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.
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).
| Soil Fertilisation. | Reaction. | Numbers of Fungi 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[25e] 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.