The micro-organisms of the soil

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Title: The micro-organisms of the soil
Author: Station, Members of the biological staff of The Rothamsted Experimental, Russell, Sir E. John
Language: English
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Transcriber’s Notes

Text printed in italics has been transcribed _between underscores_;
underlined text ~between tildes~. ^x and _{x} represent a superscript
and subscript x respectively.

Uppercase letters between square brackets (such as [A]) refer to
footnotes (to be found directly underneath the paragraph or table),
numbers between square brackets (such as [1] or [1_a_] refer to
references at the end of the chapter(s) by the same author.

More Transcriber’s Notes may be found at the end of this text.

_~THE ROTHAMSTED MONOGRAPHS ON
AGRICULTURAL SCIENCE~_

EDITED BY
SIR E. J. RUSSELL, D.Sc. (LOND.), F.R.S.

THE MICRO-ORGANISMS OF THE SOIL

THE ROTHAMSTED MONOGRAPHS ON AGRICULTURAL SCIENCE.

EDITED BY SIR E. JOHN RUSSELL, D.Sc., F.R.S.

During the past ten years there have been marked developments in
knowledge of the relations between the soil and the growing plant. The
subject involves physical, biological, and chemical considerations, and
its ramifications are now so wide that they cannot be satisfactorily
dealt with in detail in any one book. These monographs collectively
cover the whole ground. In “Soil Conditions and Plant Growth” the
general outlines are presented: in the monographs the various divisions
are fully and critically dealt with by the Heads of the Departments
concerned at Rothamsted. A homogeneous treatment is thus secured that
will, it is hoped, much facilitate the use of the series.

SOIL CONDITIONS AND PLANT GROWTH, Fourth Edition. By SIR E. JOHN
RUSSELL, F.R.S. 16_s._ net.

The following volumes are in preparation:--

MANURING OF GRASS-LANDS
FOR HAY By WINIFRED E. BRENCHLEY, D.Sc., F.Z.S.

THE MICRO-ORGANISMS OF THE
SOIL By Sir E. JOHN RUSSELL, F.R.S., and
Members of the Biological Staff of the
Rothamsted Experimental Station.

SOIL PHYSICS By B. A. KEEN, B.Sc.

SOIL PROTOZOA By D. W. CUTLER, M.A., and L. M. CRUMP,
M.Sc.

SOIL BACTERIA By H. G. THORNTON, M.A.

SOIL FUNGI AND ALGÆ By W. B. BRIERLEY, S. T. JEWSON, B.Sc.,
and B. M. ROACH (Bristol), D.Sc.

CHEMICAL CHANGES IN
THE SOIL By H. J. PAGE, B.Sc.

LONGMANS, GREEN AND CO.,
LONDON, NEW YORK, TORONTO, BOMBAY, CALCUTTA, AND MADRAS.

THE MICRO-ORGANISMS
OF THE SOIL

SIR E. JOHN RUSSELL, F.R.S.

AND

MEMBERS OF THE BIOLOGICAL STAFF OF THE
ROTHAMSTED EXPERIMENTAL STATION

_WITH DIAGRAMS_

LONGMANS, GREEN AND CO.
39 PATERNOSTER ROW, LONDON, E.C. 4
NEW YORK, TORONTO
BOMBAY, CALCUTTA and MADRAS
1923

_Made in Great Britain_

INTRODUCTION.

The purpose of this volume is to give the broad outlines of our present
knowledge of the relationships of the population of living organisms
in the soil to one another and to the surface vegetation. It is shown
that there is a close relationship with vegetation, the soil population
being dependent almost entirely on the growing plant for energy
material, while the plant is equally dependent on the activities of
the soil population for removing the residues of previous generations
of plants and for the continued production in the soil of simple
materials, such as nitrates, which are necessary to its growth. It is
also shown, however, that the soil population takes toll of the plant
nutrients and that some of its members may directly injure the growing
plant.

The soil population is so complex that it manifestly cannot be dealt
with as a whole in any detail by any one person, and at the same
time it plays so important a part in the soil economy that it must
be seriously studied. Team work therefore becomes indispensable, and
fortunately this has been rendered possible at Rothamsted.

Each group of organisms is here dealt with by the person primarily
responsible for that particular section of the work. The plan of the
book has been carefully discussed by all the authors, and the subject
matter has already been presented in a course of lectures given at
University College, London, under the auspices of the Botanical
Board of Studies of the London University. The interest shown in
these lectures leads us to hope that the subject may appeal to a
wider public, and above all to some of the younger investigators in
biological science. They will find it bristling with big scientific
problems, and those who pursue it have the satisfaction, which
increases as the years pass by, of knowing that their work is not only
of interest to themselves, but of great importance in ministering to
the intellectual and material needs of the whole community.

CONTENTS.

CHAP. PAGE

I. DEVELOPMENT OF THE IDEA OF A SOIL POPULATION 1
Sir E. JOHN RUSSELL, F.R.S., Director.

II. OCCURRENCE OF BACTERIA IN SOIL--ACTIVITIES CONNECTED WITH
THE ACQUIREMENT OF ENERGY 20
H. G. THORNTON, B.A., Head of the Department of
Bacteriology.

III. CONDITIONS AFFECTING BACTERIAL ACTIVITIES IN THE
SOIL--ACTIVITIES CONNECTED WITH THE INTAKE OF PROTEIN
BUILDING MATERIALS 39
H. G. THORNTON, B.A., Head of the Department of
Bacteriology.

IV. PROTOZOA OF THE SOIL, I. 66
D. W. CUTLER, M.A., Head of the Department of
Protozoology.

V. PROTOZOA OF THE SOIL, II. 77
D. W. CUTLER, M.A., Head of the Department of
Protozoology.

VI. SOIL ALGÆ 99
B. MURIEL BRISTOL, D.Sc., Algologist.

VII. SOIL FUNGI--THE OCCURRENCE OF FUNGI IN THE SOIL 118
W. B. BRIERLEY, D.Sc., Head of the Department of
Mycology.

VIII. SOIL FUNGI--THE LIFE OF FUNGI IN THE SOIL 131
W. B. BRIERLEY, D.Sc., Head of the Department of
Mycology.

IX. THE INVERTEBRATE FAUNA OF THE SOIL (OTHER THAN PROTOZOA) 147
A. D. IMMS, D.Sc., Head of the Department of Entomology.

X. THE CHEMICAL ACTIVITIES OF THE SOIL POPULATION AND THEIR
RELATION TO THE GROWING PLANT 164
Sir E. JOHN RUSSELL, F.R.S., Director.

INDEX 181

CHAPTER I.

THE DEVELOPMENT OF THE IDEA OF A SOIL POPULATION.

From the earliest times agriculturists have been familiar with the idea
that decomposition of vegetable and animal matter takes place in the
soil, and that the process is intimately connected with soil fertility.

By the middle of the nineteenth century three different ways were known
in which the decomposition occurred. One had been since early times
specially associated with soil fertility, in that it gave rise to
humus, the black sticky substance in farmyard manure or in soil--which
was supposed up to 1840 to be the special food of plants. No good
account of the process or of the conditions in which it occurred is,
however, given by the older writers.

A second resulted in the formation of nitrates. This process became
known as nitrification: it was described by Georgius Agricola
(1494-1555) in his book “De Re Metallica,” and it was of great
importance in the seventeenth and eighteenth centuries, because it
was used for the manufacture of gunpowder in the great wars of that
period. The conditions for the making of successful nitre beds were so
thoroughly investigated that little fresh knowledge was added to that
of 1770[A] until quite recently. This process, however, was not usually
associated with soil fertility, although both Glauber (1656) and Mayow
(1674) had insisted on the connection.

[A] See the remarkable collection of papers entitled
“Instructions sur l’établissement des nitrières,” publié par les
Régisseurs-généraux des Poudres et Salpêtre. Paris, 1777.

A third type of decomposition was brought into prominence by Liebig
in 1840.[7][B] Reviewing the decomposition of organic matter in the
light of the newer chemistry, he concluded that the process was a
slow chemical oxidation, to which he gave the name “Eremacausis.”
He recognised that humus was formed, but he regarded it only as an
intermediate product, and emphatically denied its importance in
soil fertility. The true fertility agents, in his view, were the
final products--CO₂, potassium and other alkaline salts, phosphates,
silicates, etc. He went on to argue brilliantly that instead of
applying farmyard or similar manures to the soil it was altogether
quicker and better to apply these mineral compounds obtained from
other sources than to wait for the slow process of liberation as the
result of decomposition. For some reason, difficult to understand, he
overlooked nitrification and the part that nitrates might play in soil
fertility. Lawes and Gilbert[6] were much attracted by this new idea;
they showed that it was incomplete because it took no account of the
necessity for supplying nitrogen compounds to the crop. When ammonium
salts were added to Liebig’s ash constituents the resulting mixture had
almost as good a fertilising effect as farmyard manure. Lawes at once
saw the enormous practical importance of this discovery, and set up
a factory for the manufacture of artificial fertilisers. He did not,
however, follow it up more closely on the scientific side.

[B] The numbers refer to the short bibliography on p. 18.

Both Lawes and Gilbert were in constant touch with the idea of
decomposition in the soil, and they attached so much importance to
nitrogen compounds in plant nutrition that it is not easy to understand
how they missed the connection with nitrification. But they did so, and
like other English and German workers of the day, considered that plant
roots assimilated their nitrogen as ammonia. For the first ten years of
the history of Rothamsted only few experiments with nitrates were made,
and not till thirty-five years had elapsed were they systematically
studied.

It was by Boussingault[2] and in France that the connection between
nitrification and soil fertility was first recognised. The news came
to England, but it was not accepted, although Way, one of the most
brilliant agricultural chemists of his time, showed that nitrates
were formed in soils to which nitrogenous fertilisers were added, and
that they were comparable in their fertiliser effects with ammonium
salts.[12] “The French chemists,” he wrote in 1856, “are going further,
several of them now advocating the view that it is in the form of
nitric acid that plants make use of compounds of nitrogen. With this
view I do not at present coincide, and it is sufficient here to admit
that nitric acid in the form of nitrates has at least a very high value
as manure.” Indeed, Kuhlmann went so far as to argue that the nitrates
found in the soil were there reduced to ammonia before assimilation
by plants could take place. The water-culture work of the plant
physiologists of the ’sixties finally showed the correctness of the
French view.

Even when the importance of nitrification was realised its mechanism
was not understood: some thought it was chemical, some physical.
Again the explanation came from France. Pasteur in 1862 had expressed
the view that nitrification would probably be a biological action,
since purely chemical oxidation of organic matter was of very limited
occurrence. “Pénétrés de ces idées,” as Schloesing tells us, he and
Müntz in a memorable investigation cleared up the whole problem, and
in 1877 opened the way to a most fruitful field of research.[10] The
formal description is given in his papers in the “Comptes Rendus,”
but a more lively account is given in his lectures before the _École
d’application des Manufacteurs de l’état_, which, though not printed,
were collected and issued in script by his distinguished son, and a
copy of this work is among the treasures of the Rothamsted Library.

He had been asked to study the purification of sewage, and he and Müntz
showed that it was bound up with nitrification. The process was slow in
starting, then it proceeded rapidly. Why, they asked, was the delay?
There should be none if the process were physical or chemical, and the
fact that it occurred strongly suggested biological action. The process
was stopped by chloroform vapour, but could be restarted after the
removal of the vapour by the addition of a little fresh soil.

The importance of this work in connection with soil fertility
was immediately realised by Warington, who had recently come to
Rothamsted.[11] He quickly confirmed the result, and made the valuable
discovery that two stages were involved--the conversion of ammonia to
a nitrite by one organism, and of the nitrite to nitrate by another.
He made long and persistent attempts to isolate the organisms from the
soil, using the best technique of his time, but though he found many
bacteria none of them could nitrify ammonium salts; yet the soil did
it easily. For years he continued his efforts to find the nitrifying
organism, but always failed. His health was not good, his life at
Rothamsted was not happy owing to disagreements with Gilbert, and
although his other research work was succeeding, this investigation
on which he had set his heart was not coming out; bacterial technique
was not yet sufficiently far advanced. Ten bitter, disappointing years
passed, and the crown of disappointment came when Winogradsky, a young
bacteriologist in Paris, changed the technique and succeeded at once in
isolating both the nitrite and the nitrate-forming organisms.[13]

The numerous bacteria found by Warington in the soil suggested the
presence of a soil population, and this idea was greatly strengthened
by another line of investigation which was being followed up in France.
Boussingault had shown that soils absorb oxygen and give out carbon
dioxide; Schloesing extended this discovery, as also did Wollny. It
was concluded that oxidation was the result of the activities of the
soil organisms in decomposing the organic matter of the soil, and thus
preparing the way for the nitrifying organisms.

A third important function of soil bacteria was revealed by
Berthelot.[1] It was known that considerable loss of nitrogen from the
soil took place as the result of the conversion of nitrogen compounds
into nitrates, which were subsequently washed out in the drainage
water. It followed inevitably that the stock of nitrogen compounds in
the soil must long ago have become exhausted had there been no addition
of nitrogen compounds to the soil. Berthelot argued that there must be
fixation of atmospheric nitrogen, and, following the prevailing trend
of thought in France, he attributed it to bacteria. He confirmed the
anticipation by exposing soil to air in such conditions that dust,
rain, etc., were excluded, and he found an increase in the percentage
of nitrogen.

Looking back over the work, it is difficult to understand the result.
The fixation of nitrogen is a process that absorbs energy, and
should have necessitated some source of energy, which apparently was
not supplied. But in spite of this drawback the investigation was
immediately fruitful in that it gave the key to another problem which
had long puzzled agriculturists.

It had long been known that the growth of leguminous crops, unlike that
of others, enriched the ground,[C] and Lawes and Gilbert had shown
that this was due to an increase of soil nitrogen. But no explanation
could be found till Hellriegel and Wilfarth solved the problem.[4] In
studying the nitrogen nutrition of gramineous and leguminous crops,
they discovered that the gramineous plants died in absence of nitrate,
and in its presence made growth which increased regularly with nitrate
supply; while leguminous plants sometimes died and sometimes flourished
in absence of nitrate, and behaved equally erratically with increasing
nitrate supply. When the plants flourished nodules were invariably
present on the roots, but not otherwise. They concluded, therefore,
that the nitrogen nutrition of leguminous plants differed from that
of the gramineæ, and depended on some factor which sometimes came
into their experiments and sometimes did not, and, in any case, was
associated with the nodule. Knowing that the nodules on the roots of
leguminous plants contained bacteria-like bodies, and remembering
Berthelot’s results, they explored the possibility of bacterial
fixation. They sterilised the sand and found that peas invariably
failed to develop nodules and died, but after adding a little garden
soil nodules were found and vigorous growth was obtained.

[C] “Of the leguminous plants the bean best reinvigorates the ground
... because the plant is of loose growth and rots easily, wherefore
the people of Macedonia and Thessaly turn over the ground when it
is in flower” (i.e. dig it into the ground if the soil is poor).
Theophrastus, “Enquiry into Plants,” bk. viii. 2, and bk. ix. I. This
book is of profound interest to agriculturists and botanists. An
excellent translation by Sir Arthur Hort is now available. (Loeb’s
Classical Library.)

Chemical analysis showed considerable fixation of gaseous nitrogen,
which Hellriegel associated with the nodule organism. This has proved
to be correct, and the fixation of nitrogen by bacteria is now a
well-recognised process, the conditions of which are being thoroughly
worked out. Two types of organisms are known--those associated with
leguminous plants, and those living in a free and independent state in
the soil. Of the latter the Clostridium, isolated by Winogradsky, is
anaerobic, and the Azotobacter of Beijerinck is aerobic. The essential
conditions are that a source of energy must be supplied--usually
given as sugar--that the medium must not be acid, and that sufficient
phosphate must be present.

All this brilliant work had been accomplished in the short space of the
ten years 1880 to 1890. The inspiration had in each instance come from
France, and is traceable direct to Pasteur, although coming long after
his own work on bacteriology. It is impossible for us now to realise
the thrill of wonder and astonishment with which students, teachers,
and writers of those days learned that the nutrition of plants, and
therefore the growth of crops and the feeding of themselves, was
largely the result of the activity of bacteria in the dark recesses of
the soil. It is not surprising that the ideas were pushed somewhat too
far, that the soil population was regarded as solely bacterial, and
that important chemical and physical changes were sometimes overlooked.

Gradually there came the inevitable reaction and a somewhat changed
outlook. Continued examination showed the presence in soil of almost
every kind of bacteria for which search was made. Some of them were
almost certainly in the resting condition as spores, and the new
generation of workers had an uneasy feeling that the case for the
overwhelming importance of bacteria in the economy of the soil was
not too well founded. It was shown that the decomposition of nitrogen
compounds to form ammonia would take place without micro-organisms if,
as presumably would happen, the plant enzymes continued to act after
they got into the soil. Even the oxidation of ammonia to nitrate--the
great stronghold of the biological school--was accomplished by chemical
agents. The fixation of nitrogen in soil conditions was beyond the
power of chemists to achieve, and here it was universally agreed that
bacteria were the active agents. And finally, chemists were themselves
bringing into prominence a set of bodies--the colloids--whose
remarkable properties seemed indefinitely expansible, and were in
addition sufficiently incomprehensible to the ordinary student to
attain much of the magnificence of the unknown.

All the time, however, a faithful body of workers was busy exploring
the ground already won, improving the technique, making counts of the
numbers of bacteria in the soil, and trying to measure the amount of
bacterial activity. Much of the value of this work was limited by the
circumstance that the bacteria were regarded as more or less constant
in numbers and activities, so that a single determination was supposed
to characterise the position in a given soil.

This was the condition of the subject when it was seriously taken up at
Rothamsted. Before turning to agriculture, the writer had been studying
the mechanism of certain slow chemical oxidations, and one of his first
experiments in agriculture was to examine the phenomena of oxidation
in soil. The results accorded with the biological explanation of
Schloesing: when the soil was completely sterilised oxidation almost
ceased. But the striking discovery was made, as the result of an
accident to an autoclave, that partial sterilisation increased the
rate of oxidation, and therefore presumably the bacterial activity.
This remarkable phenomenon had, however, already been observed, and
it had been shown that both bacterial numbers and soil fertility
were increased thereby. A full investigation was started in 1907 by
Dr. Hutchinson and the writer.[9] From the outset the phenomena were
recognised as dynamic and not static, and the rates of change were
always determined: thus the bacterial numbers, the nitrate and ammonia
present were estimated after the several periods. Close study of the
curves showed that the chemical and bacterial changes were sufficiently
alike to justify the view that bacteria were in the main the causes
of the production of ammonia and of nitrate; although non-biological
chemical action was not excluded, there was no evidence that it played
any great part. Thus the importance of micro-organisms in the soil was
demonstrated.

The factor causing the increased bacterial numbers after partial
sterilisation was studied by finding out what agents would, and what
would not, allow the numbers to increase, e.g. it was found that the
bacterial increases became possible when soil had been heated at 56°
C., but not at 40° C. Again, it was shown that the high numbers in
partially sterilised soils rose for a time even higher if a little
fresh untreated soil were incorporated into the partially sterilised
soil, but afterwards they fell considerably. Putting all the results
together, it appeared that some biological cause was at work depressing
the numbers of bacteria in normal soils, but not--or not so much--in
the partially sterilised soils. Studied in detail, the data suggested
protozoa as the agent keeping down bacterial numbers, and they were
found in the untreated, but not in the treated, soils. The hypothesis
was therefore put forward that bacteria are not the only members of the
soil population, but that protozoa are also present keeping them in
check, and therefore adversely affecting the production of plant food.

This conclusion aroused considerable controversy. It was maintained
that protozoa were not normal inhabitants of the soil, but only
occasional visitants, and, in any case, they were only there as cysts;
the soil conditions, it was urged, were not suitable to large organisms
like protozoa. The objection was not to be treated lightly, but, on
the other hand, the experiments seemed quite sound. As neither Dr.
Hutchinson nor the writer were protozoologists, Dr. T. Goodey and
(after he left) Mr. Kenneth R. Lewin were invited to try and find
out, quite independently of the partial sterilisation investigation,
whether protozoa are normal inhabitants of the soil, and if so, whether
they are in a trophic condition, and what is their mode of life and
their relation to soil bacteria. Had it turned out that protozoa had
nothing to do with the matter, search would have been made for some
other organism. Goodey showed that the ciliates were not particularly
important; Lewin soon demonstrated the existence of trophic amœbæ and
flagellates. Unfortunately he was killed in the war before he had got
far with the work. After the Armistice, Mr. Cutler accepted charge of
the work: he will himself relate in Chapters IV. and V. what he has
done.

At first sight it might be thought comparatively easy to settle a
question of this kind by examining soil under a microscope or by
sterilising it and introducing successively bacteria and known types
of protozoa. Unfortunately neither method is simple in practice. It
is impossible to look into the soil with a microscope, and methods of
teasing-out small pieces of soil on a slide under the high, or even the
low power, give no information, because the particles of soil have the
remarkable power of attracting and firmly retaining protozoa, and no
doubt bacteria as well; indeed, for protozoa (which have been the more
fully investigated) there seems to be something not unlike a saturation
capacity (see Fig. 9, p. 78). Further, complete sterilisation of soil
cannot be effected without at the same time altering its chemical and
physical properties, and changing it as a habitat for micro-organisms.
Cutler has, however, overcome the difficulties and shown that the
introduction of protozoa into soils sterilised and then reinfected with
bacteria considerably reduces the numbers of these organisms.

The method adopted, therefore, is to take a census of population and
of production. Counting methods are elaborated, and estimates as
accurate as possible are made of the numbers of the various organisms
in a natural field soil at stated intervals. Simultaneously, wherever
possible some measure is taken of the work done. The details of the
census are finally arranged in consultation with the Statistical
Department, to ensure that the data shall possess adequate statistical
value. From the results it is possible to adduce information of great
value as to the life of the population, the influence of external
conditions, etc.

The most important investigation of this kind carried out at Rothamsted
was organised by Mr. Cutler.[3] A team of six workers was assembled,
and for 365 days without a break they counted every day the ciliates,
the amœbæ, the flagellates, and the bacteria in a plot of arable
ground, distinguishing no less than seventeen different kinds of
protozoa. The conclusions arrived at were carefully tested by the
Statistical Department.

Of the protozoa the flagellates were found to be the most numerous,
the amœbæ came next, and the ciliates were by far the fewest. The
numbers of each organism varied from day to day in a way that
showed conclusively the essentially trophic nature of the protozoan
population. The numbers of amœbæ--especially _Dimastigamœba_ and of
a species called α--were sharply related to the numbers of bacteria:
when the amœbae were numerous the bacteria were few, and vice versa.
Detailed examination showed that the amœbæ were probably the cause
of the fluctuations in the bacterial numbers, but Mr. Cutler has not
yet been able to find why the amœbæ fluctuated; it does not appear
that temperature, moisture content, air supply or food supply were
determining causes. The flagellates and ciliates also showed large
fluctuations, amounting in one case--_Oicomonas_--to a definite
periodicity, apparently, however, not related to bacterial numbers, or,
so far as can be seen, to external conditions of moisture, temperature
and food supply, and showing no agreement with the fluctuations of the
amœbæ. However, one cannot be certain that lack of agreement between
curves expressing protozoan numbers and physical factors implies
absence of causal relationships: the observations (though the best that
can yet be made) are admittedly not complete. If we saw only the end of
the bough of a tree, and could see no connection with a trunk, we might
have much difficulty in finding relationships between its motion and
the wind; whatever the direction of the wind it would move backwards
and forwards in much the same way, and even when the wind was blowing
along the plane of its motion it would just as often move against the
wind as with it.

Meanwhile evidence was obtained that the twenty-four hour interval
adopted by the protozoological staff was too long for bacteria, and
accordingly the Bacteriological Department, under Mr. Thornton, refined
the method still further. Bacterial counts were made every two hours,
day and night, for several periods of sixty or eighty hours without
a break. The shape of the curve suggests that two hours is probably
close enough, and for the present counts at shorter intervals are not
contemplated. But there is at least one maximum and one minimum in the
day, although the bacterial day does not apparently correspond with
ours, nor can any relationship be traced with the diurnal temperature
curve.

The nitrate content of the soil was simultaneously determined by Mr.
Page and found to vary from hour to hour, but the variations did not
sharply correspond with the bacterial numbers; this, however, would not
necessarily be expected. The production of nitrate involves various
stages, and any lag would throw the nitrate and bacterial curves out
of agreement. There is a suggestion of a lag, but more counts are
necessary before it can be regarded as established.

Examination of these and other nitrate curves obtained at Rothamsted
has brought out another remarkable phenomenon. No crop is growing on
these plots, and no rain fell during the eighty hours, yet nitrate
is disappearing for a considerable part of the time. Where is it
going to? At present the simplest explanation seems to be that it is
taken up by micro-organisms. A similar conclusion had to be drawn
from a study of the nitrogen exhaustion of the soil. The whole of the
nitrate theoretically obtainable from the organic matter of the soil
is not obtained in the course of hours or even days; in one of our
experiments at Rothamsted nitrification is still going on, and is far
from complete, even after a lapse of fifty-three years. The explanation
at present offered is that part of the nitrate is constantly being
absorbed by micro-organisms and regenerated later on.

Now what organisms could be supposed to absorb nitrates from the soil?
Certain bacteria and fungi are known to utilise nitrates, and one
naturally thinks of algæ as possible agents also. Dr. Muriel Bristol
was therefore invited to study the algæ of the soil. Her account is
given in Chapter VI. She has found them not only on the surface, but
scattered throughout the body of the soil, even in the darkness of 4
inches, 5 inches, or 6 inches depth, where no light can ever penetrate,
and where photosynthesis as we understand it could not possibly take
place. Some modification in their mode of life is clearly necessary,
and it may well happen that they are living saprophytically. Dr.
Bristol has not yet, however, been able to count the algæ in the soil
with any certainty, although she has made some estimates of the numbers.

The quantitative work on the soil population indicates other
possibilities which are being investigated. There is not only a daily
fluctuation in the numbers, but so far as measurements have gone,
a seasonal one also. There seems to be some considerable uplift in
numbers of bacteria, protozoa, and possibly algæ and fungi in the
spring-time, followed by a fall in summer, a rise in autumn, and a
fall again in winter. At present we are unable to account for the
phenomenon, nor can we be sure that it is general until many more data
are accumulated.

In the cases of the protozoa and the algæ, there was a definite reason
for seeking them in the soil.

Another section of the population, the fungi, was simply found, and
at present we have only limited views as to their function. The older
workers considered that they predominated in acid soils, while bacteria
predominated in neutral soils. Present-day workers have shown that
fungi, including actinomycetes, are normal inhabitants of all soils.
The attempts at quantitative estimations are seriously complicated
by the fact that during the manipulations a single piece of mycelium
may break into fragments, each of which would count as one, while a
single cluster of spores might be counted as thousands. Little progress
has therefore been made on the quantitative lines which have been so
fruitful with protozoa. Dr. Brierley gives, in Chapters VII. and VIII.,
a critical account of the work done on fungi.

In addition to the organisms already considered there are others of
larger size. The nematodes are almost visible to the unaided eye,
most of them are free living and probably help in the disintegration
of plant residues, though a few are parasitic on living plants and do
much injury to clover, oats, and less frequently to onions, bulbs, and
potatoes. Further, there are insects, myriapods and others, the effects
of which in the soil are not fully known. Special importance attaches
to the earthworms, not only because they are the largest in size and
in aggregate weight of the soil population, but because of the great
part they play in aerating the soil, gradually turning it over and
bringing about an intimate admixture with dead plant residues, as first
demonstrated by Darwin. Earthworms are the great distributors of energy
material to the microscopic population. Systematic quantitative work
on these larger forms is only of recent date, and Dr. Imms, in Chapter
IX., discusses our present knowledge.

TABLE I.

SOIL POPULATION, ROTHAMSTED, 1922.

(The figures for algæ and fungi are first approximations only, and have
considerably less value than those for bacteria and protozoa.)

+----------------------+-----------+--------------------------------+
| | |Approximate Weight per Acre of--|
| | +---------+----------+-----------+
| | Numbers | Living |Dry Matter| Nitrogen |
| | per Gram | Organ- |in Organ- | in Organ- |
| | of Soil. | isms. | isms. | isms. |
+----------------------+-----------+---------+----------+-----------+
|_Bacteria_-- | | lb. | lb. | lb. |
| High level |45,000,000 | 50} | 2 | 0·2 |
| Low level |22,500,000 | 25} | | |
|_Protozoa_-- | | | | |
| _Ciliates_-- | | | | |
| High level | 1,000 | -- | -- | -- |
| Low level | 100 | -- | -- | -- |
| _Amœbæ_-- | | | | |
| High level | 280,000 | 320} | 12 | 1·2 |
| Low level | 150,000 | 170} | | |
| _Flagellates_-- | | | | |
| High level | 770,000 | 190} | 7 | 0·7 |
| Low level | 350,000 | 85} | | |
| _Algæ_ | | | | |
| (not blue-green)| [100,000]| 125 | 6 | 0·6 |
| Blue-green | Not known.| | Say 6 | Say 0·6 |
| _Fungi_-- | | | | |
| High level |[1,500,000]| 1700} | 60 | 6·0 |
| Low level | [700,000]| 800} | | |
| | | +----------+-----------+
| | | | 93 | 9·3 |
| | | |= 4 parts nitrogen per|
| | | |1,000,000 of soil. |
+----------------------+-----------+---------+----------------------+

+--------------------------------------
| LARGER ORGANISMS.
+-----------------+-------------------+
| | |
| | +
| | Numbers |
| | per Acre.[D] |
| +---------+---------+
| | | Unma- |
| | Manured.| nured. |
+-----------------+---------+---------+
|_Oligochaeta_ | | |
| (_Limicolae_)--| | |
| Nematoda, etc. |3,609,000| 794,000|
| Myriapoda |1,781,000| 879,000|
| Insects |7,727,000|2,475,000|
| Earthworms |1,010,000| 458,000|
+-----------------+---------+---------+
|
+--------------------------------------

+-----------------------------------------------------------+
| LARGER ORGANISMS. |
+-----------------+-----------------------------------------+
| | Approximate Weight per Acre of-- |
| +-------------+-------------+-------------+
| | Living | Dry Matter | Nitrogen in |
| | Organisms. |in Organisms.| Organisms. |
| +------+------+------+------+------+------+
| | Ma- | Unma-| Ma- | Unma-| Ma- |Unma- |
| |nured.|nured.|nured.|nured.|nured.|nured.|
+-----------------+------+------+------+------+------+------+
|_Oligochaeta_ | | | | | | |
| (_Limicolae_)--| lb. | lb. | lb. | lb. | lb. | lb. |
| Nematoda, etc. | 9 | 2 | 3 | 1 | -- | -- |
| Myriapoda | 203 | 99 | 85 | 42 | 4 | 2 |
| Insects | 34 | 16 | 14 | 6 | 1 | 1 |
| Earthworms | 472 | 217 | 108 | 50 | 10 | 5 |
+-----------------+------+------+------+------+------+------+
| Total | 210 | 99 | 15 | 9 |
+-------------------------------+------+------+------+------+

Total organic matter (dry weight) in this soil = 126,000 lb. per acre.

Total nitrogen = 5700 lb. per acre. (1 lb. nitrogen per acre = 0·4
parts per 1,000,000 of soil.)

[D] To a depth of 9 inches. The weight of soil is approximately
1,000,000 kilos.

Are there any other members of the soil population that are of
importance? As already shown, the method of investigating the soil
population in use at Rothamsted is to find by chemical methods the
changes going on in the soil; to find by biological methods what
organisms are capable of bringing about these changes; and then to
complete the chain of evidence by tracing the relationships between
the numbers or activities of these organisms and the amount of change
produced. The list as we know it to-day is given in Table I.

The method, however, does not indicate whether the account is fairly
complete, or whether there are other organisms to be found. We might,
of course, trust to empirical hunting for organisms, or to chance
discoveries such as led Goodey to find the mysterious Proteomyxan
Rhizopods, which cannot yet be cultured with certainty, so that they
are rarely found by soil workers. It is possible that there are many
such organisms, and it is even conceivable that these unknown forms far
outnumber the known. The defect of the present method is that it always
leaves us in doubt as to the completeness of the list, and so we may
have to devise another.

Reverting to Table I., it obviously serves no purpose to add the
numbers of all the organisms together. We can add up the weights of
living organisms, of their dry matter or nitrogen, so as to form some
idea of the proportion of living to non-living organic matter, and this
helps us to visualise the different groups and place them according
to their respective masses. But a much better basis for comparing the
activities of the different groups would be afforded by the respective
amounts of energy they transform, if these could be determined. It is
proposed to attempt such measurements at Rothamsted. The results when
added would give the sum of the energy changes effected by the soil
population as we know it: the figure could be compared with the total
energy change in the soil itself as determined in a calorimeter. If
the two figures are of the same order of magnitude, we shall know that
our list is fairly well complete; if they are widely different, search
must be made for the missing energy transformers. There are, of course,
serious experimental difficulties to be overcome, but we believe the
energy relationships will afford the best basis for further work on the
soil population.

Finally, it is necessary to refer to the physical conditions obtaining
in the soil. These make it a much better habitat for organisms than one
might expect. At first sight one thinks of the soil as a purely mineral
mass. This view is entirely incorrect. Soil contains a considerable
amount of plant residues, rich in energy, and of air and water. The
usual method of stating the composition of the soil is by weight, but
this is misleading to the biologist because the mineral matter has a
density some two and a half times that of water and three times that
of the organic matter. For biological purposes composition by volume
is much more useful, and when stated in this way the figures are very
different from those ordinarily given. Table II. gives the results for
two Broadbalk arable plots, one unmanured and the other dunged; it
includes also a pasture soil.

The first requirement of the soil population is a supply of energy,
without which it cannot live at all. All our evidence shows that the
magnitude of the population is limited by the quantity of energy
available. The percentage by weight of the organic matter is about
two to four or five, and the percentage by volume runs about four
to twelve. Not all of this, however, is of equal value as source of
energy. About one-half is fairly easily soluble in alkalis, and may
or may not be of special value, but about one-quarter is probably too
stable to be of use to soil organisms.

A second requirement is water with which in this country the soil is
usually tolerably well provided. Even in prolonged dry weather the soil
is moist at a depth of 3 inches below the surface. It is not uncommon
to find 10 per cent. or 20 per cent. by volume of water present, spread
in a thin film over all the particles, and completely saturating the
soil atmosphere.

TABLE II.

VOLUME OF AIR, WATER AND ORGANIC MATTER IN 100 VOLUMES OF ROTHAMSTED
SOIL.

+---+-----------------+-----------+--------------+
| | | |In Pore Space.|
| | | |Values Common-|
| | Solid Matter. | | ly Obtained. |
| +--------+--------+ +-------+------+
| |Mineral.|Organic.|Pore Space.| Water.| Air. |
+---+--------+--------+-----------+-------+------+
|(1)| 62 | 4 | 34 | 23 | 11 |
|(2)| 51 | 11 | 38 | 30 | 8 |
|(3)| 41 | 12 | 47 | 40 | 7 |
+---+--------+--------+-----------+-------+------+

(1) Arable, no manure applied to soil.

(2) Arable, dung applied to soil.

(3) Pasture.

The air supply is usually adequate owing to the rapidity with which
diffusion takes place. Except when the soil is water-logged, the
atmosphere differs but little from that of the one we breathe. There
is more CO₂, but only a little less oxygen.[8] The mean temperature is
higher than one would expect, being distinctly above that of the air,
while the fluctuations in temperature are less.[5]

The reaction in normal soils is neutral to faintly alkaline; _p_H
values of nearly 8 are not uncommon. Results from certain English soils
are shown on p. 18.

The soil reaction is not easily altered. A considerable amount of acid
must accumulate before any marked increase in intensity of _p_H value
occurs; in other words, the soil is well buffered. The same can be said
of temperature, of water, and of energy supply. Like the reaction, they
alter but slowly, so that organisms have considerable time in which to
adapt themselves to the change.

HYDROGEN ION CONCENTRATION AND SOIL FERTILITY.

A SELECTED BIBLIOGRAPHY.

[1] Berthelot, Marcellin, “Fixation directe de l’azote atmosphérique
libre par certains terrains argileux,” Compt. Rend., 1885, ci.,
775-84.

[2] Boussingault, J. B., and Léwy, “Sur la composition de l’air
confiné dans la terre végétale,” Ann. Chim. Phys., 1853, xxxvii.,
5-50.

[3] Cutler, D. W., Crump, L. M., and Sandon, H., “A Quantitative
Investigation of the Bacterial and Protozoan Population of the Soil,
with an Account of the Protozoan Fauna,” Phil. Trans. Roy. Soc.,
Series B, 1922, ccxi., 317-50.

[4] Hellriegel, H., and Wilfarth, H., “Untersuchungen über die
Stickstoffnahrung der Gramineen und Leguminosen,” Zeitsch. des
Vereins f. d. Rübenzucker-Industrie, 1888.

[5] Keen, B. A., and Russell, E. J., “The Factors determining Soil
Temperature,” Journ. Agric. Sci., 1921, xi., 211-37.

[6] Lawes, J. B., and Gilbert, J. H., “On Agricultural Chemistry,
Especially in Relation to the Mineral Theory of Baron Liebig,” Journ.
Roy. Agric. Soc., 1851, xii., 1-40.

[7] Liebig, Justus, “Chemistry in its Application to Agriculture
and Physiology,” 1st and 2nd editions (1840 and 1841), 3rd and 4th
editions (1843 and 1847); “Natural Laws of Husbandry,” 1863.

[8] Russell, E. J., and Appleyard, A., “The Composition of the Soil
Atmosphere,” Journ. Agric. Sci., 1915, vii., 1-48; 1917, viii.,
385-417.

[9] Russell, E. J., and Hutchinson, H. B., “The Effect of Partial
Sterilisation of Soil on the Production of Plant Food,” Journ. Agric.
Sci., 1909, iii., 111-14; Part II., Journ. Agric. Sci., 1913, v.,
152-221.

[10] Schloesing, Th., and Müntz, A., “Sur la Nitrification par les
ferments organisés,” Compt. Rend., 1877, lxxxiv., 301-3; 1877,
lxxxv., 1018-20; and 1878, lxxxvi., 892-5. “Leçons de chimie
agricole,” 1883.

[11] Warington, R., “On Nitrification,” Part I., Journ. Chem. Soc.,
1878, xxxiii., 44-51; Part II, Journ. Chem. Soc., 1879, xxxv.,
429-56; Part III., Journ. Chem. Soc., 1884, xlv., 637-72; Part IV.,
Journ. Chem. Soc., 1891, lix., 484-529.

[12] Way, J. T., “On the Composition of the Waters of Land Drainage
and of Rain,” Journ. Roy. Agric. Soc., 1856, xvii., 123-62.

[13] Winogradsky, S., “Recherches sur les organismes de la
nitrification,” Ann. de l’Inst. Pasteur, 1890, iv., 1^e Mémoire,
213-31; 2^e Mémoire, 257-75; 3^e Mémoire, 760-71.

“Recherches sur l’assimilation de l’azote libre de l’atmosphère par
les microbes.” Arch. des Sci. Biolog. St. Petersburg, 1895, iii,
297-352.

For further details and fuller bibliography, see E. J. Russell, “Soil
Conditions and Plant Growth,” Longmans, Green & Co.

CHAPTER II.

SOIL BACTERIA.

_A._ OCCURRENCE AND METHODS OF STUDY.

To understand the development of our knowledge of soil bacteria, it
must be remembered that bacteriology is under the disadvantage that
it started as an applied science. Although bacteria were first seen
by Leeuwenhoeck about the middle of the seventeenth century, and
some of their forms described by microscopists of the eighteenth and
early nineteenth centuries, it was only with the work of Pasteur on
fermentation, and of Duvaine, Pasteur, and their contemporaries on
disease bacteria, that bacteriology may be said to have started.
From the outset, therefore, attention has been directed mainly to
the bacteria in their specialised relationship to disease or to
fermentation and similar processes. As a result, little research was
done on the pure biology of the bacteria, so that even now many of the
most fundamental and elementary problems concerning them are quite
unsolved.

In their work on fermentations and disease bacteria, the earlier
workers were assisted by the fact that under both sets of conditions
the causative bacteria exist, as a rule, either in practically pure
culture, or else in preponderating numbers. The study and elucidation
of such a mixed micro-population as exists in the soil, became possible
only when methods had been devised for isolating the different kinds
of bacteria, and thus studying them apart from each other. It was the
development of the gelatine plate method of isolating pure cultures by
Koch[36] in 1881 that made the study of the soil bacteria practicable.
The plating method opened up two lines of research. In the first
place, it provided a simple means of isolating organisms from the mixed
population of the soil, and thus enabled a qualitative study to be made
of each organism in pure culture, and, in the second place, from it was
developed a counting technique for estimating differences in bacterial
numbers between samples of soil, from which has sprung much of our
knowledge of the quantitative side.

The earliest studies of the soil bacteria consisted of such estimations
of numbers, and showed that the soil contained a very numerous
population of bacteria. About 20,000,000 bacteria per gram of soil
is now considered a fair average number. The number and variety of
bacteria existing in the soil is so enormous that the method of
separating out all the different forms, and of discovering their
characters and functions, has proved impracticable. In practice,
therefore, the problem has been approached from the biochemical
standpoint. That is to say, the special chemical changes that the
bacteria produce in the soil have first been investigated, and this
has been followed by the isolation and study of the various groups of
bacteria that bring about the changes under investigation.

The method commonly employed in isolating the organisms that produce a
given chemical change in the soil is called the “elective” method. The
soil is inoculated into a culture medium that will especially favour
the group of bacteria to be isolated, to the exclusion of others.
For example, if it is desired to isolate the organisms that attack
cellulose, a medium is made up containing no other organic carbon
compounds except cellulose. Such a selective medium encourages the
growth of the group of organisms to be investigated, so that after
several transfers to fresh medium a culture is obtained containing
only two or three different types of organisms. These are separated by
plating and pure cultures obtained.

Another difficulty which has not yet been completely overcome is
that of adequately describing an organism when it is isolated. The
morphology of bacteria is not the constant thing that is seen in
the more stable higher organisms. In many cases the appearance of a
single strain is entirely different on different media, and may be
quite altered by such conditions as changes in acidity of the medium
or temperature of incubation. Even on a single medium remarkable
changes in morphology occur, at any rate, in some bacteria. This is
well seen in a cresol-decomposing organism under investigation at
Rothamsted. In cultures a few days old this organism develops as bent
and branching rods; these rods then break up into chains of cocci and
short rods, which separate, and in old cultures all the organisms
may be in the coccoid form (Fig. 1). It is claimed by Löhnis[47_b_]
that the possession of a complex life-cycle of changing forms is a
universal character in the bacteria. The instability of shape in many
bacteria makes it necessary to standardise very carefully the cultural
conditions under which they are kept when their appearance is described.

[Illustration:

Culture 15 hours old.

Culture 3 days old.

FIG. 1.--Change in appearance, in culture, of a cresol decomposing
bacterium.]

The inadequacy of mere morphology as a basis for describing bacteria
led to the search for diagnostic characters, based on the biochemical
changes that they produced in their culture media, and the appearance
of their growth in the mass on various media. These characters
unfortunately have also proved to be very much influenced by the exact
composition of the medium and other conditions of culture. Recently
an attempt has been made by the American Society of Bacteriologists to
standardise the diagnostic characters used in describing bacteria, and
also the media and cultural conditions under which they are grown for
the purpose of description. The need for such precautions, however,
was not sufficiently realised by the early workers, many of whose
descriptions cannot now be referred to any definite organism.

The large number of organisms found in the soil, and the difficulty
and labour of adequately describing them, is such that even now we
have no comprehensive description of the common soil bacteria that
appear on gelatine platings. A careful study based on modern methods of
characterisation has been made of certain selected groups of bacteria,
and it is hoped that the laborious systematic work of describing the
common forms will gradually be completed.

Several attempts have been made to classify the bacteria that appear
commonly on gelatine platings. This work was commenced by Hiltner and
Stormer in Germany, and continued by Chester, Harding, and Conn in
America. Conn[10],[14] found that the common organisms fell into the
following main groups:--

(1) Large spore-forming bacteria, related to _Bacillus subtilis_, which
form about 5-10 per cent. of the numbers. He adduced evidence[12],[13]
that these organisms exist in the soil mainly as spores, so that they
may not form an important part of the active soil population.

(2) Short non-sporing organisms, related to _Pseudomonas fluorescens_,
that are rapid gelatine liquefiers. These form another 10 per cent. of
the numbers.

(3) Short rod forms that liquefy gelatine slowly or not at all, and
develop colonies very slowly. These form 40-75 per cent. of the
numbers, and may therefore be of considerable importance in the soil.

(4) A few micrococci also occur.

These groups comprise the larger portion of the bacterial flora of the
soil, but, in addition to these organisms, that develop on the media
commonly used for plating, there are special and important groups
that appear only on special media, either owing to their being unable
to grow on ordinary media or because they get swamped by other forms.
Examples of such groups are the ammonia and nitrite oxidising bacteria,
the nitrogen fixing groups, the cellulose decomposing organisms, and
the sulphur bacteria.

In order that we may apply the results of the study of a definite
organism to other localities, a knowledge of the geographical
distribution of the soil bacteria is clearly needed. We have,
unfortunately, very little knowledge of the distribution of soil
organisms. The common spore-forming groups appear to be universally
distributed. Thus Barthel, in a study of the bacterial flora of soils
from Greenland and the island of Disko, obtained soil organisms
belonging to the groups of _Bacillus subtilis_, _B. amylobacter_, _B.
fluorescens_, _B. caudatus_, and _B. Zopfii_, which are common groups
in European soil, indicating that the general constitution of the
bacterial flora of the soil in arctic regions is not widely different
from that of Western Europe. Bredemann, who made an extensive study of
the _Bacillus amylobacter_ group, obtained soil samples from widely
scattered localities, and found these organisms in soil from Germany,
Holstein, Norway, Italy, Morocco, Teneriffe, Russia, Japan, China,
the East Indies, Samoa, Illinois, Arizona, German East Africa, and
the Cameroons. Some soil organisms, on the other hand, are apparently
absent from certain districts. This may be due to the conditions,
such as climatic environment, being unfavourable to them. A study has
recently been made at Rothamsted of the distribution over Great Britain
of a group of bacteria that are capable of decomposing phenol and
cresol. One of these organisms, apparently related to the acid-fast
_B. phlœi_, has an interesting distribution. It has been found in 50
per cent. of the soils samples examined from the drier region, where
the annual rainfall is less than 30 inches, but in only 20 per cent.
of the samples in the wetter parts of Britain. Another example of
limited distribution is found in the case of _Bacillus radicicola_,
the organism that produces tubercles on the roots of leguminous plants.
The distribution of the varieties of this organism follows that of
the host plants with which they are associated, so that when a new
leguminous crop is introduced into a country, nodules may not appear
on the roots unless the soil be specially inoculated with the right
variety of organism. In cases where a group of soil organisms is widely
distributed over the globe, it may yet be absent from many soils
owing to the soil conditions not suiting it. Thus, phenol decomposing
bacteria, though abundant in the neighbourhood of Rothamsted, are yet
absent from field plots that have been unmanured for a considerable
period. The occurrence of the nitrifying organisms and the nitrogen
fixing _Azotobacter_ is also very dependent on the soil conditions.

Owing to the method by which our knowledge of soil bacteria has been
acquired, by studying first the chemical changes in the soil and then
the bacteria that produce them, it is natural for us to divide them
into physiological groups according to the chemical changes that they
bring about. This grouping is the more reasonable since so little is
known as to the true relationships of the different groups of bacteria
that a classification based on morphology is well-nigh impossible. In
considering the activities of bacteria in the soil, it is convenient to
group the changes which they bring about into the two divisions into
which they naturally fall in the economy of the organisms.

In the first place, there are the changes that result in a release of
energy, which the bacteria utilise for their vital processes.

In the second place, there are the processes by which the bacteria
build up the material of their bodies. These building up processes
involve an intake of energy for their accomplishment.

It will be convenient to deal first with the release of energy for
their own use by bacteria, and its consequences.

_B._ ACTIVITIES CONNECTED WITH THE ACQUIREMENT OF ENERGY.

Unlike the green plants, most bacteria are unable to obtain the energy
that is required for their metabolism from sunlight. They must,
therefore, make use of such chemical changes as will involve the
release of energy.

As an example of the acquirement of energy in this way may be taken the
oxidation of methane by _B. methanicus_. This organism, described by
Söhngen, obtains its energy supply by the conversion of methane into
CO₂ and H₂O.

CH₄ + 2O₂ = CO₂ + 2H₂O 220 Cal.

A further example is the acetic organism that obtains its energy
through the oxidation of alcohol to acetic acid.

C₂H₆O + O₂ = C₂H₄O₂ + H₂O 115 Cal.

The decomposition processes brought about by micro-organisms in
obtaining energy are usually oxidations, but this is not necessarily
so, as can be seen in case of the fermentation of sugar into alcohol.[E]

C₆H₁₂O₆ = 2C₂H₆O + 2CO₂ 50 Cal.

[E] These examples are from Orla-Jensen (Centralblatt f. Bakt., II.,
Bd. 22, p. 305).

By far the greater part of the decomposition of organic matter is
brought about by bacteria in the process of acquiring energy. In the
soil, nearly the whole of the material utilised by bacteria as a source
of energy is derived ultimately from green plants. The energy materials
left in the soil by the plant fall into two groups, the non-nitrogenous
compounds, which are mainly carbohydrates and their derivatives, and
the nitrogenous compounds, principally derived from proteins.

(1) _Decomposition of Non-nitrogenous Compounds._

The simpler carbohydrates and starches are attacked and decomposed by
a large variety of bacteria. The addition of such substances to soil
causes a rapid increase in bacterial numbers. In nature the sugars are
in all probability among the first plant constituents to be destroyed
during the decay processes.

A large proportion of plant tissues consist of cellulose and its
derivatives. These compounds are consequently of great importance
in the soil. Unfortunately our knowledge of the processes by which
cellulose is broken down in the soil is very inadequate. The early
experimental study of cellulose decomposition, such as that of
Tappeiner[60] and Hoppe-Seyler,[33] was mostly carried out under
conditions of inadequate aeration, and the products of decomposition
were found to include methane and CO₂, and sometimes fatty acids and
hydrogen. The bacteriology of this anaerobic decomposition was studied
by Omelianski,[54] who described two spore-bearing organisms, one of
which attacked cellulose with the production of hydrogen, and the other
with the production of methane. Both species also produce fatty acids
and CO₂. It is probable that these organisms operate in the soil under
conditions of inadequate aeration. In swamp soils, in which rice is
grown, it has been shown that methane, hydrogen, and CO₂ are evolved
in the lower layers. In these soils, however, the methane and hydrogen
are oxidised when they reach the surface layers. This oxidation is
also effected by micro-organisms. Bacteria capable of deriving energy
by the oxidation of hydrogen gas have been isolated and studied by
Kaserer,[37] and by Nabokich and Lebedeff,[52] while Söhngen[57] has
isolated an organism which he named _Bacillus methanicus_, that was
capable of oxidising methane.

Under normal conditions in cultivated soils, however, the decomposition
of cellulose takes place in the presence of an adequate air supply,
and so follows a different course from that studied by Omelianski. Our
knowledge of this aerobic decomposition is very scanty. A number of
bacteria, capable of decomposing cellulose aerobically, are known. A
remarkable organism was investigated by Hutchinson and Clayton,[30]
who named it _Spirochæta cytophaga_. This organism, which they isolated
from Rothamsted soil, though placed among the _Spirochætoidea_, is
of doubtful affinities. During the active condition it exists for
the most part as thin flexible rods tapered at the extremities. This
form passes into a spherical cyst-like stage, at first thought to be
a distinct organism (Fig. 2). _Spirochæta cytophaga_ is very aerobic,
working actively, only at the surface of the culture medium. It is
very selective in its action. It appears unable to derive energy from
any carbohydrate other than cellulose. Indeed, many of the simple
carbohydrates, especially the reducing sugars, are toxic to the
organism in pure culture. An extensive study of aerobic cellulose
decomposition by bacteria was made by McBeth and Scales,[50] who
isolated fifteen bacteria having this power. Five of these were
spore-forming organisms. Unlike _Spirochæta cytophaga_, they are all
able to develop on ordinary media such as beef agar or gelatine, and
are thus not nearly so selective in their food requirements.

[Illustration: FIG. 2.--_Spirochæta cytophaga._ Changes occurring in
culture. (After HUTCHINSON and CLAYTON.)]

We are at present ignorant as to which organisms are most effective
in decomposing cellulose in the soil under field conditions, or what
are the conditions best suited to their activity. It is possible that
fungi also help in the decomposition of cellulose to a great extent.
This subject of the decomposition of cellulose offers one of the most
promising fields of research in soil bacteriology. The difficulty
of the subject is further increased by our present ignorance of the
chemical aspect of cellulose decomposition. It has been supposed that
the early decomposition products are simpler sugars, but these are
not found under conditions in which cellulose is being decomposed by
pure cultures of the bacteria mentioned above. Hutchinson and Clayton
found that their organism produced volatile acids, mucilage, and a
carotin-like pigment. The organisms isolated by McBeth and Scales also
produce acids, and in some cases yellow pigments. It is known, however,
that the decomposition products of cellulose can be utilised as energy
supply for other organisms, such as nitrogen fixing bacteria.

When plant remains decompose in the soil there are ultimately produced
brown colloidal bodies collectively known as humus. The processes by
which this humus is produced are not yet properly understood. Humus is
of great importance in the soil, in rendering the soil suitable for
the growth of crops. It affects the physical properties of the soil
to a great extent. In the first place, it improves the texture of the
soil, making heavy clay soils more friable, and loose sandy soils more
coherent. Secondly, it has great water-retaining powers, so that soils
rich in organic matter suffer comparatively little during periods of
drought. And lastly, it exerts a strong buffering effect against soil
acids. Now, it is one of the problems of present-day farming that
soil is becoming depleted of its humus. This is due to the increasing
scarcity of farmyard manure in many districts, and the consequent use
of mineral fertilisers to supply nitrogen, potash, and phosphate to
the crop. A need has therefore arisen for a substitute for farmyard
manure, by means of which the humus content of soils may be kept up in
districts where natural manure is scarce.

[Illustration: FIG. 3.--Cellulose decomposed by _S. cytophaga_ in media
with increasing amounts of nitrogen. (After HUTCHINSON and CLAYTON.)

X-axis: Milligrams of nitrogen supplied as sodium-ammonium phosphate.

Y-axis: Milligrams of cellulose decomposed in 21 days.]

It is well known that if fresh unrotted manure or straw be added to
the soil, it often produces harmful effects on the succeeding crop.
The problem, therefore, was to develop a method by which fresh straw,
before application to the soil, could be made to rot down to a mixture
of humus compounds such as occur in well-rotted farmyard manure. The
solution of this problem came as a result of an investigation by
Hutchinson and Richards,[30_b_] at Rothamsted, into food requirements
of the cellulose decomposing bacteria. They realised that since more
than 10 per cent. of the dry weight of bacteria consists of nitrogen,
it would be necessary to supply the cellulose decomposing bacteria with
a supply of nitrogen, in order that they should attain their greatest
activity. Experiments with cultures of _Spirochæta cytophaga_ showed
that the amount of cellulose decomposed depended upon an adequate
supply of nitrogen for the organism (Fig. 3). Similarly, materials such
as straw will scarcely decompose at all if wetted with pure water. An
adequate supply of nitrogen compounds is needed to enable decomposition
to take place. Hutchinson and Richards tested the effect of ammonium
sulphate, and discovered experimentally the proportion of ammonia to
straw that produced the most rapid decomposition. They found that if
a straw heap was treated with the correct proportion of ammonia, it
decomposed into a brown substance having the appearance of well-rotted
manure. This has resulted in the development of a commercial process
for making synthetic farmyard manure from straw. The method of
manufacture is as follows: A straw stack is made and thoroughly wetted
with water. The correct amount of ammonium sulphate is then sprinkled
on the top and wetted, so that the solution percolates through the
straw. The cellulose bacteria attack the straw, breaking it down
and assimilating the ammonia. This ammonia is not wasted, as it is
converted into bacterial protoplasm that eventually decays in the soil.
Field trials of this synthetic manure show that it produces an effect
closely similar to that of natural farmyard manure.

While cellulose and related carbohydrates are by far the most
important non-nitrogenous compounds left in the soil by plants, there
are other compounds whose destruction by bacteria is of special
interest. Such, for example, is the case of phenol. This compound is
produced by bacterial action as a decomposition product of certain
amino-acids. It occurs in appreciable amounts in cow urine. It is
probable that it forms a common decomposition product in soil and
also in farmyard manure. If this phenol were to persist in the soil,
it would eventually reach a concentration harmful to plant growth. It
does not, however, accumulate in the soil; indeed, if pure phenol or
cresol be added to ordinary arable soil, a rapid disappearance occurs.
This disappearance is of some practical importance, since it limits
the commercial use of these compounds as soil sterilising agents.
The cause of the disappearance has been to some extent elucidated at
Rothamsted,[58] where it was found to be in part a purely chemical
reaction with certain soil constituents, and partly due to the activity
of bacteria capable of decomposing it. A large number of soil bacteria
have now been isolated that can decompose phenol, meta-, para-, and
ortho-cresol, and are able to use these substances as the sole sources
of energy for their life processes. These organisms have a wide
distribution, having been found in soil samples taken from all over
Great Britain, from Norway, the Tyrol, Gough Island, Tristan da Cunha
and South Georgia. Soil bacteria have also been isolated that are able
to decompose and derive their energy from naphthalene and from toluene.
The ability of the bacteria to break up the naphthalene is very
remarkable, and all the more so since they can hardly have come across
this compound in the state of nature. The naphthalene organisms have a
distribution as world-wide as the phenol group.

(2) _Ammonia Production._

The second main group of products left in the soil by higher plants
are the nitrogen-containing compounds, such as the proteins and
amino-acids. Plant remains are not the only source of organic nitrogen
compounds available to soil bacteria. There are, in addition, the dead
bodies of other soil organisms, such as protozoa and algæ. The relative
importance of these sources of nitrogen is not known, but almost
certainly varies greatly with the state of activity of the various
groups of the soil population. Bacteria are able to utilise organic
nitrogen compounds as energy sources, as can be exemplified in the
oxidation of a simple amino-acid:--

H O
| //
H--C--C + 3O = 2CO₂ + H₂O + NH₃ + 152 Cal.
| \
NH₂ OH

It will be seen that, in the acquirement of energy from such a
compound, ammonia is released as a by-product. It is not certainly
known what is the exact course of the reactions brought about by
bacteria in soil during the breaking-down of organic nitrogen
compounds, but they result in the splitting off of most of the nitrogen
as ammonia. Herein lies the great importance of the process, for the
production of ammonia is an essential stage in the formation of nitrate
in the soil, and on the supply of nitrate the growth of most crops
largely depends.

[Illustration: FIG. 4.--Quantities of ammonia produced by pure cultures
from 5 grams of casein in the presence of varying quantities of
dextrose. (After DORYLAND.)

X-axis: Percentage of dextrose added.

Y-axis: Milligrams of NH₃ produced.]

It is very important to note that the production of this ammonia is
only a by-product in the economy of the bacteria, the benefit that they
derive from the reactions being due to the release of energy involved
in the decomposition. The common ammonia-producing bacteria in the
soil have been found equally capable of deriving their energy by the
oxidation of sugars and similar non-nitrogenous compounds. Fig. 4
shows an experiment by Doryland,[17] in which cultures of common soil
bacteria were grown in peptone solution, to which increasing quantities
of sugar were added. One can see that, as the amount of sugar is
increased, the production of ammonia is lowered, since the bacteria
are obtaining energy from the sugar instead of from the nitrogen
compound, peptone. Consequently, if soil contains a quantity of easily
decomposible carbohydrate material, bacteria will derive their energy
from this source, and the production of ammonia and nitrate will be
lowered. Thus the addition of sugar or unrotted straw to the soil often
lowers the nitrate production, and consequently reduces the crop yield.
If the soil is sufficiently rich in carbohydrate material, the bacteria
may multiply until the supply of organic nitrogen is used up, and
then will actually assimilate some of the ammonia and nitrate already
existing. There is thus a balance of conditions in the soil due to
varying proportions of nitrogenous and non-nitrogenous energy material.
When nitrogen compounds are the predominant energy source, the bacteria
utilise them, and ammonia is released. When a non-nitrogenous energy
source predominates, this is utilised and little or no ammonia is
released, and in extreme cases ammonia may be assimilated.

Although a large number of the common organisms in the soil produce
ammonia in culture media containing peptone, the relative importance
of these in the soil has yet to be decided. It was supposed that the
spore-forming organisms related to _Bacillus mycoides_ were of chief
importance. This supposition dates from the work of Marchal,[49] who
studied the production of ammonia by an organism of this group in
culture solution, and found it to be a very active ammonifier. As
already mentioned, however, there is some doubt as to whether the large
spore-forming organisms are very active under soil conditions.[12],[13]
The existence of rapid fluctuations in nitrate content, found to exist
in soil, may in the future indicate which are the most active of the
common bacteria in the soil itself by enabling us to observe which
types increase during periods of rapid ammonia and nitrate formation.

(3) _Nitrate Production._

The ammonia produced in the soil under normal field conditions is
rapidly oxidised successively to nitrite and to nitrate, a process
known as nitrification. The process of nitrification is more rapid than
that of ammonia production, with the consequence that no more than
traces of ammonia are able to accumulate. The rate at which nitrate is
formed in the soil is consequently set by the slower process of ammonia
production.

The work of Schloesing and of Warington showed that the oxidation
of ammonia was the work of living organisms. It is, however, to
Winogradsky’s isolation and study of the causative organisms that we
owe our present knowledge of the biology of the process. By a new
and ingenious technique, he isolated from soil two remarkable groups
of bacteria that bring about nitrification. The first group oxidises
ammonium carbonate to nitrite, and was divided by Winogradsky into
the two genera, _Nitrosomonas_, a very short rod-like organism bearing
a single flagellum, and _Nitrosococcus_, a non-motile form found in
South America. The second group oxidises nitrites to nitrates. They are
minute pear-shaped rods to which he gave the name _Nitrobacter_.

Winogradsky found that the first, or nitrite-producing group, would
live in a culture solution containing:--

2·25 grams ammonium sulphate,
2·0 „ sodium chloride,
1·0 „ magnesium carbonate,
to the litre of well water.

Nitrobacter would grow in a similar medium containing sodium nitrite
instead of ammonium sulphate. There being no organic carbon in these
media, the organisms had no source of carbon for their nutrition,
except the CO₂ of the air, or possibly that of bicarbonate in solution.
It therefore followed that the organisms must obtain their carbon
supply from one of these sources. Unlike green plants, the nitrous and
nitric organisms are able to carry on this carbon assimilation in the
dark, and must therefore obtain the energy needed for the process from
some chemical reaction. The only sources of energy in Winogradsky’s
solutions were the nitrogen compounds, and it consequently followed
that the organisms must derive their energy supply by the oxidation
of ammonia and nitrite respectively. The release of energy obtained
by these two reactions has been calculated by Orla-Jensen to be as
follows:--

(NH₄)₂CO₃ + 3O₂ = 2HNO₂ + CO₂ + 3H₂O + 148 Cals.

KNO₂ + O = KNO₃ + 22 Cals.

The exact process by which ammonium carbonate is converted into nitrite
is not at present known. The two groups of organisms are extremely
selective in their source of energy. The nitrous organisms can derive
their energy only by the oxidation of ammonia to nitrite, and the
nitric organisms only by the oxidation of nitrite to nitrate. In
culture media they are, indeed, inhibited by soluble organic compounds
such as sugars. Under natural conditions, however, they appear to
be less sensitive, since ammonium carbonate is readily nitrified
in substrata rich in organic matter. The rapid nitrification that
takes place during the purification of sewage is an example of this.
The conditions in culture, with regard to aeration and the removal
of metabolic products from the neighbourhood of the organisms, are
very different from those in the soil, and perhaps account for the
discrepancies found.

The oxidation of ammonium carbonate by nitrosomonas results in the
formation of nitrous acid. The organisms are very sensitive to acidity,
and can only operate if the nitrous acid produced is neutralised by an
available base. In normal soils calcium carbonate supplies this base,
and in acid soils the formation of nitrite is, as a rule, increased
by the addition of lime, or of calcium or magnesium carbonate. There
is evidence that in the absence of calcium carbonate, other compounds
can be used as a base. It was found by Hopkins and Whiting[32] that
in culture solution the nitrifying organisms could use insoluble rock
phosphate as a base, producing therefrom the soluble acid phosphate.
There is evidence, however, that in ordinary soil containing calcium
carbonate very little solution of phosphate takes place in this way.
The further oxidation of nitrite to nitrate by _Nitrobacter_ does not
produce acid, and requires no further neutralising base.

The nitrate produced in this way is the main source of nitrogen supply
to plants under normal conditions. Experiments have shown that a number
of plants are capable of utilising ammonia as a source of nitrogen, and
Hesselmann[34] has found forest soils in Sweden where no nitrification
was proceeding, and where, therefore, plants would presumably obtain
their nitrogen in this way, but such cases must be regarded as
exceptional.

Another group of bacteria capable of deriving their energy from
an inorganic source exists in the soil. This comprises the sulphur
bacteria, which are able to derive energy by the oxidation of sulphur,
sulphides, or thiosulphates to sulphuric acid:--

S + 3O + H₂O = H₂SO₄ + 141 Cals.

One organism studied by Waksman and Joffe[63] is able to live in
inorganic solution, deriving its carbon from carbon dioxide. The
sulphur bacteria have recently come into prominence in America owing to
their faculty for producing acid. Thus Thiospirillum will increase the
acidity of its medium to a reaction of P_{H} 1·0 before growth ceases.
The potato scab disease in America is now treated by composting with
sulphur. This treatment depends on the production of sulphuric acid by
the sulphur oxidising bacteria, which renders the soil too acid for the
parasite. There is some evidence also that acid thus produced can be
used to render insoluble phosphatic manures more available in the soil.

Analogous to the sulphur organisms are certain bacteria isolated from
sheep dig tanks in South Africa by Green,[28_b_] which can derive
energy by the oxidation of sodium arsenite to arsenate.

(4) _Anaerobic Respiration._

As is seen in the examples mentioned, energy is commonly obtained by
bacteria through an oxidation process in which free oxygen is utilised.
In water-logged soil, however, or in soil overloaded with organic
matter, anaerobic bacteria may develop, which obtain their oxygen
from oxidised compounds. Thus there are soil organisms described by
Beijerinck[2] and others which can obtain oxygen by reducing sulphates
to sulphides.

A more important source of oxygen under these conditions is nitrate,
which can supply oxygen to a larger number of bacteria. The stage to
which the reduction can be carried varies according to the organism.
A very large number of bacteria are capable of reducing nitrates to
nitrites. Many can reduce nitrate to ammonia, and some can produce
an evolution of nitrogen gas from nitrate. The effects of nitrate
reduction, therefore, appear under water-logged conditions in soils.
For example, in swamp soils in which rice is grown, it has been found
by Nagaoka,[53] in Japan, that treatment with nitrate of soda depresses
the yield, probably owing to the formation of poisonous nitrites by
reduction.

Under normal conditions of well aerated soil, however, it is unlikely
that the reduction of nitrate is of great importance. In such soils the
activities through which bacteria acquire their energy are, as we have
seen, of vital importance to the plant, resulting in the disintegration
of plant tissues, with the ultimate formation of humus, and in the
production of nitrate.

In their activities connected with the building up of their protoplasm,
bacteria may, on the other hand, compete with the plant. These
activities and their consequences will be reviewed in the following
chapter.

CHAPTER III.

SOIL BACTERIA.

_C._ ACTIVITIES CONNECTED WITH THE BUILDING-UP OF BACTERIAL PROTOPLASM.

(1) _Composition of Bacteria._

The activities of the soil bacteria that we have yet to consider are
those connected with the building-up from simpler materials of the
protoplasm of the bacterial cell. It is important to bear in mind that
this process is one requiring an expenditure of energy on the part of
the organism. The sources of energy we have already considered.

The bodies of bacteria contain the same elements common to other
living matter. Analyses of various bacteria have been made by a number
of workers. About 85 per cent. of their weight is made up of water.
This analysis of Pfeiffer’s Bacillus by Cramer[15] shows the typical
percentages of carbon, nitrogen, hydrogen, and ash in the dry matter:--

_Composition of Pfeiffer’s Bacillus (Cramer)._

C 50 per cent.
N 12·3 „
H 6·6 „
Ash 9·1 „

About 65-70 per cent. of the dry matter of bacteria consists of protein.

(2) _Sources of Carbon._

The biggest constituent of the dry matter of bacteria is therefore
carbon. In the soil, bacteria find an abundance of organic matter from
which they may derive their carbon supply. A special case, however,
is furnished by the nitrifying organisms, certain sulphur oxidising
bacteria, and others that derive their carbon from the CO₂ of the soil
atmosphere. The sources from which these special groups obtain the
necessary energy to accomplish this, we have already considered.

(3) _Assimilation of Nitrogen Compounds._

Of chief importance in its consequences are the means adopted by
bacteria to obtain their nitrogen supply.

There is some reason to believe that soil bacteria do not take up
protein and peptones as such, but must first break down these bodies
into simpler compounds. When a sufficient amount of easily decomposable
organic nitrogen is present in the soil, the ammonifying bacteria use
such compounds as sources of energy, and in this case have a nitrogen
supply exceeding their requirements.

But where there is an excess of carbohydrate or other non-nitrogenous
source of energy available in the soil, the case is different. Here
the organisms have a supply of energy which enables them to multiply
rapidly until the organic nitrogen is insufficient for their needs.
Hence they turn to the ammonia and nitrate present in the soil, and
build up their proteins from this source. Doryland[17] has shown that
many common soil ammonifiers assimilate ammonia and nitrate when
supplied with carbohydrate. There may thus be a temporary loss of
nitrate from soil when sugar, starch, straw, or such materials are
added to it.

(4) _Fixation of Free Nitrogen._

The bacteria that we have so far considered take up their nitrogen
directly from compounds containing this element. There remain, however,
a comparatively small but very important group of bacteria possessing
the power of causing elemental nitrogen to combine, and of building it
up into their proteins. This fixation of nitrogen by micro-organisms
is a vital step in the economy of nature. Losses of nitrogen from the
land are continually occurring through the washing-out of nitrates
by rain, and through the evolution of gaseous nitrogen during the
processes of decay. To maintain the supply of combined nitrogen
which is essential to living organisms, there must therefore be a
compensating process by which the supply of nitrogen compounds in the
soil is kept up.

It was discovered in the middle of the nineteenth century that if soil
were kept moist and exposed to the air, there was an increase in the
amount of nitrogen compounds present. Berthelot, in 1893, studied the
nitrogen relationships of soil, and recognised that this fixation of
nitrogen in soil was the work of micro-organisms.

Winogradsky followed up his work and isolated from soil a large
anaerobic spore-forming organism, capable of fixing nitrogen, to which
he gave the name _Clostridium pasteurianum_. In 1901 the investigations
of Beyerinck, in Holland, led to the important discovery of a group of
large aerobic organisms, which he named _Azotobacter_. These were found
to be very active in fixing free nitrogen. More recently, a number of
other nitrogen-fixing bacteria have been described, and the property
has been found to exist to a small extent in several previously
well-known organisms.

It becomes important to determine which are the groups of bacteria
whose nitrogen-fixing powers are of chief importance in the soil.

On account of its energetic fixation of nitrogen in culture media,
_Azotobacter_ has attracted the greatest attention of workers. The
evidence seems to be consistent with the view that _Azotobacter_ is of
importance in the soil. Thus the distribution of _Azotobacter_ would
appear to be world-wide. It is found all over Western Europe and the
United States. Lipman and Burgess[45] found it in soils collected from
Italy and Spain, Smyrna, Cairo, the Fayum, the Deccan in India, Tahiti,
Hawaii, Mexico, Guatemala, and Canada. C. M. Hutchinson[29] found it
to be distributed throughout India. It was found by Omelianski[55]
to be widely distributed in European and Asiatic Russia, and by
Groenewege[28] in Java. Ashby[1] at Rothamsted, isolated it from soils
from the Transvaal, East Africa, and Egypt. Also, an association has
sometimes been found between the ability of a soil to fix nitrogen and
the occurrence and vigour of its _Azotobacter_ flora. Thus Lipman and
Waynick[46] found that if soil from Kansas were removed to California,
its power to produce a growth of _Azotobacter_, when inoculated into a
suitable medium, was lost, and, at the same time, its nitrogen-fixing
power was greatly reduced. Moreover, it is known that conditions
favourable to the fixation of nitrogen by _Azotobacter_ in cultures on
the whole favour nitrogen fixation in soils. The conditions that favour
other aerobic nitrogen-fixing bacteria are, however, not sufficiently
distinct to make such evidence of great value.

It is usually found that nitrogen fixation is most active in
well-aerated soil. Thus Ashby,[1] at Rothamsted, found the
nitrogen-fixing power of a soil to decrease rapidly with depth. Similar
results were obtained in Utah by Greaves. This suggests, at first
sight, that anaerobic nitrogen fixers are unimportant under normal
soil conditions. It is, however, quite possible that they may assume
an importance when acting in conjunction with aerobic organisms.
Thus Omelianski and Salunskov[55] found that beneficial association,
or symbiosis, could occur between _Azotobacter_ and _Clostridium
pasteurianum_, the former absorbing oxygen from the surroundings, and
thus creating a suitable anaerobic environment for the _Clostridium_.

The question of symbiosis of nitrogen-fixing bacteria with each other
and with other organisms offers an inviting field for research.
There is evidence that this factor may have considerable importance.
Beijerinck and Van Delden[3] early recognised that _Azotobacter_
in mixed cultures fixed more nitrogen than in pure cultures.
_Granulobacter_, an organism which they found to be commonly associated
with _Azotobacter_ in crude cultures, appears to increase its
nitrogen-fixing powers (Krzeminiewski).[41] It was also found by
Hanzawa[31] that a greater fixation of nitrogen was obtained when two
strains of _Azotobacter_ were grown together. A symbiosis between
_Azotobacter_ and green algæ has been described, and will be further
discussed by Dr. Bristol. It is likely that this association may be of
importance under suitable conditions on the soil surface where the algæ
are exposed to light.

The combination of elemental nitrogen is an endothermic process which
requires a very considerable amount of energy for its accomplishment.
This fact is well illustrated by the various commercial processes
in use for fixation of atmospheric nitrogen. The nitrogen-fixing
bacteria obtain this energy from the carbon compounds in the soil.
A number of compounds were compared as sources of energy by Löhnis
and Pillai,[47] who tested their effect on the amounts of nitrogen
fixed by _Azotobacter_ in culture. It was found that mannitol and the
simpler sugars give the best results as sources of energy, but that
other organic compounds can also be used. Mockeridge[51] has adduced
evidence that ethylene glycol, methyl-, ethyl-, and propyl-alcohol,
lactic, malic, succinic, and glycocollic acids could also be utilised.
Since so large a part of the organic matter added to soil is in the
form of celluloses, it is of great importance to ascertain how far
these compounds and their decomposition products can be utilised in
nitrogen fixation. Stubble, corn-stalks and roots, oak leaves, lupine
and lucerne tops, maple leaves, and pine needles may all serve as
useful sources of energy to nitrogen-fixing organisms in the soil. Pure
cellulose cannot apparently be used as a source of energy, but when
acted upon by cellulose decomposing organisms, it becomes available as
a source of energy. Hutchinson and Clayton, at Rothamsted, found that
a fixation of nitrogen could be brought about by mixed cultures of
_Azotobacter_, and of the cellulose attacking _Spirochæta cytophaga_,
when grown in cultures containing pure cellulose. It is not known
how far cellulose decomposition must proceed to produce an effective
source of energy, nor what are the substances thus produced that are
utilised. This point will not be decided until something more is known
of the course of changes in the breaking-down of cellulose in the soil.

The amount of nitrogen fixed per unit of energy material decomposed
varies greatly, according to the organism and the conditions.
Winogradsky found that his _Clostridium_ assimilated 2-3 mgs. of
nitrogen per gram of sugar consumed. Lipman found that _Azotobacter_
fixed 15-20 mgs. of nitrogen per gram of mannite consumed.

[Illustration: FIG. 5.

Caption: Azotobacter. Decrease in efficiency in N fixation with age
of culture. (Koch & Seydel.)

X-axis: Days.

Y-axis: Milligrams of Nitrogen fixed per gram of dextrose consumed.]

It is found, however, that in liquid culture, the ratio of nitrogen
fixed to carbohydrates oxidised varies according to the age of the
culture, falling off rapidly as the age increases[42] (Fig. 5). This
decreasing efficiency in cultures may be due to the accumulation of
metabolic products such as would not occur under soil conditions.
Indeed, the efficiency of _Azotobacter_ in a sand culture has been
found by Krainskii[39] to be considerably greater than in solution. It
is thus probable that in soil the nitrogen-fixing organisms are less
wasteful of energy material than under the usual laboratory conditions.
It is to be hoped that future research will indicate what are the
conditions that produce the greatest economy of energy material in
nitrogen fixation.

The fixation of nitrogen in soil is depressed by the presence of
considerable amounts of nitrates. This is, in all probability, due
to the fact that nitrogen-fixing organisms are able to utilise
compounds of nitrogen where these are available. The energy needed
to build up amino-acids and proteins from nitrate or ammonia is, of
course, far less than that required to build up these substances
from elemental nitrogen. It is, therefore, not surprising that where
nitrate is available, _Azotobacter_ will use it in preference to fixing
atmospheric nitrogen.[5]

TABLE III.--ASSIMILATION OF NITRATES.

BY AZOTOBACTER IN PURE CULTURE--(_Bonazzi_).

+---------------------------+--------+-----------+------+
| | Nitrate| Organic |Total |
| | and | Nitrogen |Fixed |
| | Nitrite|and Ammonia| or |
| |Present.| Present. |Lost. |
+---------------------------+--------+-----------+------+
| | mgs. | mgs. | mgs. |
|_Culture with nitrate_-- | | | |
| At beginning | 8·55 | 0·76 | -- |
| After growth | 0·2 | 8·71 | -0·4 |
|_Culture without nitrate_--| | | |
| At beginning | -- | 0·76 | -- |
| After growth | -- | 4·50 | +3.74|
+---------------------------+--------+-----------+------+

(Growth period--24 days at 25° C.)

The chemical process by which nitrogen is fixed is quite unknown,
although a number of speculative suggestions have been made. The
appearance of considerable amounts of amino acids in young cultures of
_Azotobacter_ suggests that these may be a step in the process, but at
present the data are too inconclusive to form a basis for theorising.

_Azotobacter_ is very rich in phosphorus, an analysis of the surface
growth in _Azotobacter_ cultures, made by Stoklasa, giving about 60
per cent. of phosphoric acid in the ash. In cultures it has been found
that a considerable amount of phosphate is needed to produce full
development. As would be expected, therefore, nitrogen fixation in soil
is often greatly stimulated by the addition of phosphates. Christensen
has, indeed, found soils where lack of phosphate was the limiting
factor for _Azotobacter_ growth.

_Azotobacter_ is very intolerant of an acid medium, and is very
dependent on the presence of an available base. In cultures this
is usually provided in the form of calcium or magnesium carbonate.
Gainey[21] found that _Azotobacter_ occurred in soils having an acidity
not greater than P_{H} 6·0, and Christensen,[7],[9] in Denmark, has
found a close association between the occurrence of _Azotobacter_ in
soils and the presence of an adequate supply of calcium carbonate. So
close was this association that he devised a technique based on this
fact for detecting a deficiency of lime in a soil sample.

In addition to the groups already discussed, there is a remarkable and
important group of nitrogen-fixing bacteria that inhabit and can carry
on their functions within the root tissues of higher plants. It has
been known at least from classical times that certain leguminous plants
would, under suitable conditions, render the soil more productive. On
the roots of leguminosæ small tubercles are commonly found. These were
noted and figured by Malpighi in the seventeenth century, and for a
long time were regarded as root-galls. As was described in Chapter I.,
the true nature of these tubercles was finally elucidated by Hellriegel
and Wilfarth in 1886. As the result of a series of pot experiments,
they made the very brilliant deduction that the ability to fix
nitrogen, possessed by the legumes, was due to bacteria associated with
them in the tubercles.

These bacteria were finally isolated and studied in pure culture
by Beijerinck. Since then a very great deal of literature has
accumulated on the subject of the nodule-producing bacteria, which
it is impossible to deal with in a small space. The nodule organism,
_Bacillus radicicola_, when grown on suitable media, passes through a
number of different changes in morphology. The most connected account
of these changes is given in a paper by Bewley and Hutchinson.[4]
In a vigorous culture the commonest type is a rod-shaped bacillus
which may or may not be motile. As these get older they often become
branched, or irregular in shape, the formation of these branched forms
being perhaps due to conditions in the medium. These irregular forms,
known as “bacteroids,” are a characteristic type in the nodules. Their
production in culture media has been found to be stimulated by sugars
and organic acids such as would occur in their environment within the
host plant. In the older rods and bacteroids the staining material
becomes condensed into granules, and finally the rods disintegrate or
break up into coccoid forms. By suitable culture conditions, Bewley and
Hutchinson obtained cultures consisting almost entirely of this stage.
If such a culture be inoculated into a fresh medium rich in sugar,
the swarmer stage appears in great numbers. These swarmers are very
minute coccoid rods, ·9 × ·18 in size, that are actively motile. They
apparently develop later into the rod stage.

[Illustration: FIG. 6.--_Bacillus radicicola._ Stages in the life
cycle. (After HUTCHINSON and BEWLEY.)

Motile Rods

Vacuolated Stage

“Swarmers”

“Bacteroids”

“Pre-swarmers”]

Very little is known as to the life of the organism in the soil.
It is able, however, to fix nitrogen in cultures, and it has been
claimed[35],[48] that it can do so in the soil outside the plant, so
that it is possible that we must take it into consideration in this
connection. More knowledge is needed as to the optimum conditions for
the growth of the organism in the soil. It seems to be more tolerant of
acid soil conditions than _Azotobacter_. The limiting degree of acidity
has been found to vary among different varieties of the organism from
P_{H} 3·15 to P_{H} 4·9.

A long controversy has been held as to whether the nodule organisms
found in different host-plants all belong to one species, or whether
there are a number of separate species, each capable of infecting a
small group of host-plants. As the term “species” has at present no
exact meaning when applied to bacteria, the discussion in this form
is unlikely to reach a conclusion. The evidence seems to show that
the nodule organisms form a group that is in a state of divergent
specialisation to life in different host-plants, and that this
specialisation has reached different degrees with different hosts.
Thus the organisms from the nodule of the pea (_Pisum sativum_) will
also produce nodules on vicia, Lathyrus, and Lens, but seem to have
lost the ability normally to infect other legumes. On the other hand,
the bacteria from the nodules of the Soy Bean (_Glycine hispida_)
have become so specialised that they do not infect any other genus
of host-plant, and soy beans are resistant to infection by other
varieties of the nodule organism. Burrill and Hansen,[6] after an
extensive study, divided the nodule bacteria into eleven groups, within
each of which the host-plants are interchangeable. The existence of
different groups of nodule organisms has been confirmed by the separate
evidence of serological tests (Zipfel, Klimmer, and Kruger).[40] The
results of cross-inoculation tests have sometimes been conflicting. It
seems, indeed, that the host-plant has a variable power of resisting
infection, so that when its resistance is lowered it may be capable of
infection by a strange variety of the nodule organism. The question
that has thus arisen of the ability of the legume to resist infection
is of fundamental importance, and its elucidation should throw light on
the relation of plants to bacterial infection as a whole.

The stage of the organism that infects the plant is not at present
known. It may be supposed that it is the motile “swarmer.” The entry is
normally effected through the root-hairs. The hair is attacked close
to the tip, and an enzyme is apparently produced which causes the tip
to bend over in a characteristic manner. The organisms multiply within
the root hair and pass down it, producing a characteristic gelatinous
thread filled with bacteria, in the rod form. This “infection thread”
passes down into the cells of the root tissue, where it branches
profusely. In young stages of nodule formation the branches can be seen
penetrating cells in the pericycle layer. Rapid cell division of these
root cells is induced. In the course of this cell division abnormal
mitotic figures are sometimes found, such as occur in pathological
growths. The cells push outward the root cortical layer, and so form a
nodule.

Certain of the cells in the centre of the nodule become greatly
enlarged, and in the fully grown nodule are seen to be filled with
bacteria. Differences have been described in the morphology of the
organisms in different parts of the nodule.[62] Whether the different
stages of the organism are equally capable of fixing nitrogen, or what
is the significance of these stages within the nodule, is not certainly
known. It has been held that it is the irregular bacteroid forms that
are chiefly concerned with nitrogen fixation. In older nodules the
organisms become irregular and stain faintly, and the bacteroidal
tissue breaks down, the nodule finally decaying. In the fixation of
nitrogen that occurs in the nodules, the bacteria without doubt derive
the necessary energy from the carbohydrates of the host-plant. There
is evidence that the plant assists the process of fixation by removing
soluble metabolic products from the neighbourhood of the bacteria.
Golding[22] was able to obtain a greatly increased fixation of nitrogen
in artificial cultures by arranging a filtering device so as to remove
the products of metabolism.

The great practical importance of leguminous crops in agriculture has
led to numerous attempts being made to increase their growth, and the
fixation of nitrogen in them, by inoculating the seed or the soil with
suitable nodule-bacteria. This inoculation can be effected either with
soil in which the host-plant has been successfully grown, and which
should consequently contain the organism in fair numbers, or else pure
cultures of the organisms isolated from nodules may be used. Very
varying results have been obtained with inoculation trials.

In farm practice a leguminous crop has often been introduced into a
new area where it has never previously grown. In such soil it is very
probable that varieties of the nodule organism capable of infecting the
roots may not exist. In such cases inoculation with the right organism
or with infected soil often produces good results.

The more difficult case, however, is that in which the legume crop
has been grown for a long time in the locality, and where the soil
is already infected with right organisms. This, the more fundamental
problem, applies especially to this country. Here it would seem that
inoculation with a culture of the organism will benefit the plant only
(1) if the naturally occurring organisms are present in very small
numbers; or (2) if the organisms in the culture added are more virulent
than those already in the soil. The problem of successful inoculation
would therefore seem to be bound up with that of grading up the
infective virulence of the organism to a higher level.

Successful nodule development in a legume crop is also dependent to a
large degree on the soil conditions. The effects of soil conditions on
nodule development have been studied by numerous workers. Moisture has
been found very greatly to affect the nodule development. Certain salts
have a very definite effect on nodule formation.[64] Their effect on
the number of nodules developing has been studied, but the reason for
this effect is unusually difficult to decide. The action is usually a
complex one. Thus phosphates are known to stimulate nodule formation.
They probably act in several ways. In the first place, they may cause
the nodule organisms to multiply in the soil; in the second place, they
produce a greater root development in the plant, thus increasing the
chances of infection; and in the third place, Bewley and Hutchinson[4]
have found that phosphates cause the appearance of the motile stage
of the organism in cultures. A real understanding of the influence of
environment on nodule production will produce great improvements in our
methods of legume cropping.

_D._ THE RELATION OF BACTERIAL ACTIVITIES TO SOIL FERTILITY.

The various activities of the soil bacteria have a vital importance to
the growth of higher plants, which are dependent for their existence
on certain of these processes. In the first place, as we have seen,
bacteria decompose the tissues of higher plants and produce humus
materials, which are essential to the maintenance of good physical
properties in the soil. Then the nitrate supply on which most higher
plants depend is produced by the decomposition of organic nitrogen
compounds by bacteria in their search for energy. The depletion of the
total nitrogen content of the soil through rain and through the removal
of nitrogen in the crops, is to some extent compensated by the fixation
of atmospheric nitrogen by certain bacteria. On the other hand, in the
assimilation of nitrogen compounds to build up protein, the bacteria
are competing with higher plants for one of their essential food
constituents, and their action may, under certain conditions, cause a
temporary nitrogen starvation. One must remember, however, that large
quantities of nitrate are lost from field soils by washing-out through
rain action, especially in winter. The assimilation of nitrate and
ammonia by micro-organisms keeps some of this nitrogen in the soil, and
at certain periods may thus be beneficial.

There is another important respect in which soil bacteria influence
plant growth. Their activities result in the release of inorganic
salts, such as potash and phosphates, in a form available for the use
of plants. The release of phosphorus and potassium compounds takes
place in two ways. In the first place, organic matter containing
phosphorus and potassium, in an insoluble form, is attacked by
bacteria, resulting in these elements being set free as inorganic
salts available to the higher plant. Secondly, much of the phosphorus
supplied to the soil from rock minerals is present as insoluble
phosphates, such as apatite and iron phosphate. Much of the potassium,
too, is derived from insoluble silicate minerals. In both cases the
conversion of the insoluble minerals into soluble phosphates and
potassium compounds is brought about to a large extent by solution in
water containing carbonic and other acids. These acids are largely
produced by micro-organisms, which, in addition to carbonic acid,
produce organic acids, and in specialised cases, sulphuric and nitrous
acids. It has been found, for example, that in a compost of soil
with sulphur and insoluble phosphate, sufficient sulphuric acid may
be produced by the oxidation of the sulphur by bacteria to convert
an appreciable amount of phosphate into a soluble form. When we
consider the functions performed by soil bacteria, therefore, it is
not surprising to find that high bacterial activity in the soil is
associated, as a rule, with fertility.

_E._ CHANGES IN BACTERIAL NUMBERS AND ACTIVITIES, AND THEIR RELATION TO
EXTERNAL FACTORS.

The object of soil bacteriologists is to discover means of favouring
the activity of soil bacteria, especially those activities that are
useful to the higher plant. Knowledge is therefore needed of the
changes in numbers and activities of the soil bacteria, and of the
influence of soil conditions on them. The necessity of studying these
changes has required the development of a quantitative technique by
which the numbers of bacteria in the soil and their activities can be
estimated.

The method commonly used in counting bacteria in soil is a modification
of the plating method of Koch. In counting bacteria two difficulties
have to be overcome--their immense numbers and their small size. The
numbers of bacteria in soil are so large that the bacterial population
of a gram of soil could not, of course, be counted directly. The method
adopted, therefore, is to make a suspension of soil in sterile salt
solution, and to dilute this suspension to a convenient and known
extent, which will depend on the numbers of bacteria expected. In
ordinary field soils it is found convenient, for example, to dilute the
soil suspension so that one cubic centimeter of the diluted suspension
will contain 1/250,000th of a gram of soil. Such a volume will commonly
contain a number of bacteria sufficiently small to count. The second
difficulty is that the organisms are microscopic, and yet cannot be
readily counted under the microscope owing to the presence of soil
particles in the suspension. Hence recourse is had to plating. One
cubic centimeter of diluted suspension is placed in a petri dish and
mixed with a suitable nutrient agar medium, melted, and cooled to about
40° C. The medium sets, and after a few days’ incubation the organisms
multiply and produce colonies visible to the naked eye. By counting
these colonies we obtain an estimate of the number of bacteria in
the one cubic centimeter of suspension, it being assumed that every
organism has developed into one colony, and by multiplying this number
by the degree of dilution we obtain the numbers per gram of soil.
In practice a number of parallel platings are made from one cubic
centimeter portions of the diluted suspension and the mean number of
colonies per plate is taken. By this means the error due to the random
distribution of bacteria in the suspension is reduced, because of the
greater number of organisms counted.

In drawing conclusions from bacterial count data, it is necessary to
distinguish between the indication which the method gives of the
absolute numbers of bacteria in the soil and the accuracy with which
it enables the numbers of two soil samples to be compared. The method
cannot be used for the former purpose at present. We do not know how
far the figures obtained by this counting method fall short of the
actual number of bacteria in the soil. One reason for this is the
difficulty of effecting a complete separation of the clumps of bacteria
into discrete individuals in the suspension. Then again, there is no
known medium upon which all the physiological groups of bacteria will
develop and produce colonies. And even on a suitable medium some of the
individuals may fail to multiply.

In comparing the bacterial numbers in two soil samples, however,
the case is different. Within each bacterial group investigated the
plate method should give counts proportional to the bacterial numbers
in the soil. Thus, by the method one should be able to tell whether
the bacterial numbers are increasing or decreasing over a period of
time, or whether a certain soil treatment produces an increase or
a decrease. With this end in view the technique of the method has
been improved by recent workers. It was found that, when carefully
standardised, the process of dilution of the soil could be carried
out without significant variation in result (Table IV.), and that the
accuracy of the method is limited mainly by the variation in colony
numbers on parallel platings, due in part to random distribution of
bacteria throughout the final suspension, and partly to the uneven
development of colonies on the medium. The question of the medium was
therefore taken up with a view to improving the uniformity of results
obtained with it. Lipman, Conn, and others effected an improvement by
using chemical compounds as nutrient ingredients, thus making their
media more closely reproducible. On most agar media, an important
disturbing factor is the growth of spreading colonies, which prevent
the development of some of the other colonies. A medium has been
devised at Rothamsted on which these spreading organisms are largely
restricted.[61] A statistical examination[19] has shown that on this
medium errors due to the uneven development of colonies, except in
special cases, are prevented, so that in fact the variation in colony
numbers between parallel plates is found to be that produced merely by
random distribution of bacteria in the diluted suspension (see Table
IV.). In this case the accuracy of the counts of the bacteria in the
diluted suspension depend directly on the number of colonies counted,
and can be known with precision.

TABLE IV.--BACTERIAL COUNTS OF A SOIL SAMPLE.

PARALLEL PLATE COUNTS FROM FOUR SETS OF DILUTIONS MADE BY DIFFERENT
WORKERS.

+------------------------------------------+
| Counts of Colonies on each Plate. |
+------+--------+--------+--------+--------+
|Plate.| Set I. | Set II.|Set III.| Set IV.|
+------+--------+--------+--------+--------+
| 1 | 72 | 74 | 78 | 69 |
| 2 | 69 | 72 | 74 | 67 |
| 3 | 63 | 70 | 70 | 66 |
| 4 | 59 | 69 | 58 | 64 |
| 5 | 59 | 66 | 58 | 62 |
| 6 | 53 | 58 | 56 | 58 |
| 7 | 51 | 52 | 56 | 54 |
+------+--------+--------+--------+--------+
| Mean | 60·86 | 65·86 | 64·28 | 62·86 |
+------+--------+--------+--------+--------+

Standard deviation between the four sets = 5·62.

Standard deviation between plates within the sets = 7·76.

The knowledge obtained from counts of soil bacteria is subject to
another serious limitation. We do not know which of the bacteria
counted are the most effective in bringing about the various changes
that take place in the soil. It is not even known which of them are
active in the soil and which are in a resting condition. It is thus
possible to have two soils containing equal numbers of bacteria but
showing widely different biochemical activity, if one soil contains
organisms of a higher efficiency. Moreover, as has been pointed
out, many important groups of soil bacteria do not develop on the
plating media, and so are not counted. These considerations led to
the development of supplementary methods by which it was hoped to
estimate the actual biochemical activity of the soil microflora. The
first of these methods was developed by Remy, who attempted to study
the biochemical activity of a soil by placing weighed amounts into
sterile solutions of suitable and known composition, keeping them
under standard conditions for a definite time and then estimating the
amount of the chemical change that was being studied. Thus, to test the
activity of the organisms that produce ammonia from organic nitrogen
compounds, he inoculated soil into 1 per cent. peptone solution and
measured the amount of ammonia produced in a given time. By similar
methods the power of a soil to oxidise ammonia to nitrate, to reduce
nitrate, or to fix atmospheric nitrogen, is tested. This method has
been extensively used and developed by more recent workers. It suffers,
however, from the same serious disadvantage that it was designed to
avoid, for we cannot be certain that those bacteria that develop in
the nutrient solution are the types that are active in the soil, and,
moreover, even where the same types do function in the two conditions,
we do not know that the degree of their activity is the same in soil
and in solution cultures. For instance, _Nitrosomonas_ appears to show
very different degrees of activity in soil and in culture.

Another method, therefore, of studying the activity of soil
micro-organisms is the obvious one of estimating the chemical changes
that they produce in the soil itself. This method has obvious
advantages over the unnatural methods developed from Remy’s, but it has
a number of limitations that make its actual application difficult.
In the first place, we cannot always tell whether changes found to
occur in soil are due to the activity of micro-organisms, or are
purely chemical reactions unassisted by biological agencies. Then, if
we succeed in showing that the changes are due to micro-organisms, it
is very difficult to determine which organisms are effecting them.
This cannot be definitely tested by isolating suspected organisms and
testing their activity in sterile soil, because in sterilising soil its
nature and composition is altered. In spite of these difficulties,
however, the study of the chemical changes that take place in the soil
has produced valuable knowledge, when it has been combined with a
study of the changes in the number and variety of the micro-organisms
that accompany these reactions. This method of investigation is well
illustrated by the work of Russell and Hutchinson on the effects of
heat and volatile antiseptics on soil, where a study of the chemical
changes such as ammonia production, that occurred in these treated
soils, combined with a study of the changes in bacterial numbers, led
to the realisation that the soil micro-population was a complex one,
containing active protozoa.

A great difficulty in applying quantitative methods to bacteria in the
field is the great variation in the density of the bacterial population
over a plot of field soil, which may be so great that a bacterial count
from a single sample is quite valueless. For example, the distribution
of bacterial numbers over a plot of arable soil near Northampton was
studied by taking sixteen samples distributed over an area about
12 feet square. The result showed that in some cases the bacterial
numbers in samples taken 6 inches apart differed by nearly 100 per
cent. Fortunately, under favourable conditions, a remarkably uniform
distribution of bacterial numbers over a plot of soil can be found.

On such a plot it is possible to investigate the rapidity with which
the numbers of the soil micro-organisms alter in point of time. For
example, on the dunged plot of Barnfield, Rothamsted, which has been
cropped with mangolds for forty-seven successive years, the area
distribution of bacteria has been found to be so uniform that if a
number of samples of soil are taken from the plot at the same time, the
difference in bacterial numbers between the samples cannot be detected
by means of the counting technique (see Table V.). The work of Cutler,
Crump, and Sandon[16] on this plot showed that the bacterial numbers
vary very greatly from one day to the next, and that these fluctuations
took place over the whole plot, since two series of samples, taken in
two rows 6 feet apart, showed similar fluctuations (see Fig. 7). The
discovery of these big daily fluctuations in numbers led to an inquiry
as to how quickly bacterial numbers change, and samples from Barnfield,
taken at two-hourly intervals, showed that significant changes in
numbers took place even at such short intervals.

TABLE V.--BACTERIAL COUNTS OF FOUR SOIL SAMPLES.

FROM BARNFIELD, TAKEN SIMULTANEOUSLY.

+------------------------------------------------------+
| Counts of Colonies on each Plate. |
+------+-----------+-----------+-----------+-----------+
|Plate.| Sample I. | Sample II.|Sample III.| Sample IV.|
+------+-----------+-----------+-----------+-----------+
| 1 | 38 | 45 | 43 | 27 |
| 2 | 32 | 40 | 34 | 41 |
| 3 | 52 | 45 | 52 | 35 |
| 4 | 32 | 31 | 55 | 36 |
| 5 | 40 | 43 | 38 | 45 |
|Mean | 38·8 | 40·8 | 44·4 | 36·8 |
+------+-----------+-----------+-----------+-----------+

Standard deviation between the four samples = 7·25.

Standard deviation between parallel plates within the sets = 7·55.

[Illustration: FIG. 7.

X-axis (top): Days.

Y-axis (left): (Series A) Bacteria--millions per gramme of soil.

Y-axis (right): (Series B) Bacteria--millions per gramme.

Caption: Daily changes in bacterial numbers in field soil.

Counts from two series of soil samples taken 6 feet apart.

(After Cutler.)]

Since the bacteria involved in this fluctuation are of great importance
to the crops, being for the most part ammonia producing types, further
knowledge as to the cause of this fluctuation and of its effect on the
ammonia and nitrate in the soil is of fundamental importance. There is
evidence, which will be discussed later, that the cause is connected
with the changing activities of certain soil protozoa, since the daily
changes in the numbers of active amœbæ in the soil have been found
to be in the reverse direction to those of the bacterial numbers. It
appears, therefore, that we are dealing with an equilibrium between
the various members of the soil population, the point of equilibrium
changing at frequent intervals.

In addition to daily changes, it is possible to detect changes in the
numbers and activity of the soil population related to the season.
There is a well-marked increase in the spring and autumn (see Figs.
15, 16, pp. 89, 90). This is well seen when the fortnightly averages
of the daily bacterial and protozoal counts from Barnfield soil
are plotted. These spring and autumn increases comprise both the
bacterial and the protozoal population, and therefore differ from the
short time fluctuations in being due, not to a disturbance of the
bacteria-protozoa equilibrium, but to a general rise in activity of
both groups of organisms.

When we consider the action of external conditions on the soil
bacteria, the existence of a complex soil population and the
interdependence of its members must be borne in mind. Changes in
external conditions may affect the different components of the
population in different ways or to different degrees, thus upsetting
the equilibrium between the various groups. For example, the addition
of a mild aromatic antiseptic to the soil apparently affects the
protozoa in such a way as to disturb the bacteria-protozoa equilibrium
in favour of the bacteria, while in some cases the aromatic compound
affords a food supply to special bacteria, causing these to increase,
upsetting the equilibrium between the different bacterial groups. When
our knowledge of the effect of external factors on the soil population
becomes sufficient, it will probably be found that in nearly all
cases a change in the soil conditions produces some disturbance in
the equilibrium between the components of the soil population, though
at present there are only certain examples where this disturbance is a
probable explanation of the facts.

Since bacteria are dependent on adequate supplies of energy and food,
it is to be expected that additions of organic matter or of inorganic
food materials will greatly benefit their activities. The effect of
added farmyard manure in increasing bacterial activities has been much
studied.[27] Some of the increased bacterial numbers and activities
in this case may be due to the addition of bacteria with the manure,
but it is thought that this factor is of less importance than the
added energy and food supply which the general soil flora obtain from
it. Nutritive salts such as phosphates and salts of potassium usually
increase the bacterial activities.

The effect of alkali salts on soil bacteria has been especially studied
in the Western United States, where the existence of alkali in the
soil is a serious problem.[23] Soil bacteria are usually stimulated by
small doses of alkali salts that are toxic in higher concentration.
As a rule, chlorides are the most toxic salts, the electronegative
ion playing an important part in the effect of the salt. Salts
affect bacteria both owing to the changes in osmotic pressure which
they produce, and through their specific action on the bacterial
protoplasm.[26] When equal weights of various salts are added to soil,
their toxic action on bacteria shows so little association with their
respective osmotic pressures that we must conclude that this factor is
the less important. There is reason to suppose that the toxic action
of salts on bacteria is often connected with an effect of the specific
ions on the permeability of the bacterial cell-wall. This conclusion
is based on the changes in electrical conductivity of bacterial
suspensions in the presence of various salts.[59]

A definite antagonism between various salts has been found to exist.
It is possible that future work in this line may indicate what are
the proportions of common electrolytes which will produce a properly
“balanced” soil solution so that the harmful excess of one salt may be
antagonised.

Certain salts, such as those of arsenic[24] and manganese, seem to
exercise a stimulating action on bacterial activities; the causes of
this action are not at present understood.

The acidity of the soil has an important effect on the bacterial
processes. The acidity of soils may increase to such a point that the
decomposition of plant tissues by bacteria is hindered, a peat layer
being thus produced. The degree of acidity that is toxic varies very
greatly with different soil bacteria, some of them, like Azotobacter
and Nitrosomonas being very intolerant of acidity.

The conditions of aeration, water content, and temperature are
inter-related in field soil. Ammonifying organisms are not greatly
dependent on aeration, but this factor is sometimes a limiting one in
the case of the very aerobic nitrifying bacteria. Hence efficient soil
cultivation is beneficial to nitrification.

Many attempts have been made to correlate the temperature and moisture
of field soils with the bacterial numbers and activities. These
attempts have given very discordant results. It is generally agreed
that a plentiful moisture supply is beneficial. Thus Greaves, in Utah,
found the optimum water content for ammonia and nitrate production
to be about 60 per cent. of the water-holding capacity. On the other
hand, Prescott[56] found that the summer desiccation of soil in
Egypt was followed by increased bacterial activities. Fabricius and
Feilitzen,[18] using moor soil, found a direct relationship between
soil temperature and bacterial numbers, showing that temperature can
be a limiting factor under certain conditions. With normal arable
soils, however, no such direct effect of temperature or moisture can
be found[16] (see Fig. 8). It has even been found by Conn[11] that
freezing of the soil may cause a marked increase in bacterial numbers.
The erratic effects of temperature and moisture on the soil bacteria
probably afford instances of a disturbance of the equilibrium between
the bacteria and other components of the soil micro-population. Thus
desiccation and freezing, though they harmfully affect the bacteria,
may inhibit other micro-organisms to a greater degree, thus freeing
the bacteria from competition. It is in the investigation of this
equilibrium, and of the factors that can control it to our benefit,
that the great advances in soil biology in the future are to be
expected.

[Illustration: FIG. 8.--Effect of frost on the bacterial numbers in the
soil. (After CONN.)

X-axis: Nov.-May

Y-axis (bottom): Temperature--Degrees C.

Y-axis (top): Bacteria--Millions per Gramme of Soil.]

REFERENCES TO CHAPTERS II. AND III.

[1] Ashby, S. F., Journ. Agric. Sci., 1907, vol. ii., p. 35.

[2] Beijerinck, M. W., Centr. f. Bakt., 1900, Abt. II., Bd. 6, p. 1.

[3] Beijerinck, M. W., and Van Delden, A., Centr. f. Bakt., 1902,
Abt. II., Bd. 9, p. 3.

[4] Bewley, W. F., and Hutchinson, H. B., Journ. Agric. Sci., 1920,
vol. x., p. 144.

[5] Bonazzi, E., Journ. Bact., 1921, vol. vi., p. 331.

[6] Burrill, T. J., and Hansen, R., Illin. Exp. Sta., 1917, Bulletin
202.

[7] Christensen, H. R., Centralblatt. f. Bakt., 1915, Abt. II., Bd.
43, p. 1.

[8] Christensen, H. R., Centralblatt. f. Bakt., 1907, Abt. II., Bd.
17, pp. 109, 161.

[9] Christensen, H. R., and Larsen, O. H., Centralblatt. f. Bakt.,
1911, Abt. II., Bd. 29, p. 347.

[10] Conn, H. J., Centralblatt. f. Bakt., 1910, Abt. II., Bd. 28, p.
422.

[11] Conn, H. J., Centralblatt. f. Bakt., 1914, Abt. II., Bd. 42, p.
510.

[12] Conn, H. J., Journ. Bact., 1916, vol. i., p. 187.

[13] Conn, H. J., Journ. Bact., 1917, vol. ii., p. 137.

[14] Conn, H. J., Journ. Bact., 1917, vol. ii., p. 35.

[15] Cramer, E., Arch. f. Hyg., 1893, Bd. 16, p. 151.

[16] Cutler, W., Crump, L. M., and Sandon, H., Phil. Trans. Roy.
Soc., 1923, Series B, vol. ccxi., p. 317.

[17] Doryland, C. J. T., N. Dakota Agr. Exp. Sta., 1916, Bulletin 116.

[18] Fabricius, O., and Feilitzen, H., Centr. f. Bakt., 1905, Abt.
II., Bd. 14, p. 161.

[19] Fisher, R. A., Thornton, H. G., and Mackenzie, W. A., Ann. Appl.
Biol., 1922, vol. ix., p. 325.

[20] Fred, E. B., and Hart, E. B., Wisconsin Agr. Exp. Sta. Research,
1915, Bulletin 35.

[21] Gainey, P. L., Journ. Agric. Research, 1918, vol. xiv., p. 265.

[22] Golding, J., Journ. Agric. Sci., 1905, vol. i., p. 59.

[23] Greaves, J. E., Soil Sci., 1916, vol. ii., p. 443.

[24] Greaves, J. E., Journ. Agric. Res., 1916, vol. vi, p. 389.

[25] Greaves, J. E., Soil Sci., 1920, vol. x., p. 77.

[26] Greaves, J. E., and Lund, Y., Soil Sci., 1921, vol. xii., p. 163.

[27] Greaves, J. E., and Carter, E. G., Journ. Agric. Research, 1916,
vol. vi., p. 889.

[28] Groenewege, J., Arch. Suikerindust., 1913, Bd. 21, p. 790.

[28_b_] Green, H. H., Union of S. Africa Dept. Agr., Rept. of
Director Vet. Res., 1918, p. 592.

[29] Hutchinson, C. M., Rept. Agr. Res. Inst. and Col. of Pusa, 1912,
p. 85.

[30] Hutchinson, H. B., and Clayton, J., Journ. Agric. Sci., 1919,
vol. ix., p. 143.

[30_b_] Hutchinson, H. B., and Richards, H. H., Journ. Min. Agric.,
1921, vol. xxviii., p. 398.

[31] Hanzawa, J., Centr. f. Bakt., 1914, Abt. II., Bd. 41, p. 573.

[32] Hopkins, C. G., and Whiting, A. L., Ill. Agr. Exp. Sta., 1916,
Bulletin 190, p. 395.

[33] Hoppe-Seyler, G., Ztschr. Phys. Chem., 1886, vol. x, pp. 201,
401; 1887, vol. xi., p. 561.

[34] Hesselmann, H., Skogsvårdsför. Tidskr., 1917, No. 4, p. 321.

[35] Joshi, N. V., Mem. Dept. Agr. in India, Bact. Ser., 1920, vol.
i., No. 9.

[36] Koch, R., Mitt. Kais. Gesundh., 1881, vol. i., p. 1.

[37] Kaserer, H., Centr. f. Bakt., 1906, Abt. II., Bd. 16, p. 681.

[38] Kaserer, H., Centr. f. Bakt., 1905, Abt. II., Bd. 15, p. 573.

[39] Krainskii, A. V., Centr. f. Bakt., 1910, Abt. II., Bd. 26, p.
231.

[40] Klimmer, M., and Kruger, R., Centr. f. Bakt., 1914, Abt. II.,
Bd. 40, p. 257.

[41] Krzeminiewski, S., Centr. f. Bakt., 1909, Abt. II., Bd. 23, p.
161.

[42] Koch, A., and Seydel, S., Centr. f. Bakt., 1912, Abt. II., Bd.
31, P. 570.

[43] Lipman, C. B., Bot. Gaz., 1909, vol. xlviii., p. 106.

[44] Lipman, C. B., and Burgess, P. S., Centr. f. Bakt., 1914, Abt.
II., Bd. 41, p. 430.

[45] Lipman, C. B., and Burgess, P. S., Centr. f. Bakt., 1915, Abt.
II., Bd. 44, p. 481.

[46] Lipman, C. B., and Waynick, D. O., Soil Sci., 1916, vol. i., p.
5.

[47] Löhnis, F., and Pillai, N. K., Centr. f. Bakt., 1908, Abt. II.,
Bd. 20, p. 781.

[47_b_] Löhnis, F., and Smith, T., Journ. Agric. Res., 1914, vol.
vi., p. 675.

[48] Mackenna, J., Rept. Prog. Agric., India, 1917, p. 101.

[49] Marchal, E., Bull. Acad. Roy. Belgique, 1893, vol. xxv., p. 727.

[50] McBeth, I. G., and Scales, F. M., U.S. Dept. Ag., Bureau Plant
Indus., 1913, Bulletin 266.

[51] Mockeridge, J., Biochem. Journ., 1915, vol. ix., p. 272.

[52] Nabokich, A. J., and Lebedeff, A. F., Centr. f. Bakt., 1906,
Abt. II., Bd. 17, p. 350.

[53] Nagaoka, M., Bull. Coll. Agr., Tokyo, 1900, vol. vi., No. 3.

[54] Omelianski, W. L., Comptes Rendus Acad. Sci., 1895, vol. cxxi.,
p. 653; 1897, vol. cxxv., pp. 907, 1131; Arch. Sci. Bio., (St.
Petersburg), 1899, vol. vii., p. 411.

[55] Omelianski, W. L., and Sohmskov, M., Arch. Sci. Biol., Publ.
Inst. Imp. Med. Exp. (Petrograd), 1916, vol. xviii., pp. 327, 338,
459; vol. xix., p. 162.

[56] Prescott, J. A., Journ. Agr. Sci., 1920, vol. x., p. 177.

[57] Söhngen, N. L., Centr. f. Bakt., 1905, Abt. II., Bd. 15, p. 513.

[58] Sen Gupta, N., Journ. Agr. Sci., 1921, vol. xi., p. 136.

[59] Shearer, C., Journ. Hyg., 1919, vol. xviii., p. 337.

[60] Tappeiner, Ber. Deut. Chem. Gesell., 1883, vol. xvi., p. 1734;
Zeitsch. Biol., 1884, vol. xx., p. 52.

[61] Thornton, H. G., Ann. Appl. Biol., 1922, vol. ix., p. 241.

[62] Wallin, I. E., Journ. Bact., 1922, vol. vii., p. 471.

[63] Waksman, S. A., and Joffe, J. S., Journ. Bact., 1922, vol. vii.,
p. 239.

[64] Wilson, J. K., Cornell Agric. Exp. Sta., 1917, Bulletin 386.

CHAPTER IV.

PROTOZOA OF THE SOIL, I.

That protozoa could be isolated from the soil was a matter of common
knowledge to the biologists of the nineteenth century, but not until
the early part of the present century was it suggested that these
organisms might be playing some part in the general economy of the soil
micro-population. Of recent years a great deal of our knowledge of the
cytology of the different groups of protozoa, especially the Amœbæ, has
been obtained from the study of representatives normally living in the
soil; but unfortunately little or no knowledge has been gained of the
biology of these animals in their natural habitat.

The view that the presence of these organisms in excessive numbers may
lead to “soil sickness” was first put forward by Russell and Hutchinson
in 1909, and elaborated in their further papers dealing with “Partial
Sterilisation of the Soil.”

It is unnecessary to discuss in detail this important branch of
agriculture, but to obtain a clear idea of the development of
the study of soil protozoa it is necessary to give as briefly as
possible the conclusions deduced by Russell and Hutchinson from their
extensive experiments on soils treated with steam and various volatile
antiseptics[21],[22]:--

“(1) Partial sterilisation of the soil causes first a fall, then a
rise, in bacterial numbers, which goes on till the numbers considerably
exceed those present in the original soil.

“(2) Simultaneously there is a marked increase in the rate of
accumulation of ammonia which is formed from organic nitrogen
compounds.

“(3) The increase in bacterial numbers is the result of improvement in
the soil as a medium for bacterial growth, and not an improvement in
the bacterial flora.

“(4) The improvement in the soil brought about by partial sterilisation
is permanent, the high bacterial numbers being kept up even for 200
days or more. It is evident from (3) and (4) that the factor limiting
bacterial numbers in ordinary soil is not bacterial, nor is it any
product of bacterial activity, nor does it arise spontaneously in soils.

“(5) But if some of the untreated soil is introduced into partially
sterilised soil, the bacterial numbers, after the initial rise, begin
to fall. Thus the limiting factor can be reintroduced from untreated
soils.

“(6) Evidence of the limiting factor in untreated soils is obtained
by studying the effect of temperature on bacterial numbers. Untreated
soils were maintained at 10°, 20°, 30° C. in a well-moistened aerated
condition, and periodical counts were made of the numbers of bacteria
per gram. Rise in temperature rarely caused any increase in bacterial
numbers. But after the soil was partially sterilised the bacterial
numbers showed the normal increase with increasing temperatures.

TABLE VI.

+--------+------------------------+------------------------+
| | | Soil Treated |
| | Untreated Soil. | with Toluene. |
|Tempera-+------+-----+-----+-----+------+-----+-----+-----+
| ture of| |After|After|After| |After|After|After|
|Storage.| At | 13 | 25 | 70 | At | 13 | 25 | 70 |
| °C. |Start.|Days.|Days.|Days.|Start.|Days.|Days.|Days.|
+--------+------+-----+-----+-----+------+-----+-----+-----+
| 5°-12° | 65 | 63 | 41 | 32 | 8·5 | 73 | 101 | 137 |
| 20° | 65 | 41 | 22 | 23 | 8·5 | 187 | 128 | 182 |
| 30° | 65 | 27 | 50 | 16 | 8·5 | 197 | 145 | 51 |
| 40° | 65 | 14 | 9 | 33 | 8·5 | 148 | 52 | 100 |
+--------+------+-----+-----+-----+------+-----+-----+-----+

“(7) It is evident, therefore, that the limiting factor in the
untreated soils is not the lack of anything, but the presence of
something active. The properties of the limiting factor are:--

“(_a_) It is active and not a lack of something.

“(_b_) It is not bacterial.

“(_c_) It is extinguished by heat or poisons.

“(_d_) It can be re-introduced into soils from which it has been
extinguished by the addition of a little untreated soil.

“(_e_) It develops more slowly than bacteria.

“(_f_) It is favoured by conditions favourable to trophic life in the
soil, and finally becomes so active that the bacteria become unduly
depressed.

“It is difficult to see what agent other than a living organism can
fulfil these conditions. Search was therefore made for a larger
organism capable of destroying bacteria, and considerable numbers of
protozoa were found. The ciliates and amœbæ are killed by partial
sterilisation. Whenever they are killed the detrimental factor is found
to be put out of action; the bacterial numbers rise and maintain a high
level. Whenever the detrimental factor is not put out of action, the
protozoa are not killed. To these rules we have found no exception.”

From such premises as the above Russell and Hutchinson founded the
“protozoa theory of partial sterilisation,” and at Rothamsted there was
commenced the serious study of these soil organisms.

Goodey was one of the early workers on this new subject, and added
considerably to our knowledge of the species living in normal soils,
and of the chemical constitution of the cyst wall of ciliates. He
also made investigations on the effects of various chemicals on the
micro-population of soils, but was unable to draw very definite
conclusions.[11]

One of the first criticisms raised against the protozoa theory of
partial sterilisation was that the protozoa were not normal inhabitants
of the soil, and were present only in small numbers, all of them in the
cystic, quiescent condition. It was further held that these cysts were
carried by the wind from dried-up ponds and streams. It is difficult to
trace the origin of this view, since early observers, viz., Ehrenberg
and Dujardin, in 1841, were of the opinion that the protozoa were
living in the trophic active condition in the soil, and it was not
until 1878 that Stein showed that free living protozoa can encyst. To
Martin and Lewin, however, must be ascribed the distinction of first
proving that the soil possesses an active protozoan population, for
by a series of ingenious experiments these observers isolated several
flagellates and amœbæ in a trophic condition from certain of the
Rothamsted soils.[18] The more recent work in this country has been
in the direction of devising new quantitative methods of research,
since by this means alone is it possible to elucidate many fundamental
questions.

In America and elsewhere experiments have been devised for testing the
conclusions of Russell and Hutchinson. Cunningham and Löhnis,[2] in
America, Truffaut and Bezssonoff,[24] in France, supply evidence in
favour of the theory, but most of the American work is in opposition to
it.

Sherman[23] is perhaps the most prominent in opposing the phagocytic
action of protozoa on soil bacteria in spite of the fact that certain
of his experimental results apparently show enormous decreases in
bacterial numbers in the presence of protozoa. In many of his soil
inoculation experiments, however, it was not demonstrated that his
active cultures remained alive after entering the soil.

The experimental difficulties of dealing with soil protozoa are
considerable, and without a thoroughly sound technique investigators
may easily go astray.

CLASSIFICATION.

The animal kingdom is divided into two main groups or sub-kingdoms--the
Protozoa and the Metozoa. In the latter the characteristic feature
is that the body is composed of several units, called cells, and
consequently such animals are often spoken of as multicellular. The
Protozoa, on the other hand, are usually designated as uni-cellular,
since their bodies are regarded as being homologous to a single unit or
cell of the metozoan body. For various reasons exception has been taken
by Dobell[9] and others to the use of the term uni-cellular, for, as
Dobell says, “If we regard the whole organism as an individual unit,
then the whole protozoan is strictly comparable with a whole metozoon,
and not with a part of it. But the body of a protozoan, though it shows
great complexity of structure, is not differentiated internally into
cells, like the body of a metozoon. Consequently it differs from the
latter not in the number of its cellular constituents, but in lacking
these altogether. We therefore define the sub-kingdom of the protozoa
as the group which contains _all non-cellular animals_.”

It should be pointed out that this view does not find favour with many
zoologists, but it is useful in bringing into prominence the fact
that each protozoan is comparable as regards its functions with the
multi-cellular animals.

The protozoa are again further divided into four main classes:--

I. Rhizopoda.
II. Mastigophora.
III. Ciliophora.
IV. Sporozoa.

Of the above classes, representatives of each of the first three are
found living in the soil, but up to the present there is no evidence
that any sporozoon is capable of living an active life in the soil,
though the cysts of such organisms may be present.

The class _RHIZOPODA_ consists of those protozoa whose organs of
locomotion and food capture are _pseudopodia_, that is, temporary
extensions of the living protoplasm. The body is typically naked, that
is to say, without any cuticular membrane, though in some forms, ex.
_Amœbæ terricola_, the external layer of protoplasm is thickened to
form a pellicle. A skeleton or shell may be present.

The class is further sub-divided into various sub-classes, only
two of which concern the soil protozoologist, viz., the _Amœbæ_
and the _Mycetozoa_, of which the most important representative is
_Plasmodiophora brassicæ_, which attacks the roots of many cruciferous
plants, causing the disease familiarly known as “Fingers and Toes.”

The _Amœbæ_ are again divided into two orders:--

(_a_) _Nuda_, without shell or skeleton;

(_b_) _Testacea_, with shells often termed _Thecamœbæ_.

Representatives of the “naked” amœbæ commonly found in soils are
_Nægleria (Dimastigamœba) gruberi_, _Amœba diploidea_ (possessing
two nuclei) and _A. terricola_, the last two forms possessing a
comparatively thick skin or pellicles. _Trinema enchelys_, _Difflugia
constricta_ and _Chlamydophrys stercorea_ are examples of soil
Thecamœbæ.

The class _MASTIGOPHORA_ consists of those protozoa whose typical modes
of progression are by means of flagella, whip-like filaments which, by
their continual lashing motion, cause movement of the animal.

The body may be naked or corticate. The only organisms which concern
the soil biologist belong to the _Flagellata_ order.

The Flagellates differ considerably among themselves, both as regards
their mode of feeding, and the number of flagella, thus making their
classification difficult and outside the scope of this book. Suffice
it to say that in the soil such organisms occur possessing one, two,
three or four flagella, ex. _Oicomonas termo_, _Heteromita globosus_,
_Dallengeria_ and _Tetramitus spiralis_. Further, their mode of feeding
may be _saprophytic_ in which nourishment is absorbed by diffusion
through the body surface in the form of soluble organic substances,
_holozoic_ where solid food particles are taken in, or _holophytic_ in
which food is synthesised by the energy of sunlight. This last group
is commonly spoken of as the _Phyto flagellates_, which are to all
intents and purposes unicellular algæ, and as such will be dealt with
in Chapter VI.

The class _CILIOPHORA_ consists of those protozoa whose typical organs
of locomotion are threads or cilia. These organisms can in one sense
be regarded as the highest of the protozoa, since in no other division
does the body attain so great a complexity of structure. Moreover, they
are typically characterised by a complicated nuclear apparatus with
the vegetative and generative portions separated into distinct bodies,
the macro-nucleus and the micro-nucleus. Their mode of nutrition is
_holozoic_, though recently Peters has brought forward evidence that
certain species can obtain their nourishment saprophytically.

The sub-class Ciliata comprises four orders, all of which are
represented in the soil.

I. _Holotricha._ The cilia are equal in length and uniformly
distributed over the whole body in the primitive forms, though
restricted to special regions in the specialised forms. Typical soil
forms are _Colpoda cucullus_, _Colpidium colpoda_.

II. _Heterotricha._ There is a uniform covering of cilia, and a
conspicuous spiral zone of larger cilia forming a vibratile membrane
and leading to the mouth.

III. _Hypotricha._ The body is flattened dorso-ventrally and the cilia
are often fused to form larger appendages or cirri confined to the
ventral surface. Movement is typically a creeping one. Typical soil
forms are _Pleurotricha_, _Gastrostylis_, _Oxytricha_.

IV. _Peritricha._ Typically of a sedentary habit and the cilia are
reduced to a zone round the adoral region of the body. A typical soil
form is _Vorticella microstomum_.

The above classification is far from complete, but should be sufficient
to give an idea of the general grouping of the organisms. For a more
detailed account reference must be made to the numerous text books on
protozoa.

LIFE HISTORIES.

The life history of each species has its own characteristic features as
regards nuclear division, etc., and in many forms, notably the amœbæ,
it is impossible to identify them with certainty unless the chief
stages of the life history are known. In general, however, the soil
protozoa pass through very similar phases and develop in a perfectly
straightforward way. Broadly speaking, there are two main phases of
the life history--a period of activity often mistermed vegetative, and
a period of rest. In the former the animal moves, feeds and reproduces,
while in the latter there is secreted round the body a thick wall,
capable of resisting adverse external influences. This condition is
termed the cystic stage, and by means of it the animals are distributed
from place to place by air, water, etc. Indeed, so resistant are the
cysts that many of them are capable of withstanding the action of the
digestive juices of the intestines of animals, through which they pass
to be deposited by the fæces on fresh ground.

This cystic stage of the life history is found in practically all
free-living protozoa, though it is not formed in exactly the same
manner in every case. In the majority of instances the cyst is the
product of a single organism, round which is formed a delicate
gelatinous substance which soon hardens and gradually acquires the
peculiar characters of the wall. Concerning the chemical nature of this
wall there is little known, but Goodey,[11] working on the cysts of
_Colpoda cucullus_, found it to be formed of a carbohydrate, different
from all carbohydrates previously described, to which the name “Cytose”
was given. When in this state the animals are able to remain dormant
for considerable periods until favourable conditions once more obtain
when the wall is ruptured and the animal again resumes the active phase
of its life history. This simple process is characteristic of such
species as _Heteromita globosus_, _Cercomonas spp._, and many others.
It will be noted that no increase of numbers, i.e. reproduction,
occurs. A more complex condition is, however, sometimes found, as, for
example, in the ciliate _Colpoda steinii_, where actual reproduction
into small animals takes place within the cyst.

Finally there is the less common type of cyst formation, such as is
found in the flagellate _Oicomonas termo_ described by Martin.[19] This
flagellate, in common with all other forms, reproduces by dividing into
two; the division of the nucleus initiating the process. At certain
undetermined periods of the life history, however, conjugation occurs
between two similar animals forming a large biflagellate body known as
the zygote. After swimming about for varying periods of time, during
which the size increases and a large vacuole appears, the zygote
secretes a thick wall, loses its flagella, and becomes a cyst. While in
this condition the two gamete nuclei fuse to form one, and eventually a
single _Oicomonas_ emerges from its cyst.

Similarly in _A. diploidea_ the cysts are formed after two individuals
have come together. In the young cysts two amœbæ are found in close
association, and according to Hartmann and Nägler[12] a sexual process
occurs inside the cyst involving a “reductive” division of the nuclei.
This requires confirmation, but it is certain that only one individual
comes out of the cysts, which originally contained two amœbæ.

Such cysts have been termed by some writers “reproductive,” evidently a
misleading term, since no increase in numbers, but rather a decrease,
results from the process. A better term is, perhaps, conjugation cyst.

In soil protozoa, then, three different modes of cyst formation obtain,
and failure to make the distinction inevitably leads to confusion.

Before leaving the question of life histories, reference must be made
to a peculiar and characteristic feature of _Nægleria gruberi_. This
amœba under certain circumstances assumes a free-swimming biflagellate
stage. After variable periods of time the flagella are lost and the
ordinary amœboid condition resumed. What are the factors concerned in
the production of flagellates is unknown, but flooding the coverslips
with distilled water is an effective method for causing their
appearance.

DISTRIBUTION OF SOIL PROTOZOA.

For both the bacteria and algæ observations have been made regarding
their distribution through successive depths of the soil; little can
be said, however, about the protozoa in this connection. It is certain
that they occur throughout the first six inches of the Rothamsted
soils, though their relative frequencies in the successive inches has
not been determined, but probably they are most abundant in the 2nd to
the 4th inch.

In this country experiments have not been made to determine whether
sub-soil normally contains protozoa; but from some South African
soil, taken under sterile conditions 4 ft. down and examined in this
laboratory, large numbers of protozoa were cultivated.

This soil, however, could not, for various reasons, be regarded as a
typical sub-soil.

Kofoid records the presence of _Nægleria gruberi_ in clay and rock
talus taken from the sides of excavations of over 20 ft. depth, but the
possibility of external infection does not appear to have been excluded.

The presence of protozoa is not peculiar to British soil since they
have been found by various workers in Germany, France, the United
States, and elsewhere. In view of their probable importance in the soil
economy there has been instituted a survey of the protozoan species of
soil from all parts of the world.

This work is in charge of Mr. Sandon, to whom I am indebted for the
following summary of his as yet unpublished research.

“The majority of soil protozoa (like the fresh-water forms) appear to
be quite cosmopolitan, for the species found in such widely separated
localities as England, Spitsbergen, Africa, West Indies, Gough Island
(in the South Atlantic) and Nauru (in the Pacific) are, with few
exceptions, identical. This distribution indicates an ability to
withstand an extremely wide range of conditions, for the same species
occurring in Arctic soils, which are frozen for the greater part of
the year, are found also in soils exposed to the direct rays of the
tropical sun. Even sand from the Egyptian desert contains protozoa,
though it seems probable that in such cases they must be present only
in the encysted condition for the greater part of the time.

“Not every sample of soil, however, contains all the species capable
of living in soil, but the local conditions determining the presence
or absence of any species are at present unknown. In general the
numbers, both of species and of individuals present, follow the number
of bacteria. They are consequently most numerous in rich moist soils.
The statement sometimes made that protozoa are most numerous in peaty
soils is based solely on the number of Rhizopod shells found in such
localities; but as most of these shells are empty, their abundance is
probably due simply to the slowness with which they disintegrate in
these soils where bacterial activity is low, they do not indicate a
great protozoal activity. Active protozoa do occur even in extremely
acid soils, but their numbers in such cases are low. The common soil
protozoa, in fact, appear to be as tolerant of differences in soil
acidity as they are of differences in climate, for many of the same
forms which occur in acid soils are found also in soils containing
high percentages of chalk. It is possible that some of the less common
species may be confined within closer limits of external conditions but
the information available on this point is inadequate. All the species,
however, which in Rothamsted soils occur in the highest numbers (e.g.
_Oicomonas termo_, _Heteromita spp._, _Cercomonas crassicauda_,
_Nægleria gruberi_, _Colpoda cucullus_, _C. steinii_) occur in
practically every soil which is capable of supporting vegetation,
though, of course, in very varying numbers.”

It is evident, therefore, that the protozoa must be regarded as
constituting part of the normal micro-organic population of soils,
and as such are probably playing an important rôle. Unfortunately our
knowledge of the physiology of these organisms is extremely scant, and
much of future research must be directed towards elucidating their
functions and their responses to varying environmental conditions.

CHAPTER V.

PROTOZOA OF THE SOIL, II.

In the preceding chapter an outline has been given of the development
of the study of soil protozoa, with especial reference to its
qualitative aspects.

Here it is proposed to deal with the quantitative methods which have
been devised for studying these organisms and the results obtained.

From the beginning great difficulty has been encountered in finding
means for counting protozoa; and most of the early results have been
obtained by the use of one of the following methods: (1) direct counts
in a known volume of soil suspension by means of a microscope; (2)
dilution method as used for counting bacteria, and suggested by Rahn,
who made dilutions of the soil and determined, by examination at
periodic intervals, the one above which protozoa did not grow; (3) Agar
plating as used by Killer; (4) counting per standard loop of suspension
as devised by Müller. Of these the two last have been little used,
and for various reasons are now discarded by most workers. Direct
methods have been used extensively in the United States by Koch[13]
and others,[16] who claim to have got satisfactory results; they are,
however, highly inaccurate and should be discontinued. The present
writer[3] has shown that there exists a surface energy relationship
between the soil particles and the protozoa, so that the two are always
in intimate contact; thus rendering it impossible to count under the
microscope the number of organisms in a given weight of soil suspension
(Fig. 9). Further, in a clay soil, such as is found at Rothamsted, the
clay particles alone make it very difficult to use such methods.

The demonstration of this surface energy relationship affords an
effective rejoinder to the criticism made against Russell and
Hutchinson’s hypothesis, viz., that soil protozoa must be very few in
numbers, since it was impossible to see them on examining soil under
the microscope.

[Illustration: FIG. 9.--Showing the number of amœbæ and flagellates
withdrawn from suspensions of varying strengths by different types of
solid matter. A = clay: B = partially sterilized soil: C = ignited
soil: D = fine sand: E = waste sand. Since complete withdrawal occurs
when the numbers of organisms added are less than the capacity of the
solid matter, the first part of each of the above curves is coincident
with the ordinate. The numbers of organisms are given in thousands.
(From Journ. Agric. Soc., vol. ix.)

X-axis: Number of Organisms per c.c. left in Solution.

Y-axis: Number of Organisms per c.c. taken up by Solid Matter.]

The second or dilution method is the one, therefore, that has been most
extensively developed.

Cunningham obtained concordant results in this way, and his method,
modified by L. M. Crump, was as follows: 10 grams of soil were added
to 125 c.c. of sterile tap-water and shaken for three minutes. This
gives a 1 in 12·5 dilution. From it further dilutions were made
until a sufficiently high one was obtained. Petri dishes, containing
nutrient agar, were inoculated with 1 c.c. of each of the dilutions and
incubated. At intervals covering 28 days the plates were examined and
the presence or absence of protozoa on each recorded. In this way the
approximate number of organisms per gram of soil could be found.

By methods essentially similar to this numerous counts have been made
of the bacteria and protozoa in field soil and in partially sterilized
soils. They were, however, inconclusive; thus, on the one hand,
Goodey and several American observers, found no correlation between
the numbers of protozoa and bacteria, while Miss Crump and Cunningham
obtained evidence pointing to the reverse conclusion.

Such divergence of opinion was probably mainly due to two causes:
firstly, that the time elapsing between the successive counts was too
long, for it has been shown recently that the number of bacteria and
protozoa fluctuate very rapidly; and secondly, the method was not
completely satisfactory since only the total numbers of protozoa were
considered, no means having been found of differentiating between the
cystic and active forms. This was a particularly serious source of
error for it is possible for soil to contain large numbers of bacteria
and protozoa, of which a high percentage of the latter are in the form
of cysts. A count made on such a soil would give results apparently
opposed to the theory that protozoa act as depressors of bacteria.

This difficulty has, however, been overcome by a further modification
of the dilution method, and it is now possible in any soil sample
to count both the numbers of cysts and active forms. Also a further
advance in technique has made it possible to recognise and enumerate
the common species of protozoa, instead of simply grouping them as
Ciliates, Flagellates, and Amœbæ, as was done in the past.[7]

Briefly the method consists in dividing the soil sample into equal
portions (usually 10 grams each) one of which is counted, thus giving
the total numbers of protozoa (active + cystic) present. The second
portion is treated over-night with 2 per cent. hydrochloric acid, the
HCl used being B.P. pure 31·8 per cent. Previous experiments have shown
that such acid kills all the active protozoa, leaving viable the cysts.
The number of cysts is therefore found by counting this treated sample,
and the number obtained subtracted from the total gives the active
number.[F]

[F] The proof of the accuracy of this method will be found in the
following papers:--

(1) Cutler, D. W. (1920), Journ. Agric. Sci., vol. x., 136-143.

(2) Cutler, D. W., and Crump, L. M. (1920), Ann. App. Biol., vol.
vii., 11-24.

The discovery of this method at once puts into the hands of the
investigator a much more efficient instrument for studying the
activities of the soil micro-population, especially since at a slightly
later date Thornton’s method for counting bacteria was devised.

Early in 1920 Cutler and Crump[6] decided to make a preliminary survey
of the protozoon and bacterial populations of one of the Rothamsted
field soils (Broadbalk dunged plot). The investigation was continued
for 28 days, daily soil samples being taken. The results so obtained
showed that an extended investigation of the micro-population of field
soil would yield interesting and important results, especially as
it was evident that certain views held by soil biologists required
modification.

In July of the same year, therefore, it was decided to start an
extended investigation of the soil protozoa and bacteria. The method
adopted was to make counts of the numbers of bacteria and of six[G]
species of protozoa in soil samples taken daily direct from the field
(Barnfield dunged plot) and by statistical methods to correlate these
counts one with another and with the data for external conditions.
Observations at shorter periods than 24 hours could not be made, but it
was found possible to continue the research for 365 days.[7]

[G] Actual counts were made of six species, though, as stated on p.
10, observations were made on seventeen.

[Illustration: FIG. 10.--Daily numbers of active amœbæ (Dimastigamœba
and Species α) and bacteria in 1 gram of field soil, from August 29 to
October 8, 1920. (From Phil. Trans. Roy. Soc., vol. ccxi.)

X-axis: August September October

Y-axis (left): Amoebae Active numbers per gramme of soil

Y-axis (right): Bacteria in millions per gramme of soil

Legend: Dimastigamoeba

Species α

Bacteria]

The number of all the organisms showed large fluctuations of two kinds,
daily and seasonal. The size of the changes that took place within
so short a period as 24 hours was, perhaps, the most surprising fact
that the experiment revealed. Thus three consecutive samples gave
58·0, 14·25 and 26·25 millions of bacteria per gram respectively; and
the changes exhibited by any of the species of protozoa were at times
even larger. This fact is of extreme importance, since in the past it
has always been assumed that the number of bacteria remained fairly
constant from day to day, and investigators have not hesitated to
separate the taking of soil samples by long periods. It is now obvious
that such a procedure is of little use for comparative purposes (Fig.
10).

It has usually been assumed that the changes in the external conditions
markedly affect the density of the soil population. To test this the
environmental conditions--temperature, moisture content and rainfall
were examined; but contrary to all expectation no connection could be
traced between any of these and the daily changes in numbers of any
of the organisms investigated, and moreover the species of protozoa
appeared in the main to be living independently of one another.

It is difficult to believe that external conditions are as inoperative
as appears from the above; and in view of the known complexity of
the soil it is possible that further research will show that certain
combinations of external conditions are important agents in effecting
the changes.

[Illustration: FIG. 11.--Numbers of active amœbæ (Dimastigamœba and
Species α) and bacteria to 1 gram of field soil for typical periods in
February and April, 1921. (From Phil. Trans. Roy. Soc., vol. ccxi.)

X-axis: Feby. Feby. April

Y-axis (left): Amoebae Active numbers per gramme of soil

Y-axis (right): Bacteria millions

Legend: Dimastigamoeba

Species α

Bacteria]

In the case of the bacteria, however, the agent causing the
fluctuations is mainly the active amœbæ. This was well shown during the
year’s count, for with only 14 per cent. of exceptions, 10 per cent.
of which can be explained as due to rapid excystation or encystation,
a definite inverse relationship was established between the active
numbers of amœbæ and the number of bacteria (Figs. 11 and 12). Thus a
rise from one day to the next in the amœbic population was correlated
with a fall in the numbers of bacteria and vice versa. It must not be
supposed that the flagellates are of no account in this process; some
species, known to eat bacteria, undoubtedly induce slight depressions,
but, owing to their small size, any effect is masked by the greater
one of the amœbæ.

[Illustration: FIG. 12.--Numbers of active amœbæ (Dimastigamœba and
Species α) and bacteria in 1 gram of field soil for typical periods in
September, October, and November, 1920.

X-axis: August September October

Y-axis (left): Amoebae, thousands

Y-axis (right): Millions, Bacteria

Legend: Dimastigamoeba

Species α

Bacteria]

These experiments seem to admit of no doubt that in field soil the
active protozoa are instrumental in keeping down, below the level
they might otherwise have attained, the numbers of bacteria; but a
further proof of this contention ought to be obtained by inoculation
experiments. It should be possible, by inoculating sterile soil
with bacteria alone and with bacteria plus protozoa, to demonstrate
fluctuations in bacterial numbers in the latter, while those of the
former remained constant. This admittedly crucial test has often been
tried, but owing to difficulties in technique, etc., has always failed.
Recently, however, by using new methods confirmatory results have been
obtained.[5]

Ordinary field soil was sterilised by heat at 100° C. for 1 hour on
four successive days; it was then divided into equal portions, one of
which was inoculated with three known species of bacteria, and the
other inoculated with the same number of bacteria plus the cysts of the
common soil amœba _Nægleria gruberi_. The numbers of bacteria in each
soil were counted daily for the first eight days and then daily from
the 15th to the 21st day after the experiment started. The results are
given in Table VII. and Fig. 13.

TABLE VII.

+------------+---------+--------+
| Numbers of | Control | Control|
| Days after |(Bacteria|Bacteria|
|Inoculation.| alone).|+ Amœbæ.|
+------------+---------+--------+
| 0 | 13·0 | 12·2 |
| 1 | 48·6 | 35·4 |
| 2 | 97·6 | 117·2 |
| 3 | 127·0 | 178·4 |
| 4 | 154·8 | 154·4 |
| 5 | 196·8 | 177·0 |
| 6 | 214·4 | 151·8 |
| 7 | 193·4 | 75·6 |
| 8 | 165·2 | 65·8 |
| 15 | 169·2 | 72·8 |
| 16 | 174·8 | 30·2 |
| 17 | 175·6 | 53·2 |
| 18 | 168·4 | 82·8 |
| 19 | 160·4 | 43·8 |
| 20 | 171·2 | 70·8 |
| 21 | 176·2 | 28·2 |
+------------+---------+--------+

The numbers of bacteria are given in millions per gram of soil.

[Illustration: FIG. 13.--Numbers of bacteria counted daily in soils
containing

A. Bacteria alone.
B. Same Bacteria as in A + Amœbæ.
C. Same Bacteria as in A + Flagellates.

(From Ann. Appl. Biol., vol. x.)]

It will be noted that the numbers of bacteria in each soil rose
steadily until a maximum was reached 6-8 days after inoculation. This
is in accordance with expectation, since the reproductive rate of
bacteria is much greater than that of the amœbæ, which, until their
active forms are numerous, will not exert any appreciable influence on
the bacterial population. Further, since the protozoa were inoculated
as cysts an appreciable time would elapse before excystation took
place. The last seven days of the experiment are of particular
interest. During this period the amœbæ were known to be active in the
soil, and were depressing the bacterial numbers, for in the control
(protozoa-free) soil the variation in numbers was within experimental
error, while in the other soil the variations were considerable and
well outside experimental error. In fact the variations were comparable
with those found from day to day in untreated field soils. Finally,
the experiment shows that the bacteria in protozoa-free soil are able
to maintain high numbers for a longer period than those living in
association with protozoa.

SEASONAL CHANGES.

Superimposed on the daily variations in numbers there are seasonal
changes, as is clearly shown when fourteen day averages are made of
the numbers for each species. Bacteria have long been known to show
autumn and spring rises, but recent research has demonstrated that the
protozoan population also rises to a maximum at the end of November,
with a less marked spring rise at the end of March and beginning of
April (Figs. 14 and 15).

It has sometimes been claimed that the numbers of soil organisms are
closely linked with the soil moisture, but no support for this view
was found during the course of the experiment. Similarly, as in the
case of the daily variations, no connection could be traced between the
seasonal changes and any of the external conditions considered.

It is interesting to note, however, that the seasonal variations in the
numbers of soil organisms is very similar to those recorded for many
aquatic organisms. Miss Delf,[8] for instance, found that in ponds at
Hampstead the algæ are most numerous in spring and again in the autumn,
and like changes are recorded in British lakes by West and West[25] and
in the Illinois river by Kofoid.[14]

[Illustration: FIG. 14.--Fortnightly averages of total numbers of
Oicomonas, Species γ, and Species α, and of bacteria, moisture, and
temperature. (From Phil. Trans. Roy. Soc., vol. ccxi.)

X-axis: Fortnight beginning 1920. July. Aug. Sep^t. Oct. Nov. Dec.
Jan 1921. Feb^y. Mch. April. May. June.

Y-axis (bottom left): Percentage of moisture

Y-axis (top left): Logarithms of numbers of active protozoa per
gramme of soil

Y-axis (bottom right): Temperature F

Y-axis (top right): Bacteria in millions per gramme

Legend: Oicomonas

Species γ

Species α

Bacteria

Temperature

Moisture]

It is difficult to resist the conclusion that these annual variations
are produced by similar causes, from which it follows that the increase
in the numbers of protozoa in the soil is not wholly conditioned by an
increased food supply--the bacteria--for the algæ are not dependent on
such a form of nourishment. This is substantiated by the fact that the
numbers of protozoa, except those of _Oicomonas_, rose during March,
whereas the corresponding increase in the bacteria was delayed till the
early part of April.

[Illustration: FIG. 15.--Fortnightly averages of total numbers of
Heteromita, Cercomonas, and Dimastigamœba and of bacteria, moisture,
and temperature. (From Phil. Trans. Roy. Soc., vol. ccxi.)

X-axis: Fortnight beginning July 1920. Aug. Sep^t. Oct. Nov. Dec.
Jan. 1921. Feb. Mar. April May June

Y-axis (bottom left): Percentage of moisture.

Y-axis (top left): Logarithms of numbers of active protozoa per
gramme of soil.

Y-axis (bottom right): Temperature F

Y-axis (top right): Bacteria in millions per gramme

Legend: Heteromita

Cercomonas

Dimastigamoeba

Bacteria

Temperature

Moisture]

Owing to the variations in the numbers of both protozoa and bacteria,
little reliance can be placed on figures obtained from an isolated
count, since on one day the total numbers of flagellates may be nearly
2,000,000 per gram and drop by more than half this figure in 24 days.
It is certain, however, that the numbers recorded in the past are much
too low, since the total flagellate and amœbæ species were lumped
together in two groups. Some idea of the size of the soil population
can be obtained, nevertheless, by using the fourteen-day averages
mentioned above. In Table VIII. are tabulated the average total numbers
of flagellates, and amœbæ for the two periods of the year when the
population was at its maximum and minimum respectively. An endeavour
has also been made to strike a rough balance sheet as to the amount of
protoplasm represented by protozoa and bacteria in a ton of soil. For
this purpose it has been assumed that the organisms have a specific
gravity of 1·0 and are spheres of diameters, 6µ for the flagellates,
10µ for the amœbæ, and 1µ for the bacteria; and that they are uniformly
distributed through the top nine inches of soil. The top nine inches of
soil is taken as weighing 1000 tons.

TABLE VIII.

+-----------+---------------------------+---------------------------+
| | Maximum Period. | Minimum Period. |
| + ____________/\___________ + ____________/\___________ +
| |( )|( )|
| | No. | Weight | Weight| No. per | Weight | Weight|
| | per |in Gram |in Tons| Gram. |in Gram |in Tons|
| | Gram. | per | per | | per | per |
| | | Gram. | Acre. | | Gram. | Acre. |
+-----------+----------+--------+-------+----------+--------+-------+
|Flagellates| 770,000|0·000087| 0·087 | 350,000|0·000039| 0·039 |
|Amœbæ | 280,000|0·000147| 0·147 | 150,000|0·000078| 0·078 |
|Bacteria |40,000,000|0·000020| 0·02 |22,500,000|0·000012| 0·012 |
+-----------+----------+--------+-------+----------+--------+-------+

It must be remembered that the above figures are minimum ones, as many
species of bacteria and protozoa, known to occur in the soil, are not
included in the statement owing to their not appearing on the media
used for counting purposes.

[Illustration: FIG. 16.--Daily variations in the numbers of active
individuals of a species of flagellate, _Oicomonas termo_ (Ehrenb.)
during March, 1921. (From Phil. Trans. Roy. Soc., vol. ccxi.)

X-axis: March

Y-axis: Active numbers per gramme of soil]

Before leaving the discussion of daily variations in numbers of
protozoa, reference must be made to the flagellate species. As already
mentioned, their active numbers fluctuate rapidly, and for the most
part entirely irregularly. One species, however, _Oicomonas termo_, is
characterised by possessing a periodic change; high active numbers on
one day being succeeded by low, which are again followed by high on the
third day. This rhythm was maintained, with few exceptions, for 365
days (Fig. 16), and has been shown to take place in artificial culture
kept under controlled laboratory conditions (Fig. 17).

[Illustration: FIG. 17.--Daily variations in the numbers of active
individuals of _Oicomonas termo_ (Ehrenb.) in artificial culture media
kept at a constant temperature of 20° C. A, in hay infusion; B, in egg
albumen.

X-axis: Days

Y-axis: Thousands]

It was thought that an explanation of this phenomenon might be found
in alternate excystation and encystation, since the latter is a
constituent part of the animals’ life history (see p. 73). This,
however, does not hold, for the cyst curve is not the inverse of that
of the active; and, moreover, statistical treatment demonstrated that
cyst formation is wholly unperiodic in character.

An explanation must therefore be sought in the changes in the
organisms during the active period of their life, and the deduction
can be drawn that, increased active numbers tend to be followed by
death, conjugation, or both, while decreases in the active numbers
are followed by rises in total numbers, i.e., reproduction, and this
rhythmically.

This somewhat surprising conclusion appears to hold, in a lesser
degree, for other soil protozoa, and is of sufficient importance
to warrant further research. The direction in which this is being
pursued is by a study of the reproductive rates of pure cultures of
certain ciliates and flagellates under varying external conditions.
Space does not admit of adequate discussion of this problem, but the
results already obtained justify the view that such lines of work will
elucidate some of the baffling problems of soil micro-biology.

SOIL REACTION.

The development of the artificial fertiliser industry has in many
ways revolutionised farm practice, with the inevitable result that
new problems have arisen, not the least of which are biological in
character.

If, as seems to be indubitable, the micro-organisms of the soil are of
importance to soil fertility, it is necessary for us to know in what
way this population is affected by the application of fertilisers,
and a start has been made by investigating the effects of hydrogen
ion concentration on soil protozoa. Much has already been written
concerning this question, but almost entirely on results obtained
in artificial cultures. It is always dangerous to argue from the
artificial to the natural environment of organisms and particularly
so in respect to the soil. Also, as Collett has shown, the toxic
effects of acids are probably not entirely a function of the hydrogen
ion concentration, but that the molecules of certain acids are in
themselves toxic, an action which can, however, be diminished by the
antagonistic powers of many substances such as NaCl.

In this laboratory S. M. Nasir, by unpublished work, has shown that
the limiting value on the acid side for _Colpoda cucullus_ was P_{H}
3·3; for a flagellate (_Heteromita globosus_), 3·5; and for an amœba
(_Nægleria gruberi_), 3·9.

Also Mlle. Perey, investigating the numbers of protozoa in one of the
Rothamsted grass plots of P_{H} 3·65, found a total of 13,600 protozoa,
of which 90 per cent. were active.

The tolerance, therefore, of these organisms to varying external
conditions is greater than has formerly been supposed, a conclusion
which is becoming more evident from the researches mentioned in Chapter
IV. on soils from different parts of the world.

PROTOZOA AND THE NITROGEN CYCLE.

In partially-sterilised soil from which protozoa were absent Russell
and Hutchinson obtained an increased ammonia production, a result
also obtained by Cunningham. Hill, on the other hand, concluded that
protozoa have no effect on ammonification, but his technique is open to
criticism.

Lipman, Blair, Owen and McLean’s work[17] contains many figures
obtained by adding dried blood, tankage, soluble blood flour,
cottonseed meal, soy-bean meal, wheat flour, corn meal, etc., to soil.
It is difficult to understand how accurate results could be expected
when, to an already little understood complex substance, such as soil,
is added a series of substances whose effects are practically unknown.

Free nitrogen-fixation in soils is an important process, more
especially in soils of a light sandy nature, from which crops are
taken year after year without any application of manure. The effect of
protozoa on the organisms causing this process has in the past received
little attention. Recently, however, Nasir[20] has studied the
influence of protozoa on Azotobacter, both in artificial culture and in
sand. From a total of 36 experiments done in duplicate or triplicate,
31 showed a decided gain in nitrogen fixation over the control, while
only 5 gave negative results.

[Illustration: FIG. 18.--Showing the highest fixations of nitrogen
above the control recorded for Azotobacter in the presence of different
species of Protozoa. (From Ann. Appl. Biol., vol. ii.)

X-axis (left): Artificial Media C A F AF AC ACF

X-axis (right): Sand Cultures C A AF AC

Legend: C represents CILIATES.

A -do.- AMOEBAE.

F -do.- FLAGELLATES.]

As might be expected, the fixation figures varied from culture to
culture, the highest recorded being 36·04 per cent. above the control
and this in a sand culture (Fig. 18). Reference to the details of the
experiments shows that the criticisms made against similar work done in
the past do not hold here, and one must conclude that Azotobacter is
capable of fixing more atmospheric nitrogen in the presence of protozoa
than in their absence.

At present it is impossible to say how this occurs, but it is highly
improbable that the protozoa are themselves capable of fixing nitrogen.
A more likely explanation is that the protozoa, by consuming the
Azotobacter, kept down the numbers, and transfer the nitrogen to their
own bodies. This will tend to prevent the bacteria from reaching a
maximum density, and reproduction, involving high metabolism, will be
maintained for a longer period than would have otherwise occurred. This
and other possible explanations, are being tested.

Little has been said regarding the application of protozoology to the
question of soil partial sterilisation. As already pointed out, in
the past much work has been done, but the results were conflicting.
In view, however, of our recently acquired knowledge of the life of
protozoa in ordinary field soil, most of the early experiments require
repeating. A beginning has already been made, but the work is not
sufficiently advanced to warrant discussion.

What is urgently needed, however, is to increase our knowledge of the
general physiology of these unicellular animals. Until we know what
are the inter-relationships between the members of the micro-organic
population of normal soil it is almost impossible to hope that means
will be devised by which they can be controlled.

At present we are almost entirely ignorant of the simplest of
physiological reactions, such as the exact effect of various inorganic
salts found in the soil.

Also some experiments in Germany and the States indicate that amœbæ are
selective as regards the bacteria they ingest. If this is substantiated
it may prove of importance to economic biology.

It has been shown that the flagellates occur in the soil in large
numbers, and many of them feed on bacteria. It is probable, however,
that certain of them feed saprophytically and must therefore exert some
influence on the soil solution, though what this may be is entirely
unknown.

Finally, as Nasir has shown, the protozoa play a part in the
complicated nitrogen cycle, and work of this type needs extending.

Such, then, are a few of the outstanding problems that confront the
soil protozoologist; but he must always remember that the organisms he
studies are but a small fraction of the total, and that any influence
affecting one part of the complex will be reflected in another. As
Prof. Arthur Thomson said in his Gifford Lectures, “No creature lives
or dies to itself, there is no insulation. Long nutritive chains often
bind a series of organisms together in the very fundamental relation
that one kind eats the others.” Such nutritive chains obtain in the
soil as markedly as in other haunts of living creatures.

SELECTED BIBLIOGRAPHY.

* _Papers giving extensive bibliographies._

[1] Cunningham, A., Journ. Agric. Sci., 1915, vol. xvii., p. 49.

[2] Cunningham, A., and Löhnis, F., Centrlb. f. Bakt. Abt. II., 1914,
vol. xxxix., p. 596.

[3] Cutler, D. W., Journ. Agric. Sci., 1919, vol. ix., p. 430.

[4] Cutler, D. W., Journ. Agric. Sci., 1920, vol. x., p. 136.

[5] Cutler, D. W., Ann. App. Biol., 1923, vol. x., p. 137.

[6] Cutler, D. W., and Crump, Ann. App. Biol., 1920, vol. vii., p. 11.

[7] * Cutler, D. W., Crump and Sandon, Phil. Trans. Roy. Soc. B.,
1922, vol. ccxi., p. 317.

[8] Delf, E. M., New Phytologist, 1915, vol. xiv., p. 63.

[9] Dobell, C. C., Arch. f. Protisenk., 1911, vol. xxiii., p. 269.

[10] * Goodey, T., Roy. Soc. Proc. B., 1916, vol. lxxxix., p. 279.

[11] Goodey, T., Roy. Soc. Proc. B., 1913, vol. lxxxvi, p. 427.

[12] Hartmann, M., and Nägler, K., Sitz-Ber. Gesellsch. Naturf.
Freunde, 1908, Berlin, No. 4.

[13] Koch, G. P., Journ. Agric. Res., 1916, vol. li., p. 477.

[14] Kofoid, C. A., Bull. Illinois State Lab. Nat. Hist., 1903 and
1908.

[15] * Kopeloff, N., Lint, H. C., and Coleman, D. A., Centrlb. f.
Bakt. Abt. II., 1916, vol. xlvi., p. 28.

[16] Kopeloff, N., Lint, H. C., and Coleman, D. A., Centrlb. f. Bakt.
Abt. II., 1916, vol. xlv., p. 230.

[17] Lipman, J. G., Blair, A. W., Owen, L. L., and McLean, H. C.,
N.J. Agric. Exp. Sta. 1912, Bull., No. 248.

[18] Martin, C. H. and Lewin, K. R., Journ. Agric. Soc., 1915, vol.
vii., p. 106.

[19] Martin, C. H., Roy. Soc. Proc. B., 1912, vol. lxxxv., p. 393.

[20] * Nasir, S. M., Ann. App. Biol., 1923, vol. x., p. 122.

[21] Russell, E. J., Roy. Soc. Proc. B., 1915, vol. lxxxix., p. 76.

[22] * Russell, E. J., “Soil Conditions and Plant Growth,” 1921, 4th
ed.

[23] * Sherman, J. M., Journ. Bact., 1916, vol. i., p. 35, and vol.
ii., p. 165.

[24] Truffaut, G., and Bezssonoff, H., Compt. Rend. Acad. Sci., 1920,
vol. clxx., p. 1278.

[25] West, W., and West, G. S., Journ. Linn. Soc. Bot., 1912, vol.
xl., p. 395.

CHAPTER VI.

ALGÆ.

I. GENERAL AND HISTORICAL INTRODUCTION.

Speaking broadly, the organisms of the soil may be classified into
several distinct groups differing conspicuously in their general
characters and physiological functions and therefore in their economic
significance; among such groups may be mentioned the bacteria,
protozoa, algæ and fungi. It is found, however, that though typical
members of these groups are conspicuously different from one another,
yet there exist a number of unicellular forms which have characters
in common with more than one of these big groups, and the lines of
demarcation between them become difficult to define. It becomes
advisable, therefore, to depart a little from the systematist’s rigid
definitions and to adopt a somewhat more logical grouping of the soil
organisms based on their mode of life.

To give but a single example: _Euglena viridis_ occurs quite commonly
in soil. Through its single flagellum, its lack of a definite cellulose
wall, its changeable shape and its ability to multiply by simple
fission in the motile state it definitely belongs systematically to the
group of protozoa. But its possession of chlorophyll, in enabling it to
synthesise complex organic substances from CO₂ and water in a manner
entirely typical of plants, connects it physiologically so closely with
the lower green algæ that in studying the biology of the soil it seems
best to include it and other nearly related forms with the algæ.

On this physiological basis “soil-algæ” may be defined as those
micro-organisms of the soil which have the power, under suitable
conditions, to produce chlorophyll. Such a definition has the
advantage that it is wide enough to include the filamentous protonema
of mosses, which, though alga-like in form and in physiological
action, is nevertheless separated from the true algæ by a wide gulf.
A more accurate name for such a group of organisms would be the
“chlorophyll-bearing protophyta” of the soil; they may be classified
briefly as follows (Table IX.):--

TABLE IX.

The importance of the lower algæ from a biological standpoint has
long been recognised, since their extremely primitive organisation,
coupled with their ability to synthesise organic compounds from simple
inorganic substances, singles them out as being not very distantly
removed from the group of organisms in which life originated upon the
earth. But the possibility of their having a very much wider economic
significance was completely overlooked until about a quarter of a
century ago, when Hensen demonstrated their importance in marine
plankton as the producers of the organic substance upon which the
whole of the animal life of the ocean is ultimately dependent. In
consequence, it has been generally assumed that the growth of algæ,
since they contain chlorophyll, is entirely dependent on the action of
light. Hence the recent idea of the existence of algæ which actually
inhabit the soil has been received with a certain amount of scepticism,
though the results of modern physiological research on a number of the
lower algæ show that there is very good reason to believe that such a
soil flora is entirely possible.

In considering the alga-flora of a soil it is necessary to distinguish
between two very different sets of conditions under which the organisms
may be growing. In the first place, they may grow on the surface of the
soil, being subjected directly to insolation, rain, the deposition of
dew, the drying action of wind, relatively quick changes of temperature
and other effects of climate. Certain combinations of these conditions
present so favourable an environment for the growth of algæ that at
times there appears on the surface of the soil a conspicuous green
stratum, sometimes so dark in colour as to appear almost black. Strata
of this nature are well known, and in systematic works there are
constant references to species growing “on damp soil”; for instance, of
the 51 well-defined species of _Nostoc_ recognised by Forte, no less
than 31 are characterised as terrestrial. Such appearances, however,
seem to have been regarded as sporadic and more or less accidental,
rather than as the unusually luxuriant development of an endemic
population, and have been frequently attributed to an excessively moist
condition of the soil due to defective drainage.

In the second place, the algæ may be living within the soil itself,
away from the action of sunlight and under somewhat more uniform
conditions of moisture and temperature.

Up to the present time the greater number of the investigations carried
out in this subject have been of a systematic nature, and extremely
little direct evidence has been obtained which can throw any light on
the subject of the economic significance of the soil algæ.

The earliest systematic work was carried out by Esmarch, in 1910-11,
who investigated by means of cultures the blue-green algæ of a number
of soils from the German African Colonies, the samples being taken from
the surface and also from the lower layers of the soil. He obtained a
considerable number of species and observed that in cultivated soils
they were not confined to the surface but occurred regularly to a depth
of 10-25 cms. and occasionally as low as 40-50 cms. He attributed
their existence in the lower layers to the presence of resting spores
carried down in the processes of cultivation, since his samples from
uncultivated soils were unproductive.

Later, Esmarch extended his investigations to a far larger
number of samples, 395 in all, of soils of different types from
Schleswig-Holstein. He found that blue-green algæ were very widely
distributed in soils of certain types, though they occurred rarely
in uncultivated soils of low water-content, and he described no less
than 45 species of which 34 belonged to the _Oscillatoriaceæ_ and
_Nostocaceæ_. Certain of the commoner species were obtained from soils
of widely different types, as shown in Table X., while other forms
occurred only rarely and with a much more limited distribution.

TABLE X.--FREQUENCY OF OCCURRENCE OF CERTAIN COMMON SPECIES IN
ESMARCH’S SOIL SAMPLES.

+-------------------------+----------------------------------------+
| | Percentage of Samples |
| | containing given Alga. |
| +--------------------+-------------------+
| | Uncultivated | Cultivated |
| | Damp Sandy Soil. | Soils. |
| +------+------+------+------+-----+------+
| |Shores|Shores| | | | |
| | of | of | Sea- | | |Marsh-|
| Species. | Elbe.|Lakes.|shore.|Sandy.|Clay.| land.|
+-------------------------+------+------+------+------+-----+------+
|Anabæna variabilis | 46 | 43 | 9 | 10·3 | 60 | 46 |
|Anabæna torulosa | 31 | 14·3 | 63·6 | 27·6 | 34·3| 56·4 |
|Cylindrospermum muscicola| 23 | 28·6 | 0 | 24 | 48·6| 59 |
|Cylindrospermum majus | 0 | 14·3 | 0 | 38 | 40 | 33·3 |
|Nostoc Sp. III. | 7·7 | 0 | 0 | 38 | 37 | 48·7 |
+-------------------------+------+------+------+------+-----+------+

Taking the number of samples containing blue-green algæ as a rough
measure of their relative abundance, Esmarch obtained the following
interesting figures (Table XI.):--

TABLE XI.

+------------------------------+----------------+---------+
| | Percentage | |
| | of Samples |Number of|
| | Containing | Samples |
| Kind of Soil. |Blue-green Algæ.|Examined.|
+------------------------------+----------------+---------+
| | | |
|Cultivated marshland | 95 | 40 |
|Cultivated clay soil | 94·6 | 37 |
|Uncultivated moist sandy soils| 88·6 | 35 |
|Cultivated sandy soil | 64·4 | 45 |
| {Woodland | 12·5 | 40 |
|Uncultivated{Sandy heathland | 9 | 34 |
| {Moorland | 0 | 35 |
+------------------------------+----------------+---------+

In noting that the soils fell into two groups, those relatively rich
and those poor in blue-green algæ, Esmarch concluded that the two
chief factors governing the distribution of the _Cyanophyceæ_ on the
surface of soils are, (1) the moisture content of the soil, (2) the
availability of mineral salts, cultivated soils being especially
favoured in both of these respects. He further distinguished between
cultivated land of two kinds, viz. arable land and grass land, and
found that on all types of soil grassland was richer in species than
was arable land.

Esmarch examined, in addition, 129 samples taken from the lower layers
of the soil immediately beneath certain of his surface samples, 107 at
10-25 cms. and the rest at 30-50 cms. depth.

In cultivated soils, whether grassland or arable land, he found that
blue-green algæ occurred almost invariably in the lower layers in those
places bearing algæ on the surface and that, with rare exceptions, the
algæ found in the lower layers corresponded exactly to those on the
surface, except that with increasing depth there was a progressive
reduction in the number of species.

In uncultivated, moist, sandy soils the agreement was far less
complete, for though algæ were rarely absent from the lower layers
their vertical distribution was frequently disturbed by the action
of wind and rain. Other uncultivated soils not subject to periodic
disturbance were found to be uniformly lacking in algæ in the lower
layers, but as the limited number of samples examined came completely
from places where there were no algæ on the surface this means very
little.

By direct microscopic examination of soil Esmarch claims to have
found living filaments of blue-green algæ at various depths below the
surface. He realised, however, that there was no indication of the
length of time that such filaments had been buried, and therefore
conducted a series of experiments from which he concluded that the
period during which the algæ investigated could continue vegetatively
in the soil after burial varied with different species from 5-12 weeks,
but that during the later part of the period the algæ gradually assumed
a yellowish-green colour.

It is unfortunate that Esmarch’s investigations were directed only
towards the blue-green algæ since observations made in this country
indicate that such a series of records gives but a very incomplete
picture of the soil flora as a whole.

Petersen, in his “Danske Aërofile Alger” (1915) added considerably to
our knowledge of soil algæ, especially of diatoms. Unfortunately he
confined his investigations of the green algæ to forms growing visibly
on the surface of the ground. He observed, however, that acid soils
possessed a different flora from that commonly found on alkaline or
neutral soils, the former being dominated by _Mesotænium violascens_,
_Zygnema ericetorum_, and 2 spp. of _Coccomyxa_, while the latter were
characterised by _Mesotænium macrococcum var._, _Hormidium_, 2 spp.,
and _Vaucheria_, 3 spp.

Of diatoms he obtained no less than 24 species and varieties from
arable and garden soils, and five characteristic of marshy soils, while
from forest soils and dry heathland they appeared to be often absent.
He omitted all reference to blue-green algæ.

Meanwhile Robbins, examining a number of Colorado soils that contained
unprecedented quantities of nitrate, obtained from them 18 species of
blue-green algæ, 2 species of green algæ, and one diatom. Moore and
Karrer have demonstrated the existence of a subterranean alga-flora of
which _Protoderma viride_, the most constantly occurring species, was
shown to multiply when buried to a depth of one metre.

In this country attention was first called to the subject by Goodey and
Hutchinson of Rothamsted who, in examining certain old stored soils
for protozoa, obtained also a number of blue-green forms which were
submitted to Professor West for identification. This ability of certain
algal spores to retain their vitality for a long resting period was so
very striking that an investigation was begun at Birmingham in 1915 to
ascertain whether other forms were equally resistant. The investigation
was carried out on a large number of freshly collected samples of
arable and garden soils which were first aseptically air-dried for at
least a month and then grown in culture. No less than 20 species or
varieties of diatoms, 24 species of blue-green and 20 species of green
algæ were obtained from these cultures (Table XII.). In the majority
of the samples there was found a central group of algæ, including
_Hantzschia amphioxys_, _Trochiscia aspera_, _Chlorococcum humicola_,
_Bumilleria exilis_ and rather less frequently _Ulothrix subtilis
var. variabilis_, while moss protonema was universally present. These
species were thought to form the basis of an extensive ecological plant
formation in which, by the inclusion of other typically terrestrial
but less widely distributed species smaller plant-associations were
recognised.

In certain of the soils, associations consisting very largely of
diatoms were present, and it is to be noted that the majority of the
forms that have been described are of exceedingly small size. It is
doubtless this characteristic which enables them to withstand the
conditions of drought to which the organisms of the soil are liable to
be subjected, small organisms having been shown to be better able to
resist desiccation than are larger ones. Since the soil diatoms belong
to the pennate type, they are further adapted to their mode of life by
their power of locomotion, which enables them in times of drought to
retire to the moister layers of the soil.

In the soils examined in this work blue-green algæ were less
universally present than were diatoms or green algæ, and the species
found appeared to be more local in occurrence. There seemed to be,
however, an association between the three species, _Phormidium tenue_,
_Ph. autumnale_, and _Plectonema Battersii_, at least two of the three
species having been found together in no less than 16 of the samples,
while all three occurred in 7 of them.

TABLE XII.--ALGÆ IN DESICCATED ENGLISH SOILS. (BRISTOL.)

+---------------+-----------+------------------------------+
| | | Number of Species. |
| | Number of +-----------+-----------+------+
| | Samples | Maximum | Average | |
| Group. |Productive.|per Sample.|per Sample.|Total.|
+---------------+-----------+-----------+-----------+------+
| | per cent. | | | |
|Diatoms | 95·5 | 9 | 3·7 | 20 |
|Blue-green algæ| 77·3 | 7 | 2·5 | 24 |
|Green algæ | 100 | 7 | 4·3 | 20 |
|Moss protonema | 100 | -- | -- | -- |
+---------------+-----------+-----------+-----------+------+
| Total | -- | 20 | 10·5 | -- |
+---------------+-----------+-----------+-----------+------+

It was generally noticeable that those soils found to be rich in
blue-green algæ contained only a few species of diatoms, and vice
versa. Diatoms appeared most frequently in soils from old gardens,
whereas blue-green algæ were more characteristic of arable soils. The
green algæ and moss protonema, on the other hand, were distributed
universally.

The majority of green algæ typically found in soils are unicellular,
but a few filamentous forms occur. With the exception of _Vaucheria_
spp. these are characterised, however, by an ability to break down
in certain circumstances into unicellular or few-celled fragments, in
which condition identification is often very difficult.

It was also found by cultural examination of a number of old stored
soils from Rothamsted that germination of the resting forms of a
number of algæ could take place after an exceedingly long period of
quiescence. No less than nine species of blue-green algæ, four species
of green algæ, and one species of diatom were obtained from soils that
had been stored for periods of about forty years, the species with the
greatest power to retain their vitality being _Nostoc muscorum_ and
_Nodularia Harveyana_.

II. THE SOIL AS A SUITABLE MEDIUM FOR ALGAL GROWTH.

Were it not for the recent advances that have been made in our
knowledge of the mode of nutrition of many of the lower algæ, it would
be very difficult to account for the widespread occurrence of algæ in
the soil, for it is undoubtedly true of some of the more highly evolved
algæ that their mode of nutrition is entirely typical of that of green
plants in general. The application of bacteriological technique to the
algæ, however, by Beijerinck, by Artari, and by Chodat and his pupils,
and the introduction of pure-culture methods have led to a study of
the physiology of some of the lower algæ, in the hope of getting to
understand some of the fundamental problems underlying the nutrition
of organisms containing chlorophyll. It is impossible here to do more
than mention the names of a few of the more important of those who
have worked along these lines, such as Chodat, Artari, Grintzesco,
Pringsheim, Kufferath, Nakano, Boresch, Magnus and Schindler, and to
condense into a few sentences some of their more important conclusions.

It is now established that although in the light the algæ are able to
build up their substance from CO₂ and water containing dilute mineral
salts, yet in such conditions growth is sometimes very slow, and with
some species at any rate it is greatly accelerated by the addition of
a small quantity of certain organic compounds. The ability of the lower
algæ to use organic food materials varies specifically, quite closely
related forms often reacting very differently to the same substance,
but there have been shown to be a considerable number of forms which
can make use of organic compounds to such an extent that they can
grow entirely independently of light. In such cases the nutrition
of the organism becomes wholly saprophytic, and the chlorophyll may
be completely lost; it has frequently been observed, however, that
on suitable nutrient media, even in complete darkness, certain algæ
continue to grow and retain their green colour, provided that a
sufficient supply of a suitable nitrogenous compound is present.

_Chlorella vulgaris_, an alga frequently found in soil, has been shown
to be extremely plastic in its relations to food substances. Given only
a dilute mineral-salts solution as food source, it absorbs CO₂ from
the air, and grows in sunlight with moderate rapidity. The addition
of glucose to the medium in the light greatly increases the rate and
amount of growth and the size of the cells, while in the dark the
colonies not only remain green but have been shown to develop more
vigorously than in full daylight. The organism is also able to use
peptone as a source of nitrogen in place of nitrates.

_Stichococcus bacillaris_ and _Scenedesmus spp._, also occurring
in soils, have been shown to be almost equally adaptable, though
in these cases the organisms grow more slowly in the dark than on
the corresponding medium in the light. Liquefaction of gelatine by
the secretion of proteolytic enzymes has been shown to be a further
property of certain species, resulting in the formation of amino acids
such as glycocoll, phenylalanine, dipeptides, etc. This property is,
however, possessed by only a limited number of species and in varying
degree.

Up to the present very little work of this kind has been done upon algæ
actually taken from the soil, and our knowledge is therefore very
scanty. Of the species so far examined all show considerable increase
in growth on the addition to the medium of glucose and other sugars,
and tend to be partially saprophytic; a few have been shown to liquefy
gelatine to some extent.

Servettaz, Von Ubisch, and Robbins have also demonstrated that the
protonema of some mosses can make use of certain organic substances,
especially the sugars, and grow vigorously in the dark. It has been
shown, however, that light is essential for the development of the moss
plant.

It was thought at Rothamsted that some light might be thrown upon the
activities of the soil-algæ by making counts of the numbers present
in samples of soil taken periodically within a circumscribed area.
A dilution method similar to that in use in the protozoological
laboratory was adopted and applied to samples of arable soil taken
from the surface, and at depths of 2, 4, 6 and 12 inches vertically
beneath. A considerable number of samples were examined in this way
from two plots on Broadbalk wheat-field, viz.: the unmanured plot and
that receiving a heavy annual dressing of farmyard manure. The numbers
in the unmanured soil were observed to fall far short of those in that
containing a large amount of organic matter, while in both plots the
numbers varied considerably at different times of the year. The chief
species in both plots were identical, and their vertical distribution
was fairly uniform, but it was observed that the numbers of individuals
varied according to the depth of the sample. The 6th and 12th inch
samples contained very few individuals of comparatively few species,
but the 4th inch samples yielded numbers that were not significantly
less than those in the top inch. The 2nd inch sample was usually much
poorer in individuals than either the top or the 4th inch.

It is unfortunate that this method of counting is not really
satisfactory for the algæ, chiefly because it takes no account of the
blue-green forms. The gelatinous envelope which encloses the filaments
of these algæ prevents their breaking up into measurable units.
Assuming, as appears to be the case for the two plots investigated,
that the blue-green algæ are at least as numerous as the green forms,
the total numbers should probably be at least twice as great as those
calculated. Taking 100,000 as a rough estimate of the number of algæ
per gram of manured soil in a given sample, and assuming the cells to
be spherical and of average diameter 10µ, it has been calculated that
the volume of algal protoplasm present was at least 3 times that of
the bacteria though only one-third of that of the protozoa. This is
probably only a minimum figure for this sample.

A soil population of this magnitude can not be without effect on the
fertility of the soil. When growing on the surface of the ground
exposed to sunlight the algæ must, by photosynthesis, add considerably
to the organic matter of the soil, but when they live within the soil
itself their nutrition must be wholly saprophytic, and they can be
adding nothing either to the energy or to the food-content of the soil.
How these organisms fit into the general scheme of life in the soil is
at present undetermined, and there is a wide field for research in this
direction.

III. RELATION OF ALGÆ TO THE NITROGEN CYCLE

Probably the most important limiting factor in British agriculture is
the supply of nitrogen available for the growing crop, and it seems
likely that the soil-algæ are intimately connected with this question
in several ways.

Periodic efforts have been made during the last half century to
establish the fact that a number of the lower organisms, including
the green algæ, have the power of fixing atmospheric nitrogen and
converting it into compounds which are then available for higher
plants. This property has been definitely established for certain
bacteria, and rather doubtfully for some of the fungi, but until
recently no authentic proof had been produced that algæ by themselves
could fix nitrogen. The subject is too wide to be discussed in much
detail here.

Schramm in America, working with pure cultures of algæ, tried for
ten years to establish the fact of nitrogen fixation, and failed
completely; more recently Wann has extended Schramm’s work, and claims
to have proved indisputably that, given media containing nitrates as
a source of nitrogen and a small amount of glucose, the seven species
of algæ tested by him fixed atmospheric nitrogen to the extent of 4-54
per cent. of the original nitrogen content of the medium. So important
a result needed corroboration, and Wann’s experiment, with some slight
improvements, was therefore repeated at Rothamsted last summer.

This work has not yet been published, but in the whole series of
ninety-six cultures, with four different species, each growing on
six different media, there is no evidence that nitrogen fixation has
taken place; but there has been a total recovery at the end of the
experiment of 98·93 per cent. of the original nitrogen supplied. On
the other hand, a flaw has been detected in Wann’s method of analysing
those media containing nitrates, sufficiently great to account for the
differences he obtained between the initial and final nitrogen content
of his cultures. Hence, though one hesitates to say that the algæ are
unable, given suitable conditions, to fix atmospheric nitrogen, one
must admit that no one has yet proved that they can do so.

It is far more likely, however, that the experiments of Kossowitsch
and others throw more light on the relation of soil algæ to
nitrogen fixation. They affirm that greater fixation of nitrogen is
effected by mixtures of bacteria and certain gelatinous algæ than
by nitrogen-fixing bacteria alone, and that the addition of algæ to
cultures of bacteria produces a stimulating effect only slightly less
than that of sugar. It is probable, therefore, that the algæ, in their
gelatinous sheaths, provide easily available carbohydrates from which
the bacteria derive the energy essential to their work, and that
nitrogen fixation in nature is due to the combined working of a number
of different organisms rather than to the individual action of single
species.

Russell and Richards have shown that the rate of loss of nitrogen by
leaching from uncropped soils is far less than would be expected from
a purely chemical standpoint, and suggest that certain organisms are
present in the soil which, by absorbing nitrates and ammonium salts
as they are formed, remove them from the soil solution and so help to
conserve the nitrogen of the soil. It is probable that the soil algæ
act in this manner, though to what extent has not yet been determined.

IV. RELATION OF ALGÆ TO SOIL MOISTURE AND TO THE FORMATION OF HUMUS
SUBSTANCES.

In warmer countries than our own, especially those with an adequate
rainfall, the significance of soil algæ is perhaps more obvious to
a casual observer. Treub states that after the complete destruction
of the island of Krakatoa by volcanic eruption in 1883, the first
colonists to take possession of the island were six species of
blue-green algæ, viz., _Tolypothrix_ sp., _Anabæna_ sp., _Symploca_
sp., _Lyngbya_ 3 spp. Three years after the eruption these organisms
were observed to form an almost continuous gelatinous and hygroscopic
layer over the surface of the cinders and stones constituting the soil,
and by their death and decay they rapidly prepared it for the growth
of seeds brought to the island by visiting birds. Hence the new flora
which soon established itself upon the island can be said to have
had its origin in the alga-flora which preceded it. Fritsch has also
emphasised the importance of algæ in the colonisation of new ground in
Ceylon.

Welwitsch ascribes the characteristic colour from which the “pedras
negras” in Angola derive their name to the growth of a thick stratum of
_Scytonema myochrous_, a blue-green alga, which gradually becomes black
and completely covers the soil. At the close of the rainy season this
gelatinous stratum dries up very slowly, enabling the underlying soil
to retain its moisture for a longer period than would otherwise be the
case.

The gelatinous soil algæ are probably very important in this respect,
for their slow rate of loss of water is coupled with a capacity for
rapid absorption, and they are therefore able to take full advantage of
the dew that may be deposited upon them and increase the power of the
soil to retain moisture.

V. RELATION OF ALGÆ TO GASEOUS INTERCHANGES IN THE SOIL.

In the cultivation of rice the algæ of the paddy field have been found
to be of extreme importance. Brizi in Italy has shown that although
rice is grown under swamp conditions yet the roots of the rice plant
are typical of those of ordinary terrestrial plants and have none
of the structural adaptations to aquatic life so characteristic of
ordinary marsh plants. Hence the plants are entirely dependent for
healthy growth upon an adequate supply of oxygen to their roots from
the medium in which they are growing. A serious disease of the rice
plant, characterised by the browning and dying off of the leaves,
which was thought at first to be due to the attacks of fungi, was
found to be the effect of the inadequate aeration of the roots, while
the entry of the fungi was shown to be subsequent to the appearance
of the physiological disease. The presence of algæ in the swamp water
was found to prevent the appearance of this disease, in that they
unite with other organisms to form a more or less continuous stratum
over the surface of the ground, and add to the gases which accumulate
there large quantities of oxygen evolved during photosynthesis. The
concentration of dissolved oxygen in the water percolating through the
soil is thereby raised to a maximum, and the healthy growth of the crop
ensured.

This work has been corroborated by Harrison and Aiyer in India, and a
sufficient supply of algæ in the swamp water is now regarded as one of
the essentials for the production of a good rice crop.

From what has been said, it appears that, although our knowledge of
the soil algæ is extremely limited, and our conception of the part
they play is largely based on speculation, yet the subject is one of
enormous interest and worthy of investigation in many directions. In
its present undeveloped state, it is a little difficult to foresee
which lines of study are likely to prove most profitable, but there
is little doubt that eventually the soil algæ will be shown to play a
significant part in the economy of the soil.

SELECTED BIBLIOGRAPHY.

* _Papers giving extensive bibliographies._

I. GENERAL.

[1] Bristol, B. M., “On the Retention of Vitality by Algæ from Old
Stored Soils,” New Phyt., 1919, xviii., Nos. 3 and 4.

[2] Bristol, B. M., “On the Alga-Flora of some Desiccated English
Soils: an Important Factor in Soil Biology,” Annals of Botany, 1920,
vol. xxxiv., No. 133.

[3] Brizi, U., “Ricerche sulla Malattia del Riso detta ‘Brusone,’
Sect. IV. Influenza che le alghe verdi esercitano in risaia,”
Annuario dell Instituzione Agraria Dott. A. Ponti, Milan, 1905, vol.
vi., pp. 84-89.

[4] Esmarch, F., “Beitrag zur Cyanophyceen-Flora unserer Kolonien,”
Jahrb. der Hamburgischen wissensch. Anstalten, 1910, xxviii., 3.
Beiheft, S. 62-82.

[5] Esmarch, F., “Untersuchungen über die Verbreitung der
Cyanophyceen auf und in verschiedenen Boden,” Hedwigia, 1914, Band
lv., Heft 4-5.

[6] Fritsch, F. E., “The Rôle of Algal Growth in the Colonisation of
New Ground and in the Determination of Scenery,” Geog. Journal, 1907.

[7] Harrison, W. H., and Aiyer, P. A. Subramania, “The Gases of
Swamp Rice Soils,” Mem. Dept. Agr. in India, Chem. Ser. (I.) “Their
Composition and Relationship to the Crop,” 1913, vol. iii., No.
3; (II.) “Their Utilisation for the Aeration of the Roots of the
Crop,” 1914, vol. iv., No. 1; (IV.) “The Source of the Gaseous Soil
Nitrogen,” 1916, vol. v., No. 1.

[8_a_] Hensen, V., “Ueber die Bestimmung des Planktons oder des im
Meere treibenden Materials am Pflanzen und Thieren.” Fünfter Ber.
Komm. wiss. Unters. deutschen Meere, 1887.

[8] Moore, G. T., and Karrer, J. L., “A Subterranean Alga Flora,”
Ann. Miss. Bot. Gard., 1919, vi., pp. 281-307.

[9] Nadson, G., “Die perforierenden (kalkbohrende) Algen und ihre
Bedeutung in der Natur,” Scripta bot. hort. Univ. Imp. Petrop., 1901,
Bd. 17.

[10] Petersen, J. B., “Danske Aërofile Alger,” D. Kgl. Danske
Vidensk. Selsk. Skrifter, 7 Raekke, Naturv. og mathem., 1915, Bd.
xii., 7, Copenhagen.

[11] Robbins, W. W., “Algæ in some Colorado Soils,” Agric. Exp. Sta.,
Colorado, 1912, Bulletin 184.

[12] Treub, “Notice sur la nouvelle Flora de Krakatau,” Ann. Jard.
Bot. Buitenzorg, 1888, vol. vii., pp. 221-223.

II. RELATION OF ALGÆ TO LIGHT AND CARBON.

[13] Artari, A., “Zur Ernährungsphysiologie der grünen Algen,” Ber.
der D. bot. Ges., 1901, Bd. xix., S. 7.

[14] Artari, A., “Zur Physiologie der Chlamydomonaden (Chlam.
Ehrenbergii);” (I.) Jahrb. f. Wiss. Bot., 1913, Bd. lii., S. 410;
(II.) _Ibid._, 1914, Bd. liii., S. 527.

[15] Adjarof, M., “Recherches expérimentales sur la Physiologie de
quelques Algues vertes,” Université de Genève--Institut Botanique,
Prof. R. Chodat--1905, 6 serie, vii. fascicule, Genève.

[16] Beijerinck, M. W., “Berichte über meine Kulturen niederer Algen
auf Nährgelatine,” Centr. f. Bakt. u. Paras., 1893, Abt. I., Bd.
xiii., S. 368, Jena.

[17] Boresch, K., “Die Färbung von Cyanophyceen und Chlorophyceen in
ihrer Abhängigkeit vom Stickstoffgehalt des Substrates,” Jahrbücher
für Wiss. Botanik., 1913, lii., pp. 145-85.

[18] Chodat, R., “Étude critique et expérimentale sur le
polymorphisme des Algues,” Genève, 1909.

[19] Chodat, R., “La crésol-tyrosinase, réactif des peptides et
des polypeptides, des protéides et de la protéolyse,” Archiv. des
Sciences physiques et naturelles, 1912.

[20] Chodat, R., “Monographie d’Algues en Culture pure: Matériaux
pour la Flore Cryptogamique Suisse,” 1913, vol. iv., fasc. 2, Berne.

[21] Dangeard, P. A., “Observations sur une Algue cultivée à
l’obscurité depuis huit ans,” Compt. Rend. Acad. Sci. (Paris), 1921,
vol. clxxii., No. 5, pp. 254-60.

[22] Étard et Bouilhac, “Sur la présence de la chlorophyll dans un
Nostoc cultivé à l’abri de la lumière,” Compt. Rend., t. cxxvii, 1898.

[23] Grintzesco, J., “Recherches expérimentales sur la morphologie et
la physiologie expérimentale de _Scenedesmus acutus_,” Meyen. Bull.
herb. Boiss., 1902, Bd. ii., pp. 219-64 and 406-29.

[24] Grintzesco, J., “Contribution à l’étude des Protococcoidées:
_Chlorella vulgaris_ Beyerinck,” Revue générale de Botanique, 1903,
xv., pp. 5-19, 67-82.

[25] * Kufferath, H., “Contribution à la physiologie d’une
protococcacée nouvelle, _Chlorella luteo-viridis_ Chod. n. sp. var.,
_lutescens_ Chod. n. var.,” Recueil de l’institut bot. Léo Errera,
1913, t. ix, p. 113.

[26] Kufferath, H., “Recherches physiologiques sur les algues vertes
cultivées en culture pure,” Bull. Soc. Roy. Bot. Belgique, 1921,
liv., pp. 49-77.

[27] Magnus, W., and Schindler, B., “Ueber den Einflusz der Nährsalze
auf die Färbung der Oscillarien,” Ber. der D. Bot. Gesellschaft,
1912-13, xxx., p. 314.

[28] * Nakano, H., “Untersuchungen über die Entwicklungs- und
Ernährungsphysiologie einiger Chlorophyceen,” Journ. College of Sci.
Imp. Univ. Tokyo, 1917, vol. xl., Art. 2.

[29] Pringsheim, E., “Kulturversuche mit chlorophyll-führenden
Mikroorganismen,” Cohns Beiträge Z. Biol. d. Pflanzen. (I.)
Die Kultur von Algen in Agar, 1912, Bd. xi., S. 249; (II.) Zur
Physiologie der _Euglena gracilis_, 1913, Bd. xii., S. 1.; (III.) Zur
Physiologie der Schizophyceen, 1913, Bd. xii., S. 99.

[30] Radais, “Sur la culture pure d’une algue verte; formation de
chlorophylle à l’obscurité,” Comptes Rendus, 1900, cxxx., p. 793.

[31] Richter, O., “Zur Physiologie der Diatomeen.” (I.) Sitzber. d.
kais. Akad. d. W. in Wien, math, naturw. Kl., 1906, Bd. cxv., Abt.
I., S. 27; (II.) Denkschrift d. math. naturw. Kl. d. kais. Akad.
d. W. in Wien, 1909, Bd. lxxxiv., S. 666; (III.) Sitzber. d. Kais.
Akad., etc., 1909, Bd. cxviii., Abt. I., S. 1337.

[32] Richter, O., “Ernährung der Algen,” 1911.

[33] Robbins, W. J., “Direct Assimilation of Organic Carbon by
_Ceratodon purpureus_,” Bot. Gaz., 1918, lxv., pp. 543-51.

[34] Schindler, B., “Ueber den Farbenwechsel der Oscillarien,”
Zeitsch. f. Bot., 1913, v., pp. 497-575.

[35] Ternetz, Charlotte, “Beiträge zur Morphologie und Physiologie
der _Euglena gracilis_,” Jahrb. f. Wiss. Bot., 1912, Bd. 51, S. 435.

III. RELATION OF ALGÆ TO NITROGEN.

[36] Berthelot, “Recherches nouvelles sur les microorganismes
fixateurs de l’azote,” Comptes Rend., 1893, cxvi., pp. 842-49.

[37] Bouilhac, R., “Sur la fixation de l’azote atmosphérique par
l’association des algues et des bactéries,” Comptes Rend., 1896,
cxxiii., pp. 828-30.

[38] Bouilhac and Giustiniani, “Sur une culture de sarrasin en
présence d’un mélange d’algues et de bactéries,” Comptes Rendus,
1903, cxxxvii., pp. 1274-76.

[39] Charpentier, P. G., “Alimentation azotée d’une algue: Le
Cystococcus humicola,” Ann. Inst. Pasteur, 1903, 17, pp. 321-34.

[40] Fischer, Hugo, “Über Symbiose von Azotobacter mit Oscillarien,”
Centr. f. Bakt., 1904, xii.

[41] Frank, B., “Uber den experimentellen Nachweis der Assimilation
freien Stickstoffs durch Erdbewohnende Algen,” Ber. der D. Bot.
Gesellsch., 1889, vol. vii., pp. 34-42.

[42] Frank, B., “Ueber den gegenwärtigen Stand unserer Kenntnisse der
Assimilation elementaren Stickstoffs durch die Pflanze,” Ber. der. D.
Bot. Ges., 1889, vii., 234-47.

[43] Frank, B., and Otto, R., “Untersuchungen über Stickstoff
Assimilation in der Pflanze,” Ber. der D. Bot. Ges., 1890, viii.,
331-342.

[44] Gautier and Drouin, “Recherches sur la fixation de l’azote par
le sol et les végétaux,” Compt. Rend., 1888, cvi., pp. 1174-76;
General Conclusions, p. 1232.

[45] Kossowitsch, P., “Untersuchungen über die Frage, ob die Algen
freien Stickstoff fixiren,” Bot. Zeit., 1894, Heft 5, S. 98-116.

[46] Krüger, W., und Schneidewind, “Sind niedere chlorophyllgrüne
Algen imstande, den freien Stickstoff der Atmosphäre zu assimilieren
und Boden an Stickstoff zu bereichern?” Landwirtschaftliche Jahrb.,
1900, Bd. 29, S. 771-804.

[47] Moore, Benjamin, and T. Arthur Webster, “Studies of the
photosynthesis in f.w.a.” (I.) “The fixation of both C and N from
atmosphere to form organic tissue by green plant cell”; (II.)
“Nutrition and growth produced by high gaseous dilutions of simple
organic compounds, such as formaldehyde and methylic alcohol”; (III.)
“Nutrition and growth by means of high dilution of CO₂ and oxides of
N without access to atmosphere,” Proc. Roy. Soc., London, 1920, B.
xci., pp. 201-15.

[47_a_] Moore, B., Whiteley, Webster, T. A., Proc. Roy. Soc., London,
B., 1921; xcii., pp. 51-60.

[48] Reinke, J., “Symbiose von Volvox und Azotobacter,” Ber. der d.
Bot. Ges., 1903, Bd. xxi., S. 481.

[49] Russell, E. J., and Richards, E. H., “The washing out of
Nitrates by Drainage Water from Uncropped and Unmanured Land,” Journ.
Agric. Sci., 1920, vol. x., Part I.

[50] Schloesing, fils, and Laurent, E., “Recherches sur la fixation
de l’azote libre par les plantes,” Ann. de l’Institut Pasteur, 1892,
vi., pp. 65-115.

[51] Schramm, J. R., “The Relation of Certain Grass Green Algæ to
Elementary Nitrogen,” Ann. Mo. Bot. Gard., 1914, i., No. 2.

[52] Wann, F. B., “The Fixation of Nitrogen by Green Plants,” Amer.
Journ. Bot., 1921, viii., pp. 1-29.

CHAPTER VII.

THE OCCURRENCE OF FUNGI IN THE SOIL.

NOTE.--I am indebted to my late colleague Miss Sibyl S. Jewson,
M.Sc., for permission to include unpublished data from our
investigations on the soil fungi.

In 1886 Adametz,[1] investigating the biochemical changes occurring
in soils, isolated several species of fungi. It was, however, only
with the work of Oudemans and Koning,[17] in 1902 when forty-five
species were isolated and described, the majority as new to science,
that the real study of the fungus flora of the soil commenced. There
is now no doubt that fungi form a large and very important section
of the permanent soil population, and certain forms are found only
in the soil. Indeed, Takahashi[22] has reversed the earlier ideas by
suggesting that fungus spores in the air are derived from soil forms.
The majority of investigations on this subject fall, perhaps, into
one or more of three classes: (_a_) purely systematic studies such as
those of Oudemans and Koning,[17] Dale,[5] Jensen,[9] Waksman,[25a]
Hagem,[8c] Lendner,[12] and others, which consist in the isolation
and identification of species from various soils: (_b_) physiological
researches, such as those of Hagem[8c] on the Mucorineæ of Norway, or
the many investigations on the biochemical changes in soils produced by
fungi, such as those of Muntz and Coudon,[15] McLean and Wilson,[15]
Kopeloff,[11] Goddard,[7] McBeth and Scales,[14] and others: (_c_)
quantitative studies, such as those of Remy,[20] Fischer,[6]
Ramann,[18] Waksman,[25c] and Takahashi,[22] which involve numerical
estimates of the fungus flora in soils.

QUALITATIVE STUDY.

With very rare exceptions soil fungi cannot be examined in situ, and
the necessary basis of any qualitative research is the isolation of
the organisms in pure culture. Most soil forms belong to the _Fungi
imperfecti_, and often show considerable plasticity on artificial
media. This makes it very difficult to determine them by comparison
with type herbarium specimens or published morphological diagnoses.
In consequence many soil fungi have not infrequently been given new
specific names, as _humicola_, _terricola_, and so forth, which is
very unsatisfactory, and means that the determinations have little
significance.

Furthermore, most artificial media are slight variations on a few
common and simple themes, and are very selective, permitting the growth
of a moiety only of the fungi present. In addition, many fungi grow
so slowly that they are overwhelmed by the more rapidly germinating
or spreading forms, or on the other hand, they may be eliminated by
the metabolic products of different adjacent colonies. The extremely
selective nature of the technique commonly used is shown if one
tabulates systematically all the fungi which have been recorded or
described in soil investigations. Of _Phycomycetes_ there are fifty-six
species of eleven genera; of _Ascomycetes_ twelve species of eight
genera; and of _Fungi imperfecti_, including _Actinomycetes_ but not
sterile _Mycelia_, 197 species of sixty-two genera. Rusts and Smuts
one might not expect, but that of the multitudes of _Basidiomycetes_
growing in wood and meadow not one should have been recorded is indeed
startling. It was at first thought that many imperfect fungi might
be conidial stages of _Basidiomycetes_, but much search among forms
isolated at Rothamsted has, up to the present, failed to reveal clamp
connections in the hyphæ.

Since various species of soil fungi have different optimum temperature,
humidity and other conditions[3] one would not expect to find an even
geographic distribution. Very little is yet known of this aspect, but
_Rhizopus nigricans_, _Mucor racemosus_, _Zygorrhynchus vuilleminii_,
_Aspergillus niger_, _Trichoderma koningi_, _Cladosporium herbarum_,
and many species of _Aspergillus_, _Penicillium_, _Fusarium_,
_Alternaria_, and _Cephalosporium_ have been commonly found throughout
North America and Europe wherever soils have been examined. Species of
_Aspergillus_, however, would appear to be more common in the soils
of south temperate regions and species of _Penicillium_, _Mucor_,
_Trichoderma_, and _Fusarium_ more abundant in northern soils.

It is well known that in many plant and animal communities there
occurs a definite rhythm, various species following each other in a
regular sequence as dominants in the population. Although it is not yet
possible to make any definite statement there would seem indications
that this may also be true of the soil fungi.

Much work has been done on the distribution of species at different
depths in the soil, but the results are still confusing. Thus,
examining eighteen species, Goddard[7] found no difference in relative
distribution down to 5½ inches. Werkenthin[26] found identical species
from 1-4 inches, and then an absence of fungi from 5-7 inches, which
latter was the greatest depth he examined. Waksman[25] found little
difference in the first six inches, but very few species below 8 inches
except _Zygorrhynchus vuilleminii_, which extended down to 30 inches
and was often the only species occurring below 12 inches. Taylor[23]
has reported species of _Fusarium_ at practically every depth to 24
inches. Rathbun[19] found _Aspergillus niger_, _Rhizopus nigricans_,
and species of Fusarium and Mucor down to 34 inches, and _Oospora
lactis_, _Trichoderma koningi_, _Zygorrhynchus vuilleminii_ and species
of _Penicillium_, _Spicaria_ and _Saccharomyces_ as deep as 44 inches.
Eleven species were isolated from the alimentary canal of grubs and
worms, and Rathbun concluded that soil fungi may be spread by these
organisms.

On an unmanured grass plot at Rothamsted twenty species were isolated
from a depth of 1 inch, nineteen from 6 inches, and eleven from
12 inches, whereas on the unmanured plot of Broadbalk wheat field
twenty-six species were obtained from 1 inch, seven from 6 inches, and
five from 12 inches. There appeared to be no conspicuous differences
between the floras of the two plots save that in the Broadbalk plot
there were fewer Mucorales, and _Zygorrhynchus mœlleri_ and _Absidia
cylindrospora_ were absent. In the grass plot samples about one-half
the forms occurring at the lower levels were isolated also from the
upper levels, but in the Broadbalk sample the five forms isolated from
12 inches, and five out of seven of those at 6 inches occurred only at
those levels, i.e. each of the three levels appeared to have a specific
flora. The difference in depth distribution in these two cases may
be due to the fact that in the Broadbalk plot the stiff clay subsoil
occurs at 5-7 inches, whereas in the grass plot the depth of soil is
greater than 12 inches. Much further work needs to be done on this
aspect before any definite conclusion can be reached.

Much scattered information is available concerning the effect of
soil type, manuring, treatment, cropping, and so forth upon the
fungus content, but no clear issue as yet emerges from the results.
Hagem[8] found that cultivated soils vary greatly from forest soils
in the species of _Mucor_ present, and that certain species seem
to be associated in similar environments. Thus in pinewoods _Mucor
ramannianus_ is usually found, together with _M. strictus_, _M.
flavus_, and _M. sylvaticus_, and with this “_M. Ramannianus Society_,”
_M. racemosus_, _M. hiemalis_, and _Absidia orchidis_, are frequently
associated. The differences found by Hagem between the species of
_Mucor_ from forest and cultivated land could not, however, be
confirmed by Werkenthin.[26]

Dale,[5] examining sandy, chalky, peaty and black earth soils, found
specific differences, although many of the species were common to all.
A soil which had been manured continuously for thirty-eight years
with ammonium sulphate alone, contained twenty-two species, whereas
the same soil with the addition of lime only had thirteen species.
Both Goddard[7] and Werkenthin,[26] in their investigations, found
a constant and characteristic fungus flora regardless of soil type,
tillage, or manuring. Waksman’s[25] studies of forest soils showed
few species of _Mucor_ but many of _Penicillium_ and _Trichoderma_[2];
orchard soil contained no species of _Trichoderma_, very few of
_Penicillium_, but a large number of species of _Mucor_; species of
_Trichoderma_ were common in acid soils, whilst cultivated garden
soil contained all forms. The examination of very differently manured
plots on the Broadbalk wheat field at Rothamsted has not shown any
striking differences in the fungus flora, all the more important groups
of species being represented in every plot, but significant minor
differences are present. Thus, plot 13, manured with double ammonium
salts, superphosphate and sulphate of potash, is especially rich in
“species” of Trichoderma, whereas the unmanured plot contains large
numbers of species of green _Penicillium_, _Trichoderma_, and a species
of _Botrytis_ (pyramidalis?).

The effect of the crop upon the fungus flora is seen in cases where
the same crop is grown year after year as in certain flax areas, where
species of _Fusarium_ accumulate in the soil and tend to produce “flax
sickness.”[13]

QUANTITATIVE STUDY.

As it is not possible to count the soil fungi _in situ_, any estimation
of the numbers present in a soil must be arrived at by indirect means.
The method adopted is to make as fine a suspension as possible of
a known quantity of soil sample in a known amount of water, dilute
this to 1/5000, 1/10000, and so forth by regular gradations, incubate
cubic centimetres of the final dilution on artificial media in petri
dishes, and count the colonies of fungi developing in each plate.
Using the average figures from a series of duplicate plates, the
number of “individual” fungi in a gram of the original soil sample may
then be calculated. The very few students who have made quantitative
estimations have obtained very unsatisfactory results. In bacterial or
protozoal estimations, the shaking of the soil suspension separates the
unicellular individuals, so that in the final platings each individual
from the soil theoretically gives rise to one colony on the medium.
In the case of fungi, the organisms may be in the form of unicellular
or multicellular spores or larger or smaller masses of unicellular or
multicellular mycelium differing for each particular species or phase
of development within the single species. The organisms may be sterile
in the soil or form fruiting bodies, consisting of few or myriads
of locally or widely distributed spores. In the process of shaking
the soil-suspension fungi of different organisation or of differing
developmental stages may be broken up and moieties fragmented in
totally different ways or to very different degrees. With protozoa and
bacteria the relation of soil individual to plate colony is direct;
with fungi we do not know what is the soil “individual” nor whether it
is the same for different fungi; nor can we yet profitably discuss any
significant numerical relationship of plate colonies to soil organisms.
Thus Conn[4] has pointed out that the plate count of a fungus indicates
only the ability to produce reproductive bodies and found that the
spores of one colony of _Aspergillus_, if distributed evenly through
a kilogram of soil, could produce the average plate counts obtained
by Waksman. Abundant vegetative growth may, in some species, reduce
or inhibit spore formation, so that of two species the one giving a
lower count might really be much the more important and plentiful
in the soil. Further, the colonies developing in the final plates
represent only a selected few of the fungi present in the soil sample,
the _Basidiomycetes_, and no doubt many other forms, being absent.
In addition, different media differ among themselves in the average
number of colonies developing on the plates, each medium giving,
as it were, its own point of view. Thus, in one experiment carried
out at Rothamsted by Miss Jewson, using the same soil suspension,
twenty plates of Coon’s Agar gave 357 colonies, of Cook’s Agar 246,
of Czapek’s Agar 215, and of Prune Agar 366. Thus if one only used
Coon’s Agar and Prune Agar one would obtain a total of 723 colonies,
whereas the same suspension on Cook’s Agar and Czapek’s Agar would
give only 461, and the calculated numbers of fungi per gram of soil
would be totally different. Further, if a single medium be taken, it
is found that slight alterations in the degree of acidity may make
very considerable differences in the final numbers. Thus Coon’s Agar
acidified to a hydrogen ion concentration of 5·0 gave as the results of
four series the following average numbers of colonies per plate, 17,
23·75, 18, 23. When, however, the medium was acidified to a PH of 4·0
to 4·3, corresponding averages from three series were 38, 46·3, and
44·8; i.e. the final estimations of numbers of fungi in the soil was
about twice as great. Again, the degree of dilution of soil suspension
used in plating may also be a very serious factor. Thus, if a series of
dilutions be made of 1/80,000, 1/40,000, 1/20,000, 1/10,000, 1/5,000
and 1/2,500, the average plate numbers should be in the proportions
of 1, 2, 4, 8, 16, and 32 respectively. In an actual experiment, the
following average plate numbers were obtained, 15·4, 32·8, 59·1, 104·0,
150, 224·5, which show a very decided reduction in the higher numbers.
If, however, dilutions of a suspension of spores of a single species be
made, this reduction does not occur.

These are but three of the very numerous factors involved in the
technique of quantitative estimation, and every single factor may be
the source of errors of similar magnitude, minute fluctuations in the
operations leading to the final platings having very considerable
effect upon the numbers of colonies that develop.

By critically evaluating each particular factor in the method, and
making statistical correction, it has, however, been found possible to
obtain series of duplicate plates comparing very favourably and thus to
extract certain figures which, whilst not possessing any final value,
have yet a certain general and comparative worth. Thus, 20·0, 18·2,
and 16·8 were obtained as the averages of six plates each, of a soil
suspension divided into three parts, and the individual plate numbers
in all three series were within the range of normal distribution. The
meaning of these numerical estimates in relation to fungi per gram
of soil sample is, however, entirely hypothetical, and to have value
quantitative comparison should only be made between single species
or groups of species closely related physiologically, and where the
technique is standardised.

[Illustration: FIG. 19.--Monthly Counts of Numbers of Fungi per gramme
of Dry Soil. Broadbalk Plot 2 (Farmyard Manure), Rothamsted.

X-axis: ~Apr.~ 1921 May Jun. ~Jul.~ Aug. Sep. ~Oct.~ Nov. ~Dec.~ Jan.
1922 ~Feb.~ Mar. Apr. May ~Jun.~ Jul. Aug. ~Sep.~ ~Oct.~

Y-axis: 10.000 per Gramme of Soil]

No comparative estimations have been made of the number of fungi in
the soils of different regions. There are, however, certain figures
which show that decided seasonal differences exist. Thus, correcting
and averaging certain of Waksman’s results[25] the following numbers
of fungi per gram of soil at 4 inches deep are obtained; September,
768,000; October, 522,000; November, 310,000; January, 182,000. At
Rothamsted results have been obtained which would appear to mark
a clear seasonal rhythm, corresponding in the time of its maxima
in Autumn and Spring with the periodicities known for many other
ecological communities (Fig. 19).

The numbers of fungi at various depths in the soil show very clearly
marked differences. The distribution in the top 4-6 inches depending
probably upon the depth of soil, is more or less equal, but there is a
very rapid falling off in numbers, especially between 5-9 inches, until
at 20-30 inches fungi are either very few in number or absent. Thus
Takahashi[22] found 590,000 fungi per gram at a depth of 2 cms. and
only 160,000 at 8 cms.

TABLE XIII.--INFLUENCE OF SOIL TREATMENT UPON THE NUMBERS OF FUNGI AS
DETERMINED BY THE PLATE METHOD--(AFTER WAKSMAN).

+--------------------------+---------+-----------------+
| | |Numbers of Fungi |
| Soil Fertilisation. |Reaction.|per Gram of Soil.|
+--------------------------+---------+-----------------+
| | P.H. | |
|Minerals only | 5·6 | 37,300 |
|Heavily manured | 5·8 | 73,000 |
|Sodium nitrate | 5·8 | 46,000 |
|Ammonium sulphate | 4·0 | 110,000 |
|Minerals and lime | 6·6 | 26,200 |
|Ammonium sulphate and lime| 6·2 | 39,100 |
+--------------------------+---------+-----------------+

The type of soil and its treatment exercise a great influence over
the number of fungi present. Fischer[6] found that farmyard manure
increased the number of fungi in uncultivated “Hochmoor,” cultivated
“Grunlandmoor,” and a clay soil by two, three, and five times
respectively. Waksman’s results[25] indicate that the more fertile
soils contain more fungi, both in number and species, than the less
fertile ones, and if one averages his results, the following figures
are obtained: garden soil, 525,000 per gram; orchard soil, 250,000;
meadow soil, 750,000; and forest soil, 151,000. Recently Waksman[25_e_]
has found that manure and acid fertilisers increase the numbers of
fungi in the soil, whereas the addition of lime decreases them (Table
XIII.).

Jones and Murdock[10] examined surface and sub-surface samples of
forty-six soils representing seventeen soil types in eastern Ontario.
Molds were fairly uniform in numbers in all soils except a sandy clay
loam and sandy clay shale, in which they were absent.

It has also frequently been pointed out that acid and water-logged
soils are richer in fungus content than normal agricultural soils.
On the other hand, Brown and Halversen[2] found, examining six plots
receiving different treatment and studied through a complete year, that
the numbers of fungi were unaffected by moisture, temperature, or soil
treatment. Against this, however, must be set the work of Coleman[3]
who studied the activities of fungi in sterile soils and found such
factors as temperature, aeration and food supply to exercise a deciding
control.

Investigations at Rothamsted show that Broadbalk plot 13, receiving
double ammonium salts, superphosphate and sulphate of potash and
yielding 31 bushels per acre, and plot 2, receiving farmyard manure and
yielding 35·2 bushels, contain approximately equal numbers of fungi.
This figure is about half as high again as that for plot 3, which
is unmanured and yields 12·6 bushels, plot 10, with double ammonium
salts alone and yielding 20 bushels, and plot 11, with double ammonium
salts and superphosphate and yielding 22·9 bushels per acre. A primary
factor, however, in all considerations such as these is the equality
of distribution of fungi laterally in any particular soil. There are
probably few soils so homogeneous as the Broadbalk plots at Rothamsted,
and on plot 2 (farmyard manure since 1852) samples taken from the lower
and upper ends and the middle region gave average numbers of colonies
per plate of 24, 23, and 25 respectively. On the other hand, soil
samples taken only a few yards apart in the middle region of the plot
gave average plate counts of 33·7 and 56·8.

CONCLUSION.

Surveying generally the field covered in this chapter, one can only
be impressed with the fragmentary character of our knowledge and with
the fact that, owing to the selective nature of the technique, the
data we possess, if assumed to be representative, give an entirely
partial and erroneous picture of the soil fungi. From the qualitative
aspect, the chief impediment is the impossibility of obtaining reliable
specific determinations of very many of the soil fungi. Lists of
doubtfully-named forms from particular soils or geographic regions
are useless or a positive evil, and there is imperative need for the
systematising of selected genera by physiological criteria, such as has
been partially done for _Penicillium_, _Fusarium_, and _Aspergillus_.
Furthermore, until a standardised and non-selective technique has been
devised, or a number of standardised selective methods for particular
groups, comparative investigations into specific distribution can
give little of value. This latter criticism is also very applicable
if regard be paid to the quantitative aspect of soil work, for
progress here largely depends upon the elaboration of a standardised
fractionation technique. Every single factor in these methods needs
exact analysis, for each gives opportunity for great error, and each
error is magnified many thousand times in the final results. Much
has been done in this direction at Rothamsted, but more remains to
do. Finally, working with single species in sterilised soil under
standardised conditions, there is fundamental work to be done on the
relation of plate colony to soil “individual.”

[1] Adametz, I., “Untersuchungen über die niederen Pilze der
Ackerkrume,” Inaug. Diss., Leipzig, 1886.

[2] Brown, P. E., and Halversen, W. V., “Effect of Seasonal
Conditions and Soil Treatment on Bacteria and Molds in Soil,” Iowa
Agric. Expt. Sta. 1921, Res. Bull., 56.

[3] Coleman, D. A., “Environmental Factors Influencing the Activity
of Soil Fungi,” Soil Sci., 1916, v., 2.

[4] Conn, H. J., “The Microscopic Study of Bacteria and Fungi in
Soil,” N.Y. Agric. Expt. Sta., 1918, Bull. 64.

[5] Dale, E., (_a_) “On the Fungi of the Soil,” Ann. Mycol., 1912,
10; (_b_) “On the Fungi of the Soil,” Ann. Mycol., 1914, 12.

[6] Fischer, H., “Bakteriologisch-chemische Untersuchungen;
Bakteriologischen Teil,” Landw. Jahrb., 1909, 38.

[7] Goddard, H. M., “Can Fungi living in Agricultural Soil Assimilate
Free Nitrogen?” Bot. Gaz., 1913, 56.

[8] Hagem, O., (_a_) “Untersuchungen über Norwegische Mucorineen
I., Vidensk. Selsk, I.,” Math. Naturw. Klasse, 1907, 7; (_b_)
“Untersuchungen über Norwegische Mucorineen II., Vidensk. Selsk. I.,”
Math. Naturw. Klasse, 1910, 10.

[9] Jensen, C. N., “Fungus Flora of the Soil,” N.Y. (Cornell) Agric.
Expt. Sta., 1912, Bull. 315.

[10] Jones, D. H., and Murdock, F. G., “Quantitative and Qualitative
Bacterial Analysis of Soil Samples taken in Fall of 1918,” Soil Sci.,
1919, 8.

[11] Kopeloff, N., “The Effect of Soil Reaction on Ammonification by
Certain Soil Fungi,” Soil Sci., 1916, 1.

[12] Lendner, A., “Les Mucorinées de la Suisse,” 1908.

[13] Manns, S. F., “Fungi of Flax-sick Soil and Flax Seed,” Thesis,
N. Dak. Agric. Expt. Sta., 1903.

[14] McBeth, I. G., and Scales, F. M., “The Destruction of Cellulose
by Bacteria and Filamentous Fungi,” U.S. Dept. Agric. Bur. Plant
Indust., 1913, Bull. 266.

[15] McLean, H. C., and Wilson, G. W., “Ammonification Studies with
Soil Fungi,” N.J. Agric. Expt. Sta., 1914, Bull. 270.

[16] Muntz, A., and Coudon, H., “La fermentation ammoniaque de la
terre,” Compt. Rend. Acad. Sci. (Paris), 1893, 116.

[17] Oudemans, A. C., and Koning, C.J., “Prodrome d’une flore
mycologique, obtenue par la culture sur gelatin préparée de la terre
humeuse du Spanderswoud, près de Bussum,” Arch. Néerland. Sci. Exact
et Nat., 1902, s. ii., 7.

[18] Ramann, E., “Bodenkunde,” Berlin, 1905.

[19] Rathbun, A. E., “The Fungus Flora of Pine Seed Beds,”
Phytopath., 1918, 8.

[20] Remy, T., “Bodenbakteriologischen Studien,” Centr. f. Bakt.,
1902, ii., 8.

[21] Sherbakoff, C. D., “Fusaria of Potatoes,” N.Y. (Cornell) Agric.
Expt. Sta., 1915, Mem. 6.

[22] Takahashi, T., “On the Fungus Flora of the Soil,” Anns.
Phytopath. Soc., Japan, 1919, 1.

[23] Taylor, M. W., “The Vertical Distribution of _Fusarium_,”
Phytopath., 1917, 7.

[24] Thom, Ch., “Cultural Studies of Species of Penicillium,” U.S.
Dept. Agric. Bur. Animal Indus., 1910, Bull. 118.

[25] Waksman, S. A., (_a_) “Soil Fungi and their Activities,” Soil
Sci., 1916, 2; (_b_) “Do Fungi Actually Live in the Soil and Produce
Mycelium?” Science, 1916, 44; (_c_) “Is there any Fungus Flora of
the Soil?” Soil Sci., 1917, 3; (_d_) “The Importance of Mold Action
in the Soil,” Soil Sci., 1918, 6; (_e_) “The Growth of Fungi in the
Soil,” Soil Sci., 1922, xiv.

[26] Werkenthin, F. C., “Fungus Flora of Texas Soils,” Phytopath.,
1916, 6.

CHAPTER VIII.

THE LIFE OF FUNGI IN THE SOIL.

In the last chapter fungi were considered as so many specific but
functionless units in the soil. Unless, however, they are regarded
merely as inert spore contaminations from the air, a view which is
now no longer tenable, their very presence implies the existence
of innumerable vital relationships between the organisms and their
environment. From this point of view the studies treated in the
previous chapter are but the necessary first steps to an understanding
of the relation of soil fungi to living plants and of the part played
by them in the soil economy.

RELATION OF SOIL FUNGI TO LIVING PLANTS.

Older classifications of fungi frequently divided these organisms
into four categories--parasites, saprophytes, facultative parasites,
and facultative saprophytes, but the further mycological studies are
carried the more clearly it is seen that these groups are entirely
artificial. There are probably few fungi that cannot, under particular
conditions, invade living tissues, and it only seems a question of
time before at all events the vast majority of fungi will be grown on
synthetic media in the laboratory. From our present point of view the
importance of this lies in the fact that fungi living saprophytically
in the soil may, given the right conditions or the presence of some
particular host plant, become parasites or symbionts, and conversely
well-known pathogens may live a saprophytic existence. Thus Cucumber
Leaf Spot is caused by _Colletotrichum oligochætum_, and Bewley[3] has
repeatedly isolated this fungus from glasshouse manure and refuse of
various kinds. In his early studies, Butler[13] isolated many parasitic
species of _Pythium_ from Indian soils, and the presence of _P. de
Baryanum_ as a soil saprophyte has been confirmed by Bussey, Peters,
and Ulrich.[11] De Bruyn[17] has recently found that most species of
_Phytophthora_, including _P. erythroseptica_ and _P. infestans_ may
live as saprophytes in the soil, whilst Pratt[53] has isolated from
virgin lands and desert soils various fungi, which cause disease in
potatoes. In 1912 Jensen[29] gave a list of twenty-three “facultative
parasites” isolated from soil, and these are but a moiety of those
which could be listed to-day.

Furthermore, it was shown by Frank[24] many decades ago that forest
humus is not merely a mass of the remains of animals and plants,
but that a considerable part of its organic substance is made up of
fungus hyphæ, which ramify and penetrate in all directions. Evidence
is rapidly accumulating that this is also true of most other soils
containing organic matter. It is well known that many of the higher
plants live in symbiotic or commensal relationship with these humus
fungi, which are present in the host tissues as mycorrhiza, and further
studies only serve to show the widespread and fundamental nature
of this relationship. Thus many _Basidiomycetes_[50] (species of
_Tricholoma_, _Russula_, _Cortinarius_, _Boletus_, _Elaphomyces_, etc.)
possess a mycorrhizal relationship with various broad leaved trees,
such as beech, hazel, and birch[57] and with various conifers and
certain Ericales. Other Ericales show this relationship with species of
the genus _Phoma_,[62] many orchids, with species of _Rhizoctonia_[2]
(or _Orcheomyces_[10]), whilst _Gastrodia elata_ contains _Armillaria
mellea_.[36] Certain species of _Pteridophyta_ and _Bryophyta_ are
also known to certain mycorrhizal fungi. Of the numerous fungi taking
part in these mycorrhizal relationships, only a small number have yet
been identified, but there is little doubt that perhaps the majority
of these organisms must be regarded as true soil forms.[14],[45] The
mycological flora of the soil thus plays an important part in the life
of many higher forms of vegetation, and this relationship is a very
fruitful field for study.

RELATION OF FUNGI TO SOIL PROCESSES.

The great cycle of changes occurring in the soil whereby organic matter
is gradually transformed and again made available as plant food is
entirely dependent upon micro-organisms. Until a decade ago it was
thought that bacteria were by far the most important group concerned
in the bringing about of these changes, but recent studies have shown
that, in at all events certain arcs of this great organic cycle,
the fungi have, perhaps, an equal part to play. The life of fungi
in the soil may, for our purposes, be considered from three points
of view--their part in the decomposition of carbon compounds, their
nitrogen relationships, and their work in the mineral transformations
of the soil.

CARBON RELATIONSHIPS.

Of primary importance in the carbon relationships of soil fungi is
the part played in the decomposition of the celluloses, which compose
almost all the structural remains of plant tissues. Our first real
knowledge of this subject was given by Van Iterson[28] in 1904 when he
showed the wide extent of cellulose destruction by fungi, and devised
methods whereby fifteen cellulose-decomposing forms, many of which have
since proved to be common soil fungi, were isolated. Three years later
Appel[1] published his account of the genus _Fusarium_, and showed
that many of the species could destroy filter paper. A difficulty was
introduced in 1908 by Schellenberg,[60] who, working with common soil
forms, found that only hemicelluloses and not pure cellulose were
destroyed. This has recently been supported by Otto,[48] but from the
practical point of view the discussion is academic for the amount of
pure cellulose in plants is insignificant.

In 1913 McBeth and Scales[43] showed that a considerable number
of common soil fungi were most active cellulose destroyers, pure
precipitated cellulose and cotton being readily attacked. This was
supported by McBeth in 1916,[42] whilst Scales[59] has found that
most species of _Penicillium_ and _Aspergillus_ decompose cellulose,
especially where ammonium sulphate is the source of nitrogen.
Waksman[65] tested twenty-two soil fungi and found that eleven
decomposed cellulose rapidly and four slowly, whilst Dascewska,[16]
Waksman,[66],[67] and others have concluded that soil fungi play
a more important part in the decomposition of cellulose and in
“humification” than soil bacteria. Schmitz[61] has recently shown that
cellulose-destroying bacteria play no important part in the decay of
wood under natural conditions.

In addition to the celluloses, practically all simple and complex
organic carbon compounds are attacked by soil fungi, and in many cases
the decomposition is very rapid.[26] Many _Actinomycetes_, _Aspergilli_
and _Penicillia_ are active starch splitters, and it is of interest to
note that some of the strongest cellulose decomposers (_Melanconium
sp._, _Trichoderma sp._, and _Fusaria_) secrete little diastase.[66]
The _Mucorales_ apparently do not attack cellulose, but can only
utilise pectin bodies, monosaccharides, and partly disaccharides.[26]
Dox and Neidig[19] have shown that various species of _Aspergillus_
and _Penicillium_ are able to attack the soil pentosans. Roussy,[58]
Kohshi,[24] Verkade and Söhngen,[64] and many other workers have found
that fats and fatty acids are readily used as food by soil fungi, and
Koch and Oelsner[33] have recently shown that tannins are readily
assimilated. Klöcker,[32] Ritter,[56] and others have shown that the
utilisation of many carbon compounds is to a large extent determined by
the source of nitrogen and its concentration in the pabulum.

There would seem, therefore, no doubt that the decomposition of
celluloses and other carbon compounds is of primary importance in the
life-activities of soil fungi.

NITROGEN RELATIONSHIPS.

In this section we shall consider the problems of nitrogen fixation and
nitrification, of ammonification, and of the utilisation of nitrogenous
compounds by soil fungi.

As soil fungi form so large a part of the soil population, the question
of whether they can make use of the free nitrogen of the air is of
primary importance. During the last two decades many investigators have
attempted to solve the problem, often studying allied or identical
species; but if one consults some thirty researches published during
this period, opinion is found to be about equally divided. Even,
however, in those studies where nitrogen fixation has been recorded the
amounts are very slight, usually being below 5 mgrms. per 50 c.c. of
solution, and often being obviously within the limits of experimental
error. Latham,[37] however, working on _Aspergillus niger_, recorded
variations ranging from a nitrogen loss of 42·5 mgrms. to a nitrogen
fixation of 205·1 mgrms. per 50 c.c. of medium. Ternetz[63] found
that different strains of _Phoma radicis_ may fix from 2·5 mgrms. of
nitrogen in the lowest case, to 15·7 mgrms. in the highest per 50 c.c.
of nutrient solution. Duggar and Davis[20] report that _Phoma betæ_
may fix nitrogen in quantities of 7·75 mgrms. per 50 c.c. of medium.
The latter authors, in a very able critique of the problem, indicate
certain possible sources of error in previous work, and if one examines
the studies in which nitrogen fixation has been recorded in the light
of these criticisms, it is difficult not to think that, with the
exception of the genus _Phoma_, good evidence for nitrogen fixation
by fungi is lacking. _Phoma betæ_ is a common pathogen attacking
beets, whilst _P. radicis_ is a mycorrhizal form inhabiting various
Ericales. Apart from these exact quantitative studies, which have
given a negative verdict, there is a considerable amount of positive
but indirect evidence for nitrogen fixation by mycorrhizal fungi,[55]
and it is very unfortunate that more of these forms have not been
investigated quantitatively. As the evidence stands to-day, one must
conclude that the fungus flora does not play any part in the direct
nitrogen enrichment of the soil.

Equally obscure is the question of nitrification and denitrification
by soil fungi, but this is the result of a lack of study rather than
of a plethora of indeterminate researches. Direct nitrification or
denitrification has not been established, but the work of Laurent[38]
and a few other workers appears to show that soil fungi can reduce
nitrates to nitrites.

The second primary nitrogen relationship that we have to consider is
the process of ammonification. The ammonifying power of soil fungi was
first demonstrated by Muntz and Coudon,[46] and by Marchal[40] in 1893,
the former showing that _Mucor racemosus_ and _Fusarium Muntzii_ gave a
larger accumulation of ammonia in soil than any of the bacteria tested;
and the latter that _Aspergillus terricola_, _Cephalothecium roseum_
and other soil fungi were active ammonifiers, especially in acid
soils. Shibata,[62] Perotti,[49] Hagem,[26] Kappen,[31] Löhnis,[39]
and others, have observed that urea, dicyanamide and cyanamide are
decomposed with the liberation of ammonia; and Hagem[26] has recorded
the same process for peptones, amino acids, and other organic nitrogen
compounds in plant and animal remains in the soil. The latter author
considers soil fungi more important ammonifying agents in the soil than
bacteria, a conclusion in which McLean and Wilson,[44] and perhaps most
later workers concur. McLean and Wilson[44] found large differences in
the ammonifying powers of various soil fungi, the _Moniliaceæ_ being
the strongest ammonifiers, the _Aspergillaceæ_ the weakest. Generic and
specific differences have been confirmed by Coleman,[15] Waksman,[67]
and other authors. Waksman and Cook[70] suggested that such variations
may be due, not to innate differences in the metabolic activities of
the several organisms, but to differences in reproductive times, and
that there might be some relationship between sporogeny and the ability
to accumulate nitrogen. Kopeloff[35] has carried out experiments on
the inoculation of sterilised soil with known quantities of spores
and found that, although the amount of ammonia accumulated increased
with the number of spores the proportion was not direct but modified
by the food supply. After the first five days’ growth, the rate of
ammonia production varied markedly in a two-day rhythm which seemed
to be due to the metabolism of the fungus rather than to recurrent
stages of spore formation and germination in the life history. The
amount of ammonia liberated has been shown by recent work[66] to depend
upon the available sources of carbon and nitrogen. In the absence of
a carbohydrate supply the protein is attacked both for carbon and
nitrogen, and since more of the former is required much ammonia is
liberated. In addition, however, to the carbon and nitrogen control,
the process of ammonification by soil fungi is intimately related to
physical conditions. Working with pure cultures, McLean and Wilson,[44]
Coleman,[15] Kopeloff,[35] Waksman and Cook,[70] and other students,
have shown that the amount of ammonia accumulated depends upon such
factors as the presence of phosphates, the period of incubation of the
fungi, aeration, the moisture in the soil, the temperature, the degree
of soil acidity, the type of soil, and so forth.

That fungi take a very important place as ammonifying agents in the
soil can no longer be doubted, but the question yet remains to be
considered of the balance of profit or loss resulting from their
activities. It has usually been considered that a part of the ammonia
freed is used by the fungi themselves, but that the greater part
is liberated, and so rendered available to nitrifying organisms.
Both Neller[47] and Potter and Snyder[51] found that typical soil
fungi inoculated into sterile soil grew with a vigour approximately
equal to the growth induced by an inoculation of the entire soil
flora. This is largely to be accounted for by the fact that when
soils are sterilised by heat or by certain chemicals, breaking-down
changes occur, and substances are liberated which are peculiarly
favourable to fungus growth. This fact must be borne in mind when
interpreting ammonification and other studies where the method is that
of inoculation of fungi into sterilised soil. In many cases it tends
to nullify any application of the results to normal soils, whilst in
others the conclusions must be accepted with some reserve. In all
cases Potter and Snyder[51] found that fungi caused a diminution in
the amount of nitrates, that the ammonia was not much changed in
amount, and that there was a decrease in the quantities of soluble
non-protein nitrogen. The range of organic and inorganic nitrogenous
compounds utilisable by soil fungi is very great. Ritter[56] has shown
that certain forms can use the nitrogen of “free” nitric acid in the
medium; Ritter,[56] Hagem,[26] and others, that soil fungi can use
ammonia nitrogen equally with nitrate nitrogen, and Ehrenberg[21]
concluded that soil fungi play a more important part in the building
of albuminoids from ammonia than bacteria do. Ehrlich[22] has shown
that various heterocyclic nitrogen compounds and alkaloids can serve as
sources of nitrogen to soil fungi, whilst Ehrlich and Jacobsen[23] have
found that soil fungi can form oxy-acids from amino-acids. Hagem,[26]
Povah,[52] Bokorny,[6],[8] and others, state that for many soil forms
organic nitrogen sources are better than inorganic sources, and that
peptones, amino-acids, urea, and uric acids, etc., are very quickly
utilised by species of _Mucor_, yeasts, and so forth. Butkevitch,[12]
and Dox[18] have recently found that it depends on circumstances which
compounds of protein molecule can be utilised by particular fungi, and
that soil fungi can utilise both amino and amido complexes for the
formation of ammonia. In 1919 Boas[4] showed for _Aspergillus niger_
that if a number of nitrogenous compounds are available the fungus
absorbs the most highly dissociated.

In the welter of scattered observations on the utilisation of
nitrogenous compounds, it is difficult to trace any clear issue. That
proteins, amino-acids, and other complex organic compounds are readily
broken down to ammonia by soil fungi is clear, and, on the other hand,
it is also clear that soil fungi utilise extensively ammonia and
nitrates as sources of nitrogen. On which side the balance lies it is
yet impossible to say.

MINERAL RELATIONSHIPS.

Heinze[27] and Hagem[26] have stated that soil fungi make the insoluble
calcium, phosphorus, and magnesium compounds in soil soluble and
available for plant food; and Butkevitch[12] has used _Aspergillus
niger_ in determining the availability of the mineral constituents,
but practically no work has yet been carried out on these problems.
A further matter on which sound evidence is greatly to be desired is
the part played by soil fungi in the oxidation processes of iron and
sulphur.

A point which may be mentioned here, as it is of some considerable
practical importance, is the large quantity of oxalic, citric, and
other acids formed by certain common soil fungi. Acid formation
is partly dependent upon the species of fungus--even more the
physiological race within the species--and partly upon the substratum,
particularly the source of carbon.[5],[54] It is interesting that as
a group _Actinomycetes_ do not form acids from the carbon source but
alkaline substances from the nitrogen sources.[69]

CONTROL OF SOIL FUNGI.

In the preceding sections an attempt has been made to sketch rapidly
the chief outlines of the widespread relationships of soil fungi and
of the fundamental part that they play in the biochemical changes
occurring in the soil. It will be evident, even from this survey,
that their occurrence is of the utmost agricultural importance, both
when helpful as in mycorrhizal relationships or as agents in making
complex organic materials available as plant food, or when harmful
as when causal agents of disease in plants. It is clear that could
the soil fungi be controlled to human ends by the encouragement of
the useful forms and the elimination of the harmful, a valuable power
would be placed in the hands of the grower of plants. Certain aspects
of this control, the cruder and more destructive perhaps, are already
practicable, whilst the finer and more constructive aspects remain
possibilities of to-morrow.

Theoretically, the technique of control is selective in that it aims
to determine one or more particular fungi, leaving the remaining flora
untouched. Its highest expression is seen, perhaps, in the utilisation
of pure cultures of mycorrhizal fungi for horticultural purposes, such
as orchid cultivation, but there is no reason why this should not be
done for other purposes on a field scale similar to the way in which
cultures of special strains of the root nodule organisms of legumes
are employed. A second aspect is the direct encouragement of special
components of the fungus flora for particular purposes by selective
feeding. Thus, in a laboratory experiment, McBeth and Scales[43] record
an increase of 2000 times in cellulose-destroying and other soil fungi
by this method. It has been pointed out that soil fungus activities
such as ammonification, proteolysis and carbohydrate decomposition are
controlled by factorial equilibria, and for special purposes it would
seem feasible to weight the balance so that particular activities may
be favoured. A further step in this direction is the controlling of
particular physical conditions so that the activities of certain fungi
may be restricted. Professor L. R. Jones[30] and his colleagues at
Madison have shown the primary importance of the control of the soil
temperature in certain parasitic relationships; the work of Gillespie
and Hurst[25] and later workers has demonstrated that the parasitism
of certain species and strains of _Actinomyces_ upon the potato
is conditioned by definite ranges of soil acidity; and many other
relationships of similar nature are known. Data along such lines are
rapidly accumulating, and in certain cases are already susceptible of
practical application. In other cases, particular soil fungi are less
open to persuasive influences, and more drastic treatment needs to be
adopted. Certain chemicals mixed intimately with the soil increase or
diminish the numbers of particular fungi or groups of fungi; whilst
these organisms may be totally eliminated from the soil by wet or
dry heat for definite periods or by treatment with potent fungicides
such as formaldehyde. Although soil sterilisation and crude treatment
in other ways has been practised for decades, the possibility of a
more delicate control of soil fungi is only now being realised. Its
concrete expression will depend upon the progress that is made in exact
knowledge of the activities of soil fungi under natural and controlled
conditions, of the balance of factors in the environment which controls
any particular function and of the genetic nature of the soil fungi
which occur. Each of these aspects is a fruitful field of study.

RELATION TO SOIL FERTILITY.

From a general survey of the researches that have been carried out on
soil fungi during the past two decades certain issues emerge. It would
seem clear that fungi occupy, perhaps, a primary place as factors in
the decomposition of celluloses, and thus may be the chief agents in
the transformation of plant remains to humus and to soluble compounds
which can be used as food by the nitrogen-fixing bacteria. Furthermore,
soil fungi are very important ammonifiers, but whether the balance
of ammonia freed is utilised by the fungi themselves, or whether it
is made available to nitrifying bacteria is not yet clear. If the
latter is the case, soil fungi play a valuable indirect rôle in the
accumulation of available plant food in the soil. On the other hand, by
utilising nitrates as sources of nitrogen, fungi may play an important
part in the depletion of the nitrogenous food in the soil available to
crop plants. Thirdly, soil fungi apparently take no part in the direct
nitrogen enrichment of the soil. Thus, soil fungi would seem to be the
most important factor in the first half of that great cycle whereby
organic remains become again available as organic food.

The impression left on one’s mind by the study of the life of fungi
in the soil is of an infinitely complex series of moving equilibria,
the living activities being determined by both biological and
physico-chemical conditions. All these factors play an integral part in
the life of the soil fungi and must be considered if a true picture is
to be drawn. The principal factors may be classified into the following
groups: Most evident, perhaps, are the natures and specificities of
the fungi and the relative composition of the fungus flora. Equally
important, however, are the quantity and quality of the foods available
and the non-biological environment which results from the complex
series of physical and chemical changes occurring in the soil causally
independent of the organisms present, which interacts with the equally
vast series of changes resulting from fungus activities. Finally, one
must consider the interacting biological environment of surface animals
and plants and the microscopic fauna and flora. The complexities are
such that only the application of Baconian principles can unravel
them. A beginning has been made in the study of pure cultures of soil
fungi on synthetic media, and much valuable data have accrued, but
it is obviously not possible to apply directly to soil the results
obtained in such work. They remain possibilities; in certain cases
probabilities, but nothing more. A further step, one already taken
and of great promise, is the investigation of the changes occurring
in sterilised soils inoculated with known quantities of one or more
pure cultures of particular soil fungi. Such intensive study of single
factors in a standardised natural or artificial soil, to which has been
added a pedigreed fungus, is, perhaps, the most fruitful avenue of
progress. In all such work, however, one must bear acutely in mind the
fact that a sterilised soil and, still more, an artificial soil, is a
very different complex from a normal soil, and that results obtained
from the inoculation of such soils are not applicable directly in the
elucidation of ordinary soil processes. At present there is no method
known of completely sterilising a soil which does not destroy the
original physico-chemical balance. It is evident that the complexities
are such that chemist, physicist, and biologist must all co-operate
if the significance of the processes is to be understood, and a solid
foundation laid for future progress and for practical application.

[1] Appel, O., “Untersuchungen über die Gattung _Fusarium_,” Mitt.
Biol. Reichanst. Land- u. Forstw., 1907, 4.

[2] Bernard, N., “L’évolution dans la symbiose. Les Orchidées et
leurs Champignons commensaux,” Ann. Sci. Nat. (Bot.), Ser. 9, 1909, 9.

[3] Bewley, W. F., “Anthracnose of the cucumber under glass,” Journ.
Min. Agric., 1922, xxix.

[4] Boas, F., “Die Bildung löslicher Stärke im elektiven
Stickstoff-Stoffwechsel,” Ber. deut. bot. Ges., 1919, 37.

[5] Boas, F., und Leberle, H., “Untersuchungen über Säurenbildung bei
Pilzen und Hefen II.,” Biochem. Ztschr., 1918, 92.

[6] Bokorny, T., “Benzene derivatives as sources of nourishment,”
Zentr. Physiol., 1917, 32.

[7] Bokorny, T., “Sugar fermentation and assimilation,” Allg. Brau.
Hopfen Zeit., 1917, 57.

[8] Bokorny, T., “Verhaltung einiger organischer Verbindungen in der
lebenden Zelle,” Pflügers Archiv., 1917, 168.

[9] Brown, P. E., “Mould action in soils,” Science, 1917, 46.

[10] Burgeff, H., “Die Wurzelpilze der Orchideen,” Jena. 1909.

[11] Bussey, W., Peters, L., and Ulrich, P., “Ueber das Vorkommen
von Wurzelbranderregern im Boden,” Arb. Kais. Biol. Anst. Land- u.
Forstw., 1911, 8.

[12] Butkevitch, V. S., “Ammonia as a product of protein
transformations caused by mould fungi, and the conditions of its
formation,” Recueil d’articles dedié au Prof. C. Timiriazeff, 1916.

[13] Butler, E. J., “An account of the genus _Pythium_ and some
_Chytridiaceæ_,” Mem. Dept. Agr. India, 1907, Bot. Ser. 5, 1.

[14] Christoph, H., “Untersuchungen über die mykotrophen Verhältnisse
der Ericales und die Keimung von Pirolaceen,” Beihefte Bot. Centr.,
1921, 28.

[15] Coleman, D. A., “Environmental factors influencing the activity
of soil fungi,” Soil Sci., 1916, 2.

[16] Dascewska, W., “Étude sur la désagrégation de la cellulose dans
la terre de bruyère et la tourbe,” Univ. Genève, Inst. Bot., 1913, S.
8.

[17] De Bruyn, H. L. G., “The saprophytic life of _Phytophthora_ in
the soil,” Meded. v. d. Landbouwhoogeschool Wageningen, 1922, xxiv.

[18] Dox, A. W., “Amino acids and micro-organisms,” Proc. Iowa Acad.
Sci., 1917, 24.

[19] Dox, A. W., and Neidig, R. E., “Pentosans in lower fungi,”
Journ. Biol. Chem., 1911, 9.

[20] Duggar, B. M., and Davis, A. R., “Studies in the physiology of
the fungi. (I.) Nitrogen fixation,” Ann. Mo. Bot. Gard., 1916, 3.

[21] Ehrenberg, P., “Die Bewegung des Ammoniakstickstoffs in der
Natur,” Mitt. Landw. Inst., Breslau, 1907, 4.

[22] Ehrlich, F., “Yeasts, moulds, and heterocyclic nitrogen
compounds and alkaloids,” Biochem. Ztschr., 1917, 79.

[23] Ehrlich, F., and Jacobsen, K. A., “Über die Umwandlung von
Aminosäuren in Oxysäuren durch Schimmelpilze,” Ber. Deut. Chem.
Gesell., 1911, 44.

[24] Frank, B., “Ueber die auf Wurzelsymbiose beruhende Ernährung
gewisser Bäume durch unterirdische Pilze,” Ber. d. Deut. Bot.
Gesell., 1885, 3.

[25] Gillespie, L. J., and Hurst, L. A., “Hydrogen-ion
concentration--soil type--common potato scab,” Soil Sci., 1918, 6.

[26] Hagem, O., “Untersuchungen über Norwegische Mucorineen,”
Vidensk. Selsk. I., Math. Naturw. Klasse, 1910, 7.

[27] Heinze, B. H., “Sind Pilze imstande den elementaren Stickstoff
der Luft zu verarbeiten und den Boden an Gesamtstickstoff
anzureichen,” Ann. Mycol., 1906, 4.

[28] Van Iterson, C., “Die Zersetzung von Cellulose durch Aërobe
Mikroorganismen,” Centr. f. Bakt., 1904, ii, 11.

[29] Jensen, C. N., “Fungous flora of the soil,” Agric. Expt. Sta.
Cornell, Bull. 1912, 315.

[30] Jones, L. R., “Experimental work on the relation of soil
temperature to disease in plants,” Trans. Wisc. Acad. Sci., 1922, 20.

[31] Kappen, H., “Ueber die Zersetzung des Cyanamids durch Pilze,”
Centr. f. Bakt., 1910, ii, 26.

[32] Klöcker, A., “Contribution à la connaissance de la faculté
assimilatrice de douze espèces de levure vis-à-vis de quatre Sucres,”
Compt. Rend. Trav. Lab., Carlsberg, 1919, 14.

[33] Koch, A., und Oelsner, A., “Einfluss von Fichtenharz und Tannin
auf den Stickstoffhaushalt des Bodens und seiner physikalischen
Eigenschaften,” Centr. f. Bakt., 1916, ii, 45.

[34] Kohshi, O., “Ueber die fettzehrenden Wirkungen der Schimmelpilze
nebst dem Verhalten des Organfettes gegen Fäulnis,” Biochem. Ztschr.,
1911, 31.

[35] Kopeloff, N., “The inoculation and incubation of soil fungi,”
Soil Sci., 1916, 1.

[36] Kusano, S., “_Gastrodia elata_ and its symbiotic association
with _Armillaria mellea_,” Journ. Coll. Agric., Imp. Univ., Tokyo,
1911, iv.

[37] Latham, M. E., “Nitrogen assimilation of _Sterigmatocystis
niger_ and the effect of chemical stimulation,” Torrey Bot. Club,
Bull. 1909, 36.

[38] Laurent, “Les reduction des nitrates en nitrites par les graines
et les tubercles,” Bull. Acad. Roy. Sci. Belg., 1890, 20.

[39] Löhnis, F., “Ammonification of cyanamid,” Ztschr. f.
Gärungsphysiol., 1914, v.

[40] Marchal, E., “Sur la production de l’ammoniaque dans le sol par
les microbes,” Bull. Acad. Roy. Sci. Belg., 1893, 25.

[41] Mazé, P., Vila et Lemoigne, “Transformation de la cyanamide en
urée par les microbes du sol,” Compt. Rend. Acad. Sci., Paris, 1919,
169.

[42] McBeth, I. G., “Studies on the decomposition of cellulose in
soils,” Soil Sci., 1916, I.

[43] McBeth, I. G., and Scales, F. M., “The destruction of cellulose
by bacteria and filamentous fungi,” U.S. Dept. Agric, Bur. Pl. Ind.,
1913, Bull. 266.

[44] McLean, H. C, and Wilson, G. W., “Ammonification studies with
soil fungi,” New Jersey Agric. Expt. Sta., 1914, Bull. 270.

[45] Melin, E., “Ueber die mykorrhizenpilze von _Pinus silvestris_
(L.) und _Picea abies_ (L.), Karst.” Svensk. Botan. Tidskr., 1921, xv.

[46] Muntz, A., and Coudon, H., “La fermentation ammoniaque de la
terre,” Compt. Rend. Acad. Sci., Paris, 1893, 116.

[47] Neller, J. R., “Studies on the Correlation between the
production of carbon dioxide and the accumulation of ammonia by soil
organisms,” Soil Sci., 1918, 5.

[48] Otto, H, “Untersuchungen über die Auflösung von Zellulosen und
Zellwänden durch Pilze,” Dissert., Berlin, 1916.

[49] Perotti, B., “Uber das physiologische Verhalten des Dicyanamides
mit Rücksicht auf seinen Wert als Düngemittel,” Centr. f. Bakt.,
1907, ii, 18.

[50] Peyronel, B., “Nuovi casa di rapporti micorizici tra
Basidiomiceti e Fanerogame arboree,” Bull. Soc. Bot. Ital., 1922.

[51] Potter, R. S., and Snyder, R. S., “The production of carbon
dioxide by moulds inoculated into sterile soil,” Soil Sci., 1918, 5.

[52] Povah, A. H. W., “A critical study of certain species of
_Mucor_,” Bull. Torrey Bot. Club, 1917, 44.

[53] Pratt, O. A., “Soil fungi in relation to diseases of the Irish
potato in Southern Idaho,” Journ. Agric. Res., 1918, 13.

[54] Raistrick, H., and Clark, A. B., “On the mechanism of oxalic
acid formation by _Aspergillus niger_,” Biochem. Journ., 1919, 13.

[55] Rayner, M. C., “Nitrogen fixation in Ericaceae,” Bot. Gaz.,
1922, 73.

[56] Ritter, G. E., “Contributions to the physiology of mould fungi,”
Voronege, 1916.

[57] Rosseels, E., “L’influence des microorganismes sur la croissance
des végétaux supérieurs,” Bull. Soc. Centrale Forest. Belg., 1916, 23.

[58] Roussy, A., “Sur la vie des champignons en milieux Gras,” Compt.
Rend. Acad. Sci., Paris, 1909, 149.

[59] Scales, F. M., “The Enzymes of _Aspergillus terricola_,” Journ.
Biol. Chem., 1914, 19.

[60] Schellenberg, H. C., “Untersuchungen über das Verhalten einiger
Pilze gegen Hemizellulosen,” Flora, 1908, 98.

[61] Schmitz, H., “The relation of bacteria to cellulose fermentation
induced by fungi with special reference to the decay of wood,” Ann.
Mo. Bot. Gard., 1919, vi.

[62] Shibata, K., “Uber das Vorkommen vom Amide spaltenden Enzymen
bei Pilzen,” Beitr. Chem. Physiol. u. Path., 1904, 5.

[63] Ternetz, C., “Über die Assimilation des atmosphärischen
Stickstoffs durch Pilze,” Jahrb. f. wiss. Bot., 1907, 44.

[64] Verkade, P. E., and Söhngen, N. L., “Attackability of cis- and
trans-isomeric unsaturated acids by moulds,” Centr. f. Bakt., 1920,
ii, 50.

[65] Waksman, S. A., “Soil fungi and their activities,” Soil Sci.,
1916, 2.

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ammonia accumulation by micro-organisms,” Journ. Amer. Chem. Soc.,
1917, 39.

[67] Waksman, S. A., “Proteolytic enzymes of soil fungi and
_Actinomycetes_,” Journ. Bact., 1918, 3.

[68] Waksman, S. A., “On the metabolism of _Actinomycetes_,” Proc.
Soc. Amer. Bact. Abstract Bact., 1919, 3.

[69] Waksman, S. A., “The influence of soil reaction upon the growth
of _Actinomycetes_ causing potato scab,” Soil Sci., 1922, xiv.

[70] Waksman, S. A., and Cook, R. C., “Incubation studies with soil
fungi,” Soil Sci., 1916, 1.

CHAPTER IX.

THE INVERTEBRATE FAUNA OF THE SOIL (OTHER THAN PROTOZOA).

The micro-organisms of the soil have been fully discussed in the
preceding chapters of this volume. There now remains to be considered
the fauna of invertebrate animals, other than protozoa, which inhabit
that same medium. In the first place, it is necessary to define what
groups of invertebrate animals are to be regarded as coming under the
category of soil organisms. The latter expression has rather a wide
application and, for the present purpose, is held to mean any organism
of its kind which, in some stage or stages of its life-cycle, lives
on or below the surface of the soil. It will be obvious that, with so
comprehensive a definition, the intimacy of the association of these
animals with the soil will vary within very wide limits. Some animals
pass their whole life-cycle in the soil; others are only present during
a limited phase, and not necessarily in a trophic condition, but since
their occurrence is constant, they cannot be entirely omitted from
consideration.

Unlike the groups of organisms which have been dealt with in the
foregoing pages, the invertebrates of the soil do not admit, as a rule,
of investigation in culture media. It is, in consequence, much more
difficult to achieve in the laboratory the same control over their
environmental conditions. This fact in itself largely explains why
the interpretations of field observations in animal ecology have not
usually been subjected to the test of laboratory experimentation. The
study of animal ecology, in so far as the denizens of the soil are
concerned, is of very recent birth. It has not, as yet, passed the
preliminary stage of cataloguing empirical data, and much spade work
will be necessary before the various factors controlling the phenomena
actually observed are understood.

Owing to the paucity of information available, this chapter is
essentially based upon observations conducted at Rothamsted. Its object
is not so much to attempt to evaluate the invertebrate fauna of the
soil, as to suggest a line of ecological work demanding investigation
on land of many different types.

METHOD OF INVESTIGATING THE SOIL FAUNA.

The method adopted at Rothamsted consists in taking weekly soil samples
from a given area for a period of twelve months. Each sample is a cube
of soil, with a side dimension of nine inches, and a total content
of 729 cubic inches. The samples are taken by means of an apparatus
consisting of four iron plates, which are driven into the ground down
to the required depth so as to form a kind of box, which encloses a
cube of soil (_vide_ Morris, 1922 A). The latter is then removed in
layers, each layer being transferred to a separate bag for the purpose.
When the complete sample has been extracted, there are five bags
containing layers of soil taken from the surface to a depth of 1″, from
1″ to 3″, from 3″ to 5″, from 5″ to 7″, and 7″ to 9″ respectively.
Below a depth of 9″ no samples have been taken.

The sample obtained in this manner may be gradually worked into small
fragments by hand, and examined whenever necessary under a binocular
microscope for the smaller organisms present. This procedure, however,
is very tedious and has been replaced by the use of an apparatus
consisting of a series of three sieves, with meshes of decreasing size
(_vide_ Morris, 1922). The soil is washed through these sieves by means
of a stream of water, and the meshes of the final strainer are small
enough to retain all except the most minute organisms present, while at
the same time they allow the finest soil particles to be carried away.
When desirable, the effluent can be passed through a bag or sieve of
bolting silk, in order to collect such organisms that may have passed
through the third sieve.

In addition to the actual taking and examination of the samples, a
botanical survey of the area under investigation is made; chemical
and mechanical analyses of the soil are also required. It is further
necessary to take soil temperature readings, to determine the moisture
content of the samples taken, and the amount of organic matter which
they contain.

GROUPS OF INVERTEBRATA REPRESENTED IN THE SOIL.

The various groups of invertebrates represented in the soil may be
briefly referred to in zoological order.

_Nematoda._--The Nematoda or thread-worms are chiefly animal parasites,
nevertheless they usually lead an independent existence in the soil
in certain stages of their development. The numerous small species
belonging to the family _Anguillididæ_, or eel-worms, form a definite
constituent of the soil fauna; they are generally free-living and
non-parasitic. Certain members of this family, however, are enemies of
cultivated plants.

_Annelida._--Terrestrial Annelida are almost entirely confined to the
order _Oligochæta_, the majority of which are earthworms (_Terricolæ_),
whose whole life-cycle is passed within the confines of the soil.
The small white worms of the family _Enchytræidæ_ belong to the
aquatic section (_Limicolæ_) of the order, but they have various
representatives which are abundant in damp soil containing organic
matter.

_Mollusca._--The terrestrial Mollusca are included in the sub-order
_Pulmonata_ of the _Gastropoda_. These organisms, which include the
snails (_Helicidæ_) and slugs (_Limacidæ_), regularly deposit their
eggs in moist earth. Slugs adopt the soil as a frequent habitat, only
leaving it for feeding purposes in the presence of sufficient moisture.
They are frequent consumers of vegetation, with the exception of
_Testacella_, which is carnivorous.

_Crustacea._--The few species of Crustacea inhabiting the soil belong
to the order _Isopoda_, family _Oniscidæ_, which are popularly referred
to as “woodlice,” “slaters,” etc.

_Myriapoda._--The _Diplopoda_ or millipedes include enemies of
various crops and are common denizens of the soil. The _Chilopoda_ or
centipedes are usually less abundant and are carnivorous. The minute
_Symphyla_ are often evident but are of minor importance.

_Insecta._--Insects form the dominant element in the invertebrate
fauna. Phytophagous species devour the subterranean parts of plants,
and notable examples are afforded by the larvæ of _Melolontha_,
_Agriotes_ and _Tipula_. Saprophagous forms are abundantly represented
by the _Collembola_, and by numerous larval _Diptera_ and _Coleoptera_.
Predaceous species preying upon other members of the soil fauna
are exemplified by the _Carabidæ_ and many larval _Diptera_.
Parasitic species pass their larval stages on or within the bodies
of other organisms. The groups of _Hymenoptera_, and the dipterous
family _Tachinidæ_, which exhibit this habit, constitute, along
with predaceous forms, one of the most important natural agencies
controlling the multiplication of insect life. There are also insects
(ants, and other of the aculeate _Hymenoptera_) which utilize the
soil as a suitable medium wherein to construct their habitations or
brood chambers, without necessarily deriving their food from the soil.
Lastly, there are many insects, notably _Lepidoptera_, which only
resort to the soil for the purpose of undergoing pupation. The insect
fauna is, therefore, a closely inter-connected biological complex; for
a discussion and an enumeration of its representatives reference may be
made to papers by Cameron (1913, 1917), and Morris (1921, 1922 a).

_Arachnida._--The two principal classes represented in the soil are the
_Areinida_, or spiders, and the _Acarina_, or mites, and ticks. The
_Areinida_, which are well-known to be carnivorous, are an unimportant
constituent of the fauna. _Acarina_, on the other hand, are abundant,
and exhibit a wide range of feeding habits; most of the soil forms are
probably carnivorous, and either free-living or parasitic.

NUMBER OF ORGANISMS PRESENT AND THEIR DISTRIBUTION IN DEPTH.

In computing the number of invertebrates normally present in a given
type of soil, the method adopted consists of making individual counts
of all such organisms as occur in each sample of a series taken over
a period of twelve months. This method considerably reduces errors
due to season and to the possible deviation of one or more samples
from the average. If the total number of these organisms is known for
the samples taken, it becomes a simple procedure to arrive at their
approximate numbers per acre.

TABLE XIV.

(Based on Morris, 1922 A.)

+------------------------------+---------------+-------------+
| |Unmanured Plot.|Manured Plot.|
+------------------------------+---------------+-------------+
|Insects | 2,474,700 | 7,727,300 |
+------------------------------+---------------+-------------+
|Larger Nematoda and Oligochæta| | |
| Limicolæ | 794,600 | 3,600,400 |
+------------------------------+---------------+-------------+
|Myriapoda-- | | |
| Diplopoda | 596,000 | 1,367,000 |
| Chilopoda | 215,400 | 208,700 |
| Symphyla | 64,000 | 215,500 |
| +---------------+-------------+
| Total | 875,400 | 1,791,200 |
+------------------------------+---------------+-------------+
|Oligochæta (Terricolæ) | 457,900 | 1,010,100 |
+------------------------------+---------------+-------------+
|Arachnida-- | | |
| Acarina | 215,400 | 531,900 |
| Areinida | 20,200 | 20,200 |
| +---------------+-------------+
| Total | 235,600 | 552,100 |
+------------------------------+---------------+-------------+
|Crustacea (Isopoda) | 33,700 | 80,800 |
+------------------------------+---------------+-------------+
|Mollusca (Pulmonata) | 13,500 | 33,700 |
+------------------------------+---------------+-------------+
| Total Invertebrata | 4,885,400 | 14,795,600 |
+------------------------------+---------------+-------------+

[Illustration: FIG. 20.--Distribution in depth of the more important
groups of soil invertebrates in the manured and unmanured (or control)
plots at Rothamsted. (From Morris, “Annals of Applied Biology,” vol.
ix., nos. 3 and 4, Cambridge University Press.)]

Table XIV. represents a numerical estimate of the invertebrate fauna of
two plots of arable land at Rothamsted. The soil is clay with flints
overlying chalk, and the land in question has been devoted for eighty
years to continuous cropping with wheat; one plot (No. 3) receives an
annual dressing of farmyard manure at the rate of 14 tons per acre, and
the other plot (No. 2) receives no natural or artificial fertilizer.
The significant feature in a comparison of the fauna of the two plots
is the great numerical increase in organisms due to the addition of
manure. From the point of view of distribution in depth, Fig. 20
clearly demonstrates that the bulk of the fauna is concentrated in the
first three inches of the soil. With the exception of the _Acarina_ it
is evident that the limits of vertical distribution extend below the
depth of nine inches investigated, although the numbers of organisms
likely to be present are inconsiderable. The _Oligochæta_, or true
earthworms, occur in Rothamsted soil in numbers very much in excess of
the figures given by Darwin, who quoted observations by Hensen. The
latter authority calculated that there were 53,767 earthworms in an
acre of garden soil, and estimated that about half that number would
be present in an acre of corn field. In the Rothamsted investigations
their numbers exceeded Hensen’s estimate over 16 times in unmanured
land, and over 36 times in manured land.

In an area of pasture-land in Cheshire few insects occurred below
a depth of 2 inches, and they reached the limit of their vertical
distribution at or near 6 inches. Their number (3,586,000 per acre)
is considerably in excess of that present in unmanured arable land at
Rothamsted.

[Illustration:

1, _Collembola_; 2, _Thysanura_; 3, _Orthoptera_; 4, _Thysanoptera_;
5, _Hemiptera_; 6, _Lepidoptera_; 7, _Coleoptera_; 8, _Diptera_; 9,
_Hymenoptera_.

FIG. 21.--Number of individuals in the different orders of insects in
manured and unmanured arable land at Rothamsted. (From Morris, “Annals
of Applied Biology,” vol. ix., nos. 3 and 4, Cambridge University
Press.)]

DOMINANCE OF CERTAIN SPECIES AND GROUPS.

In Fig. 21 a numerical analysis is given of the different orders
of insects represented in Rothamsted soil. The ascendency of the
_Hymenoptera_ and _Collembola_ is almost entirely due to the occurrence
of three species in large numbers, viz., the ant _Myrmica lævinodis_
and the _Collembola_, _Onychiurus ambulans_ and _O. fimetarius_. In
the unmanured plot these two _Collembola_ constituted 13 per cent. of
the insects and the species of ant accounted for nearly 28 per cent.
In the manured plot they amounted respectively to 27 per cent. and 36
per cent. of the insects present. Next in order of numerical ascendency
are larval _Diptera_, mainly belonging to the families _Cecidomyidæ_,
_Chironomidæ_, and _Mycetophilidæ_. The _Diptera_ are followed by
the _Coleoptera_, whose most abundant representatives are larval
_Elateridæ_ (wireworms).

[Illustration:

1, _Collembola_; 2, _Thysanura_; 3, _Orthoptera_; 4, _Thysanoptera_;
5, _Hemiptera_; 6, _Lepidoptera_; 7, _Coleoptera_; 8, _Diptera_; 9,
_Hymenoptera_; 10, _Diplopoda_; 11, _Chilopoda_; 12, _Areinida_; 13,
_Acarina_.

FIG. 22.--Number of species of different orders of invertebrates
present in the manured and unmanured (or control) plots at Rothamsted.
(From Morris, “Annals of Applied Biology,” vol. ix., nos. 3 and 4,
Cambridge University Press.)]

In point of view of number of species present (Fig. 22), _Coleoptera_
take the front rank; in the unmanured plot they are very closely
approached by _Collembola_ and _Diptera_.

Passing from the insects, the next assemblage of organisms in point of
number of individuals are the smaller worms. The difficulties attending
the specific identification of these organisms are great, and, in the
present survey, the _Nematodes_ and all the smaller _Oligochætes_ have
not been separated.

The abundance of the _Myriapoda_ is mainly due to the prevalence of
_Diplopoda_, which are represented by four species. The _Chilopoda_
almost entirely consist of a single species _Geophilus longicornis_.

The dominant group of the _Arachnida_ is the _Acarine_ family
_Gamascidæ_, which are represented by about a dozen species.

+---------+-------------+-------------+------------+--------------+
| |Phytophagous.|Saprophagous.|Carnivorous.|Heterophagous.|
+---------+-------------+-------------+------------+--------------+
|Unmanured| | | | |
|plot | 14 | 48 | 13 | 20 |
|Manured | | | | |
|plot | 13 | 58 | 9 | 20 |
+---------+-------------+-------------+------------+--------------+

CLASSIFICATION OF SOIL INVERTEBRATES ACCORDING TO FEEDING HABITS.

From the point of view of the fauna as a whole, the zoological
classification of the soil invertebrates is only significant when the
various groups are analysed according to the feeding habits of their
members. All animals are directly or indirectly dependent upon plant
life for their nutrition. For the present purpose they are divided
into four categories, and the position of each class of animals in the
scheme is based upon the habits of its chief representatives in the
soil. Definite information on this subject, however, is not always
forthcoming, and it is only possible to achieve approximate estimates.
In the table above the percentages in number of individuals present in
the two plots investigated at Rothamsted are given under each type of
feeding habit.

It must be borne in mind that these estimates only apply to average
conditions; the outbreak of a plant pest in any one year must naturally
materially alter the proportions given. The phytophagous organisms are
represented by a certain number of the _Insecta_ together with the
pulmonate _Mollusca_. Carnivorous forms which are mainly beneficial
from the agricultural standpoint, include _Insecta_, together with
the _Chilopoda_, many _Acarina_ and the _Areinida_. Saprophagous
forms constitute the dominant element of the soil fauna. More than
30 per cent. of the _Insecta_ exhibit this habit, which is also the
dominant one in the _Oligochæta_, _Symphyla_, and in many of the soil
_Nematodes_. Heterophagous species include all those of somewhat
plastic habits; for the most part they are saprophagous, but, on the
other hand, a considerable proportion of the species attack growing
plants or exhibit both habits. Under this category are included a
certain number of the _Insecta_, the _Diplopoda_, _Isopoda_, and some
_Acarina_.

THE INFLUENCE OF ENVIRONMENTAL FACTORS UPON THE INVERTEBRATES OF THE
SOIL.

Since animals are endowed with powers of independent locomotion: they
are not necessarily tied to their environment to the same extent
that plants are. The investigation of the influence of environmental
factors sooner or later involves a study of the tropisms of the
animals concerned. Until these are adequately understood it is
scarcely possible to arrive at any exact conclusions relative to their
behaviour in the soil. Insects, for example, respond to the stimuli of
various, and often apparently insignificant forces, acting upon their
sensory organs. Such responses are known as chemotropism, phototropism,
hydrotropism, thermotropism, and so forth according to the nature of
the stimuli. Tropisms are automatic and, so far as they distinguish
sensations, are independent of any choice, and consequently of psychic
phenomena. Animal automatism, however, does not present the rigidity
of mechanical automatism. Differential sensibility, vital rhythms, or
periodicity, etc., are other important aspects of animal behaviour.

The environmental factors, affecting more especially the insect
population of the soil, have been discussed by Cameron (1917) and
Hamilton (1917), and certain broader aspects of animal ecology by
Adams (1915) and Shelford (1912). These factors are so numerous and
so inter-connected, that it is only possible to refer to them briefly
in the space available. As might be expected, soils that are of a
light and open texture are the ones most frequented by soil insects,
nutritional and other factors being equal. Furthermore, it has already
been shown that in arable land insects and other animals penetrate to
a greater depth than in pastures. This fact is primarily due to the
greater looseness of the soil occasioned by agricultural operations,
which ensure at the same time better drainage aeration, and greater
facilities for penetration. Hamilton found that soil insect larvæ are
very sensitive to evaporation, and especially so if the temperature
is 20° C. or over. In their natural habitat the relative humidity of
the air, in moist or wet soil, is not far below saturation, and the
temperature of the soil rarely goes above 20°-23°C., and then only in
exposed, dry, hard soil in which these larvæ do not occur.

The significance of the rate of evaporation as an environmental
factor was first emphasised by Shelford. According to him the best
and more accurate index of the varying physical conditions affecting
land animals, wholly or in part exposed to the atmosphere, is the
evaporating power of air. By means of a porous cup-atmometer, as
devised by Livingston, Shelford has carried out an important series
of experiments on the reactions of various animals to atmospheres of
different evaporation capacities, and reference should be made to his
text-book.

The importance of the organic matter present in the soil is well
illustrated in the table on p. 152. The great increase in the number of
insects and other animals is partly due to their direct introduction
along with the manure, and partly to their entry into the soil in
response to chemotropic stimuli exerted by fermentation. Organic matter
influences the fauna in other ways also; it increases the moisture
content of the soil, and it provides many species with an abundance of
food material. Also, the amount of carbon dioxide present in the soil
is partly dependent upon decaying organic matter. Hamilton conducted
experiments on the behaviour of certain soil insects in relation to
varying amounts of carbon dioxide. Although his work is of too limited
a nature to be accepted without reserve, it lends support to the
conclusions of Adams who says: “The animals which thrive in the soil
are likely to be those which tolerate a large amount of carbon dioxide,
and are able to use a relatively small amount of oxygen, at least for
considerable intervals, as when the soil is wet during prolonged rains.
The optimum soil habitat is therefore determined, to a very important
degree, by the proper ratio or balance between the amount of available
oxygen and the amount of carbon dioxide which can be endured without
injury.”

Little is known concerning the occurrence of ammonia in the soil
atmosphere, but its presence in minute quantities is probably an
important chemotropic factor in relation to saprophagous organisms
which are the largest constituent of the fauna. A great increase in
Dipterous larvæ occurs on the addition of farmyard manure, and this
is noteworthy in the light of Richardson’s experiments (1916), which
indicate that ammonia exercises a marked attraction for _Diptera_,
which spend some part of their existence in animal excrement in some
form or another.

The nature of the vegetation supported by the soil is of paramount
importance in relation to phytophagous organisms, and examples need
scarcely be instanced of certain species of soil insects being
dependent upon the presence of their specific food plants.

THE RELATION OF SOIL INVERTEBRATES TO AGRICULTURE.

The relation of these organisms to agriculture may be considered from
three points of view: (_a_) their influence upon the soil itself; (_b_)
their relation to the nitrogen cycle; and (_c_), their direct influence
upon economic plants.

(_a_) The behaviour of earthworms as a factor inducing soil fertility
is discussed by Darwin in his well-known work on the subject, and their
action may be briefly summarised as follows. In feeding habits they
are very largely saprophagous, and consume decaying vegetable matter
including humus, which they swallow, together with large quantities of
soil. Earthworms come to the surface to discharge their fæces (“worm
casts”), and in this process they are continually bringing up some of
the deeper soil to the air. Darwin estimated that earthworms annually
brought to the surface of the soil in their “casts” sufficient earth
to form a layer ·2 inch in depth, or 10 tons per acre. Their action,
along with the atmosphere, are the chief agencies which produce the
uniformity and looseness of texture of the surface soil. By means of
their burrows earthworms facilitate the penetration of air and water
into the soil, while their habit of dragging leaves and other vegetable
material into these burrows increases the organic matter present
below the surface. These facts are generally agreed upon, but it is a
disputed point whether earthworms, by devouring organic matter, aid the
conversion of the latter into plant food more rapidly than takes place
solely through the activities of micro-organisms.

Soil insects and other arthropods, by their burrowing activities,
are also instrumental in loosening the soil texture and thereby
facilitating soil aeration and the percolation of water. The action of
termites in warmer countries is discussed by Drummond in his “Tropical
Africa,” who compares the rôle of subterranean termites to that of
earthworms. The great abundance of ants renders them also significant
in this same respect, and very few species are direct enemies of the
agriculturist.

[Illustration: FIG. 23.--Diagram showing the Relation of the Soil
Invertebrata (other than Protozoa) to the Nitrogen Cycle.]

(_b_) In their relation to the nitrogen cycle (_vide_ p. 174), the
activities of the soil invertebrates may be expressed diagrammatically,
as a side-chain in the process (Fig. 23). The proteins, elaborated by
plants, are utilised as nitrogenous food by the phytophagous animals
present. The waste products of the latter, which contain the nitrogen
not used for growth or the replacement of loss by wear and tear, are
returned to the soil. Here they disintegrate, and are ultimately
converted into ammonium salts, mainly by bacterial action. The dead
bodies of these animals are also broken down by various means, becoming
eventually chemically dissociated and available as plant food. Animal
(and plant) residues serve, however, as food for the large number of
saprophagous invertebrates present in the soil. In this event the
nitrogen contained in such residues becomes “locked up,” as it were,
for the time being in their bodies. Both saprophagous and phytophagous
animals are preyed upon by carnivorous species, but ultimately the
nitrogen is returned to the soil upon the death of those organisms.
The amount present in the bodies of the whole invertebrate fauna has
been calculated by Morris (1922) upon analyses furnished by chemists
at Rothamsted. It is estimated that the fauna of manured land contains
about 7349 grm., or 16·2 lb. of nitrogen per acre, and that of
untreated land, 3490 grm., or 7·5 lb. per acre. These amounts are equal
respectively to the nitrogen content of 103·6 lb. and 48 lb. of nitrate
of soda.

The primary question affecting agriculture is, whether any notable
loss of nitrogen is occasioned by the presence of these organisms in
the soil. It has been mentioned that their nitrogenous waste material,
and their dead bodies, ultimately undergo disintegration; any loss,
if any, takes place during the latter process. With the more complex
compounds it probably consists in the production of amino-acids and
their subsequent hydrolysis or oxidation. During this process an
appreciable loss of nitrogen in the gaseous form occurs. This loss,
which is discussed on p. 173 would represent the net deficit occasioned
by the incidence of invertebrates in the soil. Against this loss must
be placed the beneficial action of such organisms as earthworms, which,
in all probability, more than counterbalances it.

(_c_) Many soil insects, on account of their phytophagous habits, are
well-known to be some of the most serious enemies of agriculture.
Certain of these, and also other classes of invertebrates, which are
likewise directly injurious, have been instanced in the earlier pages
of this chapter. Detailed information on this subject will be found in
textbooks of economic zoology, notably the volume by Reh (1913).

LITERATURE REFERRED TO.

ADAMS, C. C., “An Ecological Study of Prairie and Forest
Invertebrates,” Bull. Illin. St. Lab. Nat. Hist., 1915, xi.

CAMERON, A. E., “General Survey of the Insect Fauna of the Soil,”
Journ. Econ. Biol., 1913, viii. “Insect Association of a Local
Environmental Complex in the District of Holmes Chapel, Cheshire,”
Trans. Roy. Soc. Edin., 1917, lii.

DARWIN, C., “Vegetable Mould and Earthworms,” London, 1881.

HAMILTON, C. C., “The Behaviour of some Soil Insects in Gradients of
Evaporating Power of Air, etc.,” Biol. Bull., 1917, xxxii.

MORRIS, H. M., “Observations on the Insect Fauna of Permanent Pasture
in Cheshire,” Ann. App. Biol., 1921, vii. “On a Method of Separating
Insects and other Arthropods from Soil,” Bull. Entom. Res., 1922,
xiii. “The Insect and Other Invertebrate Fauna of Arable Land at
Rothamsted,” Ann. App. Biol., 1922 A, ix.

REH, L., In Sorauer’s “Pflanzenkrankheiten,” 1913, iii.

RICHARDSON, C. H., “The Attraction of Diptera to Ammonia,” Ann. Ent.
Soc. Amer., 1916, ix.

RUSSELL, E. J., “The Effect of Earthworms on Soil Productiveness,”
Journ. Agric. Sci., 1910, iii.

SHELFORD, V. E., “Animal Communities in Temperate America,” Chicago.
1914, “The Importance of the Measure of Evaporation in Economic
Studies of Insects,” Journ. Econ. Entom., 1912, vii.

CHAPTER X.

THE CHEMICAL ACTIVITIES OF THE SOIL POPULATION AND THEIR RELATION TO
THE GROWING PLANT.

In the preceding chapters it is shown that the soil is normally
inhabited by a very mixed population of organisms, varying in size from
the smallest bacteria up to nematodes and others just visible to the
unaided eye, on to larger animals, and finally earthworms, which can
be readily seen and handled. These organisms all live in the soil, and
therefore must find in it the conditions necessary for their growth.
We have dealt in the first chapter with the supplies of water, air,
and heat, without which life is clearly impossible. Equally necessary
is the source of energy, for the organism requires energy material as
surely as the motor engine requires petrol, and it ceases to function
unless an adequate supply is forthcoming.

All the energy comes in the first instance from the sun, if we exclude
the unknown but probably small fraction coming from radio-active
elements. But this radiant energy is not utilisable by the soil
population, excepting surface algæ; it has to be transformed into
another kind. So far, chlorophyll is the only known transformer;
it fixes the energy of sunlight and stores it up in bodies like
hemicellulose, sugar, starch, protein, etc. The transformation is
imperfect; even the heaviest yielding crops grown under glass, in
conditions made as favourable as our knowledge permits, utilise only
about 4 per cent. of the total energy available during their period of
growth; in natural conditions not more than 0·4 per cent. is utilised.
Such as it is, however, the energy fixed in the plant represents all,
indeed more than all, that the soil organisms can obtain.

In the state of Nature, vegetation dies and is left on the soil. Two
things may then happen. It may become drawn into the soil by earthworms
and other agents; the energy supply is thus distributed in the soil
to serve the needs of the varied soil population. This is the normal
case, associated with the normal soil population and the normal flora.
If, however, the mingling agents are absent, the dead vegetation lies
like a mat on the surface of the soil, only partially decomposing,
unsuitable for the growth of most seedlings, and effectually preventing
most of the vegetation below from pushing a way through: thus there
comes to be no vegetation at all, or only a very restricted and special
flora. The soil population becomes also specialised. Peats and acid
grassland afford examples.

On the neutral grass plots at Rothamsted, the dead vegetation does not
accumulate on the surface but is rapidly decomposed or drawn into the
soil, leaving the surface of the earth bare and free for the growth
of seedlings. On the acid plots dead vegetation remains long on the
surface, blotting out all new growth excepting two or three grasses
which form underground runners capable of penetrating the mat, and
sorrel, the seedling roots of which seem to have the power of boring
through a fibrous layer of this sort. It is possible to remove the
mat entirely by bacterial action alone, if sufficient lime be added
periodically to make the reaction neutral, but failing these repeated
additions the mat persists.

We shall confine ourselves to the normal case where earthworms bring
the source of energy into the soil.

Directly the energy is available, it begins to be utilised. Two laws
govern the change. The first is well-known to biologists: it states
that the total energy of the system remains constant and can neither
be increased nor diminished except from outside; in other words, that
energy can be neither created nor destroyed. The second law is less
familiar: it is that energy once transformed to heat by one organism
cannot be used again by another. It is not destroyed; it remains
intact, but is useless to the organism. One cannot have an indefinite
chain of organisms living on each other’s excretory products; there was
a certain quantity of energy in the food eaten by the first, and no
more than this quantity can be got out whether one organism obtains the
whole or whether others share it.

The outside value for the amount of energy fixed in the soil is
obtainable by combustion of the soil in a calorimeter, but much of
this is not available to the soil organisms. The normal sedimentary
soils of England still contain decomposition products of the débris
of plants and animals originally deposited with them, but in the long
course of ages much of the extractable energy has been utilised. The
soil population is thus dependent on recently grown vegetation, and it
is therefore largely confined to the layer, usually in this country
about 6 inches thick, through which the recently dead vegetation is
distributed. Below this level there may be sufficient air, water,
temperature, etc., but there is insufficient source of energy for any
large population.

Unfortunately there is no ready means for distinguishing between the
total and the actually available quantity of energy in the soil. But
it is not difficult, by adopting the Rothamsted analytical method, to
ascertain the approximate amount of energy that has been transformed in
a given period. The Rothamsted plots are periodically analysed and a
balance sheet is drawn up showing how much of each constituent has been
added to and removed from the soil in the intervening period. For two
of the Broadbalk plots the results are shown in Tables XV., XVI.

The dunged plot receives 14 tons farmyard manure per annum, a quantity
in excess of what would usually be given; the unmanured plot, on the
other hand, has received no manure for many years and is abnormally
poor. Normal soils lie somewhere between these limits, but tending
rather to the value for the dunged than for the unmanured plot. It will
be seen that each acre of the dunged land loses on an average 41,000
calories per day, while each acre of the unmanured land loses on an
average 2700 calories per day.

TABLE XV.--MATERIAL BALANCE SHEET: BROADBALK SOIL, ROTHAMSTED.

(LB. PER ACRE PER ANNUM.)

+-------------------------+-------------+---------------+
| | Farmyard | No Manure |
| |Manure Added.| Added. |
| +------+------+-------+-------+
| | C. | N. | C. | N. |
+-------------------------+------+------+-------+-------+
|Added in farmyard manure| 3600 | 200 | nil | nil |
|Added in stubble | 300 | 3 | 100 | 1 |
+-------------------------+------+------+-------+-------+
| Total added | 3900 | 203 | 100 | 1 |
|Taken from soil | nil | nil | 200 | nil |
|Stored in soil | 200 | 30 | nil | nil |
+-------------------------+------+------+-------+-------+
| Lost from soil | 3700 | 170 | 300 | nil[H]|
| Per cent. | 95 | 84 | 100 | nil |
+-------------------------+------+------+-------+-------+

Initial C : N ratio in farmyard manure, 18 : 1

Final C : N ratio in soil, 10 : 1.

[H] Gain of 6 lb. See p. 173.

TABLE XVI.--ANNUAL ENERGY CHANGES IN SOIL: BROADBALK. APPROXIMATE
VALUES ONLY.

MILLIONS OF KILO CALORIES PER ACRE PER ANNUM.

+---------------------------------+-------------+---------+
| | Farmyard |No Manure|
| |Manure Added.| Added. |
+---------------------------------+-------------+---------+
|Added in manure | 14 | nil |
|Added in stubble | 2 | 0·3 |
| +-------------+---------+
| Total added | 16 | 0·3 |
|Taken from soil | nil | 0·5-1 |
|Stored in soil | 0·5-1 | nil |
| +-------------+---------+
| Dissipated per annum | 15 | 1 |
+---------------------------------+-------------+---------+
|Per day: calories | 41,000 | 2700 |
|Equivalent to | 12 men. | ¾ man. |
|The human food grown provides for| 2 men. | ½ man. |
+---------------------------------+-------------+---------+

These numbers are interesting when we reflect that the human food
produced on the dunged land yields only 7000 calories per day, from
which it is clear that our agricultural efforts so far provide more
energy for the soil population, for which it was not intended, than for
ourselves.

The account is not complete; we have omitted all reference to the
oxidation of ammonia and of elements other than carbon. Nature
seems to be in an unexpectedly economical mood in the soil, and all
compounds which can be oxidised with liberation of energy seem to
have corresponding organisms capable of utilising them. Even phenol,
benzene, hydrogen, and marsh gas can all be oxidised and utilised as
energy sources by some of the soil population.

Even with this remarkable power the soil population has insufficient
energy to satisfy all its possibilities; our present knowledge
indicates that energy supply is, in this country at any rate, the
factor limiting the numbers of the population. Increases in the water
supply or the temperature of the soil produce no consistent effect on
the population, but directly the energy supply is increased the numbers
at once rise.

MATERIAL CHANGES.

These transformations of energy involve transformations of matter.
The original plant residues may be divided roughly into substances
forming the structure of the plant, such as the hemicelluloses, the
pentosans, gums, and the contents of the cell--the protoplasm and the
storage products, protein; in addition, there are smaller quantities of
fats and waxes and other constituents. Some of the easily-decomposable
carbohydrates never reach the soil at all, being broken down by
intracellular respiration or attack of micro-organisms. But much of the
structure material--hemicelluloses, pentosans, etc.--remains.

Once the plant residues pass through the earthworm bodies they become
completely disintegrated and lose all signs of structure.

The only visible product so far known is humus, the black sticky
substance characteristic of soil and of manure. Two modes of formation
have been suggested. Carbohydrates, sugars, pentosans, etc., are
known to yield furfuraldehyde or hydroxymethylfurfuraldehyde on
decomposition, and it has been shown at Rothamsted that this readily
condenses to form a humus-like body, if not humus itself. In the
laboratory the reaction is effected in presence of acid, but even
amino-acids suffice. All the necessary conditions occur in the soil,
and humus formation may proceed in this way.

Some of the structure material--the lignin--contains aromatic ring
groupings. Fischer and Schrader have shown that in alkaline conditions
these ring substances absorb oxygen and form something very like humus.
It is quite possible that humus formation also proceeds in the soil
in this way. Whether the two products are chemically identical is not
known.

The scheme can be represented thus:--

Cell structure material
|
+----------------+----------------+
| |
Aliphatic Aromatic
(Hemicelluloses, (Lignin, etc.,
Pentosans, etc.) in presence of
| oxygen and under
+-------+------------+ aerobic conditions)
| | |
Fatty acids Furfuraldehyde |
| or |
| Hydroxymethylfurfuraldehyde |
| (in presence of acid) |
| · |
| · |
| · |
| · |
| · |
| · |
| · |
| · |
| · |
↓ ↘ ↓
Calcium carbonate. Humus.

The disintegration of the cell and the first stages in the
decomposition of the structure material are almost certainly brought
about by micro-organisms. Whether they complete the process is not
known: purely chemical agencies could easily account for part.

The decomposition of protein in the soil has not been studied in any
detail. From what is known of the acid hydrolysis and the putrefactive
decompositions, however, it is not difficult to draw up a scheme which,
at any rate, accords with the facts at present known. It is probable
that the protein gives rise to amino-acids, which then break down by
one of the known general reactions.

Two types of non-nitrogenous products may be expected: The aliphatic
amino-acids give rise to ammonia and fatty acids; these form
calcium salts which break down to calcium carbonate. The aromatic
amino-acids--tyrosin, phenylalanine, etc.--which would account for
about 6 per cent. of the nitrogen of vegetable proteins, would be
expected to give ammonia and phenolic substances. Now phenols are
poisonous to plants and if no method existed for their removal the
accumulation would ultimately render the soil sterile. Matters would be
even worse on cultivated soils, since cows’ urine, which enters into
the composition of farmyard manure and is the chief constituent of
liquid manure, contains, according to Mooser, no less than 0·25 to 0·77
grams of _p_-cresol per litre,[I] a quantity three to ten times that
present in human urine. Fortunately this contingency never arises, for
the soil contains a remarkable set of organisms capable of decomposing
the phenols and leaving the soil entirely suitable for plant growth.
This affords an interesting case of an organism--in this case the
plant--growing well in a medium in spite of some adverse condition, not
because it is specially adapted to meet this condition, but because
some wholly different agent removes it.

[I] Mooser, Zeitschrift physiol. Chem., 1909, lxiii., 176. No phenol
was found. It is possible that the _p_-cresol is not entirely derived
from the protein, but that some comes from the glucosides in the
animals’ food.

Other ring compounds, e.g. pyrrol, arise in smaller quantity in the
decomposition of protein, but their fate in the soil is not known.

We may summarise the probable changes of the protein as follows:--

It must be admitted that the evidence is indirect. The rate of
oxidation of ammonia by bacteria in the soil is more rapid than the
rate of formation, so that ammonia is practically never found in the
soil in more than minimal amounts (1 or 2 parts per 1,000,000); indeed,
the only evidence of its formation was for a long time the fact that
no compound other than ammonia could be oxidised by the nitrifying
organism. It has, however, since been shown at Rothamsted that ammonia
accumulates in soils in which the nitrifying organism has been killed.

Nothing is known of the mechanism of the oxidation of ammonia beyond
the fact that it is biological; the reaction is not easily effected
chemically at ordinary temperatures. Possibly the organism assimilates
ammonia at one end of a chain of metabolic processes and excretes
nitrates at the other. Or, the reaction may be simply a straight
oxidation for energy purposes, the ammonia changing to hydroxylamine
and then to nitrous and nitric acids.

The nitrate does not remain long in the soil. Some is taken up by the
plant and some is washed out from the soil. Part, however, either of
the nitrate itself or of one of its precursors is converted into an
insoluble form: probably it is changed into protein by the action of
micro-organisms; it then goes through the whole process once more.

These are the general outlines; they present no particular chemical
difficulties. When we come to details, however, there is much that
cannot be understood.

First of all, there is the slow rate at which complex nitrogen
compounds disappear from the soil in comparison with the rate of
oxidation of the carbon. Thus, in the original plant residues, there
is some forty times as much carbon as nitrogen: before they have been
long in the soil there is only ten times as much carbon as nitrogen;
this seems to be the stable position. What is the reason for this
preferential oxidation of the carbon? No explanation can yet be given.

[Illustration: FIG. 24.

X-axis: 1887-8 1890-1 1900-1 1910-11

Y-axis: ℔ per acre]

An equally difficult problem arises in connection with the length of
time the process will continue. Decomposition of the nitrogen compounds
never seems to be complete in the soil; it dribbles on interminably.
In the year 1870 Lawes and Gilbert cut off a block of soil from its
surroundings and undermined it so that the drainage water could be
collected and analysed. The soil has been kept free from vegetation
or addition of nitrogen compounds from that time till now; yet it has
never failed to yield nitrates, and the annual yield falls off only
very slowly (Fig. 24). This same peculiarity is seen in the yield of
crops on unmanured land: it decreases, but very gradually; even after
eighty years the process is far from complete, and there is no sign
that it will ever come to an end.

TABLE XVII.--APPROXIMATE LOSS OF NITROGEN FROM CULTIVATED SOILS:
BROADBALK WHEAT FIELD, ROTHAMSTED, FORTY-NINE YEARS (1865-1914.)

+------------------------+---------------------+---------------------+
| | Rich Soil: Plot 2. | Poor Soil: Plot 3. |
| | Lb. per Acre. | Lb. per Acre. |
+------------------------+---------------------+---------------------+
|Nitrogen in soil in 1865|·175 per cent. = 4340|·105 per cent. = 2720|
|Nitrogen added in | | |
|manure, rain (5 lb. per | | |
|annum), and seed (2 lb. | | |
|per annum) | 10,140 | 340 |
+------------------------+---------------------+---------------------+
|Nitrogen expected in | | |
|1914 | 14,480 | 3060 |
|Nitrogen found in 1914 |·259 per cent. = 5950|·095 per cent. = 2590|
+------------------------+---------------------+---------------------+
|Loss from soil | 8530 | 470 |
|Nitrogen accounted for | | |
|in crops | 2500 | 750 |
+------------------------+---------------------+---------------------+
|Balance, being dead loss| 6030 | -280[J] |
|Annual dead loss | 123 | - 6[J] |
+------------------------+---------------------+---------------------+

[J] Gains. Possibly the result of bacterial action.

A further remarkable fact connected with the decomposition of the
nitrogen compounds is that it seems invariably to be accompanied by
an evolution of gaseous nitrogen. Apparently there are two cases.
Under anaerobic conditions many of the soil organisms have the power
of obtaining their necessary oxygen from nitrates, thereby causing
a change in the molecule which leads in some cases to liberation of
gaseous nitrogen; but the same result seems to be attained in aerobic
conditions, especially when carbon is being rapidly oxidised.

It is possible that the reaction is the same, and that in spite of the
general aerobic conditions there is locally an anaerobic atmosphere.
But it is also possible that some direct oxidation of protein or
amino-acids may yield gaseous nitrogen. However it is brought about
it affects a considerable proportion of the entire stock of nitrogen,
and it becomes more serious as cultivation is intensified. Thus, on
the Broadbalk plot receiving farmyard manure the loss is particularly
heavy; on the unmanured plot it cannot be detected. The nitrogen
balance-sheet is shown in Table XVII.

The oxidation of carbonaceous matter, however, is not invariably
accompanied by a net loss of nitrogen; in other circumstances there is
a net gain. In natural conditions there seems always to have been some
leguminous vegetation growing; the gain may, therefore, be ascribed
to the activity of the nodule organism. In pot experiments, however,
it has been found possible, by adding sugar to the soil, to obtain
gains of nitrogen where there is no leguminous vegetation, and this is
attributed to the activity of Azotobacter.

The nitrogen cycle as observed in the soil is as follows:--

By denitrifying organisms

There has been but little study of the process of decomposition of the
other compounds in plants. Part, if not all, of the sulphur is known
to appear as sulphate, and some of the phosphorus as phosphate. It is
certain that the plant constituents decompose, for there is no sign of
their accumulation in the soil. They may exert transitory effects, but
there is nothing to show permanent continuance. The toxic conditions
which cause trouble in working with pure cultures of organisms in
specific cultures media do not, so far as is known, arise in the soil.
All attempts to find bacterio-toxins or plant toxins in normal soils
have failed. The product toxic to one organism seems to be a useful
nutrient to another, and so the mixed population keeps the soil healthy
for all its members.

There is little precise knowledge as to the part played by the
different members of the soil population in bringing about these
changes.

We know in a general way that earthworms effect the distribution of
the plant residues in the soil, and serve to disintegrate them; there
is no evidence, however, that they play any indispensable part in the
decomposition. Many root and other fragments do not go through this
process; observation shows that fungi can force a way in, and they may
be followed by nematodes which continue the disintegration. Possibly
some of the flagellates help, and certainly the bacteria do. After that
nothing is certain. We cannot, with certainty, assign any particular
reaction in the decomposition to any specific organism, with the
exception of the oxidation of the phenolic substances, the conversion
of ammonia to nitrite and nitrate, and the fixation of nitrogen. With
these exceptions many organisms seem capable of bringing about the
reactions, and indeed some of the reactions may be purely chemical and
independent of biological agencies.

The relationships between the soil population and soil fertility are
readily stated in general outline, but they are by no means clear cut
when one comes to details; fertility is a complex property, and some
of its factors are independent of soil micro-organisms.

The general relationship between plants and soil organisms is one of
complete mutual interdependence. The growing plant fixes the sun’s
energy and converts it into a form utilisable by the soil organisms;
without the plant they could not exist. The plant is equally dependent
on the soil organisms in at least two directions: their scavenging
action removes the dead vegetation which would, if accumulated on the
surface of the soil, effectively prevent most plants from growing.
Further, the plant is dependent on the soil population for supplies
of nitrates. Nothing is known about the relative efficiencies of the
various soil organisms as scavengers. Numerous fungi and bacteria are
effective producers of ammonia, the precursor of nitrates; it is not
known, however, whether flagellates and such higher forms as nematodes
act in this way.

This widespread power of producing ammonia makes it impossible in our
present knowledge to regard any particular group of organisms as _par
excellence_ promoters of fertility. Indeed, it is safest not to attempt
to do so. The primary purpose of the activities of a soil organism
is to obtain energy and cell material for itself; any benefit to the
plant is purely incidental. For cell material it must have nitrogen
and phosphorus; here it competes with the plant. If it produces more
ammonia than it utilises--in other words, if it is driven to nitrogen
compounds for its energy, then the plant benefits. If, on the other
hand, it absorbs more ammonia than it produces, as happens when it
derives its energy from non-nitrogenous substances, the plant suffers.
Thus, addition of peptone to the soil or an increase in bacterial
numbers effected without addition of external energy (e.g. by partial
sterilisation) leads to increased ammonia supply, and, therefore,
to increased fertility. But addition of sugar to the soil causes so
great an increase of numbers of bacteria and other organisms that
considerable absorption of ammonia and nitrate occurs, and fertility
is for a time depressed.

Both actions proceed in soils partially sterilised by organic
substances, such as phenol, which are utilised by some of the soil
organisms; there is first a great rise in numbers of these particular
organisms with a depression of ammonia and nitrate, then a drop to the
new level, higher than the old one, and an increased production of
ammonia and nitrate resulting from the partial sterilisation effects.

We must then regard the soil population as concerned entirely to
maintain itself, and only incidently benefiting the plant, sometimes,
indeed, injuring it; always essential, yet always taking its toll, and
sometimes a heavy toll, of the plant nutrients it produces.

This effect makes it difficult to deduce simple quantitative
relationships between bacterial activity and soil fertility, and the
difficulty is increased by the fact that bacteria and plants may both
be injured or benefited by the same causes, so that high bacterial
numbers in a fertile soil would not necessarily be the cause, but might
be simply the result of fertility.

The circumstance that certain soil organisms--bacteria, algæ, and
fungi--themselves assimilate ammonia and nitrate may account for
the remarkable slowness of nitrate accumulation, to which reference
has already been made. The protein formed from the assimilated
nitrogen remains in the bodies of the organisms, living or dead, till
decomposition sets in. It is not difficult to picture a cycle of events
in which much of the nitrate formed is at once reabsorbed by other
organisms, and only little is actually thrown off into the soil. Such a
process might continue almost interminably so long as any carbonaceous
material remained.

Finally, we come to the very interesting problem--is it possible to
control the population of the soil?

The problem may seem superfluous in view of the difficulties just
mentioned. Some aspects of it, however, are fairly clearly defined.

In the first instance, some organisms appear to be wholly harmful to
the plant; among them are parasitic eelworms and fungi, and bacteria
causing disease.

Control of these organisms can be brought about by partial
sterilisation, and of all methods heat is the most effective, but it
is costly, and attempts are now being made to replace it by chemical
treatment. The results are promising, but the investigation is
laborious; the organisms show specific relationships, and in finding
a sufficiently potent and convenient poison it is necessary in each
case to make an investigation into the relationship between chemical
constitution and toxicity to the particular organism concerned.
Formaldehyde is usually potent against fungi, and the cresols, and
particularly their chlor- and chloronitro-derivatives, are potent
against animals (eelworms, etc.).

One group of organisms is wholly beneficial, those associated with
leguminous plants. Attempts have been made to increase their activities
by inoculating the soil with more vigorous strains. The practical
difficulties still remain very considerable, but there is hope that
they may be overcome.

It is also possible to shift the balance of the soil population in
certain directions. Special groups of soil organisms can be caused
to multiply temporarily, if not permanently, by satisfying their
particular requirements. Thus, when a soil has been heated above 100°
C. it becomes specially suited to the growth of fungi, and quite
unsuited to certain bacteria such as the nitrifying organisms and
others; if this heated soil is infected with a normal soil population
the fungi develop to a remarkable extent. The nodule organisms appear
to be stimulated by addition of farmyard manure and of phosphates, and
the phenol-destroying organisms by successive small additions of phenol.

Finally, quite apart from the control of disease organisms, it is
possible to alter the soil population considerably by partial
sterilisation, using a temperature of only about 60° C., or a poison
like toluene that favours few of the soil organisms. This problem has
already been discussed in Chapter I.

The control of the soil population is still only in its infancy, but
it already promises useful developments. It cannot, however, be too
strongly insisted that the only sure basis of control is knowledge, and
we cannot hope to push control further till we have learned much more
about the soil population than we know at present.

AUTHOR INDEX.

ADAMETZ, 118.

Adams, 158.

Aiyer, 113.

Appel, 133.

Artari, 107.

Ashby, 42.

BARTHEL, 24.

Beijerinck, 6, 37, 41, 42, 46, 107.

Berthelot, 5, 6, 41.

Bewley, 47, 51, 132.

Bezssonoff, 69.

Boas, 138.

Bokorny, 138.

Bonazzi, 45.

Boresch, 107.

Boussingault, 3.

Bredemann, 24.

Bristol, 106.

Brizi, 113.

Brown and Halversen, 127.

Burgess, 41.

Burrill, 48.

Bussey, Peters and Ulrich, 132.

Butkevitch, 138, 139.

Butler, 132.

CAMERON, 150, 158.

Chodat, 107.

Christensen, 46.

Clayton, 28, 43.

Coleman, 127, 136.

Conn, 23, 54, 61, 123.

Cramer, 39.

Crump, 57, 79, 80.

Cunningham, 69.

Cutler, 57, 58, 78, 80.

DALE, 118, 121.

Darwin, 153.

Dascewska, 134.

De Bruyn, 132.

van Delden, 42.

Delf, 87.

Doryland, 33, 40.

Dox, 138.

Dox and Neidig, 134.

Drummond, 160.

Duggar and Davis, 135.

Duvaine, 20.

EHRENBERG, 138.

Ehrlich and Jacobsen, 138.

Esmarch, 102, 103.

FABRICIUS, 61.

Feilitzen, 61.

Fischer, 118, 126.

Forte, 101.

Frank, 132.

Fritsch, 112.

GAINEY, 46.

Gillespie and Hurst, 140.

Goddard, 118, 120, 121.

Golding, 49.

Goodey, 68, 73, 79, 105.

Greaves, 42, 61.

Green, 37.

Grintzesco, 107.

Groenewege, 42.

HAGEM, 118, 121, 136, 138, 139.

Hamilton, 158, 159.

Hansen, 48.

Hanzawa, 43.

Harrison, 113.

Heinze, 139.

Hellriegel, 5, 6, 46.

Hensen, 100.

Hesselmann, 36.

Hill, 94.

Hiltner, 23.

Hopkins, 36.

Hutchinson, C. M., 42.

Hutchinson, H. B., 27, 43, 47, 51, 57, 105.

VAN ITERSON, 133.

JENSEN, 118, 132.

Jewson, 123.

Joffe, 37.

Jones, D. H. and Murdock, 127.

Jones, L. R., 140.

KAPPEN, 136.

Karrer, 105.

Kaserer, 27.

Klöcker, 134.

Koch, A., 44, 134.

Koch, R., 20, 53.

Kofoid, 88.

Kohshi, 134.

Kopeloff, 118, 136.

Kossowitsch, 111.

Krainskii, 44.

Krzeminiewski, 43.

Kufferath, 107.

LATHAM, 135.

Laurent, 136.

Lawes and Gilbert, 5.

Lebedeff, 27.

Leeuwenhoeck, 20.

Lendner, 118.

Lipman, C. B., 41, 42, 44, 54.

Lipman, J. G., Blair, Owen, and McLean, 94.

Löhnis, 22, 43, 69, 136.

MAGNUS, 107.

Malpighi, 46.

Marchal, 34, 136.

Martin, 73.

Martin and Lewin, 69.

McBeth, 28, 134.

McBeth and Scales, 118, 140.

McLean and Wilson, 118, 136.

Mockeridge, 43.

Moore, G. T., 105.

Morris, 150, 151, 162.

Muntz and Coudon, 118, 136.

NABOKICH, 27.

Nagaoka, 38.

Nakano, 107.

Nasir, 94, 95.

Neller, 137.

OMELIANSKI, 27, 42.

Orla-Jensen, 26, 35.

Otto, 133.

Oudemans and Koning, 118.

PASTEUR, 3, 20.

Perey, 94.

Perotti, 136.

Petersen, 104.

Pillai, 43.

Potter and Snyder, 137, 138.

Povah, 138.

Pratt, 132.

Prescott, 61.

Pringsheim, 107.

RAMANN, 118.

Rathbun, 120.

Reh, 162.

Remy, 118.

Richards, 112.

Richardson, 159.

Ritter, 138.

Robbins, W. J., 109.

Robbins, W. W., 105.

Roussy, 134.

Russell, 112.

Russell and Hutchinson, 57, 66, 94.

SALUNSKOV, 42.

Sandon, 57, 75.

Scales, 28, 134.

Schellenberg, 133.

Schindler, 107.

Schloesing, 3, 4, 34.

Schmitz, 134.

Schramm, 111.

Servettaz, 109.

Seydel, 44.

Sherman, 69.

Shibata, 136.

Söhngen, 26, 27, 134.

TAKAHASHI, 118.

Taylor, 120.

Ternetz, 135.

Treub, 112.

Truffaut, 69.

VERKADE and Söhngen, 134.

Von Ubisch, 109.

WAKSMAN, 37, 118, 120, 121, 123, 125, 126, 134, 136.

Waksman and Cook, 136.

Wann, 111.

Warington, 4, 34.

Waynick, 42.

Welwitsch, 112.

Werkenthin, 120, 121.

West, 88, 105.

Whiting, 36.

Wilfarth, 46.

Winogradsky, 4, 6, 34, 41, 44.

SUBJECT INDEX.

_Absidia_, 121.

_Acarina_, 150, 151, 157.

Acid formation by Fungi, 139.

Acidity of soil, 17; effect on Actinomyces, 140; relation to
nitrification, 36.

_Actinomycetes_, 119, 134, 139.

Aeration of soil, effect on bacteria of, 61.

_Agriotes_, 150.

Air supply in soil, 17.

Algæ, agents causing disappearance of nitrate from soil, 12;
associations of, in soil, 105, 106; blue green, 102 _sqq._ (see also
Cyanophyceæ and Myxophyceæ); colonisation of new ground by, 112;
conditions of growth for, 101, 104, 107, 108; distribution of, 102,
104, 106, 109; economic significance of, 100, 102; filamentous,
106; flora of soil, 101, 112; formation of humus substances, 112;
fragmentation of filaments, 107, 110; frequency of occurrence, 102
_sqq._; glucose, effect of, on growth, 108, 109; green, 104 _sqq._
(see also Chlorophyceæ); importance in cultivation of rice, 113;
numbers in soil of, 109, 110; nutrition of, 107, 108, 110; producers,
of organic substance, 100; pure cultures of, 107, 111; relation to
gaseous interchange in soil, 113; relation to soil moisture, 112;
seasonal changes in numbers of, 88; subterranean, 105.

Alkaloids, as source of nitrogen for fungi, 138.

_Alternaria_, 119.

Amino-acids, formation of, by algæ, 108.

Amino-compounds, decomposition of, by fungi, 136, 138.

Ammonia, assimilation of, by bacteria, 33, 40, 45; effect of partial
sterilisation on soil content of, 66; formation in soil, 170;
formation in soil by bacteria, 32 _sqq._; formation in soil by fungi,
135 _sqq._, 141; influence of physical conditions on formation of,
137; property of attracting Diptera, 159; utilisation by higher
plants, 36.

Ammonium sulphate, effect on fungi, 121, 126, 127.

_Anabæna_, 102, 112.

_Annelida_, 149.

Antagonism of salts in soil, 60.

Ants, 153.

_Arachnida_, 150, 151.

Arctic soil, bacterial flora of, 24.

_Areinida_, 150, 151, 157.

_Armillaria_, 132.

_Ascomycetes_, 119.

_Aspergillaceæ_, 136.

_Aspergillus_, 119, 120, 135, 136, 138, 139.

Azotobacter, 6, 41, 95, 96; assimilation of nitrates by, 45;
decreasing efficiency in liquid culture, 44; indicator of soil
acidity, 44.

BACILLARIACEÆ, 100 (see also Diatom).

_Bacillus amylobacter_, distribution of, 24.

_Bacillus radicicola_, 24, 46 _sqq._; inoculation of soil with, 50;
life cycle of, 47.

Bacteria, association with algæ in nitrogen fixation, 111; anærobic
respiration of, 37; effect of arsenic on, 61; cellulose destroying,
134; changes in morphology in culture, 22, 47; classification of main
groups, 23, 25; composition of cells of, 39; inverse relationship
with protozoa, 10, 79, 82 _sqq._; isolation from soil, 21; methods
of describing, 21; method of estimating numbers of, 53 _sqq._, 80;
nitrogen fixation by, 110, 111; numbers in relation to algæ, 110;
numbers in soil, 52 _sqq._; oxidation of hydrogen by, 27, 37; effect
of partial sterilisation on, 8, 9, 66, 67; part played in soil
fertility by, 7; pure cultures, isolation by plating, 20; seasonal
changes in numbers of, 59, 87 _sqq._; effect of salts on, 60; short
time changes in numbers of, 11, 57, 58; effect of temperature on, 67;
uneven distribution of, 57.

_Basidiomycetes_, 119, 123, 132.

Beets, attacked by _Phoma betæ_, 135.

_Boletus_, 132.

_Botrytis_, 122.

Bryophyta, 100, 132.

_Bumilleria_, 105.

CALCIUM compounds in soil and fungi, 139.

_Carabidæ_, 150.

Carbohydrates, decomposition by bacteria, 26 _sqq._; decomposition by
fungi, 140; decomposition in soil, 168; effect on ammonia production
in soil, 33; presence in algal sheath and bacteria, 111.

Carbon, changes in amount in soil, 167; relationships of bacteria,
27; relationships of fungi, 133; source of, for soil bacteria, 39;
sources of, for soil fungi, 139.

Carbon dioxide, assimilation by algæ, 99, 107, 108; assimilation by
soil bacteria, 35, 36, 40.

Carotin, in algæ, 100; formed by _Spirochæta cytophaga_, 29.

_Cecidomyidæ_, 155.

Cellulose, decomposition by bacteria, 27, _sqq._; decomposition by
fungi, 133, 134, 141; relation of nitrogen supply to decomposition
of, 30; decomposition in soil, 168; as source of energy for nitrogen
fixation, 43.

Centipedes, see _Chilopoda_.

_Cephalosporium_, 120.

_Cephalothecium_, 136.

_Chilopoda_, 157.

_Chironomidæ_, 155.

_Chlorella_, 108.

_Chlorococcum_, 105.

_Chlorophyceæ_, 100.

Chlorophyll, loss of, from algæ, 108.

Ciliates, classification of, 72; cyst wall of, 73.

Citric acid, formation of, by fungi, 139.

_Cladosporium_, 119.

Clamp connections in fungi, 119.

Classification, of algæ, 100; of bacteria, 23, 25; of fungi, 131; of
protozoa, 69 _sqq._

Climate, effect of, on algæ, 101.

_Clostridium_, 41, 44; as fixer of nitrogen, 6.

_Coccomyxa_, 104.

_Coleoptera_, 150, 154, 155.

_Collembola_, 150, 153, 154.

_Colletotrichum_, 131.

Commensals, 132.

_Conjugatæ_, 100.

_Cortinarius_, 132.

Cotton, destroyed by fungi, 134.

Counting, of algæ, 109; of bacteria, 53 _sqq._; of fungi, 122; of
protozoa, 77, 79, 80.

Cresol, decomposition of, by bacteria, 22, 24, 31.

Criteria, physiological, of fungi, 128.

Crop growth, effect on fungi, 122.

_Cryptomonadineæ_, 100.

Cucumber leaf spot, 131.

Cyanamide, decomposition of, by fungi, 136.

_Cyanophyceæ_, 103 (see also _Myxophyceæ_ and blue-green algæ).

_Cylindrospermum_, 102.

Cysts, 68, 73, 74.

DENITRIFICATION, by bacteria, 37; by fungi, 136.

Desiccation, resistance to, by algæ, 106.

Dew, relation to algæ, 101, 113.

Diatoms, 104 _sqq._ (see also _Bacillariaceæ_).

Dicyanamide, decomposition of, by fungi, 136.

Dipeptides, formation of, by algæ, 108.

_Diplopoda_, 157.

_Diptera_, 150, 154, 155, 159.

Disaccharides and fungi, 134.

EARTHWORMS, abundance of, in soil, 153; effect of, in soil, 13, 160,
175.

Eel-worms, 149 (see also _Nematoda_).

_Elaphomyces_, 132.

_Enchytræidæ_, 149.

Energy, laws of, 165; relationships of soils, 166; requirements of
soil organisms, 15, 16.

Energy supply, relation of bacterial activities to, 25 _sqq._, 40,
44; sources of, for soil bacteria, 26 _sqq._, 40, 43; supplies of,
for soil organisms, 111, 164, 167, 168.

Environmental conditions in soil, 16.

Eremacausis, 2.

Ericales, 132, 135.

_Euglena_, 99.

_Euglenaceæ_, 100.

Experimental error, in bacterial counts, 54; in fungal counts, 124.

FARMYARD manure, see Manure.

Fats, used by fungi, 134.

Fatty acids used by fungi, 134.

Fertility of soil, views on, 2; effect of decomposition of plant
residues on, 1, 165; effect of organisms on, 175.

Filter paper, destruction of, by fungi, 133; destruction of, by
_Spirochæta cytophaga_, 28.

Fixation of nitrogen, discovery of, by Berthelot, 5; by bacteria, 40
_sqq._; by algæ, 110, 111; by mixtures of bacteria and algæ, 111; by
fungi, 135 _sqq._ (see also Nitrogen Fixation).

_Flagellatæ_, 100.

Flax sickness and fungi, 122.

Formaldehyde, as agent for destroying fungi, 141.

Fungi, control of, in soil, 139 _sqq._; counting of, 122;
distribution of, in soil, 119 _sqq._, 127; fertilisers, effect of, on
numbers of in soil, 126; as facultative parasites, 131, 132; fruiting
bodies of, 123; destruction of hemicelluloses by, 133; individual,
122, 123; action on monosaccharides of, 134; mineral relationships
of, 139; mycorrhizal, 132, 135, 139, 140; heterocyclic nitrogen
compounds and, 138; occurrence in soil, 118; qualitative study of,
118; selective feeding of, 140; specific determination of, 119.

_Fungi imperfecti_, 119.

_Fusaria_, 134.

_Fusarium_, 119, 120, 122, 128, 133, 136.

_Gamascidæ_, 156.

Gases of swamp water (Paddy soils), 113.

_Gastrodia_, 132.

Gelatinous envelope of algæ, 109, 111.

Geographical distribution of azotobacter, 41; of soil bacteria, 24;
of protozoa, 75, 76; of soil fungi, 119, 125.

Germination, of algal spores, 107.

Glucose, use of, by algae, 108, 109, 111; use of, by moss protonema,
109.

Glycocoll, formation of, by algæ, 108.

_Granulobacter_, 42.

Greenland, bacteria in soil from, 24.

“Grunlandmoor,” fungi in, 126.

_Hantzschia_, 105.

_Hemiptera_, 154.

_Heterokontæ_, 100.

“Hochmoor,” fungi in, 126.

_Hormidium_, 104.

Humus, the food of plants, 1; formation of, by fungi, 134, 141;
formation of, in soil, 168; forest, 132; fungal hyphæ as constituent
of forest humus, 132.

Hydrogen ion concentration, in soil, 17; effect on fungi of, 124.

_Hymenoptera_, 150, 154.

_Insecta_, 150, 157.

Insects, numbers present in soil, 154.

Invertebrata, definition of, 147; method of investigating, 148;
groups represented, 149; distribution in the soil, 151; dominant
species and groups, 153; environmental factors of, 157; feeding
habits, 156; relation to agriculture, 160; relation to nitrogen
cycle, 161.

Iron compounds, oxidation by fungi, 139.

_Isopoda_, 150, 151.

_Leguminosæ_, association with bacteria, 46 _sqq._; enrichment of
ground by, 5.

_Lepidoptera_, 150, 154.

Life cycles, of bacteria, 22, 47; of protozoa, 72 _sqq._

Lime, effect on fungi in soil, 121, 126.

_Lyngbya_, 112.

MAGNESIUM compounds, effect on fungi, 139.

Manganese compounds, effect on bacteria, 61.

Manure, farmyard, effect on algæ, 109, 110; effect on numbers of
bacteria, 60; effect on numbers of fungi, 126; effect on numbers of
insects, 154, 155.

Manure, Artificial, effect on fungi, 127.

Manure, town stable, occurrence of disease organisms in, 132.

_Mastigophora_, classification of, 71; species of, 71.

Media, containing nitrates, chemical analysis of, 111; for counting
soil bacteria, 54; for counting protozoa, 79; for counting fungi,
119, 123.

_Melanconium_, 134.

_Melolontha_, 150.

Methane, oxidation of, by bacteria, 26, 27.

Millipedes, see _Diplopoda_.

Mites, see _Acarina_.

_Mollusca_, 149, 157.

_Moniliaceæ_, 136.

_Mucor_, 120, 121, 136, 138.

_Mucorales_, 121, 134.

_Mucorineæ_, 118.

_Mycetophilidæ_, 155.

Mycorrhiza, 132, 135, 139, 140.

_Myriapoda_, 150, 156.

_Myxophyceæ_, 100 (see also _Cyanophyceæ_ and blue-green algæ).

NAPHTHALENE, decomposition of, by bacteria, 31.

_Naviculoideæ_, 100.

_Nematoda_, 149, 151, 157.

Nitrate, assimilation by algæ, 105, 108, 111; assimilation by
bacteria, 33, 40, 44, 51; assimilation by fungi, 136, 138; removal
from soil, 12, 112, 171; variations in amount in soil, 11.

Nitre-beds, 1.

Nitrification, and bacteria, 34; chemical changes in, 171; and fungi,
136; energy supply in, 35; mechanism of, 1, 3; and soil fertility, 1,
3.

Nitrites and fungi, 136; formation by bacteria, 34.

_Nitrobacter_, 35.

Nitrogen, changes in amount in soil, 167; cycle in soil, 161;
fixation by bacteria, 6, 40 _sqq._; fixation by fungi, 135, 136, 141;
fixation of, in clover plant, 5; increase by protozoa of fixation
of, 94, 95 (fig.); fixation sources of energy for, 43, 49; gain of,
in soil, 174; in invertebrates, 162; loss of, by leaching, 112;
loss of, from cultivated soils, 173; relationships of fungi, 135;
relationships of algæ, 110-112; relationships of bacteria, 32 _sqq._,
40 _sqq._; relationships of insects, 162.

_Nitrosococcus_, 35.

_Nitrosomonas_, 35.

Nodule Organism of the Leguminosæ, 6, 46 _sqq._

_Nostocaceæ_, 100, 101, 102, 107.

_Oligochæta_, 149, 151, 153, 157.

_Oospora_, 120.

_Orcheomyces_, 132.

Orchid cultivation and fungi, 132, 140.

_Orthoptera_, 154.

_Oscillatoriaceæ_, 100, 102.

Osmotic pressure, influencing effect of salts on bacteria, 50.

Oxalic acid, formation of, by fungi, 139.

Oxidations effected by soil organisms; by bacteria, 26 _et seq._; by
fungi, 139.

Oxygen, absorption by soils, 4.

PARTIAL sterilisation of soil, 8, 66 _sqq._, 96, 178; influence of
organic antiseptics, 177; limiting factor in, 67, 68.

Pectin, effect of, on fungi, 134.

_Pedras negras_, 112.

_Penicillia_, 134.

Pentosans, effect of, on fungi, 134.

Peptones, decomposition of, by fungi, 136, 138; source of nitrogen
for algæ, 108.

Periodicity, of protozoa in soil, 90 _sqq._ (fig.), 92 (fig.), 93.

Phenol, decomposition of, by bacteria, 24, 25, 31.

Phenylalanine, formation of, by algæ, 108.

_Phoma_, 132.

_Phormidium_, 106.

Phosphates, availability of, influenced by bacteria, 52; by fungi,
139; effect on bacteria, 46, 51, 60.

Photosynthesis, 99, 100, 107, 110, 113.

Phycocyanin, 100.

Physical conditions in soil, 16.

Physiological criteria, of bacteria, 22; of fungi, 128.

_Phycomycetes_, 119.

_Phytophthora_, 132.

Plant disease, and fungi, 139.

Plant residues, decomposition of, in soil, 168; influence of soil
reaction on, 165; relation to soil fertility, 1, 165.

Plasticity of fungi, 119.

_Plectonema_, 106.

Potassium salts, effect on bacteria, 60; influence of bacteria on the
availability of, 52.

Protein, decomposition of, in soil, 169; decomposition by bacteria,
32; decomposition by fungi, 138, 140.

_Protococcales_, 100.

_Protoderma viride_, 105.

Protonema of mosses, 100, 105, 106, 109.

Protophyta, chlorophyll-bearing, 100.

Protozoa, inoculation into soil of, 85 _sqq._; isolation from soil,
69; classification of, 69 _sqq._; life histories of, 72 _sqq._;
species of, in soil, 70 _sqq._; distribution of, in soil, 74 _sqq._;
retention of, by soil, 78 (fig.); size of, 90; reproductive rates,
93; inverse relation with bacteria, 79 _sqq._; presence of trophic
forms in soil, 9; numbers of, in soil, 90, 96, 97; fluctuations in
numbers of, 10, 81 (fig.), 82; external conditions, effect on, 82;
seasonal changes, effect on, 87 _sqq._; weight of, 90.

_Pteridophyta_, 132.

_Pythium_, 132.

REACTION of soil, 17.

Reaction of soil, effect on bacteria, 36, 37, 46, 48, 61; effect on
protozoa, 93, 94 (see also hydrogen ion concentration).

Relationships of Fungi, commensal, 132; mycorrhizal, 132; symbiotic,
132.

_Rhizopoda_; classification of, 70, 71; species of, 70, 71.

Rhythm, supposed in ammonification by fungi, 137.

_Rhizoctonia_, 132.

_Rhizopus_, 119, 120.

Rice plant, aeration of roots, 113; physiological disease of, 113.

Rock Phosphate as base for nitrifying organisms, 36.

Rothamsted, Broadbalk plot 2 (Farmyard Manure) algæ, 109; fungi, 125,
127; Insects, 152.

Rothamsted, Broadbalk plot 3 (Unmanured) algæ, 109; fungi, 120, 122,
127; Insects, 152.

Rothamsted, Broadbalk Plots 10, 11, and 13; 122, 127.

Rothamsted, Barnfield Plot 1-0 (Farmyard Manure), Protozoa, 80.

Rothamsted, unmanured grass plot, 120.

_Russula_, 132.

Rusts, 119.

_Saccharomyces_, 120.

Saprophytes, facultative, 131.

Saprophytism and algæ, 108, 110.

_Scenedesmus_, 108.

Seasonal fluctuations in numbers of soil organisms, 12, 87 _et seq._,
125.

Selective media, use of, in isolation of soil bacteria, 21.

Serological tests, separation of varieties of _B. radicicola_ by, 48.

Slugs, see _Mollusca_.

Smuts, 119.

Snails, see _Mollusca_.

Soil; comparison of, by volume, 17; effect of depth below surface
on algæ, 101, 104, 109, 110, 113; effect of depth below surface on
insects, 151; effect of depth below surface on fungi, 121, 126, 127;
effect of various treatments on fungi, 126, 127, 132; environmental
factors in, 16; inoculation of, for leguminous plants, 50; moisture
(see Water supply); population, control of, 177 _sqq._; population,
methods of investigation, 10, 15; sterilisation and fungi, 137, 138,
141 (see Partial Sterilisation); stored, survival of algæ in, 107;
type and fungi, 121, 126, 127.

Soil conditions, effect on bacteria, 33, 36, 37, 40, 46, 48, 50, 59
_sqq._; effect on protozoa, 82.

Soil fertility, see Fertility of soil.

_Spicaria_, 120.

Spiders, see _Areinida_.

_Spirochæta cytophaga_, 28, 43.

Spore forming bacteria in soil, 23, 34.

Spore, fungus, inhibition of formation, 123; presence in air of, 118.

Standardisation of cultural methods for soil bacteria, 54 _sqq._

Starch, decomposition of, by fungi, 134.

_Stichococcus_, 108.

Straw; effect on nitrate production in soil, 33; manure, 29; rotting
of, 30.

Sulphur oxidation, by bacteria, 37; by fungi, 139.

Symbiosis, of Azotobacter with other organisms, 42, 43, see also
Mycorrhiza and Nodule organism.

_Symphyla_, 150, 151, 157.

_Symploca_, 112.

_Tachinidæ_, 150.

Tannins, used by fungi, 134.

Temperature of soil and fungi, 127, 140.

Termites, 160.

_Testacella_, 149.

_Thiospirillum_, 37.

_Thysanura_, 154.

_Thysanoptera_, 154.

_Tipula_, 150.

Toluene, decomposition by soil bacteria, 31.

_Tolypothrix_, 112.

_Trichoderma_, 119, 120, 122, 134.

_Trochiscia_, 105.

Tropisms, 157.

_Ulothrix_, 105.

_Ulotrichales_, 100.

Urea, by fungi, 136, 138.

Uric acid, utilisation of, by fungi, 138.

_Vaucheria_, 104, 106.

Vitality, retention of, by algæ and moss protonema, 105, 107.

WATER; supply in soil, 17; and algæ, 112; bacteria, 50, 61, 82;
fungi, 127; protozoa, 82.

Wireworms, 155.

Wood, decay of, 134.

Woodlice, 150; (see also _Isopoda_).

YEASTS, 138.

_Zygnema_, 104.

_Zygorrhynchus mœlleri_, 119, 120, 121.

PRINTED IN GREAT BRITAIN BY THE UNIVERSITY PRESS, ABERDEEN

Transcriber’s Notes

Inconsistent and archaic or unusual spelling, capitalisation,
italicisation, hyphenation, etc. have been retained, unless mentioned
below. The names and classifications of the organisms as used in the
book do not always conform to modern names and classifications; these
have not been changed.

Depending on the hard- and software used and their settings, not all
elements may display as intended.

Page 14, table, lower right hand cell: the data given add up to 8,
not to 9.

Page 47, ·9 × ·18 in size: the source document does not include the
units; presumably the sizes are in microns.

Page 118, endnote 8c (2×): this note does not exist.

Subject Index, entry Zygorrhynchus mœlleri: also refers to
Zygorrhynchus vuilleminii.

Changes made:

Footnotes, tables and illustrations have been moved out of text
paragraphs; some tables have been split or re-arranged.

Several minor obvious typographical, punctuation and spelling errors
(including accents) have been corrected silently. In several cases
spelling differences (mainly of proper names) between the text and
the index and endnotes have been standardised. In the indexes and in
tables some ditto marks have been replaced with the dittoed
text. Some page references have been corrected to indicate the
correct page number.

Indented text under illustrations is not present as such in the
source document, but has been transcribed from the illustration for
legibility and ease of reference. Some tables have been re-arranged
or split to fit the available width.

Page 28, 29: MacBeth changed to McBeth as elsewhere (in the Author
Index the entries MacBeth and McBeth have been merged).

Page 32, formula: 30 changed to 3O.

Page 58: From Barnfeild, ... changed to From Barnfield, ....

Page 85: closing bracket deleted after ... Table VII. and Fig. 13.

Page 90, Table VIII, column 5: 350·000 and 150·000 changed to 350,000
and 150,000.

Page 97: No creature lies or dies to itself, ... changed to No
creature lives or dies to itself, ...

Page 104: Danske Aerofile Alghe changed to Danske Aërofile Alger.

Page 114: Recherche sulla Malattia del Riso ... changed to Ricerche
sulla Malattia del Riso ....

Page 115: ... sur de polymorphisme ... changed to ... sur le
polymorphisme ....

Page 116: literature notes 38 (Robbins) and 48 (Schindler) changed to
33 and 34 respectively.

Page 120: Zygorrhynchus vuillemini changed to Zygorrhynchus
vuilleminii as elsewhere.

Page 126: references to Waksman[24] and [24_e_] changed to [25] and
[25_e_].

Page 129: ... preparée de la pres de Russum ... changed to ...
préparée de la terre humeuse du Spanderswoud, près de Bussum ....

Page 134: reference to Kohshi[24] changed to [34].

Page 143: Sämenbildung changed to Säurenbildung (entry 5);
Wurzelbranderregern im Baden changed to Wurzelbranderregern im Boden
(entry 11).

Page 144: ... Umwandlung von Aminosamen in Oxysämen ... changed to
... Umwandlung von Aminosäuren in Oxysäuren ....; ... Wirkungen der
Schimmelze ... changed to ... Wirkungen der Schimmelpilze ....;
Hydrogen-iron concentration changed to Hydrogen-ion concentration.

Page 145: Ztschr. f. Garungs. Physiol. changed to Ztschr. f.
Gärungsphysiol.

Page 146: einige Pilze gegen Hemizellulosen changed to einiger Pilze
gegen Hemicellulosen.

Page 157: Such responses are known chemotropism ... changed to Such
responses are known as chemotropism ....

Page 170: ... alphatic amino-acids ... changed to ... aliphatic
amino-acids ....

*** End of this LibraryBlog Digital Book "The micro-organisms of the soil" ***

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