The micro-organisms of the soil

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.

“(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:—

“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:—

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.