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M.Sc., for permission to include unpublished data from our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.