory of Soil Microbiology, DiJon Cedex, France
UMESH C . GUPTA (273), Research Branch, Research Station, P.O. Box 1210,
Agriculture Canada, Charlottetown, Prince Edward Island, Canada C I A 7M8
M. G. HALE (93), Departtnent of Plant Pathology and Physiology, Virginia
Polytechnic Institute and State Universiy, Blacksburg, Virginia 2406 I
L. D . MOORE (93), Departtnent of Plant Pathology and Physiology, Virginia
Polytechnic Institute and State University, Blacksburg. Virginia 24061
K . NEMETH ( 1 5 3 , Biintehof Agricultural Research Station, Biinteweg 8, 3000
Hannover 71, Federal Republic of Germany
P . H. NYE ( 2 2 5 ) , Soil Science Laboratory, Departtnent of Agricultural and
Forest Sciences, University of Oxford, Oxford, England OXI 3PF
GINETTE SIMON-SYLVESTRE ( l ) , National Institute of Agricultural Research, Department of Soil Science, Station of Soil Science, Versailles,
France
R. R. SMITH (125), United States Department of Agriculture, Madison, Wisconsin 53716
N . L. TAYLOR (125), Department oj Agronomy, University of Kentucky,
Lexington, Kentucky 40546
G . L. TERMAN* (189), Soils and Fertilizer Research Branch, National
Fertilizer Development Center, Tennessee Valley Authority, Muscle Shoals,
Alabama 35660

* Deceased
ix


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PREFACE
Agronomy as a profession has a variety of meanings. But in all cases, it has a
connotation that relates in some way to the production of crops. It is only natural
that those scientists interested in crops and their culture find through agronomy a
common interest with those interested in soils, the medium on which most crop
plants grow.
Favorable weather in most parts of the world in 1977 and 1978 has permitted
improved food production technologies to function. As a result, the yields of the
primary food staples have increased and food stocks have risen to more acceptable levels. The technologies which have made possible this state of affairs are
due in part to the work of soil and crop scientists and to their ability to communicate with each other internationally.
Contributions presented in this volume continue to give evidence of the international exchange of scientific information. The authors of the seven articles are
from five countries, and these articles relate to problems of international significance. The first, summarizing research on the effects of pesticides on soil rnicroorganisms, is in response to continued international concern with environmental quality. Likewise, a review of factors affecting the loss of ammonia from soils
has implications for environmental quality but more importantly for the rising
costs of energy required to produce nitrogen fertilizers.
Three articles are concerned with basic soil properties, their measurement, and
factors affecting them. Root exudates is the subject of one, a follow-up of an
earlier A&znces in Agronorny article on this general subject. The other two
articles are concerned with soil chemistry, one with the movement of ions in soils,
and the other with a unique way of measuring the availability of essential elements in the soil.
A review article on boron nutrition brings scientists up to date on our knowledge of this important element. In addition, the genetics of one of the world’s
most important forage crops, red clover, should be of keen interest to crop and
animal scientists alike.
Thanks must be extended to the ten authors who prepared articles for this
volume. They have done a real service to their fellow soil and crop scientists.
N . C. BRADY

xi


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ADVANCES IN AGRONOMY, VOL. 31

EFFECTS OF PESTICIDES ON THE SOIL MICROFLORA
Ginette Simon-Sylvestre* and J . 4 . Fourniert
*I.N.R.A.,Department of Soil Science, Station of Soil Science, Versailles, France
and
tl.N.R.A., Department of Soil Science, Laboratory of Soil Microbiology,
Dijon Cedex, France

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
11. Methodology Applied in the Study of the Effects of Pesticides on the Soil Microflora

111.

IV.

V.

VI.

VII.

...
A. Critical Survey of the Different Methods Used . .
B. Experimental Protocols for the Study of the lnflu
Microflora . . . . . . .
.........................................
C. Conclusions . . . . . . . . , . . . . . . . , . . . . . , . , . . , . . .
Effects of Pesticides on the Microorganisms and on th
.................
A. Counts of the Microorganisms , . . . . . . . . . . . . . . .
B. Total Activity of the Soil . . . . , . . . . . . . . , . . , . , .
C. Conclusions . . . . . , .
..............................................
Effects of Pesticides on t
iological Cycles of the Soil . . . . . . . . . . . . . . . . . . . . . . .
A. The Carbon Cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
B. The Nitrogen Cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
C. The Sulfur Cycle . . . , . . , . . . , , . . . . . , . . , . , . . . . . . . .
D. The Phosphorus Cycle.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
E. The Manganese Cycle . . , . . . . ,
...........................
.. ...
F. Conclusions . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . .
Action on Pathogenic Microorganisms . . . . . , , . . . , . . . . . , . . . . . . . . . . . . . . . . . . . . . .
A. The Influence of Pesticides on Plant Diseases., . . . . , , . . . . . . . . . . . . . . . . . . . . . .
B. Effect of Pesticides on Microorganism Resistance . . . , . . . , , . . , , . . , . . . , . . . . , .
C. Conclusion . .
..................... . . . . . . . . . . . . . . . . . . . . . . . .
Effect of Pesticides on the Microflora Responsible for Pesticide Degradation. . . . . . . .
A. Effect of Pesticides on the Microflora Responsible for Their Degradation
B. Influence of Pesticide Combinations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .. . . . . . . . . .
C. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . .
Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

II
25
26
26
33
35
35
35
37
46
46
47
47
47
48
58
62
63
63
72
80
80
81

I. Introduction

The fertility of soil-that is, its capacity to produce more or less plentiful
crops-depends not only on its physical constitution and its stock of nutrients,
but also on the intensity of the biological processes that take place within it.
Indeed, the activity of the microflora of the soil is generally favorable to
vegetation-for instance, by the fixation of atmospheric nitrogen, the production
I

Copyright 0 1979 by Academic Ress. Inc.
All rights of repmductim in any form reserved.

ISBN 012-wO731-2


2

GINE'ITE SIMON-SYLVESTRE AND J .-C. FOURNIER

of nitrates, sulfates, and carbonic anhydride, the breakdown of animal and plant
remains into compounds more easily available for the plants, and the removal
from the soil of some of the diverse products that may be added to it, such as
pesticides.
The soil appears to be a system biologically in equilibrium, but this equilibrium is a precarious one, and each disturbance of the environment presents the
risk of modifying the activity of the microflora and consequently the soil's
fertility. So the farmer, although he has no direct control over biological
phenomena, may nevertheless try to influence them, either by his choice of
farming methods or by application of various dressings (organic matter, lime,
etc.). The increasing use of pesticides, although intended to protect the crops,
may alter this equilibrium, by direct or indirect action, after short, average, or
long periods of time, depending on whether the product acts quickly or persists
longer in its initial state or in its metabolic forms. The study of these secondary
effects of pesticides on the soil microflora indicates an interest that is all the more
pronounced as the number of products offered to the farmers increases every
year; treatments are often carried out at rather short intervals, they are sometimes
superposed, and the chemical diversity of the products sold is very great. In
1972, the sale of pesticides reserved for agricultural use in France had already
amounted to 1.4 billion francs-2% of the value of the products harvested; in the
United States it reached 5%, and every five years since 1958 the consumption of
pesticides has doubled. As long as the farmer must use these products in order to
maintain the soil and its crops in a sanitary condition, there is an element of risk
for the soil microflora; this risk cannot be disregarded. It seems justifiable to give
our attention to the possible effects of pesticides on the soil microflora. The
subject is broad, and it is difficult to establish its limits.
In addition to the technical difficulties of organizing a practical research program, both in establishing the experimental protocols and in performing the
analyses, numerous other difficulties occur in the interpretation of the data. The
small doses of pesticides used, as well as the often low solubility of these
products, make their distribution in the soil very heterogeneous. The problems
that arise in determining the value of the data derive from these defects in the
conditions of experimentation. Methods of microbiological analysis often lack
precision, and the interpretation of the data then becomes difficult; moreover,
since the relationships between the various groups of microorganisms are not
well known, it is not easy to establish such relationships among all the different
effects recorded separately. The great variety of protocols used by research
workers does not simplify the problem. As in all biological research, the experimental conditions can cause perceptible modifications in the biological processes. A final difficulty must be mentioned: In the association soil-plantmicroflora to which a pesticide is applied, the distinction between the direct


EFFECTS OF PESTICIDES ON SOIL MICROFLORA

3

effects of the product on the microorganisms and the indirect effects that occur
through the plant is sometimes difficult to establish and requires particular attention.
The following bibliographic review presents the data of research carried out
during the last fifteen years, and more particularly during the last five years. The
effects of pesticides on the microflora in general (count and activity) and on the
microorganisms responsible for the major biological processes are considered in
Sections I11 and IV. A few earlier publications have dealt with the subject, but
with the increasing use of pesticides a readjustment and a broader base of information seem indispensable in determining the present state of knowledge. We do
not mean to imply, however, that this review is exhaustive, so numerous are the
papers in this field. In Sections V and VI, an innovation appears with the
discussion of the incidence of pesticides in pathogenic microflora and in decomposing microflora. Until now, these subjects have been studied separately, most
often by specialists, particularly in research concerning the pathogens. In addition, owing to the important part that technique plays in these studies of microflora in the presence of pesticides, we have presented first (Section 11) a critical
review of the various analytical methods and experimental protocols used.
II. Methodology Applied in the Study of the Effects of Pesticides on
the Soil Microflora
A . CRITICAL SURVEY OF THE DIFFERENT METHODS USED

The microorganisms of the soil are extensive. According to Dommergues and
Mangenot (1970), bacteria are the most numerous, varying in density between
lo6 and lo9bacteria per gram of soil. Actinomycetes and fungi are less dense by
factors of 10 and 100, respectively. The biomass of this population is equally
important. For the main groups, Pochon and De Barjac (1958) suggest the
following figures (given in kilograms of living matter per hectare): 500 for
bacteria, 700 for actinomycetes, and 1000-1500 for fungi. In comparison with
these figures, the amounts of pesticide applied seem insignificant, generally
about 1 ppm (= 1 mg/kg of soil), and rarely reaching 10 ppm. Moreover, the
method of application of these products and their solubility (often very low)
make their distribution into or on the soil heterogeneous, which leads to some
difficulties in sampling, not only for chemical but also for microbiological
analyses.
Apart from these general difficulties, the majority of workers studying the
effects of pesticides on the soil microflora adopt the two groups of methods most
often used by soil microbiologists: ( a ) Count methods for determining the total


4

GINETTE SIMON-SYLVESTRE AND J.-C. FOURNIER

microflora and some special microorganisms. This is the direct measurement of
the qualitative change appearing after pesticide treatments. ( b ) Indirect techniques for estimating the activity of the total microflora or of some microorganisms that play an important part in the biological cycles. We should also
mention some particular methods such as the measurement of the biomass in the
soils.

I . Composition of the Soil Microjlora: Counts of the
Microorganisms
Very rarely are counts of the microorganisms made directly with a microscope, in soil suspensions from experiments with pesticide treatments. Such
counts have the advantage of being carried out under ecological conditions, but it
is difficult to distinguish between living microorganisms and dead ones, for
evidently the dead organisms are not counted in the characterization of the
biological level of a soil. The use of fluorescence and of coloration techniques,
however, makes it possible to reduce such errors to a certain extent.
In most cases, the count of the microorganisms is carried out after they have
been grown on media favorable to their development. These are usually synthetic
organic media, either liquid or solid (with gelose) or mineral (silicogel). The
seeding is effected from inoculum suspensions, more or less diluted with water,
into the medium or on the surface. After a period of incubation, the number of
the microorganisms is estimated from the count of the colonies or from the
estimation of the highest dilution that permits the growth of the microorganisms.
In the latter case, probability tables, prepared by MacCrady (1915) and Swaroop
(1951), indicate the number of microorganisms per gram of soil on the basis of
the observed growths. This method of determination may be used on soil samples
taken directly in the field from treated experimental plots, or in trials carried out
in vitro in a laboratory, or in a greenhouse.
We have listed here some of the culture media used most often for counting the
total microflora and the main groups of microorganisms.
For counting the total microflora, the soil aqueous extract seems to be preferred, whether it is liquid or solidified by gelose (Huge, 1970; Kaiser and Reber,
1970; Kaszubiak, 1970; Lozano-Calle, 1970; Catroux and Fournier, 197 1; Karki
et al., 1973; Simon et al., 1973; Simon-Sylvestre, 1974; Oleinikov et al.,
1975). Some workers add yeast and mineral salts to this medium (for example,
Bunt and Rovira, 1955; Voets and Vandamme, 1970; Voets et al., 1974). Other
media used for the determination of the total microflora include that of Kaunat
(1965) (mineral gelose medium with casein, peptone, and glucose) used by
Kaiser and Reber, (1970); and Thoronton’s medium, used by Sharma and Saxena
(1974).


EFFECTS OF PESTICIDES ON SOIL MICROFLORA

5

For fungi, Martin's medium (1950) with rose bengal is often used (Huge,
1970a; Kaszubiak, 1970; Voets and Vandamme, 1970; Tu, 1972; Camper et al.,
1973; Karki et al., 1973; Simon et al., 1973; Sharma and Saxena, 1974;
Simon-Sylvestre, 1974). It contains streptomycin, which impedes the growth of
bacteria without having the drawbacks of the acidified media (Houseworth and
Tweedy, 1973; Focht and Josseph, 1974), in which certain fungi do not grow
because of the excessive acidity, whereas other quickly growing species overgrow all the preparations. However, some fungi (Pythiaceae, in particular) are
sensitive to streptomycin and cannot grow on media containing this antibiotic.
Finally, we should mention the work of Kaiser and Reber (1970), who use a
gelose medium with Maltea Moser and chloramphenicol.
Bacteria alone are often counted on nutritive gelose (Houseworth and Tweedy,
1973; Focht and Josseph, 1974) or on Czapek's gelose medium (Wainwright and
Pugh, 1974). Spore-forming bacteria may be separated from the others by heating the soil for 10 minutes at 90 "C (Voets et al., 19740 or by heating a soil
suspension for 15 minutes at 75°C (Oleinikov et a / . , 1975).
Actinomycetes are generally counted on media containing antibiotics: nystatine and actidione in the work of Camper et al. (1973), and albamycine and
streptomycin in the studies of Focht and Josseph (1974). Sharma and Saxena
(1974), on the other hand, use only a (nitrates + saccharose) medium.
The (gelose + sodium albuminate) medium is often chosen to evaluate bacteria and actinomycetes (Chandra, 1966; Tu, 1972; Camper, 1973). Van Faassen
(1974), however, carries out the separation of bacteria and actinomycetes on a
(soil extract + gelose) medium enriched in glycerol, asparagine, casein, glucose, and cycloheximide as antifungic.
In addition to providing estimates of the totals of the major groups of microorganisms, these methods may be used in research on the identification and evaluation of the percentages of various microorganisms. We must, however, place
restrictions on the use of Martin's medium, which often affects the morphology
of the colonies. This method of identification, which is long and dull, is suitable
only for certain microorganisms, such as the pathogenic ones.
Some criticisms may be expressed concerning methods of dilution counts:
(1) Regardless of the technique used, the number of counted microorganisms
is always much lower than that read by direct numeration, by a factor of 10-1000
on the average for the total numeration; very special organisms, such as
anaerobic bacteria, autotrophic bacteria, and strictly cellulolytic organisms, do
not grow because their feeding requirements are too specific.
(2) In spite of the standardization of the techniques of dilution, inoculation,
and incubation, the precision of the measurements remains relative, for in addition to technical errors, there are the difficulties of sampling due to the unavoid-


6

GINETTE SIMON-SYLVESTRE AND J.-C. FOURNIER

able heterogeneity of the analyzed soils (Meynel and Meynel, 1965; Ricci,
1974). Whatever the circumstances, differences lower than a half power are not
often significant.
(3) No distinction can be made between inactive microorganisms and those
really active in the soil. In fact, the dilution methods often lead to the reduction
or to the suppression of the phenomena of biostasis and synergism, which normally take place in the soil.
(4) The significance of the count of microorganisms sometimes has a restricted but far-reaching effect in the case of organisms with vegetative and
reproductive forms, such as fungi. In fact, the mycelia may be cut into several
pieces, the spores scattered and the count is not precise.
2 . Measurement of Microflora Activity
An interesting method of testing the effects of pesticides on the soil microflora
consists in measuring its biological activity. We may evaluate either the total
activity or the activity of a particular group of microflora.
Measurement of the activity of the soil microflora provides ordinary indexes of
the biological state of the soils and therefore of their fertility. We may determine
the real activity of the microflora or its potential activity-that is, its ability to
adapt to new ecological conditions, to the addition of various substances or
substrates, or to the modification of any of the environmental factors.
There are two types of techniques for determining total activity: ( a ) Classical
techniques, which allow the activity of the microflora proper to be characterized.
The treated soil may then be used just as it is, as the basis of the analysis-the
ecological conditions are then respected-or just for seeding of the media (grains
of soil, suspension dilutions). ( b )Other techniques, which determine the activity
of the microflora by measurement of enzymatic activity.
a . Classical Techniques. Measurement of biological activity. The measurement of biological activity, described by Pochon and Tardieux (1962), consists in evaluating the rapidity with which the soil microorganisms grow in the
more or less synthetic, liquid or solid media used for the counts of the microorganisms, and always in the presence of a specific substrate. The number of living
microorganisms (see the tables of MacCrady) increases as the incubation progresses. The speed of the reaction is thus known, and also its limits.
These simple methods give a first approximation of the activity of the microflora, but most of the criticisms expressed with regard to the count methods by
suspension dilutions apply here also.
These techniques are still used to study most of the major physiological groups
of the soil microflora, for example, the microorganisms active in the nitrogen and
carbon cycles: ammonifying bacteria (with tyrosine), nitrifying bacteria (with
ammonium sulfate and sodium nitrite), denitrifying bacteria (with potassium


EFFECTS OF PESTICIDES ON SOIL MICROFLORA

7

nitrate), proteolytic bacteria (with serum), and amylolytic bacteria (with starch).
They are mentioned in the reports of Huge (1970a), Kaiser and Reber (1970),
Lozano-Calle (1970), Ritter et al. (1970), Catroux and Fournier (1971), and
Simon et al. (1973).
Kinetic tneasurement of degradation of the substrates. All these kinetic
measurements are made directly on the soil itself. In incubation, a substrate is
added to the soil, which is incubated under favorable conditions of temperature
and moisture. The products formed from the added substrate are chemically
determined in the course of time. The activity curve of the microorganisms
studied can be plotted from the data. This method is particularly suitable for
studying ammonification and nitrification. The nitrogenous substrate added varies, depending on the workers. For nitrification, it is most often ammonium
sulfate (Chandra and Bollen, 1961; Balicka and Sobieszczanski, 1969; Bardiya
and Gaur, 1970; Dubey, 1969; Dubey and Rodriguez, 1970; Szernber et al.,
1973; Wainwright and h g h , 1973; Campbell and Mears, 1974; Focht and Josseph, 1974; Horowitz et al., 1974a; Voets et al., 1974; Abueva and Bagaev,
1975). Correa Salazar (1976) uses a variant of this method, in which soil suspensions take the place of soils. Sometimes the following substrates are used:
monoammonium phosphate (Shaw and Robinson, 1960; Bartha et af., 1967),
ammonia (Chandra and Bollen, 1961), urea (Vlassak and Livens, 1975), asparagine (Szember et al., 1973), cotton meal, (Eno, 1962), peanut oil cake
(Akotkar and Deshmukh, 1974), or plant remains (Bliev, 1973).
In the case of organic substrates, the breakdown of the nitrogenous compounds
allows measurement of the activity of the ammonifying as well as the nitrifying
bacteria.
A variant of this technique, percolation, is also used for nitrification studies.
Through a column filled with soil a solution of ammonium sulfate circulates in a
closed circuit; subsequent chemical analyses give information about the biological evolution of this ammonium salt (Jaques, et al., 1959; Torstensson, 1974).
Urea may also be used (Namdeo and Dube, 1973). Technical difficulties make
this method a difficult one.
Finally, measurement of the mineralization of the organic matter in the soil,
under well-controlled conditions, can give good information on biological activity (Drouineau and Lefevre, 1949; Dommergues, 1960). However, not many
papers on this technique mention research on the effects of pesticides.
Measurement of total soil respiration. Measurement of the total soil respiration (oxygen uptake and evolution of carbon dioxide) is often carried out according to Warburg’s technique, directly on the soil, treated or not-just as it is
(endogenous respiration) (Giardina et al., 1970; Tu, 1975; Weeks and Hedrick,
1971), or enriched with nutrients to measure their effects on the metabolism
(glucose: Bartha et al., 1967; Tu, 1972; mannitol: Johnson and Colmer, 1958).
This technique is suitable for short-term measurements and generally for small


8

GINETTE SIMON-SYLVESTRE AND I.-C. FOURNIER

soil samples. For long-term measurements the technique of sweeping the soil
samples with COz-free air is often used. In its passage through the soil, this air
becomes heavy with COBfrom the catabolic activities; it is then possible to trap
and to titrate the COOformed. Some workers use titrated solutions of baryta
(Elkan and Moore, 1960; Agnihotri, 1971; Smith and Weeraratna, 1975) or of
soda (Eno, 1962; Grossbard, 1971; Van Faassen, 1974).
Measurement of oxygen uptake is often carried out with electrolytic respirometers. The general principle of the operation of these apparatuses is the
incubation of soil samples in closed cells and the periodic introduction of oxygen, by electrolysis, to compensate for the soil uptake (MacGarity et a f . , 1958;
MacFayden, 1961).
The measurement of soil respiration is sometimes used in the study of particular groups of microflora. Thus, Anderson and Domsch (1973), using antifungic
or antibiotic substances, were able to distinguish the relative importance of fungi
and of bacteria in soil respiration. However, there seem to be no studies on the
combined use of pesticides.
Measurements of radioactivity. Labeled elements are helpful in making
these determinations. Thus, the radiorespirometric technique represents a neat
and practical method of measuring the rapidity of the mineralization of a radioactive substrate (labeled with 14C). Mayaudon (1973) offers a true index of the
biological activity of the soil, expressed in relation to the pmoles (lo-’’ A41
of 14C02evolved, in the course of the mineralization of dl-glutamic acid.
The tests may be repeated on treated and control plots. Mayaudon (1973)
studied the effects of the different pesticides used alone or in mixtures under
sugar beet. Domsch et a1. (1973), with a similar technique, followed the influence of benomyl on the breakdown of 14C-labeledglucose.
Other radioactive substrates may be used for biological studies, such as those
of Lauss and Danneberg (1975) on the decomposition of plant residues labeled
with 15N, in the presence of pyramin. Sometimes the pesticide itself is labeled,
but its microbial degradation is studied more often than its effects on the soil
microflora (Grossbard, 1970b, 1973).
Measurements in situ. In order to work under more ecological conditions,
some workers make their activity measurements directly in situ, either on the soil
treated with pesticides under field conditions, or on the plants. Respiration of the
soil, according to Dommergues’s technique (1968), may be measured in situ, but
not many papers have been published on pesticide research. The situation is
similar with Billes’ method (197 l), which includes titration by electrolysis of
COPdissolved in a sodium chloride solution colored by phenol-phthalein.
Cellulolysis, in contrast, has attracted several workers: its study involves
periodic checks on the behavior of strips of calico and of cellulose powder, both
buried in the treated soil. The decrease in the strength of the calico strips,
measured mechanically, and the loss of weight of the cellulose powder, enclosed


EFFECTS OF PESTICIDES ON SOIL MICROFLORA

9

in nylon bags, are dependent on the activity of cellulolytic organisms
(Klyuchnikov et a l . , 1964; Grossbard, 1974; Grossbard and Wingfield, 1975;
Grossbard and Long, 1975, oral communication).
The method of “mold soil” also gives direct measurements of activity, but the
data obtained are only qualitative. The soil serves as culture medium; it is
increased with elective substrates chosen according to the research objectives.
Microbial readings are carried out on lamellae, set on the soil. This method is
described in an earlier work on DDT and hexachlorocyclohexane (Drouineau et
a l . , 1947).
In our final example, relating to symbiotic nitrogen fixation, plants are used
for the determination of biological activity. Immediately after the rooting up of
the leguminous plants, the root nodules are counted and examined under the
microscope, and the leghemoglobin is determined. Goss and Shipton (1965)
follow this technique on plants the seeds of which have been disinfected with an
insecticide. Garcia and Jordan (1969) use the same method after spraying herbicides on a leguminous plant field.
b. Measurement of the Enzymatic Activity of Soils. We must also consider
this technique, which is based on soil enzymology. In fact, a large portion of the
enzymes present in the soil is of biological origin, both extra- and intracellular.
Several workers have endeavored to evaluate this fraction after a pesticide treatment, hoping to find a correlation between enzymatic activity and soil fertility.
Thus, some research workers have determined the presence of several enzymes
such as tryptophanase, urease, phytase, invertase, and saccharase (Voets and
Vandamme, 1970; Bliev, 1973; Zubets, 1973; Verstraete and Voets, 1974; Voets
et a l . , 1974; Karanth et a l . , 1975; Lethbridge and Bums, 1976). Others have
limited their studies to one determination only-for example, the determination
of nitrogenase in studies on atmospheric nitrogen fixation (Eisenhardt, 1975;
Neven et a l . , 1975; Peeters et al., 1975, or of dehydrogenase, which gives an
estimate of the soil’s respiratory activity (Ulasevich and Drach, 1971; Karki et
a l . , 1973; Karki and Kaiser, 1974; Van Faassen, 1974).
These enzymatic methods are attractive because of their simplicity and their
good reproducibility, as compared with the classical techniques used in soil
microbiology; yet interpretation of the data requires some prudence, in view of
the fact that at present we have limited knowledge of the enzymes-their origins,
the relationships between them, and the role they play in soil fertility.
3 . Measurement of the Biomass

This kind of measurement, which has not received much attention, provides an
estimate of the effects of pesticides on the soil microflora.
Several methods have been suggested for estimating the microbial biomass,
including measurements of ATP (MacLeod et a ! . , 1969; Lee et a!., 1971;


10

GINETTE SIMON-SYLVESTRE AND J.-C. FOURNIER

Ausmus, 1973) and measurement of muramic acid (Millar and Casida, 1970).
The biomass may also be calculated from the biovolume by multiplying the
number of organisms in a soil sample by the volume of an organism of medium
volume (Russell, 1973).
A particularly interesting method is proposed by Jenkinson and PowIson
(1976). These authors deduce the biomass of the microorganisms from the measurement of the flush of C02 evolved after fumigation of the soil with C H Q .
They note that the CO, evolved comes from the carbon mineralization in the
bodies of the microorganisms killed by the treatment; 50% of this carbon is
mineralized during the ten days after the treatment by the surviving or reinoculated microorganisms recolonizing the medium.
4 . Complementary Techniques

The preceding techniques for measuring activity are, in general, applied in
research concerning the major biological cycles, the carbon and nitrogen cycles.
They constitute the classical techniques, as opposed to less familiar methods,
which are, nevertheless, also interesting, owing to the additional information
they provide, especially with respect to the effects of pesticides on the soil
microflora.
a . Phosphorus and Sulfur Cycles. Research relating specifically to the
phosphorus and sulfur cycles includes studies on the mineralization of organic
phosphorus (Tyunyaeva et a f . , 1974, according to the technique of Menkina);
studies on the oxidation of elementary sulfur (Tu, 1970, 1972, 1973) (laboratory
experiments with incubation of powdered sulfur in the presence of pesticides);
and studies on the mineralization of organic sulfuf (Simon-Sylvestre and
Chabannes, 1975) (monthly determinations of sulfates on soil samples from
experimental plots treated under field conditions).
6 . Pure Cultures. Assays have also been carried out with pure cultures of
soil microorganisms. It is easy to follow the effects of a pesticide on these varied
organisms, but the responses are individual. Furthermore, we cannot assume that
these results would be the same for soil, where the interactions and competition
between the microorganisms are numerous.
This kind of experiment requires pure cultures of different organisms, such as
symbiotic nitrogen-fixing organisms (Brakel, 1963; Makawai and Ghaffar, 1970;
Pajewska, 1972; Mendoza, 1973; Pantera, 1974; Suriawiria, 1974; Eisenhardt,
1975; nonsymbiotic nitrogen-fixing organisms (Pajewska, 1972; Szegi et al.,
1974; Peeters et a l . , 1975); fungi (Lorinczi, 1974); Cellulolytic organisms
(Lembeck and Colmer, 1967; Szegi, 1970); Cellulolytic fungi (Grossbard,
1974); nitrifying bacteria (Garretson and San Clemente, 1968); and bacteria
(Kulinska and Romanov, 1970; Ujevic and Kovacikova, 1975). Moreover, with
this type of experiment, we must determine the action of pesticides on anti-


EFFECTS OF PESTICIDES ON SOIL MICROFLORA

11

biotic production by actinomycetes (Krezel and Leszczynska, 1970) and
bacteria (Kosinkiewicz, 1970; giberellic substance production by bacteria
(Sobieszczanski, 1970); free amino acid secretion by bacteria and actinomycetes
(Balicka et a l . , 1970); and pigment formation (Kulinska and Romanov, 1970).
We should also consider the influence of pesticides on some antagonisms and on
pathogenic microorganisms.
Finally, pure strains of microorganisms may be used as “indicators” of soil
phenomena. Most of the tests that evaluate the toxicity of substances may be
applied to the study of the effect of pesticides on the microflora. Thus, Breazeale
and Camper (1972) studied the action of a line of herbicides on the growth rate of
different microorganisms.
5 . Conclusions

All these methods of biological analysis of soil have flaws, yet they contribute
considerably to the analysis of the major biological phenomena of soil. However,
no numerical values are obtained. All the data are comparative, even for the
techniques that seem to be the most ecological; the conditions are often altered in
order to magnify the phenomena, and the optimum or potential activity is estimated rather than the actual activity. We must add to this criticism the fact that
the use of elective media allows us to study only the main groups of microorganisms; the interrelations between them in the soil are not considered, in order
to simplify the study of the problem. Therefore, it is difficult to interpret the data
obtained in the laboratory in terms of field conditions.
In short, we must emphasize comparative measurements and always work
under the same conditions, with the same techniques, on soil samples freshly
collected or stored at low temperatures. We should also prefer measurements of
activity, which give better information about the behavior of the phenomena, to
counts, which may lead to uncertain results.

B. EXPERIMENTAL PROTOCOLS FOR THE STUDY OF THE
INFLUENCE OF PESTICIDES ON THE SOIL MICROFLORA

This section will present a fairly extensive, but not exhaustive, discussion of
the characteristic conditions of experimentation described in recent studies of the
interactions between pesticides and the soil microflora. The different factors of
variation may be divided into three major groups, based on their association with
the environmental conditions, the pesticide, or the research worker.
Our purpose is to draw attention to the great diversity that exists among the
experimental protocols. As the studies concerned deal with biological processes,


12

GINETTE SIMON-SYLVESTRE AND J.-C. FOURNIER

which are very sensitive to environmental conditions, this diversity is reflected in
the variability of the effects studied.
Herbicides, which stimulated much of the research, have received the most
attention. To simplify the discussion and to give the reader a general overview of
the subject, the results are presented in tables that are very schematic and sometimes incomplete, for research workers often omit from their description of the
experimental conditions those factors affecting the results of pesticide treatment
that have already been proved.
1. Factors Associated with Environmental Conditions

Environmental conditions play an important part in the effect of pesticide
treatments on the soil microflora and therefore deserve particular attention.
a. Noncontrolled Physical Factors. In field studies-that is, in vivo-under
noncontrolled climatic conditions (Tables I and 11), where the scientist can regulate neither the rain nor the temperature, the data obtained the first year in a
specific location for a certain chemical are not necessarily repeated the next year
(Davidson and Clay, 1972; Simon-Sylvestre, 1974). Our limited knowledge of
the different climatic factors makes it difficult to demonstrate the part played by
TABLE I
Field Experiments

Pesticide

Soil type

Crop

Duration of
the trial

Authors

1 year

Mashtakov et a / . (1962)

1 year
16 weeks
2 years

Kozlova el al. (1964)
Kulinska (1967)
Bakalivanov and
Nikolova (1969)
Ulasevich and Drach
(1971)
Husarova (1972)
Kozyrev and Laptev
( 1972)
Karki er d . (1973)
Namdeo and Dube ( I 973)
Szember et ul. (1973)
Deshmukh and
Shrikhande (1974)

Simazine, atrazine,
2.4-D
Simazine, atrazine
Simazine
Simazine between
rows
Atrazine

Sod podzol, peat
podzol
Middle loam
Tchernozem
Tchernozem

Corn

Humus-bearing podzol

Corn

2 years

Alachlor, propachlor
Simazine, prometryn
2.4-D
Sodium chlorate
Dalapon, paraquat
Dinoseb
Simazine, atrazine,
cyanazine, 2,4-D,
TCA
Atrazine

Degraded tchernozem
Clayey illuvial podzol,
slightly loamy
Sandy loam
Grassland sward
Leached podzol
Clayey soil

Corn
Barley, flax,
winter rye

9 years
2 years

Grass
Rotation
Wheat

Loamy sand

Apple trees

Corn, lupine
Corn
Strawberry plants

150 days

3 years
I year

25 years

Voets et ul. (1974)


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