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withdrawn from suspensions of varying strengths by different types of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.